1. Use of reference materials As Calibrators For metrological traceability As QC control To ensure international comparability –Only common reference point for GM analysis –Only common item internationally and independent from technology used

2. Test methods DNA Protein RR-tolerance Desired effect Cause Means PCR (polymerase chain reaction) based on DNA analysis (the genes) all Protein-based techniques (ELISA, lateral-flow devices) most of current AgBiotech traits Based on phenotype (Bioassay) herbicide tolerances

3. Choice of reference materials for GMOs Artificial materials –DNA: carries information of desired trait Genomic DNA Plasmids PCR amplicons –Proteins: will produce the desired trait in plants Native plant proteins Bacterial proteins Peptides Real-life materials –Seeds: carry desired trait –Grain: carries desired trait

4. Proteins as reference materials Protein based methods are best used in unprocessed materials Native proteins –Proteins as expressed in plants –Expression level (amount produced in plants) is influenced by environment Bacterial protein/peptides –Very pure materials –Differ from native protein Affinity of antibodies Epitopes still identical? No labeling regulation sets limits in units of protein quantities

5. DNA-based standards Genomic DNA –Native DNA –Stability and purity are critical parameters –Correlation to regulations (weight-%) is unclear Plasmids, PCR amplicons –Easy to produce –Differences in amplification efficiency Purity Length Circular/linear –Correlation to regulations (weight-%) is unclear

6. Seed grain based reference materials Seed is commercialized commodity Grain is the first food product BUT –Genetics depend on plant Soybeans carry trait on both chromosomes (homozygous) all grain will be 100% GMO Corn carries trait only on one of two chromosomes (heterozygous): hybrid Grain will only be 75% GMO XX x XX XX + 2XX + XX (still 50% on DNA level) Tissue-specific genetics Seed based reference materials require huge amount of materials (1000 kernels ~ 300g) Seed based materials are produced for weight-%

7. Whole seed kernels as reference materials Purity of negative and positive material is critical No such thing as 0% or a 100% (sampling error) Probability that pool contains at least one positive kernel

8. Seed pools of 1% as target concentration

9. Seed kernels vs. flour Kernel based reference materials –Are closest to reality –Require extreme pure materials –Require excessive amount of seeds Flour based reference materials –Derived from seeds: commercial commodity –Require large amount of pure materials –Are not always easy to produce, especially for oilseeds –Process history and small and uniform particles are critical issue –Stability of analyte is critical –Are convenient to handle

10. Flour-based materials Are closest to reality (materials on the market) Analyte is likely to be affected by processing Different production conditions may easily lead to differences in measurement results Reproducibility is key requirement Are best suited to promote international comparability of measurements results Sampling considerations apply here as well –Particle size distribution needs to be known and adjusted to the recommended quantities (function of concentration)

11. Interpretation of test results How to relate test results to labeling provisions? Test results –Protein in absolute quantities –DNA in absolute or relative quantities Labeling provisions –Labeling provisions in % –No threshold (China) –No units explicitly given (Europe 1%) prevailing assumption weight-% under dispute –Weight-% (Australia)

12. More challenges Biological factors: –Protein: Expression level Degradation –DNA Zygosity/Hybrid status (Selective) Degradation (Specific) Inhibition Tissue specific issues: apoptosis, tissue-specific genetic factors, endoreduplication Measurement uncertainty –Exponential systems –Measurement unit (amount of DNA, %-DNA)

13. Stacks Breeding stack: –two parent plants carry different trait –Through allelic recombination, two traits may be on one chromosome Breeding stacks cannot be distinguished in flour from a mixture of individual events, only by single kernel analysis (e.g., YieldgardPlus) Vector stack: –Plant is transformed twice with different traits –Gene cassette contains two traits (one transformation) Vector stacks are new events easily distinguishable by PCR (e.g., BT11)

14. Need for standardization International harmonization of validated detection methods is needed Validation shall encompass all analytical procedures (incl. extraction) Validation protocols for DNA extraction need to be established Validation shall occur according to international guidelines (e.g, Harmonized protocol) Laboratories should work in compliance with ISO 17025

15. Reference materials –Build consistent basis for global harmonization –Provide basis of comparability of measurement results Reference materials need to be harmonized on an international level –Duplication is redundant and should be avoided –Process conditions may affect the analyte in a reference material Bureau of weight and measurements (BIPM) has set up a mutual recognition agreement amongst different Institutes

16. Conclusions Reference materials will continue to be the cornerstone to international comparability of measurement results The MRA of BIPM should provide an adequate measure to cross-recognize reference materials and avoid redundancy and duplication It is important that reference materials are as close as possible to the matrix/analyte to true samples We believe that only seed-based reference materials are an objective and science-based approach