International Stakeholder Panel on Alternative...

WG on Microbiological Guidelines ISPAM Mid-Year Meeting, Rockville, MD. March 20, 2012 v3

International Stakeholder Panel on Alternative Methods (ISPAM)

Working Group on Microbiological Guidelines for Alternative Methods Mid-Year Meeting

March 20, 2012 Rockville, MD, USA 10:00 am – 2:00 pm

DRAFT Meeting Minutes Attendees: Russ Flowers, Mérieux NutriSciences, ISPAM Chair Ron Johnson, BioMerieux, Sub-Group Chair Doug Abbott, Consultant Marcia Armstrong, Qiagen Stan Bacler, Health Canada Christopher Blake, Nestec Ltd. DeAnn Benesh, 3M Reginald Bennett, FDA Peter Bodnaruk, Ecolabs Joe Boison, Canadian Food Inspection Agency Philip Bronstein, USDA-FSIS Scott Burrows, Qiagen Yi Chen, FDA-CFSAN Erin Dreyling, Roka Bioscience Peter Evans, USDA FSIS Berengere Genest, BioMerieux Qian Graves, FDA-CFSAN Tom Hammack, US FDA/CFSAN Melinda Hayman, Food Safety Net Services

Anthony Hitchins, FDA-CFSAN (Retired) Paul In’t Veld, VWA, ISO Wendy Lauer, Bio-Rad Laboratories Glenn Liacouras, VWR International Xiu mei Liu, China National Center for Food Safety Risk Assessment Tara Lydick, DE Publick Health Lab/APHL Kirsten Mattison, Health Canada Michael McLaughlin, US FDA/ORA John Mills, BioMérieux Julie Moulin, Nestle Sam Mohajer, CFIA Mark Mozola, Neogen Christopher Snabes, American Proficiency Institute Danièle Sohier, ADRIA Darryl Sullivan, Covance F. Morgan Wallace, DuPont Qualicon Paul Wehling, General Mills Pamela Wilger, Cargill

Staff: Krystyna McIver Nora Marshall Tien Milor Scott Coates

2 Mid-Year ISPAM meeting Microbiological Working Group March 20, 2012 v3nlm

II. ISPAM WORKING GROUP ON MICROBIOLOGICAL GUIDELINES

ISPAM chairman, Russell Flowers, presented the top five priority areas approved by ISPAM for harmonization at the September 16, 2011 meeting, the work accomplished since the September meeting, and goals of the current meeting. (See Attachment 1).

A. Selection of Food/Category (sample matrix; Recommendation from the Sub-Group on

Food Categories - Ron Johnson, BioMérieux and Danièle Sohier, ADRIA

Ron Johnson, Sub-group Chair reported that the sub-group voted to adopt the Draft EN ISO/CD ISO 16140 – 2, Annex B, “Classification of sample types for validation studies, food categories table, Part 2,, “Microbiology of food and animal feeding stuffs – Method validation.” The group also decided to build on the food categories in ISO 16140 Annex B to include requirements for food categories from other countries, primarily United States and Canada. The revised Annex B is to be presented to ISO for Adoption as part of the revision to 16140-2. Mr. Johnson’s presentation is under Attachment 2.

Danièle Sohier, ADRIA and member of ISO TC 34/SC9, the committee revising 16140-2, presented the revised food category table as a result of the work of the sub-group. Her presentation is under Attachment 3. The revisions included food category sub-divisions and the addition of other food categories, including infant formula and cereals; a new design in the pet food and animal feed category; and the addition of the target species C. botulinum. The proposed revised Annex B to ISO 16140-2, Food Category Table is under Attachment 4. Because different regulatory authorities have slightly difference requirements as to the classification of food, Sohier reported that an informative introduction to Annex B was prepared by the sub-group with notes outlining the differences (see the Informative Annex B under Attachment 5).

After some discussion, the WG agreed with the basic food categorizations that the sub-group was proposing. The group decided, however, that a few issues remained to be discussed. For example, how to handle evolving food categories or ones that have been redefined by a regulatory agency so as to focus on a particular or emerging food pathogen in a commodity of concern; and the possibility of inserting web links to other regulatory or validation bodies requirements. Sam Mohajer, CFIA, Morgan Wallace, DuPont Qualicon, and Mark Mozola, Neogen agreed to help the sub-group finalize the food category table.

The table will be sent to the WG by April 15 for final vote in order to be submitted to ISO TC 34/SC 9 meeting for their June 27th meeting in Brussels.


B. Identifying top five top priority areas for harmonization of quantitative microbiological guidelines – Russ Flowers, Mérieux NutriSciences and Chair, ISPAM and DeAnn Benesh, 3M Microbiology Products The WG discussed a number of differences in the validation guidelines for quantitative chemistry among the different organizations. One of the issues that came up was “relative accuracy” versus “trueness,” which reflects a terminology difference between organizations; however, both terms define the same measurement. Additionally, LOD and LOQ are not for colony counts anymore. The Working Group decided that the differences in validation guidelines for quantitative methods were minor and did not warrant harmonization. The differences and similarities were outlined by Danièle Sohier in her PowerPoint presentation, “Validation Studies: Technical Protocols Harmonization: Current Global Harmonization Scheme and Perspectives” under Attachment 6.)

C. Update on project to compare the performance of reference methods – Russ Flowers, Mérieux NutriSciences and Chair, ISPAM, Julie Moulin, Nestec LTD, Nestlé Research Center and Paul Wehling, General Mills

A key piece in the harmonization effort is the acceptance of the use of different reference methods in validation studies. For that to happen, equivalency in the performance of the different reference methods needs to be demonstrated. In an attempt to determine equivalency of reference methods, AOAC conducted a call for methods from past studies conducted by kit manufacturers involving salmonella in 5 matrices. The objective was to compare reference methods on disparate studies by “anchoring” response to a common alternative method.

Data was collected from 14 ground beef, 7 ground poultry, 2 non-fat dry milk, 3 chocolate, and 3 ice cream studies. Moulin and Wehling analyzed the data and reported that not enough data with a common anchor were collected to make a determination of reference method equivalency. There were problems with different samples and concentration levels, experimental designs, and different food items with different inoculation levels. Moulin and Wehling concluded that more data was needed on a common sample that included at least 1 fractional level. In addition, data must compare the reference methods directly and not use the alternative method as the “anchor” method.

Moulin and Wehling also reported that a GovVal project conducted by AOAC RI in which test kit manufacturers were able to validate their kits against the Health Canada reference method for listeria with 5 food types at 3 levels (blank, low, and high) was not sufficient to make conclusions regarding equivalency of reference methods.


After some discussion the WG decided on the following:

1. Approved conducting a data mining analysis in the Journal of AOAC INTERNATIONAL archival issues for validation data from microbiological collaborative studies to see if statistical data using various reference methods can be collected.

2. Approved a second “call for data” from method validation studies that used multiple reference methods on the same set of samples. Range of levels must include at least 1 fractional level. The comparison will be done on the reference methods themselves.

Data is to be collected not only from kit manufacturers, but also from regulatory bodies and private laboratories and only summary data will be asked for and not raw data. Tentative deadline date is June 1, 2012.

3. Approved moving forward with a method comparison study based on the

GovVal design, if data call under #2 does not prove successful. A set of same samples will be sent to laboratories in Europe, Canada, North America, etc., with a validation study design that would compare reference method performance only. Details of the study design are to be determined but perhaps include multiple analytes; 5 food types in 5 categories.

The benefits of this approach would be the actual comparison of the performance of the reference methods. It would improve the cooperation among regulatory and method approval bodies and facilitate alternative method validations, saving time and resources, which is the ultimate goal of the harmonization efforts.

A key question for the regulatory and method validation approval bodies is whether they will accept the use of one another’s reference methods if performance equivalency can be demonstrated.

In’t Veld will propose to ISO SC9 participation in a second call for data to compare the 4

National Reference Methods; ISO, BAM, USDA and Health Canada.

D. Establishing a pilot project to develop SMPRs for potential new markets such as noroviruses – Russ Flowers, Mérieux NutriSciences and Chair, ISPAM and Scott Coates, AOAC Chief Scientific Officer

The WG discussed the possibility of developing standard method performance requirements for noroviruses. The group decided to hold off development of SMPRs


for norovirus pending a literature search on current work done in the area and the development of a long-term plan that would include a call for methods. The WG then discussed development of inclusivity, exclusivity and sensitivity panels for STEC. The group decided that this would be a worthwhile endeavor and approved moving forward. Meeting attendees who volunteered to be part of the WG on STEC included:

Sam Mohjer Kirsten Mattison Russ Flowers Morgan Wallace Ron Johnson

Tom Hammack Wendy Lauer Julie Moulin Peter Evans Pamela WIlger

DeAnn Benesh

Others may be considered at a later date. The Working Group on Microbiological Guidelines meeting adjourned at 2:00 pm.

1. Equivalence of Reference Methods

• The Sub Group recognized the essential need to determine the equivalence of reference methods and recommends that a statistical analysis be performed according to a scheme to be defined by ISO TC34/SC9 WG2.

• Using data from existing alternative method validation studies, a statistical analysis will be performed. The objective is to use the alternative methods as the anchor to compare different reference methods for equivalence.

2. Number of Levels/Samples/Fractional Positives

• Definition of Fractional Positives

• The target recovery is 50% with a range of 25%-75% based on a sample size of 20. However, in the context of the entire study, values outside of the fractional range may be considered. For example, for studies where a larger number of test portions were analyzed, (i.e., 60), a larger fractional range may be acceptable. At least one of the methods should meet this recommendation. Percent recovery outside the target range may be considered on an individual basis.

3. Selection of Food/Category (Sample Matrix)

• Deferred to sub-group for finalization

• Danièle Sohier, ADRIA

• Ron Johnson, bioMérieux

• Nuri Gras-Rebolledo, Labser, Chile

• Pamela Wilger, Cargill

• Irene Iugovaz, Health Canada

• Tom Hammack, US FDA

• Bala Jagadeesan/Julie Moulin, Nestle

4. Results analysis & Criteria/ Statistical Analysis

• The statistical analysis on data generated from

harmonized study designs will be performed by the RLOD, Chi-square, and/or POD models. As appropriate, other statistical methodologies that have been adopted by certification bodies or regulatory agencies may be applied.

5. Number of Data Sets for Qualitative Collaborative Study/Sample Size

• Data Sets: • Minimum number of collaborative data sets required is

10 valid data sets.

• Sample Size (Test Portion): • Analytical test portion size is 25g unless otherwise

specified by the methods (alternative or reference). The reference method and alternative method may have different analytical test portion sizes. The alternative test portion size cannot be smaller than the reference method test portion size.


• Number of Foods/Levels:

• Currently harmonized at 3 levels for each matrix – one negative control (n=5), one which produces fractional positive results (n=20) and one higher level (n=5). For certain commodities, a higher number of replicates may be required for specific regulatory agency acceptance.


• Number of Collaborative Study Replicates:

• A minimum of 8 replicates will be required per level

• Confirmation:

• All samples presumptively positive or negative will be confirmed

Next Steps

• March 20, 2012 ISPAM meeting in Rockville, MD USA

• ISPAM approval of Food Category Table

• Results of reference methods equivalency study for Salmonella

• If successful, consider other pathogens

Next Steps • The goal is to have stakeholders from multi-national

organizations and government agencies who participated in harmonization effort take ISPAM recommendations to their respective organization/ agency for consideration when revising their validation guidelines

• AOAC OMB approved with minor variations. Needs BoD approval

• US FDA and USDA representatives participated and will take to their leadership

• Health Canada will consider adoption

• ISO….

ISPAM Project Information

• Minutes from meetings and documents on AOAC’s website: • www.aoac.org under ISPAM

• Contact information at AOAC: Krystyna McIver [email protected]

• Executive Summary Report to leadership of international agencies and organizations early next year

• Next ISPAM meeting – Saturday, September 29, 2012 in Las Vegas, Nevada, USA

http://www.aoac.org/

mailto:[email protected]

ISPAM (International Stakeholder Panel on Alternative Methods)

Mission : Develop harmonized, internationally accepted standard validation guidelines for alternative (rapid) chemical and microbiological methods by leveraging global networks of experts to reach consensus on harmonized analytical validation protocols

AOAC sponsored Global stakeholder meetings with consensus decision making Balanced group of stakeholders from industry, government, academia and international organization working on issues of harmonization.

Subgroup on Food Categories “Our Road Map”

• Danièle Sohier, ADRIA

• Peter Evans, USDA (alternate: Victor Cook)

• Irene Iugovaz, Health Canada (alternate:

Leanne DeWinter, Health Canada )

• Tom Hammack, US FDA (alternate: Yi Chen,

FDA)


• Balamurugan (Bala) Jagadeesan and

• Julie Moulin, Nestle


Harmonized Performance

& Acceptance

Criteria

CFIA, HPB, FDA ,

USDA ,

ISO AFNOR

MicroVal AOAC

Food Manufacturers Expert Laboratories

Test Kit

Manufacturers

Method Consensus with Global Stakeholders Embodied in Subgroup on Food Categories

Solution: move to global guidelines for food Categories •Progress requires vision from ISPAM stakeholders and more involvement from regulators, food companies, test kit manufacturers and expert laboratories -ISO 16140 should be the global target for method harmonization -Best Practices in Microbiology Methods •Sustainable engagement of INTERNATIONAL stakeholders

Challenges and Opportunities

Geographical & Cultural Diversity of Foods

GLOBAL FOOD SUPPLY

AOAC PTM

CFIA Health

Canada

FDA BAM USDA FSIS

AOAC Intl

CEN

ISO

AOAC OMA AOAC PTM

Health Canada

AFNOR

MicroVal NordVAl

NMKL

Help!!

“Insanity: doing the same thing over and over again and expecting different results.”

Albert Einstein

The Weary Food Microbiologist

http://images.google.com/imgres?imgurl=http://psy1.clarion.edu/mm/RDE3/C1/c1comments/Thought_Bubble_1.GIF&imgrefurl=http://psy1.clarion.edu/mm/RDE3/C1/c1comments/c1pictorial.html&usg=__K7qSmVf8LAN2Ia-hl5SHLklzYyY=&h=410&w=464&sz=5&hl=en&start=2%20








Food Category Subgroup

“DREAM TEAM Approach”

ISO 16140 should be the global target for method harmonization

Subgroup voted to eliminate separate ISPAM draft guidelines on food

categories

Build on Food Categories in ISO 16140 Annex B (Europe regional focus)

- Geographical & Cultural Diversity of Foods (Include US & Canada)

Integrate input of North American Regulators and Food Industry

-Unique properties of foods that can inhibit recovery and detection of

food pathogens (intrinsic properties) addressed in the proposed

preamble (Annex B Informative)

- proposed new food categories, food types and foods to ISO 16140 Annex B

guidelines

Recommend approval by ISPAM. If approved this would be put forward as

ISPAM recommendations for revisions to ISO 16140 guidelines

• Danièle Sohier, ADRIA (presentation: Annex B informative and

proposed changes to the Annex B food category guidelines)

• Peter Evans, USDA (alternate: Victor Cook)

• Irene Iugovaz, Health Canada (alternate:

Leanne DeWinter, Health Canada )

• Tom Hammack, US FDA (alternate: Yi Chen,

FDA)


• Balamurugan (Bala) Jagadeesan and

• Julie Moulin, Nestle


ISPAM, 12.03.20 [email protected]

ISPAM working group

Classification of sample categories and suggested target combinations

for validation studies

ISPAM, 12.03.20

Collaboration

AOAC-RI

Method developers

FDA

Food industries

Health Canada

USDA

ISO

ISPAM, 12.03.20

Method validation &

verification

Important issue in methods harmonization deals with the

food products themselves

– AOAC-RI ISPAM working group: harmonization in the

classification of categories and types

Food, feed, environmental and primary production samples

AOAC, FDA, Food industries, Health Canada, Method developers,

USDA, and ISO

ISPAM, 12.03.20

Method validation &

verification






AOAC, FDA, Food industries, Health Canada, Method developers,

USDA, and ISO

≠ challenges

Method verification Specific part in the

revised ISO 16140 standard

Chocolate Spices

Coffee

Raw milk cheeses

Probiotic food

Fruit concentrates

Pet food

White egg

Blue cheese

ISPAM, 12.03.20

ISO 16140 Annex B

Annex B: classification of categories, types, etc….

– INFORMATIVE ANNEX

ISPAM, 12.03.20

Summary of the modifications

Meat, poultry, fish & seafood

– All of these 3 categories were divided in 2 food categories

Raw & ready to cook products

Ready-to-eat, ready-to-reheat meat products

Categories Types Items

Raw meat and ready-to-cook

meat products (except

poultry)

Fresh meats (unprocessed)

carcasses, meat cuts, carpaccio's

minced meat, meat preparations, carpaccio's

rinsates

Ready-to-cook (processed) burger meat patties

Ready-to-eat, ready-to-reheat

meat products

Cooked meat products cooked ham, pâté

Fermented or dried meat products salami

Raw cured (smoked) (Aw > 0.92) filet de sax, lard

Raw cured (smoked) (Aw < 0.92) cobourg ham, dry cured ham

Canned meat (ambient stable) corned beef

ISPAM, 12.03.20


Produces and fruits : 2 categories


Fresh produces

and fruits

Cut ready-to-eat fruits fruit mixes

Cut ready-to-eat vegetables bagged pre-cut leafy vegetables, salads, shredded carrot

Produces grown in or in contact with the ground potatoes, yams, sweet potatoes, cassava, dahlia, carrots,

cruciferous vegetables, etc.

Sprouts Soja, fenugreek, alfalfa, mung, etc.

Raw fruit/vegetable juices (unpasteurized) freshly squeezed strawberry juice, smoothies, carrot juice

Leafy greens Basil, Cilantro, Green Onions, Lettuce, and Parsley

Vegetables and fruits (unprocessed) not

described above crops

Processed

fruits and

vegetables

Heat processed fruit/vegetables juices pasteurized apple juice

Canned fruits and vegetables (ambient stable) canned ananas

Heat processed vegetables and fruits blanched spinach, frozen vegetables blanched

Fermented/acidified vegetables fermented cabbage, augurk

ISPAM, 12.03.20


Addition of one category

– Infant formula and cereals


Infant formula and infant

cereals

Probiotic ingredients Pre-blend, spray dried, culture

powders

Non probiotic ingredients Dehydrated milk, dehydrated

youghurt, dehydrated berries

Non probiotic infant formula

Whey based (dairy), soya based

(vegetable), fortification

formulation

Probiotic infant formula

Whey based (dairy), soya based

(vegetable), fortification

formulation

Non probiotic infant cereals Infant cereals

Probiotic infant cereals Probiotic infant cereals

ISPAM, 12.03.20


Pet food and animal feed: new design


Pet food and

animal feed

Animal origin ingredients Meat & bone meal, chicken & feather meal, fish meal,

animal digest, etc.

Plant origin ingredients Corn meal, soybean meal, vegetables, etc.

Other ingredients Microbial products such as yeast extracts, probiotics, etc.

Dry food (Aw ≤ 0,7) Pellets, treats

Wet food (Aw > 0,7) Fresh meat, sausages, croquettes

Canned Meat, fish

Animal feeds (bovin, ovin,

pig) Cereals, flours

Animal feeds (poultry) Cereals, flours

Animal feeds (fish) Cereals, flours

ISPAM, 12.03.20


Addition of food types in various categories

Addition of the target species C. botulinum

Modification of the suggested target combinations

ISPAM, 12.03.20


Introduction – This table outlines the classification of foods intended to guide

method developers for the relative validation of alternative methods. The intrinsic properties of foods, such as levels of indigenous microflora, fat content, pH, salt content, water activity, and the presence of antimicrobial compounds can have a substantial influence on the outcome of a method. Therefore, the intrinsic properties of foods have been considered to the extent possible in the classification of foods, but the wide variety of foods available makes this consideration difficult to apply past the level of food type.

Regulatory authorities in different jurisdictions often have slightly different requirements as to the classification of foods. These differences have been included as much as possible in the notes of the table.

ISPAM, 12.03.20

Notes

Informative annex – If relevant, some categories or items can be gathered or

split. It is the case for an ISO 16140 validation study, since

three food types shall be tested per tested categories;

the method comparison study for a qualitative method validation combines the relative detection limits determinations, as well as the relative accuracy, relative specificity and relative sensitivity study;

the method comparison study for a quantitative method validation combines the determination of the accuracy profiles, as well as the trueness study.

The most challenging categories shall be selected.

For instance, within the dairy products, raw milk based products shall be considered as one food type minimum, and the RLOD shall be determined for one raw milk based matrix (raw milk or raw milk cheese).

ISPAM, 12.03.20

Notes

Follow the regulation bodies’ specific requirements to get a regulatory approval on the validation study claim, e.g. Health Canada, Food and Drug Administration, European Regulation, United States Department of Agriculture.

ISPAM, 12.03.20

Notes

Definition and examples – Unprocessed products

– Processing

– Ready-to-eat (RTE) food

– Ready-to-cook (RTC) food

– Ready-to-reheat (RTRH) food

– Feeding stuffs

ISPAM, 12.03.20

Notes

Advices – Water used in the manufacturing process

No need of filtration

– Specific sample sizes (for instance 375 g ground beef): it is fully recommended to test a complete technical protocol in the method comparison study for this case.

– If the study target spore-formers, it is fully recommended to test both vegetative cells and spores.

– To claim infant formula and/or infant cereals including probiotics, it is fully recommended to consider this case as a full category.

ISPAM, 12.03.20

Notes

Advices – Water used in the manufacturing process

No need of filtration

– Specific sample sizes (for instance 375 g ground beef): it is fully recommended to test a complete technical protocol in the method comparison study for this case.

– If the study target spore-formers, it is fully recommended to test both vegetative cells and spores.

– To claim infant formula and/or infant cereals including probiotics, it is fully recommended to consider this case as a full category.

– To claim infant formula and/or cereals including probiotics, it is fully recommended to consider the probiotic materials as one food type, the infant formula/cereals including probiotics as an other one food type. The RLOD shall be done with a probiotic material (for instance pre-blend)

ISPAM, 12.03.20

Questions

Milks and dairy products: 2 categories

– OR Raw milk, and RTE foods ?

Categories Types

Raw milk and dairy

products

Raw milk and/or fermented/acidified milks (not heat

treated)

Raw milk based products, with high fat content

and/or high background microflora

Heat processed milk and

dairy products

Pasteurized dairy products

Sterilized or UHT dairy products

Pasteurized milk based products

Dry

ISPAM, 12.03.20

Questions

Egg products and derivates

– 2 categories (=meat, poultry, fish & seafood) Raw & ready-to-cook

RTE

Category Types Items

Eggs and

derivates

Eggs (unprocessed) whole liquid egg

Egg products (heat processed) with

additives (salt or sugar > 2%)

egg yolk, egg white,

whole liquid egg

Egg products (heat processed) without any

additives


whole liquid egg

Dry egg powder

ISPAM, 12.03.20

Questions

Egg products and derivates

Category Types Items

Raw eggs and

ready-to-cook

egg products

Eggs (unprocessed) whole liquid egg

Egg products (heat processed) with

additives (salt or sugar > 2%)


whole liquid egg

Egg products (heat processed) without

any additives


whole liquid egg

Dry egg powder

Ready-to-eat,

ready-to-reheat

egg products

Cooked egg products omelette, cooked eggs

ISPAM, 12.03.20

Questions

Target : Virus?

– It may be difficult to run validation studies according to the current guidelines and standard

– Shall we define specific technical protocol for these targets ?

ISPAM, 12.03.20 [email protected]

Thanks!

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raw milk Y Y Y Y Y Y Y

raw fermented/acidified,

raw milk yoghurts, raw

dairy based drinks

Y Y Y Y Y Y

raw butters Y Y Y Y Y Y

raw creams Y Y Y Y Y Y

hard and semi-hard

cheeses (e.g. Comté,

Beaufort)

Y Y Y Y Y Y

blue cheeses

(Roquefort)Y Y Y Y Y Y

softcheeses (Brie,

Munster)Y Y Y Y Y Y

Raw milk and dairy

products

Raw milk based products, with

high fat content and/or high

background microflora

Raw milks and/or

fermented/acidified milks (not

heat treated)

Pasteurized dairy productsmilk based desserts, ice

creams, drinks, creams,Y Y Y Y Y Y Y Y Y Y

Sterilized or UHT dairy productsUHT milks, canned milks

or creamsY Y

fermented/acidified

pasteurized milk

yoghurts, dairy based

drinks

Y Y Y Y Y Y Y Y

pasteurised milks Y Y Y Y Y Y Y Y

butters Y Y Y Y Y Y Y

creams Y Y Y Y Y Y Y

hard and semi-hard

cheeses (heat

processed) (e.g. Comté,

Emmental, Gouda)

Y Y Y Y Y Y

blue cheeses (Bleu de

Bresse)Y Y Y Y Y

soft cheeses (Brie,

Munster) Y Y Y Y Y

milk powders Y Y Y Y Y Y Y Y

powder for milk based

dessertsY Y Y Y Y Y Y Y

carcasses, meat cuts,

carpaccio'sY Y Y Y Y Y Y Y Y

minced meat, meat

preparations,

carpaccio's

Y Y Y Y Y Y Y Y Y

rinsates Y Y Y Y Y Y Y Y Y

Ready-to-cook (processed) frozen burger patties Y Y Y Y Y Y Y Y Y


Pasteurized milk based products

Dry

Heat processed

milk and dairy

products

Raw meat and

ready-to-cook meat

products (except

poultry)

Cooked meat products cooked ham, pâté Y Y Y Y Y Y Y Y Y Y Y Y

Fermented or dried meat

productssalami Y Y Y Y Y Y Y Y Y Y

Raw cured (smoked)

(Aw > 0.92)filet de sax, lard Y Y Y Y Y Y Y Y Y Y

Raw cured (smoked)

(Aw < 0.92)

cobourg ham, dry cured

hamY Y Y Y Y Y Y

Canned meat (ambient stable) corned beef Y Y

carcasses, meat cuts Y Y Y Y Y Y Y Y

carcasses swabs,

rinsatesY Y Y Y Y Y Y Y

minced meat, meat

preparationsY Y Y Y Y Y Y Y

Ready-to-cook products

(processed)

seasoned chicken

breasts Y Y Y Y Y Y Y Y

Cooked meat products cooked turkey filet Y Y Y Y Y Y Y Y Y Y Y Y

Fermented or dried meat

productschicken sausage

Y Y Y Y Y Y Y Y Y Y

Raw cured (smoked)

(Aw > 0,92)smoked turkey filet

Y Y Y Y Y Y Y Y Y Y

Canned (ambient stable)canned poultry meat,

canned duck pâté Y Y

Eggs (unprocessed) whole liquid egg Y Y Y Y Y Y Y Y

Egg products (heat processed)

with additives (salt or sugar >

2%)


whole liquid eggY Y Y Y Y Y Y Y Y Y Y

Egg products (heat processed)

without any additives


whole liquid eggY Y Y Y Y Y Y Y Y Y Y

Dry egg powder Y Y Y Y Y Y Y Y Y

Fish (unprocessed) fish Y Y Y Y Y Y Y

Shellfish (unprocessed)oyster, clam, scallop,

musselY Y Y Y Y Y Y

Crustaceans (unprocessed)shrimp, crab & crab

meat, lobsterY Y Y Y Y Y Y

Ready-to-cook fish and

seafoods (processed)Frozen fish sticks

Ready-to-eat, ready-

to-reheat meat

poultry products



to-reheat meat

products

Raw poultry and

ready-to-cook

poultry products

Raw and ready-to-

cook fish and

seafoods

(unprocssed)

Eggs and derivates

Cooked fishery products

shelled and shucked

products of cooked

crustaceans, fish and

seafood terrines

Y Y Y Y Y Y Y Y Y Y Y

Acidified and marinated fishery

productsroll herring, ansjovis Y Y Y Y Y Y Y Y Y Y Y

Smoked or cured, and other

processed products

(Aw > 0,92)

smoked fish Y Y Y Y Y Y Y Y Y Y Y

Smoked or cured, and other

processed products

(Aw < 0,92)

smoked fish, dried

(salted) fishY Y Y Y Y Y Y Y Y Y Y

Canned (ambient stable fish)canned fish, canned

crabY Y

Cut ready-to-eat fruits fruit mixes Y Y Y Y Y Y Y Y

Cut ready-to-eat vegetables

bagged pre-cut leafy

vegetables, salads,

shredded carrot Y Y Y Y Y Y Y Y

Produces grown in or in contact

with the ground

potatoes, yams, sweet

potatoes, cassava,

dahlia, carrots,

cruciferous vegetables,

etc. Y Y Y Y Y

SproutsSoja, fenugreek, alfalfa,

mung, etc. Y Y Y Y Y Y Y Y

Raw fruit/vegetable juices

(unpasteurized)

freshly squeezed

strawberry juice,

smoothies, carrot juiceY Y Y Y Y Y Y Y

Leafy greens

Basil, Cilantro, Green

Onions, Lettuce, and

Parsley Y Y Y Y Y Y Y

Vegetables and fruits

(unprocessed) not described

above

crops

Y Y Y Y Y Y Y

Heat processed fruit/vegetables

juicespasteurized apple juice

Y Y Y Y Y Y

Canned fruits and vegetables

(ambient stable)canned ananas

Y Y

Heat processed vegetables and

fruits

blanched spinach,

frozen vegetables

blanched Y Y Y Y Y Y Y Y Y

Fermented/acidified vegetablesfermented cabbage,

augurk Y Y Y Y Y Y Y Y Y

Processed fruits

and vegetables


to-reheat fishery

products

Fresh produces and

fruits

Probiotic ingredientsPre-blend, spray dried,

culture powdersY Y Y Y Y Y

Non probiotic ingredients

Dehydrated milk,

dehydrated youghurt,

dehydrated berries Y Y Y Y Y Y

Non probiotic infant formula

Whey based (dairy),

soya based (vegetable),

fortification formulation

Y Y Y Y Y Y

Probiotic infant formula

Whey based (dairy),

soya based (vegetable),

fortification formulation

Y Y Y Y Y Y

Non probiotic infant cereals Infant cereals Y Y Y Y Y Y

Probiotic infant cereals Probiotic infant cerealsY Y Y Y Y Y

Low and IMF fruits

(Aw < 0.85)

syrups, concentrates,

jams, semi-dried prunesY Y Y Y Y Y Y

Seasonings spices, herbs, peppersY Y Y Y Y Y Y

Nuts and seedsnuts, nut meats, nut

butters, seeds Y Y Y Y Y Y Y

Dried fruits and vegetables (Aw

< 0.60)freeze-dried vegetables

Y Y Y Y Y Y

Dried cerealscorn, oat, breakfast

cereals Y Y Y Y Y Y

Flours Wheat, buckwheat, oatY Y Y Y Y Y

Pâtisserie

bakery products with

custard,

confectionnaries Y Y Y Y Y Y Y Y Y Y

Dry powdered cake mixes Y Y Y Y Y Y Y

Low moisture crakers, breads, cookiesY Y Y Y Y

Dry & sugared low moisture (Aw

< 0.85)cake, pralines, marzipan

Y Y Y Y Y

Dry & sugared low moisture (Aw

< 0.65)

biscuiterie, chocolat,

confiserie, honey, sugar,

candy syrops, Y Y Y Y Y

Infant formula and

infant cereals

Dried cereals, fruits,

nuts,seeds and

vegetables

Chocolate, bakery

products and

confiserie

Composite foods with substantial

raw ingredients (excluding

pâtisserie)

refrigerated pasta

salads, sandwiches,

chocolate mousse,

bavarois


Composite processed foods

(cooked)hot meals Y Y Y Y Y Y Y Y Y Y

Ready to (re)heat food:

refrigerated

cooked chilled foods,

boiled rice or pasta, vol-

au-vent in vacuum


Ready to (re)heat food: frozenfrozen fries, pizza,

stuffed croissantsY Y Y Y Y Y Y Y Y Y

Ready to (re)heat food: ambient

stable (canned)

vol-au-vent in glass

bottlesY Y

Ready to (re)heat food: drydehydrated (instant)

soupsY Y Y Y Y Y

Mayonnaise based deli-salads

(acid) with raw ingredients

raw vegetables salads

with dressingsY Y Y Y Y Y Y Y Y Y Y Y

Mayonnaise based deli-salads

(acid) with processed ingredientssandwich spreads Y Y Y Y Y Y Y Y Y Y Y

Ambient stable acid foods (pH <

4.8)

ketchup, sauces,

dressings, mayonnaises,

mustard


Animal origin ingredients

Meat & bone meal,

chicken & feather meal,

fish meal, animal digest,

etc.

Y Y Y Y Y Y Y Y

Plant origin ingredientsCorn meal, soybean

meal, vegetables, etc.Y Y Y Y Y Y Y Y

Other ingredients

Microbial products such

as yeast extracts,

probiotics, etc.

Y Y Y Y Y Y Y Y

Dry food (Aw ≤ 0,7) Pellets, treats Y Y Y Y Y Y Y Y

Wet food (Aw > 0,7)Fresh meat, sausages,

croquettesY Y Y Y Y Y Y Y

Canned Meat, fish Y Y

Animal feeds (bovin, ovin, pig) Cereals, flours Y Y Y Y Y Y Y Y

Animal feeds (poultry) Cereals, flours Y Y Y Y Y Y Y Y

Animal feeds (fish) Cereals, flours Y Y Y Y Y Y Y Y

Equipment/production

environmentswabs, dusts, etc. Y Y Y Y Y Y Y Y Y Y Y Y

Waters used in the

manufacturing process

(recycled) washing

water, process waterY Y Y Y Y Y Y Y Y Y Y Y

Animal feces

Swab samples

(bootsocks), faeces

rectal

Y Y y

Environmental Samples and non

feces

Dust samples, Hygiene

swabs, water from

drinkers, litters, hatchery

samples

Y Y y

Multi-component

foods or meal

components

Primary production

samples (PPS)

Pet food and animal

feed

Environmental

samples (Food or

feed production)

Annex B

(Informative)

Classification of sample categories and suggested target combinations

for validation studies

This table was set up within a working group gathering AOAC-RI, FDA, Health Canada, USDA and ISO

representative members.

This table outlines the classification of foods intended to guide method developers for the relative

validation of alternative methods. The intrinsic properties of foods, such as levels of indigenous

microflora, fat content, pH, salt content, water activity, and the presence of antimicrobial

compounds can have a substantial influence on the outcome of a method. Therefore, the intrinsic

properties of foods have been considered to the extent possible in the classification of foods, but the

wide variety of foods available makes this consideration difficult to apply past the level of food type.

Regulatory authorities in different jurisdictions often have slightly different requirements as to the

classification of foods. These differences have been included as much as possible in the notes of the

table.

Notes (to be mentioned below the tables)

1. If relevant, some categories or items can be gathered or split. It is the case for an ISO 16140

validation study, since

- three food types minimum shall be tested per tested categories;

- the method comparison study for a qualitative method validation combines the relative

detection limits determinations, as well as the relative accuracy, relative specificity and

relative sensitivity study;

- the method comparison study for a quantitative method validation combines the

determination of the accuracy profiles, as well as the trueness study.

2. Follow the regulation bodies’ specific requirements to get a regulatory approval on the

validation study claim, e.g. Health Canada, Food and Drug Administration, European

Regulation, United States Department of Agriculture.

3. Selection of appropriate food categories should be relevant to the target pathogen/non-

pathogen matrix combination.

4. Unprocessed products according to the REGULATION (EC) No 852/2004:

Foodstuffs that have not undergone processing, and includes products that have been

divided, parted, severed, sliced, boned, minced, skinned, ground, cut, cleaned, trimmed,

husked, milled, chilled, frozen, deep-frozen or thawed.

It is recommended to make the distinction between raw products submitted or not to

sanitation processes.

Example: Fresh meat (REGULATION (EC) No 853/2004) means meat that has not undergone

any preserving process other than chilling, freezing or quick-freezing, including meat that is

vacuum-wrapped or wrapped in a controlled atmosphere.

Different jurisdictions have different definitions for processed and unprocessed products.

Check with the appropriate authority in your jurisdiction.

5. Processing according to the REGULATION (EC) No 852/2004

Any action that substantially alters the initial product, including heating, smoking, curing,

maturing, drying, marinating, extraction, extrusion or a combination of those processes

Processed products may contain ingredients that are necessary for their manufacture or to

give them specific characteristics.

Different jurisdictions have different definitions for processed and unprocessed products.

Check with the appropriate authority in your jurisdiction.

6. Minced meat preparations include portioned, cut or minced meat (>1% NaCl or spices)

intended to undergo a heat treatment before consumption; presented as seasoned,

marinated, coated, with herbs and spices, or other ingredients are added to improve sensory

properties or texture.

7. Poultry meat preparations include marinated and spiced meat cuts, chicken fillets, chicken

wing, i.e. intact structure either with or without skin.

8. Seafoods include live bivalve molluscs and by analogy marine gastropods, echinoderms and

tunicates.

9. Ready-to-eat (RTE) food

Food intended by the producer or the manufacturer for direct human consumption without

the need for cooking or other processing, effective to eliminate or reduce to an acceptable

level of micro-organisms of concern.

10. Ready-to-cook (RTC) food: food designed by the producer or the manufacturer as requiring

cooking or other processing effective to eliminate or reduce to an acceptable level micro-

organisms of concern.

11. Ready-to-reheat (RTRH) food: food designed by the producer or the manufacturer as suitable

for direct human consumption without the need for cooking but which may benefit in

organoleptic quality from some warming prior to consumption.

12. For definitions of Feeding stuffs refer to [9].

13. Water used in the manufacturing process

No need for filtration of samples

14. If specific sample sizes of a considered matrix are to be tested in food category, for instance

375 g ground beef, it is fully recommended to test a complete technical protocol in the

method comparison study for this case.

15. To claim infant formula and/or infant cereals including probiotics, it is fully recommended to

consider this case as a full category.

16. If the study target spore-formers, it is fully recommended to test both vegetative cells and

spores.

2012

Validation Studies:

Technical Protocols Harmonization

Current Global Harmonization Scheme

And Perspectives

2012

Core Knowledge in

Method Validation

– AOAC-RI, MicroVal and NF Validation,

– NordVal through the Method Developers

> 40% of the ISO 16140 validation studies

Combined AOAC-RI and ISO 16140 studies

2012

Core Knowledge in

Method Validation

Committees & Working Groups

– MicroVal

– NF Validation

– ISO SC9/WG3

ISO 16140 standard revision

– AOAC-RI ISPAM

International Stakeholders Panel on Method

Harmonization

2012

Core Knowledge in

Method Validation

Committees & Working Groups

– MicroVal

– NF Validation

– ISO SC9/WG3

ISO 16140 standard revision

– AOAC-RI ISPAM

International Stakeholders Panel on Method

Harmonization

Method Harmonization

2012

Current Guidelines &

Standards

AOAC INTERNATIONAL Methods Committee Guidelines for

Validation of Microbiological Methods for Food and Environmental

Surfaces (2012)

ISO 16140 Standard (2002): Microbiology of food and animal

feeding stuffs — Protocol for the validation of alternative method

Technical protocols for the performances assessment

Qualitative methods

Quantitative methods

The performances of the alternative method

demonstrates or estimates the same analyte as it is

measured by the corresponding reference method

2012


Standards

Categories Types Matrices (samples)

Meat products

Raw Meat cuts, carpaccio’s, minced meat…

Heat processed

Cooked ham, cooked meat preparations…

Cured Cured ham, bacon, etc…

Categories, types, matrices…

« A brain teaser! »

AOAC-RI Guidelines – PTM (2012)

2012


Standards


Meat products

Raw Meat cuts, carpaccio’s, minced meat…

Heat processed

Cooked ham, cooked meat preparations…


Categories, types, matrices…

« A brain teaser! »

ISO 16140 Standard (2002)

▲ ▲

▲

2012


Standards

AOAC-RI Guidelines – PTM (2012)

ISO 16140 Standard (2002)

Variety (10 matrices & 5 categories at least)

To claim a « full » validation: 5 categories, three food types per tested category - 4 food categories + Additional category - 5 food categories

Selected (5 matrices & 2 categories at least)

Selected categories

Category (5 matrices at least, 1 category)

Selected matrices or types (ground beef / raw beef meat)


Meat products Raw Meat cuts, carpaccio’s, minced meat…

Heat processed Cooked ham, cooked meat preparations…


► Claim

2012


Standards

Inter-laboratory study Required (results of 10 labs at least)

ISO 16140

Pure cultures

Detection limits 1 matrix/strain pair per tested category Minimum of 6 replicates providing fractional recovery, 6 negative & 6 positive controls, 1 lot Relative accuracy, specificity and sensitivity 60 individual samples per tested category

To-do-list AOAC-RI (PTM) Method developer & independent lab

Inclusivity & exclusivity testing

Pure cultures

Matrix study Per tested matrix: 20 replicates providing fractional recovery, 5 negative and positive controls, 1-2 lots

Robustness, stability & lot-to-lot studies

Required

To fit with the requirements of both technical protocols

Independent lab ONLY

Qualitative methods

To fit with the upper requirements per tested matrix: 20 replicates providing fractional recovery, 6 negative & 6 positive controls.

This experimental design fits with the AOAC-RI and ISO 16140 calculation requirements

: combined validation studies

2012


Standards


ISO 16140

Pure cultures

Detection limits 1 matrix/strain pair per tested category Minimum of 6 replicates providing fractional recovery, 6 negative & 6 positive controls, 1 lot Relative accuracy, specificity and sensitivity 60 individual samples per tested category



Pure cultures

Matrix study Per tested matrix: 20 replicates providing fractional recovery, 5 negative and positive controls, 1-2 lots


Required



Qualitative methods

The same requirements

Following up Paul’s

presentation

To fit with the upper requirements per tested matrix: 20 replicates providing fractional recovery, 6 negative & 6 positive controls.

This experimental design fits with the AOAC-RI and ISO 16140 calculation requirements

20 replicates providing fractional recovery, 5 negative & 5 positive controls.

This experimental design fits with the AOAC-RI (POD) and ISO 16140 calculation (RLOD) requirements

: Up coming revised ISO 16140 standard

2012


Standards


LOD & LOQ One pure culture (replicates)


ISO 16140

Pure cultures

Linearity and sensivity study 1 matrix/strain pair per tested category, 5 contamination levels, 2-10 replicates, 1 lot Relative accuracy study 10 individual samples per tested category, 2-10 replicates



Pure cultures

Matrix study Per tested matrix: 4 contamination levels, 5 replicates, 1-3 lots


Required



To fit with the upper requirements per tested matrix:

5 contamination levels, 5-10 replicates. This experimental design fits with the AOAC-RI and ISO 16140 calculation requirements

: combined validation studies

2012


LOD & LOQ One pure culture (replicates)


ISO 16140

Pure cultures

Linearity and sensivity study 1 matrix/strain pair per tested category, 5 contamination levels, 2-10 replicates, 1 lot Relative accuracy study 10 individual samples per tested category, 2-10 replicates



Pure cultures

Matrix study Per tested matrix: 4 contamination levels, 5 replicates, 1-3 lots


Required




Standards Following up Paul’s

presentation

: Up coming revised ISO 16140 standard

The same requirements

To fit with the upper requirements per tested matrix:

5 contamination levels, 5-10 replicates. This experimental design fits with the AOAC-RI and ISO 16140 calculation requirements

4 contamination levels/tested matrix, 5 replicates.

This experimental design fits with the AOAC-RI (Mean differences) and ISO 16140 (Accuracy profile) calculations

Not for colony count methods

Relative accuracy study:

18 samples/category, 1 replicate

2012

Current & revised

Guidelines & Standards

Some parts of the AOAC-RI and ISO 16140 technical protocols can

be easily adapted to suit all the concerned requirements:

– The inclusivity and exclusivity protocols which consist in testing pure strain

cultures;

– Qualitative method validation study: the same experimental design could be

used for the AOAC-RI matrix study and the ISO 16140 detection limits study;

– Quantitative method validation study: the same experimental design could be

used for the AOAC-RI matrix study and the ISO 16140 linearity/accuracy profile

studies;

The other parts should be considered as separated testing:

The AOAC-RI ruggedness and lot-to-lot studies (method

developer),

The ISO 16140 accuracy studies for the qualitative and

quantitative methods, the limit of quantification calculation of

quantitative methods, as well as the ring trial.

Added values

2012

Combining validation

studies

Helps in saving materials and human resources, i.e. time

and money!

And…

Reference methods harmonization and worldwide recognition…

2012

Method verification






AOAC, FDA, USDA, Health Canada and ISO

2012

Method verification






AOAC, FDA, USDA, Health Canada and ISO

≠ challenges

Method verification Specific part in the

revised ISO 16140 standard

Chocolate Spices

Coffee

Raw milk cheeses

Probiotic food

Fruit concentrates

Pet food

White egg

Blue cheese

2012

Thanks for your attention

[email protected] www.adria.tm.fr

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