AOAC CASP Meeting - MYM 2020

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Ca nabis Ana ytical Science Program

AOAC INTERNATIONAL

CANNABIS ANALYTICAL SCIENCE PROGRAM (CASP) MEETING

WEDNESDAY, MARCH 11, 2020 8:00AM - 2:00PM

at the

Gaithersburg Marriott Washingtonian Center 9751 Washingtonian Blvd. Gaithersburg, Maryland, 20878 SALON C/D/E

Pioneer Members

PerkinElmer R-Biopharm AG SōRSE Technology TEQ Analytical Titan Analytical

Association of Food and Drug Officials (AFDO) Applied Food Sciences BioRad MilliporeSigma

Partner Members

CV Sciences

Eurofins Scientific

Affiliate Members

Alkemist Labs BIOTECON Diagnostics Canopy Growth Corporation CEM Corporation

Charm Sciences Crystal Diagnostics Hygiena Institute of Food

Technologists (IFT)

WiFi: Marriott Conference Password: aoac2020 Sign-In Here: http://bit.ly/casp-mym

AOAC INTERNATIONAL Cannabis Analytical Science Program (CASP)

Wednesday, March 11, 2020 | 8:00AM – 2:00PM ET Gaithersburg Marriott Washingtonian Center – Salon C/D/E

MEETING AGENDA

I. WELCOME, INTRODUCTIONS AND ANNOUNCEMENTS (8:00AM – 8:15AM) David Schmidt, AOAC INTERNATIONAL

II. PROGRAM REVIEW (8:15AM – 8:30AM) Scott Coates, AOAC INTERNATIONAL

III. CONSENSUS BUILDING AT AOAC INTERNATIONAL (8:30AM – 8:45AM) Deborah McKenzie, AOAC INTERNATIONAL

IV. UPDATE ON THE USDA DOMESTIC HEMP PROGRAM (8:45AM – 9:30AM) Kerry Smith , Director, Laboratory Approval and Testing Division, Agricultural Marketing Service, USDA

V. REPORT FROM THE MICROBIAL CONTAMINANTS WORKING GROUP (9:45AM – 10:30AM) Julia Bramante, Colorado Department of Public Health & Environment

VI. REPORT FROM THE CHEMICAL CONTAMINANTS WORKING GROUP (10:30AM – 11:15AM) Susan Audino, Audino and Associates and Julie Kowalski, Consultant

VII. REPORT FROM THE CANNABOIDS IN CONSUMABLES WORKING GROUP (11:15AM – 12:00PM) Holly Johnson, American Herbal Products Association

- Lunch 12:00pm – 1:00pm -

VIII. INTRODUCTION TO TRAINING & EDUCATION WORKING GROUP (1:00PM – 1:30PM) Susan Audino, Audino and Associates and Toby Astill, PerkinElmer

IX. DISCUSSION ON NEXT STEPS FOR CASP (1:30PM – 1:45PM) Scott Coates, AOAC INTERNATIONAL

X. SUMMARY/WRAP UP (1:45PM – 2:00PM) Scott Coates & Palmer Orlandi, AOAC INTERNATIONAL

02-10-2020 Version 3 – Subject to Change Without Notice

Morning Break at 9:30AM

March 11, 2020 AOAC CASP Meeting – Presenter Bios

AOAC INTERNATIONAL CASP MEETING – SPEAKER BIOS

Susan Audino, Ph.D

Audino & Associates

Dr. Susan Audino is a chemist/chemometrician and independent consultant to chemical and biological laboratories. On behalf of Accreditation Bodies, she assesses laboratories to and is an instructor for multiple ISO/IEC standards such as ISO 17025. She is recognized as a leader in the cannabis industry, focusing on consumer safety and sound science in the development of official and consensus analytical test methods. Susan serves as a scientific advisor to several scientific organizations, regulatory bodies, and sits on expert review panels. Dr. Audino has chaired the AOAC cannabis advisory panel and currently chairs the chemical contaminants working group, and is a board member for the Center for Research on Environmental Medicine. She has provided seminars, workshops, webinars, and facilitated symposia domestically and internationally.

March 11, 2020 AOAC CASP Meeting – Presenter Bios

Julia Bramante, Ph.D.

Colorado Department of Public Health and Environment Lead Scientist

Julia began her career in the cannabis industry in 2014 at Gobi Labs, one of the first cannabis testing facilities to open in Colorado. She then transitioned to the Colorado Department of Public Health and Environment’s Marijuana Reference Laboratory where she currently serves as Lead Scientist. Julia is also the Chair of the Cannabis Chemistry Subdivision of the American Chemical Society and the Co-Chair of the AOAC CASP Microbial Contaminants Working Group.

March 11, 2020 AOAC CASP Meeting – Presenter Bios

Scott Coates, M.S.

AOAC INTERNATIONAL Senior Director of the AOAC Research Institute

Scott was appointed as the Senior Director of the AOAC Research Institute on July 1, 2018. He is responsible for daily management of and business development for the AOAC Research Institute. Scott also serves as the Program Lead for the Cannabis Analytical Science Program. Scott served as the Chief Science Office from 2009 until June 2018. In this capacity, he served as the technical lead for many AOAC projects. Scott led the writing and development of Appendix F in the Official Methods of Analysis of AOAC INTERNATIONAL that describes validation requirements and the development of Standard Method Performance Requirements . Before joining AOAC, he worked for 10 years as the Operations Manager for an in-vitro diagnostic manufacturer making medical test kits such as Strep tests and specialized bacterial culture media. Scott holds a B.S. in Microbiology (1978) and a M.S. in Biotechnology Management (1994) from the University of Maryland. Contact Scott at: scoates@aoac.org, (301) 924-7077 x137

March 11, 2020 AOAC CASP Meeting – Presenter Bios

Holly Johnson, Ph.D.

American Herbal Products Association

Chief Science Officer

Holly E. Johnson Ph.D., is the Chief Science Officer for the American Herbal Products Association (AHPA). She previously served for three years as Laboratory Director for Alkemist Labs, an ISO 17025 accredited natural products testing lab specializing in botanical dietary supplements. Dr. Johnson took her Ph.D. in Pharmacognosy at the College of Pharmacy, University of Illinois – Chicago (UIC), under renowned Pharmacognosist and researcher Dr. Norman Farnsworth. Holly was awarded a National Institutes for Health (NIH) Fellowship and trained at the UIC/NIH Center for Botanical Dietary Supplements. She was a Postdoctoral Research Fellow at the Institute for EthnoMedicine studying the etiology of neurodegenerative disease, and also worked for Waters Corporation conducting technical training and regulatory consulting for pharmaceutical and supplements companies. She is currently a Research Associate with the National Tropical Botanical Garden and serves on AOAC working groups, stakeholders’ panels, and expert review panels for Foods and Dietary Supplements. She is a member of the United States Pharmacopeia’s (USP) Medical Cannabis Expert Panel, the Editorial Board of the Journal of AOAC International, and she serves on the Advisory Boards of the American Botanical Council and the American Herbal Pharmacopeia. Holly has over 20 years’ experience working with natural products & botanicals and spent many happy years conducting research on medicinal plants and giving courses at the University of Hawaii.

March 11, 2020 AOAC CASP Meeting – Presenter Bios

Julie Kowalski, Ph.D.

jkSS llc Consultant

Julie Kowalski is a technical consultant primarily serving the cannabis and hemp testing market. She earned her graduate degree in Analytical Chemistry from Pennsylvania State University. Her professional experience includes troubleshooting, method development and validation for GC, GC-MS, LC, and LC-MS/MS in addition to pesticide residue analysis and chromatography method development. She has previously served as the President of the North American Chemical Residue Workshop, served on AOAC Expert Review Panels, the Cannabis Scientific Task Force for Washington State and is currently chairing the AOAC CASP Chemical Contaminants Working Group.

March 11, 2020 AOAC CASP Meeting – Presenter Bios

Kerry Smith, Ph.D.

United States Department of Agriculture (USDA) Director, Laboratory Approval and Testing Division

Kerry Smith, Ph.D., is the Director of the Laboratory Approval and Testing Division (LATD). LATD provides laboratory testing and approval services to facilitate domestic and international marketing of food and agricultural commodities. Kerry was critical in the reorganization and standardization of the Agricultural Marketing Services’ (Agency) laboratory services. She has led significant process and service enhancements, making LATD a leader in residue, mycotoxin, and adulteration testing. In addition, LATD serves as the scientific arm of the Agency, providing subject matter expertise in many areas, including bioengineered foods and hemp production. Kerry has worked for the United States Department of Agriculture (USDA) for 15 years.

Scott Coates Senior Director

AOAC Research Institute AOAC INTERNATIONAL

March 11, 2020

Pioneer

Association of Food and Drug Officials

Titan Analytical

Applied Food Sciences

PerkinElmer BioRad

TEQ Analytical

R- Biopharm AG

SōRSE Technology

MilliporeSigma

Partner

Eurofins Scientific

CV Sciences

Affiliate

Alkemist Labs Alkemist Labs

BIOTECON Diagnostics

CEM Corporation

Charm Sciences

Hygenia

Institute of Food Technologists (IFT)

Canopy Growth Corporation

Crystal Diagnostics

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Current CASP Working Groups and Projects

Microbiology in Cannabis  Initial focus on Aspergillus . SMPR completed. Call for methods issued. Chemical Contaminants in Cannabis  Residual Solvents in Cannabis. SMPR completed. Call for methods issued. • Heavy metals. SMPR completed. Pending review and adoption. Cannabinoids in Consumables  Initial focus on cannabinoids in hemp plant materials. SMPR completed. Call for methods issued.  Recommendation on reporting total THC (THC + THCA). Completed, in SMPR. • Recommendation on dry weight. SMPR completed. Pending review and adoption.

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Priorities March 2020 Chemical Contaminants Working Group

 Pesticides  Residual Solvents  Heavy metals  Mycotoxins

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Priorities March 2020 Cannabis in Consumables Working Group

 Hemp plant materials  Dry weight  Extracts  Foods/ beverages  Personal care products  Veterinary products

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Priorities March 2020 Microbiology in Cannabis Working Group

 Aspergillus  Listeria  Salmonella  E.coli ( STEC)

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New Working Groups

Training and Education Training Working Group • Dr. Toby Astill [Perkin Elmer] recruited to be Chair.

Proficiency Testing Working Group • To be organized in April.

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CASP Team Scott Coates, MSBTM CASP Program Lead Senior Director AOAC Research Institute scoates@aoac.org 301-924-7077 x137 Jonathan Goodwin, , SHRM-SCP, SPHR Deputy Executive Director & Chief HR Officer AOAC INTERNATIONAL jgoodwin@aoac.org 301-924- 7077 x104​​ Palmer A. Orlandi, Jr., Ph.D. Deputy Executive Director and Chief Science Officer AOAC INTERNATIONAL

Christopher Dent Manager, Standards Development & Official Methods of Analysis® AOAC INTERNATIONAL cdent@aoac.org 301-924- 7077 x119.​​ Alicia Meiklejohn Governance and Business Development

AOAC INTERNATIONAL ameiklejohn@aoac.org 301-924-7077 x101

porlandi@aoac.org 301-924- 7077 x163.​​

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AOAC Standards Development

Approval of AOAC Standards & Consensus Documents for CASP draft SMPRs

Deborah McKenzie Sr. Director, Standards & Official Methods SM AOAC INTERNATIONAL

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ANALYTICAL AOAC Products, Services, and Analytical Excellence

Standards & Methods Development

A Complete & Harmonized Quality System Through

Official Methods of Analysis SM (OMA) & Performance Tested Methods SM (PTM)

Laboratory Proficiency Testing & Quality Systems

Analytical Excellence

Publications, Training, Educational Outreach & Horizon ‐ scanning

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Examples: AOAC Consensus Products

Basic Principles

• Transparency • Openness • Balance of Interests • Due Process • Consensus • Appeals

• Performance Requirements • Guidelines

• Sampling Standards • Methods of Analysis • Best Practices • Operational Documents

AOAC Consensus & Products

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To Date – Opportunities to Participate Balance of Perspectives & Due Process

TARGETED COMMUNICATION

INVITATIONS TO SMES

EMAIL BLASTS & WEBSITE NOTIFICATIONS

PARTICIPATION IN MEETINGS – AOAC AND EXTERNAL MEETING

ASSOCIATION NEWS ARTICLES

ONLINE & WRITTEN  ‐ PUBLIC COMMENT FORMATS

BRIEFINGS & PUBLIC HEARINGS

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Global Perspectives Included

Contract Research Laboratories Independent Contractors US Rule Makers Academia Commodity Producers Product Manufacturers Instrument & Technology Providers State Regulators and Laboratories Reference Material Organizations Proficiency Testing Programs Trade Organizations Scientific Associations Rapid Method Developers Accreditation Organizations

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CASP Standards Development Activity

3 working groups launched new standards work: September ‐ October 2019

February 2020

Community Consensus via electronic consensus and approval March – April 2020

Comments on draft standard method performance requirements Comment period for all 3 documents began on February 2020 through March 2020. Online Open Comment Session held on February 17, 2020

‐ Heavy Metals ‐ Dry moisture ‐ Salmonella

Deliberate and reach consensus on a final versions of the documents WG chairs will present summaries of WG draft standards for deliberation and input March 11, 2020

Working groups developed draft documents: Working groups met to begin their work and continued drafting documents via web conference October 2019 – January 2020

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Approving the Draft Standards

Program Lead and Standards Manager will oversee the approval process

STEP 2 : Program Lead will conclude deliberation and Standards Manager will record and verify with WG chair(s) any revisions, if needed

STEP 1: WG chair(s) introduced to present the draft standard along with how comments were reconciled followed by discussion on the draft standard

STEP 3 : After final revisions are complete, the WG chair will make a motion for stakeholder acceptance of the standard and recommend approval. A second to the motion may be entertained, but is not necessary STEP 6: Standards Manager will walk attendees through the general consensus process. All attendees will be able to participate in the demonstration of consensus

STEP 4: Program lead will acknowledge the motion (and second, if offered) and offer time for discussion/questions on the motion.

STEP 5: After any due discussion, Program Lead will call for a vote on the motion.

NOTE: 2/3 vote in favor of a motion will pass a motion. 2/3 of those voting will demonstrate consensus in passing a motion Negative votes need to be recorded

If the motion is approved, a consensus standard is formed.

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Documentation and Communication

AOAC carefully documents the actions of CASP and the Working groups

AOAC will prepare summaries of the meetings Communicate summaries to the stakeholders Publish status and summaries in the Referee section of AOAC’s Inside Laboratory Management Publish documents in Journal of AOAC INTERNATIONAL, Official Methods of Analysis of AOAC INTERNATIONAL

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Questions?

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Julia Bramante Chair, CASP Microbial Contaminants Working Group SMPR Presentation

March 11, 2020 Gaithersburg Marriott Washingtonian Center 9751 Washingtonian Blvd., Gaithersburg, MD, 20878

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CASP Microbial Contaminants: Working Group Members (as of January 2020)

Organization

Organization

Organization

Organization Think20Labs LabsMart Inc,

Name

Name

Name

Name

Melissa Aldwin

Ambler Anterola

BreezeTrees, LLC

Stephen Bridgett

Goldman Graham Griffin Hansen Haskell Helbert Hildreth Hoffman Hordyk Hudalla Hughes Jayanthi Johnson Hayakawa Hom

PhytaTech

Raymundo

Lerma

Phytatech

Edward

Sawicki

Southern IllinoisUniversityCarbondale

OrganaKannalytics

Cynthia

LeVesque Lorenzen

CaligreenLaboratory

Devin

Sears

Toby Susan Brian

Astill

Perkinelmer

Todd Scott

G2Analytical

Kyle

YoungLivingEssentialOils

CARLOS Rachel Sheryl Casey Sidney SungOui Neil

SEPULVEDA

AGROLABMEXICO SoRSETechnology

Audino

S.A.Audino&Associates

BotanacorLaboratories DesignGroupCollaborative

Eva

Lynch

RockRiverLaboratory

Shegog

Beck

Microbiologics

Bradford

Lena

Madden Maqsood McKernan

Limerick InstituteofTechnology

Shepherd Silverman Simmons

NATA

Cornelius

Berka

BIOTECONDiagnostics

Darin

HawaiiDOHState LaboratoriesDivision

Madeeha

ProVerde Laboratories MedicinalGenomics

PacificStarLabs LLC

Pat

Bird

PMBBiotek SORA Labs

Yvonne

MedicinalGenomics

Kevin

Hygiena

Tammy Rafael

Blakemore Bombonato Bramante Brauninger Brodnick Campbell Boyar

Jana

SelfEmployed

Ronald Megan

Miller Murn Nelson

TherapeuticHealthChoices

Sudberg

AlkemistLabs

Curaleaf

Shannon Sherman

SteepHill

Microbiologics

Suh

ATCC

Kyle Julia

MedicinalGenomics

NJDeptofHealth,PublicHealth&Environmental LaboratMaria

AOAC

Christy

Swoboda Thomas

RomerLabs, Inc.

CDPHE

Nathan

Industrial Laboratories ProVerde Laboratories

Dustin

Newman Niehaus

Instituteof FoodSafety&Defense

Katherine

NJDepartmentofHealth VivariantLaboratories SartoriusCorporation

Roger Robert

A2LA (accreditationbody)

Chris

Gary

CrystalDiagnostics

Anand Tricia Gordon

Thota

TitanAnalytical

Daniel Srinivas

MCRLaboratories

Melissa Shawn

Nutter O'Leary

TitanAnalytical

Vail

Shari Mike

KaychaLabs

BiotechPharmacal Inc.

NJDOH

Vrdoljak

StateofCADeptofPublicHealth

Clark

BioRadLaboratories

Ron

BioMerieux

Shaun

Opie

E4Bioscience Think20Labs

Christopher

Waggener

VirginiaDCLS

Bob

Clifford

Shimadzu

James

Jursich

CenteraBioscience

Ben Jess

Orsburn Paoletti Parish Parisi Pfaller Phillips

Matthew

Ward

ColoradoDeptofPublicHealthandEnvironment

Pearl

D'Cruz

PEARLConsultingLLC

Ben

Katchman

PathogenDx

TEQAnalytical Labs

Jane

Weitzel

IndependentConsultant

Danielle

Deschene

SCLaboratories

Jason Kati Julie

Kircos

Neogen

Zachary

LevelOne Labs

Daniel Jeffrey

Wene

New JerseyDepartmentofHealthPHEL

Lori

Dodson

MarylandMedicalCannabisCommission

Kiss

ATCC

Salvatore

AlBalqaAppliedUniversity

Wigton

WesternAlternative

Wilfredo

Dominguez

3M

Kowalski

TraceAnalytics

Mike

Universityof Iowa

AnnaWilliams Williams

A2LA NIST

Janie Mike Ross

Dubois

ContractAnalytical Services

Kelsey Nikhil Robert

Kropp Kumar

CDPHE

Melissa

NIST

Walter

Wilson Wong

Esposito Franklin

MCRLabs

Canalysis Laboratories

Juan

Rodriguez Rodriguez Salfinger

GreenHillsAnalytics Lab

Seth

TEQAnalytical Laboratories/Industrial Laboratories

MCRLabs. LLC

LaBudde

Alena

Rm3Labs

Victoria Joshua Wendi

Wu

ABCTesting&MateriaMedicaLabs

Ted

Gatesy

MichiganDept.ofAgriculture

Jasen

Lavoie

U.S.CannabisPharma NeogenCorporation

Yvonne

AFDO

Wurzer Young

SCLaboratories MileHighLabs HardyDiagnostics

Quynh ‐ Nhi

Le

Nandakumara Sarma

USPharmacopeialConvention

Jessa

Youngblood

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Microbial Contaminants Working Group: Work Since Last CASP Meeting

• Six teleconferences (October 2019 – February 2020) • One SMPR drafted ( Salmonella ), one other started (STEC) • Public comment period (February – March, 2020) • SMPR made ready for review and approval

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SMPR Development

Standard Method Performance Requirements ® (SMPRs) for Detection of Salmonella in Cannabis and Cannabis Products

Applicability: Candidate methods used to detect Salmonella species and their serovars in cannabis (plants/flowers) and/or cannabis products (concentrates, infused edibles, and infused non ‐ edibles). Candidate methods may be validated for specific matrices, categories, or broader claims.

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Salmonella species • Straight rods, 0.7 – 1.5 x 2-5 μ m. • Facultative anaerobic, gram negative bacteria. • D-glucose and other carbohydrates are catabolized with the production of acid and usually gas. • Occur in humans, warm and cold blooded animals, food, and the environment. • Pathogenic for humans and many animal species. • Shown to survive well under dessication. • Causative agent of typhoid fever, enteric fevers, gastroenteritis, and septicemia.

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Definitions • POD, Probability of Detection.— The portion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration • dPOD CP – This difference in POD values between presumptive and confirmed results • LPOD – The POD value obtained from combining all valid collaborator data sets for a given matrix at a given analyte level or concentration • LCL, Lower confidence limit.— Calculated to determine 95% confidence interval of various POD values • UCL, Upper confidence limit.— Calculated to determine 95% confidence interval of various POD values • CFU, Colony forming unit.— Number of viable microorganisms, presented per a specific quantity. (ex. CFU/mL) • MPN, Most probable number.— Method used to estimate contamination levels

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Method Performance Requirements

Table 1. Validation Acceptance Criteria (Plants/Flowers, Concentrates, Infused Edibles, Infused Non ‐ Edibles)

Target Test Concentration a

Parameter

Parameter Requirements

Minimum Acceptable Results

Single Laboratory Validation with artificial contamination

Fractional positive results, 25 ‐ 75% (5 ‐ 15 positive test replicates) dPOD CP 95% CI: LCL < 0 < UCL b

Replicates per matrix: 20 Inoculation procedure: AOAC Appendix J

Low level to produce fractional positive results Ex. 0.2 ‐ 2 CFU/Test Portion

Fractional Concentration (low level)

Replicates: 5 Inoculation procedure: AOAC Appendix J

High level to produce consistently positive results Ex. 2 ‐ 10 CFU/Test Portion

High Concentration

POD of 1.00 c

Non ‐ Inoculated (Zero) concentration

Replicates: 5

0 CFU/Test Portion

POD of 0.00 c

Single Laboratory Validation with natural contamination

Fractional positive results, 25 ‐ 75% (5 ‐ 15 positive test replicates) for minimum 1 lot dPOD CP 95% CI: LCL < 0 < UCL b

Acceptable minimum detection level (low level)

2 separate lots of 20 replicates

N/A

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Method Performance Requirements

Table 1. Validation Acceptance Criteria (Plants/Flowers, Concentrates, Infused Edibles, Infused Non ‐ Edibles)

Target Test Concentration a

Parameter

Parameter Requirements

Minimum Acceptable Results

Multi Laboratory Validation

0.15   ≥  LPOD   ≥   0.85 dPOD CP 95% CI: LCL < 0 < UCL b

Replicates: 12

1 ‐ 10 CFU/Test Portion

LPOD

Replicates: 12

10 ‐ 50 CFU/ Test Portion

LPOD ≥ 0.95

Replicates: 12

0 CFU/Test Portion

LPOD ≤ 0.05

LPOD (0)

a Determined through MPN Procedures (see Table 4)

b The range between the lower and upper confidence interval should encompass 0, if not, the results must be investigated, and an explanation provided.

c If acceptance criteria is not observed, results must be investigated, and an explanation provided.

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Method Performance Requirements Table 7. Inclusivity/Exclusivity Performance Requirements

Final Test Concentration (CFU/mL)

Minimum Acceptable Results

Parameter

Parameter Requirements

Inclusivity Single ‐ laboratory validation (SLV) study: A minimum of 100 strains is required to be

10 ‐ 100 x limit of detection of the candidate method

100% positive results a

cultured by the candidate method enrichment procedure (including those detailed in Table 8).

Exclusivity SLV study: At least 30 non ‐ target organisms, cultured under optimal conditions for growth b

Overnight growth undiluted

100% negative results a

a. 100% correct analyses are expected. All unexpected results are to be retested following internationally recognized guidelines (ISO 16140, AOAC OMA Appendix J, The Compendium of Analytical Methods of Health Canada). Some unexpected results may be acceptable if the unexpected results are investigated, and acceptable explanations can be determined and communicated to method users b. In instances where an exclusivity culture produces a positive result by the candidate method, the culture may be reanalyzed after culture following the candidate method enrichment procedure. Both results (optimal growth conditions and candidate method enrichment) must be reported.

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Method Performance Requirements • Use of live (viable) cultures (liquid stressed/non-stressed, lyophilized) is required. • To screen samples for the presence or

• Final confirmation can be achieved via matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy, sequencing, or other suitable confirmatory procedures (e.g., biochemical analysis). • One matrix must contain microflora at 10x the level of the target microorganism. • A minimum three level MPN analysis to determine concentration of target microorganism. Use of matrix study replicates is encouraged.

absence of the target analyte, two methods that employ different technologies (e.g., agar plate, PCR, ELISA) must be used.

• To ensure the viability of the inoculating organism (both confirming presumptive

results or determining false negative results) a secondary enrichment followed by plating of the sample to a minimum of two types of agar plates, one of which is recommended to be chromogenic agar, is required (Table 6). Bulk inoculation of test material is required.

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Method Performance Requirements

Table 3. Acceptable Matrix Claims

Table 2. Category Test Portion Requirements

Minimum Test Portion Size a

Criteria

Category

Matrix Claim

Plants & Flowers

10 g

Number of Matrices Minimum Number of Categories

15 (minimum 3 matrices/category) ≥ 10 (minimum 2 matrices/category)

Concentrates

5 g

Broad Range

4 categories

Infused Edibles

25 g

Variety

4 categories 2 categories 1 category

Infused Non ‐ Edibles 10 g a Minimum test portion size required for validation. Alternatively, larger test portions may be validated.

Select

≥ 5 ≥ 5 ≥ 1

Specific Category Specific Matrix (s)

1 category RE: AOAC Technical Bulletin: TB02MAY2016: Acceptable Validation Claims for Proprietary/Commercial Microbiology Methods for Foods and Environmental Surfaces.

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Method Performance Requirements

Table 5. Condition of Inoculating Culture and Stabilization of Matrix Table 6: Recommended Secondary Selective Broths and Agar

Stabilization Conditions 4°C, 48 ‐ 72 h 4°C, 48 ‐ 72 h

Matrix

Inoculating Cells

Media Name

Media Type

Perishable product Heat processed perishable product

Liquid non ‐ stressed culture

Liquid heat stressed

Rappaport ‐ Vassiliadis (RV) (alternately Rappaport ‐ Vassiliadis R10)

Broth

Liquid non ‐ stressed culture (If frozen food is processed, cells must be heat stressed)

‐ 20°C, 2 weeks

Broth Broth

Tetrathionate (TT)

Frozen Product

Selenite cysteine (SC)

Ambient Temperature (20 ‐ 25 o C), 2 weeks Ambient Temperature (20 ‐ 25 o C), 2 weeks

Agar Agar Agar Agar Agar

Xylose lysine desoxycholate (XLD)

Dried culture

Hektoen enteric (HE) Bismuth sulfite (BS) Chromogenic Salmonella

Shelf stable dry product

Liquid non ‐ stressed culture (If shelf stable product is processed, cells must be heat stressed)

Shelf stable liquid product (heat processed)

MacConkey

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Comments Submitted

Comment Received

Response to Comment

Section 6 : Reference Materials Update 1st sentence to remove fungal spores

Fungal spores reference maintained in SMPR to account for potential use of fungal spores in exclusivity and/or artificial contamination.

Table 1, MLV Section, Column 2: Add Replicates in front of 12

MLV table updated to include “Replicates:” in front of 12 to provide clarity.

Table 6: Update title to read “Recommended Secondary Selective Broths and Agar Table 7, Inclusivity: Update strain requirements, Delete at least 10 strains per required Salmonella spp. Table 8: Update column 3 title to strains (not serovars) and add footnote indicating that it’s a requirement only if method claims

Table 6 title updated per comment recommendation.

Table 7, Inclusivity updated to read: A minimum of 100 strains is required to be cultured by the candidate method enrichment procedure (including those detailed in Table 8). Table 8 title updated to read: Minimum Number of Strains Included* Table 8 footnote updated to read: *Required number of strains per subspecies, per method claims

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Motion: Move to accept the Standard Method Performance Requirements ® (SMPR ® ) for Detection of Salmonella in Cannabis and Cannabis Products as presented.

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Discussion?

15

Pre-decisional draft, do not distribute

AOAC Standard Method Performance requirements 2019.XX, Version 7; March 11, 2020. Method Name: Detection of Salmonella species in Cannabis and Cannabis Products Purpose: AOAC SMPRs describe the minimum recommended performance characteristics and suggested inclusivity/exclusivity organisms to be used during the evaluation of a method. The evaluation may be an on-site verification, a single-laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC Stakeholder Panels composed of representatives from the industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC Expert Review Panels in their evaluation of validation study data for methods being considered for Performance Tested Methods or AOAC Official Methods of Analysis , and can be used as acceptance criteria for verification at user laboratories.

Approved Body: Approval Date: Final version date: 1. Intended Use :

AOAC Cannabis Analytical Science Program

Consensus-based Reference method.

2. Applicability:

Alternative methods used to detect Salmonella species and their serovars in

cannabis and cannabis products.

3. Analytical Technique : Any analytical technique that can meet the requirements. 4. Definitions :

Candidate Method . — The method submitted for validation [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] Candidate Method Presumptive Result . —Preliminary result for a test portion produced by following a candidate method’s instructions for use. Candidate Method Confirmed Result . —Final result obtained for a test portion after cultural confirmation of a candidate method. Cannabis .—genus of flowering plants within the Cannabinaceae family that commonly contain 9- tetrahydrocannabinol (THC), cannabidiol (CBD), and other cannabinoids and terpenes. Cannabis includes, but is not limited to, high-THC and high-CBD cultivars. Cannabis Concentrates . —Extracts (primarily composed of cannabinoids and/or terpenes) manufactured through the extraction and concentration of compounds derived from the cannabis plant or flower. Final products can be many forms including oils, wax, or hash (Category II). Cannabis Infused Edibles .—Food and drinks containing extracts of cannabis and/or cannabis materials (Category III).

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Draft Salmonella SMPR v6

Pre-decisional draft, do not distribute

Cannabis Infused Non-Edibles .—Products containing extracts of cannabis and/or cannabis materials intended to be applied to the human body or any part thereof. Final products can be many forms including creams, ointments, cosmetics and therapeutic pads (Category IV). Cannabis Plant and Flower . —General terms for the structural and flowering unadulterated parts of the cannabis plant (Category I). Cannabis Products .—Products (edible and non-edible) extracted or infused with compounds derived from the cannabis plant including but not limited to CBD and THC. Probability of detection (POD) .—The portion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. The difference in POD values between presumptive and confirmed results is termed dPOD CP . Exclusivity .—Study involving pure nontarget strains, which are potentially cross-reactive, that shall be not detected or enumerated by the candidate method. See Table 10 for a list of recommended nontarget strains. [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] Fractional positive .—Validation criterion that is satisfied when an unknown sample yields both positive and negative responses within a set of replicate analyses. The proportion of positive responses should fall within 25 and 75% and should ideally approximate 50% of the total number of replicates in the set. A set of replicate analyses are those replicates analyzed by one method. Only one set of replicates per matrix is required to satisfy this criterion. Inclusivity .—Study involving pure target strains that shall be detected or enumerated by the candidate method. See Tables 8 and 9 for a list of recommended target strains. [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] Laboratory probability of detection (LPOD) .—The POD value obtained from combining all valid collaborator data sets for a method for a given matrix at a given analyte level or concentration. [Appendix H: Probability of Detection (POD) as a Statistical Model for the Validation of Qualitative Methods , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA]

LCL .—Lower confidence limit.

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Salmonella Straight rods, 0.7 – 1.5 x 2- 5 μm. Gram negative. Usually motile by peritrichous flagella. Facultative anaerobic. Chemoorganotrophic, having both a respiratory and fermentative metabolism. D- glucose and other carbohydrates are catabolized with the production of acid and usually gas. Oxidase negative, catalase positive, indole and Voges-Proskauer negative, methyl red and Simmons citrate positive. Lysine and ornithine decarboxylase positive, there is a variable arginine dihydrolase reaction. H 2 S is produced, urea is not hydrolyzed, and growth on KCN and utilization of malonate are variable. Reduce nitrates. Carbohydrates usually fermented include L-arabinose, maltose, D- mannitol, D-mannose, L-rhamnose, D-sorbitol, trehalose, and D-xylose. Occur in humans, warm and cold blooded animals, food, and the environment. Pathogenic for humans and many animal species. Causative agent of typhoid fever, enteric fevers, gastroenteritis, and septicemia. 1 Test portion . —The test portion is the sample size used in most validation studies. For cannabis flower/plant and cannabis infused non-edible products a 10 g test portion is used. For cannabis concentrates, a 5 g test portion is used. For cannabis infused edibles, a 25 g test portion is used. A larger test portion can be used in validation studies when appropriate. See Table 2 for minimum test portion requirements. 5. System suitability tests and/or analytical quality control: Positive and negative controls shall be embedded in assays as appropriate. Inhibition controls should be used for method verification for each new matrix. Manufacturer must provide written justification if controls are not appropriate to an assay. 6. Reference Material(s): The use of live cultures and/or fungal spores (liquid stressed/non-stressed, lyophilized) is required for inclusivity and exclusivity testing and for inoculation of test matrices during the matrix studies. Extracted DNA is not suitable for use in validating methods against this SMPR but may be used to develop supplemental information. 7. Validation Guidance: Appendix F: Guidelines for Standard Method Performance Requirements; 19 th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_f.pdf Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces [ Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA]; or ISO 16140-2:2016. UCL . —Upper confidence limit.

United States Pharmacopeia. Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests (61), USP 40. United States Pharmacopeia.

1 Bergey's Manual of Determinative Bacteriology Ninth edition edited by John G. Holt.

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United States Pharmacopeia. Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms (62), USP 40. United States Pharmacopeia. Feng, P., Weagant, S.D., Grant, M.A., Burkhardt, W. (2017)

Bacteriological Analytical Manual: Chapter 4 Enumeration of Escherichia coli and the Coliform Bacteria

https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm064948.htm Andrews, W. H., Wang, H., Jacobson, A., Hammack, T. (2018) Bacteriological Analytical Manual Chapter 5: Salmonella https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm

At the time of the publication, no national reference method exists for the confirmation of Salmonella spp. from cannabis products. Until a suitable reference method is established the following is recommended for method developers:

To screen samples for the presence or absence of the target analyte, two methods that employ different technologies (agar plate, PCR, ELISA) must be used.

To ensure the viability of the inoculating organism (both confirming presumptive results or determining false negative results) a secondary enrichment followed by plating of the sample to a minimum of two types of agar plates, one of which is recommended to be chromogenic agar, is required (Table 6). Final confirmation can be achieved via matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy, sequencing, or other suitable confirmatory procedures (e.g., biochemical analysis).

When performing the validation, bulk inoculation of test material is required. In certain instances (e.g., therapeutic patches) individual item inoculation may be required.

For the Single Laboratory Validation with artificial contamination, matrix naturally contaminated with non-target organisms (when available) shall be used. For at least one matrix evaluated during the single laboratory validation, competing non-target microflora must be at least 10x the level of the target microorganism. If the concentration of competing microflora does not exceed 10x the target organism for any matrix, artificial contamination of one matrix with non-target organism(s) is required. A minimum three level most probable number (MPN) study should be performed to determine the concentration of the target organism used in the validation. If possible, the use of test portions included in the matrix study should be included as a level in the MPN study. See AOAC Appendix J guidelines for details on performing the MPN study.

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8. Maximum Time-To-Determination: None 9. Method Performance Requirements

See Table 1 for acceptance criteria for validation See Table 2 for category test portion requirement See Table 3 for matrix claims acceptance criteria See Table 4 for descriptions of MPN analysis See Table 5 condition of inoculating culture and stabilization of matrix for inoculation See Table 6 for selective broth and agar recommendations See Table 7 for inclusivity and exclusivity performance requirements

See Tables 8 & 9 for inclusivity organisms See Table 10 for exclusivity organisms

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Table 1. Validation Acceptance Criteria (Plants/Flowers, Concentrates, Infused Edibles, Infused Non- Edibles)

Parameter Requirements

Target Test Concentration a

Parameter

Minimum Acceptable Results

Single Laboratory Validation with artificial contamination

Low level to produce fractional positive results Ex. 0.2-2 CFU/Test Portion High level to produce consistently positive results Ex. 2-10 CFU/Test Portion 0 CFU/Test Portion

Fractional positive results, 25-75% (5-15 positive test replicates)

Replicates per matrix: 20 Inoculation procedure: AOAC Appendix J Replicates: 5 Inoculation procedure: AOAC Appendix J

Fractional Concentration (low level)

dPOD CP 95% CI: LCL < 0 < UCL b

High Concentration

POD of 1.00 c

Non-Inoculated (Zero) concentration

Replicates: 5

POD of 0.00 c

Single Laboratory Validation with natural contamination

Fractional positive results, 25-75% (5-15 positive test replicates) for minimum 1 lot

Acceptable minimum detection level (low level)

2 separate lots of 20 replicates

N/A

dPOD CP 95% CI: LCL < 0 < UCL b

Multi Laboratory Validation

0.15 ≥ LPOD ≥ 0.85 dPOD CP 95% CI: LCL < 0 < UCL b

Replicates: 12

1-10 CFU/Test Portion 10-50 CFU/ Test Portion 0 CFU/Test Portion

LPOD

Replicates: 12 Replicates: 12

LPOD ≥ 0.95 LPOD ≤ 0.05

LPOD (0)

a Determined through MPN Procedures (see Table 4) b The range between the lower and upper confidence interval should encompass 0, if not, the results must be investigated, and an explanation provided. c If acceptance criteria is not observed, results must be investigated, and an explanation provided

Table 2. Category Test Portion Requirements

Category

Minimum Test Portion Size a

Plants & Flowers Concentrates Infused Edibles

10 g

5 g

25 g

Infused Non-Edibles 10 g a Minimum test portion size required for validation. Alternatively, larger test portions may be validated.

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Table 3. Acceptable Matrix Claims

Matrix Claim

Criteria Number of Matrices 15 (minimum 3 matrices/category) ≥ 10 (minimum 2 matrices/category)

Minimum Number of Categories

Broad Range of Cannabis & Cannabis Products Variety of Cannabis & Cannabis Products Select Cannabis Products

4 categories

4 categories 2 categories 1 category

≥ 5 ≥ 5 ≥ 1

Specific Category Specific Matrix (s)

1 category RE: AOAC Technical Bulletin: TB02MAY2016: Acceptable Validation Claims for Proprietary/Commercial Microbiology Methods for Foods and Environmental Surfaces.

Table 4. Minimum Most Probable (MPN) Number Recommendation

Medium Test Portions

Inoculation Level

Large Test Portions 20 x 10 g* 5 x 10 g*

Small Test Portions

Category

Plants & Flowers Concentrates Concentrates

Low High Low High Low High Low High

3 x 5 g 3 x 5 g

3 x 1 g 3 x 1 g 3 x 1 g 3 x 1 g 3 x 5 g 3 x 5 g 3 x 1 g 3 x 1 g

20 x 5 g 5 x 5 g*

3 x 2.5 g 3 x 2.5 g 3 x 10 g 3 x 10 g

20 x 25 g* 5 x 25 g* 20 x 10 g* 5 x 10 g*

Infused Edibles

3 x 5 g 3 x 5 g

Infused Non- Edibles

*Test portions from matrix study

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Table 5. Condition of Inoculating Culture and Stabilization of Matrix

Stabilization Conditions 4°C, 48-72 h 4°C, 48-72 h

Matrix

Inoculating Cells

Liquid non-stressed culture

Perishable product Heat processed perishable product

Liquid heat stressed

Liquid non-stressed culture (If frozen food is processed, cells must be heat stressed)

-20°C, 2 weeks

Frozen Product

Ambient Temperature (20-25 o C), 2 weeks Ambient Temperature (20-25 o C), 2 weeks

Dried culture

Shelf stable dry product

Liquid non-stressed culture (If shelf stable product is processed, cells must be heat stressed)

Shelf stable liquid product (heat processed)

Table 6: Recommended Secondary Selective Broths and Agar

Media Type

Media Name

Rappaport-Vassiliadis (RV) (alternately Rappaport-Vassiliadis R10)

Broth Broth Broth Agar Agar Agar Agar Agar

Tetrathionate (TT) Selenite cysteine (SC)

Xylose lysine desoxycholate (XLD)

Hektoen enteric (HE) Bismuth sulfite (BS) Chromogenic Salmonella

MacConkey

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Table 7. Inclusivity/Exclusivity Performance Requirements

Final Test Concentration (CFU/mL)

Minimum Acceptable Results

Parameter

Parameter Requirements

Inclusivity Single-laboratory validation (SLV) study: A minimum of 100 strains is required to be cultured by the candidate method enrichment procedure (including those detailed in Table 8). Exclusivity SLV study: At least 30 non-target organisms, cultured under optimal conditions for growth b

10-100 x limit of detection of the candidate method

100% positive results a

Overnight growth undiluted

100% negative results a

a 100% correct analyses are expected. All unexpected results are to be retested following internationally recognized guidelines (ISO 16140, AOAC OMA Appendix J, The Compendium of Analytical Methods of Health Canada). Some unexpected results may be acceptable if the unexpected results are investigated, and acceptable explanations can be determined and communicated to method users b In instances where an exclusivity culture produces a positive result by the candidate method, the culture may be reanalyzed after culture following the candidate method enrichment procedure. Both results (optimal growth conditions and candidate method enrichment) must be reported.

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Tables 8 & 9: Inclusivity Panel The following are lists of required and suggested subspecies and serovars that method developers can use to validate their methods. It is recommended that method developers reference CDC’s revised Atlas on Salmonella (https://www.cdc.gov/salmonella/pdf/salmonella-atlas-508c.pdf) to incorporate as many serovars listed therein as possible. A minimum of 100 serovars are required for AOAC adoption. Additionally, the requirements in Table 8 must be met.

Table 8: Required Salmonella subspecies for Inclusivity

Minimum Number of Strains Included*

SALMONELLA

2 3 3 3 3 3

1 2 3 4 5 6 7

Salmonella bongori

Salmonella enterica subsp. arizonae Salmonella enterica subsp. diarizonae Salmonella enterica subsp. houtenae Salmonella enterica subsp. indica Salmonella enterica subsp. salamae Salmonella enterica subsp. enterica

1 strain per serovar

*Required number of strains per subspecies, per method claims

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Table 9: Suggested Salmonella serovars for Inclusivity

Center for Disease Control and Prevention Top 20**

Food and Drug Adminstration Ranking Top 40

Antigenic Properties SEROTYPE

Year OUTBREAK

SALMONELLA (serovar included)

O

H

1 2 3 4 5 6 7 8 9

66 66 47 50 53 55 56 57 58 59 60 40 51 62 63 65 35 47 48 61

z 41 :-

Salmonella bongori, Serotype Brookfield

Salmonella bongori

Salmonella enterica subsp. Salamae Salmonella enterica subsp. Salamae Salmonella enterica subsp. salamae Salmonella enterica subsp. salamae

Salmonella enterica subsp. salamae serovar Artis

Salmonella enterica subsp. salamae

Salmonella enterica subsp. salamae serovar Basel

10 Salmonella enterica subsp. salamae 11 Salmonella enterica subsp. salamae 12 Salmonella enterica subsp. Arizonae # 13 Salmonella enterica subsp. Arizonae 14 Salmonella enterica subsp. Arizonae 15 Salmonella enterica subsp. Arizonae 16 Salmonella enterica subsp. Arizonae 17 Salmonella enterica subsp. diarizonae 18 Salmonella enterica subsp. diarizonae 19 Salmonella enterica subsp. Diarizonae #

41

29

20 Salmonella enterica subsp. diarizonae serovar Eilbek 21 Salmonella enterica subsp. houtenae serovar Halmstad

3,{10}{15}{15,34}

g,s,t:-

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