Draft guidance to determine the acceptability of microbiological methods

On this page

• Food and Drug Regulations
• Purpose
• New requirements for acceptable methods
• Annex 1: Acceptable methods
  • Recognized reference methods
  • Recognized method validation standards
  • Recognized independent certification bodies
• Annex 2: Canadian interpretation of ISO 16140-2
  • Validation protocol
  • Introduction
  • General requirements
  • Technical requirements and clarification for ISO 16140-2
  • Additional requirements and technical decisions
• Annex 3: Criteria to recognize an independent certification body
  • Purpose
  • Criteria

Food and Drug Regulations

A.01.010 acceptable method means a method of analysis or examination designated by the Minister as acceptable for use in the administration of the Act and these Regulations.

A.01.012. “The Minister shall, upon request, indicate that a method is acceptable or otherwise upon its submission to him for a ruling.”

Purpose

Health Canada currently fulfills its regulatory obligations under sections A.01.010 and A.01.012 of the Food and Drug Regulations through the use of the Microbiological Methods Committee (MMC) and the Compendium of Analytical Methods to issue a ruling on the acceptability of a microbiological method upon request.

In response to stakeholder feedback and to support greater international harmonization, Health Canada is implementing new guidance for determining the acceptability of microbiological methods. This updated approach simplifies the process by aligning with internationally recognized practices for method validation, while continuing to ensure that decisions are science-based and uphold a strong food safety system in Canada.

This updated approach does not impact which methods are currently deemed acceptable. Methods published in the Compendium of Analytical Methods and reference methods recognized by Health Canada (Annex 1, Table 1) will continue to be acceptable.

New requirements for acceptable methods

Health Canada will now rely on new requirements that continue to support science-based decision-making and uphold a strong food safety system in Canada. The new requirements are as follows:

1. The method is equivalent to a microbiological reference method recognized by Health Canada (see Annex 1, Table 1) and has been validated according to recognized method validation standards (see Annex 1 Table 2); and
2. The method is certified by a recognized independent certification body (see Annex 1 Table 3) to demonstrate it meets the Canadian Requirements for Determining the Equivalence for Food Microbiological Methods of Analysis as laid out in the Division 30 of the Food and Drug Regulations.

As with the previous process, the method must still be fit for its intended purpose and application (i.e., validated for the appropriate matrix and sample size with the ability to detect and/or enumerate the target microorganism at the relevant concentration).

Following implementation, the updated approach, together with the information presented in Annexes 1 through 3, will constitute the requirements for acceptable methods.

Annex 1: Acceptable methods

A method is acceptable under section A.01.010 of the Food and Drug Regulations when it is fit for the intended purpose and application (i.e., validated for the appropriate matrix and sample size with the ability to detect and/or enumerate the target microorganism at the relevant concentration), and meets one the following criteria:

1. The method is published in the Compendium of Analytical Methods; or 
2. The method is a microbiological reference method recognized by Health Canada (Table 1 below); or 
3. The method is equivalent to a microbiological reference method recognized by Health Canada (Table 1 below) and:
   • Has been validated according to the recognized validation standards (Table 2 below); and
   • Has been certified by a recognized independent certification body (Table 3 below) to demonstrate it meets the Canadian Requirements for Determining the Equivalence for Food Microbiological Methods of Analysis as laid out in the Division 30 of the Food and Drug Regulations.

Recognized reference methods

Recognized reference methods are listed in Table 1. The most recent version of the method is to be used.

Table 1: Recognized reference methods

Target microorganism	Reference method
Aerobic colony count (ACC)	MFHPB-18 Determination of the Aerobic Colony Counts in Foods
Salmonella	MFHPB-20 Isolation and Identification of Salmonella from Food and Environmental Samples
Listeria monocytogenes	MFHPB-30 Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples MFLP-74 Enumeration of Listeria monocytogenes in foods
Escherichia coli O157:H7	MFHPB-10 Isolation of Escherichia coli O157:H7/NM from foods and environmental surface samples
Yeast and mould	MFHPB-22 Enumeration of Yeasts and Moulds in Foods
Coliforms	MFHPB-19 Enumeration of Coliforms, Faecal Coliforms and of Escherichia coli in Foods using the MPN Method MFHPB-31 Determination of Coliforms in Foods Using Violet Red Bile Agar
Faecal coliforms	MFHPB-19 Enumeration of Coliforms, Faecal Coliforms and of Escherichia coli in Foods using the MPN Method
Escherichia coli	MFHPB-19 Enumeration of Coliforms, Faecal Coliforms and of Escherichia coli in Foods using the MPN Method MFHPB-27 Enumeration of Escherichia coli in Foods by Direct Plating (DP) Method
Cronobacter spp.	ISO 22964 Microbiology of the food chain — Horizontal method for the detection of Cronobacter spp.
Bacillus cereus group	MFLP-42 Isolation and Enumeration of the Bacillus cereus Group in Foods
Clostridium perfringens	MFHPB-23 Enumeration of Clostridium perfringens in Foods
Staphylococcus aureus	MFHPB-21 Enumeration of coagulase positive Staphylococcus aureus in Foods
Vibrio parahaemolyticus	MFLP-37 Detection, isolation, and enumeration of Vibrio parahaemolyticus and/or Vibrio vulnificus in Seafood
Pseudomonas aeruginosa	ISO 16266 Water quality - Detection and enumeration of Pseudomonas aeruginosa-Method by membrane filtration
Enterobacteriaceae	ISO 21528-1 and 21528-2 Microbiology of the food chain-Horizontal method for the detection and enumeration of Enterobacteriaceae
Campylobacter	MFLP-46 Isolation of thermophilic Campylobacter from food
Yersinia enterocolitica	MFLP-48 Isolation of Yersinia enterocolitica from foods and environmental samples

Recognized method validation standards

Method validation protocols are listed in Table 2. These protocols are to be used in conjunction with the Canadian interpretation of ISO 16140-2 (see Annex 2).

Table 2: Recognized method validation standards

Organization	Validation protocol
International Organization for Standardization (ISO)	ISO 16140-1:2016 Microbiology of the food chain — Method validation Part 1: Vocabulary and associated amendments In conjunction with:  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 and associated amendments

Recognized independent certification bodies

Organizations that have successfully demonstrated to Health Canada that they meet the Criteria to recognize an independent certification body (see Annex 3) are listed in Table 3.

Table 3: Recognized independent certification body

Organization	Date of recognition by Health Canada
To be populated as independent certification bodies are recognized	To be determined

Annex 2: Canadian interpretation of ISO 16140-2

Validation protocol

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 16140-2:2016/Amd 1:2024 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.

Introduction

This interpretation is intended to complement the protocols published in ISO 16140-2 for the validation of alternative (proprietary) methods against a reference method. The instructions provided within this document are intended to clarify and/or explicitly state the requirements for demonstrating equivalence to reference methods recognized by Health Canada in the context of certifications issued by independent certification bodies.

Additionally, the interpretation provides transparent and consistent guidance for end users on implementation dates and respective transition periods for any updates or amendments to the standard or Canadian interpretation of such.

This annex is applicable for validation studies on qualitative or quantitative methods.

General requirements

The requirements and clarifications specified in Table 1 below are mandatory for routine validation studies. Where clarifications are not listed in Table 1 below, the protocols in the validation standard are to be followed as written. Exceptions to these requirements are possible on a case-by-case basis but require approval from Health Canada and the MMC.

The insert for the alternative method test kit must reflect the validation protocol, including all confirmation procedures. Confirmation procedures shall always be conducted according to the applicable microbiological reference method recognized by Health Canada as specified in Annex 1, Table 1 or approved confirmation methods.

Table 1: Technical requirements and clarifications for ISO 16140-2

5 Qualitative methods — Technical protocol for validation

5.1 Method comparison study

Subclause	Technical requirements and clarifications for ISO 16140-2
5.1.1 General Considerations	The method comparison study is performed using a reference method recognized by Health Canada.
5.1.3 Sensitivity study	5.1.3.2 Number of samples A minimum of 7 positive samples per type is required
	5.1.3.4 Calculation and interpretation for sensitivity The number of False Negatives (FN) generated by the alternative method during this study must be considered and should be <2%Footnote *
5.1.4 Relative level of Detection Study	5.1.4.2 Calculation and interpretation of the RLOD Evaluation using the AOAC probability of detection (POD) is to be included in the evaluation of this study.
Table 5.1 Footnote * Interim threshold of 2% applies pending the establishment of the ISO SC9/WG3 limits. Table 5.1 Return to footnote * referrer

6 Quantitative methods — Technical protocol for validation

6.1 Method Comparison Study

Subclause	Technical requirements and clarifications for ISO 16140-2
6.1.1 General considerations	The method comparison study is performed using a reference method recognized by Health Canada.
6.1.3.3 Calculation and interpretation of accuracy profile study	Acceptability Limits are set at ±0.5 log10 and cannot be recalculated in the event where one or the other is exceeded (i.e., implementation of 6.1.3.3 step 9 is not allowed).

Additional requirements and technical decisions

False negative (FN) threshold

• The combined number of FN results generated by the alternative method during the sensitivity and RLOD studies must be considered when determining method equivalency and should be <2%.^{Footnote 1}
• The false negative ratio (FNR %) for food types and categories should also be reviewed and, when significant, discussed/justified in the study summary report.

Environmental Samples

• Details regarding swabbing protocol (sample medium, specific neutralizer, surface area sampled, sample dilution ratio) must be clearly detailed in the study summary report.

Software/Hardware

• Software and hardware versions validated must be specified on the certificate.

Test portion sizes

• The validated test portion size of the reference method will be used in case a larger test portion size is used in the alternative method. This is independent of paired or unpaired study design. The validated test portion size of the alternative method shall be specified on the certificate.

Annex 3: Criteria to recognize an independent certification body

Purpose

This annex establishes the criteria that must be met by a certification body to be recognized by Health Canada. If Health Canada concludes that a certification body meets all the criteria mentioned below, the certification body will be included in Annex 1, Table 3 (“Recognized independent certification body”).

Criteria

• The certification body is an independent organization that facilitates the validation and certification of alternative methods in support of food safety regulations.
• The certification body has a transparent process and procedures for certification of microbiological methods.
• The certification body publishes or has available upon request the requirements for the validation and certification of microbiological methods and a mechanism to communicate changes or updates.
• The certification body provides an objective, transparent, and standardized assessment of methods, using Health Canada-recognized validation standards to establish performance relative to a Health Canada-recognized reference method.
• The certification body provides details on how it maintains oversight of the technical competency of the expert laboratories and collaborating laboratories it uses in conducting validation studies.
• The certification body ensures that the expert laboratories overseeing and performing the method validation work are ISO 17025 accredited, have the reference method in their scope of accreditation, and demonstrate technical competence in the method being validated.
• The certification body ensures that the collaborative laboratories are ISO 17025 accredited or demonstrates that the laboratory is operating under an adequate quality system.
• The certification body maintains a list of committee members and the application procedure is publicly available or available upon request.
• The certification body ensures that committee members possess and maintain the necessary technical competency and that the committee is composed of representatives with method validation expertise from varied backgrounds and sectors.
• The certification body maintains a publicly available list of certified methods and the associated validation study reports.
  • The published certificate must be available in English and French and contain at a minimum the following information:
    • the alternative method identifier and the manufacturer and supplier information;
    • the list of validated foods;
    • the title of the microbiological reference method used for the validation study;
    • the validation protocols to which the alternative method was validated according to;
    • the software and hardware validated;
    • the validated test portion size of the alternative method; and
    • the approval and expiry dates of the certificate.
• The certification body maintains a process for reassessment of validated methods and renewal of certifications at fixed intervals.
• The certification body maintains a process for complaints, appeal and technical questions.
• The certification body uses the recognized method validation standards specified in Annex 1, Table 2 when validating alternative methods.
• The certification body has established processes for seeking and obtaining technical clarification, where necessary, from the MMC on the application and interpretation of the Canadian interpretation of ISO 16140-2 and Health Canada-recognized microbiological reference methods.
• The certification body has processes in place to identify, manage, and communicate changes that may affect its continued ability to meet Health Canada’s recognition criteria, including providing advance notification to Health Canada of such changes.