AOAC GFA Working Group Documents for Review (November 2022)

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

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, 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. 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 replicate per sample. The method should produce negative results for all samples. 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 (biological replicates), 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 will be spiked with 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. Sample preparation is described in Annex B. The prepared samples/surfaces will be analyzed with the candidate method as it is designed. Test one replicate per sample. For each sample, the method should produce a positive result. In the event that the single sample replicate tests negative, it may be retested in 20 biological replicates, 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. Unless the interference can be shown to be limited to very few and specific matrices, and this claimed limitation would be of no concern to the method’s end

4 Method Validation Qualitative binary methods are those that produce one of two possible responses (as defined in AOAC Appendix N). 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. 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. From a regulatory perspective, gluten has four potential sources – wheat, rye, barley and oat grains – 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. For all studies, the sample test portions must be blind coded and randomized to the analyst, as well as to the reader (the person determining the result) if the method relies on visual determination of the test result. 4.1.2 Selectivity study Breadth The gluten source materials identified in Annex A, Table 1, should be tested at two times the lowest CDC (as long as that is equal to or below 20 mg/kg, otherwise test at 20 mg/kg) in a rice flour matrix. Only if the method’s intended use is limited to environmental surface swabbing should this study be performed on environmental surfaces. Test one replicate per sample. The claimed gluten sources (wheat (all Triticum species), rye, barley and/or oats) should all produce positive results. In the event that the single sample replicate tests negative, it may be retested in 20 biological replicates, 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