Overview - ISO 16140-2:2016/Amd 1:2024 (Method validation for microbiology of the food chain)
ISO 16140-2:2016/Amd 1:2024 updates Part 2 of the ISO 16140 series, which defines a protocol for validating alternative (proprietary) microbiological methods against an accepted reference method. The 2024 amendment revises how qualitative method-comparison studies are evaluated, updates relative level of detection calculations in interlaboratory studies, refines calculation and interpretation of relative trueness, and adds a commercial sterility testing protocol for specific sterilized or UHT dairy and plant‑based liquid products.
Key technical topics and requirements
- Purpose: Validation of an alternative method (complete test procedure and reaction system) to demonstrate equivalence or defined performance relative to a reference (standardized) method.
- Study design: Combines single‑laboratory experiments and an interlaboratory study as appropriate; supports both paired and unpaired data designs when methods use different principles or enrichment procedures.
- Data evaluation: Revised rules for interpreting qualitative comparisons (positive/negative agreement, false positives/negatives) and for documenting deviations with explanatory footnotes.
- Performance metrics: Updated approaches for calculating relative level of detection (rLOD) in interlaboratory studies and for assessing relative trueness between methods (amendment provides revised calculation and interpretation guidance).
- Test portion sizes: Alternative methods are validated for a defined test portion (e.g., 10 g, 25 g, 375 g); validated use extends to any smaller sizes if protocol (dilution, incubation) is unchanged.
- Organizing laboratory competence: The organizing lab must be competent to run both the reference and alternative methods (e.g., documented ISO/IEC 17025 compliance and training).
- Re‑validation and change control: Major technical changes to an alternative method require re-validation (and possibly re‑verification by user labs) to confirm no significant impact on performance.
- Special case: Annex J / amendment text includes a commercial sterility testing protocol for sterilized/UHT liquid products.
Practical applications and who uses this standard
- Test kit and method developers validating proprietary microbiological assays for foods and beverages.
- Accredited testing laboratories performing method validation studies and interlaboratory comparisons.
- Food manufacturers and quality teams implementing validated alternative methods for routine monitoring (e.g., sterility checks for UHT products).
- Regulators and conformity assessors reviewing evidence of method equivalence and performance claims.
- Standards bodies and method validation laboratories coordinating multi‑laboratory studies.
Related standards
- ISO 16140 series: Part 1 (vocabulary), Part 3 (verification), Part 4 (single‑lab validation), Part 5 (factorial interlaboratory validation), Part 6 (confirmation/typing procedures)
- ISO 17468 (technical rules for method development/validation)
- Examples referenced: ISO 21528-2, ISO 6579-1 (contextual relation to quantitative/reference methods)
This amendment is essential for anyone validating or implementing alternative microbiological methods in the food chain, offering updated statistical evaluation, clearer interpretation rules, and expanded scope for sterility testing of specific products.
Open preview (PDF)Microbiology of the food chain — Method validation — Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method — Amendment 1: Revision of qualitative method comparison study data evaluation, relative level of detection calculations in the interlaboratory study, calculation and interpretation of the relative trueness study, and inclusion of a commercial sterility testing protocol for specific products