Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

BIOPESTICIDES REGISTRATION ACTION DOCUMENT



### Cry1Ab and Cry1F Bacillus thuringiensis ( Bt ) Corn Plant-Incorporated Protectants

U.S. Environmental Protection Agency Office of Pesticide Programs Biopesticides and Pollution Prevention DivisionCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

2 TABLE OF CONTENTS

I. OVERVIEW...................................................................................................................

......................................... 3

A. EXECUTIVE SUMMARY ..........................................................................................................

.................. 3

B. CRY1AB AND CRY1F BT CORN PLANT-INCORPORATED PROTECTANTS ................................ 3

C. FOOD CLEARANCE/TOLER ANCE EXEMPTION LISTINGS............................................................. 6

II. SCIENCE ASSESSMENT........................................................................................................

............................ 6

A. PRODUCT CH ARACTERIZATION...................................................................................................

........ 6

1. P RODUCT C HARACTERIZATION OF B T 11 C RY 1A B C ORN (006444) .......................................................... 11

2. P RODUCT C HARACTERIZATION OF MON810 C RY 1A B C ORN (006430) .................................................... 13

3. P RODUCT C HARACTERIZATION OF P LANT O PTIMIZED (PO) C RY 1F C ORN (006481)................................. 15

4. P RODUCT C HARACTERIZATION OF M AIZE O PTIMIZED (MO) C RY 1F C ORN (006491) ............................... 19

5. P RODUCT C HARACTERIZATION OF MON 810 X TC1507 (C RY 1A B X C RY 1F) C ORN

................................ 22

B. HUMAN HEALTH ASSESSMENT....................................................................................................

........ 23

1. B ACKGROUND

............................................................................................................................... ............ 23

2. I N VITRO D IGESTIBILITY A SSAY

................................................................................................................ 24

3. A MINO A CID H OMOLOGY AND H EAT S TABILITY

....................................................................................... 25

4. A CUTE O RAL T OXICITY

............................................................................................................................. 25

5. EPA R ECOMMENDATION (2001) ............................................................................................................... 25

6. H UMAN H EALTH A SSESSMENT OF C RY 1A B C ROPS

, I NCLUDING B UT N OT L IMITED T O :........................... 25

B T 11 C RY 1A B B T C ORN (006444) AND MON810 C RY 1A B B T C ORN (006430) ........................................... 25

C. ENVIRONMENTAL ASSESSMENT...................................................................................................

...... 44

1. T IERED H AZARD AND R ISK A SSESSMENT P ROCESS

................................................................................... 45

2. E NVIRONMENTAL E XPOSURE A SSESSMENT

............................................................................................... 48

3. N ON T ARGET W ILDLIFE H AZARD A SSESSMENT

........................................................................................ 58

4. H ORIZONTAL T RANSFER OF T RANSGENES FROM B T C ROPS TO S OIL O RGANISMS

..................................... 93

5. G ENE F LOW AND W EEDINESS P OTENTIAL

................................................................................................. 94

6. I MPACTS ON T HREATENED OR E NDANGERED S PECIES

............................................................................... 99

7. P OTENTIAL I NTERACTION B ETWEEN C RY 1A B AND C RY 1F P ROTEINS

.................................................... 100 (MRID 476778 02)......................................................................................................................

................. 100

8. E COLOGICAL R ISK A SSESSMENT C ONCLUSIONS

...................................................................................... 101

9. REFERENCES.....................................................................................................................

................... 102

D. INSECT RESISTANCE MANAGEMENT.............................................................................................. 125

III. REGULATORY POSITION ON BT CORN...........................................................................................

...... 261

A. OVERVIEW (2001 CONCLUSIONS)...............................................................................................

...... 261

B. GENERAL FINDING (2001 CONCLUSIONS)....................................................................................... 264

C. INSECT RESISTANCE MANAGEMENT (IRM) PROGRAM (2001 CONCLUSIONS) ................. 265

D. 2010 UPDATE.................................................................................................................

............................. 268

E. PERIOD OF REGISTRATION ......................................................................................................

........... 269Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

3 

### I. Overview

A. EXECUTIVE SUMMARY

EPA has made the preliminary decision to amend the registrations of twenty-two (22) expiring Bt Corn PIP registrations to extend the expira tion dates. We have conducted comprehensive assessments of each of these registrations, cons idering all toxicity and environmental effects data, data from insect resistance monitoring, an d insect resistance refuge compliance reports,

received and obtained since the last comprehensive evaluation of these products in 2001. Based upon our comprehensive assessment, we have re ached significant conclusions regarding the positive environmental impact of Bt corn PI Ps, and we are proposing several actions to strengthen the insect resistance management re quirements to ensure continued success in the prevention of the evolution of resistance in target pests.

Since the commercialization of Bt crops, there have been a signi ficant number of published field studies that, combined with the post-registrati on field studies required to be submitted to the Agency, have demonstrated that non-target i nvertebrates are generally more abundant in Bt

cotton and Bt corn fields than in non-transgenic fiel ds managed with chemical insecticides. Thus, these published and registrant-produced studies demonstrate that, not only are the Bt crops not causing any unreasonable adverse effects in the environment, but, arthropod prevalence and diversity is greater in Bt crop fields. Although no effects have been observed in the environment, the Agency is requiring additional aquatic invert ebrate data in light of published laboratory studies showing reduced growth in shredding caddis flies exposed to Cry1A protein corn litter.

To strengthen insect resistance management of these corn PIPs and to address reports that compliance with the mandated refuge requireme nts has been decreasing, EPA is proposing to require enhanced compliance assurance program s (CAPs), and a phased requirement for seed bag labeling that clearly shows th e refuge requirements. Also, give n the increasing variety of PIP products and combinations, and the differing risk of resistance evolution that the various products represent, we are proposi ng to grant registrations for the corn PIP products for different timeframes, based on assessments of their likelih ood of forestalling the evolution of insect resistance. We are registering di ffering categories of products for differing time periods to reflect

the assessed level of risk of resi stance posed by the vari ous corn PIP products. The scheme that we propose to follow will include registration periods generally of five, eight, and twelve years; with the possibility of a fifteen year registration period for produc ts that are demonstrated to meet specified criteria. We retain, however, the discretion to register pr oducts for time periods differing from these defaults where circumstances warrant.

B. Cry1Ab AND Cry1F Bt CORN PLANT-INCORPORATED PROTECTANTS

1. Bt11, Cry1Ab Bt Corn

OPP Chemical Code: 006444Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

4 Pesticide Name: Bacillus thuringiensis Cry1Ab Delta-Endotoxin a nd the Genetic Material Necessary for Its Production (P lasmid Vector pZ01502) in Corn

Trade and Other Names: Bt11, YieldGard, Agrisure®, AttributeTM

Uses : Full Commercial Use in Field Corn and Sweet Corn

Registrant:

Syngenta Seeds, Inc

P.O. Box 12257 Research Triangle Park, NC 27709-2257

Registrations: 67979-1 Bt Corn Event Bt11 w ith Cry 1Ab (Field Corn) 65268-1 Bt Corn Event Bt11 with Cry 1Ab (Sweet Corn)

2. MON810, Cry1Ab Bt Corn

OPP Chemical Code: 006430

Pesticide Name: Bacillus thuringiensis Cry1Ab Delta-Endotoxin and the Ge netic Material Necessary for I

Trade and Other Names: MON 810, Yieldgard®

Uses: Full Commercial Use in Field Corn

Registrants: Monsanto Company 700 Chesterfield Parkway North St. Louis, MO 63198

Pioneer Hi-Bred International, Inc.

P.O. Box 1000 Johnston, Iowa 50131-1000

Registrations: 524-489 Bt Corn Event MON 810 Cry1Ab 29964-7 1507 (POCry1F) x MON 810 (Cry1Ab)

3. TC1507, Cry1F Bt corn

OPP Chemical Code: 006481

Pesticide Name: Bacillus thuringiensis subspecies Cry1F Protein and the Genetic Material Necessary for Its Production (Pla smid Insert PHI 8999) in CornCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

5 Trade and Other Names: HerculexTM I Insect Protection, Pioneer Brand Seed Corn with

HerculexTM I

Registrants: Mycogen Seeds c/o Dow Agrosciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054

Pioneer Hi-Bred International, Inc.

P.O. Box 1000 Johnston, Iowa 50131-1000

Monsanto Company 700 Chesterfield Parkway North St. Louis, MO 63198

Registrations: 68467-2 Bt Corn Event TC1507 with PO Cry 1F 29964-3 Bt Corn Event TC1507 with PO Cry 1F 29964-7 1507 (POCry1F) x MON 810 (Cry1Ab)

Uses: Full Commercial Use in Field Corn

5. DAS-06275-8 moCry1F Bt corn

OPP Chemical Code: 006491

Pesticide Name: Bacillus thuringiensis var. aiza wai strain PS811 Cry1 Finsecticidal crystal protein and the genetic mate rial necessary for its production (plasmid insert PHP12537) in Event DAS-06275-8 corn

Trade and Other Names: Mycogen Brand B.t. moCry1F

Registrants: Mycogen Seeds c/o Dow Agrosciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054

Registrations: 68467-4 Bt Corn Event DAS-06275-8 with MOCry1F

Uses: Full Commercial Use in Field CornCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

6 C. FOOD CLEARANCE/TOLERANCE EXEMPTION LISTINGS

40 CFR Part 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

Residues of Bacillus thuringiensis Cry1Ab protein in all plants ar e exempt from the requirement of a tolerance when used as plant-incor porated protectants in all food commodities. [72 FR 20435, Apr. 25, 2007]

40 CFR Part 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated prot ectants in the food and f eed commodities of corn; corn, field; corn, sweet; and corn, pop. [72 FR 20435, Apr. 25, 2007]



### II. Science Assessment

A. PRODUCT CHARACTERIZATION

Product characterization is criti cal to understanding the way in which the registered products were made and the unique characteristics that need to be assessed for each Bt plant-incorporated protectant. The product characte rization data provide informati on on the specific transformation systems used for each product, on the actual DNA inse rted into the plant, on the inheritance and stability of these traits in the plant, on biochemical characteristics of the Bt protein and on Bt protein expression levels for vari ous plant tissues. Specific inform ation and data for each of the registrations seeking renewal are include d in tabular and descriptive formats.

The classifications that are found for each data submission are assigned by the EPA science

reviewer and are an indication of the usefulness of the informa tion contained in the documents and if the data meet the intent of the test gui delines. A rating of “ACC EPTABLE” indicates the study is scientifically valid and has been sa tisfactorily performed according to accepted EPA guidelines or other justified cr iteria. A “SUPPLEMENTAL” rati ng indicates the data provide

some information that can be useful for risk a ssessment. However, the studies may either have certain aspects not determined to be scientifically acceptable (SUPPLEMENTAL. UPGRADABLE) or that the studies have not been done to fulfill a specific EPA guideline requirement. If a study is rated as “SUPP LEMENTAL. UPGRADABLE,” EPA always provides an indication of what is lacking or what can be provided to change the rating to “ACCEPTABLE.” If there is simp ly a “SUPPLEMENTAL” rating, th e reviewer will often state that the study is not required by current EPA guidelines or does not need to be reclassified as

“ACCEPTABLE.” Both ACCEPTABLE and SUPPLEMEN TAL studies may be used in the riskCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

7 assessment process as appropriate.The follo wing table summarizes the registered Btproteincontaining plant-incorporated prot ectant products being evaluated.

Table A1 - Bt Plant-incorporated protectant Products

Common Name and Cry Protein OPP

Chemical

Code Company Plasmid ID Plant/

Trade Name

Bt 11 Cry1Ab Bt

Corn 006444 Novartis pZO1502 YieldGard,

Attribute

MON810 Cry1Ab

Bt Corn 006430 Monsanto pvZMCT01* pZMBK07 pZMGT10** YieldGard

Cry1F Bt Corn 006481 Mycogen-Dow & Pioneer/Dupont PHI 8999 Herculex

moCry1F Bt Corn 006491 Mycogen-Dow PHP12537 Mycogen Brand Bt Cry1F Event

DAS-06275-8

Corn

* pvZMCT01 was a mixture of two plasmids ** Plasmid contains marker gene.

Transformation systems : Registered corn products were transformed using protoplast electroporation to introduce the desired DNA, methods employing bombardment of particles coated with DNA encoding the intended insert, or Agrobacterium tumefaciens.

.

Each plasmid description includes a reference to the strains of Bacillus thuringiensis used as the source of the DNA sequence for the toxin protein. In addition, the sources for marker proteins, promoters, terminators and enhancers, as we ll as the fragment size, orientation and any modifications to the original DNA sequence to enha nce expression in the plant are given. All the other DNA sequences introduced to improve or restrict expression of the in troduced traits are also described. Finally, the plas mid discussion includes a descrip tion of any modifications made to the DNA (e.g., codon modifications to improve eukaryotic expression).

Characterization of the DNA Inserted in the Plant: Inserted DNA is characterized with Southern blot data of the DNA in the plant ge nome. The analysis usually consists of DNA isolation from the transformed plant, digestion of this DNA with several different endonucleases and hybridization of these re striction endonuclease fragment s with labeled-DNA which is complementary to the introduced traits. This analysis includes not only probes specific for the entire insert, but also probes recognizing just the coding regions of the traits or DNA elements outside the coding region. Poly merase chain reaction (PCR) assa ys utilizing various specific and non-specific primers, genome walking, cosmid libraries and DNA sequenc ing have also been employed with sensitive Southern blotting techniques to more completely describe the insertedCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

8 DNA and surrounding regions. The information avai lable from these blots can indicate the presence of all the elements of the expected insert as well as in formation about the possibility of

deletions and other errors a ssociated with DNA introduction by transformation. Comparison of Southern blots of genomic DNA, digested usin g a range of restricti on endonucleases, can also reveal the copy number of the genes introdu ced and suspected linkage of the traits. Alternatively, the intensity of the radioactive label from binding the probe DNA can also estimate the number of insert copies incorporat ed in the plant genome. When the inserted DNA construct includes traits expressed only in bacteria and not expected to be expressed in the plant, data have been presented to indi cate that there is no transcription or translation of the bacterial trait (e.g., ori and amp r

- discussed further in the horizontal gene transfer section).

Inheritance and Stability after Transformation: The data generated for this endpoint examine progeny from crosses between selected elite lines with the transformed Bt expressing line, looking for the independent segregation of the in troduced traits in the progeny. Traditional breeding work done during the development of the plant line by backcr ossing can reveal the linkage of the introduced traits as well as changes in trait expression. The inheritance data is the ratio of progeny expressing the hemizygous trait based on expected Mendelian inheritance. Stability data implies an examination of either the expression of the trait or tracking of the DNA itself over several plant generations. One of the ma in concerns with stability is spontaneous loss of the inserted DNA or loss of efficacy due to gene silencing. None of the Bt plant-incorporated protectant products showed independent assortment of the introduced traits (usually the marker protein and the Bt protein were examined). This indicat es that the traits were on the same chromosome and closely linked (c rossover events between the two traits we re not detected).

The submissions that covered characterization of the actual DNA insert and stability/inheritance data are listed in the MRIDs for each product. These submissions are acc eptable and fulfill this data requirement. It should be noted that stabil ity and inheritance were not addressed with the registrations for MON810 (006430). However, c onsidering the use of these crops for several growing seasons and the lack of report s relating to loss of efficacy due to Bt protein expression, this specific endpoint can be considered to have been addressed through commercial use.

Protein Characterization and Expression: For the Bt plant-incorporated protectants, data has been presented to demonstrate that the protein expressed from the inserted DNA is similar to what was produced in the source bacterium and is active as expected agains t the intended target insect. Some protein characterization da ta demonstrate that microbially produced Bt protein is the equivalent to that expresse d in the plant. This apparent scientific tautology (where plant produced protein is the same as microbial protein is the same as the plant produced protein) has been used to justify the use of the microbially-produced protein as a test substance in toxicity tests. Because the expression level of these pr oteins is so low in plants, and the maximum hazard dose acute oral toxicity test is required as part of the human health risk assessment for these proteins, the ability to produc e the protein in an industrial mi crobe is essential. The acute oral test requires between 2000 and 5000 mg of protein per kg bodyweight of test animal. Isolating the amount of puri fied protein required to dose several animals from Bt -expressing plants would be a tremendous burden involving harv esting and processing large volumes of plant material (ecological effects testing differs and is addressed in the ecologi cal effects section ofCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

9 this document). Proper characteri zation of the equivalency between these microbial proteins and plant expressed proteins provide s an alternative to purifying the test material as the plantproduced protein from large volumes of tissue. These equivalency data were generated for all products registered to date.

Much of the characterization data describes the procedures used to isolate the protein or a highly

Bt protein enriched fraction of plant extract. The tests done to support the equivalence of microbial and plant-produced Bt protein include: molecular si zing by SDS-PAGE and western blot analysis; immunorecognition using ELISA and we stern blot analysis; N-terminal amino acid sequencing; confirmation of the lack of glyc osylation in the plant- produced protein; and bioactivity against a range of insects (often pest species includ ing the target pest). Since the issues surrounding non-target eff ects are considered essentia l for the ecological effects assessment, these non-target pest tests are also covered in the ecological effects assessment.

The Bt protein expression level in various ti ssues throughout the growing season has been

determined for each event. These data have been determined and presented (for the 2010 update), in terms of dry weight, as the amount of protein present in leaf, root, pollen, seed, and root tissue and whole plant. A lthough Bt11 Cry1Ab data have b een provided for field corn, sweet corn data on the Bt11 expression remain as a data gap.

Table A2 - Comparative expression of Cry1F prot ein in moCry1F TC6275 and poCry1F TC1507 corn tissues

Tissue Growth Stage Mean Standard

Deviation Min/Max Range Mean Standard

Deviation Min/Max Range

TC6275 TC1507

(ng/mg Tissue Dry Weight)

Leaf V9 b

R1

R4

Senescence 17.3

28.5

44.8

0.71 3.41

5.38

16.8

1.14 10.7-23.8

16.5-36.7

35.8-109.2

0-3.0.9 12.1 6.2 0-24

Root V9 b

R1

R4

Senescence 6.14

6.60

5.99

1.97 1.87

1.98

1.89

2.03 4.53-8.14

3.14-10.9

2.35-9.26

0.29-6.91

Whole Plant V9

R1

Senescence 6.22

7.16

2.47 1.16

1.45

0.41 4.98-7.87

5.32-9.57

1.95-3.07 5.2

3.6

1.6 1.9

1.1

0.6 2.6-6.8

2.5-4.7

0.9-2.4

Pollen R1 3.67 0.34 3.09-4.60 21.9 2.9 16.4-27.2

Stalk R1 11.0 2.67 6.77-16.4 5.8 1.7 3.3-10.3

Forage R4 6.26 1.09 5.05-7.77 1.7 1.1 0-3.2

Grain Maturity 1.14 0.27 0.62-1.68 2.2 0.8 0-4Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

b Recalculated Results

Table A3 - Average Cry1Ab concentration from Corn Event MON 810 corn tissues grown in four regions of the US.

Tissue (No. days post planting)

Overseason Leaf (OSL) OSL-1 (21 days) OSL-3 (40 days) OSL-4 (50 days) OSL-5 (60 days) Overseason Whole Plant (OSWP) OSWP-1 (21 days) OSWP-5 (60 days) Forage (90 days) Pollen (60 days) Overseason Root (OSR) OSR-1 (21 days) OSR-3 (40 days) OSR-4 (50 days) OSR-5 (60 days) Forage root (90 days) Grain (125 days) Cry1Ab conc. (ug/g dry weight)

120 ± 15

46 ± 5.8

61 ± 17

51 ± 17

120 ± 34

25 ± 6.3

7.6 ± 4.5

NA

42 ± 9.5

20 ± 5.0

22 ± 3.7

19 ± 8.8

16 ± 6.0

0.63 ± 0.06

NA = Not Applicable.

Residue Analytical Methods

Analytical methods and method validation ( under OPPTS Guidelines OPPTS 860.1340) for the Cry1Ab corn were required in 2001 to complete the database. Although analytical methods had been submitted for Cry1F corn, additional confir matory methods and standard EPA laboratory method validation were also necessary. The Agen cy has decided not to require validation of analytical methods by EPA’s OPP Microbiology Laboratory at Fort Meade provided the Grain Inspection, Packers and Stockyards Administration (GIPSA) of the United States Department of Agriculture (USDA) has verified the performance of an appropriate qualitative rapid test kit. In the case of Cry1Ab and Cry1F corn, the Agency has confirmed that test kits have been verified by GIPSA and, therefore, the aforementioned re quirements have been satisfied. Table A4- Cry1Ab Concentrations on a Dry Weight Basis in Event Bt11 Hybrid Plants

Developmental Stage

V9-V12 Anthesis Seed Maturity Tissue Location Hybrid 1

mean

 g Cry1Ab/gdw ± S.D. 2
(range)

Leaves Bloomington, Illinois Event Bt11 25.88 ± 1.35 (23.89—27.63) 17.82 ± 1.54 (15.99—19.74) 16.84 ± 3.31 (12.17—19.59)

Roots Bloomington, Illinois Event Bt11 9.99 ± 0.59 (9.14—10.62) 6.41 ± 0.79 (5.67—7.72) 4.32 ± 1.52 (3.32—6.99)

Kernels Bloomington, Illinois Event Bt11 N/A 3 N/A 1.45 ± 0.07 (1.39—1.56)

Pollen Mackinaw, Illinois Event Bt11 N/A 0.04 ± 0.00 (0.036—0.042) N/A

Pollen Monroeville, Indiana Event Bt11 N/A 0.06 ± 0.02 (0.048—0.079) N/A

Pollen Seward, Nebraska Event Bt11 N/A <0.037 N/ACry1F & Cry1Ab Bt Corn Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

11 1. Product Characterization of Bt 11 Cry1Ab Corn (006444)

The corn line Bt 11 was produced by transforming anothe r proprietary corn line with plasmid

PZO1502 which contained cry1Ab, pat and amp r genes. The registrant submission stated that prior to the transformation whic h resulted in Bt11, the plasmid pZO1502 was digested with the

restriction endonuclease Not I with the intention to remove the amp r gene from pZO1502. While

no data was submitted to confirm removal of the amp r gene from the transforming DNA, subsequent analysis showed that the amp r gene was not present in Bt11 corn. The cry1Ab gene was also altered to improve its GC ratio for expr ession in corn and coded for a truncated form of the original protein. Both field corn and sweet corn containing th e plant-incorporat ed protectant descend from the original Bt 11 transformant.

Data showed that the truncated Cry1Ab toxin could be extracted from corn leaf tissue and this purified material displays characteristics and activities similar to that produced in E. coli transformed to produce Cry1Ab. The purified tryptic core proteins from both plant and microbe

were shown to be similar in molecular we ight by SDS-PAGE, imm unorecognition in western blots and ELISA, partial amino acid sequence anal ysis, lack of glycosylation and bioactivity against either European corn borer or corn earw orm. This analysis justified the use of the microbially produced toxin as an analogue for th e plant produced protein in mammalian toxicity testing.

The product characterization data supporting the registration of Bacillus thuringiensis Cry1Ab delta-endotoxin and the genetic material necessary for its production (plasmid vector pZO1502)

in corn is listed below.

Study Type Result MRID #

Transformation System Corn line HE89 was transformed with plasmid pZO1502 which contains genes

for a truncated Cr y1Ab, PAT and AMP r . The cry1Ab gene was also altered to improve its GC ratio for expression in corn. (See MRID No. 437548-01 below

which indicates the absence of the amp r gene in the Bt11 and control plants.)

CLASSIFICATION: ACCEPTABLE 431308-01

Inheritance and Stability after

Transformation The linkages of the pat and cry1Ab genes were shown by examining the progeny of two selfed generations derived from a population of corn plants segregating for the desired traits. None of the 2320 plants examined showed the two traits independently assorting which indicates that the loci are tightly linked.

CLASSIFICATION: ACCEPTABLE 433526-02

Transformation System The lack of any positive probe recognition for the plant genomic DNA samples

indicate the absence of the amp r gene in the Bt11 and control plants. The positive

amp r gene probe results for the plasmid DNA digest samples confirm that a fragment of a size consistent with the 7.2 Kb pZO1502 plasmid contained the

amp r gene. This would also be appropriate for any digest which had a single restriction cut site as these enzymes di d according to the pZO1502 map. The probe results also indicate that a Not I digest would release the amp r gene from the pZO1502 plasmid CLASSIFICATION: ACCEPTABLE 437548-0112 Study Type Result MRID #

Protein

Characterization and Expression Data is presented showing that the truncated Cry1Ab toxin can be extracted from corn leaf tissue and this purified material displays characters and activities similar to that produced in E. coli . The similarities are shown in molecular weight after SDS-PAGE, immunorecogniton in western blots and ELISA of trypsin resistant core proteins, partia l amino acid sequence analysis, lack of glycosylation and bioactivity against either European corn borer of corn

earworm. CLASSIFICATION: ACCEPTABLE 433972-02

Protein Expression Event Bt11 corn plant tissues from tw o field corn hybrids were harvested, analyzed by ELISA for ex pression of Cry1Ab. Cry1Ab was detected in all transgenic plant tissues sampled and at all developmental stages. Across all plant stages, mean Cry1Ab levels measured in leaves, roots and whole plants ranged from ca. 12-154 μg/g dry wt., 9-22 μg/g dry wt., and 6-70 μg/g dry wt., respectively. Mean Cry1Ab levels meas ured in kernels at seed maturity and senescence were ca. 2 μg/g dry wt. Cry1Ab levels in pollen were below the lower limit of quantification for pollen. Control sample levels were below the limit of quantification for all stages and tissues. Therefore, the level of Cry1Ab was generally similar between hybrids for each tissue type at each time point. The estimated total Cry1Ab levels per acre and per hectar e over the growing season and across genotypes, ranged from mean levels of ca. 14/g acre (31 g/hectare) at whorl stage to ca. 125 g/acre (283 g/hectare) at kernel maturity, assuming a planting density of 26,500 plants per acre (65,500 plants/hectare).

CLASSIFICATION: ACCEPTABLE 458798-03

Protein Expression The values that are reported do not indicat e vastly different expression levels for Cry1Ab in the sweet corn varieties comp ared to the field corn Bt11 x MIR 162 varieties tested. However, no Bt11 sweet corn tissue was analyzed.

CLASSIFICATION: SUPPLEMENTAL for Bt11 478820-01

Analytical

Method The EnviroLogix Cry1Ab/Cry1Ac QuickStix TM Kit is designed to extract and detect the presence of Cry1Ab ( Bt 11 or Mon810 event) and Cry1Ac Bt proteins at the levels typically expressed in genetically modified corn grain. The sensitivity of the Cry1Ab/Cry1Ac QuickStix strips is 1% based on tests

conducted with Bt 11 corn. Blind studies with conventional corn spiked with

Bt 11 validated the 1% limit of detection. Likewise, corn grain from the following biotech events; Cry9C (StarLink), GA21 (Roundup Ready), NK603 (Roundup Ready), Cry3Bb1 and T25 (Liberty Link) were tested for cross reactivity with the QuickStix TM Kit. All results were negative indicating no cross-reactivity with the tested biot ech events. Real-time and accelerated stability data indicate the QuickStix TM Kit ( EnviroLogix Cat. #AS003BG) to be

stable for 18 months at 4-8 ° C.

CLASSIFICATION: ACCEPTAB LE, upon clarification of product stability test

data. 456867-01

Analytical

Method Further data on the analytical method as well as validation of an analytical method by EPA’s OPP Microbiology Laboratory at Fort Meade is not required since the Grain Inspection, Packers and Stockyards Administration (GIPSA) of the United States Department of Ag riculture (USDA) has verified the performance of a qualitative rapid test k it for detecting the presence of the MON 810 and Bt 11 Cry1Ab in grains and oilseeds.Cry1F & Cry1Ab Bt Corn Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

13 2. Product Characterization of MON810 Cry1Ab Corn (006430)

Monsanto's corn line MON 810 was produced by ballistically transforming another proprietary corn line with plasmid construct PV-ZMCT01. Plasmid construct PV-ZMCT01 consists of plasmids PV-ZMBK07 & PV-ZMGT 10 ballistically introduced t ogether. The MON 810 line of corn is similar to MON 801 corn in that they both were derived from transformation events utilizing PV-ZMCT01. The MON 810 only expr esses a truncated vers ion of Cry1Ab deltaendotoxin. MON801 expresses the full length vers ion of Cry1Ab and the marker gene products.

MON 810 and MON 801 were each transformed with the same plasmid construct (PVZMCT01). The MON 810 progeny expr ess a slightly truncated vers ion of Cry1Ab compared to MON 801, but the active site is still retained. The MON 810 progeny do not express detectable levels of the marker gene products found in MON 801 progeny. Some of the data used to evaluate MON810 corn was generated from MON801 co rn. To justify this bridging of data from one corn transformation event to another, the company provided product ch aracterization data to

demonstrate the similarities and differences between the two transformation events.

Study Type Result MRID #

Transformation System

Characterization of the DNA

Inserted in the Plant

Protein Characterization and Expression The digests of genomic DNA from co rn line MON 80100 revealed that the two plasmids PV-ZMBK07 and PV-ZMGT10 had been inserted apparently at two locations. Full lengt h copies of the cry1Ab, gox, nptII and cp4 epsps genes were found. Less than full length copies of all these genes were also found. Western blot analysis revealed that only Cry1Ab and CP4 EPSPS proteins were expressed at detectable levels in the corn plant.

CLASSIFICATION : ACCEPTABLE 435332-01

Protein Characterization and Expression The antiserum reactions revealed many western blot bands in both the Dipel® and the ECB resistant corn ex tracts not treated with trypsin. Nobands clearly related to the Cry1Ab toxin were seen in the nontransformed plant extracts whereas a band comigrating with the full length Cry1Ac standard (similar in size to Cry1Ab) was seen in both Dipel® and ECB resistant corn. The tryptic digests of Dipel® and ECB resistant corn extracts revealed intensified bands that comigrated with the Cry1Ab tryptic core standard. Together these data infer that the same Cry1Ab protein is being produced in ECB resistant corn plants as is found in the microbial product.

CLASSIFICATION: ACCEPTABLE 435332-03Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

14 Study Type Result MRID #

Characterization of the DNA

Inserted in the Plant The Southern blots with the two transforming plasmids PV-ZMBK07 and PV-ZMGT10 indicate that only a portion of the PV-ZMBK07 plasmid was successfully integrated. Western blots indicate that all the constructs tested (MON801, 802, 805, 809, 810, 813 and 814) produce delta endotoxin detectable as tryptic core with anti-Cry1Ac antiserum.The genes of the second plasmid used to transform the corn lines, PVZMGT10, which include CP4 EPSPS and gox , were not detected by Southern blot analysis using the PV-ZMGT10 plasmid as probe. These genes which confer glyphosate tolerance were apparently lost during development of the MON810 line since they had to be present for the original callus culture selection process but were not found in the final

line described here.

CLASSIFICATION : ACCEPTABLE 436655-01

Protein Characterization and Expression The results of the western blot show ed the trypsinized extracts of corn lines MON 802, 805, 809, 810, 813, and 814 expressed proteins that comigrated with the Cry1Ab protein as found in MON 801 and the same Cry1Ab protein purified from E. coli. These bands also reacted with antiserum #B6 specific for the tryptic core protein of Cry1Ab. These results indicate the trypsinized proteins found in all these plants were of same molecular size (63 kD) and im munoreactivity with the reference standards of Cry1Ab expressed in E. coli and corn line MON801.

CLASSIFICATION: ACCEPTABLE 436655-03

Protein Characterization and Expression The Cry1Ab protein produced in E. coli was shown by SDS-PAGE, western blot, N-terminal amino acid sequencing, glycosylation and bioactivity to be substantially equivalent to the plant produced Cry1Ab. The test results showed the tryptic core of the plant and microbial protein were of essentially identical SD S-PAGE mobility, immunoreactivity in western blot analysis and N-terminal amino acid sequence for the first 15 positions. A comparison of the dose response relationship of plant and microbial extracts against Heliothis virescens and Helicoverpa zea indicates that the tested protei ns are of similar bioactivity.

CLASSIFICATION: A CCEPTABLE. These results allow the substitution of the microbially produced Cry1Ab protein for the plant source in toxicology testing. 435332-04

Protein Expression The concentration of MON 810 Cry1Ab in the tissues was determined by ELISA and calculated on a wet- and dry-weight basis. Cry1Ab was detected in all corn tissues at ap preciable concentrations with the exception of pollen.

CLASSIFICATION: ACCEPTABLE. 458789-01

Analytical Method The independent laboratory validation is acceptable and the methodconducted in the independent laboratory validation is suitable for an inhouse performance verification at the OPP Microbiology Laboratory.

CLASSIFICATION: ACCEPTABLE. 456964-01Cry1F & Cry1Ab Bt Corn Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

15 Study Type Result MRID #

Analytical Method The OPP Microbiology Laboratory determined that the initial corn samples (event free) provided by the registrant were not suitable for the in-lab validation as set forth in the independent laboratory validation conducted by Medallion Laboratories . A second set of corn samples provided by Monsanto was tested and was also found to be unsuitable for the in-lab validation. Both sets of corn samples provided by Monsanto had background levels of Cry1Ab sufficient to interfere with the method

validation. Protocol #

PIP-2004- 01, Laboratory

Validation

of MRID 45694-01,

ILV of the

SDI Check

Bt1 Corn

Lateral Flow

Test Kit

Analytical Method Further data on the analytical method as well as validation of an analytical method by EPA’s OPP Microbiology Laboratory at Fort Meade is not required since the Grain Inspection, Packers and Stockyards Administration (GIPSA) of the United States Department of Agriculture (USDA) has verified the performance of a qualitative rapid test kit for detecting the presence of the MON 810 and Bt 11 Cry1Ab in grains and oilseeds.

3. Product Characterization of Plan t Optimized (PO) Cry1F Corn (006481)

A corn line of Pioneer Hi-Bred International and Dow Agrosciences / Mycogen was biolistically

transformed with a linear Pme I fragment from plasmid pP8999 to produce line TC1507. This plasmid contains genes cry1F

, pat and kan r encoding the delta-endotoxin from Bacillus thuringiensis var. aizawai PS811, phosphinothricin acetyl transf erase, and resistance to the antibiotic kanamycin, respectively. The Pme I fragment (6235 bp) derive d from this plasmid was purified after plasmid digesti on and used in the transforma tion process to eliminate the kan r antibiotic resistance gene. The Cry1F protein expressed in transfor med maize lines is a modified (synthetic, less than full length) fo rm as compared to that from the bacterial isolate from which it is derived. This insecticidal protein confer s resistance to the Eu ropean corn borer ( Ostrinia

nubilalis ) and feeding damage is significantly reduced or eliminated following expression of this gene in corn line TC 1507. Expression of cry1F is under the control of the maize polyubiquitin promoter in line TC1507. The CaMV 35S promoter controls expression of the pat gene in this

construct. The pat gene from Streptomyces viridochromogenes confers resistance to the herbicide glufosinate in corn lines accumulating this protei n. Hybridization patterns indicate that one full length copy each of the cry1F and pat genes was integrated into the genome of line TC1507 and

that no kan r DNA was integrated. This suggests that one Pme I fragment from pP8999 integrated into the maize genome. In addition, there are one or two partial copies of the cry1F gene integrated into the genome wh ich are most likely non-functiona l based upon the size of the fragments detected.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

16 Study Result MRID #

Protein Expression Maize plants (hybrids) from two locations grown under standard agronomic practices of the Midwestern Corn Belt were analyzed by ELISA for Cry1F protein content. The youngest leaf of expanding whorls at the V9 stage were collected from fi ve plants per entry. Values of Cry1F protein for all four hybrids were similar, ranging from 1.52 to 2.63 pg/mg dry weight. Control hybrid A M was negative for Cry1F as determined by

ELISA.

CLASSIFICATION : ACCEPTABLE 447148-04

Transformation System

Inheritance and Stability

after Transformation

Protein Characterization A modified (synthetic, less than full- length) form of the cry1Fa2 gene and the phosphinothricin acetyl transferase (p at) gene were inserted into maize plants by microprojectile bombardment. Three transformation events resulting from microprojectile bombardment will be evaluated under the proposed EUP: TC 1360, TC 1362 and TC 1507. Plants were analyzed for Cry1F by ELISA and PAT by application of glufosinate herbicide. Using a chi square analysis with a 95 % confidence interval, the expected Mendelian ratio of 1:1 was observed for both first and second generations for five inbreds with one exception; first generation TC 1632. Event TC 1507 has been analyzed for only the first generation and ratios (1:1) were as expected.CLASSIF ICATION: SUPPLEMENTARY. The registrant should clarify the source of the ubiquitin exon and intron as being from the ubiquitin gene and not the promoter region. A determination of expression of the ubiquitin exon sequence is also needed and whether it alters the sequence of Cry1F. 447148-01

Transformation System This submission represents a clarifica tion of nomenclature as presented in a previous submission and review. Labeling (in a previous submission) of the Ubi DNA fragment on the plasmid map should have indicated that it includes the Ubi ZM promoter and the first exon and intron of the Ubi ZM gene. The Ubi exon and intron are included in this construct (PHI8999), however, they have no effect on the structure of the Cry1F product, only on the expression of the gene. Exon 1 contains no ATG start site for

translation. A translation initiation se quence (Kozak consensus sequence) situated just upstream fro m the start site (first translated ATG) drives translation of the mature, spliced mRNA.

CLASSIFICATION : ACCEPTABLE 450201-17

Characterization of the

DNA Inserted in the Plant The integration pattern of cry1F and pat genes introduced into event TC 1360 was analyzed by Southern blotting. Within the Southern analysis, two types of digests are employed to determine the complexity of DNA integration into the maize genome and to determine the copy number of integrated transgenes. Analysis of four of the progeny from event TC 1360 revealed the presence of two bands hybridizing to the cry1F probe; both bands appeared to hybridize with similar intensity. Hybridization to internal controls on the blot gave an indication of single copy integration and certainly no more than two copies of the insert integrated into the maize genome. When control plant DNA was probed, no hybridization was noted. TC 1360 and control DNA probed with the kan r gene indicated no hybridization within these samples.

CLASSIFICATION : ACCEPTABLE 447148-02Cry1F & Cry1Ab Bt Corn Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

17 Study Result MRID #

Transformation System

Characterization of the

DNA Inserted in the Plant A modified (synthetic, less than full-length) form of the cry1F gene and the phosphinothricin acetyl transferase (p at) gene were inserted into maize plants by microprojectile bombardment. Digestion of the genomic DNA of maize line 1507 with NheI or HindIII and Southern hybridization with probes specific for cry1F, kan r and pat genes yielded indications of the complexity of the gene integration pattern and copy number. Hybridization patterns suggested that the copy number of introduced / integrated cry1F and pat genes is one. It is most likely that the TC 1507 line contains one functional cry1F gene and partial copies (1 or 2) of the gene which are non-functional. It is not possible with this technique, however, to discern the functionality of probed sequences. No kan r DNA was introduced into line 1507 during transformation, as indicated by the lack of signal when 1507 genomic DNA was probed with the kan r gene. There was no hybridization signal when the non-transformed maize line 13-1 was probed with pat or cry1F or kan r

. CLASSIFICATION : ACCEPTABLE 450201-02

Protein Characterization and Expression Cry1F protein from maize 1507 pollen, grain, grain-derived feeds and a microbial source was evaluated biochemically using ELISA, SDS-PAGE and Western Blotting, and for bioactivity using insect bioassays. Control maize tissues were used to prepare comparable samples. Pollen from line 1507 contained Cry1F at 31 to 33 ng / mg pollen, while no Cry1F protein was detected in pollen from non- Cry1F plants. The purified maizeexpressed Cry1F test substance was ap proximately 32 ng / mL extract. The comparable extract from non-Cry1F maize did not show any detectable Cry1F protein; the limit of detection (LOD) was 0.04 ng / mg sample. Coomassie stained gels indicated similar profiles for both control maize and Cry1F maize samples following SDS-PAGE. Antibodies directed against Cry1F detected this protein (64 kDa) in the Cry1F maize grain samples while there was no indication of any Cry1F protein in the control samples of grain. Pollen, maize-expressed Cry1F and microbially derived Cry1F were all active against the European Corn Borer larvae at the times

tested. For the Tobacco Budworm larv al bioassay, substances tested included maize grain, maize grain de rived fish feed, and maize grain derived quail feed. Samples containing Cry1F maize grain and quail feed made from this grain had identical amounts of Cry1F protein based upon

the GI 50 s calculated. Comparison of control and Cry1F fish feed over four separate bioassays indicated that ther e was no statistical difference (p = 0.05) based upon ANOVA. Preparation of the fish feed sample reduced the biological activity of the Cry1F protein below sensitivity for the assay.

CLASSIFICATION : ACCEPTABLE 450201-03Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

18 Study Result MRID #

Protein Expression Protein expression values indicated subs tantial variability in protein levels for Cry1F in the tissues sampled. No definitive conclusions could be reached from the data presented when comparing levels of Cry1F in hybrid 1507 and inbred 1507 when examining pollen, silk, stalk, leaf, grain, whole plant and senescent whole plant samples. Since these hybrids and inbreds were grown in areas of Chile w ith similar climatic extremes to the maize growing areas of the U.S., it is anticipated that these values will represent those to be expected in th e U.S. cornbelt. PAT expression was also not readily distinguishable when comparing inbred and hybrid expression values. The inability to detect PAT protein in the majority of samples, except leaf, is somewhat puzzling in that the plants demonstrated clear glufosinate tolerance at all field sites. Given the generally strong, non-tissue specific expression levels typically associated with the CaMV 35S promoter (driving pat expression), it is not readily apparent why more PAT protein was not detected in more samples. Its presence in leaf tissue was expected, however, the reason for the absence in many of these samples is less than clear.

CLASSIFICATION: ACCEPTABLE 450201-04

Analytical Method A double antibody sandwich test was developed to detect the Cry1F protein in homogenized maize grain samples using a rapid test method. A double antibody sandwich technique is used in the Lateral Flow Test Kit for Cry1F. Antibodies raised agains t the Cry1F protein are incorporated into the Lateral Flow test strip and coupled to a color reagent. When in contact with Cry1F protein, the antibodies bind Cry1F and a sandwich is formed, however, not all of the antibodi es are coupled to the color reagent. The test strips contain two zones wh erein capture of color reagent or antibodies can occur. One zone captures bound Cry1F and the othercaptures color reagent. Both zones display a reddish color when proteinantibody sandwich and / or unreacted color reagent are captured. When only one line (control ) line is present, a negative sample is indicated, while the presence of two lines indi cates the presence of Cry1F. The Cry1F Lateral Flow Test Kit accurately detected Cry1F protein in 30 of 30 corn kernels from Cry1F maize and in dicated negative reactions for the 30 control maize kernels. This finding demonstrates the utility of using the Cry1F Lateral Flow Test Kit for detection of Cry1F protein in maize grain samples. This kit allows for a rapid qualitative determination of the presence of Cry1F protein.

CLASSIFICATION : ACCEPTABLE 452793-01

Analytical Method The results of this assay validation indicate that the ELISA based assay was suitable for the analysis of Cry1 F as found in maize grain. Average recoveries from samples spiked with Cry1F protein (truncated microbial form) were between 67 and 107 %. Extractions from known Cry1F maize grain samples demonstrated that a sample as small as 50 mg could be properly extracted and quantified.

CLASSIFICATION : ACCEPTABLE 452793-02Cry1F & Cry1Ab Bt Corn Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

19 Study Result MRID #

Protein Characterization. Standard techniques of protein chemistry were used to assess similarities between the bacterial and plant sources of the Cry1F protein. Additionally, insect mortality assays were performed to determine in vitro toxicity. An

in vitro digestibility assay was done to determine that Cry1F was unstable under conditions simulating the gastric environment. This simulation of gastric conditions indicated that th e toxin (from microbial source) was readily digested by pepsin. SDS-PAGE and Western blotting of plant and

bacterial sources determined th e presence of a 65 kDa protein corresponding to the trypsinized core of the d-endotoxin. Plant extracts contained 0.158 % Cry1F as determined by ELISA; control plants were negative. N-terminal sequencing of 5 aa determined that the microbial and plant expressed protein maintained this sequence intact. Glycosylation was not evident in Cry1F from either source.

CLASSIFICATION : ACCEPTABLE 447149-03

4. Product Characterization of Maiz e Optimized (MO) Cry1F Corn (006491)

The mo cry 1F (maize-optimized) gene codes for the id entical truncated Cry1F protein as that expressed by maize plants containing the po cry 1F (plant optimized) gene (MRID 447148-01). Codon changes were made to the gene to improve expression in moCry1F maize plants, but these codon changes do not alter the amino acid sequence of the protein as compared to that expressed in poCry1F maize plants. The first 605 amino ac ids of moCry1F and poCr y1F are identical with the exception of an altered residue at positi on 604 (F604L). Because moCry1F and poCry1F proteins share an identical amino acid sequen ce, the protein equivalency and insect-pest spectrum data also support the moCry1F registration.

Specific moCry1F product characterization data(summarized below) indicate that plantproduced moCry1F protein is biologically, bioc hemically, and immunologically similar to that expressed in the source bacterium B. thuringiensis after trypsin digest. Southern blot data of restriction enzyme digests suggests that the insert in B.t. moCry1F corn line 6275 occurred as a simple integration of a partial copy of the T-DNA region (truncated at the 5' end) from plasmid PHP12537. One intact copy of bar (plant selectable marker gene, phosphinothricin acetyltransferase, bar ) was confirmed. Southern blot analyses also revealed that tetracycline and spectinomycin resistance genes were not integrat ed into corn line 6275. Southern hybridization was used to assess the genetic stab ility of the insert in multiple ge nerations of corn line TC 6275. Based on the segregation analyses , corn line 6275 exhibited stable Mendelian inheritance of the insert across the generations examined.

Study Result MRID#

Transformation System

Characterization of the DNA

Inserted in the Plant A synthetic, truncated cry 1F transgene was optimized for maize expression and transformed into maize plants using Agrobacterium tumefaciens for plant transformation (called

mo cry1F) . The mo cry 1F gene encodes a truncated, core

insecticidal toxin that is iden tical in amino acid sequence to 453186-01Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

20 Study Result MRID# the native Cry1F protein over the first 605 amino acids with the exception of an altered residue at position 604 (F604L). The codons for the remaining C-terminal (569 ) amino acids of the full-length protoxin, were removed from the transgene sequence. PCR verification of the cry1F gene indicated that the two genes segregate as a single gene.

CLASSIFICATION: ACCEPTABLE

Protein Expression A direct double antibody sandwich ELISA was developed to quantify Cry1F found in ly ophilized tissues of mo cry1F maize. Samples of leaf, pollen and grain from the two locations in Chile all produced measurable levels of Cry1F for both transformed inbred and hybrid lines when expressed on a tissue dry weight or total extractable protein basis. Leaf tissue samples indicated a higher level of Cry1F expression or accumulation as compared to pollen and grain samples. The quality control samples included in the ELISA plates yielded 73.9 to 106.2% of predicted value, which is within the realm of variation typically seen in ELISA when protein are mixed and processed along with whole tissue samples Extractions from known moCry1F corn gain samples demonstrated that a sample as small as 50 mg could be properly extracted and quantified.

CLASSIFICATION: ACCEPTABLE 453186-02

Characterization of the DNA

Inserted in the Plant The integration pattern of mo cry1F and bar genes introduced into event TC6228 plants was analyzed by Southern blotting. Control DNA spiked with PHP12537 DNA at concentrations equivalent to 1, 3 or 5 gene copies / genome was included as a positive control and a means to estimate copy number by compar ing hybridization intensity. Control DNA without plasmid DNA was also included as a negative control. Data indicate that a single integration of a complete and functional trans criptional unit, representing the T-DNA of the binary plasmid PHP12537, is present in

the modified corn line TC6228 . Two antibiotic resistance

genes, spc and tet , which are present in the region of the plasmid outside the T-DNA bord ers, were not transferred as determined by lack of hybrid ization with TC6228 DNA.

CLASSIFICATION: ACCEPTABLE 452646-01

Characterization of the DNA

Inserted in the Plant Southern blot data from restriction enzyme digests suggest that there is a single insertio n of a partial copy of the TDNA region from plasmid PHP 12537 at one locus in corn line 6275. Restriction digests of corn line 6275 indicated that

the bar gene was inserted intact and that the (mo) cry1 F transcription unit was truncated on the 5' end up to and including the restriction enzyme site. The absence of the bacterial tetracycline and spectinomycin resistance genes and regions immediately outsid e of the left and right T-DNA borders was confirmed suggesting that only DNA contained within the T-DNA borders of plasmid PHP 12537 was integrated into maize line 6275. The inserted DNA was also characterized in two distinct generations of B.t. moCry1F maize line 6275, demonstrating the stability of the inserted

DNA.

CLASSIFICATION: ACCEPTABLE 460193-01Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

21 Study Result MRID#

Characterization of the DNA

Inserted in the Plant This is a second study to char acterize the inserted synthetic
transgene cry 1F in B.t. moCry1F maize line 6275 that contains the (mo) cry 1F and bargenes (see first study, MRID#460193-01). DNA extracted from corn leaf tissue was examined by Southern blot analysis to characterize the T-DNA insert containing the (mo) cry 1F and bargenes in transgenic corn event TC6275 . Southern blot data from this study confirm a single insert ion of the T-DNA region from

PHP 12537 in event TC6275 with a T-DNA truncation or alteration of the ubiquitin promoter and intron regions Additional border fragments at the 5' end resulting from

Eco R I digestions suggested that the endonuclease restriction site at bp 1584 in the T-DNA insert was lost during integration into the corn genome.

CLASSIFICATION: ACCEPTABLE 460193-02

Protein Expression Cry1F and bar proteins were found in all transgenic lines, tissue types, and at almost all growth stages (none was detected in a few 6275H pollen samples and in most senescent leaf samples). On a dry weight basis, Cry1F levels ranged from a low of 0.71 ng/mg in senescent leaves (16.7 ng/mg in V9 leaves) in unsprayed transgenic lines to 44.8 ng/mg in R4 leaves; 3.7 ng/mg in pollen (unsprayed and sprayed transgenic lines); 10.4-11.0 ng/mg in stalks (unsprayed and sprayed transgenic lines); 5.82-6.26 ng/mg in forage tissue; 1.97 ng/mg in senescent roots (unsprayed transgenic line) to 6.60 ng/mg in R1 roots; and 1.08-1.14 ng/mg in grain (unsprayed and sprayed transgenic lines). On a dry weight basis, bar levels ranged from 0 ng/mg in senescent leaves to 682 ng/mg in R4 leaves (unsprayed transgenic line); 41 ng/mg in senescent roots to 373 ng/mg in R1 roots (sprayed transgenic line); 0-0.62 ng/mg in pollen (unsprayed and sprayed transgenic lines); 282-311 ng/mg in stalks (unsprayed and sprayed transgenic lines); 7-11 ng/mg in forage tissues (unsprayed and sprayed transgenic lines); and 23 ng/mg in grain (unsprayed and sprayed transgenic lines).

CLASSIFICATION: ACCEPTABLE 460193-03

Analytical Method This study demonstrates the high quantitative performance of the ELISA assay for the detection of the Cry1F truncated protein. The assay had a reproducible sensitivity of 0.5 ng/mL with an approximate 40-fold assay range of 0.4 to 17 ng/mL truncated Cry1F). Th e coefficient of variation (%CV) of the absorbance measurement within the curve is less than 10%. The cross-reactivity profile indicated little to no cross-reactivity or interference from a standard panel of agriculturally relevant recombinant proteins. The Cry1F assay kit was projected to be stable for approximately 1 year

at 4oC.

CLASSIFICATION: ACCEPTABLE 456856-01

Analytical Method An independent laboratory, EPL Bio-Analytical Services, validated Dow AgroSciences LLC analytical method

GRM Ε 02.13 “Determination of Cr y1F Insecticidal Crystal 456856-02Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

22 Study Result MRID# Protein in Corn Grain by Enzyme Linked Immunosorbant Assay” for accuracy, precision, and sensitivity. The LOQ of

the method was confirmed as 0. 75 μg/g (or 0.5 ng/mL) for corn grain extracts spiked with Cry1F. Average recoveries from samples spiked with Cry1F protein averaged 99 and 90 percent at the 0.075 and 0.15μg/g spike levels, respectively.

The relative standard deviatio n (RSD) of replicate recovery

measurement did not exceed 20 percent at or above the LOQ and interferences were negligible (<20% of the response of the Cry1F protein at the LOQ of 0.075 μg/g). The results of this assay validation indicate that the ELISA based assay is suitable for the analysis of Cry1F as found in corn grain. CLASSIFICATION: SUPPLEMENTAL; Upgradable with appropriate documentation that this method works to quantify Cry1F expressed in transgenic Cry1F corn.

Analytical Method Dow AgroSciences analytical residue method GRM 02.30 "Determination of Cryl F ICP in Com Tissues by ELISA" and the associated ELISA kit from Strategic Diagnostics

Inc. were demonstrated to be acceptable for the intended purpose. The concentration ra nge validated for the method was 1.0 to 10.0 ng'mL (0. I to 1.0 ng'mg) and has a validated lower limit ofquantitation in all com tissues of 0.1 ng/mg. The Cryl F protein was recovered at acceptable levels from

all tissues tested (Ieaf-V9, stalk-R I, whole plant-V9, -R I, -R4, - R6, root, pollen and grain). The assay proved to be specific for Cryl F protein and no matrix effects were detected in any ofthe com tissues tested. The Cryl F ELISA method was demonstrated to be accep table for quantitative

measurement of the CrylF protein in com tissues. 4665610-01

Analytical Method Further data on the analytical method as well as validation of an analytical method by EPA’s OPP Microbiology Laboratory at Fort Meade of an analytical method by the Fort Meade laboratory is not required since the Grain Inspection, Packers and St ockyards Administration (GIPSA) of the United States Department of Agriculture (USDA) has verified the perf ormance of a qualitative rapid test kit for detecting the presen ce of the Cry1F in grains and

oilseeds.

5. Product Characterization of MO N 810 x TC1507 (Cry1Ab x Cry1F) Corn

Study Type Result MRID #Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

23 Study Type Result MRID #

Characterization

of the DNA

Inserted in the

Plant Molecular analyses (restriction enzyme digests and Southern blots) were performed to compare the integrity of th e transgenic inserts in events TC1507, DAS-59122-7, and MON810 corn with the transgenic DNA inserts in the combination PIP 1507×59122×MON810 corn product, produced by conventional plant breeding. The Southern blot data showed the predicted DNA hybridization patterns of the cry 1F gene from TC1507 corn, the cry 35Ab1 and

cry 35Ab1 genes from DAS-59122-7 corn, and the cry 1Ab gene from MON810 corn. These data demonstrated equivale nce among events because the transgenic inserts were stably integrated and retained when the parental lines are crossed to create 1507×59122×MON810 corn. In addition, event-specific PCR (polymerase chain reaction) methods de tected and confirmed the presence of each expected transgenic DNA insert in the combination PIP corn event. This study was classified acceptable. 476778-01

Protein Expression A field study was conducted using qu antitative enzyme-linked immunosorbent assay (ELISA) methods to statistically compare the level of expression for several proteins in several combination PIP corn events. The conclusions for cry 1F and cry1Ab are summarized below: The levels of Cry1F, Cry1Ab and PAT proteins expressed in the 1507 × MON810 combination PIP corn event were compared to the individual parental ev ents. No statistical differences were observed in any plant tissue for Cry1F, PAT, and Cry1Ab protein expression when 1507 × MON810 was compared to events 1507 and MON810. Based on these results, the protein concentrations of Cry1F and Cry1Ab expressed in the

combination PIP event 1507 × MON810 ar e comparable to the respective protein concentrations of their respect ive single parental events. This study was classified acceptable. 478800-01

B. HUMAN HEALTH ASSESSMENT

1. Background

The basic premise relied on for the toxicol ogy assessment is the fact that all the Btplantincorporated protectants are prot eins. Proteins are commonly f ound in the diet and, except for a few well described phenomenona, present little risk as a mammalian hazard. In addition, for the majority of Bt proteins currently registered, the source bacterium has been a registered microbial pesticide which has been approved for use on food crops without specific restrictions. Because of their use as microbial pesticides, a long hi story of safe use is associated with many Bt products.

Several types of data are required for the Bt plant-incorporated pr otectants to provide a reasonable certainty that no harm will result from the aggregate exposure to these proteins. The

information is intended to show that the Bt protein behaves as would be expected of a dietary protein, is not structurally re lated to any known food allergen or protein toxin, and does notCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

24 display any oral toxicity when administered at high doses. These data consist of an in vitro digestion assay, amino acid sequence homology comp arisons and an acute oral toxicity test. The acute oral toxicity test is done at a maxi mum hazard dose using purifie d protein of the plantincorporated protectant as a test substance. Due to limitations of obtaining sufficient quantities of pure protein test substance fr om the plant itself, an alternat ive production source of the protein

is often used such as the Bacillus thuringiensis source organism or an industrial fermentation microbe. The justification for employing this alternative source of pure protein is the equivalence data discussed above under product char acterization.

EPA believes that protein instability in digestive fluids and the lack of adverse effects using the maximum hazard dose approach in general elim inate the need for longer-term testing of Bt protein plant-incorporated protectants. Dosing of these animals with the maximum hazard dose, along with the product characteriza tion data should identify potenti al toxins and allergens, and provide an effective means to determine the safety of these protein. The adequacy of the current testing requirements was discussed at the June 7, 2000 Scientific Advisory Panel (SAP) meeting. In their final report, the SAP agreed in princi ple with the methods used by EPA to assess the toxicity of proteins expressed in plants especially the maximum hazard dose approach.

2. In vitro Digestibility Assay

The intent of this assay is to demonstrate that the Bt protein is degraded into small peptides or amino acids in solutions that mimic digestive flui ds. Usually only gastric fluid is tested since Cry protein is known to be stable in intestinal fluid, but in the initial Bt products registered, gastric and intestinal fluids were examined se parately. In order to track the breakdown, the proteins were added to a solution of the diges tive fluids and a sample was either removed or quenched at given time points (usually at time 0, one to several minutes la ter and one hour later). The time point samples were then electrophore sed on either an SDS-PAGE gel and further analyzed by western blot or tested in a bioassay against the target pest. All were degraded in gastric fluid in 0-7 minutes. All the Bt proteins tested in intestin al fluid were not affected by trypsin digestion as would be expected since this is similar to their behavior in the insect gut. In intestinal fluid, those Bt plant-incorporated prot ectants that are expresse d as protoxin molecules broke down into the active toxin moiety and degraded no further.

As has been stated in several public fora, the in vitro digestibility test is basically a test to

confirm the biochemical characterist ic of instability of the protei n in the presence of digestive fluids. The digestibility test is not intended to provide information on the toxicity of the protein or imply that similar breakdown will happe n in all human digestive systems. The in vitro digestibility assay may also provide information about the potential of a protein to be a food allergen. The in vitro digestion assays confirm that the protein is being broken down in the presence of typical digestive fluids and is not unusually persistent in th e digestive system. One of the limitations of the test is that it usually only tracks protein breakdown to fragments still recognized by the immunologi cal reagents employed.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

25 3. Amino Acid Homology and Heat Stability

Two additional characteristics that are considered as an indication of possible relation to a food allergen are a protein's ability to withstand he at or the conditions of food processing and its amino acid sequence when compared to known food allergens.

4. Acute Oral Toxicity

One of the bases for addressing th e toxicity of proteins primaril y through the use of acute oral toxicity is that, when demonstrated to be t oxic, proteins are toxic at low doses (Sjoblad, et al

., 1992). Therefore, when no effects are shown to be caused by the protein plant-incorporated protectants, even at relatively high dose levels in the acute oral exposure, the proteins are not considered toxic. The acute oral toxicity test is performed in mice with a pure preparation of the plant-incorporated protectant protein at doses from 3280 to over 5000 mg/kg bodyweight. None of the tests performed to date have shown any significant eff ects on the treated animals.

5. EPA Recommendation (2001)

The mammalian toxicity data continue to support the registrations of the Bt products described. EPA believes the data it currently has is sufficient to support the Bt plant-incorporated protectant registrations.

6. Human Health Assessment of Cry1Ab Crops, Including But Not Limited To:

Bt11 Cry1Ab Bt Corn (006444) and MON810 Cry1Ab Bt Corn (006430)

a. Toxicology Assessment

Mammalian toxicology data are av ailable to examine the potentia l effects of Cry1Ab on human

health and assess if the data support the registration of Bacillus thuringiensis Cry1Ab deltaendotoxin and the genetic material necessary for its production (plasmid vector pZO1502) in corn (OPP PC Code 006444) and Bacillus thuringiensis Cry1Ab delta-endotoxin and the genetic material necessary for its production in corn (OPP PC code 006430). Bt microbial pesticides, containing Cry proteins other than Cry1Ab, have been applied for more than 30 years to food and feed crops consumed by the U.S. population. These data would also support other Cry1Ab plant-incorporated protectants’ human health assessments provided adequate information was submitted to show that the Cry1Ab proteins in question were biochemically and functionally similar to the proteins of the plant-in corporated protectants already examined.

1) Acute ToxicityCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

26 Study Type Result MRID #

Acute Oral Toxicity Five male and five female mice received a single dose of 3,280 mg/kg of Cry1Ab protein by oral gavage. No animals died nor were there significant clinical signs as a result of the exposure. One female failed to gain weight between day 7 and day 14. All animals gained weight by the end of the study. Males gained more weight over the study than females.

CLASSIFICATION : ACCEPTABLE Test substance is given a TOXICITY CATEGORY IV rating although highest dose administered is 3280 mg/kg due to lack of any evidence of a

dose/effect relation. 433236-08

Acute Oral Toxicity No test substance related deaths occurred. One female died within a day of BSA dose administration due to a perforated trachea. The majority of the animals failed to gain weight or showed a slight weight reduction. No treatment related trends in these losses was

apparent. CLASSIFICATION: ACCEPTABLE. Test substance is given a TOXICITY CATEGORY IV rating although highest dose administered is 4000 mg/kg due to lack of any evidence of a

dose/effect relation. 434680-01

2) Mutagenicity and Developmental Toxicity, Subchronic Toxicity, and Chronic Exposure and Oncogenicity Assessment

Data demonstrating no mammalian toxicity at high levels of exposure confirm the safety of the product at levels well above any possible maxi mum exposure levels anticipated for a plantincorporated protectant. This is similar to the Agency position regarding toxicity and the requirement of residue data for the microbial Bacillus thuringiensis products from which these plant-incorporated protecta nts were derived. [See 40 CFR Sec. 158.2130 and 158.2140.] For microbial products, further toxicity testing to veri fy the observed effects and clarify the source of the effects (Tiers II & III) and residue data ar e triggered by significant acute effects in studies

such as the mouse or al toxicity study.

The acute oral toxicity data submitted support th e determination that the Cry1Ab protein is nontoxic to humans. When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad, et al .1992). Since no effects were shown to be caused by the plant-incorporated protectants, even at relatively high dose levels, the Cry1Ab delta-endotoxin protein is not considered toxic. Because these proteins break down into their constituent amino acids, there would be no chronic exposure to the protein and therefore no need for chronic toxicity testing. Therefore, the mutagenicit y, developmental toxicit y, subchronic toxicity, chronic exposure and oncogenicity a ssessment studies were not required.

3) Effects on the Immune System

Since Cry1Ab is a protein, alle rgenic potential was c onsidered. Current scientific knowledge suggests that common food allergen s tend to be resistant to de gradation by heat, acid, andCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

27 proteases, to be glycosylated and present at hi gh concentrations in th e food. Data has been submitted which demonstrates that the Cry1Ab delta-endotoxin is degraded in two minutes by gastric fluid in vitro and is non-glycosylated. Studies s ubmitted to EPA done in laboratory animals have not indicated any pot ential for allergic reactions to B. thuringiensis or its components, including the delta-endotoxin in the crystal protein. Despite decades of widespread

use of Bacillus thuringiensis as a pesticide (it has been regist ered since 1961), there have been no confirmed reports of immediate or delayed allerg ic reactions to the delta -endotoxin itself despite significant oral, dermal and inhalation exposure to the microbial product. Several reports under FIFRA § 6(a)2 have been made for various Bacillus thuringiensis microbial products claiming dermal allergic reactions. However, the Agency determined these react ions were not due to Bacillus thuringiensis itself or any of the Cry toxins. The reported reactions were determined to be due to non-Cry proteins pr oduced during fermentation or to added formulation ingredients. Thus, the Cry1Ab protein is not expected to be a food allergen.

Allergenicity Endpoints of Cry1Ab Crops [ Bt 11 and MON810 Bt Corn (006444 & 006430)]

Study Type Result MRID #

In vitro Digestibility The Cry1Ab protein from either maize or B.t.k. HD1-9 is rapidly degraded in the presence of pepsin. Using 1/1000 strength pepsin, a time course study shows that the introduced protein from either source degr ades within 10 minutes to lack of any recognition in a western blot assay.

CLASSIFICATION: ACCEPTABLE 433236-06

In vitro Digestibility The tryptic core Cry1Ab protein is signific antly degraded by 2 minutes incubation in gastric fluid but not significantly affected by 19.5 hours in intestinal fluid as monitored by western blot. The decrease in bioactivity of these digestions against tobacco budworm is similar to its loss of immunorecogniton in western blots

CLASSIFICATION : ACCEPTABLE 434392-01

Amino Acid Sequence Homology An amino acid database was constructed containing amino acid sequences of known protein allergens and gliadins. The B.t.k. HD-1 protein was compared to this database and no significant sequence similarity was id entified. Based upon this data, there does not appear to be significant sequence similarity between HD-1 and known protein allergens and gliadins. 453849-01

Amino Acid Sequence Homology The National Center for Biotechnology GenBank database, which contains all publicly available protein sequences, was queried to determine whether the Cry1Ab protein as expressed in transgenic maize Event Bt11 has any significant amino acid homology with protein sequences identified as toxins. No significant amino acid homology between Bt11 Cry1Ab to any non- Bt Cry proteins identified as, or known to be, toxins. There was a degree of homology observed between the Bt Cry31Aa1 parasporin protein and the Bt 11 Cry1Ab protein. However, a global alignment of parasporin and Bt 11 Cry1Ab demonstrated only 20.7% identity between the twoproteins. Moreover, the Cry1Ab protein is a lepidopteran-specific toxin and is nontoxic to other non-lepidopteran species and mammals. Therefore, it is concluded that the degree of homology observed between the Bt Cry31Aa1 parasporin protein and the

Bt 11 Cry1Ab protein is not of toxicological relevance.

CLASSIFICATION: ACCEPTABLE 458798-01Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

28 Study Type Result MRID #

Amino Acid Sequence Homology Bioinformatics analyses indicate that there we re no significant similarities (defined as

≥ 35% amino acid homology) between any of the sequential Bt11 Cry1Ab 80-amino acid peptides and any entries in the Syngenta Biotechnology, Inc. (SBI) Allergen Database, which contains all known and putative protein alle rgen sequences. Additionally, there were no alignments of eight or more contiguous identical amino acids between the Bt11 Cry1Ab protein an d any of the proteins in the allergen database. Therefore, no biologically rele vant structural similarities were observed between any known or putative allergen and the Cry1Ab protein produced in corn

Event Bt11.

CLASSIFICATION: ACCEPTABLE 458798-02

Amino Acid Sequence Homology No significant sequence ho mology between any sequential MON 810 Cry1Ab amino acid peptides or alignments of eight or more contiguous identical amino acids between MON 810 Cry1Ab amino acid peptides and any entry in the AD3_1 database were identified. The closest similarity was to a wheat gamma-gliadin protein with 31.7% identity over 60 amino acid residues. No significant 8 amino acid identity stretches

were discovered. // MON 810 Cry1Ab had no significant amino acid sequence homology as defined by the Codex suggested similarity criteria of 35% amino acid identity over 80 amino acids or any 8 amino acid short sequence identities to known allergenic or gliadin proteins.

CLASSIFICATION: ACCEPTABLE 458789-02

CBI?

Amino Acid Sequence Homology The 818 amino acid sequence of event MON810 corn expressing Cry1Ab protein was sequentially searched and co mpared for protein sequence homology to known toxins contained in the NCBI Protein Sequence da tasets. The BLASTP search showed no significant amino acid sequence homologies using the Cry1Ab protein sequence as a query with any proteins known to be toxins. Therefore, the Cry1Ab protein does not share any amino acid sequence homology with any proteins known to be toxins.

Classification: ACCEPTABLE 477864-06

Amino Acid Sequence Homology No identity matches of ~ 35% over ~ 80 amino acid residues were observed for the Cry1Ab protein sequence against the pr otein sequences of known allergens. In addition, there were no 8 or greater co ntiguous identical amino acid matches observed with the Cry1Ab protein sequen ce. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted allergen homolo gy assessment for the Cry1Ab protein expressed in event MON810 corn (see MRID No. 458789-02).

Classification: ACCEPTABLE 477864-04Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

29 Study Type Result MRID #

Heat Stability This study shows that the commonly used methods of processing corn by milling reduce the level of Cry1Ab protein and lower the potential dietary exposure. Since Cry1Ab has no indications of causing adverse effects in either the acute oral study or by having similarities to known food allergen s, there is no expectation that dietary exposure will be a hazard. However, the inactivation by processing further reduces the dietary risk by lowering the expected exposure. Dien, B.S.,

R.J. Bothast,
L.B. Iten, L. Barrios,

and S.R. Eckhoff, (2002),

Fate of Bt

Protein and

Influence

of Corn Hybrid on

Ethanol Production,

Cereal Chemistry

79:582-

585.

4) Effects on the Endocrine System (Updated July 2010)

As required under FFDCA section 408(p), EP A has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (inc luding pesticide active and other ingredients) may have an effect in humans or wildlife si milar to an effect produced by a “naturally occurring estrogen, or other such endocrine effects as the Administrator may designate.” The EDSP employs a two-tiered ap proach to making the statutorily required

determinations. Tier 1 consists of a battery of 11 scr eening assays to identify the potential of a chemical substance to interact with the estrog en, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go th rough Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stag e of the EDSP where EPA will determine which, if any, of th e Tier 2 tests are necessary base d on the available data. Tier 2 testing is designed to identify a ny adverse endocrine related eff ects caused by the substance, and establish a quantitative relationship betw een the dose and the E, A, or T effect.

Between October 2009 and February 2010, EPA issued test orders/data call-ins for the first group of 67 chemicals, which contains 58 pesticid e active ingredients and 9 inert ingredients.

This list of chemicals was selected based on th e potential for human exposure through pathways such as food and water, residen tial activity, and certain post-applic ation agricultural scenarios.

This list should not be construed as a lis t of known or likely endocrine disruptors.

The Cry1Ab protein is not among the group of 58 pest icide active ingredients on the initial list to be screened under the EDSP. Under FFDCA § 4 08(p) the Agency must screen all pesticide chemicals. Accordingly, EPA anticipates i ssuing future EDSP orders/data call-ins for all Registration Review cases, including those for which EPA has already opened a Registration Review docket for a pestic ide active ingredient.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

30 For further information on the status of the ED SP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, pleas e visit our website: http://www.epa.gov/endo/

.

5) Dose Response Assessment

No toxicological endpoints we re identified, therefore a dos e response assessment was not required.

6) Dietary Risk Characterization

a) Toxicity and Allergenicity Conclusions

The data submitted and cited regarding potential health effects for the Cry1Ab protein include information on the characterization of the expr essed Cry1Ab delta-endotoxin in corn, the acute oral toxicity, and in vitro digestibility of the delta-endotoxin.

Adequate information was submitted to show that the Cry1Ab test material derived from microbial cultures were biochemically and functi onally similar to the proteins produced by the plant-incorporated protectant ingredients in corn. Production of microbially produced protein

was chosen in order to obtain su fficient material for testing.

The acute oral toxicity data submitted supports the conclusion that the Cry1Ab protein is nontoxic to humans. Therefore, because no e ffects were shown to be caused by these plantincorporated protectant s, even at relatively high dose le vels (4000 mg/kg), the Cry1Ab deltaendotoxin protein is not considered toxic. This is similar to the Agency position regarding toxicity and the requirement of residue data for the microbial Bacillus thuringiensis products from which this plant-incorporated prot ectants was derived [See 40 CFR Sec. 158.2130 and 158.2140.] Further toxicity testing to verify the observed effects and clarify the source of the effects (Tiers II & III) and residue data are only triggered by significant acute effects in studies such as the mouse oral toxicity study. Because the acute testing showed no toxicity, higher tier testing is not required.

Because Cry1Ab is a protein and the major e xposure is dietary, food allergenic potential was

considered. Current scientif ic knowledge suggests that common food allergens tend to be resistant to degradation by heat , acid, and proteases, are glyc osylated and present at highconcentrations in the food. Data has been submitted which demonstrates that the Cry1Ab deltaendotoxin is degraded in tw o minutes by gastric fluid in vitro and is non-glycosylated. After decades of widespread use of Bacillus thuringiensis as a pesticide (it has been registered since 1961), there have been no confirmed reports of i mmediate or delayed allergic reactions to the delta-endotoxin itself despite signi ficant oral, dermal and inhala tion exposure to the microbial product. Several reports under FIFRA § 6(a)(2) have been made for various Bacillus thuringiensis microbial products claiming dermal alle rgic reactions. However, the Agency

determined these reactions were not due to Bacillus thuringiensis itself or any of the Cry toxins. Thus, the Cry1Ab protein is not expected to be a food allergen.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

31 Both (1) available information concerning the di etary consumption patterns of consumers (and major identifiable subgroups of consumers includi ng infants and children) and (2) safety factors which, in the opinion of experts qu alified by scientific training and experience to evaluate the safety of food additives, are generally rec ognized as appropriate for the use of animal experimentation data were not ev aluated because the lack of mammalian toxicity at high levels of exposure demonstrates the safety of the pr oduct at levels above possible maximum exposure

levels.

The genetic material necessary for the production of the plant-incorporated protectants active ingredients are the nucle ic acids (DNA) which comprise (1) genetic material encoding these proteins and (2) their regulatory regions. “Regul atory regions” are the genetic material (termed promoters, terminators and enhancers) that control the expression of the DNA encoding proteins. DNA is common to all forms of pl ant and animal life and the Agency knows of no

instance where these nucleic acids have been a ssociated with toxic e ffects related to their consumption as a component of food. These ubi quitous nucleic acids as they appear in the subject active ingredient have been adequately characterized by the applicant. Therefore, no mammalian toxicity is anticipated from dietary expos ure to the genetic material necessary for the production of the subject active plan t pesticidal ingredients.

Residue chemistry data were not required for a human health effects assessment of the subject plant-incorporated protectant i ngredients because of the lack of mammalian toxicity in the acute

exposures.

b) Infants and Children Risk Conclusions

FFDCA section 408(b)(2)(C ) provides that EPA shall assess the available information about consumption patterns among infants and children, speci al susceptibility of infants and children to pesticide chemical residues and the cumulative e ffects on infants and children of the residues and

other substances with a co mmon mechanism of toxicity. In addition, FFDCA section 408 provides that EPA shall apply an additional tenfol d margin of exposure (safety) for infants and

children in the case of threshold effects to acco unt for pre- and post-na tal toxicity and the completeness of the database unles s EPA determines that a different margin of exposure (safety)

will be safe for infants and children.

In this instance, based on all the available in formation, the Agency conc ludes that infants and children will consume minimal residues of this pl ant-pesticide and that th ere is a finding of no toxicity.

Thus, there are no threshold e ffects of concern and, as a re sult the provision requiring an additional margin of safety does not apply. Fu rther, the provisions of consumption patterns, special susceptibility, and cu mulative effects do not apply.

c) Aggregate Exposure (Not Including O ccupational Exposure) Risk ConclusionsCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

32 The Agency has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other

related substances. These considerations include dietary expos ure under the tolerance exemption and all other tolerances or exemptions in effect for the plant-incorporated protectants chemical residue, and exposure from non-occupa tional sources. Exposure via the skin or inhalation is not likely si nce the plant-incorporated protectant s are contained within plant cells which essentially eliminates these exposure routes or reduces these exposure routes to negligible. Oral exposure, at very low levels, may occur from ingestion of pro cessed corn products anddrinking water. However, a lack of mammalian toxicity and the digestibility of the plantincorporated protectants has b een demonstrated. The use site s for Cry1Ab delta endotoxin are all agricultural for contro l of lepidopteran insects. Therefore, exposure via residential or lawn use to infants and children is not expected. Even if negligible exposure shou ld occur, the Agency concludes that such exposure would present no risk due to the lack of toxicity.

d) Cumulative Effects Risk Conclusions

The Agency has considered available informati on on the cumulative effects of such residues and

other substances that have a co mmon mechanism of toxicity. Th ese considerations included the

cumulative effects on infants and children of su ch residues and other substances with a common mechanism of toxicity. Because there is no indication of mammalian toxicity to these plantincorporated protecta nts, there are no cumulative effects.

e) Dietary Risk Conclusion

There is a reasonable certainty that no harm w ill result from aggregate exposure to the United States population, including infant s and children, to the Cry1Ab pr otein and the genetic material necessary for its production. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. We have arrived at this conclusion because, as discussed above, no toxicity to mammals has been observed for the currently registered plantincorporated protectants.

f) Occupational Exposure and Risk Characterization

Exposure via the skin or inhala tion is not likely since the plan t-incorporated protectants are contained within plant cells whic h essentially eliminates these e xposure routes or reduces these exposure routes to negligible. Worker exposure to the Cry protein via seed dust is also expected to be negligible because of th e low amount of protein expressed in transformed plants. If such exposure should occur, the Agency concludes th at such exposure would not be expected to present any risk due to the lack of toxicity. If any unreas onable adverse effects caused by exposure to Cry1Ab are identified, these effects must be reported to the Agency as required by Sec. 6(a)(2) of FIFRA.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

33 BPPD RECOMMENDATION: (2001 Conclusion)

There is a reasonable certainty that no harm will result from exposure to Bacillus thuringiensis Cry1Ab delta-endotoxin and the genetic material necessary for its pr oduction in corn. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. Therefore, EPA considers that the Cry1Ab tolerance exemption has been reassessed and meets the 408(c)(2) standard. The tolerance exempti on citation for Cry1Ab in all plants follows.

§ 174.511 Bacillus thuringiensis Cry1Ab prot ein in all plants; exemption from the requirement of a tolerance.

Residues of Bacillus thuringiensis Cry1Ab protein in all plants ar e exempt from the requirement of a tolerance when used as plant-incor porated protectants in all food commodities. [72 FR 20435, Apr. 25, 2007]

2. Human Health Assessment of Cry1F Corn

Because both poCry1F and moCry1F share the same amino acid sequence, the toxicity of the Cry1F protein expressed in moCry1F and poCry1F plants is expected to be similar. The toxicity and allergenicity data submitted in support of th e poCry1F registration are also adequate to support the registration of moCry1F corn

a. Mammalian Toxicity and Allergenicity Assessment

Data have been submitted demonstrating the lack of mammalian toxicity at high levels of exposure to the pure Cry1F protein. These data demonstrate the safety of the products at levels well above maximum possible exposure levels that are reasonably anticipated in the crops. This is similar to the Agency position regarding toxici ty and the requirement of residue data for the

microbial Bacillus thuringiensis products from which this plan t-incorporated protectant was derived. [See 40 CFR Sec. 158.2130 and 158.2140.] For microbial products, further toxicity testing and residue data are tri ggered by significant acute effects in studies such as the mouse oral toxicity study, to verify th e observed effects and cl arify the source of th ese effects (Tiers II & III).

The acute oral toxicity data submitted support th e prediction that the Cry1F protein would be non-toxic to humans. Male and female mice (5 of each) were dosed with 15 % (w/v) of the test substance, which consisted of Bacillus thuringiensis var. aizawai Cry1F protein at a net

concentration of 11.4 %. Two doses were administ ered approximately an hour apart to achieve the dose totaling 33.7 mL / kg body weight. Outwar d clinical signs and body weights were observed and recorded throughout the 14 day study. Gr oss necropsies performed at the end of the study indicated no findings of toxi city. No mortality or clinical signs were noted during the study. An LD 50 was estimated at >5050 mg / kg body weight of this microbially produced test

material. The actual dose administered containe d 576 mg Cry1F protein / kg body weight. At thisCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

34 dose, no LD 50 was demonstrated as no toxicity wa s observed. Cry1F maize seeds contain 0.0017 to 0.0034 mg of Cry1F / gram of corn kernel tissue. When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad, et al .1992). Therefore, since no effects were shown to be caused by the plantincorporated protectants, even at relatively high dose levels, the Cry1F protein is not considered toxic. Further, amino acid sequence comparis ons showed no similarity between Cry1F protein to known toxic proteins availabl e in public protein databases.

Since Cry1F is a protein, allergenic sensitivitie s were considered. Curren t scientific knowledge suggests that common food allergen s tend to be resistant to de gradation by heat, acid, and proteases, may be glycosylat ed and present at high c oncentrations in the food.

Data has been submitted which demonstrates th at the Cry1F protein is rapidly degraded by gastric fluid in vitro and is non-glycosylated. In a solution of Cry1F:pepsin at a molar ratio of 1:100, complete degradation of Cry1F to amino aci ds and small peptides occurred in 5 minutes. A heat lability study demonstrated the loss of bi oactivity of Cry1F protein to neonate tobacco

budworm larvae after 30 minutes at 75 oC. Studies submitted to EPA done in laboratory animals have not indicated any potentia l for allergic reactions to B. thuringiensis or its components, including the d-endotoxin of the crystal prot ein. Additionally, a comparison of amino acid sequences of known allergens unc overed no evidence of any homo logy with Cry1F, even at the level of 8 contiguous amino acids residues.

The potential for the Cry1F protein to be a food a llergen is minimal. Regarding toxicity to the immune system, the acute oral toxicity data submitted support the prediction that the Cry1F protein would be non-toxic to humans. When protei ns are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad, et al .1992). Therefore, since no effects were shown to be caused by the plant-incorporated prot ectants, even at relatively high dose levels, the Cry1F protein is not considered toxic.

b. Aggregate Exposures

Pursuant to FFDCA section 408( b)(2)(D)(vi), EPA considers avai lable information concerning aggregate exposures from the pesticide residue in food and all other non- occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (r esidential and other indoor uses).

The Agency has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other

related substances. These considerations includ e dietary exposure under the tolerance exemption and all other tolerances or exemptions in effect for the plant-incorporated protectant chemical residue, and exposure from non-occ upational sources. Exposure via th e skin or inhalation is not likely since the plant-incorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to neglig ible. Oral exposure, at very low levels, may occur from ingestion of pr ocessed corn products a nd, potentially, drinking water. However a lack of mammalian toxicity and the digestibility of the plant-incorporatedCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

35 protectants have been demonstrated. The use site s for the Cry1F protein are all agricultural for control of insects. Therefore, exposure via residen tial or lawn use to infants and children is not expected. Even if negligible exposure should oc cur, the Agency concludes that such exposure would present no risk due to the lack of toxicity demons trated for the Cry1F protein.

c. Cumulative Effects

Pursuant to FFDCA Section 408(b)(2)(D)(v), EPA has considered available information on the

cumulative effects of such residues and other substances that have a common mechanism of toxicity. These considerations included the cumu lative effects on infants and children of such

residues and other substances with a common mechanism of toxicity. Because there is no indication of mammalian toxicity to these plant-incorporated protec tants, we conclude that there are no cumulative effects for the Cry1F protein.

d. Determination of Safety for U.S. Population, Infants and Children

1) Toxicity and Allergenicity Conclusions

The data submitted and cited regarding potential h ealth effects for the Cry1F protein include the characterization of the expressed Cry1F protein in corn, as well as the acute oral toxicity, heat stability, and in vitro digestibility of the proteins. The resu lts of these studies were determined applicable to evaluate human risk and the validi ty, completeness, and reliability of the available

data from the studies were considered.

Adequate information was submitted to show that the Cry1F test material derived from microbial cultures was biochemically and, functionally si milar to the protein produced by the plantincorporated protectant ingred ients in corn. Production of mi crobially produced protein was

chosen in order to obtain suffi cient material for testing.

The acute oral toxicity data submitted supports the prediction that the Cry1F protein would be non-toxic to humans. When protei ns are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad, et al .1992). Since no effects were shown to be caused by Cry1F protein, even at relatively high dose le vels (>5,050 mg test substance / kg body weight; 576 mg Cry1F / kg body weight), the Cr y1F protein is not considered t oxic. This is similar to the Agency position regarding toxicity and the re quirement of residue data for the microbial Bacillus thuringiensis products from which this plant-incorpor ated protectant was derived. [See 40 CFR Sec. 158.2130 and 158.2140.] For microbial products, fu rther toxicity testing and residue data are triggered by significant acute e ffects in studies such as the mous e oral toxicity study to verify the observed effects and clarify the sour ce of these effects (Tiers II & III).

Although Cry1F expression level data was require d for an environmental fate and effects assessment, residue chemistry data were not required for a human health effects assessment of the subject plant-incorporated protectant ingred ients because of the lack of mammalian toxicity.

Both (1) available information concerning the di etary consumption patterns of consumers (andCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

36 major identifiable subgroups of consumers includi ng infants and children); and (2) safety factors which, in the opinion of experts qu alified by scientific training and experience to evaluate the safety of food additives, are generally rec ognized as appropriate for the use of animal experimentation data were not evaluated. The la ck of mammalian toxici ty at high levels of exposure to the Cry1F protein de monstrates the safety of the product at levels well above possible maximum exposure leve ls anticipated in the crop.

The genetic material necessary for the production of the plant-incorporated protectants active ingredients are the nucle ic acids (DNA, RNA) which comprise (1) genetic material encoding these proteins and (2) their regul atory regions. "Regul atory regions" are the genetic material, such as promoters, terminators, and enhancers, th at control the expression of the genetic material encoding the proteins. DNA and RNA are common to all forms of plant and animal life and the Agency knows of no instance where these nucleic aci ds have been associated with toxic effects related to their consumption as a component of food. These ubiqu itous nucleic acids, as they appear in the subject active in gredient, have been adequately characterized by the applicant. Therefore, no mammalian toxicity is anticipated from dietary e xposure to the genetic material necessary for the production of the subject active plant pe sticidal ingredients.

Study Result MRID #
Acute Oral Toxicity Dosing of ten albino mice with bacterial cell protein containing the d-endotoxin of Bacillus thuringiensis var. aizawai at > 5050 mg/kg (0.576 g/kg of Cry1F) body weight resulted in no mortality and no observed gross abnormal ities. All animals appeared normal during the study and a ll except one gained weight throughout the study.

Classification: Acceptable. Toxic ity category III based on dose given with no observable effect. 446911-01

Acute Oral Toxicity This submission represents a clarification of test substance as presented in a previous submission and review. The acute oral toxicity study dosed mice at > 5050 mg microbial protein / kg body weight. The actual dose administered contained 576 mg Cry1F protein / kg body weight. At this dose, no LD 50 was demonstrated as no toxicity was observed. The truncated form of the protein represents amino acids 28-612 of the Cry1F toxin sequence, whereas the plant-expressed form of Cry1F contains amino acids 1-605. The truncated form used in the oral toxicity study adequately represents that toxin to be found in the plant expression system. Classification: Acceptable. 450201-18Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

37 Amino Acid Sequence Homology A modified (synthetic) form of the cry1Fa2 gene and the phosphinothricin acetyl transferase (pat) gene were inserted into maize plants by microprojectile bombardment. A database of available sequenced allergens and toxins was searched for similarity to both the less than full-length Cry1F and PAT proteins such that a level of eight, contiguous amino acid homology would be detected. This number of contiguous amino acids is considered to be the smallest antigenic portion of a protein (peptide) to induce an allergic reaction based upon T-cell recognition in a sensitized indivi dual. The database search and comparison to known allergens from plant, bacterial, fungal and animal origins indicates that no significant amino acid homology exists for Cry1F or PAT with any of these proteins. For both proteins of interest, the lack of any significant amino acid homology indicates that the potential for an immunological response developing into a food allergy from consumption of these proteins is low. Classification: Acceptable. 449717-01

Amino Acid Sequence Homology The 605 amino acid sequence of event TC1507 corn expressing Cry1F protein was sequentially searched for comparisons to known toxins contained in the NCBI Protein Sequence datasets.

The BLASTP search showed no significant amino acid sequence homologies using the Cry1Ab protein sequence as a query with any proteins known to be toxins. Therefore, the Cry1F protein does not share any amino acid sequence homology with any proteins known to be toxins.

Classification: ACCEPTABLE 477864-05

Amino Acid Sequence Homology No identity matches of ~ 35% over ~ 80 amino acid residues were observed for the Cry1F protein sequence against the protein sequences of known allerg ens. In addition, there were no 8 or greater contiguous identical amino acid matches observed with the Cry1F protein sequence. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted allergen homology assess ment for the Cry1F protein expressed in event TC1507 corn in (see MRID No. 449717-01).

Classification: ACCEPTABLE 477864-01

Lack of cross-reactivity between Cry1F protein in

Herculex I maize and dust mite Der p7 protein with human sera positive for Der p7-IgE Using a six contiguous amino acid bioinformatics analytical search, a match was identified between the Cry1F protein of Herculex I (poCry1F) corn and the Der p7 protein, an allergenic protein of the dust mite, D. pteronyssinus . Thi s study examined cross-reactivity between Cry1F protein in Herculex I maize and dust mite Der p7 protein with human sera positive for Der p7-IgE. There was no cross-reactivity; th erefore, dust mite allergic individuals would not be expected to experience an allergic reaction from ingesting Cry1F.

CLASSIFICATION : ACCEPTABLE 464440-01Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

38 Heat Stability The Cry1F test substance was prepared in 10 mM potassium phosphate buffer (pH 7.5) and placed into a water bath at either 60, 75 or 90 oC for 30 minutes, or into the refrigerator at 4 ̊ C. Application of treated Cry1F to th e surface of an insect diet and measurement of growth inhibition of neonate tobacco budworm larvae, indicated that the Cry1F pr otein was labile to heat at and above 75 oC. Classification: Acceptable. 452748-01

2) Infants and Children Risk Conclusions

FFDCA section 408(b)(2)(C ) provides that EPA shall assess the available information about consumption patterns among infants and children, speci al susceptibility of infants and children to pesticide chemical residues and the cumulative e ffects on infants and children of the residues and

other substances with a co mmon mechanism of toxicity. In addition, FFDCA section 408(B)(2)(C) also provides that EPA shall apply an additional te nfold margin of safety for

infants and children in the case of threshold effect s to account for pre- an d post-natal toxicity and the completeness of the database unless EPA determin es that a different margin of safety will be

safe for infants and children.

In this instance, based on all the available in formation, the Agency concludes that there is a finding of no toxicity for the Cr y1F protein and the genetic mate rial necessary for its production. Thus, there are no threshold eff ects of concern and, as a resu lt, the provision requiring an additional margin of safety does not apply. Furt her, the provisions of consumption patterns, special susceptibility, and cu mulative effects do not apply.

3) Overall Safety Conclusion (2001 Conclusion)

There is a reasonable certainty that no harm w ill result from aggregate exposure to the U.S. population, including infants and ch ildren, to the Cry1F protei n and the genetic material necessary for its production. This includes all anticipated dietary exposures and all other exposures for which there is reliable information.

The Agency has arrived at this conclusion because , as discussed above, no toxicity to mammals has been observed for the plan t-incorporated protectants.

e. Other Considerations

1) Endocrine Disruptors (Updated July 2010)

As required under FFDCA section 408(p), EP A has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (inc luding pesticide active and other ingredients) may have an effect in humans or wildlife si milar to an effect produced by a “naturally occurring estrogen, or other such endocrine effects as the Administrator may designate.” The EDSP employs a two-tiered ap proach to making the statutorily requiredCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

39 determinations. Tier 1 consists of a battery of 11 scr eening assays to identify the potential of a chemical substance to interact with the estrog en, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go th rough Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stag e of the EDSP where EPA will determine which, if any, of th e Tier 2 tests are necessary base d on the available data. Tier 2 testing is designed to identify a ny adverse endocrine related eff ects caused by the substance, and establish a quantitative relationship betw een the dose and the E, A, or T effect.

Between October 2009 and February 2010, EPA issued test orders/data call-ins for the first group of 67 chemicals, which contains 58 pesticid e active ingredients and 9 inert ingredients.

This list of chemicals was selected based on th e potential for human exposure through pathways such as food and water, residen tial activity, and certain post-applic ation agricultural scenarios.

This list should not be construed as a lis t of known or likely endocrine disruptors.

The Cry1F protein is not among the group of 58 pest icide active ingredients on the initial list to be screened under the EDSP. Under FFDCA § 4 08(p) the Agency must screen all pesticide chemicals. Accordingly, EPA anticipates i ssuing future EDSP orders/data call-ins for all Registration Review cases, including those for which EPA has already opened a Registration Review docket for a pestic ide active ingredient.

For further information on the status of the ED SP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, pleas e visit our website: http://www.epa.gov/endo/

.

3) Codex Maximum Residue Level

No Codex maximum residue levels exists for the plant-incorporated protectants Bacillus thuringiensis Cry1F protein and the genetic material necessary for its production in corn.

f. Tolerance Exemption

The tolerance exemption citati on for Cry1F in corn follows.

§ 174.520 Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement

of a tolerance.

Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated prot ectants in the food and feed commodities of corn; corn, field; corn, sweet; and corn, pop. [72 FR 20435, Apr. 25, 2007]Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

40 3. Synergism Assessment for MON 810 x TC1507 (Cry1Ab x Cry1F) Corn

Study Result MRID #
Synergism A seven-day laboratory sensitive insect bioassay was conducted to determine if the combination PIP product 1507 x 59122 x MON810 has a synergistic effect in comparison to the individual parental events TC1507 (expressing Cry1F protein) and MON810 (expressing Cry1Ab protein) on target lepidopteran pests. The pests used in the bioassay were European corn borer (ECB,

Ostrinia nubilalis ), southwestern corn borer (SWCB, Diatraea grandiosella ), fall armyworm (FAW, Spodoptera furgiperda ), and corn earworm (CEW, Helicoverpa zea ). The observed and expected larval mortality in the 1507 x 59122 x MON810 group were similar and mean larval weig ht of the survivors exposed to 1507 x 59122 x MON810 leaf tissue was not significantly different from the single parental events or the negative control of

non- Bt maize. These results indicat e that the Cry1F and Cry1Ab proteins act independently and do not have a synergistic or antagonistic effect on the target pests, other than an additive effect. Quantitative ELISA resu lts also confirmed that the expression of each of the protei ns in the combination PIP was not affected by the presence of the other protein.

CLASSIFICATION: ACCEPTABLE 476778-02

REFERENCES

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107:575-582.

Dien, B.S., R.J. Bothast, L.B. Iten, L. Barrios, and S.R. Eckhoff, (2002), Fate of Bt Protein and Influence of Corn Hybrid on Ethanol Production, Cereal Chemistry 79:582-585.

Sjoblad, R. D., J. T. McClintock, and R. Engl er (1992) "Toxicologic al Considerations for Protein Components of Biological Pesticid e Products," Regulatory Toxicology and Pharmacology 15:3-9.

USDA. 2004. Performance verification of qualitativ e mycotoxin and biotech rapid test kits (Directive 9181.2). Available from: http://www.gipsa.usda.gov/GIPSA/docum ents/GIPSA_Documents/9181-2.pdf

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Vázquez-Padrón, R.I., L. Moreno-Fierros, L. Neri -Bazán, A.F. Martínez-Gil, G.A. de la Riva & R. López-Revilla (2000) Characterization of the mucosal and systemic response induced byCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

41 Cry1Ac protein from Bacillus thuringiensis HD 73 in mice, Brazilian Journal of Medical and Biological research 33:147-155.

43130801 Williams, D. (1994) Product Characterization: Bacillus thuringiensis var. kurstaki ( Btk ) Protein in Corn: Lab Project Numb er: NKJNV2. Unpublished study prepared by Northrup King Co. 13 p.

43352602 Williams, D. (1994) Product Characterization: Bacillus thuringiensis var. kurstaki Protein in Corn: Supplemental Informa tion: Lab Project Number: NK5PDCH. Unpublished study prepared by Northrup King Co. 6 p.

437548-01 Williams, D. (1995) Product Characterization: Bacillus thuringiensis var. kurstaki Protein in Corn: Registration Application. Unpublished study prepared by Northrup King Co. 23p.

43397202 Meeusen, R.; Mettler I. (1994) Equiva lence of Plant and Microbially Produced Bacillus thuringiensis kurstaki HD-1 Protein: Lab Project Number: 1/NK5EQ. Unpublished study prepared by Nothrup King Co.; University of Wisconsin; and Kendrick Labs. 43 p.

43533201 Keck, P.; Fromm, M.; Sanders, P.; et al . (1995) Molecular Charac terization of Insect

Protected Corn Line MON 80100: Lab Pr oject Number: MSL 13924. Unpublished study prepared by Monsanto Co. 100 p.

43533203 Lee, T.; Bailey, M.; Sanders, P. (1995) Compositional Comparison of Bacillus thuringiensis subsp. kurstaki HD-1 Protein Produced in European Corn Borer Resistant Corn and the Commercial Microbial Product, DIPEL: Lab Project Number: 94-01-39-12: MSL 13876. Unpublished study prepared by Monsanto Co. 35 p.

43665501 Levine, E.; Groth, M.; Kani a, J.; et al. (1995) Molecula r Characterization of Insect Protected Corn Line MON 810: Lab Proj ect Number: MSL 14204. Unpublished study prepared by Monsanto Co. 61 p.

43533204 Lee, T.; Bailey, M.; Sims, S. (1995) Assessment of the Equivalence of the Bacillus thuringiensis subsp. kurstaki HD-1 Protein Produced in Escherichia coli and European

Corn Borer Resistant Corn: Lab Pr oject Number: 94-01-39-09: MSL 13879. Unpublished study prepared by Monsanto Co. 94 p.

43145201 Keck, P. (1994) Determination of Copy Number and Insert Integrity for Cotton Line 531: Lab Project Number: 92 /01/36/12: 92/427/713. Unpublished study prepared by Monsanto Agricultural Group. 66 p.

43468001 Naylor, M. (1992) Ac ute Oral Toxicity Study of Btk HD-1 Tryptic Core Protein in Albino Mice: Lab Proj ect Numbers: 92069: 11985: ML92069. Unpublished study prepared by Monsanto Co. 264 p.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

42 43439201 Ream, J. (1994) Assessment of the In vitro Digestive Fate of Bacillus thuringiensis subsp. kurstaki HD-1 Protein: Lab Project Numb er: 93-01-39-04. Unpublished study prepared by Monsanto Co. 44 p.

44714801 Narva, K.; Zeph, L.; Jayne, S. (1998) Pr oduct Characterization Data for Bacillus thuringiensis var. aizawai Cry 1 F Insect Control Protein as Expressed in Maize. Unpublished study prepared by Mycogen Corporation. 335 p.

44714802 Hondred, D. (1998) Characterization of Ge ne Inserts-Bacillus thuringiensis var. aizawai Cry 1 F Insect Control Protein as Expressed in Maize: Lab Project Number: 98-01-RA-NGLP-003. Unpublished study prepared by Pioneer Hi-Bred International, Inc. 36 p.

44714804 Rivas, J.; Stauffer, C. (1998) Quantita tive ELISA Analysis of Cry 1 F Expression

Levels in Maize MPS Inbreds and Hybrid Lines 1360, 1365, 1366, and 1369. (Interim Report): Lab Project Numb er: 98-01-RA-GLP-005. Unpublished study prepared by Pioneer Hi-Bred In ternational, Inc. 95 p.

45020102 Hondred, D. (1999) Characterization of In serted Genes in Cry1F Maize Line 1507: Final Report: Lab Project Number : 98-11-RA-NGLP-013. Unpublished study prepared by Pioneer Hi-Bred In ternational, Inc. 17 p.

45020117 Meyer, T.; Shanahan, D. (1999) Supplement to MRID 44714801: Supplemental

Data-Product Characterization for Bacillus thuringiensis var. aizawai Cry1F Insect Control Protein as Expressed in Ma ize: Lab Project Number: GH-C 5034. Unpublished study prepared by Dow AgroSciences. 78 p.

45686701 Guyer, D. (2002) Analytical Method for the Detection of Cry1Ab Protein in BT11 Corn Grain: Lab Projec t Number: AS 003 BG. Unpub lished study prepared by Syngenta Seeds, Inc. 27 p.

45696401 Adams, C. (2002) Independent Lab Validat ion of the SDI Trait Bt1 Corn Lateral Flow Test Kit: An Amended Report: La b Project Number: B02-005: B02-005-A. Unpublished study prepared by Medallion Labora tories, A Division of General Mills. 32 p. {OPPTS 860.1000, 860.1340}

45878901 Ledsema, B.; Hoskins, S.; Jennings, J.; et al. (2003) Cry1Ab Prot ein Levels in Leaf, Pollen, Whole Plant, Forage, Root, and Grain Tissues from Corn Event MON 210

Produced in U.S. Field Trials in 2002 : Lab Project Number : 02-01-39-22: MSL 18508. Unpublished study prepared by Monsanto Company. 2 p.

45879803 Privalle, L. (2003) Quantification of Cry 1Ab Protein in Maize (Corn) Tissues and

Whole Plants Derived From Transformati on Event Bt11: Lab Project Number: BT11- 02-01: SSB-005-03. Unpublished study prepar ed by Syngenta Seeds, Inc. 17 p.

47882001 McDonald, J. (2009) Quantific ation of Transgenic Proteins in Maize Tissues of Bt11Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

43 x MIR162 Sweet Corn and Bt11 x MI R162 Field Corn. Project Number: BT162/08/01, SSB/020/09. Unpublished study prepared by Syngenta Biotechnology, Inc. 41 p.

47677801 Brink, K.; Dietrich, N. (2009) Molecular Characterization of Ma ize Combined Trait

Product DAS-01507-1xDAS-59122- 7xMON-00810-6xMON-00603-6 Using Southern Blot Analysis and Event-Speci fic Polymerase Chain Reaction. Project Number: PHI/2008/082. Unpublishe d study prepared by E.I. Dupont de Nemours and Company. 42 p.

47677802 Binning, R. (2009) Laboratory Characteri zation of Key Lepidopteran Pest Response to Pyramided Events. Project Number: PHI/2008/101. Unpublished study prepared by Pioneer Hi-Bred Inte rnational, Inc. 16 p.

47786401 Krauss, A.; Cressman, R. (2009) Comparison of the Amino Acid Sequence Identity Between the Cry1F Protein amd Known Protein Allergens. Project Number: PHI/2008/236. Unpublished study prepared by E.I. du Pont de Nemours and Company. 23 p.

47786404 Krauss, A.; Cressman, R. (2009) Comparison of the Amino Acid Sequence Identity Between the Cry1Ab Protein from Event MON810 and Known Protein Allergens. Project Number: PHI/2008/239. Unpublished study prepared by E.I. du Pont de Nemours and Company. 24 p.

47786405 Cressman, R.; Krauss, A. (2009) Evaluati on of the Amino Acid Sequence Similarity of the Cry1F Protein to the NCBI Prot ein Sequence Datasets. Project Number: PHI/2008/240. Unpublished study prepared by E.I. du Pont de Nemours and Company. 291 p.

47786406 Krauss, A.; Cressman, R. (2009) Evaluati on of the Amino Acid Sequence Similarity of the Cry1Ab Protein from Event MON810 to the NCBI Protein Sequence Datasets. Project Number: PHI/2008/241. Unpublished study prepared by E.I. du Pont de Nemours and Company. 354 p.

47880001 Staley, J.; Rood, T.; Johnson, T.; et al.; ( 2009) Expressed Trait Protein Concentration of Maize Lines Containing Events DAS-01507-1, DAS-59122-7, MON-00810-6, and

Combined Trait Products DAS -01507-1xMON-00810- 6, DAS-59122-7xMON00810-6, and DAS-01507-1xDAS-59122-7xMON- 00810-6: Additional Addendum. Project Number: PHI/2009/182. Unpublished study prepared by Pioneer Hi-Bred International, Inc. 110 p.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

44 C. ENVIRONMENTAL ASSESSMENT

Background

In 2001, EPA conducted an environmental reassessment of the registered Bt plant-incorporated protectants. The general topi cs covered included gene flow and the potential for weeds to develop if pollen from Bt crops plants were to fertilize othe r plants, horizontal gene transfer, expression of Bt Cry proteins in plant tissues, ecological effects, especially considering the currently available data on mona rch butterflies, and fate of Bt Cry proteins in the environment.

Information used for the reassessment include d the original data submissions for EPA registration, additional data submitted by the regist rants (including that in the response to the December 1999 Data Call-in), public literature, re sults of workshops and scientific seminars, recommendations from the SAP, additional discussi ons with scientific experts, and other public

comments.

In addition to those data, the 2010 environmenta l assessment includes all conditionally required data and new information the Agency has recei ved regarding Cry1Ab and Cry1F proteins since 2001, including long-term field studies, avian diet ary studies, and an updated assessment of the impacts of Bt proteins on mona rch butterflies.

In 2005, the Agency conducted an environmen tal effects assessment of moCry1F, the insecticidal protein as expressed in maize line TC6275 (Hill 2005). The maize-optimized (mo) Cry1F gene encodes for the identical truncated Cry1F protein as expressed by maize plants containing the plant-optimized (po) Cry1F gene (MRID No. 447148-01). Codon changes were made to the gene to improve expression in moCr y1F maize plants but these changes did not alter the amino acid sequence of the protein as compared to that expressed in poCry1F maize plants. Therefore, the specificity (i.e., t oxicity toward target pests) of the Cry1F protein expressed in moCry1F and poCry1F plants should be similar. Testing with bacterially prepared Cry1F protein at levels greatly exceeding those found in maize optimized plants resulted in no adverse effect to several beneficial species including the monarch butterfly. Expression data reveal that moCry1F plants express somewhat lower concentrations of Cry1F protein in pol len and grain and higher concentrations in stalks, leav es, and roots than the poCry1F plants, though these expression differences are not expected to significantly change the exposu re to Lepidopteran non-target organisms. Field monitoring for effects of poC ry1F corn on non-target insects confirmed the absence of adverse effects to non-target orga nisms (MRID No. 450201-13). Data illustrating the soil degradation of poCry1F protein were submitte d to the EPA in support of the registration of Herculex I (MRID No. 450201-05) . These data support the re gistration of moCry1F corn because the two proteins share an identical amino acid sequence. EPA’s assessment of the poCry1F studies submitted in support of the He rculex I registration (EPA Reg. No. 029964-3) are applicable to the moCry1F corn registration for reasons outlined above. These studies, along with those for Cry1Ab protein, will be summarized in this assessment in both a tabular format and a more descriptive format.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

45 1. Tiered Hazard and Risk Assessment Process

To minimize data requirements and avoid unnecessary tests, risk assessments are structured such

that risk is determined first from estimates of hazard under “worst-case” exposure conditions. A

lack of adverse effects under these conditions would provide e nough confidence that there is no

risk and no further data would be needed. Henc e, such screening tests conducted early in an investigation tend to be broad in scope, but re latively simple in design, and can be used to demonstrate acceptable risk under most conceivable conditions. Wh en screening studies suggest potentially unacceptable risk, additional studies ar e designed to assess risk under more realistic field exposure conditions. These later tests are mo re complex than earlier screening studies. Use of this “tiered” testing framework saves valuab le time and resources by organizing the studies in a cohesive and coherent manner and eliminating unnecessary lines of inve stigation. Lower tier, high-dose screening studies also allow tighter control over experimental variables and exposure conditions, resulting in a greater ability to produce statistically reliable re sults at relatively low

cost. 1

Tiered tests are designed to first represent unrealistic worst-case scen arios and ONLY progress

to real-world field scenarios if the earlier tier ed tests fail to indicat e adequate certainty of acceptable risk. Screening (Tier I) non-target or ganism hazard tests are conducted at exposure concentrations several times higher than the hi ghest concentrations e xpected to occur under realistic field exposure scenarios. This has allowe d an endpoint of 50% mortality to be used as a trigger for additional higher tier testing. Le ss than 50% mortality under these conditions of extreme exposure suggest that population effects are likely to be negligible given realistic field exposure scenarios.

The Environmental Protection Agency (EPA) uses a tiered (Tiers I–IV) testing system to assess the toxicity of a plant-incorporat ed protectant (PIP) to represen tative non-target organisms that could be exposed to the toxin in the field en vironment. Tier I high-dose studies reflect a screening approach to testing de signed to maximize any toxic effects of the test substance on the test (non-target) organism. The scre ening tests evaluate single speci es in a laboratory setting with mortality as the endpoint. Tiers II–IV gene rally encompass definitive hazard level determinations, longer term greenhouse or field testing, and are implemented when unacceptable effects are seen at the Tier I screening level.

Testing methods, which utilize the tiered approa ch, were last published by EPA as Harmonized Office of Prevention, Pesticides, and Toxic S ubstances (OPPTS) Testing Guidelines (now Harmonized Office of Chemical Safety and Pollu tion Prevention (OCSPP) Testing Guidelines), Series 850 and 885 (EPA 712-C-96-280, February 1996). 2 These guidelines apply to microbes

1 Non-target invertebrate hazard tests often are conducted at ex posure concentrations several times higher than the maximum con centrations expected to occur under realistic expos ure scenarios. This has customarily allowed an

endpoint of 50% mortality to be used as a trigger for additional higher tier testing. Lower levels of mortality under these con ditions of extreme exposure suggest that population effect s are likely to be negligible given realistic exposure

scenarios. Thus, it follows that the observed proportion of responding individuals can be compared to a 50% effect to determine if the observed proportion is significantly lower than 50%. For example, using a binomial approach, a

sample size of 30 individuals is sufficient to allow a treatment effect of 30% to be differentiated from a 50% effect with 95% confidence using a one-sided Z test. A one-sided test is appropriate because only effects of less than 50%

indicate that further experiments are not needed to evaluate risk.

2 General OCSPP Harmonized Testing Guidelines available from: http://www.epa.gov/ocspp/pubs/frs/home/guidelin.htm.

Series 850 Testing Guidelines available from:

http://www.epa.gov/ocspp/pubs/frs/publications/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Drafts/index.html.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

46 and microbial toxins when used as pesticides, including those that are naturally occurring and those that are strain improved e ither by natural select ion or by deliberate genetic manipulation. Therefore, plant-incorporated protectants (PIPs) containing microbial toxi ns are also covered by these testing guidelines.

The Tier I screening maximum hazard dose (MHD) approach to environmental hazard assessment is based on some factor (wheneve r possible >10) times the maximum amount of active ingredient expected to be available to te rrestrial and aqua tic non-target organisms in the environment, or the Estimated E nvironmental Concentration (EEC). 3 Tier I tests serve to identify potential hazards and are conducted in th e laboratory at high dose levels, which increase the statistical power to test the hypotheses. Elevated test dos es, therefore, add certainty to the assessment, and such tests can be well standardized. The Guidelines call for initial screening testing of a single group or severa l groups of test animals at the maximum hazard dose level. The Guidelines call for testing of one treatment group of at least thirty animals or three groups of ten test animals at the screening test concentration. The Guidelines fu rther state that the duration of all Tier I tests should be approximately 30 days. Some test species, nota bly non-target insects, may be difficult to culture and the suggested test duration has b een adjusted accordingly. Control

and treated insects should be observed for at leas t 30 days or, in cases where an insect species cannot be cultured for 30 days, until nega tive control mortalit y rises above 20%.

Failing the Tier I (10x EEC) screen ing at the MHD does not necessari ly indicate the presence of an unacceptable risk in the field, but tr iggers the need for additional testing. 4 A less than 50% mortality effect at the MHD is taken to indi cate minimal risk. Greater than 50% mortality, however, does not necessarily indicate the existen ce of unacceptable risk in the field, but it does trigger the need to collect addi tional dose-response information a nd a refinement of the exposure estimation before deciding if the risk is accepta ble or unacceptable. Where potential hazards are detected in Tier I testing (i.e., mortality is greater than 50%), a dditional information at lower test doses is required, which can serve to confirm whether any effect mi ght still be detected at more realistic field (1x EEC) concentr ations and routes of exposure. 5

When screening tests indicate a need for add itional data, the OCSPP Harmonized Guidelines call for testing at incrementally lower doses in order to establish a definitive LD 50 (i.e., dose that will kill 50% of the test organisms within a designa ted period) and to quantify the hazard. In the definitive testing, the number of doses and test organisms evaluated must be sufficient to

determine an LD 50 value and, when necessary, the Lowest Observed Adverse Effect Concentration (LOAEC), No Observed Adverse Effect Level (NOAEL), or reproductive and behavioral effects such as feeding inhibition, wei ght loss, etc. In the final analysis, a risk assessment is made by comparing the LOAEC to the EEC; when the EEC is lower than the LOAEC, a no risk conclusion is made. These tests offer greater environmen tal realism, but they may have lower statistical power . Appropriate statistical methods , and appropriate statistical

Series 885 Testing Guidelines available from: http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series885.htm.

3 The dose margin can be less than 10x where uncertainty in the sy stem is low or where high concentrations of test material are not possible to achieve due to test organism feeding habits or other factors. High-dose testing also may

not be necessary where many species are tested or tests are very sensitive, although the test concentration used must exceed 1x EEC.

4 It is notable that the 10x EEC MHD testing approach is not equivalent to what is commonly known as “testing at a 10x safety f actor,” where any adverse effect is considered signif icant. Tier I screen testing is not “safety factor

testing.” In a “10x safety factor” test, any adverse effect noted is a “level of concern,” whereas in the EPA environmental ris k assessment scenario any adverse effect is viewed as a concern only at 1x the field exposure.

5 The 1x EEC test dose is based on plant tissue content and is considered the highest dose in a worst-case scenario (sometimes referred to as the Highest Estimated Environmental Co ncentration or HEEC). Th is 1x EEC is still much

greater than any amount which any given non-target organism may be ingesting in the field because most non-target organisms do not ingest plant tissue.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

47 power, must be employed to evaluate the data from the definitive tests. Higher levels of replication, test species numbers, and/or repet ition are needed to enhance statistical power in

these circumstances.

Data that show less than 50% mortality at the maximum hazard dosage level (i.e., LC 50 , ED 50 , or
LD 50 >10x EEC) are sufficient to evaluate advers e effects, making lower field exposure dose definitive testing unnecessary. Also, the reco mmended >10x EEC maximum hazard dose level is a highly conservative factor . The published EPA Level of Con cern (LOC) is 50% mortality at 5x EEC (U.S. EPA 1998). 6

Validation: The tiered hazard assessment approach was developed for EPA by the American Institute of Biological Sciences (AIBS) and confirmed in 1996 as an acceptable method of environmental hazard assessment by a Federal Insecticide, Fungicide , and Rodenticide Act (FIFRA) Scientific Advisory Pa nel (SAP) on microbial pesticid es and microbial toxins. The December 9, 1999 SAP agreed that the tiered appro ach was suitable for use with PIPs; however, this panel recommended that, for PIPs with inse cticidal properties, a dditional testing of beneficial invertebrates closely re lated to target species and/or lik ely to be present in genetically modified (GM) crop fields s hould be conducted. Testing of Bacillus thuringiensis ( Bt ) Cry proteins on species not closely re lated to the target insect pe st was not recommended, although it is still performed to fulfill the published EPA non -target species data requirements. In October 2000, another SAP also recommended that field testing should be used to evaluate populationlevel effects on non-target or ganisms. The August 2002, SAP (a nd some public comments) generally agreed with this approach, with the additional recommendation that indicator organisms should be selected on the basis of pot ential for field exposure to the subject protein (U.S. EPA 2000, 2001a, 2002, and 2004b).

Chronic studies: Since delayed adverse effects and/or accumulation of toxi ns through the food chain are not expected to result from exposure to proteins, protein toxins are not routinely tested for chronic effects on non-target organisms. The 30-day test duration requirement, however, does amount to subchronic testing when performed at field exposure test doses. Proteins do not bioaccumulate. The biological nature of proteins makes them readily susceptible to metabolic, microbial, and abiotic degradation once they are ingested or excreted into the environment. Although there are reports that some proteins (Cry proteins) bind to soil particles, it has also been shown that these proteins are degraded rapidly by soil microbial flora upon elution from soil particles.

Conclusion: The tiered approach to test guidelines ensure s, to the greatest ex tent possible, that the Agency requires the minimum amount of data n eeded to make scientifically sound regulatory

decisions. EPA believes that maximum hazard dose Tier I screening testing presents a reasonable approach for evaluating hazards relate d to the use of biological pesticides and for identifying negative results with a high degree of confidence. The Agency expects that Tier I testing for short-term hazard assessment will be sufficient for most studies submitted in support of PIP registrations. If long-range adverse effects must be ascert ained, however, then higher tier,

6 The established peer and EPA Science Board reviewed guidance on screening test levels of concern is 50% mortality at 5x envir onmental concentration for terrestrial and 10x for aquatic species. The appropriate endpoints in highdose limit/screening testing are based on mortality of the treated, as compared to the untreated (control) non-target organisms . A single group of 30 test animals may be tested at the maximum hazard dose.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

48 longer term field testing will be required. As noted above, the October 2000, SAP and the National Academy of Sciences (NAS 2000) reco mmended testing non-target organisms directly in the field. This approach, with an emphasis on testing invertebrates found in corn fields, was also recommended by the August 2002, SAP and was supported by several public comments.

Based on these recommendations and due to the l ack of baseline data on the potential for longterm environmental effects from the cultivat ion of PIP-producing pl ants, the Agency has required long-term field studies on invertebra te populations/communities and Cry protein accumulation in soils as conditions of past PIP registrations.

Since the commercialization of Bt crops, the number of field st udies published in scientific literature, in combination with the post-registr ation field studies submitted to the Agency, hasaccumulated to a level where empirical conclusions can be made. As a result, the issue of longrange effects of cultivation of these Cry proteins on the invertebrate community structure in Bt crop fields has since been adequately addressed. Specifically, a meta-analysis 7 of the data

collected from 42 field studies indicated that non-target invertebrate s are generally more

abundant in Bt cotton and Bt maize fields than in non-transgenic fields managed with insecticides (Marvier et al. 2007). In addition, a comprehensive review of short- and long-term field studies

on the effects of invert ebrate populations in Bt corn and cotton fields indicated that no

unreasonable adverse effects are taki ng place as a result of wide-scale Bt crop cultivation (Sanvido et al. 2007). Another review of field tests publis hed to date concl uded that the largescale studies in commercial Bt cotton have not revealed any une xpected non-target effects other than subtle shifts in the arthropod community cau sed by the effective contro l of the target pests (Romeis et al. 2006). Slight reductions in some invert ebrate predator pop ulations are an inevitable result of all pest ma nagement practices, which result in reductions in th e abundance of the pests as prey.

Overall, the Agency is in agr eement with the conclusions of th ese studies and, collectively, these results provide extensive data to support that Bt crops have not caused long-term environmental effects, on a population level, to organisms not targeted by Bt proteins. Based on these considerations, regulatory testing of the specialist predators and pa rasitoids of target pests may eventually be considered unnecessary.

2. Environmental Exposure Assessment

The EPA environmental exposure assessment is base d on adverse effects at field exposure rates (1X EEC), and not on adverse effects at greater concentrations. The dose margin can be less than 10x where uncertainty in the system is low or where high concentrations of test material are not possible to achieve due to test organism f eeding habits. High dose te sting also may not be necessary when many species are tested or when tests are very sensitive, although the concentration used must exceed 1X EEC. It is impor tant to note that Tier I screen testing is not “safety factor testing.” In a tr aditional “10X safety factor” test any advers e effect noted is a “level of concern,” whereas in the EPA envir onmental risk assessment scenario any adverse

7 This research was funded by EPA grant CR-832147-01. The Bt crop non-target effects database can be found on the National Cent er for Ecological Analysis and Synthesis (NCEAS) Web Site: http://delphi.nceas.ucsb.edu/btcrops/.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

49 effect is viewed as a concern only at 1X the field exposure.

The nominal protein expression levels for Cry1A b and Cry1F, as determined by field and/or greenhouse conditions, are described below. Note that there may be variation between the Bt protein values reported by each company due to di fferences in the antibody-based reagents used for quantifying the Bt protein, or whether protei n values in tissue are re ported in fresh or dry weight. While these differences may make di rect comparisons between the tissue expression levels reported by different companies difficult, the reported dry weight levels provide enough information to be used for risk assessment purpos es especially when considered along with the reported tissue bioactivity values.

a. Cry1Ab Protein Expression in Bt11 and MON810 Corn Tissues

TABLE 1. Cry1Ab Concentrations on a Dry Weight Basis in Event Bt11 Hybrid Plants

Developmental Stage

V9-V12 Anthesis Seed Maturity Tissue Location Hybrid 1 mean μg Cry1Ab/gdw ± S.D. (range)

Leaves Bloomington, Illinois Event Bt11 25.88 ± 1.35 (23.89—27.63) 17.82 ± 1.54 (15.99—19.74) 16.84 ± 3.31 (12.17—19.59)

Roots Bloomington, Illinois Event Bt11 9.99 ± 0.59 (9.14—10.62) 6.41 ± 0.79 (5.67—7.72) 4.32 ± 1.52 (3.32—6.99)

Kernels Bloomington, Illinois Event Bt11 N/A 3 N/A 1.45 ± 0.07 (1.39—1.56)

Pollen Mackinaw, Illinois Event Bt11 N/A 0.04 ± 0.00 (0.036—0.042) N/A

Pollen Monroeville, Indiana Event Bt11 N/A 0.06 ± 0.02 (0.048—0.079) N/A

Pollen Seward, Nebraska Event Bt11 N/A <0.037 N/A

TABLE 2. Average Cry1Ab concentration from Corn Event MON 810 corn tissues grown in four regions of the US

Tissue (No. days post planting)
Overseason Leaf (OSL) OSL-1 (21 days) OSL-3 (40 days) OSL-4 (50 days) OSL-5 (60 days) Overseason Whole Plant (OSWP) OSWP-1 (21 days) OSWP-5 (60 days) Forage (90 days) Pollen (60 days) Overseason Root (OSR) OSR-1 (21 days) OSR-3 (40 days) OSR-4 (50 days) OSR-5 (60 days) Forage root (90 days) Grain (125 days) Cry1Ab conc. (μg/g dry weight)

120 ± 15

46 ± 5.8

61 ± 17

51 ± 17

120 ± 34

25 ± 6.3

7.6 ± 4.5

NA

42 ± 9.5

20 ± 5.0

22 ± 3.7

19 ± 8.8

16 ± 6.0

0.63 ± 0.06

NA = Not Applicable.

b. Cry1F Protein Expression in MoCry1F TC6275 and PoCry1F TC1507 Corn TissuesCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

50 There is significantly increased Cry1F prot ein expression in moCr y1F TC6275 stalks, whole plants, and forage sampled during the floweri ng (R1) stage in comparison to poCry1F TC1507. Conversely, there is significantly decreased Cr y1F protein expression in moCry1F pollen and grain in comparison to poCry1F. These expression differences are not expected to significantly change the exposure to lepidopter an non-target organisms. The increased expression of Cry1F protein in stalks of moCry1F hybrids could pot entially decrease the risk of resistance development by borers, while the decreased expr ession in pollen and grain should decrease the non-target exposure to Cry1F expressed in corn (Zabik et. al 2003).

TABLE 3. Comparative expression of Cry1F protein in moCry1F TC6275 and poCry1F TC1507 corn tissues (Table recreated from Zabik et al. 2003).

TC6275 (moCry1F) TC1507 (poCry1F)

Mean Standard

Deviation Min/Max Range Mean Standard

Deviation Min/Max Range Tissue Growth Stage (ng/mg Tissue Dry Weight)

Leaf V9 b

R1

R4

Senescence 17.3

28.5

44.8

0.71 3.41

5.38

16.8

1.14 10.7-23.8

16.5-36.7

35.8-109.2

0-3.0.9 12.1 6.2 0-24

Root V9 b

R1

R4

Senescence 6.14

6.60

5.99

1.97 1.87

1.98

1.89

2.03 4.53-8.14

3.14-10.9

2.35-9.26

0.29-6.91

Whole

Plant V9

R1

Senescence 6.22

7.16

2.47 1.16

1.45

0.41 4.98-7.87

5.32-9.57

1.95-3.07 5.2

3.6

1.6 1.9

1.1

0.6 2.6-6.8

2.5-4.7

0.9-2.4

Pollen R1 3.67 0.34 3.09-4.60 21.9 2.9 16.4-27.2

Stalk R1 11.0 2.67 6.77-16.4 5.8 1.7 3.3-10.3

Forage R4 6.26 1.09 5.05-7.77 1.7 1.1 0-3.2

Grain Maturity 1.14 0.27 0.62-1.68 2.2 0.8 0-4

b Recalculated Results

c. Fate in Soils and Indirect Effects on Soil Biota

Most of the Cry protei n deposited into soil by Bt crops is quickly degr aded, although a residual amount may persist in biologically active form for a much longer period of time. It has also been reported that the same degree of Bt Cry protein persistence takes pl ace in soils that have been exposed to repeat Bt spray applications as those exposed to Bt -expressing crops. Field tests of Cry protein degradation in soil unde r a range of conditions typical of Bt crop cultivation areCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

51 needed to yield relevant data on persistence and natural variation. Limite d data do not indicate that Cry proteins have any meas urable effect on microbial populat ions in the soil. Horizontal transfer from transgenic plants to soil bacteria has not been demonstrat ed. Current studies of Bt in soil show no effect on bacteria, actinomyces, fu ngi, protozoa, algae, nema todes, springtails or earthworms. In addition, new plants planted in Bt Cry protein containing soil do not take up the

Bt protein.

i. Fate of Bt Proteins in Soil Soil organisms may be exposed to δ -endotoxins from current transg enic crops through roots, incorporation of above ground plan t tissues into soil after harves t, or by pollen deposited on the soil. Root exposure may occur by feeding on living or dead roots or, theo retically, by ingestion or absorption afte r secretion of δ -endotoxin into the soil. The latt er situation is the subject of a recent brief communication in the journal Nature by Saxena et al. (1999), and is discussed in more detail below. In addition, evidence s uggests that some soil components (e.g. clays and humic acids) bind δ -endotoxins in a manner that makes them recalcitrant to degradation by soil microorganisms, without elimin ating their insect toxicity. Therefore, exposure to δ -endotoxin bound to soil particles may also be a route of exposure for some soil organisms.

Experiments addressing amounts and persistence of δ -endotoxins in the soil have been submitted by registrants and reviewed for the current condi tional registrations. In addition, a number of publications in the scientific literature have addre ssed the degradation of Cry proteins in the soil. These experiments consist of the incorporation of purified δ -endotoxin or transgenic plant material in soil in a laboratory setting. Cry protein DT 50 (time to 50% degradation) studies were submitted for registration of corn expressing Cr y1Ab and Cry1F proteins, and published studies are available for Cry1Ac cotton. Cry1Ab biodegraded in an estimated DT 50 of 1.6 days as expressed in transgenic corn tis sue and in 8.3 days for purified protein (Sims and Holden 1996). Based on a bioassay with the tobacco budworm ( Heliothis virescens ), a target species, purified Cry1F proteins incorporated into test soils biodegraded with a DT 50 of approximately 3.13 days. Data submitted by Monsanto for Cry1Ac protein and transgenic Cry1Ac in cotton report DT 50 ’s of approximately 9-20 days for the purified prot ein and 41 days for the protein in cotton tissue (MRID No. 439995-09). Published data for Cry1 Ab andy1Ac in cotton tissue or as purified protein report a DT 50 range of 2.2 to 46 days, where meas urable (in 4 of 11 experiments); DT 50 ’s for transgenic tissue were shorter than for purif ied protein in two of three experiments (Palm et
al. 1994). DT 50 ’s for purified Cry1Ac in two different non -sterile soils were 22 days and 40 days (Palm et al. 1994). None of the studies discussed above have been performed under field conditions, although most have used fi eld soil in laboratory microcosms.

Several studies indicate that Cry proteins bind to clays and humic acids (Crecchio and Stotzky 1998, Koskella and Stotzky 1997, Tapp and St otzky 1995, Tapp and Stotzky 1998, Stotzky 2000b). The results of these studies suggest that such binding slows the rate of microbial degradation of these toxins (S totzky 2000b). This protection is not absolute, however, since degradation has occurred under seve ral experimental conditions. Se veral factors influence either the affinity of binding or the ra te of degradation. In partic ular, pH near neutral generally increases degradation substantiall y. At pH above 5.8 to near neut ral, degradation of Cry protein bound to clay minerals in soil was much faster than degradation at pH 4.9-5.0 (Tapp and StotzkyCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

52 1998). For example, it was found that Cry toxin a dded to nonsterile soil containing kaolinite or montmorillonite showed little degradation even after around six months at lower pH (pH~5), while substantial degradation oc curred over this time period at higher pH (Tapp and Stotzky 1998).

Corn does not grow well below ~pH 5.6 (Aldrich et al. 1975), and therefore most corn growing soils are expected to be at a higher pH. Ther efore, under most production conditions, corn would not be grown on soils that would in hibit the rate of degradation comp ared to what is seen at near neutral pH.

Tapp and Stotzky (1998) proposed that the more rapi d degradation of Cry prot eins at near neutral pH was due to a greater amount of microbial activ ity. The authors pointed out that even at pH near neutral, protein toxin activ ity (lethal concentrations agains t a sensitive bioassay) remained after six months and they interp reted these data as evidence of prolonged persistence of Cry protein in the soil. In these experiments, substan tial degradation (loss of biological activity) typically occurred rapidly in th e first several weeks, with much slower subsequent breakdown (Tapp and Stotzky 1998). A similar pattern wa s observed in some experiments performed by others over a range of Cry1Ab and Cry1Ac protei n concentrations from ~2-700 ng toxin/g soil (Palm et al. 1994, Palm et al. 1996). These experiments suggest that testing for persistence in the field should be determined over sufficiently long periods to assure an accurate assessment of degradation.

These results must be interpreted cautiously with regard to implications for persistence in the field. Field deposition of Cry protein is associated with plant material (pollen or crop residue) or plant root exudates (e.g. carbohydrates and amino acids) which typically stimulate microbial activity and reproduction (Cheng and Coleman 1990, Griffiths et al. 1999, Jensen and Soerensen 1994, Meharg 1994). Many of the experiments exam ining persistence of Cry proteins reported in the published literature have apparently been conducted in bulk soils or soil components. Bulk soil generally does not suppor t populations of microorganisms as high as in the rhizosphere or in soils where plant residues are incorporate d. Research suggests typical ratios of 5-20 for rhizosphere to bulk soil microbe s, with rhizosphere populations commonly 100-fold higher than in bulk soil (Atlas and Bartha 1993). Therefore, degradation rates under field conditions may be higher than those shown in bulk soil experiments.

This conclusion is supported by data submitted by Sims (Sims and Holden 1996) where the DT 50 of free Cry protein alone (in bulk soil) is about 5-fold higher than in ground corn tissue added to soil (although proteases from the corn tissue might also contribute to degradation). In addition, Donegan et al. (1995) found that microbial populations increased 100-1000 fold with the addition of plant material (cotton) with or without Cry1Ab or Cry1Ac proteins. Palm et al. (1996) found more rapid degradation of tr uncated Cry1Ab or Cry1Ac proteins when incorporated with cotton crop resi dues more often than when purifie d protein was used. In these microcosm experiments, toxin DT 50 s, where possible to determine, varied greatly from 2.2 to 46 days (microbial populations in these previously dried, re-hydrate d, soils were not determined). These experiments relied on ELISA of extracted Cr y proteins to quantify residual Cry protein in the soil, so it is not clear what fraction of th e extracted protein may have retained biologicalCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

53 activity. The extraction efficiency was reported to be about 27-60%, w ith lower efficiency corresponding to higher clay and organic content, and it is unclear whet her un-extracted protein degraded at a similar rate as the extracted protei n and retained biological activity. Other research (Koskella and Stotzky 1997, Tapp and Stotzky 1995, Tapp and Stotzky 1998), however, suggests that at least some of the clay -bound fraction of Cry protein is mo re resistant to extraction and degradation while retain ing biological activity.

Some experiments that show relatively long pe rsistence of Cry proteins in the soil do not consider rates of degradation, re porting instead only the duration of protein activity or presence of protein. These experiments sometimes begin with very high concentrations of the protein compared to the amounts found in the plant. For example, Tapp and Stotzky (1995) and Koskella and Stotzky (1997) used approximately 100 μg Cry protein/g so il and approximately

100-300 μ g toxin/g soil, respectively, in their experiments, while Bt plants typically produce less

than 10 μ g/g plant tissue and the concentration in soil from incorporation is typically estimated

to be at the PPB to low PPM levels. In additi on, the bioassays or immuno assays used to detect the protein in the soil are very sensitive, able to detect the protein at concentrations of around 5- 10 PPB (ng/g). Depending on the experiment, reductions of 10 3 to 10 5 may be required to reduce the amounts of Cry protein added to soil below detect able levels. Therefore, in order to predict persistence under field conditions it is important to know starting concentrations as well as degradation rates. Other e xperiments discussed above used amounts of Cry protein more representative of many plant tissues in Bt plants.

To summarize, available data suggests that the DT 50 of Cry1Ab or Cry1Ac proteins, incorporated in corn plant residues or as free toxin in non-ster ile soil, typically range from approximately 1.6- 22 days but have been measured to be as long as 46 days and data showed DT 50 for Cry1F to be 3.13 days. As suggested by Palm et al. (1996), DT 50 may be expected to vary significantly depending on soil conditions. Conditions that favor microbial growth, however, including presence of metabolizable organic matter such as crop residues or rhiz osphere secretions, and near-neutral pH, will favor shorter DT 50 ’s. Binding of Cry protei n to clays and humic acids reduces microbial degradation rates compared to other soil components or media, but, based on current data, it cannot be concluded from thes e results that persistence is greater than demonstrated in available experiments. Furthermore, microcosm and laboratory data in nonsterile soils and near neutral pH suggest that most of the Cry protein deposited in soil may be quickly degraded, although a resi dual amount may persist in biol ogically active fo rm for a much longer period of time.

It is important to consider a number of factor s expected to influen ce persistence under actual field conditions: humic acid and clay content of the soil; clay type; pH ; moisture; soluble ion content and type; and temperature. Varyi ng conditions can affect microbial activity, composition, and population levels, along with bi nding affinity of Cry proteins for soil components. Persistence of Cry proteins coul d therefore vary consid erably. The conditions examined by the registrants, however, generally replicate common field so il conditions, although performed in a laboratory setting. Field test s of Cry protein degrad ation under a range of conditions typical of Bt crop cultivation yield relevant data on persistence and natural variation.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

54 ii. Secretion of Cry Proteins by Plant Roots

Saxena et al . (1999) reported that Cry pr otein was exuded by the roots of transgenic corn plants in laboratory studies. In this study, Bt11 and nontransgenic corn plants were grown in Hoagland’s solution or in sterile or nonsterile soil (sandy loam with 6% montmorillonite clay added, pH 6.0-6.5) (Saxena et al. 1999, Stotzky 2000d). Results of this study showed that the amount of total protein found in the medium averaged 105 μ g per plant, as determined by the Lowry method. Although it is possible that the obse rved Cry1Ab protein was the result of small pieces of plant tissue (e.g. root cells or root hairs), it is unlikely sin ce the Hoagland’s solution was centrifuged, which removed cellular debris (Sto tzky 2000a). In addition, the simple protein band pattern reported (66kDa) by Saxena et al. (1999) was the same as the Cry1Ab protein and

found in Bt corn exudates only after 7 and 15 days. N onetheless, the proce dure used to isolate the Cry protein from soil, including vortexing in extraction buffer prio r to centrifugation, could have ruptured or lysed cellular material. 8 In addition, small root mate rial is difficult to separate from the rhizosphere soil used in the subseque nt assays. The SDS-PAGE protein profile from non-sterile soil was reported to contain many more bands than from the SDS-PAGE profile from the Hoagland’s solution (Stotzky 2000d). This indicates either extrac tion of plant cellular proteins and/or microbial protei ns, possibly in addition to exudedor secreted proteins. SDSPAGE gels of extracted proteins from the st erile soil treatment were not performed (Stotzky 2000d), so such data may have been expected to resu lt in a protein profile similar to that from the Hoagland’s solution if plant cellu lar material was not included.

While the data generally suppor t the deposition of Cry protei n in the Hoagland’s solution external to corn root cells, the evidence concerning the soil experiments is not conclusive. Based on the reported methods, it is possible that both exuded and cellular protein could have

contributed to the soil results. Alternatively, Cry1Ab protein ma y not have been exuded in the soil experiments, and may be an artifact of gr owth in Hoagland’s solution experiment. This distinction is important, because it cannot currently be ruled out th at a substantial portion of the insecticidal or immunological act ivity observed after 25 days in the soil experiment was due to plant associated Cry protein, which could have been protected from microbial degradation. Rhizosphere soil, which is the re gion very close (e.g. soil adhering to plant roots) to the surface of the roots, was used in this study. The concentration of Cry protein in this soil sample was probably higher than for soil further from the roots.

Relatively long persistence of Cry proteins is not surprising when the starting amounts are high and the assays for the protein (bioassay or immunoassay) are very sensitive. Saxena et al. (1999) reported that the 66KDa band disappeared after 25 days when the soil was no longer sterile. With a single incorporation at the concentrations estimated by Saxena et al. (1999), and DT 50 estimates submitted by the registrant, it is possible that Cry protein would not be detectable after 25 days of exposure in the soil, even with sensitive bioassays or immunoassays (Sims and

8 The composition of the EnviroLogix extraction buffer is proprietary, but Karen Larkin (personal telephone communication, March 22, 2000) confirmed that it is intende d, in combination with vortexing, to lyse plant cells.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

55 Holden 1996). The presence of Cry protein activity after 25 days in nonste rile soil appears to support either the persistence or continuous exudation of Cry protein, or both.

Results of the Saxena et al. study suggest that exposure of soil organisms to Cry protein may be continuous during the growing season, as well as af ter incorporation of pl ant residues. Some proteins may be secreted from the roots of plants in significant amounts by an active export process. These amounts may be mu ch higher than the in cidental amounts that might be released by other processes (e.g. sloughing off of root cells) and could lead to continuous exposure of soil organisms to Cry proteins. Experiments indicate that leakage of cytoplasmic proteins into the soil is at most incidental (Borisjuk et al. 1999, Denecke et al. 1990). Therefore, Cry protein exuded into the soil may have different risk im plications than a single incorporation of Cry protein containing plant material.

Proteins secreted into the soil by plant roots are limited in number and specialized for that purpose, containing specific endoplasmic reticulum (ER) export signals in the form of short amino terminal amino acid sequences that target the protein to the lumen of the ER, and other short sequences targeting the protein into the apoplast (Borisjuk et al. 1999, Denecke et al. 1990, Rusch and Kendall 1995, Vitale an d Chrispeels 1992). Cry protei ns are not expected to be secreted into the soil because they are cytoplasmic proteins in Bacillus thuringiensis and, in particular, because no ER secretion peptide sequ ence has been identified in these proteins (Kostichka et al. 1996). A bacterial secre tion peptide has been found only for a CryV protein

that has been shown to be exported, or secreted, from B. thuringiensis (Kostichka et al. 1996). All other known Cry proteins, in cluding those registered, form intracellular inclusions of Cry protein, and are not secreted. Ot her transgenic cytoplasmic prot eins have been shown to be efficiently secreted only when a known ER si gnal peptide sequence is specifically added, otherwise these proteins re main cytoplasmic (Borisjuk et al. 1999, Denecke et al. 1990).

Available data for other secreted proteins suggest that the amount of a secreted protein found in culture medium may be as high or higher than the amount associated wi th plant tissue after several days growth (Borisjuk et al. 1999). In Bt11 corn roots, Cry1Ab is expressed at an average of 20.2 μ g/g total root protein. It is difficult to predict whether this level of root expression of Cry1Ab is consistent with ac tive secretion, based on the roughly estimated amounts of Cry1Ab found in the media (soil or Hoagland’s solution) reported by Saxena et al. (1999).

The corn plants used in the exudation experiments were identified as NK4640 Bt , which correspond to Syngenta Bt11 corn lines. This variety of corn contains a modified cry1Ab gene. Previous soil fate data supplied by the regi strant was from experi ments performed with nonsterile soil in the laboratory a nd consisted of replicated single incorporations of transgenic plant material or purified Cry protein. All othe r studies of Cry protei n stability in the soil performed for registration or other purposes also used a single incorporation of purified protein or protein in the transgenic plant, with subseque nt monitoring of residua l activity using sensitive bioassays or other means, such as immunoassays . Single incorporation studies were performed because it was believed that most of the exposure of soil organisms to the Cry proteins would be through the incorporation of plant residues after harvest and to a le sser extent due to pollen shed.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

56 Degradation studies were performed by Monsant o, registrant of MON 810 corn, which also

contains the cry1Ab gene. In these studies, Cry1Ab protei n degraded in non-sterile soil with a

1.6 d DT 50 when ground plant material is used, and 8.3 d for the purified Cry1Ab protein in soil. The ground tissue used in this study would be expect ed to have a signific antly increased surface area compared to crop residue incorporated at th e end of a growing season. This increase in surface area might reduce degradation times. Use of ground tissue has been criticized (NAS/NRC 2000). Cry1Ab protein bi oactivity of corn tissue inc ubated without soil decreased

with an estimated DT 50 of 25.6 days, and a DT 90 of 40.7 days. After one week in soil, approximately 1% and 10% of the original levels of B.t.k. protein remain ed in leaf and stalk tissue, respectively. After three weeks, B.t.k. protei n was still detected in the stalk tissue, but the level in transgenic leaves was si milar to the background levels seen in control leaf tissue. B.t.k. protein apparently binds to soil particles, ma king quantitative extracti on difficult. Biological activity, assessed by European corn borer bioassay, is reduced to control le vels after three weeks of incubation in soil (MRID No. 436960-01).

Degradation of Cry1F in the soil was demonstr ated in the study submitted in support of the poCry1F registration (MRID N o. 450201-05). Based on a bioassa y with the tobacco budworm ( Heliothis virescens ), a target species, purified Cry1F prot eins incorporated into test soils biodegraded with a half- life of approximately 3.13 days. The am ount of Cry1F protein in an acre of corn (if 25,000 corn plants/acre at harvest were left in the field) is approximately 20.5 g/acre. As a result, the expected maximum environmenta l concentration (EEC) for poCry1F protein was calculated to be 23 micrograms / kg dry soil (15 cm deep). Whole moCry1F plants at the R1 stage contain 7.16 ng of Cry1F/mg of tissue (dry weight) whereas poC ry1F plants at the R1 stage contain 3.6 ng of Cry1F/mg of tissu e. Therefore, we can roughly approximate that the EEC for moCry1F at the R1 stage would be 34.5 microgram s/kg dry soil (15 cm deep). Although EPA is

aware of no evidence to indicate that prolonged exposure to trace amounts of Cry protein in the soil affects non-target organisms, the Agency fe lt that the submitted data did not sufficiently address the issue of residual Cry protein accumulation in the soil.

The October 2000 Scientific Advisory Panel (SAP) concluded, in SAP Report No. 2000-07 (March 12, 2001), that published data did not, at that time, adequate ly address the persistence of Cry proteins from Bt crops in the soil. Since it is diffi cult to correlate the relevance of the published laboratory data to fiel d situations, the SAP recommende d field studies be conducted in

established Bt fields in a variety of soil types and c limatic conditions. The SAP suggested an investigation of amount, accumulation and persisten ce of biological activity of Cry proteins in the soil. The SAP also concluded, however, th at this data was not necessary for an EPA preliminary risk assessment but may be needed fo r a final assessment. In general, the Panel

believed that studies on the mechanism of how Cry proteins enter th e soil (e.g., secretion, shedding of root hairs, and degradation of biom ass pollen) were primarily of academic interest, whereas knowledge of the potenti al environmental impacts was im portant for risk assessment. EPA agreed that some of these data would be useful in completing the database for a future assessment, and required supplementary studies re garding Cry protein degradation in soil.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

57 The Agricultural Biotechnol ogy Stewardship Technical Co mmittee (ABSTC) submitted a research protocol for a Bt Cry protein soil accumulation st udy on March 15, 2002. We found the proposed protocol acceptable with the additional requirement of an ELISA (letter from USEPA to ABSTC 05/08/02). The ABSTC submitted a re sponse to the Agency’s letter, including a revised protocol, on June 5, 2002, which was acc epted by EPA with the provision that the detailed protocol of the insect bioassay also be submitted and accepted. The studies have since

been conducted and the results are summarized below.

1) Three-Year Soil Persiste nce of Cry1Ab (MRID No. 460224-01) This study, which fulfills the soil fate data requi rement for MON 810 field corn, Bt11 field corn, and Bt11 sweet corn, was submitted to EPA jointly by Syngenta Seeds, Pioneer Hi-Bred and Monsanto Company, the member companies of AB STC. Bioassay results indicated that ECB larvae fed a diet containing 15% Bt corn soil exhibited no toxic res ponse to the diet mixture, as measured through growth inhibition of test larv ae (LOD 0.03 μg/g soil). Based on these findings, the study author concluded that Cry1Ab protei n does not accumulate and persist in soil. However, Cry1Ab protein may have been present in test soil at concentra tions that were below the LOD. Consequently, it may be more accurate to state that after three years of continuous Cry1Ab field corn production, Cry1Ab protein had not accumulated in soil to a level that would elicit a toxic response from ECB larv ae, a species that is highly susceptible to Cry1Ab protein.

2) Three-Year Soil Persistence of Cry1F (MRID No. 471207-01) This study, which fulfills the soil fate data requirement for TC1507 and TC6275, was submitted to EPA by Mycogen Seeds c/o Dow AgroSciences LLC. The results showed no detectable Cry1F protein residues and no biol ogical activity observed in th e soil of Herculex I corn, demonstrating Cry1F protein rapidl y degrades in various soil matri ces after 3 years of continuous cropping with Herculex I corn and is not expected to exude from the roots or be present in the sloughed-off root cells. This fi eld study confirms previously conducted laboratory experiments [Herman et. al (5)] and the Agency’s risk and benefits assessment for Cry1F corn and the 2001

Reassessment of Bt PIPs, which predicted that Cry1F pr otein rapidly degrades in the soil.

iii. Effects on Soil Microorganisms

Numerous published studies indicate that exposure to Cry protein produced in Bt PIP crop plants does not adversely affect soil microorganisms (Sanvido et al. 2007; Oliveira et al. 2008). In addition, Bt toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil (Saxena and Stotzky 2001). Other research findings conclude no Bt -related risks have evolve d from the decomposition of Bt -corn leaves for the meso- and macr ofauna soil community (Hönemann et al . 2008). Although a

minimal transient increase and shift in microbial populations ma y result from the presence of transgenic plant tissue in soil, no adverse effect s have been attributed to the Cry protein.

In addition, there are several ongoing U.S. Depa rtment of Agricultur e and EPA Office of Research and Development funded resear ch projects evaluati ng the effects of Bt crops on soil microbial flora. If adverse effects are seen from this or any other research, the Agency will take appropriate action to mitigate potential risks.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

58 With regard to the impact of genetically engineer ed crops on soil, it is important to note that agricultural practices themselves cause large changes in soil and soil microbial composition. Furthermore, factors such as variations in seas ons and weather, plant gr owth stage, and plant varieties, independent of being ge netically engineered, are also re sponsible for sign ificant shifts in soil microbial communities. To date, most st udies with genetically engineered crops have shown minor or no effects on soil microbes beyo nd the variation caused by the factors listed

above.

3. Non Target Wildlife Hazard Assessment

Prior to registration of the first Bt plant-incorporated prot ectants in 1995, EPA conducted ecological risk assessments for all Bt Cry proteins expressed in potato, corn, and cotton. EPA evaluated studies of potential e ffects on a wide variety of non-ta rget organisms that might be exposed to the Bt protein, e.g., birds, fish, honeybees, ladybugs, parasitic wasps, lacewings, springtails, aquatic invertebrates and earthworms. Such non-target organisms are important to a healthy ecosystem, especially the predatory, para sitic, and pollinating insects. These risk

assessments demonstrated that Bt Cry proteins expressed in tr ansgenic plants do not exhibit detrimental effects to non-target organisms in populations exposed to the levels of Cry protein found in plant tissue. While EPA was aware of potential adverse effects on many species of Lepidoptera from Cry1 proteins, th e Agency did not believe that non-target Lepidoptera would be exposed to sufficient amounts of Bt protein to cause an unreas onable deleterious effect, nor

that Bt crops would threaten the long-term survival of a substantial number of individuals in the populations of these species. At that time, even though EPA knew that Bacillus thuringiensis

var. kurstaki was toxic to Lepidoptera, EPA also conc luded that threatened or endangered species of butterflies and moths would not be at risk because they would not be exposed to Bt Cry1protein in Bt crops.

Published field testing results and field test da ta voluntarily submitted to EPA by the registrants also shows minimal to undetectable changes in the abundance of benefici al and other non-target insect populations. In some cases the densities of predatory and non-targ et insects are reported to be higher on Bt crops than on non- Bt crops. These results are discussed below and are described in supporting assessment documents.

In light of recent environmental effect s concerns from commercialization of Bt crops the Agency has reviewed new and existing data rega rding non-target w ildlife effects for Bt crops with a special emphasis on Lepidoptera and monarch butterf lies and reevaluated the sufficiency of data to support continued registration of Bt crops.

Although Bt Cry proteins are very specific in their act ivity to certain insect species, EPA has examined the toxicity of the Cry proteins to bi rds, fish, honeybees and certain other beneficial

insects. Because of the extensive scientific liter ature on the susceptibility of lepidopteran species

to Bt Cry1 proteins, EPA assumes that Cry1 proteins would be toxic to butterflies if they were exposed to high levels of the protein and, therefor e, did not require lepido pteran toxicity data. EPA nevertheless required data on Collembola (spr ingtail) and earthworm species to ascertainCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

59 effects on beneficial soil invert ebrates because soil exposure to Bt Cry proteins was a possibility. In the honeybee study, effects studies on brood as well as adults were required when exposure to

the Bt Cry protein in pollen was expected. Evaluations of risk to other n on-targets that may be affected by the Bt pollen, specifically the monarch bu tterfly, were conducted in 1999, 2000 and 2001-2002 by the USDA, ABSTC and university scie ntists in the USA and Canada (ABSTC, 2001, Dively et. al. 2004). The reports from these studies are summarized below and in the supporting DCI review document (Rose 2001).

Bt delta endotoxins are proteins and, unlike inorganic chemicals, do not have the potential to bioaccumulate causing delayed effects. An accumulation through the food chain is therefore not expected to take place, and ther e are no data to support this possibility for protein substances. The basic biological properties of proteins also make Bt Cry proteins readily susceptible to metabolic, microbial, and abiotic degradation on ce they are ingested or excreted into the environment. Although there are reports of soil binding under certain circumstances, the bound Cry proteins are also reported to be rapidly degraded by microbes upon elution from soil. The same sources report that Bt proteins in the soil of Bt corn fields have no detectable effect on soil

invertebrates or culturable microbial flora. The Bt Cry proteins do not have any characteristics in common with persistent, bioaccumulative chemical s that are transferre d through the food chain. Therefore, chronic effects testing of prot ein substances is not routinely performed.

a. Summary of Non-Target Organism Toxicity Testing on Corn Bt 11 (006444) and MON810 (006430)

TABLE 4. Tabular results of non-target wildlife testing for Cry1Ab proteins

OCSPP

Guideline No. Study Results MRID No.

885.4380 Larval Honey Bee Testing Btk HD-1 protein at 20 ppm showed no toxicity to larval honey bees. An

LC 50 was not possible to calculate since this was a single dose test. Therefore, the NOEL is greater than 20 ppm. 434392-02

885.4380 Non-Target Adult Honey Bee Testing There were no statistically significant differences among the various groups. However, sizable mortality occurred in all treatments. Btk HD-1 protein at 20 ppm resulted in a mean mortality of 16.2%. Because mortality was observed at the single dose tested, a NOEL could not be determined from this study, but it was less than 20 ppm. 20 ppm was determined to be significantly higher than exposure conditions in the

environment. 434392-03

885.4380 Verification of Test Substance from Nontarget Insect and Honey Bee Testing Test substance was stable for up to 7 days in 1:1 honey:sucrose solution.

Test material was bioactive. 434680-02

885.4340 Non-Target Insect Testing - Green Lacewing Larvae Btk HD-1 protein at 16.7 ppm showed no toxicity to green lacewing larvae after 7 days. The NOEL is greater than 16.7 ppm. 434680-03

885.4340 Non-Target Insect Testing - Ladybird Beetles ( Hippodamia convergens ) Btk HD-1 protein at 20 ppm showed no toxicity to ladybird beetles ( Hippodamia convergens ). The NOEL is greater than 20 ppm. 434680-05

885.4340 Non-Target Insect - Parasitic Hymenoptera Btk HD-1 protein at 20 ppm showed no toxicity to Brachymeria

intermedia . Since this is a single dose study, an LC 50 cannot be calculated. The NOEL is greater than 20ppm. 434680-04

N/A Evaluation of Transgenic

Corn Event Bt11 in

Broiler Chickens In a six-week feeding study with 1600 young broiler chicks, diets prepared with transgenic N7070Bt co rn or N7070Bt corn that had been sprayed with Liberty herbicide produ ced growth, feed conversion rates,carcass yields, and survival rates similar to diets prepared with non456521-01Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

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transgenic corn. Birds fed N7070, N7070Bt, or N7070Bt-Liberty corn had similar body weights and feed c onversion ratios throughout the test. There were no significant differences in the carcass yield of birds (48- days old) receiving either transgenic or non-transgenic corn. The study fulfills the conditional requirement for a six-week avian study.

N/A Comparison of broiler performance when fed diets containing YieldGard® Corn, YieldGard® x Roundup Ready®, parental line, or commercial corn hybrids To compare the nutritional value of YieldGard and YieldGard xRoundup Ready to their respective parental lines, as well as to other nontransgenic corn varieties, groups of broiler chickens were fed prepared diets containing one of the corn lines for 42 days. Eight corn lines (treatments) were evaluated in a randomized complete block design. Each treatment group consisted of 10 pe ns (5 pens of 10 males each and 5 pens of 10 females each), for a to tal of 100 birds/treatment (800 birds overall). Mortality was observed thr oughout the study, and at test end survivors were examined for performance, carcass yield, and meat composition. Most of these mortaliti es were apparently due to sudden death syndrome and ascites. No mo rtalities were determined to be related to the treatment diet, and no ot her adverse effects were observed. There were no biologically significan t differences in performance, carcass yields or meat composition between any of the groups tested. 456118-01

885.4050 Avian Oral Toxicity in Northern Bobwhite Quail No treatment related mortality or di fferences in food consumption, body weight or behavior occurred in birds fed 50,000 or 100,000 ppm transgenic corn meal derived from Monsanto's MON 80187 corn line (which contains Cry1Ab protein) rela tive to birds fed corn meal made from parental corn lines which did not express Bt protein. Although this study utilized Monsanto's Bt corn for testing, the test material was considered sufficiently si milar to the Bt11 corn grain to bridge the data. 435332-05

885.4240 Corn Pollen Containing the Cry1Ab Protein: A 48-

Hour Static-Renewal Acute Toxicity Test with the Cladoceran ( Daphnia

magna ). Monsanto submitted this study to support their MON 810 corn. The study is scientifically sound and no treatment mortality or behavior change was observed between the dosed and control replicates. These

results indicate that Daphnia magna , a sensitive aquatic invertebrate species, is not affected by a 48 hour exposure to 100 mg of Cry1Ab protein containing MON810 corn po llen/L. This study adequately address potential aquatic toxicity concerns for MON 810 corn pollen expressing Cry1Ab protein. Mon 810 pollen is preferred over Bt11 pollen as a test material in studies supporting Bt11 corn. However, given the equivalent Cry1Ab expression in Bt11 and MON 810 corn (< 90 ng Cry1Ab/g dry wt.pollen) and the lack of treatment related effects seen in

any Bt corn pollen Daphnia magna studies, the data requirement is

satisfied for Bt11.

The data suggest that at the expected environmental concentration the proposed use of Cry1Ab protein in corn is not likely to have any measurable effects on aquatic invertebrates. 442715-02

885.4240 Daphnia magna Study and Bridging Rationale Novartis cited the Ciba Seeds (now Novartis Seeds) Event 176 acute 48 hr. study (MRID No. 433236-10) to support Bt11. This 48-hour static renewal toxicity study of Ev ent 176 maize pollen containing Bt Cry1Ab Cry protein was conducted using Daphnia magna . Test daphnids were

dosed at five concentration levels , including a maximum hazard dose of 150 mg/L (nominal) of water. No mo rtalities were observed at any of the

treatment levels tested. The 48-hour EC 50 was determined to be greater than 150 mg/L. The NOEC was found to be >150 mg/L. These results

indicate that Bt Cry1Ab protein expressed in corn showed not toxicity at 150 mg/L to Daphnia magna. In view of the above results, no freshwater aquatic invertebrate hazard is expect ed from the use of this product.

Bt11 pollen is preferred over Event 176 pollen as a test material in studies supporting Bt11 corn. However, given the low level of expression of Cry1Ab in Bt11 pollen [(< 0.55 micro g Cry1Ab/ g protein) or (< 90 ng Cry1Ab/g dry wt.pollen)] compared to Event 176 pollen [80.63 micro g Cry1Ab/g protein) or (12.36 micro g Cry1Ab/g dry wt. pollen)] and the lack of effects seen in the cited Daphnia magna study using Event 176 pollen, the data requirement is satisfied for Bt11. 433236-10

442742-01

885.4200 Evaluation of the European Corn Borer

Resistant corn Line MON Feed per fish, feed conversion ratios, final weight, percentage weight gain and survival were not significan tly different between fish fed the control MON 800 diet when compared to those fed the diet containing 438879-01Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

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801 as a Feed Ingredient

for Catfish. transgenic corn from the test lin e MON 801. Body composition data exhibited no significant differences in percentage moisture, fat, or ash, with higher protein content in the test fish on a dry weight basis. This difference in protein content disappe ars when one expresses the results on a wet weight basis. Data in this study are consistent with historical controls for catfish grown at the De lta Research and Extension Center. Although this study utilized Monsanto's Bt Cry1Ab corn fo r testing, the

test material was considered sufficie ntly similar to the Bt11 Cry1Ab corn grain to bridge the data.

885.4340 Effect of Cry1Ab, on

Folsomia candida and Xenylla grisea (Insecta: Collembola). In the cited study, purified Btk insecticidal proteins derived from E. coli (200 ppm), including Cry1Ab protein, had no observable toxicological effect on two species of Collembola: Folsomia candida and Xenylla grisea . The Agency has required Novartis to submit a Collembola study using leaf material rather than bacterially-derived Cry1Ab. 439416-01

885.4340 Chronic Exposure of

Folsomia candida to Corn Tissue Expressing Cry1Ab

Protein. This study determined that the LD 50 of lyophilized MON 810 corn leaf tissue containing the Cry1Ab protein to Collembola ( Folsomia candida ) over a 28-day exposure period is greater than 50% (by weight) of the diet. The no-effect-level for mortality was 50% of the diet. This same

concentration in the diet had no effect on the reproduction of Collembola. According to the spons or, the estimated concentration of Cry1Ab protein was 50.6 μg/g in lyophilized tissue and 6.27 μg/g in

fresh tissue. The control substance was lyophilized leaf tissue from the non-transgenic corn line MON 823 which has a genetic background similar to the MON 810 line but does not carry the gene responsible for the Cry1Ab protein. Thiodicarb was used as a positive control or

reference substance.

While this study is useful in charact erizing effects of Cry1Ab corn tissue on Collembola and satisfies the requi rement for MON 810 corn, it does not adequately characterize the effect of Bt -11 corn tissue on Collembola since possible treatment related effects were observed in a Bt corn Collembola study. MRID No.

442715-01

N/A Field Surveys of nonTarget Invertebrate Populations in Corn

Field Studies of NonTarget Invertebrate Populations in Bt Corn On March 15, 2002, a report titled “Field Surveys of Non-Target Invertebrate Populations in Bt Corn” was submitted on behalf of the Non-Target Organism Subcommitte e (NTO Subcommittee) of the Agricultural Biotechnology Stewards hip Technical Committee (ABSTC) members (MRID No.45652001). EPA reviewed this submission and indicated that final reports of the in-progress studies and the results of any additional research should be submitted at a later date.

On March 15, 2006, the NTO Subcommittee submitted the document “Field Studies of Non-Target Invertebrate Populations in Bt Corn” (MRID No. 467846-01). This submission included complete references

and additional information that had become available. Most papers focused on large-scale field studies using transgenic corn, including discussions of study de sign and non-target inve rtebrate populations. Overall, the published literature did not report any consistent adverse impacts on non-target invertebrates as a result of multi-year commercial

Bt

corn cultivation. Slight reductions in some invertebrate predator populations were seen; however, these ar e an inevitable result of all pest management practices which tend to result in reductions in the abundance of the pests as prey. 456520-01

467846-01

N/A Effects on Monarch Butterfly Larvae after Continuous Exposure to Cry1Ab-Expression Corn Pollen During Anthesis Effects on the monarch butterfly ( Danaus plexippus ) from continuous exposure of larvae to natural deposits of Bt and non- Bt corn pollen on milkweed ( Asclepias syriaca ) were measured in five studies usingCry1A(b)-expressing hybrids (events BT11 and MON810) and nontransgenic corn in Mary land, Iowa, and Ontario, Canada. First instars were exposed beginning at 3 to 4 and 6 to 7 days after initial anthesis. Overall mean pollen densities during la rval development in the first and second bioassays were 163 and 170 grains/cm 2 , respectively. Pollen density on milkweed plants in the non- Bt , BT11, and MON810 hybrids averaged 163, 155, and 174 grains/cm 2 , respectively, during the first bioassay and 172, 173, and 158 grains/cm 2 , respectively, in the second bioassay. There were no statistically significant differences in pollen density between assays or among hybrid types. The number of anthers on the leaves was positively correlated with the amount of pollen deposited 461620-01Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

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on the leaves. The study included th e results of a risk assessment simulation model that projected, over the range of the Corn Belt, an

increased risk of 0.6% in mortalit y to monarch larvae from prolonged

exposure to Bt corn pollen. The increased risk to monarch larvae when considering all of North America was 0.3%.

885.6200 Cry1Ab Insecticidal

Protein: An Acute Toxicity Study with the

Earthworm in Artificial

Soil Substrate The 14-Day LC 50 value for earthworms exposed to Cry1Ab insecticidal protein derived from E. coli in an artificial soil substrate was determined to be greater than 200 mg/kg (ppm), which was the single concentration tested. There were no statistically significant effects at the single dose tested, therefore the NOEL is greater than 200 ppm. Although this study was graded supplemental, Bt Cry1Ab proteins expressed in the corn plant are not expected to generate a toxic effect in the earthworm, therefore, no additional follow-up of this study is required. 438879-02

b. Summary of Non-Target Organism Toxi city Testing on Corn TC1507 (006481) and TC6275 (006491)

TABLE 5. Tabular results of non-target wildlife testing for Cry1F proteins

OCSPP

Guideline No. Study Results MRID No.

885.4150 Wild Mammal Testing,

Tier I Mammalian wildlife exposure to moCr y1F protein is considered likely; however, the mammalian toxicology data submitted for the Human Health Assessment for poCry1F indi cates that there was no significant toxicity to rodents from acute oral testing at the maximum hazard dose. Based on the bridging data in comb ination with the poCry1F rodent study, no hazard to mammalian wildlife is anticipated from moCry1F. 446911-01

885.4050 Dietary Toxicity Study

with the Northern Bobwhite ( Colinus virginianus ) The dietary LC 50 for corn grain (meal) expressing Bt var. aizawai protein in corn grain when fed to juvenile northern bobwhite quail ( Colinus virginianus ) for five days was determined to be greater than 100,000 ppm (10% of corn meal). The NOEC was 100,000 ppm and there were no treatment-related mortality or behavioral changes observed in comparison to the control replicates. These data were determined to be

insufficient to make a hazard assess ment from repeated exposure(s) to higher doses of Bt corn and a six week study with 60 to 70% corn in the diet was deemed necessary to assess hazards from chronic exposure of

wild and domesticated fowl. Theref ore, the study was determined to be supplemental. However, the additional study was submitted (see below) and the study is now upgraded to acceptable. 450201-12

Non-guideline Nutritional Equivalency

of BtCry1F MaizePoultry (Cobb x Cobb) Feeding Study In a six week study where commercial broiler chickens were fed a diet containing 54.21%-57.03% Bt Cry1F line 1507 and control diets there was no statistically significant difference found in mortality, mean body weight, mean daily weight gain, or mean food conversion. 456220-01

885.4380 Honey Bee Larva Testing, Tier I The data show that at the expected environmental exposure the proposed use of Cry1F protein in corn is not likely to have any measurable deleterious effects on the honey bee ( Apis mellifera ). There was no treatment mortality or behavior ch ange observed between the dosed and control replicates. LC 50 > 64 ng Cry1F in 2 mg pollen /larva and 640 ng Cry1F protein /larva. Based on the bridging data in combination with this study, Cry1F protein as expre ssed in corn pollen should have no detectable adverse effects on honey b ee larvae or their development into healthy adults. 450415-03,

453078-05 (supplement)

885.4340 Non-target Insect Testing, Tier I with Green Lacewing Larvae ( Chrysoperla carnea ) Green lacewing larvae fed a concentration of Bt poCry1F protein at15x the expected rate found in corn pollen (pollen expressing 32 ng Cry1F/mg pollen) resulted in no mort ality or signs of toxicity or

abnormal behavior over a 13 day period (>20% control mortality period).

The LC 50 and NOEC was determined to be >15x the concentration of poCry1F found in pollen and the was determined to be > 480 ppm a.i 450201-09,

453078-01 (supplement)Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

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(the test concentration). Mortalit y and pupation rate were comparable between the treatment and control gr oup. Based on the bridging data in combination with the poCry1F green lacewing study moCry1F should

have no detectable adverse effects on Chrysoperla carnea in the field.

885.4340 Non-target Insect Testing, Tier I with the Ladybird Beetle ( Hippodamia

convergens ) Adult lady beetles fed a concentration of Bt Cry1F protein at at 15x the expected rate found in corn pollen (pollen expressing 32 ng Cry1F/mg pollen) resulted in no mortality or sign s of toxicity over a 29 day period. Therefore, the NOEC and the LC 50 were determined to be >15x the concentration of Cry1F found in pollen determined to be > 480 ppm a.i (the test concentration). The test in sects were exposed to a dose of active ingredient approximating the amount that would be ingested by the beetles feeding on aphids under fi eld conditions. As a result, no discernible beneficial beetle popula tion effects are expected from the proposed uses of the Cry1F producing corn. 450201-10,

453078-02 (supplement)

885.4340 Non-target Insect Testing, Tier I with the Parasitic Hymenoptera ( Nasonia vitripennis ) Parasitic Hymenoptera fed a concentration of Bt Cry1F protein 10x the expected rate found in corn pollen (expressing 32 ng Cry1F/mg pollen) showed no mortality or signs of toxicity or abnormal appearance or behavior of surviving wasps in th e treatment or control group over a 12 day period. The test was terminated after 12 days because 20% mortality was reached in the negative control. The NOEC and the LC 50 were determined to be > 320 ppm a.i (10x fi eld rate when calculated for pollen expressing 32 ng Cry1F/mg pollen). No hazard to parasitic Hymenoptera at field use rates is expected from the cultivation of Cry1F containing

corn. 450201-11,

453078-03 (supplement)

885.4340 Non-target Insect Testing, Tier I with

Neonate Larvae of the Monarch Butterfly ( Danaus plexippus ) First instar Monarch larvae fed a 10, 000 ng/mL diet (the highest rate tested) showed no mortality after seve n days of feeding. There was some growth inhibition at 10,000 ng/mL diet. LC 50 > 10,000 ng/mL and NOEC <10,000 ng/mL. Since doses equivale nt to 10,000 ng/mL diet are not likely to occur in nature, it was concluded that Cry1F protein will not pose a risk to monarchs. 451311-02

885.4340 Collembola Testing, Tier I ( Folsomia candida ) Collembola ( Folsomia candida ) were fed three treatment levels (12.5, 3.1, 0.63 mg/kg) of Cry1F protein every two to three days for 28 days representing 79x, 388x, and 1560x that which would be encountered in the field with no observable treatment mortality or behavior change. Results of the study indicate that leve ls of Cry1F that might occur in the field are not expected to adversely e ffect the soil invertebrate Collembola species. LC50 and NOEL >12.5 mg Cry1F/kg soil. 450201-07

850.6200 Acute Toxicity Study

with the Earthworm ( Eisenia fetida ) in an

Artificial Soil Substrate Earthworms, Eisenia fetida , fed 2.26 mg poCry1F/kg dry soil, representing up to 100X the estimated concentration present in the top

six inches of an acre of soil follo wing the incorporation of 25,000 senescent corn plants did not have adverse effects. LC50 and NOEL >2.26 mg Cry1F/kg dry soil. Based on the results of this study, it is not likely that Cry1F transgen ic corn plantings will have adverse effects on

earthworms. 450201-06,

453078-04 (supplement)

N/A Non-target Insect Field Survey Sticky traps were set out weekly for si x weeks. In addition, ten plants in the center row were visually evaluate d for beneficial arthropods weekly

for six weeks. Beneficial insect s counted were: lady beetles ( Cycloneda

munda & Coleomegilla maculata ), predacious carabids, brown lacewings (Hemerobiidae), green lacewings ( Chrysoperla plorabunda ), minute pirate bugs ( Orius insidiosus ), assassin bugs (Reduviidae), damsel bugs (Nabidae), ichneumonids and braconids (parasitic wasps), damselflies and dragonflies (Odonata), and spid ers (Arachnida). Data included counts of adult and larval lady beetles and lacewings when appreciable

numbers were collected. Results fr om the study indicated that the transgenic corn lines TC1507 and 1360 did not adversely affect the number of beneficial arthropods in the field. In general, line TC1507 showed larger numbers of beneficial insects. The field census study adequately addressed potential concer ns for Cry1F protein expressed in corn to non-target insect populations. However, the Agency 450201-13Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

64 OCSPP

Guideline No. Study Results MRID No.

recommended that the monitoring con tinue into the first few years of commercial use of Cry1F corn crops in order to confirm the single

season A no effects @ findings and to gather long-range non-target insect

effects and abundance data.

N/A Non-target Exposure and

Risk Assessment for Environmental Dispersal of Cry1F Maize Pollen. (A probabilistic risk assessment) This study was conducted with poCry1F to consider the exposure of nontarget species including endangered Lepidoptera species to field corn pollen expressing the Cry1F delta endotoxin by evaluating pollen dissemination. The Cry1F concentration found in pollen occurring on milkweeds near the edge of Bt corn fields was predicted. Distance of pollen dispersal, levels of Cry1 F expression in pollen, milkweed distribution and biomass from the edge of the field, pollen grain physical properties, and spatial-temporal avai lability of Cry1F to monarch larvae was determined. According to a probability-log plot demonstrating lepidopteran species susceptibility to Cry1F, 99% of lepidopteran species

exhibit an LC 50 of ∃ 0.06 Φ g g -1 which is 290-fold lower than the geometric mean LC 50 (12.4 Φ g g -1 ) and lower than the most sensitive lepidopteran species. The toxicity threshold, or no effect level for monarch neonates, for the Tier 1 risk assessment was determined to be

10 Φ g g -1 diet. When fed up to 10 Φ g g -1 Cry1F microbial toxin in diet,

neonate monarch larvae were not affect ed. The toxicity threshold, or no effect level for monarch neonates, fo r the Tier 1 risk assessment was

determined to be 10 Φ g g -1 . The log-probability plot of the Bt LC 50 for lepidopteran species shows that the EEC does not exceed the LC 50 for 98% of the intergenera population beyond 1 m from the field edge. The

LC 50 is not exceeded for 90% of the population 0.2 m from the edge. For monarch larvae, the no effect level (10 Φ g g -1 ) occurs near the 50 th
percentile intergenera LC 50 . Since there is a rapid fall-off in exposure with distance, there is limited poten tial for non-target effects beyond the immediate field border. In addition, the estimated risk quotients (ratio of exposure to effect) demonstrate a lack of concern for monarchs (or other lepidopteran species) beyond 1 m from the field edge. The RQ in the corn field was 0.096. Finally, pollen from moCry1F plants express less Cry1F protein than poCry1F plants, further reducing non-target

exposure. 450415-02

885.4240 A 48-Hr Static Renewal Acute Toxicity Test with the Cladoceran ( Daphnia

magna ) There were no overt signs of toxicity to daphnids ( Daphnia magna ) exposed to 100 mg Bt -pollen/L - (maize pollen containing the Bt Cry1F delta-endotoxin). The 48-hr EC50 was > 100 mg a.i./L. The NOEC was >100 mg a.i./L. These data show that there will be no adverse effects on daphnia from incidental field expos ure to transgenic corn pollen containing Cry1F. 450201-08

885.4200 Freshwater Fish Testing,

Tier I The Agency previously waived static renewal toxicity tests for freshwater fish due to the lack of substantial exposure to poCry1F protein in runoff and corn pollen. However, the registrant submitted a study in support of the potential moCry1F registration. Juvenile rainbow trout ( Onchorhynchus mykiss ) were fed a standard fish diet containing 100 mg Cry1F ICP a.i./kg of diet for eight days with no mortality or

sublethal effects. The LD 50 was determined to be greater than 100 mg a.i./kg of diet. The actual concentration of the test material in the diet was not determined and therefore this study is supplemental. 450442-01

460193-06

850.1075 Fish Acute Toxicity Test,

Freshwater and Marine Study was not required for this pr oduct because of very low or no potential for exposure. None assigned

c. Non Target Wildlife Risk Characterization

i. Terrestrial WildlifeCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

65 1) Mammalian Wildlife

The human health data submitted to EPA indicate th at there is no significant toxicity to rodents from acute oral testing at the maximum hazard dose for Cry1Ab or poCry1F. The data submitted in support of poCry1F, combined with the bri dging data summarized in Section 2.b. of this assessment (Table 3), also indicate that no haza rd to mammalian wildlife is anticipated from moCry1F. In addition, there are reports of no adve rse effects on livestock after several years of feeding with Bt corn. Mammalian wildlife exposure to the Bt Cry proteins is considered likely; however, the mammalian toxicology information gather ed to date does not show a hazard to wild

or domesticated mammals.

2) Avian Species

When administered by oral gavage at a dosage up to 2,000 mg protein/kg body weight, Bt corn has no apparent effect upon bobwh ite quail after 14 days. A study with a non-commercial line of MON 80187 showed no mortality or differences in food consump tion, body weight, or behavior when bobwhite quail were fed 50,000 or 100,000 pp m Cry1Ab in corn meal. (Although this study utilized Monsanto's Bt corn for testing, the test material was considered sufficiently similar to the Bt11 corn grain to bridge the data.) In addition, there are reports of no adverse effects from the commercial poultry industr y after several years of using Bt corn in poultry feeds.

The dietary LC 50 value for corn grain (meal) expressing Bacillus thuringiensis var. aizawai Cry1F protein in corn grain when fed to juvenile northern bobwhite for 5 days was determined to be greater than 100,000 ppm (10 % corn meal). Th e No Observed Effect Concentration (NOEC) was also 100,000 ppm. The study is scientifically sound and no trea tment mortality or behavior change was observed between the dosed and control replicates.

Though the above data indicate that the Bt Cry1 protein produced in corn does not show a hazard to birds, EPA determined that they were not su fficient to make a final hazard assessment from repeated exposure(s) to higher doses of Bt corn. In 2001, a six-week study with 60 to 70% corn in the diet was conditionally required to assess hazards from chronic exposure of wild and

domesticated fowl. These studies have since been conducted, and the results are summarized

below.

a) Cry1Ab i) Bt11 (MRID No. 456521-01) In a six-week feeding study with 1600 young broile r chicks, diets prepared with transgenic N7070Bt corn or N7070Bt corn that had been sp rayed with Liberty herbicide produced growth, feed conversion rates, carcass yields, and surviv al rates similar to diets prepared with nontransgenic corn. Significant differences were observed, however, in survival by sex during portions of the study; males had increased mortal ity during the finishing (days 35-42) and overall (days 0-42) periods. The study author attributed this to the males being more susceptible to heat

stress. There were also some erratic differen ces in mortality by sex during the grower phase (days 21-35), but these were not seen on a cumula tive basis (days 0-42), a nd were attributed to chance. Birds fed N7070, N7070Bt, or N7070Bt-Li berty corn had similar body weights and feed conversion ratios throughout the test. Bird s fed NC2000 corn had significantly decreasedCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

66 weights for each portion of the study, and a sign ificantly decreased adjusted feed conversion ratio for the starter (days 0-21) , finishing (days 35-42), and cu mulative (days 0-42) periods. The study author attributed these decreases to an inte raction between the climatic conditions and the NC2000 diet formulation, which was more hygroscopi c than the others, thus affecting the flow of the feed into the feeder pan. Evidence of restri cted feed flow was seen in two pens during the finishing period, and these were excluded from th e statistical analysis for feed conversion ratio. There were no significant differenc es in the carcass yield of birds (48-days old) receiving either transgenic or non-transgenic corn. The Agency concurs that the diets with transgenic corn produced growth, carcass yields, and meat qual ity similar to those obtained with the nontransgenic isoline. No valid comparison to the NC2000 corn can be made, however, as it is unclear whether the superior pe rformance of the N7070-series co rns compared to that of the NC2000 was due to inferiority of the NC2000 corn or to formulation differences in the NC2000 diet. The study fulfills the conditional requirement for a six-week avian study.

ii) MON 810 (MRID No. 456118-01) YieldGard and YieldGard x Roundup Ready corn produce the Cry1A(b) protein, which confers insect protection. In additi on, YieldGard x Roundup Ready expre sses the maize EPSP synthase gene, which confers tolerance to glyphosate. To compare the nutritional value of YieldGard and YieldGard x Roundup Ready to their respective parent al lines, as well as to other non-transgenic corn varieties, groups of broiler chickens were fe d prepared diets containing one of the corn lines for 42 days. Eight corn lines (treatments) were evaluated in a randomized complete block design. Each treatment group consisted of 10 pens (5 pens of 10 males each and 5 pens of 10 females each), for a total of 100 birds/treatment (800 birds overall). At study start, two additional birds were included in each pen to compensate for potential losses due to mortality and dehydration. On study day 7, the pens were culled to 10 birds pe r pen. Mortality was observed throughout the study, and at test end survivors were exam ined for performance, carcass yield, and meat composition. Chick mortality wa s as expected during th e first 7 days of the study. Mortality during days 7-42 (6%) was slightly higher than normal, but was random across treatments, ranging from 2% to 11% for the differe nt corn lines. Most of these mortalities were apparently due to sudden death syndrome and ascite s. No mortalities were determined to be related to the treatment diet, and no other ad verse effects were observed. There were no biologically significant differences in performance, carcass yields or meat composition between any of the groups tested. Based on these resu lts, the two Cry1Ab corn lines tested do not produce adverse effects in broiler chic kens through dietary exposure.

b) Cry1F (MRID No. 456220-01) The registrant submitted a six-week study as requ ired by the EPA. Two hundred forty-five male broiler chickens (Cobb x Cobb) were fed diets co ntaining commercial corn -soy type ration with either reference yellow dent corn, Bt Cry1F maize 1507 hybrid, or a non-transgenic control hybrid corn (five replications e ach) for six weeks. The broilers were fed a starter ration (54.21% corn) for the first 20 days and then a grower ration (57.03% corn) for days 21 through 42. There were no statistically significant differences in mean percent mo rtality, mean body weight, mean daily weight gain, or mean feed conversion among any of the treatments.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

67 Furthermore, moCry1F grain contains less Cry1F protein (1.14 ng/mg) as compared to poCry1F grain which contains 2.2 ng/mg of Cry1F protein (dry weight tissue; Zabik et. al 2003). The six week broiler study, in conjunction with the in itial bobwhite quail study submitted in 2001, is

sufficient to demonstrate that there should be no discernible de trimental effects to wild or domesticated fowl from the proposed uses of Cry1F producing corn including moCry1F.

ii. Aquatic Species

There is no evidence for sensitivity of aquatic (including endangered) sp ecies to Cry proteins. Toxicity studies with aquatic organisms do not show a hazard for fish or invertebrates exposed to

either Bt corn pollen or to bacterially expressed Cry pr otein. It has also been demonstrated that

farm fish food mix made from corn seed containing the Bt protein does not contain detectable

active Bt Cry protein; therefore, farmed fish would not be exposed to Bt Cry proteins. In addition, aquatic exposure from Bt crops is extremely small. A simple standard pond scenario (1 hectare pond, 2 meters deep draining a 10 hectar e watershed planted with corn) was used to develop a worst case EEC for Cry1Ab and Cry1F pr oteins (high protein e xpression level) on the basis of corn pollen loadings from airborne pol len deposition and agricultural runoff. Airborne pollen deposition will result in water concentr ations less than 78 ng Cry1Ab protein/L when based on conservative estimates for pollen disp ersal. The contribution of Cry1Ab to the pond through agricultural runoff is comparable (66 ng L-1 based on GENECC). Thus, total water concentration of less than 144 ng Cry1Ab protei n/L is projected under worst case conditions (Wolt 2000). Airborne pollen deposition results in water concentrations of approximately 1.25 ng Cry1F/mL and the contribution of Cry1F to the pond through agricu ltural runoff is <0.15 ng/mL. Thus, total water concentration of 1.4 ng Cry1F protein/L is proj ected under worst case

conditions.

1) Aquatic Invertebrates

The major source of Bt Cry proteins in fresh water is corn pollen. Toxicity studies with corn pollen containing Cry1Ab proteins conducted using Daphnia magna show an acute EC 50 was >100 mg/L in one study and 150 mg/L in anothe r. The LOEC (lowest observed effect concentration) was found to be 150 mg/L. The amount of pollen was considered to exceed the 144 ng Cry1Ab protein /L projected aquatic exposure in the fields under worst case conditions. Toxicity studies with corn pollen containi ng Cry1F proteins conducted using the sensitive aquatic indicator species Daphnia magna show the no-mortality concentration and NOEC to be >100 mg a.i./L. There were no overt signs of toxicity to daphnids exposed to 100 mg Bt Cry1F pollen/L. The amount of pollen tested was considered to well exceed field exposure. The data for poCry1F were bridged to support the moCry1F re gistration; moCry1F polle n contains less Cry1F protein (3.67 ng/mg) as compared to poCry1F pollen, which contains 21.9 ng/mg of Cry1F protein (dry wei ght tissue; Zabik et. al 2003).

In light of recently published laboratory studies showing redu ced growth in shredding caddis flies exposed to anti-lepidopteran Cry1A protein corn litter (Rosi-Marshall, et al. 2007), additional aquatic invertebrate data are required. The submitted Daphnia magna studies are unacceptable because they are 850 Series OCSPP Guideline studies. The 48 hour duration ofCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

68 this study is not sufficient to detect mortality due to Bt proteins. It takes more than 48 hours for the target pests to succumb to the Cry proteins, therefore 48 hours is also not expected to show mortality or reproductive effects on Daphnia . A 7 to 14 day Daphnia magna study as per the 885 series OCSPP Guidelines needs to be performed. The study may be submitted as a condition of registration. Alternatively, a dietary study of the effects on an aquatic invertebrate, representing the functional group of a leaf shredde r in headwater streams, can be performed and

submitted in lieu of the Daphnia study.

Questions have been raised for using corn pollen in aquatic invert ebrate testing with Daphnia

magna because corn pollen is thought to be t oo large for ingestion by these filter feeders (EcoStrat, 2000). However, there is some obs ervational evidence that daphnids do ingest pollen. As indicated in some study reports reviewed by the Agency, daphnids were actually yellow in color, which can be indicative of ingestion of the test material, with no treatment mortality or behavior change compared to untr eated controls. Also, if the polle n is not ingested, or excreted without digestion when presented to Daphnia , then there will not be any exposure, and therefore

no risk to Daphnia in the aquatic environment.

2) Freshwater Fish

The requirement for a freshwater fish static re newal toxicity study has been waived based on a lack of any substantial exposure of fish to the Bt Cry proteins produced in Bt crops (Wolt 2000). Farm fish diets made with corn containing the Cr y proteins do not advers ely affect susceptible target insect larvae, as bioassay testing and analyses using ELISA indicate that Cry protein is not detectable in the fish feed samples. Therefor e, farm fish eating a f ood mix made from corn containing the Bt delta endotoxin would not be exposed to detectable active Bt Cry protein.

Despite acceptable data waiver justification, Dow AgroSciences submitted a freshwater fish study in support of the moCry1F registration (MRID No. 460193-06). J uvenile rainbow trout ( Onchorhynchus mykiss ) were fed a standard fish diet co ntaining 100 mg Cry1F ICP a.i./kg (100 ppm) of diet for eight days with no mortality or sublethal effects. The LD 50 was determined to be greater than 100 mg a.i./kg of di et. The study is considered s upplemental, however, because the

actual concentration of the test materi al in the diet was not determined.

In view of the lack of demonstrated toxicity and exposure, no aquatic hazard is expected from

the continued uses of Bt Cry protein in Bt 11, MON810, TC1507 and TC6275 corn crops.

iii. Plants

Since the active ingredient in this product is an insect toxin ( Bt endotoxin) and EPA is unaware of demonstrated toxicity to plants, the plant toxicity studies have been waived.

iv. Non-target Invertebrates

All of the insect studies submitted in support of the registration of Cry1F used maximum hazard dose concentrations of poCry1F (TC1507) corn pollen. As shown in Section 2.b. of this assessment (Table 3), there is higher Cry1F pr otein expression in poCry1F corn pollen (21.9Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

69 ng/mg tissue) as compared to 3.67 ng/mg tissue in moCry1F (TC6275) pollen (dry weight tissue;

Zabik et. al 2003). Therefore, the lack of discernible detrimental effects to non-target insects from poCry1F demonstrated in the studies below strongly suggests that there should also be no effects to non-target insects from moCry1F. Although moCr y1F corn does have higher Cry1F protein expression than poCry1F co rn in plant tissues most like ly to be exposed to the soil dwelling Collembola and earthworms, the Coll embola and earthworm studies conducted for poCry1F used concentrations of Cry1F protein that exceed those that would be seen from the proposed uses of either poCry1F or moCry1F corn. This sugges ts that the proposed uses of Cry1F protein in corn are not likely to have any measurable population effects on soil invertebrates including Co llembola and earthworms.

1) Honey Bees

a) Cry1Ab

Feeding tests were conducted on both honey bee larvae and adults for Cry1Ab proteins. At a single dose of Cry1Ab, 20 ppm showed no adverse effects to larval honey bees under the test conditions. The NOEL for Cry1Ab was determined to be greater than 20 ppm. In adult honey bees no statistically significant differences were seen among the various treatment and control

groups.

Concerns have been raised as to whether the honey bee larvae that were dosed with pollen containing Cry protei ns were actually exposed to the protei ns. The pollen has to be pre-digested by nurse bees (which, conversely, may also inactivate the Cry protein) in orde r to be palatable to larval honeybees (EcoStrat, 2000). However, small amounts of pollen are known to be fed directly by nurse bees in the hive (Winston, 1987). In addition, the Agency has other laboratory studies on file in which aqueous mixtures of purif ied Cry protein had been added to the diet of honeybee larvae maturing within honeycomb brood cells, or to a 1:1 (by volume) honey-water mixture for adult honeybees. No adverse effect was observed in larvae or adults. This conclusion has been confirmed by hive studies in the field.

An adult honeybee study (Schur et al. 2000) was conducted as a semi-field study in Germany using field-grown Bt Cry1Ab corn plants, and honeybee colonies placed inside tents of plastic gauze placed over areas of the cornfields. Th ree replicate tents (1 colony/tent) containing Bt corn and three replicate tents co ntaining non-transgenic corn were evaluated during the period of pollen shed, and the bee colonies were observed for an additional 30 days following pollen shed. The study showed no adverse effects of Bt corn pollen containing high levels of Cry1Ab protein on adult honeybee survival, foraging frequency, be havior or brood development during the 7-day period of pollen shed. Following the pollen expo sure period, the hives were removed from the tents and observed for an additional 30 days fo r effects on brood development. No effects on brood development were associated with field exposure to Bt Cry1Ab corn pollen.

b) Cry1FCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

70 The reviewed capped honey bee br ood cell study where larvae were fed Cry 1F corn pollen and pure Cry1F protein showed normal larval deve lopment and emergence of healthy adult honey bees. This study shows that at levels higher than the expected environmental exposure, the proposed use of Cry1F protein in corn is not like ly to have any measurab le deleterious effects on the honey bee ( Apis mellifera) . The data showed no significant difference between treatment mortality or behavior ch ange between the dosed and control re plicates. As a result, no discernible detrimental effects to honey bees are expected from the proposed uses of the Cry1F-producing corn. The data adequately addre ss potential toxicity concerns fo r foraging honey bees exposed to Cry1F protein expressed in corn pollen in the field. In addition, since corn is wind pollinated, few honey bees are expected to be exposed.

2) Lady Beetles

a) Cry1Ab

Lady beetle ( Hippodamia convergens ) predator toxicity studies submitted at the time of registration demonstrate that co rn pollen containing the anti-lep idopteran Cry pr oteins do not cause detectable adverse effects to lady beetles. Purified Cry1Ab protei n at 20 ppm also showed

no adverse effects or behavior chan ges. The test insects were expos ed to the active ingredient at approximately the dose that would be ingested by the beetles feeding on aphids under field

conditions.

b) Cry 1F

Adult lady beetles ( Hippodamia convergens) fed a concentration of Bt Cry1F protein at 15x the expected rate found in corn pollen resulted in no mortality or signs of toxicity over a 29 day period. Therefore, the NOEC was determined to be >15x the concentration of Cry1F found in pollen and the LC 50 was determined to be > 480 ppm a.i (t he test concentration). The submitted study shows that corn containing the Cry1F prot ein should not cause significant adverse effects to lady bird beetle predators. The test insects were exposed to a dose of ac tive ingredient approximating the amount that would be ingested by the beetles feeding on aphids under field conditions. As a result, no discer nible beneficial beetle population effects are expected from the proposed uses of the Cry1F producing corn. This conclusion is confirmed by adult and larval lady beetle abundance found in the field census study. These studies adequately address potential concerns for Cry1F protein expressed in corn to beneficial beetles.

3) Parasitic Hymenoptera

a) Cry1Ab

No adverse effects were observed when a maxi mum hazard dose of 20 ppm Cry 1Ab was tested

on Brachymeria intermedia. The NOEL therefore is greater than 20 ppm and no adverse effects to Hymenoptera are expected from exposure to Cry1Ab protein in the field.

b) Cry1FCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

71 Parasitic Hymenoptera (Brachymeria intermedia ) fed a concentration of Bt Cry1F protein at 10x the expected rate found in corn pollen showed no mortality or signs of toxicity over a 12 day period. Therefore, the NOEC was determined to be >10x the concentration of Cry1F found in pollen. The LC 50 was determined to be > 320 ppm a.i (the test concentration). As a result, no adverse effects to parasitic wasps are expected from field exposure to Cry1F protein producing

corn. The conclusions are also confirmed by the parasitic wa sp abundance found in a field census study submitted with the application.

4) Green Lacewing

a) Cry1Ab

The studies submitted to support the initial regist ration showed no significant adverse effects to green lacewing larvae at a maximum hazard dose of 16.7 ppm Cry1Ab protein in a 7 day feeding study. The NOEL, therefore, was greater than 1 6.7 ppm and no adverse effect to green lacewing was expected as a result of exposure to Cry1Ab protein at field concentrations.

Since that time, there have been severa l publications proposing that transgenic Bt plants may create serious impacts on non-targ et organisms that feed on pest s exposed to the transgenic proteins. The reported harmful effects of Bt corn on larvae of the be neficial predatory insect green lacewing stem largely from the work of Hilbeck et al . (1998a 1998b, 1999). EPA performed a formal review of the first two studies on the effects of Bt corn intoxicated prey and

pure Bt corn protein on lacewing (DP Barcode D236803 and D250457). If these laboratory results are taken at face value, the adverse effects are so slight as to suggest no significant impact

on beneficial insects in the field.

Hilbeck et al. (1998a) reported slightly elevated mortality and pr olonged development time in lacewing larvae reared on Bt intoxicated prey (the European corn borer - ECB). The authors subsequently reassessed these results ( Hilbeck et. al. 1998b) and reported that there are no significant reproductive effects from Bt corn protein. The authors concluded that “...surviving,

unaffected C. carnea developed at rates similar to those in the untreated contro l” and “from this, we conclude that total developmental time until adult eclosion is not an appropriate parameter for detecting Cry1Ab protein effects.” (Hilbeck, et al. 1998b). The second study (Hilbeck et al. 1998b) used defined quantities of pure Bt protein and there was signifi cant mortality only in an artificial diet test group; no significant mortality was observed when the artificial diet was supplemented with E. kuehniella eggs (a natural diet). Th erefore, this study does not demonstrate any adverse effects to lacewing la rvae under simulated field feeding habits where the lacewing larvae have a choi ce of natural diet in the fiel d. Moreover, in this study, the concentration of pure Cry protein to which the larvae were exposed was 100 micro grams /ml of diet and continuous, and therefor e not reflective of Cry1Ab exposur es that may occur under field conditions – either by exposure to plant tissues , pollen or by consumption of exposed prey species, such as ECB larvae. The dosage used in these studies is at least 30 times that found in

most corn tissues in the field.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

72 In a tritrophic study publ ished in 1999 (Hilbeck et al. , 1999) an intermediate prey not susceptible

to Bt was fed purified Bt protein in an artificial diet and then was presented to lacewing larvae. The study noted effects at no lower than 50 microgram levels, in contrast to the nanogram-level exposure which would be encountered in corn tissues in the field.

Generally, these findings do not show any detrimenta l effects at Cry protein exposure levels in the field. The laboratory results were seen only at exposure to microgram quantities, whereas in the field the exposure is only to nanogram am ounts. In addition, any surviving ECB larvae would normally be within the corn plant most of their larval life and not available for consumption by chrysopids. (ECB larvae live within the corn stalk, not on stalk surface). The

authors concluded that “...trial s investigating predation effici ency and predator performance under field conditions are necessary before c onclusions regarding the potential ecological relevance of the results presented in our paper can be drawn” (Hilbeck, et al, 1998b). Field studies have already been published on the effects of Bt crops on insect predators showing no significant differences in the de nsity of beneficial insects, including green lacewings. In addition, Pilcher et al. (1997a) showed no significant differences in growth or mortality of Coleomegilla maculata (lady beetle), O. insidiosus (minute pirate bug), and Chrysoperla carnea (green lacewing) feeding on non-transgenic and Bt -expressing pollen in the laboratory.

b) Cry1F

Green lacewing larvae fed a concentration of Bt Cry1F protein at 15x the expected rate found in corn pollen resulted in no mort ality or signs of toxicity due to feeding on Cry1F over a 13 day period. Therefore, the NOEC was determined to be >15x the concentration of Cry1F found in pollen and the LC 50 was determined to be > 480 ppm a. i (the test concentration). These laboratory findings do not show significant detrimen tal effects and provide data that show a lack

of risk to beneficial insects at Cry1F levels that will be encount ered in the field use situation. These findings confirm published field studies on the effects of B.t. crops on insect predators showing no significant differences in the density of beneficial insects, including green lacewings. The conclusions are also confirmed by the adul t and larval green lacewing abundance found in a field census study submitted with the application.

5) Soil Invertebrates

The FIFRA Scientific Advisory Panel (USEPA 2001) does not believe that Collembola and earthworms are appropriate indicator species fo r Cry1Ab testing because of the Lepidopteraspecific nature of the Cry1Ab protein. When it initially reviewed the applications for the products that were registered in 1995, EPA c onsidered requiring studies evaluating effects upon the representative beneficial soil invertebrate s Collembola and earthworms. EPA was concerned (1) that such soil organisms may be subject to long-term exposure as a result of soil incorporation of crop residues (or when crop resi dues are left on the soil surface) and (2) that adverse effects on such soil organisms could result in an accumulation of pl ant detritus in fields. Recent reports of exudation of Cry proteins by corn roots throughout the growth season add to this concern. However, EPA understands that routine agronomic practices have included the long term use of chemical insecticides, which ha ve adverse effects on soil organisms, but thereCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

73 has not been an accumulation of significant amount s of plant detritus in soils (Pimentel & Raven, 2000). Thus, Bt crops, which are expected to have less impact on these species than chemical pesticides, should not result in a ny increased build up of plant detr itus or Cry proteins at toxic levels. Supporting this conclusion are data required by the EPA that indicate that such proteins are known to degrade rapidly in field soils. Ther efore, significant soil buildup and effects to non-target soil organisms are not anticipated. This has been c onfirmed by Saxena and Stotzky (2001), who report that Bt Cry proteins released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, pr otozoa, algae, bacteria , actinomyces and fungi in soil, in spite of the fact that enough detect able Cry protein is bound to soil particles to show toxicity to the target pest. Thes e results suggest that despite its presence in soil, the Cry protein

released in root exudates of Bt corn, or from the degradation of the biomass of Bt corn, is not toxic to a variety of organisms in the soil envi ronment. Stotzky (2000) also reported that the same degree of Bt Cry protein persistence takes place in so ils that have been exposed to repeat Bt microbial spray applications. In addition, new plants grown in Bt containing soil do not take up

the Bt protein.

a) Earthworms

Earthworm feeding studies submitted to the Agency for all of the registered Cry proteins demonstrate that the Cry proteins are not toxic at the expe cted environmental concentration.

Concerns have been raised as to whet her the earthworms actually ingested the Bt Cry proteins when these are incorporat ed into the soil in the test systems used (EcoStrat, 2000). This question is mainly of academic importance. For hazard assessment purposes it is sufficient to know that

the earthworms were not harm ed when presented with the Bt Cry proteins in their soil environment. If they do not ingest it in the test soil, likewise they will no t ingest it in the field. The earthworms do, however, ingest the Bt Cry proteins with the soil without harmful effects. Saxena and Stotzky (2001) repor ted that there were no significant differences in the percent mortality and weight of earthworms af ter 40 days in soil planted with Bt or non- Bt corn or not planted, or after 45 days in soil amended with biomass of Bt or non- Bt corn or not amended. However, the toxin was present in both the casts and guts of the worms in these tests.

i) Cry 1Ab

The 14-Day LC 50 value for earthworms exposed to Cry1A b insecticidal protein derived from E.

coli in an artificial soil substr ate was determined to be greater than 200 mg/kg (ppm), which was the single concentration tested. There were no statistically signi ficant effects at the single dose tested. Although this study was graded supplemental, Bt Cry1Ab proteins expressed in the corn plant are not expected to generate a toxic effect in the earthworm; therefore, in light of recent recommendations by the FIFRA Scientific Advi sory Panel (USEPA, 2001) that invertebrates known not to be affected by the Cry proteins sp ecific for insects not be tested, no additional follow-up of this study is required.

ii) Cry1FCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

74 The submitted data show that Cry1F protein has no measurable deleterious effects on earthworms, a representative beneficial soil invert ebrate species. This suggests that the proposed uses of the Cry1F protein in corn are not like ly to have any measur able population effects on

beneficial soil invertebrates. The one limit te st concentration of 2.26 mg Cry1F/kg dry soil represented more than 100X the estimated concentr ation present in the top six inches of an acre of soil following the incorporation of 25,000 senescen t corn plants. This concentration is higher than any amount of Cry protein that may be pres ent in the soil during an y stage of the growing season (such as from root exudation). Based on th e results of this study, Cry1F transgenic corn plantings will have no adverse effects on earthworms.

b) Collembola

i) Cry1Ab

Monsanto’s original application for registra tion included a study on Collembola exposed to 200 ppm of Cry1Ab protei ns derived from E. coli . The study showed no adverse effects, but EPA classified the study as supplemental because the test substance was not leaf tissue containing Cry1Ab. Subsequently, Monsanto submitted a new study using lyophilized corn leaf tissue containing the Cry1Ab protein in the MON810 co rn line. The estimated concentration of Cry1Ab protein was 50.6 μg/g in lyophilized tiss ue and 6.27 μg/g in fresh tissue. The control substance was lyophilized leaf tissue from the non-transgenic corn line MON 823 which has a genetic background similar to the MON 810 line but does not carry the gene responsible for the Cry1Ab protein. Test substan ces included corn powder at 0.5, 5.0, and 50% of the diet. Mortality was assessed every 7 days for the durati on of the 28-day test. Additional observations were also made with respect to growth, egg production, and egg hatch. For the corn powder treatments and controls, no mortalities occurred in the treatment or control groups. Likewise, there was no significant differen ce in reproduction between the treated group and either control group. The study was scientifically sound and no tr eatment mortality or behavior change was observed between the dosed and control replicates. The study also showed th at at field use rates reproduction of the test insects will not be impaired.

The Collembola studies submitted to the Agency for most of the registered Cry proteins showed

no adverse effects at maximum hazard doses. N ovartis (now Sygenta) had cited the MON 810 leaf tissue study to suppor t their Bt11 corn plant-incorporated protectant. While this study is useful in characterizing effects of Cry1Ab corn tissue on Collembola and satisfies the requirement for MON 810 Cry1Ab co rn, it does not adequately ch aracterize the effect of Bt11 corn tissue on Collembola. The requirement for a Collembola study which includes control plant lyophilized leaf tissue from non-tr ansgenic parental corn line s and lyophilized leaf tissue containing the Bt11 plant-incorporat ed protectant is not fulfilled. However, in light of recent recommendations by the FIFRA Scientific Advi sory Panel (USEPA, 2001) that invertebrates known not to be affected by the Cry proteins specific for insects of different orders not be tested, this requirement can be waived.

ii) Cry1FCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

75 Since Collembola feed on decaying plant material in the soil, they may be exposed to Cry1F protein in corn found in the field. A study was conducted to determine if there may be adverse effects of Cry1F on Collembola. The study is scie ntifically sound and no tr eatment mortality or behavior change was observed between the dosed and control replicates after 28 days. The results of this study indicate that at levels that would reasonably be expe cted to be found in the field, collembola were not affected by chronic ex posure to Cry1F protein. The exposure rates in this study are 1560-, 388-, and 79-fold higher th an the expected field concentration. The

reviewed data show that Bacillus thuringiensis Cry1F corn protein has no measurable deleterious effects on collembola ( Folsomia candida ), a representative beneficial soil insect species. This indicates that the proposed uses of the Cry1F protei n in corn are not likely to have any measurable population effects on beneficial soil insects.

As discussed above, EPA does not believe that to date there are any va lid data demonstrating specific adverse impacts of plants expressing Bt Cry1 proteins on beneficial soil invertebrates. To the contrary, EPA believes that available sc ientific data and information indicate that

cultivation of Bt crops has a positive effect on soil flor a, when compared to the most likely alternative, use of non- selective synthetic chem ical pesticides.

6) Non-Target Insect Abundance Studies

Data available to date indicate no difference in the number of total inse cts or the numbers of specific orders between the transgenic crop plots and either the isogenic or wild type control crops. No shift in the taxonomic distribution of insects was seen, except in cases where the predators are dependent on the pest insect as prey as their major food source.

Pilcher et al. (1997b) conducted limited size field studi es in two consecutive years with Bt corn. No differences were observed in the number of predators colonizi ng either isogenic control corn

or Bt corn in 1994. In 1995 more predators were seen on Bt corn than on control corn. The

authors concluded that Bt corn pollen did not affect predator abundance. They also concluded, however, that the absence of significant differences may have resulted from plot size. Due to the small plot sizes separated by only one buffer row, pollen from Bt corn and isogenic corn may have been mixed by wind. They concluded that the inconsistent results between the two years indicate that larger scale studies are necessary for significant data.

Orr & Landis (1997), studied the oviposition of Eu ropean Corn Borer (Lepidoptera: Pyralidae) and impact of natural enemy populations in tran sgenic high pollen level Cry1Ab versus isogenic corn. No significant differences in O. nubilalis egg populations, or its predators or parasitoids were observed. Mortality factors exerted by predators were consistent in all plots. The corn type did not appear to have an impact on these fact ors. Larval parasitism was not significantly different and therefore probably density-independent.

Obrycki (1997) performed a st udy to determine the effects of transgenic corn expressing Bt Cry protein on the abundance of predatory insects in corn fields. He found that the average number of predatory insects was not si gnificantly different between th e sprayed and unsprayed plots on four of the five observation days over a seven week period. Conventional pesticide spray driftCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

76 was suspected as the reason. No significant difference in abundance was found between the Bt

and the non Bt plots on any of the five observation days. Similar num bers of Coccinellid eggs, larvae, and pupae were observed on the transgenic and the non-transformed corn plants. Higher numbers of Chrysopid eggs, Orius insidious (Anthocoridae), Nabidae, and Arachnida were

observed on the Bt corn, but not at statistic ally significant levels.

Lozzia (1999) reported a biodive rsity and structure of ground b eetle assemblages (Coleoptera Carabidae) trial in Cry1Ab Bt corn and the effects on non-target insects conducted in four trials in North Italy over a 2-year peri od. No statistical difference was evident in the total number of carabids. There was no decreasing trend in the bi odiversity indices from the first to the second year and considering the data as a whole, the two years appear comparable. The difference in biodiversity recorded for some i ndices was not due to the presence of transgenic corn. The aerial fauna as a whole for both years and both locali ties was not different. Similarly, abundance of aphids, leaf hoppers, other Homoptera, thrips, l eaf beetles, spiders, lady beetles, parasitic Hymenoptera, other Hymenoptera, a nd Diptera was not different. The number of arthropods was higher, but not significantly, in the transgenic corn. Several sampling methods and visual checking showed that there was no significan t difference in abundance, composition or biodiversity of non target arthropod s in isogenic and transgenic corn crops. The data show that the transgenic plants do not lead to an increase or decrease of any insect populations. It appears that Cry1Ab proteins do not di rectly affect the phytophagous species nor do they have “any indirect influence on other trophic levels or activities such as be havior, oviposition or predatorsprey.”

Nuessly & Hentz (1999) conducted 4 studies using Novartis (now Sygenta) Seeds' Attribute Bt11-derived Cry1Ab sweet corn hybrids and conve ntional sweet corn hybrids grown under local practices in four Florida locations . Noted in the reports were incr eases in species diversity in the corn plots, i.e. there were generally higher popu lations of beneficial a nd non-target insects as compared to the conventional control plots, asso ciated with the significa ntly decreased use of broad-spectrum insecticides (organophosphate s, carbamates and synt hetic pyrethroids).

Three single-year field surveys of non-target invertebrates in Bt Cry1F corn have been submitted to EPA, the results of which are summarized in the table below:

TABLE 6. Field surveys of no n-target invertebrates in Bt Cry1F corn.

Study # Years Study Design Taxa Collected Top Line Results

Field survey of beneficial arthropods associated with

Bt

Cry1F maize (Higgins 1999)

MRID No. 450201-13 1 year

1999 TC1507 & 1360: 2 treatments: Cry1F and non- Bt near isoline; plot size: 28 x 4 m; 1 site (Iowa) 4 reps; sampling: visual and sticky trap observations before, during, and after

anthesis lady beetles, predacious beetles, lacewings, insidious flower bugs, assassin bugs, damsel bugs, parasitic wasps, damsel or dragonflies, and spiders. Cry1F maize lines had no effect on the presence of beneficial arthropods compared with non- Bt near isolines; generally, TC1507 showed larger numbers of

beneficial insects

Field study of non-target arthropods associated with

Bt

var. aizawai Cry1F

maize

MRID No. 456520-01 1 year (2000) TC1507: 3 treatments: Cry1F,

non- Bt near isoline, and non- Bt

near isoline treat ed with foliarinsecticide (lambacyhalothrin); plot size: 24 x 6 thrips, insidious flower bugs, leafhoppers Cry1F showed no significant impact on the non-target arthropods. Insecticide treatment significantly reduced non-target arthropodCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

77 Study # Years Study Design Taxa Collected Top Line Results

m; 1 site (France) 4 reps; sampling: visual observations 7X during growing season populations

Field survey of non-target arthropods associated with

Bt

Cry1F maize

MRID No. 456480-01 1 year (2001) TC1507: 2 treatments: Cry1F

and non- Bt near isoline; plot size: 3.5 A; 1 site (Iowa); sampling: visual and sticky trap observations before, during,

and after anthesis community census Abundance of key taxa

showed no consistent reduction in the Cry1F field, although fewer parasitic hymenoptera were observed.

In October 2001, EPA approved amendments to the Bt corn registrations of Agricultural Biotechnology Stewardship Technical Committee (ABSTC) member companies (EPA Reg. Nos. 524-489, 67979-1, 65268-1, 29964-3 and 68467-2), which ex tended the registra tions of these products until October 2008. As a condition of th ese registrations, EPA required the registrants to either submit existing studies submit a protoc ol for new field survey studies. On March 15, 2002, a report titled “Field Surveys of N on-Target Invertebrate Populations in Bt Corn” was submitted on behalf of the Non-Target Organi sm Subcommittee (NTO Subcommittee) of the Agricultural Biotechnology Stewardship Tec hnical Committee (ABSTC) members (MRID No. 45652001). EPA reviewed this submission and indicat ed that final report s of the in-progress studies and the results of any additional re search should be submitted at a later date.

On March 15, 2006, the NTO Subcommittee submitte d the document “Field Studies of NonTarget Invertebrate Populations in Bt Corn” (MRID No. 467846-01). This submission included complete references and additional information that have since become available. Most papers focused on large-scale field studies using transg enic corn, including disc ussions of study design and non-target invertebrate populations. EPA re viewed the submitted information and, overall, the published literature did not report any consiste nt adverse impacts on non- target invertebrates as a result of multi-year commercial Bt corn cultivation. Slight reduc tions in some invertebrate predator populations were seen; however, these ar e an inevitable result of all pest management practices which tend to result in reductions in the abundance of the pests as prey. The continually expanding body of literature provides EPA, academia, and the public with a better understanding of the impact of transgenic crops on non-target organisms and provides useful

information and considerations for th ose conducting large-scale field studies.

As anticipated, there are reports of Bt kurstaki Cry protein toxicity to some lepidopteran species in isolated, high dose laboratory studies. At present, however, EPA is aware of no identified significant adverse effects of Bt Cry proteins on the abundanc e of non-target beneficial organisms in a population in the field, whether th ey are pest parasites, pest predators, or pollinators. Published field testing results a nd field scouting data submitted to EPA show minimal to undetectable changes in the beneficial insect abundance or diversity. Results indicate

no difference in the number of total insects or the numbers of specifi c orders between the transgenic crop plots and either the isogenic or wild type cont rol crops when these are grown without chemical pesticide treatment. In commer cial fields densities of predatory and non-target insects are generally higher on Bt crops than non- Bt crops primarily because the Bt crops are not subjected to the same number of applications of nonspecific pesticides. Generally no shift in the

taxonomic distribution of insects was seen in Bt crops, except in cases where the predators areCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

78 dependent on the pest insect as prey. In cont rast, treatment with chemical pesticides, when studied, had significant effects on the total numbers of insects and on the numbers within the specific groups. To date, the ava ilable field test data show that compared to crops treated with conventional chemical pesticides , the transgenic crops have no detrimental effect on the abundance of non-target insect populations. Howe ver, yearly insect census estimates from representative fields will continue to be required.

7) Non-Target Lepidoptera

The toxicity of Btk to butterflies is a well known and a widely published phenomenon. For the purpose of its risk assessment of Bt plant products, EPA accepted that Bt proteins could be toxic to Lepidoptera and relied exclus ively on lepidopteran exposure to Bt Cry protein. Since the exposure to butterflies and moths from the agricultural uses of Bt was not expected to be as high as in forest spraying (where no widespread/recurring or irreversib le harm to lepidopteran insects was observed), Bt crops likewise were not expected to cause widespread or irreversible harm to non-target lepidopteran insects. Published preliminary data of toxicity of high doses of Bt to monarchs in the laboratory do not translate into exposure to toxic le vels in the field. In light of the recent reports expressing concern for monarch conservation efforts, however, this conclusion

has been reevaluated and much research e ffort has been devoted to this issue.

The weight of evidence of the published and recent research da ta reviewed indicate that

milkweeds in the corn fields and to within 1 meter of cornfields are unlikely to be dusted with

toxic levels of Bt pollen from the currently registered Bt corn varieties, MON810, Bt11 and TC1507. In addition, the distribution of corn po llen within and outside of corn fields, the

distribution of milkweeds within corn habitat and othe r types of habitat, monarch oviposition and feeding behavior, limited temporal overlap between monarch larvae and pollen shed (and similar issues) in much of the corn growing regions of the United States indicate a low probability of

demonstrable adverse effects of Bt corn pollen on monarch larvae.

Based on the review of the submitted DCI data , the Agency concludes that the published monarch toxicity information is not sufficient to cause undue concern of harmful widespread

effects to monarch butterflies at this time. In the event that continuing studies demonstrate a

substantial reduction in monarch butterflies attributable to Bt corn pollen, especially as the percentage of Bt corn planting increases, and should ne w data indicate unanticipated risks to other non-target Lepidoptera, part icularly risks to threatened or endangered species, the Agency will institute appropriate risk management practices . The following is a discussion of the studies

available to date on the field t oxicity of Cry1 proteins to non -target Lepidoptera species.

a) Black Swallowtail Butterfly

Wraight et al . (2000) performed a field study to assess wh ether mortality of early instar black

swallowtails was associated either with proximity to a field of Cry1Ab Bt corn or with levels of

Bt pollen deposition on host plants. Potted host plants were infested with first instar black swallowtails and placed at interval s from the edge of a field of Bt corn (MON 810). There was no relationship between mortality an d proximity to the field or polle n deposition on host plants.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

79 Moreover, pollen from these same plants failed to cause mortality in the laboratory at the highest pollen dose tested (10,000 grains/cm 2 ), a level that far exceeded the highest pollen density observed in the field (200 grains/cm 2 ). The authors conclude that Bt pollen of the variety tested is unlikely to affect wild popul ations of black swallowtails.

b) Karner Blue Butterfly and Ot her Threatened or Endangered Species

In the preliminary BRAD EPA concluded that ther e was a possibility that off-site pollen flow

from Bt corn fields might potentially have advers e effects on federally listed threatened or endangered Lepidoptera because of the selectivit y of Cry1 proteins for certain lepidopteran species. EPA noted, however, that the majority of listed lepidopteran species have very restricted habitat ranges. Exam ination of an overlay map show ing the county level distribution of lepidopteran species relative to corn production counties in the US as listed by the U.S. Fish and Wildlife Service (USFWS, 1997) shows that as a rule, listed lepi dopteran species do not occur in agricultural areas where corn is grown nor is corn considered a host plant for these species. The map clearly indicates that any poten tial concern regarding ra nge overlap with corn production is restricted to the Karner blue butterfly (Lyceides melissa samuelis) . The butterfly is found along the northern extent of the range of wi ld lupine (its host pl ant), where there are prolonged periods of winter snow pack, primar ily in parts of Wisconsin, Michigan, Minnesota, Indiana, New Hampshire and New York. Wild lupi ne grows on dry, sandy soils in pine barrens, oak savannah, forest trails and previously dist urbed habitats such as utility rights-of-way, military installations, airports, highway corridor s, sand roads and abandoned sand pits (US Fish and Wildlife Service, 2000a, 2000b). No corn is grown in the area in New Hampshire where

Karner blue butterflies are found.

EPA concluded that because Cry1 proteins are broadly active agai nst Lepidoptera, some activity against the Karner blue would not be surprising. Toxicity testing of Karner blue larvae directly, however, is not possible due to its endangered status. Previo us studies that tested the susceptibility of lepidopterans to Cry1 proteins resulted in different LC 50 values for different species in the same genus. For example the Cry1Ab LC 50 for Spodoptera exigua is estimated at 3,180 ng/mL diet and 95,890 ng/mL diet for Spodoptera frugiperda (Luttrell et al . 1999). Herms

et al .(1997) performed a study demonstr ating that the Karner blue larvae were susceptible to a

formulated microbial Bt product based on the Bt kurstaki HD-1 strain that contains Cry1 proteins. Therefore, it can be assumed that the Karner blue ma y be susceptible to Cry1Ab, and perhaps to Cry1F, if levels of toxin in ingested pollen are high enough to adversely affect Karner blue butterflies. But, Herms et al . (1997) also showed that the Karner blue has susceptibility similar to the gypsy moth to a microbial Bt formulation containing Cr y1 proteins. Since the gypsy moth is known to be less susceptible to Cr y1Ab protein than European corn borers, and

levels of Bt pollen found in the field are not toxic to European corn borer s (the target pest), levels

toxic to the Karner blue are also not expected. Nonetheless, to be as protective as possible with respect to any potential effects on this endange red species, EPA in its preliminary BRAD and at

the October 2000 SAP considered whether registrations of Bt corn could potentially affect the

Karner blue. EPA also initiated contacts with the Fish and Wildlife Service to obtain information helpful to the Agency in assessing whether the Bt corn registrations could actually have an impact on the Karner blue. In addition to interacting with the FWS, EPA continued toCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

80 receive data and information, and to refine its analyses of whether the Bt corn registrations could

affect the Karner blue.

The Karner blue requires wild lupine (Lupinus perennis) as an oviposition substrate and larval food source. In considering the potential risk of Cry1Ab and Cr y1F proteins to Karner blue larvae, key issues to be addressed are: (1) whether the amount of corn pollen shed from Bt corn fields onto wild lupine would constitute a hazard to the Karn er blue; (2) whether there are wild lupine growing in the areas immediately adjacent to corn fields that are reestablished from fallow fields; (3) the extent of transpor t of corn pollen shed from corn fields; and (4) whether there is overlap between the period of pollen shed from corn fields with the period of Karner blue larval

emergence.

Hazard to Karner Blue. The Agency considers the most sens itive species tested to be a useful indicator of potential effects on endangered or threatened species. During the time period since EPA determined preliminarily that Bt corn could potentially affect Karner blue, the Agency has

received and obtained additional data. These data have enabled the Agency to conduct an ecological risk assessment for potential impacts to the Karner blue. Following EPA’s standard procedures for ecological risk assessment for endange red species, no effect is expected if there is a safety factor of 10X between the estimated e nvironment concentration (EEC) of the pesticide

and the LC 50 or LD 50 to the most sensitive species tested (USEPA, 1986). As described below,

EPA has determined the ratio between the EEC and LD 50 on the most sensitive species tested for Cry1Ab and Cry1F pollen protein.

Toxicity of pollen from the currently registered Cry1Ab Bt corn products to Karner blue larvae is estimated to be very low. At least 12 lepidop teran species have been tested to determine LC 50 levels for Cry1Ab (MRID 455122-00). The most sensitive species tested is the monarch butterfly. Researchers have determined th at the concentration producing no mortality whatsoever is greater th an (>) 4000 pollen grains/cm 2 of leaf surface (Hellmich, et al , 2001).

Thus the actual LD 50 for monarchs is likely to be subs tantially higher. Si nce the EEC is 300 pollen grains /cm 2 or less at the field edge, and 200 at one meter and 75 at three meters, 9 the

ratios of the EEC/LC 50 (with >4000 pollen/cm 2 as that LC 50 ) have been conservatively calculated to be 1:>13.3 for Cry1A b at the field edge, 1:>20 at one meter and 1:>53 at two meters from the field edge (Vaituzis, et al , 2001).

Similarly, toxicity of pollen from the currently registered Cry1F Bt corn products to Karner blue larvae is estimated to be very low. For Cry1F, at least 16 lepidopteran sp ecies have been tested

to determine LC 50 levels ( Wolt, 2000). The most sensit ive species tested is the diamondback moth. Because no data were submitted on toxicity of Cry1F in corn pollen, the LC 50 obtained on the diamondback moth with pure cry1F protein was converted to an LC 50 in terms of pollen grains and compared to the EEC in terms of the amount of Cry protein per gram of leaf tissue at the 300 pollen grains/cm 2 (of leaf tissue) level in the field. The ratios of the EEC/LC 50 have

9 The data collected for the DCI provide a deposition curve of pollen distribution outside a corn field. A conservative estima te of about 300 pollen grains (frequency of occurrence 0.017) per square centimeter is found at the edge of a corn field and the levels drop of f rapidly within a few meters of the corn field edge (Pleasants, et al , 2001).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

81 been calculated to be 1:172 for Cry1F at the field edge, 1:263 at one meter and 1:690 at two meters from the field edge. (Vaituzis, et al , 2001).

Overlap of wild lupine and corn. Based on its assessment of all relevant data and information, EPA has determined that the potential exposure of Karner blue to Bt corn pollen is limited because corn and lupine do not generally overlap. Wild lupine does not occur at all in corn fields. Moreover, wild lupine is not expected to grow adjacent to corn fields But, in one case brought to the attention of EPA, farm land can be taken out of production for conservation purposes in Wisconsin. Where farmland is take n out of production, and fields allowed to lie fallow, wild lupines might invade such fields. In these instances, it is possible that the Karner blue could be present on such lupine s. When EPA initially began the Bt reassessment, the Agency was concerned that lupines occurring adj acent to such reestablished corn fields could potentially contain Karner blue larvae that could possibly be a dversely affected by pollen shed

from Bt corn. EPA has received information that indica tes that the Karner blue is not expected to occur in proximity to such reestablished fields. The Wisconsin Department of Natural Resources states that most agricultural operations do not support habitat for the Karner blue, nor present a threat to the continued existence or recovery of the Karner blue in Wisconsin. Wisconsin Statewide Karner Blue Butterfly Habitat C onservation Plan and Environmental Impact Statement, Wisconsin Department of Natural Resources (2000) (http://www.dnr.state.wi.us/org/l and/er/publications/k arner/karner.htm). Moreover, while EPA received a comment to the effect that “[t]he Karn er Blue is documented as occurring adjacent to corn fields”, examination of the cited reference proves the opposite. 10 Overlap of corn pollen shed and larval emergence. Karner blue larvae are relatively less likely to be feeding during, or following the whole period of corn pollen shed. An analysis of pollen shed overlap with Karner blue larvae has been submitted to EPA and reviewed (MRID 455129- 01). This report indicates that there are 35 counties where Karner blue butterflies are found and corn is grown. EPA has received additional da ta from U.S. FWS indicating that there are 38

10 A commenter cited Andow, et al . (Andow., D.A., Lane, C.P., D.M. Olson, Use of Trichogramma in Maize - Estimating Envt’l Risk , in H.M.T. Hokkanen and J.M. Lynch, Biological Control Benefits and Risks (Cambridge U. Press 1995)) in support of the proposition that the Karner blue is “documented as occurring adjacent to corn fields.” Examination of this paper on Trichogramma demonstrates that the brief discussion of the Karner blue does not support the stated proposition. What the pape r does state is that: “the Karner blue [is] known to occur i n counties of Minnesota where maize is widely grown.” Hokkanen, p. 102. The Karner blue “occur[s] in sites near agricultural fields.” Id. (citing personal communication). “The Karner blue is a specialist feeder on w ild lupine (Lupinus perennis Fabaceae) in sandy soils intercalated in the oak savannah habitat near the Mississippi Ri ver in Minnesota. This area is surrounded by agricultural lands.” Id. Given the limited extent of pollen transport when shed from corn fields, EPA considers the term “adjacent,” when applied to corn fields in this context, to be most appropriately considered as 0-3 meters from the field edge. EPA does not agr ee that any of the quotations identified in the Andow paper is s upportive of the statement that “[t]he Karner Blue is documented as occurring adj acent to corn fields.” (Emphasi s supplied). Of greater intere st is a very brief section of the Andow paper entitled Actual distances to Karner blue habitats

.

In its entirety, this section states:

We have conducted intensive surveys of the distribution of Karner blue in Winona county. The fi ve Karner blue habitats in the area are only 0.5-0.9 km (mean 0.66 km) from the nearest agri cultural field. A more vivid picture of the proximity of Karner blue habitat to agricultural land is illustrated in fig. 10.8, which shows one of the larger populations of Karner blue in Minnesota (each square indicates the location of at least one Karner blue adult in its typical habitat of oak savanna woodlands. This population is only 0.6 km from the nearest agricultural field, which has been planted with maize. These data provide further evidence that th e potential risk from releases of T. nubilale is not negligible.

Id. at 111-12. Thus, “intensive surveys” by A ndow of the Karner blue in Minnesota demons trates that the five Karner blue populati ons identified exist from 500 to 900 meters from the nearest agricultural lands (with a mean distance of 660 meters). Given that EPA consider s that the relevant data on corn pollen transport supports a finding that “adjacent to corn fields” should be considered as the area from 0-3 meter s from the field, EPA does not consider these data to support the proposition that the Andow paper “documents” that the Karner blue “occur[s] adj acent to corn
fields.”Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

82 counties where the Karner blue is found and corn is grown. For 11 of these counties, no overlap of pollen shed for certain hybrids and Karner blue larvae is expected. For other counties, the possible overlap does not happen every year nor fo r more than a day or two in the life of the feeding larvae. For example, thes e data show that in some inst ances there might be one day of overlap every seven years. In addition, if pollen does fall on wild lupine plants, the studies done on corn pollen shed for the monarch butterfly Data Call-In (DCI), have sh own that rain and wind remove large amounts of pollen. Bt protein in corn pollen also de grades relatively rapidly in sunlight. (Pleasants, et al , 2001). The rapid removal of corn pollen from plant leaves, and the rapid degradation of Bt endotoxin in corn pollen reduces th e likelihood that Karner blue larvae

will encounter Bt endotoxin.

Thus, on the basis of new data and information received and obtained on the potential impact of

Bt corn on Karner blue, EPA has conducted an ec ological risk assessment using the best data available, and determined that there will be no effect on the Karner blue from the Bt corn registrations. This determinati on is based on a number of factor s including (1) if wild lupine were to grow adjacent to Bt corn fields, the amount of corn po llen shed from such fields onto the wild lupine would be insufficient to constitute a hazard to the Karner blue; (2) relevant data and

information indicate that there wi ll be relatively little, if any, wild lupine growing in the areas immediately adjacent to corn fields that are re established from fallow fields; (3) the amount of corn pollen shed from corn fields to adjacent area s is low; (4) available data suggest that there may be limited overlap between the period of polle n shed from corn fields with the period of Karner blue larval emergence.

As with all aspects of these registrations , however, EPA will continue to evaluate Bt corn agricultural practices, on going research, and endangered and thr eatened species implications, and will continue working with other Federal and State agencies as new information becomes

available.

EPA has also determined that there are no indire ct effects on endangere d and threatened plant species, such as impacts on lepidopteran pollinator s that are important and/or essential to an endangered or threatened plant. Working with U.S. FWS, EPA has identified an endangered bog orchid that is pollinated by a non-endangered ha wk moth. While some hawk moths might be found in and around corn fields, they feed and ovi posit on numerous plant species. Therefore, exposure of the hawk moth to Bt endotoxin is expected to be low. Moreover, even if the hawk moth is susceptible to Cry1 proteins, the number of hawk moths exposed to a lethal concentration should be insignificant to negligible based on the toxicity analysis for the most susceptible species as discu ssed above. Therefore, EPA determines that exposure to Bt corn will not sufficiently suppress the pollinator to affect the endangered plant species.

c) Monarch Butterfly

In 1999, Bt corn registrants submitte d two research reports (DP Barcode D255949) to EPA on potential effects of Bt corn pollen on mona rch butterflies ( Danaus plexippus Linneaus): Losey,

et al (Cornell) and Jesse and Obrycki (Iowa State). In the Losey et al . study, pollen collected

from Bt (Bt11 N4640 Bt corn) corn was applied by gently tapping a spatula of pollen overCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

83 milkweed leaves (Asclepias syriaca Linnaeus) which had been lightly misted with water. Pollen density was set to visually match densities on milk weed leaves collected from corn fields. Five three-day-old monarch larvae from a captive colony were placed on each leaf. The larvae reared on leaves dusted with pollen from Bt corn ate less, grew more slowly, and suffered higher mortality than larvae reared on leaves dusted with untransformed corn pollen or leaves without pollen. Larval mortality after 4 days of feeding on leaves with Bt pollen was significantly higher (44%) than the mortality either on leaves dusted with untransformed pollen or on control leaves with no pollen (both 0%).

Jesse & Obrycki used Bt field corn pollen (Event 176) covere d leaf samples taken from within and at the edge of corn fields (80-217 pollen grains/cm 2 ) to assess mortality. The samples were fed under laboratory conditions to monarch butterfly firs t instar larvae. The authors found a 19% mortality in larvae feeding on the Bt corn pollen treatment from leav es within and at the edge of the corn field within 48 hours, compared to 0% on non- Bt corn pollen exposed plants and 3% in the no pollen controls.

These reports were reviewed by the Agency. The reviews concluded that the preliminary controlled studies without exposure data are not conducive to conventional risk assessment procedures for Bt corn pollen effects on monarch butte rflies without additional field study information. The reports of Bt corn pollen toxicity to monarc h caterpillars did, however, result in a number of steps taken by the Agency to more fully assess and understand the possible effects of transgenic corn expres sing an insecticidal protein from Bacillus thuringiensis ( Bt corn) on non-target lepidopteran species, pa rticularly monarch butterflies ( Danaus plexippus ). To help identify the level of exposure and other ri sks to monarch butterflies, on December 15, 1999 EPA issued a monarch butterfly adverse effects data call-in (DCI) notice to the registrants of Bt corn products under its FIFRA Section 3(c)(2)(B) authority. On December 9, 1999 (USEPA, 2000), and again on October 18-20, 2000 (USEPA, 2001), the Agency presented current and possible new data requirements to evaluate ecological effects, including the monarch question, to a FIFRA Scientific Advisory Panel for their reco mmendations. In addition, EPA consulted with monarch butterfly experts and USDA to better understand the effect of Bt corn pollen on

monarch butterflies. Until more definitive da ta and information were available about the potential risks of Bt corn pollen to monarch but terflies and other lepido pterans, EPA requested that registrants instruct thei r customers who are planting non- Bt corn refuges (for resistance management) to place the non- Bt corn refuge between Bt corn and habitats such as prairies, forests, conservation areas, and roadsides as a precautionary measure. However, in light of the recently reviewed DCI research data showing that monarchs appear to breed on milkweed inside

corn fields and that toxic levels of Bt pollen do not accumulate outside corn fields, this recommendation no longer appears necessary.

The DCI called for information in five basic area s relating to the potential exposure of non-target lepidopterans, particularly monarchs to Bt corn pollen. These includ e: the distribution of monarch butterflies, milkweed pl ants and corn; corn pollen re lease and distribution in the environment; toxicity of Bt corn Cry proteins and Bt corn pollen to lepidopterans; monarch egg laying and feeding behavior; and monarch populati on monitoring. The Agency has reviewed the submitted DCI data and incorporated the findings into this reassessment. The DCI data is aCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

84 result of an ABSTC and USDA coordinated re search effort, with additional research by independent university and government workers.

Subchronic toxicity studies c onducted since the DCI was initia ted have shown that monarch larvae feeding on corn pollen expressing MON 810, Bt11, TC1507 or TC6275 at pollen levels

found in corn fields do not demonstrate observabl e adverse effects on survival, weight, or other fitness parameters (e.g., developmental change, weight gain, percent survival to pupation, pupal weight (mg), percent emergence from pupae, adu lt weight (mg), or adult wing length) (StanleyHorn et al . 2001). Risk from other factors such as de struction of over winter ing habitat, weather, predators, physiological stress, human act ivity (Taylor 1999) and conventional chemical insecticide use (Stanley-Horn et al . 2001) are a much greater and more widespread threat to monarch populations than the use of Bt corn. The potential reduction of insecticide use that may result from planting Bt corn will most likely benefit m onarch populations as well as other beneficial insects, especially in popcorn and sweet corn production.

The submitted data demonstrated that levels of MON 810 and Bt11 corn pollen toxic to monarchs would probably not occu r under natural field c onditions. The mean pollen density of

all the studies was found to be 170 inside the corn fields and 63 grains/cm2 at the edge. The highest average corn pollen dens ities monitored in the field we re 586 in Maryland (Stanley-Horn

et al . 2001) and 900 grains/cm2 found in one Iowa corn field (Pleasants et al . 2001). In a worst case scenario, pollen deposition when no rainfall occurred was approximately 1400 grain/cm2 (Pleasants et al . 2001). Research conducted in response to the DCI showed that Cry1Ab corn pollen densities of >4000 grains/cm2 do not show mortality to monarchs (Hellmich et al . 2001).

These studies have also shown th at the order of monarch sensitiv ity to Cry proteins is Cry1Ab > Cry1Ac > Cry9C > Cry1F. Only pollen from Event 176 corn has been shown to adversely affect growth, fitness, and mortality of monarch butterflies (Losey et al . 1999, Jesse and Obrycki 2000, Stanley-Horn et al . 2001). However, this does not create a concern for monarchs since Event 176 corn comprises less than 2% of U.S. corn acreage and will no longer be sold after the 2003 growing season.

In the report of their two year study, Stanley-Horne et al . (2001) suggested that percent monarch adult emergence warranted further investigation. Also, the field studies recording that Bt11 or MON810 pollen had no effect on survival of mo narch larvae for 14 to 22 days also needed further analysis. In addition, the authors also no ted that these studies di d not address chronic, long-term exposure of monarch larvae throughout their development cycle to determine the subtle effects of prolonged exposure to Bt toxin (Sears et al . 2001 and Stanley-Horne et al

. 2001).

As a result of these conclusions, EPA include d a statement in the 2001 EPA Registration Action

Document for Bt corn that studies on long-term exposure of monarch larvae to Bt pollen should

be considered. Researchers at th e University of Maryland (Dively, et al . 2004) conducted such studies from 2001-2002, and the results were su bmitted jointly to EPA by Monsanto Company and Syngenta Seeds, Inc. in 2003. The study include d the results of a risk assessment simulation model that projected, over the range of the Corn Belt, an increased risk of 0.6% in mortality to monarch larvae from prolonged exposure to Bt corn pollen. The increased risk to monarchCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

85 larvae when considering all of North America was 0.3%. Based on these criteria the Agency concludes that, given the other natural and man-made hazards to the monarch populations which account for up to 90% annual monarch mortality without affecting the overall monarch abundance, a 0.3% increase in mortality due to Cry1Ab Bt corn pollen will not pose an

unreasonable risks to the continue d existence of monarch populations in North America. A full summary of the study is incl uded below in subsection 4 ( Bt pollen exposure and toxicity).

1) Monarch habitat

A baseline monarch population level cannot be reasonably developed. It is difficult to develop a baseline population level using current met hodology and because the number of monarchs throughout the U.S. fluctuates be tween regions and years. Ther e are several factors such as catastrophic weather (e.g ., drought or floods) that may adversel y affect monarch population size. However, monarch populations may recover from catastrophes as is evidenced by the large

number of monarchs counted in 1994, the year after floods in the midwest. On the other hand,

warm summers result in increa sed population size in North Am erica and decreased numbers during cold summers. Among othe r factors that may affect m onarch population size are: (1) overwintering site depletion, (2) number and fitn ess of monarchs that overwinter, (3) nectar availability to adults, (4) pathogens, parasite s, parasitoids, and predators, (5) milkweed availability, (6) use of in secticides to control lepidopteran pest s, and (7) accidents (e.g., collision with automobiles). Due to these factors, it is di fficult to develop a baseli ne population size or to

determine if Bt corn pollen was a contributing factor.

There have been several attempts made to de termine monarch population levels. Swengel (1995) showed that from 1986-1994 there were signif icant changes in monarch counts including increases and decreases from five of eight year-pairs. Walton and Brower (2000) showed extreme variability in monarch counts in Cape May Point, NJ which is a major funnel point in September and October for monarchs migrating to Mexico. In Cape May, the 1999 counts were seven times greater than in 1998 and almost tw ice as high as any year since 1992 when the census began. Monarch Watch has conducted annual surveys since 1993. Surveys from 1993-

1999 are available online at www.monarchwatch.org.

Due to extreme annual swings in monarch populati on estimates, it is not reasonably possible to develop a baseline monarch butterf ly population size. However, it is possible to continue surveys such as the one conducted by the Monarch Watch to identify sudden, drastic decreases in

the number of monarchs in North America and its overwintering sites in Mexico.

The DCI addresses the potentia l of monarch exposure to Bt corn pollen in the field and whether pollen densities encountered presen t a risk to these bu tterflies. Monarch la rvae potentially feed on 14 different species of milkweeds. Seven of these milkweed species are fed on by monarchs in the Corn Belt. Common milkweed ( Asclepias syriaca ) is the predominant species oviposited and fed on by monarchs. Whorled milkweed ( Asclepias verticillata ) may also be an important resource for monarchs (Harzler and Buhler 2000). Milkweed dens ities vary and typically depend on the management practices of the habitat.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

86 Milkweeds can be found in a variety of habitats . However, non-agricultural areas are usually undisturbed supporting growth of more milkw eeds. Surveys conducted in Ontario, Maryland, Indiana, Illinois, Iowa, Nebraska , and Kansas showed that more milkweeds occur near the corn field edge, in roadsides, or in non-agricultural areas than within co rn fields (Harzler and Buhler 2000, Oberhauser et al . 2001). Roadsides that are mowed will have less milkweed than areas not mowed or tillage practices may affect densities in cultivated fields. It is difficult to control milkweed, particularly when reduced tillage is practiced, because milkweed reproduces vegetatively or by seed and is often found in clum ps (Martin and Burnside 1984). Herbicides are generally not considered to be e ffective in controlling milkweeds. However, in some instances, “good” control of milkweeds may be provided by glyphosate, halosulf uron-methyl + dicamba (2,4-D), and nicosulfuron + dicamba.

Some milkweeds occur in and near corn fiel ds, therefore, the propor tion of the migrating monarch population that may encounter Bt corn fields was considered. There are potentially 105,174 square miles (2.73 × 10 7 hectares) of field corn grown in the U.S. that may provide breeding habitat for monarchs (USDA - NASS 1997). Of this 105,174 square miles, about 26,294 square miles consist of Bt corn fields that may provide breeding sites for monarchs. The edge of corn fields constitu tes a very small area of potentia l monarch breeding habitat. Approximately 0.18% of monarch br eeding sites may occur near corn field edges. This is equivalent to 0.11% of all land in this region. It can be concluded that the near edge (within 1 meter of the field edge) of Bt corn fields constitutes a negligible portion of monarch breeding habitat. Approximately 18% of monarch habitat in the central U. S. consists of corn fields (Taylor and Shields 2000) and cu rrent approximate acreage of Bt corn is equal to approximately 26,293 square miles (25% of total U.S. corn acreage) or 5.1% of monarch habitat. The information submitted to the Agency thus far suggests that 50% of monarchs probably pass through the Corn Belt (Taylor et al . 1999).

Monarchs feeding on milkweeds in and near Bt corn fields during anthesis will potentially be exposed to Bt pollen. Time to pollination varies among hybrids and regions and is determined according to growing degree uni ts (GDU). Examples of the approximate number of GDU needed for pollination to occur in different re gions are: (1) Fargo, ND = 1130; (2) Madison, WI = 1250; (3) Lincoln, NE = 1370; (4) Champaign, IL = 1390; (5) Salisbury, MD = 1400; and (6) Lubbock, TX = 1450. Individual corn tassels typically shed polle n for two to seven days (or longer) and silks on an ear are e xposed to pollination for two to three days (Ru ssell and Hallauer 1980, Ritchie et al . 1997). A field will shed pollen for up to 15 days depending upon microclimate (Russell and Hallauer 1980).

Corn pollen grains don’t disperse far fr om its source because they are large ( < 90 to 100 microns). The majority of corn pollen stays with in corn fields and only small quantities disperse beyond 5 meters from the field edge. However, po llen levels are higher further into the corn field (e.g., 147.5 grains/cm 2 were found 25 m into the field) th an close to the edge (e.g., 55.5 grains/cm 2 were found 3 m into the field) (Pleasants et al . 2001). Raynor et al . (1972) found that 63% of corn pollen remained within fields, 88% settled within eight meters of the field edge, and 98% settled within 60 meters. They also de termined that there wa s only 0.2% of pollen deposited at 60 m from the corn field edge. Pleasants et al . (2001) found pollen de nsities at cornCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

87 field edges were 50% of the level found within th e field and densities were greater on milkweed plants within rows than between rows.

It is difficult to report one specific quantity of corn pollen that will be deposited on milkweeds within corn fields and at varying distances fr om the field edge. Many factors influence pollen deposition and retention on milkw eed leaves. Environmental fact ors such as rain and wind may increase the distance pollen will travel and may d ecrease the amount of polle n retained on leaves. Plant morphology such as leaf angle will also effect pollen deposition and retention. Upper leaves that are more upright and exposed to en vironmental factors retain less pollen than middle

and lower leaves on the m ilkweed plant (Pleasants et al . 2001).

2) Corn pollen exposure

Pleasants et al . (2001) found that levels of corn po llen deposition on milkweed leaves are influenced by wind, wind directi on, rainfall, plant arch itecture and the time period when pollen was sampled. In some instances, weather condi tions such as thunderstorms and updrafts carry some pollen grains further than usual (Emberlin et al , 1999). However, wind, rain, and other environmental factors will probab ly remove most of the pollen deposited on milkweed leaves (Pleasants et al . 2001). Rainfall has been shown to rem ove most (86 - 92%) of the pollen from milkweed leaves, thus potentially reduc ing the length of monarch exposure to Bt pollen (Pleasants et al . 2001, Stanley-Horn et al . 2001). The level of exposure of monarch larvae to Bt pollen carried to milkweed plants on exoskeleton of adults is minima l. If pollen were to adhere to monarch adults and dislodge on milkweeds, qu antities would not be great enough to adversely affect larvae feeding on these milkweeds. Since Bt Cry protein must be ingested and will not harm monarchs by contacting it’s exoskeleton, th ere is minimal risk posed from monarchs transporting pollen among milkweed plants.

Monarchs will only be exposed to Bt while it remains biologically active in pollen. Microbial enzymes, secondary plant compounds, extremes in pH, ultraviolet light, wind and rain are known to degrade Bt proteins in microbial sprays. The insecticidal activity in Bt microbial sprays has been shown to break down rapidly for two days after application and is practically nonexistent four days post application (Gelernter, 1990). These factors also affect the insecticidal activity of

Bt expressed in pollen. Head and Brown (1999) only found biologi cal activity of MON 810 in fresh pollen. Laboratory assays showed that M ON 810 activity was not detectable in pollen after seven days (Head and Brown 1999). The biological activity of Bt proteins probably decreases more rapidly in the field where it is exposed to elements such as ultraviolet light than in the laboratory. Therefore, the Cry protein may breakdown more rapidl y than seven days under field

conditions.

Monarch ovipositional and feeding behavior will also contribute to the level of milkweed pollen that the larvae will encounter. Surveys have show n that monarchs prefer to oviposit single eggs

on the underside of milkweed leaves on young, tender tissue (Urquhart 1960, Borkin 1982,

Pleasants et al . 2001). However, females may lay more than one egg per plant and may oviposit on the top of leaves, on stalks or flowers (Borki n 1982). The age of plant tissue is probably the most important influence on monarch ovipositiona l preference. Female monarchs prefer toCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

88 oviposit on young tender plant tissue (Urquhart 1960, Borkn 1982). Neonate larvae begin feeding near the area where eggs were laid which is typically the underside of leaves. As larvae mature, they may feed through leaves, on top of leaves, or on leaf veins (Urquhart 1960).

Results vary regarding monarch preference, avoidance, or i ndifference to ovipositing on milkweeds in corn fields (Tschenn et al . 2001, Oberhauser et al . 2001). In the laboratory,

Tschenn et al . (2001) showed that monarchs either do not show a preference for milkweeds with or without corn pollen dusted on them, or they avoid pollen dusted milkweeds. Field surveys conducted by Oberhauser et al . (2001) and Stanley-Horn et al . (2001) found monarch eggs on milkweeds dusted with pollen in and near corn fiel ds. In some instances monarchs may prefer to oviposit on milkweeds occurring wi thin corn fields (Oberhauser et al . 2001). Although milkweed densities are genera lly higher in nonagricultural ha bitats, surveys conducted in Minnesota, Wisconsin, and Iowa suggest that m onarchs will oviposit in corn fields 45 to 107 times more often than in nonagr icultural habita ts (Oberhauser et al . 2001).

Oberhauser et al . (2001) and Pleasants et al . (2001) showed that monarchs do occur on milkweeds in the field during pollen shed. The Oberhauser et al . (2001) study showed considerable overlap between the peak of the migratory monarch generation and pollen shed in Minnesota and Ontario. In Iowa and Maryland, the final generation of monarchs peaked prior to pollen shed. Four different mona rch breeding regions (east-centra l Minnesota and west-central Wisconsin, central Iowa, coastal Maryland, and southern Ontario) were monitored when monarchs were present.

TABLE 7. Temporal overlap of monarch larvae and corn pollination

State % Overlap of Larvae & Anthesis % Over lap of Migratory Gen. Larvae and Anthesis

Minnesota 20% to 68% 50%

Ontario 27% to 75% 50%

Maryland 0 to 36% 15%

Iowa 4% to 25% 15%

Wisconsin 50%

According to models developed by Calvin et al . (2000), overlap of monarch larval occurrence and corn pollination is negligible in southern and central parts of the Corn Belt, but there is up to 75% overlap in the northernmost area of the Corn Belt. This means that 0 to 5% of monarchs will be exposed to Bt corn pollen in most of the Corn Be lt and 10% exposure will occur in the northern region. In general, the Calvin et al . (2000) model showed that the degree of cooccurrence generally increased as latitude or elevation increased.

Since monarch eggs and larvae were found on mi lkweed plants in the northern fields when pollen was present on leaves (Oberhauser et al. 2001) it can also be assumed that monarch larvae

will consume both Bt or non- Bt pollen if it is en countered (Hellmich et al. 2001, Oberhauser et
al. 2001). Laboratory (Hellmich et al . 2001, Tschenn et al . 2001) and field studies (Oberhauser

et al . 2001) demonstrated that monarchs will not avoid feeding on plants dusted with Bt or non-Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

89 Bt corn pollen. Since eggs and larvae were f ound on milkweed plants naturally dusted in corn pollen in the field, it appears that monarchs will not avoid pollen dusted pl ants nor do they avoid

corn fields.

3) Bt Cry1Ab toxicity to monarchs

Since it has been established that mo narch larvae can encounter and feed on Bt pollen in the field, it is important to know the Bt pollen toxicity level.

TABLE 8. LC 50 s and the EC 50 s (effect-eliciting concentration) for the various monarch larval stages fed purified trypsin resistant core of Bt Cry 1Ab proteins

Instar (N) LC 50 (95% C.I.) (ng Cry1Ab/mL treated artificial diet) EC 50 (95% C.I.) (ng Cry1Ab/mL treated artificial diet)

1 st

(318) 3.29 (2.19-4.76) 0.76 (0.64-0.90)

2 nd - 3 rd (141) 35.1 (30-100) 9.60 (6.01-15.06)

3 rd - 4 th (125) > 100 (-) 18.3 (9.4-40.3)

(Hellmich et al . 2001).

LC 50 s for third and fourth instars were 30 times gr eater than first instar s and second and third

instar’s LC 50 was 11 time greater than first instar s. The Cry1Ab no observable effect concentration (NOEC) was reported as ≤ 0.3 ng/mL diet (Hellmich et al . 2001).

In nature, monarchs are not expected to get uniformly distributed doses of Bt as is observed in the laboratory. Unlike feeding on diet in the laboratory, monarchs would probably ingest varied

amounts of Bt in the field and also have th e opportunity to avoid feeding on Bt altogether. Bt activity in pollen is also expected to decline over time in the fiel d. Therefore, levels of Cry1Ab ingested by monarchs in the fiel d are expected to be lower than levels fed to them in the

Hellmich et al . (2001) laboratory study.

4) Bt pollen exposure and toxicity

Cry 1Ab in pollen of the currently registered Bt field corn hybrids (MON 810 and Bt11) is only found in trace quantities (<0.09 μg/g dry wt. pollen). In order to determine the exposure of

monarch larvae to Bt pollen on milkweed leaves, five independent surveys were conducted during the 2000 growing season in Iowa, Maryland, and Ontario (Table 9). The highest average corn pollen density monitored in the field was 586 grains/cm 2 found three meters inside a Bt11 sweet corn field (Stanley-Horn et al. 2001).

TABLE 9. Mean pollen density on milkweed leaves inside a cornfield (Pleasants et al , 2001)

Study Anthesis level Mean pollen density (cm 2 )

Maryland 1999 Near peak 65.7

Iowa 2000b 100% 425.6

Iowa 2000c Post anthesis (10days) 101.2Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

90 Study Anthesis level Mean pollen density (cm 2 )

Iowa 2000d 100% 231.4

Ontario 2000 day11 97.7

Maryland 2000 day 9 161.3

The highest pollen densities were found in a Bt sweet corn field since sweet corn produces more pollen per plant than field corn. The highest level of pollen found averaged 504-586 grains/cm 2

and occurred on milkweeds located 3 m inside Bt11 sweet corn in Maryland (Stanley-Horn et al. 2001). There was no difference in densities of Bt and non- Bt pollen found on milkweed leaves (Stanley-Horn et al. 2001). In one corn field in Iowa, Pleasants et al. (2001) found a mean of 900 pollen grains/cm 2 . However, first instar larvae feeding on milkweed leaves naturally dusted with pollen in the field resulted in no observa ble effects of MON 810 and Bt11 on survival and

fitness of monarchs.

A study conducted by Hellmich et al. (2001) involved feeding first instar monarchs no pollen or

known amounts of Bt (MON 810 and Bt11) and non- Bt pollen applied in the laboratory. Extremely high pollen leve ls (250 - 2000 grains/cm 2 for MON 810 and 150 - 4000 grains/cm 2 for Bt11) were fed to first instar monarch larvae in a controlled environment (since no pollen was removed due to environmental factors, these cond itions are considered a wo rst case scenario) and resulted in no significant eff ects on larval weight (Hellmich et al. 2001). From this data, it can be concluded that the NOEL for MON 810 is >2000 grains/cm 2 and for Bt11 is >4000 grains/cm 2
which is greater than levels that occur under natural field conditions.

However, assuming a worst case scenario where 1000 pollen/cm 2 would show weight loss effects, the effect of Bt corn pollen on monarch larvae would still be minimal since these levels are rare in the field, expected to occur at a 0.1% rate. In a ddition, monarch larvae exposed to

sub-lethal concentrations of Cr y1Ab protein have been shown to mature into healthy adults (Jesse & Obrycki, 2000).

TABLE 10. Frequency distribution of pollen deposition density on milkweed leaves (Pleasants et al , 2001):

From edge of cornfield Pollen density (cm 2 ) Inside corn field

0 meters 1 meter

0-100 0.625 0.833 0.900

100-200 0.190 0.093 0.062

200-300 0.091 0.033 0.022

300-400 0.037 0.017 0.066

400-500 0.018 0.008 0.002

500-600 0.010 0.007 0.002

600-700 0.009 0.002 0.001

700-800 0.004 0.002 0.000

800-900 0.004 0.003 0.001Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

91 From edge of cornfield Pollen density (cm 2 ) Inside corn field

0 meters 1 meter

900-1000 0.002 0.001 0.000

1000-1100 0.001 0.001 0.001

Therefore, it can be concluded that levels of MON 810 or Bt11 pollen toxi c to monarch larvae do

not occur under natura l field conditions.

Stanley-Horn et al. (2001) studied the differe nce between first instar la rvae feeding on Bt11 and

on non- Bt pollen in the field (starting six days after initiation of pollen shed ) for five days. The results showed no significant mortality, feeding, development, weight gain, % survival to pupation, days to pupation, pupal weight, emergence from pupae, adult weight and adult wing length. However, first instar monarchs feeding on milkweed sprayed with insecticides or subject to exposure from insecticide drift were adversel y affected. There was 90% to 100% mortality of monarchs feeding on milkweeds collected from w ithin the field and 21% to 45% mortality from plants outside the field. Th is study also suggests that Bt sweet corn may provide a safer habitat for monarchs than fields requiring in secticide applications (Stanley-Horn et al . 2001, Vlachos and Roegner 1997).

The 2001 EPA Registration Action document for Bt corn stated that studies on long term exposure of monarch larvae to Bt pollen should be considered due to the fact that monarch larvae hatching in corn fields may be exposed to Bt protein for periods of 12 days or longer. These studies were conducted from 2001-2002, and the results were submitted jointly by Monsanto Company and Syngenta Seeds, Inc. in 2003 (MRID 46162001) and later published in Environmental Entomology (Dively, et al. 2004). Effects on the monarch butterfly ( Danaus plexippus ) from continuous exposure of la rvae to natural deposits of Bt and non- Bt corn pollen on milkweed ( Asclepias syriaca ) were measured in five studies using Cry1A(b)-expressing hybrids (events BT11 and MON810) and non-transgen ic corn in Marylan d, Iowa, and Ontario, Canada. First instars were exposed beginning at 3 to 4 and 6 to 7 days after initial anthesis. Overall mean pollen densities during larval deve lopment in the first and second bioassays were 163 and 170 grains/cm 2 , respectively. Pollen density on milkweed plants in the non- Bt , BT11, and MON810 hybrids averaged 163, 155, and 174 grains/cm 2 , respectively, during the first bioassay and 172, 173, and 158 grains/cm 2 , respectively, in the second bioassay. There were no statistically significant differen ces in pollen density between assays or among hybrid types. The number of anthers on the leaves was positively co rrelated with the amount of pollen deposited on

the leaves.

The mean number of days for first instars to develop to eclosion was 22.7 in the laboratory reared cohorts, and 24.2 in the field reared cohorts. In the first bioassay, exposure to Bt pollen had a statistically significant effect on developmental time to pupation and to eclosion. Development to adult emergence was prolonged by 0.6 to 1.2 days over that for larvae exposed

to non- Bt pollen. Results from the sec ond bioassay were similar, wi th average delays of 0.7 to 2.4 days to pupation and 0.2 to 2.4 days to eclo sion. For both bioassays combined, exposure to Bt pollen prolonged developmental time by an average of 1.8 days.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

92 In the first bioassay, mean survival to pupation was 71.2%, 56.9%, and 50.6% for non- Bt , BT11, and MON810 pollen, respectively. Mean surv ival to eclosion wa s 66.7%, 51.3%, and 48.5%, respectively. There were no st atistical differences between the BT11 and MON810 events. Overall, 25% fewer larvae exposed to Bt pollen survived to become adults. In the second bioassay, mean survival to pupation was 59.3, 50.4, and 44.4% for non- Bt , BT11, and MON810 pollen, respectively. Mean survival to eclos ion was 58.6%, 47.8%, and 43.6%, respectively. Again, there were no statistical differences between the BT11 and MON810 events. Overall, 22% fewer larvae exposed to Bt pollen survived to become adu lts. For both bioassays combined, 23.7% fewer first instars exposed to Bt pollen reached the adult stage, compared to those exposed to non- Bt pollen. Overall survival of laboratory -reared larvae (66.1%) was significantly higher than field-reared larvae (44.6%), alt hough natural mortality factors (e.g., weather, pathogens) probably accounted for the difference.

Exposure to Bt pollen during larval development decrea sed the weight of pupae and adults. The results were statistically signifi cant for pupal weight in both bioassa ys and also for adult weight in the second bioassay. Weights of both pupae and adults reared from larvae exposed to Bt pollen were significantly reduced by an average of 5.5% compared to those exposed to non- Bt pollen.

The adults from Bt pollen-exposed larvae also had slightly shorter wing length, but the difference was not statistically significant.

A risk assessment modeling system was used to estimate the proporti on of second-generation monarchs that would be affected by exposure to Bt pollen (the first generation does not overlap with the period of anthesis in th e corn belt). A table of paramete r estimates of exposure risk and probability of harm for 15 U.S. Corn Belt st ates and Ontario was provided in MRID No.

46162001. The model results indicate that for the co rn belt area, which re presents 50% of the monarch breeding population, the risk to monarch la rvae associated with long-term exposure to

Bt corn pollen would be 0.6% additional mortality.

The Corn Belt constitutes 50% of the breeding p opulation in North America. Monarchs outside the Corn Belt are relatively unaffected due to th e low acreage of corn and a low percentage of overlap of monarch larvae with corn pollen she d. Therefore, when all of North America is considered, the risk to monarch larvae from long-term exposure to Bt11 and MON810 Bt corn pollen is an additional 0.3% mortality at the current adoption rates (37%) of Cry1Ab Bt corn. The OPP risk assessment process considers a >5 0% reduction in population (mortality) as posing a risk to the existence of a speci es (see Section 1: Tiered Hazard and Risk Assessment Process). Based on these criteria the Agency concludes that , given the other natura l and man-made hazards to the monarch populations which account for up to 90 % annual monarch mortality without affecting the overall monarch abundan ce, a 0.3 % mortality due to Cry1Ab Bt corn pollen will not pose an unreasonable risks to the continue d existence of monarc h populations in North

America.

5) Bt Cry1F toxicity to monarchsCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

93 A scientifically sound study submitted by Dow Agro Sciences showed that Cry1F does not cause mortality to neonate monarch butterfly larvae when fed a 10,000 ng/mL diet dose. [Helmich, et
al (2001) made the same observation at a 30,000 ng/mL dose level.] First instar larval weight and mortality were recorded after seven days of feeding. There was no mortality to monarchs fed 10,000 ng/mL diet, the highest rate tested. There was some gr owth inhibition at 10,000 ng/mL diet. Since pollen doses equiva lent to 10,000 ng/mL diet are not likely to occur on milkweed leaves in nature, it can be concluded that Cr y1F protein will not pose a risk to monarchs.

6) Conclusions:

MON 810 and Bt11 show relatively low toxicity to monarch larv ae and the Cry1F protein has no detectable toxicity to monarch larvae. Overall, the available information indicates a very low probability of risk to monarchs in areas beyond the near edge of corn fields. Inside corn fields and at the near edge of corn fields there is low probability of monarch larvae encountering a toxic level of pollen for the Bt corn products covered by this ri sk assessment. Consideration of factors limiting exposure, such as relatively low pollen shed and monarch breeding overlap in much of the corn belt, the distri bution of milkweed plants within corn fields compared to other milkweed habitats, the egg laying and feeding activity of monarch larvae, together with the low toxicity of the Bt corn products covered by this assessmen t indicate a low probability for adverse

effects on monarch larvae.

The weight of evidence of data ga thered to study the effects of Bt pollen on monarch larvae in

the field indicate that milkweeds in corn fields to within 1 meter of cornfields are unlikely to be

dusted with harmful levels of Bt pollen from the most widely planted corn varieties MON 810,

Bt11 and TC1507.

Based on the review of the DCI data, in combin ation with data submitted since 2001, the Agency

concludes that the curren t information on monarch toxicity an d exposure does not give sufficient cause for undue concern of widespread ri sks to monarch butterf lies at this time. 11 EPA will continue to closely monitor the re sults from further monarch butterf ly research as a part of its regulatory oversight of Bt products.

4. Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms

EPA has evaluated the potential for hor izontal gene transfer (HGT) from Bt crops to soil organisms and has considered possible risk implica tions if such a transfer were to occur. Genes that have been engineered into Bt crops are mostly found in, or have their origin in, soilinhabiting bacteria. Soil is al so the habitat of anthrax, teta nus, and botulinum toxin-producing bacteria. Transfer of these genes and/or toxins to other microorganisms or plants has not been detected. Furthermore, several experiments (publ ished in scientific journals), that were conducted to assess the likelihood of HGT, have been unable to detect gene transfer under

11 The available data can be used to make an approximation that only 0.001% of the m onarch population (1 in 100,000) may be expose d to sublethal amounts of Bt pollen in Bt corn fields (using the inform ation that 50% of the monarchs go through the corn belt, that 18 % of that habitat is corn, that 25% is Bt corn, that the maximu m overlap of anthesis and larvae is 50% in the migratory population and that in a wor st case scenario 0.1% of that population may encounter sub-lethal amounts of Bt pollen).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

94 typical environmental conditions. Horizontal gene transfer to soil organisms has only been detected with very promiscuous microbes under la boratory conditions designed to favor transfer.

As a result of these findings, whic h suggest that HGT is at most an artificial event, and the fact

that the Bt toxins engineered into Cry1Ab and Cry1F corn are derived from soil-inhabiting bacteria, EPA has concluded that there is a low probability of risk from HGT of transgenes found in Cry1Ab- and Cry1F-producing corn.

5. Gene Flow and Weediness Potential

The movement of transgenes from the host plant into weeds has been a significant concern for EPA due to the possibility of novel exposures to the pesticidal substance. This concern has been

considered for each of the B.t. plant-incorporated protectant s currently registered and EPA

believes that these concerns have been satisfactor ily addressed. The Agency has determined that there is no significant risk of ge ne capture and expression of any B.t. endotoxin by wild or weedy relatives of corn, cotton (for the duration of the Bt cotton product registrations amended as of September 29, 2001), or potato in the U.S., its possessions or territori es. In addition, the

USDA/APHIS has made this same determinatio n under its statutory authority under the Plant Pest Act. There is a possibility, however, of gene transfer from B.t. cotton to wild or feral cotton relatives in Hawaii, Florida a nd the Carribean. Where feral populat ions of sexually compatible cotton species exist in Florida and Hawaii, EP A has prohibited the sale or distribution of B.t.

cotton in these areas. These containment meas ures prevent the movement of the registered B.t.

endotoxin from B.t. cotton to wild or feral cotton relatives in Hawaii and Florida.

Under FIFRA, EPA has reviewed the potential for gene capture and expression of the B.t. endotoxins by wild or weedy relati ves of corn, cotton and potatoes in the U.S., its possessions or

territories. B.t. plant-incorporated protectants that have been registered to date have been expressed in agronomic plant speci es that, for the most part, do not have a reasonable possibility of passing their traits to wild native plants. Feral species re lated to these crops, as found within the United States, cannot be pollinated by these crops (corn, potato and cotton) due to differences in chromosome number, phenology ( i.e. , periodicity or timing of events within an organism’s life cycle as related to climate, e.g. , flowering time) and habitat. The only exception, however, is the possibility of gene transfer from B.t. cotton to wild or feral cotton relatives in Hawaii, Florida

and the Caribbean.

The FIFRA EPA Scientific Advisory Pane l meeting held on October 18-20, 2000, further discussed the matter of gene flow and offered some issues for consideration in this matter. The panel agreed that the potential fo r gene transfer between corn (maize) and any receptive plants within the U.S., its possessions and territories was of limited probability and nearly risk free. Similarly, potatoes were seen as nearly risk free with regards to gene flow in that proximity of compatible wild relatives to this crop is insuffi cient to allow for cross pollination. Some concern was expressed, however, with respect to B.t. cotton grown in areas wher e wild relatives or feral populations of the crop are known to exist.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

95 a. Gene Transfer - Gene Flow

Concern over the potential for species related to maize ( Zea mays ssp. mays ), such as Tripsacum species and the teosintes, as potential recipien ts of gene flow from genetically modified Zea

mays indicated a need for review of what is known about this subject and to reevaluate the initial Agency assessments related to gene flow potential of Zea mays . Some Zea spp., such as the teosintes, are known to be interf ertile with maize and are discu ssed as potential recipients of pollen directed gene flow from maize. This issue is of particul ar concern based upon the increased planting of genetically modified maize. Therefore, the Agency conducted a reevaluation in early 2000, the re sults of which are reported here.

b. Zea mays ssp. mays - Maize - General Biology

Zea mays is a wind-pollinated, monoecious, annual spec ies with imperfect flowers. This means that spatially separate tassels (male flowers) and silks (femal e flowers) are found on the same plant, a feature that limits inbreeding. A large variety of types are known to exist ( e.g. , dent, flint, flour, pop, sweet) and have been selected fo r specific seed characteri stics through standard breeding techniques. Maize cult ivars and landraces are known to be diploid (2n = 20) and interfertile to a large degree. However, some evidence for geneti c incompatibility exists within the species ( e.g ., popcorn x dent crosses; Mexican maize landraces x Chalco teosinte). Zea mays has been domesticated for its current use by sel ection of key agronomic characters, such as a non-shattering rachis, grain yield and resistance to pests. The origin of corn is thought to be in Mexico or Central America, based largely on arch aeological evidence of early cob-like maize in indigenous cultures approximately 7200 years ago.

A recent study has indicated that cross-pollination of commercial maize cultivars at 100 ft downwind from the source of genetically modified maize was 1 % and this proportion declined exponentially to 0.1 % at 130 ft and further declin ed to 0.03 % at 160 ft. At 1000 ft, the farthest distance measured, no cross-pollination was detected (Jemison and Vayda, 2000). For production of Foundation Seed, a distance of 660 ft has been generally required to ensure separation of pollen types. The relatively large size of corn pollen and its short viability period under most conditions preclude long distance tr ansfer for purposes of outcrossing (Schoper, personal communication,1999). Unde r conditions of high temperat ure or low humidity, corn pollen may only survive for a matter of minutes. U nder more favorable conditions in the field or with controlled handling in the laboratory, pollen life may be exte nded to several hours.

c. Tripsacum species - Gama Gr ass - General Biology

Close relatives of corn or maize are found in the genus Tripsacum. Sixteen species of Tripsacum are known worldwide and generally recognized by taxonomists and ag rostologists; most of the

16 different Tripsacum species recognized are native to Me xico, Central and South America, but

three occur within the U.S. In the Manual of Grasses of the United States, A. S. Hitchcock (revisions by Agnes Chase; 1971) reports the presence of three species of Tripsacum in the

continental United States: T. dactyloides

, T. floridanum and T. lanceolatum . Of these, T. dactyloides , Eastern Gama Grass, is the only specie s of widespread occurrence and of anyCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

96 agricultural importance. It is commonly grown as a forage grass and has been the subject of some agronomic improvement ( i.e ., selection and classical breeding). T. floridanum is known

from southern Florida and T. lanceolatum is present in the Mule Mountains of Arizona and possibly southern New Mexico.

For the species occurring in the United States, T. floridanum has a diploid chromosome number

of 2n = 36 and is native to Southern Florida; T. dactyloides includes 2n = 36 forms which are native to the central and western U.S., a nd 2n = 72 forms which extend along the Eastern seaboard and along the Gulf Coast from Florida to Texas, but which have also been found in IL and KS; these latter forms may represent te traploids (x = 9 or 18; Lambert, personal communication, 1999); and T. lanceolatum (2n = 72) which occurs in the Southwestern U.S. Tripsacum differs from corn in many respec ts, including chromosome number ( T. dactyloides n = 18; Zea mays n = 10). Many species of Tripsacum can cross with Zea , or at least some accessions of each species can cross, but only with difficulty and the resulting hybrids are primarily male and female sterile (Duvick, personal communicati on, 1999; Galinat, 1988; Wilkes, 1967). Tripsacum /maize hybrids have not been obser ved in the field, but have been accomplished in the laboratory using special techniques under highly controlled conditions.

Eastern Gama Grass is considered by some to be an ancestor of Zea mays or cultivated maize (Mangelsdorf, 1947), while others dispute this (Galinat, 1983; Il tis, 1983; Beadle 1980), based largely on the disparity in chromosome number between the two species (maize n = 10; Gama Grass x = 9 or 18, with diploid, triploid and tetraploid races ex isting; 2n = 36 or 72), as well as radically different phenotypic appearance. Albe it with some difficulty, hybrids between the two species have been made (Mangelsdorf and Reeves, 1939; Chet DeWald, personal communication;1999). In most cases these progeny have been sterile or viable only by culturing

with in vitro ‘embryo rescue’ techniques.

Even though some Tripsacum species occur in areas where maize is cultivated, gene introgression from mai ze under natural conditions is highly unlikely, if not impossible (Beadle, 1980). Hybrids of Tripsacum species with Zea mays are difficult to obtain outside of the

controlled conditions of laborator y and greenhouse. Seed obtained from such crosses are often sterile or progeny have greatly reduced fe rtility. Approximately 10 - 20% of maize- Tripsacum hybrids will set seed when backcrossed to maize, and none are able to withstand even the mildest winters. The only known case of a naturally occurring Zea
- Tripsacum hybrid is a species

native to Guatemala known as Tripsacum andersonii . It is 100% male and nearly 99% female sterile and is thought to have ar isen from gene flow to teosinte , but the lineage is uncertain (Doebley, personal communication, 2000). Zea mays is not known to harbor properties that indicate it has weedy potential and, other than occasional volunteer plants in the previous season’s corn field, maize is not c onsidered as a weed in the U.S.

In a telephone conversation w ith Dr. Chester ‘Chet’ DeWald, USDA-ARS, Woodward, OK, a geneticist working on improvement of grasses, he stated that relatively few accessions of T. dactyloides will cross with maize and the majority of progeny are not fertile or viable even in

those that do. In controlled cr osses, if the female parent is ma ize, there is a gr eater likelihood of obtaining viable seed. When th ese hybrids have been backcrosse d to maize in attempts toCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

97 introgress Tripsacum genes for quality enhancement or disease resistance, the Tripsacum chromosomes are typically lost in successi ve generations. In many instances where hybridization has been directed between these two speci es, the resultant genome is lacking in most or all of the chromosomal complements of one of the parent species in subsequent generations.

Only recently has Dr. DeWald (or anyone else) succeeded in obtaining a true Tripsacum cytoplasm with a maize nuclear background. This was done by using gama grass as the female parent and maize as the male or pollen donor. Numerous accessi ons were tested and crosses

made before this came to fruition. The Tripsacum derived mitochondrial chondrome and chloroplast plastome in these hybrid s contribute to the seed qualitie s of the plants, but the nuclear genome appears to be totally maize in origin (DeWald et al ., 1999).

Dr. DeWald concluded that the possibility of maize contributing genetic material to Eastern Gama Grass through random pollen flow in agricultu ral or natural situations is extremely remote based upon his experience trying to create hybrids unde r the best of conditions. He also felt that no other known grass species present in the con tinental U.S. would interbreed with commercial maize populations ( i.e. , be recipients of pollen- directed gene flow). This is in agreement with

Holm et al . (1979) who determined that none of the se xually compatible relatives of corn in the U.S. are considered to be serious, pr incipal, or common weeds in the U.S.

d. Zea species - Teosintes - General Biology

Teosintes, specifically Z. mays ssp. mexicana (Schrader) Iltis, Z. mays ssp. parviglumis Iltis and Doebley , Z. mays ssp. huehuetenangensis (Iltis and Doebley) Doebley , Z. luxurians (Durieu and Ascherson) Bird , Z. perennis (Hitchc.) Reeves and Mangelsdorf and Z. diploperennis Iltis, Doebley and Guzman

, have co-existed and co-evolved in close proximity to maize in the Americas over thousands of years; however, maize and teosinte maintain distinct genetic constitutions despite sporadic introgression (Doebley, 1990).

The teosintes retain a reduced cob-like fruit / in florescence that shatters more than cultivated maize, but still restricts the movement of seeds as compared to more widely dispersed weedy species. Hence, the dispersal of large numbers of seeds, as is typi cal of weeds, is not

characteristic of teosintes or maize. In their na tive habitat, some teosin tes have been observed to be spread by animals feeding on the plants. Te osintes and teosinte-maize hybrids do not survive even mild winters and could not propagate in the U.S. corn belt. Additionally, some types have strict day length requirements that pr eclude flowering within a normal season ( i.e ., they would be induced to flower in November or December) and, hence, seed production under our temperate climate (Beadle, 1980; Iltis, pe rsonal communication; 2000; Wilk es, personal communication; 2000; Wilkes, 1967).

Since both teosinte and Tripsacum are included in botanical garden s in the U.S., the possibility exists (although unlikely) that exchange of genes could occur between corn and its wild relatives. EPA is not aware, however, of any such case being reported in the United States. Gene exchange between cultivated corn and transfor med corn would be similar to what naturallyCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

98 occurs at the present time within cultivated corn hybrids and landr aces. Plant architecture and reproductive capacity of the intercrossed plants will be similar to normal corn, and the chance that a weedy type of corn will result from gene flow with cultivated corn is extremely remote.

Like corn, Zea mays ssp. mexicana (annual teosinte) and Zea diploperennis (diploid perennial teosinte) have 10 pairs of chromosomes, are wind pollinated, and tend to outcross, but are highly variable species that are often genetically compatib le and interfertile with corn, especially when maize acts as the female parent. Zea perennis (perennial teosinte) ha s 20 pairs of chromosomes and forms less stable hybrids with maize (Edwards et al ., 1996; Magoja and Pischedda, 1994). Corn and compatible species of teosinte are capab le of hybridization when in proximity to each other. In Mexico and Guatemala, teosintes ex ist as weeds around the margins of corn fields.

The F 1 hybrids have been found to vary in their fert ility and vigor. Those that are fertile are capable of backcrossing to corn. A few isolat ed populations of annual and perennial teosinte were said to exist in Florida and Texas, respectively (USDA-APHIS, 1997). The Florida populations were presumably an escape from previous use of Z. mays ssp. mexicana as a forage grass, but local botanists have not documented any natural po pulations of this species for approximately twenty-five y ears (Keith Bradley, personal communication, 2000; David Hall, personal communication, 2000; Richard W underlin, personal communication, 2000).

Consultation with botanists and ag ronomists familiar with Texas flora suggested that no teosinte populations exist in the state (Benz, personal communication, 2000; Read, personal communication, 2000; Orzell, personal comm unication, 2000; Wilson, personal communication, 2000). Further, given the day length characteristics of Z. diploperennis , it is highly unlikely a sustaining population would result fr om introduction of this species. Z. mays ssp. mexicana, Z.

mays ssp. parviglumis, Z. luxurians and Z. diploperennis may cross with maize to produce fertile hybrids in many instances (Wilkes, 1967). None of these teosinte species have, however, been shown to be aggressive weeds in their na tive or introduced habita ts (John Schoper, personal communication, 1999). Except for special plantings as noted above, teosinte is not present in the

U.S. or its territories. Its natural distributi on is limited to Mexico, Honduras, Nicaragua, El

Salvador and Guatemala.

Given the cultural and biological re lationships of various teosinte species and cultivated maize over the previous two millennia, it would appear that significant gene exchange has occurred (based upon morphological characters) between thes e two groups of plants and that no weedy types have successfully evolved as a result. Mo re recent cytogenetic, biochemical and molecular analyses have indicated that th e degree of gene exchange is far less than previously thought (Doebley, 1984; Doebley et al ., 1987; Kato, 1997a, 1997b; Smith et al ., 1985). Partial and complete gametophytic incompatibility has been documented among cultivated maize, landraces and teosinte (Kermicle, 1997). The former is demonstrated by different ial pollen growth and a skewed recovery of alleles linked to incomp atibility genes. Complete incompatibility mechanisms serve to isolate a species or subspe cies and are evidenced as pollen exclusion or non-functioning of pollen types on certain genotypes. Attempts to cross six collections of Zea

mays ssp. mexicana with U.S. maize cultivar s (W22, W23) yielded no or fe w seeds in five of the six groups (Kermicle and Allen, 1990).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

99 Based on the ability of maize to hybridize with some teosintes, the suggestion of previous genetic exchange amongst these species over cen turies, and their genera l growth habits, any introgression of genes into wild teosinte from Zea mays is not considered to be a significant agricultural or environmental risk. The growth habits of teosintes are such that the potential for serious weedy propagation and development is not biologically plausible in the United States.

e. Conclusions

The potential for pollen-directed gene flow fr om maize to Eastern Gama Grass is extremely remote. This is evidenced by the difficulty with which Tripsacum dactyloides x Zea mays hybrids are produced in structured breeding programs. Additionally, the genus does not represent any species considered as serious or pernicious weeds in the United States or its territories. Any introgre ssion of genes into this species as a result of cross fe rtilization with genetically-modified maize is not expected to re sult in a species that is weedy or difficult to control. In many instances wher e hybridization has been directed between these two species, the resultant genome is lacking in most or all of the maize chromosomal complement in subsequent generations.

Many of the Zea species loosely referred to as “teosint es” will produce viable offspring when

crossed with Zea mays ssp. mays . None of these plants are known to harbor weedy characteristics and none of the native teosinte spec ies, subspecies or races are considered to be aggressive weeds in their native or introduced habitats. In fact, many are on the brink of extinction where they are indigenous and w ill be lost without human intervention ( i.e

., conservation measures). Further, none of the landraces or cultivated lines of Zea mays are considered to have weedy potential and are genera lly considered to be incapable of survival in the wild as a result of breeding practices ( i.e ., selection) during dome stication of the crop.

6. Impacts on Threatened or Endangered Species

Toxicity data show that the only endangered species of any potential concern are in the Lepidoptera and Coleoptera group. Th e majority of endangered species in these Orders has very restricted habitat range and do not feed on, or approach the Bt crop planting areas close enough to be exposed to toxic levels of Bt pollen. Examination of an overlay map showing the county level distribution of endangered le pidopteran species relative to co rn production counties in the US shows that any potential concer n regarding range overl ap with corn production is restricted to the Karner blue butterfly. However, the Karner blue host plant, the wild lupine, does not occur in corn fields. Therefore it appears highly unl ikely that significant numbers of lupine would occur within a few (two) meters of corn field e dge, where the toxic levels of corn pollen may be present. Even using the conser vative assumption that Karner blue larvae are relatively sensitive

to all Bt proteins in all Bt corn events, the likelihood that the larvae would encounter sufficient grains of Bt corn pollen to exert toxicity is extr emely remote. Also, relevant data and

information indicate that the likelihood of wild l upines occurring adjacent to corn fields is low. Moreover, the overlap of the time of the year when corn pollen is shed with the times of the year

when Karner blue larvae are li kely to be present is limited.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

100 An examination of the endangered bird and bat sp ecies shows that their habitats are mostly nonagricultural. Of those that do encroach on agricultural fields , none would rely on cotton or potato pests as a primary food source. Corn is no t an issue, because the ECB is within the corn

stalk and is not available for bi rd predation. Bats do not prey on larvae. They rely on flying insects. Taking all of these and other pertinent issues into consideration, it becomes apparent that reduction in the target pe sts of cotton and potatoes would not have an effect on the food source of endangered birds and bats. In the rare instances where these species may feed on the target pests, the reduction in the pest species will merely cause them to rely on other plentiful insects as a source of food. Submitted and publishe d field data reviewed in this document show that a wide variety of insects is abundant in Bt crops as opposed to non- Bt crop fields when conventional insect pest cont rol practices are used. Ther efore the data show that Bt crops should actually be beneficial to bird and bat populations.

2010 Update: Current ecological effects data and EPA reviews of Cry1Ab and Cry1F support the Agency’s determination that adverse effects will not occur to nontarget organisms. Due to a

demonstrated lack of toxici ty and/or exposure, no eff ects from Cry1Ab and Cry1F are anticipated for any nontarget spec ies, including fede rally-listed threaten ed and endangered (“listed”) lepidopteran and coleopt eran species and their designate d critical habitats. EPA has

also determined that there ar e no indirect effects on endangere d and threatened plant species, such as impacts on lepidopteran pollinators that are important a nd/or essential to an endangered or threatened plant. The Agency is therefore uph olding its determination th at the registered uses of Cry1Ab and Cry1F will have “No Effect,” dire ct or indirect, on endangered or threatened terrestrial or aquatic species as listed by the U.S. Fish and Wildlife Service (USFWS) and the

National Marine Fisher ies Services (NMFS).

7. Potential Interaction Between Cry1Ab and Cry1F Proteins

(MRID 47677802)
A seven-day laboratory sensitive insect bioassay was conducted to determine if the combination PIP product 1507 x 59122 x MON810 has a synergisti c effect in comparison to the individual parental events TC1507 (expressing Cry1F prot ein) and MON810 (expressing Cry1Ab protein) on target lepidopteran pests. The pests used in the bioassay were European corn borer (ECB,

Ostrinia nubilalis ), southwestern corn borer (SWCB, Diatraea grandiosella ), fall armyworm (FAW, Spodoptera furgiperda ), and corn earworm (CEW, Helicoverpa zea ). The observed and expected larval mortality in the 1507 x 59122 x MON810 group were similar and mean larval weight of the survivors exposed to 1507 x 59122 x MON810 leaf tissue was not significantly different from the single parental ev ents or the negative control of non- Bt maize. These results indicate that the Cry1F and Cry1A b proteins act independently a nd do not have a synergistic or antagonistic effect on the target pests, other than an additive effect. Quantitative ELISA results also confirmed that the expression of each of the proteins in the combination PIP was not affected by the presence of the other protei n. This study was classified acceptable.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

101 8. Ecological Risk Assessment Conclusions

a. Direct Effects In general, the reviewed publications, recent research data, and information submitted as a result of the data call in (DCI) provide a weight of evidence assessment indicating no unreasonable

adverse effects of Bt Cry proteins expressed in plants to non-targetwildlife or beneficial invertebrates, whether they are earthworms, springt ails, parasites, predator s, pollinators or soil

microbial and invertebrate flora. Published fiel d testing results and field test data submitted to

EPA show minimal to undetectable to beneficial changes in the non-target insect populations. EPA is, however, continuing to participate in re search and review the pertinent scientific literature for the purpose of reevaluating the Agency's Ecological Risk Assessment of the Bt crop registrations in the event that unexpected long range population, community or ecosystem effects

are detected.

EPA believes that cultivation of transgenic plants expressing Bt Cry proteins may result in fewer adverse impacts to non-target organisms than resu lt from the use of chemical pesticides. Under normal circumstances, Bt crops require substantially fewer app lications of chemical pesticides. This should result in fewer adverse impacts to no n-target organisms. Many of these beneficial organisms are important integrated pest manageme nt controls (IPM) for secondary pests such as aphids and leafhoppers. The overall result of cultivation of plants expressing Bt Cry proteins is

that the number of chemical ins ecticide applications for non-target pest control is reduced for crops with multiple pest problems.

b. Indirect Effects The purpose of using PIP plants is the same as for any other pest management tactic, i.e., to reduce pest populations below economi c injury levels. As a result, the abundance of pest insects should be significantly reduced and this will have corresponding implications for those organisms that exploit these pests as prey and hosts. Thus, the potenti al for these indirect ecological effects on biological c ontrol organisms should not be re garded as a unique ecological risk associated with the PIP crop. Some reducti ons, however, should be expected if the pest management strategy is effective. Since PIP crops are often grown in vici nity with conventional crops to prevent resistance build- up by the target pest(s), speciali st antagonists can persist in these ‘refuges’, in other crops and in non-cr op habitats and retain the potential for recolonization of the PIP crop area. Based on thes e considerations, regulatory testing of the specialist predators and parasitoids of target pests may ev entually be considered unnecessary.

c. Supplemental Data Needed

EPA has sufficient information to believe that there is no risk from the registered uses Bt 11, MON810, TC1507 or TC6275 corn to non-target w ildlife, aquatic and soil organisms and domesticated fowl and animals. The Agency ha s been frequently asking the registrants to conduct post-registration long term inverteb rate population/commun ity and Cry protein

accumulation in soils studies as a condition of re gistration. The issue of long range effects of cultivation of these Cry proteins on the invertebra te community structure in corn fields has since been adequately addressed by the analysis of fi eld studies performed during the last 10 years (Marvier, et al. 2007; Sanvido, et al. 2007). No unexpected advers e effects on invertebrateCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

102 community structure were reported. The Agen cy is in agreement with these conclusions. Similarly, no unexpected accumulation of Cry protei ns in agricultural soils was seen in published studies (Icoz and Stotzky 2007; Sanvido, et al. 2007) and in numerous studies submitted directly to the EPA for the currently regist ered Cry proteins. (Milofsky, 2006).

In light of recently published laboratory studies showing redu ced growth in shredding caddis flies exposed to anti-lepidopteran Cry1A protein corn litter (Rosi-Marshall, et al. 2007), additional aquatic invertebrate data are required. The submitted Daphnia magna study is unacceptable because it is an 850 Series OSCPP Guideline study. The 48-hour duration of this study is not sufficient to detect mortality due to Bt proteins. It takes more than 48 hours for the target pests to succumb to the Cry proteins, th erefore 48 hours is also not expected to show mortality or reproductive effects on Daphnia. A 7 to 14 day Daphnia study as per the 885 Series OCSPP Guidelines needs to be performed. Th e study may be submitted as a condition of registration. Alternatively, a diet ary study of the effects on an a quatic invertebrate, representing the functional group of a leaf shredder in headwa ter streams, can be performed and submitted in

lieu of the Daphnia study.

TABLE 11. Supplemental non -target data requirements for Bt11, MON810, TC1507 an d TC6275 corn

Testing Category Type of Data

Aquatic

invertebrate A 7 to14 day Daphnia study as per the 885 Series OPPTS Guidelines has to be submitted as a condition of registration. Alternatively, a dietary study of the effects on an aquatic invertebrate, representing the functional group of a leaf shredder in headwater streams, can be performed and submitted in lieu of the Daphnia study.

9. REFERENCES

Submitted Studies by MRID Number

42932202 Rogan, G.; Andersen, J.; McCreary, J.; et al . (1993) Determination of the Expression Levels of B.t.t. and NPT II Proteins in Potato Tissues Derived from Field Grown plant: Lab Proj ect Number: 92-01-37-02: 93:081E: 12735. Unpublished study prepared by Monsanto Co. 349 p.

42932203 Bartnicki, D.; Lavrik, P.; Leimgruber, R.; et al . (1993) Equivalence of Microbially-Produced and Plant-Produced B.t.t. Protein also Called

Colorado Potato Beetle Active Protei n from Bacillus thuringiensis subsp. tenebrionis: Lab Project Nu mber: 92-01-37-07: 93-081E: 12897. Unpublished study prepared by Monsanto Co. 95 p.

42932209 Maggi, V. (1993) Evaluation of the Di etary Effect(s) of Purified B.t.t. Protein on Honey Bee Larvae: La b Project Number: CAR 188-92: 92-01- 37-03: 92-448-702. Unpublished study prep ared by California AgriculturalCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

103 Research, Inc. 52 p.

42932210 Maggi, V. (1993) Evaluation of the Di etary Effect(s) of Purified B.t.t. Protein on Honey bee Adults: La b Project Number: CAR 189-92: 92-01-37- 03: 92-448-701. Unpublished study prep ared by California Agricultural Research, Inc. 65 p.

42932211 Hoxter, K.; Smith, G. (1993) B.t.t. Protein: A Dietary Toxicity Study with Parasitic Hymenoptera (Nasonia vi tripennis): Lab Project Number: 139- 349B: WL-92-436: 93-081E. Unpublished study prepared by Wildlife International Ltd. 51 p.

42932212 Hoxter, K.; Smith, G. (1993) B.t.t. Protein: A Dietary to xicity Study with Ladybird Beetles (Hippodamia conv ergens): Lab Project Number: 139-348: WL-92-435: 93-081E. Unpublished study prepared by Wildlife International Ltd. 47 p.

42932214 Cambell, S.; Beavers, J.; Jaber, M. (1993) A Dietary Toxicity Study with Russet Burbank Potatoes in the Northern Bobwhite: Lab Project Number: 139-356: WL-93-11: 93-081E. Unpublishe d study prepared by Wildlife International Ltd. 87 p.

42932215 Campbell, S.; Beavers, J.; Jaber, M. (1993) A Dietary Toxicity Study with Russet Burbank Potatoes in the Northern Bobwhite: Lab Project Number: 139-357: WL-93-12. Unpublished study prepared by Wildlife International Ltd. 50 p.

43145205 Sims, S. (1994) Stability of the CryI A(c) Insecticidal Protein of Bacillus thuringiensis var. kurstaki (B.t.k. HD -73) in Sucrose and Honey Solutions Under Non-refrigerated Temperature Conditions: Lab Project Number: 92/01/36/15: 92/427/718: 13288. Unpublished study prepared by Monsanto Agricultural Group. 27 p.

43145206 Maggi, V. (1993) Evaluation of the Di etary Effect(s) of Purified B.t.k. Endotoxin Proteins on Honey B ee Larvae: Lab Project Number:

CAR/180/92: 92/01/36/10: 92/427/709. U npublished study prepared by California Agricultural Research, Inc. 76 p.

43145207 Maggi, V. (1993) Evaluation of the Di etary Effect(s) of Purified B.t.k. Endotoxin Proteins on Honey Bee A dults: Lab Project Number: 181/92: 92/01/36/10: 92/427/708. Unpublished study prepared by California Agricultural Research, Inc. 102 p.

43145208 Palmer, S.; Beavers, J. (1993) B.t.k. HD-73 Protein: A Dietary Toxicity Study with Parasitic Hymenoptera (N asonia vitripennis): Lab ProjectCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

104 Number: 139/369: 93/234: WL/93/234. U npublished study prepared by Wildlife International Ltd. 108 p.

43145209 Palmer, S.; Beavers, J. (1993) B.t.k. HD-73 Protein: A Dietary Toxicity Study with Ladybird Beetles (Hippodamia convergens): Lab Project

Number: 139/370: WL/93/232: 92/01/36/ 25. Unpublished study prepared by Wildlife International Ltd. 104 p.

43145210 Palmer, S.; Beavers, J. (1993) B.t.k. HD-73 Protein: A Dietary Toxicity Study with Green Lacewing Larvae (Chrysopa carnea): Lab Project

Number: 139/371: WL/93/233: 92/01/36/ 26. Unpublished study prepared by Wildlife International Ltd. 100 p.

43145211 Cambell, S.; Beavers, J. (1993) A Diet ary Toxicity Study with Cotton Seed

Meal in the Northern Bobwhite: La b Project Number: 139/358: WL/93/13. Unpublished study prepared by Wildlife International Ltd. 44 p.

43323610 Collins, M. (1994) Bt Maize Pollen (PHO176-0194): Acute Toxicity to Daphnids (Daphnia magna) under Static -Renewal Conditions: Lab Project

Number: 94/3/5217: 1781/0394/6419/110. U npublished study prepared by Springborn Laboratories, Inc. 59 p.

43397201 Hanten, J.; Meeusen, R. (1994) Determ ination of Levels of Plant-Produced Bacillus thuringiensis kurstaki HD-1 Protein in Transgenic Maize: Lab Project Number: 1/NK5LVL. Unpublished study prepared by Northrup King Co. 8 p.

43439202 Maggi, V.; Sims, S. (1994) Evaluation of the Dietary Effects of Purified B.t.k. Endotoxin Proteins on Honey Bee Larvae: Lab Project Number: IRC-91-ANA-13. Unpublished study prepared by Monsanto Co. 51 p.

43439203 Maggi, V.; Sims, S. (1994) Evaluation of the Dietary Effects of Purified B.t.k. Endotoxin Proteins on Honey Bee Adults: Lab Project Number: IRC-91-ANA-12. Unpublished study prepared by Monsanto Co. 70 p.

43468002 Sims, S. (1994) Stability of the CryI A(b) Insecticidal Protein of Bacillus thuringiensis var. kurstaki (B.t.k. HD -1) in Sucrose and Honey Solutions Under Non-refrigerated Temperature Conditions: Lab Project Numbers: IRC-91-ANA-11: MSL 13375: 13375. Unpublished study prepared by Monsanto Co. 32 p.

43468003 Hoxter, K.; Lynn, S. (1992) Activated Btk HD-1 Protein: A Dietary Toxicity Study With Green Lacewing Larvae : Lab Project Numbers: WL-92-155: 139-3388. Unpublished study prepared by Monsanto Co.; and Wildlife Int'l, Ltd. 21 p.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

105 43468004 Hoxter, K.; Lynn, S. (1992) Activated Btk HD-1 Protein: A Dietary Toxicity Study With Parasitic Hymenoptera (Brachymeria intermedia): Lab Project Numbers: WL-92-157: 139-320. Unpublished study prepared by Monsanto Co.; and Wildlife Int'l, Ltd. 24 p.

43468005 Hoxter, K.; Lynn, S. (1992) Activated Btk HD-1 Protein: A Dietary Toxicity Study With Ladybird Beetles: Lab Project Numbers: WL-92-156: 139-321. Unpublished study prepared by Monsanto Co.; and Wildlife Int'l, Ltd. 23

p.

43533205 Campbell, S.; Beavers, J. (1994) A Di etary Toxicity Study with MON 80187

Meal in the Northern Bobwhite: La b Project Number: WL94-150: 139-387. Unpublished study prepared by Wildlife International Ltd. 32 p.

43665501 Levine, E.; Groth, M.; Kania, J.; et al . (1995) Molecular Ch aracterization of Insect Protected Corn Line MON 810: Lab Project Number: MSL 14204. Unpublished study prepared by Monsanto Co. 61 p.

43665502 Sanders, P.; Elswick, E.; Groth, M.; et al . (1995) Evaluation of Insect

Protected Corn Lines in 1994 U. S. Field Test Locati ons: Lab Project Number: 94-01-39-01: 14065: 14179. Unpublished study prepared by Monsanto Co. 147 p.

43696001 Williams, D. (1995) Environmenta l Fate: Bacillus thuringiensis var.

kurstaki Protein in Corn: Lab Pr oject Number: NK5EF. Unpublished study prepared by Northrup King Co. 22 p.

43887901 Jackson, L.; Robinson, E.; Nida, D. et al . (1995) Evaluation of the European Corn Borer Resistant Corn Line MON 801 as a Feed Ingredient for Catfish: Lab Project Number: 94-01-39-16: 14066: 95-459-720. Unpublished study prepared by Mississi ppi State University Delta Research and Extension Center. 39 p.

43887902 Palmer, S.; Beavers, J. (1995) CRYIA(b) Insecticidal Protein: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate: Final Report: Lab Project Number: 139-417: WL-95-281. Unpublished study prepared by Wildlife International Ltd. 25 p.

43941601 Sims, S.; Martin, J. (1996) Effect of the Bacillus thuringiensis Insecticidal Proteins CryIA(b), CryIA(c), CryIIA , and CryIIIA on Folsomia candida and Xenylla grisea (Insecta: Collembola): Lab Project Number: 93-081E1. Unpublished study prepared by Monsanto Co. 22 p.

43999509 Stephens, M.A. (1996) Environmenta l Fate of CryIA(c) Protein: LabCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

106 Project Number DGC-96-A24. Unpublished study prepared by Monsanto Co. (formerly DEKALB Genetics Co.).

44124701 Palmer, S.; Beavers, J. (1996) Bacillus thuringiensis subsp. tenebrionis (B.t.t.) Protein: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate: Lab Proj ect Number: 139-416: WL-96-151: 96-01- 37-01. Unpublished study prepared by Wildlife International Ltd. 54 p.

44124702 Maggi, V. (1996) Evaluation of the Di etary Effect(s) of Purified B.t.t. Protein on Honey Bee Larvae: La b Project Number: CAR 111-96: XX-96- 088: 96-01-37-01. Unpublished study prepared by California Ag Research Inc. 69 p.

44161701 Halliday, W. (1996) Environmental Fate of Cry9C Protein Incorporated into Soil: Lab Project Number: 6835: 6835-96-0095-BE-002: 6835-96-0095- BE. Unpublished study prepared by Ricerca, Inc. 116 p.

44258103 MacIntosh, S. (1997) Characterization of Cry9C and PAT Protein Levels in

CBH-351 BT Corn Under Field Conditions: (Final Report): Lab Project Number: 96QZM002. Unpublished study prepared by Plant Genetic Systems N.V. 106 p.

44258106 Halliday, W. (1997) Insect Host Range Comparison of Cry9C Protein: Lab Project Number: 7094-96-0290- AC-001: 7094: 7094-96-0290-AC. Unpublished study prepared by Ricerca, Inc. 85 p.

44258110 Halliday, W. (1996) Chronic Exposure of Folsomia candida to Corn Tissue or Bacteria Expressing Cry9C Prot ein: (Final Report): Lab Project

Number: 6913-96-0158-AC-001: 6913: 6913-96-0158-AC-000. Unpublished study prepared by Ricerca, Inc. 65 p.

44258111 Palmer, S.; Beavers, J. (1997) Cry9C Protein in Corn Pollen: A Dietary Toxicity Study with the Ladybird B eetle (Hippodamia convergens): (Final Report): Lab Project Number: 452-101: 452/102496/LBLC/SUB452. Unpublished study prepared by Wildlife International Ltd. 36 p. {OPPTS 885-4350}.

44258112 Graves, W.; Swigert, J. (1997) Cry9C Protein in Corn Pollen: A 48-Hour Static-Renewal Acute Toxicity Test with the Cladoceran (Daphnia magna): Final Report: Lab Project Number: 452A-101. Unpublished study prepared by Wildlife International Ltd. 23 p.

44258113 Palmer, S.; Beavers, J. (1997) Cry9C Protein in Plant Powder: An Acute Toxicity Test with the Earthworm in an Artificial Soil Substrate: (Final Report): Lab Project Number: 452-103:Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

107 452/102496/EWSDT.WCA/SUB452. Unpublished study prepared by Wildlife International Ltd. 39 p.

44258114 Palmer, S.; Grimes, B.; Beavers, J. ( 1997) Corn Plant Powder Containing Cry9C Protein: A Dietary Toxicity Study with the Northern Bobwhite:(Final Report): Lab Project Number: 452-102: 452/102496/QLCLIM/SUB452. Unpublished study prepared by Wildlife International Ltd. 47 p.

44258115 MacIntosh, S. (1997) Effects of Cr y9C Corn on Predatory Non-Target Beneficial Insects and Endangered Species: Lab Project Number: 96QZM004. Unpublished study prepared by Plant Genetic Systems N.V. 48 p.

44271501 Halliday, W. (1997) Chronic Exposure of Folsomia candida to Corn Tissue Expressing CryIA(B) Protein: La b Project Number: 7140-97-0030-AC-001: XX-97-064: 95-152E2. Unpublished study prepared by Ricerca, Inc. 91 p.

44271502 Graves, W.; Swigert, J. (1997) Corn Pollen Containing the CryIA(b)

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44274201 Privalle, L. (1997) Comparison of CryIA(b) Levels in Transgenic BT 11- Derived Maize (Corn) Pollen and Event176-Derived Maize Pollen and Justification for Citation of Daphni a magna Toxicity Study of Event 176- Derived Pollen in Support of BT 11 Daphnia magna Data Requirement: Lab Project Number: NSB-001-97. Unpublished study prepared by Novartis Seeds, Inc. 11 p.

44384301 MacIntosh, S. (1997) Preparation and Characterization of Catfish Pellets: (Bacillus thuringiensis subsp. tolw orthi Cry9C Protein): Lab Project Number: PGS/96QZM005: 96QZM005: 97QZM002. Unpublished study prepared by Plant Genetic Systems (America) Inc. 14 p.

44384302 Palmer, S.; Krueger, H. (1997) Cry9 C Protein in Corn Pollen: A Dietary Toxicity Study with the Honey Bee (Apis mellifera): Lab Project Number: 452-104: 452/060997/BLCP/SUB452. Unpublished study prepared by Wildlife International Ltd. 38 p. {OPPTS885.4380}

45512200 McKee, M., G. Head and D. Vlatchos. 2001. Potential Risk to Endangered Lepidoptera from YieldGuard R Corn Pollen (Bt Corn Events MON810 and Bt11) with Emphasis on the Karner Blue Butterfly. Monsanto Co. and Syngenta Seeds. Submission ID 95-040E.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

108 45512901 Davis, P and L Higgins. 2001. Exposu re and risk assessment of Herculex I

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45611801 Taylor, M.; Hartnell, G.; Riordan, S. et al . (2001) Comparison of Broiler

Performance When Fed Diets Contai ning YieldGard Corn, YieldGard x Roundup Ready, Parental Line or Comm ercial Corn Hybrids: Lab Project Number: MSL-17198. Unpublished study prepared by Monsanto Co. 179 p.

45622001 Zeph, L. (2000) Nutritional Equivalen cy of B.t. Cry1F Maize--Poultry Feeding Study: Lab Project Numb er: PHI99-010: 98-PIO-25A-BB. Unpublished study prepared by PARC Institute, Inc. 24 p.

45652001 Abramson, S. (2002) Field Surveys of Non-Target Invertebrate Populations in Bt Corn. Unpublished study prepa red by Agricultural Biotechnology Stewardship Technical Committee. 106 p.

45652101 Guyer, D. (2002) Evaluation of Transgenic Corn Event Bt11 in Broiler Chicken. Unpublished study prepared by Syngenta Seeds, Inc. 25 p.

46022401 Brown, C.; Ayden, B.; Bader, B.; et . al. (2003) Assessment of the Potential

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46162001 Dively, G.; Rose, R.; Sears, M.; et. al. (2003) Effects on Monarch Butterfly Larvae After Continuous Exposure to Cry1Ab-Expressing Corn Pollen During Anthesis. Unpublished study prepared by University of Maryland, University of Guelph, and Pennsyl vania State University. 18 p.

46784601 Abramson, S. (2006) Field Surveys of Non-Target Invertebrate Populations in Bt Corn: Supplement to MRID No. 45652001. Unpublished study prepared by Agricultural Biotechno logy Stewardship Technical Committee. 239 p.

47120701 McCormick, R.; Embrey, S. (2006) So il Accumulation of Cry 1 F Protein after Three Years of Cropping with Herculex I Corn. Project Number: 020020. Unpublished study prepared by Dow Agrosciences LLC. 50 p.

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Tschenn, J., J.E. Losey, L.H. Jesse, J.J. Obrycki, and R. Hufbauer. 2001. Effects of corn plants and corn pollen on monarch butterfly (Lepi doptera: Danaidae) ovi position behavior. Environ. Entomol. 30(3): 495-500.

Urquhart, F.A. 1960. The Monarch Butterfly. Un iversity of Toronto Pr ess, Toronto, Ontario, Canada. 361pp.

USDA-NASS. 1997. U.S. Department of Agricultur e, National Agricultura l Statistics Service, Census of Agriculture Volume 1: Part 51, Chapter 2. http://www.nass.usda.gov/census/ census97/volume1/us-51/toc297.htm

U.S. EPA. 1986. Hazard Evaluati on Division, Standard Evaluation Procedure, Ecological Risk

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USEPA, 2000. SAP report No 99-06, “Sets of Sc ientific Issues being considered by the Environmental Protection Agency regarding: section I - Characteriz ation and non target organism data requirements for protein plant pesticides”. Dated February 4, 2000. Available at the EPA website: http://www. epa.gov/scipoly/sap/1999/index.htm#december

USEPA, 2001. SAP report No 2000-07, “Sets of Scie ntific Issues being considered by the Environmental Protection Agency regarding: Bt Plant Pesticides Risk and Benefits Assessments”. Dated March 12, 2001. Available at the EPA website: http://www.epa.gov/scipoly/sap/200 0/october/octoberfinal.pdf

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D. Insect Resistance Management

1. Introduction

Insect resistance management (IRM) is the term used to describe practices aimed at reducing the potential for insect pests to beco me resistant to a pesticide. Bt IRM is of great importance

because of the threat insect resist ance poses to the future use of Bt plant-incorporated protectants

and Bt technology as a whole. Specific IRM strategi es, such as the high dose/structured refugeCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

126 strategy, will mitigate insect resistance to specific Bt proteins produced in corn. Academic scientists, public inte rest groups, organic and other farmer s have expressed concern that the widespread planting of these genetically transformed plants will hasten the development of resistance to pesticidal Bt endotoxins. Effective insect resistance management can reduce this risk of resistance development. This section provides EPA's scientific assessment of Bt corn plant-incorporated protectant IR M strategies by reviewing the data and information available to the Agency. The Agency has used this asse ssment, the report of the FIFRA SAP meeting on October 18, 2000, and all public comments in its de velopment of its risk management decisions

for Bt corn plant-incorporated protectants.

The following list will assist the re ader with the acronyms for the in sect pests discussed in this

section.

Acronym Common Name Scientific Name Crop

BCW Black Cutworm Agrotis ipsilon (Hufnagel) corn

CEW Corn Earworm Helicoverpa zea (Boddie) corn

CSB Common Stalk Borer Papaipema nebris (Guen.) corn

ECB European Corn Borer Ostrinia nubilalis (Huebner) corn

FAW Fall Armyworm Spodoptera frugiperda (J. E. Smith) corn

SCSB Southern Corn Stalk Borer Diatraea crambidoides (Grote) corn

SWCB Southwestern Corn Borer Diatraea grandiosella (Dyar) corn

a. Elements of IRM Plans

To address the real concern of insect resistance to Bt proteins, EPA has imposed IRM requirements on registered Bt plant-pesticides. Sound IRM will prolong the life of Bt pesticides and adherence to the plans is to the advantage of growers, pr oducers, researchers, and the American public. EPA cons iders the development of Bt- resistant insects to constitute an adverse environmental effect. EPA's strategy to address insect resistance to Bt is two-fold: 1) mitigate any significant potential for pest resistance development in the field by instituting IRM plans, and 2) better understand the mechan isms behind pest resistance.

Scientific experts believe that a high dose and the planting of a refuge (a portion of the total acreage using non- Bt seed) will delay the development of insect resistance to Bt crops by maintaining insect susceptibilit y. In addition to a high dose a nd structured refuge, IRM plans include additional field research on pest bi ology, refuge size and de ployment, resistance monitoring for the development of resistance (and increased insect tolerance of the protein), grower education, a remedial acti on plan in case resistance is identified, annual reporting and communication. IRM plans will ch ange as more scientific data become available.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

127 Beginning with the first Bt plant-pesticide registration, the Ag ency has taken steps to manage

insect resistance to Bt with IRM plans being an important pa rt of the regulatory decision. The Agency identified (later confirme d by the 1995 SAP) seven elements that should be addressed in

a Bt plant-incorporated protectant resistance management plan: 1) knowledge of pest biology and ecology; 2) appropriate dose expr ession strategy; 3) appropriate refuge; 4) resistance monitoring and a remedial action plan should resistance oc cur; 5) employment of integrated pest management (IPM); 6) communication and educati on strategies on use of the product; and 7) development of alternative modes of action. IRM plans also include grower education and measurement of the level of compliance. Because IRM plans change as more scientific data become available, EPA has also imposed research data requirements as part of the terms and conditions of registration. EPA has also made changes to IRM requirements as the science has

evolved.

b. High Dose/Structured Refuge Strategy

The 1998 Science Advisory Panel Subpanel agreed with EPA that an appropriate resistance management strategy is necessary to mitigat e the development of insect resistance to Bt proteins expressed in transgenic crop pl ants. The 1998 Subpanel recognize d that resistance management programs should be based on the use of both a high dose of Bt and structured refuges designed to provide sufficient numbers of susceptible adult in sects. The high dose/refuge strategy assumes

that resistance to Bt is recessive and is conferred by a si ngle locus with two alleles resulting in three genotypes: susceptible homozygotes (SS), heterozygotes (RS), and resistant homozygotes (RR). It also assumes that ther e will be a low initial resistance allele frequency and that there will be extensive random mating between resist ant and susceptible adults. Under ideal circumstances, only rare RR individuals will survive a high dose produced by the Bt crop. Both SS and RS individuals will be susceptible to the Bt toxin. A structur ed refuge is a non- Bt portion of a grower’s field or set of fiel ds that provides for the production of susceptible (SS) insects that may randomly mate with rare resi stant (RR) insects surviving the Bt crop to produce susceptible RS heterozygotes that will be killed by the Bt crop. This will remove resistant (R) alleles from the insect populations and dela y the evolution of resistance. The 1998 and 2000 SAP Subpanels

noted that insect resistance mana gement strategies should also be sustainable and to the extent possible, strongly consider grower accep tance and logistical feasibility.

Although the high dose/refuge strategy is the prefe rred strategy for IRM, e ffective IRM is still possible even if the transfor med plant does not express the Bt protein at a high dose for all economically-important target pe sts (e.g., by increasing refuge size). The lack of a high dose could allow partially resistant i ndividuals (i.e., heterozygous insect s with one resist ance allele) to survive, thus increasing the fre quency of resistance genes in an insect population. For this reason, numerous IRM researchers and expert groups have concurred that non-high dose Bt expression presents a substantial resistance ri sk relative to high dose expression (Roush 1994, Gould 1998, Onstad & Gould 1998, SAP 1998, ILSI 1998, UCS 1998, SAP 2001).

The 1998 SAP Subpanel defined (and the 2000 SA P Subpanel confirmed) a high dose as “25 times the protein concentration necessary to kill susceptible larvae.” The logic for this approach is spelled out in the 1998 SAP repor t as well as in the sc ientific literature on insect resistanceCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

128 management for Bt crops. In essence, Bt cultivars must produce a high enough toxin concentration to kill nearly all of the insects that are heterozygous for resistance. The Agency has adopted the 25X definition of high dose proposed by the 1998 SAP Subpanel.

The 1998 SAP Subpanel noted that a Bt plant-incorporated protecta nt could be considered to provide a high dose if verified by at least two of the following five approaches: 1) Serial dilution bioassay with artificial diet co ntaining lyophilized tissues of Bt plants using tissues from non- Bt plants as controls; 2) Bioassays using plant lines w ith expression levels approximately 25-fold

lower than the commercial cultivar determined by quantitative ELISA or some more reliable technique; 3) Survey large numbe rs of commercial plants in th e field to make sure that the

cultivar is at the LD 99.9 or higher to assure th at 95% of heterozygotes would be killed (see Andow & Hutchison 1998); 4) Similar to #3 abov e, but would use contro lled infestation with a laboratory strain of the pest that had an LD 50 value similar to field strains; and 5) Determine if a later larval instar of the target ed pest could be found with an LD 50 that was about 25-fold higher

than that of the neonate larvae. If s o, the later stage could be tested on the Bt crop plants to determine if 95% or more of the later stage la rvae were killed. The 2000 SAP concluded that the

Bt corn PIPs registered at that time had Bt titers that would significantly exceed the 25X criteria for control European corn borer.

As an alternate definition for high dose, Caprio et al. (2000) recommend that a higher, 50-fold value be adopted (rather than 25-fold) because cu rrent empirical data suggest that a 25-fold dose may not be consistently high enough to cause high mortality among he terozygotes with known

Bt resistance alleles. The 2000 SAP Subpanel did not recommend changing the existing 25-fold definition, but noted that the “25X” definiti on is imprecise, provisional, and may require modification as more knowledge becomes availabl e about the inheritance of resistance. The Subpanel concluded that current Bt corn varieties have less than a 25-fold dose for CEW.

The size, placement, and management of the ref uge is critical to th e success of the high dose/structured refuge strategy to mitigate insect resistance to the Bt proteins produced in corn. The 1998 Subpanel defined structured re fuges to "include all suitable non- Bt host plants for a targeted pest that are planted and managed by pe ople. These refuges could be planted to offer refuges at the same time when the Bt crops are available to the pests or at times when the Bt crops are not available." Th e 1998 Subpanel suggested that a production of 500 susceptible adults in the refuge for every adult in the tr ansgenic crop area (assuming a resistance allele frequency of 5 x 10 -2 ) would be a suitable goal. The placem ent and size of the structured refuge employed should be based on the current unders tanding of the pest bi ology data and the technology. The 1998 SAP Subpanel also recognized that refuges should be based on regional pest control issues. The 2000 SAP Subpanel ech oed the 1998 SAP’s recommendations that the refuge should produce 500:1 susceptible to resi stant insects and that regional IRM working groups would be helpful in developing policies.

c. Predictive Models

EPA has used predictive models to compare IRM strategies for Bt crops. Because models cannot

be validated without actu al field resistance, models have li mitations and the information gainedCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

129 from the use of models is only a part of the weight of evidence used by EPA in assessing the risks of resistance development. It was the consensus of the 2000 SAP Subpanel that models were an important tool in determining appropriate Bt crop IRM strategies. They agreed that models were “the only sc ientifically rigorous way to integrate all of the biological information available, and that without these models, the Ag ency would have little scientific basis for choosing among alternative resistance management options.” They also recommended that models must have an agreed upon time fram e for resistance protec tion. For example, conventional growers may desire a maximum pl anning horizon of five years, while organic growers may desire an indefinite planning hor izon. The Subpanel recommended that model design should be peer reviewed and parameters validated. Models should also include such

factors as level of Bt crop adoption, level of compliance, eco nomics, fitness costs of resistance, alternate hosts, spat ial components, stochasticity, and pest population dynamics.

EPA’s Office of Research and Development (O RD), National Risk Management Research Laboratory and Office of Pesticide Programs he ld a small expert group workshop in June, 2001 that focused on model design and parameter validation for Bt corn IRM. This workshop was the first in a series of public workshops intended to provide EPA with information on developing a standardized framework for evaluating Bt corn IRM. These workshops provided direction for the development of model verifi cation and validation pr ocedures (described in Glaser et al. 2009).

A number of models have been develope d to assess resistance management for Bt cropping systems. The Agency’s use of predictive models is also discussed in Matten & Reynolds (2003) and a draft report by ORD (2009).

d. Resistance Monitoring

The need for proactive resistance detection and monitoring is critical to the survival of Bt technology. The Agency mandates that registrants monitor for insect resistance (measurement of resistance-conferring alleles) to the Bt toxins as an important ear ly warning sign to developing

resistance in the field and whether IRM strate gies are working. Grower participation (e.g., reports of unexpected damage) is also important for monitoring. Resistance monitoring is also important because it provides validation of biol ogical parameters used in models. However, resistance detection/monitoring is a difficult and im precise task. It requires both high sensitivity and accuracy. Good resistance monitoring should have well-established baseline susceptibility data prior to introduction of Bt crops. The chances of finding a resistant larva in a Bt crop depend on the level of pest pressure, the freque ncy of resistant individuals, the location and number of samples that are collected, and the se nsitivity of the detection technique. Therefore, as the frequency of resistant individuals or the number of collected samples increases, the likelihood of locating a resistant individual increases (Roush & Mill er 1986). If the phenotypic frequency of resistance is one in 1,000, then more than 3,000 individuals must be sampled to have a 95% probability of one resistant indivi dual (Roush & Miller 1986). Current sampling strategies have a target of 100 to 200 individu als per location. Prev ious experience with

conventional insecticides has shown that once re sistant phenotypes are detected at a frequency >10%, control or crop failures are common (R oush & Miller 1986). Because of samplingCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

130 limitations and monitoring technique sens itivity, resistance could develop to Bt toxins prior to it being easily detected in the field.

The 2000 SAP Subpanel concluded that resistance m onitoring programs should be peer reviewed

and used to assess the success of current IR M plans. EPA’s Office of Research and Development, National Risk Management Research Laboratory and Office of Pesticide Programs held a small expert group workshop in July, 2001 that focused on resistance monitoring plan design and detection techniques for Bt corn IRM.

Each of the following monitoring techniques desc ribed below have a number of advantages and disadvantages:

1) Grower Reports of Unexpected Damage

Growers can be encouraged to report any unsuspected control problems to a local technical expert. Toll-free telepho ne numbers and an Internet site can be provided by registrants to report any unusual control problems. A confirmed grow er report of unexpected pest damage in a Bt crop may be a way to document a control failur e and may be a useful monitoring system for determining the success or failure of existing re sistance management strategies. However, once a grower detects a control failure , and resistance has been verifi ed, the only available response may be to alter existing resistance management strategies.

2) Systematic Field Surveillance

Registrant sponsored surveys of grower Bt fields for damaged plants have been used to monitor resistance allele frequency of the development of resistance and gauge the geographic area where resistant populations exist. An in-field detection system (for quick determination of the presence or absence of Cry proteins in corn plants) has been developed for most Bt corn PIPs.

3) Discriminating Concentration Assay / Diagnostic Dose

The discriminating dose/diagnostic dose bioa ssay is currently required by the EPA. Discriminating dose bioassays are most useful wh en resistance is common or conferred by a dominant allele (resistance allele frequency >1%) (Andow & Alstad 1998). It should be considered as one of the central components of any monitoring plan, but other monitoring methods may have value in conjunction with the discriminating concentration assay.

Of the techniques available, the diagnostic dose has been the best developed and most thoroughly tested. Hawthor ne et al. (2001) consider the di agnostic dose to be less expensive

than in-field screens and the F 2 screen. It is best used when the frequency of resistance alleles is high (>10 -2 ) or the resistant allele is dominant. However, it is uncle ar (and likely pest-specific) whether resistance is carried by dominant or r ecessive alleles and wh at the frequency of resistance alleles are in pest populations. Meas urement of low resistant allele frequencies (<10 -2 ) would not be possible using the diagnostic dose without extremely large sample sizes.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

131 The October 2000 SAP Subpanel was asked whethe r the current resistance monitoring plans were adequate. They indicated that the diagnosti c or discriminating dose technique could at best detect resistance when the resistance allele freq uency has reached 1%. This is a level in which some field failure may be observed. At this lo wer level of precision, the least expensive methods are the discriminating dose assays (see U. S. EPA/USDA 1999, p. 47, Figure 3). Previous experience with conventional inse cticides has shown that once re sistant phenotypes are detected at a frequency >10%, control or crop failures are common (Roush & Miller 1986). If resistance

is carried on a recessive allele, the frequency of individua ls in a population that demonstrate resistance will equal the square of the allele fr equency. For example, if the initial resistance allele frequency is one in 1,000, then one would n eed to assay more than a million larvae to find one homozygous resistant individual. Typical ly, discriminating dose assays are based on 100- 300 larvae to detect resistance at a freque ncy of 1-3% (Roush & Miller 1986).

4) F 2 Screen

The F 2 screen may be a useful monitoring technique for Bt corn, especially for the detection of

rare recessive resistant al leles. The technique also allows fewer samples to be collected to detect potential susceptibility shifts than the discriminating dose assay. The F 2 screen may be most useful to analyze populations that are expected to be at high risk to the development of

resistance. Each isofemale line allows for char acterization of four geno mes, thus improving the sensitivity over the discriminating dose assa y by 10-fold (Andow & Alstad 1998). The F 2 screen could be an effective method for detecting changes in the allele frequency of a recessive or partially recessive allele and can be used to verify some of the assumptions underlying high dose/refuge resistance management (Andow & Alstad 1998, Andow et al. 1998). If resistance alleles are found, they can be characterized to es timate the fitness of the genotypes, determine whether there is a cost of resistance, and enable predictions of the evolu tion of resistance. The

F 2 screen is conducted by sampling mated female s from natural populations, rearing the progeny of each female as an isofemale line and sib-mating her F 1 larvae using an appropriate screening procedure such as a discriminating concentration assay or Bt crop, and performing statistical analysis. Hawthorne et al. (2001) indicate that the F 2 screen is probably the only current method

available to detect rare recessive alleles.

A number of the October, 2000 SAP S ubpanel members indicated that the F 2 screen accompanied by field screening “could be very effective for detecting low frequencies of

recessive and dominant resistance alleles.” The F 2 screen can be a powerful method for detecting rare recessive allele s in natural populations. As described by Andow and Alstad (1998), it relies on inbreeding fi eld-collected individuals so th at all recessive alleles are expressed in the F 2 generation where they can be screened for the phenotype of interest. This

method has been used to estimate the fre quency of resistance to Cry toxins from Bacillus thuringiensis in ECB (Andow et al. 1998, 2000, Bourguet et al. submitted), Scirpophaga

incertulas (Walker) (rice stem borer ) (Bentur et al. 2000), and Plutella xylostella (L.) (diamondback moth) (Zhao et al. 2001).

Andow and Alstad (1998) also pr ovide a statistical method for es timating the probability that the screen erroneously does not identify the targeted resistance allele. This is the probability of aCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

132 false negative, and its calculation is based on th e probability of inheritance of the allele, the assumption that F 1 families mate randomly, and the probability that other mortality factors may interfere with the phenotypic evaluation of the F 2 individuals.

Current insect resistance manageme nt strategies assume that resistance alleles are initially rare. That is, it is assumed that Bt resistance alleles are <10 -3 for the high dose/refuge strategies currently used for Bt crops. Studies using the F 2 screen by Andow et al. (1998, 2000) and Andow and Alstad (1998, 1999) indi cate that resistance alleles may be present at frequencies <9

x 10 -3 in southern Minnesota and <3.9 x 10 -3 in central Iowa. A F 2 screen of 1,200 isofemale

lines of ECB collected in France and in th e northern U.S. Corn Belt during 1999 and 2000 indicated that the frequency of resistan ce alleles in France was less than 1.27 x 10 -3 with 95% certainty and in the U.S. was less than 1.24 x 10 -3 with 95% certainty (Bourguet et al. 2001). These collective data support the assumption that the frequency of Bt resistance alleles in natural populations of ECB is less than 10 -3 , validating one of the ke y assumption of the high dose/refuge strategy.

Using the F 2 screen would increase the probability of detecting rare resistant alleles and the

threshold of detection would be lowered to <0.0 05. A sample of 100 female lines has a precision

of ± 0.0025 for dominant alleles and ± 0.0025 for reces sive resistance alleles. Leaving aside issues of accuracy, the theoreti cally best resolution of allele frequency is ± 0.0025 for dominant

alleles and ± 0.05 for recessive alleles for a sc reen of 100 larvae using the discriminating dose (see Andow in U.S. EPA/USDA 1999, p. 42-43).

The time-frame to respond before control failur es occur depends on the precision of monitoring and the recessivity/dominance of re sistance. If the goal of resi stance monitoring is to detect resistance at a low enough resistance allele freq uency so that changes to the insect resistance management plan can be made to increase the lo ngevity of the product and prevent field failure, then current resistance monitoring plans need refinement. The F 2 screen is one method of

refinement that can detect and meas ure resistance at frequencies of ≤ 0.005 for approximately $5000 per site. This level of precision can provide seven to 12 years to respond with alternative resistance management tactics (see Andow in U.S. EPA/USDA 1999b, p.47, Figure 1b).

A potential obstacle to the F 2 screen is that it may be labor in tensive and not suitable for routine screening purposes (Hawthorne et al. 2001). Andow has conducted a cost analysis for various monitoring techniques and has conc luded that in general the F 2 screen is more expensive than other methods for detecting dominant resistant al leles when the resistan ce allele frequency is >0.01 (see Andow in U.S. EPA/USDA 1999b, p. 49, Fi gure 3). It is estimated that 750-1200 family lines must be screened to have a 95% prob ability of detecting a do minant resistance allele that is a frequency of 10 -3 and would cost $13.90-19.70 per family line (Andow et al. 1998, 2000). However, for recessive alle les, Andow estimates that the F 2 screen is the least expensive method and can estimate resistance allele freque ncies to a high level of precision (<0.005) for under $5,000 per location (see U.S. EPA/USDA 1999b, p. 41-49). Hawthorne et al. (2001), on the other hand, estimated the cost for each F 2 screen to be $14,000 to $20,000 per population. This, they conclude, would be too expensive for r outine monitoring efforts, especially if there isCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

133 replication at each site. The area of cost and cost-effectiveness of the F 2 screen should be further

evaluated.

Hawthorne et al. (2001) c oncluded that there is a need to further evaluate the precision and accuracy of the F 2 screen by using colonies with known frequencie s of resistance a lleles. Zhao et al. (2001) also came to this same conclusion. They validated the F 2 screen using a synthetic laboratory population of the diamondback moth ( Plutella xylostella L.) for detecting the frequency of rare resistance allele s to Cry1Ac and Cry1C toxins of Bt . When using Bt broccoli as the diagnostic method, only one F 2 family was detected for Cry1Ac resistance and no family was detected for Cry1C resistance. Six familie s were detected for either Cry1Ac or Cry1C resistance using the diagnos tic diet bioassay. Four F 2 families were confirmed to contain one copy of an allele resistant to Cry1Ac in the original single -pair matings and four other F 2 families contained an allele resistant to Cry1C. These re sults suggest that transg enic plants expressing a high level of a Bt toxin in a F 2 screen may underestimate the freque ncy of resistance alleles with false negatives or fail to detect true resistance alleles. The aut hors concluded that the diagnostic diet assay was a better F 2 screen method to detect resistance alleles, especially for the Cry1Ac resistance in diamondback moth. Zhao et al. (2 001) conclude that further validation of the F 2 screen method for each insect-crop system should be conducted before the procedures used in the

F 2 screen could be used r outinely to detect rare Bt resistance alleles in field populations.

5) Screening Against Test Stocks

Gould et al. (1997) used a series of genetic crosses with test stoc ks of highly resistant tobacco budworm ( Heliothis virescens ; TBW) selected on Cry1Ac in the laboratory to estimate the resistance allele frequency in a natural popul ation. This method can identify recessive or incompletely dominant resistance alleles from fi eld-collected males. Usi ng a colony of TBW that can survive on transgenic Bt cotton producing the Cry1Ac delta endotoxin, they crossed fieldcollected males with virgin-colony females so that all F 1 progeny would be heterozygous for resistance. By using an assay that discrimina tes between heterozygotes, they could establish

which wild males carried a resistance allele. Using this allelic recove ry method, Gould et al. (1997) estimated the resistance al lele frequency to be 1.5 x 10 -3 . This method is only useful when there are previously identified resistance alleles.

6) Sentinel Bt -Crop Field Plots

Venette et al. (2000) proposed the use of an in -field screen to exam ine resistance allele frequency. This method uses Bt sweet corn to sc reen for European corn borer and corn earworm

that are resistant to the Bt protein. That is, the Bt crop is the discriminatory screen for resistant individuals. By sampling large numbers of Bt -expressing plants for live corn borer larvae, the frequency of resistance can be estimated a nd resistant individuals can be collected for documentation of resistance. For example, Venette et al. (2000) suggest that sampling ears (18- 21 days post-silking stage) for European corn borer can increase sampling efficiency by twoorders of magnitude (over splitting stalks). Late-planted sentinel Bt sweet corn would provide a highly attractive oviposition site for females and re duce the number of plants required to attain an acceptable sample size. If the Bt sweet corn is planted at the appropriate time, larval attackCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

134 will cause extensive damage, and large areas of Bt sweet corn can be sampled rapidly by examining this damage. For example, if 10 re sistant larvae are found in a sample of 5,000 Bt corn ears, and 50 larvae are rec overed from 50 plants in the non- Bt field, then the expected phenotypic frequency of resistan ce would be 0.002. If potenti al resistant individuals or populations are identified in the field then they still must be brought to the laboratory so that resistance can be documented and quantified. Hawthorne et al. (2001) commented that the infield screen coupled to a F 2 screen for verification of resistan ce might be an efficient method to detect resistance and capture resistance allele s especially in desi gnated high-risk areas. The infield screening method described by Venette et al. (2000) might be an alternative approach used for early detection of rare Bt -resistant phenotypes as well as an alternative method to estimate the initial frequency of resistance alleles.

There are potential problem s with this method that must be addressed prior to its widespread adoption as discussed by Hawthorne et al. (2001). There is a hi gh number of false positives that would reduce the efficiency and accuracy of resist ance allele measurement. One source of false positives is the occurrence of weakly or non- expressing “off-type” plants among the sampled plants. Hawthorne et al. (2001) note that GeneCheckTM strips can be used to eliminate many of these “off-types.” Another source might be surv iving susceptible larvae that are incorrectly

scored as resistant larvae because of larval movement between Bt and non- Bt off-types or weeds. A second problem is that there might not be sweet corn varieties that contain the same Bt genes as the field corn varieties. This would reduce th e efficiency of sampling. Currently, there is only BT11 Cry1Ab field corn and sweet corn. As noted by Hawthorne et al. (2001), there are also additional concerns related to the large effort needed during harvest to complete an in-field screen. This type of effo rt limits its practicality.

e. Compliance with IRM requirements

Grower compliance with refuge and IRM require ments is a critical element for resistance management. Significant non-compliance with IR M among growers may increase the risk of

resistance for Bt corn. However, it is not known what level of grower non-compliance will compromise the risk protection of current refuge requirements.

The Agency recognizes that compliance is a complex issue for Bt crops and IRM. There is currently disagreement as to the appropriate re fuge size/deployment and the level of grower compliance necessary to achie ve risk reduction. EPA considers the development of Bt -resistant

insects to constitute an adverse environmental e ffect, therefore, IRM, and subsequently grower compliance, is very important. Optimally, ref uge requirements would change over time as pest susceptibility changes. However, changes to re fuge requirements are difficult to implement. Therefore, the Agency must set safe refuge requir ements that preserve the pest(s) susceptibility and protect the benefits of Bt crops. Currently, the financia l burden of implementing these refuge requirements is borne primarily by the gr owers. Increasing refuge size and/or limiting refuge deployment to better mitigate the risk of resistance is likely to in crease costs to growers and result in a higher rate of grower non-complia nce. Grower complian ce with IRM strategies

for current Bt crops is tied into the belief that new technologies, such as plants expressing multiple Bt toxins and other new synthetic insecticid es, will reduce the risk of resistance.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

135 To minimize the effects of non-compliance, it ma y be necessary to develop a broad compliance program as part of an IRM stra tegy. Ideally this program would include four major objectives: 1) an understanding of the effect of non-complia nce on IRM; 2) identif ication of compliance mechanisms to maximize adoption of IRM requirements; 3) measurement of the level of compliance; and 4) establishment of an enforcem ent structure to ensure compliance and penalize non-compliance.

1) Effects of Non-Compliance on IRM

As a first step towards developing a complia nce program, it is necessary to understand the impact of non-compliance on the development of pest resistance (i .e., the level of noncompliance that significantly increases the like lihood of resistance). In the past, many of the

models that have been developed to evaluate refuge and resistance scenarios have assumed 100% compliance. However, based on existing surveys of grower compliance (discussed later in this section), it is unlikely that 100% compliance ca n be achieved. On the other hand, research and modeling work may show that some level of non-compliance can be tolerated without significantly increasing the risk of pest resistance. Models also tend to assume 100% adoption of

the Bt technology. Compliance and adoption are both important factors that should be considered. Ultimately, many models may need to be updated to reflect some degree of noncompliance, so that the potential impact can be more thoroughly understood. Some later simulation models submitted to the Agency in support of Bt corn registrations have incorporated variable compliance percentages an d alternate adoption scenarios.

2) Compliance Mechanisms

There have been a number of compliance mechan isms proposed by various parties (including the 2000 SAP Subpanel) to ensure gr ower conformity, reward co mpliance, and penalize noncompliance. These include such techniques as: gr ower contracts, grower certification tests, fines and other penalties, community refuge, sales incentives, crop insurance of the refuge, deposit/refund for planting refuge, databases of non-compliant growers, county/area-wide compliance goals and sales restri ctions, intensified gr ower education, and grower audits. The 2000 SAP noted that, at present, there is little information on the relative effectiveness of different compliance options and that many mechan isms have both benefits and drawbacks. The potential efficacy of compliance mechanisms may depend on the perspective of the grower. For example, if non-compliance is th e result of confusion over the requirements, increased education may be of value. However, if non-compliance is a willful act, then a punishment or incentivebased approach may be more appropriate (H urley & Mitchell 2000). The 2000 SAP consensus was that compliance would be best managed thr ough education and grower contracts, but also that sales incentives, refuge in surance, and refuge deposit/ref und programs may have value if managed properly. Also, the 2000 SAP recognized th at mechanisms that would reduce the cost of compliance will be more effective at improving compliance.

Mitchell et al. (2000) developed a model to eval uate crop (refuge) insura nce and sales incentives as potential compliance mechanisms. The cost to growers (i.e., lost yi eld, higher inputs) toCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

136 adhere to IRM requirements can be an impedi ment to compliance. Therefore, by providing growers with incentives to reduc e the cost of refuge mandates, compliance may be increased. Both insurance and sales incentives have the po tential to reduce this cost of compliance to growers, although both have drawbacks as well. Fo r refuge insurance to be profitable for private insurance companies, it would likely be too e xpensive for growers and would provide limited benefits. Sales incentives may be less costly to administer, but would require frequent, costly monitoring to ensure proper refuge implementation.

3) Measurements of Compliance

To assess the effectiveness of a compliance progra m, it is necessary to be able to accurately measure the level of grower compliance. The 2000 SAP noted several pa rties, other than the registrant alone, could verify co mpliance: 1) grower visits by industry, EPA, state authorities, USDA, or other third-parties; or 2) USDA/NASS or other third-part y grower surveys. To date, compliance has been primarily measured thr ough grower surveys conducted by industry or academics (e.g., Pilcher & Rice 1997, Rice & Pilc her 1999). However, the 2000 SAP indicated that while surveys such as these are useful for tracking grower attitudes, they are not always reliable for determining actual grower complian ce. The format of the surveys (mail or phone interviews) may encourage non-compliant growers to misrepresent their actions or “cheat” in their responses. Without confirmatory visits to individual farms (i.e., aud its), it is impossible to verify the accuracy of grower responses. The end result may be increa sed “false-positives,” which may artificially inflate estimates of grow er compliance. As such, actual non-compliance may be significantly higher than the survey result s would suggest. To resolve this problem, the 2000 SAP suggested utilizing surveys created a nd conducted by independent parties to assess grower practices. In addition to this recommendation, it may be useful to conduct some on-farm visits for firsthand verification of compliance. Such visits could be performed as part of a survey process, to evaluate the accuracy of grower su rvey responses. The use of mapping systems, such as the Global Positioning System (GPS), may al so prove useful for determining the size and position of Bt and non- Bt fields for compliance verification. The Arizona Cotton Research and Protection Council (ACRPC) has ut ilized GPS with Bt cotton gr own in Arizona in conjunction with grower visits to asse ss the level of refuge comp liance (Carrière et al. 2001).

4) Enforcement Structure / Penalties for Non-compliance

For a compliance program to be effective, a re gulatory enforcement/compliance framework will be needed. Appropriate stakeholders and regulat ory bodies will need to create clearly defined roles for compliance. At the present time, EPA’ s authority is over the product registrations and registrants but not individual grow ers. Registrants have been re sponsible for compliance at the grower level through the use of grower contracts. However, the 2000 SAP noted that EPA’s reliance on industry to monitor and enforce comp liance “was seen as a major problem.” The SAP recommended that a third party compliance monitoring program should be developed. The compliance monitoring program should be accompan ied by an appropriate enforcement program. Potential penalties for non-compliance might include : 1) sales restrictions at a county, state, regional, or national level; 2) sa les prohibitions to specific grow ers; 3) registrant fines andCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

137 warnings; and 4) increased refuge for sp ecific non-compliant gr owers (through grower contracts).

A compliance program has been developed for Bt corn as a term of regi stration. Details of this plan, including compliance surveys and enforcem ent mechanisms, are described in section

D.2.b.7.

2. Corn

A summary of the Agency’s risk assessment of insect resistance development and insect resistance management plans to mitigate resistance is provided in this section for Bt corn. Other Agency risk assessments of insect resistance ma nagement are found in A. Reynolds and R. Rose (OPP/BPPD) to M. Mendelsohn (OPP/BPPD), dated September 11, 2000. Subsequent information has been added to the Agency’s ri sk assessment of insect resistance development and IRM plans following the October 18-20, 2000 SA P meeting as new data became available.

The Agency’s IRM assessment focuses on Cry1Ab field corn, Cry1Ab sweet corn, and Cry1F

field corn. EPA has used the best available scie ntific information in its IRM assessment and has updated its IRM position as additional information and data have become available.

In 1995, at the time of the initial registrations of Bt corn, there was no scientific consensus on the details of the IRM plans necessary for prevention of the developm ent of resistance in the two primary target pests, ECB and CEW. At that time, the putative va lues for adequate refuge size ranged from 0% to 50% of non- Bt corn or other host plants per farm. While the minimum adequate refuge size or structur e could not be determined until further research was conducted, it was thought that market penetrati on of these crops would be suffici ently slow that considerable

non- Bt corn would remain to act as natural refuges while the add itional research was conducted. Thus, the initial Bt corn registrants instituted voluntary IRM plans with the requirement that these registrants must submit a refuge strate gy by April 1999. From 1995-1997, the registrants agreed to various voluntary refuge requirements in the Co rn Belt (0% to 20%).

Since 1995, all Bt corn registrations have included a re sistance monitoring plan for ECB and CEW that contained the following elements: 1) development of baseline susceptibility responses and a discriminating concentration to detect changes in sensitivity; 2) rou tine surveillance; and 3) remedial action if there is suspected resistance. One of the key purposes of resistance monitoring

is to learn whether a field control failure result ed from resistance or ot her factors that might inhibit expression of the Bt delta-endotoxin. The extent and di stribution of resistant populations can be mapped and alternative control strategies implemented in areas in which resistance has become prevalent. If monitoring techniques are sensitive enough to discriminate between resistant and susceptible indivi duals, it should be possible to detect field resistance before significant loss of efficacy and eliminate any resistan t individuals using other control tactics. In addition, EPA mandated that all regi strants must require customers to notify them of incidents of unexpected levels of target pest damage. Registrants are required to investigate these reports and identify the cause of the damage by local field sampling of the plant tissue and suspect insect populations followed by appr opriate in vitro and in planta assays. Any confirmed incidents ofCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

138 resistance are required to be reported to EPA. Based on these investigations, appropriate remedial action is required to mitigate pest resist ance. These remedial actions include: informing customers and extension agents in the affected ar eas of pest resistance, increasing monitoring in the affected areas, implementing alternative means to reduce or control pest populations in the affected areas, implementing a structured refuge in the affected areas, and cessation of sales in the affected and bordering countie s. All registrants have instru cted growers to have regular surveillance programs and report any unexpected leve ls of target pest damage. Since 1995, the Agency is aware of no evidence of ECB, CE W or SWCB field resist ance to any of the Bt proteins produced in corn (though populations of ECB have been found to be tolerant in laboratory assays – see section D.2.b.4 for information). In January 2000, the Agency required that the registrants provide a more detailed resistance monitoring plan that focused on ECB, CEW, and SWCB. The registrants provided the Agency with a revised monitoring plan in March 2000. This monitoring plan and subsequent updates are discussed in detail later in this

section.

Based on the 1998 SAP Subpanel recommendations, th e Agency began to institute mandatory refuge requirements on Bt field corn and popcorn products. In 1999, a coalition of Bt corn registrants (working with the National Corn Growers Association), the Agricultural Biotechnology Stewardship Technical Committee (ABSTC), approached EPA with a uniform IRM plan for their products. With some modifica tions to this plan, EPA put in place a consistent set of required refuge strategies for all Bt field corn products begi nning with the 2000 growing season. These requirements greatly strengthened the IRM plan to mitigate ECB, CEW, and

SWCB resistance to Bt proteins produced in field co rn. Beginning with the 2000 growing season, EPA required a 20% non- Bt field corn refuge to be plante d within 1⁄2 mile (<1/4 mile in

areas where insecticides have b een historically used to treat ECB and SWCB) (EPA letter to Bt corn registrants, 1/31/00). EPA also required a 50% non- Bt field corn (<1⁄2 mile, 1/4 mile preferred) refuge for Bt Cry1Ab field corn products in certain southern counties and states where most cotton is grown (EPA letter to Bt corn registrants, 1/31/00). The larger refuge was necessary to mitigate the development of resistance to Bt proteins in CEW populations feeding on both corn and cotton. These same refuge re quirements were mandated for the Cry1F field corn products registered in May 2001.

a. Current Insect Resistance Management (IRM) Plans for Bt Corn (2008 Amendments)

1) MON 810, BT11 (Cry1Ab) and TC 1507 (Cry1F) Field Corn

These products are known to produce a “high dose” for ECB based on the 25 x definition described by the 1998 SAP Subpanel (SAP 1998) and confirmed by the 2000 SAP Subpanel (SAP 2001). Below are EPA's current te rms and conditions for IRM for the Cry1Ab Bt corn plant-incorporated protectant , as amended in 2008 and 2009:

The required IRM program for Bt [field] corn must have the following elements:

1] Requirements relating to creation of a non- Bt corn and/or non-lepi dopteran resistant Bt corn refuge in conjunction with the planting of any acreage of Bt corn;Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

139 2] Requirements for the regist rants to prepare and require Bt corn users to sign “grower agreements” which impose bindi ng contractual obligations on the grower to comply with the refuge requirements;

3] Requirements regarding programs to e ducate growers about IRM requirements;

4] Requirements regarding programs to evalua te and promote growers’ compliance with IRM requirements;

5] Requirements regarding programs to evaluate whether there are st atistically significant and biologically relevant changes in target insect susceptibility to [Cry1Ab or Cry1F] protein in the ta rget insects;

6] Requirements regarding a “remedial ac tion plan” which contains measures the registrants would take in the ev ent that any field relevant insect resistance was detected as well as to report on activity under the plan to EPA;

7] Submit annual reports on units sold by state (units sold by county level will be made available to the Agency upon request), IR M grower agreements results, and the compliance assurance program including the education program on or before January 31st each year.

a. Refuge Requirements

These refuge requirements do not apply to se ed propagation of inbred and hybrid corn seed corn up to a total of 20,000 acres per county and up to a combined U.S. total of 250,000 acres per PIP active ingredie nt per registrant per year.

1) Corn-Belt Refuge Requirements

Field corn grown outside cotton-growing areas (e.g., the Corn Belt), grower agreements (also known as stewardship agreements) will specify that growers must adhere to the refuge requirements as described in the grower guide/product use guide and/or in supplements to the grower guide/product use guide.

• Specifically, growers must plant a stru ctured refuge of at least 20% non- Bt corn and/or non-lepidopt eran resistant Bt corn that may be treated with insecticides as needed to control lepidopteran stalk-boring and other pests.

• Refuge planting options include: separate fields, blocks within fields (e.g., along the edges or headlands), and strips across the field.

• External refuges must be planted within 1⁄2 mile.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

140 • When planting the refuge in strips across th e field, refuges must be at least 4 rows

wide.

• Insecticide treatments for control of Eu ropean corn borer (ECB), Corn earworm (CEW), Southwestern corn borer (SWCB) a nd other lepidopteran target pests listed on the label, grower guides, or other edu cational material may be applied only if

economic thresholds are reached for one or more of these target pests. Economic thresholds will be determined using me thods recommended by local or regional professionals (e.g., Extension Service agents , crop consultants). Instructions to growers will specify that microbial Bt insecticides must not be applied to non- Bt corn and/or non-lepi dopteran resistant Bt corn refuges.

2) Cotton-Growing Area Refuge Requirements for Bt Corn

For Bt field corn grown in cotton-growing ar eas, grower agreements (also known as stewardship agreements) will specify that grow ers must adhere to the refuge requirements as described in the grower guide/product use guide and/or in supplements to the grower guide/product use guide.

• Specifically, growers in these areas must pl ant a structured refuge of at least 50%

non- Bt corn and/or non-le pidopteran resistant Bt corn that may be treated with

insecticides as needed to control lepidopteran stal k-boring and other pests.

• Refuge planting options include: separate fields, blocks within fields (e.g., along the edges or headlands), a nd strips across the field.

• External refuges must be planted within 1⁄2 mile.

• When planting the refuge in strips across th e field, refuges must be at least 4 rows

wide.

• Insecticide treatments for control of E CB, CEW, SWCB, and other lepidopteran target pests listed on the labe l, grower guides, or other educational material may be applied only if economic thresholds are re ached for one or more of these target pests. Economic thresholds will be determined using methods recommended by local or regional professionals (e.g., Extens ion Service agents, crop consultants). Instructions to growers w ill specify that microbial Bt insecticides must not be applied to non- Bt corn and/or non-lepidopteran resistant Bt corn refuges.

• Cotton-growing areas include the following states: Alabama, Arkansas, Georgia, Florida, Louisiana, North Carolina, Mi ssissippi, South Carolina, Oklahoma (only the counties of Beckham, Caddo, Coma nche, Custer, Greer, Harmon, Jackson, Kay, Kiowa, Tillman, Washita), Tennessee (only the counties of Carroll, Chester, Crockett, Dyer, Fayette, Franklin, Gibson, Hardeman, Hardin, Haywood, Lake, Lauderdale, Lincoln, Madison, Obion, Ru therford, Shelby, and Tipton), TexasCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

141 (except the counties of Carson, Dallam, Hansford, Hartley, Hutchinson, Lipscomb, Moore, Ochiltree, Roberts, and Sherman), Vi rginia (only the counties of Dinwiddie, Franklin City, Greensville, Isle of Wight , Northampton, Southampton, Suffolk City, Surrey, Sussex) and Missouri (only the c ounties of Dunkin, New Madrid, Pemiscot, Scott, Stoddard).

b. Grower Agreements

1] Persons purchasing the Bt corn product must sign a grower agreement. The term “grower agreement” refers to any grower purch ase contract, license agreement, or similar legal document.

2] The grower agreement and/or specific stew ardship documents referenced in the grower agreement must clearly set forth the terms of the current IRM program. By signing the grower agreement, a grower must be contractually bound to comply with the requirements of the IRM program.

3] The registrant must maintain a system whic h is reasonably likely to assure that persons purchasing the Bt corn product will affirm annually th at they are contractually bound to comply with the requirements of the IRM program.

4] The registrant must continue to use their current grower agreement. If the registrant wishes to change any part of the grow er agreement or any specific stewardship documents referenced in the grower agreement that would affect eith er the content of the IRM program or the legal enforceability of th e provisions of the agreement relating to the IRM program, thirty days prior to implemen ting a proposed change, the registrant must submit to EPA the text of such changes to ensu re that it is consistent with the terms and

conditions of the amendment.

5] The registrant must maintain a system whic h is reasonably likely to assure that persons purchasing the Bt corn sign grower agreement(s).

6] The registrant shall maintain records of all Bt corn grower agreements for a period of three years from December 31 of the year in which the agreement was signed.

7] The registrant shall provide EPA with a report showing the numb er of units of its Bt corn seeds sold or shipped and not returned, and the number of such units that were sold to persons who have signed grower agreemen ts. The report shall cover the time frame of the twelve-month period covering th e prior August through July.

8] The registrant must allow a review of the grower agreements and grower agreement records by EPA or by a State pesticide regul atory agency if the State agency can

demonstrate that confidential business information, including names, personal information, and grower license number, will be protected.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

142 c. IRM Education and IRM Compliance Monitoring Programs

1] The registrant must maintain a co mprehensive, ongoing IRM education program designed to convey to Bt corn users the importance of co mplying with the IRM program. The program shall include information encouraging Bt corn users to pursue optional elements of the IRM program relating to refuge configuration and proximity to Bt corn fields. The education program shall involve the use of multiple media, e.g., face-to-face meetings, mailing written materials, EPA re viewed language on IRM requirements on the bag or bag tag, and electronic communications su ch as by Internet, radio, or television commercials. Copies of the materials will be provided to EPA for its records. The program shall involve at least one wr itten communication annually to each Bt corn user separate from the grower technical guide. The communication shall inform the user of the current IRM requirements. The registra nt shall coordinate its education programs with educational efforts of other registrants and other organizations, such as the National Corn Grower Association and state extension programs.

2] Annually, the registrant shall revise, and e xpand as necessary, its education program to

take into account the information collected through the compliance survey required and from other sources. The changes shall addre ss aspects of grower compliance that are not sufficiently high.

3] Annually, each January 31st, the registrant must provide EPA any substantive changes to its grower education activities as part of the overall IRM compliance assurance program report. The registrant must either submit a separate report or contribute to the report from the industry working group (ABSTC).

4] The registrant must maintain an ongoi ng IRM compliance assurance program designed

to evaluate the extent to which growers purchasing its Bt corn product are complying with the IRM program and that takes such actions as are reas onably needed to assure that growers who have not complied with the program either do so in the future or lose their

access to the Bt corn product. The registrant shall coordinate with other registrants in designing and implementing its comp liance assurance program.

5] The registrant must maintain and public ize a “phased compliance approach,” i.e., a guidance document that indicates how the registrant will address instances of noncompliance with the terms of the IRM progr am and general criteria for choosing among options for responding to any non-compliant grow ers. While recognizing that for reasons of difference in business practices there ar e needs for flexibility between different companies, all Bt corn registrants must use a consiste nt set of standards for responding to non-compliance. The options shall include with drawal of the right to purchase Bt corn for an individual grower or for all growers in a specific region. An individual grower found to be significantly out of compliance two years in a ro w would be denied sales of the product the next year. Similarly, seed de alers who are not fulf illing their obligations to inform/educate growers of their IRM oblig ations will lose their opportunity to sell Bt

corn.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

143 6] The IRM compliance assurance program sh all include an annual survey conducted by an independent third party of a statistically representative sample of growers of Bt corn borer protected products who plant the vast major ity of all corn in the U.S. and in areas in which the selection intensity is greatest. The survey shall consider only those growers who plant 200 or more acres of corn in the Corn-Belt and who plant 100 or more acres of corn in corn-cotton areas. The survey sha ll measure the degree of compliance with the IRM program by growers in different regions of the country and c onsider the potential impact of non-response. The sample size and geographical resolution may be adjusted annually, based upon input from the independent marketing research firm and academic scientists, to allow analysis of compliance be havior within regions or between regions. The sample size must provide a reasonable se nsitivity for comparing results across the

U.S.

7] The survey shall be designed to provide an understanding of a ny difficulties growers encounter in implementing IRM requirements. An analysis of the survey results must include the reasons, extent, and potential biol ogical significance of any implementation

deviations.

8] The survey shall be designed to obtain grower feedback on the usefulness of specific

educational tools and initiatives.

9] The registrant shall provide a written summar y of the results of th e prior year's survey (together with a descripti on of the regions, the methodol ogy used, and the supporting data) to EPA by January 31st of each year. The registrant shall confer with other registrants and EPA on the design and content of the survey prior to its implementation.

10] Annually, the registrant shall revise , and expand as necessary, its compliance assurance program to take into account the information collected through the compliance survey and from other sources. The changes shall address aspects of grower compliance that are not sufficiently high. The registrants must confer with the Agency prior to adopting any changes.

11] The registrant shall c onduct an annual on-farm assessment program. The registrant shall train its representatives who make on-farm visits with growers of their Bt corn borer protected products to perform assessments of compliance with IRM requirements. There is no minimum corn acreage size for this progra m. Therefore, growers will be selected for this program from across all farm sizes. In the event that any of these visits result in the identification of a grower who is not in compliance with th e IRM program, the registrant shall take appropriate action, consistent with its "phase d compliance approach," to promote compliance.

12] The registrant shall carry out a program for investigating legitimate “tips and complaints” that its growers are not in comp liance with the IRM program. Whenever an investigation results in the identification of a grower who is not in compliance with theCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

144 IRM program, the registrant sh all take appropriate action, consistent with its “phased compliance approach.”

13] If a grower, who purchases Bt corn for planting, was specifically identified as not being in compliance during the pr evious year, the registrant shall visit with the grower and evaluate whether that the grower is in compliance with th e IRM program for the

current year.

14] Each registrant shall annually provide a report to EPA summarizing the activities carried out under their compliance assurance pr ogram for the prior year and the plans for the compliance assurance program during the current year. The report will include information regarding grower interactions (i ncluding, but not limited to, on-farm visits, verified tips and complaints, grower meetings and letters), the extent of non-compliance,

corrective measures to address the non-co mpliance, and any follow-up actions taken. The registrants may elect to coordinate inform ation and report collecti vely the results of their compliance assurance programs.

15] The registrant and the seed corn dealers fo r the registrant must allow a review of the compliance records by EPA or by a State pestic ide regulatory agency if the State agency

can demonstrate that confidential business information, including the names, personal information, and grower license number of the growers will be protected.

d. Insect Resistance Monitoring

The Agency is imposing the following conditions for this product:

The registrant will monitor for resi stance to its lepidopteran-resistant Bt corn. The monitoring program shall cons ist of two approaches: (1) focused population sampling and laboratory testing; and (2) investigation of reports of le ss-than expected control of

labeled insects. Should field-relevant resistan ce be confirmed, an appropriate resistance management action plan will be implemented.

(1) Focused Population Sampling

The registrant shall annually sample and bioassay populations of the key target pests

Ostrinia nubilalis (European corn borer; ECB), Diatraea grandiosella (Southwestern corn borer; SWCB), and Helicoverpa zea (corn earworm; CEW). Sampling for the target pests will be focused in areas identified as those with the highest risk of resistance development (e.g., where lepidopteran-active Bt hybrids are plante d on a high proportion of the corn acres, and where the insect sp ecies are regarded as key pests of corn). Bioassay methods must be appropriate for the goal of detecting field- relevant shifts in population response to le pidopteran resistant Bt corn and/or changes in resistance allele frequency in response to the use of Bt corn and, as far as possible, should be consistent across sampling years to enable co mparisons with historical data.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

145 The number of populations to be collected shall re flect the regional importance of the insect species as a pest, and specific collec tion regions will be identified for each pest. For ECB, a minimum of 12 populations across the sampling region will be targeted for collection at each annual sampling. For SWCB, the target will be a minimum of six populations. For CEW, the target will be a minimum of 10 populations. Pest populations should be collected from multiple corn-growing states reflective of different geographies and agronomic conditions. To obtain sufficient sensitivity to detect resistance alleles before they become common enough to cause measurable field damage, each population collection shall attempt to ta rget 400 insect genomes (egg masses, larvae, mated females, and/or mixed-sex adults), but a successful population collection w ill contain a minimum of 100 genomes. It is recognized that it may not be possible to collect the target number of insect populations or genomes due to fact ors such as natural fluctuations in pest density, environmental conditions, and area-wide pest suppression.

The sampling program and geographic range of collections may be modified as appropriate based on changes in pest im portance and for the adoption levels of lepidopteran-resistant Bt corn. The Agency shall be cons ulted prior to the implementation

of such modifications.

The registrant will report to the Agency be fore August 31 each year the results of the population sampling and bioassay monitoring program.

Any incidence of unusually low sensitivity to the Bt protein in bioassays shall be investigated as soon as possible to understa nd any field relevance of such a finding. Such investigations shall proceed in a stepwise manner until the field relevance can be either confirmed or refuted, and results of these shal l be reported to the Agency annually before August 31. The investiga tive steps will include:

1. Re-test progeny of the collect ed population to determine wh ether the unusual bioassay response is reproducible and heri table. If it is not reproducib le and heritable, no further action is required.

2. If the unusual response is re producible and heritable, prog eny of insects that survive the diagnostic concentration will be tested using methods that are representative of

exposure to Bt corn hybrids under field conditions. If progeny do not survive to adulthood, any suspected resistance is not field relevant and no further action is required.

3. If insects survive steps 1 and 2, resistance is confirmed, and further steps will be taken to evaluate the resistance. These steps may include:

• Determining the nature of the resistance (i.e., recessive or dominant, and the level of functional dominance);

• Estimating the resistance-allele freq uency in the original population;Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

146 • Determining whether the resistance-allele Frequency is increasing by analyzing field collections in subsequent years sampled from the same site where the resistance allele(s) was originally collected;

• Determining the geographic distribution of the resistance allele by analyzing field collections in subsequent years from site s surrounding the site where the resistance allele(s) was originally collected.

Should field-relevant resistan ce be confirmed, and the resist ance appears to be increasing or spreading, the registrant will consult w ith the Agency to develop and implement a case-specific resistance management action plan.

(2) Investigation of Reports of Unexp ected Levels of Damage by the Target

Pests:

The registrant will follow up on grower, extension specialist or consultant reports of unexpected levels of damage by the lepidopteran pests listed on the pesticide label. The registrant will instruct its customers to c ontact them if such incidents occur. The registrant will investigate all legitimate reports submitted to the company or the company's representatives.

If reports of unexpected levels of damage lead to the suspicio n of resistance in any of the key target pests (ECB, SWCB, and CEW), th e registrant will implement the actions described below, based on the following definitions of suspected resistance and confirmed resistance.

Suspected resistance

EPA defines suspected resistance to mean field reports of un expected levels of insect feeding damage for which:

• The corn in question has been confirmed to be lepidopteran-active Bt corn;

• The seed used had the proper pe rcentage of corn expressing Bt protein;

• The relevant plant tissues are expressing the expected level of Bt protein; and

• It has been ruled out that sp ecies not susceptible to the protein could be responsible for the damage, that no climatic or cultu ral reasons could be responsible for the damage, and that that there could be no other reasonable causes for the damage.

The Agency does not interpret suspected resistance to mean grower reports of possible control failures or suspicious results from annual insect monitoring assays, nor does the Agency intend that extensive field studies and testing be und ertaken to confirm scientifically the presence of insects resistant to Bt corn in commerci al production fieldsCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

147 before responsive measures are undertaken.

If resistance is suspected, the registrant will instruct growers to do the following:

• Use alternative control measures in the Bt corn fields in the affected region to control the target pest during the immediate growing season.

• Destroy Bt corn crop residues in the affected re gion within one month after harvest with a technique appropriate for local production practices to minimize the possibility of resistant insects over-winte ring and contributing to the next season's target pest population.

Additionally, if possible, and pr ior to the application of alte rnative control measures or destruction of crop residue, the registrant w ill collect samples of the insect population in the affected fields for laboratory rearing a nd testing. Such rearing and testing shall be conducted as expeditiously as practical.

Confirmed resistance

EPA defines confirmed resistance to mean, in the case of field reports of unexpected levels of damage from the key target pest s, that all the following criteria are met:

• There is > 30% insect survival and commensurate insect feeding in a bioassay, initiated with neonate larvae, that uses methods that ar e representative of exposure

to Bt corn hybrids under field conditions (ECB and SWCB only).

• In standardized laboratory bioassays using diagnostic concentrations of the Bt protein suited to the target pest in ques tion, the pest exhibits resistance that has a genetic basis and the level of survivorship indicates that there may be a resistance allele frequency of ≥ 0.1 in the sampled population.

• In standardized labora tory bioassays, the LC 50 exceeds the upper limit of the 95%

confidence interval of the LC 50 for susceptible populations surveyed both in the original baselines developed for this pest species and in previous years of field monitoring.

(3) Response to Confirmed Resistance in a Key Target Pest as the Cause of Unexpected Levels of Damage in the Field

When field resistance is confirmed (as defined above), the following steps will be taken by the registrant:

• EPA will receive notification within 30 days of resistance confirmation;

• Affected customers and extension agents will be notified about confirmedCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

148 resistance within 30 days;

• Monitoring will be increased in the affected area and local target pest populations will be sampled annually to determine th e extent and impact of resistance;

• If appropriate (depending on the resistant pe st species, the extent of resistance, the timing of resistance, and the nature of resistance, and th e availability of suitable alternative control measures), alternative control measures will be employed to reduce or control target pest populations in the affected area. Altern ative control measures may include advising customers and extension agents in the affected area to incorporate crop residues into the soil following harvest to minimize the possibility of over-wintering in sects, and/or applications of chemical insecticides;

• Unless otherwise agreed with EPA, stop sale and distribution of the relevant lepidopteran-active Bt corn hybrids in the affected area immediately until an effective local mitigation plan approved by EPA has been implemented;

• The registrant will develop a case-specific resistance management action plan within 90 days according to the characteri stics of the resistance event and local agronomic needs. The registrant will consu lt with appropriate stakeholders in the development of the action plan, and the deta ils of such a plan shall be approved by EPA prior to implementation;

• Notify affected parties (e.g., growers, consultants, extension agents, seed distributors, university coopera tors and state/fede ral authorities as appropriate) in the region of the resistance situat ion and approved action plan; and

• In subsequent growing seasons, maintain sales suspension and a lternative resistance management strategies in th e affected region(s) for the Bt corn hybrids that are affected by the resistant population un til an EPA-approved local resistance management plan is in place to mitigate the resistance.

A report on results of resistance monitoring and investigations of damage reports must be submitted to the Agency annually by August 31 st each year for the duration of the conditional registration.

Table D1. Summary of Current Bt Field Corn Refuge Requirements

Active Ingredient ECB Dosage Refuge Size in

Corn Belt Refuge Size in

Cotton Areas Grower Agreement Proximity In-field strip refuges

MON 810, BT 11, &

TC 1507 High dose 20% sprayed or unsprayed 50% sprayed or unsprayed yes < 1⁄2 mile

of Bt field At least 4 rows

wide

3) BT11 Sweet Corn (Cry1Ab)Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

149 A key to understanding the resistance management issues with Attribute BT11 sweet corn is to appreciate the differences in the cultural practices of sweet corn versus field corn. Field corn is frequently grown in large blocks on farms of 500 - 1,000 acres. This results in large areas of

field corn monoculture. Conversel y, sweet corn is usually grown in blocks of 40 acres or less on farms that produce several crops that are also host plants for ECB and CEW.

In contrast to BT11 field corn, specific ref uge requirements were not mandated for this Bt sweet corn product because sweet corn harvesting occurs before insects mature and reproduce. Sweet corn is harvested 18-21 days after silking while the plant has active photosynt hesis. As a result, in transgenic sweet corn varieties, Bt protein production is high at the time of harvest. EPA mandated specific resistance monitoring requirement s for ECB, CEW, and FAW, as well as sales reporting requirements. Syngenta is required thr ough labeling and technical material to have growers destroy any Cry1Ab (BT11) sweet corn stalks that remain in the fields following harvest in accordance with local production practices. Stalk destruction is intended to reduce the possibility of any insects, includ ing resistant insects, surviving to the next generation. The major aspects insect resistance management plan for Attribute BT11 sweet corn, as amended in December, 2008, are summarized below.

The required IRM program for Bt [sweet] corn must have the following elements:

1] Requirements (except for home garden use or educational use, i.e., marketed to home gardeners or educators for use on less than 20 acres) for the regist rants to prepare and require Bt corn users to sign “grower agreemen ts” which impose binding contractual obligations on the grower to comply with the refuge requirements;

2] Requirements regarding programs to e ducate growers about IRM requirements;

3] Requirements regarding programs to evalua te and promote growers’ compliance with IRM requirements (except for home garden use or educational use, i.e., marketed to home gardeners or educators for use on less than 20 acres);

4] Requirements regarding programs to evaluate whether there are st atistically significant and biologically relevant cha nges in target insect suscepti bility to Cry1Ab protein in the target insects;

5] Requirements regarding a “remedial ac tion plan” which contains measures the registrants would take in the ev ent that any field relevant insect resistance was detected as well as to report on activity under the plan to EPA;

6] Submit annual reports on units sold by state (units sold by county level will be made available to the Agency upon request), IR M grower agreements results, and the compliance assurance program including the education program on or before January 31st each year.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

150 7] For home garden or educational use, submit annual reports on total number of estimated acres sold and the compliance assurance program including the education program on or before January 31 st each year.

a. Sweet Corn Post-Harvest Requirements

Sweet corn is harvested long before field co rn. Therefore, if the sweet corn stalks remaining in the field and any insects remaini ng in the stalks are de stroyed shortly after harvest, a refuge is not needed as a part of the IRM program fo r sweet corn. Growers must adhere to the following types of crop de struction requirements as described in the grower guide/product use guide and/or in s upplements to the grower guide/product use guide, and in the case of home gardeners on th e seed packet, in seed catalogues, and on websites offering Attribute Insect Protected Sweet Corn Hybrids for sale to home gardeners.

1. Crop destruction must occur no later than 30 days following harvest, but preferably within 14 days.

2. The allowed crop destruction methods are: rotary mowing, discing, or plow- down (or for the home garden use) by chopping up the st alks using home garden tools such as a hoe. The crop destruction methods are intended to protect against de velopment of insect

resistance

b. Grower Agreements (except for home garden use or educational use, i.e., marketed to home gardeners or educators for use on less than 20 acres)

1] Persons purchasing the Bt corn product must sign a grower agreement. The term “grower agreement” refers to any grower purch ase contract, license agreement, or similar legal document.

2] The grower agreement and/or specific stew ardship documents referenced in the grower agreement must clearly set forth the terms of the current IRM program. By signing the grower agreement, a grower must be contractually bound to comply with the requirements of the IRM program.

3] The registrant must maintain a system whic h is reasonably likely to assure that persons purchasing the Bt corn product will affirm annually th at they are contractually bound to comply with the requirements of the IRM program.

4] The registrant must continue to use their current grower agreement. If the registrant wishes to change any part of the grow er agreement or any specific stewardship documents referenced in the grower agreement that would affect eith er the content of the IRM program or the legal enforceability of th e provisions of the agreement relating to the IRM program, thirty days prior to implemen ting a proposed change, the registrant must submit to EPA the text of such changes to ensu re that it is consistent with the terms andCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

151 conditions of the amendment.

5] The registrant must maintain a system whic h is reasonably likely to assure that persons purchasing the Bt corn sign grower agreement(s).

6] The registrant shall maintain records of all Bt corn grower agreements for a period of three years from December 31 of the year in which the agreement was signed.

7] The registrant shall provide EPA with a report showing the numb er of units of its Bt corn seeds sold or shipped and not returned, and the number of such units that were sold to persons who have signed grower agreemen ts. The report shall cover the time frame of the twelve-month period covering th e prior August through July.

8] The registrant must allow a review of the grower agreements and grower agreement records by EPA or by a State pesticide regul atory agency if the State agency can

demonstrate that confidential business information, including names, personal information, and grower license number, will be protected.

c. IRM Education and IRM Compliance Monitoring Programs

1] The registrant must maintain a co mprehensive, ongoing IRM education program designed to convey to Bt corn users the importance of co mplying with the IRM program. The education program shall involve the us e of multiple media, e.g., face-to-face meetings, mailing written materials, EPA re viewed language on IRM requirements on the bag or bag tag, and electronic communications su ch as by Internet, radio, or television commercials. Copies of the materials will be provided to EPA for its records. The program shall involve at least one wr itten communication annually to each Bt corn user separate from the grower technical guide (with the exception of home gardening or educational uses). The communication sha ll inform the user of the current IRM requirements. The registrant shall coordina te its education programs with educational efforts of other registrants and other organizations, such as universities and state extension programs.

2] Annually, the registrant shall revise, and e xpand as necessary, its education program to

take into account the information collected through the compliance survey required and from other sources. The changes shall addre ss aspects of grower compliance that are not sufficiently high.

3] Annually, each January 31st, the registrant must provide EPA any substantive changes to its grower education activities as part of the overall IRM compliance assurance program report. The registrant must either submit a separate report or contribute to the report from the industry working group (ABSTC).

4] The registrant must maintain an ongoi ng IRM compliance assurance program designed

to evaluate the extent to which growers purchasing its Bt corn product are complyingCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

152 with the IRM program and that takes such actions as are reas onably needed to assure that growers who have not complied with the program either do so in the future or lose their

access to the Bt corn product (with the ex ception of home gardening or educational uses). The registrant shall coordinate with other registrants in designing and implementing its compliance assurance program.

The following IRM Education and IRM Comp liance Monitoring Programs apply to all growers who plant more than 20 acres of Bt corn:

5] The registrant must maintain and public ize a “phased compliance approach,” i.e., a guidance document that indicates how the registrant will address instances of noncompliance with the terms of the IRM progr am and general criteria for choosing among options for responding to any non-compliant grow ers. While recognizing that for reasons of difference in business practices there ar e needs for flexibility between different companies, all Bt corn registrants must use a consiste nt set of standards for responding to non-compliance. The options shall include with drawal of the right to purchase Bt corn for an individual grower or for all growers in a specific region. An individual grower found to be significantly out of compliance two years in a ro w would be denied sales of the product the next year. Similarly, seed de alers who are not fulf illing their obligations to inform/educate growers of their IRM oblig ations will lose their opportunity to sell Bt

corn.

6] The IRM compliance assurance program shall include an annual survey of all Bt sweet corn customers who purchase 5 or more ba gs of Bt11 sweet corn. The survey shall measure the degree of compliance with the IRM program, identify the response rate (e.g., the percent of Bt sweet corn acres covered by the res ponses), and consider the potential impact of non-response. An independent thir d party will participate in the design and implementation of the survey. Data and information derived from the annual survey will be audited by an independent third party.

7] The survey shall be designed to provide an understanding of a ny difficulties growers encounter in implementing IRM requirements. An analysis of the survey results must include the reasons, extent, and potential biol ogical significance of any implementation

deviations.

8] The survey shall be designed to obtain grower feedback on the usefulness of specific

educational tools and initiatives.

9] The registrant shall provide a written summar y of the results of th e prior year’s survey (together with a description of the methodol ogy used, and the supporting data) to EPA by January 31 of each year. The registrant shall confer with EPA on changes to the design and content of the survey prior to its implementation.

10] Annually, the registrant shall revise , and expand as necessary, its compliance assurance program to take into account the information collected through the complianceCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

153 survey and from other sources. The changes shall address aspects of grower compliance that are not sufficiently high. The registrants must confer with the Agency prior to adopting any changes.

11] The registrant shall train its repres entatives who make on-farm visits with Bt corn growers to perform assessments of compliance with IRM requirements. In the event that any of these visits result in the identificati on of a grower who is not in compliance with the IRM program, the registrant shall take a ppropriate action, consis tent with its "phased compliance approach," to promote compliance.

12] The registrant shall carry out a program for investigating legitimate “tips and complaints” that its growers are not in comp liance with the IRM program. Whenever an investigation results in the identification of a grower who is not in compliance with the IRM program, the registrant sh all take appropriate action, consistent with its “phased compliance approach.”

13] If a grower, who purchases Bt corn for planting, was specifically identified as not being in compliance during the pr evious year, the registrant shall visit with the grower and evaluate whether that the grower is in compliance with th e IRM program for the

current year.

14] Each registrant shall annually provide a report to EPA summarizing the activities carried out under their compliance assurance pr ogram for the prior year and the plans for the compliance assurance program during the current year. The report will include information regarding grower interactions (i ncluding, but not limited to, on-farm visits, verified tips and complaints, grower meetings and letters), the extent of non-compliance,

corrective measures to address the non-co mpliance, and any follow-up actions taken. The registrants may elect to coordinate info rmation and report collect ively the results of their compliance assurance programs.

15] The registrant and the seed corn dealers fo r the registrant must allow a review of the compliance records by EPA or by a State pestic ide regulatory agency if the State agency

can demonstrate that confidential business information, including the names, personal information, and grower license number of the growers will be protected.

d. Insect Resistance Monitoring

The Agency is imposing the following conditions for this product:

The registrant will monitor for resistance to its lepidopteran-resistant Bt corn. The monitoring program shall cons ist of two approaches: (1) focused population sampling and laboratory testing; and (2) investigation of reports of le ss-than expected control of

labeled insects. Should field-relevant resistan ce be confirmed, an appropriate resistance management action plan will be implemented.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

154 (1) Focused Population Sampling

The registrant will develop and ensure the implementation of a plan for resistance monitoring for Spodoptera frugiperda (fall armyworm or FAW) in counties in which CrylAb sweet corn acreage exceeds 5,000 acres a nd the pest is capable of overwintering in that county. The registrant should c onsult with academic and USDA experts in developing the monitoring plan and will pr ovide EPA with a copy of its proposed resistance monitoring plan for EPA’s appr oval prior to implementation. This proposed FAW monitoring plan must be submitted to EPA by January 31 of the year following that in which Cry lAb sweet corn acreage exceeds the trigger specified in this requirement (i.e., greater than 5,000 acres in any county in which FAW overwinters). The proposed plan must be implemented the season fo llowing the acreage trigger being met. The proposed plan will remain in place until an EPA approved plan can be implemented.

The registrant shall annually sample and bioassay populations of the key target pests

Ostrinia nubilalis (European corn borer; ECB) and Helicoverpa zea (corn earworm; CEW). Sampling for the target pests will be fo cused in areas identified as those with the highest risk of resistance devel opment (e.g., where lepidopteran-active Bt hybrids are planted on a high proportion of the corn acres, and where the insect species are regarded as key pests of corn). Bioassay methods mu st be appropriate for the goal of detecting field-relevant shifts in populati on response to lepidopteran resistant Bt corn and/or changes in resistance allele fre quency in response to the use of Bt corn and, as far as possible, should be consiste nt across sampling years to enable comparisons with

historical data.

The number of populations to be collected shall re flect the regional importance of the insect species as a pest, and specific collec tion regions will be identified for each pest. For ECB, a minimum of 12 populations across the sampling region will be targeted for collection at each annual sampling. For CEW, the target will be a minimum of 10 populations. Pest populations should be co llected from multiple corn-growing states reflective of different geographies and ag ronomic conditions. To obtain sufficient sensitivity to detect resistance alleles be fore they become common enough to cause measurable field damage, each population collection shall attempt to target 400 insect genomes (egg masses, larvae, mated females, a nd/or mixed-sex adults ), but a successful population collection will contain a minimum of 100 genomes. It is recognized that it may not be possible to collect the target numbe r of insect populations or genomes due to factors such as natural fluctuations in pe st density, environmental conditions, and areawide pest suppression.

The sampling program and geographic range of collections may be modified as appropriate based on changes in pest im portance and for the adoption levels of lepidopteran-resistant Bt corn. The Agency shall be cons ulted prior to the implementation

of such modifications.

The registrant will report to the Agency before August 31 each year the results ofCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

155 the population sampling and bioassay monitoring program.

Any incidence of unusually low sensitivity to the Bt protein in bioassays shall be investigated as soon as possible to understa nd any field relevance of such a finding. Such investigations shall proceed in a stepwise manner until the field relevance can be either confirmed or refuted, and results of these shal l be reported to the Agency annually before August 31. The investiga tive steps will include:

1. Re-test progeny of the collect ed population to determine wh ether the unusual bioassay response is reproducible and heri table. If it is not reproducib le and heritable, no further action is required.

2. If the unusual response is re producible and heritable, prog eny of insects that survive the diagnostic concentration will be tested using methods that are representative of exposure to Bt corn hybrids under field c onditions. If progeny do not survive to adulthood, any suspected resistance is not field relevant and no further action is required.

3. If insects survive steps 1 and 2, resistance is confirmed, and further steps will be taken to evaluate the resistance. These steps may include:

• Determining the nature of the resistance ( i.e., recessive or dominant, and the level of functional dominance);

• Estimating the resistance-allele freq uency in the original population;

• Determining whether the resistance-allele Frequency is increasing by analyzing field collections in subsequent years sampled from the same site where the resistance allele(s) was originally collected;

• Determining the geographic distribution of the resistance allele by analyzing field collections in subsequent years from site s surrounding the site where the resistance allele(s) was originally collected.

Should field-relevant resistan ce be confirmed, and the resist ance appears to be increasing or spreading, the registrant will consult w ith the Agency to develop and implement a case-specific resistance management action plan.

(2) Investigation of Reports of Unexpect ed Levels of Damage by the Target

Pests:

The registrant will follow up on grower, extension specialist or consultant reports of unexpected levels of damage by the lepidopteran pests listed on the pesticide label. The registrant will instruct its customers to c ontact them if such incidents occur. The registrant will investigate all legitimate reports submitted to the company or the company's representatives.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

156 If reports of unexpected levels of damage lead to the suspicio n of resistance in any of the key target pests (ECB, SWCB, CEW, and FAW), the registrant will implement the actions described below, based on the following definitions of suspected resistance and confirmed resistance.

Suspected resistance

EPA defines suspected resistance to mean field reports of un expected levels of insect feeding damage for which:

• The corn in question has been confirmed to be lepidopteran-active Bt corn;

• The seed used had the proper pe rcentage of corn expressing Bt protein;

• The relevant plant tissues are expressing the expected level of Bt protein; and

• It has been ruled out that sp ecies not susceptible to the protein could be responsible for the damage, that no climatic or cultu ral reasons could be responsible for the damage, and that that there could be no other reasonable causes for the damage.

The Agency does not interpret suspected resistance to mean grower reports of possible control failures or suspicious results from annual insect monitoring assays, nor does the Agency intend that extensive field studies and testing be und ertaken to confirm scientifically the presence of insects resistant to Bt corn in commerci al production fields before responsive measures are undertaken.

If resistance is suspected, the registrant will instruct growers to do the following:

• Use alternative control measures in the Bt corn fields in the affected region to control the target pest during the immediate growing season.

• Destroy Bt corn crop residues in the affected re gion within one month after harvest with a technique appropriate for local production practices to minimize the possibility of resistant insects over-winte ring and contributing to the next season's target pest population.

Additionally, if possible, and pr ior to the application of alte rnative control measures or destruction of crop residue, the registrant w ill collect samples of the insect population in the affected fields for laboratory rearing a nd testing. Such rearing and testing shall be conducted as expeditiously as practical.

Confirmed resistance

EPA defines confirmed resistance to mean, in the case of field reports of unexpectedCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

157 levels of damage from the key target pest s, that all the following criteria are met:

• There is > 30% insect survival and commensurate insect feeding in a bioassay, initiated with neonate larvae, that uses methods that ar e representative of exposure

to Bt corn hybrids under field conditions (ECB only).

• In standardized laboratory bioassays using diagnostic concentrations of the Bt protein suited to the target pest in ques tion, the pest exhibits resistance that has a genetic basis and the level of survivorship indicates that there may be a resistance allele frequency of ≥ 0.1 in the sampled population.

• In standardized labora tory bioassays, the LC 50 exceeds the upper limit of the 95%

confidence interval of the LC 50 for susceptible populations surveyed both in the original baselines developed for this pest species and in previous years of field monitoring.

(3) Response to Confirmed Resistance in a Key Target Pest as the Cause of Unexpected Levels of Damage in the Field

When field resistance is confirmed (as defined above), the following steps will be taken by the registrant:

• EPA will receive notification within 30 days of resistance confirmation;

• Affected customers and extension agents will be notified about confirmed resistance within 30 days;

• Monitoring will be increased in the affected area and local target pest populations will be sampled annually to determine th e extent and impact of resistance;

• If appropriate (depending on the resistant pe st species, the extent of resistance, the timing of resistance, and the nature of resistance, and th e availability of suitable alternative control measures), alternative control measures will be employed to reduce or control target pest populations in the affected area. Altern ative control measures may include advising customers and extension agents in the affected area to incorporate crop residues into the soil following harvest to minimize the possibility of over-wintering in sects, and/or applications of chemical insecticides;

• Unless otherwise agreed with EPA, stop sale and distribution of the relevant lepidopteran-active Bt corn hybrids in the affect ed area immediately until an effective local mitigation plan approved by EPA has been implemented;

• The registrant will develop a case-specific resistance management action plan within 90 days according to the characteri stics of the resistance event and local agronomic needs. The registrant will consu lt with appropriate stakeholders in theCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

158 development of the action plan, and the deta ils of such a plan shall be approved by EPA prior to implementation;

• Notify affected parties (e.g., growers, consultants, extension agents, seed distributors, university coopera tors and state/fede ral authorities as appropriate) in the region of the resistance situat ion and approved action plan; and

• In subsequent growing seasons, maintain sales suspension and a lternative resistance management strategies in th e affected region(s) for the Bt corn hybrids that are affected by the resistant population un til an EPA-approved local resistance management plan is in place to mitigate the resistance.

A report on results of resistance monitoring and investigations of damage reports must be submitted to the Agency annually by August 31 st each year for the duration of the conditional registration.

b. Analysis of the Risks Associated with Current IRM Plans and Alternatives

The risk that insect pests may become resistant to Bt plant-incorporated protectants and Bt microbial sprays has been acknowledged by many organizations and indi viduals including EPA's Scientific Advisory Panel (SAP) and Pestic ide Program Dialogue Committee (PPDC). SAP meetings and reports in 1995, 1998, and 2000 have c onfirmed that EPA's approach and elements required in an insect resistance management pl an are appropriate. EPA believes that pest biology and the dose of the Bt protein expressed in the variou s plant tissues influence the size and placement needed for an effective refuge. This section is a summary of the key elements of several options for IRM plans for corn and compar es the level of risk of resistance development for each scenario. An additional Agency assessment of the IRM plan for Bt corn can be found in the Agency’s memorandum, A. Reynolds a nd R. Rose (OPP/BPPD) to M. Mendelsohn (OPP/BPPD), dated September 11, 2000. IRM for TC 1507 was reviewed in R.Rose (OPP/BPPD) memorandum to M. Mendelsohn (OPP/BPPD) dated January 24, 2001. Subsequent information has been added to the Agency’s risk assessment of insect resistance development following the October 18-20, 2000 SAP mee ting and as new data became available. The Agency’s IRM program for Bt corn has also been described in Matten et al. (2004). This section has been updated (2010) to incorporate resistance monitori ng, compliance, and other data submitted since 2001 for the Cry1Ab and Cry1F corn registrations.

1) Pest Biology

Knowledge of pest biology is cr itical for the development of e ffective IRM strategies and to increase confidence that the IRM plans will be effective at reducing th e likelihood that insects

will become resistant to Bt proteins.

a) European Corn Borer (Primary Target Pest)Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

159 European Corn Borer (ECB, Ostrinia nubilalis ) is a major pest of corn throughout most of the United States. The pest has 1-4 generations pe r year, with univoltine (i.e., one generation per year) populations in the far North (i.e., all of North Dakota, northern South Dakota, northern Minnesota, and northern Wisconsin ), bivoltine (i.e., two gene rations per year) populations throughout most of the Corn Belt, and multivoltine (3-4 generations) populations in the South (Mason et al. 1996). The February, 1998 SAP m eeting on IRM identified a number of areasneeding additional research including larval movement, adult movement, mating behavior, preand post-mating dispersal, oviposi tional behavior, fitness, and ove rwintering habitat (SAP 1998). Since the first registrations of Bt corn hybrids in 1995, a significant amount of research has been undertaken in many of these areas, although addi tional work could enhance the knowledge base for this pest. A summary of key aspects of ECB biology that relate to IR M is presented below:

i. Larval Movement

ECB larvae are capable of significant, plant-to-pla nt movement within corn fields. Research conducted in non-transgenic corn sh owed that the vast majority of larvae do not move more than two plants within a row (Ross & Ostlie 1990). However, in transgenic corn, unpublished data (used in modeling work) from F. Gould (cite d in Onstad & Gould 1998) indicates that approximately 98% of susceptible ECB neona tes move away from plants containing Bt . Recent multi-year studies by Hellmich (1996, 1997, 1998) have attempted to quantify the extent of plant-to-plant larval movement. It was observed th at 4th instar larvae we re capable of movement up to six corn plants within a row and six co rn plants across rows from a release point. Movement within a row was much more likely th an movement across rows (not surprising, due to the fact that plants within are row are more likely to be “tou ching” as opposed to those across rows). In fact, the vast majority of across row movement was limited to one plant. This type of information has obvious implications for op timal refuge design. Larvae moving across Bt and

non- Bt corn rows may be exposed to sublethal do ses of protein, increasing the likelihood of resistance (Mallet & Porter 1992). Given the ex tent of ECB larval movement between plants,

seed mixes have been determined to be an in ferior refuge option (M allet & Porter 1992, SAP 1998, Onstad & Gould 1998).

ii. Adult Movement

Information on movement of adult ECB (post- pupal eclosion) is ne cessary to determine appropriate proximity guidelines for refuges. Refuges must be esta blished within the flight rangeof newly emerged adults to help ensure the potential for random mating. An extensive, multiyear project to investigate ECB adult disper sal has been undertaken by the University of Nebraska (Hunt et al. 1997, 1998a). Results from these mark and recapture studies (with newly emerged, pre-mated adults) showed that the major ity of ECB adults did not disperse far from their emergence sites. The percentage recaptured was very low (< 1%) and the majority of those that were recaptured were caught within 1500 feet of the release site. Few moths were captured

outside of 2000 feet. These resu lts have specifically led to recommendations and guidelines for refuge proximity and deployment (dis cussed later in this document).

iii. Mating BehaviorCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

160 In addition to patterns of adult movement, ECB ma ting behavior is an impor tant consideration to insure random mating between susceptible and potent ially resistant moths. In particular, it is important to determine where newly emerged female s mate (i.e., near the site of emergence or after some dispersal).

It is well established that many ECB take advantage of aggregati on sites (usually clusters of weeds or grasses) near corn fi elds for mating. Females typica lly mate the second night after pupal eclosion (Mason et al. 1996). One recent study suggested that it may be possible to manipulate aggregation sites to increase the likelihood of random mati ng between susceptible and potentially resistant ECB (H ellmich et al. 1998). Another recent study (mark/recapture studies with newly eclosed ECB) conducted by the University of Nebraska showed that relatively few unmated females moved out of the corn field from which they emerged as adults (Hunt et al. 1998b). This was especia lly true in irrigated (i.e., attr active) corn fields. In addition, a relatively high proportion of fema les captured close to the releas e point (within 10 feet) were mated. This work suggests that females mate ve ry close to the point of emergence and that refuges may need to be placed very close to Bt fields (or as in-field refuges) to maximize the probability of random mating.

In terms of male mating behavior, a study by Shower s et al. (2001) looked at male dispersal to locate mates. The study was carried out using mark-recapture techniques with pheromone-baited traps placed at 200, 800, 3200, and 6400 m from a releas e point. Results showed that males in search of mates were trapped more frequently at traps placed at 200 m fr om the release site. However, significant numbers were also trappe d at 800 m or greater fr om the release site (Showers et al. 2001). Similar to Hunt et al., th is work suggests that refuges may need to be placed relatively close to Bt fields to maximize random mating.

iv. Ovipositional Behavior

ECB ovipositional (egg-laying) behavior is impo rtant for refuge design. For instance, if oviposition within a corn field is not random, certain types of refuge (i.e., in-field strips) may not

be effective.

After mating, which occurs primaril y in aggregation sites, female s move to find suitable corn hosts for oviposition. Most females will oviposit in corn fields near the aggregation sites, provided there are acceptable corn hosts. Ovi position begins after mating and occurs primarily at night. Eggs are laid in clusters of up to sixty eggs (one or more clusters is deposited per night) (Mason et al. 1996).

It is known that females generally prefer taller and more vigorous corn fields for oviposition (Beck 1987). This has implications for refuge design. To avoid potential host discrimination among ovipositing females, the non- Bt corn hybrid selected for re fuge should similar to the Bt hybrid in terms of growth, matur ity, yield, and management practi ces (i.e., planting date, weed management, and irrigation). It should be noted that research has shown no significant difference in ovipositional preferences between Bt and non- Bt corn (derived from the sameCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

161 inbred line) when phenological and management characteristics are similar (Orr & Landis 1997, Hellmich et al. 1999). Within a corn field suitab le for egg laying, oviposition is thought to be

random and not restricted to border rows near aggregation sites (She lton et al. 1986, Calvin 1998).

v. Host Range

ECB is a polyphagous pest known to infest over 200 species of plants. Among the ECB plant hosts are a number of species of common weeds, wh ich has led some to speculate that it may be possible for weeds to serve as an ECB refuge for Bt corn. In response to this, a number of recent research projects have investigated the feasib ility of weeds as refuge. Studies conducted by Hellmich (1996, 1997, 1998) have shown that weeds are capable of producing ECB, although the

numbers were variable and too inconsistent to be a reliable source of ECB refuge. This conclusion was also reached by the 1998 SAP Subpanel on IRM. In addition to weeds, a number of grain crops (e.g., wheat, sorghum, oats) ha ve been investigated for potential as a Bt corn ECB refuge (Hellmich 1996, 1997, 1998, Mason et al. 1998). In these studies, small grain crops generally produced less ECB than corn (popcorn or field corn) and ar e unlikely to produce enough susceptible adult insects.

b) Corn Earworm

As was the case with ECB, the 1998 SAP identif ied a number of research areas that need additional work with corn earworm (CEW, Helicoverpa zea ). In addition to increased knowledge regarding larval/adult movement, ma ting behavior, and ovipositional behavior, a better understanding of movement between corn/cotton and long di stance migration is also needed (SAP 1998). Additional research re garding CEW biology has occurred since 1998. These data have been submitted as part of the annual research reports required as a condition of registration. The Agency has reviewed these data and has concluded that additional information would be useful for effective long-term impr ovements of IRM strategies to mitigate CEW

resistance.

i. Host Range and Corn to Cotton Movement

CEW is a polyphagous insect (3-4 generations pe r year), feeding on a number of grain and vegetable crops in addition to weeds and other wild hosts. Typically, it is thought that CEW feeds on wild hosts and/or corn for two generati ons (first generation on w horl stage corn, second generation on ear stage corn). After corn sene scence, CEW moves to othe r hosts, notably cotton, for 2-3 additional generations. By utilizing mu ltiple hosts within the same growing season, CEW presents a challenge to Bt resistance management in that there is the potential for double

exposure to Bt protein in both Bt corn and Bt cotton (potentially up to five generations of exposure in some regions).

Given the wide host range of CEW, it has been speculated that wild hos ts (weeds) and other nonBt crops (e.g., soybean) may be able to serve as refuge for CEW. Howeve r, research into the

value of these alternate hosts as reliable producers of CEW is s till lacking (1998 SAP).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

162 ii. Overwintering Behavior

CEW are known to overwinter in the pupal stag e. Although it is know n that CEW migrate northward during the growing season to corn-g rowing regions (i.e., the U.S. Corn Belt and Canada), CEW typically are not capable of overwintering in these regions. Rather, CEW are known to overwinter in the South, often in cotton fields. Temperature, moisture, and cultivation practices are all thought to play some role in the overwinter ing survival of CEW (Caprio & Benedict 1996).

Overwintering is an important consideration for IR M--resistant insects must survive the winter to pass their resistance genes on to future generati ons. In the Corn Belt, for example, CEW incapable of overwintering should not pose a resi stance threat. Given th at different refuge strategies may be developed based upon where CEW is a resistance th reat, accurate sampling data would help to precisely predic t suitable CEW overwintering areas.

iii. Adult Movement and Migration

CEW is known to be a highly mobile pest, capab le of significant long distance movement. Mark/recapture studies have shown that CEW mo ths are capable of dispersing distances ranging from 0.5 km (0.3 mi.) to 160 km (99 mi.) (some migration up to 750 km (466 mi.) was also noted) (Caprio & Benedict 1996). The general pa ttern of migration is a northward movement, following prevailing wind patterns, with moths originating in southern overwintering sites moving to corn-growing regions in the northern U.S. and Canada.

It has been assumed that CEW migration proceeds progressively northward through the course of the growing season. However, observations made by Dr. Fred Gould (N.C. State University) indicate that CEW may also m ove southward from corn-growing regions back to cotton regions in the South (described in remarks made at the 1999 EPA/USDA Workshop on Bt Crop Resistance Management in Cotton, Memphis, TN 8/26/99). If this is true (and more investigation is needed for confirmation of th is effect), the result may be additional CEW

exposure to Bt crops. In addition, the assumptions rega rding CEW overwintering may need to be revisited--moths that were thought to be incapable of winter surv ival (and thus not a resistance threat) may indeed be moving south to suitable overwintering sites.

Most CEW flight movement is lo cal, rather than migratory. He liothine moths move primarily at night, with post-eclosion moths typically flying short distance s of less than 200 m (Caprio & Benedict 1996). However, as was indicated by the 1998 SAP, additional research would be useful, particularly as it pertai ns to CEW and optimal refuge design. On the other hand, given the long distance movements typical of CEW and the lack of high dose in Bt corn hybrids, the 2000 SAP noted that refuge placement for this pest is of less importance than with other pests (e.g., ECB) (SAP 2001).

iv. Mating/Ovipositional BehaviorCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

163 Dr. Michael Caprio (entomologist , Mississippi State University) has indicated that there is significant localized mating among females (i.e ., within 600 m (1969 ft.) of pupal eclosion), typically with males that emerged nearby or m oved in prior to female eclosion (Caprio 1999). CEW females typically deposit eggs singly on hosts. A recent st udy (conducted in cotton fields) found that 20% of the eggs found from releas ed CEW females were within 50-100 m (164-328 ft.) of the release point, indicating some locali zed oviposition. However, males were shown to be able to move over 350 m (1148 ft.) to mate w ith females (Caprio 2000). These data indicate that, in terms of CEW, refuges may not have to be embedded or immediately adjacent to a Bt field to be effective (although the data do not exclude these options). Additional research with mating and ovipositional behavior would provi de useful information for CEW IRM.

v. Larval Movement

CEW larvae, particularly later instars, are cap able of plant-to-plant movement. At the recommendation of the SAP (1998), EPA has not eval uated seed mixes as a viable refuge option

for CEW.

c) Southwestern Corn Borer and Other Secondary Pests

Some southwestern corn borer (SWCB, Diatraea grandiosella ) pest biology data have been provided as part of the annual research reports re quired as a condition of re gistration. However, there is still relatively limited information available and more data on SWCB pest biology would be beneficial to help develop IRM strategies for regions in which SWCB and ECB are both pests

of economic concern. The 1998 SAP also noted th e relative lack of information for SWCB, concluding that “[c]ritical resear ch is needed for SWCB...includi ng: short-term movement, longdistance migration, mating behavior relative to movement (i.e. does mating occur before or after migration)...” Because of this, it is unknown wh ether IRM strategies designed for ECB (another corn boring pest) will also function optimally for SWCB.

SWCB is an economic pest of corn in some areas (i.e., SW Kansas, SE Colorado, north Texas, west Oklahoma) and can require regular manage ment. Like ECB, SWCB has 2-4 generations and similar feeding behavior. First generation larvae feed on whorl tissu e before tunneling into stalks before pupation, while late r generations feed on ear tissue before tunneling into stalks. Females typically mate on the night of emer gence and can lay 250-350 eggs (Davis 2000).

Research to investigate the m ovement patterns of SWCB has been initiated (Buschman et al. 1999). In this mark/recapture study, the followi ng observations were made regarding SWCB from the 1999 data: 1) more males than female s were captured at greater distances from the release point (similar to ECB); 2) most recaptures of SWCB were within 100 feet of the release site, although some were also noted at 1200 fee t; and 3) the moth movement patterns for ECB and SWCB appear to be similar in mo st regards. Given these results, it is likely that this part of the IRM strategy (refuge proximity guidelines esta blished for ECB) will also be applicable to SWCB. However, the 1999 results were hamper ed by low SWCB numbers available for testing

and the authors have indicated that this work will continue during the 2000 season.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

164 Research for other secondary pests (e.g., black cutworm, fall armyworm, southern corn stalk borer, others) is also lacking and could be usef ul for specific regions in which these pests may pose an additional concern. However, the 1998 SAP indicated that CEW and SWCB should have the highest priority for biology res earch among the secondary corn pests.

2) High Dose

A high level of Bt protein expression (termed “high dose”) is considered to be an essential aspect of high dose/structure refuge strategy to mitigate the risk of Bt resistance. The lack of a high dose could allow partially resist ant (i.e., heterozygous insects with one resistance allele) to survive, thus increasing the fre quency of resistance genes in an insect population. For this reason, numerous IRM researchers and expert groups have concurred that non-high dose Bt expression presents a substantial resistance ri sk relative to high dose expression (Roush 1994, Gould 1998, Onstad & Gould 1998, SAP 1998, ILSI 1998, UCS 1998). To mitigate the

additional resistance ri sk of a non-high dose Bt corn product, alternate refuge strategies (i.e., larger refuges) may need to be developed.

The 1998 and 2000 SAPs defined high dose as “25 times the protein concentration necessary to kill susceptible larvae” and provided five techniques to verify high dose (defined earlier in this document). However, the 2000 SAP noted that this definition is imprecise , provisional, and may require modification as more knowledge becomes av ailable about the inheri tance of resistance (SAP 2001). It is also important to consider protein expression over the course of the growing

season as some Bt corn hybrids may not maintain a steady level of protein expression over the

season. The 1998 SAP noted these concerns indica ting that the “toxin concentration encountered by the pest” should be the true measure.

Among the currently registered Bt corn products, most have been evaluated to determine high dose (via the 1998 SAP verification te chniques) for ECB (the primary target pest). It is likely that BT11, MON 810, and TC 1507 corn have a high dose for ECB. It is also known that none of the currently registered Bt corn products expresses a high dose for CEW (CEW is known to be less susceptible to Bt proteins than other targ eted lepidopteran pests). High dose evaluations for other secondary pests (i.e., SWCB , FAW, etc.) have been sporadic. Ideally, high dose could be evaluated for all susceptible pests, so that appr opriate resistance manageme nt strategies could be developed. However, verification of the high dose using the 1998 SAP Subpanel techniques may

be best directed at th e major target pests of Bt corn (ECB, CEW, and SWCB ), due to the fact that these pests play a larger role in the formula tion of IRM strategies. Below, each registered Bt corn product is discussed individually in rega rd to high dose (as defined by the 1998 SAP) for each of the labeled target pests. It is not expected that label clai ms of “control” or “suppression” for individual target pests are indicative of high dose.

a) Syngenta BT11 Cry1Ab Corn

According to grower guides, BT11 corn is targ eted against ECB (claims of “control”), SWCB (“control”), CEW (“control” of 1st generation, “suppression ” of 2nd gen.), FAW (“suppression”), and SCSB (“suppression”).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

165 Syngenta has not submitted any data to the Ag ency to confirm high dose, via the 1998 SAP guidelines, for any of the targeted pests. Howe ver, the Agency is able to conclude that BT11 probably produces a season-long high dose for ECB based on the review of all available data submitted to the Agency. Submitted studies have shown consistent control of ECB from the whorl stage to kernel maturi ty (VanDuyn et al. 1997, Catangui & Berg 1998). BT11 has also been shown to be effective against la te instar ECB (Walker et al. 2000).

For CEW, several submitted stud ies suggest that BT11 does not c ontain a season-long high dose.

These studies revealed excellent control of fi rst generation CEW on whorl stage BT11 but also showed significant survival of second gene ration CEW on BT11 corn ears (Dively & Horner 1997, VanDuyn et al. 1997). However, in both studies, surviving second generation CEW showed fitness costs (i.e., reduced weight and de layed developmental time). Other research has

shown similar results (VanDuyn et al. 1998).

For SWCB, no information on the potential for hi gh dose has been submitted to the Agency. For FAW, one submitted study with BT11 showed good control during whorl stage, but significant infestation during ear stage (Bened ict et al. 1998). It is theref ore unlikely that BT11 contains a full season high dose for FAW. For SCSB, one st udy with a limited data set has been submitted, showing good control (VanDuyn 1998). With addi tional data, it may be possible to confirm whether BT11 contains a high dose for SCSB.

b) Monsanto MON 810 Cry1Ab Corn

According to grower guides and product labels , MON 810 is targeted against ECB (claim of “control”), SWCB (“control”), SCSB (“control”), CEW (“supp ression”), CSB (“suppression”), and FAW (“suppression”).

For ECB, Monsanto has submitted information to verify (with the 1998 SAP guidelines) that MON 810 expresses a high dose (reviewed by EP A, R.Rose/S.Matten memo to M.Mendelsohn, 5/30/99). SAP techniques #2, 3, and 5 were ut ilized to confirm the high dose expression.

For SCSB, submitted research showed that MON 810 provided good control versus non- Bt corn (VanDuyn et al. 1997, VanDuyn 1998, VanDuyn et al. 1998), although there was not enough information (due to low pest pressu re in the tests) to determine if there is a high dose expression. With additional data, it may be possible to dete rmine whether there is a high dose expression for

control of SCSB.

For CEW, submitted studies have shown signi ficant larval survival on MON 810 corn, particularly in ear stage corn (Dively et al. 1997, Dively & Horner 1997, VanDuyn et al. 1997, Benedict et al. 1998, VanDuyn et al . 1998). Therefore, it is un likely that MON 810 expresses a season-long high dose for CEW. For FAW, MON 810 was found to have good whorl stage control, but significant ear infest ation later in the seas on (Benedict et al. 1998). Given this, and the known lower sensitivity of FAW to Cry1A proteins, it is unlikely that MON 810 has a season-long high dose for FAW. High dose has no t been verified for SWCB or CSB with theCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

166 1998 SAP techniques. With additional data, it may be possible to verify whether there is a high dose expression for control of SWCB or CSB.

c) Pioneer and Dow TC 1507 Cry1F Field Corn

TC 1507 is targeted against ECB, BCW, FAW, a nd SWCB (label claims “control” of these pests).

For ECB, data have been submitted to demonstrate high dose (using the 1998 SAP criteria - techniques #4 and #5) (MRID# 451311-01; re viewed in R.Rose memo to M.Mendelsohn, 1/24/01).

Other submitted data showed that TC 1507 pr ovided good protection against SWCB and FAW, although insufficient information was submitte d to determine high dose (MRID# 450201-14; reviewed in R.Rose memo to M.Mendelsohn, 1/24/ 01). This same data also showed some damage to TC 1507 plants from CEW and BCW. It is unlikely that TC 1507 expresses a high dose for these pests.

d) Syngenta Attribute Cry1Ab Sweet Corn

Attribute sweet corn is targeted against ECB, CEW, and FAW. Attribute contains the same Bt gene as the BT11 hybrid.

For ECB, like BT11, it is probable that Attribut e sweet corn expresses a high dose, although it

has not been verified with the SAP criteria. Re search submitted to EPA specifically for Bt sweet corn has shown virtually no surviv al of ECB (Dively & Linduska 1998).

For FAW and CEW, it is less likely that Bt sweet corn will express a high dose. Several submitted studies have shown (limited) FAW and CEW survival and damage on Attribute Bt sweet corn (Dively & Linduska 1998, Whal en & Spellman 1999, Lynch et al. 1999).

The current knowledge base for high dose expre ssion is summarized in the following table.

Table D2. High Dose Summary

SEASON-LONG HIGH DOSE FOR CORN PESTS

HYBRID ECB CEW SWCB FAW SCSB CSB

BT11 Probable NO Unknown NO Unknown Unknown *

Bt

Sweet Corn (BT11) Probable NO Unknown* NO Unknown * Unknown *

MON 810 YES NO Unknown NO Unknown Unknown

TC 1507 YES NO Unknown Unknown Unknown * Unknown *

YES = high dose verified with 1998 SAP recommended techniques; NO = information indicates that no high dose is likely; Probable = information indicates high dose likely (but not verified by SAP guidelines); Unknown = no or insufficient information available for high dose determination; * = untargeted pestCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

167 3) Refuge

The February 1998 and October 2000 FIFRA SAP Subpanels agreed that a high dose/refuge strategy is necessary to mitigat e target insect resistance to Bt field corn (SAP 1998, 2001). A structured refuge should be planted and managed to produce 500 insects susceptible to Bt for every one potentially resistant insect. Refuge options should address regional differences and varying levels of the dose of Bt in the crop that effect refuge management as well as the need for feasibility and flexibility for the growers. Howe ver, if there is not a high dose for the primary target pests, the risk of resi stance increases. Larger refuges, increased monitoring, and possible sales restrictions may be used to m itigate some or all of this risk.

a) Deployment of Refuges

There have been a number of approaches propos ed for the optimal design of refuges for Bt corn. These include external blocks, in-field strips, seed mixes, temporal refuge strategies, and noncorn hosts. A number of resear ch projects have been undertaken to identify the most appropriate refuge design.

i. Hosts for the Refuge

Non- Bt field corn should provide the best refuge to increase the probability that susceptible insects will mate with potentially resistant ECB from the Bt corn. Non- Bt corn hybrids used as refuges should be selected for growth, maturity, fertility, irri gation, weed management, planting date, and yield traits similar to the Bt corn hybrid. Hybrids that are not agronomically similar may result in different developmental times in corn pests that could lead to assortive (nonrandom) mating between plants in refuge and Bt fields.

Research has shown that temporal and alternate ho st, non-corn refuges (e.g., weeds, oats, alfalfa, soybeans) are inadequate stra tegies (Rice et al. 1997, Ostlie et al. 1997b, Calvin et al. 1997, Mason et al. 1998, Hellmich 1998). In addition, non- Bt popcorn may also be viable as refuge for

Bt corn (Hellmich 1998).

ii. Seed Mixes vs. In-Field Strips vs. External Blocks

The NC-205 group has recommended three options for refuge placement relative to Bt corn: blocks planted adjacent to fields , blocks planted within fields, or strips planted within fields (Ostlie et al. 1997). In general, refuges may be deployed as external blocks on the edges or headlands of fields or as strips within the Bt corn field.

Research has shown that ECB larvae are capable of moving up to six co rn plants within or between rows with the majority of movement occurring within a si ngle row. Later instar (4th and 5th) ECB are more likely to move within ro ws than between rows (Hellmich 1998). This is a cause for concern because heterozygous (partia lly resistant) ECB larv ae may begin feeding on

Bt plants, then move to non- Bt plants (if planted nearby) to complete development, thusCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

168 defeating the high dose strategy and increasing the risk of resistance. For this reason, seed mixes (refuge created by mixing seed in the hopper) have been discouraged as possible ECB refuges (Mallet & Porter 1992, Buschman et al. 1997).

Buschman et al. (1997) suggested that the within field refuge is the id eal strategy for an IRM program. Since the ECB larvae tend to move with in rows, the authors sugge st intact corn rows as an acceptable refuge. Narrow (filli ng one or two planter boxes with non- Bt corn seed) or wide strips (filling the en tire planter with non- Bt seed) may be used as in-fie ld refuges. Data indicate that in-field strips may provide th e best opportunity for ECB produced in Bt corn to mate with

ECB from non- Bt corn. Since preliminary data suggest s that the refuge should be within 100

rows of the Bt corn, Buschman et al. (1997) recommende d alternating strips of 96 rows of non- Bt

corn and 192 rows of Bt corn. This would result in a 33% re fuge that is within 100 rows of the

Bt corn.

In-field strips (planted as complete rows) should extend the full length of the field and include a minimum of six rows planted with non- Bt corn alternating with a Bt corn hybrid. NC-205 has recommended planting six to 12 rows of non- Bt corn when implementing the in-field strip refuge strategy (NC 205 Supplement 1998). The 2000 SAP also agreed that, due to larval movement, wider refuge strips ( ≥ 6 rows) are superior to narrower stri ps, although planter sizes may restrict strip sizes for some smaller growers (SAP 2001). In -field strips may offer the greatest potential to ensure random mating between susceptible and resistant adults because they can maximize adult genetic mixing. Modeling indicat es that strips of at least six rows wide are as effective for ECB IRM as adjacent blocks when a 20% refuge is used (Onstad & Guse 1999). However, strips that are only two rows wide might be as effective as blocks, but may be more risky than either blocks or wider strips given our incomp lete understanding of differences in survival between susceptible borers and he terozygotes (Onstad & Gould 1998).

Given the concerns with larval movement and n eed for random mating, either external blocks or in-field strips (across the entire field, at leas t 6 rows wide) are the refuge designs which may provide the most reduction in risk of resistance development. Research indicates that random mating is most likely to occur with in-field strip refuges.

iii. Proximity

The issue of refuge proximity is a critical variab le for resistance management. Refuges must be located so that the potential for random mating between susceptible moths (from the refuge) and possible resistant survivors (from the Bt field) is maximized. Therefor e, pest flight behavior is a critical variable to consider when discussing refuge proximity. Refuges planted as external blocks should be adjacent or in close proximity to the Bt corn field (Onstad & Gould 1998, Ostlie et al. 1997b). NC-205 initially recommended that refuges should be planted within 1⁄2 sections (320 acres) (NC-205 Supplement 1998). Subseq uently, the recommendation was revised to specify that non- Bt corn refuges should be pla ced within 1/2 mile of the Bt field (1/4 mile

“would be even bett er”) (Ortman 1999).

Hunt et al. (1997) completed a st udy which suggests that the major ity of ECB do not disperse farCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

169 from their pupal emergence sites. According to this mark-recapture study, the majority of ECB may not disperse more than 1500 to 2000 feet. A majority (70-98%) of recaptured ECB were trapped within 1500 feet of the release point. However, in an addendum to the 1997 study, the

authors caution that the 1500 foot distance does not necessarily represent the maximum dispersal distance for ECB (H unt et al. 1998a).

Another mark-recapture ECB project was devoted to within-field movement of emerging ECB (in particular unmated females) (Hunt et al. 1998b). Relatively few unmated females were recaptured (10 over the entire e xperiment), although the majority of those were found within 85 ft of the release point. This suggests that unmated females may not disperse far from the point of pupal eclosion (this was especially true in the irrigated field) . In addition, a relatively high proportion of mated females (31%) in irrigated fields were trapped within 10 feet of the release point, suggesting that mating occurr ed very close to the point of emergence. Both of these observations indicate that many emerging ECB female s may not disperse outside of their field of origin. With respect to resistance management and refuge proximity, thes e results suggest that refuges should be placed in close proximity to Bt corn fields (or as in-fie ld refuge) to increase the chance of random mating (especia lly for irrigated fields).

In terms of male ECB dispersal, another mark-re capture study by Showers et al. (2001) showed that males dispersing in search of mates may mo ve significant distances (> 800 m). However, a greater percentage of males were trapped at closer distances (200 m) to the release point. Based on this research, the authors suggest that, in terms of male movement, the current refuge proximity guidelines of 1⁄2 mile should be adequa te to ensure mating between susceptible moths and any resistant survivors from the Bt field.

While it is clear that ECB dispersal decrea ses further from pupal emergence points, the quantitative dispersal behavior of ECB has not been fully determined. However, in terms of optimal refuge placement, it is critical that refuge proximity be selected to maximize the potential for random mating. Based on Hunt et al. data, the closer the refuge is to the Bt corn, the

lower the risk of resistance. Si nce the greatest number of ECB were captured within 1500 feet of the field and most females may mate within ten f eet of the field, placing refuges as close to the

Bt fields as possible should increase the chan ce of random mating and decrease the risk of resistance. The proximity requirement for Bt corn refuge was initially established as 1⁄2 mile (1/4

mile in areas where insecticides have been hist orically used to treat ECB and SWCB) (EPA letter

to Bt corn registrants, 1/31/00). The 2000 SAP agreed with this gui deline, stating that “...refuges

should be located no further than a half mile (within 1/4 mile if possible) from the Bt corn field” (SAP 2001).

In 2008 and 2009, the Bt corn registrations were amended to simplify the distance proximity requirement to read “external re fuges must be planted within 1⁄2 mile” (see EPA review in A. Reynolds memo to M. Mendelsohn, 7/9/08).

iv. Temporal and Spatial Refuge

The use of temporal and spatial mosaics has recei ved some attention as alternate strategies toCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

170 structured refuge to delay resi stance. A temporal refuge, in theory, would manipulate the life cycle of ECB by having the Bt portion of the crop planted at a time in which it would be most attractive to ECB. For example, Bt corn fields would be planted several weeks before conventional corn. Because ECB are thought to pr eferentially oviposit on ta ller corn plants, the hope is that the Bt corn will be infested instead of the s horter, less attractive conventional corn. However, there are indications from experts in th e field that temporal refuges are an inferior alternative to structured refuge s (SAP 1998). Research has shown that planting date cannot be used to accurately pr edict and manipulate ECB oviposition ra tes (Calvin et al. 1997, Rice et al. 1997, Ostlie et al. 1997b, Calvin 1998). Local clim atic effects on corn phenology make planting date a difficult variable to manipulate to mana ge ECB. Additional studies will have to be conducted under a broad range of conditions to fu lly answer this question. In addition, a temporal mosaic may lead to assortive mating in which resistant moths from the Bt crop mate with each other because their developmental time differs from susceptible moths emerging from the refuge (Gould 1994).

Spatial mosaics involve the planting of two separate Bt corn events with different modes of action. The idea is that insect populations will be exposed to multiple proteins, reducing the likelihood of resistance to any one protein. However, because many of the registered Bt corn products only express one protein and the primar y pests of corn (ECB, CEW, SWCB) generally remain on the same plant throughout the larval feed ing stages, individual insects will be exposed to only one of the proteins. In the absence of structured refuges produci ng susceptible insects, resistance may still have the poten tial to develop in such a system as it would in a single protein

monoculture.

v. CEW North to South Movement and Refuge Issues

It is known that during the growing season CEW move northward from southern overwintering sites to corn-growing regions in th e Corn Belt. However, as disc ussed in the pest biology section (D.2.b.1.b.iii), observations of CEW north to sout h migration (from corn-growing regions to cotton-growing regions) have been noted. Altho ugh more research is needed for confirmation, this phenomenon could result in additional exposure to Bt crops and increased selection pressure

for CEW resistance. This effect is compounded by the fact that neither Bt cotton nor any registered single trait Bt corn event contains a high dose for CEW. As such, it may be necessary to consider additional mitigation measures for CEW.

In considering this issue, the 2000 SAP indicated that CEW refuge is best considered on a regional scale (instead of structur ed refuge on an individual farm basis) due to the long distance movements typical of this pest (i.e., refuge proxi mity is not as important for CEW). According to the SAP, a 20% refuge (per farm) would be adequate for CEW, provided the amount of Bt corn in the region does not exceed 50% of the total corn crop. If the regional Bt corn crop exceed 50%, however, additional structured refuge may be necessary (SAP 2001). However, the SAP did not define what a “region” should be (i.e., county, state, or other division).

Based on the last available acreage data for Bt corn, it should be noted that a number of counties

in the Corn Belt exceed the 50% threshold recognized by the 2000 SAP. Because of this, thereCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

171 may be additional risk for CEW resistance. As a condition of registra tion (EPA letters to Bt corn registrants, October 15, 2001), Bt corn registrants were required to investigate CEW north-south movement as it relates to resistance manageme nt and submit a report to EPA. To accomplish this task, the Agricultural Stewardship Tec hnical Committee (ABSTC, a consortium representing

Bt corn registrants) conducted 1) a review of studies on the extent of CEW north-south migration and 2) computer modeling to evaluate the effect of north-south migration on the risk of CEW adaptation to Bt corn and Bt cotton in a mixed cropping system. This information was submitted to the Agency in a report dated May 8, 2003 (no MRID number).

Fitt (1989) reviewed evidence for long-range mi gration of CEW in North America. Other noctuids, black cutworm ( Agrotis ipsilon ) and fall armyworm ( Spodoptera frugiperda ) have also been shown to migrate from southern overwin tering areas to the Corn Belt. Long distance migration in all of these insect s was associated with passage in weather fronts. Showers (1997) provided evidence for the return migration of A. ipsilon on southerly air mass flows in the fall

from Iowa to Louisiana and Texas. Pair et al. (1987) presented evidence that CEW return migration can occur from north Texas to the Lower Rio Grande Valley.

Gould et al. (2002) provided indi rect evidence for migration of CEW moths from the other corngrowing regions to the cotton-grow ing regions. Data were collected from two locations – Bossier Parish, Louisiana and College Station, Texas. The timing and extent of reverse migration appears to vary considerably from year to year (Gould et al. 2002, figs. 2, 3, and 4). Gould et al. (2002) pointed out that at the time of apparent re verse migration, there is very little vegetation that is capable of supporting larval development in Texas. This m eans that it is unlikely that any migrating moths (from the north) can contri bute to the local ove r-wintering population. However, if the moths can move further south into more tropical areas in which they would

contribute to the local over-win tering population then their ge nes could contribute to the persisting population. There may be locally suitable host plants in cotton-growing regions (other than in the examined area of Texas) such th at a migrating population could be important.

ABSTC noted that Gould et al. (2002) did not addre ss the point that cotton in the areas investigated is treat ed for CEW either by Bt PIPs, applied chemical insecticides, or both. Bt PIPs and insecticides are very effec tive at reducing boll damage and CEW larval survival by 80% or

more. This means that the number of moths produced in cotton would be relatively small compared to the number of eggs laid in cotton. The converse situation is true for Midwest corn, up to 25% of which is Bt corn, which is seldom if ever trea ted for CEW. ABST C suggested that care should be taken when interp reting the Gould et al . (2002) data that the relative importance

of selection in the Corn Belt versus the Cotton Belt not be overstated.

For the second part of the anal ysis, ABSTC used a computer model to quantify how migration may impact adaptation rates under a range of di fferent circumstances. The spatially-explicit model of CEW adaptation to corn and cotton (Storer et al. 2003) was modified to incorporate south-north migration in the spring and north-sou th migration in the summer. Effectively, two models were run in parallel for the summer ge nerations: one for the cotton-growing region and one for the corn-growing region. The Storer et al. (2003) model focused on CEW adaptation in eastern North-Carolina; however, alte rnations to the adaptation risk in that region are likely to beCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

172 quantitatively similar to the alternations to the ri sk in other regions. The scenarios modeled were

based on the available data and the conclusions of Gould et al. (2002).

The results of the modeling showed no significant interaction between the percent of the late summer adult CEW population in the south that is made up of immigrants and the date at which return migrants actually return. This means that even with 50-60% CEW migrating from the north (as inferred by Gould et al. 2002) and prod ucing pupae, there was no effect of return migration on the resistance gene frequency.

There was, however, a weak intera ction between the percentage of Bt corn planted in the north and return migration. At 80% Bt corn in the north, return migr ation increased the 15-year gene frequency from 2.2e -3 to 2.4e -3 , an increase in adaptati on rate of 2%. At 30% Bt corn in the north, return migration decreased the 15-year gene frequency from 2.3e -3 to 2.1e -3 , a decrease in adaptation rate of 4%. Thus, even with extreme adoption rates of Bt corn of 80%, return migration had very little effect on th e CEW adaptation rate in the south.

There was a trend for return migration to slow resistance evolution when investigating the percentage of insects moving north. This is due to the returning population having a lower resistance (r) allele frequency th an the resident population. The ma in decrease in adaptation rate due to return migration was 5%.

Two of the parameters had significant main eff ects on the 15-year r-allele frequency: increasing the percentage of Bt corn or Bt cotton in the south increased the 15-year r-allele frequency. These effects are expected because the percentage of Bt corn and Bt cotton determine the intensity of selection exerted on the insects feedi ng in these two crops. At the highest level of Bt cotton deployment simulated (95%), selection in the south is most intense and return migrants are expected to reduce the adapta tion rate by introducing a populat ion that has experienced lower selection. However, this model suggests that even under this s cenario, return migration would not significantly affect the rate of adaptation.

EPA reviewed ABSTC’s report (S. Matten memo to M. Mendelsohn, 3/23/04) and agreed with ABSTC’s analysis and conclusions. Base d on the modeling studies submitted by ABSTC parameterized using the data in Gould et al. (2002), CEW reverse migration has no significant impact (0.05<P) on CEW adaptation to Bt crops. Under current levels of Bt crop deployment (30% Bt corn and 60% Bt cotton), return migration is expected to slow adaptation by about four percent (4%). If Bt corn reached its maximum allowable le vel of 80% adoption in the Corn Belt, reverse migration would only increase the rate of adaptation by about two percent (2%) if Bt cotton was at 60% adoption (the low end of the range investigated). Even if extreme parameters were used in the model, reverse migration w ould be predicted to have a 10% impact on CEW adaptation. However, it is extremely unlikely th at all of these parameter conditions would be met in the field year after year. Modeling studies indicated that the percentage of Bt cotton and

Bt corn in the south significantly increased the 15-year r-allele frequency, but return migration did not significantly affect the rate of adaptation even when Bt cotton was at 95% adoption.

b) Refuge OptionsCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

173 This analysis of refuge options pertains only to single toxin PIPs expressing Cry1Ab or Cry1F. Refuge options for pyramided or stacked products containing these (and other) toxins are not

discussed here.

i. High Dose Events ; MON 810, BT11, TC 1507 (Field Corn)

Non-Cotton Growing Regions That Don't Spray Insecticides on a Regular Basis (e.g., Corn Belt)

This region encompasses most of the Corn Belt eas t of the High Plains. The original USDA NC205 refuge recommendations include d a 20-30% untreated structured refuge or a 40% refuge that

could be treated with non- Bt insecticides (Ost lie et al. 1997a). In the case of ECB, the primary pest of corn for most of the U.S., it is know n that on average less th an 10% of growers use

insecticide treatment to control this pest (National Center for Food and Agriculture Policy 1999). Due to the fact that many growers do not re gularly treat for ECB, NC-205 modified their position in a May 24, 1999 letter to Dr . Janet Andersen (Director, B PPD). In this letter, NC-205

amended their recommendation to a 20% non- Bt corn refuge that may be treated with insecticides and should be deployed within 1/2 mile (1/4 mile is better) of the Bt corn. Specific recommendations in the letter were: “1) insect icide treatment of refuges should be based on scouting and accepted economic thre sholds, 2) treatment should be with a product that does not

contain Bt or Cry toxin, 3) records shoul d be kept of treated refuges and shared with the EPA, 4) the potential impact of sprayed refuges should be monitored closely and evaluated annually, and 5) monitoring for resistance should be most in tense in higher risk areas, for example where refuges are treated with in secticides” (Ortman 1999).

Since most growers (>90%) do not typically treat fi eld corn with insecticides to control ECB, a refuge of 20% non- Bt corn that may be sprayed with non- Bt insecticides if ECB densities exceed

economic thresholds should be viable for the Corn Belt. Refuges can be treated as needed to control lepidopteran stal k-boring insects with non- Bt insecticides or other appropriate IPM practices. Insecticide use should be based on scou ting using economic thresholds as part of an IPM program.

Non-Cotton Growing Regions That Spray Insecticides on a Regular Basis (e.g., the High Plains for SWCB)

NC-205 (1998) noted that there ar e some areas that regularly re quire insecticide treatment (e.g., the High Plains for SWCB or spider mites) and that separate refuge strategies may be needed for these regions. This is because highly effective insecticides may significantly reduce the number of susceptible adults emerging from the refuge. In a May 1999 letter sent to Dr. Andersen (BPPD Division Director), NC-205 stated: “A refuge management strategy that is more conservative than the one applied across the greater Corn Belt, yet less restrictive than the one proposed for areas growing both corn and cotton, ma y be most appropriate in the heavily treated areas jointly infested with SW CB and ECB” (Ortman 1999). The size of the refuge is based on

the amount of non- Bt corn needed to produce 500 susceptibl e insects for every resistant insect. When insecticide sprays are used on the refuge , fewer susceptible insects are produced and the refuge area may need to be la rger to produce the 500:1 ratio.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

174 Entomologists from Kansas State University (Dr. Randy Higgins, Dr. Lawrence Buschman, and Dr. Phillip Sloderbeck) have indicated that the fr equent use of highly effective insecticides in

areas that are co-infeste d with both SWCB and ECB is the issu e of concern rather than the mere presence of SWCB. Using highly effective insec ticides in these areas will decrease the number of susceptible insects emerging from the ref uge and reduce refuge efficacy (Buschman and Sloderbeck 1999; Higgins 1999). The 2000 SAP rationa lized that a 20% refuge treated with an insecticide with high efficacy (> 90% kill) will be equivalent to a 2% unsprayed refuge (SAP 2001). As a result of the Agency’s new IRM requirements for Bt corn products for the year 2000, areas that are routinely treated with inse cticides were specifi cally identified by the Agricultural Biotechnology Stewardship Technical Committee (ABSTC) in a letter to Dr. Janet Andersen dated March 31, 2000. This area incl udes counties in southwest Kansas, southeast Colorado, and the Texas/Oklahoma Panhandle.

After reviewing the insecticid e issue, the 2000 SAP conclude d that insecticide use may negatively impact IRM if only the refuge (and not the Bt crop) is treated. The panel did not, however, reach a consensus on whether additional measures would be needed to mitigate the potential risk. Some panel members felt that add itional refuge is needed in these areas, while others thought that current refuge requirements (20%) are adequate and would help maintain compliance. Another potential mitigation alternative proposed by the panel was to restrict insecticide use to allow for only those treatments that provide <70% kill. The panel also noted

that additional information would be needed to define these areas and that the NC-205 group was looking at the issue and was planning to survey grower insecticide use practices (SAP 2001).

To address concerns about risks to IRM strategies, registrants of Bt field corn were required to investigate the potential impact of in secticide use on the effectiveness on non- Bt corn refuges as a condition of registration (EPA letters to Bt corn registrants, 10/15/01) . A protocol was required to be submitted by March 15, 2002, followed by an in terim report in 2003 and the final report in 2004. ABSTC submitted a protocol which wa s accepted by the Agency on May 8, 2002 (EPA letter to ABSTC). An interim report was s ubsequently submitted on May 8, 2003 and the final report was submitted on May 7, 2004 ( no MRID number was assigned).

ABSTC employed two separate techni ques to address the issue of ins ecticide use in refuges. The first was to conduct a survey of crop consultants to assess insecticide us e in regions known to frequently treat for corn borers while the second method was to utilize models to simulate the

effects of insecticide use on th e effectiveness of refuges.

For the survey (conducted in 2002), a total of 220 questionnaires we re mailed to crop consultants in three states where insecticides are frequently used (Nebraska, Kansas , and Colorado) with a response rate of 45% (96 total). Responses we re divided by reporting re gions and survey state quarters as follows: Southwest (SW Kansas, SE Kansas, and SE Colorado), West (NW Kansas, SW Nebraska, and NW Nebraska), and East (NE Kansas, SE Nebraska, and NE Nebraska). The southwest region experiences regu lar control issues with ECB, SWCB, and spider mites. The west region experiences ECB and spider mite in festations (but little SWCB), while the east region primarily has ECB control issues (and little SWCB or spider mite problems).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

175 The results showed that the vast majority of co rn grown in all three re gions is irrigated with consultants overseeing an average of 9,978 acres. Consultants manage most of the corn in all three regions. The percentage of Bt corn acreage was lowest in th e west region (31%) and higher in the southwest (47% irrigated, 29% non-irrigated) and highest in the east (54%). Insecticide use patterns were as follows:

Early Whorl Stage: Insecticides were applied on ear ly whorl stage, irrigated corn on 3.4% of Bt corn acreage and 7.5% of non- Bt corn acreage during the 2002 season. On non-irrigated corn, insecticide us e was slightly less: 1.9% ( Bt ) and 3.8% (non- Bt ). Treatments were applied on more acreage in th e east region than the southwest or west regions, although the differences between region s were not significant. The historical (1998-2002) patterns were similar with 0.7% irrigated Bt corn, 9.6% irrigated non- Bt corn, 1.2% non-irrigated Bt corn, and 3.6% of non-irrigated, non- Bt corn acreage treated (the east region reported the highest percentage of treated acres).

Late Whorl Stage: Insecticide use was significantly higher on irrigated, late-whorl stage corn. Overall, 26.7% of Bt corn and 44.9% of non- Bt corn acres were treated during 2002, with the highest insecticide use in the southwest region. For non-irrigated corn,

insecticide use was much less: 1.5% Bt acreage and 1.0% non- Bt acreage. The survey

authors attributed the lower in secticide use in non- irrigated corn to drought conditions. Of the treated acreage, a minority was treate d exclusively for corn borers (most reported treating for both borers and ot her pests or exclusively for other pests) and single treatments were more common than repeated applications. The historical (1998-2002) patterns reflected the 2002 data, with 15.4% irrigated Bt corn, 31.2% irrigated non- Bt corn, 2.0% non-irrigated Bt corn, and 9.2% of non-irrigated, non- Bt corn acreage treated (the southwest region reported the highest per centage of treated acres). In both the 2002 and historical survey data, consultants ge nerally reported treating small (i.e., <50%) percentages of their total acreage (most claime d to have treated <30%). However, the

number of consultants who described treati ng 90-100% of their to tal acreage was highest in the southwest region.

In the second part of ABSTC’s analysis of the effects of insecticide use on Bt corn refuges, the data generated in the crop consultant survey was incorporated into two resistance management

models. ABSTC indicated that two models we re used: Guse et al. (2002), which includes variables for irrigation and pest biology (dispersal) and Onstad et al. (2002) which allows for the evaluation of insecticide use in refuges. Howeve r, data were reported only for the Onstad model

in the submission.

Data regarding insecticide efficacy and use fre quency was obtained from the consultant survey and applied to the models. In terms of efficac y, a value of 70% was assigned based on the lower

effectiveness of insecticides on co rn-boring lepidoptera. In practi ce, eggs and neonate larvae are more likely to be exposed and killed by insec ticide treatments, while later instar larvae frequently lodge themselves inside corn plan ts and escape exposure. For insecticide use frequency, a value of 0.25 was assigned (i.e., 25% of refuges treated). This value was derivedCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

176 from the consultant survey which indicated that less than 50% of non- Bt corn is treated and that the majority of those treatments are targeted at second generation corn borers. Therefore, with

50% of non- Bt corn acres treated for the second ge neration (i.e., 50% of the annual borer generations), the total net effect is 25% treatment of the functional refuge within a growing season. Non-irrigated corn was not included in the simulations due to the infrequent use of

insecticides on those acres.

For dose expression, the definitions used by Onst ad et al. (2002) for theoretical high dose (recessive resistance) and practical high dose (partially recessive re sistance) were included in the simulation. Refuge sizes were set between 20 and 50% (the 50% value was derived from the

total amount of non- Bt corn planted in the survey regions). Biological parameters for ECB and SWCB dispersal were also incorporated into the models.

Simulations were run for both SWCB and ECB. For SWCB, the Onstad model predicted that normal patterns of insecticide use (i.e., those identified in the cr op consultant survey) would have no impact on refuge effectiveness. Effects were noted only if resistan ce was assumed to be

dominant or insecticides were extremely effective (i.e., 99.9%), scenarios that are extremely unlikely for SWCB and Bt corn.

For ECB, the picture was somewhat more compli cated. When the Onstad model was run with parameters discussed above (spray frequency = 0.25, insecticide efficacy = 70%), the time to resistance was reduced by a third (30 years to 20 years). However, the use of irrigated corn was a key factor in the simulations run in the Onst ad model. ECB dispersal is more limited in irrigated corn than dryland corn, as demonstrat ed by Hunt et al. (2001). When the reduced ECB dispersal characteristic of irriga ted fields was incorporated into the model, refuge treatment had no impact on resistance over a 100 year period. This revised simulation also utilized more conservative values for spray frequency (50% - all refuges treated for second generation ECB) and spray efficacy (90%).

EPA reviewed the data submitted by ABSTC (A. Reynolds memo to M. Mendelsohn, 7/6/04) on the IRM impacts of insecticides in refuges. Ba sed on the crop consultant survey and simulation model (Onstad et al. 2002), it is unlikely that refuges for SWCB will be negatively affected by insecticide use practices. For ECB, the model pr edicted that sprays on non-irrigated corn could decrease the time to resistance by a third. Ho wever, according to the consultant survey, nonirrigated corn is rarely treated (<10% of acreage) in the survey re gions. For irrigated corn (more likely to be treated), detrimental effects on refuge from insecticide treatments were not observed, largely due to increased ECB dispersal. Overa ll, it is likely that any impact on potential ECB resistance from insecticide use in “high sp ray” regions will be small or negligible.

ii. High Dose (MON 810, BT11, and TC 1507) Field Corn Events in Cotton-Growing Regions

As part of their April 19 99 and January 2000 submissions, the NCGA/Industry Coalition requested growers be required to plant a minimum of 20% non- Bt corn in the northern portion of the corn/cotton region. The northern corn/co tton region corresponds to northern Arkansas,Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

177 Missouri Bootheel, northern Texas, and the stat es of North Carolina, Oklahoma, Tennessee and Virginia. A minimum 50% refuge of non- Bt corn was suggested for the southern portion of the corn/cotton-growing region. The so uthern corn/cotton region corres ponds to the entire states of Alabama, Florida, Georgia, Louisiana, Mississi ppi, South Carolina, as well as southern Texas

and southern Arkansas.

Cotton-growing regions represent a higher risk for resistance due to the potential double exposure of CEW to both Bt corn (Cry1Ab, Cry1F) and Bt cotton (Cry1Ac) during the same growing season. Dr. Mike Caprio (Mississipp i State University) de veloped a corn-cotton ecosystem model for resistance evolution in CEW to Bt -endotoxins expressed in plants to examine the movement of CEW between corn, cotton, soybean, and othe r wild hosts (Caprio 1997). In the model, the presence of Bt cotton (160 fields) and the ratio of Bt corn/non- Bt corn fields (120 total fields) are importa nt factors. As the ratio of non- Bt corn decreases relative to Bt corn, the time to resistance also decreases; meaning that less non- Bt corn planted as a refuge results in quicker resistance. This eff ect was most pronounced when the percent of Bt to non- Bt corn exceeded 50%. Caprio’s model suggests that even without cross-resi stance as a variable, a sizable proportion of non- Bt corn (at least 50%) should be planted with Bt corn in Bt cotton growing regions to avoid the quick evolution of resist ance. The years to resistance are also impacted by the percent of Bt cott on relative to Bt corn. A second model, developed by Storer et al. (1999), has also examined CEW resistance in corn/cotton regions (represented by eastern North Carolina). This model showed that resist ance can develop rapidly wh en the percentage of

Bt cotton is high relative to Bt corn (which is true for some northern cotton growing regions), underscoring the need for robust refuge in these regions.

In terms of the proposed “northern cotton- growing region,” a signi ficant increase in Bt cotton in these areas has been observed over the past several growing seasons. From 1996 to 1999, the percent Bollgard acreage increased in North Ca rolina from 3% to 19% (total increase: 250,000 acres), in Oklahoma from 7% to 20% (total in crease: 57,773 acres), in Tennessee from 2% to 68% (total increase: 380,000 acres), and in Virginia from 1% to 7% (total increase: 6,214 acres) (MRID# 450294-01). This shows that the Bt cotton acreage cannot be predicted accurately and may not be an appropriate jus tification for reduced refuge.

Dr. Fred Gould (North Carolina State University) has also identified resistance risk issues in southern cotton growing regions (described in remarks made at the 1999 EPA/USDA Workshop

on Bt Crop Resistance Management in Cotton, Memphi s, TN 8/26/99). According to Dr. Gould, CEW are thought to feed on corn in Mexico in the early spring before moving to cotton in the southern U.S. and ultimately corn in more norther n areas. If these CEW diapause in the northern areas and die over the winter, they pose no resistan ce problem. However, some indirect evidence

has indicated that at least some CEW move fr om northern areas to southern cotton growing regions to overwinter. CEW that move from the north to south to overwinter could be exposed for four generations or more to Bt crop hosts. Along these lines, data were developed by ABSTC

to address this concern and are discussed in th e previous section. Based on the modeling studies submitted by ABSTC parameterized using the da ta in Gould et al. (2002), CEW reverse migration has no significant impact on CEW adaptation to Bt crops.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

178 Drs. Caprio, Van Duyn, and Gould recommend a minimum of a 50% non- Bt corn refuge that may be treated only as necessary with non- Bt insecticides is needed in all cotton-growing regions

to reduce the risk of resistance. Smaller refuges may present a greater risk and may result in a more rapid evolution of resistance. Since cott on is a preferred overwintering site for CEW, postharvest plowing of Bt cotton fields to destroy potentiall y overwintering CEW pupae may also be

an effective tool to decrease the risk of resistance, but further research is necessary.

iii. Non-High Dose Events

Non-Cotton Growing Regions That Do Not Spray Insecticides on a Regular Basis (e.g., Corn Belt)

As indicated earlier, there ar e no specific non-high dose produc ts for ECB that have been

considered in this scientific assessment. It is also clear that a high dose/refuge strategy is preferred for IRM with Bt crops. However, an assessment of non-high dose is included here to provide a comprehensive revi ew of all possibilities.

Research regarding ref uge size for non-high dose Bt events is limited. In general, non-high dose

Bt corn hybrids pose a higher risk (approximately five times higher) of resistance than high dose events (Onstad & Gould 1998). The International Life Sc iences Institute/Health and Environmental Sciences Institute (ILSI/HESI) recommended larger refuges (e.g., 40% unsprayed in the North) for non-high dose or high risk va rieties (ILSI 1998). Th e Union of Concerned Scientists (UCS) also suggested that a separa te resistance management strategy should be developed for varieties that do not meet the high dose refuge strategy. UCS recommended a 50% refuge that should not be sprayed with insecticides for Bt corn varieties that do not contain a high dose (UCS 1998).

For non-high dose events, larger refuges ma y be necessary (Gould 1998, ILSI 1998, UCS 1998). Based on the ILSI and UCS repor ts, at least a 40% unsprayed refuge in non-cotton growing regions (Corn Belt) would be needed to mitigate the threat of resistance. According to the National Center for Food and Agriculture Polic y (1999), the percent in secticide use for ECB control in U.S. field corn is on average < 10%. Since most refuges will not be routinely sprayed and some growers need the option of spraying if pests reach economic injury levels, mandating an unsprayed refuge should not be necessary. Th e risk of insect resistance to the non-high dose events could also be limited by restricting sales (e.g., a total sales cap or in areas where ECB are univoltine). Since ECB exposure to Bt is limited in areas where th ere is one generation per year, restricting the use of non-high dos e events to these areas will li kely decrease the risk of

resistance.

Non-Cotton Growing Regions That Spray Insecticides on a Regular Basis (e.g., the High Plains for SWCB)

Non-high dose plants have an increased risk of insect resistance which is compounded if the refuge is sprayed with insecticides. The IL SI panel has recommended larger refuges for nonhigh dose or “high” risk Bt corn varieties. For areas where the refuge will be sprayed withCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

179 insecticides, the ILSI recommended an 80% non- Bt corn refuge (ILSI 1999). Since there may be

an increased risk of resistance in areas that ar e routinely sprayed with insecticides, restricting sales of non-high dose events could reduce the risk. In addition to planting restrictions, larger refuges (e.g., the ILSI Panel's recommended 80% in secticide treatable refuge) are an option that could be implemented to mitigate the risk of resistance.

4) Monitoring

a) Monitoring Strategies

A monitoring program for Bt corn is useful to evaluate the effectiveness of resistance

management programs. Detecting shifts in the frequency of resistance genes through resistance monitoring can be an aggressive method to detect the onset of resistance before widespread crop

failure occurs.

In general, resistance monitoring plans should in clude a detailed sampling strategy for all pests susceptible to the expressed Bt proteins regardless of whether th ey are stated on the label. For Bt field corn and sweet corn, the susceptible pe sts would include, but are not limited to: ECB, SWCB, and CEW. To be effective, the monito ring for resistance should be undertaken in areas where the pests are known to regu larly overwinter. For FAW a nd BCW (target pests of TC 1507 Cry1F corn), resistance monitoring is less of a concern. These secondary corn pests overwinter in the south (FAW overwinters only in south Texas, south Fl orida, and the Caribbean) and migrate north during the growing season. Both FAW and BCW are also polyphagous insects that feed on a variety of other crops and weeds and corn is not necessarily a primary host for these pests. Therefore, resistance to Bt corn is not likely and a specific resistance monitoring plan should not be necessary. However, if large amounts of Bt corn (particularly Cry1F corn targeting FAW) were to be planted in areas in which FAW overwinters (e.g., >1000 acres), selection pressure for resistance may increase and a resistance monitoring plan could be warranted. Other secondary corn pests such as SCSB and CSB may also need to be monitored (on a case-by-case basis), as these pests ma y be of local or regional significance.

The resistance monitoring plan should not be tied to specific sales thresholds but be based on sampling areas in which selection pressure for ECB resistance development is the greatest. Samples should be distributed throughout all co rn-growing areas but can be concentrated in higher resistance risk areas (SAP 1998, 2001).

Dr. Blair Siegfried (entomologist, University of Nebraska) has indi cated that at least 100 or more insects, with a target of 500-1000 insects, should be collected pe r location (noted at the June 18,

1999 EPA/USDA Bt Crop Insect Resistance Management Workshop in Chicago, IL). Sampling

locations should be selected to reflect all crop production practic es and should be separated by a

sufficient distance to reflect distinct populations. More intensively planted Bt corn areas in which selection pressure is expected to be higher should also be targeted.

The utilization of sensitive and effective resistance monitoring t echniques is critical to the success of an IRM plan. The following monitoring techniques can be cons idered as part of aCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

180 tiered approach to monitoring: 1) Grower repor ts of unexpected damage; 2) Systematic field surveying of Bt corn; 3) Discriminating c oncentration assay; 4) F 2 screen; 5) Screening against resistant colonies; and 6) Sentinel Bt -crop field plots. These techniques were discussed in detail

in the Introducti on (section D.1).

b) Agricultural Biotechnology Stewards hip Technical Committe e's (ABSTC) Tiered Approach

i. 2000 ABSTC Monitoring Plan

In response to requirements detailed in Agency letters to Bt corn registrants (12/20/99 and 1/31/00), the ABSTC submitted (March 31, 2000) a refined Bt field corn resistance monitoring plan for ECB, SWCB, and CEW for the 2000 grow ing season. The ABSTC plan was designed to concentrate resistance monitoring in areas where Bt corn market penetrati on is highest as well as areas with the highest inse cticide use. The plan include d the identification of counties growing more than 50,000 acres of field corn ( Bt and non- Bt ) to focus monitoring efforts. ABSTC's plan was intended to detect resistance when it reaches 1-5% (a level that may allow for

detection of resistance before field failures occu r). Four corn-growing regions were identified with monitoring for each pest to occur in the re gions in which the pests are prevalent. ABSTC proposed a sampling goal of 4-6 locations in Regi ons I and III and 2-3 locations in Regions II

and IV. When possible, at least 200 fi rst or second flight adults (100 females), 100 second flight egg masses, or 100 diapausing larvae per site were to be collected in each region, though insect population levels may limit the number collecte d. It should be noted that the ABSTC plan applied to both Cry1Ab (MON 810 and BT 11) and Cry1F (TC 1507) Bt field corn hybrids.

The October, 2000 SAP concluded that it did not have enough detail ed information to adequately evaluate the current resistance monitoring plans. The SAP Subpanel suggested that there be a “careful peer review to a ssess the adequacy of all Bt resistance monitoring programs.”

A number of the October, 2000 SA P members indicated that the F 2 screen accompanied by field screening “could be very effec tive for detecting low frequenc ies of recessive and dominant

resistance alleles.” The F 2 screen can be a powerful method for detecting rare recessive alleles in natural populations and is described in de tail in the Introduc tion (section D.1.d.4). The time-frame to respond before control failur es occur depends on the precision of monitoring and the recessivity or dominance of resistance. If the goal of re sistance monitoring is to detect resistance at a low enough resistan ce allele frequency so that ch anges to the insect resistance management plan can be made to increase the lo ngevity of the product and prevent field failure, then the ABSTC resistance monitoring plan needs further consideration. The F 2 screen can detect and measure resistance at frequencies of less than or equal to 0.005 for approximately $5000 per site. This level of precision can provide seven to 12 years to respond with alternative resistance management tactics (see U.S. EP A/USDA 1999, p.47, Figure 1b). Hawthorne et al. (2001) concluded that there is a need to further evaluate th e precision and accuracy of the F 2

screen by using colonies with known freque ncies of resistance alleles. Zhao et al. (2001) also

came to this same conclusion.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

181 The October, 2000 SAP Subpanel indicated that th e diagnostic or discri minating dose technique could at best, detect resistance when the resistan ce allele frequency has reached 1%. This is a level in which some field failure may be observe d. At this lower leve l of precision, the least expensive methods are the discriminating dos e assays (see U.S. EPA/USDA 1999, p. 47, Figure 1b).

One performance standard to consider is that a resistance monitoring plan could be designed so

that there is at least a 95% confidence level in detecting resistance and th at there is also a 95%

confidence level that resistance will not go undetected. The chance of finding a resistant larva

in a Bt crop depends on the level of pest pressure, th e frequency of resistant individuals, and the number of samples that are collected. Therefore, as the frequency of resi stant individuals or the number of collected samples increases, the likel ihood of locating a resistant individual increases (Roush & Miller 1986). If the phenotypic frequency of resistance is one in 1,000, then more than 3,000 individuals must be sampled to have a 95% probability of one resistant individual (Roush & Miller 1986). The ABSTC strategy proposes to detect resistance alleles once they reach a frequency of one in 100. This level of detec tion may not be low enough to detect resistance alleles prior to some field failure. Previous ex perience with conventional insecticides has shown than once resistant phenotypes are detected at a frequency >10%, contro l or crop failures are common (Roush & Miller 1986). Using the F 2 screen could increase the probability of detecting rare resistant alleles, so that the threshold of dete ction would be lowere d to <0.005 or 50-fold more sensitive than the diagnos tic or discriminating dose assay.

The October, 2000 SAP agreed that sampling efforts must be concentrated in areas of high risk in which high usage of a Bt crop would be used as an interim definition. This is also the same recommendation made by the February, 1998 SAP (SAP 1998). The 2000 ABSTC resistance monitoring plan identified t hose counties that are >50% Bt corn sales with at least 50,000 acres

of Bt and non- Bt corn. Based on the 1999 sales data, th ere were approximately 40-50 counties that exceeded this level of market penetrati on. Most of these counties were located in Minnesota, Iowa, and South Dakota (Region I as defined by the ABSTC plan). The ABSTC resistance monitoring plan had a goal of 4-6 samp ling locations in Region I. The October, 2000 SAP Subpanel indicated that it w ould be difficult to determine how many areas of high risk should be sampled, but that genetic differentiatio n of insect samples over large transects could help answer that question. Further evaluati on of the ABSTC’s sampling strategy including statistical analysis and detection sensitivity is recommended.

ii. 2003 Revised ABSTC Monitoring Plan

As part of the terms of cond itions of the amended 2001 registrations for Cry1Ab and Cry1F corn PIPs, registrants were required to submit a revised resistance monitoring plan by 2003 (see part

V - “ Bt Corn Confirmatory Data and Terms and Cond itions of the Amendment” in this BRAD). To accomplish this, the registration terms specified that “ABSTC will convene an advisory panel of academic experts from NC-205, USDA, and EPA to examine the current monitoring program and methodology and to consider enhancements to the current monitoring program for implementation in 2002.” In a ddition, the terms mandate that registrants must follow up on reports of unexpected pest damage in the fiel d for the major target pests (ECB, SWCB, andCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

182 CEW).

Pursuant to these conditions, ABSTC convened a meeting in May, 2002 with representatives from industry, academia, USDA, and EPA to discuss refinements for Bt corn resistance monitoring. Based on these discu ssions, a revised monitoring plan (“Updated Monitoring Plan

for Bt Corn” - no MRID number was assigned) was submitted on January 31, 2003 by ABSTC. This plan is similar in subs tance to the plan developed by ABSTC in 2000 and is based on two tiers of monitoring: report s of unexpected damage from growers and random population sampling.

The sampling strategy utilizes the geographic regions that were identified in the original 2000 ABSTC monitoring plan. For ECB and SWCB, four regions have been identified in corngrowing areas. A map of these regions was provi ded in ABSTC’s submission and is attached to the end of this review. For CEW, monitoring is to occur in the south (10-12 locations), primarily in areas with the greatest Bt cotton plantings.

For ECB/SWCB monitoring, Region 1 (ECB colle ctions only) is defined as southwest Minnesota, eastern South Dakota, southeast North Dakota, and north west Iowa. The target will be to sample 4-6 ECB populations from Re gion 1. Region 2 (ECB and SWCB collections)

covers southwest Kansas and the Texas/Okla homa panhandle. From this region, 4-6 ECB populations and 3-4 SWCB (at least one from Texas) will be ta rgeted for sampling. Region 3 (ECB only) consists of central/southeastern Iowa and north-central Illinois (target sampling of 4- 6 ECB populations). Region 4 (SWCB only) is focused on the Missouri Bootheel, western Kentucky, western Tennessee, and the southern tip of Illinois (target sampling of 3-4 SWCB populations). Individual sampling sites are to be determined by pest population size and will be taken at distances greater than 1⁄2 mile from Bt cornfields to minimize the effects of “elevated resistance gene frequencies misrepre sentative of the population average.”

For each insect population to be sampled, a target of 200 larvae, 200 adults, 100 mated females, or 100 egg masses will be collected for an ove rall goal of 400 genomes per population (egg masses are assumed to have at least 4 genomes). In the event of low pest numbers, the minimum population sizes to be collected wi ll be 50 larvae, 50 adults, 25 mate d females, or 25 egg masses. The specific life stage to be sampled will depend on the insect and the practicality of the collection method. For example, pheromone traps may prove to be the most efficient way to sample CEW. Most collections will occur at the peak flights of second or later generations (first generation flights may not produce sufficient numbers for sampling).

To conduct the resistance assays, neonates are n eeded. Therefore, all collected larvae and (unmated) adults will be reared a nd mated in the laboratory to obtain F 1 progeny. Neonates from collected egg masses will be directly used in the assays. In the event of small samples, populations may be reared for multiple generations to obtain sufficient numbers of neonates for testing.

The primary bioassays to be uti lized to detect potential increases in resistance allele frequency will be assessments of baseline susceptibility (LC 50 ) and the discriminating dose assay. TheCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

183 discriminating dose assay utilizes arti ficial diet with a high dose (LC 99 or EC 99 ) of Bt toxin such that only “resistant” insects will be detect ed – i.e., homozygous resistant (for recessive resistance) or heterozygous resistant (for dominant/incomplete recessive resistance). Discriminating dose assays will be conducted with 500 neonates in four replicates. Based on the number of genomes to be sampled (400 per popul ation), the assays are designed to detect resistance allele frequenc ies at 1) 0.01 for high dose Bt corn and incomplete recessive resistance (probability of detecting a re sistance allele in 400 genomes = 98.2%); 2) 0.002 for a moderate dose and dominant resistance (pr obability of detecting a resist ance allele = 55.0%); 3) 0.075 for recessive resistance (probability of detec ting homozygous resistance = 88.0%, assuming mating of collected insects). Scenarios 1 and 3 apply to ECB and SWCB (high dose in Bt corn, resistance is likely to be recessive or incomp letely recessive) and scen ario 2 applies to CEW (non-high dose, resistance likely to be dominant). The resistance detection thresholds are based on an analysis of population surv ival and a time to resistance of 5 generations (2 1⁄2 growing seasons) after resistance allele detection. The data generated from the assays may be combined for populations within a region or across multiple years to increase the statistical power of the

resistance allele estimates.

The Agency reviewed ABSTC’s revised 2003 plan (A. Reynolds memo to M. Mendelsohn, 2/4/04) and determined that it was adequate to address the term s and conditions of the amended

Bt corn registrations (as of 10/15/ 01). However, questions remain as to the overall effectiveness of the monitoring strategy for suspected or c onfirmed resistance in that it is unknown whether any monitoring plan can sufficiently sample all corn-growing regions to proactively detect resistance before field failure. Further, th e review recommended that ABSTC continue to research and develop technique s to improve the sensitivity and precision of the current monitoring strategy. Such techniques coul d include improved (larger) sampling and development of the F 2 screen and/or DNA markers. Greater sensitivity could allow for more opportunities to adjust the IRM plan to mitigate potential resistance.

iii. 2008 Revisions to the Monitoring Program

In 2008, ABSTC amended the Bt corn registrations to make minor adjustments to the resistance monitoring program. Specifically, changes were requested in the sampling program for ECB, CEW, and SWCB as well as adjustments to the procedures to confirm resistance and mitigate

resistance. ABSTC’s submissi on (MRID# 474070-01) contains a co mplete text of the revised monitoring strategy.

In terms of sampling, ABSTC proposed to maintain the overall numerical targets for each of the monitored corn pests. For ECB, at least 12 populations will be coll ected annually, six for SWCB, and 10 for CEW. Additionally, the ove rall target for each population of 400 genomes (consisting of egg masses, larvae, adults, or mated females) w ith a minimum of 100 genomes will remain the same. As with the past sampling strategy, the goal will be to sample in areas with greater potential for resi stance development (i.e., areas with large proportions of Bt corn adoption and pest pressure). However, AB STC proposed to remove the specific sampling regions that had been established for ECB a nd SWCB monitoring. Four regions had been established for different parts of the Corn Belt including two regions for ECB sampling, oneCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

184 region for SWCB sampling, and one region for bo th ECB and SWCB collections. Rather, the collections of populations will be taken from area s of “regional importance of the insect species as a pest,” but without specifically-defined sampli ng zones. ABSTC also pr oposed to be able to modify the sampling program “based on changes in pest importance and/or the adoption levels of lepidopteran-resistant Bt corn.”

Should resistance monitoring bioassays detect a population with lo w susceptibility to Bt , a series of follow-up steps are to be initiated. These steps follow the procedures in the previouslydeveloped 2003 ABSTC monitoring strategy a nd include the following elements: 1)

Confirmation that the resistance is heritable; 2) Confirmation that the resistance will be observed in the field (i.e., on live Bt corn plants); 3) Determination of the nature of resistance (dominant, recessive); 4) Estimation of the resistance allele fr equency; 5) Analysis of whether the resistance allele frequency is increasing; 6) Determination of the geographic ex tent of the resistance allele distribution; and 7) Design of a remedial acti on plan if the resistan ce allele frequency is spreading. ABSTC’s new proposal included caveats that if the trait is found to be either not heritable or incapable of conf erring the ability to survive on Bt plant tissue (i.e., #1 and 2 above), that the registrants will cease further investigative activity sinc e the observed tolerance is not

relevant for Bt corn in the field. In addition, ABSTC i ndicated that the analytical work will be initiated “as soon as practical” after lo w sensitivity is noted in the bioassays.

EPA reviewed ABSTC’s proposal (A. Reynolds me mo to M. Mendelsohn, 7/9/08) and agreed with ABSTC on the need for flexibility: 1) to id entify areas (i.e., outside of the four previous collection regions) in which Bt corn adoption may be high or incr easing; and 2) to be able to sample in areas with known high pest pressure. Both of these scenario s may be indicative of high selection pressure for resistance and w ould warrant sampling for resistance monitoring. Overall, the minimum number of populations collected would remain the same (12 for ECB, 10 for CEW, and 6 for SWCB). However, the review cautioned that samples should not be “bunched” in only one or two states. While it may be convenient to group samples, the collections that are taken should be representa tive of corn-growing regions in the U.S. Therefore, it was recommended that a caveat be a dded to the sampling plan to ensure that pest populations are collected from multiple corn-growi ng states reflective of different geographies and agronomic conditions.

ABSTC’s 2008 revised monitoring plan keeps larg ely intact the follow- up investigations for populations with low sensitivity to Bt toxin(s). The Agency belie ves these procedures are a crucial component of the overall monitoring prog ram and are needed to verify potential field resistance. On multiple occasions in the pa st (ECB collections in Kandiyohi, MN, Hamilton County, IA, and Jefferson County, NE) these follow- up steps have been implemented to assess populations demonstrating tolerance to Cry1Ab and/or Cry1F in laboratory bioassays (see discussions in the next section) . However, it has also been recommended that ABSTC initiate the investigative work “as soon as possible” after low sensitivity is detected, rather than “as soon as practical” as proposed by ABSTC. Confirma tion of resistance can be an important, timesensitive process that should be completed quic kly enough to ensure effective implementation of remedial action plans (if warranted).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

185 c) Monitoring Results

EPA currently mandates that both baseline sus ceptibility and a discriminating concentration assay be employed for certain primary target pe sts including ECB, SECB and CEW. Baseline susceptibility data have been collected for each labeled/target pest although consideration should be given for all potentially susceptible pest s (e.g., BCW, FAW, SCSB) with focus on major economic pests. This information is essent ial to managing resistance in pest populations, especially in assessing whether a field control fa ilure was due to actual resistance or other factors affecting expression of the Bt protein. These baseline data are helpful in documenting the extent and distribution of resistant populat ions. Continued monitoring effo rts are needed to provide the Agency with standardized information to dete rmine whether resistance is developing to the registered Bt toxins. ABSTC has submitted a nnual reports to EPA on resistance monitoring results for ECB, CEW, and SWCB with Cry1A b and Cry1F as required by the terms of registration. The results of these monitoring re ports are detailed below and are complete through the 2008 growing season.

i. ECB

Cry1Ab

Dr. Blair Siegfried (University of Nebraska) has coordinated a standardized monitoring program for ECB (since 1995) and CEW (through 2000) involving LC 50 susceptibility determinations and diagnostic concentration (LC 99 ) bioassays to determine susceptibility levels to Bt corn. In terms of baseline susceptibility (LC 50 ), bioassays have been conducted for ECB (Siegfried et al. 1999a, Siegfried & Spencer 2000) and CEW (Siegfried et al. 2000, see CEW di scussion in the next section). For 1999, ECB were collect ed from 14 separate sites and F 1 and/or F 2 generations were bioassayed to determine LC 50 s. Bioassays utilized dilutions of purified Cry1Ab obtained from Bt

kurstaki strain HD1-9 (provided by N ovartis) spread on artificial diet. Neonate larvae were exposed to the diet less than 24 hours after ha tching and mortality and larval weight were recorded seven days later. For 2000, 13 ECB popul ations were sampled using similar procedures with formulated Cry1Ab protein (CellCap, provid ed by Dow/Mycogen). This CellCap source of Cry1Ab was used for ECB monitoring from 2000 to 2003. ECB are more sensitive to the CellCap Cry1Ab formulation, therefore, suscepti bility results during this time period are not directly comparable with those from 1995-1999. Starting in 2004, a new source of Cry1Ab toxin was used in the assays, which has been used in each subsequent year of testing. The new toxin (obtained from Monsanto) was purified, trypsin-r esistant Cry1Ab core protein and produced results similar to the purified toxin used fr om 1995-1999. Monitoring results for ECB through the 2008 season are displayed in Table D3 and show no significant change in ECB susceptibility (LC 50 ) to Cry1Ab over 13 years of testing (1995 - 2008).

Table D3. Mean Susceptibility of ECB to Cry1 Ab from 1995 to 2008 (created from data in Siegfried et al. 1999a; Siegfried & Spencer 2000, 2001a, 2001b, 2002a, 2003a; Siegfried et al. annual reports 2005 to 2009)

Year LC 50 (ng Cry1Ab/cm 2 ) ± SEM Disc. Dose (% mortality)Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

186 1995 4.34 ± 0.68 ---

1996 6.25 ± 1.25 > 99

1997 2.12 ± 0.53 > 99

1998 2.57 ± 0.28 100

1999 4.01 ± 0.49 99.9 - 100

2000 1 (0.12 - 0.49) 99.7 - 100

2001 1 (0.14 - 1.34) 2 97.8 - 100 3

2002 1 (0.18 - 0.47) 99.1 - 100

2003 1 (0.06 - 0.21) 99.6 - 100

2004 (1.74 - 19.86) 4 50.7 - 100 4

2005 (1.33 – 4.45) 99.4 - 100

2006 (1.18 - 4.28) 99.5 - 100

2007 (1.70 - 4.26) 100

2008 (1.49 - 2.94) 100

1 Data collected for 2000 - 2003 were obtained using a different Cry1Ab formulation (CellCap) that is more toxic to ECB. As such, results from these years are not dir ectly comparable with results from other years. LC 50 values for 2000 - 2008 are given as a range (without SEM).

2 2001 included one outlier population with an LC 50 of 1.34 ng/cm 2 . The range for the other populations was 0.14 - 0.46 ng/cm 2

. 3 Three populations from 2001 had < 99% mortality at the diagnostic dose.

4 2004 included one tolerant population with an LC 50 of 19.86 ng/cm 2 and a 50.68% mortality at the diagnostic concentration. The other tested populations had LC 50 values of 1.74 - 5.24 ng/cm 2 and discriminating dose

mortalities > 99.6%.

For 1999 diagnostic concen tration analysis (LC 99 ), baseline susceptibil ity studies conducted by Marçon et al. (2000) were used to determine th e discriminating concentration for ECB. These tests with the discriminating concentrations were conducted in a similar manner to the bioassays

to determine LC 50 values. For 2000, a new discriminating dose (10 ng/cm 2 ) was established for the CellCap Cry1Ab formulation. The result s (for both 1999 and 2000 populations) showed nearly 100% mortality for ECB at the discriminating dose (LC 99 ) (Siegfried et al. 1999a, Siegfried & Spencer 2000). For the change in toxin source during 2004, a revised concentration (LC 99 ) was calculated (55 ng/cm 2 Cry1Ab) based on dose/response assays done for ECB populations from Nebraska. This diagnostic concentration has b een used subsequently for ECB monitoring. Results of the diagnostic assays through the 2008 season are summarized in Table

D3.

Since 2001, there have been two ECB populations that showed less than expected mortality (i.e. < 99%) on the diagnostic concentration. Survival on diagnostic concentra tions can be a sign of some degree of resistance to the toxin and triggers follow-up investigations. In 2001, one population from Kandiyohi, MN was shown to have 98.4% mortality in th e discriminating dose assay. The second population was collected from Hamilton County, IA and was shown to have only 50.68% mortality to the Cry1Ab discrimina ting concentration (t his population also demonstrated high tolerance to Cry1F – see the di scussion in the next sect ion). Procedures forCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

187 follow-up testing included the follo wing: 1) Rearing additional generations from the populations (F 2 and F 3 ) for further discriminating dose testing; 2) Pooling and rearing survivors from the discriminating dose assays for additional testing (F 3 ); 3) Testing with leaf discs (F 3 ); 4)

Continued selection of survivors at the diagnostic concentration (F 4 - F 7 ); and 5) Testing on whorl stage Bt corn (F 7 ).

For the Kandiyohi population, F 2 larvae were retested at the di agnostic concentration and showed 99.9% mortality. F 3 larvae showed significant survival (48.8%) on Bt leaf discs, but those survivors were shown to have very low weights relative to th e control group (non- Bt corn leaf discs). The colony was also test ed on whole, whorl stage MON 810 plants in a greenhouse test. Individual corn plants were infe sted with Kandiyohi egg masses (F 7 , each generation selected with a diagnostic concentration) and were assessed for feeding da mage and larval survival after

three weeks. The Bt corn plants were found to be undamaged and no larval survival was noted (there was no difference between the Kandiyohi strain and a control strain). Given the results of the follow-up testing, successfully conducted with in the allotted two year time frame, the Kandiyohi population was determined not to be fi eld resistant (reviewed in A. Reynolds memo to M. Mendelsohn, 2/4/04). Further, resistan ce monitoring in the Kandiyohi area in the following years (2002 - 2004) did not reveal any in stances of decreased ECB susceptibility to Cry1Ab.

For the Hamilton County, IA population, the or iginal colony was reared for additional generations and subjected to diagnostic and dose response bi oassays with Cry1Ab protein (described in MRID# 468749-01). Surviving larvae from the initial Cry1Ab diagnostic assays were separately pooled and rear ed as independent colonies. These “survivor colonies” were subjected to purified protein, leaf disc, and on-plant assays. Resu lts of the bioassays indicated that the Hamilton ECB population c ontinued to exhibit significantly lower susceptibility to the Cry1Ab protein compared to the non-resistant control colonies. Mortality at the diagnostic

concentration was 15.2 and 11.9 for the F 7 and F 8 generations, respectively. However, the F 6 and

F 8 generation from this colony exhibited no su rvival on leaf discs obtained from Cry1Ab expressing plants compared with high rates of survival on non-expres sing leaf discs from non- Bt isoline plants. Separately, the Cry1Ab survi vor colony showed interm ediate (47.9%) survival and severe stunting on leaf discs from Cry1F -expressing plants when tested at the F 6 generation. In the last set of tests, the survivor colony was unable to de velop on Cry1Ab-expressing whorlor reproductive-stage plants – only one live (severely stunted) larva was found among the 10 Cry1Ab-expressing plants infested with the colony.

Three new populations of ECB were collected fr om Hamilton Co. in 2005 and results from these assays indicated that the newl y sampled populations did not have reduced susceptibility to Cry1Ab compared to control colonies. Furt her collections taken from Hamilton in 2006, 2007, and 2008 have also shown no signs of elevated tolerance to Cry1Ab and no incidents of field

level failure have been identified in the re gion. Given these resu lts, the Hamilton, IA 2004 population was determined not to be field rele vant despite the high su rvival noted in the diagnostic concentration assay (Ham ilton, IA data were reviewed in 1) A. Reynolds memo to M. Mendelsohn, 1/23/06; 2) T. Milofsky memo to M. Mendeloshn, 3/12/07; 3) A. Reynolds memo to M. Mendelsohn, 4/2/08; and 4) S. Borges memo to M. Mendelsohn, 4/8/09).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

188 Cry1F Resistance monitoring for Cry1F and ECB was initiated with the 2000 season. Similar to Cry1Ab, the program for Cry1F has been coordi nated by Dr. Blair Siegfried (University of Nebraska) since the initial 2000 season. The sa mpling strategy and bioassays used for Cry1Ab (i.e., baseline susceptibility and diagnostic concentrations) are also employed for Cry1F.

Baseline susceptibility (LC 50 ) results for ECB ranged from 0.17 μg Cry1F/g diet (1st instar) to 10.67 μg Cry1F/g diet (4th instar) (MRID# 453077-01; reviewed in R.Rose memo to M.Mendelsohn, 1/24/01). As a condi tion of registration, Bt corn re gistrants were required to develop a diagnostic concentration for E CB, CEW, and SWCB. ABSTC’s monitoring submissions from the 2001 and 2002 growing seasons reported testing and validation of a ECB diagnostic concentration of 60 ng/cm 2 Cry1F. This concentration ha s been used subsequently for ECB Cry1F monitoring and adequately a ddressed the condition of registration.

ABSTC uses the same sampled ECB populations for both Cry1Ab and Cry1F monitoring. To illustrate, in 2001 ECB collections were made from populations at 10 locations from four states, including Nebraska (2 locations), Illinois (4 locations), Iowa (2 locations), and Minnesota (2 locations). In 2002, samples were obtained from populations at 14 locations from six states. Similar numbers of populations from locations in the Corn Belt have also been sampled in succeeding years. The toxin used in the assays since 2004 has been purified and truncated Cry1F protein obtained from recombinant Pseudomonas fluorescens (provided from Dow AgroSciences). A summary of the susceptibility (LC 50 ) and diagnostic concentration assay data through the 2008 growing season is detailed in Table D4 below.

Table D4. Monitoring Results for ECB and Cry1F Corn from 2000 to 2008 (Siegfried & Spencer 2001c, 2002b, 2003b; Siegfried et al. annual reports 2005 to 2009)

Year LC 50 range (ng Cry1F/cm 2 ) Disc. Dose (% mortality range)

2000 2.35 - 6.26 98.66 - 100

2001 1.23 - 3.68 97.47 - 100

2002 1.04 - 5.32 99.50 - 100

2003 2.33 - 8.45 99.10 - 100

2004 1,2 3.76 - 48.35 48.35 - 100

2005 1 2.96 – 13.8 99.2-100

2006 1,3 1.59 - 9.81 97.89 - 100

2007 1 2.06 – 8.00 100

2008 1 3.78 - 6.72 99.23 - 100

1 A new source of Cry1F toxin (purified and truncated toxin obtained from recombinant Pseudomonas fluorescens ) has been used since the 2004 bioassays

2 2004 included one tolerant population with an LC 50 of 45.42 ng/cm 2 and a 48.35% mortality at the diagnostic concentration. The other tested populations had LC 50 values of 3.76 - 9.73 ng/cm 2 and discriminating dose

mortalities > 96.47%.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

189 3 One population collected in 2006 had < 99% mortality at the diagnostic concentration.

As with the Cry1Ab monitoring, two populations of ECB have shown greater than expected survival (>1%) in diagnostic concentration tests. The first was the same population collected in Hamilton County, IA during the 2004 season that s howed high tolerance to Cry1Ab (discussed in the next section below). A second populati on sampled in Jefferson County, Nebraska, was

observed to have somewhat reduced susceptibility to Cry1F in 2006.

ECB collected from the Jefferson County population were observed to have lower than expected mortality (97.89%) to the Cry1F diagnostic concentration (expect ed mortality > 99%). ABSTC initiated a follow-up investigation on this populati on (using the same procedures described in the Cry1Ab section above). This testing suggested some heritability of the trait (32.8% of F 2 progeny survived at the same diagnostic concentr ation). However, low survival (10%) with growth stunting was noted on separate tests with Cry1F plant tissue and testing with F 3 larvae on a higher (10x) diagnostic concentration produced 100% mortality. Given the low level of tolerance displayed by the Jefferson County population, it is unlikely that the trait detected in the monitoring assays could have conferred field resistance. Despite the 2006 data, Jefferson County was not sampled by ABSTC in 2007 or 2008. This area shoul d be included as a collection site for Cry1F bioassa ys in subsequent gr owing seasons and monitored for unexpected pest damage.

Hamilton County, IA Cry1F-Tolerant ECB Population

The Hamilton County (HC) population collected in 2004 exhibited substantially higher tolerance to Cry1F than the other tested ECB popul ations from that year with a Cry1F LC 50 of 45.42 ng/cm 2 (compared to a range of 3.76 - 9.73 ng/cm 2 for the other sampled populations). Additionally, the population had only 48.35% morta lity on the Cry1F diagnostic concentration. Follow-up testing (conducted by Dow AgroScie nces and described in MRID# 466958-01 and 470112-01) focused on four major objectives: 1) The level and heritabi lity of the potential resistance; 2) Survival on Bt (Cry1F) expressing plants; 3) The genetics of the potential resistance; and 4) The frequency of resist ance in field populations from Hamilton County.

Heritability testing was initially conducted on F 7 and F 8 generation of the HC ECB colony. Bioassays were performed with artificial diet and a diagnostic concentration of 60 ng Cry1F/cm 2
(based on the LC 99 of ECB), similar to the procedures used for the routin e annual resistance monitoring of Bt corn. When exposed to this co ncentration in the trial, few F 7 (2.7%) or F 8 (0.9%) larvae were killed. Thes e reported mortalities were subs tantially lower than the 48.3% mortality that was observed with the F 3 generation in the original monitoring work. With such high survival, the investigators were unable to generate a dose -response curve to calculate LC 50 or EC 50 values. Subsequent bioassays were conducted on F 13 and F 18 generations using higher diagnostic concentrations of Cry1F. F 13 larvae (previously unexpos ed to Cry1F since the F 3 generation) were exposed to a diagno stic concentration of 600 ng Cry1F/cm 2 (10x the standard concentration used for monitoring). These larv ae also demonstrated high tolerance to Cry1F, with only 4.1% mortality (compared with 3.9% mortality in an unexposed control group). A

final test of the F 18 generation (used as a control group for the reciprocal cross test described later in this review) was conducted at concentrations up to 12,000 ng Cry1F/cm 2 (200-foldCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

190 greater than the standard diagnosti c concentration). No detectable mortality or growth inhibition was observed at even the highest 12,000 ng Cry1F/cm 2 concentration. Considering the extremely low mortality (< 5%) to the diagnostic concentrations observed in the generations (F 7 - F 18 ) after initial (F 3 ) selection, Dow concluded that the Cry1 F tolerance trait was heritable in the HC ECB colony. Overall, tested ECB exhibited a resistance to Cry1F levels that exceeded 2,000 to 6,000 times the LC 50 for susceptible ECB.

Dow also investigated the ability of th e HC ECB colony to survive on Herculex Bt corn plants. Pest populations showing tolerance to Bt toxins are generally not c onsidered “resistant” unless they are able to develop into adults on Bt expressing plants. Without the ability to survive on Bt plants, tolerant populations will no t be able to reproduce and will not proliferate in the field. Prior to conducting the whole plant assays, Dow challenged the HC population on leaf disks taken from Herculex (Cry1F) and non-expressing isoline plants. The results showed that 79.2% of the HC ECB survived after four days exposur e to the Cry1F leaf disks (survival on the non- Bt leaf disks was 90.6%). Larval weights of th e HC colony averaged 0.42 mg for those on Cry1F disks and 0.71 mg for the non- Bt disks. By comparison, the uns elected control colony had no survival on the Cry1F disks and 97.8% on the non- Bt leaf disks.

Whole plant assays were also conducted in gr eenhouse settings and were done with different generations (F 7 -F 8 and F 11 ) from the HC ECB colony. In the first set of assays, F 7 and F 8 generation larvae were infested (30-40 per plant) on whorl and ear stage corn plants (Cry1F and isoline). With whorl stage corn, the HC ECB (F 7 ) were found in approximately equal numbers on both Cry1F and isoline corn plants, although the overall number of larvae recovered was small (<0.5 per plant). In contrast, high num bers of larvae from the control colony were recovered on isoline corn (>3 per plant), bu t only one larvae was f ound on Cry1F corn. HC larvae collected on Cry1F plants weighed less on av erage than those from is oline plants (~ 30 mg Cry1F vs. ~ 50 mg isoline). For reproductive stag e corn (i.e., ear stage), similar numbers of larvae from the HC colony (F 8 ) were recovered on both Cry1F and isoline corn, although unlike the whorl stage results high numbers were recovere d for both treatments (~ 7 per plant). Similar to the whorl stage corn test, the recovered HC larvae from Cry1F ear stage corn weighed less than those collected from the isoline corn (~ 40 mg Cry1F vs ~ 60 mg isoline). High survival was noted for the control colony (~ 9 per plant) on isoline corn, while fe w larvae (<1 per plant) were found on the Cry1F corn plants. The i nvestigators noted that the experiment was “inconclusive” because the surviving larvae were not allowed to fully develop into pupae and

adults.

A second whole plant test was performed on the F 11 and F 12 generations from the HC colony. As with the first test, whorl and ear stage Cry1F corn plants were infested with 30 neonates per plant from the HC colony or an unselected control group. After three weeks, the plants were assessed for damage and living larvae were collected (from diapause) and for ear stage corn ultimately

reared to adults. The results of this experiment showed that on whorl stage corn, few HC F 11 larvae were recovered from either Cry1F or isoline corn. A to tal of four HC larvae were recovered from isoline corn and only one larva from Cry1F plants. This compared with 1.4 larvae/plant from the control colony on isoline corn (no control larvae were recovered on Cry1F plants). Cry1F corn plants showed some da mage from HC feeding (2.3 avg. Guthrie score)Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

191 though not to the extent of the isoline plants ( 4.5). In comparison, the unselected control group had Guthrie ratings of 5.3 on isoline corn and 1.0 (no visible feeding) on Cry1F plants. For ear stage corn, equal numbers of F 12 HC larvae (68 total) were rec overed on Cry1F and isoline corn, while only three larvae from the control col ony were found on Cry1F corn (114 were found on isoline corn). Of the 68 HC larvae found on Cry1F corn, 45 completed diapause, 36 pupated, and 26 emerged as adults (38% of the total re covered larvae). Fewer HC larvae completed development on isoline corn (38% diapause, 31 % pupae, and 28% eclosi on) than on the Cry1F hybrids.

Based on the results from the greenhouse studies, Do w surmised that the Cr y1F tolerance trait in the HC population allows larvae to grow and complete development on Herculex Cry1F corn plants. Although, it was noted that there ar e differences between greenhouse and field environments, the company concluded that the abili ty of HC larvae to survive Cry1F in the field is a “strong possibility.”

The third goal of the HC studies was to determine the genetic st ructure of the Cry1F resistance trait. Diet bioassays and cro sses with known susceptible (laborat ory) ECB colonies were utilized

to evaluate dominance/recessiveness of the tr ait and potential sex lin kage. Crosses were conducted with HC (F 13 and F 18 generations) and cont rol (Cry1F-susceptible) ECB colonies to produce F 1 larvae for the bioassays. Four groups were established: 1) HC; 2) HC male x control female; 3) HC female x control male; and 4) control. The F 13 generation HC was crossed with a laboratory control colony, while the F 18 were bred with a field-co llected control colony. Progeny obtained from the crosses were then exposed to a range of Cry1F concentrations in diet bioassays (up to a maximum of 12,000 ng/cm 2 ). Both sets of crosses re sulted in similar responses to Cry1F: HC ECB crossed with the susceptible col onies remained sensitive to the toxin while the uncrossed HC group was highly tolerant to Cry1F. The dose response curves of the two crosses (HC ♂ x CC ♀ and HC ♀ x CC ♂ ) were similar to that of th e uncrossed contro l colony, although

the crosses showed some increase d ability to tolerate Cry1F rela tive to the control group. An

EC 50 or LC 50 for the uncrossed HC groups could not be calculated because no mortality or growth inhibition was observed at th e highest test concentration (12,000 ng/cm 2 ). Given the results from the reciprocal crosses, Dow concl uded that the Cry1F tolerance trait in the HC colony is recessive but autosomal and not sex-linked.

The final step in Dow’s follow-up investigations of the Cry1F tolerant ECB from HC was an estimation of the prevalence of the trait in fiel d populations from the county. To accomplish this objective, Dow (via ABSTC) conducted ECB samp ling of the HC area during the 2005 growing season as part of the annual resistance mon itoring work. Three populations were collected directly from HC (543 total ECB) and their F 1 progeny were screened ag ainst the standard Cry1F diagnostic concentration (60 ng/cm 2 ). None of the tested HC progeny survived the diagnostic Cry1F concentration, indicating that the Cry1F tolerance trait was below the level of detection. Subsequent sampling in 2006, 2007, and 2008 has also failed to produce any survivors to the diagnostic concentration and there have been no reports of field failures in this area.

EPA reviewed the HC data (A. Reynolds memo to M. Mendelsohn, 8/7/07) and concluded that the collected 2004 HC colony was resi stant to the Cry1F toxin. Th e colony met a ll of the majorCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

192 criteria for resistance: the trait was heritable (determined to be a single recessive gene), the trait conferred survival to high levels of Cry1F, and ECB with the trait were capable of developing to adults on Cry1F-expressing corn plants. This app eared to be the first documented case of pest resistance to a high dose Bt toxin in corn; though it should al so be noted that no documented

cases of field failure or resistance with ECB have been detected in HC or elsewhere. Given that the sampling strategy for the annual resistance monitoring program covers only a small portion of corn-growing regions, it is possible that such traits are relatively common. Other cases may remain undetected if significant fi eld-level effects are not observed.

Dow contends that because the tr ait was not detected in 2005 (a nd beyond), the resistance allele frequency was below the level of concern and a re medial action plan was not needed. To verify this conclusion, additional monitoring in HC was recommended during subsequent growing seasons. ABSTC has conducted monitoring in this county since 2005 (through 2008, the last season tabulated in this document) with no incide nts of increased toleran ce or field failure to Cry1F corn. Additional EPA revi ews of HC monitoring data can be found in 1) T. Milofsky memo to M. Mendeloshn, 3/12/07; 2) A. Reynolds memo to M. Mendelsohn, 4/2/08; and 3) S. Borges memo to M. Mendelsohn, 4/8/09).

ii. CEW

Cry1Ab

Since the 2001 season, resistance monitoring as says have been conducted by Custom BioProducts (Maxwell, IA). Similar to ECB, mon itoring is based on suscep tibility bioassays and diagnostic concentration tests.

For CEW, baseline susceptibility (LC 50 ) values to Cry1Ab ranged from 70.3 ng/cm 2 (lab colony) to 221.3 ng/cm 2 (field colony) (Siegfried et al. 2000) . A separate diagnostic concentration analysis (using similar methods to those used for ECB) was conducted for CEW (using a dose of 6600 ng/cm 2 ), which showed nearly 100% mortality (S iegfried et al. 1999b). This diagnostic

dose has been revised since this initial assessme nt, largely due to the fact that CEW is less sensitive to Cry1Ab then other le pidoptera. The insect freque ntly exhibits highly variable responses to the toxin, such that it has been difficult to determine a reliable diagnostic

concentration based on an LC 99 . Monitoring in 2001-2 used a concentration of 40 ng Cry1Ab/cm 2 . In 2004, ABSTC increased the Cry1Ab diagnostic concentrations to 80 ng/cm 2 for the CEW monitoring. Despite this increase, a number of populati ons have continued to survive at levels greater than 1%.

The Cry1Ab toxin used in the diet bioassays has been provided by Dow AgroSciences since

2001. CEW are collected as larvae, pupae, or eggs from fiel d sites and shipped to Custom BioProducts for establishment of test colonies. Bioassays are conducted with neonate larvae from

the established colonies. Baseline susceptibility (LC 50 and EC 50 ) and diagnostic concentration bioassays are performed in accordance with th e methods used by Siegfried et al. (2000).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

193 The results of LC 50 and diagnostic concentration assays are summarized in Table D5 below. Susceptibility data (LC 50 ranges) have been reasonably c onsistent over the tested growing seasons and support the conclusion that there ha s been no documented CEW field resistance to Cry1Ab. The range of LC 50 values from 2007 was less varied and closer to the higher end of the ranges from previous years, though this could be an artifact of the small number of populations that were assayed that year. Four populations in 2008 also exhibited LC 50 values on the high end of the historical range (i.e., > 8.0 ng Cry1Ab/cm 2 ), though most of the populations collected during this season had LC 50 s of < 4.5 ng/cm 2 . In addition to the monitoring assays, ABSTC has reported that no incidents of field failure or unexpected CEW damage have been observed through the 2008 growing season. Th e diagnostic concentration that has been used since 2004 (80 ng Cry1Ab/cm 2 ) also appears to have remained functional (i.e., mortality > 99% in susceptible CEW colonies) and is an improvement over the previously us ed concentration of 40 ng Cry1Ab/cm 2 , though several populations in 2008 had slightly less than 99% mortality.

Table D5. Monitoring Results for CEW a nd Cry1Ab and Cry1F Corn from 2001 - 2008 (Custom Bio-Products annual reports 2002 through 2009)

Cry1Ab Cry1F

Year LC 50 range (ng Cry1Ab/cm 2 ) Disc. Dose (% mortality range) a LC 50 range (ng Cry1F/cm 2 ) Disc. Dose (% mortality range) b

2001 1.37 - 4.98 ≥ 99 89.68 - 2301.48 94 - 97

2002 1.09 - 8.08 97.77 - 99.85 269.45 - 2151.99 85.27 - 100

2003 2.35 - 8.94 90.8 - 100 671.39 - 1366.61 82.9 - 99.0

2004 0.15 - 8.93 99.4 - 100 45.30 - 2828.28 99.1 - 100

2005 1.68 - 11.45 97.5 - 100 306.21 - 2804.42 62.2 - 96.1

2006 0.73 - 8.40 99.6 - 100 65.29 - 806.90 87.5 - 100

2007 6.00 - 7.99 99.4 - 100 1129.12 - 1555.88 48.7 - 99.6

2008 0.78 - 10.55 96.7 - 100 87.10 - 2259.55 69.0 - 100

a Cry1Ab discriminating concentration for 2001-3 = 40 ng Cry1Ab/cm 2 ; for 2004 - 2007, concentration = 80 ng Cry1Ab/cm 2

b Cry1F discriminating concentration for 2001 = 5000 ng Cry1F/cm 2 ; for 2002-3, concentration = 7500 ng Cry1F/cm 2 ; for 2004, concentration = 54,675 ng Cry1F/cm 2 ; for 2005-8, high concentration = 4,000 ng Cry1F/cm 2

Cry1F

Resistance monitoring for Cry1F and CEW has also been conducted by Custom Bio-Products using the same paradigm for Cry1Ab. Both th e susceptibility and diagnostic concentration testing with Cry1F have shown br oad variability in response by CEW. In general, the pest has been less susceptible to Cry1F than Cry1Ab. As such, it has proven difficult to determine a reliable diagnostic concentration for year -to-year screening of field populations.

The initial diagnostic concentration for monitoring in 2001 was set 5000 ng Cry1F/cm 2 but was increased in 2002 to 7500 ng Cry1F/cm 2 , based on the results from 2001. However, even theCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

194 higher concentration failed to consistently pr oduce mortalities > 99% in collected populations indicating that the concentrati on was actually less than the LC 99 for CEW. In 2004, the dose was further increased to 54,675 ng/cm 2 , a level at which all sampled populations had >99% mortality. However, in 2005 the dose was reduced to 4000 ng/cm 2 without explanation from ABSTC. This concentration has been used since 2005 (thr ough the 2008) season but has frequently produced mortality below 99% in the bioassays. No follo w-up testing has been reported for any of the populations in the diagnostic assa ys. In light of these results , it is apparent that 4,000 ng/cm 2 is not acting as a functional diagnostic concentration (i.e., a benchmark that triggers investigation of potential resistance) and has lim ited value as a monitoring tool. It has been recommended that

ABSTC either reestab lish the higher test c oncentration (54,675 ng/cm 2 ) or work to develop an effective alternate technique for monitoring CEW in future testing so that potentially resistant

individuals can be identified.

The results of Cry1F monitoring for CEW are su mmarized in Table D5 above. Given the high variability in LC 50 ranges, it is difficult to discern y ear-to-year trends, though there does not appear to be a significant decrease (if any) in the susceptibility of CEW to Cry1F. Additionally, no incidents of field failure due to CEW damage to Cry1F corn have been reported to the Agency.

iii. SWCB

Cry1Ab and Cry1F

Additional monitoring work has been done with SWCB. Based on collections from 1998 and 1999, a study was conducted by Trisyono and Ch ippendale (1999) to determine SWCB susceptibility to Cry1Ab and establish a diagnos tic concentration. A bioassay was conducted that established a diagnostic concentration for SWCB of 110 ng Cry1Ab protein/g diet. Susceptibility data (LC 50 s and EC 50 s), determined after 7 and 14 days of exposure to Cry1Ab, are

summarized in Table D6 below. SWCB monito ring has been conducted since the 2000 growing season by Dr. Qisheng Song at the University of Missouri using simila r methodology (Song et al. 2000) to obtain susceptibility data (LC 50 s and EC 50 s) and perform diagno stic concentration assays. A diagnostic concentra tion assay was performed (7 day test dose = 0.35 μg Cry1Ab/g diet, 14 day test dose = 5 μg Cry1Ab/g diet), wh ich resulted in 100% mortality for all tested populations. The 5 μg Cry1Ab/g concentration ha s been used as the diagnostic dose since 2000.

For Cry1F, baseline susceptibility assays provided an estimate for the SWCB LC 50 of 0.70 μg Cry1F/cm 2 diet (MRID# 450201-01; reviewed in R. Rose memo to M. Mendelsohn, 1/24/01). Annual Cry1F monitoring formally started w ith the 2001 growing season. The 2001 SWCB susceptibility tests resulted in a LC 50 range of 3.69 - 5.73 μg Cry1F/ml (7 day exposure) and 0.79 - 1.63 μg Cry1F/ml (14 day exposure) for field popul ations; a lab colony wa s also tested (7-day

LC 50 = 27.20 μg Cry1F/ml; 14-day LC 50 = 7.05 μg Cry1F/ml). Discriminating doses of 216.4 μg Cry1F/g (7 days) and 68.6 μg Cry1F/g (14 days) were used in the initi al testing and produced 100% mortalities for both exposure periods. The 14 day diagnostic dose 68.6 μg/g has been used for SWCB monitoring since 2001.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

195 SWCB monitoring data are summarized in Ta ble D6 below. Taken together, the SWCB monitoring results show that, to date, no apprecia ble increase in susceptibility has resulted from exposure to the Cry1Ab or Cry1F toxins. Also, the laboratory colonies evaluated as control groups have been less susceptible to Cry1Ab than the field collect ed populations. Furthermore, there have been no survivors to the 14 day diagnos tic concentration in any of the years tested or reports of unexpected pest damage in Cry1F corn due to SWCB. It is note d that the results from 1998 and 1999 indicated that a bioassay using growth inhibition is more sensitive than one based on larval mortality. Trisyono and Chippendale (1999) suggested that bioassays based on growth

inhibition rather than larval mo rtality may have greater benefits because they require a smaller

amount of Bt protein, sublethal effects can be observed, the time of observation is flexible (weight gain is being compar ed to a control), and variation may be minimized.

Table D6. SWCB Susceptibility (LC 50 ) to Cry1Ab and Cry1F from 1998 to 2008 (Trisyono and Chippendale 1999; Song et al. annual reports 2000 to 2009)

Cry1Ab Cry1F

Year LC 50 ( μ g Cry1Ab/ml diet) - field populations Discriminating Dose (5 μg Cry1Ab/g) (percent mortality) LC 50 ( μ g Cry1F/g diet) - field populations Discriminating Dose (68.6 μg Cry1F/g) (percent mortality)

1998 1 7-day: 0.22 - 1.09 14-day: 0.04 - 0.09 -- – --

1999 1 7-day: 0.07 - 0.17 14-day: 0.02 - 0.05 -- – –

2000 7-day: 0.08 - 0.15 14-day: 0.04 - 0.09 100 -- --

2001 7-day: 0.09 - 0.22 14-day: 0.05 - 0.12 100 7-day: 3.69 - 5.73 14-day: 0.79 - 1.63 100

2002 14-day: 0.07 - 0.12 100 14-day: 1.04 - 2.14 100

2003 14-day: 0.06 - 0.21 100 14-day: 0.98 - 2.34 100

2004 14-day: 0.10 - 0.28 100 14 day: 0.87 - 2.57 100

2005 14-day: 0.07 - 0.41 100 14-day: 2.02 - 6.04 100

2006 14-day: 0.06 - 0.28 100 14-day: 1.64 - 3.18 100

2007 14-day: 0.08 - 0.25 100 14-day: 1.90 - 4.03 100

2008 14-day: 0.12 - 0.47 100 14-day: 2.48 - 4.29 100

1 The units for the 1998 and 1999 data are μ g Cry1Ab/g diet for LC 50 values.

iv. FAW

There is also a monitoring program for FAW as part of the Bt sweet corn registration. The status of this program is described in the Bt sweet corn section (D.2.b.10).

FAW Resistance to Cry1F in Puerto RicoCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

196 Separately, Dow AgroSciences and Pioneer Hi-Bred reported to EPA that unexpected Spodoptera frugiperda (fall armyworm) damage had occu rred on Cry1F TC1507 maize (also known as Herculex® Insect Protection Maize) in Puerto Rico during the 2006 season. Damage was reported in corn fields by three customers as well as in Dow (Mycogen Seeds) research plots on the island (deta iled in MRID# 471760-01).

Dow AgroSciences’ personnel performed follow-up testing on the Puerto Rican populations. The following conclusions were reached from these investigations:

• Testing using ELISA lateral flow membrane strips confirmed that the TC1507 maize expressed the Cry1F protein.

• Testing confirmed that the unexpected dama ge was caused by FAW larvae feeding on TC1507 maize (company expert).

• Testing of the susceptibility to the Cry1F protein indicated that the two FAW larval populations collected from VEH Farms (a commercial farm) and Mycogen Research Farm (SI) had significantly lowe r susceptibility to the Cry1Fa protein than the control, Benzon colony. The screening level assessm ent of the Cry1Fa-sensitivity of F 1 progeny from the field-collected larvae at 10,000 ng Cry1F/cm 2 revealed no significant mortality

of either Puerto Rico co llection on the Cry1F-treated diet. Testing of the F 2 progeny did not show a significant concentration re sponse to Cry1F, nor di d these progeny show a significant reduction in growth. The re sistance ratio was >167, but an exact level

could not be calculated for the Puer to Rico populations because the LC 50 s could not be

determined.

A specific remedial action plan for FAW resi stance was not required under the terms and conditions of the Cry1F Bt (TC1507) maize registrations (EPA registration numbers 68467-2 and 29964-3) because FAW is considered to be a sec ondary pest. Therefore, Dow AgroSciences and Pioneer Hi-Bred formulated a specific remedial action plan designed to reduce the selection pressure for FAW resistance to TC1507 maize. Th is plan is analogous to that required by EPA for the primary target pests of TC1507 maize (ECB, SWCB, and CEW). The following steps were and are being taken by the registrants following confirmati on of FAW resistance Puerto Rico (described in a letter from Dow AgroSciences to EPA, dated April 23, 2009):

• Existing customers were notified and recommen ded to use insecticides (as needed) to control FAW damage on exis ting TC1507 commercial fields.

• The two registrants took steps to disconti nue commercial sales of TC1507 maize in Puerto Rico by terminating grower agr eements and revising product use guides to prohibit plantings in Puerto Rico.

• FAW populations were managed in maize resear ch and production fields in Puerto Rico

with insecticides.

• Before any commercial reintroduction of TC1507 (Cry1F) maize in Puerto Rico, Dow/Pioneer will confirm the susceptibility of FAW. Dow’s letter indicated that there are currently no plans to reintrodu ce Cry1F corn to Puerto Rico.

• Additional FAW sampling was conducted dur ing 2007 and 2008 in Puerto Rico, whichCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

197 also showed similar tolerance to Cry1F.

Several unique factors li kely contributed to the developmen t of Cry1F-resistant FAW in Puerto

Rico. These factors include:

• An island setting provides an isolated ecosy stem that reduces insect migration;

• A tropical climate allows for year-rou nd maize production and year-round insect

pressure;

• TC1507 maize was heavily adopted in Puerto Rico for highly effective control of FAW (a major pest on the island);

• More than 50 FAW generations were con tinually exposed to Cry1F maize (TC1507)

after its introduction into Puer to Rico in 2003 through 2006; and

• A high population and drought conditions in 2006 funneled a large percentage of insects through irrigated TC1507 maize (FAW larvae migrate from declining quality host plants).

There are a number of limiting factors that redu ce the likelihood of FAW resistance in the continental U.S. FAW is a migratory seasonal pe st. The species cannot de velop at temperatures below 50°F and, as a tropical species, do not diap ause. FAW only survive winter in the extreme south of Texas and Florida and thus, selecti on for Cry1F-resistance in maize-growing areas exerts no long-term selection pr essure. In the continental U. S., there are only one or two generations per year on maize. Alternate hos t crops, which include primarily grasses, are abundant. FAW is not a key target pest of Bt maize, except for Bt sweet corn. Both Cry1F- and Cry1Ab-expressing maize are planted in the contin ental U.S. and this market mix reduces the selection pressure on either pr otein. Finally, there have been no reports of FAW performance problems on TC1507 maize in the continental U.S.

Based on the results of the screening level bioa ssay and concentration-dependent bioassay, EPA concluded that the unexpected pe rformance failures observed in 2006 in Puerto Rico were due to Cry1F-resistant FAW feeding on TC1507 maize (reviewed in S. Matten memo to M. Mendelsohn, 8/24/07). This represented the firs t documented case of field failure associated

with insect resistance to a Bt crop. Since FAW is the most important pest of maize in Puerto Rico, Herculex® I (TC1507) Insect Protection Maize cannot be used to effectively control FAW in corn grown on the island.

5) Remedial Action

Remedial action plans are a potential respons e measure should resistance develop to Bt crops. Since resistance may develop in “localized” pest populations, it may be possible to contain the resistance outbreak before it becomes widesprea d. A specific remedial action plan should clearly indicate what actions the registra nt will take in cases of “suspe cted” resistance (i.e., unexpected damage) and “confirmed” resistance. The remedi al action plan can also include appropriate adaptations for regional variati on and the inclusion of appropriate stakeholders. To fully mitigate resistance, a critical element of any remedial action plan should be that once pestCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

198 resistance is confirmed, sales of all Bt corn hybrids that express a si milar protein or a protein in which cross-resistance potential has been demons trated would be ceased in the affected region.

A remedial action plan was proposed by ABSTC for Bt corn (applicable to MON 810, BT11, and TC 1507) consisting of two elements: 1) Strategies for unexpected damage; and 2) Strategies for confirmed resistance. Both components are discussed below.

a) Actions to be Taken if Unexpected Levels of Insect Damage Occur

ABSTC proposed a strategy for unexpected pest damage in Bt corn in the “Industry Insect Resistance Management for Cry1A Plant-Expre ssed Protectants in Field Corn” (submitted 4/19/99). The language of th e ABSTC plan is as follows:

“Customers (growers and seed distributors) will be instructed to contact the registrant or authorized distributor if incide nts of unexpected levels of target insect damage occur during use of the registrant's Bt corn products. Registrants (or their authorized distributors) will investigate and identify the cause for this damage by local field sampling of plant tissue from corn hybrids that contain the Bt corn plant-expressed protectant and sampling of local pest populatio ns, followed by appropriate in vitro and in planta assays. Upon confirmation by i mmunoassay that the plants contain the appropriate Cry1A/Cry1F protein, bioassays wi ll be conducted to determine whether the collected insect population exhib its a resistant phenotype.

Where available and validated for a target pe st species, a discriminating concentration assay will be employed to define a confirme d instance of resistance. For other target pests, until such time that a discriminati ng concentration assay is established and validated, registrants will utilize the followi ng to define a confirme d instance of insect

resistance:

Progeny from the sampled pest population will be considered resistant if they exhibit BOTH of the following charac teristics in bioassays initiated with

neonates:

1. An LC 50 in a standard diet bioassay (incorporating the appropriate Cry1A/Cry1F protein) that exceeds the upper limit of the 95% confidence interval of the mean historical LC 50 for susceptible pest populations, as established by the ongoing baseline monitoring program.

2. > 30% survival and > 25% leaf area dam aged in a five-day bioassay using the appropriate Cry1A/Cry1F-positive leaf tissue under controlled laboratory

conditions.

Based upon continued experience and resear ch, this working definition of confirmed resistance may warrant further refinement. In the event that the registrants find itCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

199 appropriate to alter the criteria specified in the working definition, the registrants will obtain Agency approval in establishing a more suitable definition.”

In the January 31, 2000 letter to Bt corn registrants, the Agency ag reed with this strategy and the working definition of “confirmed resistance.” The letter also clarified the Agency’s interpretation of “suspected” resistance to be:

“...in the case of reported product failure, that corn in question has be en confirmed to be

Bt corn, that the seed used had the proper percentage of corn expressing Bt protein, that the relevant plant tissues are ex pressing the expected level of Bt protein, that it has been ruled out that species not susceptible to the protein could be responsible for the damage,

that no climatic or cultural reasons could be responsible for the damage, and that other reasonable causes for the observed product failu re have been ruled out. The Agency does not interpret ‘suspected resistance’ to mean grower reports of possible control failures, nor does the Agency intend that extensive field studies and testing to fully scientifically confirm insect resistance be completed be fore responsive measures are undertaken.”

Two other elements that could further mitigate th e risk of resistance in the event of unexpected damage (i.e., these measures could be undertaken while the cause of the suspected resistance is investigated) are:

1) The immediate use of alternate control m easures to control the pest suspected of

resistance to Bt corn in the affected region.

2) The destruction of crop residues in the a ffected region immediatel y after harvest (i.e., within one month) with a technique appr opriate for local production practices to minimize the possibility of resistant insect s overwintering and contributing to the next season’s pest population.

A panelist on the 2000 SAP also noted that given the logistics of monito ring, it may take two years from resistance detection to remedial action plan implem entation. During this period of “suspected” resistance, the panelist noted that increasing refuge size could help to prolong susceptibility (SAP 2001).

b) Remedial Measures in Confirme d Cases of Insect Resistance

In cases of “confirmed” resistance (as defi ned in section a) above), ABSTC proposed the following strategy for Bt corn hybrids:

“The registrant will report all instances of confirmed pest resistance, as defined above, to the Agency within 30 days. Upon identifica tion of a confirmed instance of resistance, registrants will take the following immediate mitigation measures:

1. Notify customers and extension agents in the affected area,Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

200 2. Recommend to customers and extension agents in the affected area the use of

alternative control measures to reduce or control the lo cal target pest population,

and

3. Where appropriate, recommend to cust omers and extension agents in the affected area that crop residues be incor porated into the soil following harvest, to minimize the possibility of overwintering insects.

Within 90 days of a confirmed instance of pe st resistance, as defin ed above, registrants

will:

1. Notify the Agency of the immediat e mitigation measures that were implemented,

2. Submit to the Agency a proposed long-te rm resistance management action plan for the affected area,

3. Work closely with the Agency in as suring that an appropriate long-term resistance management action plan for the affected area is implemented, and

4. Implement an action plan that is approved by EPA and that cons ists of some or all the following elem ents, as warranted:

a. Informing customers and extension age nts in the affected area of pest
resistance,

b. Increasing monitoring in the affe cted area, and ensuring that local target pest populations are sampled on an annual basis,

c. Recommending alternative measures to reduce or control target pest populations in the affected area,

d. Implementing intensified local IRM me asures in the affected area based on the latest research results. The impl ementation of such measures will be coordinated by the Agency with other registrants; and

e. If the above elements are not e ffective in mitigating resistance, registrants will voluntarily cease sale of all Bt corn hybrids subject to the Industry IRM Plan in the county expe riencing loss of pr oduct efficacy and in the bordering counties until an effective local management plan approved by EPA has been implemente d. During the voluntary suspension period, registrants may sell and distri bute in these counties only after obtaining EPA approval to study resistance management in those counties. The implementation of such a strategy will be coordinated by the Agency with other registrants and stakeholders.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

201 If EPA agrees that an effecti ve local resistance management plan has been implemented which mitigates resistance, the registrants can resume sales in the affected county(ies).”

The Agency agreed with this strategy for c onfirmed resistance, with the condition that once resistance has been confirmed, the sale and distribution of Bt corn in the affected counties must be halted until an EPA-approved mitigat ion plan is in place. In addition, Bt corn registrants assumed responsibility for resistance mitigation actions (EPA letter to Bt corn registrants, 1/31/00).

In addition to the remedial strategy for confirme d resistance developed by ABSTC, the following elements could further mitigate the risk of resistance development:

1) Immediate suspension of the sale of Bt corn hybrids expressing the same or similar Bt protein (i.e. same mode of action, cross -resistant varieties) as the suspected Bt corn hybrid harboring the resistant population in the affected regi on (this was mandated in the 1/31/00 letter).

2) The mandatory use of a lternate control measures a nd post-harvest crop residue destruction in the affected region (the ABSTC plan “recommends” these measures).

3) For mitigation of resistance in the growing season(s) following a confirmed resistance incident(s), use of the following procedures:

a) Maintenance of the sales suspension of all Bt corn hybrids (with the same protein or similar Bt proteins as the Bt corn hybrids with the resistant population) in the affected region, which would rema in in place until resistance has been

determined to have return ed to acceptable levels.

b) The development and use of alternativ e resistance management strategies for controlling the resistan t pest(s) on corn in the affected region.

c) Notification of all rele vant personnel (e.g., growers, consultants, extension agents, seed distributors, processors, university cooperators, and state/federal authorities) in the affected regi on of the resistance situation.

d) Intensified monitoring and surveillance in the affected region(s) for resistance

and definition of the boundaries of the a ffected region. These studies could also include assays to track the decline of resistance in the field and determine the potential for cross-resistance in the resistant population.

In discussing remedial action, the 2000 SAP sugges ted that eradication of a resistance gene (as part of a remedial action plan) may prove to be too difficult. Rather, a plan based on slowing the spread of resistance genes (and possibly causi ng their decline) may prove more practical. As part of a plan to slow resistance genes, the SA P suggested the following elements: 1) EducationCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

202 of growers/crop consultants to look for unexpe cted pest damage; 2) Monitoring for plant damage, pest susceptibility, and resistance allele frequency (with rapid ve rification and alternate control strategies for verified resistance); 3) Sa les suspensions of the affected product in the region until it can be shown that the product’s bene fits will outweigh its risks; 4) Continual monitoring to determine the effectiveness of the remedial action plan; and 5) An assessment of how the resistance problem occurred (SAP 2001).

c) 2008 Revisions to the Remedial Action Plan

In 2008, ABSTC amended the remedial action pl an to adjust the pr ocess for confirming resistance (reviewed in A. Reynolds memo to M. Mendelsohn, 7/9/08). Under the revised plan, to confirm resistance a pest population must dem onstrate: 1) 30% surv ival and commensurate insect feeding in a bioassay re presentative of field exposure to Bt corn for ECB and SWCB only (CEW was removed); 2) survival on a laboratory diagnostic concentration that demonstrates a genetic basis for the tolerance a nd a resistance allele frequency ≥ 0.1; 3) a LC 50 in a standardized laboratory bioassay that exceeds the upper 95% LC 50 confidence interval for a susceptible population. For the first standard, ABSTC’s revi sed plan removed CEW, removed the explicit requirement to test on Bt corn plant tissue, and eliminated crit eria for > 25% leaf feeding. Steps two and three are essentially the same as in the original remedial action plan. The revised plan maintained the existing definitions of “suspect ed” and “confirmed” resistance and the follow-up procedures for suspected resistance.

Further revisions to the remedial action plan were made for the steps to be implemented in the event of confirmed resistance. The previous (200 0) plan included requirements to be undertaken within 30 days, 90 days, and 1 year from the even t in an effort to mitigate the spread of

resistance. ABSTC’s 2008 revised plan removed the time dependent responses in favor of a simplified list of procedures that include the following elements (taken from the terms and

conditions for Bt corn registrations as amended in 2008 and 2009):

• EPA will receive notification within 30 days of resistance confirmation;

• Affected customers and extension agents will be notified about confirmed resistance within 30 days;

• Monitoring will be increased in the affected area and local target pest populations will be sampled annually to determine the extent and impact of resistance;

• If appropriate (depending on the resistant pe st species, the extent of resistance, the timing of resistance, and the nature of resistance, and th e availability of suitable alternate control measures), alternative control measures will be employed to reduce or control target pest populations in the affected area. Alte rnative control measures may include advising customers and extension agen ts in the affected area to incorporate crops residues into the soil following ha rvest to minimize the possibility of overwintering insects, and/or applications of chemical insecticides;

• Unless otherwise agreed with EPA, sale a nd distribution of the relevant lepidopteranactive Bt corn hybrids will stop in the affected ar ea immediately until an effective local mitigation plan approved by EPA has been implemented;

• [The registrant] will develop a case-specific resistance management action plan withinCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

203 90 days according to the characteristics of the resistance event and local agronomic needs. The registrant will consult with appr opriate stakeholders in the development of the action plan, and the details of such a plan shall be approved by EPA prior to implementation;

• Notification of affected part ies (e.g., growers, consultant s, extension agents, seed distributors, university cooperato rs and state/federal authoriti es as appropriate) in the region of the resistance situat ion and approved action plan; and

• In subsequent growing seasons, sales suspen sion and alternative resistance management strategies will be maintained in the affected region(s) for the Bt corn hybrids that are affected by the resistant population un til an EPA-approved local resistance management plan is in place to mitigate the resistance.

6) Cross-Resistance

Cross-resistance is an area of ma jor concern for resistance management and poses risks to both transgenic Bt crops and microbial Bt insecticides. Cross-resistance occurs when a pest becomes

resistant to one Bt protein, which then allows the pe st to resist other, separate Bt proteins. The threat of cross-resistance is particularly acute with Bt corn, since there are multiple Bt proteins and hybrids currently registered and commercially available (Cr y1Ab and Cry1F are assessed in this document, though other Bt toxins have been re gistered for use in corn ). In addition, some pests of corn are also pests of other crops for which Bt transgenic varieties are or may soon be available or of crops on which microbial Bt insecticides may be used (e.g., CEW on cotton, FAW on tomato). Cross-resistance also poses a risk to pyramid strategies, in which multiple proteins targeting the same pest comple x are deployed simultaneously in the same hybrid. However, it should be noted that, to date, the development of cross-resistance has not been shown in insect pests exposed in the field to Bt crops producing different Bt proteins.

In general, it is possible for resistance to Bt proteins to occur through a number of different mechanisms, some of which may result in cross- resistance to other proteins. The most well

documented mechanism of resistance is reduced (midgut) binding affinity to Bt proteins. Different Cry proteins may bind to distinct receptors in an ins ect gut. Modifications to these insect crystalline protein receptors have been im plicated in resistance to Cry proteins. Other mechanisms that may lead to resistance (and ultimately cross-resistance) include protease inhibition, metabolic adaptations, gut recover y, and behavioral adap tations (Heckel 1994, Tabashnik 1994).

Regarding binding sites, cross-re sistance may result if two protei ns share the same binding site (receptor) in the insect midgut. Therefore, if exposure to one Bt protein results in a modification of the receptor, other proteins sharing this site w ill be affected as well. An example of a possible shared binding site resu lting in cross-resistance was observe d with tobacco budworm (TBW). In this case, TBW selected for resistance to Cry1Ac were also found to be re sistant to the Cry1Aa, Cry1Ab, and Cry1F proteins (Gould et al. 1995).

Cross-resistance patterns in ECB, the major pest of corn, have proven to be complicated. The binding of three Bt insecticidal crystal proteins to the midgut epithelium of ECB larvae wasCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

204 characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections (Denolf et al. 1993). Results de monstrated that two independent insecticidal crystal protein rece ptors were present in the brush border of ECB gut epithelium. From competition binding experiments, it was concluded that Cry1Ab and Cry1Ac are recognized by the same receptor. Also, the Cry1B protein did not compete for the binding site of Cry1Ab and Cry1Ac and was determined to have a different receptor. Cry1D and Cry1E, two proteins that are not toxic to ECB, were not bound to the gut ep ithelial cells. Ot her experiments using laboratory-selected resistant strains to pred ict survival and cross-re sistance in the field on

Bt corn with ECB have provided different results . A Cry1Ac-resistant ECB strain (produced by Dr. Hutchinson, University of Minnesota) and a Cry1Ab-resistant ECB strain (produced by Dr. Keil, University of Delaware) had a moderate level of resistance, about 30 to 60X. None of the

resistant larvae survived on Bt corn beyond the second instar. It is interesting to note that the Cry1Ac-resistant ECB were not cr oss-resistant to Cry1Ab and that Cry1Ab-resistant ECB are not cross-resistant to Cry1Ac (Hutchison, persona l communication, reviewed in U.S. EPA 1998). Based on receptor binding studies, one would have expected both resistant strains to survive on

Bt corn. It can be concluded that although two proteins are closely related, there may be different binding mechanisms or binding affinity in ECB relative to other pest s, such as DBM or

TBW.

Based upon the binding properties of Cry1A and Cry2A proteins in CEW, TBW, and ECB larvae, there appears to be a much lower proba bility of cross-resist ance developing to Cry2A delta endotoxins from resistance to Cry1Ab or Cr y1Ac. Because the Cry1A and Cry2A proteins exhibit different binding characte ristics and very low amino aci d homology, they likely possess different modes of action. However, there is so me evidence for the development of broad crossresistance to Cry1 and Cry2A in at least two la boratory-selected strains: beet armyworm (BAW) (Moar et al. 1995) and TBW (Gould et al. 1992).

Binding studies have also been conducted with Cry1F (expressed in TC 1507 field corn) to

determine cross-resistance potential with other Bt toxins including Cry1A b, Cry1Ac, and Cry9C in ECB. The results showed that Cry1Ab likel y recognizes multiple binding sites in ECB brush border membrane vessicles (BBMV), one of which may be shared by Cry1F (MRID# 450201- 15; reviewed in R. Rose memo to M. Mende lsohn, 1/24/01). However, a second published study to assess midgut receptor binding patterns for Cry1Ab and Cry1F in E CB (Hua et al. 2001) suggested that Cry1F and Cry1Ab only weakly co mpete for BBMV binding sites. As in the previous study, ligand blotting analysis show ed that Cry1Ab and Cry1F recognize multiple binding proteins in ECB. Both toxins rec ognized binding proteins of 154 and 220 kDas and Cry1Ab also recognized 145 and 167 kDa proteins. Despite these shared binding sites, preincubation with Cry1F did not inhibit Cry1Ab binding to BBMV in surface plasmon resonance studies, though Cry1Ab-incubated BBMV did reduce Cry1F binding. Cry1F reduced Cry1Ab BBMV binding in radioligand assa ys, but only at the highest test ed concentrations of Cry1F. The study authors concluded that the “results are explained if Cry1F has low affinity for the Cry1Ab binding site.” Another set of studies using resistant coloni es of ECB showed little cross resistance potential between Cry1F and Cry1Ab. In the first study, four ECB colonies selected for resistance to Cry1Ab were shown to have lo w levels (< 5 fold) of cross resistance with Cry1F, but not Cry9C (Siqueira et al. 2004). Subsequently, a Cr y1F resistant ECB strain wasCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

205 used to assess cross resistance to Cry1Ab, Cry1Ac, and Cry9C (Pereira et al. 2008). This strain possessed 3,000 fold resistance to Cry1F but was s till highly susceptible to Cry1Ab and Cry9C. Cry1F was only slightly (7 fold) cross resistant with Cry1Ac.

Collectively, laboratory-selected strains and isol ated field populations indicate that there is a genetic potential for Bt cross-resistance to develop to multiple or single Cry delta endotoxins in a number of corn pests from exposure to Cry1A b. However, cross-resistance patterns and physiological mechanisms are complex and unpredicta ble, even within related groups of proteins and susceptible pests. Research has suggested that Cry1Ab and Cry1Ac may share binding sites in several tested insect species, although this may not necessarily result in cross-resistance in the field. Still, areas in which Bt corn (expressing Cry1Ab) and Bt cotton (Cry1Ac) are grown may pose additional selection pressure for resistance in CEW, a pest of both corn and cotton.

Given the unpredictability of cr oss-resistance among pest species, it would be useful to generate cross-resistance data for SWCB, SCSB, CSB, BCW, and other secondary pests, to gain a more complete understanding of the implications for Bt corn.

7) Compliance

a) ABSTC Compliance Program

As a term of the amended Bt corn registrations, registrants we re required to develop and submit to EPA a compliance assurance program (CAP ) to ensure grower adherence to IRM requirements (EPA letters to Bt corn registrants, 10/ 15/01). The terms of registration mandated a number of components for the compliance program including:

• Grower Agreements: Contractual arrangement between the registrant and grower to obligate adherence to IRM requirements.

• Annual IRM survey: The survey (conducted anonymously by an independent research firm) is intended to provide a statistically representative sample of growers from various corn-growing regions in the U.S. Re sults from the survey should assess levels of grower compliance with refuges as we ll as grower motivations, attitudes, and reasons for non-compliance.

• On-farm assessments: Registrants are requi red to develop an on-site assessment program in which trained personnel from each company make visits to farms growing

Bt corn. During these visits, compliance w ith refuge requirements is assessed and growers out of compliance are identifie d for corrective action under the Phased Compliance Approach.

• Tips and complaints: Registrants must establish a means for the reporting and investigation of incidences of refuge non-compliance.

• Phased Compliance Approach (PCA): A c onsistent set of procedures (for all Bt corn registrants) to be empl oyed to address non-compliance among growers and seed

dealers.

The Bt corn registrants (under ABSTC) submitted a proposed CAP to the EPA in 2002. AfterCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

206 review, EPA approved the cap (letter to Bt corn registrants dated November 1, 2002) to be fully implemented for the 2003 growing season. Subsequently, ABSTC submitted revised versions of the CAP in 2004 and 2005 in response to EPA review s of annual growing seas on reports. Issues

identified in these reviews are discussed in the sections below specific to each element of the CAP. EPA reviews of CAP reports include the following documents:

• 2002 and 2003 growing season reports: T. Milofsky memo to M. Mendelsohn, 7/15/04;

• 2004 growing season report and CAP amendments: S. Matten memo to M. Mendelsohn, 3/23/05);

• 2005 growing season report: S. Mattten memo to M. Mendelsohn, 11/2/06;

• 2006 growing season report: T. Milo fsky memo to M. Mendelsohn, 8/30/07

• 2007 and 2008 growing season report: J. Ma rtinez memo to M. Mendelsohn, 4/15/09

b) Grower Surveys (1996-2001)

Several surveys and estimates of th e level of grower compliance for Bt corn IRM were conducted prior to the development and implem entation of the 2002 ABSTC CAP. Dr. Marlin Rice (Iowa State University) conducted regular gr ower surveys to measure grower attitudes towards various aspects of Bt corn, including compliance with IRM guidelines. These surveys showed that the great majority of growers unders tood and were receptive to the need for refuge and resistance management. However, they also demonstrated that some level of noncompliance must be expected. The results from the 1996 grower survey showed that 23.5% of sampled growers would follow a prescribed IRM st rategy, 57.1% would if co mpatible with their growing practices, 7.2% would not follow IR M, and 12.2% “didn’t know” (Pilcher & Rice 1997). Results from the 1998 grower survey show ed that 25.5% of growers would implement recommended IRM, 58.9% would if compatible with their growing practices, 2.6% would not follow IRM recommendations, and 12.9% “didn’t know” (Rice & Pilcher 1999).

In terms of compliance information submitted by industry, ABSTC (representing Bt corn registrants) conducted a compliance survey for the 2000 growing season (MRID# 453205-03). The ABSTC compliance plan consisted of grower contracts, intensified education for regions showing low compliance, and restrictions on future use of Bt corn for individual growers repeatedly out of compliance. The compliance survey was conducted by a marketing research firm and included telephone survey s of 501 total growers, each farm ing at least 200 acres. This survey did not involve visits to individual farm s (i.e., grower audits). Compliance was assessed

for two Bt corn IRM requirements: percent refuge (re quired to be 20% or greater) and refuge proximity (required to be within 1⁄2 mile of the Bt field). Survey respondents indicated that 87% planted an appropriate amount of refuge (at least 20%), while 13% had less than the required amount or no refuge. In terms of proximity, 82% of growers reported re fuges planted within 1⁄2

mile of the Bt field (18% reported refuges plante d greater than 1⁄2 mile from the Bt field). When both refuge percentage and proxi mity are considered together, 71% of growers were in total compliance. It should be noted that growers we re sampled in southern cotton growing regions, where a 50% refuge is required. It is unclear from the survey whether these growers were counted as compliant for pl anting a refuge of less than 50%, but greater than 20%.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

207 Collectively, these surveys indicate that 100% co mpliance is not likely and that some level of non-compliance must be expected. However, the 2000 SAP indicated that while surveys such as these are useful for tracking grower attitudes, they are not reliable for determining actual grower compliance (SAP 2001). The format of the surv eys (mail or phone interviews) may encourage non-compliant growers to misrepresent their actio ns or “cheat” in their responses. Without confirmatory visits to individual farms (i.e., aud its), it may be impossible to verify the accuracy of grower responses. The end re sult could be increased “false-pos itives,” which may artificially inflate estimates of grower compliance. As such, actual non-compliance may be significantly higher than the survey results would suggest. To resolve this problem, the 2000 SAP suggested utilizing surveys created and conducted by independe nt parties to assess gr ower practices (SAP 2001). In addition to this recommendation, it may be useful to conduct some on-farm visits for firsthand verification of compliance. Such visits could be performed as part of a survey process, to evaluate the accuracy of grower survey responses.

c) Grower Surveys (2001- 2008)

As a term and condition of the Bt Corn product re gistrations in 2001, regist rants were required to perform an “annual survey of a statis tically representative sample of Bt corn growers conducted by an independent third pa rty” (EPA letters to Bt corn registrants, 10/15/01). The grower survey functions to measure compliance adherence to refuge size and distance requirements at a regional level and to identify edu cational opportunities in these four regions to increase grower compliance with IRM requirements.

Beginning with the 2002 growing season, >500 growers from four separate regions have been anonymously surveyed. The methodology for c onducting the grower survey has remained largely unchanged since it was first conducted by Market Horizons, Inc in 2000 for ABSTC (MRID# 453205-03). However, in 2007 Marketing Hori zons started to utili ze an internet-based survey approach due to an incr easing complexity of growers’ Bt corn planting practices and a need to standardize the grower survey acro ss insect-protected traits. Consistency in methodology is important because it allows for y ear-to-year comparisons of the results. There are four sampling regions that diffe r by pest and level of adoption: a) regions with high rates of

Bt corn adoption (150 samples from the Eastern Iowa, Northern Illinois region and 200 samples from the South Dakota, Minnesota , Nebraska, Western Iowa region) ; b) areas where insecticides have historically been sprayed for control of Lepidopteran pests (100 sa mples from the Kansas, Oklahoma, Texas Panhandle region); and c) areas where Bt corn and Bt cotton may be grown simultaneously (100 samples from the North Ca rolina, South Carolina, Southeast Missouri, Tennessee, Mississippi region). Resu lts are weighted to reflect th e actual distribution of corn acres in each region.

Data from the 2002 through 2008 compliance survey s are summarized in Table D7 below. In 2007 and 2008, the online survey results were not br oken down for the four regions, as was done in previous years, and therefor e, it is impossible to determine which region had greater noncompliance with refuge requirements. It has been recommended that this information be provided in future reports so th at it can be determined in whic h farming regions the registrantsCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

208 should focus their educational outreach (see EPA review in J. Martinez memo to M. Mendelsohn, 4/15/09).

Overall, the survey results through 2008 show that grower compliance with refuge size requirements for Bt corn has declined and is now at th e lowest level since initiation of the surveys in 2000. A report issued by the Center fo r Science in the Public Interest (CSPI) that analyzed the same data set also highlighted th is declining trend in refuge compliance (Jaffe 2009). Most growers continue to adhere to the refuge requirements and a portion of the noncompliant growers likely have planted some refu ge (though not large enough in size). However, decrease in refuge compliance is a concern and can increase the risk of resistance by reducing the availability of susceptible insects to mate with any resistant survivors of Bt corn. It is noted that the observed decrease has occurred despite increa sed efforts by registrants and the National Corn Grower Association (NCGA) to provide more out reach tools, improved educational efforts, and more avenues of communication with growers. In addition, grower awareness of IRM requirement has consistently in creased since 2002 through 2008, sugge sting that there is a degree of willful non-compliance by some growers. Th e reasons for the increase in non-compliance are

not clear but could include financia l incentives (i.e., increased yield on Bt acres), logistical difficulties with planting refuges, lack of availability of refuge seed, or ignorance of refuge requirements and IRM obligations.

Table D7. Summary of Telephone/Online Survey Results for Corn Borer-Protected Bt Corn Growers from 2002 to 2008 (Data fr om ABSTC annual reports 2002 through 2008) 1

Survey Year

2002 2003 2004 2005 2006 2007 2008 Survey Question

-----------% respondents-----------

Adherence to Refuge Size

Weighted average across a ll four regions surveyed 86 92 91 92 89 80 78

NC/SC/SE MO/TN/MS

( 50% refuge in cotton-growing areas) 77 82 78 82 80

E IA/N IL (20% refuge) 88 96 95 94

SD/MN/NE/W IA (20% refuge) 85 93 90 91 89-99

KS/OK/TX (20% refuge) [High Plains] 81 82 93 95 82 N/A N/A

Adherence to Distance Requirements

Weighted average across a ll four regions surveyed 89 93 96 96 96 88 5 88 5

NC/SC/SE MO/TN/MS [cotton-growing region] 78 83 84 80 80

E IA/N IL 95 96 96 97

SD/MN/NE/W IA 89 94 98 97

KS/OK/TX [High Plains] 79 92 94 94 89-99 N/A N/ACry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

209 Survey Year

2002 2003 2004 2005 2006 2007 2008 Survey Question

-----------% respondents-----------

Awareness of IRM Requirements

All growers 88 93 92 92 92 96 98

Unaided Recall of IRM Requirements

Refuge size

(weighted average across all four regions surveyed) 42 52 53 59 N/A 63 4 79 4

Refuge distance

(weighted average across all four regions surveyed) 37 49 55 53 49 79 4 89 4

Refuge size (Corn Belt and High Plains) 2 61 N/A N/A

Refuge size (cotton-growing region) 3 31 N/A N/A

1 The data in this table include only growers planting single trait Bt corn registrations expressing Cry1Ab and Cry1F. Growers with stacked products containing Cry1Ab or Cry1F (and other toxins for rootworm control) are not

included.

2 Corn Belt and high plains = Iowa, Illinois, South Dakota, Minnesota, Nebraska, Kansas, Oklahoma, Texas, i.e., three of four regions surveyed

3 Cotton-growing region = North Carolina, South Carolina, south-eastern Missouri, Tennessee, and Mississippi

4 Cotton Belt numbers are not separated from Corn Belt survey results

5 Percent reflects only those growers that could remember the layout of all their Bt fields; 447 out of 467 (95.7%) in 2007; 298 out of 317 (94%) in 2008.

ABSTC has focused the survey on growers who plan t more than 200 corn acres in the Corn Belt and 100 cotton-growing areas. A nnually, these larger growers plant more than 85% of the Bt corn acreage. EPA’s initial review of the survey (see T. Milofsky memo to M. Mendelsohn, 7/15/04 and EPA letter to ABSTC, 8/19/04) noted concerns associat ed with the use of farm size as a component of the grower survey participant selection criteria. First, the terms and conditions of the registration state that the a nnual survey shall cons ist of a “s tatistically representative sample of Bt corn growers.” Since corn borer-p rotected corn is grown on farms of all sizes, a survey that excludes responses fr om smaller growers would fail to provide a “statistically representative” picture of all grow ers and their compliance. It would exclude a body of growers who plant approximately 15% of the Bt corn acres. A 2003 CSPI report (Jaffe 2003) using USDA/National Agricu ltural Statistics Survey (NASS) data from 10 corn-growing states found substantially lowe r IRM compliance on small farms (<200 acres). This finding may have been indicative of inadequate gr ower education among smaller farmers.

In response to EPA’s concerns, ABSTC proposed to amend the terms of the registration to limit the surveys to growers planting more than 200 acres (100 total acres in the cotton-growing region) (amendment requests from Bt corn registrants, Octobe r and November, 2005). ABSTC provided four reasons as to why the third-party survey should con tinue to exclude growers with low acreage:Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

210 1. The farm size restriction ensures that the survey covers the la rgest number of corn

acres. 2002 USDA/NASS statistics i ndicated that nearly 88% of all Bt corn in the 10 states sampled in 2002 was planted on fa rms with greater than 200 total acres of

corn. 2. The farm size restriction has been in place since 2000 and ensures consistency in methodology for year-to-year statistical comparisons.

3. Inclusion of smaller farms would dilute the number of acres surveyed if current sample sizes remained constant.

4. The cost of the survey would increase signi ficantly if sample size was increased and/or extra efforts were made to survey smaller farms. The extra cost would not be justified and inclusion of small fa rmers would have limited impact on the potential for resistance development.

After review (see S. Matten memo to M. Mendelsohn, 2/15/06), EPA accepted ABSTC’s rationale for focusing the third-party survey on gr owers with larger acreag es. It was recognized that the grower survey is not a direct measurem ent of individual compliance and that the on-farm assessment program (intended to detect non-co mpliant individuals) includes all growers regardless of size. An analysis of the 2003 NASS data in dicated that there would have been only a difference of 2.3% in compliance (range of 1.7% to 5.7% for individual states) if both larger

and smaller farms were included in the NASS assessm ent. According to this same data set, less

than 5% of farms in the 10 states would have been classified as poten tially contributing to a localized cluster of small farmers growing Bt corn that could have le d to higher local selection pressure for resistance. It is unlikely that such farms could form clusters that were not interspersed amongst the other 95% of larger farm s. Further, even if refuge adherence on

smaller farms was found to be lower than on larger farms, the overall acreage of Bt corn not protected by the required refuge would be extremely small. Therefore, the lower overall amount

of Bt corn on smaller farms would not likely incr ease the risk of resi stance evolution to Bt corn.

d) On-Farm Assessments

The on-farm assessment program is the portion of the CAP that iden tifies individual noncompliant growers (regardless of farm size) for remedial IRM education, follow-up reassessments, and other activities as part of the phased compliance approach (PCA). It can also serve as a tool to enhance the registrant’s understanding of the obsta cles growers face in implementing IRM requirements. The mandatory on-farm assessment program was fully implemented for the first time in 2003 and ha s typically encompassed approximately 2,000 - 2,200 growers. Company represen tatives are trained to conduc t on-farm assessments. Topics covered in these trainings include IRM requireme nts, messages that should be communicated to growers, follow-up actions if gr owers are found to be out of co mpliance and how to use the IRM assessment form. All assessment forms contain the same questions, but the introductory paragraphs and company represen tative sections are customiz ed to suit the needs of each registrant. Registrant s respond to all compliance deviations identified in the assessments according to the common set of standards outlined in the PCA.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

211 Data from the on-farm assessments (2003 thr ough 2008) of corn borer-p rotected corn are

summarized in Table D8 below. The assessments do not have the statistical power associated with the consistently stratifie d and randomized telephone/on-line surveys and are not used to measure representative rates of non-compliance. Fewer on farm assessments were made in 2008 relative to previous years, though this coincided with an increase in the on-farm assessments for growers of stacked Bt corn PIPs for lepidopteran and corn rootworm control. In 2007 and 2008, no information was provided regarding specific n on-compliance with refuge size and distance. This information should be provided in future repor ts to be consistent with previously collected data and to illustrate how grow ers are out of compliance (see EPA review in J. Martinez memo to M. Mendelsohn, 4/15/09).

Table D8. Cumulative Results (2003-2008) fo r the First-Time On-Farm Assessments of

Corn Borer-Protected Bt Corn Growers (Data from ABSTC annual reports 2003 through 2008) 1

Number of growers

2003 2004 2005 2006 2007 2008

Growers assessed 1961 2130 2215 2020 2083 1312

Compliant growers 1,789

(91%) 2032

(95.4%) 2089

(94.3%) 1930

(95.5%) 1895

(91.0%) 1132

(86.3%)

Noncompliant growers 2 172

(9%) 98

(4.6%) 126

(5.7%) 90

(4.5%) 188

(9.0%) 180

(13.7%)

Refuge size deviations 64 (3%) 27 (1.3%) 33 (1.5%) 33 (1.6%) N/A N/A

Refuge distance deviations 127 (7%) 69 (3.2%) 68 (3.1%) 64 (3.2%) N/A N/A

Insignificant deviations 68 (4%) 59 (2.8%) 51 (2.3%) 45 (2.2%) 59 (2.8%) 46 (3.5%)

Significant deviations 3 104 (5%) 39 (1.8%) 75 (3.4%) 45 (2.2%) 129 (6.2%) 134 (10.2%)

1 The data in this table include only growers planting single trait Bt corn registrations expressing Cry1Ab and Cry1F. Growers with stacked products containing Cry1Ab or Cry1F (and other toxins for rootworm control) are not included.

2 Some growers had compliance deviations othe r than refuge size or distance or some growers may have had both a refuge size and a refuge distance deviation; thus, the total of refuge distance and size de viations does not equal the number of non-compli ant

growers.

3 Significant deviations are defined as: A Bt corn grower who planted less than 15% non- Bt corn refuge (except in certain cotton growing areas in which case it would be less than 40% non- Bt corn refuge); or planted fewer than 2/3 of the Bt corn fields are planted within 2 mile of a non- Bt corn refuge.

e) Tips and Complaints

As required by the terms of registration, Bt corn registrants must ha ve a “tips and complaints” system as a mechanism for individuals (e.g., growers, sales representatives, etc.) to report alleged instances of IRM noncompliance. A system wa s developed by ABSTC and implemented for the first full-season in 2003. It allows each registra nt to develop a “tips and complaints” system that is “compatible with their busin ess operations...” The registrants have developed mechanisms (e.g., customer service numbers) to receive alleged instances of non-compliance with the IRM requirements. The tips and complaints numbe r is publicized through a variety of IRM communications mechanisms (e.g., technical bulle tins, grower guides, letters, seed catalogs, newsletters, etc.).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

212 The number of tips and complaints (for all Bt corn registrations) received through 2008 is

summarized in Table D9 below. Each of th ese growers identified through the tips and complaints mechanism were visited as part of the on-farm assessment program. However, it is not possible to determine whether any of the non- compliant growers identified via the tips and complaints route were subject to the Phased Compliance Approach.

EPA’s initial review of the tips and complaints system identified a number of flaws that could make it difficult to report instances of non-compliance (see T. Milofsky memo to M. Mendelsohn, 7/15/04). This was highlighted by the fact that there were no legitimate tips and complaints in 2003 or 2004. In response, ABSTC stated that “the companies are planning to modify language in Bt corn product use guides and other grow er communications to clearly state how growers can register a tip or complaint ab out an allegedly non-comp liant grower by calling a toll free number or by contacting a company re presentative” (October 4, 2004 letter to EPA). Since the 2005 growing season anonymous tips ha ve been reported, though they have been relatively rare.

Table D9. Anonymous Tips and Compla ints about Non-Compliance with IRM requirements (Data from ABSTC annual reports 2003 through 2008)

Year Number of Tips and Complaints

2003 0

2004 0

2005 5

2006 3

2007 14

2008 5

f) Phased Compliance Approach

ABSTC’s 2002 CAP included a standa rd set of procedures (shown in Table D10) known as the Phased Compliance Approach (PCA), which is to be used by registrants when responding to instances of grower noncomplian ce with the IRM requirements. The PCA also established a tiered approach for non-compliance with “significant” deviations and “other” deviations. For refuge requirements of 20% (Corn Belt) and 50% (cotton-growing re gions) signifi cant deviations

were defined as: a Bt grower planted less than 15% non- Bt corn refuge (in cotton growing areas

less than 40% non- Bt corn refuge); or fewer than 2/3 of the Bt corn fields were planted within 1⁄2

mile of a non- Bt corn refuge. Under the PCA, sales are to be suspended to individual growers for one year after two years of significant deviations. Following the one-year suspension, growers will need to requalify to purchase seeds.

Table D10. Phased Compliance Approach (PCA) – Standards for BtCorn Refuge NonCompliance (submitted with the ABSTC 2002 CAP)Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

213 Mandatory Responses Additional Responses

Significant Deviations • IRM education.

• Warning letter.

• Compliance assistance contact prior to planting.

• Compliance assessment contact for the following growing season.

• Deny access to the Bt corn product for any significant deviation two years in a

row.

Other Deviations • IRM education.

• Letter and/or compliance assistance contact prior to planting.

• Compliance assessment contact in the following growing season. • Invoice monitoring.

• Technical assistance.

• Grower IRM training.

• Reaffirmation of IRM obligations.

• Deny access to the Bt corn product for other

deviations that are repeated over a period of

years.

Grower identified as non-complia nt (significant or other devia tions) are required to receive a “compliance assessment contact” the following year under the PCA. Non-compliant growers are typically identified through the on-farm assess ment program (see discussion in the on-farm assessment section below). Table D11 summa rizes the numbers of non-compliant growers reassessed under the PCA and the growers still f ound to be out of compliance. As of the 2008 growing season, two growers have been denied access to Bt corn technology due to consecutive years of significan t non-compliance.

Table D11. Reassessment of Non-Compliant Corn Borer-Protected Corn Growers Under the Phased Compliance Approach (take n from ABSTC annua l CAP reports) 1

Year Reassessments 2 Significant

Deviations 3 Loss of Access to Technology

2004 172 0 0

2005 98 0 0

2006 126 2 2

2007 90 0 0

2008 188 0 0

1 The data in this table include only growers planting single trait Bt corn registrations expressing Cry1Ab and Cry1F. Growers with stacked products containing Cry1Ab or Cry1F (and other toxins for rootworm control) are notCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

214 included.

2 Reassessments of growers identified with deviations (significant and other) to refuge requirements the previous growing season.

3 Significant deviations recorded the following season. Two successive years of significant deviations results in loss of access to Bt corn technology.

EPA’s reviews of the PCA and annual reports id entified several concer ns with the overall approach (see T. Milofsky memo to M. Me ndelsohn, 7/15/04). First, one year of seed suspension may be insufficient to reform noncomp liant growers. Second, it is unclear how the PCA will address repeat violators. However, th e PCA results indicate that the reassessment process and the penalty for repeated non-complian ce in the second year ha ve been effective in bringing non-compliant growers back into comp liance. Through 2008, only two growers (out of 674 assessed) have lost access to Bt corn due to repeated years of significant non-compliance.

8) Grower Education

Growers are perhaps the most essential elemen t for the implementation and success of any IRM plan as they will ultimately be responsible fo r ensuring that refuges are planted according to guidelines and that Bt fields are monitored for unexpected pest damage. Therefore, a program that educates growers as to the necessity of IRM and provides guidance as to how to deploy IRM should be an integral part of any resistance management strategy. The 2000 SAP also suggested that a comprehensive education program ma y help increase IRM compliance (SAP 2001). Ideally, the educational messages presented to growers should be consistent (among different registrants) and reflect the most current resistan ce management guidelines. Specific examples of education tools for growers can include grower guides, technical bulletins, sales materials, training sessions, Internet sites, toll-free num bers for questions or further information, and educational publications.

9) Annual Reports

Written reports on various aspects of IRM, submitted on an annual basis to EPA, are of great aid

in the evaluation of the success of resistance management for Bt corn. The Agency has received annual reports from Bt corn registrants (as a requ irement of registration) on Bt corn sales/market penetration, IRM-related research, grower education, grower compliance and resistance monitoring. It is particularly us eful to receive reports from Bt corn registrants on grower compliance and resistance monitoring.

10) Bt Sweet Corn IRM

Attribute Bt sweet corn is a BT11 hybrid and expresses the Cry1Ab protein. It is thought that Attribute, like BT11 field corn, contains a high do se for ECB. The other targeted pests, for which there is not a high dose, are CEW and FAW.

Refuge for Bt sweet corn was not recommended for the following reasons: 1) sweet corn is typically harvested earlier than field corn (18-21 ) days after silking (bef ore most lepidopteran larvae complete development); and 2) all Bt sweet corn residues were to be destroyed within oneCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

215 month of harvest (a practi ce that presumably would destroy any live larvae le ft in corn stalks). The 2000 SAP agreed that this approach should be sufficient to mitigate pest resistance to Bt sweet corn. Several panelists, however, suggested a shorter crop destruction period (i.e., 14 days instead of one month) (SAP 2001).

The terms and conditions of the Bt sweet corn registration stipul ate that, based on IRM concerns, the product is for commercial use only and is not av ailable to growers planti ng less than 40 acres. However, should smaller growers (i.e., those pl anting less than 40 acres ) adhere to the crop destruct requirements for Bt sweet corn (to destroy any overwinte ring insects), it is unlikely these growers will pose a threat to pest resistance give n the limited acreage involved. As such, from an IRM perspective, it sh ould be possible to lift the acreage re strictions on smaller growers for Bt

sweet corn.

Regarding crop destruction, it is possible that th e crop destruct requirement may not be adequate in itself to mitigate the threat of resistance for ECB. Specifically, there are data (Mason et al. 1983) that show variance among di fferent crop destruct technique s in terms of the number of surviving ECB. The variation in the efficacy of crop destruct techniques may increase the risk

for ECB resistance in Bt sweet corn. This risk may be m itigated by either: 1) Prescribing a specific and effective crop destruct technique; or 2) Utilizing structur ed refuge. Regarding option #1, it should be noted that co rn cultivation practices vary (i .e., plow vs. no-till) and certain crop destruct techniques may not be compatible with all practices. Furthermore, additional research could help to verify the mo st appropriate crop destruct technique.

The threat of resistance for CEW and FAW in sweet corn should be lower than ECB due to the fact that CEW and FAW typically complete deve lopment in corn ears (unlike stalk-boring ECB), which are mostly harvested and removed from th e field prior to crop destruction (Lynch et al. 1999). Also, FAW is known to overwinter only in south Florida, south Texas, and the

Caribbean.

As part of the registration, a FAW monitori ng program has been developed to determine susceptibility to Cry1Ab (other Bt sweet corn target pests, E CB and CEW, are part of the monitoring program for Bt field corn, described in the Monitoring section - D.2.b.4). Susceptibility was determined with diet assays utilizing toxin overlay s for FAW populations collected from four geographic lo cations in 1998 and 1999. For 1998, the LD 50 range was 0.90 - 1.50 μg Cry1Ab/cm 2 and for 1999, the LD 50 range was 2.14 - 10.22 μg Cry1Ab/cm 2 (Lynch et al. 2000). Susceptibility data for FAW and Cry1 Ab are summarized in Table D12 below. The decreased susceptibility observed in 1999 was not likely an increase in toleran ce to the toxin (the lab colony used as a control showed similar tren ds) and presumably was the result of population variability or experimental effects.

It should be noted that for FAW, resistance monitoring is less of a concern due to the fact that

resistance is not lik ely. However, should there be significant Bt sweet corn acreage in areas where FAW overwinters (south Florida and south Te xas), it would be beneficial for FAW to be

monitored for resistance. As a term of registration, FAW mon itoring was required for Attribute Bt sweet corn in counties where acreage exceeds 5,000 acres and FAW is capable ofCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

216 overwintering. However, FAW data have not been submitted since the 2000 growing season because this 5,000 acre/county requirement has not b een met. Given the low acreage, there has likely been low pest pressure for resistance with Bt sweet corn and it is unlikely FAW tolerance to Cry1Ab has increased due to the planting of Bt sweet corn.

Table D12. FAW Susceptibility (LC 50 ) to Cry1Ab (Lynch et al. 2000, Hamm et al. 2001)

Year Susceptibility (LC 50 - μg Cry1Ab/cm 2 )

1998 0.90 - 1.50

1999 2.14 - 10.22

2000 0.27 - 0.94

c. Stacked and Pyramided PIPs Containing Cry1Ab and/or Cry1F

Since the initial registrations of Cry1Ab and Cry1F, these traits have been “stacked” (i.e., combined with one or more other toxins for control of multiple pest complexes) or “pyramided” (i.e., combined with one or more other toxins for control of the same target pest) into additional PIP products.

Both Cry1Ab and/or Cry1F have been combined with other Bt toxins for control of corn rootworm ( Diabrotica sp.) in stacked PIPs. For these products, IRM must be addressed for lepidopteran and corn rootworm by deploying either separate refuges (for each pest complex) or a combined refuge that addresses both pest comp lexes. Complete discussions of corn rootworm IRM (as well as combination refuges for lepi doptera and corn rootworm) can be found in the regulatory documents (i.e., BRADs) for the relevant corn rootworm PIP traits. The following products with Cry1Ab or Cry1F have been stacked with corn rootworm traits (some have also been pyramided with multiple lepidopteran toxins):

• MON 863 x MON 810 (YieldGard Plus) – EPA Reg. No. 524-545 (Cry1Ab, Cry3Bb1)

• MON 88017 x MON 810 (YieldGard VT Plus) – EPA Reg. No. 524-552 (Cry1Ab, Cry3Bb1)

• DAS-59122-7 x TC1507 (Herculex Xtra ) – EPA Reg. No. 68467-6 (Cry1F, Cry34/35Ab1)

• MIR 604 x Bt11 (Agrisure CB/RW) – EP A Reg. No. 67979-8 (Cry1Ab, Cry3A)

• Bt11 x MIR 162 x MIR 604 – EPA Reg. N o. 67979-13 (Cry1Ab, Vip3Aa20, mCry3A)

• MON 89034 x TC1507 x MON 88017 x DAS-59 122-7 (SmartStax) – EPA Reg. No. 524-581 and 68467-7 (Cry1F, Cry1A.105, Cry2Ab2, Cry3Bb1, Cry34/35Ab1)

• DAS-59122-7 x TC1507 (Herculex Xtra) + TC 1507 (Herculex) (Optimum AcreMax 1 Seed Blend) – EPA Reg. No. 29964-6 (Cry1F, Cry34/35Ab1)

• TC1507 x DAS-59122-7 x MON 810 – EP A Reg. No. 29964-8 (Cry1F, Cry1Ab, Cry34/35Ab1)

• DAS-59122-7 x MON 810 – EPA Reg. No. 29964-9 (Cry1Ab, Cry34/35Ab1)Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

217 Pyramided products for enhanced lepidopteran contro l have also been registered. Such products (with no stacked toxins for rootworm control) include the following:

• Bt11 x MIR 162 – EPA Reg. No. 67979-12 (Cry 1Ab, Vip3Aa20) – discussed in the BRAD for Vip3Aa20

• TC1507 x MON 810 – EPA Reg. No. 29964-7 (C ry1F, Cry1Ab) – discussed below

TC1507 x MON 810 (EPA Reg. No. 29964-7)

TC1507 x MON 810 was registered by Pioneer a nd expresses both Cry1Ab and Cry1F for lepidopteran control. The product was registered with the same refuge requirements and IRM plan as the single toxin Cry1A b (MON 810) and Cry1Ab (TC1507) re gistrations (see details on the IRM plan in section D.1.a of this document).

For stacked and pyramided products of previous ly-registered toxins de veloped by conventional breeding, IRM data typically cons ists of efficacy and protein e xpression data. These data are used to confirm that the stacked/pyramided produc ts will have comparable dose profiles as the single toxin constituent products. Along these li nes, Pioneer submitted protein expression data and field efficacy data for the major target pests.

Protein expression data (MRI D# 475109-04) were generated for the pyramided product (1507 x MON 810) and the single toxin constituent PIPs . Studies were conduc ted in greenhouses and plant tissues were analyzed by ELI SA for protein expression. Pi oneer sampled leaf, stalk, root, whole plant, and grain tissues for the analyses (pollen tissue was also collected but was often below the limit of detection). Corn tissues we re sampled during the R1 corn growth stage, except for grain which was taken from the R6 stage. The results from the ELISA assays showed that Cry1F and Cry1Ab, protein we re expressed at comparable le vels in the pyramided product as the single toxin constituent products. Expressi on was consistent throug hout all tissues -- only small differences in protein levels were noted between the pyramid a nd single toxin products which were not statistically significant.

Field efficacy data (MRID# 476778-03) were ge nerated for ECB, FAW, and SWCB with TC1507 x 59122 x MON 810 (a PIP that also contai ns a corn rootworm toxin). Efficacy for ECB was tested for both first and second generation using artificial infest ation in five states (Illinois, Indiana, Iowa, Minneso ta, and Nebraska). First gene ration ECB were assessed with a “reversed” Guthrie 1-9 damage scale (i.e., 1 = heavy damage, 9 = no damage). Second generation ECB damage was assessed by sampling stalks from infested plots and measuring tunnel (feeding) damage (in cm). The results for first generation ECB were consistent across test

locations. Untreated controls (isoline corn) experienced modera te ECB feeding, with a damage ratings range of 4.8 to 7.9. The stacked pr oduct (1507 x 59122 x MON 810) suffered no damage (rating 9 for all locations). A similar pattern was also observe d for second generation ECB. Stalk tunneling in the untreated control groups was moderate to high (average tunnel length 7.5 to 26.2 cm) while damage was very low in Bt corn, with average tunne l lengths generally less than 0.2 cm (two locations had 0.6 and 0.8 cm avg. tunneling). SWCB testing was conductedCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

218 similarly to ECB but using natura l infestation at two sites in Te nnessee and Kansas. Damage at the R4-R5 reproductive stage was assessed throu gh measurements of stalk tunneling. Results

showed that non- Bt corn (isoline) experienced moderate damage (average 10.9 cm tunnel length)

while the Bt treatments had little or no tunneling (0 to 1.33 cm avg. length). FAW tests were conducted using artificial infest ation on whorl stage corn (V6) . FAW feeding damage was

scored after three weeks of infestation with a 1 to 9 scal e (1 = heavy damage, 9 = no damage) much like the one used for ECB testing. Data fr om the field trials show ed that FAW inflicted significant damage (average damage ratings of 2.22 to 3.10) on isoline corn but the Bt corn treatments experienced very slight to no da mage from FAW (avg. ratings of 8.93 to 9.0).

Both MON 810 (Cry1Ab) and Herculex (Cry1F) ha ve been considered “high dose” for ECB and likely SWCB, but not for CEW. Given the confir matory protein expression and efficacy studies, the stacked products can also be considered high dose for ECB and SWCB and non-high dose for CEW. With similar dose pr ofiles, the IRM assessments and refuge plans developed for the single toxin PIPs (i.e., TC1507 and MON 810 can be applied to the pyramided PIP (1507 x MON 810). Therefore, the submitted data support ed the use of the ex isting lepidopteran IRM plan including a 20% refuge (50% in cotton gr owing regions) for TC1507 x MON 810 corn (see EPA review in A. Reynolds memo to A. Sibold, 1/7/10).

d. Information to Improve the Risk Assessment

Although the Agency has considered the most up-to -date scientific information in this risk assessment, resistance management is a developi ng field. Therefore, the IRM strategies may be improved with the collection of additional inform ation, the results of which can be submitted in annual research reports. As part of the 2001 Bt crops reassessment, a number of areas were

identified that would benefit from additional res earch. These data goals are summarized in Table D13 below with a 2010 status update.

Table D13. Summary of Data That Coul d Improve Insect Resistance Management Strategies for Bt Corn Products
Data Need (As Identified in 2001) Pests 2010 Status

Pest Biology: e.g., larval movement, adult movement,mating behavior, pre- and postmating dispersal, ovipositional behavior, fitness, and overwintering habi tat and survival ECB, CEW, SWCB No additional data were required

but research has been conducted and reported in the public

literature. No information has

been submitted to EPA that would change this risk assessment for Cry1Ab or Cry1F corn.

North to South Movement CEW Data were required as a condition of registration in 2001. A summary of th e data and

conclusions can be found in

section D.2.b.3 of this document. High Dose Verification (using 1998 SAP techniques) ECB and SWCB No additional data have been received, although it is likely that both Cry1Ab (MON 810, BT11) and Cry1F (TC 1507) corn expressCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

219 Data Need (As Identified in 2001) Pests 2010 Status

a high dose for both of these

insects. Resistance Allele Frequency ECB, CEW, SWCB, FAW (Bt sweet corn) Some additional information has

been received for ECB and FAW populations of interest as part of the resistance monitoring data (see section D.2.b.4) Cross-Resistance - Cry1F, Cry2A, Cry1A proteins ECB, CEW, SWCB Additional data have been developed for Cry1F and Cry1Ab (see section D.2.b.6). Data for Cry2A, Cry1F, and Cry1A proteins are further discussed in

the BRAD for MON 89034 Bt

corn. Evaluation (field studies and models) of Refuge Options (20% external refuge (sprayable) v. 20% in-field) - [Issues to consider: production of susceptible insects (500:1 ratio) in insecticide treated

and non-insecticide treated refuges, adequacy of size, structure, and deployment of the refuge, rotation of refuge.] ECB, CEW, SWCB Data on the impacts of insecticide

use on Bt corn refuges were required as a condition of registration. These data are

discussed in section D.2.b.3 of this

document.

Models: development, validation, refinement of existing and new

models ECB, SWCB, CEW New models have been conducted for stacked and pyramided products containi ng Cry1Ab and Cry1F and are discussed in the regulatory documents for those products. New modeling has been conducted for single trait Cry1Ab and Cry1F products to evaluate insecticide use in refuges (see section D.2.b.3).

Collection of Baseline Susceptibility Data and Validation of Discriminating /Diagnostic Dose ECB, SWCB, CEW, FAW (Bt sweet corn) These data have been required as conditions of registration.

Submitted information is

discussed in section D.2.b.4 of this

document.

Evaluation of Resistance Monitoring Techniques, e.g., discriminating v. diagnostic dose,

F 2 screen, sentinel plots, gene mapping ECB, CEW, SWCB No additional data were required

or submitted to EPA.

Grower Compliance - more detailed information on refuge (%, deployment, and management), impact of non-compliance ECB, CEW, SWCB Development of a compliance program was required as a condition of registration. A full discussion of this program can be

found in section D.2.b.7 of this

document.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

220 e. References

Andow, D. A. and D. N. Alstad, 1998. The F 2 screen for rare resist ance alleles. J. Econ. Entomol., 91: 572-578.

Andow, D. A. and W. D. Hutchison, 1998. Bt corn resistance management. In Now or never: Serious new plans to save a natural pest control. M. Mellon and J. Rissl er [eds.]. Union of Concerned Scientists, Two Brattle Square, Cambridge, MA.

Andow, D. A., D. N. Alstad, Y.-H. Pang, P. C. Bolin, and W. D. Hutchison, 1998. Using a F 2 screen to search for resistance alleles to Bacillus thuringiensis toxin in European corn borer (Lepidoptera: Crambidae). J. Econ. Entomol. 91: 579-584.

Beck, S.D., 1987. Developmental and season biology of Ostrinia nubilalis . Agricultural Zoology Review. 2: 59-96.

Benedict, J., D. Fromme, J. Cosper, C. Correa, G. Odvody, and R. Parker, 1998. Efficacy of Bt corn events MON 810, BT11, and Event 176 in controlling corn earworm, fall armyworm, sugarcane borer, and aflatoxin. Unpublished study submitted to EPA (part of Monsanto’s 1998 IRM research report). MRID # 447753-01.

Bentur, J. S., D. A. Andow, M. B. Cohen, A. M. Romena, and F. Gould, 2000. Frequency of alleles conferring resistance to a Bacillus thuringiensis toxin in a Philippine population of Scripophaga incertulas (Lepidoptera: Pyralidae). J. Econ. Entomol. 93: 1515-1521.

Bing, J., 1997. Establishment and survival of se cond generation European corn borer on Event 176 hybrids. Unpublished study submitted to EP A (part of Mycogen’s 1997 IRM research report). MRID # 445436-02.

Bourguet, D., A. Genissel, and M. Raymond, 2000. Insecticide resistance and dominance levels.

J. Econ. Entomol. 93: 1588-1595.

Bourguet, D., J. Chaufaux, M. Seguin, C. Buisson, J. L. Hinton, T. J. Stodola, L. Buschman, P. Porter, G. Cronholm, and D. A. Andow, 2001. Is resistance to Bt maize rare enough to delay pest adaptation of the European corn borer? Submitted.

Buschman, L., P. Sloderbeck, and R. Higgins, 1997. Evaluation of refuge planting patterns for corn borer resistance management in Bt-corn. Unpublished study submitted to EPA (part of

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Song, Q., C. Luppens, and X. Gan, 2002b. Monitori ng the susceptibility of the southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis toxin Cry1F: 2002 data summary. Unpublished study submitted to EPA - No MRID #.

Song, Q., C. Luppens, and X. Gan, 2003a. Monitori ng the susceptibility of the southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis toxin Cry1Ab: 2003 data summary. Unpublished study submitted to EPA - No MRID #.

Song, Q., C. Luppens, and X. Gan, 2003b. Monitori ng the susceptibility of the southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis toxin Cry1F: 2003 data summary. Unpublished study submitted to EPA - No MRID #.

Song, Q., S. Wang, and Y. Sun, 2004a. Monitoring the susceptibility of th e southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis toxin Cry1Ab: 2004 data summary. Unpublished study submitted to EPA - No MRID #.

Song, Q., S. Wang, and Y. Sun, 2004b. Monitoring th e susceptibility of th e southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis Cry1F protein: 2004 data summary. Unpublished study submitted to EPA - No MRID #.

Song, Q., S. Wang and Y. Sun, 2006a. Monitoring th e susceptibility of th e Southwestern Corn
Borer, Diatraea grandiosella , to Bacillus thuringiensis Cry1F Protein. 2005 Data Summary. Unpublished study submitted to EPA - No MRID #.

Song, Q., S. Wang and Y. Sun, 2006b. Monitoring the susceptibility of th e southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis Cry1Ab protein. 2005 data summary. Unpublished study submitted to EPA - No MRID #.

Song, Q., S. Wang and Y. Sun, 2007a. Monitoring th e susceptibility of th e Southwestern Corn
Borer, Diatraea grandiosella , to Bacillus thuringiensis Cry1F Protein. 2006 Data Summary. Unpublished study submitted to EPA - Included with MRID # 471184-01.

Song, Q., S. Wang and Y. Sun, 2007b. Monitoring the susceptibility of th e southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis Cry1Ab protein. 2006 data summary. Unpublished study submitted to EPA - Included with MRID # 471184-01.

Song, Q., S. Wang and Y. Sun, 2008. Monitoring the susceptibility of th e southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis Cry1Ab and Cry1F proteins. 2007 data summary. Unpublished study submitted to EPA - Included with MRID # 474139-01.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

232 Song, Q., S. Wang and S. An, 2009. Monitoring the susceptibility of th e southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis Cry1Ab and Cry1F proteins. 2008 data summary. Unpublished study submitted to EPA - Included with MRID # 478418-01.

Storer, N.P., F. Gould, and G.G. Kennedy, 1999. E volution of regional-wide resistance in cotton

bollworm to Bt cotton as influenced by Bt corn: Identification of ke y factors through computer simulation. 1999 Proceedings of the Beltwide Cotton Conference. National Cotton Council. 2:

952-956.

Storer, N. P., S. L. Peck, F. Gould, J. W. Van Duyn, and G. G. Kennedy. 2003. Spatial processes in the evolu tion of resistance in Helicoverpa zea (Lepidoptera: Noctuidae) to Bt transgenic corn and cotton in a mixed agroeco system: a biology-rich stochastic simulation

model. J. Econ. Entomol. 96: 156-172.

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

39: 47-79.

Trisyono, A and M. Chippendale, 1999. Monitoring th e susceptibility of the Southwestern corn borer, Diatraea grandiosella , to Bacillus thuringiensis . Unpublished study submitted to EPA (part of Monsanto’s 1999 IRM re search report). MRID # 450369-02.

U.S. Environmental Protection Agency, 1998. Th e Environmental Protection Agency's White paper on Bt Plant-Pesticide Resi stance Management. U.S. EPA, Biopesticides and Pollution Prevention Division (7511W), 401 M Street, SW , Washington, D.C. 14 January 1998. [EPA Publication 739-S-98-001]

U.S. Environmental Protection Agency and U.S. Department of Agriculture, 1999a. EPA/USDA Position Paper on Insect Resist ance Management for Bt Crops. http://www.epa.gov/pesticides/biopesticides/o therdocs/bt_position_paper_618.htm (posted June 18, 1999; minor revisions July 12, 1999).

U.S. Environmental Protection Agency and U.S. Department of Agriculture, 1999b. Report of the EPA/USDA Workshop on Bt Crop Resistance Management in Corn, Rosemont, IL, June 18,

1999. P.A. Stewart, ed. 88 p. American Farmland Trust, Center for Agriculture in the Environment. (Posted at http://www.epa.gov/pesticides/biopesticides).

Van Duyn, J., W. Modlin, and N. Storer, 1997. Large scale replicated field trials for representative commercial Bt and non-Bt corn hybrids to establish in sect resistance, yield performance, and commercial value. Unpublis hed study submitted to EPA by Novartis. MRID

# 443474-01.

Van Duyn, J., W. Modlin, and A. Summerlin, 1998. Evaluations of Bt corn hybrids against selected corn pests: 1998. Unpublished study submitted to EPA (part of Novartis’ 1998 IRM research report). MRID # 447439-01.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

233 Van Duyn, J., 1998. Large scale replicated field tr ials of representative commercial Bt and nonBt corn hybrids to establish relative insect resist ance, yield performance, and commercial value. Unpublished study submitted to EPA (part of Myc ogen’s 1998 IRM research report). MRID #

448545-01.

Venette, R.C., W.D. Hutchison, a nd D.A. Andow, 2000. An in-field screen for early detection and monitoring of insect resistance to Bacillus thuringiensis in transgenic crops. J. Econ. Entomol. 94(4): 1055-1064.

Walker, K., R. Hellmich, and L. Lewis, 2000. Injury by late-instar European corn borer (Lepidoptera: Crambidae) on Bacillus thuringiensis (Bt) transgenic corn. J. Econ. Entomol. 93(4): 1276-1285.

Whalen, J. and M. Spellman, 1999. Insect contro l in late planted Bt sweet corn - 1998. Unpublished study submitted to EPA (part of N ovartis’ 1999 IRM research report). MRID #

450568-01.

Zhao J-Z., Y-X. Li, H. L. Collins, and A. M. Shelton, 2001. Examination of the F 2 screen for

rare resistant alleles to Bacillus thuringiensis toxins in the diamondback moth (Lepidoptera: Plutellidae). Submitted to Journal of Economic Entomology.

Additional studies identified by MRID number:

44347401 Stein, J. (1997) Research Reports Regarding the Interaction Between Corn Earworm ( Helicoverpa zea ) and Novartis Seeds' Insect Resistant Corn. Unpublished study prepared by Novartis Seeds, Inc. 151 p.

44475401 Stein, J. (1998) 1997 Growing Season Report for Event 176-Derived and BT11- Derived Corn. Unpublished study prepar ed by Novartis Seeds, Inc. 269 p.

44743901 Stein, J. (1999) 1998 Growing Season Report for Event 176-Derived Field Corn,

BT11-Derived Field Corn and BT11-De rived Sweet Corn. Unpublished study prepared by Novartis Seeds, Inc.-Field Crops-NAFTA. 202 p.

44775301 Gustafson, K. (1999) MON 810 1998 Re search Progress and Growing Season Sales Report: Lab Project Number: 99-088E. Unpublished study prepared by Monsanto Company. 405 p.

44543602 Bing, J. (1997) Establishment and Surv ival of Second Generation European Corn Borer on Event 176 Corn Hybrids: Lab Project Number: JB-97-02. Unpublished study prepared by Mycogen Seeds. 5 p.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

234 44854501 Bystrak, P. (1999) 1998 Progress Repor t Activities Relating to Resistance Management in Event 176 Maize: Lab Pr oject Number PB-98-01: PB-98-02: BS98-01. Unpublished study prepared by Mycogen Seeds and University of Nebraska. 158 p.

45020101 Herman, R.A. and Korjagin, V.A. (1999) Microbial Bt Cry1F (truncated) DeltaEndotoxin: Maize Insect Pest Susceptib ility Study. Unpublished study prepared by Dow and Pioneer.

45020114 Moellenbeck, D. and M. Peters (1999) Efficacy of Cry1F Events TC 1360 and TC 1507. Unpublished study prepared by Dow and Pioneer.

45020115 Masson, L., M. Adang, and G. Schwab (1999) Cry1F Binding Studies. Unpublished study prepared by Dow and Pioneer.

45036902 Stone, T. (2000) 1999 Research Progress and Growing Season Sales Report for MON 810 YieldGard Corn: Research Repor ts: Lab Project Number: 00-CR-001E. Unpublished study prepared by Monsanto Company. 76 p.

45056801 Stein, J. (2000) 1999 Growing Season Report for Event 176-Derived Field Corn,

BT 11-Derived Field Corn and BT 11-Deri ved Sweet Corn: Lab Project Number. Unpublished study prepared by Novartis Seeds, Inc.-Field Crops-NAFTA. 138 p.

45131101 Bystrak, P., L. Higgins, D. Moellenbeck (2000) High Dose Demonstrations for Cry1F Events TC 1360 and TC 1507: Eur opean Corn Borer. Unpublished study prepared by Dow and Pioneer.

45320503 Stone, T. (2001) Monsanto 2000 MON 810 Resistance Management Grower Education Program. Unpublished study prepared by Monsanto Co. 43 p.

46695801 Storer, N.; Owens, E. (2005) Slide Pr esentation Summarizing European Corn Borer and Cry1F Resistance Monitori ng Update. Project Number: GH/C/5826. Unpublished study prepared by Dow Agro Sciences LLC and Pioneer Hi-Bred International. 27 p.

46874901 Owens, E. (2006) Monitoring the Suscepti bility of Corn Lepidoptera Pests to Cry1Ab and Cry1F Proteins: 2005 M onitoring Results. Unpublished study prepared by Agricultural Biotechnol ogy Stewardship Technical Committee, University of Missouri and University of Nebraska. 58 p.

47011201 Storer, N.; Owens, E. (2006) Research Results on 2004 European Corn Borer Collection from Hamilton County, Io wa: Cry1F. Project Number: 061061. Unpublished study prepared by Mycogen S eeds, Pioneer Hi-Bred International Inc. and Dow AgroSciences 30 p.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

235 47176001 Storer, N.; Babcock, J.; Schlenz, M.; et al. (2007) TC1507 Maize and Fall Armyworm in Puerto Rico. Project Number: 071147. Unpublished study prepared by Dow AgroSciences, LLC. 36 p.

47407001 Storer, N. (2008) Proposed Revisions to IRM-Related Registration Requirements for Cry1 Plant-Incorporated Protectan ts in Field Corn. Project Number: IRM/2008/1. Unpublished study prepar ed by Agricultural Biotechnology Stewardship Technical Committee. 18 p.

47339601 Bailey, L. (2008) 2007 Insect Resistan ce Management Compliance Assurance Program Report for Corn Borer-Protected Bt corn, Corn Rootworm-Protected Bt corn, and Corn Borer/ Corn Rootworm -Protected Stacked Bt corn. Project Number: CAP/2007. Unpublished study prep ared by Agricultural Biotechnology Stewardship Technical Committee. 17 p.

47510904 Werning, M. (2008) Expressed Trait Pr otein Concentration of Maize Lines Containing Events DAS-01507-1, DAS -59122-7, MON-00810-6, and DAS01507-1xDAS-59122-7xMON-00810-6. Proj ect Number: PHI/2008/006, PHI/2008/006/012, PHI/2008/006/011. Unpublis hed study prepared by Pioneer Hi-Bred International, Inc. 174 p.

47663301 Bailey, L. (2009) 2008 Insect Resistan ce Management Compliance Assurance Program Report for Corn Borer-Protected Bt Corn, Corn Rootworm-Protected Bt Corn, and Corn Borer/Corn Rootworm-P rotected Stacked Bt Corn. Project Number: CAP/2008. Unpublished study prep ared by Agricultural Biotechnology Stewardship Techn. Committee. 15 p.

47677803 Alves, A.; Bailey, L.; Cressman, R.; et al. (2009) Field Efficacy, Allergen/Toxin Homology and Trait Durability Supplemental Information for 1507x59122xMON810, 1507xMON810 and 59122xMON8 10. Project Number: PHI/2009/025. Unpublished study prepared by Pi oneer Hi-Bred International, Inc. 40 p.

E. Benefits Assessment (2001 Bt Corn Assessment)

Bt Corn Plant-Incorporated Protectants

a. Usage Estimates

There have been two grower benefit studies of Bt corn (field corn) (Marra, Carlson & Hubbell, 1998 and Carpenter & Gianessi, 1999) and both us ed essentially the same information about yield advantages due to reduced insect damage , technology fee and reduc tions in conventional pesticide use. As discussed above, only a very small portion of field corn acres are treated with

foliar insecticides.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

236 Field corn is the most widely grown crop in the 48 contiguous states and USDA/NASS reports acres planted for all of these states. Table E.1. contains the states whic h average in excess of 1 million acres planted to field cor n. These 16 states account for about 90 percent of field corn acres and 93 percent of the va lue of field corn grown for gr ain. EPA estimates of planted Bt corn are based on registrant submissions of annual sales. Since the fi rst registrations in 1995, planting

of Bt corn increased to a maximum of almost 20 million acres in 1999 and decreased slightly in

2000 to 19.5 million acres. USDA estimates of acres planted to Bt field corn for those states covered by the corn estimating program represen ts about 15 million acres versus the 19.5 million acres estimated by this Agency. The states in the corn estimating pr ogram were not readily available and USDA has estimates published for 1999 and 2000. We have compared state level usage estimates from the registrants (which is claimed to be Confidential Business Information)

with the USDA estimates for those states wh ere USDA has published adoption estimates and they do not agree. The USDA estimate is base d on a survey while the EPA estimate is based on

sales data. Each method has its potential problems a nd we have no basis to prefer one over the other except that the EPA estimate covers all corn producing states. If the USDA estimates covered more states (those states listed in Table E.1 below), we would have reason to prefer the

USDA estimates. The EPA estimate is used as the basis for the benefit calc ulation. However, we recognize that this could present an overestimate of benefits.

The Agency has recently registered an additiona l plant-incorporated pr otectant known as Cry1F which has efficacy against those pests currently c ontrolled plus some control against the black cutworm and armyworms. This product does not provide any control of the corn rootworm which is the primary soil borne inse ct pest of field cor n. There would have been little, if any, commercial planting of Cry1F in 2001 so we do not know actual farmer experience with this

material.

Table E. 1. Acres Plante d to Field Corn and Crop Value for Selected States

State Acres planted Crop value Percent Bt corn

1,000 1,000 dollars

Colorado 1,253 306,309 NA

Illinois 10,867 3,007,964 14

Indiana 5,767 1,506,910 7

Iowa 12,300 3,119,481 25

Kansas 3,200 811,489 26

Kentucky 1,317 291,899 NA

Michigan 2,183 449,236 8

Minnesota 7,167 1,674,917 30Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

237 State Acres planted Crop value Percent Bt corn

Missouri 2,667 568,464 22

Nebraska 8,633 2,109,197 26

New York 1,087 127,698 NA

Ohio 3,517 896,085 6

Pennsylvania 1,533 237,273 NA

South Dakota 3,933 649,020 37

Texas 2,150 465,761 NA

Wisconsin 3,600 718,233 14

U.S. total 79,032 18,215,745

Percent of U.S. total

in these States 90 93 89

Source: Crop Production 2000 Annual Summary, Crop Values 2000 Annual Summary, and Acreage. NA indicates USDA did not estimate Bt corn usage.

b. Insect Pests

The Bt protein expressed by the field corn plant targ ets Lepidopteran insects. This protein is effective in controlling the Europ ean corn borer, the Southwestern corn borer and provides some control of the corn earworm. The European co rn borer and the Southwestern corn borer are difficult to control since they bore into the corn stalk where most conventional pesticides do not provide any control. Some control can be achieved with foliar applications prior to entry into the stalk but control is not adequate since the pests usually arrive on the plant over a period two to

three weeks. Bt corn presents a method to control virtua lly all of these pests. Corn borers consume plant energy and weaken th e corn stalks so they are mo re likely to lodge (blow down) under windy conditions. Lodged stalks are difficult, if not impossible, to mechanically harvest.

The corn earworm enters the ea r via the silk and feeds on the ear s. Again once the insect larvae

is in the ear under cover of the husk, it is difficult to contro l with conventional insecticides. These pests populations are not c onstant from year to year na tionwide nor within regions. Varying population levels results in varying pest pressure, varying need for pest control and

variation in the benefit a farmer will gain from controlling the pest.

The populations of these pests vary geographica lly and from year to year depending onCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

238 environmental conditions including farming practi ces. This includes the past years planting of Bt corn. The planting of Bt corn on a significant portion of th e corn acreage in a region probably would reduce the populations of co rn borers available to survive the winter and reproduce the next year. This means that a farmer is not gua ranteed to be making a wise investment with a decision to plant Bt corn. Some areas ha ve historically high levels of European corn borer or the

Southwestern corn borer. These include sout hwestern Kansas plus parts of Colorado, New Mexico, Texas and Oklahoma for the Southweste rn corn borer and Iowa, Minnesota, Nebraska and South Dakota for the European Corn Borer. There is some probability that some farmers have adopted Bt corn when their actual damage did not warrant the additional expense. We have not been able to estimate the magnitude of th is. It is reasonable to expect farmers to be examining losses in their mandated refuge acres to determine whether they should continue to plant Bt corn and that if damage does not reach economic thresholds, they would not plant Bt corn the next year.

Major states where these pests are regularly considered economic pests recommend farmers examine yields for previous years and projectio ns for the current year (Nebraska 2001). We

understand Bt corn is less expensive to plant than the cost of non Bt seed plus spraying a conventional pesticide. If a farmer were to appl y two sprays, the cost would be significantly

lower. It needs to be noted that something less than about 20 percent of Bt field corn acres were likely to have been treated with an insecticide wh ich could target corn bo rers (this is discussed below).

Bt corn has been on the market for a number of years and the levels of reported damage have been down from the historical average. It is not clear if there is just an unusually long period of reduced pest pressure or if the use of Bt corn has resulted in a loweri ng of insect pressure. Either way, it is logical to expect some farmers will ma ke a decision to reduce the portion of their corn acres planted with Bt corn unless the monitoring of damage in refuge acres implies continued planting of Bt corn is financially justified.

The addition of Cry1F Bt corn provides some degree of cont rol against the black cutworm and armyworms in addition to the European corn borer and the Southwestern corn borer.

c. Potential to Replace Conventional Pesticides

Insecticide use on field corn is largely to c ontrol the soil pest complex, rather than the Bt corn target pests. As discussed above, those pests targeted by Bt corn, are difficult to control with conventional pesticides. Therefor e, little conventional pesticide has been used on field corn. Five to eight percent of field co rn acres may have been treated with conventional pesticides to control these pests implying that 6.3 million of t hose 79 million acres planted to field corn could

have been treated with conventional insec ticides. A look at changes from 1995 and 2000

indicates a reduction of around 3.9 million acr e treatments due to all causes.

Cry1F Bt corn has the potential to replace conventiona l pesticides in areas where corn growers rotate corn with a crop such as soybeans which is not a host to the corn rootworm and their crop

land is river bottomland. These fa rmers are unlikely to also have corn rootworm and it may be aCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

239 wise economic decision to plant Cry1F Bt corn and would be part of the 6.3 million acres planted

to field corn discussed above. Some of these farmers probably adopted other Bt corn products but if they have, they may have used conventio nal pesticides to contro l the black cutworm. Where there are cutworms, farmers ma y make the decision to plant Cry1F Bt corn and be able to

reduce or eliminate use of conventional insecticid es. While we can predict there will be some reduction, we do not have data to accurately estimate the magnitude of the potential reduction.

Assuming there was a 3.9 million acre treatment reduction due to the corn borer pests, this equates to 0.2 acre treatments per Bt corn acre (all 19.5 million acres). The majority of field corn growers who adopted Bt corn did not consider reductions in cost of conventional pesticides applied when they made the decision to plant or not plant Bt corn because they had not been applying conventional pesticides to control these pests.

d. Benefits for Field Corn

Field corn is grown on a average (1998-2000) of about 79 million acres with production close to 10 billion bushels having a market value of about $18 billion (Table E.2). The 1994-1996 average for acres planted was 76.6 million acres with production of 8.9 billion bushels and a market value of $24.6 billion, reflecti ng the fact that higher yields have been more than offset by lower prices. Other market factor s may have been contributing to th e change in market value.

Table E.2. 1998-2000 National Field Corn Data

Item Unit of

measure 1998 1999 2000 Average

Acres planted 1,000 acres 80,165 77,386 79,545 79,032

Acres of Bt

corn 1,000 acres 14,500 19,800 19,500 17,933

Corn production 1,000 bushels 9,758,685 9,430,612 9,968,358 9,719,218

Yield Bushels/acre 134.4 133.8 137.1 135.1

Value 1,000 dollars 18,922,084 17,103,991 18,621,160 18,215,745

Price per

bushel dollars 1.94 1.82 1.85 1.87

Source: Crop Production, 2000 Annual Summary, USDA/NASS Crop Value, 2000 Annual Summary, USDA/NASS

Acres of Bt corn are EPA estimates obtained from industry sales reports.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

240 Two studies estimated farmer level benefits from the adoption of Bt field corn. Marra, Carlson and Hubbell (1998) utilized a 4 to 8 percent increase in yield, no minal reduction in pesticide use and a technology fee of about $11 per acre. They discussed economic thre sholds for adoption of

Bt corn but did not estimate overall benefits. Gianessi and Carpenter (1999) estimated benefits for 1997 through 1999. They estimated farmers had significant gains in 1997 and a net loss in 1998 and 1999 because yield gains went from 12 bushels in 1998 to 4.2 in 1998 and 3.3 in 1999 and the price of field corn went down. The decr ease in prices was due to a combination of market forces largely unrelat ed to the introduction of Bt corn.

We conducted a partial budgeting analysis for grower benefits of field corn using three most recent year averages and obtained estimates of benefits from $38 milli on (about $2 per acre to $219 million (about $12 per acre) per year (table 3) . Thirty-eight million dollars per year across

19.5 million acres of Bt corn implies benefits of less than $2 per acre which may be too close to the margin to make economic sense for many farmers. It is likely that benefits would be about $38 million or less in years of lo w insect pressure and benefits would be in the area of $219 million in years of high insect pressure. It may be clearer to state that benefits of Bt corn are likely to be less than a maximum of $219 milli on per year. They would be around $38 million or less in years of low infestations. There is c onsiderable uncertainty re garding the magnitude of yield changes and the amount of the technology fee. The technology fee is charged by the seed dealer and can be subject to discounts due to market factors. It is lik ely that farmers who find planting of Bt corn to be a profitable move will lose money planting Bt corn in years of low insect pressure but that the e xpected gain (the returns from averaging out impacts over the years of high to low pest pressure) warrant planting Bt corn or that Bt corn is viewed as insurance against the high infestation years.

This partial budget estimate does not include the cost of the insect refuge. Acres planted to refuge would not obtain the gains of $12 per acre. That is, the grower would plan for the gain on up to 80 percent of field corn acreage.

The introduction of Cry1F Bt corn may result in additi onal acres being planted to Bt corn and/or

could result in a shift in acres from another Bt corn products to Cry1F. It is reasonable to expect some corn growers have not planted Bt corn because the economic gain from control of European corn borers or Southwes tern corn borers did not warrant the cost, but Cry1F Bt corn controls additional pests and its use may be warranted. We are not projecting the numbers of corn acres that may be impacted.

The partial budgeting estimates assume there are no price effects wh ich would affect the

distribution of benefits. T ypically, if growers increase pr oduction, value per unit goes down implying some of the benefit is being passed to processors or the consumer resulting in less benefit to the growers. The other implication is non-adopters of the technology also will receive less money per acre (or per bushel) for their cr op. These effects apply to any new technology which results in increased efficiency.

Table E. 3. Grower Benefits of Bt Field Corn (Partial Budgeting Approach)Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

241 Item Unit of measure Low Insect Pest

Pressure** High Insect Pest

Pressure

Acres planted 1/ 1,000 79,032 79,032

Yield 1/ bushels/acre 135.1 135.1

Acres Bt corn 1/ 1,000 17,933 17,933

Yield increase 2/ bushels/acre 5.4 10.81

Value of yield change 3/ dollars/acre 10.11 20.21

Technology fee 4/ dollars/acre 8.00 8.00

Per acre benefit 5/ dollars 2.11 12.21

Total grower benefit

6/ 1,000 dollars 37,758 218,979

1/ Three Year Average from Table E. 2. 2/ based on 4 to 8 percent yield increase from Marra et al. (1998) and from Carpenter and Gianessi (1999). 3/ change in yield times average grower price received of $1.87/ bushel (Table E. 2.). 4/ The 1999 technology fee as estimated by Carpenter & Gianessi (1999) 5/ value of yield incr ease less technology fee. 6/ per acre benefit times acres Bt corn. **This is intended to be repres entative of the lower bound benef its. Various conditions could

result in actual benefits below th is lower bound estimate for some years.

e. Mycotoxin Reduction

Mycotoxins are chemicals produced by fungi, that are toxic or carcinogenic to animals and humans. The most commonly occurring mycotoxi ns on corn are produced by the fungal genus

Fusarium , and are known as fumonisins (Munkvold, 2000) . There are several different kinds of

fumonisins: FB 1 , FB 2 , FB 3 , FB 4 , FA 1 , and FA 2 (Marasas, 1996; Ross et al., 1992). Another class of corn mycotoxins are those produced by the genus Aspergillus , including the notorious aflatoxins. The economic impact of aflatoxins is greater than that of other mycotoxins because they can be passed into milk if dairy cows eat contaminated grain (Munkvold et al., 1999).

Damage by insect pests such as the European corn borer can be an important factor for mycotoxin development in corn. Insect pests pr omote the growth of my cotoxin producing fungi in two ways: 1) They carry fungal spores from the plant surface to the surfaces of damaged kernels, and 2) They create entr y wounds on the kernels for the fungi. Even when the insect pestsCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

242 do not directly carry fungal spores to the corn wounds, ambient spores deposited later on tissue wounded by pest feeding are more likely to in fect the plant (Munkvold, 1999). Field studies have shown that damage due to southwestern co rn borer (SWCB) can increase aflatoxin levels (Windham, et.al. 1999).

When mycotoxin contamination occurs in co rn, the potential damages can be both economic costs to growers and health risk s to humans and livestock. Corn grain that contains mycotoxins above a certain level is more likely to be reject ed in the market, forcing growers to accept the lower price for non-food uses. In particular , the FDA’s new proposed guidelines about recommended levels of fumonisins in grain ma y have a significant im pact on amount of corn sold at the better food/feed pric es. While these FDA guidelines fo r fumonisin levels are not yet set as action levels, they have been proposed to industry as safety threshol ds (Randall A. Lovell, Center for Veterinary Medicine/ FDA, personal communicati on). The guidelines in human food

and animal feed are shown in Tables E1 and E2.

Table E1. FDA guidelines for total fumonisins in human foods (FDA, 2001a).

Product Total Fumonisins (FB 1 +FB 2 +FB 3 ) parts per million (ppm)

Degermed dry milled corn products (e.g., flaking grits, corn grits, corn meal, corn flour with fat content of < 2.25 %, dry weight basis) 2 ppm

Whole or partially degermed dry milled corn products (e.g. flaking grits, corn grits, corn meal, corn flour with 4 ppm

Dry milled corn bran 4 ppm

Cleaned corn intended for masa production 4 ppm

Cleaned corn intended for popcorn 3 ppmCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

243 Table E2. FDA guidelines for total fumonisins in animal feed (FDA, 2001b).

Animal or Class Recommended Maximum

Level of Total Fumonisins in Corn and Corn By-Products (ppm 1 ) Feed

Factor 2 Recommended Maximum

Level of Total Fumonisins in the Total Ration (ppm 1 )

Horse 3 5 0.2 1

Rabbit 5 0.2 1

Catfish 20 0.5 10

Swine 20 0.5 10

Ruminants 4 60 0.5 30

Mink 5 60 0.5 30

Poultry 6 100 0.5 50

Ruminant, Poultry & Mink Breeding

Stock 7 30 0.5 15

All Others 8 10 0.5 5

1 total fumonisins = FB 1 + FB 2 + FB 3

. 2 fraction of corn or corn by-pr oduct mixed into the total ration.

3 includes asses, zebras and onagers.

4 cattle, sheep, goats and ot her ruminants that are > 3 months old and fed for slaughter.

5 fed for pelt production.

6 turkeys, chickens, ducklings a nd other poultry fed for slaughter.

7 includes laying hens, roosters, lactating dairy cows and bulls.

8 includes dogs and cats.

Fumonisins are toxic to livestock, especially horses, swine, and cattle; and are carcinogenic in laboratory animals. The 1989 US corn crop had part icularly high levels of fumonisins, resulting in dramatic increases in the horse disease eq uine leukoencephalomalacia (ELEM) and the swine disease porcine pulmonary edema (PPE) (Marasas , 1996; Ross et al., 1992). At the time of the 1989 mycotoxicosis outbreaks, FB1 concentrations in suspect swine feeds were 20-360 ppm, and in equine feeds were 8-117 pp m. Non-problem feeds containe d concentrations below 8 ppm (Ross et al., 1992). Epidemio logical studies have linked consumption of fumonisin-Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

244 contaminated grain with elevated esophageal cancer incidence in humans (Marasas, 1996). A definitive link between fumonisin levels and human cancer is not possible from these studies due to the presence of possibly confounding effects in the study. Other documented toxicological

effects of fumonisins in labor atory studies include toxicity and carcinogenicity in rats, cytotoxicity to mammalian cell cultures, and phyto toxicity to weeds and other plants including tomatoes (inhibiting growth and ch lorophyll synthesis) (Marasas, 1996).

One of the benefits of Bt corn (a genetically modified, pest-protected corn) is that it has demonstrated drastically reduced occurrences of contamination by the mycotoxin fumonisin. This is because Bt corn is far less prone to insect injury, which in turn prevents the growth of fumonisin producing fungi. Certai n events of Bt corn, such as MON810 and BT11, can reduce fumonisin levels by as much as 90% (Munkvold, 2000). This implies both private and social

benefits: economic returns on corn sales would increase, and ther e would be potential reductions in mortality and morbidity among livestock and, presumably, humans.

Munkvold (2000) estimated that, if the current F DA guidelines for fumonisins in food were to become action levels, about 160 million bushels of corn in just the states of Iowa, Illinois, Missouri, and Nebraska would be at risk of rejection – an annual loss of value in the tens of millions of dollars in just these states. Depending on the amount of Bt corn planted in these states in lieu of conventional corn, the savings that Bt corn would afford mi ght also range in the tens of millions. Vardon (2000) made similar predic tions on potential economic losses due to fumonisins in corn, estimating an annual loss of $11 million. Hence, the economic benefits from Bt corn by this estimation is in the several millions. One of the reasons this value is lower than Munkvold’s estimates (in the tens of millions of benefits) is that Vardon’s model assumes that the corn that is rejected for f ood is still acceptable for animal f eed. This in fact may no longer be the case, as FDA’s most recent prop osed guidelines in animal feed are at about the same level as for human food. Neither study calculates the costs of the fumonisin mycotoxin to human health, acknowledging the difficulty of extrapolating from available epidemiological studies directly to

human health benefits.

Aflatoxins are known carcinogens to laboratory animals and presumably man; hence, the presence of aflatoxins in foods is restricted to the minimum levels practically attainable by modern processing techniques. Hist orically, aflatoxin levels in co rn have been highest in the Southeastern states. Corn from anywhere in the US may be affected, however, depending on the growth, harvesting and storage c onditions involved, as was the cas e with aflatoxin contamination in the Midwest in 1988 and Texas in 1987 (FDA, 1999).

Currently, the action level for af latoxins in corn grain for human food is 20 ppb (FDA, 1994; Munkvold, 1999). When dairy animals consume feed containing high levels of aflatoxin, one of the metabolized aflatoxins (B 1 ) may be secreted into the an imals’ milk as aflatoxin M 1 . Dairy cattle consuming corn feed that contains le ss than the FDA action level of 20 ppb total aflatoxins, however, should produce milk under the 0.5 ppb action level for aflatoxin M 1 in milk (FDA, 1999). In 1969, the FDA had established the action level of 20 ppb afla toxins in all foods, including animal feed; however, subsequent tests showed that aflatoxin levels above 20 ppb could be fed to certain food-producing animals without endangering either these animals or consumers of food derived from the animals (F DA, 1994). The action levels for aflatoxins inCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

245 corn are summarized in Table E3:

Table E3. FDA’s action levels for corn aflatoxins in human and animal foods (FDA, 1994)

.

Product or animal Aflatoxin action level (ppb)

Human food 20

Milk 0.5

Beef cattle 300

Swine over 100 lbs 200

Breeding beef cattle, swine, or mature poultry 100

Immature animals 20

Dairy animals 20

FDA compliance monitoring program from 1990 to 1996 indicate that 6 .6 percent of corn samples exceeded the aflatoxin action level for food (Vardon, 2000). The potential value of crops lost because of aflatoxin contamination has been estimated to be $47 million per year in food crops (corn and peanuts) and $225 million per year in feed corn. The cost of livestock morbidity was estimated at $4 million per year (Vardon, 2000).

Studies comparing Bt with non Bt hybrids have usually show no signi ficant difference in aflatoxin levels. The variability in aflatoxin le vels due to environmental factors overwhelms the

beneficial effects related to insect control seen in the current Bt products (Odvody 2001, personal communication). Even though insect damage ratings are lower for Bt hybrids, apparently the amount of insect feeding is sufficient for A. flavus establishment and subsequent aflatoxin contamination (Windham, et.al 1999). Studi es across 10 states in 2000 found little or no aflatoxin to begin with, and in cases of substantial aflato xin contamination, no significant

differences were seen between Bt and non Bt hybrids (Headrick, 2001). Two studies in Alabama

in 1999 also showed no difference in aflatoxin levels while yields were significantly higher for the Bt hybrids (Delamar, et. al, 1999).

A study in 1999 in Corpus Christi, Texas act ually showed that under conditions of extreme drought and artificial inoculation with A. flavus , Bt corn hybrids had higher aflatoxin levels compared with non-Bt isolines (Odvody 2000). The study was expanded in 2000 to include

more locations with mixed result s in terms of aflatoxin contam ination levels between Bt and non-Bt isolines. However, Bt corn hybrids ha d less aflatoxin contamination than the non-Bt hybrids, on average in 2000 when the comparis on is done excluding one of the 9 Bt/non-Bt hybrids (Pioneer 3394). The author concludes that differences in individual hybrid susceptibility to infection by A. flavus was the primary factor influe ncing aflatoxin accumulation (OdvodyCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

246 2001, personal communication). The reasons for th e negative performance of the one particular

commercial line are not known. Fact ors that were forwarded as hypothesis by the researcher are the particular adaptability of a hybrid in a region, differences in the test ma terial (i.e., not true isolines), or unintended effects fr om the insertion of the Bt gene to the plant’s natural defense system against infection. Better pest control is viewed as only one of many defenses in the attempt to develop hybrids with improved perf ormance against aflatoxin contamination (Odvody 2001, personal communication).

e. Future Benefits

This analysis has used the benefits which have occurred from the adoption of Bt corn as a basis to project future benefits. It is expected that benefits will continue at about the magnitude of those for the period of the analysis. Individual growers will have more experience with Bt corn and should have the experience to determine wh ether the reduced damage warrants the additional cost for the technology. The European corn bor er and the Southwestern corn borer cause significant damage in certain regi ons of this country most years. Those growers in these areas with regular infestations, will co ntinue to utilize this technology and others with significant damage will adopt the technology. Those whose infest ations is not serious, will not continue to utilize the technology. It will be interesting to see whether for ecasting of insect problems can become sufficiently sophisticated to enable gr owers throughout major corn growing areas to know enough about the probability of an economic infestation to plant Bt corn only in those years when the problem will warrant it.

4. Bt Sweet Corn Plant-Incorporated Protectant

In 1998, EPA approved the registration of S yngenta’s (formerly Novartis) Cry1Ab ( Bt 11) sweet corn. Major pests controlled are European co rn borer (ECB), corn earworm (CEW), and fall armyworm (FAW). Approximately 742,000 acres of sweet corn is grown in the United States, including processed and fresh corn. EPA recently registered Cry1F Bt sweet corn which has control of the Black cutworm as well as the ECB, CEW and FAW. This material has not been available for a sweet corn growing season. Th erefore, while the addi tion of Cry1F has the potential to increase acres planted to Bt sweet corn, we have not f actored this new registration into the analysis.

Table E. 4 . Top States Growing Sweet Corn (Acres Planted in 1999)

State Processed Fresh Total Sweet Corn

Minnesota 127,400 0 127,400

Wisconsin 107,100 8,900 116,000Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

247 State Processed Fresh Total Sweet Corn

Washington 99,400 2,100 101,500

New York 33,100 35,900 69,000

Oregon 44,200 6,900 51,100

Florida 0 38,900 38,900

California 0 31,000 31,000

Illinois 16,600 7,600 24, 200

Pennsylvania 2,800 20,800 23,600

Georgia 0 22,000 22,000

Ohio 0 17,200 17,200

Idaho 15,900 0 15,800

Michigan 0 11,500 11,500

New Jersey 0 10,500 10,500

Selected States 446,400 213,300 659,700

Source: NASS, USDA, 2000

a. Potential to Replace Chemical Insecticides

Commercial field data studies for Bt sweet corn submitted by Synge nta suggest the potential to achieve equivalent yields to trad itional varieties while reducing th e quantity of insecticides used to control these pests. Accordi ng to NASS data, about 3.3 million acre treatments of insecticide are applied annually to sweet co rn. Based on the pest complex being targeted, the potential

market for Bt sweet corn is 2.0 million acres, or 60% of total acre treatments (Doane, 1998). The major chemical insecticide alternatives ar e cyhalothrin-lambda, permethrin, and methomyl with esfenvalerate, carbaryl, chlorpyrif os, cyfluthrin, and methyl parathion. Bt microbial spraysCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

248 are used to a lesser extent. (Doane, 1998).

b. Benefits for Sweet Corn

The majority of sweet corn acres are planted to processed corn while the value per acre of fresh corn is over 3 times the market value of processed corn.

Table E. 5. Value of Pro cessed and Fresh Sweet Corn

Year 1997 1998 1999

Processed

Acres Planted 478,900 486,400 473,400

Value ($000's) 250,329 238,748 234,448

Value/acre 522.72 490.85 495.24

Fresh

Acres Planted 254,900 255,700 268,300

Value ($000's) 418,617 452,410 458,632

Value/acre 1,642.28 1,769.30 1,709.40

Total

Acres Planted 733,800 742,100 741,700

Value ($000's) 668,946 691,158 693,080

Value/acre 911.62 931.35 934.45

Source: NASS, USDA, 2000Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

249 On average, sweet corn is treate d for all insect pests 5.5 times pe r year: 4.3 times for processed corn and 8.6 times for sweet, although the va riability is quite significant among states.

Table E. 6. Fresh Sweet Corn Insectic ide Treatments, 1998 (thousands of acres)

State Acres Planted Acre Trea tments No. of Applications/Yr

California 31.0 389.4 12.56

Florida 38.9 657.6 16.9

Georgia 22.0 115.8 5.26

Illinois 7.6 30.3 3.99

Michigan 11.5 50.4 4.39

New Jersey 10.5 82.2 7.83

New York 35.9 136.2 3.79

Oregon 6.9 5.8 0.84

Washington 2.1 11.3 5.4

Wisconsin 8.9 36.7 4.12

Total for Top States 175.3 1,479.0 8.65

Source: NASS, USDA, 2000

A simulation model based on a demand curve for Bt sweet corn shows an average net benefit/acre of $3.55 for processed corn and $5.75 for fresh corn. Upper limits benefits for Bt sweet corn are based on savings from reduced insecticide applica tions. An upper limit application savings of $45/acre is based on 9 appl ications per year, 60% (5.4) of which target BtCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

250 pests, and each application costs an average of $8.25 per acre (Doane, 1998). The source for

market share estimates for Bt sweet corn is USDA’s Pest Mana gement Practices 1999 summary. The USDA estimated 4% of vegetables in 1999 were planted with genetical ly modified seed to

resist insects and sweet corn is the only crop with a registered pl ant-incorporated protectant. However, Syngenta Seeds considers their market share information for Bt sweet corn
information to be confidential business information. Information available from USDA indicates the quantity of Bt plant-incorporated protectant on a ll vegetables for 2000 was too small to quantify ( Bt plant-incorporated protecta nts for vegetables are only re gistered for use on potatoes and sweet corn). If we assume less than 5% of sweet corn is Bt sweet corn, seed premium cost $30/acre (personal communication: Warnick, Debra, Novartis Seeds, Inc [year]), upper limit benefits $45/acre, and upper limit Bt specific costs are $58/acre (whi ch is 6.2% of the average value per acre grown in 1999) . Net benefits are $5.38/acre.

Table E. 7 Estimated Benefits for Bt Sweet Corn

Item Unit of Measure Value

Acres planted acres 739,200

Average benefits to Bt adopters dollars per acre 40

Average other costs to Bt adopters dollars per acre 5

Technology fee dollars per acre 30

Net benefit dollars per acre 5

Insecticide treatments saved per Bt acre 4.8Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

251 Source: Acres planted-average from Table E.5

Average benefit to Bt adopters was estimated by the simu lation model using the subset of

observations for which benefits exceeded all costs.

Other costs (insect resistant management, di scounts for marketability, and underlying hybrid yield) equals the average cost to the adopters using the subs et of observations for which

benefits exceeded all costs. Other costs have been estimated indirectly by the model.

Technology fee is the seed premium.

Net benefit equals average benefit less other costs le ss technology fee.

Insecticide treatments saved equals average be nefit divided by average treatment cost ($8.25 per treatment) based on the assumption that the principal benefits are reduction in treatment

costs.

The average Bt sweet corn user must cover all costs (the seed cost premium & other costs), and if benefits are mainly to reduce co st, then use reduction can be deduced from the average benefits plus seed cost premium divided by the chemical cost per acre. At a cost per treatment of $8.25 and average benefit of $40.00/acre, the use reduction of 4.8 treatment s per year. Applied to the 29,600 acres assumed treated with Bt plant pesticides, total pesticide use reduction is estimated to be 142,000 acre treatments for 1999.

c. Environmental Benefits of Bt Sweet Corn

A number of comments a ddressed the potential envi ronmental benefits of Bt sweet corn. Because of the low adoption rates, potential benef its have not been realized. Biorationals with novel modes of action have not significantly re placed the more acutely toxic organophosphate and pyrethroid insecticides. Bt sweet corn allows a transition to more selective toxins and increase the beneficial arthropod community (Fle isher, 2000). The benefits of reducing toxic

insecticide use are as follows:

1. Sweet corn in Florida is still mostly hand picked and packed in the field. Detasseling operations also bring workers in to direct contact with sweet corn (Nuessley, 2000). 2. Maryland growers of sweet corn and potatoes are concerned about worker exposure

risks and believe that the Bt technology offers an alternative to toxic insectic ides (Dively, 2000). 3. Studies in Maryland conducted in 1999 clearly showed that BT11 corn had significantly less fumonism contamination of up to 96% compared to its non-transgenic isoline (Dively, 2000). 4. Adoption of Bt corn (field and sweet corn) may he lp an areawide suppression of the corn earworm since corn serves as the prim ary nursery for recruitment of CEW populations,Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

252 which later in the season infest soybeans, lim a bean and tomatoes. Further insecticide use

reductions could therefore occur in these ot her crops as well as corn (Dively, 2000).

d. Future Benefits

Information available suggests adoption of Bt sweet corn has not grown as expected which implies fear of consumer rejecti on or that the technology is not working as expected. We expect the seed companies marketing this technol ogy will resolve any technology problems and consumers will accept the product in time.

5. Summary of Results

a. General Findings

EPA believes that significant benefits accrue to growers, the public, and the environment from the availability and use of certain Bt plant-incorporated protectants. Direct benefits to growers

for all Bt products is estimated to be less than $350 million in 2000. Indirect or environmental benefits occur as improved pest resistance decr eases corn diseases that result from insect interactions. Insect pests that damage ears, ke rnels or stalks are causative agents for mycotoxin development by carrying fungal spores to the su rfaces of damaged kernels and by creating entry wounds on the plant. Common mycotoxins on corn are fumonisins and aflatoxins. Fumonisins are toxic to livestock, especially horses, swine, and cattle; and are carc inogenic to humans and

animals. Aflatoxins are known car cinogens to laboratory animals and presumably man. Growers must accept lower prices if mycotoxin levels ex ceed FDA food standards or total loss if the

lower feed standards are not met. The public costs of mycotoxins to human health has not been quantified due to the difficulty of extrapolat ing from available epidemiological studies.

There are several Bt corn plant-incorporated protectant products registered by three basic registrants with more than 19 million acres planted to Bt corn. The per acre benefits are modest but there are a large number of acres where the only control before Bt corn was a hybrid with

corn stalks with some re sistance to corn borers. Bt corn provides season long control and became a viable control. Annual benefits are estimated to be up to $220 million.

6. References

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DeLamar, Flanders, Bowen, Burc h, Pegues and Wells, Efficacy of Transgenic Corn Against Southern Insect Pests, Fairhope and Head land, Alabama 1999, Auburn Univeristy, Wiregrass Research and Extension Center, Gulf Coast Research and Extension Center.

Dively, Galen. 2000. Public comments, Oct ober 20, 2001 FIFRA SAP Meeting. OPP Docket

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Doane Marketing Research, Inc. 1998. Multi Client Studies.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

253 Duvick, Jon, Environmental Health Pers pectives Volume 109, Supplement 2, May 2001

Prospects for Reducing Fumonisin Contamina tion of Maize through Ge netic Modification

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Falck-Zepeda, Traxler, Nelson. 2000. Surplu s Distribution from the Introduction of Biotechnology Innovation. American J. Agr. Econ. May 2000.

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254 Headrick, John “Aflatoxin Concentrations in YieldGard MON810 and non-Bt Corn Hybrids at 17 Locations across 10 states,” Monsanto Company, Sept. 2001.

T. Kuiper-Goodman, P.M. Scott, N. P. McEwen, G.A. Lombaert, W. Ng , Approaches to the Risk Assessment of Fumonisins in Corn-Based Foods in Canada in Fumonisins in Food , L. Jackson, ed., New York, Plenum Press, 1996.

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F. Benefits Assessment for MoCry1F Corn (2005 Conclusion)

Benefit Claims Made in Maize-optimized Cry1F-protected Corn Event TC6275 Public Interest Document (Zabik et al., 2003; MRID# 460193-12)

Dow AgroSciences (Dow) believes that the maize- optimized Cry1F-protected corn is clearly in the public interest and provides data to support the following claims:

1. Maize-optimized Cry1F-protected corn pr ovides highly efficacious control of key Lepidopteran pests of field corn

2. Maize-optimized Cry1F-protected corn has the potential to provide co mparable or superior pest control to existing commercial Bt corn pr oducts for key corn pests and affords a broader spectrum of pest control than do corn products expressing the Cry1A(b) in sect resistance trait.

3. The use of moCry1F-protected corn is expected to reduce th e use of chemical insecticides.

4. Yields of moCry1F-protected corn varieties will be significantly greater than the yields of the

non-Bt varieties.

5. Economic models show that moCry1F-prote cted corn maximizes economic benefits as compared to the application of conventional chem ical insecticides to control Lepidopteran pests that are susceptible to Cry1F.

6. Maize-optimized Cry1F-protected corn represents a competitive choice for insect control.

7. Maize-optimized Cry1F-protected corn will solidify and extend the benefits of insecticide use

reduction that have been es tablished for plant-optimized Cry1F-protected corn.

8. Cry1F expressed in corn poses no foreseeable risks to human health or the environment.

Field EfficacyCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

257 Field efficacy trials were conducted over multip le locations and years. EPA reviewed the moCry1F-protected corn field efficacy data submitted by Dow AgroSciences (Babcock and Bing, 2003) and found these data to be A acceptable @ (see EPA review, Hill, 2004). Dow has submitted sufficient field efficacy data to de monstrate that event TC6275 (maize-optimized Cry1F-protect corn) offers excellent control of European corn borer ( Ostrinia nubilalis (Huebner), ECB), southwestern corn borer ( Diatraea grandiosella (Dyar), SWCB), fall armyworm ( Spodoptera frugiperda (J.E. Smith), FAW), black cutworm ( Agrostis ipsilon (Hufnagel), BCW), West ern bean cutworm ( Richia albicosta (Smith), WBCW), and suppression of the corn earworm( Helicoverpa zea (Boddie), CEW). TC6275 performed comparably to TC1507 (plant-optimized Cry1F-p rotected corn, Herculex I 7 ) and showed significantly less infestation than the non-Bt isogenic hybrid. No additional data are requ ired at this time.

Comparative Product Performance

Dow submitted an analysis of the comparative efficacy of event TC6275 (moCry1F) corn against several commercial standards (conventional and transgenic) for insect control in corn. A no treatment (no Cry1F) control was also incl uded. Based on this analysis, TC6275 provides comparable performance relativ e to TC1507. TC6275 performed bette r or comparably with the commercial standards against ECB, SW CB, BCW, FAW, CEW, and WBCW.

Yield and Economic Benefits

The primary economic benefit of corn hybrids co ntaining Cry1F insect re sistance trait (mo and po Cry1F) is the protection of yield. At the indivi dual farm level, there will be cost savings from reduced field scouting and applying fewer chemical insecticides. The two factors that drive a farmer to choose a Bt corn hybrid versus conventi onal hybrids treated with chemical insecticides are : 1) the higher level of field efficacy that Bt corn hybrids, such as Cry1F corn hybrids, offer in comparison to current chemical insecticides and 2) the A insurance @ factor of a Bt corn hybrid as a prophylactic control measure. Both of these f actors result in potential yield benefits to the

farmer.

Yield and Agronomic Characteristics

Previously, EPA concluded that Event TC1507 co rn hybrids expressing plant-optimized Cry1F were competitive with other Bt hybrids as well as with non- Bt corn varieties (EPA, 2001b). Event TC6275 corn hybrids expressing maize-optimi zed Cry1F was also competitive with plantoptimized Cry1F corn hybrids (Event TC1507) as well as non- Bt corn varieties.

Agronomic performance traits of TC6275 hybrids were compared to isogenic hybrids containing TC1507 and isogenic hybrids containi ng no transgenic trait in field trials for early (11 locations) and late (15 locations) season hybrids in 2002. Th e comparison of both late and early maturity hybrids found no statistically signi ficant differences within ei ther maturity group among the TC6275 hybrid, the TC1507 hybrid, and non-transgenic isogenic hybrid for grain density, plantCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

258 stature, emergence vigor, root lodging, and dropp ed ears (see Tables 5 and 6 in Zabik et al., 2003). There were no statistically significant yi eld differences between either early or late maturity comparisons of TC6275 and TC1507 hybr ids. Both the early maturity TC6275 and TC1507 hybrids were significantl y higher yielding than the ea rly maturity non-transgenic isogenic hybrid. This is most likely due to the protection o ffered by Cry1F against European corn borer-induced yield loss. Both early and late maturity TC6275 hybri ds had significantly better top integrity scores than the non-transgenic hybrids. Although there were some statistically significant differences found in other parameters , these differences were not considered to biologically or commercially signi ficant. Based on this analysis , there would be no expectation

of increased likelihood of weediness due to the presence of the mo -Cry1F transgene.

Economic Benefits

EPA has previously analyzed the economic benef its resulting from Cry1F-protected corn (Event TC1507) (EPA, 2001b). At product maturity, grower benefits of Cry1F-protected corn are estimated to be between $28 and $81 million per year on 7.3 to 12.5 million acres of field corn. The range depends upon the technology fee, fr om $7.50 to $13.13/acre. Bt-related costs are assumed to be $10/acre. These costs cover refuge requirements and marketability concerns and apply to situations where Cry1F replaces chemi cal control or no contro l. Acres at risk are estimated to be 25 million acres, based on the states affected and the extent of area infested. Grower benefits could vary by an average of $3.90/acre to $6.51/acre. Th e very wide range is due to the wide range of the proposed technology fee. It should be noted that these annual benefits would occur at product maturity, or 3 to 5 years after commercialization. The analysis does not consider possible stacked products which offer multiple protections and efficiencies, the effect of new competitor products, or the imp act of increased competition on overall market equilibrium conditions. Increased competition sh ould offer growers more choice and lower the cost of pest control. The benef its are the incremental improvement to grower profits compared to current practice. All costs are eventually passed along to consum ers in the long run, but this analysis did not deal with when that would occur.

The economic benefit to the grower who plants corn va rieties containing Event TC6275 (moCry1F) was evaluated on the basis of data on yield and efficacy as re ported in Zabik et al. (2003) using the Herculex Value Calculat or. The Herculex Value Calculator, http://www.dowagro.com/h erculex/calc/index.htm, is a tool that allows growers to evaluate the advantages/disadvantages of using corn varieties using the Cry1F insect resistance trait. Three scenarios using both early and late maturity hybrid s were used to illustrate the economic benefit associated with Event TC6275 (see Table 7, p. 33, in Zabik et al. 2003). As summarized in Table 7 of Zabik et al. (2003), the value of the Event TC6275 corn ranged from $40 to $98 per acre. The economic benefit of ECB protection a ccounts from from $24 to $81 per acre of this

total with an added benefit of from $16 to $17 per acre for BCW protection.

Marketing Issues

TC6275 (moCry1F) and TC1507 (poCry1F) are pos itioned to compete against the following

chemical alternatives: bifenthr in, carbofuran, chlorpyrifos, cy halothrin-lambda , permethrin,Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

259 fipronil, tebuprimiphos/cyluthri n, tefluthrin, terbufos, and zeta- cypermethrin and against the Cry1Ab-protected corn hybrids. The registered chem ical alternatives comm only used to treat the target pest complex protected by Cry1F are re stricted use for the most part. They have precautionary label statements such as extremely toxic to fish and aquatic organisms, wildlife and require protective clothing for workers. The specific organophos phate and pyrethroid pesticides likely to be replaced are ranked in the top 15 of all pest icides with respect to reported incidents of mortality to non-targ et wildlife. Many of these produc ts also control corn rootworm, which is the most significant pest of corn and is frequently treated along with the lepidopteran target pest complex of Cry1F. Cry1F performa nce data have shown performance equal to or better than any of the conventiona l chemical alternatives or othe r Bt corn hybrids against ECB, BCW, SWCB, FAW, WBCW, and CEW. Grow ers may be more likely to choose Cry1F protected corn due to better product performance and broade r spectrum of control. Cry1F protected corn is also expect ed to be economical on some unp rotected fields and provide insurance against the risk of crop loss and the need to repl ant. But without rootworm protection, the use of Cry1F to reduce conventional pesticide use is somewhat limited.

Human Health and Environmental Risks and Benefits

The Agency

= s human health and environmental safety assessments attest to the safety of the Cry1F protein (both as poCry1F and moCry1F) that it is expe cted to pose no unreasonable

adverse effects to human health or the envi ronment (Matten, 2005a and b; Hill, 2005; EPA 2001 a and b). Based on the Agency

= s previous evaluation of the benefits of Event TC1507 (EPA, 2001a and b), Event TC6275 can also substantiall y reduce the health and environmental risks

associated with the use of traditional chemical insecticides. Cry1F-protected corn varieties will potentially decrease the reliance on conventional pesticides when used as part of an integrated pest management program. Reductions in the use of conventional pesticides would eliminate the need to transport, mix, apply, and dispose of these pesticides, reduce spray-drift and run-off associated with some of the registered altern atives, and reduce potential adverse effects to nontarget organisms. Increased use of Cry1F-prot ected corn would also improve worker protection as compared to chemical insecticides.

The data submitted show that poCry1F and moCr y1F corn produce the same Cry1F protein and therefore have the same expected toxicity to target pests. Data also demonstrate that Cry1F is not heat labile (MRID 452748-01), is rapidly digested in simulate d gastric fluid (MRID 447149-03), and does not share any amino acid sequence simi larity to known allergens (MRID 449717-01). These data indicate there is no likely potential for the Cry1F protein to be a food allergen.

Expression data reveal that moCr y1F plants express lower concentrations of Cry1F protein in pollen and grain than poCry1F plants (1/6 and1 /2, respectively) while moCry1F plants express the Cry1F protein three to eight tim es greater in the leaf than th e poCry1F plants (Zabik et al. 2003, see p. 36, Table 8). Increased expression in the stalks and l eaves should decrease the risk of resistance development by bor ers (a higher, high dose) whils t the decreased expression in pollen and grain should decrease the non-target exposure to Cry1F expressed in corn (Zabik et.
al 2003).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

260 Similarly, testing with bacterially prepared Cry1 F protein at levels gr eatly exceeding those found in maize optimized plants resulted in no effect with several beneficial species including the monarch butterfly. Additionally, field monitoring for effects of poCry1F corn on non-target

insects confirmed the absence of adverse e ffects to non-target organisms (MRID 450201-13, see Table 1). Data were also pr ovided regarding the rapid soil de gradation of poCry1F protein, approximately 3.13 days (MRID 450201-05), although the Agency required additional data to study long-term soil degradation (EPA, 2001b).

F. REFERENCES

Babcock, J.M. and J. Bing. 2003. Field efficacy of Maize-optimized Cry1F (TC6275) for the control of Lepidoptera pests of corn. U npublished study submitted by Dow AgroSciences

LLC. MRID No. 460630-04.

Hill, H. 2004. EPA review of field efficac y, high dose studies, and insect resistance management plan of maize-optimized Cry1F (T C6275) for the control of Lepidoptera pests of corn, EPA Reg. No. 68467-U. Memorandum dated April 19, 2004.

Hill, H. 2005. Environmental effects assessment for Bacillus thuringiensis moCry1F (maizeoptimized) insecticidal protein as expressed in maize line 6275 as part of Dow AgroSciences LLC Application for a FIFRA Section 3 Re gistration, EPA Reg. No.68467-U. Memorandum dated January 4, 2005.

Matten, S. 2005a. EPA Review of Waiver Ra tionales for Human Health Safety Data in Support of the Section 3 Application for the Mycogen Brand B.T. Cry1F Event TC6275 Corn (a.k.a. maize-optimized Cry1F corn or moCry1F corn), Dow AgroSciences, EPA Reg. No. 68467-U. Memorandum dated January 13, 2005.

Matten, S. 2005b. EPA Review of Product Character ization and Human Health Safety Data in Support of the Section 3 Application for the Mycogen Brand B.T. Cry1F Event TC6275 Corn (a.k.a. maize-optimized Cry1F corn or moCry1F corn), Dow AgroSciences, EPA Reg. No. 68467-U. Memorandum dated January 13, 2005.

Matten, S. 2005c. EPA Review of Public Inte rest Document in Support of the Section 3 Registration of the Mycogen Brand B.T. Cr y1F Event TC6275 (a.k.a. maize-optimized Cry1F corn or moCry1F corn), Dow AgroSciences (Reg. No. 68467-U). Memorandum dated February 8, 2005.

Scientific Advisory Panel (SAP), Subpanel on Bacillus thuringiensis (Bt) Plant-Pesticides (February 9- 10, 1998), 1998. Transmittal of the fina l report of the FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) Plant-Pesticides and Resistance Management, Meeting held on February 9-10, 1998. Report dated, April 28, 1998. (Docket Number: OPPTS-00231).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

261 Scientific Advisory Panel (SAP), Subpanel on Insect Resistance Management (October 18-20, 2000), 2001. Report: sets of scientific issu es being considered by the Environmental Protection Agency regarding: Bt plant-pesticides risk and bene fit assessments. Report dated, March 12, 2001. (Pp. 5-33)

U.S. Department of Agriculture (USDA), Anim al Plant Health Inspection Service (APHIS). 2004. Approval of Mycogen Seeds c/o Dow AgroSciences LLC Request (03-181-01p) Seeking Extension of Determination of Non-regul ated Status for Bt Cry1F Insect Resistant,

Glufosinate Tolerant Corn Line 6275. Findi ng of no significant impact. October 20, 2004.

U.S. Environmental Protection Agency (EPA). 2001a. Biopesticides Registration Action

Document: Bacillus thuringiensis Cry1F Corn (August 2001). http://www.epa.gov/pesticides/biopestic ides/ingredients/t ech_docs/brad_006481.pdf.

U.S. Environmental Protection Agency (E PA) 2001b. Biopesticides Registration Action

Document: Bacillus thuringiensis (Bt) Plant-incorporated Protectants (October 15, 2001). http://www.epa.gov/pesticides/biopesticides/pips/bt_brad.htm.

Wozniak, C. 2002. EPA Memorandum: Review ofproduct characterization data for maizeoptimized Cry1F insect control pr otein as expressed in maize, a Bacillus thuringiensis var. aizawai -based plant-incorporated protectant. April 2, 2002.

Zabik, J. M., J. D. Wolt, D.J. Borgmeier, N. Storer. 2003. Public inte rest document for maizeoptimized Cry1F-protected corn event TC6275. GH-C 5659. MRID 460193-12.



### III. Regulatory Position on Bt Corn

A. Overview (2001 Conclusions)

Currently registered Bt corn products were conditionally approved for commercial use in August 1996 (Bt11 Cry1Ab field corn amendment), December 1996 (MON810 Cry1Ab field corn registration), February 1998 (Bt 11 Cry1Ab sweet corn registration), May 2001 (Cry1F field corn registration) under FIFRA Section 3(c)(7)(B). The data reviewed for the initial commercial approvals as well as new data and reports r eceived, results of public meeting, hearings, workshops, forums, and Scientific Advisory Pa nel meetings, and public comments received regarding the Bt crops reassessment have been taken in to consideration. The scientific assessment has included product characterization, human health effects, gene flow, effects on non-target organisms, ecological exposure, insect resistance management, and benefits. Over the last five years, new data and information have been provided to the Agency in each of these areas and these data have been incorporated into the science assessment and has been taken into account in making regulatory decisions.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

262 Tests have shown no toxicity to mammals from the Cry1Ab and Cry1F prot eins; the proteins are readily digestible in gastric fluids and are non-glycosylated, the prot eins are inactivated by typical food processing, and anticipa ted exposure of farm workers to the proteins is negligible. The Cry1Ab protein acute oral toxicity data subm itted demonstrated no effects at the relatively high dose level of 4,000 mg/kg. The Cry1F pr otein acute oral toxicity data submitted demonstrated no effects at the relatively high dose level of 5,050 mg/kg. The Cry1Ab and Cry1F proteins were readily degraded in gastric fluid in vitro . Exposure via the skin or inhalation is not likely since the Cry1Ab a nd Cry1F proteins are contained within corn plant cells which essentially eliminates or reduces exposure routes to negligible. Oral exposure, at very low levels, may occur from ingestion of processed products and drinking water. Worker exposure to the Cry protein via seed dust is also expected to be negligible because of the low amount of protein expressed in seeds of the transformed plants . Taken in total, these data allow the Agency to make a determination that for human health, there is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, including infant s and children, to the Cry1Ab and Cry1F proteins and the genetic mate rial necessary for their production. Thus, EPA

concludes that there are no adve rse effects on human health fro m the use of Cry1Ab or Cry1F proteins expressed in corn.

EPA has also reviewed the original data base and the new data, information, and comments regarding ecological effects. EPA has reviewed the potential for gene ca pture and expression of the Cry1Ab/Cry1F endotoxin in corn by wild or weedy relatives of corn in the United States, its possessions or territories. The Agency has determ ined that there is no significant risk of gene capture and expression of any B.t. endotoxin by wild or weedy relatives of corn product registrations in the U.S., its possessions or te rritories. In addition, the USDA/APHIS has made

this same determination under its statut ory authority under the Plant Pest Act. The Agency has concluded that based on the weight of evidence there are no unreasonable adverse effects of Cry1Ab or Cry1F protein expressed in corn to non-target wildlife or beneficial invertebrates. However, EPA is requiring insect census estimates from representative fields to determine if there are long-term adverse impacts from the use of Bt corn, field tests of Cry1Ab and Cry1F protein accumulation and/or persistence in soil under a range of conditions typical of

Bt crop cultivation as confirmatory data, and chronic avian data.

In the Cry1Ab ecological effects testing done, no treatment related effects were observed in Bobwhite quail or catfish fed Cry1Ab corn as part of their diet. No measurable deleterious effects from the Cry1Ab protein on honey bee larvae, honey bee adults , parasitic wasps, Ladybird beetles, green lacewings, Collembola (springtails), and Daphnia were observed in

submitted studies.

In the Cry1F ecological effects testing done, no treatment related effects were observed in Bobwhite quail fed Cry1Ab corn as part of their diet. No measurable deleterious effects from the Cry1F protein on honey bees, parasitic wasps, Ladybird beetles, green lacewings, Collembola (springtails), earthworms, Daphnia, and Monarc h butterflies were observed in submitted studies.

MON 810 and Bt11 show relatively low toxicity to monarch larvae and the Cry1F protein has no detectable impact on monarch larvae. Overall, the available information indicates a very lowCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

263 probability of risk to monarchs in areas beyond the near edge of corn fields. Inside corn fields and at the near edge of corn fields there is low probability of monarch larvae encountering a toxic level of pollen for the Bt corn products covered by this risk assessment.

Limited data do not indicate th at Cry proteins have any m easurable effect on microbial populations in the soil. Horizontal transfer from transgenic plants to soil bacteria has not been demonstrated. Cry1Ab protein bioactivity from Cry1Ab corn tissue added to the soil decreased

with an estimated DT 50 (Degradation Time) of 1.6 days and an estimated DT 90 of 15 days. The bioactivity of purified Cry1Ab protein in soil decreased with an estimated DT 50 of 8.3 days and a

an estimated DT 90 of 32.5 days. The bioactivity of purifie d Cry1F protein in soil decreased with

an estimated DT 50 of 3.13 days.

The issue of insect resistance management has generated more data, meetings, and public

comments than all of the other sections covered in this BRAD. Insect resistance management (IRM) is the set of practices aime d at reducing the potential for in sect pests to become resistant to a pesticide. Bt IRM is of great importance because of th e threat insect resistance poses to the

future use of Bt plant-pesticides and Bt technology as a whole. EPA considers protection of insect (pest) susceptibility of Bt to be in the “public good.” EPA has determined that development of resistant insects would constitute an adverse environmental effect. In order to delay the development of insect resistance to Bt field corn by maintaini ng insect susceptibility, growers must choose at least one of structured refuge (a porti on of the total acreage using non- Bt seed) options listed in Section V.B.4.a. above.

For Bt sweet corn, no specific refuge requirements ar e necessary because sweet corn is typically harvested much earlier than field corn, 18-21 days after silking, and before most lepidopteran larvae complete development. However, to m itigate the development of resistance, EPA has determined that crop residue destruction is necessa ry within 30 days. Th is practice will likely destroy any live larvae left in Bt sweet corn stalks and preven t overwintering of any resistant

insects.

The IRM program for Bt field and sweet corn also require: 1) anyone purchasing Bt corn to sign a grower agreement which contra ctually binds the grower to co mply with the IRM program and that there will be a mechanism by the year 2003 by which every grower affirms, annually, their contractual obligations to comp ly with the IRM program, 2) an IRM education program, 3) an IRM compliance monitoring program including a th ird party compliance survey and mechanisms to address non-compliance, 4) an insect resist ance monitoring program for each target insect pest, 5) remedial action plans to be implemented if resistan ce does develop, and 6) annual reporting of the IRM (and other) activities. No other pesticide products than the Bt crop products have such extensive IRM requirements.

In addition to assessing the risks from the use of Cry1Ab and Cry1F expressed in corn, EPA has evaluated the benefits from the use of these prod ucts. Direct grower be nefits include improved yield and profitability, improved crop management effectiveness, reduction in farming risk, and improved opportunity to grow field corn in case of severe pest infestation. Total annual monetary grower benefits from the use of Bt field corn are less than $219 million annually. The magnitudeCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

264 of benefits for any year is largely a function of the level of lepidopteran in sect pressure in that year. That is, other things bei ng equal, the higher the insect pr essure, the higher the benefits. The major environmental benefit is potential redu ction in mycotoxins. EPA believes that use of Bt sweet corn would result in significant reductions in the use of chemical pesticides. However, the current use of Bt sweet corn is very low.

Pursuant to FIFRA Section 3(c)(7)(A), EPA may conditionally amend the registration of a pesticide if the Agency determines (i) that th e pesticide and proposed used are identical or substantially similar to a currently registered pe sticide and use thereof, or differs only in ways that would not significantly increa se the risk of unreasonable adve rse effects on the environment, and (ii) approving the amendment in the ma nner proposed by the applicant would not significantly increase the risk of unreasonable adverse effect on th e environment. FIFRA defines

“unreasonable adverse effects on the environment” in pertinent part as: “an y unreasonable risk to man or the environment, taking into account th e economic, social, and environmental costs and benefits of the use of any pesticide . . . .” Thus, the FIFRA unr easonable adverse effects standard requires EPA to balance the risks and benefits of using the pe sticide in reaching its regulatory decision. EPA finds that the use of Cry1Ab or Cry1F expr essed in corn will not significantly increase the

risk of unreasonable adverse effects on the enviro nment. This finding, however, applies only to the use of Cry 1Ab or Cry1F protein expre ssed corn under the terms and conditions of registration specified below, and only for th e limited time period of 7 additional years (to October 15, 2008). The following sections set fort h the basis for EPA’s finding in general, and

the basis for the decision to a pprove the registration subject to the specific terms and conditions

identified below.

B. General Finding (2001 Conclusions)

EPA’s finding that Cry1Ab or Cry1F protein as expressed in corn will not significantly increase

the risk of unreasonable adverse effects on the envi ronment is based on the analysis contained in the preceding sections of this BRAD and the specific terms and conditions that are imposed upon this registration, as set forth in Section V. In general terms, EPA concludes that use of Cry1Ab or Cry1F expressed in corn is effective at co ntrolling significant lepi dopteran pests of corn including European corn borer, corn earworm, and southwestern corn borer. Therefore, these products have clear benefits for users. Beyond these economic benefits, EPA determines that Cry1Ab and Cry1F corn hybrids, to the extent they are an alternative to the use of other corn insecticides, will provide benefits in that use of Cry1Ab or Cry1F protein expressed in corn

results in less human and environmental risk than chemical alternatives. In addition, EPA finds that the use of these products, s ubject to the specific terms and c onditions set forth below, would not pose risks to human health or to non-target species. EPA also concludes that the use of Cry1Ab or Cry1F corn hybrids expressed in corn rais es concerns with respect to insect resistance management. As discussed below, the registratio ns for Cry1Ab and Cry1F proteins expressed in corn is subject to specific terms and conditions th at effectively restrict the use of the product in ways that EPA determines will adequately mitigate these concerns. Therefore, EPA determines

that the allowed use will not si gnificantly increase the risk of unreasonable adverse effects on theCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

265 environment. Finally, EPA has identified the n eed for certain confirma tory data on potential accumulation of Cry1Ab and Cry1F proteins in soil and field impacts of Cry1Ab and Cry1F proteins on non-target species. Th e registration of these products is specifically conditioned on

submission of these data.

By this reassessment, EPA has completed its tolerance reassessment for Cry1Ab (180.1173) under 408(q) of the FFDCA. The tolerance ex emption for Cry1F (180.1217) does not require

reassessment at this time.

C. Insect Resistance Management (IRM) Program (2001 Conclusions)

Rationale for IRM Requirements:

In deciding on the size, proximity, conf iguration, and management of the non- Bt corn refuge, EPA has taken into account empirical data on the pest biology and ecology of the three primary target pests, European corn borer ( Ostrinia nubilalis (Huebner)), corn earworm ( Helicoverpa zea (Boddie)), and southwes tern corn borer ( Diatreae grandiosella (Dyar)), models that predict the estimated time that resistance would develop to compare the effectiveness of various IRM strategies, economics, sustainabi lity, and grower feasibility.

Beginning with the first Bt plant-incorporated pesticide registration, the Agency has taken steps to manage insect resistance to Bt with IRM plans being an impor tant part of the regulatory decision. The Agency identified (later confir med by the 1995 SAP) seven elements that should

be addressed in a Bt plant-incorporated protectant resistan ce management plan : 1) knowledge of pest biology and ecology; 2) a ppropriate dose expression strategy; 3) appropriate refuge; 4) resistance monitoring and a remedial action plan should resistance occur; 5) employment of integrated pest management (IPM); 6) communi cation and education strategies on use of the product; and 7) development of alternative mode s of action. IRM plans also include grower

education and measurement of the level of compliance.

The Agency has determined that the 20% non- Bt field corn refuge requirements for Bt corn grown in the Corn-Belt and the 50% non- Bt corn refuge requirements for Bt corn grown in cotton-growing areas are scientifically-sound, protect ive, feasible, sustainable, and practical to growers. Models have been developed by scientis ts in academia to predict the estimated time

that insect resistance would devel op to compare IRM strategies for Bt field corn. For example, if a high dose is achieved to control ECB (as it is for the currently registered Bt corn products), then these models predict that ECB will not evol ve resistance for at least 99 years if a 20% refuge is implemented in the Corn Belt. Models are also used to pred ict the evolution of CEW

resistance have also been used. These models indicate that 50% non- Bt field corn refuge in cotton-growing areas is sufficient to delay CEW resistance for at least the time frame of the registrations. A 20% non- Bt field corn refuge in the Corn Belt is sufficient to delay CEW

resistance because CEW do not overwinter in the Corn Belt. EPA believes that the use of these models provides confidence that resistance will not evolve under the time frame of the registrations.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

266 However, it should be noted that these predictive models cannot be validated without actual field resistance. They have limitations and the inform ation gained from the us e of such models can only be used as a part of the weight of evid ence determination conducted EPA to assess the risks of resistance developing in target pest populations. EPA agr ees with the October 2000 SAP that models are an important tool in determining appropriate Bt crop IRM strategies and that model design should be peer reviewed a nd parameters validated. In th e absence of field resistance, EPA agrees with the October 2000 SAP that models are “the only scientifically rigorous way to integrate all of the biological information available, and that without these models, the Agency

would have little scientific basis for choos ing among alternative resistance management options.” While the absolute number of years to resistance is not precisely determined from the models, the relative difference in effectiveness be tween refuge options can be determined. Thus, the utility of the models is not that they make accurate quantitative predictions, rather, it is that they enable the Agency to make informed judg ments of the potential e ffects of using various refuge options. In addition to assessing the likelihood of resi stance, EPA has mandated specific requirements for annual resistance monitoring to determine, in a pr o-active fashion, whethe r insect susceptibility has changed or whether resistance is likely to occur (or is occu rring). After five years of analyzing resistance monitoring da ta (1996-2000), there is no evidence of European corn borer, corn earworm, or southwestern corn borer resi stance developing in the fi eld to the Cry1Ab deltaendotoxins produced by current registered Bt corn products. There are no resistance monitoring reports yet available for Cry1F field corn produc ts because they were just registered in 2001. Therefore, EPA believes that resistance is not oc curring in the field based on the available data. The Agency is mandating enhancements to th e resistance management programs that will improve the certainty of detection of resistan ce. If insect resistance occurs, EPA has also mandated a specific remedial acti on plan that will c ontain (and perhaps eradicate) resistance prior to the occurrence of any widespread, endemic resistance.

In addition, to the use of biological data, pr edictive models, and resistance monitoring information, EPA also weighed practical consider ations in deciding which refuge options to allow. Grower education and compliance with ref uge options are essential to the success of any IRM strategy. Growers must be able to implemen t the refuge options within the constraints of their farming operations. Based upon the currently available scientific da ta and information and understanding of farming operations, EPA believes that the 20% non- Bt field corn refuge options

in the Corn-Belt and the 50% non- Bt field corn refuge options in the cotton-growing areas provide an adequate time-to-resistance for Bt field corn and are prac tical, sustainable, and feasible to growers. If the 20% non- Bt field corn refuge options in the Corn-Belt and the 50%

non- Bt field corn refuge options ar e deployed correctly then, ther e is a very limited chance of insect resistance evolving over the next seven years of the registration of these products.

EPA has determined that a mandatory refuge strategy was not necessary for Bt sweet corn products to reduce the likelihood of resistance for the following reasons: 1) sweet corn is typically harvested earlier than field corn (18-21 ) days after silking (bef ore most lepidopteran larvae complete development); and 2) all Bt sweet corn residues are mandated by the terms and conditions of the registration to be destroyed wi thin one month of harvest (a practice thatCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

267 presumably would destroy any live larvae left in corn stalks). The 2000 SAP agreed that

destruction of Bt sweet corn residues would be sufficient to mitigate pest resistance to Bt sweet

corn.

To strengthen the IRM strategies for Bt field and sweet corn, EP A has mandated that the registrants have grower agreemen ts that contractually bind the gr ower to the IRM requirements, ongoing grower education programs, ongoing resear ch programs, ongoing resistance monitoring programs, and a multi-faceted compliance monitori ng program (including an annual third-party compliance survey) to ensure that IRM strategies are deployed correctly. EPA will be obtaining annual reports on grower agreements, grower education programs, resistance monitoring programs, research programs, and complian ce monitoring programs. EPA has asked for

additional data on the effect of inse cticides on refuge effectiveness for Btfield corn and northsouth movement of CEW to further enhance th e IRM strategies. Part of the compliance monitoring program (to be developed as part of the terms and conditions of registration) includes specific actions for growers or growers in a regi on who are found to be non-compliant with IRM requirements. As noted above, if resistance were to occur, EPA has mandated refinements to specific remedial action plans for the Bt corn products to contai n (and perhaps eradicate)

resistance.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

268 D. 2010 Update

Section 3(c)(7)(A) of FIFRA provides for the regi stration or amendment of a pesticide when the pesticide and proposed use “...are id entical or substantially simila r to any currently registered pesticide and use thereof, or differ only in ways that would not significantly increase the risk of

unreasonable adverse effects on the environmen t, and (ii) approving the registration or amendment in the manner proposed by the applicant would not significantly increase the risk of any unreasonable adverse effect on the environment.” Unreasonable adverse effects on the environment are defined under section 2(bb) of FI FRA as “... any unreasonable risk to man or the environment, taking into account the economic, social, and environmental costs and benefits of the use of any pesticide...” Thus, pursuant to section 3(c)(7)(A), EPA may conditionally register a pesticide if (1) the pesticide and its proposed use are identical or substantially similar to a currently registered pesticide; or (2) the pesticide and its proposed use differ only in ways that would not significantly increa se the risk of unreasonable a dverse effects; and (3) approving the registration would not significantly increase the risk of any unreasonable adverse effect.

The Agency concludes that the following Cry1Ab and Cry1F Bt corn product registrations, set to expire in September or October 2010 and described in-depth throughout this BRAD, meet both criteria (1) and (2):

1) Reg. # 67979-1 Bt Corn Event Bt11 with Cry 1Ab (Field Corn) 2) Reg. # 65268-1 Bt Corn Event Bt11 with Cry 1Ab (Sweet Corn) 3) Reg. # 524-489 Bt Corn Event MON 810 Cry1Ab 4) Reg. # 68467-2 Bt Corn Event TC1507 with PO Cry 1F 5) Reg. # 29964-3 Bt Corn Event TC1507 with PO Cry 1F 6) Reg. # 68467-4 Bt Corn Event DAS-06275-8 with MOCry1F 7) Reg. # 29964-7 1507 (POCry1F) x MON 810 (Cry1Ab)

All of these Bt corn products are identical in both composition and use (corn) to plantincorporated protectants that are currently registered. Thus, criterion (1) has been fulfilled.

With regard to criterion (2), the Agency maintains, as was previously determined for the original registration of these particular products, that cultiv ation of Cry1Ab or Cry1F -containing corn

will not cause unreasonable adverse effects on the environment. The conditional environmental effects data, submitted in response to terms a nd conditions of registration and summarized in section II(C) of this BRAD, strengthen the Agency ’s initial position and al so confirm that longterm effects on non-target organi sms are not anticipated. Lastl y, the continued use of these products will likely still provide ma ny of the benefits as were eval uated in section II(E) and II(F) of this BRAD to support the 2001 Bt crops reasse ssment of Cry1Ab and Cry1F Bt corn and the 2005 registration of moCry1F corn (e.g., reduction in use of conventional insecticides that are highly toxic to both humans and the environment).Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

269 In conclusion, as the expiring Cry1Ab and Cry1F products have met the required criteria under section 3(c)(7)(A) of FIFRA, the Agency is pr oposing to amend these registrations to extend their respective expiration dates a as follows:

Product (EPA Reg. No.) Proposed Expiration Date

67979-1 Bt Corn Event Bt11 with Cry 1Ab (Field Corn) 5 years after amendmen t (~September 2015)

65268-1 Bt Corn Event Bt11 with Cry 1Ab (Sweet Corn) 5 years after amendmen t (~September 2015)

524-489 Bt Corn Event MON 810 Cry1Ab 5 years after amendmen t (~September 2015)

68467-2 Bt Corn Event TC1507 with PO Cry 1F 5 years after amendmen t (~September 2015)

29964-3 Bt Corn Event TC1507 with PO Cry 1F 5 years after amendmen t (~September 2015)

68467-4 Bt Corn Event DAS-06275-8 with MOCry1F 5 years after amendmen t (~September 2015)

29964-7 1507 (POCry1F) x MON 810 (Cry1Ab) 12 years after amend ment (~September 2022)

Although data provided were satisfactory to make the determinations required by section 3(c)(7)(A) of FIFRA, they we re not sufficient to support an unconditional registration under FIFRA section 3(c)(5). Additional data, specifically in relation to aquatic invertebrate testing, Bt11 sweet corn expression data on a dry weight basis, and ins ect resistance management, are necessary for a finding of registrability under FIFR A section 3(c)(5) and will remain as terms or conditions for the purposes of the proposed amendments.

E. Period of Registration

In the 2001 BtCorn reassessment, EPA determined that it was appropriate to amend the thenexisting registrations to extend the period of regi stration of those products to an expiration date of October 15, 2008. All of the products being as sessed at that time were efficacious against lepidopteran pests. EPA based this action on the finding that use of Cry1Ab or Cry1F expressed in corn will not significantly in crease the risk of unreasonable a dverse effects on the environment “for the limited time period of 7 additional ye ars (to October 15, 2008).” These registrations were later amended to extend the period of regi stration to an expirati on date of September 30, 2010. EPA subsequently granted time-limited regi strations to products efficacious against coleopteran corn rootworm pests. For exam ple, EPA registered Cry3Bb1 on February 24, 2003, to May 1, 2004, and extended that registration twice, to February 24, 2008, and September 30,

2010.

a See section III(C) of this BRAD for an explanation describing how the proposed expiration dates were determined.Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

270 As set forth elsewhere in this docum ent, EPA’s primary concern for the Bt protected transgenic corn products is the possibility that target pest s will develop resistance to one or more of the plant-incorporated protectant (PIP) t oxins. Development of resistance to a Bt toxin would be a grave adverse effect, and, for over 15 years, EPA has imposed stringent requirements intended to countermand the potential development of resist ance. Registrants similarly have been busily developing various products, product mixes (i.e., so-called “pyramids” and “stacks”), and resistance strategies, to maximize agronomic bene fits and address resistance management issues. The result has been a vast array of product combin ations and, occurring over the past couple of years, a re-emergence of varying refuge requirements for different products.

As discussed in the 2001 Bt PIP BRAD (at IID13), the earliest Bt corn registrations did not include mandatory refuge requirements. There was a lack of scientific consensus as to what the appropriate refuge requirement should be, and, it was assumed that the limited market penetration of these early crops would be so lo w as to guarantee that ad equate natural refuges would be available from neighboring non- Bt corn fields. From 1995 to 1997, Bt corn registrations included voluntary re fuge requirements of 0% to 20% in the Corn Belt. In 1999, the Agricultural Biotechnology Stewardship Technical Committee (ABSTC), in conjunction with the

National Corn Growers Associat ion, proposed uniform insect resistance management (IRM) requirements for Bt corn registrations. With some modifica tions, this proposal, put in place for the 2000 growing season, formed the baseline IRM requirements for almost all Bt corn registrations for the better part of a decade: fa rmers were required to plant a 20% refuge that could be treated for insects, or a 50% treated re fuge in cotton-growing ar eas; all refuges to be planted within one-half mile of the Bt corn field.

These uniform requirements brought certainty and consistency to the market after the initial period where many Bt corn products had different refuge requirements. Recently, however, as product developers have begun to conceive of products with differe nt combinations of “pyramided” products (i.e., products containing two or more toxins efficacious against the same pest) and “stacked” products (i.e., products combin ing toxins efficacious ag ainst different pests), the refuge requirements have begun to vary. For example, certain products require a 20% external refuge; some products permit a 5% ex ternal refuge; one produc t incorporates a 10% seed blend refuge; we have applications in proc ess for products that prop ose to incorporate a 5% seed blend refuge; and other permutations are possible.

Given the profusion of various toxin combinati ons and refuge options, we can no longer proceed on the basis that, as concerns insect resistance management, a ll products are equal. It was a relatively simple proposition when the default requi rement of a 20% sprayed refuge applied to

almost all of the Bt corn crops in the market. Under thos e circumstances, the relative durability of products against the development of resist ance was functionally equivalent, and, as a consequence, imposing functionally equivalent registra tion periods was appropriate. That is now no longer the case.

As part of our continually e volving regulatory approach to th e continually evolving product mix wrought by developers, we think it appropriate to revise ou r regulatory requirements in scientifically defensible ways to reflect the actual le vel of risks posed by the products that weCry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document

271 regulate. Here, for example, where we’ve determin ed that a particular product, or category of products, likely will pose less risk of insect resistance developing to a particular PIP protein, we think it appropriate to grant that particular pr oduct, or category of products, a registration for a period greater than that grante d a corresponding product that poses a greater risk of insect resistance developing. This approa ch is reflective of complementar y principles: first, to ensure that we apply our limited resources to the products that pose greater risk of adverse effects to the environment; and, second, to conserve the resources that registrants and applicants must expend in amending the registrations of products that pose less risk of adverse effects to the

environment.

The scheme that we propose to follow will include registration periods of five, eight, and twelve years; a fifteen-year registrati on period will also be available, if adequately supported by our science assessment. In this scheme, (i) a produc t with a single PIP toxin, and a 20% external refuge, qualifies for a five-year registration; (ii) a product with pyramided PIP toxins (i.e., two or more toxins with distinct, non- cross reacting modes of action) , that are non-high dose (the definition for a high dose product remains unchanged) , with either a seed blend or external refuge, qualifies for an eight-year registration; (i ii) a product with pyramided PIP toxins (i.e., two or more toxins with distinct, non-cross reacting modes of action), that are high-dose, with either a seed blend or external refuge, qualifies for a twelve-year registrati on; (iv) a product with pyramided PIP toxins (i.e., two or more toxins with distinct non- cross reacting modes of actions), with either a seed blend or external refuge , that has been determined by EPA’s science

assessment to be 150% as durable as the base line single toxin product with a 20% external refuge, would qualify for a fifteen-year regist ration. Products determined by EPA’s science

assessment to be less than 100% as durable as the baseline single toxin product with a 20% external refuge would not qualify for a five-year registration and the registration period for such products will be determined on a case-by-case basis consistent with the leve l of risk they pose. Similarly, instances where other risk issues ma y arise, or where novel resistance concerns may be present, would also be dete rmined on a case-by-case basis.