BIOPESTICIDE REGISTRA TION ACTION DOCUMENT

Bacillus thuringiensis Cry1Ac Protein and the Genetic Mate rial (Vector PV-GMIR9) Necessary for Its Production in MON 87701 (OECD Un ique Identifier: MON 877Ø1-2) Soybean [PC Code 006532]

U.S. Environmental Protection Agency Office of Pesticide Programs Biopesticides and Pollution Prevention DivisionBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

2 TABLE of CONTENTS

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

A. EXECUTIVE SUMMARY

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

B. USE PROFILE

............................................................................................................................... ......................... 4

C. REGULATORY HISTORY

............................................................................................................................... ... 5

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

A. PRODUCT CHARACTERIZATION

B. HUMAN HEALTH ASSESSMENT

D. ENVIRONMENTAL ASSESSMENT FOR MON 89034 ..................................................................................... 15

E. INSECT RESISTANCE MANAGEMENT (IRM) ............................................................................................. 33

III. TERMS AND CONDITIO NS OF REGI STRATION...................................................................................... ..... 38

IV. REGULATORY POSITION FOR CRY1AC SOYBEAN.................................................................................... 3 8Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

3 BIOPESTICIDE REGISTRATION ACTION TEAM

Office of Pesticide Programs:

Biopesticides and Pollution Prevention Division

Product Characterization and Human Health

John Kough, Ph.D. Chris Wozniak, Ph.D.

Environmental Fate and Effects

Zigfridas Vaituzis, Ph.D. Shannon Borges, M.S.

Insect Resistance Management

Alan Reynolds, M.S. Jeannette Martinez, M.S.

Registration Support

Mike Mendelsohn

I. OVERVIEW

A. EXECUTIVE SUMMARY

Monsanto Company (hearafter, “Monsanto”) has s ubmitted an application to register the soybean product MON 87701, which expresses the Bacillus thuringiensis ( Bt ) subsp. kurstaki insecticidal protein Cry1Ac as a Plant Incorpor ated Protectant (PIP). Cry1Ac provides protection from feeding damage by target lepidopteran pests, including the velvetbean caterpillar ( Anticarsia gemmatalis ), soybean looper ( Pseudoplusia includens ), soybean axil borer ( Epinotia aporema ), and sunflower looper ( Rachiplusia nu ). Monsanto has applied for a FIFRA Section 3 registration for breeding and seed increase activities to suppor t commercial production of MON 87701 in tropical and subtropical regions outside the U.S. and its territories.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

4 Product Characterization

MON 87701, which was developed by Agrobacterium -mediated transformation of soybean ( Glycine

max ) using the 2T-DNA plasmid vector PV- GMIR9, produces one Bacillus thuringiensis protein, Cry1Ac. This protein is intende d to provide protection from feed ing damage caused by a number of lepidopteran pests, such as the soybean looper, beet armyworm, green cloverworm and velvetbean caterpillar.

Mammalian Toxicity and Allergenicity Assessment

Cry1Ac protein is a δ -endotoxin from B. thuringiensis that has been used extensively in both microbial and plant-incorporated protectants as a means of insect pest management. An existing exemption from the requirement of a toleran ce for Cry1Ac (CFR 40 Section 174.510; 72 FR 20435, April 25, 1997) in food and feed commodities precluded the need for a separate tolerance action in conjunction with this review of product char acterization and human health assessment.

Environmental Assessment

Adverse effects to nontarget organisms, incl uding birds, wild mamm als, freshwater and marine/estuarine fish and invertebrates, nontarge t insects, honey bees, soil invertebrates, and terrestrial and aquatic plants, are not anticipated. Horizontal gene transf er, gene flow, and the development of weediness are also not anticipa ted. Based on these findings and information on habitat requirements of federally-listed threatened and endangered lepidopteran species, the Agency

also makes a No Effect determination for direct and indirect effects to threatened and endangered species and their habitats.

Insect Resistance Management

Given the low acreage permitted for MON 87701 (<15,000 total acres per year), it is unlikely that there will be a significant risk of resistance to the main soybean ta rget pests in the United States, soybean looper and velvetbean caterpillar. This conclusion is further su pported by the biology of the target insects, which do not overwinter in non-tropical areas and are polyphagous (feeding on a number of wild hosts and cultivated crops).

The use of MON 87701 on a limited basis (as descri bed by Monsanto in thei r submissions) should not impact the natural refuge strategy in place fo r Bt cotton PIPs that express Cry1Ac. The total proposed acreage for MON 87701 would represent a sm all fraction of total soybean acres in each state and should not significantly reduce the am ount of natural refuge. Further, MON 87701 will not be planted in states (i .e., Mississippi Delta states) with lower natural refuge.

The Agency is requiring adopti on of a resistance mon itoring plan, remedial action strategy, and annual sales reporting for MON 87701.

B. USE PROFILEBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

5 Active Ingredient Name: Bacillus thuringiensis Cry1Ac Protein and th e Genetic Material (Vector PV-GMIR9) Necessary for Its Production in MON 87701 (OECD Unique Identifier: MON 877Ø1-2) Soybean

Trade and Other Name(s): MON 87701

OPP Chemical Code s: 006532

Basic Manufacturer: Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167

Type of Pesticide: Plant-incorporated Protectant

Uses: Soybean. Limited to seed increase and to a total of 15,000 acres per year in the

Atlantic Coast States within the Continental United States with no more than 1,000 acres per

county per year.

Target Pests for Active Ingredient: Velvetbean caterpillar ( Anticarsia gemmatalis ), soybean looper ( Pseudoplusia includens ), soybean axil borer ( Epinotia apoerema ), and sunflower looper ( Rachiplusia nu )

C. REGULATORY HISTORY

Monsanto previously submitted EUP requests for event MON 87701 in order to conduct field evaluations and gather da ta. Cry1Ac protein is a δ -endotoxin from B. thuringiensis and has been used extensively in both microbial and plant-incorporated protectants as a means of insect pest management. An existing exemption from the requirement of a tolerance for Cry1Ac (CFR 40 Section 174.510; 72 FR 20435, April 25, 1997) in food and feed commodities precludes the need for a separate tolerance action in conjunction wi th this review of pr oduct characterization and

human health assessment.

On April 14, 2010, EPA announced its receipt of this application pursuant to FIFRA secion 3(c)(4) in the Federal Register and in regulations .gov under docket number EPA-HQ-OPP-2010-0023. No comments were received in response to this notice.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

6 II. SCIENCE ASSESSMENT

A. PRODUCT CHARACTERIZATION

MON 87701 was developed by Agrobacterium -mediated transformation of soybean using the 2TDNA plasmid vector PV- GMIR9 and produces Bacillus thuringiensis protein δ -endotoxin, Cry1Ac. This protein is intended to provide protecti on from feeding damage caused by a number of lepidopteran pests.

Transformation System and Genetic Elements : PV- GMIR9 is a binary vector containing two separate transfer DNAs (2T-DNAs). The first TDNA (T-DNA I) contains the cry1Ac gene under the direction of the RbcS4 (small subunit

RuBISCO from Arabidopsis thaliana ) promoter and termination sequences from T-7S ά (3’ region

of the Sphas gene of Glycine max encoding the 7S ά seed storage protein conglycinin). The chloroplast transit peptide coding sequence from Arabidopsis thaliana from RbcS4 small subunit 1A was placed at the 5’ end of the gene (N-ter minus) to direct accumulation of the insecticidal protein to the chloroplast. In the Cry1Ac prot ein produced in MON 87701, 4 additional amino acids are present at the N-terminus of the protein as a result of the use of th is targeting sequence.

The second T-DNA, designated as T-DNA II, contains the cp4-epsps gene under the direction of the Figwort Mosaic Virus 35S promoter. During transformation, both T-DNAs were inserted into the soybean genome independently. The cp4-epsps gene was used as the selectable marker (tolerance to glyphosate) to sel ect transformed cells and plants. After the transformed cells and subsequent regenerated pl ants were identified, the cp4-epsps selectable marker gene was no longer needed. Therefore, traditional breeding and segregati on were employed to isolat e plants that contain only the cry1Ac expression cassette (T-DNA I), produci ng marker-free MON 87701 plants lacking

the cp4-epsps selectable marker gene.

Analysis of the insert junction of the single copy T-DNA-I into MON 87701 genomic DNA indicated that the entire sequen ce, as present in T-DNA-I from PV - GMIR9, is intact in the final transformant. Sequencing of the insertion site indi cated a loss of 32 bp as well as an addition of 14 bp at the junction with soybean genomic DNA. This has been previously noted in the literature regarding Agrobacterium -mediated integration events (Gorbunova and Levy, 1997; Salomon and Puchta, 1998).

Protein Characterization : Protein characterization data de monstrate that the plant-produ ced Cry1Ac has biochemical and

functional activities that are similar to those of the E. coli -produced protein that was used in several toxicity studies. The following techniques were used to characterize and compare the plantproduced and the E. coli -produced proteins: sodium dod ecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot anal ysis, densitometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) / mass spectrometry, glyc osylation analysis, Nterminal amino acid sequencing, a nd insecticidal activ ity bioassays. Glycoslyation analysis indicated that the protein is not glycoslyated. These analyses demonstrated the structural andBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

7 functional similarity between the plant-produced and the E. coli -produced Cry1Ac protein and justified the use of E. coli -produced protein in toxicity studies.

Soybean MON 87701 derived Cry1Ac protein contains an additiona l 4 amino acids derived from the chloroplast transit peptide (CTP1) sequence at the N-terminus, as deduced by N-terminal sequencing. While this addition differs fr om the protein that was produced in E. coli for the oral toxicity study and the insect bioassays with corn earworm, it should be noted that the Cry1Ac protein is processed at both the N- and C- term ini during maturation of the protein into its active form (tryptic core) in the insect gut. The CTP1 residual 4 amino acid residue s would be cleaved off by protease action as part of that maturation process in situ . The equivalency of the protein used as a test substance and that as produced in planta were reviewed in this risk assessment and found to be acceptable.

Analytical Detection Methods : An exemption from the requirement of a food tolera nce is already in existe nce (since 1997) for this protein and an appropriate analyt ical detection method already revi ewed and in place for Cry1Ac. Hence, there is no separate review of an anal ytical method in this ri sk assessment summary.

Protein Expression : Expression level data were provide d for Cry1Ac in different plant ti ssues and at different growth stages. The data were produced using ELISA me thods for this protein. Summary results are provided below in Table 1. Table 2 provides summa ries of the product char acterization studies and data provided.

TABLE 1. Cry1Ac protein le vels in MON 87701 tissues

Tissue type 1 Mean (SD) 2,4

(μg/g fwt) Range 5

(μg/g fwt) Mean (SD) 3,4

(μg/g dwt) Range 5

(μg/g dwt) OSL-1 30 (8.5) 12-40 220 (70) 110-350 OSL-2 38 (16) 18-80 260 (100) 130-500 OSL-3 34 (17) 14-77 240 (110) 94-480 OSL-4 53 (36) 15-110 340 (290) 78-960

Root <LOD <LOD NA 6 NA 6

Forage 9.0 (8.8) 2.5-32 34 (36) 8.2-140 Seed 4.2 (0.73) 3.1-5.0 4.7 (0.79) 3.4-5.7 Pollen 2.3 (0.58) 1.8-3.1 NA NA

Data from pp. 17 and 18 of 20, MRID 47841703

1 Tissues were collected at the following growth stages: OSL-1: V3-V4; OSL-2: V6-V8; OSL-3: V10-V12; OSL-4: V14- V16; Root: R6; Forage: R6; Seed: R8, harvested at or dried to 10-15% moisture; Pollen: R2.

2 Protein levels expressed as μg of protein/g of tissue on a fresh weight basis.

3 Protein levels expressed as μg/g on a dry weight basis, calculated by dividing the fresh weight by the dry weight. conversion factors obtained from moisture analysis data. Moisture analysis was not performed for pollen.

4 The mean and standard deviation were calculated across sites (n=15, except OSL-1 n=13 and pollen n=4).

5 Minimum and maximum values were determined for each tissue type across sites.

6 Protein levels <LOD on a fresh weight basis were not converted to dry weight values.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

8 Table 2. Product Characterization Data Submitted

Study Type/Title Summary MRID #

Human Health and

Environmental

Assessment of the Plant-Incorporated

Protectant Bacillus thuringiensis Cry1Ac Protein

Produced in InsectProtected Soybean

MON 87701 MON 87701 is an insect-protected soybean that produces the Cry1Ac insecticidal protein derived from Bacillus thuringiensis subsp. kurstaki . Cry1Ac provides protection from feeding damage by target lepidopteran pests. Characterization studies have confirmed that the gene tic modification in MON 87701 contains a single insert with th e intended sequence, and that the insert is stable over multiple generations. The mean Cry1Ac

content in MON 87701 tissues (dry weight basis) was determined to be 4.7 μg/g in harvested seed, 34 μg/g in forage, 220 to 340 μg/g in leaves, 2.3 μg /g in pollen/anthers (fresh weight basis), and below the lim it of detection in roots. MON 87701 was effective against major lepidopteran pests of soybean in screenhouse and open field trials at multiple locations in the US and Argentina. The Cry1Ac prot ein has a history of safe use in cotton and corn products. No structurally relevant similarity exists between Cry1Ac and any kno wn toxic or biologically active proteins or known allergens that would be harmful to human or animal health. MON 87701 is not expected to produce adverse effects in non-target organisms, including threatened or endangered species, based on tests with mice, two avian species, soil decomposers, and beneficial insects. Tests on aquatic species were

not conducted since there is no meaningful, ecologically relevant exposure to aquatic organisms fr om soybean. A laboratory soil degradation study showed that 72% to 96% of the Cry1Ac in

MON 87701 biomass inco rporated into soil degraded within six months, indicating Cry1Ac is unlik ely to persist or accumulate

in the environment.

Classification: ACCEPTABLE 47841701

Assessment of the Cry1Ac Protein Levels in Soybean

Tissues Collected

from MON 87701

Produced in U.S. Field Trials During

2007 MON 87701 is an insect-protected soybean that produces the Cry1Ac insecticidal protein derived from Bacillus thuringiensis subsp. kurstaki . Cry1Ac provides protection from feeding damage by the target lepidopteran pests. An enzyme linked immunosorbent assay method was used to determine the level of Cry1Ac protein expressed in MON 87701 tissues from plants grown in field trials conducted in Alabama, Arkansas, Georgia, Illinois, and North Carolina in 2007. The mean Cry1Ac levels across all sites we re 4.7 μg/g dry weight in harvested seed, 34 μg/g dry wei ght in forage, and below the 47841703Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

9 Study Type/Title Summary MRID #
limit of detection (LOD) in root s. The mean Cry1Ac level in leaves harvested during differen ce stages of the growing season ranged from 220-340 μg/g dry weight. The mean Cry1Ac level in pollen from the single site sampled was 2.3 μg/g fresh weight.

Classification: ACCEPTABLE

Characterization of the Cry1Ac protein purified from the

harvested seed of

MON 87701 soybean and comparison of the physicochemical

and functional properties of the

MON 87701- produced and E

coli -produced Cry1Ac protein The information and data presen ted are in accord with the conclusion of the study author that MON-87701-derived and E.

coli -derived Cry1Ac are equiva lent proteins. Relative molecular mass measurements derived through SDS-PAGE and fragment analysis through MALD I-TOF / MS clearly indicate that the proteins derived from in planta expression in MON 87701 and from expression in E. coli are of the same mass. Nterminal sequencing of the first 7 amino acids and their identity to the Cry1Ac sequence aided in verifying that the protein of

interest was consistent with what is known of the insecticidal protein, along with the ambiguous cysteine residue present in accord with the use of the CTP1 peptide at the terminus. Reactivity to antiserum directed against Cry1Ac also indicates the sequence similarities between the two proteins and any products of catalysis as noted on the blots.

An absence of glycosylation in both microbial and plant produced forms of Cry1Ac is as expected for this protein and further suggests the homology be tween the proteins from the

two sources. The EC 50 values for the two proteins, when tested against the corn earworm, identify the insecticidal activity of the two proteins as so similar in value as to be the same. The weight of evidence from the variety of testing parameters

indicates that the two sources of the Cry1ac test substance protein are identical and inte rchangeable for the testing performed.

Classification: ACCEPTABLE 47841704

Functional Equivalence of E.

coli -Produced Full Length Cry1Ac

Protein and the Cry1Ac Tryptic

Core Protein The functional activity of the 131.7 kDa full-length E. coli

produced Cry1Ac protein (protoxin) was compared to that of

the 57.8 kDa E. coli -produced Cry1Ac tryptic core (toxin). The

EC 50 values from 18 previous dietary bioassays of corn
earworm ( Helicoverpa zea ) exposed to the Cry1Ac tryptic core were compared to the EC 50 values from 12 previous studies
exposing corn earworm to full length Cry1Ac. To correct for differences in the molecular mass, the EC 50 values (μg/mL) for
each set of bioassays were conve rted to molar concentrations, 47841706Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

10 Study Type/Title Summary MRID #
and the means were compared statistically. There was no statistically significant differe nce (p = 0.39) in the mean EC 50 values for the tryptic core and full length Cry1Ac protein expressed on a molar basis, indicating that both forms are functionally equivalent in potency against susceptible Lepidoptera. Therefore, results of safety and efficacy studies using the tryptic core can be used to evaluate the safety and insecticidal activity of the full length protein.

Classification: ACCEPTABLE

Molecular Analysis

of Insect-Protected Soybean MON

87701 – Amended Report for

MSL0022176 (Amendment 3) MON 87701 contains one copy of the insert at a single integration locus and all expression el ements were present in the TDNA I; MON 87701 does not cont ain detectable plasmid

backbone or selectable marker sequences; and the integrity of

the inserted cry1Ac expression cassette within the inserted sequences confirmed and identifi ed the 5' and 3' insert togenomic DNA junctions. The insertion site in conventional soybean confirmed the flanking genomic DNA is native to the soybean genome and was maintained through five generations of breeding.

Classification: ACCEPTABLE 47873801

B. HUMAN HEALTH ASSE SSMENT OF CRY1AC AS EXPRESSED IN MON 87701

Toxicological Profile

EPA has reviewed the available scie ntific data and other relevant information in support of this

action and considered its validit y, completeness and reliability and the relationship of this

information to human health risk

The human health studies submitted for Cry1Ac are summarized in Table 3 below.

Table 3. Summary of Cry1Ac Human Health Data

Study Type/Title Summary MRID #

Immunodetection of Cry1Ac protein in MON 87701 ground seed following heat

treatment. Heating of MON 87701 ground seed in a manner that simulates baking results in the loss of im munodetectable Cry1Ac protein below the LOD, indicating a decrease ≥ 94% of the Cry 1Ac relative to the protein in unheated MON 87701 samples.

Classification: ACCEPTABLE 47841705Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

11 Study Type/Title Summary MRID #
An Acute Study of Cry1AcProtein Administered by the Oral (Gavage) Route to Mice;

Human Healthand

Environmental

Assessment In an acute oral toxicity study, twenty male and twenty female fasted, approximately 8-10 week old CD-1 mice were given an

oral dose of control Bovine serum albumin (BSA) or Cry1Ac

Protein at doses of 1280 or 1290 mg protein/kg body weight, respectively. In an additional study phase, twenty male fasted, approximately 6 week old CD-1 mice were given an oral dose of control BSA or Cry1Ac Protein at dose of 1620 or 1460 mg protein/kg body weight, respectiv ely. The control and test material were administered as two doses separated by approximately 4 hours. The animals were observed for 14 days. The Oral LD 50 for female mice was > 1290 mg / kg BW and for male mice it was > 1460 mg / kg BW. Based on the results of this study, Cry1Ac Protein is in EPA Toxicity Category III. All animals survivedthe study. No test materialrelated clinical signs, body weight effects, and food consumption effects were noted during the study. No test material-related gross abnormalities were noted in any animal

at necropsy.

Classification: ACCEPTABLE 47841707

Assessment of the

in vitro digestibility of the Cry1Ac protein in simulated gastric and

simulated intestinal

fluids The data presented in this evalua tion of protein di gestibility in simulated gastric and simulated inte stinal fluids clearly indicate a trend toward digestion of the Cry1Ac protein in a rapid

manner. Within the limits of detection of the colloidal Coomassie Brilliant Blue protei n binding dye (between 10 - 20

ŋ g) following SDS-PAGE, the Cr y1Ac protein was degraded (i.e., not visible) within 30 sec of treatment with SGF. Treatment with trypsin (SIF) re sults in a peptide fragment representing the tryptic core of Cry1Ac. This tryptic core fragment is typical of δ -endotoxins from B. thuringiensis . With sequential treatment of SGF and SIF, Cry1Ac was rapidly degraded suggesting that under conditions typical of a monogastric mammalian stomac h, the Cry1Ac protein would not enter the small intestine in intact form, but rather as small, peptide fragments. Similar resu lts were obtained using Western blotting techniques with chemilu minescent detection; Cry1Ac protein was degraded within 30 seconds of SGF treatment with a limit of detection of approximately 1 ŋ g. A small stained band of approximately 4 kDa was noted on the SDS-PAGE gelfollowing SGF digestion. This band was examined by Nterminal sequencing and determined to consist of two peptides from Cry1Ac. Following treatment with SIF for 5 min, this band of two peptides was no l onger detected. The study results 47841708Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

12 Study Type/Title Summary MRID #
show that full-length Cry1Ac protei n is rapidly digested in the gastrointestinal digestive system.

Classification: ACCEPTABLE

Bioinformatics

evaluation of the Cry1Ac protein present in MON 87701 soybean utilizing the AD8, TOXIN6, and

PROTEIN

databases No significant sequence homo logy between any sequential MON 87701 Cry1Ac protein sequences and known allergens, toxins, or reactive proteins was found as based upon FASTA alignment procedures and an 8 amino acid sliding window comparison.

Classification: ACCEPTABLE 47841709

HUMAN HEALTH ASSESSMENT Cry1Ac in MON 87701

. Mammalian Toxicity and Allergenicity Assessment

Monsanto has submitted acute oral toxicity data demonstrating the lack of mammalian toxicity at high levels of exposure (ingestion) to the pure Cry1Ac protein. Th ese data demonstrate the safety of the product at a level well above maximum po ssible consumption levels that are reasonably anticipated as expressed in the crop. Basing this conclusion on acute oral toxicity data without requiring further toxicity testi ng and residue data is similar to the Agency position regarding toxicity testing and the requirement of residue data for the microbial Bacillus thuringiensis products from which this plant-incorporat ed protectant was deri ved (See 40 CFR Sec. 158.740(b)(2)(i)). For microbial products , further toxicity testing (Tiers II & III) and residue data are triggered by significant adverse acute effects in studies, such as the acute oral toxicity study, to verify the observed adverse effects and clarify the source of these effects.

An acute oral toxicity study in mice (MRID 47841707) indicated that Cry1Ac is non-toxic to humans and other mammals. Three groups of ten male and ten female mice were dosed by oral gavage with 1290 or 1460 mg/kg bodyweight of micr obially-produced Cry1Ac protein. A negative control group was also included in the study: bovine serum albumin protein control. There were no significant differences between th e test and control groups; theref ore, the Cry1Ac protein does not appear to cause any significant adverse effects at an exposure le vel of > 1460 mg/kg bodyweight.

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 acute effects were show n to be caused by Cry1Ac, even at relatively high dose levels, th e Cry1Ac protein is not consider ed toxic. Further, amino acidBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

13 sequence comparisons showed no similarities that would raise a safety concern between the Cry1Ac protein and known toxic proteins in protein databases.

Since Cry1Ac is a protein, allergenic potential wa s also considered. Currently, no definitive tests for determining the allergenic potential of novel protei ns exist. Therefore, EPA uses a weight-ofevidence approach where the following factors ar e considered: source of the trait; amino acid sequence comparison with known allergens; and bioc hemical properties of the protein, including in

vitro digestibility in simulated gastric fluid (SGF) fo llowed by simulated intestinal fluid (SIF), and glycosylation. This approach is consistent with the approach outlined in the Annex to the Codex

Alimentarius “Guideline for the Conduct of F ood Safety Assessment of Foods Derived from Recombinant-DNA Plants.” The allerg enicity assessment for Cry1Ac follows:

1. Source of the trait. Bacillus thuringiensis is not considered to be a source of allergenic proteins.

2. Amino acid sequence. A comparison of th e amino acid sequence of Cry1Ac with known allergens showed no significant overall sequence similarity or identity at the level of eight contiguous amino acid residues.

3. Digestibility. The Cry1Ac protei n was digested within 30 seconds in simulated gastric fluid containing pepsin. Small peptides remaining following gastric simulated digestion were completely degraded to amino acid residues in SIF upon contact.

4. Glycosylation. Cry1Ac expressed in soyb ean was shown not to be glycosylated.

5. Conclusion. Considering all of the availabl e information, EPA has concluded that the potential for Cry1Ac to be a food allergen is minimal.

Although Cry1Ac was only shown not to be glycosylated in corn, it is unlikely to be glycosylated in any other crops because in order for a protein to be glycoslyated, it must contain specific recognition sites for the enzymes involved in gl ycosylation, and the mechanisms of protein glycosylation are similar in differe nt plants (Lero uge et al., 1998).

Overall Safety Conclusions The continued use of Cry1Ac protein in pl ant expression systems (plant-incorporated protectants) and in microbial biope sticides is fully supported by th e information presented in this registration submission relative to human and an imal health concerns. The lack of mammalian toxicity and allergenicity eff ects following thorough examination of pertinent information, as well as the efficacy noted in the insect bioa ssays, indicates that th e specificity of the δ -endotoxin Cry1Ac as an insect management mechanism is sa fe as proposed for use in soybean intended for cultivation and human and animal consumption.

Endocrine Disruptors As required under FFDCA section 408(p), the Ag ency has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (inc luding pesticide active and other ingredients) may have an effect in humans or wildlife si milar to an effect produced by a “naturally occurring estrogen, or other such endocrine effects as the Administrator may designate.” The EDSP employs a two-tiered ap proach to making the statutorily requiredBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

14 determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a chemical substance to interact with the estrog en, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go th rough Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stag e of the EDSP where the Agency will determine which, if any, of the Ti er 2 tests are necessary based on the available data. Tier 2 testing is designe d to identify any adverse endocr ine-related effects caused by the substance, and establish a quantit ative relationship between the dos e and the E, A, or T effect.

Between October 2009 and February 2010, the Agency 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 sele cted based on the potential for human exposure through pathways such as food and water, resi dential activity, and cer tain post-application agricultural scenarios. Th is list should not be construed as a list of known or likely endocrine disruptors.

The Cry1Ac protein is not among the group of 58 pest icide active ingredients on the initial list to

be screened under the EDSP. Under FFDCA section 408(p), the Agency must screen all pesticide chemicals. Accordingly, the Agency anticipates issuing future EDSP orders/data call-ins for all pesticide active ingredients.

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

.

REFERENCES:

Gorbunova, V. and A.A Levy, (1997) Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions.

Nucleic Acids Res. , 25:4650–4657.

Lerouge, P. Cabanes-Macheteau, M., Rayon, C., Fichette-Lainé, A-C., Gomord, V., and Faye, L., (1998) N-Glycoprotein biosynthesis in plants: recent developments and future trends. Plant Molecular Biology 38: 31-48.

Salomon, S. and H. Puchta (1998) Captur e of genomic and T-DNA sequences during double-strand break repair in somatic pl ant cells. The EMBO Journal, 17:6086–6095.

Sjoblad, Roy D., et al., (1992) Toxicological Considerations for Pr otein Components of Biological Pesticide Products. Regulat ory Toxicology and Pharmacology 15:3-9.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

15 C. ENVIRONMENTAL ASSESSMENT for MON 87701

Overall Conclusions

Adverse effects to nontarget organisms, incl uding birds, wild mamm als, freshwater and marine/estuarine fish and invertebrates, nontarge t insects, honey bees, soil invertebrates, and terrestrial and aquatic plants, are not anticipated. Horizontal gene transf er, gene flow, and the development of weediness are also not anticipa ted. Based on these findings and information on habitat requirements of federally-listed threat ened and endangered lepidopteran species, the Agency also makes a No Effect determination for direct and indirect effects to threatened and endangered species and their habitats.

1. Nontarget Organism Tiered Testing and Risk Assessment Process for PIPs

The paragraphs below describe the process a nd rationale developed by BPPD for evaluating hazard of PIPs to nontarget organisms. This process is described in several of BPPD’s documents and is presented again here as background information.

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

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

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

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

cost. 1

1 Non-target invertebrate hazard tests ofte n are conducted at exposure concentrations several times higher than the maximum concentra tions 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 negl igible given re alistic exposure scenarios. Thus, it follows that the observ ed proportion of respondi ng individuals can be compared to a 50% effect to determine if th e 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 differe ntiated 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 ar e not needed to evaluate risk.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

16 Tiered tests are designed to firs t represent unrealistic worst case scenarios and ONLY progress to

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

The EPA uses a tiered (Tiers I-IV ) testing system to assess the toxi city of a 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 appro ach to testing designed to maximi ze any toxic effects of the test substance on the test (non-target ) organism. The screening test s evaluate single species in a laboratory setting with mortality as the end point . Tiers II – IV generally encompass definitive hazard level determinations, longer term greenhous e or field testing, and are implemented when unacceptable effects are seen at the Tier I screening level.

Testing methods which utilize the tiered appr oach were last published by the EPA as Harmonized OPPTS Testing Guidelines, Seri es 850 and 885 (EPA 712-C-96-280, February 1996). 2 These guidelines, as defined in 40 CFR 152.20, apply to microbes and microbial toxins when used as pesticides, including those that are naturally occurring, and those that are strainimproved, either by natural selection or by delibe rate genetic manipulation. Therefore, PIPs containing microbial toxins are also covered by thes e testing guidelines.

The Tier I screening maximum hazard dose (MHD) approach to environmental hazard assessment is based on some factor (wheneve r possible >10) times the maximum amount of active ingredient expected to be available to te rrestrial and aqua tic non-target organisms in the environment (EEC). 3 Tier I tests serve to identify poten tial hazards and are conducted in the laboratory at high dose levels that increase the statistical power to test the hypotheses. Elevated test doses, therefore, add certainty to the asse ssment, and such tests can be well standardized.

The Guidelines call for initial screening testing of a single gr oup or several groups of test

animals at the maximum hazard dose level. The Gu idelines call for testing of one treatment group of at least 30 animals or th ree groups of 10 test animals at the screening test concentration.

The Guidelines further state that the duration of all Tier I test s should be approximately 30 days.

2 OPPTS Testing Guidelines, Series 850 and 885 website: http://www.epa.gov/opptsfrs/publications/O PPTS_Harmonized/885Microbial_Pesticide_ Test_Guidelines/Series

3 The dose margin can be less than 10x where uncertainty in the system is low or where high concentrations of test mate rial 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.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

17 Some test species, notably non-targ et insects, may be difficult to culture and the suggested test duration has been adjusted accordingly. Control a nd treated insects should be observed for at least 30 days, or in cases where an insect spec ies cannot be cultured fo r 30 days, until negative control mortality rises above 20 percent.

Failing the Tier I (10 X EEC) sc reening at the MHD dose does not necessarily indicate the presence of an unacceptable risk in the field but it 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 th e existence of unacceptable risk in the field, but it does trigger the need to collect additional dose-response in formation and a refinement of the exposure estimation before deciding if the risk is acceptable or unaccep table. Where potential hazards are detected in Tier I testing (i.e. morta lity is greater than 50%), additional information at lower test doses is required which can serv e to confirm whether any effect might still be

detected at more realistic field [1X EEC] concentrati ons and routes of exposure. 5

When screening tests indicate a need for additio nal data, the OPPTS Harmonized Guidelines call for testing at incrementally lower doses in order to establish a definitive LD 50 and to quantify the

hazard. In the definitive testi ng, the number of doses and test organisms evaluated must be

sufficient to determine an LD 50 value and, when necessary, the Lowest Observed Effect Concentration (LOEC), No Observed Adverse Effect Level (NOAEL) , or reproductive and behavioral effects such as feeding inhibition, we ight loss, etc. In the final analysis, a risk assessment is made by comparing the LOAEC to the EEC; when the EEC is lower than the LOAEC, a no risk conclusion is made. These tests offer greater environmen tal realism, but they may have lower statistical power . Appropriate statistical methods , and appropriate statistical power, must be employed to evaluate the data from the definitive tests. Higher levels of replication, the number of test species, and/or re petition are needed to e nhance statistical power

in these circumstances.

Data that show less than 50 % mortality at the maximum hazard dosage level – (i.e. LC 50 , ED 50 , or LD 50 >10 X EEC) are sufficient to evaluate adve rse effects, making lower field exposure dose definitive testing unnecessary. It is also notable that the recomme nded >10X EEC maximum

4 It is notable that that the 10 X EEC MHD tes ting approach is not e quivalent 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 tes ting’. 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 ti ssue content and is c onsidered a high worst case dose (sometimes referred to as HEEC). This 1X EEC is still much greater than any amount which any given non-target organism may be i ngesting in the field because most non-target organisms do not inge st plant tissue.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

18 hazard dose level is a highly conservative factor . The published EPA Level of Concern [LOC] is 50% mortality at 5X EEC (US EPA, 1998). 6

Validation: The tiered hazard assessment approach was developed for the EPA by the American Institute of Biological Sciences (AIBS) and confirmed in 1996 as an acceptable method of environmental hazard assessment by a FIFRA Sc ientific Advisory Panel (SAP) on microbial pesticides and microbial toxins . The December 9, 1999, SAP agreed that the Tiered approach was suitable for use with Plant-Incorporated Protectan ts (PIPs); however, this panel recommended that, for PIPs with insecticidal propertie s, additional testing of beneficial invertebrates closely related to target species and/or likely to be present in GM crop fields should be conducted. Testing of Bt Cry proteins on species not closely re lated to the target insect pest was not recommended, although it is still performed to fulfill the publis hed EPA non-target species data requirements. In October 2000,

another SAP also recommended that field testin g should be used to evaluate population-level 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 expo sure to the subject pr otein (US EPA, 2000, 2001a, 2002, and 2004).

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

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

decisions. EPA believes that maximum hazard dose Tier I screening testing presents a reasonable approach for evaluating hazards related to the us e of biological pesticides and for identifying negative results with a high degree of confidence. The Agency expects that Tier 1 testing for short-term hazard assessment will be sufficient for most studies submitted in support of PIP registrations. But, if long range adverse effects must be ascertained, then higher-tier longer-term field testing will be required. As noted above, the October 2000 SAP and the National Academy of Sciences (NAS, 2000) recommended te sting non-target organi sms directly in the

6 The established peer and EPA Science Board reviewed guidance on screening test

levels of concern is 50% mortal ity at 5X environmental concen tration. The appropriate endpoints in high dose limit/screening testing are based on mo rtality of the treated, as compared to the untreated (control) non-target orga nisms. A single group of 30 test animals may be tested at the

maximum hazard dose.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

19 field. This approach, with an emphasis on testi ng invertebrates found in corn fields, was also recommended by the August 2002 SAP and was s upported by several public comments. Based on these recommendations, the Agency has requir ed field studies on long term invertebrate population/community and Cry protein accumulation in soils as a condition of registration due to

the lack of baseline data on th e potential for long-term environmental effects from the cultivation of PIP-producing plants.

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

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

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

on the effects of invert ebrate populations in Bt corn and cotton fields indicated that no unreasonable adverse effects are taking place as a result of wide scale Bt crop cultivation (Sanvido, et al. 2007). Another review of field tests publis hed to date concl uded that the largescale studies in commercial Bt cotton have not revealed any une xpected non-target effects other than subtle shifts in the arthropod community cau sed by the effective contro l of the target pests (Romeis et al ., 2006). Slight reductions in some inve rtebrate predator populations are an inevitable result of all pest ma nagement practices, which result in reductions in th e abundance of the pests as prey.

Overall, the Agency is in agreement with the co nclusions 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 pa rasitoids of target pests may eventually be considered unnecessary.

2. Nontarget Organism Risk Assessment for Cry1Ac as Expressed in MON 87701 Soybeans

a. Exposure Assessment

Two separate SAP reports (October 2000 and Augus t 2002) recommended that non-target testing

of Bt Cry proteins should focus on invertebrate species exposed to the crop being registered. Following SAP recommendations, the EPA determ ined that non-target organisms with the

7 This research was funded by Environmental Protection Agency grant CR-832147-01. The Bt crop non-target effects database can be found on the National Center for Ecological Analysis and Synthesis (NCEAS). Website. ( http://delphi.nceas.u csb.edu/btcrops/ ).Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

20 greatest exposure potential to Cr y proteins in transgenic crop fields are beneficial insects, especially those that feed on pollen and nectar , and soil invertebrates. While EPA’s risk

assessments of Bt PIP crops have focused primarily on these taxa, BPPD recognizes that exposure to other nontarget organisms can occu r and has required test ing on representative species.

The EPA risk assessment is centered only on adve rse effects at the fiel d exposure rates, which for PIPs are typically based on protein expression levels with in the plant. Although it is recommended that non-target testing be conduc ted at a test dose 10X the EEC whenever possible, the test 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. BPPD may also allow for testing at lower doses in cases where many species are tested or tests are very sensitiv e, although the concentration used must exceed the EEC. For the purposes of the non-target organism studies submitted in support of MON 87701 soybeans, the

test material dose levels were based on an esti mated concentration of Cr y1Ac protein expressed in the tissue(s) to which the assessed taxa are likely to be exposed. For nontarget insects and honeybees, tests were performed at concentrations of 19X to 322X the expression level in MON 87701 pollen. For soil invertebrates, Monsanto tested at 15X to 18X the estimated soil concentration based on the expected amount resulti ng from plant incorporation in the soil. For birds, tests were performed using raw soybeans. Details of these dosing amounts are discussed further in the summary of the toxi city studies presented below.

b. Ecological Effects Data

As discussed above, in the absence of PIP-speci fic risk assessment data requirements, EPA requires applicants for PIP registrations to meet the 40 CFR Part 158 data requirements for microbial toxins. These requirements include tes ting on birds, mammals, nontarget insects, honey bee, plants, and aquatic species, and information ha s been submitted to address these requirements. Limit dose testing and dietary studies were perf ormed on representative organisms from several taxa in support of the MON 87701 Soybean Section 3 registration. As stated above, BPPD’s risk assessments focus heavily on beneficial nontarget i nvertebrates, since they are most closely related to organisms susceptible to the insecticidal action of Bt toxins. The Cry1Ac protein is meant to target species within the order Le pidoptera (moths and butterflies). Bt toxins are known typically to have a limited host range; howev er, to address any unforeseen change in activity spectrum as a result of laboratory protein synthesis and to fu lfill the published registra tion data requirements EPA requires that test species used for non-target insect eval uations should include several invertebrate species that are not related to the target pests, as well as tests on birds and other vertebrates as appropriate. Earthworm studies are al so recommended. Data to evaluate the toxicity of Cry1Ac on several species of invertebrates were submitted, including the lady beetle, honey bee, and earthworm.

Since exposure may also occur to other nontarget organisms, EPA has received data to comply with the Agency’s published non-target data re quirements on other nontarget organisms. ABacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

21 study to determine soil degradation of Cry1Ac has also been submitted, as well as a description of the MON 87701 soybean Cry1Ac in soil. The i ndividual results for nontarget organism and soil degradation testing for Cry1Ac are summarized in Table 1. The studies are described in

more detail within the risk assessment conclusion s below, and full reviews of each study for each

event can be found in the individu al Data Evaluation Records.

The October 2000 SAP recommended that while act ual plant material is the preferred test material, bacteria derived protein is also a valid test substance, particularly in scenarios where test animals do not normally consume soybean plant tissue and where large amounts of Cry protein are needed for maximum hazard dose testing. In support of the MON 87701 soybean registration, test substances used in the submitte d studies included bacterially produced purified Cry1Ac protein. Comparative analyses have been performed to verify the equivalence of the bacterially produced and purified Cry1Ac a nd the Cry1Ac protein produced in MON 87701 soybean plants (MRID 47841701). These studies s how that the bacteria produced protein is equivalent to those produced by MON 87701 soybean plants.

Table 4. Summary of environmental effects studi es for Cry1Ac as expressed in MON 87701 Soybean

Data Requirement OPPTS

Guideline Test Substance Results Summary and Classification MRID

No.

Avian dietary testing, broiler chicken,

Gallus domesticus 885.4050 MON 87701 Soybean meal A 42-day dietary study showed no adverse affects to broiler chickens when fed a diet composed of approximately 30% MON 87701 soybean meal containing Cry1Ac. The presence of active Cry protein in the test material was not confirmed. Classification: Unacceptable 47841712

Avian dietary testing,

Northern bobwhite, Colinus virginianus 885.4050 Raw ground MON 87701 soybean An 8-day dietary study showed no adverse affects to Northern bobwhite fed a diet composed of 20% raw ground MON 87701 soybean. This exposure level is consistent with previously accepted studies for other

PIPs. Classification: Acceptable 47841711

Non-target insect testing, ladybird beetle, Coleomegilla

maculata 885.4340 Microbiallyproduced Cry1Ac No adverse effects on survival, development to adult, or mean adult weight were observed in Coleomegilla

maculata larvae exposed to 60 μg Cry1Ac protein/g of diet for 20 days. The LC 50 was determined to be >60 μg Cry1Ac/g of diet and the NOAEC is ≥ 60 μg Cry1Ac/g of diet. Classification: Acceptable 47841715

Non-target insect testing, parasitic

wasp, Pediobius foveolatus 885.4340 Microbiallyproduced Cry1Ac No adverse effects on survival were observed in adult Pediobius foveolatus fed 250 μg Cry1Ac protein/g of diet in a 21-day laboratory bioassay. The nominal

LC 50 was determined to be >250 μg Cry1Ac/g diet

and the NOAEC is ≥ 250 μg Cry1Ac/g diet. Classification: Acceptable 47841716

Honeybee testing, Honeybee larvae, Apis mellifera 885.4380 Microbiallyproduced Cry1Ac No adverse effects on survival were observed in honey bee larvae treated in natal cells with 410 μg/mL (4.1 μg/cell) solution of Cry1Ac protein. The

LC 50 was determined to be > 410 μg Cry1Ac/mL or >4.1 μg/cell. 47841713Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

22 Data Requirement OPPTS

Guideline Test Substance Results Summary and Classification MRID

No.

Classification: Acceptable

Honeybee testing, Honeybee adult, Apis mellifera 885.4380 Microbiallyproduced Cry1Ac No adverse effects on survival attributable to the test material were observed in honey bee adults fed 175 μg Cry1Ac protein/mL for 10 days. The LC 50 was determined to be > 175μg Cry1Ac/mL and the

NOAEC was determined to be ≥ 175 μg Cry1Ac/mL. Classification: Acceptable 47841714

Earthworm toxicity, Eisenia foetida 850.6200 Microbiallyproduced Cry1Ac No adverse effect on survival or burrowing time were observed in adult earthworms exposed to 250 mg Cry1Ac protein/kg dry soil for 14 days. The LC 50 was determined to be >250 mg Cry1Ac protein/kg dry soil and the NOAEC was determined to be ≥ 250 mg Cry1Ac protein/kg dry soil. Classification: Acceptable 47841717

Aerobic soil degradation 885.5200 Lypohilized MON 87701 soybean plants Based on the measured bioactivity in corn earworm ( Helicoverpa zea ), the DT 50 for Cry1Ac in three different soils was ranged from 0.4 –13 days. Classification: Acceptable 47841718

3. Risk Assessment Conclusions for MON 87701 Soybeans

a. Effects to Nontarget Wild life, Invertebrates, and Plants

1. Avian Wildlife

The primary routes of exposure of birds to Cry1Ac expressed in MON 87701 soybean plants are expected to occur through consumption of MON 87701 plant material (e.g., seeds) and invertebrates that feed on MON 87701 soybean plant material. Exposure via consumption of soybean seeds may be limited, however, by compounds that interfere with digestion and nutrient uptake, such as trypsin inhibitors. Other routes of exposure (e.g., inhalation) are not expected to be significant, since the Cry1Ac protein is contained primarily w ithin the plant.

The registrant has submitted two toxicity studies fo r use in determining risks to birds. In a 42- day feeding study, broiler chickens (MRI D 47841712) exposed in a diet containing approximately 30% processed soybean meal showed no adverse effects. We note, however, that soybean meal processing involves heat, which c ould inactivate the Cry1Ac protein. Given that confirmation of the presence of the Cry protei n in the diet was not performed, it is unknown whether the test animals were exposed to Cry1Ac, so this study may not be reliable for risk assessment. Confirmation of the presence of the Cry1Ac in the diet would resolve this uncertainty. An 8-day feeding study with Nort hern bobwhite (MRID 47841711) is available, in which the birds were fed a diet of 20% raw gr ound soybeans. As mentioned above, consumption of raw soybeans by birds can have detrimental eff ects; however, the inclusion of raw soybean in

the diet at this level without significant effects was supporte d by an additional non-guideline study (MRID 47896601). This study shows that effect s are not expected when birds consume aBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

23 diet containing 20% MON 87701 soybeans. Additi onally, no adverse effect s were observed in a study of birds exposed to Cry1A c expressed in Monsanto’s Bollgard cotton (USEPA 2001b) tested an exposure of 10% cottonseed in the diet . The level of exposure to the birds in these

studies is consistent with the Agency’s previous assumptions of EECs of PIPs for birds (e.g., USEPA 2001b, USEPA 2005a, USEPA 2005b, USEPA 200 8), and based on those assumptions

adverse effects to birds are not anticipated. A refined estimate of exposure to birds in the field for soybeans has not been determined (e.g., based on their potential avoidance of soybean seeds and/or consumption of other foods, such as invertebrates). Therefor e, a feeding test at a higher concentration of Cry1Ac or a limit dose test at the MHD would increase the certainty of risk

conclusions.

Cry1Ac has been registered as a PIP in several crops with no identified concerns for birds, and it has a history of use with no reported incidents in volving birds in the field, including use in a crop that is typically ingested by bird s (corn). Therefore, based on this history, current toxicity data, and the BPPD’s assumptions of avian exposure, adverse effects to bird s as a result of the registration of MON 87701 s oybeans are not expected

2. Wild Mammals

Wild mammals could be exposed to Cr y1Ac produced by MON 87701 soybean plans by consumption of plant material a nd invertebrates that feed on M ON 87701 soybean plant material. As with birds, these are expected to be the primary sources for exposure.

Data are available with which to determine the risk of Cry1Ac from MON 87701 soybeans to

wild mammals. The results from the acute oral toxicity studies showed no significant toxicity to mice from acute oral testing with the Cry1A c protein, with a NOAEL that exceeds 1290 mg/kg body weight for females and 1460 mg/kg for males (MRID 47841707). These data are sufficiently representative of t oxicity that would be expected in wild mammals, and based on the results of this study risk to wild mammals resulting from exposure to Cry1Ac expressed in MON 87701 soybean is not expected.

3. Freshwater Animals

The Agency has previously determined that the exposure of aquatic systems to registered Cry proteins produced in their respective crops is mi nimal. Off-field pollen movement is a possible mechanism by which Cry proteins may be transpor ted to aquatic habitats. But, given that soybeans are primarily self-pollinated and cultiv ated soybean plants release pollen prior to flower opening (Abud et al. 2007, Cavi ness 1966), off-site movement of pollen in this case is not expected. The Agency has conclude d that plant litter from both Bt corn and Bt cotton crops are not deposited in amounts high enough to result in adverse effects (U SEPA 2001b). Moreover, given that soybean produces less above-ground re sidue under cultivation (Green and Blackmer 1995), this conclusion is also made for MON 87701 soybeans. Monsanto submitted data waiver rationales that are consistent with the Ag ency’s position on aquatic exposure with Bt PIPs.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

24 Based on the expected lack of exposure, risk to fr eshwater fish as a result of the registration of Cry1Ac in MON 87701 soybeans is not expected. While exposure is expected to be very low in aquatic habitats, and effects on freshwater inve rtebrates is not expected, a published laboratory study with lepidopteran-active Cry proteins has revealed that th e leaf shredding (caddis fly) trichopteran, Lepidostoma liba , had 50% lower growth rate when fed Btcorn litter (RosiMarshall, et al. 2007). Two previ ous field study reports by the same authors did not find adverse

effects on head stream invertebrates. Because of concerns raised in this study, EPA has been requiring registrants of Bt PIPs to submit a 7-10 day freshwat er invertebrate toxicity study for each PIP to reduce uncertainty regarding this issue.

4. Estuarine and marine animals

BPPD typically waives the requirement of studies with estuarine/marine animals for Bt Cry proteins because of an expected lack of exposure in these environm ents. Therefore, data were not required for Cry1Ab for the registration of MON 87701 soybeans, and based on expected lack of exposure, adverse effects to thes e species are not anticipated.

5. Terrestrial and aquatic plant species

BPPD typically waives nont arget plant testing for Bt Cry proteins, since the active ingredient is an insect toxin ( Bt δ -endotoxin) that has never shown any t oxicity to plants. Therefore, BPPD has concluded that adverse effects of Cry1Ac expressed in MON 87701 s oybeans to terrestrial and aquatic plants are not anticipated. 6. Invertebrate species

BPPD assumes that nontarget in sects receive exposur e primarily through consumption of MON 87701 soybean pollen and/or nectar, though they ma y also consume pest insects that feed on soybean plant tissue or occasionally consume ot her soybean plant tissues themselves. The principal route of exposure to soil-dwelling invert ebrates, such as collembola, earthworms, and rove beetles, is assumed to be consumption of decomposing plant tissue, and also possibly plant exudates, in soil during feeding.

Monsanto submitted studies with which to determine the effects of Cry1Ac expressed in MON 87701 to nontarget insects and soil invertebrates. Studies were submitted with a lady bird beetle and a parasitic wasp species, each of which showed that adverse effects di d not occur to the test

insects at >60 μ g/g diet and >250 μ g/g diet, respectively. As suming a maximum expected

environmental concentration of 3.1 μ g/g diet (see MRID 47841701) based on the estimated expression level in pollen, adverse effects would not be expected at concen trations up to 19X and

80X this assumed maximum EEC for lady bird beetle and parasitic wasp, respectively. Additionally, a study was submitted with earthworm s that showed no effects on mortality or burrowing time resulting from exposure to Cry1Ac pr esent in artificial soil at approximately 18XBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

25 maximum EEC. The maximum EEC of the soil was determined based on incorporation of entire soybean plants into the top six inches of soil, and is described in MRID 47841717.

Two studies that tested the effect of Cry1Ac expressed in MON 87701 soybean in honey bee were also submitted. One study tested the effects in honey bee larvae at 410 μ g Cry1Ac/mL test

solution or 4.1 μ g Cry1Ac/cell (larva). This study te sted effects at 132X EEC based on the

concentration of the test soluti on. No adverse effects were obse rved in the larvae in this study, and the number emerging to adults in the gr oup exposed to Cry1Ac was not significantly different from the controls. The other bee study te sted the effects of oral exposure to Cry1Ac in adult bees. In this study, adult bees were fed a solution containing 175 μ g Cry1Ac/mL test solution (56X EEC). The test so lution contained a buffer, which was shown to cause a decrease

in survival. A decrease in survival was also observed in the group exposed to Cry1Ac, but these

effects were determined to be st atistically similar to those in th e buffer control group. Therefore, the effects observed on adult honey bees in the Cry1Ac group were determined to be due to the buffer and not the Cry1Ac.

The Agency has additional information for nont arget insects on Cry1Ac submitted to support the registration of Bollgard cotton. The Cry1A c protein produced in MON 87701 soybeans is identical to the Cry1Ac produced in Bollgard co tton, with the exception of four additional Nterminal amino acids derived from a chloroplast transit peptide targeting sequence that is not expected to impact activity or toxicity because of excision of the N-terminus during prototoxin activation (MRID 47841701). Studies with th e Cry1Ac protein produced in Bollgard additionally showed no adverse e ffects in two Collembola spp. ( Folsomia candida and Xenylla grisea ) and minute pirate bug ( Orius albidipennis ) (USEPA 2001b).

Based on the studies submitted for MON 87701, as well as other information for Cry1Ac, risk to nontarget insects, soil in vertebrates, and honey bees resultin g from registration of Cry1Ac in MON 87701 soybean is not expected.

b. Fate of Cry1Ac in Soil

A study was submitted to show that Cry1Ac expr essed in MON 87701 soybean plants degrades quickly and does not accumulate in soil (MRID 47841718). Lyophilized MON 87701 soybean plants (leaves + stems + root s), containing 63 μg of Cry1Ac/ g of soybean biomass were incorporated into soils collected in three locations with soybe an-growing regions. Soils were incubated for 181 days, and samples collected weekly were subjected to su sceptible insect (corn earworm [ Helicoverpa zea ]) bioassays and ELISA to determine Cry1Ac content and bioactivity. Both the ELISA and the bioassays showed that Cry1A c degraded in all three soils, with the most rapid degradation occurring during the first 16 days. At test e nd, 72% to 96% of the Cry1Ac had degraded regardless of soil texture, pH, clay content, or the method us ed to analyze the soil (ELISA or bioassay), and the DT 50 of Cry1Ac in the soils was ranged from 0.4 – 13 days. These results indicate that Cry1Ac from MON 87701 br eaks down rapidly in soil and is unlikely to persist or accumulate in the environment after continuous cultivation. Because currentlyBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

26 registered PIPs have not been shown to persis t in soil, the Agency has determined that no additional long-term field studies are required for MON 87701 soybeans.

c. 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). 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, the soil degradation study discussed above shows th at the Cry1Ac protein from MON 87701 plants degrades quickly in soil.

With regard to the impact of genetically engineer ed crops on soil, it is important to note that agricultural practices themselves cause large changes in soil and soil microbial composition. Furthermore, factors such variations in seasons and weather, plant gr owth stage, and plant varieties, independent of being ge netically engineered, are also re sponsible for sign ificant shifts

in soil microbial communities. Most studies with genetically engineered crops to date have shown minor or no effects on soil microbes beyo nd the variation caused by the factors listed above. If reports of adverse effects became availa ble, the Agency will take appropriate action to mitigate potential risks.

d. Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms

The EPA has evaluated the potential fo r horizontal gene transfer (HGT) from Bt crops to soil organisms and has considered possible risk implica tions if such a transfer were to occur. Genes that have been engineered into Bt crops are mostly found in, or have their origin in, soilinhabiting bacteria. Soil is al so the habitat of anthrax, teta nus and botulinum toxin-producing bacteria. Transfer of these genes and/or toxins to other microorganisms or plants has not been detected. Furthermore, several published experi ments, that were conducted to assess the likelihood of HGT, have been unable to detect gene transfer under typical environmental conditions. Horizontal gene tr ansfer to soil organisms has only been detected with very promiscuous microbes under laboratory c onditions designed to favor transfer. As a result of these findings and the fact that the Bt toxins engineered into MON 87701 were derived from soil-inhabiting bacteria, the EPA ha s concluded that HGT of transgenes found in Cry1Ac producing soybean is not expected.

e. Gene Flow and Weediness Potential

Movement of transgenes from crop plants into we eds is a significant concern, due to uncertainty regarding the effect that a new pest resistance gene may have on plant populations in the wild. Under FIFRA, the Agency has reviewed the poten tial for gene capture an d expression of Cry proteins in commercial Bt soybeans by wild or weedy relatives of cotton in the United States, its possessions or territories.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

27 An Experimental Use Permit (EUP) registrati on was granted for MON 87701 soybeans in 2007. At that time, BPPD examined the potential for gene flow and the weediness potential of MON 87701 soybean. Since no known wild or weedy relatives exist in the U.S. with which Glycine

max (L.) Merr. can form viable hybrids in nature, and because soybean is not weedy in character or invasive, BPPD determined that there is no si gnificant risk of gene ca pture and expression of

any Bt endotoxin by wild or weedy rela tives of soybean in the U.S., its possessions or territories (BPPD 2007).

f. Impacts on Endangered Species

Because of the selectivity of Cr y1Ac for lepidopteran species and lack of evidence of effects on other nontarget species, the Agen cy has investigated concerns fo r federally-listed threatened and endangered insect species in the order Lepidoptera. Because soyb ean pollen is not expected to move beyond the planted soybean field and its immediate margins, as discussed above, any exposure to lepidopterans would be expect ed to occur within those areas.

Exposure could occur via direct consumption of MON 87701 soybean plants or consumption of MON 87701 pollen that falls on non -soybean plants within the s oybean field and its immediate margins. However, exposure to significant amounts of Cry1Ac via pollen consumption is not likely. Little pollen is expected to be released from soybean fl owers, since soybean plants are primarily self-pollinated and anthers usually de hisce and release pollen before flowers open. Airborne pollen concentrations have been measur ed at very low levels within soybean fields (mean of 0.18 grains/cm 2 /day) (Yoshimura et al. 2006). To examine the potential exposure from pollen falling on food plants, BPPD performed a similar analysis as that used in Hellmich et al. (2001), which was also employed in the Bt cr ops reassessment BRAD (U SEPA 2001b) and the assessment of nontarget lepidopteran risk for Wi deStrike cotton (USEPA 2005a). Based on the

most sensitive EC 50 value reported for CEW (used in confir matory tests) in nontarget organism studies submitted for MON 87701 soybean (0.0022 μ g Cry1Ac/mL diet), an expression level of

3.1 μ g Cry1Ac/g for pollen, and assumptions of leaf consumption and pollen weight as given in Hellmich et al. (2001), BPPD determined that an estimated number of pollen grains resulting in

the EC 50 is 3.51 grains/day. Based on the assumed daily food consumption, 18.49 grains pollen/cm 2 /day must be present on potential food plan ts to result in an amount equal to the EC 50 for the sensitive species. This amount is 100X the mean levels of pollen measured in soybean fields. The Hellmich et al. analys is was performed for corn. Corn pollen is recognized as being relatively heavy; however, since corn is wind polli nated, soybean pollen is lik ely to be heavier. However, even if a soybean pollen grain weighs 5x that of corn pollen, the estimated number of pollen grains resulting in the EC 50 would still be 20x the amount of pollen measured in soybean fields. These are expected to be conservative es timates, since higher pollen weights for corn are reported (e.g., 2.0 million grains/gram [Mascarenha s et al. 1984], 3.5 million grains/gram [RosiMarshall et al. 2007]). Additiona lly, based on the mean amount of pollen measured in soybean fields, and assuming a 14 day flowering period for a soybean field, 2.52 grains/cm 2 would be expected on potential food plants at the end of the flowering period if they are entirelyBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

28 undisturbed and no other organisms consume the po llen, and the amount needed to result in the equivalent EC 50 would not be attained. Based on this analysis, BPPD conc ludes that exposure resulting from pollen falling on potential non-soyb ean food plants in the field and immediate margins is not sufficient to cause effects in lis ted lepidopterans. Ther efore, any significant exposure would have to occur through consum ption of the MON 87701 soybean plants on the

field.

A search of EPA’s LOCATES database indicates th at three species of li sted lepidopterans are present in U.S. counties in which soybeans are grown. These are the Ka rner blue butterfly ( Lycaeides melissa samuelis ), St. Francis’s Satyr Butterfly ( Neonympha mitchellii fransisci ), and Mitchell’s Satyr Butterfly ( Neonympha mitchellii mitchellii ).

The potential effects of Bt PIPs in corn on the Karner blue bu tterfly was extensively analyzed in

BPPD’s Bt Crops Reassessment (USEPA 2001b). This species requires a specific wild lupine species ( Lupinis perrenis ) as a site of oviposition and larv al food source, and the concern for exposure in the case of Bt corn resulted from the possibility of pollen falling on this required plant species. As shown in the analysis above, soybean pollen is not expected to be deposited on plants in or around soybean fi elds in amounts sufficient to cause effects in sensitive lepidopterans. Additionally, EPA has previously reviewed information on the proximity of Karner blue habitats, and none are known to ex ist immediately adjacent to agricultural fields (“adjacent to” was defined as 0-3 m for corn fi elds, which is reasonably applied to soybean fields) (USEPA 2001b). Therefore, effects to th e Karner blue butterfly via consumption of pollen on its food/host plant are not expected. Ad ditionally, because the Karner blue butterfly has an obligate relationship with a specific host plant, it will not be exposed to Cry1Ac via direct consumption of MON 87701 soybeans. Therefore, effects to this specie s as a result of the proposed registration of MON 87701 soybeans are not expected.

Mitchell’s satyr butterfly has a strict reliance on fen habitats, which are characterized as stable, highly alkaline, and nutrient poor wetland habitats. Fen habitats can occur near agricultural areas; however this species is considered to be a strict habitat speci alist (USFWS 1998, USFWS 2009). Szymanski et al. (2004) describes habitat for this species as “mosaics of open shrubby and forested communities and associated ecotones.” They also note that all active habitats for this species are sedge-dominated, and contain scattered deciduous or coniferous shrubs, and observed that individuals stay w ithin their habitat patch. Edge habitat and scattered shrubs appear to be crucial habitat components for these species, and vegeta tion structure is as important as the presence of suitable host plants (Lee 2000, Shuey 1997). The species has a strong preference for habitats with dense stan ds of sedges (USFWS 1998), and meadows and

fens without shrubs are consid ered to be unsuitable habitat (Shuey 1997). In a study by Barton and Bach (2005) captures of adu lts at their study sites showed clear preference for edge and

scattered shrub cover and avoidan ce by this species of open areas a nd habitats other than suitable fen habitats, including agricultural fields. Host plants have been determined to be almost certainly sedges, and Carex stricta is considered likely to be their primary host plant (USFWS 1998, USFWS 2009), although they have been observed ovipositing on other small (<5 cm)Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

29 herbaceous plants (Lee 2000, Szymanski et al. 2004) , reported to be located under dense stands of sedge (Szymanski et al. 2004). Sources c ited within Barton and Bach (2005) observed ovipositioning within 1 m of the n earest tree or shrub. The species is also considered to be sedentary and a weak flyer (USFWS 1998, USFWS 2009, Szymanski et al. 2004, Lee 2000), although some populations may be less sedentar y than others (Barton and Bach 2005, USFWS 1998). Corridors containing suitable habitat are considered to be critical for dispersal and conservation (Barton and Bach 2005, Szymanski et al . 2004), so an open, agricultural field is not likely to be used for this purpose. Adults are generally not observed f eeding, and the butterflies

do not live for more than a few weeks in this life stage. U.S. Fish and Wildlife Service (FWS) documents describing habitat for this species do not associate it with agricultural fields (USFWS 1998, USFWS 2009), though the extent to which such habitats have been surveyed for this species is not reported. Mitchell’s satyr butterfly is recognized as a st rict specialist of fen habitats that cannot be provided by soybean cultivation. Based on the life history described in the documents and studies examined, BPPD concl udes that the occurrence of Mitchell’s satyr in MON 87701 soybean fields is extremely unlikely and its use of MON 877 01 soybean plants as ovipositioning sites or larval food pl ants will not occur. Therefore, effects to this species as a result of the registration of MON 87701 soybeans are not anticipated.

The St. Francis’s satyr bu tterfly is closely related to the Mitchell’s satyr, but occupies bog habitats that tend to be more transitory compared to fens. This species is also considered to be a strict habitat specialist, and is most closely associated with Carex spp. The St. Francis’s satyr

has habits similar to those described for Mitchell’s satyr above, in that it relies heavily on early successional habitats dominated by sedges, is sede ntary, and is a weak flyer. All occupied habitats have been created by some kind of distur bance (e.g., beaver activit y, fire). Dispersal has been recorded rarely over unsui table habitats. Dispersing indivi duals show a strong tendency to fly along streams, remaining within riparian vege tation, and have been observed almost always turning back to the colony on ce the edge of the wetland is re ached. Movements >1 km are probably rare (NatureServe 2009). The St. Franci s’s satyr butterfly exists in limited number of small populations (one metapopulati on). It is currently known to occupy only a small area (10- 20 ha) within Department of Defense (DOD) lands in the sandhills regi on of North Carolina (Bartel and Sexton 2009, Haddad et al. 2008, Kuefler et al. 2008, USFWS 1996), which substantially restricts its potenti al for exposure. Since this sp ecies is recognized as a strict habitat specialist, and because it has not been detected outside of DOD lands , despite surveys of surrounding areas, cultivation of MON 87701 soybean pl ants will not result in effects to the St. Francis’s satyr.

Based on the above analysis, the Agen cy determines that there will be no direct effect to listed lepidopteran species as a result of the cultiv ation of MON 87701 soybeans as proposed. Obligate relationships between insectivorous listed species with lepidopterans that are expected to be found in soybean fields, especially pest species that feed on MON 87701 pl ants, are not currently known. Since the Cry1Ac in MON 87701 soybeans targ ets only lepidopteran insects, loss of the pest insects as a result of MON 87701 are expected to be offset by the presence of other insects that could act as food sources for listed species, including beneficial insect s that are known not toBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

30 be affected by Cry1Ac. Effects on species other th an insects have also be en determined to be very unlikely because of the specificity of Cry1Ac. Therefore, no effects to listed species due to

indirect effects or effects on habita t are anticipated to occur.

4. Data Needed to Confirm MON 87701 S oybean Non-Target Hazard Assessment

BPPD has sufficient information to believe that there is no risk from the proposed uses of MON 87701 soybean to non-target wildlife a nd terrestrial invertebrates, fi sh and aquatic invertebrates, plants and soil organisms. As indicated above, the Agency has b een alerted to possible concerns

of effects of Bt PIPs on freshwater aquatic invertebrates. As a result, the Agency is requiring registrants of Bt PIPs to submit a 7 -10 day freshwater invertebrate toxicity study to reduce uncertainty with this issue. C onsistent with this determination, this study must be submitted to support a full commercial registration of Cry1Ac in MON 87701 soybeans.

CONCLUSION

The environmental risk assessment indicates that , based on information submitted to support this proposed registration, as well as previous assessments for Cry1Ac, cultivation of MON 87701 soybeans as intended will not result in unreasonabl e adverse environmental effects. The Agency additionally concludes that M ON 87701 soybeans will have no dire ct or indirect effect on endangered and/or threatened species listed by the FWS and the Nati onal Marine Fisheries

Service or their habitats.

References

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33 aizawai Cry1F and the Genetic Material (Fro m the Insert of Plasmid PGMA281) Necessary for its Production in Cotton and Bacillus thuringiensis var. kurstaki Cry1Ac and the Genetic Material (From the Insert of Plasmid PMYC3006) Necessary for its

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D. INSECT RESISTANCE MANAGEMENT (IRM)

1. Overall Conclusions

a) Given the low acreage projected by Monsanto for MON 87701 (<15,000 to tal acres per year), it is unlikely that there will be a significant risk of resistance to the main soybean target pests in the United States, soybean looper and velvetbean caterpillar. This conclusion is further supported by the biology of the target insects, which do not overwinter in non-tropical areas and are polyphagous (feeding on a number of wild hosts and cultivated crops).

b) The use of MON 87701 on a limited basis should not impact the natural refuge strategy in place for Bt cotton PIPs. The total proposed acreage for MON 87701 would represent a smallBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

34 fraction of total soybean acres in each state and should not si gnificantly reduce the amount of natural refuge. Further, MON 87701 will not be pl anted in states (i.e., Mississippi Delta states) with lower natural refuge.

c) BPPD is requiring adoption of a resistan ce monitoring plan, remedial action strategy, and annual sales reporting for MON 87701.

2. Insect Resistance Management Assessment

Based on the materials submitted to support re gistration, BPPD does not have sufficient information to completely assess the risk of re sistance to the primary target pests for a full commercial registration of MON 87701 soybean. However, Monsan to’s proposal to limit the overall acreage and distribution of MON 87701 in the U.S. should be sufficient to mitigate potential resistance development.

a. Target Pests

Soybean looper (SL, Pseudoplusia includens ) and velvetbean caterpillar (VBC, Anticarsia gemmatalis ) are the primary target pests of MON 87701 in the U.S., but only cursory information on pest biology was provided in th e submission. These insects have not been targeted by other registered Bt crops (e.g. Bt cotton or Bt corn) and have not been assessed as part of any other IRM plan. The other two in sects targeted by MON 87701, soybean axil borer ( Epinotia apoerema ), and sunflower looper ( Rachiplusia nu ), have South Amer ican distributions

and do not occur in the U.S.

Soybean looper occurs throughout the U.S. wher e soybeans are grown, bu t overwinters only in tropical regions migrating annua lly from Mexico, Central Americ a, and the Caribbean (Pedigo, 1999). The insect is multivoltine with two (northern regions) to seven (southern states) generations per season. In addition to soybea n, SL is known to feed on a wide range of vegetables, field crops (including corn and cotton), and weeds (Capinera, 2005).

Velvetbean caterpillar, similar to SL, is a tropical species that migrates annually northward into the U.S. It can range as far north as New England, but overwinte rs only in the southern end of the Florida peninsula (Pedigo, 1999; Barbara, 20 08). VBC is also polyphagous and feeds on a number of field crops (soybean, alfalfa, co tton, peanut, other legumes) and weeds (Barbara, 2008).

b. IRM Risk Considerations

To assess the risk of resistance for new PIPs, BPPD typically consider s (in addition to pest biology) information on dose expr ession and simulation modeling. Regarding the dose of MON 87701, Monsanto referenced a published article (M cRae et al., 2005) that concluded the tested soybean cultivar (TIC107 with a cry1A gene) exhibited high dose contro l against the target pests.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

35 A copy of this article was not provided in th e submission, though BPPD was able to obtain a reprint. This research showed that the tested soybean line appears to meet the criteria for high dose using two of the techniques established by the 1998 SAP, including serial dilutions with lyophilized plant tissue (technique #1) and field trials to dem onstrate high efficacy against potential heterozygotes (technique #3). But, B PPD cannot verify the high dose claims from the

article because it is unclear whether the test ed variant (TIC107) and toxin (a Cry1A toxin described as being “similar” to Cr y1Ac) are equivalent to MON 87701.

Simulation modeling was not conducted fo r MON 87701 and a refuge-based resistance management strategy was not proposed by Monsant o. Rather, the company has indicated that a

number of factors should reduce th e risk of resistance so that a formalized refuge strategy is unnecessary. These factors include the distribu tion of the target pests (SL and VBC), the abundance of natural refuge for these insects, and anticipated limited plantings of MON 87701 for seed increase purposes.

BPPD believes that additional information on pe st biology, dose, simulation modeling, and cross resistance would be necessary to assess the risk s for an unlimited commercial registration of MON 87701. But, for a limited “seed increase” registration, BPPD agrees with Monsanto’s rationale regarding the low risk of resistance for the target pests.

Biological attributes of SL and VBC reduce the overall likelihood of resistance development. Both insects are polyphagous and feed on numer ous other wild and cultivated hosts, which should provide a source of natura l refuge to reduce se lection pressure for Cry1Ac resistance. Monsanto’s submission also indicated that soybea ns have relatively small acreage (ca. 13% of U.S. soybean acres) in the southeast, where these two insects are most prevalent. Further, SL and VBC overwinter only in trop ical areas (e.g., Florida in th e U. S.); populations migrate annually from the Caribbean, Mexico and Central America northward through the U.S. Because of this, it is unlikely that any resistant indivi duals that might evolve during the growing season north of tropical areas would be able to successfully overwinter and propagate the resistant trait to future generations.

Monsanto suggested in their submission that the proposal for MON 87701 is similar to the requirements in place for “breeding and seed multip lication activities” with other registered PIPs. Seed production is often not compa tible with refuges due to the n eed for purity in seed stock. For IRM purposes, the seed activity is geographically limited to re duce the selection pressure for resistance. To illustrate, an annual per county limit of 20,000 acres for seed production has been imposed for Bt corn PIPs. Also, refuges have not generally been used with PIP Experimental Use Permits because of smaller and dispersed acreages.

Based on the proposed acreage-limited (seed increas e) registration and in consideration of the biological aspects of the target pests discussed above, BPPD concl udes the risk of resistance to Cry1Ac should be low for SL and VBC. Therefore, a refuge strategy is not recommended at this

time for MON 87701. BPPD notes that Monsanto’s submission provided an estimate of theBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

36 geographic limitations for MON 87701. Projecte d acreage is not expected to exceed 15,000 (U.S.) total and 1,000 per county. A follow-up subm ission from Monsanto (April 8, 2010 letter) indicated that a maximum of 15,000 acres would be planted in Atlantic Coast states (Maryland through Georgia). Monsanto’s submission also indicated, howe ver, that “MON 87701 plantings....will be initially limited to breeding and seed mu ltiplication activitie s...” (emphasis added). Should any request be made to e xpand the scope of MON 87701 to a commercial registration (with large or unlimited acreage), B PPD will require a formal IRM assessment to better evaluate the resistance risk and develop ap propriate mitigation measures. Such a review should include appropriate data for dose expres sion, simulation modeling, and cross resistance.

c. Natural Refuge Considerations

In addition to the primary target pests of soyb ean, BPPD is also concerned about other insect pests that are targeted by Bt cotton but may al so be exposed to Bt soybean. These insects, tobacco budworm (TBW, Heliothis virescens ) and cotton bo llworm (CBW, Helicoverpa zea ), are similar to SL and VBC in that they are high ly polyphagous and are known to exploit numerous crop and wild plant hosts. Cry1Ac is expresse d in both MON 87701 and Bt cotton (Bollgard and WideStrike varieties).

The established resistance manageme nt plan for Bt cotton involves th e use of “natural refuge” in which non-PIP hosts (such as weeds and other non -cotton cultivated crops) provide sources of susceptible insects to dilute any potential resistan ce genes arising from transgenic cotton. One of the non-cotton crops included in calculations of natural refuge is soyb ean. Should a significant portion of the soybean crop be devoted to a Bt variety, the potential amou nt of natural refuge

available to TBW and CBW could be reduced.

In response to BPPD’s concerns, Monsanto (A pril 8, 2010 Submission) provided a rationale for low resistance based on geographic limitations for MON 87701. The company intends to plant limited acres (<15,000) in areas with high amounts of natural refuge. Wh ile Monsanto did not provide revised natural refuge calculations or modeling to support MON 87701, BPPD agrees

that there should be low resist ance risk potential for TBW a nd CBW based on the low acreage projections. The areas intende d for plantings of MON 87701 ha ve high proportions (>90%) of natural refuge including numerous wild hosts and cultivated crops (s ee reviews in BPPD 2006, 2007). As described in Monsanto ’s submission, 15,000 acres of Bt soybean in these areas would represent only a small fraction of total soybean acres -- to i llustrate, if all 15,000 acres were planted in one state and assuming the lowest USDA acreage figure (430,000), Bt soybean would represent 3.5% of tota l cultivation. Given the small overa ll amount of MON 87701 projected for planting, it is unlikely that the amount of natu ral refuge available to TBW and CBW will be significantly reduced. Further, even if natural refuge was lowered due MON 87701 there would still be abundant sources of other plants hos ts (e.g., peanuts, tobacco, vegetable crops, and weeds) such that it is unlik ely the resistance risk of TBW and CBW to Cry1Ac would appreciably increase.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

37 BPPD’s assessment of the impact of MON 87701 on Bt cotton natural refuge is contingent upon the acreage projections provided by Monsanto (as discussed in th e previous section). A full commercial registration of M ON 87701 without acreage limitati ons would require a more detailed assessment that likely would include revised natural re fuge calculations and simulation modeling.

d. Stewardship Activities

BPPD recommends that other aspects of IRM in cluding resistance monito ring, a remedial action plan, and annual sales reporting be implemented for MON 87701. A full resistance monitoring plan with pest sampling and detection bioassays may not be warranted for a limited registration; rather an approach based on investigations and follow-ups on reports of unexpected pest damage may be appropriate. A remedial action plan, in the event of document resistance, could mimic those in place for Bt corn or cotton or could rely on a “stop sale” approach if resistance develops. An annual sales report detailing total acreage by state is also reco mmended. Provided MON 87701 is registered without a structured refuge requirement, a compliance assurance plan and grower education program should not be necessary.

References

Barbara, K.A., 2008. University of Florida, Department of Entomology and Nematology informational web page for velvetbean caterpillar ( Anticarsia gemmatalis ): http://entnemdept.ufl.edu/cr eatures/field/velvetbean.htm . Publication Number: EENY151.

BPPD, 2006. Review of data and support materials submitted by Monsanto to amend the insect resistance management requirements for Bollg ard II Bt cotton. A. Reynolds and S. Matten memorandum to L. Cole, May 16, 2006.

BPPD, 2007. Scientific review of a proposal by Monsanto to re place structured refuge with natural refuge for Bollgard II Bt cotton (resubmitted amendment request to address the concerns of the FIFRA SAP). A. Re ynolds and S. Matten memorandum to M. Mendelsohn, May 3, 2007.

Capinera, J.L., 2005. University of Florida, Department of Entomology and Nematology informational web page for soybean looper ( Pseudoplusia includens ): http://entnemdept.ufl.edu/creatu res/veg/leaf/cab bage_looper.htm . Publication Number:

EENY-116.

MacRae T.C., M.E. Baur, D.J. Boethel, B.J. Fitzpatrick, A.G. Gao, J.C. Gamundi, L.A. Harrison, V.T. Kabuye, R.M. McPherson, J.A. Miklos, M. S. Paradise, A.S. Toedebusch, A. Viegas, 2005. Laboratory and field evaluations of transgenic soybean exhibiting high-doseBacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

38 expression of a synthetic Bacillus thuringiensis cry1A gene for control of Lepidoptera. J

Econ Entomol. 98 (2): 577-87.

Pedigo, L.P., 1999. Entomology and Pest Manageme nt (Third Edition). Prentice Hall: New Jersey. 691 pp.

Scientific Advisory Panel (SAP), 1998. Transmitta l of the final report of the FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) Plant-Pesticides and Resistance Management, Meeting held February 9 and 10, 1998. Report dated April 28, 1998.

Available at http://www.epa.gov/scipoly/sa p/meetings/1998/0298_mtg.htm

III. TERMS AND CONDITIONS OF REGISTRATION

1) The subject registration will automatical ly expire on midnight September 30, 2012.

2) The subject registration is limited to seed incr ease and to a total of 15,000 acres per year in the Atlantic Coast States of Georgia, S outh Carolina, North Ca rolina, Virginia, and Maryland within the Continental United Stat es with no more than 1,000 acres per county

per year.

3) Monsanto must submit IRM monitoring and reme dial action plans to EPA for approval by January 31, 2011 and reports on such annually by August 31 st

.

4) Monsanto must provide EPA annual reports on the acreage and States where MON 88017 has been grown by January 31 st

.

IV. REGULATORY POSITI ON FOR Cry1Ac Soybean

Pursuant to FIFRA section 3( c)(5), EPA may unconditionally register a pesticide if EPA determines that, when used in accordance with widespread and commonly recognized practice, it will not generally result in unreasonable adverse effects to the environment. Monsanto has

submitted or cited data sufficient for EPA to determine that an unconditional time-limited registration of Bacillus thuringiensis Cry1Ac Protei n and the Genetic Material (Vector PVGMIR9) Necessary for Its Production in MO N 87701 (OECD Unique Identifier: MON 877Ø1-2) Soybean under FIFRA 3(c)(5) will not result in unreas onable adverse effects to the environment. Monsanto submitted and/or cited satisfactory data pertaining to the proposed use. The human health effects data and nontarge t organism effects data are cons idered sufficient for the period, limited acreage, and geographic limitations of the unconditional registration. These data

demonstrate that no foreseeable human health hazar ds or ecological effects are likely to arise from the use of the product and that the risk of resistance developing to Cry1Ac protein, during the limited registration period is not expected to be significant.Bacillus thuringiensis Cry1Ac in MON 87701 Soybean Biopesticide Registration Action Document

39 The expiration date of this registration has b een set to September 30, 2012. The registration is

limited to seed increase and to a total of 15,000 acres per year in the Atlantic Coast States of Georgia, South Carolina, North Carolina, Virgin ia, and Maryland within the Continental United States with no more than 1,000 acres per county per year.