Risk assessment and risk management of LMOs with stacked genes or traits (part I)

In Austria we have recently undertaken a project on the risk assessment of LMO products with stacked events. In this context we have identified some shortcomings concerning the approach to and the available guidance for the assessment of such applications. Most of the controversy is about requirements in addition to the data submitted for the parental LMO events.

Our study identifies open questions and the need for further clarification and research concerning the risk assessment of stacked events (Spök et al. 2007).

Some of the key issues identified appear to be less controversial or not controversial at all:
• Risk assessment of stacked events can draw on the assessment of parental LMO plants and should consider the results of such assessments.
• Requirement for a molecular characterisation in order to confirm the preservation of the inserted traits and to compare the expression of transgenes between parental LMO events and the stacked event.
• Comparative analysis of the stacked event including a standard set of compositional and agronomic parameters.
• Need to consider any potential interaction of combined traits in the stacked events.

On the other hand, some issues are debated more intensively and require further clarification, discussion and research:

• Can the methods used for molecular characterisation be considered sufficiently precise to achieve demonstration of identity with parental events?
• Should the parental LMO events be included in the comparative analysis of plant compounds, agronomic traits, and expression of the transgenes along other comparator lines?
• How should the potential interaction of traits be assessed and at what levels (genetic, protein, metabolic)?
• What would trigger the need to conduct whole food toxicity studies with the stacked event?

At present the latter questions are neither appropriately addressed in the risk assessment dossiers nor covered adequately by available guidance documents. The Guidance Document available at the EU level is the EFSA guidance document on the risk assessment of stacked events (EFSA 2007). According to this document a case-by-case flexibility as to which requirements listed for the risk assessment of single event LMOs would apply for the assessment of stacked events is necessary. The assessment should focus on issues related to
• Stability
• Expression of traits
• Potential synergistic or antagonistic effects resulting from the combination of traits.

Specifically the last issue presents challenges regarding experimental investigation and appropriate methodology. In current practice such assessments only rely on the theoretical discussion of the lack of probability of interactive effects in stacked events , although there is evidence from the published literature for such interactive (synergistic, antagonistic) effects, for example of plant-produced proteins and toxins such as Bt toxins (Spök et al. 2008). From a precautionary point of view, it is necessary to conduct specific assessments with the whole stacked event LMO plant in order to test for potential interactive effects of the traits on the molecular, the protein or the metabolic level. The parental, single event LMOs should be used as comparators in these tests in addition to a non-LMO comparator.

In notifications of stacked events introduced in the EU the stacked event itself is only assessed experimentally in the comparative analysis of plant compounds, the characterization of agronomic traits and the expression of the transgenic traits. Controversy exists with regard to the need for further whole plant studies for food safety and environmental aspects (for details see Spök et al. 2007).

In the context of the large numbers of combinations of different proteins, toxins or other gene products which can be obtained by gene stacking, the general aim should be to assess potential effects of combined traits in stacked events in a case-by-case assessment under realistic environmental conditions. This objective should be supported by detailed and scientifically robust guidance.


EFSA (2007). Guidance document of the Scientific Panel on genetically modified organisms for the risk assessment of genetically modified plants containing stacked transformation events. Adopted on 16 May 2007. The EFSA Journal 512, 1-5.

Spök A., Dolezel M., Eckerstorfer M., Freigassner M., Gaugitsch H., Heissenberger A., Karner S., Klade M., Proksch M., Schneider L., Treiber F. & M. Uhl (2008a). Assessment of toxic and ecotoxic properties of novel proteins in GMOs. Bundesministerium für Gesundheit, Familie und Jugend, Forschungsberichte der Sektion IV, Band 1/2008.

Spök A., Eckerstorfer M., Heissenberger A. & H. Gaugitsch (2007). Risk Assessment of stacked events. Untersuchungen zur Risikoabschätzung von „stacked events“. Bundesministerium für Gesundheit, Familie und Jugend, Forschungsberichte der Sektion IV, Band 2/2007.

This is more response to the previous comments.

The evaluation of the stacked lines shall be principally with the RA for the original LMOs.
But again, the reproductive nature and genetics of the species, especially plant species with outcrossing and heterozygous status of the parental lines could have a large segregation besides the transgene per se.  With the new genotype together with the transgene, assessments shall be made accordingly to if  trasngene x new background genotype change could alter the traits including stability. My comments may correspond mostly with the Dr. Helmut, but my point could be more on plant breeding with outcrossing species.  While it is true that focus shall be made on the behavior of transgene in the stacked line, always genetic segregation is the major basis of plant breeding, attention shall be made on the trait performance in the progeny line (stacked line).

regards,

As already pointed out by others on this discussion group, there are some key issues that are mostly agreed upon and then again there are other key issues that still require further discussion and probing.

I want to add some arguments to the second group of key issues.

There has been a tendency to focus on trait-specific risk assessment and not paying sufficient attention to the assessment of unintended changes (both DNA level as well as metabolic pathway level) or looking for unpredicted or unpredictable effects. This includes for example the assessment of transformation induced mutations (both genome-wide mutations as well as insert site specific mutations), which have subtantial potential for giving rise to unpredicted effects and where further research is still urgently required (Wilson et al. 2006).

Concerning gene stacking I regard it as crucial to not only look at the traits as a matter of adding them up individually, but also to assess what mutational burdens have been introduced through/by the stacking process (whether this is by additional transformation or cross breeding of previous GM events).

Furthermore, toxicology has taught us, that if consuming substance A is safe when eaten on its own and substance B is safe when eaten on its own, a completely different scenario may arise when substance A and B are consumed together – they may indeed be toxic together. Whilst such synergistic effects are widely recognised now in toxicology and even made use of in vaccines and chemotherapy when utilising adjuvants, we seem to find it difficult to apply the same standards to genetically engineered organisms (LMOs).

Until otherwise proven safe, our starting point for risk assessment of stacked genes should be treating the particular LMO as a brand new LMO and carry out risk assessment accordingly, including metabolic studies, field trials and feeding trials. Of course, the assessments of the parental lines are going to be of use in this process, but only as a starting point.

With regards to the stacking of multiple insecticidal genes, there is also the question of cumulative affects as well as increased or new allergenicity, for both ingestion as well as inhalation or touch.


Literature cited:

Wilson AK, Latham JR & Steinbrecher RA (2006). Transformation-induced mutations in transgenic plants: Analysis and biosafety implications. Biotechnology and Genetic Engineering Reviews 23: 209-234.