AOAC Gluten Qualitative Validation Guidance-FINAL (July 2023)

Q UALITATIVE G LUTEN M ETHOD V ALIDATION G UIDELINES PG . 5

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

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