# SAP Minutes No. 2014- 02

A Set of Scientific Issues Being Considered by the Environmental Protection Agency Regarding:

RNAi Technology: Program Formulation for Human Health and Ecological Risk Assessment

January 28, 2014 FIFRA Scientific Advisory Panel Meeting

Held at One Potomac Yard Arlington, Virginia2 TABLE OF CONTENTS

NOTICE ................................................................................................................................................................ 4
PANEL ROSTER ................................................................................................................................................. 6
COMMONLY USED TERMS AND ACRONYMS .......................................................................................... 8
INTRODUCTION .............................................................................................................................................. 10
PUBLIC COMMENTERS ................................................................................................................................ 12
OVERALL SUMMARY .................................................................................................................................... 14
EXECUTIVE SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS ............................ 16
OVERVIEW OF THE RNA INTERFERENCE PATHWAY ....................................................................... 26 DETAILED RESPONSES TO CHARGE QUESTIONS ............................................................................... 27 Q UESTION 1. P LEASE DISCUSS THE NATURE AND EXTENT OF UNCERTAINTY IN THE SPECIFICITY OF LONG SEQUENCES OF DS RNA TARGETED AT PEST SPECIES , IF BIOINFORMATIC ANALYSIS SHOWS NO SIGNIFICANT SIMILARITY TO MAMMALIAN GENES ...................................................................... 27 Q UESTION 2. B ASED ON DATA INDICATING DEGRADATION OF THE MAJORITY OF DS RNA IN THE DIGESTIVE SYSTEM , PLEASE DISCUSS THE STRENGTHS AND LIMITATIONS IN CONCLUDING THERE WILL NOT BE SIGNIFICANT ABSORPTION OF DS RNA WITH POSSIBLE MAMMALIAN EFFECTS ON ORAL EXPOSURE ................................................................................................ ..............................29 Q UESTION 3. T O WHAT EXTENT DOES THE SPECIFIC STRUCTURE OF DS RNA, IF IT IS SUPER COILED OR IN A HAIRPIN STRUCTU RE , MAKE IT MORE LIKELY TO SURVIVE DEGRADATION IN THE GUT AND LEAD TO POSSIBLE MAMMALIAN EFFECTS WITH ORAL EXPOSURE ?

.................................................. 38 Q UESTION 4. E NVIRONMENTAL F ATE ................................................................ ................................... 42
4.a.i) To what extent will data on degradation in soil inform EPA on nontarget exposure to dsRNA PIPs? ...... 42
4.a.ii) What additional information, if any, would EPA need to assess environmental fate of dsRNA PIPs? ..... 45
4.b. In section IV.B.4., EPA proposed environmental fate data needs for exogenously applied dsRNAs. To what extent is the proposed testing sufficiently robust to inform nontarget organism exposure for these dsRNAs?47
4.c. What analytical methods are available to accurately and precisely measure dsRNAs in diverse plant, soil, and water matrices? ....................................................................................................................................... 48
Q UESTION 5. P RIMARY R OUTE OF E XPOSURE FOR N ONTARGET O RGANISMS TO DS RNA

................. 48
5.a.i) In addition to the conditions of the gut environment and enzymes influencing digestion, what other factors may play a role in uptake within the gut and potentially limit exposure to dsRNA? ..................................... 49
5.a.ii) Please comment on how these barriers can be generalized across all nontarget taxa that are considered in EPA’s risk assessments (e.g., birds, plants, fish, etc.) for both dsRNA PIPs and exogenously applied dsRNA. ....................................................................................................................................................................... 503

5.b.i) Please comment on the importance of the contact route of exposure for nontarget risk assessments for each of these types of dsRNAs ................................................................................................................................ 51
5.b.ii) What barriers are likely to exist for this route of exposure for both terrestrial and aquatic organisms, and how can these be generalized across nontarget taxa? ................................................................................... 51
Q UESTION 6. U NINTENDED E FFECTS OF DS RNA

.................................................................................. 52 6.a. Please comment on the unintended effects that EPA might reasonably anticipate in nontarget organisms exposed to dsRNAs, the likelihood of such unintended effects, and the biological significance of these effects in nontarget organisms, should they occur. ................................................................................................... 52
6.b. To the extent that additional information would reduce uncertainty in addressing these issues, please describe specifically the nature of additional information that EPA may need and the degree to which this information would reduce uncertainty in the ecological risk assessment. ..................................................... 56
Q UESTION 7. F RAMEWORK OF T ESTING FOR N ONTARGET E FFECTS ................................................... 57 7. a. Please comment on how each of EPA’s current PIP and biochemical frameworks for nontarget effects testing will inform risk assessment for dsRNA PIPs and exogenously applied dsRNAs. In providing a response, please address the potential for unintended effects as described in Question 7, as well as the three issues outlined as concerns for nontarget testing .......................................................................................... 57
7.b. What additional nontarget effects testing, if any, should EPA consider to gain a full understanding of the potential for dsRNAs to cause effects to nontarget organisms? .................................................................... 60
7.c. What other approaches, such as bioinformatics analysis, may be used to address concerns for effects on nontarget species and reduce the set of data requirements? ......................................................................... 60
7.d. In providing answers to the above subquestions, please be specific in discussing the extent to which additional information would reduce the nature and magnitude of these specific areas of uncertainty. ...... 60

REFERENCES ................................................................................................................................................... 654

NOTICE

These meeting minutes have been written as part of the activities of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), Scientific Advisory Panel (SAP). The meeting minutes represent the views and recommendations of the FIFRA SAP, not the United States Environmental Protection Agency (EPA or the Agency). The content of the meeting minutes does not represent information approved by the Agency. The meeting minutes have not been reviewed for approval by the Agency and, hence, the contents of these meeting minutes do not necessarily represent the views and policies of the Agency, nor of other agencies in the Executive Branch of the Federal government, nor does mention of trade names or commercial products constitute a recommendation for use.
The FIFRA SAP is a Federal advisory committee operating in accordance with the Federal Advisory Committee Act and established under the provisions of FIFRA as amended by the Food Quality Protection Act (FQPA) of 1996. The FIFRA SAP provides advice, information, and recommendations to the Agency Administrator on pesticides and pesticide-related issues regarding the impact of regulatory actions on health and the environment. The Panel serves as the primary scientific peer review mechanism of the Environmental Protection Agency (EPA), Office of Pesticide Programs (OPP), and is structured to provide balanced expert assessment of pesticide and pesticide-related matters facing the Agency. FQPA Science Review Board members serve the FIFRA SAP on an ad hoc basis to assist in reviews conducted by the FIFRA SAP. Further information about FIFRA SAP reports and activities can be obtained from its website at http://www.epa.gov/scipoly/sap/ or the OPP Docket at (703) 305-5805. Interested persons are invited to contact Sharlene R. Matten, Ph.D., SAP Designated Federal Official, via e-mail at matten.sharlene@epa.gov

. This report was reviewed by Laura E. Bailey, M.S., FIFRA SAP Executive Secretary.
In preparing these meeting minutes, the Panel carefully considered all information provided and presented by EPA, as well as information presented in public comment. This document addresses the information provided and presented by EPA within the structure of the charge.

# SAP Minutes No. 2014-02

A Set of Scientific Issues Being Considered by the Environmental Protection Agency Regarding:

RNAi Technology as a Pesticide: Problem Formulation for Human Health and Ecological Risk Assessment

January 28, 2014 FIFRA Scientific Advisory Panel Meeting Held at

Daniel Schlenk, Ph.D. FIFRA SAP Chair 

# One Potomac Yard Arlington, Virginia



# a:;_cientific Advisory 

# Panel Sharlene R. Matten, Ph.D. Designated Federal Official FIFRA Scientific AdvisoryPanel Staff

# Da~:4~/**f** Date:6

PANEL ROSTER

FIFRA SAP Chair
Daniel Schlenk, Ph.D. Professor of Aquatic Ecotoxicology & Environmental Toxicology Department of Environmental Sciences University of California, Riverside Riverside, CA
Designated Federal Official Sharlene R. Matten, Ph.D. U.S. Environmental Protection Agency Office of Science Coordination & Policy FIFRA Scientific Advisory Panel EPA East Building, MC 7201M 1200 Pennsylvania Avenue, NW Washington, DC 20460 Tel: 202-564-8450, E-mail: matten.sharlene@epa.gov
FIFRA Scientific Advisory Panel Members
K. Barry Delclos, Ph.D. Pharmacologist Division of Biochemical Toxicology National Center for Toxicological Research U.S. Food & Drug Administration Jefferson, AR
Marion F. Ehrich, Ph.D., D.A.B.T., A.T.S. Professor of Pharmacology and Toxicology Department of Biomedical Science and Pathobiology Virginia-Maryland Regional College of Veterinary Medicine Virginia Polytechnic Institute and State University Blacksburg, VA
Stephen J. Klaine, Ph.D. Professor and Director Clemson University Institute of Environmental Toxicology Department of Biological Sciences Pendleton, SC James L. McManaman, Ph.D. Professor and Chief Section of Basic Reproductive Sciences Departments of Obstetrics and Gynecology & Physiology and Biophysics University of Colorado-Denver Aurora, CO
Martha S. Sandy, Ph.D., M.P.H. Supervising Toxicologist and Chief Reproductive & Cancer Hazard Assessment Branch Office of Environmental Health Hazard Assessment California Environmental Protection Agency Oakland, CA7

FQPA Science Review Board Members
George P. Cobb, Ph.D. Professor and Chair Department of Environmental Science Baylor University Waco, TX
Brian D. Gregory, Ph.D. Assistant Professor of Biology Department of Biology University of Pennsylvania Philadelphia, PA
Jonathan G. Lundgren, Ph.D. Research Entomologist North Central Agricultural Research Laboratory United States Department of Agriculture Brookings, SD Brenda S. Oppert, Ph.D. Research Molecular Biologist Center for Grain and Animal Health Research U.S. Department of Agriculture Manhattan, KS
Guy Smagghe, Ph.D. Professor Department of Crop Protection Ghent University Ghent, Belgium
Kenneth W. Witwer, Ph.D. Assistant Professor Department of Molecular and Comparative Pathobiology Johns Hopkins University School of Medicine Baltimore, MD8

COMMONLY USED TERMS AND ACRONYMS

Term /Acronym Description
Argonaute Members of the Argonaute protein family are the part of RNA

- induced silencing complex responsible for cleavage of the mRNA as directed by small RNAs.

Bt Bacillus thuringiensis , a soil microorganism

DNases D eoxyribonuclease s

Dicer Dicer is a ribonuclease RNase III

- like enzyme that processes long double

- stranded RNA (dsRNA) or pre

- micro R NA hairpin precursors into small interfering RNAs (siRNAs) or micro RNAs (miRNAs).

dsRNA Double

- stranded RNA

GI tract Gastrointestinal tract

mRNA Messenger RNA

miRNA M icro RNA

Non

- PIP Non plant

- incorporated protectant

nt Nucleotide

PIP Plant

- incorporated p rotectant is a term used by the EPA to describe the pesticidal active ingredient and the genetic material necessary for its product ion in a plant.
qPCR Quantitative reverse transcription PCR assays

RDRPs RNA

- dependent RNA polymerases

RISC RNA

- induced silencing complex

RNA Ribonucleic acid

RNA interference Gene silencing process initiated by dsRNA

RNAi RNAi interference9

Term /Acronym Description

RNA

- Seq An approach to transcriptome profiling that uses deep

- sequencing technologies

RNases Ribonucleases

RNPs ribonucleoprotein complexes

rRNA Ribosomal RNA

siRNA Small interfering RNA

ssRNA Single

- stranded RNA

SID

- 1 Transport protein that mediates cellular uptake of dsRNA

Sid1 Gene encoding SID

- 1

SID

- 2 Transport protein that mediates cellular uptake of dsRNA

Sid

- 2 Gene encoding SID

- 2

SNP Single nucleotide polymorphism10

INTRODUCTION
The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) has completed its report of the SAP meeting regarding scientific issues associated with “ RNAi Technology as a Pesticide: Problem Formulation for Human Health and Ecological Risk Assessment

. ” Advance notice of the SAP meeting was originally announced in the Federal Register on August 15, 2013 (78 FR 49750)

. Following the exten ded government

- wide shutdown, the Agency issued a notice of cancellation in the Federal Register on October 28, 2013 (78 FR 64211). The meeting was rescheduled for January 28, 2014 and announced in the Federal Register on November 15, 2013 (78 FR 68836).

The SAP reviewed the Agency’s charge in an open Panel meeting on January 28, 2014 at One Potomac Yard, Arlington, Virginia. Materials for this meeting are available in the Office of Pesticide Programs (OPP) public docket or via www.regulations.gov
, Docket No. EPA

- HQ

- OPP

- 2013

- 0485

.
Daniel Schlenk, Ph.D., chaired the meeting. Sharlene Matten, Ph.D., served as t h e Designated Federal Official. Steven Bradbury, Ph.D., Director, Office of Pesticide Programs (OPP)
, and Robert McNally, Director, Biopesticides and Pollution Prevention Divisi on, OPP, provided introductory remarks at the meeting

. Technical presentations were provided by the following individuals : Chris Wozniak, Ph.D., John Kough, Ph.D., Shannon Borges, and Russell Jones, Ph.D, all from the Biopesticides and Pollution Prevention Division, OPP.

Interference or suppression of gene expression by naturally occurring double stranded ribonucleic acid

- based mechanisms was largely unknown or misunderstood until the work of Andrew Fire and Craig Mello (Fire et al., 1998) which earned them the Nobel Prize in Physiology or Medicine in 2006. Working with Caenorhabditis elegans , a common saprobic nematode, the laboratories of Andrew Fire and Craig Mello ascertained th at injected double

- stranded RNA ( dsRNA ) sequences corresponding to known gene sequences in the nematode genome w ere quite effective at gene silencing (Fire et al., 1998; Grishok and Mello, 2002). Evidence for naturally occurring gene silencing, and that resulting from introduction of foreign DNA or RNA sequences, has been noted in numerous prokaryotic and eukaryotic

o rganisms to date. Long dsRNA molecules are thought to be critical to triggering RNA interference (RNAi) . The long strands of dsRNA that trigger RNA interference can originate outside the cell or be internally produced. Small interfering RNA s (siRNA s ) are t he product of Dicer (a ribonuclease III

- like enzyme ) activity on longer molecules of dsRNA. The siRNA molecules produced by D icer are also double

- stranded from 21

- 24 nucleotides (nt) in size with a 2 nt overhang at the 3’end.

In the past several years, a number of pharmaceutical and agricultural products based on RNA i have been developed. Most of these products are pharmaceuticals aimed at turning off aberrant protein expression (e.g., cancer genes or macular degeneration), but a few agricultural products utilizing RNA interference have also been developed for p est control. Development of new RNAi based pesticidal products led the EPA to convene this SAP meeting to consider aspects of the current biopesticide risk
assessment framework.11

The current framewor k for the risk assessment of biopesticides is found in the EPA’s RNAi issue paper ( EPA
, 2013). The approach for mammalian safety assessment of plant

- incorporated protectants ( PIPs ) and biochemical pesticides can be foun d at the start of section III. The fr amework for ecological safety assessment for PIPS and biochemical pesticides can be found in section IV A and B. Biopesticide products are likely to be developed utilizing RNAi technology as both dsRNA PIPs expressed in plants and non

- PIP dsRNA end

- products that may be externally applied for pest control.

The focus of this SAP meeting was on the problem formulation phase of risk assessment of pesticidal products based on RNA interference (RNAi). Problem formulation is the first step in the risk

as sessment process and guides the subsequent questions about hazard and exposure (EPA 1998 ). In problem formulation, available information is used to define assessment endpoints and to develop a preliminary understanding of potential risks (i.e., develop a r isk hypothesis and conceptual model) associated with the use of a pesticide. Problem formulation also serves as an opportunity to identify missing information and uncertainties that may limit the assessment and any assumptions that may be made in the absen ce of such data.

EPA consulted with the SAP on scientific issues that might be unique to RNAi and how they could fit under the existing risk assessment framework. The SAP addressed seven charge questions divided into human health considerations (questions 1

- 3) and ecological risk considerations (questions 4

- 7) as shown below.

Human Health Considerations :

1. Nature and extent of uncertainty in the specificity of long sequences of dsRNA targeted at pest species and relatedness to human sequences ; 2. Factors that may limit exposure to dsRNA: ext ent of digestion of dsRNA and effect on absorption ; 3. Effect of the s tructure of dsRNA on deg radation in the gut and exposure to dsRNA

. Ecological Risk Considerations :

4. The environmental fate of the dsRNA; 5. Factors that may limit non

- t arget organism exposure to dsRNA ; 6. Potential unintended effects in no n

- target organism s expos ed to dsRNA ; 7. Framework of testing for non

- target effects

.12

PUBLIC COMMENTERS
Oral statements were presented by:

1. Taiwo Koyejo, Ph.D., Manager, Regulatory Affairs, Plant Biotechnology, CropLife International 2. Raymond Layton, Ph.D., on behalf of the Agricultural Biotechnology Stewardship Technical Committee (ABSTC)

- Non

- Target Organism Subcommittee 3. Jay Petrick , Ph.D.
, David Carson , Ph.D., and Steven Levine , Ph.D. on behalf of Monsanto Company 4. Craig Mello, Ph.D., Howard Hughes Medical Institute and Department of Molecular Medicine, University of Massachusetts Medical School (The Nobel Prize in Physiology or Medicine 20 06 was awarded jointly to Andrew Z. Fire and Craig C. Mello "for their discovery of RNA interference

- gene silencing by double

- stranded RNA ." ) 5. Alaina Sauv é
, M.S., Syngenta 6. James White, Ph.D. Regulatory Affairs Manager, Citrus Research Development Foundation 7. Michael Barrett, Ph.D., Weed Science Society of America 8. Scott Slaughter, Center for Regulatory Effectiveness 9. Stephen McFadden, M.S., Independent Scientific Research Advocates (ISRA)

Written statements were provided by:

1. Jack A. Heinemann, P h.D. University of Canterbury, Christchurch, New Zealand 2. Doug Kornbrust, Ph.D., D.A.B.T. President, Preclinsight 3. Brett Adee and Dave Hackenburg, Co

- Chairs, National Honey Bee Advisory Board 4. Belinda Martineau, Ph.D., University of California

- Davis 5. Step hen Whyard, Ph.D., Department of Biological Sciences, University of Mannitoba 6. James D. Thompson, Ph.D., Vice President, Development, Quark Pharmaceuticals 7. Andrew F. Roberts, Ph.D., Deputy Director, Center for Env ironmental Risk Assessment, International Life Sciences Institute (ILSI) Research Foundation 8. Subba Palli, Ph.D., Department of Entomology, University of Kentucky 9. Robert Sears, President, Ea stern Missouri Beekeepers Association 10. private citizen anonymous 11. Dan Jenkins on behalf of Monsanto Company 12. Stephen Chan, M.D., Ph.D., Harvard Medical School 13. E. Keith Menchey, Manager, Science & Environmental Issues, National Cotton Council (NCC) 14. Diana L. Cox

- Foster, Ph.D., Department of Entomology, The Pennsylvania State University 15. Nancy B. Beck, Ph.D., D.A. B.T., Senior Director, Regulatory and Technical Affairs, American Chemistry Council (ACC) 16. James White, Ph.D. Regulatory Affairs Manager, Citrus Research Development Foundation 17. Craig Mello, Ph.D., Howard Hughes Medical Institute and Department of Molecular Medicine, University of Massachusetts Medical School 18. James Carrington, Ph.D., President, Donald Danforth Plant Sciences Center13

19. Raymond Layton, Ph.D. on behalf of the Agricultural Biotechnology Stewardship Technical Committee (ABSTC)

- Non

- Target Organism Subcommittee 20. Randy Oliver, contributor to the American Bee Journal and site manager of Scientific Beekeeping.com 21. Taiwo Koyejo, Ph.D., Manager, Regulatory Affairs, Plant Biotechnology, CropLife International 22. Ray Gaesser, President, American Soybean Associ ation 23. Ian MacLachlan, Ph.D., Executive Vice

- President and Chief Technology Officer, Tekmira Pharmaceuticals Corp. 24. Lee Van Wychen, Ph.D., Director of Science Policy, Weed Science Society of America 25. Barbara Glenn, Ph.D., Senior Vice President, Science & Regu latory Affairs, CropLife America 26. Cyndi Heintz, Executive Director, Project Apis m. 27. Wenonah Hauter, Executive Director, Food & Water Watch 28. Martin Barbre, President, National Corn Growers Association14

OVERALL SUMMARY

The focus of this SAP meeting was on the problem formulation phase of the human health and environmental risk assessment of pesticidal products using RNA interference technology (RNAi). EPA consulted with the SAP on scientific issues that might be unique to RNAi and how they could fit unde r the existing risk assessment framework. The SAP addressed seven charge questions divided into human health considerations (questions 1

- 3) and ecological risk considerations (questions 4

- 7). The Panel provided the following overall summary of the major co nclusions and recommendations detailed in the report.

Human Health Considerations

– Major Conclusions and Recommendations

The Panel agreed that bioinformatic analysis can be used to identify specific nucleotide identity in long sequences of dsRNA or processed shorter products that could bind in a siRNA

- like fashion, provided the sequences are in the database and the appropriate algorithm is used. Sequence identity does not mean that the dsRNA would provoke an RNAi response, but should caution the potential for unintended interactions and further review.

The Panel agreed with EPA’s assessment that the primary route of exposure is thro ugh the oral ingestion of dsRNA PIP s in food plants or food plants treated with exogenously applied dsRNAs end

products. Dietary RNA is extensively degraded in the mammalian digestive s ystem by a combination of ribonucleases (RNases) and acids that are likely to ensure that all structural f orms of RNA are degraded throughout the digestive process. There is no convincing evidence that ingested dsRNA is absorbed from the mammalian gut in a form that causes physiologically relevant adverse effects. Evidence of any dietary uptake plant mi cro RNA s (miRNAs) in mammals is nominal and non

- specific.

Recommendations

 The Panel recommended collection of data on dsRNAs PIP abundance and tissue distribution to investigate factors that may affect absorption and effects of dietary dsRNAs.

 The Panel recommended experimental testing of the mammalian blood and exposed tissues be done to ensure that the siRNAs processed from the PIP dsRNAs are not present, since these could have off

- target effects after human consumption.

 The Panel recommended that stability of different structural forms of dsRNAs be addressed for dermal and inhalation routes of exposure.

 The Panel recommended that the stability of dsRNA in individuals that manifest diseases
, immune compromised, elderly, o r children be investigated.

Ecological Risk Considerations

– Major Conclusions and Recommendations
Overall, the Panel agreed with the concerns raised by the EPA regarding the inadequacies of the current environmental fate and non

- target effects testing fr ameworks for dsRNA PIPs and exogenously applied dsRNA products. Uncertainties in the potential modes of action in non

- target species, potential15

for chronic and sublethal effects, and potential unintended consequences in the various life stages of

non

- targe t organisms are sufficient justification to question whether the current Agency framework for ecological effects testing is applicable to dsRNA PIPs or exogenously applied non

- PIP end

- use products. Due to the modes of action of RNAi , no one set of test spe cies will serve as an adequate representation of non

- target species for all pesticidal products using RNAi technology. The classic approach of developing and assembling effects data for a standard set of test species will likely not work well for this tech nology.
Current requirements for soil degradation studies will provide some insight for non

- target testing, but are inadequate to address the environmental fate of dsRNA PIPs or exogenously applied dsRNA products and potential exposures to non

- target orga nisms. The inability to predict dsRNA exposure to

non

- target organisms using the current non

- target testing framework challenges the evaluation of potential non

- target effects.

The Panel concluded that additional data are needed to reduce uncertainty in t he environmental fate and ecological risk assessments; however, the task of prescribing additional tests or additional test species cannot be done without a better understanding of exposure to dsRNA PIPs or exogenously applied dsRNA products , and the RNAi modes of action

. Recommendations

 The Panel recommended an exposure

- based conceptual model to reduce the uncertainties in the ecological risk assessment of dsRNA PIPs and dsRNA non

- PIP end

- products. This approach would identify non

- target organisms that mi ght be exposed and narrow the spectrum of non

- target organisms that would need to be tested for effects. Using the exposure

- based conceptual model for ecological risk assessment will further the understanding of the likelihood of adverse outcomes in non

- ta rget species, facilitate the selection of endpoints, and identify potential data gaps that would require further study.

 The Panel recommended the following information be collected to address uncertainties in the environmental fate and ecological risk assessments:  Determine soil degradation kinetics for both dsRNA PIPs and exogenously applied dsRNA end

- products. Collect data representing different soil types, soil biology, and abiotic factors such as weather, soil temperature and moisture under which ds RNA PIP crops are grown or where dsRNAs products are applied.  Determine plant degradation kinetics for dsRNA PIPs and consider plant materials (above and below ground), off

- site movement of plant materials, and in planta exposure for organisms that directl y ingest plant materials.  Determine environmentally

- relevant dosages, the importance of physical barriers in non

target organisms concerning degradation and cellular uptake of dsRNA, and off

- target effects to non

- target organisms.  Evaluate the potential f or chronic and sublethal effects and unintended consequences in the various life stages of non

- target organisms.  Consider possible immunostimulatory effects and impacts on food webs.  Evaluate the potential for pest resistance.16

EXECUTIVE SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS

Human Health Considerations
Question 1. Please discuss the nature and extent of uncertainty in the specificity of long sequences of dsRNA targeted at pest species, if bioinformatic analysis shows no significant similarity to mammalian genes

. Summary
The Panel agreed that bioinformatic analysis can be used to identify specific nucleotide identity in long sequence s of dsRNA or processed shorter products that could bind in a siRNA

- like fashion, provided the sequences are in th e database and the appropriate algorithm is used. The certainty in the matches is dependent on the reliability and completeness of the sequence information in existing databases, the algorithm used, and the presence of single nucleotide polymorphisms (SNPs ). It is important to emphasize that this is a predictive tool, but not absolute. Sequence i dentity does not mean that the dsRNA would provoke a n RNAi response, but should caution the potential for unintended interactions and further review. The Panel suggested that dsRNA sequence

- specific factors may increase target specificity and reduce the likelihood of unintended effects. If the charge questio n is interpreted more broadly to include miRNA

- like effectors and modes of binding, sequence predictions are much less reliable.

Question 2. Based on data indicating degradation of the majority of dsRNA in the digestive system, please discuss the strengt hs and limitations in concluding there will not be significant absorption of dsRNA with possible mammalian effects on oral exposure

.

Summary
Dietary RNA is extensively degraded in the mammalian digestive system. In contrast with some other organisms, mech anisms for RNA uptake from the environment, RNAi amplification by RNA

dependent RNA polymerases (RdRPs) , and systemic RNAi are not known or are not robust in mammals. The Panel concluded that available evidence supports the conclusion of no significant absorption of dsRNA in mammals and minimal likelihood of adverse effects. Th is evidence consists of:

 Observations that ingested dsRNAs undergo rapid degradation in the mammalian digestive tract,

 The history of safe consumption of plants containing RNA by humans and other mammals
,

 T he history of challenges developing effective oral RNAi therapeutics which can withstand the hostil e environment of the human gut,

 Findings from three published studies of no significant mammalian uptake of dietary miRNA

re sulting in functional consequences . These findings call into question the study of L. Zhang et al. (2012) , which reported mammalian uptake of dietary miRNA resulting in an effect on cholesterol metabolism

.17

However, the Panel stressed that data are lackin g in this area and there are no published PIP

specific studies. The Panel recommended that additional data on dsRNA PIP abundance and tissue distribution is needed, and that factors that affect absorption and effects of dietary RNA should be investigated f

urther. The Panel recommended experimental testing of the mammalian blood and exposed tissues be done to ensure that the siRNAs processed from the PIP dsRNAs are not present, since these could have off

- target effects after human consumption.
Question 3. To what extent does the specific structure of dsRNA, if it is super coiled or in a hairpin structure, make it more likely to survive degradation in the gut and lead to possible mammalian effects with oral exposure?

Summary

The combination of RNases and acid s found in the human digestive system are likely to ensure that all forms of RNA structure are degraded throughout the digestive process. RNA levels are below the limit of detection even when using very sensitive PCR amplification methods following treatme nts of total RNA samples from both plants and animals with a mixture of both ss

- and ds

- RNases (Li et al., 2012 a, b; Silverman and Gregory, 2013; Silverman et al., 2014; Zheng et al., 2010). Thus, this 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. However, the Panel recognized that other issues concerning the stability of dsRNA

s hould be addressed and made the following recommendations:

 The question of the ability of different structural forms of dsRNAs to survive degradation in the gut should also be addressed for dermal and inhalation routes of exposure.

 The stability of dsRNA should be tested in individuals that manifest specific diseases (e.g.
, Crohn’s, colitis, irritable bowel syndrome, etc.), the immune compromised, elderly, as well as children. These individuals may have compromised digestion or increased sensitivity to ds RNA exposure

.

 Modifications may be used to stabilize dsRNAs as exogenously

- applied end

- products. Possible effects on the uptake of dsRNA should be evaluated

.
Environmental Considerations: Questions directed toward dsRNA expressed

as part of a PIP and applied to the plant as a biochemical

Question 4. EPA needs a clear understanding of the environmental fate of dsRNAs in terrestrial and aquatic environments. In sections IV.A.1. and IV.B.3, respectively, EPA has presented potential scenarios for dsRNA mo vement within the environment that may result from their pesticidal uses as PIPs or as exogenously applied dsRNAs. Environmental fate of dsRNAs is not well understood; however, EPA has frameworks in place for PIPs and Biochemicals to obtain data related to degradation and movement of pesticides in the environment. These data can be used to refine environmental exposure estimates to dsRNAs.18

a. While nontarget exposure to dsRNA PIPs may result primarily from consumption of PIP crop plant tissue, EPA must also assess the exposure of nontarget organisms to dsRNA that may enter the environment through plant exudates and plant tissue breakdown. In section IV.A.2.a, EPA presented the current approach for testing to assess environmental fate for PIPs, which focuses

o n degradation within soil.

i) To what extent will data on degradation in soil inform EPA on nontarget exposure to dsRNA PIPs?

Summary
The Panel commended the Agency for initiating this critical dialogue and for the meticulous background research done to provide the Panel with useful information. Panel members agreed that additional information is needed to understand the persistence and stability of dsRNA PIPs in soil. There are few studies in the published literature that describe the stability and effe cts of dsRNA and degraded products (if present) in the environment . The Panel emphasized the importance of understanding how de gradation of plant materials proceeds as part of efforts to determine the extent to which in planta dsRNA and degraded products ( if present) are released over time. Duration of dsRNA persistence in soil will define the season (s) during which dsRNA or any active transformation products might be present in soils and soil associated organisms. Fields with summer crops that produce or a re treated with dsRNA may have enhanced microbial or abiotic processes to degrade dsRNA compared to fields in the winter

. The Panel made the following recommendations:

 Depending on the degradation profiles in planta and cropping practices, tests of multiple soil temperatures and soil moistures may be needed to evaluate degradation occurring as plant material overwinters (above or below soil surfaces).

 Degradation of root material and any above ground par ts that may remain in the field should be evaluated.

 In addition, b oth fresh and dry plant materials should be evaluated.

The Panel added that it is important to determine if products of degradation in environmental and biological samples are bioactive in target and non

- target organisms, e.g., siRNA.
ii) What additional information, if any, would EPA need to assess environmental fate of dsRNA PIPs?

Summary
The Panel agreed that the proposed testing scheme used for Bacillus thuringiensis ( Bt ) PIPs presented covers a portion of the information needed to understand environmental fate, but is insufficient to address all potential risks associated with the use of dsRNA. The Panel stated that there will be significant differences in evaluations of PIP dsRNA s , dsRNA in plant tissue and in the rhizosphere , and e xogenously applied dsRNA end

- products , e.g., sprays . Plant tissue may be desiccated and remain in fields during quite cold intervals. Thus, dsRNA may remain in plant material for long19

periods of time allowing slow release into the environment. Double

- stranded RNA used as a PIP may be exuded into the rhizosphere, where biotic and abiotic processes can vary quite significantly between growing and dormant seasons. The Panel noted that roots of annual plants will decomp ose under soil chemistry conditions (nutrients, oxygen, temperature, moisture, microbial activity) that may be dissimilar to growing season conditions. The Panel emphasized that additional data are critical to understanding the stability of dsRNA in soil a nd aquatic environments. While there is a reasonable body of literature describing the environmental persistence of nucleic acids in soil, there is little information on the persistence of dsRNA and small RNA (siRNAs and miRNAs). The Panel recommended coll ection of environmental fate data representing different soil types, water bodies, or weather conditions under which crops expressing dsRNA PIP s may be cultivated. The Panel also recommended significant changes to the overall risk assessment paradigm as di scussed in the response to question 7.

b. In section IV.B.4, EPA proposed environmental fate data needs for exogenously applied dsRNAs. To what extent is the proposed testing sufficiently robust to inform nontarget organism exposure for these dsRNAs?

Summ ary The Panel agreed that EPA’s standard testing paradigm represents a good beginning for data sets needed to understand soil degradation and environmental fate of dsRNAs in the environment; however, there are deficiencies in the testing regime. Non

- target species are more likely to be exposed to exogenously applied dsRNA end

- products than to dsRNA PIPs . Non

- PIP dsRNA exposures are likely multi

- route while dsRNA PIP exposure is more likely via diet

. The Panel stressed the importance of assessing the availability of the dsRNA to non

- target organisms as one of the earliest steps in the environmental fate evaluation process (a first tier evaluation). Data are lacking on cellular uptake of ds RNA at various taxonomic levels. In the absence of data, t he Pan el recommended that the present focus should be on evaluating uptake in non

- target organisms closely related to the target (e.g., species in the same genus and related genera in the same family ). The Panel indicated that the life stage exposed to dsRNAs sh ould be considered in the design of

non

- target tests and recommended that multiple species be evaluated for some of the required tests. Test species might include organisms not currently listed in the testing paradigm. Special consideration should be given to non

- target amphibian testing as part of a tier 1 testing regime. See the Panel’s response to Question 7.

c. For both dsRNA PIPs that may be free in the environment and exogenously applied dsRNAs, knowledge of the initial residue burden, and the dissipation rate of residues, is essential for developing environmental exposure estimates, particularly if models of environmental fate are used to arrive at these estimates. What analytical methods are available to accurately and precisely measure dsRNAs in diverse plant, soil, and water matrices?20

Summary
The Panel indicated that existing RNA extraction methods and subsequent use of quantitative PCR, high throughput sequencing, or hybridization analysis are the most current tools to assess the presence of a particular small RNA within a sample. The Panel emphasized the critical nature of using a reliable method for extracting and mea suring the dsRNA to track the environmental fate of the dsRNA in soil.

Question 5. The primary route of exposure for nontarget organisms to dsRNA PIPs is assumed to be ingestion, either of dsRNAs contained in plant tissue or free in the environment. Howev er, some evidence also indicates the potential for exposure by direct contact in nematodes and some arthropods. For dsRNAs that are applied to plants and/or the environment, the primary routes of exposure are expected to be both ingestion and direct conta ct.

a. In section III.C. and IV.A.1.c. of the issue paper, EPA discusses potential barriers to uptake of dsRNA in the gut of mammals and arthropods; however, little information exists for other taxa.

i) In addition to the conditions of the gut environment and enzymes influencing digestion, what other factors may play a role in uptake within the gut and potentially limit exposure to dsRNA?

Summary

The Panel stated that additional information is neede d to understand what factors may play a role in uptake within the insect gut (or other organisms) and potentially limit non

- target insect exposure to dsRNA and small RNAs. The published literature indicates there is a wide range of cellular uptake mechanis ms that vary from species to species and across taxa. For example, cellular uptake of dsRNA is mediated by transmembrane proteins, SID

- 1 and SID

- 2, or through endocytosis ( Hannon , 2002 ; Huvenne and Smagghe
, 2010 )

. Factors that influence cellular uptake and activity include, but are not lim ited to: tissue

- or cell

- specific expression, transport proteins, dose, dsRNA sequence length, and degradation.

ii) Please comment on how these barriers can be generalized across all nontarget taxa that are considered in EPA’s risk assessments (e.g., bir ds, plants, fish, etc.) for both dsRNA PIPs and exogenously applied dsRNA.

Summary
The published literature indicates there is a wide range of environmental uptake mechanisms that vary from species to species and across taxa. The Panel stated that there is insufficient understanding of the uptake mechanisms of dsRNAs (PIPs or non

- PIPs) and potential barriers to make any generalizations

across non

- target taxa. The Panel also commented that the ability to cross physiological barriers has to be combine d with binding in the gut (where there is an actual target to be affected). The Panel21

recommended collection of additional information to evaluate the types of cellular uptake mechanisms that exist and whether there are any patterns among different species , genera, or families , for example. For now, the Panel recommended a case

- by

- case evaluation of cellular uptake mechanisms for non

target taxa , as needed, based on exposure.

b. The degree of exposure by direct contact is likely to differ between nontarget ris k assessments for dsRNA PIPs and nontarget risk assessments for exogenously applied dsRNAs.

i) Please comment on the importance of the contact route of exposure for nontarget risk assessments for each of these types of dsRNAs

Summary
There are insufficient data on the contact route of exposure to comment on the importance of this route in non

- target risk assessment. The Panel found only two studies that examined direct contact as route of exposure to dsRNA. The limited information avai lable indicates that the dietary route of exposure was probably the primary route of entry leading to activity and not the contact route of exposure. The Pan el recommended collection of additional information to evaluate the importance of this route of exp osure. For now, the Panel recommended a case

- by

- case evaluation to determine whether this is a likely route of exposure before pursuit of an in

- depth study.

ii) What barriers are likely to exist for this route of exposure for both terrestrial and aquatic organisms, and how can these be generalized across nontarget taxa?

Summary

The Panel indicated that there are insufficient data on the contact route of exposure for both terrestrial and aquatic organisms to make any comparisons or generalizations across non

- target taxa. See response to question 5.b. i

. For now, t he P anel recommended that each dsRNA PIP o r exogenously applied dsRNA end

- product should be eva luated on a case

- by

- case basis, as needed, based on exposure

.

Question 6. In the issue paper, EPA discussed possible effects other than silencing of the target gene in the target organism that may occur as a result of exposure to dsRNA. These unintended effects of dsRNA include off

- target effects, silencing the target gene in nontarget organisms, degradation of non

- targeted mRNA by transitive RNA, and effects resulting from immune stimulation and saturat ion of the RNAi machinery. EPA has little information to estimate the range of unintended effects that may occur and their probability of occurrence as a result of exposure to dsRNA in the environment.

a. Please comment on the unintended effects that EPA might reasonably anticipate in nontarget organisms exposed to dsRNAs, the likelihood of such unintended effects, and the biological significance of these effects in nontarget organisms, should they occur.22

Summary

The Panel stated that there is uncertaint y in defining the spectrum of insecticidal activity. One

concern expressed is that the potential scale for use of pesticidal RNAi in the agroecosystem (in particular) will increase the potential of unintended effects in non

- target organisms. The Panel disc ussed several possible unintended effects that may result from exposure to dsRNA

. For example, dsRNA used to silence the target gene may also silence the same conserved gene in non

- target organisms. But one of the advantages of RNAi technology is that the RNAi effect is sequence and target gene dependent. Therefore, it is possible to design the dsRNA sequence to induce highly species specific gene suppression which would reduce the potential for off

- target effects. Saturation of RNAi machinery and immunostimulation have b een observed in animals (in mice or in cell cultures), but both require a fairly high dose to cause deleterious effects. The dose required to affect the RNAi machinery and induce immunostimulation in insects is unknown. There is also a lack of information on the risk of insect resistance to RNAi

- mediated gene suppression. The knowledge gaps make it difficult to predict with any certainty whether unintended effects will occur in non

- target species as a result of exposure to dsRNA. More data to close these gaps will increase the certainty in predicting the likelihood of unintended effects.

b. To the extent that additional information would reduce uncertainty in addressing these issues, please describe spec ifically the nature of additional information that EPA may need and the degree to which this information would reduce uncertainty in the ecological risk assessment.

Summary
As discussed in the response to Question 6.a., there is uncertainty in how to def ine the spectrum of insecticidal activity. The spectrum of activity depends on the target gene, dsRNA chosen, where and how much the dsRNA PIP is expressed in the plant, and the persistence and stability of the effect. What is a realistic level of non

- targ et exposure based on the stability and persistence of the dsRNA? While the Panel noted that t he mode of action of RNAi (by siRNAs) is, by definition, very specific (see discussion in response to q uestion 1) , there was some concern that unintended effects could occur across multiple insect orders. There could also be unique effects for each non

- target organism. The question is how do you decide what non

- target should be tested? For example, insect species representing 10 families and four insect orders wer e tested for DvSnf7 dsRNA activity (Bachman et al., 2013). Uncertainty in the spectrum of activity makes it more challenging to understand the environmental exposure levels and potential uptake pathways, important information that can narrow the scope of n on

- target organism testing for an ecological risk assessment.

The Panel concluded that the proposed ecological risk paradigm should be modified to reduce uncertainty in the ris k assessment. In addition, data are needed in several areas. The Panel recomme nded

an exposure

- based decision framework for ecological risk assessment of dsRNA PIPs and dsRNA non

PIP end

- products (see response to question 7).23

Questi on 7. In sections IV.A.2.a. and IV.B.4. of the issue paper, EPA presents the current framework of testing for determining nontarget effects resulting from exposure to PIPs and biochemicals, respectively. In section IV.A.2.c, EPA also raised potential issues related to nontarget testing w ith dsRNAs that may arise given their unique mode of action, which included 1) the potential influence of latent effects on results of nontarget testing, 2) the appropriate life stage for testing, and 3) the possibility for chronic effects.

a. Please comment on how each of EPA’s current PIP and biochemical frameworks for nontarget effects testing will inform risk assessment for dsRNA PIPs and exogenously applied dsRNAs. In providing a response, please address the potential for unintended effects as des cribed in Question 7, as well as the three issues outlined as concerns for nontarget testing listed above.

Summary

The Panel agreed with the concerns the Agency expressed regarding application of current protocols for identifying non

- target effects and c haracterizing environmental fate. Uncertainties in the potential mode s of action in non

- target species, potential for chronic and sublethal effects, and potential unintended consequences in the various life stages of non

- target organisms are sufficient jus tification to question whether the current Agency framework for environmental fate and ecological effects testing is applicable to dsRNA PIPs or exogenously applied non

- PIP end

- use products

. Due to the mode s of action of this technology, no one set of test species will serve as an adequate representation of non

- target species for all pesticidal products using RNAi technology. Hence, the Panel concluded that the classic approach of developing and assembling ef fects data for a standard set of test species will likely not work well for this technology.

The Panel discussed the following limitations in the current ecological risk assessment framework :

 Spectrum of activity

- d etermination of non

- target organisms that might be exposed.

 Maximum hazard dose assays alone may not be sufficient for assessing off

- target effects associated with the use of RNAi technology

.

 Lack of consideration for d ifferent life stages of a non

- target organism and under dif ferent enviro nmental conditions.

 The current Tier 1 testing scheme does not provide insight into potential chronic effects and limited insight to the potential for sublethal effects.

 Inability to determine possible synergistic effects (of co

- exposures to other PIPs ) w ith respect to target and non

- target organisms

 Lack of consideration of other endpoints, e.g., immune response, potential for saturation of the RNAi machinery in a non

- target organism.

b. What additional nontarget effects testing, if any, should EPA consider to gain a full understanding of the potential for dsRNAs to cause effects to nontarget organisms?24

c. What other approaches, such as bioinformatics analysis, may be used to address concer ns for effects on nontarget species and reduce the set of data requirements?

d. In providing answers to the above subquestions, please be specific in discussing the extent to which additional information would reduce the nature and magnitude of these specifi c areas of uncertainty.

Summary

The Panel p rovided a combined response to q uestions 7b.

- d.

The Panel found it difficult to prescribe additional tests or additional test species without a better understanding of RNA

- based modes of action, spectrum of activity, degradation kinetics of dsRNA in soil, and what non

- target organisms are likely to be exposed to dsRNA PIPs or dsRNA non

- PIPs

. To address the limitations of the proposed ecological risk assessment framework, the Panel developed an exp osure

- based decision framework or conceptual network model (figure 7

- 1). Data needs are linked to various steps in the framework.

One important outcome of the proposed framework is identification of non

- target organisms that might be realistically expose d to dsRNA PIPs or dsRNA non

- PIPs. This result will eliminate the need to test a battery of standard species (current tier 1) many of which will either not be exposed or be insensitive. The Panel suggested that using this framework would further the unders tanding of the likelihood of adverse outcomes in non

- target species, facilitate the selection of endpoints, and identify potential data gaps that would require further study

. The Panel provided the following general recommendations for data needed in the ecological risk
assessment:

 Before any testing, identify what non

- target organisms will be exposed to the dsRNA PIP or exogenously

- applied dsRNA end

- products. See exposure

- based conceptual model for ecological risk assessment (figure 7

- 1).

 Bioassays should include doses that mimic the in planta expression levels of the PIP and where possible, use plant tissue.

 Mortality is one of many possible endpoints that should be considered. The potential for chronic effects and for sublethal effects should be considered.

 The Panel recommended the evaluation of entire life cycles, while simultaneously examining multiple life history parameters (intrinsic population growth rate is a robust metric) rather than simply relying on toxicity testing. The P anel recommended that25

strategic research is need to determine under what conditions and what life stage s should be assayed for RNAi activity.

 The Panel recommended inclusion of an immunological endpoint in the non

- target organism bioassays.

 The Panel a lso recommended an endpoint to measure the potential for saturation of the RNAi machinery in a non

- target organism

.

 The Panel recommended that EPA consider possible synergistic effects (of co

- exposures to other dsRNA PIPs ) with respect to target and non

- target organisms.

 The Panel also recommended that EPA consider the evolution of pest resistance.

 One panelist identified potential scenarios that could minimize the concern for non

- target exposure and effects:

 When the dsRNA PIP is very specific without homology to any of the sequence databases ;  When the dsRNA PIP is not modified and therefore likely to degrade rapidly ;  When the dsRNA is expressed at extremely low levels and is tissue specific (e .g., in the
roots )

.26

O VERVIEW OF THE RNA INTERFERENC E PATHWAY

RNA interference (RNAi) is nearly u biquitous in plants, animals, fungi, and microorganisms. Using the model nematode, Caenorhabditis elegans , Fire et al. (1998) discovered that injected dsRNA sequences matching known gene sequences in the nematode genome were highly effective at silencing these loci. RNAi processes are known in most eukaryotes. RNAi describes a group of related processes whereby small RNA molecule s and their associated protein partners interfere with the production of specific genetic transcripts by binding in a partly or completely sequence

- specific manner to RNA transcripts (see f

igure I

- 1 for a simple illustration of this process). This process depends on the degree of sequence homology , protein partners, and cellular context . S pecific binding may lead to translational suppression, mRNA degradation, or target sequestration (Wilson et al., 2013; Bartel 2009). The unifying feature of the RNAi mecha nisms is the use of entirely or partially double

- stranded precursors to produce single stranded effector molecules that recognize sequences in target RNAs

. Particular attention has been given to RNAi triggered by introduced long dsRNA that is cleaved by a n RNase III enzyme called Dicer into 20

- 25 nucleotide (nt) siRNAs with a two base overlap overhang at the 3’ end. The siRNAs are incorporated into the multi

- protein RNA

- induced silencing complex (RISC) which removes the non

guide strand and retains the guid e strand. The Argonaute protein of the RISC complex cleaves a single

stranded messenger RNA (mRNA) with sequence complementary to the guide strand and lea ds to destruction of the mRNA. Therefore, the mRNA can no longer be translated into a protein product, i.e., interference in translation. Additional discussion on RNAi in eurkaryotic organisms is found in the response to question 6.a.

Figure I

- 1. Simple illustration of the RNAi process . Abbreviations: RISC = RNA

- induced silencing complex; dsRNA = double

- stranded R NA; siRNA = small interfering RNA; Dicer = a family of RNA endonuclease III enzymes; m RNA = messenger RNA; Agronaute = family of proteins that cleave mRNA dsRNA sequence dsRNA ingested cellular uptake dsRNA cleaved into siRNA by Dicer siRNA into RISC Argonaute directs guide strand to homologous mRNA Gene suppression27

DETAILED RESPONSES TO CHARGE QUESTIONS

Human Health Consideratio ns

Question 1. Please discuss the nature and extent of uncertainty in the specificity of long sequences of dsRNA targeted at pest species, if bioinformatic analysis shows no significant similarity to mammalian genes

.

Panel Response
The Panel agreed that bioinformatic analysis can be used to identify specific nucleotide identity in a long sequence of dsRNA or process ed shorter products that could bind in a siRNA

- like fashion
, provided the sequences are in the database and the appropriate algorithm is used. The certainty in the matches is dependent on the reliability and completeness of the sequence information in existing databases, the algorithm used, and the presence of single nucleotide polymorphisms (SNPs). The Panel emphasized that bioinformatics analysis is a usefu l, but not absolute, predictive tool. Sequence i dentity does not mean that the dsRNA would provoke an RNAi response, but should caution the potential for unintended interactions and further review. The Panel suggested that dsRNA sequence

- specific factors

m ay increase target specificity and reduce the likelihood of unintended effects. If the charge question is interpreted more broadly to include miRNA

- like effectors and modes of binding, sequence predictions are much less reliable

.

The Panel discussed the following major points concerning certainty of bioinformatic analysis to identify sequences of dsRNA targeted at pest species that are homologous to mammalian genes. Specific discussion with respect to non

- m ammalian organisms is found in q uestion 6.

1 ) Bioinformatic analysis can reduce uncertainty in sequence similarity

The Panel stated that coverage of nearly all of the human transcriptome is now sufficient to allow accurate identification of nucleotide (nt) sequences with identity to "long sequences o f dsRNA" or even short process ed products , such as
~ 20

- 25

- mer s, that could bind in a siRNA

- like fashion. Algorithms can be used to determine if there is overlap to mammalian sequences with the goal of minimiz ing unintended effects

. While “long” dsRNA may have no similarity to mammalian genes, processing of dsRNA into shorter siRNAs may present additional issues if these siRNAs have a high degree of similarity to sequences in non

- target species including mammals . Algorithms such as Bl astn (Altschul et al., 1990), ClustalW (Thompson et al., 1994), and MegAlign (DNAStar, Madison, WI) can be used to identify shared sequence identity and scan for 21 nt matches among databases, but certainty in the matches will depend on the reliability and completeness of the sequence information in existing databases, the algorithm used, and the presence of single nucleotide polymorphisms (SNPs).28

2) To what d egree does the siRNA have to match the target genetic sequence to trigger RNAi ?

The Panel indica ted that current evidence suggests that a high degree of homology between the siRNA and target mRNA is necessary for induction of RNAi effects (Sabariegos et al., 2006)

. However, other studies question this dogma (see discussion in Question 6)

. Sequence identity in the end of the guide strand of siRN A, known as the seed region (located 2

- 8 nts from the 5’ end ) of the siRNA sequence and loaded into the RISC is critical for target gene silencing, but may also be responsible for silencing off

- target genes ( Jackson et al., 2006, Jackson and Linsley, 2010). For example, s ubstituting the second base of the seed region with O

- methyl ribosyl can reduce off

- target siRNA binding within the target organism (Jackson and Linsley, 2010) , which may reduce the potential for non

- target effects, but there are no data to confirm this conclusion.

3 ) Choice of dsRNA sequence makes a d ifference to target specificity

The Panel discussed how the choice of dsRNA sequences can limit target specificity and minimize the likelihood of unintended effects in non

- target organisms

. See also related discussion in response to Question 6.

 Choose dsRNA sequences with perfect or near perfect sequence homology to regions of the target gene

. Some regions may work better than others and have to be determined experimentally. The more specific the dsRNA sequence is to the target sequence , the greater the insurance will be against unintended effects. For example, s elect dsRNA sequences that target regions of genes with no shared 21 nt sequences wit h other species

.

 Choose target genes that are unique to the genome and physiology of the target pest, if possible.

 An ideal target gene en codes a protein with a short half

- life (see discussion in Scott et al. 2013); this is because the duration of the ef fect of RNAi will be shorter to achieve the desired phenotypic suppression. Phenotypic analysis of gene function at any life stage becomes more difficult if the protein product of the target gene has a long half

- life.

 Careful selection of siRNA sequences that favor incorporation of the antisense strand into the RISC may improve efficacy and specificity of RNAi (Aronstein et al., 2011).

 Reducing the size of the dsRNA to the minimal effective length will likely result in fewer siRNA fragments in the mammalian gut and lower the subsequent “dose” (Scott et al.
, 2013). For the purpose of target specificity, the shorter the sequence, the better; however, this has to also be balanced with efficacy (in which case, the longer the sequence, the better). At le ast 100 copies of siRNA are needed to induce RNAi in mammalian cells (Brown et al., 2007)

.

 Screen for sequences that have been demonstrated to induce innate immune response (Judge29

et al., 2005) and eliminate th em from further consideration. See also discu ssion in response to q uestion s 2 and 6.

4) miRNA

- like molecules : less certainty in sequence predictions

The Panel noted that if the database for bioinformatics screening is increased more broadly to include miRNA

- like effector sequences , predictions of non

- target effects against mammals may be much less reliable . In contrast to siRNA s that require high sequence complementarity to the target mRNA with few off

- target exceptions, miRNAs have less exact complementarity to the target mRNA . A single miRNA coul d be specific to a single sequence or could target hundreds of mRNAs (off

- target possibilities). De pending on the in silico target prediction algorithm used, up to thousands of binding sites may be predicted for miRNA in the 3' untranslated regions of known transcripts alone. This number increases if 5' untranslated regions ( UTRs ) and coding sequences are included in the search

. Micro RNAs are also known to bind to each other, ribosomal RNAs, long non

- coding RNAs, transfer RNAs, and other forms of RNA in the cell (Helwak et al

. , 2013). The factors governing miRNA binding are not well defined.

Various algorithm targeting rules invoke perfect or near

- perfect "seed" sequences at the 5' end of the miRNA, but these are imperfect. For example, there are predicted high

- scoring target interactions that cannot be validated in the lab oratory (even under non

- physiologic conditions), and conversely, there

are laboratory validated interactions that are not predicted by the algorithms (e.g., Ha et al

. , 1996; Lal et al

. , 2009). A recent technical advance apparently provides direct evidence of small RNA

- target in teraction, which suggests that a large proportion of miRNA

- target pairings are non

- canonical (Helwak et al

. , 201 3). Thus, the Panel indicated that it would be a difficult task to predict all possible miRNA

- like interactions. This means that the effect of a miRNA on a particular transcript, even when direct binding is confirmed by in vitro assays may be meager and, hundreds or thousands of copies of miRNA must be present in the cell to exert an observed effect (Brown e t al., 2007; Mullokandov et al., 2012).
Question 2. Based on data indicating degradation of the majority of dsRNA in the digestive system, please disc uss the strengths and limitations in concluding there will not be significant absorption of dsRNA with possible mammalian effects on oral exposure

.

Panel Response The focus of this charge question is on the potential dietary exposure to RNAi PIPs through oral ingestion. The Panel addressed the following areas in its discussion

.

 Safe consumption of RNA

 RNA degra da tion in the mammalian digestive system

 Substantial systemic distribution of dietary RNA as input i nto RNAi pathways is not predicted for mammals by comparative biology

 Inflammatory responses to plant RNAi molecules

 Evidence for uptake and distribution of dsRNA or processing products in mammals

 Human disease and RNA uptake/clearance.30

 Dose, s toichiometry and delivery: diet

- derived miRNAs and gene regulation

 Other routes of exposure
1) Safe consumption of RNA

The Panel stated that to date there appears to be no convincing evidence in the literature that ingested dsRNA is absorbed from the mammalian gut in a form th at produces biological effects

. Although, some panel members argued that there is also no convincing evidence to the contrary

. As noted in t he public comments and in the literature , the history of safe mammalian consumption of RNA in plants suggests that nominal uptake of dietary RNA by mammals does not lead to adverse effects
, despite the fact that siRNAs and miRNAs are abundant in soybean, corn, and rice, some with exact matches to human gene sequ ences (Ivashuta et al., 2008, 2009). The Panel pointed out that a perfect sequence match does not always correlate with gene silencing
, one of the fa lse positive issues with algorithms. The Panel added that t he FDA considers RNA as GR AS (“Generally Regarded As Safe”)
, with the only concern that ingestion of high levels of purines resulting from nucleic acid degradation ( e.g. , through consumption of hig h levels of RNA

- rich foods like brewer’s yeast supplements) could aggravate gout. The P anel point ed out that t his technology was developed because of the promise of improved efficacy and minimal unintended effects compared to traditional pesticides.

2 ) RNA degradation in the mammalian digestive system
The mammalian digestive system functions to break apart first tissues, then cells, then subcellular components including ribonucleoprotein complexes (RNPs) . Breakdown of food into constituent nutrients begins with masticat ion and exposure to degradative enzymes in saliva, followed by further digestion in the acidic environment of the stomach. Passage into the gut is marked by increased pH, continued digestion (including by resident microorganisms), and absorpti on of liberated nutrients. Digestive strateg ies vary with diet: mammals that consume chiefly plant materials with tough cell walls have evolved more complex digestive processes than others.
In the digestive tract and elsewhere, RNA is subject to both non

- e nzymatic and enzymatic degradation. On the non

- enzymatic side, in contrast with DNA, the 2′ hydroxyl group on the ribose moiety of RNA renders RNA sensitive to non

- enzymatic hydrolysis. RNAs of all structural forms are relatively labile compared with DNA i n the sense that RNA is more susceptible to non

- enzymatic hydrolysis. However, all forms of RNA are not equally susceptible to certain nucleases. Even RNA that has been purified in the laboratory to exclude RNase enzymes, stored in properly buffered soluti ons, and maintained at extremely low temperatures will degrade over time.
The physiological conditions of the mammalian gut are barriers to the uptake of foreign dsRNA. The digestive system of mammalian organisms is replete with enzymes such as nucleases (Fort et al., 1969) . As presente d in detail in the response to q uestion 3, these include RNases. Of course, RNA is often found in complex with proteins and lipids and this greatly affects stability. Extracellular RNA may also be carried within vesicles tha t are mobile in planta or are the product of tissue destruction during feeding. RNA contained within such protect packaging can be liberated by lipases and proteases.31

Pancreatic and intestinal nucleases and enzymes eventually metabolize RNA to mono

- nucleot ides and subsequent nucleosides and bases, which are primarily absorbed in the upper small intestine (Carver and Walker, 1995; Hoerter et al., 2011; Rehman et al., 2011; Sorrentino et al., 2003)

. In fact, the RNA

- hostile nature of the various microenvironments of the mammalian digestive system, extreme acidic pH and RNA nucleases in the stomach, has hampered the development of dsRNA for oral therapies in humans (Petrick et al., 2013) . Members of the Panel, pub lic commenters, as well as evidence in the published literature referred to the difficulties in the development of orally active RNAi therapeutics as compelling arguments for the safety of oral exposure to dsRNA and derivatives. For example, l imited succes s has been reported for oral siRNA delivery using encapsulation into engineered nanoparticles (Aouadi et al., 2009; Ballarin

- Gonzalez et al., 2013)

. However, these approaches may be generally more effective for distribution to cells of the gut rather than across the gut and into the general circulation (Kriegel et al., 2013) . Indeed, for candidate RNAi therapeutics to survive the extra

- and intracellular biological environments, special modifications and/or formulations are needed even when molecules are in jected into mammals at high concentrations. However, miRNAs that are resistant to low pH and RNases have been reported in milk. T hese miRNAs appear to be resistant based on their incorporation into extracellular vesicles (Admyre et al., 2007; Hata et al., 2010; Lasser et al., 2011; Zhou et al., 2012)

. Although there has been speculation as to the biological effects of these milk miRNAs, direct demonstration of such effects or transfer of RNA to the infant, to our knowledge, has not been done

. The extent to which dsRNA PIP products could be similarly protected within “ plant

specific packaging” is lacking and should be evaluated.
One panel member noted that some insects have high levels of cysteine proteases in the gut (e.g., some coleopterans), but there is no consistent response following oral ingestion of dsRNA. In contrast to insects, mammals lack cysteine proteases in the gut.
The Panel also discussed possible effects of ingested RNA on the gut microbiome. Dietary composition, e.g. fiber or fat content, have been reported to alter the microbiome, but there is no evidence that specific dietary RNA sequences have any such effects. As noted above, RNA import by microbes is generally followed by b reakdown to provide nutrition. While bacteria and archaea have RNA

- based regulatory systems, the machinery, RNA sequences, and binding behavior for these systems differ fro m those in eukaryotic systems. In addition, there is no current evidence that eukaryotic dsRNA is amplified by bacteria.
3) Barriers to uptake and systemic distribution of dietary RNA in mammals vs. other organisms

The Panel discussed barriers to the exogenous uptake and systemic distribution of dietary RNA into RNAi pathways in humans. Mechanisms for exogenous dsRNA import into RNAi pathways are no t known in mammals, and reports of carrier

- unmediated small RNA uptake await confirmation

. In contrast, C. elegans and some plants have evolved mechanisms for uptake and use of exogenous dsRNA in sequence

- specific RNA silencing processes that function as a type of defense against parasites and pathogens. Cellular uptake and distribution of dsRNA have been most extensively studied in C. elegans (e.g., Fire et al., 1998)

. These features render these species attractive targets for PIPs or non

- PIP topical dsRNA pesticides, while limiting the impact of RNAi control measures on non

- target organisms in32

which these mechanisms are lacking or are not as robust, including mammals. Features include efficient up take of ds RNA from the environment, amplification of RNAi by generation of secondary effector molecules, and RNAi spread throughout the organism. Ancillary processes that make RNAi particularly sensitive and effective in C. elegans do not appear to be shar ed by mammal s or even some closely related nematodes (Felix, 2008)

. a. Uptake of dsRNA from the environment for input into the RNAi pathway. C. elegans assimilates dsRNA contained in dietary materials (Timmons and Fire, 1998) and when soaked in dsRNA sol ution (Tabara et al., 1998) . Dietary uptake occurs when dsRNA of 60 nt or more in length (McEwan et al., 2012) is recognized by a single

- pass trans membrane protein, SID

- 2 (McEwan et al., 2012; Winston et al., 2007) in the acidic environment of the gut. Bin ding leads to endocytosis, followed by import across the cell membrane via a multipass channel protein known as SID

- 1 (Hunter et al., 2006; Winston et al., 2002)

. Gene homologs of sid

- 1 can be found in many organisms
, but with varying comparative functionality. In contrast, the sid

- 2 gene is less conserved across organisms. Experiments in C. elegans
, demonstrated that dsRNA uptake in refractory nematode species may become permissive when the C. elegans SID

- 2 gene is in troduced (Nuez and Felix, 2012; Winston et al., 2007)

. While the sid

- 2 gene is poorly conserved across organisms
, there is some evidence for alternative uptake mechanisms. C. elegans , for example, does not require SID

- 2 (Liu et al., 2012)

. Such pathways ma y also exist in other organisms (or cell culture systems) that lack functional SID

- 2 but appear to import dsRNA (Saleh et al., 2006; Ulvila et al., 2006) , e.g., through endocytosis

- initiated processes (Price and Gatehouse, 2008)

. b. RNAi amplification. A second ancillary process that provides sensitivity to RNAi in some plants and animals is the production of “secondary” RNAi effectors (Axtell et al., 2006; Pak and Fire, 2007; Ruby et al., 2006; Sijen et al., 2001; Sijen et al., 2007)

. In C. elegans , RNA

- d ependent RNA polymerases are primed by binding of siRNA to a target mRNA

. The small RNA s formed through this process are multiple secondary siRNA molecules, each of which recognizes the target (Parrish and Fire, 2001)

. Additional amplification of dsRNA is also known to occur in some plants (Axtell et al., 2006; Sarkies and Miska, 2013) . Even very low copy numbers of imported dsRNA may lead to a robust RNAi response in any organism with RDRPs that function in RNAi (e.g., C. elegans
, some plants ) . However, m any organisms , e.g., mammals, do not appear to have RDRP

- mediated RNAi amplification (Duan et al., 2013; Lipardi et al., 2001)

. For mammals, each copy of a hypothetical PIP or non

- PIP RNAi effector would need to come from the diet since RDRP

- mediated RNAi amplification is absent.

c. Conservation of cellular transport proteins

. As mentioned above, the SID

- 1 transmembrane channel transfers long dsRNA across the cell membrane (Feinberg and Hunter, 2003; Hunter et al., 2006; Shih and Hunter, 2011; Winston et al ., 2002) . In C. elegans , this allows long dsRNA to silence target genes throughout the organism (systemic RNAi). Systemic distribution of dsRNA is also a key feature of plant RNAi (Brosnan and Voinnet, 2011; Voinnet, 2009) . However, despite conservation of SID

- 1 sequences across many organisms (Hunter et al., 2006) , effective systemic distribution seems to be the exception rather than the rule in animal species. Systemic RNAi of the type observed in C. elegans has not been reported in mammals. Cell

- to

- cell communication by extracellular vesicles has33

been observed in mammalian cell culture systems, in some cases reportedly mediated by RNA cargo (Pegtel et al., 2010; Raposo and Stoorvogel, 2013; Skog et al., 2008 ; Valadi et al., 2007) . However, its
extent in vivo is not yet well known (Sverdlov, 2012; Turchinovich et al., 2012) . Ties to dietary input, i.e., findings of vesicles that arrive from the diet, have not yet been reported.

d. Barriers in mammals vs. insects. One panelist stated that the depth and breadth of human barriers to foreign RNA uptake and ultimate incorporation into cellular regulatory processes was previously advanced as an argument against off

- target effects in insects. Yet, RNAi works in s ome insects. Why would there be variable effects of RNAi in insects versus humans, or observed effects in some insects and not others? In response, panel members remarked that a mongst insects, there is a wide range of gut pH; diet composition and feeding p ractices; conservation and function of RNA receptors and transmembrane channels including, but not limited to, the SID proteins; and activity of RN a ses in digestive fluids and hemolymph. These factors may contribute to species differences in foreign RNAi

e ffectiveness. A complete discussion of potential barriers to foreign uptake in insects in discuss ed in the Panel’s responses to q uestions 5 and 6.

4 ) Evidence for uptake and distribution of dsRNA or processing products in mammals
Mechanisms for exogenous dsRNA import into RNAi pathways are not known in mammals, and reports of carrier

- unmediated small RNA uptake await confirmation. The Panel stated that there is currently a lack of substantiated evidence that exogenous dsRNA and/or its processing products t raverse the gut, are distributed throughout the organism in a manner analogous to plant or C. elegans RNAi processes, or are processed by native RNAi machinery to yield functional RNAi effector molecules. Of the various forms of RNA in dietary materials, A rgonaute

- bound and vesicle

- protected small RNAs may well be among the most stable and thus the strongest cand idates for survival and uptake. Small RNAs would be the ultimate effectors in tissue of hypothetical ingested and subsequently processed longer RNA s. The question of potential dietary uptake of miRNAs arose after the publication of a recent study by L. Zhang et al. (2012). L. Zhang et al. (2012) reported that dietary plant miRNAs enter the mammalian bloodstream and regulate cholesterol metabolism in the ingesting animal through LDLRAP1 (a low

density lipoprotein). Four plant miRNAs were found in all samples, two of them occasionally reaching levels similar to those of abundant end ogenous miRNAs. It was also reported that one plant miRNA, Mi R168a, targeted an endogenous transcript involved in cholesterol metabolism, LDLRAP1, and raised circulating cholesterol counts. The Panel questioned the findings of L. Zhang and colleagues (L. Z hang et al., 2013) and identified the following issues:

 S e quencing data are currently unavailable for this study and thus the data cannot be independently analyzed

.

 Despite the varied and abundant plant small RNAs in the human diet, only four plant miRNA s were detected in all pools of human sera and only two were abundant (Witwer, 2012)

.34

 There was striking variability of read numbers despite a sample pooling strategy.

 At least one of the detected miRNAs is routinely used as a spike

- in control in high

throughput sequencing library preparation, raising questions of possible cross

contamination. The presence of miRNA in pollen (Wei et al., 2011) also supports the notion of inadvertent contamination. Analysis of public sequence reads from numerous animal studies turned up no consistent evidence of high levels of plant miRNA in animal blood, cells, or tissues, except for what was described as an ap parent over

- representation of Mi R168, suggesting contami nation or other artifact(s) (Y. Zhang et al., 2012)

.

 The timing and magnitude of changes of both RNA and target in mouse feeding studies have been questioned (Witwer et al., 2014 ). No murine biological effects have been detected in subsequent feeding stu dies (Dickinson et al.
, 2013). If murine biological effects were to be confirmed in future studies, translation to human risk would need to account for gastric pH differences from mice to humans.

Three subsequent studies (Snow et al. , 2013; Witwer et al.
, 2013, and Dickinson et al., 2013) failed to replicate the finding of high plant miRNA concentrations in mammals. Each study found negligible uptake of plant dietary miRNAs.
Snow et al. (2013) found that t ransfer of plant miRNAs from miRNA

- replete diets i n human and murine subjects, as well as adult bees was negligible, below the limit of detection of sensitive, amplification

- based quantitative reverse transcription PCR assays (qPCR)

. This same study also reported that animal miR

- 21 in a lard diet was unde tected in a miR

- 21 knockout mouse that completely lack this animal encoded miRNA
, which implies that barriers to dietary uptake are not specific to plant RNAi effectors.

In a time

- course study of non

- human primates , Witwer et al. (2013) concluded that plant miRNAs

were undetectable or nearly so in plasma before and after feeding, and that any low

- level positive findings may be due to non

- specific amplification

. The miRNAs examined in both studies included highly abundant and less abundant miRNAs in the food sources (Witwer et al., 2013; Snow et al., 2013).
Miragen r eplicated the original mouse feeding study of L. Z hang et al. (2012) and found no consistently detectable plant miRNA from rice (Dickinson, et al., 2013) . By administering seve ral rice

containing diets to separate groups of animals, the investigators also uncovered evidence that the cholesterol results reported in the initial study (L. Zhang et al., 2012) may have been precipitated b y dietary insufficiency, not MiR 168

- mediated inhibition of LDLRAP1 (Dickinson et al., 2013)

. In addition to these feeding studies, high

- throughput sequencing was used to measure plant miRNA in human blood (Wang, et al., 2012) and in a mouse model of liver damage (Wang et al., 2013)

. Onl y one plant miRNA, Mi R168 (see comments above regarding this miRNA), was detected, but at very low levels, <10 ppm in blood in the human study and <1 ppm in blood in the mouse study. Such low levels of apparent reads are difficult to distinguish from backg round. Public commenters stated that35

additional, unpublished negative studies exist, and the Panel suggested that publication of these data would be helpful

. The Panel concluded that available evidence supports the conclusion of no significant absorption of dsRNA in mammals and minimal likelihood of adverse effects. Evidence of a ny dietary uptake plant miRNAs in mammals is nominal and non

- specific. However, the Panel stressed that data are lacking in this area and there are no published PIP

- specific studie s. The Panel recommended collection of data on dsRNAs PIP abundance and tissue distribution to investigate factors that may affect absorption and effects of dietary dsRNAs. The Panel also recommended experimental testing of the mammalian blood and exposed tissues should be done to ensure that the siRNAs processed from the PIP dsRNAs are not present, since these could have off

- target effects after human consumption. One panelist added that these evaluations should consider dietary exposure over an extended t imeframe.

5 ) Inflammatory and immune responses to plant RNAi molecules
Mammals respond to the detection of foreign RNA in a strong general fashion. Mammalian cells sense foreign RNA by means of pattern recognition receptors including several Toll

- like receptors (TLRs) and cytoplasmic sensors such as MDA5 and RIG

- I (Dixit et al. 2013, Loo et al. 2011, Kawai et al., 2008 ). Evidence also exists for diffe rences in innate immune responses to dsRNA from commensal and pathogenic bacteria (Kawashima, et al., 2013)

. The Panel commented that r ecognition of foreign RNA triggers innate immune responses: degradation of RNA transcripts, shutdown of protein expressio n, and secretion of inflammatory cytokines. During acute viral infections, these responses might present as “flu

- like symptoms.” While such reactions may protect against infection, depending on their strength and longevity, tissue damage may occur. The Pan el concluded that factors governing inflammatory response s to some foreign RNA
, but not others
, are incompletely understood.

a. Could miRNA in plant extracellular vesicles or lipid

- bound tissue breakdown products be relatively resistant to degradation, all owing delivery to gut cells and detection by pattern recognition receptors?
Mammals regularly ingest plant material replete with all species of RNA without reported induction of innate immune responses. Additionally, although limited experimental evidence has been reported, oral administration of purified plant

- derived nanovesicle s in a murine

- induced colitis model did not cause inflammation, but instead protected against inflammation (B. Wang, et al., 2013) resulting in significantly decreased production of pro

- inflammatory cytokines such as IL

- 6. If active dsRNA PIPs are at relat ive abundance as low as indicated by industry representatives during the public comment session, the Panel thought it would be unlikely that PIPs would be a special case, differing from other plant RNA in their ability to trigger inflammatory responses. Ho wever, the absence of PIP

- specific data suggests to some panel members that the potential of orally delivered plant

- derived RNA to activate host innate immune mechanisms should be evaluated.

b. Exosomes

. The Panel indicated that exosomes might be a method to encapsulate dsRNA and then it could be released inside an organism, but this is speculation. For example, An et al. (2007)36

provided evidence that exosomes could be released upon fungal penetration (An et al., 2007). Regente et al. (2012) provided indire ct evidence of exosomes in sunflower seeds.

c. Possibility of plant RNA

- stimulated immune responses in mammals

. The Panel noted that there were a few published studies that raise questions about the possibility of plant RNA

- stimulated innate responses in mammals.

 Experimental injection of animals with large quantities of foreign RNA may lead to systemic inflammation and damage to organs including the gut (Zhou et al., 2007)

.

 A report that fluorescent label from parasite extracellular vesicles was internalized by cultured intestinal epithelial cells (Marcilla et al., 2012) provides support for uptake and potential concentration of biologically active substances at the gut su rface, potentially contributing to immune activation.

 An orally administered adenoviral vector engineered to overexpress dsRNA induced an inflammatory response in mice and ferrets (Scallan et al., 2013) . The inclusion of the dsRNA enhan ced systemic antibody responses, likely through interaction with T oll

- like receptor 3 on dendritic or intestinal epithelial cells
, protected the animals from lethal challenge with antigen. The Panel commented that presentation of antigen and adjuvant would not be expected to occur in the RNAi pesticide applications . However, S callan et al. (2013) illustrated the potential issue of species differences in gastric pH. Specifically, while the mice were vaccinated by gavage because of their higher gastric pH, th e ferrets (more like humans) required the use of an endoscope to administer the vaccine to the duodenum because of the lower gastric pH.

6 ) Stoichiometry , d elivery, and estimated dietary e xposure

a. Stoichiometry

. Measurable silencing of a particular target gene does not occur unless the stoichiometry of targeting molecules and target or targets is favorable. Hundreds to thousands of targeting RNAs per cell appear to be necessary for observable RNAi to occur (Brown et al., 2007)

. Many predicted binding sites for plant RNAs have been found in mammalian genes (Ivashuta et al., 2009)
, but lack of regulation of these genes by dietary intake suggests that insufficient copy numbers of foreign RNA are transported through th e gut and then into cells to silence these targets. The Panel stated
t hat the history of safe mamm alian consumption of plants indicates that nominal uptake of dietary RNA by mammals is insufficient to lead to regulatory effects (see related discussion late r in th is response to this question). This conclusion is in agreement with statements by public commenters and fi ndings of published studies.
As noted in the response to question 1 , miRNAs have less exact complementarity to the target mRNA than siRNAs

. However, a large number of targets will also dilute out the effects of any miRNA

like binding. Regardless of the type of off

- target binding, measurable off

- target effects are not necessarily expected even when large copy numbers of the effector are present . Studies that have reported regulation of mammalian transcripts by foreign sequences have usually involved in vitro37

transfections of non

- physiologic levels of nucleic acid. For example, transfection of several endogenous bacterial RNA sequences into mammalian cells altered cellular gene expression directly or indirectly (Wang et al., 2012) . T hese researchers astutely noted, however, that RN A was transfected at a concentration many orders of magnitude higher than those at which any exogenous RNA species would have been present in the blood (Wang et al., 2012)

. b. Delivery. The form in which diet

- derived RNA would exist in blood is not known, and it is unclear if these RNAs could be imported into cells in a manner consistent with regulatory function. Despite the growing evidence for endogenous RNA transfer by mammalian extracellular vesicles, it is unclear whether or how natively packaged matur e miRNAs or other short ssRNA effectors shuttle between cells and influence gene regulatory programs in vivo . In C. elegans , long dsRNA and shorter dsRNA processing intermediates move from cell to cell and undergo ultimate processing in the destination cel l (Jose et al., 2011)

. Single

- stranded RNAi effector molecules (derived from processed short dsRNA) and single

- stranded secondary molecules from the RNA

- dependent RNA polymerase

mediated RNAi amplification pathway do not appear to spread with the same efficiency (Jose et al., 2011)

.

c . Estimated human exposure to diet

- derived RNAi

. The Panel indicated that more detailed information is needed (more than th at provided in the EPA background documents) to estim ate human dietary exposure. This would include abundance data for different tissues. Where is the PIP expressed in the plant? What is the level of expression? Does expression change over time? Is the dsRNA expressed constitutively or transiently? Is the ds RNA stable or degraded in planta ? The Panel recommended the collection of data on the abundance of PIP dsRNA relative to other ds RNAs in PIP plants.
Some panel members expressed concern that PIPs could be engineered to greatly over express RNAi effectors i n comparison with other forms of RNA. Drastic over expression of specific dsRNAs would likely overwhelm the small RNA processing machinery in plant cells. In mammals, this can lead to loss of viability (Grimm et al., 2006)

. Responding to direct questions from the P anel during the public comme nt period
, Monsanto representatives indicated that levels of a dsRNA PIP under development for corn rootworm control were in the parts per billion of total RNA in planta , and that estimat ed human exposure to dsRNA PIPs in food materials would be in the nanograms per kilogram level

. Panel members noted that published theoretical estimates of the human dietary intake necessary to achieve minimally relevant levels of foreign RNAi effectors wo uld be physiologically impossible to achieve in mammals, e.g., 33 kg per day of rice (Petrick et al., 2013) or several thousand pounds of raw fruit (Snow et al., 2013)

. The Panel encouraged publication of industry data on abundance and recommended independent verification of these data.38

Question 3. To what extent does the specific structure of dsRNA, if it is super coiled or in a hairpin structure, make it more likely to survive degradation in the gut and lead to possible mammali an effects with oral exposure?

Panel Response

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. RNA levels are below the limit of de tection even when using very sensitive PCR amplification methods following treatments of total RNA samples from both plants and animals with a mixture of both ss

- and ds

- RNases (Li et al., 2012 a, b; Silverman and Gregory, 2013; Silverman et al., 2014; Zhe ng et al., 2010). Thus, this 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.

1 ) RNA structure

All RNA molecules have an intrinsic structure. This structure is the collection of intricate folding patterns that an RNA molecule forms through specific base pairing interactions encoded within its primary sequence (Buratti et al., 2004; Cooper et al., 2009; Cruz and Westhof, 2009; Sharp, 2009). Many RNA molecules cannot properly function without the formation of an extremely precise secondary structure (Buratti et al., 2004; Cooper et al., 2009; Cruz and Westhof, 2009; Sharp, 2009) . For i nstance, ribosomal RNAs (rRNAs) must form structural folds that enable interactions with the correct ribosomal subunits at specific locations along their length, thereby allowing the formation of functional ribosomes (Trappl and Polacek, 2011) . Additionall y, the structure of long non

- coding RNAs (lncRNAs), not their primary sequence, drives their function in regulating gene expression (Hindorff et al., 2009; Khalil and Rinn, 2011)

. Structural elements also affect the overall steady state abundance and stability of many eukaryotic mRNAs (Goodarzi et al, 2012; Li et al., 2012 a, b) . Finally, other post

transcriptional processes such as protein translation (Kozak, 1980; Kozak, 1986) and RNA

- mediated silencing (Baulcombe, 2004; Jones

- Rhoades and Bartel, 2004) are also tightly contr olled by structural features within the RNA transcript. Thus, the structure of RNAs is often required for their functionality and regulation in diverse cellular and regulatory processes.

2 ) RNA stability

RNA is an intrinsically unstable molecule even in normal aqueous conditions no matter what structural confirmation (single

- stranded or double

- stranded) it assumes. This is due to its chemical nature, where the additional hydroxyl group at the 2’ position o n the ribose sugar ring provides the destabilizing moiety through intra

- hydrolytic degradation. Both acidic and basic conditions can drive intra

- strand hydrolysis of RNA chains irrespective of the structural confirmation of that molecule (Lilley, 2011) . Ad ditionally, numerous ribonuclease enzymes (RNases) are encoded by both prokaryotic and eukaryotic organisms ( Sorrentino, 2010 )

. RNases are a type of nuclease that catalyzes the degradation of RNA into single nucleotides. They can be divided into endoribonu cleases and39

exoribonucleases, and comprise several sub

- classes of enzymes. In fact, RNases are classified based on their structural preference. Specific RNases prefer single

- stranded RNAs (single

- stranded RNases (ssRNases)), and others target double

- strand ed RNAs (double

- stranded RNases (dsRNases)). Thus, there are RNases that can destroy all types of RNA molecules regardless of their structural confirmation expressed in both prokaryotes and eukaryotes (Sorrentino, 2010).

3 ) RNases in humans (with a foc us on the gastrointestinal (GI) tract)

All organisms studied to date contain many RNases of many different classes, demonstrating that RNA degradation is a very ancient and important process. Humans specifically contain a large repertory of RNase enzymes, many of which are expressed and function throughout the GI tract. In fact, RNases are expressed and functional in saliva in the human mouth, throughout the esophageal tract, as well as in the stomach and small intestine. The RNases expressed and active in the human GI tract include both ss

and dsRNases ( e.g., RNase A, T1s, T2s, Us, U

- likes, etc.) (Sorrentino, 2010; Goo and Cho, 2013; Economopoulou et al., 2007; Uchida et al., 1970) and are present to degrade all types of RNAs (with their varied structures ) present in foods consumed. The degradation of RNAs by RNases allow the breakdown of all types and forms of RNAs to be broken down to single nucleotides, which can be further degraded into their components or directly scavenged and used in cells throughou t the body. This collection of RNAs can break down all structural conformations of both PIP and non

- PIP RNAs that are ingested by humans. Overall, RNAs present in foods consumed by human beings are subjected to RNase exposure from the time that they enter the human body through the mouth during consumption (Sorrentino, 2010; Goo and Cho, 2013; Economopoulou et al., 2007; Uchida et al., 1970).

In addition to the RNases encoded within the human genome, there are likely numerous others present and functional (especially in the GI tract) provided by the collection of microorganisms that colonize the body (microbiome). Given the huge diversity of bacteria that constitute the microbiomes of the human GI tract, the RNases present in the human GI tract are likely to encompass a collection of enzymes that can degra de all RNA types no matter the structural confirmation (Hattori and Taylor, 2009) . Thus, the collection of RNases in the human GI tract is incredibly varied and is present to function in breaking down RNAs from all consumed food products into their primary components without regard to their structure.

4 ) Naked RNA in the human gut

RNA is a fundamental constituent of all living cells. Therefore, humans unavoidably ingest natural dsRNA every day (with every meal) as a significant component of their diet. As mentioned previously
, RNA is readily hydrolyzed under both acidic and basic co nditions (Lilley, 2011) . In the human GI tract, RNA encounters hydrochloric acid (HCl) within the human stomach, which is the acid released for digestion of consumed foods. The HCl of the stomach lowers the pH of the stomach to acidic conditions to aid in food digestion. This lower pH also favors hydrolysis of both PIP and non

- PIP RNA molecules. The pH is then raised to ~8 when food moves into the small intestine for the completion of its digestion. These alkaline conditions strongly favor hydrolytic destru ction of RNA molecules. Thus, no matter the structural confirmation in which an RNA molecule arrives into the human GI tract the non

- specific40

hydrolysis of RNA molecules is likely to result in significant degradation of both ss

- and dsRNAs. These include a ll ss

- and dsRNAs from PIP and non

- PIP materials.

Additionally, collections of RNases that result in the degradation of all forms of RNA structure (both secondary and tertiary) are present throughout the human digestive tract (Sorrentino, 2010; Goo and Ch o, 2013; Economopoulou et al., 2007; Uchida et al., 1970). Both PIP and non

- PIP dsRNAs will first be confronted with a broad collection of RNases in the mouth, where they are found in human saliva. In both the stomach and small intestine a large battery of RNases that will degrade all types and structural confirmations of RNAs are also released during the digestion of consumed food products [ 16

- 19 ]

. The combination of RNases and acids found in the hum an digestive system are likely to ensure the all forms of RNA structure are degraded throughout the digestive process. This hypothesis is also supported by other work that has demonstrated that after treatment of total RNA samples from both plants and anim als with a cocktail of both ss

- and dsRNases for more than 2 hours, RNA levels are below the limit of detection even when using very sensitive PCR (amplification

- based) methods ( Li e t al., 2012a, b; Zheng et al., 2010 ; Silverman et al., 2013; Silver et al., 2014
, and Gregory, B.D., unpublished results). Thus, this 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
str uctural status when entering the human digestive system.

5 ) Studies on potential dietary up t ake of miRNAs
The question of potential dietary uptake of miRNAs arose after the publication of a recent study by L. Zhang et al. (2012). The Panel discussed this study and subsequent studies in detail in response to q uestion 2. A brief summary is included here. A recent study by L

. Zhang et al. (2012) reported that dietary plant miRNAs enter the mammalian bloodstream and regulate cholesterol metabolism in the ingesting animal through LDLRAP1 (a low

- density lipoprotein). Three subsequent feeding studies (Snow et al.
, 2013, Witwer et al.
, 2013, and Dickinson et al.
, 2013) reported that up take of foreign miRNAs from the diet is negligible at best. Given the available evidence , the Panel s trongly suggested that non

- modified dietary RNAs (PIP or non

- PIP) regardless of structural status are not transmitted at regulation

- relevant levels out of the GI tract. Although this is likely true, experimental testing of the mammalian blood and exposed tissues should be done to ensure that the siRNAs processed from the PIP dsRNAs are not present, since these could have off

- target effects after huma n consumption.

6 ) RNase resistant RNAs

There is evidence that microRNAs and other RNAs incorporated into microvesicles, for example as in milk, have been reported to be stabilized to degradation by acid conditions and ribonucleases (Hata et al., 2010; Izu mi et al., 2012). Furthermore, the addition of specific proteins, including viral packaging or certain phage RNA

- binding proteins, as well as other chemical modifications has been demonstrated to stabilize RNAs against digestion by RNases and from degrada tion in general (Pasloske et al., 1998; Ghidini et al., 2013; Sczepanski and Joyce, 2013; Li et al., 2014 ). The Panel indicated that it is conceivable that some exogenously applied dsRNA pesticide end

- products will be formulated to increase the stability and protection of these products. Therefore, the Panel recommended further testing41

of stabilizers used in exogenously applied ds RNA end

- products applied to food plants that will be consumed by humans.

7 ) Influence of human health conditions on susceptibi lity to RNA uptake/clearance

The Panel commented on the potential influence of human health conditions (that, e.g., influence digestion conditions, intestinal permeability, or glomerular filtration) on uptake, distribution, and persisten ce of exogenous RNA in the body. As noted by one of the public commenters and further discussed by the Panel, reaction to ingested dsRNA may not be uniform among the population, and certain medical conditions may result in atypical responses. Susceptible subpopulations could include

some individuals with ( 1) limited ability to degrade dsRNA in the digestive system, such as individuals with achlorhydria (producing lower than normal levels of stomach acid), as a result of genetics, disease or inf ections (e.g., Helicobacter pylori ), or medication (e.g., proton pump in hibitors such as omeprazole), ( 2) increased intestinal permeability, facilitating uptake of dsRNA from the intestine, such as individuals with cel iac disease and Crohn’s disease or ( 3) individuals with reduced levels of RNase activity.
Although few studies are available, one panel member indicated that in one study comparing detection of foreign RNA in serum (human) of healthy controls, colitis patients, and individuals with colon canc er, there were no apparent differences in detection of the only plant miRNA (Wang et al., 2012). This panel member noted that Mi R168 was previously reported to be a potential artifact or contaminant of sensitive high

- throughput sequencing studies, and that it was detected in human blood at less than 10 reads per million. Studies on the effects of dietary PIP and individuals with special dietary conditions are not currently available. Given this situation, there are uncertainties in the assumptions of the degradation, uptake, and possible effects of dsRNAs in the absence of species

- and susceptible population

- specific data.

T he Panel re cognized that other issues concerning the stability of dsRNA s hould be addressed and made the following recommendations:

 This charge question focused on the stabilit y of various structural forms of dsRNAs when ingested into the human gut. The Panel agreed that the dietary route of exposure is the most likely route of exposure to dsRNA PIPs, but other routes of exposure, such as dermal or intranasal (pollen and dermal) should also be considered as part of th e human health risk
assessment.

 T he Panel recommended that t he stability of dsRNA should be tested in individuals that manifest specific diseases (e.g. colitis, irritable bowel syndrome, etc.), the immune compromised, elderly, as well as children. These individuals may have compromised di gestion or increased sensitivity to dsRNA exposure

.

 Exogenously applied dsRNA end

- products may use stabilizers to stabilize dsRNA. The Panel recommended that possible effects of stabilizers on uptake of dsRNA should be evaluated

.42

Environmental Considerations: Questions directed toward dsRNA expressed as

part of a PIP and applied to the plant as a biochemical

Question 4. Environmental Fate

EPA needs a clear understanding of the environmental fate of dsRNAs in terrestrial and aquatic environments. In sections IV.A.1. and IV.B.3, respectively, EPA has presented potential scenarios for dsRNA movement within the environment that may result from their pesticidal uses as PIPs or as exogenously applied dsRNAs. Environmental fate of dsRNAs is not well understood; however, EPA has frameworks in place for PIPs and Biochemicals to obtain data related to degradation and movement of pesticides in the environment. These data can be used to refine environmental exposure estimates to dsRNAs.
While nontarget exposure to dsRNA PIPs may res ult primarily from consumption of PIP crop plant tissue, EPA must also assess the exposure of nontarget organisms to dsRNA that may enter the environment through plant exudates and plant tissue breakdown. In section IV.A.2.a, EPA presented the current appr oach for testing to assess environmental fate for PIPs, which focuses on degradation within soil.

4.a. i) To what extent will data on degradation in soil inform EPA on nontarget exposure to dsRNA PIPs?

Panel Response

The Panel noted that few studies in the published literature describe the degradation, persistence, and bioavailability of dsRNA and siRNA in the environment. The Panel recommended that environmental persistence of dsRNA and any active RNA i effectors ( e.g., siRNA capable of binding Argonaut e and forming RISC) should be documented as part of the environmental fate assessment . This documentation should include soil sampling representing all soil types, temperatures, and moisture content across the targeted pest

- crop agroecosystem that will be affected by the proposed dsRNA pest management strategies. Binding of dsRNA to soil organic matter may decrease degradation, but such binding may also decre ase availability to organisms. Both the persistence in soil and bioavailability should be evaluated in soil degradation studies and pesticidal activity studies. Measurement of the de gradation of plant materials should be included to determine the extent to which in planta dsRNA and activated RNAi effectors are released over time. Results of these tests c ould inform and direct non

- target effect testing (see response to question 7 ).

The Panel recognized that there will be significant differences in evaluations of dsRNA PIPs and exogenously applied dsRNA, e.g., dsRNA in plant tissue and in sprays . Plant tis sue may be desiccated and remain in fields during quite cold intervals. Thus, dsRNA may remain in plant material for long periods of time allowing slow release into the environment. Double

- stranded RNA used as a PIP may be exuded into the rhizosphere, wher e biotic and abiotic processes can vary quite significantly between43

growing and dormant seasons. The Panel noted that roots of annual plants will experience different soil chemistry conditions (nutrients, oxygen, temperature, moisture, microbial activity) that may be dissimilar to growing season conditions.

The Panel provided the following comments and recommendations on the proposed environmental fate testing scheme. Many of these recommendations apply to both PIP and exogenously applied dsRNA.

1) Bioavailability

The Panel stressed the importance of assessing the bioavailability of the dsRNA to non

- target organisms as one of the earliest steps in the environmental fate evaluation process (part of a tier 1 evaluation). Panel members agreed that pers istence of dsRNA PIPs must be better understood to evaluate exposure potential. Persistence of dsRNA in th e soil will be affected by a number of factors, e.g., temperature, soil structure and type , abiotic and biotic factors. For example, f ield s with summe r crops that express dsRNA PIPs or are sprayed with dsRNA may have enhanced microbial or abiotic processes to degrade dsRNA as compared to fields in the winter. When conducting studies with plant tissue, care s hould be taken to ensure that studies of dsRNA degradation in above ground or root portions of plants are done in such a way that degradation/release from plant tissue is being measured and not simple degradation in soil. The Panel recommended studies be conducted to evaluate dsRNA degradation in the environment and bioavailability for uptake by unintended organisms. An example of the type of soil degradation study to be conducted was provided by one of the public commenters.

Soil dwelling invertebrates comprise a significant portion of the diet in adults and nestlings of selected avian species. For example
, the primary route of avian exposure to pesticides may be through ingestion o f earthworms. Thus , data on the soil degradation of dsRNA and degraded products (if present) would be usefu l to inform the bioavailability of environmental dsRNAs for uptake by soil dwelling invertebrates.
Some panel members commented that nanotechnology might be a potential delivery system for exogenously applied dsRNAs applied in or sorbed to nanoparticles.
2) dsRNA stability in soils

Little is known about the dsRNA stability and degradation kinetics of dsRNA PIPs and derivatives in soil. The Panel stated that specific data on dsRNA kinetics in soil and in plant material are needed to minimize uncertainties in exposure assessments . The importance of abiotic and biotic factors in the soil on the rate of degradation is discussed later in the response to this question.

I n the absence of RNA stability data in dried tissues, the Panel looked at the relative stability data for RNA versus DNA in dried forensic samples

. Results in H art e veld et al. ( 2013 ) indicated that saliva RNA can be more stable than saliva DNA in periods of up to 7 days at temperatures up to 20 o C and for 1 day at temperatures of 37 o C . At col der temperatures, approximately 50% of RNA remained after 1 day at 2 o C and 20% remained after 7 days. In the same study, d r y i ng blood samples st abilized RNA. The44

Panel suggested that this information p rovides some insight on RNA stability in desiccated and cold plant materials above and below ground during fall and winter months.
Using in vitro cell cultures, Hoerter et al. (2011) found that dsRNA may be more stable in epithelial cells than serum. Furthermore, this study found th at dsRNA is more stable than RNA/DNA hybrids in epithelial cells. The Panel suggested that this study addresses stability in animal tissues (potential non

- target organisms) or in food items of carnivorous/omnivorous non

- target species.

In contrast to these two studies indicating the relative stability of RNA vs. DNA , studies in aphids demonstrated that dsRNA is rapidly degraded in a manner of hours (Yu et al., 2013)

. 3 ) Bioactive degradate s
The Panel added that it is important to determine if products of degradation in the environmental and biological samples are bioactive in target and non

- target organisms

. Recognizing that testing all RNAi processes for all degradation products is improbable, investigations should strive to identify dsRNA tra nsformation products that have moderate to high likelihood of exhibiting RNAi activities in target and non

- target organisms.

4 ) Uptake by non

- target organisms

If the dsRNA is bioavailable to target and non

- target organisms (either as the parent dsRNA or siRNA)
, the next step is to determine the potential exposure to non

- target organisms. The EPA background document (EPA, 2013) discussed the possibility that some organisms have the ability to uptake environmental siRNA and amplify these siRNAs within the organism that may lead to additional exposure

. For example, C. elegans can accomplish this through RdRP amplification (Sijen et al., 2001). Environmental uptake and RdRP amplification has al so been noted for some plants and fungi (Dillin, 2003). If cellular uptake is not possible then environmental exposure would be irrelevant. RdRP amplification pathways are not present in mammals, insects, and bacteria. The presence of other amplification p athways over a wider range of organisms should be investigated if there is a potential for transfer. The Panel discussed amplification mechanisms in response to questions 2 and 6 and cellular uptake mechanisms in response to question 5.

5 ) Trophic connections

The Panel discussed exposures due to possible trophic connections (e.g., predation, herbivory, mycophagy, etc.)

. The focus was on the connection between soil and above ground organisms, e.g., rhizosphere microbes and invertebrates that can be consumed by above ground vertebrates and invertebrates. Given these trophic linkages, the Panel suggested that the potential of dsRNA transfer between soil and above ground organisms be investigated if the potential for transfer of dsRNA is determined in the previous studies

. In these cases, t he Panel also recommended that the uptake of dsRNA in herbivorous insects and non

- target insects should be evaluated because of the possible impacts on pr edatory insects and arachnids. The Panel discussed amplificatio n mechanisms in response to questions 2 and 6 and cellular uptake mechanisms in response to question 5.45

6 ) Non

- target tox icity testing

The current non

- target testing paradigm for PIPs, most of which express proteins derived from the bacterium, Bacillus thuringiensis ( Bt)
, is based on modes of action that are fund amentally different from RNAi. The Panel indicated that that EPA’s proposed ecological risk assessment framework should be modified to address the unique environmental fate and exposure scenarios posed by dsRNA PIPs

. In any non

- target testing paradigm , multiple non

- target species should be tested ; however, a standard suite of species may not be appropriate for all RNAi products. The life stage of potentially exposed non

- target species should be considered in toxicity testing paradigms. For example, if toxicity is observed for vertebrates, juveniles should be considered as a sensitive alternative to the standard test design. Juveniles are most likely to be present at the time when insecticidal activity is needed and as such they are likely to be exposed at a susceptible life stage. Fossorial or other seasonally burrowing organisms (e.g., amphibians) may be exposed dermally. This could be particularly important for amphibians in the playa or prairie pothole regions. The Panel suggested having amphibian testing earlier as part of a tier 1 testing scheme. Much more descriptive information on the suitability of the current ecological risk a ssessment is found in the response to question 7.

4.a. ii) What additional information, if any, would EPA need to assess environmental fate of dsRNA PIPs?

Panel Response
The Panel agreed with EPA that dsRNA PIPs pose unique cha llenges not seen for other PIPs (see discussion in EPA’s issue paper, pp. 13

- 19). There will be significant differences in evaluations of dsRNA PIPs , e.g., dsRNA in plant tissue and in the rhizosphere, and exogenously applied dsRNA end

products, e.g., sprays

.

The Panel stated that c urrent requirements for soil degradation studies will provide some insight for non

- target testing, but additional data are needed to assess the environmental fate of dsRNA PIPs and potential exposures to non

- target organis ms, i.e., plant degradation (above and below ground), off

site movement of plant materials, in planta exposure for organisms that directly ingest plant materials. Plant tissue may be desiccated and remain in fi elds during quite cold periods

. As a result, d sRNA may remain in plant material for long periods of time allowing slow release into the environment. Double

stranded RNA PIPs may be exuded into the rhizosphere, where biotic and abiotic processes can vary quite significantly between growing and dormant seasons. The Panel noted that roots of annual plants will decompose under soil chemistry conditions (nutrients, oxygen, temperature, moisture, microbial activity) that may be dissimilar to growing season conditions. The Panel emphasized that additional da t a are critical to understand the stability of dsRNA in soil and aquatic environments. While there is a reasonable body of literature describing the environmental persistence of nucleic acids in soil, there is little informa tion on the persistence of dsRNA and small RNA (siRNAs and miRNAs). The Panel recommended the collection of environmental fate data for different soil types, water bodies, and weather conditions under which crops with dsRNA PIP may be cultivated.46

1) Soil type
Persistence in the soil is affected by a range of soil qualities and environmental conditions. The Panel suggested that persistence be measured for different structural forms of the RNAi molecules (dsRNA, siRNAs and hairpin RNAs) across a range of temperat ures, moistures, and soil type. Whether the dsRNA PIP bound to organic matter renders them unavailable for uptake or degraded is not known. Thus, in studies of RNA degradation, it will be important to indicate when and where synthetic RNA molecules are use d, and what differences, if any, there may be between these substrates and the native plant materials. These might include chemical modifications, sequence differences, protein packaging,
etc. The contribution of soil type on degradation kinetics of dsRNA PIPs expressed in crops should be further evaluated for multiple soil types and at various temperatures, etc.
During th e public comment period, representatives from Monsanto presented unpublished data from preliminary soil degradation studies of a dsRNA P IP for corn rootworm control under development

. One panelist was concerned that low PIP concentrations in planta in Monsanto’s studies were insufficient to allow de tection in degradation studies. In response, representatives from Monsanto stated that the active dsRNA PIPs are at such low concentration that in their soil studies, vast quantities of synthetic ds RNAs were added to biological samples in order to be able to measure the RNA. The Panel stated that these data should be made publicly available 1

. 2) Soil biology
An important and overlooked component in EPA’s list of required tests is the contribution of abiotic and biotic factors on the rate of soil degradation . For example, biodegradation of DNA is mediated by m icrobial

- produced deoxyribonuclease s ( DNases ) ( Levy

- Booth et al., 2007). The degree to which biota contribute to the degradation rate of ds RNAs in the soil is poorly understood, and as such, represents an important data gap that should be addressed. The Panel recommended that the contribution of soil biota on degradation kinetics of dsRNA PIPs expressed in crops (e.g., degrading plant biomass in the presence or absence of other organic matter) should be evaluated using multiple soil types, temperatures, etc. conducted to refine exposure assessments.
3) Viability of dsRNA, siRNA transfer to other organisms

The viability of dsRNA (siRNA) of uptake by other or ganisms is an issue that needs to be addressed. If the dsRNA (siRNA) is bioavailable then transfer to other organisms might be possible if there is cellular uptake . If uptake is not possible then the issue of viability is irrelevant. See also the discussion of cellular uptake in response to question 4.a. i.2 and 5.

4) Predictive value of models

1 Note: These data were published subsequent to the SAP meeting as Dubelman et al. (2014). The Panel used only the information presented by Monsanto during the meeting.47

Models are good tools if the model has sufficiently precise and accurate empirical data defining input parameters. However, this is not the case for dsRNA P IPs. The Panel stated that uncertainties involved in input parameters of the EPA and other stability/persistence models may be sufficiently large to provide outputs that are of little or no predictive use. For this reason, the Panel urges caution in using EPA models ( discussed on p . 15 of EPA’s issue paper ) until more empirical data are available. This shortcoming can only be overcome with sufficient empirical data that describe PIP environmental fate and transport, persistence
, bioavailability and biological effects.

4. b. In section IV.B.4., EPA proposed environmental fate data needs for exogenously applied dsRNAs. To what extent is the proposed testing sufficiently robust to inform nontarget organism exposure for these dsRNAs?

Panel Response

As noted for dsRNA PIPs, there are also unique challenges to the assessment of environmental fate of and exposure to exogenously applied dsRNA products. The response to question 4.a. i. regarding environmental fate data needs for dsRNA PIPs (soil type, soil biology, viability of dsRNA (siRNA) transfer to other organisms, and applicability of models) is also generally applicable to answer questions concerning environmental fate data needs for exogenously applied dsRNAs; however, there are som e differences. Non

- target species are more likely to be exposed to exogenously applied dsRNA end

products than to dsRNA PIPs

. Future end

- products might fit into several different pest control categories (p. 24, EPA issue paper)

. Non

- PIP dsRNA exposures are likely multi

- route while dsRNA PIP exposure is more likely via diet

. Exposure may be through airborne particles, diet , or dermal/cuticle penetration depending on the end

- product. However, the dsRNA in an applied product (e.g., spray application) may be mo re exposed to environmental factors, e.g., UV light. As such, more species may be exposed to exogenously applied dsRNA products, although this concern may be balanced by the likely increased instability of the sprayed product (duration of the RNAi).

The P anel agreed that EPA’s standard testing paradigm (shown below) represents a good beginning for data sets needed to understand soil degradation and environmental fate of dsRNAs in the environment; however, additional data are needed to address exposure scen arios unique to exogenously applied dsRNA products (see also EPA’s discussion of exposure scenarios on pp. 26

- 28 of the issue paper).

 Sediment and soil adsorption/desorption for parent and degradates

 Soil column leaching

 Hydrolysis

 Aerobic soil metabolism

 Photodegradation in water

 Photodegradation on soil

 Anaerobic soil metabolism

 Aerobic aquatic metabolism

 Anaerobic aquatic metabolism48

The Panel stressed the importance of assessing the bio availability of the dsRNA to non

- target organisms as one of the ear liest steps in the environmental fate evaluation process (a first tier evaluation). Data are lacking on cellular uptake of ds RNA at various taxonomic levels. In the absence of data, t he Panel recommended that the present focus should be on evaluating uptake in non

- target organisms closely related to the target (e.g., species in the same genus and related genera in the same family ). See additional discussion in response to question 5. Special consideration should be given to

non

- target amp hibian testing as part of a tier 1 testing regime. The Panel indicated that the life stage exposed to dsRNAs should also be considered in the design of non

- target tests. Early life stages often do not have protective enzymes or other detoxifying mechanisms . The Panel recommended that multiple species be evaluated for some of the required tests

. Test species might include organisms not currently listed in the non

- target testing paradigm. See the Panel’s response to question 7.
For both dsRNA PIPs that may b e free in the environment and exogenously applied dsRNAs, knowledge of the initial residue burden, and the dissipation rate of residues, is essential for developing environmental exposure estimates, particularly if models of environmental fate are used to arrive at these estimates.

4.c. What analytical methods are available to accurately and precisely measure dsRNAs in diverse plant, soil, and water matrices?
Panel Response

The Panel indicated that existing RNA extraction methods and subsequent quantitative PCR, high throughput sequencing, or hybridization analysis are the most current tools to assess the presence of a particular small RNA within a sample

. The Panel stressed the importanc e of standardizing t he extraction and isolation protocols and having accurate and precise analytical methods to quantify if active degradation products, dsRNA and associated small RNA s , are present in the biotic or abiotic samples.
The Panel emphasized the critical nature of using a reliable method for extracting the dsRNA to track the environmental fate of the dsRNA in soil . Each PCR is run on a VERY small quantity of soil or tissue, and thus it becomes very important to conduct power analyses to assess how many samples will be necessary in order to reliably detect the substance within environmental samples. Recovery of material from an environmental sample may be limited. The use of small sample sizes (mass/volume) may introduce significant intra sample variance due to sampl e heterogeneity. For example
, metal distribution in soils can be extremely variable

. With these considerations, extremely large numbers of samples may be required for a reliable analysi s of agricultural systems under different field conditions

. Question 5. Primary Route of Exposure for Nontarget Organisms to dsRNA.

The primary route of exposure for nontarget organisms to dsRNA PIPs is assumed to be ingestion, either of dsRNAs contained in plant tissue or free in the environment. However, some evidence also indicates the potential for exposure by direct contact in nematodes and some arthropods. For49

dsRNAs that are applied to plants and/or the environment, the primary routes of exposure are expected to be both ingestion and direct contact.

5. a .i ) In addition to the conditions of the gut environment and enzymes influencing digestion, what other factors may play a role in uptake within the gut and potentially limit exposure to dsRNA?

Panel Response
The published literature indicat es there is a wide range of cellular uptake mechanisms that vary from species to species and across taxa. For example, cellular uptake of dsRNA is mediated by transmembrane proteins, SID

- 1 and SID

- 2, or through endocytosis ( Hannon , 2002 ; Huvenne and Smagghe
, 2010 )

. It seems likely that other pathways for dsRNAs to enter cells will be discovered in the future. Factors that influence cellular uptake and activity include: tissue

- or cell

- specific expression, transport proteins, dose, dsRNA sequence length, and degradation. The Panel recommended additional research to study this issue.

The Panel commented on the following f actors that influence cellular uptake and activity :

 Tissues. Gene expression may be tissue or cell specific. It remains unclear what processes make tissues amenable or not to RNAi. Indeed different tissues, even in target organisms, lack the ability to uptake dsRNA from the surrounding environment. The basis of this difference is still unknown, but may be due to fundamental developmental differences between tissue types, such as cell ploidy, or due to differences in gene expression required for the uptake and transport of dsRNA. The gut consists of different cell types
, columnar cells with typical microvillar pole , goblet cells, endocrine cells, and stem cells. Data is lacking on cellular uptake in different cell types or in cellular uptake in different organisms ( Hakim et al., 2010).

 Transport proteins. The impact of different transport proteins is not clear , e.g., SID

- 1, SID

- 1 like/SID

- 2, clathrin

- mediated and/or caveolae

- mediated endocytosis, and lipophorins can all bind dsRNA . For example, l ipophorins can adhere to dsRNA, bacteria and fungi present in the insect hemolym ph (Wynant et al., 2014) and assist in the cellular u ptake of dsRNA in some insects ( Terenius et al., 2011; Scott et al., 2013; Swevers and Smagghe, 2012). The Panel stressed that a generalization of physical barriers for environmental uptake of dsRNAs (from PIPs or exogenously applied) acr oss non

- target taxa (e.g., targeted versus non

- targeted taxa or humans versus arthropods) is not possible at the present time due to the lack of information across species. The published literature indicates there is a wide range of environmental uptake

me chanisms that vary from species to species and across taxa. No definitive conclusions can be made concerning specific physiological barriers at this time. The Panel also commented that ability to cross physiological barriers also has to be combined with th e binding in the gut; i.e., whether there is an actual target to be affected. There is no predictable conserved pattern of transport mechanisms among all insects.50

 dsRNA length. There is some information that dsRNA length impacts cellular uptake, but there is no general ized pattern among insect taxa.

 Dose. Dose information is needed for the assessment of exposure and effects of exogenous dsRNA. What dose of dsRNA will trigger effective gene silencing?

 Formulation of applied dsRNA end

- products. Polymer s and agents used in an end

- product formulation to increase uptake/penetration kinetics to overcome physical b arriers should be investigated for potential effects on non

- target organisms.

Other factors suggested by some panel members:

 Metabolic degradation of dsRNA in insects. One panel member suggested that rapid degradation of dsRNA in some insects would minimize exposure to exogenous dsRNA. There are only two papers so far that confirm enzymatic degradation of dsRNA in insects (Lygus and aphi ds) in the saliva and in the gut (Allen and Walker, 2012; Christiaens et al., 2014). Additional information is ne eded for other insects before any generalization can be made regarding the role of degradation in reducing non

- target exposure to exogenous dsR NAs (PIPs

or non

- PIPs)

 Peritrophic matrix. One panel member mentioned that the peritrophic matrix lining the midgut in many insects might act as a physical barrier, but no informati on exists to verify this role. Another panel member responded that exogenous dsRNA will readily pass through the peritroph ic matrix because of its size.

. 5. a

. ii) Please comment on how these barriers can be generalized across all nontarget taxa that are considered in EPA’s risk assessments (e.g., birds, plants, fish, etc. ) for both dsRNA PIPs and exogenously applied dsRNA.
Panel Response The published literature indicates there is a wide range of environmental uptake mechanisms that vary from species to species and across taxa. The strategy of using dsRNA as a species

- specific pesticide against insects depends at least in part on dietary uptake that varies from species to species ( see references including , but not limited to: Whyard et al., 2009; Zhou et al., 2008; Terenius et al ., 2011; Bachman et al ., 2013; Zhang et al., 2013)

. For example, a mongst insects, there is a wide range of gut pH; diet composition and feeding practices; conservation and function of RNA receptors and transmembrane channels including, but not limited to, the SID proteins; and activity of RNAses in digestive fluids and hemolymph. These factors may contribute to species differences in foreign RNAi effectiveness. Gut conditions among organisms are variable . A low pH of 1

- 2 occurs in the human stomach, while t he stomach pH is higher in mice . I n Lepidoptera the midgut pH can reach 12. There is51

no indication of interaction of insect dsRNAs with the gut microbiome communities (e.g., degradation, accumulation, amplification).

The Panel stated that there is insufficient understanding of the uptake mechanisms of dsRNAs (PIPs

or non

- PIPs) and potential barriers to make any generalizations across non

- target taxa. The Panel also commented that the ability to cross physiological barriers has to be combine d with binding in the gut (where there is an actual target to be affected). The Panel recommended the collection of additional information to evaluate the types of cellular uptake mechanisms that exist and whether there are any patterns among different spe cies, genera, or families , for example. For now, the Panel recommended a

case

- by

- case evaluation of cellular uptake mechanisms for non

- target taxa , as needed (based on exposure).

5.b. The degree of exposure by direct contact is likely to differ between no ntarget risk assessments for dsRNA PIPs and nontarget risk assessments for exogenously applied dsRNAs.

5.b. i) Please comment on the importance of the contact route of exposure for nontarget risk assessments for each of these types of dsRNAs

.

Panel Response
There are insufficient data on the contact route of exposure to comment on the importance of this route in non

- target risk assessment s

. The Panel found only two studies that examined direct contact as a route of exposure to dsRNA. The limited info rmation available indicates that the dietary route of exposure was probably the primary route of entry leading to activity and not the contact route of exposure. The Panel recommended the collection of additional information to evaluate the importance of
t his route of exposure. For now, the Panel recommended a case

- by

- case evaluation to determine whether this is a likely route of exposure before pursuit of an in

- depth study.

In both studies
, insects were soaked or treated topically with a solution of dsRNA. In the first study (Singh et al., 2012)
, mosquito larvae ( Aedes aegypti ) were soaked in 0.5 μg/μL dsRNA solution for 2 hours. The mRNA transcripts for β

- tubulin
, chitin synthase

- 1 and

- 2
, and heat shock protein 83 were reduced between 30 and 50% at three days post

- dsRNA treatment. Based on the Panel’s review of the data, and as reported by the authors, the gut was probably the primary route of entry for the dsRNAs leading to activity. In th e second case (Wang et al. 2011), newly hatched Ostrinia fumalalis larvae (a lepi d opteran) sprayed with 50 ng/ml dsRNAs of the genes DS10 and DS28 resulted in around 40

- 50% mortality

. In this situation, uptake happened via the gut as caterpillars crawl ed over each other

. The Panel remarked that in C. elegans , soaking is a common practice (Tabara et al., 1998). Here, as in the other described cases, the probable route of dsRNA uptake is through the digestive system and not through the integument itself. Th e Panel recommended that this route of exposure should be evaluated

on a case by case basis , as indicated (based on exposure)

. 5.b. ii) What barriers are likely to exist for this route of exposure for both terrestrial and aquatic organisms, and how can these be generalized across nontarget taxa?52

The Panel indicated that there are not enough experimental data available on the contact route of exposure and uptake for both terrestrial and aquatic organisms to make any comparisons or generalizations across non

- target taxa. For now, t he P anel recommended that each dsRNA PIP or exogenously applied dsRNA end

- product should be evaluated on a case

- by

- case basis (based on exposure)

. See response to question 5.b.i.
Question 6. Unintended Effects of dsRNA

In the issue paper, EPA discussed possible effects other than silencing of the target gene in the target organism that may occur as a result of exposure to dsRNA. These unintended effects of dsRNA include off

- target effects, silencing the target gene in nontarget organisms, degradation of

non - targeted mRNA by transitive RNA, and effects resulting from immune stimulation and saturation of the RNAi machinery. EPA has little information to estimate the range of unintended effects that may occur and their probability of occurrence as a result of exposure to dsRNA in the environment.

6

. a. Please comment on the unintended effects that EPA might reasonably anticipate in nontarget organisms exposed to dsRNAs, the likelihood of such unintended effects, and the biological s ignificance of these effects in nontarget organisms, should they occur.

Panel Response

Note: The Panel focused the discussion on unintended effects of dsRNA on non

- target insects.

The Panel discussed several possible unintended effects that may result fr om exposure to dsRNA. One concern is that the potential scale for use of pesticidal RNAi in the agroecosystem (in particular) will increase the potential of unintended effects. For example, dsRNA used to silence the target gene may also silence the same co nserved gene in non

- target organisms. But one of the advantages of RNAi technology is that the RNAi effect is sequence and target gene dependent. Therefore, it is possible that dsRNA sequence used to trigger RNAi can be designed to be highly spe cies specif ic which should help reduce possible non

- target effects

. Saturation of RNAi machinery and immunostimulation has been observed in animals (in mice or in cell cultures) , but both require a fairly high dose to cause deleterious effects. The dose required to affect the RNAi machinery and induce immunostimulation in insects is unknown. There is also a lack of information on the risk of insect resistance to RNAi

- mediated gene suppression. The knowledge gaps make it difficult to predict with any certainty whether unintended effects will occur in non

- target species as a result of exposure to dsRNA

. More data to close these gaps will increase the certainty in predicting the likelihood of unintended effects.
1) A standard suite of molecular machinery is present in R NAi

- active eukaryotes
The introduction to the report provided an overview of the elements involved in dsRNA

- mediated gene interference. This section focuses on eukaryotic RNAi. There are four essential steps that potentially dictate which species will be a ffected by this technology ( Agrawal et al.
, 2003 ) . First, a dsRNA must be produced (either endogenously or exogenously) that has some minimal level of53

specificity for a targeted mRNA. Next, this relatively long dsRNA (<1,000 bp) must be cleaved into small ( 20

- 25 nt) siRNAs usin g an enzym e in the Dicer family ( Elbashir et al.
, 2001 ; Siomi and Siomi 2009 )

. These siRNAs dissociate into single strands after integration into RISC. Finally, the target mRNA molecule is either cleaved ( by an enzyme in the Argonaute family) or removed from translation through bindin g with the siRNA. Silencing in the absence of cleavage may result if the RISC unit simply binds to mRNA, thereby restricting its translation ( Alemán et al.
, 2007 ) . The amplified siRNAs are

som etimes then transported to other cells of eukaryotic hosts via the SID

- 2 transport protein; this process is dubbed systemic RNAi. Although these steps appear to be universally present in eukaryotes that use RNAi, there are slig ht differences in the machine ry, which potentially restrict the activity spectrum of a given dsRNA for non

- target organisms ( Agrawal et al.
, 2003 ; Reynolds, 2004; Leake et al.
, 2004 ; Alemán et al., 2007; Doench et al. 2007 )

. In plants, the amplification process is regulated by RNA

- directed RNA polymerase s (RdRP). In contrast, a n Rd RP amplification process or an equivalent has not been identified in insects (a primar y target of RNAi

- based genetically

- modified crops) or in mammals (see discussion in response to question 2) . An exception is embryonic Drosophila melanogaster ( Agrawal et al.
, 2003 ; Dillin
, 2003 ; Gordon and Waterhouse
, 2007 ; Price and Gatehouse
, 2008 ) . A lthough RNAi functions at low molar quantities in these organisms and many cell types and tissues remain to be examined. As such, some mechanism that facilitates the proliferation of siRNA within a target insect or mammal may be discovered upon further exploration.

2 ) Unintended gene suppression by pesticidal RNAs (non

- and off

- target effects)

Most of the work on off

- target silencing is related to functional genomics within a cell culture or a single organism, and so the question of how the choice of dsRNA sequence affect target and off

- target genes in non

- target organisms that are potentially phylogenetically distant from the target pest has received very little attention. Nevertheless, substan tial research has been conducted in this area from the perspective of functional genomics and gene therapy that can be used to inform the likelihood of this effect. Most of the experience with this technology is in cell cultures or sick individuals.

The potential scale for use of dsRNA technology as pesticides in agroecosystem s (in particular) warrants exploration of the potential for unintended ecological effects

. The Panel discussed several possible unintended effects that might occur if non

- target orga nisms are exposed to d sRNA PIPs or exogenously applied dsRNA end

- products. The knowledge gaps make it difficult to predict with any certainty whether unintended effects will occur in non

- target species, and their severity should they occur, as a result of exposure to dsRNA. Additional information is needed to close these gaps and reduce uncertainty in predicting the likelihood of unintended effects.

a. Silencing the target gene in a non

- target organism using pesticidal RNA can be partially mitigated through design of the pesticidal RNAs. Carefully targeting genes that have species

- level divergence in sequence will help to hone the specificity of RNAi to the target pest. Through the use of dsRNAs that produce multiple, distinct functional siRNAs, the g enes of non

- target organisms that are the focus of RNAi can have substantial sequence divergence from the target species’ gene and still experience gene suppression. For example, in developing genetically

- modified maize plants resistant to Diabrotica virgi

fera virgifera (Western corn rootworm), Baum et a l. ( 2007 ) also examined the effects of54

a few of the dsRNAs identified for plant transformation on several other beet le species. The y found that the dsRNAs that targeted D. virgifera v

- ATPase A and E also reduced survival of Diabrotica undecimpunctata (spotted cucumber beetle) and Leptinotarsa decemlineata (Colorado potato beetle) significantly, even though these pests shared only 79 and 83% sequence homologies in these genes with D. virgifera . Whyard et al. ( 2009 ) did not find increased mortality when Drosophila spp. ingested dsR NAs designed to suppress a congener’s tubulin gene in laboratory feeding assays. These results were echoed when other, more phylogenetically distant, insect taxa ingested dsRNAs aimed at repressing other species’ γ

- tubulin or v

- ATPase expression, although mRNA knockdown for the latter gene was minimal even for the targeted insect species.

Each organism’s genome presents a unique set of potential off

- target gene sequences ( Qiu et al.
, 2005 ) . The likelihood of off

- target binding increases with the size of the genome, and so reduced hazard is posed to non

- target species with relatively small genomes ( Qiu et al.
, 2005 ) . The length of the dsRNA is also positively correlated with off

- target binding ( Lew

- Tabor et al.
, 2011 ) , and using strictly siRNAs can substantially reduce the likelihood of off

- target effects ( Qiu et al.
, 2005 ) . Structural characteristics of the siRNA, especially in the seed region (2’ location of the guide strand) is particularly important in reducing off

- target binding, and pesticidal RNAs can be engineered rationally (examples would be methylating the 2’ position of the small RNA ( Caffrey et al.
, 2011 ) , or producing “bulge” RNA s) ( Dua et al., 2011 ) . Chances of off

- target binding increase as the siRNA becomes shorter and if sequence s mismatches between target and off

- target sites occur ( Qi u et al.
, 2005 ; Seinen et al., 2011 ) . Small inducing RNAs are more likely to produce off

- target effects when a higher dose than is needed is administered ( Caffrey et al.
, 2011 )

. b

. Sequence homology and off

- target gene sil

encing. In silico searches commonly reveal that 10

- 20% (but potentially up to 80%) of randomly generated siRNA sequences will bind to unintended mRNAs within a single organism ( Qiu et al.
, 2005 ; Kulkarni e t al.
, 2006 ) . There is also experimental evidence of off

- target binding ( Jackson et al., 2003 ; Lin et al.
, 2005
, Ma et al., 2006
, Davidson and McCray
, 2011 ; Dua e t al.
, 2011 ; Lew

- Tabor et al., 2011 ; Seinen et al.
, 2011 ; Jarosch and Moritz
, 2012 )

. Rates of mRNA and protein suppression may be correlated with rates of off

- target binding predicted by in silico searches for sequence homologies with siRNAs ( Lew

- Tabo r et al.
, 2011
, Seinen et al.
, 2011 )
, especially when considering the sequences of the seed region rather than complete sequence of the siRNA ( Birmingham et al., 2006 )

. Given the diversity of organisms pre sent in and around agroecosystems and uncertainty in defining the spectrum of activity and non

- target organisms exposed to potential dsRNA PIPs or exogenously applied dsRNA end

- pr oducts, the Panel suggested that in silico searches may help identify potential non

- target organisms that could be further evaluated (see related discussion in response to question 7). As stated in the response to question 1, in silico searches may be

use ful screening tools to identify potential non

- target organisms, but should not be used as an absolute predictor of effects.
Evidence suggests that o ff

- target binding can change the phenotypes of affected organisms, sometimes unpredictably. Suppression of m RNA by off

- target binding reduces some phenotypes ( Saxena et al.
, 2003 ; Lin et al., 2005 ) , although RNAi effects on off

- target protein levels tend to be less studied than mRNA regulation. Federov et al

. ( 2006 ) found that 29% of off

- target suppression of mRNAs resulted in the death of transfected cells. Off

- target binding of siRNAs resulted in reduced55

protein production in 7 of 30 cases involving cult ure cells ; surprisingly this off

- target suppression of genes was not accompanied by mRNA cleavage, but by binding of the siRNA and RISC unit with the targeted mRNA ( Alemán et al.
, 2007 ) . Thus, looking only at mRNA levels may overlook some off

target gene silencing ( Saxena et al.
, 2003 ; Alemán et al.
, 2007 ) . These off

- target effects become particularly important when proteins within metabolic cascades or biochemical pathways; numerous phenotypes could be affected by disruptions of individual mRNA ( Wheeler et al., 2013 )

. The Panel noted that off

- target gene silencing will only affect phenotypes of the transcribed genes, and so it may be more effective to predict off

- target effects by examining comprehensive transcriptomes rather than full genomes. Transcriptome changes would need to be related to apical effects (growth, development, survival, e tc.) to confirm the prediction. This issue is also discussed below and in the response to question 7.

3 ) Saturation of RNAi machinery can have severe consequences
Saturation of RNAi machinery could have severe c onsequences for cellular functions, although it is unclear what dose is necessary to harm non

- target organisms. High levels of exogenous siRNAs can saturate a cell’s RNAi machinery, and thereby reduce the efficiency at which a cell regulates endogenous gene expression with miRNA ( Agrawal i et al.
, 2003 ; Dillin
, 2003 ; Grimm et al.
, 2010 )

. Dicer was one of the first componen ts of RNAi machinery that was demonstrated to be saturated by high levels of siRNA. Essentially, there are limited number of RISCs present within a cell, and if the augmented siRNAs saturate these complexes, then health and performance of the cell may be

c ompromised ( Kahn et al., 2009 ) . In particular, Exportin

- 5 and Argonaute proteins (especially Ago

- 2) seem to be particularly limiting components of the RNAi machinery that have been demonstrated to be saturated by exogenous siRNAs ( Grimm
, 2011 ) . Saturation occurred when hundreds of thousands of siRNA sequences were being produced per cell ( Grimm et al.
, 2010 ) , and dose is clearly an important component of this risk factor. Jackson and Lindley ( Jackson and Linsley
, 2010 ) found eviden ce that small RNAs could have “ global effects on the exp ression of genes predicted to be under the control of endogenous microRNAs.” This process of saturation is better documented with shRNA (small hairpin RNAs), although it is known from siRNA as well. One question that needs to be answered first is whether i t is possible to deliver enough pesticidal dsRNA to saturate an organism’s RNAi machinery. On e panel member commented that saturation will not matter if the amount will be so m inim al that it is inconsequential.

4 ) Immune stimulation
Innate immune systems of higher organisms rely on pattern recognition proteins and other factors to identify potentially pathogenic invaders, and these defenses recognize and eliminate dsRNAs that are potential pathogens ( Elbashir e t al.
, 2001 ; Kulkarni et al.
, 2006 ) . Immunostimulation is more likely to occur with molecules smaller than 28 nt in length. Generally speaking, siRNAs are able to trigger mammalian endosomal immune cascades (e.g. Toll receptors) or cytoplasmic pathways (e.g., RIG

- 1, Mda

- 5, PKR) ( Robbins et al.
, 2009 ; Forsbach et al.
, 2011 ; Meng et al.
, 2013 ) . Immune stimulation appears to be seque nce dependent, and the length of the siRNA does not apparently affect immunostimulation ( Forsbach et al.
, 2011 ) . In mice the immuno stimulation of RNAi led to reduced lymphocytes and platelet cells, largely correlated with cytokine response to the siRNA ( Judg e et al.
,56

2005 ) . Although there are some similarities in the innate immune response of insects and mammals ( Saleh et al., 2009 ; Lundgre n and Jurat

- Fuentes , 2012 ) , it is unclear how the immune systems of other organisms will react to an influx of small RNAs. Nor is it known how this immuno stimulation will affect the fitness of non

- target organisms. Slight changes in nucleotide sequence, particularly at the 2’ position of the guid e strand, can reduce the immuno stimulatory effects in a given organism ( Jackson and Linsley
, 2010 ; Meng et al.
, 2013 )

. The Panel concluded that there is no clear evidence that small RNAs could trigger the immune systems of non

- mammalian non

- targ et species or what dose is necessary to trigger a response. See also the Pan el’s discussion in response to q uestion 2.
4) Concern for insect resistance
While the general consensus in the scientific community is that resistance to PIPs is unlikely, this has not been fully investigated. The idea of resistance (the selection of a genetically different population of insects from the target population that ha ve a survival advantage when exposed to the biopesticide) is unlikely because it is assumed that the PIP would be designed to a highly conserved region of a gene that has low/no sequence polymorphisms. However, this information may not be available for dif ferent populations of the target pest, and failures could occur if SNPs were found in the gene for the critical target region of the dsR NA/siRNA (Scott et al., 2013). Therefore, caution is warranted in evaluating the potential for resistance in multiple fi eld populations prior to release of the event, and for continued monitoring once the event is released for commercialization.

6

. b. To the extent that additional information would reduce uncertainty in addressing these issues, please describe specifically t he nature of additional information that EPA may need and the degree to which this information would reduce uncertainty in the ecological risk assessment.

Panel Response

As discussed in the response to Question 6.a., there is uncertainty in how to define the spectrum of insecticidal activity. The spectrum of activity depends on the target gene, dsRNA chosen, where and how much the dsRNA PIP is expressed in the plant, and t he persistence and stability of the effect. What is a realistic level of non

- target exposure based on the stabilit y and persistence of the dsRNA? The Panel noted that t he mode of action of RNAi (by siRNAs) is, by definition, very specific (see discussion in response to q uestion 1)

. However, there is some concern that unintended effects could occur across multiple insect orders. There could be unique effects for each non

- target organism. The que stion is how do you decide what non

- target should be tested? Fo r example, insect specie s representing 10 families and four insect orders were tested for DvSnf7 dsRNA activity (Bachman et al.
, 2013). Uncertainty in the spectrum of activity make s it more challenging to understand the environmental exposure levels and potential uptake pathways
, important information that can narrow the scope of non

- target organism testing for an ecological risk assessment.

The Panel suggested an exposure

- based conceptual model to reduce the uncertainty in the ecological risk assessmen t of dsRNA PIPs and dsRNA non

- PIP end

- products

. This approach would57

identify non

- target organisms that might be exposed and narrow the spectrum of non

- target organisms that would need to be tested for effects. The conceptual model is explained in the Panel ’s response to q uestion 7.

One panel member suggested a screening process to identify a diversity of responses at a “network level.” C ell lines from different tissues could be used to screen for differential gene expression. Test endpoints could be better defined.
Q uestion 7. Framework of Testing for Nontarget Effects

In sections IV.A.2.a. and IV.B.4. of the issue paper, EPA presents the current framework of testing for determining nontarget effects resulting from exposure to PIPs and biochemicals, respectively. In section IV.A.2.c

. , EPA also raised potential issues related to nontarget testing with dsRNAs that may arise given their unique mode of action, which included 1) the potential influence of latent effects on result s of nontarget testing, 2) the appropriate life stage for testing, and 3) the possibility for chronic effects.

7. a. Please comment on how each of EPA’s current PIP and biochemical frameworks for nontarget effects testing will inform risk assessment for dsRNA PIPs and exogenously applied dsRNAs. In providing a response, please address the potential for unintended effects as described in Question 7, as well as the three issues outlined as concerns for nontarget testing listed above.

Panel Response

The P an el appreciates all the time and effort spent by both the Agency and the public commenters in preparation and presentation of t he materials for this meeting. Use of RNAi technology in pesticidal products poses some unique challenges to environment fate and ecological risk assessment.
The Panel agreed with concerns the Agency expressed regarding inadequacies of the current environmental fate and non

- target effects testing frameworks for ecological risk assessment dsRNA PIPs and exogenously applied dsRNA produ cts. As stated in Secti on IV of the EPA issue paper ( p. 23, EPA
, 2013):

 Environmental fate and effects of dsRNA are poorly understood and present unique

challenges for ecological risk assessment that have not yet been encountered in assessments for t raditional chemical pesticides.

 Unlike PIPs, however, the Agency has not, to date, assessed the hazards or risks of dsRNA applied directly to the environment as components of end

- use products intended for pest

con trol under Section 3 of FIFRA.

 The scree ning level assessments currently used for traditional chemical pesticides may not be applicable due to the unique modes of acti on of dsRNA active ingredients.
Current requirements for soil degradation studies will provide some insight for non

- target testing, b ut additional data are needed to address the environmental fate of dsRNA PIPs and potential exposures58

to non

- target organisms, i.e., plant degradation (above and below ground), off

- site movement of plant materials, in planta exposure for organism s that directly ingest plant materials. Additional data are also needed to address the environmental fate of exogenously applied dsRNA products. Non

- target species are more likely to be exposed to exogenously applied dsRNA end

- products than to dsRNA PIPs

. Future end

- products might fit into several different pest control categories. Non

- PIP dsRNA exposures are likely multi

- route while dsRNA PIP exposure is more likely via diet

. Exposure may be through airborne particles, diet , or dermal/cuticle penetration depending on the end

- product. However, the dsRNA in a n applied product (e.g., spray) may be more exposed to environmental factors, e.g., UV light. As such, more species may be exposed to exogenously applied dsRNA products, although this concern may be bala nced by the likely increased instability of the sprayed product (duration of the RNAi). The Panel discussed these issues in detail in response to question 4.

The Panel indicated that the classic approach of developing and assembling effects data will not

w ork well for this technology d ue to the mode s of action of RNAi. N o one set of test species will serve

as an adequate representation of non

- target species at risk for every dsRNA PIP or exogenously applied dsRNA products

. The Panel discussed the following limitations in the current ecological risk assessment framework

.

 dsRNA PIP and dsRNA non

- PIP characterization including spectrum of activity. There is limited environmental fate information for dsRNA PIPs or dsRNA non

- PIPs to determine exposure and what non

- target organisms might need to be tested. The specificity of control is dependent on the target gene selected (gene specificity), dsRNA sele cted and resulting siRNAs (sequence specificity), m echanisms of action
, plant tissue expressed (dsRNA PIPs) , dose needed to trigger RNAi , amplificat ion, and duration of the effect

. This is why the approach to defining the spectrum of activity for RNAi PIPs or RNAi end

- products is critical for the assessment of

non

- target effects.

 Dose considerations in bioassays. Maximum hazard dose assays alone may not be sufficient for assessing off

- target effects associated with the use of RNAi technology. There is lim ited information on the dose and duration of exposure needed to trigger a nontarget effect. Given these limitations, the Panel observed that it will be difficult to determine the range of doses to test and the duration of the bioassays. The Panel recommend ed that bioassays for dsRNA PIPs include doses that mimic the in planta expression levels of the PIP and where possible, use plant tissue.

 Consideration of c hronic and sublethal effects. Mortality is one of several endpoints. Maximum hazard dose and measuring mortality offers limited information needed for risk assessment dsRNA PIPs and non

- PIP dsRNAs. The current Tier 1 testing scheme does not provide insight into potential chronic effect s and limited insight to the potential for sublethal effects.

 Prediction of non

- target effects and exposure. In the Panel’s response to Question 6.a., the following potential unintended effects were identified: silencing the target gene in non

- target59

spe cies, silencing off

- target genes in non

- target organisms, saturating the RNAi machinery in

non

- target organisms, and stimulating immune responses in non

- target organisms. The proposed testing scheme should include a process assessing the potential and seve rity of unintended effects if a non

- target organism was exposed to the dsRNA PIP. Thus, an early step in the non

- target testing scheme is determining what organisms are exposed to the dsRNA.

T he Panel remarked that the most difficult effect to predict wi ll be off

- target binding of pesticidal RNAs to mRNAs that share a minimal level of sequence homology with the target gene. A complicating factor is whether potential effects of RNAi can only be observed if an off

target gene is expressed during the time an d conditions of the observations

. For example, different genes are up

- regulated during different life stages of a non

- target organism (this would also be true for the target organism), and under different environmental conditions. Therefore, the Panel indicated that strategic research will be important to determine under what conditions and what life stage should be assayed for RNAi activity.

 Genomics and sequence specificity. The Panel encouraged sequencing full genomes to expand existing databases of known genomes, but also recognized that associated costs may limit this application. The Panel encouraged sequencing full genomes to expand existing databases of known genomes. In silico searches could be performed using existing (and potentially expanded) databases of genomes to curtail th e list of potential non

- target organisms to be tested. The Panel also re

- emphasized that bioinformatics analysis is a useful, but not an absolute, predictive tool (see related discussion in response to question 1) . Sequence identity does not mean that the dsRNA would provoke an RNAi response, but should caution the potential for unintended interactions and further review. Results from in silico searches could be used to help identify potential non

- target organisms that might be affected by off

- target bindin g.

 Potential synergism. The Panel observed that ther e might be synergism between dsRNA PIP

s and other PIPs expressed in a crop plant. The Panel recommended that EPA consider possible synergistic effects (of co

- exposures) with respect to target and non

- target organisms.

 Potential for evolution of resistance. The Panel stressed the importance of assessing the potential for the evolution of pest resistance to RNAi, as well as cross

- resistance (or multiple resistance) to different modes of action (e.g ., RNAi and Bt PIPs ).

 Potential scenarios that could minimize the concern for non

- target exposure and effects. One panelist suggested three potential scenarios that could minimize the concern for non

- target exposure and effects :  When the dsRNA PIP is very specific without homology to any of the sequence databases ;  When the dsRNA PIP is not modified and therefore likely to degrade rapidly in the environment ; and  When the dsRNA is expressed at extremely low levels and is tissue specific (i.e., in the roots f or corn rootworm).60

In conclusion, the uncertainties in our understanding of the potential modes of action in non

- target species, potential for chronic and sublethal effects, and potential unintended consequences in the various life stages of non

- target or ganisms are sufficient justification to question the applicability of the current Agency framework for assessing the environmental fate and ecological effects of dsRNA PIPs or exogenously applied non

- PIP end

- use products. As a consequence, u se of RNAi tech nology as dsRNA PIPs or exogenously applied dsRNA products poses some new (not insurmountable) challenges for risk
assessment.

7 .b

. What additional nontarget effects testing, if any, should EPA consider to gain a full understanding of the potential for dsR NAs to cause effects to nontarget organisms?

7.c. What other approaches, such as bioinformatics analysis, may be used to address concerns for effects on nontarget species and reduce the set of data requirements?

7.d. In providing answers to the above subq uestions, please be specific in discussing the extent to which additional information would reduce the nature and magnitude of these specific areas of uncertainty.

Panel Response

The Panel provided a combined response to question s 7b

- d.
1) Data needs
Based on the responses to questions 4

- 6, the Panel concluded that additional data are needed to reduce uncertainty in the environmental fate and ecological risk assessments ; however, the task of prescribing additional tests or additional test species canno t be done without a better understanding of the RNAi mode s of action and what non

- target organisms are likely to be exposed to dsRNA PIPs or exogenously applied dsRNA products

. The inability to easily predict what non

- target organisms might be exposed to the specific dsRNAs using the current framework challenges the evaluation of potential

non

- target effects.
The Panel provided the following general recommendations for data needed in the ecological risk
assessment:

 Bioassays should include doses that mimic the in planta expression levels of the PIP and where possible, use plant tissue.

 Mortality is one of many possible endpoints. The potential for chronic effects and for s ublethal effects should be considered.

 The Panel recommended evaluating entire life cycles, while simultaneously examining multiple life history parameters (intrinsic population growth rate is a robust metric) rather than simply relying on toxicity testi ng. These tests should be conducted under realistic conditions (as much as possible) to ensure that genes in operation in normal developmental conditions are61

not suffering from off

- target binding. Therefore, the Panel indicated that strategic research will be important to determine under what conditions and what life stage should be assayed for RNAi activity.

 In light of the discussion on potential immune system interactions in the EPA issue paper (EPA 2013) and during the meeting, the Panel recommended i nclusion of an immunological endpoint in the non

- target organism bioassays.

 The Panel also recommended an endpoint to measure the potential for saturation of the RNAi machinery in a non

- target organism . Saturation of RNAi machinery could reduce the effic iency at which a cell regulates endogenous gene expression.

 The Panel recommended that EPA consider possible synergistic effects (of ds RNA PIP co

exposures to other PIPs ) with respect to target and non

- target organisms.

 The Panel also recommended that EPA consider the evolution of pest resistance.

2) Proposed exposure

- based conceptual model for assessment of ecological risk posed by dsRNA PIPs or exogenously applied dsRNA end

- products (figure 7

- 1)

To address the limitations in the EPA’s proposed ecological risk assessment framework, the Panel designed an exposure

- based conceptual model for use in ecological risk assessment of RNAi PIPs and

non

- RNAi PIPs (see figure 7

- 1 ). The Panel proposed a network approach that incorporates mode of action, expo sure, species sensitivity, various endpoints, and adverse outcome pathway components (see Ankley et al., 2010) at the organism and population levels

. Only non

- target organisms that might be realistically exposed to RNAi PIPs or non

- RNAi PIPs will be tested . This result will eliminate the need to test a battery of standard species (current tier 1) many of which will either not be exposed or be insensitive. Integration of the exposure

- based conceptual model to non

- target organism testing will further the unde rstanding of the likelihood of adverse outcomes in non

- target species, facilitate the selection of endpoints, and identify potential data gaps that would require further study

. The Panel provided a detailed discussion of the framework for the proposed conc eptual model.
Box 1: Agent

1. Characterize the dsRNA (“Agent”)

a. What is the dsRNA sequence? b. What is the gene targeted and what portion ?

c. What is the expected phenotypic effect? How was it measured? How long does it last? d. What are the target organisms?

e. What is the dose to trigger an effect in the target organism? f. For PIPs, what is the expression level

– systemic or tissue

- specific? g. For sprays, what is the concentration?62

2. What is the dsRNA degradation kinetics i n decaying plant tissue, soil ?

Box 2

- Identify non - target species

1. Agent studies define which species are likely exposed. Use this information to define the community that overlaps in space and time with the siRNAs (e.g., those organisms that are most likely to be exposed).

2. Based on the route of expos ure (e.g., topical, ingestion), curtail the number of species of interest . For example, establish trophic linkages to the dsRNA PIP plant and determine the species likely to ingest plant

.

3. Of this curtailed community define a set of species that will be te sted in vitro based on relative abundance, importance in food web structure (e.g., trophic interactions), phylogenetic plac ement, and functional significance.

4. Perform in silico searches on the narrowed list of non

- target species to help focus specific in vitro studies . Is there sequence complementarity with gene sequences in other organisms? As noted previously, i n silico searches are not a predictive of effects. The Panel discussed different options to focus the in silico searches.

a. Perform in silico s earches using a database of known genomes. b. If full genomes do not exist, s equence full genomes of this curtailed non

- target list and perform in silico searches for gene sequences with high complementarity to the dsRNA.

c. Perform RNA

- seq of organisms fed on modified and unmodified plants so that only organisms with significantly changed gene expression would need to be considered further in downstream analysis

. d. Use primers specific for dsRNA to attempt to amplify sequences from non

- target species; no amplification would be evidence of lack of sequence conservation in non

- target.

Box 3: Model feeding studies

1. Conduct in vitro feeding bio assays on non

- target organisms based on the in silico searches. Measure activity at vario us endpoints, e.g., mortality
, immune response. Measure protein production. Examine different life stages and tissues rather than simply standard toxicity testing. What is the dose to achieve an effect? Can the dose be saturated? What is the consequence? C onduct these tests under realistic exposure conditions. Results will help elucidate potential mechanisms of action and inform selection of sublethal endpoints.

2. For those species that have reduced protein production of off

- target genes, determine whether predicted phenotypic effects of the off

- target gene suppression are measureable. When off target gene function is unknown, or multiple off target sites are identified, a comprehensive laboratory examination that examin es lethal ity and a battery of subletha l effects should be63

undertaken . These should form a composite fitness assessment, rather than each component being analyzed individually.

3. Determine possible synergistic effects (of ds RNA PIP co-exposures) with respect to target and non -target organisms.

4. Determine the potential for insect resistance and recommend resistance management strategies.

Box 4: Cellular and molecular studies

1. Is mortality, toxicity, sublethal effect the result of differential gene regulation? 2. Which gene(s) and what dose? 3. How persistent is the effect? 4. Is the response due to RNAi? 5. Is the response related to immunostimulation? 6. If RNAi is confirmed, what is the mechanism? 7. How is RNA taken up by cells, e.g., transporters? Other?

Box 5: Population effects

1. If necessary, conduct field tests over multiple years to determine how RNAi-based pesticides may affect biological networks and food web functioning under realistic conditions. 2. Field reports of pest resistance 3. Are there disturbances in the food web?

Box 6: Mitigation

1. Could non-target uptake be inhibited? 2. Could dsRNA be re-designed to minimize off-target effects? Resistance?Figure 7.1. Proposed exposure-based conceptual model for assessment of ecological risk posed by dsRNA PIPs or exogenously

applied dsRNA end-products

64REFERENCES

Admyre, C., Johansson, S.M., Qazi, K.R., Filen, J.-J., Lahesmaa, R., Norman, M., Neve, E.P.A., Scheynius, A., Gabrielsson, S. (2007) Exosomes with immune modulatory features are present in human breast milk. J Immunol 179:1969-1978. Agrawal, N., Dasaradhi, P.V.N., Mohmmed, A., Malhotra, P., Bhatnagar, R.K., Mukherjee, S.K. (2003) RNA interference: Biology, mechanism, and applications. Microbiology and Molecular Biolog y Reviews 67:657-685. Alemán, L.M., Doench, J., Sharp, P.A. (2007) Comparison of siRNA-induced off-target RNA and protein effects. RNA 13:385-395. Allen, M.L., Walker, W.B. (2012) Saliva of Lygus lineolaris digests double stranded ribonucleic acids. J Insect Physiol 58:391-396.
Altschul, S.F ., Gish, W., Miller, W., Myers, E.W., Lipman, D.J. (1990) Basic local alignment search tool. J Mol Biol 215:403-410. An, Q., van Bel, A.J., Huckelhoven, R. (2007) Do plant cells secrete exosomes derived from multivesicular bodies? Plant Signal Behav 2:4-7. Ankley, G.T., Bennett, R.S., Erickson, R.J., Hoff, D.J., Hornung, M.W., Johnson, R.D., Mount, D.R., Nichols, J.W., Russom, C.L., Schmieder, P.K., Serrrano, J.A., Tietge, J.E., Villeneuve, D.L. (2010) Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. Environ Toxicol Chem 29:730-741. Aouadi, M., Tesz, G.J., Nicoloro, S.M., Wang, M., Chouinard, M., Soto, E., Ostroff, G.R., Czech, M.P. (2009) Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature 458:1180-1184. Aronstein, K., Oppert, B., Lorenzen, M., RNAi in agriculturally-important arthropods. In RNA Processing , Grabowski, P., Ed. InTech: 2011. Axtell, M.J., Jan, C., Rajagopalan, R., Bartel, D.P. (2006) A two-hit trigger for siRNA biogenesis in plants. Cell 127:565-577. Bachman, P.M., Bolognesi, R., Moar, W.J., Mueller, G.M., Paradise, M.S., Ramaseshadri, P., Tan, J., Uffman, J.P., Warren, J
., Wigg ins, B .E., Levine, S.L. (2013) Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm ( Diabrotica virgifera virgifera LeConte). Transgenic Res 22:1207-1222. Ballarin-Gonzalez, B., Dagnaes-Hansen, F., Fenton, R.A., Gao, S., Hein, S., Dong, M., Kjems, J., Howard, K.A. (2013) Protection and s ystemic t ranslocation of siRNA f ollowing o ral a dministration of

c hitosan/siRNA n anoparticles. Mol Ther Nucleic Acids 2:e76.

B artel, D.P. (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215-233. Baulcombe, D. (2004) RNA silencing in plants. Nature 431:356-363.

6566

Baum, J.A., Bogaert, T., Clinton, W., Heck, G.R., Feldmann, P., Ilagan, O., Johnson, S.D., Plaetinck, G., Munyikwa, T., Pleau, M., Vaughn, T., Roberts, J. (2007) Control of coleopteran insect pests through RNA interference. Nature Biotechnology 25:1322-1326. Birmingham, A., Anderson, E.M., Reynolds, A., Ilsley-Tyree, D., Leake, D., Fedorov, Y., Baskerville, S., Maksimova, E., Robinson, K., Karpilow, J., Marshall, W.S., Khvorova, A. (2006) 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nature Methods 3:199-204. Brosnan, C.A., Voinnet, O. (2011) Cell- to -cell and long-distance siRNA movement in plants: mechanisms and biological implications. Curr Opin Plant Biol 14:580-587. Brown, B.D., Gentner, B., Cantore, A., Colleoni, S., Amendola, M., Zingale, A., Baccarini, A., Lazzari, G., Galli, C., Naldini, L. (2007) Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nat Biotechnol 25:1457-1467. Buratti, E., Muro, A.F., Giombi, M., Gherbassi, D., Iaconcig, A., Baralle, F.E. (2004) RNA folding affects the recruitment of SR proteins by mouse and human polypurinic enhancer elements in the fibronectin EDA exon. Mol Cell Biol 24:1387-1400. Caffrey, D.R., Zhao, J.Z., Song, Z., Schaffer, M.E., Haney, S.A., Subramanian, R.R., Seymour, A.B., Hughes, J.D. (2011) siRNA off-target effects can be reduced at concentrations that match their individual potency. PLoS One 6:e21503. Carver, J.D., Walker, W.A. (1995) The role of nucleotides in human nutrition. J Nutr Biochem 6:58-72. Christiaens, O., Swevers, L., Smagghe, G. (2014) DsRNA degradation in the pea aphid ( Acyrthosiphon pisum ) associated with lack of response in RNAi feeding and injection assay. Peptides. Cooper, T.A., Wan, L., Dreyfuss, G. (2009) RNA and disease. Cell 136:777-793. Cruz, J.A., Westhof, E. (2009) The dynamic landscapes of RNA architecture. Ce ll 136:604-609. Davidson, B.L., McCray, P.B.J. (2011) Current prospects for RNA interference-based therapies. Nature Reviews: Genetics 12:329-340. Dickinson, B., Zhang, Y., Petrick, J.S., Heck, G., Ivashuta, S., Marshall, W.S. (2013) Lack of detectable oral bioavailability of plant microRNAs after feeding in mice. Nat Biotechnol 31:965-967. Dillin, A. (2003) The specifics of small interfering RNA specificity. Proceedings of the National Academy of Sciences of the U.S.A. 100:6289-6291. Dixit, E., Kagan, J.C. (2013) Intracellular pathogen detection by RIG-I-like receptors. Adv Immunol 117:99-9125. Dua, P., Yoo, J.W., Kim, S., Lee, D.-k. (2011) Modified siRNA structure with a single nucleotide bulge overcomes conventional siRNA-mediated off-target silencing. Molecular Therapy 19:1676-1687. Duan, G., Saint, R.B., Helliwell, C.A., Behm, C.A., Wang, M.-B., Waterhouse, P.M., Gordon, K.H.J. (2013) C. elegans RNA-dependent RNA polymerases rrf-1 and ego-1 silence Drosophila transgenes by differing mechanisms. Cell Mol Life Sci 70:1469-1481. Dubelman, S., Fischer, J., Zapata, F., Huizinga, K., Jiang, C., Uffman, J., Levine, S., Carson, D. (2014) Environmental fate of double-stranded RNA in agricultural soils. PloS One 9:e93155.67

Economopoulou, M.A., Fragoulis, E.G., Sideris, D.C. (2007) Molecular cloning and characterization of the human RNase kappa, an ortholog of Cc RNase. Nucleic Acids Res 35:6389-6398. Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., Tuschl, T. (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO Journal 20:6877-6888. Federov, Y., Anderson, E.M., Birmingham, A., Reynolds, A., Karpilow, J., Robinson, K., Leake, D., Marshall, W.S., Khvorova, A. (2006) Off-target effects by siRNA can induce toxic phenotype. RNA 12:1188-1196. Feinberg, E.H., Hunter, C.P. (2003) Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301:1545-1547. Felix, M.-A. (2008) RNA interference in nematodes and the chance that favored Sydney Brenner. J Biol 7:34-34. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

. Nature 391:806-811.
Forsbach, A., Müller, C., Montino, C., Kritzler, A., Curdt, R., Benahmed, A., Jurk, M., Vollmer, J. (2011) Impact of delivery systems on siRNA immune activation and RNA interference. Immunology Letters 141:169-180. Fort, L., Taper, H.S., Brucher, J.M. (1969) Nucleases activity in different segments of the human digestive tube compared to the incidence of carcinomas (histochemical study). Histochemie 20:150-158. Ghidini, A., Ander, C., Winqvist, A., Stromberg, R. (2013) An RNA modification with remarkable resistance to RNase A. Chem Commun (Camb) 4 9:9036-9038. Goo, S.M., Cho, S. (2013) The expansion and functional diversification of the mammalian ribonuclease a superfamily epitomizes the efficiency of multigene families at generating biological novelty. Genome Biol Evol 5:2124-2140. Goodarzi, H., Najafabadi, H.S., Oikonomou, P., Greco, T.M., Fish, L., Salavati, R., Cristea, I.M., Tavazoie, S. (2012) Systematic discovery of structural elements governing stability of mammalian messenger RNAs. Nature 485:264-268. Gordon, K.H.J., Waterhouse, P.M. (2007) RNAi for insect-proof plants. Nature Biotechnology 25:1231- 1232. Grimm, D. (2011) The dose can make the poison: lessons learned from adverse in vivo toxicities caused by RNAi overexpression. Silence 2:8. Grimm, D., Streetz, K.L., Jopling, C.L., Storm, T.A., Pandey, K., Davis, C.R., Marion, P., Salazar, F., Kay, M.A. (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441:537-541. Grimm, D., Wang, L., Lee, J.S., Schürmann, N., Gu, S., Börner, K., Storm, T., Kay, M.A. (2010) Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver. Journal of Clinical Investigation 120:3106-3119.68

Ha, I., Wightman, B., Ruvkun, G. (1996) A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation. Genes Dev 10:3041-3050. Hakim, R.S., Baldwin, K., Smagghe, G. (2010) Regulation of midgut growth, development, and metamorphosis. Annu Rev Entomol 55:593-608. Hannon, G.J. (2002) RNA interference. Nature 418:244-251. Harteveld, J., Lindenbergh, A., Sijen, T. (2013) RNA cell typing and DNA profiling of mixed samples: Can cell types and donors be associated? Science and Justince 53:261-269.
Hata, T., Murakami, K., Nakatani, H., Yamamoto, Y., Matsuda, T., Aoki, N. (2010) Isolation of bovine milk -derived microvesicles carrying mRNAs and microRNAs. Biochem Biophys Res Commun 396:528-533. Hattori, M., Taylor, T.D. (2009) The human intestinal microbiome: a new frontier of human biology. DNA Res 16:1-12. Helwak, A., Kudla, G., Dudnakova, T., Tollervey, D. (2013) Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153:654-665. Hindorff, L.A., Sethupathy, P., Junkins, H.A., Ramos, E.M., Mehta, J.P., Collins, F.S., Manolio, T.A. (2009) Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A 106:9362-9367. Hoerter, J.A., Krishnan, V., Lionberger, T.A., Walter, N.G. (2011) siRNA-like double-stranded RNAs are specifically protected against degradation in human cell extract. PLoS One 6:e20359. Hunter, C.P., Winston, W.M., Molodowitch, C., Feinberg, E.H., Shih, J., Sutherlin, M., Wright, A.J., Fitzgerald, M.C. (2006) Systemic RNAi in Caenorhabditis elegans. Cold Spring Harb Symp Quant Biol 71:95-9100. Huvenne, H., Smagghe, G. (2010) Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. Journal of Insect Physiology 56:227-235.
Ivashuta, S.I., Petrick, J.S., Heisel, S.E., Zhang, Y., Guo, L., Reynolds, T.L., Rice, J.F., Allen, E., Roberts, J.K. (2009) Endogenous small RNAs in grain: semi-quantification and sequence homology to human and animal genes. Food Chem Toxicol 47:353 -360. Izumi, H., Kosaka, N., Shimizu, T., Sekine, K., Ochiya, T., Takase, M. (2012) Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions. J Dairy Sci 95:4831-4841. Jackson, A.L., Bartz, S.R., Schelter, J., Kobayashi, S.V., Burchard, J., Mao, M.K., Li, B., Cavet, G., Linsley, P.S. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology 21:635-638. Jackson, A.L., Burchard, J., Schelter, J., Chau, B.N., Cleary, M., Lim, L., Linsley, P.S. (2006) Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity. RNA 12:1179-1187. Jackson, A.L., Linsley, P.S. (2010) Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov 9 :57-67.69

Jarosch, A., Moritz, R.F.A. (2012) RNA interference in honeybees: off-target effects caused by dsRNA. Apidologie 43:128-138. Jones-Rhoades, M.W., Bartel, D.P. (2004) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14:787-799.
Jose, A.M., Garcia, G.A., Hunter, C.P. (2011) Two classes of silencing RNAs move between Caenorhabditis elegans tissues. Nat Struct Mol Biol 18:1184-1188.Judge, A.D., Sood, V., Shaw, J.R., Fang, D., McClintock, K., MacLachlan, I. (2005) Sequencedependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 23:457-462.Judge, A.D., Sood, V., Shaw, J.R., Fang, D., McClintock, K., MacLachlan, I. (2005) Sequencedependent stimulation of the mammalian innate immune response by synthetic siRNA. Nature Biotechnology 23:457-462. Kahn, A.A., Betel, D., Miller, M.L., Sander, C., Leslie, C.S., Marks, D.S. (2009) Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs. Nature Biotechnology 27:549-555. Kawai, T., Akira, S. (2008) Toll-like receptor and RIG-I-like receptor signaling. Ann N Y Acad Sci 1143:1-20. Kawashima, T., Kosaka, A., Yan, H., Guo, Z., Uchiyama, R., Fukui, R., Kaneko, D., Kumagai, Y., You, D. -J., Carreras, J., Uematsu, S., Jang, M.H., Takeuchi, O., Kaisho, T., Akira, S., Miyake, K., Tsutsui, H., Saito, T., Nishimura, I., Tsuji, N.M. (2013) Double-stranded RNA of intestinal commensal but not pathogenic bacteria triggers production of protective interferon- β. Immunity 38:1187-1197. Khalil, A.M., Rinn, J.L. (2011) RNA-protein interactions in human health and disease. Semin Cell Dev Biol 22:359-365. Kozak, M. (1980) Influence of mRNA secondary structure on binding and migration of 40S ribosomal subunits. Cell 19:79-90. Kozak, M. (1986) Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A 83:2850-2854. Kriegel, C., Attarwala, H., Amiji, M. (2013) Multi-compartmental oral delivery systems for nucleic acid therapy in the gastrointestinal tract. Adv Drug Deliv Rev 65:891-901. Kulkarni, M.M., Booker, M., Silver, S.J., Friedman, A., Hong, P., Perrimon, N., Mathey-Prevot, B. (2006) Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cellbased assays. Nature Methods 3:833-838. Kusaba, M. (2004) RNA interference in crop plants. Current Opinions in Biotechnology 15:139-143. Lal, A., Navarro, F., Maher, C.A., Maliszewski, L.E., Yan, N., O'Day, E., Chowdhury, D., Dykxhoorn, D.M., Tsai, P., Hofmann, O., Becker, K.G., Gorospe, M., Hide, W., Lieberman, J. (2009) miR-24 Inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to "seedless" 3'UTR microRNA recognition elements. Mol Cell 35:610-625.70

Lasser, C., Alikhani, V.S., Ekstrom, K., Eldh, M., Paredes, P.T., Bossios, A., Sjostrand, M., Gabrielsson, S., Lotvall, J., Valadi, H. (2011) Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med 9:9-9. Levy-Booth, D. J. , Campbell R. G. , Gulden R. H. , Hart M

. M. , Powell J. R. , et al. (2007) Cycling of extracellular DNA in the soil environment. Soil Biology and Biochemistry 39 2977

– 2991. Lew-Tabor, A.E., Kurscheid, S., Barrero, R., Gondro, C., Moolhuijzen, P.M., Valle, M.R., Morgan, J.A.T., Covacin, C., Bellgard, M.I. (2011) Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus. International Journal for Parasitology 41:1001-1014. Li, F., Zheng, Q., Ryvkin, P., Dragomir, I., Desai, Y., Aiyer, S., Valladares, O., Yang, J., Bambina, S., Sabin, L.R., Murray, J.I., Lamitina, T., Raj, A., Cherry, S., Wang, L.S., Gregory, B.D. (2012a) Global analysis of RNA secondary structure in two metazoans. Cell Rep 1:69-82. Li, F., Zheng, Q., Vandivier, L.E., Willmann, M.R., Chen, Y., Gregory, B.D. (2012b) Regulatory impact of RNA secondary structure across the Arabidopsis transcriptome. Plant Cell 24:4346-4359. Li, J., Sun, Y., Jia, T., Zhang, R., Zhang, K., Wang, L. (2014) Messenger RNA vaccine based on recombinant MS2 virus-like particles against prostate cancer. Int J Cancer 134:1683-1694. Lilley, D.M. (2011) Mechanisms of RNA catalysis. Philos Trans R Soc Lond B Biol Sci 366:2910-2917. Lin, X., Ruan, X., Anderson, M.G., McDowell, J.A., Kroeger, P.E., Fesik, S.W., Shen, Y. (2005) siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Research 33:4527-4535. Lipardi, C., Wei, Q., Paterson, B.M. (2001) RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107:297-307. Liu, H., Wang, X., Wang, H.-D., Wu, J., Ren, J., Meng, L., Wu, Q., Dong, H., Wu, J., Kao, T.-Y., Ge, Q., Wu, Z.-x., Yuh, C.-H., Shan, G. (2012) Escherichia coli noncoding RNAs can affect gene expression and physiology of Caenorhabditis elegans. Nat Commun 3:1073-1073. Loo, Y.-M., Gale, M. (2011) Immune signaling by RIG-I-like receptors. Immunity 34:680-692. Lundgren, J.G., Jurat-Fuentes, J.L., Physiology and ecology of host defense against microbial invaders. In Insect Pathology 2 nd Edition , Vega, F.E.; Kaya, H.K., Eds. Elsevier: London, UK, 2012; pp 461-480. Ma, Y., Creanga, A., Lum, L., Beachy, P.A. (2006) Prevalence of off-target effects in Drosophila RNA interference screens. Nature 443:359-363. Marcilla, A., Trelis, M., Cortes, A., Sotillo, J., Cantalapiedra, F., Minguez, M.T., Valero, M.L., Sanchez del Pino, M.M., Munoz-Antoli, C., Toledo, R., Bernal, D. (2012) Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells. PLoS One 7. McEwan, D.L., Weisman, A.S., Hunter, C.P. (2012) Uptake of extracellular double-stranded RNA by SID-2. Mol Cell 47:746-754. Meng, Z., Zhang, X., Wu, J., Pei, R., Xu, Y., Yang, D., Roggendorf, M., Lu, M. (2013) RNAi induces innate immunity through multiple cellular signaling pathways. PLoS ONE 8:e64708.71

Mullokandov, G., Baccarini, A., Ruzo, A., Jayaprakash, A.D., Tung, N., Israelow, B., Evans, M.J., Sachidanandam, R., Brown, B.D. (2012) High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries. Nat Methods 9:840-846. Nuez, I., Felix, M.-A. (2012) Evolution of susceptibility to ingested double-stranded RNAs in Caenorhabditis nematodes. PLoS One 7. Pak, J., Fire, A. (2007) Distinct populations of primary and secondary effectors during RNAi in C. elegans . Science 315:241-244. Parrish, S., Fire, A. (2001) Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans

. RNA 7:1397-1402. Pasloske, B.L., Walkerpeach, C.R., Obermoeller, R.D., Winkler, M., DuBois, D.B. (1998) Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards. J Clin Microbiol 36:3590-3594. Pegtel, D.M., Cosmopoulos, K., Thorley-Lawson, D.A., van Eijndhoven, M.A.J., Hopmans, E.S., Lindenberg, J.L., de Gruijl, T.D., Wurdinger, T., Middeldorp, J.M. (2010) Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A 107:6328-6333. Petrick, J.S., Brower-Toland, B., Jackson, A.L., Kier, L.D. (2013) Safety assessment of food and feed from biotechnology-derived crops employing RNA-mediated gene regulation to achieve desired traits: A scientific review. Regul Toxicol Pharmacol. Price, D., Gatehouse, J. (2008) RNAi-mediated crop protection against insects. Trends Biotechnol 26:393-400. Qiu, S., Adema, C.M., Lane, T. (2005) A computational study of off-target effects of RNA interference. Nucleic Acids Research 33:1834 -1847. Raposo, G., Stoorvogel, W. (2013) Extracellular vesicles: Exosomes, microvesicles, and friends. J Cell Biol 200:373-383. Regente, M., Pinedo, M., Elizalde, M., de la Canal, L. (2012) Apoplastic exosome-like vesicles: a new way of protein secretion in plants? Plant Signal Behav 7:544-546. Rehman, A., Sina, C., Gavrilova, O., Hasler, R., Ott, S., Baines, J.F., Schreiber, S., Rosenstiel, P. (2011) Nod2 is essential for temporal development of intestinal microbial communities. Gut 60:1354-1362. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S., Khvorova, A. (2004) Rational siRNA design for RNA interference. Nature Biotechnology 22:326-330. Robbins, M., Judge, A., MacLachlan, I. (2009) siRNA and innate immunity. Oligonucleotides 19: 89

- 101.Ruby, J.G., Jan, C., Player, C., Axtell, M.J., Lee, W., Nusbaum, C., Ge, H., Bartel, D.P. (2006) Largescale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans . Cell 127:1193-1207. Sabariegos, R., Gimenez-Barcons, M., Tàpia, N., Clotet, B., Martinez, M.A. (2006) Sequence homology required by human immunodeficiency virus type 1 to escape from short interfering RNAs. J Virol 80:571-577.72

Saleh, M.-C., Tassetto, M., van Rij, R.P., Goic, B., Gausson, V., Berry, B., Jacquier, C., Antoniewski, C., Andino, R. (2009) Antiviral immunity in Drosophila requires systemic RNA interference spread. Nature 458:346-350. Saleh, M.-C., van Rij, R.P., Hekele, A., Gillis, A., Foley, E., O'Farrell, P.H., Andino, R. (2006) The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat Cell Biol 8:793-802. Sarkies, P., Miska, E.A. (2013) RNAi pathways in the recognition of foreign RNA: antiviral responses and host-parasite interactions in nematodes. Biochem Soc Trans 41:876 -880. Saxena, S., Jónsson, Z.O., Dutta, A. (2003) Small RNAs with imperfect match to endogenous mRNA repress translation. Journal of Biological Chemistry 278:44312-44319. Scallan, C.D., Tingley, D.W., Lindbloom, J.D., Toomey, J.S., Tucker, S.N. (2013) An adenovirus-based vaccine with a double-stranded RNA adjuvant protects mice and ferrets against H5N1 avian influenza in oral delivery models. Clin Vaccine Immunol 20:85-94. Scott, J.G., Michel, K., Bartholomay, L.C., Siegfried, B.D., Hunter, W.B., Smagghe, G., Zhu, K.Y., Douglas, A.E. (2013) Towards the elements of successful insect RNAi. J Insect Physiol 59:1212-1221. Sczepanski, J.T., Joyce, G.F. (2013) Binding of a structured D-RNA molecule by an L-RNA aptamer. J Am Chem Soc 135:13290-13293.
Seinen, E., Burgerhof, J.G.M., Jansen, R.C., Sibon, O.C.M. (2011) RNAi-induced off-target effects in Drosophila melanogaster : frequencies and solutions. Briefings in Functional Genomics 10:206-214. Sharp, P.A. (2009) The centrality of RNA. Cell 136:577-580. Shih, J.D., Hunter, C.P. (2011) SID-1 is a dsRNA-selective dsRNA-gated channel. RNA 17:1057-1065. Sijen, T., Fleenor, J., Simmer, F., Thijssen, K.L., Parrish, S., Timmons, L., Plasterk, R.H., Fire, A. (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465-476. Sijen, T., Steiner, F.A., Thijssen, K.L., Plasterk, R.H. (2007) Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science 315:244-247. Silverman, I.M., Li, F., Alexander, A., Goff, L., Trapnell, C., Rinn, J.L., Gregory, B.D. (2014) RNasemediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome. Genome Biol 15:R3. Silverman, I.M., Li, F., Gregory, B.D. (2013) Genomic era analyses of RNA secondary structure and RNA-binding proteins reveal their significance to post-transcriptional regulation in plants. Plant Sci 205-206:55-62. Singh, A.D., Wong, S., Ryan, C.P., Whyard, S. (2013) Oral delivery of double-stranded RNA in larvae of the yellow fever mosquito, Aedes aegypti : implications for pest mosquito control. J Insect Sci 13:69. Siomi, H., Siomi, M.C. (2009) On the road to reading the RNA-interference code. Nature 457:396-404. Skog, J., Wurdinger, T., van Rijn, S., Meijer, D.H., Gainche, L., Sena-Esteves, M., Curry, W.T., Carter, B.S., Krichevsky, A.M., Breakefield, X.O. (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10:1470-1476.73

Snow, J.W., Hale, A.E., Isaacs, S.K., Baggish, A.L., Chan, S.Y. (2013) Ineffective delivery of dietderived microRNAs to recipient animal organisms. RNA Biol 10:1107-1116. Sorrentino, S. (2010) The eight human "canonical" ribonucleases: molecular diversity, catalytic properties, and special biological actions of the enzyme proteins. FEBS Lett 584:2194-2200. Sorrentino, S., Naddeo, M., Russo, A., D'Alessio, G. (2003) Degradation of double-stranded RNA by human pancreatic ribonuclease: crucial role of noncatalytic basic amino acid residues. Biochemistry 42:10182-10190. Sverdlov, E.D. (2012) Amedeo Avogadro's cry: what is 1 microg of exosomes? Bioessays 34:873-875. Tabara, H., Grishok, A., Mello, C.C. (1998) RNAi in C. elegans : soaking in the genome sequence. Science 282:430-431. Terenius, O., Papanicolaou, A., Garbutt, J.S., Eleftherianos, I., Huvenne, H., Kanginakudru, S., Albrechtsen, M., An, C., Aymeric, J.L., Barthel, A., Bebas, P., Bitra, K., Bravo, A., Chevalier, F., Collinge, D.P., Crava, C.M., de Maagd, R.A., Duvic, B., Erlandson, M., Faye, I., Felfoldi, G., Fujiwara, H., Futahashi, R., Gandhe, A.S., Gatehouse, H.S., Gatehouse, L.N., Giebultowicz, J.M., Gomez, I., Grimmelikhuijzen, C.J., Groot, A.T., Hauser, F., Heckel, D.G., Hegedus, D.D., Hrycaj, S., Huang, L., Hull, J.J., Iatrou, K., Iga, M., Kanost, M.R., Kotwica, J., Li, C., Li, J., Liu, J., Lundmark, M., Matsumoto, S., Meyering-Vos, M., Millichap, P.J., Monteiro, A., Mrinal, N., Niimi, T., Nowara, D., Ohnishi, A., Oostra, V., Ozaki, K., Papakonstantinou, M., Popadic, A., Rajam, M.V., Saenko, S., Simpson, R.M., Soberon, M., Strand, M.R., Tomita, S., Toprak, U., Wang, P., Wee, C.W., Whyard, S., Zhang, W., Nagaraju, J., Ffrench-Constant, R.H., Herrero, S., Gordon, K., Swevers, L., Smagghe, G. (2011) RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design. J Insect Physiol 57:231-245. Thompson, J.D., Higgins, D.G., Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673-4680. Timmons, L., Fire, A. (1998) Specific interference by ingested dsRNA. Nature 395:854-854. Trappl, K., Polacek, N. (2011) The ribosome: a molecular machine powered by RNA. Met Ions Life Sci 9:253-275. Turchinovich, A., Weiz, L., Burwinkel, B. (2012) Extracellular miRNAs: the mystery of their origin and function. Trends Biochem Sci 37:460-465. Uchida, T., Arima, T., Egami, F. (1970) Specificity of RNase U2. J Biochem 67:91-102. Ulvila, J., Parikka, M., Kleino, A., Sormunen, R., Ezekowitz, R.A., Kocks, C., Ramet, M. (2006) Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells. J Biol Chem 281:14370-14375. United States Environmental Protection Agency (EPA). (2013) White Paper on RNAi technology as a pesticide : Problem formulation for human health and ecological risk assessment. October 29, 2013. Accessible in the Office of Pesticide Programs public regulatory e-docket, EPA

- HQ -OPP-2013 -0485: http://www.regulations.gov/#%21documentDetail;D=EPA- HQ -OPP-2013 -0485 -0011

.74

Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J.J., Lotvall, J.O. (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654-659. Voinnet, O. (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136:669-687. Wang, B., Zhuang, X., Deng, Z.-B., Jiang, H., Mu, J., Wang, Q., Xiang, X., Guo, H., Zhang, L., Dryden, G., Yan, J., Miller, D., Zhang, H.-G. (2014) Targeted drug delivery to intestinal macrophages by bioactive nanovesicles released from grapefruit. Mol Ther 22:522-534. Wang, K., Li, H., Yuan, Y., Etheridge, A., Zhou, Y., Huang, D., Wilmes, P., Galas, D. (2012) The complex exogenous RNA spectra in human plasma: an interface with human gut biota? PLoS One 7. Wang, K., Yuan, Y., Li, H., Cho, J.-H., Huang, D., Gray, L., Qin, S., Galas, D.J. (2013) The spectrum of circulating RNA: a window into systems toxicology. Toxicol Sci 132:478-492. Wang, Y., Zhang, H., Li, H., Miao, X. (2011) Second-generation sequencing supply an effective way to screen RNAi targets in large scale for potential application in pest insect control. PLoS One 6:e18644. Wei, L.Q., Yan, L.F., Wang, T. (2011) Deep sequencing on genome-wide scale reveals the unique composition and expression patterns of microRNAs in developing pollen of Oryza sativa . Genome Biol 12. Wheeler, M.M., Ament, S.A., Rodriguez-Zas, S.L., Robinson, G.E. (2013) Brain gene expression changes elicited by peripheral vitellogenin knockdown in the honeybee. Insect Molecular Biology 22:562-573. Whyard, S., Singh, A.D., Wong, S. (2009) Ingested double-stranded RNAs can act as species-specific insecticides. Insect Biochem Mol Biol 39:824-832. Wilson, R.C., Doudna, J.A. (2013) Molecular mechanisms of RNA interference. Annu Rev Biophys 42:217-239. Winston, W.M., Molodowitch, C., Hunter, C.P. (2002) Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science 295:2456-2459. Winston, W.M., Sutherlin, M., Wright, A.J., Feinberg, E.H., Hunter, C.P. (2007) Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proc Natl Acad Sci U S A 104:10565-10570. Witwer, K.W. (2012) XenomiRs and miRNA homeostasis in health and disease: Evidence that diet and dietary miRNAs directly and indirectly influence circulating miRNA profiles. RNA Biol 9. Witwer, K.W., Hirschi, K.D. (2014) Transfer and functional consequences of dietary microRNAs in vertebrates: Concepts in search of corroboration: Negative results challenge the hypothesis that dietary xenomiRs cross the gut and regulate genes in ingesting vertebrates, but important questions persist. Bioessays 36:394-406. Witwer, K.W., McAlexander, M.A., Queen, S.E., Adams, R.J. (2013) Real-time quantitative PCR and droplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs: limited evidence for general uptake of dietary plant xenomiRs. RNA Biol 10:1080- 1086.75

Zhang, H., Li, H.-C., Miao, X.-X. (2013) Feasibility, limitation and possible solutions of RNAi-based technology for insect pest control. Insect Sci 20:15-30. Zhang, L., Hou, D., Chen, X., Li, D., Zhu, L., Zhang, Y., Li, J., Bian, Z., Liang, X., Cai, X., Yin, Y., Wang, C., Zhang, T., Zhu, D., Zhang, D., Xu, J., Chen, Q., Ba, Y., Liu, J., Wang, Q., Chen, J., Wang, J., Wang, M., Zhang, Q., Zhang, J., Zen, K., Zhang, C.-Y. (2012) Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res 22:107- 126.Zhang, Y., Wiggins, B.E., Lawrence, C., Petrick, J., Ivashuta, S., Heck, G. (2012) Analysis of plantderived miRNAs in animal small RNA datasets. BMC Genomics 13:381-381. Zheng, Q., Ryvkin, P., Li, F., Dragomir, I., Valladares, O., Yang, J., Cao, K., Wang, L.S., Gregory, B.D.(2010) Genome-wide double-stranded RNA sequencing reveals the functional significance of basepaired RNAs in Arabidopsis. PLoS Genet 6:e1001141. Zhou, Q., Li, M., Wang, X., Li, Q., Wang, T., Zhu, Q., Zhou, X., Wang, X., Gao, X., Li, X. (2012) Immune-related microRNAs are abundant in breast milk exosomes. Int J Biol Sci 8:118-123. Zhou, R., Wei, H., Sun, R., Tian, Z. (2007) Recognition of double-stranded RNA by TLR3 induces severe small intestinal injury in mice. J Immunol 178:4548-4556. Zhou, X., Wheeler, M.M., Oi, F.M., Scharf, M.E. (2008) RNA interference in the termite Reticulitermes flavipes through ingestion of double-stranded RNA. Insect Biochem Mol Biol 38:805-815.