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 Division

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

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TABLE OF CONTENTS

I. OVERVIEW............................................................................................................................................................3

A. EXECUTIVE SUMMARY ............................................................................................................................3

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

C. FOOD CLEARANCE/TOLERANCE EXEMPTION LISTINGS.............................................................6

II. SCIENCE ASSESSMENT....................................................................................................................................6

A. PRODUCT CHARACTERIZATION...........................................................................................................6

1. PRODUCT CHARACTERIZATION OF BT 11 CRY1AB CORN (006444) ..........................................................11

2. PRODUCT CHARACTERIZATION OF MON810 CRY1AB CORN (006430) ....................................................13

3. PRODUCT CHARACTERIZATION OF PLANT OPTIMIZED (PO) CRY1F CORN (006481).................................15

4. PRODUCT CHARACTERIZATION OF MAIZE OPTIMIZED (MO) CRY1F CORN (006491) ...............................19

5. PRODUCT CHARACTERIZATION OF MON 810 X TC1507 (CRY1AB X CRY1F) CORN ................................22

B. HUMAN HEALTH ASSESSMENT............................................................................................................23

1. BACKGROUND ...........................................................................................................................................23

2. IN VITRO DIGESTIBILITY ASSAY ................................................................................................................24

3. AMINO ACID HOMOLOGY AND HEAT STABILITY.......................................................................................25

4. ACUTE ORAL TOXICITY.............................................................................................................................25

5. EPA RECOMMENDATION (2001) ...............................................................................................................25

6. HUMAN HEALTH ASSESSMENT OF CRY1AB CROPS, INCLUDING BUT NOT LIMITED TO:...........................25

BT11 CRY1AB BT CORN (006444) AND MON810 CRY1AB BT CORN (006430) ...........................................25

C. ENVIRONMENTAL ASSESSMENT.........................................................................................................44

1. TIERED HAZARD AND RISK ASSESSMENT PROCESS...................................................................................45

2. ENVIRONMENTAL EXPOSURE ASSESSMENT...............................................................................................48

3. NON TARGET WILDLIFE HAZARD ASSESSMENT ........................................................................................58

4. HORIZONTAL TRANSFER OF TRANSGENES FROM BT CROPS TO SOIL ORGANISMS.....................................93

5. GENE FLOW AND WEEDINESS POTENTIAL.................................................................................................94

6. IMPACTS ON THREATENED OR ENDANGERED SPECIES...............................................................................99

7. POTENTIAL INTERACTION BETWEEN CRY1AB AND CRY1F PROTEINS ....................................................100

(MRID 47677802).......................................................................................................................................100

8. ECOLOGICAL RISK ASSESSMENT CONCLUSIONS ......................................................................................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 .................................................................................................................269

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

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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 expiration dates. We have conducted comprehensive 

assessments of each of these registrations, considering all toxicity and environmental effects 

data, data from insect resistance monitoring, and 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 reached significant conclusions regarding the 

positive environmental impact of Bt corn PIPs, and we are proposing several actions to 

strengthen the insect resistance management requirements 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 significant number of published field 

studies that, combined with the post-registration field studies required to be submitted to the 

Agency, have demonstrated that non-target invertebrates are generally more abundant in Bt 

cotton and Bt corn fields than in non-transgenic fields 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 invertebrate 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 requirements has been decreasing, EPA is proposing to 

require enhanced compliance assurance programs (CAPs), and a phased requirement for seed 

bag labeling that clearly shows the refuge requirements. Also, given the increasing variety of PIP 

products and combinations, and the differing risk of resistance evolution that the various 

products represent, we are proposing to grant registrations for the corn PIP products for different 

timeframes, based on assessments of their likelihood of forestalling the evolution of insect 

resistance. We are registering differing categories of products for differing time periods to reflect 

the assessed level of risk of resistance posed by the various 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 products that are demonstrated to 

meet specified criteria. We retain, however, the discretion to register products 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: 006444 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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Pesticide Name: Bacillus thuringiensis Cry1Ab Delta-Endotoxin and the Genetic Material 

Necessary for Its Production (Plasmid 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 with 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 Genetic 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 (Plasmid Insert PHI 8999) in Corn 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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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. aizawai strain PS811 Cry1 Finsecticidal 

crystal protein and the genetic material 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 Corn 

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

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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 are exempt from the requirement 

of a tolerance when used as plant-incorporated 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 protectants in the food and feed 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 critical 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 characterization data provide information on the specific transformation 

systems used for each product, on the actual DNA inserted 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 various plant tissues. Specific information and data for each of the 

registrations seeking renewal are included 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 information contained in the documents 

and if the data meet the intent of the test guidelines. A rating of “ACCEPTABLE” indicates the 

study is scientifically valid and has been satisfactorily performed according to accepted EPA 

guidelines or other justified criteria. A “SUPPLEMENTAL” rating indicates the data provide 

some information that can be useful for risk assessment. 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 “SUPPLEMENTAL. UPGRADABLE,” EPA always provides 

an indication of what is lacking or what can be provided to change the rating to 

“ACCEPTABLE.” If there is simply a “SUPPLEMENTAL” rating, the 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 SUPPLEMENTAL studies may be used in the risk 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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assessment process as appropriate.The following table summarizes the registered Bt proteincontaining plant-incorporated protectant products being evaluated. 

Table A1 - Bt Plant-incorporated protectant Products

Common Name 

and Cry Protein 

OPP 

Chemical 

Code 

Company Plasmid ID Plant/ 

 Trade Name 

Bt11 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 well as the fragment size, orientation and any 

modifications to the original DNA sequence to enhance expression in the plant are given. All the 

other DNA sequences introduced to improve or restrict expression of the introduced traits are 

also described. Finally, the plasmid discussion includes a description 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 genome. The analysis usually consists of DNA 

isolation from the transformed plant, digestion of this DNA with several different endonucleases 

and hybridization of these restriction endonuclease fragments 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. Polymerase chain reaction (PCR) assays utilizing various specific and 

non-specific primers, genome walking, cosmid libraries and DNA sequencing have also been 

employed with sensitive Southern blotting techniques to more completely describe the inserted 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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DNA and surrounding regions. The information available from these blots can indicate the 

presence of all the elements of the expected insert as well as information about the possibility of 

deletions and other errors associated with DNA introduction by transformation. Comparison of 

Southern blots of genomic DNA, digested using a range of restriction endonucleases, can also 

reveal the copy number of the genes introduced 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 incorporated 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 indicate that there is no transcription or translation of the bacterial 

trait (e.g., ori and ampr - 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 introduced traits in the progeny. Traditional 

breeding work done during the development of the plant line by backcrossing 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 main 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 indicates that the traits were on the same 

chromosome and closely linked (crossover events between the two traits were 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 acceptable and fulfill this 

data requirement. It should be noted that stability and inheritance were not addressed with the 

registrations for MON810 (006430). However, considering the use of these crops for several 

growing seasons and the lack of reports 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 against the intended target 

insect. Some protein characterization data demonstrate that microbially produced Bt protein is 

the equivalent to that expressed 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 proteins 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 produce the protein in an industrial microbe is essential. The acute 

oral test requires between 2000 and 5000 mg of protein per kg bodyweight of test animal. 

Isolating the amount of purified protein required to dose several animals from Bt-expressing 

plants would be a tremendous burden involving harvesting and processing large volumes of plant 

material (ecological effects testing differs and is addressed in the ecological effects section of 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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this document). Proper characterization of the equivalency between these microbial proteins and 

plant expressed proteins provides 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 sizing by SDS-PAGE and western 

blot analysis; immunorecognition using ELISA and western blot analysis; N-terminal amino acid 

sequencing; confirmation of the lack of glycosylation in the plant-produced protein; and 

bioactivity against a range of insects (often pest species including the target pest). Since the 

issues surrounding non-target effects are considered essential 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 tissues 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. Although Bt11 Cry1Ab data have been provided for field corn, 

sweet corn data on the Bt11 expression remain as a data gap. 

Table A2 - Comparative expression of Cry1F protein 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 V9b

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 V9b

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

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 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 confirmatory methods and standard EPA laboratory 

method validation were also necessary. The Agency 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 requirements 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 Hybrid1

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/A3 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 
Cry1F & Cry1Ab Bt Corn Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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

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

PZO1502 which contained cry1Ab, pat and ampr genes. The registrant submission stated that 

prior to the transformation which resulted in Bt11, the plasmid pZO1502 was digested with the 

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

no data was submitted to confirm removal of the ampr gene from the transforming DNA, 

subsequent analysis showed that the ampr gene was not present in Bt11 corn. The cry1Ab gene 

was also altered to improve its GC ratio for expression in corn and coded for a truncated form of 

the original protein. Both field corn and sweet corn containing the plant-incorporated 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 weight by SDS-PAGE, immunorecognition in western 

blots and ELISA, partial amino acid sequence analysis, lack of glycosylation and bioactivity 

against either European corn borer or corn earworm. This analysis justified the use of the 

microbially produced toxin as an analogue for the 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 Cry1Ab, PAT and AMPr

. 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 ampr

 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 ampr

 gene in the Bt11 and control plants. The positive 

ampr

 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 

ampr

 gene. This would also be appropriate for any digest which had a single 

restriction cut site as these enzymes did according to the pZO1502 map. The 

probe results also indicate that a Not I digest would release the ampr

 gene from 

the pZO1502 plasmid CLASSIFICATION: ACCEPTABLE

437548-01 
 

12

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, partial 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 two field corn hybrids were harvested, 

analyzed by ELISA for expression 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 measured 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 hectare 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 indicate vastly different expression levels for 

Cry1Ab in the sweet corn varieties compared 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 QuickStixTM Kit is designed to extract and 

detect the presence of Cry1Ab (Bt11 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 Bt11 corn. Blind studies with conventional corn spiked with 

Bt11 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 QuickStixTM Kit. All results were negative indicating no 

cross-reactivity with the tested biotech events. Real-time and accelerated 

stability data indicate the QuickStixTM Kit ( EnviroLogix Cat. #AS003BG) to be 

stable for 18 months at 4-8°C. 

CLASSIFICATION: ACCEPTABLE, 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 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. 
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-ZMGT10 ballistically introduced together. 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 expresses a truncated version of Cry1Ab deltaendotoxin. MON801 expresses the full length version 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 express a slightly truncated version 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 corn. To justify this bridging of data from 

one corn transformation event to another, the company provided product characterization 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 corn line MON 80100 revealed that 

the two plasmids PV-ZMBK07 and PV-ZMGT10 had been inserted 

apparently at two locations. Full length 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 extracts not treated with trypsin. No 

bands 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 

435332-03 
Cry1Ab 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 showed 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 immunoreactivity 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 SDS-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 proteins are of similar bioactivity. 

CLASSIFICATION: ACCEPTABLE. 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 appreciable concentrations with the 

exception of pollen.

CLASSIFICATION: ACCEPTABLE. 

458789-01 

Analytical Method The independent laboratory validation is acceptable and the method 

conducted in the independent laboratory validation is suitable for an inhouse performance verification at the OPP Microbiology Laboratory. 

CLASSIFICATION: ACCEPTABLE. 

456964-01 
Cry1F & 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 Plant Optimized (PO) Cry1F Corn (006481) 

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

transformed with a linear PmeI fragment from plasmid pP8999 to produce line TC1507. This 

plasmid contains genes cry1F, pat and kanr

 encoding the delta-endotoxin from Bacillus 

thuringiensis var. aizawai PS811, phosphinothricin acetyl transferase, and resistance to the 

antibiotic kanamycin, respectively. The PmeI fragment (6235 bp) derived from this plasmid was 

purified after plasmid digestion and used in the transformation process to eliminate the kanr

antibiotic resistance gene. The Cry1F protein expressed in transformed maize lines is a modified 

(synthetic, less than full length) form as compared to that from the bacterial isolate from which it 

is derived. This insecticidal protein confers resistance to the European corn borer (Ostrinia 

nubilalis) and feeding damage is significantly reduced or eliminated following expression of this 

gene in corn line TC1507. 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 protein. 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 kanr DNA was integrated. This suggests that one PmeI 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 which are most likely non-functional 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 five 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 AM 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 (pat) 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.CLASSIFICATION: 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 clarification 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 sequence (Kozak consensus sequence) 

situated just upstream from 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

447148-02
Cry1F & 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 (pat) 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, kanr

 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 kanr DNA was introduced into 

line 1507 during transformation, as indicated by the lack of signal when 

1507 genomic DNA was probed with the kanr

 gene. There was no 

hybridization signal when the non-transformed maize line 13-1 was probed 

with pat or cry1F or kanr

. 

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 approximately 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 larval bioassay, substances tested 

included maize grain, maize grain derived 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 GI50s calculated. Comparison of control and Cry1F fish feed over four 

separate bioassays indicated that there 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

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

18 

Study Result MRID #

Protein Expression Protein expression values indicated substantial 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 with 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 the 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 against 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 antibodies are coupled to the color reagent. 

The test strips contain two zones wherein capture of color reagent or 

antibodies can occur. One zone captures bound Cry1F and the other 

captures 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 indicates the presence of Cry1F. The 

Cry1F Lateral Flow Test Kit accurately detected Cry1F protein in 30 of 30 

corn kernels from Cry1F maize and indicated 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 Cry1F 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

452793-02
Cry1F & 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 the toxin (from microbial source) was 

readily digested by pepsin. SDS-PAGE and Western blotting of plant and 

bacterial sources determined the 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 Maize Optimized (MO) Cry1F Corn (006491) 

The mocry1F (maize-optimized) gene codes for the identical truncated Cry1F protein as that 

expressed by maize plants containing the pocry1F (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 acids of moCry1F and poCry1F are identical with 

the exception of an altered residue at position 604 (F604L). Because moCry1F and poCry1F 

proteins share an identical amino acid sequence, 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, biochemically, 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 integrated into corn line 6275. Southern hybridization 

was used to assess the genetic stability of the insert in multiple generations 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 cry1F transgene was optimized for 

maize expression and transformed into maize plants using 

Agrobacterium tumefaciens for plant transformation (called 

mocry1F). The mocry1F gene encodes a truncated, core 

insecticidal toxin that is identical in amino acid sequence to 

453186-01 
Cry1Ab 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 lyophilized tissues of mocry1F

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 comparing 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 transcriptional 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 borders, were not transferred as 

determined by lack of hybridization 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 insertion 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)cry1F 

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 outside 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 

460193-01 
Cry1Ab 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 characterize the inserted synthetic 

transgene cry1F in B.t. moCry1F maize line 6275 that 

contains the (mo)cry1F and bar genes (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)cry1F and bar genes in 

transgenic corn event TC6275 . Southern blot data from this 

study confirm a single insertion 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 

EcoR 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). The 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 

GRMΕ02.13 “Determination of Cry1F Insecticidal Crystal 

456856-02 
Cry1Ab 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 deviation (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 range 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 acceptable 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 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 Cry1F in grains and 

oilseeds. 

5. Product Characterization of MON 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 the 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 cry1F gene from TC1507 corn, the cry35Ab1 and 

cry35Ab1 genes from DAS-59122-7 corn, and the cry1Ab gene from MON810 

corn. These data demonstrated equivalence 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 detected 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 quantitative 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 events. 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 are comparable to the respective protein 

concentrations of their respective single parental events. 

This study was classified acceptable. 

478800-01 

B. HUMAN HEALTH ASSESSMENT 

1. Background 

The basic premise relied on for the toxicology assessment is the fact that all the Bt plantincorporated protectants are proteins. Proteins are commonly found 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 history of safe use is associated with many Bt

products. 

Several types of data are required for the Bt plant-incorporated protectants 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 related to any known food allergen or protein toxin, and does not 
Cry1Ab 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 comparisons and an acute oral toxicity test. 

The acute oral toxicity test is done at a maximum hazard dose using purified protein of the plantincorporated protectant as a test substance. Due to limitations of obtaining sufficient quantities 

of pure protein test substance from the plant itself, an alternative 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 characterization. 

EPA believes that protein instability in digestive fluids and the lack of adverse effects using the 

maximum hazard dose approach in general eliminate 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 characterization data should identify potential 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 principle 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 fluids. 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 separately. In order to track the breakdown, the 

proteins were added to a solution of the digestive fluids and a sample was either removed or 

quenched at given time points (usually at time 0, one to several minutes later and one hour later). 

The time point samples were then electrophoresed 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 intestinal 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 protectants that are expressed 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 characteristic of instability of the protein 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 happen 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 the digestive system. One 

of the limitations of the test is that it usually only tracks protein breakdown to fragments still 

recognized by the immunological 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 heat 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 the toxicity of proteins primarily through the use of acute oral 

toxicity is that, when demonstrated to be toxic, 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 effects 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 available to examine the potential 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-incorporated protectants already examined. 

1) Acute Toxicity
Cry1Ab 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 maximum 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 protectants were derived. [See 40 CFR Sec. 158.2130 and 158.2140.] For 

microbial products, further toxicity testing to verify the observed effects and clarify the source of 

the effects (Tiers II & III) and residue data are triggered by significant acute effects in studies 

such as the mouse oral toxicity study. 

The acute oral toxicity data submitted support the 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 mutagenicity, developmental toxicity, subchronic toxicity, 

chronic exposure and oncogenicity assessment studies were not required. 

3) Effects on the Immune System

Since Cry1Ab is a protein, allergenic potential was considered. Current scientific knowledge 

suggests that common food allergens tend to be resistant to degradation by heat, acid, and 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

27 

proteases, to be glycosylated and present at high concentrations in the 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 submitted to EPA done in laboratory 

animals have not indicated any potential 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 registered since 1961), there have been no 

confirmed reports of immediate or delayed allergic 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 reactions 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 produced during fermentation or to added formulation ingredients. 

Thus, the Cry1Ab protein is not expected to be a food allergen. 

Allergenicity Endpoints of Cry1Ab Crops [Bt11 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 degrades 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 significantly 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 identified. 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 Bt11 Cry1Ab protein. However, a global alignment of 

parasporin and Bt11 Cry1Ab demonstrated only 20.7% identity between the two 

proteins. 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 

Bt11 Cry1Ab protein is not of toxicological relevance. 

CLASSIFICATION: ACCEPTABLE 

458798-01 
Cry1Ab 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 were 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 allergen sequences. 

Additionally, there were no alignments of eight or more contiguous identical amino 

acids between the Bt11 Cry1Ab protein and any of the proteins in the allergen 

database. Therefore, no biologically relevant 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 homology 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 compared for protein sequence homology 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 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 protein sequences of known allergens. 

In addition, there were no 8 or greater contiguous identical amino acid matches 

observed with the Cry1Ab protein sequence. Therefore, the updated amino acid 

homology search verifies no changes in the results and conclusions of the 

previously submitted allergen homology assessment for the Cry1Ab protein 

expressed in event MON810 corn (see MRID No. 458789-02). 

Classification: ACCEPTABLE

477864-04 
Cry1Ab 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 allergens, 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), EPA has developed the Endocrine Disruptor 

Screening Program (EDSP) to determine whether certain substances (including pesticide active 

and other ingredients) may have an effect in humans or wildlife similar 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 approach to making the statutorily required 

determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a 

chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal 

systems. Chemicals that go through Tier 1 screening and are found to have the potential to 

interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA 

will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 

testing is designed to identify any adverse endocrine related effects caused by the substance, and 

establish a quantitative relationship between 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 pesticide active ingredients and 9 inert ingredients. 

This list of chemicals was selected based on the potential for human exposure through pathways 

such as food and water, residential activity, and certain post-application agricultural scenarios. 

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

The Cry1Ab protein is not among the group of 58 pesticide active ingredients on the initial list to 

be screened under the EDSP. Under FFDCA § 408(p) the Agency must screen all pesticide 

chemicals. Accordingly, EPA anticipates issuing 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 pesticide active ingredient. 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

30 

For further information on the status of the EDSP, the policies and procedures, the list of 67 

chemicals, the test guidelines and the Tier 1 screening battery, please visit our website: 

http://www.epa.gov/endo/. 

5) Dose Response Assessment

No toxicological endpoints were identified, therefore a dose 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 expressed 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 functionally similar to the proteins produced by the 

plant-incorporated protectant ingredients in corn. Production of microbially produced protein 

was chosen in order to obtain sufficient material for testing. 

The acute oral toxicity data submitted supports the conclusion that the Cry1Ab protein is nontoxic to humans. Therefore, because no effects were shown to be caused by these plantincorporated protectants, even at relatively high dose levels (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 protectants 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 exposure is dietary, food allergenic potential was 

considered. Current scientific knowledge suggests that common food allergens tend to be 

resistant to degradation by heat, acid, and proteases, are glycosylated and present at high 

concentrations in the food. Data has been submitted which demonstrates that the Cry1Ab deltaendotoxin is degraded in two 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 immediate or delayed allergic 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 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 dietary consumption patterns of consumers (and 

major identifiable subgroups of consumers including infants and children) and (2) safety factors 

which, in the opinion of experts qualified by scientific training and experience to evaluate the 

safety of food additives, are generally recognized as appropriate for the use of animal 

experimentation data were not evaluated because the lack of mammalian toxicity at high levels 

of exposure demonstrates the safety of the product at levels above possible maximum exposure 

levels. 

The genetic material necessary for the production of the plant-incorporated protectants active 

ingredients are the nucleic acids (DNA) which comprise (1) genetic material encoding these 

proteins and (2) their regulatory regions. “Regulatory 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 plant and animal life and the Agency knows of no 

instance where these nucleic acids have been associated with toxic effects related to their 

consumption as a component of food. These ubiquitous 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 exposure to the genetic material necessary for the 

production of the subject active plant pesticidal ingredients. 

Residue chemistry data were not required for a human health effects assessment of the subject 

plant-incorporated protectant ingredients 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, special susceptibility of infants and children to 

pesticide chemical residues and the cumulative effects on infants and children of the residues and 

other substances with a common mechanism of toxicity. In addition, FFDCA section 408 

provides that EPA shall apply an additional tenfold margin of exposure (safety) for infants and 

children in the case of threshold effects to account for pre- and post-natal toxicity and the 

completeness of the database unless EPA determines that a different margin of exposure (safety) 

will be safe for infants and children. 

In this instance, based on all the available information, the Agency concludes that infants and 

children will consume minimal residues of this plant-pesticide and that there is a finding of no 

toxicity. 

Thus, there are no threshold effects of concern and, as a result the provision requiring an 

additional margin of safety does not apply. Further, the provisions of consumption patterns, 

special susceptibility, and cumulative effects do not apply. 

c) Aggregate Exposure (Not Including Occupational Exposure) Risk Conclusions
Cry1Ab 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 exposure under the tolerance 

exemption and all other tolerances or exemptions in effect for the plant-incorporated protectants 

chemical residue, and exposure from non-occupational sources. Exposure via the skin or 

inhalation is not likely since the plant-incorporated protectants 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 processed corn products and 

drinking water. However, a lack of mammalian toxicity and the digestibility of the plantincorporated protectants has been demonstrated. The use sites for Cry1Ab delta endotoxin are 

all agricultural for control of lepidopteran insects. Therefore, exposure via residential or lawn 

use to infants and children is not expected. Even if negligible exposure should 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 information on the cumulative effects of such residues and 

other substances that have a common mechanism of toxicity. These considerations included the 

cumulative 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 plantincorporated protectants, there are no cumulative effects. 

e) Dietary Risk Conclusion

There is a reasonable certainty that no harm will result from aggregate exposure to the United 

States population, including infants and children, to the Cry1Ab protein 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 inhalation is not likely since the plant-incorporated protectants are 

contained within plant cells which essentially eliminates these exposure 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 the low amount of protein expressed in transformed plants. If such 

exposure should occur, the Agency concludes that such exposure would not be expected to 

present any risk due to the lack of toxicity. If any unreasonable 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 production 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 exemption citation for Cry1Ab in all 

plants follows. 

§ 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 are exempt from the requirement 

of a tolerance when used as plant-incorporated 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 the 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 toxicity and the requirement of residue data for the 

microbial Bacillus thuringiensis products from which this plant-incorporated protectant was 

derived. [See 40 CFR Sec. 158.2130 and 158.2140.] For microbial products, further toxicity 

testing and residue data are triggered by significant acute effects in studies such as the mouse 

oral toxicity study, to verify the observed effects and clarify the source of these effects (Tiers II 

& III). 

The acute oral toxicity data submitted support the 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 administered approximately an hour apart to achieve 

the dose totaling 33.7 mL / kg body weight. Outward clinical signs and body weights were 

observed and recorded throughout the 14 day study. Gross necropsies performed at the end of the 

study indicated no findings of toxicity. No mortality or clinical signs were noted during the 

study. An LD50 was estimated at >5050 mg / kg body weight of this microbially produced test 

material. The actual dose administered contained 576 mg Cry1F protein / kg body weight. At this 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

34 

dose, no LD50 was demonstrated as no toxicity was 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 comparisons showed no similarity between Cry1F protein 

to known toxic proteins available in public protein databases. 

Since Cry1F is a protein, allergenic sensitivities were considered. Current scientific knowledge 

suggests that common food allergens tend to be resistant to degradation by heat, acid, and 

proteases, may be glycosylated and present at high concentrations in the food. 

Data has been submitted which demonstrates that 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 acids and small peptides occurred in 5 minutes. 

A heat lability study demonstrated the loss of bioactivity 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 potential for allergic reactions to B. thuringiensis or its components, 

including the d-endotoxin of the crystal protein. Additionally, a comparison of amino acid 

sequences of known allergens uncovered no evidence of any homology with Cry1F, even at the 

level of 8 contiguous amino acids residues. 

The potential for the Cry1F protein to be a food allergen 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 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 plant-incorporated protectants, 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 available 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 (residential 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 include 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-occupational sources. Exposure via the 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 negligible. Oral exposure, at 

very low levels, may occur from ingestion of processed corn products and, potentially, drinking 

water. However a lack of mammalian toxicity and the digestibility of the plant-incorporated 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

35 

protectants have been demonstrated. The use sites for the Cry1F protein are all agricultural for 

control of insects. Therefore, exposure via residential or lawn use to infants and children is not 

expected. Even if negligible exposure should occur, the Agency concludes that such exposure 

would present no risk due to the lack of toxicity demonstrated 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 cumulative 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 protectants, 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 health 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 results of these studies were determined 

applicable to evaluate human risk and the validity, 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 similar to the protein produced by the plantincorporated protectant ingredients in corn. Production of microbially produced protein was 

chosen in order to obtain sufficient material for testing. 

The acute oral toxicity data submitted supports the prediction that the Cry1F protein would be 

non-toxic 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 

Cry1F protein, even at relatively high dose levels (>5,050 mg test substance / kg body weight; 

576 mg Cry1F / kg body weight), the Cry1F 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 protectant was derived. [See 40 CFR 

Sec. 158.2130 and 158.2140.] For microbial products, further toxicity testing and residue data 

are triggered by significant acute effects in studies such as the mouse oral toxicity study to verify 

the observed effects and clarify the source of these effects (Tiers II & III). 

Although Cry1F expression level data was required 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 ingredients because of the lack of mammalian toxicity. 

Both (1) available information concerning the dietary consumption patterns of consumers (and 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

36 

major identifiable subgroups of consumers including infants and children); and (2) safety factors 

which, in the opinion of experts qualified by scientific training and experience to evaluate the 

safety of food additives, are generally recognized as appropriate for the use of animal 

experimentation data were not evaluated. The lack of mammalian toxicity at high levels of 

exposure to the Cry1F protein demonstrates the safety of the product at levels well above 

possible maximum exposure levels anticipated in the crop. 

The genetic material necessary for the production of the plant-incorporated protectants active 

ingredients are the nucleic acids (DNA, RNA) which comprise (1) genetic material encoding 

these proteins and (2) their regulatory regions. "Regulatory regions" are the genetic material, 

such as promoters, terminators, and enhancers, that 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 acids have been associated with toxic effects 

related to their consumption as a component of food. These ubiquitous 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 exposure to the genetic material 

necessary for the production of the subject active plant pesticidal 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 abnormalities. All animals appeared 

normal during the study and all except one gained weight 

throughout the study. 

Classification: Acceptable. Toxicity 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 LD50 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.

450201-18
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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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 individual. 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 allergens. 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 assessment 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. This 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; therefore, dust mite allergic 

individuals would not be expected to experience an allergic 

reaction from ingesting Cry1F. 

CLASSIFICATION: ACCEPTABLE 

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

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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 the surface of an insect diet and 

measurement of growth inhibition of neonate tobacco budworm 

larvae, indicated that the Cry1F protein 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, special susceptibility of infants and children to 

pesticide chemical residues and the cumulative effects on infants and children of the residues and 

other substances with a common mechanism of toxicity. In addition, FFDCA section 

408(B)(2)(C) also provides that EPA shall apply an additional tenfold margin of safety for 

infants and children in the case of threshold effects to account for pre- and post-natal toxicity and 

the completeness of the database unless EPA determines that a different margin of safety will be 

safe for infants and children. 

In this instance, based on all the available information, the Agency concludes that there is a 

finding of no toxicity for the Cry1F protein and the genetic material necessary for its production. 

Thus, there are no threshold effects of concern and, as a result, the provision requiring an 

additional margin of safety does not apply. Further, the provisions of consumption patterns, 

special susceptibility, and cumulative effects do not apply. 

 

3) Overall Safety Conclusion (2001 Conclusion)

There is a reasonable certainty that no harm will result from aggregate exposure to the U.S. 

population, including infants and children, to the Cry1F protein 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 plant-incorporated protectants. 

e. Other Considerations 

1) Endocrine Disruptors (Updated July 2010)

As required under FFDCA section 408(p), EPA has developed the Endocrine Disruptor 

Screening Program (EDSP) to determine whether certain substances (including pesticide active 

and other ingredients) may have an effect in humans or wildlife similar 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 approach to making the statutorily required 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

39 

determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a 

chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal 

systems. Chemicals that go through Tier 1 screening and are found to have the potential to 

interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA 

will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 

testing is designed to identify any adverse endocrine related effects caused by the substance, and 

establish a quantitative relationship between 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 pesticide active ingredients and 9 inert ingredients. 

This list of chemicals was selected based on the potential for human exposure through pathways 

such as food and water, residential activity, and certain post-application agricultural scenarios. 

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

The Cry1F protein is not among the group of 58 pesticide active ingredients on the initial list to 

be screened under the EDSP. Under FFDCA § 408(p) the Agency must screen all pesticide 

chemicals. Accordingly, EPA anticipates issuing 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 pesticide active ingredient. 

For further information on the status of the EDSP, the policies and procedures, the list of 67 

chemicals, the test guidelines and the Tier 1 screening battery, please 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 citation 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 protectants 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 

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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 weight 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 indicate 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 results also confirmed that the 

expression of each of the proteins in the combination PIP was not 

affected by the presence of the other protein. 

CLASSIFICATION: ACCEPTABLE 

476778-02 

REFERENCES

Bernstein, I.L., J.A. Bernstein, M. Miller, S. Tierzieva, D. I. Bernstein, Z. Lummus, M.K. 

Selgrade, D.L. Doerfler & Verner L. Seligy (1999) Immune Responses in Farm Workers after 

Exposure to Bacillus thuringiensis Pesticides, Environmental Health Perspectives Volume 

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. Engler (1992) "Toxicological Considerations for 

Protein Components of Biological Pesticide Products," Regulatory Toxicology and 

Pharmacology 15:3-9. 

USDA. 2004. Performance verification of qualitative mycotoxin and biotech rapid test kits 

(Directive 9181.2). Available from: 

http://www.gipsa.usda.gov/GIPSA/documents/GIPSA_Documents/9181-2.pdf. 

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 by 
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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 Number: NKJNV2. Unpublished study prepared by 

Northrup King Co. 13 p. 

 43352602 Williams, D. (1994) Product Characterization: Bacillus thuringiensis var. kurstaki

Protein in Corn: Supplemental Information: 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) Equivalence 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 Characterization of Insect 

Protected Corn Line MON 80100: Lab Project 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.; Kania, J.; et al. (1995) Molecular Characterization of Insect 

Protected Corn Line MON 810: Lab Project 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 Project 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) Acute Oral Toxicity Study of Btk HD-1 Tryptic Core Protein in 

Albino Mice: Lab Project Numbers: 92069: 11985:ML92069. Unpublished study 

prepared by Monsanto Co. 264 p. 
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 43439201 Ream, J. (1994) Assessment of the In vitro Digestive Fate of Bacillus thuringiensis

subsp. kurstaki HD-1 Protein: Lab Project Number: 93-01-39-04. Unpublished study 

prepared by Monsanto Co. 44 p. 

 

44714801 Narva, K.; Zeph, L.; Jayne, S. (1998) Product 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 Gene 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) Quantitative ELISA Analysis of Cry 1 F Expression 

Levels in Maize MPS Inbreds and Hybrid Lines 1360, 1365, 1366, and 1369. 

(Interim Report): Lab Project Number: 98-01-RA-GLP-005. Unpublished study 

prepared by Pioneer Hi-Bred International, Inc. 95 p. 

45020102 Hondred, D. (1999) Characterization of Inserted Genes in Cry1F Maize Line 1507: 

Final Report: Lab Project Number: 98-11-RA-NGLP-013. Unpublished study 

prepared by Pioneer Hi-Bred International, 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 Maize: 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 Project Number: AS 003 BG. Unpublished study prepared by 

Syngenta Seeds, Inc. 27 p. 

45696401 Adams, C. (2002) Independent Lab Validation of the SDI Trait Bt1 Corn Lateral 

Flow Test Kit: An Amended Report: Lab Project Number: B02-005: B02-005-A. 

Unpublished study prepared by Medallion Laboratories, A Division of General Mills. 

32 p. {OPPTS 860.1000, 860.1340} 

45878901 Ledsema, B.; Hoskins, S.; Jennings, J.; et al. (2003) Cry1Ab Protein 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 Transformation Event Bt11: Lab Project Number: BT11-

02-01: SSB-005-03. Unpublished study prepared by Syngenta Seeds, Inc. 17 p. 

47882001 McDonald, J. (2009) Quantification of Transgenic Proteins in Maize Tissues of Bt11 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

43 

x MIR162 Sweet Corn and Bt11 x MIR162 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 Maize Combined Trait 

Product DAS-01507-1xDAS-59122-7xMON-00810-6xMON-00603-6 Using 

Southern Blot Analysis and Event-Specific Polymerase Chain Reaction. Project 

Number: PHI/2008/082. Unpublished study prepared by E.I. Dupont de Nemours and 

Company. 42 p. 

47677802 Binning, R. (2009) Laboratory Characterization of Key Lepidopteran Pest Response 

to Pyramided Events. Project Number: PHI/2008/101. Unpublished study prepared 

by Pioneer Hi-Bred International, 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) Evaluation of the Amino Acid Sequence Similarity 

of the Cry1F Protein to the NCBI Protein 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) Evaluation 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. 
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C. ENVIRONMENTAL ASSESSMENT 

Background 

In 2001, EPA conducted an environmental reassessment of the registered Bt plant-incorporated 

protectants. The general topics covered included gene flow and the potential for weeds to 

develop if pollen from Bt crops plants were to fertilize other plants, horizontal gene transfer, 

expression of Bt Cry proteins in plant tissues, ecological effects, especially considering the 

currently available data on monarch butterflies, and fate of Bt Cry proteins in the environment. 

Information used for the reassessment included the original data submissions for EPA 

registration, additional data submitted by the registrants (including that in the response to the 

December 1999 Data Call-in), public literature, results of workshops and scientific seminars, 

recommendations from the SAP, additional discussions with scientific experts, and other public 

comments. 

In addition to those data, the 2010 environmental assessment includes all conditionally required 

data and new information the Agency has received regarding Cry1Ab and Cry1F proteins since 

2001, including long-term field studies, avian dietary studies, and an updated assessment of the 

impacts of Bt proteins on monarch butterflies. 

In 2005, the Agency conducted an environmental 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 moCry1F 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., toxicity 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 pollen and grain and higher 

concentrations in stalks, leaves, and roots than the poCry1F plants, though these expression 

differences are not expected to significantly change the exposure to Lepidopteran non-target 

organisms. Field monitoring for effects of poCry1F corn on non-target insects confirmed the 

absence of adverse effects to non-target organisms (MRID No. 450201-13). Data illustrating the 

soil degradation of poCry1F protein were submitted to the EPA in support of the registration of 

Herculex I (MRID No. 450201-05). These data support the registration 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 Herculex 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 enough confidence that there is no 

risk and no further data would be needed. Hence, such screening tests conducted early in an 

investigation tend to be broad in scope, but relatively simple in design, and can be used to 

demonstrate acceptable risk under most conceivable conditions. When screening studies suggest 

potentially unacceptable risk, additional studies are designed to assess risk under more realistic 

field exposure conditions. These later tests are more complex than earlier screening studies. Use 

of this “tiered” testing framework saves valuable time and resources by organizing the studies in 

a cohesive and coherent manner and eliminating unnecessary lines of investigation. 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 results at relatively low 

cost.1 

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

to real-world field scenarios if the earlier tiered tests fail to indicate adequate certainty of 

acceptable risk. Screening (Tier I) non-target organism hazard tests are conducted at exposure 

concentrations several times higher than the highest concentrations expected to occur under 

realistic field exposure scenarios. This has allowed an endpoint of 50% mortality to be used as a 

trigger for additional higher tier testing. Less 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-incorporated protectant (PIP) to representative non-target organisms that 

could be exposed to the toxin in the field environment. Tier I high-dose studies reflect a 

screening approach to testing designed to maximize any toxic effects of the test substance on the 

test (non-target) organism. The screening tests evaluate single species in a laboratory setting with 

mortality as the endpoint. Tiers II–IV generally 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 approach, were last published by EPA as Harmonized 

Office of Prevention, Pesticides, and Toxic Substances (OPPTS) Testing Guidelines (now 

Harmonized Office of Chemical Safety and Pollution 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 exposure concentrations several times higher than the maximum concentrations expected to occur under realistic exposure 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 conditions of extreme exposure suggest that population effects 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 either by natural selection or by deliberate genetic manipulation. 

Therefore, plant-incorporated protectants (PIPs) containing microbial toxins 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 (whenever possible >10) times the maximum amount of 

active ingredient expected to be available to terrestrial and aquatic non-target organisms in the 

environment, or the Estimated Environmental Concentration (EEC).3 Tier I tests serve to 

identify potential hazards and are conducted in the laboratory at high dose levels, which increase 

the statistical power to test the hypotheses. Elevated test doses, 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 several 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 further state that the duration of 

all Tier I tests should be approximately 30 days. Some test species, notably non-target insects, 

may be difficult to culture and the suggested test duration has been adjusted accordingly. Control 

and treated insects should be observed for at least 30 days or, in cases where an insect species 

cannot be cultured for 30 days, until negative control mortality rises above 20%. 

Failing the Tier I (10x EEC) screening at the MHD does not necessarily indicate the presence of 

an unacceptable risk in the field, but triggers the need for additional testing.4 A less than 50% 

mortality effect at the MHD is taken to indicate minimal risk. Greater than 50% mortality, 

however, does not necessarily indicate the existence of unacceptable risk in the field, but it does 

trigger the need to collect additional dose-response information and a refinement of the exposure 

estimation before deciding if the risk is acceptable or unacceptable. Where potential hazards are 

detected in Tier I testing (i.e., mortality is greater than 50%), additional information at lower test 

doses is required, which can serve to confirm whether any effect might still be detected at more 

realistic field (1x EEC) concentrations and routes of exposure.5 

When screening tests indicate a need for additional data, the OCSPP Harmonized Guidelines call 

for testing at incrementally lower doses in order to establish a definitive LD50 (i.e., dose that will 

kill 50% of the test organisms within a designated period) and to quantify the hazard. In the 

definitive testing, the number of doses and test organisms evaluated must be sufficient to 

determine an LD50 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, weight 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 environmental 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 system 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 factor,” where any adverse effect is considered significant. 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 risk 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 Concentration or HEEC). This 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 repetition are needed to enhance statistical power in 

these circumstances. 

Data that show less than 50% mortality at the maximum hazard dosage level (i.e., LC50, ED50, or 

LD50 >10x EEC) are sufficient to evaluate adverse effects, making lower field exposure dose 

definitive testing unnecessary. Also, the recommended >10x EEC maximum hazard dose level 

is a highly conservative factor. The published EPA Level of Concern (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 Panel (SAP) on microbial pesticides and microbial toxins. The 

December 9, 1999 SAP agreed that the tiered approach was suitable for use with PIPs; however, 

this panel recommended that, for PIPs with insecticidal properties, additional testing of 

beneficial invertebrates closely related to target species and/or likely to be present in genetically 

modified (GM) crop fields should be conducted. Testing of Bacillus thuringiensis (Bt) Cry 

proteins on species not closely related to the target insect pest 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 organisms. The August 2002, SAP (and some public comments) 

generally agreed with this approach, with the additional recommendation that indicator 

organisms should be selected on the basis of potential 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 toxins 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 ensures, to the greatest extent possible, that 

the Agency requires the minimum amount of data needed to make scientifically sound regulatory 

decisions. EPA believes that maximum hazard dose Tier I screening testing presents a 

reasonable approach for evaluating hazards related 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 ascertained, 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 environmental 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 

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longer term field testing will be required. As noted above, the October 2000, SAP and the 

National Academy of Sciences (NAS 2000) recommended 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 lack of baseline data on the potential for longterm environmental effects from the cultivation of PIP-producing plants, the Agency has 

required long-term field studies on invertebrate populations/communities and Cry protein 

accumulation in soils as conditions of past PIP registrations. 

Since the commercialization of Bt crops, the number of field studies published in scientific 

literature, in combination with the post-registration field studies submitted to the Agency, has 

accumulated 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-analysis7 of the data 

collected from 42 field studies indicated that non-target invertebrates 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 invertebrate populations in Bt corn and cotton fields indicated that no 

unreasonable adverse effects are taking place as a result of wide-scale Bt crop cultivation 

(Sanvido et al. 2007). Another review of field tests published to date concluded that the largescale studies in commercial Bt cotton have not revealed any unexpected non-target effects other 

than subtle shifts in the arthropod community caused by the effective control of the target pests 

(Romeis et al. 2006). Slight reductions in some invertebrate predator populations are an 

inevitable result of all pest management practices, which result in reductions in the abundance of 

the pests as prey. 

Overall, the Agency is in agreement with the conclusions of these 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 parasitoids of target pests may 

eventually be considered unnecessary. 

2. Environmental Exposure Assessment 

The EPA environmental exposure assessment is based 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 feeding habits. High dose testing 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 important to note that Tier I screen testing is not 

“safety factor testing.” In a traditional “10X safety factor” test any adverse effect noted is a 

“level of concern,” whereas in the EPA environmental 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 Center 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 

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

The nominal protein expression levels for Cry1Ab 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 differences in the antibody-based reagents used 

for quantifying the Bt protein, or whether protein values in tissue are reported in fresh or dry 

weight. While these differences may make direct 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 purposes 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 Hybrid1

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/A3 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 Tissues
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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There is significantly increased Cry1F protein expression in moCry1F TC6275 stalks, whole 

plants, and forage sampled during the flowering (R1) stage in comparison to poCry1F TC1507. 

Conversely, there is significantly decreased Cry1F protein expression in moCry1F pollen and 

grain in comparison to poCry1F. These expression differences are not expected to significantly 

change the exposure to lepidopteran non-target organisms. The increased expression of Cry1F 

protein in stalks of moCry1F hybrids could potentially decrease the risk of resistance 

development by borers, while the decreased expression 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 

Tissue Range Growth 

Stage (ng/mg Tissue Dry Weight) 

Leaf 

V9b

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 

V9b

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 protein deposited into soil by Bt crops is quickly degraded, 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 place 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 under a range of conditions typical of Bt crop cultivation are 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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needed to yield relevant data on persistence and natural variation. Limited data do not indicate 

that Cry proteins have any measurable effect on microbial populations in the soil. Horizontal 

transfer from transgenic plants to soil bacteria has not been demonstrated. Current studies of Bt

in soil show no effect on bacteria, actinomyces, fungi, protozoa, algae, nematodes, 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 transgenic crops through roots, 

incorporation of above ground plant tissues into soil after harvest, or by pollen deposited on the 

soil. Root exposure may occur by feeding on living or dead roots or, theoretically, by ingestion 

or absorption after secretion of δ-endotoxin into the soil. The latter 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 suggests 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 eliminating 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 conditional registrations. In addition, a number of 

publications in the scientific literature have addressed 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 DT50 (time to 50% degradation) studies were 

submitted for registration of corn expressing Cry1Ab and Cry1F proteins, and published studies 

are available for Cry1Ac cotton. Cry1Ab biodegraded in an estimated DT50 of 1.6 days as 

expressed in transgenic corn tissue 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 DT50 of approximately 3.13 days. 

Data submitted by Monsanto for Cry1Ac protein and transgenic Cry1Ac in cotton report DT50’s 

of approximately 9-20 days for the purified protein and 41 days for the protein in cotton tissue 

(MRID No. 439995-09). Published data for Cry1Ab andy1Ac in cotton tissue or as purified 

protein report a DT50 range of 2.2 to 46 days, where measurable (in 4 of 11 experiments); DT50’s 

for transgenic tissue were shorter than for purified protein in two of three experiments (Palm et 

al. 1994). DT50’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 field 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 Stotzky 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 (Stotzky 2000b). This protection is not absolute, however, since 

degradation has occurred under several experimental conditions. Several factors influence either 

the affinity of binding or the rate of degradation. In particular, pH near neutral generally 

increases degradation substantially. At pH above 5.8 to near neutral, degradation of Cry protein 

bound to clay minerals in soil was much faster than degradation at pH 4.9-5.0 (Tapp and Stotzky 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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1998). For example, it was found that Cry toxin added to nonsterile soil containing kaolinite or 

montmorillonite showed little degradation even after around six months at lower pH (pH~5), 

while substantial degradation occurred 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. Therefore, under most production conditions, corn would 

not be grown on soils that would inhibit the rate of degradation compared to what is seen at near 

neutral pH. 

Tapp and Stotzky (1998) proposed that the more rapid degradation of Cry proteins at near neutral 

pH was due to a greater amount of microbial activity. The authors pointed out that even at pH 

near neutral, protein toxin activity (lethal concentrations against a sensitive bioassay) remained 

after six months and they interpreted these data as evidence of prolonged persistence of Cry 

protein in the soil. In these experiments, substantial degradation (loss of biological activity) 

typically occurred rapidly in the first several weeks, with much slower subsequent breakdown 

(Tapp and Stotzky 1998). A similar pattern was observed in some experiments performed by 

others over a range of Cry1Ab and Cry1Ac protein 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 examining persistence of Cry proteins reported 

in the published literature have apparently been conducted in bulk soils or soil components. 

Bulk soil generally does not support populations of microorganisms as high as in the rhizosphere 

or in soils where plant residues are incorporated. Research suggests typical ratios of 5-20 for 

rhizosphere to bulk soil microbes, 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 DT50

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 truncated Cry1Ab or Cry1Ac proteins when 

incorporated with cotton crop residues more often than when purified protein was used. In these 

microcosm experiments, toxin DT50s, where possible to determine, varied greatly from 2.2 to 46 

days (microbial populations in these previously dried, re-hydrated, soils were not determined). 

These experiments relied on ELISA of extracted Cry proteins to quantify residual Cry protein in 

the soil, so it is not clear what fraction of the extracted protein may have retained biological 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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activity. The extraction efficiency was reported to be about 27-60%, with lower efficiency 

corresponding to higher clay and organic content, and it is unclear whether un-extracted protein 

degraded at a similar rate as the extracted protein 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 more resistant to extraction and 

degradation while retaining biological activity. 

 

Some experiments that show relatively long persistence of Cry proteins in the soil do not 

consider rates of degradation, reporting 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 soil and approximately 

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

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

to be at the PPB to low PPM levels. In addition, the bioassays or immunoassays 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 103

 to 105 may be required to reduce 

the amounts of Cry protein added to soil below detectable levels. Therefore, in order to predict 

persistence under field conditions it is important to know starting concentrations as well as 

degradation rates. Other experiments discussed above used amounts of Cry protein more 

representative of many plant tissues in Bt plants. 

To summarize, available data suggests that the DT50 of Cry1Ab or Cry1Ac proteins, incorporated 

in corn plant residues or as free toxin in non-sterile soil, typically range from approximately 1.6-

22 days but have been measured to be as long as 46 days and data showed DT50 for Cry1F to be 

3.13 days. As suggested by Palm et al. (1996), DT50 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 rhizosphere secretions, and 

near-neutral pH, will favor shorter DT50’s. Binding of Cry protein 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 these 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 residual amount may persist in biologically active form for a much 

longer period of time. 

It is important to consider a number of factors expected to influence persistence under actual 

field conditions: humic acid and clay content of the soil; clay type; pH; moisture; soluble ion 

content and type; and temperature. Varying conditions can affect microbial activity, 

composition, and population levels, along with binding affinity of Cry proteins for soil 

components. Persistence of Cry proteins could therefore vary considerably. The conditions 

examined by the registrants, however, generally replicate common field soil conditions, although 

performed in a laboratory setting. Field tests of Cry protein degradation 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 

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ii. Secretion of Cry Proteins by Plant Roots

Saxena et al. (1999) reported that Cry protein 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 μg per plant, as determined by the 

Lowry method. Although it is possible that the observed Cry1Ab protein was the result of small 

pieces of plant tissue (e.g. root cells or root hairs), it is unlikely since the Hoagland’s solution 

was centrifuged, which removed cellular debris (Stotzky 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. Nonetheless, the procedure used to isolate 

the Cry protein from soil, including vortexing in extraction buffer prior to centrifugation, could 

have ruptured or lysed cellular material.8

 In addition, small root material is difficult to separate 

from the rhizosphere soil used in the subsequent 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 extraction of plant cellular 

proteins and/or microbial proteins, possibly in addition to exuded or secreted proteins. SDSPAGE gels of extracted proteins from the sterile soil treatment were not performed (Stotzky 

2000d), so such data may have been expected to result in a protein profile similar to that from the 

Hoagland’s solution if plant cellular material was not included. 

While the data generally support the deposition of Cry protein 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 may not have been exuded in the 

soil experiments, and may be an artifact of growth in Hoagland’s solution experiment. This 

distinction is important, because it cannot currently be ruled out that a substantial portion of the 

insecticidal or immunological activity 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 region 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 DT50

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 intended, 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 nonsterile 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 after incorporation of plant residues. Some 

proteins may be secreted from the roots of plants in significant amounts by an active export 

process. These amounts may be much higher than the incidental 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 implications 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 and Chrispeels 1992). Cry proteins 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 sequence has been identified in these proteins 

(Kostichka et al. 1996). A bacterial secretion 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, including those registered, form intracellular inclusions of Cry 

protein, and are not secreted. Other transgenic cytoplasmic proteins have been shown to be 

efficiently secreted only when a known ER signal peptide sequence is specifically added, 

otherwise these proteins remain 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 with plant tissue after 

several days growth (Borisjuk et al. 1999). In Bt11 corn roots, Cry1Ab is expressed at an 

average of 20.2 μg/g total root protein. It is difficult to predict whether this level of root 

expression of Cry1Ab is consistent with active 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 NK4640Bt, which 

correspond to Syngenta Bt11 corn lines. This variety of corn contains a modified cry1Ab gene. 

Previous soil fate data supplied by the registrant was from experiments performed with 

nonsterile soil in the laboratory and consisted of replicated single incorporations of transgenic 

plant material or purified Cry protein. All other studies of Cry protein 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 subsequent monitoring of residual 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 lesser extent due to pollen shed. 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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Degradation studies were performed by Monsanto, registrant of MON 810 corn, which also 

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

1.6 d DT50 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 expected to have a significantly increased surface 

area compared to crop residue incorporated at the 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 bioactivity of corn tissue incubated without soil decreased 

with an estimated DT50 of 25.6 days, and a DT90 of 40.7 days. After one week in soil, 

approximately 1% and 10% of the original levels of B.t.k. protein remained in leaf and stalk 

tissue, respectively. After three weeks, B.t.k. protein was still detected in the stalk tissue, but the 

level in transgenic leaves was similar to the background levels seen in control leaf tissue. B.t.k. 

protein apparently binds to soil particles, making quantitative extraction difficult. Biological 

activity, assessed by European corn borer bioassay, is reduced to control levels after three weeks 

of incubation in soil (MRID No. 436960-01). 

Degradation of Cry1F in the soil was demonstrated in the study submitted in support of the 

poCry1F registration (MRID No. 450201-05). Based on a bioassay with the tobacco budworm 

(Heliothis virescens), a target species, purified Cry1F proteins incorporated into test soils 

biodegraded with a half-life of approximately 3.13 days. The amount 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 environmental 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 poCry1F plants at the R1 stage 

contain 3.6 ng of Cry1F/mg of tissue. Therefore, we can roughly approximate that the EEC for 

moCry1F at the R1 stage would be 34.5 micrograms/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 felt 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, adequately address the persistence of 

Cry proteins from Bt crops in the soil. Since it is difficult to correlate the relevance of the 

published laboratory data to field situations, the SAP recommended field studies be conducted in 

established Bt fields in a variety of soil types and climatic conditions. The SAP suggested an 

investigation of amount, accumulation and persistence of biological activity of Cry proteins in 

the soil. The SAP also concluded, however, that this data was not necessary for an EPA 

preliminary risk assessment but may be needed for a final assessment. In general, the Panel 

believed that studies on the mechanism of how Cry proteins enter the soil (e.g., secretion, 

shedding of root hairs, and degradation of biomass pollen) were primarily of academic interest, 

whereas knowledge of the potential environmental impacts was important 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 regarding Cry protein degradation in soil. 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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The Agricultural Biotechnology Stewardship Technical Committee (ABSTC) submitted a 

research protocol for a Bt Cry protein soil accumulation study 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 response to the Agency’s letter, including a 

revised protocol, on June 5, 2002, which was accepted 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 Persistence of Cry1Ab (MRID No. 460224-01) 

This study, which fulfills the soil fate data requirement 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 ABSTC. Bioassay results indicated that ECB 

larvae fed a diet containing 15% Bt corn soil exhibited no toxic response to the diet mixture, as 

measured through growth inhibition of test larvae (LOD 0.03 μg/g soil). Based on these findings, 

the study author concluded that Cry1Ab protein does not accumulate and persist in soil. 

However, Cry1Ab protein may have been present in test soil at concentrations 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 larvae, 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 biological activity observed in the soil of Herculex I corn, 

demonstrating Cry1F protein rapidly degrades in various soil matrices 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 field 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 protein 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 evolved from the decomposition of Bt-corn 

leaves for the meso- and macrofauna soil community (Hönemann et al. 2008). Although a 

minimal transient increase and shift in microbial populations may result from the presence of 

transgenic plant tissue in soil, no adverse effects have been attributed to the Cry protein. 

In addition, there are several ongoing U.S. Department of Agriculture and EPA Office of 

Research and Development funded research projects evaluating 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 

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With regard to the impact of genetically engineered 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 seasons and weather, plant growth stage, and plant 

varieties, independent of being genetically engineered, are also responsible for significant shifts 

in soil microbial communities. To date, most studies with genetically engineered crops have 

shown minor or no effects on soil microbes beyond the variation caused by the factors listed 

above. 

3. Non Target Wildlife Hazard Assessment 

Prior to registration of the first Bt plant-incorporated protectants in 1995, EPA conducted 

ecological risk assessments for all Bt Cry proteins expressed in potato, corn, and cotton. EPA 

evaluated studies of potential effects on a wide variety of non-target 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, parasitic, and pollinating insects. These risk 

assessments demonstrated that Bt Cry proteins expressed in transgenic 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, the Agency did not believe that non-target Lepidoptera would 

be exposed to sufficient amounts of Bt protein to cause an unreasonable 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 concluded 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 data voluntarily submitted to EPA by the registrants 

also shows minimal to undetectable changes in the abundance of beneficial and other non-target 

insect populations. In some cases the densities of predatory and non-target 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 effects concerns from commercialization of Bt crops the Agency 

has reviewed new and existing data regarding non-target wildlife effects for Bt crops with a 

special emphasis on Lepidoptera and monarch butterflies and reevaluated the sufficiency of data 

to support continued registration of Bt crops. 

Although Bt Cry proteins are very specific in their activity to certain insect species, EPA has 

examined the toxicity of the Cry proteins to birds, fish, honeybees and certain other beneficial 

insects. Because of the extensive scientific literature 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, therefore, did not require lepidopteran toxicity data. 

EPA nevertheless required data on Collembola (springtail) and earthworm species to ascertain 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

59 

effects on beneficial soil invertebrates 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 non-targets that may be 

affected by the Bt pollen, specifically the monarch butterfly, were conducted in 1999, 2000 and 

2001-2002 by the USDA, ABSTC and university scientists 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 there 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 once 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 chemicals that are transferred through the food chain. 

Therefore, chronic effects testing of protein 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 

LC50 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 LC50 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 corn or N7070Bt corn that had been 

sprayed with Liberty herbicide produced growth, feed conversion rates, 

carcass yields, and survival rates similar to diets prepared with non456521-01 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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OCSPP 

Guideline No. Study Results MRID No. 

transgenic corn. Birds fed N7070, N7070Bt, or N7070Bt-Liberty corn 

had similar body weights and feed conversion 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 x 

Roundup 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 pens (5 pens of 10 males each and 

5 pens of 10 females each), for a total of 100 birds/treatment (800 birds 

overall). Mortality was observed throughout the study, and at test end 

survivors were examined for performance, carcass yield, and meat 

composition. Most of these mortalities were apparently due to sudden 

death syndrome and ascites. No mortalities were determined to be 

related to the treatment diet, and no other adverse effects were observed. 

There were no biologically significant 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 differences 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) relative 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 similar 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 pollen/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 Event 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 mortalities were observed at any of the 

treatment levels tested. The 48-hour EC50 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 expected 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 significantly different between fish fed the 

control MON 800 diet when compared to those fed the diet containing 

438879-01 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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OCSPP 

Guideline No. Study Results MRID No. 

801 as a Feed Ingredient 

for Catfish. 

transgenic corn from the test line 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 disappears when one expresses the results 

on a wet weight basis. Data in this study are consistent with historical 

controls for catfish grown at the Delta Research and Extension Center. 

Although this study utilized Monsanto's Bt Cry1Ab corn for testing, the 

test material was considered sufficiently 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 LD50 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 sponsor, 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 characterizing effects of Cry1Ab corn tissue 

on Collembola and satisfies the requirement 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 Subcommittee (NTO Subcommittee) of the 

Agricultural Biotechnology Stewardship 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 design and non-target invertebrate 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 are 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 using 

Cry1A(b)-expressing hybrids (events BT11 and MON810) and nontransgenic corn in Maryland, 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 development in the first and 

second bioassays were 163 and 170 grains/cm2, respectively. Pollen 

density on milkweed plants in the non-Bt, BT11, and MON810 hybrids 

averaged 163, 155, and 174 grains/cm2, respectively, during the first 

bioassay and 172, 173, and 158 grains/cm2, 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 

461620-01 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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OCSPP 

Guideline No. Study Results MRID No. 

on the leaves. The study included 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 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 LC50 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 Toxicity 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 moCry1F protein is considered likely; 

however, the mammalian toxicology data submitted for the Human 

Health Assessment for poCry1F indicates that there was no significant 

toxicity to rodents from acute oral testing at the maximum hazard dose. 

Based on the bridging data in combination 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 LC50 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 assessment 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. Therefore, 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 Bt Cry1F 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 change observed between the dosed and 

control replicates. LC50 > 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 expressed in corn pollen should have no 

detectable adverse effects on honey bee 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 mortality or signs of toxicity or 

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

The LC50 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 

63 

OCSPP 

Guideline No. 

Study Results MRID No. 

(the test concentration). Mortality and pupation rate were comparable 

between the treatment and control group. 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 signs of toxicity over a 29 day period. 

Therefore, the NOEC and the LC50 were determined to be >15x the 

concentration of Cry1F found in pollen determined to be > 480 ppm a.i 

(the test concentration). The test insects were exposed to a dose of active 

ingredient approximating the amount that would be ingested by the 

beetles feeding on aphids under field conditions. As a result, no 

discernible beneficial beetle population 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 the 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 LC50 were 

determined to be > 320 ppm a.i (10x field 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 seven days of feeding. There was some 

growth inhibition at 10,000 ng/mL diet. LC50 > 10,000 ng/mL and NOEC 

<10,000 ng/mL. Since doses equivalent 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 levels of Cry1F that might occur in the 

field are not expected to adversely effect 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 following 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 transgenic 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 six weeks. In addition, ten plants in 

the center row were visually evaluated for beneficial arthropods weekly 

for six weeks. Beneficial insects 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 spiders (Arachnida). Data included 

counts of adult and larval lady beetles and lacewings when appreciable 

numbers were collected. Results from 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 concerns for Cry1F protein expressed in 

corn to non-target insect populations. However, the Agency 

450201-13 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

64 

OCSPP 

Guideline No. 

Study Results MRID No. 

recommended that the monitoring continue into the first few years of 

commercial use of Cry1F corn crops in order to confirm the single 

season Ano 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 Cry1F expression in pollen, milkweed 

distribution and biomass from the edge of the field, pollen grain physical 

properties, and spatial-temporal availability 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 LC50 of ∃0.06 Φg g-1 which is 290-fold lower than the 

geometric mean LC50 (12.4 Φg 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 Φg g-1 diet. When fed up to 10 Φg g-1 Cry1F microbial toxin in diet, 

neonate monarch larvae were not affected. The toxicity threshold, or no 

effect level for monarch neonates, for the Tier 1 risk assessment was 

determined to be 10 Φg g-1. The log-probability plot of the Bt LC50 for 

lepidopteran species shows that the EEC does not exceed the LC50 for 

98% of the intergenera population beyond 1 m from the field edge. The 

LC50 is not exceeded for 90% of the population 0.2 m from the edge. For 

monarch larvae, the no effect level (10 Φg g-1) occurs near the 50th

percentile intergenera LC50. Since there is a rapid fall-off in exposure 

with distance, there is limited potential 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 exposure 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 LD50 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 product because of very low or no 

potential for exposure. 

None 

assigned 

c. Non Target Wildlife Risk Characterization 

i. Terrestrial Wildlife 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

65 

1) Mammalian Wildlife 

The human health data submitted to EPA indicate that 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 bridging data summarized in Section 2.b. of this 

assessment (Table 3), also indicate that no hazard to mammalian wildlife is anticipated from 

moCry1F. In addition, there are reports of no adverse 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 gathered 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 bobwhite quail after 14 days. A study with a non-commercial line of 

MON 80187 showed no mortality or differences in food consumption, body weight, or behavior 

when bobwhite quail were fed 50,000 or 100,000 ppm 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 industry after several years of using Bt corn in poultry feeds. 

The dietary LC50 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). The No Observed Effect Concentration (NOEC) 

was also 100,000 ppm. The study is scientifically sound and no treatment 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 sufficient 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 broiler chicks, diets prepared with transgenic 

N7070Bt corn or N7070Bt corn that had been sprayed with Liberty herbicide produced growth, 

feed conversion rates, carcass yields, and survival 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 mortality 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 differences in mortality by sex during the grower phase 

(days 21-35), but these were not seen on a cumulative basis (days 0-42), and were attributed to 

chance. Birds fed N7070, N7070Bt, or N7070Bt-Liberty corn had similar body weights and feed 

conversion ratios throughout the test. Birds fed NC2000 corn had significantly decreased 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

66 

weights for each portion of the study, and a significantly decreased adjusted feed conversion 

ratio for the starter (days 0-21), finishing (days 35-42), and cumulative (days 0-42) periods. The 

study author attributed these decreases to an interaction between the climatic conditions and the 

NC2000 diet formulation, which was more hygroscopic than the others, thus affecting the flow 

of the feed into the feeder pan. Evidence of restricted feed flow was seen in two pens during the 

finishing period, and these were excluded from the statistical analysis for feed conversion ratio. 

There were no significant differences 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 quality 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 performance of the N7070-series corns 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 addition, YieldGard x Roundup Ready expresses 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 parental lines, as well as to other non-transgenic 

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 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 per pen. Mortality was 

observed throughout the study, and at test end survivors were examined for performance, carcass 

yield, and meat composition. Chick mortality was as expected during the 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 different corn lines. Most of these mortalities were 

apparently due to sudden death syndrome and ascites. No mortalities were determined to be 

related to the treatment diet, and no other adverse 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 results, the two Cry1Ab corn lines tested do not 

produce adverse effects in broiler chickens through dietary exposure. 

b) Cry1F (MRID No. 456220-01) 

The registrant submitted a six-week study as required by the EPA. Two hundred forty-five male 

broiler chickens (Cobb x Cobb) were fed diets containing 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 each) 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 mortality, 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 initial bobwhite quail study submitted in 2001, is 

sufficient to demonstrate that there should be no discernible detrimental 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) species 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 protein. 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 hectare watershed planted with corn) was used to 

develop a worst case EEC for Cry1Ab and Cry1F proteins (high protein expression level) on the 

basis of corn pollen loadings from airborne pollen deposition and agricultural runoff. Airborne 

pollen deposition will result in water concentrations less than 78 ng Cry1Ab protein/L when 

based on conservative estimates for pollen dispersal. 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 protein/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 agricultural runoff is <0.15 

ng/mL. Thus, total water concentration of 1.4 ng Cry1F protein/L is projected 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 EC50 was 

>100 mg/L in one study and 150 mg/L in another. 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 containing 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 registration; moCry1F pollen contains less Cry1F 

protein (3.67 ng/mg) as compared to poCry1F pollen, which contains 21.9 ng/mg of Cry1F 

protein (dry weight tissue; Zabik et. al 2003). 

In light of recently published laboratory studies showing reduced 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 of 
Cry1Ab 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 shredder in headwater streams, can be performed and 

submitted in lieu of the Daphnia study. 

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

magna because corn pollen is thought to be too large for ingestion by these filter feeders 

(EcoStrat, 2000). However, there is some observational 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 untreated controls. Also, if the pollen 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 renewal 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 Cry proteins do not adversely affect susceptible 

target insect larvae, as bioassay testing and analyses using ELISA indicate that Cry protein is not 

detectable in the fish feed samples. Therefore, farm fish eating a food 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). Juvenile rainbow trout 

(Onchorhynchus mykiss) were fed a standard fish diet containing 100 mg Cry1F ICP a.i./kg (100 

ppm) of diet for eight days with no mortality or sublethal effects. The LD50 was determined to be 

greater than 100 mg a.i./kg of diet. The study is considered supplemental, however, because the 

actual concentration of the test material 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 protein expression in poCry1F corn pollen (21.9 
Cry1Ab 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 moCry1F corn does have higher Cry1F 

protein expression than poCry1F corn in plant tissues most likely to be exposed to the soil 

dwelling Collembola and earthworms, the Collembola 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 suggests that the proposed uses of 

Cry1F protein in corn are not likely to have any measurable population effects on soil 

invertebrates including Collembola 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 proteins were actually exposed to the proteins. The pollen has to be pre-digested 

by nurse bees (which, conversely, may also inactivate the Cry protein) in order 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 purified 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. Three replicate tents (1 colony/tent) containing Bt

corn and three replicate tents containing 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, behavior or brood development during the 7-day 

period of pollen shed. Following the pollen exposure period, the hives were removed from the 

tents and observed for an additional 30 days for effects on brood development. No effects on 

brood development were associated with field exposure to Bt Cry1Ab corn pollen. 

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

70 

The reviewed capped honey bee brood cell study where larvae were fed Cry 1F corn pollen and 

pure Cry1F protein showed normal larval development 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 likely to have any measurable deleterious effects on 

the honey bee (Apis mellifera). The data showed no significant difference between treatment 

mortality or behavior change between the dosed and control replicates. As a result, no discernible 

detrimental effects to honey bees are expected from the proposed uses of the Cry1F-producing 

corn. The data adequately address potential toxicity concerns for 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 corn pollen containing the anti-lepidopteran Cry proteins do not 

cause detectable adverse effects to lady beetles. Purified Cry1Ab protein at 20 ppm also showed 

no adverse effects or behavior changes. The test insects were exposed 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 LC50 was determined to be > 480 ppm a.i (the test concentration). The submitted 

study shows that corn containing the Cry1F protein should not cause significant adverse effects 

to lady bird beetle predators. The test insects were exposed to a dose of active ingredient 

approximating the amount that would be ingested by the beetles feeding on aphids under field 

conditions. As a result, no discernible 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 maximum 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) Cry1F
Cry1Ab 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 LC50 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 wasp abundance found in a field 

census study submitted with the application. 

 

4) Green Lacewing

a) Cry1Ab

The studies submitted to support the initial registration 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 16.7 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 several publications proposing that transgenic Bt plants may 

create serious impacts on non-target organisms that feed on pests exposed to the transgenic 

proteins. The reported harmful effects of Bt corn on larvae of the beneficial 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 prolonged 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 control” 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 significant 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). Therefore, this study does not 

demonstrate any adverse effects to lacewing larvae under simulated field feeding habits where 

the lacewing larvae have a choice of natural diet in the field. 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 therefore not reflective of Cry1Ab exposures 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. 
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In a tritrophic study published 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 detrimental 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 amounts. 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 “...trials investigating predation efficiency and predator performance 

under field conditions are necessary before conclusions 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 density 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 mortality 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 LC50 was determined to be > 480 ppm a.i (the test concentration). These 

laboratory findings do not show significant detrimental effects and provide data that show a lack 

of risk to beneficial insects at Cry1F levels that will be encountered 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 adult 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 for 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 considered requiring studies evaluating effects upon 

the representative beneficial soil invertebrates 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 residues are left on the soil surface) and (2) that 

adverse effects on such soil organisms could result in an accumulation of plant 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 have adverse effects on soil organisms, but there 
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has not been an accumulation of significant amounts 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 any increased build up of plant detritus 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. Therefore, significant soil buildup and effects to 

non-target soil organisms are not anticipated. This has been confirmed 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, protozoa, algae, bacteria, actinomyces and fungi 

in soil, in spite of the fact that enough detectable Cry protein is bound to soil particles to show 

toxicity to the target pest. These 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 environment. Stotzky (2000) also reported that the 

same degree of Bt Cry protein persistence takes place in soils 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 expected environmental concentration. 

Concerns have been raised as to whether the earthworms actually ingested the Bt Cry proteins 

when these are incorporated 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 harmed when presented with the Bt Cry proteins in their soil 

environment. If they do not ingest it in the test soil, likewise they will not ingest it in the field. 

The earthworms do, however, ingest the Bt Cry proteins with the soil without harmful effects. 

Saxena and Stotzky (2001) reported that there were no significant differences in the percent 

mortality and weight of earthworms after 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 LC50 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. 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 Advisory Panel (USEPA, 2001) that invertebrates 

known not to be affected by the Cry proteins specific for insects not be tested, no additional 

follow-up of this study is required. 

ii) Cry1F
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The submitted data show that Cry1F protein has no measurable deleterious effects on 

earthworms, a representative beneficial soil invertebrate species. This suggests that the proposed 

uses of the Cry1F protein in corn are not likely to have any measurable population effects on 

beneficial soil invertebrates. The one limit test concentration of 2.26 mg Cry1F/kg dry soil 

represented more than 100X the estimated concentration present in the top six inches of an acre 

of soil following the incorporation of 25,000 senescent corn plants. This concentration is higher 

than any amount of Cry protein that may be present in the soil during any stage of the growing 

season (such as from root exudation). Based on the results of this study, Cry1F transgenic corn 

plantings will have no adverse effects on earthworms.

b) Collembola 

i) Cry1Ab

Monsanto’s original application for registration included a study on Collembola exposed to 200 

ppm of Cry1Ab proteins 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 corn line. 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. Test substances included corn powder at 0.5, 5.0, and 50% of the diet. 

Mortality was assessed every 7 days for the duration 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 difference in reproduction between the treated group and either control 

group. The study was scientifically sound and no treatment mortality or behavior change was 

observed between the dosed and control replicates. The study also showed that 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. Novartis (now Sygenta) had cited the MON 810 

leaf tissue study to support 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 corn, it does not adequately characterize the effect of Bt11 

corn tissue on Collembola. The requirement for a Collembola study which includes control plant 

lyophilized leaf tissue from non-transgenic parental corn lines and lyophilized leaf tissue 

containing the Bt11 plant-incorporated protectant is not fulfilled. However, in light of recent 

recommendations by the FIFRA Scientific Advisory 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) Cry1F
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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 scientifically sound and no treatment 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 expected to be found in the 

field, collembola were not affected by chronic exposure to Cry1F protein. The exposure rates in 

this study are 1560-, 388-, and 79-fold higher than 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 protein 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 valid data demonstrating 

specific adverse impacts of plants expressing Bt Cry1 proteins on beneficial soil invertebrates. 

To the contrary, EPA believes that available scientific data and information indicate that 

cultivation of Bt crops has a positive effect on soil flora, when compared to the most likely 

alternative, use of non-selective synthetic chemical pesticides. 

 

6) Non-Target Insect Abundance Studies 

Data available to date indicate no difference in the number of total insects 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 studies in two consecutive years with Bt corn. 

No differences were observed in the number of predators colonizing 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 European Corn Borer (Lepidoptera: Pyralidae) 

and impact of natural enemy populations in transgenic 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 factors. Larval parasitism was not significantly 

different and therefore probably density-independent. 

 

Obrycki (1997) performed a study 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 significantly different between the sprayed and unsprayed plots on 

four of the five observation days over a seven week period. Conventional pesticide spray drift 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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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 numbers 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 statistically significant levels. 

Lozzia (1999) reported a biodiversity and structure of ground beetle 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 period. No statistical difference was evident in the total number of 

carabids. There was no decreasing trend in the biodiversity 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 indices was not due to the presence of transgenic corn. The aerial 

fauna as a whole for both years and both localities was not different. Similarly, abundance of 

aphids, leaf hoppers, other Homoptera, thrips, leaf beetles, spiders, lady beetles, parasitic 

Hymenoptera, other Hymenoptera, and 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 significant difference in abundance, composition or 

biodiversity of non target arthropods 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 directly affect the phytophagous species nor do they have “any 

indirect influence on other trophic levels or activities such as behavior, oviposition or predatorsprey.” 

Nuessly & Hentz (1999) conducted 4 studies using Novartis (now Sygenta) Seeds' Attribute 

Bt11-derived Cry1Ab sweet corn hybrids and conventional sweet corn hybrids grown under local 

practices in four Florida locations. Noted in the reports were increases in species diversity in the 

corn plots, i.e. there were generally higher populations of beneficial and non-target insects as 

compared to the conventional control plots, associated with the significantly decreased use of 

broad-spectrum insecticides (organophosphates, carbamates and synthetic 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 non-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 treated with foliar 

insecticide (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 arthropod 
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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 extended the registrations of these 

products until October 2008. As a condition of these registrations, EPA required the registrants 

to either submit existing studies submit a protocol for new field survey studies. 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 Subcommittee (NTO Subcommittee) of the 

Agricultural Biotechnology Stewardship 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 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 transgenic corn, including discussions of study design 

and non-target invertebrate populations. EPA reviewed the submitted information and, 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 are 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 those 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 abundance of non-target beneficial 

organisms in a population in the field, whether they are pest parasites, pest predators, or 

pollinators. Published field testing results and 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 specific orders between the 

transgenic crop plots and either the isogenic or wild type control crops when these are grown 

without chemical pesticide treatment. In commercial 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 are 
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dependent on the pest insect as prey. In contrast, 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 available 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. However, 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 exclusively 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 irreversible 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 levels in the field. In light of 

the recent reports expressing concern for monarch conservation efforts, however, this conclusion 

has been reevaluated and much research effort has been devoted to this issue. 

The weight of evidence of the published and recent research data 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 pollen within and outside of corn fields, the 

distribution of milkweeds within corn habitat and other 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 new data indicate unanticipated risks to 

other non-target Lepidoptera, particularly 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 toxicity of Cry1 proteins to non-target Lepidoptera species. 

a) Black Swallowtail Butterfly

Wraight et al. (2000) performed a field study to assess whether 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 intervals from the edge of a field of Bt corn (MON 810). There was 

no relationship between mortality and proximity to the field or pollen deposition on host plants. 
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Moreover, pollen from these same plants failed to cause mortality in the laboratory at the highest 

pollen dose tested (10,000 grains/cm2), a level that far exceeded the highest pollen density 

observed in the field (200 grains/cm2). The authors conclude that Bt pollen of the variety tested 

is unlikely to affect wild populations of black swallowtails. 

b) Karner Blue Butterfly and Other Threatened or Endangered Species

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

from Bt corn fields might potentially have adverse effects on federally listed threatened or 

endangered Lepidoptera because of the selectivity of Cry1 proteins for certain lepidopteran 

species. EPA noted, however, that the majority of listed lepidopteran species have very 

restricted habitat ranges. Examination of an overlay map showing 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 lepidopteran 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 potential concern regarding range 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 wild lupine (its host plant), where there are 

prolonged periods of winter snow pack, primarily in parts of Wisconsin, Michigan, Minnesota, 

Indiana, New Hampshire and New York. Wild lupine grows on dry, sandy soils in pine barrens, 

oak savannah, forest trails and previously disturbed habitats such as utility rights-of-way, 

military installations, airports, highway corridors, 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 against 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. Previous studies that tested the 

susceptibility of lepidopterans to Cry1 proteins resulted in different LC50 values for different 

species in the same genus. For example the Cry1Ab LC50 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 demonstrating 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 may 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 Cry1 proteins. Since the 

gypsy moth is known to be less susceptible to Cry1Ab protein than European corn borers, and 

levels of Bt pollen found in the field are not toxic to European corn borers (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 endangered 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 to 
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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 Cry1F 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 Karner 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 transport 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 sensitive 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 endangered species, no effect is expected if there is 

a safety factor of 10X between the estimated environment concentration (EEC) of the pesticide 

and the LC50 or LD50 to the most sensitive species tested (USEPA, 1986). As described below, 

EPA has determined the ratio between the EEC and LD50 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 lepidopteran species have been tested to determine LC50

levels for Cry1Ab (MRID 455122-00). The most sensitive species tested is the monarch 

butterfly. Researchers have determined that the concentration producing no mortality 

whatsoever is greater than (>) 4000 pollen grains/cm2

 of leaf surface (Hellmich, et al, 2001). 

Thus the actual LD50 for monarchs is likely to be substantially higher. Since the EEC is 300 

pollen grains /cm2 or less at the field edge, and 200 at one meter and 75 at three meters,9 the 

ratios of the EEC/LC50 (with >4000 pollen/cm2

 as that LC50) have been conservatively 

calculated to be 1:>13.3 for Cry1Ab 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 species have been tested 

to determine LC50 levels ( Wolt, 2000). The most sensitive species tested is the diamondback 

moth. Because no data were submitted on toxicity of Cry1F in corn pollen, the LC50 obtained on 

the diamondback moth with pure cry1F protein was converted to an LC50 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/cm2

 (of leaf tissue) level in the field. The ratios of the EEC/LC50 have 

 

9

 The data collected for the DCI provide a deposition curve of pollen distribution outside a corn field. A conservative estimate 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 off rapidly within a 

few meters of the corn field edge (Pleasants, et al, 2001). 
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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 taken 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 lupines. When EPA initially began the Bt reassessment, the 

Agency was concerned that lupines occurring adjacent to such reestablished corn fields could 

potentially contain Karner blue larvae that could possibly be adversely affected by pollen shed 

from Bt corn. EPA has received information that indicates 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 Conservation Plan and Environmental Impact 

Statement, Wisconsin Department of Natural Resources (2000) 

(http://www.dnr.state.wi.us/org/land/er/publications/karner/karner.htm). Moreover, while EPA 

received a comment to the effect that “[t]he Karner 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 data 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 paper does state is that: “the Karner blue [is] known to occur in 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 wild lupine (Lupinus perennis Fabaceae) in sandy soils intercalated in the oak 

savannah habitat near the Mississippi River 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 agree that any of the quotations identified in the Andow paper is supportive of the 

statement that “[t]he Karner Blue is documented as occurring adjacent to corn fields.” (Emphasis supplied). Of greater interest 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 five Karner blue habitats in 

the area are only 0.5-0.9 km (mean 0.66 km) from the nearest agricultural 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 the potential risk from releases of T. nubilale is not negligible. 

Id. at 111-12. Thus, “intensive surveys” by Andow of the Karner blue in Minnesota demonstrates that the five Karner blue populations identified 

exist from 500 to 900 meters from the nearest agricultural lands (with a mean distance of 660 meters). Given that EPA considers 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 meters from the field, 

EPA does not consider these data to support the proposition that the Andow paper “documents” that the Karner blue “occur[s] adjacent to corn 

fields.” 
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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 for more than a day or two in the life of the 

feeding larvae. For example, these data show that in some instances 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 shown that rain and wind 

remove large amounts of pollen. Bt protein in corn pollen also degrades 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 the 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 ecological 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 determination is based on a number of factors including (1) if wild lupine 

were to grow adjacent to Bt corn fields, the amount of corn pollen 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 will be relatively little, if any, wild lupine growing in the areas 

immediately adjacent to corn fields that are reestablished from fallow fields; (3) the amount of 

corn pollen shed from corn fields to adjacent areas is low; (4) available data suggest that there 

may be limited overlap between the period of pollen 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, ongoing research, and endangered and threatened 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 indirect effects on endangered and threatened plant 

species, such as impacts on lepidopteran pollinators 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 hawk moth. While some hawk moths might be 

found in and around corn fields, they feed and oviposit 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 discussed 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 submitted two research reports (DP Barcode D255949) to EPA on 

potential effects of Bt corn pollen on monarch 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 over 
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milkweed leaves (Asclepias syriaca Linnaeus) which had been lightly misted with water. Pollen 

density was set to visually match densities on milkweed 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) covered leaf samples taken from within 

and at the edge of corn fields (80-217 pollen grains/cm2) to assess mortality. The samples were 

fed under laboratory conditions to monarch butterfly first instar larvae. The authors found a 19% 

mortality in larvae feeding on the Bt corn pollen treatment from leaves 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 butterflies without additional field study 

information. The reports of Bt corn pollen toxicity to monarch 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 expressing an insecticidal protein from Bacillus thuringiensis (Bt corn) 

on non-target lepidopteran species, particularly monarch butterflies (Danaus plexippus). To help 

identify the level of exposure and other risks 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 recommendations. 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 data and information were available about the 

potential risks of Bt corn pollen to monarch butterflies and other lepidopterans, EPA requested 

that registrants instruct their 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 areas relating to the potential exposure of non-target 

lepidopterans, particularly monarchs to Bt corn pollen. These include: the distribution of 

monarch butterflies, milkweed plants and corn; corn pollen release 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 population monitoring. The Agency has reviewed the 

submitted DCI data and incorporated the findings into this reassessment. The DCI data is a 
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result of an ABSTC and USDA coordinated research effort, with additional research by 

independent university and government workers. 

Subchronic toxicity studies conducted since the DCI was initiated 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 observable 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, adult weight (mg), or adult wing length) (StanleyHorn et al. 2001). Risk from other factors such as destruction of over wintering habitat, weather, 

predators, physiological stress, human activity (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 monarch 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 occur under natural field conditions. 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 densities monitored in the field were 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 that the order of monarch sensitivity 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 monarch larvae for 14 to 22 days also needed 

further analysis. In addition, the authors also noted that these studies did 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 included 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 the University of Maryland (Dively, et al. 2004) conducted such 

studies from 2001-2002, and the results were submitted jointly to EPA by Monsanto Company 

and Syngenta Seeds, Inc. in 2003. The study included 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 monarch 
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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 continued existence of monarch populations in North America. A full 

summary of the study is included 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 methodology and because the number of monarchs 

throughout the U.S. fluctuates between regions and years. There are several factors such as 

catastrophic weather (e.g., drought or floods) that may adversely 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 increased population size in North America and decreased numbers 

during cold summers. Among other factors that may affect monarch population size are: (1) 

overwintering site depletion, (2) number and fitness of monarchs that overwinter, (3) nectar 

availability to adults, (4) pathogens, parasites, parasitoids, and predators, (5) milkweed 

availability, (6) use of insecticides to control lepidopteran pests, and (7) accidents (e.g., collision 

with automobiles). Due to these factors, it is difficult to develop a baseline population size or to 

determine if Bt corn pollen was a contributing factor. 

There have been several attempts made to determine monarch population levels. Swengel (1995) 

showed that from 1986-1994 there were significant 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 twice 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 population estimates, it is not reasonably possible to 

develop a baseline monarch butterfly 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 potential of monarch exposure to Bt corn pollen in the field and whether 

pollen densities encountered present a risk to these butterflies. Monarch larvae 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 densities vary and typically depend 

on the management practices of the habitat. 
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Milkweeds can be found in a variety of habitats. However, non-agricultural areas are usually 

undisturbed supporting growth of more milkweeds. 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 corn 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 clumps (Martin and Burnside 1984). Herbicides are 

generally not considered to be effective in controlling milkweeds. However, in some instances, 

“good” control of milkweeds may be provided by glyphosate, halosulfuron-methyl + dicamba 

(2,4-D), and nicosulfuron + dicamba. 

 

Some milkweeds occur in and near corn fields, therefore, the proportion of the migrating 

monarch population that may encounter Bt corn fields was considered. There are potentially 

105,174 square miles (2.73 × 107 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 constitutes a very small area of potential monarch breeding habitat. 

Approximately 0.18% of monarch breeding 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 current 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 units (GDU). Examples of the approximate number of GDU 

needed for pollination to occur in different regions 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 pollen for two to seven days (or 

longer) and silks on an ear are exposed to pollination for two to three days (Russell 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 from its source because they are large (<90 to 100 

microns). The majority of corn pollen stays within corn fields and only small quantities disperse 

beyond 5 meters from the field edge. However, pollen levels are higher further into the corn 

field (e.g., 147.5 grains/cm2

 were found 25 m into the field) than close to the edge (e.g., 55.5 

grains/cm2 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 determined that there was only 0.2% of pollen 

deposited at 60 m from the corn field edge. Pleasants et al. (2001) found pollen densities at corn 
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field edges were 50% of the level found within the 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 from the field edge. Many factors influence pollen 

deposition and retention on milkweed leaves. Environmental factors such as rain and wind may 

increase the distance pollen will travel and may decrease the amount of pollen 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 environmental factors retain less pollen than middle 

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

 

2) Corn pollen exposure

Pleasants et al. (2001) found that levels of corn pollen deposition on milkweed leaves are 

influenced by wind, wind direction, rainfall, plant architecture and the time period when pollen 

was sampled. In some instances, weather conditions such as thunderstorms and updrafts carry 

some pollen grains further than usual (Emberlin et al, 1999). However, wind, rain, and other 

environmental factors will probably remove most of the pollen deposited on milkweed leaves 

(Pleasants et al. 2001). Rainfall has been shown to remove most (86 - 92%) of the pollen from 

milkweed leaves, thus potentially reducing 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 minimal. If pollen were to adhere 

to monarch adults and dislodge on milkweeds, quantities 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, there 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 biological activity of MON 810 in 

fresh pollen. Laboratory assays showed that MON 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 rapidly 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 shown 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 (Borkin 1982). The age of plant tissue is probably the 

most important influence on monarch ovipositional preference. Female monarchs prefer to 
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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 indifference 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 fields. In some instances monarchs may prefer to 

oviposit on milkweeds occurring within corn fields (Oberhauser et al. 2001). Although 

milkweed densities are generally higher in nonagricultural habitats, surveys conducted in 

Minnesota, Wisconsin, and Iowa suggest that monarchs will oviposit in corn fields 45 to 107 

times more often than in nonagricultural habitats (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 monarch breeding regions (east-central 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 % Overlap 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 Belt 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 milkweed 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 encountered (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-
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Bt corn pollen. Since eggs and larvae were found on milkweed plants naturally dusted in corn 

pollen in the field, it appears that monarchs will not avoid pollen dusted plants nor do they avoid 

corn fields. 

3) Bt Cry1Ab toxicity to monarchs

Since it has been established that monarch larvae can encounter and feed on Bt pollen in the 

field, it is important to know the Bt pollen toxicity level. 

TABLE 8. LC50s and the EC50s (effect-eliciting concentration) for the various monarch larval stages fed 

purified trypsin resistant core of Bt Cry 1Ab proteins 

Instar (N) LC50 (95% C.I.) 

(ng Cry1Ab/mL treated artificial diet) 

EC50 (95% C.I.) 

(ng Cry1Ab/mL treated artificial diet) 

1st (318) 3.29 (2.19-4.76) 0.76 (0.64-0.90) 

2nd - 3rd (141) 35.1 (30-100) 9.60 (6.01-15.06) 

3rd - 4th (125) > 100 (-) 18.3 (9.4-40.3) 

(Hellmich et al. 2001). 

LC50s for third and fourth instars were 30 times greater than first instars and second and third 

instar’s LC50 was 11 time greater than first instars. 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 the opportunity to avoid feeding on Bt altogether. Bt

activity in pollen is also expected to decline over time in the field. Therefore, levels of Cry1Ab 

ingested by monarchs in the field 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/cm2

 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 (cm2) 

Maryland 1999 Near peak 65.7 

Iowa 2000b 100% 425.6 

Iowa 2000c Post anthesis (10days) 101.2 
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Study Anthesis level Mean pollen density (cm2) 

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/cm2

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/cm2. However, first instar larvae feeding on milkweed leaves naturally dusted 

with pollen in the field resulted in no observable 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 levels (250 - 2000 grains/cm2 for MON 810 and 150 - 4000 grains/cm2 for 

Bt11) were fed to first instar monarch larvae in a controlled environment (since no pollen was 

removed due to environmental factors, these conditions are considered a worst case scenario) and 

resulted in no significant effects on larval weight (Hellmich et al. 2001). From this data, it can be 

concluded that the NOEL for MON 810 is >2000 grains/cm2

 and for Bt11 is >4000 grains/cm2

which is greater than levels that occur under natural field conditions. 

However, assuming a worst case scenario where 1000 pollen/cm2 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 addition, monarch larvae exposed to 

sub-lethal concentrations of Cry1Ab 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): 

Pollen density (cm From edge of cornfield 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 0.001 
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Pollen density (cm From edge of cornfield 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 toxic to monarch larvae do 

not occur under natural field conditions. 

 

Stanley-Horn et al. (2001) studied the difference between first instar larvae 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 adversely affected. There was 90% to 100% mortality of 

monarchs feeding on milkweeds collected from within the field and 21% to 45% mortality from 

plants outside the field. This study also suggests that Bt sweet corn may provide a safer habitat 

for monarchs than fields requiring insecticide 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 larvae 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-transgenic corn in Maryland, 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 development in the first and second bioassays were 

163 and 170 grains/cm2, respectively. Pollen density on milkweed plants in the non-Bt, BT11, 

and MON810 hybrids averaged 163, 155, and 174 grains/cm2, respectively, during the first 

bioassay and 172, 173, and 158 grains/cm2, 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 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 second bioassay were similar, with average delays of 0.7 to 

2.4 days to pupation and 0.2 to 2.4 days to eclosion. For both bioassays combined, exposure to Bt

pollen prolonged developmental time by an average of 1.8 days. 
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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 survival to eclosion was 66.7%, 51.3%, and 48.5%, 

respectively. There were no statistical 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 eclosion 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 adults. 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%), although natural mortality factors (e.g., weather, 

pathogens) probably accounted for the difference. 

Exposure to Bt pollen during larval development decreased the weight of pupae and adults. The 

results were statistically significant for pupal weight in both bioassays 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 proportion 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 the corn belt). A table of parameter estimates of exposure risk and 

probability of harm for 15 U.S. Corn Belt states and Ontario was provided in MRID No. 

46162001. The model results indicate that for the corn belt area, which represents 50% of the 

monarch breeding population, the risk to monarch larvae associated with long-term exposure to 

Bt corn pollen would be 0.6% additional mortality. 

The Corn Belt constitutes 50% of the breeding population in North America. Monarchs outside 

the Corn Belt are relatively unaffected due to the low acreage of corn and a low percentage of 

overlap of monarch larvae with corn pollen shed. 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 >50% reduction in population (mortality) as posing 

a risk to the existence of a species (see Section 1: Tiered Hazard and Risk Assessment Process). 

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 % mortality due to Cry1Ab Bt corn pollen will 

not pose an unreasonable risks to the continued existence of monarch populations in North 

America. 

5) Bt Cry1F toxicity to monarchs

 
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A scientifically sound study submitted by Dow AgroSciences 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 growth inhibition at 10,000 ng/mL 

diet. Since pollen doses equivalent to 10,000 ng/mL diet are not likely to occur on milkweed 

leaves in nature, it can be concluded that Cry1F protein will not pose a risk to monarchs. 

6) Conclusions:

MON 810 and Bt11 show relatively low toxicity to monarch larvae 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 risk assessment. Consideration of 

factors limiting exposure, such as relatively low pollen shed and monarch breeding overlap in 

much of the corn belt, the distribution 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 assessment indicate a low probability for adverse 

effects on monarch larvae. 

The weight of evidence of data gathered 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 combination with data submitted since 2001, the Agency 

concludes that the current information on monarch toxicity and exposure does not give sufficient 

cause for undue concern of widespread risks to monarch butterflies at this time.11 EPA will 

continue to closely monitor the results from further monarch butterfly 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 horizontal gene transfer (HGT) from Bt crops to soil 

organisms and has considered possible risk implications 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 also the habitat of anthrax, tetanus, and botulinum toxin-producing 

bacteria. Transfer of these genes and/or toxins to other microorganisms or plants has not been 

detected. Furthermore, several experiments (published in scientific journals), that were 

conducted to assess the likelihood of HGT, have been unable to detect gene transfer under 

 

11The available data can be used to make an approximation that only 0.001% of the monarch population (1 in 100,000) may be exposed to sublethal amounts of Bt pollen in Bt corn fields (using the information that 50% of the monarchs go through the corn belt, that 18% of that habitat is 

corn, that 25% is Bt corn, that the maximum overlap of anthesis and larvae is 50% in the migratory population and that in a worst case scenario 

0.1% of that population may encounter sub-lethal amounts of Bt pollen). 
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typical environmental conditions. Horizontal gene transfer to soil organisms has only been 

detected with very promiscuous microbes under laboratory conditions designed to favor transfer. 

As a result of these findings, which 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 protectants currently registered and EPA 

believes that these concerns have been satisfactorily addressed. The Agency has determined that 

there is no significant risk of gene 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 territories. In addition, the 

USDA/APHIS has made this same determination 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 and the Carribean. Where feral populations of sexually compatible 

cotton species exist in Florida and Hawaii, EPA has prohibited the sale or distribution of B.t.

cotton in these areas. These containment measures 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 relatives 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 species that, for the most part, do not have a reasonable possibility 

of passing their traits to wild native plants. Feral species related 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 Panel 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 for 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 insufficient to allow for cross pollination. Some concern 

was expressed, however, with respect to B.t. cotton grown in areas where wild relatives or feral 

populations of the crop are known to exist.

 
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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 recipients 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 interfertile with maize and are discussed as potential recipients of 

pollen directed gene flow from maize. This issue is of particular concern based upon the 

increased planting of genetically modified maize. Therefore, the Agency conducted a 

reevaluation in early 2000, the results of which are reported here. 

b. Zea mays ssp. mays - Maize - General Biology 

Zea mays is a wind-pollinated, monoecious, annual species with imperfect flowers. This means 

that spatially separate tassels (male flowers) and silks (female 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 for specific seed characteristics through standard 

breeding techniques. Maize cultivars and landraces are known to be diploid (2n = 20) and 

interfertile to a large degree. However, some evidence for genetic 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 selection 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 archaeological 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 declined 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 transfer for purposes of outcrossing (Schoper, 

personal communication,1999). Under conditions of high temperature or low humidity, corn 

pollen may only survive for a matter of minutes. Under more favorable conditions in the field or 

with controlled handling in the laboratory, pollen life may be extended to several hours. 

c. Tripsacum species - Gama Grass - 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 agrostologists; most of the 

16 different Tripsacum species recognized are native to Mexico, 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 species of widespread occurrence and of any 
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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., and 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 tetraploids (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 respects, 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 communication, 1999; Galinat, 1988; 

Wilkes, 1967). Tripsacum/maize hybrids have not been observed 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; Iltis, 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 existing; 2n = 36 or 72), as well as 

radically different phenotypic appearance. Albeit 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 maize 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 laboratory and greenhouse. Seed obtained from such crosses are often 

sterile or progeny have greatly reduced fertility. 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 arisen 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 considered as a weed in the U.S. 

In a telephone conversation with 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 crosses, if the female parent is maize, there is a greater likelihood of 

obtaining viable seed. When these hybrids have been backcrossed to maize in attempts to 
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introgress Tripsacum genes for quality enhancement or disease resistance, the Tripsacum

chromosomes are typically lost in successive generations. In many instances where 

hybridization has been directed between these two species, 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 accessions were tested and crosses 

made before this came to fruition. The Tripsacum derived mitochondrial chondrome and 

chloroplast plastome in these hybrids contribute to the seed qualities 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 agricultural or natural situations is extremely remote 

based upon his experience trying to create hybrids under the best of conditions. He also felt that 

no other known grass species present in the continental 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 sexually compatible relatives of corn in the 

U.S. are considered to be serious, principal, 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 / inflorescence 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 typical of weeds, is not 

characteristic of teosintes or maize. In their native habitat, some teosintes have been observed to 

be spread by animals feeding on the plants. Teosintes 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 preclude 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, personal communication; 2000; Wilkes, personal communication; 

2000; Wilkes, 1967). 

Since both teosinte and Tripsacum are included in botanical gardens 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 transformed corn would be similar to what naturally 
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occurs at the present time within cultivated corn hybrids and landraces. 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 compatible and interfertile with corn, especially when 

maize acts as the female parent. Zea perennis (perennial teosinte) has 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 capable of hybridization when in proximity to each 

other. In Mexico and Guatemala, teosintes exist as weeds around the margins of corn fields. 

The F1 hybrids have been found to vary in their fertility and vigor. Those that are fertile are 

capable of backcrossing to corn. A few isolated 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 populations of this species for 

approximately twenty-five years (Keith Bradley, personal communication, 2000; David Hall, 

personal communication, 2000; Richard Wunderlin, personal communication, 2000). 

Consultation with botanists and agronomists familiar with Texas flora suggested that no teosinte 

populations exist in the state (Benz, personal communication, 2000; Read, personal 

communication, 2000; Orzell, personal communication, 2000; Wilson, personal communication, 

2000). Further, given the day length characteristics of Z. diploperennis, it is highly unlikely a 

sustaining population would result from 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 native or introduced habitats (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 distribution is limited to Mexico, Honduras, Nicaragua, El 

Salvador and Guatemala. 

Given the cultural and biological relationships 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 these two groups of plants and that no weedy 

types have successfully evolved as a result. More recent cytogenetic, biochemical and molecular 

analyses have indicated that the 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 differential pollen growth and a 

skewed recovery of alleles linked to incompatibility genes. Complete incompatibility 

mechanisms serve to isolate a species or subspecies 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 cultivars (W22, W23) yielded no or few seeds in five of the 

six groups (Kermicle and Allen, 1990). 
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Based on the ability of maize to hybridize with some teosintes, the suggestion of previous 

genetic exchange amongst these species over centuries, and their general 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 from 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 introgression of genes into this species as a result of cross fertilization with 

genetically-modified maize is not expected to result in a species that is weedy or difficult to 

control. In many instances where 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 “teosintes” 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 species, 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 will 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 generally considered to be incapable of survival in 

the wild as a result of breeding practices (i.e., selection) during domestication 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. The 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 lepidopteran species relative to corn production counties in the 

US shows that any potential concern regarding range overlap 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 unlikely that significant numbers of lupine would 

occur within a few (two) meters of corn field edge, where the toxic levels of corn pollen may be 

present. Even using the conservative 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 extremely remote. Also, relevant data and 

information indicate that the likelihood of wild lupines 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 likely to be present is limited. 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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An examination of the endangered bird and bat species 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 not an issue, because the ECB is within the corn 

stalk and is not available for bird 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 pests 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 published 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 control practices are used. Therefore 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 toxicity and/or exposure, no effects from Cry1Ab and Cry1F are 

anticipated for any nontarget species, including federally-listed threatened and endangered 

(“listed”) lepidopteran and coleopteran species and their designated critical habitats. EPA has 

also determined that there are no indirect effects on endangered and threatened plant species, 

such as impacts on lepidopteran pollinators that are important and/or essential to an endangered 

or threatened plant. The Agency is therefore upholding its determination that the registered uses 

of Cry1Ab and Cry1F will have “No Effect,” direct 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 Fisheries 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 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 

weight 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 

indicate 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 results 

also confirmed that the expression of each of the proteins in the combination PIP was not 

affected by the presence of the other protein. This study was classified acceptable. 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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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-target wildlife or beneficial 

invertebrates, whether they are earthworms, springtails, parasites, predators, pollinators or soil 

microbial and invertebrate flora. Published field 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 research 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 result from the use of chemical pesticides. Under 

normal circumstances, Bt crops require substantially fewer applications of chemical pesticides. 

This should result in fewer adverse impacts to non-target organisms. Many of these beneficial 

organisms are important integrated pest management 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 insecticide 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 economic 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 potential for these indirect 

ecological effects on biological control organisms should not be regarded as a unique ecological 

risk associated with the PIP crop. Some reductions, however, should be expected if the pest 

management strategy is effective. Since PIP crops are often grown in vicinity with conventional 

crops to prevent resistance build-up by the target pest(s), specialist antagonists can persist in 

these ‘refuges’, in other crops and in non-crop habitats and retain the potential for recolonization of the PIP crop area. Based on these considerations, regulatory testing of the 

specialist predators and parasitoids of target pests may eventually be considered unnecessary. 

c. Supplemental Data Needed 

EPA has sufficient information to believe that there is no risk from the registered uses Bt11, 

MON810, TC1507 or TC6275 corn to non-target wildlife, aquatic and soil organisms and 

domesticated fowl and animals. The Agency has been frequently asking the registrants to 

conduct post-registration long term invertebrate population/community and Cry protein 

accumulation in soils studies as a condition of registration. The issue of long range effects of 

cultivation of these Cry proteins on the invertebrate community structure in corn fields has since 

been adequately addressed by the analysis of field studies performed during the last 10 years 

(Marvier, et al. 2007; Sanvido, et al. 2007). No unexpected adverse effects on invertebrate 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

102 

community structure were reported. The Agency is in agreement with these conclusions. 

Similarly, no unexpected accumulation of Cry proteins 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 registered Cry proteins. (Milofsky, 2006).

 

In light of recently published laboratory studies showing reduced 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, therefore 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. 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 shredder in headwater streams, can be performed and submitted in 

lieu of the Daphnia study. 

TABLE 11. Supplemental non-target data requirements for Bt11, MON810, TC1507 and 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 NPTII Proteins in Potato Tissues Derived 

from Field Grown plant: Lab Project 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 Protein from Bacillus thuringiensis subsp. 

tenebrionis: Lab Project Number: 92-01-37-07: 93-081E: 12897. 

Unpublished study prepared by Monsanto Co. 95 p. 

42932209 Maggi, V. (1993) Evaluation of the Dietary Effect(s) of Purified B.t.t. 

Protein on Honey Bee Larvae: Lab Project Number: CAR 188-92: 92-01-

37-03: 92-448-702. Unpublished study prepared by California Agricultural 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

103 

Research, Inc. 52 p. 

42932210 Maggi, V. (1993) Evaluation of the Dietary Effect(s) of Purified B.t.t. 

Protein on Honey bee Adults: Lab Project Number: CAR 189-92: 92-01-37-

03: 92-448-701. Unpublished study prepared 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 vitripennis): 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 toxicity Study with 

Ladybird Beetles (Hippodamia convergens): 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. Unpublished 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 CryIA(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 Dietary Effect(s) of Purified B.t.k. 

Endotoxin Proteins on Honey Bee Larvae: Lab Project Number: 

CAR/180/92: 92/01/36/10: 92/427/709. Unpublished study prepared by 

California Agricultural Research, Inc. 76 p. 

43145207 Maggi, V. (1993) Evaluation of the Dietary Effect(s) of Purified B.t.k. 

Endotoxin Proteins on Honey Bee Adults: 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 (Nasonia vitripennis): Lab Project 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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Number: 139/369: 93/234: WL/93/234. Unpublished 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 Dietary Toxicity Study with Cotton Seed 

Meal in the Northern Bobwhite: Lab 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. Unpublished study prepared by 

Springborn Laboratories, Inc. 59 p. 

43397201 Hanten, J.; Meeusen, R. (1994) Determination 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 CryIA(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 

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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 Dietary Toxicity Study with MON 80187 

Meal in the Northern Bobwhite: Lab 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 Characterization 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 Locations: Lab Project 

Number: 94-01-39-01: 14065: 14179. Unpublished study prepared by 

Monsanto Co. 147 p. 

43696001 Williams, D. (1995) Environmental Fate: Bacillus thuringiensis var. 

kurstaki Protein in Corn: Lab Project 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 Mississippi 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) Environmental Fate of CryIA(c) Protein: Lab 
Cry1Ab 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 Project 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 Dietary Effect(s) of Purified B.t.t. 

Protein on Honey Bee Larvae: Lab 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 Protein: (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 Beetle (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 Cry9C 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: Lab 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) 

Protein: A 48-Hour Static-Renewal Acute Toxicity Test with the 

Cladoceran (Daphnia magna): Final Report: Lab Project Number: WL-96-

322: 139A-201: 95-152E2. Unpublished study prepared by Wildlife 

International Ltd. 23 p. 

44274201 Privalle, L. (1997) Comparison of CryIA(b) Levels in Transgenic BT11-

Derived Maize (Corn) Pollen and Event176-Derived Maize Pollen and 

Justification for Citation of Daphnia magna Toxicity Study of Event 176-

Derived Pollen in Support of BT11 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. tolworthi 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) Cry9C 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 YieldGuardR 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 

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45512901 Davis, P and L Higgins. 2001. Exposure and risk assessment of Herculex I 

Bt field corn pollen to the Karner blue butterfly (Ly caides melissa 

samuelis). Study ID PHI-2001-054. Unpublished study by Dow 

AgroSciences. 397 p. 

45611801 Taylor, M.; Hartnell, G.; Riordan, S. et al. (2001) Comparison of Broiler 

Performance When Fed Diets Containing YieldGard Corn, YieldGard x 

Roundup Ready, Parental Line or Commercial Corn Hybrids: Lab Project 

Number: MSL-17198. Unpublished study prepared by Monsanto Co. 179 p. 

45622001 Zeph, L. (2000) Nutritional Equivalency of B.t. Cry1F Maize--Poultry 

Feeding Study: Lab Project Number: 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 prepared 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 

for Persistance and Accumulation of Cry1Ab Protein in Soil as a Result of 

Sustained Bt Corn Use. Project Number: 02/01/39/16, MSL/18547. 

Unpublished study prepared by Monsanto Company, Agvise Inc., and 

Syngenta Crop Protection . 110 p. 

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 Pennsylvania 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 Biotechnology Stewardship Technical Committee. 

239 p. 

47120701 McCormick, R.; Embrey, S. (2006) Soil 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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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 become resistant to a pesticide. Bt IRM is of great importance 

because of the threat insect resistance poses to the future use of Bt plant-incorporated protectants 

and Bt technology as a whole. Specific IRM strategies, such as the high dose/structured refuge 
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strategy, will mitigate insect resistance to specific Bt proteins produced in corn. Academic 

scientists, public interest groups, organic and other farmers 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 IRM strategies by reviewing the data and information available to 

the Agency. The Agency has used this assessment, the report of the FIFRA SAP meeting on 

October 18, 2000, and all public comments in its development of its risk management decisions 

for Bt corn plant-incorporated protectants. 

The following list will assist the reader with the acronyms for the insect 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, producers, researchers, and the 

American public. EPA considers 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 mechanisms 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 susceptibility. In addition to a high dose and structured refuge, IRM plans 

include additional field research on pest biology, refuge size and deployment, resistance 

monitoring for the development of resistance (and increased insect tolerance of the protein), 

grower education, a remedial action plan in case resistance is identified, annual reporting and 

communication. IRM plans will change as more scientific data become available. 
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Beginning with the first Bt plant-pesticide registration, the Agency has taken steps to manage 

insect resistance to Bt with IRM plans being an important part of the regulatory decision. The 

Agency identified (later confirmed 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 expression strategy; 3) appropriate refuge; 4) resistance monitoring 

and a remedial action plan should resistance occur; 5) employment of integrated pest 

management (IPM); 6) communication and education 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 mitigate the development of insect resistance to Bt proteins 

expressed in transgenic crop plants. The 1998 Subpanel recognized 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 insects. The high dose/refuge strategy assumes 

that resistance to Bt is recessive and is conferred by a single locus with two alleles resulting in 

three genotypes: susceptible homozygotes (SS), heterozygotes (RS), and resistant homozygotes 

(RR). It also assumes that there will be a low initial resistance allele frequency and that there 

will be extensive random mating between resistant 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 structured refuge is a non-Bt portion 

of a grower’s field or set of fields that provides for the production of susceptible (SS) insects that 

may randomly mate with rare resistant (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 delay the evolution of resistance. The 1998 and 2000 SAP Subpanels 

noted that insect resistance management strategies should also be sustainable and to the extent 

possible, strongly consider grower acceptance and logistical feasibility. 

Although the high dose/refuge strategy is the preferred strategy for IRM, effective IRM is still 

possible even if the transformed plant does not express the Bt protein at a high dose for all 

economically-important target pests (e.g., by increasing refuge size). The lack of a high dose 

could allow partially resistant individuals (i.e., heterozygous insects with one resistance allele) to 

survive, thus increasing the frequency 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 risk 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 SAP 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 report as well as in the scientific literature on insect resistance 
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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 protectant 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 containing lyophilized tissues of Bt plants using tissues from non-Bt

plants as controls; 2) Bioassays using plant lines with expression levels approximately 25-fold 

lower than the commercial cultivar determined by quantitative ELISA or some more reliable 

technique; 3) Survey large numbers of commercial plants in the field to make sure that the 

cultivar is at the LD99.9 or higher to assure that 95% of heterozygotes would be killed (see 

Andow & Hutchison 1998); 4) Similar to #3 above, but would use controlled infestation with a 

laboratory strain of the pest that had an LD50 value similar to field strains; and 5) Determine if a 

later larval instar of the targeted pest could be found with an LD50 that was about 25-fold higher 

than that of the neonate larvae. If so, the later stage could be tested on the Bt crop plants to 

determine if 95% or more of the later stage larvae 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 current empirical data suggest that a 25-fold dose 

may not be consistently high enough to cause high mortality among heterozygotes with known 

Bt resistance alleles. The 2000 SAP Subpanel did not recommend changing the existing 25-fold 

definition, but noted that the “25X” definition is imprecise, provisional, and may require 

modification as more knowledge becomes available 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 refuge is critical to the success of the high 

dose/structured refuge strategy to mitigate insect resistance to the Bt proteins produced in corn. 

The 1998 Subpanel defined structured refuges to "include all suitable non-Bt host plants for a 

targeted pest that are planted and managed by people. 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." The 1998 Subpanel suggested that a production of 500 susceptible 

adults in the refuge for every adult in the transgenic crop area (assuming a resistance allele 

frequency of 5 x 10-2) would be a suitable goal. The placement and size of the structured refuge 

employed should be based on the current understanding of the pest biology data and the 

technology. The 1998 SAP Subpanel also recognized that refuges should be based on regional 

pest control issues. The 2000 SAP Subpanel echoed the 1998 SAP’s recommendations that the 

refuge should produce 500:1 susceptible to resistant 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 actual field resistance, models have limitations and the information gained 
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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 scientifically rigorous way to integrate all of the biological information 

available, and that without these models, the Agency would have little scientific basis for 

choosing among alternative resistance management options.” They also recommended that 

models must have an agreed upon time frame for resistance protection. For example, 

conventional growers may desire a maximum planning horizon of five years, while organic 

growers may desire an indefinite planning horizon. 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, economics, fitness costs of resistance, 

alternate hosts, spatial components, stochasticity, and pest population dynamics. 

EPA’s Office of Research and Development (ORD), National Risk Management Research 

Laboratory and Office of Pesticide Programs held 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 verification and validation procedures (described in Glaser et al. 

2009). 

A number of models have been developed 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 early warning sign to developing 

resistance in the field and whether IRM strategies 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 biological parameters used in models. However, 

resistance detection/monitoring is a difficult and imprecise 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 frequency of resistant individuals, the location and 

number of samples that are collected, and the sensitivity 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 & 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). Current sampling 

strategies have a target of 100 to 200 individuals per location. Previous experience with 

conventional insecticides has shown that once resistant phenotypes are detected at a frequency 

>10%, control or crop failures are common (Roush & Miller 1986). Because of sampling 
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limitations and monitoring technique sensitivity, resistance could develop to Bt toxins prior to it 

being easily detected in the field. 

The 2000 SAP Subpanel concluded that resistance monitoring programs should be peer reviewed 

and used to assess the success of current IRM 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 described 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 telephone numbers and an Internet site can be provided by registrants to report 

any unusual control problems. A confirmed grower report of unexpected pest damage in a Bt

crop may be a way to document a control failure and may be a useful monitoring system for 

determining the success or failure of existing resistance management strategies. However, once 

a grower detects a control failure, and resistance has been verified, 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 bioassay is currently required by the EPA. 

Discriminating dose bioassays are most useful when 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. Hawthorne et al. (2001) consider the diagnostic dose to be less expensive 

than in-field screens and the F2 screen. It is best used when the frequency of resistance alleles is 

high (>10-2) or the resistant allele is dominant. However, it is unclear (and likely pest-specific) 

whether resistance is carried by dominant or recessive alleles and what the frequency of 

resistance alleles are in pest populations. Measurement of low resistant allele frequencies (<10-2) 

would not be possible using the diagnostic dose without extremely large sample sizes. 
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The October 2000 SAP Subpanel was asked whether the current resistance monitoring plans 

were adequate. They indicated that the diagnostic or discriminating dose technique could at best 

detect resistance when the resistance allele frequency has reached 1%. This is a level in which 

some field failure may be observed. At this lower 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 insecticides has shown that once resistant 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 individuals in a population that demonstrate 

resistance will equal the square of the allele frequency. For example, if the initial resistance 

allele frequency is one in 1,000, then one would need to assay more than a million larvae to find 

one homozygous resistant individual. Typically, discriminating dose assays are based on 100-

300 larvae to detect resistance at a frequency of 1-3% (Roush & Miller 1986). 

4) F2 Screen

The F2 screen may be a useful monitoring technique for Bt corn, especially for the detection of 

rare recessive resistant alleles. The technique also allows fewer samples to be collected to detect 

potential susceptibility shifts than the discriminating dose assay. The F2 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 characterization of four genomes, thus improving the 

sensitivity over the discriminating dose assay by 10-fold (Andow & Alstad 1998). The F2

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 estimate the fitness of the genotypes, determine 

whether there is a cost of resistance, and enable predictions of the evolution of resistance. The 

F2 screen is conducted by sampling mated females from natural populations, rearing the progeny 

of each female as an isofemale line and sib-mating her F1 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 F2 screen is probably the only current method

available to detect rare recessive alleles. 

A number of the October, 2000 SAP Subpanel members indicated that the F2 screen 

accompanied by field screening “could be very effective for detecting low frequencies of 

recessive and dominant resistance alleles.” The F2 screen can be a powerful method for 

detecting rare recessive alleles in natural populations. As described by Andow and Alstad 

(1998), it relies on inbreeding field-collected individuals so that all recessive alleles are 

expressed in the F2 generation where they can be screened for the phenotype of interest. This 

method has been used to estimate the frequency 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 provide a statistical method for estimating the probability that the 

screen erroneously does not identify the targeted resistance allele. This is the probability of a 
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false negative, and its calculation is based on the probability of inheritance of the allele, the 

assumption that F1 families mate randomly, and the probability that other mortality factors may 

interfere with the phenotypic evaluation of the F2 individuals. 

Current insect resistance management 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 F2 screen by Andow et al. (1998, 2000) and 

Andow and Alstad (1998, 1999) indicate 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 F2 screen of 1,200 isofemale 

lines of ECB collected in France and in the northern U.S. Corn Belt during 1999 and 2000 

indicated that the frequency of resistance 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 key assumption of the high 

dose/refuge strategy. 

Using the F2 screen would increase the probability of detecting rare resistant alleles and the 

threshold of detection would be lowered to <0.005. A sample of 100 female lines has a precision 

of ± 0.0025 for dominant alleles and ± 0.0025 for recessive resistance alleles. Leaving aside 

issues of accuracy, the theoretically best resolution of allele frequency is ± 0.0025 for dominant 

alleles and ± 0.05 for recessive alleles for a screen 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 failures occur depends on the precision of monitoring 

and the recessivity/dominance of resistance. If the goal of resistance monitoring is to detect 

resistance at a low enough resistance allele frequency so that changes to the insect resistance 

management plan can be made to increase the longevity of the product and prevent field failure, 

then current resistance monitoring plans need refinement. The F2 screen is one method of 

refinement that can detect and measure 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 F2 screen is that it may be labor intensive and not suitable for routine 

screening purposes (Hawthorne et al. 2001). Andow has conducted a cost analysis for various 

monitoring techniques and has concluded that in general the F2 screen is more expensive than 

other methods for detecting dominant resistant alleles when the resistance allele frequency is 

>0.01 (see Andow in U.S. EPA/USDA 1999b, p. 49, Figure 3). It is estimated that 750-1200 

family lines must be screened to have a 95% probability of detecting a dominant 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 alleles, Andow estimates that the F2 screen is the least expensive 

method and can estimate resistance allele frequencies 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 F2 screen to be $14,000 to $20,000 per population. 

This, they conclude, would be too expensive for routine monitoring efforts, especially if there is 
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replication at each site. The area of cost and cost-effectiveness of the F2 screen should be further 

evaluated. 

Hawthorne et al. (2001) concluded that there is a need to further evaluate the precision and 

accuracy of the F2 screen by using colonies with known frequencies of resistance alleles. Zhao 

et al. (2001) also came to this same conclusion. They validated the F2 screen using a synthetic 

laboratory population of the diamondback moth (Plutella xylostella L.) for detecting the 

frequency of rare resistance alleles to Cry1Ac and Cry1C toxins of Bt. When using Bt broccoli 

as the diagnostic method, only one F2 family was detected for Cry1Ac resistance and no family 

was detected for Cry1C resistance. Six families were detected for either Cry1Ac or Cry1C 

resistance using the diagnostic diet bioassay. Four F2 families were confirmed to contain one 

copy of an allele resistant to Cry1Ac in the original single-pair matings and four other F2 families 

contained an allele resistant to Cry1C. These results suggest that transgenic plants expressing a 

high level of a Bt toxin in a F2 screen may underestimate the frequency of resistance alleles with 

false negatives or fail to detect true resistance alleles. The authors concluded that the diagnostic 

diet assay was a better F2 screen method to detect resistance alleles, especially for the Cry1Ac 

resistance in diamondback moth. Zhao et al. (2001) conclude that further validation of the F2

screen method for each insect-crop system should be conducted before the procedures used in the 

F2 screen could be used routinely 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 stocks of highly resistant tobacco 

budworm (Heliothis virescens; TBW) selected on Cry1Ac in the laboratory to estimate the 

resistance allele frequency in a natural population. This method can identify recessive or 

incompletely dominant resistance alleles from field-collected males. Using 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 F1 progeny would be heterozygous for 

resistance. By using an assay that discriminates between heterozygotes, they could establish 

which wild males carried a resistance allele. Using this allelic recovery method, Gould et al. 

(1997) estimated the resistance allele 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 examine resistance allele 

frequency. This method uses Bt sweet corn to screen 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 and 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 reduce the number of plants required to attain 

an acceptable sample size. If the Bt sweet corn is planted at the appropriate time, larval attack 
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will cause extensive damage, and large areas of Bt sweet corn can be sampled rapidly by 

examining this damage. For example, if 10 resistant larvae are found in a sample of 5,000 Bt

corn ears, and 50 larvae are recovered from 50 plants in the non-Bt field, then the expected 

phenotypic frequency of resistance would be 0.002. If potential 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 F2 screen for verification of resistance might be an efficient method to 

detect resistance and capture resistance alleles especially in designated 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 problems with this method that must be addressed prior to its widespread 

adoption as discussed by Hawthorne et al. (2001). There is a high number of false positives that 

would reduce the efficiency and accuracy of resistance 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 surviving 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 the 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 effort limits its practicality. 

e. Compliance with IRM requirements 

Grower compliance with refuge and IRM requirements is a critical element for resistance 

management. Significant non-compliance with IRM 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 refuge size/deployment and the level of grower 

compliance necessary to achieve risk reduction. EPA considers the development of Bt-resistant 

insects to constitute an adverse environmental effect, therefore, IRM, and subsequently grower 

compliance, is very important. Optimally, refuge requirements would change over time as pest 

susceptibility changes. However, changes to refuge requirements are difficult to implement. 

Therefore, the Agency must set safe refuge requirements that preserve the pest(s) susceptibility 

and protect the benefits of Bt crops. Currently, the financial burden of implementing these 

refuge requirements is borne primarily by the growers. Increasing refuge size and/or limiting 

refuge deployment to better mitigate the risk of resistance is likely to increase costs to growers 

and result in a higher rate of grower non-compliance. Grower compliance 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 insecticides, will reduce the risk of resistance. 
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To minimize the effects of non-compliance, it may be necessary to develop a broad compliance 

program as part of an IRM strategy. Ideally this program would include four major objectives: 

1) an understanding of the effect of non-compliance on IRM; 2) identification of compliance 

mechanisms to maximize adoption of IRM requirements; 3) measurement of the level of 

compliance; and 4) establishment of an enforcement structure to ensure compliance and penalize 

non-compliance. 

1) Effects of Non-Compliance on IRM

As a first step towards developing a compliance 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 likelihood 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 can 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 and alternate adoption scenarios. 

2) Compliance Mechanisms

There have been a number of compliance mechanisms proposed by various parties (including the 

2000 SAP Subpanel) to ensure grower conformity, reward compliance, and penalize noncompliance. These include such techniques as: grower 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 restrictions, intensified grower 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 mechanisms 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 the 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 (Hurley & Mitchell 2000). The 2000 SAP consensus 

was that compliance would be best managed through education and grower contracts, but also 

that sales incentives, refuge insurance, and refuge deposit/refund programs may have value if 

managed properly. Also, the 2000 SAP recognized that mechanisms that would reduce the cost 

of compliance will be more effective at improving compliance. 

Mitchell et al. (2000) developed a model to evaluate crop (refuge) insurance and sales incentives 

as potential compliance mechanisms. The cost to growers (i.e., lost yield, higher inputs) to 
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adhere to IRM requirements can be an impediment to compliance. Therefore, by providing 

growers with incentives to reduce the cost of refuge mandates, compliance may be increased. 

Both insurance and sales incentives have the potential to reduce this cost of compliance to 

growers, although both have drawbacks as well. For refuge insurance to be profitable for private 

insurance companies, it would likely be too expensive 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 program, it is necessary to be able to accurately 

measure the level of grower compliance. The 2000 SAP noted several parties, other than the 

registrant alone, could verify compliance: 1) grower visits by industry, EPA, state authorities, 

USDA, or other third-parties; or 2) USDA/NASS or other third-party grower surveys. To date, 

compliance has been primarily measured through grower surveys conducted by industry or 

academics (e.g., Pilcher & Rice 1997, Rice & Pilcher 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 compliance. 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., audits), it is impossible to 

verify the accuracy of grower responses. The end result may be increased “false-positives,” 

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 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 survey responses. The use of mapping systems, such 

as the Global Positioning System (GPS), may also 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 utilized GPS with Bt cotton grown in Arizona in conjunction 

with grower visits to assess the level of refuge compliance (Carrière et al. 2001). 

4) Enforcement Structure / Penalties for Non-compliance

For a compliance program to be effective, a regulatory enforcement/compliance framework will 

be needed. Appropriate stakeholders and regulatory 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 growers. Registrants have been responsible 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 compliance “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 accompanied by an appropriate enforcement program. 

Potential penalties for non-compliance might include: 1) sales restrictions at a county, state, 

regional, or national level; 2) sales prohibitions to specific growers; 3) registrant fines and 
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warnings; and 4) increased refuge for specific non-compliant growers (through grower 

contracts). 

 

A compliance program has been developed for Bt corn as a term of registration. Details of this 

plan, including compliance surveys and enforcement 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 management 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 risk assessment of insect resistance development 

and IRM plans following the October 18-20, 2000 SAP 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 scientific 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 development of resistance in the two 

primary target pests, ECB and CEW. At that time, the putative values 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 structure could not be determined until further research was conducted, it 

was thought that market penetration of these crops would be sufficiently slow that considerable 

non-Bt corn would remain to act as natural refuges while the additional research was conducted. 

Thus, the initial Bt corn registrants instituted voluntary IRM plans with the requirement that 

these registrants must submit a refuge strategy by April 1999. From 1995-1997, the registrants 

agreed to various voluntary refuge requirements in the Corn Belt (0% to 20%). 

Since 1995, all Bt corn registrations have included a resistance 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) routine 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 resulted from resistance or other factors that might 

inhibit expression of the Bt delta-endotoxin. The extent and distribution 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 individuals, it should be possible to detect field resistance before 

significant loss of efficacy and eliminate any resistant individuals using other control tactics. In 

addition, EPA mandated that all registrants 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 appropriate in vitro and in planta assays. Any confirmed incidents of 
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resistance are required to be reported to EPA. Based on these investigations, appropriate 

remedial action is required to mitigate pest resistance. These remedial actions include: informing 

customers and extension agents in the affected areas 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 counties. All registrants have instructed growers to have regular 

surveillance programs and report any unexpected levels of target pest damage. Since 1995, the 

Agency is aware of no evidence of ECB, CEW or SWCB field resistance 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, the 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 modifications to this plan, EPA put in place a consistent 

set of required refuge strategies for all Bt field corn products beginning 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 corn. Beginning with the 2000 growing 

season, EPA required a 20% non-Bt field corn refuge to be planted within 1⁄2 mile (<1/4 mile in 

areas where insecticides have been 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 requirements 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 terms 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-lepidopteran resistant Bt 

corn refuge in conjunction with the planting of any acreage of Bt corn; 
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2] Requirements for the registrants to prepare and require Bt corn users to sign “grower 

agreements” which impose binding contractual obligations on the grower to comply with 

the refuge requirements; 

3] Requirements regarding programs to educate growers about IRM requirements; 

4] Requirements regarding programs to evaluate and promote growers’ compliance with 

IRM requirements; 

5] Requirements regarding programs to evaluate whether there are statistically significant 

and biologically relevant changes in target insect susceptibility to [Cry1Ab or Cry1F] 

protein in the target insects; 

6] Requirements regarding a “remedial action plan” which contains measures the 

registrants would take in the event 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), IRM 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 seed 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 ingredient 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 structured refuge of at least 20% non-Bt corn 

and/or non-lepidopteran 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. 
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• When planting the refuge in strips across the field, refuges must be at least 4 rows 

wide. 

• Insecticide treatments for control of European corn borer (ECB), Corn earworm 

(CEW), Southwestern corn borer (SWCB) and other lepidopteran target pests listed 

on the label, grower guides, or other educational material may be applied only if 

economic thresholds are reached for one or more of these target pests. Economic 

thresholds will be determined using methods 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-lepidopteran resistant Bt corn refuges. 

2) Cotton-Growing Area Refuge Requirements for Bt Corn 

For Bt field corn grown in cotton-growing areas, 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 in these areas must plant a structured refuge of at least 50% 

non-Bt corn and/or non-lepidopteran 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. 

• When planting the refuge in strips across the field, refuges must be at least 4 rows 

wide. 

• Insecticide treatments for control of ECB, CEW, SWCB, and other lepidopteran 

target pests listed on the label, grower guides, or other educational material may be 

applied only if economic thresholds are reached for one or more of these target 

pests. Economic thresholds will be determined using methods 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-lepidopteran resistant Bt corn refuges. 

• Cotton-growing areas include the following states: Alabama, Arkansas, Georgia, 

Florida, Louisiana, North Carolina, Mississippi, South Carolina, Oklahoma (only 

the counties of Beckham, Caddo, Comanche, 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, Rutherford, Shelby, and Tipton), Texas 
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(except the counties of Carson, Dallam, Hansford, Hartley, Hutchinson, Lipscomb, 

Moore, Ochiltree, Roberts, and Sherman), Virginia (only the counties of Dinwiddie, 

Franklin City, Greensville, Isle of Wight, Northampton, Southampton, Suffolk City, 

Surrey, Sussex) and Missouri (only the counties 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 purchase contract, license agreement, or similar 

legal document. 

2] The grower agreement and/or specific stewardship 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 which is reasonably likely to assure that persons 

purchasing the Bt corn product will affirm annually that 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 grower agreement or any specific stewardship 

documents referenced in the grower agreement that would affect either the content of the 

IRM program or the legal enforceability of the provisions of the agreement relating to the 

IRM program, thirty days prior to implementing a proposed change, the registrant must 

submit to EPA the text of such changes to ensure that it is consistent with the terms and 

conditions of the amendment. 

5] The registrant must maintain a system which 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 number 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 agreements. The report shall cover the time frame of 

the twelve-month period covering the 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 regulatory agency if the State agency can 

demonstrate that confidential business information, including names, personal 

information, and grower license number, will be protected. 
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c. IRM Education and IRM Compliance Monitoring Programs

1] The registrant must maintain a comprehensive, ongoing IRM education program 

designed to convey to Bt corn users the importance of complying 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 reviewed language on IRM requirements on the 

bag or bag tag, and electronic communications such 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 written 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 registrant 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 expand as necessary, its education program to 

take into account the information collected through the compliance survey required and 

from other sources. The changes shall address 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 ongoing 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 reasonably 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 compliance assurance program. 

5] The registrant must maintain and publicize 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 program and general criteria for choosing among 

options for responding to any non-compliant growers. While recognizing that for reasons 

of difference in business practices there are needs for flexibility between different 

companies, all Bt corn registrants must use a consistent set of standards for responding to 

non-compliance. The options shall include withdrawal 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 row would be denied sales of 

the product the next year. Similarly, seed dealers who are not fulfilling their obligations 

to inform/educate growers of their IRM obligations will lose their opportunity to sell Bt

corn. 
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6] The IRM compliance assurance program shall 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 majority 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 shall measure the degree of compliance with the 

IRM program by growers in different regions of the country and consider 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 behavior within regions or between regions. 

The sample size must provide a reasonable sensitivity for comparing results across the 

U.S. 

7] The survey shall be designed to provide an understanding of any difficulties growers 

encounter in implementing IRM requirements. An analysis of the survey results must 

include the reasons, extent, and potential biological 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 summary of the results of the prior year's survey 

(together with a description of the regions, the methodology 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 conduct 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 program. 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 the IRM program, the registrant 

shall take appropriate action, consistent 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 compliance with the IRM program. Whenever an 

investigation results in the identification of a grower who is not in compliance with the 
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IRM program, the registrant shall 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 previous year, the registrant shall visit with the grower 

and evaluate whether that the grower is in compliance with the 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 program for the prior year and the plans for 

the compliance assurance program during the current year. The report will include 

information regarding grower interactions (including, 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-compliance, and any follow-up actions taken. 

The registrants may elect to coordinate information and report collectively the results of 

their compliance assurance programs. 

15] The registrant and the seed corn dealers for the registrant must allow a review of the 

compliance records by EPA or by a State pesticide 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 consist of two approaches: (1) focused population sampling 

and laboratory testing; and (2) investigation of reports of less-than expected control of 

labeled insects. Should field-relevant resistance 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 planted on a high proportion 

of the corn acres, and where the insect species are regarded as key pests of corn). 

Bioassay methods must be appropriate for the goal of detecting field-relevant shifts in 

population response to lepidopteran 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 comparisons with historical data. 
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The number of populations to be collected shall reflect the regional importance of the 

insect species as a pest, and specific collection 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 target 400 insect genomes (egg masses, larvae, mated females, 

and/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 number 

of insect populations or genomes due to factors 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 importance and for the adoption levels of 

lepidopteran-resistant Bt corn. The Agency shall be consulted prior to the implementation 

of such modifications. 

The registrant will report to the Agency before 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 understand 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 shall be reported to the Agency annually before 

August 31. The investigative steps will include: 

1. Re-test progeny of the collected population to determine whether the unusual bioassay 

response is reproducible and heritable. If it is not reproducible and heritable, no further 

action is required. 

2. If the unusual response is reproducible and heritable, progeny 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 frequency in the original population; 
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• 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 sites surrounding the site where the resistance 

allele(s) was originally collected. 

Should field-relevant resistance be confirmed, and the resistance appears to be increasing 

or spreading, the registrant will consult with the Agency to develop and implement a 

case-specific resistance management action plan. 

(2) Investigation of Reports of Unexpected 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 contact 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 suspicion of resistance in any of the 

key target pests (ECB, SWCB, and CEW), 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 unexpected 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 percentage of corn expressing Bt protein; 

• The relevant plant tissues are expressing the expected level of Bt protein; and 

• 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 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 undertaken to confirm 

scientifically the presence of insects resistant to Bt corn in commercial production fields 
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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 region within one month after harvest 

with a technique appropriate for local production practices to minimize the 

possibility of resistant insects over-wintering and contributing to the next season's 

target pest population. 

Additionally, if possible, and prior to the application of alternative control measures or 

destruction of crop residue, the registrant will collect samples of the insect population in 

the affected fields for laboratory rearing and 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 pests, 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 are 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 question, 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 laboratory bioassays, the LC50 exceeds the upper limit of the 95% 

confidence interval of the LC50 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 
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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 pest species, the extent of resistance, the 

timing of resistance, and the nature of resistance, and the availability of suitable 

alternative control measures), alternative control measures will be employed to 

reduce or control target pest populations in the affected area. Alternative 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 insects, 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 characteristics of the resistance event and local 

agronomic needs. The registrant will consult with appropriate stakeholders in the 

development of the action plan, and the details of such a plan shall be approved by 

EPA prior to implementation; 

• Notify affected parties (e.g., growers, consultants, extension agents, seed 

distributors, university cooperators and state/federal authorities as appropriate) in 

the region of the resistance situation and approved action plan; and 

• In subsequent growing seasons, maintain sales suspension and alternative resistance 

management strategies in the affected region(s) for the Bt corn hybrids that are 

affected by the resistant population until 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 31st 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) 
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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. Conversely, 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 refuge 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 photosynthesis. As a result, 

in transgenic sweet corn varieties, Bt protein production is high at the time of harvest. EPA 

mandated specific resistance monitoring requirements for ECB, CEW, and FAW, as well as sales 

reporting requirements. Syngenta is required through 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, including 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 registrants to prepare and 

require Bt corn users to sign “grower agreements” which impose binding contractual 

obligations on the grower to comply with the refuge requirements; 

2] Requirements regarding programs to educate growers about IRM requirements; 

3] Requirements regarding programs to evaluate 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 statistically significant 

and biologically relevant changes in target insect susceptibility to Cry1Ab protein in the 

target insects; 

5] Requirements regarding a “remedial action plan” which contains measures the 

registrants would take in the event 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), IRM grower agreements results, and the 

compliance assurance program including the education program on or before January 

31st each year. 
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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 31st each year. 

 

a. Sweet Corn Post-Harvest Requirements 

Sweet corn is harvested long before field corn. Therefore, if the sweet corn stalks 

remaining in the field and any insects remaining in the stalks are destroyed shortly after 

harvest, a refuge is not needed as a part of the IRM program for sweet corn. Growers 

must adhere to the following types of crop destruction requirements as described in the 

grower guide/product use guide and/or in supplements to the grower guide/product use 

guide, and in the case of home gardeners on the 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 stalks using home garden tools such as a 

hoe. The crop destruction methods are intended to protect against development 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 purchase contract, license agreement, or similar 

legal document. 

2] The grower agreement and/or specific stewardship 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 which is reasonably likely to assure that persons 

purchasing the Bt corn product will affirm annually that 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 grower agreement or any specific stewardship 

documents referenced in the grower agreement that would affect either the content of the 

IRM program or the legal enforceability of the provisions of the agreement relating to the 

IRM program, thirty days prior to implementing a proposed change, the registrant must 

submit to EPA the text of such changes to ensure that it is consistent with the terms and 
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conditions of the amendment. 

5] The registrant must maintain a system which 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 number 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 agreements. The report shall cover the time frame of 

the twelve-month period covering the 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 regulatory 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 comprehensive, ongoing IRM education program 

designed to convey to Bt corn users the importance of complying with the IRM program. 

The education program shall involve the use of multiple media, e.g., face-to-face 

meetings, mailing written materials, EPA reviewed language on IRM requirements on the 

bag or bag tag, and electronic communications such 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 written communication annually to each Bt corn user 

separate from the grower technical guide (with the exception of home gardening or 

educational uses). The communication shall inform the user of the current IRM 

requirements. The registrant shall coordinate 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 expand as necessary, its education program to 

take into account the information collected through the compliance survey required and 

from other sources. The changes shall address 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 ongoing IRM compliance assurance program designed 

to evaluate the extent to which growers purchasing its Bt corn product are complying 
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with the IRM program and that takes such actions as are reasonably 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 exception 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 Compliance Monitoring Programs apply to all 

growers who plant more than 20 acres of Bt corn: 

 

5] The registrant must maintain and publicize 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 program and general criteria for choosing among 

options for responding to any non-compliant growers. While recognizing that for reasons 

of difference in business practices there are needs for flexibility between different 

companies, all Bt corn registrants must use a consistent set of standards for responding to 

non-compliance. The options shall include withdrawal 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 row would be denied sales of 

the product the next year. Similarly, seed dealers who are not fulfilling their obligations 

to inform/educate growers of their IRM obligations 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 bags 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 responses), and consider the potential 

impact of non-response. An independent third 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 any difficulties growers 

encounter in implementing IRM requirements. An analysis of the survey results must 

include the reasons, extent, and potential biological 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 summary of the results of the prior year’s survey 

(together with a description of the methodology 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 compliance 
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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 representatives 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 identification of a grower who is not in compliance with 

the IRM program, the registrant shall take appropriate action, consistent 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 compliance 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 shall 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 previous year, the registrant shall visit with the grower 

and evaluate whether that the grower is in compliance with the 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 program for the prior year and the plans for 

the compliance assurance program during the current year. The report will include 

information regarding grower interactions (including, 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-compliance, and any follow-up actions taken. 

The registrants may elect to coordinate information and report collectively the results of 

their compliance assurance programs. 

15] The registrant and the seed corn dealers for the registrant must allow a review of the 

compliance records by EPA or by a State pesticide 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 consist of two approaches: (1) focused population sampling 

and laboratory testing; and (2) investigation of reports of less-than expected control of 

labeled insects. Should field-relevant resistance be confirmed, an appropriate resistance 

management action plan will be implemented. 
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(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 and the pest is capable of overwintering 

in that county. The registrant should consult with academic and USDA experts in 

developing the monitoring plan and will provide EPA with a copy of its proposed 

resistance monitoring plan for EPA’s approval 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 following 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 focused in areas identified as those with the 

highest risk of resistance development (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 must be appropriate for the goal of detecting 

field-relevant shifts in population response to lepidopteran 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 comparisons with 

historical data. 

The number of populations to be collected shall reflect the regional importance of the 

insect species as a pest, and specific collection 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 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 target 400 insect 

genomes (egg masses, larvae, mated females, and/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 number of insect populations or genomes due to 

factors such as natural fluctuations in pest 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 importance and for the adoption levels of 

lepidopteran-resistant Bt corn. The Agency shall be consulted prior to the implementation 

of such modifications. 

The registrant will report to the Agency before August 31 each year the results of 
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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 understand 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 shall be reported to the Agency annually before 

August 31. The investigative steps will include: 

1. Re-test progeny of the collected population to determine whether the unusual bioassay 

response is reproducible and heritable. If it is not reproducible and heritable, no further 

action is required. 

2. If the unusual response is reproducible and heritable, progeny 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 frequency 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 sites surrounding the site where the resistance 

allele(s) was originally collected. 

Should field-relevant resistance be confirmed, and the resistance appears to be increasing 

or spreading, the registrant will consult with the Agency to develop and implement a 

case-specific resistance management action plan. 

(2) Investigation of Reports of Unexpected 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 contact them if such incidents occur. The 

registrant will investigate all legitimate reports submitted to the company or the 

company's representatives. 
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If reports of unexpected levels of damage lead to the suspicion 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 unexpected 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 percentage of corn expressing Bt protein; 

• The relevant plant tissues are expressing the expected level of Bt protein; and 

• 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 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 undertaken to confirm 

scientifically the presence of insects resistant to Bt corn in commercial 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 region within one month after harvest 

with a technique appropriate for local production practices to minimize the 

possibility of resistant insects over-wintering and contributing to the next season's 

target pest population. 

Additionally, if possible, and prior to the application of alternative control measures or 

destruction of crop residue, the registrant will collect samples of the insect population in 

the affected fields for laboratory rearing and 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 
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levels of damage from the key target pests, 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 are 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 question, 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 laboratory bioassays, the LC50 exceeds the upper limit of the 95% 

confidence interval of the LC50 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 the extent and impact of resistance; 

• If appropriate (depending on the resistant pest species, the extent of resistance, the 

timing of resistance, and the nature of resistance, and the availability of suitable 

alternative control measures), alternative control measures will be employed to 

reduce or control target pest populations in the affected area. Alternative 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 insects, 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 characteristics of the resistance event and local 

agronomic needs. The registrant will consult with appropriate stakeholders in the 
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development of the action plan, and the details of such a plan shall be approved by 

EPA prior to implementation; 

• Notify affected parties (e.g., growers, consultants, extension agents, seed 

distributors, university cooperators and state/federal authorities as appropriate) in 

the region of the resistance situation and approved action plan; and 

• In subsequent growing seasons, maintain sales suspension and alternative resistance 

management strategies in the affected region(s) for the Bt corn hybrids that are 

affected by the resistant population until 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 31st 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 individuals including EPA's 

Scientific Advisory Panel (SAP) and Pesticide Program Dialogue Committee (PPDC). SAP 

meetings and reports in 1995, 1998, and 2000 have confirmed that EPA's approach and elements 

required in an insect resistance management plan are appropriate. EPA believes that pest 

biology and the dose of the Bt protein expressed in the various 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 compares 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 and 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 meeting 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 monitoring, compliance, and other data 

submitted since 2001 for the Cry1Ab and Cry1F corn registrations. 

 

1) Pest Biology 

Knowledge of pest biology is critical for the development of effective IRM strategies and to 

increase confidence that the IRM plans will be effective at reducing the likelihood that insects 

will become resistant to Bt proteins. 

a) European Corn Borer (Primary Target Pest)
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European Corn Borer (ECB, Ostrinia nubilalis) is a major pest of corn throughout most of the 

United States. The pest has 1-4 generations per 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 generations 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 meeting on IRM identified a number of areas 

needing additional research including larval movement, adult movement, mating behavior, preand post-mating dispersal, ovipositional behavior, fitness, and overwintering 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 additional work could enhance the knowledge base 

for this pest. A summary of key aspects of ECB biology that relate to IRM is presented below: 

i. Larval Movement 

ECB larvae are capable of significant, plant-to-plant movement within corn fields. Research 

conducted in non-transgenic corn showed 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 (cited in Onstad & Gould 1998) indicates that 

approximately 98% of susceptible ECB neonates 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 that 4th instar larvae were capable of movement 

up to six corn plants within a row and six corn plants across rows from a release point. 

Movement within a row was much more likely than movement across rows (not surprising, due 

to the fact that plants within are row are more likely to be “touching” 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 optimal refuge design. Larvae moving across Bt and 

non-Bt corn rows may be exposed to sublethal doses of protein, increasing the likelihood of 

resistance (Mallet & Porter 1992). Given the extent of ECB larval movement between plants, 

seed mixes have been determined to be an inferior refuge option (Mallet & Porter 1992, SAP 

1998, Onstad & Gould 1998). 

ii. Adult Movement

Information on movement of adult ECB (post-pupal eclosion) is necessary to determine 

appropriate proximity guidelines for refuges. Refuges must be established within the flight range 

of newly emerged adults to help ensure the potential for random mating. An extensive, multiyear project to investigate ECB adult dispersal 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 majority 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 results have specifically led to recommendations and guidelines for 

refuge proximity and deployment (discussed later in this document). 

iii. Mating Behavior
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In addition to patterns of adult movement, ECB mating behavior is an important consideration to 

insure random mating between susceptible and potentially resistant moths. In particular, it is 

important to determine where newly emerged females mate (i.e., near the site of emergence or 

after some dispersal). 

It is well established that many ECB take advantage of aggregation sites (usually clusters of 

weeds or grasses) near corn fields for mating. Females typically 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 mating between susceptible 

and potentially resistant ECB (Hellmich 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 especially true in irrigated (i.e., attractive) corn fields. In addition, 

a relatively high proportion of females captured close to the release point (within 10 feet) were 

mated. This work suggests that females mate very 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 Showers 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 release point. Results showed that males in 

search of mates were trapped more frequently at traps placed at 200 m from the release site. 

However, significant numbers were also trapped at 800 m or greater from the release site 

(Showers et al. 2001). Similar to Hunt et al., this 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 important 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 primarily in aggregation sites, females 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. Oviposition 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 refuge should similar to the Bt

hybrid in terms of growth, maturity, yield, and management practices (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 same 
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inbred line) when phenological and management characteristics are similar (Orr & Landis 1997, 

Hellmich et al. 1999). Within a corn field suitable for egg laying, oviposition is thought to be 

random and not restricted to border rows near aggregation sites (Shelton 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, which 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 feasibility 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) have 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 are unlikely to produce 

enough susceptible adult insects. 

b) Corn Earworm

As was the case with ECB, the 1998 SAP identified a number of research areas that need 

additional work with corn earworm (CEW, Helicoverpa zea). In addition to increased 

knowledge regarding larval/adult movement, mating behavior, and ovipositional behavior, a 

better understanding of movement between corn/cotton and long distance migration is also 

needed (SAP 1998). Additional research regarding 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 improvements of IRM strategies to mitigate CEW 

resistance. 

i. Host Range and Corn to Cotton Movement

CEW is a polyphagous insect (3-4 generations per 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 generations (first generation on whorl stage corn, second 

generation on ear stage corn). After corn senescence, CEW moves to other hosts, notably cotton, 

for 2-3 additional generations. By utilizing multiple 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 hosts (weeds) and other nonBt crops (e.g., soybean) may be able to serve as refuge for CEW. However, research into the 

value of these alternate hosts as reliable producers of CEW is still lacking (1998 SAP). 
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ii. Overwintering Behavior

CEW are known to overwinter in the pupal stage. Although it is known that CEW migrate 

northward during the growing season to corn-growing 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 overwintering survival of CEW (Caprio & 

Benedict 1996). 

Overwintering is an important consideration for IRM--resistant insects must survive the winter to 

pass their resistance genes on to future generations. In the Corn Belt, for example, CEW 

incapable of overwintering should not pose a resistance threat. Given that different refuge 

strategies may be developed based upon where CEW is a resistance threat, accurate sampling 

data would help to precisely predict suitable CEW overwintering areas. 

iii. Adult Movement and Migration

CEW is known to be a highly mobile pest, capable of significant long distance movement. 

Mark/recapture studies have shown that CEW moths 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 pattern 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 move 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 this effect), the result may be additional CEW 

exposure to Bt crops. In addition, the assumptions regarding CEW overwintering may need to be 

revisited--moths that were thought to be incapable of winter survival (and thus not a resistance 

threat) may indeed be moving south to suitable overwintering sites. 

Most CEW flight movement is local, rather than migratory. Heliothine moths move primarily at 

night, with post-eclosion moths typically flying short distances of less than 200 m (Caprio & 

Benedict 1996). However, as was indicated by the 1998 SAP, additional research would be 

useful, particularly as it pertains 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 Behavior
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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 moved in prior to female eclosion (Caprio 1999). 

CEW females typically deposit eggs singly on hosts. A recent study (conducted in cotton fields) 

found that 20% of the eggs found from released CEW females were within 50-100 m (164-328 

ft.) of the release point, indicating some localized oviposition. However, males were shown to 

be able to move over 350 m (1148 ft.) to mate with 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 provide useful information for CEW IRM. 

v. Larval Movement

CEW larvae, particularly later instars, are capable of plant-to-plant movement. At the 

recommendation of the SAP (1998), EPA has not evaluated 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 required as a condition of registration. 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 the relative lack of information for SWCB, 

concluding that “[c]ritical research is needed for SWCB...including: 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 whether 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 management. Like ECB, SWCB has 2-4 generations 

and similar feeding behavior. First generation larvae feed on whorl tissue before tunneling into 

stalks before pupation, while later generations feed on ear tissue before tunneling into stalks. 

Females typically mate on the night of emergence and can lay 250-350 eggs (Davis 2000). 

Research to investigate the movement patterns of SWCB has been initiated (Buschman et al. 

1999). In this mark/recapture study, the following observations were made regarding SWCB 

from the 1999 data: 1) more males than females 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 feet; and 3) the moth movement patterns for ECB 

and SWCB appear to be similar in most regards. Given these results, it is likely that this part of 

the IRM strategy (refuge proximity guidelines established for ECB) will also be applicable to 

SWCB. However, the 1999 results were hampered by low SWCB numbers available for testing 

and the authors have indicated that this work will continue during the 2000 season. 
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Research for other secondary pests (e.g., black cutworm, fall armyworm, southern corn stalk 

borer, others) is also lacking and could be useful 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 research 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 resistant (i.e., heterozygous insects with one resistance allele) to 

survive, thus increasing the frequency 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 risk relative to high dose expression (Roush 1994, 

Gould 1998, Onstad & Gould 1998, SAP 1998, ILSI 1998, UCS 1998). To mitigate the 

additional resistance risk 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 available about the inheritance 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 indicating 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 techniques) 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 targeted 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 appropriate resistance management strategies could be 

developed. However, verification of the high dose using the 1998 SAP Subpanel techniques may 

be best directed at the major target pests of Bt corn (ECB, CEW, and SWCB), due to the fact that 

these pests play a larger role in the formulation of IRM strategies. Below, each registered Bt

corn product is discussed individually in regard to high dose (as defined by the 1998 SAP) for 

each of the labeled target pests. It is not expected that label claims 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 targeted against ECB (claims of “control”), SWCB 

(“control”), CEW (“control” of 1st generation, “suppression” of 2nd gen.), FAW 

(“suppression”), and SCSB (“suppression”). 
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Syngenta has not submitted any data to the Agency to confirm high dose, via the 1998 SAP 

guidelines, for any of the targeted pests. However, 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 maturity (VanDuyn et al. 1997, Catangui & Berg 1998). BT11 has also 

been shown to be effective against late instar ECB (Walker et al. 2000). 

For CEW, several submitted studies suggest that BT11 does not contain a season-long high dose. 

These studies revealed excellent control of first generation CEW on whorl stage BT11 but also 

showed significant survival of second generation 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 delayed developmental time). Other research has 

shown similar results (VanDuyn et al. 1998). 

For SWCB, no information on the potential for high 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 (Benedict et al. 1998). It is therefore unlikely that BT11 contains a 

full season high dose for FAW. For SCSB, one study with a limited data set has been submitted, 

showing good control (VanDuyn 1998). With additional 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 (“suppression”), 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 EPA, R.Rose/S.Matten memo to M.Mendelsohn, 

5/30/99). SAP techniques #2, 3, and 5 were utilized 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 pressure in the tests) to determine if there is a high dose expression. 

With additional data, it may be possible to determine whether there is a high dose expression for 

control of SCSB. 

For CEW, submitted studies have shown significant 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 unlikely 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 infestation later in the season (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 not been verified for SWCB or CSB with the 
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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, and 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; reviewed in R.Rose memo to M.Mendelsohn, 

1/24/01). 

Other submitted data showed that TC 1507 provided good protection against SWCB and FAW, 

although insufficient information was submitted 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 Attribute sweet corn expresses a high dose, although it 

has not been verified with the SAP criteria. Research submitted to EPA specifically for Bt sweet 

corn has shown virtually no survival 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, Whalen & Spellman 1999, Lynch et al. 1999). 

The current knowledge base for high dose expression 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 pest 
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3) Refuge

The February 1998 and October 2000 FIFRA SAP Subpanels agreed that a high dose/refuge 

strategy is necessary to mitigate 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. However, if there is not a high dose for the primary 

target pests, the risk of resistance increases. Larger refuges, increased monitoring, and possible 

sales restrictions may be used to mitigate some or all of this risk. 

a) Deployment of Refuges 

There have been a number of approaches proposed 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 research 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, irrigation, 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 host, non-corn refuges (e.g., weeds, oats, alfalfa, 

soybeans) are inadequate strategies (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 corn plants within or 

between rows with the majority of movement occurring within a single row. Later instar (4th 

and 5th) ECB are more likely to move within rows than between rows (Hellmich 1998). This is 

a cause for concern because heterozygous (partially resistant) ECB larvae may begin feeding on 

Bt plants, then move to non-Bt plants (if planted nearby) to complete development, thus 
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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 ideal strategy for an IRM 

program. Since the ECB larvae tend to move within rows, the authors suggest intact corn rows 

as an acceptable refuge. Narrow (filling one or two planter boxes with non-Bt corn seed) or wide 

strips (filling the entire planter with non-Bt seed) may be used as in-field refuges. Data indicate 

that in-field strips may provide the best opportunity for ECB produced in Bt corn to mate with 

ECB from non-Bt corn. Since preliminary data suggests that the refuge should be within 100 

rows of the Bt corn, Buschman et al. (1997) recommended alternating strips of 96 rows of non-Bt

corn and 192 rows of Bt corn. This would result in a 33% refuge 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 strips, 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 indicates 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 incomplete understanding of differences in survival 

between susceptible borers and heterozygotes (Onstad & Gould 1998). 

Given the concerns with larval movement and need for random mating, either external blocks or 

in-field strips (across the entire field, at least 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 variable 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. Therefore, 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). Subsequently, the recommendation was revised to 

specify that non-Bt corn refuges should be placed within 1/2 mile of the Bt field (1/4 mile 

“would be even better”) (Ortman 1999). 

Hunt et al. (1997) completed a study which suggests that the majority of ECB do not disperse far 
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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 (Hunt 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 experiment), 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 occurred very close to the point of emergence. Both of these 

observations indicate that many emerging ECB females may not disperse outside of their field of 

origin. With respect to resistance management and refuge proximity, these results suggest that 

refuges should be placed in close proximity to Bt corn fields (or as in-field refuge) to increase the 

chance of random mating (especially for irrigated fields). 

In terms of male ECB dispersal, another mark-recapture study by Showers et al. (2001) showed 

that males dispersing in search of mates may move 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 adequate to ensure mating between susceptible moths 

and any resistant survivors from the Bt field. 

While it is clear that ECB dispersal decreases 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. Since the greatest number of ECB were captured within 1500 feet of 

the field and most females may mate within ten feet of the field, placing refuges as close to the 

Bt fields as possible should increase the chance 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 historically used to treat ECB and SWCB) (EPA letter 

to Bt corn registrants, 1/31/00). The 2000 SAP agreed with this guideline, 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 refuges 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 received some attention as alternate strategies to 
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structured refuge to delay resistance. 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 preferentially oviposit on taller corn plants, the 

hope is that the Bt corn will be infested instead of the shorter, less attractive conventional corn. 

However, there are indications from experts in the field that temporal refuges are an inferior 

alternative to structured refuges (SAP 1998). Research has shown that planting date cannot be 

used to accurately predict and manipulate ECB oviposition rates (Calvin et al. 1997, Rice et al. 

1997, Ostlie et al. 1997b, Calvin 1998). Local climatic effects on corn phenology make planting 

date a difficult variable to manipulate to manage ECB. Additional studies will have to be 

conducted under a broad range of conditions to fully 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 primary pests of corn (ECB, CEW, SWCB) generally 

remain on the same plant throughout the larval feeding stages, individual insects will be exposed 

to only one of the proteins. In the absence of structured refuges producing susceptible insects, 

resistance may still have the potential 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 the Corn Belt. However, as discussed in the pest biology section 

(D.2.b.1.b.iii), observations of CEW north to south migration (from corn-growing regions to 

cotton-growing regions) have been noted. Although 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 structured refuge on an individual farm basis) due to the long distance 

movements typical of this pest (i.e., refuge proximity 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, there 
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may be additional risk for CEW resistance. As a condition of registration (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 management and submit a report to EPA. To accomplish 

this task, the Agricultural Stewardship Technical 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 migration of CEW in North America. Other 

noctuids, black cutworm (Agrotis ipsilon) and fall armyworm (Spodoptera frugiperda) have also 

been shown to migrate from southern overwintering areas to the Corn Belt. Long distance 

migration in all of these insects 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 indirect evidence for migration of CEW moths from the other corngrowing regions to the cotton-growing 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 reverse migration, there is very little vegetation 

that is capable of supporting larval development in Texas. This means that it is unlikely that any 

migrating moths (from the north) can contribute to the local over-wintering population. 

However, if the moths can move further south into more tropical areas in which they would 

contribute to the local over-wintering population then their genes 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 that a migrating population could be important. 

ABSTC noted that Gould et al. (2002) did not address the point that cotton in the areas 

investigated is treated for CEW either by Bt PIPs, applied chemical insecticides, or both. Bt PIPs 

and insecticides are very effective 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 treated for CEW. ABSTC suggested that 

care should be taken when interpreting 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 analysis, ABSTC used a computer model to quantify how migration 

may impact adaptation rates under a range of different 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-south migration in the summer. Effectively, two 

models were run in parallel for the summer generations: 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, alternations to the adaptation risk in that region are likely to be 
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quantitatively similar to the alternations to the risk 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 producing pupae, there was no effect of return 

migration on the resistance gene frequency. 

There was, however, a weak interaction between the percentage of Bt corn planted in the north 

and return migration. At 80% Bt corn in the north, return migration increased the 15-year gene 

frequency from 2.2e-3 to 2.4e-3, an increase in adaptation 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 the 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 than the resident population. The main decrease in adaptation rate 

due to return migration was 5%. 

Two of the parameters had significant main effects 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 feeding 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 adaptation rate by introducing a population that has experienced lower 

selection. However, this model suggests that even under this scenario, 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. Based 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 level 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 would be predicted to have a 10% impact on CEW 

adaptation. However, it is extremely unlikely that 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 Options
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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 east of the High Plains. The original USDA NC205 refuge recommendations included a 20-30% untreated structured refuge or a 40% refuge that 

could be treated with non-Bt insecticides (Ostlie et al. 1997a). In the case of ECB, the primary 

pest of corn for most of the U.S., it is known that on average less than 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 regularly treat for ECB, NC-205 modified their 

position in a May 24, 1999 letter to Dr. Janet Andersen (Director, BPPD). 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) insecticide treatment of refuges should be based on 

scouting and accepted economic thresholds, 2) treatment should be with a product that does not 

contain Bt or Cry toxin, 3) records should 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 intense in higher risk areas, for example where 

refuges are treated with insecticides” (Ortman 1999). 

Since most growers (>90%) do not typically treat field 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 stalk-boring insects with non-Bt insecticides or other appropriate IPM 

practices. Insecticide use should be based on scouting 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 are some areas that regularly require 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, may be most appropriate in the heavily treated 

areas jointly infested with SWCB and ECB” (Ortman 1999). The size of the refuge is based on 

the amount of non-Bt corn needed to produce 500 susceptible 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 larger to produce the 500:1 ratio. 
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Entomologists from Kansas State University (Dr. Randy Higgins, Dr. Lawrence Buschman, and 

Dr. Phillip Sloderbeck) have indicated that the frequent use of highly effective insecticides in 

areas that are co-infested with both SWCB and ECB is the issue of concern rather than the mere 

presence of SWCB. Using highly effective insecticides in these areas will decrease the number 

of susceptible insects emerging from the refuge and reduce refuge efficacy (Buschman and 

Sloderbeck 1999; Higgins 1999). The 2000 SAP rationalized 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 insecticides were specifically identified by the 

Agricultural Biotechnology Stewardship Technical Committee (ABSTC) in a letter to Dr. Janet 

Andersen dated March 31, 2000. This area includes counties in southwest Kansas, southeast 

Colorado, and the Texas/Oklahoma Panhandle. 

After reviewing the insecticide issue, the 2000 SAP concluded 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 additional 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 insecticide 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 interim report in 2003 and the final report in 

2004. ABSTC submitted a protocol which was accepted by the Agency on May 8, 2002 (EPA 

letter to ABSTC). An interim report was subsequently submitted on May 8, 2003 and the final 

report was submitted on May 7, 2004 (no MRID number was assigned). 

ABSTC employed two separate techniques to address the issue of insecticide use in refuges. The 

first was to conduct a survey of crop consultants to assess insecticide use 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 the effectiveness of refuges. 

For the survey (conducted in 2002), a total of 220 questionnaires were 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 were divided by reporting regions 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 regular control issues with ECB, SWCB, and spider mites. The 

west region experiences ECB and spider mite infestations (but little SWCB), while the east 

region primarily has ECB control issues (and little SWCB or spider mite problems). 
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The results showed that the vast majority of corn grown in all three regions 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 the 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 early 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 use was slightly less: 1.9% (Bt) and 3.8% (non-Bt). 

Treatments were applied on more acreage in the east region than the southwest or west 

regions, although the differences between regions 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 insecticide use in non-irrigated corn to drought conditions. 

Of the treated acreage, a minority was treated exclusively for corn borers (most reported 

treating for both borers and other 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 percentage of treated acres). In both the 2002 

and historical survey data, consultants generally reported treating small (i.e., <50%) 

percentages of their total acreage (most claimed to have treated <30%). However, the 

number of consultants who described treating 90-100% of their total 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 were 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. However, data were reported only for the Onstad model 

in the submission. 

Data regarding insecticide efficacy and use frequency was obtained from the consultant survey 

and applied to the models. In terms of efficacy, a value of 70% was assigned based on the lower 

effectiveness of insecticides on corn-boring lepidoptera. In practice, eggs and neonate larvae are 

more likely to be exposed and killed by insecticide treatments, while later instar larvae 

frequently lodge themselves inside corn plants and escape exposure. For insecticide use 

frequency, a value of 0.25 was assigned (i.e., 25% of refuges treated). This value was derived 
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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 generation (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 Onstad et al. (2002) for theoretical high dose 

(recessive resistance) and practical high dose (partially recessive resistance) 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 crop consultant survey) would have 

no impact on refuge effectiveness. Effects were noted only if resistance 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 complicated. 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 Onstad model. ECB dispersal is more limited in 

irrigated corn than dryland corn, as demonstrated by Hunt et al. (2001). When the reduced ECB 

dispersal characteristic of irrigated 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. Based 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 predicted that sprays on non-irrigated corn could 

decrease the time to resistance by a third. However, according to the consultant survey, nonirrigated corn is rarely treated (<10% of acreage) in the survey regions. For irrigated corn (more 

likely to be treated), detrimental effects on refuge from insecticide treatments were not observed, 

largely due to increased ECB dispersal. Overall, it is likely that any impact on potential ECB 

resistance from insecticide use in “high spray” 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 1999 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/cotton region corresponds to northern Arkansas, 
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Missouri Bootheel, northern Texas, and the states 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 southern corn/cotton region corresponds to the entire states of 

Alabama, Florida, Georgia, Louisiana, Mississippi, 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 (Mississippi State University) developed 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 other 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 important 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 effect was most pronounced when the percent of Bt to non-Bt

corn exceeded 50%. Caprio’s model suggests that even without cross-resistance 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 resistance. The years to resistance are also 

impacted by the percent of Bt cotton 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 resistance can develop rapidly when 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 significant 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 Carolina from 3% to 19% (total increase: 250,000 

acres), in Oklahoma from 7% to 20% (total increase: 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 justification 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, Memphis, 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 northern areas. If these CEW diapause in the northern 

areas and die over the winter, they pose no resistance problem. However, some indirect evidence 

has indicated that at least some CEW move from 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 the previous section. Based on the modeling studies 

submitted by ABSTC parameterized using the data in Gould et al. (2002), CEW reverse 

migration has no significant impact on CEW adaptation to Bt crops. 
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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 cotton is a preferred overwintering site for CEW, postharvest plowing of Bt cotton fields to destroy potentially 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 are no specific non-high dose products 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 review of all possibilities. 

Research regarding refuge 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 Sciences 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 varieties (ILSI 1998). The Union of Concerned 

Scientists (UCS) also suggested that a separate 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 may be necessary (Gould 1998, ILSI 1998, UCS 1998). 

Based on the ILSI and UCS reports, 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 Policy (1999), the percent insecticide 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. The 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 there is one generation per year, 

restricting the use of non-high dose events to these areas will likely 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 ILSI panel has recommended larger refuges for nonhigh dose or “high” risk Bt corn varieties. For areas where the refuge will be sprayed with 
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insecticides, the ILSI recommended an 80% non-Bt corn refuge (ILSI 1999). Since there may be 

an increased risk of resistance in areas that are 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% insecticide 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 include a detailed sampling strategy for all pests 

susceptible to the expressed Bt proteins regardless of whether they are stated on the label. For Bt

field corn and sweet corn, the susceptible pests would include, but are not limited to: ECB, 

SWCB, and CEW. To be effective, the monitoring for resistance should be undertaken in areas 

where the pests are known to regularly overwinter. For FAW and 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 Florida, 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 may 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 corn-growing areas but can be concentrated in 

higher resistance risk areas (SAP 1998, 2001). 

Dr. Blair Siegfried (entomologist, University of Nebraska) has indicated that at least 100 or more 

insects, with a target of 500-1000 insects, should be collected per 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 practices 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 techniques is critical to the 

success of an IRM plan. The following monitoring techniques can be considered as part of a 
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tiered approach to monitoring: 1) Grower reports of unexpected damage; 2) Systematic field 

surveying of Bt corn; 3) Discriminating concentration assay; 4) F2 screen; 5) Screening against 

resistant colonies; and 6) Sentinel Bt-crop field plots. These techniques were discussed in detail 

in the Introduction (section D.1). 

b) Agricultural Biotechnology Stewardship Technical Committee'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 growing season. The ABSTC plan was designed 

to concentrate resistance monitoring in areas where Bt corn market penetration is highest as well 

as areas with the highest insecticide use. The plan included 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 occur). Four corn-growing regions were identified 

with monitoring for each pest to occur in the regions in which the pests are prevalent. ABSTC 

proposed a sampling goal of 4-6 locations in Regions I and III and 2-3 locations in Regions II 

and IV. When possible, at least 200 first 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 collected. 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 detailed information to adequately 

evaluate the current resistance monitoring plans. The SAP Subpanel suggested that there be a 

“careful peer review to assess the adequacy of all Bt resistance monitoring programs.” 

A number of the October, 2000 SAP members indicated that the F2 screen accompanied by field 

screening “could be very effective for detecting low frequencies of recessive and dominant 

resistance alleles.” The F2 screen can be a powerful method for detecting rare recessive alleles in 

natural populations and is described in detail in the Introduction (section D.1.d.4). 

The time-frame to respond before control failures occur depends on the precision of monitoring 

and the recessivity or dominance of resistance. If the goal of resistance monitoring is to detect 

resistance at a low enough resistance allele frequency so that changes to the insect resistance 

management plan can be made to increase the longevity of the product and prevent field failure, 

then the ABSTC resistance monitoring plan needs further consideration. The F2 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. EPA/USDA 1999, p.47, Figure 1b). Hawthorne et al. 

(2001) concluded that there is a need to further evaluate the precision and accuracy of the F2

screen by using colonies with known frequencies of resistance alleles. Zhao et al. (2001) also 

came to this same conclusion. 
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The October, 2000 SAP Subpanel indicated that the diagnostic or discriminating dose technique 

could at best, detect resistance when the resistance allele frequency has reached 1%. This is a 

level in which some field failure may be observed. At this lower level of precision, the least 

expensive methods are the discriminating dose 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 that 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, the frequency of resistant individuals, and the 

number of samples that are collected. Therefore, as the frequency of resistant individuals or the 

number of collected samples increases, the likelihood 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 detection may not be low enough to detect resistance 

alleles prior to some field failure. Previous experience with conventional insecticides has shown 

than once resistant phenotypes are detected at a frequency >10%, control or crop failures are 

common (Roush & Miller 1986). Using the F2 screen could increase the probability of detecting 

rare resistant alleles, so that the threshold of detection would be lowered to <0.005 or 50-fold 

more sensitive than the diagnostic 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 those 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, there were approximately 40-50 counties 

that exceeded this level of market penetration. 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 sampling locations in Region I. The October, 2000 

SAP Subpanel indicated that it would be difficult to determine how many areas of high risk 

should be sampled, but that genetic differentiation of insect samples over large transects could 

help answer that question. Further evaluation 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 conditions 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 Conditions 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 addition, the terms mandate that registrants must follow up on 

reports of unexpected pest damage in the field for the major target pests (ECB, SWCB, and 
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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 discussions, 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 substance to the plan developed by ABSTC in 2000 and is based on two 

tiers of monitoring: reports 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 provided 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 collections only) is defined as southwest 

Minnesota, eastern South Dakota, southeast North Dakota, and northwest Iowa. The target will 

be to sample 4-6 ECB populations from Region 1. Region 2 (ECB and SWCB collections) 

covers southwest Kansas and the Texas/Oklahoma panhandle. From this region, 4-6 ECB 

populations and 3-4 SWCB (at least one from Texas) will be targeted 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 misrepresentative 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 overall 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 will be 50 larvae, 50 adults, 25 mated 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 needed. Therefore, all collected larvae and 

(unmated) adults will be reared and mated in the laboratory to obtain F1 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 utilized to detect potential increases in resistance allele frequency 

will be assessments of baseline susceptibility (LC50) and the discriminating dose assay. The 
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discriminating dose assay utilizes artificial diet with a high dose (LC99 or EC99) of Bt toxin such 

that only “resistant” insects will be detected – 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 population), the assays are designed to detect 

resistance allele frequencies at 1) 0.01 for high dose Bt corn and incomplete recessive resistance 

(probability of detecting a resistance allele in 400 genomes = 98.2%); 2) 0.002 for a moderate 

dose and dominant resistance (probability of detecting a resistance allele = 55.0%); 3) 0.075 for 

recessive resistance (probability of detecting 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 incompletely recessive) and scenario 2 applies to CEW 

(non-high dose, resistance likely to be dominant). The resistance detection thresholds are based 

on an analysis of population survival 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 terms 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 confirmed 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, the review recommended that ABSTC continue to 

research and develop techniques to improve the sensitivity and precision of the current 

monitoring strategy. Such techniques could include improved (larger) sampling and 

development of the F2 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 submission (MRID# 474070-01) contains a complete 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 collected annually, six for 

SWCB, and 10 for CEW. Additionally, the overall target for each population of 400 genomes 

(consisting of egg masses, larvae, adults, or mated females) with 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 resistance development (i.e., areas with large proportions of Bt corn 

adoption and pest pressure). However, ABSTC proposed to remove the specific sampling 

regions that had been established for ECB and SWCB monitoring. Four regions had been 

established for different parts of the Corn Belt including two regions for ECB sampling, one 
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region for SWCB sampling, and one region for both ECB and SWCB collections. Rather, the 

collections of populations will be taken from areas of “regional importance of the insect species 

as a pest,” but without specifically-defined sampling zones. ABSTC also proposed 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 low 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 and 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 frequency; 5) Analysis of whether the resistance 

allele frequency is increasing; 6) Determination of the geographic extent of the resistance allele 

distribution; and 7) Design of a remedial action plan if the resistance allele frequency is 

spreading. ABSTC’s new proposal included caveats that if the trait is found to be either not 

heritable or incapable of conferring the ability to survive on Bt plant tissue (i.e., #1 and 2 above), 

that the registrants will cease further investigative activity since the observed tolerance is not 

relevant for Bt corn in the field. In addition, ABSTC indicated that the analytical work will be 

initiated “as soon as practical” after low sensitivity is noted in the bioassays. 

EPA reviewed ABSTC’s proposal (A. Reynolds memo to M. Mendelsohn, 7/9/08) and agreed 

with ABSTC on the need for flexibility: 1) to identify areas (i.e., outside of the four previous 

collection regions) in which Bt corn adoption may be high or increasing; and 2) to be able to 

sample in areas with known high pest pressure. Both of these scenarios may be indicative of 

high selection pressure for resistance and would 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 representative of corn-growing regions in the U.S. 

Therefore, it was recommended that a caveat be added to the sampling plan to ensure that pest 

populations are collected from multiple corn-growing states reflective of different geographies 

and agronomic conditions. 

ABSTC’s 2008 revised monitoring plan keeps largely intact the follow-up investigations for 

populations with low sensitivity to Bt toxin(s). The Agency believes these procedures are a 

crucial component of the overall monitoring program and are needed to verify potential field 

resistance. On multiple occasions in the past (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. Confirmation of resistance can be an important, timesensitive process that should be completed quickly enough to ensure effective implementation of 

remedial action plans (if warranted). 
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c) Monitoring Results

EPA currently mandates that both baseline susceptibility and a discriminating concentration 

assay be employed for certain primary target pests 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 pests (e.g., BCW, FAW, SCSB) with focus on major 

economic pests. This information is essential to managing resistance in pest populations, 

especially in assessing whether a field control failure 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 populations. Continued monitoring efforts are needed to provide the 

Agency with standardized information to determine whether resistance is developing to the 

registered Bt toxins. ABSTC has submitted annual reports to EPA on resistance monitoring 

results for ECB, CEW, and SWCB with Cry1Ab and Cry1F as required by the terms of 

registration. The results of these monitoring reports 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 LC50 susceptibility determinations and 

diagnostic concentration (LC99) bioassays to determine susceptibility levels to Bt corn. In terms 

of baseline susceptibility (LC50), bioassays have been conducted for ECB (Siegfried et al. 1999a, 

Siegfried & Spencer 2000) and CEW (Siegfried et al. 2000, see CEW discussion in the next 

section). For 1999, ECB were collected from 14 separate sites and F1 and/or F2 generations were 

bioassayed to determine LC50s. Bioassays utilized dilutions of purified Cry1Ab obtained from Bt

kurstaki strain HD1-9 (provided by Novartis) spread on artificial diet. Neonate larvae were 

exposed to the diet less than 24 hours after hatching and mortality and larval weight were 

recorded seven days later. For 2000, 13 ECB populations were sampled using similar procedures 

with formulated Cry1Ab protein (CellCap, provided 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, susceptibility 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-resistant Cry1Ab core protein and produced 

results similar to the purified toxin used from 1995-1999. Monitoring results for ECB through 

the 2008 season are displayed in Table D3 and show no significant change in ECB susceptibility 

(LC50) to Cry1Ab over 13 years of testing (1995 - 2008). 

Table D3. Mean Susceptibility of ECB to Cry1Ab 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 LC50 (ng Cry1Ab/cm2) ± SEM Disc. Dose (% mortality) 
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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 

20001 (0.12 - 0.49) 99.7 - 100 

20011 (0.14 - 1.34) 2

 97.8 - 100 3 

20021 (0.18 - 0.47) 99.1 - 100 

20031 (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 directly comparable with results from other years. LC50 values for 

2000 - 2008 are given as a range (without SEM). 

2

 2001 included one outlier population with an LC50 of 1.34 ng/cm2. The range for the other populations was 0.14 - 

0.46 ng/cm2

. 3

 Three populations from 2001 had < 99% mortality at the diagnostic dose. 

4

 2004 included one tolerant population with an LC50 of 19.86 ng/cm2 and a 50.68% mortality at the diagnostic 

concentration. The other tested populations had LC50 values of 1.74 - 5.24 ng/cm2

 and discriminating dose 

mortalities > 99.6%. 

For 1999 diagnostic concentration analysis (LC99), baseline susceptibility studies conducted by 

Marçon et al. (2000) were used to determine the discriminating concentration for ECB. These 

tests with the discriminating concentrations were conducted in a similar manner to the bioassays 

to determine LC50 values. For 2000, a new discriminating dose (10 ng/cm2) was established for 

the CellCap Cry1Ab formulation. The results (for both 1999 and 2000 populations) showed 

nearly 100% mortality for ECB at the discriminating dose (LC99) (Siegfried et al. 1999a, 

Siegfried & Spencer 2000). For the change in toxin source during 2004, a revised concentration 

(LC99) was calculated (55 ng/cm2 Cry1Ab) based on dose/response assays done for ECB 

populations from Nebraska. This diagnostic concentration has been 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 concentrations 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 the discriminating dose 

assay. The second population was collected from Hamilton County, IA and was shown to have 

only 50.68% mortality to the Cry1Ab discriminating concentration (this population also 

demonstrated high tolerance to Cry1F – see the discussion in the next section). Procedures for 
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follow-up testing included the following: 1) Rearing additional generations from the populations 

(F2 and F3) for further discriminating dose testing; 2) Pooling and rearing survivors from the 

discriminating dose assays for additional testing (F3); 3) Testing with leaf discs (F3); 4) 

Continued selection of survivors at the diagnostic concentration (F4 - F7); and 5) Testing on 

whorl stage Bt corn (F7). 

For the Kandiyohi population, F2 larvae were retested at the diagnostic concentration and showed 

99.9% mortality. F3 larvae showed significant survival (48.8%) on Bt leaf discs, but those 

survivors were shown to have very low weights relative to the control group (non-Bt corn leaf 

discs). The colony was also tested on whole, whorl stage MON 810 plants in a greenhouse test. 

Individual corn plants were infested with Kandiyohi egg masses (F7, each generation selected 

with a diagnostic concentration) and were assessed for feeding damage 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 within the allotted two year time frame, the 

Kandiyohi population was determined not to be field resistant (reviewed in A. Reynolds memo 

to M. Mendelsohn, 2/4/04). Further, resistance monitoring in the Kandiyohi area in the 

following years (2002 - 2004) did not reveal any instances of decreased ECB susceptibility to 

Cry1Ab. 

For the Hamilton County, IA population, the original colony was reared for additional 

generations and subjected to diagnostic and dose response bioassays with Cry1Ab protein 

(described in MRID# 468749-01). Surviving larvae from the initial Cry1Ab diagnostic assays 

were separately pooled and reared as independent colonies. These “survivor colonies” were 

subjected to purified protein, leaf disc, and on-plant assays. Results of the bioassays indicated 

that the Hamilton ECB population continued 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 F7 and F8 generations, respectively. However, the F6 and 

F8 generation from this colony exhibited no survival on leaf discs obtained from Cry1Ab 

expressing plants compared with high rates of survival on non-expressing leaf discs from non-Bt

isoline plants. Separately, the Cry1Ab survivor colony showed intermediate (47.9%) survival 

and severe stunting on leaf discs from Cry1F-expressing plants when tested at the F6 generation. 

In the last set of tests, the survivor colony was unable to develop 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 from Hamilton Co. in 2005 and results from these 

assays indicated that the newly sampled populations did not have reduced susceptibility to 

Cry1Ab compared to control colonies. Further 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 region. Given these results, the Hamilton, IA 2004 

population was determined not to be field relevant despite the high survival noted in the 

diagnostic concentration assay (Hamilton, 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). 
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Cry1F

Resistance monitoring for Cry1F and ECB was initiated with the 2000 season. Similar to 

Cry1Ab, the program for Cry1F has been coordinated by Dr. Blair Siegfried (University of 

Nebraska) since the initial 2000 season. The sampling strategy and bioassays used for Cry1Ab 

(i.e., baseline susceptibility and diagnostic concentrations) are also employed for Cry1F. 

Baseline susceptibility (LC50) 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 condition of registration, Bt corn registrants were required to 

develop a diagnostic concentration for ECB, 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/cm2 Cry1F. This concentration has been used subsequently for 

ECB Cry1F monitoring and adequately addressed 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 (LC50) 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 LC50 range (ng Cry1F/cm2) 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 LC50 of 45.42 ng/cm2 and a 48.35% mortality at the diagnostic 

concentration. The other tested populations had LC50 values of 3.76 - 9.73 ng/cm2 and discriminating dose 

mortalities > 96.47%. 
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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 showed high tolerance to Cry1Ab (discussed in 

the next section below). A second population 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 (expected mortality > 99%). ABSTC 

initiated a follow-up investigation on this population (using the same procedures described in the 

Cry1Ab section above). This testing suggested some heritability of the trait (32.8% of F2

progeny survived at the same diagnostic concentration). However, low survival (10%) with 

growth stunting was noted on separate tests with Cry1F plant tissue and testing with F3 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 should be included as a 

collection site for Cry1F bioassays in subsequent growing 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 populations from that year with a Cry1F LC50 of 45.42 

ng/cm2 (compared to a range of 3.76 - 9.73 ng/cm2 for the other sampled populations). 

Additionally, the population had only 48.35% mortality on the Cry1F diagnostic concentration. 

Follow-up testing (conducted by Dow AgroSciences and described in MRID# 466958-01 and 

470112-01) focused on four major objectives: 1) The level and heritability of the potential 

resistance; 2) Survival on Bt (Cry1F) expressing plants; 3) The genetics of the potential 

resistance; and 4) The frequency of resistance in field populations from Hamilton County. 

Heritability testing was initially conducted on F7 and F8 generation of the HC ECB colony. 

Bioassays were performed with artificial diet and a diagnostic concentration of 60 ng Cry1F/cm2

(based on the LC99 of ECB), similar to the procedures used for the routine annual resistance 

monitoring of Bt corn. When exposed to this concentration in the trial, few F7 (2.7%) or F8

(0.9%) larvae were killed. These reported mortalities were substantially lower than the 48.3% 

mortality that was observed with the F3 generation in the original monitoring work. With such 

high survival, the investigators were unable to generate a dose-response curve to calculate LC50

or EC50 values. Subsequent bioassays were conducted on F13 and F18 generations using higher 

diagnostic concentrations of Cry1F. F13 larvae (previously unexposed to Cry1F since the F3

generation) were exposed to a diagnostic concentration of 600 ng Cry1F/cm2 (10x the standard 

concentration used for monitoring). These larvae 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 F18 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/cm2 (200-fold 
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greater than the standard diagnostic concentration). No detectable mortality or growth inhibition 

was observed at even the highest 12,000 ng Cry1F/cm2 concentration. Considering the 

extremely low mortality (< 5%) to the diagnostic concentrations observed in the generations (F7 - 

F18) after initial (F3) selection, Dow concluded that the Cry1F 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 LC50 for susceptible ECB. 

Dow also investigated the ability of the HC ECB colony to survive on Herculex Bt corn plants. 

Pest populations showing tolerance to Bt toxins are generally not considered “resistant” unless 

they are able to develop into adults on Bt expressing plants. Without the ability to survive on Bt

plants, tolerant populations will not 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 exposure to the Cry1F leaf disks (survival on the non-Bt

leaf disks was 90.6%). Larval weights of the HC colony averaged 0.42 mg for those on Cry1F 

disks and 0.71 mg for the non-Bt disks. By comparison, the unselected 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 greenhouse settings and were done with different 

generations (F7-F8 and F11) from the HC ECB colony. In the first set of assays, F7 and F8

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 (F7) 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 numbers of larvae from the control colony were 

recovered on isoline corn (>3 per plant), but only one larvae was found on Cry1F corn. HC 

larvae collected on Cry1F plants weighed less on average than those from isoline plants (~ 30 mg 

Cry1F vs. ~ 50 mg isoline). For reproductive stage corn (i.e., ear stage), similar numbers of 

larvae from the HC colony (F8) were recovered on both Cry1F and isoline corn, although unlike 

the whorl stage results high numbers were recovered 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 few larvae (<1 per plant) 

were found on the Cry1F corn plants. The investigators 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 F11 and F12 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 F11

larvae were recovered from either Cry1F or isoline corn. A total 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 damage from HC feeding (2.3 avg. Guthrie score) 
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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 F12 HC larvae (68 total) were recovered on Cry1F and isoline corn, 

while only three larvae from the control colony 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 recovered larvae). Fewer HC larvae completed 

development on isoline corn (38% diapause, 31% pupae, and 28% eclosion) than on the Cry1F 

hybrids. 

Based on the results from the greenhouse studies, Dow surmised that the Cry1F tolerance trait in 

the HC population allows larvae to grow and complete development on Herculex Cry1F corn 

plants. Although, it was noted that there are differences between greenhouse and field 

environments, the company concluded that the ability 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 structure of the Cry1F resistance 

trait. Diet bioassays and crosses with known susceptible (laboratory) ECB colonies were utilized 

to evaluate dominance/recessiveness of the trait and potential sex linkage. Crosses were 

conducted with HC (F13 and F18 generations) and control (Cry1F-susceptible) ECB colonies to 

produce F1 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 F13 generation HC was crossed with a 

laboratory control colony, while the F18 were bred with a field-collected 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/cm2). Both sets of crosses resulted in similar responses to 

Cry1F: HC ECB crossed with the susceptible colonies 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 the uncrossed control colony, although 

the crosses showed some increased ability to tolerate Cry1F relative to the control group. An 

EC50 or LC50 for the uncrossed HC groups could not be calculated because no mortality or 

growth inhibition was observed at the highest test concentration (12,000 ng/cm2). Given the 

results from the reciprocal crosses, Dow concluded 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 field populations from the county. To accomplish this 

objective, Dow (via ABSTC) conducted ECB sampling of the HC area during the 2005 growing 

season as part of the annual resistance monitoring work. Three populations were collected 

directly from HC (543 total ECB) and their F1 progeny were screened against the standard Cry1F 

diagnostic concentration (60 ng/cm2). 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 resistant to the Cry1F toxin. The colony met all of the major 
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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 appeared to be the first documented case of pest 

resistance to a high dose Bt toxin in corn; though it should also 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 field-level effects are not observed. 

Dow contends that because the trait was not detected in 2005 (and beyond), the resistance allele 

frequency was below the level of concern and a remedial 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 incidents of increased tolerance or field failure to 

Cry1F corn. Additional EPA reviews 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 assays have been conducted by Custom BioProducts (Maxwell, IA). Similar to ECB, monitoring is based on susceptibility bioassays and 

diagnostic concentration tests. 

For CEW, baseline susceptibility (LC50) values to Cry1Ab ranged from 70.3 ng/cm2 (lab colony) 

to 221.3 ng/cm2 (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/cm2), which showed nearly 100% mortality (Siegfried et al. 1999b). This diagnostic 

dose has been revised since this initial assessment, largely due to the fact that CEW is less 

sensitive to Cry1Ab then other lepidoptera. The insect frequently exhibits highly variable 

responses to the toxin, such that it has been difficult to determine a reliable diagnostic 

concentration based on an LC99. Monitoring in 2001-2 used a concentration of 40 ng 

Cry1Ab/cm2. In 2004, ABSTC increased the Cry1Ab diagnostic concentrations to 80 ng/cm2 for 

the CEW monitoring. Despite this increase, a number of populations 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 field sites and shipped to Custom BioProducts for establishment of test colonies. Bioassays are conducted with neonate larvae from 

the established colonies. Baseline susceptibility (LC50 and EC50) and diagnostic concentration 

bioassays are performed in accordance with the methods used by Siegfried et al. (2000). 
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The results of LC50 and diagnostic concentration assays are summarized in Table D5 below. 

Susceptibility data (LC50 ranges) have been reasonably consistent over the tested growing 

seasons and support the conclusion that there has been no documented CEW field resistance to 

Cry1Ab. The range of LC50 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 LC50 values on the high end 

of the historical range (i.e., > 8.0 ng Cry1Ab/cm2), though most of the populations collected 

during this season had LC50s of < 4.5 ng/cm2. 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. The diagnostic concentration that has been used since 2004 

(80 ng Cry1Ab/cm2) also appears to have remained functional (i.e., mortality > 99% in 

susceptible CEW colonies) and is an improvement over the previously used concentration of 40 

ng Cry1Ab/cm2, though several populations in 2008 had slightly less than 99% mortality. 

Table D5. Monitoring Results for CEW and Cry1Ab and Cry1F Corn from 2001 - 2008 

(Custom Bio-Products annual reports 2002 through 2009) 

Cry1Ab Cry1F

Year 

LC50 range (ng 

Cry1Ab/cm2) 

Disc. Dose (% 

mortality range) a

LC50 range (ng 

Cry1F/cm2)

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/cm2; for 2004 - 2007, concentration = 80 ng 

Cry1Ab/cm2

b

 Cry1F discriminating concentration for 2001 = 5000 ng Cry1F/cm2; for 2002-3, concentration = 7500 ng 

Cry1F/cm2; for 2004, concentration = 54,675 ng Cry1F/cm2; for 2005-8, high concentration = 4,000 ng Cry1F/cm2

Cry1F

Resistance monitoring for Cry1F and CEW has also been conducted by Custom Bio-Products 

using the same paradigm for Cry1Ab. Both the susceptibility and diagnostic concentration 

testing with Cry1F have shown broad 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/cm2 but was 

increased in 2002 to 7500 ng Cry1F/cm2, based on the results from 2001. However, even the 
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higher concentration failed to consistently produce mortalities > 99% in collected populations 

indicating that the concentration was actually less than the LC99 for CEW. In 2004, the dose was 

further increased to 54,675 ng/cm2, a level at which all sampled populations had >99% mortality. 

However, in 2005 the dose was reduced to 4000 ng/cm2 without explanation from ABSTC. This 

concentration has been used since 2005 (through the 2008) season but has frequently produced 

mortality below 99% in the bioassays. No follow-up testing has been reported for any of the 

populations in the diagnostic assays. In light of these results, it is apparent that 4,000 ng/cm2 is 

not acting as a functional diagnostic concentration (i.e., a benchmark that triggers investigation 

of potential resistance) and has limited value as a monitoring tool. It has been recommended that 

ABSTC either reestablish the higher test concentration (54,675 ng/cm2) 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 summarized in Table D5 above. Given the high 

variability in LC50 ranges, it is difficult to discern year-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 Chippendale (1999) to determine SWCB 

susceptibility to Cry1Ab and establish a diagnostic concentration. A bioassay was conducted 

that established a diagnostic concentration for SWCB of 110 ng Cry1Ab protein/g diet. 

Susceptibility data (LC50s and EC50s), determined after 7 and 14 days of exposure to Cry1Ab, are 

summarized in Table D6 below. SWCB monitoring has been conducted since the 2000 growing 

season by Dr. Qisheng Song at the University of Missouri using similar methodology (Song et al. 

2000) to obtain susceptibility data (LC50s and EC50s) and perform diagnostic concentration 

assays. A diagnostic concentration assay was performed (7 day test dose = 0.35 μg Cry1Ab/g 

diet, 14 day test dose = 5 μg Cry1Ab/g diet), which resulted in 100% mortality for all tested 

populations. The 5 μg Cry1Ab/g concentration has been used as the diagnostic dose since 2000. 

For Cry1F, baseline susceptibility assays provided an estimate for the SWCB LC50 of 0.70 μg 

Cry1F/cm2 diet (MRID# 450201-01; reviewed in R. Rose memo to M. Mendelsohn, 1/24/01). 

Annual Cry1F monitoring formally started with the 2001 growing season. The 2001 SWCB 

susceptibility tests resulted in a LC50 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 populations; a lab colony was also tested (7-day 

LC50 = 27.20 μg Cry1F/ml; 14-day LC50 = 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 initial 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. 
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SWCB monitoring data are summarized in Table D6 below. Taken together, the SWCB 

monitoring results show that, to date, no appreciable 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 collected populations. Furthermore, 

there have been no survivors to the 14 day diagnostic concentration in any of the years tested or 

reports of unexpected pest damage in Cry1F corn due to SWCB. It is noted 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 mortality 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 compared to a control), and variation may be minimized. 

Table D6. SWCB Susceptibility (LC50) to Cry1Ab and Cry1F from 1998 to 2008 (Trisyono 

and Chippendale 1999; Song et al. annual reports 2000 to 2009) 

Cry1Ab Cry1F 

Year LC50 (μg Cry1Ab/ml

diet) - field 

populations 

Discriminating Dose 

(5 μg Cry1Ab/g) 

(percent mortality) 

LC50 (μg Cry1F/g

diet) - field 

populations 

Discriminating Dose 

(68.6 μg Cry1F/g) 

(percent mortality) 

19981 7-day: 0.22 - 1.09 

14-day: 0.04 - 0.09 -- – -- 

19991 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 μg Cry1Ab/g diet for LC50 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 Rico
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Separately, Dow AgroSciences and Pioneer Hi-Bred reported to EPA that unexpected 

Spodoptera frugiperda (fall armyworm) damage had occurred 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 (detailed 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 damage 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 lower susceptibility to the Cry1Fa protein than the control, 

Benzon colony. The screening level assessment of the Cry1Fa-sensitivity of F1 progeny 

from the field-collected larvae at 10,000 ng Cry1F/cm2 revealed no significant mortality 

of either Puerto Rico collection on the Cry1F-treated diet. Testing of the F2 progeny 

did not show a significant concentration response to Cry1F, nor did these progeny show 

a significant reduction in growth. The resistance ratio was >167, but an exact level 

could not be calculated for the Puerto Rico populations because the LC50s could not be 

determined. 

A specific remedial action plan for FAW resistance 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 secondary 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. This 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 confirmation of FAW resistance Puerto 

Rico (described in a letter from Dow AgroSciences to EPA, dated April 23, 2009): 

• Existing customers were notified and recommended to use insecticides (as needed) to 

control FAW damage on existing TC1507 commercial fields. 

• The two registrants took steps to discontinue commercial sales of TC1507 maize in 

Puerto Rico by terminating grower agreements and revising product use guides to 

prohibit plantings in Puerto Rico. 

• FAW populations were managed in maize research 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 reintroduce Cry1F corn to Puerto Rico. 

• Additional FAW sampling was conducted during 2007 and 2008 in Puerto Rico, which 
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also showed similar tolerance to Cry1F. 

Several unique factors likely contributed to the development of Cry1F-resistant FAW in Puerto 

Rico. These factors include: 

• An island setting provides an isolated ecosystem that reduces insect migration; 

• A tropical climate allows for year-round 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 continually exposed to Cry1F maize (TC1507) 

after its introduction into Puerto 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 reduce the likelihood of FAW resistance in the 

continental U.S. FAW is a migratory seasonal pest. The species cannot develop at temperatures 

below 50°F and, as a tropical species, do not diapause. FAW only survive winter in the extreme 

south of Texas and Florida and thus, selection for Cry1F-resistance in maize-growing areas 

exerts no long-term selection pressure. In the continental U.S., there are only one or two 

generations per year on maize. Alternate host 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 continental U.S. and this market mix reduces the 

selection pressure on either protein. 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 bioassay and concentration-dependent bioassay, EPA 

concluded that the unexpected performance 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 first 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 response 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 widespread. A specific remedial action plan should clearly 

indicate what actions the registrant will take in cases of “suspected” resistance (i.e., unexpected 

damage) and “confirmed” resistance. The remedial action plan can also include appropriate 

adaptations for regional variation and the inclusion of appropriate stakeholders. To fully 

mitigate resistance, a critical element of any remedial action plan should be that once pest 
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resistance is confirmed, sales of all Bt corn hybrids that express a similar protein or a protein in 

which cross-resistance potential has been demonstrated 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-Expressed Protectants in Field Corn” (submitted 

4/19/99). The language of the ABSTC plan is as follows: 

“Customers (growers and seed distributors) will be instructed to contact the registrant or 

authorized distributor if incidents 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 populations, followed by appropriate in vitro and in 

planta assays. Upon confirmation by immunoassay that the plants contain the 

appropriate Cry1A/Cry1F protein, bioassays will be conducted to determine whether the 

collected insect population exhibits a resistant phenotype. 

Where available and validated for a target pest species, a discriminating concentration 

assay will be employed to define a confirmed instance of resistance. For other target 

pests, until such time that a discriminating concentration assay is established and 

validated, registrants will utilize the following to define a confirmed instance of insect 

resistance: 

Progeny from the sampled pest population will be considered resistant if they 

exhibit BOTH of the following characteristics in bioassays initiated with 

neonates: 

1. An LC50 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 LC50 for susceptible pest populations, as established by the 

ongoing baseline monitoring program. 

2. > 30% survival and > 25% leaf area damaged in a five-day bioassay using the 

appropriate Cry1A/Cry1F-positive leaf tissue under controlled laboratory 

conditions. 

Based upon continued experience and research, this working definition of confirmed 

resistance may warrant further refinement. In the event that the registrants find it 
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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 agreed 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 been confirmed to be 

Bt corn, that the seed used had the proper percentage of corn expressing Bt protein, that 

the relevant plant tissues are expressing 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 failure 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 before responsive measures are undertaken.” 

Two other elements that could further mitigate the 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 measures to control the pest suspected of 

resistance to Bt corn in the affected region. 

2) The destruction of crop residues in the affected region immediately after harvest (i.e., 

within one month) with a technique appropriate for local production practices to 

minimize the possibility of resistant insects overwintering and contributing to the next 

season’s pest population. 

A panelist on the 2000 SAP also noted that given the logistics of monitoring, it may take two 

years from resistance detection to remedial action plan implementation. During this period of 

“suspected” resistance, the panelist noted that increasing refuge size could help to prolong 

susceptibility (SAP 2001). 

b) Remedial Measures in Confirmed Cases of Insect Resistance 

In cases of “confirmed” resistance (as defined 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 identification of a confirmed instance of resistance, 

registrants will take the following immediate mitigation measures: 

1. Notify customers and extension agents in the affected area, 
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2. Recommend to customers and extension agents in the affected area the use of 

alternative control measures to reduce or control the local target pest population, 

and 

3. Where appropriate, recommend to customers and extension agents in the 

affected area that crop residues be incorporated into the soil following harvest, to 

minimize the possibility of overwintering insects. 

Within 90 days of a confirmed instance of pest resistance, as defined above, registrants 

will: 

1. Notify the Agency of the immediate mitigation measures that were 

implemented, 

2. Submit to the Agency a proposed long-term resistance management action plan 

for the affected area, 

3. Work closely with the Agency in assuring 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 consists of some or 

all the following elements, as warranted: 

a. Informing customers and extension agents in the affected area of pest 

resistance, 

b. Increasing monitoring in the affected 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 measures in the affected area based 

on the latest research results. The implementation of such measures will 

be coordinated by the Agency with other registrants; and 

e. If the above elements are not effective in mitigating resistance, 

registrants will voluntarily cease sale of all Bt corn hybrids subject to the 

Industry IRM Plan in the county experiencing loss of product efficacy and 

in the bordering counties until an effective local management plan 

approved by EPA has been implemented. During the voluntary suspension 

period, registrants may sell and distribute 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. 
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If EPA agrees that an effective 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 confirmed 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 mitigation 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 confirmed 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 region (this was mandated in the 

1/31/00 letter). 

2) The mandatory use of alternate control measures and 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 remain in place until resistance has been 

determined to have returned to acceptable levels. 

b) The development and use of alternative resistance management strategies for 

controlling the resistant pest(s) on corn in the affected region. 

c) Notification of all relevant personnel (e.g., growers, consultants, extension 

agents, seed distributors, processors, university cooperators, and state/federal 

authorities) in the affected region of the resistance situation. 

d) Intensified monitoring and surveillance in the affected region(s) for resistance 

and definition of the boundaries of the affected 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 suggested 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 causing their decline) may prove more practical. As 

part of a plan to slow resistance genes, the SAP suggested the following elements: 1) Education 
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of growers/crop consultants to look for unexpected pest damage; 2) Monitoring for plant 

damage, pest susceptibility, and resistance allele frequency (with rapid verification and alternate 

control strategies for verified resistance); 3) Sales suspensions of the affected product in the 

region until it can be shown that the product’s benefits 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 plan to adjust the process 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 demonstrate: 1) 30% survival and commensurate 

insect feeding in a bioassay representative 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 and a resistance allele frequency ≥ 0.1; 3) a LC50 in a standardized 

laboratory bioassay that exceeds the upper 95% LC50 confidence interval for a susceptible 

population. For the first standard, ABSTC’s revised plan removed CEW, removed the explicit 

requirement to test on Bt corn plant tissue, and eliminated criteria 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 “suspected” 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 (2000) plan included requirements to be undertaken 

within 30 days, 90 days, and 1 year from the event 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 pest species, the extent of resistance, the 

timing of resistance, and the nature of resistance, and the availability of suitable 

alternate control measures), alternative control measures will be employed to reduce or 

control target pest populations in the affected area. Alternative control measures may 

include advising customers and extension agents in the affected area to incorporate 

crops residues into the soil following harvest to minimize the possibility of overwintering insects, and/or applications of chemical insecticides; 

• Unless otherwise agreed with EPA, sale and distribution of the relevant lepidopteranactive Bt corn hybrids will stop 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 
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90 days according to the characteristics of the resistance event and local agronomic 

needs. The registrant will consult with appropriate 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 parties (e.g., growers, consultants, extension agents, seed 

distributors, university cooperators and state/federal authorities as appropriate) in the 

region of the resistance situation and approved action plan; and 

• In subsequent growing seasons, sales suspension 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 until an EPA-approved local resistance 

management plan is in place to mitigate the resistance. 

6) Cross-Resistance

Cross-resistance is an area of major 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 pest 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 (Cry1Ab and Cry1F are assessed in 

this document, though other Bt toxins have been registered 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 complex 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 insect gut. Modifications to these 

insect crystalline protein receptors have been implicated in resistance to Cry proteins. Other 

mechanisms that may lead to resistance (and ultimately cross-resistance) include protease 

inhibition, metabolic adaptations, gut recovery, and behavioral adaptations (Heckel 1994, 

Tabashnik 1994). 

Regarding binding sites, cross-resistance may result if two proteins 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 will be affected as well. An example of a possible 

shared binding site resulting in cross-resistance was observed with tobacco budworm (TBW). In 

this case, TBW selected for resistance to Cry1Ac were also found to be resistant 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 was 
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204 

characterized by performing binding experiments with both isolated brush border membrane 

vesicles and gut tissue sections (Denolf et al. 1993). Results demonstrated that two independent 

insecticidal crystal protein receptors 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 epithelial cells. Other experiments 

using laboratory-selected resistant strains to predict survival and cross-resistance 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 cross-resistant to Cry1Ab and that Cry1Ab-resistant ECB are not 

cross-resistant to Cry1Ac (Hutchison, personal 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 pests, 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 probability of cross-resistance developing to Cry2A 

delta endotoxins from resistance to Cry1Ab or Cry1Ac. Because the Cry1A and Cry2A proteins 

exhibit different binding characteristics and very low amino acid homology, they likely possess 

different modes of action. However, there is some evidence for the development of broad crossresistance to Cry1 and Cry2A in at least two laboratory-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 Cry1Ab, Cry1Ac, and Cry9C 

in ECB. The results showed that Cry1Ab likely 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. Mendelsohn, 1/24/01). However, a second published study 

to assess midgut receptor binding patterns for Cry1Ab and Cry1F in ECB (Hua et al. 2001) 

suggested that Cry1F and Cry1Ab only weakly compete for BBMV binding sites. As in the 

previous study, ligand blotting analysis showed that Cry1Ab and Cry1F recognize multiple 

binding proteins in ECB. Both toxins recognized 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 assays, but only at the highest tested 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 colonies 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 low levels (< 5 fold) of cross resistance with 

Cry1F, but not Cry9C (Siqueira et al. 2004). Subsequently, a Cry1F resistant ECB strain was 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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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 still highly susceptible to Cry1Ab and Cry9C. 

Cry1F was only slightly (7 fold) cross resistant with Cry1Ac. 

Collectively, laboratory-selected strains and isolated 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 Cry1Ab. However, cross-resistance patterns and 

physiological mechanisms are complex and unpredictable, 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 cross-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 were 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. Results from the survey should assess levels 

of grower compliance with refuges as well as grower motivations, attitudes, and 

reasons for non-compliance. 

• On-farm assessments: Registrants are required 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 with refuge requirements is assessed and 

growers out of compliance are identified 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 consistent set of procedures (for all Bt corn 

registrants) to be employed to address non-compliance among growers and seed 

dealers. 

The Bt corn registrants (under ABSTC) submitted a proposed CAP to the EPA in 2002. After 
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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 reviews of annual growing season 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. Milofsky memo to M. Mendelsohn, 8/30/07 

• 2007 and 2008 growing season report: J. Martinez memo to M. Mendelsohn, 4/15/09 

b) Grower Surveys (1996-2001) 

Several surveys and estimates of the level of grower compliance for Bt corn IRM were 

conducted prior to the development and implementation of the 2002 ABSTC CAP. Dr. Marlin 

Rice (Iowa State University) conducted regular grower 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 understood 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 strategy, 57.1% would if compatible with their 

growing practices, 7.2% would not follow IRM, and 12.2% “didn’t know” (Pilcher & Rice 

1997). Results from the 1998 grower survey showed 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 surveys of 501 total growers, each farming at least 200 acres. This 

survey did not involve visits to individual farms (i.e., grower audits). Compliance was assessed 

for two Bt corn IRM requirements: percent refuge (required 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 refuges planted within 1⁄2 

mile of the Bt field (18% reported refuges planted greater than 1⁄2 mile from the Bt field). When 

both refuge percentage and proximity are considered together, 71% of growers were in total 

compliance. It should be noted that growers were 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 planting a refuge of less than 50%, but greater than 20%. 
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Collectively, these surveys indicate that 100% compliance 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 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., audits), it may be impossible to verify the accuracy 

of grower responses. The end result could be increased “false-positives,” 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 independent parties to assess grower 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 registrations in 2001, registrants were required to 

perform an “annual survey of a statistically representative sample of Bt corn growers conducted 

by an independent third party” (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 educational 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 conducting 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 Horizons started to utilize an internet-based 

survey approach due to an increasing complexity of growers’ Bt corn planting practices and a 

need to standardize the grower survey across insect-protected traits. Consistency in 

methodology is important because it allows for year-to-year comparisons of the results. There 

are four sampling regions that differ 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 samples 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 Carolina, South Carolina, Southeast Missouri, 

Tennessee, Mississippi region). Results are weighted to reflect the actual distribution of corn 

acres in each region. 

Data from the 2002 through 2008 compliance surveys are summarized in Table D7 below. In 

2007 and 2008, the online survey results were not broken down for the four regions, as was done 

in previous years, and therefore, 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 that it can be determined in which farming regions the registrants 
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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 the lowest level since initiation of the 

surveys in 2000. A report issued by the Center for Science in the Public Interest (CSPI) that 

analyzed the same data set also highlighted this 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 refuge (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 increased efforts by registrants and the National Corn 

Grower Association (NCGA) to provide more outreach tools, improved educational efforts, and 

more avenues of communication with growers. In addition, grower awareness of IRM 

requirement has consistently increased since 2002 through 2008, suggesting that there is a degree 

of willful non-compliance by some growers. The reasons for the increase in non-compliance are 

not clear but could include financial 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 from ABSTC annual reports 2002 through 2008)1

Survey Year

Survey Question 2002 2003 2004 2005 2006 2007 2008 

-----------% respondents----------- 

Adherence to Refuge Size

Weighted average across all 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 all four regions surveyed 89 93 96 96 96 885 885

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/A 
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Survey Year

Survey Question 2002 2003 2004 2005 2006 2007 2008 

-----------% 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 634 794

Refuge distance 

(weighted average across all four regions surveyed) 

37 49 55 53 49 794 894

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 plant more than 200 corn acres in the Corn Belt 

and 100 cotton-growing areas. Annually, 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 associated 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 annual survey shall consist of a “statistically 

representative sample of Bt corn growers.” Since corn borer-protected corn is grown on farms of 

all sizes, a survey that excludes responses from smaller growers would fail to provide a 

“statistically representative” picture of all growers 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 Agricultural Statistics Survey (NASS) data from 10 corn-growing 

states found substantially lower IRM compliance on small farms (<200 acres). This finding may 

have been indicative of inadequate grower 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, October and November, 2005). ABSTC 

provided four reasons as to why the third-party survey should continue to exclude growers with 

low acreage: 
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1. The farm size restriction ensures that the survey covers the largest number of corn 

acres. 2002 USDA/NASS statistics indicated that nearly 88% of all Bt corn in the 

10 states sampled in 2002 was planted on farms 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 significantly 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 farmers 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 growers with larger acreages. It was recognized 

that the grower survey is not a direct measurement of individual compliance and that the on-farm 

assessment program (intended to detect non-compliant individuals) includes all growers 

regardless of size. An analysis of the 2003 NASS data indicated 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 assessment. According to this same data set, less 

than 5% of farms in the 10 states would have been classified as potentially contributing to a 

localized cluster of small farmers growing Bt corn that could have led 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 farms. 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 increase the risk of resistance evolution to Bt corn. 

d) On-Farm Assessments 

The on-farm assessment program is the portion of the CAP that identifies 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 obstacles growers face in 

implementing IRM requirements. The mandatory on-farm assessment program was fully 

implemented for the first time in 2003 and has typically encompassed approximately 2,000 - 

2,200 growers. Company representatives are trained to conduct on-farm assessments. Topics 

covered in these trainings include IRM requirements, messages that should be communicated to 

growers, follow-up actions if growers are found to be out of compliance and how to use the IRM 

assessment form. All assessment forms contain the same questions, but the introductory 

paragraphs and company representative sections are customized to suit the needs of each 

registrant. Registrants respond to all compliance deviations identified in the assessments 

according to the common set of standards outlined in the PCA. 
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Data from the on-farm assessments (2003 through 2008) of corn borer-protected corn are 

summarized in Table D8 below. The assessments do not have the statistical power associated 

with the consistently stratified 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 non-compliance with refuge size and distance. 

This information should be provided in future reports to be consistent with previously collected 

data and to illustrate how growers are out of compliance (see EPA review in J. Martinez memo 

to M. Mendelsohn, 4/15/09). 

Table D8. Cumulative Results (2003-2008) for 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 other 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 deviations does not equal the number of non-compliant 

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 have 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 was developed by ABSTC and implemented for the 

first full-season in 2003. It allows each registrant to develop a “tips and complaints” system that 

is “compatible with their business 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 number is publicized through a variety of IRM 

communications mechanisms (e.g., technical bulletins, grower guides, letters, seed catalogs, 

newsletters, etc.). 
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The number of tips and complaints (for all Bt corn registrations) received through 2008 is 

summarized in Table D9 below. Each of these 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 grower communications to clearly state 

how growers can register a tip or complaint about an allegedly non-compliant grower by calling 

a toll free number or by contacting a company representative” (October 4, 2004 letter to EPA). 

Since the 2005 growing season anonymous tips have been reported, though they have been 

relatively rare. 

Table D9. Anonymous Tips and Complaints 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 standard 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 noncompliance 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 regions) significant 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 Bt Corn Refuge NonCompliance (submitted with the ABSTC 2002 CAP)
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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-compliant (significant or other deviations) are required to receive a 

“compliance assessment contact” the following year under the PCA. Non-compliant growers are 

typically identified through the on-farm assessment program (see discussion in the on-farm 

assessment section below). Table D11 summarizes the numbers of non-compliant growers 

reassessed under the PCA and the growers still found 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 significant non-compliance. 

Table D11. Reassessment of Non-Compliant Corn Borer-Protected Corn Growers Under 

the Phased Compliance Approach (taken from ABSTC annual 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 not 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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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 identified several concerns with the overall 

approach (see T. Milofsky memo to M. Mendelsohn, 7/15/04). First, one year of seed 

suspension may be insufficient to reform noncompliant growers. Second, it is unclear how the 

PCA will address repeat violators. However, the PCA results indicate that the reassessment 

process and the penalty for repeated non-compliance in the second year have been effective in 

bringing non-compliant growers back into compliance. 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 element for the implementation and success of any IRM 

plan as they will ultimately be responsible for 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 may help increase IRM compliance (SAP 2001). 

Ideally, the educational messages presented to growers should be consistent (among different 

registrants) and reflect the most current resistance management guidelines. Specific examples of 

education tools for growers can include grower guides, technical bulletins, sales materials, 

training sessions, Internet sites, toll-free numbers 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 requirement of registration) on Bt corn sales/market 

penetration, IRM-related research, grower education, grower compliance and resistance 

monitoring. It is particularly useful 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 dose 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 (before most lepidopteran 

larvae complete development); and 2) all Bt sweet corn residues were to be destroyed within one 
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215 

month of harvest (a practice that presumably would destroy any live larvae left 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 stipulate that, based on IRM concerns, 

the product is for commercial use only and is not available to growers planting less than 40 acres. 

However, should smaller growers (i.e., those planting less than 40 acres) adhere to the crop 

destruct requirements for Bt sweet corn (to destroy any overwintering insects), it is unlikely these 

growers will pose a threat to pest resistance given the limited acreage involved. As such, from 

an IRM perspective, it should be possible to lift the acreage restrictions on smaller growers for Bt

sweet corn. 

Regarding crop destruction, it is possible that the 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 different crop destruct techniques 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 mitigated by either: 1) Prescribing a 

specific and effective crop destruct technique; or 2) Utilizing structured refuge. Regarding 

option #1, it should be noted that corn 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 most 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 development in corn ears (unlike stalk-boring ECB), 

which are mostly harvested and removed from the 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 monitoring program has been developed to determine 

susceptibility to Cry1Ab (other Bt sweet corn target pests, ECB 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 overlays for FAW populations 

collected from four geographic locations in 1998 and 1999. For 1998, the LD50 range was 0.90 - 

1.50 μg Cry1Ab/cm2

 and for 1999, the LD50 range was 2.14 - 10.22 μg Cry1Ab/cm2 (Lynch et 

al. 2000). Susceptibility data for FAW and Cry1Ab are summarized in Table D12 below. The 

decreased susceptibility observed in 1999 was not likely an increase in tolerance to the toxin (the 

lab colony used as a control showed similar trends) 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 likely. However, should there be significant Bt sweet corn acreage in areas 

where FAW overwinters (south Florida and south Texas), it would be beneficial for FAW to be 

monitored for resistance. As a term of registration, FAW monitoring was required for Attribute 

Bt sweet corn in counties where acreage exceeds 5,000 acres and FAW is capable of 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

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overwintering. However, FAW data have not been submitted since the 2000 growing season 

because this 5,000 acre/county requirement has not been 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 (LC50) to Cry1Ab (Lynch et al. 2000, Hamm et al. 2001)

Year Susceptibility (LC50 - μg 

Cry1Ab/cm2) 

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 complexes. Complete discussions of corn rootworm 

IRM (as well as combination refuges for lepidoptera 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) – EPA Reg. No. 67979-8 (Cry1Ab, Cry3A) 

• Bt11 x MIR 162 x MIR 604 – EPA Reg. No. 67979-13 (Cry1Ab, Vip3Aa20, mCry3A) 

• MON 89034 x TC1507 x MON 88017 x DAS-59122-7 (SmartStax) – EPA Reg. No. 

524-581 and 68467-7 (Cry1F, Cry1A.105, Cry2Ab2, Cry3Bb1, Cry34/35Ab1) 

• DAS-59122-7 x TC1507 (Herculex Xtra) + TC1507 (Herculex) (Optimum AcreMax 1 

Seed Blend) – EPA Reg. No. 29964-6 (Cry1F, Cry34/35Ab1) 

• TC1507 x DAS-59122-7 x MON 810 – EPA 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 control 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 (Cry1Ab, Vip3Aa20) – discussed in the 

BRAD for Vip3Aa20 

• TC1507 x MON 810 – EPA Reg. No. 29964-7 (Cry1F, Cry1Ab) – discussed below 

TC1507 x MON 810 (EPA Reg. No. 29964-7) 

TC1507 x MON 810 was registered by Pioneer and expresses both Cry1Ab and Cry1F for 

lepidopteran control. The product was registered with the same refuge requirements and IRM 

plan as the single toxin Cry1Ab (MON 810) and Cry1Ab (TC1507) registrations (see details on 

the IRM plan in section D.1.a of this document). 

For stacked and pyramided products of previously-registered toxins developed by conventional 

breeding, IRM data typically consists of efficacy and protein expression data. These data are 

used to confirm that the stacked/pyramided products will have comparable dose profiles as the 

single toxin constituent products. Along these lines, Pioneer submitted protein expression data 

and field efficacy data for the major target pests. 

Protein expression data (MRID# 475109-04) were generated for the pyramided product (1507 x 

MON 810) and the single toxin constituent PIPs. Studies were conducted in greenhouses and 

plant tissues were analyzed by ELISA for protein expression. Pioneer 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 were 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 were expressed at comparable levels in the pyramided product 

as the single toxin constituent products. Expression was consistent throughout all tissues -- only 

small differences in protein levels were noted between the pyramid and single toxin products 

which were not statistically significant. 

Field efficacy data (MRID# 476778-03) were generated for ECB, FAW, and SWCB with 

TC1507 x 59122 x MON 810 (a PIP that also contains a corn rootworm toxin). Efficacy for 

ECB was tested for both first and second generation using artificial infestation in five states 

(Illinois, Indiana, Iowa, Minnesota, and Nebraska). First generation 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 moderate ECB feeding, with a damage 

ratings range of 4.8 to 7.9. The stacked product (1507 x 59122 x MON 810) suffered no damage 

(rating 9 for all locations). A similar pattern was also observed 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 tunnel lengths generally less 

than 0.2 cm (two locations had 0.6 and 0.8 cm avg. tunneling). SWCB testing was conducted 
Cry1Ab and Cry1F Bt Plant-Incorporated Protectants August 2010 Biopesticides Registration Action Document 

218 

similarly to ECB but using natural infestation at two sites in Tennessee and Kansas. Damage at 

the R4-R5 reproductive stage was assessed through 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 infestation on whorl stage corn (V6). FAW feeding damage was 

scored after three weeks of infestation with a 1 to 9 scale (1 = heavy damage, 9 = no damage) 

much like the one used for ECB testing. Data from the field trials showed 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 damage from FAW (avg. ratings of 8.93 to 9.0). 

Both MON 810 (Cry1Ab) and Herculex (Cry1F) have been considered “high dose” for ECB and 

likely SWCB, but not for CEW. Given the confirmatory 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 profiles, 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 supported the use of the existing lepidopteran IRM 

plan including a 20% refuge (50% in cotton growing 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 developing field. Therefore, the IRM strategies may be 

improved with the collection of additional information, 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 research. These data goals are summarized in Table 

D13 below with a 2010 status update. 

Table D13. Summary of Data That Could 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 habitat 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 the 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 express 
Cry1Ab 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 containing 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, 

F2 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 

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Song, Q., C. Luppens, and X. Gan, 2001a. Monitoring the susceptibility of the southwestern 

corn borer, Diatraea grandiosella, to Bacillus thuringiensis toxin Cry1Ab: 2001 data summary. 

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Song, Q., C. Luppens, and X. Gan, 2001b. Monitoring the susceptibility of the southwestern 

corn borer, Diatraea grandiosella, to Bacillus thuringiensis toxin Cry1F: 2001 data summary. 

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Song, Q., C. Luppens, and X. Gan, 2002a. Monitoring the susceptibility of the southwestern 

corn borer, Diatraea grandiosella, to Bacillus thuringiensis toxin Cry1Ab: 2002 data summary. 

Unpublished study submitted to EPA - No MRID #. 

Song, Q., C. Luppens, and X. Gan, 2002b. Monitoring the susceptibility of the southwestern 

corn borer, Diatraea grandiosella, to Bacillus thuringiensis toxin Cry1F: 2002 data summary. 

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Song, Q., C. Luppens, and X. Gan, 2003a. Monitoring the susceptibility of the southwestern 

corn borer, Diatraea grandiosella, to Bacillus thuringiensis toxin Cry1Ab: 2003 data summary. 

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Song, Q., C. Luppens, and X. Gan, 2003b. Monitoring the susceptibility of the southwestern 

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borer, Diatraea grandiosella, to Bacillus thuringiensis Cry1F protein: 2004 data summary. 

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Song, Q., S. Wang and Y. Sun, 2006a. Monitoring the susceptibility of the Southwestern Corn 

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Song, Q., S. Wang and Y. Sun, 2006b. Monitoring the susceptibility of the 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 the susceptibility of the 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 the 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 the 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. 
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Song, Q., S. Wang and S. An, 2009. Monitoring the susceptibility of the southwestern corn 

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39: 47-79. 

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U.S. Environmental Protection Agency, 1998. The Environmental Protection Agency's White 

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Van Duyn, J., 1998. Large scale replicated field trials of representative commercial Bt and nonBt corn hybrids to establish relative insect resistance, yield performance, and commercial value. 

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93(4): 1276-1285. 

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Zhao J-Z., Y-X. Li, H. L. Collins, and A. M. Shelton, 2001. Examination of the F2 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 prepared 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-Derived Sweet Corn. Unpublished study 

prepared by Novartis Seeds, Inc.-Field Crops-NAFTA. 202 p. 

44775301 Gustafson, K. (1999) MON 810 1998 Research 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 Survival 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. 
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44854501 Bystrak, P. (1999) 1998 Progress Report Activities Relating to Resistance 

Management in Event 176 Maize: Lab Project 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 Susceptibility 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 Reports: 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-Derived 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: European 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 Presentation Summarizing European Corn 

Borer and Cry1F Resistance Monitoring Update. Project Number: GH/C/5826. 

Unpublished study prepared by Dow AgroSciences LLC and Pioneer Hi-Bred 

International. 27 p. 

46874901 Owens, E. (2006) Monitoring the Susceptibility of Corn Lepidoptera Pests to 

Cry1Ab and Cry1F Proteins: 2005 Monitoring Results. Unpublished study 

prepared by Agricultural Biotechnology 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, Iowa: Cry1F. Project Number: 061061. 

Unpublished study prepared by Mycogen Seeds, Pioneer Hi-Bred International 

Inc. and Dow AgroSciences 30 p. 
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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 Protectants in Field Corn. Project Number: 

IRM/2008/1. Unpublished study prepared by Agricultural Biotechnology 

Stewardship Technical Committee. 18 p. 

47339601 Bailey, L. (2008) 2007 Insect Resistance 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 prepared by Agricultural Biotechnology 

Stewardship Technical Committee. 17 p. 

47510904 Werning, M. (2008) Expressed Trait Protein Concentration of Maize Lines 

Containing Events DAS-01507-1, DAS -59122-7, MON-00810-6, and DAS01507-1xDAS-59122-7xMON-00810-6. Project Number: PHI/2008/006, 

PHI/2008/006/012, PHI/2008/006/011. Unpublished study prepared by Pioneer 

Hi-Bred International, Inc. 174 p. 

47663301 Bailey, L. (2009) 2008 Insect Resistance 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/2008. Unpublished study prepared 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 59122xMON810. Project Number: 

PHI/2009/025. Unpublished study prepared by Pioneer 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 used essentially the same information about 

yield advantages due to reduced insect damage, technology fee and reductions in conventional 

pesticide use. As discussed above, only a very small portion of field corn acres are treated with 

foliar insecticides. 
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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 which average in excess of 1 

million acres planted to field corn. These 16 states account for about 90 percent of field corn 

acres and 93 percent of the value of field corn grown for grain. EPA estimates of planted Bt corn 

are based on registrant submissions of annual sales. Since the first 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 represents about 15 million acres versus the 19.5 million 

acres estimated by this Agency. The states in the corn estimating program 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 where USDA has published adoption estimates and 

they do not agree. The USDA estimate is based on a survey while the EPA estimate is based on 

sales data. Each method has its potential problems and 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 calculation. However, we 

recognize that this could present an overestimate of benefits. 

The Agency has recently registered an additional plant-incorporated protectant known as Cry1F 

which has efficacy against those pests currently controlled 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 insect pest of field corn. 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 Planted 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 30 
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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 targets Lepidopteran insects. This protein is 

effective in controlling the European corn borer, the Southwestern corn borer and provides some 

control of the corn earworm. The European corn 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 virtually all of these pests. Corn borers 

consume plant energy and weaken the corn stalks so they are more likely to lodge (blow down) 

under windy conditions. Lodged stalks are difficult, if not impossible, to mechanically harvest. 

 The corn earworm enters the ear via the silk and feeds on the ears. Again once the insect larvae 

is in the ear under cover of the husk, it is difficult to control with conventional insecticides. 

These pests populations are not constant from year to year nationwide 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 geographically and from year to year depending on 
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environmental conditions including farming practices. This includes the past years planting of Bt

corn. The planting of Bt corn on a significant portion of the corn acreage in a region probably 

would reduce the populations of corn borers available to survive the winter and reproduce the 

next year. This means that a farmer is not guaranteed to be making a wise investment with a 

decision to plant Bt corn. Some areas have historically high levels of European corn borer or the 

Southwestern corn borer. These include southwestern Kansas plus parts of Colorado, New 

Mexico, Texas and Oklahoma for the Southwestern 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 this. 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 projections 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 apply 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 which could target corn borers (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 lowering of insect pressure. Either 

way, it is logical to expect some farmers will make 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 control 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 control 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. Therefore, little conventional pesticide has been used on field corn. 

Five to eight percent of field corn acres may have been treated with conventional pesticides to 

control these pests implying that 6.3 million of those 79 million acres planted to field corn could 

have been treated with conventional insecticides. A look at changes from 1995 and 2000 

indicates a reduction of around 3.9 million acre treatments due to all causes. 

Cry1F Bt corn has the potential to replace conventional 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 farmers are unlikely to also have corn rootworm and it may be a 
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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 conventional pesticides to control the black cutworm. 

Where there are cutworms, farmers may make the decision to plant Cry1F Bt corn and be able to 

reduce or eliminate use of conventional insecticides. 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, reflecting the fact that higher yields have been more than offset by 

lower prices. Other market factors may have been contributing to the 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. 
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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, nominal reduction in pesticide use 

and a technology fee of about $11 per acre. They discussed economic thresholds 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 decrease in prices was due to a combination of 

market forces largely unrelated 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 million (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 low 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 million per year. They would be around $38 million or 

less in years of low infestations. There is considerable uncertainty regarding 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 likely 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 expected 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 additional 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 Southwestern 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 which would affect the 

distribution of benefits. Typically, if growers increase production, 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 crop. These effects apply to any new technology 

which results in increased efficiency. 

Table E. 3. Grower Benefits of Bt Field Corn (Partial Budgeting Approach) 
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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 increase less technology fee. 

6/ per acre benefit times acres Bt corn. 

**This is intended to be representative of the lower bound benefits. Various conditions could 

result in actual benefits below this 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 mycotoxins on corn are produced by the fungal genus 

Fusarium, and are known as fumonisins (Munkvold, 2000). There are several different kinds of 

fumonisins: FB1, FB2, FB3, FB4, FA1, and FA2 (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 promote the growth of mycotoxin producing fungi 

in two ways: 1) They carry fungal spores from the plant surface to the surfaces of damaged 

kernels, and 2) They create entry wounds on the kernels for the fungi. Even when the insect pests 
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do not directly carry fungal spores to the corn wounds, ambient spores deposited later on tissue 

wounded by pest feeding are more likely to infect the plant (Munkvold, 1999). Field studies 

have shown that damage due to southwestern corn borer (SWCB) can increase aflatoxin levels 

(Windham, et.al. 1999). 

When mycotoxin contamination occurs in corn, the potential damages can be both economic 

costs to growers and health risks to humans and livestock. Corn grain that contains mycotoxins 

above a certain level is more likely to be rejected 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 may have a significant impact on amount of corn 

sold at the better food/feed prices. While these FDA guidelines for fumonisin levels are not yet 

set as action levels, they have been proposed to industry as safety thresholds (Randall A. Lovell, 

Center for Veterinary Medicine/ FDA, personal communication). 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 

(FB1+FB2+FB3) 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 ppm 

 
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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 

(ppm1

)

Feed 

Factor2

Recommended Maximum 

Level of Total Fumonisins 

in the Total Ration (ppm1)

Horse3 5 0.2 1 

Rabbit 5 0.2 1 

Catfish 20 0.5 10 

Swine 20 0.5 10 

Ruminants4 60 0.5 30 

Mink5 60 0.5 30 

Poultry6 100 0.5 50 

Ruminant, Poultry 

& Mink Breeding 

Stock7

30 0.5 15 

All Others8 10 0.5 5 

1

 total fumonisins = FB1 + FB2 + FB3. 2

 fraction of corn or corn by-product mixed into the total ration. 

3

 includes asses, zebras and onagers. 

4 cattle, sheep, goats and other ruminants that are > 3 months old and fed for slaughter. 

5

 fed for pelt production. 

6

 turkeys, chickens, ducklings and 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 particularly high levels of fumonisins, resulting 

in dramatic increases in the horse disease equine 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 ppm. Non-problem feeds contained concentrations below 8 ppm 

(Ross et al., 1992). Epidemiological studies have linked consumption of fumonisin-
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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 laboratory studies include toxicity and carcinogenicity in rats, 

cytotoxicity to mammalian cell cultures, and phytotoxicity to weeds and other plants including 

tomatoes (inhibiting growth and chlorophyll 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. Certain 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 there would be potential reductions 

in mortality and morbidity among livestock and, presumably, humans. 

Munkvold (2000) estimated that, if the current FDA 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 might also range in the tens of 

millions. Vardon (2000) made similar predictions 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 food is still acceptable for animal feed. This in fact may no longer be 

the case, as FDA’s most recent proposed 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. Historically, aflatoxin levels in corn have been highest in the 

Southeastern states. Corn from anywhere in the US may be affected, however, depending on the 

growth, harvesting and storage conditions involved, as was the case with aflatoxin contamination 

in the Midwest in 1988 and Texas in 1987 (FDA, 1999). 

Currently, the action level for aflatoxins 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 (B1) may be secreted into the animals’ milk as aflatoxin M1. Dairy 

cattle consuming corn feed that contains less than the FDA action level of 20 ppb total 

aflatoxins, however, should produce milk under the 0.5 ppb action level for aflatoxin M1 in milk 

(FDA, 1999). In 1969, the FDA had established the action level of 20 ppb aflatoxins 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 (FDA, 1994). The action levels for aflatoxins in 
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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 significant difference in 

aflatoxin levels. The variability in aflatoxin levels 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). Studies across 10 states in 2000 found little or no 

aflatoxin to begin with, and in cases of substantial aflatoxin 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 actually 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 results in terms of aflatoxin contamination levels between Bt and 

non-Bt isolines. However, Bt corn hybrids had less aflatoxin contamination than the non-Bt 

hybrids, on average in 2000 when the comparison 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 influencing aflatoxin accumulation (Odvody 
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2001, personal communication). The reasons for the negative performance of the one particular 

commercial line are not known. Factors that were forwarded as hypothesis by the researcher are 

the particular adaptability of a hybrid in a region, differences in the test material (i.e., not true 

isolines), or unintended effects from 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 performance 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 whether the reduced damage warrants the additional 

cost for the technology. The European corn borer and the Southwestern corn borer cause 

significant damage in certain regions of this country most years. Those growers in these areas 

with regular infestations, will continue to utilize this technology and others with significant 

damage will adopt the technology. Those whose infestations is not serious, will not continue to 

utilize the technology. It will be interesting to see whether forecasting of insect problems can 

become sufficiently sophisticated to enable growers 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 Syngenta’s (formerly Novartis) Cry1Ab (Bt11) sweet 

corn. Major pests controlled are European corn 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. Therefore, while the addition of Cry1F has the 

potential to increase acres planted to Bt sweet corn, we have not factored 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 8,900 116,000
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 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 Syngenta suggest the potential to 

achieve equivalent yields to traditional varieties while reducing the quantity of insecticides used 

to control these pests. According to NASS data, about 3.3 million acre treatments of insecticide 

are applied annually to sweet corn. 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 are cyhalothrin-lambda, permethrin, and methomyl 

with esfenvalerate, carbaryl, chlorpyrifos, cyfluthrin, and methyl parathion. Bt microbial sprays 
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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 Processed 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, 2000 
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On average, sweet corn is treated for all insect pests 5.5 times per year: 4.3 times for processed 

corn and 8.6 times for sweet, although the variability is quite significant among states. 

Table E. 6. Fresh Sweet Corn Insecticide Treatments, 1998 (thousands of acres) 

 State Acres Planted Acre Treatments 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 applications. An upper limit 

application savings of $45/acre is based on 9 applications per year, 60% (5.4) of which target Bt
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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 Management Practices 1999 summary. 

The USDA estimated 4% of vegetables in 1999 were planted with genetically modified seed to 

resist insects and sweet corn is the only crop with a registered plant-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 all vegetables for 2000 was too small to 

quantify (Bt plant-incorporated protectants for vegetables are only registered 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 (which 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 4.8 
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Source: Acres planted-average from Table E.5 

Average benefit to Bt adopters was estimated by the simulation model using the subset of 

observations for which benefits exceeded all costs. 

Other costs (insect resistant management, discounts for marketability, and underlying hybrid 

yield) equals the average cost to the adopters using the subset 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 less technology fee. 

Insecticide treatments saved equals average benefit 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 cost, 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 treatments 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 addressed the potential environmental benefits of Bt sweet corn. 

Because of the low adoption rates, potential benefits have not been realized. Biorationals with 

novel modes of action have not significantly replaced the more acutely toxic organophosphate 

and pyrethroid insecticides. Bt sweet corn allows a transition to more selective toxins and 

increase the beneficial arthropod community (Fleisher, 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 into 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 insecticides (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 help an areawide suppression of the 

corn earworm since corn serves as the primary nursery for recruitment of CEW populations, 
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which later in the season infest soybeans, lima bean and tomatoes. Further insecticide use 

reductions could therefore occur in these other 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 rejection or that the technology is not working as expected. We expect 

the seed companies marketing this technology 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 decreases corn diseases that result from insect 

interactions. Insect pests that damage ears, kernels or stalks are causative agents for mycotoxin 

development by carrying fungal spores to the surfaces 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 carcinogenic to humans and 

animals. Aflatoxins are known carcinogens to laboratory animals and presumably man. Growers 

must accept lower prices if mycotoxin levels exceed 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 extrapolating 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 resistance 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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moniliforme and its phytotoxin, fumonisin,” Weed Science 39, 1991, 673-677. 

DeLamar, Flanders, Bowen, Burch, Pegues and Wells, Efficacy of Transgenic Corn Against 

Southern Insect Pests, Fairhope and Headland, Alabama 1999, Auburn Univeristy, Wiregrass 

Research and Extension Center, Gulf Coast Research and Extension Center. 

Dively, Galen. 2000. Public comments, October 20, 2001 FIFRA SAP Meeting. OPP Docket 

OPP-00678. 

Doane Marketing Research, Inc. 1998. Multi Client Studies. 
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Duvick, Jon, Environmental Health Perspectives Volume 109, Supplement 2, May 2001 

Prospects for Reducing Fumonisin Contamination of Maize through Genetic Modification 

http://ehpnet1.niehs.nih.gov/docs/2001/suppl-2/337-342duvick/abstract.html 

Falck-Zepeda, Traxler, Nelson. 2000. Surplus Distribution from the Introduction of 

Biotechnology Innovation. American J. Agr. Econ. May 2000. 

FDA, “Background Paper in Support of Fumonisin Levels in Corn and Corn Products 

Intended for Human Consumption,” http://vm.cfsan.fda.gov/~dms/fumonbg1.html, June 

6, 2000a. 

FDA, “Background Paper in Support of Fumonisin Levels in Animal Feed,” 

http://vm.cfsan.fda.gov/~dms/fumonbg2.html, June 6, 2000b. 

FDA, “Food Compliance Program: Mycotoxins in Domestic Foods,” 

http://vm.cfsan.fda.gov/~comm/cp07001.html, March 11, 1999. 

FDA/ORA, Sec. 683.100 Action Levels for Aflatoxins in Animal Feeds (CPG 7126.33), 

http://www.fda.gov/ora/compliance_ref/cpg/cpgvet/cpg683-100.html, last revised 

August 28, 1994 

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Fernandez-Cornejo, Jorge, USDA/ERS (Personal Communication 5/30/2000. Tel. 202-694-

5537) 

Fernandez-Cornejo and McBride, 2000. Genetically Engineered Crops for Pest Management in 

U.S. Agriculture: Farm Level Effects, Resource Economics Division, Economic Research 

Service, U.S. Department of Agriculture. Agricultural Economic Report No. 786. 

Fleischer, Shelby. 2000. Public comments, October 20, 2001 FIFRA SAP Meeting. OPP Docket 

OPP-00678. 

Frisvold, George F., Russell Tronstad and Jorgen Mortensen. 2000. Adoption of Bt cotton: 

Regional differences in Producer Costs and Returns. P 337-340. 2000 Proceedings Beltwide 

Cotton Conferences. 2000. 

Gianessi and Carpenter, 1999. National Center for Food and Agricultural Policy (NCFAP), 

Agricultural Biotechnology: Insect Control Benefits. 

Gianessi, L.P. and J.E. Carpenter, 2000. “Agricultural Biotechnology: Insect Control benefits,” 

National Center for Food and Agricultural Policy, updated to 2000. 
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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. 

Lovell, Randall A. 2001. Center for Veterinary Medicine. U.S. Food and Drug Administration. 

Personnel Communication. June 14, 2001. 

Marra, Michelle, Gerald Carlson and Gerald Carlson. 1998. Economic Impacts of the First Crop 

Biotechnologies. Http:// www.ag-econ.ncsu.edu/faculty/marra/firstcrop/sld001.htm.

Ontario Ministry of Agriculture, Food and Rural Affairs 1997. European Corn Borer in Field 

Corn, Factsheet. www.gov.on.ca/OMAFRA/english/crops/facts/97-021.htm

Mason, Charles E. et al.1996. European Corn Borer Ecology and Management. NCR Publication 

327. Iowa State University. Ames, Iowa. 

W. Marasas, Fumonisins: History, World-Wide Occurrence and Impact in Fumonisins in Food, 

L. Jackson, ed., New York, Plenum Press, 1996. 

G.P. Munkvold, R.L. Hellmich, “Genetically modified, insect resistant corn: Implications for 

disease management,” APSnet Feature, 1999, www.apsnet.org/online/feature/btcorn, last 

accessed May 2001. 

G.P. Munkvold, “Statement to US Environmental Protection Agency FQPA Scientific Advisory 

Panel: Public meeting on Bt plant pesticides risk and benefit assessments,” Arlington, VA, 20 

October 2000, Docket # OPP-00678. 

Nuessly, Gregg. 2000. Public comments, October 20, 2001 FIFRA SAP Meeting. OPP Docket 

OPP-00678. 

Ostlie, K.R., W.D. Hutchinson and R.L. Hellmich, 1997. Bt Corn & European Corn Borer. NCR 

publication 602. University of Minnesota, St. Paul, MM. 

Rice, Marlin. Personal communication 8-31-2000, Iowa State Entomology, Tel. 515-294-1101. 

Rice, Marlin. 1998. Advantages and Disadvantages of Bt corn, Iowa State. 

Http://www.ipm.iastate.edu/ipm/icm/1997/3-3-1997/btcorn.html 

P.F. Ross, L.G. Rice, G.D. Osweiler, P.E. Nelson, J.L. Richard, T.M. Wilson, “A review and 

update of animal toxicoses associated with fumonisin-contaminated feeds and production of 

fumonisins by Fusarium isolates,” Mycopathologia 117: 109-114, 1992. 

Title 7 CFR Part 205 section 202(c). United States Government Printing Office. Washington 

D.C. July 2001. 

University of Guelph, 2000. Biotechnology Benefits

 Http//:www.plant.uoguelph.ca/plant/safefood/gmo/bt-survey/bt-backgrounder.htm.
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USDA-ARS. 1997. Fungus, Corn Plants Team Up To Stymie Borer Pest. U.S. Department of 

Agriculture, Agricultural Research Service. 

http://www.ars.usda.gov/is/ar/archive/nov97/fung1197.htm

USDA-ERS. 2001. Economic Issues in Agricultural Biotechnology. Robbin Shoemaker. Ed. 

Agriculture Information Bulletin #762. http://www.ers.usda.gov/publications/aib762/ . 

USDA-ERS. 2000. Biotechnology: Implications for U.S. Corn & Soybean Trade. Agricultural 

Outlook. April 2000. U.S. Department of Agriculture. Economic Research Service. 

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Washington, DC. 

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Department of Agriculture, National Agricultural Statistics Service. May, 2001. 

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Agricultural Statistics Service. June 2000. 

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Agricultural Statistics Service. June 2000. 

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Agricultural Statistics Service. 

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last accessed June 2001. 

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of Agriculture, National Agricultural Statistics Service. 

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National Agricultural Statistics Service. 

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National Agricultural Statistics Service. 

Turner, Larry. 2000. Personal communication and email : 7-15-00. EPA/OPP/FEAD. 

P. Vardon, “Potential Economic Costs of Mycotoxins in the United States,” Center for Food 

Safety and Applied Nutrition, FDA, 2000. 

Warnick, Debra. 2000. Personal Communication on Sweet Corn benefits, Novartis Seeds, Inc.-

NAFTA-Vegetables, 208 327-9368. 

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G.L. Windham, W.P. Williams, and F.M. Davis, “Effects of Southwestern Corn Borer on 

Aspergillus flavus Kernel Infection and Aflatoxin Accumulation in Maize Hybrids”, Plant 

Disease, June 1999. 

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Bt Transgenic Corn Hybrid. University of Nebraska. 

http:// www.ianr.unl.edu/pubs/fieldcrops/nf409.htm

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 provides highly efficacious control of key 

Lepidopteran pests of field corn 

2. Maize-optimized Cry1F-protected corn has the potential to provide comparable or superior 

pest control to existing commercial Bt corn products for key corn pests and affords a broader 

spectrum of pest control than do corn products expressing the Cry1A(b) insect resistance trait. 

3. The use of moCry1F-protected corn is expected to reduce the 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-protected corn maximizes economic benefits as 

compared to the application of conventional chemical 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 established for plant-optimized Cry1F-protected corn. 

8. Cry1F expressed in corn poses no foreseeable risks to human health or the environment. 

Field Efficacy 
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Field efficacy trials were conducted over multiple 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 Aacceptable@ (see EPA review, Hill, 2004). Dow has 

submitted sufficient field efficacy data to demonstrate 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), Western bean cutworm (Richia albicosta (Smith), WBCW), and suppression 

of the corn earworm(Helicoverpa zea (Boddie), CEW). TC6275 performed comparably to 

TC1507 (plant-optimized Cry1F-protected corn, Herculex I7) and showed significantly less 

infestation than the non-Bt isogenic hybrid. No additional data are required 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 included. Based on this analysis, TC6275 provides 

comparable performance relative to TC1507. TC6275 performed better or comparably with the 

commercial standards against ECB, SWCB, BCW, FAW, CEW, and WBCW. 

Yield and Economic Benefits 

The primary economic benefit of corn hybrids containing Cry1F insect resistance trait (mo and 

po Cry1F) is the protection of yield. At the individual 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 conventional 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 Ainsurance@ factor of a Bt corn hybrid 

as a prophylactic control measure. Both of these factors result in potential yield benefits to the 

farmer. 

Yield and Agronomic Characteristics 

Previously, EPA concluded that Event TC1507 corn 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-optimized 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 containing no transgenic trait in field trials for early (11 locations) 

and late (15 locations) season hybrids in 2002. The comparison of both late and early maturity 

hybrids found no statistically significant differences within either maturity group among the 

TC6275 hybrid, the TC1507 hybrid, and non-transgenic isogenic hybrid for grain density, plant 
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stature, emergence vigor, root lodging, and dropped ears (see Tables 5 and 6 in Zabik et al., 

2003). There were no statistically significant yield differences between either early or late 

maturity comparisons of TC6275 and TC1507 hybrids. Both the early maturity TC6275 and 

TC1507 hybrids were significantly higher yielding than the early maturity non-transgenic 

isogenic hybrid. This is most likely due to the protection offered by Cry1F against European 

corn borer-induced yield loss. Both early and late maturity TC6275 hybrids 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 significant. 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 benefits 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, from $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 chemical control or no control. 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. The 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 impact of increased competition on overall market 

equilibrium conditions. Increased competition should offer growers more choice and lower the 

cost of pest control. The benefits are the incremental improvement to grower profits compared to 

current practice. All costs are eventually passed along to consumers in the long run, but this 

analysis did not deal with when that would occur. 

The economic benefit to the grower who plants corn varieties containing Event TC6275 

(moCry1F) was evaluated on the basis of data on yield and efficacy as reported in Zabik et al. 

(2003) using the Herculex Value Calculator. The Herculex Value Calculator, 

http://www.dowagro.com/herculex/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 hybrids 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 accounts 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 positioned to compete against the following 

chemical alternatives: bifenthrin, carbofuran, chlorpyrifos, cyhalothrin-lambda, permethrin, 
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fipronil, tebuprimiphos/cyluthrin, tefluthrin, terbufos, and zeta-cypermethrin and against the 

Cry1Ab-protected corn hybrids. The registered chemical alternatives commonly used to treat the 

target pest complex protected by Cry1F are restricted 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 organophosphate and pyrethroid 

pesticides likely to be replaced are ranked in the top 15 of all pesticides with respect to reported 

incidents of mortality to non-target wildlife. Many of these products 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 performance data have shown performance equal to or 

better than any of the conventional chemical alternatives or other Bt corn hybrids against ECB, 

BCW, SWCB, FAW, WBCW, and CEW. Growers may be more likely to choose Cry1F 

protected corn due to better product performance and broader spectrum of control. Cry1F 

protected corn is also expected to be economical on some unprotected fields and provide 

insurance against the risk of crop loss and the need to replant. 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 expected to pose no unreasonable 

adverse effects to human health or the environment (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 substantially 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 alternatives, and reduce potential adverse effects to nontarget organisms. Increased use of Cry1F-protected corn would also improve worker protection 

as compared to chemical insecticides. 

The data submitted show that poCry1F and moCry1F 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 simulated gastric fluid (MRID 447149-03), 

and does not share any amino acid sequence similarity 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 moCry1F 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 times greater in the leaf than the poCry1F plants (Zabik et al. 

2003, see p. 36, Table 8). Increased expression in the stalks and leaves should decrease the risk 

of resistance development by borers (a higher, high dose) whilst the decreased expression in 

pollen and grain should decrease the non-target exposure to Cry1F expressed in corn (Zabik et. 

al 2003). 
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Similarly, testing with bacterially prepared Cry1F protein at levels greatly 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 effects to non-target organisms (MRID 450201-13, see 

Table 1). Data were also provided regarding the rapid soil degradation 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. Unpublished study submitted by Dow AgroSciences 

LLC. MRID No. 460630-04. 

Hill, H. 2004. EPA review of field efficacy, high dose studies, and insect resistance 

management plan of maize-optimized Cry1F (TC6275) 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 Registration, EPA Reg. No.68467-U. Memorandum 

dated January 4, 2005. 

Matten, S. 2005a. EPA Review of Waiver Rationales 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 Characterization 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 Interest Document in Support of the Section 3 

Registration of the Mycogen Brand B.T. Cry1F 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 final 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). 
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Scientific Advisory Panel (SAP), Subpanel on Insect Resistance Management (October 18-20, 

2000), 2001. Report: sets of scientific issues being considered by the Environmental 

Protection Agency regarding: Bt plant-pesticides risk and benefit assessments. Report dated, 

March 12, 2001. (Pp. 5-33) 

U.S. Department of Agriculture (USDA), Animal 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-regulated Status for Bt Cry1F Insect Resistant, 

Glufosinate Tolerant Corn Line 6275. Finding 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/biopesticides/ingredients/tech_docs/brad_006481.pdf.

U.S. Environmental Protection Agency (EPA) 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 of product characterization data for maizeoptimized Cry1F insect control protein 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 interest 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 (Bt11 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 received, results of public meeting, hearings, 

workshops, forums, and Scientific Advisory Panel meetings, and public comments received 

regarding the Bt crops reassessment have been taken into 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. 
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Tests have shown no toxicity to mammals from the Cry1Ab and Cry1F proteins; the proteins are 

readily digestible in gastric fluids and are non-glycosylated, the proteins are inactivated by 

typical food processing, and anticipated exposure of farm workers to the proteins is negligible. 

The Cry1Ab protein acute oral toxicity data submitted demonstrated no effects at the relatively 

high dose level of 4,000 mg/kg. The Cry1F protein 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 and 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 infants and children, to the 

Cry1Ab and Cry1F proteins and the genetic material necessary for their production. Thus, EPA 

concludes that there are no adverse effects on human health from 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 capture 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 determined 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 territories. In addition, the USDA/APHIS has made 

this same determination under its statutory 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 Monarch 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 low 
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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 that Cry proteins have any measurable 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 DT50 (Degradation Time) of 1.6 days and an estimated DT90 of 15 days. The 

bioactivity of purified Cry1Ab protein in soil decreased with an estimated DT50 of 8.3 days and a 

an estimated DT90 of 32.5 days. The bioactivity of purified Cry1F protein in soil decreased with 

an estimated DT50 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 aimed at reducing the potential for insect pests to become resistant 

to a pesticide. Bt IRM is of great importance because of the 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 maintaining insect susceptibility, 

growers must choose at least one of structured refuge (a portion 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 are 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 mitigate the development of resistance, EPA has 

determined that crop residue destruction is necessary within 30 days. This practice will likely 

destroy any live larvae left in Bt sweet corn stalks and prevent 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 contractually binds the grower to comply 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 comply with the IRM program, 2) an IRM education program, 3) an 

IRM compliance monitoring program including a third party compliance survey and mechanisms 

to address non-compliance, 4) an insect resistance monitoring program for each target insect 

pest, 5) remedial action plans to be implemented if resistance 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 products. Direct grower benefits 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 magnitude 
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of benefits for any year is largely a function of the level of lepidopteran insect pressure in that 

year. That is, other things being equal, the higher the insect pressure, the higher the benefits. 

The major environmental benefit is potential reduction 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 the pesticide and proposed used are identical or 

substantially similar to a currently registered pesticide and use thereof, or differs only in ways 

that would not significantly increase the risk of unreasonable adverse effects on the environment, 

and (ii) approving the amendment in the manner proposed by the applicant would not 

significantly increase the risk of unreasonable adverse effect on the environment. FIFRA defines 

“unreasonable adverse effects on the environment” in pertinent part 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, the FIFRA unreasonable adverse effects 

standard requires EPA to balance the risks and benefits of using the pesticide in reaching its 

regulatory decision. 

EPA finds that the use of Cry1Ab or Cry1F expressed in corn will not significantly increase the 

risk of unreasonable adverse effects on the environment. This finding, however, applies only to 

the use of Cry 1Ab or Cry1F protein expressed corn under the terms and conditions of 

registration specified below, and only for the limited time period of 7 additional years (to 

October 15, 2008). The following sections set forth the basis for EPA’s finding in general, and 

the basis for the decision to approve 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 environment 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 controlling significant lepidopteran 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, subject to the specific terms and conditions 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 raises concerns with respect to insect resistance 

management. As discussed below, the registrations for Cry1Ab and Cry1F proteins expressed in 

corn is subject to specific terms and conditions that 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 significantly increase the risk of unreasonable adverse effects on the 
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environment. Finally, EPA has identified the need for certain confirmatory data on potential 

accumulation of Cry1Ab and Cry1F proteins in soil and field impacts of Cry1Ab and Cry1F 

proteins on non-target species. The 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 exemption 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, configuration, 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 southwestern corn borer (Diatreae grandiosella (Dyar)), models that predict the 

estimated time that resistance would develop to compare the effectiveness of various IRM 

strategies, economics, sustainability, 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 important part of the regulatory 

decision. The Agency identified (later confirmed 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 expression strategy; 3) appropriate refuge; 4) 

resistance monitoring and a remedial action plan should resistance occur; 5) employment of 

integrated pest management (IPM); 6) communication and education 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. 

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, protective, feasible, sustainable, and practical to 

growers. Models have been developed by scientists in academia to predict the estimated time 

that insect resistance would develop 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 evolve resistance for at least 99 years if a 20% 

refuge is implemented in the Corn Belt. Models are also used to predict 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. 
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However, it should be noted that these predictive models cannot be validated without actual field 

resistance. They have limitations and the information gained from the use of such models can 

only be used as a part of the weight of evidence determination conducted EPA to assess the risks 

of resistance developing in target pest populations. EPA agrees 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 and parameters validated. In the 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 choosing 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 between 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 judgments of the potential effects of using various 

refuge options. 

In addition to assessing the likelihood of resistance, EPA has mandated specific requirements for 

annual resistance monitoring to determine, in a pro-active fashion, whether insect susceptibility 

has changed or whether resistance is likely to occur (or is occurring). After five years of 

analyzing resistance monitoring data (1996-2000), there is no evidence of European corn borer, 

corn earworm, or southwestern corn borer resistance developing in the field to the Cry1Ab deltaendotoxins produced by current registered Bt corn products. There are no resistance monitoring 

reports yet available for Cry1F field corn products because they were just registered in 2001. 

Therefore, EPA believes that resistance is not occurring in the field based on the available data. 

The Agency is mandating enhancements to the resistance management programs that will 

improve the certainty of detection of resistance. If insect resistance occurs, EPA has also 

mandated a specific remedial action plan that will contain (and perhaps eradicate) resistance 

prior to the occurrence of any widespread, endemic resistance. 

In addition, to the use of biological data, predictive models, and resistance monitoring 

information, EPA also weighed practical considerations in deciding which refuge options to 

allow. Grower education and compliance with refuge options are essential to the success of any 

IRM strategy. Growers must be able to implement the refuge options within the constraints of 

their farming operations. Based upon the currently available scientific data 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 practical, 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 are deployed correctly then, there 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 (before 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 within one month of harvest (a practice that 
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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, EPA has mandated that the 

registrants have grower agreements that contractually bind the grower to the IRM requirements, 

ongoing grower education programs, ongoing research programs, ongoing resistance monitoring 

programs, and a multi-faceted compliance monitoring 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 compliance monitoring programs. EPA has asked for 

additional data on the effect of insecticides on refuge effectiveness for Bt field corn and northsouth movement of CEW to further enhance the 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 region 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 contain (and perhaps eradicate) 

resistance. 
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D. 2010 Update 

 

Section 3(c)(7)(A) of FIFRA provides for the registration or amendment of a pesticide when the 

pesticide and proposed use “...are identical or substantially similar 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 environment, 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 FIFRA 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 increase the risk of unreasonable adverse 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 cultivation 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 and conditions of registration and summarized in 

section II(C) of this BRAD, strengthen the Agency’s initial position and also confirm that longterm effects on non-target organisms are not anticipated. Lastly, the continued use of these 

products will likely still provide many of the benefits as were evaluated in section II(E) and II(F) 

of this BRAD to support the 2001 Bt crops reassessment 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). 
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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 proposing to amend these registrations to extend 

their respective expiration datesa as follows: 

Product (EPA Reg. No.) Proposed Expiration Date 

67979-1 Bt Corn Event Bt11 with Cry 1Ab (Field 

Corn) 5 years after amendment (~September 2015) 

65268-1 Bt Corn Event Bt11 with Cry 1Ab (Sweet 

Corn) 5 years after amendment (~September 2015) 

524-489 Bt Corn Event MON 810 Cry1Ab 5 years after amendment (~September 2015) 

68467-2 Bt Corn Event TC1507 with PO Cry 1F 5 years after amendment (~September 2015) 

29964-3 Bt Corn Event TC1507 with PO Cry 1F 5 years after amendment (~September 2015) 

68467-4 Bt Corn Event DAS-06275-8 with MOCry1F 5 years after amendment (~September 2015) 

29964-7 1507 (POCry1F) x MON 810 (Cry1Ab) 12 years after amendment (~September 2022) 

Although data provided were satisfactory to make the determinations required by section 

3(c)(7)(A) of FIFRA, they were 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 insect resistance management, are 

necessary for a finding of registrability under FIFRA 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 Bt Corn reassessment, EPA determined that it was appropriate to amend the thenexisting registrations to extend the period of registration of those products to an expiration date 

of October 15, 2008. All of the products being assessed 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 increase the risk of unreasonable adverse effects on the environment 

“for the limited time period of 7 additional years (to October 15, 2008).” These registrations 

were later amended to extend the period of registration to an expiration date of September 30, 

2010. EPA subsequently granted time-limited registrations to products efficacious against 

coleopteran corn rootworm pests. For example, 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. 
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As set forth elsewhere in this document, EPA’s primary concern for the Bt protected transgenic 

corn products is the possibility that target pests will develop resistance to one or more of the 

plant-incorporated protectant (PIP) toxins. 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 resistance. Registrants similarly have been busily 

developing various products, product mixes (i.e., so-called “pyramids” and “stacks”), and 

resistance strategies, to maximize agronomic benefits and address resistance management issues. 

The result has been a vast array of product combinations 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 low as to guarantee that adequate natural refuges 

would be available from neighboring non-Bt corn fields. From 1995 to 1997, Bt corn 

registrations included voluntary refuge 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 Association, proposed uniform insect resistance management (IRM) 

requirements for Bt corn registrations. With some modifications, 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: farmers were required to plant a 20% refuge that 

could be treated for insects, or a 50% treated refuge in cotton-growing areas; 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 different combinations of 

“pyramided” products (i.e., products containing two or more toxins efficacious against the same 

pest) and “stacked” products (i.e., products combining toxins efficacious against different pests), 

the refuge requirements have begun to vary. For example, certain products require a 20% 

external refuge; some products permit a 5% external refuge; one product incorporates a 10% 

seed blend refuge; we have applications in process for products that propose to incorporate a 5% 

seed blend refuge; and other permutations are possible. 

Given the profusion of various toxin combinations and refuge options, we can no longer proceed 

on the basis that, as concerns insect resistance management, all products are equal. It was a 

relatively simple proposition when the default requirement of a 20% sprayed refuge applied to 

almost all of the Bt corn crops in the market. Under those circumstances, the relative durability 

of products against the development of resistance was functionally equivalent, and, as a 

consequence, imposing functionally equivalent registration periods was appropriate. That is now 

no longer the case. 

As part of our continually evolving regulatory approach to the continually evolving product mix 

wrought by developers, we think it appropriate to revise our regulatory requirements in 

scientifically defensible ways to reflect the actual level of risks posed by the products that we 
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regulate. Here, for example, where we’ve determined 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 product, or category of products, a registration for a 

period greater than that granted a corresponding product that poses a greater risk of insect 

resistance developing. This approach is reflective of complementary 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 registration period will also be available, if adequately supported by our 

science assessment. In this scheme, (i) a product 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; (iii) 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 registration; (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 baseline single toxin product with a 20% external 

refuge, would qualify for a fifteen-year registration. 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 level of risk they pose. 

Similarly, instances where other risk issues may arise, or where novel resistance concerns may 

be present, would also be determined on a case-by-case basis.