Color Additives from Natural Sources Meeting Book AM23

AOAC INTERNATIONAL Annual Meeting 2023 Color Additives from Natural Sources Working Group Monday, August 28, 2023 3 :30 – 5 :00PM CDT Draft Agenda

Moderator: Bhakti Petigara Harp I.

WELCOME & INTRODUCTIONS Allison Baker (AOAC) will welcome attendees and lead introductions . II. OVERVIEW OF AOAC EXPERT REVIEW PANELS (3:30PM – 3:35PM) Allison Baker, AOAC INTERNATIONAL, will provide an overview of the Expert Review Panel processes, and review how attendees can get involved.

III.

SUBGROUP UPDATES • PESTICIDE RESIDUES (3:35PM – 3:55PM)

Joe Konschnik, PlusOne Consulting, LLC, representing Restek Corporation, will provide an update on the Pesticide Residues Subgroup and an overview of the subgroup’s SMPR and public comment period • RESIDUAL SOLVENTS (3:55PM – 4:10PM) David Bolliet, Kalsec, Inc. will provide an update on the Residual Solvents Subgroup and an overview of the subgroup’s SMPR. John Mills, bioMérieux, will present on emerging technology for predictive microbiology for natural food colors. He will also provide an overview on the background and current challenges, for the Microbial Contaminants Subgroup V. The Role of FCC in Protecting the Integrity of Food Colors from Natural Sources (4:30PM – 5:00PM) Eric Schwartz, US Pharmacopeia, and Tongtong Xu, US Pharmacopeia, will present on the role of FCC in protecting the integrity of natural good colors IV. EMERGING TECHNOLOGY FOR NATURAL COLORS (4:10PM – 4:30PM)

VI.

ADJOURNMENT

Bhakti Petigara Harp, FDA CFSAN Dr. Bhakti Petigara Harp has been with FDA's Center for Food Safety and Applied Nutrition (CFSAN) as a research chemist since 2000. Her current responsibilities include research and methods development for color additives and other components using various analytical for the agency’s color certification program. She also is a technical expert and troubleshooter to support the FDA

district laboratories in regulatory actions concerning the analysis of atypical samples of food, drugs and cosmetics containing organic color additives. In addition, Bhakti performs chemistry reviews of data establishing chemical identity, composition, stability, and manufacture of color additives submitted in support of color additive petitions. She serves as a volunteer in two AOAC working groups and communities.

Joe Konschnik, Plus One Consulting LLC

As Principal Consultant for Plus One Consulting LLC Joe applies his 40 years of analytical chemistry knowledge and experience to support his clients in the analytical testing industry. Prior to retiring from RESTEK in January, 2023 Joe was RESTEK’s Business Development Manager for the global Food & Agriculture market where he identified market needs, new technologies, new product opportunities and coordinated technical content development for new workflow solutions. Prior to RESTEK Joe spent 19 years

working for some of the nation’s largest environmental testing laboratory companies as a bench chemist and in technical and laboratory operations senior management roles. Joe has been an invited speaker on sampling and analytical chemistry topics globally. He serves as a volunteer in multiple AOAC working groups and communities and previously served in volunteer leadership roles for the Independent Laboratories Institute (ILI), the American Council of Independent Labs (ACIL)’s Food Sciences Section and The North American Chemical Residue Workshop (NACRW). Direct correspondence at joe@plusone.llc.

David Bolliet, Kalsec ® After completing his undergraduate degree in analytical chemistry at CPE Lyon (formerly Lyon Superior School of Chemistry), Lyon, France, David Bolliet earned a Master of Science degree in Analytical Chemistry under the guidance of Dr. Colin Poole at Wayne State University, Detroit, MI, where he studied and modeled the effects of solvent, temperature, and pH on the HPLC retention mechanisms of

polymeric stationary phases.

Currently, David holds a position of Senior R&D Manager & Analytical Platform Lead at Kalsec ® , Kalamazoo, MI, a family-owned and B Corp certified global company that provides a full portfolio of high quality, natural food and beverage ingredients for savory food and brewed beverage applications.

In his spare time, David enjoys downhill skiing, mountain biking, rock climbing, and kayaking.

John Mills, bioMérieux John Mills is an Associate Director in the Industry Scientific Affairs division of bioMérieux, Inc, and has been with bioMérieux for 27 years. John has worked in Food and Cannabis Safety for the majority of that time. John is currently responsible for support and application activities in St. Louis involving the TEMPO, VIDAS, GENE-UP, VITEK 2 Compact, and VITEK MS systems. John has a significant

amount of experience with Quality Indicator testing, Pathogen testing and identification of bacteria and yeasts. John also works extensively with AOAC, NCIMS, and Health Canada regarding the approval and validation of bioMérieux products. John is a graduate of Southeast Missouri State University with a Bachelor of Science degree in Microbiology and a Masters of Natural Science.

Eric Schwartz, Ph.D., Senior Scientist I I , Food Standards Eric joined United States Pharmacopeia (USP) and Food Chemicals Codex (FCC) in March of 2020. He serves as a scientific liaison between the USP Food Ingredients Expert Committee (FIEC) and FCC staff to usher standards through the standard setting process. He also serves as coordinator of the Foods lab projects and staff lead to the FCC Dietary Proteins Expert Panel. Prior to working at FCC, Eric

worked as a Senior Scientist at the Flavor Research and Education Center (FREC) at the Ohio State University Department of Food Science and Technology where his research involved method development, isolation, and characterization of flavor active compounds. Eric received his doctorate degree in medicinal chemistry and pharmacognosy at the Ohio State University where his research was focused on the synthesis and optimization of bioactive natural products.

Tongtong Xu, Ph.D. Senior Scientist II, for the Food Chemicals Codex Tongtong is a Senior Scientist supporting the Food Chemicals Codex (FCC) managing development and modernization of FCC standards for food ingredients, as well as the development of general tests detecting potential adulterants in vulnerable food ingredients. Part of her job is to develop monographs for color additives from natural sources. She also leads the High Value Food Oil Expert Panel to develop new FCC standards for fats.

Color Additives from Natural Sources Meeting Monday, August 28 | 3:30PM – 5:00PM CDT

Thank you to our Advisory Panel Members for supporting the current scope of work in the AOAC Natural Colors Working Group!

Scan to sign into the meeting and get access to meeting materials!

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Today’s Agenda:  Expert Review Panel Overview  Subgroup Updates

 Pesticide Residues  Residual Solvents  Microbial Contaminants  Using Emerging Technology for Predictive Microbiology for Natural Food Colors  The Role of FCC in Protecting the Integrity of Natural Food Colors

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Expert Review Panels

What are ERPs?

AOAC Expert Review Panels (ERPs) are responsible for adopting candidate methods as First Action Official Methods and making recommendations for Final Action status to the AOAC Official Methods Board (OMB).

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Expert Review Panels

Who are ERPs Composed of?

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA Potential expert reviewers are vetted by the OMB and if selected, undergo orientation to be fully prepared to review First Action or Final Action Methods. ERPs are composed of qualified subject matter experts representing various perspectives. ERP candidates are obtained via a Call for Experts from AOAC.

Expert Review Panels

Wait… what is a Call for Experts?

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA During this time SMEs can apply to join the ERP. All applications will be screened and submitted to the AOAC Official Methods Board, who will vet the candidates and form the ERP roster. Once a candidate method is submitted to AOAC, the Corresponding ERP is notified. If an ERP does not already exist, AOAC will publicize a Call for Experts.

Expert Review Panels

What is the ERP Review Process?

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA Method author(s) are invited to present their method during ERP sessions and to respond to comments. AOAC facilitates and coordinates the ERP review of each method against collaborative study protocols and/or Standard Method Performance Requirements (SMPRs) submitted with the method.

Expert Review Panels

What is the ERP Review Process?

During ERP meetings, the ERP may require changes and/or revisions to the manuscript to be updated in real time. At the conclusion of its review of the method, the ERP will render a decision to either grant or withhold AOAC First Action Official Method status

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Interested in Joining an ERP for Natural Colors?

Scan the QR Code to show your interest or contact Allison Baker at abaker@aoac.org

Link to Interest Form

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Residual Solvents Consensus Form (Color Addi�ves from Natural Sources)

JotForm Link: htps://form.jo�orm.com/232135221806143

Microbial Contaminants Interest Form

htps://form.jo�orm.com/232343816410044

Update on Progress for the Natural Colors Community Pesticides Working Group Joe Konschnik, Plus One Consulting LLC representing Restek Corporation

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update

• Review Final SMPR • Responses to Public Comments • Call for ERP Members • Questions

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update: Review Final SMPR Standard Method Performance Requirements (SMPRs) for Selected Pesticides in Color Additives from Crop Based Sources

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update: Review Final SMPR Standard Method Performance Requirements (SMPRs) for Selected Pesticides in Color Additives from Crop Based Sources

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update: Review Final SMPR Standard Method Performance Requirements (SMPRs) for Selected Pesticides in Color Additives from Crop Based Sources

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update: Review Final SMPR Standard Method

Performance Requirements (SMPRs) for Selected Pesticides in Color Additives from Crop Based Sources

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update: Review Final SMPR Standard Method Performance Requirements (SMPRs) for Selected Pesticides in Color Additives from Crop Based Sources

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update: Review Final SMPR Standard Method Performance Requirements (SMPRs) for Selected Pesticides in Color Additives from Crop Based Sources

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update: Public Comments Table 3: Provide concentrations in mg/kg (ppm) - Change "10-100ppb" to "0.01 - 0.1) Line 79: Define CRM

Line 80: Which ISO guide? Table 1: LOQs are not listed

Table 1: Phthalimide (metabolite of folpet) is also a metabolite of phosmet. It is also produced from other sources (without folpet or phosmet present). See publication by Bundesverband Naturkost Naturwaren on the subject. Additionally, only the EU has phthalimide listed in the residual definition of folpet.

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update: Public Comments Table 2: What are the numbers in front of the matrix? Table 3: If the analyte can recover within 60-120% and there is a LOQ of 0.01 mg/kg, shouldn't the analytical range cover got to at least 60% of 0.01 mg/kg? Table 3: Should a footnote be added that recoveries outside of 60-120% are acceptable if the RSD results are acceptable (according to SANTE guidelines)? Additionally, if poor recovery is observed, would it be beneficial to have a matrix effects study to determine that it is indeed matrix effects and not a preparation issue? What is the rationale for selecting the pesticides in Table 1? are they most frequently detected or for the compliance?

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update Call for ERP Members Please contact Allison Baker abaker@aoac.org if you are interested in serving on the ERP.

Join us at our community meeting immediately following this session if you have questions before volunteering.

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Pesticides WG Update Questions?

Thank you!

Joe Konschnik Restek Corporation joe.konschnik@restek.com

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Update on Progress for the Natural Colors Community Residual Solvents Working Group

David Bolliet Kalsec

Monday, August 28, 2023

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

In the beginning…

• Lab work • AOAC poster & presentation • Proof of concept

Fontana, A.; Kotani, M.; Konschnik, J.; Poster presented at 2019 AOAC Annual Meeting Fontana, A.; Kotani, M.; Oral presentation at 2020 AOAC Annual Meeting

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

2019 poster – Static headspace GC-MS

Fontana, A.; Kotani, M.; Konschnik, J.; Poster presented at 2019 AOAC Annual Meeting

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Residual solvents working group

• Tasked with developing analytical methods to measure residual solvents in color additives from natural sources • USP <467> list of 36 authorized solvents narrowed to 12 at first

Fontana, A.; Kotani, M.; Konschnik, J.; Poster presented at 2019 AOAC Annual Meeting * Singly or in combination

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

SMPR – Current version

• Subgroup meetings

• Standard Method Performance Requirement (SMPR®) o 20 analytes o 16 matrices o Method performance requirements

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Solvents methanol

CAS

67-56-1 64-17-5 67-63-0 71-36-3 67-64-1

ethanol

20 analytes

isopropanol (2-propanol)

n-butanol acetone hexanes a heptanes a

110-54-3 b 142-82-5 c 141-78-6 110-19-0 78-93-3 96-47-9 75-09-2 107-06-2 79-01-6 67-66-3 71-43-2 108-88-3 1330-20-7 d

• Selection of solvents both authorized and unauthorized

ethyl acetate

isobutyl acetate

methyl ethyl ketone

methyloxolane (methyltetrahydrofuran)

methylene chloride

ethylene dichloride (1,2-dichloroethane) trichloroethylene (1,1,2-trichloroethene)

chloroform

benzene toluene xylenes a

a - Reported as sum of isomers b - CAS# for n-hexane. (Isomers with CAS# listed in SMPR) c - CAS# for n-heptane. (Isomers with CAS# listed in SMPR) d - CAS# for mixed xylene isomers. (Isomers with CAS# listed in SMPR)

diethyl ether

60-29-7

methyl tert-butyl ether

1634-04-4

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

16 matrices

CFR

Target matrix

Manufacturing process

Source

73.30 Annatto extract

Solvent extraction Dehydration of beets

Annatto seed

73.40 Dehydrated beets (beet powder) *

Beets

73.95 Beta-carotene

Natural, not synthetic, product

Yellow, orange, and green leafy fruits and vegetables; algae; fungi

73.125 Sodium copper chlorophyllin

Processed to add copper

Alfalfa

73.169 Grape color extract *

Aqueous extraction Aqueous extraction Aqueous extraction Aqueous extraction

Concord grapes

73.170 Grape skin extract (enocianina) *

Grapes

73.250 Fruit juice * 73.260 Vegetable juice *

Fruit

Vegetables

73.300 Carrot oil 73.340 Paprika *

Solvent extraction from carrots Ground dried pod of capsicum Solvent extraction from paprika Dried stigma of Crocus sativus L.

Carrots

Pod of capsicum

73.345 Paprika oleoresin

Capsicum

73.500 Saffron *

Dried stigma of Crocus sativus L.

73.530 Spirulina Extract

Aqueous extraction

Arthrospira platensis

73.585 Tomato lycopene extract; tomato lycopene concentrate * Ethyl acetate extraction from tomato pulp Tomato 73.600 Turmeric * Ground rhizome of Curcuma longa L.

Rhizome of curcuma Rhizome of curcuma

73.615 Turmeric oleoresin

Solvent extraction of turmeric

* - Natural source only

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Method performance requirements Solvents Analytical Range (ppm) % Recovery LOQ (ppm) % RSD r % RSD R min max ≤ 1 0 ppm > 10 ppm methanol 2 100 2

ethanol

2 2 2 2 2 2 2 2 2 2 2 2 2

400 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

2 2 2 2 2 2 2 2 2 2 2 2 2

isopropanol (2-propanol)

n-butanol acetone hexanes* heptanes*

ethyl acetate

methyl ethyl ketone methyloxolane methylene chloride ethylene dichloride trichloroethylene

50 - 150 75 - 125

≤ 20 ≤ 30

chloroform benzene toluene xylenes* diethyl ether

0.5 0.5 0.5 0.5 0.5

0.5 0.5 0.5 0.5 0.5

methyl tert-butyl ether

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

SMPR – Analytical technique

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

SMPR – Reference materials

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Review period

• Call for public comments period o June 14 – July 28, 2023 o No comments

• Next step: Voting

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Open for voting

• Review SMPR and vote https://form.jotform.com/232135221806143 (Open through Friday, September 1, 2023)

• Next step: call for methods

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Thank you!

• AOAC International • Kalsec • Dr. Bhakti Petigara • Residual Solvents Subgroup members • Dr. Doug Williams

Dr. Doug Williams, retired

AOAC INTERNATIONAL 137 th Annual Meeting & Exposition August 25 – 30, 2023 • New Orleans, LA

Using Emerging Technology for Predictive Microbiology of Natural Food Colors 2023 John Mills

P I ONEER I NG D I AGNOST I CS

A MULTI-TIER APPROACH TO PROBLEM SOLVING

Genomics + Molecular Biology + Data Science + Data Management

Identify contaminants 1

Track and mitigate

3

2

Predict/Prevent

Targeted screening

4

Better information, faster decisions

B I O M É R I E U X

STRICTLY CONFIDENTIAL

IDENTIFY CONTAMINANTS

B I O M É R I E U X

METAGENOMICS TO ID CAUSE OF SPOILAGE

• Many spoilers are non-culturable • Enrichment bias

Traditional Enrichment & Plating

→ Shallow Shotgun Metagenomics Whole microbiome, no bias

B I O M É R I E U X

PROBLEM – SEASONAL BLOWN SOUP PACKAGES

Blown Packages

SOUP

SOUP

4 o C

Could Not ID Spoilage

Traditional Enrichment & Plating

5

B I O M É R I E U X

SOLUTION – PSYCHROPHILIC YEAST

Extreme Psychrophilic Yeast: Mrakia

SOUP

SOUP

6

B I O M É R I E U X

STRICTLY CONFIDENTIAL

TARGETED SCREENING

B I O M É R I E U X

CUSTOM MOLECULAR ASSAY

Custom Molecular Assay

B I O M É R I E U X

EXAMPLE

9

B I O M É R I E U X

STRICTLY CONFIDENTIAL

TRACK AND MITIGATE

B I O M É R I E U X

TESTING WORKFLOW

Negative

Results

PCR

Sample

Release

Positive

True or lab cross contamination?

HOLD Time for questions and decisions

?

Persistent strain/biofilm?

Biocide resistance?

Where is it coming from?

11

B I O M É R I E U X

TRACK AND MITIGATE – MAPPING (TYPING)

WE CAN TELL RELATIONSHIPS

WE CAN TELL TRANSMISSION

WE CAN TELL RESISTANCE

Sample ID Cluster Colistine Beta-Lactam Quinolone Aminoglycoside Sulfonamides Chloramphenicol Trimethoprim Fosfomycin Tetracycline 303727 S1 + + + + 303728 S1 + + + + 304067 S2 + 303811 M1 + + ? + + 303814 M1 + + ? + + 303820 M1 + + ? + + 303822 M1 + + ? + + 303827 M1 + + ? + + 303853 M1 + + + + 303854 M1 + + + 305366 M1 + + ? + + 303857 M1 + + ? + + 305246 M2 + ? + + + + 305173 H1 + + + + 305244 H1 + + + + 305247 H1 + + + + 305281 A1 + 304188 A1 + 303716 K1 303721 K1 303729 K1 304052 P1 303807 P1 304062 Se1

• Source (from the plant, supplier or lab) • Likely date of introduction • Persistence at factory and where

• Resistance prevalence per species or serotype • Validation against phenotypic resistance

• Geographic transmission (people & equipment) • Potential hygiene issues

12

B I O M É R I E U X

CONTAMINANT MAPPING

28 Cronobacter Positives • Is it the same problem we can’t resolve? • Or something new every time?

Is It Staying In the Same Place? • Is the recurring strain in the same place or spreading?

Recurring Despite Mitigation • Why do we find the same strain over time? • Why is it spreading through the plant?

Cronobacter turicensis

2020-10 2020-11 2020-12 2021-01 October sample November sample December sample January sample

1

1

1

High hygiene

3

Cronobacter dublinensis

1

Cronobacter turicensis

1

1

Cronobacter sakazakii

Basic hygiene

Medium hygiene

Cronobacter turicensis

• Multiple sampling locations in medium hygiene zone – spreading within an area • Multiple sampling locations in high hygiene zone – spreading across plant (including hygiene zones)

• Strain is resistant to multiple metals, including silver (often used as antimicrobial coating on surfaces)

• 1 strain is recurring with 22 related samples found over 4 months • 5 strains appear transient with resolution (not seen over time)

B I O M É R I E U X

ACTIONABLE DATA IN EM SOFTWARE

15

B I O M É R I E U X

STRICTLY CONFIDENTIAL

PREDICT AND PREVENT

B I O M É R I E U X

PREDICTIVE MODELING

HOW CAN I PREDICT SAFETY OR QUALITY ISSUES FROM SUPPLIERS? IN WHICH CONDITIONS IS THE ISSUE MOST LIKELY TO APPEAR?

Framing the question

Testing and PDIC data

Data Collection & validation, Analysis & modeling

Other internal data

External data

Predictive model delivery

17

B I O M É R I E U X

ILLUSTRATION CULTURE MEDIA MANUFACTURING

Site snapshot • Production activity: Culture Media (550 refs / 45 MUnits), Chemunex instruments (70 units) & reagents (130 refs / 1.5 MUnits), Blue Line (2500 units) , LabGuard (10000 units) • 200 FTE, 40.000 m², certified ISO 9001 / 17025

18

B I O M É R I E U X

SMPR UPDATE

1. Reference methods/compendial methods (culture-based methods vs. rapid methods) 2. Standards: How are new methods validated? 3. How are screened positive samples confirmed? 4. Implementation strategies for new methods (lab validation/verification) 5. What is the sampling plan for examining raw material lots? a. What is a representative sample? b. How many samples are tested per lot? 1. Sample size requirements (# grams) for testing each lot of raw materials? 2. Is composite sample testing acceptable? a. Number and size of samples composited 1. Which pathogens are tested for in raw materials? a. Bacteria pathogens? b. Viral pathogens? c. Parasitic protozoan pathogens? d. Microbial toxins? 1. Are the acceptance criteria zero tolerance (absence) for all pathogens in natural color raw materials (i.e., <1 CFU per 25g for Salmonella, Listeria, STEC)? a. Are any target pathogen levels quantified in incoming raw materials? 1. Are raw materials tested for microbial limits (quantification) of specific quality indicators a. Total aerobic counts b. Total yeast and mold 1. What are the mitigation strategies for eliminating unacceptable levels of microbial contaminants (i.e., irradiating and retesting of raw materials)? 2. What are the standard (general) categories of Natural Color raw materials? 3. Are suppliers testing the raw materials or sending to expert labs for analysis? 4. Are there established reference labs for testing Natural Color raw materials? 5. Are there Lab Proficiency Micro testing programs for Natural Color raw materials?

19

B I O M É R I E U X

P I O N E E R I N G D I A G N O S T I C S

B I O M É R I E U X

AOAC Annual Meeting August 28, 2023

The Role of FCC in Protecting the Integrity of Food Colors from Natural Sources Eric Schwartz Ph.D., eric.schwartz@usp.org Food Chemicals Codex at U.S. Pharmacopeia

Overview

• FCC introduction and scope • Importance of standards on colors from natural sources • FCC development of a new guideline appendix • Current tools in FCC to protect the integrity of colors from natural sources

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Food Chemicals Codex

A compendium of internationally recognized standards for the identity and purity of food ingredients

Created by the US-FDA and the US Institute of Medicine in 1966

A fully independent source of food

>1250 standards for additives, ingredients, and

Currently published by USP, a non-profit organization

Standards are developed by expert volunteers

ingredient standards

other food chemicals

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Food Chemicals Codex Scope

• Monographs (>1250) • Identity Standards • Appendices • General Tests and Assays (>150) • Guidelines • Developing and Validating Non-Targeted Testing • Olive Oil Guidance, Methods and Applicable Resources • Food Fraud Mitigation

Protecting public health and the integrity of the food supply worldwide

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Food Integrity is a Major Public Health Challenge

Food Fraud

Food Defense

Food Safety

Food Quality

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY Elements of food integrity

Source: GSFI

Importance of Standards in Maintaining Ingredient Integrity

• Food supply chains are complex, non-linear, and subject to sudden disruption which can affect food integrity • While traceability of an ingredient may be known, without proper oversight and controls, the potential for fraud exists • Standards are integral to the reduction of an ingredient's vulnerability to food fraud and protection of ingredient integrity. • especially when the methods and specifications in the standard well characterize the ingredient and contain appropriate validated methods for adulteration detection.

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Importance of Quality and Safety for Colors from Natural Sources

• With the rising demand for natural ingredients, quality and safety are extremely important topics and opens the door for both unintentional and intentional economic adulteration • Up to 85% of consumer buying decisions are potentially influenced by color, underscoring that appropriate application of color additives and their safety is critical • Globally, colors from natural sources are used more often in food and beverage launches compared to artificial colors or whole foods that add color

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY Source: Innova Market Insights Simon , J.E., De cke r , E.A., Fe rruzzi, M.G., Gius t i, M.M., Me jia , C.D., Go ldschm id t , M. and Ta lco t t , S.T. (2017), Es tab lish ing Standa rds on Co lors from Na tu ra l Sou rce s . Jou rna l of Food Science , 82: 2539-2553.

The Need for Industry Standards and Guidance on Colors from Natural Sources

• Review article published in Journal of Food Science • Simon JE, Decker EA, Ferruzzi MG, Giusti MM, Mejia CD, • Objectives of Review Article • Make recommendations for quality and product safety standards for the natural colors industry • Promote standardization of methods used to test natural colors • “Raise the bar” on the quality and safety of natural colors • Development of “white paper” Goldschmidt M, Talcott ST. Establishing Standards on Colors from Natural Sources. J Food Sci. 2017 Nov;82(11):2539-2553.

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

FCC’s Efforts to Provide Stakeholder Guidelines on Colors from Natural Sources • FCC in collaboration with the AOAC working group is developing a new guideline appendix on quality and safety specifications for Colors Exempt from Certification (Colors from Natural Sources) • Appendix references “Establishing safety standards on colors from natural sources,” J. of Food Science , Nov 2017 • Establishing recommended specifications • Metals • Microbial contaminants • Residual solvents • Pesticides • Adulteration • Appendix XXI: Guidance and Best Practices for the Use of Standards on Colors from Natural Sources, including recommended specifications is targeted for submission to the June 2024 FCC Forum

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Outline of New FCC Guideline Appendix XXI

Appendix XXI: Guidance and Best Practices for the Use of Standards on Colors from Natural Sources 1. Introduction and Background • Description, quality control considerations, purpose of guidance 2. Regulatory • Definitions, quality requirements, list of exempt colors 3. Types of Colors from Natural Sources and manufacturing processes 4. Intentional Adulteration 5. Guidelines for Setting Specifications for Quality Control • Identification • Contaminant specifications and methods o Microbial, Residual Solvents, Elemental Impurities, Pesticides 6. Risk Management • Proposed risks and mitigation strategies • Reference to USP Food Fraud Mitigation Guidance

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Determination of Recommended Specifications for Selected Contaminants

HEAVY METALS

J. Food Sci. 21 CFR HC (Canada) EC 231/2012 JFSA (Japan) GB (China) < 2 ppm < 10 ppm < 10 ppm < 2 ppm < 2 ppm < 10 ppm < 3 ppm < 3 ppm < 3 ppm < 3 ppm < 2 ppm < 1 ppm

USP <561> for botanical raw materials

Lead (as Pb) Arsenic (as As) Mercury (as Hg) Cadmium (as Cd)

< 5 ppm < 2 ppm < 1 ppm < 0.5 ppm

< 1 ppm <1 ppm

< 1 ppm < 1 ppm

< 1 ppm

< 1 ppm

MICROBIAL CONTAMINANTS

RESIDUAL SOLVENTS

USP <2021>/<2022> DS

USP <2023> Botanical raw materials

J. Food Sci.

21 CFR 173

EU Japan Korea

Methanol

< 50 ppm < 50 ppm < 50 ppm < 50 ppm < 50 ppm

J. Food Sci.

FCC XV/XI

Use 10 g sample Use 10 g sample

Ethanol

< 250 ppm < 250 ppm < 50 ppm NA

NA

Total aerobic plate count

< 1000 cfu/g Use 10 g sample

<10^5

Dichloromethane

< 30 ppm < 10 ppm NA 10-30 ppm < 30 ppm < 50 ppm < 30 ppm < 50 ppm < 50 ppm < 50 ppm < 50 ppm 30-50 ppm < 25 ppm < 50 ppm < 25 ppm < 25 ppm

Total Yeast and Molds

< 100 cfu/g Use 10 g sample

Acetone

<10^3

Propan-2-ol

Total coliforms Escherichia coli

<3 cfu/g Use 25 g sample

Hexane

Negative in 10 g Negative in 25 g Negative in 10 g Negative in 10 g Negative in 10 g Negative in 25 g Negative in 10 g Negative in 10 g Negative in 10 g Negative in 50 g Negative in 10 g Negative in 10 g

Ethyl acetate

< 100 ppm < 50 ppm NA

NA

Salmonella

Ethylene dichloride

< 30 ppm NA

NA

NA

Staphylococcus aureus

Trichloroethylene

Listeria monocytogenes Negative in 10 g Negative in 50 g Aflatoxins Total mycotoxins < 20 ppb

< 30 ppm NA < 30 ppm NA

n-butanol

NA

< 50 ppm NA

NA

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

FCC Appendix XIII: Adulterants and Contaminants in Food Ingredients • Effective text published in FCC Appendix XIII • Non-targeted tests for the detection of undeclared colors in paprika, chili powder, turmeric, curry, and sumac • Thin-layer chromatography screening method • Three different validated procedures for detection of a total of 17 colors

N O - O N N

HO

Na +

O 2 N

HO

O

O

N

N

O

N N

NH 2

N HN

O

N

O

O

N

- O

N

Na +

O

- O

N

S

S

Na + O O O - S

O

O

Na +

- O

Na +

S

S

O -

N H

O

S O O O -

S

S

O

Na +

O

- O

- O

O

Na +

+

Na +

O

Na

O

O

SO 3 Na

NaO 3 S

Tartrazine

Ponceau 4R

Acid Black 1

Allura Red AC

Orange II

OH

- O

Na +

HO

N N

- O

S O O

N +

O

O

S

N

S O O

O

N

Na +

HN

O

N NH

S O O

ONa

- O

O

N

N

N

NH

S O O O -

N

NaO

OH

S O O

O

N

N

OH

S

O -

Metanil Yellow Na +

N

N

O - Na +

O

Sudan I

O

Na +

S

S O O O -

O

- O

Na +

Na +

Para Red

Azorubine

Sunset Yellow FCF

Brilliant Black PN

O -

N +

N

O

O

Na +

N

HO S

N

NH

O

-

N

N

Cl

S O O

N

OH O

- O

HO

N N

N

HO

HO

N

N

Na +

HCl

O

Sudan II

S

Sudan IV

Na + 1

N N

O -

O

Rhodamine B

Auarmine O

Sudan III

Amaranth

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

FCC Appendix XIII: Adulterants and Contaminants in Food Ingredients • Current Work (Targeted for submission to December 2023 FCC Forum)

• Targeted test for the detection of undeclared colors in paprika, chili powder, turmeric, curry, and sumac • LC-MS/MS validated screening method for the detection of 25 undeclared colors • Involves matrix matched calibration curves for quantitation

RT (min.)

Parent (m/z)

Cone (V)

Daughter 1 (m/z)

Collision Energy 1 (V)

Daughter 2 (m/z)

Collision Energy 2 (V)

Recovery (%)

Compound Name

Basic Red 9 Safranin O Auramin O

4.9 6.3 6.9 7.2 7.4 7.8 8.2 8.9 9.0 9.0 9.1 9.2 9.2 9.2 9.8 9.8

289 315 268 307 322 241 329 274 238 443 443 213 215 373 238 385

60 60 60 60 60 60 60 60 60 60 80 60 34 60 60 60 42 34 25 30 20 30 50 34 50

151 210 147 170 136 157 208 105 165 341 355

50 55 30 35 25 25 40 30 35 50 60 30 25 50 35 55 30 34 55 28 40 20 25 30 44

195 237 252 292 160 185 313 228 223 415 399 121 122 356 223 341 121 156 108 156 152 156 197 183 224

30 45 35 25 15 22 35 35 25 35 42 15 20 40 25 40 25 18 35 20 10 20 19 20 22

75.6 75.5 88.9 90.9 89.9 93.1 91.2 79.8 84.2 92.4 92.5 99.2 86.6 91.6 85.5 92.5 92.6 78.2 81.6 91.8 93.5 99.4 99.4 94 81

Basic Yellow 13 Basic Yellow 28

Alizarin

Malachite Green Quinoline Yellow Disperse Orange 11

Rhodamine 6G Rhodamine B Chrysodine G Sudan orange G Crystal Violet

77 93

340 167 297

1-Methyl Amino anthraquinone

Brilliant Green Butter Yellow

10.7 226 11.0 294 11.5 279 11.6 249 11.6 309 12.9 277 13.5 353 14.2 380 14.4 381

77

Para Red

128

Sudan Red G

80

Sudan I

128 138 121 156 169 106

Citrus Red II

Sudan II Sudan III

Sudan Red 7B

Sudan IV

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY Colors, retention times, MRM transitions, and recovery of 25 colors in chili at 10 ppm

FCC Appendix XIII: Adulterants and Contaminants in Food Ingredients

Data from commercial chili powder (rejected samples) in ppm

Stacked spectra of 25 colors spiked in chili powder

Color

Source-1 Source-2 Source-3 Source-4 Source-5Source-6Source-7

Butter Yellow ND 3.41

6.91

ND ND ND ND

1672.67 1095.17 1209.78 1455.98 ND ND 100.11

Sudan I

ND ND ND ND 14.73 ND ND

Sudan II Para Red

ND ND ND 0.48

ND ND ND

Malachite Green ND ND ND ND ND ND 0.47

1.74

11.94 12.09 21.62 ND ND 1.41

Sudan III

Sudan Red 7B

0.5

ND ND ND ND ND 0.24

Sudan IV 212.74 101.87 90.54 114.73 0.74 10.68 ND

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Summary

• Maintaining food and ingredient integrity is a major challenge evidenced by numerous food safety incidents throughout history • Consistent industry-wide safety standards are needed to address the manufacturing, processing, application, and international trade of colors from natural sources • FCC in collaboration with the color community and AOAC working group is developing a new guidance appendix based on the article published in the Journal of Food Science • FCC has tools in place to protect the integrity of colors from natural sources - ingredient monographs, tests, and guidline appendices • Next steps include development of new standards on colors from natural sources and further development of guidance appendix

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Food Chemicals Codex (FCC) | FCC | Online

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Questions? Contact: eric.schwartz@usp.org

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

AOAC Annual Meeting August 28, 2023

The Role of FCC in Protecting the Integrity of Food Colors from Natural Sources Tongtong Xu Ph.D., tongtong.xu@usp.org Food Chemicals Codex at U.S. Pharmacopeia

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Outline

• Introduction • Relevant FCC work • New FCC monographs • New FCC general tests and assays • FCC monograph modernization

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Food Chemicals Codex

A compendium of internationally recognized standards for the identity and purity of food ingredients

Created by the US-FDA and the US Institute of Medicine in 1966

Currently published by USP, a non-profit organization

A fully independent source of food

>1250 standards for additives, ingredients, and

Standards are developed by expert volunteers

ingredient standards

other food chemicals

SAFEGUARDING THE INTEGRITY OF THE FOOD SUPPLY

Ingredient standard is…

FD&C Act Sec 201

21 CFR Sec. 73.450 Riboflavin "(a) Identity. (1) The color additive riboflavin is the riboflavin defined in the food chemicals codex... Specifications . Riboflavin shall meet the specifications given in the food chemicals codex ..."

4

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

The content of an FCC standard

Impurities

Assay

Description

Identification

Chemical Information

Specific Tests

5

An example of an FCC standard – Riboflavin

6

An example of an FCC standard – Riboflavin

7

An example of an FCC standard – Riboflavin

8

FCC Work on Natural Food Colors

New monograph: Jagua (genipin-glycine) blue

 Monograph section - Identification  Visible Absorption Spectrum  UV-Vis spectrophotometer (maximum 590-594 nm)  Infrared Spectra

FCC13 1S

10

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

New monograph: Jagua (genipin-glycine) blue

 Monograph section - Assay  Jagua (Genipin-Glycine) Blue Polymer Standard preparation Method: HPLC Detector: UV-Vis/PDA 590 nm

 Total Color

FCC Appendix IIIC Color Determination Acceptance criteria: 25%– 54.4%, calculated on the dried basis

Acceptance criteria: 20%– 43.5% (as the blue polymer), calculated on the dried basis

11

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

New monograph: Jagua (genipin-glycine) blue

 Monograph section - Impurities  Arsenic NMT 1 mg/kg  Lead NMT 2 mg/kg  Cadmium NMT 1 mg/kg FCC Appendix IIIC ICP Method

 Monograph section - Specific Test  Genipin Method: HPLC

Detector: UV-Vis 240 nm Acceptance criteria: NMT 0.3%, calculated on the dried basis

12

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

New monograph: Paprika oleoresin

 Monograph section - Identification  Color reaction

 UV-Visible Absorption Spectrum  Chromatographic profile for β - carotene, capsanthin diester peak 1, and capsanthin diester peak 2 (Chromatographic system in Assay )  Approximate Relative Retention Time of the peaks for carotenoids (Chromatographic system in Assay )

13

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

New monograph: Paprika oleoresin

 Monograph section - Assay  Capsanthin and capsorubin Method: HPLC Detector: Vis 450 nm

 Monograph section - Specific Tests  Color Value

Acceptance criteria: NLT 500 units, as specified on the label

Column:4.6-mm × 250-mm; packing of octadecylsilane chemically bonded to 5- μm porous silica or ceramic micro - particles. Use a suitable C18 guard column. Acceptance criteria: NLT 30%

 Total Capsaicinoids Content Acceptance criteria:

Color applications: NMT 200 mg/kg Flavor applications: NMT 0.5%

14

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

New monograph: Paprika oleoresin

 Monograph section - Impurities  Arsenic NMT 3mg/kg  Lead NMT 1 mg/kg  Residual Solvents

 Total chlorinated hydrocarbons: NMT 0.003% as the total of dichloromethane, trichloroethylene, and ethylene dichloride when used singly or in combination  Acetone: NMT 0.003%

 Isopropanol: NMT 0.005%  Methanol: NMT 0.005%  Hexane: NMT 0.0025%

15

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

FCC appendix XIII: Residual solvents in food colors

Chromatographic parameters Mode: Headspace GC Detector: FID

Column: Fused silica, 30-m × 0.25-mm (id) with a 1.4-µm coating of 6% cyanopropylphenyl–94% dimethylpolysiloxane stationary phase Gas flow Air: 400 mL/min Hydrogen: 40 mL/min Nitrogen column flow: 1.5 mL/min Makeup flow: 25 mL/min (nitrogen) Injection type: Split injection, split ratio of 1:1

16

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

FCC appendix XIII: Residual solvents in food colors

• Effective as of Dec 2021 • Method developed in USP internal lab • Fully validated in the food color matrices:

• Annatto extracts • Tagetes extract • Sodium Copper Chlorophyllins • Paprika Oleoresins

17

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

Monograph modernization

The Residual Solvent test method is modernized to FCC Appendix XIII method Effective as of Dec 2021

Tagetes extract Acceptance criteria: NMT 0.005% acetone, hexane, ethanol, ethyl methyl ketone, isopropanol, and methanol, individually or in combination

Annatto extracts

Acceptance criteria: Acetone: NMT 0.003% Ethylene dichloride: NMT 0.003% Hexanes: NMT 0.0025%

Isopropyl alcohol, ethanol, and ethyl acetate: NMT 0.005%, individually or in combination Methyl alcohol: NMT 0.005% Trichloroethylene and Dichloromethane: NMT 0.003%, individually or in combination

18

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

Monograph modernization

The Residual Solvent test method is added based FCC Appendix XIII method June 2023 FCC Forum

Lutein Acceptance criteria: Hexane: NMT 0.005% Methanol: NMT 0.001%

Free access to FCC Forum : https://www.foodchemicalscodex.org/fcc-forum

19

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

Future FCC work

Targeted Dec 2023 FCC Forum

One Monograph Modernization: • Carmine: propose to replace the two specific absorption maxima in the Acceptance criteria in Identification test with two ranges of max wavelength

Targeted June 2024 FCC Forum

Two New Monographs: • Chlorophyllins, Copper Complexes Sodium and Potassium Salts • Cochineal Extract

20

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

Summary

• Maintaining food and ingredient integrity is a major challenge evidenced by numerous food safety incidents throughout history • Consistent industry-wide safety standards are needed to address the manufacturing, processing, application, and international trade of colors from natural sources • FCC in collaboration with the color community and AOAC working group is developing a new guidance appendix based on the article published in the Journal of Food Science • FCC has tools in place to protect the integrity of colors from natural sources - ingredient monographs, tests, and guidance appendices

21

SAFEGUARDING THE INTEGRITY OF THE GLOBAL FOOD SUPPLY

Tha nk you

Food Chemicals Codex (FCC) | FCC | Online

Sta y Conne cte d

tongtong.xu@usp.org