Ensure Border Acceptance, Avoid

Border Rejection through Mycotoxin Awareness

Danrey Toth

June 6-8, 2017

Contents

Regulation RASFF What is Aflatoxin and Ochratoxin How to test aflatoxin and ochratoxin A Sampling issue

EC Regulation for Aflatoxin (PPB)

Commodity B1 Total M1

Processing

Almond, pistachios and apricots Kernels 12 15

Peanuts, Hazel and Brazil nuts 8 15

Other tree nuts, dried fruits, rice ,maize 5 10

Direct consum

ption

Cereals, rice ,maize ,peanuts, other tree nuts & dried fruit 2 4

Almond, pistachios and apricots 8 10

Hazel and Brazil nuts 5 10

Spices (capsicum, pepper, nutmeg, ginger, turmeric) 5 10

Dried Fig 6 10

Baby/Infant food, formula and dietary supplement 0.1 0.025

All Liquid milk 0.05

EC Regulation for Ochratoxin A

Commodity OTA(ppb)

Wine, Alcohol drinks, Grape juice 2.0

All Cereals for direct consumptions 3.0

All unprocessed Cereals, Roasted coffee 5.0

Wheat gluten not sold directly to the consumer 8.0

Dried vine fruit, Soluble coffee 10.0

Spices (pepper, nutmeg, ginger, turmeric) 15.0

Liquorice root, Capsicum (including chillies, chilli powder, cayenne and paprika), Ingredient for herbal infusion

20.0

Liquorice extract , for use in food in particular beverages and confectionary

80.0

Baby/Infant food, formula and dietary supplement 0.50

Regulation for mixture of nuts and mixtures of nuts and dried fruit

Example: sample of 20 kg from a mixture with hazelnuts, cashews, walnuts, shelled Brazil nuts, pistachios (kernels), almonds, peanuts and dried raisins.

After grouping of the nuts and dried fruits with the same levels following result was obtained: - pistachios and almonds: 5,3 kg - shelled Brazil nuts and hazelnuts: 4.8 kg - peanuts, cashews, walnuts and raisins: 9.9 kg The maximum level aflatoxin B1 applicable is: [(5.3 x 8) + (4.8 x 5) + (9.9 x 2)]/20 = (42.4 + 24 + 19.8) / 20 = 4.31 μg/kg The maximum level aflatoxin total applicable is: [(5.3 x 10) + (4.8 x 10) + (9.9 x 4)]/20 = (53 + 48 + 39.6) / 20 = 7.03 μg/kg Thereafter the 20 kg sample is completely mixed and then afterwards subdivided into two laboratory samples and both laboratory samples have to comply with the above mentioned calculated maximum levels.

The Rapid Alert System for Food and Feed (RASFF)

was put in place to provide food and feed control authorities with an effective tool to exchange information about measures taken responding to serious risks detected in relation to food or feed.

This exchange of information helps Member States to act more rapidly and in a coordinated manner in response to a health threat caused by food or feed.

More in-depth analysis of RASFF performance is available in annual reports.

History of RASFF

How Does RASFF Work

How Does RASFF Work

Types of Notifications

RASFF 2015 Annual Report

2015 US (O) Nuts and Seed, 38 cases

1. Pistachio had more issues than other nuts for aflatoxin in 2015, 29 out of 38

2. About 90% total Aflatoxin in total Pistachio is B1, Except couple of exception(?)

3. There was one Ochratoxin A in nuts

Commodity B1 Total ratio

Processing Almond, Pistachios and Apricots 12 15 80

Peanuts, Hazel and Brazil nuts 8 15 53

Other tree nuts 5 10 50

Direct

Peanuts, other tree nuts 2 4 50

Almond, pistachios and apricots 8 10 80

Hazel and Brazil nuts 5 10 50

2017 US (O) Nuts and Seed

1. Pistachio had more issues than other nuts for aflatoxin by March 2017, 12 out of 16 , ratio remain same

2. There was three Ochratoxin A in Pistachio in first quarter.

US (O) Nuts and Seed 2014 2015 2016 2017 to May

19

Almond 4 16% 2 5.3% 2 4.3% 4 61.9%

Pecan N/A N/A 1 2.6% 3 6.5% N/A N/A

Peanuts 6 24% 4 10.5% 27 58.7% 4 19%

Pistachio 15 60% 29 76.3% 14 30.4% 13 61.9%

Walnuts N/A N/A 2 5.3% N/A N/A N/A N/A

Total # case 25 38 46 21

• Mykes: Greek for fungus/mold • Toxicum: Latin for poison/toxin

• Mycotoxins are metabolic products of food spoilage fungi that induce toxic responses

when consumed by animals or people. • Hundreds of mycotoxins have been identified; They will fall into many different chemical

classes, and induce a wide variety of toxic responses.

Mycotoxins

Major Classes

http://www.mycotoxins.info

Health Effects

http://www.mycotoxins.info

Aflatoxins • Produced by Aspergillus flavus and

A. parasiticus molds • Four key aflatoxins: B1, B2, G1, G2

• Found in many commodities

• Grow in soil and decaying vegetation

• Severe liver damage • IARC -class 1 human carcinogen

• One of the most carcinogenic substances known to man

Aflatoxins

Can be ingested, inhaled, and B1 can even permeate the skin Stunted growth, delayed Development, liver damage,

immune suppression2, and cancer – there is no antidote.

www.hopkinsmedicine.org

www.medicalpicturesinfo.com

1Fratamico, PM et al. (editors) (2008). Foodborne Pathogens: Microbiology and Molecular Biology. Horizon Scientific Press. ISBN 978-1-898486-52-7 2Jolly, P.E.; Inusah, S.; Lu, B.; Ellis, W.O.; Nyarko, A.; Phillips, T.D.; Williams, J.H. (2013). "Association between high aflatoxin B1 levels and high viral load in HIV-positive people". World Mycotoxin Journal 6 (3): 255–261.

Ochratoxin A

Produced by Aspergillus and Penicillium molds One of the most abundant mycotoxins and found in many food and agricultural

commodities Nephrotoxic, immune suppression, neurotoxic, teratogenic

– Humans have the longest half-life to elimination of any species studied Group 2B Suspect Carcinogen by IARC, mutagenic

Can act synergistically with other mycotoxins, such as Citrinin

Ochratoxin A disturbs cellular physiology in multiple ways – enzymes involved in phenylalanine

metabolism, mostly by inhibiting the enzyme involved in the synthesis of the phenylalanine-tRNA complex

– inhibits mitochondrial ATP production – stimulates lipid peroxidation.

Ochratoxin A

Based on FIGURE 1 Topographical distribution of Balkan endemic nephropathy geographical foci from the article Balkan endemic nephropathy and associated urothelial cancer - Vladisav Stefanovic and Zoran Radovanovic, Nature Clinical Practice Urology (2008) 5, 105-112

Mycotoxin Economic and Health Risks

Biological Factors Susceptible Crop +

Compatible, Toxigenic Fungus

Environmental Factors Temperature

Moisture Mechanical Injury

Insect/Bird Damage Fungus

Harvesting Crop Maturity Temperature

Moisture Detection/Diversion

Storage Temperature

Moisture Detection/Diversion

Distribution-Processing Detection/Diversion

Humans

Animals Animal Products

Factors affecting mycotoxin occurrence in the food chain (Pestka and Casale, 1989).

How to Test Aflatoxins and Ochratoxin A ?

25

Technology Pyramid/Market Dynamics • Official Gov. & Commercial Labs

• Finished Products

• Commercial & Gov. Laboratories • Some Transit Points & Processors

• Receiving Points • Processors • Commercial Labs • Transit Points

LC/MS 1%

LC 39%

Fluorometer, Strips,

ELISA 60%

Methods

Confirmatory Method LC or LC-MS

Rapid Method ELISA, Later flow, fluorometer,

Aflatoxin-protein

Anti-Antibody

No Test Line

Reaction zone

Sample Extraction Aflatoxin

Sponge

Control Line

Gold-Antibody Complex

Principle of Lateral Flow Test

Aflatoxin-protein Anti-Antibody Reaction zone

Sample Extraction

No Aflatoxin

Sponge

Control Line

Gold-mAb Complex

Visible Test Line

Lateral Flow Tests

Sample preparation and pre-concentration

1.Solvent extraction 2. Solid-phase extraction 3. Other sample preparation techniques

-Non-specific stationary phases: reverse-phase, normal-phase, ion-exchange, activated carbon, -Immunoaffinity Column (IAC),

Pros and Cons Low-End Testing; ELISA, Strip test

Lower cost per test (labor, space, equipment) Rapid Easy to Use, less technically demand Minimal Consumables Consistent data less accurate and less sensitive.

High End Testing HPLC/UPLC or LC/MS/MS Sensitive, low LOD Accurate and confirmatory Expensive Cumbersome Technically well trained personnel More time per test, unless batch samples.

HPLC vs. UPLC

HPLC Chromatogram for spiked Almond

UPLC chromatogram for Spiked Cereal

Official Methods

AOAC®Official Methods vs. AOAC RI

Performance Criteria* for Aflatoxins Confirmatory Methods

*COMMISSION REGULATION (EU) No 519/2014

Notes: • Values to apply to both B1 and sum of B1 + B2 + G1 + G2. • The detection limits of the methods used are not stated since the precision values are given at the concentrations of interest • The precision values are calculated from the Horwitz equation,

Performance Criteria for Ochratoxin A Confirmatory Methods

Requirements for Semi-Quantitative Screening Methods

Bioanalytical methods based on immuno-recognition or receptor binding (ELISA, dip-sticks/lateral flow test with reader/devices, immuno-sensors) Physicochemical methods based on

chromatography or direct detection by mass spectrometry, LC-MS Other methods, TLC

Requirements for Qualitative Screening Methods

Qualitative Binary Method A method of analysis with two possible outcomes.

Probability of Detection (POD) The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. POD is concentration dependent.

Follow AOAC Guidelines for Validation of Binary Qualitative Chemistry Methods from March, 2013

The biggest challenge in mycotoxin analysis is still the sampling issue. Despite recent available guidance (2), it is still a difficult and tedious task to obtain a representative sample.

What are the biggest challenges in mycotoxin analysis?

The Importance of Sampling

Effective schemes to test for mycotoxins depend not only upon sound analytical methods, but also on well designed and implemented sampling plans. The consequences of a poorly designed sampling plan on the reliability of the measured levels of mycotoxins, possibly resulting in legal disputes and barriers to trade.

Thomas.B. Whitaker, North Carolina State University, Raleigh,

Laying Down the Methods of Sampling and Analysis for the Official Control of the Levels of Mycotoxins in Foodstuffs*

*Commission Regulation (EC) No 401/2006 of 23 February 2006

Sampling < 15 tonnes (peanuts, other oilseeds, apricot kernels and tree nuts

Weight of the aggregate sample = 20 kg which has to be mixed thoroughly (but not ground) and only afterwards to be divided into two equal laboratory samples of 10 kg before grinding and homogenisation.

Sampling of dried figs for human consumption

Sampling of dried figs for human consumption*

*Spain and Germany because of their additional national legislation

Sampling of groundnuts, other oil seeds, apricot kernels and tree nuts for direct human consumption

In cases where the aggregate sample weights are less than 20 kg, • ≥12 kg: division into two laboratory samples • < 12 kg: no division into laboratory samples Each laboratory sample must be separately ground finely to achieve complete homogenisation

Sampling of groundnuts, other oil seeds, apricot kernels and tree nuts for direct human consumption

Calculation of proportion of shell/kernel of whole nuts

Approximately 100 whole nuts are taken at random from the lot and kept separately from the sample or are to be taken by the laboratory from each aggregate sample and be put aside.

The ratio nut shell/kernel can vary from 25/75 (hard shell almonds), to 40/60 (soft shell almonds), 50/50 (pistachios)

Example: the ratio nut shell/nut kernel is 50/50 and if the analytical result in the test material is 1.5 μg/kg(PPB) of aflatoxin B1, The edible part of aflatoxin B1 = 1.5 μg x 2 = 3 μg/kg .

Notes

1. Sample slurry preparation for dried fruit Weigh the laboratory sample received and record the weight. The sample may be minced to break it up. Add water in the proportion of five parts fruit to four parts water. Homogenize the sample and water for at least 30 min or until a slurry of a smooth consistency is achieved. In all instances if the sample has been frozen allow it to thaw completely before sampling. Stir slurried samples thoroughly before removing an analytical test portion. 2. Immunoaffinity column cleanup Do not exceed the maximum specified flow rates. Extra care is needed when a vacuum manifold is used 3. Preparation of the sample test solution eluate with water (or water with 2 % acetic acid)

one pistachio sample was contaminated with OTA at 890 ng/g, while the others contained less than 3 ng/g OTA. In the case of the highly contaminated pistachio sample, it is possible that that sample contained a relatively small number of very highly contaminated nuts, which then contaminated the sample as a whole once it was homogenized by grinding, Likewise,contaminated figs and dates contained less than 4 ng/g OTA.

Can be used in a field environment - rugged

Established methods being used for many

years

Easy- Anyone can be trained quickly to run

the test

Will give a numerical result on a digital

screen, with printout

AOAC and USDA Official methods

No refrigeration needed for AflaTest,

OchraTest or AflaOchra

Method times vary from 7 minutes to

20 minutes depending on the toxin

Rapid, Accurate Methods for Mycotoxin Testing