EFSA Risk Assessment Biotoxinas

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1ICMSS09 – Nantes, France – June 2009

www.symposcience.org

European risk assessments of marine biotoxins

Benford Diane J.1 , Eskola Mari

2 , van Leeuwen Rolaf

3

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, Italy3 National Institute for Public Health and the Environment (RIVM), Bilthoven, The

Netherlands

* Corresponding author: [email protected] , 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 requestfrom the European Commission. Opinions have been adopted on theokadaic acid-, azaspiracid-, yessotoxin-, saxitoxin- and pectenotoxin-group toxins and on domoic acid. EFSA followed the establishedparadigm for toxicological risk assessments. Due to the lack of long termtoxicity studies it was not possible to establish tolerable daily intakes(TDIs) for any of these marine biotoxins. However because they exhibitacute toxicity, it was considered appropriate to establish acute referencedoses (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, andassuming that this amount of toxin could be contained in a single largeportion of shellfish, EFSA advised on concentrations of toxin in shellfishthat would not result in risks to the consumer. EFSA also proposedtoxicity equivalency factors (TEFs) to allow the toxicity of the differentanalogues within a toxin group to be summed, but noted that moreinformation is needed on the relative toxicity by the oral route for relevanttoxins in each group in order to establish robust TEFs. The EFSA

assessments suggest that consumption of shellfish containing the okadaicacid-, azaspiracid- and saxitoxin-group toxins and DA at the current EUregulatory 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

ce of processing on the levels of lipophilicEFSA statement on the influen

1

Biotoxin working group members: Jan Alexander, Tore Aune, Diane Benford,Luis Botana, Peter Fürst, Gerhard Heinemeyer, Philipp Hess, Sophie Krys, Angelika Preiss-Weigert, Hans van Egmond, Rolaf van Leeuwen (chair), PhilippeVerger.

EFSA staff: Mari Eskola, Stefan Fabiansson, Claudia Heppner, FrancescoVernazza

mailto:[email protected]

mailto:[email protected]





marine biotoxins in bivalve molluscs and a summary of the opinions on thecurrently regulated marine biotoxins have also been published.

Keywords: Marine biotoxins, shellfish, okadaic acid, azaspiracid,yessotoxin, saxitoxin, domoic acid, acute reference dose

IntroductionThe 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 inresponse to a request from the European Commission (EC) to assess the current EuropeanUnion (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 remainingopinions 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) hazardcharacterisation, (iii) exposure assessment, and (iv) risk characterisation. In line with theapproach to other types of chemicals in food, the aim is to establish a health-basedguidance value as an output of steps (i) and (ii) and then to compare it with estimatedexposure 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 thedose 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 anuncertainty factor, which is commonly the multiple of:

- 10 to allow for possible greater sensitivity of humans, if the key data are derivedfrom 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 “theamount, expressed on a body weight basis, that can be ingested daily over a lifetimewithout appreciable health risk”. For substances that exhibit acute toxicity, an acutereference 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





period of 24 hours or less without appreciable health risk”. If relevant human data areavailable, 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 inindividuals who became ill, such as whether shellfish available for analysis was

representative of that consumed, the impact of cooking on the toxin levels, the analyticaltechniques 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 weremostly 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 theamount and quality of the data available. For the YTX-group and PTX-group toxins, onlylimited data in laboratory animals were available. Table 1 shows the derivation of theARfDs for the currently regulated marine biotoxins.

Table 1. ARfDs established by the CONTAM Panel for the regulated biotoxins

Toxin group Endpoint NOAEL/LOAEL (μg/kg b.w.) ARfD (μg/kg b.w.)

AZA Gastrointestinal 1.9 (human LOAEL) 0.2 AZA1 eq.

OA Gastrointestinal 0.8 (human LOAEL) 0.3 OA eq.

STX Neurotoxicity 1.5 (human LOAEL) 0.5 STX eq.

PTX Intestinal 250 (mouse LOAEL) 0.8 PTX2 eq.

YTX Cardiotoxicity 5000 (mouse NOAEL) 25 YTX eq.

DA Neurotoxicity 900 (human LOAEL) 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 tothe total toxin content. If the analogues exert toxicity by the same mode of action, then itis 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 1for PTX1, PTX2, PTX3, PTX4, PTX6 and PTX11. More information is needed on the





relative toxicity by the oral route for relevant toxins in each group in order to establishrobust 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 needfor 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 Azaspiracids Saxitoxins Yessotoxins

OA =1 *

AZA1 = 1 * STX = 1 * YTX = 1 *

DTX1 = 1 AZA2 = 1.8 NeoSTX = 1 45-OH-YTX = 1

DTX2 = 0.6 AZA3 = 1.4 GTX1 = 1 1a-homoYTX = 1

DTX3 = as for AZA4 = 0.4 GTX2 = 0.4 45-OH-la-homoYTX = 0.5

unesterified parenttoxin (OA, DTX1 or

DTX2) AZA5 = 0.2 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

3. Exposure assessment

Dietary exposure assessment needs to take into account the levels of toxins present inshellfish and the amount of shellfish that is consumed. The occurrence data were

submitted by European Countries in response to a request from EFSA. Consumption datawere available from national dietary surveys conducted in France, Germany, Italy, the Netherlands and the United Kingdom. Because the toxins have acute effects arising froma 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





shellfish to not exceed the ARfD for the relevant toxin. Based upon these comparisons itcan 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 thecurrent 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 STX-group toxins, these exposures are respectively 4-fold and 10-fold higher than the ARfDsand 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 inexposures 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 referencemethod for the analysis of the currently regulated marine biotoxins in shellfish, with theexception of DA. However, the bioassays have limited capability to detect OA-, AZA-and 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 theconsumption 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 YTX-group toxins. In addition there are animal welfare issues related to the use of thesemethods.

Table 3. Comparison of the estimated exposures to the biotoxins with the ARfDs

ToxinConcentration of toxin

(μg/kg shellfish)

Intake

(μg toxin per

400g portion)

Intake

(μg toxin per

kg bw)

Approximate

comparison with the

ARfD

OA-

group

160 (EU limit value)240 (95th percentile)

45 (based on ARfD)

6496

18

11.6

0.3

3-times higher 5-times higher

At the ARfD

AZA-

group

160 (EU limit value)40 (95th percentile)

30 (based on ARfD)

6416

12

10.25

0.2

5-times higher Slight exceedance

At the ARfD

STX-

group

800 (EU limit value)75 (based on the ARfD

32030

5.30.5

10-times higher At the ARfD

YTX-

group

1000 (EU limit value)315 (Norway) 799 (Italy)

(95th percentile)3750 (based on ARfD)

400126 (Norway)

320 (Italy)1500

6.72.1 (Norway)

5.3 (Italy)25

4 times lower 10 times lower

5 times lower At the ARfD

PTX-

group

160 (EU limit value *)79 (95th percentile)

120 (based on ARfD)

6432

48

10.5

0.8

Slight exceedanceBelow the ARfD

At the ARfD

DA

20,000 (EU limit value)

2500 (95th percentile)4500 (based on ARfD)

8000

10001800

130

1730

4-times higher

Half of the ARfDAt the ARfD

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 adifferent mechanism of toxicity and should be considered separately.





The CONTAM Panel reviewed the alternative methods under development in order toadvise 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 canoccur.

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 toxinsand DA at the current EU regulatory limits could cause illness in some consumers. Incontrast, for YTX-group toxins, the evidence shows no concern for consumer health atlevels up to about 4 times higher than the maximum levels. It is now for the EuropeanCommission, in consultation with Member States, to consider whether the maximumlevels should be revised in view of the EFSA opinions. The remaining EFSA evaluations

are due to be completed by the end of 2009.

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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), adoptedon 13 August 2009 [online] 2009e. http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1211902812884.htm (page consulted 30/11/2009).



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