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, Virginia 
2

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

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 ......................................................................27

QUESTION 2. BASED ON DATA INDICATING DEGRADATION OF THE MAJORITY OF DSRNA IN THE

DIGESTIVE SYSTEM, PLEASE DISCUSS THE STRENGTHS AND LIMITATIONS IN CONCLUDING THERE

WILL NOT BE SIGNIFICANT ABSORPTION OF DSRNA WITH POSSIBLE MAMMALIAN EFFECTS ON

ORAL EXPOSURE ..............................................................................................................................29

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? ..................................................38

QUESTION 4. ENVIRONMENTAL FATE ...................................................................................................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

QUESTION 5. PRIMARY ROUTE OF EXPOSURE FOR NONTARGET ORGANISMS TO DSRNA .................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.

 ....................................................................................................................................................................... 50
3

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

QUESTION 6. UNINTENDED EFFECTS OF DSRNA ..................................................................................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

QUESTION 7. FRAMEWORK OF TESTING FOR NONTARGET EFFECTS...................................................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 ................................................................................................................................................... 65
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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: 

4/28/14
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, CA
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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, MD 
8

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 Deoxyribonucleases

Dicer Dicer is a ribonuclease RNase III-like enzyme 

that processes long double-stranded RNA 

(dsRNA) or pre-micro RNA hairpin precursors 

into small interfering RNAs (siRNAs) or micro

RNAs (miRNAs).

dsRNA Double-stranded RNA

GI tract Gastrointestinal tract

mRNA Messenger RNA

miRNA Micro RNA

Non-PIP Non plant-incorporated protectant

nt Nucleotide

PIP Plant-incorporated protectant is a term used by 

the EPA to describe the pesticidal active 

ingredient and the genetic material necessary for 

its production 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 interference
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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 polymorphism
10

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 extended 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 the Designated 

Federal Official. Steven Bradbury, Ph.D., Director, Office of Pesticide Programs (OPP), and Robert 

McNally, Director, Biopesticides and Pollution Prevention Division, 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 that injected double-stranded RNA (dsRNA) sequences corresponding to

known gene sequences in the nematode genome were 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 

organisms 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 RNAs (siRNAs) are the product of Dicer (a ribonuclease III-like 

enzyme) activity on longer molecules of dsRNA. The siRNA molecules produced by Dicer 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 RNAi

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 pest 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 framework 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 found at the start of section III. The framework 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 

assessment 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 risk 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 absence 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: extent of digestion of dsRNA and effect on 

absorption;

3. Effect of the structure of dsRNA on degradation in the gut and exposure to dsRNA.

Ecological Risk Considerations:

4. The environmental fate of the dsRNA;

5. Factors that may limit non-target organism exposure to dsRNA;

6. Potential unintended effects in non-target organisms exposed to dsRNA;

7. Framework of testing for non-target effects.
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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 2006 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 Sauvé, 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, Ph.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. Stephen 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 Environmental Risk Assessment, 

International Life Sciences Institute (ILSI) Research Foundation

8. Subba Palli, Ph.D., Department of Entomology, University of Kentucky 

9. Robert Sears, President, Eastern 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 Center
13

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 Association

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 & Regulatory 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 Association
14

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 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). The Panel 

provided the following overall summary of the major conclusions 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 through the oral 

ingestion of dsRNA PIPs in food plants or food plants treated with exogenously applied dsRNAs endproducts. Dietary RNA is extensively degraded in the mammalian digestive system by a combination of 

ribonucleases (RNases) and acids that are likely to ensure that all structural forms 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 micro RNAs (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, or 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 frameworks for dsRNA PIPs and exogenously 

applied dsRNA products. Uncertainties in the potential modes of action in non-target species, potential 
15

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

non-target 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 species 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 

technology. 

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 organisms. 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 the 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 might 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-target 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 dsRNA 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 directly ingest plant materials. 

 Determine environmentally-relevant dosages, the importance of physical barriers in nontarget organisms concerning degradation and cellular uptake of dsRNA, and off-target 

effects to non-target organisms.

 Evaluate the potential for 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.
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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 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. 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 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 suggested that dsRNA sequence-specific factors may 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. 

Question 2. Based on data indicating degradation of the majority of dsRNA in the digestive 

system, please discuss the strengths 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, mechanisms for RNA uptake from the environment, RNAi amplification by RNAdependent 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. This 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, 

 The history of challenges developing effective oral RNAi therapeutics which can withstand 

the hostile environment of the human gut,

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

resulting 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 lacking in this area and there are no published PIPspecific 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 further. 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 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 detection 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; 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 

should 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 dsRNA 

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 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. 
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 

on 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 effects of dsRNA and degraded 

products (if present) in the environment. The Panel emphasized the importance of understanding how 

degradation 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 are 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 parts that may remain in the field 

should be evaluated. 

 In addition, both 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 dsRNAs, dsRNA in plant tissue and in the 

rhizosphere, and exogenously 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 long 
19

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 decompose 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 and 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 

collection of environmental fate data representing different soil types, water bodies, or weather 

conditions under which crops expressing dsRNA PIPs may be cultivated. The Panel also recommended 

significant changes to the overall risk assessment paradigm as discussed 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? 

Summary

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 dsRNA at various taxonomic levels. In the absence of 

data, the 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). The Panel indicated that the life stage exposed to dsRNAs should 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 measuring 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. However, 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 contact. 

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 needed 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 

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). Factors that influence cellular uptake and activity include, but are not 

limited 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., birds, 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 combined with binding in the gut (where there is an actual target to be affected). The Panel 
21

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 nontarget taxa, as needed, based on exposure.

b. The degree of exposure by direct contact is likely to differ between nontarget risk 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 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 collection of additional information to evaluate the importance of 

this 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. 

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, the Panel recommended that each dsRNA PIP or exogenously 

applied dsRNA end-product should be evaluated 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 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. 

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 uncertainty 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 discussed 

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 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.

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. 

Summary

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 the persistence and stability of the effect. What 

is a realistic level of non-target exposure based on the stability and persistence of the dsRNA? While the 

Panel noted that the mode of action of RNAi (by siRNAs) is, by definition, very specific (see discussion 

in response to question 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 were 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 non-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 risk assessment. In addition, data are needed in several areas. The Panel recommended 

an exposure-based decision framework for ecological risk assessment of dsRNA PIPs and dsRNA nonPIP end-products (see response to question 7).
23

Question 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 with 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 described 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 characterizing environmental fate. Uncertainties in 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 organisms are sufficient justification 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 modes 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 effects 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 - determination 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 different life stages of a non-target organism and under different 

environmental 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) with 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 concerns 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 specific areas of 

uncertainty.

Summary

The Panel provided a combined response to questions 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 

exposure-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 exposed 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 understanding 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 Panel recommended that 
25

strategic research is need to determine under what conditions and what life stages should be 

assayed for RNAi activity. 

 The Panel recommended inclusion 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. 

 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

OVERVIEW OF THE RNA INTERFERENCE PATHWAY

RNA interference (RNAi) is nearly ubiquitous 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 molecules 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 figure I-1 for a simple illustration of this process). This process depends on the degree of 

sequence homology, protein partners, and cellular context. Specific binding may lead to translational 

suppression, mRNA degradation, or target sequestration (Wilson et al., 2013; Bartel 2009). The unifying 

feature of the RNAi mechanisms 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 an 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 nonguide strand and retains the guide strand. The Argonaute protein of the RISC complex cleaves a singlestranded messenger RNA (mRNA) with sequence complementary to the guide strand and leads 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 RNA; siRNA = small interfering RNA; Dicer = a family of RNA 

endonuclease III enzymes; mRNA = 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 

suppression
27

DETAILED RESPONSES TO CHARGE QUESTIONS

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.

Panel Response

The Panel agreed that bioinformatic analysis can be used to identify specific nucleotide identity in 

a long sequence 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. 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 useful, but not absolute, predictive tool. 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 suggested that dsRNA sequence-specific factors 

may 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-mammalian organisms is found in question 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 of dsRNA" or even 

short processed products, such as ~20-25-mers, 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 minimizing 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 Blastn (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 degree does the siRNA have to match the target genetic sequence to trigger 

RNAi?

The Panel indicated 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 siRNA, 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, substituting 

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 difference 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, select dsRNA sequences that 

target regions of genes with no shared 21 nt sequences with other species.

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

possible.

 An ideal target gene encodes a protein with a short half-life (see discussion in Scott et al. 

2013); this is because the duration of the effect 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 least 

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 (Judge 
29

et al., 2005) and eliminate them from further consideration. See also discussion in response 

to questions 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 siRNAs 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 could be specific to a single sequence or could target hundreds of mRNAs (off-target 

possibilities). Depending 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. MicroRNAs 

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 laboratory (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 

interaction, which suggests that a large proportion of miRNA-target pairings are non-canonical (Helwak

et al., 2013). 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 et al., 2007; Mullokandov et al., 2012). 

Question 2. Based on data indicating degradation of the majority of dsRNA in the digestive 

system, please discuss 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 degradation in the mammalian digestive system

 Substantial systemic distribution of dietary RNA as input into 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, stoichiometry 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 that produces biological effects. 

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

noted in the 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 sequences (Ivashuta et al., 2008, 2009). The Panel pointed out that a perfect 

sequence match does not always correlate with gene silencing, one of the false positive issues with 

algorithms. The Panel added that the FDA considers RNA as GRAS (“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 high levels of RNA-rich foods like brewer’s yeast supplements) could 

aggravate gout. The Panel pointed out that this 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 mastication 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 absorption of liberated nutrients. 

Digestive strategies 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-enzymatic 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 in 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 solutions, 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 presented in detail in the response to question 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 that 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-nucleotides 

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, public 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, limited success 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 injected into mammals at high concentrations. However, miRNAs that 

are resistant to low pH and RNases have been reported in milk. These 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 “plantspecific 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 breakdown to provide nutrition. While bacteria and archaea have 

RNA-based regulatory systems, the machinery, RNA sequences, and binding behavior for these systems 

differ from 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 not 

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 in 
32

which these mechanisms are lacking or are not as robust, including mammals. Features include efficient 

uptake of dsRNA 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 shared by mammals 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 

solution (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 transmembrane protein, SID-2 (McEwan et al., 2012; 

Winston et al., 2007) in the acidic environment of the gut. Binding 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 introduced (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 may 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-dependent RNA 

polymerases are primed by binding of siRNA to a target mRNA. 

The small RNAs 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, many 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 has 
33

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 some

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 amongst 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. A complete discussion of potential barriers to foreign uptake in insects in discussed in the 

Panel’s responses to questions 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 traverse 

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, Argonaute-bound and vesicle-protected small RNAs may 

well be among the most stable and thus the strongest candidates for survival and uptake. Small RNAs 

would be the ultimate effectors in tissue of hypothetical ingested and subsequently processed longer 

RNAs. 

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 lowdensity lipoprotein). Four plant miRNAs were found in all samples, two of them occasionally reaching 

levels similar to those of abundant endogenous miRNAs. It was also reported that one plant miRNA, 

MiR168a, 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. Zhang 

et al., 2013) and identified the following issues:

 Sequencing 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 

miRNAs 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 highthroughput sequencing library preparation, raising questions of possible crosscontamination. 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 apparent over-representation of 

MiR168, suggesting contamination 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 studies (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 transfer of plant miRNAs from miRNA-replete diets in 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 undetected 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 replicated the original mouse feeding study of L. Zhang et al. (2012) and found no 

consistently detectable plant miRNA from rice (Dickinson, et al., 2013). By administering several ricecontaining 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 by 

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). 

Only one plant miRNA, MiR168 (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 background. Public commenters stated that 
35

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 any 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 studies. 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 timeframe. 

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 differences in innate immune responses to dsRNA from commensal 

and pathogenic bacteria (Kawashima, et al., 2013).

The Panel commented that recognition of foreign RNA triggers innate immune responses: 

degradation of RNA transcripts, shutdown of protein expression, 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 Panel concluded that factors governing inflammatory responses 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, allowing 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 nanovesicles 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 relative 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. However, 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 indirect 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 surface, 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 enhanced systemic antibody responses, likely through interaction with Toll-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, Scallan 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, the 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, delivery, and estimated dietary exposure

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 the gut and then into cells to silence these targets. The Panel stated 

that the history of safe mammalian consumption of plants indicates that nominal uptake of dietary RNA 

by mammals is insufficient to lead to regulatory effects (see related discussion later in this response to 

this question). This conclusion is in agreement with statements by public commenters and findings 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 miRNAlike 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 vitro
37

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). These researchers astutely noted, however, that RNA 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 mature 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 cell (Jose et al., 2011). Single-stranded RNAi effector molecules (derived from processed 

short dsRNA) and single-stranded secondary molecules from the RNA-dependent RNA polymerasemediated 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 that provided in the EPA background documents) to estimate 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 dsRNA stable or degraded in planta? The Panel recommended the 

collection of data on the abundance of PIP dsRNA relative to other dsRNAs in PIP plants.

Some panel members expressed concern that PIPs could be engineered to greatly over express 

RNAi effectors in 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 Panel during the public comment 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 estimated 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 would 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 mammalian 

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 detection 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; 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. 

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 

instance, 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). Additionally, 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 posttranscriptional processes such as protein translation (Kozak, 1980; Kozak, 1986) and RNA-mediated 

silencing (Baulcombe, 2004; Jones-Rhoades and Bartel, 2004) are also tightly controlled 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 on 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). Additionally, 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 endoribonucleases and 
39

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-stranded 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 focus 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 ssand 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 throughout 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 degrade 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 conditions (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 destruction of RNA molecules. Thus, no matter the 

structural confirmation in which an RNA molecule arrives into the human GI tract the non-specific 
40

hydrolysis of RNA molecules is likely to result in significant degradation of both ss- and dsRNAs. 

These include all 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 Cho, 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 human 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 animals 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 et 

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 

structural status when entering the human digestive system. 

5) Studies on potential dietary uptake 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 

question 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 uptake of foreign 

miRNAs from the diet is negligible at best. Given the available evidence, the Panel strongly 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 human 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; Izumi 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 degradation 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 testing 
41

of stabilizers used in exogenously applied dsRNA end-products applied to food plants that will be 

consumed by humans. 

7) Influence of human health conditions on susceptibility 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 

persistence 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 infections (e.g., Helicobacter pylori), or medication (e.g., proton pump inhibitors such as 

omeprazole), (2) increased intestinal permeability, facilitating uptake of dsRNA from the intestine, such 

as individuals with celiac 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 cancer, there were no apparent differences in detection of the only plant miRNA (Wang et al., 

2012). This panel member noted that MiR168 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.

The Panel recognized that other issues concerning the stability of dsRNA should be addressed and 

made the following recommendations:

 This charge question focused on the stability 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 the human health risk 

assessment.

 The Panel recommended that the 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 digestion 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 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 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 RNAi effectors (e.g., siRNA capable of binding 

Argonaute 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 decrease availability to organisms. Both the persistence in soil and bioavailability 

should be evaluated in soil degradation studies and pesticidal activity studies. Measurement of the 

degradation 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 could 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 tissue 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, where biotic and abiotic processes can vary quite significantly between 
43

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 persistence of dsRNA PIPs must be better understood to 

evaluate exposure potential. Persistence of dsRNA in the soil will be affected by a number of factors, 

e.g., temperature, soil structure and type, abiotic and biotic factors. For example, fields with summer 

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 should 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 of earthworms. Thus, data on the soil degradation of dsRNA and degraded products (if 

present) would be useful 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.

In 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 Harteveld 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 20oC and for 1 

day at temperatures of 37oC. At colder temperatures, approximately 50% of RNA remained after 1 day 

at 2oC and 20% remained after 7 days. In the same study, drying blood samples stabilized RNA. The 
44

Panel suggested that this information provides 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 that 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 degradates

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 transformation 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 also 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 pathways 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, the Panel also recommended that the uptake of 

dsRNA in herbivorous insects and non-target insects should be evaluated because of the possible 

impacts on predatory insects and arachnids. The Panel discussed amplification mechanisms in response 

to questions 2 and 6 and cellular uptake mechanisms in response to question 5.
45

6) Non-target toxicity 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 fundamentally 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 assessment 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 challenges 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 endproducts, e.g., sprays. 

The Panel stated that current 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 organisms, i.e., plant degradation (above and below ground), offsite movement of plant materials, in planta exposure for organisms that directly ingest plant materials. 

Plant tissue may be desiccated and remain in fields during quite cold periods. As a result, dsRNA may 

remain in plant material for long periods of time allowing slow release into the environment. Doublestranded 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 data 

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 information 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 temperatures, 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 used, 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 the public comment period, representatives from Monsanto presented unpublished data

from preliminary soil degradation studies of a dsRNA PIP for corn rootworm control under 

development. One panelist was concerned that low PIP concentrations in planta in Monsanto’s studies

were insufficient to allow detection 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 dsRNAs were added to biological samples in order to be able to measure the RNA. The 

Panel stated that these data should be made publicly available1.

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 microbial-produced deoxyribonucleases (DNases) (Levy-Booth et al., 2007). The degree to 

which biota contribute to the degradation rate of dsRNAs 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 organisms 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 PIPs. 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 some 

differences. Non-target species are more likely to be exposed to exogenously applied dsRNA endproducts 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 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 balanced by the likely increased 

instability of the sprayed product (duration of the RNAi).

The Panel 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 scenarios 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 metabolism
48

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 (a first tier 

evaluation). Data are lacking on cellular uptake of dsRNA at various taxonomic levels. In the absence of 

data, the 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 amphibian 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 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. 

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 importance of standardizing the extraction 

and isolation protocols and having accurate and precise analytical methods to quantify if active 

degradation products, dsRNA and associated small RNAs, 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 sample 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 analysis 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. For 
49

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 indicates 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 factors 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, lipophorins can adhere to dsRNA, bacteria and fungi present in the

insect hemolymph (Wynant et al., 2014) and assist in the cellular uptake 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) across 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 

mechanisms 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 the 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 generalized 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. Polymers and agents used in an end-product 

formulation to increase uptake/penetration kinetics to overcome physical barriers 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 

aphids) in the saliva and in the gut (Allen and Walker, 2012; Christiaens et al., 2014).

Additional information is needed for other insects before any generalization can be made 

regarding the role of degradation in reducing non-target exposure to exogenous dsRNAs (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 information exists to verify this role. 

Another panel member responded that exogenous dsRNA will readily pass through the 

peritrophic 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, amongst 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 the stomach pH is higher in mice. In Lepidoptera the midgut pH can reach 12. There is
51

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 combined 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 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).

5.b. The degree of exposure by direct contact is likely to differ between nontarget 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 assessments. The Panel found only two studies that examined direct contact as a

route of exposure to dsRNA. The limited information 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 

this 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 the second case (Wang et al. 2011), newly hatched Ostrinia fumalalis larvae (a 

lepidopteran) 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 crawled 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. The 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, the Panel 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 significance 

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 from 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 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 that 

dsRNA sequence used to trigger RNAi can be designed to be highly species specific 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 RNAi-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 affected by this technology (Agrawal et al., 2003). First, a 

dsRNA must be produced (either endogenously or exogenously) that has some minimal level of 
53

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

small (20-25 nt) siRNAs using an enzyme 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 binding 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 

sometimes 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 slight differences in the machinery, 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 polymerases (RdRP). In 

contrast, an RdRP amplification process or an equivalent has not been identified in insects (a primary 

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). Although 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, substantial 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 agroecosystems (in particular) 

warrants exploration of the potential for unintended ecological effects. The Panel discussed several 

possible unintended effects that might occur if non-target organisms are exposed to dsRNA 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 genes 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 virgifera virgifera (Western corn rootworm), Baum et al. (2007) also examined the effects of 
54

a few of the dsRNAs identified for plant transformation on several other beetle species. They 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 

dsRNAs 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” RNAs) (Dua 

et al., 2011). Chances of off-target binding increase as the siRNA becomes shorter and if sequences 

mismatches between target and off-target sites occur (Qiu 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 silencing. 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 et 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 et 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-Tabor 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 present 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-products, 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 

useful screening tools to identify potential non-target organisms, but should not be used as an absolute 

predictor of effects.

Evidence suggests that off-target binding can change the phenotypes of affected organisms, 

sometimes unpredictably. Suppression of mRNA 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 reduced 
55

protein production in 7 of 30 cases involving culture 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 offtarget 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, etc.) 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 consequences 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 components 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 

compromised (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 evidence that 

small RNAs could have “global effects on the expression 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 it is possible to deliver enough pesticidal dsRNA to saturate an organism’s RNAi machinery.

One panel member commented that saturation will not matter if the amount will be so minimal 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 et 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; Menget al., 2013). Immune stimulation 

appears to be sequence dependent, and the length of the siRNA does not apparently affect 

immunostimulation (Forsbach et al., 2011). In mice the immunostimulation of RNAi led to reduced 

lymphocytes and platelet cells, largely correlated with cytokine response to the siRNA (Judge et al.,
56

2005). Although there are some similarities in the innate immune response of insects and mammals 

(Saleh et al., 2009; Lundgren 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 immunostimulation will 

affect the fitness of non-target organisms. Slight changes in nucleotide sequence, particularly at the 2’ 

position of the guide strand, can reduce the immunostimulatory 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-target species or what dose is necessary to trigger a response. See also 

the Panel’s discussion in response to question 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 have 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 different 

populations of the target pest, and failures could occur if SNPs were found in the gene for the critical 

target region of the dsRNA/siRNA (Scott et al., 2013). Therefore, caution is warranted in evaluating the 

potential for resistance in multiple field 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 the 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 the persistence and stability of the effect. What 

is a realistic level of non-target exposure based on the stability and persistence of the dsRNA? The Panel 

noted that the mode of action of RNAi (by siRNAs) is, by definition, very specific (see discussion in 

response to question 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 question is how 

do you decide what non-target should be tested? For example, insect species representing 10 families 

and four insect orders were 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 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 assessment of dsRNA PIPs and dsRNA non-PIP end-products. This approach would 
57

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

question 7.

One panel member suggested a screening process to identify a diversity of responses at a “network 

level.” Cell lines from different tissues could be used to screen for differential gene expression. Test 

endpoints could be better defined. 

Question 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 results 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 Panel appreciates all the time and effort spent by both the Agency and the public commenters in 

preparation and presentation of the 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 products. As stated in Section 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 traditional 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 

control under Section 3 of FIFRA. 

 The screening level assessments currently used for traditional chemical pesticides may not 

be applicable due to the unique modes of action of dsRNA active ingredients.

Current requirements for soil degradation studies will provide some insight for non-target testing, 

but additional data are needed to address the environmental fate of dsRNA PIPs and potential exposures 
58

to non-target organisms, i.e., plant degradation (above and below ground), off-site movement of plant 

materials, in planta exposure for organisms 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 an

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 

balanced 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 

work well for this technology due to the modes of action of RNAi. No 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 selected and resulting siRNAs

(sequence specificity), mechanisms of action, plant tissue expressed (dsRNA PIPs), dose needed 

to trigger RNAi, amplification, 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 limited 

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 recommended 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 chronic 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 effects 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-target 
59

species, 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 severity 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.

The Panel remarked that the most difficult effect to predict will 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 offtarget gene is expressed during the time and 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 the 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 binding.

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

sand 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 for 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 organisms 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, use of RNAi technology 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 dsRNAs 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 subquestions, 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 questions 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 cannot be done without a better understanding of 

the RNAi modes 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 sublethal 

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 testing. These tests should be conducted under realistic conditions 

(as much as possible) to ensure that genes in operation in normal developmental conditions are 
61

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 inclusion 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 efficiency 

at which a cell regulates endogenous gene expression. 

 The Panel recommended that EPA consider possible synergistic effects (of ds RNA PIP coexposures 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, exposure, 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 understanding 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 conceptual 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 in 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 exposure (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 tested in vitro based on 

relative abundance, importance in food web structure (e.g., trophic interactions),

phylogenetic placement, 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, in silico searches are not a predictive of effects. The Panel discussed 

different options to focus the in silico searches.

a. Perform in silico searches using a database of known genomes.

b. If full genomes do not exist, sequence 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 bioassays on non-target organisms based on the in silico searches. 

Measure activity at various 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? Conduct 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 examines lethality and a battery of sublethal effects should be 
63 

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

64
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