Simposio Internacional sobre Detección de OGM 25 y 26 de octubre 2011, México

1. Implementation of the ISO and JRC standards in GMO analysis laboratories Mathieu Rolland 1 - Emmanuelle Pic 2 1 GEVES - BioGEVES, 2 Anses - Plant Health Laboratory Simposio Internacional sobre Detección de OGM 25 y 26 de octubre 2011, México

2. The European regulatory framework :

3. The European regulatory framework : • Main regulations : • - Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environmentof genetically modified organisms and repealing Council Directive 90/220/EEC • - Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed • - Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003 concerning the traceability and labellingof genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive 2001/18/EC

4. The European regulatory framework : Petitioner : submission of application National Competent Authority To submit an application, the petitioner has to provide: - The sequence of the event - The description of a detection method (based on qPCR) - Some reference material

5. The European regulatory framework : Petitioner : submission of application National Competent Authority European Food Safety Authority Validation of the detection method by the European reference laboratory Member States consultation (3 months) 6 months Scientific opinion of EFSA Decision of the EU : authorization or refusal

6. The European regulatory framework : Petitioner : submission of application National Competent Authority European Food Safety Authority Validation of the detection method by the European reference laboratory European Network of GMO Laboratories France : A network of three official laboratories

7. The European regulatory framework : Petitioner : submission of application National Competent Authority European Food Safety Authority Validation of the detection method by the European reference laboratory European Network of GMO Laboratories France : Plant Health Laboratory

8. The French Network of official laboratories :

9. GEVES: Group for the study and control of varieties and seeds Ministry for Food, Agriculture and Fisheries (National Institute for Agricultural Research) (The French Association for Seeds and Seedlings) • A public Interest Group (GIP) • An official and unique organization in France

10. BioGEVES: Laboratory of molecular biology and biochemistry of GEVES Two main tasks • Analysis of 4 different types : • Detection of GMOs • Detection of pathogens • - Genetic marking of varieties • The analysis of biochemical constituents Participation in research programs: - Expertise at the international level : adaptation, optimization and validation of protocols - Participate in and monitor inter-laboratory networks - Scientific publications, posters, oral communications… Accreditation ISO 17025 cofrac since 2004 ACCREDITATION N°1-1540 WWW.COFRAC.FR

11. French Agency for Food, Environmental and Occupational Health & Safety Ministry for ecology and sustainable development Ministry for labour Ministry for health Ministry for food, agriculture and fisheries Ministry for consumer affairs 5 supervisory ministries State goals & resources contract Involvement in several national plans: environmental health, occupational health, Ecophyto 2018, Cancer Plan, etc. Vote of the annual grant, annual report Parliament

12. Anses contributes to ensuring : - human health and safety in the fields of environment, work and food, - protection of animal health and welfare, as well as plant health. Assess nutritional and health risks and benefits Recommend public health measures Provide laboratory reference missions Monitor health safety Provide training and information and contribute to public debate Conduct, coordinate and initiate research projects Coordinate the R 31 network of scientific organisations Authorise marketing of veterinary medicinal products Collaborate with European agencies (EFSA, ECHA , EEA, EU-OSHA, ECDC and EMA)

13. Anses contributes to ensuring : - human health and safety in the fields of environment, work and food, - protection of animal health and welfare, as well as plant health. Assess nutritional and health risks and benefits Recommend public health measures Provide laboratory reference missions Monitor health safety Provide training and information and contribute to public debate Conduct, coordinate and initiate research projects Coordinate the R 31 network of scientific organisations Authorise marketing of veterinary medicinal products Collaborate with European agencies (EFSA, ECHA , EEA, EU-OSHA, ECDC and EMA)

14. 12 reference and research laboratories Plant Health Laboratory : 6 sites (Montpellier, Nancy, Rennes, Saint-Pierre (Reunion Island), Clermont-Ferrand and Angers) Bacteriology, Virology and GMO unit (Angers) Accreditation ISO 17025 by the COFRAC for the detection and quantification of some GMO events

15. Standards for the detection of genetically modified organisms:

16. ISO standards : - ISO 24276:2006 "Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - General requirements and definitions" - ISO 21571:2005 "Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction"

17. ISO standards : - ISO 21569:2005 "Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Qualitative nucleic acid based methods" - ISO 21570:2005 "Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Quantitative nucleic acid based methods"

18. In France : guides to help apply the ISO standards - Guide technique d'accréditation COFRAC LAB GTA 02, March 2008 - Guide d'application AFNOR GA V03-042, November 2006

19. Implementation of a new method

20. Methods description : - Compendium of reference methods for GMO analysis - http://gmo-crl.jrc.ec.europa.eu/gmomethods/ Intellectual Property aspects

21. Methods description : - Compendium of reference methods for GMO analysis - http://gmo-crl.jrc.ec.europa.eu/gmomethods/ Intellectual Property aspects CropLife International  licences

22. Certified Reference Materials : Unit of certification : mass/mass or copy number/copy number

23. Qualitative analysis : Specificity : - Theoretical evaluation (FastA, Blast etc. on GenBank, EMBL etc.) - Experimental evaluation (preferably on CRM) :

24. Qualitative analysis : Limit of Detection (LOD) : - Independent dilutions (each level) of GMO DNA in non-GMO DNA - LOD = lowest level where 6 (or 10) repetitions give a positive result - 0.1 copy level : the number of positive results must be  1 (control of the method used to quantify DNA)

25. Quantitative analysis : Specificity and LOD : idem qualitative analysis. Limit of Quantification (LOQ) : Same principle as for LOD estimation but : - target copy numbers ~ 10 times higher - LOQ = level where RSDr 25 %

26. Quantitative analysis : • Linearity : • At least 5 levels prepared by independent dilutions of GMO DNA in non-GMO DNA (The dynamic range must cover the values corresponding to the expected use) • For each level, 4 independent experiments and 2 PCR by experiment. R2 = 1 y = -3.22 x + 49.5 • Calculation of : • R2 • slope Performance of the method : - R2 ≥ 0.98 - -3.1 ≥ slope ≥ -3.6 (ideally -3.32)

27. Quantitative analysis : Accuracy (trueness and precision) : Definition of minimum performance requirements for analytical methods of GMO testing 10/2008 (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm)

28. Quantitative analysis : • Accuracy (trueness and precision) : • Verification of analytical methods for GMO testing when implementing interlaboratory validated methods 07/2011 (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) • Trueness • Procedure: The trueness should be determined at a level close to the level set in legislation (e.g. 0.9 % • threshold), or according to the intended use of the method, and additionally at a level close to the • LOQ. • Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testing • of samples. At least results from 16 PCR replicates should be obtained. • Acceptance criterion: The trueness shall be within ± 25 % of the accepted reference value or a Z-score • within the range of 2 and -2 should be obtained. • Precision - Relative Repeatability Standard Deviation (RSDr) • Procedure:Repeatability can be determined in a similar way as described under Trueness. It is • calculated from PCR replicates run under repeatability conditions (see Terminology). Repeatability • should be available for all tested GM-levels. • Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testing • of samples. At least 16 single test results should be obtained. • Acceptance criterion: The relative repeatability standard deviation should be ≤ 25%, over the dynamic • range of the method.

29. Quantitative analysis : • Accuracy (trueness and precision) : • Verification of analytical methods for GMO testing when implementing interlaboratory validated methods 07/2011 (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) • Trueness • Procedure: The trueness should be determined at a level close to the level set in legislation (e.g. 0.9 % • threshold), or according to the intended use of the method, and additionally at a level close to the • LOQ. • Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testing • of samples. At least results from 16 PCR replicates should be obtained. • Acceptance criterion: The trueness shall be within ± 25 % of the accepted reference value or a Z-score • within the range of 2 and -2 should be obtained. • Precision - Relative Repeatability Standard Deviation (RSDr) • Procedure:Repeatability can be determined in a similar way as described under Trueness. It is • calculated from PCR replicates run under repeatability conditions (see Terminology). Repeatability • should be available for all tested GM-levels. • Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testing • of samples. At least 16 single test results should be obtained. • Acceptance criterion: The relative repeatability standard deviation should be ≤ 25%, over the dynamic • range of the method. The trueness should be determined at a level close to the level set in legislation (e.g. 0.9 % threshold), or according to the intended use of the method, and additionally at a level close to the LOQ.

30. Quantitative analysis : • Accuracy (trueness and precision) : • Verification of analytical methods for GMO testing when implementing interlaboratory validated methods 07/2011 (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) • Trueness • Procedure: The trueness should be determined at a level close to the level set in legislation (e.g. 0.9 % • threshold), or according to the intended use of the method, and additionally at a level close to the • LOQ. • Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testing • of samples. At least results from 16 PCR replicates should be obtained. • Acceptance criterion: The trueness shall be within ± 25 % of the accepted reference value or a Z-score • within the range of 2 and -2 should be obtained. • Precision - Relative Repeatability Standard Deviation (RSDr) • Procedure:Repeatability can be determined in a similar way as described under Trueness. It is • calculated from PCR replicates run under repeatability conditions (see Terminology). Repeatability • should be available for all tested GM-levels. • Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testing • of samples. At least 16 single test results should be obtained. • Acceptance criterion: The relative repeatability standard deviation should be ≤ 25%, over the dynamic • range of the method. The trueness shall be within ± 25 % of the accepted reference value or a Z-score within the range of 2 and -2 should be obtained.

31. Summary Context of low contamination levels Favorable context for the EU official labs : - Validation of the methods by the JRC - Access to the methods - Access to certified reference materials - In France: access to guides helping the Implementation of new methods But : only applies to events for which an application has been submitted.

32. Thank you very much ! Muchas gracias ! mathieu.rolland@geves.fr emmanuelle.pic@anses.fr www.anses.fr