5. Living modified organisms containing stacked events

Dear Colleagues,

Just a gentle reminder that the first week of discussions on Living Modified Organisms (LMOs) containing stacked events will close on Monday, 28 April at 11:00 a.m. (Montreal time).

This is a valuable opportunity to contribute your insights to help shape a deeper understanding for risk assessments of LMOs containing stacked events. Your perspective could make a real difference in enriching the dialogue for everyone involved.

We truly value your insight in this conversation and look forward to hearing from you soon.

Anita Anthonysamy

In contribution to the discussion on Question 1 and 2.

LMO stacks especially in plants are globally being adopted because of its ability to offer multiple benefits in a single product. However the possible challenges to risk assessment as may be faced by biosafety regulatory systems will be in the intensity and complexity of the "case by case" scenarios of Plant LMOs with stacked genes. Based on the understanding that combination of traits (stacked traits) can be achieved either through conventional breeding (breeding stacks) or molecular technique (molecular stacks), the case by case risk assessment methodology must be adhered to avoid complexities for decision making. This case by case assessment are usually on:
a) Stacking of GM events through conventional breeding where all the single events in the stack have been separately assessed and approved by the regulatory body. In this case, the higher order stack (final stacked product) for some regulatory systems, may be seen as a new GM product.
b) Stacking of GM events through conventional breeding where one or more of the single events in the stack have not been assessed and approved by the regulatory body. In this case, for some regulatory systems, much concern on the higher order stack will be pronounced.
c) Stacking of GM events through conventional breeding where none of the single events in the stack have been assessed and approved by the regulatory body. In this case, the higher order stack follows full risk assessment
d) Molecular stacks where molecular technique such as co-transformation, re-transformation, multi-gene cassettes etc. are used. Risk assessment is fully approached.

In view of these scenarios, the specific challenge is on the capacity of the risk assessors to understand the scenarios and being able to apply the specific case by case approach. Also some biosafety laws have also made assessment of scenario "a" much as easier that it may be considered as "already assessed" and may not pass through any form of risk assessment review while some laws have also made it difficult that the final stacked product is a new GM event that must be assessed.

In which ever case, the special understanding of the case by case consideration of LMO stacks must be prioritized and perhaps even harmonized for effectiveness. For instance, in scenario "a", assessment may be limited to:
i) an assessment of intactness and stability of inserted genetic elements
ii) an assessment of the potential interaction between combined events and the resulting phenotypic traits, and
iii) an assessment of potential interaction of the stacked events on conservation and sustainable use of biodiversity in the likely receiving environment, also taking into account risk to human health.

However, for other breeding stacks scenarios, the outcome of the risk assessment of the higher order stack should cover for the intermediates (sub-stacks) and other singles, where applicable (even still on case by case basis). In such that, if they are found safe following risk assessment review, the intermediates and singles should also be considered to be safe.

In view of the above contribution, highlighting possible challenges for risk assessment, priority for capacity especially in developing countries for risk assessment of LMO stacks must be considered through special financing or initiation of triangular cooperation mechanisms. Furthermore, these countries should also be supported to have in place regulatory framework to address LMO stacks in-line with their biosafety laws or processes.


Onyeka Nwosu
Nigeria

Living Modified Organisms containing stacked events
Moussa Savadogo, Burkina Faso
In contributing to questions 1, 2, and partially 3

Products from numerous genetically modified (GM) crops with stacked genes have been available in the global market for several years. These products have received approval from current biosafety systems and have been consumed without any officially reported harm. Additionally, several countries and organizations have developed and published individual guidelines to effectively guide the evaluation and decision-making processes for GM crop products containing stacked genes. Nigeria, Ghana, and other developing African countries have issued such guidelines. With the support of AUDA-NEPAD, more countries will soon establish their national guidelines on GM crops enhanced with stacked genes. For the sake of multilateralism and harmonization, in the spirit of the Convention on Biological Diversity (CBD) and its Cartagena Protocol, developing guidelines that will incorporate some compilation of the existing individual guidelines will be helpful. 
The discussions surrounding the risk assessment of GM crops with stacked genes primarily reveal a lack of consensus regarding the potential interactions among the involved genes. This uncertainty may sometimes jeopardize the approval of transboundary movements in some regions and the use of living modified organisms containing stacked genes and their products, which could undermine the primary goal of the Cartagena Protocol on Biosafety.
Although numerous references indicate that the existing framework for risk assessment, primarily based on Annex III of the Cartagena Protocol on Biosafety, can be readily applied to assessing the safety of crop varieties with stacked genes, some voices continue to express concerns about the potential interactions between the introduced genes and with the endogenous genes of the modified crop plants.
While some argue that interactions between the stacked genes and with the endogenous genes in the plant are complex to anticipate, others believe that in-depth knowledge of individual genes and their metabolic pathways makes it possible to predict possible interactions. The guidelines should indicate whether Annex III of the Cartagena Protocol is sufficient for the information needed to predict the gene interactions, or if additional information is needed.
Others also admit that knowledge of genes and their interactions may not be sufficient. In this case, data and information gaps could be filled during the pre-marketing phase by carrying out appropriate tests as part of field trials. The guidelines should help determine the acceptable level of data and information deficit to guide regulators in deciding whether to authorize the next steps for field trials. It is also crucial to provide recommendations to harmonize data collection in the field, aimed at filling the gaps identified.

Moussa Savadogo,
Burkina Faso

Esteemed colleagues,

In contribution to discussion I would like to comment on question 1:

1. It is considered that there is already a substantial number of resources available on this topic, including guidelines, studies, and regulatory frameworks. Therefore, developing new voluntary guidelines or horizon scanning studies for living modified organisms containing stacked events may not be the most appropriate path to pursue. However, taking into account a potential challenge namely, that in some countries the approval of individual events leads to the automatic approval of stacked events containing them, while in others, separate risk assessments are conducted for each stacked event even if the individual events are already approved, it may be more relevant to focus on discussions around regulatory harmonization. From such discussions, maybe a document with suggestions and recommendations could arise to support partial or full harmonization for the risk assessments of stacked events between Parties.

Gabriel Mutis
Colombia

In contribution to the discussion on questions 2 and 3:

LMOs with stacked events are widely used and being developed in crops around the world. Therefore, it is imperative to continue efforts to regulate and assess probable risks to human health and the environment. As there are many ways of generating LMOs and stacked genes, a case-by-case approach, as stated before, needs to be taken. Stacked LMOs resulting from traditional breeding techniques are usually regarded as safe as their parentals, even when those are genetically modified [1]. It has therefore been concluded by many regulatory agencies and independent studies that they are safe and do not need further risk assessment if their parents have already passed a risk screening. However, recalling the principle of a case-by-case approach, it is important to note that, especially with the rise of new genomic techniques that can be used in the parentals of stacking elements by breeding, there is a risk of non-targeted events and unexpected interactions that could have detrimental effects on the environment if released, potentially affecting the allelic diversity of the natural populations [2]. 

Even more important to consider are the stacked events produced by molecular techniques, i.e., the editing of more than one gene or trait within a single individual. The 2016 Report of the AHTEG on Risk Assessment and Risk Management did not address stacked LMOs from molecular techniques, only those from breeding (this includes gene-edited parentals), and no further guidelines regarding stacked LMOs from molecular techniques have been generated. As new genomic techniques are increasingly used, new regulations and screening protocols are needed to ensure that their products are safe for biodiversity.

The assessment of stacked LMOs on a case-by-case basis should then be carried out based on the nature of the LMO, specifically, the direct gene product interactions, gene expression patterns, and metabolic products of the transgenes [3].

Additionally, as mentioned before, countries have different regulations for the risk assessment of stacked products, and these varying approaches can lead to inconsistencies in the approval process [1]. There is a clear need for unified regulations to ensure effective and harmonized risk assessments globally.

Equally important is the need to recognize the significant risks that the introduction of any kind of LMOs poses to food sovereignty and the indigenous practices of local communities, particularly women. Women are traditionally the ones who carry the knowledge of local biodiversity and its uses, and they bear the adverse effects of losing that knowledge.

[1] https://croplife.org/wp-content/uploads/2021/01/Stacked-Traits-Goodwin-et-al.pdf
[2] https://enveurope.springeropen.com/articles/10.1186/s12302-023-00734-3
[3] https://pmc.ncbi.nlm.nih.gov/articles/PMC3613440/#s9

Sol Carolina Parra Santos

Dear colleagues
Thank you for the opportunity to contribute to this forum and to Ms Anthonysamy for her capable moderation. It is a pleasure to read familiar names and I look forward to meeting new members of the forum.

Questions 1-3 (with a few references relevant to 4)

1. Challenges and solutions
Global experience with environmental release of organisms with stacked events is predominantly if not exclusively restricted to plants. I echo Ms Parra Santo’s intervention [#12290]. I find it difficult to be confident that when existing guidance was written, there was adequate consideration given to how or if the guidance would satisfactorily apply to all organisms, including bacteria, fungi, and animals, and all ways of making stacks. A potential solution is to include this topic in the work of an AHTEG composed of the right diversity of biological experts.

2. Specific challenges
Global experience with environmental release of organisms with stacked events is predominantly with plants expressing multiple exotic proteins. A much larger variety of stacks can now be envisioned. These include stacks of: gene silencing cassettes; gene editing and non-RNAi gene silencing cassettes, e.g. CRISPR arrays; mixtures of these events and mixtures of these events and events where exotic proteins are produced.

3. Challenges to existing RA frameworks
As a biologist I am acutely aware that the simple story of an enzyme and its target substrate, a dsRNA and its target, or an RNA guide and its target, is inadequate for assessing risk. No enzyme can be assumed to have no secondary activity, and no oligonucleotide can be assumed to base-pair with only one other sequence of nucleotides or the same particular sequences under all conditions. Because each of these categories of interventions in a genome will create unintended effects, and because of their diverse biochemical actions, it is difficult for me to see how existing guidance would be sufficient. For example:
Benevenuto, R.F., Zanatta, C.B., Waßmann, F. et al. Integration of omics analyses into GMO risk assessment in Europe: a case study from soybean field trials. Environ Sci Eur 35, 14 (2023). https://doi.org/10.1186/s12302-023-00715-6

3.1-3.2
Existing frameworks do not in my opinion provide the needed baseline descriptions of risk assessment based on a non-comparative approach. That kind of approach is needed when the outcome is not an incremental change from a parental or even ancestral comparator with which there is substantial experience. For the stacked events I described above, and in the diversity of organisms that may be constructed with them, the comparative risk assessment is not a validated approach.
Solutions could include:
• a strategy to test the hypothesis that a comparative approach applied to a large range of organisms with different complex stacked events is adequate. This would be consistent also with Mr. Savadogo’s #12283 suggestion. Rejection of the hypothesis would then necessitate developing alternative guidance; or
• evaluating and adopting methodologies that are less vulnerable to the weaknesses of a comparative approach. Here I recall the encouragement of the US National Academies of Science, Engineering, and Medicine to routinely include “omics” methodologies. (NASEM. (2016) Genetically Engineered Crops: Experiences and Prospects, The National Academies Press. https://nap.nationalacademies.org/catalog/23395/genetically-engineered-crops-experiences-and-prospects).

3.3
Guidance on stacked events should be developed to account not only for the diversity of organisms that may have multiple independent events in their genomes, but also for minimizing safety and trade implications of stacked events in non-target organisms.

Stacked events can occur by design or unintentionally. Intentional stacking may arise as others in this thread have discussed (eg Mr. Nwosu #12275), but also as mentioned by Ms. Parra Santos #12290, from multiplex or sequential applications of gene editing. Unintentional stacking could arise from insertions of DNA not intended to be part of the process. For example, would the Recombinetics “polled cattle” created through use of gene editing later found to have the vector inserted into the genome be a gene edited animal with an off-target insertion, or a stack of a gene edit and an insertion? (Solomon, S.M. (2020) Genome Editing in Animals: Why FDA Regulation Matters. Nat Biotech 38, 142-143.) For risk assessment, this is probably an irrelevant difference, as would be the case when an organism has both an insertion of the intended cassette and a partial insertion of the same cassette.

Concerningly, and also pertinent to Week Two topics 1 and 2, new chemical and biological vectors make it possible to use techniques of modern biotechnology outside of specialist laboratories or without the need for trained personnel. Therefore, organisms from many different biological kingdoms could be unintentionally but unavoidably exposed to the transformation process (eg, microorganisms including bacteria, microscopic plants and animals). These organisms may also have multiple independent (“stacked”) events. They could be immediately released into the environment should countries make it possible (as has at least one non-Party country) to use techniques of modern biotechnology without regulatory provisions for containment or registration.

The effect is to create new potential for accidental release of an organism that may cause adverse effects on human health or the environment and which may be difficult if not impossible to keep from disseminating across national borders. 
https://ir.canterbury.ac.nz/server/api/core/bitstreams/0e1aa118-5e68-4b43-b395-2a4487d90aa4/content, https://ir.canterbury.ac.nz/server/api/core/bitstreams/fb5002ba-2e21-4a45-be4e-56d6259b4571/content, https://ir.canterbury.ac.nz/server/api/core/bitstreams/05dd6485-82e0-4f54-844b-8860e8548b68/content

In conclusion, I think there is a need for guidance on LMOs with stacked events. Apologies for the long intervention.

Greetings,

Replying to Question 1
Stacked events present a tight stretch on existing risk assessment frameworks. This is especially so when it comes to molecular stacks. Many risk assessment frameworks focus on the presence or not of interactions between genes or traits in a stack event. But given the complexity and interconnectedness, both direct and indirect, of biological systems, one must wonder if this kind of risk assessment is enough.
I believe there’s need for in-depth risk assessment of stack events beyond what’s done for single events, with special emphasis on field testing across diverse environmental conditions to cater for as many scenarios as possible. For example, different epigenetic changes may be triggered by the presence or absence of some or all of the stresses addressed in the stack.
Could there be unexpected responses of a stack LMO to new or increased stresses? For example, how would higher than normal temperatures affect the expression of other genes, in addition to HSPs, in a multi-gene cassette that has a heat-activated gene promoter?

There’s need to test/ observe the behaviour of breeding stacks, and ensure sufficient regulatory safeguards, before commercialization. This is because as much as crossing is something that can and does happen naturally, it doesn’t always result in desirable traits, often resulting in unexpected phenotypes.

Replying to Question 2
One of the challenges maybe in making assumptions based on insufficient knowledge of the intricacies of biological networks. There’s need to collect and consider more data on the possibilities of less direct interactions in different organisms.

Replying to Question 4
Several documents exist that guide on this issue, but they do not all say the same thing. It’d be helpful to have a guidance document on risk assessment of stacks, that addresses different potential scenarios without the underlying assumption that if single events are safe then the resulting stacks should be safe too, especially concerning molecular stacks.

Dear colleagues,

Thank you, Ms. Anita Anthonysamy, for moderating this discussion. I would like to express my appreciation to the colleagues who have shared insights on risk assessment considerations for stacked events. I agree with the concerns raised and acknowledge the various challenges in assessing their safety under existing regulatory frameworks.

Currently, there are 386 living modified organisms (LMOs) registered in the Biosafety Clearing-House (BCH) that contain stacked events, which accounts for one-third of the total database. However, it is known that the majority of GM crops grown globally are stacked events. The primary concern is the complete lack of institutional capacity and response to address the risk assessment of this type of LMO at the global level. We are at least ten years behind in this regard.

Regarding question #1, in addition to the issues already mentioned by others, I would like to add the following points:
• Identification of Stacked Events: Existing detection methods used in regulatory contexts cannot identify stacked events in samples containing multiple individuals (e.g., food or feed samples, crushed grains, bulk leaves, environmental samples for GM microorganisms, etc). The lack of capacity to identify stacked events presents additional challenges for risk monitoring.
• Synergistic and Antagonistic Effects of Multiple Transgenes: Current risk assessment methodologies fail to address unintended synergistic and antagonistic effects arising from stacking transgenes. Our research group has dedicated several years to studying untargeted molecular profiling techniques to better understand negative metabolic impacts in plants in support of dedicated risk hypothesis.
References:
Agapito-Tenfen, S.Z., Vilperte, V., Benevenuto, R.F. et al. Effect of stacking insecticidal cry and herbicide tolerance epsps transgenes on transgenic maize proteome. BMC Plant Biol 14, 346 (2014). https://doi.org/10.1186/s12870-014-0346-8
Zanatta, C.B., Benevenuto, R.F., Nodari, R.O. et al. Stacked genetically modified soybean harboring herbicide resistance and insecticide rCry1Ac shows strong defense and redox homeostasis disturbance after glyphosate-based herbicide application. Environ Sci Eur 32, 104 (2020). https://doi.org/10.1186/s12302-020-00379-6

Regarding questions #2 and #3, other key challenges include:
• Lack of Analytical Identification of Stacked Events: This deficiency poses significant challenges in risk assessment and environmental monitoring, as well as food and feed safety.
• Lack of Risk Assessment Methodologies for Combinatorial Effects: Current risk assessment frameworks often focus on single-event analyses, failing to account for the complexities that arise when considering multiple genetic modifications. The absence of established methodologies for assessing combinatorial effects can leave significant gaps in understanding how these stacked traits may impact biodiversity, ecosystem functioning, and human health.

In regard to question #4, there is a specific section on the Guidance on Risk Assessment of Living Modified Organisms and Monitoring in the Context of Risk Assessment that serves as a good resource for setting the scope and conducting risk assessments. However, this section requires significant support from updated scientific methodologies to perform the necessary studies. Key considerations extracted from the Guidance document include:
• Effects of the use of pesticides, chemicals, or agricultural practices commonly used in the cultivation of the parental LM plants.
• Phenotypic characteristics compared to the parent LM plants and to non-modified recipient organisms.
• Interactions between the stacked transgenes or their products, as well as interactions among the physiological pathways in which the transgenes are involved, taking into account the possibility that these interactions could result in potentially harmful substances (e.g., anti-nutritional factors), some of which may persist or accumulate in the environment (e.g., via the food chain).
• Combinatorial and cumulative effects arising from the presence of two or more insecticidal proteins, which could lead to increased toxicity to non-target organisms or accelerate the development of resistance in target organisms.
• Effects on native and local biodiversity.


Best regards,
Sarah Agapito

My name is Christoph Then and I am a member of ENSSER (The European Network of Scientists for Social and Environmental Responsibility) and representing Testbiotech (http://www.testbiotch.org) in this discussion.

I address the four questions as raised by the moderator:

1. Combinatorial and cumulative effects can be called a ‘dark matter’ of risk assessment. There is no guidance, no methodology and lack of empirical data that would allow to assess their real impact on biodiversity and human health.

2. Stacked events are a reality for example in transgenic crops since more 20 years. In the EU, around 100 events of transgenic plants are allowed to import for food and feed, nearly half of it are stacked events. Many of the authorisations for stacked events are extending to several sub-combinations.

The stacking may imply several resistances to herbicide or produce several toxins against insects, in various combinations (see https://www.testbiotech.org/en/projects/plantgenerisk-database/)

The experience shows that current practice of risk assessment is still very much focused on single events, while potential combinatorial effects are largely ignored (see Testbiotech 2021). There are a number of plausible and relevant hypotheses that were never or only poorly investigated and assessed in regard to combinatorial effects and risk assessments of stacked events: (i) the higher toxicity of Bt toxins if combined with residues from spraying; (ii) the higher toxicity and immunogenicity of Bt toxins if combined with protease inhibitors; (iii) the higher toxicity and/or immunogenicity of a combination of traits (several Bt toxins or several herbicide resistances and combinations thereof ) if combined in stacked events or mixed in a diet; (iv) changes in the intestinal flora after long-term consumption of food and feed derived from herbicide-resistant or Bt producing plants, or combinations thereof; (v) changes in the soil flora after long term cultivation of a stacked crops with herbicide-resistant and insecticidal traits; (vi) effects of outcrossing of stacked events to wild relatives or of spontaneous stacking by outcrossing.

3. All four questions can be answered with yes.


4. Stacked events and cumulative effects are considered in EU GMO regulation, but poorly assessed in practice. The European Food Safety Authority (EFSA) is still following a logic of assessing and testing single components to conclude on the overall risks of LMO plants. This causes its opinions to be incomplete, inconclusive and also potentially wrong in their findings. Being aware of a steadily rising number of applications for stacked events, this problem should be followed with some priority.


Testbiotech (2021) Risk assessment of GE plants in the EU: Taking a look at the ‘dark side of the moon’, https://www.testbiotech.org/publikation/risk-assessment-ge-plants-eu-taking-look-dark-side-moon/

Living Modified Organisms containing stacked events (or simply "stacked LMOs") refer to organisms — primarily crops, but increasingly also trees, fish, or microbes — that carry multiple genetically engineered traits combined either through cross-breeding of separate LMOs or by simultaneous insertion of multiple genes into a single organism.
Stacked traits may include combinations like insect resistance + herbicide tolerance, or disease resistance + drought tolerance.
The increasing use of stacked LMOs in agriculture and biotechnology raises new biosafety, ecological, and regulatory challenges that differ from those associated with single-trait LMOs.
1. How does this topic potentially pose challenges to the existing risk assessment frameworks, guidance, and methodologies?
Challenges:
Assumption of Trait Independence:
Many existing frameworks assess stacked LMOs by evaluating the individual traits separately. However, interactions among stacked genes at molecular, physiological, or ecological levels may create new, unforeseen risks not captured by simple additive assessments (EFSA, 2021).

Complexity of Molecular and Phenotypic Effects:
Multiple transgenes may interact synergistically or antagonistically, altering expression levels, metabolic pathways, or stress responses in ways that are hard to predict.

Higher Environmental and Ecological Uncertainty:
Stacked traits may amplify ecological impacts, such as combined resistance to multiple pests or herbicides leading to ecosystem-level shifts.

Cumulative and Long-Term Effects:
Existing methodologies often fail to account for cumulative impacts of multiple traits over several growing seasons, potentially affecting soil microbiomes, non-target species, and food webs.

Challenges in Monitoring and Detection:
Identifying and tracking stacked LMOs post-release is more complex because multiple genetic elements must be monitored simultaneously.

Increased Potential for Gene Flow:
Stacked LMOs may increase the range of potential hybridization partners in the wild, facilitating gene flow of multiple traits to non-modified or wild relatives.

Regulatory Fragmentation:
Some jurisdictions regulate stacked LMOs as new LMOs requiring full risk assessment, while others treat them as simple combinations of previously assessed traits — leading to regulatory inconsistency (UNEP, 2021).

Unpredictable Effects in Marginal Environments:
Environmental stresses (e.g., drought, salinity, nutrient deprivation) could trigger unexpected interactions between stacked traits, influencing plant behavior or survival in ways not observed under normal testing conditions.

Public Perception and Acceptance Challenges:
Stacked LMOs often exacerbate public concerns about "unnaturalness" or corporate control over seeds and agriculture, even when regulatory science focuses only on technical risk.

2. What could be the specific challenges related to this issue?
Trait Interaction Effects:
Unintended pleiotropic effects — where one gene influences multiple traits — could be exacerbated by stacking.

Resistance Evolution Acceleration:
Stacked pest-resistance traits may accelerate the evolution of resistant insect populations or superweeds more rapidly than single-trait LMOs.

Data Gaps in Multi-Trait Risk Assessment:
Very few long-term, multi-generational studies exist evaluating ecological impacts of stacked LMOs under real field conditions.

Trade and Traceability Issues:
The complex genetic profiles of stacked LMOs complicate international trade regulations, labelling requirements, and traceability in food and feed chains.

Post-Market Monitoring Complexity:
Monitoring unintended environmental or health effects becomes more challenging when multiple traits — possibly interacting with multiple environmental factors — must be evaluated.

3. Specific Issues Concerning LMOs with Stacked Events
(i) Potential to Cause Serious or Irreversible Adverse Effects on Biodiversity
Yes.

Stacked traits may impact non-target species (e.g., beneficial insects, soil organisms) more severely than single-trait LMOs.
Gene flow to wild relatives could result in wild species with multiple advantageous traits, potentially altering local biodiversity permanently.

(ii) Potential for Introduction into the Environment
Yes.
Stacked LMOs are deliberately introduced in agriculture, forestry, aquaculture, and increasingly in new contexts such as bioenergy production.
(iii) Potential for Dissemination Across National Borders
Yes.
Seeds, pollen, or organisms containing stacked traits can easily move across borders via trade, natural dispersal, or accidental release.
(iv) Commercialization or Current Use
Yes, widespread and growing.
Stacked LM crops such as Bt corn (insect resistance + herbicide tolerance) and cotton dominate global GMO agriculture.
Emerging stacked LMOs include climate-resilient crops combining multiple abiotic stress tolerances (e.g., heat + drought resistance) under development.

4. Are there existing resources on similar issues that can address this issue or resources that could be adapted to address this need?
Resources includes:

EFSA Guidance on Risk Assessment of GM Plants with Stacked Events (2021):

Highlights the need for case-by-case assessments considering potential interactions between stacked traits.
OECD Consensus Documents (2022 Update):
Technical background on the compositional analysis and safety considerations for stacked traits.

UNEP (2021):
Frontiers in Synthetic Biology Report.
Discusses complex genetic constructs, including stacked traits and synthetic multi-gene insertions.
Recent Research Contributions:

Schaart et al. (2021), New Plant Breeding Techniques and Risk Assessment, Trends in Plant Science.

Domingo & Bordonaba (2021), A Literature Review on Stacked GMO Safety, Environmental Sciences Europe.

Conclusion
Living Modified Organisms containing stacked events represent an evolution in modern biotechnology, offering significant productivity and resilience advantages.
However, the complex genetic, ecological, and regulatory challenges associated with stacked traits demand enhanced, multi-dimensional risk assessment methodologies.
Risk assessments must move beyond simple additive approaches, embracing systems biology, ecological modeling, and long-term post-release monitoring.
International harmonization and greater public transparency will be essential to ensure the safe deployment of stacked LMOs while maintaining trust and protecting biodiversity.

References
EFSA Panel on Genetically Modified Organisms (2021). Guidance for Risk Assessment of GM Plants with Stacked Events. EFSA Journal, 19(6), e06620.
Schaart, J.G., et al. (2021). New Plant Breeding Techniques and Risk Assessment Considerations. Trends in Plant Science, 26(3), 223–234.
Domingo, J.L., & Bordonaba, J.G. (2021). A Literature Review on Stacked Genetically Modified Organisms (GMO) Safety. Environmental Sciences Europe, 33(1), 1–12.
UNEP (2021). Frontiers in Synthetic Biology: Environmental Opportunities and Challenges. Nairobi: UNEP.
OECD (2022). Safety Considerations for Modern Biotechnology and Stacked Traits in GM Plants.

Ossama AbdelKawy
Egypt National Focal Point of the Cartagena Protocol
Microbiology and Biotechnology Professor, British University in Egypt
Research Professor, Egyptian Atomic Energy Authority
Science Advisor, Egyptian Ministry of Environment

Dear colleagues, it is a pleasure to join this important discussion and share perspectives with you all.

The risk assessment of LMOs requires a critical update of the current regulatory and methodological frameworks under the Cartagena Protocol. The case of the experimental release of stacked GM cotton events in Mexico illustrates this challenge: stacked events, such as the quintuple stack (MON-887Ø2-4 x MON-15985-7 x SYN-IR1Ø2-7 x MON-887Ø1-3 x MON-88913-8), combine multiple genes, increasing biological complexity and the risk of unforeseen synergistic effects. Current assessments, focused on individual events, are insufficient, and the lack of an integrated evaluation of associated technological packages (including the intensive use of herbicides such as glyphosate, dicamba, and glufosinate) represents a significant methodological gap, given their impacts on human health and biodiversity.

Mexico faces risks of transgene introgression into native and wild cottons, with the presence of volunteer and feral cottons and natural seed dispersion already documented (Wegier et al., 2011; Arteaga, 2021). This poses risks such as: (i) impacts on endemic species and unique ecosystems, (ii) deliberate or accidental release of transgenic seeds, (iii) social dissemination of seeds through agricultural laborers (Castiñeiras et al., 2009), and (iv) international commercial pressure for local adoption of GM cotton, which undermines the precautionary principle.

To strengthen risk assessment, Mexico has recently undertaken efforts that include the comprehensive evaluation of technological packages, recognizing herbicide impacts as an inseparable part of GM crops; the application of an integral biosafety approach considering socioeconomic, biocultural, and environmental factors; the robust application of the precautionary principle in the face of relevant scientific uncertainties, prioritizing environmental protection and human rights; and the guarantee of the right to free, prior, and informed consultation with Indigenous and Afro-Mexican peoples.

References:
Arteaga, R. A.E. (2021). Análisis del posible uso del algodón silvestre (Gossypium hirsutum) como material de construcción en nidos de aves en Yucatán y Oaxaca, México. Tesis que para obtener el título de Licenciada en Biología. Facultad de Ciencias, Universidad nacional Autónoma de México. http://132.248.9.195/ptd2021/julio/0813672/Index.html
Castiñeiras, L., Cristóbal, R., Pinedo, R., Collado, L., & Arias, L. (2009). Redes de abastecimiento de semillas y limitaciones que enfrenta el sistema informal. Cómo conservan los agricultores sus semillas en el trópico húmedo de Cuba, México y Perú, 73-84. https://lc.cx/IyhCO4
Wegier, A., Piñeyro-Nelson, A., Alarcón, J., Gálvez-Mariscal, A., Alvarez-Buylla, E. R., & Piñero, D. (2011). Recent long-distance transgene flow into wild populations conforms to historical patterns of gene flow in cotton (Gossypium hirsutum) at its centre of origin. Molecular Ecology, 20(19), 4182–4194. https://doi.org/10.1111/j.1365-294X.2011.05258.x

Dear colleagues, I thank you for the opportunity to participate in this Forum.

As a center of origin for critical crops such as maize and cotton, Mexico acknowledges that Living Modified Organisms (LMO) containing stacked events represent a critical risk to the conservation of biodiversity and to the food sovereignty.

Below, I present our position in response to the questions posed:

Question 1
The issue of living modified organisms (LMOs) containing stacked events presents serious challenges to existing regulatory frameworks, guidelines, and risk assessment methodologies. Traditionally, these frameworks were designed to evaluate single events and do not adequately address the potential synergistic or antagonistic interactions that can occur between multiple transgenes in a single organism. Studies such as Agapito-Tenfen et al. (2014) have demonstrated that stacking insecticidal and herbicide tolerance genes in maize significantly alters the proteome, generating unexpected metabolic profiles. This shows that combinatorial effects are not merely additive but can generate new risks.
Therefore, Mexico considers that stacked events must not be automatically deemed safe simply because their individual components have been approved previously. Each combination must undergo specific case-by-case risk assessments. Furthermore, we propose that this issue be incorporated into the update of Annex 3 of the Cartagena Protocol, and that an international program to strengthen capacities in risk assessment for stacked events be developed, particularly targeting developing countries and centers of origin.

Question 2
Among the specific challenges is the difficulty in identifying emerging risks resulting from new genetic combinations. For example, Zanatta et al. (2020) documented that genetically modified soybeans with stacked events showed severe disturbances in their defense systems after glyphosate application, a phenomenon not observed in single events. This indicates that combinations of transgenes can produce unforeseen cumulative or synergistic effects, both metabolically and ecologically.
Additionally, in Mexico, studies like Pérez-López et al. (2023) on insects associated with wild transgenic cotton show residual impacts on the gut microbiota of non-target organisms. These changes may alter local trophic networks and have long-term ecological effects, which current risk assessments often fail to consider adequately.
Detecting unintended combinations of transgenes in the environment, conducting case-by-case risk assessments and not assuming that a stacked event is safe simply because its individual events have been previously assessed, as highlighted by Mr. Onyeka Kingsley Nwosu ([#12275]). And lack of molecular detection capacity for stacked events in many developing countries.

Question 3
There are serious and potentially irreversible risks associated with living modified organisms (LMOs) containing stacked events, particularly in centers of origin such as Mexico. The introgression of transgenes into wild species has already been documented, as in the case of wild cotton (Gossypium hirsutum), where the expression of transgenic genes has been shown to alter native ecological interactions (Vazquez-Barrios et al., 2021;).
(i) There is strong evidence that stacked LMOs can cause serious or irreversible adverse effects on biodiversity. In Mexico, gene flow from genetically modified crops to wild relatives has been documented in both maize (Quist, D., & Chapela, I. H.,2001; Piñeyro-Nelson et al., 2009) and cotton (Wegier et al., 2011), compromising the genetic integrity of key species and affecting indigenous peoples' food sovereignty.
(ii) The introduction into the environment, either deliberate or accidental, is highly probable. Dyer et al. (2009) demonstrated how the exchange of seeds through traditional farming networks facilitates the dispersal of transgenes in traditional maize populations, a phenomenon likely to increase with stacked events.
(iii) There is also a real risk of transboundary spread of stacked LMOs, since seed and pollen flows do not respect political borders. Baltazar et al. (2005) showed that gene flow between maize and teosinte occurs naturally in Mexico, implying a serious risk of international propagation of stacked transgenes.
(iv) Stacked LMOs are already commercialized and used in various regions of the world, as evidenced by the European Food Safety Authority (EFSA) database and the reports from Testbiotech (2021), where almost half of the GMO authorizations correspond to stacked events.

Question 4
There are several relevant resources that could be adapted to address the need for deeper risk analysis of stacked events. Testbiotech (2021) emphasizes the need to incorporate specific methodologies to assess cumulative and combinatorial effects, moving away from evaluations that only consider single events. Similarly, the studies by Vázquez-Barrios et al. (2021) provide an ecological model for assessing the impacts of transgenes in wild populations.

Additionally, Mexico strongly supports:
The proposal of Mr. Nwosu ([#12275]) to prioritize capacity building, especially in developing countries, through special financing or triangular cooperation mechanisms. The intervention of Ms. Sol Parra Santos ([#12290]), recognizing the significant risks to food sovereignty and the traditional knowledge of local communities, particularly women. The critical assessment made by Mr. Christoph Then ([#12312]) regarding the urgent need to improve risk assessment methodologies for stacked events.
It is recommended that risk assessment methodologies not only address environmental and human health aspects in isolation, but also analyse socio-economic and human rights considerations, especially those of indigenous peoples, who are the guardians of biodiversity in centres of origin. We formally request the creation of a global capacity-building plan for the evaluation of stacked events, with a differentiated approach for developing countries and those that are centers of origin as in case of Mexico.

References:
Agapito-Tenfen, S. Z., Vilperte, V., Benevenuto, R. F., Rover, C. M., Traavik, T. I., & Nodari, R. O. (2014). Effect of stacking insecticidal cry and herbicide tolerance epsps transgenes on transgenic maize proteome. BMC plant biology, 14, 1-19. https://link.springer.com/article/10.1186/s12870-014-0346-8
Baltazar, B. M., de Jesús Sánchez-Gonzalez, J., de la Cruz-Larios, L., & Schoper, J. B. (2005). Pollination between maize and teosinte: an important determinant of gene flow in Mexico. Theoretical and Applied Genetics, 110, 519-526. https://link.springer.com/article/10.1007/s00122-004-1859-6
Dyer, G. A., Serratos-Hernández, J. A., Perales, H. R., Gepts, P., & Pineyro-Nelson, A. (2009). Dispersal of Transgenes through Maize Seed Systems in. https://psfaculty.plantsciences.ucdavis.edu/gepts/Dyer%20et%20al.%20Transgene%20dispersal%20&%20seed%20systems%20MEX.%202009.pdf
Pérez-López, J., Alavez, V., Cerritos, R., Andraca-Gómez, G., Fornoni, J., & Wegier, A. (2023). Residual effects of transgenic cotton on the intestinal microbiota of Dysdercus concinnus. Microorganisms, 11(2), 261. https://www.mdpi.com/2076-2607/11/2/261
Piñeyro-Nelson, A., van Heerwaarden, J., Perales, H. R., Serratos -Hernández, J. A., Rangel, A., Hufford, M. B., ... & Álvarez‐Buylla, E. R. (2009). Transgenes in Mexican maize: molecular evidence and methodological considerations for GMO detection in landrace populations. Molecular ecology, 18(4), 750-761. https://pubmed.ncbi.nlm.nih.gov/19143938/
Quist, D., & Chapela, I. H. (2001). Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico. Nature, 414(6863), 541-543. https://www.nature.com/articles/35107068
Testbiotech. (2021). Risk assessment of GE plants in the EU: Taking a look at the ‘dark side of the moon’. https://www.testbiotech.org/publikation/risk-assessment-ge-plants-eu-taking-look-dark-side-moon/
Vazquez-Barrios, V., Boege, K., Sosa-Fuentes, T. G., Rojas, P., & Wegier, A. (2021). Ongoing ecological and evolutionary consequences by the presence of transgenes in a wild cotton population. Scientific Reports, 11(1), 1959. https://www.nature.com/articles/s41598-021-81567-z 
Wegier, A., Piñeyro-Nelson, A., Alarcón, J., Gálvez-Mariscal, A., Álvarez‐Buylla, E. R., & Piñero, D. (2011). Recent long‐distance transgene flow into wild populations conforms to historical patterns of gene flow in cotton (Gossypium hirsutum) at its centre of origin. Molecular ecology, 20(19), 4182-4194. https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-294X.2011.05258.x
Zanatta, C. B., Benevenuto, R. F., Nodari, R. O., & Agapito-Tenfen, S. Z. (2020). Stacked genetically modified soybean harboring herbicide resistance and insecticide rCry1Ac shows strong defense and redox homeostasis disturbance after glyphosate-based herbicide application. Environmental Sciences Europe, 32(1), 1-17. https://link.springer.com/article/10.1186/s12302-020-00379-6

Cinthia Soberanes, México

Dear colleagues,

The discussion on living modified organisms containing stacked events is very rich and profund. I thank and support the information shared in comments #12291 #12306 #12312 #12325 #12327. In complement to these, I would like to point that since there are no experimental methods nor protocols for detecting GM events with multiple events, a largely un answered question in the scope of safety of the food and feed with stacked events.  Are there genomic/epigenomic/proteomic/metabolic effects-impacts due to the expression of recombinant regulatory elements derived from evolutionary distant species and with very different modes of action (i.e. RNA i) in a totally biologically different receptor organism?

Proportionately speaking, the issue of the current risk assessment guidance and protocols for single event LM organisms, resembles to that of the actual commercial formulation of herbicide -glyphosate- while the risk assessment and most of the toxicological studies were performed on the single active ingredient. Moreover, could these potential effects act synergistically, or in an additive manner to the already published effects of the merely process of genetic transformation (via transgenesis, via genomic edition)? This is a challenge for the current risk assessment guidance.

In a publication from our group (https://doi.org/10.1080/21683565.2017.1372841) aimed to detect and identify transgenic sequences in food products with maize as a principal ingredient, we identified several combinations of transgenes that are not registered in the Biosafety Clearing House (#12306). This issue couldn’t allow us to determine which specific GM maize (most probably, stacked events) were present in the food consumed by a large amount of Mexican population, which should be considered in the updating process of risk assessment guidance.

References:
Unintended Genomic Outcomes in Current and Next Generation GM Techniques: A Systematic Review https://doi.org/10.3390/plants11212997
The present and potential future methods for delivering CRISPR/Cas9 components in plants https://doi.org/10.1186/s43141-020-00036-8
The complex architecture and epigenomic impact of plant T-DNA insertions https://doi.org/10.1371/journal.pgen.1007819
Stacked genetically modified soybean harboring herbicide resistance and insecticide rCry1Ac shows strong defense and redox homeostasis disturbance after glyphosate-based herbicide application https://doi.org/10.1186/s12302-020-00379-6
Transgene Flow: Challenges to the On-Farm Conservation of Maize Landraces in the Brazilian Semi-Arid Region
https://doi.org/10.3390/plants11050603
Transgene behavior in Zea mays L. crosses across different genetic backgrounds: Segregation patterns, cry1Ab transgene expression, insecticidal protein concentration and bioactivity against insect pests https://doi.org/10.1371/journal.pone.0238523
Alterations in genetically modified crops assessed by omics studies: Systematic review and meta-analysis https://doi.org/10.1016/j.tifs.2022.01.002

Thank you very much to the  Secretariat, and the moderators.

The issue of stacked events is in my view, an increasingly important one given the trajectory of GMO crop cultivation towards stacked traits in recent years. The rapid proliferation of weed species developing resistance to herbicides since the adoption of herbicide tolerant crops, as well as rising insect resistance to Bt crops has resulted in a significant decline in single trait varieties, which are being increasingly replaced by stacked combinations of these two dominant traits.

Q1: This raises specific biosafety considerations with regard to the increasingly complex combinations of genes, traits as well as their associated herbicides. With stacked varieties currently entrenching the use of herbicide tolerant traits, the assessment of impacts of herbicide cocktails (and in combination with Bt crops) is an aspect that could indeed be strengthened. The rise in stacked varieties, as highlighted in previous posts, also poses challenges to detection and monitoring, with implications for trade, transboundary movement, farmer and consumer rights, food sovereignty and transparency with regard to traceability and labelling.

To answer question 3 (iv), industry figures for 2019 (ISAAA) state that 45 % of GM crops planted were stacked varieties (with 88 % (both stacked or individual), carrying herbicide tolerant traits).  Since 2019, more phase outs of single traits have occurred. For example, USDA figures for 2024 state that 83 % of corn crops are stacked traits  https://www.ers.usda.gov/data-products/adoption-of-genetically-engineered-crops-in-the-united-states/recent-trends-in-ge-adoption.

The increasing use of genome editing may also exacerbate both the introduction of stacked traits and the planting of herbicide tolerant crops. For example, in the case if INARI, they are developing edited traits into existing herbicide tolerant varieties. Their latest trial application in the EU (Spain) involves the expression of CRISPR machinery into glyphosate tolerant soybeans.

This raises specific biosafety considerations with regard to the increasingly complex combinations of genes, traits as well as their associated herbicides. With stacked traits  entrenching the use of herbicide tolerant traits, the assessments of impacts of herbicide cocktails is an aspect that could indeed be strengthened when assessing stacked traits.

Q2: The existing Guidance on Risk Assessment of LMOs and Monitoring in the Context of Risk Assessment, produced under the Cartagena Protocol on Biosafety provides a precautionary starting point for risk assessment, taking into consideration the additional complexities that are involved e.g. potential for increased genetic instability, potential for interactions of stacked genes, potential for combinatorial or cumulative effects, potential for stacked events to arise in the environment, and complexities around detection. However, as stated in the previous post, new developments suggest that it needs to be updated and expanded upon in order to address all the potential issues that stacked events raise, the increasingly complex combinations of introduced traits, techniques and processes involved, and species being manipulated, which are posing specific challenges to current protocols.

Moreover, in order to adhere to a precautionary approach, in line with Annex III of the Cartagena Protocol, unintended effects cannot be reliably excluded based on predicted intended effects of introduced traits. Any limitation or harmonisation of assessments to intended effects (e.g. by excluding potential interactions without empirical testing) has the potential to miss capturing the full spectrum of risks stacked traits pose to the sustainable use of biodiversity, taking into account human health.

Q3: Unintended effects could occur and indeed some have already manifested, with direct implications for biodiversity, e.g. synergistic and combinatorial effects in insecticidal ‘Bt’ crops that express multiple toxins in a single plant, including stronger effects on non-target organisms https://doi.org/10.3389/fenvs.2015.00071; altered expression levels of transgenes in stacked varieties in comparison to their respective parental lines, as well as altered compositional profiles has been observed https://doi.org/10.1186/s12870-014-0346-8, as well as increasing the application of herbicides in crops designed to be tolerant to more than one https://doi.org/10.13140/RG.2.1.1947.7847;

As such it is my view, that full risk assessments are warranted for all stacked products, including those that derive from parental varieties that have previously been approved, or vis versa, and that these are appropriately updated to incorporate the full spectrum of risks and uncertainties that have been so clearly highlighted in previous posts. Such assessments warrant updating in line with the risks and challenges raised in this very interesting discussion.

Thank you very much.

I would like to thank Ms Anita Anthonysamy for moderating this discussion, the Secretariat for facilitating it and especially the participants for their very informative contributions.
My name is Dr Werner Schenkel, I have been working as a risk assessor for the German Competent Authority for about 20 years and have been involved in Cartagena Protocol related issues since 2017.
As several contributions (#12275, #12283. #12306) rightly point out LMOs with stacked events have been in use and registered worldwide for many years and in many countries. This indicates, that there is considerable experience with this group of LMOs that have been successfully assessed with existing risk assessment frameworks. Their regulation and associated risk assessment requirements may differ between Parties to the Cartagena Protocol according to national circumstances.
In the European Union LMOs with stacked events are currently regulated as individual LMOs requiring a separate risk assessment. Implementing Regulation (EU) No 503/2013 details the requirements for applications for food and feed use of both single and stacked event. EFSA Guidance on risk assessment of genetically modified plants containing stacked transformation events (EFSA 2007) has been replaced by relevant provisions in the “Guidance for risk assessment of food and feed from genetically modified plants” (EFSA 2011). In addition, risk assessment aspects of LMOs with stacked events are covered by the “Guidance on the environmental risk assessment of genetically modified plants” (EFSA 2010) and the “Guidance on the environmental risk assessment of genetically modified animals” (EFSA 2013).
I assume that the challenges and specific issues that arise in the risk assessment of LMOs with stacked events have been observed and subsequently largely addressed by many Parties to the Protocol according to their national circumstances and regulations.
I do not believe that there is a lack of guidance on the risk assessment of LMOs with stacked events and therefore the development of such guidance should not be a priority.

Werner Schenkel
Germany
Implementing Regulation (EU) No 503/2013: https://bch.cbd.int/en/database/BCH-LAW-EU-115173-1
EFSA 2011: https://bch.cbd.int/en/database/BCH-LAW-EU-115262-1
EFSA 2010: https://bch.cbd.int/en/database/BCH-LAW-EU-115257-1
EFSA 2013: https://bch.cbd.int/en/database/BCH-LAW-EU-115260-1

Dear colleagues,

I am Clementina Johnson from Nigeria.

In contributing to the ongoing insightful discussions, Q1,2 and 3 in particular, from some Risk Assessment Guidelines available, LMOs with stacked events are evaluated on: case by case basis, by the adoption of previous safety studies of the individual traits or by the evaluation of  unapproved trait(s) in the stack. Going forward, the provisions of these regulatory regimes requires more precautionary measures to avert  for instance indications of increased spread, establishment and invasion in natural environments or other areas outside agricultural areas resulting from spillage of seed during transport or processing. In areas where there are natural wild relatives in which hybridization would occur and cultivation of maize is not limited for example, what wholistic control measures are or should be put in place?

Presence of multiple transgenes from the context of complex molecular effects either in part or whole can be difficult to trace or detect. There are various factors which negates existing Risk Assessment Guidelines on LMOs with stacked events, some of these factors include but not limited to: ecological, regulatory, trade disruptions.

Summarily, from whatever angle a risk is assessed with the use of LMOs with stacked events, the call is for the review of existing guidelines to comprehensively address risk assessment, regulatory oversight and environmental safety. The  effectiveness of these guidelines lie  in consistency across Regulations  and the evaluation of stacked events not just individually but also in combination, considering potential interactions between traits.

Thank you.

Dear colleagues,

Regarding question 3.iv, many stacked events in plants have been authorised for food and feed in the EU. The complete list is available in the Community register of GMO at this link: https://ec.europa.eu/food/food-feed-portal/screen/gmo/search.

Best regards,
Alexandre

My name is Samson Simon, I work for the German Federal Agency for Nature Conservation (BfN) as a biosafety expert.
For my comment I will differentiate between breeding stacks (BS) and stacked events (SE; i.e. adding several traits or proteins as single event).

1. Assessing combinatorial effects from single or breeding stacks is challenging and would strongly benefit from harmonization for all relevant experiments and protection goals.


For SE the case-by-case approach is inevitable (Schrijver et al. 2015) and the risk assessment needs to focus – amongst other things – on unintended effects from the genetic modification. For SE the combinatoric effect of the different traits and changes have to be assessed. As many phenotypic tests are based on plant material (e.g. animal feeding studies for human health effects of effects on livestock) the necessary experiments will have to be carried out.
In the case of BS the situation becomes more difficult, because by default experiments with the single events usually exist. Here regulators may have different opinions on whether and which toxicity, ecotoxicity or phenotypic tests may be necessary for BS crops. So, when different procedures exist cross border transport (by purpose or by accident) may pose a challenge and this issue has already been raised by Moussa Savadogo #122823.
Scientific evidence suggests that SE and BS need a separate assessment for toxins. The reason is, that combinatoric effects, at least for Bt proteins, cannot be predicted sufficiently without testing (Hilbeck und Otto 2015; Schrijver et al. 2015). In addition, there is no guidance on how to specifically test for combinatorial effects. Such a guidance, which needs to be specified according to the different experimental setups and protection goals (human health, animal health, non-target organisms, phenotype and invasiveness).
I agree with Sarah Agapito-Tenfen #12306 that that the lack of risk assessment concepts and methodologies for combinatorial effects challenge the current risk assessments.

References:
Hilbeck, Angelika; Otto, Mathias (2015): Specificity and Combinatorial Effects of Bacillus Thuringiensis Cry Toxins in the Context of GMO Environmental Risk Assessment. In: Frontiers in Environmental Science 3, S. 71. DOI: 10.3389/fenvs.2015.00071.
Schrijver, A. de; Clercq, P. de; Maagd, R. A. de; van Frankenhuyzen, Kees (2015): Relevance of Bt toxin interaction studies for environmental risk assessment of genetically modified crops. In: Plant Biotechnology Journal 13 (9), S. 1221–1223. DOI: 10.1111/pbi.12406.

3. Stacks may increase environmental load without need to do so
Higher order stacks containing insect resistance and herbicide resistance traits increases the environmental load with pesticides (both plant integrated and externally applied). The use of multiple herbicide resistant, multiple insect resistant crops can be counteracting to efforts in minimizing effects on the environment and on biodiversity. In many cases multiple resistances may not be needed or the internal concentrations of pesticides (pore-forming toxins such as Bt, VIP and others) are many times higher than required for a sufficient control of the target pests. So, guidance shall ideally consider best practices on how not to overuse the pesticides contained in current and future stacked events marketed in e.g. maize or cotton.

POSTED ON BEHALF OF MS. PERLA PATRICIA GODOY, ARGENTINA:
*****

No me voy a meter con lo que dice al final sobre los pueblos indígenas, porque desconozco esa realidad y posiblemente (o no) sea así. Pero todo lo que dice antes, (No sé en otros marcos regulatorios) pero en Argentina se tiene todo en cuenta. Si en otros países evalúan distinto que Argentina (si están evaluados los individuales no evalúan el acumulado) de todos modos nunca hubo ningún incidente desde fines de los 90 a la fecha. Cuando se mete con EG, bueno, si es GM se evalúa bajo la normativa de OGM, si no bajo las normativas de No GM

Dear All,

First, I apologize for the delayed response.

My name is Eder Toppa, and I have been working at the Brazilian Ministry of Agriculture and Livestock for the past decade. I currently serve as the Head of the Biosafety Service and am a member of the National Biosafety Commission.

This topic should be discussed during the third week of the online forum on simplified procedures related to Article 13 and agreements and arrangements under Article 14.

For now, single GM events are subject to rigorous risk assessments, including molecular characterization, toxicological and allergenicity evaluations, and environmental impact studies. These assessments provide a robust baseline for evaluating stacked events, reducing the need for redundant testing when no new genetic material is introduced. The traits in stacked GM plants typically function independently, with no evidence of significant synergistic or antagonistic interactions that would alter their safety profile. The safety of each single event is well-characterized, and combining them does not generally introduce unforeseen effects.

Requiring full risk assessments for stacked GM plants — equivalent to those for single events — increases regulatory costs and causes delays without proportional safety benefits. Streamlining assessments by leveraging existing data from single events can facilitate innovation and market access while maintaining safety standards.

In summary, we do not need new guidelines to assess GM stacked events; rather, we should focus on streamlining their risk assessment based on Article 13 and Article 14 of the CP.

Eder Victor Braganti Toppa
Federal Inspector
Head of the GMO Biosafety Service
Plant Health Department
Ministry of Agriculture and Livestock

First, let me thank Ms. Anthonysamy for moderating the discussion and the CBD Secretariat for hosting it an allowing me to contribute.

As many have pointed out, stacked events (particularly for LMO plants) have been the rule, rather than the exception in regulatory submissions for quite some years now.  Regulatory decisions taken by Parties and non-Parties are available for reference and review, and of course countries apply their own domestic regulatory frameworks, including their risk assessment requirements and their domestic policy on whether to review single traits or combinations of traits.  What we don't seem to have are examples of emergent risks being realized in stacked trait LMOs that are not predicted (or predictable) in risk assessment for single traits. 

I see quite a lot of speculative risk being mentioned here - that is the assertion that there may be things we don't know and that therefore existing risk assessment frameworks are inadequate - a lot of appeal to "complexity," without attempting to explain why this complexity matters and how we might address it in risk assessment.  Frankly, none of these speculations are different than the same posts that might be made by the same individuals or organizations for risk assessment of single event LMOs (and have, numerous times in the past).  Trying to construct guidance from a foundation of speculative risk is not a productive activity.  Guidance must consist of the identification of a concern (i.e. a potential harm - not a potential phenomenon), identification of how information might be collected to inform an assessment of the likelihood that that potential harm would be realized, and then a consideration of when or how such an assessment is required (i.e. is the assessment proportionate to our understanding of the potential harm).  I am not seeing new ideas that have not previously been considered during problem formulation for LMOs, including LMOs with stacked events.

I would therefore strongly endorse the conclusion offered by post #12336 that pursuing guidance on LMOs containing stacked events should not be a priority at this time.

Dear participants,

My name is Luciana Ambrozevicius, I´m a regulator and a risk assessor at the Brazilian Biosafety Commission. Thank you for the opportunity to participate in the on line forum. For the proposed topic “LMOs containing stacked events” I would like to make the following considerations regarding the criteria for new topics established in the annex I to decision CP-9/13:

1. How does this topic potentially pose challenges to the existing risk assessment frameworks, guidance and methodologies? Do solutions exist?
I do not see any potential challenge for stacked events that are being already evaluated by decades, approved at different countries, consumed by billions and cultivated in million of hectares.

2. What could be the specific challenges to related to this issue?
I agree with #12283 about the utility of compilation of the existing individual guidelines about RA of stack events. Although it’s not specifically about regulatory approaches for stack events, there is an initiative at APEC about the "Regulatory Cooperation for Biotechnology" that can be mentioned, including a case study database - https://biotechpolicyportal.org/case-studies/. Regarding on the benefits of such approach it can be highlighted for developing countries: efficiency gains in the assessment process; improved synchronization of authorizations; mutual capacity building and learning and a regulatory environment that supports innovation. Those benefits could also be extrapolate to a simplified procedure for stacked events.

I also agree with #12287 “that in some countries the approval of individual events leads to the automatic approval of stacked events containing them, while in others, separate risk assessments are conducted for each stacked event even if the individual events are already approved, it may be more relevant to focus on discussions around regulatory harmonization”.

In this case the specific challenge related with RA can’t be identified, the challenge is related with regulatory harmonization. The countries have access to information and experience with RA of stacked events, further guidance will add no value in the current context.

3. What are the specific issues concerning this topic?
There are no specific issues as the experience gained through decades from assessing several traits or events (i.e., different GMO modifications) and the same event multiple times (i.e., repeated assessments of a particular GMO across different contexts) could be used by countries. The implementation of a simplified procedure for those events could be pursued based on the assessment of effects of the potential interactions between the newly expressed proteins when the GM parental lines have already been positively assessed for their biosafety. The experience and rational of RA of stacked crops can be used to assess the risk of other stacked organisms with history of safe use.

4. Are there existing resources on similar issues that can address this issue or resources that could be adapted to address this need?
There are various sources of regulatory approaches to stack events evaluations at different countries   (BCH, FAO GM Foods Platform, BioTrack Product Database) and also scientific articles including: 
- Comparing agronomic and phenotypic plant characteristics between single and stacked events in soybean, maize, and cotton (DOI:10.1371/journal.pone.0231733)
- Advancing ecological risk assessment on genetically engineered breeding stacks with combined insect-resistance traits (DOI:10.1007/s11248-019-00185-8)
- Evolution of risk assessment strategies for food and feed uses of stacked GM events (DOI: https://doi.org/10.1111/pbi.12551)    
- Plants with stacked genetically modified events: to assess or not to assess? (DOI:https://doi.org/10.1016/j.tibtech.2013.12.001)   
- Risk assessment of GM stacked events obtained from crosses between GM events (DOI: https://doi.org/10.1016/j.tifs.2006.09.002)   
- Regulatory policy on genetically modified breeding stack in key countries and the current status in Korea (DOI: 10.1007/s10068-021-01004-9)   
- Consideration of familiarity accumulated in the confined field trials for environmental risk assessment of genetically modified soybean (Glycine max) in Japan. (DOI: https://doi.org/10.1007/s11248-020-00193-z(0123456789().,-volV()0123456789().,-volV)  
- Modernizing and harmonizing regulatory data requirements for genetically modified crops - perspectives from a workshop (DOI: https://doi.org/10.3389/fbioe.2024.1394704)  

Based on the above, I consider that this topic does not fulfill the criteria established for the process for the identification and prioritization of specific issues regarding risk assessment of living modified organisms, according with the decision CP-9/13.

Best regards,
Luciana P. Ambrozevicius

Dear Colleagues,

First, let me thank the moderators for their work in moderating the discussion, and the CBD Secretariat for hosting it and allowing me to contribute.

My name is Andrés Frankow. I am a biologist, and I have been working in risk assessment of genetically modified organisms since 2004. I am currently part of the Coordination of Innovation and Biotechnology in the Secretariat of Agriculture, Livestock, and Fisheries.

Given that there are well-established regulatory frameworks, guidelines, and methodologies for the assessment of living modified organisms (LMOs), no significant challenges have been identified regarding this topic.
There are specific considerations for the assessment of stacked events, both within environmental risk assessment and the analysis for human and animal consumption.
Stacked events and their derived products have been present on the market for many years, and no issues have been reported by competent authorities regarding impacts on agroecosystems or consumers.
Robust and updated regulatory frameworks provide for science-based, case-by-case analyses for each of the stacked events and the genes involved. The evaluation focuses on the potential interaction between the expression products present in the stack, including all possible combinations in intermediate stacked events.
Environmental risk assessments for stacked events are specific in their approach: while they draw upon information from individual event assessments, they focus on a case-by-case analysis of potential interactions.
All evaluations must be science-based, utilizing appropriate problem formulation and the correct risk hypothesis, while identifying the values to be protected.
This approach ensures a thorough and science-based evaluation, contributing to the protection of biodiversity, human health, and the environment.

Therefore, there is no evidence to support the need for developing a specific guide on this topic.

Best regards,

Andrés

Dear colleagues
Thank you for the opportunity to contribute to this forum. Below are my comments in response to Questions 1, 2 and 4.

Question 1
Living modified organisms (LMOs) containing stacked events are increasingly being developed and used in crops globally, highlighting the importance of continued efforts to regulate and assess potential risks to human health and the environment. Given the various methods (molecular and breeding) by which stacked traits can be introduced, a case-by-case risk assessment remains essential, which is currently an established principle within the risk assessment framework in Annex III of the Cartagena Protocol on Biosafety (CPB).

Living modified organisms (LMOs) containing stacked events introduce additional layers of complexities due to potential interactions between gene products, additive or synergistic effects, and cumulative environmental exposures. However, these complexities do not undermine the adequacy of the existing risk assessment framework outlined in Annex III of CPB. The methodology stipulated in Annex III of CPB explicitly acknowledges that “the process of risk assessment may… give rise to a need for further information about specific subjects, which may be identified and requested during the assessment process”. This provision is particularly relevant in the context of stacked events, where the need to evaluate potential gene-gene interactions has been identified. Importantly, the request for such additional information does not modify the established structure of risk assessment—which includes hazard identification, hazard characterization, exposure assessment, risk characterization, and risk management. Instead, the evaluation of potential interactions between gene products aligns with steps 8(b) and 8(c) of Annex III and serves to enhance the robustness of risk characterization under step 8(d).

Question 2
In view of the above, the specific challenge related to LMOs containing stacked events borders around how to determine, assess, understand, and prevent any potential interactions between/among gene products including assessment of modes of action, metabolic pathways, cellular localization, and potential structural effects, epistatic effects, expression studies, bioinformatics analysis, and the characterization of any identified effects (synergistic, additive, antagonistic, or silencing).
The challenge is due to the following:
• Limited expertise and infrastructure in some countries to conduct nuanced assessments of complex stacked events.
• Lack of harmonized data requirements and assessment methodologies across countries, leading to inconsistent regulatory decisions.
• Data access issues, where risk assessors may lack full access to proprietary studies submitted to other regulatory authorities.
• Limited post-market monitoring capacity in some countries, limiting the ability to detect and respond to potential unintended effects over time.

Question 4
Yes, some countries have established specific guidelines for the regulation of LMOs containing stacked events, recognizing the growing global trend in their development, evaluation, approval, and use in crop production. While some countries have established guidelines on this subject matter, including African countries such as Ghana, Kenya, Malawi and Nigeria, with knowledge-based advisory support from AUDA-NEPAD; experience among others is still evolving, with some in the process of drafting guidelines. As such, this topic has been identified, by AU member states, as a priority area, to help build regulatory capacity and ensure informed decision-making.

AUDA-NEPAD continues to provide support to AU member states through services including capacity building, development of risk-proportionate policy guidelines and enabling access to a suite of relevant resources. A range of resources is available globally that offer guidance and set important precedents for the risk assessment of stacked events. These can be leveraged by other countries to inform and strengthen their regulatory approaches. Additionally, there exist validated methodologies for assessing potential interactions between gene products as well as related peer-reviewed publications.

While Annex III offers a robust and adaptable framework for risk assessment, it would be highly beneficial to encourage countries with established guidelines and experience in assessing stacked events, as well as researchers and developers familiar with applicable methodologies, to share relevant resources and practical insights. This would enable Parties with limited expertise or experience to learn from existing practices and, where appropriate, adapt them to their national contexts. In addition, Parties should be encouraged to share any challenges, limitations, or gaps encountered in the process of developing or regulating LMOs containing stacked events. Such experience-sharing initiatives should be augmented with targeted capacity-building efforts, particularly for Parties that are still developing their regulatory competencies on this subject matter.

Modupe Adeyemo
AUDA-NEPAD

Dear participants,

Thank you for the opportunity to contribute to these discussions. I wish to address the questions posed by our moderator from the perspective of technology developers that have experience with the commercialization of biotech crops with stacked traits. As pointed out by previous contributors (#12275, #12283, #12336, #12351), LMOs containing stacked events are not new – biotech crops with stacked traits (referred to as “stacked trait products” or “stacks”) have been in commercial production for at least 30 years. Stacks therefore do not present new challenges for risk assessment. We agree with previous contributors that the criteria of CP-9/13 are not fulfilled for with this topic (#12362), that there is no demonstrated need for the development of additional voluntary guidance materials (#12236, #12350, #12362), and that many of the “challenges” raised are speculative and repetitive of that raised over the past 30 years in relation to biotech crops more generally (#12351).

Stacks are developed in two ways: (1) by conventional plant breeding, where parents containing single events are crossed resulting in progeny that contain two or more events (also known as “breeding stacks”), or (2) by using molecular methods to simultaneously or sequentially introduce two or more traits (also known as a “molecular stacks”) (Goodwin et al 2021: available at https://croplife.org/wp-content/uploads/2021/01/Stacked-Traits-Goodwin-et-al.pdf). Some of the earliest commercial biotech crops (from the mid-1990s) are molecular stacks, e.g. constructs containing two genes, one conferring herbicide tolerance and the other conferring insect resistance. Breeding stacks have been in commercial production since the early 2000s.

To commercialize these products, technology developers seek a “global“ registration – this involves regulatory assessment and authorization in the jurisdictions where the crops are intended to be cultivated, as well as the jurisdictions where the products will be use in food, feed or processing. These are different types of regulatory assessments and authorizations, with some of the contributions in this online discussion combining elements of both – we limit our comments to environmental risk assessment per the applicable Cartagena Protocol decisions (CP-11/7 and CP-9/13).

Questions 1 and 2
Environmental risk assessment for stacked trait products is undertaken using established risk assessment approaches, consistent with the science-based case-by-case principles of Annex III of the Cartagena Protocol. Given their long history of safe use, specific challenges are not related to the conduct of the risk assessment per se, but rather the lack of harmonization across jurisdictions in what requires assessment (as mentioned by #12287, #12290, #12362).

More specifically, whether a stacked trait product requires environmental risk assessment depends on how the regulatory authority defines a new event, with this is inconsistent across jurisdictions. For example, certain jurisdictions do not require another risk assessment for a stack where the parental events have already been assessed and approved as the risk of the stack is considered to be equivalent to that of the parental events, while other jurisdictions may require a new risk assessment for the stack as though it is a new event, or they may require a streamlined risk assessment.

In our view, where a stack contains events that have been assessed and approved by regulatory authorities previously, additional regulatory assessment is not required when the potential for adverse trait interactions can be scientifically excluded. Any additional regulatory assessment (and associated data requirements) should be predicated on a plausible and testable hypothesis of the potential for adverse gene or gene product interactions specific to the modes of action of the single event components. This requires a problem formulation approach to risk assessment, which can address "challenges" listed in previous posts in this discussion (e.g. #12291, #12325) in a scientifically sound manner, consistent with Annex III.

In response to the challenges mentioned regarding detection methods for stacks (raised in #12306, #12312, #12325, #12327, #12333), we would like to point out that for commercial events full descriptions of validated DNA-based methods are provided by developers of biotech crops in a searchable database hosted by CropLife International: https://detection-methods.com/. These methods are developed for the detection of single events (which can include molecular stacks), and they continue to be the method of choice for breeding stacks (see explanation in: https://detection-methods.com/content/uploads/Implications-of-using-PCR-detection-methods-with-stacked-products_Final-draft.pdf).

Question 3
Biotech crops containing stacked events are in commercial production around the world and have been for 30 years. Collectively, these represent more than 360 “cultivation” regulatory authorizations granted in 18 countries (AgbioInvestor GM Monitor, available at https://gm.agbioinvestor.com/approvals-database, accessed 27 April 2025). While not the scope of the present discussion, elements relevant to food safety assessment have been raised so it is worth pointing out that over 1100 food/feed safety assessments and regulatory authorizations have been granted for stacks (AgbioInvestor GM Monitor, accessed 28 April 2025). These figures demonstrate that stacks are not new to risk assessors, and that risk assessments have been conducted for stacks for as long as biotech crops have been commercialized.

Question 4
Given the experience with stacked biotech crops there are ample relevant resources, and we emphasize the many published risk assessments by regulatory authorities in the jurisdictions where biotech crops are cultivated. The wealth of knowledge and experience that has accumulated over the past 30 years for plants can guide risk assessment for other types of organisms, and the work of some of these regulatory authorities extends beyond plants to other types of organisms that have been mentioned in this context.

Other useful resources include the OECD 2023 Consensus document on Environmental Considerations for Risk/safety Assessment for the Release of Transgenic Plants (available at https://one.oecd.org/document/ENV/CBC/MONO(2023)30/en/pdf) that was developed by risk assessors and describes a problem formulation approach. This approach is also described in the “Additional voluntary guidance materials to support case-by-case risk assessments of living modified organisms containing engineered gene drives” (CBD/CP/MOP/11/9; available at https://www.cbd.int/doc/c/175e/90b0/89c0c71660cccc1539adf34f/cp-mop-11-09-en.pdf). While the latter was developed for a particular application in a living modified insect, the approach is broadly applicable to any type of organism.

Dear Participants,

Thank you kindly for your valuable interventions and active engagement during the first week of discussions.

Week 1 of the Open-Ended Online Forum is now closed.

Please return for Week 2. The discussions will open in an hour (12 pm (noon) Montreal time).

Best regards,

The Secretariat