Page 2 of 29 Fungicide, and Rodenticide Act ( FIFRA ) Scientific Advisory Panel ( SAP ) meeting held on January 28,

2014

.

CONCLUSION:

T he Monsanto Compan y submitted product characterization data in support for a seed i ncrease 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 tr aditional breeding methods, with other registered corn events to provide protec tion against both above

- ground and below ground maize pests, as well as tolerance to

herbicides. T he new PIP technology express es the Bt Cry3Bb1 protein and DvSnf7 dsRNA to cont rol 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 suppress ion 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 casset te present in MON 88017

. Since t he protein is

identical to those in the currently registered PIP product (MON 88017)

, s everal comparative studies

were submitted to demonstrate the biochemical and functional protein s equivalence of Cry3B b1 and

CP4 EPSPS in PIP MON 87411 mai ze 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. T he results of a 28 day oral toxicity study indicate that DvSnf7 is

not toxic to mamm als 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 include d : 28 day oral study in mice ; 90 day feeding study in rats; Southern blot analyses for confirmation of the molecular identity ; amino acid sequenc e analyses ; Western blot analyses for d etermination of the protein molecular weight s , immunoreactivity, functional activity and glycosylation status ; Northern blot analys es for determination of molecular equi valence 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 submit ted 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. Collective ly, the results support the physico chemical and functional equivalence for the Cry3Bb1 and CP4 EPSPS proteins expressed in

MON 874 11 maize to the corresponding proteins expressed in PIP MON 88017

.

The data submitted an d reviewed for Cry3B b1 and CP4 EPSPS proteins as expressed in M ON 874 11 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 t he 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, t here is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, includi ng infants and childr en, to the dsRNA transcript compris ing a DvSnf7 inverted repeat sequence and the Cry3Bb1 protein and the ge ne tic material necessary for its production in MO N

874 11 corn . This includes all anticipated dietary exposures and all other exposures for which ther e 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 h as been confirmed as sufficiently equiv alent to currently registered plant

- incorporated
protectant s

.

RECOMMENDATION:

The submitted product characterization and toxicity data are suf ficient to support Monsanto’s application for a seed increase Sec. 3 Registration of MON 874 11 maize as a pl ant

- incorporated
protectant.Page 4 of 29 DATA REVIEW RECORD Active Ingredients: DvSnf7 ds RNA and Bacillus thuringiensis ( Bt ) Cry 3 B b1 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 20 11

- 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 a nd 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

- P roduced 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 E valuation 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 Te chnology 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 Sp r ague 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.

49 576101 Response to the Request for Additional Information for MRID Nos: 49505804 and 49505806 (Re: Mon 87411)

BACKGROUND

M onsanto Company has developed a biotechnology

- derive d maize, MON 87411 (OECD unique

identifier MON 87411

- 9) , that confer protect ion 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 virgife ra
, 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 87 411 is recognized by the CRW’s

RNA interfer ence (RNAi ) machinery resulting in the down

- regulation of the targeted Dv Snf7 gene leading to CRW mortality

. MON 87411 also contains a cry3Bb1 coding sequence that produce s a modified Bacillus thuringiensis (subs p. 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 , whic h confers tolerance to glyphosate, the active ingredient in Roundup 1 ® 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 f amily 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 expressi on cassette present in MON 88017

.

The following sections summarize the regulatory information for the currently registered PIP events that will be used to suppor t 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 >9 5% amino sequence homology. The Cry3Bb1 protein is also pro duced 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 C ry3Bb1 protein from this cassette was c onfirmed by SDS

- PAGE and determination of

the N

- terminus (# amino acids sequenced) to be identical to corn event MON 86 3 . The Cry3Bb1 protein

does not exhibit similarity to known mammalian toxins or allergens, is rea dily 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 87411 corn 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 f ood /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 plant

incorporated protectant inert ingr edient in all food commodities ( 40 CFR § 174 .523 )

. Therefore

, the existing Federal Food, Drug and Cosmetic Act ( FFDCA ) Food Safety Assessment s 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 lo ng 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 insec ticidal 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. U pon 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 B acillus thuringiensis (subsp.

kumamotoensis ) Cry3Bb1 protein to protect against CRW larval feeding. In addition, MON 87411 produce s the 5

- enolpyruvylshikimate

- 3

- phosphate synthase protein from Agrobacterium sp . Strain CP4 (CP4 EPSPS), which confers tolera nce 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 tumef aciens 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 th e 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 c ontain s an inverted repeat sequence (named DvSnf7 ) derived from Diabrotica virgifera , the West ern 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 mechani sms 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 sho rt interfering siRNAs by the nuclease Dicer (Fire A, et al , 1998). The siRNAs then bind to a complex of protein s know n 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.

D v 49 dsRNA is 240 base pair s (bp) long . Dv49 siRNA is 21

- 24 mer. High molecular weight (HMW) northern blot show ed all detectable Dv49 sequences are double stran ded. 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 produce s 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 (gene rated 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 Dv Snf7 suppression cass ette, 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 ide ntified 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 sta bility 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 bacteria l 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 proteinsPage 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 c haracterized 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 as sisted 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

wa s 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 pro tein. 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. Pu rity 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 showe d 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 LC 50 of 0.77 μ g/ml diet using C olorado 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 cha racteristics, 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 cont ained 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 equi valence 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 purifi ed 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 (MALD I

- 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 pepti de 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 cha racterized 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 fr eezer (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 Exp ression

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 (Dv Snf 7_968 RNA) in order to generate sufficient quantities of DvSnf7 RNA for safety assessments. The demon stration of the molecular equivalence

between DvSnf7 RNA in MON 87411 and Dv Snf 7_968 RNA justifies the use of Dv Snf7 _968 RNA to demonstrate the safety of DvSnf7 RNA in MON 87411. DvSnf7 RNA in MON 87411 was characterized by sequencing its transcript as we ll 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 Dv Snf 7 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 Dv Snf 7

_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 puri fied 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 (op en 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 searc h, using the open reading frames/ c oding regions search criteria for endogenous corn small RNAs did not produce any matches between DvSnf7 RNA andPage 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 a cid component of MON 8 7411 . 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 af firmed ( 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 syst ems 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 sensiti ve 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 presen t 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 a l., 2015) . In addition, t he Agency reserves the right to require information on stability in blood on more complex dsRNA structures if warranted

.

The submitted studies verify the Cry3B b1 protein ex pressed in MON 87411 are biochemically and functionally equivalent with the Cry protein expressed in currently regis tered PIP MON 88017 corn. Therefore, the data submitted a nd 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 Cry3 Bb1 [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, t here is a reasonable certainty that no harm will resul t from aggregate exposure to the U.S. population, includi ng infants and children, to DvSnf7 dsRNA, Cry3B b1 and CP4 EPSPS protein s and the

ge netic material necessary for their production in MON 87411 . This includes all anticipated dietary exposures and all other exposures for which th ere is reliable information. The Agency has arrived at this

conclusi on because no toxicity t o 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 S ec. 3 registration of PIP

MON 87411 corn expressing Cry3B b1 and CP4 EPSPS proteins are summarized in Table 1. For detai led 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 . Pr oduct Characterization D ata for PIP MON 87411 Maize expressing Bt Cry34Bb1 and CP4 (CP4 EPSPS) proteins [EPA Reg. No. 524

- A RI ]

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 con tains 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 ne xt

- generation sequence data (comprehensively covering the genome) was used to characterize the insert number of DNA sequences from the transformation plasmid vector PV

ZMIR10871. The data indicated that MON 87411 contains a single integration

locus of PV

- Z MIR10871 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 th e 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

sequen cing data also identified the 5' and 3' insert

- to

- flank junctions in

MON 87411 and characterized the insertion site in c onventional maize. Furthermore, bioinformatic analysis of next

- generation sequence data (comprehensively covering the genome) was used t o 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 fiv e 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 wei ght (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 49315104Page 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 i n 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 MO N 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 eq uivalence 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 funct ional 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 Physicochemic al 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 su fficient 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

. col i

- 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 pur ified 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 N

terminal 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 t he 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 prote in 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

functio nal 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 LC 50 , of 0.77 μg/ml diet using Colorado Potato Beetle. Finally, assessment of the purity and appar ent 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. 49315107Page 14 of 29 STUDY TYPE/ TITLE SUMMARY MRID NO

The equivalence of the MON 87411

- produced and E. coli

- produced Cr y3Bb1 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 isolate d 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

- Produc ed

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 curren t 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 sa fety 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 a nd 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 a mino 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

- produc ed 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 a nd 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 Te chnology 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 yea st 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 do se 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 con trols). 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 b etween 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 con trol 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 t o 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 tre atment

- 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 49 505804

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). Evalua ted parameters included clinical signs, mortality, functional observation battery (FOB), body weight, food consumption, clinical pathology, organ weights, and gross and histopathology 49505806

49576101Page 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. Func tional 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 a nd 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 substance

treated groups.

All test substance

- treated group animals survived to the scheduled nec ropsy. 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 administrat ion.

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 n ot 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 id entify 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 o f 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%. T he Cry3Bb1 protein in MON 863 has been termed “Cry3Bb1.11098 49553303Page 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 enc oded 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 Nter minal end of the protein is blocked while new technology has

allowed demonstration that the first methionine is removed from Cry3Bb1 in planta.

CLASSIFICATION: ACCEPTABLEPage 18 of 29 Per the recommendation in the report from the EPA’s FIFRA SAP meeting h eld 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 recommendat ions 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 c assette 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 alterin g 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 produce s 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

.

Mon santo Response to Request # 2.

E xtensive 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 b iological barriers include nucleases in the saliva and gastrointestinal tract, acidicPage 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 Dv Snf7 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 μ g/gram fresh weight or 91 parts per trillion) and 95th percentile consumption values in non

- n ursing 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 bioav ailable (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 (Molito ris 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 muc h 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 exposur es 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 10 8 (based on 95 th 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 app reciable 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 assessme nt 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 barr iers 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 syst ems 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 sensiti ve 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 presen t 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 a l., 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 f rames/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 considera tion 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 ap plications 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

bioin formatics 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 furt her 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 ( r eviewed by Petrick et al. 2013). However, these studies have utilized in vitro models employing high doses of RNAPage 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 exce edingly 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 i n 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%

identit y 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 m olecules 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, C T) 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 clo ning 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

u sed 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 RNA s 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 diet ary 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 eva luating 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 no t 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 t he 240 bpPage 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 RN A and hu man 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 addi tional 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 S hort 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 5 00 447 150

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 bioinformati cs 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 : E PA 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 sequen ces 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 ameliorat es 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 d egradation 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). H owever, 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 co nstruct

- 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 fo r 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 a nd 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, n egligible 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 respirabilit y 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 t o 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 RNAPage 24 of 29 following any exposure to the skin or lung in agricultural workers, thus precluding any meaningful hazard or risk to exposed individuals.

Monsa nto’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 r eviewed 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 m embrane, 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 req uires 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 facil itate 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 stud ies 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 uptak e 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, W ang 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 no t 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 barri ers 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 genePage 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 10 8 , 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 Comme nt : 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 expec ted 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 pro vided for commercial release. The Agency reserves the right to require information on stability in blood on more complex dsRNA

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