Page 2 of 29

Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) meeting held on January 28, 

2014.

CONCLUSION: 

The Monsanto Company submitted product characterization data in support for a seed increase Sec. 3 

Registration of MON 87411. MON 87411 will not be offered for commercial use as a stand- alone 

product, but will be combined, through traditional breeding methods, with other registered corn events 

to provide protection against both above-ground and below ground maize pests, as well as tolerance to 

herbicides. The new PIP technology expresses the Bt Cry3Bb1 protein and DvSnf7 dsRNA to control the 

coleopteran corn rootworm (CRW) pests. MON 87411 contain a suppression cassette that expresses an 

inverted repeat sequence designed to match the sequence of western corn rootworm (WCR; Diabrotica 

virgifera virgifera). The expression of the suppression cassette results in the formation of a double 

stranded RNA (dsRNA) transcript. Upon consumption, the plant-produced dsRNA is recognized by the 

CRW’s RNA interference (RNAi) machinery resulting in the down-regulation of the targeted Snf7 gene 

expression and eventual CRW mortality. The Cry3Bb1 protein present in MON 87411 has over 99% 

amino acid identity to the Cry3Bb1 protein produced in MON 863 and the deduced amino acid sequence 

is identical to that produced from the expression cassette present in MON 88017. Since the protein is

identical to those in the currently registered PIP product (MON 88017), several comparative studies 

were submitted to demonstrate the biochemical and functional proteins equivalence of Cry3Bb1 and 

CP4 EPSPS in PIP MON 87411 maize to the corresponding Cry proteins expressed individually in PIP 

event MON 88017. 

In addition, the toxicity data from a 90 day toxicity study (MRID 49505806) with corn grain from MON 

87411 at up to 33% in the diet (Mertens, 2013) and a 28 day toxicity study (MRID 49505804) with 

purified DvSnf7 at doses up to 100 mg/kg (Wahle, 2014) did not indicate adverse effects in the animals 

receiving test diet or test substance. The results of a 28 day oral toxicity study indicate that DvSnf7 is 

not toxic to mammals after 28 consecutive days of repeat oral exposure. Collectively, these results show 

the absence of any dietary hazard associated with DvSnf7 RNA at very high doses. 

The submitted data included: 28 day oral study in mice; 90 day feeding study in rats; Southern blot 

analyses for confirmation of the molecular identity; amino acid sequence analyses; Western blot 

analyses for determination of the protein molecular weights, immunoreactivity, functional activity and 

glycosylation status; Northern blot analyses for determination of molecular equivalence and functional 

activity, bioinformatics review for allergenicity, and ELISA analyses of protein expression levels in 

various plant tissues at key developmental growth stages. In addition, information was submitted to the 

Agency in order to address the 75-day deficiency letter sent to Monsanto, based on recommendations in 

the report from the EPA’s FIFRA SAP meeting held on January 28, 2014.

The Agency has reviewed these data and found them ACCEPTABLE. Collectively, the results support 

the physicochemical and functional equivalence for the Cry3Bb1 and CP4 EPSPS proteins expressed in 

MON 87411 maize to the corresponding proteins expressed in PIP MON 88017. 

The data submitted and reviewed for Cry3Bb1and CP4 EPSPS proteins as expressed in MON 87411

corn also support their inclusion in the existing exemptions from the requirement of tolerance for 

Bacillus thuringiensis Cry3Bb1 [40 CFR § 174.518] protein, when used as a PIP in food and feed 

commodities of corn; and the CP4 EPSPS protein [40 CFR § 174.523], when used as a PIP inert 

ingredient in all feed/food commodities.
Page 3 of 29

In conclusion, there is a reasonable certainty that no harm will result from aggregate exposure to the 

U.S. population, including infants and children, to the dsRNA transcript comprising a DvSnf7 inverted 

repeat sequence and the Cry3Bb1 protein and the genetic material necessary for its production in MON 

87411 corn. This includes all anticipated dietary exposures and all other exposures for which there is 

reliable information. The Agency has arrived at this conclusion because no toxicity to mammals has

been observed, nor any indication of allergenicity potential for a plant-incorporated protectant

(Cry3Bb1) that has been confirmed as sufficiently equivalent to currently registered plant-incorporated 

protectants. 

RECOMMENDATION: 

The submitted product characterization and toxicity data are sufficient to support Monsanto’s

application for a seed increase Sec. 3 Registration of MON 87411 maize as a plant-incorporated 

protectant.
Page 4 of 29

DATA REVIEW RECORD

Active Ingredients: DvSnf7 dsRNA and Bacillus thuringiensis (Bt) Cry3Bb1 insecticidal 

crystal protein and the genetic material necessary for their production in

Plant-Incorporated Protectant MON 87411 maize

Product Name: MON 87411 corn [EPA Reg. No 524-ARI; OECD Unique Identifier:

 MON-87411-9] 

Company Name: Monsanto Company

PC Code: 006566 and 006580

Decision Number: 487782 and 963404

DP Barcode: 420481, 424377, 426179 and 426500

Submission No. 947461, 960120, 964669 and 963404

MRID No. /Study Titles: 

49315101 Dietary Risk Assessment of the Cry3Bb1 and CP4 EPSPS Proteins and Exposure 

Assessment of the DvSnf7 Construct-Derived RNA from Consumption of MON 87411 

Maize in the U.S. (Amended MSL0024893) 

49315103 Amended Report for MSL0025048. Molecular Characterization of MON 87411. 

49315104 Assessment of DvSnf7 RNA Levels in Maize Tissues Collected from MON 87411 

Produced in Argentina Field Trials during 2011-2012.

49315105 Assessment Cry3Bb1 and CP4 EPSPS Protein levels in Corn Tissues Collected from 

MON 87411 Produced in Argentina Field Trials during 2011-2012.

49315106 Characterization of DvSnf7 RNA Extracted from MON 87411 and Comparison of 

Molecular and Functional Properties of Plant-Produced and in- vitro- Produced DvSnf7 

RNA. 

49315107 Characterization of Cry3Bb1 Protein from the Maize Grain of MON 87411 and 

Comparison of the Physicochemical and Functional Properties of Plant-Produced and E. 

coli- Produced Cry3Bb1 Proteins.

49315108 Characterization of CP4 EPSPS Protein from the Maize Grain of MON 87411 and 

Comparison of the Physicochemical and Functional Properties of Plant-Produced and E. 

coli- Produced CP4 EPSPS Proteins.

49315109 Bioinformatics Evaluation of DNA Sequences Flanking the 5” and 3” Junctions of 

Inserted DNA in MON 87411: Assessment of Putative Polypeptides

49505801 Monsanto Company (2014) Response to the Charge Questions Presented to the SAP 

Panel January 28, 2014 FIFRA SAP on RNAi Technology as a Pesticide: Problem 

Formulation for Human Health and Ecological Risk Assessment. 

49505804 A 28 day Oral Study in the Mouse with DvSnf7_ 968 RNA. 

49505806 A 90 day Feeding Study in Sprague Dawley rats with Grain from MON 87411 Corn. 

49553301 Response to Address EPA Request for Further Information Related to Human Health 

Considerations.

49553303 MON 88017 and MON 87411 Express Identical Cry3Bb1 Proteins.
Page 5 of 29

49553305 Bioinformatic Comparison of DvSnf7 versus a Human Transcriptome Using STELLAR.

49576101 Response to the Request for Additional Information for MRID Nos: 49505804 and 

49505806 (Re: Mon 87411)

BACKGROUND

Monsanto Company has developed a biotechnology- derived maize, MON 87411 (OECD unique 

identifier MON 87411-9), that confer protection against corn rootworm (CRW; Diabrotica spp.) and 

tolerance to the herbicide glyphosate. MON 87411 contain a suppression cassette that expresses an

inverted repeat sequence designed to match the sequence of western corn rootworm (WCR; Diabrotica 

virgifera, virgifera). The expression of the suppression cassette results in the formation of a double 

stranded RNA (dsRNA) transcript containing a 240 bp fragment of the WCR Snf7 gene (DvSnf7). Upon 

consumption by the CRW pest, the plant-produced dsRNA in MON 87411 is recognized by the CRW’s 

RNA interference (RNAi) machinery resulting in the down-regulation of the targeted DvSnf7 gene

leading to CRW mortality. MON 87411 also contains a cry3Bb1 coding sequence that produces a 

modified Bacillus thuringiensis (subsp. Kumamotoensis) Cry3Bb1 protein to protect against CRW larval 

feeding. In addition, MON 87411 contains the cp4 epsps coding sequence from Agrobacterium sp. 

strain CP4 that encodes for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein, which 

confers tolerance to glyphosate, the active ingredient in Roundup1® agricultural herbicides.

MON 87411 builds upon the current Bt protein- based mode-of-action (MOA) for CRW control by the 

addition of a new RNA-mediated MOA that offers enhanced control to target insect pests and prolonged 

durability of existing Bt technologies designed to control CRW. MON 87411 expresses the Cry3Bb1

protein and DvSnf7 dsRNA to control coleopteran corn rootworm (Diabrotica spp.) pests. The 

Cry3Bb1 protein belongs to a family of Cry proteins from B. thuringiensis that has been used 

commercially in the U.S. to produce microbial-derived products with insecticidal activity. The Cry3Bb1 

protein present in MON 87411 has over 99% amino acid identity to Cry3Bb1protein produced in MON 

863 and the deduced amino acid sequence is identical to that produced from the expression cassette 

present in MON 88017.

The following sections summarize the regulatory information for the currently registered PIP events that 

will be used to support this seed increase Sec. 3 Registration of MON 87411: 

Event MON 88017: The Cry3Bb1 protein produced in MON 88017 (EPA Reg No. 524-551), is a 

member of the Cry3Bb class of proteins that share >95% amino sequence homology. The Cry3Bb1

protein is also produced in MON 87411. The transformation plasmid

(PV-ZMIR10871), used to produce MON 87411, contains expression cassette for Cry3Bb1 protein that 

has the identical amino acid sequences to the Cry3Bb1 produced in MON 88017. The expression and 

identity of the Cry3Bb1 protein from this cassette was confirmed by SDS-PAGE and determination of 

the N-terminus (# amino acids sequenced) to be identical to corn event MON 863. The Cry3Bb1 protein 

does not exhibit similarity to known mammalian toxins or allergens, is readily digested in simulated 

 1 ®Roundup and Roundup Ready are registered Trademarks of Monsanto Technology LLC.
Page 6 of 29

gastric fluid and is not immunodetectable in corn grain after heat treatment (U.S. EPA, 2010b). The EPA

previously reviewed the safety assessment of the Cry3Bb1 protein and granted an exemption from the 

requirement of a tolerance in corn in 2004 (40 CFR §174.518). 

The Cry protein expressed in MON 87411corn is already covered by the existing exemption from the 

requirement of a tolerance for Cry3Bb1 [40 CFR § 174.518] protein when used as a plant-incorporated 

protectant in corn food/feed commodities. MON 87411 corn also expresses the CP4 EPSPS protein, 

which is also covered by the existing exemption from the requirement of a tolerance when used as plantincorporated protectant inert ingredient in all food commodities (40 CFR § 174.523). Therefore, the 

existing Federal Food, Drug and Cosmetic Act (FFDCA) Food Safety Assessments established for

Cry3Bb1 protein can be bridged to support the safety of the corresponding Cry protein residues in MON 

87411 feed/food corn commodities, as long as all data submitted in support for its registration are

reviewed as acceptable.

SUMMARY OF DATA SUBMITTED: 

PRODUCT CHARACTERIZATION

The Monsanto Company has developed corn event MON 87411 which express DvSnf7 dsRNA, 

Cry3Bb1 and CP4 EPSPS insecticidal proteins. MON 87411 contain a suppression cassette with an 

inverted repeat sequence DvSnf7 derived from Diabrotica virgifera. The expression of the DvSnf7

suppression cassette results in the formation of a double stranded RNA (dsRNA) transcript. Upon 

consumption, the plant-produced DvSnf7 dsRNA in MON 87411 is recognized by the CRW’s RNA 

interference (RNAi) machinery resulting in the down-regulation of the targeted Snf7 gene expression

and eventual CRW mortality. MON 87411 also produce a modified Bacillus thuringiensis (subsp. 

kumamotoensis) Cry3Bb1 protein to protect against CRW larval feeding. In addition, MON 87411 

produces the 5- enolpyruvylshikimate-3-phosphate synthase protein from Agrobacterium sp. Strain CP4 

(CP4 EPSPS), which confers tolerance to glyphosate. Cry3Bb1 is the same protein in the existing EPA 

registered PIP event MON 88017.

A. Transformation System

The DvSnf7 suppression cassette, cry3Bb1 and cp4 epsps genes were transferred into the genome of 

corn cells by Agrobacterium tumefaciens mediated transformation utilizing the plant transformation 

vector PV-ZMIR10871. The transformation vector, PV-ZMIR10871, used to develop MON 87411 is a 

vector that contains well-characterized DNA segments required for selection and replication of the 

plasmid vector in bacteria and transfer of the T-DNA into plant cells. The inserted T-DNA from this 

plasmid contains three gene cassettes.

1) The first cassette contains an inverted repeat sequence (named DvSnf7) derived from Diabrotica 

virgifera, the Western Corn rootworm (WCR). The WCR Snf7 ortholog (DvSnf7) is a component of 

the ESCRT-III complex (endosomal sorting complex required for transport), which is involved in 

essential biological processes including sorting of cell membrane receptors (Sweeney NT, et al, 
Page 7 of 29

2006; Vaccari T, et al, 2009; Kim DW, et al, 2011). Due to its vital cellular function resulting in 

WCR mortality at relatively low concentrations when targeted by dsRNA, DvSnf7 was selected as 

the target mRNA to investigate the molecular mechanisms leading to WCR mortality after feeding 

on dsRNA (Baum JA, et al, 2007). Once the dsRNA is ingested by WCR, it is thought to be taken up 

by midgut cells and processed by native RNAi machinery. The RNAi pathway is initiated by 

cleavage of dsRNA into short interfering siRNAs by the nuclease Dicer (Fire A, et al, 1998). The 

siRNAs then bind to a complex of proteins known as the RNA induced silencing complex (RISC) 

which leads to specific suppression of the target mRNA. The specific suppression can cause 

lethality if the target mRNA encodes a protein with an essential function within the insect. 

Dv49 dsRNA is 240 base pairs (bp) long. Dv49 siRNA is 21-24 mer. High molecular weight 

(HMW) northern blot showed all detectable Dv49 sequences are double stranded. Low molecular 

weight (LMW) northern blot showed siRNA production. The dsRNA with 240 bp is the active 

molecule that kills CRW in diet bioassay, however, it is processed to small interfering RNAs 

intracellularly which are biologically active. Single stranded and small siRNA are not active against 

CRW in diet bioassays. 

2) The second cassette contains cry3Bb1 is a modified Bacillus thuringiensis (subsp. kumamotoensis) 

protein to protect against corn rootworm larval feeding.

3) The third cassette produces the 5-enolpyruvylshikimate-3-phosphate synthase protein from

Agrobacterium sp. strain CP4 (CP4 EPSPS), which confers tolerance to glyphosate, the active 

ingredient in Roundup® agricultural herbicides. 

B. Characterization of the DNA Inserted in the Plant and Inheritance and Stability

MON 87411 was produced by Agrobacterium-mediated transformation of maize tissue using the 

transformation vector PV-ZMIR10871. This plasmid vector contains one T-DNA (transfer DNA), which 

is delineated by Right and Left Border sequences. The T-DNA contains the DvSnf7 suppression cassette, 

the cry3Bb1 expression cassette, and the cp4 epsps expression cassette. The data indicated that MON 

87411 contains a single integration locus of PV-ZMIR10871 DNA. Directed sequence data (generated 

from PCRs targeted at a specific locus) was used to demonstrate that the single DNA insert in 

MON 87411 contains a single copy of the T-DNA from the transformation plasmid vector PVZMIR10871 and confirmed the integrity of the DvSnf7 suppression cassette, the cry3Bb1 expression 

cassette, and the cp4 epsps expression cassette in the T-DNA insert. No vector backbone sequences from 

the transformation plasmid PV-ZMIR10871 were found in MON 87411 as determined by ..... The 

directed sequencing data also identified the 5' and 3' insert-to-flank junctions in MON 87411 and 

characterized the insertion site in conventional maize. Furthermore, bioinformatic analysis of nextgeneration sequence data (comprehensively covering the genome) was used to evaluate the stability of 

the DNA insert and demonstrated that the DNA insert in MON 87411 was stably maintained across five 

generations.

C. Protein Characterization

1) The level of Cry3Bb1 protein in the grain of MON 87411 is relatively low. Therefore, the protein 

was expressed in a bacterial expression system in order to produce a sufficient quantity of the 

protein for safety testing. An E. coli-produced Cry3Bb1protein was engineered to match the 

MON 87411-produced Cry3Bb1protein. Demonstration of physicochemical and functional 

equivalence between the MON 87411-produced and the E. coli-produced Cry3Bb1 proteins 
Page 8 of 29

justifies the use of the E. coli-produced protein to demonstrate the safety of the MON 87411-

produced Cry3Bb1 protein.

MON 87411-produced Cry3Bb1 protein was characterized as follows. The total protein 

concentration of the purified MON 87411-produced Cry3Bb1 protein was measured using a BioRad protein assay and was determined to be 0.34 mg/ml. Identity was confirmed by N-terminal 

sequence analysis and matrix assisted laser desorption/ionization time of flight mass spectrometry 

(MALDI-TOF MS) analysis of the trypsin digested Cry3Bb1 protein. The N-terminal sequencing

did not yield any observable sequence for the 77.0 kDa band presumably because the N-terminus 

was blocked. Several peptide masses consistent with expected peptide masses for the trypsin 

digested Cry3Bb1 protein were observed. The identified peptide masses yielded coverage of 73% 

of the expected sequence (478 out of 652 amino acids) of the Cry3Bb1 protein. Additionally, the 

identity of the MON 87411-produced Cry3Bb1 protein was confirmed by western blot analysis 

carried out with previously characterized anti-Cry3Bb1 antibodies. Anti-Cry3Bb1 antibodies 

detected the MON 87411-produced Cry3Bb1 protein. Purity and apparent molecular weight (MW) 

of the MON 87411-produced Cry3Bb1 protein were determined using SDS-PAGE analysis. Purity 

of the MON 87411-produced Cry3Bb1 protein was 97% and the apparent MW was 77.0 kDa. 

Analysis of the glycosylation status showed that the MON 87411-produced Cry3Bb1 protein was 

not glycosylated. The functional activity of the MON 87411-produced Cry3Bb1 was determined 

using an insect bioassay. The MON 87411-produced Cry3Bb1 protein was shown to be active, 

with a LC50 of 0.77 μg/ml diet using Colorado Potato Beetle larvae / adults as the test insect. 

Finally, assessment of the purity and apparent MW of the protein after storage in a -80 °C freezer 

(30 days) by SDS-PAGE analysis demonstrated that the MON 87411-produced Cry3Bb1 protein

was stable throughout the experimental phase.

The equivalence of the MON 87411-produced and E. coli-produced Cry3Bb1 proteins was 

assessed by comparing their apparent MW, immunoreactivity, functional activity, and 

glycosylation status. Based on these characteristics, the characterized E. coli-produced protein was 

established to be equivalent to this protein isolated from the grain of MON 87411.

2) The MON 87411-produced CP4 EPSPS protein was engineered to match the protein sequence of 

the CP4 EPSPS contained in other Roundup Ready® crops.1 In addition, the MON 

87411-produced CP4 EPSPS matches the protein sequence of the current and historical lots of E. 

coli-produced CP4 EPSPS that have been used for safety assessment studies. Demonstration of 

the equivalence between the E. coli-produced and MON 87411-produced proteins justifies 

application of the existing CP4 EPSPS protein safety data set to the CP4 EPSPS protein produced 

by MON 87411. Hence, the purpose of this study was to characterize the CP4 EPSPS protein 

isolated from the grain of MON 87411 and assess its equivalence to the E. coli-produced CP4 

EPSPS reference standard (lot 10000739).

MON 87411-produced CP4 EPSPS protein was characterized as follows. The total protein 

concentration of the purified MON 87411-produced CP4 EPSPS protein was measured using the 

Bio-Rad Assay and was determined to be 0.28 mg/ml. Identity was confirmed by N-terminal 

sequence analysis and matrix assisted laser desorption/ionization time of flight mass spectrometry

(MALDI-TOF MS) analysis of the trypsin digested CP4 EPSPS protein. The expected N-terminal 

sequence was observed, and several peptide masses consistent with expected peptide masses for 

 1 Roundup and Roundup ready are registered trademarks of Monsanto Technology LLC.
Page 9 of 29

the trypsin digested CP4 EPSPS protein were observed. The identified peptide masses yielded 

coverage of 67.3% of the expected sequence (306 out of 455 amino acids) of the CP4 EPSPS 

protein. Additionally, the identity of the MON 87411-produced CP4 EPSPS protein was 

confirmed by western blot analysis conducted with previously characterized anti-CP4 EPSPS

antibodies. Anti-CP4 EPSPS antibodies detected the MON 87411-produced CP4 EPSPS. Purity 

and apparent molecular weight (apparent MW) of the MON 87411-produced CP4 EPSPS protein 

were determined using SDS-PAGE analysis. Purity of the MON 87411-produced CP4 EPSPS 

protein was 94% and the apparent MW was 42.9 kDa. Analysis of the glycosylation status showed 

that the MON 87411-produced CP4 EPSPS protein was not glycosylated. The functional activity 

of the MON 87411-produced CP4 EPSPS was determined by an inorganic phosphate release assay. 

The MON 87411-produced CP4 EPSPS protein was shown to be active, with a specific activity of 

5.78 U/mg. Finally, assessment of the purity and apparent MW of the protein after storage in 

a -80 °C freezer (17 days) by SDS-PAGE analysis demonstrated that the MON 87411-produced 

CP4 EPSPS protein was stable throughout the experimental phase.

The equivalence of the MON 87411-produced and E. coli-produced CP4 EPSPS proteins was 

assessed by comparing their apparent MW, immunoreactivity, functional activity, and 

glycosylation status. Based on these characteristics, the characterized E. coli-produced protein 

was established to be equivalent to this protein isolated from the grain of MON 87411.

D. Protein Expression

The levels of DvSnf7 RNA in MON 87411 leaf tissues are very low (Song, et al., 2012). Therefore, 

RNA was produced by in vitro transcription (DvSnf7_968 RNA) in order to generate sufficient 

quantities of DvSnf7 RNA for safety assessments. The demonstration of the molecular equivalence 

between DvSnf7 RNA in MON 87411 and DvSnf7_968 RNA justifies the use of DvSnf7_968 RNA to 

demonstrate the safety of DvSnf7 RNA in MON 87411. DvSnf7 RNA in MON 87411 was 

characterized by sequencing its transcript as well as identifying the presence of the dsRNA region which 

has been shown to be the active region of DvSnf7 (Bolognesi, et al., 2012). Additionally, a functional 

activity assay was performed. The equivalence of the DvSnf7 RNA in MON 87411 and DvSnf7_968 

RNA was assessed by comparing their sequences, the presence and size of dsRNA regions in each, via 

northern blot analyses and by comparing their functional activity. Based on these molecular and 

functional characteristics, the DvSnf7 RNA in MON 87411 and DvSnf7_968 RNA were shown to be 

equivalent.

E. Mammalian Toxicity and Allergenicity Assessment

A 90 day toxicity study (MRID 49505806) with corn grain from MON 87411 at up to 33% in the diet 

(Mertens, 2013) and a 28 day toxicity study (MRID 49505804) with purified DvSnf7 at doses up to 100 

mg/kg (Wahle, 2014) did not indicate adverse effects in the animals receiving test diet or test substance. 

The results of a 28 day oral toxicity study indicate that DvSnf7 is not toxic to mammals after 28 

consecutive days of repeat oral exposure. Collectively, these results show the absence of any dietary 

hazard associated with DvSnf7 RNA at very high doses.

In addition, bioinformatic searches was conducted to determine DvSnf7 dsRNA’s sequence match or 

other human genes (open reading frames/coding regions). Monsanto conducted a search of all of the 21 

base pair small RNAs encoded by the 240 bp dsRNA DvSnf7 sequence to evaluate whether there were

any matches to human transcripts. This search, using the open reading frames/ coding regions search

criteria for endogenous corn small RNAs did not produce any matches between DvSnf7 RNA and 
Page 10 of 29

human transcripts (Table 2). This bioinformatics dataset provides further support for the history of safe 

consumption of endogenous plant small RNAs with 100% sequence identity to human transcripts and 

demonstrates that MON 87411 does not contribute any additional small RNAs to the diet that have 

identity to human transcripts. These data also further illustrate the important role that barriers to

exogenous RNA molecules serve in protecting higher organisms from these ubiquitous dietary 

components and suggest that matches between small RNAs in the diet and human transcripts do not 

constitute a safety concern. This is further illustrated by empirical toxicology data showing the absence 

of any dietary hazard associated with MON 87411 (Mertens 2013) or DvSnf7 RNA (Wahle 2014), in the 

unlikely event of any appreciable systemic exposure.

F. Overall Safety Conclusion

DvSnf7 is an insecticidal nucleic acid component of MON 87411. Nucleic acids (i.e., RNA, dsRNA, and

DNA) are present in all living organisms and are routinely consumed as a part of human and animal 

diets with no apparent adverse effects (Parrott et al, 2010; Ivashuta et al., 2009). Nucleic acids are 

considered to be “generally recognized as safe” (GRAS) by the U.S. Food and Drug Administration 

(FDA) (U.S. FDA 1992) and exempt from the requirement of a tolerance under the Federal Food Drug 

and Cosmetic Act by the U.S. EPA. The EPA recently affirmed (U.S. EPA, 2010b) that “DNA and RNA 

are common to all forms of plant and animal life, and the Agency knows of no instance where these 

nucleic acids have been associated with toxic effects related to their consumption as a component of 

food”.

Nevertheless, the FIFRA Scientific Advisory Panel (SAP) is requesting information on the stability of 

DvSnf7 dsRNA in animal blood and tissue. EPA agrees with Monsanto that there are extensive and 

redundant systems that can be expected to degrade free nucleic acids from an oral exposure. The intent 

of this request is to confirm that components in blood would indeed degrade the DvSnf7 RNA molecule, 

if any were to be present in the bloodstream given that a sensitive analytical system were available to 

specifically detect the intact molecule. While the theoretical argument is sufficient to justify the seed 

increase registration, a confirmatory study adding DvSnf7 dsRNA to whole blood and tracking its decay 

over time should be provided for full commercial release. While RNAse and various scavenging 

mechanisms capable of effecting destruction or excretion of RNA molecules are known from 

mammalian blood, other recent evidence suggests that RNAs, including miRNAs, are present in blood 

and are transported to various target tissues and organs as part of normal homeostasis (Wang et al., 

2012). It remains to be determined if these small RNAs are endogenous in origin or influenced by 

dietary uptake (Weiland et al., 2012; Yang et al., 2015). In addition, the Agency reserves the right to 

require information on stability in blood on more complex dsRNA structures if warranted.

The submitted studies verify the Cry3Bb1 protein expressed in MON 87411are biochemically and 

functionally equivalent with the Cry protein expressed in currently registered PIP MON 88017 corn. 

Therefore, the data submitted and reviewed for Cry3Bb1 and CP4 (CP4 EPSPS) proteins as expressed in 

MON 87411 corn justify bridging the existing findings and conclusions from the human health 

assessments conducted for PIP MON 88017 corn (EPA 524-551) and support their inclusion in the 

existing exemptions from the requirement of tolerance for Bacillus thuringiensis Cry3Bb1 [40 CFR § 

174.518] when used as a PIP in food and feed commodities of corn; and CP4 (CP4 EPSPS) protein [40 

CFR § 174.523] when used as a PIP inert ingredient in all feed/food commodities.
Page 11 of 29

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

population, including infants and children, toDvSnf7 dsRNA, Cry3Bb1 and CP4 EPSPS proteins and the 

genetic material necessary for their production in MON 87411. This includes all anticipated dietary 

exposures and all other exposures for which there is reliable information. The Agency has arrived at this 

conclusion because no toxicity to mammals has been observed, and there is no indication of allergenicity 

potential for the plant-incorporated protectant from available information.

G. Supporting Data

The submitted studies to support the registrant’s request for the seed increase Sec. 3 registration of PIP 

MON 87411 corn expressing Cry3Bb1 and CP4 EPSPS proteins are summarized in Table 1. For detailed 

information of each study, please refer to the individual studies reviewed in the Data Evaluation Reports 

(DERs) according to its respective assigned MRID number. 
Page 12 of 29

TABLE 2. Product Characterization Data for PIP MON 87411 Maize expressing Bt Cry34Bb1 and CP4 

(CP4 EPSPS) proteins [EPA Reg. No. 524-ARI]

STUDY TYPE/TITLE SUMMARY MRID NO

Molecular 

Characterization of 

MON 87411; Amended 

Report MSL0025048

MON 87411 was produced by Agrobacterium-mediated transformation of 

maize tissue using the transformation vector PV-ZMIR10871. This plasmid 

vector contains one T-DNA (transfer DNA), which is delineated by Right and 

Left Border sequences. The T-DNA contains the DvSnf7 suppression 

cassette, the cry3Bb1 expression cassette, and the cp4 epsps expression 

cassette.

In this study, bioinformatic analysis of next-generation sequence data 

(comprehensively covering the genome) was used to characterize the insert 

number of DNA sequences from the transformation plasmid vector PVZMIR10871. The data indicated that MON 87411 contains a single integration 

locus of PV-ZMIR10871 DNA. Directed sequence data (generated from PCRs 

targeted at a specific locus) was used to demonstrate that the single DNA insert 

in MON 87411 contains a single copy of the T-DNA from the transformation 

plasmid vector PV-ZMIR10871 and confirmed the integrity of the DvSnf7 

suppression cassette, the cry3Bb1 expression cassette, and the cp4 epsps 

expression cassette in the T-DNA insert; the data showed that MON 87411 does 

not contain any sequences from the backbone of PV-ZMIR10871. The directed 

sequencing data also identified the 5' and 3' insert-to-flank junctions in 

MON 87411 and characterized the insertion site in conventional maize. 

Furthermore, bioinformatic analysis of next-generation sequence data 

(comprehensively covering the genome) was used to evaluate the stability of the 

DNA insert and demonstrated that the DNA insert in MON 87411 was stably

maintained across five generations.

CLASSIFICATION: ACCEPTABLE

49315103

Assessment of DvSnf7 

dsRNA levels in Maize 

Tissues Collected from 

MON 87411 Produced 

in Argentina Field 

Trials during 2011-

2012.

This study determined the DvSnf7 RNA expression levels in the following 

tissue types of MON 87411 using a validated QuantiGene® Plex 2.0 Assay: 

over season leaf (OSL1, OSL2, OSL3, and OSL4), over season root (OSR1, 

OSR2, OSR3, and OSR4), over season whole plant (OSWP1, OSWP2, 

OSWP3, and OSWP4), forage root, forage, senescent root, stover, pollen, 

silk, and grain. Tissue samples were collected from MON 87411 plants 

produced in Argentina field trials at five sites during 2011-2012. The DvSnf7 

RNA level in each tissue type was calculated on a microgram (μg) per gram 

(g) of fresh weight (fw) basis. Moisture content was measured for each tissue 

type and all the DvSnf7 RNA levels were converted to a μg/g dry weight 

(dw) basis. The DvSnf7 RNA levels (μg/g) on both fw and dw basis were 

reported.

DvSnf7 RNA levels were determined in all nineteen tissue types. The results 

obtained for each sample by the QuantiGene® Plex 2.0 Assay were averaged 

across the five sites and are summarized. The individual DvSnf7 RNA levels 

in MON 87411 across all samples analyzed from all sites ranged from lower 

than the limit of detection (<LOD) to 213 × 10-3 μg/g dw. The mean DvSnf7 

RNA level for each tissue type was highest in OSWP1 at 84.8 × 10-3 μg/g dw 

and lowest in grain at 0.104 × 10-3 μg/g dw. Samples with an expression level 

<LOD or lower than the limit of quantitation (<LOQ) were not included in 

the mean determinations.

CLASSIFICATION: ACCEPTABLE 

49315104
Page 13 of 29

STUDY TYPE/TITLE SUMMARY MRID NO

Characterization of 

DvSnf7 Extracted from 

MON 87411 and 

Comparison of the 

Molecular and 

Functional Properties 

of Plant-Produced and 

in vitro-Produced 

DvSnf7

The levels of DvSnf7 RNA in MON 87411 leaf tissues are very low (Song, et 

al., 2012). Therefore, RNA was produced by in vitro transcription 

(DvSnf7_968 RNA) in order to generate sufficient quantities of DvSnf7 RNA 

for safety assessments. The demonstration of the molecular equivalence 

between DvSnf7 RNA in MON 87411 and DvSnf7_968 RNA justifies the 

use of DvSnf7_968 RNA to demonstrate the safety of DvSnf7 RNA in 

MON 87411. Hence, the purpose of this study was to characterize the DvSnf7 

RNA in MON 87411 and to assess the equivalence of DvSnf7 RNA in MON 

87411 and the previously characterized DvSnf7_968 RNA.

DvSnf7 RNA in MON 87411 was characterized by sequencing its transcript 

as well as identifying the presence of the dsRNA region which has been 

shown to be the active region of DvSnf7 (Bolognesi, et al., 2012). 

Additionally, a functional activity assay was performed. The equivalence of 

the DvSnf7 RNA in MON 87411 and DvSnf7_968 RNA was assessed by 

comparing their sequences, the presence and size of dsRNA regions in each, 

via northern blot analyses and by comparing their functional activity. Based 

on these molecular and functional characteristics, the DvSnf7 RNA in 

MON 87411 and DvSnf7_968 RNA were shown to be equivalent.

CLASSIFICATION: ACCEPTABLE

49315106

Characterization of the 

Cry3Bb1 Protein 

Purified from the 

Maize Grain of 

MON 87411 and 

Comparison of the 

Physicochemical and 

Functional Properties 

of the Plant-Produced 

and E. coli-Produced 

Cry3Bb1 Proteins

The level of Cry3Bb1 protein in the grain of MON 87411 is relatively low. 

Therefore, the protein was expressed in a bacteria expression system in order 

to produce a sufficient quantity of the protein for safety testing. An E. coliproduced Cry3Bb1protein was engineered to match the MON 87411-

produced Cry3Bb1protein. Demonstration of physicochemical and functional 

equivalence between the MON 87411-produced and the E. coli-produced 

Cry3Bb1 proteins justifies the use of the E. coli-produced protein to 

demonstrate the safety of the MON 87411-produced Cry3Bb1 protein. 

MON 87411-produced Cry3Bb1 protein was characterized as follows. The 

total protein concentration of the purified MON 87411-produced Cry3Bb1 

protein was measured using a Bio-Rad protein assay and was determined to be 

0.34 mg/ml. Identity was confirmed by N-terminal sequence analysis and 

matrix assisted laser desorption/ionization time of flight mass spectrometry

(MALDI-TOF MS) analysis of the trypsin digested Cry3Bb1 protein. The Nterminal did not yield any observable sequence for the 77.0 kDa band 

presumably because the N-terminus was blocked. Several peptide masses 

consistent with expected peptide masses for the trypsin digested Cry3Bb1 

protein were observed. The identified peptide masses yielded coverage of 73% 

of the expected sequence (478 out of 652 amino acids) of the Cry3Bb1 protein. 

Additionally, the identity of the MON 87411-produced Cry3Bb1 protein was 

confirmed by western blot analysis carried out with previously characterized 

anti-Cry3Bb1 antibodies. Anti-Cry3Bb1 antibodies detected the MON 87411-

produced Cry3Bb1 protein. Purity and apparent molecular weight (MW) of the 

MON 87411-produced Cry3Bb1 protein were determined using SDS-PAGE 

analysis. Purity of the MON 87411-produced Cry3Bb1 protein was 97% and 

the apparent MW was 77.0 kDa. Analysis of the glycosylation status showed 

that the MON 87411-produced Cry3Bb1 protein was not glycosylated. The 

functional activity of the MON 87411-produced Cry3Bb1 was determined 

using an insect bioassay. The MON 87411-produced Cry3Bb1 protein was 

shown to be active, with a LC50, of 0.77 μg/ml diet using Colorado Potato 

Beetle. Finally, assessment of the purity and apparent MW of the protein after 

storage in a -80 °C freezer (30 days) by SDS-PAGE analysis demonstrated that 

the MON 87411-produced Cry3Bb1 protein was stable throughout the 

experimental phase.

49315107
Page 14 of 29

STUDY TYPE/TITLE SUMMARY MRID NO

The equivalence of the MON 87411-produced and E. coli-produced Cry3Bb1 

proteins was assessed by comparing their apparent MW, immunoreactivity, 

functional activity, and glycosylation status. Based on these characteristics, the 

characterized E. coli-produced protein was established to be equivalent to this 

protein isolated from grain of MON 87411.

CLASSIFICATION: ACCEPTABLE

Characterization of the 

CP4 EPSPS Protein 

Purified from the 

Maize Grain of 

MON 87411 and 

Comparison of the 

Physicochemical and 

Functional Properties 

of the Plant-Produced 

and E. coli-Produced 

CP4 EPSPS Proteins

The MON 87411-produced CP4 EPSPS protein was engineered to match the 

protein sequence of the CP4 EPSPS contained in other Roundup Ready®

crops.1

 In addition, the MON 87411-produced CP4 EPSPS matches the 

protein sequence of the current and historical lots of E. coli-produced CP4 

EPSPS that have been used for safety assessment studies. Demonstration of 

the equivalence between the E. coli-produced and MON 87411-produced 

proteins justifies application of the existing CP4 EPSPS protein safety data 

set to the CP4 EPSPS protein produced by MON 87411. 

MON 87411-produced CP4 EPSPS protein was characterized as follows. The 

total protein concentration of the purified MON 87411-produced CP4 EPSPS 

protein was measured using the Bio-Rad Assay and was determined to be 

0.28 mg/ml. Identity was confirmed by N-terminal sequence analysis and 

matrix assisted laser desorption/ionization time of flight mass spectrometry

(MALDI-TOF MS) analysis of the trypsin digested CP4 EPSPS protein. The 

expected N-terminal sequence was observed, and several peptide masses 

consistent with expected peptide masses for the trypsin digested CP4 EPSPS 

protein were observed. The identified peptide masses yielded coverage of 

67.3% of the expected sequence (306 out of 455 amino acids) of the CP4 

EPSPS protein. Additionally, the identity of the MON 87411-produced CP4 

EPSPS protein was confirmed by western blot analysis conducted with 

previously characterized anti-CP4 EPSPS antibodies. Anti-CP4 EPSPS

antibodies detected the MON 87411-produced CP4 EPSPS. Purity and 

apparent molecular weight (apparent MW) of the MON 87411-produced CP4 

EPSPS protein were determined using SDS-PAGE analysis. Purity of the 

MON 87411-produced CP4 EPSPS protein was 94% and the apparent MW 

was 42.9 kDa. Analysis of the glycosylation status showed that the MON 

87411-produced CP4 EPSPS protein was not glycosylated. The functional 

activity of the MON 87411-produced CP4 EPSPS was determined by an 

inorganic phosphate release assay. The MON 87411-produced CP4 EPSPS 

protein was shown to be active, with a specific activity of 5.78 U/mg. Finally, 

assessment of the purity and apparent MW of the protein after storage in 

a -80 °C freezer (17 days) by SDS-PAGE analysis demonstrated that the MON 

87411-produced CP4 EPSPS protein was stable throughout the experimental 

phase.

The equivalence of the MON 87411-produced and E. coli-produced CP4 

EPSPS proteins was assessed by comparing their apparent MW, 

immunoreactivity, functional activity, and glycosylation status. Based on these 

characteristics, the characterized E. coli-produced protein was established to 

be equivalent to this protein isolated from grain of MON 87411.

These data provide a detailed characterization of the CP4 EPSPS protein 

isolated from MON 87411 and establish the equivalence of the 

E. coli-produced CP4 EPSPS protein to this plant-derived protein.

49315108

 1 Roundup and Roundup ready are registered trademarks of Monsanto Technology LLC.
Page 15 of 29

STUDY TYPE/TITLE SUMMARY MRID NO

CLASSIFICATION: ACCEPTABLE

28 Day Oral Toxicity 

Study in Mice

In a 28-day oral toxicity study (MRID 49505804), DvSnf7_968 RNA (100% 

a.i., Lot# 11350669) was administered to 10 CD-1 mice/sex/dose by gavage 

at dose levels of 0 (vehicle control- nuclease free water), 1.06, 11.0, or 105 

mg/kg bw/day. A group of 10 CD-1 mice/sex served as negative controls and 

received 104 mg/kg bw/day of Torula yeast RNA. Animals were examined 

for clinical signs, mortality, body weight, food consumption, clinical 

pathology, organ weights, and gross and histopathology examination. 

Apparent statistical changes were noted in the thyroid of the males (mid and 

high dose treatment groups) and ovaries in the females (all treatment groups), 

but these changes were not considered to be attributable to the test substance.

Decreased thyroid weight was noted in 10 (↓24.4%) and 100 (↓23.3%) mg/kg 

males (compared to vehicle controls). The mouse thyroid is extremely 

difficult to trim (due to very small size) and was weighed following fixation, 

both of which can introduce variation. A clear dose response to increasing 

dose was also lacking, as the thyroid weights were comparable between the 

10 and 100 mg/kg males. With the exception of a single incidence in a 

negative control animal (cyst), microscopic changes in the thyroid were not 

observed. Therefore, the observed thyroid weight differences were not 

considered treatment-related.

Decreased ovary weights were noted in the negative control (NC) and all 

treated groups (↓26%, ↓23%, ↓27.4% and ↓16.7% in the NC, low, mid and 

high dose groups respectively, compared to vehicle controls). The decrease 

may be more of a function of the control ovary weights being considerably 

heavier (even compared to the negative control group (↓26%)). Ovaries are 

also very small tissues that are very difficult to trim (can induce a certain 

degree of variability) and there was no consistent dose response to increasing 

dose. In addition, ovary weights in the test substance treated groups were 

comparable to the negative control group, thereby indicating that the 

observed differences between the test substance and control groups did not 

represent a change from the normal range expected for this parameter. With 

the exception of lesions in the ovaries of two negative control animals (cyst), 

microscopic changes in the ovary were not observed. Therefore, the observed 

ovary weight differences were not considered treatment-related.

There were no treatment-related effects on clinical signs, mortality, body 

weight parameters, food consumption or gross and histologic pathology. The 

NOAEL is 105 mg/kg/day. The LOAEL was not established. 

CLASSIFICATION: ACCEPTABLE

49505804

49576101

Subchronic 90 Day 

Oral Toxicity Study in 

Rats

In a 90-day oral toxicity study (MRID 49505806), MON 87411 (purity not 

provided; Lot# 1815707-101), a biotechnology-derived corn conferring 

resistance to corn rootworm and containing a modified Bacillus thuringiensis 

Cry3Bb1 protein, was administered to 16 Sprague Dawley rats/sex in the diet 

at 33% (w/w) (equivalent to 1899 and 2303 mg/kg cage body weight/day for 

males, and female, respectively). The mean DvSnf7 expression value in grain 

was 0.091 x 10-3 μg/g fresh weight (SD= 0.028 x 10-3 μg/g). Control animals 

(16/sex) received conventional ground corn grain in the diet at 33% (w/w) 

(equivalent to 1924 and 2168 levels of mg/kg cage body weight/day for 

males, and female, respectively). Evaluated parameters included clinical 

signs, mortality, functional observation battery (FOB), body weight, food 

consumption, clinical pathology, organ weights, and gross and histopathology 

49505806

49576101
Page 16 of 29

STUDY TYPE/TITLE SUMMARY MRID NO

examination. 

All animals were observed twice daily for mortality and moribundity. 

Clinical examinations were performed daily, and detailed physical 

examinations were performed weekly (± 2 days). Individual body weights 

were recorded weekly (± 2 days) and food weights were recorded weekly (± 2 

days) on a per cage basis. Functional observational battery (FOB) and 

locomotor activity data were recorded for all animals during acclimation 

(following randomization) and near the end of the dosing period (study week 

12). Ophthalmic examinations were performed during study weeks -2 and 

12. Clinical pathology parameters (hematology, coagulation, serum 

chemistry, and urinalysis) were analyzed for all rats at the scheduled (study 

week 13) necropsy. Complete necropsies were conducted on all animals, and 

selected organs (per OECD Guideline 408) were weighed at the scheduled 

necropsy. Selected tissues (per OECD Guideline 408) were

examined microscopically from all animals in the control and test substancetreated groups.

All test substance-treated group animals survived to the scheduled necropsy. 

There were no test substance-related effects on clinical observations, body 

weight, food consumption, or clinical pathology. No macroscopic or 

histologic findings or differences in organ weights were considered a result of 

test substance administration.

There were no treatment-related effects on mortality, clinical signs, FOB, 

body weight, body weight gain, food consumption, food efficiency, clinical 

pathology, organ weights, gross pathology, or microscopic pathology. 

The LOAEL for MON 87411 is not established. The NOAEL is 1899 and 

2303 mg/kg total cage body weight/day for males and females, 

respectively. 

CLASSIFICATION: ACCEPTABLE

Bioinformatic 

Comparison of DvSnf7 

versus a Human 

Transcriptome Using 

STELLAR

This report was conducted to document the results of a sequence similarity 

search between the 240 nucleotide DvSnf7 sequence from MON 87411 and 

human transcriptome. 

A search of the human transcriptome was conducted using STELLAR version 

1.3 (Kehr et al., 2011) to identify all alignments of 21 nucleotides or greater 

in length and displaying zero mismatches. 

The human transcriptome database contains 98,650 sequences extracted from 

the RefSeq archive file downloaded on March 10, 2014 from:

ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/mRNA_Prot/human.rna.fna.gz

The DvSnf7 sequence was used as a query to search a human transcriptome 

database containing 98,650 sequnces. No alignments of 21 nucleotides or 

greater displaying 100% identify were found. 

CLASSIFICATION: ACCEPTABLE

49553305

MON 88017 and MON 

87411 Express Identical 

Cry3Bb1 Proteins

There are two commercial Monsanto corn events which contain the Cry3Bb1 

protein. The events are MON 863 (YieldGard rootworm) and MON 88017 

(CCR). These events have been stacked to generate other commercial 

products (not discussed here). The Cry3Bb1 protein in these two events 

differ by only one amino acid and share identity of greater than 99%. The 

Cry3Bb1 protein in MON 863 has been termed “Cry3Bb1.11098

49553303
Page 17 of 29

STUDY TYPE/TITLE SUMMARY MRID NO

(Q349R)”. The Cry3Bb1 protein in MON 88017 has been termed 

“Cry3Bb1.pvzmir39”.

The Cry3Bb1 expression cassette in MON 87411 codes for an amino acid 

sequence that is 100% identical to the one encoded in Cry3Bb.1 expression 

cassette presence in MON 88017 and therefore both are the 

Cry3Bb1.pvzmir39 variant. 

Characterization studies on the Cry3Bb1 isolated from MON 87411

(MSL0024872) and MON 88017 (MSL0018816) have shown that in both 

cases the Nterminal end of the protein is blocked while new technology has 

allowed demonstration that the first methionine is removed from Cry3Bb1 in 

planta.

CLASSIFICATION: ACCEPTABLE
Page 18 of 29

Per the recommendation in the report from the EPA’s FIFRA SAP meeting held on January 28, 

2014; Response to EPA Request for Further Information Related to Human Health 

Considerations.

On December 15, 2014 the EPA, OPP, BPPD sent a 75 day deficiency letter to the Monsanto Company, 

pertaining to the conclusions and recommendations set forth in the Meeting Minutes from the January 

28, 2014, Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP)

on Human Health, Environmental Fate and Non-Target Effects. The questions and Monsanto’s response 

pertaining to Human Health Effects are address in this Memorandum. 

EPA’s Request #1: Structure of DvSnf7 dsRNA (e.g., hairpins, super coil).

Monsanto Response to Request #1.

In MON 87411, the predominant RNA transcript produced from the suppression cassette was identified 

as being 968 nucleotides (nt) in length. The DvSnf7 suppression cassette contains two DvSnf7 

sequences (240 nt each) in an inverted orientation separated by 150 nt of intervening sequence. When 

the suppression cassette is transcribed, the expressed RNA forms a hairpin loop consisting of the 240 

base pair double stranded DvSnf7 RNA with a 150 nt loop region (Urquhart et al. 2013).

The DvSnf7 RNA expressed in MON 87411 is a native RNA sequence, having no synthetic 

modifications altering its stability. As indicated in the SAP panel report (U.S. EPA 2014), “The 

combination of RNases and acids found in the human digestive system are likely to ensure that all forms 

of RNA structure are degraded throughout the digestive process.” Furthermore, the panel report 

discusses susceptibility of plant and animal RNA samples to RNAses and concludes that such evidence 

“supports the likelihood that PIP and non-PIP RNAs expressed in plant material consumed by humans 

are likely to be degraded no matter the type of RNA or its structural status when entering the human

digestive system.” Therefore, there are no potential hazards indicated by the structure of the DvSnf7 

RNA expressed in MON 87411.

Monsanto’s Conclusion: Transcription of the DvSnf7 RNA produces a 240 bp dsRNA in a hairpin 

loop, separated by 150 nucleotides of intervening sequence.

EPA’s Comment: The single hairpin structure of the dsRNA for DvSnf7 is one of the simpler structures 

expected for RNAi inducing molecules. EPA does not expect this structure to present any unique 

stability to RNA degrading enzymatic attack. The Agency reserves the right to require stability 

information on more complex dsRNA structures if warranted.

EPA’s Request #2: Stability of DvSnf7 dsRNA in animal blood.

Monsanto Response to Request #2.

Extensive physical and biochemical barriers present a significant challenge to oral delivery of nucleic 

acids (Lawrence 2014) and these barriers would preclude DvSnf7 RNA absorption and also stability in 

animal blood. These biological barriers include nucleases in the saliva and gastrointestinal tract, acidic 
Page 19 of 29

conditions in the stomach, and multiple membrane barriers, which collectively limit the delivery of

ingested RNA into the blood. Anticipated human dietary exposures to DvSnf7 RNA from consumption 

of MON 87411 are extremely low, on the order of ≤ 0.4 ng/kg/day, based on the very low expression of 

DvSnf7 RNA in corn grain (0.000091 μg/gram fresh weight or 91 parts per trillion) and 95th percentile 

consumption values in non-nursing infants derived from DEEM-FCID 

(http://www.epa.gov/pesticides/science/deem/; Calculated in Petrick (2014)). These exceedingly low 

dietary exposures assume 100% of DvSnf7 RNA is orally bioavailable (e.g. absorbed), yet we know 

from the literature that biological barriers greatly limit this uptake, making uptake about 0.3 % for 

unformulated oligonucleotides (Nicklin et al. 1998). Even oral formulations limit uptake to about 1-

10% for nucleic acid drugs (Raoof et al. 2004; Tillman et al. 2008). Based on DvSnf7 RNA being an 

unmodified, native RNA sequence, our assumption of 100% bioavailability/uptake in this exposure 

assessment is highly conservative, as not enough of this RNA would be absorbed or distributed to enable 

a potential for biological activity.

Nucleic acids in circulation are subjected to extracellular nuclease activity in the blood (Houck 1958), 

along with rapid renal filtration (within minutes) and excretion by the kidneys (Molitoris et al. 2009; 

Vaishnaw et al. 2010). Intravenously administered chemically stabilized siRNAs (a route that bypasses 

all of the barriers limiting oral absorption), were shown to have a half-life of less than 10 minutes, being 

rapidly broken down into much shorter sequences and individual nucleosides too small to initiate RNAi

(Christensen et al. 2013). Although we have not generated empirical data concerning the stability of 

DvSnf7 RNA in animal blood, the scientific literature provides both in vitro and in vivo evidence that 

like any other native RNA (Petrick et al. 2013), DvSnf7 should be rapidly degraded in blood. 

Additionally, native RNA is inherently more susceptible to degradation than stabilized RNA, and 

therefore, the work by Christensen et al., (2013) described above demonstrates that bioaccumulation is 

highly unlikely to occur. Thus, the weight of the scientific evidence supports the conclusion of rapid 

metabolism and elimination of any DvSnf7 RNA that could be absorbed following very low exposures

from MON 87411. Furthermore, the toxicology data from a 90 day toxicity study (MRID 49505806)

with corn grain form MON 87411 at up to 33% in the diet (Mertens 2013) and a 28 day toxicity study 

(MRID 49505804) with purified DvSnf7 RNA at doses up to 100 mg/kg (Wahle 2014) did not indicate 

adverse effects in the animals receiving test diet or test substance. The results of the 28 day study 

indicate that at a margin of exposure of at least 2.3 x 108 (based on 95th percentile consumption of MON 

87411 in non-nursing infants from DEEM), DvSnf7 is not toxic to mammals after 28 consecutive days 

of repeat oral exposure. Collectively, these results show the absence of any dietary hazard associated 

with DvSnf7 RNA at very high doses, in the unlikely event of any appreciable systemic exposure or 

stability. Thus even if stability were presumed as a worst case scenario, the absence of any hazard when

DvSnf7 RNA is administered at high doses to a rodent demonstrates that this endpoint is not informative 

for risk assessment or hazard identification.

The SAP report concluded that, “available evidence supports the conclusion of no significant absorption 

of dsRNA in mammals and minimal likelihood of adverse effects” and that “ingested dsRNAs undergo 

rapid degradation in the mammalian digestive tract”, calling into question the value of the requested 

stability data for hazard identification or risk assessment (U.S. EPA 2014). Although the panel also 

stressed a lack of dsRNA PIP studies exist and asked for testing of blood and tissues on a precautionary 

basis, this recommendation is inconsistent with their above conclusions. Most importantly, the

aforementioned toxicology tests on MON 87411 and DvSnf7 RNA that were not available at the time of 

the panel demonstrate conclusively that there is no identifiable hazard in mammals from consumption 

and that additional data on stability in blood would provide no additional value from a hazard 

identification or risk assessment perspective.
Page 20 of 29

Monsanto’s Conclusion: Extensive biological barriers and lack of toxicity after repeat oral dosing 

make DvSnf7 RNA unlikely to be stable and demonstrate the lack of hazard posed by high levels of 

exposure to this sequence in mammals.

EPA’s Comment: EPA agrees that there are extensive and redundant systems that can be expected to 

degrade free nucleic acids from an oral exposure. The intent of this request was to confirm that 

components in blood would indeed degrade the DvSnf7 RNA molecule, if any were to be present in the 

bloodstream given that a sensitive analytical system were available to specifically detect the intact 

molecule. While the theoretical argument is sufficient to justify the seed increase registration, a 

confirmatory study adding DvSnf7 dsRNA to whole blood and tracking its decay over time should be 

provided for commercial release. While RNAse and various scavenging mechanisms capable of 

effecting destruction or excretion of RNA molecules are known from mammalian blood, other recent 

evidence suggests that RNAs, including miRNAs, are present in blood and are transported to various 

target tissues and organs as part of normal homeostasis (Wang et al., 2012). It remains to be determined 

if these small RNAs are endogenous in origin or influenced by dietary uptake (Weiland et al., 2012; 

Yang et al., 2015). The Agency reserves the right to require information on stability in blood on more 

complex dsRNA structures if warranted.

EPA’s Request #3: Bioinformatic searches to determine DvSnf7 dsRNA’s sequence match to other 

human genes (open reading frames/coding regions).

Monsanto Response to Request #3.

We disagree that bioinformatics should be a tool for assessing potential hazards in mammals from oral 

exposure to RNA derived from RNAi based products. Sequence identity is a criterion for consideration 

of potential for gene suppression of a given RNA molecule. However, the length, context, biophysical 

properties, and other criteria related to the match are also quite relevant in assessing the potential for a 

sequence to mediate gene suppression (Liu et al. 2013). Furthermore, the relevance of sequence identity

presupposes that exposure at the site of action is suitable for modulation of gene expression. This 

exposure scenario is highly unlikely, as evidenced by the literature regarding therapeutic applications of 

RNAi. For example, as discussed by Snow and colleagues (Snow et al. 2013), 100 copies per cell are

needed to modulate gene expression (Snow and colleagues report orders of magnitude fewer copies than 

this in vivo following oral exposure), and this is unlikely to occur upon RNA exposure from MON 

87411 due to exceedingly low dietary exposures (described above), limited uptake, biological barriers, 

and the need for a suitable match to mediate gene regulation. Although the potential value of 

bioinformatics for hazard characterization was not a topic of discussion by the SAP, their report 

acknowledges, “Sequence identity does not mean that the dsRNA would provoke an RNAi response, but 

should caution the potential for unintended interactions and further review (U.S. EPA 2014).” We have 

addressed this further below with empirical data by providing bioinformatics datasets on endogenous 

corn small RNAs as well as the DvSnf7 RNA.

We acknowledge that there is a body of literature discussing the potential for off-target gene suppression 

mediated by small RNAs through partial complementarity to a target or through complementarity 

between the seed region of a small RNA and the 3' untranslated region (UTR) of a gene (reviewed by 

Petrick et al. 2013). However, these studies have utilized in vitro models employing high doses of RNA 
Page 21 of 29

and transfection reagents. Therefore, they have very limited predictive potential for in vivo impact in 

mammals or other consuming organisms following dietary exposures, given the exceedingly low

systemic exposures and the absence of formulations necessary to facilitate any appreciable 

systemic/cellular uptake of RNA. Vaishnaw and colleagues also note that unformulated siRNAs have 

generally been well tolerated at i.v. doses >100 mg/kg in preclinical safety studies, thus indicating that 

potential changes in transcriptional profiles (e.g. target or off-target gene suppression) does not appear to 

impact safety in vivo (Vaishnaw et al. 2010). This is also underscored by the work of Thompson and

colleagues who safely administered doses to rats of up to 200 mg/kg (i.v.) of a stabilized RNA targeting 

an endogenous gene (Thompson et al. 2012).

In order to demonstrate the safe consumption of small RNAs (encoded by longer dsRNAs) with 100% 

identity to human transcripts, the presence of such small RNAs in corn grain were analyzed by 

Monsanto using a bioinformatics analysis comparing endogenous corn grain small RNAs to the human 

transcriptome. A combined dataset containing small double stranded RNA molecules from corn grain 

collected in eight separate sequencing experiments was generated from three sequencing experiments of

RNA extracted from mature corn grain. Small RNA fractions were isolated from total RNA after 

separation on a polyacrylamide gel, followed by sequential ligation of cloning adaptors to the small 

RNAs in these extracts (as described by Llave et al., 2002). This material was reverse transcribed and 

the cDNA libraries generated in this process were sent to 454 Life Sciences (Branford, CT) for deep 

sequencing via pyrosequencing. Computer algorithms written in the Perl programming language (Perl 

scripts) were used to identify small RNA inserts within the raw sequence data through identification and 

removal of sequences representing the cloning adaptors. The sequences from the eight experiments were

combined and duplicates were removed. A total of 386,557 small RNA sequence reads (includes 

sequences 21-26 nucleotides in length) were obtained from the corn grain small RNA libraries and were 

used for subsequent analysis. The above corn endogenous small RNA library was compared to a library 

of 98,650 human transcript sequences (transcriptome).

The initial alignment data, generated using the STELLAR algorithm (Kehr et al. 2011), revealed a total

of 3540 alignments with endogenous corn grain small RNAs. This dataset was filtered to remove 

database sequences that are unlikely to be protein coding mRNAs (Table 2). The following types of 

sequences were removed: ribosomal sequences, small nuclear RNAs, and sequences encoding predicted 

genes and pseudogenes. Following filtering, a total of 500 alignments representing 447 unique human 

mRNA sequences that aligned with 150 short corn RNAs were identified. Thus, there are 150 

endogenous corn grain small RNAs that have 100% sequence identity (21 out of 21 nucleotides or 

greater) to a total of 500 likely protein coding human transcripts. This bioinformatics dataset

provides further support for the history of safe consumption of endogenous plant small RNAs (and their 

dsRNA precursors), including those with 100% sequence identity to human transcripts. These data 

illustrate the key functional role that barriers to exogenous RNA molecules (described above) play in 

higher organisms that consume these ubiquitous dietary components and suggest that matches between 

small RNAs in the diet (be they from endogenous or transgene-derived sources) and human transcripts 

do not constitute a safety concern. If bioinformatics were applied as a hazard characterization

tool for evaluating the safety of conventional corn, the data presented herein would indicate a potential 

hazard due to the more than 500 human transcript matches, and would call into question the safety of 

this widely consumed and safe staple crop.

Although we do not think the weight of the scientific evidence suggests any relevance of

bioinformatics searches to hazard characterization or risk assessment to humans (or vertebrates) for 

MON 87411, we conducted a search of all of the 21 base pair small RNAs encoded by the 240 bp 
Page 22 of 29

dsRNA DvSnf7 sequence to evaluate whether there were any matches to human transcripts. This search, 

using the same algorithms and search criteria described above for endogenous corn small RNAs did not 

produce any matches between DvSnf7 RNA and human transcripts (Table 3). This bioinformatics 

dataset provides further support for the history of safe consumption of endogenous plant small

RNAs with 100% sequence identity to human transcripts and demonstrates that MON 87411 does not 

contribute any additional small RNAs to the diet that have identity to human transcripts. These data also 

further illustrate the important role that barriers to exogenous RNA molecules serve in protecting higher 

organisms from these ubiquitous dietary components and suggest that matches between small RNAs in 

the diet and human transcripts do not constitute a safety concern. This is further illustrated by empirical

toxicology data showing the absence of any dietary hazard associated with MON 87411 (Mertens 2013) 

or DvSnf7 RNA (Wahle 2014), in the unlikely event of any appreciable systemic exposure.

Table 2. Exact 21 or greater nucleotide matches between endogenous corn small RNAs and the human 

transcriptome.

Total alignments Unique Human database 

Sequence identified

Unique Short Corn RNA 

Sequences

Primary search results 3540 1011 1254

Ribosomal 2086 9 1043

Non ribosomal 1454 1002 212

 Small Nuclear RNA 376 48 29

 Predicted 479 432 106

 Pseudogene 23 9 22

 Non-coding RNA 76 66 46

 Likely protein coding 500 447 150

Table 3. Exact 21 Nucleocide matches between the 240 bp DvSnf7 dsRNA and the Human 

Transcriptome.

Total alignments Unique Human Database 

sequence identified

Primary search results 0 0

Monsanto’s Conclusion: We disagree that bioinformatics represents a hazard assessment tool for 

ingested RNA. We have provided additional data showing many small RNAs in conventional corn

match human transcripts. DvSnf7 RNA does not encode any small RNAs with identity to human 

transcripts.

EPA’s Comment: EPA disagrees at this time that bioinformatics analyses provide no useful 

information in hazard assessment. The Agency at this time recognizes that there is the possibility of 

significant sequence alignment in endogenous small RNAs in crop plants to sequences in the human 

transcriptome that indicate little probability of induced RNAi from dietary consumption. The other 

aspects to be considered are related to exposure due to dsRNA expression levels in plant tissue, dsRNA 

concentration in a formulated product and the potential to interact with the identified target sequence in 

expected exposure scenarios. EPA agrees that absence of any significant alignments in the human 

transcriptome and the low dietary exposure expected for the DvSnf7 RNA molecule ameliorates hazard 

concerns. The Agency reserves the right to require bioinformatics analysis on future dsRNA constructs.
Page 23 of 29

EPA’s Request #4: Stability of DvSnf7 dsRNA via the dermal and inhalation routes of exposure. 

In doing so, you must verify whether the same degradation for DvSnf7 dsRNA occurs for these 

routes of exposure as presented for the dietary route of exposure.

Monsanto Response to Request #4.

Clinical trials have been initiated with siRNA drugs delivered via the dermal route (Vaishnaw et al. 

2010). However, the skin is generally impermeable to high molecular weight charged molecules such as 

RNA (Lewin et al. 2005) and these drug trials involve the use of specialized delivery agents to promote 

absorption (Geusens et al. 2009). The mean expression of construct-derived DvSnf7 RNA in MON 

87411 among six tissue types (forage root, forage, scenescent root, stover, pollen, silk, and grain) from a 

2011-2012 field trial was highest in forage, at 0.00128 μg/g fresh weight and was lowest in grain, at 

0.000091 μg/g fresh weight (Song et al. 2013). Thus, from an occupational safety standpoint, dermal 

exposures to agricultural dusts and pollen containing naked dsRNA molecules expressed in 

biotechnology derived crops are negligible and present little or no opportunity for systemic exposure and 

thus minimal hazard and/or risk to those in the field.

The pharmaceutical industry has explored the inhalation route for localized delivery of RNAi-based 

drugs to the upper respiratory tract (Bitko et al. 2005; Vaishnaw et al. 2010). Inhaled RNA 

oligonucleotide drugs have only localized activity in the upper respiratory tract, as they are not 

systemically absorbed to any meaningful extent (DeVincenzo et al.2008). Particle size is the key 

determinant for pulmonary exposure. Large particles typically deposit in the upper airways, resulting in 

clearing and swallowing, leading to secondary oral exposure rather than deposition in the deep lung 

tissue.

Agricultural workers can be exposed to plant material due to inhalation of pollen and agricultural dusts. 

Corn pollen is not respirable, as it consists of spherical particles ranging in size from 90 to 100 μm 

(Goldstein et al. 2004), in contrast with respirable particles that are less than 10 μm. In the case of 

agricultural dusts derived from activities such as planting, cultivation, and harvest, these particles also 

tend to be nonrespirable sizes (Goldstein et al. 2004). Exposure to RNA through agricultural dusts 

would also be limited as noncoding RNAs (e.g. dsRNAs, siRNAs) are located intracellularly. This 

information, taken together with extremely low expression of construct-derived RNA in plant tissues 

from MON 87411 indicates that there will be negligible inhalation exposures to these plant-derived 

RNAs. Exposure will primarily be secondary oral exposure after swallowing. Based on significant 

barriers to oral uptake of RNA and lack of oral toxicity of DvSnf7 RNA described previously, secondary 

oral exposure after breathing agricultural dusts and pollen containing construct-derived RNA from MON 

87411 would not present a health hazard to agricultural workers.

As noted above, negligible exposure to DvSnf7 RNA via the dermal and inhalation routes of exposure is 

expected due to vanishingly low levels in plant tissues, poor permeability of high molecular weight 

charged molecules such as RNA in the skin, and the limited respirability of agricultural dusts and pollen. 

Due to limited environmental exposures, there would be limited local or systemic exposures to DvSnf7 

RNA after dermal contact or inhalation of materials containing MON 87411. Although several of the 

biological barriers to ingested RNA are not relevant to dermal or inhalation exposure, nucleases and

various membrane barriers present a formidable challenge to any potential biological impact of 

exogenous RNA, both due to degradation and poor transport into and across tissues. The limited 

exposure, combined with nucleases in blood and tissues would ensure the degradation of DvSnf7 RNA 
Page 24 of 29

following any exposure to the skin or lung in agricultural workers, thus precluding any meaningful 

hazard or risk to exposed individuals.

Monsanto’s Conclusion: We have provided a thorough discussion outlining why DvSnf7 RNA from 

MON 87411 will not undergo dermal absorption nor inhalation based on its size, physicochemical 

properties, and biological barriers.

EPA’s Comment: The argument for low potential for significant exposure from dermal or inhalation 

exposure to the DvSnf7 dsRNA molecule expressed in corn is persuasive. The Agency reserves the 

right to require information on dermal or inhalation exposures for dsRNA in stabilized formulations if 

warranted.

EPA’s Request #5: Uptake of DvSnf7 dsRNA in human scenarios with altered absorption or 

digestion.

Monsanto Response to Request #5.

The extensive physical and biochemical barriers described in our previous submission (Lawrence 2014) 

and reviewed by Petrick and colleagues (Petrick et al. 2013) are multifaceted, consisting of pH, 

nucleases, and many membrane barriers (e.g. into GI cells, across GI cell to endothelium, across 

endothelium to blood, across endothelium to tissue, across tissue membrane, etc.). Thus, no single 

barrier is responsible for limiting functional uptake of exogenous dsRNA from the diet. Functional 

uptake implies ability not to reach the blood, but to reach a target tissue at a sufficient concentration to 

mediate the RNAi process. Although there are individuals that may have higher intestinal pH or

more permeable GI epithelium, there are still a multitude of other barriers that would preclude functional 

uptake of ingested RNAs. As indicated by Snow and colleagues, this requires 100 copies per cell in vitro 

and in healthy humans, orders of magnitude fewer copies are present following ingestion (Snow et al. 

2013). Given exceedingly low exposures from consumption of MON 87411, and the absence of 

formulations necessary to facilitate any appreciable systemic/ cellular uptake of RNA, even in the case 

of altered absorption or digestion, other physical and biochemical barriers would likely preclude

sufficient exposures to mediate potential biological activity.

Although several studies have indicated the potential for uptake of small RNAs from the diet in humans 

(Wang et al. 2012; Zhang et al. 2012a), these studies did small RNA profiling and did not characterize 

small RNA levels in diets and then look for uptake.

When dietary uptake was evaluated directly, data demonstrated that small RNAs are not taken up from 

the diet (Snow et al. 2013). Contamination is a likely source for detection of supposed diet derived small 

RNAs (Lusk 2014; Tosar et al. 2014; Zhang et al. 2012b). However, Wang and colleagues claim uptake 

from the diet in humans, yet, they do not show an increase in this purported uptake among colitis or 

colon cancer patients relative to healthy individuals (Wang et al. 2012), indicating that alterations in the 

GI tract do not lead to differences in exogenous blood small RNAs.

Pharmaceutical studies demonstrate a very short half-life for injected RNAs that have been chemically 

stabilized (Christensen et al. 2013), implying that absorption from the GI tract and digestive barriers are 

not the only critical barriers to potential activity of ingested RNAs. In addition, injection of a stabilized 

but unformulated RNA targeting a mouse gene did not demonstrate biodistribution to or gene 
Page 25 of 29

suppression in the liver or jejunum, despite an i.v. dose of 50 mg/kg (Soutschek et al. 2004), indicating 

that barriers outside the GI tract are sufficient to preclude activity of exogenous naked nucleic acids.

Such a dose is millions of times higher than anticipated dietary exposure to DvSnf7 RNA from MON 

87411. The short half-life and limited biodistribution of exogenous RNAs injected directly into the 

bloodstream and the very low exposures predicted for DvSnf7 RNA, coupled with no appreciable 

sequence similarity to human transcripts make humans with altered absorption or digestion unlikely to 

have any appreciable risk from exposures to DvSnf7 RNA. Lastly, susceptible individuals can be 

accounted for utilizing standard safety factors that are typically incorporated into risk assessments 

conducted by US EPA. Given the above information and the favorable safety profile identified for

DvSnf7 RNA in a repeat dose mouse oral toxicology study at 100 mg/kg (Wahle 2014), producing 

margins of exposure >1 x 108

, the weight of the evidence supports the conclusion that any humans with 

digestive deficiencies are not at risk from exposure to DvSnf7 RNA or MON 87411.

Monsanto’s Conclusion: DvSnf7 will not undergo appreciable uptake in compromised individuals due 

to redundancy of biological barriers. Although some barriers may be compromised in certain 

individuals, many others exist to preclude potential for oral activity. Risk assessment calculations 

indicate no adverse effects in rodents at DvSnf7 MOEs in excess of 108 times anticipated human 

exposures.

EPA’s Comment: EPA agrees that there are extensive and redundant systems that degrade free nucleic 

acids from an oral exposure. The intent of this request was to confirm that components in blood would 

indeed degrade the DvSnf7 RNA molecule, if any were to be present in the bloodstream due to altered 

membrane integrity of the gastrointestinal tract. Given a sensitive analytical system is available to 

specifically detect the intact molecule, a study with whole blood could address both degradation in blood 

and the expected lack of exposure even in cases of altered GI tract integrity. While the theoretical 

argument is sufficient to justify the seed increase registration, a confirmatory study adding DvSnf7 

dsRNA to whole blood and tracking its decay over time should be provided for commercial release. The 

Agency reserves the right to require information on stability in blood on more complex dsRNA 

structures if warranted.
Page 26 of 29

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