EFSA Releases Expert Opinion on Marine Biotoxin Testing

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EFSA Releases Expert Opinion on Marine Biotoxin Testing

Sherry Ward, Contributing Editor

Published: October 23, 2008

The European Food Safety Authority (EFSA) released a report October 8 from the scientific panel evaluating the safety testing for Azaspiracid shellfish biotoxins (AZAs) in the European Union (EU) (EFSA, 2008). The EFSA experts concluded that an analytical method, which is already being used in Germany, is a potential replacement for the animal test method currently required by European Union (EU) regulatory authorities.

The AZAs are one of a number of marine biotoxins found worldwide that are toxic to humans when marine organisms, primarily shellfish, are consumed as food. Marine biotoxins are produced by marine algae, and are concentrated in the shellfish during the process of filter feeding. AZAs were recently found to be produced by marine dinoflagellates. Consumption of AZA-contaminated shellfish can result in nausea and other gastrointestinal symptoms. Some marine biotoxins cause neurological symptoms, including paralysis and even death. Information on the acute toxicity of AZAs is limited.

The mouse or rat LD50 assay (bioassay) is the official method for monitoring AZAs in shellfish in the EU. The mouse bioassay involves injection of an extract of the potentially contaminated marine organism tissue into the abdomen of the rodent followed by monitoring for death over several days. The EFSA panel “noted that both [animal] methods have shortcomings e.g. they are not specific and not quantitative, and that method performance characteristics for AZAs have not been established for the mammalian assays” (EFSA, 2008). The EFSA report also indicated that the animal bioassay is not sufficiently sensitive in detecting the toxin for the protection of human health.

A press release by the Dr Hadwen Trust noted that hundreds of animals per bioassay are subjected to severe suffering for testing marine biotoxins, and it referenced a Parliamentary Question response stating “a total of 6,468 animals were used in the relevant procedures during 2004 [in the UK].”

Scientists have been investigating the use of alternative methods for detecting marine biotoxins. However, alternatives to the animal bioassay for AZAs have not been sufficiently evaluated in the type of inter-laboratory studies needed to demonstrate their scientific validity. The EFSA concluded that an instrumental method called liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) has demonstrated the greatest potential as a replacement for the animal bioassay. The LC-MS/MS methods are more sensitive than the LD50 assay in detecting AZAs, and can also be used simultaneously to detect the presence of other types of marine biotoxins.

Dr. Horst Spielmann, former Director of the Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET) in Germany commented that “the positive evaluation of in vitro alternatives to the mouse bioassay by the EFSA is a major scientific and regulatory breakthrough for alternatives.” He explained that ZEBET and the European Center for the Validation of Alternative Methods (ECVAM) as well as the animal welfare movement in Europe have fought for the past six years to reach this goal.

Germany uses sensitive analytical methods rather than the mouse bioassay for marine biotoxin detection. In 2004, the EU Commission initiated official action against Germany for not conducting the official EU mouse bioassay. The chemicals regulatory authority in Germany, the Federal Institute for Risk Assessment (BfR), released a scientific opinion paper on marine biotoxin methods in 2005, which “recommends using chemico-physical analyses like the LC/MS method as the reference method for the determination of algae toxins in mussels” (BfR, 2005). Their approach is to first test for marine biotoxins using the analytical methods, and then use the mouse bioassay only if those results do not provide a clear answer that protects the public health. The BfR began advocating in 2005 for the EFSA to replace “the mouse bioassay with the LC/MS method as the reference method because it offers greater consumer protection and, at the same time, contributes to animal welfare” (BfR, 2005).

Reports from ECVAM and the World Health Organization (WHO) have also condemned the mouse bioassay for marine biotoxin testing (Hess, 2006; WHO, 2004). WHO recommended that member states develop and validate improved biotoxin test methods. WHO’s member states extend beyond the EU, so other countries are expected to follow EU efforts to replace this animal test.

Researchers in Spain have developed in vitro cell-based assays that can be used to detect marine biotoxins. At the recent ESTIV 2008 meeting, Caillaud, et al. (2008) reported that cell viability assays “proved efficient to quantify the potency of numerous marine toxins.”

A previous AltTox.org In the Spotlight article described New Zealand researchers’ development of a human protein-based assay for shellfish toxin testing as an alternative to the mouse bioassay.

The take home message, according to Dr. Spielmann, is that “eating shellfish is safe in Europe without conducting the mouse bioassay.” Safety testing for marine biotoxins provides a good example of how animal alternative methods are starting to be considered for regulatory testing purposes. The in vitro methods are the better scientific approach, and are more sensitive than the mouse bioassay.

References
BfR. (2005). Mouse bioassay not suitable as a reference method for the regular analysis of algae toxins in mussels (BfR Expert Opinion No. 032/2005 of 2005-05-26). Available here.

Caillaud, A., Cañete, E., de la Iglesia, P., et al. (2008). In vitro cell toxicity evaluation for seafood risk assessment. Abstract. ESTIV 2008. Available here.

Dr Hadwen Trust. (2008). Dr Hadwen Trust welcomes European Food Safety Authority report (Press Release, Oct. 9, 2008). Available here.

EFSA. (2008). Marine biotoxins in shellfish – Azaspiracid group – Scientific Opinion of the Panel on Contaminants in the Food Chain. Available here.

Hess, P., Grune, B., Anderson, D.B., et al. (2006). Three Rs Approaches in Marine Biotoxin Testing. ECVAM Workshop Report 55. Altern. Lab. Anim. 34, 193-224.

WHO (2004). Report of the Joint FAO/IOC/WHO ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs. Oslo, Norway, Sept. 26-30, 2004. Available here.