The International Seed Testing Association GMO PT Program AIEC Fall Meeting October 15-16 2014 New Orleans, Louisana

What is the International Seed Testing Association? An international, non-profit association of persons and seed testing laboratories from the public and the private sector Under the Governance of the Governments of member countries and distinct economies An Association standardizing the determination of seed quality to facilitate international trade Explain the strategy used by ista.

Ordinary Meeting Executive Committee Secretariat Voting delegates from 72 Member Governments Executive Committee 11 Members from all Continents of the World nominated by Governments Secretariat 11 professional employees 18 Technical Committees and Boards Over 200 experts from all over the world working voluntarily Experts from the public/private sectors, from member/non-member laboratories

ISTA Executive Committee 2013-2016: President Vice President 9 Members-at-Large Joël Léchappé, France Craig McGill, New Zeeland Mable Simwanza, Zambia Steve Jones, Canada Alexander Malko, Russia Berta Killerman, Germany Masatoshi Sato, Japan Grethe Tarp, Denmark Margus Friedenthal, Estonia Rita Zecchinelli, Italy Cecilia Jones, Uruguay

ISTA Secretariat Benjamin Kaufman Secretary General Rasha El-Khadem Name: Role: Benjamin Kaufman Secretary General Jonathan Taylor Publication Specialist Nadine Ettel TCOM Coordinator Rasha El-Khadem Head of Accreditation Dept. Branislava Opra System Auditor Florina Palada Patricia Muschick Head of Administration Dept. Cannice Gubser Membership Administration Agnes Hegedüs Sales- and Office Administrator Sejal Patel Administrator Olga Stockli

ISTA Technical Committees: ADVANCED TECHNOLOGIES COMMITTEE BULKING AND SAMPLING COMMITTEE FLOWER SEED COMMITTEE FOREST TREE AND SHRUB SEED COMMITTEE GERMINATION COMMITTEE GMO COMMITTEE MOISTURE COMMITTEE NOMENCLATURE COMMITTEE PROFICIENCY TEST COMMITTEE PURITY COMMITTEE RULES COMMITTEE SEED HEALTH COMMITTEE STATISTICS COMMITTEE SEED STORAGE COMMITTEE TETRAZOLIUM COMMITTEE VARIETY COMMITTEE VIGOUR COMMITTEE EDITORIAL BOARD (SST)

Why is ISTA relevant to seed stakeholders? The vision of ISTA: Uniformity in Seed Quality Evaluation Worldwide

International standardization of reporting results International standardization of methodologies International standardization of laboratory performance International exchange of scientific results

International exchange of scientific results: ISTA Publications and Symposia Editing and issuing the Scientific Journal: Seed Science and Technology Organize Seed Symposium as part of the tri-annual ISTA Seed Congress

International standardization of reporting results International standardization of methodologies International standardization of laboratory performance International exchange of scientific results

2013: New Chapter added to the ISTA Rules!! Chapter 19-GMO Testing International Standardization of Methodologies: The International Rules for Seed Testing -> ISTA has been publishing the international rules for seed testing since 1933. Most recently Rules chapter: 19 - GMO testing The ISTA Rules can be modified and amended annually Proposals to modify or amend current ISTA International Rules for Seed Testing can be made by: members, non-members, public/private laboratories, persons working in the area of seed science and technology.

Chapter 19: GMO Testing Objective: Why develop a new chapter? To formalize the ISTA approach for GMO testing within the ISTA Rules To describe a system that will ensure uniformity in GM seed testing Why develop a new chapter? ISTA has been working to address challenges related to testing GM seed since 2001! GMO testing is an increasing activity for seed trade Laboratories need guidance about ISTA‘s principles and technical aspects of GMO testing. Answer ISTA‘s strategy decision (2001: to establish a GMO chapter) Key Considerations: This chapter remains general on all aspects of GMO seed testing This chapter is based on already existing ISTA‘s papers Doesn‘t provide protocols (follows the Performance Based Approach) Is linked to a Handbook for the detailed technical considerations (in development!) Is not creating new ISTA position / statement about GMO analysis Established TF in 2001, incorporated process for “sprecified trait testing” into chapter 8… TF became Technical Committee in 2010, rules development followed from 2010-13; Came following 10 year’s of experience in working within the system and recognizing what worked and where gaps were. Next most important step is development of the handbook – which provides technical guidance for labs wanting to establish gmo testing and becom eaccreditted.

International standardization of reporting results International standardization of methodologies International standardization of laboratory performance International exchange of scientific results

International Standardization of Laboratory Performance: The ISTA Accreditation Program: Open to : Seed Testing Laboratories from the public and the private sector official language: English all relevant documents can be downloaded from the ISTA website at www.seedtest.org

International standardization of reporting results International standardization of methodologies International standardization of laboratory performance International exchange of scientific results

International Standardization of Reporting Results: Facilitation of International Seed Trade ISTA Certificates have international recognition Indicate the sampling and testing has been carried out in accordance with the ISTA Rules Sampling and testing methods used have been validated, internationally harmonised and voted on by the ISTA membership.   The International Seed Analysis Certificates are available only for accredited member laboratories of ISTA. They must only be issued in accordance with the ISTA Rules currently in force. Blue Certificates are test reports for seed samples. They do not represent a seed lot; therefore, sampling is the responsibility of the customer. We can issue these certificates on any sample, regardless of origin or lot location. Orange Certificates are test reports that represent whole seed lots. To ensure that the certificate accurately represents the seed lot, the lab is responsible for both sampling and testing.

International standardization of reporting results International standardization of methodologies International standardization of laboratory performance International exchange of scientific results

The ISTA Accreditation Programme ISTA Rules ISTA Accreditation Programme Proficiency Testing 120 participating laboratories world wide The laboratory‘s performance is directly linked to its accreditation status Three test rounds per year ISTA Membership Q-Audit Seed Testing Laboratory ISTA Certificate Quality assurance system according to the ISTA Accreditation Standard ISTA audit every three years Audit by two ISTA auditors

The ISTA Proficiency Test Program Generally: you are accredited  obligatory, not accredited  voluntary What they all have in common: measure competency. We are not talking about comparison tests to evaluate a method. Voluntary and obligatory - explain Basic Tests Seed Health Tests GMO Tests

The ISTA Proficiency Test Program Goal: measure and monitor laboratory‘s proficiency Proficiency Test fee is covered in yearly Membership fee Participation is mandatory for accredited labs, for the tests/crop group/events for which they have been accredited Voluntary participation possible for all member laboratories, regardless if accredited or not Non members are welcome to participate (service fee will be charged) Why voluntary participation? Gain experience with other species Compare your laboratory‘s testing performance with the performance of accredited laboratories Your participation will be rated, but the rating has no influence on your accreditation

The ISTA Proficiency Test Program: Several rounds per year Labs test ‘blind’ samples in accordance with determined methods* Submission of results Review and Statistical evaluation Evaluation of the laboratory‘s proficiency Detection of systematic errors and performance trends Results influence accreditation status (only labs for which participation is mandatory) Accreditation maintained? þ

The ISTA Proficiency Test Program: Ratings Ratings are assigned: A, B, C and BMP (Below Minimum Performance) for each test in each round After six rounds of participation calculation of overall rating Only mandatory test results are included in overall rating Performance of a laboratory influences their accreditation: Overall rating of C  warning letter Overall rating of BMP  suspension Failure to participate in 2 rounds out of 6  suspension A & B: good ratings, C and BMP require CA

GMO Proficiency Testing: Challenges! ISTA’s performance based approach for GMO testing means the way development and execution of a PT used for Basic Seed Tests and Seed Health Tests must be modified in order to have a Proficiency Testing program for GMO testing

GMO TESTING: Performance Based Approach The ISTA approach has targeted the uniformity in GMO testing results, not by the uniformity in testing methodology, by using a “Performance Based Approach (PBA)”. The “Performance Based Approach” requires that laboratories demonstrate that the GMO detection, identification or quantification methods they are using on seed samples for reporting results on the ISTA International Seed Lot Certificate, meets acceptable standards set by ISTA. This standard includes, among others, sampling, reporting and test accuracy. This requirement will ensure reliability of the analysis and the final test result reported on the ISTA International Seed Lot Certificate. The Performance Based Approach gives seed testing laboratories the choice to use different technological approaches, e.g. bioassays, protein based methods and DNA based methods. 24 24

What does this mean? Basic Seed Tests and Seed Health Tests must use ISTA approved protocols… but…. …the Performance Based Approach gives seed testing laboratories the choice to use different technological approaches, e.g. bioassays, protein based methods and DNA based methods. The GMOPT must be able to accommodate evaluation and rating of results obtained from many test types!

GMO PT Program basics: 1 species per round, 1-3 Events Qualitative and Quantitative evaluations Spiking levels between 0-4% 10-12 samples of 1800-3000 seeds each Two panels per year offered Matrix is SEED

GMO PT19: Design of Round

Announcement and Technical Protocol: Announcement is made on the ISTA Website and also sent to member labs via e.mail Technical protocol provides information needed to develop the test plan Reporting sheets are provided for results to be returned http://seedtest.org/en/proficiency-tests-_content---1--1157.html

ISTA GMO PT Database Data from the past 20 rounds are contained. The data is evaluated to improve the program and also by a group within the publications WG of the GMO TCOM for future publication. The PT process is tracked and executed through the use of a specially developed Access database which is used for registration to sample prep, shipping, participant tracking over time, data recording and analysis, etc.

Results Evaluation: Qualitative Determination The rating for the presence/absence ( yes/no - qualitative) results is based on a % of misclassified samples/total samples. Misclassification may either be a false positive or a false negative result. Missing results for individual samples are considered as misclassification.

Results Evaluation: Quantitative Determination The quantitative rating is based on the quantification results for the positive samples and their reference value (samples with zero spiking level are not used in quantification rating). The reference value is either the # of GM seeds in %, the mass of the GM seeds in % or the median of the results reported by the participants in the unit ‘%DNA copies’. Which of the reference values is chosen is determined by the panel of experts appointed for each round, the guiding principle being: Sub-sampling quantification: %number Results reported in %number: %number Results reported in %mass: %mass Results reported in any other unit, such as number DNA copies: median of the results

ISTA GMO Proficiency Test – Ratings The rating system is based on the system established for basic and seed health tests which are accredited by ISTA – A, B, C, BMP A No problem has been detected in this test B There are small problems, but no specific look or action is suggested to the participant C Problems, ISTA indicates there might be things to consider by the laboratory to explain or correct things BMP Below Minimum of Performance, ISTA indicates that the results were poor and the laboratory need to find explanations and to improve/correct

GMO PT19: Results/Evaluation Sample sets were sent to 46 laboratories; 39 participants submitted results; 8 provided qualitative results only For this PT round, a change in the procedure has been made in order to better take into account recognized standards regarding the precision of a quantitative result. Therefore, in the definition of the Reference intra-laboratory standard-deviation used in the computation of the z-scores, we have introduced the following change (using the same notations as in the reference document in ISTA News Bulletin No. 130): If (reference lab stdev)/(true level) is lower than 0.25, use 0.25 x (true level) as the Reference intra-laboratory standard deviation.

GMO PT: Qualitative and Quantitative Rating Systems Procedure updated STI 128 – 2004: https://www.seedtest.org/upload/cms/user/STI128Oct2004.pdf STI 130:-2005: https://www.seedtest.org/upload/cms/user/STI130Oct2005.pdf

Success of the Performance Based Approach for GMO Testing within ISTA: 13 laboratories are accredited for GMO Testing Performance Trends: Qualitative Detection Quantification Quantitation results not necessarily due to poor performance of labs, but seems to be due to increased accuracy – a statistical artefact of a low z-score (JLL/KR)

GMO PT Program: Participant Stats: About half the participants are ISTA labs The majority of participants are from Europe

1 round of the GMO PT program: Complete process: 1 round of the GMO PT program: The execution of a single round of the GMO PT Program is complex and involves significant organization, coordination, and administration from all parties involved in it’s delivery. Steps include: 1. Program Planning and Seed procurement 2. Purity testing of sample seed material 3. Announcement of PT Round 4. Initiation of a new PT in the PTDB 5. Registration of participants and invoicing Adding to the complexity – there are always 3 PT rounds ongoing at any time, all at different stages of completion! 6. Sample preparation and shipment 7. Participant Results 8. Evaluation of Results and Ratings 9. Generation of Final results reports 10. Record keeping

How is this work accomplished? Working Groups Lead Members Work Description and Projects GMO Proficiency Test: Administration and Sample Preparation ISTA TCORD Nadine Ettel Christoph Haldemann, Jean-Louis Laffont and Cheryl Dollard (Expert Team); Andrea Jonitz (Sample Prep) Purpose: Execution of the GMO PT ; Lead: Coordinate administration of the PT program from within the ISTA secretariat; Expert Team: Develop PT Plans, Evaluate Results and Ratings, address technical questions, assist with administration of program as described in the SOP for administration of the GMO PT Program. Sample prep: Purity check of material and preparation of PT Samples as described in the GMO PT Program Sample Prep SOP GMO Proficiency Test: Resource Procurement Ray Shilito Elizabeth Bates (Bayer), Bruno Zaccomer (Monsanto), Sofia Ben Tahar (Limagrain), Purpose: Resource procurement in support of the GMO PT Program: Secure reference materials (GM and non-GM) from Industry; Coordinate financial support for PT program from Industry/corporate partners; Action items: solicit WG members from other Industry areas: Syngenta, Pioneer, etc Secretariat contact: ISTA TCOM Coordinator (Nadine Ettel)

GMO PT Program: Procedures established Two SOPS developed describing all steps required to execute a single PT round: “Administration of GMO PT program” “Sample preparation for GMO PT program” Significant challenges: Sourcing GM and Non-GM Seed; Funding to cover associated costs Finding labs willing to prepare the samples

One Round of the GMO PT ..Mostly! K. Studer

Sample Preparation A sample preparation laboratory prepares the blind samples, packages and ships lots to participants. Requires Seed Lot Purity Checks: GM and Non-GM Seed Preparation and blind coding of the spiked samples (10 samples x 50 participants – 500) Arrangement of phytos Rounds 1-19: LTZ (Germany), Round 20: Eurofins

Expert Team Group of experts who are involved in statistical analysis and interpretation of the results for each round They address the activities which cannot involve other committee or WG members in order to ensure secrecy of the program, and to protect confidentiality of participants and results. Includes: finalizing details and logistics to addressing questions and issues that arise and are unique in each PT.

Expert Team: A snapshot… Development of PT test plans (computing percentages/spiking levels/seed requirements/statistical verification). Provision of support for Administrative functions carried out in the Secretariat as required (preparation/review of documents/files, and database use, generation of reports, etc). Preparation of the PT schedule Coordination of activities and problem solving issues related to seed procurement, including addressing Industry questions regarding seed used for the PT program. Verification of seed check procedure; analyse data for approval of lots; revision of test plans when necessary. Provided input for development of new GMODB; beta tested versions during development process. Review of raw results, ratings computation and determination of final ratings. Provision of advice and answers to the Secretariat, Sample prep lab and members with questions. Review of data for trends that signal potential issues which could require modification of the Program. ** “Corporate Memory” ** Addressing issues related to the PT: These are often complex and require technical analysis, research, etc.

# spiking levels (incl. 0) 20 rounds and counting… PT Crop # of Events # of participants # of samples in a set # seeds per sample # spiking levels (incl. 0) PT01 Maize 2 46 30 300 4 PT02 1 50 10 3000 3 PT03 42 12 1500 7 PT04 Soybean 52 PT05 58 5 PT06 Canola 3300 PT07 67 2000 6 PT08 53 14 PT09 Maize* 64 12+2 2000/50 11 PT10 Cotton** 2+1 2500 9 PT11 49 PT12 60 8 PT13 31 PT14 40 PT15 Flax**** 35 PT16 Maize***** 1800 PT17 45 PT18 Maize+ 48 PT19 Soybean+ PT20 Cotton++ in progress PT09 Included Trait Purity samples- 12 samples for AP, 2 samples for Purity ** PT10 Cotton round used 3 GM varieties - 2 varieties were single Event and 1 variety was a double stack (stack included same Events as those in the single Trait varieties) *** # of spiking levels calculated to include 0%; double spiked samples (i.e. those which had 2 Events spiked into the same sample) are calculated as 2 spiking levels - this is to get an accurate estaimate of the work involved **** Seed material (GM and NON) as well as purity testing was done by CFIA - no charge to ISTA ***** non-GM seed had to be replaced, Lot and purity testing was done by Bayer - no Charge to ISTA + Seed for PT18 and 19 - most purity testing was not required as leftover seed from PT16 and 17 was able to be used again ++ Seed for PT20 - purity testing to be done by Provider - no charge to ISTA

Ordinary Meeting Executive Committee Secretariat Voting delegates from 72 Member Governments Executive Committee 11 Members from all Continents of the World nominated by Governments Secretariat 11 professional employees 18 Technical Committees and Boards Over 200 experts from all over the world working voluntarily in the Technical Committees Experts from the public/private sectors, from member/non-member laboratories

Conclusion ISTA GMO PT program is a unique tool for achieving uniform and reliable seed quality determination for GMO testing It is possible to evaluate and compare test performance internationally in the dynamic field of GM testing internationally while still achieving uniformity of results reporting that is ISTA’s aim.

Thank you for your attention www.seedtest.org

Questions ?