Bayer CropScience LP CR282-20U1 1

Petition for the Determination of Nonregulated Status for Lepidopteran-Protected Maize

MON 95379

The undersigned submits this petition under 7 CFR § 340.6 to request that the Administrator make 

a determination that the article should not be regulated under 7 CFR Part 340

July 21, 2020

(Revised January 24, 2024)

OECD Unique Identifier: MON-95379-3

Bayer Petition number: CR282-20U1

Submitted by:

Heather M. Anderson, M.Sc.

Bayer U.S. – Crop Science

700 Chesterfield Pkwy West

Chesterfield, MO 63017

E-mail: heather.anderson@bayer.com

Prepared by:

P. Asiimwe, Ph.D., M. Bedair, Ph.D., J. Calcaterra, Ph.D., J. Fischer, Ph.D., G. Frierdich, B.S., 

D.A.B.T., I. Husic, Ph.D., W. Urquhart, Ph.D.

Contributors and/or Principal Investigators:

L. Barberis, B.S., E. Bretsnyder, M.S., C. Brown, Ph.D., G. Brown, B.S., B. Chen, Ph.D., E.

Dickinson, B.S., T. Edrington, Ph.D., R. Dobert, Ph.D., J. Fridley, B.S., C. Jiang, Ph.D., S. Joshi, 

Ph.D., C. Kessenich, M.S., H. Kaur Bal Ph.D., D. Kendrick M.S., S. Levine, Ph.D., G.

MacQuarrie, M.A., W. Li, Ph.D., S. Martinelli, Ph.D., M. Maven, M.S., G. Mueller, M.A., 

J. Nyangulu Ph.D., S. Reyna, M.S., K. Robinson, Ph.D., A. Silvanovich, Ph.D , K. Skottke, 

Ph.D., J. Tan, Ph.D., F. Zapata, M.S.

Contains no CBI.

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Bayer CropScience LP CR282-20U1 2

RELEASE OF INFORMATION 

Bayer is submitting the information in this petition for review by the USDA as part of the 

regulatory process. Bayer understands that the USDA complies with the provisions of the 

Freedom of Information Act (FOIA). In the event the USDA receives a FOIA request, pursuant 

to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of the information in this petition, Bayer 

expects that, in advance of the release of the document(s), USDA will provide Bayer with a copy 

of the material proposed to be released and the opportunity to object to the release of any 

information based on appropriate legal grounds, e.g., responsiveness, confidentiality, and/or 

competitive concerns. Bayer understands that a CBI-deleted copy of this information may be made 

available to the public in a reading room and upon individual request as part of a public comment 

period. Bayer also understands that when deemed complete, a copy of the petition may be posted 

to the USDA-APHIS BRS website or other U.S. government websites (e.g., 

www.regulations.gov). Except in accordance with the foregoing, Bayer does not authorize the 

release, publication or other distribution of this information without Bayer's prior notice and 

consent. 

© 2024 Bayer Group. All Rights Reserved. 

This document is protected under national and international copyright law and intellectual property 

rights treaties. This document and any accompanying materials are for use only by the regulatory 

authority to which it has been submitted by the Bayer Group, including all subsidiaries and 

affiliated companies, and only in support of actions requested by the Bayer Group. Any other use, 

copying, or transmission, including internet posting, of this document and the materials described 

in or accompanying this document, without prior consent of the Bayer Group, is strictly prohibited; 

except that the Bayer Group hereby grants such consent to the regulatory authority where required 

under applicable law or regulation. The intellectual property, information and materials described 

in or accompanying this document are owned by the Bayer Group, who has filed for or been 

granted patents on those materials. By submitting this document and any accompanying materials, 

the Bayer Group does not grant any party or entity any right or license to the information, materials 

or intellectual property described or contained in this submission. 
Bayer CropScience LP CR282-20U1 3

CERTIFICATION

The undersigned certifies that, to the best knowledge and belief of the undersigned, this petition 

includes all information and views on which to base a determination, and that it includes all 

relevant data and information known to the petitioner that are unfavorable to the petition.

_________________________________________

Heather M. Anderson, M.Sc.

Senior Global Regulatory Manager

Address:

Bayer U.S. – Crop Science

700 Chesterfield Pkwy W, 

Chesterfield, MO 63017

Tel: (636) 236-4499

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Bayer CropScience LP CR282-20U1 4

EXECUTIVE SUMMARY 

The Animal and Plant Health Inspection Service (APHIS) of the United States (U.S.) Department 

of Agriculture (USDA) has responsibility under the Plant Protection Act (Title IV Pub. L. 106-

224, 114 Stat. 438, 7 U.S.C. § 7701-7772) to prevent the introduction and dissemination of plant 

pests into the U.S. 

APHIS prior regulations codified in 7 CFR § 340.6 provides that an applicant may petition APHIS 

to evaluate submitted data to determine that a particular regulated article does not present a plant 

pest risk and no longer should be regulated. If APHIS determines that the regulated article does 

not present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction of 

the article. Newly promulgated 7 CFR § 340 regulations (issued May 18, 2020) state, in §340.1(c), 

that “All plants determined by APHIS to be deregulated pursuant to § 340.6 as that section was set 

forth prior to August 17, 2020 will retain their nonregulated status under these regulations.” Thus, 

this petition seeks to gain a deregulation determination under those prior regulations and thus be 

exempt from the newly issued 7 CFR § 340 regulations. 

Bayer is submitting this request to APHIS for a determination of nonregulated status for the new 

biotechnology-derived maize product, MON 95379, any progeny derived from crosses between 

MON 95379 and conventional maize, and any progeny derived from crosses of MON 95379 with 

biotechnology-derived maize that have previously been granted nonregulated status under 7 CFR 

Part 340 or determined in the future to not pose a plant pest risk via a regulatory status review or 

otherwise deemed to be exempt from 7 CFR Part 340. 

Product Description 

Insect-protected maize MON 95379 was developed to produce two insecticidal proteins, 

Cry1B.868 and Cry1Da_7, which protect against feeding damage caused by targeted lepidopteran 

insect pests. Cry1B.868 is a chimeric protein comprised of domains I and II from Cry1Be (Bacillus 

thuringiensis, Bt), domain III from Cry1Ca (Bt subsp. aizawai) and C-terminal protoxin domain 

from Cry1Ab (Bt subsp. kurstaki). Cry1Da_7 is a modified Cry1Da protein derived from Bt

subsp. aizawai. 

MON 95379 was developed to provide growers in South America an additional tool for controlling 

target lepidopteran maize pests, including fall armyworm resistant to current Bt technologies. 

MON 95379 will be combined through traditional breeding with other deregulated traits to provide 

protection against both above-ground and below-ground maize pests, as well as herbicide 

tolerance. These next generation combined-trait maize products will offer broader grower choice, 

improved production efficiency, increased pest control durability, and enhanced grower profit 

potential. 

MON 95379 will not be commercialized in the U.S., but is intended to only be cultivated in smallscale breeding, testing, and seed increase nurseries to develop seed of products that will be sold in 

other countries, primarily in South America. These intended cultivation uses will be subject to the 

terms and conditions of an EPA seed increase registration, which we have proposed to be limited 

to no more than 100 acres per growing season across Nebraska, Hawaii, and Iowa. In the course 

of developing these products containing MON 95379, Bayer may also develop breeding stacks in 
Bayer CropScience LP CR282-20U1 5

which MON 95379 will be combined with other traits for commercialization outside the United 

States. To support subsequent global regulatory submissions of these stack products, from time to 

time Bayer may conduct confined, small-scale field trials that will be planted in additional 

counties/states that are assessed under the standard USDA-BRS permitting process. Trials and trial 

material will be managed according to the conditions of applicable BRS permits and terms of an 

EPA seed increase registration, if applicable.

Data and Information Presented Confirms the Lack of Plant Pest Potential and the Food 

and Feed Safety of MON 95379 Compared to Conventional Maize 

The data and information presented in this petition demonstrate MON 95379 is agronomically, 

phenotypically, and compositionally comparable to commercially cultivated maize, with the 

exception of the introduced trait. Moreover, the data and information presented demonstrate 

MON 95379 is not expected to pose a greater plant pest risk, including weediness, or adverse 

affects on non-target organisms beneficial to agriculture or maize agronomic practices compared 

to conventional maize. The food, feed, and environmental safety of MON 95379 was confirmed 

based on multiple, well-established lines of evidence: 

x Maize is a familiar crop that does not possess any of the attributes commonly associated 

with weeds and has a history of safe consumption. The conventional control used for the 

transformation process was included in studies to serve as an appropriate basis of 

comparison for MON 95379. 

x A detailed molecular characterization of the inserted DNA demonstrates a single, intact 

copy of the T-DNA insert in a single locus within the maize genome. 

x Extensive evaluation of the Cry1B.868 and Cry1Da_7 proteins expressed in MON 95379 

confirms they are unlikely to be human and other mammalian toxins or allergens.

x A compositional assessment supports the conclusion that MON 95379 grain and forage is 

compositionally equivalent to grain and forage of conventional maize. 

x An extensive evaluation of MON 95379 phenotypic and agronomic characteristics and 

environmental interactions demonstrates MON 95379 has no increased plant pest risk 

potential compared to conventional maize. 

x A non-target organism (NTO) assessment supports the conclusion that Cry1B.868 and 

Cry1Da_7 proteins are specific for lepidopteran species and MON 95379 maize would not 

be expected to adversely impact NTOs beneficial to agriculture at levels expected to be 

encountered in the field. As MON 95379 maize will not be commercialized in the U.S., a 

threatened and endangered species assessment focused on lepidopteran species in the states 

of Nebraska, Hawaii, and Iowa, where MON 95379 will be cultivated for small-scale 

breeding, testing, and seed increase activities and small-scale, confined field trials

potentially in other locations to collect needed regulatory data to support global regulatory 

submissions of future products stacked with MON 95379. Results of the assessment 

support a conclusion of no effect for currently listed lepidopteran species. 
Bayer CropScience LP CR282-20U1 6

x MON 95379 will not be commercialized in the U.S. and therefore will not impact U.S. 

commercial maize agricultural practices. It will only be cultivated in small-scale breeding, 

testing, and seed increase nurseries in the U.S. with typical production practices to produce 

high quality seed and/or data. Evaluation of the agronomic and phenotypic characteristics 

of MON 95379 support that, with the exception of the introduced trait, MON 95379 maize 

is phenotypically similar to conventional maize and is not expected to have an effect 

on maize agronomic practices under its intended use. 

Maize is a Familiar Crop Lacking Weedy Characteristics 

Maize is grown extensively throughout the world and is the largest cultivated grain crop followed 

by wheat (Triticum sp.) and rice (Oryza sativa L.) in total global production. In the U.S., maize is 

grown in almost all states and is the largest crop grown in terms of net value. Maize has been 

studied extensively, and the initial steps in its domestication can be traced back to approximately 

9,000 years ago in southern Mexico. Although grown extensively throughout the world, maize is 

not considered a threat to invade natural or agricultural ecosystems because it does not establish 

self-sustaining populations outside of cultivation. This lack of weediness may reflect its poor 

competitive ability, lack of seed dormancy, and barriers to seed dispersal, as maize cobs retain 

seed and are covered in a husk. Several other characteristics common in weeds, such as rapid 

flowering following emergence, are lacking in maize. Traits often associated with weediness are 

typically not selected for during domestication and subsequent breeding and selection, and 

similarly, the history of maize breeding and production in the U.S. does not indicate there are any 

changes in the characteristics of maize that would increase the weediness of the crop. Although 

maize seed can overwinter in a rotation with soybeans or other crops, mechanical and chemical 

measures are routinely used to control maize volunteers. Maize is not sexually compatible with 

plant species occurring in the U.S. other than teosinte, an introduced wild relative. However, gene 

introgression from maize into teosinte is unlikely in the U.S. due to barriers to crossing, including 

morphological and developmental differences and limited geographical distribution of teosinte 

populations. 

Conventional Maize LH44 is an Appropriate Comparator to MON 95379 

Conventional control materials developed for use as comparators in safety assessment studies were 

based on the type of study conducted and the genetic background of the test material. The 

conventional control materials included the original transformation line (LH244) and LH244 

crossed to a conventional line (HCL617) to create F1 starting control materials. LH244 was used 

as the control in molecular characterization studies. LH244 × HCL617 was used as the control in 

compositional analysis studies and in phenotypic, agronomic and environmental interactions 

assessments. Where appropriate, commercial hybrid maize materials (reference hybrids) were also 

used to establish a range of variability or responses representative of commercial maize in the U.S. 

Molecular Characterization Verified the Integrity and Stability of the Inserted DNA in 

MON 95379 

MON 95379 was produced by Agrobacterium-mediated transformation of maize tissue using the 

transformation vector PV-ZMIR522223. This vector contains a single T-DNA (transfer DNA), 

that is delineated by Right and Left Border regions. The T-DNA contains the cry1B.868 and
Bayer CropScience LP CR282-20U1 7

cry1Da_7 expression cassettes. The T-DNA that was inserted initially contained a cp4 epsps

selectable marker cassette flanked by two excision targeting sequences called lox sites. After 

MON 95379 was selected as an acceptable transformant, the selectable marker cassette was 

excised by crossing MON 95379 with a Cre recombinase expressing line (the “Cre line” was 

transformed with the vector PV-ZMOO513642). Subsequently, traditional breeding, segregation, 

selection, and screening were used to isolate those plants that contained the cry1B.868 and

cry1Da_7 expression cassettes, and lacked the cp4 epsps selectable marker and any sequences 

from the cre gene containing plasmid, PV-ZMOO513642. 

Characterization of the DNA insert in MON 95379 was conducted using a combination of 

sequencing, polymerase chain reaction (PCR), and bioinformatics. The results of this 

characterization demonstrate that MON 95379 contains one copy of the intended T-DNA 

containing the cry1B.868 and cry1Da_7 expression cassettes that is stably inherited over multiple 

generations and segregates according to Mendelian principles. These conclusions are based on 

several lines of evidence: 

x Molecular characterization of MON 95379 by Next Generation Sequencing (NGS) 

demonstrated that MON 95379 contained a single intended DNA insert. These wholegenome analyses provided a comprehensive assessment of MON 95379 to determine the 

presence and identity of sequences derived from PV-ZMIR522223 and demonstrated that 

MON 95379 contained a single T-DNA insert, no detectable backbone or cp4 epsps

selectable marker sequence from PV-ZMIR522223 or any sequences from 

PV-ZMOO513642. 

x Directed sequencing (locus-specific PCR, DNA sequencing and analyses) performed on 

MON 95379 was used to determine the complete sequence of the single DNA insert from 

PV-ZMIR522223, the adjacent flanking genomic DNA, and the 5' and 3' insert-to-flank 

junctions. This analysis confirmed that the sequence and organization of the DNA is 

identical to the corresponding region in the PV-ZMIR522223 T-DNA and lacks the cp4 

epsps selectable marker. 

x Furthermore, the genomic organization at the insertion site was assessed by comparing the 

sequences flanking the T-DNA insert in MON 95379 to the sequence of the insertion site 

in conventional maize. This analysis determined there was a 160 bp deletion upon 

T-DNA integration in MON 95379. 

x Generational stability analysis by NGS demonstrated that the single PV-ZMIR522223 

T-DNA insert in MON 95379 has been maintained through five breeding generations, 

thereby confirming the stability of the T-DNA in MON 95379. 

x Segregation analysis corroborates the insert stability demonstrated by NGS and 

independently establishes the nature of the T-DNA as a single chromosomal locus that 

shows an expected pattern of inheritance. 

Taken together, the characterization of the genetic modification in MON 95379 demonstrates that 

a single copy of the intended T-DNA was stably integrated at a single locus of the maize genome 
Bayer CropScience LP CR282-20U1 8

and that no PV-ZMIR522223 plasmid backbone, selectable marker, or PV-ZMOO513642 

sequences are present in MON 95379. 

Data Confirms Cry1B.868 and Cry1Da_7 Protein Safety 

A multistep approach was used to characterize and assess the safety of the MON 95379 Cry1B.868 

and Cry1Da_7 proteins. The expression level of the Cry1B.868 and Cry1Da_7 proteins in selected 

tissues of MON 95379 was determined and exposure to humans and other mammals through diet 

was evaluated. In addition, the donor organism for the cry1B.868 and cry1Da_7

coding sequences, Bacillus thuringiensis, is ubiquitous in the environment and is not commonly 

known for human or mammalian pathogenicity or allergenicity. Bioinformatics analysis 

determined that the Cry1B.868 and Cry1Da_7 proteins lack structural similarity to known 

allergens, gliadins, glutenins, or protein toxins which could have adverse effect to human or 

mammalian health. The Cry1B.868 and Cry1Da_7 proteins are rapidly digested in simulated 

digestive fluids and demonstrate no acute oral toxicity in mice at the levels tested that far exceed 

anticipated exposure by humans and mammals. Hence, the consumption of the Cry1B.868 and 

Cry1Da_7 proteins from MON 95379 or its progeny poses no risk to human and mammalian 

health. 

MON 95379 is Compositionally Equivalent to Conventional Maize 

Safety assessments of biotechnology-derived crops include a comparative safety assessment in 

which the composition of grain and/or other raw agricultural commodities of the biotechnologyderived crop are compared to the appropriate conventional control that has a history of safe 

use. Maize is not known to have any endogenous toxicants or anti-nutrients associated with overall 

plant pest potential. 

Compositional analysis was conducted on grain and forage of MON 95379 grown at five sites 

representative of typical agricultural regions for maize production in the U.S. in 2018. The 

compositional analysis provided a comprehensive comparative assessment of the levels of key 

nutrients and anti-nutrients in grain and forage of MON 95379 and the conventional control and 

followed considerations relevant to the compositional quality of maize as defined by the OECD 

consensus document (OECD, 2002a). Grain and forage samples were analyzed for levels of key 

nutrients including proximates, carbohydrates by calculation, fiber, amino acids, and linoleic acid. 

In addition, grain samples were analyzed for anti-nutrient levels of phytic acid and raffinose. Key 

nutrients in forage samples were analyzed for levels of protein, total fat, ash, carbohydrates by 

calculation, and fiber. In total, 25 different components were assayed (6 in forage and 19 in grain). 

The results of the compositional assessment found that there were no compositional differences 

that were biologically meaningful between MON 95379 and conventional control and support the 

conclusion that MON 95379 maize is compositionally equivalent to the conventional control. 

These results support the overall food and feed safety of MON 95379. 

 
Bayer CropScience LP CR282-20U1 9

MON 95379 Will Not Negatively Affect NTOs Including Those Beneficial to Agriculture 

An evaluation of the impacts of MON 95379 on non-target organisms (NTOs) beneficial to 

agriculture is a component of a comprehensive environmental assessment. MON 95379 maize will 

not be commercialized in the U.S. and small-scale breeding, testing, and seed increase activities 

would be limited to a maximum total of 100 acres per growing season across three states 

(Nebraska, Hawaii and Iowa) as proposed in an EPA seed increase registration application, and 

small-scale, confined field trials potentially in other locations to collect needed regulatory data to 

support global regulatory submissions of future products stacked with MON 95379. Therefore, 

exposure of populations of non-target organisms will be low due to limited acreage and geography. 

Prior to conducting a risk assessment for MON 95379 maize to beneficial NTOs, the proposed 

areas for cultivation of MON 95379 maize, the mode-of-action (MOA) and the spectrum of 

insecticidal activity of Cry1B.868 and Cry1Da_7 proteins, as well as expression analyses of the 

Cry1B.868 and Cry1Da_7 proteins produced by MON 95379 tissues were evaluated. The results 

of the activity spectrum studies indicate that, similar to previously commercialized Cry1 proteins, 

activity of Cry1B.868 and Cry1Da_7 proteins is limited to lepidopteran insects and effects are not 

expected on NTOs beneficial to agriculture. An evaluation of potential off-crop exposure to 

MON 95379 pollen indicated that significant exposure to non-target lepidopteran species is not 

anticipated and therefore MON 95379 poses minimal risk to non-target Lepidoptera. Results from 

Tier I NTO testing demonstrated no effects on survival from exposure to the Cry1B.868 and 

Cry1Da_7 proteins at levels well above those found in MON 95379 maize and in the environment. 

Calculated margins of exposure (MOE) based on LC50 values and conservative environmental 

concentrations were !11 for all species tested with the exception of C. maculata where the MOE 

for Cry1Da_7 was !7, indicating low risk to NTOs at field expression levels. A threatened and 

endangered species assessment focused on listed Lepidoptera in the states of Nebraska, Hawaii, 

and Iowa, where MON 95379 will primarily be cultivated, resulted in the conclusion of no effect 

for currently listed lepidopteran species. 

MON 95379 Does Not Change Maize Plant Pest Potential or Environmental Interactions 

Plant pest potential of a biotechnology-derived crop is assessed from the basis of familiarity that 

the USDA recognizes as an important underlying concept in risk assessment. The concept of 

familiarity is based on the fact that the biotechnology-derived plant is developed from a 

conventional plant hybrid or variety whose biological properties and plant pest potential are well 

known. Familiarity considers the biology of the plant, the introduced trait, the receiving 

environment, and the interactions among these factors. This provides a basis for the comparative 

risk assessment between a biotechnology-derived plant and the conventional control. Thus, the 

phenotypic, agronomic, and environmental interaction assessment of MON 95379 included the 

genetically similar conventional control as a comparator. This evaluation used a weight of 

evidence approach and considered statistical differences between MON 95379 and the 

conventional control with respect to reproducibility, magnitude, and directionality. Comparison to 

a range of commercial references grown concurrently established the range of natural variability 

for maize, and provided a context from which to further evaluate any statistical differences. 

Assessments included laboratory evaluation of seed germination and dormancy characteristics, as 

well as phenotypic and agronomic characteristics and plant responses to abiotic stressors, diseases, 

and arthropod pests in the field. Results from the germination, phenotypic, agronomic, and 
Bayer CropScience LP CR282-20U1 10

environmental interactions assessments indicated that MON 95379 does not possess enhanced 

weediness characteristics, increased susceptibility or tolerance to specific abiotic stressors, 

diseases, non-target arthropod pests, or characteristics that would confer an increased plant pest 

risk compared to conventional maize. 

Deregulation of MON 95379 is Not Expected to Have Effects on Maize Agronomic 

Practices 

MON 95379 was developed to provide growers in South America an additional, effective tool for 

controlling target lepidopteran pests, including those resistant to current Bt technologies. As 

MON 95379 will not be commercialized in the U.S, no impact on agronomic or cultivation 

practices used in commercial maize production are expected. 

While MON 95379 (and possible stack products containing MON 95379) may be grown on small 

acreage breeding/seed increase nurseries and agronomic/regulatory testing trials in limited 

geographies, subject to an EPA seed increase registration and applicable BRS permits, the 

cultivation of MON 95379 is not expected to significantly change current maize agronomic 

practices under the intended use. This conclusion is supported by the assessment of phenotypic, 

agronomic, and environmental interactions of MON 95379 that included the assessment of seed 

germination and dormancy characteristics, as well as phenotypic and agronomic characteristics 

and plant responses to abiotic stressors, diseases, and arthropod pests in the field under a range of 

environmental conditions. Results demonstrated that MON 95379 is similar to conventional maize 

in its agronomic and phenotypic characteristics and susceptibility or tolerance to specific abiotic 

stressors, diseases, or non-lepidopteran arthropod pests. Therefore, no changes in agronomic 

practices are anticipated for the cultivation of MON 95379 compared to conventional maize, 

particularly those that can significantly impact plant diseases or pests or their management. 

Furthermore, maize breeding and seed production nurseries are typically closely managed 

following best practices to control weed and insect pests to produce high quality data and seed. 

Based on this assessment, the intended use of MON 95379 is not expected to result in changes or 

impacts to current maize agronomic practices. 

Conclusion 

Based on the data and information presented in this petition, it is concluded that MON 95379 maize 

is unlikely to pose a greater plant pest risk than conventional maize. Therefore, in acknowledgment 

of the limitations that the EPA sets on the seed increase registration for MON 95379, Bayer 

requests a determination from USDA-APHIS that MON 95379 and any progeny derived from 

crosses between MON 95379 and conventional maize or deregulated biotechnology-derived maize 

be granted nonregulated status under 7 CFR Part 340. 
Bayer CropScience LP CR282-20U1 11 

TABLE OF CONTENTS 

I. RATIONALE FOR THE DEVELOPMENT OF MON 95379......................................35

I.A. Basis for the Request for a Determination of Nonregulated Status under 

7 CFR § 340.6 ...............................................................................................................35

I.B. Rationale for the Development of Lepidopteran-Protected Maize MON 95379 ...35

I.C. Submissions to Other Regulatory Agencies ..........................................................36

I.C.1. Submission to U.S. EPA ..............................................................................36

I.C.2. Submission to U.S. FDA .............................................................................37

I.C.3. Submissions to Foreign Government Agencies ..........................................37

II. THE BIOLOGY OF MAIZE ........................................................................................38

II.A. Maize as a Crop ....................................................................................................38

II.B. Characteristics of the Recipient Plant ...................................................................39

II.C. Maize as a Test System in Product Safety Assessment ........................................40

III. DESCRIPTION OF THE GENETIC MODIFICATION............................................41

III.A. Description of Transformation Plasmid PV-ZMIR522223 ................................41

III.A.1. Marker Removal Through Cre/lox Recombination System .....................42

III.B. Description of the Transformation System .........................................................43

III.C. The cry1B.868 Coding Sequence and Cry1B.868 Protein .................................50

III.D. The cry1Da_7 Coding Sequence and Cry1Da_7 Protein ...................................52

III.E. Regulatory Sequences .........................................................................................54

III.F. T-DNA Border Regions ......................................................................................54

III.G. Genetic Elements Outside the T-DNA Border Regions .....................................55

IV. CHARACTERIZATION OF THE GENETIC MODIFICATION .............................56

IV.A. Description of Methodology Used to Characterize MON 95379 .......................56

IV.B. Characterization of the DNA Inserts in MON 95379 .........................................60

IV.B.1. Next Generation Sequencing for MON 95379 and Conventional Control 

Genomic DNA .......................................................................................................60

IV.B.2. Selection of Sequence Reads Containing Sequence of the PVZMIR522223 and PV-ZMOO513642....................................................................65

IV.B.3. Determination of T-DNA Copy Number and Presence or Absence of 

Plasmid Vector Backbone ......................................................................................65
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IV.B.4. Determination of Absence of Plasmid Vector PV-ZMOO513642 ...........69

IV.C. Organization and Sequence of the Insert and Adjacent DNA in MON 95379 ..72

IV.D. Sequencing of the MON 95379 Insertion Site ....................................................74

IV.E. Determination of Insert Stability over Multiple Generations of MON 95379 ...76

IV.F. Inheritance of the Genetic Insert in MON 95379 ................................................77

IV.G. Characterization of the Genetic Modification Summary and Conclusion ..........81

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE Cry1B.868 AND 

Cry1Da_7 PROTEINS PRODUCED IN MON 95379 ......................................................82

V.A. Identity and Function of the Cry1B.868 and Cry1Da_7 Proteins from MON 

95379 .............................................................................................................................82

V.B. Characterization and Equivalence of Cry1B.868 and Cry1Da_7 Proteins from 

MON 95379 ...................................................................................................................84

V.C. Cry1B.868 and Cry1Da_7 Donor Organism and History of Safe Use .................85

V.C.1. The Donor Organism has a History of Safe Use ........................................85

V.C.2. Cry Proteins have a History of Safe Use ....................................................86

V.C.3. Cry1B.868 Protein has a History of Safe Use ............................................87

V.C.4. Cry1Da_7 Protein has a History of Safe Use .............................................88

V.D. Cry1B.868 and Cry1Da_7 Proteins in MON 95379 are Not Homologous to 

Known Allergens or Toxins ..........................................................................................89

V.E. Cry1B.868 and Cry1Da_7 Proteins in MON 95379 are Susceptible to 

Degradation in in vitro Digestion Assays ......................................................................90

V.F. Expression Levels of Cry1B.868 and Cry1Da_7 Proteins in MON 95379 ..........90

V.F.1. Expression Levels of Cry1B.868 Protein ...................................................91

V.F.2. Expression Levels of Cry1Da_7 Protein ....................................................93

V.G. Cry1B.868 and Cry1Da_7 Proteins are Not Acutely Toxic .................................94

V.H. Assessment of Potential Allergenicity of the Cry1B.868 and Cry1Da_7 Proteins94

V.I. Mammalian Exposure to the Cry1B.868 and Cry1Da_7 Proteins in MON 9537995

V.J. Cry1B.868 and Cry1Da_7 Proteins Characterization and Safety Conclusion ......95

VI. COMPOSITIONAL ASSESSMENT OF MON 95379 ..............................................96

VI.A. Compositional Equivalence of MON 95379 Grain and Forage to Conventional 

Maize .............................................................................................................................97

VI.B. Compositional Assessment of MON 95379 Conclusion.....................................98
Bayer CropScience LP CR282-20U1 13

VII. ASSESSMENT OF POTENTIAL IMPACTS ON NON-TARGET ORGANISMS 

BENEFICIAL TO AGRICULTURE AND THREATENED AND ENDANGERED 

SPECIES ..........................................................................................................................106

VII.A. Mode of Action and Spectrum of Insecticidal Activity ...................................106

VII.B. Exposure Assessment ......................................................................................110

VII.B.1. Expression of Cry1B.868 and Cry1Da_7 in MON 95379 Tissues .......111

VII.B.2. Estimated Environmental Concentrations of Cry1B.868 and Cry1Da_7112

VII.C. Hazard Assessment ..........................................................................................126

VII.C.1. Effects of Cry1B.868 on NTOs.............................................................128

VII.C.2. Effects of Cry1Da_7 on NTOs..............................................................133

VII.D. Effect of MON 95379 Maize on Threatened and Endangered Species ...........137

VII.E. Conclusion .......................................................................................................140

VIII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL INTERACTIONS 

ASSESSMENT ................................................................................................................141

VIII.A. Characteristics Measured for Assessment .....................................................141

VIII.B. Interpretation of Phenotypic and Environmental Interaction Data ................144

VIII.B.1. Interpretation of Phenotypic and Agronomic Data ..............................144

VIII.B.2. Interpretation of Environmental Interactions Data ..............................146

VIII.C. Comparative Assessments of the Phenotypic, Agronomic, and Environmental 

Interaction Characteristics of MON 95379 .................................................................146

VIII.C.1. Seed Dormancy and Germination Characteristics ...............................146

VIII.C.2. Field Phenotypic, Agronomic, and Environmental Interactions 

Characteristics ......................................................................................................147

VIII.C.2.1. Field Phenotypic and Agronomic Characteristics ...................................................... 148 

VIII.C.2.2. Environmental Interaction Assessments ................................................................... 149 

VIII.D. Conclusions for Phenotypic, Agronomic, and Environmental Interactions 

Evaluation ....................................................................................................................150

IX. U.S. AGRONOMIC PRACTICES ............................................................................152

IX.A. Introduction .......................................................................................................152

IX.B. Maize Breeding and Seed Production ...............................................................152

IX.B.1. Management of Insect Pests ...................................................................152

IX.B.2. Management of Diseases and Other Pests ..............................................153
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IX.B.3. Weed Management .................................................................................153

IX.B.4. Crop Rotation and Volunteer Management Practices in Maize .............153

IX.B.5. Stewardship of MON 95379 Maize ........................................................154

IX.C. Summary and Conclusion: Impact of the Introduction of MON 95379 Maize on 

Agricultural Practices ..................................................................................................154

X. PLANT PEST ASSESSMENT ...................................................................................155

X.A. Introduction ........................................................................................................155

X.B. Plant Pest Assessment of MON 95379 and Expressed Cry1B.868 and Cry1Da_7 

Proteins ........................................................................................................................155

X.B.1. Characteristics of the Genetic Insert and Expressed Protein ....................156

X.B.1.1. Genetic Insert ................................................................................................................. 156 

X.B.1.2. Mode-of-Action .............................................................................................................. 156 

X.B.2. Protein Safety and Expression Levels ......................................................156

X.B.3. Compositional Characteristics ..................................................................157

X.B.4. Assessment of Potential Impacts on Non-Target Organisms Beneficial to 

Agriculture and Threatened and Endangered Species .........................................158

X.B.5. Phenotypic, Agronomic, and Environmental Interaction Characteristics 158

X.B.5.1. Seed Dormancy and Germination .................................................................................. 159 

X.B.5.2. Phenotypic and Agronomic Assessment ........................................................................ 159 

X.B.5.3. Response to Abiotic Stressors, Diseases, and Arthropod Pests ..................................... 159 

X.C. Weediness Potential of MON 95379 ..................................................................159

X.D. Potential for Pollen Mediated Gene Flow and Introgression .............................160

X.D.1. Hybridization with Cultivated Maize .......................................................160

X.D.2. Hybridization with Teosinte .....................................................................163

X.D.3. Hybridization with Tripsacum .................................................................164

X.E. Transfer of Genetic Information to Species with which Maize Cannot Interbreed 

(Horizontal Gene Flow) ...............................................................................................164

X.F. Potential Impact on Maize Agronomic Practices ................................................165

X.G. Conventional Breeding with Other Biotechnology-derived or Conventional 

Maize ...........................................................................................................................165

X.H. Summary of Plant Pest Assessments ..................................................................166

XI. ADVERSE CONSEQUENCES OF INTRODUCTION ...........................................168
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Appendix A : USDA Notifications and Permits ..............................................................189

Appendix B : Overview, Materials, Methods and Supplementary Results for Molecular 

Analyses of MON 95379 .................................................................................................193

B.1. Test Substance .....................................................................................................193

B.2. Control Substances ..............................................................................................193

B.3. Reference Substance ............................................................................................193

B.4. Characterization of Test, Control, and Reference Substances .............................194

B.5. Genomic DNA Isolation ......................................................................................194

B.6. DNA Quantification .............................................................................................194

B.7. Paired End Library Preparation ...........................................................................194

B.8. Next-Generation Sequencing ...............................................................................195

B.9. Read Mapping and Junction Identification ..........................................................195

B.9.1. Mapping and Junction Detection ......................................................................195

B.9.2. Effective Sequencing Depth Determination .....................................................195

B.9.3. Summary NGS Data for the Control and Test Substances ...............................196

B.9.4. Positive Control ................................................................................................196

B.10. PCR and DNA Sequence Analyses to Examine the Insert and Flanking 

Sequences in MON 95379 ...........................................................................................196

B.11. PCR and DNA Sequence Analyses to Examine the Integrity of the DNA 

Insertion Site in MON 95379 ......................................................................................197

B.12. Protocol Amendments and Deviations ..............................................................197

B.13. Rejected Data .....................................................................................................198

Appendix C : Characterization of Cry1B.868 Protein Produced in MON 95379 ...........200

C.1. Characterization of the Cry1B.868 Protein .........................................................200

C.1.1. Cry1B.868 Protein Identity and Equivalence ...................................................200

C.1.2. Results of the N-Terminal Sequencing Analysis ..............................................202

C.1.3. Results of Mass Fingerprint Analysis ...............................................................203

C.1.4. Results of Western Blot Analysis of the Cry1B.868 Protein Isolated from the 

Grain of MON 95379 and Immunoreactivity Comparison to Bt-produced Cry1B.868 

Protein .........................................................................................................................213

C.1.5. Results of the Cry1B.868 Protein Molecular Weights and Purity Analysis .....215

C.1.6. Cry1B.868 Glycosylation Equivalence ............................................................217
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C.1.7. Cry1B.868 Functional Activity ........................................................................219

C.1.8. Cry1B.868 Protein Identity and Equivalence Conclusion ................................219

C.2. Materials and Methods for Characterization of Cry1B.868 Protein Produced in 

MON 95379 .................................................................................................................220

C.2.1. Materials ...........................................................................................................220

C.2.2. Protein Purification ...........................................................................................220

C.2.3. Methods for Characterization ...........................................................................222

C.2.3.1. N-Terminal Sequencing/Mass Fingerprint Analysis .....................................222

C.2.3.2. Western Blot Analysis-Immunoreactivity .....................................................223

C.2.3.3. Apparent Molecular Weight and Purity Determination by SDS-PAGE .......223

C.2.3.4. Glycosylation Analysis ..................................................................................224

C.2.3.5. Functional Activity ........................................................................................224

C.2.3.6. Prediction Intervals as Acceptance Criteria ..................................................225

Appendix D : Characterization of Cry1Da_7 Protein Produced in MON 95379 ...........229

D.1. Characterization of the Cry1Da_7 Protein ..........................................................229

D.1.1. Cry1Da_7 Protein Identity and Equivalence ....................................................229

D.1.2. Results of the N-Terminal Sequencing Analysis ..............................................231

D.1.3. Results of Mass Fingerprint Analysis...............................................................231

D.1.4. Results of Western Blot Analysis of the Cry1Da_7 Protein Isolated from the 

Grain of MON 95379 and Immunoreactivity Comparison to Bt-produced Cry1Da_7 

Protein .........................................................................................................................243

D.1.5. Results of the Cry1Da_7 Protein Molecular Weights and Purity Analysis ......245

D.1.6. Cry1Da_7 Glycosylation Equivalence .............................................................247

D.1.7. Cry1Da_7 Functional Activity .........................................................................249

D.1.8. Cry1Da_7 Protein Identity and Equivalence Conclusion .................................250

D.2. Materials and Methods for Characterization of Cry1Da_7 Protein Produced in 

MON 95379 .................................................................................................................250

D.2.1. Materials ...........................................................................................................250

D.2.2. Protein Purification ...........................................................................................251

D.2.3. Methods for Characterization ...........................................................................252

D.2.3.1. N-Terminal Sequencing/Mass Fingerprint Analysis .....................................252

D.2.3.2. Western Blot Analysis-Immunoreactivity .....................................................253
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D.2.3.3. Apparent Molecular Weight and Purity Determination by SDS-PAGE .......254

D.2.3.4. Glycosylation Analysis ..................................................................................254

D.2.3.5. Functional Activity ........................................................................................255

D.2.3.6. Prediction Intervals as Acceptance Criteria ..................................................256

Appendix E : Materials and Methods Used for the Analysis of the Levels of Cry1B.868 

and Cry1Da_7 Proteins Expressed in MON 95379 .........................................................260

E.1. Materials ..............................................................................................................260

E.2. Characterization of the Materials .........................................................................260

E.3. Field Design and Tissue Collection .....................................................................260

E.4. Tissue Processing and Protein Extraction ............................................................260

E.5. ELISA Reagents and Methods .............................................................................263

E.5.1. Cry1B.868 Antibodies ......................................................................................263

E.5.2. Cry1Da_7 Antibodies .......................................................................................263

E.5.3. Cry1B.868 ELISA Method ...............................................................................263

E.5.4. Cry1Da_7 ELISA Method ................................................................................264

E.6. Data Analyses ......................................................................................................264

Appendix F : Digestive Fate of the Cry1B.868 and Cry1Da_7 Proteins .........................266

F.1. Digestive Fate of the Cry1B.868 and Cry1Da_7 Proteins ...................................266

F.1.1. Degradation of Cry1B.868 and Cry1Da_7 Proteins in the Presence of Pepsin 266

F.1.1.1. Degradation of Cry1B.868 Protein in the Presence of Pepsin .......................267

F.1.1.2. Degradation of Cry1Da_7 Protein in the Presence of Pepsin ........................271

F.1.2. Degradation of Cry1B.868 and Cry1Da_7 Proteins in the Presence of Pancreatin275

F.1.2.1. Degradation of Cry1B.868 Protein in the Presence of Pancreatin .................275

F.1.2.2. Degradation of Cry1Da_7 Protein in the Presence of Pancreatin ..................277

F.1.3. Degradation of Cry1B.868 and Cry1Da_7 Proteins by Pepsin Followed by 

Pancreatin ....................................................................................................................279

F.1.3.1. Degradation of Cry1B.868 Protein by Pepsin Followed by Pancreatin.........279

F.1.3.2. Degradation of Cry1Da_7 Protein by Pepsin Followed by Pancreatin..........281

F.1.4. Degradation of Cry1B.868 and Cry1Da_7 Proteins Conclusion ......................283

F.1.4.1. Degradation of Cry1B.868 Protein Conclusion .............................................283

F.1.4.2. Degradation of Cry1Da_7 Protein Conclusion ..............................................283
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F.2. Materials and Methods Used in Assessing Stability of Cry1B.868 and Cry1Da_7 

Proteins Proteins in Pepsin and Pancreatin .................................................................284

F.2.1. Test Substances .................................................................................................284

F.2.1.1. Cry1B.868 Test Substance .............................................................................284

F.2.1.2. Cry1Da_7 Test Substance ..............................................................................284

F.2.2. Characterization of Test Substance ...................................................................284

F.2.2.1. Characterization of the Cry1B.868 Test Substance .......................................284

F.2.2.2. Characterization of the Cry1Da_7 Test Substance ........................................284

F.3. Experimental Design ............................................................................................285

F.3.1. Preparation of the Pepsin Stock Solution ..........................................................285

F.3.2. Degradation of the Cry1B.868 and Cry1Da_7 Proteins by Pepsin ...................285

F.3.2.1. Degradation of the Cry1B.868 Protein by Pepsin ..........................................285

F.3.2.2. Degradation of the Cry1Da_7 Protein by Pepsin ...........................................286

F.3.2.3. Pepsin Degradation Experimental Controls ...................................................287

F.3.3. Preparation of the Pancreatin Stock Solution ....................................................287

F.3.3.1. Degradation of the Cry1B.868 and Cry1Da_7 Proteins by Pancreatin .........287

F.3.3.1.1. .............................................................. Degradation of the Cry1B.868 Protein by 

Pancreatin ....................................................................................................................287

F.3.3.1.2. ............................................................... Degradation of the Cry1Da_7 Protein by 

Pancreatin ....................................................................................................................288

F.3.3.2. Pancreatin Degradation Experimental Controls .............................................290

F.3.4. Degradation of the Cry1B.868 and Cry1Da_7 Proteins by Pepsin Followed by 

Pancreatin ....................................................................................................................290

F.3.4.1. Degradation of the Cry1B.868 Protein by Pepsin Followed by Pancreatin ...290

F.3.4.2. Degradation of the Cry1Da_7 Protein by Pepsin Followed by Pancreatin ....292

F.3.4.3. Pepsin Followed by Pancreatin Degradation Experimental Controls ............293

F.4. Analytical Methods ..............................................................................................293

F.4.1. Pepsin Activity Assay .......................................................................................293

F.4.2. Pancreatin Activity Assay .................................................................................294

F.4.3. SDS-PAGE and Colloidal Brilliant Blue G Staining ........................................294

F.4.4. Western Blot Analysis .......................................................................................295

F.4.4.1. Western Blot Analysis of Cry1B.868 Protein ................................................295
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F.4.4.2. Western Blot Analysis of Cry1Da_7 Protein .................................................296

Appendix G : Materials and Methods for Seed Germination and Dormancy Assessment 

of MON 95379 .................................................................................................................299

G.1. Materials ..............................................................................................................299

G.2. Characterization of the Materials ........................................................................299

G.3. Germination Testing Facility and Experimental Methods ..................................299

G.4. Statistical Analysis ..............................................................................................300

Appendix H : Material and Methods for Phenotypic, Agronomic, and Environmental 

Interaction Assessment of MON 95379 under Field Conditions .....................................302

H.1. Materials ..............................................................................................................302

H.2. Characterization of the Materials ........................................................................302

H.3. Field Sites and Plot Design ..................................................................................302

H.4. Planting and Field Operations .............................................................................302

H.5. Phenotypic Characteristic Assessments ..............................................................305

H.6. Environmental Interaction Assessments ..............................................................305

H.7. Statistical Analysis/Data Summarization ............................................................305

H.8. Detailed Results for Environmental Interactions Assessments for MON 95379 306

Appendix I : Materials and Methods for Compositional Analysis of MON 95379 Maize 

Grain and Forage..............................................................................................................311

I.1. Materials ..............................................................................................................311

I.2. Characterization of the Materials .....................................................................311

I.3. Field Production of the Samples ........................................................................311

I.4. Summary of Analytical Methods ..........................................................................311

I.5. Acid Detergent Fiber ..........................................................................................312

I.6. Amino Acids ........................................................................................................312

I.7. Ash ........................................................................................................................313

I.8. Carbohydrate ......................................................................................................313

I.9. Fat by Acid Hydrolysis .......................................................................................313

I.10. Fat by Soxhlet Extraction .................................................................................313

I.11. Linoleic Acid .......................................................................................................314

I.12. Moisture ..............................................................................................................314

I.13. Neutral Detergent Fiber ......................................................................................314
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I.14. Phytic Acid ..........................................................................................................314

I.15. Protein .................................................................................................................315

I.16. Raffinose .............................................................................................................315

I.17. Data Processing and Statistical Analysis ............................................................315

Appendix J : Materials, Methods, and Results for Tier I NTO Testing...........................318

J.1. Evaluation of the Potential for Interaction between the Cry1Da_7 and Cry1B.868 

Proteins Expressed in MON 95379 with Fall Armyworm (Spodoptera frugiperda) ..318

J.1.1. Protein Expression Analysis and Tissue Preparation ........................................321

J.1.2. FAW Bioassay Testing Procedure .....................................................................322

J.1.3. Data Analysis and Interpretation .......................................................................325

J.1.4. Hazard Index Assessment for CryB.868 and Cry1Da_7 ...................................327

J.2. Methods for Aerobic Soil Degradation of Cry1B.868 and Cry1Da_7 Protein from 

MON 95379 Maize ......................................................................................................329

J.3. Evaluation of the potential effects of Cry1B.868 Protein on Larvae of the Ladybird 

Beetle, Coleomegilla maculata ...................................................................................331

J.4. Evaluation of the Potential Effects of Cry1B.868 Protein on Larvae of the Carabid 

Beetle, Poecilus cupreus .............................................................................................333

J.5. Evaluation of the Potential Effects of Cry1B.868 Protein on Nymphs of the BigEyed Bug, Geocoris punctipes ....................................................................................336

J.6. Evaluation of the Potential Effects of Cry1B.868 Protein on Adults of the 

Parasitoid Wasp, Pediobius foveolatus .......................................................................339

J.7. Evaluation of the Potential Effects of Cry1B.868 Protein on Adults of the Green 

Lacewing, Chrysoperla carnea ...................................................................................341

J.8. Evaluation of the potential effects of Cry1B.868 Protein on Larvae of the Green 

Lacewing, Chrysoperla rufilabris ...............................................................................343

J.9. Evaluation of the Potential Effects of Cry1B.868 Protein on Adults of the Green 

Lacewing, Chrysoperla rufilabris ...............................................................................346

J.10. Evaluation of the Potential Effects of Cry1B.868 Protein on the Springtail, 

Folsomia candida ........................................................................................................348

J.11. Evaluation of the Potential Effects of Cry1B.868 Protein on the Earthworm, 

Eisenia andrei ..............................................................................................................351

J.12. Evaluation of the Potential Effects of Cry1B.868 Protein on Adults of the Honey 

Bee, Apis mellifera ......................................................................................................354
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J.13. Evaluation of the Potential Effects of Cry1B.868 Protein on Larvae of the Honey 

Bee, Apis mellifera ......................................................................................................356

J.14. Evaluation of the Potential Effects of Cry1B.868 Protein on the Northern 

Bobwhite Quail, Colinus virginianus ..........................................................................358

J.15. Evaluation of the Potential effects of Cry1B.868 Protein on the Mouse, Mus 

musculus ......................................................................................................................360

J.16. Evaluation of the Potential Effects of Cry1Da_7 Protein on Larvae of the 

Ladybird Beetle, Coleomegilla maculata ....................................................................362

J.17. Evaluation of the Potential Effects of Cry1Da_7 Protein on Larvae of the Carabid 

Beetle, Poecilus cupreus .............................................................................................365

J.18. Evaluation of the Potential effects of Cry1Da_7 Protein on Nymphs of the BigEyed Bug, Geocoris punctipes ....................................................................................368

J.19. Evaluation of the Potential Effects of Cry1Da_7 Protein on Adults of the 

Parasitoid Wasp, Pediobius foveolatus .......................................................................370

J.20. Evaluation of the Potential Effects of Cry1Da_7 Protein on Adults of the Green 

Lacewing, Chrysoperla carnea ...................................................................................372

J.21. Evaluation of the Potential Effects of Cry1Da_7 Protein on Larvae of the Green 

Lacewing, Chrysoperla rufilabris ...............................................................................374

J.22. Evaluation of the Potential Effects of Cry1Da_7 Protein on the Springtail, 

Folsomia candida ........................................................................................................377

J.23. Evaluation of the Potential effects of Cry1Da_7 Protein on the Earthworm, 

Eisenia andrei ..............................................................................................................380

J.24. Evaluation of the Potential Effects of Cry1Da_7 Protein on Adults of the Honey 

Bee, Apis mellifera ......................................................................................................383

J.25. Evaluation of the Potential Effects of Cry1Da_7 Protein on Larvae of the Honey 

Bee, Apis mellifera ......................................................................................................385

J.26. Evaluation of the Potential Effects of Cry1Da_7 Protein on the Northern 

Bobwhite Quail, Colinus virginianus ..........................................................................387

J.27. Evaluation of the Potential Effects of Cry1Da_7 Protein on the Mouse, Mus 

musculus ......................................................................................................................390

Appendix K : Read Mappings and Associated Metrics for all Test, Control, and 

Reference Substances in the Molecular Characterization of MON 95379 .....................395

Appendix L : Supplemental Information ..................................................................418

L.1. Additional Information Regarding Total Quality and Quality of Sequence 

Data Generated for the Characterization of the Genetic Modification (Section IV)418
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L.2. Additional Information Regarding Generations Analyzed and Junction Site 

Consistency in the Characterization of the DNA Inserts in MON 95379 (Section IV.B)418

L.3. Additional Information Regarding Consistency of Read Mapping Methodology 

used in the Characterization of the DNA Inserts in MON 95379 (Section IV.B) .......419

L.4. Additional Information Regarding the Endogenous Maize Zm.Ubq Element in the 

Characterization of the DNA Inserts in MON 9539 (Section IV.B) ...........................419

L.5. Additional Information Regarding Flank Capture and Coverage in the 

Characterization of the DNA Inserts in MON 95379 (Section IV.B) .........................426

L.6. Additional Information Regarding Sequencing Utilized in the Mapping Process 

for the Conformation of the Absence of Cre Recombinase (Section IV.B.4) .............427

L.6.1. Clarification of Whole Genome Sequence Analysis (Appendix B) .................427

L.6.2. Clarification on B.13 Rejected Data .................................................................427

L.7. Additional Information Regarding Binding Domains Located in the Sequences of 

Cry1B.868 and Cry1Da_7 (Appendix C.1.3 and D.1.3) .............................................428

L.8. Additional Clarification Regarding the Least-Squares Means and Standard Errors 

(SE) Presented in Table VI-1 of the Compositional Assessment of MON 95379 ......428

L.9. Additional Information Regarding Insect Activity Spectrum Testing for 

Cry1B.868 and Cry1Da_7 (Section VII) .....................................................................429

L.10. Additional Information Regarding Potential Exposure to Cry1B.868 and 

Cry1Da_7 Through Prey (Section VII.B.2) ................................................................430

L.11. Additional Information Regarding Environmental Fate of Cry1B.868 and 

Cry1Da_7 in Soil (Section VII.B.2) ............................................................................431

L.12. Further Discussion of the Cry1Da_7 Results from the Soil Dissipation Study 

(Section VII.B.2) .........................................................................................................433

L.13. Additional Information on Potential Exposure of Cry1B.868 and Cry1Da_7 to 

Collembola (Section VII.B.2) .....................................................................................434

L.14. Additional Information Regarding Reported Endpoints from Tier I Lacewing 

Tests with Cry1B.868 (Section VII.C.1) .....................................................................435

L.15. Additional Information Regarding Assay Validity Criteria for the Larval 

Lacewing Studies (Appendix J) ..................................................................................435

L.16. Additional Information Regarding Sublethal Endpoints from Earthworm Studies 

with Cry1B.868 and Cry1Da_7 (Appendix J) .............................................................435

L.17. Additional Information Regarding Environmental Interactions and the Criteria 

for ‘No Overlap’ Between Stressor Ratings (Appendix H) ........................................436
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L.18. Additional Information Regarding Plot Size and Measured Stressors for the 

Environmental Interaction Assessment (Appendix H) ................................................437

L.19. Additional Information Regarding Agrochemical Use and Relative Timing of 

Field Observations of Arthropod and Disease Stressors for Environmental Interaction 

Assessment (Appendix H) ...........................................................................................438

L.20. Additional Information Regarding Trial Management and Assessment of Abiotic 

Stressors for Environmental Interaction Assessment (Appendix H) ...........................442

L.21. Additional Information Regarding the Threatened and Endangered Species 

Assessment for MON 95379 Maize (VII.B.2 and VII.D) ...........................................447

L.21.1. Background ...................................................................................................447

L.21.2. Listed Lepidoptera Will Have Minimal Exposure to MON 95379 Corn 

Pollen ..........................................................................................................................450

L.21.3. Standard Agricultural Practices to Generate High Quality Seed are 

Protective of Listed Lepidoptera ..............................................................................453

L.21.4. Species-Specific Information to Support the Threatened and Endangered 

Species Assessment ....................................................................................................454

a. ...................................................................................................................... Mitchell’s satyr 

butterfly ......................................................................................................................454

b. ................................................................................................................... St. Francis’ satyr 

butterfly ......................................................................................................................456

c.................................................................................................................................. Poweshiek 

skippering ...................................................................................................................460

d. ............................................................................................................................ Karner blue 

butterfly ......................................................................................................................465

e........................................................................................................................................ Dakota 

skipper ........................................................................................................................468

L.21.5. Conclusion .....................................................................................................469

L.21.6. Refined Species Range Maps .......................................................................470

 
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LIST OF TABLES 

Table IV-1. Summary of Genetic Elements in MON 95379 ............................................61

Table IV-2. Unique Junction Sequence Class Results ......................................................66

Table IV-3. Unique Junction Sequence Class Results ......................................................70

Table IV-4. Junction Sequence Classes Detected .............................................................76

Table IV-5. Segregation Results for MON 95379 from the F4F2, F4F3, and F4F4 ........80

Table V-1. Summary of Cry1B.868 Protein Levels in Maize Tissues Collected from 

MON 95379 Produced in United States Field Trials in 2018 ............................................92

Table V-2. Summary of Cry1Da_7 Protein Levels in Maize Tissues Collected from 

MON 95379 Produced in United States Field Trials in 2018 ............................................93

Table VI-1. Summary of Maize Grain Proximates, Linoleic Acid, Carbohydrates by 

Calculation and Fiber for MON 95379 and the Conventional Control .............................99

Table VI-2. Summary of Maize Grain Essential Amino Acids for MON 95379 and 

Conventional Control .......................................................................................................100

Table VI-3. Summary of Maize Grain Anti-Nutrients for MON 95379 and Conventional 

Control .............................................................................................................................102

Table VI-4. Summary of Maize Forage Proximates, Carbohydrates by Calculation and 

Fiber for MON 95379 and Conventional Control ............................................................103

Table VI-5. Literature and ILSI Database Ranges for Components in Maize Grain and 

Forage ..............................................................................................................................104

Table VII-1. Activity Spectrum Results from Feeding Assays with Cry1B.868 Protein in 

Invertebrates .....................................................................................................................108

Table VII-2. Activity Spectrum Results from Feeding Assays with Cry1Da_7 Protein in 

Invertebrates. ....................................................................................................................109

Table VII-3. Fresh Weight 95th Percentile Expression of Cry1Da_7 and Cry1B.868 

Proteins in Maize Tissues Collected from MON 95379 Produced in United States Field 

Trials in 2018 ...................................................................................................................112

Table VII-4. Summary of Estimated Environmental Concentrations (EECs) for Exposure 

of Invertebrate NTOs to Cry1B.868 and Cry1Da_7 in MON 95379 Maize ...................114

Table VII-5. Predicted Environmental Concentrations (PECs) and Dissipation Kinetics 

of Cry1B.868 and Cry1Da_7 in Soil................................................................................117

Table VII-6. First-Order Accumulation Model to Predict Maximum Expected 

Concentration of Cry1Da_7 Protein in Sandy Loam Soil ...............................................120
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Table VII-7. Summary of Dose-Based Estimated Environmental Concentrations (EECs) 

for Exposure of Terrestrial Vertebrate NTOs to Cry1B.868 and Cry1Da_7 in MON 

95379 Maize.....................................................................................................................124

Table VII-8. Estimated Environmental Concentrations (EECs), Median Lethal 

Concentrations (LC50) from Non-target Terrestrial Invertebrate Studies and Margins of 

Exposure (MOEs) for the Cry1B.868 Protein..................................................................131

Table VII-9. Dose-Based Estimated Environmental Concentrations (EECs), Median 

Lethal Concentrations (LC50) from Terrestrial Vertebrate Studies and Margins of 

Exposure (MOEs) for the Cry1B.868 Protein..................................................................133

Table VII-10. Estimated Environmental Concentrations (EECs), Median Lethal 

Concentrations (LC50), from Non-target Terrestrial Invertebrate Studies and Margins of 

Exposure (MOEs) for the Cry1Da_7 Protein...................................................................135

Table VII-11. Dose-Based Estimated Environmental Concentrations (EECs), Median 

Lethal Concentrations (LC50) from Terrestrial Vertebrate Studies and Margins of 

Exposure (MOEs) for the Cry1Da_7 Protein...................................................................137

Table VII-12. Spatial Overlap of Threatened and Endangered Species Habitat and Maize 

Cultivation in Nebraska, Hawaii, and Iowa .....................................................................139

Table VIII-1. Phenotypic, Agronomic, and Environmental Interaction Characteristics 

Evaluated in U.S. Field Trials and Laboratory Studies ...................................................142

Table VIII-2. Germination Characteristics of MON 95379 and the Conventional Control147

Table VIII-3. Phenotypic and Agronomic and Environmental Interactions Sites for 

MON 95379 during 2018 .................................................................................................149

Table VIII-4. Combined-Site Analysis of Phenotypic and Agronomic Characteristics of 

MON 95379 Compared to the Conventional Control in 2018 U.S. Field Trials .............149

Table VIII-5. Summary of Qualitative Environmental Interactions Assessments for 

MON 95379 during 2018 .................................................................................................150

Table X-1. Summary of Published Literature on Maize Cross Pollination ....................161

Table A-1. USDA Notifications and Permits Approved for MON 95379 and Status of 

Trials Planted under These Notifications .........................................................................189

Table C-1. Summary of MON 95379 Cry1B.868 Protein Identity and Equivalence .....201

Table C-2. Summary of the Tryptic Masses Identified for the MON 95379-Produced 

Cry1B.868 Using LC-MS/MS1

 ........................................................................................204

Table C-3. Summary of the Tryptic Masses Identified for the Bt-Produced Cry1B.868 

Using LC-MS/MS1...........................................................................................................207
Bayer CropScience LP CR282-20U1 26

Table C-4. Immunoreactivity of the MON 95379-Produced and Bt-Produced Cry1B.868 

Proteins ............................................................................................................................215

Table C-5. Apparent Molecular Weight and Purity Analysis of the 

MON 95379-Produced Cry1B.868 Protein......................................................................217

Table C-6. Apparent Molecular Weight Comparison Between the MON 95379-Produced 

Cry1B.868 and Bt-Produced Cry1B.868 Proteins ...........................................................217

Table C-7. Functional Activity of MON 95379-Produced and Bt-Produced Cry1B.868 

Proteins ............................................................................................................................219

Table C-8. Individual Assay Data and 95% Prediction Interval Generated for the 

Apparent MW of Cry1B.868 Protein for One Future Assay ...........................................225

Table C-9. Estimated EC50 Values from 24 Dose-Response Diet-Incorporation Insect 

Bioassays of the Cry1B.868 Protein ................................................................................227

Table D-1. Summary of MON 95379 Cry1Da_7 Protein Identity and Equivalence ......230

Table D-2. Summary of the Tryptic Masses Identified for the MON 95379-Produced 

Cry1Da_7 Using LC-MS/MS1

 .........................................................................................233

Table D-3. Summary of the Tryptic Masses Identified for the Bt-Produced Cry1Da_7 

Using LC-MS/MS1

...........................................................................................................237

Table D-4. Immunoreactivity of the MON 95379-Produced and Bt-Produced Cry1Da_7 

Proteins ............................................................................................................................245

Table D-5. Apparent Molecular Weight and Purity Analysis of the 

MON 95379-Produced Cry1Da_7 Protein.......................................................................247

Table D-6. Apparent Molecular Weight Comparison Between the MON 95379-Produced 

Cry1Da_7 and Bt-Produced Cry1Da_7 Proteins .............................................................247

Table D-7. Functional Activity of MON 95379-Produced and Bt-Produced Cry1Da_7 

Proteins ............................................................................................................................249

Table D-8. Individual Assay Data and 95% Prediction Interval Generated for the 

Apparent MW of Cry1Da_7 Protein for One Future Assay ............................................257

Table D-9. Estimated EC50 Values from 24 Dose-Response Diet Incorporation Insect 

Bioassays of the Cry1Da_7 Protein .................................................................................258

Table E-1. Cry1B.868 Extraction Parameter 1 .................................................................261

Table E-2. Cry1Da_7 Extraction Parameter 1 .................................................................262

Table E-3. Summary of the Tissue-Specific Moisture Conversion Factors1 ..................265

Table G-1. Starting Seed for Germination and Dormancy Assesment of MON 95379 .300
Bayer CropScience LP CR282-20U1 27

Table H-1. Starting Seed for Phenotypic, Agronomic, and Environmental Interactions 

Assessment of MON 95379 .............................................................................................303

Table H-2. Field Information for Phenotypic, Agronomic, and Environmental 

Interactions Assessment of MON 95379 .........................................................................304

Table H-3. Summary of Abiotic Stressor Response Evaluations for MON 95379 and the 

Conventional Control in 2018 U.S. Field Trials ..............................................................307

Table H-4. Summary of Arthropod Damage Evaluations for MON 95379 and the 

Conventional Control in 2018 U.S. Field Trials ..............................................................308

Table H-5. Summary of Disease Damage Evaluations for MON 95379 and the 

Conventional Control in 2018 U.S. Field Trials ..............................................................309

Table I-1. Re-expression Formulas for Statistical Analysis of Composition Data .........316

Table J-1. Summary of test and control substances .........................................................321

TableJ-2. Summary of protein levels in leaf tissue with the fresh weight values converted 

to dry weight values. ........................................................................................................322

Table J-3. Summary of protein levels in leaf tissue with the fresh weight values converted 

to dry weight values. ........................................................................................................324

Table J-4. Relative Concentrations of Cry1B.868 and Cry1Da_7 in Lyophilized Leaf 

Tissue from MON 95379 Maize ......................................................................................325

Table J-5. Observed seven-day FAW GI50 values and 95% confidence intervals (CI) for 

MON 95379, Cry1B.868-single, and Cry1Da_7-single and the predicted GI50 value and 

95% CI for MON 95379. .................................................................................................326

Table J-6. Calculated Risk Quotients (RQ) and Hazard Index (HI) Values for Cry1B.868 

and Cry1Da_7 as Expressed in MON 95379 Maize ........................................................328

Table K-1. Summary of total Generated NGS Data for the Control and Test Substances415

Table K-2. Summary Coverage Data for the T-DNA region of Control and Test 

Substances ........................................................................................................................416

Table L-1. Cry1Da_7 concentrations in three soil types as measured by ELISA ..........434

Table L-2. Summary of Insect Control Measures Applied and Timing of Field 

Observations of Arthropod Stressors for MON 95379 and the Conventional Control 

Across in 2018 U.S. Field Trials ......................................................................................440

Table L-3. Summary of Disease Control Measures Applied and Timing of Field 

Observations of Disease Stressors for MON 95379 and the Conventional Control Across 

in 2018 U.S. Field Trials ..................................................................................................441
Bayer CropScience LP CR282-20U1 28

LIST OF FIGURES 

Figure III-1. Schematic of the Development of MON 95379 ...................................................... 44

Figure III-2. Circular Map of PV-ZMIR522223........................................................................... 45

Figure III-3. Deduced Amino Acid Sequence of the Cry1B.868 Protein .................................... 51

Figure III-4. Deduced Amino Acid Sequence of the Cry1Da_7 Protein ..................................... 53

Figure IV-1. Molecular Characterization using Sequencing and Bioinformatics ........................ 57

Figure IV-2. Five Types of NGS Reads....................................................................................... 59

Figure IV-3. Schematic Representation of the Insert and Flanking Sequences in MON 95379 . 63

Figure IV-4. Breeding History of MON 95379 ........................................................................... 64

Figure IV-5. Read Mapping of Conventional Maize LH244 Versus PV-ZMIR522223 ............. 67

Figure IV-6. Read Mapping of MON 95379 (F4) Versus PV-ZMIR522223 .............................. 68

Figure IV-7. Read Mapping of MON 95379 (F4) Versus PV-ZMOO513642 ............................ 70

Figure IV-8. Read Mapping of Conventional Maize LH244 Versus PV-ZMOO513642 ........... 71

Figure IV-9. Overlapping PCR Analysis across the Insert in MON 95379................................. 73

Figure IV-10. PCR Amplification of the MON 95379 Insertion Site.......................................... 75

Figure IV-11. Breeding Path for Generating Segregation Data for MON 95379 ........................ 79

Figure V-1. Schematic Representation of the Primary Domain Architecture of the Cry1B.868 

Protein ........................................................................................................................................... 83

Figure V-2. Schematic Representation of the Primary Domain Architecture of the Cry1Da_7 

Protein ........................................................................................................................................... 84

Figure VII-1. Relevant Routes of Exposure to Non-Target Organisms from Cultivation of MON 

95379 Maize................................................................................................................................ 110

Figure VII-2. A First-Order Soil Accumulation Model Based on Highly Conservative Parameter 

Estimates Predicts Limited Accumulation of Cry1Da_7 in Sandy Loam Soil ........................... 121

Figure C-1. N-Terminal Sequence of the MON 95379-Produced Cry1B.868 Protein .............. 202

Figure C-2. Peptide Map of the MON 95379-Produced Cry1B.868 and Bt-produced Cry1B.868 

Proteins ....................................................................................................................................... 212
Bayer CropScience LP CR282-20U1 29

Figure C-3. Western Blot Analysis and Immunoreactivity of MON 95379-Produced and 

Bt-Produced Cry1B.868 Proteins ................................................................................................ 214

Figure C-4. Purity and Apparent Molecular Weight Analysis of the MON 95379-Produced 

Cry1B.868 Proteins ..................................................................................................................... 216

Figure C-5. Glycosylation Analysis of the MON 95379-Produced and Bt-Produced Cry1B.868 

Proteins ....................................................................................................................................... 218

Figure D-1. N-Terminal Sequence of the MON 95379-Produced Cry1Da_7 Protein .............. 231

Figure D-2. Peptide Map of the MON 95379-Produced Cry1Da_7 and Bt-Produced Cry1Da_7

..................................................................................................................................................... 242

Figure D-3. Western Blot Analysis and Immunoreactivity of MON 95379-Produced and 

Bt-Produced Cry1Da_7 Proteins................................................................................................. 244

Figure D-4. Purity and Apparent Molecular Weight Analysis of the MON 95379-Produced 

Cry1Da_7 Proteins ...................................................................................................................... 246

Figure D-5. Glycosylation Analysis of the MON 95379-Produced and Bt-Produced Cry1Da_7 

Proteins ....................................................................................................................................... 248

Figure F-1. SDS-PAGE Analysis of the Degradation of Cry1B.868 Protein by Pepsin ........... 268

Figure F-2. Western Blot Analysis of the Degradation of Cry1B.868 Protein by Pepsin ......... 270

Figure F-3. SDS-PAGE Analysis of the Degradation of Cry1Da_7 Protein by Pepsin ............ 272

Figure F-4. Western Blot Analysis of the Degradation of Cry1Da_7 Protein by Pepsin .......... 274

Figure F-5. Western Blot Analysis of the Degradation of Cry1B.868 Protein by Pancreatin ... 276

Figure F-6. Western Blot Analysis of the Degradation of Cry1Da_7 Protein by Pancreatin .... 278

Figure F-7. SDS-PAGE and Western Blot Analysis of the Degradation of Cry1B.868 Protein by 

Sequential Digestion ................................................................................................................... 280

Figure F-8. SDS-PAGE and Western Blot Analysis of the Degradation of Cry1Da_7 Protein by 

Sequential Digestion ................................................................................................................... 282

Figure J-2. Tissue from MON 95379, Cry1B.868-single, and Cry1Da_7-single resulted in 

concentration-dependent growth inhibition in seven-day diet-incorporation FAW bioassays 

(GraphPad, 2019). ....................................................................................................................... 327

Figure K-1. Read Mapping of MON 95379 (F5) Versus PV-ZMIR522223 ............................. 395
Bayer CropScience LP CR282-20U1 30

Figure K-2. Read Mapping of MON 95379 (F4F1) Versus PV-ZMIR522223 ......................... 396

Figure K-3. Read Mapping of MON 95379 (F5F1) Versus PV-ZMIR522223 ......................... 397

Figure K-4. Read Mapping of MON 95379 (F6F1) Versus PV-ZMIR522223 ......................... 398

Figure K-5. Read Mapping of Conventional Maize LH244 Versus PV-ZMIR522223 ............ 399

Figure K-6. Read Mapping of Conventional Maize LH244 × HCL617 Versus PV-ZMIR522223

..................................................................................................................................................... 400

Figure K-7. Read Mapping of Conventional Maize LH244 Versus PV-ZMOO513642 ........... 401

Figure K-8. Read Mapping of MON 95379 (F4) Versus pdc3 .................................................. 402

Figure K-9. Read Mapping of MON 95379 (F5) Versus pdc3 .................................................. 403

Figure K-10. Read Mapping of MON 95379 (F4F1) Versus pdc3 ............................................ 404

Figure K-12. Read Mapping of MON 95379 (F6F1) Versus pdc3 ............................................ 406

Figure K-13. Read Mapping of LH244 Conventional Maize Versus pdc3 ............................... 407

Figure K-14. Read Mapping of LH244 × HCL617 Conventional Maize Versus pdc3 ............. 408

Figure K-17. Read Mapping of the PV-ZMOO513642 NGS Library (1 genome equivalent) .. 411

Figure K-18. Read Mapping of the PV-ZMOO513642 NGS Library (1/10th genome equivalent)

..................................................................................................................................................... 412

Figure K-19. Junction Sequences Detected: All Breeding Generations ................................... 414

Figure L-1. Alignment A. The alignment Between an Endogenous Maize Zm.Ubq Sequence and 

the MON 95379 T-DNA Zm.Ubq Element................................................................................. 419

Figure L-2. (Derived from Figure IV-5) Read Mapping of Conventional Maize LH244 Versus 

PVZMIR522223 with Supplemental Annotations ...................................................................... 424

Figure L-3. Isolated Un-Paired Read Regions Corresponding to the Zm.Ubq Elements in MON 

95379 Sample F4 and the LH244 Conventional Control............................................................ 425

Figure L-4. Side by Side Annotated comparison of Unpaired Read and Depth Plots of the MON 

95379 Insertion and the LH244 Conventional Control............................................................... 426

Figure L-5. Framework of the Approach to Provide Geographic, Biological, and Habitat 

Information to Refine the Threatened and Endangered Species Assessment for MON 95379 .. 450

Figure L-6. Current Species Range for MSB (accessed 9/27/2021) ........................................... 470
Bayer CropScience LP CR282-20U1 31

Figure L-7. Current Species Range for SFSB (accessed on 9/24/2021) ..................................... 471

Figure L-8. Current National-Level Species Range for Poweshiek Skipperling (accessed 

9/27/2021) ................................................................................................................................... 472

Figure L-9. Michigan Unit 1 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021)473

Figure L-10. Michigan Unit 5 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021)

..................................................................................................................................................... 474

Figure L-11. Michigan Unit 3 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021)

..................................................................................................................................................... 475

Figure L-12. Michigan Unit 4 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021)

..................................................................................................................................................... 476

Figure L-13. Michigan Unit 6 Critical Habitat for Poweshiek Skipperling (9/23/2021) ............ 477

Figure L-14. Michigan Unit 7 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021)

..................................................................................................................................................... 478

Figure L-15. Michigan Unit 9 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021)

..................................................................................................................................................... 479

Figure L-16. Wisconsin Unit 2 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021)

..................................................................................................................................................... 480

Figure L-17. Current Species Range for Karner Blue Butterfly (accessed 9/27/2021) .............. 481

Figure L-18. Current Critical Habitat Range for Dakota Skipper (FESTF Gopher ;accessed 

9/28/2021) ................................................................................................................................... 482

Figure L-19. Current Species Range for Dakota Skipper (FESTF Gopher; accessed 9/28/2021)

..................................................................................................................................................... 483
Bayer CropScience LP CR282-20U1 32 

ABBREVIATIONS AND DEFINITIONS1

aadA Aminoglycoside-modifying enzyme, 3''(9) –O–

nucleotidyltransferase from the transposon Tn7

Act Actin 

ADF Acid Detergent Fiber 

APHIS Animal and Plant Health Inspection Service 

aroA 5-enolpyruvylshikimate-3-phosphate synthetase gene from 

Agrobacterium sp. Strain CP4 

ANOVA Analysis of Variance 

AOSA Association of Official Seed Analysts 

BIO Biotechnology Innovation Organization

bp Basepair 

BRS Biotechnology Regulatory Service 

Bt Bacillus thuringiensis

bw Body Weight 

CBI Confidential Business Information 

CEW Corn Earworm 

CFR Code of Federal Regulations 

COA Certificate of Analysis 

COMPARE COMprehensive Protein Allergen REsource 

CP4 EPSPS Agrobacterium tumefaciens strain CP4, 5-enolpyruvlshikimate-3-

phosphate synthase protein 

Cry Crystalline Proteins 

Cry1 Subspecies of Cry Proteins 

Cry1B.868

Cry1B.868 is a chimeric protein comprised of domains I and II 

from Cry1Be (Bt), domain III from Cry1Ca (Bt subsp. aizawai) 

and C-terminal protoxin domain from Cry1Ab 

(Bt subsp. kurstaki) 

Cry1Da_7 Modified Cry1Da protein derived from Bt subsp. aizawai

CTP Chloroplast Transit Peptide 

CTAB Hexadecyltrimethylammonium Bromide 

d Day(s) 

DNA Deoxyribonucleic Acid 

DT50 50% Dissipation Time 

dw Dry Weight 

1

 Alred, G.J., C.T. Brusaw, and W.E. Oliu. 2003. Handbook of Technical Writing, 7th edn., pp. 2-7. Bedford/St. 

Martin's, Boston, MA. 
Bayer CropScience LP CR282-20U1 33

EC50 Median Effective Concentration 

E. coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

EEC Estimated Environmental Concentrations 

ELISA Enzyme-linked Immunosorbent Assay 

ELN Electronic Lab Notebook 

EPA Environmental Protection Agency 

ETS Excellence Through Stewardship 

FAO Food and Agriculture Organization 

FAW Fall Armyworm 

FDA Food and Drug Administration (U.S.) 

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FIR Food Intake Rate 

FMOC fluorenylmethyl chloroformate 

FMV Figwort Mosaic Virus 

FOIA Freedom of Information Act 

fw Fresh Weight 

GE Genetically Engineered 

GLP Good Laboratory Practice 

HESI Health and Environmental Sciences Institute 

IAC Immunoaffinity Chromatography 

ICP Insect Control Proteins 

ISF International Seed Foundation 

kb Kilobase 

kDa Kilodalton 

lbs Pounds 

LC50 Median Lethal Concentration 

LC/MS Liquid chromatography-mass spectrometry 

LOD Limit of Detection 

LOQ Limit of Quantitation 

LTP Lipid Transfer Protein 

mg/kg Milligram per Kilogram 

MOA Mode of Action 

MOE Margins of Exposure 

mRNA Messenger RNA 

MMT Million Metric Ton 

MW Molecular Weight 

NCBI National Center for Biotechnology Information 

NDF Neutral Detergent Fiber 

NGS Next Generation Sequencing 

NOAEC No Observed Adverse Effect Concentration 

NOAEL No Observed Adverse Effects Level 

NOEC No Observed Effect Concentration 

NTO Non-Target Organism 

OECD Organization for Economic Co-operation and Development 
Bayer CropScience LP CR282-20U1 34

OPA o-phthalaldehyde 

OR Origin of Replication 

ORF Open Reading Frame 

OSL Over Season Leaf 

OSR Over Season Root 

OT Optimum Temperature 

PCR Polymerase Chain Reaction 

PEC Predicted environmental concentration 

PIPs Plant Incorporated Protectants 

PPA Plant Protection Act 

SCB Sugarcane Borer 

SDS-PAGE Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis 

S.E. Standard Error 

ShkG 5-enolpyruvylshikimate-3-phosphate synthase from 

Arabidopsis thaliana

SOP Standard Operating Procedure 

SOT Suboptimum Temperature 

T-DNA Transfer Deoxyribonucleic Acid 

TIP Tonoplast Intrinsic Protein 

Tub Tubulin 

μg/g Microgram per Gram 

Ubq Ubiquitin gene 

U.S. United States 

USDA United States Department of Agriculture 

UTR Untranslated Region 

WHO World Health Organization 
Bayer CropScience LP CR282-20U1 35 

I. RATIONALE FOR THE DEVELOPMENT OF MON 95379 

I.A. Basis for the Request for a Determination of Nonregulated Status under 7 CFR § 340.6 

The Animal and Plant Health Inspection Service (APHIS) of the United States (U.S.) Department 

of Agriculture (USDA) has responsibility, under the Plant Protection Act (Title IV Pub. L. 106-

224, 114 Stat. 438, 7 U.S.C. § 7701-7772) to prevent the introduction and dissemination of plant 

pests into the U.S. APHIS regulations at 7 CFR § 340.6, effective as of the date this petition was 

filed, provides that an applicant may petition APHIS to evaluate submitted data to determine that 

a particular regulated article does not present a plant pest risk and no longer should be regulated. 

If APHIS determines that the regulated article does not present a plant pest risk, the petition is 

granted, thereby allowing unrestricted introduction of the article. Newly promulgated 7 CFR § 340 

(issued May 18, 2020) states, in §340.1(c), that “All plants determined by APHIS to be deregulated 

pursuant to § 340.6 as that section was set forth prior to August 17, 2020 will retain their 

nonregulated status under these regulations.” Because this petition was filed before October 1, 

2021 (see 7 CFR 340.1(c)(2)), Bayer seeks deregulation pursuant to the regulations in effect prior 

to August 17, 2020. 

Bayer is submitting this request to APHIS for a determination of nonregulated status for the new 

biotechnology-derived maize product, MON 95379, any progeny derived from crosses between 

MON 95379 and conventional maize, and any progeny derived from crosses of MON 95379 with 

biotechnology-derived maize that have previously been granted nonregulated status under 7 CFR 

Part 340 or determined in the future to not pose a plant pest risk via a regulatory status review or 

otherwise deemed to be exempt from 7 CFR Part 340. 

I.B. Rationale for the Development of Lepidopteran-Protected Maize MON 95379 

Lepidopteran-protected maize MON 95379 was developed to provide growers in South America 

with new options for protection against the feeding damage of targeted lepidopteran pests, 

including fall armyworm (FAW; Spodoptera frugiperda), sugarcane borer (SCB; Diatraea 

saccharalis) and corn earworm (CEW; Helicoverpa zea). The larval feeding behavior of these 

species typically limits the efficacy of synthetic insecticides by creating additional difficulties for 

the sprayed active ingredients to reach the insects (Burtet et al., 2017; Grimi et al., 2018; ReayJones, 2019). MON 95379 produces two insecticidal proteins, Cry1B.868 and Cry1Da_7, which 

protect against feeding damage caused by these lepidopteran pests. Cry1B.868 is a chimeric 

protein comprised of domains I and II from Cry1Be (Bacillus thuringiensis, Bt), domain III from 

Cry1Ca (Bt subsp. aizawai) and the C-terminal protoxin domain from Cry1Ab (Bt subsp. kurstaki). 

Cry1Da_7 is a modified Cry1Da protein derived from Bt subsp. aizawai. 

Fall Armyworm is the main pest of maize in most of South America. In warmer regions such as 

Brazil, where maize can be grown during two growing seasons (summer crop and winter crop), 

FAW populations can build up considerably under conditions favoring multiple and overlapping 

generations (Omoto et al., 2016). Synthetic insecticides have been the historic management tactic 

against FAW in maize and other crops in South America. Resistance to several distinctive classes 

of insecticides have been documented in Brazil (Carvalho et al., 2013; Diez-Rodríguez and Omoto, 
Bayer CropScience LP CR282-20U1 36

2001; Nascimento et al., 2016; Okuma et al., 2018). Therefore, in this challenging environment 

for FAW management, Bt maize has become highly adopted by growers for FAW management in 

South America. Within nine years of Brazil’s first introduction of insect-protected Bt maize in 

2008, ~85% of the cultivated maize contained one or more insect-protection traits that expressed 

Bt proteins (ISAAA, 2017). Due to the appearance of resistance in FAW, growers in South 

America are in need of new tools to protect against larval feeding damage caused by lepidopteran 

pests. Resistance to Bt traits has already been observed for FAW to the proteins Cry1F and Cry1Ab 

and SCB to the proteins Cry1F and Cry1A.105 in South America (Bernardi et al., 2015a; Farias et 

al., 2014; Omoto et al., 2016). Currently, Vip3Aa20 is the only fully effective Bt protein against 

FAW available in the market in many regions in South America. However, an allele that confers 

resistance of FAW to Vip3Aa20 was already identified in Brazilian field populations (Amaral et 

al., 2020; Bernardi et al., 2016; Bernardi et al., 2015b), and is indicative of an increasing potential 

of resistance developing to this protein in FAW. The receptors that Cry1B.868 and Cry1Da_7 

proteins in MON 95379 bind are different from each other in FAW and from other commercially 

available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3 indicating that they would 

provide enhanced durability of this Bt maize technology in the field against this economically 

important pest (Wang et al., 2019). 

Bayer will not seek a commercial registration for MON 95379 from the EPA since it will not be 

commercialized in North America. Under the proposed terms of an EPA seed increase registration 

application, MON 95379 will be cultivated for small-scale breeding, testing, and seed increase 

nurseries in Nebraska, Hawaii, and Iowa with a maximum total acreage of 100 acres per growing 

season. These activities will support the development of future commercial products for South 

America. In the course of developing these products containing MON 95379, Bayer may also 

develop breeding stacks in which MON 95379 will be combined with other traits for 

commercialization outside the United States. To support subsequent global regulatory 

submissions of these stack products, from time to time, Bayer may conduct confined, small-scale 

field trials that will be planted in additional counties/states that are assessed under the standard 

USDA-BRS permitting process. Trials and trial material will be managed according to the 

conditions of applicable BRS permits and terms of an EPA seed increase registration, if applicable. 

I.C. Submissions to Other Regulatory Agencies 

Under the Coordinated Framework for Regulation of Biotechnology (USDA-APHIS, 1986), the 

responsibility for regulatory oversight of biotechnology-derived maize falls primarily on three U.S. 

agencies: U.S. Food and Drug Administration (FDA), the United States Department of Agriculture 

(USDA), and in the case of plant incorporated protectants (PIPs), the Environmental Protection 

Agency (EPA). Deregulation of MON 95379 by USDA constitutes only one component of the 

overall regulatory oversight and review of this product by the three relevant U.S. regulatory 

agencies. 

I.C.1. Submission to U.S. EPA 

Substances that are pesticides, as defined under the Federal Insecticide, Fungicide and Rodenticide 

Act (FIFRA) [7 U.S.C. §136(u)], are subject to regulation by U.S. EPA. Pesticides produced in 

planta, referred to as PIPs, are also subject to regulation by U.S. EPA under FIFRA. 
Bayer CropScience LP CR282-20U1 37

Bayer submitted an application to U.S. EPA on May 1, 2020 requesting a FIFRA Section 3 seed 

increase registration (PRIA code B884) of the plant-incorporated protectant Bacillus thuringiensis

Cry1B.868 and Cry1Da_7 proteins and the genetic material (Vector 

PV-ZMIR522223) necessary for their production in MON 95379. A maximum total U.S. acreage 

of 100 acres per growing season for breeding and seed increase purposes in the states of Nebraska, 

Hawaii and Iowa was requested. Thus MON 95379 will not be commercialized in the U.S., and 

Bayer will not seek a commercial registration (Section 3) for MON 95379 from the U.S. EPA. 

In addition, a petition for a permanent exemption from the requirement of a tolerance for the plantincorporated protectant (PIP) proteins Cry1B.868 and Cry1Da_7 in or on the food and feed 

commodities of maize was also submitted. 

I.C.2. Submission to U.S. FDA 

MON 95379 falls within the scope of the 1992 FDA policy statement concerning regulation of 

products derived from new plant varieties, including those developed through biotechnology (U.S. 

FDA, 1992). In compliance with this policy, Bayer will initiate a formal consultation with the FDA 

and will be submitting a Food/Feed Safety and Nutritional Assessment Summary document to 

FDA in the near future. 

I.C.3. Submissions to Foreign Government Agencies 

Consistent with our commitments to the Biotechnology Innovation Organization (BIO) and 

Excellence Through Stewardship® (ETS)2, Bayer will meet applicable regulatory requirements for 

MON 95379 in the country(ies) of intended production and for key import countries identified in 

a trade assessment process that have functioning regulatory systems to assure global compliance 

and support the flow of international trade. Bayer will continue to monitor other countries that are 

key importers of maize from the intended commercial production countries, for the development 

of new formal biotechnology approval processes. If new functioning regulatory processes are 

developed, Bayer will re-evaluate its stewardship plans and make appropriate modifications to 

minimize the potential for trade disruption. 

 

® Excellence Through Stewardship is a registered trademark of Excellence Through Stewardship, Washington, DC. 

2 http://www.excellencethroughstewardship.org/. 
Bayer CropScience LP CR282-20U1 38

II. THE BIOLOGY OF MAIZE 

The biology of maize has been well documented (Anderson and de Vicente, 2010; Farnham et al., 

2003; OGTR, 2008). In addition to the wealth of information on maize biology, the Organisation 

for Economic Co-operation and Development authored a Consensus Document (OECD, 2003) on 

the biology of maize. This document provides key information regarding: 

x general description of maize biology, including taxonomy and morphology and 

use of maize as a crop plant 

x agronomic practices in maize cultivation 

x geographic centers of origin 

x reproductive biology 

x cultivated maize as a volunteer weed 

x inter-species/genus introgression into relatives and interactions with other 

organisms 

x a summary of the ecology of maize 

Additional information on the biology and uses of maize has also been compiled by the Australian 

Office of the Gene Technology Regulator (OGTR, 2008), and the Canadian Food Inspection 

Agency (CFIA, 2020). The taxonomic information for maize is available in the USDA’s PLANTS 

Profile (USDA-NRCS, 2019). 

To support the evaluation of the plant pest potential of MON 95379 relative to conventional maize, 

additional information regarding several aspects of maize biology can be found elsewhere in this 

petition. This includes: agronomic practices for maize in Section IX; volunteer management of 

maize in Section IX.B and inter-species/genus introgression potential in Section X.D. 

II.A. Maize as a Crop 

Maize is widely grown in nearly all areas of the world and is the largest grain crop in the world, 

ahead of both wheat (Triticum sp.) and rice (Oryza sativa L.), in total metric ton production 

(FAOSTAT, 2020). In the 2019/2020 marketing year, world maize area was approximately 192 

million hectares (ha) with a total grain production of approximately 1,115 million metric tons 

(MMT) (USDA-FAS, 2020). The top five production regions were: USA (347 MMT), China (261 

MMT), Brazil (101 MMT), the European Union (67 MMT), and Argentina (50 MMT) (USDAFAS, 2020). In the U.S., maize is grown in most states and in 2019, its production value of over 

$53 billion was the highest of any crop (USDA-NASS, 2020). 

In industrialized countries maize has two major uses: (1) as animal feed in the form of grain, forage 

or silage; and (2) as a raw material for wet- or dry-milled processed products such as high fructose 

maize syrup, oil, starch, glucose, dextrose and ethanol. By-products of the wet- and dry- mill 

processes are also used as animal feed. These processed products are used as ingredients in many 

industrial applications and in human food products. Most maize produced in industrialized 

countries is used as animal feed or for industrial purposes, but maize remains an important food 

staple in many developing regions, especially sub-Saharan Africa and Central America, where it 

is frequently the mainstay of human diets (Morris, 1998). 
Bayer CropScience LP CR282-20U1 39

Maize is a familiar plant that has been rigorously studied due to its use as a staple food/feed and 

the economic opportunity it brings to growers. Archaeological and genetic evidence suggests that 

maize domestication began in southern Mexico approximately 9,000 years ago, and that it was 

derived from Balsas teosinte, Zea mays subsp. parviglumis (Kistler et al., 2018; Matsuoka et al., 

2002; Piperno et al., 2009). Although grown extensively throughout the world, maize is not 

considered a threat to invade natural or agricultural ecosystems. Maize does not establish selfsustaining populations outside of cultivation (Crawley et al., 2001; OECD, 2003; Raybould et al., 

2012). This lack of weediness may reflect its poor competitive ability (Olson and Sander, 1988), 

lack of seed dormancy, and barriers to seed dispersal, as maize cobs retain seed and are covered in 

a husk (Wilkes, 1972). A number of other characteristics common in weeds, such as rapid 

flowering following emergence, are lacking in maize (Keeler, 1989). Today, the majority of U.S. 

maize acreage is planted to hybrids, a practice that started in the 1920s (Wych, 1988). Maize 

hybrids have advantages in yield and plant vigor associated with heterosis, also known as hybrid 

vigor (Duvick, 1999). 

Conventional plant breeding results in selection of desirable characteristics in a plant through the 

generation of unique combinations of genes obtained by intra- and inter-specific crossing, mutation 

breeding or utilization of other traditional breeding methodologies. However, there is a limit to 

the genetic diversity that is available for use and selection with conventional plant breeding. 

Biotechnology, as an additional tool to conventional breeding, offers access to greater genetic 

diversity than conventional breeding alone, resulting in expression of traits that are highly 

desirable to growers and downstream crop users.

II.B. Characteristics of the Recipient Plant 

The MON 95379 transformation was conducted with inbred maize line LH244, a maize line 

assigned to Holden’s Foundation Seeds, LLC in 2001 (U.S. Patent #6,252,148). LH244 is a 

medium season yellow dent maize line of Stiff Stalk background that is best adapted to the central 

regions of the U.S. corn belt. 

Following transformation of immature LH244 embryos, selected transformants were selfpollinated to increase seed supplies. A Cre recombination system was used to remove the 

selectable marker starting with the selected events at the R2 generation. A selected inbred line 

homozygous for the presence of the T-DNA and lacking the selectable marker cassette was used 

to produce other MON 95379 materials for product testing, safety assessment studies, and 

commercial hybrid development. The non-transformed LH244 was used to produce conventional 

maize comparators (hereafter referred to as conventional controls) in the safety assessment of 

MON 95379. 

 
Bayer CropScience LP CR282-20U1 40

II.C. Maize as a Test System in Product Safety Assessment 

In studies utilizing hybrid maize, the test was a hybrid of LH244 containing MON 95379 

(expressing the Cry1B.868 and Cry1Da_7 proteins) x HCL617 unless otherwise noted (Figure 

IV-4). LH244 x HCL617 was used as near isogenic, conventional control for the hybrid studies 

conducted (hereafter referred to as conventional control). LH244 was used as the conventional 

control in molecular characterization studies. LH244 x HCL617 was used as the conventional 

control in compositional analysis and in phenotypic, agronomic and environmental interactions 

assessments. Where appropriate, conventional commercial maize hybrids (hereafter referred to as 

reference hybrids) were used to establish ranges of natural variability or responses representative 

of commercial maize hybrids. Reference hybrids used at each field trial location were selected 

based on their availability and agronomic fit for the respective geographic regions. 

To conduct the studies reported in this petition, appropriate MON 95379 test materials were 

generated for the molecular characterization (Sections III and IV), protein characterization and 

expression analysis (Section V), compositional analysis (Section VI), assessment of potential 

impacts on non-target organisms beneficial to agriculture and threatened and endangered (Section 

VII) and phenotypic, agronomic and environmental interactions assessment (Section VIII). The 

molecular characterization studies and initiation of commercial breeding efforts were conducted 

with the F4 generation (Figure IV-4). Protein characterization and expression analysis, 

composition analysis, and phenotypic, agronomic and environmental interactions assessment were 

conducted with MON 95379 breeding generation F5F1. 

 
Bayer CropScience LP CR282-20U1 41

III. DESCRIPTION OF THE GENETIC MODIFICATION 

This section provides a description of the transformation process and plasmid vector used in the 

development of MON 95379 (Figure III-1 and Figure III-2). Molecular analyses are an integral 

part of the characterization of maize products with new traits introduced by methods of 

biotechnology. Vectors and methods are selected for transformation to achieve high probability 

of obtaining the trait of interest and integration of the introduced DNA into a single locus in the 

plant genome. This helps ensure that only the intended DNA encoding the desired trait(s) is 

integrated into the plant genome and facilitates the molecular characterization of the product. 

Information provided here allows for the identification of the genetic material present in the 

transformation vector delivered to the host plant and an analysis of the data supporting the 

characterization of the DNA inserted in the plant found in Section V. 

III.A. Description of Transformation Plasmid PV-ZMIR522223 

Plasmid Vector PV-ZMIR522223 was used for the transformation of conventional maize to 

produce MON 95379 and its plasmid map is shown in Figure III-2. A description of the genetic 

elements and their prefixes (e.g., B, P, TS, CS, T, I, E, and OR) in PV-ZMIR522223 is provided 

in Table III-1. PV-ZMIR522223 is approximately 21.6 kb and contains one T-DNA (transfer 

DNA), that is delineated by Right and Left Border regions. The T-DNA contains the cp4 epsps

expression cassette, cry1B.868 expression cassette, and the cry1Da_7 expression cassette. During 

transformation, the T-DNA was inserted into the maize genome. Following transformation, 

traditional breeding, Cre/lox recombination (described in Section III.A), segregation, molecular 

screening and selection were used to isolate those plants that contained the cry1B.868 and 

cry1Da_7 expression cassettes and did not contain the backbone sequences from the 

transformation vector, the cp4 epsps selectable marker or any sequence from the 

cre gene-containing vector PV-ZMOO513642. 

The cry1B.868 coding sequence in MON 95379 is under the regulation of the promoter, 5' 

untranslated region (UTR) and intron for a ubiquitin gene (Ubq) from Zea mays subsp. Mexicana

(Mexican teosinte) that directs transcription in plant cells (Cornejo et al., 1993). The cry1B.868

coding sequence also utilizes the 3′ UTR sequence of the Lipid Transfer Protein-like gene (LTP) 

from Oryza sativa (rice) that directs polyadenylation of mRNA (Hunt, 1994). 

The codon optimized cry1Da_7 coding sequence in MON 95379 is under the regulation of the 

promoter and 5' UTR from Setaria italica (foxtail millet) tonoplast membrane integral protein

(Tip) gene (Hernandez-Garcia and Finer, 2014). In addition to the promoter and 5' UTR, the 

cry1Da_7 coding sequence is regulated by the enhancer from the 35S RNA of figwort mosaic 

virus (FMV) (Richins et al., 1987) that enhances transcription in most plant cells (Rogers, 2000) 

and the intron and flanking UTR sequence from the Actin 15 (Act 15) gene from Oryza sativa

(rice) that is involved in regulating gene expression (Rose, 2008). The cry1Da_7 coding sequence 

also utilizes 3' UTR sequence from the GOS2 gene encoding a translation initiation factor from 

Oryza sativa (rice) that directs polyadenylation of mRNA (Hunt, 1994). 

The cp4 epsps selectable marker cassette is also part of the originally inserted T-DNA in 

MON 95379. The selectable marker cassette is under the regulation of the promoter, 5' UTR, 

intron and 3′ UTR sequences of the OsTubA gene family from Oryza sativa (rice) encoding 
Bayer CropScience LP CR282-20U1 42

α-tubulin (Jeon et al., 2000) that directs transcription and polyadenylation of mRNA in plant cells. 

The cp4 epsps expression cassette was excised from progeny plants using the Cre/lox 

recombination system for marker removal as described in Section III.A.1 (Hare and Chua, 2002; 

Zhang et al., 2003). 

The backbone region of PV-ZMIR522223, located outside of the T-DNA, contains two origins of 

replication for maintenance of the plasmid vector in bacteria (oripBR322 and ori V) and a bacterial 

selectable marker gene (aadA). 

III.A.1. Marker Removal Through Cre/lox Recombination System 

The use of the Cre/lox recombination system for marker removal has been previously described 

(Hare and Chua, 2002; Russell et al., 1992; Zhang et al., 2003). The Cre/lox recombination system 

is derived from the bacteriophage P1 and consists of the Cre recombinase and a stretch of DNA 

flanked by two copies of the loxP sites. The loxP site is 34bp in length and consists of two 13-bp 

inverted repeats and an asymmetrical 8-bp spacer. The 13-bp inverted repeats are the 

Cre recombinase binding sequence, and the 8-bp spacer is essential for the recombination reaction. 

Cre recombinase binds to the inverted repeat sequence in the loxP site, catalyzing a crossover in 

the 8-bp spacer regions of the two loxP sites. The results of this crossover are two-fold: one is the 

excision of the DNA fragment flanked by the two half loxP sites (in MON 95379 this is the cp4 

epsps selectable marker cassette) forming a circular extra-genomic DNA fragment, the other is the 

recombination of linear DNA between the remaining two half loxP sites within the maize genome 

(Gilbertson, 2003). 

As reviewed (Gilbertson, 2003), one of the advantages of the Cre/lox system is the specificity of 

the enzyme for the wild-type loxP 34-bp recognition sequence. The frequency of Cre recombinasemediated DNA recombination can be significantly reduced with even a single nucleotide change 

in specific regions of the loxP sequence (Hartung and Kisters-Woike, 1998; Hoess et al., 1986; 

Lee and Saito, 1998). Therefore, neither the specific DNA insert nor the usage of the Cre/lox 

system was expected to negatively influence the stability of the T-DNA in MON 95379 across 

breeding generations, which has been confirmed, and is described in Section IV.E. of this petition. 

This technology was previously reviewed by USDA in the petition for LY038 maize (USDAAPHIS petition #04-229-01p). 

A maize line expressing Cre recombinase (developed with the transformation plasmid vector 

PV-ZMOO513642) was crossed with lines transformed with PV-ZMIR522223. In the resulting 

hybrid plants, the cp4 epsps selectable marker cassette that was flanked by the loxP sites was 

excised. The excised cp4 epsps selectable marker cassette (circular extra-genomic DNA) was 

subsequently not maintained during cell division. The cre gene and associated genetic elements 

were subsequently segregated away from the cry1B.868 and cry1Da_7 expression cassettes by 

conventional breeding to produce the MON 95379 product lacking the cp4 epsps gene cassette. 

The absence of both the cp4 epsps gene and cre gene were confirmed in the F4 generation (Figure 

IV-4). Since the cp4 epsps gene and sequence derived from PV-ZMOO513642 were eliminated 

through conventional breeding, the resulting progeny only contain the genes of interest (cry1B.868

and cry1Da_7) and not the gene used for selection. 
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III.B. Description of the Transformation System 

MON 95379 was developed through Agrobacterium tumefaciens mediated transformation of 

immature maize embryos based on the method described by Sidorov and Duncan (2009) utilizing 

PV-ZMIR522223. Immature embryos were excised from a post-pollinated maize ear of LH244. 

After co-culturing the excised immature embryos with Agrobacterium carrying the plasmid vector, 

the immature embryos were placed on selection medium containing glyphosate and carbenicillin 

disodium salt in order to inhibit the growth of untransformed plant cells and excess Agrobacterium, 

respectively. Once transformed callus developed, the callus was placed on media conducive to 

shoot and root development. The rooted plants (R0) with normal phenotypic characteristics were 

selected and transferred to soil for growth and further assessment. As demonstrated in this petition, 

the use of disarmed Agrobacterium tumefaciens strain ABI, a designated plant pest, as the 

transformation vector has not imparted plant pest characteristics to MON 95379. 

The R0 plants were self-pollinated to produce R1 seed. Subsequently, the R1 population was 

screened for the presence of T-DNA and absence of vector backbone sequences by construct-level 

PCR assay and Southern blot analysis. Only plants that were homozygous positive for 

T-DNA and negative for vector backbone were selected for further development and their 

progenies were subjected to further molecular and phenotypic assessments. As is typical of a 

commercial event production and selection process, hundreds of different transformation events 

(regenerants) were generated in the laboratory using PV-ZMIR522223. Selected R2 events were 

crossed with a line expressing the Cre recombinase protein and screened for the absence of cp4 

epsps and the cre genes. After careful selection and evaluation of these events in the laboratory, 

greenhouse and field, MON 95379 was selected as the lead event based on superior trait efficacy, 

agronomic, phenotypic, and molecular characteristics according to the general process described 

in Prado et al. (2014). Studies on MON 95379 were initiated to further characterize the genetic 

insertion and the expressed product, and to establish the food, feed, and environmental safety 

relative to conventional maize. The major steps involved in the development of MON 95379 are 

depicted in Figure III-1. The result of this process was the production of MON 95379 maize with 

the cry1B.868 and cry1Da_7 expression cassettes. 
Bayer CropScience LP CR282-20U1 44

Figure III-1. Schematic of the Development of MON 95379

Transformed LH244 (a maize line for more efficient transformation) 

immature embryos with PV-ZMIR522223 in Agrobacterium tumefaciens

Selected transformants (R0 plants) containing the selectable marker (cp4

epsps expression cassette) and generated rooted shoots from the 

transformed callus tissues. 

Identified MON 95379 as lead event and further evaluated its progeny 

in laboratory and field assessments for T-DNA insert integrity, trait 

efficacy, agronomic/phenotypic characteristics and absence of all other 

vector DNA and PV-ZMOO513642 DNA

Evaluated R1 plants by PCR and selected the transformed plants for the 

homozygous presence of the T-DNA

Evaluated plants (subsequent generations) for insert integrity (via

molecular analyses), insect control efficacy and other phenotypic 

characteristics

Assembled Agrobacterium binary plasmid vector PV-ZMIR522223 and

transferred to Agrobacterium tumefaciens strain ABI

Selected R2 plants were crossed with the Cre recombinase expressing 

line 

Identified plants lacking the cp4 epsps and cre genes in F1 and F2 plants 

respectively
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Figure III-2. Circular Map of PV-ZMIR522223 

A circular map of PV-ZMIR522223 used to develop MON 95379 is shown. PV-ZMIR522223 contains 

one T–DNA. Genetic elements are shown on the exterior of the map.

21637 bp
Bayer CropScience LP CR282-20U1 46 

Table III-1. Summary of Genetic Elements in Plasmid Vector PV-ZMIR522223 

Genetic Element Location in 

Plasmid 

Vector

Function (Reference)

T-DNA

B1-Left Border 

Region

1-442 DNA region from 

Agrobacterium tumefaciens containing the 

left border sequence used for transfer of the 

T–DNA (Barker et al., 1983). 

Intervening Sequence 443-477 Sequence used in DNA cloning 

loxP 478-511 Sequence from Bacteriophage P1 for the 

loxP recombination site recognized by the 

Cre protein (Russell et al., 1992). 

Intervening Sequence 512-517 Sequence used in DNA cloning 

T4-TubA 518-1099 3' UTR sequence of the OsTubA gene from 

Oryza sativa (rice) encoding α–tubulin (Jeon 

et al., 2000) that directs polyadenylation of 

mRNA. 

Intervening Sequence 1100-1106 Sequence used in DNA cloning 

CS3-cp4 epsps 1107-2474 Coding sequence of the aroA gene from 

Agrobacterium sp. strain CP4 encoding the 

CP4 EPSPS protein that provides herbicide 

tolerance (Barry et al., 2001; Padgette et al., 

1996). 

TS5-CTP2 2475-2702 Targeting sequence of the ShkG gene from 

Arabidopsis thaliana encoding the EPSPS 

transit peptide region that directs transport of 

the protein to the chloroplast (Herrmann, 

1995; Klee et al., 1987). 

Intervening Sequence 2703-2706 Sequence used in DNA cloning 

P2-TubA 2707-4887 Promoter, 5' UTR and intron sequences of 

the OsTubA gene family from Oryza sativa

(rice) encoding α-tubulin (Jeon et al., 2000) 

that directs transcription in plant cells. 

Intervening Sequence 4888-4893 Sequence used in DNA cloning 

loxP 4894-4927 Sequence from Bacteriophage P1 for the 

loxP recombination site recognized by the 

Cre protein (Russell et al., 1992). 

 
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Table III-1. Summary of Genetic Elements in Plasmid Vector PV-ZMIR522223 

(Continued) 

 

Intervening Sequence 4928-5038 Sequence used in DNA cloning 

T4-Ltp 5039-5338 3' UTR sequence of a Lipid Transfer 

Protein-like gene (LTP) from Oryza 

sativa (rice) that directs polyadenylation 

of mRNA (Hunt, 1994). 

Intervening Sequence 5339-5347 Sequence used in DNA cloning 

CS3-Cry1B.868 5348-8947 Coding sequences of three domains and 

a protoxin sourced from Bacillus 

thuringiensis (Bt) parental proteins 

arranged as a single chimeric pesticidal 

protein (Cry1B.868) that confers 

protection against lepidopteran insects 

via insect midgut disruption (Wang et 

al., 2019). 

Intervening Sequence 8948-8973 Sequence used in DNA cloning 

P2-Zm.Ubq 8974-10981 Promoter, 5' UTR and first intron 

sequences of the ubiquitin (Ubq) gene 

from Zea mays subsp. Mexicana

(Mexican teosinte) that directs 

transcription in plant cells (Cornejo et 

al., 1993). 

Intervening Sequence 10982-11008 Sequence used in DNA cloning 

E9-FMV 11009-11545 Enhancer from the 35S RNA of Figwort 

mosaic virus (FMV) (Richins et al., 

1987) that enhances transcription in 

most plant cells (Rogers, 2000). 

Intervening Sequence 11546-11556 Sequence used in DNA cloning 

P2-Tip 11557-12537 Promoter and 5' UTR sequences from 

Setaria italica (foxtail millet) tonoplast 

membrane integral protein (Tip) gene 

(Hernandez-Garcia and Finer, 2014). 

Intervening Sequence 12538-12545 Sequence used in DNA cloning 

I7-Act15 12546-13838 Intron and flanking UTR sequence from 

the Actin 15 (Act 15) gene from Oryza 

sativa (rice) that is involved in 

regulating gene expression (Rose, 

2008). 

Intervening Sequence 13839-13856 Sequence used in DNA cloning 
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Table III-1. Summary of Genetic Elements in Plasmid Vector PV-ZMIR522223 

(Continued) 

1

 B, Border 

2

 P, Promoter 

3

 CS, Coding Sequence 

4

 T, Transcription Termination Sequence 

5

 TS, Targeting Sequence 

6

 L, Leader 

7

 I, Intron 

8

 OR, Origin of Replication 

CS3-Cry1Da_7 13857-17357 Codon optimized coding sequence of 

cry1Da_7 from Bacillus 

thuringiensis (Bt) encoding a modified 

Cry1Da_7 protein that confers 

protection against lepidopteran insects 

via insect midgut disruption (Wang et 

al., 2019). 

Intervening Sequence 17358-17373 Sequence used in DNA cloning 

T4-GOS2 17374-17841 3' UTR sequence from the GOS2 gene 

encoding a translation initiation factor 

from Oryza sativa (rice) that directs 

polyadenylation of mRNA (Hunt, 1994). 

Intervening Sequence 17842-18045 Sequence used in DNA cloning 

B1-Right Border 

Region

18046-18376 DNA region from

Agrobacterium tumefaciens containing 

the right border sequence used for 

transfer of the T–DNA (Depicker et al., 

1982; Zambryski et al., 1982). 

Vector Backbone 

Intervening Sequence 18377-18520 Sequence used in DNA cloning 

aadA 18521-19409 Bacterial promoter, coding sequence, 

and 3' UTR for an aminoglycosidemodifying enzyme, 3''(9) –O–

nucleotidyltransferase from the 

transposon Tn7 (Fling et al., 1985) that 

confers spectinomycin and streptomycin 

resistance 

Intervening Sequence 19410-19943 Sequence used in DNA cloning 

OR8-ori-pBR322 19944-20532 Origin of replication from plasmid 

pBR322 for maintenance of plasmid in 

E. coli (Sutcliffe, 1979). 

Intervening Sequence 20533-21154 Sequence used in DNA cloning 

OR8-ori V 21155-21551 Origin of replication from the broad host 

range plasmid RK2 for maintenance of 

plasmid in Agrobacterium (Stalker et al., 

1981). 

Intervening Sequence 21552-21637 Sequence used in DNA cloning 
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9 E,Enhancer
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III.C. The cry1B.868 Coding Sequence and Cry1B.868 Protein 

The cry1B.868 expression cassette in MON 95379 encodes a 127 kDa Cry1B.868 protein 

consisting of a single polypeptide of 1199 amino acids (Figure III-3.). As is typical for plantexpressed proteins, the lead methionine of the Cry1B.868 protein is not present. The cry1B.868 

coding sequence is the coding sequence of domains I and II from Cry1Be, domain III from Cry1Ca, 

and the C-terminal domain from Cry1Ab from various subspecies of soil bacterium B. 

thuringiensis. The presence of Cry1B.868 protein in maize provides protection against 

lepidopteran pests. 

 
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 1 MTSNRKNENE IINALSIPAV SNHSAQMNLS TDARIEDSLC IAEGNNIDPF 

 51 VSASTVQTGI NIAGRILGVL GVPFAGQIAS FYSFLVGELW PRGRDPWEIF 

 101 LEHVEQLIRQ QVTENTRDTA LARLQGLGNS FRAYQQSLED WLENRDDART 

 151 RSVLYTQYIA LELDFLNAMP LFAIRNQEVP LLMVYAQAAN LHLLLLRDAS 

 201 LFGSEFGLTS QEIQRYYERQ VEKTREYSDY CARWYNTGLN NLRGTNAESW 

 251 LRYNQFRRDL TLGVLDLVAL FPSYDTRVYP MNTSAQLTRE IYTDPIGRTN 

 301 APSGFASTNW FNNNAPSFSA IEAAVIRPPH LLDFPEQLTI FSVLSRWSNT 

 351 QYMNYWVGHR LESRTIRGSL STSTHGNTNT SINPVTLQFT SRDVYRTESF 

 401 AGINILLTTP VNGVPWARFN WRNPLNSLRG SLLYTIGYTG VGTQLFDSET 

 451 ELPPETTERP NYESYSHRLS NIRLISGNTL RAPVYSWTHR SADRTNTISS 

 501 DSINQIPLVK GFRVWGGTSV ITGPGFTGGD ILRRNTFGDF VSLQVNINSP 

 551 ITQRYRLRFR YASSRDARVI VLTGAASTGV GGQVSVNMPL QKTMEIGENL 

 601 TSRTFRYTDF SNPFSFRANP DIIGISEQPL FGAGSISSGE LYIDKIEIIL 

 651 ADATFEAESD LERAQKAVNE LFTSSNQIGL KTDVTDYHID QVSNLVECLS 

 701 DEFCLDEKKE LSEKVKHAKR LSDERNLLQD PNFRGINRQL DRGWRGSTDI 

 751 TIQGGDDVFK ENYVTLLGTF DECYPTYLYQ KIDESKLKAY TRYQLRGYIE 

 801 DSQDLEIYLI RYNAKHETVN VPGTGSLWPL SAPSPIGKCA HHSHHFSLDI 

 851 DVGCTDLNED LGVWVIFKIK TQDGHARLGN LEFLEEKPLV GEALARVKRA 

 901 EKKWRDKREK LEWETNIVYK EAKESVDALF VNSQYDRLQA DTNIAMIHAA 

 951 DKRVHSIREA YLPELSVIPG VNAAIFEELE GRIFTAFSLY DARNVIKNGD 

1001 FNNGLSCWNV KGHVDVEEQN NHRSVLVVPE WEAEVSQEVR VCPGRGYILR 

1051 VTAYKEGYGE GCVTIHEIEN NTDELKFSNC VEEEVYPNNT VTCNDYTATQ 

1101 EEYEGTYTSR NRGYDGAYES NSSVPADYAS AYEEKAYTDG RRDNPCESNR 

1151 GYGDYTPLPA GYVTKELEYF PETDKVWIEI GETEGTFIVD SVELLLMEE 

Figure III-3. Deduced Amino Acid Sequence of the Cry1B.868 Protein 

The amino acid sequence of the MON 95379 Cry1B.868 protein was deduced from the full-length coding 

nucleotide sequence present in PV-ZMIR522223 (See Table III-1 for more details). The lead methionine 

(boxed with solid line) of the Cry1B.868 protein produced in MON 95379 is cleaved in vivo. 

 
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III.D. The cry1Da_7 Coding Sequence and Cry1Da_7 Protein 

The cry1Da_7 expression cassette in MON 95379 encodes a 132 kDa Cry1Da_7 protein consisting 

of a single polypeptide of 1166 amino acids (Figure III-4). The lead methionine of the Cry1Da_7 

protein is cleaved in vivo. The cry1Da_7 coding sequence is a modified version of the cry1Da

gene in soil bacterium Bacillus thuringiensis that encodes the Cry1Da protein. The Cry1Da_7 

protein expressed by MON 95379 is highly homologous (approximately 99.7% sequence 

similarity) to the amino acid sequence of wild-type Cry1Da from B. thuringiensis, with one amino 

acid addition (alanine in position 2) and three amino acid substitutions (S282V, Y316S, I368P). 

The presence of Cry1Da_7 protein in maize provides protection against lepidopteran pests. 

 
Bayer CropScience LP CR282-20U1 53

 1 MAEINNQNQC VPYNCLSNPK EIILGEERLE TGNTVADISL GLINFLYSNF 

 51 VPGGGFIVGL LELIWGFIGP SQWDIFLAQI EQLISQRIEE FARNQAISRL 

 101 EGLSNLYKVY VRAFSDWEKD PTNPALREEM RIQFNDMNSA LITAIPLFRV 

 151 QNYEVALLSV YVQAANLHLS ILRDVSVFGE RWGYDTATIN NRYSDLTSLI 

 201 HVYTNHCVDT YNQGLRRLEG RFLSDWIVYN RFRRQLTISV LDIVAFFPNY 

 251 DIRTYPIQTA TQLTREVYLD LPFINENLSP AAVYPTFSAA ESAIIRSPHL 

 301 VDFLNSFTIY TDSLARSAYW GGHLVNSFRT GTTTNLIRSP LYGREGNTER 

 351 PVTITASPSV PIFRTLSYPT GLDNSNPVAG IEGVEFQNTI SRSIYRKSGP 

 401 IDSFSELPPQ DASVSPAIGY SHRLCHATFL ERISGPRIAG TVFSWTHRSA 

 451 SPTNEVSPSR ITQIPWVKAH TLASGASVIK GPGFTGGDIL TRNSMGELGT 

 501 LRVTFTGRLP QSYYIRFRYA SVANRSGTFR YSQPPSYGIS FPKTMDAGEP 

 551 LTSRSFAHTT LFTPITFSRA QEEFDLYIQS GVYIDRIEFI PVTATFEAEY 

 601 DLERAQKVVN ALFTSTNQLG LKTDVTDYHI DQVSNLVACL SDEFCLDEKR 

 651 ELSEKVKHAK RLSDERNLLQ DPNFRGINRQ PDRGWRGSTD ITIQGGDDVF 

 701 KENYVTLPGT FDECYPTYLY QKIDESKLKA YTRYQLRGYI EDSQDLEIYL 

 751 IRYNAKHEIV NVPGTGSLWP LSVENQIGPC GEPNRCAPHL EWNPDLHCSC 

 801 RDGEKCAHHS HHFSLDIDVG CTDLNEDLGV WVIFKIKTQD GHARLGNLEF 

 851 LEEKPLLGEA LARVKRAEKK WRDKRETLQL ETTIVYKEAK ESVDALFVNS 

 901 QYDRLQADTN IAMIHAADKR VHRIREAYLP ELSVIPGVNA AIFEELEERI 

 951 FTAFSLYDAR NIIKNGDFNN GLLCWNVKGH VEVEEQNNHR SVLVIPEWEA 

1001 EVSQEVRVCP GRGYILRVTA YKEGYGEGCV TIHEIENNTD ELKFNNCVEE 

1051 EVYPNNTVTC INYTATQEEY EGTYTSRNRG YDEAYGNNPS VPADYASVYE 

1101 EKSYTDRRRE NPCESNRGYG DYTPLPAGYV TKELEYFPET DKVWIEIGET 

1151 EGTFIVDSVE LLLMEE 

Figure III-4. Deduced Amino Acid Sequence of the Cry1Da_7 Protein 

The amino acid sequence of the MON 95379 Cry1Da_7 protein was deduced from the full-length coding 

nucleotide sequence present in PV-ZMIR522223 (See Table III-1 for more details). The lead methionine 

(boxed with solid line) of the Cry1Da_7 protein produced in MON 95379 is cleaved in vivo. 

 
Bayer CropScience LP CR282-20U1 54

III.E. Regulatory Sequences 

The cry1B.868 coding sequence in MON 95379 is under the regulation of the promoter, 5' 

untranslated region (UTR) and intron for a ubiquitin gene (Ubq) from Zea mays subsp. Mexicana

(Mexican teosinte) that direct transcription in plant cells (Cornejo et al., 1993). The cry1B.868

coding sequence also utilizes the 3′ UTR sequence of the Lipid Transfer Protein-like gene (LTP) 

from Oryza sativa (rice) that directs polyadenylation of mRNA (Hunt, 1994). 

The codon optimized cry1Da_7 coding sequence in MON 95379 is under the regulation of the 

enhancer from the 35S RNA of Figwort mosaic virus (FMV) (Richins et al., 1987) that enhances 

transcription in most plant cells (Rogers, 2000). In addition to the enhancer the cry1Da_7 coding 

sequence is regulated by the promoter and 5' UTR from Setaria italica (foxtail millet) tonoplast 

membrane integral protein (Tip) gene (Hernandez-Garcia and Finer, 2014) and the intron and 

flanking UTR sequence from the Actin 15 (Act 15) gene from Oryza sativa (rice) that is involved 

in regulating gene expression (Rose, 2008). The cry1Da_7 coding sequence also utilizes 3' UTR 

sequence from the GOS2 gene encoding a translation initiation factor from Oryza sativa (rice) that 

directs polyadenylation of mRNA (Hunt, 1994). 

The cp4 epsps selectable marker cassette is also part of the originally inserted T-DNA in 

MON 95379. The selectable marker cassette is under the regulation of the promoter, 5' UTR, intron 

and 3′ UTR sequences of the OsTubA gene family from Oryza sativa (rice) encoding α-tubulin 

(Jeon et al., 2000) that directs transcription and polyadenylation of mRNA in plant cells. The cp4 

epsps gene was eliminated from subsequent progeny using the Cre/lox recombination system for 

marker removal (Hare and Chua, 2002; Zhang et al., 2003). The cp4 epsps cassette was flanked by 

loxP sites that allowed the cassette to be excised by Cre recombinase when plants were crossed 

with maize plants expressing the cre gene (the “Cre line” was developed with the transformation 

plasmid vector PV-ZMOO513642). The cre gene was subsequently segregated out by 

conventional breeding to produce the MON 95379 product from which the cp4 epsps gene was 

eliminated. The absence of both the cp4 epsps gene, the cre gene and their associated genetic 

elements were confirmed in the F4 generation (Figure IV-4). Since the cp4 epsps gene and 

sequence derived from PV-ZMOO513642 were eliminated through conventional breeding, the 

resulting progeny only contain the genes of interest (cry1B.868 and cry1Da_7) and not the gene 

used for selection. 

III.F. T-DNA Border Regions 

PV-ZMIR522223 contains Left and Right Border regions (Figure III-2 and Table III-1) that were 

derived from A. tumefaciens plasmids. The border regions each contain a nick site that is the site 

of DNA exchange during transformation (Barker et al., 1983; Depicker et al., 1982; Zambryski et 

al., 1982). The border regions separate the T-DNA from the plasmid backbone region and are 

involved in the efficient transfer of T-DNA into the maize genome. 

 
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III.G. Genetic Elements Outside the T-DNA Border Regions 

Genetic elements that exist outside of the T-DNA border regions are those that are essential for 

the maintenance or selection of PV-ZMIR522223 in bacteria and are referred to as plasmid 

backbone. The origin of replication, ori V, is required for the maintenance of the plasmid in 

Agrobacterium and is derived from the broad host range plasmid RK2 (Stalker et al., 1981). The 

origin of replication, ori-pBR322, is required for the maintenance of the plasmid in E. coli and is 

derived from the plasmid vector pBR322 (Sutcliffe, 1979). The selectable marker aadA is the 

coding sequence for an aminoglycoside-modifying enzyme, 3''(9)–O–nucleotidyltransferase from 

the transposon Tn7 (Fling et al., 1985) that confers spectinomycin and streptomycin resistance in 

E. coli and Agrobacterium during molecular cloning. Because these elements are outside the 

border regions, they are not expected to be transferred into the maize genome. The absence of the 

backbone and other unintended plasmid sequence in MON 95379 was confirmed by sequencing 

and bioinformatic analyses (Section IV.B). 
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IV. CHARACTERIZATION OF THE GENETIC MODIFICATION 

This section describes the methods and results of a comprehensive molecular characterization of 

the genetic modification present in MON 95379. It provides information on the DNA insertion(s) 

into the plant genome of MON 95379, and additional information regarding the arrangement and 

stability of the introduced genetic material. 

IV.A. Description of Methodology Used to Characterize MON 95379 

A schematic representation of the next generation sequencing (NGS) methodology and the basis 

of the characterization using NGS and PCR sequencing are illustrated in Figure IV-1 below. 

Appendix B defines the test, control and reference substances, and provides an additional overview 

of these techniques, their use in DNA characterization in maize plants and the materials and 

methods. 
Bayer CropScience LP CR282-20U1 57

Figure IV-1. Molecular Characterization using Sequencing and Bioinformatics 

Genomic DNA from the test and the conventional control was sequenced using technology that produces a 

set of short, randomly-distributed sequence reads that comprehensively cover test and control genomes 

(Step 1). Utilizing these genomic sequence reads, bioinformatics searches are conducted to identify all 

sequence reads that are significantly similar to the transformation plasmid (Step 2). These captured reads 

are then mapped and analyzed to determine the presence/absence of transformation plasmid backbone 

sequences, identify insert junctions, and to determine the insert and copy number (Step 3). Overlapping 

PCR products are also produced which span any insert and their wild type locus (Step 4 and Step 5, 

respectively); these overlapping PCR products are sequenced to allow for detailed characterization of the 

inserted DNA and insertion site. 

 

Step 1: Next Generation Sequencing (NGS) of 

genomic DNA samples. A collection of read 

sequences are generated which comprehensively 

cover the test and control sample genomes

Step 2: Selection of the reads containing 

sequence significantly similar to that of the 

transformation plasmid

Step 3: Bioinformatic analysis to detect and 

characterize all read sequences originating from 

the transgenic insertion(s)

1. Presence or absence of backbone insertions:

No unintended backbone or T-DNA

sequences detected (Read Mapping)

2. Insert and copy number determined:

Junctions located and characterized

Step 4: Directed sequencing across the insertion 

from the 5’ flank to the 3’ flank

Step 5: Directed sequencing across the wild type 

insertion site

3. Exact sequence of insert(s)

4. Organization and intactness of genetic 

elements in the insert sequence is confirmed

5. Integrity and organization of insertion site(s)

Experimental Stage Resultant Molecular Characterization

NGS

Directed 

Sequencing
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The NGS method was used to characterize the genomic DNA from MON 95379 and the 

conventional control by generating short (~150 bp) randomly distributed sequence fragments 

(sequencing reads) generated in sufficient number to ensure comprehensive coverage of the sample 

genomes. It has been previously demonstrated that whole genome sequencing at 75× depth of 

coverage is adequate to provide comprehensive coverage and ensure detection of inserted DNA 

(Kovalic et al., 2012). A comprehensive analysis of NGS as a characterization method 

demonstrated that coverage depth as low as 11× is sufficient to detect both intended transgenes as 

well as unintended inserted vector-derived fragments as small as 25 base pairs in length (Cade et 

al., 2018). Therefore, 75× coverage is a robust level of sequencing for the complete 

characterization of both homozygous and hemizygous transgenes, and, well in excess of the levels 

which have been demonstrated as sufficient for identifying unintended inserted fragments. To 

confirm sufficient sequence coverage of the genome, the 150 bp sequence reads were analyzed to 

determine the coverage of a known single-copy endogenous maize gene. This establishes the depth 

of coverage (the median number of times each base of the genome is independently sequenced). 

Furthermore, the sensitivity of the method was assessed by sequencing the transformation plasmid 

and then sampling the data to represent a single genome equivalent dataset and a 1/10th genome 

equivalent dataset. This confirms the method’s ability to detect any sequences derived from the 

transformation plasmid. Bioinformatics analysis was then used to select sequencing reads that 

contained sequences similar to the transformation plasmid, and these were analyzed in depth to 

determine the number of DNA inserts. NGS was run on five breeding generations of MON 95379 

and the appropriate conventional controls. Results of NGS are shown in Sections IV.B and IV.E. 

The DNA inserts of MON 95379 was determined by mapping of sequencing reads relative to the 

transformation plasmid and identifying junctions and unpaired read mappings adjacent to the 

junctions. Examples of five types of NGS reads are shown in Figure IV-2. The junctions of the 

DNA insert and the flanking DNA are unique for each insertion (Kovalic et al., 2012). Therefore, 

insertion sites can be recognized by analyzing for sequence reads containing such junctions. 

Directed sequencing (locus-specific PCR and DNA sequencing analyses, Figure IV-1, Step 4) 

complements the NGS method. Sequencing of the insert and flanking genomic DNA determined 

the complete sequence of the insert and flanks by evaluating if the sequence of the insert was 

identical to the corresponding sequence from the T-DNA in PV-ZMIR522223, and if each genetic 

element in the insert was intact. It also characterizes the flank sequence beyond the insert 

corresponding to the genomic DNA of the transformed maize. Results are described in Sections 

IV.B, IV.C and IV.D; methods are presented in Appendix B. 

The stability of the T-DNA present in MON 95379 across multiple breeding generations was 

evaluated by NGS as described above. This information was used to determine the number and 

identity of the DNA inserts in each generation. For a single copy T-DNA insert, two junction 

sequence types are expected. In the case of an event where a single locus is stably inherited over 

multiple breeding generations, two identical junction sequences would be detected across all the 

breeding generations tested. Results are described in Section IV.E; methods are presented in 

Appendix B. 

Segregation analysis of the T-DNA was conducted to determine the inheritance and generational 

stability of the insert in MON 95379. Segregation analysis corroborates the insert stability 
Bayer CropScience LP CR282-20U1 59

demonstrated by NGS and independently establishes the genetic behavior of the T-DNA. Results 

are described in Section IV.F. 

Figure IV-2. Five Types of NGS Reads 

NGS yields data in the form of read pairs where sequence from each end of a size selected DNA fragment 

is returned. Depicted above are five types of sequencing reads/read pairs generated by NGS sequencing 

which can be found spanning or outside of junction points. Sequence boxes are color-filled if it matches 

with plasmid sequence, and empty if it matches with genomic sequence. Grey highlighting indicates 

sequence reads spanning the junction. Junctions are detected by examining the NGS data for reads having 

portions of plasmid sequences that span less than the full read, as well as reads mapping adjacent to the 

junction points where their mate pair does not map to the plasmid sequence. The five types of sequencing 

reads/read pairs being (1) Paired and unpaired reads mapping to genomic sequence outside of the insert, 

greater than 99.999% of collected reads fall into this category and are not evaluated in this analysis, (2) 

Paired reads mapping entirely to the transformation plasmid sequence, such reads reveal the presence of 

transformation sequence in planta, (3) Paired reads where one read maps entirely within the inserted DNA 

and the other read maps partially to the insert (indicating a junction point), (4) Single read mapping partially 

to the transformation plasmid DNA sequence (indicating a junction point) where its mate maps entirely to 

the genomic flanking sequence and (5) Single read mapping entirely to the transformation plasmid DNA 

sequence where its mate maps entirely to genomic flanking sequence, such reads are part of the junction 

signature.
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IV.B. Characterization of the DNA Inserts in MON 95379 

The number of inserted DNA sequences from PV-ZMIR522223 in MON 95379 was assessed by 

generating a comprehensive collection of reads via NGS of MON 95379 genomic DNA using the 

F4 generation (Figure IV-4). A plasmid map of PV-ZMIR522223 is shown in Figure III-2. Table 

IV-1 provides descriptions of the genetic elements present in MON 95379. A schematic 

representation of the insert and flanking sequences in MON 95379 is shown in Figure IV-3. For 

full details on materials and methods see Appendix B. 

IV.B.1. Next Generation Sequencing for MON 95379 and Conventional Control Genomic 

DNA 

Genomic DNA from five distinct breeding generations of MON 95379 (Figure IV-4) and 

conventional controls were isolated from seed and prepared for sequencing. For material and 

method details see Appendix B. These genomic DNA libraries were used to generate short 

(~150 bp) randomly distributed sequencing reads of the maize genome (Figure IV-1, Step 1). 

To demonstrate sufficient sequence coverage the ~150 bp sequence reads were analyzed by 

mapping all reads to a known single copy endogenous gene (Zea mays pyruvate decarboxylase 

(pdc3), GenBank Accession: AF370006.2) in each of the five distinct breeding generations. The 

analysis of sequence coverage plots showed that the depth of coverage (i.e., the median number of 

times any base of the genome is expected to be independently sequenced) was 82× or greater for 

the five generations of MON 95379 (F4, F5, F4F1, F5F1, and F6F1) and the conventional controls 

(Appendix B, Table B-1). It has been previously demonstrated that whole genome sequencing at 

75× depth of coverage provides comprehensive coverage and ensures detection of inserted DNA 

(Cade et al., 2018; Kovalic et al., 2012). Total sequencing generated, renderings of mappings 

across subsequent generations, controls, and reference substances, and summary statistics of 

T-DNA coverage are included in Appendix K. 

To demonstrate the method’s ability to detect any sequences derived from the PV-ZMIR522223 

transformation plasmid or the Cre recombinase-containing transformation plasmid vector 

PV-ZMOO513642, a sample of PV-ZMIR522223 and PV-ZMOO513642, were sequenced by 

NGS following the same processes outlined for all samples in Appendix B. The resulting 

PV-ZMIR522223 and PV-ZMOO513642 reads were randomly selected to achieve a depth of one 

and 1/10th genome equivalent (relative to the median coverage of the LH244 conventional control). 

In all cases, 100% coverage of the known PV-ZMIR522223 or PV-ZMOO513642 sequences were 

observed (Appendix B, Table B-2). This result demonstrates that all nucleotides of 

PV-ZMIR522223 and PV-ZMOO513642 are detectable by the sequencing and bioinformatic 

assessments performed and that a detection level of at least 1/10th genome equivalent was achieved 

for the plasmid DNA sequence assessment. 
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Table IV-1. Summary of Genetic Elements in MON 95379 

Genetic Element1 Location in 

Sequence2

Function (Reference)

5' Flanking DNA 1-1000 DNA sequence flanking the 5' end of the 

insert 

B3-Left Border Region

r1

1001-1186 DNA region from 

Agrobacterium tumefaciens containing the 

left border sequence used for transfer of 

the T–DNA (Barker et al., 1983). 

Intervening Sequence 1187-1221 Sequence used in DNA cloning 

loxP 1222-1255 Sequence from Bacteriophage P1 for the 

loxP recombination site recognized by the 

Cre recombinase (Russell et al., 1992). 

Intervening Sequence 1256-1366 Sequence used in DNA cloning 

T4-Ltp 1367-1666 3' UTR sequence of a Lipid Transfer 

Protein-like gene (LTP) from Oryza sativa

(rice) that directs polyadenylation of 

mRNA (Hunt, 1994). 

Intervening Sequence 1667-1675 Sequence used in DNA cloning 

CS5-Cry1B.868 1676-5275 Coding sequences of three domains and a 

protoxin sourced from Bacillus 

thuringiensis (Bt) parental proteins 

arranged as a single chimeric pesticidal 

protein (Cry1B.868) that confers 

protection against lepidopteran insect 

pests via insect midgut disruption (Wang 

et al., 2019). 

Intervening Sequence 5276-5301 Sequence used in DNA cloning 

P6-Zm.Ubq 5302-7309 Promoter, 5' UTR and first intron 

sequences of the ubiquitin (Ubq) gene 

from Zea mays subsp. Mexicana (Mexican 

teosinte) that directs transcription in plant 

cells (Cornejo et al., 1993). 

Intervening Sequence 7310-7336 Sequence used in DNA cloning 

E7-FMV 7337-7873 Enhancer from the 35S RNA of figwort 

mosaic virus (FMV) (Richins et al., 1987) 

that enhances transcription in most plant 

cells (Rogers, 2000). 

Intervening Sequence 7874-7884 Sequence used in DNA cloning 

 
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Table IV-1. Summary of Genetic Elements in MON 95379 (continued) 

P-Tip 7885-8865 Promoter and 5' UTR sequences from 

Setaria italica (foxtail millet) tonoplast 

membrane integral protein (Tip) gene 

(Hernandez-Garcia and Finer, 2014). 

Intervening Sequence 8866-8873 Sequence used in DNA cloning 

I8-Act15 8874-10166 Intron and flanking UTR sequence from 

the Actin 15 (Act 15) gene from Oryza 

sativa (rice) that is involved in 

regulating gene expression (Rose, 2008). 

Intervening Sequence 10167-10184 Sequence used in DNA cloning 

CS-Cry1Da_7 10185-13685 Codon optimized coding sequence of 

cry1Da_7 from Bacillus 

thuringiensis (Bt) encoding a modified 

Cry1Da_7 protein that confers 

protection against lepidopteran insect 

pests via insect midgut disruption (Wang 

et al., 2019). 

Intervening Sequence 13686-13701 Sequence used in DNA cloning 

T-GOS2 13702-14169 3' UTR sequence from the GOS2 gene 

encoding a translation initiation factor 

from Oryza sativa (rice) that directs 

polyadenylation of mRNA (Hunt, 1994). 

Intervening Sequence 14170-14322 Sequence used in DNA cloning 

3' Flanking DNA 14323-15322 DNA sequence flanking the 3' end of the 

insert 

1

 Although flanking sequences and intervening sequences are not functional genetic elements, they 

comprise a portion of the sequence. 

2

 Numbering refers to the sequence of the insert in MON 95379 and adjacent DNA 

3

 B, Border 

4

 T, Transcription Termination Sequence 

5

 CS, Coding Sequence 

6

 P, Promoter 

7

 E, Enhancer 

8

 I, Intron 

r1 Superscript in Left indicate that the sequence in MON 95379 was truncated compared to the sequences 

in PV-ZMIR522223 
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Figure IV-3. Schematic Representation of the Insert and Flanking Sequences in MON 95379 

DNA derived from T-DNA of PV-ZMIR522223 integrated in MON 95379. Right-angled arrows indicate the ends of the integrated T-DNA and the 

beginning of the flanking sequence. Identified on the map are genetic elements within the insert. This schematic diagram may not be drawn to 

scale. r1 Superscript in Left Border Regions indicate that the sequence in MON 95379 was truncated compared to the sequences in PV-ZMIR522223 
Bayer CropScience LP CR282-20U1 64

Figure IV-4. Breeding History of MON 95379 

The generations used for molecular characterization and insert stability analyses are indicated in bold text. 

R0 corresponds to the transformed plant, 8 designates self-pollination. 

1

Generations used to confirm insert stability. 

2 Generation used for molecular characterization. 

3 Generation used for commercial development of MON 95379.

4 The F2 generation was screened for plants lacking the cre gene. Only those plants lacking the cre gene 

were self-pollinated to create a F3 population of plants lacking the cre gene.

 

R0

R1

R2

× HCL617

× LH244 Cre

F1 

F24

F3

F41,2,3 F4F11

F5 F5F11 × HCL617

F6 F6F11 × HCL617

1
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IV.B.2. Selection of Sequence Reads Containing Sequence of the PV-ZMIR522223 and 

PV-ZMOO513642 

The transformation plasmid, PV-ZMIR522223, was transformed into the parental variety LH244 

to produce MON 95379. Consequently, any DNA inserted into MON 95379 will consist of 

sequences that are similar to the PV-ZMIR522223 DNA sequence. Therefore, to fully characterize 

the DNA from PV-ZMIR522223 inserted in MON 95379, it is sufficient to completely analyze 

only the sequence reads that have similarity to PV-ZMIR522223 

(Figure IV-1, Step 2). Similarly, to confirm the absence of the Cre-containing transformation 

plasmid vector, PV-ZMOO513642, in MON 95379 it is sufficient to completely analyze only the 

sequence reads that have similarity to PV-ZMOO513642. 

Using established criteria (described in the materials and methods, Appendix B), any sequence 

reads similar to PV-ZMIR522223 and PV-ZMOO513642 were selected from MON 95379 

sequence datasets (PV-ZMOO513642 selection was only conducted on the F4 generation) and 

were then used as input data for bioinformatic junction sequence analysis. PV-ZMIR522223 and 

PV-ZMOO513642 sequences were also compared against the conventional control sequence 

datasets. 

IV.B.3. Determination of T-DNA Copy Number and Presence or Absence of Plasmid Vector 

Backbone 

Mapping sequence reads relative to the transformation plasmid allows for the identification of 

junction signatures, the presence or absence of plasmid backbone sequence and the number of 

T-DNA insertions. For a single copy T-DNA insert sequence at a single genomic locus and the 

complete absence of plasmid vector backbone, a single junction signature pair and few if any reads 

aligning with the transformation plasmid backbone sequences are expected. 

When reads from the LH244 dataset were aligned with the transformation plasmid sequence, a 

number of reads mapped to the T-DNA promoter element Zm.Ubq sequence (Figure IV-5). The 

alignment of these sequence reads is the result of an endogenous maize sequence that is 

homologous to the T-DNA encoded promoter element Zm.Ubq sequence. No other regions of 

homology were identified between the PV-ZMIR522223 transformation plasmid reference 

sequence and the conventional control. 

When reads from the MON 95379 (F4) dataset were aligned with the transformation plasmid 

sequence, large numbers of reads mapped to the intended T-DNA sequence, one paired read was 

identified which aligned to the transformation plasmid backbone, a number of unpaired reads 

aligned to the T-DNA promoter element Zm.Ubq, and no reads mapped to the selectable marker 

cassette (Figure IV-6). The presence of unpaired reads aliging to this Zm.Ubq are indicative of 

sequences from an endogenous maize element mapping to the transformation vector 

(Appendix L). This is most readily observed by comparing and contrasting the unpaired reads 

aligning to the Zm.Ubq region to those observed in the LH244 conventional control dataset (Panel 

1, Figures IV-5 and IV-6). A comparison of mapped reads from F4 and the conventional control 

reveals that mapped reads are present at sample equivalent levels and show virtually identical 

mapping signatures. This, in conjunction with an absence of junction sites, indicates that all 

additional reads and subsequent relative increases of depth across this region are entirely derived 
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from the endogenous maize Zm.Ubq homolog, and therefore not indicative of any additional 

T-DNA insertion (partial or whole). The mapping of large numbers of reads from the MON 95379 

(F4) dataset to the intended T-DNA sequence is expected and fully consistent with the presence of 

the inserted DNA. 

A single pair of reads was found to align with OR-ori-pBR322 sequences (Figure IV-6). The 

sporadic low-level detection of plasmid sequences such as OR-ori-pBR322 has previously been 

described (Zastrow-Hayes et al., 2015), and reported (see Supplemental Figure S1 in Yang et al. 

(2013)), and is due to the presence of environmental bacteria in tissue samples used in the 

preparation of genomic DNA used for library construction. The presence of this sequence from 

environmental bacteria does not indicate the presence of backbone sequence in the MON 95379 

(F4) generation. This analysis indicates that MON 95379 (F4) does not contain inserted sequence 

from the transformation plasmid backbone. 

No reads mapped to the selectable marker cassette (T-TubA, TS-CTP2, CS- cp4 epsps, and PTubA). This result is expected as MON 95379 was crossed with a Cre recombinase expressing 

line that enable the removal of the cp4 epsps gene cassette positioned between two excision 

targeting lox sites (Hare and Chua, 2002; Zhang et al., 2003). After excision, a single lox site 

remained, as expected. The absence of the selectable marker cassette results in a gap in reads as 

illustrated in Figure IV-6. 

To determine the insert number in MON 95379 (F4), selected reads mapping to T-DNA as 

described above were analyzed to identify junctions. This bioinformatic analysis is used to find 

and classify partially matched reads characteristic of the ends of insertions. The number of unique 

junctions determined by this analysis are shown in Table IV-2. 

Table IV-2. Unique Junction Sequence Class Results 

Sample Junctions Detected 

MON 95379 (F4) 2 

LH244 0 

Detailed mapping information of the junction sequences is shown in Figure IV-6. The location and 

orientation of the junction sequences relative to T-DNA insert determined for MON 95379 are 

illustrated in Figure IV-6, panels 1 and 2. As shown in the figure, there are two junctions identified 

in MON 95379. Both junctions contain the T-DNA border sequence joined to flanking genomic 

sequence, indicating that they represent the sequences at the junctions of the intended T-DNA 

insert and the maize genome. As described earlier, no junctions were detected in any of the 

conventional maize control samples. 

Considered together, the absence of plasmid backbone and the presence of two junctions (joining 

T-DNA borders and flanking sequences) indicate a single intended T-DNA at a single locus in the 

genome of MON 95379. Both of these junctions originate from the same locus of the MON 95379 

genome and are linked by contiguous, previously determined and expected DNA sequence (with 

the exception of the selectable marker cassette which was excised as described earlier). This is 
Bayer CropScience LP CR282-20U1 67

demonstrated by complete coverage of the sequenced reads spanning the interval between the 

junctions and the directed sequencing of overlapping PCR products described in Section IV.C. 

Based on the comprehensive NGS and junction identification it is concluded that MON 95379 

contains one copy of the T-DNA inserted into a single locus. This conclusion is confirmed by the 

sequencing and analysis of overlapping PCR products from this locus as described in Section IV.C. 

Figure IV-5. Read Mapping of Conventional Maize LH244 Versus PV-ZMIR522223

Panel 1 shows the location of unpaired mapped reads, Panel 2 shows paired mapped reads, and Panel 3 

shows a representation of combined raw read depth for unpaired and paired reads across the reference. 

Vertical lines show genetic element boundaries. 

 
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Figure IV-6. Read Mapping of MON 95379 (F4) Versus PV-ZMIR522223 

Panel 1 shows the location of unpaired mapped reads. Panel 2 shows paired mapped reads and Panel 3 

shows a representation of combined raw read depth for unpaired and paired reads across the reference 

sequence. Vertical lines show genetic element boundaries. The region of flank junction sequences that align 

with the transformation plasmid are shown in red. Comparable results were observed when read mapping 

the F4F1, F5, F5F1, and F6F1 generations of MON 95379 versus PV-ZMIR522223 (Appendix K). 
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IV.B.4. Determination of Absence of Plasmid Vector PV-ZMOO513642 

At the R2 generation (Figure IV-4), MON 95379 was crossed with a Cre recombinase expressing 

line. The Cre/lox technology enables the removal of the selectable marker cassette (T-TubA, TSCTP2, CS-cp4 epsps, and P-TubA) which was inserted during the transformation as part of the 

T-DNA insertion that also included the cry1B.868 and cry1Da_7 trait cassettes. The resulting F1 

progeny were self-pollinated and plants from the F2 generation were screened for the absence of 

the cre gene and associated genetic elements (and other sequences from plasmid 

PV-ZMOO513642), allowing for selection of lines lacking the Cre recombinase cassette that were 

used as the progenitor of subsequent generations and the final product. 

To confirm the absence of the Cre recombinase cassette and any part of the cre gene containing 

transformation vector, MON 95379 was assessed by utilizing the comprehensive collection of 

generation F4 NGS reads and subsequently mapping them to the cre gene containing 

transformation plasmid (PV-ZMOO513642) sequence Figure IV-7. In the absence of any 

PV-ZMOO513642 insertions there should be zero junction signature pairs and limited reads 

aligning with the PV-ZMOO513642 sequences. 

When reads from the MON 95379 (F4) dataset were aligned with the PV-ZMOO513642 sequence, 

a number of reads aligned to the left border region (B-Left Border Region). Only a small number 

of reads mapping to the promoter element Ract1 mapped to T-DNA, and only one paired read was 

identified which aligned to the PV-ZMOO513642 backbone (Figure IV-7). 

The mapping of a number of reads from the MON 95379 (F4) dataset to the left border region (BLeft Border Region) is expected since PV-ZMOO513642 and PV-ZMIR522223 share the same 

left border region sequence and PV-ZMIR522223’s left border region is present in MON 95379 

(Figure IV-6). The small number of reads mapping to the promoter element Ract1 are also present 

in the LH244 control background. This is fully consistent with the presence of a homologous 

sequence being present in the LH244 control background (Figure IV-8) and does not indicate the 

presence of PV-ZMOO513642 T-DNA in MON 95379. Additionally, a single pair of reads was 

found to align with OR-ori-pBR322 sequences (Figure IV-7). The sporadic low level detection of 

plasmid sequences such as OR-ori-pBR322 has previously been described (Zastrow-Hayes et al., 

2015), and reported (see Supplemental Figure S1 in Yang et al. (2013)), and is due to the presence 

of environmental bacteria in tissue samples used in the preparation of genomic DNA used for 

library construction. As such, the incidence of this sequence from environmental bacteria does not 

indicate the presence of backbone sequence in the MON 95379 (F4) generation. Considered 

together, the limited reads aligning with the PV-ZMOO513642 sequences and the absence 

junctions (joining T-DNA borders and flanking sequences; Table IV-3) indicate that MON 95379 

(F4), and subsequent generations, do not contain inserted sequence from the cre gene containing 

transformation plasmid, PV-ZMOO513642. 

 
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Table IV-3. Unique Junction Sequence Class Results 

Sample Junctions Detected 

MON 95379 (F4) 0 

LH244 0 

Figure IV-7. Read Mapping of MON 95379 (F4) Versus PV-ZMOO513642 

Panel 1 shows the location of unpaired mapped reads. Panel 2 shows paired mapped reads and Panel 3 

shows a representation of combined raw read depth for unpaired and paired reads across the reference 

sequence. Vertical lines show genetic element boundaries. 

 
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Figure IV-8. Read Mapping of Conventional Maize LH244 Versus PV-ZMOO513642 

Panel 1 shows the location of unpaired mapped reads, there are no paired mapped reads, and Panel 2 shows 

a representation of combined raw read depth for unpaired and paired reads across the reference sequence. 

Vertical lines show genetic element boundaries. 

 
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IV.C. Organization and Sequence of the Insert and Adjacent DNA in MON 95379 

The organization of the elements within the DNA insert and the adjacent genomic DNA was 

assessed using directed DNA sequence analysis (refer to Figure IV-1, Step 4). PCR primers were 

designed to amplify two overlapping regions of the MON 95379 genomic DNA that span the entire 

length of the insert and the adjacent DNA flanking the insert (Figure IV-9). The amplified PCR 

products were subjected to DNA sequencing analyses. The results of this analysis confirm that the 

MON 95379 insert is 13322 bp and that each genetic element within the T-DNA is intact compared 

to PV-ZMIR522223, with the exception of the border regions. The right border region was absent 

and the left border region contained small terminal deletions with the remainder of the inserted 

border region being identical to the sequence in PV-ZMIR522223. The sequence and organization 

of the insert was also shown to be identical to the corresponding T-DNA of PV-ZMIR522223 as 

intended. As noted, in Section IV.B.3, the selectable marker cassette (T-TubA, TS-CTP2, CS-cp4 

epsps, and P-TubA) and one loxP site (bases 478 through 4893 of the PV-ZMIR522223 sequence) 

were excised by Cre recombinase, and as expected, are not present in the MON 95379 sequence. 

This analysis also shows that only T-DNA elements (described in Table IV-1) were present. In 

addition, 1000 base pairs flanking the 5′ end of the MON 95379 insert (Table IV-1, bases 1-1000) 

and 1000 base pairs flanking the 3′ end of the MON 95379 insert (Table IV-1, bases 14323-15322) 

were determined. 

 
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Figure IV-9. Overlapping PCR Analysis across the Insert in MON 95379 

PCR was performed on both conventional control genomic DNA and genomic DNA of the F4 generation 

of MON 95379 using two pairs of primers to generate overlapping PCR fragments from MON 95379 for 

sequencing analysis. To verify the PCR products, 2 μl of each of the PCR reactions was loaded on the gel. 

The expected product size for each amplicon is provided in the illustration of the insert in MON 95379 that 

appears at the bottom of the figure. This figure is a representative of the data generated in the study. Lane 

designations are as follows: 

Lane 

1 1 Kb Plus DNA Ladder 

2 No template control 

3 LH244 Conventional Control 

4 MON 95379 

5 No template control 

6 LH244 Conventional Control 

7 MON 95379 

8 1 Kb Plus DNA Ladder 

Arrows on the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained from the 

1 Kb Plus DNA Ladder (Invitrogen) on the ethidium bromide stained gel. 

r1 Superscript in Left Border Regions indicate that the sequence in MON 95379 was truncated compared 

to the sequences in PV-ZMIR522223. 

1 2 3 4 5 6 7 8

12

11

6.0

5.0

4.0

12

11

6.0

5.0

4.0

Product A Product B
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IV.D. Sequencing of the MON 95379 Insertion Site 

PCR and sequence analysis were performed on genomic DNA extracted from the conventional 

control to examine the insertion site in conventional maize (see Figure IV-1, Step 5). The PCR 

was performed with one primer specific to the genomic DNA sequence flanking the 5’ end of the 

MON 95379 insert paired with a second primer specific to the genomic DNA sequence flanking 

the 3’ end of the insert (Figure IV-10). A sequence comparison between the PCR product 

generated from the conventional control and the sequence generated from the 5’ and 3’ flanking 

sequences of MON 95379 indicates that 160 bases of maize genomic DNA were deleted during 

integration of the T-DNA. Such changes are common during plant transformation (Anderson et 

al., 2016) and these changes presumably resulted from double stranded break repair mechanisms 

in the plant during Agrobacterium-mediated transformation process (Salomon and Puchta, 1998). 

The remainder of the maize genomic DNA sequences flanking the insert in MON 95379 are 

identical to the conventional control. 

 
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Figure IV-10. PCR Amplification of the MON 95379 Insertion Site 

PCR analysis was performed to evaluate the insertion site. PCR was performed on conventional control 

DNA using Primer A, specific to the 5′ flanking sequence, and Primer B, specific to the 3′ flanking 

sequence of the insert in MON 95379. The DNA generated from the conventional control PCR was used 

for sequencing analysis. This illustration depicts the MON 95379 insertion site in the conventional control 

(upper panel) and the MON 95379 insert (lower panel). Approximately 2 μl of each of the PCR reactions 

was loaded on the gel. This figure is representative of the data generated in the study. Lane designations 

are as follows: 

Lane 

1 1 Kb Plus DNA Ladder 

2 No template control 

3 LH244 Conventional Control 

4 1 Kb Plus DNA Ladder 

Arrows on the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained from the 

1 Kb Plus DNA Ladder (Invitrogen) on the ethidium bromide stained gel. 

r1 Superscript in Left Border Regions indicate that the sequence in MON 95379 was truncated compared 

to the sequences in PV-ZMIR522223. 

Primer A

Primer A

Primer B

Primer B

1 2 3 4 

3.0

2.0

3.0

2.0
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IV.E. Determination of Insert Stability over Multiple Generations of MON 95379 

In order to demonstrate the stability of the T-DNA present in MON 95379 through multiple 

breeding generations, NGS was performed using DNA obtained from five breeding generations of 

MON 95379. The breeding history of MON 95379 is presented in Figure IV-4, and the specific 

generations tested are indicated in the figure legend. The MON 95379 (F4) generation was used 

for the molecular characterization analyses discussed in Sections IV.B-IV.D and shown in Figure 

IV-4. To assess generational stability of the T-DNA, four additional generations were evaluated 

by NGS as previously described in Section IV.B, and compared to the fully characterized F4 

generation. The conventional controls used for the generational stability analysis included LH244, 

with similar background genetics to the F4 and F5 generations and represents the original 

transformation line; and LH244 × HCL617, a hybrid with similar background genetics to the F4F1, 

F5F1, F6F1 hybrids. Genomic DNA isolated from each of the selected generations of MON 95379 

and conventional control was used for NGS, mapping, and subsequent junction identification 

(Table IV-4). 

Table IV-4. Junction Sequence Classes Detected 

Sample 

Junction Sequence 

Classes Detected

MON 95379 (F4) 2 

MON 95379 (F5) 2 

MON 95379 (F4F1) 2 

MON 95379 (F5F1) 2 

MON 95379 (F6F1) 2 

LH244 0 

LH244 × HCL617 0 

As shown by alignment to the full flank/insert sequence obtained from directed sequencing, a 

single conserved pair of junctions linked by contiguous known and expected DNA sequence is 

present in MON 95379 (F4). Two identical junctions are found in each of the breeding generations 

(F5, F4F1, F5F1, and F6F1), confirming the insertion of a single copy of PV-ZMIR522223 TDNA at a single locus in the genome of MON 95379, and the consistency of these junctions in the 

mapping data across all generations tested demonstrates that this single locus is stably maintained 

throughout the MON 95379 breeding process. 

These results demonstrate that the single locus of integration characterized in the F4 generation of 

MON 95379 is found in five breeding generations of MON 95379, confirming the stability of the 

insert. This comprehensive NGS and bioinformatic analysis of NGS data from multiple 

generations supports the conclusion that MON 95379 contains a single, stable insert T-DNA. 

 
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IV.F. Inheritance of the Genetic Insert in MON 95379 

The MON 95379 T-DNA resides at a single locus within the maize genome and therefore should 

be inherited according to Mendelian principles of inheritance. During development of lines 

containing MON 95379, phenotypic and genotypic segregation data were recorded to assess the 

inheritance and stability of the MON 95379 T-DNA using Chi square (χ2) analysis over several 

generations. The χ2 analysis is based on comparing the observed segregation ratio to the expected 

segregation ratio according to Mendelian principles. 

The MON 95379 breeding path for generating segregation data is described in Figure IV-11. The 

transformed R0 plant was self-pollinated to generate R1 seed. An individual homozygous positive 

plant for the MON 95379 T-DNA was identified in the R1 segregating population by Real-Time 

TaqMan®3PCR assay. 

The homozygous positive R1 plant was self-pollinated to give rise to R2 seed. At the R2 

generation, plants were crossed with a Cre recombinase expressing line. The Cre/lox technology 

enables the removal of DNA sequence positioned between two excision targeting sequences called 

lox sites (Hare and Chua, 2002; Zhang et al., 2003). The Cre recombinase enzyme facilitates the 

excision of the selectable marker cassette which was inserted during the transformation as part of 

the T-DNA insertion that also included the gene of interest cassettes. After excision, a single lox

site remains in the F1 generation. The resulting F1 progeny were self-pollinated and the F2 

generation were screened for the absence of the cre gene (and other sequences from plasmid PVZMOO513642), allowing for selection of lines lacking the Cre recombinase cassette from 

subsequent generations and the final product. The F2 plants lacking the cre gene were selfpollinated to produce F3 seed. The homozygous positive F3 plant was self-pollinated to give rise 

to F4 seed. The homozygous positive F4 plants were crossed via traditional breeding techniques 

to a proprietary elite inbred parent that does not contain the cry1B.868 and cry1Da_7 coding 

sequences to produce hemizygous F4F1 seed. The hemizygous F4F1 plants were self-pollinated 

to produce F4F2 seed. The F4F2 generation was tested for the presence of MON 95379 T-DNA 

by Real Time TaqMan® PCR assay for cry1B.868. Hemizygous positive F4F2 plants were selfpollinated to produce F4F3 seed. The F4F3 generation was tested for the presence of the T-DNA 

by Real Time TaqMan® PCR assay for cry1B.868. Hemizygous positive F4F3 plants were selfpollinated to produce F4F4 seed. The F4F4 generation was tested for the presence of the T-DNA 

by Real Time TaqMan® PCR assay for cry1B.868. 

The inheritance of the MON 95379 T-DNA was assessed in the F4F2, F4F3, and F4F4 

generations. At all generations, the MON 95379 T-DNA was predicted to segregate at a 1:2:1 ratio 

(homozygous positive: hemizygous positive: homozygous negative) according to Mendelian 

inheritance principles. 

3

 TaqMan® is a registered trademark of Roche Molecular Systems, Inc. 
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A Pearson’s chi square (χ2

) analysis was used to compare the observed segregation ratios of the 

MON 95379 T-DNA I coding sequence to the expected ratios. 

The Chi square was calculated as: 

χ 2 = ∑ [( | o – e | )2

 / e] 

where o = observed frequency of the genotype or phenotype and e = expected frequency of the 

genotype or phenotype. The level of statistical significance was predetermined to be 5% 

(α = 0.05). 

The results of the χ2 analysis of the segregating progeny of MON 95379 are presented in Table 

IV-5. The χ2 value in the F4F2, F4F3, and F4F4 generations indicated no statistically significant 

difference between the observed and expected segregation ratios of MON 95379 T-DNA. These 

results support the conclusion that the MON 95379 T-DNA resides at a single locus within the 

maize genome and is inherited according to Mendelian principles of inheritance. These results are 

also consistent with the molecular characterization data indicating that MON 95379 contains a 

single intact copy of the T-DNA inserted at a single locus in the maize genome (Sections IV.BIV.E). 

 
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Figure IV-11. Breeding Path for Generating Segregation Data for MON 95379

*Chi-square analysis was conducted on segregation data from F4F2, F4F3, and F4F4 generations (bolded 

text).

8: Self-Pollinated

1 The F2 generation was screened for plants lacking the cre gene cassette. Only those plants lacking the

cre gene cassette were self-pollinated to create a F3 generation lacking the cre gene cassette.
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Table IV-5. Segregation Results for MON 95379 from the F4F2, F4F3, and F4F4 

1:2:1 Segregation 

Generation Total Plants

Observed # Plant Homozygous Positive 

Observed # Plant Hemizygous Positive 

Observed # Plant Homozygous Negative 

Expected # Plant Homozygous Positive

Expected # Plant Hemizygous Positive 

Expected # Plant Homozygous Negative 

χ 2 Probability 

F4F2 200 60 85 55 50 100 50 4.75 0.093 F4F3 247 62 125 60 61.75 123.5 61.75 0.07 0.966 F4F4 246 74 109 63 61.5 123 61.5 4.17 0.124 
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IV.G. Characterization of the Genetic Modification Summary and Conclusion 

As described in this section, characterization of the genetic modification in MON 95379 was 

conducted using a combination of sequencing, PCR, and bioinformatics. The results of this 

characterization demonstrate that MON 95379 contains a single copy of the intended T-DNA 

containing the cry1B.868 and cry1Da_7 expression cassettes that is stably integrated at a single 

locus and is inherited according to Mendelian principles over multiple generations. These 

conclusions are based on the following: 

x Molecular characterization of MON 95379 by NGS demonstrated that MON 95379 

contained a single intended DNA insert. These whole-genome analyses provided a 

comprehensive assessment of MON 95379 to determine the presence and identity of 

sequences derived from PV-ZMIR522223 and demonstrated that MON 95379 contains a 

single T-DNA insert, with no detectable backbone or cp4 epsps selectable marker from 

sequences from PV-ZMIR522223 or any sequences from PV-ZMOO513642. 

x Directed sequencing (locus-specific PCR, DNA sequencing and analyses) performed on 

MON 95379 was used to determine the complete sequence of the single DNA insert from 

PV-ZMIR522223, the adjacent flanking DNA, and the 5' and 3' insert-to-flank junctions. 

This analysis confirmed that the sequence and organization of the DNA is identical to the 

corresponding region in the PV-ZMIR522223 T-DNA. Directed sequencing also 

confirmed that the cp4 epsps selectable marker cassette which was excised, along with one 

loxP site, by Cre recombinase, is not present in the MON 95379 sequence. Furthermore, 

the genomic organization at the insertion site in MON 95379 was assessed by comparing 

the sequences flanking the T-DNA insert in MON 95379 to the sequence of the insertion 

site in conventional maize. This analysis determined that 160 bases were deleted at the 

insertion site in MON 95379 upon DNA integration. 

x Generational stability analysis by NGS demonstrated that the single PV-ZMIR522223 

T-DNA insert in MON 95379 has been maintained through five breeding generations, 

thereby confirming the stability of the T-DNA in MON 95379. 

x Segregation data confirm that the inserted T-DNA segregated according to Mendelian 

inheritance patterns, which corroborates the insert stability demonstrated by NGS and 

independently establishes the nature of the T-DNA at a single chromosomal locus. 

Taken together, the characterization of the genetic modification in MON 95379 demonstrates that 

a single copy of the intended T-DNA was stably integrated at a single locus of the maize genome 

and that no PV-ZMIR522223 plasmid backbone, cp4 epsps selectable marker, or 

PV-ZMOO513642 sequences are present in MON 95379. 
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V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE Cry1B.868 

AND Cry1Da_7 PROTEINS PRODUCED IN MON 95379 

Characterization of the introduced protein(s) in a biotechnology-derived crop is important to 

establishing food, feed, and environmental safety. As described in Section IV, MON 95379 

contains cry1B.868 and cry1Da_7 expression cassettes that, when transcribed and translated, result 

in the expression of the Cry1B.868 and Cry1Da_7 proteins, respectively. 

A multistep approach to the safety assessment of the MON 95379 Cry1B.868 and Cry1Da_7 

proteins was conducted according to guidance established by the Codex Alimentarius Commission 

(Codex Alimentarius, 2009) and OECD, which embody the principles and guidance of the FDA’s 

1992 policy on foods from new plant varieties. The assessment includes: 1) characterization of the 

physicochemical and functional properties of each expressed protein; 2) documenting the history 

of safe consumption of the expressed protein or its structural and functional homology to proteins 

that lack adverse effects on human or mammalian health; 3) examination of the similarity of each 

expressed protein to known allergens, toxins or other biologically active proteins known to have 

adverse effects on humans and other mammals; 4) evaluation of the susceptibility of each 

expressed protein to the digestive enzymes pepsin and pancreatin; 5) quantification of each 

expressed proteins’ expression in plant tissues; 6) a confirmatory evaluation of potential 

mammalian toxicity. The safety assessment completed for MON 95379 maize supports the 

conclusion that exposure to the Cry1B.868 and Cry1Da_7 proteins derived from MON 95379 

would not pose any risk to human or mammalian health. 

V.A. Identity and Function of the Cry1B.868 and Cry1Da_7 Proteins from MON 95379 

The MON 95379 Cry1B.868 and Cry1Da_7 proteins belong to the Cry three domain family of 

proteins that has a well-documented mode of action (Gill et al., 1992; OECD, 2007; Schnepf et 

al., 1998; Vachon et al., 2012). Ingestion of Cry proteins by the target insect pest exposes the 

protein to the alkaline conditions and proteases in the insect midgut, resulting in proteolytic 

cleavage of the protein’s protoxin domain, thus solubilizing the parasporal inclusions and 

converting the protein to the active insecticidal toxin. Following activation, this protease-resistant 

core protein is comprised of three distinct structural domains that function in a step-wise 

mechanism of binding to specific membrane-embedded receptors, oligomerization at the 

membrane interface, insertion into the plasma membrane and pore formation leading to loss of cell 

integrity followed by delayed development or insect death (Bravo et al., 2007; Deist et al., 2014). 

Cry1B.868 is a protein that comprises a single polypeptide of 1199 amino acids with an apparent 

molecular weight of approximately 127 kDa. Like other Cry proteins, Cry1B.868 is first 

synthesized as a prototoxin that upon exposure to the midgut of target organisms is cleaved by 

digestive enzymes to yield an approximately 60 kDa activated protein (Bravo et al., 2007). 

Cry1B.868 is a chimeric three domain protein that consists of domains I and II from Cry1Be, 

domain III from Cry1Ca, and the C-terminal domain from Cry1Ab (Figure V-1). Cry1A, Cry1C 

and Cry1B proteins are from a family of insecticidal proteins derived from various subspecies of 

the soil bacterium Bacillus thuringiensis (Bt) which have been used extensively in formulation for 

commercial biopesticides (Betz et al., 2000; Bravo et al., 2011; EFSA, 2012). Cry1B.868 was 

designed using a domain exchange strategy to achieve high levels of activity against target 
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lepidopteran insect pests. Domain exchange is a well-known mechanism in nature, resulting in 

diversities in Cry protein functional activity that have been described extensively in the literature 

(de Maagd et al., 2003; de Maagd et al., 2001). By utilizing modern molecular biological tools, a 

domain exchange strategy has previously been used successfully to switch the functional domains 

of Cry1 proteins to develop microbial biopesticides with improved specificity to lepidopteran 

insect pests. (Baum, 1998; Baum et al., 1999; Gao et al., 2006). Similarly, by exchanging Domain 

III, Cry1B.868 has been engineered to have an enhanced specificity for fall armyworm relative to 

Cry1Be (Wang et al., 2019). Domains I and II of Cry1B.868 are 100% identical to the respective 

domains of Cry1Be. Domain III of Cry1B.868 is 100% identical to domain III of Cry1Ca. The 

C-terminal region of Cry1B.868 is 100% identical to that of Cry1Ab. 

Figure V-1. Schematic Representation of the Primary Domain Architecture of the 

Cry1B.868 Protein 

The domain architecture of the chimeric Cry1B.868 protein is schematically presented. Different shades 

are used to differentiate the origin of domains. For simplicity, the lengths of the domains in this illustration 

are not in proportion to the lengths of amino acid sequence of the respective domains. 

Cry1Da_7 in MON 95379 is a protein that comprises a single polypeptide of 1166 amino acids, 

with an apparent molecular weight of approximately 132 kDa. Like other Cry proteins, it is first 

synthesized as a prototoxin that upon exposure to the midgut of target organisms is cleaved by 

digestive enzymes to yield an approximately 60 kDa activated protein (Bravo et al., 2007). 

Cry1Da_7 is a modified version of the Cry1Da protein that shares approximately 99.7% sequence 

identity to its parent protein (Figure V-2). Cry1Da is an insecticidal protein derived from the soil 

bacterium Bt, which has been used extensively in the formulation of commercial biopesticides 

(Bravo et al., 2011; EFSA, 2012). Cry1Da_7 has four distinct amino acid differences relative to 

Cry1Da. In Domain I, the protein has an additional alanine at position 2. This alanine was added 

to optimize the codon for translation of the protein in planta. In Domain II, there are three amino 

acid substitutions: serine 282 to valine, tyrosine 316 to serine, and isoleucine 368 to proline. These 
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amino acid substitutions result in an improvement in activity towards corn earworm relative to 

Cry1Da while maintaining fall army worm activity (Wang et al., 2019). 

Figure V-2. Schematic Representation of the Primary Domain Architecture of the 

Cry1Da_7 Protein 

Domain architecture of the Cry1Da_7 protein, which differs from its parent protein by only four amino 

acids (an alanine is added in position two in Domain I and 3 single amino acid positions in Domain II; V = 

valine, S = serine, Y = tyrosine, P = proline, I = isoleucine). For simplicity, the lengths of the domains in 

this illustration are not in proportion to the lengths of amino acid sequence of the respective domains. 

V.B. Characterization and Equivalence of Cry1B.868 and Cry1Da_7 Proteins from 

MON 95379 

The safety assessment of crops derived through biotechnology includes characterization of the 

physicochemical and functional properties of the protein(s) produced from the inserted DNA, and 

confirmation of the safety of the protein(s). The expression level of Cry1B.868 and Cry1Da_7 in 

MON 95379 is low, and insufficient for use in the subsequent safety evaluations. Therefore, 

recombinant Cry1B.868 and Cry1Da_7 proteins were produced in Bt, using expression vectors 

with cry1B.868 or cry1Da_7 coding sequences that matched those of the cry1B.868 or cry1Da_7

coding sequences in MON 95379. The physicochemical and functional characteristics of the MON 

95379-produced Cry1B.868 and Cry1Da_7 proteins were determined and shown to be equivalent 

to the Bt-produced Cry1B.868 and Cry1Da_7 proteins, respectively. A summary of the analytical 

results for the characterization and evaluation of the introduced proteins is shown below and the 

details of the materials, methods, and results are described in Appendix C, Appendix D and 

Appendix E.

The MON 95379-produced Cry1B.868 and Cry1Da_7 proteins were purified from the grain of 

MON 95379 and the physicochemical and functional properties were characterized using a panel 

of analytical tests and compared to the Bt-produced version of each respective protein to establish 

equivalence: 1) N-terminal sequence analysis of MON 95379-produced Cry1B.868 and 

Cry1Da_7 identified the expected N-terminal sequence; 2) liquid chromatography-mass 
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spectrometry (LC/MS) analysis yielded peptide masses consistent with the expected peptide 

masses from the theoretical trypsin or Asp-N digest of the MON 95379-produced Cry1B.868 and 

Cry1Da_7 sequences; 3) western blot analysis with antibodies specific for Cry1B.868 and 

Cry1Da_7 proteins demonstrated that the immunoreactive properties of each respective 

MON 95379-produced and Bt-produced protein were equivalent; 4) SDS-PAGE analysis showed 

that the molecular weight based on electrophoretic mobility of each respective 

MON 95379-produced and Bt-produced protein was equivalent; 5) the MON 95379-produced and 

Bt-produced MON 95379 Cry1B.868 and Cry1Da_7 proteins were determined to be nonglycosylated; and 6) insect bioassay data was used to demonstrate equivalent functional activity 

for each MON 95379-produced protein and its corresponding Bt-produced protein (See Appendix 

C and Appendix D). 

Taken together, these data provide a detailed characterization of the MON 95379-produced 

Cry1B.868 and Cry1Da_7 proteins and establish their equivalence to the Bt-produced Cry1B.868 

and Cry1Da_7 proteins, respectively. Demonstration of equivalency justifies the use of the 

Bt-produced Cry1B.868 and Cry1Da_7 proteins in studies to establish the safety of the Cry1B.868 

and Cry1Da_7 proteins expressed in MON 95379. 

V.C. Cry1B.868 and Cry1Da_7 Donor Organism and History of Safe Use 

V.C.1. The Donor Organism has a History of Safe Use 

The coding sequences for the Cry1B.868 and Cry1Da_7 proteins are derived from the bacterium 

Bacillus thuringiensis: 

Kingdom: Bacteria 

Phylum: Firmicutes 

Class: Bacilli 

Order: Bacillales 

Family: Bacillaceae 

Genus: Bacillus 

The Cry1B.868 and Cry1Da_7 proteins are derived from genetic elements that code for crystalline 

proteins (Cry) that are expressed as parasporal inclusions (or δ-endotoxins) in the ubiquitous grampositive bacterium Bacillus thuriengensis (Bt) (Gill et al., 1992; Schnepf et al., 1998; Vachon et 

al., 2012). Bt isolates have a long, documented history of safe use in agriculture and safe human 

consumption. Since the first Bt isolate was registered as a pesticide in 1961, over 180 microbial Bt

products have been registered in the United States (U.S.), with more than 120 microbial products 

registered in the European Union (EU) (Hammond, 2004). Bt microbial biopesticides have been 

safely and directly applied to consumed agricultural commodities including berry crops, cabbage, 

grapes, tomatoes, celery, lettuce, and spinach (U.S. EPA, 1998a). For certain crops, a significant 

percentage of the total U.S. grown crop has been treated with Bt crystal/spore preparations (e.g.,

blackberries (50%), celery (46%), and cabbage (39%)) (U.S. EPA, 1998a). In Europe, residual 

levels of Bt microbials of up to 100,000 CFUs (colony forming units) were observed on fresh 

vegetables following application of Bt microbial pesticides (Frederiksen et al., 2006). Thus, the 

use of Bt microbials for pest control in agriculture, including in organic farming, provides a 50-

year history of safe consumption of food crops sprayed with Bt microbial pesticides. Due to the 
Bayer CropScience LP CR282-20U1 86

high efficacy and safety, global demand for Bt biopesticides, as a preferred substitution for 

chemical pesticides, is expected to increase 5.3% from 2018 to 2022 (Chen, 2018) and is projected 

to continue increasing (Damalas and Koutroubas, 2018; Seiber et al., 2014). 

Several different Bt subspecies, including Bt subsp. kurstaki and Bt subsp. aizawai, have been 

subjected to toxicity testing and showed no evidence for adverse effects on human health (Baum 

et al., 1999; Betz et al., 2000; Federici and Siegel, 2008; Hammond, 2004; McClintock et al., 1995; 

Mendelsohn et al., 2003; Siegel, 2001; U.S. EPA, 1986; 2001a). These subspecies are extensively 

used in formulations for commercial biopesticides and express a diverse array of Cry1 proteins 

(e.g., Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D and Cry1B proteins) (Betz et al., 2000). 

Additionally, direct toxicity assessments of Bt microbial biopesticide formulations containing 

these strains, such as Dipel®4, Cutlass® OF5, Crymax®6, Xentari®7 WG, Turex® 50 WG8 and 

Thuricide®9 have shown no evidence for adverse effects on human or mammalian health (Betz et 

al., 2000; Fisher and Rosner, 1959; Hadley et al., 1987; McClintock et al., 1995; U.S. EPA, 1986; 

1996b; VKM, 2016). 

Taken together, the long history of safe use in agriculture and the comprehensive toxicity testing 

of Bt subspecies and Bt-derived biopesticides provides strong support for the conclusions that the 

Cry1B.868 and Cry1Da_7 donor organism (Bt) presents no health hazard to mammals when 

present in food or feed or in the environment to non-target organisms beneficial to agriculture. 

V.C.2. Cry Proteins have a History of Safe Use 

The mode of action of Cry proteins has been extensively studied and is well-documented (Gill et 

al., 1992; OECD, 2007; Schnepf et al., 1998; Vachon et al., 2012). Insecticidal proteins in this 

family confer insect control by forming pores in the insect intestinal tract leading to developmental 

delay or insect death. Ingestion of Cry proteins by the target insect pest exposes the protein to the 

alkaline conditions and proteases in the insect midgut, which results in proteolytic cleavage of the 

protein’s protoxin domain(s), thus solubilizing the parasporal inclusions and converting the protein 

to the active insecticidal toxin. Target insect specificity is mediated in part by specific insect 

midgut proteases that activate the Cry protein (Bravo et al., 2007; Federici and Siegel, 2008; Koch 

et al., 2015; Wang et al., 2018). Following activation, this protease-resistant core protein is 

comprised of three distinct structural domains that function in a step-wise mechanism of binding 

to specific membrane-embedded receptors, oligomerization at the membrane interface, insertion 

into the plasma membrane and pore formation (Bravo et al., 2007; Deist et al., 2014; Federici and 

Siegel, 2008). The specificity of Cry proteins is mediated in part by their activation by midgut 

4

 Dipel is a registered trademark of Abbott, Inc. 

5

 Cutlass is a registered trademark of Certis USA 

6

 Crymax is a registered trademark of Ecogen, Inc. 

7

 Xentari is a registered trademark of Valent USA 8

 Turex is a registered trademark of Certis USA 

9

 Thuricide is a registered trademark of Certis USA 
Bayer CropScience LP CR282-20U1 87

proteases and their binding to specific receptors on the brush-border membrane within the insect 

midgut (Deist et al., 2014; Farmer et al., 2017; Federici and Siegel, 2008; Gill et al., 1992; OECD, 

2007; Schnepf et al., 1998; Vachon et al., 2012). These specific receptors are not present in humans 

or other mammals, nor in most of non-target insects; therefore, there is limited potential hazard 

related to exposure in humans and animals including the majority of non-target insects (Farmer et 

al., 2017; Koch et al., 2015). 

Several Bt-derived Cry proteins that confer insect protection have been used in genetically 

engineered (GE) plants. Consistent with the specific mode of action of the Cry proteins, no adverse 

effects have been reported in mammals, birds, amphibians or reptiles after consumption of Cry 

proteins or Cry-protein expressing, biotechnology-derived plants (OECD, 2007; Schnepf et al., 

1998). Furthermore, there are no confirmed cases of allergic reactions due to exposure or 

consumption of Cry proteins either through GE crops expressing Cry proteins or microbial-derived 

Bt insecticides during nearly 60 years of use (Federici and Siegel, 2008; Mendelsohn et al., 2003; 

Siegel, 2001; U.S. EPA, 2018). After an extensive review of the safety data on Bt microbial 

formulations the World Health Organization (WHO, 1999) concluded that: 

“Owing to their specific mode of action, Bt products are unlikely to pose any hazard 

to humans or other vertebrates or to the great majority of non-target vertebrates 

provided they are free from non-Bt microorganisms and biologically active products 

other than ICPs (insect control proteins).”

The US EPA has established separate tolerance exemptions for various Cry proteins (e.g., Cry1Ab, 

Cry1Ac, Cry1A.105, Cry1F, Cry2Ab2 and Cry3A, Cry3Bb1, Cry34/35, Cry51Aa2) expressed in 

GE crops (U.S. EPA, 1995a; b; 1996a; 2004a; b; c; 2005b; a; 2006a; b; 2018). The USDA has 

deregulated GE crops containing these Cry proteins. Similarly, many global regulatory agencies 

have reviewed and affirmed the safety of numerous Cry proteins (ILSI-CERA, 2010; 2011; 2013b; 

a; c; 2014; Koch et al., 2015). Due to a lack of adverse toxic and allergenic effects of a wide array 

of Bt spray formulations, crops derived from biotechnology and Cry proteins in food or feed, there 

is a global consensus of reasonable certainty of no harm when Cry proteins are consumed by 

mammals and other terrestrial vertebrates. 

V.C.3. Cry1B.868 Protein has a History of Safe Use 

The Cry1B.868 protein in MON 95379 is a chimeric protein containing domains derived from 

wild-type Cry1 proteins of the Cry1A, Cry1B, and Cry1C subclasses (Figure V-1, Panel A) which 

are expressed natively in Bt and are therefore present in biopesticide formulations (e.g., Cry1A 

and Cry1C proteins in Xentari® and CryMax®) (Akhurst et al., 2003; Sanahuja et al., 2011). The 

Cry1B.868 protein provides enhanced specific activity towards fall armyworm (FAW) relative to 

the parental Cry1Be protein, primarily due to the substitution of Domain III with the corresponding 

domain from Cry1C that is recognized as having high Spodoptera spp. activity. The genetic 

recombination of sequences encoding domains from different Cry proteins is a naturally occurring 

biological process (Bravo et al., 2013; de Maagd et al., 2001). Domain recombination, or “domainswapping”, can also generate chimeric Cry proteins that possess increased insecticidal efficacy 

and/or expanded spectrum of activity while maintaining a common mode of action (Wang et al., 

2018). For example, the chimeric Cry1A.105 protein, expressed in the current commercialized GE 
Bayer CropScience LP CR282-20U1 88

maize products containing MON 89034 (FDA BNF No. 107, 200710) including VT Double PRO®

and Genuity SmartStax®, is an engineered chimeric Cry protein comprised of domains from 

Cry1Ab, Cry1Ac (from Bt subsp. kurstaki) and Cry1F (from Bt subsp. aizawai). A detailed 

comparison of the physicochemical, structural and functional properties of Cry1A.105 to its donor 

proteins, along with a review of the acute oral toxicity assessments of each donor protein and the 

chimeric protein, supports that the history of safe use of donor protein domains are applicable to a 

conclusion of safety for chimeric proteins (Wang et al., 2018). 

The donor proteins from which the Cry1B.868 protein domains are derived have a strong safety 

profile. These proteins or proteins from the same subclass of Cry1 proteins, have been directly 

assessed or are present in the Bt microbial products that have been directly assessed for mammalian 

toxicity. The results from these toxicity assessments indicated that there was no evidence for 

toxicity at high doses relative to anticipated human and mammalian exposures (Betz et al., 2000; 

James, 2006; Koch et al., 2015; McClintock et al., 1995; Pellegrino et al., 2018; U.S. EPA, 1986; 

2007). In addition, it is well-documented that Cry1 proteins become denatured and lose their 

functional activity when exposed to temperatures commonly used during food/feed processing and 

are rapidly degraded upon exposure to gastrointestinal proteases (Hammond and Jez, 2011; Wang 

et al., 2018). Thus, processing and digestion of foods and/or feeds further diminish exposure of 

functional, intact Cry1 proteins. 

V.C.4. Cry1Da_7 Protein has a History of Safe Use 

To improve the insecticidal activity of Cry1Da, Cry1Da_7 was designed with minor amino acid 

changes. Improved insecticidal activity due to minor amino acid differences has been previously 

reported (BenFarhat-Touzri et al., 2019; Gowda et al., 2016). The Cry1Da_7 protein in 

MON 95379 shares a high (99.7%) level of amino acid sequence identity to the Cry1Da protein. 

Proteins from the Cry1Da subclass are co-expressed natively along with Cry1Ab and Cry1Ca in 

Bt subsp. aizawai (Chang et al., 2001), which is the active ingredient in several commercial 

biopesticide formulations (notably, Xentari® and Turex®) (Chang et al., 2001; Sanahuja et al., 

2011; VKM, 2016). As shown schematically in Figure V-2, the Cry1Da_7 protein differs from the 

wild-type Cry1Da protein by only four amino acids: an additional alanine is present at amino acid 

position two of the protein and three amino acid substitutions are present in Domain II (serine 282 

to valine, tyrosine 316 to serine, isoleucine 368 to proline). These single amino acid variations 

resulted in a 50-fold improvement in activity towards Helicoverpa zea (corn earworm, CEW) 

while not reducing activity towards Spodoptera frugiperda (fall armyworm, FAW). 

A recent assessment comparing the weight of evidence for the protein safety of two variants of 

Cry51Aa2.834, another Cry protein, demonstrated that minor amino acid sequence modifications 

resulted in an increase in insecticidal activity but did not alter the protein’s hazard to mammals 

10 https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=Biocon&id=MON-89034-3
Bayer CropScience LP CR282-20U1 89

(Farmer et al., 2017; Gowda et al., 2016; Koch et al., 2015). Given the highly-specific mode of 

action of Cry proteins these results are not surprising, as the specific receptors for Cry proteins are 

not found in taxa outside of insects (Gill et al., 1992; OECD, 2007; Schnepf et al., 1998; Vachon 

et al., 2012). 

V.D. Cry1B.868 and Cry1Da_7 Proteins in MON 95379 are Not Homologous to Known 

Allergens or Toxins 

Bioinformatics analyses were performed to assess the potential for allergenicity or toxicity of the 

Cry1B.868 and Cry1Da_7 proteins. The allergen, gliadin, and glutenin protein sequence database 

(AD_2019) was obtained as the "COMprehensive Protein Allergen REsource" (COMPARE) 

database from the Health and Environmental Sciences Institute (HESI). The AD_2019 curated 

database contains 2,081 sequences. 

The allergen bioinformatic results demonstrated there were no biologically relevant sequence 

similarities to allergens when the Cry1B.868 and Cry1Da_7 protein sequences were used as a 

query for a FASTA search of the publicly available allergen (AD_2019) database. Furthermore, 

no short (eight amino acid) polypeptide matches were shared between the Cry1B.868 and 

Cry1Da_7 protein sequences and proteins in the allergen database. These data show that the 

Cry1B.868 and Cry1Da_7 protein sequences lack both structurally and immunologically relevant 

similarities to known allergens, gliadins, and glutenins. 

FASTA bioinformatic alignment searches using the Cry1B.868 and Cry1Da_7 amino acid 

sequences were performed with a toxin database to identify possible homology with proteins that 

may be harmful to human and animal health. The toxin database, TOX_2019, is a subset of 

sequences derived from the PRT_2019 database (which is an all protein GenBank release 

downloaded from NCBI11), that was selected using a keyword search and filtered to remove likely 

non-toxin proteins. The TOX_2019 database contains 34,642 sequences and has been curated to 

remove Bt insecticidal Cry proteins. The results of the bioinformatic analyses demonstrated that 

no relevant similarity exists between the Cry1B.868 and Cry1Da_7 proteins and any sequence in 

the TOX_2019 database. 

These analyses demonstrated that the Cry1B.868 and Cry1Da_7 proteins do not share amino acid 

sequence similarities with known allergens, gliadins, glutenins, or protein toxins which could have 

adverse effects on human or animal health. 

11 https://www.ncbi.nlm.nih.gov/search/all/?term=GenBank 
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V.E. Cry1B.868 and Cry1Da_7 Proteins in MON 95379 are Susceptible to Degradation in 

in vitro Digestion Assays 

One characteristic of several food allergens is their ability to withstand proteolytic digestion by 

enzymes present in the gastrointestinal tract (Astwood et al., 1996; Moreno et al., 2005; 

Vassilopoulou et al., 2006; Vieths et al., 1999). The enzymatic degradation of an ingested protein 

by exposure to gastric pepsin and/or intestinal pancreatic proteases (e.g., pancreatin) makes it 

highly unlikely that either the intact protein or protein fragment(s) will reach the absorptive 

epithelial cells of the small intestine where antigen processing cells reside (Moreno et al., 2005). 

The susceptibility of the MON 95379-produced Cry1B.868 and Cry1Da_7 to degradation by 

pepsin and pancreatin was assessed using the Bt-produced Cry1B.868 and Cry1Da_7 proteins, 

which were shown to be equivalent to the MON 95379-produced Cry1B.868 and Cry1Da_7 (see 

Appendix F). The results indicated that the full-length, Bt-produced Cry1B.868 and Cry1Da_7 

proteins are readily degraded by pepsin and pancreatin. Peptide fragments of ~4 kDa that were 

resistant (i.e., present over the course of the 60 min digestive reaction) to pepsin degradation were 

observed for both proteins. To better understand the fate of the detected peptide fragments, 

sequential degradation of the Cry1B.868 and Cry1Da_7 proteins in pepsin followed by pancreatin 

was conducted. The results indicated that the fragments are transient as they were readily degraded 

by sequential digestion. As expected, Cry1B.868 and Cry1Da_7 subjected to pancreatin were each 

processed to a trypsin-resistant core that was stable throughout the pancreatin digestion period. 

These results are consistent with observations for these and other Cry proteins subjected to 

pancreatin digestion. Thus, evidence supports the conclusion that gastrointestinal digestion is 

sufficient to degrade the intact Cry1B.868 and Cry1Da_7 proteins and any fragments thereof 

making it highly unlikely that intact or large peptide fragments of Cry1B.868 and Cry1Da_7 

proteins would be absorbed in the small intestine and have the potential to impact human or 

mammalian health. 

V.F. Expression Levels of Cry1B.868 and Cry1Da_7 Proteins in MON 95379 

Cry1B.868 and Cry1Da_7 protein levels determined in MON 95379 are used to assess potential 

exposure to the introduced proteins via food or feed ingestion or potential environmental exposure. 

Section VII.C provides specific results of Tier I toxicity testing on NTOs with Cry1B.868 and 

Cry1Da_7 as well as the MON 95379 environmental risk assessment. To support environmental 

exposure scenarios, the most appropriate tissues to evaluate Cry1B.868 and Cry1Da_7 protein 

levels are leaf [over season leaf 1 (OSL1) and over season leaf 4 (OSL4)], root [over season root 

1 (OSR1) and forage root] silk, pollen, forage and grain tissue samples. Levels of the introduced 

proteins were determined in forage and grain tissue to evaluate food and feed exposure of each 

protein to humans and mammals. Cry1B.868 and Cry1Da_7 protein levels in various tissues of 

MON 95379 relevant to the characterization and environmental assessment were determined by a 

validated enzyme-linked immunosorbent assay (ELISA) assay as part of the characterization of 

MON 95379 (Appendix E). 

Tissues of MON 95379 were collected from four replicate plots planted in a randomized complete 

block design during the 2018 growing season from five field sites in the United States that were 

representative of maize-producing regions. OSL1, OSR1, OSL4, silk, pollen, forage, forage root 

and grain tissue samples were collected from each replicated plot at all field sites. 
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V.F.1. Expression Levels of Cry1B.868 Protein 

Cry1B.868 protein levels were determined in leaf, root, silk, pollen, forage and grain tissues. The 

results obtained from the ELISA are summarized in Table V-1 and the details of the materials and 

methods are described in Appendix E. The expression of Cry1B.868 in MON 95379 maize was 

determined and reported on a dry weight basis. Dry weight (dw) values were converted to a 

μg/g fresh weight (fw) using a moisture conversion factor (Appendix E). The mean Cry1B.868 

protein levels in MON 95379 across all sites was highest in OSL1 at 630 μg/g dw and lowest in 

forage root at 22 μg/g dw. 

 
Bayer CropScience LP CR282-20U1 92

Table V-1. Summary of Cry1B.868 Protein Levels in Maize Tissues Collected from 

MON 95379 Produced in United States Field Trials in 2018 

Tissue 

Type

Development 

Stage1

Mean (SE) 

Range 

(μg/g fw)2

Mean (SE) 

Range 

(μg/g dw)2

LOD/LOQ 

(μg/g dw)3

OSL1 V2-V4 94 (3.3)

46 – 110

630 (22)

310 - 760 0.348/0.625 

OSR1 V2-V4 12 (0.90) 

7.3 - 23

110 (8.2) 

67 - 210 0.254/0.625 

OSL4 VT-R1 49 (2.6)

28 - 69 

210 (11)

120 - 300 0.348/0.625 

Silk R1 6.7 (0.23) 

5.3 - 8.6

67 (2.3)

53 - 86 0.380/0.625 

Pollen R1 52 (1.2)

42 - 63 

91 (2.1)

73 - 110 0.323/0.625 

Forage R5 32 (2.5)

15 - 52 

110 (8.2) 

50 - 170 0.455/0.625 

Forage Root R5 4.3 (0.67) 

1.3 - 14

22 (3.5)

6.7 - 76 0.254/0.625 

Grain R6 23 (3.1)

6.9 - 68

26 (3.5)

7.8 - 77 0.420/0.625 

1

 The crop development stage at which each tissue was collected (Abendroth et al., 2011). 

2

 Protein levels are expressed as the arithmetic mean and standard error (SE) as microgram (μg) of protein 

per gram (g) of tissue on a fresh weight (fw) and dry weight basis (dw). The means, SE, and ranges 

(minimum and maximum values) were calculated for each tissue across all sites (n = 20). 

3 LOQ = limit of quantitation, LOD = limit of detection. 

 
Bayer CropScience LP CR282-20U1 93

V.F.2. Expression Levels of Cry1Da_7 Protein 

Cry1Da_7 protein levels were determined in leaf, root, silk, pollen, forage and grain tissues. The 

results obtained from the ELISA are summarized in Table V-2 and the details of the materials and 

methods are described in Appendix E. The expression of Cry1Da_7 in MON 95379 maize was 

determined and reported on a dry weight basis. Dry weight (dw) values were converted to a 

μg/g fresh weight (fw) using a moisture conversion factor (Appendix E). The mean Cry1Da_7 

protein levels in MON 95379 across all sites was highest in OSL1 at 92 μg/g dw and lowest in 

pollen at less than the limit of quantification (LOQ) μg/g dw. 

Table V-2. Summary of Cry1Da_7 Protein Levels in Maize Tissues Collected from 

MON 95379 Produced in United States Field Trials in 2018 

Tissue 

Type

Development 

Stage1

Mean (SE) 

Range 

(μg/g fw)2

Mean (SE) 

Range 

(μg/g dw)2

LOD/LOQ 

(μg/g dw)3

OSL1 V2-V4 14 (0.78) 

8.4 - 21

92 (5.2)

56 - 140 0.061/0.125 

OSR1 V2-V4 4.7 (0.34) 

2.8 - 7.9

43 (3.1)

26 - 72 0.065/0.125 

OSL4 VT-R1 10 (0.72) 

6.6 - 16

44 (3.1)

29 - 68 0.061/0.125 

Silk R1 0.91 (0.045) 

0.58 – 1.2 

9.1 (0.45) 

5.8 - 12 0.080/0.125 

Pollen R1 <LOQ <LOQ 0.065/0.125 

Forage R5 7.8 (0.63) 

3.8 - 15

26 (2.1)

13 - 50 0.078/0.125 

Forage Root R5 1.8 (0.12) 

0.99 – 2.9 

9.6 (0.64) 

5.2 - 15 0.065/0.125 

Grain R6 0.22 (0.028) 

0.11 – 0.56 

0.25 (0.032) 

0.13 - 0.64 0.037/0.050 

1

 The crop development stage at which each tissue was collected (Abendroth et al., 2011). 

2 Protein levels are expressed as the arithmetic mean and standard error (SE) as microgram (μg) of protein 

per gram (g) of tissue on a fresh weight (fw) and dry weight basis (dw). The means, SE, and ranges 

(minimum and maximum values) were calculated for each tissue with values above the LOQ across all 

sites (n = 20). 

3 LOQ = limit of quantitation, LOD = limit of detection. 
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V.G. Cry1B.868 and Cry1Da_7 Proteins are Not Acutely Toxic 

Acute oral toxicology studies with Cry1B.868 and Cry1Da_7 proteins were conducted to provide 

further confirmation of the mammalian safety of these proteins. There was no evidence of acute 

toxicity in mice when dosed orally at a limit dose of 5,000 mg/kg body weight (bw) with either 

the Cry1B.868 or the Cry1Da_7 protein, which far exceeds any exposure scenario (Appendix J, 

Section J.15 and J.27, respectively). Based on an absence of toxicity in the acute oral toxicity 

studies with the Cry1B.868 and Cry1Da_7 proteins and the relatively low dietary exposure to the 

protein (i.e., low expression levels of the Cry1B.868 and Cry1Da_7 proteins present in 

MON 95379 grain and forage (Section V.F.1; Table V-1 and Table V-2) the risk to humans and 

mammals following dietary exposure to Cry1B.868 and Cry1Da_7 proteins from consumption of 

food or feeds derived from MON 95379 is considered to be negligible. 

V.H. Assessment of Potential Allergenicity of the Cry1B.868 and Cry1Da_7 Proteins 

The allergenic potential of an introduced protein is assessed by comparing the biochemical 

characteristics of the introduced protein to biochemical characteristics of known allergens (Codex 

Alimentarius, 2009). Using a weight of evidence approach, a protein is not likely to be associated 

with allergenicity if: 1) the protein is from a non-allergenic source; 2) the protein represents a small 

portion of the total plant protein; 3) the protein does not share structural similarities to known 

allergens based on the amino acid sequence; and 4) the protein shows susceptibility to pepsin and 

pancreatin treatments. 

The Cry1B.868 and Cry1Da_7 proteins have been assessed for potential allergenicity according to 

these safety assessment guidelines. 

1) Cry1B.868 and Cry1Da_7 proteins originate from Bt, an organism that has not been 

reported to be a source of known allergens. 

2) Cry1B.868 and Cry1Da_7 proteins represent a small portion of the total protein in the grain 

that could be consumed from MON 95379 maize due to very low expression in grain. 

3) Bioinformatics analyses demonstrated that the Cry1B.868 and Cry1Da_7 proteins do not 

share amino acid sequence similarities with known allergens and, therefore, are highly unlikely to 

contain immunologically cross-reactive allergenic epitopes. 

4) Finally, in vitro experiments conducted with the Cry1B.868 and Cry1Da_7 proteins 

demonstrated that the proteins are rapidly digested by proteases found in the human 

gastrointestinal tract (pepsin and pancreatin) under physiological conditions. 

Taken together, these data support the conclusion that the Cry1B.868 and Cry1Da_7 proteins 

produced in MON 95379 do not pose a significant allergenic risk to humans or mammals. 

 
Bayer CropScience LP CR282-20U1 95

V.I. Mammalian Exposure to the Cry1B.868 and Cry1Da_7 Proteins in MON 95379 

The ubiquitous donor organism Bt and Cry protein family have a long, documented history of safe 

use in food and feed. The MON 95379 Cry1B.868 and Cry1Da_7 proteins lack structural similarity 

to known allergens or mammalian toxins. Cry1B.868 and Cry1Da_7 are susceptible to the 

digestive enzymes pepsin and pancreatin and because of the relatively low expression levels of 

Cry1B.868 and Cry1Da_7 proteins present in MON 95379 grain, there is relatively low dietary 

exposure to the proteins. In addition, there was an absence of toxicity for the Cry1B.868 and 

Cry1Da_7 proteins in a confirmatory acute oral toxicity study. Based on these factors, it is unlikely 

that exposure to the Cry1B.868 and Cry1Da_7 proteins in food and feed products derived from 

MON 95379 pose a risk to human and other mammals. 

V.J. Cry1B.868 and Cry1Da_7 Proteins Characterization and Safety Conclusion 

The Cry1B.868 and Cry1Da_7 proteins are members of the three-domain Cry family of 

insecticidal proteins which have a well-documented history of safe use and mode of action. The 

physicochemical and functional characteristics of the Cry1B.868 and Cry1Da_7 proteins from 

MON 95379 were determined and shown to be equivalent to those of their Bt-produced 

counterparts. This equivalence justifies the use of the Bt-produced Cry1B.868 and Cry1Da_7 

proteins as test substances in the protein safety studies, including acute oral toxicity and 

digestibility studies. Expression studies using immunoassay demonstrated that Cry1B.868 and 

Cry1Da_7 were expressed at low levels in grain, forage and other tissues evaluated. The 

Cry1B.868 and Cry1Da_7 proteins lack structural similarity to allergens, toxins or other proteins 

known to have adverse effects on mammals. In addition, the donor organism for the Cry1B.868 

and Cry1Da_7 coding sequences, B. thuringiensis, is ubiquitous in the environment and not known 

for human or mammalian pathogenicity or allergenicity. The Cry1B.868 and Cry1Da_7 proteins 

are rapidly digested by proteases found in the human gastrointestinal tract (pepsin and pancreatin) 

and demonstrated no acute oral toxicity in mice at the highest level tested, which far exceeds any 

exposure scenario. Taken together, these findings support the conclusion that the consumption of 

the Cry1B.868 and Cry1Da_7 proteins in food and feed derived from MON 95379 or its progeny 

poses no risk to human and mammalian health. 

The protein safety data presented herein support the overall conclusion that food and feed products 

containing MON 95379 or derived from MON 95379 are as safe for human and mammalian 

consumption as maize currently on the market and expression of the Cry1B.868 and Cry1Da_7 

proteins do not impact the plant pest risk of MON 95379 maize. 

 
Bayer CropScience LP CR282-20U1 96

VI. COMPOSITIONAL ASSESSMENT OF MON 95379 

For MON 95379, the introduced Cry1B.868 and Cry1Da_7 proteins confer protection from 

lepidopteran pests and lack catalytic activity that is intended or expected to affect the plant’s 

metabolism. Given the nature of these introduced traits and the overall lack of meaningful 

unintended compositional characteristics observed for biotechnology-derived products 

characterized to date, compositional changes that would affect the levels of nutritional components 

in MON 95379 maize were not expected. 

Food and feed safety assessments of biotechnology-derived crops follow the comparative safety 

assessment process (Codex Alimentarius, 2009) in which the composition of grain and/or other 

raw agricultural commodities of the biotechnology-derived crop are compared to the appropriate 

conventional control that has a history of safe use. Maize is not known to have any endogenous 

toxicants or anti-nutrients associated with overall plant pest potential (OECD, 2002a). For maize, 

assessments are performed using the general principles outlined in the OECD consensus document 

for maize composition (OECD, 2002a). 

A review of compositional assessments conducted according to OECD guidelines, that 

encompassed a total of seven biotechnology-derived crop varieties, nine countries, and eleven 

growing seasons, concluded that incorporation of biotechnology-derived agronomic traits has had 

little impact on natural variation in crop composition. Most compositional variation is attributable 

to growing region, agronomic practices, and genetic background (Harrigan et al., 2010; Zhou et 

al., 2011). Numerous scientific publications have further documented the extensive variability in 

the concentrations of crop nutrients, anti-nutrients, and secondary metabolites that reflect the 

influence of environmental and genetic factors as well as extensive conventional breeding efforts 

to improve nutrition, agronomics, and yield (Harrigan et al., 2010; Harrigan et al., 2009; Ridley et 

al., 2011; Zhou et al., 2011). 

Compositional equivalence between biotechnology-derived and conventional crops supports an 

“equal or increased assurance of the safety of foods derived from genetically modified plants” 

(OECD, 2002b). OECD consensus documents on compositional considerations for new crop 

varieties emphasize quantitative measurements of essential nutrients and known anti-nutrients. 

These quantitative measurements effectively discern any compositional changes that imply 

potential nutritional or safety (e.g., anti-nutritional) concerns. Levels of the components in grain 

and/or other raw agricultural commodities of the biotechnology-derived crop product are 

compared to: 1) corresponding levels in a conventional comparator, a genetically similar 

conventional line, grown concurrently under similar field conditions, and 2) natural ranges 

reported in ILSI Crop Composition Database and from data published in the scientific literature 

and from data published in the scientific literature. The comparison to data published in the 

literature places any potential differences between the assessed crop and its comparator in the 

context of the well-documented variation in the concentrations of crop nutrients and anti-nutrients. 

This section provides a summary of the analyses conducted to evaluate the levels of key nutrients 

and anti-nutrients in grain and forage of MON 95379 compared to that of a conventional control 

maize (LH244 x HCL617) grown and harvested under similar conditions. The production of 

materials for compositional analyses used a sufficient variety of field trial sites, robust 

experimental design (randomized complete block design with four blocks), and sensitive analytical 
Bayer CropScience LP CR282-20U1 97

methods that allow accurate assessments of compositional characteristics over a range of 

environmental conditions typical for corn production. See Appendix I for details on composition 

methods. 

VI.A. Compositional Equivalence of MON 95379 Grain and Forage to Conventional Maize

Grain and forage samples were collected from MON 95379 and a conventional control at five sites 

grown in the United States during the 2018 season. The field sites were planted in a randomized 

complete block design with four blocks per site. MON 95379 and the conventional control were 

grown under agronomic field conditions typical for the different growing regions. 

Compositional analyses of grain and forage samples are reported for the key components listed in 

the maize OECD consensus document (OECD, 2002a). Harvested grain samples were assessed for 

moisture and levels of key nutrients, including proximates (protein, total fat and ash; Table VI-1), 

essential amino acids (10 components; Table VI-2), the essential fatty acid linoleic acid (Table 

VI-1), carbohydrates by calculation and fiber (acid detergent fiber (ADF); and neutral detergent 

fiber (NDF) Table VI-1). Grain samples were also assessed for levels of 

anti-nutrients (phytic acid and raffinose; Table VI-3). Harvested forage samples were assessed for 

moisture and key levels of nutrients, including proximates (protein, total fat and ash; Table VI-4), 

carbohydrates by calculation and fiber (ADF and NDF; Table VI-4). In all, 27 different 

components were analyzed. Moisture values for grain and forage were measured to enable the 

conversion of components from fresh to dry weight but were not statistically analyzed. Therefore, 

25 components were statistically analyzed. 

The statistical comparison of MON 95379 and the conventional control was based on 

compositional data that was combined across all field sites. Statistically significant differences 

were identified at the 5% level (α = 0.05). A statistically significant difference between 

MON 95379 and the conventional control does not necessarily imply biological relevance from a 

food and feed perspective. Therefore, statistically significant differences observed between 

MON 95379 and the conventional control were evaluated further to determine whether the 

detected difference indicated a biologically relevant compositional change or supported a 

conclusion of compositional equivalence, as follows: 

Step 1 – Determination of the Magnitude of Difference between MON 95379 and Conventional 

Control Means 

The difference in mean values between MON 95379 and the conventional control was determined 

for use in subsequent steps. 

Step 2 – Assessment of the Difference in the Context of Natural Variation within the Conventional 

Control 

The relative impact of MON 95379 was evaluated in the context of variation within the 

conventional control germplasm grown across multiple sites. This step assessed the mean 

difference between MON 95379 and the conventional control in the context of the conventional 

control range value (maximum value minus the minimum value). When a mean difference is less 

than the variability seen due to natural environmental variation within the conventional control, 

the difference is typically not a food or feed safety concern (Venkatesh et al., 2014). 
Bayer CropScience LP CR282-20U1 98

Step 3 – Assessment of the Difference in the Context of Natural Variation Due to Multiple Factors 

The relative impact of MON 95379 on composition was also evaluated in the context of sources 

of natural variation such as environmental and germplasm influences. This assessment determined 

whether the component mean value of MON 95379 was within the natural variability defined by 

the literature values and/or the ILSI Crop Composition Database (ILSI-CCDB, 

Table VI-5) values. This naturally occurring variability is important in assessing the biological 

relevance of statistically significant differences in composition between MON 95379 and the 

conventional control. 

These evaluations of natural variation are important as crop composition is known to be greatly 

influenced by environment and variety (Harrigan et al., 2010). Although used in the comparative 

assessment process, detection of statistically significant differences between MON 95379 and the 

conventional control mean values does not imply a meaningful contribution by MON 95379 to 

compositional variability. Only if the impact of MON 95379 on levels of components was large 

relative to natural variation inherent to conventional maize would further assessments be required 

to establish whether the change in composition would have an impact from a food and feed safety 

and nutritional perspective. The steps reviewed in this assessment therefore describe the process 

for determining whether the differences between MON 95379 and the conventional control are 

meaningful from a food and feed safety perspective or whether they support a conclusion of 

compositional equivalence. 

VI.B. Compositional Assessment of MON 95379 Conclusion 

Compositional analysis was conducted on grain and forage of MON 95379 and the conventional 

control grown at five sites in the United States during the 2018 field season. Of the 25 components 

statistically assessed, 17 showed no statistically significant differences (p<0.05) between 

MON 95379 and the conventional control. A total of 8 components in grain (protein, 

carbohydrates, isoleucine, leucine, methionine, phenylalanine, threonine, and valine) showed a 

statistically significant difference (p<0.05) between MON 95379 and the conventional control. 

For these components, the mean difference in component values between MON 95379 and the 

conventional control was less than the range of the conventional control values (Table VI-1 and 

Table VI-2). The MON 95379 mean component values were within the range of values observed 

in the literature and/or the ILSI-CCDB (Table VI-5). 

These results support the overall conclusion that MON 95379 maize was not a major contributor 

to variation in component levels in grain or forage and confirmed the compositional equivalence 

of MON 95379 to the conventional control in levels of key nutrients and anti-nutrients in grain 

and forage. The statistically significant differences observed were not compositionally meaningful 

from a food and feed safety perspective. In addition, these results support the overall food and feed 

safety of MON 95379. 
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Table VI-1. Summary of Maize Grain Proximates, Linoleic Acid, Carbohydrates by Calculation and Fiber for MON 95379 and the Conventional Control 

Difference (MON 95379 minus Control) 

Component 

MON 95379 

Mean (S.E.)1

Range 

Control 

Mean (S.E.) 

Range 

Control Range 

Value2

Mean 

(S.E.) p-Value 

Protein (% dw)3 9.61 (0.56) 

7.97 - 12.47 

9.18 (0.56) 

7.54 - 11.58 

4.04 0.43 (0.16) 0.012 

Total fat (% dw) 3.86 (0.038) 

3.69 - 4.08 

3.80 (0.038) 

3.59 - 4.06 

0.47 0.059 (0.039) 0.204 

Linoleic acid (% Total FA)4

55.24 (0.37) 

53.67 - 56.93 

54.99 (0.37) 

52.94 - 56.19 

3.26 0.25 (0.16) 0.126 

Ash (% dw) 1.29 (0.024) 

1.14 - 1.42 

1.28 (0.024) 

1.12 - 1.41 

0.29 0.012 (0.022) 0.607 

Carbohydrates by calculation (% dw) 

85.23 (0.61) 

82.24 - 87.10 

85.73 (0.61) 

83.22 - 87.53 

4.31 -0.50 (0.16) 0.005 

ADF (% dw) 3.65 (0.098) 

2.92 - 4.55 

3.51 (0.098) 

2.71 - 4.17 

1.45 0.14 (0.12) 0.317 

NDF (% dw)3 9.26 (0.15) 

7.54 - 10.28 

9.26 (0.15) 

8.67 - 10.52 

1.86 -0.0022 (0.22) 0.992 

1Mean (S.E.) = least-square mean (standard error) obtained from the linear mixed model analysis 2Maximum value minus minimum value for the control maize hybrid 3dw = dry weight 4FA = Fatty Acid 
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Table VI-2. Summary of Maize Grain Essential Amino Acids for MON 95379 and Conventional Control 

Difference (MON 95379 minus Control) 

Component 

(% dw)1

MON 95379 

Mean (S.E.)2

Range 

Control 

Mean (S.E.)

Range 

Control Range 

Value3

Mean 

(S.E.) p-Value 

Arginine 0.48 (0.019) 

0.40 - 0.56 

0.46 (0.019) 

0.40 - 0.53 

0.13 0.016 (0.0082) 0.062 

Histidine 0.25 (0.013) 

0.20 - 0.32 

0.25 (0.013) 

0.21 - 0.31 

0.11 0.0063 (0.0048) 0.204 

Isoleucine 0.34 (0.023) 

0.28 - 0.45 

0.33 (0.023) 

0.27 - 0.43 

0.16 0.015 (0.0059) 0.019 

Leucine 1.24 (0.10) 

0.93 - 1.74 

1.17 (0.10) 

0.91 - 1.62 

0.71 0.070 (0.026) 0.012 

Lysine 0.26 (0.0062) 

0.21 - 0.30 

0.26 (0.0062) 

0.22 - 0.31 

0.087 0.0011 (0.0067) 0.874 

Methionine 0.22 (0.013) 

0.18 - 0.29 

0.21 (0.013) 

0.17 - 0.27 

0.098 0.012 (0.0042) 0.008 
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Table VI-2. Summary of Maize Grain Essential Amino Acids for MON 95379 and Conventional Control (continued) 

Difference (MON 95379 minus 

Control) 

Component 

(% dw)1

MON 95379 

Mean (S.E.)2

Range 

Control 

Mean (S.E.) 

Range 

Control Range 

Value3

Mean 

(S.E.) p-Value 

Phenylalanine 0.49 (0.035) 

0.40 - 0.67 

0.47 (0.035) 

0.38 - 0.63 

0.25 0.023 (0.0093) 0.025 

Threonine 0.34 (0.018) 

0.30 - 0.44 

0.33 (0.018) 

0.28 - 0.41 

0.12 0.011 (0.0052) 0.048 

Tryptophan 0.077 (0.0036) 

0.065 - 0.097 

0.074 (0.0036) 

0.066 - 0.090 

0.025 0.0027 (0.0013) 0.106 

Valine 0.45 (0.024) 

0.39 - 0.57 

0.43 (0.024) 

0.36 - 0.53 

0.17 0.016 (0.0076) 0.038 

1dw = dry weight 2Mean (S.E.) = least-square mean (standard error) obtained from the linear mixed model analysis 3Maximum value minus minimum value for the control maize hybrid 
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Table VI-3. Summary of Maize Grain Anti-Nutrients for MON 95379 and Conventional Control 

Difference (MON 95379 minus 

Control) 

Component (% dw)1

MON 95379 

Mean (S.E.)2

Range 

Control 

Mean (S.E.) 

Range 

Control Range 

Value3

Mean 

(S.E.) p-Value 

Phytic acid 0.74 (0.034) 

0.46 - 0.91 

0.70 (0.034) 

0.49 - 0.92 

0.43 0.045 (0.045) 0.351 

Raffinose 0.21 (0.014) 

0.17 - 0.27 

0.19 (0.014) 

0.16 - 0.26 

0.11 0.011 (0.0053) 0.105 

1dw = dry weight 2 Mean (S.E.) = least-square mean (standard error) obtained from the linear mixed model analysis 3Maximum value minus minimum value for the control maize hybrid
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Table VI-4. Summary of Maize Forage Proximates, Carbohydrates by Calculation and Fiber for MON 95379 and Conventional Control 

Difference (MON 95379 minus 

Control) 

Component (% dw)1

MON 95379 

Mean (S.E.)2

Range 

Control 

Mean (S.E.) 

Range 

Control Range 

Value3

Mean 

(S.E.) p-Value 

Protein 6.95 (0.38) 

5.52 - 8.69 

6.64 (0.38) 

4.68 - 9.01 

4.32 0.32 (0.24) 0.203 

Total fat 2.82 (0.12) 

2.17 - 3.63 

2.69 (0.12) 

1.56 - 3.53 

1.97 0.12 (0.15) 0.473 

Carbohydrates by calculation 

86.19 (0.58) 

83.16 - 88.79 

86.98 (0.58) 

82.69 - 89.53 

6.84 -0.79 (0.41) 0.064 

ADF 22.04 (0.67) 

18.18 - 28.00 

21.26 (0.67) 

16.83 - 25.04 

8.22 0.78 (0.70) 0.282 

NDF 34.07 (0.81) 

27.29 - 43.83 

33.99 (0.81) 

27.87 - 40.38 

12.51 0.080 (1.08) 0.941 

Ash 4.02 (0.26) 

2.71 - 6.27 

3.72 (0.26) 

2.46 - 4.87 

2.41 0.29 (0.20) 0.155 

1dw = dry weight 2Mean (S.E.) = least-square mean (standard error) obtained from the linear mixed model analysis 3Maximum value minus minimum value for the control maize hybrid 
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Table VI-5. Literature and ILSI Database Ranges for Components in Maize Grain and 

Forage 

Tissue Components1 Literature Range2 ILSI Range3

Grain Nutrients 

Proximates 

protein (% dw) 8.27-13.33a; 9.17-12.19b 5.72-17.26 

total fat (% dw) 2.95-4.40a; 3.18-4.23b 1.363-7.830 

ash (% dw) 1.17-2.01a; 1.27-1.63b 0.616-6.282 

Amino Acids 

alanine (% dw) 0.60-1.04a; 0.68-0.96b 0.40-1.48 

arginine (% dw) 0.34-0.52a; 0.34-0.50b 0.12-0.71 

aspartic acid (% dw) 0.52-0.78a; 0.59-0.76b 0.30-1.21 

cystine (% dw) 0.19-0.26a; 0.20-0.26b 0.12-0.51 

glutamic acid (% dw) 1.54-2.67a; 1.71-2.44b 0.83-3.54 

glycine (% dw) 0.33-0.43a; 0.33-0.42b 0.184-0.685 

histidine (% dw) 0.25-0.37a; 0.27-0.34b 0.14-0.46 

isoleucine (% dw) 0.30-0.48a; 0.32-0.44b 0.18-0.69 

leucine (% dw) 1.02-1.87a; 1.13-1.65b 0.60-2.49 

lysine (% dw) 0.26-0.33a; 0.28-0.31b 0.129-0.668 

methionine (% dw) 0.17-0.26a; 0.16-0.30b 0.11-0.47 

phenylalanine (% dw) 0.43-0.72a; 0.45-0.63b 0.24-0.93 

proline (% dw) 0.74-1.21a; 0.78-1.11b 0.46-1.75 

serine (% dw) 0.39-0.67a; 0.43-0.6b 0.15-0.77 

threonine (% dw) 0.29-0.45a; 0.31-0.39b 0.17-0.67 

tryptophan (% dw) 0.047-0.085a; 0.042-0.07b 0.027-0.215 

tyrosine (% dw) 0.13-0.43a; 0.12-0.41b 0.10-0.73 

valine (% dw) 0.42-0.62a; 0.45-0.58b 0.27-0.86 

Fatty Acids 

palmitic acid (% Total FA) 8.80-13.33a; 9.84-12.33b 6.81-26.55 

palmitoleic acid (% Total FA) 0.059-0.23a 0.067-0.453 

stearic acid (% Total FA) 1.36-2.14a; 1.3-2.1b 1.02-3.83 

oleic acid (% Total FA) 19.50-33.71a; 19.59-29.13b 16.38-42.81 

linoleic acid (% Total FA) 49.31-64.70a; 56.51-65.65b 34.27-67.68 

linolenic acid (% Total FA) 0.89-1.56a; 1.03-1.38b 0.55-2.33 

arachidic acid (% Total FA) 0.30-0.49a; 0.30-0.41b 0.267-0.993 

eicosenoic acid (% Total FA) 0.17-0.29a; 0.17-0.27b 0.098-1.952 

behenic acid (% Total FA) 0.069-0.28a; 0.059-0.18b 0.093-0.417 

Carbohydrates By Calculation 

carbohydrates by calculation (% dw) 81.31-87.06a; 82.10-85.98b 77.4-89.7 

Fiber 

ADF (% dw) 1.82-4.48a; 1.83-3.39b 1.41-11.34 

NDF (% dw) 6.51-12.28a; 6.08-10.36b 4.28-24.30 

TDF (% dw) 10.65-16.26a; 10.57-14.56b 5.78-35.31 

Minerals 

calcium (% dw) 0.0036-0.0068a; 0.0035-0.007b 0.001-0.101 
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Table VI-5. Literature and ILSI Database Ranges for Components in Maize Grain and Forage 

(continued) 

Tissue Components1 Literature Range2 ILSI Range3

copper (mg/kg dw) 0.85-3.54c 0.55-21.20 

iron (mg/kg dw) 14.17-23.40a; 15.90-24.66b 9.51-191.00 

magnesium (% dw) 0.091-0.14a; 0.1-0.14b 0.06-0.19 

manganese (mg/kg dw) 4.83-8.34a; 4.78-9.35b 1.69-14.30 

phosphorus (% dw) 0.24-0.37a; 0.27-0.38b 0.13-0.55 

potassium (% dw) 0.29-0.39a; 0.36-0.43b 0.18-0.60 

zinc (mg/kg dw) 16.78-28.17a; 18.25-30.44b 6.5-42.6 

Vitamins 

vitamin A (mg/kg dw) 0.14-11.27d 0.19-80.20 

vitamin B1 (mg/kg dw) 2.33-4.17a; 2.71-4.33b 1.26-40.00 

vitamin B2 (mg/kg dw) 0.94-2.42a; 1.64-2.81b 0.50-7.35 

vitamin B3 (mg/kg dw) 15.07-32.38a; 13.64-42.06b 7.42-46.94 

vitamin B6 (mg/kg dw) 4.93-7.53a; 4.97-8.27b 1.18-12.14 

vitamin B9 (mg/kg dw) 0.19-0.35a; 0.23-0.42b 0.09-3.50 

vitamin E (mg/kg dw) 5.96-18.44a; 2.84-15.53b 0.84-68.67 

Grain Other

Anti-Nutrients 

phytic acid (% dw) 0.69-1.09a; 0.60-0.94b 0.111-1.940 

raffinose (% dw) 0.079-0.22a; 0.061-0.15b 0.020-0.466 

Secondary Metabolites 

ferulic acid (μg/g dw) 1205.75-2873.05a; 1011.40-2539.86b 291.93-4397.30 

p-coumaric acid (μg/g dw) 94.77-327.39a; 66.48-259.68b 53.4-820.0 

Forage Nutrients 

Proximates 

protein (% dw) 5.80-10.24a; 5.56-9.14b 2.37-16.32 

total fat (% dw) 1.28-3.62a; 0.20-1.76b 0.296-6.755 

ash (% dw) 2.67-8.01a; 4.59-6.9b 0.66-13.20 

Carbohydrates By Calculation 

carbohydrates by calculation (% dw) 81.88-89.26a; 84.11-87.54b 73.3-92.9 

Fiber 

ADF (% dw) 19.11-30.49a; 20.73-33.39b 5.13-47.39 

NDF (% dw) 27.73-49.62a; 31.81-50.61b 18.30-67.80 

Minerals 

calcium (% dw) 0.12-0.33a; 0.21-0.41b 0.04-0.58 

phosphorus (% dw) 0.090-0.26a; 0.13-0.21b 0.07-0.44 1dw = dry weight; FA = Fatty Acid; mg/kg/ dw 

2Literature range references: a

(Harrigan et al., 2009) (see U.S. Field data);b(Harrigan et al., 2009) (see Chile 

field data);c

(Ridley et al., 2011);d(Egesel et al., 2003) 3ILSI range is from ILSI Crop Composition Database, version 7, 2019 (Accessed January 25th, 2019). 

 
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VII. ASSESSMENT OF POTENTIAL IMPACTS ON NON-TARGET 

ORGANISMS BENEFICIAL TO AGRICULTURE AND THREATENED AND 

ENDANGERED SPECIES 

Evaluation of the potential risks to NTOs is an important component of the risk assessment of a 

biotechnology-derived crop. Since risk is a function of hazard and exposure, it is critical to 

determine the potential hazard as well as routes and levels of exposure. MON 95379 maize will 

not be commercialized in the United States. Under the proposed terms of an EPA seed increase 

registration application, MON 95379 maize will be cultivated for small-scale breeding, testing, 

and seed increase-related activities in three U.S. states including Nebraska, Hawaii, and Iowa with 

a maximum combined total acreage of 100 acres per growing season and small-scale confined field 

trials (potentially in other locations) to collect needed regulatory data to support global regulatory 

submissions of future products stacked with MON 95379. Environmental exposure to Cry1B.868 

and Cry1Da_7 is expected to be extremely limited based on the combined total acreage and 

therefore, the cultivation of MON 95379 maize is unlikely to result in unacceptable risk to 

populations of NTOs beneficial to agriculture. 

The results of Tier I toxicity testing with NTOs representing key ecological functions is 

summarized within this section. The risk assessment plan was informed by the mode of action 

(MOA) of the proteins, the spectrum of insecticidal activity for the Cry1B.868 and Cry1Da_7 

proteins, the expression profile of these proteins in relevant tissues, the potential routes of exposure 

to NTOs, and the biology and familiarity with maize. A conceptual model was developed and 

identified species representing key ecological functions with relevant direct or indirect dietary 

exposure to Cry1B.868 and Cry1Da_7 proteins expressed in MON 95379 maize. An exposure 

assessment was conducted to calculate conservatively estimated environmental concentrations 

(EECs) relevant to NTOs representing key ecological functions identified in the conceptual model. 

The calculated EEC values were compared to endpoints from the laboratory NTO testing with 

Cry1B.868 and Cry1Da_7 proteins to calculate margins of exposure (MOE) and characterize risk. 

Finally, the potential risk of limited cultivation of MON 95379 maize to threatened and endangered 

species was evaluated. 

VII.A. Mode of Action and Spectrum of Insecticidal Activity 

Cry1B.868 and Cry1Da_7 both belong to the Cry1 family of insecticidal proteins, which have a 

long history of safe use (Wang et al., 2019). The mode of action of these proteins is similar to 

previously commercialized Cry proteins and involves the same general steps of ingestion, 

solubilization, oligomerization and formation of selective ion channels (commonly referred to as 

pore-formation), which leads to cell lysis in the midgut and ultimately mortality (Pigott and Ellar, 

2007; Wang et al., 2019). All the steps mentioned above are required for biological activity to 

occur, hence the specific nature of the biological activity of Cry proteins. Cry1 family proteins 

have been expressed in crops for specific management of lepidopteran pests (Koch et al., 2015). 

Cry1B.868 and Cry1Da_7 proteins were individually tested in initial laboratory screening assays 

against a range of agronomically and ecologically relevant taxa to characterize their spectrum of 

insecticidal activity (Appendix L). Diet incorporation bioassays for test organisms were conducted 

and these bioassays were designed to: (1) provide continuous exposure of each protein to the test 
Bayer CropScience LP CR282-20U1 107

species and (2) provide a sufficient duration of exposure to evaluate the potential effects of each 

protein on survival. Proteins were provided in diet at or above expected concentrations in the 

highest expressing tissue in MON 95379 plants (i.e., early-stage leaf). Details regarding general 

methodologies for activity spectrum assays have been published previously (Bachman et al., 

2013). While Monarch butterfly (Danaus plexippus) was not tested previously, the Monarch 

butterfly activity spectrum tests conducted with Cry1B.868 and Cry1Da_7 were based on the 

methodologies of lepidopteran tests described in the Bachman publication. 

The results from bioassays with three lepidopteran insect pests, fall armyworm (Spodoptera 

frugiperda), corn earworm (Helicoverpa zea), and European corn borer (Ostrinia nubilalis) 

indicate that Cry1B.868 and Cry1Da_7 proteins are active against these lepidopteran pest species 

(Table VII-1 and Table VII-2). Biological activity was also detected in the other lepidopteran 

species evaluated, Monarch butterfly. No toxic effects were observed at the highest dose tested in 

the activity spectrum evaluations in species outside of Lepidoptera, including coleopteran species 

western corn rootworm (Diabrotica virgifera virgifera), southern corn rootworm (Diabrotica 

undecimpunctata howardi), Colorado potato beetle (Leptinotarsa decimlineata), or Mexican bean 

beetle (Epilachna varivestis), or hemipteran species of western tarnished plant bug (Lygus 

hesperus) and neotropical brown stink bug (Euschistus heros). The results of these initial screening 

studies indicated that the spectrum of activity of Cry1B.868 and Cry1Da_7 was limited to 

Lepidoptera. 
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Table VII-1. Activity Spectrum Results from Feeding Assays with Cry1B.868 Protein in Invertebrates 

Order Family Genus Species Representative Function EC50 or LC50 (μg/mL diet) Activity 

Lepidoptera Noctuidae Spodoptera frugiperda Herbivore 0.15 YesbLepidoptera Noctuidae Helicoverpa zea Herbivore 120 YesbLepidoptera Crambidae Ostrinia nubilalis Herbivore 9.8 YesbLepidoptera Nymphalidae Danaus plexippus Herbivore 0.077 Yesc

Coleoptera Chrysomelidae Diabrotica virgifera virgifera Herbivore >301a NodColeoptera Chrysomelidae Diabrotica undecimpunctata howardi Herbivore >837a NodColeoptera Chrysomelidae Leptinotarsa decemlineata Herbivore >1708a NodColeoptera Coccinellidae Epilachna varivestis Herbivore >837a Nod

Hemiptera Miridae Lygus hesperus Herbivore >700a NoeHemiptera Pentatomidae Euschistus heros Herbivore >700a Nof

a The most conservative (i.e. lowest) concentration observed at the highest treatment level is reported based on diet stability analysis conducted at 

multiple times during exposure. The measured concentrations were above the 95th percentile values of Cry1B.868 protein expression (μg/g fw) in 

MON 95379 leaf tissue (Table VII-3). 

b EC50 value was estimated in a 7-day exposure to Cry1B.868 protein treated diets for ≥5 concentrations. c LC50 value was estimated in a 7-day exposure to Cry1B.868 protein treated diets for 7 concentrations. d Activity was measured for survival in a 7-day feeding exposure to Cry1B.868 protein.e Activity was measured for survival in a 6-day feeding exposure to Cry1B.868 protein. f Activity was measured for survival in a 5-day feeding exposure to Cry1B.868 protein. 
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Table VII-2. Activity Spectrum Results from Feeding Assays with Cry1Da_7 Protein in Invertebrates. 

Order Family Genus Species Representative Function EC50 or LC50 (μg/mL diet) Activity 

Lepidoptera Noctuidae Spodoptera frugiperda Herbivore 0.096 YesbLepidoptera Noctuidae Helicoverpa zea Herbivore 0.042 YesbLepidoptera Crambidae Ostrinia nubilalis Herbivore 11 YesbLepidoptera Nymphalidae Danaus plexippus Herbivore 0.016 Yesc

Coleoptera Chrysomelidae Diabrotica virgifera virgifera Herbivore >106a NodColeoptera Chrysomelidae Diabrotica undecimpunctata howardi Herbivore >65a NodColeoptera Chrysomelidae Leptinotarsa decemlineata Herbivore >58a NodColeoptera Coccinellidae Epilachna varivestis Herbivore >65a Nod

Hemiptera Miridae Lygus hesperus Herbivore >50a NoeHemiptera Pentatomidae Euschistus heros Herbivore >50a Nof

a The most conservative (i.e. lowest) concentration observed at the highest treatment level is reported based on diet stability analysis 

conducted at multiple times during exposure. The measured concentrations were above the 95th percentile values of Cry1Da_7 protein 

expression (μg/g fw) in MON 95379 leaf tissue (Table VII-3). 

b EC50 value was estimated in a 7-day exposure to Cry1Da_7 protein treated diets for ≥5 concentrations. c LC50 value was estimated in a 7-day exposure to Cry1Da_7 protein treated diets for 7 concentrations. d Activity was measured for survival in a 7-day feeding exposure to Cry1Da_7 protein.e Activity was measured for survival in a 6-day feeding exposure to Cry1Da_7 protein. f Activity was measured for survival in a 5-day feeding exposure to Cry1Da_7 protein. 
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VII.B. Exposure Assessment 

MON 95379 maize will not be commercialized in the United States. Under the proposed terms of 

an EPA seed increase registration application, cultivation of MON 95379 maize will be limited to 

three U.S. states, including Nebraska, Hawaii, and Iowa, with a maximum combined acreage of 

100 acres per growing season and small-scale, confined field trials in additional locations to collect 

needed regulatory data to support global regulatory submissions of future products stacked with 

MON 95379. Therefore, environmental exposure to Cry1B.868 and Cry1Da_7 is expected to be 

extremely limited and the cultivation of MON 95379 maize is unlikely to result in adverse effects 

to NTO populations. 

An exposure assessment was conducted, and illustrated in Figure VII-1, to characterize potential 

routes of exposure of Cry1B.868 and Cry1Da_7 proteins expressed in MON 95379 maize to 

NTOs. Direct exposure to NTOs is expected to be through consumption of maize tissues (e.g. leaf, 

pollen, grain) expressing the Cry1B.868 and Cry1Da_7 proteins (Figure VII-1). The conceptual 

model identified pollinators, facultative predators and parasitoids, wild and managed vertebrates 

and organisms involved in decomposition and soil nutrient cycling as species with relevant direct 

or indirect dietary exposure to Cry1B.868 and Cry1Da_7 proteins expressed in MON 95379 maize. 

Only non-pest species were considered relevant for an assessment of invertebrate herbivores (e.g. 

Monarch butterfly). Cry proteins are not known to possess contact toxicity in sensitive or nontarget species and this route of exposure was not considered further. 

Figure VII-1. Relevant Routes of Exposure to Non-Target Organisms from Cultivation of 

MON 95379 Maize 

The above conceptual model depicts relevant routes of exposure to non-target organisms from 

cultivation of MON 95379 maize. Relevant tissues expressing Cry1B.868 and Cry1Da_7 proteins 

were identified as pollen, leaf, forage, and grain. Key non-target organism groups with expected 

direct or indirect exposure to Cry1B.868 and Cry1Da_7 include pollinators, facultative predators 

and parasitoids, wild and managed vertebrate species and organisms involved in decomposition 
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and soil nutrient cycling. Only non-pest species were consider relevant for an assessment of 

invertebrate herbivores (e.g., monarch butterfly). 

For the exposure assessment described below, conservatively estimated environmental 

concentration (EEC) values were calculated to characterize exposure to NTOs and were based on 

expression of Cry1B.868 and Cry1Da_7 evaluated in multiple tissues and growth stages. For tissue 

types where multiple growth stages were analyzed, the growth stage with highest mean expression 

was identified and the upper 95th percentile fresh weight expression values were then calculated. 

Maximum predicted environmental concentrations of Cry1B.868 and Cry1Da_7 proteins in soil 

were calculated based on a soil incorporation model and soil dissipation results. Finally, Food 

Intake Rate (FIR) and the concetrations of the proteins in diet were used to estimate dietary 

exposure to Cry1B.868 and Cry1Da_7 to terrestrial vertebrates such as birds and small mammals. 

VII.B.1. Expression of Cry1B.868 and Cry1Da_7 in MON 95379 Tissues 

The expression of Cry1B.868 and Cry1Da_7 in MON 95379 maize was measured with lyophilized 

tissue and reported on a dry weight basis (Section V.F). Since fresh tissue is most relevant for 

exposure to NTOs, a conversion factor was used to calculate the amount of Cry1B.868 and 

Cry1Da_7 in fresh weight tissues and the 95th percentiles of the mean fresh weight expression 

values were used to provide conservative EEC values for ecological exposure. These values served 

as the basis for high-end estimates of exposure levels of Cry1B.868 and Cry1Da_7 to target pests 

and non-target organisms. Given that the highest expression levels for both Cry1B.868 and 

Cry1Da_7 were measured in V2-V4 stage leaves (OSL1) of MON 95379 maize, all concentrations 

tested in the activity spectrum assessment (Table VII-1 and Table VII-2) were based upon 20.9 μg 

Cry1Da_7/g fresh weight and 111.4 μg Cry1B.868/g fresh weight leaf tissue (Table VII-3). In 

addition to leaf tissue, the 95th percentile of fresh weight expression levels of Cry1B.868 and 

Cry1Da_7 in tissues deemed most relevant to NTO exposure (e.g. pollen, forage, and grain) were 

also determined (Table VII-3). Levels of Cry1Da_7 could not be quantified in pollen and therefore 

the assay LOQ (0.125 μg Cry1Da_7/g dw pollen) was used as worst-case estimate of exposure to 

Cry1Da_7 via pollen. 

 
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Table VII-3. Fresh Weight 95th Percentile Expression of Cry1Da_7 and Cry1B.868 

Proteins in Maize Tissues Collected from MON 95379 Produced in United States Field 

Trials in 2018 

Tissue 

Typea

Development 

Stageb

Cry1Da_7 

(μg/g fw)c

Cry1B.868 

(μg/g fw)c

OSL1 V2-V4 20.9 111.4 

pollen R1 <LOQd 61.9 

forage R5 11.1 48.8 

grain R6 0.5 41.2 

a

OSL = over season leaf 

b

The crop development stage at which each tissue was collected. In cases where multiple development 

stages were analyzed, the growth stage with the maximum observed value was used for calculating EECs. 

c

Protein levels are expressed as the 95th percentile of the mean expression expressed in microgram (μg) of 

protein per gram (g) of tissue on a fresh weight basis (fw). 

d

LOQ = limit of quantitation. The LOQ for Cry1Da_7 in pollen was 0.125 μg/g dry weight. 

VII.B.2. Estimated Environmental Concentrations of Cry1B.868 and Cry1Da_7 

Predators and Parasitoids 

The concentrations of Cry1B.868 and Cry1Da_7 in leaves taken from plants in the V2-V4 growth 

stage were used to determine potential exposure of predator and parasitoids to Cry1B.868 and 

Cry1Da_7. Growth stage V2-V4 leaf was the stage and tissue-type with the highest observed 

expression for both proteins and serves as a worst-case exposure level. The expression values were 

20.9 μg Cry1Da_7 and 111.4 μg Cry1B.868 per gram of fresh weight leaf tissue (Table VII-3). 

For predators and parasitoids, the most relevant exposure scenario is predation of herbivore prey 

exposed to MON 95379 tissues. The degree of such exposure would be predicated on the 

concentrations of Cry1B.868 and Cry1Da_7 acquired in prey species. Herbivores generally contain 

lower levels of Cry proteins than what is expressed in the tissues being consumed. Minimal 

concentrations of Cry proteins have been found in aphids and other phloem-feeding insects (Obrist 

et al., 2006); reviewed in Romeis et al. (2014). Lepidopteran pest larvae and thrips have been 

shown to contain between 0.1 and 0.35 times the amount of Cry1Ab protein expressed in maize 

tissue (reviewed in (Raybould et al., 2007)). The highest concentrations of Cry protein found in 

herbivores have been observed in spider mites where observed levels vary, but may be similar to 

those expressed in maize leaf tissues (Obrist et al., 2006). 

Given the broad diversity of prey taxa likely to be consumed by beneficial predators in the agroecosystem, leaf expression levels for Cry1B.868 and Cry1Da_7 are sufficiently conservative to 
Bayer CropScience LP CR282-20U1 113

account for indirect exposure to generalist predators feeding on herbivore prey (Table VII-4; (U.S. 

EPA, 2016; Appendix L). Leaf expression is likely a very conservative estimate of indirect 

exposure through prey and previously published risk assessments have proposed a value of 0.2-

times leaf expression as a more realistic estimate of prey exposure to predators and parasitoids 

(Raybould et al., 2007). Some predators and parasitoids will also consume pollen and nectar to 

supplement nutrition during specific stages in development or when preferred prey is scarce (Li et 

al., 2008; Li et al., 2010). As maize does not produce nectar, 95th percentile expression values for 

Cry1B.868 and Cry1Da_7 in pollen were used to estimate this route of exposure (Table VII-4). 
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Table VII-4. Summary of Estimated Environmental Concentrations (EECs) for Exposure of Invertebrate NTOs to Cry1B.868 and Cry1Da_7 in MON 95379 Maize 

aFor beneficial predator and parasitoids where herbivore prey is the most relevant route of exposure, 95th percentile leaf expression was used as a 

worst-case exposure level. 

bCry1Da_7 could note be quantified in pollen so the LOQ (0.125 μg/g) was used to derive the EEC. c Calculation of EEC values for Cry1B.868 and Cry1Da_7 are described in the section on soil invertebrates and are derived from PEC values 

(Table VII-5). The EEC for Cry1Da_7 was derived from the maximum predicted concentration assuming a DT50 of 1 year in sandy loam soil 

(Table VII-6). 

dValues expressed as μg protein per g of tissue or soil. 

NTO Group Test Species Exposure route used to derive EEC 

value 

Cry1B.868 EEC 

(μg/gd) 

Cry1Da_7 EEC 

(μg/gd) 

Predators & 

Parasitoids Coleomegilla maculata Diet 100% MON 95379 leavesa 111.4 20.9 

Poecilus cupreus Diet 100% MON 95379 leaves 111.4 20.9 

Geocoris punctipes Diet 100% MON 95379 leaves 111.4 20.9 

Pediobius foveolatus Diet 100% MON 95379 leaves 111.4 20.9 

Chrysoperla sp. (larva) Diet 100% MON 95379 leaves 111.4 20.9 Chrysoperla sp. (adult) Diet 100% MON 95379 pollen 61.9 0.1b 

Soil organisms Folsomia candida Soil incorporation of MON 95379 forage 0.4 0.2c 

Eisenia andrei Soil incorporation of MON 95379 forage 0.4 0.2c 

Pollinators Apis mellifera (adult) Diet 100% MON 95379 pollen 61.9 0.1b 

Apis mellifera (larva) Diet 100% MON 95379 pollen 61.9 0.1b 
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Non-target Lepidoptera 

The spectrum of activity for Cry1B.868 and Cry1Da_7 was limited to Lepidoptera and therefore 

consideration was given to potential exposure to non-target lepidopteran species. Non-target 

lepidopteran (i.e., non-pest) species are generally not known to feed on maize tissue as their 

feeding and reproductive ecology is typically tightly associated with a preferred host plant (U.S. 

EPA, 2001a). Standard agricultural practices for weed management would typically minimize the 

growth of such host plants within the boundaries of the maize field. Therefore, exposure to nontarget lepidopteran species would most likely be through the off-crop movement and deposition of 

pollen on preferred host plants adjacent to the field. Previous work with maize pollen and 

deposition on milkweed, a host plant for monarch butterfly larvae, has demonstrated that density 

of pollen deposition decreases exponentially with increasing distance from the field edge. 

Pleasants et al. (2001) reported that at 4 to 5 meters from the edge of a maize field, the maize 

pollen density on milkweed leaves was expected to be less than 25 grains per cm2 95% of the time; 

a density less than 10% of the 95th percentile measured at the field edge. Based on these results, 

at 10 meters from the field edge, expected density of deposition would be less than 2 grains per 

cm2 indicating there is essentially no exposure to pollen beyond 10m. Standard agronomic 

management practices commonlyutilized for the small-scale field plantings of MON 95379 will 

result in negligible pollen exposure to non-target Lepidoptera (see Appendix L.21). 

Under the proposed terms of an EPA seed increase registration application, MON 95379 maize 

will be cultivated for small-scale breeding, testing, and seed increase-related activities in three 

U.S. states including Nebraska, Hawaii, and Iowa with a maximum combined total acreage of 100 

acres per growing season (as well as potential small-scale confined field trials for stack products 

in other locations, as described earlier). Cultivation of MON 95379 under the terms of the proposed 

EPA registration minimizes the potential exposure of MON 95379 pollen to non-target 

Lepidoptera populations. Furthermore, the extent of off-crop movement of maize pollen is limited 

temporally as peak production of pollen from a maize field typically occurs within a 4-day interval 

(Abendroth et al., 2011). In addition, wind and rain events during or following pollen shed can 

significantly reduce pollen densities on non-target lepidopteran host plants near the field. In a 

combined analysis of six pollen deposition trials across four regions where maize is cultivated, at 

least one rainfall event occurred during anthesis in all but one trial and these rain events resulted 

in 54-86% reduction in densities of pollen deposition (Pleasants et al., 2001). Analysis of 

prevailing wind patterns also indicated the potential exposure from off-crop pollen deposition was 

limited to the downwind side of the field. Standard agronomic practices used to produce high 

quality seed/data in breeding and seed increase nurseries (e.g., weed control and buffer rows) 

further limit movement of MON 95379 pollen off-crop and minimize exposure to non-target 

Lepidoptera. Based on these reasons, minimal off-crop exposure of non-target Lepidoptera to 

Cry1B.868 and Cry1Da_7 in MON 95379 pollen is expected and this exposure is further 

minimized by the limited acreage for intended cultivation. Therefore, the expected absence of 

significant exposure of MON 95379 pollen supports the conclusion of minimal risk to non-target 

Lepidoptera. 

 
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Pollinators 

Exposure of species responsible for pollination services to Cry proteins is typically the result of 

pollinators foraging for pollen and nectar in crops expressing the Cry protein of interest. The honey 

bee (Apis mellifera) serves as a representative species for protecting bee diversity and pollination 

services as honey bees will forage nectar and pollen for nutrition and support of the hive and 

developing larva. Since maize does not produce nectar, exposure to honey bees is limited to the 

presence of Cry1B.868 and Cry1Da_7 in pollen. Analysis of expression data indicated that the 95th

percentile concentration of Cry1B.868 in pollen is 61.9 μg/g pollen. Cry1Da_7 was not 

quantifiable by ELISA in pollen and therefore the assay LOQ value of 0.125 μg/g pollen was used 

as a worst-case estimate (Table VII-4). 

Soil Invertebrates

The most relevant route of exposure for MON 95379 to soil invertebrates is via tillage of late 

season maize tissue into the top 6 inches of soil (Appendix L). To calculate predicted 

environmental concentrations (PECs) of Cry1B.868 and Cry1Da_7 in soil, the amount of biomass 

(stover) expected to remain in the field following cultivation and harvest of maize was determined. 

The mass of maize stover biomass is related to yield by harvest index and the relationship is 

defined as: 

= ݔ݊݀݁ܫ ݐݏ݁ݒݎܽܪ

௦௟௕ܽ݅݊ݎܩ

 (௦௟௕ݎ݁ݒ݋ݐܵ + ௦௟௕ܽ݅݊ݎܩ)

Using the 5-year national average for grain yield in the United States from 2014-2018 (173.4 Bu/A) 

and a typical harvest index value of 0.5 for maize results in a value of 4408.5 kg stover 

biomass/acre (Chakwizira et al., 2016; Ertl, 2013; Fischer et al., 2020; Ion et al., 2015; Li et al., 

2015; Zwahlen et al., 2003; Zwahlen et al., 2007). For late season tissue, 95th percentile expression 

in forage was used to calculate the PEC values for Cry1B.868 and Cry1Da_7 in soil (Table VII-3) 

as expression of these proteins in forage tissue was about 5-fold higher than that found in forage 

root. Assuming incorporation of the entire calculated post-harvest biomass into the top 6 inches of 

soil with a bulk density of 1 g/cm3, the 1u field load was calculated to be 7.3 g fw tissue/kg soil, 

and based on expression levels in forage tissue, soil concentrations for Cry1B.868 and Cry1Da_7 

were calculated to be 0.35 μg/g soil and 0.08 μg/g soil respectively (Table VII-5). 
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Table VII-5. Predicted Environmental Concentrations (PECs) and Dissipation Kinetics of 

Cry1B.868 and Cry1Da_7 in Soil 

a

n.a. = not applicable; DTx = X% dissipation time 

b

The dissipation parameter could not be determined as it fell outside of the exposure period of the study. The Cry1Da_7 

protein concentrations remaining at the end of the study expressed as percentage of maximum protein concentrations 

detected were 66%, 25%, and 22% in Sandy Loam, Silt Loam, and Clay Loam soils respectively.

Soil organisms may be exposed to the Cry1B.868 and Cry1Da_7 proteins by incorporation of 

above ground plant tissues (stover) into soil after harvest, and to a much lesser extent, by pollen 

deposited on the soil, or by root exudation. Exposure may occur by feeding on incorporated maize 

biomass or by ingestion or absorption of the Cry proteins to soil particles after their release from 

maize tissues into the soil. Clay soils, often associated with higher cation-exchange capacities, can 

generally bind macromolecules such as Cry proteins and nucleic acids more tightly than other soil 

types like sand and silt (Gruber et al., 2012; Icoz and Stotzky, 2008). Binding to clay particles can 

render proteins less available to enzymatic degradation and result in prolonged dissipation. 

However, other factors can also influence dissipation rates including protein characteristics, 

organic matter content, robustness of the microbial communities, soil moisture, and pH. Given the 

variety of environmental factors that influence degradation, it is not unexpected that relative 

degradation rates across soil types would vary across studies and proteins. Generally, microbial 

activity is thought to be a principle factor in degradation rates of free protein (Icoz and Stotzky, 

2008). Radiolabeled 14C-Cry1Ab applied to soils resulted in rapid CO2 evolution and the 14C-label 

was incorporated into the microbial biomass indicating that the applied Cry1Ab protein was being 

utilized as a microbial nutrition source (Valldor et al., 2015). This result is consistent with the 

notion that proteins are typically amendable to biodegradation in the environment. Guidance from 

EPA test guideline OPPTS 835.4100 recommends that soil dissipation tests generally not be 

conducted beyond 120 days due to the potential decline of microbial communities in test systems 

that are isolated from the environment and natural replenishment. For this reason, laboratory 

studies represent a more conservative approach to assess the potential for persistence of Cry 

Parameter Soil Type Cry1B.868 Cry1Da_7 

PEC (μg protein/g soil) n.a.a 0.35 0.08 

DT50 (d)a Sandy Loam 19 >213b 

DT90 (d) Sandy Loam 49 >213b 

DT50 (d) Silt Loam 9 40 

DT90 (d) Silt Loam 24 >213b 

DT50 (d) Clay Loam 8 36 

DT90 (d) Clay Loam 28 >213b
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proteins in soils. Indeed, published field monitoring studies with other Bt crops have shown no 

persistence or accumulation of Cry proteins in fields where corn (Cry1Ab, Cry1F, Cry3Bb1) or 

cotton (Cry1Ac, Cry2Ab2) were grown for one or more years (Ahmad et al., 2005; Dubelman et 

al., 2005; Gruber et al., 2012; Head et al., 2002; Shan et al., 2008). In a comprehensive multi-year 

field survey, Gruber and colleagues evaluated soils for the presence of Cry1Ab in fields that were 

planted with MON 810 maize for nine consecutive years (Gruber et al., 2012). Over the course of 

the 9-year trial, Cry1Ab was detected in soil above detectable levels at a single site 6 weeks after 

the 8th consecutive growing season. Notably, no Cry1Ab protein was detected at any site in any of 

the spring samplings prior to planting indicating that seasonal decreases in soil temperature did 

not result in persistence or accumulation of Cry1Ab in soil. The predicted environmental 

concentrations of Cry1B.868 and Cry1Da_7 in soil are 0.35 and 0.08 μg/g soil, respectively (Table 

VII-5). With cultivation of MON 95379 maize in the U.S. limited to a maximum of 100 acres per 

growing season, this further supports a conclusion of minimal exposure of Cry1B.868 and 

Cry1Da_7 to soil organism populations. Further discussion of the fate of Cry proteins in soil can 

be found in Appendix L. 

Although exposure is limited by the small area of intended planting, a study was conducted to 

estimate the dissipation rate of Cry1B.868 and Cry1Da_7 in agricultural soils (Appendix J). 

Lyophilized and ground MON 95379 leaf tissue, collected at the V2-V4 growth stage, was 

incorporated into agriculturally relevant soils at a rate of 75 mg/g soil (75 g/kg soil). This rate is 

approximately 10-fold higher than the 1u field load (7.3 g fw tissue/kg soil) calculated above. Soil 

microcosms were incubated in the dark at approximately 22 °C for 213 days and samples were 

collected at multiple sampling times during the duration of the study. The concentrations of 

Cry1B.868 and Cry1Da_7 proteins in collected soil samples were measured with specific ELISA 

assays. In a complimentary analysis, the temporal change in the biological activity of incorporated 

MON 95379 maize tissue in collected soil samples was evaluated using a sensitive bioassay with 

Spodoptera frugiperda (fall armyworm). 

Results from the ELISA analysis indicated that Cry1B.868 dissipated rapidly in soils with DT50

values of 19, 9, and 8 days in sandy loam, silt loam, and clay loam soils respectively (Table VII-5). 

Calculated DT90 values for Cry1B.868 in soil were determined to be 49, 24, and 28 days in sandy 

loam, silt loam, and clay loam, respectively, indicating that Cry1B.868 is unlikely to persist or 

accumulate in agricultural soils. Therefore, the EEC for Cry1B.868 (Table VII-4) was the same as 

the PEC (Table VII-5) rounded to one significant digit. 

Cry1Da_7 demonstrated a more prolonged dissipation profile compared to Cry1B.868. The DT50

values for Cry1Da_7 were determined to be 40 and 36 days in silt loam and clay loam soils, 

respectively. In sandy loam soil, the DT50 was not captured in the study (>213d) and the percent 

of maximum protein concentration remaining on day 213 was 66%, though it should be noted that 

the percent of maximum protein concentration remaining at the penultimate sampling time (121d) 

was 46% (Appendix L). Ninety percent dissipation of Cry1Da_7 was not reached in any soil type 

within the 213 day duration of the study. The relatively prolonged dissipation profile of Cry1Da_7 

is supported by results with the insect bioassay that showed a gradual loss of biological activity 

over time. 

Since a DT50 value could not be determined for Cry1Da_7 in sandy loam soil, a first-order soil 

accumulation model was used to estimate the effect of potential accumulation of Cry1Da_7 if 
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MON 95379 maize was grown once a year in the same location (FOCUS, 1997) without rotation. 

The model assumed a single planting per year, and a starting concentration of 0.08 μg Cry1Da_7/g 

soil. The model was then run under three different assumptions for the DT50 of Cry1Da_7 in sandy 

loam soil: 183d (1/2y), 274d (3/4y), and 365d (1y). An estimated DT50 of 365d in sandy loam soil 

is expected to be highly conservative as the observed DT50 values for Cry1Da_7 in silt loam and 

clay loam soils were 40 and 36 days respectively, greater than 50% dissipation was observed at 

day 121 in sandy loam soil, and no biological activity was detected in sandy loam soil samples 

containing MON 95379 maize tissue at the final sampling time of 213 days. The first order soil 

accumulation model is given by: 

଴ହ்(஽/)ଶ (୪୬ି(݁)1 − ݐ)ܥ + ଴ܥܧܲ = (ݐ)ܥ

ଷ଺ହ )

C(t) = amount of Cry1Da_7 protein remaining after soil incorporation in year t

PEC0 = 0.08 μg Cry1Da_7/g soil 

DT50 = DT50 in days 

Under the highly conservative scenario assuming MON 95379 maize is planted in the same field 

without rotation for 25 consecutive years and using the most conservative estimated DT50 value, 

the soil accumulation model indicates that peak soil concentrations of Cry1Da_7 would plateau at 

about 2-fold above the initial PEC value for Cry1Da_7 in soil (Figure VII-2; Table VII-6). 

Therefore, the Cry1Da_7 EEC for soil organisms was conservatively calculated to be 0.16 μg/g 

soil; or 2u the initial PEC value for Cry1Da_7 in soil (Table VII-4). 
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Table VII-6. First-Order Accumulation Model to Predict Maximum Expected Concentration of Cry1Da_7 Protein in Sandy Loam Soil 

Cry1Da_7 (μg/g soil) Cry1Da_7 (μg/g soil) 

Year DT50 = 365d DT50 = 274d DT50 = 183d Year DT50 = 365d DT50 = 274d DT50 = 183d 

1 0.08 0.08 0.08 14 0.16 0.13 0.11 2 0.12 0.11 0.10 15 0.16 0.13 0.11 3 0.14 0.12 0.11 16 0.16 0.13 0.11 4 0.15 0.13 0.11 17 0.16 0.13 0.11 5 0.16 0.13 0.11 18 0.16 0.13 0.11 6 0.16 0.13 0.11 19 0.16 0.13 0.11 7 0.16 0.13 0.11 20 0.16 0.13 0.11 8 0.16 0.13 0.11 21 0.16 0.13 0.11 9 0.16 0.13 0.11 22 0.16 0.13 0.11 

10 0.16 0.13 0.11 23 0.16 0.13 0.11 11 0.16 0.13 0.11 24 0.16 0.13 0.11 12 0.16 0.13 0.11 25 0.16 0.13 0.11 13 0.16 0.13 0.11 
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Figure VII-2. A First-Order Soil Accumulation Model Based on Highly Conservative Parameter Estimates Predicts Limited Accumulation of Cry1Da_7 in Sandy Loam SoilAssuming a single yearly planting of MON 95379 and a conservative DT50 estimate of 1 year for Cry1Da_7 in sandy loam soil indicates that soil 

concentrations would peak at 2u the initial soil PEC value.

0.180.160.140.120.100.080.060.040.020.00

0 5 10 15 20 25

g Cry1Da_7 / g soilμ

Years

Maximum Estimated Accumulation of Cry1Da_7 in Sandy Loam Soil

DT50 = 365d DT50 = 274d DT50 = 183d
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The observed dissipation kinetics for Cry1Da_7 are generally similar to those of Cry1Ac in 

MON 87701 soybean, which had DT90 values in silt and sandy soils that exceeded 181d. However, 

field monitoring surveys have demonstrated that Cry1 proteins, including Cry1Ac, do not persist 

or accumulate in the environment (Dubelman et al., 2005; Gruber et al., 2012; Head et al., 2002). 

This is likely due to additional factors and abiotic stressors present in the environment that include 

variations in temperature, rain events, exposure to sunlight as well as potentially higher numbers 

of microorganisms in field soil that could promote protein degradation. This is consistent with 

previous conclusions from EPA (U.S. EPA, 2001a): 

“Field deposition of Cry protein would be associated with plant material (pollen or 

crop residue) or plant root exudates (e.g. carbohydrates and amino acids) which 

typically stimulate microbial activity and reproduction (Cheng and Coleman 1990, 

Griffiths et al. 1999, Jensen and Soerensen 1994, Meharg 1994). Many of the 

experiments examining persistence of Cry proteins reported in the published 

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

generally does not support populations of microorganisms as high as in the 

rhizosphere or where plant residues are incorporated into the soil. Other work 

suggests typical ratios of 5-20 for rhizosphere to bulk soil microbes, with rhizosphere 

populations commonly 100 fold higher than in bulk soil (Atlas and Bartha 1993). 

Therefore, degradation rates under field conditions may be higher than those shown 

in bulk soil experiments.”

Even if there is some persistence of Cry1Da_7, ultimate soil concentrations are not expected to 

result in adverse effects to soil organisms and this conclusion is consistent with previous 

statements by EPA SAP (U.S. EPA, 2001c): 

“Nevertheless, it must be emphasized that accumulation and even long-term 

persistence of Cry proteins in soil does not necessarily result in adverse 

environmental effects.”

Taken together, it is unlikely that Cry1B.868 and Cry1Da_7 will persist or accumulate in the 

environment and any potential accumulation of Cry1Da_7 would result in, at maximum, a 2-fold 

relative increase in soil concentrations. Despite the potential for limited accumulation of Cry1Da_7 

in soil, cultivation of MON 95379 in the U.S. limited to the proposed maximum of 100 acres per 

growing season further supports a conclusion of minimal exposure of Cry1B.868 and Cry1Da_7 

to soil organism populations. 

Terrestrial Vertebrates 

Wild birds are not known to appreciably feed on maize leaves and therefore would most likely be 

exposed to Cry1B.868 and Cry1Da_7 by consuming MON 95379 grain and recently germinated 

seedlings. Birds such as crows (Corvus brachyrhynchos), grackles (Quiscalus quiscula), and 

sandhill cranes (Grus canadensis) will uproot and feed on recently germinated maize seedlings 

(Blackwell et al., 2001; Raybould et al., 2007; Sterner et al., 2003). Blackbirds will feed on 

developing kernels as well as spilled kernels in maize fields (Linz et al., 2003; Raybould et al., 

2007). Similarly, the primary route of exposure of Cry1B.868 and Cry1Da_7 to wild mammals is 

through consumption of MON 95379 maize kernels. Rodent species including ground squirrels 
Bayer CropScience LP CR282-20U1 123

(Spermophilus tridecemlineatus), deer mice (Peromyscus maniculatus), house mice (Mus 

musculus), and voles (Microtus spp.) are known to feed on germinating maize seeds (Raybould et 

al., 2007; Sterner et al., 2003). Indirect exposure to wild birds and mammals could occur via 

predation of arthropod herbivores that have fed on MON 95379 maize tissues. However, the 

concentrations in herbivore prey are typically less than levels found in leaf tissue (discussed above 

in Predators and Parasitoids; (Raybould et al., 2007). Therefore, direct exposure to terrestrial 

herbivore vertebrates via consumption of leaf tissue is sufficiently conservative to account for 

indirect exposure from preying on herbivores that have consumed MON 95379 leaf tissue. 

Since endpoints from Tier I toxicity tests in birds and mammals are normalized to body weight, a 

diet-based exposure normalized to body weight was calculated for birds and mammals. 

Consumption of MON 95379 germinating seedlings results in potential exposure to grain and 

vegetative tissues. Therefore, potential exposures of Cry1B.868 and Cry1Da_7 to wild birds and 

mammals representing herbivore and granivore groups were determined. Food Intake Rate (FIR) 

values for birds and mammals of different classes of body weights were calculated using the 

empirically derived equations (U.S. EPA, 1993): 

Avian food consumption: FIR = (଴.଺ସ଼×ୠ୵)బ.లఱభ

(ଵି୛)

and 

Mammalian food consumption: FIR = (଴.଺ଶଵ×ୠ୵)బ.ఱలర

(ଵି୛)

where:

FIR = food intake rate in grams of fresh weight per day (g/day) 

bw = body mass of animal (g) 

W = mass fraction of water in food (U.S. EPA value = 0.8 for herbivores and 0.1 for granivores) 

These calculations are the same that are used to estimate FIRs for avian and small mammal species 

in the current version of EPA’s Terrestrial Residue EXposure model12 (T-REX v1.5.2). The 

calculated FIR values were multiplied by the 95th percentile fresh weight expression values for 

Cry1B.868 and Cry1Da_7 in leaf and grain tissue (Table VII-3) to determine diet-based exposure 

levels to birds and small mammals (Table VII-7). These values are likely highly conservative as 

they assume 100% of an animal’s diet consists of MON 95379 maize. Previous studies have 

indicated that maize kernels can comprise up to 50% of blackbird and grackle diets and 73% of 

wild rodent diets (Ellis et al., 1998; Homan et al., 1994; Houtcooper, 1978; McNichol et al., 1979; 

Raybould et al., 2007). 

12 https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/t-rex-version-15-users-guide-calculatingpesticide#Section3_1_1 (accessed June 29, 2020). 
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Table VII-7. Summary of Dose-Based Estimated Environmental Concentrations (EECs) for Exposure of Terrestrial Vertebrate NTOs to Cry1B.868 and Cry1Da_7 in MON 95379 Maize 

aFIR = Food Intake Rate is the calculated dietary intake for each dietary and body weight class of birds and mammals (U.S. EPA TREX v.1.5.2) based on U.S. EPA (1993). bTo calculate dose-based EECs, FIR values were multiplied by the 95th percentile protein expression in leaf for herbivores and grain for granivores 

and then divided by the nominal body weight values for each dietary and size class of birds and mammals. 

NTO Group Test Species Dietary Class Body Weight (g) 

FIRa 

(g diet/day) 

Cry1B.868 EEC 

(mg/kg b.w.) 

Cry1_Da7 EEC 

(mg/kg b.w.) 

Wild Birds Colinus virginianus Herbivores 20 100 1000 22.8 64.9 290.8 126.9 72.4 32.4 23.8 13.6 6.1 

Granivores 

20 

100 

1000 

5.1 

14.4 64.6 

10.4 

6.0 2.7 

0.1 0.1 

<0.1 

Wild 

Mammals Mus musculus Herbivores 

15 35 

1000 

14.3 23.1 

152.8 

106.2 

73.4 17.0 

19.9 13.8 

3.2 

Granivores 

15 35 

1000 

3.2 5.1 

34.0 

8.7 6.0 1.4 

0.1 0.1 

<0.1 
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Aquatic Organisms 

Exposure of organisms in aquatic environments to Cry proteins through erosion of soil containing 

bound protein, surface runoff containing soluble protein, aerial deposition of pollen, and aerial 

deposition of crop dust can largely be ruled out (Carstens et al., 2012). Corn tissue can enter the 

aquatic environment immediately after harvest, but peak input typically occurs weeks or months 

later (Jensen et al., 2010). Exposure to aquatic organisms is predicted to be very low, since 

concentrations of Cry proteins in late-season crop tissues are typically less than what is measured 

during vegetative growth stages (Nguyen and Jehle, 2007). 

Plant tissues must be conditioned in the aquatic environment for a period of approximately two 

weeks before it is utilized by aquatic organisms, considered to be “shredders”, as a food source 

(Jensen et al., 2010). During this period of conditioning, soluble organic molecules rapidly leach 

from the tissue and the tissue surface is colonized by resident microorganisms (Carstens et al., 

2012; Webster and Benfield, 1986). Using a sensitive insect bioassay, Jensen et al. (2010) did not 

detect Cry protein biological activity in senescent maize tissue two weeks after entering the aquatic 

environment. Cry3Bb1 was shown to dissipate rapidly from maize tissue in sediment microcosms, 

displaying a half-life within tissue of less than 3 days and was not detectable in the water or 

sediment phases over the course of the 30-day study (Prihoda and Coats, 2008). Cry1Ab has been 

shown to dissipate rapidly from maize tissue in aquatic microcosms with an estimated half-life of 

about two hours (Griffiths et al., 2017; Strain and Lydy, 2015). Recent work has shown that 

DvSnf7 RNA dissipates rapidly from maize tissue in aquatic microcosms with comparable kinetics 

to Bt proteins (Fischer et al., 2020). This suggests that the conclusion of rapid dissipation from 

maize tissue in aquatic environments can be generally applied to PIPs. It is therefore likely that 

the senescent maize tissue in an aquatic environment no longer retains Cry protein activity by the 

time it has been appropriately conditioned to be consumed by an aquatic shredder. Furthermore, 

these proteins are unlikely to persist or accumulate in aquatic environments resulting in minimal 

exposure to aquatic organisms. 

Given the proposed growing areas of MON 95379 maize is limited to a maximum combined 100 

acres per growing season, exposure to freshwater, estuarine, and marine environments is expected 

to be minimal. The reported rapid dissipation of Cry proteins in aquatic environments further 

supports the conclusion that expression of Cry1B.868 and Cry1Da_7 in MON 97539 maize poses 

minimal risk to aquatic organisms. Since exposure of MON 95379 maize to aquatic organisms is 

expected to be minimal, the risk to aquatic NTOs was not considered further in this assessment. 

 
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VII.C. Hazard Assessment 

Tier-Based Approach for NTO Testing 

Testing for potential effects of Cry1B.868 and Cry1Da_7 expressed in MON 95379 maize on nontarget organisms (NTOs) was done according to U.S. regulatory guidelines for NTO testing and 

risk assessment of insect-protected crops (i.e., crops expressing PIPs). These guidelines were 

developed by USDA and EPA and suggest that testing and assessment be conducted based on a 

tier-based system (U.S. EPA, 2007). In this tiered approach, risk (a function of hazard and 

exposure) is evaluated within different levels or “tiers”. Testing progresses from the lower tiers 

reflecting worst-case scenarios to increasingly more realistic exposure scenarios if the lower-tiered 

tests fail to indicate adequate certainty of acceptable risk. Additionally, the EPA has convened 

several Scientific Advisory Panel meetings to gather recommendations and provide guidance for 

NTO testing and risk assessment for PIPs (U.S. EPA, 2001b; c). Following the tiered approach, 

the Tier I study represents a worst-case exposure scenario to estimate hazard using an exposure 

pathway that is usually not realistic due to the high level of continuous exposure (e.g. no-choice 

laboratory test designs using artificial diets). Typically, an exposure factor of 10 times, a highly 

conservative exposure estimate, is administered in a Tier I test. EPA and USDA have established 

that “an endpoint of 50% mortality to be used as a trigger for additional higher tier testing” (U.S. 

EPA, 2007). Less than 50% mortality under these conditions of extreme exposure suggest that 

population effects are likely to be negligible given realistic field exposure scenarios (U.S. EPA, 

2007; 2010b; a). However, if an adverse effect (i.e. greater than 50% mortality) is observed under 

these conditions, this does not necessarily indicate the PIP poses an unacceptable risk in the field, 

but it does trigger the need for a better understanding of potential hazards at lower test 

concentrations using more realistic exposure conditions reflective of field concentrations (e.g. 1u

the amount of the PIP expected to be available to the NTO under field conditions). 

Test organisms were selected based on characteristics of the crop (maize) and the trait (insect 

protection). Based on the results from the activity spectrum assessment (Section VII.A) and known 

expression levels of the Cry1B.868 and Cry1Da_7 proteins in MON 95379 maize (Section VII.B), 

an evaluation of the potential toxicity to NTOs was conducted. Surrogate beneficial species used 

in Tier I laboratory tests were selected based on the results of the activity spectrum assessment and 

to ensure representation of different taxonomic groups, habitats and functions in the agroecosystem (maize fields), the characteristics of the crop (maize), the trait (insect protection) and 

the availability of robust Tier I test methods. The lack of adverse effects to surrogate NTOs 

representing an ecosystem function indicates that the ecosystem function will be maintained in the 

maize agro-ecosystem (U.S. EPA, 2007). The use of surrogate species in an NTO assessment is 

also in line with the strategy for tier-based testing for the effects of PIPs on non-target invertebrates 

described by the USDA and EPA (U.S. EPA, 2007), in which it was stated that “it is impossible 

to test all species that are potentially present...”. 

Tier I testing to assess the hazard of MON 95379 maize included laboratory toxicity testing against 

a representative pollinator [honey bee larvae and adults (Apis mellifera)], six beneficial insect 

species that represent biocontrol species [parasitic wasp (Pediobius foveolatus), ladybird beetle 

(Colleomegilla maculata), carabid beetle, (Poecilus cupreus), green lacewing (Chrysoperla 

carnea and Chrysoperla rufilabris), big-eyed bug (Geocoris punctipes)], and two representative 

soil taxa [earthworm (Eisenia andrei) and Collembola (Folsomia candida)]. 
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The Tier I NTO studies performed with Cry1B.868 and Cry1Da_7 proteins followed methods 

based on either established EPA/OECD test guidelines or Bayer protocols that were sufficiently 

powered to detect adverse effects and evaluated biologically relevant endpoints. The Tier I 

assessment consisted of laboratory testing using intended exposure levels at least 10 times above 

conservative EEC values relevant for a given NTO species (Table VII-4). Ecologically relevant 

endpoints of survival and/or growth and development observations were assessed and tests were 

of sufficient duration to detect adverse effects based on the mode of action of the Cry1B.868 and 

Cry1Da_7 proteins. Acute studies were conducted with vertebrate NTOs as Cry proteins exhibit 

an acutely toxic MOA in sensitive species and there is low potential for chronic effects in 

vertebrate organisms (Koch et al., 2015). All studies met specific acceptance criteria to ensure 

their validity. All studies were conducted using purified Cry1B.868 and Cry1Da_7 proteins 

produced in a Bt expression system. These purified Cry1B.868 and Cry1Da_7 proteins were shown 

to be equivalent to the Cry1B.868 and Cry1Da_7 proteins expressed in MON 95379 maize based 

on apparent molecular weight, immunoreactivity, functional activity, and glycosylation, thus 

making them suitable surrogates to the plant-produced proteins for NTO testing. 

The Cry1B.868 and Cry1Da_7 proteins have been shown to bind to different receptors and 

therefore act independently in lepidopteran insect pests (Wang et al., 2019). Laboratory bioassays 

with the sensitive fall armyworm species (Spodoptera frugiperda) demonstrated that the combined 

activity of Cry1B.868 and Cry1Da_7 is additive, further demonstrating these proteins act 

independently (Appendix J). When two or more proteins act independently and environmental 

concentrations of the proteins are below their no-effect levels, then the proteins can be assessed 

individually (U.S. EPA, 2009b; a); reviewed in Koch et al. (2015). Since Cry1B.868 and 

Cry1Da_7 have been shown to act independently when provided in combination to a sensitive 

species (Appendix J; (Wang et al., 2019)) interaction in NTOs at concentrations expected to be 

encountered in the field is not anticipated (Levine and Borgert, 2018; Levine et al., 2019). 

Furthermore, a supplementary analysis with a highly conservative hazard index approach indicates 

the additive activities of Cry1B.868 and Cry1Da_7 are below the levels of concern for NTOs 

(Appendix J). Therefore, Cry1B.868 and Cry1Da_7 proteins were assessed individually for the 

NTO assessment. 

With the exception of the acute earthworm study, all studies on invertebrates utilizing the purified 

proteins included a diet analysis using a sensitive insect (FAW) bioassay to confirm that the 

Cry1B.868 and Cry1Da_7 proteins incorporated in the test diet were biologically active and had 

the expected level of biological activity. Additionally, where appropriate based upon the diet 

matrix, the homogeneity and stability of the Cry proteins in diet under test conditions and frozen 

storage was also confirmed. A dose confirmation is typically not necessary for the earthworm 

study because the test design is an acute study with a single application of test protein as opposed 

to continuous dietary exposure provided in other invertebrate Tier I tests. Dose confirmations in 

earthworm studies are also often challenging due to poor recovery from the artificial soil as well 

as temporal decline in soil concentrations commonly exhibited by Cry proteins (Ahmad et al., 

2005; Dubelman et al., 2005; Head et al., 2002); reviewed in Icoz and Stotzky (2008). Details on 

study methods and results for all NTO tests are provided in Appendix J. 

To characterize risk of Cry1B.868 and Cry1Da_7 to NTOs, a deterministic approach was used 

whereby MOEs were defined as the ratio of the median lethal concentration (LC50) values to the 

EECs. Calculated MOEs were based on survival endpoints as mortality has been identified as the 
Bayer CropScience LP CR282-20U1 128

primary endpoint in Tier I testing (U.S. EPA, 2007; 2010b). When necessary, sublethal endpoints 

were put into context of the ERA by comparing no observed adverse effect concentration

(NOAEC) values to the EECs. Median lethal concentration and NOAEC values were derived from 

study results that are summarized in Appendix J. An EEC that is lower than the level required to 

cause an adverse effect is generally indicative of negligible risk and U.S. regulatory guidance states 

that only adverse effects to NTOs at field exposure levels (1u are viewed as an environmental risk 

(U.S. EPA, 2007; 2010b; a). Since the study results from Tier I tests indicated no adverse effects 

at field exposure levels, higher tier studies were not conducted (U.S. EPA, 1998b; 2007). 

VII.C.1. Effects of Cry1B.868 on NTOs 

Predator and Parasitoids 

Six species representing four different insect orders of predators and parasitoids were evaluated in 

Tier I toxicity tests where Cry1B.868 protein was provided under continuous dietary exposure. 

These species included Coleomegilla maculata (Coleoptera), Poecilus cupreus (Coleoptera),

Geocoris punctipes (Hemiptera), Chrysoperla carnea and Chrysoperla rufilabris (Neuroptera), 

and Pediobius foveolatus (Hymenoptera). For C. maculata, P. cupreus, G. punctipes, and P. 

foveolatus, no effects on survival were observed at all treatment levels including the highest 

concentration tested (3500 μg/g). The MOEs for these species derived from LC50 values and 

conservative EECs based on leaf expression was determined to be !31 indicating minimal risk to 

these tested predators and parasitoids (Table VII-8). 

While no adverse effects were observed in a Tier I test with adult G. punctipes at 3500 μg/g, a 

statistically significant increase in development time (<1d) was observed at the lower 700 μg/g 

treatment level compared to the control (Appendix J). The magnitude of treatment-related effects 

based on the MOA of Cry proteins should be proportional to the test concentration. Since there 

was no dose-response evident between the treatment levels with no effects on development 

observed at a 5-fold higher concentration, it was determined that the less than 1 day effect on 

development time at the lower 700 μg/g test concentration was not treatment-related.

For C. maculata (ladybird beetle) a statistically significant decrease in adult body weight (<10%) 

at test completion was observed at 3500 μg/g, but not 700 μg/g when compared to the control 

group (Appendix J). An MOE for growth and development of C. maculata derived from a 

conservative EEC based on leaf expression and a NOAEC of 700 μg/g was determined to be t6. 

This is a highly conservative margin of exposure because the EC50 for adult body weight was 

>3500 μg/g. When prey is scarce, ladybird beetles may feed on pollen as a supplemental nutrition 

source and can constitute a significant portion of their diet (Berkvens et al., 2008; Duan et al., 

2002). An MOE for growth and development based on exposure through pollen and a NOAEC of 

700 μg/g was determined to be t11 indicating that Cry1B.868 is unlikely to have adverse sublethal 

effects on ladybird beetles at field-exposure levels. 

Initial Tier I testing with adult C. carnea green lacewings resulted in statistically significant 

increased mortality following continuous dietary exposure to Cry1B.868 (Appendix J). When 

correcting for 15% control mortality, corrected mortality was 23.5% at the 3500 μg/g treatment 

level and 29.4% at the 700 μg/g. While statistically significant compared to the control, there was 
Bayer CropScience LP CR282-20U1 129

no dose-response in mortality between the treatment levels and the estimated LC50 for C. carnea 

adults was >3500 μg/g and a NOAEC could not be determined. 

In the absence of an observable dose-response, it is unclear whether the observed effects on 

survival in C. carnea were due to Cry1B.868 activity. Activity spectrum screening demonstrated 

that Cry1B.868 activity was limited to Lepidoptera and previous work with Cry1-family proteins 

have shown these proteins do not have adverse effects on green lacewing (reviewed in Romeis et 

al. (2014); Appendix L). It should be noted that the only ecologically relevant route of exposure 

for adult green lacewings is through consumption of pollen (Li et al., 2008; Li et al., 2010), as only 

the larval stages are predatory. The observed effects in this study occurred at concentrations well 

above those expected to be found in pollen (!11u; Table VII-4). These results indicate that it is 

unlikely that the mortality that was observed was due to toxicity from the Cry1B.868 protein. 

While guidance indicates that <50% effect at 10u a conservative EEC in Tier I testing is unlikely 

to result in risk under field conditions (U.S. EPA, 2007), without a finite LC50 or NOAEC defined, 

additional studies were conducted with lacewing larvae and adults to more fully evaluate potential 

effects of Cry1B.868 protein on green lacewings. Given delays in study initiation encountered with 

C. carnea due to challenges in getting sufficient numbers of quality insects and that species-level 

differences in lacewing sensitivity to Cry proteins is not anticipated, the additional larval and adult 

green lacewing studies were conducted with C. rufilabris; a species highly relavent to North 

American agriculture and commonly used in laboratory testing. A summary of the green lacewing 

studies and characterization of risk based on the results of these studies is described below. 

A relevant route of exposure to green lacewing is consumption of herbivore prey that has fed on 

MON 95379 by green lacewing larvae, and larvae generally represent a more sensitive life-stage 

compared to adults (U.S. EPA, 2007). Therefore, to more fully characterize the potential effects 

of Cry1B.868 on green lacewing, a Tier I test was conducted with C. rufilabris larvae 

(Appendix J). C. rufilabris larvae were fed continuously on an artificial diet with Cry1B.868 

protein incorporated at three treatment levels including 3500, 700, and 110 μg/g and were 

evaluated for pre-imaginal survival, development time to adult emergence, and weight at 

emergence. No effects on survival and growth and development of juvenile lacewings as well as 

emerged adult body weights were observed at all treatment levels including the highest 

concentration tested (3500 μg/g). The lack of effects on the lethal and sublethal endpoints at all 

concentrations tested, including those that far exceed the EEC, indicate that Cry1B.868 poses 

minimal risk to green lacewing larvae. The MOE for predatory C. rufilabris larvae, derived from 

a conservative EEC value based on leaf expression was determined to be !31 indicating minimal 

risk to green lacewing larvae (Table VII-8). 

To further evaluate potential effects of Cry1B.868 on green lacewing adults, an additional Tier I 

study with C. rufilabris adult green lacewings was conducted. Newly emerged C. rufilabris adult 

green lacewings were fed continuously an artificial diet with Cry1B.868 incorporated at three 

treatment levels including 700, 350, and 110 μg/g and were evaluated for survival (Appendix J). 

The lower concentrations in the C. rufilabris adult test compared to the C. carnea test were chosen 

to capture a range of exposure levels more closely reflective of those expected to be found from 

exposure through pollen. The test design was more conservative than the C. carnea adult study 

described above in that the test diet was provided to organisms in a no-choice environment. No 

effects were observed on survival of C. rufilabris adults at any of the treatment levels including 
Bayer CropScience LP CR282-20U1 130

the highest concentration tested (700 μg/g; Appendix L). Given that green lacewing adults would 

be exposed to Cry1B.868 solely through consumption of pollen, the MOE derived from an EEC 

value based on pollen expression is !11 indicating that Cry1B.868 as expressed in pollen poses 

minimal risk to green lacewing adults (Table VII-8). 

The testing conducted with C. rufilabris covered the entire lacewing lifespan, included multiple 

lethal and sublethal endpoints, and evaluated multiple treatment levels including concentrations 

comparable to, as well as, far-exceeding those expected to be encountered in the field. 

Furthermore, exposure of lacewing larvae to Cry1B.868 is expected to be minimal as their 

preferred aphid prey have been shown to not efficiently take up Cry proteins expressed in maize 

(Romeis and Meissle, 2011); reviewed in Romeis et al. (2014). The temporally limited availability 

of maize pollen also minimizes exposure to green lacewing adults (Abendroth et al., 2011). In 

addition to the limited acreage under the proposed registration, the results of these analyses 

indicate that Cry1B.868 as expressed in MON 95379 maize poses minimal risk to green lacewing 

populations. 
Bayer CropScience LP CR282-20U1 131

Table VII-8. Estimated Environmental Concentrations (EECs), Median Lethal Concentrations (LC50) from Non-target 

Terrestrial Invertebrate Studies and Margins of Exposure (MOEs) for the Cry1B.868 Protein 

Test Organism Common Name Order EEC LC50 MOE 

Coleomegilla maculata Ladybird beetle Coleoptera 111.4 μg/g leaf >3500 μg/g !31 

Poecilus cupreus Carabid beetle Coleoptera 111.4 μg/g leaf >3500 μg/g !31 

Geocoris punctipes Big-eyed bug Hemiptera 111.4 μg/g leaf >3500 μg/ml !31 

Pediobius foveolatus Parasitic wasp Hymenoptera 111.4 μg/g leaf >3500 μg/g !31 

Chrysoperla rufilabris (larva) Green lacewing Neuroptera 111.4 μg/g leaf >3500 μg/g !31 Chrysoperla rufilabris (adult) Green lacewing Neuroptera 61.9 μg/g pollen >700 μg/g !11 

Folsomia candida Springtail Collembola 0.4 μg/g soil >3500 μg/g !8000 

Eisenia andrei Earthworm Haplotaxida 0.4 μg/g soil >3500 mg/kg soil !8000 

Apis mellifera (adult) Honey bee Hymenoptera 61.9 μg/g pollen >900 μg/g !14 Apis mellifera (larva) Honey bee Hymenoptera 61.9 μg/g pollen >900 μg/g !14 
Bayer CropScience LP CR282-20U1 132

Pollinators 

To test for potential effects of Cry1B.868 on pollinators, honey bee adults and larva (Apis 

mellifera) were tested in chronic Tier I toxicity tests. Cry1B.868 was provided continuously in diet 

for 10d to honey bee adults and no adverse effects were observed at any treatment level including 

the highest concentration tested (900 μg/g). To evaluate potential effects on honey bee larva, 

Cry1B.868 protein was provided in diet in a chronic honeybee larval test. No adverse effects were 

observed at any treatment level including the highest concentration tested (900 μg/g). Using a 

conservative EEC based on the expression of Cry1B.868 in pollen, the MOEs for adult and larval 

honeybees were determined to be >14 indicating minimal risk of Cry1B.868 to pollinators (Table 

VII-8). 

Soil Organisms 

Potential effects on soil organisms were evaluated in Tier I tests with earthworm (Eisenia andrei) 

and Collembola (Folsomia candida). No adverse effects were observed in earthworms and 

Collembola when Cry1B.868 was provided at 3500 μg/g in soil and diet, respectively. The MOEs 

were calculated by comparing the LC50 values to the EEC derived from the PEC of Cry1B.868 in 

soil. The MOEs for earthworm and Collembola were >8000 indicating minimal risk to soil 

organisms (Table VII-8). 

Terrestrial Vertebrates 

An avian acute oral toxicity test was conducted to test the effects of Cry1B.868 on avian species. 

Lyophilized Cry1B.868 protein was provided to quail at a nominal dose of 3500 mg protein/kg 

body weight. The LC50 of Cry1B.868 in this study was determined to be >3500 mg/kg. Using the 

smallest body weight as a worst-case dose-based EEC (Table VII-7), the MOEs for Cry1B.868 in 

wild bird species were determined to be !27 for herbivores and !336 for granivores (Table VII-9). 

The results from an acute oral toxicity test with mice were used to characterize risk to wild 

mammal populations. The LC50 for Cry1B.868 protein in mice was determined to be >5000 mg 

protein/kg body weight. Using the smallest body weight as a worst-case dose-based EEC for wild 

mammals (Table VII-7), the MOEs for Cry1B.868 were determined to be !47 for herbivores and 

!574 for granivores (Table VII-9). The estimated exposure level of Cry1B.868 to wild bird and 

mammal species is far below the highest levels tested where no adverse effects on survival were 

observed. These results support the conclusion that dietary exposure of Cry1B.868 poses 

negligible risk to wild bird and small mammals. 
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Table VII-9. Dose-Based Estimated Environmental Concentrations (EECs), Median Lethal 

Concentrations (LC50) from Terrestrial Vertebrate Studies and Margins of Exposure 

(MOEs) for the Cry1B.868 Protein 

Test Organism 

(NTO group) Dietary Class EECa LC50 MOE 

Colinus virginianus 

(birds) 

Herbivore 126.9 mg/kg b.w. >3500 mg/kg b.w. !27 

Granivore 10.4 mg/kg b.w. >3500 mg/kg b.w. !336 

Mus musculus 

(mammals)

Herbivore 106.2 mg/kg b.w. >5000 mg/kg b.w. !47 

Granivore 8.7 mg/kg b.w. >5000 mg/kg b.w. !574 

a

The EEC value for the smallest body-weight vertebrate (Table VII-7) was used as a worst-case for risk 

characterization. 

VII.C.2. Effects of Cry1Da_7 on NTOs 

Predators and Parasitoids 

Six species representing four different insect orders were evaluated in Tier I toxicity tests where 

Cry1Da_7 protein was provided under continuous dietary exposure. These species included 

Coleomegilla maculata (Coleoptera), Poecilus cupreus (Coleoptera), Geocoris punctipes

(Hemiptera), Chrysoperla carnea and Chrysoperla rufilabris (Neuroptera), and Pediobius 

foveolatus (Hymenoptera). No adverse effects on survival were observed in all predators and 

parasitoids tested at the highest treatment level in provided diets (500 μg/g for all species with 

exception of 400 μg/g for P. cupreus and 150 μg/g corrected concentration for C. maculata). The 

MOEs for these species derived from LC50 values and conservative EECs based on leaf expression 

was determined to be >7. Consistent with EPA and USDA guidance, MOE values >1 support the 

conclusion of minimal risk to these tested predators and parasitoids (Table VII-8; (U.S. EPA, 

2007)). 

An increase in development time (<1 day) was observed in C. maculata (ladybird beetle) at both 

treatment levels (Appendix J). While statistically significant, such a small effect in development 

time is not anticipated to result in adverse effects in ladybird beetle populations under field 

conditions. EPA and USDA guidance states that <50% effect at 10u the EEC in a Tier I test is 

indicative of minimal risk (U.S. EPA, 2007). While it is not straight-forward to interpret a percent 

effect with endpoints such as development time, the magnitude of effect at the highest treatment 

concentration was 0.8d; a duration representing <4% of the total development time in the buffer 

control group with no apparent dose response between the two treatment levels. Additionally, there 

were no significant effects at any treatment level for the other endpoints in the study including 

survival and body weight of emerged adults. Lastly, the observed development times for the 

Cry1Da_7 treatments were less than those of the control groups in a companion study conducted 

with Cry1B.868. In other words, the difference in development time of the control groups between 
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the two studies was larger than the observed effect with Cry1Da_7. Therefore, the weight-ofevidence indicates that the effect observed in the development time endpoint will not adversely 

affect ladybird beetle populations. 

While no adverse effects were observed in a Tier I test with adult C. carnea (green lacewing) at 

500 μg/g, a significant increase in mortality was observed at the lower 50 μg/g treatment level (27 

μg/g measured concentration) compared to the control. Since there was no dose-response evident 

between the treatment levels with no effects on survival observed at a >10-fold higher 

concentration, it was determined that the effects on survival at 27 μg/g were not treatment-related.

To fully confirm that Cry1Da_7 will not have adverse effects on green lacewing, the effects of 

Cry1Da_7 were evaluated on C. rufilabris larvae, which represent a potentially more sensitive and 

ecologically relevant growth stage (Appendix J). Cry1Da_7 was incorporated into an artificial diet 

at 500 and 50 μg/g diet and fed continuously to C. rufilabris larva. Effects on pre-imaginal 

survival, development time to adult emergence, and weight at emergence were evaluated. 

Following continuous dietary exposure of Cry1Da_7 protein, no adverse effects were observed on 

survival, growth and development of C. rufilabris larvae. The results of these tests indicate that 

Cry1Da_7 will not have adverse effects on green lacewing. 

When the LC50 values from Tier I tests with species representing predators and parasitoids were 

compared to conservative EEC values, the MOEs were determined to be !19 with the exception 

of C. maculata, where the MOE was !7 (Table VII-10). The MOE for C. maculata was relatively 

lower compared to other tested predators and parasitoids because of a correction for measured 

activity in test diets and not due to an observed adverse effect. Although leaf expression values 

were conservatively used to calculate EEC for C. maculata, it should be noted that the preferred 

prey for ladybird beetles are aphids (Tian et al., 2015). Exposure of C. maculata to Cry1Da_7 

through aphid prey is expected to be minimal as aphids have been shown to not efficiently take up 

Cry proteins expressed in maize (Romeis and Meissle, 2011; Tian et al., 2015); reviewed in Romeis 

et al. (2014). The other potential route of exposure is consumption of pollen as a supplemental 

nutrition source and the MOE for C. maculata based on pollen exposure is !1500 as Cry1Da_7 is 

not detectable in pollen. Therefore, the results of these Tier I studies and risk characterization 

indicate that adverse effects from Cry1Da_7 as expressed in MON 95379 maize on beneficial 

predators and parasitoids is not anticipated. 
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Table VII-10. Estimated Environmental Concentrations (EECs), Median Lethal Concentrations (LC50), from Non-target 

Terrestrial Invertebrate Studies and Margins of Exposure (MOEs) for the Cry1Da_7 Protein 

Test Organism Common Name Order EEC LC50 MOE

Coleomegilla maculata Ladybird beetle Coleoptera 20.9 μg/g leaf >150 μg/ga !7 

Poecilus cupreus Carabid beetle Coleoptera 20.9 μg/g leaf >400 μg/g !19

Geocoris punctipes Big-eyed bug Hemiptera 20.9 μg/g leaf >500 μg/ml !23

Pediobius foveolatus Parasitic wasp Hymenoptera 20.9 μg/g leaf >500 μg/g !23

Chrysoperla rufilabris (larva) Green lacewing Neuroptera 20.9 μg/g leaf >500 μg/g !23

Chrysoperla carnea (adult) Green lacewing Neuroptera 0.1 μg/g pollen >500 μg/g !5000 

Folsomia candida Springtail Collembola 0.2 μg/g soil >500 μg/g !2500 

Eisenia andrei Earthworm Haplotaxida 0.2 μg/g soil >500 mg/kg soil !2500 

Apis mellifera (adult) Honey bee Hymenoptera 0.1 μg/g pollen >500 μg/g !5000 Apis mellifera (larva) Honey bee Hymenoptera 0.1 μg/g pollen >500 μg/g !5000 

aNo adverse effects on survival observed at nominal concentration of 415 μg/g. The nominal concentration for Cry1Da_7 was corrected for 

measured activity in test diet. 
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Pollinators 

To evaluate potential effects of Cry1Da_7 on pollinators, honey bee adults and larvae (Apis 

mellifera) were tested in chronic Tier I toxicity tests. Cry1Da_7 was provided continuously in diet 

for 10d to honey bee adults and no adverse effects were observed at any treatment level including 

the highest concentration tested (500 μg/g). To evaluate potential effects on honey bee larvae, 

Cry1Da_7 protein was provided in diet in a chronic honeybee larval test. No adverse effects were 

observed at any treatment level, including the highest concentration tested (500 μg/g). Since the 

concentration of Cry1Da_7 in pollen is below the limits of quantitation for the ELISA assay, a 

value equivalent to the method LOQ was used as a conservative estimate of exposure. The MOEs 

for adult and larval honeybees were !5000 indicating minimal risk of Cry1Da_7 to pollinators 

(Table VII-10). 

Soil Organisms 

Potential effects on soil organisms were evaluated in Tier I tests with earthworm (Eisenia andrei) 

and Collembola (Folsomia candida). No adverse effects were observed in earthworms and 

Collembola when Cry1Da_7 was provided at 500 μg/g in soil and diet, respectively. The MOEs 

were calculated comparing the LC50 values to the EEC derived from the PEC for Cry1Da_7 in 

soil. The MOEs for both species were !2500 indicating minimal risk to soil organisms (Table 

VII-10). 

Terrestrial Vertebrates 

An avian acute oral toxicity test was conducted to test the effects of Cry1Da_7 on avian species. 

Lyophilized Cry1Da_7 protein was provided to quail at a nominal dose of 1000 mg protein/kg 

body weight. No adverse effects were observed over the course of the study and the LC50 of 

Cry1Da_7 was determined to be >1000 mg/kg body weight. Using the smallest body weight as a 

worst-case dose-based EEC (Table VII-7), the MOEs for Cry1Da_7 in wild birds were determined 

to be !42 for herbivores and !10,000 for granivores (Table VII-11). The results from an acute oral 

toxicity test with mice were used to characterize risk to wild mammal populations. The LC50 of 

Cry1Da_7 protein was determined to be >5000 mg protein/kg body weight. Using the smallest 

body weight as a worst-case dose-based EEC for wild mammals (Table VII-7), the MOEs for 

Cry1Da_7 were determined to be !250 for herbivores and !50,000 for granivores (Table VII-11). 

The estimated exposure level of Cry1Da_7 to wild bird and mammal species is far below the 

highest levels tested where no adverse effects were observed. These results support the conclusion 

that dietary exposure of Cry1Da_7 poses negligible risk to birds and small mammals. 
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Table VII-11. Dose-Based Estimated Environmental Concentrations (EECs), Median 

Lethal Concentrations (LC50) from Terrestrial Vertebrate Studies and Margins of 

Exposure (MOEs) for the Cry1Da_7 Protein 

Test Organism

(NTO group) Dietary Class EECa LC50 MOE 

Colinus virginianus 

(birds) 

Herbivore 23.8 mg/kg b.w. >1000 mg/kg b.w. !42

Granivore 0.1 mg/kg b.w. >1000 mg/kg b.w. !10,000 

Mus musculus 

(mammals)

Herbivore 19.9 mg/kg b.w. >5000 mg/kg b.w. !250 

Granivore 0.1 mg/kg b.w. >5000 mg/kg b.w. !50,000 

a

The EEC value for the smallest body-weight vertebrate (Table VII-7) was used as a worst case for risk 

characterization. 

VII.D. Effect of MON 95379 Maize on Threatened and Endangered Species 

A comprehensive assessment on the potential effects of MON 95379 maize on listed threatened 

and endangered species was conducted. While MON 95379 will not be commercialized in the 

U.S., limited breeding and seed increase related activities are intended to occur in Nebraska, 

Hawaii, and Iowa on up to a combined maximum U.S. total of 100 acres per growing season and 

small-scale, confined field trials may potentially occur in other locations to collect needed 

regulatory data to support global regulatory submissions of future products stacked with 

MON 95379. Since the results of activity spectrum screening and Tier I testing indicated effects 

on survival from Cry1B.868 and Cry1Da_7 are limited to Lepidoptera, a comparison of the county 

level distribution of endangered lepidopteran species in these three states relative to maize 

production counties identified three species for further evaluation (Table VII-12). These species 

include Blackburn’s sphinx moth (Manduca blackburni) in Hawaii, Dakota skipper (Hesperia 

dacotae) and the Poweshiek skipperling (Oarisma Poweshiek) in Iowa and no species in Nebraska. 

Evaluation of critical habitats and updated ranges for these species supports the conclusion that 

Blackburn’s sphinx moth, Dakota skipper, and Poweshiek skipperling would have no significant 

exposure to MON 95379 maize. In support of our assessment on likely exposure, a recent 

assessment of potential effects on threatened and endangered species was conducted by U.S. EPA 

for MON 89034 u TC1507 u MON 87411 u DAS-59122-7 maize, that expresses lepidopteranactive Cry1A.105, Cry2Ab2, and Cry1F proteins, resulted in a No Effect determination for listed 

lepidopteran species, including Blackburn’s sphinx moth, due to habitat requirements that exclude 

them from maize fields (U.S. EPA, 2017). Recent surveys by U.S. FWS indicate that Dakota 

skipper and Poweshiek skipperling have not been observed in Iowa since the time of listing (2014) 

and are no longer believed to be present in Iowa (USFWS, 2018a; b). The specificity of Cry1B.868 

and Cry1Da_7 for Lepidoptera and negligible expected exposure based on the proposed limited 

acreage and use of standard agronomic management practices for small scale field trials results in 
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the conclusion that MON 95379 will have no effect on currently listed threatened and endangered 

species (see also Appendix L.21). 
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Table VII-12. Spatial Overlap of Threatened and Endangered Species Habitat and Maize Cultivation in Nebraska, Hawaii, and Iowa 

Common Name Species Name Reference Habitat 

Overlap of Species 

Presence with MaizeProduction Countiesa 

Blackburn's sphinx moth Manduca blackburni USFWS, 2005 Mesic shrubland Counties: 2 

HI: Hawaii, Maui 

Dakota skipper Hesperia dacotae USFWS, 2018a Prairie grasslands Counties: 1b 

IA: Dickinson

Poweshiek skipperling Oarisma powershiek USFWS, 2018b Prairie grasslands Counties: 7c 

IA: Cerro Gordo, 

Dickinson, Emmet, 

Hancock, Howard, 

Kossuth, Osceola 

aCounty-level maize production estimates derived from USDA-NASS 2017 Agricultural Census (USDA-NASS, 2017). bRecent update by USFWS indicate this species is no longer believed to be present in Iowa (USFWS, 2018a). cSpecies has not been observed in Iowa since the time of listing and is no longer believed to be present in the United States outside of Michigan 

(USFWS, 2018b). 
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VII.E. Conclusion 

MON 95379 maize expresses Cry1B.868 and Cry1Da_7 whose activity is limited to Lepidoptera 

under field conditions. Cry1B.868 and Cry1Da_7 are members of the Cry1 family of Bt proteins. 

Cry1B.868 and Cry1Da_7 have a MOA shared with previously commercialized Cry1 proteins that 

have a long history of safe use (Koch et al., 2015; Wang et al., 2019). Under the proposed terms 

of an EPA seed increase registration application, MON 95379 maize will be cultivated for smallscale breeding, testing, and seed increase-related activities in the three U.S. states of Nebraska, 

Hawaii, and Iowa with a maximum combined total acreage of 100 acres per growing season and 

small-scale, confined field trials potentially in other locations to collect needed regulatory data to 

support global regulatory submissions of future products stacked with MON 95379. This limited 

geography significantly limits exposure to NTOs beneficial to agriculture. Tier I toxicity testing 

on NTOs representing key ecological functions demonstrated no adverse effects on survival from 

Cry1B.868 and Cry1Da_7 at levels expressed in MON 95379 maize. Calculated MOEs based on 

conservative estimates of exposure were !11 for all species tested with the exception of C. 

maculata where the MOE for Cry1Da_7 was !7. These results indicate no adverse effects to NTOs 

are anticipated at field expression levels. Therefore, the results of the assessment based on the 

studies described herein support the conclusion that intended cultivation of MON 95379 maize 

under the proposed limited acreage poses minimal risk to NTO beneficial to agriculture. An 

evaluation focused on listed Lepidoptera species in the states of Nebraska, Hawaii, and Iowa, 

where MON 95379 will be primarily cultivated, resulted in the conclusion of no effect for currently 

listed threatened and endangered species. 
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VIII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL INTERACTIONS 

ASSESSMENT 

This section provides a comparative assessment of the phenotypic, agronomic, and environmental 

interactions characteristics of MON 95379 compared to the conventional control. The data support 

a conclusion that MON 95379 is unlikely to pose a greater plant pest risk than conventional maize. 

This conclusion is based on the results of multiple evaluations including field and laboratory 

assessments. MON 95379 has been planted in the U.S. under permit/notification since 2015 

(Appendix A). 

Phenotypic, agronomic, and environmental interactions characteristics of MON 95379 were 

evaluated in a comparative manner to assess plant pest potential. These assessments included seed 

germination and dormancy characteristics and as well as phenotypic and agronomic characteristics 

and plant responses to abiotic stressors, diseases, and arthropod pests in the field. Results from 

these assessments demonstrate that MON 95379 does not possess 1) increased weediness 

characteristics; 2) increased susceptibility or tolerance to specific abiotic stressors, diseases, or 

arthropod pests; or 3) characteristics that would confer an increased plant pest risk compared to 

conventional maize. 

VIII.A. Characteristics Measured for Assessment 

In the phenotypic, agronomic, and environmental interactions assessment of MON 95379, data 

were collected to evaluate altered plant pest potential. A detailed description of the regulated 

article phenotype is requested as part of the petition for determination of nonregulated status in the 

previous regulations codified in 7 CFR § 340.6 including differences from the unmodified 

recipient organism that would “substantiate that the regulated article is unlikely to pose a greater 

plant pest risk than the unmodified organism from which it was derived.” As part of the 

characterization of MON 95379, data were collected to provide a detailed description of the 

phenotypic, agronomic, and environmental interactions characteristics of MON 95379. A subset 

of these data represents specific characteristics that are typically associated with altered plant pest 

potential (e.g., seed dormancy, lodging, seed loss, and environmental interactions data). 

The plant characterization of MON 95379 encompassed five general data categories: 1) seed 

germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive development; 4) 

lodging and seed retention on the plant; and 5) plant responses to abiotic stressors, diseases, and 

arthropod pests. An overview of the characteristics assessed is presented in Table VIII-1. 

The phenotypic, agronomic, and environmental interactions data were evaluated from a basis of 

familiarity (OECD, 1993) and were comprised of a combination of field and laboratory studies 

conducted by scientists who are familiar with the production and evaluation of maize. In each of 

these assessments, MON 95379 was compared to an appropriate conventional control that had a 

genetic background similar to MON 95379 but did not possess the lepidopteran-protected trait. In 

addition, multiple commercial maize hybrids developed through conventional breeding and 

selection (see Appendix G-Appendix I and Tables G-1, H-1, and I-1) were included to provide a 

range of comparative values for each characteristic that are representative of the variability in 

existing commercial maize hybrids. Data collected for the various characteristics from the 

commercial reference maize hybrids provide context for interpreting experimental results. 
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Table VIII-1. Phenotypic, Agronomic, and Environmental Interaction Characteristics 

Evaluated in U.S. Field Trials and Laboratory Studies 

Data category 

Characteristic 

measured

(section where 

discussed) 

Evaluation timing (setting of 

evaluation) 

Evaluation description 

(measurement endpoints) 

Germination, 

dormancy, and 

emergence

Germinated 

(VIII.C.1)

Day 4 and 7 at OT1 

Day 4, 7, and 11 at SOT1 

(Laboratory) 

Percentage of seed with a radicle 

protruding through the seed coat 

and greater than 1 mm in length 

Dead 

(VIII.C.1)

Day 4 and 7 at OT 

Day 4, 7, and 11 at SOT 

(Laboratory)

Percentage of seed that had visibly 

deteriorated and become soft to the 

touch (also included non-viable 

hard and non-viable firm-swollen 

seed) 

Viable firm-swollen 

(VIII.C.1) 

Day 7 at OT 

Day 11 at SOT (Laboratory) 

Percentage of seed that imbibed 

water and were firm to the touch 

but lacked any evidence of growth 

(viability determined by a 

tetrazolium test) 

Viable hard 

(VIII.C.1)

Day 7 at OT 

Day 11 at SOT (Laboratory) 

Percentage of seed that did not 

imbibe water and remained hard to 

the touch (viability determined by a 

tetrazolium test) 

Early stand count 

(VIII.C.2.1) 

V2 – V4 growth stage 

(Field) 

Number of plants per m2

Vegetative 

growth

Plant height 

(VIII.C.2.1) 

R1 – R6 (Field) Distance from the soil level to the 

flag leaf collar 

Days to maturity 

(VIII.C.2.1) 

R6 (Field) Number of days from planting to 

kernel black layer 

Final stand count 

(VIII.C.2.1) 

Pre-harvest (Field) Number of plants per m2

1

 Optimum temperature OT = 25°C for seven days, Suboptimum temperature SOT = 10°C for seven days 

followed by 25°C for four days. 

 
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Table VIII-I. Phenotypic, Agronomic, and Environmental Interactions Characteristics 

Evaluated in U.S. Field Trials and Laboratory Studies (continued) 

Data category 

Characteristic 

measured

(section where 

discussed) 

Evaluation timing (setting 

of evaluation) 

Evaluation description 

(measurement endpoints) 

Reproductive 

development

Days to flowering 

(VIII.C.2.1) 

R1 (Field) Days from planting until 50% of the 

plants have begun to shed pollen 

Grain moisture 

(VIII.C.2.1) 

Harvest (Field) Percentage moisture of harvested 

grain 

Seed weight 

(VIII.C.2.1) 

Harvest (Field) Mass of 100 mature seeds, adjusted 

to 15.5% moisture content 

Yield 

(VIII.C.2.1) 

Harvest (Field) Mass of harvested grain per hectare, 

adjusted to 15.5% moisture content 

Lodging and seed 

retention 

Lodging 

(VIII.C.2.1) 

Pre-harvest (Field) Percentage of plants leaning >45° 

from vertical or broken below the ear 

Seed loss 

(VIII.C.2.1) 

Pre-harvest (Field) Number of ears completely detached 

from plants in two rows 

Environmental 

interactions

Abiotic stress 

response (VIII.C.2.2) 

Four times during growing 

season (Field)

Qualitative assessment of each plot, 

with categorical scale of increasing 

severity (none, slight, moderate, 

severe) 

Disease damage 

(VIII.C.2.2) 

Four times during growing 

season (Field)

Qualitative assessment of each plot, 

with categorical scale of increasing 

severity (none, slight, moderate, 

severe) 

Arthropod damage 

(VIII.C.2.2) 

Four times during growing 

season (Field)

Qualitative assessment of each plot, 

with categorical scale of increasing 

severity (none, slight, moderate, 

severe) 

1

 Optimum temperature OT = 25°C for seven days, Suboptimum temperature SOT = 10°C for 

seven days followed by 25°C for four days. 

 
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VIII.B. Interpretation of Phenotypic and Environmental Interaction Data 

Plant pest risk assessments for biotechnology-derived crops are comparative assessments and are 

considered from a basis of familiarity. The concept of familiarity is based on the fact that the 

biotechnology-derived plant is developed from a well-characterized conventional maize whose 

biological properties and plant pest potential are well-known. Familiarity considers the biology of 

the crop, the introduced traits, the receiving environment and the interaction of these factors, and 

provides a basis for comparative environmental risk assessment between a biotechnology-derived 

plant and its conventional counterpart. 

Expert knowledge and experience with conventionally bred maize was the basis for selecting 

appropriate endpoints and estimating the range of responses that would be considered typical for 

maize. As such, MON 95379 was compared to the conventional control in the assessment of 

phenotypic, agronomic, and environmental interactions characteristics. Based on all of the data 

collected, an assessment was made to determine if MON 95379 could be expected to pose an 

increased plant pest risk compared to conventional maize. 

VIII.B.1. Interpretation of Phenotypic and Agronomic Data 

Comparative plant characterization data between a biotechnology-derived crop and the 

conventional control are interpreted in the context of contributions to increased weediness or plant 

pest risk. Under the framework of familiarity, characteristics for which no differences are detected 

support a conclusion of no increased weediness or plant pest risk of the biotechnology-derived 

crop compared to the conventional crop. Characteristics for which statistically significant 

differences are detected are considered in a step-wise method (Figure VIII-1) or in a similar 

fashion. All detected differences for a characteristic are considered in the context of whether or 

not the difference would increase the crop’s pest/weed potential. Ultimately, a weight of evidence 

approach considering all characteristics and data is used for the overall risk assessment of 

differences and their significance. Figure VIII-1 illustrates the stepwise assessment process 

employed. 

 
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Figure VIII-1. Interpretation of Statistical Differences 

Step 1 – Evaluate Detected Statistically Significant Differences 

Data on each measured characteristic are statistically analyzed. A combined-site analysis is used for multisite data. All statistically significant differences are evaluated and considered in the context of a change in 

weediness or plant pest risk. Any difference detected is further assessed. 

Step 2 – Evaluate Differences in the Context of Commercial Reference Materials Included in the Study 

If a difference for a characteristic is detected then the mean value of the biotechnology-derived crop for the 

characteristic is assessed relative to the range of variation of the commercial reference materials included 

in the study (e.g., reference range). 

Step 3 – Evaluate Differences in the Context of the Crop 

If the mean value of the characteristics for a biotechnology-derived crop is outside the variation of the 

commercial reference materials included in the study, the mean value of the biotechnology-derived crop is 

assessed relative to known values common for the crop (e.g., published values). 

Step 4 – Relevance of Difference to Weediness or Plant Pest Risk 

If the mean value of the characteristics for a biotechnology-derived crop is outside the range of values 

common for the crop, the difference is then assessed for whether or not it is meaningful in terms of 

weediness or plant pest risk. 

Step 5 – Conduct Risk Assessment on Identified Hazard 

If an adverse effect (hazard) is identified, a risk assessment on the difference is conducted. The risk 

assessment considers contributions to enhanced weediness or plant pest risk of the crop itself, the impact 

of differences detected in other measured characteristics, and potential for and effects of trait introgression 

into any populations growing outside of cultivated environments or into a sexually compatible species. 

 
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VIII.B.2. Interpretation of Environmental Interactions Data 

The environmental interactions data consisting of plant responses to abiotic stressors, diseases, 

and arthropod pests are categorical and were considered to differ in susceptibility or tolerance if 

the range of injury symptoms did not overlap between the biotechnology-derived crop and the 

conventional control across all four replications within an observation at a site. 

Observations for which no differences are observed support a conclusion of no increased 

weediness or plant pest risk. Observations for which differences were detected are not considered 

to indicate an increase in weediness or plant pest risk if the biotechnology-derived crop stressor 

responses and damage ratings are within the reference range or are not consistently observed in 

multiple environments in which the same stressor occurred. 

VIII.C. Comparative Assessments of the Phenotypic, Agronomic, and Environmental 

Interaction Characteristics of MON 95379 

This section provides the results of comparative assessments conducted in replicated laboratory 

and/or multi-site field experiments to provide a detailed phenotypic, agronomic, and 

environmental interactions description of MON 95379. The characteristics for MON 95379 

evaluated in these assessments included: seed germination and dormancy characteristics (Section 

VIII.C.1), plant phenotypic, agronomic, and environmental interactions observations under field 

conditions (Section VIII.C.2). Additional details for each assessment are provided in 

Appendix G and Appendix H. 

VIII.C.1. Seed Dormancy and Germination Characteristics 

USDA-APHIS considers the potential for weediness to constitute a plant pest factor (previously 7 

CFR § 340.6). Seed germination and dormancy mechanisms vary with species and their genetic 

basis tends to be complex. Seed dormancy (e.g., hard seed) is an important characteristic that is 

often associated with plants that are considered weeds (Anderson, 1996; Lingenfelter and Hartwig, 

2007). Information on germination and dormancy characteristics is therefore useful when 

assessing a plant for increased weediness potential. To assess germination characteristics, 

standardized germination assays are available and routinely used. The Association of Official Seed 

Analysts (AOSA), an internationally recognized seed testing organization, recommends a 

temperature regime of 25°C for testing the germination and dormancy characteristics of maize 

seed (AOSA, 2018b; a). The AOSA further recognizes a temperature regime of constant 10°C for 

seven days followed by 25°C for four days for cold testing of maize (AOSA, 2009). 

A comparative assessment of seed germination and dormancy characteristics was conducted for 

MON 95379 and the conventional control. The seed lots for MON 95379 and the conventional 

control were harvested from one 2018 field production site in Kihei, Hawaii. Four reference maize 

hybrids were obtained from commercial sources. MON 95379 was compared to the conventional 

control for percentages of germinated, dead, viable firm-swollen, and viable hard seed using two 

temperature regimes: optimum treatment was performed at 25oC for 7 days; suboptimum treatment 

was performed at 10oC for 7 days, followed by 4 days at 25oC. The assay for each temperature 

regime was conducted using a randomized complete block design with four replications. 

Descriptions of the evaluated germination and dormancy characteristics and the timing of the 
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evaluations are listed in Table VIII-1. The materials and experimental methods are further 

discussed in Appendix G. 

In the analyses of the dormancy and germination data, no statistically significant differences 

(α = 0.05) were detected between MON 95379 and the conventional control in either the optimum 

(25°C) or suboptimum (10/25°C) temperature regimes for any of the evaluated characteristics, 

including viable hard seed (Table VIII-2). These results support the overall conclusion that the 

introduction of lepidopteran-protected trait does not result in increased weediness or plant pest risk 

of MON 95379 compared to conventional maize. 

Table VIII-2. Germination Characteristics of MON 95379 and the Conventional Control 

Temperature 

Treatment2 Characteristic3 Mean % (SE)1

Test Control Reference Range4

Optimum Germinated 99.5 (0.50) 100.0 (0.00) 98.8 – 99.3 

Dead 0.5 (0.50) 0.0 (0.00) 0.5 - 1.3 

Viable Firm-Swollen 0.0 (0.00) 0.0 (0.00) 0.0 - 0.5 

Viable Hard 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 

Suboptimum Germinated 99.3 (0.48) 100.0 (0.00) 99.3 – 99.5 

Dead 0.0 (0.00) 0.0 (0.00) 0.0 - 0.5 

Viable Firm-Swollen 0.8 (0.48) 0.0 (0.00) 0.0 - 0.5 

Viable Hard 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0 

Note: The experimental design was a randomized complete block with 4 replicates. 1

Values represent means with standard error (SE) in parentheses.

2

Optimum treatment was performed at 25oC for 7 days; suboptimum treatment was performed at 10oC for 7 days, 

followed by 4 days at 25oC. 

3

Statistical comparisons were performed using Fisher’s Exact Test. No statistical comparisons were made if test and 

control values were 0. 

4

Minimum and maximum mean values observed among the reference materials. 

VIII.C.2. Field Phenotypic, Agronomic, and Environmental Interactions Characteristics 

Phenotypic and agronomic characteristics were evaluated under field conditions as part of the plant 

characterization assessment of MON 95379. These data were developed to provide USDA-APHIS 

with a detailed description of MON 95379 relative to the conventional control and reference maize 

hybrids. According to previous regulations codified in 7 CFR § 340.6, as part of the petition to 

seek deregulation, a petitioner must submit “a detailed description of the phenotype of the 

regulated article.” This information is being provided to assess whether there are phenotypic 

differences between MON 95379 and the conventional control that may impact its weediness or 

plant pest risk. Specific characteristics that are typically associated with weediness (e.g., lodging 

and seed loss) were used to assess whether there is a potential increase in weediness of 

MON 95379 compared to conventional maize. 

USDA-APHIS considers the environmental interaction of the biotechnology-derived crop 

compared to its conventional control to determine the potential for increased plant pest 
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characteristics. Evaluations of environmental interactions were conducted as part of the plant 

characterization for MON 95379. In the 2018 U.S. field trials conducted to evaluate the phenotypic 

and agronomic characteristics of MON 95379, data were also collected on plant responses to 

abiotic stressors, diseases, and arthropod pests. 

Data were collected from eight 2018 field sites within maize production regions of the U.S. (Table 

VIII-3). The test material MON 95379, the conventional control, and four commercial reference 

hybrids were planted at each site in a randomized complete block design with four replications. 

Seventeen unique references were included among the sites. Additional details on the materials 

and methods are presented in Appendix H. 

VIII.C.2.1. Field Phenotypic and Agronomic Characteristics 

MON 95379 was compared to the conventional control in a combined-site analysis for nine 

phenotypic and agronomic characteristics: early stand count, days to flowering, plant height, days 

to maturity, lodging, final stand count, moisture, seed weight, and yield. Descriptive statistics are 

provided for an additional characteristic, seed loss, that had insufficient variability for formal 

statistical analysis. Descriptions of the evaluated phenotypic and agronomic characteristics and the 

timing of the evaluations are listed in Table VIII-1. The materials and methods are further 

discussed in Appendix H. 

The means for MON 95379 and the conventional control for seed loss, which was excluded from 

formal analysis, were low and comparable. The mean for MON 95379 was within the reference 

range. These results suggest that this characteristic does not contribute to a biological change for 

MON 95379 in terms of plant pest potential. 

In the combined-site analysis of phenotypic characteristics of the MON 95379 compared to the 

conventional control, no statistically significant differences (α = 0.05) were detected between the 

test and the conventional control for 7 of the 9 analyzed phenotypic characteristics. The 

MON 95379 test had significantly higher final stand and lower seed weight compared to the 

conventional control. However, the mean value(s) of MON 95379 were within respective 

reference ranges of conventional maize for these characteristics (Table VIII-4). These results, 

together with those for seed loss, support the overall conclusion that the introduction of the 

lepidopteran-protected trait does not result in increased weediness or plant pest risk of MON 95379 

compared to conventional maize. 

 
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Table VIII-3. Phenotypic and Agronomic and Environmental Interactions Sites for 

MON 95379 during 2018 

Site Code County, State 

IAEH Shelby, Iowa 

IAOG Boone, Iowa 

IARL Jefferson, Iowa 

ILHY Clinton, Illinois 

ILJO Champaign, Illinois 

INSH Clinton, Indiana 

NEYO York, Nebraska 

OHTR Miami, Ohio 

Table VIII-4. Combined-Site Analysis of Phenotypic and Agronomic Characteristics of 

MON 95379 Compared to the Conventional Control in 2018 U.S. Field Trials 

Characteristic (units) Mean (S.E.)1Reference Range2

MON 95379 Control 

Early stand count (plants/m2) 8.6 (0.10) 8.5 (0.08) 7.6 – 9.4

Days to flowering 58.5 (0.62) 57.9 (0.60) 51.0 – 63.2

Plant height (cm) 263.0 (2.07) 257.2 (2.19) 225.3 – 267.0

Days to maturity 119.2 (1.34) 120.4 (1.36) 106.0 – 126.0

Lodging (%) 12.9 (2.37) 9.4 (1.68) 0.4 – 18.9

Seed loss (ears)† 0.0 (0.03) 0.0 (0.03) 0.0 – 0.5

Final stand count (plants/m2) 8.2 (0.10)* 8.0 (0.10) 7.3 – 8.9

Moisture (%) 17.1 (0.24) 17.1 (0.22) 15.2 – 19.1

Seed weight (g) 34.1 (0.51)* 35.8 (0.48) 29.3 – 40.5

Yield (t/ha) 13.2 (0.34) 13.3 (0.35) 12.0 – 18.6

Notes: *Indicates a statistically significant difference between MON 95379 and the conventional control (α = 0.05) in 

the combined-site, linear mixed model analysis. All plots at sites INSH and NEYO were thinned to promote uniform 

density following early stand count. 

†

 Indicates that p values could not be generated because the data did not meet the normality and/or common variance 

assumptions of the linear mixed model. 

1

 N = 32 for means. S.E. = standard error. 

2 Minimum and maximum mean values among 17 references, where each mean was combined over all the sites at 

which the reference hybrid was planted. 

VIII.C.2.2. Environmental Interaction Assessments 

Environmental interaction assessments evaluate the potential for unintended effects by seeking to 

determine whether the biotechnology derived plant demonstrates an increase in susceptibility or 

tolerance to abiotic stressors, diseases and arthropod pests. Plant responses to abiotic stressors, 

diseases, and arthropod pests were assessed at natural levels, i.e., no artificial infestation or 

imposed abiotic stress; therefore, these levels typically varied between observations at a site and 

among sites. These data were collected from each plot using a categorical scale (none, slight, 

moderate, and severe) of increasing severity of observed damage for each stressor. This scale was 

utilized to allow for the evaluation of the wide variety of potential abiotic stressors, diseases, and 
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arthropod pests occurring across the season and across sites. These data were summarized and not 

subjected to ANOVA statistical evaluation. For a particular stressor, all comparisons of the range 

of responses for MON 95379 to the range of responses for the conventional control across all 

observation times and sites are reported. Descriptions of the evaluated environmental interactions 

characteristics and the timing of the evaluations are listed in Table VIII-1. The materials, methods, 

additional details concerning the qualitative environmental interactions assessments, and detailed 

results of the qualitative data comparisons are further discussed and presented in Appendix H and 

Appendix L. 

In an assessment of plant responses to abiotic stressors, diseases, and arthropod pests, no 

differences were observed between MON 95379 and the conventional control for any of the 288 

observations (including 96 abiotic stressor, 96 disease, and 96 arthropod pest observations) among 

the eight sites (Table VIII-5). These results support no increased susceptibility or tolerance to 

specific abiotic stressors, diseases or non-target arthropod pests of MON 95379 compared to 

conventional maize. 

Table VIII-5. Summary of Qualitative Environmental Interactions Assessments for 

MON 95379 during 2018 

Stressor Number of observations 

across all sites 

Number of observations with no 

differences between MON 95379 and the 

conventional control across all sites 

Abiotic stressors 96 96 

Diseases 96 96 

Arthropod pests 96 96 

Total 288 288 

No differences were observed between MON 95379 and the conventional control during any of 

the 96 observations (8 sites, 4 timepoints, 3 stressors) for each stressor category. MON 95379 and 

the conventional control were considered different in susceptibility or tolerance if the range of 

injury symptoms across four replications did not overlap between MON 95379 and the 

conventional control. 

VIII.D. Conclusions for Phenotypic, Agronomic, and Environmental Interactions 

Evaluation 

Comparative plant characterization data between a biotechnology-derived crop and the 

conventional control are interpreted in the context of contributions to increased plant pest potential 

as assessed by USDA-APHIS. Under the framework of familiarity, characteristics for which no 

statistically significant differences are detected support a conclusion of no increased plant pest 

potential of the biotechnology-derived crop compared to the conventional crop. Ultimately, a 

weight of evidence approach that considers all characteristics and data is used for the overall risk 

assessment of differences and their significance. 

An extensive and robust set of phenotypic, agronomic, and environmental interactions data were 

used to assess whether the introduction of the lepidopteran-protected trait altered the plant pest 

potential of MON 95379 compared to the conventional control, considered within the context of 

the variation among the reference maize hybrids. These assessments included five general data 
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categories: 1) seed germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive 

development; 4) lodging and seed retention on the plant; and 5) plant responses to abiotic stressors, 

diseases, and arthropod pests. Within these data categories, specific characteristics typically 

associated with weediness were also assessed to determine whether there was a potential increase 

in weediness of MON 95379 compared to conventional maize. 

Results from these assessments comparing MON 95379 and the conventional control support the 

conclusion that MON 95379 does not possess: 1) increased weediness characteristics; 2) increased 

susceptibility or tolerance to specific abiotic stressors, diseases, or arthropod pests; or 3) 

characteristics that would confer a plant pest risk compared to conventional maize. Therefore, 

based on the results of multiple assessments discussed above and presented in the appendices, the 

weight of evidence supports the overall conclusion that the introduction of the lepidopteranprotected trait does not result in increased weediness or plant pest risk of MON 95379 compared 

to conventional maize. 
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IX. U.S. AGRONOMIC PRACTICES 

IX.A. Introduction 

As part of the plant pest assessment required by the previous regulations codified in 7 CFR § 

340.6(c)(4), impacts to agricultural and cultivation practices must be considered. MON 95379 will 

not be commercialized in the U.S. and therefore will not impact U.S. commercial maize 

agricultural practices. It is intended to be cultivated in small scale breeding, testing, and seed 

increase nurseries to develop seed for future products in South America. In the future, MON 95379 

stack products may also be planted in confined, small-scale field trials that would support 

subsequent global regulatory submissions. 

Information presented in Section VIII.C demonstrated that MON 95379 maize is no more 

susceptible to diseases or pests than commercially cultivated maize. Additionally, data presented 

in this petition support that, with the exception of the introduced trait, MON 95379 maize is 

phenotypically similar to conventional maize and is not expected to pose a greater plant pest risk 

than conventional maize. This section will focus on the potential impacts to agricultural and 

cultivation practices considering the intended use of small-scale breeding and seed increase. It is 

not anticipated that breeding and seed increase activities in the U.S. would have a notable impact 

on current practices such as the management of weeds, diseases, and insects except for the 

management of lepidopteran pests. 

IX.B. Maize Breeding and Seed Production 

An overview of U.S. maize production in the context of commercial production was provided in 

Bayer’s petition for nonregulated status of MON 87429 maize (USDA-APHIS Petition #19-316-

01p p.125). However, MON 95379 will not be commercialized in the U.S. but is intended to be 

cultivated in small-scale breeding, testing, and seed increase nurseries to develop seed for future 

products in South America as well as potential small-scale confined field trials for stack products 

in other locations, as described earlier.

Maize nurseries typically used for breeding and seed increase activities utilize practices to 

minimize pollen flow in order to produce high quality seeds. Those practices may include, but are 

not limited to: 660 feet spatial isolation or 21 day temporal isolation from other maize, planting 

border around nurseries (10 - 15 ft), maintaining fallow area (up to 50 ft) and hand-bagging 

(isolating) male flower (tassels) of plants targeted to be self-pollinated or used as a pollen donor. 

In hybrid seed production fields (commonly called isolated crossing blocks) female parental inbred 

rows (MON 95379 is expected to be deployed on female side of the pedigree) would be de-tasseled 

or utilize male sterility technologies. 

Pre-season, in-season and post-harvest agronomic practices, including insect, disease or weed 

control management, crop rotation or volunteer management, will not differ for MON 95379 seed 

production when compared to the current maize practices implemented by nursery sites. Seed 

produced will not be used for commercial grain production in the United States. 

IX.B.1. Management of Insect Pests 
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Bayer summarized major issues associated with the management of insect pests in its petition for 

nonregulated status for corn rootworm-protected maize MON 87411 maize (USDA-APHIS 

Petition #13-290-01p p166). In brief, insect pests are a significant source of damage to maize and 

are commonly addressed by biotechnology-derived insect-protection traits, insecticide treatment 

of seeds, soil or foliar application of insecticides, or use of crop rotation or other integrated pest 

management practices. The EPA under the Federal Insecticide, Fungicide and Rodenticide Act 

(FIFRA), regulates the distribution, sale, use and testing of pesticidal substances (including those 

produced in plants), that are intended to control insect pests. 

Although MON 95379 contains the coding sequences for the Cry1B.868 and Cry1Da_7 proteins 

that provide protection against certain lepidopteran insect pests present in the U.S., Bayer is only 

seeking to cultivate MON 95379 in small-scale breeding, testing, and seed increase nurseries to 

develop seed for future products in South America as well as potential small-scale confined field 

trials for stack products in other locations, as described earlier. 

IX.B.2. Management of Diseases and Other Pests 

Bayer summarized major issues associated with the management of diseases and non-insect pests 

in its petition for nonregulated status for corn rootworm-protected maize MON 87411 maize 

(USDA-APHIS Petition #13-290-01p p171). MON 95379 does not contain disease protection 

traits, therefore the information on this subject is incorporated here by reference (USDA-APHIS 

Petition #13-290-01p p. 171). 

MON 95379 was developed to improve the management of targeted lepidopteran insect pests and 

has no unique attributes for control of diseases or other pests. Environmental interactions 

observations in field studies indicated that the presence of the lepidopteran-protected trait did not 

meaningfully alter the susceptibility of MON 95379 maize to diseases or other pests (Section 

VIII.C). Therefore, no changes in current management practices for diseases or other pests in 

maize are anticipated for the intended use of MON 95379 maize relative to conventional maize. 

IX.B.3. Weed Management 

MON 95379 does not contain herbicide tolerance trait and is therefore no different from other nonherbicide tolerant maize in terms of its weed management considerations. However, the 

information on this subject is incorporated here by reference from its petition for nonregulated 

status for MON 87429 maize (USDA-APHIS Petition #19-316-01p p.130). In brief, annual and 

perennial weeds are considered to be the greatest pest problem in maize production (Aref and Pike, 

1998). Weed control in maize is essential for optimizing yield because weeds compete with maize 

for light, nutrients, and moisture and can lead to reductions in yield (Knake et al., 1990). 

IX.B.4. Crop Rotation and Volunteer Management Practices in Maize 

MON 95379 activities in the United States will be limited to breeding and seed increase activities 

and will not be commercialized in the U.S. These breeding and seed production activities will take 

place at research and breeding facilities following typical recommended practices employed to 

achieve high quality seed and control volunteer maize as needed to maintain and assure product 

quality. The intended use of MON 95379 maize in the U.S. is not expected to impact crop rotation 

practices or volunteer management practices in maize. 
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IX.B.5. Stewardship of MON 95379 Maize 

Bayer develops effective products and technologies that deliver value to growers and conserve 

resources that agriculture depends on and is committed to assuring that its products and 

technologies are safe and environmentally responsible. Bayer demonstrates this commitment by 

implementing product stewardship processes throughout the lifecycle of a product and by 

participation in the Excellence Through Stewardship® (ETS) Program13 (BIO, 2010). These 

policies and practices include rigorous field compliance and quality management systems and 

verification through auditing. 

As an integral component of fulfilling this global stewardship commitment, Bayer will seek 

biotechnology regulatory approvals for MON 95379 maize in all important maize import countries 

with a functioning regulatory system to assure global compliance and support the flow of 

international trade. These actions will be consistent with the ETS Guide for Product Launch 

Stewardship of Biotechnology-Derived Plant Products (ETS, 2018) and Biotechnology Innovation 

Organization (BIO) Policy on Product Launches (BIO, 2012). 

Bayer is also committed to following industry best practices on seed quality assurance and control 

to ensure the purity and integrity of MON 95379 maize hybrid seed. As with all of Bayer’s 

products, before commercializing MON 95379 maize in any country, a MON 95379 maize 

detection method will be available via a third-party vendor to maize producers, processors, and 

buyers regardless of whether they have purchased MON 95379 or not. 

IX.C. Summary and Conclusion: Impact of the Introduction of MON 95379 Maize on 

Agricultural Practices 

MON 95379 was developed to improve the management of targeted lepidopteran insect pests for 

growers in South America and has no unique attributes for control of diseases or other pests. 

MON 95379 will not be commercialized in the U.S. and is therefore not expected to have adverse 

impacts on current agronomic, cultivation and management practices for commercial grain or 

hybrid maize seed production. Activities will be limited to small-scale breeding, testing and seed 

increases in the U.S., with typical production practices to produce high quality seed and/or data. 

Evaluation of the agronomic and phenotypic characteristics of MON 95379 support that, with the 

exception of the introduced trait, MON 95379 maize is phenotypically similar to conventional 

maize and is not expected to have an effect on practices used in small scale breeding and seed 

activities. 

13 www.excellencethroughstewardship.org/
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X. PLANT PEST ASSESSMENT 

X.A. Introduction 

This section provides a brief review and assessment of the plant pest potential of MON 95379 and 

its potential impact on agronomic practices and the environment. USDA-APHIS has 

responsibility, under the Plant Protection Act (PPA) (7 U.S.C. § 7701-7772), to prevent the 

introduction and dissemination of plant pests into the U.S. Prior regulations codified in 7 CFR § 

340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that 

a particular regulated article does not present a plant pest risk and should no longer be regulated. 

If APHIS determines that the regulated article is unlikely to pose a greater plant pest risk than 

conventional maize, the petition is granted, thereby allowing introduction without the need for 

permit or notification. 

According to the PPA, the definition of “plant pest” includes the living stage of any of the 

following, or a similar article, that can directly or indirectly injure, damage, or cause disease in 

any plant or plant product: (A) a protozoan; (B) a nonhuman animal; (C) a parasitic plant; (D) a 

bacterium; (E) a fungus; (F) a virus or viroid; or (G) an infectious agent or other pathogens (7 

U.S.C. § 7702[14]). 

The regulatory endpoint under the USDA-APHIS regulations for biotechnology-derived crop 

products is not zero risk, but rather a determination that deregulation of the article in question is 

not expected to pose a greater plant pest risk than conventional maize. Information in this petition 

related to plant pest risk characteristics includes: 1) mode-of-action and changes to plant 

metabolism; 2) composition; 3) expression and characteristics of the gene product; 4) potential for 

weediness of the regulated article; 5) potential impacts to NTOs beneficial to agriculture and no 

effect on threatened and endangered species; 6) abiotic stressor, disease and pest susceptibilities; 

7) impacts on agronomic practices; and 8) impacts on the weediness of any other plant with which 

it can interbreed, as well as the potential for gene flow. Using the assessment above, the data and 

analysis presented in this petition led to a conclusion that MON 95379 is unlikely to pose a greater 

plant pest risk than conventional maize, and therefore should no longer be subject to regulation 

under 7 CFR § 340. 

X.B. Plant Pest Assessment of MON 95379 and Expressed Cry1B.868 and Cry1Da_7 

Proteins 

This section summarizes the details of the genetic insert, characteristics of the genetic 

modification, and safety and expression of the Cry1B.868 and Cry1Da_7 protein expressed in 

MON 95379 used to evaluate the food, feed, and environmental safety of MON 95379. 

 
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X.B.1. Characteristics of the Genetic Insert and Expressed Protein 

X.B.1.1. Genetic Insert 

As described in Section III.B, MON 95379 was developed by Agrobacterium-mediated 

transformation of maize embryos using plasmid vector PV-ZMIR522223. Characterization of the 

DNA insert in MON 95379 was conducted using a combination of sequencing, PCR, and 

bioinformatics methods. The results of this characterization demonstrate that MON 95379 contains 

one copy of the intended transfer DNA containing the cry1B.868 and cry1Da_7 expression 

cassettes that is stably integrated at a single locus and is inherited according to Mendelian 

principles over multiple breeding generations. These methods also confirmed that no vector 

backbone or other unintended plasmid sequences (from PV-ZMIR522223 or PV-ZMOO513642 

(the “Cre line” transformation vector)) are present in MON 95379. Furthermore it was confirmed 

that the cp4 epsps selectable marker cassette, excised by Cre recombinase, is not present in 

MON 95379. Additionally, the genomic organization at the insertion site was assessed by 

comparing the sequences flanking the T-DNA insert in MON 95379 to the sequence of the 

insertion site in conventional maize. This analysis determined that no major DNA rearrangement 

occurred at the insertion site in MON 95379 upon DNA integration. 

X.B.1.2. Mode-of-Action 

The mode of action of Cry proteins has been extensively studied and is well-documented (Gill et 

al., 1992; OECD, 2007; Schnepf et al., 1998; Vachon et al., 2012). The mode of action for 

Cry1B.868 and Cry1Da_7 have been assessed (Wang et al., 2019) and follows the same general 

steps as other Bt insecticidal proteins currently in commercial use for insect crop protection (Jerga 

et al., 2016). The uptake, activation, receptor binding, and pore formation steps that lead to insect 

toxicity are conserved in Cry1B.868 and Cry1Da_7. The receptor binding of Cry1B.868 and 

Cry1Da_7 confers specificity to the targeted lepidopteran insect pests (Wang et al., 2019). 

X.B.2. Protein Safety and Expression Levels 

The safety and expression of the Cry1B.868 and Cry1Da_7 proteins are detailed in Section V. 

MON 95379 Cry1B.868 and Cry1Da_7 protein levels in leaf, root, silk, pollen, forage, forageroot and grain of MON 95379 were determined using an enzyme-linked immunosorbent assay 

(ELISA) as described in Appendix E. The mean Cry1B.868 protein level in MON 95379 across 

all sites was the highest in leaf tissue (OSL 1) at 630 μg/g dw and lowest in forage root at 22 

μg/g dw. The mean Cry1Da_7 protein levels in MON 95379 across all sites was highest in leaf 

(OSL1) at 92 μg/g dw and lowest in grain at <LOQ μg/g dw. 

The Cry1B.868 and Cry1Da_7 proteins expressed by MON 95379 maize are PIPs and therefore 

are regulated by the U.S. Environmental Protection Agency (EPA). The data for the safety 

assessment of Cry1B.868 and Cry1Da_7 proteins in MON 95379 were provided to EPA in a 

petition to establish a permanent exemption from the requirement of a tolerance for Cry1B.868 

and Cry1Da_7 proteins in or on food and feed commodities of maize. The physicochemical and 

functional characteristics of the MON 95379-produced Cry1B.868 and Cry1Da_7 proteins were 

determined and equivalence between MON 95379-produced Cry1B.868 and Cry1Da_7 and 

Bt-produced Cry1B.868 and Cry1Da_7 proteins was demonstrated. Therefore, Bt-produced 
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Cry1B.868 and Cry1Da_7 are appropriate test substances for the human, mammalian and NTO 

safety studies. Expression studies demonstrated that Cry1B.868 and Cry1Da_7 proteins are 

expressed at low levels in grain. Assessments of the allergenic and toxic potential of the Cry1B.868 

and Cry1Da_7 proteins found that the Cry1B.868 and Cry1Da_7 proteins do not pose a significant 

allergenic risk and were not similar to known toxins. In addition, the donor organism for the 

MON 95379 Cry1B.868 and Cry1Da_7 coding sequence is from Bacillus thuringiensis which is 

ubiquitous in the environment and is not known for allergenicity and human or animal 

pathogenicity. The Cry1B.868 and Cry1Da_7 proteins are rapidly digested by proteases found in 

the human gastrointestinal tract (pepsin and pancreatin) and demonstrate no acute oral toxicity in 

mice at the highest level tested, which far exceeds any exposure scenario. Based on the above 

information, the consumption of the Cry1B.868 and Cry1Da_7 proteins from MON 95379 or its 

progeny is considered safe for humans and mammals. Taken together, the results of these analyses 

support a determination that MON 95379 is no more likely to pose a plant pest risk than 

conventional maize. 

X.B.3. Compositional Characteristics 

Compositional comparisons of MON 95379 to conventional control followed considerations 

relevant to the compositional quality of maize as defined by the OECD consensus document 

(OECD, 2002a) were presented in Section VI. Grain samples were analyzed for levels of key 

nutrients including proximates (protein, fat, ash, moisture), essential amino acids (10 components), 

linoleic acid, carbohydrates by calculation, acid detergent fiber (ADF) and neutral detergent fiber 

(NDF). The anti-nutrients analyzed in grain were phytic acid and raffinose. Forage samples were 

analyzed for levels of proximates, carbohydrates by calculation, and fiber (ADF, NDF). In all, 27 

different components were analyzed. Moisture values for grain and forage were measured for 

conversion of components from fresh to dry weight but were not statistically analyzed. Therefore, 

25 different components were statistically analyzed. 

Of the 25 components statistically assessed, 17 showed no significant differences between 

MON 95379 and the conventional control. Eight components in grain (protein, carbohydrate, 

isoleucine, leucine, methionine, phenylalanine, threonine and valine) showed a significant 

difference between MON 95379 and the conventional control. For these eight components, the 

mean difference in the component values between MON 95379 and the conventional control was 

less than the range value of the conventional control. The MON 95379 mean component value was 

also within the range of values observed in the literature and the ILSI-CCDB. These data indicated 

that the statistically significant differences in grain were not compositionally meaningful from a 

food and feed safety perspective. There were no statistically significant differences (p < 0.05) 

between MON 95379 and the conventional control forage components observed. 

These results support the overall conclusion that MON 95379 was not a major contributor to 

variation in key nutrient and anti-nutrient levels in maize grain and forage and confirmed the 

compositional equivalence of MON 95379 to the conventional control in levels of these 

components. 

 
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X.B.4. Assessment of Potential Impacts on Non-Target Organisms Beneficial to Agriculture 

and Threatened and Endangered Species 

Prior to conducting a risk assessment for MON 95379 maize to beneficial NTOs, the proposed 

areas for cultivation of MON 95379 maize, the MOA and the spectrum of insecticidal activity of 

Cry1B.868 and Cry1Da_7 proteins, as well as expression analyses of the Cry1B.868 and 

Cry1Da_7 proteins produced by MON 95379 tissues were evaluated (Section VII.A). The results 

of the activity spectrum studies indicate that, similar to previously commercialized Cry1 proteins, 

activity of Cry1B.868 and Cry1Da_7 proteins is limited to lepidopteran insects and effects are not 

expected on NTOs beneficial to agriculture. An evaluation of potential off-crop exposure to 

MON 95379 pollen indicated significant exposure to non-target lepidopteran species is not 

anticipated and therefore poses minimal risk to non-target Lepidoptera. Results from Tier I NTO 

testing demonstrated no effects on survival from exposure to the Cry1B.868 and Cry1Da_7 

proteins at levels well above those found in MON 95379 maize and in the environment (Section 

VII.C). Calculated margins of exposure based on LC50 values and conservative environmental 

concentrations were !11 for all species tested with the exception of C. maculata where the MOE 

for Cry1Da_7 was !7, indicating low risk to NTOs at field expression levels. A threatened and 

endangered species assessment focused on listed Lepidoptera in the states of Nebraska, Hawaii, 

and Iowa, where MON 95379 will primarily be cultivated, resulted in the conclusion of no effect 

for currently listed lepidopteran species. 

X.B.5. Phenotypic, Agronomic, and Environmental Interaction Characteristics 

An extensive set of comparative plant characterization data were used to assess whether the 

introduction of lepidopteran-protected trait altered the plant pest potential of MON 95379 

compared to the conventional control (Section VIII). Phenotypic, agronomic, and environmental 

interactions characteristics of MON 95379 were evaluated and compared to those of the 

conventional control. As described previously, these assessments included seed germination and 

dormancy characteristics in the laboratory as well as phenotypic and agronomic characteristics and 

plant responses to abiotic stressors, diseases, and arthropod pests in the field. Results from all 

phenotypic, agronomic, and environmental interaction assessments demonstrated that 

MON 95379 does not possess increased weedy characteristics or increased susceptibility or 

tolerance to specific abiotic stressors, diseases, or arthropod pests compared to the conventional 

control. Taken together, the assessments support a determination that MON 95379 is no more 

likely to pose a plant pest risk than conventional maize. 

 
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X.B.5.1. Seed Dormancy and Germination 

A comparative assessment of seed germination and dormancy characteristics was conducted on 

MON 95379 and the conventional control. The results of this assessment, including the lack of 

significant differences and particularly the lack of increased hard seed, support the conclusion that 

the introduction of the lepidopteran-protected trait does not result in increased weediness or plant 

pest risk of MON 95379 compared to conventional maize. 

X.B.5.2. Phenotypic and Agronomic Assessment 

Evaluations of phenotypic and agronomic characteristics in the field are useful for assessing 

characteristics typically associated with weediness, such as lodging and seed loss. The 

characteristics early stand count, days to flowering, plant height, days to maturity, lodging, seed 

loss, final stand count, moisture, seed weight, and yield were assessed. The lack of observed 

differences between MON 95379 and the conventional control for phenotypic and agronomic 

characteristics across sites supports the overall conclusion that the introduction of the lepidoptern 

protected trait does not result in increased weediness or plant pest risk of MON 95379 compared 

to conventional maize. 

X.B.5.3. Response to Abiotic Stressors, Diseases, and Arthropod Pests 

In an assessment of plant response to abiotic stressors, diseases, and arthropod pests, no differences 

were observed between MON 95379 and the conventional control for any of the 96 observations 

for each stressor category across eight sites (Section VII.C.2). The lack of observed differences 

between MON 95379 and the conventional control for plant responses to abiotic stressors, 

diseases, and arthropod pests in multiple field environments supports the overall conclusion that 

the lepidopteran-protected trait does not result in increased plant pest risk of MON 95379 

compared to conventional maize. 

X.C. Weediness Potential of MON 95379 

Although grown extensively throughout the world, maize is not considered a threat to invade 

natural or agricultural ecosystems. Maize does not establish self-sustaining populations outside of 

cultivation (Crawley et al., 2001; OECD, 2003; Raybould et al., 2012). This lack of weediness 

may reflect its poor competitive ability (Olson and Sander, 1988), lack of seed dormancy, and 

barriers to seed dispersal, as maize cobs retain seed and are covered in a husk (Wilkes, 1972). A 

number of other characteristics common in weeds, such as rapid flowering following emergence, 

are lacking in maize (Keeler, 1989). Traits often associated with weediness are typically not 

selected for during domestication and subsequent breeding and selection. Similarly, the history of 

maize breeding and production does not indicate there are any changes in the characteristics of 

maize that would increase the weediness of the crop. Even if kernels of maize are distributed within 

a field or along transportation routes from the fields to storage or processing facilities, selfsustaining volunteer maize populations are not found growing in fields, fence rows, ditches, or 

roadsides. 

In comparative studies between MON 95379 and a conventional control, germination and 

dormancy, phenotypic and agronomic, environmental interaction were evaluated (Section VIII) 
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for changes that could impact the plant pest potential, and in particular, plant weediness potential. 

Results of these evaluations show that there were no compositional differences that were 

biologically meaningful between MON 95379 and the conventional control for characteristics 

(percentage viable hard seed, lodging, and seed loss) typically associated with weediness. 

Furthermore, field observations of MON 95379 and the conventional control indicated no 

differences in plant responses to abiotic stressors, diseases, or arthropod pests. Collectively, these 

findings support the conclusion that MON 95379 is no more likely to become a weed than 

conventional maize. 

X.D. Potential for Pollen Mediated Gene Flow and Introgression 

Pollen mediated gene flow (often referred to as cross pollination) occurs when pollen of one plant 

fertilizes ovules of a second plant. Pollen mediated gene flow is affected by both biotic and abiotic 

factors such as plant biology, pollen biology/volume, plant phenology, overlap of flowering times, 

proximity of the pollen source and sink, ambient conditions such as temperature and humidity, and 

field architecture. Pollen mediated gene flow is a natural biological process, and therefore does 

not constitute an environmental risk in and of itself. 

Introgression is a process whereby one or more genes successfully incorporate into the genome of 

a recipient plant. Pollen mediated gene flow and gene introgression must be considered in the 

context of the transgenes inserted into the biotechnology-derived plant, and the likelihood that the 

presence of the transgenes and their subsequent transfer to recipient plants and plant populations 

will result in increased plant pest potential. 

Under the proposed terms of an EPA seed increase registration application, MON 95379 maize 

will be cultivated for small-scale breeding, testing, and seed increase-related activities as well as 

potential small-scale confined field trials for stack products in other locations, as described 

earlier. . The potential for gene flow and introgression from deregulation of MON 95379 is 

discussed in greater detail below. 

X.D.1. Hybridization with Cultivated Maize 

Maize is a wind pollinated species with plant morphology that facilitates cross pollination. 

Therefore, relatively high levels of pollen-mediated gene flow can occur in this species at short 

distances (Jones and Brooks, 1950). Some biotic and abiotic factors that may influence the amount 

of pollen-mediated gene flow in maize include: (1) wind direction and speed; (2) distance between 

the pollen-source and pollen-recipient plants; (3) environmental factors that may impact pollen 

viability and dispersal (e.g. temperature and relative humidity); (4) duration of pollen shed and (5) 

floral synchrony between pollen donor and pollen recipient. 

The results from several studies conducted on the extent of pollen-mediated gene flow between 

maize fields demonstrate consistent trends regardless of the experimental design, world region, or 

detection method. The amount of pollen-mediated gene flow is greatest within the first few meters 

and decreases sharply with increasing distance from the pollen source (Table X-1). The 

distance >200 m (660 feet) is used for managing gene flow during breeding, seed production, 

identity preservation or other applications; in addition, it forms the basis for the USDA-APHIS 

performance standards for maize. All U.S. testing and production of regulated MON 95379 seed 
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or grain have been conducted under USDA notification or release permits according to these 

standards. Since no meaningful differences were observed for MON 95379 compared to 

conventional maize in composition or nutritional value, or in pest/weed potential in field 

evaluations, no adverse effects are expected from gene flow from small-scale MON 95379 

breeding or seed production nurseries to other maize. 

Table X-1. Summary of Published Literature on Maize Cross Pollination 

Pollinator 

Distance 

(m) 

Reported 

Outcrossing 

(%) 

Comments Country Reference 

1 19.0 Three-year study with two to three sites per 

year. Outcrossing was detected by yellow 

seeds in the white-seeded pollen recipient 

Values are averaged over site-years for the 

downwind direction. 

Canada (Ma et al., 

5 2.6 2004) 

10 2.0 

14 0.6 

19 0.4 

24 0.3 

28 0.5 

33 0.3 

36 0.1 

24–32 0.69 Two-year study with two sites per year. 

Outcrossing was detected by purple seeds in 

the yellow-seeded pollen recipient. Values 

are the greatest observed between years for 

the site where outcrossing was most 

prevalent. 

USA (Halsey et 

60–62 0.23 al., 2005) 

123–125 0.08 

244–254 0.02 

486–500 0.005 

743–745 0.002 

1 29.9 Two-year study with one site per year. 

Outcrossing was detected by yellow seeds in 

the non-transgenic white-seeded pollen 

recipient and confirmed by tests for a 

glyphosate tolerance gene in the yellow 

seeds. Values are the greatest between years 

as averaged across all directions from the 

pollen source. 

USA (Goggi et 

10 2.5 al., 2006) 

35 0.4 

100 0.05 

150 0.03 

200 0.03 

250 0.03 
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Table X-1. Summary of Published Literature on Maize Cross Pollination (continued) 

Pollinator 

Distance 

(m) 

Reported 

Outcrossing 

(%) 

Comments Country Reference 

0 12.65 One-year study with one site. Outcrossing 

was detected by yellow seeds in the whiteseeded pollen recipient. Values are the 

greatest reported at listed distances from the 

edge of two sampling areas, one adjacent to 

and one 10 m from the pollen source. The 

values are half of those observed to reflect 

flow of a hemizygous gene. 

Spain (Pla et al., 

2 8.81 2006) 

5 2.33 

10 3.69 

20 1.05 

40 0.74 

80 0.65 

0 0.74 Three-year study with a total of 55 sites. 

Outcrossing was quantified on ground 

samples of nontransgenic pollen recipient 

grain using a PCR assay for the pat herbicide 

tolerance gene. Values are means for 

percentage GM DNA for distances with >30 

samples. 

UK (Weekes et 

2 0.66 al., 2007) 

5 0.35 

10 0.27 

20 0.16 

25 0.14 

50 0.12 

100 0.10 

150 0.12 
Bayer CropScience LP CR282-20U1 163

Table X-1. Summary of Published Literature on Maize Cross Pollination (continued) 

Pollinator 

Distance 

(m) 

Reported 

Outcrossing 

(%) 

Comments Country Reference 

1 44 Two-year study with one site per year. 

Outcrossing was detected by herbicide 

tolerance and PCR tests for an herbicide 

tolerance transgene in progeny of the 

non-transgenic pollen recipient. Values are 

the greatest between years as averaged across 

all directions from the pollen source. 

China (Zhang et 

5 16 al., 2011) 

10 10 

15 5 

30 1 

60 1 

100 0 

150 0 

200 0 

300 0 

1 12.9 Three-year study with a total of eight sites. 

Outcrossing was detected by yellow seeds in 

the white-seeded pollen recipient. Values are 

averaged over all directions and sites. 

Mexico (Baltazar 

et al., 

2015) 

2 4.6 

4 2.7 

8 1.4 

12 1.0 

16 0.8 

20 0.5 

25 0.5 

X.D.2. Hybridization with Teosinte 

For gene flow to occur by typical sexual transmission, the following conditions must exist: (1) the 

two parents must be sexually compatible; (2) there must be flowering synchrony between the 

pollen source and pollen recipient; (3) the plants must be within sufficient proximity to each other; 

and (4) suitable environmental factors, such as relative humidity, temperature, or wind, must be 

present. 

Maize is sexually compatible with certain species or subspecies of teosinte. Although teosinte is 

not native to the U.S., a compatible subspecies of annual teosinte (Zea mays subsp. mexicana) is 

reported to have feral populations in Florida, Alabama, and Maryland (USDA-NRCS, 2019). 

Hybrids of maize and teosinte are not expected in the United States. In a study of maize and Zea 

mays subsp. mexicana, very few ovules (approximately 1-2%) produced seed after hand 

pollination with pollen from the other subspecies (Baltazar et al., 2005). Natural hybridization 

rates for maize and Zea mays subsp. mexicana have also been reported to be low (≤0.2% of 

progeny) (Ellstrand et al., 2007). A genetic barrier to hybridization of maize and teosinte has been 

described (Evans and Kermicle, 2001) and if present may limit hybridization rates of maize and 

Zea mays subsp. mexicana. In addition, differences in developmental and morphological factors 
Bayer CropScience LP CR282-20U1 164

(Baltazar et al., 2005), potential differences in flowering time, and limited geographical 

distribution of teosinte make natural crosses and gene introgression from maize into teosinte 

unlikely in the U.S. 

X.D.3. Hybridization with Tripsacum 

Tripsacum is a genus with 15 recognized species (Zuloaga et al., 2003). There are three species of 

Tripsacum that are native to the U.S.: T. floridanum (Florida gamagrass), T. lanceolatum (Mexican 

gamagrass), and T. dactyloides (Eastern gamagrass) (USDA-NRCS, 2019). Two additional species 

have introduced populations: T. latifolium (wideleaf gamagrass) and T. fasciculatum (Guatemalan 

gamagrass) (USDA-NRCS, 2019). 

Tripsacum floridanum (Florida gamagrass) naturally grows in the extreme southern Florida 

counties of Miami-Dade, Collier, Martin, and Monroe (USDA-NRCS, 2019; Wunderlin et al., 

2019). Florida gamagrass has been described as rare and occurring in “low, rocky pinelands” 

(Blakey et al., 2007) and is categorized as a threatened species in Florida (USDA-NRCS, 2019). 

Tripsacum lanceolatum (Mexican gamagrass) has been reported in Arizona and New Mexico 

(USDA-NRCS, 2019) and is found on “stream banks or moist cliffs” (de Wet and Harlan, 1978). 

Tripsacum dactyloides (eastern gamagrass) is found primarily throughout the eastern U.S. It is 

categorized as endangered in Massachusetts and Pennsylvania, threatened in New York, and 

special concern in Rhode Island (USDA-NRCS, 2019). Tripsacum fasciculatum (Guatemalan 

gamagrass) and T. latifolium (wideleaf gamagrass) and are present as introduced populations in 

Puerto Rico (USDA-NRCS, 2019). 

To our knowledge, hybrids between maize and Tripsacum do not occur in nature. The formation 

of hybrids between maize and Tripsacum species requires human intervention under specific 

controlled laboratory conditions, and the hybrids exhibit male sterility that is not resolved by 

several backcrosses to maize (Russell and Hallauer, 1980). Empirical data showed that “no 

evidence of gene flow from transgenic maize to eastern gamagrass in nature was observed even 

though the two species have grown in close proximity for years and have had ample opportunities 

for outcrossing” (Lee et al., 2017). Thus, no species of Tripsacum is expected to form viable hybrid 

progeny with maize under natural conditions. 

In summary, although hybrids between maize and Tripsacum have been produced using 

specialized laboratory techniques, there is no evidence of hybrid existence in nature. Therefore, 

gene flow from maize to any Tripsacum species is extremely unlikely. Under natural conditions, 

as is the case with conventional maize, pollen-mediated gene flow from MON 95379 to any 

species of Tripsacum is not expected. 

X.E. Transfer of Genetic Information to Species with which Maize Cannot Interbreed 

(Horizontal Gene Flow) 

Bayer is unaware of any reports regarding the unaided transfer of genetic material from maize 

species to other sexually-incompatible plant species. The likelihood for horizontal gene flow to 

occur is exceedingly small, a conclusion also reached by USDA (APHIS-BRS Final PPRA, p. 31 

for Petition #15-113-01p). Therefore, potential ecological risk associated with horizontal gene 

flow from MON 95379 due to the presence of lepidopteran-protected trait are not expected. The 
Bayer CropScience LP CR282-20U1 165

consequence of horizontal gene flow of the MON 95379 trait into other plants that are sexuallyincompatible is negligible since, as data presented in this petition confirm, the genes and traits 

confer no increased plant pest potential to maize. Thus, in the highly unlikely event that horizontal 

gene transfer was to occur, the presence of the MON 95379 trait would not be expected to increase 

pest potential in the recipient species. 

X.F. Potential Impact on Maize Agronomic Practices 

An assessment of current maize agronomic practices was conducted to determine whether the 

cultivation of MON 95379 has the potential to impact current maize management practices 

(Section IX). MON 95379 will not be commercialized in the U.S. and therefore will not impact 

U.S. commercial maize agricultural practices. It is intended to be cultivated in small scale breeding, 

testing, and seed increase nurseries to develop seed for future products in South America. In the 

future, MON 95379 stack products may also be planted in confined, small-scale field trials that 

would support subsequent global regulatory submissions.Maize fields utilized for small-scale 

breeding, testing and seed increase activities are typically highly managed agricultural areas that 

are dedicated to these purposes. Other than the specific insertion of the Cry1B.868 and Cry1Da_7 

coding sequence that provide protection against targeted lepidopteran insect pests MON 95379 is 

similar to other conventional maize hybrids. 

The data presented demonstrate that MON 95379 is similar to conventional maize in its phenotypic 

and agronomic characteristics, and has levels of susceptibility to abiotic stressors, diseases, and 

arthropod pests comparable to other conventional maize. Based on this assessment, the intended 

use of MON 95379 for breeding and seed increase activities, MON 95379 will not have an impact 

on current U.S. maize agronomic or cultivation practices, and is not likely to have adverse impacts 

on practices used in small-scale breeding/seed increase activities, or lead to an increased plant pest 

potential compared to conventional maize. 

X.G. Conventional Breeding with Other Biotechnology-derived or Conventional Maize 

Several biotechnology-derived maize products have been deregulated or are under consideration 

for deregulation. Once deregulated, MON 95379 may be bred with these deregulated 

biotechnology-derived maize products, as well as with conventional maize, creating new improved 

hybrids. APHIS has determined that none of the individual biotechnology-derived maize products 

it has previously deregulated displays increased plant pest characteristics. APHIS has also 

concluded that any progeny derived from crosses of these deregulated biotechnology-derived 

maize products with conventional or previously deregulated biotechnology-derived maize are 

unlikely to exhibit new plant pest properties. This presumption, that combined-trait biotechnology 

products are unlikely to exhibit new characteristics that would pose new plant pest risks or 

potential environmental impacts not observed in the single event biotech product, is based upon 

several facts, namely: 1) stability of the genetic inserts is confirmed in each approved biotechderived maize product across multiple generations (See Section IV.E for MON 95379 data); 2) 

stability of each of the introduced traits is continually and repeatedly assessed by plant breeders as 

new combined-trait hybrids are created and tested over multiple seasons prior to 

commercialization; 3) combined-trait products are developed using the well-established process 

of conventional breeding that has been safely used for thousands of years to generate new varieties 

(Cellini et al., 2004; NRC, 2004; WHO, 1995); 4) worldwide organizations, such as World Health 
Bayer CropScience LP CR282-20U1 166

Organization, Food and Agriculture Organization/World Health Organization, International Seed 

Federation, CropLife International and U.S. FDA, conclude that the safety of the combined-trait 

product can be based on the safety of the parental GE events (CLI, 2005; FAO-WHO, 1996; ISF, 

2005; U.S. FDA, 2001; WHO, 1995); and 5) practical applications in the field have shown that 

two unrelated biotechnology traits combined together by conventional breeding do not display new 

characteristics or properties distinct from those present in the single event biotech products 

(Brookes and Barfoot, 2012; Clawson et al., 2019; James, 2010; Jose et al., 2020; Lemaux, 2008; 

Pilacinski et al., 2011; Sankula, 2006). 

Therefore, based on the considerations above and the conclusion that MON 95379 is no more 

likely to pose a plant pest risk than conventional maize, it can be concluded that any progeny 

derived from crosses between MON 95379 and conventional maize or other maize with 

deregulated biotechnology-derived events are no more likely to pose a plant risk than conventional 

maize. 

X.H. Summary of Plant Pest Assessments 

A plant pest, as defined in the Plant Protection Act, is the living stage of any of the following that 

can directly or indirectly injure, damage, or cause disease in any plant or plant product: (A) a 

protozoan; (B) a nonhuman animal; (C) a parasitic plant; (D) a bacterium; (E) a fungus; (F) a virus 

or viroid; (G) an infectious agent or other pathogens; or (H) any article similar to or allied with 

any of the articles specified in the preceding subparagraphs (7 U.S.C. § 7702[14]). 

Characterization data presented in Sections III through IX of this petition confirm that 

MON 95379, with the exception of the lepidopteran-protected trait, is not meaningfully different 

from conventional maize in terms of plant pest potential. Bayer is not aware of any other study 

results or observations associated with MON 95379 that would suggest an increased plant pest risk 

would result from its limited cultivation for small-scale breeding, testing, and seed increase-related 

activities. 

The plant pest assessment was based on multiple lines of evidence developed from a detailed 

characterization of MON 95379 compared to conventional maize, followed by a risk assessment 

on detected differences. The plant pest risk assessment in this petition was based on the following 

lines of evidence: 1) insertion of a single functional copy of the Cry1B.868 and Cry1Da_7 cassette; 

2) characterization and safety of the expressed products; 3) compositional equivalence of 

MON 95379 grain and forage compared to a conventional control; 4) phenotypic, agronomic, and 

environmental interactions characteristics demonstrating no increased plant pest potential 

compared to conventional maize; 5) minimal risk to NTOs including organisms beneficial to 

agriculture and no effect on threatened and endangered species 6) familiarity with maize as a 

cultivated crop and 7) no impact on U.S. commercial maize agricultural practices as MON 95379 

will not be commercialized in the U.S. and no greater likelihood to impact practices used in smallscale breeding, testing and seed increase activities, agronomic practices, cultivation practices, or 

the management of weeds, diseases and pests, than conventional maize. 

Based on the data and information presented in this petition, it is concluded that MON 95379 is 

unlikely to pose a greater plant pest risk than conventional maize. Therefore, Bayer Company 

requests a determination from USDA-APHIS that MON 95379 and any progeny derived from 
Bayer CropScience LP CR282-20U1 167

crosses between MON 95379 and other de-regulated maize events be granted nonregulated status 

under 7 CFR part 340. 
Bayer CropScience LP CR282-20U1 168 

XI. ADVERSE CONSEQUENCES OF INTRODUCTION 

Bayer knows of no study results or observations associated with MON 95379 indicating that there 

would be adverse consequences from the intended use of MON 95379 for breeding and seed 

increase activities. MON 95379 produces the Cry1B.868 and Cry1Da_7 proteins. The safety of 

the MON 95379 Cry1B.868 and Cry1Da_7 proteins, which have been fully characterized, has been 

thoroughly assessed in this submission. The data for the safety assessment of Cry1B.868 and 

Cry1Da_7 and MON 95379 is being provided to EPA to support the establishment of a tolerance 

exemption for residues of Cry1B.868 and Cry1Da_7 in food and feed. As demonstrated by field 

results and laboratory tests, the only phenotypic differences between MON 95379 and 

conventional maize is presence of lepidopteran-protected traits which protect against feeding 

damage caused by targeted lepidopteran insect pests. 

The data and information presented in this petition demonstrate that MON 95379 is unlikely to 

pose a greater plant pest risk compared to conventional maize. This conclusion is reached based 

on multiple lines of evidence developed from a detailed characterization of the product compared 

to conventional maize, followed by risk assessment on detected differences. The characterization 

evaluations included molecular analyses, which confirmed the insertion of one copy of the 

intended DNA containing the cry1B.868 and cry1Da_7 expression cassette that is stably integrated 

at a single locus and is inherited according to Mendelian principles over multiple generations. 

Analysis of key nutrients and anti-nutrients of MON 95379 demonstrate that MON 95379 is 

compositionally equivalent to conventional maize. The phenotypic evaluations, including an 

assessment of seed germination and dormancy characteristics, phenotypic and agronomic 

characteristics, environmental interactions also indicated MON 95379 is unchanged compared to 

conventional maize. There is no indication that MON 95379 would have an adverse impact on 

non-target organisms (NTOs), including those beneficial to agriculture, and would have no effect 

on threatened or endangered species under the intended use of the product. Therefore, based on 

the lack of increased pest potential compared to conventional maize, the risks for humans, animals, 

and other NTOs from MON 95379 are negligible. 

MON 95379 will not be commercialized in the United States and will therefore have no impact on 

U.S. commercial maize agricultural practices. The intended use of MON 95379 for small-scale 

breeding, testing and seed increase activities will not adversely impact cultivation practices or the 

management of weeds, diseases, and pests in maize seed production systems. Activities will be 

limited to breeding and seed increase in Nebraska, Hawaii and Iowa on a maximum total of 100 

acres per growing season and small-scale, confined field trials (in other locations, as described 

earlier) to collect needed regulatory data to support global regulatory submissions of future 

products stacked with MON 95379. Seed production activities will take place at research and 

breeding facilities following typical recommended practices employed to achieve high quality 

seed. Based on the data and information presented in this petition, it is concluded that, like 

previously deregulated biotechnology-derived maize, MON 95379 is unlikely to pose a greater 

plant pest risk than conventional maize. 
Bayer CropScience LP CR282-20U1 169 

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Appendix A: USDA Notifications and Permits 

Field trials of MON 95379 have been conducted in the U.S. since 2015. The protocols for these 

trials include field performance, breeding and observation, agronomics, and generation of field 

materials and data necessary for this petition. In addition to the MON 95379 phenotypic 

assessment data, observational data on pest and disease stressors were collected from these product 

development trials. The majority of the final reports have been submitted to the USDA. However, 

some final reports, mainly from the 2019 season, are still in preparation. A list of trials conducted 

under USDA notifications or permits and the status of the final reports for these trials are provided 

in Table A-1. 

Table A-1. USDA Notifications and Permits Approved for MON 95379 and Status of 

Trials Planted under These Notifications 

Field Trial 

Year USDA No. 

Effective 

Date Trial Status 

Release 

State Sites 

2015

15-028-

101rm 6/1/2015 Submitted to USDA 

HI 1 

PR 1 

15-120-

112rm 9/1/2015 

Submitted to 

USDA HI 2 

2016

15-120-

112rm 9/1/2015 Submitted to USDA 

HI 1 

PR 1 

15-289-

113rm 3/2/2016 Submitted to USDA 

HI 2 

PR 1 

15-306-

101rm 3/15/2016 Submitted to USDA

IA 4 

IL 5 

IN 1 

KS 3 

15-306-

107rm 3/15/2016 Submitted to 

USDA

AR 1 

IL 6 

NE 4 

TN 2 

16-117-

104rm 9/1/2016 

Submitted to 

USDA HI 1 

16-182-

104rm 12/1/2016 

Submitted to 

USDA HI 1 

 
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Table A-1. USDA Notifications and Permits Approved for MON 95379 and Status 

of Trials Planted under These Notifications (continued) 

2017

16-117-

112rm 9/1/2016 

Submitted to 

USDA PR 1 

16-302-

110rm 3/1/2017 

Submitted to 

USDA HI 1 

16-302-

111rm 3/1/2017 

Submitted to 

USDA HI 1 

16-315-

101rm 3/15/2017 Submitted to USDA 

IA 10 

IL 18 

16-315-

105rm 3/15/2017 Submitted to 

USDA

AR 2 

KS 5 

NE 6 

TN 2 

17-038-

102n 3/7/2017 Submitted to 

USDA

AL 1 

NC 1 

TX 2 

17-115-

108rm 9/1/2017 

Submitted to 

USDA HI 1 

17-115-

111rm 9/1/2017 

Submitted to 

USDA HI 1 

 
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Table A-1. USDA Notifications and Permits Approved for MON 95379 and Status 

of Trials Planted under These Notifications (continued)

2018

17-304-

105rm 3/1/2018 

Submitted to 

USDA HI 2 

17-304-

107rm 3/1/2018 

Submitted to 

USDA HI 1 

17-304-

108rm 3/1/2018 

Submitted to 

USDA HI 1 

17-320-

101rm 3/15/2018 Submitted to USDA 

IL 2 

NE 1 

17-320-

102rm 3/15/2018 Submitted to 

USDA

IA 6 

IL 8 

IN 1 

NE 2 

18-065-

105n 4/5/2018 Submitted to USDA

IA 2 

IL 1 

IN 1 

NC 1 

NE 1 

OH 1 

PA 1 

TX 1 

18-065-

106n 4/5/2018 Submitted to USDA

IA 1 

IL 2 

NE 1 

18-122-

103rm 9/1/2018 

Submitted to 

USDA HI 1 

18-122-

106rm 9/1/2018 

Submitted to 

USDA HI 2 

18-144-

104n 6/25/2018 

Submitted to 

USDA GA 1 

18-318-

102rm 11/30/2018 

Submitted to 

USDA IA 1 

 
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Table A-1. USDA Notifications and Permits Approved for MON 95379 and Status 

of Trials Planted under These Notifications (continued)

2019

18-122-

101rm 9/1/2018 

Submitted to 

USDA HI 1 

18-207-

101rm 12/1/2018 

Submitted to 

USDA HI 1 

18-305-

104rm 3/1/2019 In Progress HI 1 

18-318-

104rm 3/15/2019 In Progress IL 1 

18-347-

107rm 4/1/2019 In Progress

IA 3 

IL 3 

IN 1 

NC 2 

NE 1 

OH 1 

PA 1 

19-022-

102rm 6/1/2019 In Progress HI 1 

19-115-

101rm 9/1/2019 In Progress HI 2 
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Appendix B: Overview, Materials, Methods and Supplementary Results for 

Molecular Analyses of MON 95379 

B.1. Test Substance 

The test substance in this study was MON 95379. Five breeding generations of MON 95379 were 

used to assess the stability of the T-DNA insert. Genomic DNA for use in this study was extracted 

from seed tissue listed in the table below. 

MON 95379 Breeding Generation Seed ID 

F4 11479057 

F5 11479060 

F4F1 11478830 

F5F1 11479279 

F6F1 11493009 

B.2. Control Substances 

The control substance is the conventional crop variety which is the same genetic background as the 

test substances. Genomic DNA was extracted from seed as listed in the table below. 

Control Substance Seed ID Breeding 

Generations 

LH244 11479052 F4, F5 

LH244 × HCL617 11479278 F4F1, F5F1, F6F1 

B.3. Reference Substance 

The reference substances were plasmid vector PV-ZMIR522223 and PV-ZMOO513642, which 

were used to develop MON 95379 and a Cre recombinase expressing line. Whole plasmid DNA 

and its sequence served as a positive control for sequencing and bioinformatic analyses. The 

identity of the reference plasmid was confirmed by sequencing within the study. Documentation 

of the confirmation of the plasmid vector identity was archived with the raw data. Appropriate 

molecular weight markers from commercial sources were used for size estimations on agarose 

gels. The unique identity of the molecular weight markers was documented in the raw data. 
Bayer CropScience LP CR282-20U1 194 

B.4. Characterization of Test, Control, and Reference Substances 

The Study Director reviewed the chain-of-custody forms to confirm the identity of the test and 

control substances prior to the use of these materials in the study. Further confirmation of test and 

control substances identity was determined by sequencing in the study. Test, control, and reference 

DNA as well as sequencing library substances were considered stable during storage if they yield 

interpretable signals in sequencing experiments, or did not appear visibly degraded on the stained 

gels (DNA) or sufficient cluster amplification was observed during flow cell generation 

(sequencing libraries). 

B.5. Genomic DNA Isolation 

For sequencing library construction and PCR reactions, genomic DNA was isolated from seed 

tissues of the test and control substances. First the seeds were decontaminated by vigorously 

agitating them by hand for 30 seconds with 0.05% (v/v) Tween-20, followed by a tap water rinse. 

The seeds were then vigorously agitated with 0.5% (w/v) NaOCl, allowed to stand for one minute 

at room temperature, and rinsed with tap water. The seeds were then vigorously agitated with 

1% (v/v) HCl, allowed to stand for one minute at room temperature, and rinsed with tap water. 

The 1% (v/v) HCl rinse was repeated one time, and then the seeds were rinsed with distilled water 

and air dried. The dried seeds were ground to a fine powder in a GenoGrinder® SPEX 2010 

homogenizer (SPEX SamplePrep, LLC). Genomic DNA was extracted using a Qiagen® DNeasy®

Plant Mini Kit (Qiagen GmbH), according to manufacturer’s instructions. All extracted DNA was 

stored in a 4°C refrigerator or a -20°C freezer.

B.6. DNA Quantification 

Plasmid DNA and extracted genomic DNA were quantified using a NanodropTM

Spectrophotometer (Thermo Scientific) or a Qubit Fluorometer (Invitrogen). 

B.7. Paired End Library Preparation 

DNA from the test, control, and reference samples, including transformation plasmids, were 

submitted to Nucleic Acid Technologies (Bayer) for library preparation and whole genome 

sequencing. 

The DNA samples were fragmented by sonication using a Covaris sonicator (Covaris). Following 

fragmentation, the test, control, and reference DNA were made into sequencing libraries with 

TruSeq DNA UD Indexes using a KAPA Hyper library preparation kit following the manufactures 

instructions with the following exceptions. 1) The sample cleanup steps utilized BioChain 

magnetic beads. 2) The library amplification step utilized a PCR program with the following 

cycling conditions: 1 cycle at 98qC for 30 seconds; 5 cycles at 98qC for 10 seconds, 60qC for 30 

seconds, 72qC for 30 seconds; and 1 cycle at 72qC for 5 minutes. 3) Library size selection (~ 500 

bp) was performed with a 1.5% agarose gel cassette run on a BluePippin Size Selection system 

(Sage Science). 
Bayer CropScience LP CR282-20U1 195 

After preparation, libraries were analyzed using a Fragment Analyzer QC (Advanced Analytical 

Technologies, Inc.) according to the manufacturer’s instructions. All purified library DNA was 

stored in a -20°C freezer. 

B.8. Next-Generation Sequencing 

The library samples described above were sequenced by Bayer’s Nucleic Acid Technologies using 

Illumina short read technology that produced short sequence reads (~150 bp long). Sufficient 

numbers of these sequence reads were obtained to comprehensively cover the entire genomes of 

the test samples, conventional controls, and reference substances (transformation vectors) (Cade 

et al., 2018; Kovalic et al., 2012). More details can be found in Appendix K; Table KI. 

Furthermore, a transformation plasmid spike was sequenced to >75× to assess method sensitivity 

through modelling of 1/10th and one full genome equivalent plasmid spike. 

B.9. Read Mapping and Junction Identification 

High-throughput sequence reads were captured by aligning to the PV-ZMIR522223 

transformation plasmid sequence using the read alignment software Bowtie2 version 2.3.4.3 

(Langmead and Salzberg, 2012) in order to collect all reads that were sourced from the plasmid as 

well as reads with sequences representing integration points (parameters: bowtie2 --very-fastlocal -X 1000 --gbar 10 --dovetail --no-unal). No additional read quality filtering was applied. The 

high-throughput sequence reads from F4 and conventional LH244 were also aligned to the 

PV-ZMOO513642 transformation plasmid sequence utilizing the same steps and software outlined 

above. All software versions were documented in the archived data package and the software 

versions which were used in this study have been archived. 

B.9.1. Mapping and Junction Detection 

Captured reads from all test and control samples were mapped to the complete PV-ZMIR522223 

transformation plasmid sequence and captured reads from F4 and conventional LH244 were 

mapped to the complete PV-ZMOO513642 plasmid sequence in order to detect junction sequences 

using the Bowtie2 (version 2.3.4.3) program. Reads with partial matches to the transformation 

plasmid of at least 30 bases and 96.6% or greater identity were also identified as potential junction 

sequences (Kovalic et al., 2012). 

B.9.2. Effective Sequencing Depth Determination 

A single copy locus from the native plant genome (Zea mays) pyruvate decarboxylase (pdc3, 

GenBank accession AF370006.2) was used to estimate the sequence depth coverage. All reads 

with at least 30 bases match and 96.6% identity were considered as reads sourced from this locus 

and used to calculate the final sequencing depth. Further details, mapping figures, and depth 

metrics can be found in Appendix K. 
Bayer CropScience LP CR282-20U1 196 

B.9.3. Summary NGS Data for the Control and Test Substances 

For each sample, the raw data produced are presented in terms of total nucleotide number. The 

reads generated for this study were a maximum of 151bp in length. Estimated sequencing depth is 

provided in Appendix K, Table K1. Effective depth of coverage for this study was determined by 

mapping and alignment of all raw data to a single copy locus within the Zea mays genome 

(AF370006.2) and is shown for F4 in Figure IV-5, and all subsequent samples in Appendix K 

Figures K8-K14. Appendix Table K1 provides a comparison of total generated data, hypothetical 

coverage, and effective median coverage for all samples. 

A graph of positional depths across samples relative to the transformation construct are shown in 

panel 3 of Figure IV-6 and Appendix K Figures K1-K6. Coverage statistics derived from the 

T-DNA regions of these samples are further provided in Appendix K Table K2. 

B.9.4. Positive Control 

To produce the positive control sample for sequencing, a plasmid DNA library was created for 

PV-ZMIR522223 and PV-ZMOO513642 as described in Section B.7 and was loaded at 

approximately ~0.04 % and ~0.02 %, respectively, of the total sequenced libraries. The collected 

data were sampled to represent a single genome equivalent dataset and a 1/10th genome equivalent 

dataset. 

B.10. PCR and DNA Sequence Analyses to Examine the Insert and Flanking Sequences in 

MON 95379 

Overlapping PCR products, denoted as Product A and Product B were generated that span the 

insert and adjacent 5′ and 3′ flanking DNA sequences in MON 95379. For each fragment 

generation, experimental conditions were chosen to successfully produce on-target amplifications. 

These products were analyzed to determine the nucleotide sequence of the insert in MON 95379, 

as well as that of the DNA flanking the 5′ and 3′ ends of the insert, as depicted in Figure IV-9. 

The PCR analyses for Product A was conducted using approximately 50 ng of genomic DNA 

template in a 50 Pl reaction volume. The reaction contained a final concentration of 0.2 PM of 

each primer, 0.2 mM of each dNTP, and 1.25 units/reaction of PrimeSTAR GXL Polymerase 

(TaKaRa Bio Inc.). 

The PCR amplification of Product A was performed under the following cycling conditions: 1 

cycle at 98qC for 1 minute; 35 cycles at 98qC for 10 seconds, 68qC for 10 minutes. 

The PCR amplification of Product B was performed under the following cycling conditions: 1 

cycle at 98qC for 30 seconds; 35 cycles at 98qC for 10 seconds, 64qC for 20 seconds, 72qC for 

2.5 minutes and 1 cycle at 72qC for 5 minutes. 
Bayer CropScience LP CR282-20U1 197 

Aliquots of each PCR product were separated on a 1.0% (w/v) agarose gel and visualized by 

ethidium bromide staining to verify that the products were the expected sizes. Each PCR product 

was purified with Agencourt AMPure XP (Beckman Coulter Life Sciences). Approximately 

200 ng of purified amplicon was submitted to Bayer’s Nucleic Acid Technologies for library 

preparation and MiSeq sequencing. 

A consensus sequence was generated by compiling captured reads from MiSeq sequencing of the 

overlapping PCR products. This consensus sequence was aligned to the PV-ZMIR522223 

sequence to determine the integrity and organization of the integrated DNA and the 5′ and 

3′ insert-to-flank DNA junctions in MON 95379. 

B.11. PCR and DNA Sequence Analyses to Examine the Integrity of the DNA Insertion Site 

in MON 95379 

To examine the MON 95379 T-DNA insertion site in LH244 conventional maize, PCR and 

sequence analyses were performed on genomic DNA from the LH244 conventional control as 

depicted in Figure IV-10. 

The primers used in this analysis were designed from the DNA sequences flanking the insert in 

MON 95379. A forward primer specific to the DNA sequence flanking the 5′ end of the insert 

was paired with a reverse complement primer specific to the DNA sequence flanking the 3′ end 

of the insert. 

The PCR reactions were conducted using approximately 50 ng of genomic DNA template in a 

50 Pl reaction volume. The reaction contained a final concentration of 0.2 PM of each primer, 

0.2 mM of each dNTP, and 1.25 units/reaction of PrimeSTAR GXL Polymerase. The PCR 

amplification was performed under the following cycling conditions: 1 cycle at 98qC for 1 minute; 

35 cycles at 98qC for 10 seconds, 68qC for 2.5 minutes. 

Aliquots of each PCR product were separated on a 1.0% (w/v) agarose gel and visualized by 

ethidium bromide staining to verify that the products were the expected sizes. Each PCR product 

was purified with Agencourt AMPure XP (Beckman Coulter Life Sciences). Approximately 

200 ng of purified amplicon was submitted to Bayer’s Nucleic Acid Technologies for library 

preparation and MiSeq sequencing. 

A consensus sequence was generated by compiling sequences from MiSeq sequencing reactions 

of the verified PCR product. This consensus sequence was aligned to the 5′ and 3′ sequences 

flanking the MON 95379 insert to determine the integrity and any rearrangement of the insertion 

site. 

B.12. Protocol Amendments and Deviations 

During the study, changes were made to the original protocol. The changes were documented as 

protocol amendments and are summarized below. These changes positively impacted the quality 

and integrity of the study data and conclusions. 
Bayer CropScience LP CR282-20U1 198 

1. The protocol was amended to include PV-ZMOO513642 as a reference material. 

2. The protocol was amended to clarify the algorithms used to analyze the next generation 

sequencing data. 

3. The protocol was also amended to update the breeding generations to be tested. 

B.13. Rejected Data 

Rejected data were assessed to have no effect on the quality, integrity, or conclusions of the study 

and are retained in the study package (Appendix L). 

 
Bayer CropScience LP CR282-20U1 199 

References for Appendix B

Cade, R., K. Burgin, K. Schilling, T.-J. Lee, P. Ngam, N. Devitt and D. Fajardo. 2018. Evaluation 

of whole genome sequencing and an insertion site characterization method for molecular 

characterization of GM maize. Journal of Regulatory Science 6:1-14. 

Kovalic, D., C. Garnaat, L. Guo, Y. Yan, J. Groat, A. Silvanovich, L. Ralston, M. Huang, Q. Tian, 

A. Christian, N. Cheikh, J. Hjelle, S. Padgette and G. Bannon. 2012. The use of next generation 

sequencing and junction sequence analysis bioinformatics to achieve molecular characterization 

of crops improved through modern biotechnology. The Plant Genome 5:149-163. 

Langmead, B. and S.L. Salzberg. 2012. Fast gapped-read alignment with Bowtie 2. Nature 

Methods 9:357-359. 

 
Bayer CropScience LP CR282-20U1 200 

Appendix C: Characterization of Cry1B.868 Protein Produced in MON 95379 

C.1. Characterization of the Cry1B.868 Protein 

C.1.1. Cry1B.868 Protein Identity and Equivalence 

The safety assessment of crops derived through biotechnology includes characterization of the 

physicochemical and functional properties and confirmation of the safety of the introduced 

protein(s). For the safety data generated using the Bt-produced Cry1B.868 protein to be applied 

to the MON 95379-produced Cry1B.868 protein (plant-produced Cry1B.868), the equivalence of 

the plant- and Bt-produced proteins must first be demonstrated. To assess the equivalence between 

the MON 95379-produced and Bt-produced Cry1B.868 proteins, a small quantity of the 

MON 95379-produced Cry1B.868 protein was purified from MON 95379 grain. The 

MON 95379-produced Cry1B.868 protein was characterized and the equivalence of the 

physicochemical characteristics and functional activity between the MON 95379-produced and 

Bt-produced Cry1B.868 proteins was assessed using a panel of analytical tests; as shown in Table 

C-1. Taken together, these data provide a detailed characterization of the MON 95379-produced 

Cry1B.868 protein and establish the equivalence of the MON 95379-produced and Bt-produced 

Cry1B.868 proteins. Based on this established equivalence, conclusions derived from digestibility, 

heat susceptibility, acute oral toxicology study and non-target organism studies conducted with 

Bt-produced Cry1B.868 protein are applicable to MON 95379-produced Cry1B.868 protein. 

 
Bayer CropScience LP CR282-20U1 201 

Table C-1. Summary of MON 95379 Cry1B.868 Protein Identity and Equivalence 

Analytical Test Assessment Analytical Test Outcome 

N-terminal sequence Identity The expected N-terminal sequence for 

MON 95379-produced Cry1B.868 

protein was observed by Nano LCMS/MS1

Nano LC-MS/MS1 Identity Nano LC-MS/MS1 analysis of trypsin 

or Asp-N digested peptides from 

MON 95379-produced Cry1B.868 

protein yielded peptide masses 

consistent with expected peptide 

masses from the theoretical trypsin or 

Asp-N digest of the amino acid 

sequence

Western blot analysis Identity 

and

Equivalence 

MON 95379-produced Cry1B.868 

protein identity was confirmed using a 

western blot probed with antibodies 

specific for Cry1B.868 protein 

Immunoreactive properties of the 

MON 95379-produced Cry1B.868 and 

the Bt-produced Cry1B.868 proteins 

were shown to be equivalent 

Apparent molecular 

weight (MW)

Equivalence Electrophoretic mobility and apparent 

molecular weight of the 

MON 95379-produced Cry1B.868 and 

the Bt-produced Cry1B.868 proteins 

were shown to be equivalent

Glycosylation analysis Equivalence Glycosylation status of 

MON 95379-produced Cry1B.868 and 

Bt-produced Cry1B.868 proteins were 

shown to be equivalent

Functional activity Equivalence Functional activity of the 

MON 95379-produced Cry1B.868 and 

the Bt-produced Cry1B.868 proteins 

were shown to be equivalent by insect 

bioassay

1 Nano LC-MS/MS = Nanoscale liquid chromatography-tandem mass spectrometry 
Bayer CropScience LP CR282-20U1 202 

The details of the materials and methods for the panel of analytical tests used to evaluate and 

compare the properties of the MON 95379-produced Cry1B.868 and Bt-produced Cry1B.868 

proteins are described at the end of Appendix C. A summary of the data obtained to support a 

conclusion of protein equivalence is provided below. 

C.1.2. Results of the N-Terminal Sequencing Analysis 

The identification of N-terminal sequence was conducted by LC-MS/MS analysis of peptide 

fragments produced through enzymatic digestion of the MON 95379-produced Cry1B.868 protein 

with Asp-N. The expected N-terminal sequence for the Cry1B.868 protein deduced from the 

Cry1B.868 gene present in maize of MON 95379 was observed, except that the N-terminal 

methionine was cleaved in vivo from the MON 95379-produced Cry1B.868 by methionine 

aminopeptidase (see Experimental Sequence, Figure C-1). The cleavage of the N-terminal 

methionine from proteins in vivo by methionine aminopeptidase is common in many organisms 

(Bradshaw et al., 1998; Wang et al., 2016). The N-terminal sequence for MON 95379-produced 

Cry1B.868 protein was consistent with the N-terminal sequence for the Bt-produced Cry1B.868 

protein observed by LC-MS/MS (Figure C-1). An additional minor form of the MON 95379-

produced Cry1B.868 protein with the N-terminal methionine was also observed. Hence, the 

sequence information confirms the identity of the Cry1B.868 protein isolated from the grain of 

MON 95379. 

Amino Acids 

Residue # from 

the N-terminus → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 

Bt-produced 

Cry1B.868 

sequence 

→ - T S N R K N E N E I I N A L S 

Expected 

Cry1B.868 

Sequence 

→ M 

│

T 

│

S 

│

N 

│

R 

│

K 

│

N 

│

E 

│

N 

│

E 

│

I 

│

I 

│

N 

│

A 

│

L 

│

S 

MON 95379 

Experimental 

Sequence → - 

│

T 

│

S 

│

N 

│

R 

│

K 

│

N 

│

E 

│

N 

│

E 

│

I 

│

I 

│

N 

│

A 

│

L 

│

S 

Figure C-1. N-Terminal Sequence of the MON 95379-Produced Cry1B.868 Protein 

The experimental sequence obtained from the MON 95379-produced Cry1B.868 was compared to the 

expected sequence deduced from the cry1B.868 gene present in MON 95379. Bt-produced Cry1B.868 

protein sequence above was derived from the reference substance Certificate of Analysis (COA) (lot 7565). 

The single-letter International Union of Pure and Applied Chemistry - International Union of Biochemistry 

(IUPAC-IUB) amino acid code is M, methionine; T, Threonine; S, Serine; N, Asparagine; R, Arginine; K, 

Lysine; E, Glutamic acid; I, Isoleucine; A, Alanine; L, Leucine. 

 
Bayer CropScience LP CR282-20U1 203 

C.1.3. Results of Mass Fingerprint Analysis 

Peptide mass fingerprint analysis is a standard technique used for confirming the identity of 

proteins. The identity of the MON 95379-produced Cry1B.868 protein was confirmed by 

LC-MS/MS analysis of peptide fragments produced through enzymatic digestion of the 

MON 95379-produced Cry1B.868 protein. 

There were 110 unique peptides identified that correspond to the masses expected to be produced 

by trypsin digestion of the MON 95379-produced Cry1B.868 protein (Table C-2). The identified 

peptides were used to assemble a sequence coverage map of the entire Cry1B.868 protein (Figure 

C-2, Panel A). The experimentally determined coverage of the MON 95379-produced Cry1B.868 

protein was 91% (Figure C-2, Panel A, 1088 out of 1199 amino acids). This analysis further 

confirms the identity of MON 95379-produced Cry1B.868 protein. 

There were 145 unique peptides identified that correspond to the masses expected to be produced 

by trypsin digestion of the Bt-produced Cry1B.868 protein (Table C-3) by LC-MS/MS analysis 

during the protein characterization. The identified peptides were used to assemble a coverage map 

of the entire Cry1B.868 protein (Figure C-2, Panel B). The experimentally determined coverage 

of the Bt-produced Cry1B.868 protein was 96% (Figure C-2, Panel B, 1156 out of 1199 amino 

acids). 

 
Bayer CropScience LP CR282-20U1 204 

Table C-2. Summary of the Tryptic Masses Identified for the MON 95379-Produced 

Cry1B.868 Using LC-MS/MS1 

Experimental 

Mass2

Calculated 

Mass3 Diff4 Fragment5 Sequence6

3136.5591 3136.5571 0.0020 6 - 34 KNEN...TDAR 

3008.4627 3008.4621 0.0006 7 - 34 NENE...TDAR 

3261.5882 3261.5823 0.0059 35 - 65 IEDS...IAGR 

2935.6012 2935.6000 0.0012 66 - 92 ILGV...LWPR 

2136.1137 2136.1116 0.0021 93 - 109 GRDP...QLIR 

1922.9901 1922.9890 0.0011 95 - 109 DPWE...QLIR 

974.4775 974.4781 -0.0006 110 - 117 QQVTENTR 

1601.8130 1601.8121 0.0009 110 - 123 QQVT...ALAR 

645.3443 645.3446 -0.0003 118 - 123 DTALAR 

990.5252 990.5247 0.0005 124 - 132 LQGL...NSFR 

1650.7638 1650.7638 0.0000 133 - 145 AYQQ...LENR 

2107.9605 2107.9559 0.0046 133 - 149 AYQQ...DDAR 

2800.4884 2800.4873 0.0011 152 - 175 SVLY...FAIR 

2518.4144 2518.4093 0.0051 176 - 197 NQEV...LLLR 

1983.9540 1983.9538 0.0002 198 - 215 DASL...EIQR 

629.2809 629.2809 0.0000 216 - 219 YYER 

502.2752 502.2751 0.0001 220 - 223 QVEK 

1249.6222 1249.6203 0.0019 234 - 243 WYNT...NNLR 

1032.4997 1032.4988 0.0009 244 - 252 GTNA...SWLR 

726.3454 726.3449 0.0005 253 - 257 YNQFR 

2263.2201 2263.2212 -0.0011 258 - 277 RDLT...YDTR 

2107.1237 2107.1201 0.0036 259 - 277 DLTL...YDTR 

1379.6874 1379.6867 0.0007 278 - 289 VYPM...QLTR 

1062.5349 1062.5346 0.0003 290 - 298 EIYT...PIGR 

5243.6491 5243.6469 0.0022 299 - 346 TNAP...VLSR 

1840.8100 1840.8104 -0.0004 347 - 360 WSNT...VGHR 

503.2703 503.2703 0.0000 361 - 364 LESR 

2619.2933 2619.2889 0.0044 368 - 392 GSLS...FTSR 

551.2703 551.2704 -0.0001 393 - 396 DVYR 

2888.5213 2888.5185 0.0028 393 - 418 DVYR...PWAR 

2355.2594 2355.2587 0.0007 397 - 418 TESF...PWAR 

621.3022 621.3023 -0.0001 419 - 422 FNWR 

1415.7410 1415.7422 -0.0012 419 - 429 FNWR...NSLR 

812.4502 812.4504 -0.0002 423 - 429 NPLNSLR 

4407.0833 4407.0819 0.0014 430 - 468 GSLL...YSHR 

601.3545 601.3547 -0.0002 469 - 473 LSNIR 

872.5075 872.5080 -0.0005 474 - 481 LISGNTLR 
Bayer CropScience LP CR282-20U1 205 

Experimental 

Mass2

Calculated 

Mass3 Diff4 Fragment5 Sequence6

1115.5512 1115.5512 0.0000 482 - 490 APVY...WTHR 

447.2077 447.2077 0.0000 491 - 494 SADR 

2158.1219 2158.1230 -0.0011 491 - 510 SADR...PLVK 

1728.9273 1728.9258 0.0015 495 - 510 TNTI...PLVK 

2089.1224 2089.1168 0.0056 495 - 513 TNTI...KGFR 

2349.2223 2349.2230 -0.0007 511 - 533 GFRV...DILR 

1989.0345 1989.0320 0.0025 514 - 533 VWGG...DILR 

2145.1336 2145.1331 0.0005 514 - 534 VWGG...ILRR 

2405.2430 2405.2452 -0.0022 534 - 554 RNTF...ITQR 

2249.1461 2249.1441 0.0020 535 - 554 NTFG...ITQR 

582.2760 582.2762 -0.0002 561 - 565 YASSR 

2325.2765 2325.2726 0.0039 569 - 592 VIVL...PLQK 

1249.5978 1249.5972 0.0006 593 - 603 TMEI...LTSR 

1379.6156 1379.6146 0.0010 607 - 617 YTDF...FSFR 

2890.4515 2890.4600 -0.0085 618 - 645 ANPD...YIDK 

4906.4687 4906.4651 0.0036 618 - 663 ANPD...DLER 

2034.0166 2034.0157 0.0009 646 - 663 IEII...DLER 

1947.0454 1947.0425 0.0029 664 - 681 AQKA...IGLK 

1619.8520 1619.8519 0.0001 667 - 681 AVNE...IGLK 

732.4017 732.4017 0.0000 709 - 714 KELSEK 

604.3070 604.3068 0.0002 710 - 714 ELSEK 

774.3987 774.3984 0.0003 720 - 725 RLSDER 

618.2979 618.2973 0.0006 721 - 725 LSDER 

1715.8592 1715.8591 0.0001 721 - 734 LSDE...PNFR 

1115.5730 1115.5723 0.0007 726 - 734 NLLQ...PNFR 

458.2602 458.2601 0.0001 735 - 738 GINR 

530.2812 530.2813 -0.0001 739 - 742 QLDR 

1551.7420 1551.7417 0.0003 746 - 760 GSTD...DVFK 

4150.9422 4150.9245 0.0177 746 - 781 GSTD...LYQK 

590.2911 590.2911 0.0000 782 - 786 IDESK 

509.2598 509.2598 0.0000 789 - 792 AYTR 

578.3175 578.3176 -0.0001 793 - 796 YQLR 

1825.9111 1825.9098 0.0013 797 - 811 GYIE...YLIR 

494.2489 494.2489 0.0000 812 - 815 YNAK 

2819.4554 2819.4606 -0.0052 812 - 838 YNAK...PIGK 

2343.2223 2343.2223 0.0000 816 - 838 HETV...PIGK 

1024.5414 1024.5414 0.0000 869 - 877 IKTQ...GHAR 

783.3622 783.3624 -0.0002 871 - 877 TQDGHAR 

2097.1480 2097.1470 0.0010 878 - 896 LGNL...ALAR 
Bayer CropScience LP CR282-20U1 206 

Experimental 

Mass2

Calculated 

Mass3 Diff4 Fragment5 Sequence6

502.2863 502.2863 0.0000 899 - 902 RAEK 

474.2802 474.2802 0.0000 900 - 903 AEKK 

1550.7981 1550.7980 0.0001 909 - 920 EKLE...IVYK 

1293.6601 1293.6605 -0.0004 911 - 920 LEWE...IVYK 

1969.9390 1969.9381 0.0009 921 - 937 EAKE...QYDR 

1641.7639 1641.7635 0.0004 924 - 937 ESVD...QYDR 

1610.8088 1610.8086 0.0002 938 - 952 LQAD...AADK 

1766.9090 1766.9097 -0.0007 938 - 953 LQAD...ADKR 

766.4563 766.4562 0.0001 953 - 958 RVHSIR 

610.3547 610.3551 -0.0004 954 - 958 VHSIR 

2615.3490 2615.3482 0.0008 959 - 982 EAYL...LEGR 

1302.6615 1302.6608 0.0007 983 - 993 IFTA...YDAR 

1756.9508 1756.9512 -0.0004 983 - 997 IFTA...NVIK 

472.3006 472.3009 -0.0003 994 - 997 NVIK 

2078.9832 2078.9844 -0.0012 994 - 1011 NVIK...WNVK 

1624.6944 1624.6940 0.0004 998 - 1011 NGDF...WNVK 

1432.6460 1432.6443 0.0017 1012 - 1023 GHVD...NNHR 

1955.0016 1955.0000 0.0016 1024 - 1040 SVLV...QEVR 

620.3647 620.3646 0.0001 1046 - 1050 GYILR 

580.3217 580.3221 -0.0004 1051 - 1055 VTAYK 

2969.3562 2969.3600 -0.0038 1051 - 1076 VTAY...DELK 

2407.0481 2407.0485 -0.0004 1056 - 1076 EGYG...DELK 

6460.6970 6460.6802 0.0168 1056 - 1110 EGYG...YTSR 

4071.6560 4071.6422 0.0138 1077 - 1110 FSNC...YTSR 

2742.1708 2742.1681 0.0027 1111 - 1135 NRGY...YEEK 

2472.0260 2472.0241 0.0019 1113 - 1135 GYDG...YEEK 

3135.3217 3135.3217 0.0000 1113 - 1141 GYDG...TDGR 

681.3079 681.3082 -0.0003 1136 - 1141 AYTDGR 

837.4118 837.4093 0.0025 1136 - 1142 AYTDGRR 

2730.2378 2730.2344 0.0034 1142 - 1165 RDNP...YVTK 

2574.1331 2574.1333 -0.0002 1143 - 1165 DNPC...YVTK 

1600.7776 1600.7773 0.0003 1151 - 1165 GYGD...YVTK 

1269.5754 1269.5765 -0.0011 1166 - 1175 ELEY...ETDK 

4001.9276 4001.9271 0.0005 1166 - 1199 ELEY...LMEE 

1All imported values were rounded to 4 decimal places. 

2Only experimental masses that matched calculated masses with the highest scores are listed in the table. 3 The calculated mass is the exact molecular mass calculated from the matched peptide sequence. 4 The calculated difference = experimental mass – calculated mass. 5 Position refers to amino acid residues within the predicted MON 95379-produced Cry1B.868 sequence as depicted in Figure C2, Panel A. 

6 For peptide matches greater than nine amino acids in length, the first 4 residues and last 4 residues are show separated by three 

dots (...). 
Bayer CropScience LP CR282-20U1 207 

Table C-3. Summary of the Tryptic Masses Identified for the Bt-Produced Cry1B.868 

Using LC-MS/MS1

Experimental 

Mass2

Calculated 

Mass3 Difference4 Fragment5 Sequence6

3594.7858 3594.7808 0.0050 2 - 34 TSNR...TDAR 

3136.5591 3136.5571 0.0020 6 - 34 KNEN...TDAR 

3008.4640 3008.4621 0.0019 7 - 34 NENE...TDAR 

3260.6014 3260.5983 0.0031 35 - 65 IEDS...IAGR 

6178.1936 6178.1878 0.0058 35 - 92 IEDS...LWPR 

2935.6029 2935.6000 0.0029 66 - 92 ILGV...LWPR 

2136.1155 2136.1116 0.0039 93 - 109 GRDP...QLIR 

3092.5798 3092.5792 0.0006 93 - 117 GRDP...ENTR 

1922.9911 1922.9890 0.0021 95 - 109 DPWE...QLIR 

974.4786 974.4781 0.0005 110 - 117 QQVTENTR 

1601.8121 1601.8121 0.0000 110 - 123 QQVT...ALAR 

645.3440 645.3446 -0.0006 118 - 123 DTALAR 

990.5245 990.5247 -0.0002 124 - 132 LQGL...NSFR 

1650.7633 1650.7638 -0.0005 133 - 145 AYQQ...LENR 

2107.9515 2107.9559 -0.0044 133 - 149 AYQQ...DDAR 

2365.1092 2365.1047 0.0045 133 - 151 AYQQ...ARTR 

3057.6331 3057.6361 -0.0030 150 - 175 TRSV...FAIR 

2800.4818 2800.4873 -0.0055 152 - 175 SVLY...FAIR 

7266.8344 7266.8293 0.0051 152 - 215 SVLY...EIQR 

2518.4161 2518.4093 0.0068 176 - 197 NQEV...LLLR 

4484.3640 4484.3526 0.0114 176 - 215 NQEV...EIQR 

1983.9531 1983.9538 -0.0007 198 - 215 DASL...EIQR 

1113.5454 1113.5454 0.0000 216 - 223 YYERQVEK 

1319.5563 1319.5564 -0.0001 224 - 233 TREY...YCAR 

1062.4082 1062.4076 0.0006 226 - 233 EYSDYCAR 

1249.6207 1249.6203 0.0004 234 - 243 WYNT...NNLR 

2264.1086 2264.1086 0.0000 234 - 252 WYNT...SWLR 

1032.4995 1032.4988 0.0007 244 - 252 GTNA...SWLR 

1896.9329 1896.9343 -0.0014 244 - 258 GTNA...QFRR 

882.4483 882.4460 0.0023 253 - 258 YNQFRR 

2263.2196 2263.2212 -0.0016 258 - 277 RDLT...YDTR 

2107.1212 2107.1201 0.0011 259 - 277 DLTL...YDTR 

3468.7981 3468.7963 0.0018 259 - 289 DLTL...QLTR 

4513.3151 4513.3203 -0.0052 259 - 298 DLTL...PIGR 

1379.6881 1379.6867 0.0014 278 - 289 VYPM...QLTR 

1062.5338 1062.5346 -0.0008 290 - 298 EIYT...PIGR 

5243.6491 5243.6469 0.0022 299 - 346 TNAP...VLSR 
Bayer CropScience LP CR282-20U1 208 

Experimental 

Mass2

Calculated 

Mass3 Difference4 Fragment5 Sequence6

7066.4556 7066.4467 0.0089 299 - 360 TNAP...VGHR 

1840.8100 1840.8104 -0.0004 347 - 360 WSNT...VGHR 

2326.0712 2326.0701 0.0011 347 - 364 WSNT...LESR 

2989.5168 2989.5217 -0.0049 365 - 392 TIRG...FTSR 

2619.2933 2619.2889 0.0044 368 - 392 GSLS...FTSR 

3152.5479 3152.5487 -0.0008 368 - 396 GSLS...DVYR 

5489.7927 5489.7968 -0.0041 368 - 418 GSLS...PWAR 

2888.5197 2888.5185 0.0012 393 - 418 DVYR...PWAR 

2355.2601 2355.2587 0.0014 397 - 418 TESF...PWAR 

1415.7423 1415.7422 0.0001 419 - 429 FNWR...NSLR 

5804.8251 5804.8135 0.0116 419 - 468 FNWR...YSHR 

812.4504 812.4504 0.0000 423 - 429 NPLNSLR 

5201.5275 5201.5218 0.0057 423 - 468 NPLN...YSHR 

4407.0847 4407.0819 0.0028 430 - 468 GSLL...YSHR 

1455.8525 1455.8521 0.0004 469 - 481 LSNI...NTLR 

2553.3958 2553.3928 0.0030 469 - 490 LSNI...WTHR 

872.5078 872.5080 -0.0002 474 - 481 LISGNTLR 

1970.0517 1970.0486 0.0031 474 - 490 LISG...WTHR 

1115.5515 1115.5512 0.0003 482 - 490 APVY...WTHR 

2158.1246 2158.1230 0.0016 491 - 510 SADR...PLVK 

1728.9264 1728.9258 0.0006 495 - 510 TNTI...PLVK 

2089.1186 2089.1168 0.0018 495 - 513 TNTI...KGFR 

4060.1354 4060.1382 -0.0028 495 - 533 TNTI...DILR 

2349.2274 2349.2230 0.0044 511 - 533 GFRV...DILR 

2505.3200 2505.3241 -0.0041 511 - 534 GFRV...ILRR 

1989.0338 1989.0320 0.0018 514 - 533 VWGG...DILR 

2145.1384 2145.1331 0.0053 514 - 534 VWGG...ILRR 

2405.2503 2405.2452 0.0051 534 - 554 RNTF...ITQR 

2249.1415 2249.1441 -0.0026 535 - 554 NTFG...ITQR 

2325.2740 2325.2726 0.0014 569 - 592 VIVL...PLQK 

1249.5969 1249.5972 -0.0003 593 - 603 TMEI...LTSR 

1783.8307 1783.8318 -0.0011 604 - 617 TFRY...FSFR 

1379.6152 1379.6146 0.0006 607 - 617 YTDF...FSFR 

2890.4637 2890.4600 0.0037 618 - 645 ANPD...YIDK 

4906.4672 4906.4651 0.0021 618 - 663 ANPD...DLER 

5233.6603 5233.6557 0.0046 618 - 666 ANPD...RAQK 

1947.0437 1947.0425 0.0012 664 - 681 AQKA...IGLK 

1619.8526 1619.8519 0.0007 667 - 681 AVNE...IGLK 

3243.4239 3243.4224 0.0015 682 - 708 TDVT...LDEK 
Bayer CropScience LP CR282-20U1 209 

Experimental 

Mass2

Calculated 

Mass3 Difference4 Fragment5 Sequence6

3371.5164 3371.5174 -0.0010 682 - 709 TDVT...DEKK 

3957.8241 3957.8136 0.0105 682 - 714 TDVT...LSEK 

732.4015 732.4017 -0.0002 709 - 714 KELSEK 

831.4702 831.4702 0.0000 710 - 716 ELSEKVK 

774.3982 774.3984 -0.0002 720 - 725 RLSDER 

1871.9588 1871.9602 -0.0014 720 - 734 RLSD...PNFR 

1715.8591 1715.8591 0.0000 721 - 734 LSDE...PNFR 

2156.1103 2156.1086 0.0017 721 - 738 LSDE...GINR 

1115.5726 1115.5723 0.0003 726 - 734 NLLQ...PNFR 

1555.8231 1555.8219 0.0012 726 - 738 NLLQ...GINR 

970.5311 970.5308 0.0003 735 - 742 GINRQLDR 

929.4830 929.4831 -0.0001 739 - 745 QLDRGWR 

4549.1413 4549.1424 -0.0011 743 - 781 GWRG...LYQK 

1551.7441 1551.7417 0.0024 746 - 760 GSTD...DVFK 

4149.9435 4149.9405 0.0030 746 - 781 GSTD...LYQK 

2616.2074 2616.2094 -0.0020 761 - 781 ENYV...LYQK 

831.4697 831.4702 -0.0005 782 - 788 IDESKLK 

1322.7212 1322.7194 0.0018 782 - 792 IDES...AYTR 

750.4382 750.4388 -0.0006 787 - 792 LKAYTR 

1069.5662 1069.5668 -0.0006 789 - 796 AYTRYQLR 

1825.9099 1825.9098 0.0001 797 - 811 GYIE...YLIR 

2302.1537 2302.1481 0.0056 797 - 815 GYIE...YNAK 

4627.3601 4627.3598 0.0003 797 - 838 GYIE...PIGK 

2819.4571 2819.4606 -0.0035 812 - 838 YNAK...PIGK 

2343.2218 2343.2223 -0.0005 816 - 838 HETV...PIGK 

3520.6328 3520.6293 0.0035 839 - 868 CAHH...VIFK 

1024.5414 1024.5414 0.0000 869 - 877 IKTQ...GHAR 

783.3623 783.3624 -0.0001 871 - 877 TQDGHAR 

2862.5031 2862.4988 0.0043 871 - 896 TQDG...ALAR 

2097.1474 2097.1470 0.0004 878 - 896 LGNL...ALAR 

1706.8983 1706.8991 -0.0008 908 - 920 REKL...IVYK 

1550.8030 1550.7980 0.0050 909 - 920 EKLE...IVYK 

1878.9717 1878.9727 -0.0010 909 - 923 EKLE...KEAK 

1293.6614 1293.6605 0.0009 911 - 920 LEWE...IVYK 

1621.8354 1621.8351 0.0003 911 - 923 LEWE...KEAK 

1969.9380 1969.9381 -0.0001 921 - 937 EAKE...QYDR 

1641.7602 1641.7635 -0.0033 924 - 937 ESVD...QYDR 

3390.6601 3390.6626 -0.0025 924 - 953 ESVD...ADKR 

1610.8082 1610.8086 -0.0004 938 - 952 LQAD...AADK 
Bayer CropScience LP CR282-20U1 210 

Experimental 

Mass2

Calculated 

Mass3 Difference4 Fragment5 Sequence6

1766.9128 1766.9097 0.0031 938 - 953 LQAD...ADKR 

766.4567 766.4562 0.0005 953 - 958 RVHSIR 

4492.3474 4492.3430 0.0044 954 - 993 VHSI...YDAR 

2615.3494 2615.3482 0.0012 959 - 982 EAYL...LEGR 

3900.0148 3899.9985 0.0163 959 - 993 EAYL...YDAR 

1302.6617 1302.6608 0.0009 983 - 993 IFTA...YDAR 

1756.9504 1756.9512 -0.0008 983 - 997 IFTA...NVIK 

1432.6447 1432.6443 0.0004 1012 - 1023 GHVD...NNHR 

3369.6337 3369.6338 -0.0001 1012 - 1040 GHVD...QEVR 

1954.9977 1955.0000 -0.0023 1024 - 1040 SVLV...QEVR 

2524.2738 2524.2744 -0.0006 1024 - 1045 SVLV...CPGR 

1189.6406 1189.6390 0.0016 1041 - 1050 VCPG...YILR 

1182.6760 1182.6761 -0.0001 1046 - 1055 GYIL...TAYK 

2968.3764 2968.3760 0.0004 1051 - 1076 VTAY...DELK 

4069.6770 4069.6742 0.0028 1077 - 1110 FSNC...YTSR 

4339.8198 4339.8182 0.0016 1077 - 1112 FSNC...SRNR 

2742.1699 2742.1681 0.0018 1111 - 1135 NRGY...YEEK 

3405.4638 3405.4657 -0.0019 1111 - 1141 NRGY...TDGR 

2472.0208 2472.0241 -0.0033 1113 - 1135 GYDG...YEEK 

3135.3206 3135.3217 -0.0011 1113 - 1141 GYDG...TDGR 

3291.4220 3291.4228 -0.0008 1113 - 1142 GYDG...DGRR 

681.3083 681.3082 0.0001 1136 - 1141 AYTDGR 

837.4092 837.4093 -0.0001 1136 - 1142 AYTDGRR 

1146.4839 1146.4836 0.0003 1142 - 1150 RDNP...ESNR 

2729.2520 2729.2504 0.0016 1142 - 1165 RDNP...YVTK 

990.3831 990.3825 0.0006 1143 - 1150 DNPCESNR 

2573.1489 2573.1493 -0.0004 1143 - 1165 DNPC...YVTK 

1600.7778 1600.7773 0.0005 1151 - 1165 GYGD...YVTK 

1269.5777 1269.5765 0.0012 1166 - 1175 ELEY...ETDK 

4001.9221 4001.9271 -0.0050 1166 - 1199 ELEY...LMEE 

1

 All imported values were rounded to 4 decimal places. 2

 Only experimental masses that matched calculated masses with the highest scores are listed in table. 3

 The calculated mass is the exact molecular mass calculated from the matched peptide sequence. 4

 The calculated difference = experimental mass – calculated mass. 5

 Position refers to amino acid residues within the predicted Bt-produced Cry1B.868 sequence as depicted in Figure 

C-2, Panel B. 

6

 For peptide matches greater than nine amino acids in length the first 4 residues and last 4 residues are shown separated 

by dots (...).

 
Bayer CropScience LP CR282-20U1 211 

(A) 

 1 MTSNRKNENE IINALSIPAV SNHSAQMNLS TDARIEDSLC IAEGNNIDPF 

 51 VSASTVQTGI NIAGRILGVL GVPFAGQIAS FYSFLVGELW PRGRDPWEIF 

 101 LEHVEQLIRQ QVTENTRDTA LARLQGLGNS FRAYQQSLED WLENRDDART 

 151 RSVLYTQYIA LELDFLNAMP LFAIRNQEVP LLMVYAQAAN LHLLLLRDAS 

 201 LFGSEFGLTS QEIQRYYERQ VEKTREYSDY CARWYNTGLN NLRGTNAESW 

 251 LRYNQFRRDL TLGVLDLVAL FPSYDTRVYP MNTSAQLTRE IYTDPIGRTN 

 301 APSGFASTNW FNNNAPSFSA IEAAVIRPPH LLDFPEQLTI FSVLSRWSNT 

 351 QYMNYWVGHR LESRTIRGSL STSTHGNTNT SINPVTLQFT SRDVYRTESF 

 401 AGINILLTTP VNGVPWARFN WRNPLNSLRG SLLYTIGYTG VGTQLFDSET 

 451 ELPPETTERP NYESYSHRLS NIRLISGNTL RAPVYSWTHR SADRTNTISS 

 501 DSINQIPLVK GFRVWGGTSV ITGPGFTGGD ILRRNTFGDF VSLQVNINSP 

 551 ITQRYRLRFR YASSRDARVI VLTGAASTGV GGQVSVNMPL QKTMEIGENL 

 601 TSRTFRYTDF SNPFSFRANP DIIGISEQPL FGAGSISSGE LYIDKIEIIL 

 651 ADATFEAESD LERAQKAVNE LFTSSNQIGL KTDVTDYHID QVSNLVECLS 

 701 DEFCLDEKKE LSEKVKHAKR LSDERNLLQD PNFRGINRQL DRGWRGSTDI 

 751 TIQGGDDVFK ENYVTLLGTF DECYPTYLYQ KIDESKLKAY TRYQLRGYIE 

 801 DSQDLEIYLI RYNAKHETVN VPGTGSLWPL SAPSPIGKCA HHSHHFSLDI 

 851 DVGCTDLNED LGVWVIFKIK TQDGHARLGN LEFLEEKPLV GEALARVKRA 

 901 EKKWRDKREK LEWETNIVYK EAKESVDALF VNSQYDRLQA DTNIAMIHAA 

 951 DKRVHSIREA YLPELSVIPG VNAAIFEELE GRIFTAFSLY DARNVIKNGD 

1001 FNNGLSCWNV KGHVDVEEQN NHRSVLVVPE WEAEVSQEVR VCPGRGYILR 

1051 VTAYKEGYGE GCVTIHEIEN NTDELKFSNC VEEEVYPNNT VTCNDYTATQ 

1101 EEYEGTYTSR NRGYDGAYES NSSVPADYAS AYEEKAYTDG RRDNPCESNR 

1151 GYGDYTPLPA GYVTKELEYF PETDKVWIEI GETEGTFIVD SVELLLMEE 
Bayer CropScience LP CR282-20U1 212 

(B) 

 1 MTSNRKNENE IINALSIPAV SNHSAQMNLS TDARIEDSLC IAEGNNIDPF 

 51 VSASTVQTGI NIAGRILGVL GVPFAGQIAS FYSFLVGELW PRGRDPWEIF 

 101 LEHVEQLIRQ QVTENTRDTA LARLQGLGNS FRAYQQSLED WLENRDDART 

 151 RSVLYTQYIA LELDFLNAMP LFAIRNQEVP LLMVYAQAAN LHLLLLRDAS 

 201 LFGSEFGLTS QEIQRYYERQ VEKTREYSDY CARWYNTGLN NLRGTNAESW 

 251 LRYNQFRRDL TLGVLDLVAL FPSYDTRVYP MNTSAQLTRE IYTDPIGRTN 

 301 APSGFASTNW FNNNAPSFSA IEAAVIRPPH LLDFPEQLTI FSVLSRWSNT 

 351 QYMNYWVGHR LESRTIRGSL STSTHGNTNT SINPVTLQFT SRDVYRTESF 

 401 AGINILLTTP VNGVPWARFN WRNPLNSLRG SLLYTIGYTG VGTQLFDSET 

 451 ELPPETTERP NYESYSHRLS NIRLISGNTL RAPVYSWTHR SADRTNTISS 

 501 DSINQIPLVK GFRVWGGTSV ITGPGFTGGD ILRRNTFGDF VSLQVNINSP 

 551 ITQRYRLRFR YASSRDARVI VLTGAASTGV GGQVSVNMPL QKTMEIGENL 

 601 TSRTFRYTDF SNPFSFRANP DIIGISEQPL FGAGSISSGE LYIDKIEIIL 

 651 ADATFEAESD LERAQKAVNE LFTSSNQIGL KTDVTDYHID QVSNLVECLS 

 701 DEFCLDEKKE LSEKVKHAKR LSDERNLLQD PNFRGINRQL DRGWRGSTDI 

 751 TIQGGDDVFK ENYVTLLGTF DECYPTYLYQ KIDESKLKAY TRYQLRGYIE 

 801 DSQDLEIYLI RYNAKHETVN VPGTGSLWPL SAPSPIGKCA HHSHHFSLDI 

 851 DVGCTDLNED LGVWVIFKIK TQDGHARLGN LEFLEEKPLV GEALARVKRA 

 901 EKKWRDKREK LEWETNIVYK EAKESVDALF VNSQYDRLQA DTNIAMIHAA 

 951 DKRVHSIREA YLPELSVIPG VNAAIFEELE GRIFTAFSLY DARNVIKNGD 

1001 FNNGLSCWNV KGHVDVEEQN NHRSVLVVPE WEAEVSQEVR VCPGRGYILR 

1051 VTAYKEGYGE GCVTIHEIEN NTDELKFSNC VEEEVYPNNT VTCNDYTATQ 

1101 EEYEGTYTSR NRGYDGAYES NSSVPADYAS AYEEKAYTDG RRDNPCESNR 

1151 GYGDYTPLPA GYVTKELEYF PETDKVWIEI GETEGTFIVD SVELLLMEE 

Figure C-2. Peptide Map of the MON 95379-Produced Cry1B.868 and Bt-produced 

Cry1B.868 Proteins 
Bayer CropScience LP CR282-20U1 213 

(A). The amino acid sequence of the MON 95379-produced Cry1B.868 protein was deduced from 

the cry1B.868 gene present in MON 95379. Boxed regions correspond to peptides that were 

identified from the MON 95379-produced Cry1B.868 protein sample using LC-MS/MS. In total, 

91% coverage (1088 out of 1199 amino acids) of the expected protein sequence was covered by 

the identified peptides. Gray highlighted regions correspond to the receptor binding domain see 

Appendix L. 

(B). The amino acid sequence of the Bt-produced Cry1B.868 protein was deduced from the 

cry1B.868 gene that is contained on the expression plasmid pMON236906. Boxed regions 

correspond to peptides that were identified from the Bt-produced Cry1B.868 protein sample using 

LC-MS/MS. In total, 96% coverage (1156 out of 1199 amino acids) of the expected protein 

sequence was covered by the identified peptides. Gray highlighted regions correspond to the 

receptor binding domain see Appendix L. 

C.1.4. Results of Western Blot Analysis of the Cry1B.868 Protein Isolated from the Grain 

of MON 95379 and Immunoreactivity Comparison to Bt-produced Cry1B.868 Protein 

Western blot analysis was conducted using rabbit anti-Cry1B.868 polyclonal antibody as 

additional means to confirm the identity of the Cry1B.868 protein isolated from the grain of MON 

95379 and to assess the equivalence of the immunoreactivity of the MON 95379-produced and 

Bt-produced Cry1B.868 proteins. 

The results showed that immunoreactive bands with the same electrophoretic mobility were 

present in all lanes loaded with the MON 95379-produced and Bt-produced Cry1B.868 proteins 

(Figure C-3). For each amount loaded, comparable signal intensity was observed between the 

MON 95379-produced and Bt-produced Cry1B.868 protein bands. As expected, the signal 

intensity increased with the increasing amounts of protein loaded, thus, supporting identification 

of MON 95379-produced Cry1B.868 protein. 

To compare the immunoreactivity of the MON 95379-produced and Bt-produced Cry1B.868 

proteins, densitometric analysis was conducted on the bands that migrated at the expected apparent 

MW for Cry1B.868 proteins (~ 130 kDa). The signal intensity (reported in OD) of the band of 

interest in lanes loaded with MON 95379-produced and Bt-produced Cry1B.868 proteins was 

measured. Because the mean signal intensity of the MON 95379-produced Cry1B.868 protein was 

within 35% of the mean signal intensity of the Bt-produced Cry1B.868 protein (Table C-4), the 

MON 95379-produced Cry1B.868 and Bt-produced Cry1B.868 proteins were determined to have 

equivalent immunoreactivity. 

 
Bayer CropScience LP CR282-20U1 214 

Figure C-3. Western Blot Analysis and Immunoreactivity of MON 95379-Produced and 

Bt-Produced Cry1B.868 Proteins 

Aliquots of the MON 95379-produced and Bt-produced Cry1B.868 proteins were subjected to SDS-PAGE 

and electro-transferred to a PVDF membrane. Proteins were detected using rabbit anti-Cry1B.868 

polyclonal antibodies as the primary antibodies. Immunoreactive bands were visualized using HRPconjugated secondary antibodies and an ECL system. The 1-minute exposure is shown. The approximate 

MW (kDa) of the standards are shown on the left. Lane designations are as follows: 

Lane Sample Amount (ng) 

1 Precision Plus ProteinTM Standards - 

2 Blank - 

3 MON 95379-produced Cry1B.868 5 

4 MON 95379-produced Cry1B.868 5 

5 MON 95379-produced Cry1B.868 10 

6 MON 95379-produced Cry1B.868 10 

7 MON 95379-produced Cry1B.868 15 

8 MON 95379-produced Cry1B.868 15 

9 Blank - 

10 Bt-produced Cry1B.868 5 

11 Bt-produced Cry1B.868 5 

12 Bt-produced Cry1B.868 10 

13 Bt-produced Cry1B.868 10 

14 Bt-produced Cry1B.868 15 

15 Bt-produced Cry1B.868 15 
Bayer CropScience LP CR282-20U1 215 

Table C-4. Immunoreactivity of the MON 95379-Produced and Bt-Produced Cry1B.868 

Proteins 

Mean Signal Intensity from 

MON 95379-Produced 

Cry1B.868 1 

(OD) 

Mean Signal Intensity from 

Bt-Produced Cry1B.868 1 

(OD) 

Acceptance Limits2

(OD) 

2156.21 2527.92 1643.15 – 3412.69 

1

 Each value represents the mean of six values (n = 6). 

2

 The acceptance limits are for the MON 95379-produced Cry1B.868 protein and are based on the interval 

between -35% (2527.92 x 0.65 = 1643.15) and +35 % (2527.92 x 1.35 = 3412.69) of the mean of the Bt-produced 

Cry1B.868 signal intensity across all loads. 

C.1.5. Results of the Cry1B.868 Protein Molecular Weights and Purity Analysis 

For apparent MW and purity determination, the MON 95379-produced Cry1B.868 and the Btproduced Cry1B.868 proteins were subjected to SDS-PAGE. Following electrophoresis, the gel 

was stained with Brilliant Blue G-Colloidal stain and analyzed by densitometry. The 

MON 95379-produced Cry1B.868 protein (Figure C-4, lanes 3-8) migrated to the same position 

on the gel as the Bt-produced Cry1B.868 protein (Figure C-4, lane 2) and the apparent MW was 

calculated to be 126.8 kDa (Table C-5). Because the experimentally determined apparent MW of 

the MON 95379-produced Cry1B.868 protein was within the acceptance limits for equivalence 

(Table C-6), the MON 95379-produced Cry1B.868 and Bt-produced Cry1B.868 proteins were 

determined to have equivalent apparent molecular weights. 

 
Bayer CropScience LP CR282-20U1 216 

Figure C-4. Purity and Apparent Molecular Weight Analysis of the MON 95379-Produced 

Cry1B.868 Proteins 

Aliquots of the MON 95379-produced and the Bt-produced Cry1B.868 proteins were subjected to SDSPAGE and the gel was stained with Brilliant Blue G-Colloidal stain. The MWs (kDa) are shown on the left 

and correspond to the standards loaded in lanes 1 and 9. Lane 10 was cropped from the image. The intact 

Cry1B.868 protein is indicated with an arrow in the image. Lane designations are as follows:

Lane Sample Amount (μg) 

 1 Broad Range MW Standard 5.0 

 2 Bt-produced Cry1B.868 1.0 

 3 MON 95379-produced Cry1B.868 1.0 

 4 MON 95379-produced Cry1B.868 1.0 

 5 MON 95379-produced Cry1B.868 2.0 

 6 MON 95379-produced Cry1B.868 2.0 

 7 MON 95379-produced Cry1B.868 3.0 

 8 MON 95379-produced Cry1B.868 3.0 

 9 Broad Range MW Standard 5.0 

 10 Blank 

 
Bayer CropScience LP CR282-20U1 217 

Table C-5. Apparent Molecular Weight and Purity Analysis of the MON 95379-Produced 

Cry1B.868 Protein 

Apparent MW1 (kDa) Purity2 (%) 

Average (n = 6) 126.8 97 

1

Final MW was rounded to one decimal place. 

2

Average % purity was rounded to the nearest whole number. 

Table C-6. Apparent Molecular Weight Comparison Between the MON 95379-Produced 

Cry1B.868 and Bt-Produced Cry1B.868 Proteins 

Apparent MW 

of MON 95379-Produced 

Cry1B.868 Protein (kDa) 

Apparent MW 

of Bt-Produced 

Cry1B.868 Protein1 (kDa) 

Acceptance 

Limits2

(kDa) 

126.8 129.6 118.5 – 140.7 1

 As reported on the COA of the Bt-produced Cry1B.868 protein (lot 7565). 2

 Data obtained for the Bt-produced Cry1B.868 protein were used to generate the prediction interval. Values in this 

column represent a 95% prediction interval developed from eight individual assays with Bt-produced Cry1B.868 

protein (Section C.2.3.6. and Table C-8). 

C.1.6. Cry1B.868 Glycosylation Equivalence 

Some eukaryotic proteins are post-translationally modified by the addition of carbohydrate 

moieties (Rademacher et al., 1988). To test whether the Cry1B.868 protein was glycosylated when 

expressed in the maize grain of MON 95379, the MON 95379-produced Cry1B.868 protein was 

analyzed using an ECLTM glycoprotein detection method. Transferrin, a glycosylated protein, was 

used as a positive control in the assay. To assess equivalence of the MON 95379-produced and 

Bt-produced Cry1B.868 proteins, the Bt-produced Cry1B.868 protein was also analyzed. 

A clear glycosylation signal was observed at the expected molecular weight (~ 80 kDa) in the lanes 

containing the positive control (transferrin) and the band intensity increased with increasing 

concentration (Figure C-5, Panel A). In contrast, no glycosylation signal was observed in the lanes 

containing the Bt-produced Cry1B.868 protein or MON 95379-produced Cry1B.868 protein 

(Figure C-5, Panel A). 

To confirm that MON 95379-produced Cry1B.868 and Bt-produced Cry1B.868 proteins were 

appropriately loaded for glycosylation analysis, a second membrane with identical loadings and 

transfer time was stained with Coomassie Blue R250 for protein detection. Both the 

MON 95379-produced and Bt-produced Cry1B.868 proteins were detected (Figure C-5, Panel B). 

These data indicate that the glycosylation status of MON 95379-produced Cry1B.868 protein is 

equivalent to that of the Bt-produced Cry1B.868 protein and that neither is glycosylated. 

 
Bayer CropScience LP CR282-20U1 218 

 A. B. 

Figure C-5. Glycosylation Analysis of the MON 95379-Produced and Bt-Produced 

Cry1B.868 Proteins 

Aliquots of the transferrin (positive control), MON 95379-produced and Bt-produced Cry1B.868 proteins 

were subjected to SDS-PAGE and electro-transferred to a PVDF membrane. The MWs (kDa) correspond 

to the Precision Plus ProteinTM Standards. The arrows show the expected migration of the MON 

95379-produced and Bt-produced Cry1B.868 proteins and transferrin. (A) Where present, the labeled 

carbohydrate moieties were detected by addition of streptavidin conjugated to HRP followed by a luminolbased detection using ECL reagents and exposure to HyperfilmTM. The 1.5-minute exposure is shown. (B) 

An equivalent blot was stained with Coomassie Blue R250 to confirm the presence of proteins. Lane 

designations are as follows: 

Lane Sample Amount (ng) 

 1 Precision Plus ProteinTM Standards - 

 2 Blank - 

 3 Transferrin (positive control) 100 

 4 Transferrin (positive control) 200 

 5 Blank - 

 6 MON 95379-produced Cry1B.868 100 

 7 MON 95379-produced Cry1B.868 200 

 8 Blank - 

 9 Bt-produced Cry1B.868 100 

10 Bt-produced Cry1B.868 200 

 
Bayer CropScience LP CR282-20U1 219 

C.1.7. Cry1B.868 Functional Activity 

The MON 95379-produced Cry1B.868 and Bt-produced Cry1B.868 proteins were considered to 

have equivalent functional activity if the activities of both proteins (measured by EC50 values) were 

within the acceptance limits of 0.024 to 0.272 μg of Cry1B.868 protein/mL diet, which is the 

prediction interval calculated from a data set of Bt-produced Cry1B.868 protein activity. 

The experimentally determined functional activity for the MON 95379-produced and Bt-produced 

Cry1B.868 proteins are presented in Table C-7. The mean EC50s values of MON 95379-produced 

and Bt-produced Cry1B.868 proteins were 0.15 and 0.20 μg of Cry1B.868 protein/mL diet, 

respectively. Because both EC50s fall within the preset acceptance limits (Table C-7), the 

MON 95379-produced Cry1B.868 protein was considered to have equivalent functional activity 

to that of the Bt-produced Cry1B.868 protein. 

Table C-7. Functional Activity of MON 95379-Produced and Bt-Produced Cry1B.868 

Proteins 

Replicates 

MON 95379-Produced 

 Cry1B.868 EC50 1

(μg protein/ml diet) 

Bt-Produced

 Cry1B.868 EC50 1 

(μg protein/ml diet) 

EC50

Acceptance 

Limits2 

(μg protein/ml 

diet) 

1 0.18 0.25 

2 0.15 0.21 0.024 – 0.272 

3 0.12 0.14 

Mean 0.15 0.20 - 

1 Value refers to mean EC50 calculated based on three independent assays (n = 3).

2 Data obtained for the Bt-produced Cry1B.868 proteins were used to generate a prediction interval for 

setting the acceptance limits. Values in this column represent a 95% prediction interval developed from 24 

individual bioassays with Bt-produced Cry1B.868 protein (Section C.2.3.6. and Table C-9). 

C.1.8. Cry1B.868 Protein Identity and Equivalence Conclusion 

The MON 95379-produced Cry1B.868 protein purified from MON 95379 grain was 

characterized, and a comparison of the physicochemical and functional properties between the 

MON 95379-produced and the Bt-produced Cry1B.868 proteins was conducted following a panel 

of analytical tests: 1) N-terminal sequence analysis established the same identity for the 

MON 95379-produced and the Bt-produced Cry1B.868 proteins; 2) Nano LC-MS/MS analysis 

yielded peptide masses consistent with the expected peptide masses from the theoretical trypsin 

digest of the cry1B.868 gene product present in MON 95379; 3) the MON 95379-produced and 

the Bt-produced Cry1B.868 proteins were both detected on a western blot probed with antibodies 

specific for Cry1B.868 protein and the immunoreactive properties of both proteins was shown to 
Bayer CropScience LP CR282-20U1 220 

be equivalent; 4) the electrophoretic mobility and apparent molecular weight of the MON 95379-

produced and the Bt-produced Cry1B.868 proteins were shown to be equivalent; 5) 

the glycosylation status of MON 95379-produced and the Bt-produced Cry1B.868 proteins was 

determined to be equivalent; and 6) the functional activity of the MON 95379-produced and the 

Bt-produced Cry1B.868 was demonstrated to be equivalent. These results demonstrate that the 

MON 95379-produced and the Bt-produced Cry1B.868 protein are equivalent. 

The ability of the Cry1B.868 to bind to its target receptor is dependent on the confirmation of the 

Cry1B.868 receptor binding domain. Protein structure, or confirmation, is determined by the 

specific sequence of amino acids that comprise the different structural and functional elements. It 

can inferred that once properly folded, proteins that possess equivalent amino acid sequences 

would have equivalent functional activity or receptor binding. The MON 95379- and Bt-produced 

Cry1B.868 proteins possess equivalent amino sequences, and thereby equivalent receptor binding 

domains, as evidenced by the very high amino acid sequence similarity seen by mass fingerprint 

analysis where >90% coverage was observed. The equivalency of the protein sequences was also 

confirmed indirectly through sequencing the of genes of the expressed MON 95379- and Btproduced Cry1B.868 proteins. Finally, the equivalent functional activity of the protein isolated 

from both sources indicates that the confirmation of the receptor binding domains in both proteins 

are equivalent. 

Taken together, the equivalency data provided in support of MON 95379 Cry1B.868 confirms that 

the Bt-produced Cry1B.868 protein is appropriate surrogate for use in the evaluation of the safety 

of the MON 95379-produced Cry1B.868 protein. 

C.2. Materials and Methods for Characterization of Cry1B.868 Protein Produced in 

MON 95379 

C.2.1. Materials 

The test substance was the MON 95379-produced Cry1B.868 protein (lot 8210) purified from 

grain of MON 95379 (lot 11479279). The grain was produced by Bayer Company. The 

MON 95379-produced Cry1B.868 protein was stored in a -80 oC freezer in a buffer containing 20 

mM CAPS, pH 11.5, 10 mM DTT, 240 mM NaCl, and 1 mM Benzamidine. 

The reference substance was the Bt-produced Cry1B.868 (lot 7565, historical Orion lot 11479349). 

The Cry1B.868 reference substance was generated from the cell paste produced by fermentation 

of Bt containing the pMON236906 expression plasmid. The DNA sequence encoding Cry1B.868 

protein that is contained on the expression plasmid was confirmed both prior to and following 

fermentation. 

C.2.2. Protein Purification 

The plant-produced Cry1B.868 protein used in this equivalence study was purified from the grain 

of MON 95379 maize. The purification procedure was not performed under a GLP plan; however, 

all procedures were documented in ELN and, where applicable, SOPs were followed. Because of 
Bayer CropScience LP CR282-20U1 221 

the physicochemical similarity between Cry1B.868 and Cry1Da_7 proteins, a purification process 

was developed to co-isolate these two proteins in the front purification steps, i.e., extraction from 

the maize flour followed by Q Sepharose Fast Flow (QSFF; GE Healthcare, Piscataway, NJ) 

chromatography. The resulting fractions from the QSFF chromatography were applied to 

immuno-affinity chromatography (IAC) to purify Cry1B.868 and Cry1Da_7 separately with their 

respective antibodies. A detailed description of the purification procedure specific for Cry1B.868 

protein was archived under lot 8210 in the Bayer Regulatory Archives and a brief purification 

procedure was described below. All purification steps were performed at ~4 °C, except where 

specifically stated. 

Grain of MON 95379 was ground to fine powder in the presence of dry ice and stored at -80 °C 

until use. A total of ~30 kg of ground powder was processed in three different runs. In each run, 

~10 kg of ground powder was mixed with 100 L of extraction buffer (50 mM Na2CO3, pH ~11.3, 

5 mM DTT, 2 mM EDTA, 2 mM Benzamine-HCl, 0.5 mM AEBSF, and 0.15 M NaCl) for ~2 hr. 

The slurry was clarified by centrifugation at 13,500 × g for 1 h. The supernatant (~90 L) was 

loaded onto a ~3 L QSFF column that was equilibrated with buffer A (50 mM 

Carbonate/Bicarbonate, pH 9.0, 5 mM DTT, 2 mM EDTA, 2 mM benzamidine-HCl, and 0.1 mM 

AEBSF). After a washing step with 0.1 M NaCl, bound proteins were eluted with a linear salt 

gradient from 0.1 M to 1.0 M NaCl over a volume of ~30 L. The fraction pool (~7 L) that contained 

both Cry1B.868 and Cry1Da_7 was then concentrated and buffer-exchanged to the IAC binding 

buffer (50 mM Carbonate/Bicarbonate, pH 9.0, 2 mM EDTA, 2 mM benzamidine-HCl, and 0.15 

M NaCl,), using a 30,000 NMWC Hollow Fiber Cartridge (GE Healthcare). The end volume was 

~1L. 

To purify Cry1B.868 and Cry1Da_7 from the buffer-exchanged QSFF fraction pool (~1 L), the 

pool was first loaded onto a Cry1Da_7 immuno-affinity column. The Cry1Da_7 protein bound on 

the column was recovered through a separate procedure. The flow-through with un-bound 

Cry1B.868 was collected and loaded onto a Cry1B.868 immuno-affinity column of ~50 ml 

MabSelect Protein A resin (GE Healthcare, Cat #: 17-5199-02) cross-linked with ~100 mg of antiCry1B.868 monoclonal antibody. After washing with a buffer containing 50 mM Tris, pH 8, 0.75 

M (NH4)2SO4, 2 mM EDTA and 2 mM benzamidine, Cry1B.868 was then eluted with polyol 

buffer [50 mM Tris, pH 8.0, 0.75 M (NH4)2SO4, 40% propylene glycol, 2 mM EDTA, and 2 mM 

benzamidine]. Fractions containing Cry1B.868 protein as identified by SDS-PAGE and western 

blot were pooled and stored in -80°C until further process. 

The purified Cry1B.868 protein samples from the three runs were separately concentrated and 

buffer exchanged to storage buffer (20 mM CAPS, pH 11.5, 10 mM DTT, 240 mM NaCl, and 

1 mM Benzamidine) using 15 ml Amicon Ultracel-30K centrifugal concentration device 

(Millipore, Tullagreen, Ireland). The resulting samples were then combined as the final Cry1B.868 

protein product. This Cry1B.868 protein, purified from the grain of MON 95379, was aliquoted, 

assigned lot 8210, and stored in a -80 °C freezer. 
Bayer CropScience LP CR282-20U1 222 

C.2.3. Methods for Characterization 

C.2.3.1. N-Terminal Sequencing/Mass Fingerprint Analysis 

The test substance was analyzed for N-terminal amino acid sequence and peptide mass fingerprint 

by LC-MS/MS. Peptide fragments produced by digestion with trypsin or Asp-N protease were 

analyzed. An aliquot of the test substance was subjected to SDS-PAGE and Cry1B.868 was 

visualized on the gel with InstantBlueTM (Expedeon, Cambridgeshire, UK) staining followed by 

de-staining with Milli-Q water. Gel bands containing Cry1B.868 protein (~4 μg in each gel band) 

were excised, de-stained, incubated in ~200 μl of 100 mM ammonium bicarbonate for 30 min at 

room temperature (RT) for each gel band. The protein in each gel band was reduced by incubation 

in 100 μl of 10 mM DTT at ~37°C for 1 hr, alkylated by incubation in the dark for 30 min at RT 

with 100 μl of 20 mM iodoacetamide, and then incubated in 200 μl of 25 mM ammonium 

bicarbonate for ~15 min with agitation at RT for three times. Each gel band was dried down, 

rehydrated in 20 μl of either 20 μg/ml of trypsin or 20 μg/ml of Aps-N protease solution and 

incubated at RT for 1 hr to allow the solution to diffuse into the gel band. After incubation, 

removed excess liquid; 40 μl of 25 mM ammonium bicarbonate/10% acetonitrile solution was 

added to each gel band and the reaction was incubated at 37°C for ~17 hr. After incubation, 1 μl 

of formic acid was added to stop the digestion; each tube was sonicated for 6 min and the 

supernatant was collected and transferred to a microcentrifuge tube. The digested gel band was 

extracted with 30 μl of 70% acetonitrile/0.1% formic acid (FA) twice. The samples were 

completely dried using vacuum centrifugation and then solubilized in 10 μl of 0.1 % FA for LCMS/MS analysis. 

The LC-MS/MS data acquisition was performed on a Dionex 3000 Ultimate nano LC- system 

(Dionex) interfaced to an orbitrap fusion mass spectrometer (Thermo Scientific), which is 

equipped with a nano-ESI (electrospray ionization) source. The sample was loaded and separated 

online using an Acclaim PepMap100 C18 nano column (75 μm id × 150 mm, 2 μm, 100 Å, 

Dionex). The separation of the digests was achieved at a 300 nl/min flow rate using an acetonitrile 

gradient as follows: 0-4 min sustaining 2% solvent B, 4-80 min ramping solvent B 2-40%, 80-

85 min ramping solvent B 40-90%. The column was washed/equilibrated between injections as 

follows: 85-90 min maintaining solvent B at 90%, 90-90.1 min decreasing solvent B 90-2%, and 

90.1-100 min maintaining solvent B at 2%. Solvent A was 0.1% FA in water. Solvent B was 0.1% 

FA in acetonitrile. 

The orbitrap fusion mass spectrometer was operated with data dependent acquisition method. The 

first scan event was a full Fourier transform mass spectrometry scan with a mass resolution of 

120,000 at m/z of 200. The second scan event was a tandem mass spectrometry scan of fragments 

from collision induced dissociation (CID) of precursor ions from the first scan event with an 

isolation width of 2.0 m/z. 

The LC-MS/MS dataset were analyzed using Mascot (version 2.5, Matrix Science). MS/MS 

spectra were searched against the theoretical Cry1B.868 protein sequence. The mass tolerances 

of MS1 mass and MS/MS mass were set as 5 ppm and 0.5 Da, respectively. Carboxymethylation 

of cysteine was set as a fixed modification. Methionine oxidation and N-terminal amino acid 
Bayer CropScience LP CR282-20U1 223 

acetylation were set as variable modifications. A maximum of two missed cleavages for the 

protease used were allowed. The mass coverage was accepted if it is greater than 40% of the 

protein sequence. 

C.2.3.2. Western Blot Analysis-Immunoreactivity 

Western blot analysis was performed as follows to confirm the identity of the 

MON 95379-produced Cry1B.868 protein and to compare the immunoreactivity of the 

MON 95379-produced and Bt-produced Cry1B.868 protein. MON 95379-produced and 

Bt-produced Cry1B.868 proteins were diluted to 1 ng/μl (purity-corrected intact Cry1B.868 

protein concentration) in 5 × LB and water to obtain a final concentration of 1 × LB and heated to 

95-105 °C for 5 min. Three amounts (~5, ~10, and ~15 ng) of the intact test substance (total 

protein concentration × purity of the intact Cry1B.868 protein) and the intact reference substance 

(total protein concentration × purity of the intact Cry1B.868 protein) were loaded in duplicate onto 

a pre-cast Tris-glycine 4-20% polyacrylamide mini-gel (Invitrogen). Pre-stained molecular weight 

standards (Precision Plus Protein StandardsTM, Bio-Rad) were loaded on the gel for molecular 

weight reference and to verify electrotransfer of the proteins to the membrane. Following 

electrophoresis at a constant voltage, proteins were electrotransferred to a PVDF membrane 

(Invitrogen). 

The western blotting procedure was performed using an iBindTM Western System apparatus (Life 

Technologies, Grand Island, NY). The membrane was blocked with 1 × iBindTM Solution (Life 

Technologies) and incubated with rabbit anti-Cry1B.868 polyclonal antibody (lot 29121) at a 

dilution of 1:1000 in 1 × iBindTM Solution. After washing with 1 × iBindTM Solution, the 

membrane was next incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG 

(Vector Laboratories, Burlingame, CA) at a dilution of 1:1000 in 1 × iBindTM Solution and washed 

again with 1 × iBindTM Solution. Immunoreactive bands were visualized using the enhanced 

chemiluminescence (ECL) detection system (GE Healthcare) and exposed to HyperfilmTM ECL 

high performance chemiluminescence film (GE Healthcare). The film was developed using a 

Konica SRX-101A automated film processor (Konica, Tokyo, Japan). 

Quantification of the bands on the film was performed on a Bio-Rad GS-900 densitometer with 

the supplied Image Lab 5.0 Security Edition software using the volume tool. The signal intensity 

of the immunoreactive bands migrating at the expected position for the intact Cry1B.868 protein 

was quantified as adjusted volume values. The immunoreactivity was reported in OD, which 

equals to the total sum of the intensity of the pixels inside the volume boundary. 

C.2.3.3. Apparent Molecular Weight and Purity Determination by SDS-PAGE 

MON 95379-produced and Bt-produced Cry1B.868 proteins were diluted in 5 × loading buffer 

[LB, 0.31 M Tris-Cl, pH 7.5, 10% SDS, 50% glycerol, 25% (v/v) 2-mercaptoethanol, 0.025% 

(w/v) Bromophenol blue] and Milli-Q water and heated to 95-105°C for 5 min. The 

MON 95379-produced Cry1B.868 protein was loaded in duplicate at ~1.0, ~2.0, and ~3.0 μg based 

on total protein concentration, onto a Tris-glycine 4-20% polyacrylamide mini-gel (Invitrogen, 

Carlsbad, CA) in lanes 3, 4, 5, 6, 7 and 8. The Bt-produced Cry1B.868 protein was loaded at 
Bayer CropScience LP CR282-20U1 224 

~1.0 μg total protein in lane 2. Broad Range Molecular Weight Standards (Bio-Rad) were 

prepared and loaded in lanes 1 and 9 on the gel. Following electrophoresis at a constant voltage, 

proteins were briefly fixed in 40% (v/v) methanol, 7% (v/v) acetic acid and stained for 18 ± 2 hr 

with Brilliant Blue G-Colloidal stain (Sigma, St. Louis, MO). Gels were briefly destained in 

10% (v/v) acetic acid, 25% (v/v) methanol followed by 8 ± 2 hr in 25% (v/v) methanol. Analysis 

of the gel was performed using a Bio-Rad GS-900 densitometer supplied with Image Lab 5.0 

Security Edition software. The molecular weight of the test substance was calculated based on the 

mean of the molecular weight of the major band of Cry1B.868 (~130 kDa) in all six lanes 

containing the test substance. The purity of the test substance was calculated based on multiple 

stained bands corresponding to the Cry1B.868 protein including a major band (~130 kDa) and 

three minor bands (~120, ~110, and ~100 kDa) recognized by western blot analysis. The purity 

was reported as an average of all 6 lanes containing the MON 95379-produced Cry1B.868 protein. 

C.2.3.4. Glycosylation Analysis 

An ECL glycoprotein detection method (GE Healthcare) was used for glycoprotein detection. The 

MON 95379-produced Cry1B.868 protein, the Bt-produced Cry1B.868 protein and a positive 

control, transferrin (Sigma) were diluted in 1 × loading buffer and then heated to 95 -105 °C for 

5 min. Two amounts (~ 100 ng and ~ 200 ng) of the intact MON 95379-produced Cry1B.868 

protein (purity-corrected), the intact Bt-produced Cry1B.868 protein (purity-corrected), and the 

positive control were loaded onto a pre-cast Tris-glycine 4-20% polyacrylamide mini-gel 

(Invitrogen). Pre-stained molecular weight markers (Precision Plus ProteinTM Standards, Bio-Rad) 

were also loaded for molecular weight reference and to verify electrotransfer of the proteins to the 

membrane. Following electrophoresis at a constant voltage, proteins were electrotransferred to a 

PVDF membrane (Invitrogen). 

Glycosylation analysis was performed on the PVDF membrane at room temperature using the ECL 

Glycoprotein Detection Module (GE Healthcare) as directed by the manufacturer. The labeled 

carbohydrate moieties were detected by addition of streptavidin conjugated to HRP followed by a 

luminol-based detection using ECL reagents (GE Healthcare) and exposure to HyperfilmTM (GE 

Healthcare). The film was developed using a Konica SRX-101A automated film processor (Konica 

Minolta). An identical gel was run and electrotransferred to a PVDF membrane in parallel. 

Proteins were stained with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad) and then 

destained with equivalent chemical reagents to 1u Coomassie Brilliant Blue R-250 Destaining 

Solution (Bio-Rad). After washing with water, the blot was scanned using a Bio-Rad GS-900 

densitometer with the supplied Image Lab 5.0 Security Edition Software. 

C.2.3.5. Functional Activity 

Insects. Fall armyworm (FAW, Spodoptera frugiperda) is an appropriate test system due to its 

susceptibility to the Cry1B.868 protein. FAW were obtained from Bayer (Union City, TN). Insect 

eggs were incubated at temperatures ranging from 10°C to 27°C, to achieve the desired hatch time. 

Bioassays. FAW larvae were used to measure the functional activity of the MON 95379-produced 

Cry1B.868 and the Bt-produced Cry1B.868 in accordance with ME-0044-04. The MON 95379-
Bayer CropScience LP CR282-20U1 225 

produced Cry1B.868 and the Bt-produced Cry1B.868 substances were analyzed concurrently in 

each bioassay replicate. Bioassay replicates were initiated on different days, each using a separate 

batch of insects. Each bioassay replicate consisted of a series of six dilutions yielding a dose series 

with a two-fold separation factor ranging from 0.012 – 0.4 μg Cry1B.868/ml diet for the Btproduced Cry1B.868 and 0.014 – 0.47 Cry1B.868/ml diet for the MON 95379-produced 

Cry1B.868 and an assay (buffer) control. Dosing solutions (5.0 ml) were prepared by diluting the 

protein sample with purified water and incorporating the dilution into an agar-based FAW diet 

(Southland, Village Lake, AR) to a final volume of 25 ml. Each diet mixture was dispensed in 1 

mL aliquots into 16 wells of a 128-well tray (Frontier Agricultural Services). Each well was 

targeted to be infested with a single FAW larva (≤ 30 hours after first observation of hatching). 

Larvae were allowed to feed for a period of seven days in an environmental chamber programmed 

at 27oC, 60% relative humidity, and a lighting regime of 14 hours light: 10 hours dark. The number 

of FAW larvae infested, the number of surviving larvae, and the combined mass of the surviving 

larvae were recorded for each treatment at the end of the seven-day incubation period. 

The functional activity, measured as an EC50, of the test substance and reference substance was 

characterized using a fall armyworm insect bioassay. The EC50 value is defined as the 

concentration that results in 50% growth The EC50 value is defined as the concentration that results 

in 50% growth inhibition. Logistic regression was used to estimate the EC50 values for growth 

inhibition for each bioassay. The insect bioassay was considered acceptable if less than or equal 

to 20% mortality was observed for the vehicle control and if >50% growth inhibition was observed 

in the highest tested protein concentration level. 

C.2.3.6. Prediction Intervals as Acceptance Criteria 

Acceptance criteria (acceptance limits) based on prediction intervals were used to assess the 

equivalence of the MON 95379-produced and Bt-produced Cry1B.868 proteins for apparent MW 

and functional activity. A prediction interval is an estimate of an interval in which a randomly 

selected future observation from a population will fall, with a certain degree of confidence, given 

what has already been observed; i.e., prediction intervals are generated based on statistical analysis 

of existing data (Urquhart et al., 2015). 

The source of the data used to generate the prediction intervals for apparent MW and functional 

activity for Cry1B.868 protein equivalence assessment are provided in Table C-8 and Table C-9, 

respectively. The data used were generated under GLP guidelines and included apparent MW and 

functional activity estimates for the reference Bt-produced Cry1B.868 protein. 

The two-sided 95% prediction interval (PI) for one future assay was calculated using SAS software 

or an ELN template validated against SAS (2012, Software Release 9.4 (TS1M4), SAS Institute) 

according to the guidance document of Ramírez (2009). 

Table C-8. Individual Assay Data and 95% Prediction Interval Generated for the 

Apparent MW of Cry1B.868 Protein for One Future Assay 

Assay Number1 Apparent MW of Cry1B.868 Protein 

(kDa)2
Bayer CropScience LP CR282-20U1 226 

1 130.9 

2 134.4 

3 133.7 

4 133.4 

5 124.6 

6 124.8 

7 124.1 

8 131.1 

Mean 129.6 

Standard Deviation 4.4 

95% Prediction Interval 118.5 – 140.7 

99% Prediction Interval 113.2 – 146.0 1 Assay 1-8 represents the MW data from the initial characterization of Bt-produced Cry1B.868 (lot 7565). 2

 The values in the table represent the mean of six (n = 6) data points within each assay. With 95% or 99% confidence, 

the mean of the 6 data points from the next single assay of the population will fall within the stated interval. Mean and 

prediction interval values rounded to one decimal point. 

 
Bayer CropScience LP CR282-20U1 227 

Table C-9. Estimated EC50 Values from 24 Dose-Response Diet-Incorporation Insect 

Bioassays of the Cry1B.868 Protein 

Assay Number1 EC50 (μg protein/ml diet) 

1 0.095

2 0.067

3 0.129

4 0.050

5 0.072

6 0.040

7 0.062

8 0.091

9 0.042

10 0.054

11 0.089

12 0.172

13 0.083

14 0.103

15 0.070

16 0.186

17 0.051

18 0.038

19 0.084

20 0.097

21 0.138

22 0.062

23 0.091

24 0.187

Mean 0.081 

95% Prediction Interval 0.024 – 0.272 

99% Prediction Interval 0.017 – 0.379 1 Assay 1-24 represents functional activity data from the characterization of Bt-produced Cry1B.868 (lot 7565). 

 
Bayer CropScience LP CR282-20U1 228 

References for Appendix C 

Bradshaw, R. A., Brickey, W. W. and K. W. Walker. 1998. N-terminal processing: the methionine 

aminopeptidase and Nɑ -acetyl transferase families. Trends in Biochemical Sciences 23: 263-7. 

Rademacher, T.W., R.B. Parekh and R.A. Dwek. 1988. Glycobiology. Annual Review of 

Biochemistry 57: 785-838. 

Ramírez, J.G. 2009. Statistical Intervals: Confidence, Prediction, Enclosure. JMP White Paper 

(SAS Institute, Inc., Cary, NC). http://www.sas.com/reg/wp/corp/4430. 

Urquhart, W., G.M. Mueller, S. Carleton, Z. Song, T. Perez, J.P. Uffman, P.D. Jensen, S.L. Levine 

and J. Ward. 2015. A novel method of demonstrating the molecular and functional equivalence 

between in vitro and plant-produced double-stranded RNA. Regulatory Toxicology and 

Pharmacology In Press. 

Wang, C., K. C. Glenn, C. Kessenich, E. Bell, L.A. Burzio, M.S. Koch, B. Li and A. Silvanovich. 

2016. Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various 

crops. Regulatory Toxicology Pharmacology 81: 171-182. 
Bayer CropScience LP CR282-20U1 229 

Appendix D: Characterization of Cry1Da_7 Protein Produced in MON 95379 

D.1. Characterization of the Cry1Da_7 Protein 

D.1.1. Cry1Da_7 Protein Identity and Equivalence 

The safety assessment of crops derived through biotechnology includes characterization of the 

physicochemical and functional properties and confirmation of the safety of the introduced 

protein(s). For the safety data generated using the Bt-produced Cry1Da_7 protein to be applied to 

the MON 95379-produced Cry1Da_7 protein (plant-produced Cry1Da_7), the equivalence of the 

plant- and Bt-produced proteins must first be demonstrated. To assess the equivalence between the 

MON 95379-produced and Bt-produced Cry1Da_7 proteins, a small quantity of the MON 95379-

produced Cry1Da_7 protein was purified from MON 95379 grain. The MON 95379-produced 

Cry1Da_7 protein was characterized and the equivalence of the physicochemical characteristics 

and functional activity between the MON 95379-produced and Bt-produced Cry1Da_7 proteins 

was assessed using a panel of analytical tests; as shown in Table D-1. Taken together, these data 

provide a detailed characterization of the MON 95379-produced Cry1Da_7 protein and establish 

the equivalence of the MON 95379-produced and Bt-produced Cry1Da_7 proteins. Based on this 

established equivalence, conclusions derived from digestibility, heat susceptibility and oral acute 

toxicology, and non-target organism studies conducted with Bt-produced Cry1Da_7 protein are 

applicable to MON 95379-produced Cry1Da_7 protein. 

 
Bayer CropScience LP CR282-20U1 230 

Table D-1. Summary of MON 95379 Cry1Da_7 Protein Identity and Equivalence 

Analytical Test Assessment Analytical Test Outcome 

N-terminal sequence Identity The expected N-terminal sequence for 

MON 95379-produced Cry1Da_7 

protein was observed by Nano LCMS/MS1

Nano LC-MS/MS1 Identity Nano LC-MS/MS1 analysis of trypsin 

digested peptides from 

MON 95379-produced Cry1Da_7 

protein yielded peptide masses 

consistent with expected peptide 

masses from the theoretical trypsin 

digest of the amino acid sequence

Western blot analysis Identity 

and

Equivalence 

MON 95379-produced Cry1Da_7 

protein identity was confirmed using a 

western blot probed with antibodies 

specific for Cry1Da_7 protein 

Immunoreactive properties of the 

MON 95379-produced Cry1Da_7 and 

the Bt-produced Cry1Da_7 proteins 

were shown to be equivalent

Apparent molecular 

weight (MW) 

Equivalence Electrophoretic mobility and apparent 

molecular weight of the 

MON 95379-produced Cry1Da_7 and 

the Bt-produced Cry1Da_7 proteins 

were shown to be equivalent

Glycosylation analysis Equivalence Glycosylation status of 

MON 95379-produced Cry1Da_7 and 

Bt-produced Cry1Da_7 proteins were 

shown to be equivalent

Functional activity Equivalence Functional activity of the 

MON 95379-produced Cry1Da_7 and 

the Bt-produced Cry1Da_7 proteins 

were shown to be equivalent by insect 

bioassay
Bayer CropScience LP CR282-20U1 231 

1 Nano LC-MS/MS = Nanoscale liquid chromatography-tandem mass spectrometry 

The details of the materials and methods for the panel of analytical tests used to evaluate and 

compare the properties of the MON 95379-produced Cry1Da_7 and Bt-produced Cry1Da_7 

proteins are described at the end of Appendix D. A sumary of the data obtained to support a 

conclusion of protein equivalence is provided below. 

D.1.2. Results of the N-Terminal Sequencing Analysis 

The expected N-terminal sequence for the Cry1Da_7 protein deduced from the cry1Da_7 gene 

present in maize of MON 95379 was observed by LC-MS/MS, except that the N-terminal 

methionine was cleaved in vivo from MON 95379 produced Cry1Da_7 by methionine 

aminopeptidase or other aminopeptidases (see Experimental Sequence, Figure D-1). The cleavage 

of the N-terminal methionine from proteins in vivo by methionine aminopeptidase is common in 

many organisms (Bradshaw et al., 1998; Wang et al., 2016). The N-terminal sequence for 

MON 95379-produced Cry1Da_7 protein was consistent with the N-terminal sequence for the Btproduced Cry1Da_7 protein observed by LC-MS/MS (Figure D-1). Hence, the sequence 

information confirms the identity of the Cry1Da_7 protein isolated from the grain of MON 95379. 

Amino Acids 

Residue # from 

the N-terminus → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 

Bt-produced 

Cry1Da_7 

sequence 

→ - A E I N N Q N Q C V P Y N C L 

Expected 

Cry1Da_7 

Sequence

→ M 

│

A 

│

E 

│

I 

│

N 

│

N 

│

Q 

│

N 

│

Q 

│

C 

│

V 

│

P 

│

Y 

│

N 

│

C 

│

L 

MON 95379 

Experimental 

Sequence

→ - 

│

A 

│

E 

│

I 

│

N 

│

N 

│

Q 

│

N 

│

Q 

│

C 

│

V 

│

P 

│

Y 

│

N 

│

C 

│

L 

Figure D-1. N-Terminal Sequence of the MON 95379-Produced Cry1Da_7 Protein 

The experimental sequence obtained from the MON 95379-produced Cry1Da_7 was compared to the 

expected sequence deduced from the cry1Da_7 gene present in MON 95379. Bt-produced Cry1Da_7 

protein sequence above was derived from the reference substance COA (lot 7799). The single letter 

International Union of Pure and Applied Chemistry - International Union of Biochemistry (IUPAC-IUB) 

amino acid code is M, Methionine; A, Alanine; E, Glutamic acid; I, Isoleucine; N, Asparagine; Q, 

Glutamine; C, Cysteine; V, Valine; P, Proline; Y, Tyrosine; L, Leucine. 

D.1.3. Results of Mass Fingerprint Analysis 

Peptide mass fingerprint analysis is a standard technique used for confirming the identity of 

proteins. The identity of the MON 95379-produced Cry1Da_7 protein was confirmed by LCMS/MS analysis of peptide fragments produced by the trypsin digestion of the 

MON 95379-produced Cry1Da_7 protein. 
Bayer CropScience LP CR282-20U1 232 

There were 113 unique peptides identified that corresponded to the masses expected to be produced 

by trypsin digestion of the MON 95379-produced Cry1Da_7 protein (Table D-2, Panel A). The 

identified masses were used to assemble a coverage map of the entire Cry1Da_7 protein (Figure 

D-2, Panel A). The experimentally determined coverage of the MON 95379-produced Cry1Da_7 

protein was 89% (Figure D-2, Panel A, 1049 out of 1166 amino acids). This analysis further 

confirms the identity of MON 95379-produced Cry1Da_7 protein. 

There were 136 unique peptides identified that corresponded to the masses expected to be produced 

by trypsin digestion of the Bt-produced Cry1Da_7 protein (Table D-3) by LC-MS/MS analysis 

during the protein characterization. The identified masses were used to assemble a coverage map 

of the entire Cry1Da_7 protein (Figure D-2, Panel B). The experimentally determined coverage of 

the Bt-produced Cry1Da_7 protein was 89% (Figure D-2, Panel B, 1049 out of 1166 amino acids). 

This analysis further confirms the identity of Bt-produced Cry1Da_7 protein. 

 
Bayer CropScience LP CR282-20U1 233 

Table D-2. Summary of the Tryptic Masses Identified for the MON 95379-Produced 

Cry1Da_7 Using LC-MS/MS1 

Experimental 

Mass2

Calculated 

Mass3

Difference4 Fragment5 Sequence6

2304.0267 2304.0263 0.0004 2 - 20 AEIN...SNPK 

3243.5301 3243.5288 0.0013 2 - 28 AEIN...GEER 

957.5145 957.5131 0.0014 21 - 28 EIILGEER 

763.3866 763.3864 0.0002 88 - 93 IEEFAR 

687.3664 687.3664 0.0000 94 - 99 NQAISR 

1035.5602 1035.5600 0.0002 100 - 108 LEGL...NLYK 

535.3120 535.3118 0.0002 109 - 112 VYVR 

1745.8390 1745.8373 0.0017 113 - 127 AFSD...PALR 

2291.0670 2291.0640 0.0030 113 - 131 AFSD...EEMR 

882.4554 882.4559 -0.0005 120 - 127 DPTNPALR 

1427.6810 1427.6827 -0.0017 120 - 131 DPTN...EEMR 

2063.0863 2063.0874 -0.0011 132 - 149 IQFN...PLFR 

2712.5042 2712.4962 0.0080 150 - 173 VQNY...SILR 

3601.9240 3601.9256 -0.0016 150 - 181 VQNY...FGER 

907.4401 907.4400 0.0001 174 - 181 DVSVFGER 

1309.6070 1309.6051 0.0019 182 - 192 WGYD...INNR 

2868.3482 2868.3501 -0.0019 193 - 216 YSDL...QGLR 

3024.4491 3024.4512 -0.0021 193 - 217 YSDL...GLRR 

629.3609 629.3609 0.0000 217 - 221 RLEGR 

1311.6614 1311.6612 0.0002 222 - 231 FLSD...VYNR 

1614.8294 1614.8307 -0.0013 222 - 233 FLSD...NRFR 

2379.2982 2379.2951 0.0031 234 - 253 RQLT...YDIR 

2223.1952 2223.1940 0.0012 235 - 253 QLTI...YDIR 

1391.7412 1391.7409 0.0003 254 - 265 TYPI...QLTR 

3409.7462 3409.7445 0.0017 266 - 296 EVYL...AIIR 

2295.1538 2295.1535 0.0003 297 - 316 SPHL...SLAR 

1492.7231 1492.7211 0.0020 317 - 329 SAYW...NSFR 

975.5357 975.5349 0.0008 330 - 338 TGTT...NLIR 

691.3647 691.3653 -0.0006 339 - 344 SPLYGR 

2170.1388 2170.1382 0.0006 345 - 364 EGNT...PIFR 

2978.4638 2978.4621 0.0017 365 - 392 TLSY...TISR 

537.2912 537.2911 0.0001 393 - 396 SIYR 

665.3863 665.3860 0.0003 393 - 397 SIYRK 

2841.3900 2841.3933 -0.0033 397 - 423 KSGP...YSHR 

2713.2996 2713.2984 0.0012 398 - 423 SGPI...YSHR 

1145.5647 1145.5652 -0.0005 424 - 432 LCHA...FLER 

528.3017 528.3020 -0.0003 433 - 437 ISGPR 
Bayer CropScience LP CR282-20U1 234 

Experimental 

Mass2

Calculated Mass3 Difference4 Fragment5 Sequence6

1273.6564 1273.6568 -0.0004 438 - 448 IAGT...WTHR 

1230.5843 1230.5840 0.0003 449 - 460 SASP...SPSR 

983.5802 983.5804 -0.0002 461 - 468 ITQIPWVK 

2119.2160 2119.2153 0.0007 461 - 480 ITQI...SVIK 

1153.6452 1153.6455 -0.0003 469 - 480 AHTL...SVIK 

2325.2455 2325.2441 0.0014 469 - 492 AHTL...ILTR 

1189.6101 1189.6092 0.0009 481 - 492 GPGF...ILTR 

1076.5298 1076.5284 0.0014 493 - 502 NSMG...GTLR 

679.3655 679.3653 0.0002 503 - 508 VTFTGR 

1699.9055 1699.9046 0.0009 503 - 516 VTFT...YYIR 

1038.5499 1038.5498 0.0001 509 - 516 LPQSYYIR 

1082.5652 1082.5621 0.0031 517 - 525 FRYA...VANR 

779.3924 779.3926 -0.0002 519 - 525 YASVANR 

566.2813 566.2813 0.0000 526 - 530 SGTFR 

1469.7206 1469.7191 0.0015 531 - 543 YSQP...SFPK 

2628.2534 2628.2530 0.0004 531 - 554 YSQP...LTSR 

1176.5445 1176.5445 0.0000 544 - 554 TMDA...LTSR 

1724.8899 1724.8886 0.0013 555 - 569 SFAH...TFSR 

2044.9717 2044.9742 -0.0025 570 - 586 AQEE...YIDR 

2142.0511 2142.0521 -0.0010 587 - 604 IEFI...DLER 

1931.0804 1931.0840 -0.0036 605 - 622 AQKV...LGLK 

1603.8943 1603.8934 0.0009 608 - 622 VVNA...LGLK 

3185.4190 3185.4169 0.0021 623 - 649 TDVT...LDEK 

3341.5160 3341.5180 -0.0020 623 - 650 TDVT...DEKR 

774.3980 774.3984 -0.0004 661 - 666 RLSDER 

618.2972 618.2973 -0.0001 662 - 666 LSDER 

1715.8601 1715.8591 0.0010 662 - 675 LSDE...PNFR 

1115.5731 1115.5723 0.0008 667 - 675 NLLQ...PNFR 

458.2603 458.2601 0.0002 676 - 679 GINR 

514.2499 514.2500 -0.0001 680 - 683 QPDR 

1551.7422 1551.7417 0.0005 687 - 701 GSTD...DVFK 

4133.9082 4133.9092 -0.0010 687 - 722 GSTD...LYQK 

2600.1822 2600.1781 0.0041 702 - 722 ENYV...LYQK 

831.4703 831.4702 0.0001 723 - 729 IDESKLK 

1322.7187 1322.7194 -0.0007 723 - 733 IDES...AYTR 

578.3178 578.3176 0.0002 734 - 737 YQLR 

1825.9126 1825.9098 0.0028 738 - 752 GYIE...YLIR 
Bayer CropScience LP CR282-20U1 235 

Experimental 

Mass2

Calculated Mass3 Difference4 Fragment5 Sequence6

494.2490 494.2489 0.0001 753 - 756 YNAK 

3631.7841 3631.7841 0.0000 753 - 785 YNAK...EPNR 

3155.5468 3155.5458 0.0010 757 - 785 HEIV...EPNR 

2050.8506 2050.8560 -0.0054 786 - 801 CAPH...CSCR 

2480.0419 2480.0420 -0.0001 786 - 805 CAPH...DGEK 

1024.5408 1024.5414 -0.0006 836 - 844 IKTQ...GHAR 

783.3623 783.3624 -0.0001 838 - 844 TQDGHAR 

2111.1646 2111.1626 0.0020 845 - 863 LGNL...ALAR 

1592.8764 1592.8774 -0.0010 875 - 887 RETL...IVYK 

1436.7744 1436.7762 -0.0018 876 - 887 ETLQ...IVYK 

1764.9519 1764.9509 0.0010 876 - 890 ETLQ...KEAK 

1969.9379 1969.9381 -0.0002 888 - 904 EAKE...QYDR 

1641.7638 1641.7635 0.0003 891 - 904 ESVD...QYDR 

1610.8084 1610.8086 -0.0002 905 - 919 LQAD...AADK 

1766.9092 1766.9097 -0.0005 905 - 920 LQAD...ADKR 

2956.5439 2956.5545 -0.0106 924 - 949 IREA...LEER

2687.3701 2687.3694 0.0007 926 - 949 EAYL...LEER 

1302.6612 1302.6608 0.0004 950 - 960 IFTA...YDAR 

486.3167 486.3166 0.0001 961 - 964 NIIK 

1649.7636 1649.7620 0.0016 965 - 978 NGDF...WNVK 

1446.6600 1446.6600 0.0000 979 - 990 GHVE...NNHR 

1969.0179 1969.0156 0.0023 991 - 1007 SVLV...QEVR 

587.2851 587.2850 0.0001 1008 - 1012 VCPGR 

1189.6378 1189.6390 -0.0012 1008 - 1017 VCPG...YILR 

620.3646 620.3646 0.0000 1013 - 1017 GYILR 

580.3221 580.3221 0.0000 1018 - 1022 VTAYK 

3010.3846 3010.3866 -0.0020 1018 - 1043 VTAY...DELK 

4094.7379 4094.7422 -0.0043 1044 - 1077 FNNC...YTSR 

2807.2318 2807.2310 0.0008 1078 - 1102 NRGY...YEEK 

2537.0892 2537.0870 0.0022 1080 - 1102 GYDE...YEEK 

640.2822 640.2816 0.0006 1103 - 1107 SYTDR 

796.3828 796.3828 0.0000 1103 - 1108 SYTDRR 

1316.6012 1316.6003 0.0009 1108 - 1117 RREN...ESNR 

1160.4985 1160.4992 -0.0007 1109 - 1117 RENP...ESNR 

1004.3982 1004.3981 0.0001 1110 - 1117 ENPCESNR 

1600.7768 1600.7773 -0.0005 1118 - 1132 GYGD...YVTK 
Bayer CropScience LP CR282-20U1 236 

Experimental 

Mass2

Calculated Mass3 Difference

4

Fragment5 Sequence6

1269.5765 1269.5765 0.0000 1133 - 1142 ELEY...ETDK 

4001.9309 4001.9271 0.0038 1133 - 1166 ELEY...LMEE 

2750.3644 2750.3612 0.0032 1143 - 1166 VWIE...LMEE 1 All imported values were rounded to 4 decimal places. 2 Only experimental masses that matched calculated masses with the highest scores are listed in the table. 3 The calculated mass is the exact molecular mass calculated from the matched peptide sequence. 4 The calculated difference = experimental mass – calculated mass. 5 Position refers to amino acid residues within the predicted MON 95379-produced Cry1Da_7 sequence as depicted 

in Figure D-2, Panel A. 

6 For peptide matches greater than nine amino acids in length, the first 4 residues and last 4 residues are show 

separated by three dots (...). 

 
Bayer CropScience LP CR282-20U1 237 

Table D-3. Summary of the Tryptic Masses Identified for the Bt-Produced Cry1Da_7 

Using LC-MS/MS1 

Experimental 

Mass2

Calculated 

Mass3

Difference4 Fragment5 Sequence6

2262.015 2262.0157 -0.0007 2 - 20 AEIN...SNPK 

3201.5143 3201.5182 -0.0039 2 - 28 AEIN...GEER 

957.5144 957.5131 0.0013 21 - 28 EIILGEER 

763.3872 763.3864 0.0008 88 - 93 IEEFAR 

687.3666 687.3664 0.0002 94 - 99 NQAISR 

1704.9171 1704.9158 0.0013 94 - 108 NQAI...NLYK 

2222.2168 2222.2171 -0.0003 94 - 112 NQAI...VYVR 

1035.5602 1035.56 0.0002 100 - 108 LEGL...NLYK 

1745.8373 1745.8373 0.0000 113 - 127 AFSD...PALR 

2291.0721 2291.064 0.0081 113 - 131 AFSD...EEMR 

882.4562 882.4559 0.0003 120 - 127 DPTNPALR 

1427.6821 1427.6827 -0.0006 120 - 131 DPTN...EEMR 

2063.089 2063.0874 0.0016 132 - 149 IQFN...PLFR 

2712.4942 2712.4962 -0.0020 150 - 173 VQNY...SILR 

907.44 907.44 0.0000 174 - 181 DVSVFGER 

1309.6056 1309.6051 0.0005 182 - 192 WGYD...INNR 

2868.3523 2868.3501 0.0022 193 - 216 YSDL...QGLR 

1311.6612 1311.6612 0.0000 222 - 231 FLSD...VYNR 

1614.8332 1614.8307 0.0025 222 - 233 FLSD...NRFR 

1770.932 1770.9318 0.0002 222 - 234 FLSD...RFRR 

2379.2964 2379.2951 0.0013 234 - 253 RQLT...YDIR 

2223.1949 2223.194 0.0009 235 - 253 QLTI...YDIR 

1391.7414 1391.7409 0.0005 254 - 265 TYPI...QLTR 

4783.4748 4783.4748 0.0000 254 - 296 TYPI...AIIR 

3409.7481 3409.7445 0.0036 266 - 296 EVYL...AIIR 

2295.1527 2295.1535 -0.0008 297 - 316 SPHL...SLAR 

1492.7235 1492.7211 0.0024 317 - 329 SAYW...NSFR 

2450.2467 2450.2455 0.0012 317 - 338 SAYW...NLIR 

3123.5982 3123.6003 -0.0021 317 - 344 SAYW...LYGR 

975.536 975.5349 0.0011 330 - 338 TGTT...NLIR 

691.3659 691.3653 0.0006 339 - 344 SPLYGR 

2170.1405 2170.1382 0.0023 345 - 364 EGNT...PIFR 

2978.4662 2978.4621 0.0041 365 - 392 TLSY...TISR 

3497.7438 3497.7427 0.0011 365 - 396 TLSY...SIYR 

3625.8386 3625.8376 0.0010 365 - 397 TLSY...IYRK 

2841.3951 2841.3933 0.0018 397 - 423 KSGP...YSHR 

2713.3017 2713.2984 0.0033 398 - 423 SGPI...YSHR 
Bayer CropScience LP CR282-20U1 238 

Experimental 

Mass2

Calculated Mass3 Difference4 Fragment5 Sequence6

1145.5655 1145.5652 0.0003 424 - 432 LCHA...FLER 

1655.8571 1655.8566 0.0005 424 - 437 LCHA...SGPR 

2996.5252 2996.5217 0.0035 433 - 460 ISGP...SPSR 

1273.6562 1273.6568 -0.0006 438 - 448 IAGT...WTHR 

2486.2324 2486.2302 0.0022 438 - 460 IAGT...SPSR 

1230.5847 1230.584 0.0007 449 - 460 SASP...SPSR 

2196.1544 2196.1539 0.0005 449 - 468 SASP...PWVK 

983.5805 983.5804 0.0001 461 - 468 ITQIPWVK 

2119.2152 2119.2153 -0.0001 461 - 480 ITQI...SVIK 

3290.8122 3290.814 -0.0018 461 - 492 ITQI...ILTR 

1153.6462 1153.6455 0.0007 469 - 480 AHTL...SVIK 

2325.2484 2325.2441 0.0043 469 - 492 AHTL...ILTR 

1189.6085 1189.6092 -0.0007 481 - 492 GPGF...ILTR 

2248.1292 2248.127 0.0022 481 - 502 GPGF...GTLR 

2909.4823 2909.4818 0.0005 481 - 508 GPGF...FTGR 

1076.5288 1076.5284 0.0004 493 - 502 NSMG...GTLR 

1737.8858 1737.8832 0.0026 493 - 508 NSMG...FTGR 

679.3647 679.3653 -0.0006 503 - 508 VTFTGR 

1699.905 1699.9046 0.0004 503 - 516 VTFT...YYIR 

1038.5506 1038.5498 0.0008 509 - 516 LPQSYYIR 

2103.1018 2103.1013 0.0005 509 - 525 LPQS...VANR 

779.3924 779.3926 -0.0002 519 - 525 YASVANR 

1327.6578 1327.6633 -0.0055 519 - 530 YASV...GTFR 

1469.7193 1469.7191 0.0002 531 - 543 YSQP...SFPK 

2628.2613 2628.253 0.0083 531 - 554 YSQP...LTSR 

1176.5456 1176.5445 0.0011 544 - 554 TMDA...LTSR 

2883.4187 2883.4225 -0.0038 544 - 569 TMDA...TFSR 

1724.8919 1724.8886 0.0033 555 - 569 SFAH...TFSR 

2044.9735 2044.9742 -0.0007 570 - 586 AQEE...YIDR 

4169.0169 4169.0157 0.0012 570 - 604 AQEE...DLER 

2142.0526 2142.0521 0.0005 587 - 604 IEFI...DLER 

1931.0842 1931.084 0.0002 605 - 622 AQKV...LGLK 

1603.8943 1603.8934 0.0009 608 - 622 VVNA...LGLK 

3185.4193 3185.4169 0.0024 623 - 649 TDVT...LDEK 

3341.5189 3341.518 0.0009 623 - 650 TDVT...DEKR 

3927.8037 3927.8143 -0.0106 623 - 655 TDVT...LSEK 

760.4078 760.4079 -0.0001 650 - 655 RELSEK 
Bayer CropScience LP CR282-20U1 239 

Experimental 

Mass2

Calculated Mass3 Difference4 Fragment5 Sequence6

831.4701 831.4702 -0.0001 651 - 657 ELSEKVK 

774.3987 774.3984 0.0003 661 - 666 RLSDER 

1871.9602 1871.9602 0.0000 661 - 675 RLSD...PNFR 

1715.8562 1715.8591 -0.0029 662 - 675 LSDE...PNFR 

1115.5729 1115.5723 0.0006 667 - 675 NLLQ...PNFR 

954.499 954.4995 -0.0005 676 - 683 GINRQPDR 

913.4513 913.4518 -0.0005 680 - 686 QPDRGWR 

1950.9428 1950.9436 -0.0008 684 - 701 GWRG...DVFK 

1551.7398 1551.7417 -0.0019 687 - 701 GSTD...DVFK 

4133.909 4133.9092 -0.0002 687 - 722 GSTD...LYQK 

2600.1793 2600.1781 0.0012 702 - 722 ENYV...LYQK 

831.4704 831.4702 0.0002 723 - 729 IDESKLK 

1322.7204 1322.7194 0.0010 723 - 733 IDES...AYTR 

750.4387 750.4388 -0.0001 728 - 733 LKAYTR 

1069.5683 1069.5668 0.0015 730 - 737 AYTRYQLR 

1825.9098 1825.9098 0.0000 738 - 752 GYIE...YLIR 

3631.788 3631.7841 0.0039 753 - 785 YNAK...EPNR 

3155.5482 3155.5458 0.0024 757 - 785 HEIV...EPNR 

5188.3761 5188.3913 -0.0152 757 - 801 HEIV...CSCR 

2050.8556 2050.856 -0.0004 786 - 801 CAPH...CSCR 

1024.5416 1024.5414 0.0002 836 - 844 IKTQ...GHAR 

783.3623 783.3624 -0.0001 838 - 844 TQDGHAR 

2111.1649 2111.1626 0.0023 845 - 863 LGNL...ALAR 

1592.8784 1592.8774 0.0010 875 - 887 RETL...IVYK 

1921.0559 1921.052 0.0039 875 - 890 RETL...KEAK 

1436.7766 1436.7762 0.0004 876 - 887 ETLQ...IVYK 

1764.9496 1764.9509 -0.0013 876 - 890 ETLQ...KEAK 

3388.7085 3388.7038 0.0047 876 - 904 ETLQ...QYDR 

1969.9387 1969.9381 0.0006 888 - 904 EAKE...QYDR 

1641.7646 1641.7635 0.0011 891 - 904 ESVD...QYDR 

3234.563 3234.5615 0.0015 891 - 919 ESVD...AADK 

3390.6575 3390.6626 -0.0051 891 - 920 ESVD...ADKR 

1610.8088 1610.8086 0.0002 905 - 919 LQAD...AADK 

1766.9087 1766.9097 -0.0010 905 - 920 LQAD...ADKR 

2159.1381 2159.1382 -0.0001 905 - 923 LQAD...RVHR 

2956.5539 2956.5545 -0.0006 924 - 949 IREA...LEER 

2687.3712 2687.3694 0.0018 926 - 949 EAYL...LEER 

3972.025 3972.0196 0.0054 926 - 960 EAYL...YDAR 
Bayer CropScience LP CR282-20U1 240 

Experimental 

Mass2

Calculated Mass3 Difference4 Fragment5 Sequence6

1302.6615 1302.6608 0.0007 950 - 960 IFTA...YDAR 

1649.7618 1649.762 -0.0002 965 - 978 NGDF...WNVK 

1446.6599 1446.66 -0.0001 979 - 990 GHVE...NNHR 

3397.666 3397.6651 0.0009 979 - 1007 GHVE...QEVR 

1969.016 1969.0156 0.0004 991 - 1007 SVLV...QEVR 

1189.6389 1189.639 -0.0001 1008 - 1017 VCPG...YILR 

1182.6756 1182.6761 -0.0005 1013 - 1022 GYIL...TAYK 

2968.3751 2968.376 -0.0009 1018 - 1043 VTAY...DELK 

2406.0655 2406.0645 0.0010 1023 - 1043 EGYG...DELK 

4094.7467 4094.7422 0.0045 1044 - 1077 FNNC...YTSR 

2807.2285 2807.231 -0.0025 1078 - 1102 NRGY...YEEK 

2537.0816 2537.087 -0.0054 1080 - 1102 GYDE...YEEK 

3159.3576 3159.3581 -0.0005 1080 - 1107 GYDE...YTDR 

796.3828 796.3828 0.0000 1103 - 1108 SYTDRR 

1316.6022 1316.6003 0.0019 1108 - 1117 RREN...ESNR 

1160.4985 1160.4992 -0.0007 1109 - 1117 RENP...ESNR 

2743.267 2743.266 0.0010 1109 - 1132 RENP...YVTK 

1004.3996 1004.3981 0.0015 1110 - 1117 ENPCESNR 

2587.1674 2587.1649 0.0025 1110 - 1132 ENPC...YVTK 

1600.7776 1600.7773 0.0003 1118 - 1132 GYGD...YVTK 

2852.3411 2852.3432 -0.0021 1118 - 1142 GYGD...ETDK 

1269.5768 1269.5765 0.0003 1133 - 1142 ELEY...ETDK 

4001.9295 4001.9271 0.0024 1133 - 1166 ELEY...LMEE 

2750.3631 2750.3612 0.0019 1143 - 1166 VWIE...LMEE 

1

 All imported values were rounded to 4 decimal places. 2

 Only experimental masses that matched calculated masses with the highest scores are listed in table. 3

 The calculated mass is the exact molecular mass calculated from the matched peptide sequence. 4

 The calculated difference = experimental mass – calculated mass. 5

 Position refers to amino acid residues within the predicted Bt-produced Cry1Da_7 sequence as depicted in Figure 

D-2, Panel B. 

6

 For peptide matches greater than nine amino acids in length the first 4 residues and last 4 residues are shown separated 

by dots (...).
Bayer CropScience LP CR282-20U1 241 

(A) 

 1 MAEINNQNQC VPYNCLSNPK EIILGEERLE TGNTVADISL GLINFLYSNF 

 51 VPGGGFIVGL LELIWGFIGP SQWDIFLAQI EQLISQRIEE FARNQAISRL 

 101 EGLSNLYKVY VRAFSDWEKD PTNPALREEM RIQFNDMNSA LITAIPLFRV 

 151 QNYEVALLSV YVQAANLHLS ILRDVSVFGE RWGYDTATIN NRYSDLTSLI 

 201 HVYTNHCVDT YNQGLRRLEG RFLSDWIVYN RFRRQLTISV LDIVAFFPNY 

 251 DIRTYPIQTA TQLTREVYLD LPFINENLSP AAVYPTFSAA ESAIIRSPHL 

 301 VDFLNSFTIY TDSLARSAYW GGHLVNSFRT GTTTNLIRSP LYGREGNTER 

 351 PVTITASPSV PIFRTLSYPT GLDNSNPVAG IEGVEFQNTI SRSIYRKSGP 

 401 IDSFSELPPQ DASVSPAIGY SHRLCHATFL ERISGPRIAG TVFSWTHRSA 

 451 SPTNEVSPSR ITQIPWVKAH TLASGASVIK GPGFTGGDIL TRNSMGELGT 

 501 LRVTFTGRLP QSYYIRFRYA SVANRSGTFR YSQPPSYGIS FPKTMDAGEP 

 551 LTSRSFAHTT LFTPITFSRA QEEFDLYIQS GVYIDRIEFI PVTATFEAEY 

 601 DLERAQKVVN ALFTSTNQLG LKTDVTDYHI DQVSNLVACL SDEFCLDEKR 

 651 ELSEKVKHAK RLSDERNLLQ DPNFRGINRQ PDRGWRGSTD ITIQGGDDVF 

 701 KENYVTLPGT FDECYPTYLY QKIDESKLKA YTRYQLRGYI EDSQDLEIYL 

 751 IRYNAKHEIV NVPGTGSLWP LSVENQIGPC GEPNRCAPHL EWNPDLHCSC 

 801 RDGEKCAHHS HHFSLDIDVG CTDLNEDLGV WVIFKIKTQD GHARLGNLEF 

 851 LEEKPLLGEA LARVKRAEKK WRDKRETLQL ETTIVYKEAK ESVDALFVNS 

 901 QYDRLQADTN IAMIHAADKR VHRIREAYLP ELSVIPGVNA AIFEELEERI 

 951 FTAFSLYDAR NIIKNGDFNN GLLCWNVKGH VEVEEQNNHR SVLVIPEWEA 

1001 EVSQEVRVCP GRGYILRVTA YKEGYGEGCV TIHEIENNTD ELKFNNCVEE 

1051 EVYPNNTVTC INYTATQEEY EGTYTSRNRG YDEAYGNNPS VPADYASVYE 

1101 EKSYTDRRRE NPCESNRGYG DYTPLPAGYV TKELEYFPET DKVWIEIGET 

1151 EGTFIVDSVE LLLMEE 
Bayer CropScience LP CR282-20U1 242 

(B) 

 1 MAEINNQNQC VPYNCLSNPK EIILGEERLE TGNTVADISL GLINFLYSNF 

 51 VPGGGFIVGL LELIWGFIGP SQWDIFLAQI EQLISQRIEE FARNQAISRL 

 101 EGLSNLYKVY VRAFSDWEKD PTNPALREEM RIQFNDMNSA LITAIPLFRV 

 151 QNYEVALLSV YVQAANLHLS ILRDVSVFGE RWGYDTATIN NRYSDLTSLI 

 201 HVYTNHCVDT YNQGLRRLEG RFLSDWIVYN RFRRQLTISV LDIVAFFPNY 

 251 DIRTYPIQTA TQLTREVYLD LPFINENLSP AAVYPTFSAA ESAIIRSPHL 

 301 VDFLNSFTIY TDSLARSAYW GGHLVNSFRT GTTTNLIRSP LYGREGNTER 

 351 PVTITASPSV PIFRTLSYPT GLDNSNPVAG IEGVEFQNTI SRSIYRKSGP 

 401 IDSFSELPPQ DASVSPAIGY SHRLCHATFL ERISGPRIAG TVFSWTHRSA 

 451 SPTNEVSPSR ITQIPWVKAH TLASGASVIK GPGFTGGDIL TRNSMGELGT 

 501 LRVTFTGRLP QSYYIRFRYA SVANRSGTFR YSQPPSYGIS FPKTMDAGEP 

 551 LTSRSFAHTT LFTPITFSRA QEEFDLYIQS GVYIDRIEFI PVTATFEAEY 

 601 DLERAQKVVN ALFTSTNQLG LKTDVTDYHI DQVSNLVACL SDEFCLDEKR 

 651 ELSEKVKHAK RLSDERNLLQ DPNFRGINRQ PDRGWRGSTD ITIQGGDDVF 

 701 KENYVTLPGT FDECYPTYLY QKIDESKLKA YTRYQLRGYI EDSQDLEIYL 

 751 IRYNAKHEIV NVPGTGSLWP LSVENQIGPC GEPNRCAPHL EWNPDLHCSC 

 801 RDGEKCAHHS HHFSLDIDVG CTDLNEDLGV WVIFKIKTQD GHARLGNLEF 

 851 LEEKPLLGEA LARVKRAEKK WRDKRETLQL ETTIVYKEAK ESVDALFVNS 

 901 QYDRLQADTN IAMIHAADKR VHRIREAYLP ELSVIPGVNA AIFEELEERI 

 951 FTAFSLYDAR NIIKNGDFNN GLLCWNVKGH VEVEEQNNHR SVLVIPEWEA 

1001 EVSQEVRVCP GRGYILRVTA YKEGYGEGCV TIHEIENNTD ELKFNNCVEE 

1051 EVYPNNTVTC INYTATQEEY EGTYTSRNRG YDEAYGNNPS VPADYASVYE 

1101 EKSYTDRRRE NPCESNRGYG DYTPLPAGYV TKELEYFPET DKVWIEIGET 

1151 EGTFIVDSVE LLLMEE

Figure D-2. Peptide Map of the MON 95379-Produced Cry1Da_7 and Bt-Produced 

Cry1Da_7 

(A). The amino acid sequence of the MON 95379-produced Cry1Da_7 protein was deduced from 

the cry1Da_7 gene present in MON 95379. Boxed regions correspond to peptides that were 

identified from the MON 95379-produced Cry1Da_7 protein sample using LC-MS/MS. In total, 

89% coverage (1049 out of 1166 amino acids) of the expected protein sequence was covered by 

the identified peptides. Gray highlighted regions correspond to the receptor binding domain (see 

Appendix L). 
Bayer CropScience LP CR282-20U1 243 

(B). The amino acid sequence of the Bt-produced Cry1Da_7 protein was deduced from the 

cry1Da_7 gene that is contained on the expression plasmid pMON417133. Boxed regions 

correspond to peptides that were identified from the Bt-produced Cry1Da_7 protein sample using 

LC-MS/MS. In total, 89% coverage (1049 out of 1166 amino acids) of the expected protein 

sequence was covered by the identified peptides. Gray highlighted regions correspond to the 

receptor binding domain (see Appendix L). 

D.1.4. Results of Western Blot Analysis of the Cry1Da_7 Protein Isolated from the Grain of 

MON 95379 and Immunoreactivity Comparison to Bt-produced Cry1Da_7 Protein 

Western blot analysis was conducted using goat anti-Cry1Da_7 polyclonal antibody as additional 

means to confirm the identity of the Cry1Da_7 protein isolated from the grain of MON 95379 and 

to assess the equivalence of the immunoreactivity of the MON 95379-produced and Bt-produced 

Cry1Da_7 proteins. 

The results showed that immunoreactive bands with the same electrophoretic mobility were 

present in all lanes loaded with the MON 95379-produced and Bt-produced Cry1Da_7 proteins 

(Figure D-3). For each amount loaded, comparable signal intensity was observed between the 

MON 95379-produced and Bt-produced Cry1Da_7 protein bands. As expected, the signal 

intensity increased with increasing load amounts of the MON 95379-produced and Bt-produced 

Cry1Da_7 proteins, thus, supporting identification of MON 95379-produced Cry1Da_7 protein. 

To compare the immunoreactivity of the MON 95379-produced and Bt-produced Cry1Da_7 

proteins, densitometric analysis was conducted on the bands that migrated at the expected apparent 

MW for Cry1Da_7 proteins (~ 130 kDa). The signal intensity (reported in OD) of the band of 

interest in lanes loaded with MON 95379-produced and Bt-produced Cry1Da_7 proteins was 

measured (Table D-4). Because the mean signal intensity of the MON 95379-produced Cry1Da_7 

protein was within 35% of the mean signal intensity of the Bt-produced Cry1Da_7 protein, the 

MON 95379-produced Cry1Da_7 and Bt-produced Cry1Da_7 proteins were determined to have 

equivalent immunoreactivity. 

 
Bayer CropScience LP CR282-20U1 244 

Figure D-3. Western Blot Analysis and Immunoreactivity of MON 95379-Produced and 

Bt-Produced Cry1Da_7 Proteins 

Aliquots of the MON 95379-produced and Bt-produced Cry1Da_7 proteins were subjected to 

SDS-PAGE and electrotransferred to a nitrocellulose membrane. Proteins were detected using 

goat anti-Cry1Da_7 polyclonal antibodies as the primary antibodies. Immunoreactive bands were 

visualized using HRP-conjugated secondary antibodies and an ECL system. The 3-minute 

exposure is shown. The approximate MW (kDa) of the standards are shown on the left. Lane 

designations are as follows: 

Lane Sample Amount (ng) 

1 Precision Plus ProteinTM Standards - 

2 Bt-produced Cry1Da_7 10 

3 Bt-produced Cry1Da_7 10 

4 Bt-produced Cry1Da_7 15 

5 Bt-produced Cry1Da_7 15 

6 Bt-produced Cry1Da_7 20 

7 Bt-produced Cry1Da_7 20 

8 MON 95379-produced Cry1Da_7 10 

9 MON 95379-produced Cry1Da_7 10 

10 MON 95379-produced Cry1Da_7 15 

11 MON 95379-produced Cry1Da_7 15 

12 MON 95379-produced Cry1Da_7 20 

13 MON 95379-produced Cry1Da_7 20 

14 Blank - 

15 Blank - 
Bayer CropScience LP CR282-20U1 245 

Table D-4. Immunoreactivity of the MON 95379-Produced and Bt-Produced Cry1Da_7 

Proteins 

Mean Signal Intensity from 

MON 95379-Produced 

Cry1Da_7 1 

(OD) 

Mean Signal Intensity from 

Bt-Produced Cry1Da_7 1 

(OD) 

Acceptance Limits2

(OD) 

1260.28 1175.99 764.39 – 1587.59 

1

 Each value represents the mean of six values (n = 6). 

2

 The acceptance limits are for the MON 95379-produced Cry1Da_7 protein and are based on the interval 

between -35% (1175.99 x 0.65 = 764.39) and +35 % (1175.99 x 1.35 = 1587.59) of the mean of the Bt-produced 

Cry1Da_7 signal intensity across all loads. 

D.1.5. Results of the Cry1Da_7 Protein Molecular Weights and Purity Analysis 

For apparent MW and purity determination, the MON 95379-produced Cry1Da_7 and the 

Bt-produced Cry1Da_7 proteins were subjected to SDS-PAGE. Following electrophoresis, the gel 

was stained with Brilliant Blue G-Colloidal stain and analyzed by densitometry. The 

MON 95379-produced Cry1Da_7 protein (Figure D-4, lanes 3-8) migrated to the same position on 

the gel as the Bt-produced Cry1Da_7 protein (Figure D-4, lane 2) and the apparent MW was 

calculated to be 132.1 kDa (Table D-5). Because the experimentally determined apparent MW of 

the MON 95379-produced Cry1Da_7 protein was within the acceptance limits for equivalence 

(Table D-6), the MON 95379-produced Cry1Da_7 and Bt-produced Cry1Da_7 proteins were 

determined to have equivalent apparent molecular weights. 

 
Bayer CropScience LP CR282-20U1 246 

Figure D-4. Purity and Apparent Molecular Weight Analysis of the MON 95379-Produced 

Cry1Da_7 Proteins 

Aliquots of the MON 95379-produced and the Bt-produced Cry1Da_7 proteins were subjected to 

SDS-PAGE and the gel was stained with Brilliant Blue G-Colloidal stain. The MWs (kDa) are 

shown on the left and correspond to the standards loaded in lanes 1 and 9. Lane 10 was cropped 

from the image. The intact Cry1Da_7 protein is indicated with an arrow in the image. Lane 

designations are as follows: 

Lane Sample Amount (μg) 

 1 Broad Range MW Standard 5.0 

 2 Bt-produced Cry1Da_7 1.0 

 3 MON 95379-produced Cry1Da_7 1.0 

 4 MON 95379-produced Cry1Da_7 1.0 

 5 MON 95379-produced Cry1Da_7 2.0 

 6 MON 95379-produced Cry1Da_7 2.0 

 7 MON 95379-produced Cry1Da_7 3.0 

 8 MON 95379-produced Cry1Da_7 3.0 

 9 Broad Range MW Standard 5.0 

 10 Blank 
Bayer CropScience LP CR282-20U1 247 

Table D-5. Apparent Molecular Weight and Purity Analysis of the MON 95379-Produced 

Cry1Da_7 Protein 

Apparent MW1 (kDa) Purity2 (%) 

Average (n = 6) 132.1 52 

1

Final MW was rounded to one decimal place. 

2

Average % purity was rounded to the nearest whole number. 

Table D-6. Apparent Molecular Weight Comparison Between the MON 95379-Produced 

Cry1Da_7 and Bt-Produced Cry1Da_7 Proteins 

Apparent MW 

of MON 95379-Produced 

Cry1Da_7 Protein (kDa) 

Apparent MW 

of Bt-Produced 

Cry1Da_7 Protein1 (kDa) 

Acceptance 

Limits2

(kDa) 

132.1 126.5 119.9 – 133.1 1

 As reported on the COA of the Bt-produced Cry1Da_7 protein (lot 7799). 2

 Data obtained for the Bt-produced Cry1Da_7 protein were used to generate the prediction interval. Values in this 

column represent a 95% prediction interval developed from eight individual assays with Bt-produced Cry1Da_7 

protein (Section D.2.3.6. and Table D-8). 

D.1.6. Cry1Da_7 Glycosylation Equivalence 

Some eukaryotic proteins are post-translationally modified by the addition of carbohydrate 

moieties (Rademacher et al., 1988). To test whether the Cry1Da_7 protein was glycosylated when 

expressed in the maize grain of MON 95379, the MON 95379-produced Cry1Da_7 protein was 

analyzed using an ECLTM glycoprotein detection method. Transferrin, a glycosylated protein, was 

used as a positive control in the assay. To assess equivalence of the MON 95379-produced and 

Bt-produced Cry1Da_7 proteins, the Bt-produced Cry1Da_7 protein was also analyzed. 

A clear glycosylation signal was observed at the expected molecular weight (~80 kDa) in the lanes 

containing the positive control (transferrin) and the band intensity increased with increasing 

concentration (Figure D-5, Panel A). In contrast, no glycosylation signal was observed in the lanes 

containing the Bt-produced Cry1Da_7 protein or MON 95379-produced Cry1Da_7 protein (Figure 

D-5, Panel A). 

To confirm that MON 95379-produced Cry1Da_7 and Bt-produced Cry1Da_7 proteins were 

appropriately loaded for glycosylation analysis, a second membrane with identical loadings and 

transfer time was stained with Coomassie Blue R250 for protein detection. Both the 

MON 95379-produced and Bt-produced Cry1Da_7 proteins were detected (Figure D-5, Panel B). 

These data indicate that the glycosylation status of MON 95379-produced Cry1Da_7 protein is 

equivalent to that of the Bt-produced Cry1Da_7 protein and that neither is glycosylated. 

 
Bayer CropScience LP CR282-20U1 248 

 A. B. 

Figure D-5. Glycosylation Analysis of the MON 95379-Produced and Bt-Produced 

Cry1Da_7 Proteins 

Aliquots of the transferrin (positive control), Bt-produced Cry1Da_7 and MON 95379-produced 

Cry1Da_7 proteins were subjected to SDS-PAGE and electrotransferred to a PVDF membrane. 

The MWs (kDa) correspond to the Precision Plus ProteinTM Standards. The arrows show the 

expected migration of the MON 95379-produced and Bt-produced Cry1Da_7 proteins and 

transferrin. (A) Where present, the labeled carbohydrate moieties were detected by using ECL 

reagents and exposure to HyperfilmTM. The 4-minute exposure is shown. (B) An equivalent blot 

was stained with Coomassie Blue R250 to confirm the presence of proteins. Lane designations 

are as follows: 

Lane Sample Amount (ng) 

 1 Precision Plus ProteinTM Standards - 

 2 Blank - 

 3 Transferrin (positive control) 100 

 4 Transferrin (positive control) 200 

 5 Blank - 

 6 Bt-produced Cry1Da_7 100 

 7 Bt-produced Cry1Da_7 200 

 8 Blank - 

 9 MON 95379-produced Cry1Da_7 100 

10 MON 95379-produced Cry1Da_7 200 

 
Bayer CropScience LP CR282-20U1 249 

D.1.7. Cry1Da_7 Functional Activity 

The MON 95379-produced Cry1Da_7 and Bt-produced Cry1Da_7 proteins were considered to 

have equivalent functional activity if the activities of both proteins (EC50 values) were within 

acceptance limits of 0.076 to 0.293 μg of Cry1Da_7 protein/mL diet, which is the prediction 

interval calculated from a data set of Bt-produced Cry1Da_7 protein activity. 

The experimentally determined functional activity for the MON 95379-produced and Bt-produced 

Cry1Da_7 proteins are presented in Table D-7. The functional activities of MON 95379-produced 

and Bt-produced Cry1Da_7 proteins were 0.17 and 0.12 μg of Cry1Da_7 protein/mL diet, 

respectively. Because the functional activities of MON 95379-produced and Bt-produced 

Cry1Da_7 proteins fall within the preset acceptance limits (Table D-7), the MON 95379-produced 

Cry1Da_7 protein was considered to have equivalent functional activity to that of the Bt-produced 

Cry1Da_7 protein. 

Table D-7. Functional Activity of MON 95379-Produced and Bt-Produced Cry1Da_7 

Proteins 

Replicates 

MON 95379-Produced 

 Cry1Da_7 EC50 1 

(μg protein/mL diet) 

Bt-Produced 

 Cry1Da_7 EC50 1

(μg protein/mL diet) 

EC50 Acceptance 

Limits2 

(μg protein/mL diet) 

1 

2 

3 

0.20 

0.14 

0.17 

0.11 

0.076 

0.16 

0.076 – 0.293 

 

Mean 0.17 0.12 - 

1

 Value refers to mean EC50 calculated based on three independent assays (n = 3). 2

 Data obtained for the Bt-produced Cry1Da_7 proteins were used to generate a prediction interval for setting the 

acceptance limits for EC50 values of 3 future independent assays. Values in this column represent a 95% prediction 

interval developed from 24 individual bioassays with Bt-produced Cry1Da_7 protein (Section D.2.3.6. and Table D9). 

 
Bayer CropScience LP CR282-20U1 250 

D.1.8. Cry1Da_7 Protein Identity and Equivalence Conclusion 

The MON 95379-produced Cry1Da_7 protein purified from MON 95379 grain was characterized, 

and a comparison of the physicochemical and functional properties between the MON 95379-

produced and the Bt-produced Cry1Da_7 proteins was conducted following a panel of analytical 

tests: 1) N-terminal sequence analysis established the same identity for the MON 95379-produced 

and the Bt-produced Cry1Da_7 proteins; 2) Nano LC-MS/MS analysis yielded peptide masses 

consistent with the expected peptide masses from the theoretical trypsin digest of the cry1Da_7

gene product present in MON 95379; 3) the MON 95379-produced and the Bt-produced 

Cry1Da_7 proteins were both detected on a western blot probed with antibodies specific for 

Cry1Da_7 protein and the immunoreactive properties of both proteins was shown to be equivalent; 

4) the electrophoretic mobility and apparent molecular weight of the MON 95379-produced and 

the Bt-produced Cry1Da_7 proteins were shown to be equivalent; 5) the glycosylation status of 

MON 95379-produced and the Bt-produced Cry1Da_7 proteins was determined to be equivalent; 

and 6) the functional activity of the MON 95379-produced and the Bt-produced Cry1Da_7 was 

demonstrated to be equivalent. These results demonstrate that the MON 95379-produced and the 

Bt-produced Cry1Da_7 protein are equivalent. 

The ability of the Cry1Da_7 to bind to its target receptor is dependent on the confirmation of the 

Cry1Da_7 receptor binding domain. Protein structure, or confirmation, is determined by the 

specific sequence of amino acids that comprise the different structural and functional elements. It 

can inferred that once properly folded, proteins that possess equivalent amino acid sequences 

would have equivalent functional activity or receptor binding. The MON 95379- and Bt-produced 

Cry1Da_7 proteins possess equivalent amino sequences, and thereby equivalent receptor binding 

domains, as evidenced by the very high amino acid sequence similarity seen by mass fingerprint 

analysis where >90% coverage was observed. The equivalency of the protein sequences was also 

confirmed indirectly through sequencing the of genes of the expressed MON 95379- and Btproduced Cry1Da_7 proteins. Finally, the equivalent functional activity of the protein isolated 

from both sources indicates that the confirmation of the receptor binding domains in both proteins 

are equivalent. 

Taken together, the equivalency data provided in support of MON 95379 Cry1Da_7 confirms that 

the Bt-produced Cry1Da_7 protein is appropriate surrogate for use in the evaluation of the safety 

of the MON 95379-produced Cry1Da_7 protein. 

D.2. Materials and Methods for Characterization of Cry1Da_7 Protein Produced in 

MON 95379 

D.2.1. Materials 

The test substance was the MON 95379-produced Cry1Da_7 protein (lot 8206) purified from grain 

of MON 95379 (lot 11479279). The MON 95379-produced Cry1Da_7 protein was stored in 

a -80 oC freezer in a buffer containing 25 mM sodium carbonate, pH 10.5. 
Bayer CropScience LP CR282-20U1 251 

The reference substance was the Bt-produced Cry1Da_7 (lot 7799, historical Orion lot 11479575). 

The Cry1Da_7 reference substance was generated from cell paste produced by fermentation of Bt

containing the pMON417133 expression plasmid. The DNA sequence encoding Cry1Da_7 protein 

that is contained on the expression plasmid was confirmed both prior to and following 

fermentation. 

D.2.2. Protein Purification 

The plant-produced Cry1Da_7 protein used in this equivalence study was purified from the grain 

of MON 95379 maize. The purification procedure was not performed under a GLP plan; however, 

all procedures were documented in ELN and, where applicable, SOPs were followed. A unique 

purification process was developed to combine both Cry1B.868 and Cry1Da_7 isolations into one 

single process, as they share similar physicochemical properties. Both Cry1B.868 and Cry1Da_7 

were purified from an extract of ground grain using a combination of anion-exchange 

chromatography and immunoaffinity chromatography (IAC). A detailed description of the 

purification procedure specific for Cry1Da_7 protein was archived under lot 8206 in the Bayer 

Regulatory Archives and a brief purification procedure was described below. All purification steps 

were performed at ~4 °C, except where specifically stated. 

Grain of MON 95379 was ground to fine powder in the presence of dry ice and stored at -80 °C 

until use. A total of ~47 kg of ground powder was processed by 5 different runs. For each run, ~ 

10 kg of ground powder was mixed with 100 L of Extraction Buffer (50 mM Na2CO3, 5 mM DTT, 

2 mM EDTA, 2 mM Benzamine-HCl, 0.5 mM AEBSF, 0.15 M NaCl, pH ~11.3) at 4°C for ~2 hr. 

The slurry was clarified by centrifugation at 13,500 x g for 1 h at ~4°C, and about 90 L supernatants 

were collected, filtered, and loaded onto a ~3 L Q Sepharose Fast Flow (QFF; GE Healthcare, 

Piscataway, NJ) column (15 cm x 20 cm) after equilibrated with Binding Buffer A for QFF (50 

mM Carbonate/Bicarbonate Buffer, 5 mM DTT, 2 mM EDTA, 2 mM benzamidine-HCl, 0.1 mM 

AEBSF, pH 9.0). Proteins were eluted with a linear salt gradient that increased from 0.1 M to 1.0 

M NaCl in Elution Buffer B for QFF (50 mM Carbonate/Bicarbonate Buffer, 5 mM DTT, 2 mM 

EDTA, 2 mM benzamidine-HCl, 0.1 mM AEBSF, 1 M NaCl, pH 9.0) over a volume of ~30 L 

(~10 x column volume) after the column was washed with 10 % Elution Buffer B for QFF. 

Fractions containing both Cry1B.868 and Cry1Da_7, identified by western blot analysis, were 

pooled to a final volume of ~7 L. This pooled sample was concentrated and buffer-exchanged to 

Wash and Binding Buffer A for IAC (50 mM Carbonate/Bicarbonate Buffer, 2 mM EDTA, 2 mM 

benzamidine-HCl, 0.15 M NaCl, pH 9.0), using an ultrafiltration system sequentially equipped 

with a 30,000 NMWC Hollow Fiber Cartridge (8400 cm2 surface area) (GE Healthcare). About 

1 L of final sample was collected and stored on wet ice for next step IAC. 

For 1st, 2nd, 4th, and 5th production runs, the following steps were conducted for IAC. About 1 L 

of protein sample collected after buffer exchange was loaded onto a ~75-ml of MabSelect Protein 

A resin (GE healthcare, Cat #: 17-5199-02) cross-linked with ~150 mg of anti-Cry1Da_7 

monoclonal antibody (mAb) by BS3 following the manufacturer’s instruction and equilibrated with 

Wash and Binding Buffer for IAC. Cry1Da_7 protein was bound by the resin, whereas Cry1B.868 

was not bound by the resin (confirmed by western blot analysis). The flow through samples from 

1st and 2nd production runs were collected and used for the further purification of Cry1B.868 
Bayer CropScience LP CR282-20U1 252 

protein, but the flow through samples from 4th and 5th production runs were discarded since a 

sufficient amount of Cry1B.868 protein was already purified. Cry1Da_7 protein was eluted from 

the immunoaffinity column by Polyol Buffer (50 mM Tris, pH 8.0, 0.75 M (NH4)2SO4, 40% 

propylene glycol, 2 mM EDTA, 2 mM Benzamidine-HCl) after the column was washed with Wash 

Buffer (50 mM Tris, pH 8.0, 0.75 M (NH4)2SO4, 2 mM EDTA, 2 mM Benzamidine-HCl). 

Fractions containing Cry1Da_7 protein were identified by SDS-PAGE and western blot analysis, 

pooled together, and stored at -80°C for final concentration and buffer exchange. 

For 3rd production run, the following steps were conducted for IAC. About 1 L of final sample 

collected after buffer exchange was sequentially loaded onto the same anti-Cry1Da_7 mAb 

column described earlier and a ~100 ml anti-Cry1B.868 mAb column (two mAb columns were 

tandem-connected). Cry1Da_7 protein was bound by the anti-Cry1Da_7 mAb resin, whereas 

Cry1B.868 protein was bound by the anti-Cry1B.868 mAb resin (confirmed by western blot 

analysis). After sample load was completed, both columns were disconnected. The anti-Cry1Da_7 

mAb resin/column bound with Cry1Da_7 protein was used for next step Cry1Da_7 protein 

purification following the steps described earlier. The anti-Cry1B.868 mAb resin/column bound 

with Cry1B.868 protein was used for Cry1B.868 protein purification later. 

After 5 individual runs were completed, each final pooled protein sample (~200 ml) was 

concentrated and run buffer exchange to Storage Buffer for Cry1Da_7 (25 mM Sodium Carbonate, 

pH 10.5) using 4 x 15 ml Amicon Ultracel-30K centrifugal concentration device (Millipore, 

Tullagreen, Ireland), and further concentrated using 4 x 0.5 ml Amicon Ultracel-30K centrifugal 

concentration device (Millipore) at 4°C, to a final volume of 0.4 ml. All final protein samples after 

being concentrated and buffer exchanged were pooled together and that was the final Cry1Da-7 

sample. Buffer composition of the final sample was the same as Storage Buffer for Cry1Da_7. 

This Cry1Da_7 protein purified from the grain of MON 95379 was aliquoted, assigned lot 8206, 

and stored in a -80 °C freezer. 

D.2.3. Methods for Characterization 

D.2.3.1. N-Terminal Sequencing/Mass Fingerprint Analysis 

The test substance was analyzed for N-terminal amino acid sequence and peptide mass fingerprint 

by LC-MS/MS. An aliquot of the test substance was subjected to SDS-PAGE and proteins were 

visualized on the gel with InstantBlueTM (Expedeon, Cambridgeshire, UK) staining followed by 

de-staining with Milli-Q water. Gel bands containing Cry1Da_7 (~1 μg in each gel band) were 

excised, de-stained, incubated in ~200 μl of 100 mM ammonium bicarbonate for 30 min at room 

temperature (RT) for each gel band. The protein in each gel band was reduced by incubation in 

100 μl of 10 mM DTT at ~37°C for 1 hr, alkylated by incubation in the dark for 30 min at RT with 

100 μl of 20 mM iodoacetamide, and incubated in 200 μl of 25 mM ammonium bicarbonate for 

~15 min with agitation at RT for three times. Each gel band was dried down, rehydrated in 20 μl 

of 20 μg/ml trypsin protease solution and incubated at RT for 1 hr to allow the solution to diffuse 

into the gel band. After the excess liquid was removed, 40 μl of 25 mM ammonium 

bicarbonate/10% acetonitrile solution was added to each gel band and the reaction was incubated 

at 37°C for ~17 hr. After each tube was sonicated for 6 min, the supernatant was collected. The 
Bayer CropScience LP CR282-20U1 253 

digest was extracted with 30 μl of 70% acetonitrile/0.1% formic acid (FA) twice. The samples 

were combined and completely dried using vacuum centrifugation and then solubilized in 10 μl of 

0.1 % FA for LC-MS/MS analysis. 

The LC-MS/MS data acquisition was performed on a Dionex 3000 Ultimate nanoLC- system 

(Dionex) interfaced to an orbitrap fusion mass spectrometer (Thermo Scientific), which is 

equipped with a nano-ESI (electrospray ionization) source. The sample was loaded and separated 

online using an Acclaim PepMap100 C18 nano column (75 μm id × 150 mm, 2 μm, 100 Å, 

Dionex). The separation of the digests was achieved at a 300 nl/min flow rate using an acetonitrile 

gradient as follows: 0-4 min sustaining 2% solvent B, 4-80 min ramping solvent B 2-40%, 80-

85 min ramping solvent B 40-90%. The column was washed/equilibrated between injections as 

follows: 85-90 min maintaining solvent B at 90%, 90-90.1 min decreasing solvent B 90-2%, and 

90.1-100 min maintaining solvent B at 2%. Solvent A was 0.1% FA in water. Solvent B was 0.1% 

FA in acetonitrile. 

The orbitrap fusion mass spectrometer was operated with two scan events. The first scan event 

was a full Fourier transform mass spectrometry scan with a range of m/z from 200 to 1800 and a 

mass resolution of 120,000 at m/z of 200. The second scan event was a tandem mass spectrometry 

scan of fragments from collision induced dissociation (CID) of precursor ions from the first scan 

event with an isolation width of 2.0 m/z. 

The LC-MS/MS dataset were analyzed using Mascot (version 2.5, Matrix Science). MS/MS 

spectra were searched against the theoretical Cry1Da_7 protein sequence. The mass tolerances of 

MS1 mass and MS/MS mass were set as 5 ppm and 0.5 Da, respectively. Carboxymethylation of 

cysteine was set as a fixed modification. Methionine oxidation and N-terminal amino acid 

acetylation were set as variable modifications. A maximum of two missed cleavages for trypsin 

were allowed. The mass coverage was accepted if it is greater than 40% of the protein sequence. 

D.2.3.2. Western Blot Analysis-Immunoreactivity 

Western blot analysis was performed as follows to confirm the identity of the 

MON 95379-produced Cry1Da_7 protein and to compare the immunoreactivity of the 

MON 95379-produced and Bt-produced Cry1Da_7 protein. MON 95379-produced and 

Bt-produced Cry1Da_7 proteins were diluted to 2 ng/μl (purity-corrected intact Cry1Da_7 protein 

concentration) in 5 × LB and 6 M urea to obtain a final concentration of 1 × LB and heated to 

100°C for 5 min. Three amounts (~10, ~15, and ~20 ng) of the intact test substance (total protein 

concentration × purity of the intact Cry1Da_7 protein) and the intact reference substance (total 

protein concentration × purity of the intact Cry1Da_7 protein) were loaded in duplicate onto a 

pre-cast Tris-glycine 4-20% polyacrylamide mini-gel (Invitrogen). Pre-stained molecular weight 

standards (Precision Plus Protein StandardsTM, Bio-Rad) were loaded on the gel for molecular 

weight reference and to verify electrotransfer of the proteins to the membrane. Following 

electrophoresis at a constant voltage, proteins were electrotransferred to a nitrocellulose membrane 

(Bio-Rad). 

The western blotting procedure was performed using an iBindTM Western System apparatus (Life 

Technologies, Grand Island, NY). The membrane was blocked with 1 × iBindTM Solution (Life 
Bayer CropScience LP CR282-20U1 254 

Technologies) and incubated with goat anti-Cry1Da_7 polyclonal antibody (lot G1496) at a 

dilution of 1:1000 in 1 × iBindTM Solution. After washing with 1 × iBindTM Solution, the 

membrane was next incubated with a horseradish peroxidase (HRP)-conjugated anti-goat IgG 

(Vector Laboratories, Burlingame, CA) at a dilution of 1:2000 in 1 × iBindTM Solution and washed 

again with 1 × iBindTM Solution. Immunoreactive bands were visualized using the enhanced 

chemiluminescence (ECL) detection system (GE Healthcare) and exposed to HyperfilmTM ECL 

high performance chemiluminescence film (GE Healthcare). The film was developed using a 

Konica SRX-101A automated film processor (Konica, Tokyo, Japan). 

Image acquisition and quantification of the bands on the film were performed on a Bio-Rad 

GS-900 densitometer with the supplied Image Lab 5.0 Security Edition software using the volume 

tool. The signal intensities of the immunoreactive bands migrating at the expected position for the 

Cry1Da_7 protein were quantified as adjusted volume values. The immunoreactivity was reported 

in OD, which equals to the total sum of the intensities of the pixels inside the volume boundary. 

D.2.3.3. Apparent Molecular Weight and Purity Determination by SDS-PAGE 

MON 95379-produced and Bt-produced Cry1Da_7 proteins were diluted in 5 × loading buffer 

[LB, 0.31 M Tris-Cl, pH 7.5, 10% SDS, 50% glycerol, 25% (v/v) 2-mercaptoethanol, 0.025% 

(w/v) Bromophenol blue] and 6 M urea and heated to 95-105°C for 3-5 min. The MON 

95379-produced Cry1Da_7 protein was loaded in duplicate at ~1.0, ~2.0, and ~3.0 μg based on 

total protein concentration, onto a Tris-glycine 4-20% polyacrylamide mini-gel (Invitrogen, 

Carlsbad, CA) in lanes 3, 4, 5, 6, 7 and 8. The Bt-produced Cry1Da_7 protein was loaded at ~1.0 μg 

total protein in lane 2. Broad Range Molecular Weight Standards (Bio-Rad) were prepared and 

loaded in lanes 1 and 9 on the gel. Following electrophoresis at a constant voltage, proteins were 

briefly fixed in 40% (v/v) methanol, 7% (v/v) acetic acid and stained for 18 ± 2 hr with Brilliant 

Blue G-Colloidal stain (Sigma, St. Louis, MO). Gels were briefly destained in 10% (v/v) acetic 

acid, 25% (v/v) methanol followed by 8 ± 2 hr in 25% (v/v) methanol. Analysis of the gel was 

performed using a Bio-Rad GS-900 densitometer supplied with Image Lab 5.0 Security Edition 

software. The molecular weight of the test substance was calculated based on the mean of the 

molecular weight of the major band of Cry1Da_7 (~130 kDa) in all six lanes containing the test 

substance. The purity of the test substance was calculated based on multiple stained bands 

corresponding to the Cry1Da_7 protein including a major band (~130 kDa) and three minor bands 

(~120, ~110, and ~90 kDa) recognized by western blot analysis. The purity was reported as an 

average of all 6 lanes containing the MON 95379-produced Cry1Da_7 protein. 

D.2.3.4. Glycosylation Analysis 

An ECL glycoprotein detection method (GE Healthcare) was used for glycoprotein detection. The 

MON 95379-produced Cry1Da_7 protein and the Bt-produced Cry1Da_7 protein were diluted in 

5 × LB and 6 M urea while a positive control, transferrin (Sigma) was diluted in 5 × LB and water 

and then heated to 100.6°C for 5 min. Two amounts (~ 100 ng and ~ 200 ng) of the intact MON 

95379-produced Cry1Da_7 protein (purity-corrected), the intact Bt-produced Cry1Da_7 protein 

(purity-corrected), and the positive control were loaded onto a pre-cast Tris-glycine 4-20% 

polyacrylamide mini-gel (Invitrogen). Pre-stained molecular weight markers (Precision Plus 
Bayer CropScience LP CR282-20U1 255 

ProteinTM Standards, Bio-Rad) were also loaded for molecular weight reference and to verify 

electrotransfer of the proteins to the membrane. Following electrophoresis at a constant voltage, 

proteins were electrotransferred to a PVDF membrane (Invitrogen). 

Glycosylation analysis was performed on the PVDF membrane at room temperature using the ECL 

Glycoprotein Detection Module (GE Healthcare) as directed by the manufacturer. The labeled 

carbohydrate moieties were detected by addition of streptavidin conjugated to HRP followed by a 

luminol-based detection using ECL reagents (GE Healthcare) and exposure to HyperfilmTM (GE 

Healthcare). The film was developed using a Konica SRX-101A automated film processor 

(Konica Minolta). An identical gel was run and electrotransferred to a PVDF membrane in 

parallel. Proteins were stained with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad) 

and then destained with equivalent chemical reagents to 1u Coomassie Brilliant Blue R-250 

Destaining Solution (Bio-Rad). After washing with water, the blot was scanned using a Bio-Rad 

GS-900 densitometer with the supplied Image Lab 5.0 Security Edition software. 

D.2.3.5. Functional Activity 

Insects. Fall armyworm (FAW, Spodoptera frugiperda) is an appropriate test system due to its 

susceptibility to the Cry1Da_7 protein. FAW were obtained from Bayer (Union City, TN). Insect 

eggs were incubated at temperatures ranging from 10o C to 27o C, to achieve the desired hatch 

time. 

Bioassays. FAW larvae were used to measure the functional activity of the MON 95379-produced 

Cry1Da_7 and the Bt-produced Cry1Da_7 in accordance with ME-0044-04. The MON 95379-

produced Cry1Da_7 and the Bt-produced Cry1Da_7 substances were analyzed concurrently in 

each bioassay replicate. Bioassay replicates were initiated on different days, each using a separate 

batch of insects. Bioassay replicate one consisted of a series of six dilutions yielding a dose series 

with a two-fold separation factor ranging from 0.05 – 1.6 μg Cry1Da_7/ml diet for the MON 

95379-produced Cry1Da_7 and the Bt-produced Cry1Da_7, as well as, an assay (buffer) control. 

Bioassay replicates two and three consisted of a series of six dilutions yielding a dose series with 

a two-fold separation factor ranging from 0.025 – 0.8 μg Cry1Da_7/ml diet for the MON 95379-

produced Cry1Da_7 and the Bt-produced Cry1Da_7, as well as, an assay (buffer) control. Dosing 

solutions (5.0 ml) were prepared by diluting the protein sample with purified water and 

incorporating the dilution into an agar-based FAW diet (Southland, Village Lake, AR) to a final 

volume of 25 ml. Each diet mixture was dispensed in 1 mL aliquots into 16 wells of a 128-well 

tray (Frontier Agricultural Services). Each well was targeted to be infested with a single FAW 

larva (≤ 30 hours after first observation of hatching). Larvae were allowed to feed for a period of 

seven days in an environmental chamber programmed at 27 oC, 60% relative humidity, and a 

lighting regime of 14 hours light: 10 hours dark. The number of FAW larvae infested, the number 

of surviving larvae, and the combined mass of the surviving larvae were recorded for each 

treatment and concentration level at the end of the seven-day incubation period. 

The functional activity of the test substance and reference substance were characterized using a 

fall armyworm insect bioassay. The biological activity of the test substance and reference 

substance were measured as a 50% effective concentration (i.e. EC50 value). The EC50 value is 
Bayer CropScience LP CR282-20U1 256 

defined as the concentration that results in 50% growth inhibition. Logistic regression was used to 

estimate the EC50 values for growth inhibition for each bioassay. The insect bioassay was 

considered acceptable if less than or equal to 20% mortality was observed for the vehicle control 

and if >50% growth inhibition was observed relative to the vehicle control in the highest tested 

protein concentration level. 

D.2.3.6. Prediction Intervals as Acceptance Criteria 

Acceptance criteria (acceptance limits) based on prediction intervals were used to assess the 

equivalence of the MON 95379-produced and Bt-produced Cry1Da_7 proteins for apparent MW 

and functional activity. A prediction interval is an estimate of an interval in which a randomly 

selected future observation from a population will fall, with a certain degree of confidence, given 

what has already been observed; i.e., prediction intervals are generated based on statistical analysis 

of existing data. 

The source of the data used to generate the prediction intervals for apparent MW and functional 

activity for Cry1Da_7 protein equivalence assessment are provided in Table D-8 and Table D-9, 

respectively. The data used were generated under GLP guidelines and included apparent MW and 

functional activity estimates for the reference Bt-produced Cry1Da_7 protein. 

The two-sided 95% prediction interval (PI) for one future assay was calculated using JMP software 

(version 9.4, SAS Institute, Inc., Cary, NC) according to the guidance document of Ramírez 

(2009). 

 
Bayer CropScience LP CR282-20U1 257 

Table D-8. Individual Assay Data and 95% Prediction Interval Generated for the 

Apparent MW of Cry1Da_7 Protein for One Future Assay 

Assay Number1 Apparent MW of Cry1Da_7 Protein 

(kDa)2

1 128.645 

2 127.876 

3 127.103 

4 129.681 

5 123.739 

6 124.134 

7 122.535 

8 128.281 

Mean 126.5 

Standard Deviation 2.65 

95% Prediction Interval 119.9 – 133.1 

99% Prediction Interval 116.7 – 136.3 1

 Assay 1-8 represents the MW data from the initial characterization of Bt-produced Cry1Da_7 (lot 7799). 2

 The values in the table represent the mean of six (n = 6) data points within each assay. With 95% confidence, the 

mean of the 6 data points from the next single assay of the population will fall within the stated interval. Mean and 

prediction interval values rounded to one decimal point. 

 
Bayer CropScience LP CR282-20U1 258 

Table D-9. Estimated EC50 Values from 24 Dose-Response Diet Incorporation Insect 

Bioassays of the Cry1Da_7 Protein 

Assay Number1 Functional Activity of Cry1Da_7 

Protein (U/mg) 

1 0.135

2 0.112

3 0.169

4 0.127

5 0.135

6 0.141

7 0.132

8 0.167

9 0.250

10 0.156

11 0.184

12 0.125

13 0.200

14 0.118

15 0.092

16 0.165

17 0.179

18 0.095

19 0.197

20 0.194

21 0.226

22 0.141

23 0.122

24 0.141

Mean 0.149 

95% Prediction Interval 0.076 – 0.293 

99% Prediction Interval 0.064 – 0.351 1

 Assay 1-24 represents the functional activity from the characterization of Bt-produced Cry1Da_7 protein (lot 7799). 

 
Bayer CropScience LP CR282-20U1 259 

References for Appendix D 

Bradshaw, R. A., Brickey, W. W. and K. W. Walker. 1998. N-terminal processing: the methionine 

aminopeptidase and Nɑ -acetyl transferase families. Trends in Biochemical Sciences 23: 263-7. 

Rademacher, T.W., R.B. Parekh and R.A. Dwek. 1988. Glycobiology. Annual Review of 

Biochemistry 57: 785-838. 

Ramírez, J.G. 2009. Statistical Intervals: Confidence, Prediction, Enclosure. JMP White Paper 

(SAS Institute, Inc., Cary, NC). http://www.sas.com/reg/wp/corp/4430. 

Wang, C., K. C. Glenn, C. Kessenich, E. Bell, L.A. Burzio, M.S. Koch, B. Li and A. Silvanovich. 

2016. Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various 

crops. Regulatory Toxicology Pharmacology 81: 171-182. 
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Appendix E: Materials and Methods Used for the Analysis of the Levels of 

Cry1B.868 and Cry1Da_7 Proteins Expressed in MON 95379 

E.1. Materials 

OSL1, OSR1, OSL4, silk, pollen, forage, forage-root and grain tissue samples from five sites in 

MON 95379 were harvested from United States Field sites during the 2018 growing season from 

starting seed lot 11479279. ELISA Cry1B.868 (7565) and Cry1Da_7 (7799) was used as the 

analytical reference standard. 

E.2. Characterization of the Materials 

The identity of the test substance was confirmed by analysis of the starting seed DNA by an eventspecific polymerase chain reaction method. 

E.3. Field Design and Tissue Collection 

The test substance was planted in four replicated plots at five sites using randomized complete 

block design. 

E.4. Tissue Processing and Protein Extraction 

The Cry1B.868 and Cry1Da_7 proteins were extracted from tissue samples as described in Tables 

E-1 and E-2, respectively. 

 
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Table E-1. Cry1B.868 Extraction Parameter 1 

Sample Type Target Tissue to Buffer 

Ratio Extraction Buffer

OSL1 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

OSR1 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

OSL4 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Silk 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Pollen 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Forage 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Forage Root 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Grain 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

1

 Cry1B.868 protein was extracted by adding the appropriate volume of extraction buffer, metal beads, and shaking 

in a Geno Grinder (SPEX Inc., Metuchen, NJ). The extracted samples were clarified by centrifugation 

2

 100 mM Na2CO3/ 0.2% (v/v) Tween 20, 0.2% (v/v) N-laurylsarcosine Sodium salt/ 0.2% (w/v) ascorbic 

acid/0.2mM DTT/ 2 mM benzamidine/ 2mM PMSF 

 
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Table E-2. Cry1Da_7 Extraction Parameter 1 

Sample Type Target Tissue to Buffer 

Ratio Extraction Buffer

OSL1 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

OSR1 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

OSL4 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Silk 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Pollen 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Forage 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Forage Root 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

Grain 1:100 

100 mM Sodium Carbonate Extraction 

Solution2

1

 Cry1Da_7 protein was extracted by adding the appropriate volume of extraction buffer, metal beads, and shaking 

in a Geno Grinder (SPEX Inc., Metuchen, NJ). The extracted samples were clarified by centrifugation 

2

 100 mM Na2CO3/ 0.2% (v/v) Tween 20, 0.2% (v/v) N-laurylsarcosine Sodium salt/ 0.2% (w/v) ascorbic 

acid/0.2mM DTT/ 2 mM benzamidine/ 2mM PMSF 

 
Bayer CropScience LP CR282-20U1 263 

E.5. ELISA Reagents and Methods 

E.5.1. Cry1B.868 Antibodies 

Mouse anti- Cry1B.868 monoclonal antibody (clone 9E8; lot 658272A) is used as the capture 

antibody. This antibody was purified using Protein G affinity chromatography and stored in 

phosphate buffered saline, pH 7.2. The concentration of the purified antibody was determined to 

be 3.21 mg/mL by spectrophotometric methods. 

Mouse anti- Cry1B.868 antibody (clone 3B8; lot 634038) was purified using Protein G affinity 

chromatography. The purified antibodies were coupled with biotin (Thermo Fisher Scientific), 

according to the manufacturer's instructions and stored in phosphate buffered saline, pH 7.2. 

The detection reagent was NeutrAvidin conjugated to horseradish peroxidase (Thermo Fisher 

Scientific). 

E.5.2. Cry1Da_7 Antibodies 

Mouse anti-Cry1Da_7 monoclonal antibody (clone 5231-6; lot 617720) is used as the capture 

antibody. This antibody was purified using Protein A affinity chromatography and stored in 

phosphate buffered saline, pH 7.2. The concentration of the purified antibody was determined to 

be 3.59 mg/mL by spectrophotometric methods. 

Protein A affinity chromatography purified monoclonal antibody (mouse anti-Cry1Da_7, (clone 

5231-18; lot 617506) was coupled with biotin (Thermo Fisher Scientific), per the manufacturer’s 

instructions and stored in phosphate buffered saline, pH 7.2. 

The detection reagent was NeutrAvidin conjugated to horseradish peroxidase (Thermo Fisher 

Scientific). 

E.5.3. Cry1B.868 ELISA Method 

The Cry1B.868 ELISA was performed according to the current version of method 

ME-2206. Mouse anti-Cry1B.868 capture antibody, was diluted in a coating buffer (0.015 M 

Na2CO3 and 0.035 M NaHCO3 with 150mM NaCl) to 3 μg/ml and immobilized onto microtiter 

plates followed by incubation in a 4oC refrigerator for ≥8 h. Prior to each step in the assay, plates 

were washed with 1X phosphate buffered saline containing 0.05% (v/v) Tween 20. Cry1B.868 

protein standard or sample extract diluted in assay buffer (0.1% (w/v) bovine serum albumin in 

1X phosphate buffered saline containing 0.05% (v/v) Tween 20) was added incubated for 60 ± 10 

minutes at 37 oC. Biotinylated mouse anti-Cry1B.868 was added and incubated for 60 ± 10 

minutes at 37 oC. NeutrAvidin-horseradish peroxidase conjugate was added and incubated for 60 

± 10 minutes at 37 oC. Plates were developed by adding horseradish peroxidase substrate, 

3,3',5,5'- tetramethylbenzidine (TMB, Sigma-Aldrich). The enzymatic reaction was terminated by 

the addition of 6 M H3PO4. Quantification of Cry1B.868 protein was accomplished by 

interpolation from a Cry1B.868 protein standard curve that ranged from 0.625 – 20 ng/ml. 
Bayer CropScience LP CR282-20U1 264 

E.5.4. Cry1Da_7 ELISA Method 

The Cry1Da_7 ELISA was performed per the current version of method ME-2203. The capture 

antibody, mouse anti-Cry1Da_7 monoclonal antibody, was diluted in a coating buffer (0.015 M 

Na2CO3 and 0.035 M NaHCO3 with 150mM NaCl) and immobilized onto a microtiter plate at 4 

μg/ml followed by incubation in a 4oC refrigerator for ≥8 h. Prior to each step in the assay, plates 

were washed with 1X phosphate buffered saline containing 0.05% (v/v) Tween 20. Cry1Da_7 

protein standard or sample extract diluted in assay buffer (0.1% (w/v) bovine serum albumin in 

1X phosphate buffered saline containing 0.05% (v/v) Tween 20) was added to the plate and 

incubated for 60 ± 10 minutes at 37 oC. Biotinylated mouse anti-Cry1Da_7 monoclonal antibody 

was added to the plate and incubated for 60 ± 10 minutes at 37 oC. NeutrAvidin-horseradish 

peroxidase conjugate was added at to the plate and incubated for 60 ± 10 minutes at 37 oC. Plates 

were developed by adding horseradish peroxidase substrate, 3,3',5,5'- tetramethylbenzidine (TMB, 

Sigma-Aldrich). The enzymatic reaction was terminated by the addition 6 M H3PO4. 

Quantification of Cry1Da_7 protein was accomplished by interpolation from a Cry1Da_7 protein 

standard curve that ranged from 0.250 – 8 ng/ml. 

E.6. Data Analyses 

Cry1B.868 and Cry1Da_7 ELISA plates were analyzed on a SPECTRAmax Plus 384 (Molecular 

Devices) microplate spectrophotometer, using a dual wavelength detection method. Protein 

concentrations were determined by optical absorbance at a wavelength of 450 nm with a 

simultaneous reference reading of 620 nm. Data reduction analyses were performed using 

Molecular Devices SOFTmax PRO GxP software. Absorbance readings and protein standard 

concentrations were fitted with a five-parameter curve fit. 

Following the interpolation from the standard curve, for data that were determined to be greater 

than or equal to the LOQ, the protein levels (ng/ml) in the tissues were converted to a μg/g dw 

value. For each protein, this conversion utilized a sample dilution factor and a tissue-to-buffer ratio 

and an extraction efficiency correction factor. All values were also converted to a μg/g fw using 

moisture conversion factor (MCF) (Table E-3). 
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Table E-3. Summary of the Tissue-Specific Moisture Conversion Factors1

Tissue 

Type N Average MCF Standard Deviation

OSL1 56 0.15 0.02 

OSR1 60 0.11 0.02 

OSL4 31 0.23 0.03 

Silk 21 0.10 0.01 

Pollen 56 0.57 0.11 

Forage 51 0.30 0.04 

Forage Root 55 0.19 0.04 

Grain 56 0.88 0.02 

1

 Universal moisture conversion factor for specific tissues based on historical data for the 

estimation of fw protein expression levels from protein expression levels reported on μg/g dw basis.

Core LIMS (Core Informatics) and its supporting tools were used to calculate the protein levels in 

maize tissues. The sample means, standard errors (SEs), and ranges were calculated by R code. 

All protein expression levels were rounded to two significant figures 

 
Bayer CropScience LP CR282-20U1 266 

Appendix F: Digestive Fate of the Cry1B.868 and Cry1Da_7 Proteins 

F.1. Digestive Fate of the Cry1B.868 and Cry1Da_7 Proteins 

As part of a comprehensive evaluation, introduced proteins in biotechnology-derived crops are 

evaluated for their safety for human and animal consumption. Proteins are an essential dietary 

component for humans and animals, and most are rapidly degraded to the component amino acids 

for nutritional purposes (Hammond and Jez, 2011). Although the vast majority of ingested 

proteins are non-allergenic, a small set of proteins or their fragments have been associated with a 

variety of gastrointestinal and systemic manifestations of immune-mediated allergy. One 

characteristic of several food allergens is their ability to withstand proteolytic digestion by 

enzymes present in the gastrointestinal tract (Astwood et al., 1996; Moreno et al., 2005; 

Vassilopoulou et al., 2006; Vieths et al., 1999), although exceptions to the correlation can be 

identified (Fu et al., 2002). The enzymatic degradation of an ingested protein by exposure to 

gastric pepsin and/or intestinal pancreatic proteases (e.g., pancreatin) makes it highly unlikely that 

either the intact protein or protein fragment(s) will reach the absorptive epithelial cells of the small 

intestine where antigen processing cells reside (Moreno et al., 2005). Therefore, the susceptibility 

of Cry1B.868 and Cry1Da_7 to the presence of pepsin was evaluated using an assay protocol that 

has been standardized based on results obtained from an international, multi-laboratory ring study 

(Thomas et al., 2004). The susceptibility of proteins in the presence of pancreatin has also been 

used as a separate test system to assess the digestibility of food components (Okunuki et al., 2002; 

Yagami et al., 2000). 

F.1.1. Degradation of Cry1B.868 and Cry1Da_7 Proteins in the Presence of Pepsin 

Degradation of the Cry1B.868 and Cry1Da_7 proteins by pepsin was evaluated over time by 

analyzing digestion mixtures incubated for targeted time intervals following a standardized 

protocol validated in an international, multi-laboratory ring study (Thomas et al., 2004) collected 

at targeted incubation time points. The specific methods used to assess the digestive 

fate/degradation of the Cry1B.868 and Cry1Da_7 proteins in pepsin are summarized below and 

detailed in Appendix F.2. The study showed that the results of in vitro pepsin digestion assays 

using this protocol were reproducible and consistent for determining the digestive susceptibility of 

a protein. This standardized in vitro pepsin digestion protocol utilized a physiologically relevant 

acidic buffer appropriate for pepsin activity. The susceptibility of Cry1B.868 and Cry1Da_7 

proteins to pepsin degradation was assessed by visual analysis of a Brilliant Blue G Colloidal 

stained SDS-PAGE gel and by visual analysis of a western blot probed with anti-Cry1B.868 or 

anti-Cry1Da_7 polyclonal antibodies. Both visualization methods were run concurrently with 

separate SDS-PAGE and western blot analyses to estimate the limit of detection (LOD) of the 

Cry1B.868 and Cry1Da_7 proteins for each method. 

 
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F.1.1.1. Degradation of Cry1B.868 Protein in the Presence of Pepsin 

For SDS-PAGE analysis of the digestibility of the Cry1B.868 protein in pepsin, the gel was loaded 

with 1 μg of total test protein (based on pre-digestion protein concentrations) for each of the 

digestion samples (Figure F-1, Panel A). The SDS-PAGE gel for the digestibility assessment was 

run concurrently with a separate SDS-PAGE gel to estimate the LOD of the Cry1B.868 protein 

(Figure F-1, Panel B). The LOD of intact Cry1B.868 protein was approximately 1.6 ng (Figure F1, Panel B, lane 9). Visual examination of SDS-PAGE data showed that the intact Cry1B.868 

protein was digested within 0.5 min of incubation in pepsin (Figure F-1, Panel A, lane 5). 

Therefore, based on the LOD, more than 99.8% (100% - 0.2% = 99.8%) of the intact Cry1B.868 

protein was digested within 0.5 min of incubation in pepsin. Transiently-stable peptide fragments 

at ~4-kDa were observed throughout the course of the digestion. 

No change in the Cry1B.868 protein band intensity was observed in the absence of pepsin in the 0 

min No Pepsin Control and 60 min No Pepsin Control (Figure F-1, Panel A, lanes 3 and 12). This 

indicates that the degradation of the Cry1B.868 protein was due to the proteolytic activity of pepsin 

and not due to instability of the protein while incubated in 10 mM HCl, 2 mg/ml NaCl, pH ~1.2 

for 60 min. 

The 0 min No Test Protein Control and 60 min No Test Protein Control (Figure F-1, Panel A, lanes 

2 and 13) demonstrated that the pepsin is stable throughout the experimental phase. 

 
Bayer CropScience LP CR282-20U1 268 

 

Figure F-1. SDS-PAGE Analysis of the Degradation of Cry1B.868 Protein by Pepsin 

Colloidal Brilliant Blue G stained SDS-PAGE gels were used to assess the degradation of Cry1B.868 

protein by pepsin. Molecular weights (kDa) are shown on the left of each gel and correspond to the markers 

loaded. In each gel, the Cry1B.868 protein migrated to approximately 129.6 kDa and pepsin to 

approximately 38 kDa. Empty lanes and molecular weight markers that were not visible on the film were 

cropped from the images.

A: Cry1B.868 protein degradation in the presence of pepsin. Based on pre-reaction protein concentrations, 

1 μg of test protein was loaded in each lane containing Cry1B.868 protein. 

B: LOD determination. Indicated amounts of the test protein from the Pepsin Treated T0 sample were 

loaded to estimate the LOD of the Cry1B.868 protein. 

A B 

Lane Sample Incubation 

Time (min) Lane Sample Amount (ng) 

1 Mark12 MWM - 1 Mark12 MWM - 

2 0 min No Test Protein Control 0 2 Pepsin Treated T0 200 

3 0 min No Pepsin Control 0 3 Pepsin Treated T0 100 

4 Pepsin Treated T0 0 4 Pepsin Treated T0 50 

5 Pepsin Treated T1 0.5 5 Pepsin Treated T0 25 

6 Pepsin Treated T2 2 6 Pepsin Treated T0 12.5 

7 Pepsin Treated T3 5 7 Pepsin Treated T0 6.25 

8 Pepsin Treated T4 10 8 Pepsin Treated T0 3.1 

9 Pepsin Treated T5 20 9 Pepsin Treated T0 1.6 

10 Pepsin Treated T6 30 10 Pepsin Treated T0 0.8 

11 Pepsin Treated T7 60 11 Pepsin Treated T0 0.4 

12 60 min No Pepsin Control 60 12 Mark12 MWM - 

13 60 min No Test Protein Control 60 13 Empty - 

14 Mark12 MWM - 14 Empty - 

15 Empty - 15 Empty - 

For western blot analysis of Cry1B.868 pepsin susceptibility, the Cry1B.868 protein was loaded 

with approximately 40 ng per lane of total protein (based on pre-reaction total protein 

concentrations) for each reaction time point examined. The western blot used to assess Cry1B.868 

protein degradation (Figure F-2, Panel A) was run concurrently with the western blot used to 
Bayer CropScience LP CR282-20U1 269 

estimate the LOD (Figure F-2, Panel B). The LOD of the Cry1B.868 protein was approximately 

0.63 ng (Figure F-2, Panel B, Lane 9). Western blot analysis demonstrated that the intact 

Cry1B.868 protein was degraded below the LOD within 0.5 min of incubation in the presence of 

pepsin (Figure F-2, Panel A, Lane 6). Based on the western blot LOD for the Cry1B.868 protein, 

more than 98.4% (100% - 1.6% = 98.4%) of the intact Cry1B.868 protein was degraded within 0.5 

min. No peptide fragments were detected at the 0.5 min and beyond time points in the western blot 

analysis. 

No change in the Cry1B.868 protein band intensity was observed in the absence of pepsin in the 0 

min No Pepsin Control and 60 min No Pepsin Control (Figure F-2, Panel A, lanes 3 and 12). This 

indicates that the degradation of the Cry1B.868 protein was due to the proteolytic activity of pepsin 

and not due to instability of the protein while incubated in 2 mg/ml NaCl, 10 mM HCl, pH ~1.2 

for 60 min. The transiently-stable fragments at ~4 kDa that were observed by SDS-PAGE were 

not recognized by the antibody used in this western blot. 

No immunoreactive bands were observed in 0 min No Protein Control and 60 min No Protein 

Control (Figure F-2, Panel A, lanes 3 and 14). This result indicates that there was no non-specific 

interaction between the pepsin solution and the Cry1B.868-specific antibody under these 

experimental conditions. 

 
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Figure F-2. Western Blot Analysis of the Degradation of Cry1B.868 Protein by Pepsin 

Western blots probed with an anti-Cry1B.868 antibody were used to assess the degradation of 

Cry1B.868 by pepsin. Molecular weights (kDa) are shown on the left of each gel and correspond 

to the MagicMarkTM molecular weight marker. Empty lanes and molecular weight markers that 

were not visible on the film were cropped from the images. A 1.5 min-exposure is shown. 

A: Cry1B.868 protein degradation by pepsin. Based on pre-reaction protein concentrations, 40 ng 

of test protein was loaded in each lane containing Cry1B.868 protein. 

B: LOD determination. Indicated amounts of the test protein from the Pepsin Treated T0 sample 

were loaded to estimate the LOD of the Cry1B.868 protein. 

A B 

Lane Sample Incubation 

Time (min) Lane Sample Amount (ng) 

1 Precision Plus MWM - 1 Precision Plus MWM - 

2 MagicMark MWM - 2 MagicMark MWM - 

3 0 min No Test Protein Control 0 3 Pepsin Treated T0 40 

4 0 min No Pepsin Control 0 4 Pepsin Treated T0 20 

5 Pepsin Treated T0 0 5 Pepsin Treated T0 10 

6 Pepsin Treated T1 0.5 6 Pepsin Treated T0 5 

7 Pepsin Treated T2 2 7 Pepsin Treated T0 2.5 

8 Pepsin Treated T3 5 8 Pepsin Treated T0 1.25 

9 Pepsin Treated T4 10 9 Pepsin Treated T0 0.63 

10 Pepsin Treated T5 20 10 Pepsin Treated T0 0.31 

11 Pepsin Treated T6 30 11 Pepsin Treated T0 0.16 

12 Pepsin Treated T7 60 12 Pepsin Treated T0 0.08 

13 60 min No Pepsin Control 60 13 MagicMark MWM - 

14 60 min No Test Protein Control 60 14 Precision Plus MWM - 

15 MagicMark MWM - 15 Empty - 

 
Bayer CropScience LP CR282-20U1 271 

F.1.1.2. Degradation of Cry1Da_7 Protein in the Presence of Pepsin 

For SDS-PAGE analysis of the digestibility of the Cry1Da_7 protein in pepsin, the gel was loaded 

with 1 μg of total test protein (based on pre-digestion protein concentrations) for each of the 

digestion samples (Figure F-3, Panel A). The SDS-PAGE gel for the digestibility assessment was 

run concurrently with a separate SDS-PAGE gel to estimate the LOD of the Cry1Da_7 protein 

(Figure F-3, Panel B). The LOD of intact Cry1Da_7 protein was approximately 6.3 ng (Figure F3, Panel B, lane 8). Visual examination of SDS-PAGE data showed that the intact Cry1Da_7 

protein was digested within 0.5 min of incubation in pepsin (Figure F-3, Panel A, lane 5). 

Therefore, based on the LOD, more than 99.4% (100% - 0.6% = 99.4%) of the intact Cry1Da_7 

protein was digested within 0.5 min of incubation in pepsin. Transiently-stable peptide fragments 

at ~4-kDa were observed throughout the course of the digestion. 

No change in the Cry1Da_7 protein band intensity was observed in the absence of pepsin in the 0 

min No Pepsin Control and 60 min No Pepsin Control (Figure F-3, Panel A, lanes 3 and 12). This 

indicates that the degradation of the Cry1Da_7 protein was due to the proteolytic activity of pepsin 

and not due to instability of the protein while incubated in 10 mM HCl, 2 mg/ml NaCl, pH ~1.2 

for 60 min. 

The 0 min No Test Protein Control and 60 min No Test Protein Control (Figure F-3, Panel A, lanes 

2 and 13) demonstrated that the pepsin is stable throughout the experimental phase. 

 
Bayer CropScience LP CR282-20U1 272 

Figure F-3. SDS-PAGE Analysis of the Degradation of Cry1Da_7 Protein by Pepsin 

Colloidal Brilliant Blue G stained SDS-PAGE gels were used to assess the degradation of Cry1Da_7 protein 

by pepsin. Molecular weights (kDa) are shown on the left of each gel and correspond to the markers loaded. 

In each gel, the Cry1Da_7 protein migrated to approximately 126.5 kDa and pepsin to approximately 38 

kDa. Empty lanes and molecular weight markers that were not visible on the film were cropped from the 

images.

A: Cry1Da_7 protein degradation in the presence of pepsin. Based on pre-reaction protein concentrations, 

1 μg of test protein was loaded in each lane containing Cry1Da_7 protein. 

B: LOD determination. Indicated amounts of the test protein from the Pepsin Treated T0 sample were 

loaded to estimate the LOD of the Cry1Da_7 protein. 

A B 

Lane Sample Incubation 

Time (min) Lane Sample Amount (ng) 

1 Mark12 MWM - 1 Mark12 MWM - 

2 0 min No Test Protein Control 0 2 Pepsin Treated T0 400 

3 0 min No Pepsin Control 0 3 Pepsin Treated T0 200 

4 Pepsin Treated T0 0 4 Pepsin Treated T0 100 

5 Pepsin Treated T1 0.5 5 Pepsin Treated T0 50 

6 Pepsin Treated T2 2 6 Pepsin Treated T0 25 

7 Pepsin Treated T3 5 7 Pepsin Treated T0 12.5 

8 Pepsin Treated T4 10 8 Pepsin Treated T0 6.3 

9 Pepsin Treated T5 20 9 Pepsin Treated T0 3.1 

10 Pepsin Treated T6 30 10 Pepsin Treated T0 1.6 

11 Pepsin Treated T7 60 11 Pepsin Treated T0 0.8 

12 60 min No Pepsin Control 60 12 Pepsin Treated T0 0.4 

13 60 min No Test Protein Control 60 13 Mark12 MWM - 

14 Mark12 MWM - 14 Empty - 

15 Empty - 15 Empty - 

 
Bayer CropScience LP CR282-20U1 273 

For western blot analysis of Cry1Da_7 pepsin susceptibility, the Cry1Da_7 protein was loaded 

with approximately 40 ng per lane of total protein (based on pre-reaction total protein 

concentrations) for each reaction time point examined. The western blot used to assess Cry1Da_7 

protein degradation (Figure F-4, Panel A) was run concurrently with the western blot used to 

estimate the LOD (Figure F-4, Panel B). The LOD of the Cry1Da_7 protein was approximately 

1.25 ng (Figure F-4, Panel B, Lane 7). Western blot analysis demonstrated that the intact 

Cry1Da_7 protein was degraded below the LOD within 0.5 min of incubation in the presence of 

pepsin (Figure F-4, Panel A, Lane 6). Based on the western blot LOD for the Cry1Da_7 protein, 

more than 96.9% (100% - 3.1% = 96.9%) of the intact Cry1Da_7 protein was degraded within 0.5 

min. No peptide fragments were detected at the 0.5 min and beyond time points in the western 

blot analysis. 

No change in the Cry1Da_7 protein band intensity was observed in the absence of pepsin in the 0 

min No Pepsin Control and 60 min No Pepsin Control (Figure F-4, Panel A, lanes 3 and 12). This 

indicates that the degradation of the Cry1Da_7 protein was due to the proteolytic activity of pepsin 

and not due to instability of the protein while incubated in 2 mg/ml NaCl, 10 mM HCl, pH ~1.2 

for 60 min. The transiently-stable fragments at ~4 kDa that were observed by SDS-PAGE were 

not recognized by the antibody used in this western blot. 

No immunoreactive bands were observed in 0 min No Protein Control and 60 min No Protein 

Control (Figure F-4, Panel A, lanes 3 and 14). This result indicates that there was no non-specific 

interaction between the pepsin solution and the Cry1Da_7-specific antibody under these 

experimental conditions. 

 
Bayer CropScience LP CR282-20U1 274 

Figure F-4. Western Blot Analysis of the Degradation of Cry1Da_7 Protein by Pepsin 

Western blots probed with an anti-Cry1Da_7 antibody were used to assess the degradation of Cry1Da_7 by 

pepsin. Molecular weights (kDa) are shown on the left of each gel and correspond to the MagicMarkTM

molecular weight marker. Empty lanes and molecular weight markers that were not visible on the film 

were cropped from the images. A 1.5 min exposure is shown. 

A: Cry1Da_7 protein degradation by pepsin. Based on pre-reaction protein concentrations, 40 ng of test 

protein was loaded in each lane containing Cry1Da_7 protein. 

B: LOD determination. Indicated amounts of the test protein from the Pepsin Treated T0 sample were 

loaded to estimate the LOD of the Cry1Da_7 protein. 

A B 

Lane Sample Incubation 

Time (min) Lane Sample Amount (ng) 

1 Precision Plus MWM - 1 Precision Plus MWM - 

2 MagicMark MWM - 2 MagicMark MWM - 

3 0 min No Test Protein Control 0 3 Pepsin Treated T0 20 

4 0 min No Pepsin Control 0 4 Pepsin Treated T0 10 

5 Pepsin Treated T0 0 5 Pepsin Treated T0 5 

6 Pepsin Treated T1 0.5 6 Pepsin Treated T0 2.5 

7 Pepsin Treated T2 2 7 Pepsin Treated T0 1.25 

8 Pepsin Treated T3 5 8 Pepsin Treated T0 0.63 

9 Pepsin Treated T4 10 9 Pepsin Treated T0 0.31 

10 Pepsin Treated T5 20 10 Pepsin Treated T0 0.16 

11 Pepsin Treated T6 30 11 Pepsin Treated T0 0.08 

12 Pepsin Treated T7 60 12 MagicMark MWM - 

13 60 min No Pepsin Control 60 13 Precision Plus MWM - 

14 60 min No Test Protein Control 60 14 Empty - 

15 MagicMark MWM - 15 Empty - 

 
Bayer CropScience LP CR282-20U1 275 

F.1.2. Degradation of Cry1B.868 and Cry1Da_7 Proteins in the Presence of Pancreatin 

F.1.2.1. Degradation of Cry1B.868 Protein in the Presence of Pancreatin 

The degradation of the Cry1B.868 protein by pancreatin was assessed by western blot analysis 

(Figure F-5). The western blot used to assess the Cry1B.868 protein degradation (Figure F-5, 

Panel A) was run concurrently with the western blot used to estimate the LOD (Figure F-5, Panel 

B) of the Cry1B.868 protein. The LOD of the Cry1B.868 protein was observed at approximate 

0.16 ng protein loading (Figure F-5, Panel B, lane 11). The LOD was used to calculate the 

maximum relative amount of Cry1B.868 protein that could remain visually undetected after 

digestion, which corresponded to approximately 0.4% of the total protein loaded. 

The Cry1B.868 protein was loaded with approximately 40 ng per lane of total protein (based on 

pre-reaction total protein concentrations) for each reaction time point examined. Western blot 

analysis demonstrated that the intact Cry1B.868 protein was degraded below the LOD within 5 

min of incubation in the presence of pancreatin (Figure F-5, Panel A, lane 5). Based on the western 

blot LOD for the Cry1B.868 protein, more than 99.6% (100% - 0.4% = 99.6%) of the intact 

Cry1B.868 protein was degraded within 5 min. Bands of ~60 kDa corresponding to fragments of 

Cry1B.868 were present over the course of the assessment (Figure F-5, Panel A, lanes 5-12). The 

Cry1B.868 demonstrated typical characteristics that are present in many other Cry proteins, such 

as Cry1A.105 (Wang et al 2018, USDA-APHIS Petition #13-337-01p p75) and Cry2Ab2 (USDAAPHIS Petition #13-337-01p p75). 

No obvious change in the intact Cry1B.868 (~129.6 kDa) band intensity was observed in the 

absence of pancreatin in the 0 min No Pancreatin Control and 24 hour No Pancreatin Control 

(Figure F-5, Panel A, lanes 3 and 13). This indicates that the degradation of all immunoreactive 

forms of the Cry1B.868 protein was due to the proteolytic activity of pancreatin and not due to 

instability of the protein when incubated in 50 mM KH2PO4, pH 7.5 over the course of the 

experiment. 

No immunoreactive bands were observed in the 0 min No Test Protein Control and 24 hour No 

Test Protein Control (Figure F-5, Panel A, lanes 2 and 14), demonstrating the absence of nonspecific antibody interactions with the pancreatin solution.

 
Bayer CropScience LP CR282-20U1 276 

 

Figure F-5. Western Blot Analysis of the Degradation of Cry1B.868 Protein by Pancreatin 

Western blots probed with an anti-Cry1B.868 antibody were used to assess the degradation of Cry1B.868 

by pancreatin. Molecular weights (kDa) are shown on the left of each gel and correspond to the 

MagicMarkTM molecular weight marker. Empty lanes and molecular weight markers that were not visible 

on the film were cropped from the images. A 1.0 min-exposure is shown.

A: Cry1B.868 protein degradation by pancreatin. Based on pre-reaction protein concentrations, 40 ng of 

test protein was loaded in each lane containing Cry1B.868 protein. 

B: LOD determination. Indicated amounts of the test protein from the Pancreatin Treated T0 sample were 

loaded to estimate the LOD of the Cry1B.868 protein. 

A B 

Lane Sample Incubation 

Time Lane Sample Amount (ng) 

1 MagicMark MWM - 1 Precision Plus MWM - 

2 0 min No Test Protein Control 0 2 MagicMark MWM - 

3 0 min No Pancreatin Control 0 3 Pancreatin Treated T0 40 

4 Pancreatin Treated T0 0 4 Pancreatin Treated T0 20 

5 Pancreatin Treated T1 5 min 5 Pancreatin Treated T0 10 

6 Pancreatin Treated T2 15 min 6 Pancreatin Treated T0 5 

7 Pancreatin Treated T3 30 min 7 Pancreatin Treated T0 2.5 

8 Pancreatin Treated T4 1 h 8 Pancreatin Treated T0 1.25 

9 Pancreatin Treated T5 2 h 9 Pancreatin Treated T0 0.63 

10 Pancreatin Treated T6 4 h 10 Pancreatin Treated T0 0.31 

11 Pancreatin Treated T7 8 h 11 Pancreatin Treated T0 0.16 

12 Pancreatin Treated T8 24 h 12 Pancreatin Treated T0 0.08 

13 24 h No Pancreatin Control 24 h 13 MagicMark MWM - 

14 24 h No Test Protein Control 24 h 14 Precision Plus MWM - 

15 Precision Plus MWM - 15 Empty - 

 
Bayer CropScience LP CR282-20U1 277 

F.1.2.2. Degradation of Cry1Da_7 Protein in the Presence of Pancreatin 

The degradation of the Cry1Da_7 protein by pancreatin was assessed by western blot analysis 

(Figure F-6). The western blot used to assess the Cry1Da_7 protein degradation (Figure F-6, Panel 

A) was run concurrently with the western blot used to estimate the LOD (Figure F-6, Panel B) of 

the Cry1Da_7 protein. The LOD of the Cry1Da_7 protein was observed at approximate 1.25 ng 

protein loading (Figure F-6, Panel B, lane 7). The LOD was used to calculate the maximum 

relative amount of Cry1Da_7 protein that could remain visually undetected after digestion, which 

corresponded to approximately 3.1% of the total protein loaded. 

The Cry1Da_7 protein was loaded with approximately 40 ng per lane of total protein (based on 

pre-reaction total protein concentrations) for each reaction time point examined. Western blot 

analysis demonstrated that the intact Cry1Da_7 protein was degraded below the LOD within 5 min 

of incubation in the presence of pancreatin (Figure F-6, Panel A, lane 5). Based on the western 

blot LOD for the Cry1Da_7 protein, more than 96.9% (100% - 3.1% = 96.9%) of the intact 

Cry1Da_7 protein was degraded within 5 min. A band of ~60 kDa corresponding to a fragment 

of Cry1Da_7 was present over the course of the assessment (Figure F-6, Panel A, lanes 5-12). The 

Cry1Da_7 demonstrated typical characteristics that are present in many other Cry proteins, such 

as Cry1A.105 (Wang et al 2018, USDA-APHIS Petition #13-337-01p p75) and Cry2Ab2 (USDAAPHIS Petition #13-337-01p p75). 

No obvious change in the intact Cry1Da_7 (~126.5 kDa) band intensity was observed in the 

absence of pancreatin in the 0 min No Pancreatin Control and 24 hour No Pancreatin Control 

(Figure F-6, Panel A, lanes 3 and 13). This indicates that the degradation of all immunoreactive 

forms of the Cry1Da_7 protein was due to the proteolytic activity of pancreatin and not due to 

instability of the protein when incubated in 50 mM KH2PO4, pH 7.5 over the course of the 

experiment. 

No immunoreactive bands were observed in the 0 min No Test Protein Control and 24 hour No 

Test Protein Control (Figure F-6, Panel A, lanes 2 and 14), demonstrating the absence of nonspecific antibody interactions with the pancreatin solution.

 
Bayer CropScience LP CR282-20U1 278 

Figure F-6. Western Blot Analysis of the Degradation of Cry1Da_7 Protein by Pancreatin 

Western blots probed with an anti-Cry1Da_7 antibody were used to assess the degradation of Cry1Da_7 by 

pancreatin. Molecular weights (kDa) are shown on the left of each gel and correspond to the MagicMarkTM

molecular weight marker. Empty lanes and molecular weight markers that were not visible on the film 

were cropped from the images. A 1.5 min exposure is shown.

A: Cry1Da_7 protein degradation by pancreatin. Based on pre-reaction protein concentrations, 40 ng of 

test protein was loaded in each lane containing Cry1Da_7 protein. 

B: LOD determination. Indicated amounts of the test protein from the Pancreatin Treated T0 sample were 

loaded to estimate the LOD of the Cry1Da_7 protein. 

A B 

Lane Sample Incubation 

Time Lane Sample Amount (ng) 

1 MagicMark MWM - 1 Precision Plus MWM - 

2 0 min No Test Protein Control 0 2 MagicMark MWM - 

3 0 min No Pancreatin Control 0 3 Pancreatin Treated T0 20 

4 Pancreatin Treated T0 0 4 Pancreatin Treated T0 10 

5 Pancreatin Treated T1 5 min 5 Pancreatin Treated T0 5 

6 Pancreatin Treated T2 15 min 6 Pancreatin Treated T0 2.5 

7 Pancreatin Treated T3 30 min 7 Pancreatin Treated T0 1.25 

8 Pancreatin Treated T4 1 h 8 Pancreatin Treated T0 0.63 

9 Pancreatin Treated T5 2 h 9 Pancreatin Treated T0 0.31 

10 Pancreatin Treated T6 4 h 10 Pancreatin Treated T0 0.16 

11 Pancreatin Treated T7 8 h 11 Pancreatin Treated T0 0.08 

12 Pancreatin Treated T8 24 h 12 MagicMark MWM - 

13 24 h No Pancreatin Control 24 h 13 Precision Plus MWM - 

14 24 h No Test Protein Control 24 h 14 Empty - 

15 Precision Plus MWM - 15 Empty - 

 
Bayer CropScience LP CR282-20U1 279 

F.1.3. Degradation of Cry1B.868 and Cry1Da_7 Proteins by Pepsin Followed by Pancreatin 

To better understand the fate of the transiently-stable peptide fragments at ~4 kDa that were 

observed in the reaction mixtures throughout the course of the pepsin digestion of Cry1B.868 and 

Cry1Da_7, sequential digestibility of the Cry1B.868 and Cry1Da_7 proteins were conducted. This 

sequential digestibility was assessed both by visual analysis of a Colloidal Brilliant Blue G stained 

SDS-PAGE gel, and visual analysis of a western blot probed with an anti Cry1B.868-polyclonal 

and Cry1Da_7-polyclonal antibodies.

F.1.3.1. Degradation of Cry1B.868 Protein by Pepsin Followed by Pancreatin 

For the sequential degradation assay, the Cry1B.868 protein was incubated with pepsin for 2 min, 

followed by incubation with pancreatin. For the Colloidal Brilliant Blue G stained SDS-PAGE 

assessment, the gel was loaded with 1 μg of Cry1B.868 protein (based on pre-digestion protein 

concentrations) for each of the digestion samples. Examination of SDS-PAGE data showed that 

the intact Cry1B.868 protein was digested within 2 min of incubation in pepsin (Figure F-7, Panel 

A, lane 3) and the small transient fragments at ~4 kDa was completely digested within 0.5 min of 

pancreatin exposure (Figure F-7, Panel A, lane 7). 

No change in the fragment band intensities was observed in the absence of pancreatin in the SEQ 

0 min No Pancreatin Control and SEQ 2 hour No Pancreatin Control (Figure F-7, Panel A, lanes 

5 and 14). This indicates that the digestion of the fragments was due to the proteolytic activity of 

pancreatin and not due to instability of the fragment when incubated in 50 mM KH2PO4 at pH 7.5 

over the course of the experiment. 

The SEQ 0 min No Test Protein Control and SEQ 2 hour No Test Protein Control (Figure F-7, 

Panel A, lanes 4 and 15) demonstrated the integrity of the pancreatin over the course of the 

experiment. The intensity of some pancreatin bands decreased during the course of the experiment, 

most likely due to auto-digestion. This is not expected to adversely impact the pancreatin 

degradation results, as the transiently stable fragments were digested within 0.5 min of exposure 

to pancreatin. 

The sequential digestion of the Cry1B.868 protein was also assessed by western blot (Figure F-7, 

Panel B), with 40 ng of the test protein (based on total protein pre-digestion concentrations) loaded 

per lane. No bands were detected in the 2 min Pepsin Treated sample (Figure F-7, Panel B, lane 

3).

 
Bayer CropScience LP CR282-20U1 280 

Figure F-7. SDS-PAGE and Western Blot Analysis of the Degradation of Cry1B.868 

Protein by Sequential Digestion 

SDS-PAGE and western blot analysis were used to assess the degradation of Cry1B.868 in sequential 

digestion. Molecular weights (kDa) are shown on the left of each gel and correspond to the markers loaded. 

A: Colloidal Brilliant Blue G stained SDS-PAGE gel analysis of Cry1B.868 in sequential digestion. Based 

on pre-digestion protein concentrations, 1 μg of test protein was loaded in each lane containing Cry1B.868 

protein. 

B: Western blot analysis of Cry1B.868 in sequential digestion. Based on pre-digestion protein 

concentrations, 40 ng of test protein was loaded in each lane containing Cry1B.868 protein. A 1.0 minexposure is shown. 

A B 

Lane Sample Incubation 

Time Lane Sample Incubation Time 

1 Mark12 MWM - 1 Precision Plus MWM - 

Pepsin Degradation Pepsin Degradation

2 0 min Pepsin Treated 0 min 2 0 min Pepsin Treated 0 min 

3 2 min Pepsin Treated 2 min 3 2 min Pepsin Treated 2 min 

Pancreatin Degradation Pancreatin Degradation

4 SEQ 0 min No Test Protein 

Control 0 min 4 SEQ 0 min No Test Protein Control 0 min 

5 SEQ 0 min No Pancreatin Control 0 min 5 SEQ 0 min No Pancreatin Control 0 min 

6 SEQ T0 0 min 6 SEQ T0 0 min 

7 SEQ T1 0.5 min 7 SEQ T1 0.5 min 

8 SEQ T2 2 min 8 SEQ T2 2 min 

9 SEQ T3 5 min 9 SEQ T3 5 min 

10 SEQ T4 10 min 10 SEQ T4 10 min 

11 SEQ T5 30 min 11 SEQ T5 30 min 

12 SEQ T6 1 h 12 SEQ T6 1 h 

13 SEQ T7 2 h 13 SEQ T7 2 h 

14 SEQ 2 h No Pancreatin Control 2 h 14 SEQ 2 h No Pancreatin Control 2 h 

15 SEQ 2 h No Test Protein Control 2 h 15 SEQ 2 h No Test Protein Control 2 h 

 
Bayer CropScience LP CR282-20U1 281 

F.1.3.2. Degradation of Cry1Da_7 Protein by Pepsin Followed by Pancreatin 

For the sequential degradation assay, the Cry1Da_7 protein was incubated with pepsin for 2 min, 

followed by incubation with pancreatin. For the Colloidal Brilliant Blue G stained SDS-PAGE 

assessment, the gel was loaded with 1 μg of Cry1Da_7 protein (based on pre-digestion protein 

concentrations) for each of the digestion samples. Examination of SDS-PAGE data showed that 

the intact Cry1Da_7 protein was digested within 2 min of incubation in pepsin (Figure F-8, Panel 

A, lane 3) and the small transient fragments at ~4 kDa was completely digested within 0.5 min of 

pancreatin exposure (Figure F-8, Panel A, lane 7). 

No change in the fragment band intensities was observed in the absence of pancreatin in the SEQ 

0 min No Pancreatin Control and SEQ 2 hour No Pancreatin Control (Figure F-8, Panel A, lanes 

5 and 14). This indicates that the digestion of the fragments was due to the proteolytic activity of 

pancreatin and not due to instability of the fragment when incubated in 50 mM KH2PO4 at pH 7.5 

over the course of the experiment. 

The SEQ 0 min No Test Protein Control and SEQ 2 hour No Test Protein Control (Figure F-8, 

Panel A, lanes 4 and 15) demonstrated the integrity of the pancreatin over the course of the 

experiment. The intensity of some pancreatin bands decreased during the course of the experiment, 

most likely due to auto-digestion. This is not expected to adversely impact the pancreatin 

degradation results, as the transiently stable fragments were digested within 0.5 min of exposure 

to pancreatin. 

The sequential digestion of the Cry1Da_7 protein was also assessed by western blot (Figure F-8), 

with 40 ng of the test protein (based on total protein pre-digestion concentrations) loaded per lane. 

No bands were detected in the 2 min Pepsin Treated sample (Figure F-8, Panel B, lane 3).

 
Bayer CropScience LP CR282-20U1 282 

Figure F-8. SDS-PAGE and Western Blot Analysis of the Degradation of Cry1Da_7 

Protein by Sequential Digestion 

SDS-PAGE and western blot analysis were used to assess the degradation of Cry1Da_7 in sequential 

digestion. Molecular weights (kDa) are shown on the left of each gel and correspond to the markers loaded. 

A: Colloidal Brilliant Blue G stained SDS-PAGE gel analysis of Cry1Da_7 in sequential digestion. Based 

on pre-digestion protein concentrations, 1 μg of test protein was loaded in each lane containing Cry1Da_7 

protein. 

B: Western blot analysis of Cry1Da_7 in sequential digestion. Based on pre-digestion protein 

concentrations, 40 ng of test protein was loaded in each lane containing Cry1Da_7 protein. A 1.0 min 

exposure is shown. 

A B 

Lane Sample Incubation 

Time Lane Sample Incubation Time 

1 Mark12 MWM - 1 Precision Plus MWM - 

Pepsin Degradation Pepsin Degradation

2 0 min Pepsin Treated 0 min 2 0 min Pepsin Treated 0 min 

3 2 min Pepsin Treated 2 min 3 2 min Pepsin Treated 2 min 

Pancreatin Degradation Pancreatin Degradation

4 SEQ 0 min No Test Protein 

Control 0 min 4 SEQ 0 min No Test Protein Control 0 min 

5 SEQ 0 min No Pancreatin Control 0 min 5 SEQ 0 min No Pancreatin Control 0 min 

6 SEQ T0 0 min 6 SEQ T0 0 min 

7 SEQ T1 0.5 min 7 SEQ T1 0.5 min 

8 SEQ T2 2 min 8 SEQ T2 2 min 

9 SEQ T3 5 min 9 SEQ T3 5 min 

10 SEQ T4 10 min 10 SEQ T4 10 min 

11 SEQ T5 30 min 11 SEQ T5 30 min 

12 SEQ T6 1 h 12 SEQ T6 1 h 

13 SEQ T7 2 h 13 SEQ T7 2 h 

14 SEQ 2 h No Pancreatin Control 2 h 14 SEQ 2 h No Pancreatin Control 2 h 

15 SEQ 2 h No Test Protein Control 2 h 15 SEQ 2 h No Test Protein Control 2 h 

 
Bayer CropScience LP CR282-20U1 283 

F.1.4. Degradation of Cry1B.868 and Cry1Da_7 Proteins Conclusion 

F.1.4.1. Degradation of Cry1B.868 Protein Conclusion 

The ability of Cry1B.868 protein to be degraded by pepsin and by pancreatin was evaluated in this 

study. The results showed that at least 99.8% of the intact Cry1B.868 protein was degraded by 

pepsin within 0.5 min when analyzed by SDS-PAGE and 98.4% of the intact Cry1B.868 was 

degraded by pepsin within 0.5 min when analyzed by western blot using a Cry1B.868-specific 

antibody. SDS-PAGE analysis showed that transient peptide fragments at ~4 kDa were observed 

throughout the course of the pepsin digestion. At least 99.6% of the intact Cry1B.868 protein was 

degraded by pancreatin within 5 min when analyzed by western blot. These results show that the 

full-length Cry1B.868 is rapidly degraded by pepsin and pancreatin. The transient fragments at ~4 

kDa were rapidly degraded by sequential digestion. Rapid degradation of the intact Cry1B.868 

protein by pepsin or pancreatin alone and complete degradation by pepsin followed by pancreatin 

indicates that the Cry1B.868 protein is highly unlikely to pose any safety concern to human or 

animal health.

F.1.4.2. Degradation of Cry1Da_7 Protein Conclusion 

The ability of Cry1Da_7 protein to be degraded by pepsin and by pancreatin was evaluated in this 

study. The results showed that at least 99.4% of the intact Cry1Da_7 protein was degraded by 

pepsin within 0.5 min when analyzed by SDS-PAGE and 96.9% of the intact Cry1Da_7 was 

degraded by pepsin within 0.5 min when analyzed by western blot using a Cry1Da_7-specific 

antibody. SDS-PAGE analysis showed that transient peptide fragments at ~4 kDa were observed 

throughout the course of the pepsin digestion. At least 96.9% of the intact Cry1Da_7 protein was 

degraded by pancreatin within 5 min when analyzed by western blot. These results show that the 

full-length Cry1Da_7 is rapidly degraded by pepsin and pancreatin. The transient fragments at ~4 

kDa were rapidly degraded by sequential digestion. Rapid degradation of the intact Cry1Da_7 

protein by pepsin alone and pancreatin alone and complete degradation by pepsin followed by 

pancreatin indicates that the Cry1Da_7 protein is highly unlikely to pose any safety concern to 

human or animal health. 

 
Bayer CropScience LP CR282-20U1 284 

F.2. Materials and Methods Used in Assessing Stability of Cry1B.868 and Cry1Da_7 

Proteins Proteins in Pepsin and Pancreatin 

F.2.1. Test Substances 

F.2.1.1. Cry1B.868 Test Substance 

The test substance was the Cry1B.868 protein (lot # 7565, also known as 11479349) purified from 

Bacillus thuringiensis (Bt) transformed with the pMON236906 plasmid (Bt-produced Cry1B.868, 

referred to in this document as Cry1B.868 protein). The Cry1B.868 protein is stored in a -80 °C 

freezer in a buffer solution containing 20 mM CAPS, pH 11.5, 10 mM DTT, 240 mM NaCl, 1 mM 

Benzamidine (storage buffer).

F.2.1.2. Cry1Da_7 Test Substance 

The test substance was the Cry1Da_7 protein (lot # 7799, also known as 11479575) purified from 

Bacillus thuringiensis (Bt) transformed with the pMON417133 plasmid (Bt-produced Cry1Da_7, 

referred to in this document as Cry1Da_7 protein). The Cry1Da_7 protein is stored in a -80 °C 

freezer in a buffer solution containing 25 mM sodium carbonate, pH 10.5 (storage buffer).

F.2.2. Characterization of Test Substance 

F.2.2.1. Characterization of the Cry1B.868 Test Substance 

The characterization of the test substance was performed under characterization study CHR-2018-

0276 at Bayer. The test protein (lot 7565) had a purity of 91%, a total protein concentration of 

2.8 mg/ml, and an apparent molecular weight of 129.6 kDa.

F.2.2.2. Characterization of the Cry1Da_7 Test Substance 

The characterization of the test substance was performed under characterization study CHR-2018-

0365 at Bayer. The test protein (lot 7799) had a purity of 92%, a total protein concentration of 

3.6 mg/ml, and an apparent molecular weight of 126.5 kDa. 

 
Bayer CropScience LP CR282-20U1 285 

F.3. Experimental Design 

Assays designed to assess the degradation of Cry1B.868 and Cry1Da_7 proteins by pepsin and by 

pancreatin were performed independently. Because protein fragments attributable to the 

Cry1B.868 and Cry1Da_7 proteins were identified after 10 min of pepsin degradation, a sequential 

digestion of the Cry1B.868 and Cry1Da_7 proteins with pepsin followed by pancreatin were also 

performed.

F.3.1. Preparation of the Pepsin Stock Solution 

High purity pepsin (Sigma catalog number P6887; specific activity of 3546 U/mg, 96% protein, 

purity-corrected specific activity of 3404 U/mg) was dissolved in 10 mM HCl, 2 mg/ml NaCl, 

pH ~1.2 to a concentration of ~2,632 U/ml. The amount of powder used to prepare the pepsin stock 

solution was calculated by using the purity-corrected pepsin specific activity value of 3404 U/mg. 

Activity was assessed using a pepsin activity assay (Section F.4.1). 

F.3.2. Degradation of the Cry1B.868 and Cry1Da_7 Proteins by Pepsin 

F.3.2.1. Degradation of the Cry1B.868 Protein by Pepsin 

Degradation of the Cry1B.868 protein by pepsin was evaluated over time by analyzing samples 

collected at targeted incubation time points. A numerical code using the numbers 0 through 7 was 

used to distinguish incubation time points as follows: 

Targeted Incubation Time Point Sample Code 

0 min 0 min No Test Protein Control 

0 min 0 min No Pepsin Control 

0 min Pepsin Treated T0 

0.5 min Pepsin Treated T1 

2 min Pepsin Treated T2 

5 min Pepsin Treated T3 

10 min Pepsin Treated T4 

20 min Pepsin Treated T5 

30 min Pepsin Treated T6 

60 min Pepsin Treated T7 

60 min 60 min No Pepsin Control 

60 min 60 min No Test Protein Control 

The reaction mixture was prepared by adding 531.9 μl of pre-heated (38.3 oC, 10 min) pepsin stock 

solution to a tube containing 50 μl of Cry1B.868 protein, which corresponded to 140 μg of 

Cry1B.868 protein and 1400 U of pepsin. The tube contents were mixed and immediately returned 

to the water bath (38.3 oC). Samples (58.2 μl) were removed at 0.5, 2, 5, 10, 20, 30 and 60 min 

(corresponding to Pepsin Treated T1 through Pepsin Treated T7). Each 58.2 μl sample was placed 

immediately in a tube containing quenching mixture consisting of 20.4 μl of 0.7 M sodium 
Bayer CropScience LP CR282-20U1 286 

carbonate buffer and 19.6 μl of 5u loading buffer (5u LB; ~310 mM Tris-HCl, 25% (v/v) 2-

mercaptoethanol, 10% (w/v) sodium dodecyl sulfate, 0.025% (w/v) bromophenol blue, 50% (v/v) 

glycerol, pH 6.8), heated at 95-100 oC for 5-10 min, and frozen on dry ice. 

The Pepsin Treated T0 sample was prepared in a separate tube. The pepsin stock solution (53.2 μl) 

was quenched by the addition of 0.7 M sodium carbonate buffer (20.4 μl) and 5u LB (19.6 μl) and 

heated at 95-100 oC for 5-10 min prior to the addition of the Cry1B.868 protein (5.0 μl). 

All quenched samples were frozen on dry ice and stored in a -80 oC freezer until analysis.

F.3.2.2. Degradation of the Cry1Da_7 Protein by Pepsin 

Degradation of the Cry1Da_7 protein by pepsin was evaluated over time by analyzing samples 

collected at targeted incubation time points. A numerical code using the numbers 0 through 7 was 

used to distinguish incubation time points as follows: 

Targeted Incubation Time Point Sample Code 

0 min 0 min No Test Protein Control 

0 min 0 min No Pepsin Control 

0 min Pepsin Treated T0 

0.5 min Pepsin Treated T1 

2 min Pepsin Treated T2 

5 min Pepsin Treated T3 

10 min Pepsin Treated T4 

20 min Pepsin Treated T5 

30 min Pepsin Treated T6 

60 min Pepsin Treated T7 

60 min 60 min No Pepsin Control 

60 min 60 min No Test Protein Control 

The reaction mixture was prepared by adding 683.9 μl of pre-heated (37.7oC, 5 min) pepsin stock 

solution to a tube containing 50 μl of Cry1Da_7 protein, which corresponded to 180 μg of 

Cry1Da_7 protein and 1800 U of pepsin. The tube contents were mixed and immediately returned 

to the water bath (38.0oC). Samples (73.4 μl) were removed at 0.5, 2, 5, 10, 20, 30 and 60 min 

(corresponding to Pepsin Treated T1 through Pepsin Treated T7). Each 73.4 μl sample was placed 

immediately in a tube containing quenching mixture consisting of 25.7 μl of 0.7 M sodium 

carbonate buffer and 24.8 μl of 5u loading buffer (5u LB; ~310 mM Tris-HCl, 25% (v/v) 2-

mercaptoethanol, 10% (w/v) sodium dodecyl sulfate, 0.025% (w/v) bromophenol blue, 50% (v/v) 

glycerol, pH 6.8), heated at 95-100 oC for 5-10 min, and frozen on dry ice. 

The Pepsin Treated T0 sample was prepared in a separate tube. The pepsin stock solution (68.4 μl) 

was quenched by the addition of 0.7 M sodium carbonate buffer (25.7 μl) and 5u LB (24.8 μl) and 
Bayer CropScience LP CR282-20U1 287 

heated at 95-100 oC for 5-10 min prior to the addition of the Cry1Da_7 protein (5.0 μl). The 

sample was then heated again at 95-100 oC for 5-10 min. 

All quenched samples were frozen on dry ice and stored in a -80 oC freezer until analysis.

F.3.2.3. Pepsin Degradation Experimental Controls 

Experimental control samples were prepared to determine the stability of the Cry1B.868 and 

Cry1Da_7 proteins in 10 mM HCl, 2 mg/ml NaCl, pH ~1.2, the solution used to prepare the pepsin 

stock solution. The 0 min No Pepsin Control was prepared in a similar manner as described in 

Section F.3.2.2 for Pepsin Treated T0, except that 10 mM HCl, 2 mg/ml NaCl, pH 1.2 was added 

instead of the pepsin stock solution. The 60 min No Pepsin Control was prepared in a manner 

similar to 0 min No Pepsin Control, except the protein and 10 mM HCl, 2 mg/ml NaCl, pH 1.2 

were incubated for 60 min at 38oC before quenching with 0.7 M sodium carbonate buffer and 

5× LB. 

Experimental control samples were also prepared to determine the stability of the pepsin stock 

solution lacking the Cry1B.868 and Cry1Da_7 proteins. The 0 min No Test Protein Control was 

prepared in a similar manner as described in Section F.3.2.2 for Pepsin Treated T0, except that 

protein storage buffer (25 mM sodium carbonate, pH 10.5) was added in place of the Cry1B.868 

and Cry1Da_7 proteins. The 60 min No Test Protein Control was prepared in a manner similar to 

0 min No Test Protein Control, except that protein storage buffer and the pepsin stock solution 

were incubated for 60 min at 38oC before quenching with 0.7 M sodium carbonate buffer and 

5× LB. 

All quenched samples were frozen on dry ice and stored in a -80 oC freezer until analysis.

F.3.3. Preparation of the Pancreatin Stock Solution 

Pancreatin contains a mixture of proteolytic enzymes and was prepared based on the method 

described in The United States Pharmacopoeia (USP, 1995). The pancreatin was obtained from 

Alfa Aesar (catalog number J62162) and was dissolved in 50 mM potassium phosphate buffer 

(pH 7.5) to a concentration of 10 mg of pancreatin powder/ml. Activity was assessed using a 

pancreatin activity assay (Section F.4.2).

F.3.3.1. Degradation of the Cry1B.868 and Cry1Da_7 Proteins by Pancreatin 

F.3.3.1.1. Degradation of the Cry1B.868 Protein by Pancreatin 

Degradation of the intact Cry1B.868 protein by pancreatin was evaluated over time by analyzing 

samples at multiple incubation time points. A numerical code using the numbers 0 through 8 was 

used to distinguish incubation time points as follows: 
Bayer CropScience LP CR282-20U1 288 

Targeted Incubation Time Point Sample Code

0 min 0 min No Test Protein Control 

0 min 0 min No Pancreatin Control

0 min Pancreatin Treated T0

5 min Pancreatin Treated T1

15 min Pancreatin Treated T2

30 min Pancreatin Treated T3

1 h Pancreatin Treated T4

2 h Pancreatin Treated T5

4 h Pancreatin Treated T6

8 h Pancreatin Treated T7

24 h Pancreatin Treated T8

24 h 24 h No Pancreatin Control

24 h 24 h No Test Protein Control

The reaction mixture was prepared by adding 774.2 μl of pre-heated (37.7 oC, 10 min) pancreatin 

stock solution to a tube containing 50 μl of the Cry1B.868 protein, corresponding to 140 μg of 

Cry1B.868 protein and 7,742 μg of pancreatin. The tube contents were vortex mixed and 

immediately returned to the water bath (37.6 oC). Digestion samples (82.4 μl) were removed at 5, 

15, 30 min, 1, 2, 4, 8, and 24 h (corresponding to time points Pancreatin Treated T1 through 

Pancreatin Treated T8) and immediately placed in a tube containing 26.1 μl of 5u LB, heated at 

95-100 oC for 5-10 min, and frozen on dry ice. 

The Pancreatin Treated T0 sample was prepared in a separate tube by first quenching 77.4 μl of 

pancreatin stock solution with 20.6 μl of 5u LB buffer and heating to 95-100 oC for 5-10 min prior 

to the addition of 5.0 μl of the Cry1B.868 protein. Samples were heated again to 95-100 oC for 5-

10 min. 

All quenched samples were frozen on dry ice and stored in a -80 oC freezer until analysis. 

F.3.3.1.2. Degradation of the Cry1Da_7 Protein by Pancreatin 

Degradation of the intact Cry1Da_7 protein by pancreatin was evaluated over time by analyzing 

samples at multiple incubation time points. A numerical code using the numbers 0 through 8 was 

used to distinguish incubation time points as follows: 
Bayer CropScience LP CR282-20U1 289 

Targeted Incubation Time Point Sample Code

0 min 0 min No Test Protein Control 

0 min 0 min No Pancreatin Control

0 min Pancreatin Treated T0

5 min Pancreatin Treated T1

15 min Pancreatin Treated T2

30 min Pancreatin Treated T3

1 h Pancreatin Treated T4

2 h Pancreatin Treated T5

4 h Pancreatin Treated T6

8 h Pancreatin Treated T7

24 h Pancreatin Treated T8

24 h 24 h No Pancreatin Control

24 h 24 h No Test Protein Control

The reaction mixture was prepared by adding 995.4 μl of pre-heated (37.8oC, 5.5 min) pancreatin 

stock solution to a tube containing 50 μl of the Cry1Da_7 protein, corresponding to 180 μg of 

Cry1Da_7 protein and 9,954 μg of pancreatin. The tube contents were vortex mixed and 

immediately returned to the water bath (37.7oC). Digestion samples (104.5 μl) were removed at 

5, 15, 30 min, 1, 2, 4, 8, and 24 h (corresponding to time points Pancreatin Treated T1 through 

Pancreatin Treated T8) and immediately placed in a tube containing 26.1 μl of 5u LB, heated at 

95-100 oC for 5-10 min, and frozen on dry ice. 

The Pancreatin Treated T0 sample was prepared in a separate tube by first quenching 99.5 μl of 

pancreatin stock solution with 26.1 μl of 5u LB buffer and heating to 95-100 oC for 5-10 min prior 

to the addition of 5.0 μl of the Cry1Da_7 protein. The sample was then heated again at 95-100 oC 

for 5-10 min. 

All quenched samples were frozen on dry ice and stored in a -80 oC freezer until analysis. 

 
Bayer CropScience LP CR282-20U1 290 

F.3.3.2. Pancreatin Degradation Experimental Controls 

Experimental control samples were prepared to determine the stability of the Cry1B.868 and 

Cry1Da_7 proteins in 50 mM KH2PO4, pH 7.5, the buffer used to prepare pancreatin stock 

solution. The 0 min No Pancreatin Control was prepared in a similar manner as described in 

Section F.3.3.1.2 for Pancreatin Treated T0, except that 50 mM KH2PO4, pH 7.5 was added 

instead of the pancreatin stock solution. The 24 hour No Pancreatin Control was also prepared in 

a similar manner, except the protein and 50 mM KH2PO4, pH 7.5 were incubated for 24 hour at 

38oC before quenching with 5× LB and heating. 

Experimental control samples were also prepared to characterize the test system lacking the 

Cry1B.868 and Cry1Da_7 proteins. The 0 min No Test Protein Control was prepared in a similar 

manner as described in Section F.3.3.1.2 for Pancreatin Treated T0, except that protein storage 

buffer (25 mM sodium carbonate, pH 10.5) was added instead of Cry1B.868 and Cry1Da_7 

proteins. The 24 hour No Test Protein Control was also prepared in a similar manner, except the 

protein storage buffer and pancreatin stock solution were incubated for 24 hour at 38oC before 

quenching with 5× LB and heating. 

All quenched samples were frozen on dry ice and stored in a -80 oC freezer until analyzed.

F.3.4. Degradation of the Cry1B.868 and Cry1Da_7 Proteins by Pepsin Followed by 

Pancreatin 

F.3.4.1. Degradation of the Cry1B.868 Protein by Pepsin Followed by Pancreatin 

Degradation of the Cry1B.868 protein by sequential digestion was evaluated by first incubating 

the Cry1B.868 protein with pepsin for 2 min, quenching the pepsin degradation reaction, and then 

incubating the reaction mixture with pancreatin. The sequential digestion of the Cry1B.868 protein 

was evaluated over time by analyzing samples at multiple incubation time points with a numerical 

code using the number 0 through 7 to distinguish incubation time points as follows: 
Bayer CropScience LP CR282-20U1 291 

Targeted Incubation Time Point Sample Code 

Pepsin Degradation

0 min 0 min Pepsin Treated 

2 min 2 min Pepsin Treated 

Pancreatin Degradation

0 min SEQ 0 min No Test Protein Control 

0 min SEQ 0 min No Pancreatin Control 

0 min SEQ T0 

0.5 min SEQ T1 

2 min SEQ T2 

5 min SEQ T3 

10 min SEQ T4 

30 min SEQ T5 

1 h SEQ T6 

2 h SEQ T7 

2 h SEQ 2 h No Pancreatin Control 

2 h SEQ 2 h No Test Protein Control 

In the pepsin degradation phase, 531.9 μl of pre-heated (38.4 oC, 10 min) pepsin solution (2632 

units of pepsin activity/ml) was added to 50 μl of the Cry1B.868 protein, corresponding to 140 μg 

of Cry1B.868 protein and 1,400 U of pepsin. The tube contents were mixed by vortexing and 

immediately returned to the water bath (38.3 oC). The tube was removed at a targeted time of 2 

min, and immediately quenched by adding 203.7 μl of 0.7 M sodium carbonate buffer. The 2 min 

Pepsin Treated sample was prepared by mixing 120 μl of the quenched sample with 30 μl of 5u

LB, heated at 95-100 oC for 5-10 min, and frozen on dry ice. 

In the pancreatin degradation phase, the quenched 2 min pepsin treated sample was digested in 

pancreatin. For this phase, 398.2 μl of pre-heated (38.3 oC) pancreatin solution was added to 400 

μl of quenched 2 min pepsin treated sample, corresponding to 72 μg total Cry1B.868 protein 

(based on predigested concentration) and 3,982 μg of pancreatin. The tube contents were mixed 

by vortexing and immediately returned to the water bath (38.3 oC). Digestion samples (79.8 μl) 

were removed from the tube at targeted times of 30 sec, 2, 5, 10, 30 min, 1, and 2 h (corresponding 

to specimen codes SEQ T1 through SEQ T7) and immediately quenched by placing in a tube 

containing 20.0 μl of 5u LB, heated at 95-100 oC for 5-10 min, and frozen on dry ice. 

The zero incubation time point for the pepsin degradation phase (0 min Pepsin Treated) was 

prepared in a separate tube. The pepsin solution (53.2 μl) was quenched by the addition of 0.7 M 

sodium carbonate buffer (20.4 μl) and 5u LB (19.6 μl) and heated at 95-100 oC for 5-10 min prior 

to the addition of the Cry1B.868 protein (5.0 μl). 

The zero incubation time point for the pancreatin digestion phase (SEQ T0) was prepared in a 

separate tube. The pancreatin solution (39.8 μl) was quenched with 5u LB (20.0 μl) and heated at 

95-100 oC for 5-10 min prior to the addition of the quenched 2 min pepsin treated Cry1B.868 (40.0 

μl). 
Bayer CropScience LP CR282-20U1 292 

All quenched samples were frozen on dry ice and stored in a -80 oC freezer until analyzed. 

F.3.4.2. Degradation of the Cry1Da_7 Protein by Pepsin Followed by Pancreatin 

Degradation of the Cry1Da_7 protein by sequential digestion was evaluated by first incubating the 

Cry1Da_7 protein with pepsin for 2 min, quenching the pepsin degradation reaction, and then 

incubating the reaction mixture with pancreatin. The sequential digestion of the Cry1Da_7 protein 

was evaluated over time by analyzing samples at multiple incubation time points with a numerical 

code using the number 0 through 7 to distinguish incubation time points as follows: 

Targeted Incubation Time Point Sample Code 

Pepsin Degradation

0 min 0 min Pepsin Treated 

2 min 2 min Pepsin Treated 

Pancreatin Degradation

0 min SEQ 0 min No Test Protein Control 

0 min SEQ 0 min No Pancreatin Control 

0 min SEQ T0 

0.5 min SEQ T1 

2 min SEQ T2 

5 min SEQ T3 

10 min SEQ T4 

30 min SEQ T5 

1 h SEQ T6 

2 h SEQ T7 

2 h SEQ 2 h No Pancreatin Control 

2 h SEQ 2 h No Test Protein Control 

In the pepsin degradation phase, 683.9 μl of pre-heated (37.6oC, 5 min) pepsin solution (2632 units 

of pepsin activity/ml) was added to 50 μl of the Cry1Da_7 protein, corresponding to 180 μg of 

Cry1Da_7 protein and 1,800 U of pepsin. The tube contents were mixed by vortexing and 

immediately returned to the water bath (37.7oC). The tube was removed at a targeted time of 2 

min, and immediately quenched by adding 256.9 μl of 0.7 M sodium carbonate buffer. The 2 min 

Pepsin Treated sample was prepared by mixing 120 μl of the quenched sample with 30 μl of 5u

LB, heated at 95-100 oC for 5-10 min, and frozen on dry ice. 

In the pancreatin degradation phase, the quenched 2 min pepsin treated sample was digested in 

pancreatin. For this phase, 497.7 μl of pre-heated (37.6oC) pancreatin solution was added to 500 

μl of quenched 2 min pepsin treated sample, corresponding to 90 μg total Cry1Da_7 protein (based 

on predigested concentration) and 4,977 μg of pancreatin. The tube contents were mixed by 

vortexing and immediately returned to the water bath (37.6oC). Digestion samples (99.8 μl) were 

removed from the tube at targeted times of 30 sec, 2, 5, 10, 30 min, 1, and 2 h (corresponding to 

specimen codes SEQ T1 through SEQ T7) and immediately quenched by placing in a tube 

containing 25.0 μl of 5u LB, heated at 95-100 oC for 5-10 min, and frozen on dry ice. 
Bayer CropScience LP CR282-20U1 293 

The zero incubation time point for the pepsin degradation phase (0 min Pepsin Treated) was 

prepared in a separate tube. The pepsin solution (68.4 μl) was quenched by the addition of 0.7 M 

sodium carbonate buffer (25.7 μl) and 5u LB (24.8 μl) and heated at 95-100 oC for 5-10 min prior 

to the addition of the Cry1Da_7 protein (5.0 μl). 

The zero incubation time point for the pancreatin digestion phase (SEQ T0) was prepared in a 

separate tube. The pancreatin solution (49.8 μl) was quenched with 5u LB (25.0 μl) and heated at 

95-100 oC for 5-10 min prior to the addition of the quenched 2 min pepsin treated Cry1Da_7 (50.0 

μl). 

All quenched samples were frozen on dry ice and stored in a -80 oC freezer until analyzed. 

F.3.4.3. Pepsin Followed by Pancreatin Degradation Experimental Controls 

The SEQ 0 min No Pancreatin Control was prepared in a similar manner as described for SEQ T0, 

except that 50 mM KH2PO4, pH 7.5 was added instead of pancreatin solution. The SEQ 2 hour 

No Pancreatin Control was also prepared in a similar manner, except the protein and 50 mM 

KH2PO4 were incubated for 2 hours at 38°C before quenching with 5× LB and heating. 

The SEQ 0 min No Test Protein Control was prepared in a similar manner as described for SEQ 

T0, except that protein storage buffer (25 mM sodium carbonate, pH 10.5) was added instead of 

Cry1B.868 or Cry1Da_7 protein. The SEQ 2 hour No Test Protein Control was also prepared in 

a similar manner, except the protein storage buffer and pancreatin solution were incubated for 2 

hours at 38oC before quenching with 5× LB and heating. 

All quenched samples were frozen on dry ice and stored in a -80 oC freezer until analyzed.

F.4. Analytical Methods 

F.4.1. Pepsin Activity Assay 

Pepsin activity was determined by measuring the degradation of denatured hemoglobin. To 

conduct the assay, the pepsin stock solution (Section F.3.1), was diluted to 0.03 mg of powder per 

ml with 10 mM HCl, 2 mg/ml NaCl, pH ~1.2 [dilution factor (DF) = 25.67]. Five ml of acidified 

hemoglobin (2% (w/v)) was added to three test sample tubes and three blank tubes and all were 

pre-warmed for 5 min in a water bath at 37.7-38.0qC. Diluted pepsin stock solution (1 ml) was 

added to the three test sample tubes (test samples) and all six tubes were incubated for 10 min in 

the water bath. Ten ml of 5% (v/v) chilled trichloroacetic acid (TCA) was added to each of the six 

tubes and 1 ml diluted pepsin stock solution was then added to the three blank samples. Samples 

were mixed and then incubated for another 5 min in the water bath. Precipitated protein was 

removed from each sample using 0.45 μm syringe filters and the absorbance of the test and blank 

sample filtrates at 280 nm were measured using a SpectraMax M2 plate reader (Molecular 

Devices). One unit of pepsin produces a change in absorbance at 280 nm of 0.001 per min at 

37 ± 2 oC, pH 1.2. The units of pepsin in 1 ml of the stock solution were calculated using the 

following equation: 
Bayer CropScience LP CR282-20U1 294 

௠௡଴଼ଶ஺݈ܽ݊݇ܤ ݁ܽ݊ܯ − ௠௡଴଼ଶ஺ݐݏܶ݁ ݁ܽ݊ܯ

ܨܦ × ݈݉ 1 × min 10 × 0.001

where 0.001 is the change in the absorbance at 280 nm per min produced by one unit of pepsin 

activity at 37 ± 2 oC, pH 1.2; 10 min is the reaction time; 1 ml is the volume of the diluted pepsin 

stock solution added to the reaction; and DF is the dilution factor for the pepsin stock solution. 

The activity of pepsin was converted from units/ml to units/mg. Acceptable specific activity 

(units/mg pepsin powder) for the pepsin solution was equal to the specific activity determined by 

the manufacturer ± 1,000 units/mg.

F.4.2. Pancreatin Activity Assay 

The pancreatin activity was determined by measuring the degradation of resorufin-labeled casein 

(Roche Life Science). To conduct the assay, the pancreatin stock solution (Section F.3.3), was 

diluted to 0.05× with 50 mM KH2PO4, pH 7.5. Fifty μl of 0.4% (w/v) resorufin-labeled casein and 

50 μl of incubation buffer (200 mM Tris, pH 7.8, 20 mM CaCl2) were added to three test sample 

tubes and three blank tubes and all were pre-warmed for 1.5-2.0 min in a water bath at 37.8°C. To 

initiate the reaction, 100 μl of 0.05× pancreatin stock solution was added to each of the three test 

sample tubes while 100 μl of 50 mM KH2PO4, pH 7.5 was added to each of the three blank tubes. 

All six tubes were incubated for 15 min in the water bath. Reactions were quenched by adding 

480 μl of 5% (w/v) TCA to each tube, followed by vortex mixing. The samples were incubated in 

the water bath for an additional 10 min. The supernatants recovered after centrifugation (400 μl) 

were neutralized by the addition of 600 μl assay buffer (500 mM Tris-HCl, pH 8.8) and the 

absorbance of the test and blank supernatants was read at 574 nm using a SpectraMax M2 plate 

reader (Molecular Devices). One unit of pancreatin produces a change in the absorbance at 574 nm 

of 0.001 per min at 37 ± 2 oC. The units of pancreatin in the stock solution were calculated using 

the following equation: 

௠௡ସ଻ହ஺݈ܽ݊݇ܤ ݁ܽ݊ܯ − ௠௡ସ଻ହ஺ݕݐ݅ݒ݅ݐܿܣ ݁ܽ݊ܯ

0.001 × 15 min× 0.005 ݈݉

where 0.001 is the change in the absorbance at 574 nm per min at 37 ± 2 oC produced by one unit 

of pancreatin activity, 15 min is the reaction time, 0.1 ml is the volume of diluted pancreatin stock 

solution added to the reaction, and 0.005 ml is the volume of the pancreatin stock solution used in 

the assay (0.05× Pancreatin solution, 0.1 ml). An acceptable specific activity for the pancreatin 

stock solution was defined as 11,000 ± 3,000 U/ml. 

F.4.3. SDS-PAGE and Colloidal Brilliant Blue G Staining 

Pepsin-treated samples and associated control samples were subjected to SDS-PAGE using precast Tricine 10-20% (w/v) polyacrylamide gradient mini-gels and Tricine SDS running buffer 

(Invitrogen). The Cry1B.868 and Cry1Da_7 proteins were loaded at 1 μg per lane based on total 

pre-digestion protein concentration. The experimental controls were loaded at the same volumes 

as those containing Cry1B.868 and Cry1Da_7 proteins so that they would be comparable. 

Mark12TM molecular weight marker (Invitrogen) was loaded to estimate the relative molecular 
Bayer CropScience LP CR282-20U1 295 

weight of proteins and peptides. Electrophoresis was performed at a constant voltage of 125-150 

volts for 75-95 minutes. After electrophoresis, gels were fixed in a solution containing 7% (v/v) 

acetic acid and 40% (v/v) methanol for 25-60 min and stained for 18-20 hours in 1u Brilliant Blue 

G-Colloidal stain solution (Sigma), destained for 30 sec in 10% (v/v) acetic acid, 25% (v/v) 

methanol and then destained for 4.0-6 hours in a 25% (v/v) methanol solution. The gels were 

scanned using a Bio-Rad GS-900 densitometer (Bio-Rad) to produce digitized images to be used 

as figures for reporting purposes. 

To estimate the LOD of the Cry1B.868 and Cry1Da_7 proteins, dilutions of the Pepsin Treated T0 

samples were loaded on a second SDS-PAGE gel and the gel was run and processed exactly as the 

gel used to assess Cry1B.868 and Cry1Da_7 protein degradation in the presence of pepsin. Loads 

per lane were approximately 0.4 to 200 ng or 400 ng of the Cry1B.868 and Cry1Da_7, respectively. 

The LOD was determined as the lowest amount of the Cry1B.868 and Cry1Da_7 proteins that was 

visible on the gel with Colloidal staining. 

Sequential digestion samples were also analyzed by SDS-PAGE as described above for pepsin 

degradation samples.

F.4.4. Western Blot Analysis 

Pepsin-treated, pancreatin-treated, and sequential digestion samples, and the associated control 

samples, were subjected to SDS-PAGE using pre-cast Tricine 10-20% (w/v) polyacrylamide 

gradient mini-gels. The Cry1B.868 and Cry1Da_7 proteins were loaded at approximately 40 ng 

per lane based on total pre-digestion protein concentration. The experimental controls were loaded 

to equal the digestion samples. Electrophoresis was performed at a constant voltage of 125-150 

volts for 75-95 minutes. After electrophoresis, the proteins were electrotransferred to PVDF 

membranes (Invitrogen). Pre-stained molecular weight markers (Precision Plus ProteinTM

Standards Dual Color Standard, Bio-Rad; MagicMarkTM XP, Invitrogen) were used to verify 

electrotransfer of the proteins to the membranes and estimate the relative molecular weight of 

proteins. Proteins transferred to PVDF membranes were analyzed by western blot. The western 

blotting procedure was performed using an iBindTM Western System apparatus (Life 

Technologies). 

F.4.4.1. Western Blot Analysis of Cry1B.868 Protein 

The Cry1B.868 protein membranes were blocked with 1× iBindTM Solution (Life Technologies) 

and incubated with rabbit anti-Cry1B.868 antibody (lot 29121) at a dilution of 1:2,000 in 1× 

iBindTM Solution. After washing with 1× iBindTM Solution, the membrane was incubated with 

horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Vector Laboratories) at a dilution 

of 1:2,000 in 1× iBindTM Solution and washed again with 1× iBindTM Solution. Immunoreactive 

bands were visualized using the enhanced chemiluminescence (ECL) detection system (GE 

Healthcare) and exposed to HyperfilmTM ECL high performance chemiluminescence film (GE 

Healthcare). The film was developed using a Konica SRX-101A automated film processor 

(Konica). The films were scanned using a Bio-Rad GS-900 densitometer to produce electronic 

images to be used as figures for reporting purposes. 
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To estimate the LOD of the Cry1B.868 protein, dilutions of either the Pepsin Treated T0 or the 

Pancreatin Treated T0 samples were subjected to western blot analysis run and processed exactly 

as the western blot used to assess Cry1B.868 protein degradation in the presence of pepsin or 

pancreatin, respectively. Loads per lane were approximately 0.08 to 40 ng of the Cry1B.868 

protein. The LOD was determined as the lowest amount of the Cry1B.868 protein that was visible 

on the film. 

F.4.4.2. Western Blot Analysis of Cry1Da_7 Protein 

The Cry1Da_7 protein membranes were blocked with 1× iBindTM Solution (Life Technologies) 

and incubated with goat anti-Cry1Da_7 antibody (lot G1496) at a dilution of 1:1,000 in 1× 

iBindTM Solution. After washing with 1× iBindTM Solution, the membrane was incubated with 

horseradish peroxidase (HRP)-conjugated horse anti-goat IgG (Vector Laboratories) at a dilution 

of 1:1,000 in 1× iBindTM Solution and washed again with 1× iBindTM Solution. Immunoreactive 

bands were visualized using the enhanced chemiluminescence (ECL) detection system (GE 

Healthcare) and exposed to HyperfilmTM ECL high performance chemiluminescence film (GE 

Healthcare). The film was developed using a Konica SRX-101A automated film processor 

(Konica). The films were scanned using a Bio-Rad GS-900 densitometer to produce electronic 

images to be used as figures for reporting purposes. 

To estimate the LOD of the Cry1Da_7 protein, dilutions of either the Pepsin Treated T0 or the 

Pancreatin Treated T0 samples were subjected to western blot analysis run and processed exactly 

as the western blot used to assess Cry1Da_7 protein degradation in the presence of pepsin or 

pancreatin, respectively. Loads per lane were approximately 0.08 to 20 ng of the Cry1Da_7 

protein. The LOD was determined as the lowest amount of the Cry1Da_7 protein that was visible 

on the film.

 
Bayer CropScience LP CR282-20U1 297 

References for Appendix F 

Astwood, J.D., J.N. Leach and R.L. Fuchs. 1996. Stability of food allergens to digestion in vitro. 

Nature Biotechnology 14:1269-1273. 

Fu, T.-J., U.R. Abbott and C. Hatzos. 2002. Digestibility of food allergens and nonallergenic 

proteins in simulated gastric fluid and simulated intestinal fluid - A comparative study. Journal of 

Agricultural and Food Chemistry 50:7154-7160. 

Hammond, B.G. and J.M. Jez. 2011. Impact of food processing on the safety assessment for 

proteins introduced into biotechnology-derived soybean and corn crops. Food and Chemical 

Toxicology 49:711-721. 

Moreno, F.J., F.A. Mellon, M.S.J. Wickham, A.R. Bottrill and E.N.C. Mills. 2005. Stability of 

the major allergen Brazil nut 2S albumin (Ber e 1) to physciologically relevant in vitro 

gastrointestinal digestion. FEBS Journal 272:341-352. 

Okunuki, H., R. Techima, T. Shigeta, J. Sakushima, H. Akiyama, Y. Goda, M. Toyoda and J. 

Sawada. 2002. Increased digestibility of two products in genetically modified food (CP4 EPSPS 

and Cry1Ab) after preheating. Journal of the Food Hygienic Society of Japan 43:68-73. 

Thomas, K., M. Aalbers, G.A. Bannon, M. Bartels, R.J. Dearman, D.J. Esdaile, T.J. Fu, C.M. Glatt, 

N. Hadfield, C. Hatzos, S.L. Hefle, J.R. Heylings, R.E. Goodman, B. Henry, C. Herouet, M. 

Holsapple, G.S. Ladics, T.D. Landry, S.C. MacIntosh, E.A. Rice, L.S. Privalle, H.Y. Steiner, R. 

Teshima, R. van Ree, M. Woolhiser and J. Zawodny. 2004. A multi-laboratory evaluation of a 

common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. 

Regulatory Toxicology and Pharmacology 39:87-98. 

USP. 1995. Test solutions. Page 2053 in The National Formulary. United States Pharmacopeia, 

Washington, D.C. 

Vassilopoulou, E., N. Rigby, F.J. Moreno, L. Zuidmeer, J. Akkerdaas, I. Tassios, N.G. 

Papadopoulos, P. Saxoni-Papageorgiou, R. van Ree and C. Mills. 2006. Effect of in vitro gastric 

and duodenal digestion on the allergenicity of grape lipid transfer protein. Journal of Allergy and 

Clinical Immunology 118:473-480. 

Vieths, S., J. Reindl, U. Müller, A. Hoffmann and D. Haustein. 1999. Digestibility of peanut and 

hazelnut allergens investigated by a simple in vitro procedure. European Food Research and 

Technology 209:379-388. 

Wang, C., W. Li, C.R. Kessenich, J.S. Petrick, T.J. Rydel, E.J. Sturman, T.C. Lee, K.C. Glenn 

and T.C. Edrington. 2018. Safety of the Bacillus thuringiensis-derived Cry1A.105 protein: 

Evidence that domain exchange preserves mode of action and safety. Regulatory Toxicology and 

Pharmacology 99:50-60 
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Yagami, T., Y. Haishima, A. Nakamura, H. Osuna and Z. Ikezawa. 2000. Digestibility of allergens 

extracted from natural rubber latex and vegetable foods. Journal of Allergy and Clinical 

Immunology 106:752-762. 
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Appendix G: Materials and Methods for Seed Germination and Dormancy 

Assessment of MON 95379 

G.1. Materials 

Seed germination and dormancy characteristics were assessed on seed from MON 95379 and the 

conventional control harvested from one 2018 field production site in Kihei, Hawaii, and on four 

reference maize hybrids obtained from commercial sources (Table G-1). 

G.2. Characterization of the Materials 

The presence of the MON 95379 event in the starting seed of the test material and the absence of 

the MON 95379 event in the starting seed of the conventional control were verified by eventspecific polymerase chain reaction (PCR) analyses. 

G.3. Germination Testing Facility and Experimental Methods 

Germination and dormancy evaluations were conducted at SGS North America, Inc, Brookings, 

SD. The Principal Investigator was qualified to conduct seed germination and dormancy testing 

consistent with the standards established by the Association of Official Seed Analysts (AOSA, 

2009; 2018a; 2018b). 

The seed lots of MON 95379, the conventional control, and four reference hybrids were tested 

under two temperature regimes. The optimum temperature regime consisted of constant 

temperatures of approximately 25°C maintained for seven days (AOSA, 2018 a; b) . The 

suboptimum temperature regime consisted of constant temperatures of approximately 10°C for 

seven days followed by 25°C for four days (AOSA, 2009). One germination chamber was used 

for each temperature regime. Each chamber was maintained dark. The temperature inside each 

germination chamber was monitored and recorded throughout the duration of the study. 

Rolled germination towels were assembled for each material by placing approximately 50 seeds 

on a pre-moistened and labeled germination towel. An additional pre-moistened germination towel 

was placed on top of the seed, and the towels and seed were rolled up. Approximately 100 seeds 

(a set of two germination towel rolls secured together with a rubber band) of each material were 

placed into a bucket to form a replication. Each temperature regime constituted a separate 

experiment that was conducted using a randomized complete block design with four replications 

(i.e., buckets). 

Each set of rolled germination towels in each temperature regime was assessed at prescribed 

timings for germinated, dead, firm swollen (viable and nonviable), and hard (viable and nonviable) 

seed. Additional details for each germination characteristic evaluated and the timing of evaluations 

are presented in Table VIII-1. Seeds placed under the optimum temperature regime were evaluated 

according to AOSA standards for testing of maize (AOSA, 2018a; b). Seeds placed under the 

suboptimum temperature regime were evaluated according to AOSA standards for cold testing of 

maize (AOSA, 2009). 
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Within both the optimum and suboptimum temperature regimes, firm-swollen and hard seeds 

remaining at the final evaluation date were subjected to a tetrazolium (Tz) test for viability 

according to AOSA standards (AOSA/SCST, 2010). The numbers of non-viable firm-swollen and 

non-viable hard seeds were added to the number of dead seed counted on all collection dates to 

determine the total number of dead seed. Total numbers of viable firm-swollen and viable hard 

seed were determined from the Tz test. 

The percentage of seed in each assessment category was based on the number of seeds evaluated. 

Across temperature regimes, the total number of seeds evaluated from each set of rolled 

germination towels was approximately 100. 

G.4. Statistical Analysis 

For each temperature regime, ANOVA was conducted according to a randomized complete block 

design using SAS® (SAS, 2012) to compare MON 95379 to the conventional control for the 

germination and dormancy characteristics. If analysis of variance assumptions were not satisfied, 

Fisher’s Exact test was conducted for that characteristic using SAS (SAS, 2012). The level of 

statistical significance was predetermined to be 5% (α = 0.05). The reference range for each 

characteristic was determined from the minimum and maximum mean values among the four 

references. 

Table G-1. Starting Seed for Germination and Dormancy Assesment of MON 95379 

Material Name 

Regulatory 

Lot Number

Phenotype Material Type 

MON 95379 11493962 Insect Protected Test 

LH244+HCL617 11493959 Conventional Control 

Dekalb DKC65-18 11464652 Conventional Reference 

Dekalb DKC61-52 11479327 Conventional Reference 

Golden Harvest G07F23 11490232 Conventional Reference 

Mycogen 2H721 11490229 Conventional Reference 

Note: starting seed of test and control materials were produced at Kihei, HI. Reference materials were commercial 

sources.
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References for Appendix G 

AOSA. 2009. Seedling evaluation. Rules for Testing Seeds. Volume 4. Association of Official 

Seed Analysts, Ithaca, New York. 

AOSA. 2017a. AOSA Rules for testing seeds. Volume 1: Principles and procedures. Association 

of Official Seed Analysts, Washington, D.C. 

AOSA. 2017b. AOSA Rules for testing seeds. Volume 4: Seedling evaluation. Association of 

Official Seed Analysts, Washington, D.C. 

AOSA. 2018a. AOSA Rules for testing seeds. Volume 1: Principles and procedures. Association 

of Official Seed Analysts, Washington, D.C. 

AOSA. 2018b. AOSA Rules for testing seeds. Volume 4: Seedling evaluation. Association of 

Official Seed Analysts, Washington, D.C. 

AOSA/SCST. 2010. Tetrazolium testing handbook. Association of Official Seed Analysts and the 

Society of Commercial Seed Technologists, Ithaca, New York. 

SAS. 2012. Software Release 9.4 (TS1M4). Copyright 2002-2012 by SAS Institute, Inc., Cary, 

North Carolina. 

 
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Appendix H: Material and Methods for Phenotypic, Agronomic, and 

Environmental Interaction Assessment of MON 95379 under Field Conditions 

H.1. Materials 

Agronomic, phenotypic, and environmental interaction characteristics were assessed for 

MON 95379, the conventional control, and reference hybrids grown under similar agronomic 

conditions. Four reference hybrids were planted per site (Table H-1). A total of 17 unique reference 

hybrids were evaluated among the eight sites. 

H.2. Characterization of the Materials 

The presence of the MON 95379 event in the starting seed of the test material and the absence of 

the MON 95379 event in the starting seed of the conventional control were verified by eventspecific polymerase chain reaction (PCR) analyses. 

H.3. Field Sites and Plot Design 

Field trials in 2018 at eight sites provided a range of environmental and agronomic conditions 

representative of U.S. maize growing regions. The Principal Investigator at each site was familiar 

with crop growth and production and evaluation of crop characteristics. The study was established 

at each site in a randomized complete block design with four replications. Plot and row dimensions 

are listed in Table H-2. 

H.4. Planting and Field Operations 

Planting information, soil description, and cropping history of the sites are listed in Table H-2. The 

Principal Investigator at each site followed local agronomic practices including those related to 

seed bed preparation and trial maintenance such as application of agricultural chemicals, fertilizer, 

and irrigation. All maintenance operations were performed uniformly across all plots within each 

site. 

 
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Table H-1. Starting Seed for Phenotypic, Agronomic, and Environmental Interactions 

Assessment of MON 95379 

Site 

Code1 Material Name Regulatory Lot Number Phenotype Material Type 

All MON 95379 11479279 Insect Resistant Test 

All LH244xHCL617 11479278 Conventional Control 

IAEH Dekalb DKC61-52 11427255 Conventional Reference 

IAEH Lewis 1407 11427264 Conventional Reference 

IAEH Golden Harvest G12J11-A 11446928 Conventional Reference 

IAEH LG Seeds LG2549 11446934 Conventional Reference 

IAOG Dekalb DKC61-52 11427255 Conventional Reference 

IAOG Dekalb DKC62-06 11427256 Conventional Reference 

IAOG Golden Harvest G09C43 11446927 Conventional Reference 

IAOG Agrigold A6267 11446941 Conventional Reference 

IARL Lewis 1613 11427265 Conventional Reference 

IARL Stone 5820 11427266 Conventional Reference 

IARL Agrigold A6472 11446939 Conventional Reference 

IARL Agrigold A6574 11446940 Conventional Reference 

ILHY NH6769 11380173 Conventional Reference 

ILHY Golden Harvest G09C43 11446927 Conventional Reference 

ILHY LG Seeds LG2636 11446935 Conventional Reference 

ILHY Agrigold A6472 11446939 Conventional Reference 

ILJO Dekalb DKC64-85 11427257 Conventional Reference 

ILJO Lewis 1613 11427265 Conventional Reference 

ILJO Mycogen Seeds MY09V40 11446930 Conventional Reference 

ILJO Agrigold A6472 11446939 Conventional Reference 

INSH Kruger K-0708 11427267 Conventional Reference 

INSH Mycogen Seeds 2H721 11446932 Conventional Reference 

INSH LG Seeds LG2549 11446934 Conventional Reference 

INSH LG Seeds LG2636 11446935 Conventional Reference 

NEYO Dekalb DKC61-52 11427255 Conventional Reference 

NEYO Dekalb DKC64-85 11427257 Conventional Reference 

NEYO Golden Harvest G12J11-A 11446928 Conventional Reference 

NEYO LG Seeds LG2549 11446934 Conventional Reference 

OHTR Dekalb DKC62-06 11427256 Conventional Reference 

OHTR Dekalb DKC64-85 11427257 Conventional Reference 

OHTR Kruger K-0708 11427267 Conventional Reference 

OHTR Golden Harvest G09C43 11446927 Conventional Reference 

Note: Starting seed of test and control materials were produced in Kihei, HI in 2017 1 Site codes: IAEH = Shelby County, IA; IAOG = Boone County, IA; IARL = Jefferson County, IA; ILHY = Clinton 

County, IL; ILJO = Champaign County, IL; INSH = Clinton County, IN; NEYO = York County, NE; OHTR = Miami 

County, OH; 
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Table H-2. Field Information for Phenotypic, Agronomic, and Environmental Interactions Assessment of MON 95379 

Site Code1 

Planting 

Date2

Harvest 

Date2

Planting 

Rate

(seeds/m2) 

Rows/ 

Plot 

Interrow 

Distance 

(m) 

Row 

Length 

(m) 

Plot 

Size 

(m2) 

Soil Texture

Organic 

Matter 

(%) 

Previous 

Crop2017

IAEH 05/18/2018 10/24/2018 9.5 8 76 6.1 37.2 Silty Clay Loam 3.1 Soybeans IAOG 05/17/2018 10/23/2018 9.5 8 76 6.1 37.2 Loam 3.6 Soybeans IARL 05/07/2018 09/25/2018 9.5 8 76 6.1 37.2 Silty Clay Loam 3.1 Soybeans ILHY 05/22/2018 10/03/2018 9.5 8 76 6.1 37.2 Silt Loam 1.8 Soybeans ILJO 05/17/2018 10/01/2018 8.6 8 76 6.2 38.1 Silty Clay Loam 4.7 Maize INSH 05/08/2018 10/11/2018 9.5 8 76 6.1 37.2 Silty Clay Loam 3.4 Maize NEYO 05/04/2018 10/17/2018 9.5 8 76 6.1 37.2 Silty Clay Loam 2.4 Soybeans OHTR 05/09/2018 10/09/2018 9.5 8 76 6.4 39.0 Loam 2.3 Wheat 

1 Site codes: IAEH = Shelby County, IA; IAOG = Boone County, IA; IARL = Jefferson County, IA; ILHY = Clinton County, IL; ILJO = Champaign County, IL; 

INSH = Clinton County, IN; NEYO = York County, NE; OHTR = Miami County, OH. 

2 Date format = mm/dd/yyyy. 
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H.5. Phenotypic Characteristic Assessments 

Phenotypic characteristics assessed and the timing of each assessment are listed in Table VII-1. 

H.6. Environmental Interaction Assessments 

The test, conventional control, and commercial references were evaluated at each site for 

differences in plant responses to abiotic stressors, diseases, and arthropod pests. Evaluations were 

performed four times during the growing season at the following growth stages: V5 – V8, V12 –

R1, R1 – R3, and R4 – R5. 

The Field Co-operator at each site identified abiotic stressors, diseases, and arthropod pests that 

were either actively causing plant injury in the plots or likely to occur in maize during a given 

observation period. Recognizing that damage symptoms for certain stressors can be similar and 

sometimes difficult to identify to specific taxonomic levels, Field Cooperators experienced in 

agricultural research identified stressors to the best of their ability based on knowledge of what 

would most likely be occurring at that site and at that time of the season. Stressors assessed often 

varied among observations and sites. 

Ratings were made using the categorical scale of increasing severity listed below: 

Category Severity of plant damage 

None No symptoms observed 

Slight Symptoms not damaging to plant development (e.g., minor feeding or minor 

lesions); mitigation likely not required

Moderate Intermediate between slight and severe; likely requires mitigation1

Severe Symptoms damaging to plant development (e.g., stunting or death); 

mitigation unlikely to be effective

1

Pest management is based on pest pressure threshold to represent commercial crop production 

practices that would produce an agronomically acceptable crop (Appendix L). 

H.7. Statistical Analysis/Data Summarization 

A combined-site analysis was conducted according to a randomized complete block design using 

SAS® (SAS, 2012) to compare the test to the conventional control for the phenotypic 

characteristics listed in Table VIII-1. The level of statistical significance was predetermined to be 

5% (α = 0.05). Descriptive statistics are provided for one characteristic, seed loss, that had 

insufficient variability for formal statistical analysis. The reference range for each phenotypic 

characteristic was determined from the minimum and maximum mean values among the 17 
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references, where each mean was combined over all the sites at which the reference was planted. 

There were no plots for which data were excluded from the study. 

The environmental interactions data consisting of plant responses to abiotic stressors, diseases, 

and arthropod pests are categorical and were considered different in susceptibility or tolerance if 

the range of injury symptoms did not overlap between the biotechnology-derived crop and the 

conventional control across all four replications within an observation (see Appendix L for 

additional context regarding assessment of potential differences between the test and conventional 

control). 

H.8. Detailed Results for Environmental Interactions Assessments for MON 95379 

No differences were observed between the test and the conventional control for any of the 96 

comparisons for the assessed abiotic stressors: cold temperatures, excessive rain, hail, high 

temperatures, high winds, mineral toxicity, nutrient deficiency, soil compaction, and sun scald 

(Table H-3). 

No differences were observed between the test and the conventional control for any of the 96 

comparisons for the assessed arthropod pests: aphids, billbugs, corn flea beetles, corn rootworm 

beetles, grape colaspis, grasshoppers, Japanese beetles, leafhoppers, sap beetles, slugs, spider 

mites, stink bugs, and thrips (Table H-4). 

No differences were observed between the test and the conventional control for any of the 96 

comparisons for the assessed diseases: anthracnose, common rust, common smut, corn stunt, ear 

root, eyespot, Goss’s bacterial wilt, gray leaf spot, northern leaf blight, northern leaf spot, 

physoderma brown spot, southern rust, stalk root, and Stewart’s wilt (Table H-5). 

The results of the assessed environmental interactions support the conclusion that MON 95379 is 

not expected to pose increased plant pest potential compared to conventional maize. 

 
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Table H-3. Summary of Abiotic Stressor Response Evaluations for MON 95379 and the 

Conventional Control in 2018 U.S. Field Trials 

Abiotic Stressor

Total 

Observations 

across Sites

Number of Observations 

without Differences between the 

Test and the Conventional 

Control 

Total 96 96 

Cold Temperatures 3 3 

Excessive Rain (i.e. waterlogging) 14 14 

Hail 13 13 

High Temperatures 10 10 

High Winds (lodged plants) 25 25 

Mineral toxicity 2 2 

Nutrient deficiency 15 15 

Soil compaction 8 8 

Sun scald 6 6 

Notes: No differences were observed between the test and the conventional control during any observation 

for responses to any of the assessed abiotic stressors. Categorical data were summarized across sites and 

observation times. 

 
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Table H-4. Summary of Arthropod Damage Evaluations for MON 95379 and the 

Conventional Control in 2018 U.S. Field Trials 

Arthropod

Total 

Observations 

across Sites 

Number of Observations 

without Differences 

between the Test and the 

Conventional Control 

Total 96 96 

Aphids (Aphididae) 15 15 

Billbugs (Curculionidae) 1 1 

Corn flea beetles (Chrysomelidae) 10 10 

Corn rootworm beetles (Chrysomelidae) 13 13 

Grape colaspis (Chrysomelidae) 2 2 

Grasshoppers (Acrididae) 19 19 

Japanese beetles (Scarabaeidae) 14 14 

Leafhoppers (Cicadellidae) 4 4 

Sap beetles (Nitidulidae) 2 2 

Slugs1 1 1 

Spider mites (Tetranychidae) 6 6 

Stink bugs (Pentatomidae) 8 8 

Thrips (Thripidae) 1 1 

Notes: No differences were observed between the test and the conventional control during any 

observation for damage caused by any of the assessed arthropods. Categorical data were 

summarized across sites and observation times. 

1

 Slugs are not arthropods but are occasional pests in maize 

 
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Table H-5. Summary of Disease Damage Evaluations for MON 95379 and the 

Conventional Control in 2018 U.S. Field Trials 

Disease

Total 

Observations 

Across Sites 

Number of 

Observations Without 

Differences Between 

MON 95379 and the 

Conventional Control 

Total 96 96 

Anthracnose (Colletotrichum graminicola) 13 13 

Common rust of maize (Puccinia sorghi) 16 16 

Common smut of maize (Ustilago maydis) 9 9 

Corn stunt (Spiroplasma kunkelii) 3 3 

Eyespot (Aureobasidium zeae) 9 9 

Goss's bacterial wilt (Clavibacter michiganensis

subsp. nebraskensis) 4 4 

Gray leaf spot (Cercospora zeae-maydis) 19 19 

Northern leaf blight of maize (Exserohilum 

turcicum) 15 15 

Northern leaf spot (Bipolaris zeicola) 2 2 

Physoderma brown spot (Physoderma maydis) 1 1 

Southern rust of maize (Puccinia polysora) 1 1 

Stalk Rot (includes Colletotrichum graminicola, 

Erwinia chrysanthemi pv. zeae, Macrophomina 

phaseolina, Stenocarpella maydis, Fusarium spp., 

Pythium aphanidermaturm) 

2 2 

Stewart's wilt (Pantoea stewartii) 2 2 

Notes: No differences were observed between the test and the conventional control during any observation 

for damage caused by any of the assessed diseases. Categorical data were summarized across sites and 

observation times. 

 
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References for Appendix H 

SAS. 2012. Software Release 9.4 (TS1M4). Copyright 2002-2012 by SAS Institute, Inc., Cary, 

North Carolina. 

 
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Appendix I: Materials and Methods for Compositional Analysis of MON 95379 

Maize Grain and Forage 

Compositional comparisons between MON 95379 and the conventional control maize hybrid were 

performed using the principles and analytes outlined in the OECD consensus document for maize 

composition (OECD, 2002). These principles are accepted globally and have been employed 

previously in assessments of maize products derived through biotechnology. The compositional 

assessment was conducted on grain and forage samples harvested from multiple U.S. field sites 

during 2018 grown under typical agronomic field conditions practices. 

I.1. Materials 

Harvested grain and forage from MON 95379 and a conventional control that has similar genetic 

background to that of MON 95379 hybrid were compositionally assessed. 

I.2. Characterization of the Materials 

The identities of MON 95379 and the conventional control were confirmed prior to use in the 

compositional assessment. 

I.3. Field Production of the Samples 

Grain and forage samples were harvested from MON 95379 and the conventional control grown 

in the United States during the 2018 season. The field production was conducted at five sites. The 

field sites were planted in a randomized complete block design with four replicates per site. 

MON 95379 and the conventional control were grown under normal agronomic field conditions 

for their respective growing regions. 

Grain was harvested at physiological maturity and shipped at ambient temperature from the field 

sites to Bayer. Forage was harvested at R5 and shipped on dry ice from the field sites to Bayer. A 

subsample for compositional analysis was obtained from each tissue sample collected. These 

subsamples were ground and stored in a freezer set to maintain 20qC until their shipment on dry 

ice to Eurofins Food Integrity & Innovation Laboratories Inc. (Madison, Wisconsin) for analysis. 

I.4. Summary of Analytical Methods 

Nutrients analyzed in this study included protein, total fat, ash, amino acids, linoleic acid, 

carbohydrates by calculation, fiber (acid detergent fiber (ADF), neutral detergent fiber (NDF)) in 

the grain, protein, total fat, ash, carbohydrates by calculation and fiber (ADF and NDF) in the 

forage. The anti-nutrients assessed in grain included phytic acid and raffinose. 

All compositional analyses were performed at Eurofins Food Integrity & Innovation Laboratories, 

Inc. (Madison, Wisconsin). Methods for analysis were based on internationally-recognized 
Bayer CropScience LP CR282-20U1 312 

procedures and literature publications. Brief descriptions of the methods utilized for the analyses 

are described below. 

I.5. Acid Detergent Fiber 

Sample aliquots were weighed into pre-weighed filter bags. Samples were placed in an ANKOM 

Fiber Analyzer and treated with an acid detergent solution containing sulfuric acid with cetyl 

trimethylammonium bromide, then filtered to remove proteins, starches, simple sugars, pectins, 

and ash. Fats and pigments were removed via an acetone wash leaving cellulose and lignin 

fractions. The remaining residue was the acid detergent fiber and was determined gravimetrically. 

The results are reported on a fresh weight (fw) basis. The limit of quantitation was calculated as 

1.00%. 

I.6. Amino Acids 

The following 10 amino acids were analyzed: 

Total arginine Total methionine 

Total histidine Total phenylalanine 

Total isoleucine Total threonine 

Total leucine Total tryptophan 

Total lysine Total valine 

The samples were hydrolyzed in 6N hydrochloric acid for approximately 24 hours at 

approximately 106-118oC. Tryptophan was hydrolyzed from proteins by heating at approximately 

110oC in 4.2N sodium hydroxide for approximately 20 hours. 

The samples were analyzed by HPLC after pre-injection derivatization. The primary amino acids 

were derivatized with o-phthalaldehyde (OPA) and the secondary amino acids were derivatized 

with fluorenylmethyl chloroformate (FMOC) before injection. The amino acids were quantified 

using external standards. The results are reported on a fresh weight basis. The limit of quantitation 

for this study was 0.100 mg/g. 

 
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Reference Standards: 

Component Manufacturer Lot No. Purity (%)

L-Arginine Monohydrochloride Sigma-Aldrich SLBT2948 100 

L-Histidine Monohydrochloride Monohydrate Sigma-Aldrich SLBS4669 100 

L-Isoleucine Sigma-Aldrich SLBS6030 100 

L-Leucine Sigma-Aldrich SLBS4968 100 

L-Lysine Monohydrochloride Sigma-Aldrich SLBM5436V 100 

L-Methionine Sigma-Aldrich SLBS7026 100 

L-Phenylalanine Sigma-Aldrich SLBQ7928V 100 

L-Threonine Sigma-Aldrich SLBT5994 100 

L-Tryptophan Sigma-Aldrich BCBT8640 100 

L-Valine Sigma-Aldrich SLBT4145 99 

I.7. Ash 

All organic matter was driven off when the samples were ignited at approximately 550oC in a 

muffle furnace for at least 5 hours. The remaining inorganic material was determined 

gravimetrically and referred to as ash. The results are reported on a fresh weight basis. The limit 

of quantitation was 0.100%. 

I.8. Carbohydrate 

The total carbohydrate level was calculated by difference using the fresh weight-derived data and 

the following equation: 

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash) 

The limit of quantitation was calculated as 0.100% on a fresh weight basis. 

I.9. Fat by Acid Hydrolysis 

The samples were hydrolyzed with hydrochloric acid. The fat was extracted using ether and 

hexane. The extracts were dried down and filtered through a sodium sulfate column. The 

remaining extracts were then evaporated, dried, and weighed. The limit of quantitation was 

calculated as 0.100% on a fresh weight basis. 

I.10. Fat by Soxhlet Extraction 

The samples were weighed into a cellulose thimble containing sodium sulfate and dried to remove 

excess moisture. Pentane was dripped through the samples to remove the fat. The extract was then 
Bayer CropScience LP CR282-20U1 314 

evaporated, dried, and weighed. The results are reported on a fresh weight basis. The limit of 

quantitation was calculated as 0.100%. 

I.11. Linoleic Acid 

The lipid was extracted and saponified with 0.5N sodium hydroxide in methanol. The 

saponification mixture was methylated with 14% boron trifluoride in methanol. The resulting 

methyl esters were extracted with heptane containing an internal standard. The methyl esters of 

the fatty acids were analyzed by gas chromatography using external standards for quantitation. 

The results were converted to their triglyceride equivalent and reported on a fresh weight basis. 

The Methyl Linoleate analytical reference standard was purchased from Nu-Chek Prep. The lot 

numbers were Z-2 and X-2 with a purity of 99.8%. The limit of quantitation was calculated as 

0.00245%. 

I.12. Moisture 

The samples were dried in a vacuum oven at approximately 100qC. The moisture weight loss was 

determined and converted to percent moisture. The results are reported on a fresh weight basis. 

The limit of quantitation was calculated as 0.100%. 

I.13. Neutral Detergent Fiber 

Sample aliquots were weighed into pre-weighed filter bags. Samples were placed in an ANKOM 

Fiber analyzer and treated with a neutral detergent solution containing EDTA. Samples were then 

filtered to remove proteins, simple sugars, pectins, and ash. Fats and pigments were removed via 

an acetone wash leaving hemicellulose, cellulose, and lignin fractions. Starches were removed 

with a heat stable alpha amylase soak. The remaining residue was the neutral detergent fiber and 

was determined gravimetrically. The results are reported on a fresh weight basis. The limit of 

quantitation was calculated as 1.00%. 

I.14. Phytic Acid 

The samples were extracted using hydrochloric acid and sonication, purified using a silica-based 

anion exchange column, concentrated and injected onto a high-performance liquid 

chromatography (HPLC) system with a refractive index detector. The Phytic Acid Sodium Salt 

Hydrate analytical reference standard was purchased from Sigma-Aldrich. The lot number was 

BCBQ7037V with a purity of 79.909%. The results are reported on a fresh weight basis. The limit 

of quantitation was calculated as 0.125%. 
Bayer CropScience LP CR282-20U1 315 

I.15. Protein 

The protein and other organic nitrogen in the samples were converted to ammonia by digesting the 

samples with sulfuric acid containing a catalyst mixture. The acid digest was made alkaline. The 

ammonia was distilled and then titrated with a previously standardized acid. Instrumentation was 

used to automate the digestion, distillation and titration processes. The percent nitrogen was 

calculated and converted to equivalent protein using the factor 6.25. The results are reported on a 

fresh weight basis. The limit of quantitation was calculated as 0.100%. 

I.16. Raffinose 

Sugars in the samples were extracted with a 50:50 water: methanol solution. Aliquots were taken, 

dried under inert gas, and then reconstituted with a hydroxylamine hydrochloride solution in 

pyridine containing phenyl-β-D-glucopyranoside as the internal standard. The resulting oximes 

were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid 

treatment, and then analyzed by gas chromatography using a flame ionization detector. The D-

(+)-Raffinose pentahydrate analytical reference standard was purchased from Sigma-Aldrich. The 

lot number was WXBC5581V with a purity of 99.1%. The results are reported on a fresh weight 

basis. The limit of quantitation was calculated as 0.0500%. 

I.17. Data Processing and Statistical Analysis 

After compositional analyses were performed, data spreadsheets containing individual values for 

each analysis were sent to Bayer for review. Data were then transferred to the Bayer Regulatory 

Statistics Team where they were converted into the appropriate units and statistically analyzed. 

The following formulas were used for re-expression of composition data for statistical analysis 

(Table I-1): 

 
Bayer CropScience LP CR282-20U1 316 

Table I-1. Re-expression Formulas for Statistical Analysis of Composition Data 

Component From (X) To Formula1

Proximates, Fiber, Phytic Acid, 

Raffinose % fw % dw X/d 

Amino Acids (AA) mg/g fw % dw X/(10d) 

Fatty Acids (FA), Linoleic Acid % fw % Total FA 

(100)Xj/6X, for 

each FAj where 

6X is over all the 

FA 

1

‘X’ is the individual sample value; d is the fraction of the sample that is dry matter.

In order to complete a statistical analysis for a compositional constituent in this compositional assessment, at least 

50% of all the values for an analyte in grain or forage had to be greater than the assay limit of quantitation (LOQ). 

No analytes with more than 50% of observations below the assay LOQ were observed. 

The following linear mixed model was used for the combined-site analysis. 

Yijk = μ + Si + R(S)j(i) + Mk + (SM)ik + εijk (1) 

where: 

Yijk is the observed response for the kth substance in the jth replicate of the ith site; 

μ is the overall mean;

Si is the random effect of the ith site; 

R(S)j(i) is the random effect of the jth replicate nested with the ith site; 

Mk is the fixed effect of the kth substance; 

(SM)ik is the random effect of the interaction between the ith site and kth substance; 

εijk is the residual error. 

SAS PROC MIXED was used to fit model (1) separately for each component to conduct the statistical analysis. 

Studentized residuals were obtained to detect potential outliers in the dataset. Studentized residuals tend to have a 

standard normal distribution when outliers are absent. Thus, most values are expected to be between ± 3. Data points 

that are outside of the ± 6 studentized residual ranges are considered as potential outliers. No value had a studentized 

residual outside of the ± 6 range for this study. 

The linear mixed model (1) assumes that the experimental errors, εijk, are independent, normallydistributed, and have a common variance. In this analysis, independence of the errors was 

controlled by the randomized complete block design. The normality and common variance 

assumptions were checked by visual examination of residual plots and histograms. No extreme 

violations were observed for any characteristic. Comparisons between MON 95379 and 

conventional control were defined within the model (1) and tested using t-tests. 

 
Bayer CropScience LP CR282-20U1 317 

References for Appendix I

OECD. 2002. Consensus document on compositional considerations for new varieties of maize 

(Zea mays): Key food and feed nutrients, anti-nutrients and secondary plant metabolites. OECD 

ENV/JM/MONO (2002)25. Organisation of Economic Co-Operation and Development, Paris, 

France. 

 
Bayer CropScience LP CR282-20U1 318 

Appendix J: Materials, Methods, and Results for Tier I NTO Testing 

J.1. Evaluation of the Potential for Interaction between the Cry1Da_7 and Cry1B.868 

Proteins Expressed in MON 95379 with Fall Armyworm (Spodoptera frugiperda) 

Introduction 

The stacking of Cry proteins that confer distinct modes of action in combined trait products is an 

essential tool to help to control key insect pests while delaying the onset of insect resistance. 

Evaluation of the potential interaction between multiple Cry proteins is conducted as an early step 

of the safety assessment for Cry proteins produced by conventional breeding or as a vector stack. 

In 2009, EPA published a codified framework and data requirements for assessing the safety of 

combined trait products (U.S. EPA, 2009). These data are used to bridge to the existing risk 

assessments for the individual Cry proteins. The first two requirements are to demonstrate that the 

composition of the inserted genetic material is unchanged and the expression (i.e., exposure) level 

Cry proteins in the single and combined trait products are comparable. Neither of these data 

requirements apply to MON 95379 maize as both proteins are expressed as a vector stack. 

The third component of a combined trait bridging assessment is to assess for a potential 

toxicological interaction between the Cry proteins in a sensitive species (U.S. EPA 2009). 

Historically, EPA has referred to this study as the “synergy” study. The term “synergism” can 

sometimes have negative or confounding connotations in other applications; therefore, the more 

precise term “Greater than Additive” (GTA) will be used. Additionally, the term “Less than 

additive” (LTA) interaction will be used (e.g., 5 + 5 = 1) rather than the term antagonism and does 

not constitute a safety concern for Cry proteins. A GTA interaction (e.g., 5 + 5 = 50) could impact 

conclusions of the risk assessment and if the level of GTA activity is sufficiently high, additional 

testing with a mixture of the Cry proteins could be required. Recommended guidance from EPA 

indicates that only levels of combined activity in a sensitive species >10-fold above that expected 

through additivity would constitute a need for additional testing in non-target organisms (EPA, 

2009). This recommended threshold is based on margins of exposure t10 that are generally 

achieved in NTO testing. 

The design of an interaction study with two proteins should be based on the null hypothesis of no 

interaction. Results from resistant insect bioassays, disabled insecticidal protein bioassays, and 

cell-based assays using insect cell lines expressing individual receptors indicate that CryB.868 and 

Cry1Da_7 utilize receptors that are distinct from each other as well as other commercialized Bt 

proteins (Wang et al., 2019). This makes Cry1B.868 and Cry1Da_7 amenable to use in combined 

trait products both together and with other commercialized Bt proteins making them a potentially 

valuable tool for Insect Resistance Management programs. While Wang et al. does not directly 

address the potential for GTA activity in a sensitive species, the results demonstrate that 

Cry1B.868 and Cry1Da_7 act through distinct receptors supporting the hypothesis that Cry1B.868 
Bayer CropScience LP CR282-20U1 319 

and Cry1Da_7 act would be expected to act independently. Therefore, when expressed together in 

MON 95379, their activities would be predicted to be additive against a species that is susceptible 

to both proteins (e.g., 5 + 5 = 10). 

The chosen test species for an interaction study must be sensitive to at least one protein with the 

underlying notion that an interaction between the two proteins is most likely to be observed in a 

sensitive species where responses can be quantifiably measured. This approach is consistent with 

the expectation that for NTOs, where exposure levels for proteins with different modes of action 

(i.e., act independently) are below those needed to elicit adverse effects, a response from the 

combination of proteins is not predicted (e.g., 0 + 0 = 0 or NOEC + NOEC = NOEC; EPA 2009; 

Levine and Borgert, 2018). However, if the margins of exposure for an NTO are low, (e.g., effects 

observed at near field expression levels), and a GTA response is observed in a sensitive species, 

then additional studies may be needed to evaluate the impact of a mixture of both proteins on that 

NTO species. 

To assess for a potential interaction between Cry1B.868 and Cry1Da_7, Fall armyworm (FAW) 

was selected as a test species because it is sensitive to both Cry1B.868 and CryDa_7 in laboratory 

diet-incorporation bioassays. In the interaction study described below, the 50% effect levels for 

Cry1B.868 and Cry1Da_7 alone and in combination were evaluated and their combined predicted 

activity was calculated with the concentration addition model. The concentration addition model 

provides results generally similar to other mixture models like response addition, but in some 

circumstances, can be more conservative by estimating higher combined activity (Finney, 1971; 

Levine and Borgert, 2018). Therefore, the concentration addition model is EPA’s default model 

for evaluation of mixtures of toxicants with similar modes of action (EPA, 1986, Tabashnik et al., 

1994). 

 
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Using growth-inhibition as a sensitive endpoint with FAW, the concentration addition model is 

given by: 

ଵ

௉௥௘ௗ௜௖௧௘ௗ ீூఱబ

= 

గ௔

ீூఱబ௔

+ గ௕

ீூఱబ௕

 

πa and πb are the proportions of the relative expression of Cry1B.868 and 

Cry1Da_7 proteins in MON 95379 maize tissue. 

GI50a and GI50b represent the observed GI50 values obtained with the 

Cry1B.868 and Cry1Da_7 single-expressing lines. 

If the predicted combined activity (GI50) falls within the 95% confidence interval (CI) of the 

observed combined activity for Cry1B.868 and Cry1Da_7 it can be concluded that combined 

activity is consistent with additivity. Interpretation of mixture toxicity calculations are commonly 

based on constructing a CI around the fitted effect of the response and then analyzing whether the 

predicted effect is captured by this CI (Tabashnik, 1992; Kudsk and Mathiassen, 2004; Levine and 

Borgert, 2018; Levine et al., 2016; Levine et al., 2019). A CI quantifies the degree of uncertainty 

around an estimate of a predicted or observed endpoint of interest. The advantage of this approach 

is that it considers the uncertainty in the predicted and/or observed response. 

Results of the interaction study indicate that the combined activity of Cry1B.868 and Cry1Da_7 is 

additive, and this conclusion is consistent with the other lines of evidence showing that the two 

proteins act independently. Details of the study design, analysis and interpretation in context of 

the NTO assessment are provided below. Finally, additive activity of Cry1B.868 and Cry1Da_7 

are conservatively assessed with a supplementary Hazard Index analysis adding together risk 

quotients from the independent assessments for each protein. The results of this analysis confirm 

the conclusion that adverse effects on NTOs from exposure to Cry1B.868 and Cry1Da_7 at the 

levels expressed in MON 95379 maize are not anticipated. 

Purpose 

The purpose of this study was to evaluate the potential for a toxicological interaction between the 

lepidopteran-active Cry1B.868 and Cry1Da_7 proteins in MON 95379. 

Experimental Design 

The study was designed to test the null hypothesis of no interaction between Cry1B.868 and 

Cry1Da_7 and was assessed using a concentration addition model. Cry protein expression was 

determined in tissues collected from Cry1B.868 and Cry1Da_7 single events as well as 

MON 95279 tissues using a quantitative ELISA. This analysis was conducted such that activities 

of Cry1B.868 and Cry1Da_7 could be expressed with respect to protein concentration to determine 
Bayer CropScience LP CR282-20U1 321 

the relative proportions of Cry1B.868 and Cry1Da_7 expressed in MON 95379 tissue. The activity 

of the Cry1B.868 and Cry1Da_7 in single event tissues as well as MON 95379 tissue was assessed 

with diet-incorporation bioassays using the sensitive species, FAW. The bioassay was repeated on 

three separate days using different batches of insects. The predicted combined activity was 

calculated based on the activity of Cry1B.868 and Cry1Da_7 single events and was compared to 

the observed activity of MON 95379 tissue. Specifically, the predicted combined activity of 

Cry1B.868 and Cry1Da_7 was compared to the 95% confidence interval for the observed activity 

of MON 95379 maize to determine whether to reject the null hypothesis of no interaction. 

Test Materials 

The test and control substances were finely-ground lyophilized tissue from the materials listed in 

Table J-1. Leaf tissue was harvested when all plants reached the V8 growth stage, and then stored 

in a -80 °C freezer before and after processing. 

Table J-1. Summary of test and control substances 

Substance Name 

(Treatment)1 IP Protein2 Phenotype

MON 95379 Cry1B.868 & Cry1Da_7 Insect-protected 

Cry1B.868-single Cry1B.868 Insect-protected 

Cry1Da_7-single Cry1Da_7 Insect-protected 

Conventional Control 

(Assay Control) None Conventional 

1 All test and control substances have a common germplasm. 

2 IP = Insect-protected 

J.1.1. Protein Expression Analysis and Tissue Preparation 

Protein levels in MON 95379, the Cry1B.868-single, and Cry1Da_7-single tissues were quantified 

using ELISA. These values were used to convert the tissue amounts of each test and control 

treatment into μg of total protein/mL diet. Additionally, the ratio of each individual protein in 

MON 95379 was used in the concentration addition model to calculate the predicted GI50 value 

for the combined trait product. The protein analysis data summary results are provided in Table J2. Since lyophilized tissues were used in this study, the fresh weight values determined by ELISA 

were converted into dry weight values (Table J-2). Tissue samples utilized in this study were 

primary milled, freeze dried, and ground again prior to study initiation. 

 
Bayer CropScience LP CR282-20U1 322 

TableJ-2. Summary of protein levels in leaf tissue with the fresh weight values converted to 

dry weight values. 

Protein Material Name 

Mean Bt

Concentration 

(μg/g fwt)

Moisture 

Conversion 

Factor for 

OSL-1 Corn 

Leaf Tissue 

Mean Bt

Concentration 

(μg/g dwt) 

Cry1B.868 

Conventional <LOQ1 0.15 <LOQ 

Cry1Da_7-single <LOQ1 0.15 <LOQ 

Cry1B.868-

single 81 0.15 540 

MON 95379 86 0.15 573 

Cry1Da_7 

Conventional <LOQ1 0.15 <LOQ 

Cry1Da_7-single 4.7 0.15 31 

Cry1B.868-

single <LOQ1

 0.15 <LOQ

MON 95379 17 0.15 113 

1

LOQ = Limit of quantitation 

J.1.2. FAW Bioassay Testing Procedure 

Prior to the start of the assay, FAW eggs were placed into covered hatch-boxes and held at a target 

temperature of 10°C prior to being incubated for hatching at a target temperature of 27°C. A dietincorporation insect bioassay procedure was used to characterize the biological activity of the 

treatments shown in Table J-1. 

Treatments were prepared by mixing a dosing solution that contained finely-ground lyophilized 

leaf tissue and purified water with an agar-based multiple-species diet (Southland) to achieve a 

final volume and vortex-mixed until visually homogenous. A repeat pipettor was used to dispense 

16, 1.0 mL aliquots of each treated diet mixture into individual wells of 128-well bioassay trays. 

The diet was allowed to cool and solidify before adding one newly hatched FAW to each well. 

Each bioassay replicate contained a target number of 16 individually housed FAW larvae per 

treatment which were covered with a ventilated adhesive cover. Three conventional control 

treatments, each containing 10.0 mg of conventional control tissue/ml of diet, were included with 

each of three bioassays replicates. The treatment concentrations were chosen to appropriately 

characterize concentration-response relationships for each of the test substances. The original 

treatment concentrations were mg tissue/mL diet. However, prior to statistical analysis, the 
Bayer CropScience LP CR282-20U1 323 

concentrations for each treatment were converted to μg Bt protein/mL diet using the dry-weight 

converted values from the tissue expression analysis (Table J-2 and Table J-3). For the 

MON 95379 treatments, six diet concentrations (0.054, 0.11, 0.21, 0.43, 0.86 and 1.72 μg total Bt

protein (Cry1B.868 and Cry1Da_7)/mL diet) were used, for the Cry1B.868-single treatment, six 

diet concentrations (0.042, 0.084, 0.16, 0.34, 0.68, and 1.35 μg Cry1B.868/mL diet) were used, 

and for the Cry1Da_7-single treatment, six diet concentrations (0.0097, 0.019, 0.039, 0.078, 0.16 

and 0.31 μg Cry1Da_7/mL diet) were used in each of the three bioassay replicates. To ensure each 

treatment level replicate contained an approximately equal amount of tissue, conventional control 

tissue was added to each treatment level as necessary to obtain a final tissue concentration of 

approximately 10.0 mg tissue/mL of diet. Bioassay trays were incubated at a target temperature of 

27°C, 60% relative humidity and a 14h:10h light:dark photoperiod. The bioassay was replicated 

three times on separate days using separate batches of insects. 

At the end of each seven-day incubation period for each bioassay replicate, the actual number of 

larvae infested, the number of surviving larvae, and the combined larval mass of the surviving 

insects were recorded for each treatment concentration. 

For each bioassay replicate, the test and control treatments were run concurrently on the same day 

with the same batch of insects. The bioassay was replicated three times on separate days with 

separate batches of insects. The bioassay was considered valid if the mean mortality was <20% 

and the calculated GI50 for each treatment group was bracketed by at least one treatment level. 

 
Bayer CropScience LP CR282-20U1 324 

Table J-3. Summary of protein levels in leaf tissue with the fresh weight values converted to 

dry weight values. 

Treatment Description 

mg of 

Tissue 

(dwt)/ml 

of Diet 

Mean Bt

Concentration 

in Tissue 

(μg/mg dwt)1

μg Bt 

Protein/ml of 

Diet 

MON 95379 Tissue 0.078 0.686 0.054 

MON 95379 Tissue 0.156 0.686 0.11 

MON 95379 Tissue 0.313 0.686 0.21 

MON 95379 Tissue 0.625 0.686 0.43 

MON 95379 Tissue 1.25 0.686 0.86 

MON 95379 Tissue 2.5 0.686 1.72 

Cry1B.868-single Tissue 0.078 0.540 0.042 

Cry1B.868-single Tissue 0.156 0.540 0.084 

Cry1B.868-single Tissue 0.313 0.540 0.16 

Cry1B.868-single Tissue 0.625 0.540 0.34 

Cry1B.868-single Tissue 1.25 0.540 0.68 

Cry1B.868-single Tissue 2.5 0.540 1.35 

Cry1Da_7-single Tissue 0.313 0.031 0.0097 

Cry1Da_7-single Tissue 0.625 0.031 0.019 

Cry1Da_7-single Tissue 1.25 0.031 0.039 

Cry1Da_7-single Tissue 2.5 0.031 0.078 

Cry1Da_7-single Tissue 5.0 0.031 0.16 

Cry1Da_7-single Tissue 10 0.031 0.31 

1

Mean Bt concentration in MON 95379 is the sum of Cry1B.868 and Cry1Da_7 in MON 95379 

from Table J-2.
Bayer CropScience LP CR282-20U1 325 

J.1.3. Data Analysis and Interpretation 

GI50 values for the concentration-response of the test substances were estimated along with their 

95% CIs by logistic regression (Table J-5). The data from the three replicate bioassays performed 

on separate days was analyzed in a combined analysis. 

Under the concentration addition model, the activity of Cry1B.868 (GI50a) and Cry1Da_7 (GI50b) 

were added to predict the activity of MON 95379 (Finney 1971; Tabashnik 1992). The predicted 

GI50 values were calculated as follows: 1/predicted GI50 = πa/GI50a + πb/GI50b, where πa and πb 

are the proportions of the two single trait products expressed in MON 95379. Using the expression 

data in Table J-3 to convert from mg tissue /ml diet to ug Bt protein/ml diet the proportions of the 

single proteins in the combined trait were calculated as: 

Table J-4. Relative Concentrations of Cry1B.868 and Cry1Da_7 in Lyophilized Leaf 

Tissue from MON 95379 Maize 

Protein Type Protein Expression 

(μg/g dwt) Proportion 

Cry1B.868 573 πa = 573/(573+113) or 0.835 

Cry1Da_7 113 πb = 113/(573+113) or 0.165 

Results 

Bioassay replicates 1 and 2 both had no mortality and replicate 3 had two dead insects out of 45 

infested for 4.4% mortality thereby meeting the acceptance criteria of less than or equal to 20% 

mean mortality for the assay control treatment. All treatments in all replicates had at least one 

growth inhibition response between both 10% - 50% and 50% - 90% relative to the mean control 

response. 

An assessment of potential interaction between Cry1B.868 and Cry1Da_7 was evaluated with the 

concentration addition model. The concentration addition model is a well-established model to 

assess deviations from additivity and is based on the premise that the biological activity of the 

combined trait product can be predicted directly from the biological activity of the individual 

components. Under this model, a conclusion of additivity (i.e., no interaction) can be made if the 

predicted GI50 value calculated from the individual components is within the 95% CI of the 

observed GI50 value for the mixture (Levine et al.,2016; Tabashnik, 1992).

In seven-day diet incorporation bioassays with tissue from MON 95379, Cry1B.868-single, and 

Cry1Da_7-single, FAW demonstrated concentration-dependent responses for growth inhibition 

(Figure J-1). The observed GI50 values, from the combined analyses of three bioassay replicates, 
Bayer CropScience LP CR282-20U1 326 

for MON 95379, Cry1B.868-single and Cry1Da_7-single were 0.35 μg total protein (Cry1B.868 

and Cry1Da_7)/ml diet, 0.34 μg Cry1B.868/ml diet, and 0.27 μg Cry1Da_7/ml diet, respectively 

(Table J-4). 

Predicted and observed GI50 values for combined activity of Cry1B.868 and Cry1Da_7 were 

similar (Table J-5). The predicted GI50 value for Cry1B.868 and Cry1Da_7 using the concentration 

addition model was 0.33 μg total protein (Cry1B.868 and Cry1Da_7)/ml diet and was captured 

within the 95% CI for the observed GI50 value for MON 95379 (95% CI 0.29 to 0.42 μg total 

protein (Cry1B.868 and Cry1Da_7)/ml diet) indicating that combined activity was consistent with 

additivity. Additionally, the 95% confidence intervals for both the observed and predicted GI50

values for MON 95379 overlap with one another [95% CI 0.29 to 0.42 and 0.27 to 0.38 μg total 

protein (Cry1B.868 and Cry1Da_7)/ml diet)]. 

Table J-5. Observed seven-day FAW GI50 values and 95% confidence intervals (CI) for 

MON 95379, Cry1B.868-single, and Cry1Da_7-single and the predicted GI50 value and 

95% CI for MON 95379. 

Substance Name (Treatment) Estimated GI50 Value 

(μg Bt Protein/ml Diet)1

95% CI 

(μg Bt Protein/ml 

Diet) 

MON 95379 (Observed) 0.35 0.29 – 0.42 

Cry1B.868-single 0.34 0.28 – 0.40 

Cry1Da_7-single 0.27 0.20 – 0.34 

Substance Name (Treatment) Predicted GI50 Value 

(μg Protein/ml Diet)1,2

95% CI 

(μg Bt Protein/ml 

Diet)3

MON 95379 (Predicted) 0.33 0.27 – 0.38 

1

The slope parameter for concentration response curves was shared for each regression since the 

slopes were determined to not be different for each treatment. 

2

Predicted GI50 value for MON 95379: 1/Predicted GI50 = 0.835/0.34 + 0.165/0.27 

3

Calculated using the delate method.
Bayer CropScience LP CR282-20U1 327 

Figure J-2. Tissue from MON 95379, Cry1B.868-single, and Cry1Da_7-single resulted in 

concentration-dependent growth inhibition in seven-day diet-incorporation FAW bioassays 

(GraphPad, 2019). 

Study Conclusions 

These results demonstrate that the activity of Cry1B.868 and Cry1Da_7 is consistent with 

additivity (i.e., no interaction) in the combined trait product, MON 95379. Predicted and observed 

activity levels differed by only 6% (with overlapping 95% confidence intervals). The lack of 

interaction in addition to large margins of exposure and lack of adverse effects demonstrated in 

the NTO assessment support the conclusion that Cry1B.868 and Cry1Da_7 can be assessed 

independently. 

J.1.4. Hazard Index Assessment for CryB.868 and Cry1Da_7 

A supplementary assessment was conducted using the Hazard Index (HI) approach that 

conservatively evaluates the combined risk of Cry1B.868 and Cry1Da_7 to NTOs. Based on 

previous guidance and results from the interaction study, the expectation would be that MOE 

values established in the individual assessments of Cry1B.868 and Cry1Da_7 would be sufficiently 

protective and effects from the additive activity of Cry1B.868 and Cry1Da_7 would not be 

0.0 0.5 1.0 1.5 2.0

0

20

40

60

80

100

120

140

ug Bt Protein/ml Diet

Mean Insect Mass (mg)

Cry1B.868-single

Cry1Da_7-single

MON 95379
Bayer CropScience LP CR282-20U1 328 

anticipated. The HI represents a highly conservative approach to assess the potential impact of 

toxicant mixtures when their activities have been demonstrated to be additive (U.S. EPA, 2000). 

It should be noted that HI has been criticized in some contexts as overly conservative and should 

not be applied to substances below their no-effect levels because it could falsely identify risk where 

none exists. The HI approach is based on the concentration model and calculated as the summation 

of risk quotients (RQs): 

ܪܽݖܽݎ݀ ܫ݊݀݁ݔ ෍ = ா௦௧௜௠௔௧௘ௗ ா௡௩௜௥௢௡௠௘௡௧௔௟ ஼௢௡௖௘௡௧௥௔௧௜௢௡

ே௢ ெ௢௥௧௔௟௜௧௬ ௅௘௩௘௟

௡

௜ୀଵ

 

Adapting this approach for MON 95379 maize, the RQs of Cry1B.868 and Cry1Da_7 were 

calculated for each NTO as the estimated environmental concentrations (EECs) divided by the 

highest concentration tested for which there was no observed effect on survival. The survival 

endpoint was selected for this analysis because it was the endpoint that was common for all NTO 

studies. The RQs for each protein were summed to calculate the HI for each NTO and is shown in 

the Table J-6 below: 

Table J-6. Calculated Risk Quotients (RQ) and Hazard Index (HI) Values for Cry1B.868 

and Cry1Da_7 as Expressed in MON 95379 Maize 

Test Organism Protein EEC 

(ppm)

No Mortality 

Level (ppm) RQ HI

Coleomegilla maculata Cry1B.868 111.4 3500 0.0318 0.1712 Cry1Da_7 20.9 150 0.1393 

Poecilus cupreus Cry1B.868 111.4 3500 0.0318 0.0841 Cry1Da_7 20.9 400 0.0523 

Geocoris punctipes Cry1B.868 111.4 3500 0.0318 0.0736 Cry1Da_7 20.9 500 0.0418 

Pediobius foveolatus Cry1B.868 111.4 3500 0.0318 0.0736 Cry1Da_7 20.9 500 0.0418 

Chrysoperla rufilabris 

(larva) 

Cry1B.868 111.4 3500 0.0318 0.0736 Cry1Da_7 20.9 500 0.0418 

Chrysoperla sp. (adult) Cry1B.868 61.9 700 0.0884 0.0887 Cry1Da_7 0.125 500 0.0003 

Folsomia candida Cry1B.868 0.4 3500 0.0001 0.0005 Cry1Da_7 0.2 500 0.0004 

Eisenia andrei Cry1B.868 0.4 3500 0.0001 0.0005 Cry1Da_7 0.2 500 0.0004 

Apis mellifera (adult) Cry1B.868 61.9 900 0.0688 0.0690 Cry1Da_7 0.125 500 0.0003 

Apis mellifera (larva) Cry1B.868 61.9 900 0.0688 0.0690 Cry1Da_7 0.125 500 0.0003 
Bayer CropScience LP CR282-20U1 329 

USDA and EPA guidance states that only effects at 1u field expression levels environmental 

constitute risk (U.S. EPA, 2007). Based on this guidance, only HI values >1 would indicate 

potential risk from the additive activity of Cry1B.868 and Cry1Da_7. The HI values for nearly all 

tested species were <0.1 and thus, indicate minimal risk of additive activity to NTOs. The lone 

exception was C. maculata where the HI was >0.1 (but less than 1.0) due to the RQ for Cry1Da_7. 

The RQ for Cry1Da_7 was relatively higher because the test concentrations were adjusted to 

account for observed biological activity in test diet, but there was no adverse effect on survival of 

C. maculata exposed to Cry1Da_7. Applying the HI approach to Cry1B.868 and Cry1Da_7 is 

expected to be conservative because no adverse effects on survival were observed in Tier I testing 

resulting in unbounded no mortality levels (e.g., no lowest observed effect concentrations (LOECs) 

were determined). Thus, RQs calculated from test concentrations where effects on survival would 

actually occur (e.g., LOEC, LC50, etc.) are likely quite smaller. 

Conclusions 

The study described above demonstrates that Cry1B.868 and Cry1Da_7 act independently, with 

additive activity in a species that is susceptible to both proteins. This result is consistent with 

published observations that Cry1B.868 and Cry1Da_7 act through distinct receptors (Wang et al., 

2019). Since Cry1B.868 and Cry1Da_7 act independently and have high margins of exposure to 

NTOs, they can be assessed independently. Furthermore, the additive activities of Cry1B.868 and 

Cry1Da_7 as expressed in MON 95379 maize is not expected to pose a risk to NTOs. 

J.2. Methods for Aerobic Soil Degradation of Cry1B.868 and Cry1Da_7 Protein from 

MON 95379 Maize 

The purpose of this study was to estimate the aerobic degradation rate of the Cry1B.868 and 

Cry1Da_7 proteins derived from MON 95379 lyophilized maize leaf tissue in agricultural soils. 

The soils were exposed to Cry1B.868 and Cry1Da_7 through the incorporation of MON 95379 

leaf tissue in soil. To serve as a negative control, conventional maize leaf tissue, which does not 

produce either Cry1B.868 or Cry1Da_7 protein, was incorporated into soil at the same rate as 

MON 95379 tissue. Three different soil types with diverse physicochemical characteristics were 

evaluated and were classified as sandy loam, silt loam and clay loam. 

MON 95379 and conventional leaf tissues were incorporated into soils at the relative incorporation 

rate of 10-fold above those levels expected in the field. The amount of lyophilized leaf tissue to be 

incorporated into the soil was determined by calculating the amount of biomass present in a field 

that could enter the soil under standard cultivation conditions (1u field load) and is described in 

Section VII.B. 

Vessels containing soil and incorporated maize tissue were incubated at an approximate 

temperature of 22 °C with soil moisture maintained at approximately 86% field moisture capacity. 

Samples were collected at 9 time points during a total incubation period of 213 days. Incubated 
Bayer CropScience LP CR282-20U1 330 

soil samples were extracted and analyzed for Cry1B.868 and Cry1Da_7 concentrations using 

enzyme-linked immunosorbent assays (ELISA) specific for each protein. 

In addition, insecticidal activity of MON 95379 incorporated into soil was determined by a diet 

incorporation insect bioassay against a target lepidopteran species, fall armyworm (Spodoptera 

frugiperda). Fall armyworm (FAW) was selected as a test species because of its high sensitivity 

to the Cry1 class of proteins. Because the bioassay cannot distinguish activity from Cry1B.868 

versus Cry1Da_7, the results were used qualitatively to evaluate temporal changes in activity and 

the soil concentrations from the ELISA analysis were used to calculate dissipation rate estimates. 

The times to 50% dissipation (DT50) and 90% dissipation (DT90) for Cry1B.868 and Cry1Da_7 in 

each soil type were calculated from the ELISA results using a modified three-parameter logistic 

model and were applied at the sampling interval where maximum soil concentrations were 

observed (Section VII.B). 

 
Bayer CropScience LP CR282-20U1 331 

J.3. Evaluation of the potential effects of Cry1B.868 Protein on Larvae of the Ladybird 

Beetle, Coleomegilla maculata 

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 protein 

Description: Clear dark amber solution 

Purity corrected concentration: 28.7 mg/mL 

Reference and/or positive control: Potassium arsenate 

Dietery concentration: 200 μg/g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the diet to achieve the same 

concentration of buffer per g diet as used for the 

highest test substance treatment 

Test organisms: 

Species: Coleomegilla maculata 

Age: First instar, <24 hour old 

Diet/Food: Artificial diet provided ad libitum 

Environmental conditions: 

Temperature: 27 oC 

Relative Humidity: 60% 

Photoperiod: 14 hours light / 10 hours dark 

Study Design and Methods 

Experimental treatments: Cry1B.868 protein (in a buffer vehicle) was incorporated into an 

artificial agar-based diet at concentrations of 700 and 3500 μg Cry1B.868 protein/g diet. 

Additionally, a negative control (purified water only), a buffer control (25 mM sodium carbonate 

buffer solution, pH 11.5 incorporated into the diet at the same level as in the 3500 μg Cry1B.868 

protein diet), and a positive control diet containing potassium arsenate at 200 μg/g diet were 

included in the study. Each treatment was incorporated into a diet medium and provided ad libitum

and continuously to individually housed C. maculata larvae (target n = 30 per treatment) until the 

test larvae develop into adults. Freshly-defrosted aliquots of the appropriate treatment diet were 

provided in each test arena and renewed every 48 hours. Each test arena consisted of an inverted 

60 mm × 15 mm Petri dish containing a filter paper, moistened with approximately two drops of 

deionized water, and a Post-it® flag (25.4 mm x 43.2 mm). Freshly-defrosted treatment diet 
Bayer CropScience LP CR282-20U1 332 

(approximately one 1/8 section of a 1 mL diet aliquot) was placed on Post-it® flag in each test 

arena and renewed every 48 to 72 hours. A targeted two additional drops of water were added to 

each arena daily. The other test arena contents (e.g. frass) were removed or replaced as needed. 

There were approximately equal numbers of larvae per treatment per day. 

Observations: Following the initiation of the diet exposures, daily observations were made to 

record larval survival and development, and bw of the newly-emerged adults. 

Statistical calculations: Fisher’s Exact test was performed to compare the survival between each 

treatment with the buffer control. Treatment effects on the time of development to adult and adult 

body mass were analyzed with a linear model using SAS PROC Mixed. Pair-wise comparisons 

with the buffer control were defined within the linear model and tested using t-tests with significant 

difference at α = 0.05. 

Results

C. maculata larval survival was 90% in the negative control and 93% in the buffer control diet 

treatments, compared with 83.3% and 93.1% survival in the 700 and 3500 μg Cry1B.868 protein/g 

diet concentrations, respectively. There were no significant differences in survival between either 

test treatment level and the buffer control (p>0.05). The positive control treatment resulted in 

100% mortality. 

The mean development time was 21.4d in the negative control and 21.3d in the buffer control, 

compared with 20.8d and 21.6d in the 700 and 3500 μg Cry1B.868 protein/g diet concentraions, 

respectively. There were no significant differences in development time between either test 

treatment level and the buffer control (p>0.05). No insects developed into adults in the positive 

control treatment. 

The mean adult weight was 10.0 mg in the negative control and 10.0 mg in the buffer control, 

compared with 10.0 mg and 9.2 mg in the 700 and 3500 μg Cry1B.868 protein/g diet 

concentraions, respectively. There was no significant difference in mean adult weight between the 

700 μg Cry1B.868/g treatment level and the buffer control (p>0.05). There was a significant 

decrease in mean adult weight between the 3500 μg Cry1B.868/g, treatment level and the buffer 

control (p = 0.046). 

Assay Validity Criteria 

The survival of C. maculata larvae was 90% in the negative control and 93.3% in the buffer 

control, indicating there was no adverse effect from the buffer on survival. These results met 

acceptance criteria of having d20% mortality in the control. The positive control treatment resulted 

in 100% mortality demonstrating sensitivity of the test species. 
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The results of the diet-incorporation insect bioassay analysis demonstrate that the Cry1B.868 

protein incorporated into the C. maculata diet had the expected level of biological activity, was 

homogenous through the test diet, was stable under test conditions and frozen storage. 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous dietary exposure to 

Cry1B.868 protein at concentrations of either 700 or 3500 μg Cry1B.868 protein/g diet had no 

effects on either the survival or development time of the C. maculata larvae. There was also no 

effect on mean adult mass for C. maculata exposed to 700 μg Cry1B.868/g diet; however, at 3500 

μg Cry1B.868/g diet, a statistically significant reduction (<10%) in mean mass was observed. The 

LC50 was >3500μg Cry1B.868 protein/g diet, the highest concentration tested, and the NOAEC 

was 700 μg Cry1B.868/g diet. 

J.4. Evaluation of the Potential Effects of Cry1B.868 Protein on Larvae of the Carabid 

Beetle, Poecilus cupreus

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 protein 

Description: Clear dark amber solution 

Purity corrected concentration: 28.7 mg/mL 

Reference and/or positive control: Teflubenzuron Pestanal (Sigma-Aldrich) 

Dietery concentration: 0.032 mg per g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the diet at the same rate as for the 

3500 μg/g test substance diet 

Test organisms: 

Species: Poecilus cupreus larvae 

Age: 1-2 d 

Diet/Food: Artificial diet provided ad libitum 

Acclimation Period: 11 days after hatching 

Environmental conditions: 

Temperature: 18-19.8 oC 

Relative humidity: 64-83% 

Photoperiod: 16 hours light / 8 hours dark 

Illumination: 250 lux 
Bayer CropScience LP CR282-20U1 334 

Study Design and Methods 

Experimental treatments: Cry1B.868 protein (in a buffer vehicle) was incorporated into a base 

diet mixture at two separate treatment concentrations, equivalent to 700 and 3500 μg 

Cry1B.868 protein/g diet. Additionally, a negative control (purified water only), a buffer control 

(25 mM sodium carbonate, pH 10.5 incorporated into the diet at the same rate as for the 3500 μg/g 

test substance diet), and a positive control of teflubenzuron at 32 μg/g diet to confirm the 

effectiveness of the dietary feeding were included in the study. The test arenas comprised lidded 

clear plastic tubes, each containing one beetle larva placed onto the surface of 25 g (dry weight) 

of a standard sandy soil moistened to 25% ± 5% of its maximum water-holding capacity (WHC). 

For the study, the soil-filled plastic tubes were stored upright in plastic boxes (one box per 

treatment), with lids containing 8 holes covered with nylon netting for ventillation and floor lined 

with capillary matting to raise the relative humidity around the tubes. Freshly-defrosted aliquots 

of appropriate treatment diets were provided daily to beetle larvae (n = 40 per treatment), starting 

with 24-48 hour old larvae, until all larvae had pupated or 21 days had passed. 

Observations: The endpoints of the study were pre-imaginal mortality of the test insects, 

development time to adult emergence, and bw of the emerged adult beetles. The condition of 

visible beetle larvae was assessed three times per week for the first three weeks of the study, and 

twice each week thereafter until all larvae had pupated. Fresh bodyweight of adult beetles was 

recorded after the elytra had hardened. The study ended 45 DAT when it was determined that no 

more adults would emerge. 

Statistical calculations: The overall percentage pre-imaginal mortality in each treatment was 

determined and the data corrected for any control deaths using Abbott’s formula (Abbott, 1925). 

Multiple sequentially-rejective Fisher test after Bonferroni-Holm correction (one-sided, > control, 

α = 0.05) was used to compare mortalities in the test substance and the buffer control treatment. 

The positive control treatment was compared to the buffer control by Fisher’s exact binomial test 

(one-sided, > control, α = 0.05). Adult bw was evaluated with a Dunnett’s multiple t-test (onesided, < control α = 0.05) comparing test treatments to the buffer control. Effects on development 

time were evaluated with a Two-sample Mann Whitney U-test (one-sided, > control α = 0.05) 

comparing test treatments to the buffer control. 

Results

Pre-imaginal mortality was 12.5% in the negative control and 7.5% in the buffer control, compared 

with 22.5% and 12.5% mortality in the 700 and 3500 μg Cry1B.868 protein/g diet treatment 

concentrations, respectively. When adjusted for the deaths in the buffer control treatment, the test 

substance treatments resulted in 16.2% and 5.4% corrected mortality in the 700 and 

3500 μg Cry1B.868 protein/g diet treatment concentrations, respectively. There were no 

significant differences in mortality between either test treatment level and the buffer control 

(p>0.05). No abnormal larvae, pupae or adults were seen, in any of the treatments. The positive 
Bayer CropScience LP CR282-20U1 335 

control treatment resulted in 100% pre-imaginal mortality (100% corrected) and this treatment 

differed significantly from the buffer control (p<0.05). 

The mean emergence time for beetles surviving was 44.3 days in the negative control and 44.4 

days in the buffer control, compared with 44.7 days and 44.6 days in the 700 and 3500 μg 

Cry1B.868 protein/g diet treatment concentrations, respectively. There was no significant 

difference in development time between either test treatment level and the buffer control (p>0.05). 

The mean bw of individual beetles taken soon after their emergence was 63.5 mg in the negative 

control and 63.6 mg in the buffer control, compared to 61.2 mg and 62.8 mg in the 700 and 3500 

μg Cry1B.868 protein/g diet treatment, respectively. There was no significant difference in adult 

bw between either test treatment level and the buffer control (p>0.05). 

Assay Validity Criteria 

The mortality of P. cupreus was 12.5% in the negative control and 7.5% in the buffer control, 

indicating there was no adverse effect from the buffer on survival. These results met assay 

acceptance criteria of having d20% mortality in the control. The positive control had 100% 

mortality, demonstrating sensitivity of the test species. 

The results of the diet analysis demonstrated the Cry1B.868 test substance incorporated into the 

test treatment diets had the expected level of biological activity and was stable under test and 

frozen storage conditions. The homogeneity of the Cry1B.868 protein throughout the diet could 

not be confirmed, however, the concentrations detected in the test diet were never less than 

3500 μg Cry1B.868 protein/g diet and, therefore, supports the conclusion that insects were 

exposed to the Cry1B.868 protein at the nominal test concentration.

Conclusion 

Under laboratory test conditions, Cry1B.868 protein had no statistically significant effects on the 

survival of larvae, development or bw of the ground-dwelling carabid beetle, Poecilus cupreus,

when provided orally at concentrations of 700 μg and 3500 μg Cry1B.868/g diet. The LC50

was >3500 μg Cry1B.868 protein/g diet and the NOAEC was 3500 μg Cry1B.868/g diet, the 

highest concentration tested. 
Bayer CropScience LP CR282-20U1 336 

J.5. Evaluation of the Potential Effects of Cry1B.868 Protein on Nymphs of the Big-Eyed 

Bug, Geocoris punctipes

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 protein 

Description: Clear dark amber solution 

Purity corrected concentration: 28.7 mg/mL 

Reference and/or positive control: Potassium arsenate 

Dietery concentration: 100 μg/g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the diet to achieve the same 

concentration of buffer per g diet as used for the 

highest test substance treatment 

Test organisms: 

Species: Geocoris punctipes 

Age: 1 d 

Diet/Food: Ephestia kuehniella eggs and a slice of green bean 

pod provided ad libitum 

Acclimation Period: 1 d 

Environmental conditions: 

Temperature: 25 oC 

Relative Humidity: 50% 

Photoperiod: 14 hours light / 10 hours dark 

Study Design and Methods 

Experimental treatments: Cry1B.868 protein (in a buffer vehicle) was incorporated into a base 

diet mixture at two separate treatment rates, equivalent to 700 and 3500 μg 

Cry1B.868 protein/g diet each encapsulated to form a dome stored at -20°C. Additionally, a 

negative control (purified water only), a buffer control (25 mM sodium carbonate, pH 11.5 

incorporated into the diet at the same rate as for the 3500 μg/g test substance diet), and a positive 

control diet containing potassium arsenate at 100 μg/g diet were included in the test. Two freshlydefrosted encapsulated diets (domes) of the appropriate treatment diet were provided in each test 

arena and renewed every 48 hours. The nymphs were allowed to feed ad libitum throughout the 

test duration. The nymphs after one-day acclimation were individually transferred into test arenas 
Bayer CropScience LP CR282-20U1 337 

(inverted Falcon petri dishes, 60×15mm, Corning, NY) and impartially assigned to each of the 

treatments by providing appropriate treatment diets. Each diet treatment consisted of a total of 30 

nymphs individually housed in inverted petri dishes lined with a piece of filter paper on the bottom. 

One drop of distilled water was added on the filter paper every day at the time of observation to 

moisten the filter paper. 

Observations: Daily observations were made to record test nymph survival, the time taken for the 

nymphs to develop into adulthood, and the fresh bw of the newly-emerged adults. 

Statistical calculations: Fisher’s Exact test was performed to compare the survival between 

predefined treatments. Treatment effects on the time of development to adult and adult body mass 

were analyzed with a linear model using SAS PROC Mixed. Pair-wise comparisons with the buffer 

control were defined within the linear model and tested using t-tests with significant difference at 

α = 0.05. 

Results 

G. punctipes nymph survival was 86.7% in the negative control and 90% in the buffer control diet 

treatments, compared with 92.9% and 100% survival in the 700 and 3500 μg Cry1B.868 protein/g 

diet treatment concentrations, respectively. There were no significant differences in mortality 

between either test treatment level and the buffer control (p>0.05). The positive control treatment 

resulted in 100% mortality. 

All surviving nymphs completed the development into adulthood. The mean nymph development 

time to adults was 31.0 days in the negative control and 29.4 days in the buffer control, compared 

with 30.3 days and 30.0 days in the 700 and 3500 μg Cry1B.868 protein/g diet treatment 

concentrations, respectively. There was a significant difference between the 700 μg Cry1B.868/g 

diet treatment level and the buffer control (p = 0.0288). However, there was no significant 

difference btween the 3500 μg Cry1B.868/g diet treatment level and the buffer control (p = 

0.1281). 

The mean bw of newly-emerged adults was 4.4 mg in the negative control and 5.1 mg in the buffer 

control, compared to 4.7 mg in both Cry1B.868 treatment levels. There were no significant 

differences in bw between either test treatment level and the buffer control (p>0.05). 

 
Bayer CropScience LP CR282-20U1 338 

Assay Validity Criteria 

G. punctipes nymph survival was 86.7% in the negative control and 90% in the buffer control diet 

treatments, indicating there was no adverse effect from the buffer on survival. These results met 

assay acceptance criteria of having t80% survival The positive control treatment resulted in 100% 

mortality, demonstrating sensitivity of the test species. 

The results of the diet analysis demonstrated that the Cry1B.868 protein in the treatment diet had 

the expected level of biological activity and was stable under test and frozen storage conditions. 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous dietary exposure to 

Cry1B.868 protein at 3500 μg Cry1B.868 protein/g diet had no effects on the survival, growth, or 

development of the G. punctipes nymphs. The LC50 was >3500 μg Cry1B.868 protein/g diet and 

the NOAEC was 3500 μg Cry1B.868 protein/g diet, the highest concentration tested. 

 
Bayer CropScience LP CR282-20U1 339 

J.6. Evaluation of the Potential Effects of Cry1B.868 Protein on Adults of the Parasitoid 

Wasp, Pediobius foveolatus

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 protein 

Description: Clear dark amber solution 

Purity corrected concentration: 28.7 mg/mL 

Reference and/or positive control: Potassium arsenate 

Dietery concentration: 200 μg/ml diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the diet to achieve the same 

concentration of buffer per g diet as used for the 

highest test substance treatment 

Test organisms: 

Species: Pediobius foveolatus 

Age: 1 d 

Diet/Food: 30% honey/water (v/v) solution

Acclimation Period: 24 h 

Environmental conditions: 

Temperature: 25 oC 

Relative Humidity: 70% 

Photoperiod: 14 hours light / 10 hours dark 

Study Design and Methods 

Experimental treatments: Cry1B.868 protein (in a buffer vehicle) was incorporated into 30% 

honey/purified water solution at two nominal concentrations of 700 and 3500 Pg/ml 30% 

honey/purified water solution, respectively. Additionally, a negative control (purified water only), 

a buffer control (25 mM sodium carbonate, pH 10.5 incorporated into the diet at the same rate as 

for the 3500 μg/ml test substance diet), and a positive control diet containing potassium arsenate 

at 200 μg/ml diet were included in the study. The newly emerged P. foveolatus adults produced in 

laboratory from the parasitized E. varivestis larvae were supplied with 30% honey/water (v/v) 

solution and were impartially transferred into 162 cm2 cell culture flasks. After approximately 24 

hours of acclimation, 15 adults in each flask were impartially assigned to the five treatments and 

provided with appropriate treatment diet in two screened feeding dishes, replaced every two days. 
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Each treatment included four replicates. The adult wasps were allowed to feed ad libitum

throughout the 21 days feeding period. 

Observations: All wasps in all treatments were observed for mortality/survival every two days at 

the time of diet replacement. Dead wasps, if any, were removed from the test arenas and recorded. 

Statistical calculations: Statistical analyses were not performed since no mortality was recorded 

in either Cry1B.868 treatment level as well as the negative and buffer controls and 100% mortality 

was recorded in positive control treatment. 

Results

P. foveolatus adult survival was 100% in both 700 and 3500 μg Cry1B.868 protein/ml diet 

treatment levels as well as the negative and buffer controls. The positive control treatment resulted 

in 100% mortality (0% survival). 

Assay Validity Criteria 

The survival of P. foveolatus larvae in both the negative (water) and buffer control groups was 

100%, indicating there was no adverse effect from the buffer on survival, and met the assay 

acceptance criteria of having t80% survival in the control. The positive control treatment resulted 

in 100% mortality demonstrating sensitivity of the test species. 

The results of the diet analysis demonstrated that the Cry1B.868 protein in the treatment diet had 

the expected level of biological activity and was stable under test and frozen storage conditions. 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous dietary exposure to 

Cry1B.868 protein at concentrations of either 700 or 3500 μg Cry1B.868 protein/ml diet for 21 

days had no effects on the survival of the P. foveolatus adults. The LC50 was >3500 μg Cry1B.868 

protein/ml diet, the highest concentration tested. 
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J.7. Evaluation of the Potential Effects of Cry1B.868 Protein on Adults of the Green 

Lacewing, Chrysoperla carnea

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 protein 

Description: Clear dark amber solution 

Purity corrected concentration: 28.7 mg/mL 

Reference and/or positive control: dimethoate 

Dietery concentration: 0.04 μg/g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the diet at the same rate as for the 

3500 μg/g test substance diet 

Test organisms: 

Species: Chrysoperla carnea

Age: 24 h 

Sex: 10 males/10 females per treatment group 

Diet/Food: None

Environmental conditions: 

Temperature: 23.9-25.6 oC 

Relative humidity: 67-76% 

Photoperiod: 16 hours light / 8 hours dark 

Illumination: 3100-4100 lux 

Study Design and Methods 

Experimental treatments: Chrysoperla carnea adults, less than 24 h old, were exposed to five 

treatments, Cry1B.868 protein at 3500 μg protein/g diet, Cry1B.868 μg protein at 700 

protein/g diet, a negative control (purified water only), buffer control consisting of test substance 

buffer (25 mM sodium carbonate, pH 11.5) at a volume equivalent to the highest test substance 

concentration, and a positive control of dimethoate at 0.04 μg/g diet to confirm the effectiveness 

of the dietary feeding. The test arenas comprised clear polystyrene boxes with close-fitting lids. 

The dishes of the treated diet, and dishes of honey-water and water-only, were placed on the floor 

of each box. For each treatment there were two replicate boxes, each containing 20 adult lacewings 

(i.e. 40 per treatment) with 10 female and 10 male lacewings impartially assigned to each test arena 

except positive control treatment arenas that receievd 11 female and 9 male lacewings. Any insects 
Bayer CropScience LP CR282-20U1 342 

that appeared to have been damaged during the transfer process were replaced immediately. 

Freshly-defrosted aliquots of appropriate treatment diets were provided to newly-emerged adult 

lacewings three times per week, for two weeks. 

Observations: The endpoint of the study was an assessment of the adult mortality.. The adults 

were assessed every 1-3 days for 14 days and finally at 14 days after treatment, from which the 

overall percentage mortality in each treatment was calculated. 

Statistical calculations: The percentage adult mortality in each treatment was determined and the 

data corrected for any insect deaths in the buffer control using Abbott’s formula (Abbott, 1925). 

Multiple sequentially-rejective Fisher test after Bonferroni-Holm correction (one-sided, > control, 

α = 0.05) was used to compare mortalities in the test substance and the buffer control treatment. 

The positive control treatment was compared to the buffer control by Fisher’s exact binomial test 

(one-sided, > control, α = 0.05). 

Results

Mortality was 15% in the negative control and 15% in the buffer control, compared with 40% and 

35% mortality in the 700 μg and 3500 μg Cry1B.868 protein/g diet treatment concentrations, 

respectively. There was a significant increase in mortality for both Cry1B.868 treatment levels 

compared to the buffer control (p<0.05). The positive control treatment resulted in 62.5% mortality 

(55.9% corrected) and this treatment differed significantly from the buffer control (p<0.05). 

Assay Validity Criteria 

Mortality was 15% in the negative control and 15% in the buffer control, indicating the was no 

adverse effect from the buffer on survival. These results met assay acceptance criteria of d20% 

mortality in the control. The positive control had 62.5% mortality, demonstrating sensitivity of the 

test species. 

The results of the diet analysis by sensitive insect bioassay demonstrated that the Cry1B.868 

protein incorporated into diet had the expected level of biological activity, was considered 

homogenous and was stable under test and frozen conditions. 

Conclusion 

Under laboratory test conditions, continuous dietary exposure of Cry1B.868 protein to adults of 

the green lacewing, C. carnea, for 14 days resulted in statistically significant reduced survival at 

both the 700 and 3500 μg protein/g diet concentrations. The LC50 was >3500 μg Cry1B.868 

protein/g diet and the NOAEC could not be determined in the study. 
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J.8. Evaluation of the potential effects of Cry1B.868 Protein on Larvae of the Green 

Lacewing, Chrysoperla rufilabris

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 protein 

Description: Clear dark amber solution 

Purity corrected concentration: 28.7 mg/mL 

Reference and/or positive control: Potassium arsenate 

Dietery concentration: 0.1 mg/g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the diet at the same rate as for the 

3500 mg/kg test substance diet 

Test organisms: 

Species: Chrysoperla rufilabris

Age: Second instar larvae 

Diet/Food: Untreated diet and water soaked cotton balls

Acclimation Period: 24 hours 

Environmental conditions: 

Temperature: 24-26oC 

Relative humidity: 52-69% 

Photoperiod: 16 hours light / 8 hours dark 

Illumination: 550-710 lux 

Study Design and Methods 

Experimental treatments: Second instar Chrysoperla rufilabris larvae (3-5 days from egg hatch) 

were exposed to six treatments, Cry1B.868 protein at 110 μg protein/g diet, Cry1B.868 protein at 

700 μg protein/g diet, Cry1B.868 protein at 3500 μg protein/g diet, a negative control consisting 

of meat-based diet without any test substance or buffer added, buffer control consisting of test 

substance buffer (25 mM sodium carbonate buffer solution, pH 11.5) at a volume equivalent to the 

highest test substance concentration, and a positive control of potassium arsenate at 0.1 mg/g diet 

to confirm the effectiveness of the dietary feeding for 24 days. The test vessels comprised Petri 

dishes with lids. Lacewing larvae were acclimated for 24 hours prior to test initiation and were 

impartially allocated to the test vessels. Each treatment and control had 40 replicate test vessels 

with each vessel containing one test organism. Individual vessels were randomly assigned to the 
Bayer CropScience LP CR282-20U1 344 

treatments using computer generated random numbers. Test vessels were then randomly placed 

within the environmental chamber during the exposure. At study initiation, one freshly-defrosted 

50 μL pellet of the appropriate control or treatment level was added to each test vessel along with 

one moistened small cotton ball with both being replaced at every 48-hour interval until organisms 

reached pupal state, when organisms do not actively feed on diet. The study was terminated on 

day 24 and pupae that had not emerged were considered dead. 

Observations: The endpoints of the study were percentage pre-imaginal survival, development 

time from larvae to emerged adult, and adult weight at emergence. The health of the organisms 

was observed and recorded daily. Pupation and adult emergence were recorded when observed. 

When adult emergence was observed, the replicate was removed from the study and placed in a 

freezer for euthanization. 

Statistical calculations: The treatment data were tested for normality and homogeneity of variance 

using the appropriate qualifying test. Fisher’s exact test with Bonferroni-Holm correction was used 

to compare survival between the treatment and negative control groups. Effects on development 

time were evaluated with a Dunnett’s multiple comparison test comparing test treatments to the 

negative control. Adult bw was evaluated with a Jonckheere-Terpstra’s Step-down test comparing 

the test treatments and the negative control. All comparisons were made at ≥95% level of certainty 

(D= 0.05) and compared on a per replicate basis. 

Results

The mean pre-imaginal survival was 80% in the negative control and 78% in the negative control, 

compared with 70, 73, and 75% in the 110, 700, and 3500 mg/kg treatment levels, respectively. 

There were no significant reductions in pre-imaginal survival between any test treatment level and 

the negative control (p>0.05). 

The mean weight for adults at emergence was 0.0079 g in the negative control and 0.0073 g in the 

buffer control, compared with 0.0076 g, 0.0074 g, and 0.0074 g in the 110, 700, and 3500 mg/kg 

treatment levels, respectively. There were no significant reductions in adult body weight between 

any test treatment level and the negative control (p>0.05). 

The mean development time from larvae to emerged adult was 19 days in the negative control and 

20 days in the buffer control, compared with 20 days for all Cry1B.868 test treatment levels. There 

were no significant reductions in development time between the test treatment levels and the 

negative control (p>0.05). 

 
Bayer CropScience LP CR282-20U1 345 

Assay Validity Criteria 

Larval mortality was 8% in the negative control and 3% in the buffer control, indicating there was 

no adverse effect from the buffer on survival. These results met assay acceptance criteria of d20% 

mortality in the control. The positive control had 100% larval mortality, demonstrating sensitivity 

of the test species. 

The results of the dose confirmation demonstrated that the Cry1B.868 protein incorporated into 

diet had the expected level of biological activity, was homogeneous throughout the test diet, and 

was stable under test and frozen storage conditions. 

Conclusion

Under laboratory test conditions, Cry1B.868 protein had no statistically significant effects on the 

survival, development of larvae, and adult weight of the the green lacewing, Chrysoperla rufilabris

at concentrations of 110 μg, 700 μg, and 3500 μg Cry1B.868/g diet fed continuously during their 

development for 24 days. The LC50 was >3500 μg Cry1B.868 protein/g diet and the NOAEC was 

3500 μg Cry1B.868/g diet, the highest concentration tested. 

 
Bayer CropScience LP CR282-20U1 346 

J.9. Evaluation of the Potential Effects of Cry1B.868 Protein on Adults of the Green 

Lacewing, Chrysoperla rufilabris

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 protein 

Description: Clear dark amber solution 

Purity corrected concentration: 28.7 mg/mL 

Reference and/or positive control: Potassium arsenate 

Dietery concentration: 500 μg/g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the diet at the same rate as for the 

test substance diet 

Test organisms: 

Species: Chrysoperla rufilabris

Age: < 48 h 

Diet/Food: 40% honey/water (v/v) solution provided ad libitum 

Acclimation period 24 h 

Environmental conditions: 

Temperature: 25 oC 

Relative humidity: 75% 

Photoperiod: 16 hours light / 8 hours dark 

Study Design and Methods 

Experimental treatments: Chrysoperla rufilabris adults, less than 48 h old, were exposed to six 

treatments, Cry1B.868 protein at 110 μg protein/g diet, Cry1B.868 μg protein at 350 protein/g diet, 

Cry1B.868 μg protein at 700 protein/g diet, a negative control diet consisting of purified water 

only, buffer control consisting of test substance buffer (25 mM sodium carbonate, pH 11.5) at a 

volume equivalent to the highest test substance concentration, and a positive control of potassium 

arsenate at 500 μg/g diet to confirm the effectiveness of the dietary feeding. Each treatment was 

replicated three times with targeted 15 adults per replicate. The test arenas comprised 10.8-cup 

plastic food storage container with two feeding stations and the treatment diets were replaced every 

48 hours. The newly emerged adults (< 48 h) in each test arena were provided with 40% 

honey/water (v/v) solution in two feeding stations and acclimated for 24 hours prior to inclusion 

in feeding test. The adults in each replicate allowed to feed ad libitum on the treatment diet for 14 
Bayer CropScience LP CR282-20U1 347 

days. Freshly-defrosted aliquots of treatment diet were provided to adult lacewings on seven 

occasions over a 14-day period in two feeding stations placed in each replicate test arena. 

Observations: The endpoint of the bioassay was a daily assessment of the adult mortality for 14 

days, from which the percentage mortality in each treatment was calculated. 

Statistical calculations: SAS PROC Freq was used to conduct the Fisher’s exact test to compare 

survival rate between treatments at significance α = 0.05. SAS PROC MEANS was used to 

calculate the survival (%) across replicates for each treatment. 

Results

Survival of C. rufilabris adults was 97.8% in the negative control and 95.6% in the buffer control, 

compared with 91.1%, 95.7% and 100.0% survival in the 110, 350 and 700 μg Cry1B.868 protein 

/g diet treatment concentrations, respectively. There were no significant differences in mortality 

between either test treatment level and the buffer control (p>0.05). The positive control treatment 

resulted in 100% mortality. 

Assay Validity Criteria 

Survival of C. rufilabris adults was 97.8% in the negative control and 95.6% in the buffer control, 

indicating there was no adverse effect from the buffer on survival. These results met assay 

acceptance criteria in having t80% survival in the control. The positive control treatment resulted 

in 100% mortality, demonstrating sensitivity of the test species. 

The results of diet analysis indicated that the Cry1B.868 protein in diet samples had the expected 

level of biological activity, was homogeneous in diet, and was stable under test and frozen storage 

conditions. 

Conclusion 

Under laboratory test conditions, continuous dietary exposure of Cry1B.868 protein to adults of 

the green lacewing, C. rufilabris for 14 days resulted in no significant effect on survival at 110, 

350 and 700 μg protein/g diet treatments. The LC50 was >700 μg Cry1B.868 protein/g diet, the 

highest concentration tested. 

 
Bayer CropScience LP CR282-20U1 348 

J.10. Evaluation of the Potential Effects of Cry1B.868 Protein on the Springtail, Folsomia 

candida

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 protein 

Description: Clear dark amber solution 

Purity corrected concentration: 28.7 mg/mL 

Reference and/or positive control: Teflubenzuron 

Dietery concentration: 0.2 mg per g diet (see: Study plan deviations) 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the diet to achieve the same 

concentration of buffer per g diet as used for the 

highest test substance treatment 

Test organisms: 

Species: Folsomia candida 

Age: 12 d 

Diet/Food: Artificial diet provided ad libitum 

Acclimation Period: 11 days after hatching 

Environmental conditions: 

Temperature: 19.3-20.8 oC 

Photoperiod: 16 hours light / 8 hours dark 

Illumination: 600-780 lux 

Study Design and Methods 

Experimental treatments: Cry1B.868 protein (in a buffer vehicle) was incorporated into a diet 

medium of inactivated yeast at two separate treatment concentrations, equivalent to 3500 and 

700 μg Cry1B.868 protein/g diet. For the study, these two dietary treatments were compared to a 

buffer control diet to which 25 mM sodium carbonate buffer solution (pH 11.5) was added at an 

equivalent concentration as in the Cry1B.868 protein treatments. A negative control diet (purified 

water only) and a positive control diet treated with a technical-grade sample of teflubenzuron, at a 

rate equivalent to 0.2 mg/g diet were included in the study. Freshly-defrosted aliquots of 

appropriate treatment diets were provided continuously to batches of confined springtails over a 

28-day period. The test arenas comprised lidded jars, the bases of which were lined with a plasterof-Paris and charcoal substrate. Ten juvenile springtails (12 days old) were placed in each jar (n = 
Bayer CropScience LP CR282-20U1 349 

4 per treatment) and were fed ad libitum throughout the study. Freshly-treated aliquots of 

appropriate treatment diet were provided on the day of study initiation (Day 0) and additional 

freshly-defrosted aliquots were then provided every 2-3 days. 

Observations: For the study, the numbers both of surviving adults and F1 progeny in each 

replicate arena were recorded at 28 days after treatment (DAT). It was assumed that any adult 

springtails that were recovered would have been those originally introduced and that any shortfall 

in the original number was an indication that they had died during the study. The mean number of 

juveniles produced at 28 DAT, and the standard deviation and the coefficient of variation were 

calculated from the replicates in each treatment. The numbers of surviving springtails were used 

to calculate the percentage mortality of the springtails originally introduced in each treatment. 

Statistical calculations: The 28-day mortality data for the individual test substance treatments 

were compared to those for the buffer control using multiple sequentially-rejective Fisher test after 

Bonferroni-Holm correction (one-sided greater, α = 0.05). Following a check for normal 

distribution of the data (Shapiro-Wilk test, α = 0.05) and for equality of variances (Levene’s test, 

α = 0.05), test treatments were compared to the buffer control by multiple sequentially-rejective 

t-test after Bonferroni-Holm correction (one-sided smaller, α = 0.05). The positive control 

treatment was compared to the buffer control by Student t-test for homogeneous variances (onesided smaller, α = 0.05). 

Results

At 28 days, there was 5% mortality in both the negative and buffer control diet treatments, 

compared with 5% mortality in both the 700 and 3500 μg Cry1B.868 protein/g diet treatment 

concentrations. When adjusted for the deaths in the buffer control treatment, both the test substance 

treatments resulted in 0% corrected mortality. There was no significant difference in mortality 

between either test treatment level and the buffer control (p>0.05 for both concentrations). The 

positive control treatment resulted in 93% mortality (92% corrected) and this treatment therefore 

differed significantly from the buffer control (p<0.05). 

The mean number of progeny produced per treatment was 331 in the negative control and 358 in 

the buffer control, compared with 330 and 304 in the 700 and 3500 μg Cry1B.868 protein/g diet 

treatment concentrations, respectively. There were no significant differences in reproduction 

between either test treatment level and the buffer control (p>0.05). 

 
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Assay Validity Criteria 

The mortality of F. candida was 5% in both the negative and buffer controls, indicating there was 

no adverse effect from the buffer on survival. These results met the assay acceptance criteria of 

having d20% mortality in the control. The mean number of juveniles recorded in the negative 

control treatment was at least 100 per replicate at the end of the study and the coefficient of 

variation of reproduction in the negative control was d30% meeting acceptance criteria. The 

positive control treatment resulted in 93% mortality, demonstrating sensitivity of the test species. 

The results of the diet analysis demonstrated that the test substance, Cry1B.868 protein in test diet 

had the expected level of biological activity, was homogenous throughout the freshly-prepared test 

diet and was stabile under test and storage conditions. 

Conclusion

Under laboratory test conditions, it was demonstrated that continuous exposure to Cry1B.868 

protein for 28 days at concentrations of either 700 or 3500 μg Cry1B.868 protein/g diet had no 

effects on either the survival or reproduction rate of the springtail Folsomia candida. The LC50

was >3500 μg Cry1B.868 protein/g diet and the NOAEC was 3500 μg Cry1B.868 protein/g diet, 

the highest concentration tested. 

 
Bayer CropScience LP CR282-20U1 351 

J.11. Evaluation of the Potential Effects of Cry1B.868 Protein on the Earthworm, Eisenia 

andrei

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 protein 

Description: Clear dark amber solution 

Purity corrected concentration: 28.7 mg/mL 

Vehicle and/or positive control: 2-chloroacetamide (tested in a separate study) 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the soil to achieve the same 

concentration of buffer per g soil as used for the test 

substance treatments 

Test organisms: 

Species: Eisenia andrei 

Age: ~8 months 

Diet/Food: None

Acclimation Period: 1 day 

Environmental conditions: 

Temperature: 20.2-22.0 oC 

Photoperiod: 24 hours light 

Illumination: 490-550 lux 

Study Design and Methods 

Experimental treatments: The Cry1B.868 protein held in a buffer solution was incorporated into 

an artificial soil medium at two separate treatment concentrations, equivalent to 3500 and 700 μg 

Cry1B.868 protein/g soil dry weight. For the study, these two treatments were compared to a 

control soil treated with either 25 mM sodium carbonate buffer solution, pH 11.5 at the same 

concentration as used for the test substance treatments (buffer control) or with purified water 

(negative control). To confirm sensitivity of the test species, a positive control treatment of 2-

chloroacetamide was evaluated in a separate, GLP-compliant study within 2 months of the study. 

The artificial soil substrate (containing 10% w/w peat), was moistened to 50% of its predetermined maximum water-holding capacity at the time of treatment. The treated soil was held 

within 1-L-capacity lidded jars (n = 4 per treatment) and 10 adult E. andrei (approx. 8 months old, 

with an individual fresh weight of 358-598 mg and with a visible clitellum) were weighed and 
Bayer CropScience LP CR282-20U1 352 

introduced into each jar immediately after treatment application. Each treatment was replicated 

four times for a total of 40 earthworms exposed per treatment. No food was provided throughout 

the duration of the study. 

Observations: For the study, the numbers of surviving adult earthworms in each test arena were 

recorded at 7 and 14 DAT. It was assumed that any adult earthworms that were recovered would 

have been those originally introduced and that any shortfall in the original number was an 

indication that they had died during the study. The mean percentage change in weight of the 

earthworms in the controls and test-substance treatments over 14 days was calculated. The 

earthworms were gently cleaned using purified water and individually re-weighed at 14 DAT after 

removing excess moisture to determine the mean weight of individuals in each replicate. The 

percentage change in mean weight per replicate was derived by comparing the mean weight 

recorded at 0 DAT with that measured again at 14 DAT for earthworms in each replicate. The 

earthworms were also examined for any other harmful effects (e.g. behavioral abnormalities or 

open wounds) at 0, 7 and 14 DAT. 

Statistical calculations: Since there was no mortality in the controls, it was unnecessary to correct 

for mean control mortality using Abbott’s formula. Fisher’s Exact binomial test with Bonferroni 

correction (one-sided, > control, α = 0.05) was used to compare mortalities in the individual test 

treatments with the buffer control treatment. Following a check for normal distribution of the data 

(Shapiro-Wilk test, α = 0.05) and for equality of variances (Levene’s test, α = 0.05), test treatments 

were compared to the buffer control by multiple sequentially-rejective t-test after Bonferroni-Holm 

correction (one-sided smaller, α = 0.05). 

Results

At 14 days, there was no mortality in both the negative and buffer control treatments, compared 

with 3% and 0% mortality in 700 and 3500 μg Cry1B.868 protein/g soil dry weight treatment 

concentrations, respectively. There were no significant differences in mortality between either test 

treatment level and the buffer control (p>0.05). 

The mean percentage change in earthworm weight per treatment was equivalent to a decrease of 

7.9% in the negative control and 4.4% in the buffer control, compared with an increase of 4.4% 

and an increase of 43.3% in the 700 and 3500 μg Cry1B.868 protein/g soil dry weight treatment 

concentrations, respectively. There was not a significant decrease in bw between either test 

treatment level and the buffer control (p>0.05). At both 7 and 14 DAT, all of the earthworms in 

all of the treatments appeared healthy and active. 

Assay Validity Criteria 

The mortality of Eisenia andrea was 0% in the negative control and 0% in the buffer control group, 

indicating there was no adverse effect of the buffer on survival. These results met the assay 
Bayer CropScience LP CR282-20U1 353 

acceptance criteria of d10% mortality in the control. The mean post-treatment weight of the 

negative control group decreased by <20% compared to the start of the study, meeting acceptance 

criteria. 

As this was an acute study with a single application of the Cry1B.868 protein, no direct 

measurement was made of test substance homogeneity in the soil. However, care was taken to mix 

the treated soil thoroughly. No measurement was made of the stability of the test substance in the 

soil, but the test earthworms were placed onto the soil immediately after the treatment of each 

arena. 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous exposure to Cry1B.868 

protein for 14 days in an artificial soil containing 10% w/w peat at concentrations of either 700 or 

3500 μg Cry1B.868 protein/g soil dry weight had no significant treatment effects on the survival 

or biomass of the earthworm Eisenia andrei. Additionally, no abnormal behaviors were observed 

in any of the treatments. The LC50 was >3500 μg Cry1B.868 protein/g soil dry weight and the

NOAEC was 3500 μg Cry1B.868 protein/g soil dry weight, the highest concentration tested.

 
Bayer CropScience LP CR282-20U1 354 

J.12. Evaluation of the Potential Effects of Cry1B.868 Protein on Adults of the Honey Bee, 

Apis mellifera

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 

Description: Liquid 

Purity corrected concentration: 28.7 mg/mL 

Positive control: Dimethoate 

Dietery concentration: 0.65 mg/kg diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 11.5) 

incorporated into the diet to achieve the same 

concentration of buffer per g diet as used for the 

highest test substance treatment 

Test organisms: 

Species: Apis mellifera L. 

Age: d2d at dosing 

Diet/Food: 50% sucrose/purified water solution provided ad 

libitum

Acclimation Period: 1 d 

Environmental conditions: 

Temperature: 33°C 

Relative Humidity: 50-70 % 

Photoperiod: Maintained in the dark thoughout duration of test 

Study Design and Methods 

Experimental treatments: Honey bee adults, ≤2 days old, were exposed to five treatments, 

Cry1B.868 protein at 180 μg/g in combination with 50% sucrose/purified water solution, 

Cry1B.868 protein at 900 μg/g in combination with 50% sucrose/purified water solution, a 

negative control consisting of 50% sucrose/purified water solution only, buffer control consisting 

of test substance buffer (25 mM sodium carbonate buffer solution, pH 11.5) at a volume equivalent 

to the highest test substance concentration, and a positive control of dimethoate at 0.65 mg/kg to 

confirm the effectiveness of the dietary feeding. Five cages (replicates) for each five treatments 

comprised a total of 25 cages. Individual cages contained 10 adult bees obtained from a local hive 

mainatined by Eurofins that was apparently healthy, queen-right, and had not been treated for 
Bayer CropScience LP CR282-20U1 355 

control of Varroa or pathogens within four weeks of the study. Once the honey bee adults were 

caged impartially, each cage was assigned to a treatment/replicate in a non-systematic fashion. 

After all adult bees were acclimated under red light with 50% (w/v) sucrose solution provided ad 

libitum, each cage was provided with the appropriate treatment diet solution through two feeders 

(syringes, each containing ~2.5 mL diet) inserted through the lid of the test chamber and bees were 

allowed to feed ad libitum. Feeders were replaced daily and were weighed prior to placing them 

into the cages and again after removal 24 ± 2 hours later to measure the amount of diet consumed 

in each replicate. The study duration was 10 days with adult bees being observed daily for 

mortality, behavioral and toxicological responses. 

Observations: All treatments were observed for mortality, behavioral and toxicological responses 

once within the first four hours after initial treatment and at approximately 24-hour intervals (from 

dosing) thereafter. Abnormal behavior was determined by comparing honey bees in the treatment 

groups with those in the negative control group. Dead honey bee adults remained in their respective 

cages until study completion to avoid unnecessary disturbances. Consumption per bee in each 

replicate was calculated for each day using the consumption data adjusted for evaporative loss and 

the total number of bees alive in each replicate at the start of each feeding period (day). The 

consumption per bee values for all the replicates within a treatment level were then averaged for 

each day. Replicates were no longer included in the average consumption calculations after 

reaching 100% mortality. 

Statistical calculations: The NOEC value was empirically determined by visual observation of 

the mortality data because mortality in the highest level was lower than in the negative control. 

Results 

Mortality of Apis mellifera adults was 12% in the negative control and 6% in the buffer control, 

compared to 22% and 8% mortality in the 180 μg Cry1B.868 protein/g and 900 μg Cry1B.868 

protein/g groups, respectively. All surviving bees appeared normal at the end of the study. Positive 

control group mean mortality was 100%. 

Average daily consumption over the 10-day exposure period were 28, 27, 30, 27 and 13 mg 

diet/bee/day in the negative control, 180 μg Cry1B.868 protein/g, 900 μg Cry1B.868 protein/g, 

buffer control and positive control groups, respectively. These results demonstrate that diet 

consumption was comparable in all groups, except positive control. The daily doses were 

calculated based on the average daily consumption values and were 4.89 and 26.6 μg Cry1B.868 

protein/bee/day during the study for the 180 μg Cry1B.868 protein/g and 900 μg Cry1B.868 

protein/g treatment levels, respectively. 

 
Bayer CropScience LP CR282-20U1 356 

Assay Validity Criteria 

Mortality of Apis mellifera adults was 12% in the negative control and 6% in the buffer control, 

indicating there was no adverse effect from the buffer on survival. The positive control had 100% 

mortality, demonstrating sensitivity of the test species. 

The results of the diet analysis demonstrated the Cry1B.868 test substance incorporated into the 

test treatment diets had the expected level of biological activity, was stable for 1 day under test 

condtions, 4 days at 4 °C, and under frozen storage conditions. 

Conclusion

Following oral exposure of adult honey bees under laboratory test conditions to Cry1B.868 protein 

for 10 days, the LC50 was >900 μg Cry1B.868 protein/g diet and the NOAEC was 900 μg 

Cry1B.868 protein/g, the highest concentration tested. 

J.13. Evaluation of the Potential Effects of Cry1B.868 Protein on Larvae of the Honey Bee, 

Apis mellifera

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 

Description: Liquid 

Purity corrected concentration: 28.7 mg/mL 

Positive control: Dimethoate 

Dietery concentration: 7.42 μg/bee 

Test organisms: 

Species: Apis mellifera L. 

Age: ~6 d at dosing 

Diet/Food: Artificial diet (OECD 239) 

Acclimation Period: 2 d after grafting 

Environmental conditions: 

Average Temperature: 34.47°C (Larval exposure phase) 

34.27°C (Pupal transfer to emergence) 

Average Relative Humidity: 92.88 % (Larval exposure phase) 

76.80 % (Pupal transfer to emergence) 

Photoperiod: Maintained in the dark thoughout duration of study 
Bayer CropScience LP CR282-20U1 357 

Study Design and Methods 

Experimental treatments: First instar larvae from three hives were transferred (grafted) to well 

plates containing untreated artificial diet and held in an incubator. Starting at two days after 

grafting, artificial diets containing the test or control substance were provided to larvae. Test diets 

were provided for a total of four days. Larvae were then held for up to 14 days after the completion 

of dosing in order to allow emergence of adult bees. Negative control (untreated artificial diet), 

buffer control (25 mM sodium carbonate buffer solution, pH 11.5 at a volume equivalent to the 

highest test substance concentration) and positive control (dimethoate, 7.42 μg/bee) groups were 

maintained concurrently. Each treatment and control group contained 16 larvae from each of the 

three hives, for a total of 48 larvae per treatment group. Nominal test levels were 180 and 900 μg 

Cry1B.868/g diet. 

Observations: Starting on the first day of dosing (Day 3), larvae were observed daily until either 

mortality or adult emergence occurred. Cell cups containing dead larvae were removed from well 

plates after mortality was recorded. Observations of sublethal effects, including the presence of 

uneaten diet and larvae with reduced body size, were recorded on Days 7 and 8. Emerged bees 

from each replicate were checked to evaluate sublethal effects on morphology of emerged bees. 

By Day 20, approximately 14 days after final dosing, all bees had either emerged or died. 

Statistical calculations: Dunnett’s multiple comparison test was used to compare mortalities in 

the individual test treatments to the negative control. Mean larval survival and pupal survival were 

calculated but not be analyzed statistically. Sublethal effects were reported but not statistically 

analyzed. 

Results

Mean mortality was 10% in the negative control 21% in the buffer control and no significant 

difference was detected according to t-test, (p>0.05). Mean mortality values in the 180 and 900 μg 

Cry1B.868 protein/g treatment groups were 17 and 21%, respectively. There were no significant 

differences in mortality between the test treatments and the negative control (p>0.05). At the end 

of the study all adult bees appeared healthy with no morphological deformities observed, except 

one bee in the buffer control. 

Assay Validity Criteria 

Cumulative larval mortality was less than 15% across replicates, adult emergence was higher than 

70% on Day 20 in the negative control group, and larval mortality was higher than 50% by day 8 

in the positive control group, meeting acceptance criteria. 

The results of the diet analysis demonstrated the Cry1B.868 test substance incorporated in diets 

had the expected level of biological activity, was homogeneous throughout the diet, and was stable 

under test and frozen storage conditions.
Bayer CropScience LP CR282-20U1 358 

Conclusion 

Following dietary exposure of larval honey bees under laboratory test conditions to Cry1B.868 

protein, the LC50 was >900 μg Cry1B.868 protein/g diet and the NOAEC for larval honey bees 

was 900 μg Cry1B.868 protein/g, the highest concentration tested. 

J.14. Evaluation of the Potential Effects of Cry1B.868 Protein on the Northern Bobwhite 

Quail, Colinus virginianus

Materials and Methods 

Materials 

Test material: 

Test item: Cry1B.868 lyophilized powder 

Description: Solid 

Purity corrected concentration: 0.74 mg/mg lyophilized powder 

Control: Bovine serum albumin (BSA) lyophilized powder 

Test organisms: 

Species: Northern bobwhite (Colinus virginianus) 

Age: 27 weeks 

Sex: 5 males/5 females per treatment group 

Weight: 189-223 g 

Diet/Food: Cargill game bird ration, ad libitum 

Acclimation Period: 8 weeks 

Environmental conditions: 

Temperature: 23.9°C (Range: 22.4-25.6°C) 

Humidity: 62% (Range: 37-75%) 

Photoperiod: 8 hours light / 16 hours dark 

Illumination: 625 lux 

Study Design and Methods 

Experimental treatments: After an acclimation period of 8 weeks, ten adult northern bobwhite 

were indiscriminately assigned to the treatment and control groups. The test dose substance (TDS) 

and control dose substance (CDS) was dosed using capsules at a rate of 3500 mg product/kg of 

bw. Nominal dosages used in this study were 0 mg/kg, 3500 mg of CDS/kg and 3500 mg TDS/kg. 

The birds were fasted for approximately 16.5 hours prior to dosing and individually weighed and 

dosed on the basis of mg of a.i/kg of bw. At the experimental start, two capsules containing either 
Bayer CropScience LP CR282-20U1 359 

the CDS or TDS was orally inserted into the crop of each bird. A third capsule was dosed after 

approximately four hours following the initial dosing. The negative control birds received three 

empty capsules of the same size used for the test birds and at the same intervals as the test birds. 

Two pens containing either 5 male or 5 female quail were used for each treatment and control 

group. 

Observations: After dosing, the birds were observed multiple times on Day 0, and then twice 

daily for 14 days following dosing for mortality, toxicity symptoms and abnormal behavior. In 

addition, birds were observed for any signs of regurgitation. Body weights were measured 

individually the day prior to dosing (Day -1) and on Day 3, 7, and 14 of the test. Feed consumption 

was determined by pen for approximately 24-hour intervals from Day 0 to Day 1, Day 1 to Day 2 

and Day 2 to Day 3. Average daily feed consumption was determined by pen for the dosage group 

and the control group for Days 3–7, and 7–14. At study termination, three birds from each group 

were subjected to a gross necropsy. 

Statistical calculations: An LD50 value was not able to be calculated since no treatment group 

resulted in greater than 50% mortality. The bw and bw change by interval was compared between 

the TDS group and the two control groups by Student’s t-test using Microsoft Excel®. The sample 

unit was the individual bird within each experimental group and the comparison was made by sex 

and by group. 

Results

There were no mortalities in the control or treatment groups. When compared to the negative 

control group, there were no apparent treatment-related differences in mean bw, mean bw changes, 

or feed consumption for the males and females in 3500 mg/kg dosage level, except for significantly 

higher (Student’s t-test, p<0.01) mean body weight change for the males in the TDS group than 

the males in the negative control group from Day -1 to Day 14 of the study. There were no 

remarkable findings for the birds subjected to gross necropsy from any group. 

Validity criteria 

There was no evidence of regurgitation by any bird in any of the treatment groups. All birds in the 

negative control group, CDS group and TDS group were normal in appearance and behavior for 

the duration of the study. 

All validity criteria according to OCSPP 850.2100 were fulfilled, as no mortalities were observed 

in the control groups. 

Results from the dose confirmation demonstrated that the Cry1Da_7 protein was homogeneous 

and stable under conditions of administration. 

 
Bayer CropScience LP CR282-20U1 360 

Conclusion 

The acute oral LD50 value for northern bobwhite quail exposed to formulated Cry1B.868 protein 

as a single oral dose was determined to be greater than 3500 mg Cry1B.868 protein/kg, the highest 

dosage level tested. The no-mortality level was 3500 mg Cry1B.868 protein/kg bw. 

J.15. Evaluation of the Potential effects of Cry1B.868 Protein on the Mouse, Mus musculus 

Materials and Methods 

Materials 

Test material and test dose solution: 

Test item: Cry1B.868 protein 

Description: Light brown liquid 

Purity corrected concentration: 86.5 mg/mL 

Control: Bovine serum albumin (BSA) 

Vehicle control: Permeate collected during concentration of the test 

substance was thoroughly dialized into 10 mM sodium 

carbonate/bicarbonate buffer. Dialized permeate was 

used as vehicle control for this study. 

Test organisms: 

Species: CD-1 mice (Mus musculus) 

Age: 8 weeks 

Sex: 10 males/10 females per treatment group 

Weight: 29.6-39.5 g males/ 22.9-31.2 g females 

Diet/Food: PMI Nutrition International Certified Rodent Chow No. 

5002, ad libitum 

Acclimation Period: 6 days 

Environmental conditions: 

Temperature: 21°C 

Humidity: 48-57% 

Photoperiod: 12 hours light / 12 hours dark 

Study Design and Methods 

Experimental treatments: After an acclimation period of 6 days in their designated housing, ten 

male and ten female mice were indiscriminately assigned to the treatment and control groups. 

Dosages used in this study were 0 mg of vehicle dose substance (VDS) per kg, 5000 mg of control 

(BSA) dose substance (CDS) per kg and 5000 mg of test (MON 95379) dose substance (TDS) per 
Bayer CropScience LP CR282-20U1 361 

kg. Any assigned animal considered unsuitable for use in the study was replaced by an alternate 

animal obtained from the same shipment and maintained under the same environmental conditions. 

The animals were housed individually throughout the study in polycarbonate cages containing 

appropriate bedding equipped with an automatic watering valve. Animals were provided with 

enrichment items and edible treats for psychological/environmental enrichment, except when 

interrupted by study procedures/activities. On Day 0, the animals chosen for use on study were 

weighed and fasted approximately 3 to 4 hours prior to dose administration. The test, control, and 

vehicle dosing solutions were administered in 2 doses (approximately 3 to 4 hours apart), each 

with a dose volume of 33.3 mL/kg body weight by oral gavage on Day 0 to the appropriate group 

of 10 males and 10 females. Individual doses were calculated based on the animal’s non-fasted 

(Day 0) body weght. The animals were fasted between doses and were returned to feed following 

the second dose. 

Observations: After dosing, the animals were observed for general health/mortality and 

moribundity twice daily, once in the morning and afternoon, throughout the study. The following 

parameters and end points were evaluated in this study: clinical signs, body weights, body weight 

changes, food consumption, and gross necropsy findings. Each animal was observed for clinical 

signs once prior to study start, once prior to dosing on Day 0, a minimum of 2 times postdose on 

Day 0 (1 within 1 hour of the first dose and 1 approximately 2 hours following the second dose), 

and daily thereafter (Days 1 to 14). A final detailed clinical observation was performed for each 

animal on the day of scheduled euthanasia (Day 14). Body weights were measured individually 

once prior to study start, prior to fasting (Day 0), prior to dosing (Day 0, fasted), and on Days 7 

and 14 of the test. Feed consumption was determined for each animal on Days 0, 7, and 14 and 

was reported for the intervals from Study Days 0 to 7 and 7 to 14. At study termination, three 

animals from each group were subjected to a gross necropsy. 

Statistical calculations: Body weight, body weight change and food consumption were compared 

between the three treatment groups using an overall one-way ANOVA F-test or Kruskal-Wallis 

test (if parametric assumptions were not met) at the 5% significance level, but excluded semiquantitative data, and any group with less than 3 observations. Levene’s test was used to assess 

the homogeneity of group variances parametric assumption at the 5% significance level. Pairwise 

comparisons were conducted between TDS and the control groups using a two-sided Dunnett’s or 

Dunn’s test, respectively, if the overall test was significant. All significant pairwise comparisons 

were reported at the 0.1%, 1%, and 5% significance levels. 

Results

There were no mortalities in the control or treatment groups throughout the study. There were no 

test substance-related clinical signs during the study. Clinical findings included piloerection in 

3/10 TDS males. This finding was considered incidental as there were no other correlating 

observations. All other clinical findings noted were considered normal for animals of this age and 
Bayer CropScience LP CR282-20U1 362 

strain. There were no test substance-related effects on body weights, body weight gains, food 

consumption or gross necropsy findings noted during the study. 

Observations 

The analytical tests established stability and homogeneity of the test and control dosing solutions 

administered in the mouse oral acute gavage toxicity with Cry1B.868 protein. 

Conclusion 

There were no test substance-related adverse effects of Cry1B.868 protein when administered by 

oral gavage at a dose of 5000 mg/kg body weight in male and female CD-1 mice on mortality, 

clinical signs, body weights, body weight gains, food consumption or gross pathology findings. 

Therefore, the NOAEL for Cry1B.868 protein was 5000 mg/kg body weight. 

J.16. Evaluation of the Potential Effects of Cry1Da_7 Protein on Larvae of the Ladybird 

Beetle, Coleomegilla maculata 

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7 protein 

Description: Clear colorless solution 

Purity corrected concentration: 3.3 mg/mL 

Reference and/or positive control: Potassium arsenate 

Dietery concentration: 100 μg/g diet 

Buffer control: 25 mM sodium carbonate buffer solution, 

(pH 10.5) incorporated into the diet to achieve the 

same concentration of buffer per g diet as used for 

the highest test substance treatment 

Test organisms: 

Species: Coleomegilla maculata 

Age: First instar, <24 hour old 

Diet/Food: Artificial diet provided ad libitum 

Environmental conditions: 

Temperature: 27 oC 

Relative Humidity: 60% 

Photoperiod: 14 hours light / 10 hours dark 
Bayer CropScience LP CR282-20U1 363 

Study Design and Methods 

Experimental treatments: Cry1Da_7 protein (in a buffer vehicle) was incorporated into an 

artificial agar-based diet at concentrations of 50 and 415.1 μg Cry1Da_7 protein/g diet. 

Additionally, a negative control (purified water only), a buffer control (25 mM sodium carbonate 

buffer solution, pH 10.5 incorporated into the diet at the same level as in the 415.1 μg Cry1Da_7 

protein diet), and a positive control diet containing potassium arsenate at 100 μg/g diet were 

included in the test. Each treatment was incorporated into a diet medium and provided ad libitum

and continuously to individually housed C. maculata larvae (target n = 30 per treatment) until the 

test larvae develop into adults. Each test arena consisted of an inverted 60 mm × 15 mm Petri dish 

containing a filter paper, moistened with approximately two drops of deionized water, and a Postit® flag (25.4 mm x 43.2 mm). Freshly-defrosted treatment diet (approximately one 1/8 section of 

a 1 mL diet aliquot) was placed on Post-it® flag in each test arena and renewed every 48 to 72 

hours. A targeted two additional drops of water were added to each arena daily. The other test 

arena contents (e.g. frass) were removed or replaced as needed. There were approximately equal 

numbers of larvae per treatment per day. 

Observations: Following the initiation of the diet exposures, daily observations were made to 

record larval survival and development, and body weight of the newly-emerged adults. 

Statistical calculations: Fisher’s exact test was performed to compare the survival between each 

treatment with the buffer control. Treatment effects on the time of development to emerged adult 

and adult body weight were analyzed with a linear model using SAS PROC Mixed. Pair-wise 

comparisons with the buffer control were defined within the linear model and tested using t-tests 

with significant difference at α = 0.05. 

Results 

C. maculata larval survival was 93% in the negative control and 93% in the buffer control, 

compared with 86.2% and 100.0% survival in the 50 and 415.1 μg Cry1Da_7 protein/g diet 

treatment concentrations, respectively. There were no significant differences in survival between 

either test treatment level and the buffer control (p>0.05). The positive control treatment resulted 

in 100% mortality. 

The mean adult body weight was 10.5 mg in the negative control and 10.5 mg in the buffer control, 

compared with 11.1 and 10.1 mg in the 50 and 415.1 μg Cry1Da_7 protein/g diet treatment 

concentrations, respectively. There were no significant differences in mean adult mass between 

either treatment level and the buffer control (p>0.05). No insects developed into adults in the 

positive control treatment. 

The mean development time was 20.1 days in the negative control and 20.2 days in the buffer 

control, compared with 21.0 days in both the 50 μg Cry1Da_7/g and 415.1 μg Cry1Da_7/g protein 
Bayer CropScience LP CR282-20U1 364 

treatment levels. There was a significant increase in development time (<1 day) between both test 

treatment levels and the buffer control (p<0.05). 

Assay Validity Criteria 

The survival of C. maculata larvae in both the negative (water) and buffer control groups was 

93.3%, indicating there was no adverse effect from the buffer on survival. These results met assay 

acceptance criteria of having t80% survival in the control. The positive control treatment resulted 

in 100% mortality, demonstrating sensitivity of the test species. 

The results of the diet analysis confirmed that the Cry1Da_7 protein in the 415.1 μg Cry1Da_7/g 

diet treatment level was homogeneous and was stable under test and frozen storage conditions. 

However, the targeted concentration could not be confirmed in the 415.1 μg Cry1Da_7/g diet. 

Because the Cry1Da_7 test substance incorporated into the C. maculata diet had lower than the 

expected level of biological activity in the 415.1 μg Cry1Da_7/g diet, the concentration was 

corrected based on the difference in activity between the reference and combined Day 0 test diet 

observed in the dose confirmation study. The effective concentration was calculated to be 150 μg 

Cry1Da_7/g diet. While diets from the 50 μg Cry1Da_7/g diet treatment level were not evaluated 

for dose, homogeneity, and stability, the same correction factor was applied to this treatment level 

resulting in a calculated effective concentration of 18 μg Cry1Da_7/g diet. 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous dietary exposure to 

Cry1Da_7 protein at concentrations of either 18 or 150 μg/ml diet had no effects on the survival 

or adult mass of C. maculata. A statistically significant increase in development time (<1d) was 

observed for the Cry1Da_7 treatments at both concentrations. However, given the relatively small 

magnitude of the effect and the lack of effects in other study endpoints, the increase in development 

time was not considered to be adverse. The LC50 was >150 μg Cry1Da_7 protein/g diet and the 

NOAEC was 150 μg Cry1Da_7 protein/g diet, the highest concentration tested. 

 
Bayer CropScience LP CR282-20U1 365 

J.17. Evaluation of the Potential Effects of Cry1Da_7 Protein on Larvae of the Carabid 

Beetle, Poecilus cupreus 

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7 protein 

Description: Clear colorless solution 

Purity corrected concentration: 3.3 mg/mL 

Reference and/or positive control: Teflubenzuron Pestanal (Sigma-Aldrich) 

Dietery concentration: 32 μg/g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 10.5) 

incorporated into the diet at the same rate as for the 

400 μg/g test substance diet 

Test organisms: 

Species: Poecilus cupreus larvae 

Age: 1-2 d 

Diet/Food: Artificial diet provided ad libitum 

Acclimation Period: 11 days after hatching 

Environmental conditions: 

Temperature: 19.5-21.6 oC 

Relative humidity: 71-80% 

Photoperiod: 16 hours light / 8 hours dark 

Illumination: 742 lux 

Study Design and Methods 

Experimental treatments: Cry1Da_7 protein (in a buffer vehicle) was incorporated into a base 

diet mixture at two separate treatment concentrations, equivalent to 50 and 400 μg 

Cry1Da_7 protein/g diet. Additionally, a negative control (purified water only), a buffer control 

(25 mM sodium carbonate, pH 10.5 incorporated into the diet at the same rate as for the 400 μg/g 

test substance diet), and a positive control of teflubenzuron at 32 μg/g diet to confirm the 

effectiveness of the dietary feeding, were included in the study. The test arenas comprised lidded 

clear plastic tubes, each containing one beetle larva placed onto the surface of 25 g (dry weight) 

of a standard sandy soil moistened to 25% ± 5% of its maximum water-holding capacity (WHC). 

For the study, the soil-filled plastic tubes were stored upright in plastic boxes (one box per 

treatment), with lids containing 8 holes covered with nylon netting for ventillation and floor lined 
Bayer CropScience LP CR282-20U1 366 

with capillary matting to raise the relative humidity around the tubes. Freshly-defrosted aliquots 

of appropriate treatment diets were provided to 24-48 hour old P. cupreus larvae (n = 40 per 

treatment) and diets were refreshed daily until all larvae had pupated or 21 days had passed. 

Observations: The endpoints of the study were pre-imaginal mortality of the test insects, 

development time to adult emergence, and body weight of the emerged adult beetles. The condition 

of visible beetle larvae was assessed three times per week for the first three weeks of the study, 

and twice each week thereafter until all larvae had pupated. Fresh body weight of adult beetles was 

recorded after the elytra had hardened. The study ended 45 DAT when it was determined that no 

more adults would emerge. 

Statistical calculations: The overall percentage pre-imaginal mortality in each treatment was 

determined and the data corrected for any control deaths using Abbott’s formula (Abbott, 1925). 

Multiple sequentially-rejective Fisher test after Bonferroni-Holm correction (one-sided, > control, 

α = 0.05) was used to compare mortalities in the test substance and the buffer control treatment. 

The positive control treatment was compared to the buffer control by Fisher’s exact binomial test 

(one-sided, > control, α = 0.05). Adult body weight was evaluated with a Dunnett’s multiple t-test 

(one-sided, < control α = 0.05) comparing the test treatments to the buffer control. Effects on 

development time were evaluated with a two-sample Mann Whitney U-test (one-sided, > control 

α = 0.05) comparing the test treatments to the buffer control. 

Results

Pre-imaginal mortality was 15% in the negative control and 10% in the buffer control, compared 

with 5% and 7.5% mortality in the 50 and 400 μg Cry1Da_7 protein/g diet treatment 

concentrations, respectively. There were no significant differences in mortality between either test 

treatment level and the buffer control (p>0.05). No abnormal larvae, pupae or adults were seen, in 

any of the treatments. The positive control treatment resulted in 100% pre-imaginal mortality 

(100% corrected) and this treatment differed significantly from the buffer control. 

The mean emergence time for beetles surviving was 39.1 days in the negative control and 38.4 

days in the buffer control, compared with 39.3 days and 37.3 days in the 50 and 400 μg Cry1Da_7 

protein/g diet treatment concentrations, respectively. There were no significant differences in 

development time between either test treatment level and the buffer control (p>0.05). 

The mean body weight of individual beetles taken soon after their emergence was 68.0 mg in the 

negative control and 69.4 mg in the buffer control, compared with 68.2 and 69.8 mg in the 50 and 

400 μg Cry1Da_7 protein/g diet treatment concentrations, respectively. There were no significant 

differences in mean body weight between either test treatment level and the buffer control 

(p>0.05). 

Assay Validity Criteria 
Bayer CropScience LP CR282-20U1 367 

The mortality of P. cupreus was 15% in the negative control and 10% in the buffer control, 

indicating there was no adverse effect from the buffer on survival. These results met the assay 

acceptance criteria of having d20% mortality in the control. The positive control had 100% 

mortality, demonstrating sensitivity of the test species. 

The results of the diet analysis performed for the 400 μg Cry1Da_7 protien/g diet demonstrated 

the Cry1Da_7 test substance incorporated into diet had the expected level of biological activity, 

was considered homogeneous, and was stable under test and frozen storage conditions. 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous dietary exposure to 

Cry1Da_7 protein at concentrations of either 50 μg or 400 μg Cry1Da_7/g diet had no effects on 

on the survival, development time or adult body weight of the ground-dwelling carabid beetle, 

Poecilus cupreus. The LC50 was >400 μg Cry1Da_7 protein/g diet and the NOAEC was 400 μg 

Cry1Da_7/g diet, the highest concentration tested. 

 
Bayer CropScience LP CR282-20U1 368 

J.18. Evaluation of the Potential effects of Cry1Da_7 Protein on Nymphs of the Big-Eyed 

Bug, Geocoris punctipes

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7 protein 

Description: Clear colorless solution 

Purity corrected concentration: 3.3 mg/mL 

Reference and/or positive control: Potassium arsenate 

Dietery concentration: 100 μg/g diet 

Buffer control: 25 mM sodium carbonate buffer solution, 

(pH 10.5) incorporated into the diet to achieve the 

same concentration of buffer per g diet as used for 

the highest test substance treatment 

Test organisms: 

Species: Geocoris punctipes 

Age: 1 d 

Diet/Food: Ephestia kuehniella eggs and a slice of green bean 

pod provided ad libitum 

Acclimation Period: 1 d 

Environmental conditions: 

Temperature: 25 oC 

Relative Humidity: 50% 

Photoperiod: 14 hours light / 10 hours dark 

Study Design and Methods 

Experimental treatments: Cry1Da_7 protein (in a buffer vehicle) was incorporated into a base 

diet mixture at two separate treatment concentrations, equivalent to 50 and 500 μg

Cry1Da_7 protein/g diet each encapsulated to form a dome stored at -20°C. Additionally, a 

negative control (purified water only), a buffer control (25 mM sodium carbonate, pH 10.5 

incorporated into the diet at the same rate as for the 500 μg/g test substance diet), and a positive 

control diet containing potassium arsenate at 100 μg/g diet were included in the study. The nymphs 

after one-day acclimation were individually transferred into test arenas (inverted Falcon petri 

dishes, 60×15mm, Corning, NY) and impartially assigned to each of the treatments by providing 

appropriate treatment diets. Two freshly-defrosted encapsulated diets (domes) of the appropriate 
Bayer CropScience LP CR282-20U1 369 

treatment diet were provided in each test arena and renewed every 48 hours. The nymphs were 

allowed to feed ad libitum throughout the study duration. Each diet treatment consisted of a total 

of 30 nymphs individually housed in inverted petri dishes lined with a piece of filter paper on the 

bottom. One drop of distilled water was added on the filter paper every day at the time of 

observation to moisten the filter paper. 

Observations: Daily observations were made to record nymph survival, the time taken for the 

nymphs to develop into adulthood, and the fresh body weight of the newly-emerged adults. 

Statistical calculations: Fisher’s Exact test was performed to compare the survival between 

predefined treatments. Treatment effects on the time of development to adult and adult body mass 

were analyzed with a linear model using SAS PROC Mixed. Pair-wise comparisons with the buffer 

control were defined within the linear model and tested using t-tests with significant difference at 

α = 0.05. 

Results

G. punctipes nymph survival was 86.7% in the negative control and 96.7% in the buffer control 

diet treatments, compared with 89.7% and 93.3% survival in the 50 and 500 μg Cry1Da_7 

protein/g diet treatment concentrations, respectively. There were no significant differences in 

mortality between either test treatment level and the buffer control (p>0.05). The positive control 

treatment resulted in 100% mortality. 

All surviving nymphs completed the development into adulthood. The mean nymph development 

time to adults was 31.0 days in the negative control and 30.3 days in the buffer control, compared 

with 30.3 days and 30.5 days in the 50 and 500 μg Cry1Da_7 protein/g diet treatment 

concentrations, respectively. There were no significant differences in development time between 

either test treatment level and the buffer control (p>0.05). 

The mean body weight of newly-emerged adults was 4.4 mg in the negative control, 4.3 mg in the 

buffer control, compared with 4.5 mg in both test treatment levels. There was no significant 

differences in mean body weight between either test treatment level and the buffer control 

(p>0.05). 

Assay Validity Criteria 

The survival of G. punctipes nymphs was 86.7% in the negative control and 96.7% in the buffer 

control, indicating there was no adverse effect from the buffer on survival. These results met the 

assay acceptance criteria of having t80% survival in the control. The positive control resulted in 

100% mortality demonstrating sensitivity of the test species. 

The results of the diet analysis demonstrated that the Cry1Da_7 protein in the treatment diet had 

the expected level of biological activity and was stable under test and frozen storage conditions. 
Bayer CropScience LP CR282-20U1 370 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous dietary exposure to 

Cry1Da_7 protein at concentrations of either 50 or 500 μg Cry1Da_7 protein/g diet had no effects 

on either the survival or development of the G. punctipes nymphs. The LC50 was >500 

μg Cry1Da_7 protein/g diet and the NOAEC was 500 μg Cry1Da_7 protein/g diet, the highest 

concentration tested. 

J.19. Evaluation of the Potential Effects of Cry1Da_7 Protein on Adults of the Parasitoid 

Wasp, Pediobius foveolatus

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7 protein 

Description: Clear colorless solution 

Purity corrected concentration: 3.3 mg/mL 

Reference and/or positive control: Potassium arsenate 

Dietery concentration: 200 μg/ml diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 10.5) 

incorporated into the diet to achieve the same 

concentration of buffer per g diet as used for the 

highest test substance treatment 

Test organisms: 

Species: Pediobius foveolatus 

Age: 1 d 

Diet/Food: 30% honey/water (v/v) solution

Acclimation Period: 24 h 

Environmental conditions: 

Temperature: 25 oC 

Relative Humidity: 70% 

Photoperiod: 14 hours light / 10 hours dark 
Bayer CropScience LP CR282-20U1 371 

Study Design and Methods 

Experimental treatments: Cry1Da_7 protein (in a buffer vehicle) was incorporated into 30% 

honey/purified water solution at two nominal concentrations of 50 and 500 Pg/ml 30% 

honey/purified water solution, respectively. Additionally, a negative control (purified water only), 

a buffer control (25 mM sodium carbonate, pH 10.5 incorporated into the diet at the same rate as 

for the 500 μg/ml test substance diet), and a positive control diet containing potassium arsenate at 

200 μg/ml diet were included in the study. The newly emerged P. foveolatus adults produced in 

laboratory from the parasitized E. varivestis larvae were provided a 30% honey/water (v/v) 

solution and were impartially transferred into 162 cm2 cell culture flasks. After approximately 24 

hours of acclimation, 15 adults in each flask were impartially assigned to the five treatments and 

provided with appropriate treatment diet in two screened feeding dishes, replaced every two days. 

Each treatment included four replicates. The wasp adults were allowed to feed ad libitum

throughout the 21 days feeding period. 

Observations: All wasps in all treatments were observed for mortality/survival every two days at 

the time of diet replacement. Dead wasps, if any, were removed from the test arenas and recorded. 

Statistical calculations: Statistical analyses were not performed since no mortality was recorded 

in either Cry1Da_7 treatment level as well as the negative and buffer controls and 100% mortality 

was recorded in positive control treatment. 

Results

P. foveolatus adult survival was 100% in both 50 and 500 μg Cry1Da_7 protein/ml diet treatment 

levels as well as the negative and buffer controls. The positive control treatment resulted in 100% 

mortality (0% survival). 

Assay Validity Criteria 

The survival of P. foveolatus larvae in both the negative control and buffer control was 100%, 

indicating there was no adverse effect from the buffer on survival. These results met assay 

acceptance criteria of having t80% survival in the control. The positive control treatment resulted 

in 100% mortality, demonstrating sensitivity of the test species. 

The results of the diet analysis demonstrated that the Cry1Da_7 protein in the treatment diet had 

the expected level of biological activity, and was stable under test and frozen storage conditions. 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous dietary exposure to 

Cry1Da_7 protein at concentrations of either 50 or 500 μg Cry1Da_7 protein/ml diet for 21 days 
Bayer CropScience LP CR282-20U1 372 

had no effects on the survival of the P. foveolatus adults. The LC50

was >500 μg Cry1Da_7 protein/ml diet, the highest concentration tested. 

J.20. Evaluation of the Potential Effects of Cry1Da_7 Protein on Adults of the Green 

Lacewing, Chrysoperla carnea

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7 protein 

Description: Clear colorless solution 

Purity corrected concentration: 3.3 mg/mL 

Reference and/or positive control: Dimethoate 

Dietery concentration: 0.04 μg a.s./g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 10.5) 

incorporated into the diet at the same rate as for the 

500 μg/g test substance diet 

Test organisms: 

Species: Chrysoperla carnea

Age: 24 h 

Sex: 10 males/10 females per treatment group 

Diet/Food: None

Environmental conditions: 

Temperature: 23.8-25.4 oC 

Relative humidity: 70-78% 

Photoperiod: 16 hours light / 8 hours dark 

Illumination: 2000-4800 lux 

Study Design and Methods 

Experimental treatments: Cry1Da_7 protein (in a buffer vehicle) was incorporated into an 

artificial agar-based diet at concentrations of 50 and 500 μg Cry1Da_7 protein/g diet. Additionally, 

a negative control (purified water only), a buffer control (25 mM sodium carbonate buffer solution, 

pH 10.5 incorporated into the diet at the same level as in the 500 μg Cry1Da_7 protein diet), and 

a positive control diet containing dimethoate at 0.04 μg/g diet to confirm the effectiveness of the 

dietary feeding, were included in the test. The test arenas comprised clear polystyrene boxes with 

close-fitting lids. The dishes of the treated diet, and dishes of honey-water and water-only, were 
Bayer CropScience LP CR282-20U1 373 

placed on the floor of each box. For each treatment there were two replicate boxes, each containing 

20 Chrysoperla carnea adult lacewings (i.e. 40 per treatment) with 10 female and 10 male 

lacewings impartially assigned to each test arena. Any insects that appeared to have been damaged 

during the transfer process were replaced immediately. Freshly-defrosted aliquots of appropriate 

treatment diets were provided to newly-emerged adult lacewings three times per week, for two 

weeks. 

Observations: The endpoint of the study was an assessment of the adult mortality. The adults 

were assessed every 1-3 days for 14 days and finally at 14 days after treatment, from which the 

overall percentage mortality in each treatment was calculated. 

Statistical calculations: The percentage adult mortality in each treatment was determined and the 

data corrected for any insect deaths in the buffer control using Abbott’s formula (Abbott, 1925). 

Mortality was evaluated with a multiple sequentially-rejective Fisher test after Bonferroni-Holm 

correction (one-sided, > control, α = 0.05) comparing mortalities in the test substance and the 

buffer control. The positive control treatment was compared to the buffer control by Fisher’s exact 

binomial test (one-sided, > control, α = 0.05). 

Results

Mortality of C. carnea adults was 17.5% in the negative control and 10% mortality in the buffer 

control, compared with 30% and 7.5% mortality in the 50 μg and 500 μg Cry1Da_7 protein/g diet 

treatment levels, respectively. When corrected against the buffer control, mortality was 22.2% 

and -2.8% in the 50 μg and 500 μg Cry1Da_7 protein/g diet treatment levels, respectively. There 

was no significant difference in mortality between the 500 μg Cry1Da_7/g diet treatment level and 

the buffer control (p>0.05). The 50 μg Cry1Da_7 protein/g diet treatment showed significantly 

increased mortality compared to the buffer control (p = 0.024). However, since no adverse effects 

were observed in the 500 μg Cry1Da_7 protein/g diet treatment and the concentration in diet was 

confirmed by sensitive insect assay, it was considered that the statistically significant increase in 

mortality at the 50 μg Cry1Da_7 protein/g diet treatment level was not due to a treatment effect. 

The positive control treatment resulted in 77.5% mortality and this treatment differed significantly 

from the buffer control (p<0.05). 

Assay Validity Criteria 

Mortality of C. carnea was 17.5% in the negative control and 10.0% in the buffer control, 

indicating there was no adverse effect of the buffer on survival. These results met assay acceptance 

criteria of having d20% mortality in the control. The positive control had 77.5% mortality, 

demonstrating sensitivity of the test species. 

The results of the diet analysis by sensitive insect bioassay demonstrated that the Cry1Da_7 protein 

at the 500 μg Cry1Da_7/g diet treatment level was biologically active, homogenous throughout 

the test diet and was stable under test and frozen storage conditions. For the 50 μg Cry1Da_7/g 
Bayer CropScience LP CR282-20U1 374 

diet treatment level, the Cry1Da_7 protein was confirmed to be homogenous throughout the test 

diet and was stable under test conditions, but did not have the expected level of biological activity. 

Therefore, 50 μg Cry1Da_7/g diet treatment level was corrected to an effective concentration of 

27 μg Cry1Da_7 protein/g of diet. 

Conclusion 

Under laboratory test conditions, continuous dietary exposure of Cry1Da_7 protein to adults of the 

green lacewing, C. carnea over 14 days resulted in no treatment-related adverse effects on survival 

at either 27 (dose-corrected) or 500 μg protein/g diet. The LC50 was >500 μg Cry1Da_7 protein/ml 

diet, the highest concentration tested. 

J.21. Evaluation of the Potential Effects of Cry1Da_7 Protein on Larvae of the Green 

Lacewing, Chrysoperla rufilabris

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7 protein 

Description: Clear colorless solution 

Purity corrected concentration: 3.3 mg/mL 

Reference and/or positive control: Potassium arsenate 

Dietery concentration: 0.243 mg/g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 10.5) 

incorporated into the diet at the same rate as for the 

500 mg/kg test substance diet 

Test organisms: 

Species: Chrysoperla rufilabris

Age: Second instar larvae 

Diet/Food: Untreated diet and water soaked cotton balls

Acclimation Period: 24 hours 

Environmental conditions: 

Temperature: 24-26oC 

Relative humidity: 55-65% 

Photoperiod: 16 hours light / 8 hours dark 

Illumination: 420-790 lux 

 
Bayer CropScience LP CR282-20U1 375 

Study Design and Methods 

Experimental treatments: Second instar Chrysoperla rufilabris larvae (3-5 days from egg hatch) 

were exposed to six treatments, Cry1Da_7 protein at 500 μg protein/g diet, Cry1Da_7 protein at 

100 μg protein/g diet, Cry1Da_7 protein at 20 μg protein/g diet, a negative control consisting of 

meat-based diet without any test substance or buffer added, buffer control consisting of 25 mM 

sodium carbonate buffer solution (pH 11.5) incorporated into the diet at the same rate as for the 

500 μg/g test substance diet, and a positive control of potassium arsenate at 0.243 μg product/g 

diet to confirm the effectiveness of the dietary feeding for 24 days. The test vessels comprised 

Petri dishes with lids. Lacewing larvae were acclimated for 24 hours prior to study initiation and 

were impartially allocated to the test vessels. Each treatment and control had 40 replicate test 

vessels with each vessel containing one lacewing larva. Individual vessels were randomly assigned 

to the treatments using computer generated random numbers. Test vessels were then randomly 

placed within the environmental chamber during the exposure. At study initiation, one freshlydefrosted 50 μL pellet of the appropriate control or treatment level was added to each test vessel 

along with one moistened small cotton ball with both being replaced at every 48-hour interval until 

organisms reached pupal state, when organisms do not actively feed on diet. The study was 

terminated on day 24 and pupae that had not emerged were considered dead. 

Observations: The endpoints of the study were percentage pre-imaginal survival, development 

time from larvae to emerged adult, and adult weight at emergence. The health of the organisms 

was observed and recorded daily. Pupation and adult emergence were recorded when observed. 

Statistical calculations: The treatment data were tested for normality and homogeneity of 

variance using the appropriate qualifying test. Fisher’s exact test with Bonferroni-Holm correction 

was used to compare survival between the treatment and negative control groups. Effects on 

development time were evaluated with a Dunnett’s multiple comparison test comparing test 

treatments to the negative control. Adult bw was evaluated with a Jonckheere-Terpstra’s Stepdown test comparing the test treatments and the negative control. All comparisons were made at 

≥95% level of certainty (D= 0.05) and compared on a per replicate basis. 

Results

The mean pre-imaginal survival was 85% in the negative control and 88% in the buffer control, 

compared with 75, 75, and 73% in the 20, 100, and 500 mg/kg treatment levels, respectively. There 

were no significant reductions in pre-imaginal survival among organisms exposed to the test 

treatment levels compared to the negative control (p>0.05). 

The mean weight for adults at emergence was 0.0076 in the negative control and 0.0077 in the 

buffer control, compared with 0.0072, 0.0071, and 0.0076 g in the 20, 100, and 500 mg/kg 

treatment levels, respectively. There were no significant reductions in emergence weight among 

organisms exposed to the test treatment levels compared to the negative control (p>0.05). 
Bayer CropScience LP CR282-20U1 376 

The mean development time from larvae to emerged adult was 19 for the negative control, buffer 

control, 20, 100, and 500 mg/kg treatment levels. There were no significant reductions in time 

from larvae to emerged adult among organisms exposed to the test treatment levels compared to 

the negative control (p>0.05). 

Assay Validity Criteria 

The survival of C. rufilabris was 85% in the negative control and 88% in the buffer control group, 

indicating there was no adverse effect from the buffer on survival. These results met acceptance 

criteria of having t80% survival in the control. The positive control had 100% larval mortality 

demonstrating sensitivity of the test species. 

The results of the diet analysis demonstrate that the Cry1Da_7 test substance incorporated into the 

test substance treatment diets had the expected level of biological activity, was homogeneous 

throughout the test substance diet, was stable under test and frozen storage conditions. 

Conclusion 

Under laboratory test conditions, Cry1Da_7 protein had no statistically significant effects on the 

survival and development of larvae or adult weight of the green lacewing, Chrysoperla rufilabris

at concentrations of 20 μg, 100 μg, and 500 μg Cry1Da_7/g diet fed continuously during their 

development for 24 days. The LC50 was >500 μg Cry1Da_7 protein/g diet and the NOAEC was 

500 μg Cry1Da_7/g diet, the highest concentration tested. 

 
Bayer CropScience LP CR282-20U1 377 

J.22. Evaluation of the Potential Effects of Cry1Da_7 Protein on the Springtail, Folsomia 

candida

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7 protein 

Description: Clear colorless solution 

Purity corrected concentration: 3.3 mg/mL 

Reference and/or positive control: Teflubenzuron 

Dietery concentration: 0.1 mg per g diet 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 10.5) 

incorporated into the diet to achieve the same 

concentration of buffer per g diet as used for the 

highest test substance treatment 

Test organisms: 

Species: Folsomia candida 

Age: 12 d 

Diet/Food: Artificial diet provided ad libitum 

Acclimation Period: 11 days after hatching 

Environmental conditions: 

Temperature: 19.3-21.0 oC 

Photoperiod: 16 hours light / 8 hours dark 

Illumination: 560-710 lux 

Study Design and Methods 

Experimental treatments: Cry1Da_7 protein (in a buffer vehicle) was incorporated into a diet 

medium of inactivated yeast at two separate treatment concentrations, equivalent to 50 and 500 μg 

Cry1Da_7 protein/g diet. Additionally, a buffer control diet to which 25 mM sodium carbonate 

buffer solution (pH 10.5) was added at an equivalent concentration as in the 500 μg Cry1Da_7 

protein/g treatment, a negative control diet to which only purified water was added, and a positive 

control diet treated with a technical-grade sample of teflubenzuron, at a concentration equivalent 

to 0.1 mg/g diet were included in the study. Freshly-defrosted aliquots of appropriate treatment 

diets were provided continuously to batches of confined springtails over a 28-day period. The test 

arenas comprised lidded jars, the bases of which were lined with a plaster-of-Paris and charcoal 

substrate. Ten juvenile springtails (12 days old) were placed in each jar (n = 4 per treatment) and 
Bayer CropScience LP CR282-20U1 378 

were fed ad libitum throughout the study. Freshly-treated aliquots of appropriate treatment diet 

were provided on the day of study initiation (Day 0) and additional freshly-defrosted aliquots were 

then provided every 2-3 days. At Day 28, the numbers of surviving springtails and their offspring 

(progeny) were recorded for each replicate arena. 

Observations: For the study, the numbers both of surviving adults and F1 progeny in each test 

arena were recorded at 28 days after treatment (DAT). It was assumed that any adult springtails 

that were recovered would have been those originally introduced and that any shortfall in the 

original number was an indication that they had died during the study. The numbers of surviving 

springtails were used to calculate the percentage mortality of the springtails originally introduced 

in each treatment. 

Statistical calculations: The overall percentage mortality in each treatment was determined and 

the data corrected for any control deaths using Abbott’s formula (Abbott 1925). Multiple 

sequentially-rejective Fisher test after Bonferroni-Holm correction (one-sided greater, α = 0.05) 

was used to compare mortalities in the individual test substance treatments with the buffer control. 

Following a check for normal distribution of the data (Shapiro-Wilk test, α = 0.05) and for equality 

of variances (Levene’s test, α = 0.05), test substance treatments were compared to the buffer 

control by multiple sequentially-rejective t-test after Bonferroni-Holm correction (one-sided 

smaller, α = 0.05). The positive control treatment was compared to the buffer control by Student 

t-test for homogeneous variances (one-sided smaller, α = 0.05). 

Results

After 28 days, mortality was 0% in both the negative and buffer controls, compared with 3% and 

0% mortality in the 50 and 500 μg Cry1Da_7 protein/g diet treatment concentrations, respectively. 

When adjusted for the buffer control treatment, the test substance treatments resulted in 3% and 

0% corrected mortality in the 500 and 50 μg Cry1Da_7 protein/g diet treatment concentrations, 

respectively. There were no significant differences in mortality between either test treatment level 

and the buffer control (p>0.05). The positive control treatment resulted in 100% mortality (100% 

corrected) and this treatment differed significantly from the buffer control (p>0.05). 

The mean number of progeny produced per treatment was 557 in the negative control and 560 in 

the buffer control, compared with 531 and 547 in the 50 and 500 μg Cry1Da_7 protein/g diet 

treatment concentrations, respectively. There were no significant differences in reproduction 

between either Cry1Da_7 treatment level and the buffer control (p>0.05). 

Assay Validity Criteria 

The mortality of F. candida was 0% in both the negative and buffer controls, indicating there was 

no adverse effect from the buffer on survival. These results met the assay acceptance criteria of 

having d20% mortality in the control. The mean number of juveniles recorded in the negative 
Bayer CropScience LP CR282-20U1 379 

control treatment was at least 100 per replicate at the end of the study and the coefficient of 

variation of reproduction in the negative control was d30% meeting acceptance criteria. The 

positive control treatment resulted in 100% mortality, demonstrating sensitivity of the test species. 

The results of the diet analysis demonstrated that the test substance, Cry1Da_7 protein in test diet 

had the expected level of biological activity, was homogenous throughout the freshly-prepared test 

diet and was stabile under test and storage conditions. 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous exposure to Cry1Da_7 

protein for 28 days at concentrations of either 50 or 500 μg Cry1Da_7 protein/g diet had no effects 

on either the survival or reproduction rate of the springtail Folsomia candida. The LC50 was >500 

μg Cry1Da_7 protein/g diet and the NOAEC was 500 μg Cry1Da_7 protein/g diet, the highest 

concentration tested. 

 
Bayer CropScience LP CR282-20U1 380 

J.23. Evaluation of the Potential effects of Cry1Da_7 Protein on the Earthworm, Eisenia 

andrei 

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7protein 

Description: Clear colorless solution 

Purity corrected concentration: 3.3 mg/mL 

Vehicle and/or positive control: 2-chloroacetamide (tested in a separate study) 

Buffer control: 25 mM sodium carbonate buffer solution, (pH 10.5) 

incorporated into the soil to achieve the same 

concentration of buffer per g soil as used for the test 

substance treatments 

Test organisms: 

Species: Eisenia andrei 

Age: ~7.5 months 

Diet/Food: None

Acclimation Period: 1 day 

Environmental conditions: 

Temperature: 20.2-20.9 oC 

Photoperiod: 24 hours light 

Illumination: 520-600 lux 

Study Design and Methods 

Experimental treatments: The Cry1Da_7 protein held in a buffer solution was incorporated into 

an artificial soil medium at two separate treatment rates, equivalent to 50 and 500 μg 

Cry1Da_7protein/g soil dry weight. For the study, these two treatments were compared to a control 

soil treated with either 25 mM sodium carbonate buffer solution, pH 10.5 at the same concentration 

as used for the test substance treatments (buffer control) or with purified water (negative control). 

To confirm sensitivity of the test species, a positive control treatment of 2-chloroacetamide was 

evaluated in a separate, GLP-compliant study within 2 months prior to the start of the study. The 

artificial soil substrate (containing 10% w/w peat), was moistened to 50% of its pre-determined 

maximum water-holding capacity at the time of treatment. The treated soil was held within 1-Lcapacity lidded jars (n = 4 per treatment) and 10 adult E. andrei (approx. 7.5 months old, with an 

individual fresh weight of 358-598 mg and with a visible clitellum) were weighed and introduced 
Bayer CropScience LP CR282-20U1 381 

into each jar immediately after treatment application. Each treatment was replicated four times for 

a total of 40 earthworms exposed per treatment. No food was provided throughout the duration of 

the study. 

Observations: For the study, the numbers of surviving adult earthworms in each test arena were 

recorded at 7 and 14 DAT. It was assumed that any adult earthworms that were recovered would 

have been those originally introduced and that any shortfall in the original number was an 

indication that they had died during the study. The mean percentage change in weight of the 

earthworms in the controls and test-substance treatments over 14 days was calculated. The 

earthworms were gently cleaned using purified water and individually re-weighed at 14 DAT after 

removing excess moisture to determine the mean weight of individuals in each replicate. The 

percentage change in mean weight per replicate was derived by comparing the mean weight 

recorded at 0 DAT with that measured again at 14 DAT for earthworms in each replicate. The 

earthworms were also examined for any other harmful effects (e.g. behavioral abnormalities or 

open wounds) at 0, 7 and 14 DAT. 

Statistical calculations: Since there was no mortality in the controls, it was unnecessary to correct 

for mean control mortality using Abbott’s formula. Fisher’s Exact binomial test with Bonferroni 

correction (one-sided, > control, α = 0.05) was used to compare mortalities in the individual test 

treatments with the buffer control treatment. Following a check for normal distribution of the data 

(Shapiro-Wilk test, α = 0.05) and for equality of variances (Levene’s test, α = 0.05), test treatments 

were compared to the buffer control by multiple sequentially-rejective t-test after Bonferroni-Holm 

correction (one-sided smaller, α = 0.05). 

Results

After 14 days, mortality was 3% in the negative control and 0% in the buffer control treatments, 

compared with no mortality in both the 50 and 500 μg Cry1Da_7protein/g soil dry weight 

treatment concentrations. Therer were no significant differences in mortality between either test 

treatment level and the buffer control (p>0.05). 

The mean percentage change in earthworm weight per treatment was equivalent to a decrease of 

5.4% in the buffer control and 5.6% in the negative control, compared with an increase of 4.9% 

and a decrease of 1.4% in the 50 and 500 μg Cry1Da_7protein/g soil dry weight treatment 

concentrations, respectively. There was no significant decrease in body weight between the test 

treatments and the buffer control (p>0.05). At both 7 and 14 DAT, all of the earthworms in all of 

the treatments appeared healthy and active. 

 
Bayer CropScience LP CR282-20U1 382 

Assay Validity Criteria 

The mortality of Eisenia andrea was 3% in the negative control and 0% in the buffer control group, 

indicating there was no adverse effect of the buffer on survival. These results met the assay 

acceptance criteria of d10% mortality in the control. The mean post-treatment weight of the 

negative control group decreased by <20% compared to the start of the study, meeting acceptance 

criteria. 

As this was an acute study with a single application of the Cry1Da_7 protein, no direct 

measurement was made of test substance homogeneity in the soil. However, care was taken to mix 

the treated soil thoroughly. No measurement was made of the stability of the test substance in the 

soil, but the test earthworms were placed onto the soil immediately after the treatment of each 

arena. 

Conclusion 

Under laboratory test conditions, it was demonstrated that continuous exposure to Cry1Da_7 

protein for 14 days in an artificial soil containing 10% w/w peat at concentrations of either 50 or 

500 μg Cry1Da_7 protein/g soil dry weight had no significant adverse treatment effects on the 

survival or biomass of the earthworm Eisenia andrei. Additionally, no abnormal behaviors were 

observed in any of the treatments. The LC50 was >500 μg Cry1Da_7 protein/g soil dry weight and 

the NOAEC was 500 μg Cry1Da_7 protein/g soil dry weight, the highest concentration tested. 

 
Bayer CropScience LP CR282-20U1 383 

J.24. Evaluation of the Potential Effects of Cry1Da_7 Protein on Adults of the Honey Bee, 

Apis mellifera

Materials 

Test material: 

Test item: Cry1Da_7 

Description: Liquid 

Purity corrected concentration: 3.3 mg/mL 

Positive control: Dimethoate 

Dietery concentration: 0.65 mg/kg diet 

Buffer control: 25 mM sodium carbonate buffer solution (pH 10.5) 

incorporated into the diet to achieve the same 

concentration of buffer per g diet as used for the 

highest test substance treatment 

Test organisms: 

Species: Apis mellifera L. 

Age: d2d at dosing 

Diet/Food: 50% sucrose/purified water solution provided ad 

libitum

Acclimation Period: 1 d 

Environmental conditions: 

Temperature: 33°C 

Relative Humidity: 50-61 % 

Photoperiod: Maintained in the dark thoughout duration of study 

Study Design and Methods 

Experimental treatments: Honey bee adults, ≤2 days old, were exposed to five treatments, 

Cry1Da_7 protein at 10 μg/g in combination with 50% sucrose/purified water solution, Cry1Da_7 

protein at 500 μg/g in combination with 50% sucrose/purified water solution. In addition, a 

negative control consisting of 50% sucrose/purified water solution only, buffer control consisting 

of test substance buffer (25 mM sodium carbonate buffer solution, pH 10.5) at a volume equivalent 

to the highest test substance concentration, and a positive control of dimethoate at 0.65 mg/kg to 

confirm the effectiveness of the dietary feeding, were included in the study. Five cages (replicates) 

for each five treatments comprised a total of 25 cages. Individual cages contained 10 adult bees 

obtained from a local hive mainatined by Eurofins that was apparently healthy, queen-right, and 

had not been treated for control of Varroa or pathogens within four weeks of the test. Once the 

honey bee adults were caged impartially, each cage was assigned to a treatment/replicate in a nonsystematic fashion. After all adult bees were acclimated under red light with 50% (w/v) sucrose 
Bayer CropScience LP CR282-20U1 384 

solution provided ad libitum, each cage was provided with the appropriate treatment diet solution 

through two feeders (syringes, each containing ~2.5 mL diet) inserted through the lid of the test 

chamber and bees were allowed to feed ad libitum. Feeders were replaced daily and were weighed 

prior to placing them into the cages and again after removal 24 ± 2 hours later to measure the 

amount of diet consumed in each replicate. The study duration was 10 days with adult bees being 

observed daily for mortality, behavioral and toxicological responses. 

Observations: All treatments were observed for mortality, behavioral and toxicological responses 

once within the first four hours after initial treatment and at approximately 24-hour intervals (from 

dosing) thereafter. Abnormal behavior was determined by comparing honey bees in the treatment 

groups with those in the negative control group. Dead honey bee adults remained in their respective 

cages until study completion to avoid unnecessary disturbances. Consumption per bee in each 

replicate was calculated for each day using the consumption data adjusted for evaporative loss and 

the total number of bees alive in each replicate at the start of each feeding period (day). The 

consumption per bee values for all the replicates within a treatment level were then averaged for 

each day. Replicates were no longer included in the average consumption calculations after 

reaching 100% mortality. 

Statistical calculations: Dunnett’s test was used to compare mortalities in the test treatments with 

the negative control. 

Results

Mortality was 8% in the negative control and 4% in the buffer control, compared to 20% and 12% 

mortality in the 10 and 500 μg Cry1Da_7 protein/g diet treatment concentrations, respectively. 

There were no significant differences in mortality between test treatment levels and the negative 

control (p>0.05). All surviving bees appeared normal in the end of the study. Positive control group 

mean mortality was 100% at the end of the study. 

Average daily consumption over the 10-day exposure period were 28, 29, 26, 30, and 11 mg 

diet/bee/day in the negative control, buffer control, 10 μg Cry1Da_7 protein/g, 500 μg Cry1Da_7 

protein /g, and positive control groups, respectively. These results demonstrate that diet 

consumption was comparable in all groups, except positive control. The daily doses were 

calculated based on the average daily consumption values and were 0.26 and 15 μg Cry1Da_7 

protein/bee/day during the test for the 10 μg Cry1Da_7 protein/g and 500 μg Cry1Da_7 protein/g 

treatment levels, respectively. 

Assay Validity Criteria 

The mortality of Apis mellifera adults was 8% in the negative control and 4% in the buffer control 

group, indicating there was no adverse effect from the buffer on survival. These results met assay 

acceptance criteria of d20% mortality in the control. The positive control had 100% mortality 

demonstrating sensitivity of the test species. 
Bayer CropScience LP CR282-20U1 385 

The results of the diet analysis demonstrated the Cry1Da_7 protein incorporated into the test 

treatment diets had the expected level of biological activity, was stable for 1 day under test 

conditions, was stable at 4 °C for 3 days, and was stable under frozen storage conditions.

Conclusion 

Following oral exposure of adult honey bees to Cry1Da_7 protein for 10 days under laboratory 

test conditions, the LC50 was >500 μg Cry1Da_7 protein/g diet and the NOAEC value was 500 μg 

Cry1Da_7 protein/g, highest concentration tested. 

J.25. Evaluation of the Potential Effects of Cry1Da_7 Protein on Larvae of the Honey Bee, 

Apis mellifera

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7 

Description: Liquid 

Purity corrected concentration: 3.3 mg/mL 

Positive control: Dimethoate 

Dietery concentration: 7.42 μg/bee 

Test organisms: 

Species: Apis mellifera L. 

Age: ~6 d at dosing 

Diet/Food: Artificial diet (OECD 239) 

Acclimation Period: 2 d after grafting 

Environmental conditions: 

Average Temperature: 34.47°C (Larval exposure phase) 

34.27°C (Pupal transfer to emergence) 

Average Relative Humidity: 92.88 % (Larval exposure phase) 

76.80 % (Pupal transfer to emergence) 

Photoperiod: Maintained in the dark thoughout duration of study 

 
Bayer CropScience LP CR282-20U1 386 

Study Design and Methods 

Experimental treatments: Cry1Da_7 protein (in a buffer vehicle) was incorporated into an 

artificial diet at concentrations of 10 and 500 μg Cry1Da_7 protein/g diet. Additionally, a negative 

control (purified water only), a buffer control (25 mM sodium carbonate buffer solution, pH 10.5 

incorporated into the diet at the same level as in the 500 μg Cry1Da_7 protein diet), and a positive 

control diet containing dimethoate at 52.8 μg/ml diet to confirm the effectiveness of the dietary 

feeding, were included in the test. First instar larvae from three hives were transferred (grafted) to 

well plates containing untreated artificial diet and held in an incubator. Starting at two days after 

grafting, artificial diets containing the test or control substance were provided to larvae. Test diets 

were provided for a total of four days. Larvae were then held for up to 14 days after the completion 

of dosing in order to allow emergence of adult bees. Each treatment and control group contained 

16 larvae from each of the three hives, for a total of 48 larvae per treatment group. Nominal test 

levels were 10 and 500 μg Cry1Da_7/g diet.

Observations: Starting on the first day of dosing (Day 3), larvae were observed daily until either 

mortality or adult emergence occurred. Cell cups containing dead larvae were removed from well 

plates after mortality was recorded. Observations of sublethal effects, including the presence of 

uneaten diet and larvae with reduced body size, were recorded on Days 7 and 8. Emerged bees 

from each replicate were checked to evaluate sublethal effects on morphology of emerged bees. 

By Day 20, approximately 14 days after final dosing, all bees had either emerged or died. 

Statistical calculations: Dunnett’s multiple comparison test was used to compare mortalities in 

the individual test treatments to the negative control. Mean larval survival and pupal survival were 

calculated but not be analyzed statistically. Sublethal effects were reported but not statistically 

analyzed. 

Results

Mean mortality was 10% in the negative control and 17% in the buffer control and no significant 

difference was detected according to t-test (p>0.05). Mean mortality in the 10 and 500 μg a.i./g 

treatment groups were 15 and 13%, respectively. There was no significant differences in mortality 

between either test treatment level and the buffer control (p>0.05). In the end of the study, all 

emerged adult bees appeared healthy with no morphological deformities observed, except one bee 

with deformed wings in the buffer control. 

Assay Validity Criteria 

Cumulative larval mortality was less than 15% across replicates, adult emergence was higher than 

70% on Day 20 in the negative control group, and larval mortality was higher than 50% by day 8 

in the positive control group, meeting acceptance criteria. 
Bayer CropScience LP CR282-20U1 387 

The results of the diet analysis demonstrated the Cry1Da_7 protein incorporated into the test 

treatment diets had the expected level of biological activity, was homogeneous throughout the diet, 

was stable under test and frozen storage conditions. 

Conclusion 

Following dietary exposure of larval honey bees to Cry1Da_7 protein under laboratory test 

conditions, the LC50 was >500 μg Cry1Da_7 protein/g diet and the NOAEC value was 500 μg 

Cry1Da_7 protein/g, the highest concentration tested. 

J.26. Evaluation of the Potential Effects of Cry1Da_7 Protein on the Northern Bobwhite 

Quail, Colinus virginianus

Materials and Methods 

Materials 

Test material: 

Test item: Cry1Da_7 lyophilized powder 

Description: Solid 

Purity corrected concentration: 0.72 mg/mg lyophilized powder 

Control: Bovine serum albumin (BSA) lyophilized powder 

Test organisms: 

Species: Northern bobwhite (Colinus virginianus) 

Age: 27 weeks 

Sex: 5 males/5 females per treatment group 

Weight: 188-232 g 

Diet/Food: Cargill game bird ration, ad libitum 

Acclimation Period: 8 weeks 

Environmental conditions: 

Temperature: 23.7°C (Range: 22.1-25.6°C) 

Humidity: 59% (Range: 37-72%) 

Photoperiod: 8 hours light / 16 hours dark 

Illumination: 369 lux 

 
Bayer CropScience LP CR282-20U1 388 

Study Design and Methods 

Experimental treatments: After an acclimation period of 8 weeks, ten adult northern bobwhite 

were indiscriminately assigned to the treatment and control groups. The test dose substance (TDS) 

and control dose substance (CDS) was dosed using capsules at a rate of 1000 mg product/kg of 

body weight. Nominal dosages used in this study were 0 mg/kg, 1000 mg of CDS/kg and 1000 mg 

TDS/kg. The birds were fasted for approximately 19 hours prior to dosing and individually 

weighed and dosed on the basis of mg of product/kg of body weight. At the experimental start, a 

capsule containing either the CDS or TDS was orally inserted into the crop of each bird. The 

negative control birds received an empty capsule of the same size used for the test birds. Two pens 

containing either 5 male or 5 female quail were used for each treatment and control group. 

Observations: After dosing, the birds were observed multiple times on Day 0, and then twice 

daily for 14 days following dosing for mortality, toxicity symptoms and abnormal behavior. In 

addition, birds were observed for any signs of regurgitation. Body weights were measured 

individually the day prior to dosing (Day -1) and on Day 3, 7, and 14 of the study. Feed 

consumption was determined by pen for approximately 24-hour intervals from Day 0 to Day 1, 

Day 1 to Day 2 and Day 2 to Day 3. Average daily feed consumption was determined by pen for 

the dosage group and the control group for Days 3–7, and 7–14. At study termination, three birds 

from each group were subjected to a gross necropsy. 

Statistical calculations: An LD50 value was not able to be calculated since no treatment group 

resulted in greater than 50% mortality. The body weight and body weight change by interval was 

compared between the TDS group and the two control groups by Student’s t-test using Microsoft®

Excel®. The sample unit was the individual bird within each experimental group and the 

comparison was made by sex and by group. 

Results

There were no mortalities in the control or treatment groups. When compared to the negative 

control group and the CDS group, there were no apparent treatment-related differences in mean 

body weight, mean body weight changes, or feed consumption for the males and females in TDS 

group at 1000 mg/kg dosage level. The mean body weight change was significantly higher 

(Student’s t-test, p<0.01) and lower (Student’s t-test, p<0.01) for the females in the TDS group 

compared to the females in the negative control group from Day -1 to Day 3 and Day 3 to Day 7 

of the test, respectively. There were no remarkable findings for the birds subjected to gross 

necropsy from any group, except one male bird of the three birds necropsied from the TDS group 

with feather loss and small lesions on the rump. 

 
Bayer CropScience LP CR282-20U1 389 

Assay Validity Criteria 

There was no evidence of regurgitation by any bird in any of the treatment groups. All birds in the 

negative control group, CDS group and TDS group were normal in appearance and behavior for 

the duration of the study, except one male in the TDS group that was noted as being aggressive to 

pen mates on Day 3 of the study. 

All validity criteria according to OCSPP 850.2100 were fulfilled, as no mortalities were observed 

in the control groups. 

Results from the dose confirmation demonstrated that the Cry1Da_7 protein was homogeneous 

and stable under conditions of administration. 

Conclusion 

The acute oral LD50 value for northern bobwhite exposed to Cry1Da_7 protein as a single oral 

dose was determined to be greater than 1000 mg Cry1Da_7 protein/kg, the highest dosage level 

tested. The no-mortality level and the no-observed-adverse-effect level (NOAEL) were 1000 mg 

Cry1Da_7 protein/kg bw. 

 
Bayer CropScience LP CR282-20U1 390 

J.27. Evaluation of the Potential Effects of Cry1Da_7 Protein on the Mouse, Mus musculus 

Materials and Methods 

Materials 

Test material and test dose solution: 

Test item: Cry1Da_7 protein 

Description: Light brown liquid 

Purity corrected concentration: 80.8 mg/mL 

Control: Bovine serum albumin (BSA) 

Vehicle control: 10 mM sodium carbonate/bicarbonate, 5 mM cysteine 

pH 10 with the permeate collected during concentration 

of the test substance 

Test organisms: 

Species: CD-1 mice (Mus musculus) 

Age: 8 weeks 

Sex: 10 males/10 females per treatment group 

Weight: 32.8-39.1 g males/ 24.8-33.1 g females 

Diet/Food: PMI Nutrition International Certified Rodent Chow No. 

5002, ad libitum 

Acclimation Period: 7 days 

Environmental conditions: 

Temperature: 21-22°C 

Humidity: 47-57% 

Photoperiod: 12 hours light / 12 hours dark 

Study Design and Methods 

Experimental treatments: After an acclimation period of 7 days in their designated housing, ten 

male and ten female mice were indiscriminately assigned to the treatment and control groups. 

Dosages used in this study were 0 mg of vehicle dose substance (VDS) per kg, 5000 mg of control 

(BSA) dose substance (CDS) per kg and 5000 mg of test (MON 95379) dose substance (TDS) per 

kg. Any assigned animal considered unsuitable for use in the study was replaced by an alternate 

animal obtained from the same shipment and maintained under the same environmental conditions. 

The animals were housed individually throughout the study in polycarbonate cages containing 

appropriate bedding equipped with an automatic watering valve. Animals were provided with 

enrichment items and edible treats for psychological/environmental enrichment, except when 

interrupted by study procedures/activities. On Day 0, the animals chosen for use on study were 

weighed and fasted approximately 3 to 4 hours prior to dose administration. The test, control, and 

vehicle dosing solutions were administered in 2 doses (approximately 3 to 4 hours apart), each 

with a dose volume of 33.3 mL/kg body weight by oral gavage on Day 0 to the appropriate group 
Bayer CropScience LP CR282-20U1 391 

of 10 males and 10 females. Individual doses were calculated based on the animal’s non-fasted 

(Day 0) body weight. The animals were fasted between doses and were returned to feed following 

the second dose. 

Observations: After dosing, the animals were observed for general health/mortality and 

moribundity twice daily, once in the morning and afternoon, throughout the study. The following 

parameters and end points were evaluated in this study: clinical signs, body weights, body weight 

changes, food consumption, and gross necropsy findings. Each animal was observed for clinical 

signs once prior to study start, once prior to dosing on Day 0, a minimum of 2 times postdose on 

Day 0 (1 within 1 hour of the first dose and 1 approximately 2 hours following the second dose), 

and daily thereafter (Days 1 to 14). A final detailed clinical observation was performed for each 

animal on the day of scheduled euthanasia (Day 14). Body weights were measured individually 

once prior to study start (Day -5), prior to fasting (Day 0), prior to dosing (Day 0, fasted), and on 

Days 7 and 14 of the study. Feed consumption was determined for each animal on Days 0, 7, and 

14 and was reported for the intervals from Study Days 0 to 7 and 7 to 14. At study termination, 

three birds from each group were subjected to a gross necropsy. 

Statistical calculations: Body weight, body weight change and food consumption were compared 

between the three treatment groups using an overall one-way ANOVA F-test or Kruskal-Wallis 

test (if parametric assumptions were not met) at the 5% significance level, but excluded semiquantitative data, and any group with less than 3 observations. Levene’s test was used to assess 

the homogeneity of group variances parametric assumption at the 5% significance level. Pairwise 

comparisons were conducted between TDS and the control groups using a two-sided Dunnett’s or 

Dunn’s test, respectively, if the overall test was significant. All significant pairwise comparisons 

were reported at the 0.1%, 1%, and 5% significance levels. 

Results

There were no test substance-related on survival or clinical signs during the study, except one 

female in the CDS group that was considered incidental and not CDS related as the cause of death 

was not determined. 

There were no significant differences noted in mean body weights, body weight gains or food 

consumption for males. There were significant decreases in mean bw and mean bw gain for the 

TDS females compared to VDS females for Study Days 7 and 14, and Days 0 to 14, respectively. 

Since the mean body weight and mean body weight gain values were within the historical control 

data range, these differences were not considered adverse. Food consumption for TDS females 

when compared to VDS females was significantly decreased for Study Days 0 to 7. However, this 

was a transient decrease as food consumption was not decreased for Study Days 7 to 14. In 

addition, the mean food consumption value for the females from Days 0 to 7 were within the 

historical control data range for both VDS and CDS females. Therefore, due to the variability in 
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food consumption values for mice, this difference was considered incidental and not treatment 

related. 

There were no test substance-related effects on gross necropsy findings. A single finding of an 

ovarian cyst was noted on a TDS female, however, this lesion has been noted in control groups in 

previous studies at an incidence level of 10-20%. Therefore, this finding was considered incidental 

and not treatment related. 

Assay Validity Criteria 

The analytical tests established stability and homogeneity of the test and control dosing solutions 

administered in the mouse oral acute gavage toxicity with Cry1Da_7 protein. 

Conclusion 

There were no test substance-related adverse effects of Cry1Da_7 protein when administered by 

oral gavage at a limit dose of 5000 mg/kg body weight in male and female CD-1 mice on mortality, 

clinical signs, body weights, body weight gains, food consumption or gross pathology findings. 

Therefore, the NOAEL for Cry1Da_7 protein was 5000 mg/kg body weight. 

 
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References for Appendix J 

Finney, D.J. 1971. Probit analysis: A statistical treatment of the sigmond response curve. The 

Syndics of the Cambridge University Press, London, England. 

GraphPad. 2019. GraphPad Prism version 8.2.0. GraphPad Software, Inc, LaJolla, California. 

Kudsk, P. and S.K. Mathiassen. 2004. Joint action of amino acid biosynthesis-inhibiting 

herbicides. Weed Research 44:313-322. 

Levine, S.L. and C.J. Borgert. 2018. Review and recommendations on criteria to evaluate the 

relevance of pesticide interaction data for ecological risk assessments. Chemosphere 209:124-136. 

Levine, S.L., J.M. Fridley and J.P. Uffman. 2019. Assessing the potential for interaction in 

insecticidal activity between MOM 87751 × MON 87701 produced by conventional breeding. 

Enviornmental Entomology 48:1241-1248. 

Levine, S.L., G.M. Mueller and J.P. Uffman. 2016. Assessing the potential for interaction between 

the insecticidal activity of two genetically engineered cotton events combined by conventional 

breeding: An example with COT102 × MON 15985. Regulatory Toxicology and Pharmacology

79:35-41. 

Tabashnik, B.E. 1992. Evaluation of synergism among Bacillus thuringiensis toxins. Applied and 

Environmental Microbiology 58:3343-3346. 

U.S. EPA. 1986. Guidelines for the health risk assessment of chemical mixtures. Federal Register 

51(185):34014-34025. U.S. Environmental Protection Agency, Washington, D.C. 

U.S. EPA. 2000. Supplementary guidance for conducting health risk assessment of chemical 

mixtures. U.S. Environmental Protection Agency, Washington, D.C. 

U.S. EPA. 2007. White paper on tier-based testing for the effects of proteinaceous insecticidal 

plant-incorporated protectants on non-target arthopods for regulatory risk assessments. U.S. 

Environmental Protection Agency, Washington, D.C. 

U.S. EPA. 2009a. Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel 

Meeting Held February 25-26, 2009 on the Data Required to Register Plant-Incorporated 

Protectants. SAP Minutes No.2009-04. U.S. Environmental Protection Agency, Washington, D.C. 

Wang, Y., J. Wang, X. Fu, J.R. Nageotte, J. Silverman, E.C. Bretsnyder, D. Chen, T.J. Rydel, G.J. 

Bean, K.S. Li, E. Kraft, A. Gowda, A. Nance, R.G. Moore, M.J. Pleau, J.S. Milligan, H.M. 

Anderson, P. Asiimwe, A. Evans, W.J. Moar, S. Martinelli, G.P. Head, J.A. Haas, J.A. Baum, F. 

Yang, D.L. Kerns and A. Jerga. 2019. Bacillus thuringiensis Cry1Da_7 and Cry1B.868 protein 
Bayer CropScience LP CR282-20U1 394 

interactions with novel receptors allow control of resistant fall armyworm, Spodoptera frugiperda

(J.E. Smith). Applied and Environmental Microbiology 85:e00579-00519. 
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Appendix K: Read Mappings and Associated Metrics for all Test, Control, and 

Reference Substances in the Molecular Characterization of MON 95379 

Figure K-1. Read Mapping of MON 95379 (F5) Versus PV-ZMIR522223 

Panel 1 shows the location of unpaired mapped reads, Panel 2 shows paired mapped reads, and Panel 3 

shows a representation of combined raw read depth for unpaired and paired reads across the reference. 

Vertical lines show genetic element boundaries. The region of flank junction sequences that align with 

the transformation plasmid are shown in red.
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Figure K-2. Read Mapping of MON 95379 (F4F1) Versus PV-ZMIR522223 

Panel 1 shows the location of unpaired mapped reads, Panel 2 shows paired mapped reads, and Panel 3 

shows a representation of combined raw read depth for unpaired and paired reads across the reference. 

Vertical lines show genetic element boundaries. The region of flank junction sequences that align with 

the transformation plasmid are shown in red.
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Figure K-3. Read Mapping of MON 95379 (F5F1) Versus PV-ZMIR522223 

Panel 1 shows the location of unpaired mapped reads, Panel 2 shows paired mapped reads, and Panel 3 

shows a representation of combined raw read depth for unpaired and paired reads across the reference. 

Vertical lines show genetic element boundaries. The region of flank junction sequences that align with the 

transformation plasmid are shown in red. 
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Figure K-4. Read Mapping of MON 95379 (F6F1) Versus PV-ZMIR522223 

Panel 1 shows the location of unpaired mapped reads, Panel 2 shows paired mapped reads, and Panel 3 

shows a representation of combined raw read depth for unpaired and paired reads across the reference. 

Vertical lines show genetic element boundaries. The region of flank junction sequences that align with the 

transformation plasmid are shown in red. 
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Figure K-5. Read Mapping of Conventional Maize LH244 Versus PV-ZMIR522223 

Panel 1 shows the location of unpaired mapped reads, Panel 2 shows paired mapped reads, and Panel 3 

shows a representation of combined raw read depth for unpaired and paired reads across the reference. 

Vertical lines show genetic element boundaries. 
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Figure K-6. Read Mapping of Conventional Maize LH244 × HCL617 Versus 

PV-ZMIR522223 

Panel 1 shows the location of unpaired mapped reads, Panel 2 shows paired mapped reads, and Panel 3 

shows a representation of combined raw read depth for unpaired and paired reads across the reference. 

Vertical lines show genetic element boundaries. 

 
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Figure K-7. Read Mapping of Conventional Maize LH244 Versus PV-ZMOO513642 

Panel 1 shows the location of unpaired mapped reads, there are no paired mapped reads, and Panel 2 shows 

a representation of combined raw read depth for unpaired and paired reads across the reference. Vertical 

lines show genetic element boundaries. 
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Figure K-8. Read Mapping of MON 95379 (F4) Versus pdc3

Panel 1 shows the location of reads that are unpaired when mapped, Panel 2 shows reads that are paired 

when mapped and Panel 3 shows a representation of combined raw read depth for unpaired and paired reads 

across the reference. Vertical lines represent 1000 nucleotide intervals. 
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Figure K-9. Read Mapping of MON 95379 (F5) Versus pdc3

Panel 1 shows the location of reads that are unpaired when mapped, Panel 2 shows reads that are paired 

when mapped and Panel 3 shows a representation of combined raw read depth for unpaired and paired reads 

across the reference. Vertical lines represent 1000 nucleotide intervals. 
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Figure K-10. Read Mapping of MON 95379 (F4F1) Versus pdc3

Panel 1 shows the location of reads that are unpaired when mapped, Panel 2 shows reads that are paired 

when mapped and Panel 3 shows a representation of combined raw read depth for unpaired and paired reads 

across the reference. Vertical lines represent 1000 nucleotide intervals. 
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Appendix Figure K11. Read Mapping of MON 95379 (F5F1) Versus pdc3

Panel 1 shows the location of reads that are unpaired when mapped, Panel 2 shows reads that are paired 

when mapped and Panel 3 shows a representation of combined raw read depth for unpaired and paired reads 

across the reference. Vertical lines represent 1000 nucleotide intervals. 
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Figure K-12. Read Mapping of MON 95379 (F6F1) Versus pdc3

Panel 1 shows the location of reads that are unpaired when mapped, Panel 2 shows reads that are paired 

when mapped and Panel 3 shows a representation of combined raw read depth for unpaired and paired reads 

across the reference. Vertical lines represent 1000 nucleotide intervals. 
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Figure K-13. Read Mapping of LH244 Conventional Maize Versus pdc3 

Panel 1 shows the location of reads that are unpaired when mapped, Panel 2 shows reads that are paired 

when mapped and Panel 3 shows a representation of combined raw read depth for unpaired and paired reads 

across the reference. Vertical lines represent 1000 nucleotide intervals. 
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Figure K-14. Read Mapping of LH244 × HCL617 Conventional Maize Versus pdc3 

Panel 1 shows the location of reads that are unpaired when mapped, Panel 2 shows reads that are paired 

when mapped and Panel 3 shows a representation of combined raw read depth for unpaired and paired reads 

across the reference. Vertical lines represent 1000 nucleotide intervals. 

 
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Figure K-15. Read Mapping of the PV-ZMIR522223 NGS Library (1 genome equivalent) 

PV-ZMIR522223 sequence reads mapping to the plasmid reference sequence were randomly 

sampled to yield a 1 genome equivalent number of reads. Panel 1 shows the location of reads 

that are unpaired when mapped. Panel 2 shows reads that are paired when mapped. Panel 3 

shows a representation of combined raw read depth for all reads. Vertical lines show genetic 

element boundaries.
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Figure K-16. Read Mapping of the PV-ZMIR522223 NGS Library (1/10th genome 

equivalent) 

PV-ZMIR522223 sequence reads mapping to the plasmid reference sequence were randomly 

sampled to yield a 1/10th genome equivalent number of reads. Panel 1 shows the location of reads 

that are unpaired when mapped. Panel 2 shows reads that are paired when mapped. Panel 3 shows 

a representation of combined raw read depth for all reads. Vertical lines show genetic element 

boundaries. 
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Figure K-17. Read Mapping of the PV-ZMOO513642 NGS Library (1 genome equivalent) 

PV-ZMOO513642 sequence reads mapping to the plasmid reference sequence were randomly 

sampled to yield a 1 genome equivalent number of reads. Panel 1 shows the location of reads that 

are unpaired when mapped. Panel 2 shows reads that are paired when mapped. Panel 3 shows a 

representation of combined raw read depth for all reads. Vertical lines show genetic element 

boundaries. 
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Figure K-18. Read Mapping of the PV-ZMOO513642 NGS Library (1/10th genome 

equivalent) 

PV-ZMOO513642 sequence reads mapping to the plasmid reference sequence were randomly 

sampled to yield a 1/10th genome equivalent number of reads. Panel 1 shows the location of reads 

that are unpaired when mapped. Panel 2 shows reads that are paired when mapped. Panel 3 shows 

a representation of combined raw read depth for all reads. Vertical lines show genetic element 

boundaries. 
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Junction A Alignment (All Generations) 

Directed_Seq_5p GGCCGGCGCCTGATCGACTTCTCTTCCGCCGCCTTCGCTAGCTATAGCTTGGTAGTAGTC 

F4 -----------GATCGACTTCTCTTCCGCCGCCTTCGCTAGCTATAGCTTGGTAGTAGTC 

F5 -----------GATCGACTTCTCTTCCGCCGCCTTCGCTAGCTATAGCTTGGTAGTAGTC 

F4F1 --------------CGACTTCTCTTCCGCCGCCTTCGCTAGCTATAGCTTGGTAGTAGTC 

F5F1 ----------------------------------TCGCTAGCTATAGCTTGGTAGTAGTC 

F6F1 ---------------------------------------AGCTATAGCTTGGTAGTAGTC 

Directed_Seq_5p ACCAACAACGACCTAGGGAGCTAGCGTACGTTCGAGGTACCTGTCCTGCAAGCAAATGTG 

F4 ACCAACAACGACCTAGGGAGCTAGCGTACGTTCGAGGTACCTGTCCTGCAAGCAAATGTG 

F5 ACCAACAACGACCTAGGGAGCTAGCGTACGTTCGAGGTACCTGTCCTGCAAGCAAATGTG 

F4F1 ACCAACAACGACCTAGGGAGCTAGCGTACGTTCGAGGTACCTGTCCTGCAAGCAAATGTG 

F5F1 ACCAACAACGACCTAGGGAGCTAGCGTACGTTCGAGGTACCTGTCCTGCAAGCAAATGTG 

F6F1 ACCAACAACGACCTAGGGAGCTAGCGTACGTTCGAGGTACCTGTCCTGCAAGCAAATGTG 

Directed_Seq_5p TGGTAGTGGT^CCAATTTTTTTTCAATTCAAAAATGTAGATGTCCGCAGCGTTATTATAA 

F4 TGGTAGTGGT^CCAATTTTTTTTCAATTCAAAAATGTAGATGTCCGCAGCGTTATTATAA 

F5 TGGTAGTGGT^CCAATTTTTTTTCAATTCAAAAATGTAGATGTCCGCAGCGTTATTATAA 

F4F1 TGGTAGTGGT^CCAATTTTTTTTCAATTCAAAAATGTAGATGTCCGCAGCGTTATTATAA 

F5F1 TGGTAGTGGT^CCAATTTTTTTTCAATTCAAAAATGTAGATGTCCGCAGCGTTATTATAA 

F6F1 TGGTAGTGGT^CCAATTTTTTTTCAATTCAAAAATGTAGATGTCCGCAGCGTTATTATAA 

Directed_Seq_5p AATGAAAGTACATTTTGATAAAACGACAAATTACGATCCGTCGTATTTATAGGCGAAAGC 

F4 AATGAAAGTACATTTTGATAAAACGACAAATTACGATCCGTCGTATTTATAGGCGAAAGC 

F5 AATGAAAGTACATTTTGATAAAACGACAAATTACGATCCGTCGTATTTATAGGCGAAAGC 

F4F1 AATGAAAGTACATTTTGATAAAACGACAAATTACGATCCGTCGTATTTATAGGCGAAAGC 

F5F1 AATGAAAGTACATTTTGATAAAACGACAAATTACGATCCGTCGTATTTATAGGCGAAAGC 

F6F1 AATGAAAGTACATTTTGATAAAACGACAAATTACGATCCGTCGTATTTATAGGCGAAAGC 

Directed_Seq_5p AATAAACAAATTATTCTAATTCGGAAATCTTTATTTCGACGTGTCTACATTCAC 

F4 AATAAACAAATTATTCTAATTCGGAAATCTTTATTTCGACGT------------ 

F5 AATAAACAAATTATTCTAATTCGGAAATCTTTATTTC----------------- 

F4F1 AATAAACAAATTATTCTAATTCGGAAATCTTTA--------------------- 

F5F1 AATAAACAAATTATTCTAATTCGGAAATCTTTATTTCGAC-------------- 

F6F1 AATAAACAAATTATTCTAATTCGGAAATCTTTATTTC----------------- 

Junction B Alignment (All Generations)

Directed_Seq_3p GCTTCAGTTAATTAAAAGTAAGGGTCCCAAGTAAGGCCGGCCAAGTAACGGTCCGAAGTA 

F4 ----------ATTAAAAGTAAGGGTCCCAAGTAAGGCCGGCCAAGTAACGGTCCGAAGTA 

F5 -----------TTAAAAGTAAGGGTCCCAAGTAAGGCCGGCCAAGTAACGGTCCGAAGTA 

F4F1 -----------TTAAAAGTAAGGGTCCCAAGTAAGGCCGGCCAAGTAACGGTCCGAAGTA 

F5F1 ---------------AAGTAAGGGTCCCAAGTAAGGCCGGCCAAGTAACGGTCCGAAGTA 

F6F1 ------------TAAAAGTAAGGGTCCCAAGTAAGGCCGGCCAAGTAACGGTCCGAAGTA 
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Directed_Seq_3p AGGCGCGGGTACCGTCGGTCCGGGCCTAGTAGGCCAAGCAGGACGTGGCGCGCCAAGAAG 

F4 AGGCGCGGGTACCGTCGGTCCGGGCCTAGTAGGCCAAGCAGGACGTGGCGCGCCAAGAAG 

F5 AGGCGCGGGTACCGTCGGTCCGGGCCTAGTAGGCCAAGCAGGACGTGGCGCGCCAAGAAG 

F4F1 AGGCGCGGGTACCGTCGGTCCGGGCCTAGTAGGCCAAGCAGGACGTGGCGCGCCAAGAAG 

F5F1 AGGCGCGGGTACCGTCGGTCCGGGCCTAGTAGGCCAAGCAGGACGTGGCGCGCCAAGAAG 

F6F1 AGGCGCGGGTACCGTCGGTCCGGGCCTAGTAGGCCAAGCAGGACGTGGCGCGCCAAGAAG 

Directed_Seq_3p AACGATTGGCAAACAGCTATTATGGGTATTATGGGTAGGC^ACATGGGAATATAGTGGGA 

F4 AACGATTGGCAAACAGCTATTATGGGTATTATGGGTAGGC^ACATGGGAATATAGTGGGA 

F5 AACGATTGGCAAACAGCTATTATGGGTATTATGGGTAGGC^ACATGGGAATATAGTGGGA 

F4F1 AACGATTGGCAAACAGCTATTATGGGTATTATGGGTAGGC^ACATGGGAATATAGTGGGA 

F5F1 AACGATTGGCAAACAGCTATTATGGGTATTATGGGTAGGC^ACATGGGAATATAGTGGGA 

F6F1 AACGATTGGCAAACAGCTATTATGGGTATTATGGGTAGGC^ACATGGGAATATAGTGGGA 

Directed_Seq_3p GGCAGAGGCGTGCCTGTGCCTGCTCTTAATTTGAGCTCCTCCCCTGGCCCTGATAGGGCA 

F4 GGCAGAGGCGTGCCTGTGCCTGCTCTTAATTTGAGCTCCTCCCCTGGCCCTGATAGGGCA 

F5 GGCAGAGGCGTGCCTGTGCCTGCTCTTAATTTGAGCTCCTCCCCTGGCCCTGATAGGGCA 

F4F1 GGCAGAGGCGTGCCTGTGCCTGCTCTTAATTTGAGCTCCTCCCCTGGCCCTGATAGGGCA 

F5F1 GGCAGAGGCGTGCCTGTGCCTGCTCTTAATTTGAGCTCCTCCCCTGGCCCTGATAGGGCA 

F6F1 GGCAGAGGCGTGCCTGTGCCTGCTCTTAATTTGAGCTCCTCCCCTGGCCCTGATAGGGCA 

Directed_Seq_3p TGTGCATGCGCACAATAATCATTGGAGCTGCCTGAATGATTTGCATGCGCTC 

F4 TGTGCATGCGCACAATAATCATTGGAGCTGCCTGAATGATTT---------- 

F5 TGTGCATGCGCACAATAATCATTGGAGCTGCCTG------------------ 

F4F1 TGTGCATGCGCACAATAATCATTGGAGCTG---------------------- 

F5F1 TGTGCATGCGCACAATAATCATTGGAGCT----------------------- 

F6F1 TGTGCATGCGCACAATAATCATTGGAGCTGCCTGAATGATTT---------- 

Figure K-19. Junction Sequences Detected: All Breeding Generations 

Full consensus sequence for Junction A and Junction B showing exact alignment to the 

independently determined in planta locus specific sequence (labeled “Directed_Seq” in the 

figure), the individual consensus sequences for each of the five generations are labeled according 

to their generation (F4, F5, F4F1, F5F1, and F6F1). Double underlined text indicates plasmidderived T-DNA sequence, single underlined text indicates flank sequence, and the caret character 

“^” indicates the junction point between the insert and the flank. Dash characters “-” indicate 

positions past the end of the consensus sequence for a particular generation. 

 
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Table K-1. Summary of total Generated NGS Data for the Control and Test Substances 

Sample

Total Nucleotide 

Number (Gb)1

Hypothetical Genome 

Coverage (×-fold)2

Effective Median Depth 

of Coverage (×-fold)3

LH244 202.18 88× 100× 

LH244 × HCL617 246.48 107× 90× 

MON 95379 (F4) 191.54 83× 96× 

MON 95379 (F5) 314.87 137× 152× 

MON 95379 (F4F1) 241.51 105× 105× 

MON 95379 (F5F1) 193.37 84× 82× 

MON 95379 (F6F1) 247.22 107× 116× 

1

 For each sample, the raw data produced are presented in terms of total nucleotide number in 

gigabases (Gb). Notably, the read lengths generated in this experimate range from 35 to 151bp. 

2

 The genome coverage of the sequencing events was estimated based on the Zea mays genome 

sized (approximately 2.3 Gb, Schnable et al., 2009). 

3

 Effective depth of coverage is determined by mapping and alignment of all raw data to a single 

copy locus within the Zea mays genome (pdc3, GenBank accession AF370006.2). The median 

effective depth of coverage as aligned against this locus is shown for all samples. The positional 

raw read depths for all samples can be observed in panel 3 of Appendix Figures K8 through K14. 

 
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Table K-2. Summary Coverage Data for the T-DNA region of Control and Test Substances 

A summary of the coverage data across the T-DNA insert regions1 in panel 3 of the mapping 

figures (Appendix K, Figures K1-K6) for the control libraries and test substance generations. 

Sample Minimum Maximum Average (x-fold) Median (x-fold) 

LH244 0 94 2.51× 0× 

LH244 × HCL617 0 113 6.7× 0× 

MON 95379 (F4) 26 200 94.44× 94× 

MON 95379 (F5) 51 351 156.32× 156× 

MON 95379 (F4F1) 10 225 68.41× 67× 

MON 95379 (F5F1) 13 183 56.59× 53× 

MON 95379 (F6F1) 14 218 71.99× 71× 

1

Insert region corresponds to positions 258-514 and 4895-17996 relative to the reference used to 

generate Figure IV-5, Figure IV-6 and Appendix K Figures K1 through K6. 
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References for Appendix K 

Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, 

Graves TA, Minx P. 2009. The B73 maize genome: complexity, diversity, and dynamics. 

Science. 326:1112-5. 
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Appendix L: Supplemental Information 

This appendix expands on several areas of the petition, and are provided here for reference. 

L.1. Additional Information Regarding Total Quality and Quality of Sequence Data 

Generated for the Characterization of the Genetic Modification (Section IV) 

Notably, in sequencing runs it is possible for a proportion of reads to be returned with a reduced 

length relative to the targeted sequence length. Although much of the data generated herein is 151 

base pairs in length, a portion of the reads is shorter. As such we have reported as the total number 

of nucleotides in giga-bases (Gb) generated alongside hypothetical coverage base on genome size 

as it is a directly quantifiable and therefore translative metric regarding total sequencing generated. 

This data has been provided in Appendix K (Table K-1). Appendix K further includes data 

summarizing total sequencing generated and coverage metrics for all generations (F4F1, F5, F5F1, 

and F6F1) and controls (Tables K-1 and K-2), and includes coverage across the T-DNA regions. 

The molecular characteristics, including a) the number of inserts, b) the presence or absence of 

plasmid backbone, and c) the DNA sequence of inserted transformation construct, can be 

determined using bioinformatics by mapping all reads to the plasmid sequence and selecting reads 

mapped to the junctions of the insert DNA. Mapping reads back to the entire reference genome is 

not necessary to determine these desired endpoints of molecular characterization. Furthermore, 

estimated genome coverage can be calculated using total sequence data generated and supported 

by the observed coverage of inserted DNA and single copy controls. Data supporting these 

conclusions are included in the Appendix K, Tables K-1 and K-2. 

All reads were mapped to the reference sequences without any exclusion or filtering based on per 

base quality metrics. However, only mapped reads that meet Bowtie2 mapping criteria (reported 

in Section B.9.) were displayed in the read mapping figures and used to calculate the effective 

median coverage against the single copy locus as well as the T-DNA insert coverage. Additionally, 

reads with partial matches to the plasmid sequence of at least 30 bases and 96.6% or greater 

identity were classified as potential junction spanning reads and used to determine the immediate 

T-DNA to flanking genomic DNA sequence at the integration point (reported in Section B.9.1). 

L.2. Additional Information Regarding Generations Analyzed and Junction Site 

Consistency in the Characterization of the DNA Inserts in MON 95379 (Section IV.B) 

Five distinct generations were characterized, with those designated with a secondary XXF1 

designation representing those crossed with HCL617 making them hybrid generations. A breeding 

history demonstrating these crosses is provided in Figure IV-4. The total sequencing generated, 

hypothetical genome coverage, and mapped coverage to the T-DNA for each of the generations 

are provided in Appendix K (Tables K-1 and K-2). 

A consensus of the junction sequences was aligned across all generations alongside an 

independently sequenced PCR product derived from the same site and is provided in Appendix K 
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(Figure K-19). The consensus sequences for the two junction sites of the single insert were 

generated by compiling the corresponding group of junction reads which share the same junction 

point and common flanking sequence for each of the generations. These sequences are 

representative of the reads that support the corresponding junction site. The alignment of these 

sequences demonstrated that they were identical across the entirety of their captured sequence for 

all samples, supporting the generational stability of the single insert event. 

L.3. Additional Information Regarding Consistency of Read Mapping Methodology used in 

the Characterization of the DNA Inserts in MON 95379 (Section IV.B) 

The sequencing herein utilized NGS platforms (Illumina) to conduct whole genome sequencing 

on multiple generations and controls, as well as transformation plasmids which are described as a 

“reference substances” to establish the molecular characterization endpoints. Identical library 

preparation methods, and mapping parameters were used on all samples, including all 

transformation vectors and controls. Identical software and parameters were used in all mappings 

generated regardless of the substance/generation characterized, and the mapping parameters used 

are provided in Section B.9. 

L.4. Additional Information Regarding the Endogenous Maize Zm.Ubq Element in the 

Characterization of the DNA Inserts in MON 9539 (Section IV.B) 

As discussed in Section IV.B.3 and observed in Figure IV-5, it can be observed that a number of 

reads align to the Zm.Ubq element in the read mapping of the conventional maize LH244 to PVZMIR522223. It is important to note that the similarity of endogenous maize Zm.Ubq to that 

present in the MON 95379 T-DNA is unnecessary to establish the endpoints of molecular 

characterization. Insert copy number is determined by junction site identification and further 

supported by the relative depth of sequencing, and the use of controls to account for the presence 

or absence of any other shared feature between the transformed and conventional lines as described 

in Section IV.B.3. Nevertheless, an alignment of common elements may in some cases provide 

some additional context as to what is being observed, and is provided in 

Figure L-1. 

Figure L-1. Alignment A. The alignment Between an Endogenous Maize Zm.Ubq Sequence 

and the MON 95379 T-DNA Zm.Ubq Element. 

# fasta36 P-Zm-Ubq.fasta /data/databases/2020/ZMA_2020/GCF_000005005.2_B73_RefGen_v4_genomic.fna 

FASTA searches a protein or DNA sequence data bank

version 36.3.5d Aug, 2012(preload8)

Please cite:

W.R. Pearson & D.J. Lipman PNAS (1988) 85:2444-2448

 Query: P-Zm-Ubq.fasta

 1>>>MON95379_P-Zm-Ubq - 2008 nt

Library: /data/databases/2020/ZMA_2020/GCF_000005005.2_B73_RefGen_v4_genomic.fna

 2135083061 residues in 267 sequences

 Statistics: Expectation_n fit: rho(ln(x))= 18.8188+/- 0.222; mu= -88.5427+/-26.544

mean_var=459.1494+/-454.864, 0's: 0 Z-trim(101.4): 2119 B-trim: 0 in 0/9
Bayer CropScience LP CR282-20U1 420 

Lambda= 0.059855

statistics sampled from 3448 (5210) to 3448 sequences

Algorithm: FASTA (3.7 Nov 2010) [optimized]

Parameters: +5/-4 matrix (5:-4), open/ext: -12/-4

ktup: 6, E-join: 0.25 (0.412), E-opt: 0.05 (0.248), width: 16

Scan time: 202.060

 The best scores are: opt bits E(14397)

NC_024463.2 Zea mays cultivar B73 chromosome 5 (223902240) [r] 4439 402.6 2.7e-109

 >>NC_024463.2 Zea mays cultivar B73 chromosome 5, B73 Re (223902240 nt)

rev-comp initn: 5036 init1: 1852 opt: 4439 Z-score: 2069.1 bits: 402.6 E(14397): 2.7e-109

banded Smith-Waterman score: 4989; 86.1% identity (86.1% similar) in 1422 nt overlap (1385-1:84633633-

84635038)

 1410 1400 1390 1380 1370 1360 

MON95- CTACTCCGATCTAGAACGACCCTTTATCCTTTAATAAATCCACCAGGTAGTTTGAAACAG

 ::::::::::::::::::::::::::::::

NC_024 TATGTATGGCACACACATACAGATCCAAAATTAATAAATCCACCAGGTAGTTTGAAACAG

 84633610 84633620 84633630 84633640 84633650 84633660 

 1350 1340 1330 1320 1310 1300 

MON95- TATTCTACTCCGATCTAGAACGACCGCCCAACCAGACCATATCATCACAACCAAGCGAGA

 :::::::::::::::::::::::::::::::::::::: ::::::::::::::: : :

NC_024 AATTCTACTCCGATCTAGAACGACCGCCCAACCAGACCACATCATCACAACCAAGACA-A

 84633670 84633680 84633690 84633700 84633710 84633720 

 1290 1280 1270 1260 1250 

MON95- AAAAAAGCATCTCTGTATATGCATCAGTAAAAC--CCGCATCAACATGTAT--ACCTATC

 ::::::::: : : ::: : : : :::: ::: : ::: ::: : :: 

NC_024 AAAAAAGCA--TGAAAAGATG-ACCCGACAAACAAGTGCA-CGGCATATATTGAAATAAA

 84633730 84633740 84633750 84633760 84633770 

 1240 1230 1220 1210 1200 

MON95- CTAGATCGATATTTCCATCCAT---CATCTTAA-ACTCGTAACTATGAA---GAT---GT

 : : : : : : :: : :: : :: : :: ::::: ::: ::

NC_024 GGAAAAGGGCAAACCAAACCCTATGCAACGAAACAAAAAAAATCATGAAATCGATCCCGT

 84633780 84633790 84633800 84633810 84633820 84633830 

 1190 1180 1170 1160 1150 1140 

MON95- ATGGCACACACATACAGATA---CAAAATT-----AATAAATCCACCAGGTAGCTTG-AA

 :: :: :: :: : :: ::: :: : : ::: : :: : : ::

NC_024 CTGCGGAACGGCTAGAGCCATCCCAGGATTCCCCAAAGAGAAACACTGGCAAGTTAGCAA

 84633840 84633850 84633860 84633870 84633880 84633890 

 1130 1120 1110 1100 1090 

MON95- ACAGTATCCTACTCCGATCTAGAACGACCGCCCAACCA-GACCAC-ATCATCACAAACAT

 ::: : : :: :: :: : : :: : :: : : : :: ::: : :

NC_024 TCAGAA--CGTGTCTGACGTACAGGTCGCATCCGTGTACGAACGCTAGCAGCACGGATCT

 84633900 84633910 84633920 84633930 84633940 84633950 

 1080 1070 1060 1050 1040 1030 

MON95- GAACATGAACATGTTTGCTCTAACACAAACATGAACAGAAGTAGAACTACCGGGCCCTAA

 :::: :::: : : ::::::::::::::::::::::::::::::::::::::::::

NC_024 -AACACAAACACG---GATCTAACACAAACATGAACAGAAGTAGAACTACCGGGCCCTAA

 84633960 84633970 84633980 84633990 84634000 84634010 

 1020 1010 1000 990 980 970 

MON95- CCATGGACCGGATCGCCGATCTAGAGAAGGTAGAGAGAGGGGGGGGGGGAGGATGAGCGG

 :::::::::::: :::::::::::::::::::::::: ::::::::::::::: ::::::
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NC_024 CCATGGACCGGAACGCCGATCTAGAGAAGGTAGAGAG-GGGGGGGGGGGAGGACGAGCGG

 84634020 84634030 84634040 84634050 84634060 84634070

 960 950 940 930 920 910 

MON95- CGTACCTTGAAGCGGAGGTGCCGACGGCTGGATTTGGGGGAGATCTGGTTGCGTGTGTGT

 ::::::::::::::::::::::::::: ::::::::::::::::::::::: ::::::::

NC_024 CGTACCTTGAAGCGGAGGTGCCGACGGGTGGATTTGGGGGAGATCTGGTTGTGTGTGTGT

 84634080 84634090 84634100 84634110 84634120 84634130

 900 890 880 870 860 850 

MON95- GCGCTCCGAACGAACACGAGGTTGGGGAAAGAGGGTGTGGAGGGGGTGTCTATTTATTAC

 ::::::::::: ::::::::::::::::::::::::::::::::::::::::::::::::

NC_024 GCGCTCCGAAC-AACACGAGGTTGGGGAAAGAGGGTGTGGAGGGGGTGTCTATTTATTAC

 84634140 84634150 84634160 84634170 84634180 

 840 830 820 810 800 790 

MON95- GGCGGGCGAGGAAGGGAAAGCGAAGGAGCGGTGGGAAAGGAATCCCCCGTAGCTGCCGGT

 ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

NC_024 GGCGGGCGAGGAAGGGAAAGCGAAGGAGCGGTGGGAAAGGAATCCCCCGTAGCTGCCGGT

84634190 84634200 84634210 84634220 84634230 84634240 

 780 770 760 750 740 730 

MON95- GCCGTGAGAGGAGGAAGAGGCCGCCTGCCGTGCCGCCTCACGTCTGCCGCTCCGCCACGC

 ::::::::::::::: ::::::::::::::::::: ::::::::::::::::::::::::

NC_024 GCCGTGAGAGGAGGAGGAGGCCGCCTGCCGTGCCGGCTCACGTCTGCCGCTCCGCCACGC

84634250 84634260 84634270 84634280 84634290 84634300 

 720 710 700 690 680 670 

MON95- AATTTCTGGATGCCGACAGCGGAGCAAGTCCAACGGTGGAGCGGAACTCTCGAGAGGGGT

 ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

NC_024 AATTTCTGGATGCCGACAGCGGAGCAAGTCCAACGGTGGAGCGGAACTCTCGAGAGGGGT

84634310 84634320 84634330 84634340 84634350 84634360 

 660 650 640 630 620 610 

MON95- CCAGAGGCAGCTACAGAGATGCCGTGCCGTCTGCTTCGCTTGGCCCGACGCGACGCTGCT

 ::::::::::: ::::::::::::::::::::::::::::::::::::::::::::::::

NC_024 CCAGAGGCAGCGACAGAGATGCCGTGCCGTCTGCTTCGCTTGGCCCGACGCGACGCTGCT

84634370 84634380 84634390 84634400 84634410 84634420 

 600 590 580 570 560 550 

MON95- GGTTCGCTGGTTGGTGTCCGTTAGACTCGTCGACGGCGTTGAACAGCCTGTCATTATCTA

 :::::::::::::::::::::::::::::::::::::::: ::::: ::: :::::::::

NC_024 GGTTCGCTGGTTGGTGTCCGTTAGACTCGTCGACGGCGTTTAACAGGCTGGCATTATCTA

84634430 84634440 84634450 84634460 84634470 84634480 

 540 530 520 510 500 490 

MON95- CTCGAAACAAGAAAAATGTTTGCTTAGTTTTTTT-ATTTCTTAAAGGGTATTTGTTTTAT

 ::::::::::::::::::::: :::::::::::: :::::::::::::::::::::: ::

NC_024 CTCGAAACAAGAAAAATGTTTCCTTAGTTTTTTTAATTTCTTAAAGGGTATTTGTTTAAT

84634490 84634500 84634510 84634520 84634530 84634540 

 480 470 460 450 440 430 

MON95- TTGTAGTCAATTTATTTTATTTCATTTTATATCTAAATTATTAAAT-AAAAAACTAAAAT

 :: :::::: ::::::::::: ::::::::::::::::::::::: :::::::::::::

NC_024 TTTTAGTCACTTTATTTTATTCTATTTTATATCTAAATTATTAAATAAAAAAACTAAAAT

84634550 84634560 84634570 84634580 84634590 84634600 

 420 410 400 390 380 370 

MON95- AGAGTTTTAGTTTTAAAAAATTTAGAGACTAAAA-AGAATAAAATGGATGTACT--AAAA

 :::::::::::::: ::::::::: :::::: :::::::::: :::::::: ::::
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NC_024 AGAGTTTTAGTTTT-CTTAATTTAGAGGCTAAAATAGAATAAAATAGATGTACTAAAAAA

84634610 84634620 84634630 84634640 84634650 84634660 

 360 350 340 330 320 310 

MON95- ATTAGTCTATAGAAACCATCAACCCTAAATCCTAAATGGATGTACTAATAAAATGGATGA

 ::::::::::: ::::::: ::::::::: ::::::::::::::::::::::::::::::

NC_024 ATTAGTCTATAAAAACCATTAACCCTAAACCCTAAATGGATGTACTAATAAAATGGATGA

84634670 84634680 84634690 84634700 84634710 84634720 

 300 290 280 270 260 250 

MON95- AGTATTATATAGGTCAAGCTATTTGCAAAAAAAACAGAGAGATCACATGCACACTAAAAA

 :::::::::::::: ::::::::::::::::::: :: :::: :::::::::::::::::

NC_024 AGTATTATATAGGTGAAGCTATTTGCAAAAAAAA-AG-GAGAACACATGCACACTAAAAA

84634730 84634740 84634750 84634760 84634770 84634780 

 240 230 220 210 200 

MON95- GATAAAACTGTAGA------TTGTCAAAATATTCAATTATCCTTTAGACCATGTCTAGCA

 :::::::::::::: ::::::::::: :::::: :::::::::::::::::: : 

NC_024 GATAAAACTGTAGAGTCCTGTTGTCAAAATACTCAATTGTCCTTTAGACCATGTCTAACT

 84634790 84634800 84634810 84634820 84634830 84634840 

 190 180 170 160 150 140 

MON95- GTTTATTTATATGATCCTCTAAAACACTAATATTATTTTAGTATTATAGACTATATTATT

 ::: ::::::::::: :::::::::::: :::::::: ::::: :::::: :::::::::

NC_024 GTTCATTTATATGATTCTCTAAAACACTGATATTATTGTAGTACTATAGATTATATTATT

 84634850 84634860 84634870 84634880 84634890 84634900 

 130 120 110 100 90 80 

MON95- TGTAGAGTGAAGTTTAAATATATGTATAGAGATAGATAAACTACACTTCAAATAAGTGTG

 ::::::: ::::::::::::::::::: ::::::::::::: ::::::::: :::::::

NC_024 CGTAGAGTAAAGTTTAAATATATGTATAAAGATAGATAAACTGCACTTCAAACAAGTGTG

 84634910 84634920 84634930 84634940 84634950 84634960 

 70 60 50 40 30 20 

MON95- AC-AAAAAAATATGTGGTAATTTTTTATACTTTAGACATGCAATGCTCTTTATCTCTAGA

 :: :::::::::::::::::::::::::: ::::::::::::::::: :::::::::::

NC_024 ACAAAAAAAATATGTGGTAATTTTTTATAACTTAGACATGCAATGCTCATTATCTCTAGA

 84634970 84634980 84634990 84635000 84635010 84635020 

 10 

MON95- GAGGGGCACGAC 

 :::::::::::: 

NC_024 GAGGGGCACGACCGGGTCACGCTGCACTGCAGTCTACCTTGCCATGCCGCCGGATTCAGC

 84635030 84635040 84635050 84635060 84635070 84635080 

>-- 

rev-comp initn: 1922 init1: 1113 opt: 2507 Z-score: 1167.5 bits: 235.8 E(14397): 4.5e-59

banded Smith-Waterman score: 2507; 94.9% identity (94.9% similar) in 568 nt overlap (2007-

1451:84633065-84633628)

 2000 1990 1980 

MON95- GACCTGCAGAAGTATCACCAAACAACAGGGT

 : ::::::::::: ::::::::::::::::

NC_024 TTGCCAGTCAGGGTTTTCACAAAGATCTGCATCTGCAGAAGTAACACCAAACAACAGGGT

 84633040 84633050 84633060 84633070 84633080 84633090 

 1970 1960 1950 1940 1930 1920 

MON95- GAGCATCGGACAAAAGAAACAGTACCAAGCAAATAAATAGCGTATGAAGGCAGGGCT-AA

 ::::::: ::::::::::::::::::::::::::::::::::::::::::::::::: ::

NC_024 GAGCATC-GACAAAAGAAACAGTACCAAGCAAATAAATAGCGTATGAAGGCAGGGCTAAA
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 84633100 84633110 84633120 84633130 84633140 84633150 

 1910 1900 1890 1880 1870 1860 

MON95- AAAATCCACATATAGCTGCTGCATATGCCATCATCCAAGTATATCAAGATCAAAATAATT

 ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

NC_024 AAAATCCACATATAGCTGCTGCATATGCCATCATCCAAGTATATCAAGATCAAAATAATT

 84633160 84633170 84633180 84633190 84633200 84633210 

 1850 1840 1830 1820 1810 1800 

MON95- ATAAAACATACTTGTTTATTATAATAGATAGGTACTCAAGGTTAGAGCATATGAATAGAT

 ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

NC_024 ATAAAACATACTTGTTTATTATAATAGATAGGTACTCAAGGTTAGAGCATATGAATAGAT

 84633220 84633230 84633240 84633250 84633260 84633270 

 1790 1780 1770 1760 1750 1740 

MON95- GCCGCATATGCCATCATGTATATGCATCAGTAAAACCCACATCAACATGTATACCTATCC

 :: :::::::::::::::::::::::::::::::::::::::::::::::::::::::::

NC_024 GCTGCATATGCCATCATGTATATGCATCAGTAAAACCCACATCAACATGTATACCTATCC

 84633280 84633290 84633300 84633310 84633320 84633330 

 1730 1720 1710 1700 1690 1680 

MON95- TAGATCAATATTTCCATCCATCATCTTAAACTCGTAACTATGAAGATGTATGGCACACAC

 :::::: ::::::::::: ::::::::::::::::::::::::::::::: :::::::

NC_024 TAGATCGATATTTCCATC---CATCTTAAACTCGTAACTATGAAGATGTATGACACACAC

 84633340 84633350 84633360 84633370 84633380 84633390

 1670 1660 1650 1640 1630 1620 

MON95- ATAAAGATACAAAATTAATAAATCCACCAGGTAGTTTGAAACAGTATTCTACTCCGATCT

 ::: :: : :::::::::::::: ::::::::::::::::::::::::::::::::::::

NC_024 ATACAGTTCCAAAATTAATAAATACACCAGGTAGTTTGAAACAGTATTCTACTCCGATCT

 84633400 84633410 84633420 84633430 84633440 84633450

 1610 1600 1590 1580 1570 

MON95- A--------GAACGACCGCCCAACCAGACCACATCATCACAACCAAGCGAA-AAAAAGCA

 : ::::::::::::::::: :::::::::::::::::::::::: ::::::::

NC_024 AGAACGAATGAACGACCGCCCAACCACACCACATCATCACAACCAAGCGAACAAAAAGCA

 84633460 84633470 84633480 84633490 84633500 84633510

 1560 1550 1540 1530 1520 1510 

MON95- TCTCTGTATATGCATCAGTAAAACCCGCATCAACATGTATACCTATCCTAGATCGATATT

 ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

NC_024 TCTCTGTATATGCATCAGTAAAACCCGCATCAACATGTATACCTATCCTAGATCGATATT

 84633520 84633530 84633540 84633550 84633560 84633570

 1500 1490 1480 1470 1460 1450 

MON95- TCCATCCATCATCTTAAACTCGTAACTATGAAGATGTA-GGCACATACATACAGATCCTT

 ::::::::::::::: :: ::::::::::::: ::::: :::::: :::::::::::: 

NC_024 TCCATCCATCATCTTCAATTCGTAACTATGAATATGTATGGCACACACATACAGATCCAA

 84633580 84633590 84633600 84633610 84633620 84633630

 1440 1430 1420 1410 1400 1390 

MON95- TAATAAATCCACCAGGTAGTTTGAAACAGTATTCTACTCCGATCTAGAACGACCCTTTAT

 

NC_024 AATTAATAAATCCACCAGGTAGTTTGAAACAGAATTCTACTCCGATCTAGAACGACCGCC

 84633640 84633650 84633660 84633670 84633680 84633690 
Bayer CropScience LP CR282-20U1 424 

The alignment between the MON 95379 Zm.Ubq element and the corresponding region of a 

conventional Zea mays chromosome five, which encodes the endogenous homolog, is broken into 

two main alignments derived from T-DNA element residues 1-1385, and 1451-2007 (Figure L-1). 

Each of these regions of alignment required numerous gaps and mismatches to align and displayed 

86.1% and 94.9% identity respectively (Figure L-1). This level of alignment fragmentation along 

with the internal gaps and mismatches within the alignments, is consistent with the non-uniform 

coverage and over representation of unpaired reads observed in the conventional control dataset 

(Figure L-2). This is the result of some quantity of endogenously sourced reads having sufficient 

similarity to map to portions of the T-DNA Zm.Ubq element, while their mate pairs are of 

insufficient identity for placement, resulting in an over-representation of unpaired reads (Figure 

L-2, panel 1). 

This is fragmentation and dissimilarity further substantiated in the conventional control mapping, 

where it can be observed that reads are unable to align with uniform coverage across the entirety 

of the Zm.Ubq element (Figure L-2, panel 3). Another indication that these reads are sourced form 

an endogenous element and not a secondary insert is that the mapped reads are constrained 

specifically to the Zm.Ubq element, which would be highly unlikely in the event of a secondary 

insert fragment (Figure L-2). 

Figure L-2. (Derived from Figure IV-5) Read Mapping of Conventional Maize LH244 

Versus PVZMIR522223 with Supplemental Annotations 
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This LH244 conventional control mapping demonstrates the expected level and areas of mapping that 

might be expected due to the presence of a maize native element (Zm.Ubq) within the T-DNA. Notably, 

all reads are constrained to the known common element boundaries, and coverage is non-uniform across 

the element indicating some level of sequence deviation relative to that of the 

T-DNA element.

When comparing the mapped reads from the conventional control sequencing back to that of the 

MON 95379 sample F4 T-DNA mapping it becomes clear that the additional unpaired reads 

captured across the Zm.Ubq element result in virtually superimposable signatures (Figure L-3). 

Figure L-3. Isolated Un-Paired Read Regions Corresponding to the Zm.Ubq Elements in 

MON 95379 Sample F4 and the LH244 Conventional Control. 

Panel A corresponds to MON 95379 sample F4 (Figure IV-6), and Panel B to LH244 control mapping 

(Figure IV-5). Notably the control and samples demonstrate qualitatively identical signatures 

indicating that all commonality across these regions is derived from the endogenous LH244 Zm.Ubq

homolog.

This identical presence and relative quantity of unpaired reads along with the isolated regions of 

increased depth seen in the characterized MON 95379 generation (Figure L-4) shows that all 

additional sequencing derived from this location is both expected due to the presence of the 

endogenous Zm.Ubq homolog in conventional maize, and not due to a secondary insertion. In 

contrast, if a secondary copy of a portion of the T-DNA were present, it would result in depth of 

sequencing being doubly present across the entire secondary insert/insert fragment, with a tripling 

of coverage in the areas sourced from the endogenous copy.
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Figure L-4. Side by Side Annotated comparison of Unpaired Read and Depth Plots of the 

MON 95379 Insertion and the LH244 Conventional Control. 

Section A displays panels 1 and 3 from Figure IV-6. Section B displays panels 1 and 3 from Figure 

IV-5. Red boxed areas represent location of Zm.Ubq element. Arrows pointing to areas of greatest 

depth observed and corresponding peak observable unpaired and paired (not pictured) reads in the 

LH244 control dataset. 

Although some DNA from the LH244 control was identified, its presence was expected due to the 

shared element Zm.Ubq. A review of this identified region between the control and test samples 

demonstrates highly congruent levels of capture and is therefore accounted for in the LH244 

conventional control dataset. Overall, these mapping signatures and lack of identified secondary 

junction sites supports a single T-DNA insertion in MON 95379. 

L.5. Additional Information Regarding Flank Capture and Coverage in the 

Characterization of the DNA Inserts in MON 95379 (Section IV.B) 

The NGS mapping portion of the experiment is designed to identify any and all plasmid insertions 

within a genome, and simultaneously absence of vector backbone, by mapping to the entirety of 

the transformation construct. The inclusion of the flanking genomic regions in the analysis is not 

feasible because it 1) pre-supposes knowledge of these insertion sites and their respective junctions 

and would confound our ability to detect potential secondary or higher order inserts, and 2) would 

impact the plasmid “backbone” region of the figure, confounding the ability to conclusively 

determine the absence of any backbone sequence. However, the coverage over the flanking 

genomic regions as captured by the NGS experiments are analyzed over five breeding generations 

as part of a generational stability analysis, and the alignment of the consensus sequences of the 

flank-junction spanning reads are illustrated in Appendix K; Figure K-19. Notably, the flank depth 

is at minimum the same depth of all truncated reads terminating at the junction point. The Y-Axis 

in panel 3 of the mapping represents the raw depth of reads across the plasmid sequence. Appendix 

K also includes 1) mapping figures for the subsequent generations (F4F1, F5, F5F1, and F6F1), 

controls, and reference substances, and 2) tables summarizing coverage statistics for all samples 

including controls (Tables K-1 and K-2). 

A. B.
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L.6. Additional Information Regarding Sequencing Utilized in the Mapping Process for the 

Conformation of the Absence of Cre Recombinase (Section IV.B.4) 

The reads used to confirm the absence of the Cre recombinase cassette are the same reads generated 

and used to confirm the presence of the single copy of the T-DNA (generation F4). Likewise, the 

process and parameters used are identical to those used for all samples as described in Section B.9. 

The difference between the processes to show the absence of the Cre recombinase cassette and the 

characterization of the inserted T-DNA is the transformation vector used as a reference sequence 

during the mapping process. Read mappings to the transformation vector containing the Cre 

recombinase machinery (PV‐ZMOO513642) are provided in Figure IV-7, and total sequencing 

generated for F4 is provided in Appendix K (Table K-1). Notably the total sequencing generated 

for this sample, which was then mapped to both of these transformation vectors, was 191.54 

gigabases (Gb). 

L.6.1. Clarification of Whole Genome Sequence Analysis (Appendix B) 

Although whole genome sequencing was done on multiple generations, these data were generated 

to be used in a read mapping experiment. As such no de-novo assembly was conducted on this 

data, and therefore no assembly software was used. 

L.6.2. Clarification on B.13 Rejected Data 

Data was rejected in three instances during the molecular characterization of MON 95379: 1) 

During the preparation of PCR amplicons for the insertion site analysis. 2) During the trimming 

and assembly of insert and insertion site sequences. 3) During the whole genome sequencing of 

MON 95379. The context and impact of these rejections are explained in greater detail below: 

1. In order to determine the insertion site sequence a PCR is conducted on LH244 DNA 

(conventional control). In one instance the amplification was not sufficient to support 

the downstream purification and sequencing of the amplicon causing the samples were 

rejected, and the PCR to be repeated. 

2. The analysis of the insert and insert sequence is conducted using CLC workbench (a 

commercially available software). During the analysis, some sequences were 

automatically rejected by the CLC software at two different stages of the analysis 

process, read trimming and read mapping. The reads that failed to meet default quality 

settings were automatically rejected during the trimming process. During the mapping 

process, additional reads were automatically rejected by the CLC workbench software if 

they failed to map to the reference sequence, if the sequence files contained no data, or 

if the sequence data was of insufficient quality. 

3. A limited number of whole genome sequencing runs were rejected for MON 95379 

generation F4F1 and F6F1 because of either the presence of PV-ZMIR522223 
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contamination or insufficient coverage. Additional sequencing runs were generated from 

the same starting materials to reach the targeted read depths. 

In all instances, the rejected data were assessed to have no effect on the quality, integrity, or 

conclusions of the study. 

L.7. Additional Information Regarding Binding Domains Located in the Sequences of 

Cry1B.868 and Cry1Da_7 (Appendix C.1.3 and D.1.3) 

Mass fingerprint analysis is a standard technique used to confirm the identity of a protein in a 

matrix. The design and intent of the mass fingerprint analyses provided to support the 

characterization of Cry1B.868 and Cry1Da_7 as expressed in MON 95379 was not to obtain full 

amino acid sequence coverage of the proteins. Rather, the purpose of this analysis was to confirm 

the identity of each protein and thereby validate the subsequent equivalency assessment. The 

identity of the MON 95379- and Bt-produced Cry1B.868 and Cry1Da_7 proteins was confirmed 

by mass fingerprint analysis, where very high sequence coverage was observed for all the proteins 

tested. 

The Cry1B.868 and Cry1Da_7 proteins are classical 3-domain Cry proteins. As such, the receptor 

binding sites are located in domains II and III (BenFarhat-Touzri et al., 2019; Bravo et al., 2013; 

Pigott and Ellar, 2007; Wang et al., 2019). Domains II and III fell between amino acid position 

242 to 606 for Cry1B.868 and 225 to 567 for Cry1Da_7. Despite the technical limitation of the 

mass spectrometry technology, there were significant coverages within those amino acid ranges 

for both Cry1B.868 and Cry1Da_7 proteins. Based upon the peptide maps in Figures C-2, 96% of 

the amino acid sequence in the region that contains the receptor binding domain was confirmed 

for both MON 95379- and Bt-produced Cry1B.868. Based upon the peptide maps in Figures D-2, 

100% of the amino acid sequence in the region that contains the receptor binding domain was 

confirmed for both MON 95379- and Bt-produced Cry1Da_7. The high percentage coverage of 

the mass fingerprint analyses provide clear support for the sequence similarity between the MON 

95379- and Bt-produced proteins. In addition, the DNA sequences of genes of the expressed MON 

95379 (Section IV.C) and DNA obtained from the Bt cell paste from which the proteins were 

purified (mentioned in Sections C.2.1 and D.2.1) had 100% coverage and the sequences aligned 

perfectly. It is evident from these data that the receptor binding domains between the MON 95379- 

and Bt-produced proteins are equivalent. 

L.8. Additional Clarification Regarding the Least-Squares Means and Standard Errors 

(SE) Presented in Table VI-1 of the Compositional Assessment of MON 95379 

The least-squares means and standard errors (SE) presented in Table VI-1 are model-based 

estimates obtained from the linear mixed model analysis of composition data. The SE values are 

calculated using the model-based estimates of the random effects and residual variance, as well as 

the sample sizes of the treatments (Milliken and Johnson, 2009). Under the model assumption of 

homogeneity of variance, all treatments are assumed to have the same residual variance. 
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Additionally, the sample size of each treatment is equal, i.e. four replicates per site and treatment. 

Therefore, the SE values are identical for all treatments. 

L.9. Additional Information Regarding Insect Activity Spectrum Testing for Cry1B.868 

and Cry1Da_7 (Section VII) 

Activity spectrum screening is not intended to provide a comprehensive assessment of specificity 

or cover all ecological functions or guilds, but rather provides a reasonable early indication of 

activity at levels expressed in the plant that contributes to problem formulation and informs NTO 

safety testing (Bachman et al., 2017; Bachman et al., 2013; Romeis et al., 2008). When 

characterizing activity of these proteins, the activity spectrum data are not interpreted in a vacuum. 

Information gathered for problem formulation provides a strong history of safe use, including 

limited insecticidal activity spectra for Cry1 proteins (Romeis et al., 2006) . Multiple species from 

the target order are typically selected to understand the variability in potency within the target 

order. Additional species outside of the target order are typically selected for activity spectrum 

screening and taxa selection is based on multiple criteria including representation of other key 

orders, assay availability, historical precedence, and relatedness to the target order. For 

MON 95379 maize, selected taxa were representative of the breadth of testing for previous familiar 

Bt proteins (e.g., MON 89034 maize). Four lepidopteran, four coleopteran species, and two 

hemipteran species were selected based on such criteria and, as such, activity spectrum testing is 

not intended to serve as a comprehensive safety assessment accounting for all feeding guilds and 

functions (Section VII.A). In the case of Cry1B.868, there was information from the literature that 

Cry1B may have activity against chrysomelids and therefore, focus on chrysomelids provides 

especially useful information. Also, chrysomelids have often been susceptible to coleopteranactive Bt proteins. Additionally, information from the subsequent tier I NTO studies also feeds 

back into an evergreen problem formulation further contributing to our understanding of “activity 

spectrum” in a broad sense. Conclusions of risk for Coleoptera are ultimately driven by margins 

of exposure established with the coccinellid and carabid NTO studies. 

Interactions are unlikely to result in effects to species when the individual components are present 

below their no-effect level (Levine and Borgert, 2018). Results of the interaction study supports 

the conclusion that Cry1B.868 and Cry1Da_7 act independently and thus can be assessed 

independently (Appendix J). This supports the conclusion that the combination of Cry1B.868 and 

Cry1Da_7 is unlikely to exhibit a different spectrum activity from those of the individual proteins. 

The reference to activity spectrum screening results, in the context of assessing results from adult 

lacewing testing, was part of a weight-of-evidence approach to reiterate what was known about 

potential activity of Cry1B.868 based on information developed during problem formulation 

(Section VII.C.1). As stated above, the primary purpose of activity spectrum is not to provide a 

comprehensive assessment on specificity and safety, but rather to inform the species selection in 

the NTO studies that drive the safety assessment. The interpretation of activity spectrum results 

with respect to Neuroptera is not based on phylogenetic distance, but rather that in initial screening, 

effects were only observed in Lepidoptera, consistent with the history of safe use developed with 

familiar Cry1-family proteins and their typical spectra of insecticidal activity (e.g., Lepidoptera). 
Bayer CropScience LP CR282-20U1 430 

For clarity, activity spectrum was not used to make conclusions of risk to Neuroptera. The result 

observed in C. carnea was addressed through additional testing, including a neuropteran species 

where a more sensitive larval life-stage could be evaluated. Conclusions of risk were based on 

minimal exposure from cultivation of MON 95379 maize in combination with results from the 

additional testing in green lacewing larvae and adults. 

In conclusion, activity spectrum results are used as part of a weight-of-evidence approach along 

with mode-of-action, exposure assessments, and laboratory NTO testing to assess potential 

impacts on organisms beneficial to agriculture. Ultimately, risk conclusions are driven by the 

margins of exposure established during laboratory NTO testing of species representing key 

ecological functions with relevant exposure to MON 95379 maize. 

L.10. Additional Information Regarding Potential Exposure to Cry1B.868 and Cry1Da_7 

Through Prey (Section VII.B.2) 

With respect to the estimates on potential exposure to beneficial predators through herbivore prey 

(Section VII.B.2), the maximum levels of Cry1Ac observed in spider mites by Obrist and 

colleagues correspond to approximately 1.5-fold above that expressed in plant tissue. A broader 

survey of the literature indicates that relative concentrations in mites can vary significantly 

between different Cry proteins and experiments and can typically range from 0.1u to 1.5u of that 

expressed in plants (Kim et al., 2020; Obrist et al., 2006b; Svobodavá et al., 2017). Spider mites 

are typically considered a worst-case scenario with respect to potential exposure to beneficial 

predators through prey. However, Cry protein concentrations in prey species such as aphids and 

juvenile lepidoptera are much lower than those found in spider mites. Given the generalist nature 

of predation by beneficial predators in the agricultural ecosystem, the magnitude of exposure to 

Cry proteins would be expected to be less than the worst-case scenario: a diet 100% consisting of 

spider mites feeding on MON 95379 maize. This would be consistent with previous published risk 

assessments that advocate for a value of 0.2u plant expression to reflect a realistic exposure 

scenario for generalist predators in the agricultural ecosystem (Raybould and Vlachos, 2011). 

Therefore, based on the potential variability in experiments, prey diversity, uptake of different Cry 

proteins, plant expression values were considered to be a reasonably conservative estimate of 

exposure through prey. However, it should be noted that focusing the assessment on an absolute 

worst-case scenario would not change the conclusions of the risk assessment for beneficial 

arthropods exposed via this route. Applying a 1.5-fold exposure factor to EECs for beneficial 

predators results in MOEs >20 for Cry1B.868 and >12 for Cry1Da_7 (with the exception of C. 

maculata where the MOE is >4 due to a correction to the test concentration, but no adverse effects 

on survival were observed). Therefore, the margins of exposure determined from Tier I NTO 

testing are sufficient to account for a worst-case exposure scenario of 1.5-fold above plant 

expression. 
Bayer CropScience LP CR282-20U1 431 

L.11. Additional Information Regarding Environmental Fate of Cry1B.868 and Cry1Da_7 

in Soil (Section VII.B.2) 

The calculations for the predicted environmental concentrations (PECs) for Cry1Da_7 and 

CryB.868 are provided at the beginning of the Soil Invertebrates exposure section (Section 

VII.B.2). The PEC values serve as a starting point in the exposure assessment for soil organisms 

in providing a conservative estimate of soil concentrations assuming the entire biomass of a field 

is incorporated into the top 6 in (15 cm) of soil The PEC values do not take into account other 

factors that may influence soil concentrations such as degradation. The results of the soil 

degradation study were used to refine potential exposure to soil organisms. For Cry1B.868, which 

exhibited rapid dissipation in soil, the conclusion was made that this protein was unlikely to persist 

or accumulate and thus the EEC for Cry1B.868 is the same as the PEC (0.35 μg/g; compare Table 

VII-2 and Table VII-5; please note that the EEC was rounded to one significant digit). Results 

from the laboratory study indicated Cry1Da_7 did show potential for persistence in sandy loam 

soil. Therefore, the accumulation model described in Section VIII.B.2 was used to account for 

potential accumulation of Cry1Da_7 under very conservative assumptions for both half-life 

duration (i.e., up to 1 year) and 25 consecutive years of planting MON 95379 without rotation. 

The 2-fold increase in soil concentration predicted by the model was then applied to the PEC to 

generate the EEC for Cry1Da_7. While soil incorporation was deemed the most ecologically 

relevant route of exposure to evaluate risk to soil organisms, test concentrations were deliberately 

selected to be sufficiently conservative to account for more direct exposure scenarios if necessary. 

Several factors influence the potential degradation rate of proteins in the environment including 

temperature, rain events, sunlight, and interaction with biotic factors such as robustness of the 

microbial community. The soil degradation study is based on general principles outlined in the 

EPA test guideline OPPTS 835.4100. The purpose of this study is to characterize the likelihood of 

persistence in soils given the characteristics of the proteins and soil properties while minimizing 

variability in the environment. It is reasonable to expect fluctuation in temperatures could impact 

enzymatic processes involved in degradation, but as noted above, it is only one of several factors. 

Environmental and soil temperatures are also cyclical in nature and even if periods of cold or 

frozen soil could slow degradation, inevitable seasonal warming would eventually lead to 

conditions amenable for degradation. This conclusion would be consistent with results from field 

monitoring studies where Bt proteins would be exposed to a full array of environmental factors 

and these studies have shown that Bt proteins do not persist or accumulate in the soil environment 

(Ahmad et al., 2005; Dubelman et al., 2005; Gruber et al., 2012; Head et al., 2002; Shan et al., 

2008). Persistence in soil and dissipation rates are determined by a number of factors. Clay soils, 

often associated with higher cation-exchange capacities, can generally bind macromolecules such 

as Cry proteins and nucleic acids more tightly than other soil types like sand and silt (Gruber et 

al., 2012; Icoz and Stotzky, 2008). Binding to clay particles can render proteins less available to 

enzymatic degradation and result in prolonged dissipation. However, other factors can also 

influence dissipation rates including protein characteristics, organic matter content, robustness of 

the microbial communities, soil moisture, and pH. Given the variety of environmental factors that 

influence degradation, it is not unexpected that relative degradation rates across soil types would 
Bayer CropScience LP CR282-20U1 432 

vary across studies and proteins. Generally, microbial activity is thought to be a principle factor in 

degradation rates of free protein (Icoz and Stotzky, 2008). Radiolabeled 14C-Cry1Ab applied to 

soils resulted in rapid CO2 evolution and the 14C-label was incorporated into the microbial biomass 

indicating that the applied Cry1Ab protein was being utilized as a microbial nutrition source 

(Valldor et al., 2015). This result is consistent with the notion that proteins are typically amendable 

to biodegradation in the environment. Guidance from EPA test guideline OPPTS 835.4100 

recommends that soil dissipation tests generally not be conducted beyond 120 days due to the 

potential decline of microbial communities in test systems that are isolated from the environment 

and natural replenishment. For this reason, laboratory studies represent a more conservative 

approach to assess the potential for persistence of Cry proteins in soils. 

The likelihood that test conditions resulted in an underestimate of exposure in soil is low. 

Cry1B.868 dissipated rapidly in soil with DT50 values comparable to other proteins that have been 

shown to not persistent in the environment. Cry1Da_7 also had DT50 values in two soil types that 

were comparable to previously commercialized Bt proteins (Icoz and Stotzky, 2008). For sandy 

loam soil, a first-order accumulation model based on highly conservative assumptions was used to 

generate a worst-case exposure levels to soil organisms in this soil type. These assumptions 

included a conservative estimated DT50 of 1 year, well beyond the duration of the study, and does 

not account for crop rotation. This calculation does not consider other environmental factors that 

would be expected to influence degradation rates. The result of the model was used to apply an 

exposure factor to the Cry1Da_7 PEC to generate a conservative EEC for soil organisms to 

characterize risk. Therefore, the assumptions applied provide a reasonable worst-case estimate and 

it is unlikely the results from the laboratory soil degradation study would lead to an underestimate 

of soil concentrations. Furthermore, the margins of exposure for Cry1Da_7 generated in Tier I 

testing for soil organisms was >2500, after calculating highly conservative exposure estimates, 

and is sufficiently protective of soil organisms. 

A similar dissipation profile was observed for Cry1Ac (expressed in MON 87701 soybean), which 

was tested in a soil degradation study to evaluate potential persistence in soil as part of the 

registration with EPA (U.S. EPA, 2010b). At the end of the 181-day incubation period, 72% to 

96% of the Cry1Ac had degraded regardless of soil texture, pH, clay content, or the method used 

to analyze the soil. The DT50 of Cry1Ac in soils was ranged from 0.4 – 13 days. However, 

modeling of the dissipation curves indicated that the DT90 was >181d for all three soil types, 

including a sandy soil. Despite the inability to calculate DT90 times in the laboratory study, field 

studies indicate that the Cry1Ac protein does not persist in sandy loam and silt loam soils under 

field conditions following multiple consecutive years of planting Bollgard cotton (Head et al., 

2002). Results from Cry1Ac in soybean and cotton indicate that while Cry1 proteins can exhibit 

relatively longer dissipation times, including in sandy soils under laboratory conditions, it is 

unlikely to result in persistence in the field. This further supports the conclusion that concentrations 

of Cry1Da_7 protein in sandy loam soil are expected to be low (d0.08 μg/g) and Cry1Da_7 is 

unlikely persist or accumulate in the environment. 

Environmental factors that impact potential degradation are taken into account in open field trials 

and published field surveys have demonstrated that Cry proteins do not persist or accumulate in 
Bayer CropScience LP CR282-20U1 433 

the environment (Ahmad et al., 2005; Dubelman et al., 2005; Gruber et al., 2012; Head et al., 2002; 

Shan et al., 2008). In a comprehensive multi-year field survey, Gruber and colleagues evaluated 

soils for the presence of Cry1Ab in fields that were planted with MON 810 maize for nine 

consecutive years (Gruber et al., 2012). Over the course of the 9-year trial, Cry1Ab was detected 

in soil above detectable levels at a single site 6 weeks after the 8th consecutive growing season. 

Notably, no Cry1Ab protein was detected at any site in any of the spring samplings prior to planting 

indicating that seasonal decreases in soil temperature did not result in persistence or accumulation 

of Cry1Ab in soil. 

In review of the results with Cry1B.868 and Cry1Da_7, Cry1B.868 dissipated rapidly in all soil 

types and Cry1Da_7 dissipated in clay and silt soils with kinetics comparable to other Bt proteins 

(reviewed in (Icoz and Stotzky, 2008)). While a DT50 could not be calculated for Cry1Da_7 in 

sandy soil, it should be noted that no biological activity was measured in sandy loam soil at the 

end of the study using a sensitive bioassay with FAW. Furthermore, the study was conducted at a 

field incorporation rate of 10-fold above a 1u maximum field load scenario. An exposure factor 

derived from a conservative accumulation model was applied to account for the longer dissipation 

profile for Cry1Da_7 in sandy soil and margins of exposure were still >2500 indicating minimal 

risk of Cry1Da_7 to soil organisms, regardless of soil type. 

L.12. Further Discussion of the Cry1Da_7 Results from the Soil Dissipation Study (Section 

VII.B.2) 

ELISA analysis completed on samples generated to establish dissipation rates for Cry1B.868 and 

Cry1Da_7 in MON 95379 in the soil dissipation study was conducted under GLP using a fully 

validated method with documented protocols. All assays contain positive and negative controls 

and assays have accept and reject type criteria as quality control measures. 

Assay to assay variation likely played a role in sample variability observed when comparing 

sample concentrations on Day 121 to Day 213. Samples were run on two separate assays on 

separate days. The first set contained all samples up to and including Day 121. The second set of 

samples contained the samples collected at Day 213 only. It was deemed necessary to generate 

the Day 213 sample to more fully characterize the dissipation rates for Cry1Da_7. 

Sample to sample variability is low as shown in the table below. Reps were from independent 

vessels. Calculated standard error values for the MON 95379 soil dissipation study are low and 

represent acceptable variation found when subsampling from multiple vessels. 

 
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Table L-1. Cry1Da_7 concentrations in three soil types as measured by ELISA 

Cry1Da_7 

incubation time (days) 

Sandy Loam Silt Loam Clay Loam 

sand 

mean 

μg/g 

(SD) silt

mean 

μg/g 

(SD) clay 

mean 

μg/g 

(SD) 

90 1.08 0.90 0.48 0.37 0.31 0.28 

90 0.88 (0.17) 0.41 (0.14) 0.35 (0.09) 

90 0.74 0.22 0.18 

120 0.69 0.61 0.43 0.32 0.16 0.24 

120 0.53 (0.08) 0.28 (0.09) 0.32 (0.11) 

120 0.61 0.25 <LOQ1

213 1.04 0.88 0.24 0.40 0.25 0.23 

213 0.83 (0.14) 0.41 (0.15) 0.22 (0.02) 

213 0.78 0.54 0.21 

LOQ for Cry1Da_7 is 0.063 μg protein /g sandy loam and silt loam soil, and 0.125 μg protein /g clay 

loam soil 

L.13. Additional Information on Potential Exposure of Cry1B.868 and Cry1Da_7 to 

Collembola (Section VII.B.2) 

Soil incorporation of post-harvest maize tissue represents a standard approach for early tier 

assessment of risk to soil organisms (Raybould and Vlachos, 2011; U.S. EPA, 2008; 2018b). This 

approach considers exposure from tilling the field into the top layers or soil and also accounts for 

any leaching up free protein from the degrading biomass on the soil surface into the field. The diet 

of Collembola is principally composed of soil hummus, mycorrhizae, and other fungal material 

and higher plant litter represents a relatively small proportion of the total diet (Ponge, 2000). Based 

on the results of laboratory testing, direct exposure of Collembola to maize litter is sufficiently 

accounted for in the risk assessment (Sections VII.C.1 and VII.C.2). The 95th percentile expression 

in forage provides a highly conservative estimate of direct exposure to terrestrial detritovores. 

Based on the results of the Collembola test, the margins of exposure to surface detritivores would 

be >71 for Cry1B.868 ((>3500 μg/g)/(48.8 μg/g)) and >45 for Cry1Da_7 ((>500 μg/g)/(11.1 

μg/g)). These risk estimates are likely highly conservative as they do not take into account any 
Bayer CropScience LP CR282-20U1 435 

degradation of the Bt proteins within senescing tissue and make an unrealistic assumption that the 

diet of Collembola consists of 100% MON 95379 forage tissue. 

L.14. Additional Information Regarding Reported Endpoints from Tier I Lacewing Tests 

with Cry1B.868 (Section VII.C.1) 

Section VII.C.1 describes the result of the Tier I test with Chrysoperla rufilabris and Cry1B.868 

where highest concentration tested was 700 μg/g diet. In initial testing with Chrysoperla carnea 

adults, 3500 μg/g was evaluated and a statistically significant difference in mortality was observed. 

Due to multiple reasons outlined in Section VII.C.1, including to the availability of a larval toxicity 

test, the relevance of C. rufilabris to North American agro-ecosystems, and the difficulties in 

acquiring sufficient C. carnea adults to run the test, additional studies were conducted with C. 

rufilabris. When no effects were observed in the larval test, an additional confirmatory test with 

C. rufilabris adults was conducted. The only relevant route of exposure for adult green lacewings 

to Cry1B.868 is through direct consumption of pollen so test concentrations with C. rufilabris 

were chosen to more closely reflect the magnitude of exposure from pollen consumption. Margins 

of exposure based on the 700 μg/g endpoint were >11 and thus sufficiently protective of green 

lacewing adults. 

L.15. Additional Information Regarding Assay Validity Criteria for the Larval Lacewing 

Studies (Appendix J) 

Assay validity for the green lacewing larval study is determined, in part, by mortality observed at 

the conclusion of the larval stage, prior to pupation (Appendix J). This assessment is distinct from 

the mortality observed at the end of the study reflected by adult emergence and is one of the apical 

endpoints evaluated in the study. This approach is consistent with established criteria outlined in 

OECD 239 Test Guidance for the chronic larval honey test, which requires assessment of survival 

at the end of the larval stage (Day 8 or 9), prior to pupation. This larval survival assessment is 

what’s provided in the 1st paragraph of the assay validity section (Appendix J.8 and J.21). Preimaginal survival reported in the results section is assessed at the end of the assay when adult 

emergence is evaluated. Mortality that occurs in the pupal and adult emergence stages result in 

anddifferences between larval and pre-imaginal survival values. 

L.16. Additional Information Regarding Sublethal Endpoints from Earthworm Studies 

with Cry1B.868 and Cry1Da_7 (Appendix J) 

As described in Appendix J, no food is provided in the acute earthworm study and typically a 

weight loss is observed over the 14-day duration of the study. Individuals in the water control 

groups from the Cry1B.868 and Cry1Da_7 studies exhibited mean weight losses of 7.9% and 

5.6%, respectively. For earthworms exposed to Cry1B.868, a 43% increase was observed at the 

3500 μg/g treatment level and a 4% increase was observed at 700 μg/g. For Cry1Da_7, a 1% 

increase was observed at the 500 μg/g treatment level and a 5% decrease was observed at the 50 

μg/g treatment level. The relationship between test concentrations and changes in body weight 
Bayer CropScience LP CR282-20U1 436 

observed in both studies indicate that the earthworms are using the Cry proteins as a nutrient source 

in the study. Specifically, higher test concentrations corresponded to increases in body weight. 

These results are consistent with the observations that earthworms can consume Bt proteins in soil 

from incorporated maize tissue and root exudates (reviewed in (Icoz and Stotzky, 2008)). While 

the 43% weight gain in worms exposed to Cry1B.868 at 3500 μg/g was not interpreted to be 

adverse as this treatment level far exceeds levels expected to be encountered in the environment, 

the <5% increase in body weight at 700 μg/g indicates that Cry1B.868 as expressed in MON 95379 

is not expected to result in adverse effects to earthworms. 

L.17. Additional Information Regarding Environmental Interactions and the Criteria for 

‘No Overlap’ Between Stressor Ratings (Appendix H)

An environmental interaction assessment is a part of a plant characterization study that is used to 

assess plant interactions with the receiving environment under typical agronomic management 

conditions. The environmental interaction assessment is not conducted to assess for specific pests 

or pathogens due to a lack of a specific risk hypothesis indicating that the traits would interact with 

non-target herbivorous insects or pathogens in an unexpected way. However, it is used to add 

weight of evidence to an assessment of plant pest potential and the already long history of safe use 

of GE crops that have demonstrated that GE plants interact with the receiving environment in a 

similar manner as conventional plants with the exception of the intended effects of the introduced 

traits. Specific risk hypotheses related to insect-resistance traits are most thoroughly addressed by 

the non-target organism (NTO) data conducted according to the tiered approach (U.S. EPA, 2007). 

The environmental interaction assessment demonstrate that the plants were closely monitored 

throughout the growing season and the data are primarily used to assess for potential gross 

differences between the GE plant and the conventional control. Overall, this methodology provides 

a structure and general process by which experienced agricultural researchers evaluate plants for 

biotic and abiotic stressors across the growing season. 

For individual site assessments, qualitative comparisons for environmental interactions 

assessments were made only between MON 95379 and the conventional control, and the two were 

considered different if their within-site ranges of raw values did not overlap. Raw data were 

summarized only within individual observations and sites due to the variation in temporal activity 

and geographical distribution of the stressors. The ranges for the damage ratings of each stressor 

were determined from the minimum and maximum raw damage rating values across four 

replications for each treatment within a site. These ranges are compared to the reference range 

obtained from commercial reference entries in the study to determine the relevance of any 

differences between the test and control. Detecting meaningful differences is a multi-layered 

approach. For example, the strength of the assessment lies in the comprehensiveness of approach 

which is obtained by collecting qualitative data 4 times across the growing season and across 

multiple sites that allows for robust comparative assessment of plant response to abiotic and biotic 

stressors. As such, any observed difference between the test and control would need to be 

examined in the context of data collected at other timepoints and from other sites. Also, a reference 

range data provides additional context to the comparative assessment. Data from the reference 

ranges within and across sites would further clarify the potential relevance of an observed 
Bayer CropScience LP CR282-20U1 437 

difference regarding whether it was a meaningful difference. Other information supporting an 

assessment of potential differences include in-season communication with field researchers and 

in-season real time QC of data. Stressors ratings showing any difference would trigger 

communication between the field researcher and the BCS Study Director to enable both impact on 

the trial at that study site as well as to evaluate the apparent differences between test and control 

leveraging all potential context as discussed above. Ultimately, an observed differencewould result 

in a site visit by the Study Director or delegate to further investigate. Therefore, even if there is no 

overlap between ratings, these multiple layers of comparative assessment inherent in the 

methodology would ensure that meaningful differences would be detected. 

Overall, the strength of environmental interactions assessment is not in statistical robustness, 

which is not the intent of the method due to lack of plausible risk hypotheses against specific pests 

or pathogens. Instead, strength of the assessment is related to the comprehensiveness of the 

assessment that facilitates close observation of the plants across the season and across sites with 

flexibility for experienced agricultural researchers to assess for any potential stressor that may be 

causing damage at a given time, the context provided by the reference range, and close connection 

of the Study Director to the field researchers and data during the season. The approach as submitted 

is aligned with the intended qualitative nature of the assessment, provides the desired comparative 

assessment of the appropriate level of resolution, and avoids over-interpretation or over-analysis 

of the data. Also, this is published method (Horak et al., 2015) and has been consistently used by 

BCS in the past submissions. 

L.18. Additional Information Regarding Plot Size and Measured Stressors for the 

Environmental Interaction Assessment (Appendix H)

The study is conducted across 8 sites that are representative of large-scale agriculture with diverse 

geographical and environmental conditions. Plot size was 37-39 m2 with an overall trial area of 

approximately 0.7 acres. The qualitative environmental interaction data collected from the given 

plot size are sufficient for a fit-for-purpose assessment of environmental interactions of the GE 

crop intended to identify gross differences. For example, a relatively small plot size is appropriate 

since stressor ratings are done on plant damage symptoms as opposed to arthropod collections, 

which would typically require larger plot size to account for insect mobility. Relatively small plots 

are effectively used to differentiate between treatments in efficacy trials using damage symptoms 

under natural infestation conditions (Reisig et al., 2015; Souza et al., 2016). Additionally, the plot 

size used is part of a consistent published approach (Horak et al., 2015) and used historically in 

BCS submissions. 

The stressors for any given site and timepoint combination were not predetermined. Measured 

stressors are based on the crop, growth stage, and geographical location. Prior to each data 

collection, the border rows in proximity of the study area were surveyed to select stressors that 

were actively causing damage to the crop for the subsequent evaluation in the study plots. A 

comprehensive list of potential stressors based on the stressors that are relevant to the crop, growth 

stage and geographical location of the trial was provided to experienced field researchers for data 

collection. Researchers selected stressors that were either actively causing damage at the time, or 
Bayer CropScience LP CR282-20U1 438 

in the absence of that, they were selected based on what would typically be occurring at that 

location and time. In addition, researchers had the flexibility to define and rate a relevant stressor 

not included in the above-mentioned list. The benefit of collecting data in the absence of damage 

provides positive confirmation that the assessment was done and that researchers were closely 

monitoring the plots. Recognizing that damage symptoms for certain stressors can be similar and 

sometimes difficult to identify to specific taxonomic levels, researchers identified stressors to the 

best of their ability based on knowledge of what would most likely be occurring at that site and at 

that time of the season. Therefore, expert local knowledge and experience with the crop was the 

basis for selecting appropriate stressors and estimating the range of responses that would be 

considered typical for maize. 

L.19. Additional Information Regarding Agrochemical Use and Relative Timing of Field 

Observations of Arthropod and Disease Stressors for Environmental Interaction 

Assessment (Appendix H)

Pest management was based on pest pressure threshold to maintain trial quality and yield, 

mimicking commercial crop production practices (Section H.6). Based on the study protocol, field 

researchers apply commercially-registered agrichemicals at labeled rates for control of arthropods, 

diseases and weeds when they reach levels that can cause economic injury to the crop, to produce 

an agronomically acceptable crop. In addition to expert local knowledge and experience of field 

researchers with conventionally bred maize used as the basis for selecting appropriate pest 

management measures, the field researchers also consult with BCS regarding the type and timing 

of pest management. Pest management operations were performed uniformly across all plots 

within each site. 

Pest control measures applied and timing of field observations of arthropod and disease stressors 

across sites are given in Tables L-2 and L-3, respectively. In summary, five out of eight sites did 

not receive any insecticide application. Two sites received one in-season insecticide application 

and one site received two insecticide applications one of which was a pre-emergent insecticide 

application only. Six out of eight sites did not receive any fungicide application. Only two sites 

received one in-season fungicide application. Therefore, relatively few insecticide and fungicide 

applications were used across sites so any potential effect on stressor ratings was minimal. 

When pest management is applied, field researchers are expected to collect field observations on 

biotic and abiotic stressors either prior to the pesticide application or approximately 10 days after 

pesticide application to minimize impact of pesticides on environmental interaction assessment. 

Most of the field observations were collected 10 days after pesticide application except at site 

NEYO, where an environmental interaction assessment was done six days after 

insecticide/fungicide application due to oversight and was captured as a protocol deviation. The 

assessment of arthropod and disease damage “near to application time” is still useful for a 

comparative assessment because all plots received uniform applications of the pesticide to avoid 

potential confounding effects from these pest management practices. Furthermore, this visual 

assessment is not a measure of abundance or presence of actual arthropods that would likely be 

impacted following insecticide application. Additionally, any insecticide/fungicide application 
Bayer CropScience LP CR282-20U1 439 

was often triggered by damage symptoms by insects/diseases, thus allowing comparative 

assessment of arthropod/disease damage between the test MON 95379 and the conventional 

control. Therefore, comparative environmental interaction assessment of arthropod/disease 

stressors between MON 95379 and the conventional control is meaningful in this study since 

relatively few pesticides were applied and when they were applied, assessments were conducted 

before or at least 10 days after pest control measures occurred (with the exception of one protocol 

deviation), thus minimizing any potential effects of the pesticide treatments. 
Bayer CropScience LP CR282-20U1 440 

Table L-2. Summary of Insect Control Measures Applied and Timing of Field Observations of Arthropod Stressors for MON 95379 and the Conventional Control Across in 2018 U.S. Field Trials 

Site Code1

Date Applied2

Commercial Product3

Active Ingredient(s) 

Application Rate (g a.i./ha)

Carrier Volume (L/ha)4

Adjuvant Type

Adjuvant Rate (%)5

Application Method

Growth Stage (V to R)

EI Observation Date2

ILHY 05/25/2018 Warrior II Lambda- cyhalothrin 27 140 – – On Soil Pre- emergence 06/20/2018 ILJO 05/17/2018 Force 3G Tefluthrin 111 –† – – In soil Preemergence 06/14/2018 ILJO 07/24/2018 Fastac CS Alpha- cypermethrin 22 28 – – On vegetation R2 08/06/2018 NEYO‡ 07/24/2018 Brigade 2EC Bifenthrin 105 

281 Nonionic surfactant 0.3 On vegetation R2 07/30/2018 NEYO‡ 07/24/2018 Warrior II Lambda- cyhalothrin 29 R2 07/30/2018 

1Site code: ILHY= Clinton County, IL; ILJO= Champaign County, IL; NEYO=York County, NE. 2Date format: mm/dd/yyyy.3All product names are the property of their respective owners.4Carrier volume associated with multiple commercial products indicates that the products were applied as a tank mix.5Volume of adjuvant as a percentage of volume of spray solution.†Applied as granule.

‡Environmental interaction assessment was done 6 days after insecticide application (instead of 10 days intended) due to oversight (discussed 

in response to technical review comment ‘P. 299, 2nd paragraph’). 
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Table L-3. Summary of Disease Control Measures Applied and Timing of Field Observations of Disease Stressors for MON 95379 and the Conventional Control Across in 2018 U.S. Field Trials 

Site Code1

Date Applied2

Commercial Product3

Active Ingredient(s)

Application Rate (g a.i./ha)

Carrier Volume (L/ha)4

Adjuvant Type

Adjuvant Rate (%)5

Application Method

Growth Stage (V to R) 

EI Observation Date2

ILJO 07/24/2018 Headline AMP Pyraclostrobin 107 28 – – On vegetation R2 08/06/2018 Metconazole 40 NEYO‡ 07/24/2018 Trivapro 

Benzovindiflupyr Azoxystrobin Propiconazole 

30 110125

281 Nonionic surfactant 0.3 On vegetation R2 07/30/2018 

1Site code: ILHY= Clinton County, IL; ILJO= Champaign County, IL; NEYO=York County, NE. 2Date format: mm/dd/yyyy. 3All product names are the property of their respective owners. 4Carrier volume associated with multiple commercial products indicates that the products were applied as a tank mix.5Volume of adjuvant as a percentage of volume of spray solution. ‡Environmental interaction assessment was done 6 days after insecticide application (instead of 10 days intended) due to oversight (discussed in 

response to technical review comment ‘P. 299, 3rd paragraph’). 
Bayer CropScience LP CR282-20U1 442 

L.20. Additional Information Regarding Trial Management and Assessment of Abiotic 

Stressors for Environmental Interaction Assessment (Appendix H) 

The Principal Investigator at each site followed local agronomic practices including those related 

to seed bed preparation and trial maintenance such as application of agricultural chemicals, 

fertilizer, and irrigation. All maintenance operations were performed uniformly across all plots 

within each site. In accordance with typical commercial crop production practices, most sites did 

not receive irrigation and fertilization during the growing season. Only 2 out of 8 sites received 

irrigation during the growing season. All sites received initial (pre-planting) fertilizer application 

and only the OHTR site received an in-season fertilizer application. All maintenance operations 

were performed uniformly across all plots within a site to avoid potential confounding effects from 

management practices. Therefore, in practice, most sites did not receive irrigation and fertilization 

during the growing season. However, even though irrigation and fertilizer treatments are 

implemented, drought and nutrient deficiency could still occur during the season and potentially 

be influenced by factors such as geographical location, season dynamics and climate. Additionally, 

the environmental interaction assessment methodology provided flexibility to experienced 

agricultural researchers to evaluate plants for any relevant stressor that could be present or was 

actively causing damage to the crop at the time of evaluation at their site. A total of 15 observations 

for nutrient deficiency were recorded during the season across sites (Table H-3). Seven out of eight 

sites recorded nutrient deficiency as one of the three abiotic stressors during the growing season. 

Nutrient deficiency was given a value other than none only in one case so, in practice, nutrient 

deficiency was not a commonly occurring stressor in these trials. Nutrient deficiency occurred at 

site ILJO in all six maize entries, consisting of the test MON 95379, the control, and four 

references at the third environmental interaction evaluation corresponding to R3 growth stage. The 

degree of severity of damage caused by nutrient deficiency recorded as per the categorical rating 

scale (categories: none, slight, moderate, or severe) given in section H.6 was slight in test plots 

and ranged from none to slight in control and reference plots. Although specific symptoms of 

nutrient deficiency were not recorded, it was likely due to nitrogen and potassium typical for that 

site and growth stage of the crop as per communication with the field researcher. However, no 

differences were observed between 

MON 95379 and the conventional control due to the overlap between the range of injury symptoms 

between MON 95379 and the conventional control across all four replications within the 

observation.The remaining stressor assessments from the other six sites had nutrient deficiency 

with ratings of “none”, which reflects the structure of the environmental interactions assessment 

methodology. 

Field researchers rated for three stressors in each category (insect, disease, and abiotic) that are 

either actively causing damage or, in the absence of damage symptoms, are typical for that location 

and time of the season. Including stressors in the absence of damage symptoms adds value as part 

of a consistent method that contributes to the overall knowledge of what stressors were occurring 

(or not) at a given site/timepoint combination. Assessment of stressors rated as “none” also 

provides positive confirmation that the complete assessment was done and facilitates active 

monitoring of the plots by field researchers. 
Bayer CropScience LP CR282-20U1 443 

Therefore, drought and nutrient deficiency are useful to include as potential stressors given that 

study sites are typically rain-fed and fertilizer application largely occur only prior to planting. In 

addition, drought and nutrient deificiency are useful stressors to provide flexibility to researchers 

rating any potential stressor that could be occurring. Furthermore, comparative environmental 

interaction assessment of drought and nutrient deficiency between MON 95379 and conventional 

control is meaningful since symptoms from these stressors can still be observed despite use of 

irrigation and fertilizer treatments. 

 
Bayer CropScience LP CR282-20U1 444 

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Yang, D.L. Kerns and A. Jerga. 2019. Bacillus thuringiensis Cry1Da_7 and Cry1B.868 protein 

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Bayer CropScience LP CR282-20U1 447 

L.21. Additional Information Regarding the Threatened and Endangered Species 

Assessment for MON 95379 Maize (Section VII.B.2 and Section VII.D) 

L.21.1. Background

Insect-protected maize MON 95379 produces two insecticidal proteins, Cry1Da_7 and 

Cry1B.868, which protect against feeding damage caused by targeted lepidopteran insect 

pests. Cry1Da_7 is a modified Cry1Da protein derived from Bacillus thuringiensis (Bt subsp. 

aizawai). Cry1B.868 is a chimeric protein comprised of domains I and II from Cry1Be (Bt), 

domain III from Cry1Ca (Bt subsp. aizawai) and C-terminal protoxin domain from Cry1Ab (Bt

subsp. kurstaki). 

MON 95379 was developed to provide growers in South America an additional tool for controlling 

target lepidopteran pests, including fall armyworm resistant to current Bt technologies. 

MON 95379 will not be commercialized in the United States, but is intended to only be cultivated 

in small-scale breeding, testing, and seed increase nurseries to develop seed of products that will 

be sold in other countries, primarily in South America. These intended cultivation uses will be 

subject to the terms and conditions of an EPA breeding registration, which Bayer has proposed to 

be limited to no more than 100 acres per growing season across Nebraska, Hawaii, and Iowa. 

In July 2020, Bayer submitted a petition for deregulation of MON 95379 to USDA (USDA-APHIS 

Petition Number: 20-205-01p). Included in the petition, Bayer provided a comprehensive 

ecological risk assessment (ERA) demonstrating MON 95379 poses minimal risk to organisms 

beneficial to agriculture, including non-target Lepidoptera, under the proposed conditions of use 

(e.g., small-scale breeding, testing, and seed increase nurseries). In addition to the ERA, an 

assessment on the potential effects of MON 95379 on listed threatened and endangered species 

was conducted considering the proposed conditions of use in a limited geography. Since the results 

of activity spectrum screening and Tier 1 testing indicated effects on survival from Cry1B.868 and 

Cry1Da_7 are limited to Lepidoptera, a comparison of the county level distribution of endangered 

lepidopteran species and corn cultivation was conducted for the three proposed states for 

cultivation. The results of this county-level overlap analysis identified three species for further 

evaluation. These species identified were Blackburn’s sphinx moth (Manduca blackburni) in 

Hawaii, Dakota skipper (Hesperia dacotae) and the Poweshiek skipperling (Oarisma Poweshiek) 

in Iowa and no species in Nebraska. Evaluation of critical habitats and updated ranges for these 

species supported the conclusion that Blackburn’s sphinx moth, Dakota skipper, and Poweshiek 

skipperling would have no exposure to MON 95379 corn. A recent assessment of potential effects 

on threatened and endangered species was conducted by the U.S. EPA for MON 89034  ́ TC1507 

 ́ MON 87411  ́ DAS-59122-7 corn expressing lepidopteran-active Cry1A.105, Cry2Ab2, and 

Cry1F proteins, resulted in a No Effect determination for all listed lepidopteran species due to 

habitat requirements that exclude them from corn fields (U.S. EPA, 2017). Updated species range 

information published by USFWS indicate that Dakota skipper and Poweshiek skipperling have 

not been observed in Iowa since the time of listing (2014) and are no longer believed to be present 

in Iowa (USFWS, 2018a; b). Therefore, based on the taxonomic specificity of Cry1B.868 and 

Cry1Da_7 for Lepidoptera and lack of exposure based on critical habitat and current species range 
Bayer CropScience LP CR282-20U1 448 

maps resulted in the conclusion that MON 95379 will have no effect on currently listed threatened 

and endangered species. 

In June 2021, it was communicated to Bayer that as part of the threatened and endangered species 

assessment for MON 95379, USDA sought additional information on five lepidopteran species. 

The purpose of this assessment is to provide additional information to support the threatened and 

endangered species evaluation for five species identified by USDA whose ranges generally overlap 

with corn-growing regions of the United States. The species evaluated were Mitchell’s satyr 

butterfly (Neonympha mitchellii mitchellii), St. Francis’ satyr butterfly (Neonympha mitchellii 

francisci), Poweshiek skipperling (Oarisma poweshiek), Karner blue butterfly (Lycaeides melissa 

samuelis), and Dakota skipper (Hesperia dacotae). 

Approach 

For this assessment, information is provided to address the identified species collectively as well 

as individually (Figure L-5). Listed Lepidoptera (i.e., non-pest) species are generally not known 

to feed on corn tissue as their feeding and reproductive ecology is typically tightly associated with 

a preferred host plant (U.S. EPA, 2001a). Standard agricultural practices for weed management 

typically preclude the growth of preferred host plants within the boundaries of corn fields. 

Therefore, exposure to listed Lepidoptera would only occur by deposition of MON 95379 pollen 

on preferred host plants adjacent to the field. To refine the qualitative exposure assessment for 

non-target Lepidoptera provided in the submitted petition for MON 95379, laboratory toxicity 

information with the most sensitive lepidopteran species tested, monarch butterfly (Danaus 

plexippus), was incorporated with an established pollen deposition model to quantitatively 

characterize exposure and potential risk at the field edge. Consideration of a proposed product use, 

and how this use could impact potential exposure, is part of the initial step for developing scenarios 

to assess risk to listed species (EPA, 2021). The potential for exposure at the field edge was put 

into context considering standard agronomic practices used to generate high quality seed by 

limiting pollen flow in seed increase and corn breeding activities. Results and conclusions from 

the field-edge characterization and product use considerations would apply to all listed 

Lepidoptera. 

The risk assessment for the five species of interest was refined following the general principles 

outlined in the EPA revised guidance for evaluation of Threatened and Endangered species (EPA, 

2020). For each species, county-level comparisons were made between the species ranges provided 

by the USFWS ECOS database and corn acreage provided in the 2017 USDA Ag census. This 

overlap analysis was then further refined based on updated state-level and county-level species 

range information provided in recent 5-Year Reviews and Species Status Updates published by 

USFWS. 

For species such as Karner blue and Mitchell’s satyr butterfly, where current species ranges are 

available only at the county level, no further range refinement was conducted. For St. Francis’ 

satyr butterfly and Poweshiek skipperling, shapefiles downloaded from the USFWS ECOS 

database were uploaded into USDA CropScape and the percent of the species range occupied by 
Bayer CropScience LP CR282-20U1 449

corn based on the 2020 Crop Data Layers (CDLs) was determined. For Poweshiek skipperling, the 

current USFWS range map has sub-county species ranges in Michigan and Wisconsin where the 

species is still present and county-level range information for Iowa, Minnesota, South Dakota, and 

North Dakota where the species is no longer present. To refine the potential exposure for this 

species, new polygons were constructed for each individual critical habitat corresponding to the 

species range in Wisconsin and Michigan and a corn CDL overlay analysis was conducted for each 

location. The total acres of critical habitat and overlapping corn acres for these sites were then 

summed to determine the total amount of corn acres overlapping with the most current species 

range information for Poweshiek skipperling. For the sub-county assessment of Dakota skipper, 

the current USFWS range map files are too large for USDA CropScape so shapefiles were 

uploaded into the FESTF Gopher utility (NatureServe and its natural heritage member programs). 

For the overlap analysis, the current species range and critical habitat information for Dakota 

skipper was compared to the EPA Use Data Layer for corn. It should be noted that potential 

exposure and risk is limited to off-field areas due to the lack of host plants within corn fields. 

Resolution of CDL/UDL-based overlap analyses is typically limited to 30 m and therefore does 

not provide enough precision to account for the typical off-field transport distances for corn pollen 

(Pleasants et al., 2001). Therefore, the overlap of the species ranges with the in-field corn 

CDL/UDLs was determined to be a reasonable, albeit likely highly conservative estimate for 

potential exposure of listed Lepidoptera to corn pollen.

Following species range refinement, potential exposure of each species was further characterized 

based on individual species biology and critical habitat considerations. Information for this 

refinement step was derived from USFWS, NatureServe, and public literature sources.
Bayer CropScience LP CR282-20U1 450 

Figure L-5. Framework of the Approach to Provide Geographic, Biological, and Habitat 

Information to Refine the Threatened and Endangered Species Assessment for MON 95379

L.21.2. Listed Lepidoptera Will Have Minimal Exposure to MON 95379 Corn Pollen 

The results of the ecological risk assessment in the submitted petition have shown that the activity 

of the two Bt proteins expressed by MON 95379 are highly specific for lepidopteran insects. Listed 

Lepidoptera (i.e., non-pest) species are generally known to not feed on corn tissue as their feeding 

and reproduction is often dependent on a preferred host plant (e.g., wild lupine for Karner blue 

butterfly and bluestem grasses for Dakota skipper; 2018). Previous work evaluating drift of corn 

pollen and deposition at the field edge demonstrated that density of pollen deposition on the surface 

of host plants decreases exponentially with increasing distance from the edge of the field. For 

example, at a distance of 4 to 5 meters from the edge of a corn field, corn pollen density on host 

plants is predicted to be less than 10% of the density at the field edge (Pleasants et al. 2001). 

Consequently, significant exposure of MON 95379 corn pollen to listed Lepidoptera will only 

occur in areas near the field edge. 

In addition, movement of corn pollen is limited to a short interval of time. Peak production of 

pollen from a corn field typically occurs within a 4-day interval and pollen shed is generally 

confined to a period of about two weeks (Abendroth et al., 2011). Therefore, sensitive larvae would 

only be potentially impacted by consumption of MON 95379 pollen if they were feeding on a host 

plants in close proximity to a MON 95379 field at the time of pollen shed. Rain events that occur 

during or following pollen shed would significantly reduce pollen densities on host plants near the 

field. In an analysis of six pollen deposition trials across four regions in the U.S. mainland where 

corn is cultivated, at least one rainfall event occurred during anthesis in all but one trial and these 

rain events resulted in 54-86% reduction on deposited pollen densities (Pleasants et al., 2001). 

Prevailing wind patterns also influence where pollen deposition will occur in open-pollinated 

fields. Analysis of prevailing wind patterns in mainland U.S. trials indicated the potential exposure 

at the field edge from pollen deposition was limited to the downwind side of the field (Pleasants, 

et al., 2001). Therefore, any habitat present up-wind of a MON 95379 corn field would be expected 

to have minimal exposure to corn pollen. Additionally, as wind distributes pollen from the field 

edge, it also disperses the pollen from the surface of host plant leaves where corn pollen has settled 

and would reduce the expected density of deposition at distances further from the field edge. This 

would result in further minimizing concentrations of pollen on off-field host plant leaves. 

To estimate potential risk of Cry1B.868 and Cry1Da_7 to listed Lepidoptera at the field edge, a 

quantitative risk analysis was conducted. Cry1Da_7 is not expressed at quantifiable levels in 

MON 95379 pollen. Therefore, the assessment focused on potential exposure and effects from 

Cry1B.868. Risk can be conservatively characterized using the U.S. EPA approach for assessing 

potential risk to endangered animal species and a non-target Lepidoptera risk assessment model 

incorporating design aspects of confined field trials, such as distance from experimental corn to 

off-field vegetation. 
Bayer CropScience LP CR282-20U1 451 

The U.S. EPA (U.S. EPA. 1992; U.S. EPA, 2004) outlined a conservative approach for assessing 

potential risk to endangered animal species. For terrestrial animal species, a 10-fold safety factor 

based on the median lethal concentration (LC50) for the most sensitive species tested is used to 

accommodate uncertainty in the risk assessment process for the particular terrestrial endangered 

species. Consequently, a concern for terrestrial endangered animal species would be indicated if 

the ratio of the LC50 concentration for the most sensitive animal species tested to the maximum 

potential exposure were less than 10. Results from activity screening with Cry1B.868 indicated 

that monarch butterfly was the most sensitive lepidopteran species tested. 

The model developed by Pleasants et al. (2001) and Dively et al. (2004) to assess the risk of Bt

corn to monarch butterfly larvae was used to determine a safety factor for corn events such as 

MON 95379 expressing Cry1B.868. The safety factor is derived from model parameters such as 

toxicity of the protein (i.e., hazard) and exposure considerations such as pollen deposition amounts, 

and actual amounts of protein encountered by Lepidoptera as they feed on pollen dusted host plant 

leaves. 

The LC50 value used for the assessment was determined from results of a laboratory dietincorporation bioassay with the most sensitive lepidopteran species (monarch butterfly) tested 

against the Cry1B.868 protein (Table L-4). Exposure to Cry1B.868 from deposition of pollen was 

calculated for increasing distances from the field edge based on results from published pollen 

deposition trials (Pleasants et al., 2001). 

At a distance of 6 m from the field edge, the margin of safety for Cry1B.868 was calculated to be 

10 indicating minimal risk to non-target Lepidoptera (Table L-4). This margin of safety is likely 

to be highly conservative based on several factors. The estimated exposure levels are derived using 

95th percentile values for Cry1B.868 expression as well as 95th-centile values for pollen deposition. 

This characterization of risk is also highly conservative in that it does not consider host plant 

distribution in the environment and what proportion of host plants are at the field edge compared 

to other agricultural and non-agricultural landscapes. The model above is derived from pollen 

deposition densities on milkweed plants that have horizontally positioned broad leaf architectures 

and are thus relatively conducive to holding deposited pollen. In contrast, the species of interest 

feed on grasses (little bluestem for Dakota skipper and Poweshiek skipperling and Carex spp. for 

St. Francis’ and Mitchell’s satyr butterflies) or wild lupine in the case of Karner blue. The preferred 

host grasses, discussed further below for each listed species, have relatively narrow (<1cm) leaf 

blades arranged in a more vertical orientation compared to milkweed leaves and thus would not be 

expected to collect and maintain significant amounts of corn pollen. Likewise, leaflets of wild 

lupine are significantly smaller than common milkweed leaves indicating that calculated exposure 

levels in the milkweed model are likely to considerably overestimate realistic exposure levels. 
Bayer CropScience LP CR282-20U1 452 

Table L-4. Estimated Exposure and Hazard as well as the Calculated Margin of Safety for 

the Cry1B.868 Protein in Pollen Deposited on Host-Plant Leaf Surfaces using Milkweed as 

a Model System1.

Distance from 

edge of the 

corn field (m)

95th centile 

pollen 

deposition on 

host leaf surface 

(grains/cm2)

Maximum dietary 

exposure for 

Cry1B.868

(μg/g diet) 2

7-day LC50 for 

MBF (μg/g diet) 3

Margin of 

safety 4

0 300 0.232 0.076 <1 

1 200 0.155 0.076 <1 

2 75 0.058 0.076 1 

3 54 0.042 0.076 2 

4-5 25 0.019 0.076 4 

65 10 0.008 0.076 10 

75 6 0.004 0.076 17 

85 3 0.002 0.076 31 1 The model used for these calculations is taken, in part, from Pleasants et al., 2001 and Dively et al., 2004. Pollen 

exposure is based on 95th centile exposure values from Pleasants et al., 2001. 2 The maximum exposure concentration for both Cry proteins in a pollen-dusted food substrate at a given distance 

from the edge was calculated by the equation: 

{[(a/b)*c]/d} 

where: 

a = number of the pollen grains deposited per cm2 on the surface of milkweed leaves (surrogate food 

substrate) at various distances from a cornfield (Pleasants, 2001); 

b = number of pollen grains per gram of fresh weight corn pollen [≈ 4 x 106

 grains according to Miller 

(1985)]; 

c = the upper 95th percentile Cry protein concentration of 61.9 μg/g fresh weight: corn pollen; 

d = fresh weight (g) of the food plants per cm2 [≈ 0.02 g per cm2

 of fresh milkweed leaves according to the 

published information in Hellmich et al. (2001)]. 

 3 For convenience, each mL of diet is assumed to weigh approximately one gram, and therefore the LC50 is expressed 

as μg/g of diet. 4 The margin of safety was calculated as the quotient of the 7-day LC50 value for D. plexippus divided by the maximum 

exposure concentration for the Cry1B.868 protein. For this calculation, the upper 95th percentile Cry1B.868 

concentration in pollen of 61.9 μg/g fresh weight was used. 5 Predicted pollen deposition at 3m and beyond 5 m from the field edge was calculated using regression analysis of 

distances from field edge and log-transformed deposition values (Pleasants et al., 2001; GraphPad 8 Prism). 

There are several examples of commercialized and deregulated corn products cultivated on broad 

acres with detectable expression of lepidopteran-active proteins in pollen. For example, 

Cry1A.105 in MON 89034 u TC1507 u MON 88017 u DAS-59122-7 corn has a maximum 

reported expression of 21 μg/g in pollen (EPA, 2009). Similarly, Cry1F has a maximum reported 

expression of 35 μg/g in 4114 corn (EPA, 2010) and Vip3A20 in MIR162 corn has reported 
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maximum expression in pollen of 49.7 μg/g (EPA, 2009). Using a most sensitive species surrogate 

approach, these products would be expected to have risk profiles at the field edge comparable to 

MON 95379 (EPA, 2009; Wolt et al., 2005). However, threatened and endangered species 

conclusions for these products have been largely based on lack of habitat in agricultural areas 

(EPA, 2009a; 2010; 2017). Consistent with this, USDA concluded MON 89034 corn expressing 

Cry1A.105 would have no effect on listed Lepidoptera, including specifically St. Francis’ satyr 

butterfly and Mitchell’s satyr butterfly, due to a lack of breeding habitat in agricultural areas. 

(USDA, 2008a). The general lack of overlap between critical habitat and corn fields and the 

conservative nature of the pollen deposition model indicate cultivation of MON 95379 corn is 

unlikely to cause adverse effects to listed Lepidoptera under realistic field conditions. 

L.21.3. Standard Agricultural Practices to Generate High Quality Seed are Protective of 

Listed Lepidoptera 

Environmental risk mitigation measures are a common consideration in defining conditions of safe 

use for many pesticides. Standard mitigating measures for MON 95379 corn are derived from 

typical practices used to limit pollen movement outside of the field and would also be expected to 

minimize exposure to listed Lepidoptera. A combination of standard agronomic practices (Table 

L-5) commonly used in seed production and other small-scale breeding and testing nurseries 

permitted under the proposed EPA registration are expected to further mitigate potential risk to 

listed Lepidoptera.

Table L-5. Typical Agronomic Practices Expected to Mitigate Off-Field Exposure to Listed 

Lepidoptera from MON 95379 Pollen During Anthesis. 

Agronomic Practice Mitigating Measure Benefits 

Pre-pollen shed 

detasseling

Detasseled plants within field 

prior to pollen shed

Reduces pollen flow off field and eliminates 

potential exposure to nontarget Lepidoptera 

habitat.

Border rows 4 to 6 border rows (10 to 15 ft) Reduces pollen flow off-field to non-target 

Lepidoptera habitat.

Maintenance of fallow 

areas surrounding plots 

Removal of flowering weeds at 

the field edge

Reduces Lepidoptera habitat within the field and 

at field margin and increases distance between 

field edge and adjacent habitat.

Closed pollination Bagging of tassels Limits pollen movement off-field. 

Cultivation in controlled 

environments

Cultivating in greenhouse and 

screenhouse facilities

Limits pollen flow to non-target Lepidoptera 

habitat
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For example, utilizing a minimum of four rows of conventional corn buffer (10 ft) and a 15 ft 

fallow area would be protective of non-target Lepidoptera as no effects would be expected at 

distances >20 ft (~6m) from the field edge under a conservative worst-case exposure scenario 

(Table L-4). Buffer rows of conventional corn that commonly surround experimental corn plots 

additionally serve as barriers to pollen flow (Ireland et al., 2006), thus limiting potential deposition 

on off-field host plants. For these reasons, minimal off-field exposure of non-target Lepidoptera 

from MON 95379 corn pollen is expected and this exposure is further minimized by standard 

agronomic practices used to generate high quality seed with activities permitted under the proposed 

EPA registration. 

L.21.4. Species-Specific Information to Support the Threatened and Endangered Species 

Assessment 

a. Mitchell’s satyr butterfly

Species Range 

The current range for Mitchell’s satyr butterfly (MSB) is limited to counties within the states of 

Alabama, Indiana, Michigan, Mississippi, Ohio, and Virginia (Table L-6). While the current 

species range overlaps counties with corn production, MSB is not present in Hawaii, Iowa, or 

Nebraska where MON 95379 is intended for cultivation. 

 
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Table L-6. County-Level Overlap of Corn Acres with Mitchell’s Satyr Butterflya

State County 

Alabama Bibb, Greene, Hale, Perry, Tuscaloosa 

Indiana LaGrange 

Michigan Barry, Berrien, Branch, Cass, Jackson, Kalamazoo, St. Joseph, Van 

Buren, Washtenaw 

Mississippi Itawamba, Monroe, Prentiss 

Ohio Portage 

Virginia Floyd 

a County-level species range derived from USFWS ECOS (accessed 8/10/2021). County-level corn acres 

derived from USDA AgCensus, 2017.

Species range refinement 

MSB is currently believed to exist in 9 counties between Michigan and Indiana in fens ranging in 

size between 0.5 and 130Ha for a total of 504 Ha. At the time of listing, MSB was known to exist 

in a single county in Ohio, but no extant populations were identified in surveys spanning 1985-

1990 and as of 2013, the species is believed to be extirpated from Ohio. Twenty-eight sites are 

currently occupied by MSB in Alabama, most within the protected lands of the Oakmulgee Ranger 

District of the Talledega National Forest. Previous surveys identified 11 extant colonies withing 

the protected Natchez Trace Parkway in Mississippi and 17 sites within the Blue Ridge Mountains 

region of Virginia (USFWS, 2021b). 

The range maps for MSB provided by USFWS are at the county level and thus, no further 

refinement of the species range was conducted (Figure L-6). 

Biology and habitat 

The life cycle of MSB consists of a single annual brood where hatched larvae feed through the 

summer before entering diapause through the winter and resume feeding the following spring. In 

late May to early June, larvae form a chrysalis and adults emerge mid-June through late July. While 

females may oviposition on a variety of plant species, hatched larvae migrate to food plants 

including those that do not support full development. Full development and maturation are 

believed to be supported by the preferred host plant, Carex stricta, a sedge that serves as the 

primary food plant (USFWS, 2021b). 

C. stricta is restricted to sedge meadows, shallow marshes, fens, shores, streambanks, and ditches, 

consistent with the known habitat for MSB: 
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“Although MSB habitat requirements are not yet fully understood, the butterfly 

appears to be restricted to calcareous wetlands that range along a continuum from 

open fen, wet prairie, sedge meadow, shrub-carr, tamarack savanna, and numerous 

variations and combinations of these community types (Shuey 1997, Szymanski 

1999, Hyde et al. 2001). It appears that the MSB occupies areas in these fen 

communities where woody and herbaceous vegetation occurs as a mosaic 

(Szymanski and Shuey 2002). Important structural components of the habitat 

include presence of peat or muck soil (Shuey 1997), scattered deciduous shrubs or 

coniferous trees (Shuey 1997), seeps (McKinnon and Albert 1996) and a 

herbaceous community dominated by C. stricta. MSB habitat also appears to 

exhibit large variability in vegetative structure and composition at the habitat patch 

scale, suggesting the importance of habitat heterogeneity (Szymanski 1999). Recent 

research has further reinforced the importance of the edge component; in the later 

part of the adult flight period (i.e., during the time of oviposition), males and 

females tend to be found within one meter of a tree or shrub (Barton 2003).”

-NatureServe, 2018 

The perennial sedge, Carex stricta, also known as an upright or hummock sedge, is typically found 

in sedge meadows, shallow marshes, fens, shores, streambanks, and ditches. Leaves are typically 

2-6 mm in width (USDA, 2008b) extending relatively vertically from the base of the plant. Based 

on the plant architecture and relatively narrow leaf blades, levels of corn pollen retained on the 

leaf surface would be expected to be far less than those observed with common milkweed or 

artificial pollen traps (Pleasants et al., 2001) thus further minimizing potential exposure to MSB 

larvae. 

While MSB populations can be found in counties that have corn production, MSB exhibits 

preference for sedge grasses and wetland habitats that are not expected to be amenable to corn 

production. Furthermore, several of the populations currently exist within protected lands. 

Consistent with this, USDA concluded for MON 89034 corn that there would be no effect on MSB 

due to a lack of proximity of habitat to cultivated corn (USDA, 2008a). Given the habitat and host 

plant preferences of MSB, no significant exposure to MON 95379 pollen is expected. 

b. St. Francis’ satyr butterfly

Species Range 

The current range for St. Francis’ satyr butterfly (SFSB) is limited to counties within the state of 

North Carolina (Table L-7). While the current species range overlaps counties with corn 

production, the species is not present in Hawaii, Iowa, or Nebraska where MON 95379 is intended 

for cultivation. 

 
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Table L-7. County-level overlap of corn acres with St. Francis’ satyr butterflya

State County 

North Carolina Cumberland, Harnett, Hoke, Moore a

County-level species range derived from USFWS ECOS (accessed 8/10/2021). County-level corn acres 

derived from USDA AgCensus, 2017. 

Species Range Refinement 

As of 2020, no populations have been observed outside of Department of Defense lands at the Ft. 

Bragg Army installation since the time of listing despite extensive surveys. To confirm anticipated 

minimal exposure of SFSB to cultivated corn, an overlap analysis was conducted at the sub-county 

level using the refined current USFWS species range for SFSB (Figure L-7). The landscape of the 

current SFSB range is predominantly forest, woody wetlands, and developed land covers 

associated with the military installation. Analysis of the CDLs within the species range indicate 

that corn represents approximately 0.3% of the land use and is limited to the very outer edges of 

the expected species range (Table L-8 Figure L-7). The results of the analysis confirm minimal 

overlap between the SFSB species range and cultivated corn. 

 
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Table L-8. Refined Overlap of the Corn CDL and the Current Species Range of SFSB 

CDL ID Value Category Acreage 

142 Evergreen Forest 124908.4 

190 Woody Wetlands 20729.2 

121 Developed/Open Space 18156.7 

122 Developed/Low Intensity 12096.7 

131 Barren 10606.9 

152 Shrubland 10075.4 

176 Grass/Pasture 3181.4 

141 Deciduous Forest 2435.9 

123 Developed/Medium Intensity 2039.1 

143 Mixed Forest 2035.1 

5 Soybeans 1662 

37 Other Hay/Non Alfalfa 1615.5 

111 Open Water 1584.1 

1 Corn 687.2 

61 Fallow/Idle Cropland 578.2 

2 Cotton 116.5 

195 Herbaceous Wetlands 106.7 

124 Developed/High Intensity 79.2 

59 Sod/Grass Seed 75.8 

26 Dbl Crop WinWht/Soybeans 71.8 

27 Rye 34.2 

24 Winter Wheat 31.6 

44 Other Crops 24 

29 Millet 18 

4 Sorghum 12.9 

28 Oats 7.1 

10 Peanuts 5.6 

53 Peas 5.6 

46 Sweet Potatoes 5.1 

67 Peaches 4.2 

11 Tobacco 3.1 

69 Grapes 3.1 

242 Blueberries 1.3 

47 Misc Vegs & Fruits 1.1 

48 Watermelons 0.9 

74 Pecans 0.9 

50 Cucumbers 0.7 
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237 Dbl Crop Barley/Corn 0.4 

205 Triticale 0.2 

221 Strawberries 0.2 

226 Dbl Crop Oats/Corn 0.2 

236 Dbl Crop WinWht/Sorghum 0.2 

254 Dbl Crop Barley/Soybeans 0.2 

Total 213002.6

Biology and habitat 

The life cycle of the St. Francis’ satyr butterfly (SFSB) consists of two brood cycles per season 

where the first flight adults emerge late May to early June and then again in July through early 

August. The last brood overwinters in the larval stage before resuming feeding and completing 

development in the spring. While caterpillars have been difficult to identify in surveys, the two 

that have been found have been associated with the presumed preferred host plant sedge, Carex 

mitchelliana. The species is generally sedentary, and any movement would be expected to be 

between proximal subpopulations (USFWS, 2020). 

The preferred habitat for SFSB is described as: 

“The habitat occupied by this satyr consists primarily of wide wet meadows 

dominated by a high diversity of sedges (Carex spp.) and other wetland graminoids. 

In the North Carolina sandhills, such meadows are often relicts of beaver activity. 

Saint Francis’ satyr has also been observed in pitcher plant (Sarracenia flava) 

swales, with cane (Arundinaria tecta), and with the rare plants rough-leaved 

loosestrife (Lysimachia asperulaefolia, federally listed as endangered) and pocosin 

lily (Lilium iridollae, a species of Federal concern). It is, however, unknown 

whether the satyr uses such swale habitat for feeding, breeding, and perching, or 

simply as a dispersal corridor. Unlike the habitat of Mitchell’s satyr, the North 

Carolina species’ habitat cannot properly be called a fen because the waters of this 

sandhills region are extremely poor in inorganic nutrients (USFWS, 1996). Known 

only from a few sedge wetlands in close proximity. Habitat apparently open 

seepage areas dominated with Carex. Habitat is successional or disclimax with 

both beaver and fires being apparently critical factors in maintaining it 

(Natureserve, 2015). Clumped spatial arrangement and narrow environmental 

specificity are based on specific habitat requirements of the species as are high 

ecological integrity and site fidelity and low tolerance range. (USFWS, 1996; 

NatureServe, 2015).” 

-NatureServe, 2015 

The preferred host plant for SFSB larvae is not currently known. Surveys and limited captive 

rearing have shown SFSB larvae to feed on Carex spp. including Carex mitchelliana and Carex 
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atlantica. USFWS notes that it is possible for larvae to feed on more than one host plant over the 

course of each year due to the fact that C. mitchelliana senesces relatively later in the summer 

compared to other Carex species (USFWS, 2020). Carex spp. leaves are typically 2-6 mm in width 

and extend relatively vertically from the base of the plant (USDA, 2008b). Based on the plant 

architecture and relatively narrow leaves, levels of deposited and retained pollen on the leaf surface 

would be expected to be far less than those observed with common milkweed or artificial pollen 

traps (Pleasants et al., 2001), thus further minimizing potential exposure to SFSB larvae. 

While SFSB populations can be found in counties that have corn production, SFSB exhibits 

preference for sedge grasses and wetland habitats that are not expected to be amenable to corn 

production. Furthermore, all populations currently exist within DoD lands with minimal overlap 

to commercial corn cultivation. Consistent with this, USDA concluded for MON 89034 corn that 

there would be no effect on SFSB due to a lack of proximity of habitat to cultivated corn (USDA, 

2008a). Considering critical habitat and host plant preferences of SFSB, no significant exposure 

to MON 95379 pollen is expected. 

c. Poweshiek skippering 

Species Range 

The range for Poweshiek skipperling (PS) provided by a query of the USFWS ECOS database is 

limited to counties within the states of Iowa, Michigan, Minnesota, North Dakota, South Dakota, 

Wisconsin (Table L-9). 

Table L-9. County-level overlap of corn acres with Poweshiek skipperlinga

State County 

Iowa Cerro Gordo, Dickinson, Emmet, Hancock, Howard, Kossuth, Osceola 

Michigan Hillsdale, Jackson, Lenawee, Livingston, Oakland, Washtenaw

Minnesota Polk 

North Dakota Richland, Sargent 

South Dakota Brookings, Clark, Codington, Day, Deuel, Grant, Marshall, Roberts 

Wisconsin Green Lake, Waukesha a County-level species range derived from USFWS ECOS (accessed 8/10/2021). County-level corn acres 

derived from USDA AgCensus, 2017.

 
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Species Range Refinement 

As part of the 2018 5-year review published by USFWS for the Poweshiek skipperling, the 

updated species range for each state identified in the ECOS database is as follows: 

Michigan: 

“All six sites where the Poweshiek skipperling was considered extirpated at the 

time of listing are still considered extirpated...Five Michigan sites are currently 

classified as present, out of the nine that had present status at the time of listing. 

However, the numbers of individuals detected at these remaining present sites have 

decreased since listing, with high daily counts of 1, 2, and 9 individuals in 2018 at 

Halstead Lake Fen, Holly Fen (Brandt Road), and Buckhorn Lake (Big Valley), 

respectively. The stronghold of Long Lake Fen (2 sites, including the Eaton Road 

site) has not had a high daily count above 49 since listing, with a high daily count 

of 28 in 2018 (compared to multiple counts in the hundreds in the five years 

preceding listing). Similarly, numbers are down for the maximum number of 

Poweshiek skipperlings observed per minute at these sites relative to the recent 

years before listing (Figure L-5). Belitz and colleagues (2019, p. 645) estimated 

the adult Poweshiek abundance in Michigan prairie fens to be 231 (95% CI 160-

332). Furthermore, no additional sites have been found, even though a habitat 

model identified approximately 33 sites that may have significant potential to be 

inhabited by Poweshiek skipperling. Of the potential sites surveyed thus far, no new 

Poweshiek skipperling sites have been found (MNFI 2017, unpublished).”

-USFWS, 2018b 

Wisconsin: 

“At the time of listing, there were three sites with unknown occupancy and one site 

where Poweshiek skipperling were present. The three sites with previously 

unknown occupancy are now all considered extirpated. The site with Poweshiek 

skipperling presence, Puchyan Prairie, is still considered to be present. Since 2012, 

no more than three Poweshiek skipperlings have been observed in a given year at 

that site. In both 2017 and 2018, there was one individual sighted, however no 

photo documentation confirms these sightings.”

-USFWS, 2018b 

Minnesota: 

“Poweshiek skipperling was once widespread and abundant in Minnesota; 

however there have been no confirmed sightings of the species in the state since 

2007 (U.S. Fish and Wildlife Service 2019, unpub. data). One unconfirmed sighting 

in 2013 occurred at a prairie complex owned and managed primarily by the 

Minnesota Department of Natural Resources (MNDNR) in the Chicog Wildlife 
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Management Area (WMA). This area has had recent adult observations over 

multiple years (2004-2007, and unconfirmed in 2013). Follow-up surveys since in 

2014 and 2016 resulted in no detections of the species at Chicog WMA (MNDNR 

2017, unpub. data).”

-USFWS, 2018b 

Iowa, North Dakota, South Dakota: 

“Since the time of listing, there have been no sightings in Indiana, Illinois, Iowa, 

North Dakota, and South Dakota. There are no sites where the Poweshiek 

skipperling is currently considered present in those states.”

-USFWS, 2018b 

Since the Minnesota observations are unconfirmed and located within wildlife management areas, 

additional refinement on the current range for PS was conducted by focusing on the two states 

where PS is still considered present: Michigan and Wisconsin. The current species range was 

recently confirmed in a published draft Biological Opinion for Malathion that indicates PS is now 

only present in two states (USFWS, 2021). The species range maps for Michigan and Wisconsin 

correspond to areas designated as critical habitat in these states. The 2018 5-year review was used 

to screen for critical habitat units within Michigan and Wisconsin where PS was still believed to 

be present (Table L-10). 

Table L-10. Locations of Current Species Range for PS in Michigan and Wisconsin 

State Unit Name Status1 

MI 1 Holly Fen Present 

MI 2 Halstead Lake Present 

MI 3 Long Lake Present 

MI 4 Buckhorn Lake Present 

MI 5 Bullard Lake Extirpated 

MI 6 Park Lydon Unknown 

MI 7 Goose Creek Unknown 

MI 8 Liberty Fen Extirpated 

MI 9 Liberty Bowl Fen Possibly Extirpated 

WI 1 Waukesha Co Present 

WI 2 . Green Lake Co. Extirpated 

1

Current species statues provided by USFWS in the 2018 5-year review for Poweshiek 

skipperling (USFWS, 2018b). 

 
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To quantitatively refine the potential overlap between corn acres and the current PS species range, 

each critical habitat unit where PS was either present, unknown, or possibly extirpated was queried 

within the USDA CropScape against the 2020 CDLs (Figures L-8 – L-16). When the CDL layers 

were evaluated, the total corn acres accounted for 0.3% of the critical habitat acres in Michigan 

and Wisconsin indicating minimal exposure of PS to cultivated corn (Table L-11). 

Table L-11. Refined Overlap of the Corn CDL and the Current Species Range of PS 

State Unit CDL ID Value Category Acreage 

MI 1 121 Developed/Open Space 1.1 

MI 1 141 Deciduous Forest 5.6 

MI 1 190 Woody Wetlands 19.6 

MI 2 1 Corn 0.2 

MI 2 61 Fallow/Idle Cropland 0.2 

MI 2 111 Open Water 3.6 

MI 2 121 Developed/Open Space 0.7 

MI 2 141 Deciduous Forest 4.2 

MI 2 143 Mixed Forest 0.4 

MI 2 190 Woody Wetlands 60.7 

MI 2 195 Herbaceous Wetlands 1.3 

MI 3 1 Corn 1.3 

MI 3 5 Soybeans 0.7 

MI 3 36 Alfalfa 0.2 

MI 3 61 Fallow/Idle Cropland 2 

MI 3 111 Open Water 50.3 

MI 3 121 Developed/Open Space 4.2 

MI 3 122 Developed/Low Intensity 1.3 

MI 3 141 Deciduous Forest 79.4 

MI 3 143 Mixed Forest 0.4 

MI 3 176 Grass/Pasture 2.7 

MI 3 190 Woody Wetlands 258.9 

MI 3 195 Herbaceous Wetlands 2.4 

MI 4 1 Corn 0.9 

MI 4 4 Sorghum 0.2 

MI 4 61 Fallow/Idle Cropland 1.1 

MI 4 111 Open Water 15.1 

MI 4 121 Developed/Open Space 2.9 

MI 4 122 Developed/Low Intensity 0.2 

MI 4 141 Deciduous Forest 48 

MI 4 176 Grass/Pasture 4.4 

MI 4 190 Woody Wetlands 198.2 

MI 4 195 Herbaceous Wetlands 1.1 

MI 6 61 Fallow/Idle Cropland 0.2 

MI 6 111 Open Water 12.7 

MI 6 141 Deciduous Forest 26 

MI 6 143 Mixed Forest 0.7 

MI 6 176 Grass/Pasture 9.3 

MI 6 190 Woody Wetlands 211.5 
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MI 6 195 Herbaceous Wetlands 0.9 

MI 7 1 Corn 1.3 

MI 7 5 Soybeans 0.7 

MI 7 61 Fallow/Idle Cropland 1.1 

MI 7 111 Open Water 0.2 

MI 7 121 Developed/Open Space 0.7 

MI 7 122 Developed/Low Intensity 0.9 

MI 7 141 Deciduous Forest 2.4 

MI 7 143 Mixed Forest 0.2 

MI 7 176 Grass/Pasture 4 

MI 7 190 Woody Wetlands 111.4 

MI 7 195 Herbaceous Wetlands 0.2 

MI 9 141 Deciduous Forest 0.9 

MI 9 190 Woody Wetlands 33.8 

WI 2 141 Deciduous Forest 6.9 

WI 2 143 Mixed Forest 0.2 

WI 2 190 Woody Wetlands 2.2 

WI 2 195 Herbaceous Wetlands 188.6 

Total 1390.5 

Biology and habitat 

PS have a single flight per year that occurs middle of June through the middle of July. The flight 

period for PS individuals lasts one to two weeks where mating and egg laying occurs. Eggs 

typically hatch late summer whereby larvae feed and develop through early fall. Larvae then 

overwinter before completing development in the spring. 

The historical habitat for PS has been described as prairie grasslands and meadows where there 

preferred host plants would be found. Only 4% of the original native tall grass prairies remain in 

small isolated sites that limit movement of PS. Presently, the known remaining PS populations are 

present on either protected conservation lands or wetland environments in Michigan and 

Wisconsin. 

“The disjunct populations of Poweshiek skipperlings in Michigan have more 

narrowly defined habitat preferences, variously described as wet marshy meadows 

(Holzman 1972, p. 114), bog fen meadows or carrs (Shuey 1985, p. 181), sedge 

fens (Bess 1988, p. 13), and prairie fens (Michigan Natural Features Inventory 011, 

unpubl. data; Michigan Natural Features Inventory 2012, unpubl. data). Bess 

(1988, p. 13) found the species primarily in the drier ortions of Liberty Fen, Jackson 

County, dominated by ‘‘low sedges’’ and an abundance of nectar sources. 

Summerville and Clampitt (1999, p. 231) noted that the population was 

concentrated in areas dominated by spikerush and that only 10–15 percent of the 

fen area was occupied despite the abundance of nectar sources throughout. 

Poweshiek skipperling have been described as occupying peat domes within larger 

prairie fen complexes in areas either dominated by mat muhly or prairie dropseed 

(Cuthrell 2013a, pers. comm.)... Puchyan Prairie consists of wet-mesic prairie that 
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grades lower into sedge meadow (WI DNR Web site http://dnr.wi.gov/topic/ 

Lands/naturalareas/ index.asp?SNA=172; Swengel 2013, pers. comm.) and adult 

Poweshiek Skipperlings have been observed in wet prairie there, although it is not 

known if these areas function as successful larval habitat (Swengel 2013, pers. 

comm.). Like the Dakota skipper, it has been hypothesized that Poweshiek 

skipperling larvae may be vulnerable to desiccation during dry summer months 

(Borkin 2012a, pers. comm.) and require movement of shallow groundwater to the 

soil surface or wet low areas to provide relief from high summer temperatures or 

dry conditions (Royer et al. 2008, pp. 2, 16; Borkin 2012a, pers. comm.).”

-NatureServe, 2021

As described above, the habitats represented in the current range of PS are described principally 

as fens or wetland prairies, which would not be suitable for cultivation of corn, and this conclusion 

is consistent with the results of the refined overlay analysis. 

Larvae feed on native grass species, however little bluestem (Schizachyrium scoparium) is a 

preferred food source. Grass species such as little bluestem provide the larvae with a dense cluster 

of erect blades in proximity to an abundance of edible leaf tissue (USFWS, 2018b). Little bluestem 

is a warm-season perennial grass with broad distribution in the U.S. and Canada. Leaf blades are 

relatively vertically positioned, 5-30 cm long and 1.5-6 mm wide (USDA, 2013). Based on the 

plant architecture and relatively narrow leaves, levels of deposited and retained pollen on the leaf 

surface would be expected to be far less than those observed with common milkweed or artificial 

pollen traps (Pleasants et al., 2001), thus further minimizing potential exposure to PS larvae. 

While PS populations can be found in counties with reported corn acreage, the current known 

species range is limited to Michigan and Wisconsin in wetland habitats that are not expected to be 

in close proximity to corn production. Therefore, given the current species range, preferred habitat 

and host plant specificity, no significant exposure to MON 95379 pollen is expected. 

d. Karner blue butterfly 

Species Range 

The current range for Karner blue butterfly (KBB) is limited to counties within the states of Illinois, 

Indiana, Michigan, Minnesota, New Hampshire, New York, Ohio, and Wisconsin (Table L-12). 

Based on the 2017 AgCensus, no corn acres were produced for grain in Merrimack county, NH. 

Merrimack county is the only county in NH where KBB is believed to be present and thus the state 

was excluded from the county-level overlap table. While the current species range overlaps in 

counties with corn production at the national level, the species is not currently found in Hawaii, 

Iowa, or Nebraska where MON 95379 is intended for cultivation. 

 
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Table L-12. County-Level Overlap of Corn Acres with Karner Blue Butterflya

State County 

Illinois Lake 

Indiana Lake, Porter 

Michigan Allegan, Ionia, Kent, Lake, Mason, Mecosta, Monroe, Montcalm, 

Newaygo, Oceana 

Minnesota Winona 

New York Albany, Saratoga, Schenectady 

Ohio Lucas 

Wisconsin Adams, Barron, Burnett, Chippewa, Clark, Columbia, Dane, Dunn, 

Eau Claire, Green Lake, Iowa, Jackson, Marquette, Menominee, 

Monroe, Oconto, Portage, Richland, Sauk, Shawano, St. Croix, 

Vernon, Waupaca, Waushara, Wood a

County-level species range derived from USFWS ECOS (accessed 8/10/2021). County-level 

corn acres derived from USDA AgCensus, 2017. 

Species range refinement 

In the 2019 5-year review for KBB, USFWS notes of the 8 states with KBB at the time of listing 

in 1992, KBBs are likely no longer present in Illinois, Minnesota, and Indiana (USFWS, 2019). 

The current range maps for this species are at the county level for the largest population in 

Wisconsin and therefore, no sub-county refinement could be conducted (Figure L-17). 

Biology and habitat 

The potential for risk to KBB as a result of exposure to Bt corn has been previously assessed 

(Mendelsohn et al., 2003; Peterson et al., 2006). KBBs are found along the northern range of wild 

lupine, comprising parts of Wisconsin, Michigan, Minnesota, Indiana, New Hampshire and New 

York where packed snow occurs late into the spring (Peterson et al., 2006; US EPA, 2001b; 

USFWS, 2003). Wisconsin supports the largest population of KBB, which is found in 25 

Wisconsin counties. There are two complete generations of KBB per year. Larvae from the firstgeneration hatch from eggs in mid-April, well before corn pollen shed in these geographic areas. 

The adults emerge in late May to early June. Larvae from the second-generation feed through midJuly and the adults emerge through mid-August. Eggs from the second generation are the 

overwintering stage. 

Wild lupine is the only known host for KBB larvae. Wild lupine grows on dry, sandy soils in pine 

barrens, oak savannahs, and disturbed habitats such as forest trails, roadsides, military training 
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areas, airports, highway corridors, sand roads and abandoned sand pits (USFWS, 2003; Peterson 

et al., 2006). The preferred habitat of KBB is best described as broken or scattered tree canopies 

that vary from 0 to 50% and up to 80% canopy cover, with a mixture of grasses and forbs in the 

open areas. Compared to milkweed, the leaflets of wild lupine are relatively small; typically 2” 

long and only half an inch wide (Minnesota Wildflowers, 2021). Therefore, the pollen deposition 

rates based on milkweed leaves is likely a highly conservative estimate of exposure to KBB larvae 

feeding on wild lupine. Habitat destruction, through development and land practices, is the major 

cause of the decline of KBB populations (USFWS, 2003). 

Wild lupine does not occur in corn fields. Data from Peterson et al. (2006) show that <10% of all 

lupine sites are even within 100 meters of a corn field and indeed, approximately 80% of all lupine 

sites are more than 500 meters from a corn field. There is also evidence that many extant KBB 

populations are spatially separated from production agriculture areas by large bodies of water, 

urban areas or publicly owned land. \ The Wisconsin Department of Natural Resources has 

concluded: "Most agricultural operations do not appear to support habitat for the Karner blue 

butterfly or present a threat to the continued existence or recovery of the Karner blue butterfly in 

Wisconsin." (Wisconsin Dept. Natural Resources, 2010). This assertion is supported by the recent 

work of Peterson et al. (2006). 

KBB larvae use wild lupine exclusively as a host plant; therefore, the only route of exposure of 

Cry1B.868 and Cry1Da_7 to Karner blue larvae is through deposition of pollen from corn 

MON 95379 onto lupine plants (USFWS, 2003; Peterson et al., 2006). In order for KBB larvae to 

be exposed to pollen from MON 95379, lupine plants must first be in and around corn fields and, 

more importantly, pollen shed must occur onto the lupine at the time KBB larvae are feeding. In 

the northern corn maturity zones where KBB is present, corn plants (field corn, sweet corn or 

popcorn) typically shed pollen after mid to late July or early August (USFWS, 2003). Thus, even 

if wild lupine plants occurred in or close to corn fields, it is highly unlikely that corn pollen would 

be present at the time KBB larvae are actively feeding. Degree-day effects that might result in 

earlier or later pollen shed would also tend to correspondingly affect the rate of KBB development, 

and therefore not contribute to any greater likelihood of Bt pollen exposure. An analysis based on 

degree-day modeling of KBB butterfly phenology and corn pollen shed (Peterson et al., 2006) 

showed that there are only two Wisconsin locations (counties) in the Midwestern corn belt where 

there is a potential temporal overlap between KBB larvae and corn pollen shed. However, there 

was no evidence that KBB larvae were exposed to corn pollen in these two locations (Peterson et 

al., 2006). Based on the lack of overlap of critical habitat and host plants in proximity to corn 

fields, significant exposure of MON 95379 to KBB is not anticipated. 

 
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e. Dakota skipper 

Species Range 

The current range for the Dakota skipper (DS) is limited to counties within the states of Minnesota, 

North Dakota, and South Dakota (Table L-13). While the current species range overlaps in 

counties with corn production, the species is not currently found in Hawaii, Iowa, or Nebraska 

where MON 95379 is intended for cultivation. 

Table L-13. County-Level Overlap of Corn Acres with Dakota Skippera

State County 

Minnesota Big Stone, Chippewa, Clay, Lac qui Parle, Lincoln, Norman, 

Pipestone, Stevens, Swift, Traverse 

North Dakota Barnes, Benson, Billings, Bottineau, Burke, Burleigh, Cass, Dickey, 

Divide, Dunn, Eddy, Emmons, Foster, Griggs, Kidder, Lamoure, 

Logan, McHenry, McKenzie, McLean, Mercer, Morton, Mountrail, 

Nelson, Oliver, Pierce, Ramsey, Ransom, Renville, Richland, Rolette, 

Sargent, Sheridan, Stark, Steele, Stutsman, Traill, Ward, Wells, 

Williams 

South Dakota Brookings, Brown, Clark, Codington, Day, Deuel, Edmunds, Grant, 

Hamlin, Kingsbury, Lake, Marshall, McCook, McPherson, 

Minnehaha, Moody, Roberts, Spink, Turner a

County-level species range derived from USFWS ECOS (accessed 9/22/2021). County-level 

corn acres derived from USDA AgCensus, 2017. 

Species range refinement 

The states of Minnesota, North Dakota, and South Dakota contain the only known remaining 

populations of Dakota skipper in the United States. An overlap analysis was conducted with the 

UDL for corn and the critical habitat and species range for DS within the FESTF Gopher utility. 

Based on the results of the overlap analysis, 12.7% of the species range and 2.2% of the DS critical 

habitat overlaps with corn acres (Figure L-18 and L-19). 

Biology and habitat 

DS have a single flight per year that occurs middle of June through the end of July. The flight 

period for DS individuals lasts two to four weeks where mating and egg laying occurs. Flight 

distances are relatively limited as DS are believed to not travel more than 1km between habitat 

patches. Eggs are laid on broadleaf plants and grasses. Eggs typically hatch within 14 days 

whereafter larvae crawl to the base of grass plants at the soil surface. Larvae develop to 4th or 5th

instars where they then overwinter before completing development in the spring. 
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Larvae feed on native grass species, but little bluestem (Schizachyrium scoparium) is a preferred 

food source. Grass species such as little bluestem provide the larvae with a dense cluster of erect 

blades in proximity to an abundance of edible leaf tissue (USFWS, 2018a). Little bluestem is a 

warm-season perennial grass with broad distribution in the U.S. and Canada. Leaf blades are 

relatively vertically positioned, 5-30 cm long and 1.5-6 mm wide (USDA, 2013). Based on the 

plant architecture and relatively narrow leaves, levels of deposited and retained pollen on the leaf 

surface would be expected to be far less than those observed with common milkweed or artificial 

pollen traps (Pleasants et al., 2001), thus further minimizing potential exposure to DS larvae. 

DS habitat can be classified into one of two types. Type A habitat consists of low wet-mesic prairie 

that occurs on near-shore glacial lake deposits. This type of habitat common in North Dakota and 

parts of South Dakota may be subject to flooding and is dominated by bluestem grasses. The 

second type of habitat (Type B) can be found in Minnesota and occurs on rolling terrain over 

gravelly glacial moraine deposits. Present-day populations of DS are now found in remnant nativeprairie habitats that have soils typically unsuitable for agriculture and steep topography (USFWS, 

2018a). Both factors would be expected to significantly limit exposure of DS larvae to cultivated 

corn fields and is consistent with the minimal overlap between DS critical habitat and corn acres. 

Therefore, significant exposure of DS to MON 95379 is not anticipated. 

L.21.5. Conclusion

Under the proposed EPA registration, cultivation of MON 95379 is limited to a combined total of 

100 acres per growing season in Nebraska, Hawaii, or Iowa. An overlay analysis at the county 

level comparing areas of corn cultivation with the most current species range information for the 

five listed lepidopteran species of interest confirm that these species are not present within the 

intended states of cultivation. Quantitative characterization of risk at the field edge indicate 

adverse effects would not be anticipated outside at distances of 6 m or greater from the field edge, 

but it should be noted that this characterization relied on highly conservative assumptions for 

protein expression, pollen deposition, and landscape distribution of host plants. The 

conservativeness of this assessment is further demonstrated by characterization of preferred host 

plant architectures indicating these host plants are much less likely to retain deposited pollen 

compared to common milkweed used in the field-edge characterization model. The results of the 

field-edge assessment when placed into context of the intended product use indicate that typical 

agronomic practices used to generate high quality seed in breeding and seed increase activities 

permitted under the proposed EPA registration would limit off-field pollen flow and thus be 

protective of listed Lepidoptera. Analyses of current species range maps, species biology and 

critical habitats support the conclusion that these species occupy habitat landscapes distinct from 

areas of corn production. Therefore, the above considerations in addition to the intended use for 

MON 95379 corn are expected to be protective of listed Lepidoptera. 
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L.21.6. Refined Species Range Maps 

Figure L-6. Current Species Range for MSB (accessed 9/27/2021) 

Current species range is indicated by blue polygons and the CDL for corn is indicated in yellow. 
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Figure L-7. Current Species Range for SFSB (accessed on 9/24/2021) 

Current species range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-8. Current National-Level Species Range for Poweshiek Skipperling (accessed 9/27/2021) Historical and current species ranges are indicated by blue polygons and the CDL for corn is indicated in yellow. Species is no longer 

present in IA, ND, and SD (USFWS, 2018).
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Figure L-9. Michigan Unit 1 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021) The specified critical habitat range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-10. Michigan Unit 5 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021) The specified critical habitat range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-11. Michigan Unit 3 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021) The specified critical habitat range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-12. Michigan Unit 4 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021) The specified critical habitat range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-13. Michigan Unit 6 Critical Habitat for Poweshiek Skipperling (9/23/2021) The specified critical habitat range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-14. Michigan Unit 7 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021) The specified critical habitat range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-15. Michigan Unit 9 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021) The specified critical habitat range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-16. Wisconsin Unit 2 Critical Habitat for Poweshiek Skipperling (accessed 9/23/2021) The specified critical habitat range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-17. Current Species Range for Karner Blue Butterfly (accessed 9/27/2021) The current species range is indicated by blue polygons and the CDL for corn is indicated in yellow.
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Figure L-18. Current Critical Habitat Range for Dakota Skipper (FESTF Gopher ;accessed 9/28/2021) 

Range for DS critical habitat is indicated in purple and Use Data Layer (UDL) for Corn is indicated in green. Data Source: NatureServe and its natural heritage member programs. 
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Figure L-19. Current Species Range for Dakota Skipper (FESTF Gopher; accessed 9/28/2021) 

Species range for DS is indicated in purple and Use Data Layer (UDL) for Corn is indicated in green. Data Source: NatureServe and its natural heritage member programs.
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