Shellfish toxin testing without using animals

Shell

The freedom to enjoy a feed of shellfish has been an important part of New Zealanders' culture since humans first arrived here. Shellfish also play an important part in our economy, generating hundreds of millions of export dollars. In certain conditions, naturally occurring toxins in the sea can be picked up by shellfish. While not harmful to the shellfish, they can be poisonous to people who eat them. Testing for these toxins is therefore vitally important for the protection of human health and our export markets. Large numbers of mice have had to be used for these tests, but a New Zealand-led development could see the use of animals for shellfish toxin testing phased out completely.

There are several marine phytoplankton in New Zealand that can produce toxic compounds. These phytoplankton can bloom under favourable environmental conditions and the biotoxins concentrated by shellfish as they filter feed. People can become seriously ill if they eat shellfish that are sufficiently contaminated with these biotoxins.

Toxic terminology

  • Biotoxin: a poisonous compound produced by a living thing.
  • Phytoplankton: the microscopic plants making up some of the plankton in our oceans.
  • Bioassay: a test that uses a living animal or animal organs. A mouse bioassay uses mice or a preparation of mouse tissue.

There are eight toxin groups of concern (based on chemical structure) and each toxin group contains multiple toxin analogues (chemicals with a similar structure but slightly different composition). They produce a range of symptoms, including vomiting, headaches, diarrhoea, neurological problems and, in extreme cases, death.

Biotoxin problem relatively recent

Prior to 1993, New Zealand had no recorded incidence of shellfish biotoxins of public health significance. Following a significant outbreak of poisoning cases in 1993, a comprehensive national management plan was implemented, including the routine monitoring of shellfish for marine biotoxins.

Many other countries have adopted similar monitoring programmes, in order to protect public health. Most of these countries stipulate the use of the mouse bioassay for detecting the presence of marine biotoxins in shellfish tissues. New Zealand does not require the use of the mouse bioassay for testing all marine biotoxins. In fact, New Zealand is leading the world in the adoption of improved non-mouse test methods.

Shortcomings of mouse bioassay

The mouse bioassays used to detect and quantify marine biotoxins are non-validated procedures, which are prone to interferences, and entail the use of large numbers of animals. In New Zealand, this testing is considered to fall into the 'very severe suffering' category. Nearly 12,000 mice were used in this category in 2005. Of these, 77.6 percent were used for testing, including shellfish toxin testing. Essentially, the test requires administration of a shellfish extract to the mice.

It is accepted internationally that the mouse bioassays for the diarrhetic (producing diarrhoea) and paralytic (producing paralysis) shellfish poison groups (DSP and PSP) are also deficient for various reasons, animal welfare impacts notwithstanding. For example, the mouse bioassay for the DSP toxins is not consistently able to meet the regulated limit for DSP toxins in shellfish.

Various types of non-animal-based methods for the detection of toxins have been developed and validated world-wide. These involve direct measurement of the level of toxin in extracts of shellfish using several new methods. In addition, monitoring for phytoplankton levels and environmental conditions for potential blooms has been enhanced and is often used in concert with the other tests.

World first

In 2000 and 2001, the Cawthron Institute (Nelson, New Zealand) developed, validated and implemented a liquid chromatography mass spectrometry (LCMS)-based method for the detection of 17 toxins in four of the eight toxin groups. In 2001, the New Zealand Food Safety Authority (NZFSA) approved the Cawthron LCMS method for regulatory use in New Zealand – this was the first such approval issued world-wide. The LCMS method has proved to be faster, more sensitive, and more specific than the mouse bioassay. The introduction of this LCMS method in New Zealand has seen a significant reduction in the number of mouse bioassays being undertaken (see Figure 1).

While New Zealand has developed and implemented alternative testing methods, progress in many other countries has taken a different tack. Changes in the United Kingdom in recent years have mainly focused on reducing the number of animals used for each test and the refinement of the mouse bioassay so that animals suffer less (by the use of euthanasia at an earlier stage in the test). Within the EU, only Germany has abandoned the mouse bioassay for testing shellfish. A workshop organised by the European Commission in 2005 recommended that: "Since non-animal methods providing equivalent or better public health provision are available, these can serve as monitoring methods, which should be used by the competent authorities, thereby leading to a significant reduction in animal tests."

International endorsement for non-animal method

During a recent international conference on molluscan shellfish safety in Blenheim, a round table discussion on international regulatory changes in marine biotoxin standards was held with key experts from Europe, Canada, the United States and New Zealand. A unanimous outcome of this session was that non-animal methods are validated and available for use and that these should be implemented on a country-by-country basis.

There are several obstacles to the implementation of these methods:

  • lack of harmonised validation criteria for judging method acceptability
  • limited availability of certified reference materials
  • lack of cohesion between regulators and scientists
  • lack of toxic equivalence factors (a means of comparing the toxicity of very different compounds) for calculating the potency of toxin analogues.

In New Zealand, the export focus of New Zealand's shellfish industry requires NZFSA to ensure compliance with the regulations of importing nations. Nonetheless, the NZFSA has a strong commitment to the implementation of non-animal-based testing methods.

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Graph
Number of mice used per year in support of
the New Zealand marine biotoxin monitoring.
Graph kindly provided by Paul McNabb, Cawthron
Institute, Nelson, New Zealand.

Page last updated: 30 April 2008