Overview of the ISO 16140 series

Prepared by ISO/TC 34/SC 9/WG 3 “Method validation” (version: 20210322) 1

Overview of the ISO 16140 series – standards for validation and verification of microbiology methods

Version: 22 March 2021


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


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?


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’


European Commission Regulation

COMMISSION REGULATION (EC) No 2073/2005of 15 November 2005

on microbiological criteria for foodstuffs

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.

Specific rules for testing and sampling

Article 5

n c m M

1.1. Ready-to-eat foods intended for infants and

ready-to-eat foods for special medical purposes4 Listeria monocytogenes 10 0 EN/ISO 11290-1Products placed on the market during their

shelf-life

5 0 EN/ISO 11290-2 Products placed on the market during their

5 0 EN/ISO 11190-1

Before the food has left the immediate

control of the food buiness operator,who as

produced it

Stage where the criterion applies

100 cfu/g

Absence in 25 g

1.2. Ready-to-eat foods able to support the

growth of L. monocytogenes, other than those

intended for infants and for special medical

purposes

Listeria monocytogenes

Limits2 Sampling-plan1

Absence in 25 g

Food categoryMicro-organisms/their

toxins, metabolites

Analytical reference

method3


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)


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


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

Who participated in creation of these standards?


Many opportunities for global review and input

Preliminary Work Item (PWI)

New Work Item Proposal (NWIP)

Working Draft (WD)

Committee Draft (CD)

Draft International Standard (DIS)

Final Draft International Standard (FDIS)

International Standard (IS)

Ideas

Comments

and/or votes

Consensus

based

Market driven


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

https://www.iso.org/standard/59858.html

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


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’

https://www.iso.org/standard/54870.html


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).’;


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

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


Why do we need to validate and verify methods?


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


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.

Scope of the ISO 16140 series


Provides definitions for the terms used in the ISO 16140 series

Three clauses:

1. Scope

2. Terms and definitions (83)

3. Bibliography

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


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.


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)

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’


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

Categories

Raw milk and

dairy products

Heat-processed

milk and dairy

products

Raw meat and

ready-to-cook

meat products

(except poultry)

Ready-to-eat,

ready-to-reheat

meat products

Raw poultry and

ready-to-cook

poultry products

Ready-to-eat,

ready-to-reheat

meat poultry

products

Eggs and egg

products

(derivatives)

Raw and ready-

to-cook fish and

seafoods

(unprocessed)

Ready-to-eat,

ready-to-reheat

fishery products

Fresh produce

and fruits

Processed fruits

and vegetables

Dried cereals,

fruits, nuts,

seeds and

vegetables

Infant formula

and infant

cereals

Chocolate,

bakery products

and

confectionary

Multi-component

foods or meal

components

Pet food and

animal feed

Environmental

samples (food or

feed production)

Primary

production

samples (PPS)

*Same categories are provided in ISO 16140-3:2021, Table A.1, for verification studies.


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

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


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

RLOD study:

• artificially contaminated samples

• 1 (food) item per category, 20 samples

Inclusivity/exclusivity study

• use of 50/30 strains (Salmonella 100 strains)

Interlaboratory study

• 1 (food) item

• 10 data sets


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

Accuracy profile study:

• evaluation of precision and trueness (= accuracy)

• 6 samples each, 5 replicates /category

LOQ study:

• measurement principle is not enumeration

Inclusivity/exclusivity study

• use of 50/30 strains

Interlaboratory study

• 1 (food) item

• 8 data sets


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’

2.83 Verificationdemonstration 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

2.81 Validationestablishment of the performance characteristics of a method and provision of

objective evidence that the performance requirements

for a specified intended use are fulfilled


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)


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

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


Scope of Method vs Validation vs Laboratory application

Method

It specifies the

(group of)

products

(categories or

types or items) for

which the method

is claimed to be

applicable

Validation

It specifies the

(group of)

products

(categories or

types or items) for

which the method

is claimed to be

validated

It specifies the

(group of)

products

(categories or

types or items) for

which the method

is claimed to be

used by the

laboratory and are

within the scope

of validation

Laboratory


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


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)


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)


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


Flow chart for application of the ISO 16140 series


ISO 16140 series of standards and main users

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

Parts 2-6

Certification bodies

User laboratories

User laboratories

User laboratories

Certification bodies


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

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



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Overview of the ISO 16140 series

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