1. TESTING AND REGULATION (free from foods) Dr Mike Bromley Genon Laboratories Specialists in immunological and genetic testing for safety and authenticity Delivering customer service excellent from scientific experts

2. Legislation governing allergenic foods in the European Union

3. Importance of legislation Primary importance for public safety Help manufacturers adhere to standards Aid regulators in assessing compliance

4. Legislation governing allergenic foods in the European Union Disclaimer: I aim to provide a concise background to the EU law, but I do not intend to provide detailed legal advice. Anyone requiring detailed legal advice must contact a properly qualified legal practitioner. While I have taken every care to provide accurate information, I cannot be held legally responsible for the content of these slides or for any misunderstandings or misinterpretations.

5. Legislation governing allergenic foods in the European Union The Labelling Directive (Directive 2000/13/EC) and its later amendments specifically refers to allergenic foods. –Directive 2003/89/EC introduced Annex IIIa –Directive 2007/68/EC has the most recent amendment of Annex IIIa Directive 2000/13/EC –requires manufacturers to declare all ingredients present in pre- packaged foods sold in the EU with very few exceptions

6. Directive 2003/89/EC : Annex IIIa –No indication of thresholds or action levels Legislation governing allergenic foods in the European Union –Cereals containing gluten –Crustaceans –Eggs –Fish –Peanuts –Soybeans –Milk (including lactose) –Nuts –Celery –Mustard –Sesame –Sulphur dioxide/sulphites at a concentration of more than 10ppm

7. Legislation governing allergenic foods in the European Union Directive 2007/68/EC –Modification of Annex IIIa to permanently exclude certain products derived from gluten containing grains, milk, fish and soybean. –Included Lupin and Molluscs –Still no indication of thresholds or action levels!!!

8. Legislation concerning foodstuffs suitable for people intolerant to gluten Commission Regulation 41/2009 –Finally adopted the codex standards 20ppm (mg/kg) gluten in ‘gluten free’ food 100ppm (mg/kg) gluten in ‘very low gluten’ food –No prescriptive method

9. Other relevant legislation General Food Law: Regulation (EC) No 178/2002 –Imposes an obligation to provide safe food –Traceability –Withdrawal and Recall Hygiene of foodstuffs Regulation (EC) No 852/2004 –Businesses must implement procedures to prevent unsafe foods

10. Impending legislation Proposal for a Regulation of the European Parliament and of the Council on the provision of food information to consumers –Merger of 2000/13/EC and 90/496/EEC (Nutrition Labelling of Foodstuffs) –Expands the legislation to cover unpackaged foods and foods served by catering establishments –Annex IIIa will become Annex II –But still no guidance on threshold levels

11. Towards establishing thresholds for allergenic foods

12. Why is specifying ‘free from’ so difficult? What criteria do we use to determine safety? –An allergy sufferer may say – a safe level for me –A member of the food industry may suggest – a risk management strategy –A regulator may suggest – a level to protect the majority of the population that can be effectively monitored by testing

13. What is a safe level of allergen? How do we assess the safe level for a population? –Historically a wide variety of methods and food matrices have been used in human tests –Harmonised protocol needed Double-Blind Placebo-Controlled Food Challenge (DBPCFC) Europrevall Crevel et al 2008 Allergy 63: 597-609

14. What is a safe level of allergen? Safety thresholds exist at both a population and individual level Results to date indicate a wide range of doses over which allergenic people respond Due to limitations of studies in humans obtaining an absolute safe threshold for everybody is effectively impossible

15. Peanut: a case study Taylor et al 2010 Food and Chemical Toxicology 48 814–819

16. What is a safe level of allergen? The solution, a minimum dose? –An amount that protects the majority of the population –Acknowledges the fact that one cannot provide zero risk food

17. So when will we get defined thresholds Nothing imminent Most data available for peanut however as yet no EU proposal in place A working group of the GSACI and the AGA have proposed: –10–100 mg/kg of the allergenic food or –1–10 mg/kg of the protein fraction of the allergenic food Vieths et al 2006 Allergo J 15:114–123

18. Analytical testing: Problems and Pitfalls

19. Analytical testing Relationship between clinical thresholds, analytical results and allergic episodes Lack of standardised reference materials Lack of standardised protocols for diagnostics

20. Reference materials Variation between cultivars Processed vs Unprocessed material

21. Monaci et al 2011 Food Chemistry 127: 669–675 Effect of heat processing on milk protein

22. Reference materials Variations between cultivars Processed vs unprocessed material Comparison between clinical and analytical data

23. Detection methods ELISA (Enzyme Linked Immunosorbent Assay) Detects protein to low ppm level (but not necessarily the allergenic ones) Quantitative Can be affected by sample matrix Extraction methodologies limited Takes time to develop assays PCR (Polymerase Chain Reaction) Allergen not directly detected Sensitive Relatively quick to develop Qualitative Sample matrix issues Specificity Mass Spectrometry (MS) At development stage Potential for multi-allergen detection Animal Models Ethically questionable Expensive

24. The Enzyme Linked Immunosorbent Assay (ELISA) Capture antibody Antigen (soya) Enzyme linked detection antibody Substrate

25. Variability between ELISA assays: Gluten

27. Variability between ELISA assays: Soya Supplier Assay name Antibody Detection range Platform Suitable for processed foods? Neogen Veratox Soy Allergen Test (#8410) polyclonal against denatured protein 10 to 100 ppmSandwichYes Neogen Veratox Soy Flour Allergen Test (#8490) polyclonal against soy flour 2.5 to 25 ppmSandwichNo Elisa Systems Soy protein residue polyclonal against soy protein 2.5 to 25 ppmSandwichNo Biokits (Neogen) Soya protein assay kit polyclonal against denatured protein 700-14000 ppmSandwichYes Diagnostic Innovations Nutrilinia polyclonal against micronized soy flour 1 to 20 ppm Competitive indirect Yes

28. Variability between ELISA assays: Soya Pedersen et al 2008 Mol. Nutr. Food Res. 52: 1486 – 1496

29. Veratox Soy Allergen vs Soy Flour Assay Soy AllergenSoy Flour

30. Interpretation of results Reporting as ppm protein or ppm foodstuff? –Use of conversion factors Allergen hotspots Uncertainty of measurement

31. Situation ISituation IISituation IIISituation IV The analytical result is less than the maximum value by an amount greater than the measurement uncertainty. All authorities will consider the sample as being compliant without any hesitation. The analytical result is below the maximum level by less than the measurement uncertainty. The sample should be considered to be compliant, but one would probably be wary of future samples. The analytical result exceeds the maximum level by less than the measurement uncertainty The effect of the draft codex guidelines on measurement uncertainty is that all authorities should accept the result as being compliant (i.e. the result is not non- compliant “beyond reasonable doubt”). The analytical result together with the measurement uncertainty exceeds the maximum level. The sample should be considered as being non-compliant. Uncertainty of measurement

32. Concluding remarks We require effective and more prescriptive legislation Unfortunately clinical data will take time Many of the issues surrounding variability in analytical techniques can be addressed Effective communication between analytical laboratories and their customers

33. Further information The Institute of Food Science & Technology Food allergy statement EuroPrevall –http://www.foodallergens.infohttp://www.foodallergens.info –http://www.europrevall.org/http://www.europrevall.org/

34. Thank you for listening www.genonlabs.co.uk Dr Mike Bromley Specialists in immunological and genetic testing for safety and authenticity Delivering customer service excellence from scientific experts