Commission Regulation (EU) No 15/2011
of 10 January 2011
amending Regulation (EC) No 2074/2005 as regards recognised testing methods for detecting marine biotoxins in live bivalve molluscs
(Text with EEA relevance)

THE EUROPEAN COMMISSION,
Having regard to the Treaty on the Functioning of the European Union,
Having regard to Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food of animal origin, and in particular Article 11(4) thereof,
OJ L 139, 30.4.2004, p. 55.
Having regard to Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption, and in particular Article 18(13)(a) thereof,
OJ L 139, 30.4.2004, p. 206.
Whereas:
(1) Regulation (EC) No 854/2004 lays down specific rules for the organisation of official controls on products of animal origin and Regulation (EC) No 853/2004 lays down specific requirements concerning hygiene rules for food of animal origin. Implementing measures for those Regulations as regards recognised testing methods for marine biotoxins are set out in Commission Regulation (EC) No 2074/2005 of 5 December 2005 laying down implementing measures for certain products under Regulation (EC) No 853/2004 of the European Parliament and of the Council and for the organisation of official controls under Regulation (EC) No 854/2004 of the European Parliament and of the Council and Regulation (EC) No 882/2004 of the European Parliament and of the Council, derogating from Regulation (EC) No 852/2004 of the European Parliament and of the Council and amending Regulations (EC) No 853/2004 and (EC) No 854/2004. It is necessary to modify those implementing measures in the light of new scientific evidence.
OJ L 338, 22.12.2005, p. 27.
(2) In July 2006 the Commission requested the European Food Safety Authority (EFSA) to provide a scientific opinion to assess the current limits and methods of analysis with regard to human health for various marine biotoxins as established in the Community legislation, including new emerging toxins. The last of a series of opinions was published on 24 July 2009.
(3) The mouse bioassay (MBA) and the rat bioassay (RBA) are the official methods for the detection of lipophilic biotoxins. The Panel on Contaminants in the Food Chain of EFSA noted that these bioassays have shortcomings and are not considered an appropriate tool for control purposes because of the high variability in results, the insufficient detection capability and the limited specificity.
(4) Recently developed alternatives to the biological methods for the determination of the marine biotoxins with lower limits of detection (LOD) have successfully been tested in prevalidation studies.
(5) A liquid chromatography-mass spectrometry (LC-MS/MS) method was validated under the coordination of the European Union Reference Laboratory on marine biotoxins (EU-RL) in an inter-laboratory validation study carried out by the Member States. This method is publicly available for consultation in the web page of the EU-RL (http://www.aesan.msps.es/en/CRLMB/web/home.shtml). This validated technique of liquid chromatography (LC) mass spectrometry (MS) should be applied as the reference method for the detection of lipophilic toxins and used as matter of routine, both for the purposes of official controls at any stage of the food chain and own-checks by food business operators.
(6) Any other recognised method, different from the liquid chromatography (LC) mass spectrometry (MS), could be applied for the detection of lipophilic toxins provided that they fulfil the method performance criteria stipulated by the EU-RL. Such methods should be intra-laboratory validated and successfully tested under a recognised proficiency test scheme. If the results are challenged, the reference method shall be the EU-RL LC-MS/MS method.
(7) To allow Member States to adapt their methods to the chemical method, the biological methods should continue to be used for a limited period of time. After this period, the biological methods should be used not as a matter of routine and only during the periodic monitoring of production areas for detecting new or unknown marine toxins.
(8) Therefore, Regulation (EC) No 2074/2005 should be amended accordingly.
(9) The measures provided for in this Regulation are in accordance with the opinion of the Standing Committee on the Food Chain and Animal Health,
HAS ADOPTED THIS REGULATION:

Article 1 Annex III to Regulation (EC) No 2074/2005 is amended in accordance with the Annex to this Regulation.
Article 2 This Regulation shall enter into force on the 20th day following its publication in the Official Journal of the European Union.
It shall apply from 1 July 2011.
This Regulation shall be binding in its entirety and directly applicable in all Member States.
Done at Brussels, 10 January 2011.
For the Commission
The President
José Manuel Barroso
ANNEX In Annex III to Regulation (EC) No 2074/2005, Chapter III is replaced by the following:

‘CHAPTER III LIPOPHILIC TOXIN DETECTION METHODS

A. Chemical methodology

(1) The EU-RL LC-MS/MS method shall be the reference method for the detection of marine toxins as referred to in Chapter V(2)(c), (d) and (e) of Section VII of Annex III, to Regulation (EC) No 853/2004. This method shall determine at least the following compounds:

— okadaic acid group toxins
OA, DTX1, DTX2, DTX3 including their esters,

— pectenotoxins group toxins
PTX1 and PTX2,

— yessotoxins group toxins
YTX, 45 OH YTX, homo YTX, and 45 OH homo YTX,

— azaspiracids group toxins
AZA1, AZA2 and AZA3.
(2) Total toxicity equivalence shall be calculated using toxicity equivalent factors (TEFs) as recommended by EFSA.
(3) If new analogues of public health significance are discovered, they should be included in the analysis. Total toxicity equivalence shall be calculated using toxicity equivalent factors (TEFs) as recommended by EFSA.

(4) Other methods, such as liquid chromatography (LC) mass spectrometry (MS) method, high-performance liquid chromatography (HPLC) with appropriate detection, immunoassays and functional assays, such as the phosphatase inhibition assay, can be used as alternatives or supplementary to the EU-RL LC-MS/MS method, provided that:
(a) either alone or combined they can detect at least the analogues as identified in point A(1) of this Chapter; more appropriate criteria shall be defined when necessary;
(b) they fulfil the method performance criteria stipulated by the EU-RL. Such methods should be intra-laboratory validated and successfully tested under a recognised proficiency test scheme. The EU-RL shall support activities toward inter-laboratory validation of the technique to allow for formal standardisation;
(c) their implementation provides an equivalent level of public health protection.

B. Biological methods
(1) To allow Member States to adapt their methods to the LC-MS/MS method as defined in point A(1) of this Chapter, a series of mouse bioassay procedures, differing in the test portion (hepatopancreas or whole body) and in the solvents used for extraction and purification, may be still used until 31 December 2014 for detecting marine toxins as referred to in Chapter V(2)(c), (d) and (e) of Section VII of Annex III to Regulation (EC) No 853/2004.
(2) Sensitivity and selectivity depend on the choice of solvents used for extraction and purification and this should be taken into account when a decision is made on the method to be used in order to cover the full range of toxins.
(3) A single mouse bioassay involving acetone extraction may be used to detect okadaic acid, dinophysistoxins, azaspiracids, pectenotoxins and yessotoxins. This assay may be supplemented, if necessary, with liquid/liquid partition steps with ethyl acetate/water or dichloromethane/water to remove potential interferences.
(4) Three mice shall be used for each test. Where two out of three mice die within 24 hours of inoculation with an extract equivalent to 5 g hepatopancreas or 25 g whole body, this shall be considered a positive result for the presence of one or more toxins as referred to in Chapter V(2)(c), (d) and (e) of Section VII of Annex III to Regulation (EC) No 853/2004 at levels above those laid down.
(5) A mouse bioassay with acetone extraction followed by liquid/liquid partition with diethylether may be used to detect okadaic acid, dinophysistoxins, pectenotoxins and azaspiracids but it cannot be used to detect yessotoxins as losses of these toxins may take place during the partition step. Three mice shall be used for each test. Where two out of three mice die within 24 hours of inoculation with an extract equivalent to 5 g hepatopancreas or 25 g whole body, this shall be considered a positive result for the presence of okadaic acid, dinophysistoxins, pectenotoxins and azaspiracids at levels above those laid down in Chapter V(2)(c) and (e) of Section VII of Annex III to Regulation (EC) No 853/2004.
(6) A rat bioassay may be used to detect okadaic acid, dinophysistoxins and azaspiracids. Three rats shall be used for each test. A diarrhetic response in any of the three rats shall be considered a positive result for the presence of okadaic acid, dinophysistoxins and azaspiracids at levels above those laid down in Chapter V(2)(c) and (e) of Section VII of Annex III to Regulation (EC) No 853/2004.
C. After the period established in point B(1) of this Chapter, the mouse bioassay shall be used only during the periodic monitoring of production areas and relaying areas for detecting new or unknown marine toxins on the basis of the national control programmes elaborated by the Member States.’