10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.

1. Federal Department of Economic Affairs,
Education and Research EAER
Agroscope
Ecological assessment of
topically applied dsRNA-based
products
Jörg Romeis,
Franco Widmer
Agroscope, Zurich, Switzerland

2. RNAi for pest control
In planta
- Genetic engineering of crop plants to
produce dsRNA
Non-transformative strategies
- dsRNA uptake of plants through
- roots (irrigation water)
- injections (woody plants)
- dsRNA delivery via
- food baits
- GM bacteria
- spray products

3. RNAi for pest control
Plant protection products require an environmental risk
assessment: EC 1107/2009, EU approval of active
substances
Article 4, 3(e) "it shall have no unacceptable effects
on the environment, having particular regard to […] its
impact on biodiversity and the ecosystem"

4. Problem formulation
What do we not want to see harmed? What must be
protected?
Protection goals
Gray (2012) Collection of Biosafety Reviews 6, 10-65

5. Protection goals
Millennium Ecosystem Assessment (2015)
(conducted under the auspices of the United Nations)
Regulating services
• Biological control of arthropod pests
• Pollination
Cultural services
• Protected butterflies
Supporting services
• Nutrient cycling, decomposition
Devos et al. (2015) EMBO Reports 16, 1060-1063

6. Problem formulation
What do we not want to see harmed? What must be
protected?
Protection goals
Can we envision a way in which they could be
harmed?
Pathway to harm
How can we assess whether they are likely to be
harmed?
Development of risk hypotheses and a plan
to test them
Gray (2012) Collection of Biosafety Reviews 6, 10-65

7. Pathway to harm
Application of dsRNA-containing spray product
Biological control function is disrupted
E
X
P
O
S
U
R
E
H
A
Z
A
R
D

8. Pathway to harm
Predators consume
herbivores
Herbivores contain
dsRNA
Application of dsRNA-containing spray product
Predators covered by spray
Predators groom
Predators uptake (active) dsRNA
Biological control function is disrupted
dsRNA penetrates
body wall
E
X
P
O
S
U
R
E
H
A
Z
A
R
D

9. Pathway to harm
dsRNA causes adverse effects
Predators consume
herbivores
Herbivores contain
dsRNA
Application of dsRNA-containing spray product
Predators covered by spray
Predators groom
Predators uptake (active) dsRNA
Biological control function is disrupted
Population of predators is reduced
dsRNA penetrates
body wall
E
X
P
O
S
U
R
E
H
A
Z
A
R
D

10. Testable risk hypotheses
Focus Exposure
• Predators not covered by spray
• No dermal uptake of dsRNA
• Predators do not groom
• Herbivores do not contain (active) dsRNA

11. Exposure
• Dermal uptake of dsRNA leading to an RNAi response
has (rarely) been reported
Pridgeon et al. 2008; Wang et al. 2011
• Uptake can be enhanced/ affected by carriers
He et al. 2013; Yu et al. 2013
• dsRNA may stay biologically active after ingestion
Garbian et al. 2012
• dsRNA applied as sprays appears to be uptaken by
leaves to some extent
Mitter et al. 2017
Oral route of exposure is most important
Exposure routes will depend on formulation of the product

12. Testable risk hypotheses
Focus Exposure
• Predators not covered by spray
• No dermal uptake of dsRNA
• Predators do not groom
• Herbivores do not contain (active) dsRNA
Focus Hazard/ effect
• Predators are not affected by environmental dsRNA
• Effects do not result in population declines
• Population declines do not lead to a disruption of the
biological control function

13. Ingested dsRNA could cause …
• off-target effects
• silencing of any gene in a non-target organism
• ? general immune stimulation
• ?? saturation of the RNAi machinery as consequence
of the ingestion of high doses of dsRNA
Roberts et al. (2015) Front. Plant Sci. 6, 958
 Conduct feeding studies with dsRNA, similar to
insecticidal Cry proteins
 Consider mortality and sublethal endpoints

14. Romeis et al. (2008) Nature Biotechnology 26, 203-208
Tiered risk assessment

15. Selection of test species for PPP
• Data requirements are provided in Commission Regulation
(EU) No 283/2013 and 284/2013 for the approval of active
substances and the authorisation of plant protection products
• Commission Regulation (EU) 546/2011 and Regulation (EC)
1107/2009 of the European Parliament and of the Council
define uniform principles for evaluation and authorisation of
plant protection products incl. defined set of test parameters
and endpoints!
• SANCO, Draft Guidance Document on Terrestrial
Ecotoxicology, 2002
• ESCORT 2, SETAC, Guidance Document on Regulatory
Testing and Risk Assessment Procedures for Plant Protection
products with Non-target Arthropods, 2001
 All applicable for contact toxicants

16. Selection of test species for PPP
Fixed set of NTO test organisms
(birds, mammals, earthworms, fish, aquatic invertebrates)
Terrestrial arthropods
• Honey bees (pollinator; Hymenoptera)
• Beneficial arthropods other than bees
• Aphidius rhopalosiphi (parasitoid; Hymenoptera)
• Typhlodromus pyri (predatory mite; Acarina)

17. Selection of test species for PPP
If adverse effects cannot be excluded, more species
need to be tested. Protocols are established for
• Lacewings (Neuroptera)
• Flower bugs (Hemiptera)
• Hoverflies (Diptera)
• Ladybird beetles (Coleoptera)
• Ground beetles (Coleoptera)
• Rove beetles (Coleoptera)
• Wolf spiders (Aranea)

18. Laboratory toxicity studies
• Opinion does not mention RNAi
• Address oral exposure of NTAs to PPP
• Standardised tests for addressing oral exposure are missing
• Test systems should
• cover exposure routes particular to the active substance
• allow detection of effects resulting from novel modes of action

19.  representative of valued taxa and functional
groups that are most likely to be exposed
 species most likely to be sensitive to the test
compound
 Amenability and availability to testing
Romeis et al. 2013, Chemosphere 90, 901-909
Carstens et al. 2014, GM Crops & Food 5, 11-15
Wach et al. 2016, Transgenic Research 25, 499-505
Selection of test species for GM plants
"case-by-case" approach

20. Selection of test species for GM plants
NTOs most likely to be sensitive:
• Arthropod orders differ in their sensitivity to dietary
RNAi. Coleoptera are more sensitive then other insect
orders (Baum and Roberts, 2014, Adv. Insect Physiol. 47, 249-295)
• Pylogenetic relationship of NTO to target is important
(Bachmann et al., 2013, Transgenic Res. 22, 1207-1222)
• Bioinformatics help predict NTO effects: analyse
sequence complementarity between pool of siRNA and
the NTO genome (Roberts et al., 2015, Front. Plant Sci. 6, 958)

21. Bioinformatics: Spectrum of activity

22. Main areas to consider when designing
laboratory NTO studies
(Romeis et al. 2011, Transgenic Research 20, 1-22)
• Test substance characterization and formulation
• Worst-case exposure conditions
Early-tier studies

23. Main areas to consider when designing
laboratory NTO studies
(Romeis et al. 2011, Transgenic Research 20, 1-22)
• Test substance characterization and formulation
• Worst-case exposure conditions
Early-tier studies
High doses may cause sequence-independent
adverse effects (Haller et al. 2019)

24. Main areas to consider when designing
laboratory NTO studies
(Romeis et al. 2011, Transgenic Research 20, 1-22)
• Test substance characterization and formulation
• Worst-case exposure conditions
• Measure suitable endpoints
• Test duration
Early-tier studies

25. Measurement endpoints
(used for Cry proteins)
Non-target species Parameters recorded
Orius insidiosus/ laevigatus Survival, development time
Coleomegilla maculata Survival, development time, adult
weight, fecundity
Coccinella septempunctata Survival, development time, adult
weight
Hippodamia convergens Survival
Poecilus cupreus Survival, development time, weight
Aleochara bilineata Fecundity
Chrysoperla carnea Survival
Pediobius foveolatus Survival
Apis mellifera Larval survival, development time,
adult emergence
Folsomia candida Survival, fecundity
Duan et al. 2002, Ent Exp Appl 104, 271-280; Duan et al. 2006, Env Ent 35, 135-142; Stacey et al. 2006, IOBC/WPRS Bull 29(5), 165-172; Raybould et
al. 2007, J Appl Entomol 131: 391-399; Duan et al. 2008, Env Ent 37, 838-844; Raybould & Vlachos 2011, Transgenic Res 20, 599-611; Devos et al.
2012, Transgenic Res 21,1191-1214; De Schrijver et al. 2016, Transgenic Res 25, 395-411; Bachmann et al. 2017, PLoS ONE 12, e0169409)

26. Main areas to consider when designing
laboratory NTO studies
(Romeis et al. 2011, Transgenic Research 20, 1-22)
• Test substance characterization and formulation
• Worst-case exposure conditions
• Measure suitable endpoints
• Test duration
• Negative/ positive control treatments
Early-tier studies

27. Control treatments
Known toxicant E-64 confirms that test organisms have
ingested the test substances.

28. Control treatments
Adalia bipunctata
Haller et al. (2019)
Pest Management Science, doi: 10.1002/ps.5370
sigf.
sigf.
Survival(%)
Treatment
dsRNA targeting vATPase A
of D. v. virgifera
Positive control
dsRNA targeting vATPase A
of A. bipunctata
Negative control
dsRNA targeting GFP
Control
Sucrose solution
Control GFP Ab Dvv
dsRNA treatments

29. Open questions
Exposure
• Fate of dsRNA
• Importance of tri-trophic exposure
• Formulations for delivery define routes of exposure
Hazard
• Factors affecting off-target effects
• 21-nt matches necessary ? How many ?
• no. of mismatches allowed ?
• value of bioinformatics
• Sensitivity of arthropod orders/ families to dietary RNAi
• Uncertainty about occurrence/ severity of sequence-
independent effects

30. Conclusions
• The established NTO test list for PPP is not sufficient
to test for non-target effects of dsRNA spray products
• Oral route of exposure is most important
• To detect non-target effects lethal and sublethal
endpoints might need to be recorded
• Validated test systems are available
• Include appropriate controls to avoid false positives/
negatives

31. Thank you for your attention
Drawing by Simone Haller
joerg.romeis@agroscope.admin.ch