Second Workshop on Food Allergen Methodologies

October 27-29, 2003
Ottawa, Ontario, Canada

The Food Directorate's Bureau of Chemical Safety, in collaboration with the Canadian Food Inspection Agency (CFIA) organized the second workshop on food allergen methodologies from October 27-29, 2003.
This second session was meant to be a follow-up to the 2002 workshop organized by Health Canada, the CFIA and scientists from the US Food and Drug Administration (CFSAN). The workshop aimed mostly at expanding the consultation, information exchange and harmonization of allergen methodologies.

The 2003 session gathered more than 75 participants: scientists, chemists and analysts from government agencies, university, industry and consumer associations. Issues related to the detection, identification and characterization of allergens in foods were presented and discussed:

• Method development /evaluation and validation
• Risk assessment / risk management - Impact on methodology development
• Development of reference material
• Confirmatory techniques
• Harmonization of methodologies

A keynote address was presented by Dr. Sue Hefle, Co-Director of the Food Allergen Research and Resource Program (FARRP) from the University of Nebraska in the U.S., and focused on "Issues Surrounding Food Allergen Detection in the U.S.". An overview of the regulatory environment and the application of methodologies of detection of allergens for enforcement and compliance purposes were discussed. Several presentations tackled aspects of methodology development and validation aiming at the detection of allergens in foods. Discussions about method harmonization and the use of such methodologies to investigate allergenicity of proteins resulting from genetically modified crops were also addressed.

Health Canada's Food Allergen Research Program presented its latest achievements in developing and transferring ELISA-based methods targeting hazelnut proteins, crustacean tropomyosin as well as a multi-residue method for peanut and four tree nuts, to CFIA laboratories. A program of method evaluation ,developed jointly with CFIA laboratories, completed its first interlaboratory evaluation on a method developed in house at Health Canada for the detection of markers of hazelnut. Results from this evaluation are to be posted in the Health Canada Compendium of Food Allergen Methodologies. This compendium is being developed under the auspices of the HC/CFIA Allergen Methods Committee (AMC). Efforts to develop confirmatory techniques were also illustrated by the latest findings on an LC-MS/MS method to identify protein / peptide markers of peanut allergens. The CFIA allergen testing laboratories reported on their increasing efforts to introduce new allergen methodologies, upgrading their capabilities in compliance testing for allergens.

Several diagnostic kit manufacturers presented their latest ready-to-use assays covering priority allergens such as egg test kits, gluten/gliadin tests and milk protein assays. The US FDA and the AOAC Research Institute (AOAC RI) reported on the validation of three commercially available peanut assays that they initiated. Details on the outcomes of similar initiatives in the EU were presented by Dr. Elke Anklam deputy Director of the IRMM / EU JRC. Highlights of emerging techniques to be used for allergen testing as well as, confirmatory techniques of ELISA methods were illustrated by PCR-based methods and immunosensor assays targeting markers of food allergens. An ATP-based assay, to be possibly used in the context of food processing line sanitation, was also presented. Other presentations targeted food allergen characterization as well as techniques to investigate the allergenicity of novel proteins in genetically engineered foods. A summary of each presentation, made as part of the technical program will soon be available in both official languages on the Health Canada website.

The meeting hosted a special discussion group session of the AOAC International Presidential Taskforce on food allergens, dealing with reference materials and validation of methodologies.

Need for reference materials to evaluate / validate allergen testing methodologies

There was a consensus on the need to develop reference materials for priority allergens, in order to be used in measuring the performance of allergen analytical methodologies and to further promote efforts of method development for allergens. Discussions were held on how to define a reference material for food allergens and whether there should be one or more than one material for each identified priority allergen.
The Canadian Allergen Method Committee (AMC) shared its current definition for a "designated reference material" to be used in the context of method evaluation / validation: " a material representative of the allergenic food commodity, that is well characterized, and that could be produced/supplied with reproducible characteristics" Using this starting point the AMC would actively contribute to all efforts of harmonization and development of such materials where applicable. Such efforts are illustrated by the use of a characterized peanut butter provided by NIST for the validation of commercial peanut kits, and by the latest efforts of IRMM/EU JRC to develop and characterize a coarse material of peanut, taking into account major grown varieties and considering various processing conditions. Some of the action items that were identified throughout the discussions are as follows:

• To prioritize the development of materials based on on-going efforts for allergen method validation.
• To establish a database for existing reference materials already developed by reference institutes (NIST, IRMM, Agriculture Canada etc..) that could fulfill the criteria of reference materials for priority allergens.
• Where no reference material is identified, initiate appropriate actions of development of such material and devote collaborative resources to expedite such development.

Validation of allergen analytical methodologies

The need to develop consistent criteria for validation of food allergen analytical methods was reiterated. Such criteria are meant to clarify and better utilize results generated by various allergen methods, and properly identify the risks faced by consumers.

The recent efforts of validation of commercial peanut kits by the AOAC Research Institute, were discussed. The Canadian AMC gave its current view, on how allergen methodologies are being evaluated / validated, to be used for compliance testing by the CFIA laboratories. On top of internal validation approaches used by each CFIA laboratory, as part of its QA activities, the launch of the allergen compendium of methodologies has lead to the development of a set of criteria for the introduction of methodologies into the compendium. The AMC shared the general principles behind this set of criteria:

• The compendium of methods will aim at reporting the performance of methods that were evaluated according to the set protocol. It will not endorse or approve methods.
• The evaluation protocol will be designed and implemented in total collaboration with the method developer, whether a test kit manufacturer, a private, university or government laboratory.
• Two levels of evaluation will be targeted: an entry level, where a method is evaluated by a single AMC-affiliated laboratory and that of the method developer, and a full evaluation level, where results are generated by at least three AMC-affiliated laboratories and that of the method developer/author.
• A designated reference material should be defined for the method to be evaluated and must be of some validity of use for more than the targeted methodology, leading therefore to evaluate the performance of several methods based on the same or a similar material.
• Levels of fortification and matrices to be targeted will be defined according to the claims of the method author: typically, 3 levels of fortification will be evaluated, a blank, 2xLOQ and 5xLOQ.
• The number of replicates has been defined so that the evaluation / validation process does not impact negatively on activities of the participating laboratories, whose primary mission is to deliver on compliance activities rather than to be involved in endless evaluation of methodologies.
• The report of performance on evaluated methods will be posted on the Health Canada web-based allergen compendium of methodologies and will therefore make available more data on existing methodologies, allowing users from the analytical community to make their choice according to their needs.
• The generated data will be shared in agreement with all involved parties, including the method developer/author and will enable a better understanding of data provided by a third party based on evaluated methods.
• Federal laboratories are free to use whatever testing methodologies, they consider more appropriate, to fulfill the requirements of allergen testing, among evaluated methodologies. Such information will not be posted on the web-based application, as it is subject to change and it may be regarded as an endorsement of a particular method.

The issue of whether the current "Canadian Process" would duplicate international efforts of method harmonization such as that being led by the AOAC RI was raised. The AMC has adopted a co-operative approach that favors further collaboration and contribution to these efforts. Any validation process of allergen methods, to which AMC-affiliated laboratories are associated, will be acknowledged and will therefore be used to increase the number of methods entering the compendium. The AOAC-RI led process to evaluate peanut commercial kits, was implemented with the participation of an AMC-affiliated laboratory, and according to a protocol that was fully acceptable to the AMC. The corresponding methods will therefore be entered into the compendium, once the corresponding authorizations are obtained.

Further discussions considered establishing a priority for concerted efforts of method validation under the AOAC taskforce on food allergens as well as increasing the investigation on alternative methods to ELISA techniques. It was decided that the co-chairs of the AOAC taskforce will nominate working groups for these activities and will send a call for participation to all taskforce members.

The Canadian AMC has announced that it will carry-on its initiative of organizing a regular workshop to report on advances in the area of food allergen methodologies, in collaboration with national and international partners among regulatory agencies and the analytical community, most notably FAARP. A tentative date for the next workshop was set for Spring 2005.