**Draft BIOPEST**  **I CIDE**  **REGISTR A**  **TION ACTION DOCUM E**  **N T** 

**PLANT -**  **INCORPORATED PROTECTANTS** Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 Protein s and the Genetic Material (Vector PV GMIR13196) Necessary

For Their Production in MON 87751 (OECD Unique ID. MON - 87751 - 7) Soy bean [ PC Code 006614 and PC Code 006527 ]

U.S. Environm ental Protection Agency Office of Pesticide Program s Biopesticides and Pollution Prevention Division March 5, 2015Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

2 Table of Contents I. Overview ................................ ................................ ................................ ................................ .................. 4 A. Executive S ummary ................................ ................................ ................................ ........................... 4 B. Use Profile ................................ ................................ ................................ ................................ ......... 6 C. Regulatory History ................................ ................................ ................................ ............................ 6 II. Science Assessment ................................ ................................ ................................ ................................ . 7 A. Pro duct Characterization ................................ ................................ ................................ .................... 7 B. Transformation System ................................ ................................ ................................ ...................... 7 C. Characterization of the DNA Inserted in the Plant and its Inheritance and Stability ......................... 7 D. Protein Characterization and Equivalence ................................ ................................ ......................... 7 E. Protein Expression ................................ ................................ ................................ ............................. 9 F. Analy tical Method ................................ ................................ ................................ ............................ 12 G. Overall Safety

Conclusion ................................ ................................ ................................ ............... 12 H. Supporting Data ................................ ................................ ................................ ............................... 12 III. Human Health Assessment of Cry 1A.105 and Cry2Ab2 as Expressed in Mon 87751 Soy bean ........ 18 A. Mammalian Toxicity

and Allergenicity

Assessment - Cry 1A.105 ................................ ................... 18 B. Mammalian Toxicity

and Allergenicity

Assessment Cry

2Ab2 ................................ ........................ 21 C. Determination of Safety for U.S. Population, Infants and Children Toxicity and Allergenicity Conclusions (Cry 1A.105 and Cry 2Ab2) ................................ ................................ ........................ 23 D. Overall Safety

and Conc lusion (Cry 1A.105 and Cry 2Ab2) ................................ ............................ 23 E. Other Consideration (Cry 1A.105 and Cry 2Ab2) ................................ ................................ ............. 24 IV . References ................................ ................................ ................................ ................................ ............ 29 V. Environmental Risk Assessment for Mon 87751 ................................ ................................ .................. 30 A. Overall Conclusions ................................ ................................ ................................ ......................... 30 B. Nontarget Organism Tiered Testing and Risk Assessment Process for PIPs ................................ ... 30 C. Nontarget Organism Risk Assessment for MON 87751 Soy beans ................................ ................ 35 VI . Risk Assessment Conclusions for MON 87751 Soy beans ................................ ................................ . 43 A. Effects to Nontarget Wildlife, Invertebrates, and Plants ................................ ................................ .. 43 B. Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms ................................ ............. 44 C. Gene Flow and Weediness Potential ................................ ................................ ............................... 44 D . Impacts on Endangered Species ................................ ................................ ................................ ...... 45 E. Conclusions ................................ ................................ ................................ ................................ ..... 46 VII. Insect Resistance Management ................................ ................................ ................................ ........... 46 VIII. References ................................ ................................ ................................ ................................ ......... 48 IX . Terms an d Conditions of Registration ................................ ................................ ................................ . 51 X. Regulatory

Posit i on for Mon 87751 (Cry 1A.105 and Cry2Ab2) Soy bean ................................ ........... 51Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

3 **BIOPEST I**  **CIDE REGISTRATION ACTION TEAM Office**  **o f Pesticide Pro**  **g rams:**  Biopestici d es and Pollution Prevention Division Product Characteri z ation and Human Health Judy Facey , Ph.D. John Kough, Ph.D. Environmental Fate an d Effects In - So o n You, Ph.D. Shannon Borges, M.S. Insect Resi s tance Man a gement Alan Rey nolds, M.S. Jeannette M artinez, Ph.D. Registration Support Kenneth Hay

mes, Ph.D. Alan Rey nolds, M.S.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

4 **I. Overview**  A . Executive S ummary The United States Environmental Protection Agency (U.S. EPA) proposes to register two new Bacillus thuringiensis (

Bt) soy bean plant - incorporated protectant (PIP) active ingredients as described in this Biopesticide Registration Action Document (BRAD). Monsanto Company

(hereafter, “Monsanto”) has submitted an application for a FIFRA Section 3 Seed In crease Registration of Event MON 87751 (MON 87751), a new Bt soy bean ( Glycine max (L.) Merr.), which produces Bt- derived insecticidal proteins Cry 1A.105 and Cry2Ab2 as Plant Incorporated Protectants (PIPs). A PIP is defined as a pesticidal substance that is intended to be produced and used in a living plant, and the genetic material necessary for production of such a pesticidal substance (40 CFR Part 174.3). Two new active ingredients (a.i.s), Cry1A.105 and Cry2Ab2 insect control proteins expressed in MON 87751 soy bean, prov ide protection from feeding damage caused by key lepidopteran soy bean pests, including the bean shoot moth ( Crocidosema aporema ), sunflower looper (Rachiplusia nu ) and fall army

worm ( Spodoptera frugiferda) . Cry 1A.105 and Cry2Ab2 proteins are also active ag ainst other lepidopteran soy bean pests such as the velvetbean caterpillar ( Anticarsia gemmatalis) , soy bean looper ( Chrysodeixis includens) and corn earworm ( Helicoverpa zea ). Product Characterization MON 87751 was produced by Agrobacterium tumefaciens - mediated transformation of soy bean ( Glycine max) tissue using the 2T - DNA transformation plasmid vector PV - GMIR13196. The Cry 1A.105 and Cry2Ab2 proteins provide protection from feeding damage caused by targeted lepidopteran insect pests. Mammalian Toxicity and Allergenicity Assessment Monsanto has developed insect - protected soy bean MON 87751 that produces the Cry 1A.105 and Cry2Ab2 insecticidal crystal (Cry) proteins (δ - endotoxin) derived from Bacillus thuringiensis ( Bt) subsp. kurstaki . EPA will establi sh an exemption from the require m ent of a tolerance for Cry 1A.105 and Cry2Ab2 protein s in soy bean when used as a plant - incorporated protectant. Based on the review and consideration of all the available information, there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of the Cry 1A.105 and Cry2Ab2 protei ns and the genetic material necessary for their production in soy

bean. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency has arrived at this conclusion because no toxicity to mammal s has been observed, and there is no indication of allergenicity potential for the plant - incorporated protectant from available information.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

5 Environm ental Assessment EPA does not anticipate a dverse effects to nontarget organis m s, including birds, wild m amm als, freshwater and m arine/estuarine fish and invertebrates, nontarget insects, honey bees, soil invertebrates, and ter r estri a l an d aquatic pl ants . EPA also does not anticipate horizontal gene transfer, gene flow, and the develop m ent of weediness. Based on these findings and infor m ation on habit at requ ire m ents of federa lly- li s ted thr e aten e d and endan gered lepid o pteran s p ecies, the Age n cy also m akes a No Effect deter mination for direct and indirect effects to threatened and endangered species and their ha b itat s . Insect Re s i st ance Mana g e m ent Given the l o w acreage p er mitted for MON 87751 (<15,000 t o tal acres p er year), it is unlikely that there will be a significant risk of resistance to the m ain soy bean target pests in the United States, soy bean looper and velvetbean caterpillar. This conclusion is further s u pported by

the biology of the target insects, which do not overwinter in non - tropical areas and are poly phagous (feeding on a num ber of wild hosts and cultivated crops). In addition, the registration will have an acreage limitation which will further reduce the risk of resistance. Public Participation On October 1, 2009, the EPA announced a policy to provide a more meaningful opportunit y for the public to participate in major registration decisions before they occur. According to this policy , the EPA provides a public comment period prior to making a registration decision for the following typ es of applications: new active ingredients; first food uses; first outdoor uses ; first residential uses; or an y other registration actions for which the EPA believes there may be significant public interest. Consistent with the policy of making registration actions more transparent, the proposed plant incorporated protectant contain ing the Cry 1A.105 and Cry2Ab2 proteins as new active ingredients, will be subject to a comment period. The docket identification number, which is associated with this registration action and can be accessed through http://www.regulations.gov , is EPA - HQ - OPP - 2014 - 0456 . The following document is available for comment in EPA - HQ - OPP - 2014 - 0456 : (1) draft Bacillus Thuringiensis Cry 1A.105 and Cry2Ab2 Proteins and the Genetic Material (Vector PV - GM IR 13196) Necessary

For Th eir Production in MON 87751 (OECD Unique ID. MON - 87751 - 7) Soy bean [PC Code 006614 and PC Code 006527] . While a final decision on registration is contingent upon review and consideration of public comments, the EPA presently believes that, based upon the ri sk assessment and the supporting material, it is appropriate to issue this registration. The basis for this preliminary decision can be found in the risk assessment for Mon 87751 Soy bean, which is characterized throughout this BRAD.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

6 **B. Use Profile**  **Active Ingredient:** Bacillus thuringiensis Cry1A.105 and Cry2Ab2 insecticidal proteins and the genetic material necessary for its production in soy

bean **Trade and Other Name(s):** MON 87751 **OPP Chemical Codes (PC Codes):** 006614 and 006615 **Company Name:** Mons anto C o m pan y 800 North L indbergh Blvd. St. Lo uis, MO 63167 **Type of Pesticide:** Plant - i n corporated Protectant **Uses** : Soy bean, Limited to seed increase and a total of 15,000 acres for breeding purposes, agronomic testing, and producing seed in the United States and the Commonwealth of Puerto Rico. **Target Pests for Acti v**  **e Ingredient:**  Lepidopteran soy bean pests, including the bean shoot moth ( Crocidosema aporema ), sunflower looper ( Rachiplusia nu ) and fall arm yworm (Spodoptera frugiferda) , velvetbean cater pillar ( Anticarsia gemmatalis) , soy bean looper ( Chrysodeixis includens) and corn earworm ( Helicoverpa zea ). **C. Regulatory History** O n August 28, 2014 (79 FR 63596) , EPA published a Notice of Receipt in the Federal Register announcing that Monsanto submitted an application to register two new active ingredient s , Bt Cry 1A.105 and Cry2Ab2 insecticidal proteins expressed in soy bean (Mon 87751 Soy

bean; EPA File S ymbol 524 - 619 ) not included in an y currentl y registered pesticide products. Concurrentl y with the FIFRA section 3 application, Monsanto submitted two petition s ( PP 4F8275 and PP 4F8276 ) to establish a permanent exemption from the requirement of a tolerance for residues of Bacillus thuringiensis Cry1A.105 and Cry2Ab2 protein s in soy bean. EPA published a Notice of Filing in the Federal Register, initially on October 24, 2014 (79 FR 63596) and again on December 17, 2014 for Cry 1A.105 (79 FR 75111) and on January 28, 2015 for Cry2Ab2 (80 FR 4527) and the public was given a 30 - day comment period. One public comment was received in response to this publication.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

7 **I I**  **. Science Assessment A.**  **Product Characterization** Monsanto has developed insect - protected soy bean MON 87751 that produces the Cry 1A.105 and Cry2Ab2 insecticidal crystal (Cry) proteins (δ - endotoxin) derived from Bacillus thuringiensis ( Bt) subsp. kurstaki . The Cry1A.105 and Cry2Ab2 proteins provide protection from feeding damage caused by targeted lepidopteran insect pests. **B. Transformation System**  MON 87751 was produced by Agrobacterium tumefaciens - mediated transformation of soy bean tissue using the 2T - DNA transformation plasmid vector PV - GMIR13196. PV - GMIR13196 contains two separate T - DNAs that are each del ineated b y l eft and right border regions. The first T - DNA, designated as T - DNA I, contains the CrylA.105 coding sequence regulated by the Act2 promoter and RbcS4 terminator; and the Cry2Ab2 coding sequence regulated by the Mt promoter and Ptl terminator. T he second T - DNA, designated as T - DNA II, contains the aadA coding sequence regulated b y FMV enhancer, EF

- Iα promoter and E9 terminator; and the splA coding sequence regulated b y the Usp promoter and nos terminator. During transformation, both T - DNAs were inserted into the soy bean genome. Subsequently , traditional breeding, segregation, selection and screening were used to isolate those plants that contain the CrylA.105 and Cry2Ab2 expression cassettes (T - DNA I) and do not contain the aadA and splA expressi on cassettes (T - DNA II), resulting in the production of marker - free, MON 87751. **C .**  **Characterization of the DNA Inserted in the Plant and its Inheritance and Stability** Bioinformatics anal ys is of next - generation sequence data (comprehensivel y covering the genome) was used to characterize the insert number of any DNA sequences from the transformation plasmid vector PV - GMIR13196. The data indicated that MON 87751 contains a single integration locus for the DNA insert. Directed sequence data (generated from PCRs targeted at a specific locus) was used to demonstrate that the single DNA insert in MON 87751 contains a single copy of T - DNA 1 from the transformation plasmid vector PV - GMIR13196 and confirmed the integrity of the CrylA.105 and Cry2Ab2 expression cassettes in the T - DNA I insert. The data also showed that MON 87751 does not contain an y sequences from the vector backbone or T - DNA II sequences of PV - GMIR13196. The direc ted sequencing data also identified the 5' and 3' insert - to - flank junctions in MON 87751 and characterized the insertion site in conventional soybean. Furthermore, bioinformatics anal ys is of next generation sequence data (comprehensively covering the genom e) was used to evaluate the stability of the DNA insert and demonstrated that the DNA insert in MON 87751 was stably maintained across five generations. **D . Protein Characterization and Equivalence**  **1. Cry1A. 105**  : The level of the Cry 1A.105 protein in the seed of MON 87751 soy bean is relatively

low. Therefore, theBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

8 protein was produced in a bacterial expression sy stem in order to yield a sufficient quantity of the protein for safety testing. The total protein concentration of the purified MON 87751 - produce d CrylA.105 protein was measured using the Bio - Rad Protein Assay and was determined to be 0.12 mg/ml. Apparent molecular weight (MW) and Purity of the MON 87751 - produced CrylA.105 protein were determined using SDS - PAGE anal ys is. The apparent MW was 132.9 kDa and the purity of the MON 87751 - produced CrylA.105 protein was 60%. Identity was confirmed b y N - terminal sequence anal ys is, matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI - TOF MS) anal ys is of try psin digested CrylA .105 protein, and western blot analy sis. The expected N - terminal sequence was observed. Several peptide masses consistent with expected peptide masses for the try psin digested CrylA.105 protein were observed. The identified peptide masses yielded coverage of 53.5% (632 out of 1181 amino acids) of the expected sequence of the CrylA.105 protein. Anti - Cry 1A.105 antibodies recognized the MON 87751 - produced CrylA.105. The functional activity of the MON 87751 - produced CrylA.105 was determined using a corn earworm diet incorporation assay . The MON 87751

– produced CrylA.105 was shown to be active, with an EC 50 of 0.0035 μg CrylA.105/ml diet. Analy sis of the gl ycos yl ation status showed that the MON 87751 - produced CrylA.105 protein was not gl ycos yl ated. The equivale nce of the MON 87751 - produced and E. coli - produced CrylA.105 protein was assessed by comparing their apparent MW, immunoreactivity , functional activity , and glycos yl ation status. Based on these characteristics, the characterized E. coli - produced protein w as established to be equivalent to the Cry 1A.105 protein isolated from seed of MON 87751 soy bean. **2. Cry2Ab2**  The level of the Cry2Ab2 protein in the seed of MON 87751 soy bean is relatively

low. Therefore, the protein was made in a bacterial expression sy stem in order to produce a sufficient quantity of the protein for safety testing. The total protein concentration of the purified MON 87751 - produced Cry2Ab2 protein was measured using the Bio - Rad Protein Assay and was determined to be 0.40 mg/ml. Apparent molecular weight (MW) and purity of the MON 87751 - produced Cry2Ab2 protein were determined using SDS - PAGE anal ys is. The apparent MW of the MON 87751 - produced Cry2Ab2 protein was 61.4 kDa, and the purity

of the MON 87751 - produced Cry2Ab2 protein was 29%. Identity was confirmed by N - terminal sequence analy sis, matrix assisted laser desorption/ionization time of flight mass spectrometry

(MALDI - TOF MS) anal ys is of the try psin digested Cry2Ab2 protein, and western blot anal ys is. The N - terminal sequence was confirmed using automated Edman degradation chemistry and MALDI - TOF MS anal ys is. MALDI - TOF MS analy sis also produced several peptide masses consistent with expected peptide masses for the try psin digested Cry2Ab2 protein. The identif ied peptide masses yi elded coverage of 66% (406 out of 619 amino acids) of the expected sequence of the Cry2Ab2 protein. Anti - Cry2Ab2 antibodies detected the MON 87751 - produced Cry2Ab2. The functional activity of the MON 87751 - produced Cry2Ab2 was determin ed using a corn earworm diet incorporation assay . The MON 87751 - produced Cry2Ab2 protein was shown to be active, with an EC 50 of 0.0734 μg Cry2Ab2/ml diet. Analy sis of the gl yc osylation status showed that the MON 87751 - produced Cry2Ab2 protein was not gl yc os yl ated.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

9 The equivalence of the MON 87751 - produced and E. coli - produced Cry2Ab2 protein was assessed by comparing their apparent MW, immunoreactivity , functional activity , and glycos yl ation status. Based on these characteristics, the characterized E. coli produced protein was established to be equivalent to the Cry2Ab2 protein isolated from seed of MON 87751. **E .**  **Protein Expression** Enzy me - linked Immunosorbent Assay (ELISA) analy ses quantified the amount of Cry 1A.105 and Cry2Ab2 protein concentrations expr essed in Over season leaf (OSL1 - 4), root, seed, and forage tissue samples collected from MON 87751 Soy bean at various growth stages. The results of the ELISA anal ys is across field sites showed the majorit y of the mean Cry1A.105 protein level in MON 87751 as highest in OSL4 at 790 μg/g dwt and lowest in root which was below limit of detection (<LOD). The mean CrylA.105 protein level in MON 87751 was 2.4 μg/g dry weight (dwt) in seed. The mean CrylA.105 protein level in MON 87751 was 11 μg/g fresh weight ( fwt) in pollen/anther. The mean Cry2Ab2 protein level in MON 87751 across all sites was highest in OSL3 at 32 μg/g dwt and lowest in seed at 4.0 μg/g dwt. The mean Cry2Ab2 protein level in MON 87751 was 7.7 μg/g fwt in pollen/anther. Summary results are p rovided below in Tables 1 and 2.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

10 **Table 1. Summary of Cry1A.105 Protein Levels in S oybean Tissues from MON**  **87751 Produced in United States Field Trials During 2012**  **Tissue Type 1**  **Development**

**Stage2 Mean(SD)** 

**Range (μg/g fwt)**  **3 Mean(SD)** 

**Range (μg/g dwt)**  **4 LOQ/**  **LOD**

**(μg/g fwt) 5**  OSL 1 **V3**
**– V4**  130 (50) 580 (250) 1.500/0.406 61

– 220 260

– 1100 OSL2 **V5**
**– V7**  120 (54) 590 (270) 1.500/0.406 13

– 220 68

– 1100 OSL 3 **R2**
**– R3**  79 (45) 400 (220) 1.500/0.406 8.5

– 160 50

– 780 OSL4 **R6** 230 (82) 790 (280) 1.500/0.406 120

– 480 430

– 1600 Root **R6** <LOD (N/A) N/A (N/A) 0.563/0.322 N/A

– N/A N/A

– N/A Forage **R6** 62 (21) 230 (91) 1.500/0.524 31

– 110 110

– 440 Seed **R8** 2.1 (0.46) 2.4 (0.50) 0.900/0.226 1.5

– 2.9 1.7

– 3.2 Pollen/Anther **R2** 11 (N/A) N/A (N/A) 1.500/N.D. 6 N/A

– N/A N/A

– N/A 1. OSL= over season leaf 2. The crop development stage each tissue was collected. 3. Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of protein per gram (g) of tissue on a fresh weight basis (fwt). The means, SD, and ranges (minimum and maximum values) were calculated for each tissue across all sites (n=20 except for OSL1 where n=19 due to one sample expressing <LOD and pollen where n=1). N/A: N ot Applicable 4. Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of protein per gram (g) of tissue on a dry weight basis (dwt). The dry weight values were calculated by dividing the μg/g fwt b y the dry we ight conversion factor obtained from moisture anal ys is data except for pollen tissue. 5. L OQ = limit of quantitation; L OD = limit of detection. 6. N.D. = Not determined. Pollen/anther LOD was not determined due to an insufficient amount of tissue.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

11 **Tabl e 2. Summary of Cry2Ab2 Protein Levels in Soybean Tissues from MON 87751 Produced in**  **United States Field Trials During 2012 Tissue Type**  **1 Development** 

**Stage 2**  **Mean(SD)**

**Range (μg/g fwt)**  **3 Mean(SD)** 

**Range (μg/g dwt)**  **4 LOQ/LOD** 

**(μg/g fwt) 5**  OSL 1 V3

– V4 5.4 (0.74) 24 (5.9) 0.625/0.034 4.4

– 6.8 17

– 37 OSL2 V5

– V7 5.2 (0.70) 26 (3.1) 0.625/0.034 4.0

– 6.6 20

– 33 OSL 3 R2

– R3 6.3 (0.80) 32 (5.2) 0.625/0.034 5.2

– 8.0 25

– 43 OSL4 R6 6.9 (0.79) 24 (2.7) 0.625/0.034 5.5

– 8.5 18

– 29 Root R6 4.6 (1.0) 15 (2.7) 1.250/1.241 3.1

– 7.1 11

– 22 Forage R6 3.9 (0.60) 14 (2.2) 0.313/0.060 3.0

– 5.1 11

– 18 Seed R8 3.6 (0.71) 4.0 (0.77) 0.313/0.094 2.3

– 4.7 2.6

– 5.1 Pollen/Anther R2 7.7 (N/A) N/A (N/A) 0.313/N.D. 6 N/A

– N/A N/A

– N/A 1. OSL= over season leaf 2. The crop development stage each tissue was collected. 3. Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of protein per gram (g) of tissue on a fresh weight ba sis (fwt). The means, SD, and ranges (minimum and maximum values) were calculated for each tissue across all sites (n=20 except for OSL1 where n=19 due to one sample expressing <LOD and pollen/anther where n=1). N/A: Not Applicable 4. Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of protein per gram (g) of tissue on a dry weight basis (dwt). The dry weight values were calculated by dividing the μg/g fwt b y the dry weight conversion factor obtained from moisture anal ys is data except for pollen tissue. 5. L OQ = limit of quantitation; L OD = limit of detection 6. N.D. = Not determined. Pollen/anther LOD was not determined due to an insufficient amount of tissue.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

12 **F .**  **Analytical Method** ELISA plates were analy zed on a SPECTRAmax Plus 384 (Molecular Devices, Sunny vale, CA) microplate spectrophotometer, using a dual wavelength detection method. P rotein concentrations were determined by

o ptical absorbance at a wavelength of 450 nm with a si multaneous reference reading of 620 nm. Data reduction anal ys es were performed using Molecular Devices SOFTmax PRO GxP version 5.4 software. Absorbance readings and protein standard concentrations were fitted with a four - parameter logistic curve fit for Cry1A.105 protein. Absorbance readings and protein standard concentrations were fitted with a five - parameter logistic curve fit for Cry2Ab2 protein. F ollowing the interpolation from the standard curve, the amount of protein (ng/ml) in the tissue was repo rted on a “  g/ g fwt” basis for data that were greater than or equal to the LOQ. T his conversion utilized a sample dilution factor and a tissue - to - buffer ratio. T he protein values in “  g/g fwt” were also converted to “  g/ g dwt” by a pplying the DWCF, excep t for expression levels in pollen/anther. **G .**  **Overall Safety Conclusion** Cry proteins have been found as components of topical Bt microbial pesticides for over 45 ye ars and are widely considered to be non - toxic to humans and other mammalian species (Betz e t al., 2000; EPA, 2000; OECD, 2007). The CrylA.105 and Cry2Ab2 proteins produced by MON 89034 are closel y related to the Cry proteins that have been used as the active ingredients in existing Bt microbial pesticides and/or biotechnology- derived crops. MON 87751 produces the Cry 1A.105 and Cry2Ab2 insecticidal proteins. The Cry 1A.105 and Cry2Ab2 proteins are individually expressed in currently

registered PIP event MON 89034 maize (EPA Reg. No. 524 - 575). In addition, the safety data provided for the Cry 1A.105 and Cry2Ab2 proteins demonstrate that neither protein exhibits amino acid sequence similarities to know allergenic or toxic proteins, both proteins are degraded in simulated gastric fluid, and both proteins lose functional activity

after heating. The data submitted and reviewed for the Cry 1A.105 and Cry2Ab2 proteins as expressed in MON 87751soy bean also support their inclusion in the existing exemptions from the requirement of tolerance for Bacillus thuringiensis Cry 1A.105 [40 CFR § 174.502] and Cry2Ab2 [40 CFR § 174.519] proteins, when used as a PIP in food and feed commodities of soy bean. Thus, there is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, including infants and children, to the Cry 1A.105 and Cry2Ab2 proteins and the genetic material necessary

for their production in event MON 87751 soy bean. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency has arrived at this conclusion because no toxicity to mammals has been observed, nor any indication of allergenicity potent ial for a plant - incorporated protectant that has been confirmed as sufficiently equivalent to currentl y registered plant - incorporated protectants. **H .**  **Supporting Data** The submitted studies to support the registrant’s request for an Experimental Use Patter n and Sec. 3Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

13 registration of PIP event MON 87751 soy bean expressing Cry 1A.105 and Cry2Ab2 proteins are summarized in Table 1. For detailed information of each study, please refer to the individual studies reviewed in the Data Evaluation Reports (DERs) acco rding to its respective assigned MRID number.

**Table 3. Product Characterization Data Submitted for MON 87751 Soybean STUDY TITLE**  **STUDY SUMMARY MRID NO.**  Molecular Characterization of Insect Protected Soy bean (MON 87751) MON 87751 was produced by Agrobacterium

tumefaciens - mediated transformation of soy bean tissue using the 2T - DNA transformation plasmid vector PV - GMIR13196. PV - GMIR13196 contains two separate T - DNAs that are each delineated by

l eft and right border regions. T he first T - DNA, designated as T - DNA I, c ontains the CrylA.105 coding sequence regulated by

the Act2 promoter and RbcS4 terminator; and the Cry2Ab2 coding sequence regulated by the Mt promoter and Pt1 terminator. T he second T - DNA, designated as T - DNA II, contains the aadA coding sequence regulated by F MV enhancer, EF

- 1α promoter and E9 terminator; and the splA coding sequence regulated by the Usp promoter and nos terminator. D uring transformation, both T - DNAs were inserted into the soy bean genome. Subsequently , tradition al breeding, segregation, selection and screening were used to isolate those plants that contain the CrylA.105 and Cry2Ab2 expression cassettes (T - DNA I) and do not contain the aadA and splA expression cassettes (T - DNA II), resulting in the production of m arker - free, MON 87751. Inthis study , bioinformatic anal ys is of next - generation sequence data (comprehensivel y covering the genome) was used to characterize the insert number of any D NA sequences from the transformation plasmid vector PV - GMIR13196. T he data indicated that MON 87751 contains a single integration locus of DNA insert. D irected sequence data (generated from PCRs targeted at a specific locus) was used to demonstrate that the single DNA insert in MON 87751 contains a single copy o f T - DNA I from the transformation plasmid vector PV - GMIR13196 and confirmed the integrity of the CrylA.105 and Cry2Ab2 expression cassettes in the T - DNA I insert; the data showed that MON 87751 does not contain an y sequences from the backbone or T - DNA II sequences of PV - GMIR13196. T he directed sequencing data also identified the 5' and 3' i nsert - to - flank junctions in MON 87751 and characterized the insertion site in conventional soy bean. Furthermore, bioinformatic anal ys is of next - generation sequence data (comprehensiv el y covering the genome) was used to evaluate the stability

o f the DNA insert and demonstrated that the DNA insert in MON 87751 was stably m aintained across five **49332003**Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

14 **STUDY TITLE STUDY SUMMARY**  **MRID NO.**

generations. **CLASSIFICATION: ACCEPTABLE** Assessment of Cry 1A.105 and Cry2Ab2 Protein Levels in Soy bean Tissues Collected from MON 87751 Produced in U.S. Field Trails during 2012 The purpose of this study was to determine the levels of Cry 1A.105

and Cry2Ab2 proteins in soy bean tissues of MON 87751 grown in United States field trials during 2012. Over season leaf (OSL1 - 4), root, seed, and forage tissue samples were collected as outlined in Production Plan PL C - 2012 - 0022 and pollen/anther samples were collected as outlined in Production Plan PPN - 2012 - 0041. The expression levels of C ry 1A.105 and Cry2Ab2 proteins were determined in OSL1 - 4, root, seed, forage and pollen/anther tissue by v alidated enzy

me - linked immunosorbent assay s (E LISAs). Protein levels for OSL1 - 4, root, forage, seed and pollen/anther tissues were calculated on a mic rogram (μg) per gram (g) fresh weight (fwt) basis. M oisture content was then measured for all tissue ty pes, except pollen/anther, and all protein levels, except for those in pollen/anther, were converted and reported on a dry w eight (dwt) basis. The mean Cry 1A.105 protein level in MON 87751 across all sites was highest in OSL4 at 790 μg/g dwt and lowest in root which was below limit of detection (<LOD). T he mean Cry 1A.105 protein level in MON 87751 was 2.4 μg/g dwt in seed. T he mean Cry 1A.105 protein le vel in MON 87751 was 11 μg/g fwt in pollen/anther. The mean Cry2Ab2 protein level in MON 87751 across all sites was highest in OSL 3 at 32 μg/g dwt and lowest in seed at 4.0 μg/g dwt. The mean Cry2Ab2 protein level in MON 87751 was 7.7 μg/g fwt in pollen/anther. **CLASSIFICATION: ACCEPTABLE 49332004** 

Protein Characterization: Characterization of the Cry 1A.105 Protein Purified from the Soy

bean Seed of MON 87751 and Comparison of the Phy siochemical The level of Cry 1A.105 protein in the MON 87751 seed is

relativel y low. Therefore, the protein was made in a bacterial expression sy stem in order to produce a sufficient quantity of the protein for safety test ing. An Escherichia coli ( E. coli) - produced Cry 1A.105 protein was engineered to have the same amino acid sequence as the Cry 1A.105 protein detected in MON 87751. Demonstration of ph ys icochemical and functional equivalence between the MON 87751 - produced and the E . coli - produced Cry 1A.105 proteins justifies the use of the E. coli - produced protein to demonstrate the safety of the MON 87751 - produced Cry 1A.105 protein. The purpose of this study was to characterize the **49332005**Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

15 **STUDY TITLE STUDY SUMMARY**  **MRID NO.** and Functional Properties of the Plant - Produced and Escherichia coli - Produced Cry 1A.105 Protein. Cry 1A.105 protein isolated from MON 8775 1 seed and to assess

the equivalence of the MON 87751 - produced and the E . coli - produced Cry 1A.105 proteins. MON 87751 - produced Cry 1A.105 protein was characterized as follows: The total protein concentration of the purified MON 87751 - produced Cry 1A.105 protein was measured using the Bio - Rad Protein Assay

and was determined to be 0.12 mg/ml. Apparent molecular weight (MW) and Purity of the MON 87751 - produced Cry 1A.105 protein were determined using SDS - PAGE analy sis. The apparent MW was 132.9 kDa and the purity of the MON 87751 - produced Cry 1A.105 protein was 60%. Id entity was confirmed by

N - terminal sequence analy sis, matrix assisted laser desorption/ionization time of flight mass spectrometry

(MALDI - TOF MS) anal ys is of try psin digested Cry 1A.105 protein , and western blot anal ys is. The expected N terminal sequence was observed. Several peptide masses consistent with expected peptide masses for the try psin digested Cry 1A.105 protein were observed. The identified peptide masses yielded coverage of 53.5% (632 out of 1181 amino acids) of the expected sequence of the Cry 1A.105 protein. Anti - Cry 1A.105 antibodies detected the MON 87751 - produced Cry 1A.105. The functional activity of the MON 87751 - produced Cry 1A.105 was determined using a corn earworm diet inc orporation assay . The MON 87751 - produced Cry 1A.105 was shown to be active, with an EC 50 of 0.0035 μg Cry 1A.105/ml diet. Analy sis of the gl ycos yl ation status showed that the MON 87751 - produced Cry 1A.105 protein was not gl ycos yl ated. Finall y, assessment o f the purity and apparent MW of the protein after storage in a - 80 °C freezer (49 day s) b y SDS - PAGE anal ys is demonstrated that the MON 87751 - produced Cry 1A.105 protein was stable throughout the experimental phase . The equivalence of the MON 87751 - produce d and E. coli - produced Cry 1A.105 proteins was assessed by comparing their apparent MW, immunoreactivity , functional activity , and glycos yl ation status. Based on these characteristics, the E. coli - produced protein was established to be equivalent to the Cry1A.105 protein isolated from seed of MON 87751 . These data provide a detailed characterization of the Cry 1A.105Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

16 **STUDY TITLE STUDY SUMMARY**  **MRID NO.**

protein isolated from MON 87751 and establish the equivalence of the MON 87751 - produced Cry 1A.105 protein to the E. coli - produced Cry 1A.105 protein **CLASSIFICATION: ACCEPTABLE** Protein Characterization: Characterization of the Cry2Ab2 Protein Purified from the Soy

bean Seed of MON 87751 and Comparison of the Phy sicochemical and Functional Properties of the Plant - Produced and Escheric hia coli - Produced Cry2Ab2 Protein The level of the Cry2Ab2 protein in the seed of MON 87751 is

relativel y low. T herefore, the protein was made in a bacterial expression sy stem in order to produce a sufficient quantity o f the protein for safety testing. An Escherichia coli ( E. coli) - produced Cry2Ab2 protein was engineered to have the same amino acid sequence as the Cry2Ab2 protein detected in MON 87751. Demonstration of phy sicochemical and functional equivalence between the MON 87751 - produced and the E . coli - produced Cry2Ab2 proteins justifies the use of the E. coli - produced protein to demonstrate the safety o f the MON 87751 - produced Cry2Ab2 pro tein. H ence, the purpose of this study w as to characterize the Cry2Ab2 protein isolated from seed of MON 87751 and to assess the equivalence of the MON 87751 - produced and the E . coli - produced Cry2Ab2 proteins. MON 87751 - produced Cry2Ab2 protein was characterized as follows. T he total protein concentration of the purified MON 87751 - produced Cry2Ab2 protein was measured using the Bio - Rad Protein Assay and was determined to be 0.40 mg/ml. Apparent molecular weight (MW) and purity o f the MON 87751 - produced Cry2Ab2 protein were determined using SDS - PAGE anal ys is. T he apparent MW of the MON 87751 - produced Cry2Ab2 protein was 61.4 kDa, and the purity o f the MON 87751 - produced Cry2Ab2 protein was 29%. Id entity w as confirmed by

N - terminal sequence analy sis, matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI - TOF MS) anal ys is of the try psin digested Cry2Ab2 protein, and western blot analy sis. T he N - terminal sequence was confirmed using automated Edman degradation chemistry and MA LD I - TOF MS anal ys is. M ALDI - TOF MS anal ys is produced several peptide masses consistent with expected peptide masses for the try psin digested Cry2Ab2 protein. T he identified peptide masses yi elded coverage of 66% (406 out of 619 amino acids) of the expecte d sequence of the Cry2Ab2 protein. Anti - Cry2Ab2 antibodies detected the MON 87751 - produced Cry2Ab2. T he functional activity of the MON 87751 - produced Cry2Ab2 was determined using a corn earworm diet incorporation assay . T he MON 87751 - produced Cry2Ab2 protein was shown to be active, with an EC 50 of 0.0734  g Cry2Ab2/ml diet. Anal ys is of **49332006**Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

17 **STUDY TITLE STUDY SUMMARY**  **MRID NO.**

the gl ycos yl ation status showed that the MON 87751 - produced Cry2Ab2 protein was not gl ycos yl ated. F inall y, assessment of the apparent MW and purity o f the protein after storage in a - 80 °C freezer (42 day s) by

S DS - PAGE analy sis demonstrated that the MON 87751 - produced Cry2Ab2 protein was stable throughout the experimental phase. The equivalence of the MON 87751 - produced and E. coli - produced Cry2Ab2 proteins was assessed by comparing their apparent MW, immunoreactivity , functional activity , and glycosylation status. Based on these characteristics, the characterized E. coli - produced protein was established to be equivalent to the Cry2Ab2 protein isolated from seed of MON 87751. These data provide a detailed characterization of the Cry2Ab2 protein isolated from MON 87751 and establish the equivalence of the E. coli - produced Cry2Ab2 protein to this plant - derived protein. **CLASSIFICATION: ACCEPTABLE** Comparison of the Functional activity of two E. Coli - Produced Cry 1A.105 Protein L ots (10000776 and 11349124) Against a susceptible Lepidopteran Species. The purpose of this study w as to compare the biological (insecticidal) activity

( EC 50 values) of two Escherichia coli
produced Cry 1A.105 protein lots (10000776 and 11349124) in a 7 - day diet incorporation assay w ith the corn earworm (CEW), Helicoverpa zea . The EC 50 value is defined as the concentration that results in 50% growth inhibition relative to the control and is a reliable and relevant measure of biological activity . In the 7 - day

diet incorporation bioassays for both E. coli - produced Cry 1A.105 lots (10000776 and 11349124), CEW demonstrated nearl y identical concentration - dependent responses in terms of shape an d location for growth inhibition. Mean EC 50 values for lots 10000776 and 11349124 were 0.0042 μg Cry 1A.105/ml diet and 0.0040 μg Cry 1A.105/ml diet, respectively, and differ by l ess than a 5%. Mean EC 50 values for both lots fall well within the calculated 9 5% prediction interval of 0.0030

– 0.0099 μg Cry 1A.105/ml diet, which was constructed from historical mean EC 50 values for lot number 10000776. Therefore, the two Cry1A.105 lots are concluded to have functionally e quivalent biological activity and provide th e required information to bridge to the assessments performed with the Cry 1A.105 protein in support of MON 89034. **CLASSIFICATION: ACCEPTABLE**  **49332007**Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

18 **STUDY TITLE STUDY SUMMARY**  **MRID NO.** Comparison of the Functional Activity of two E. coli
- Produced Cry2Ab2 Protein Lots (11346423 and 11351 673) Against a Susceptible Lepidopteran Species The purpose of this study w as to compare the biological (insecticidal) activity

( EC 50 values) of two Escherichia coli
produced Cry2Ab2 protein lots (11346423 and 11351673) in a 7 - day diet incorporation assay with the corn earworm (CEW), Helicoverpa zea . The EC 50 value is defined as the concentration that results in 50% growth inhibition relative to the control and is a reliable and relevant measure of biological activity . Inthe 7 - day

diet incorporation bioassay s for both E. coli - produced Cry2Ab2 lots (11346423 and 11351673), CEW demonstrated nearl y identical concentration - dependent responses in terms of shape and location for growth inhibition. Mean EC 50 values for lots 11 346423 and 11351673 were 0.065 μg Cry2Ab2/ml diet and 0.079 μg Cry2Ab2/ml diet, respectively . Mean EC 50 values for both lots fall within the calculated 95% prediction interval of 0.043

– 0.25 μg Cry2Ab2/ml diet, which was constructed from historical mean EC 5 0 values for lot number 11346423. Therefore, the two Cry2Ab2 lots are concluded to have functionall y equivalent biological activity and provide the required information to bridge to the assessments performed with Cry2Ab2 in support of MON 89034. **CLASSIFICA TION: ACCEPTABLE**  **49332008**

**III. Human Health Assessment**  **o f**  **Cry1A.105 and Cry2Ab2 as Expressed in Mon 87751 Soybean**  EPA performs a number of anal ys es to determine the risks from aggregate exposure to pesticide residues. First, EPA determines the toxicity of pesticides. Second, EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings. **A. Mammalian Toxicity and Allergenicity Assessment**  **- Cry1A.105**  **1. Toxicological Profile**  Consistent with section 408(b) (2) (D) of the FFDCA, EPA has reviewed the available scientific data and other relevant information in support of this action and considered its validity , completeness and reliability and the relationship of this information to human risk. EPA has also considered available information concerning the variability of the sensi tivities of major identifiable subgroups of consumers, including infants and children. **2. Mammalian Toxicity Assessment**  Monsanto has submitted acute oral toxicity data demonstrating the lack of mammalian toxicity at high levels of exposure to the pure Cry 1A.105 protein. These data demonstrate the safety of the product at a level well above maximum possible exposure levels that are reasonabl y anticipated in the crop.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

19 An acute oral toxicity

study in mice (MRID No. 46694603) indicated that Cry 1A.105 is non - toxic to humans. Cry 1A.105 produced from microbial culture was dosed by gavage as two doses separated by 4 hours (±20 mi nutes) to 10 females and 10 males (2072 mg/kg/ body weight). Two control groups were also included in the study: a bovine serum albumin protein control, and a vehicle control. One male in the test protein group was moribund and sacrificed on day 1 due to a mechanical dosing error; this death was not attributed to the test material. All other mice survived the study. There were no significant differences in body weight or body weight change among the three groups during the study, and no treatment - related gross pathological findings were observed. The oral LD50 for males, females, and combined mice was greater than 2072 mg/kg. When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad, Roy

D., et al., “Toxic ological Considerations for Protein Components of Biological Pesticide Products,” Regulatory

Toxicology

and Pharmacology

15, 3 - 9 (1992)). Therefore, since no acute effects were shown to be caused by Cry 1A.105, even at relatively high dose levels, the Cry 1A .105 protein is not considered toxic. Further, amino acid sequence comparisons showed no similarities between the Cry 1A.105 and known toxic proteins in protein databases that would raise a safety concern. **3. Allergenicity Assessment**  Since Cry 1A.105 is a protein, allergenic potential was also considered. Currently , no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight - of - evidence approach where the following factors are considered: source of the trait; amino acid sequence comparison with known allergens; and biochemical properties of the protein, including in - vitro digestibility

in simulated gastric fluid (SGF) and glycos yl ation. This approach is consistent with the approach outlined in the Annex to the Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant - DNA Plants.” The allergenicity assessment for Cry 1A.105 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 the amino acid sequence of Cry 1A.105 with known allergens showed no significant overall sequence similarity or identity

at the level of eight contiguous amino acid residues. 3. **Digestibil**

**ity.** The Cry 1A.105 protein was rapidly digested in less than 30 seconds in simulated mammalian gastric fluid containing pepsin. 4. **Glycosylation.** The Cry 1A.105 protein expressed in soy bean was shown not to be gl ycos yl ated. **5. Conclusion.**  Considering all of the available information, EPA has concluded that the potential for Cry 1A.105 to be a food allergen is minimal. **4. Aggregate Exposures**  In examining aggregate exposure, EPA considers available information concerning exposures from the pesticide residue in food and all other non - occupational exposures, including drinking water fromBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

20 ground water or surface water and exposure through pestici de use in gardens, lawns, or buildings (residential and other indoor uses). The Agency

has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue an d to other related substances. First, with respect to other related substances, the Cry 1A.105 protein is a chimeric Bacillus thuringiensis Cry 1A protein with overall amino acid sequence identit y of 93.6%, 90.0%, and 76.7% to the Cry 1Ac, Cry 1Ab, and Cry 1F p roteins, respectively . All are registered PIPs that were previously assessed as having a lack of mammalian toxicity at high levels of exposure. Exemptions from the requirement of a tolerance already have been established for Cry 1Ab, Cry 1Ac and Cry 1F [see 40 CFR § 174.511, effective Apr. 25, 2007, 40 CFR § 174.510, effective Apr. 25, 2007, and 40 CFR § 174.504, effective Apr. 25, 2007, respectivel y] . Second, and specific to the Cry 1A.105 protein, these considerations include dietary exposure under the toler ance exemption and all other tolerances or exemptions in effect for the plant - incorporated protectant chemical residue and exposure from non occupational sources. Exposure via the skin or inhalation is not likely since the plant - incorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to negligible. In addition, even if exposure can occur through inhalation, the potential for Cry 1A.105 to be an allergen is low, as discussed previously . Although the allergenicity assessment focused on the Cry 1A.105 protein’s potential to be a food allergen, the data also indicated a low potential for Cry 1A.105 to be an inhalation allergen. Exposure via residential or lawn use to infants and children is also not expected because the use sites for the Cry 1A.105 protein are all agricultural for control of insects. Oral toxicity testing done at a dose of 2072 mg/kg showed no adverse effects. Furthermore, the expected dietary exposure from soy

bean is several orders of magnitude lower than the amounts of Cry 1A.105 protein shown to have no toxicity . Therefore, even if negligible aggregate exposure should occur, the Agency concludes that such exposure would present no harm due to the lack of mammalian toxicity

and the rapid digestibility demonstrated for the Cry1A.105 protein. **5. Cumulative Effects**  Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify , or revoke a tolerance, the Agency consider “available information” concerning the cumulative effects of a particular pesticide’s residues and “other substances that have a common mechanism of toxicity .” Since Cry 1A.105 is not considered toxic, EPA has not found Cry 1A.105 to share a common mechanism of toxici ty

with an y other substances, and Cry 1A.105 does not appear to produce a toxic metabolite produced b y other substances. For the purposes of this tolerance action, therefore, EPA has assumed that Cry 1A.105 does not have a common mechanism of toxicity with o ther substances. Following from this, therefore, EPA concludes that there are no cumulative effects associated with Cry 1A.105 that need be considered. For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see EPA’s website at http://www.epa.gov/pesticides/cumulative
.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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21 **B . Mammalian Toxicity and Allergenicity Assessment**  **Cry2Ab2 1.**  **Toxicological Profile** Consistent with section 408(b) (2) (D) of the FFDCA, EPA has reviewed the available scientific data and other relevant information in support of this action and considered its validity , completeness and reliability and the relations hip of this information to human risk. EPA has also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children. **2. Mammalian Toxicity Assessment**  Monsanto has submitted acute oral toxicity data demonstrating the lack of mammalian toxicity at high levels of exposure to the pure Cry2Ab2 protein. These data demonstrate the safety of the product at a level well above maximum possible exposure levels that are reasona bl y anticipated in the crop. An acute oral toxicity

study in mice (MRID 44966602) indicated that Cry2Ab2 is non - toxic to humans. Three groups of ten male and ten female mice were dosed b y oral gavage with 30, 300, or 1000 mg/kg body weight of microbially produced Cry2Ab2 protein. Two negative control groups were also included in the study : bovine serum albumin protein control, and a vehicle control (purified water). There were no significant differences between the test and control groups; therefore, the C ry2Ab2 protein does not appear to cause an y significant adverse effects at an exposure level of up to 1000 mg/kg bodywei ght. When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels. Therefore, given that no acute effects were shown to be caused b y Cry2 Ab2, even at relativel y high dose levels, the Cry2Ab2 protein is not considered toxic. Further, amino acid sequence comparisons showed no similarities between the Cry2Ab2 protein and known toxic proteins in protein databases that would raise a safety concern. **3. Allergenicity Assessment**  Since Cry2Ab2 is a protein, allergenic potential was also considered. Currently , no definitive te sts for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight - of - evidence approach where the following factors are considered: source of the trait; amino acid sequence comparison with known allergens; and biochemical p roperties of the protein, including in vitro digestibility

in simulated gastric fluid (SGF) and glycos yl ation. This approach is consistent with the approach outlined in the Annex to the Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant - DNA Plants.” The allergenicity assessment for Cry2Ab2 follows:Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

22 1. **Source of the trait.** Bacillus thuringiensis is not considered to be a source of allergenic proteins. 2. **Amino acid sequence.** A comparison of the amino acid sequence of Cry2Ab2 with known allergens showed no significant overall sequence similarity or identity

at the level of eight contiguous amino acid residues. 3. **Digestibility.** The Cry2Ab2 protein was rapidly digested in 15 seconds in simulated mammalian gas tric fluid containing pepsin. 4. **Glycosylation.** The Cry2AB2 protein expressed in soy bean was shown not to be gl ycos yl ated. 5. **Conclusion.** Considering all of the available information, EPA has concluded that the potential for Cry2Ab2 to be a food allergen is minimal. **4. Aggregate Exposures**  Pursuant to FFDCA section 408(b)(2)(D)(vi), EPA considers available information concerning aggregate exposures from the pesticide residue in food and all other non - occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses). The Agency considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other related substances. These considerations include dietary exposure under the to lerance exemption and all other tolerances or exemptions in effect for the PIP ’s chemical residue, and exposure from non - occupational sources. Exposure via the skin or inhalation is not likely since the plant incorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces exposure b y these routes to negligible. In addition, even if exposure can occur through inhalation, the potential for Cry2Ab2 protein to be an allergen is low, as previously discussed. Although the allergenicity assessment focused on Cry2Ab2 protein’s potential to be a food allergen, the data also indicated a low potential for Cry2Ab2 to be an inhalation allergen. Exposure to infants and children via residential or lawn use is also not expected because the use sites for the Cry2Ab2 protein is agricultural. Oral exposure, at very

low levels, may

occur from ingestion of corn , cotton and soy bean products and, theoreticall y, drinking water. However, oral toxicity testing in laboratory

mammal s showed no adverse effects. **5. Cumulative Effects**  Pursuant to FFDCA section 408(b)(2)(D)(v), EPA has considered available information on the cumulative effects of such residues and other substances that have a common mechanism of toxicity . These considerat ions included the cumulative effects on infants and children of such residues and other substances with a common mechanism of toxicity . Because there is no indication of mammalian toxicity from the plant incorporated protectant, we conclude that there are no cumulative effects for the Cry2Ab2 protein.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

23 C . **Determination of Safety for U.S. Population, Infants and Children Toxicity and Allergenicity Conclusions (Cry1A.105 and Cry2Ab2)**  The data submitted and cited regarding potential health effects for the Cry 1A.105 and Cry2Ab2 proteins include the characterization of the expressed Cry 1A.105 and Cry2Ab2 proteins in soy bean, as well as the previous submitted acute oral toxicity , heat stab ility , and in vitro digestibility of the proteins. The results of these studies were used to evaluate human risk, and the validity , completeness, and reliability of the available data from the studies were also considered. As discussed above, the previou s submitted acute oral toxicity data supports the prediction that the Cry 1A.105 and Cry2Ab2 proteins would be nontoxic to humans. Moreover, Cry 1A.105 and Cry2Ab2 showed no sequence similarity to an y known toxin. Because of this lack of demonstrated mammalian toxicity , no protein residue chemistry data for Cry1A.105 and Cry2Ab2 proteins were required for a human health effects assessment. Even so, preliminary expression level anal ys is showed the Cry1A.105 and Cry2Ab2 proteins are present at relativel y low levels. Dietary exposure is expected to be correspondingl y low. Since Cry 1A.105 and Cry2Ab2 are proteins, its potential allergenicity is also considered as part of the toxicity assessment. Data considered as part of the allergenicity assessment include that the Cry 1A.105 and Cry2Ab2 proteins came from Bacillus thuringiensis which is not a known allergenic source, showed no sequence similarity to known allergens, was readily degraded b y pepsin, and was not gl ycos yl ated when expressed in the plant. Therefo re, there is a reasonable certainty that Cry 1A.105 and Cry2Ab2 proteins will not be an allergen. Considered together, the lack of mammalian toxicity at high levels of exposure to the Cry 1A.105 and Cry2Ab2 proteins and the minimal potential for the protei ns to be a food allergen demonstrate the safety of the products at levels well above possible maximum exposure levels anticipated in the crop. Finally , and specificall y in regards to infants and children, FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about consumption patterns among infants and children, special susceptibility

of infants and children to pesticide chemical residues, and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity . In addition, FFDCA section 408(b)(2)(C) provides that EPA shall apply an additional tenfold margin of safety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the data base unless EPA determines that a different margin of safety will be safe for infants and children. **D .**  **Overall Safety and Conclusion (Cry1A.105 and Cry2Ab2** ) Based on the review and consideration of all the available information, as discussed in more detail above, there is a reasonable certainty that no harm will result to the U.S. population, including infantsBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

24 and children, from aggregate exposure to residues of the Cry 1A.105 and Cry2Ab2 proteins and the genetic material necessary for their production in soy bean. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency has arrived at this conclusion because no toxicity to mammals has been observed, and there is no indication of allergenicity potential for the plant - incorporated protectant from available information. **E .**  **Other Consideration (Cry1A.105 and Cry2Ab2** ) **1. Endocrine Disruptors**  As required under FFDCA section 408(p), EPA has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (including pesticide active and other ingredients) may

have an effect in humans or wildlife similar to an effect produced b y a “naturall y occurring estrogen, or other such endocrine effects as the Administrator may designate.” The EDSP employ s a two - tiered approach to making the statutorily required determinations. Tier 1 consists of a battery of 11 screening assay s to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal sy stems. Chemicals that go through Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal sy st ems will proceed to the next stage of the EDSP where EPA will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 testing is designed to identify any adverse endocrine related effects caused b y the substance, and establish a dose - response relationship between the dose and the E, A, or T effect. Between October 2009 and February 2010, EPA issued test orders/data call - ins for the first group of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients. This list of chemicals was selected based on the potential for human exposure through pathways such as food and water, residential activity , and certain post - application agricultural scenarios. This list should not be construed as a list of known or likely endocrine disruptors. The proposed PIP, Cry 1A.105 and Cry2Ab2 proteins from MON 87751 (as expressed in soy bean) is not among the group of 58 pesticide active ingredients on the initial list to be screened under the EDSP. Under FFDCA sec. 408(p) the Agency must screen all pesticide chemicals. Accordingl y, EPA anticipates issuing future EDSP test orders/data call - ins for all pesticide active ingredients. For further info rmation on the status of the EDSP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, please visit our website: http://www.epa.gov/endo/ . **2. Codex Maximum**  **Residue Level** In making its tolerance decisions, EPA seeks to harmonize U.S. tolerances with international standards whenever possible, consistent with U.S. food safety standards and agricultural practices. In this context,Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

25 EPA considers the international maximum residue limits (MRL s) established by the Codex Alimentarius Commission (Codex), as required b y FFDCA section 408(b)(4). The Codex Alimentarius is a joint U.N. Food and Agriculture Organization/World Health Organization food standards program, and it is recognized as an international food safety standards - setting organization in trade agreements to which the United States is a party . EPA may establish a tolerance that is different from a Codex MRL ; however, FFDCA section 408(b)(4) requires that EPA explain the reasons for departing from the Codex level. The Codex has not established a MRLfor Bacillus thuringiensis Cry 1A.105 and Cry2Ab2 proteins in soy bean.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

26 **Table 4**  **. Human Health**  **Data Submitted for MON 87751 Soybean** Assessment of the in vitro Digestibility

of C ry 1 A .105 P rotein in S imulated G astric and S imulated Intestinal F luids Inthis study , the in vitro digestibility o f the cry 1a.105 protein was assessed in simulated gastric fluid (sgf) containing the proteol yt ic enzy me, pepsin, and in simulated intestinal fluid (sif) containing pancreatin, a mixture of enz ym es. T he digestibility o f the cry1a.105 protein was also assessed in a sequential digestion of the protein, i.e. Brief digestion in sgf followed by digestion in sif. Digestibility

o f the cry 1a.105 protein was assessed using sodium dodecyl sulfate - polyacrylamide gel (sds - page) and/or western blot anal ys is. T he extent of digestion of the cry1 a.105 protein was evaluated by

v isual analy sis of scanned colloidal brilliant blue g stained p olyacrylamide gels or by v isual analy sis of scanned developed western blot x - ray films. Greater than 98.7% of the full - length cry 1a.105 protein was digested in sgf within 0.5 min when analy zed using colloidal brilliant blue g stained sds - page, and at least 98.4% of the full length cry1a.105 protein was digested within 0.5 min when anal yz ed by

w estern blot using a cry 1a.105 - specific antibody . Colloidal brilliant blue g stained sds - page anal ys is showed that a transient peptide fragment of ~60 kda was present at 0.5 min but gone by 2 m in and a transiently- stable fragment under 5 kda was observed for 20 min but was gone by 3 0 min in the sgf digestion. When the cry1a.105 protein was subjected to a sequential digestion, the transientl y - stable fragments observed at earl y time points in the sg f anal ysis were degraded within 0.5 min upon exposure to sif. Greater than 98.4% of the full - length cry 1a.105 protein was digested in sif within 5 min when analy zed by w estern blot using a cry 1a.105 - specific antibody . A s exp ected, a try psin resistant core (~55 kda) was observed throughout the course of the sif digestion. The results show that full - length cry1a.105 protein is rapidly digested in sgf and sif. Rapid digestion of the full - length cry1a.105 protein in sgf, and of all visible cry 1a.105 protein fragments in sequential digestion indicates that it is highly unlikely that the cry1a.105 protein will pose an y safety concern to human or animal health. **CLASSIFICATION: ACC EPTABLE**  **49332009**

Assessment of the in vitro Diges tibility

of Cry2Ab2 protein In this study , the in vitro digestibility o f the Cry2Ab2 protein was assessed in simulated gastric fluid (SGF) containing the proteol yt ic enzy me pepsin, and in simulated int estinal fluid (SIF) containing pancreatin, a mixture of enzy mes. D ig estibility of the Cry2Ab2 **493302010**Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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27 in Simulated Gastric and Simulated Intestinal Fluids. protein was assessed using sodium dodecyl sulfate - polyacrylamide

gel (SDS - PAGE) and/or western blot analy sis. T he extent of digestion of the Cry2Ab2 protein was evaluated by v isual anal ys is of scanned Colloidal Brilliant Blue G stained polyacrylamide gels or by v isual anal ys is of scanned developed western blot X - ray films. Greater than 99.4% of the full - length Cry2Ab2 protein was digested in SGF within 0.5 min when analyzed using Colloidal Brilliant Blue G stained SDS - PAGE and at least 98.4% of the full - length Cry2Ab2 protein was digested within 0.5 min when anal yz ed by western blot using a Cry2Ab2 - specific antibody . Colloidal Brilliant Blue G stained SDS - PAGE analy sis showed that peptide fragments of ~4 - 5 kDa were observed in the 0.5 min time points in SGF, but were gone b y 2 min. Greater than 96.8% of the full - length Cry2Ab2 protein was digested in SIF within 5 min when anal yz ed by w ester n blot using a Cry2Ab2 - specific antibody . A s expected, a trypsin - resistant core (~50 kDa) was observed throughout the course of the SIF digestion. Other fragments ranging in size from ~57 kDa to just under 10 kDa were transientl y present during SIF diges tion, but all were gone by 4 hr. These results show that the full - length Cry2Ab2 is rapidly dig ested in SGF and SIF. R apid and complete digestion of the Cry2Ab2 protein in SGF indicates that the Cry2Ab2 protein is highly unlikely to pose an y safety concern to human or animal health. **CLASSIFICATION: ACCEPTABLE** Effect of Heat treatment on the Functional Activity of Escherichia coli ( E. coli ) - produced MON 87751 Cry 1A.105 Protein The purified E. coli - produced MON 87751 Cry1A.105 protein

(referred to elsewhere in this document as Cry 1A.105 protein) was heated to 25, 37, 55, 75, and 95 °C for either 15 or 30 minutes. Heated samples and an unheated control sample of the protein (maintained on wet ice) were anal yz ed by an insect bioassay

t o assess the impact of temperature on the functional activity of the Cry 1A.105 protein. A dditionally , all samples were anal yz ed by SDS - PAGE to assess Cry 1A.105 protein intactness following the heat treatment. Results of this study

d emonstrated that the Cry1A.105 prot ein is functionally

a ctive following heat treatment at 25 oC, 37 oC and 55 oC for 15 or 30 min. C ry 1A.105 protein heated to 75 oC and 95 oC demonstrated no activity at the highest dose level tested in the insect bioassay . SDS - PAGE anal ys is demonstrated that treatment at 25, 37, 55, 75 and 95 oC for 15 minutes or 25, 37, 55 and 75 oC for 30 minutes did not significantly c hange the band intensity o f the **49332011**Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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28

Cry 1A.105 protein. When treated at 95 oC for 30 minutes, a slight reduction in band intensity o f the Cry 1 A.105 protein was observed. These data demonstrate that the Cry 1A.105 protein behaves with a predictable tendency

t owards loss of functional activity at elevated temperatures. **CLASSIFICATION: ACCEPTABLE** Effect of Heat treatment on the Functional Activity of Escherichia coli ( E. coli ) - produced MON 87751 Cry2Ab2 Protein The purified Escherichia coli ( E. coli) - produced MON 87751 Cry2Ab2 protein (referred to elsewhere in this document as Cry2Ab2 protein) was heated to 25, 37, 55, 75, or 95 °C for either 15 or 30 minutes. H eated samples and an unheated control sample of the protein (maintained on wet ice) were analy zed by an insect bioassay t o assess the impact of temperature on the functional activity o f the Cry2Ab2 protein. A dditionally , all samples were anal yz ed by

S DS - PAGE to assess Cry2Ab2 protein intactness following the heat treatment. Results of this study

d emonstrated that the Cry2Ab2 protein is functionally

a ctive following heat treatment at 25 oC and 37 oC for 15 or 30 minutes. When the Cry2Ab2 protein was heated at a temperature of 55 oC for 15 minutes, a 96% reduction of functional activity and a 99% reduction at 55 oC for 30 minutes was observed relative to the control substance. F urthermore, when the Cry2Ab2 protein was heat ed at a temperature 75 and 95 oC for 15 or 30 minutes, a >99% loss of functional activity w as observed relative to the control. SDS - PAGE anal ys is demonstrated that treatment at 25, 37, or 55 oC for 15 or 30 minutes did not change the band intensity of the Cry2Ab2 protein relative to control substance. When treated at 75 oC for 15 minutes a reduction in band intensity of the Cry2Ab2 protein was observed. T he reduction in the band intensity b ecame more pronounced for the Cry2Ab2 protein heated at 75 oC for 30 minutes and 95 oC for 15 minutes, and the band intensity w as reduced further to only s lightl y higher than a 10% reference standard when heated at 95 oC for 30 minutes. T hese data demonstrate that Cry2Ab2 behaves with a predictable tendency towards loss of functional activity at elevated temperatures. **CLASSIFICATION: ACCEPTABLE 49332012** Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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29 **IV. References**  Baum, J.A. 1998. Transgenic Bacillus thuringiensis . Phy toprotection 79: 127 - 130. Baum, J.A., T.B. Johnson and B.C. Carlton. 1999. Bacillus thuringiensis . Natural and recombinant bioinsecticide products, Pages 189 - 209 in Methods in Biotechnology : Biopesticides: Uses and Delivery . Volume 5. F.R. Hall and J.J. Menn (eds.). Humana Press Inc. Totowa, New Jersey . Betz, F.S., B.G. Hammond and R. L. Fuchs. 2000. Safety and advantages of Bacillus thuringiensis protected plants to control insect pests. Regulatory Toxicology and Pharmacology

32: 156 - 173. Gill, S.S., E.A. Cowles and P.V. Pietrantonio. 1992. The mode of action of Bacillus thuringi ensis protected plants to control insect pests. Regulatory Toxicology and Pharmacology

32: 156 - 173. OECD. 2007. OECD Environment, Health and Safety Publications. Series on Harmonization of Regulatory

Oversight in Biotecnology . No. 42. Consensus Document on Safety Information on Transgenic Plants Expressing Bacillus thuringiensis - Derived Insect Control Proteins. Environment Directorate. Organization for Economic Cooperation and Development. Paris 2007. Enj/jm/mono (2007)14. Richter, S. and G.K. Lamp pa. 1998. A chloroplast processing enz ym e functions as the general stromal processing peptidase. Proceedings of the National Academy of Sciences of the United States of America 95:7463 - 7468. Sjoblad, Roy

D., et al., 1992. Toxicological Consideration for Protein Components of Biological Pesticide Products. Regulatory Toxicology

and Pharmacology

15, 3 - 9, 1992. US EPA. 2010. Biopesticides registration action document: Bacillus thuringiensis Cry 1A.105 and Cry2Ab2 insecticidal proteins and the genetic materi al necessary for their production in corn [PC Codes 006515 (Cry2Ab2), 006514 (Cry 1A.105)]. US Environmental Protection Agency , Office of Pesticide Programs, Biopesticides and Pollution Prevention Division, Washington, D.C. U.S. EPA. 2000. FIFRA Scientific Advisory Panel on Sets of scientific issues being considered by the Environmental Protection Agency regarding: Bt plant - insecticides risk and benefit assessments, October 18 - 20, 2010. Widner, W.R. and H.R. Whiteley . 1989. Two highl y related insecticidal crys tal protein of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. Journal of Bacteriology 171: 965 - 974.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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30 **V. Environmental Risk Assessment for Mon 87751**  **A. Overall Conclusions**  Monsanto has applied for a FIFRA Section 3 seed increase registration to support seed production of MON 87751 for further development of combined - trait soy bean products. EPA has also completed an assessment of the environmental risks of the proposed FIFRA Section 3 seed increase registration of M ON 87751, and has examined the effects of the soy bean PIP consisting of insecticidal proteins Cry 1A.105 and Cry2Ab2 and the genetic material (PV - GMIR13196) necessary for their production in MON 87751 soy

bean on avian and mammalian wildlife, aquatic animals , nontarget insects (including honey bees), and nontarget plants. Gene flow, development of weediness, horizontal gene transfer, and fate in the environment have also been examined, as well as effects on federally listed threatened and endangered species. Monsanto submitted no new guideline testing data for the ecological risk assessment of MON 87751 PIP expressed in soy

bean for a Section 3 seed increase registration, but requested that the data developed for a previousl y registered PIP (event MON 89034 in corn, EPA Reg. No. 524 - 575), containing functionally equivalent active ingredients be used to support the registration of MON 87751 in soy bean. Based on the previous assessment conducted b y the Agency for MON 89034 (US EPA, 2010a) and in comparison of the levels of the insecticidal proteins expressed in MON 87751 to those of the proteins expressed in MON 89034 in corn, no significant increase in risk of nontarget organisms to the insecticidal proteins expressed by

event MON 87751 in soy bean is expected to o ccur. For the proposed seed increase registration of MON 87751, adverse effects to nontarget organisms, including birds, wild mammals, aquatic animals, nontarget insects, honey bees, soil invertebrates, and nontarget plants, are not likely to occur. Horiz ontal gene transfer, gene flow, and the development of weediness are also not likely to occur. Based on these findings and information on habitat requirements of federally- listed threatened and endangered lepidopteran species, the Agency also makes a No E ffect determination for direct and indirect effects to federally listed threatened and endangered species and their habitats. **B .**  **Nontarget Organism Tiered Testing and Risk Assessment Process for PIPs** The paragraphs below describe the process and rational e developed b y BPPD for evaluating hazard of PIPs to nontarget organisms. This process has been described in several of BPPD’s documents and is presented again here as background information. To minimize data requirements and avoid unnecessary tests, ris k assessments are structured such that risk is determined first from estimates of hazard under “worst - case” exposure conditions. A lack of adverse effects under these conditions would provide enough confidence that there is no risk and no further data wou ld be needed. Hence, such screening tests conducted earl y in an investigation tend to be broad in scope but relatively simple in design, and can be used to demonstrate acceptable risk under mostBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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31 conceivable conditions. When screening studies suggest potentiall y unacceptable risk, additional studies are designed to assess risk under more realistic field exposure conditions. These later tests are more complex than earlier screening studies. Use of this “tiered” testing framework saves time and resources by organizing the studies in a cohesive and coherent manner and eliminating unnecessary lines of investigation. Lower tier, high dose screening studies also allow tighter control over experimental variables and exposure conditions, resulting in a greater ab ility to produce statistically

reliable results at relativel y low cost 1

. Tiered tests are designed to first represent unrealistic worst case scenarios and would progress to real world field scenarios if the earlier tiered tests fail to indicate adequate certainty of acceptable risk. Screening (Tier I) non - target organism hazard tests are conducted at exposure concentrations several times higher than the highest concentrations expected to occur under realistic field exposure scenarios. This has allowed an endpoint of 50% mortality to be used as a trigger for additional higher - tier testing. Less than 50% mortality

under these conditions of extreme exposure suggest that population effects are likely to be negligible given realistic field exposure scenarios. The EPA uses a tiered (Tiers I - IV ) testing sy stem to assess the toxicity of a PIP to representative non target organisms that could be exposed to the PIP in the field environment. Tier I high dose studies reflect a screening approach to testing designed to maximize any toxic effects of the PIP on the tested non - target organism. The screening tests evaluate single species in a laboratory setting with mortality

as the end point. Tiers II

– IVgenerally encompass definitive hazard level determinations, longer term greenhouse or field testing, and are implemented when unacceptable effects are seen at the Tier I screening level. Testing methods which utilize the tiered approach were last published b y the EPA as Harmonized OSCPP Testing Guidelines, Series 850 and 885 (EPA 712 - C - 96 - 280, February 1996) 2 . These guidelines, as defined in 40 CFR 152.2150, apply to microbes and microbial toxins when used as pesticides, including those that are naturally occurring, and those that are strain - improved, either by natural selection or b y deliberate genetic manipulation. Therefore, PIPs consisting of microbial toxins (e.g., Cry proteins) and the genes encoding the toxins are also covered b y these testing guidelines. The Tier I screening maximum hazard dose (MHD) approach to environmental hazard assessment is
1 Non - target invertebrate hazard tests often are conducted at exposure concentrations several times higher than the maximum concentrations expected to occur under realistic exposure scenarios. This has customarily allowed an endpoint of 50% mo rtality to be used as a trigger for additional higher - tier testing. Lower levels of mortality under these conditions of extreme exposure suggest that population effects are likely to be negligible given realistic exposure scenarios. Thus, it follows th at the observed proportion of responding individuals can be compared to a 50% effect to determine if the observed proportion is significantly lower than 50%. For example, using a binomial approach, a sample size of 3 0 individuals is sufficient to allow a treatment effect of 30% to be differentiated from a 50% effect with 95% confidence using a one - sided Z test. A one - sided test is appropriate because only effects of less than 50% indicate that further experiments are not needed to evaluate risk. 2 OSCPP Testing Guideli nes, Series 850 and 885 we bsite: http://www.epa.gov/ OSCPP frs/publications/ OSCPP _Harmonized/ 885Microbial_Pesticide_Test_Guidelines/SeriesBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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32 based on some factor (whenever possible >10) times the maximum amount of active ingredient expected to be available to terrestrial and aquatic non - target organ isms in the environment (estimated environmental concentration or EEC) 3 . Tier I tests serve to identify potential hazards and are conducted in the laboratory

at high dose levels which increase the statistical power to test the hy potheses. Elevated test do ses, therefore, add certainty to the assessment, and such tests can be well standardized. The Guidelines call for initial screening testing of a single group or several groups of test animals at the maximum hazard dose level. The Guidelines call for testin g of one treatment group of at least 30 animals or three groups of 10 test animals at the screening test concentration, including proper controls, to be observed for approximately 30 day s. In cases where an insect species cannot be cultured for 30 days, te sting be continued until negative control mortality rises above 20 percent. Failing the Tier I (e.g., 10 X EEC) screening at the MHD dose does not necessaril y indicate the presence of an unacceptable risk in the field but it triggers the need for additio nal testing. 4 A less than 50% mortality effect at the MHD is taken to indicate minimal risk. However, greater than 50% mortality does not necessaril y indicate the existence of unacceptable risk in the field, but it does trigger the need to collect additio nal dose - response information and a refinement of the exposure estimation before deciding if the risk is acceptable or unacceptable. Where potential hazards are detected in Tier I testing (i.e. mortality is greater than 50%), additional information at low er test doses is required which can serve to confirm whether an y effect might still be detected at more realistic field [1X EEC] concentrations and routes of exposure 5

. When screening tests indicate a need for additional data, the OSCPP Harmonized Guide lines call for testing at incrementall y lower doses in order to establish a definitive LD 50 and to quantify the hazard. In the definitive testing, the number of doses and test organisms evaluated must be sufficient to determine an LD 50 value and, when nec essary , the Lowest Observed Effect Concentration (L OEC), No Observed Adverse Effect Level (NOAEL) , or reproductive and behavioral effects such as feeding inhibition, weight loss, etc. In the final anal ys is, a risk assessment is made by comparing the LOA EC (Low Observed Adverse Effect Concentration) to the EEC; when the EEC is lower than the LOAEC, a no risk conclusion is made. These tests offer greater environmental realism, but they may have lower statistical power. Appropriate statistical methods, and appropriate statistical power, must be employed to evaluate the data from the definitive tests. Higher levels of replication, the number of test species, and/or repetition are needed to enhance statistical power in these circumstances.
3 The dose margin can be less than 10x where uncertainty in the system is low or where high concentrations of test material are not possible to achieve due to test organism feeding habits or other factors . High dose testing also may not be necessary where many species are tested or tests are ve ry sensitive, although the test concentration used must exceed 1X EEC . 4 It is notable that that the 10 X EEC MHD testing approach is not equivalent to what is commonly known as “testing at a 10X SAFETY FACTOR” where any adverse effect is considered signif icant. Tier I screen testing is not ‘safety factor testing’. In a “ 10X safety factor” test any adverse effect noted is a “level of concern”, whereas in the EPA environmental risk assessment scenario any adverse effect is viewed as a concern only at 1X the field exposure. 5 The 1X EEC test dose is based on plant tissue content and is considered 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 ingesting in the field because most non - target organisms do not ingest plant tissue.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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33 Data that shows le ss than 50 % mortality at the maximum hazard dosage level

– (i.e. L C 50 , ED 50 , or LD 50 >10 X EEC) is sufficient to evaluate adverse effects, making lower field exposure dose definitive testing unnecessary . It is also notable that the recommended >10X EEC maximum hazard dose level is a highly conservative factor. The published EPA L evel of Concern [L OC] is 50% mortality at 5X EEC 6 (US EPA, 1998). **1. Validation**  The tiered hazard assessment approach was developed for the EPA b y the American Institute of Biological Sciences (AIBS) and confirmed in 1996 as an acceptable method of environmental hazard assessment by a FIFRA Scientific Advisory Panel (SAP) on microbial pesticides and microbial toxins. The December 9, 1999 SAP agreed that the Tiered approach wa s suitable for use with PIPs; however, this panel recommended that, for PIPs with proteinaceous insecticidal properties (e.g., Bt - derived PIPs), additional testing of beneficial invertebrates closel y related to target species and/or likely to be present in genetically engineered (GE) crop fields should be conducted. Testing of Bt Cry proteins on species not closel y related to the target insect pest was not recommended, although it is still performed to fulfill the published EPA non - target species data requi rements. In October 2000, another SAP also recommended that field testing should be used to evaluate population - level effects on non - target organisms. The August 2002 SAP, and some public comments, generall y agreed with this approach, with the additional recommendation that indicator organisms should be selected on the basis of potential for field exposure to the subject protein (US EPA, 2000, 2001, 2002, and 2004). **2. Chronic studies**  Since delayed adverse effects and/or accumulation of toxins through the food chain are not expected to result from exposure to proteins, protein toxins are not routinely tested for chronic effects on non - target organisms. However, the 30 day test duration requirement does amount to subchronic testing when performed at field e xposure 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 ingested or excreted into the environment. Although there are reports that some proteins (e.g.,Bt Cry proteins) from both plants and micro - organisms bind to some substances in soil, thus increasing the duration of their persistence in soil (Stotzky , 2000, Saxena et al., 2002a and 2002b), no adverse effects have been reported in this increase in exposure (OECD, 2007). **3. Conclusion** 

6 Th e established peer and EPA Science Board reviewed guidance on screening test levels of concern is 50% mortality at 5X environmental concentration . The appropriate endpoints in high dose limit/screening testing are based on mortality of the treated, as compared to the untreated (control) non - target organisms. A single group of 30 test animals may be tested at the maximum hazard dose .Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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34 The tiered approach to test guidelines ensures, to the extent possible, that the Agency requires the minimum amount of data needed to make science - based regulatory decisions. The EPA believes that maximum hazard dose Tier I screening testing presents a reasonable approach for evaluating hazards related to the use of biopesticides and for identifying negative results with a high degree of confidence. The Agency

expects that Tier 1 testing for short - ter m hazard assessment will be sufficient for most studies submitted in support of PIP registrations. However, 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 Academ y of Sciences (NAS, 2000) recommended testing of non - target organisms directly in the field. This approach, with an emphasis on testing invertebrates found in corn fields, was also recommended b y the August 2002 SAP and was support ed b y several public comments. Based on these recommendations, the Agency has required field studies on long term invertebrate population/community

and Bt Cry

protein persistence in soils as a condition of registration due to the lack of baseline data on the potential for long - term environmental effects from the cultivation of PIP producing plants. Since the commercialization of Bt PIP - producing plants, the number of field studies published in scientific literature in combination with the post - registration field studies submitted to the Agency has accumulated to a level where empirical conclusions can be made. As a result, the issue of long range effects of cultivation of these PIP plants containing Bt Cry proteins on the invertebrate community structure i n Bt crop fields has since been adequately addressed. Specificall y, a meta - anal ys is 7 of the data collected from 42 field studies indicated that non - target invertebrates are generall y more abundant in Bt cotton and Bt corn fields than in non - Bt crop fields managed with insecticides (Marvier et al., 2007). In addition, a comprehensive review of short and long term field studies on the effects of invertebrate populations in Bt corn and cotton fields indicated that no unreasonable adverse effects are taking p lace as a result of wide scale Bt crop cultivation (Sanvido et al
. , 2007). Another review of field tests published concluded that the large - scale studies in commercial Bt cotton have not revealed an y unexpected non target effects except subtle shifts in the arthropod community caused b y the effective control of the target pests (Romeis et al ., 2006). Slight reductions in some invertebrate predator populations are an inevitable result of all pest management practices, which result in reductions in the abun dance of the pests as prey . The Agency

is generally

in agreement with the conclusions of the above studies and collectivel y, these results provide sufficient data to support that Bt crops registered to date have not caused long term environmental effects on a population level to organisms not targeted b y Bt
- derived Cry proteins. These available field studies further support the tiered testing approach in that the absence of hazard in the Tier 7 This research was funded by Environmental Prote ction Agency grant CR - 83214 Bt 7 - 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.ucsb.edu/btcrops/).Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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35 I toxicity studies is predictive of an absence of ecological harm in the field. These results are also consistent with earlier studies of Bt strains used as microbial insecticides, which have shown no significant detrimental impacts on populati ons of nontarget organisms that were studied. Based on these information, the tiered testing approach serves as an appropriate method for assessing environmental risk of Bt crops while ensuring, to the extent possible, that the Agency requires the minimum amount of data needed to make science - based regulatory decisions. **C .**  **Nontarget Organism Risk Assessment for MON 87751 Soybeans 1.**  **Environmental Exposure Assessment** Two separate FIFRA SAP reports (US EPA, 2000 & 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 determined that non - target organisms with the greatest exposure potential to Bt Cry 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, it recognizes that exposure to other nontarget organisms can occur and has continued to require testing on representative species. The EPA risk assessment is focused on adverse effects at the field exposure rates, which for PIPs are ty picall y based on protein expression levels within the plant. Although it is recommended that non - target testing be conducted at a test dose 10X the EEC whenever possible, the test dose m argin can be less than 10X where uncertainty

in the sy stem is low or where high concentrations of test material are not possible to achieve due to test organism feeding habits. The Agency may also allow for testing at lower doses in cases where man y speci es are tested or tests are very sensitive, although the concentration used must exceed 1X EEC. T he exposure assessment is based mainly on the exposure which was determined from the expression data using the MON 87751 soy bean containing Cry 1A.105 and Cry2 Ab2 proteins (MRID No. 49332002). Further details can also be found in Table 5 of this document . **2. Ecological Effects Data**  As discussed above, in the absence of PIP - specific risk assess m ent data require m ents, EPA requires applicants for P IP registrations to m eet the 40 CFR Part 158 data require m ents for mi crobial pesticides. These require m ents include te s ting on birds, m amm als, nontarget insects, honey bee, plants, and aquatic species, and in f

or m ation has been sub mitted to a d dress th ese r equire m ent s . No new guideline testing data using the MON 87751 soy bean plant parts or soy

bean plant residues containing both the insecticidal proteins (Cry 1A.105 and Cry2Ab2) were submitted. In stead, the applicant has requested to bridge to the Agency database of previ ousl y reviewed toxicity studies of selectBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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36 nontarget organisms that supported the currentl y registered MON 89034 in corn ( EPA Reg. No. 524 - 575 ). The October 2000 SAP recomm end e d that while ac tual plant m aterial is the preferred test m aterial, bacteria derived protein is also a valid test substance, particularly in scenari o s where test ani m als do not normally consume soy

bean plant tissue and where large am ounts of Cry protein are needed for maxi m um haz ard dose testing. For the MON 89034 corn registration , test substances used in the submitted studies included bacteriall y produced purified Cry1A.105 and Cry2Ab2 proteins (see Table 5). Comparative analyses were performed to verify

the equivalence of the bacterially produced and purified Cry 1A.105 and Cry2Ab2 and the Cry 1A.105 and Cry2Ab2 proteins produced in MON 89034 corn (see Table 3) . These anal ys es showed that the two bacterially produced insecticidal proteins were equivalent to the corresponding proteins produced b y the MON 89034 corn (US EPA, 2010a). The toxicity

of Cry 1A.105 and Cry2Ab2 proteins has been previously evaluated on several species of invertebrates including: lady beetle ( Coleomegilla maculata ), honey bee, adult and larvae (

Apis mellifera ), collembola (

Folsomia candida ), minute pirate bu g ( Orius insidiosus ), parasitic wasp ( Ichneumon promissorius ) and earthworm ( Eisenia fetida ). Two dietary toxicity studies were submitted in support for the currently

registered MON 89034 corn, which included broiler chicken ( Gallus domesticus ) and a freshwater invertebrate (

Daphnia magna )
. The individual results for the cited studies and a newl y submitted environmental risk assessment prepared by the applicant in support for the Section 3 seed increase registration of MON 87751 are summarized in Table 5 . The evaluation of these studies are described in more detail in the environmental risk assessment for Cry 1A.105 and Cry2Ab2 expressed in MON 89034 corn (US EPA, 2010a). EPA conducted an environmental risk assessment based mainly on the following lines of evidence to support the seed increase registration of MON 87751 soy bean: a) Determination of protein equivalency of Cry 1A.105 and Cry2Ab2 proteins produced in MON 87751 soy bean to the previousl y characterized corn, cotton or microbe - derived Cry 1A.105 and Cry2Ab2 protein test substances; and b) Finding of no significant increase i n exposure to nontarget organisms based on calculated margins of exposure for protein concentrations in MON 87751 soy bean plant tissue matrices relative to the dose concentrations used in the nontarget organisms laboratory toxicity tests conducted on Cry1 A.105 and Cry2Ab2 for MON 89034.
**a .**  **Finding of No Increase in Exposure to Nontarget Organisms** The Agency

previousl y assessed MON 89034, a PIP corn containing insecticidal genes cry 1A.105 and cry2Ab2, which were initially derived from PV - ZMIR245, for its ecological effects of the insecticidal proteins Cry 1A.105 and Cry2Ab2, singl y or in combination, on nontarget organisms. A summary of the assessment is described in Table 5 (see below) which was in turn derived from the Biopesticide Registration Action Do cument (BRAD) entitled “Bacillus thuringiensis Cry1A.105 and Cry2Ab2Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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37 Insecticidal Proteins and the Genetic Material Necessary for Their Production in Corn” (US EPA, 2010a). Finding of no significant increase in exposure to nontarget organisms is based on calculated margins of exposure (MOEs which are based on the ratio of the NOEC to EEC) for protein concentrations in MON 87751 soy bean plant tissue matrices (per MRID No. 49332002) relative to the dose concentrations used in the non - target organisms laborat ory toxicity tests conducted on Cry 1A.105 and Cry2Ab2 for MON 89034. **Table 5**  **. Summary**  **of the assessment of the environmental effects data based on previously submitted data for plant**  **- or microbe**  **- produced Cry1A.105 and/or Cry2Ab2 (e.g., the MON 89034**  **corn produced insecticidal proteins) in support of data requirements publ ished in 40 CFR PART**  **158.2150.** The summary was prepared based on a previous assessment carried out by the Agency (US EPA, 2010a), and also on an environmental risk assessment prepared by the registrant (MRID No 49332002) in support of the registration of event MON 87751 in soybean. **Data Requirement**  **OSCPP**

**Guideline Test Substance**  **Results Summary and Classification**  **MRID No.**  Avian dietary testing, broiler chicken, Gallus domesticus 885.4050* MON 89034 corn grain** A 42 - day dietary study showed no adverse effects to broiler chickens. **Classification: Acceptable 46951412** 

Avian acute oral 850.2100 MON 89034 corn grain An 8 - day dietary study showed that the LC50 for MON 89034 is >500,000 ppm in the diet of Northern bobwhite. **Classification: Acceptable for MON 89034. Acceptable for MON 87751, conditional on**  **acceptance of protein equivalence data**  **46951427**

Avian inhalation testing 885.4100 N/A **Not Required N/A** 
Wild mammal testing 885.4150 N/A **Acceptable waiver rationale N/A**  Freshwater fish testing, 885.4200 N/A **Acceptable waiver rationale N/A** Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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38 **Data Requirement**  **OSCPP**

**Guideline Test Substance**  **Results Summary and Classification**  **MRID No.**  Freshwater invertebrate testing, Daphnia magna 885.4240 MON 89034 corn pollen A 48 - hour static renewal limit bioassay resulted in 17% mortality compared with 0% mortality in the control groups when exposed to 120 mg/liter. The acute EC50 was estimated to be >120 mg/liter and NOEC was 100 mg/liter. However, The Guideline for 885.4240 calls for a 7 - 14 day study, not 48 hour acute study. **Classification: Unacceptable for MON 89034 -** 

**--------------------------------------------------------** A new study was sub mitted for Mon 89034. The study was acceptable and satisfies the condition of registration for additional aquatic invertebrate toxicity testing. **Classification: Acceptable for MON 89034 A**  **cceptable for MON 87751 46951417** 

**------------ 47838801** 

Estuarine and marine animal testing 885.4280 N/A **Acceptable waiver rationale N/A** 
Non - target plant testing 885.4300 N/A **Acceptable waiver rationale N/A** 
Non - target insect testing, collembolan Folsomia candida 885.4340 MON 89034 corn leaf tissue (80ug Cry1A.105 and 70 ug Cry2Ab2/g diet

Lyophilized MON 89034 corn leaf tissue for NOEC determinations, but MON 87751 soybean root for EEC determinations. Collembola were fed a diet containing 50% Brewer’s yeast and 50% lyophilized leaf tissue for 28 days. No statistically significant effects on survival or reproduction were found among the test and negative control groups. The NOEC for Folsomia candida is at least 80 ug Cry1 A.105 and 70 ug Cry2Ab2 per gram of diet. **Classification: Acceptable for MON 89034** The EEC and NOEC for Folsomia candida we re <0.322 ug Cry1A.105/g fw root and >80 ug/g, respectively. The EEC and NOEC for Folsomia candida we re 7.5 ug Cry2Ab2/g dry root w t and >70 ug/g, respectively. (It is noted that the above NOECs were calculated as 50% of the maximum Cry1A.105 and Cry2Ab2 expressed in the MON 89034 corn leaves.) Margins of Exposure (MOEs***) were >248 for Cry1A.105 and >9.3 for Cry2Ab2. **A cceptable for**  **MON 87751 . Note that this**  **was not a new study, but the registrant 46951416** 

**------------ 49332002** Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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39 **Data Requirement**  **OSCPP**

**Guideline Test Substance**  **Results Summary and Classification**  **MRID No.**  **calculated a risk quotient using the expression data from** **MON 87751 soybean and the study previously submitted for MON**  **89034.** Non - target insect testing, ladybird beetle, Coleomegilla maculata 885.4340 Purified Cry1A.105 protein (Lot No. 20 - 100073)

----------------------- MON 87751 soybean pollen (fresh) N o statistically significant differences in survival, development to adult, or mean adult weight were observed in Coleomegilla maculata larvae when exposed to a diet containing 240 μg Cry1A.105 protein/g of diet fo r 14 days . **Classification: Acceptable for MON 89034**

**--** -------------------------------------------------------- The EEC and NOEC for Coleomegilla maculata were stated to be 11 μg/g fw pollen and >240 ug/g for Cry1A.105, respectively. The MOE wa s 21. **Classification: A**  **cceptable for MON 87751** . See above. **46951421**

**------------ 49332002** 

Non - target insect testing, ladybird beetle, Coleomegilla maculate 885.4340 Purified Cry2Ab2 protein (Lot No. 20 - 100071)

MON 87751 soybean pollen (fresh) N o statistically significant differences in survival, or development to adult were observed between the test and control groups when Coleomegilla maculata larvae were exposed to a diet containing 120 Cry2Ab2.105 protein/g of diet for 14 days. **Classification: Acceptable for MON 89034**

**----------------------------------------------------------** The EEC and NOEC for Coleomegilla maculata were 7.7 μg/g fw pollen and >120 ug/g for Cry2Ab2, respectively. The MOE was >15. **Classification: A**  **cceptable for MON 87751** . **See above. 46951422** 

**- -----------**  **49332002**Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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40 **Data Requirement**  **OSCPP**

**Guideline Test Substance**  **Results Summary and Classification**  **MRID No.**  Honeybee testing, Honeybee larvae, Apis mellifera 885.4380

margin Purified Cry1A.105 (Lot

No. 20 - 100073)

MON 87751 soybean pollen (fresh) Two - to - three day old honeybee larvae in brood frames were administered a single 10 ul dose of Cry1A.105 protein brood cell (equivalent to 12 ug total protein/cell. On day 18 after dosing, mean survival of the test group was 95%. . The NOAEC was determined to be at least 12 μg Cry1A.105 protein/mL. **Classification: Acceptable for MON 890 34**  The EEC and NOEC for Aphis mellifera were 0.022ug and >12 μg Cry1A.105 /larva fw pollen, respectively. The MOE was >545. **Classification: A**  **cceptable for MON 87751** . S ee above. **46951420**

**------------ 49332002**  Honeybee testing, Honeybee larvae, Apis mellifera 885.4380 MON 87751 soybean pollen (fresh) The EEC and NOEC for Aphis mellifera were 0.0154 Cry2Ab2 ug/fw pollen and >0.60 ug/larva , respectively. The MOE was >38. **Classification: A**  **cceptable for MON 87751 1.**  See above. **49332002**

Honeybee testing, Honeybee adult, Apis mellifera 885.4380 Purified Cry1A.105 (Lot No. 20 - 100073)

MON 87751 soybean pollen (fresh) 87751No statistically significant differences in mo rtality were observed between the test group and negative controls when adult honey bees were fed a 30% sucrose solution containing 550 μg Cry1A.105 protein/mL for 19 days. The NOAEC was determined to be at least 550 μg Cry1A.105 protein/mL. **Classification: Acceptable for MON 89034** The EEC and NOEC for Aphis mellifera were 11 ug/g fw pollen and >550 μg/ml for Cry1A.105, respectively. The MOE was >50. **Classification: A**  **cceptable for MON 87751 .**  See above. **4695140**

**------------ 49332002**  Honeybee testing, Honeybee adult, Apis mellifera 885.4380 MON 87751 soybean pollen ( fw) The EEC and NOEC for Aphis mellifera were 7.7 ug/g fw pollen and >68 Cry2Ab2 ug ml, respectively. The MOE was 8.8. **Classification: A**  **cceptable for MON 87751 .**  See above. **49332002**Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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41 **Data Requirement**  **OSCPP**

**Guideline Test Substance**  **Results Summary and Classification**  **MRID No.**  Non - target insect testing, minute pirate/insidious flower bug Orius insidiosus 885.4340 Purified Cry2Ab2 protein (Lot No. 20 - 100071)

MON 87751 soybean pollen (fresh) Orius nymphs were fed a diet containing 100 ug Cry2Ab2 protein/diet for 14 days. No adverse effects were observed. **Classification: Acceptable for MON 8903 4** 
The EEC and NOEC for Orius insidiosus were 7.7 Cry2Ab2 ug and >100 ug/g fw pollen, r espectively. The MOE was >12. **Classification: A**  **cceptable for MON 87751 .**  See above. **46951424**

**------------ 49332002** 

Non - target insect testing, minute pirate/insidious flower bug Orius insidiosus 885.4340 Purified Cry1A.105protein (Lot No. 20 - 100073)

MON 87751 soybean pollen (fresh) Orius nymphs were fed a diet containing 30 to 240 ug Cry1A.105/g diet for for 14 days. In an initial maximum dose test (240 ug) the survival rate was 47% compared to 88% in the control groups. In the three subsequent dose - response tests, the mean survival rate of the 240 ug group wa s 55% compared to 91% and 89% in the control grou

ps. N o statistically significant effects on survival or development were observed at concentrations less than or equal to 120 ug Cry Cry1A.105/g diet. **Classification: Acceptable for MON 89034** The EEC and NOEC for Orius insidiosus were 11 Cry1A.105 ug and 120 ug/g fw pollen, r espectively. The MOE was 10.9. **Classification: A**  **cceptable for MON 87751 46951423** 

**------------ 49332002** 
Non - target insect testing, parasite wasp, Ichneumon promissorius 885.4340 Purified Cry2Ab2 protein (Lot No. 20 - 100071)

----------------------- MON 87751 soybean pollen (fresh) Adult female wasps were fed a sucrose solution containing 100 ug Cry2Ab2 protein/ml for 21 days. Mortality in the Cry2Ab2 group was 3% and the LC50 was determined to be >100 ug/L **Classification: Acceptable for MON 89034** 

**--------------------------- -------------------------------**  The EEC and NOEC for Ichneumon promissorius were 7.7ug/g fw pollen and >100ug/g. 11 for Cry2Ab2, r espectively. The **46951426**

**------------ 49332002** Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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42 **Data Requirement**  **OSCPP**

**Guideline Test Substance**  **Results Summary and Classification**  **MRID No.**  MOE was >12. **Classification: A**  **cceptable for MON 87751** See above. Non - target insect testing, parasite wasp, Ichneumon promissorius 885.4340 Purified Cry1A.105protein (Lot No. 20 - 100073) MON 87751 soybean pollen (fesh) Adult female wasps were fed a sucrose solution containing 240 ug Cry1A.105 protein/ml for 21 days. Mortality in the Cry2Ab2 group was 7% and the LC50 was determined to be >240 ug/L **Classification: Acceptable for MON 89034** The EEC and NOEC for Ichneumon p romissorius were 11 Cry1A.105 ug/g fw pollen and >240 ug/ml, r espectively. The MOE was >21. **Classification: A**  **cceptable for MON 87751 .**  See above. **46951425**

**------------ 49332002** 

Earthworm toxicity, Eisenia fetida 850.6200 Purified Cry1A.105 (Lot No. 20 - 100073)

MON 87751 soybean root (fresh) Adult earthworms were exposed to artificial soil containing 178 mg Cry1A.105protein/kg dry soil for 14 days. The 14 - day LC 50 for earthworms was determined to be > 178 mg Cry1A.105 protein/kg dry soil and the NOAEC wa s determined to be ≥ 178 mg Cry1A.105 protein/kg dry soil. **Classification: Acceptable for MON 89034: A**  **cceptable for MON 87751** The EEC and NOEC for Eisenia fetidas we re <0322 ug/g fw root and > 178 mg/kg dry soil for Cry1A.105, r espectively. The MOE was >552. **Classification: A**  **cceptable for MON 87751 .**  See above. **46951418**

**------------ 49332002** 

Earthworm toxicity, Eisenia fetida 850.6200 MON 87751 soybean root (fresh) The EEC and NOEC for Esenia fetida we re 22 ug/g dw root and >330 mg/kg dry soil for Cry2Ab2, r espectively. The MOE was >15. **Classification: A**  **cceptable for MON 87751** Aerobic soil degradation 885.5200 Purified Cry1A.105 (Lot No. 20 - 100073) and Cry2Ab2 protein (Lot No. 20 - 100071) Results of this degradation study indicate that Cry1A.105 and Cry2Ab2 proteins do not persist in soil beyond about three weeks **Classification: Acceptable for 89034 PIP corn:**  **46951428**Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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43 **Data Requirement**  **OSCPP**

**Guideline Test Substance**  **Results Summary and Classification**  **MRID No.**  **A cceptable for MON 87751**  Environmental Risk Assessm ent for the Cry1A.105 and Cry2Ab2 Proteins as expressed in MON 87751 Soy bean Non guideline N/A The applicant submitted voluntarily an environmental risk assessment (ERA) for the registration of the current submission, MON 87751 soybean containing cry1A.105 and cry 2Ab2 genes encoding Cry1A.105 and Cry2Ab2 insecticidal proteins. Monsanto requested data bridging to the previously conducted testing on the effects of Cry1A.105 and Cry2Ab2 proteins produced in MON 89034 corn( or other organisms) on various nontarget organisms. In the ERA, Cry1A.105 and Cry2Ab2 protein levels in select soybean tissues were presented in order to calculate expected environmental concentrations (EECs) from MON 87751. EECs, no observed effect concentrations (NOECs) for nontarget organism studies and margins of exposure (MOEs) for Cry1A.105 and Cry2Ab2 proteins were summarized. **Classification** : **Supplemental** for risk assessment purposes **49332002**

**Note** : Earthworm and honey bee studies for Cry2Ab2 protein were submitted and reviewed with previously

registered products (US EPA, 2005). The interaction study between Cry 1A.105 and Cry2Ab2 was reviewed for the MON 89034 Experimental Use Permit (US EPA, 2007) * OSCPP Microbial test gu idelines **Cry 1A.105 and Cry2Bb2 are the active ingredients in MON 89034 *** Margins of Exposure (MOEs) were calculated based on the ratio of the NOEC to EEC. The MOE was determined based on the expression level of the Cry 1A.105 and Cry Ab2 proteins in the tissue of MON 87751 deemed relevant to nontarget insect exposure. Classification fo **r** MON 87751 is acceptable for prot ein equivalence data **VI. Risk Assessment Conclusions for MON 87751 Soybeans**  **A. Effects to Nontarget Wildlife, Invertebrates, and Plants** The active ingredients of the MON 87751 soy bean are Cry 1A.105 and Cry2Ab2 insecticidal proteins which are very

similar to the Cry 1A.105 and Cry2Ab2 proteins produced in the registered MON 89034 corn. It is stated that the Cry 1A.105 and Cry2Ab2 proteins in the MON 87751 share greater than 99% and 98% identity , respectively , with the Cry 1A.105 and Cry2Ab2 proteins in MON89034 in their deduced amino acid sequences. The Cry 1A.105 and Cry2Ab2 proteins produced in MON 87751 areBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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44 determined to be functionally equi valent to those produced in MON 89034 (MRID No. 49332002). Monsanto submitted no new testing data for the FIFRA Section 3 seed increase registration for MON 87751 soy

bean, but referenced the data/information submitted previously for the MON 88034 registration (see Table 5 above and also refer to the BRAD for MON 89034, US EPA, 2010a). The Agency ’s previous environmental risk assessment of the MON 89034 corn (US EPA, 2010a) is briefl y described as follows: Maximum hazard dose (MHD) testing on representative nontarget organisms was performed in support of the registration of the MON 89034 corn producing Cry 1A.105 and Cry2Ab2 proteins. Avian oral toxicity testing data were submitted. The toxicity of the insecticidal Cry 1A.105 and/or Cry2Ab2 proteins was e valuated on several species of invertebrates, including the lady bird beetle, minute pirate bug, parasitic wasp, Daphnia , honey bee, Collembola , and earthworm. Developmental observations were also made in the ladybird beetle, minute pirate bug and honey bee studies. Possible reproductive effects were also examined. Earthworm studies were provided to the Agency to evaluate the potential effects of the Cry1A.105 and/or Cry2Ab2 proteins on soil decomposer species. As shown in Table 5 above, the test substanc e used for the studies provided to the Agency in support of the registration of the MON 89034 corn included bacteriall y- produced, purified Cry 1A.105 and Cry2Ab2 proteins, MON 89034 corn leaf tissue, pollen, and grain. Even though actual plant material is the preferred test material, bacteriall y derived protein is also a valid test substance, especiall y when test animals do not normally

consume specific plant tissue and large amounts of Cry protein are needed for maximum hazard dose testing (US EPA, 2000). **B. Horizontal Transfer of Transgenes from**  **Bt Crops to Soil Organisms**  The EPA has evaluated the potential for horizontal gene transfer (HGT) from Bt crops to soil organisms and has considered possible risk implications if such a transfer were to occur. Genes that have been engineered into Bt crops are mostly found in, or have their origin in, soil - inhabiting bacteria. Soil is also the habitat of anthrax, tetanus and botulinum toxin - producing bacteria. Transfer of these genes and/or toxins to other microorganisms or plants has not been detected. Furthermore, several published experiments, that were conducted to assess the likelihood of HGT, have been una ble to detect gene transfer under typical environmental conditions. Horizontal gene transfer to soil organisms has only been detected with very

promiscuous microbes under laboratory conditions designed to favor transfer. As a result of these findings and the fact that the Bt toxins engineered into MON 87751 were derived from soil - inhabiting bacteria, the EPA has concluded that HGT of transgenes found in Cry 1A.105 and Cry2Ab2 producing soy

bean is not expected. **C. Gene Flow and Weediness Potential**  Movement of transgenes from crop plants into weeds is a significant concern, due to uncertaintyBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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45 regarding the effect that a new pest resistance gene may have on plant populations in the wild. Under FIFRA, the Agency

has reviewed the potential for gene capture and expression of Cry proteins in commercial Bt soy

beans by wild or weedy relatives of cotton in the United States, its possessions or territories. The potential for gene flow and the weediness potential of MON 87751 soybean is expected to be low sin ce no known wild or weedy relatives exist in the North America with which soybean ( Glycine max (L .) Merr.) can form viable hy brids in nature (OECD, 2000), and because soy bean is not weedy in character or invasive, BPPD determined that there is no significa nt risk of gene capture and expression of an y Bt endotoxin by

wild or weedy relatives of soybean in the U.S., its possessions or territories (US EPA, 2007). **A. Impacts on Endangered Species**  Because of the selectivity of Cry 1A.105 and Cry2Ab2 insecticidal p roteins for lepidopteran species and lack of evidence of effects on other nontarget species, the Agency has investigated concerns for federally- listed threatened and endangered insect species in the order Lepidoptera. Because soy bean pollen is not expecte d to move beyond the planted soy bean field and its immediate margins, any exposure to lepidopterans would be expected to occur within those areas (US EPA, 2010b & 2014). Exposure could occur via direct consumption of MON 87751 soy bean plants or consumpti on of MON 87751 pollen that falls on non - soy bean plants within the soy bean field and its immediate margins. However, exposure to significant amounts of Cry 1A.105 and Cry2Ab2 via pollen consumption is not likely . Little pollen is expected to be released f rom soy bean flowers, since soy

bean plants are primarily self - pollinated and anthers usually dehisce and release pollen before flowers open. Airborne pollen concentrations have been measured at very low levels within soy

bean fields (mean of 0.18 grains/cm 2 /day) (Yoshimura et al., 2006). Based on previous anal ys es for Bt soy beans (e.g., see USEPA 2010b and 2014), EPA concludes that exposure resulting from pollen falling on potential non - soy bean food plants in the field and immediate margins is not sufficient to cause effects in federall y listed lepidopterans. Therefore, any significant exposure would have to occur through consumption of the MON 87751 soy

bean plants on the field. A search of EPA’s LOCATES database indicates that four species of federally l isted lepidopterans are present in U.S. counties in which soy beans are grown. These are the Karner blue butterfl y ( Lycaeides melissa samuelis ), St. Francis’s Satyr Butterfl y (Neonympha mitchellii fransisci ), Mitchell’s Saty r Butterfl y (

Neonympha mitchelli i mitchellii ), and Uncompahgre Fritillary Butterfly ( Boloria acrocnema ). The potential effects of Bt PIPs in soy bean on the Karner blue butterfl y, Mitchell’s satyr butterfl y, and St. Francis’s satyr butterfly were previously

anal yzed in the endangered species assessment section in the BRAD for event MON 87701 soy bean expressing Cry 1Ac insecticidal prote in (US EPA, 2010b) andBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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46 event DAS - 81419 - 2 soy

bean expressing Cry 1Ac and Cry 1F insecticidal proteins (US EPA, 2014). Based on their biology

and habitat requirements, EPA determined that these species would not be exposed to the Cry proteins expressed in the MON 87701 or DAS - 81419 - 2 soy bean plants. Since MON 87751 soy beans would be expected to be grown in the same locations as soy beans expressing previousl y registered Bt PIPs, these conclusions would also apply to the Cry 1A.105 and Cry2Ab2 proteins expressed i n MON 87751 soy beans. According to the LOCATES database, the Uncompahgre Fritillary Butterfly ( Boloria acrocnema ) is present in one county (Delores) in Colorado in which soy beans are also grown (US FWS, 2009). However, all known populations of this spe cies are associated with large patches of snow willow above 12,400 feet which provide food and cover. The species has been located only on northeast - facing slopes, which are the coolest and wettest microhabitat available. Snow willow serves as the larval food, while the adult butterflies take nectar from a wide range of alpine plants. Movement of the Uncompahgre Fritillary Butterfl y is limited to their habitat patches. Based on its limited habitat in high altitudes, effects to this species as a result of the proposed seed increase registration of MON 87751 soy beans are not expected, since this butterfly species will not be exposed. Based on the above anal ysis, the Agency determines that there will be no direct effect to federall y listed lepidopteran spe cies as a result of the cultivation of MON 87751 so ybeans as proposed. Obligate relationships between insectivorous listed species with lepidopterans that are expected to be found in soy bean fields, especially

pest species that feed on MON 87751 plants, are not currentl y known. Since the Cry 1A.105 and Cry2Ab2 in MON 87751 soy beans target only

lepidopteran insects, loss of the pest insects as a result of MON 87751 are expected to be offset b y the presence of other insects that could act as food sources for listed species, including beneficial insects that are known not to be affected by Cry 1A.105 and Cry2Ab2. Effects on species other than insects have also been determined to be unlikely because of the specificity of Cry1A.105 and Cry2Ab2. Therefore, no e ffects to listed species due to indirect effects or effects on habitat are anticipated to occur. **E .**  **Conclusions** The environmental risk assessment indicates that, based on information submitted to support this proposed registration, will not result in unreasonable adverse environmental effects. The Agency additionally

concludes registering MON 87751 soy beans will have no direct or indirect effect on endangered and/or threatened species listed by the FWS and National Marine Fisheries Service or their hab itats. **VII . Insect Resistance Management**  An insect resistance management (IRM) assessment was not conducted spec ificall y for MON 87751 soy bean. However, BPPD did conduct an IRM review (BPPD 2010) for MON 87701 soy bean, whichBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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47 expresses Cry 1Ac protein. MON 87751 expresses both Cry 1A.105 and Cry2Ab2 proteins. Li ke MON 87751, MON 87701 is a “breeding onl y” registration and was limited to 15,000 acres per ye ar in the U.S. Both products target lepidopteran so ybean pests, including so ybean looper and velvetb ean caterpillar. BPPD’s review for MON 87701 concluded that there is not likely to be a significant risk of resistance in the U.S. to the lepidopt eran target pests. This conclusion was supported by the approved low acreage (maximum 15,000 per year, with n o more than 1,000 in any individual county) and the biology of the target pests, which do not overwinter in non - tropical areas and are pol yphagous (feeding on a number of non - soy bean wild hosts and cultivated crops). BPPD also evaluated the potential impac t of Bt so ybean on the natural refuge strategy in place for Bt cotton PIPs in the southeastern U.S. The review noted that the total annual acreage for MON 87701 is a small fraction of the of the total soy bean acres in each state and should not significantl y reduce the amount of natural refuge available for Bt cotton. The IRM review conclusions for MON 87701 are also applicable to MON 87751 soy bean. Both will be grown on comparable acreage and targ et the same lepidopteran pests. In addition, MON 87751 is a py ramided product that expresses two Bt proteins (Cry 1A.105 and Cry2Ab2), while MON 87701 expresses a single toxin (Cry 1Ac) . Py ramided PIPs are inherently more durable than single toxin varieties (Roush 1998); therefore, it is expected that the risk of res istance will be lower for MON 87751 than for MON 87701. As part of the IRM strategy for MON 87751 soy

bean, a resistance monit oring program will be required. Similar to the approach in place for MON 87701, the monitoring plan can be based on investigations of unexpected p est damage in MON 87751 fields. An associated remedial action plan should be applied in the event of confirmed resistance (similar to the remedial action plans used for Bt corn PIPs). It should be noted that BPPD’s IRM assessment is applic able onl y to an acreage - limited, breeding registration. A separate evaluation will be necessary if a full commercial product (i.e., without acreage restrictions) is proposed for registration. Data needed to support such a review would include additional in formation about pest biology , dose, simulation model ing, and resistance monitoring. Further information to evaluate the impact on natural refuge for Bt cotton will also be necessary .Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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48 **VIII .**  **References** BPPD, 2010. Review of Insect Resistance Management Considerations for Bt Soy bean Event MON 87701. A. Rey

nolds memorandum to M. Mendelsohn, June 1, 2010. Marvier, M., McCreedy, C., Regetz, J. & Kareiva, P. (2007). A meta - anal ysis of effects of Bt cotton and maize on nontarget invertebrates. Science 316: 1475

– 1477. Monsanto. 2014. Environmental Risk Assessment for the Cry 1A.105 and Cry 2Ab2 Proteins as Expressed in MON 87751 Soy bean. MRID No. 49332002. National Academ y of Science (NAS). 2000. Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation is available from the National Academy Press, 2101 Constitution Avenue, N.W., L ockbox 285, Washington, DC 20055; (800) 624 - 6242 or (202) 334 - 3313 (in the Washington metropolitan area); http://www.nap.edu . OECD. 2000. Consensus Document on the Biology of Glycine max (L.) Merr. (Soy bean). ENV/JM/MONO(2000)9. OECD. 2007. Consensus Document on Safety In formation on Transgenic Plants Bacillus thuringiensis Derived Insect Control Protein. ENV/JM/MONO(2007)14. Romeis, J., Meissle, M. and Bigler, F. 2006. Transgenic Crops expressing Bacillus thuringiensis toxins and biological control. Nature Biotechnology 24: 63 - 71. Roush, R.T., 1998. Two toxin strategies for management of insecticidal transgenic crops: will py ramiding succeeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond. 353: 1777 - 1786. Sanvido, O., Romeis , J., Bigler, F. 2007. Ecological Impacts of Genetically Modified Crops: Ten Years of Field Research and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107: 235

–278. Saxena, D.S. Flores, and G. Stotzky . 2002a. Bt toxin released in root exudates from 12 transgenic corn hy brids representing three transformation events. Soil Biol. Biochem. 34:133 - 137. Saxena, D.S. Flores, and G. Stotzky . 2002b. Vertical movement in soil of insecticidal Cry 1Ab protein from Bacillus thuringiensis . Soil Biol. Biochem. 34:111 - 120. Stotzky , G. 2000. Persistence and biological activity in soil of insecticidal proteins from Bacillus thuringiensis and of bacterial DNA on clay s and humic acids. J. Environ. Qual. 29:691 - 705. United States Environmental Protection Agency (U S EPA). “Guidelines for Ecological RiskBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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49 Assessment.” EPA 630/R - 95 - 002F. Washington, DC, USA. [Federal Register, May 14, 1998. 63(93): 26846 - 26924.] US EPA. 2000. SAP report No 99 - 06. Sets of scientific issues being considered b y the Environmental Protec tion Agency

regarding: Section I - Characterization and Nontarget Organism Data Requirements for Protein Plant Pesticides. Dated February 4, 2000. Available at the EPA website: http://www.epa.gov/scipoly /sap/1999/index.htm#december US EPA. 2001. SAP Report No. 2000 - 07. Sets of scientific issues being considered by the Environmental Protection Agency regarding: Bt plant - pesticides risk and benefit assessments. Dated March 12, 2001. Web site: http://www.epa.gov/scipoly /sap/2000/october/octoberfinal.pdf US E PA. 2002. SAP Report No. 2002 - 05. A set of scientific issues being considered b y the Environmental Protection Agency regarding: Soybean rootworm plant - incorporated protectant nontarget insect and insect resistance management issues. Dated November 6, 2002 . http://www.epa.gov/scipoly /sap/2002/august/august2002final.pdf US EPA. 2004. SAP Report No.2004 - 05. Product characterization, human health risk, ecological risk, and insec t resistance management for Bt cotton products. Dated August 19, 2004. http://www.epa.gov/scipoly /sap/meetings/2004/june/final1a.pdf US EPA. 2005. Biopesticides Registration Action Document: Bacillus thuringiensis Cry2Ab2 Protein and its Genetic Material Necessary for its Production in Cotton. Washington, DC. http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006487.pdf US EPA. 2007. Ecological risk assessment for Monsanto’s MON 87701 Bt soy bean EUP. Memorandum from T. Milofsky

to M. Hunter. US EPA. 2010a. Biopes ticides Registration Action Document: Bacillus thuringiensis Cry 1A.105and Cry2Ab2 Insecticidal Proteins and the Genetic Material Necessary for Their Production in Corn. Washington, DC. http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006514 - US EPA. 2010b. Biopesticides Registration Action Document: Bacillus thuringiensis Cry 1Ac Protein and the Genetic Material Necessary for its Production in Event MON 87701 Soy bean. US Environmental protection Agency , Washington, D.C. http://www.epa.gov/pesticides/biopesticides/pips/bt - cry1ac - protein.pdfBacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

50 US EPA. 2014. Biopesticides Registration Action Document (BRAD) Plant - In corporated protectants: Bacillus thuringiensis Cry1Ac protein and the Genetic Material Necessary for its Production (PC Code 006527) and Bacillus thuringiensis Cry 1F Protein and the Genetic Material Necessary for its Production (PC Code 006528) as Expressed in Event DAS - 814 Soy bean. Washington, DC. http://www.regulations.gov/# !documentDetail;D=EPA - HQ - OPP - 2013 - 0703 - 0003 U.S. Fish and Wildlife Service (US FWS). 2009. Uncompahgre Fritillary Butterfl y. Grand Junction, CO. http://ecos.fws.gov/speciesProfile/profile/speciesProfile?spcode=I 01Q Yoshimura, Y., K. Matsuo, and K. Yasuda. 2006. Gene flow from GM gl yphosate - tolerant to conventional soy

beans under field conditions in Japan. Environmental Biosafet y Research 5:169 - 173.Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

51 **IX .**  **Terms and Conditions o**  **f Registration** 1) The subject registration is limited to a combined yearl y total of 15,000 acres for breeding purposes, agronomic testing, and producing seed in the United States and the Commonwealth of Puerto Rico. 2) Monsanto must submit reports on IRM monitoring and remedial action plans to EPA annually by August 31 st each year. 3) Upon request, Monsanto must provide EPA by January 31 st each ye ar annual reports on the acreage where MON 877 5 1 has been grown b y States and territory . 4) Harvested seed are not allowed for sale as commercial seed in the U.S. under the conditions of this registration, but an y non - treated seed containing MON 877 5 1 may be sold as grain. **X .**  **Regulatory Posit i**  **on f or**  **Mon 87751 (Cry1A.105 and Cry2Ab2) Soybean**  Pursuant to FIFRA section 3 ( c)(5), EPA m ay unconditionally register a pesticide if EPA deter mines that, when used in acc o rd ance with widespread an d commonly recognized practice, it will not g e n erall y r e sult in unreaso na

ble adv e rse e ff

ects to th e environm ent. Monsanto has sub mitted or cited d ata s ufficient for EPA to deter

mine that an unconditi o nal breeding registration of Bacillus Thuringiensis Cry 1A.105 and Cry2Ab2 Protein s and the Genetic Material (Vector PV - GMIR13196) Necessary For Their Production in MON 87751 (OECD Unique ID. MON - 87751 - 7) Soy bean under FIFRA 3(c)(5) will not result in unrea s onable adverse effects to the environm ent. Monsanto s ub mitted an d/or c ited satis f act o ry d ata pe rt ai n i ng to the p rop o sed use. The hum an health effects data and nontarget organism effects data are considered sufficient for the li mited acreage, and geographic li mitations of the unconditional registration. These data de m onstrate that no foreseeable human health hazards or ecological effects are likel y to arise from the use of the product and that the risk of resistance developing to Cry1A.105 and Cry2Ab2 protein, during this li mited registration is not expected to be significant. The registration is li mited to seed increase and to a total of 15,000 acres per ye ar , with no more than 1,000 acres per county per year.