Scientific Opinion on marine biotoxins in shellfish Cyclic imines (spirolides, gymnodimines, pinnatoxins and pteriatoxins) 1

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1 EFSA Journal 2010; 8(6):1628 SCIENTIFIC OPINION Scientific Opinion on marine biotoxins in shellfish Cyclic imines 1 EFSA Panel on Contaminants in the Food Chain (CONTAM) 2, 3 European Food Safety Authority (EFSA), Parma, Italy This scientific opinion replaces the earlier version published on 7 June ABSTRACT The EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) assessed the risks to human health related to the consumption of spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs) in shellfish. They are cyclic imines (CIs), a family of marine biotoxins. SPXs and GYMs are produced by the dinoflagellates Alexandrium ostenfeldii and Karenia selliformis, respectively. The organism producing PnTXs has not been identified but has been described as a peridinoid dinoflagellate. PtTXs are suggested to be bio-transformed from PnTXs in shellfish. No information has been reported linking CIs to poisoning events in humans. SPXs have been detected in Europe while GYMs have not been found. Recently PnTXs were identified for the first time in shellfish in Europe but PtTXs have not been detected. There are no regulatory limits for CIs in shellfish. The toxicological database for SPXs, GYMs, PnTXs and PtTXs is limited, comprising mostly acute toxicity studies. In view of the acute toxicity and the lack of chronic toxicity data for CIs, the CONTAM Panel considered that an acute reference dose should be established but due to the lack of data this was not possible. By comparing the lowest lethal dose (LD 50 ) values for SPXs (50 and 500 µg/kg body weight (b.w.) administered by gavage or in feed, respectively) and the estimated 95 th percentile of exposure (0.06 µg/kg b.w.) a margin of exposure in the range of was calculated. The mouse bioassay has traditionally been used to detect CIs. However, due to poor specificity and ethical concerns it is not considered an appropriate method. The receptor-based fluorescence polarisation method has been developed as alternative, but it needs further development. Liquid chromatography-tandem mass spectrometry methods would be of value for the quantification of CIs, but certified reference standards and reference materials are needed to allow method development and (inter-laboratory) validation. KEY WORDS Marine biotoxins, cyclic imines (CIs), spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs), pteriatoxins (PtTXs), shellfish, methods of analysis, human health, risk assessment. 1 On request from the European Commission, Question No EFSA-Q F, adopted on 19 May Panel members: Jan Alexander, Diane Benford, Alan Boobis, Sandra Ceccatelli, Jean-Pierre Cravedi, Alessandro Di Domenico, Daniel Doerge, Eugenia Dogliotti, Lutz Edler, Peter Farmer, Metka Filipič, Johanna Fink-Gremmels, Peter Fürst, Thierry Guerin, Helle Katrine Knutsen, Miroslav Machala, Antonio Mutti, Josef Schlatter and Rolaf van Leeuwen. Correspondence: contam@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on marine biotoxins for the preparation of this opinion: Jan Alexander, Diane Benford, Luis Botana, Peter Fürst, Gerhard Heinemeyer, Philipp Hess, Angelika Preiss-Weigert, Hans van Egmond and Rolaf van Leeuwen, and EFSA s staff members Jean-Lou Dorne, Mari Eskola and Francesco Vernazza for the support provided to this EFSA scientific output. 4 Chemical structures of pinnatoxins (Figure 3) and pteriatoxins (Figure 4) were revised. The changes do not affect the overall conclusions of the opinion. Suggested citation: EFSA Panel on Contaminants in the Food Chain (CONTAM); Scientific Opinion on marine biotoxins in shellfish Cyclic imines. EFSA Journal 2010; 8(6):1628. [39 pp.]. doi: /j.efsa Available online: European Food Safety Authority,

2 SUMMARY Spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs) are cyclic imines (CIs), and are a family of marine biotoxins present in shellfish. SPXs are produced by the dinoflagellate Alexandrium ostenfeldii, whereas GYMs are produced by the dinoflagellate Karenia selliformis. The organism producing PnTXs has not been identified yet but has been described as a peridinoid dinoflagellate. It has been suggested that PtTXs are bio-transformed from PnTXs in shellfish. SPXs have been identified in a number of European countries bordering the Mediterranean Sea, Atlantic coast and the North Sea. To date GYMs have not been reported in shellfish produced in Europe, but they have been found in products imported from outside of Europe. Only recently PnTXs were identified for the first time in shellfish in Europe. They have so far only been identified in Norway. The other European countries have not conducted surveys to this end. The recent findings suggest that PtTXs are transformed from PnTXs in shellfish but PtTXs have not been reported in shellfish in Europe. Although SPXs, GYMs, PnTXs and PtTXs are now known to occur in microalgae and/or shellfish in several parts of the world, no information has been reported linking these toxin groups to poisoning events in humans. SPXs, GYMs, PnTXs and PtTXs, like other CIs such as prorocentrolides and spiro-prorocentrimines are macrocyclic compounds with imine (carbon-nitrogen double bond) and spiro-linked ether moieties. Due to similarities in chemical structure and toxicity in mice these different groups of toxins have been grouped together. SPXs are the largest group of the CIs. Presently 12 SPX-analogues are known of which 13-desmethyl SPX C is the most commonly found in shellfish. The chemical structures of three GYM-analogues have been characterised (GYM A-C). PnTXs are the CIs which are most closely related to the chemical structure of SPXs. Seven PnTX-analogues (PnTX A-G) have been chemically characterised. PtTXs and PnTXs are almost structurally identical and the latest results suggest that PnTX-analogues F and G are progenitors of all the known PnTXs and PtTXs via metabolic and hydrolytic transformation in shellfish. The chemical structures of PtTX A, B and C have been characterised. Currently there are no regulatory limits for CIs in shellfish in Europe or in other regions of the world. The toxicological database for CIs is limited and comprises only studies on their acute toxicity following intraperitoneal (i.p.) administration and oral (gavage, feed) administration. Based on the available information it can be concluded that CIs have the ability to bind and block acetylcholine receptors in the central- and peripheral nervous systems, including neuromuscular junctions. There are no long term studies on CIs in experimental animals that would allow establishing a tolerable daily intake (TDI). In view of the acute toxicity of CIs the Panel on Contaminants in the Food Chain (CONTAM Panel) considered that an acute reference dose (ARfD) should be established for the different groups of CIs, but due to the lack of adequate quantitative data on acute oral toxicity (i.e. no-observed-adverse-effect levels (NOAELs)) this was not possible. The CONTAM Panel calculated a margin of exposure (MOE) between the lowest lethal dose (LD 50 ) values for SPXs (in the region of 50 and 500 µg/kg body weight (b.w.) when administered by gavage or in feed, respectively) and the estimated 95 th percentile of exposure (0.06 µg/kg b.w.) from consumption of shellfish currently on the market. The MOE is in the range of The lower end of this range was based on the LD 50 by gavage in fasted mice. The higher end of the range was based on the LD 50 following administration of SPXs in the feed and is therefore more likely to be of relevance for the assessment of the risk of consumption of shellfish contaminated with SPXs. Taking also into account the steep dose response relationship observed in mice, and that mice given sublethal doses of SPXs are reported to make a rapid and full recovery, the CONTAM Panel concluded that EFSA Journal 2010; 8(6):1628 2

3 current estimated exposure to SPXs does not raise concern for the health of the consumer. The CONTAM Panel stresses, however, that this conclusion for SPXs is based on very limited toxicity data. Since exposure to other groups of CIs (GYMs, PnTXs and PtTXs) could not be estimated from the available data, no conclusions can be drawn with respect to any possible risk to consumers for these groups of CIs. The mouse bioassay (MBA) has traditionally been used to detect CIs in shellfish. However, for reasons of animal welfare there is a growing concern with respect to its use. Due to its poor specificity it is not considered an appropriate detection method for CIs. The receptor-based fluorescence polarisation method has been shown to be able to detect GYM A and 13-desmethyl SPX C at relevant levels in shellfish but it needs further development. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods allow specific detection of individual CIs and they would be of value for their quantification in shellfish. None of the current methods of analysis to determine CIs in shellfish has been formally validated in interlaboratory studies. The CONTAM Panel noted that certified reference standards and reference materials for toxicologically relevant CIs need to be provided to allow method development and (inter-laboratory) validation. EFSA Journal 2010; 8(6):1628 3

4 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 4 Background as provided by the European Commission... 5 Terms of reference as provided by the European Commission... 9 Assessment Introduction Chemical characteristics Regulatory status Methods of analysis Supply of appropriate reference material Mouse bioassay Biochemical assays Chemical methods LC-MS/MS methods HPLC-UV method Proficiency tests Summary of methods Occurrence of CIs Data collection CI concentration in shellfish SPXs PnTXs Difference between shellfish species Influence of processing Human consumption of shellfish Exposure assessment Deterministic estimate of dietary exposure to SPXs Probabilistic estimate of dietary exposure to SPXs Toxicokinetics Toxicity data Mechanistic considerations Effects in laboratory animals Acute toxicity Toxicity following intraperitoneal (i.p.) administration Toxicity following oral administration Relative potency of analogues Observations in humans Hazard characterisation Risk characterisation Uncertainty Conclusions and Recommendations References Abbreviations EFSA Journal 2010; 8(6):1628 4

5 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Marine biotoxins, also commonly known as shellfish toxins, are mainly produced by algae or phytoplankton. Based on their chemical structure, the toxins have been classified into eight groups, namely, the azaspiracid (AZA), brevetoxin (BTX), cyclic imine (CI), domoic acid (DA), okadaic acid (OA), pectenotoxin (PTX), saxitoxin (STX) and yessotoxin (YTX) groups, as agreed at the Joint FAO/IOC/WHO ad hoc Expert Consultation held in Two additional groups, palytoxins (PlTX) and ciguatoxins (CTX), may also be considered. STX and its derivatives cause Paralytic Shellfish Poisoning (PSP), and DA causes Amnesic Shellfish Poisoning (ASP). Diarrhetic Shellfish Poisoning (DSP) is caused by OA-group toxins (OA and dinophysis toxins (DTX)), and AZA group toxins cause Azaspiracid Shellfish Poisoning (AZP). These toxins can all accumulate in the digestive gland (hepatopancreas) of filter-feeding molluscan shellfish, such as mussels, oysters, cockles, clams and scallops, and pose a health risk to humans if contaminated shellfish are consumed. Marine biotoxinrelated illness can range from headaches, vomiting and diarrhoea to neurological problems, and in extreme cases can lead to death. To protect public health, monitoring programmes for marine biotoxins have been established in many countries, which often stipulate the use of animal models (for example, the mouse bioassay (MBA) and the rat bioassay (RBA)), for detecting the presence of marine biotoxins in shellfish tissues. In the European Union (EU), bioassays are currently prescribed as the reference methods. Various stakeholders (regulators, animal welfare organisations, scientific organisations) have expressed their concerns about the current legislation in Europe, not only with regard to the use of large numbers of animals, involving procedures which cause significant pain and suffering even though non-animal based methods are available, but also since the scientific community argues that the animal test may not be suitable for all classes of toxins and that the state-of-the-art scientific methodology for the detection and determination of marine biotoxins is not fully reflected in current practices. 1. Legal framework In 2004, the purported EU Hygiene Package of regulations, bringing together and replacing the existing hygiene regulations for the food sector previously contained in numerous individual vertical Directives was published. In Annex II Section VII Chapter V (2) to Regulation 853/2004/EC 6, are established maximum levels for ASP, PSP and DSP toxins. Annex III of Commission Regulation No 2074/2005/EC 7 of 5 December 2005 lays down the recognised testing methods for detecting marine biotoxins. Annex II Chapter II (14) to Regulation (EC) 854/2004 8, gives the monitoring authorities in the EU Member States the mandate to examine live molluscs for the presence of marine biotoxins. The EU Hygiene Package came into effect on 1 January The Council Directive 86/609/EEC Council Directive 86/609/EEC 9 makes provision for laws, regulations and administrative provisions for the protection of animals used for experimental and other scientific purposes. This includes the use of live vertebrate animals as part of testing strategies and programmes to detect identify and quantify 5 ftp://ftp.fao.org/es/esn/food/biotoxin_report_en.pdf 6 OJ L 139, , pp OJ L 338, , pp OJ L 139, , pp OJ L 358, , pp EFSA Journal 2010; 8(6):1628 5

6 marine biotoxins. Indeed, the scope of Article 3 of the Directive includes the use of animals for the safety testing of food, and the avoidance of illness and disease. Directive 86/609/EEC sets out the responsibilities that Member States must discharge. As a result of this use of prescriptive language, Member States have no discretion or flexibility, and most of the provisions of the Directive must be applied in all cases. It is clear that Member States have to ensure that: the number of animals used for experimental and other scientific purposes is reduced to the justifiable minimum; that such animals are adequately cared for; and that no unnecessary or avoidable pain, suffering, distress or lasting harm are caused in the course of such animal use. Member States may not (Article 7, 2) permit the use of live animals in procedures that may cause pain, suffering, distress or lasting harm: if another scientifically satisfactory method of obtaining the result sought and not entailing the use of live animals is reasonably and practicably available. When animal use can be justified, Directive 86/609/EEC specifies a range of safeguards that Member States must put in place to avoid or minimise any animal suffering that may be caused. All justifiable animal use should be designed and performed to avoid unnecessary pain, suffering, distress and lasting harm (Article 8). Member States must ensure (Article 19, 1) that user establishments undertake experiments as effectively as possible, with the objective of obtaining consistent results, whilst minimising the number of animals and any suffering caused. This latter requirement necessitates the use of minimum severity protocols, including appropriate observation schedules, and the use of the earliest humane endpoints that prevent further suffering, once it is clear that the scientific objective has been achieved, that the scientific objective cannot be achieved, or that the suffering is more than can be justified as part of the test procedure. The EC and Member States are also required (Article 23, 1) to encourage research into, and the development and validation of, alternative methods that do not require animals, use fewer animals, or further reduce the suffering that may be caused, whilst providing the same level of scientific information. 3. Recognised testing methods for marine biotoxins and maximum levels Commission Regulation (EC) No. 2074/ specifies a mouse bioassay (MBA) for the determination of paralytic shellfish poisoning toxins (PSP) and a MBA or the rat bioassay (RBA) for lipophilic marine biotoxins. Alternative test methods can be applied if they are validated following an internationally recognised protocol and provide an equivalent level of public health protection. Besides paralytic shellfish poisoning toxins, okadaic acid, dinophysistoxins, pectenotoxins, azaspiracids and yessotoxins, also cyclic imines, (gymnodimine, spirolides and others which are currently not regulated in the EU), all give a positive response in MBAs. The reference method for the domoic acid group (the causative agent of ASP) is based on highperformance liquid chromatography (HPLC). Chapter V (2) (c) and (e) of Section VII of Annex III to Regulation (EC) No 853/ establishes that food business operators must ensure that live bivalve molluscs placed on the market for human consumption must not contain marine biotoxins in total quantities (measured in the whole body or any part edible separately) that exceed the following limits: 800 micrograms per kilogram for paralytic shellfish poison (PSP), 20 milligrams of domoic acid per kilogram for amnesic shellfish poison (ASP), EFSA Journal 2010; 8(6):1628 6

7 160 micrograms of okadaic acid equivalents 10 per kilogram for okadaic acid, dinophysistoxins and pectenotoxins in combination, 1 milligram of yessotoxin equivalents per kilogram for yessotoxins, 160 micrograms of azaspiracid equivalents per kilogram for azaspiracids. 4. Joint FAO/IOC/WHO ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs (Oslo, September ) Based on the available information, the Joint FAO/IOC/WHO ad hoc Expert Consultation suggested provisional acute reference doses (ARfDs) 11 for the AZA, OA, STX, DA, and YTX-group toxins, respectively (summarized in the Table 1). The Expert Consultation considered that the database for the cyclic imines, brevetoxins and pectenotoxins was insufficient to establish provisional ARfDs for these three toxin groups. In addition, guidance levels were derived comparing results based on the consumption of 100 g, 250 g or 380 g shellfish meat by adults. However, the Expert Consultation noted that the standard portion of 100 g, which is occasionally used in risk assessment, is not adequate to assess an acute risk, whereas a portion of 250 g would cover 97.5 % of the consumers of most countries for which data were available. Available methods of analysis were reviewed for the 8 toxin groups and recommendations made for choice of a reference method, management of analytical results and development of standards and reference materials. The Joint FAO/IOC/WHO ad hoc Expert Consultation, however, did not have sufficient time to fully evaluate epidemiological data and to assess the effects of cooking or processing for deriving the provisional guidance levels/maximum levels for several toxin groups (especially the AZA and STX groups). The Consultation encouraged Member States to generate additional toxicological data in order to perform more accurate risk assessments and to facilitate validation of toxin detection methods in shellfish. 10 Equivalents: the amount of toxins expressed as the amount of okadaic acid that gives the same toxic response followed intraperitoneal administration to mice. This applies similarly for the group of yessotoxins and azapiracids, respectively. 11 The acute reference dose is the estimate of the amount of substance in food, normally expressed on a body-weight basis (mg/kg or µg/kg of body weight), that can be ingested in a period of 24 hours or less without appreciable health risk to the consumer on the basis of all known facts at the time of evaluation (JMPR, 2002). EFSA Journal 2010; 8(6):1628 7

8 Table 1: Summary data used in the derivation of the acute reference dose (ARfD) and current guidance levels. Group toxin LOAEL(1) NOAEL(2 ) µg/kg body weight AZA 0.4 (1) 10 (H) BTX Cyclic Imines DA 1,000 (1) 10 (H) OA 1 (1) 3 (H) PTX Safety Factor (Human data (H) Animal data (A)) Provisional ARfD 11 Derived Guidance Level/ Maximum Level based on consumption of 100g (1), 250g (2) and 380g (3) 0.04 µg/kg mg/kg SM (1) 2.4 µg/adult (a) mg/kg SM (2) mg/kg SM (3) N/A N/A 60 mg/kg SM (1) 100 µg/kg 6 mg/adult (a) 24 mg/kg SM (2) 16 mg/kg SM (3) 0.2 mg/kg SM (1) 0.33 µg/kg 20 µg/adult (a) 0.08 mg/kg SM (2) 0.05 mg/kg SM (3) N/A Limit Value currently implemented in EU legislation 0.16 mg/kg SM 20 mg/kg SM 0.16 mg/kg SM 0.16 mg OA equivalents/kg SM STX 2 (1) 3 (H) 0.42 mg/kg SM (1) 0.7 µg/kg 42 µg/adult (a) 0.17 mg/kg SM (2) 0.11 mg/kg SM (3) 0.8 mg/kg SM YTX 5,000 (2) 100 (A) 30 mg/kg SM (1) 50 µg/kg 3 mg/adult (a) 12 mg/kg SM (2) 8 mg/kg SM (3) 1 mg/kg SM SM: shellfish meat; LOAEL: lowest-observed-adverse-effect level; NOAEL: no-observed-adverse-effect level; N/A: not available; EU: European Union; OA okadaic acid (a): Person with 60 kg body weight (b.w.) The Joint FAO/IOC/WHO ad hoc Expert Consultation also indicated that there were discrepancies between different risk assessments, especially for determining methods of analysis for certain marine biotoxins and in relation to established maximum limits. Test methods for the eight toxin groups were reviewed and recommendations for Codex purposes made. Mouse bioassays are widely used for shellfish testing but for technical and ethical reasons it is highly desirable to move to new technologies which can meet Codex requirements more adequately. Most currently available methods do not meet fully the strict criteria for Codex type II 12 or III 13 methods and have therefore not been widely used in routine shellfish monitoring. However, the recommendations made by the Expert Consultation represent the best currently available methods. Liquid chromatography-mass spectrometry (LC-MS) has much potential for multi-toxin analysis and has been recommended for consideration and recommendation by Codex. The Joint FAO/IOC/WHO ad hoc Expert Consultation is of the opinion that the complexity and chemical diversity of some toxin 12 A Type II method is the one designated Reference Method where Type I methods do not apply. It should be selected from Type III methods (as defined below). It should be recommended for use in cases of dispute and for calibration purposes. 13 A Type III Method is one which meets the criteria required by the Codex Committee on Methods of Analysis and Sampling for methods that may be used for control, inspection or regulatory purposes. EFSA Journal 2010; 8(6):1628 8

9 groups is such that validated quantitative methods to measure all toxins within a group will be extremely difficult. Thus the implementation of a marker compound concept and the use of functional assays should be explored. 5. Working Group Meeting to Assess the Advice from the Joint FAO/IOC/WHO ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs, Ottawa, Canada, April 10-12, 2006 The working group (WG) discussed available reference methods in particular and concluded that they should be highly specific, highly reproducible, and not prone to false positives or false negatives. The methods are expected to be definitive and may well result in significant rejections of products and must therefore withstand the most robust legal and scientific scrutiny. In considering their weaknesses and merits, the meeting noted that the various mouse bioassays should be discussed individually since the level of performance and success differs markedly between the official method for PSP by mouse bioassay, the American Public Health Association (APHA) method for brevetoxins and the multiple mouse bioassay DSP procedures employed for the other lipophilic toxins such as okadaic acid, azaspiracids and others. Recognizing that the majority of the currently available methods do not meet all Codex criteria for reference methods (Type II), the WG concluded that Codex Committee for Fish and Fishery Products (CCFFP) should consider a variety of biotoxin analytical methods. Wherever possible, reference methods should not be based on animal bioassays. Functional methods, biochemical/immunological and chemical-analytical methods currently in use, and considered to be validated according to Codex standards, should be recommended by CCFFP to the Codex Committee on Methods of Analysis and Sampling (CCMAS) for review and designation as Type II or Type III methods. Because the Expert Consultation has offered 3 different guidance limits associated with three levels of consumption (100 g, 250 g and 380 g) for most toxin groups, it is important to determine which consumption level is appropriate for the protection of consumers. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION In accordance with Art. 29 (1) (a) of Regulation (EC) No 178/2002, the Commission asks EFSA to assess the current EU limits with regard to human health and methods of analysis for various marine biotoxins as established in the EU legislation, including new emerging toxins, in particular in the light of - the report of the Joint FAO/IOC/WHO ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs (Oslo, September ), including the ARfDs and guidance levels proposed by the Expert Consultation, - the conclusions of the CCFFP working group held in Ottawa in April 2006, - the publication of the report and recommendations of the joint European Centre for the Validation of Alternative Methods (ECVAM)/DG SANCO Workshop, January 2005, - the report from CRL Working group on Toxicology in Cesenatico October 2005, - any other scientific information of relevance for the assessment of the risk of marine biotoxins in shellfish for human health. EFSA Journal 2010; 8(6):1628 9

10 ASSESSMENT 1. Introduction Spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs) are cyclic imines (CIs), and are a family of marine biotoxins present in shellfish. They have only been discovered during the last few decades. The main producer of SPXs is the dinoflagellate Alexandrium ostenfeldii. This dinoflagellate has also been reported to produce saxitoxin (STX)-group toxins (Mackenzie et al., 1996; Cembella, 1998; Gribble et al., 2005; Kremp et al., 2009). SPXs were first isolated and characterised in scallops and mussels harvested in Nova Scotia, Canada in early 1990s (Hu et al., 1995). Due to environmental conditions, the profile of SPXs produced by A. ostenfeldii varies remarkably between the geographical locations (Cembella et al., 2000; Otero et al., 2010a). Also a first GYM analogue (GYM A) was isolated and characterised in the beginning of 1990s in New Zealand oysters (Mackenzie et al., 1996; Seki et al., 1995, 1996) and later two additional GYManalogues were isolated (Miles et al., 2000, 2003). Presently it is known that GYMs are produced by dinoflagellate Karenia selliformis (formerly Gymnodinium selliforme) (Seki et al., 1995; Miles et al., 2003; Haywood et al., 2004). MacKenzie et al. (2002) noticed that since GYM persisted in New Zealand oysters for several years after the initial contamination event, GYM A in shellfish might be a long term residue from the previous bloom of K. selliformis. The organism producing PnTXs has not been identified yet but has been described as a peridinoid dinoflagellate (Rhodes et al., 2010). In 1995 a finding of the first PnTX-analogue (PnTX A) was reported in Japanese shellfish (Uemura et al., 1995). In the following year PnTX D was isolated from shellfish in Japan (Chou et al., 1996a). The isolation of PnTX-analogues B and C turned out to be more difficult due to their trace amounts in shellfish and therefore they were successfully isolated only in 2001 (Takada et al., 2001a). Recently new PnTX-analogues E, F and G were isolated from oysters in New Zealand and Australia (Selwood et al., 2010). The latest results suggest that PnTXanalogues F and G are produced separately in different dinoflagellates and are progenitors of all the known PnTXs and PtTXs via metabolic and hydrolytic transformation in shellfish (Selwood et al., 2010). Three analogues of PtTXs were isolated in shellfish in Japan in 2001 (Takada et al., 2001b), and it has been suggested that they are bio-transformed from PnTXs in shellfish (Selwood et al., 2010). CIs were discovered in shellfish because of their high acute toxicity in mice upon intraperitoneal (i.p.) injections of lipophilic extracts. When they are present in shellfish at elevated levels, they rapidly kill mice, and they may interfere with the mouse bioassay (MBA) for okadaic acid (OA)-, brevetoxin (BTX)-, yessotoxin (YTX)- and azaspiracid (AZA)-group toxins (FAO/IOC/WHO, 2004). In Europe SPXs have only recently been found in their producer dinoflagellate A.ostenfeldii in Scotland (John et al., 2003), Italy (Ciminiello et al., 2006), Denmark (MacKinnon et al., 2006) and in Ireland (Touzet et al., 2008). They have also been found in shellfish in Norway (Aasen et al., 2005), Spain (Villar González et al., 2006) and in Italy (Pigozzi et al., 2008). To date GYMs have not been reported in shellfish produced in Europe. They have only been detected in shellfish imported from Tunisia. Biré et al. (2002) and Marrouchi et al. (2009) reported GYM A and B in Tunisian clams. In Europe PnTXs have only recently been found in mussels in various parts of the Norwegian coast (Miles et al., 2010). Other European countries have not conducted surveys on PnTXs in shellfish to this end. PtTXs have not been reported in shellfish in Europe. EFSA Journal 2010; 8(6):

11 Although SPXs, GYMs, PnTXs and PtTXs are now known to occur in microalgae and/or shellfish in several parts of the world (Canada, Denmark, New Zealand, Norway, Scotland, Tunisia, USA and Japan), no information has been reported linking these toxin groups to poisoning events in humans (FAO/IOC/WHO, 2004; Cembella and Krock, 2008; Rhodes et al., 2010; Selwood et al., 2010). In addition to SPXs, GYMs, PnTXs and PtTXs, the CI group comprises prorocentrolides and spiroprorocentrimines. Due to similarities in chemical structure and toxicity in mice these different groups of toxins have been grouped together. SPXs, GYMs and PnTXs are dealt with in this scientific opinion as they have been reported to occur in shellfish produced in Europe or imported into Europe (Cembella and Krock, 2008; Miles et al., 2010). Although PtTXs have not been reported in shellfish in Europe they are included in this opinion because recent findings suggest that they are biotransformation products of PnTXs in shellfish (Selwood et al., 2010). To date prorocentrolides and spiro-prorocentrimines have not been reported in shellfish in Europe. 2. Chemical characteristics SPXs, GYMs, PnTXs and PtTXs, like other CIs, (prorocentrolides and spiro-prorocentrimines) are macrocyclic compounds with imine (carbon-nitrogen double bond) and spiro-linked ether moieties. Comparison of the chemical structures of the six CI toxin groups shows a high degree of structural similarity e.g. approximately 70 % homology between PnTXs and SPXs (Cembella and Krock, 2008). SPXs, PnTXs, GYMs and PtTXs are soluble in organic solvents such as methanol, acetone, chloroform and ethyl acetate. SPXs are the largest group of the CIs and there are several studies on mass spectrometric (MS) and nuclear magnetic resonance (NMR) methods which have been used for structure elucidation of SPX analogues (Hu et al., 1995, 1996, 2001; Falk et al., 2001; Sleno et al., 2004a, b; Aasen et al., 2005; Sleno and Volmer, 2005; Ciminiello et al., 2006, 2007; MacKinnon et al., 2006; Krock and Cembella, 2007). Based on their chemical structure SPXs can be divided into three classes. The first, SPXs has a polyether ring system in addition to the heptacyclic imine ring. This class includes SPXs A, B, C, D, 13-desmethyl SPX C (also known as SPX 1), 13,19-didesmethyl SPX C, 13-desmethyl SPX D and 27-hydroxy 13,19-didesmethyl SPX C (Figure 1). The second SPX class has the same polyether ring system but the heptacyclic imine ring has opened to a keto amine (Figure 1). This class comprises SPX E and F which are shellfish metabolites of SPX A and SPX B, respectively. Because they have a hydrolysed cyclic imine group the SPX E and F are biologically inactive (Hu et al., 1996, 2001). The third class includes SPX G and its analogue 20-methyl SPX G (Figure 1). Both of them have a heptacyclic imine ring and an unusual trispiroketal ring system which has not been found in other marine biotoxin groups (MacKinnon et al., 2006; Otero et al., 2010b). SPX C, SPX D, 13,19-didesmethyl SPX C, 13-desmethyl SPX C and SPX G, containing an additional methyl group on the imine ring when compared to SPX A and B, are suspected to be resistant to acidic and enzymatic hydrolysis in shellfish, and therefore they may be biologically active (Hu et al., 2001; Christian et al., 2008). It has also been reported that like other marine biotoxins e.g. okadaic acid (OA)-group toxins and pectenotoxin (PTX)-group toxins, SPXs can be metabolised in shellfish to fatty acid esters (Aasen et al., 2006; Doucet et al., 2007). The structures of GYMs have also been characterised by applying NMR and MS techniques (Seki et al., 1995; Stewart et al., 1997; Miles et al., 2000; Miles et al., 2003). They are the smallest molecules of the group of CIs. The chemical structures of GYM A, GYM B and GYM C are known now (Figure 2). All GYM analogues have a six-membered imino ring. Their macrocycle contains 16 carbon units and one ether bridge. GYM-C is an oxidised analogue of GYM-A and is isomeric with GYM B at C- 18. Possibly GYM C and GYM B originate from a precursor 18-deoxy GYM B, which is an analogue of GYM A formed through oxidation (Miles et al., 2000, 2003). EFSA Journal 2010; 8(6):

12 Spirolide (SPX) R 1 R 2 R 3 R 4 Δ 2,3 MW SPX A H CH 3 CH 3 H Δ 2,3 691 SPX B H CH 3 CH 3 H 693 SPX C CH 3 CH 3 CH 3 H Δ 2,3 705 SPX D CH 3 CH 3 CH 3 H desmethyl SPX C CH 3 H CH 3 H Δ 2, ,19-didesmethyl SPX C CH 3 H H H Δ 2, desmethyl SPX D CH 3 H CH 3 H hydroxy 13,19- didesmethyl SPX C CH 3 H H CH 3 Δ 2,3 691 SPX E H CH 3 CH 3 H Δ 2,3 709 SPX F H CH 3 CH 3 H 711 SPX G H methyl SPX G CH MW: molecular weight; Δ 2,3 : Double bond between C2-C3 Figure 1: Chemical structures of spirolides (SPXs) (modified from Otero et al., 2010b). Gymnodimine (GYM) R 1 R 2 MW GYM A H - (a) 507 GYM B H OH 523 GYM C OH H 523 (a): Double bond between C17-C18; MW: molecular weight Figure 2: Chemical structures of gymnodimines (GYMs) (modified from Cembella and Krock, 2008). EFSA Journal 2010; 8(6):

13 NMR and MS techniques have been applied to elucidate the chemical structures of PnTXs (Uemura et al., 1995; Chou et al., 1996a,b; Takada et al., 2001a; Selwood et al. 2010). Of the group of CIs the chemical structure of PnTXs is most closely related to that of SPXs. PnTXs have a polyether ring system and they have an additional bicyclic ether moiety in the macrocycle (Figure 3). PnTXanalogues vary in the length of their cyclohexenyl side chains. While PnTX A has a carboxylic group at R1, PnTX B and PnTX C have a 2-amino acetic acid group at R1 and they are pairs of diastereoisomers at C-34. PnTX D has a C4 γ-ketobutyric acid moiety (Figure 3). For the novel PnTX-analogues E, F and G, Selwood et al., (2010) suggested that PnTX E has a carboxylic acid in the side chain and that it is the reduced analogue of PnTX D. The defined structure of PnTX E is as presented in Figure 3 (Selwood et al., 2010). The side chain of PnTX F was defined to have a lactone ring which readily hydrolyses to hydroxyl acid to form PnTX E. Selwood et al. (2010) suggested that the side chain of PnTX G was formed by an ethenyl group. The latest results suggest that the novel PnTX F and G are progenitors of all the known PnTXs and PtTXs that are produced via metabolic transformation (e.g. hydrolysis) in shellfish (Selwood et al., 2010). It is probable that PnTX F is metabolised to PnTX D and E, while PnTX G is metabolised to PnTXs A-C and PtTXs A-C (Selwood et al., 2010). Pinnatoxin (PnTX) R 1 R 2 R 3 R 4 MW PnTX A OH H H 711 PnTX B, C OH H H 740 PnTX D H OH CH PnTX E H OH CH PnTX F H OH CH PnTX G OH H H 693 MW: molecular weight Figure 3: Chemical structures of pinnatoxins (PnTXs) (modified from Miles et al. 2010; Selwood et al., 2010). The chemical structures of the PtTX-analogues A, B and C were determined based on NMR and MS spectral analysis by Takada et al. (2001b) and Hao et al. (2006) (Figure 4). PtTXs and PnTXs are almost structurally identical with the difference that the cyclohexenyl side chain of PtTXs ends in a cysteine moiety. PtTXs have one acidic and two basic functional groups. PtTX B and C are epimers of each other at C-34. Just as PnTXs, PtTXs have a free carbocyclic group at the cyclohexenyl side chain while other CI compounds have lactones (Takada et al., 2001b; Cembella and Krock, 2008). EFSA Journal 2010; 8(6):

14 Pteriatoxin (PtTX) R 1 R 2 R 3 R 4 MW PtTX A OH H H 830 PtTX B, C OH H H 830 MW: molecular weight Figure 4: Chemical structures of pteriatoxins (PtTXs) (modified from Miles et al. 2010). 3. Regulatory status Currently there are no regulations on CIs in shellfish in Europe or in other regions of the world. However, the toxicology working group of the EU Community Reference Laboratory for marine biotoxins (CRLMB) has proposed a guidance level of 400 µg sum of SPXs/kg shellfish meat (CRLMB, 2005; Pigozzi et al., 2008). 4. Methods of analysis Several published methods exist for the determination of CIs. The mouse bioassay (MBA) is considered a simple way to detect CIs, but for ethical and scientific reasons there are growing concerns with respect to its use. Some functional assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have also been developed. None of the methods to determine SPXs, GYMs, PnTXs and PtTXs have been formally validated in interlaboratory validation studies Supply of appropriate reference material Certified reference standards are available for 13-desmethyl SPX C and for GYM A (National Research Council Canada 14 ), but there are no certified standards for other CIs. Certified reference materials are not available for shellfish containing SPXs, GYMs, PnTXs or other CIs Mouse bioassay The MBA has traditionally been used to detect CIs in shellfish. SPXs were identified as a source of false positive results in the classical MBA developed by Yasumoto et al. (1978) for lipophilic toxins. Their signs of toxicity prompted the use of the term fast acting toxins (Cembella and Krock, 2008), to emphasise their action (neurological signs, convulsions, cramps) within a few minutes after the injection and death within 30 minutes. The reason for this response in the MBA is the high solubility of SPXs in polar organic solvents, such as methanol, aqueous ethanol or acetone, hence being readily incorporated into the same fraction as the other lipophilic toxins (Cembella and Krock, 2008; Otero et 14 EFSA Journal 2010; 8(6):

15 al., 2010 a,b). The classical MBA was also applied to identify GYM A for the first time in clams in Tunisia (Biré et al., 2002). The period of time when death occurred has not been reported for GYM A. Assuming that an extract from 25 g shellfish is injected into a 20 g mouse, the i.p. LD 50 of 6.9 µg/kg b.w. for 13-desmethyl SPX C (Munday, unpublished studies; Munday, 2008) indicates a limit of detection (LOD) for this compound in the MBA of about 5.6 µg/kg shellfish. For SPX C, the i.p. LD 50 of 8.0 µg/kg b.w. (Munday, unpublished studies; Munday, 2008) indicates an LOD in the MBA of about 6.4 µg/kg shellfish. For GYM A, the i.p. LD 50 of 96 µg/kg b.w. (Munday et al., 2004), indicates an LOD of about 77 µg/kg shellfish in the MBA. For PnTx E, F and G, the i.p. LD 50 values of about 50, 45 and 16 µg/kg b.w. (Selwood et al., 2010), indicate LODs in the MBA of 40, 36 and 13 µg/kg shellfish, respectively. The main advantages of the MBA are: the provision of a measure of total toxicity based on the biological response of the animal to the toxin(s); it does not require complex or expensive analytical equipment. The main disadvantages of the MBA are: it is not quantitative; it does not differentiate easily from high levels of other lipophilic marine biotoxins; no specific information is provided on individual toxins; it cannot be automated; it requires specialised animal facilities and expertise; the inherent variability in results between laboratories due to e.g. specific animal characteristics (strain, sex, age, weight, general state of health, diet, stress); it has not been validated; in many countries the use of the MBA is considered undesirable for ethical reasons Biochemical assays The selective interaction of GYMs and SPXs with cholinergic receptors, where they act as blockers, has prompted the development of functional methods based on this interaction. Vilariño et al. (2009) developed an inhibition assay method based on the competition of GYM A and 13-desmethyl SPX C with fluorescent-labeled alpha-bungarotoxin for the nicotinic acetyl choline receptor (nachr) of the electric organ of the electric ray Torpedo marmorata, with quantification using fluorescence polarisation. Whereas the method was developed using GYM A and 13-desmethyl SPX C, it is selective for any analogue of CIs that may interact with nachr. The method has been shown to be adequate for the analysis of GYM A and 13-desmethyl SPX C in various shellfish species such as mussels, clams, cockles and scallops as reported by Fonfría et al. (2010). The limit of quantification (LOQ) of the method is 80 µg/kg for GYM A and 85 µg/kg for 13-desmethyl SPX C in shellfish. The applicability of the receptor-based fluorescence polarisation method for the other CIs than GYM A and 13-desmethyl SPX C has not been reported. The binding of GYM A and 13-desmethyl SPX C to acetylcholine-binding proteins (AchBPs) in two snail species: Aplysia californica and Lymnaea EFSA Journal 2010; 8(6):

16 stagnalis has also been shown recently using stopped-flow instrumentation and conventional binding competition (Bourne et al., 2010). This AChBP-based binding assay could also be used to quantitatively detect CIs in shellfish but more research is needed. The main advantages of the receptor-based fluorescence polarisation method are: it is technically easy to perform; the necessary equipment is inexpensive; the method provides fast results; it detects any analogue of the CIs that may interact with nachr. The main disadvantages of the receptor-based fluorescence polarisation method are: it is dependent on availability of receptors from Torpedo marmorata; the method has not been validated Chemical methods The imino moiety of CI-molecules is a very good proton acceptor and therefore LC-MS/MS methods can detect CIs at low levels and have high selectivity. LC-MS/MS methods are currently the methods of choice for analysis of SPXs, GYMs, PnTXs and PtTXs in shellfish. CIs lack chromophores and therefore the optical detection methods such as ultraviolet (UV) do not provide sufficient selectivity. Recently multitoxin LC-MS/MS methods have been developed for comprehensive phycotoxin monitoring of lipophilic marine biotoxins (Fux et al, 2007; Gerssen et al., 2009a,b) LC-MS/MS methods Several publications have reported LC-MS/MS methods for SPXs, GYMs, PnTXs and PtTXs (Cembella et al., 1999; Quilliam et al., 2001; Stirling, 2001; Takada et al., 2001a, b; Biré et al., 2002; MacKenzie et al., 2002; Aasen et al., 2005; Ciminiello et al., 2006; Villar González et al., 2006; Fux et al., 2007; Gerssen et al., 2009a, b; Álvarez et al., 2010, Miles et al., 2010; Selwood et al., 2010). These methods are based on reversed-phase LC coupled with electrospray ionization MS used in either the selected ion monitoring (SIM) or selected reaction monitoring (SRM) modes. Shellfish tissues are usually extracted with (aqueous) methanol. If necessary, the crude extract may be further partitioned with chloroform. Alternatively, a solid phase extraction (SPE) procedure can be used for purification. In order to reduce the matrix effect from shellfish in the LC-MS/MS analysis, SPE purification prior to acidic and basic HPLC separation conditions were studied for several lipophilic marine biotoxins including 13-desmethyl SPX C and GYM A by Gerssen et al. (2009a, b). The reported recoveries of the SPE purification for 13-desmethyl SPX C and GYM A in mussel extract were 90 % (Gerssen et al., 2009a) and LODs were reported to be 0.8 µg/kg shellfish meat for 13-desmethyl SPX C and 3.7 µg/kg shellfish meat for GYM A (Gerssen et al., 2009b). LODs vary with procedures and LC-MS/MS instruments used. Higher LODs for multiresidue methods were reported for GYM A (at levels µg/kg shellfish) (MacKenzie et al. (2002) and for 13-desmethyl SPX C (approximately 7 µg/kg shellfish) (Álvarez et al., 2010). LODs for the LC-MS/MS methods used for analysis of PnTXs in shellfish were not reported, but the lowest concentration measured for PnTX G was reported to be 5 µg/kg shellfish (Miles et al., 2010). For the LC-MS/MS method used for EFSA Journal 2010; 8(6):

17 structural characterisation of PtTXs isolated from shellfish, the LODs were not determined (Takada et al., 2001b). An inter-laboratory study of an LC-MS/MS method for determination of various lipophilic toxins in shellfish extracts was carried out by McNabb et al. (2005). The method was first subjected to a full single-laboratory validation and then to a limited interlaboratory study between 8 laboratories in 7 countries. GYM A, SPX A, SPX D and 13-desmethyl SPX C were measured in the study but the method was quantitative only for GYM A. For SPXs the relative response factors were used to estimate their concentrations. The LOD for GYM A was 1 µg/kg shellfish meat. The betweenlaboratories reproducibility values for lipophilic toxins including GYM A were compared with the Horwitz criterion and ranged between HORRAT R values of 0.8 to (McNabb et al., 2005). The main advantages of the LC-MS/MS methods are: they are very specific and thus superior for use as confirmatory methods; their LODs for SPXs, GYMs and PnTXs are lower than with the other methods. The main disadvantages of LC-MS/MS methods are: they require costly equipment and highly trained personnel; there is a lack of reference materials of the various analogues of SPXs, GYMs, PnTXs and PtTXs; no formal interlaboratory validation studies have been published for GYMs, SPXs, PnTXs or PtTXs and detailed performance characteristics have not been reported HPLC-UV method Recently a reversed-phase HPLC-UV method was developed for routine analysis of GYMs in shellfish (Marrouchi et al., 2009). UV detection at wavelength of 210 nm was used to detect GYM A and GYM B in clam samples. The LOD was reported to be 2.4 µg GYM A/kg digestive gland of clams and the results were confirmed by LC-MS/MS. No other HPLC methods applying optical detection techniques either for GYMs, SPXs, PnTXs or PtTXs were identified in the literature. The main disadvantages of the HPLC-UV method are: due to low UV absorption of CIs the method is not specific and confirmation by LC-MS/MS is needed; there is a lack of reference materials of the various analogues of SPXs, GYMs, PnTXs and PtTXs; 15 HORRAT R : The observed relative standard deviation calculated from results generated under reproducibility conditions (RSD R ) divided by the RSD R value calculated from the Horwitz equation. The Horwitz equation is a generalised precision equation which has been found to be independent of analyte and matrix but solely dependent on concentration for most routine methods of analysis. EFSA Journal 2010; 8(6):

18 no formal interlaboratory validation studies have been carried out for SPXs, GYMs, PnTXs or PtTXs, and detailed performance characteristics have not been reported Proficiency tests For CIs there are no ongoing proficiency tests Summary of methods The MBA has traditionally been used to detect CIs in shellfish but for reasons of animal welfare there is growing concern with respect to its use and it has shown poor specificity as other lipophilic toxins interfere in the analysis. The receptor-based fluorescence polarisation method has been shown to be able to detect GYM A and 13-desmethyl SPX C at relevant levels in shellfish. CIs lack chromophores and therefore the optical detection methods such as ultraviolet HPLC-UV methods do not provide sufficient selectivity. Therefore recent studies have focussed primarily on the development of multiresidue LC-MS/MS methods for determination of SPXs, GYMs and PnTXs in shellfish. Some of these studies strongly suggest that the LC-MS/MS methods would be of value for the quantification of SPXs, GYMs, PnTXs and PtTXs. However, the optimisation of these methods, their (inter-laboratory) validation and the development of standards and reference materials are necessary. 5. Occurrence of CIs 5.1. Data collection Following a request by the European Food Safety Authority (EFSA), France, Italy, the Netherlands and Spain provided data on the occurrence of CIs in shellfish. The results for a total of 1821 samples analysed by LC-MS/MS were submitted. The number of samples differs considerably between the reporting countries, ranging between 20 samples from Spain and 852 samples from France. The reported results are over the years Although some small surveillance studies have been conducted in the four countries, GYMs have not been detected. Hence they were not included in the data analysis and statistics were calculated only for SPXs. Very recently PnTX G and A have been found in blue mussels in Norway (Miles et al., 2010). In addition Miles et al., (2010) reported occurrence results for SPXs in Norway. These data were, however, not available for inclusion in the set of occurrence data submitted to EFSA. Table 2 shows a summary of the number of data submitted by four European countries including the reason for testing, analytical method applied, LOD and LOQ of the methods. Table 2: Data submissions from European countries for SPXs in the period from 2002 to Country Year(s) of harvesting Number of samples Purpose of testing (a) Method of testing LOD (µg/kg) LOQ (µg/kg) France PreMC LC-MS/MS Italy PreMC LC-MS/MS 2 5 The Netherlands PreMC LC-MS/MS Spain PreMC LC-MS/MS Total Pre-MC: pre-market control; LOD: limit of detection; LOQ: limit of quantification (a): PreMC are samples collected at the place of origin, before or during harvesting. EFSA Journal 2010; 8(6):