European risk assessments of marine biotoxins

Benford Diane J.1, Eskola Mari2, van Leeuwen Rolaf3

On behalf of the biotoxin working group of the EFSA Panel on Contaminants in the Food Chain (CONTAM) 1 1 Food Standards Agency (FSA), London, UK 2 European Food Safety Authority (EFSA), Parma, Italy 3 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands * Corresponding author: mari.eskola@efsa.europa.eu , Fax + 39 0521 0360 890;

The European Food Safety Authority (EFSA) has published a series of risk assessments on marine biotoxins in shellfish in response to a request from the European Commission. Opinions have been adopted on the okadaic acid-, azaspiracid-, yessotoxin-, saxitoxin- and pectenotoxingroup toxins and on domoic acid. EFSA followed the established paradigm for toxicological risk assessments. Due to the lack of long term toxicity studies it was not possible to establish tolerable daily intakes (TDIs) for any of these marine biotoxins. However because they exhibit acute toxicity, it was considered appropriate to establish acute reference doses (ARfDs), i.e. the amount of toxin that can be ingested in a period of 24 hours or less without appreciable health risk. Based on the ARfD, and assuming that this amount of toxin could be contained in a single large portion of shellfish, EFSA advised on concentrations of toxin in shellfish that would not result in risks to the consumer. EFSA also proposed toxicity equivalency factors (TEFs) to allow the toxicity of the different analogues within a toxin group to be summed, but noted that more information is needed on the relative toxicity by the oral route for relevant toxins in each group in order to establish robust TEFs. The EFSA assessments suggest that consumption of shellfish containing the okadaic acid-, azaspiracid- and saxitoxin-group toxins and DA at the current EU regulatory limits could cause illness in some consumers. In contrast, for yessotoxin-group toxins, the evidence shows no concern for consumer health at levels up to about 4 times higher than the maximum levels. A EFSA statement on the influence of processing on the levels of lipophilic

Biotoxin working group members: Jan Alexander, Tore Aune, Diane Benford, Luis Botana, Peter Frst, Gerhard Heinemeyer, Philipp Hess, Sophie Krys, Angelika Preiss-Weigert, Hans van Egmond, Rolaf van Leeuwen (chair), Philippe Verger. EFSA staff: Mari Eskola, Stefan Fabiansson, Claudia Heppner, Francesco Vernazza 1 ICMSS09 Nantes, France June 2009 www.symposcience.org
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marine biotoxins in bivalve molluscs and a summary of the opinions on the currently regulated marine biotoxins have also been published. Keywords: Marine biotoxins, shellfish, okadaic acid, yessotoxin, saxitoxin, domoic acid, acute reference dose azaspiracid,

Introduction
The European Food Safety Authority (EFSA) panel on contaminants in the food chain (CONTAM) has carried out a series of risk assessments on marine biotoxins in shellfish in response to a request from the European Commission (EC) to assess the current European Union (EU) limits with regard to human health and methods of analysis for various marine biotoxins as established in EU legislation, including new emerging toxins. So far opinions have been adopted on the major classes of biotoxins: okadaic acid (OA)-group toxins (EFSA, 2007), azaspiracid (AZA)-group toxins (EFSA, 2008a), yessotoxin (YTX)group toxins (EFSA 2008b), saxitoxin (STX)-group toxins (EFSA 2009a), pectenotoxin (PTX)-group toxins (EFSA, 2009b) and domoic acid (DA) (EFSA 2009c). The remaining opinions will be on palytoxins, cyclic imines, and finally brevetoxins. A statement on the influence of processing on the levels of lipophilic marine biotoxins in bivalve molluscs (EFSA, 2009d) and a summary of the opinions on the currently regulated marine biotoxins (EFSA, 2009e) have also been published. The EFSA risk assessments were conducted according to the established paradigm for toxicological risk assessments, which consists of (i) hazard identification, (ii) hazard characterisation, (iii) exposure assessment, and (iv) risk characterisation. In line with the approach to other types of chemicals in food, the aim is to establish a health-based guidance value as an output of steps (i) and (ii) and then to compare it with estimated exposure in order to characterise the risk.

1. Health-based guidance values

Health-based guidance values are derived by first identifying the most sensitive (critical) adverse effect that is considered to be relevant to human exposure, selecting a point on the dose response relationship, such as a no observed adverse effect level (NOAEL) or lowest observed adverse effect level (LOAEL). The NOAEL or LOAEL is then divided by an uncertainty factor, which is commonly the multiple of: - 10 to allow for possible greater sensitivity of humans, if the key data are derived from toxicological studies in experimental animals; - 10 to allow for greater sensitivity of some individuals compared to average; - An additional factor if a NOAEL has not been identified, or if there are important gaps in the available information. This leads to establishment of a Tolerable Daily Intake (TDI), which is defined as the amount, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable health risk. For substances that exhibit acute toxicity, an acute reference dose (ARfD) may be established. This is the estimate of the amount of a substance in food or drink, expressed on a body weight basis, that can be ingested in a 2 ICMSS09 Nantes, France June 2009 www.symposcience.org

period of 24 hours or less without appreciable health risk. If relevant human data are available, they provide a more relevant and preferred basis for deriving the TDI or ARfD. However there can be major uncertainties in ascertaining the amounts of toxin ingested in individuals who became ill, such as whether shellfish available for analysis was representative of that consumed, the impact of cooking on the toxin levels, the analytical techniques used to measure them, and the amount of shellfish consumed. Few toxicity data were available for the biotoxins, which is mainly due to the fact that purified toxins have been available only for a limited number of analogues and in very low amounts and that not all relevant toxins have been purified. Acute toxicity data were mostly from studies in mice with the toxin injected into the intraperitoneal cavity, although some oral studies were also available for some toxins. There were very few, if any, repeated dose toxicity studies. Due to the lack of long term toxicity studies it was not possible to establish TDIs for any marine biotoxins. However because they exhibit acute toxicity, it was considered appropriate to establish ARfDs. Reports of human poisoning were used as the basis for setting an ARfD for the OA-group, AZA-group and STX-group toxins, with different uncertainty factors depending on the amount and quality of the data available. For the YTX-group and PTX-group toxins, only limited data in laboratory animals were available. Table 1 shows the derivation of the ARfDs for the currently regulated marine biotoxins.
Table 1. ARfDs established by the CONTAM Panel for the regulated biotoxins Toxin group AZA OA STX PTX YTX DA Endpoint Gastrointestinal Gastrointestinal Neurotoxicity Intestinal Cardiotoxicity Neurotoxicity
*

NOAEL/LOAEL (g/kg b.w.) 1.9 (human LOAEL) 0.8 (human LOAEL) 1.5 (human LOAEL) 250 (mouse LOAEL) 5000 (mouse NOAEL) 900 (human LOAEL)

ARfD (g/kg b.w.) 0.2 AZA1 eq. 0.3 OA eq. 0.5 STX eq. 0.8 PTX2 eq. 25 YTX eq. 30 (sum of DA and epi-DA) *

b.w. bodyweight; eq. - equivalents; Because DA can be converted to epi-DA during storage, the ARfD applies to the sum of DA and epi-DA.

2. Toxicity equivalency factors (TEFs)

When considering the different toxin analogues within a toxin group, it is necessary to bear in mind that they can occur together in shellfish and that the toxicity will be due to the total toxin content. If the analogues exert toxicity by the same mode of action, then it is assumed that the total toxicity of two or more analogues is additive with respect to dose (see EFSA, 2008c for discussion of the basic principles). Toxicity equivalency factors (TEFs) allow the toxicity to be summed up taking into account the toxic potency of each analogue relative to the best characterised analogue. Derivation of TEFs would ideally be based on oral data, but only intraperitoneal data were available for marine biotoxins, which were therefore used in proposing the TEFs shown in Table 2. Due to the lack of data on the PTX-group toxins, the CONTAM Panel proposed provisional TEF values of 1 for PTX1, PTX2, PTX3, PTX4, PTX6 and PTX11. More information is needed on the 3 ICMSS09 Nantes, France June 2009 www.symposcience.org

relative toxicity by the oral route for relevant toxins in each group in order to establish robust TEFs that would be more relevant for assessing the risk to health of consumers. Furthermore these TEFs only apply to toxin analogues within a group and there is a need for information on the combined toxicity of toxins of different groups that co-occur.
Table 2. TEFs established by the CONTAM Panel for the regulated biotoxins OA-group OA =1 * DTX1 = 1 DTX2 = 0.6 DTX3 = as for unesterified parent toxin (OA, DTX1 or DTX2) Azaspiracids AZA1 = 1 * AZA2 = 1.8 AZA3 = 1.4 AZA4 = 0.4 AZA5 = 0.2 Saxitoxins STX = 1 * NeoSTX = 1 GTX1 = 1 GTX2 = 0.4 GTX3 = 0.6 GTX4 = 0.7 GTX5 and 6 = 0.1 C2 and 4 = 0.1 dc-STX = 1 dc-NeoSTX = 0.4 Dc GTX2 = 0.2 GTX3 = 0.4 11 OH-STX = 0.3 * TEFs for other analogues are relative to the first member in each group, and the total toxin content is expressed in equivalents to this analogue (i.e. OA eq., AZA1 eq., STX eq., YTX eq.) DTX: Dinophysis toxins; GTX: Gonyautoxin Yessotoxins YTX = 1 * 45-OH-YTX = 1 1a-homoYTX = 1 45-OH-la-homoYTX = 0.5

3. Exposure assessment
Dietary exposure assessment needs to take into account the levels of toxins present in shellfish and the amount of shellfish that is consumed. The occurrence data were submitted by European Countries in response to a request from EFSA. Consumption data were available from national dietary surveys conducted in France, Germany, Italy, the Netherlands and the United Kingdom. Because the toxins have acute effects arising from a single eating occasion, it is important to identify the size of large portion that could be eaten by EU consumers. From the available data, 400g shellfish was identified as a high portion size to be used in acute exposure assessments.

4. Risk characterisation
Table 3 compares the estimated exposures with the ARfDs and indicates the concentration of toxin in shellfish that should not be exceeded in order for consumption of 400g 4 ICMSS09 Nantes, France June 2009 www.symposcience.org

shellfish to not exceed the ARfD for the relevant toxin. Based upon these comparisons it can be seen for OA- and AZA-group toxins that the dietary exposures corresponding to the consumption of a single 400 g portion of shellfish meat containing the toxins at the current EU limit values are respectively 3-fold and 5-fold higher than the ARfDs. Such exposures could exert gastrointestinal effects in susceptible consumers. For DA and STXgroup toxins, these exposures are respectively 4-fold and 10-fold higher than the ARfDs and are considered a concern for human health due to possible neurotoxic effects. Hence, the current EU regulatory limit values for the above mentioned toxins could result in exposures considered of human health concern, and thus could be not sufficiently protective for more sensitive individuals.

5. Analytical reference methods

The mouse bioassay (or in some instances also rat bioassay) is the official reference method for the analysis of the currently regulated marine biotoxins in shellfish, with the exception of DA. However, the bioassays have limited capability to detect OA-, AZAand YTX- group toxins at the current EU regulatory limits and they are not capable of detecting OA-, AZA- and STX- group toxins at the calculated concentrations at which the consumption of a 400 g portion would not lead to an exceedance of the ARfD (Table 3). Bioassays are also limited in specificity and are not quantitative for OA-, AZA- and YTXgroup toxins. In addition there are animal welfare issues related to the use of these methods.
Table 3. Comparison of the estimated exposures to the biotoxins with the ARfDs Toxin OAgroup AZAgroup STXgroup YTXgroup Concentration of toxin (g/kg shellfish) 160 (EU limit value) 240 (95th percentile) 45 (based on ARfD) 160 (EU limit value) 40 (95th percentile) 30 (based on ARfD) 800 (EU limit value) 75 (based on the ARfD 1000 (EU limit value) 315 (Norway) 799 (Italy) (95th percentile) 3750 (based on ARfD) 160 (EU limit value *) 79 (95th percentile) 120 (based on ARfD) 20,000 (EU limit value) 2500 (95th percentile) 4500 (based on ARfD) Intake (g toxin per 400g portion) 64 96 18 64 16 12 320 30 400 126 (Norway) 320 (Italy) 1500 64 32 48 8000 1000 1800 Intake (g toxin per kg bw) 1 1.6 0.3 1 0.25 0.2 5.3 0.5 6.7 2.1 (Norway) 5.3 (Italy) 25 1 0.5 0.8 130 17 30 Approximate comparison with the ARfD 3-times higher 5-times higher At the ARfD 5-times higher Slight exceedance At the ARfD 10-times higher At the ARfD 4 times lower 10 times lower 5 times lower At the ARfD Slight exceedance Below the ARfD At the ARfD 4-times higher Half of the ARfD At the ARfD

PTXgroup DA

b.w. bodyweight; * The current regulation considers OA and PTX-group toxins together with the limit value expressed as OA-equivalents; however the CONTAM Panel concluded that PTX has a different mechanism of toxicity and should be considered separately.

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The CONTAM Panel reviewed the alternative methods under development in order to advise on those with the greatest potential for replacing the bioassay(s). However all assays need validation at relevant toxin levels.

Concluding remarks
The CONTAM Panel highlighted key uncertainties in the risk assessments in relation to: reporting of outbreaks of human illness and associated toxin exposure; the toxicity of different analogues by the oral route, including setting of robust TEFs; effects of repeated exposure; the relevant high portion size for different types of shellfish; the effects of processing/cooking; and on the combined effects of different classes of toxins that can occur. EFSA has now published 6 of the 9 planned risk assessments of marine biotoxins. These suggest that consumption of shellfish containing the OA-, AZA- and STX-group toxins and DA at the current EU regulatory limits could cause illness in some consumers. In contrast, for YTX-group toxins, the evidence shows no concern for consumer health at levels up to about 4 times higher than the maximum levels. It is now for the European Commission, in consultation with Member States, to consider whether the maximum levels should be revised in view of the EFSA opinions. The remaining EFSA evaluations are due to be completed by the end of 2009.

References
EFSA, Marine biotoxins in shellfish okadaic acid and analogues, Scientific Opinion of the Panel on Contaminants in the Food chain, (Question No EFSA-Q-2006-065A), adopted on 27 November 2007 [online], 2007. http://www.efsa.europa.eu/EFSA/efsa_locale1178620753812_1178682985887.htm (page consulted 30/11/2009). EFSA, Marine biotoxins in shellfish Azaspiracid group, Scientific Opinion of the Panel on Contaminants in the Food chain, (Question No EFSA-Q-2006-065B), adopted on 9 June 2008 [online] 2008a. http://www.efsa.europa.eu/EFSA/efsa_locale1178620753812_1211902121673.htm (page consulted 30/11/2009). EFSA, Marine biotoxins in shellfish Yessotoxin group, Scientific Opinion of the Panel on Contaminants in the Food chain, (Question No EFSA-Q-2006-065D), adopted on 2 December 2008 [online] 2008b. http://www.efsa.europa.eu/EFSA/efsa_locale1178620753812_1211902314590.htm (page consulted 30/11/2009). EFSA, Opinion of the Scientific Panel on Plant Protection products and their Residues to evaluate the suitability of existing methodologies and, if appropriate, the identification of new approaches to assess cumulative and synergistic risks from pesticides to human health with a view to set MRLs for those pesticides in the frame of Regulation (EC) 396/20051, Scientific Opinion of the Panel on Plant Protection Products and their Residues (PPR) (Question N EFSA-Q-2006-160), adopted on 15 April 2008 [online] 2008c. http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1178712607885.htm (page consulted 01/03/2010). EFSA, Marine biotoxins in shellfish Saxitoxin group, Scientific Opinion of the Panel on Contaminants in the Food Chain. (Question No EFSA-Q-2006-065E), adopted on 25 March 2009 [online] 2009a. http://www.efsa.europa.eu/EFSA/efsa_locale1178620753812_1211902452476.htm (page consulted 30/11/2009). EFSA, Marine biotoxins in shellfish Pectenotoxin group. Scientific Opinion of the Panel on Contaminants in the Food chain. (Question No EFSA-Q-2006-065C), adopted on 27 May 2009 [online] 2009b. http://www.efsa.europa.eu/EFSA/efsa_locale1178620753812_1211902599809.htm (page consulted 30/11/2009).

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EFSA, Marine biotoxins in shellfish Domoic acid. Scientific Opinion of the Panel on Contaminants in the Food Chain, (Question No EFSA-Q-2006-065H), adopted on 2 July 2009 [online] 2009c. http://www.efsa.europa.eu/EFSA/efsa_locale1178620753812_1211902707355.htm (page consulted 30/11/2009). EFSA, Influence of processing on the levels of lipophilic marine biotoxins in bivalve molluscs, Statement of the Panel on Contaminants in the Food Chain, (Question No EFSA-Q-200900203), adopted on 25 March 2009 [online] 2009d. http://www.efsa.europa.eu/EFSA/efsa_locale1178620753812_1211902424332.htm (page consulted 30/11/2009). EFSA, Marine biotoxins in shellfish Summary on regulated marine biotoxins, Scientific Opinion of the Panel on Contaminants in the Food Chain, (Question No EFSA-Q-2009-00685), adopted on 13 August 2009 [online] 2009e. http://www.efsa.europa.eu/EFSA/efsa_locale1178620753812_1211902812884.htm (page consulted 30/11/2009).

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