Overview of the ISO 16140 series

– standards for validation and verification of microbiology methods

Prepared by ISO/TC 34/SC 9/WG 3 “Method validation”

22 March 2021 1
(version: 20210322)
Version:
Objectives of this presentation

• Background on validation standard development

• Familiarize you with each of the six published standards

• Outline when to choose the use of each standard

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Need for validated food microbiology methods

Recognized more than 30 years ago - proprietary methods:

• Provide faster results versus traditional culture methods

• Cost effective
• More efficient
• Becoming more sensitive

Acceptance by regulatory authorities?

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European Committee for Standardization (CEN)

• European Eureka R&D programme requested CEN to develop

technical protocols for validation of methods

• Technical protocols were transformed into a standardized protocol

as an International Standard by ISO/TC 34/SC 9 ‘Food products –
Microbiology’

ISO 16140:2003 ‘Protocol for the validation of alternative methods’

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European Commission Regulation
COMMISSION REGULATION (EC) No 2073/2005
of 15 November 2005
on microbiological criteria for foodstuffs
1
Micro-organisms/their Sampling-plan Limits2 Analytical reference
Food category Stage where the criterion applies
toxins, metabolites n c m M method3
1.1. Ready-to-eat foods intended for infants and Products placed on the market during their
Listeria monocytogenes 10 0 Absence in 25 g EN/ISO 11290-1
ready-to-eat foods for special medical purposes 4 shelf-life
1.2. Ready-to-eat foods able to support the 5 0 100 cfu/g EN/ISO 11290-2 Products placed on the market during their
growth of L. monocytogenes, other than those Before the food has left the immediate
Listeria monocytogenes
intended for infants and for special medical 5 0 Absence in 25 g EN/ISO 11190-1 control of the food buiness operator,who as
purposes produced it

Article 5
Specific rules for testing and sampling
The use of alternative analytical methods is acceptable when the methods are validated against
the reference method in Annex I and if a proprietary method, certified by a third party in
accordance with the protocol set out in EN/ISO standard 16140 or other internationally accepted
similar protocols, is used.

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ISO 16140:2003 – Needed several updates

2005:
• Need for revision of ISO 16140
• Need for more standards on method validation

2006:
• ISO/TC 34/SC 9 in cooperation with related CEN/TC started a
Working Group for standards development on method validation
• Established objectives for this Working Group (WG 3)

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WG 3 ‘Method validation’: several objectives

• Revise ISO 16140

• Develop a standard on method verification
• Develop standard for validation of reference methods
• Develop standard for singe-laboratory validation
• Develop standard on intermediate validation
• Develop standard for validation of confirmation methods

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Who participated in creation of these standards?

WG 3 ‘Method validation’:
• > 100 experts coming from 23 countries and liaison organizations,
representation of:
o government
o industry
o laboratories
o academic and research bodies
o method developers and validation bodies
• developed 7 standards and more standards will follow

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Many opportunities for global review and input

Preliminary Work Item (PWI)

Market driven
New Work Item Proposal (NWIP)
Ideas
Working Draft (WD)

Committee Draft (CD)

Consensus
Draft International Standard (DIS)
based
Final Draft International Standard (FDIS)
Comments
and/or votes International Standard (IS)

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Method validation – Reference methods
ISO 17468:2016 ‘Microbiology of the food chain — Technical requirements
and guidance on establishment or revision of a standardized reference
method’

https://www.sciencedirect.com/journal/international-journal-of-food-microbiology/vol/288/suppl/C

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Method validation – Alternative methods
ISO 16140-2:2016
‘Microbiology of the food chain —
Method validation — Part 2: Protocol
for the validation of alternative
(proprietary) methods against a
reference method’

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COMMISSION REGULATION (EU) 2019/229 of 7 February 2019
amending Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs
Article 1
Amendments to Regulation (EC) No 2073/2005
(1) in Article 2 the following points are inserted after point (m):
‘(n) “a broad range of foods”, as referred to in EN ISO 16140-2, means food as defined by the first
subparagraph of Article 2 of Regulation (EC) No 178/2002 of the European Parliament and of the Council
(*);

(o) “independent certification body” means a body which is independent from the organisation that
manufactures or distributes the alternative method, and which provides a written assurance, in the form of
a certificate, testifying that the validated alternative method meets the requirements of EN ISO 16140-2;

(p) “production process assurance of the manufacturer” means a production process whose
management system guarantees that the validated alternative method remains conform to the
characteristics required by EN ISO 16140-2 and ensures that mistakes and defects in the alternative
method are prevented;
(*)Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general
principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in
matters of food safety (OJ L 31, 1.2.2002, p. 1).’;

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Certification bodies: using ISO 16140-2 and ISO 16140-6

Method validation certificates and reports on their websites:

• https://microval.org/en/issued-certificates/
• https://nf-validation.afnor.org/en/food-industry/#discover-certified-methods
• https://www.nmkl.org/index.php/en/nordval

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Why do we need to validate and verify methods?

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ISO 16140 series of standards

1. Terminology

2. Validation of alternative (proprietary) methods

3. Method verification in a single laboratory

4. Method validation in a single laboratory

5. Factorial interlaboratory validation for non-proprietary methods

6. Validation for confirmation and typing methods

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Scope of the ISO 16140 series

Standards apply to validation or verification of methods for the

analysis of microorganisms in:

• products intended for human consumption;

• products intended for animal feeding;

• environmental samples in the area of food and feed production, handling;

• samples from the primary production stage.

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ISO 16140-1:2016 ‘Microbiology of the food chain –
Method validation – Part 1: Vocabulary’

Provides definitions for the terms used in the ISO 16140 series

Three clauses:
1. Scope
2. Terms and definitions (83)
3. Bibliography

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ISO 16140-1:2016 – Important definitions

2.59 reference method

internationally recognized and widely accepted method

2.4 alternative method (method submitted for validation)

method of analysis that detects or quantifies, for a given category of products, the
same analyte as is detected or quantified using the corresponding reference
method (2.59)
Note 1 to entry: The method can be proprietary. The term ‘alternative’ is used to refer to the entire ‘test
procedure and reaction system’. This term includes all ingredients, whether material or otherwise, required for
implementing the method.

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ISO 16140-2:2016 ‘Microbiology of the food chain –
Method validation – Part 2: Protocol for the validation of
alternative (proprietary) methods against a reference method’

Provides a protocol for validation of alternative (proprietary) methods

• Compared to the corresponding reference method

• Applicable to qualitative and quantitative methods

• Succeeds the first version of ISO 16140 (ISO 16140:2003)

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ISO 16140-2: Annex A
Classification of (food) categories and suggested target combinations for validation studies

Categories
Raw milk and Heat-processed Raw meat and Ready-to-eat, Raw poultry and Ready-to-eat,
dairy products milk and dairy ready-to-cook ready-to-reheat ready-to-cook ready-to-reheat
products meat products meat products poultry products meat poultry
(except poultry) products

Eggs and egg Raw and ready- Ready-to-eat, Fresh produce Processed fruits Dried cereals,
products to-cook fish and ready-to-reheat and fruits and vegetables fruits, nuts,
(derivatives) seafoods fishery products seeds and
(unprocessed) vegetables

Infant formula Chocolate, Multi-component Pet food and Environmental Primary

and infant bakery products foods or meal animal feed samples (food or production
cereals and components feed production) samples (PPS)
confectionary

*Same categories are provided in ISO 16140-3:2021, Table A.1, for verification studies.
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Part 2: Protocol for the validation of alternative (proprietary)
methods against a reference method

Part 2 consists of two steps:

• Methods comparison study (MCS) – testing diversity of samples / (food) items

• Interlaboratory study (ILS) – establishing reproducibility of the method using a

single (food) item

• Data is evaluated using preset criteria [acceptability limits (AL)]

• Alternative methods performing better than the reference method

are accepted

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Part 2: Protocol for the validation of alternative (proprietary)
methods against a reference method – Qualitative

Method comparison study

Sensitivity study:
• (naturally) contaminated samples
• ≥ 5 food categories with ≥ 60 samples each
Interlaboratory study
• 1 (food) item
RLOD study: • 10 data sets
• artificially contaminated samples
• 1 (food) item per category, 20 samples

Inclusivity/exclusivity study
• use of 50/30 strains (Salmonella 100 strains)

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Part 2: Protocol for the validation of alternative (proprietary)
methods against a reference method – Quantitative
Method comparison study
Relative trueness study:
• ≥ 5 food categories,15 samples /category
• graphical interpretation of the data Interlaboratory study
Accuracy profile study: • 1 (food) item
• evaluation of precision and trueness (= accuracy) • 8 data sets
• 6 samples each, 5 replicates /category
LOQ study:
• measurement principle is not enumeration
Inclusivity/exclusivity study
• use of 50/30 strains
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ISO 16140-3:2021 ‘Microbiology of the food chain –
Method validation – Part 3: Protocol for the verification of reference
and validated alternative methods implemented in a single laboratory’

2.81 Validation
establishment of the performance characteristics of a method and provision of
objective evidence that the performance requirements
for a specified intended use are fulfilled

2.83 Verification
demonstration that a validated method performs, in the user’s hands, according
to the method’s specifications determined in the validation study and is fit for its
intended purpose

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So why do methods also need to be verified?

The airplane “works”

(is validated for use)

But would you want to fly

with someone who didn’t
know how to properly use it?
(proper use is verified)

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Part 3: Protocol for the verification of reference and validated
alternative methods implemented in a single laboratory

Verification is a 2-step procedure:

1. Implementation verification
• Demonstrate the user laboratory can run the method correctly
• Verify using ONE (food) item
2. (Food) item verification
• Demonstrate the user laboratory can run the method with the (food) items
claimed by the user laboratory (laboratory application)
• Verify using categories tested in your laboratory

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Scope of Method vs Validation vs Laboratory application

Method Validation Laboratory

It specifies the
(group of)
It specifies the It specifies the
products
(group of) (group of)
(categories or
products products
types or items) for
(categories or (categories or
which the method
types or items) for types or items) for
is claimed to be
which the method which the method
used by the
is claimed to be is claimed to be
laboratory and are
applicable validated
within the scope
of validation

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Part 3: Protocol for the verification of reference and validated
alternative methods implemented in a single laboratory

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ISO 16140-4:2020 ‘Microbiology of the food chain –
Method validation – Part 4: Protocol for method validation in a
single laboratory’

Addresses method validation within a single laboratory:

• Validation with or without a reference method
• Results are only valid in the laboratory which conducted the study
• Method verification (Part 3) is not required

Validation can be conducted using:

• Conventional method validation design (Part 2)
• Factorial method validation design (Part 5)

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ISO 16140-5:2020 ‘Microbiology of the food chain –
Method validation – Part 5: Protocol for factorial
interlaboratory validation for non-proprietary methods’

Method validation requires interlaboratory studies:

• Challenging to find laboratories to participate in collaborative studies

ISO 16140-5 provides a protocol that:

• Reduces # of laboratories required, using factorial design
• Applies only to methods that have been fully specified and optimized, because:
o Several factors are altered simultaneously (technician, culture medium)
o Method is used in a range of different factor settings (time, temperature)

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ISO 16140-6:2019 ‘Microbiology of the food chain – Method validation
– Part 6: Protocol for the validation of alternative (proprietary)
methods for microbiological confirmation and typing procedures’

Different from other parts of ISO 16140 series:

• Specific to validation of the confirmation or typing procedure of a method

• Confirmation advances a suspected (presumptive) result to a confirmed result

• Validation includes comparison to the reference confirmation procedure

• Intended for “full” validation of an alternative (proprietary) method through

confirmation and typing = alternative confirmation method

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Flow chart for application of the ISO 16140 series

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ISO 16140 series of standards and main users

1. Terminology Parts 2-6

2. Validation of alternative (proprietary) methods Certification bodies

3. Method verification in a single laboratory User laboratories

4. Method validation in a single laboratory User laboratories

5. Factorial interlaboratory validation for non-proprietary methods User laboratories

6. Validation for confirmation and typing methods Certification bodies

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WG 3 ‘Method validation’: achieved objectives

Objectives Published standards

Revision of ISO 16140:2003 ISO 16140-1:2016
ISO 16140-2:2016
Method verification ISO 16140-3:2021
Validation of reference methods ISO 17468:2016
Single-laboratory method validation ISO 16140-4:2020
Intermediate method validation ISO 16140-5:2020
Validation of confirmation methods ISO 16140-6:2019

More information related to these International Standards can be found on:

https://committee.iso.org/home/tc34sc9

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Questions?

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