Symptoms of ASP include gastrointestinal symptoms (vomiting, diarrhoea or abdominal cramps) and/or neurological symptoms (confusion, loss of memory, or other serious signs such as seizure or coma) occurring within 24-48 hours after consuming contaminated shellfish.
DA is a water-soluble cyclic amino acid mainly produced by marine red algae of the genus Chondria and diatoms of the genus Pseudo-nitschia. The first confirmed outbreak of ASP occurred in Canada in 1987 and was related to mussels affected by a bloom of the Pseudonitzschia f. multiseries.
DA isomers have also been detected in shellfish in the United States and in a number of European countries. Although several isomers of DA (diastereoisomer epi-domoic acid (epi-DA) and isodomoic acids (iso-DAs)) have been identified data on the occurrence only of DA and epi-DA (expressed as sum DA) have been reported.
The toxicological database for DA is limited, comprising mostly studies on the acute toxicity in rodents and Cynomolgus monkeys following administration by parenteral routes and with few studies with oral administration. Neurotoxicity is the critical toxicological effect identified in experimental animals as well as in humans. The toxic effects of DA are mediated through its high affinity binding and agonist activity on some forms of glutamate receptors particularly in certain regions (e.g. hippocampus) of the brain. The few data available indicate that Cynomolgus monkeys are more sensitive than rodents.
Following oral administration DA shows low systemic absorption. Absorbed DA is rapidly cleared from the body by renal excretion and impaired renal function increases the susceptibility to DA.
Data on the genotoxicity of DA are inconclusive. No data on the chronic effects of DA in animals are available, therefore the Panel on Contaminants in the Food Chain (CONTAM Panel) could not establish a tolerable daily intake (TDI). In view of the acute toxicity of DA the CONTAM Panel decided to establish an acute reference dose (ARfD) based on the available human data on acute toxicity.
The few data on exposure to DA associated with adverse effects in humans (9 individuals) in ASP outbreaks indicate that severe and irreversible effects occurred at about 4 mg/kg bodyweight (b.w.) and the lowest-observed-adverse-effect-level (LOAEL) for mild signs and symptoms was 0.9 mg/kg b.w. Although the acute oral toxicity is not well characterised, the CONTAM Panel considered it appropriate to base the establishment of the ARfD on this LOAEL.
Taking into account the steep dose-response relationship, the CONTAM Panel decided to apply a factor of 3 for extrapolation from a LOAEL to a no-observed-adverse-effect level (NOAEL). The CONTAM Panel concluded that a factor of 10 should be applied to allow for human variability and also for fact that sensitive methods for the detection of neurotoxic effects had not been used in the investigation of affected individuals.
The CONTAM Panel therefore established an ARfD of 30 µg DA/kg b.w. by applying the overall uncertainty factor of 30 to the LOAEL of 0.9 mg/kg b.w. Because DA can be converted to epi-DA during storage, the ARfD applies to the sum of DA and epi-DA.
In order to ensure protection against the acute effects of DA, it is important to use a large portion size rather than the long-term average consumption in the health risk assessment of shellfish consumption. Consumption data for shellfish species across the European Union (EU) were limited, therefore the European Food Safety Authority (EFSA) requested the Member States to provide information on consumption of relevant shellfish species. Based on data provided by five Member States, the CONTAM Panel identified 400 g of shellfish meat as a large portion size to be used in the acute risk assessment of marine biotoxins.
Consumption of a 400 g portion of shellfish meat containing DA and epi-DA at the current EU limit of 20 mg DA/kg shellfish meat would result in a dietary exposure of 8 mg DA (equivalent to about 130 µg DA/kg b.w. for a 60 kg adult). This is about four times higher than the ARfD of 30 µg DA/kg b.w. (equivalent to 1.8 mg DA per portion for a 60 kg adult) and is considered to constitute a potential health risk. Based on current consumption and occurrence data there is a chance of about 1 per cent of exceeding the ARfD of 30 μg DA/kg b.w. when consuming shellfish currently available on the European market.
The CONTAM Panel concluded that in order for a 60 kg adult to avoid exceeding the ARfD of 30 µg DA/kg b.w. a 400 g portion of shellfish should not contain more than 1.8 mg DA corresponding to 4.5 mg DA/kg shellfish meat. Of the currently available occurrence data for samples in compliance with the EU regulatory limit of 20 mg/kg shellfish meat, 3.5 per cent exceed this value of 4.5 mg/kg shellfish meat.
DA is heat stable and cooking does not destroy the toxin, although normal home cooking processes, such as boiling and steaming, could reduce the amount of DA in shellfish meat due to partial leaching of the toxin into the cooking fluids. In scallops, redistribution of DA from the hepatopancreas into the other tissues could occur. For other types of shellfish it is unlikely that processing would have a major effect on the DA concentration in shellfish meat.
Several analytical methods are available for the determination of DA and its isomers. High performance liquid chromatography-ultraviolet detection (HPLC-UV) methods are widely used. One HPLC-UV method has been validated (AOAC method 991.26) and standardised (CEN method 14176).
In addition, a method based on enzyme-linked immunosorbent assay (ELISA) has been validated (AOAC method 2006.02) and is officially allowed to be used in the EU for screening purposes. These methods have limits of detection that are sufficiently low to adequately detect DA at the concentration of 4.5 mg/kg shellfish meat, for which consumption of a large portion would not lead to the ARfD being exceeded.
The available evidence suggests that liquid chromatography-mass spectrometry (LC-MS), particularly using electrospray ionisation in the tandem mode, can also be a valuable tool for rapid and selective determination of DA and its isomers in crude extracts. Other chemical methods have been developed but they are only rarely used and they have not been validated in interlaboratory studies.
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