RNA Interference – Problem Formulation for Human Health and Ecological Risk 

Assessment: Charge Questions for the SAP 

The intent of this meeting is to solicit scientific expertise for 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 to pose about hazard and 

exposure (EPA, 1998b). The framework for the risk assessment of biopesticides is found in the RNAi 

issue paper in two sections. The current approach for mammalian safety assessment of 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.

EPA has found that the current approach utilizing reduced data sets and tiered approaches for more 

involved studies works well for the risk assessment of products based on biological materials. EPA 

recognizes that biopesticides tend to be more specific in their activity and present a lower risk profile 

than some alternative pest control measures. Biopesticide products are likely to be developed utilizing 

RNAi phenomena as both dsRNA PIPs expressed in plants and typical sprays and other products based 

on isolated dsRNA molecules. EPA is asking the following questions to ascertain the issues unique to 

RNAi and how they could fit under the existing risk assessment framework.

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?

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?

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?

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. 
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?

ii) What additional information, if any, would EPA need to assess environmental fate of 

dsRNA PIPs? 

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? 

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? 

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? 

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.

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.

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?

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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.

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. 

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.

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? 

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.

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