1. March 3 and 4, 2005 Wiertz-Eggert-Jörissen 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Nitrofurans as an example - How to control zero tolerance? Katrin Hoenicke and Robert Gatermann Eurofins Analytik GmbH Wiertz-Eggert-Jörissen Hamburg, Germany

2. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium The Situation Council Regulation (EC) No. 2377/90, Annex IV: Pharmacologically active substances for which no Maximum Residue Limits (MRLs) are fixed No ADI due to their nature of toxicity or a lack of data No safe limit, unacceptable at any concentration Accepted to be banned Controled with zero-tolerance

3. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Zero-Tolerance What does zero-tolerance mean? The respective residue should not be detectable Not detectable is based on the limit of detection (LOD) Nowadays: LOD has decrased drastically Significance of regulations must sometimes be questioned with regards to the toxicity of residues determined in the lower ppb-level

4. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Zero-tolerance Basic Hypothesis of the zero-tolerance concept: Residues of pharmacologically active substances in food of animal origin are a result of the use of medicines in food producing animals Anti-Thesis: A concentration level close to zero could be caused by other sources than the use of medicines in food producing animals

5. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Possible other sources Cross contamination from former use Cross contamination from use in human medicine Environmental contamination Natural occurrence / formation To discuss: The limitation of a zero-tolerance concept which is dependent on the sophistication of analytical equipment

6. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Nitrofurans as an example Nitrofurans: Banned within the EU in the 90ies (zero-tolerance) In those days: Control was limited by the analytical technique Nowadays: Technical developments in LC/MS-MS lowered LODs for nitrofuran metabolites more than 4 orders of magnitude Minimum Required Performance Limit (MRPL) for nitrofuran metabolites was set to 1 µg/kg (Commission Decision 2002/657/EC)

7. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Semicarbazide – more than a nitrofuran metabolite Semicarbazide (SEM): Characteristic marker of the nitrofuran nitrofurazone Zero-tolerances for SEM in food are in force Toxicological studies of SEM: Weak mutagenic activity, mainly in the absence of a metabolic activating system (EFSA, 2003) nitrofurazone SEM

8. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Other possible sources for SEM in food products: Azodicarbonamide (ADC) Identification of SEM in food packaged in glass jars where ADC is used as a blowing agent in the gaskets of the lids (EFSA, 2003) Identification of SEM in flour to which ADC was added as an improver (Pereira et al., 2004) Hypochlorite Treatment Identification of SEM in carrageenan (E 470a) due to a bleaching step using hypochlorite (Hoenicke et al., 2004) Semicarbazide – more than a nitrofuran metabolite

9. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Other possible sources for SEM in food products: Heat treatment Identification of SEM in egg white powder due to a pasteurisation step (Gatermann et al., 2004) Natural occurrence Identification of SEM in dried marine products (crayfish, algae) (Saari and Peltonen, 2004, Hoenicke et al., 2004) Numerous food products have been destroyed erroneously!!! Semicarbazide – more than a nitrofuran metabolite

10. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Consequences for SEM testing November 2003, Community Reference Laboratory (CRL), Fougères: Illegal use of nitrofurazone can be detected by targeting the bound residues of SEM But: SEM from other sources was also shown to be rapidely bound December 2004, Standing Comittee on the Food Chain and Animal Health (SCFCAH): SEM in the animal fraction of composite food may result from the use of nitrofurazone in live animals, but may also result from other sources or chemical reactions during processing Appropriate statement should accompany the analytical test report

11. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Commission Decision 2005/34/EC – Progress in regulation Harmonised standards for handling with zero-tolerances The MRPL correspond to the average limit above which the detection of a substance or its residue can be construed as methodologically meaningful Introduction of action limits MRPL shall be used as reference point for action Only test results at or above the MRPL shall be considered non- compliant with Community legislation Residues below the MRPL should be construed as not of immediate concern Useful tool to handle with zero tolerances

12. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Commission Decision 2005/34/EC – Ongoing problems MRPLs shall be used as reference points for action irrespective of the matrix tested In the case of composite or processed food other possible sources should be considered

13. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium Conclusion Introduction of MRPLs as action limits is the only acceptable compromise to deal with zero-tolerances Use of MRPLs as action limits for all matrices without any prove is not possible especially in the case of composite or processed food products

14. Wiertz-Eggert-Jörissen March 3 and 4, 2005 3rd AOAC Europe - Eurachem Symposium Brussels, Belgium References European Food Safety Authority (EFSA) (2003) Statement of the Scientific Panel on Additives, Flavourings, Processing Aids and Materials in Contact with Food updating the advice on available semicarbazide in packaged foods. EFSA/AFC/FCM/17-final, 1 October 2003. Pereira, A.S., Donato, J.L., De Nucci, G. (2004) Implications of the use of semicarbazide as a metabolic target of nitrofurazone contamination in coated products, Food Addit. Contam. 21, 63-69. Hoenicke, K., Gatermann, R., Hartig, L., Mandix, M., Otte, S. (2004) Formation of semicarbazide (SEM) in food by hypochlorite treatment: is SEM a specific marker for nitrofurazone abuse? Food Addit. Contam. 21, 526-537. Gatermann, R., Hoenicke, K., Mandix, M. (2004) Formation of semicarbazide (SEM) from natural compounds in food by heat treatment, Czech J. Food Sci. 22, 353-354. Saari, L., Peltonen, K. (2004) Novel source of semicarbazide: levels of semicarbazide in cooked crayfish samples determined by LC/MS/MS, Food Addit. Contam. 21, 825-32.