AOAC Gluten Qualitative Validation Guidance-FINAL (July 2023)

AOAC INTERNATIONAL

STAKEHOLDER PROGRAM ON GLUTEN & FOOD ALLERGENS (GFA) GLUTEN QUALITATIVE VALIDATION GUIDELINES

FINAL REVIEW

(JULY 2023)

Delia Boyd AOAC INTERNATIONAL 2275 Research Blvd., Suite 300 Rockville, Maryland 20850 Tel: 240-801-8668 Ext. 126 Fax: +1-301-924-7089 Internet Email: aoac@aoac.org Web Site: www.aoac.org

Guidelines for Validation of Qualitative Gluten Methods, with Specific Examples for Lateral-Flow Devices

Contents 1 Scope ...............................................................................2 2 Applicability .....................................................................2 3 Terms and Definitions ......................................................2 4 Method Validation ...........................................................5 4.1 Method Developer Validation Study.........................5 4.1.1 Scope..................................................................5 4.1.2 Selectivity study .................................................5 4.1.3 Matrix / POD study ............................................6 4.1.4 Data analysis and reporting for selectivity and matrix / POD studies ...................................................7 4.1.5 Intended use statement.....................................7 4.1.6 Robustness study ...............................................7 4.1.7 High-Dose Hook Effect Study .............................7 4.1.8 Method Instructions ..........................................7 4.1.9 Product consistency and stability study.............8 4.2 Independent Lab Validation Study............................8 4.2.1 Scope..................................................................8 4.2.2 Matrix Study.......................................................8 4.2.3 Data Analysis and Reporting ..............................9 4.3 Collaborative Study ...................................................9 4.3.1 Scope..................................................................9 4.3.2 Number of Collaborators ...................................9 4.3.3 Matrix / POD study ............................................9 4.3.4 Data analysis and reporting ...............................9 4.3.5 Collaborator comments .....................................9 4.4 Matrix Extension .....................................................10 4.4.1 Matrix Extension for single lab validation studies .................................................................................. 10 4.4.2 Matrix Extension for multi-site collaborative studies.......................................................................10

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AOAC Appendix H: Probability of Detection (POD) as a Statistical Model for the Validation of Qualitative Methods AOAC Appendix N: ISPAM Guidelines for Validation of Qualitative Binary Chemistry Methods CLSI EP07-A2, Interference Testing in Clinical Chemistry CLSI EP12-A2, User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline CODEX STAN 118-1979: Standard for foods for special dietary use for persons intolerant to gluten FDA ORA-LAB 5.4.5, 2020, Volume II —Methods, Method Verification and Validation, Document No IV-02, Version 2, Section 2—Microbiology. ISO/IEC Guide 99:2007, International vocabulary of metrology—Basic and general concepts and associated terms (VIM) ISO 3534-2:2006 , Statistics—Vocabulary and symbols—Part 2: Applied statistics ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results—Part I: General principles and definitions ISO 6206:1979, Chemical products for Industrial Use – Sampling – Vocabulary ISO/TS 16393, Molecular biomarker analysis – Determination of the performance characteristics of qualitative measurement methods and validation of methods ISO 17511:2020, In vitro diagnostic medical devices— Measurement of quantities in biological samples— Metrological traceability of values assigned to calibrators and control materials ISO/TS 27878:2023, Reproducibility of the level of detection (LOD) of binary methods in collaborative and in house validation studies. JAOAC – INSERT UPCOMING POD STATISTICS REFERENCE USP 31:2008, U.S. Pharmacopeia General Information/ Validation of Alternative Microbiological Methods

1 Scope

The purpose of this document is to provide comprehensive technical guidelines for method developers conducting validation studies for qualitative gluten methods, for example methods submitted for AOAC INTERNATIONAL (AOAC) Performance Tested Methods SM (PTM) status and/or for AOAC Official Methods of Analysis SM (OMA) status. This document is not intended to describe requirements for laboratories using commercial immunoassay methods for gluten analysis, though for these laboratories it would assist their understanding of the consensus-based approach, the terminology used and what information they can expect to receive from method developers. The requirements for method developer (single laboratory) validation studies, independent validation studies, and collaborative validation studies are described. Specific examples are provided for lateral-flow devices. These guidelines are intended to be applicable to the validation of candidate qualitative gluten methods, whether proprietary or non-proprietary, including those that may be submitted to AOAC for OMA status or PTM certification. Unforeseen circumstances may necessitate divergence from these guidelines in certain cases, and these must be reviewed by AOAC or another appropriate agency (other than the method developer). The AOAC PTM Program requires a method developer (single-laboratory) validation (SLV), and an independent laboratory study. The AOAC OMA Program requires an SLV (also known as the pre-collaborative study) and a collaborative study to achieve Final Action status. A harmonized PTM-OMA Program can be followed in which PTM certification is sought and, if successful, serves as the SLV phase of the OMA Program. 2 Applicability Where appropriate, definitions have been taken from international standards and the source is cited. Sources of definitions include the following: AAFCO Good Samples and Good Test Portions: https://www.aafco.org/resources/guides-and manuals/good-test-portions-and-goodsamples-resources/ AOAC Appendix F: Guidelines for Standard Method Performance Requirements, Official Methods of Analysis 3 Terms and Definitions References

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and 0.5M NaCl (CODEX STAN 118-1979). Throughout this document, the word ‘wheat’ refers to all Triticum species, such as durum wheat, spelt and khorasan wheat, and their hybrids and crossbred varieties such as Triticale. 3.11 High-Dose Hook Effect The result of a suboptimal antigen-antibody reaction in which either the antibody or antigen is in excess, incomplete, or blocks an optimal reaction (CLSI EP12-A2). Also known as Prozone Effect or Overload Effect. 3.12 Incurred Test Material A test material prepared from a food matrix into which a gluten source (e.g. flour) has been incorporated prior to subjecting the matrix to a given food processing operation. 3.13 Independent Testing Site A testing site not owned, operated or controlled by the same entity as the method developer. 3.14 Interference Study The examination of matrices expected to be tested with the method, to demonstrate that they do not interfere with detection of the analyte. 3.15 Lateral Flow Device (LFD) An analytical method characterized by use of an immunochromatography platform for the detection of specific analyte. 3.16 LPOD Composite POD pooled across laboratories. Includes between-laboratory variation in addition to variation inherent in the binomial mature of binary probabilities (INSERT UPCOMING JAOAC REF). 3.17 Matrix The totality of components of a material system except the analyte (ISO 17511). For example, the food, beverage, or environmental surface material to be included in the validation as per the intended use of the method. 3.18 Measurand The quantity intended to be measured (the specification of the measurand should be sufficiently detailed to avoid any ambiguity). See also Analyte . 3.19 Measurement interference: A cause of significant bias in the measured analyte concentration due to the effect of another component or property of the sample which may result from non specificity of the detection system, suppression of an

Definitions

3.1 Analyte Chemical / molecular entity or entities, which may be surrogate(s), measured by the measurement system (see also Measurand ). 3.2 Candidate Method The method submitted for validation 3.3 Candidate Method Result The final result of the qualitative analysis for the candidate method. 3.4 Claimed Detection Capability (CDC) An analyte concentration, expressed in mg/kg of gluten (or µ g per surface area), that demonstrates a Probability of Detection (POD) of at least 0.95. This may be claimed as one level that achieves a POD of 0.95 across all validated matrices, or individually per matrix and/or gluten source. The claimed detection capability must be verified empirically during method validation. 3.5 Collaborator An intended user who participates in the collaborative study. 3.6 Cross-reactivity A measurable positive response, above the LOD or CDC of the method, to a material other than the target analyte. 3.7 Cross-reactivity Study The examination of matrices that do not contain the claimed analyte, which are potentially cross-reactive, to determine that they are not detected by the method. 3.8 Data Set Results from a combination of an analytical instrument, device, or equipment and an operator, technician or analyst. Results from a single test site may serve as multiple data sets dependent on nonredundant operators and acceptable separation of effort. 3.9 Fractional POD Range Validation criterion that is satisfied when a test material yields both positive and negative responses within a set of replicate analyses. See also POD. 3.10 Gluten A protein fraction from wheat, rye, barley, oats 1 or their crossbred varieties and derivatives thereof, to which some persons are intolerant and that is insoluble in water

1 Oats can be tolerated by most but not all people who are intolerant to gluten. Therefore, the allowance of oats that are not contaminated with wheat, rye or barley in foods […] may be determined at the national level (CODEX STAN 118-1979)

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indicator reaction, or inhibition of the analyte. (CLSI EP07 A2) An interference can be endogenous, present in the sample, or exogenous, introduced into the sample during the measurement process. 3.20 Method A procedure that includes test material processing (e.g. analyte extraction), assay and data interpretation. 3.21 Probability of Detection (POD) The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given gluten level or concentration. POD is concentration dependent. 3.22 Qualitative Binary Method A method of analysis with two possible outcomes. (AOAC Appendix N) 3.23 Repeatability Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator (in the same laboratory) and repeating during a short time period. 3.24 Reproducibility Variation arising when identical test materials are analyzed in different laboratories by different operators on different instruments. 3.25 Robustness Study A study which tests the capacity of a method to remain unaffected by small but deliberate variations in method parameters and which provides an indication of its reliability during normal usage (USP 31). This includes an analysis of the stability and variation of production lots (for methods sold as kits). 3.26 Selectivity The degree to which the method can detect the analyte in the presence of other substances, matrices, or potentially interfering materials. Often measured by analyzing a variety of common matrices for cross-reactivity (3.6) and interference (3.14). 3.27 Spiked Test Material A food matrix into which a gluten source (e.g. flour) has been incorporated after all relevant food processing operations have been completed. 3.28 Test Material A material used for method validation that either contains gluten at a given concentration in the context of a food or environmental matrix or is a blank matrix free of gluten.

3.29 Test Portion The quantity of material taken from the test material (or, if both are the same, from the laboratory sample) and on which the test or observation is actually carried out. (ISO 6206:1979) 3.30 Test Sites Sites that simulate where the method is intended to be used, such as a traditional laboratory (e.g. public health or commercial laboratory) or non-laboratory indoor facility (e.g. manufacturing site, restaurant).

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Each gluten source material identified in Annex A, Table 1 must be tested in a rice flour matrix. For claimed gluten sources the test level is two times the CDC (as long as that is equal to or below 20 mg/kg, otherwise test at 20mg/kg) For unclaimed gluten sources the test level is twice the lowest CDC among the claimed gluten sources (as long as that is equal to or below 20 mg/kg, otherwise test at 20mg/kg.). Only if the method’s intended use is limited to environmental surface swabbing should this study be performed on environmental surfaces. Analyze one test portion of each prepared test material. The claimed gluten sources (wheat, rye, barley and/or oats) should all produce positive results. In the event that the single test portion tests negative, it may be retested in 20 additional test portions, with no failures allowed, to rule out an insufficient response to that gluten source. All data must be reported, and retests must be explained. Any gluten sources that do not meet these criteria cannot be claimed, and must be reported in the method instructions. A method claiming wheat must give a positive response to all listed Triticum species and Triticale, otherwise a claim for the specific Triticum species must be made. For methods claiming any Triticum species, only common wheat ( Triticum aestivum ) should be used in all other studies described in this guidance. Cross Reactivity The matrices identified in Annex A, Table 2, at full, undiluted concentration (with some exceptions as noted), will be prepared and analyzed with the candidate method as it is designed for testing food products. If any of the potential gluten sources from Table 1 are not claimed as a source for the candidate method, and are not being used as a matrix in the matrix studies, then they shall be included at full, undiluted concentration, in the list of matrices in Table 2. Only if the method’s intended use is limited to environmental surface swabbing should the cross-reactivity study be performed on environmental surfaces. Test one test portion per matrix. The method should produce negative results for all matrices. In the event that an unclaimed matrix tests positive, it or another example of the same matrix may be retested in six independent test portions, with no positive results, to rule out cross-reactivity. Any cross-reactive matrix must be reported to end users as part of the method limitations. All data must be reported, and retests must be explained. Interference The matrices identified in Annex A, Table 2 (including any unclaimed gluten sources from Table 1) will be spiked with

4 Method Validation Qualitative binary methods are those that produce one of two possible responses. This guidance has been developed for use with candidate methods that are designed to detect gluten. If a candidate method’s intended use is not covered by this document or existing standard method performance requirements (SMPRs), the standing AOAC expert review panel (ERP) for gluten, or other qualified agency, may determine the appropriate cross-reactivity/interference panels, and performance requirements. Method developers may prepare study test materials in house for the single laboratory validation (method developer study), but all test materials and test portions must be blind-coded and randomized. Analyses conducted by the method developer must be performed by an independent analyst without prior knowledge of the test materials undergoing analysis. Ideally, all test materials for the independent laboratory and collaborative studies should be prepared by an external entity independent from the method developer. At least one incurred test material for the independent laboratory and collaborative studies must be prepared by an external entity independent from the method developer. In situations where an independent entity is unavailable to prepare all of the test materials for the independent laboratory and collaborative studies, or their use is impractical for all test materials, method developers may produce and distribute test materials as long as detailed information is provided on procedures used to prevent bias (preparation, coding, etc.), and justification is provided for failing to use an independent entity to prepare all of the test materials. 4.1 Method Developer Validation Study 4.1.1 Scope The method developer validation study is intended to determine the performance of a method under the controlled conditions of a laboratory. The study is designed to evaluate performance parameters including cross reactivity, interference, POD at regulatory level(s), robustness, between-device variation, lot-to-lot variability and product stability. Gluten has multiple potential sources – wheat, rye, barley, oats and their hybrids and crossbreeds – and multiple regulatory levels. Developers must determine which of these sources and levels their method is intended to detect, and perform interference and POD studies for each claimed gluten source. 4.1.2 Selectivity study

Breadth

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gluten from each claimed gluten source at 20 mg/kg (or less, depending on the regulatory threshold of the target market/country), or two times the environmental surface detection limit for methods limited to environmental surface testing. Test material preparation is described in Annex B. The prepared test materials/surfaces will be analyzed with the candidate method as it is designed. Analyze one test portion per test material/surface. For each test portion/surface, the method should produce a positive result. In the event that the single test portion tests negative, it may be retested in 20 test portions, with no failures allowed, to rule out interference. Findings that certain matrices interfere with gluten detection should be investigated further, using additional similar matrices, to determine the full scope of interference.. Any interfering matrices must be reported in the method instructions. 4.1.3 Matrix / POD study The purpose of the laboratory matrix study is to measure the probability of detection at the claimed CDC(s), and below and above this level(s), in a controlled laboratory setting for all gluten sources, matrices and surfaces claimed in the method’s intended use statement. A matrix/POD study must be performed in each claimed matrix. In order to ensure that each claimed gluten source is represented, the gluten sources must be rotated across the claimed matrices as shown in Tables 1 and 2. The single matrix in which all gluten sources are tested, listed in Tables 1 and 2 as Matrix 1, should be the most highly processed matrix used in the validation study.

Number of Matrices Claimed

1

2

3

4

5

Wheat Barley Rye Oats

Wheat Barley Rye Oats Wheat

Wheat Barley Rye Oats Wheat

Wheat Barley Rye Oats Wheat

Wheat Barley Rye Oats Wheat

Matrix A Matrix B Matrix C Matrix D Matrix E

Barley

Barley

Barley

Rye

Rye

Oats Table 2. Rotation of gluten sources across claimed matrices for methods claiming to detect wheat, rye, barley and oats. The rotation of single gluten sources would continue for six matrices and greater. Alternatively, a matrix/POD study for a matrix category may be performed by testing each claimed gluten source, per the rotation shown in Table 1 or 2, in at least 5 examples from the category, equally distributed across each available type of processing (Annex C). Test materialss under each type of processing must be incurred. As an example, a method wishing to make a claim for the “Cereals (Not Fermented, Hydrolyzed or Fractionated)” category would need to test one matrix from each of the five provided processing categories, and in each instance, gluten would need to be added to the matrix prior to the described processing step. If a method developer was unable to access suitable equipment for preparing incurred test materials in the Pressure/Extruded type of processing, but was able to make incurred test materials for all other types of processing, they could not claim the “Cereals (Not Fermented, Hydrolyzed or Fractionated)” category. However, they could make a limited claim for “Raw, Processed, Baked, Fried and Dehydrated Cereals”. Method developers with the ability to produce fermented, hydrolyzed or fractionated matrix test materials that were incurred with gluten prior to these processes may make individual claims based on the fermentation organism, hydrolyzing agent or fractionation process. Example claims would be “Soy Tempeh fermented with Rhizopus oligosporus ”, “Modified corn starch hydrolyzed with sodium hydroxide”, or “Wheat starch fractioned with water”. For each gluten source-matrix combination, the developer must analyze a blank (no gluten) test material, one test material per each CDC, one test material at half of the lowest CDC, and one test material at least 1.5 times the highest CDC, prepared as described in Annex B. If the test material at half the lowest CDC produces a POD of 0%, an

Number of Matrices Claimed

1

2

3

4

5

Wheat Barley Rye

Wheat Barley Rye Wheat

Wheat Barley Rye Wheat

Wheat Barley Rye

Wheat Barley Rye

Matrix A Matrix B Matrix C Matrix D Matrix E

Wheat Wheat

Barley

Barley

Barley

Rye

Rye

Wheat

Table 1. Rotation of gluten sources across claimed matrices for methods claiming to detect wheat, rye and barley. The rotation of single gluten sources would continue for six matrices and greater.

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Robustness should be analyzed with a blank and a test material at the lowest CDC for one claimed gluten source, in a single incurred matrix. The matrix chosen should be one of the more challenging matrices (most highly processed) from the Matrix/POD study. Test ten (10) test portions for each factorial treatment combination. Data should be reported and analyzed as described in Annex D. Any parameter change that has a significant effect on the POD (p < .05) should be reported in the method instructions. 4.1.7 High-Dose Hook Effect Study To determine whether the method is adversely affected by high concentrations of the analyte, a “hook effect” study must be performed. For each gluten source claimed, perform an extraction of a whole flour (or higher concentrate if commercially available, such as vital wheat gluten) per the kit instructions. Dilute this extract 1:10, 1:100, 1:1000 and 1:10,000 in the kit extraction buffer. Follow the kit instructions to prepare these extracts for testing and analysis. Repeat this process four more times, resulting in five separate test portions for the whole gluten source and each dilution. All results are expected to be positive (or potentially invalid, for methods that have a ‘hook line’ indicating an excessive amount of analyte). In the event of any negative result, additional extracts may be tested. Negative results at any level may indicate a hook effect, and any gluten concentration where a hook effect is seen must be reported in the kit instructions. 4.1.8 Method Instructions Following the cross-reactivity, interference, matrix / POD, intended use, and robustness studies, the method developer should finalize the method instructions, taking into account any information learned from the validation. The method instructions should describe the method in detail. For antibody-based/binding methods this would include 1) information about the antibodies (or other equivalent binding agents) used, 2) whether the method targets one or multiple proteins 3) whether the antibodies used are monoclonal or polyclonal, and 4) the protein(s) used to generate the antibodies, and whether those were modified, fractionated or synthesized. Instructions must include specific qualifications or training required to perform the method, as well as links or references to this training. The method instructions should also describe, or provide diagrams/pictures describing, the interaction of the test

additional test material must be prepared and analyzed with a concentration midway between the half-lowest CDC and lowest CDC test materials. If the test material at half the lowest CDC produces a POD of 100%, an additional test material must be prepared and analyzed with a concentration midway between the blank (no gluten) and half-lowest-CDC test material. For environmental swabbing assays and CIP/rinse water validations, the developer will treat each surface and/or CIP/rinse water solution as a separate matrix, and analyze the prepared gluten source matrix materials as described above. Analyze twenty (20) test portions of each gluten source matrix-spike level test material. For environmental swabbing, perform (20) replicates of each gluten source matrix-spike level test material on each surface claimed. The test materials at and above the CDC must produce a POD of 0.95 or higher. The blank test materials must produce a POD of 0.05 or lower. 4.1.4 Data analysis and reporting for selectivity and matrix / POD studies A table or tables should be prepared for the results of the cross-reactivity and interference studies, listing the cross reactivity test results, as well as the test results for each matrix, gluten-source and spike level used in the interference study. In the case that multiple gluten sources are claimed, a CDC should be declared for each source. For the matrix / POD study, analyze the data for positive or negative responses, and prepare a table listing the matrix, gluten source, gluten level, number of test portions analyzed ( N ), number of positive responses ( x ), POD and 95% lower confidence limit (LCL) and upper confidence limit (UCL) of the POD, as well as POD curves (see AOAC Appendix H). Gluten concentrations at and above the CDC must produce a POD of 0.95 or greater. Blanks test materials must produce a POD of 0.05 or lower. 4.1.5 Intended use statement The method developer must provide a statement of the expected context(s) of use, expected matrices and expected analytical goals of the method. 4.1.6 Robustness study The method developer, in conjunction with the AOAC or other independent validation manager, is expected to make a good faith effort to determine which, and to what magnitude, parameters are most likely to vary in the hands of an end user. Each parameter should be varied both up and down by at least 20%. These parameters should be tested in a factorial or Plackett-Burman design, as described in Annex D.

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material. Test materials should consist of a blank, as well as a test material spiked at the lowest CDC of the kit for one claimed gluten source. 20 replicates of a single test material extract (one test portion extract, or one pooled extract from multiple test portions) should be performed on each kit lot, following the method instructions. Data should be reported in a table listing the test kit lot, age of the lot (real time and accelerated), the individual results for each test portion, as well as the POD and 95% POD confidence interval for each set of 20 replicates. The CDC test material must produce a POD of 0.95 or greater, and the blank test material must produce a POD of 0.05 or less, in each kit. Alternatively, method developers may provide internal lot to-lot and stability data for review, provided they meet the criteria of the combined study described above. 4.2 Independent Lab Validation Study 4.2.1 Scope The independent validation study, conducted under PTM guidance, should verify the analytical results obtained in the method developer study in a controlled laboratory setting. Independent labs must receive two types of training prior to performing the study. They shall receive training on the processes for independent studies (e.g. AOAC Method Validation Training Course), followed by training on the specific test method, which will be provided by the method developer. 4.2.2 Matrix Study An independent laboratory, approved by AOAC or the appropriate agency, will perform a matrix / POD study for each gluten source in at least one incurred matrix per claimed matrix category, or in at least one matrix per every five individual matrices claimed, as shown below:

material and each reagent used, and the method by which they produce a result. Step-by-step instructions for test portion preparation and performance of the method are required. Pictorial examples are encouraged. Details should be provided on result interpretation. For example, in kits where color change is a factor, end users should be directed on when to call a result positive based on color intensity. In this area as well, pictorial examples are encouraged. Include details on the reporting of results (positive, negative, < > the CDC, detected/not detected at the CDC, etc.), name of method, the units (wheat protein, gluten, gliadin, etc.), and the method for converting all results to gluten values. Final results should be reported in mg/kg gluten. The instructions must clearly outline any known limitations of the method, including any sources of gluten that are not detected by the method, any known under- or over sensitivity to specific gluten sources, cross-reactivity or interference from any matrices, and information on specific fractions/proteins targeted. Appropriate storage conditions must be provided. The kit lot number and expiration date should be clearly indicated on product packaging. 4.1.9 Product consistency and stability study If the test method is sold as a kit or device prepared in lots or batches, a product consistency and stability study must be performed to ensure that the performance of the product is consistent from lot-to-lot and over time under normal storage conditions for the shelf life of the product. Lot-to-lot consistency and product stability can be measured in the same set of experiments. A minimum of three separate product lots must be evaluated. The product lots should span the shelf life of the kit. For example, if the kit shelf life is 12 months, an approximately 12-month-old kit, six-month-old kit and recently produced kit should be evaluated. For an initial single lab validation, accelerated aging may be used if kits at the end of their shelf life are not available - if this is done, then lot-to-lot stability should still be performed across 3 recent lots. Kits should be aged using increased temperature storage as described in ASTM F1980-16 or CLSI EP25-A. Real time data is needed for validations such as AOAC Official Method applications, and prior to the first AOAC Performance Tested Method renewal. Test materials used in the evaluation should be made in any one matrix claimed for the method, or using a stable control

Claimed Matrices Matrices tested by Ind. Lab

1-5

6-10

11-15

16-20

1

2

3

4

Table 3 . Number of matrices to be tested by the independent laboratory, as related to the number of claimed matrices. The matrix/POD study will be performed as described in 4.1.3, including the rotation of claimed gluten sources shown in Tables 1 or 2, depending on the method claims. The independent laboratory must analyze at least one environmental surface for every 5 environmental surfaces claimed. The selection of which matrices/surfaces are

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gluten sources shown in Tables 1 or 2, depending on the method claims. If the method developer study consisted of only individual matrices, rather than matrix categories, then the collaborator study will test at least one incurred matrix for every 5 matrices tested in the method developer study, as shown below:

analyzed should be reflective of the range of difficulty associated with the claimed matrices. 4.2.3 Data Analysis and Reporting For the matrix / POD study, analyze the data for positive or negative responses, and prepare a table listing the matrix, gluten source, gluten level, number of test portions analyzed ( N ), number of positive responses ( x ), POD and 95% lower confidence limit (LCL) and upper confidence limit (UCL) of the POD, as well as POD curves (INSERT UPCOMING JAOAC REFERENCE). Gluten concentrations at and above the CDC must produce a POD of 0.95 or greater. Blanks test materials must produce a POD of 0.05 or lower. The collaborative study evaluates the performance of the method across multiple collaborators. The collaborative study is a requirement of the AOAC OMA program, and is not required for the AOAC PTM program. Collaborators should receive training on the specific test method, which will be provided by the method developer. The method developer shall provide a data submission form for the collaborator sites. It is recommended that a trial run be conducted prior to the initiation of the validation study. The purpose of the trial run is to ensure that logistics, test material handling and data reporting processes are worked out and understood by all of the collaborators. A small test material set (2-4 materials) should be analyzed. The trial run should be conducted under the same conditions as the validation study. A period of time should be allotted for troubleshooting after completion of the trial run, including a discussion with each collaborator to address issues and answer questions. The data from this trial run should not be analyzed or included in the validation report. 4.3.2 Number of Collaborators The minimum number of collaborators is based on AOAC Appendix N guidelines. Presently these call for usable data from a minimum of ten independent testing sites, so it is recommended to use more than ten to ensure at least ten usable data sets. 4.3.3 Matrix / POD study The collaborator sites will perform the matrix / POD studies for each claimed gluten source in at least one of the incurred matrices for each matrix category claimed in the method developer study, following the rotation of claimed 4.3 Collaborative Study 4.3.1 Scope

Claimed Matrices

1-5

6-10

11-15

16-20

Matrices tested by Collaborators

1

2

3

4

Table 4 . Number of matrices to be tested by each collaborator site, as related to the number of claimed matrices. The selection of the specific matrices used in the collaborative studies should be reflective of the range of difficulty and matrix category associated with the claimed matrices. Test materials to be analyzed will include, for each matrix, a blank test material, a test material at half the lowest CDC (or the fractional range of the POD, if found), a test material at each CDC, and a test material at least 1.5 times the highest CDC. The collaborator sites will be provided with, and test, a minimum of eight test portions of each test material. All test materials must be blinded when sent to the collaborator sites. 4.3.4 Data analysis and reporting For each gluten source-matrix-contamination level test material, calculate the POD for each collaborator. Then for each gluten source-matrix-contamination level, calculate the LPOD (mean POD across all collaborators) and standard deviation of collaborator PODs (sPOD). (Wehling et al 2011 – REPLACE WITH UPCOMING JAOAC REF) Report the LPOD estimates with 95% confidence intervals, and the POD curves. Calculate repeatability and reproducibility as described in Wehling et al. 2011 (REPLACE WITH UPCOMING JAOAC REF). Additional guidance for calculating reproducibility and limit of detection from the collaborative data can be found in ISO/TS 27878:2023. Gluten concentrations at and above the CDC must produce a LPOD of 0.95 or greater. Blanks test materials must produce a LPOD of 0.05 or lower. 4.3.5 Collaborator comments Comments on the candidate method should be encouraged from all collaborators, and any comments should be reported in the collaborative study report.

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4.4 Matrix Extension 4.4.1 Matrix Extension for single lab validation studies A single lab matrix/POD study must be performed as described in 4.1.3 . A matrix/POD study must also be completed by an independent laboratory as described under 4.2.2 and reported as described under 4.2.3. 4.4.2 Matrix Extension for multi-site collaborative studies A single lab matrix/POD study must be performed as described in 4.1.3 . A minimum of ten collaborator sites will perform the matrix / POD studies as described under 4.3.3 and reported as described under 4.3.4 .

Annex A

ANNEX A SELECTIVITY STUDY

*These may be omitted if they are being used as a gluten source in the validation matrix studies. # Oats are not regulated as a gluten source in all countries. Please see the definition of “Gluten”, and the related footnote, in the main guidance document. If oats are not considered a gluten source for the test method undergoing validation, then oats should be moved to Table 2 and treated as a commodity for the cross-reactivity and interference studies. + Oats that are not comingled with wheat, rye or barley may be difficult to source. Whole oat groats may need to be ground to generate a pure oat flour sample. ^For all minor wheat species and Triticale, 20 ppm samples can be prepared using the protein-to-gluten conversion factor in Annex B, or the method developer may use the cited wet chemistry method to determine their own conversion factor. All methodology and findings must be included in the study report. Table 2. Commodities for Cross-Reactivity and Interference Studies (materials should be tested as normally purchased/used. Any processing should be described (roasting, irradiation, etc.). Adapted from Koerner et al (2013) JAOAC 96, 1033-1040 Almond flour ( Prunus dulcis ) Faba bean flour ( Vicia faba ) Quinoa flour ( Chenopodium quinoa ) Amaranth flour ( Amaranthus spp.) Flax seed flour/ meal ( Linum usitatissimum ) Salmon ( Oncorhynchus spp.) Arrowroot ( Maranta arundinacea ) Garbanzo Bean/Chickpea flour ( Cicer arietinum ) Sesame flour ( Sesamum indicum ) Black bean flour ( Phaseolus vulgaris ) Green pea flour ( Pisum sativum ) Sorghum flour ( Sorghum bicolor ) Table 1. Gluten Sources (materials should be tested at two times the CDC, as long as that is equal to or below 20 mg/kg, in rice flour) Wheat Flour* ( Triticum aestivum ) Wheat Flour^ ( Triticum compactum ) Khorasan Wheat Flour^ ( Triticum turanicum ) Oat Flour* #+ ( Avena sativa ) Durum Wheat Flour^ ( Triticum durum ) Spelt Wheat Flour^ ( Triticum spelta ) RyeFlour* ( Secale cereale ) Einkorn Wheat Flour^ ( Triticum monococcum ) Triticale Flour^ (x Triticosecale ) Barley Flour* ( Hordeum vulgare ) Emmer Wheat Flour^ ( Triticum dicoccon )

Guar gum, dilute 1:10 in rice flour ( Cyamopsis tetragonoloba )

Beef Meat ( Bos taurus )

Soya flour ( Glycine max )

Hazelnut flour ( Corylus avellana )

Spices (at least one from Table 3) Sweet rice flour ( Oryza sativa glutinosa ) Tapioca flour/starch ( Manihot esculenta ) Tea, Ground ( Camellia sinensis )

Brown rice flour ( Oryza sativa )

Buckwheat flour ( Fagopyrum esculentum )

Lentil Flour ( Lens culinaris )

Lima bean flour ( Phaseolus lunatus )

Carob ( Ceratonia siliqua )

Chestnut flour ( Castanea sativa ) Lupin Flour ( Lupinus spp. )

Annex A

Chicken Meat ( Gallus gallus domesticus )

Milk powder, cow ( Bos taurus )

Teff Flour ( Eragrostis tef)

Millet flour ( Panicum miliaceum )

Cocoa

Walnuts ( Juglans spp.)

Oat Flour* + ( Avena sativa ), if not a claimed gluten source.

White bean flour ( Phaseolus vulgaris var. humilis )

Coconut flour ( Cocos nucifera )

Ground coffee ( Coffea arabica or Coffea canephora )

Pea protein ( Pisum sativum )

White rice flour ( Oryza sativa )

Yellow pea flour ( Lathyrus aphaca )

Corn meal ( Zea mays )

Peanuts ( Arachis hypogaea )

Xanthan gum, dilute 1:10 in rice flour (from Xanthomonas campestris )

Dried fruits or raisins ( Vitis vinifera ) Egg powder, chicken ( Gallus gallus domesticus )

Pork sausage ( Sus domesticus )

Potato flour/starch ( Solanum tuberosum )

**subject to further research, this may be of interest as a gluten-like source Buy from reputable sources and ensure that you are getting the actual material, and that it’s gluten free. This can be done by testing using an appropriate validated method. If you have information on the specific varietal tested, include that information in the validation report, as well as including the part(s) of the material that is tested (skin, flesh, stone, pit, etc.). For a multi-component matrix like pork sausage, provide all ingredients. Table 3. Possible Additional Commodities (materials should be tested as normally purchased/used - any processing should be described (roasting, irradiation, etc.) Carrageenan (dilute 1:10 in rice flour) Hemp ( Cannabis sativa ) Rye Grass ( Lolium perenne )** Cauliflower (Brassica oleracea var. botrytis ). Kidney Bean Flour ( Phaseolus vulgaris ) Romano Bean Flour ( Phaseolus coccineus ) Chia ( Salvia hispanica ) Marjoram ( Origanum majorana ) Sage ( Salvia officinalis ) Cinnamon ( Cinnamomum verum ) Paprika ( Capsicum annuum ) Sunflower Kernels ( Helianthus annuus ) Clove ( Syzygium aromaticum ) Parsley flakes ( Petroselinum crispum ) Thyme ( Thymus vulgaris ) Coriander seed ( Coriandrum sativum ) Poppy Seeds ( Papaver spp .) Turmeric ( Curcuma longa ) Cumin ( Cuminum cyminum ) Protein Sources (e.g. Duckweed ( Lemna minor ), insect, algal, fungal), Urad Dal Flour ( Vigna mungo ) Ginger Powder ( Zingiber officinale )

Annex B

ANNEX B PREPARATION OF MATERIALS FOR GLUTEN METHOD VALIDATION Until such time as a reference materials are available, the gluten source for all prepared test materials should be commercial, unbleached whole flours from each claimed gluten source. The chosen flour should be analyzed for Dumas or Kjeldahl nitrogen. Convert to percent crude protein by multiplying the nitrogen value by 5.83. Then convert to percent gluten by multiplying the crude protein value by the following factors, depending on the grain: • Barley 0.78 • Oats* 0.15 * Oats are not regulated as a gluten source in all countries. Please see the definition of “Gluten”, and the related footnote, in the main guidance document. These conversion factors are suggestions, and may vary across different grain materials. The factors come from two publications (1,2); the conversion factors for wheat rye and barley are based on the wet chemistry method described in Wehling and Scherf (2). Method developers may also use the wet chemical method in Wehling and Scherf (2) to arrive at the gluten content for their own wheat, rye and barley flours. (1) Schalk K, Lexhaller B, Koehler P, Scherf KA (2017) Isolation and characterization of gluten protein types from wheat, rye, barley and oats for use as reference materials. PLoS ONE 12(2): e0172819. doi:10.1371/journal.pone.0172819 (2) Wehling, P and Scherf, KA (2020) Preparation of Validation Materials for Estimating Gluten Recovery by ELISA According to SMPR 2017.21. Journal of AOAC International 103(1): 210-215. Finally, convert the percent gluten to mg/kg (ppm) gluten by multiplying the result by 10,000. As an example, a barley flour is tested and found to have a Dumas nitrogen level of 1.5%. This is multiplied by 5.83 to attain a crude protein level of 8.75%. Using the conversion factor for barley, the 8.75% crude protein is multiplied by 0.78 to obtain the gluten percent of 6.83%. This percent value is then multiplied by 10,000 to estimate the mg/kg (ppm) value at 68,300. This is equivalent to 68.3 mg of gluten per gram of flour. Making Spiked Materials Bulk spiked materials may be prepared for the selectivity, stability and lot-to-lot studies, and bulk spikes of raw materials are often made prior to the processing steps when making incurred test materials. These methods can be used for any material that has a small particle size or uniform consistency, including flours, baking mixes, spices, meats, sauces, dressings, ice cream (melted), etc. They can also be used in other matrices that can be dried and ground to a flour-like consistency, such as nuts, seeds, and bread crumbs. Thorough blending is key to a successful trial. For dry materials like flours, or for liquid consistencies, blending can be done in a blender or tumbler-style mixer, or even by manual tumbling of material in a zippered plastic bag. Add the spike material uniformly within the matrix, rather than adding it all in one • Wheat 0.74 • Rye 0.52

Annex B

location prior to blending it in. Making spikes in very fine matrices with small particle size can be difficult, and re-milling of the matrix and spike may be necessary to achieve particle size homogeneity. While gluten is not water soluble, it can be uniformly dispersed in sauces, dressings and other liquids by either spiking directly with flour, or making a suspension of gluten in the matrix, mixing it thoroughly to achieve uniformity, and using this to make the spikes. Make sure to mix the material again before any test portions are taken from it. For paste-like items and meats, spread the matrix out on aluminum foil, parchment or other non-stick surface, sprinkle the spike material uniformly across the top, and then recombine the matrix and mix by kneading. Extremely high-speed or high-heat mixing can alter the gluten results, so mechanical blending should be done in short pulses, and only for the duration needed to achieve sufficient uniformity. Liquid suspensions made in the kit extraction buffer can be used to spike individual test portions for the interference portion of the selectivity study prior to extraction. Liquid spiking of test portions may not be used for the matrix or other studies. If this method is used, state in the validation report that the selectivity study only tests for analytical interference, not interference with the extraction. Options for adding gluten to the matrix, either as a spike or prior to processing of an incurred matrix, include (see Figure 1): 1. Creation of a mid or high-level stock followed by serial dilution. The gluten concentration in the stock should be chosen to allow the largest volume of stock material to be used in the preparation of each spike level. 2. Creation of mid or high-level stock used to then make each individual bulk preparation. 3. Creating bulk spike level test materials directly from the source material (flour). 4. A combination of the above, in which spikes are made directly from the flour source for higher levels, then diluted to achieve lower levels) The method for creation of each test material must be described in the report. Any suitable validated quantitative method can be used to assess test material homogeneity. Assessing homogeneity of the high or mid level stock can be a good initial step before preparing lower level spikes. Homogeneity should be assessed for every bulk test material, or at least as many as needed to confirm that the mixing procedure is adequate to minimize distributional variance. Homogeneity should be assessed by testing 10 test portions, taken from throughout the material, individually extracted and run according to the method instructions of any validated quantitative assay (e.g., use 2 wells if the method calls for it). The preferred CV from the homogeneity data will depend on the method performance requirements, with the homogeneity SD below the required repeatability SD. Higher CVs may be expected at lower analyte concentrations. Use the stocks for testing on the same day if possible. Test materials made in dry matrices, like flours, can be stored at room temperature for several days, remixing each stock thoroughly before use. Test Materials made in perishable matrices (dairy products, meats) should be refrigerated for no more than 2 days, remixing each stock thoroughly before use. Test Materials may also be stored frozen in working aliquot sized portions for an extended period.

Annex B

Making Incurred Materials The section above, Making Spiked Materials, describes the initial steps in making an incurred material. The spiking must occur prior to the major processing step in order for the end product to be considered an incurred matrix. Further considerations for common types of processing are provided below Baked, Fried or Dehydrated Materials Baking, frying and dehydrating are processing methods that can be reasonably replicated at a small scale, in a laboratory. The same process applies for each. When possible, weigh the incurred material before and after processing. Any change in the analyte concentration above or below the expected value should be accounted for by the change in mass. When exact ppm values are needed, for example for a quantitative method, the moisture/weight change from processing must be accounted for in determining the amount of spike material to be added. If the entirety of the material cannot be weighed before and after processing, additional analyses can be performed to determine the potential analyte gain or loss, such as moisture content, protein, or zinc/other metals. If moisture/weight change results in a slightly higher ppm value than intended, higher level incurred test materials can be mixed with blank, processed material to achieve various concentrations. The lowest concentration achieved in this way should not be less than 10% of the concentration of the high-level incurred material. Larger discrepancies require a second incurred matrix to be made at a lower level. Pressure Treated/High Heat/Extruded These are processes that cannot normally be replicated outside of a manufacturing facility. If a manufacturer is particularly interested in the development of the assay, the kit developer may be able to partner with them to make gluten spikes on a pilot scale, using a similar method as described above for baked, fried and dehydrated products. In the absence of access to a manufacturing plant, some highly processed matrices can be incurred through “fortification”. An example would be a whole wheat puffed/extruded breakfast cereal. A pilot plant could create a mid-level spike (100 ppm, for example), which could be diluted down in a similarly processed blank matrix to create lower concentrations. Any validated method can be used to verify the absence of gluten in the non-gluten-containing matrix. Making Environmental Surface Test Materials Determine the surface area that’s expected to be swabbed. Typical area is 25 cm 2 – 100 cm 2 (approx. 4 in. 2 - 16 in. 2 ) Make suspensions from the flour in the kit extraction solution, or 60% ethanol solution. Create solutions at gluten concentrations ( µ g/ml) around the expected sensitivity level of the method, as described in the validation requirements.