1. ISTA’s Position, Experience and Activities in the Field of Biotech Trait Detection ISTA GMO TF B.R. Kahlert S. Grégoire, C. Haldemann, K. Remund, J.L. Laffont and many others International Seed Testing Association

2. Outline What is ISTA? ISTA and of GM Seed Testing Accreditation Proficiency Tests Performance Data Evaluation Audit ISTA can help, ISTA helps to share Workshops, Seedcalc, Website

3. Objectives of the International Seed Testing Association The primary purpose of the Association is to develop, adopt and publish standard procedures for sampling and testing seeds, and to promote uniform application of these procedures for evaluation of seeds moving in international trade. The secondary purposes of the Association are actively to promote research in all areas of seed science and technology, to participate in conferences and training courses aimed at furthering these objectives, and to establish and maintain liaison with other organisations having common or related interests in seed.

4. More than: 160 laboratories, 200 personal members, originating from 70 countries supporting the association (Government mandates) ISTA: Laboratories Worldwide

5. ISTA’s major Products & Services International Rules for Testing Seed –Sampling, Purity, Other Seeds, Germination, Tetrazolium, Seed Health, Species and Variety Testing, Moisture, Coated Seed, Vigour, X-ray –seeds of agricultural & vegetable; flowers, spice, herb & medicinal; tree & shrubs Proficiency Test Programme Laboratory Accreditation Programme International Seed Lot Certificates

6. ISTA’s Vision & Strategy in Testing of GM Seeds ISTA Seed Testing Laboratories are competence centres for detection, identification and quantification of GM seeds ISTA focuses its activity on developing and maintaining a system that ensures reliability and accuracy of results all over the world, when testing for GM seeds – Performance Based Approach defined in the Position Paper: ISTA’s Strategy Regarding Methods for the Detection, Identification and Quantification of Genetically Modified Seeds in Conventional Seed Lots adopted by the Executive Committee Nov. 2001

7. ISTA’s Actions in Testing of Seed with Specified Trait(s) 1.Chapter in ISTA Rules for testing of seeds with specified trait(s) 2.Implementation of Laboratory Accreditation under Performance Based Approach 3.Organising proficiency tests on detection and quantification of GM seeds in conventional seed 4.Establishing a system for Performance Data Evaluation for the Presence of Seed with Specified Trait(s) in Seed Lots and Specified Trait Purity 5.Provide technical help for the laboratories (training courses, workshops, Seedcalc7,...)

8. 1. Chapter in the ISTA Rules Rules for the testing of seeds with specified traits including the detection, identification and quantification of GM seeds came into force in February 1 st, 2006 The Rules do not contain specific methods, but define the general principles in testing and reporting results of such tests Laboratories having methods for GM seed testing in their scope of accreditation can report the test results on the International Seed Analysis Certificate

9. 2. Conditions for ISTA Accreditation under the Performance Based Approach Laboratory... Has established method(s) Defines scope of accreditation Participates successfully in the ISTA Proficiency Test Programme Submits performance data Has an audit

10. 3. Organisation of Proficiency Tests AIMS to evaluate the performance of participating laboratories to collect data to be used to set performance standards To increase experience for ISTA and participating laboratories Satisfactory performance in proficiency tests is a prerequisite for attaining or maintaining the status as an accredited laboratory Laboratories can participate to ISTA proficiency tests, even if they do not seek accreditation

11. Design of the 6 th Proficiency Test crop:Brassica napus L. # of samples per lab: 10 # of negative samples: 3 # of positive samples: 7 0.3%: 2 0.6%: 3 1.2%: 2 event:GT73 sample size:3300 seeds Qualitative and Quantitative tests the laboratory chooses the method

12. Qualitative Test: The detection of GM seeds in samples 49 labs classified each of the 10 samples as either positive or negative. 4 laboratories reported false results. These are 8% of the laboratories. Sample classified as false... 2.4% 5.4% 1.2% 2.0% 0.7% 1.0%

13. Sub-sampling Quantification Laboratory’s estimated results and its mean Estimate of % GM seeds 12 laboratories reported the test plan... the size of sub-samples (in number of seeds) the number of sub-samples tested the number of positive sub-samples  used to compute the result Spiking level 0.3%Spiking level 0.6%Spiking level 1.2%

14. Quantitative Test 23 laboratories reported the GM content of the positive test samples in % of GM seed by number or by mass or in % DNA copies

15. Summary PT06 PT06 - Qualitative Results: The percentage of laboratories reporting correct results for all samples is higher as in the previous tests >90% of labs made no missclassification, >97% of samples are reported correctly PT06 - Quantitative Results: The results show similar variation for the the spiking level (VC = ~50%) Most laboratories overestimated the true level

16. Design of the ISTA Proficiency Tests 110 laboratories from 42 countries

17. PT01 – PT06: Qualitative Tests Percentage of laboratories reporting results correctly or falsely: Percentage of samples reported correctly or falsely: PT01: maize, MON810 & T25; 1% PT02: maize; MON810; 0.7% & 1.4% PT03: maize; MON810 & T25; 0.2%, 2% & 4% PT04: soybean; GTS40; 0.1%, 0.5% & 1.0% PT05: soybean; GTS40 & A2704; 0.2%, 0.5%, 1.0%, 1.5% PT06: rapeseed; GT70; 0.3%, 0.6% & 1.2%

18. PT01 – PT06: Quantitative Test (Overall) mean of the quantitative test results for each spiking level

19. PT01 – PT06: Quantitative Test Variation coefficient of the quantitative test results

20. Summary PT01-PT06 Qualitative Results: Better results when only one transgenic event involved transgenic events are well known Better results for negative samples than for positive samples Quantitative Results: Reported values close to the true value when spiking level between 0.2% and 1.5% when transgenic event and reference material are well known

21. How to evaluate the performance of laboratories in ISTA Proficiency Tests? ANo problem has been detected in this test BThere are small problems, but no specific look or action is suggested to the participant CProblems, ISTA indicates there might be things to consider by the laboratory to explain or correct things BMPBelow Minimum of Performance, ISTA indicates that the results were poor and the laboratory need to find explanations and to improve/correct

22. In-round rating: Presence/absence 3 suggested rating systems RateFixed Number of misclassified samples A0 errors B1 or 2 errors C3 errors BMPMore than 3 errors RatePercentage of misclassified samples A0% - 5% B>5% - 10% C>10% - 20% BMP>20% RatePercentage of misclassified samples A0% - 6% B>6% - 20% C>20% - 30% BMP>30% Example with 10 samples in a Test System1System2 System3 2 mistakes 2/10=20%

23. In-round Rating: Principles for Quantification

24. Qualitative Test (49 labs)Quantitative and Sub-sampling Quantitative Tests (35 labs) PT06 Rating RS1: seed number RS2: mass of seed RS3: median

25. % of laboratories which reported an A, B, C or BMP Qualitative ResultsQuantitative Results PT01-PT06: In-round Rating

26. Rating System for 6 PTs based on the in-round rating values Qualitative Results Quantitative Results PT01-PT06: Overall Rating

27. 4. Performance Data Evaluation for presence of seed with specified trait(s) in seed lots and specified trait purity Laboratory can choose an established and reliable method The «Performance Based Approach» is used instead of «one ISTA validated method» (not published in the ISTA Rules) Laboratory needs to demonstrate to ISTA accreditation team its ability to perform well with the method routinely used

28. Performance approved method to be included in lab’s scope of accreditation must be defined in terms of method used, the species tested, and the specified trait is to be determined Performance Data Evaluation Scope of ISTA Accreditation

29. Evaluation of Performance Data for each combination species, test method and trait! For some combinations a PD has to be performed (on seeds, defined number of samples, defined spiking levels) For other combinations suitable alternative Performance Data may be used (any other data providing effidence that accuracy and repeatability criteria are met) Performance Data Evaluation Relevant PD Documents

30. Quantification of Trait(s) Performance Data Evaluation Range: 0.1% to 3% 7 levels * 4 samples

31. Accuracy: departure from true value Repeatability: variability of results by level Performance Data Evaluation 7

32. Example of use of the data sent by the laboratory

33. Accuracy: Departure from true value

34. Repeatability: variability of results by level

35. Deviation and variability by level

36. Grading performance Performance Data Evaluation 8.2 8.3

37. Audit Auditors assess whether implementation aspects are appropriately addressed and whether practice complies with the documented procedures To that effect, training, equipment management, facilities, reporting and running the overall quality managements system will be assessed A complete practical demonstration of any performance approved method will not be required

38. 5. ISTA Workshops on GM Seed Testing periodworkshop titlelocation 2002 – 2006 ISTA/FAO Training Course on Varietal Verification and GMO Detection (8) Argentina, South Africa, Thailand, Slovenia, Egypt, China, Jamaica, Turkey 2004 – 2005 ISTA Workshop on Statistical Aspects of GMO Detection (3) France, USA, Argentina 2003 ISTA Variety Testing Workshop - Way to the future ISTA Open Forum Discussion on (Semi)-Quantitative GMO Testing Switzerland

39. ISTA Web site http://www.seedtest.org