Reports

FAO/IAEA Consultants Meeting on Analytical Methods for Determining Mycotoxin Contamination of Food/Feed in Trade Vienna, Austria 28 July - 1 August 1997

1. Introduction

The Meeting was one in a series of meetings being convened by the FAO/IAEA Training and Reference Centre for Food and Pesticide Control under the Centre´s mission "to assist Member States and their institutions to fulfill requirements to support the implementation of international standards/agreements relevant to food safety and control, the safe use of pesticides and sanitary and phytosanitary measures by providing training, quality assurance services and technology transfer." The Meeting was held at IAEA from 28 July to 1 August 1997.

Mr. Paisan Loaharanu, Head, Food and Environmental Protection Section welcomed the participants on behalf of Dr. J.D. Dargie, Director, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. He stressed the need for strengthening the analytical capabilities of laboratories in developing countries to meet the quality and safety requirements of food in trade, based on the Agreement on Application of Sanitary and Phytosanitary Measures (SPS) being implemented by the World Trade Organization (WTO). He also referred to compliance with pesticide control legislation and international agreements, in particular the Code of Conduct on the Distribution and Use of Pesticides, which requires analytical capacities to monitor the quality of pesticide products in trade and the level of pesticide residues in food and in the environment.

Dr. Ramesh Bhat and Dr. Alison Williams were invited to serve as Chairman and Rapporteur of the Meeting, respectively. The Meeting agenda is attached as Annex 1. A list of participants is attached as Annex 2.

2. Background

Mycotoxins are the toxic products of certain microscopic fungi which, in some circumstances, develop on or in agricultural commodities and may be present in animal-derived food as a result of contaminated animal feed or direct contamination. They are ubiquitous and wide-spread at all levels of the food chain. They are found in food produced at all latitudes save the polar regions. Although more than 300 mycotoxins have been identified, produced by some 350 fungal species, only about 20 have been shown to occur frequently in agricultural commodities. Among these, aflatoxins, deoxynivalenol, fumonisins, ochratoxin A, patulin, and zearalenone are considered major food contamination problems and hence are of potential concern.

Fungal growth and mycotoxin production in agricultural commodities can occur during cultivation, harvest and storage. The conditions in which fruits such as apples and pears are harvested and handled may induce the production of patulin in fruit and lead to contamination in fruit juices. In dairy products, the contamination may occur directly from moulds (especially on cheese) or indirectly from mycotoxins in the feed of dairy cattle. For example, about 1% of the aflatoxin B₁ contained in animal feed reappears as aflatoxin M₁ in cow's milk. Industrial processing (roasting, baking, fermentation, pasteurisation, etc.) and household cooking in most cases only partially inactivate mycotoxins in foods.

Although the reported incidents of acute toxicity due to mycotoxins are rare, long-term effects give cause for concern. Aflatoxin B₁, for example, is a hepatocellular carcinogen in animals and humans. Epidemiological studies show that humans are more at risk in regions with specific environmental conditions that favor the growth of aflatoxin-producing moulds and where the population has a high prevalence of hepatitis B infection. Regular consumption of corn containing high levels of aflatoxins has resulted in high dietary exposure of millions of people. In addition, a variety of mycotoxins such as deoxynivalenol, zeralenone, ochratoxin, ergot alkaloids and fumonisins can have adverse effects on various organs, including kidney and liver as well as on sustems of the body such as gastrointestinal, nervous, uro-genital and vascular systems. Thus mycotoxins are of considerable public health significance.

Both the FAO/WHO International Conference on Nutrition (Rome, 1992) and the United Nations Conference on Environment and Development (Rio, 1992) recognized the importance of safe food for human health and the potential hazards posed by chemicals in the food supply. In April/May 1996, the UN Commission on Sustainable Development approved a work programme on indicators of sustainable development which included mycotoxins in food as one of the components of an indicator related to protection and promotion of human health (see Annex 3).

In terms of their economic implications, mycotoxins are one of the most important contaminants of the food chain. According to FAO estimates, world losses of foodstuffs due to mycotoxin contamination are in the range of 1000 million tonnes per year, a figure which illustrates the importance of the problem.

Based on Global Environment Monitoring System - Food Contamination Monitoring and Assessment Programme (GEMS/Food) data and other national data on mycotoxin contamination, mycotoxins are a widespread problem of the food supplies in most countries. As a result, many countries have enacted regulations to control the level of mycotoxins in the national food supply as well as in food moving in international trade. At the international level, the Codex Alimentarius Commission, through its Committee on Food Additives and Contaminants and relevant commodity committees, has considered the establishment of international guideline levels for various mycotoxins based on risk assessments performed by the Joint FAO/WHO Expert Committee on Food Additives. Codex activities are of particular importance in view of the WTO SPS Agreement as well as on the WTO´s Agreement on Technical Barriers to Trade. The SPS Agreement specifically refers to Codex standards, guidelines and recommendations as representing the international consensus on health and safety requirements for food based on sound scientific risk assessment. This will require national authorities to give greater attention to the development of consistent and standardized approaches to regulations and their enforcement, including sampling and methods of analysis.

In the past, FAO, the International Agency for Research on Cancer (IARC), WHO and other international organizations have carried out a number of activities and programmes pertaining to capacity building of developing countries in monitoring, prevention and control of mycotoxin contamination. These programmes included the development of relevant training materials and manuals as well as the training of analysts and implementation of analytical quality assurance activities. However, these activities have been carried out on an ad hoc basis and have been insufficient to meet the needs of developing countries, particularly in the area of quality assurance which has been given greater emphasis due to the requirements of the SPS Agreement. Therefore, continuous training and assistance in quality assurance for mycotoxin analysis is urgently needed.

3. Objective and Scope

The objective of the Meeting was to provide recommendations to FAO and IAEA in planning, implementation and evaluation of activities of the Centre pertaining to training, quality assurance services and technology transfer in the area of mycotoxin analysis.

In fulfilling this objective, the Meeting considered various issues, including target groups and institutions for collaboration; priority mycotoxins to be considered in training and quality assurance activities; methods of analysis to be used and relevant commodities; sampling methodology and sample preparation; proficiency testing and assistance in accreditation; provision of reference materials and standards; training modules; and databases. In addition, the Meeting considered the development of project proposals for training and for quality assurance, as well as a proposal for a coordinated research programme on analytical methodology.

4. Discussion and Conclusions

The Meeting recognized the need for the Centre to have the necessary resources under the Regular Programmes of FAO and IAEA to carry out core activities on mycotoxins. Such resources could be complemented by extra-budgetary funds to expand the Centre's activities at central and regional levels.

The Meeting noted that additional physical facilities, equipment and technical manpower are needed to enable the Centre to perform the proposed mycotoxin-related activities. In particular, provision of a separate area for sample preparation, specific laboratory equipment for mycotoxin analysis and essential safety devices are needed in view of the very toxic nature of mycotoxins.

In addition, the Meeting considered the following specific topics:

A. Target Groups and Institutions

The Meeting agreed that there are a number of laboratories that would benefit from the services of the Centre and identified the following:

i. food control laboratories engaged in analytical services and routine monitoring of mycotoxins;

ii. laboratories engaged in analysis of mycotoxins in commodities moving in international trade; and

iii. laboratories engaged in surveillance, research and prevention, in the field of mycotoxins.

B. Priority Mycotoxins

In identifying the mycotoxins to be given priority in the Centre's activities, the Meeting gave consideration to the following criteria:

i. health significance as determined by well-established risk assessment procedures, and

ii. mycotoxins for which regulations or guidelines have been established at regional, national and international levels for commodities moving in international trade.

The Meeting agreed that aflatoxins including aflatoxin M₁), deoxynivalenol, fumonisins, ochratoxin A, patulin, and zearalenone met the above criteria. Some of these mycotoxins can occur in various animal feeds and feed ingredients. They can also occur in foods of animal origin such as milk. There is considerable international trade in feed ingredients. In view of this, analysis of animal feed ingredients such as peanut meal and corn assumes importance. As such, this aspect needs to be included not only in training programmes but also in analytical quality assurance and reference material programmes.

C. Methods of Analysis

The Meeting agreed that basic as well as advanced methodologies are needed for mycotoxin analysis. However, any such method needs to be validated and well established. Coordination and/or collaboration by the Centre in method validation trials could facilitate the development of new valid methods.

Considering the expertise available in the FAO/IAEA Division on the use of isotopes in research, and considering the potential use of isotope-labelled mycotoxins to evaluate mycotoxin recovery by various analytical methods, the Meeting agreed that the Centre would be in an excellent position to study the application of these techniques in method optimization. Radiolabelled mycotoxins could be provided to collaborating laboratories for other applications.

While validated methods such as minicolumn and thin-layer chromatography (TLC) are useful for surveillance/monitoring and prevention programmes in some countries, methods based on gas chromatography (GC), High performance liquid chromatography (HPLC) and, more recently, enzyme-linked immunosorbent assays (ELISA), need to be considered for analysing commodities moving in international trade. Examples of such methods are provided in Annex 4.

D. Sampling and Sample Preparation

The Meeting recognized the importance of sampling methodology and sample preparation in mycotoxin analysis and agreed that emphasis needs to be given to these aspects. The extreme variability of contamination levels in the same lot of foodstuffs poses serious problems when sampling for mycotoxin analysis. For aflatoxins in peanuts, up to 80% of the distribution in multiple analyses of a single lot may be ascribed to sampling, and 20% to the analytical method. While definite sampling plans for aflatoxins in peanuts and corn moving in trade and for pre-packaged foods in general have been published by Codex, information on sampling plans appropriate for national surveillance and monitoring programmes is scarce. However, the sampling plans suggested in the FAO/UNEP/WHO publication "Guidelines for establishing or strengthening national food contamination monitoring programmes" could be useful for this purpose. Since analysts trained by the Centre could guide food inspectors in their respective countries in proper sampling procedures, the Meeting agreed that the latter subject should be given emphasis in the training curriculum.

E. Proficiency Testing and Accreditation

The Meeting agreed that one of the most important activities of the Centre should be the organization of appropriate check sample studies on mycotoxins. In order to develop this capacity in the Centre, the Meeting felt that appropriate training of existing personnel and provision of additional equipment, including safety devices, are required. Initially, collaboration with more experienced laboratories is necessary to develop the expertise in the preparation of reference materials and in the organization of check sample studies.

Regular check sample studies should be organized for participating laboratories, at least annually. These studies could also be open to private laboratories on a fee-paying basis. Appropriate statistical analysis of results has to be carried out and promptly communicated to the participants. Where necessary, follow-up actions should be undertaken.

The Meeting agreed that there is a need for the Centre to obtain accreditation for the methods of analysis used in preparation of mycotoxin reference materials. Immediate action for this purpose must include training of its staff. The Meeting felt that the Centre needs to also develop criteria for accreditation that are pertinent to mycotoxins and seek endorsement of these criteria by recognized accreditation bodies. This process would assist laboratories interested in obtaining accreditation from such recognized bodies. The Meeting also agreed that there is a need to assist laboratories in their efforts to obtain accreditation in mycotoxin methods.

F. Reference Materials and Standards

Reference materials for selected mycotoxins in specific foods need to be prepared by the Centre and provided to participating laboratories when required. Reference standards for various mycotoxins are commercially available, but difficulties have been experienced in the past by many laboratories in developing countries in their procurement. In view of this, the Meeting agreed that assistance needs to be provided in procurement of such standards.

G. Training

The Meeting recognized the need to organize the training programme in mycotoxin analysis at two levels: a basic course and an advanced course. The basic courses (4 weeks duration each for up to 15 participants) should be held initially at the Centre and later be organized at the regional level in collaboration with recognized regional centres of excellence. The advanced level courses (2 weeks duration each for up to 10 participants) should be organized at the Centre's Seibersdorf Laboratory.

The participants in the Centre's training programme could be selected among applicants from laboratories identified among the target groups and institutions mentioned earlier. Considering past experiences of international training programmes in the analysis of mycotoxins, the Meeting was of the opinion that clear guidelines on the background and qualifications of analysts to be trained need to be provided when seeking nominations so that similarly qualified groups can be trained. The Meeting felt that participants in the training programmes should be involved in the Centre's check sample studies.

Training courses to be implemented by the Centre need to be announced well in advance so that participants can be identified and supported by the Centre. Additional participants may be supported by other agencies and institutions, subject to availability of space. The fellowship training programmes on mycotoxins that are envisaged by other UN agencies such as FAO, WHO, and UNEP, need to be coordinated with the Centre's programme.

Modules for the basic course should include an overview of mycotoxin contamination and fundamental principles of sampling, sample preparation and analytical methodologies (e.g. minicolumn, TLC, HPLC, GC and ELISA). Appropriate hands-on experience should be offered.

Modules for the advanced course should include, in addition to advanced instrumental methods, such aspects as sampling plans, method validation, analytical quality assurance and procedures for accreditation.

Manuals developed by FAO and other agencies, such as WHO and IARC, for various training programmes could be used in the initial stages with appropriate additional inputs. The Meeting agreed that, in due course, a comprehensive training manual on mycotoxins needs to be developed by the Centre.

H. Database

The Meeting recognized that mycotoxin databases exist already in several areas and are available through agencies, such as FAO and WHO. A specific database on analytical methodologies is, however, lacking. The Meeting agreed that there is a need for the Centre to establish and maintain such a database using information available from laboratories participating in the check sample studies and elsewhere.

I. Project Proposals

The Meeting developed outlines of the following project proposals:

1. Training in Mycotoxin Analysis (see Annex 5).

2. Reference Material Production and Check Sample Studies (see Annex 6).

J. Coordinated Research Programme

The Meeting considered a protocol on the evaluation and validation of methods of analysis of mycotoxins in foods (Annex 7).

5. Recommendations

The Meeting made the following recommendations to FAO and IAEA through their Joint FAO/IAEA Division in implemeting mycotoxin-related activities of the Centre:

1. The Centre should be provided with the necessary resources under the Regular Programme to carry out core activities on mycotoxins, especially in the areas of training, quality assurance and technology transfer. Such resources should be complemented by extra-budgetary funds to expand the Centre's activities at central and regional levels. The Centre should develop the project proposals outlined in Annexes 5 and 6 and seek funding from potential donors.

2. Additional physical facilities, equipment and technical manpower should be provided to enable the Centre to perform mycotoxin-related activities. In particular, provision of a separate area for sample preparation, specific laboratory equipment for mycotoxin analysis and essential safety devices are needed in view of the extreme toxic nature of mycotoxins.

3. Aflatoxins (including aflatoxin M₁), deoxynivalenol, fumonisins, ochratoxin A, patulin and zearalenone should be given priority in the Centre's work. Foods that may contribute to human exposure to these mycotoxins and agricultural commodities contaminated with these mycotoxins and moving in international trade, including feed ingredients, should be considered as priority matrices.

4. Basic as well as advanced analytical methodologies used by the Centre should be validated. When necessary, the Centre should coordinate or collaborate in method validation trials.

5. The Centre should study the application of tracer techniques in the optimization of mycotoxin analytical methods. Radiolabelled mycotoxins could be provided to collaborating laboratories for various applications.

6. The Centre should organize regular check sample studies for participating laboratories, at least annually. These studies could also be open to private laboratories on a fee-paying basis. Appropriate statistical analysis of results should be carried out and promptly communicated to the participants. Where necessary, follow-up actions should be pursued.

7. The Centre should obtain accreditation for the analysis of the mycotoxins included in the reference materials that it will prepare. Immediate action for this purpose should include training of its staff.

8. The Centre should develop criteria for accreditation that are pertinent to mycotoxins and seek endorsement by recognized accreditation bodies for these criteria. The Centre should consider means for assisting laboratories in their efforts to obtain accreditation in mycotoxin methods.

9. The Centre should prepare reference materials on selected mycotoxins in specific foods and provide them to participating laboratories as required. Whenever possible, the Centre should provide assistance in the procurement of mycotoxin standards.

10. The Centre should organize a training programme on mycotoxin analysis at two levels: a basic course and an advanced course. The training programme should give emphasis to sampling methodology and sample preparation. Modules for the basic course should include an overview of mycotoxin contamination and fundamental principles of sampling, sample preparation and analytical methodologies. Basic courses should be held initially at the Centre and later be organized at the regional level in collaboration with recognized regional centres of excellence. Modules for the advanced course should include, in addition to advanced instrumental methods, such aspect as sampling plans, method validation, analytical quality assurance and procedures of accreditation. Hands-on experience should be offered.

11. A comprehensive training manual on mycotoxin analysis should be developed by the Centre based on existing manuals and taking into account recent developments in the field.

12. The Fellowship Programmes of FAO and IAEA should be open to candidates interested in mycotoxin analysis.

13. The Centre should establish and maintain a database on analytical methodologies using information available from laboratories participating in check sample studies and elsewhere.

14. FAO and IAEA are encouraged to implement the Coordinated Research Programme for Evaluation and Validation of Methods of Analysis of Mycotoxins in Food at the earliest opportunity (see Annex 7).

ANNEX 1

FAO/IAEA Consultants Meeting on
Analytical Methods for Determining Mycotoxin Contamination
of Food/Feed in Trade

Vienna, Austria
28 July - 1 August, 1997

Agenda

Monday, 28 July

9.00-12:00 Opening

FAO/IAEA Training and Reference Centre for Food and Pesticide Control (P. Loaharanu, Head, Food and Environmental Protection Section, Joint FAO/IAEA Division, Vienna)

Role of FAO in co-ordinating studies on mycotoxin contamination of food/feed in developing countries (E. Boutrif, FAO)

GEMS Food and other WHO activities related to mycotoxins (G. Moy, WHO)

12:00-13:30 Lunch

13:30-17:00

Intercomparison studies on mycotoxin contamination of food in Latin America (S. Resnik, Argentina)

Problems of mycotoxin contamination of food/feed in India (R. Bhat, India)

Accreditation of laboratories for mycotoxin analysis (A. Williams, U.K.)

Status of mycotoxin analysis in Austria (R. Krska, Austria)

Tuesday, 29 July

9:00-12:00

Discussion of proposed activities related to mycotoxin analysis to be carried out by the FAO/IAEA Training and Reference Centre for Food and Pesticide Control

12:00-13:30 Lunch

16:00

Visit to the FAO/IAEA Agricultural and Biotechnology Laboratories (Seibersdorf)

Wednesday, 30 July - Friday, 1 August

Discussion and preparation of report on the role of the FAO/IAEA Training and Reference Centre for Food and Pesticide Control in the training, analytical quality assurance and technology transfer related to analytical methods for mycotoxins.

ANNEX 2

FAO/IAEA Consultants Meeting on
Analytical Methods for Determining Mycotoxin Contamination
of Food/Feed in Trade

Vienna, Austria 28 July - 1 August, 1997

LIST OF PARTICIPANTS

ANNEX 3

Mycotoxins identified for priority monitoring in specific foodstuffs depending on a country´s stage of development

Source: Adapted from "Indicators of sustainable development; framework and methodologies." U.N., New York, 1996, pp. 115-117.

ANNEX 4

Commodity Standard Provision Method Principle

• Milk Aflatoxin M1 IDF STD.171:1995 Immunoaffinity
  0.05 mg/kg column & LC
• Milk & dried milk Aflatoxin M1 IDF STD.111A:1990 TLC/LC
  A-5 (milk powder) 0.05 mg/kg
• Fluid milk Aflatoxin M1 AOAC 986.16 HPLC
  0.05 g/kg
• Peanuts (intended for Aflatoxin, total AOAC 975.36 Romer mini
  further processing 15 mg/kg (Step 6) column
• Peanuts (intended for Aflatoxin, total AOAC 979.18 Holaday-Velasco
  further processing 15 mg/kg (Step 6) mini column
• Corn Aflatoxin, total AOAC979.13 Holaday-Velasco
  15 mg/kg mini column
• Peanuts Aflatoxin, total AOAC 990.34 ELISA
  15 mg/kg (Step 6)
• Peanuts & peanut products Aflatoxin, total AOAC 968.22 CB Method
  15 mg/kg (Step 6)
• Peanuts & peanut products Aflatoxin, total AOAC 970.45 BF Method
  15 mg/kg (Step 6)
• Peanuts (Raw) Aflatoxin, total AOAC 993.17 TLC
  15 mg/kg (Step 6)
• Peanuts (Raw) Aflatoxin, total AOAC 991.31 Immunoaffinity
  15 mg/kg (Step 6) column (Aflatest)
• Corn Aflatoxin, total AOAC 990.34 ELISA
• Cotton seed Aflatoxin, total AOAC 990.34 ELISA

Source: Codex Committee on Methods of Analysis and Sampling.

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