1. www.jrc.ec.europa.eu Serving society Stimulating innovation Supporting legislation 1 The EU Reference Laboratory for GMO detection Missions and interactions with Member States reference laboratories Joint Research Centre The European Commission’s in-house science service

2. The Joint Research Centre (JRC) The JRC is a Directorate-General of the European Commission under the responsibility of the European Commissioner for Science and Research

3. JRC structure: 7 institutes in 5 Member States to provide customer-driven scientific and technical support for the conception, development, implementation and monitoring of EU policies…….the JRC functions as a reference centre of science and technology for the Union.

4. 4 European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) 1.Regulation (EC) No 1829/2003  validation of methods for detection and quantification of GM events as part of the authorisation dossier 2.Regulation (EC) No 882/2004  official controls applied to ensure the verification of compliance with feed and food law

5. Official mandate in the EU regulatory process, based upon recognition of existing expertise in JRC/ENGL Operations are carried out in parallel with the European Food Safety Authority (EFSA) It has a central role in assessment and validation of methods that are “fit for the purpose of regulatory compliance” It has a key role in disputes between MS and in response to emergencies Prepares and distributes control samples to NRLs The EU-RL GMO: tasks as outlined by Article 32 of Reg. (EC) No 1829/2003

6. 6 The EU-RL GMO: tasks as outlined by Article 32 of Reg. (EC) No 882/2004 Assisting the National Reference Laboratories (NRLs) in their duties to monitoring the European market in a context of health and consumer protection with three main objectives: Solving scientific issues related to harmonisation and communication of scientific data among laboratories Monitoring the quality levels of the analytical laboratories for GMO detection Building capacities through training, workshops and any common scientific normative tool available

7. 7 National Reference Laboratories GMO List of National reference laboratories assisting the EU-RL for testing and validation of methods for detection (72 labs annexed to COMMISSION REGULATION (EC) No 1981/2006 of 22 December 2006) National Reference Laboratories nominated by MS under Regulation (EC) No 882/2004 (38 labs)

8. 8 Preparation and distribution of appropriate control samples Organisation of Comparative Testing Bi-annual workshops Ad-hoc training sessions (e.g. on ISO 17025, implementation of Regulations and Decisions) Provision of Reference Methods of analysis (Compendium) Constant technical assistance through e.g. the ENGL (WGs, Guideline documents, re-testing of problematic samples) Some examples of support to NRLs by the EU-RL GMFF

9. 9 An official laboratory shall be assessed and be accredited in accordance with the European EN ISO/IEC 17025 standard “General requirements for the competence of testing and calibration laboratories” (Article 12 of Regulation EC No 882/2004 of official food and feed controls) Requirements for laboratories assisting the EU-RL GMFF in validation studies (Regulation EC No 1981/2006): a) be accredited according to EN ISO/IEC 17025 b) provide assurance that their staff respect the confidential nature of subjects, data, results or communications NRLs: quality requirements

10. Comparative testing (CT) Article 32: Regulation (EC) No 882/2004 The EU-RLs for feed and food shall be responsible for: Coordinating application by the NRLs of analytical methods, in particular by organising comparative testing and by ensuring appropriate follow-up of such comparative testing Proficiency testing (PT) = Comparative testing EU-RL accredited ISO 17043

11. 11 Aim of proficiency testing For participating laboratories: Evaluation of laboratory performance Identification of problems in laboratories Education of participating laboratories For customers, regulators and accreditation bodies: Ongoing confidence in laboratory performance ISO 17025 accredited laboratories need to show proof of participation in proficiency testing schemes to accreditation body

12. 12 Distribution of participants ILC-CRL-GMFF-CT-02/10

13. 13 Management of underperforming NRLs Laboratories exhibiting an unsatisfactory z-score are asked to repeat the analyses in a short time-frame Technical assistance is given to underperforming laboratories – on-site visits offered In case of unsatisfactory z-scores in two consecutive comparative testing rounds DG SANCO is informed Delisting the NRL as possible action

14. 14 The European Network of GMO Laboratories (ENGL) 2 plenaries and several WG meeting group meetings per year cover sampling, interpretation of thresholds, expression of GM percentage, method uncertainty, reference materials, unapproved GMOs, decision trees etc. Chaired by the EC and managed by a Steering Committee (1 participant per MS) Provides extensive support to the EU-RL GMFF, e.g. in method validation, acceptance criteria etc. Links with ISO – CEN – Codex Alimentarius All 27 EU (+ Norway, Switzerland) are members and member states’ national networks are associated (> 100 participating laboratories) Observers from Tunisia, Morocco, Turkey, China, Malaysia, International seed testing association

15. 15 National Reference Laboratories GMO ENGL 97 members NRLs Reg. 1981/2006 NRLs Reg. 882/2004

16. 16 ENGL Structure National reference laboratories Steering Committee JRC

17. RLA-OGM Red Latino Americana de laboratorios de detección de OGM ASEAN GM Food Testing Network Brunei, Indonesia, Malaysia, Myanmar, Philippines, Singapore, Thailand, Vietnam, Cambodia, Lao, SANGL Botswana, Namibia, Madagascar, Malawi, Mozambique, Swaziland, South Africa, Tanzania, Zambia, Zimbabwe The next step : a Global Network of GMO Laboratories

18. Global GMO Networking Forum (GGNF) Date: 16-17 October 2012 Location: Brussels (EC Buildings) General Objective: to "network the GMO networks" Participants: approx 100 delegates from all regions including representatives from - EU Commission - Regional Networks - International Organisations - Individual Countries Roadmap 2009 - 2012

19. "Low Level Presence " Legislation

20. From: The global pipeline of new GM crops. JRC Reference Report, EUR 23486 EN - 2009

22. LLP legislation is based upon: -No deviation from the “zero tolerance policy”; -No redefinition/modifications of the EU regulatory system on GMOs; -Application only to feed; -A “technical solution” that would alleviate the potential problems of LLP, including trade disruption, departure from “mirror policy”, meeting EU feed stocking needs; -Discussions on the Minimum Required Performance Limit (MRPL), quantification strategies, sampling.

23. Salient points of Regulation (EU) 619/2011: Only GMOs can be considered that: - are authorised for commercialisation in a non-EU country; -have a valid EFSA application or have an expired authorisation; -a quantitative method of analysis has been validated and published by the EU-RL; -certified reference material are available to EU countries and third parties

24. Measurement Uncertainty (from the EU-RL to the lab)

25. The EURL GMFF accepts only methods when the applicant proves that the RSDr at the level of 0.1 % related to mass fraction of GM material ≤ 25%; this value will be published in the validation reports The EURL GMFF will determine in-house the RSDr at the level of 0.1% related to mass fraction of GM material and will publish that data in the validation report. Following a ring-trial, the EURL GMFF calculates again the RSDr, this time according to ISO standard 5725. This value has been and will continue to be published in the validation reports. In order to be fit for the purpose of meeting the requirements of the LLP regulation, all RSDr values mentioned above have to be below 25%. Method precision at 0.1% (1/2)

26. If the method allows to quantify at the 0.1% level within the stated level of precision (i.e. RSDr ≤ 25%) at all previous steps, the method may be considered to be included in the list of GMOs that can benefit from the LLP legislation (SANCO); otherwise, the method will not be included in that list; however, it will be part of the EFSA overall opinion in the context of the authorisation of GMOs for food and feed (Reg. (EC) No 1829/2003) Method precision at 0.1% (1/2)

27. EURL-GMFF precision data around 0.1% GM-event GM-level (%)(*) RSDrGM-level (%)RSDr TC15070.1180.512 T25 #0.15260.422 DAS591220.1180.413 H7-10.1160.514 281-24-2360.1220.416 3006-210-23 #0.1300.420 LLRice620.15210.412 LL25 cotton #0.15230.428 MIR6040.1240.417 Bt110.09170.413 A2704-120.1130.48 GA210.09230.517 40-3-2 #0.1290.426 MON897880.1160.422 EU-RL precision data for validated methods, at the lower end of the dynamic range

28. ISO 17025: “The laboratory shall confirm that it can properly operate standard methods before introducing the tests or calibrations” At time zero (t 0 ): − CRM is available. − NRLs test the RSDr of the method on the 0.1% − RSDr on CRM should be ≤ 25% Establishing method precision at NRLs

29. The analytical procedure in GMO analysis based on the real-time PCR consists of two steps (Reg. (EC) No 641/2004): - DNA extraction ENGL Guidance Document to Applicants and the next-to-release ENGL document on ‘method verification’ explain that DNA extracts should pass ‘quality criteria’ to access the qPCR step. - qPCR Fully covered by Method Acceptance and Method Performance documents MU on the whole analytical method

30. Tendency for Interlaboratory Precision in the GMO Analysis Method Based on Real-Time PCR TAKASHI KODAMA, YASUNORI KUROSAWA, KAZUMI KITTA, and SHIGEHIRO NAITO JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010, p 734 – 749 They analysed data of 53 coll. trials, including several EURL; Validations done including and excluding the DNA extraction module.

31. Some conclusions of the JP study: “In the GMO quantitative methods using relative quantification based on the simplex real-time PCR, we found that S R (Reproducability Standard Deviation) and S r (Repeatability Standard Deviation) were expressed by a function only of the GMO amount (%).” “These relationships were independent of GM crops and evaluation procedure steps. The collaborative data analyzed in this study indicated that the SD of the GMO amount (%) derived from the DNA extraction step would be negligibly small or considerably smaller than that of the PCR quantitation step.” The authors propose that S R (S r ) is a function only of the GMO amount (%).

32. Accept? Reject?

33. Testing of feed samples (1/3) To ensure a level of confidence of approximately 95%, the outcome of the analysis shall be reported as x +/– U whereby x is the analytical result and U is the expanded measurement uncertainty. U = k * u(x) 1) The standard uncertainty u(x) corresponds to the relative repeatability standard deviation (RSDr) of test results; U is obtained by multiplying u(x) by a coverage factor k. The coverage factor k is a function of the number of replicates and can be approximated to 2 when the number of test replicates is at least 10 (the EU-RL applies 15).

34. A feed shall be considered as non compliant with Regulation (EC) No 1829/2003 when the analytical result (x) minus the expanded measurement uncertainty (U) equals or exceeds the level of 0.1 % related to mass fraction of GM material: x – U ≥ 0.1% or x ≥ 0.1% + U(2) Testing of feed samples (2/3)

35. In practice, assuming the maximum RSDr allowed of 25% and an applied coverage factor of 2, the rejection criterion for low level presence of unauthorized GMO in feed concerns any GMO concentration larger than 0.15%, since: 0.1% + U = 0.1% + (50% * 0.1%) = 0.15% (expressed in mass) Testing of feed samples (1/3)

36. Unit of Measurement

37. Measurement results calibrated with a calibrant of a known mass fraction (i.e. a CRM), lead to measurement results expressed in mass fraction. Measurement results calibrated with a calibrant of a known copy number ratio (i.e. a CRM and/or a dual target plasmid), leads to measurement results expressed in copy number ratios. Unit of Measurement (1/3)

38. − The validation exercise is run following the provision of Annex I to Reg. (EC) No 641/2004 and the ENGL method performance requirements; − The results will be expressed in DNA mass fraction of GM material; − The control samples provided should be of homogeneous origin and well described in terms of zigosity, GM origin (paternal, maternal) etc. − In the Validation Report results will be also provided in copy number ratio, following the analysis by digital PCR. Unit of Measurement (2/3)

39. Biological factors impacting the accuracy of GMO quantification ♀♂ embryo 11 endosperm 21 seedcoat 20 seedcoat (2n) Maize: Embryo ≈ 48 % of total DNA Endosperm ≈ 49 % of total DNA Seedcoat ≈ 3 % of total DNA From Trifa & Zhang J. Agric. Food Chem. 52: 1044-1048 (2004) endosperm (3n) embryo (2n) This relationship needs to be verified by dPCR and published in the validation report; ====> Single copy endogenous reference gene always required! GM ♀ X non-GM ♂ GM = 24 + 33 + 3 % = 60 % of total DNA Non-GM ♀ X GM ♂ GM = 24 + 16 + 0 % = 40 % of total DNA GM ♀ X GM ♂ GM = 48 + 49 + 3 % = 100 % of total DNA Effect of parental contributions

40. Considering that the maize commodity market consists mainly of hybrid maize, and considering the uncertainty related to the biology estimates to vary approximately between 40 to 60%, the proposed relationship to deal with the biological uncertainty for heterozygous single inserts in maize is: GM % in DNA copy number = 50% [GM% in mass fraction] The proposed relationship to deal with the biological uncertainty for homozygous single inserts in soya is: GM % in DNA copy number = 100% [GM% in mass fraction] This is similar to the equation accepted by the European Network for GMO laboratories in the case of seeds. Guidance on conversion factor

41. Stacks

42. About stacks in LLP “…the outcome of the analysis shall be reported as x +/– U whereby x is the analytical result for one transformation event” The LLP applies to each GM-target singularly

43. AxB AxBx C AxC B Assume A approved, B and C not approved: If B or C are at or above the MRPL (as defined in accordance with the rules of interpretation), the feed shall be considered as non- compliant A

45. 4527 March 2012 Thank you for your attention!