​Study to investigate the effect of General Anaesthesia on the Paralytic Shellfish Poisioning (PSP) Bioassay

Summary

Biomathematics and Statistics Scotland, Rowett Research Institute
Holtrop, G ;
FRS Marine Laboratory
Petrie, J;
Rowett Research Institute
Dennison, N

1.1 Background

Currently shellfish toxin monitoring for Paralytic Shellfish Poison (PSP) in Europe requires the use of the mouse bioassay as described in European Union Directive 91/492/EEC laying down the health conditions for the production and the placing on the market of live bivalve molluscs. The Directive stipulates that the limit for total PSP must not exceed 80 mg/100g shellfish flesh.

The extract from shellfish is injected into the unanaesthetised (conscious) mouse and the time of death is monitored. The time of death is translated into toxicity of the shellfish sample using a conversion table (Sommers table), which was created almost 70 years ago during the development of the mouse bioassay to calculate PSP toxin levels (Sommer and Meyer (1937)).

The ethical issues associated with the mouse bioassay have placed increasing pressure on regulatory bodies to develop and validate alternative analytical and in vitro methods for detecting PSP in shellfish. However, to date, no single chemical test has been approved to replace the mouse bioassay for the monitoring of shellfish for PSP in Europe.

The Food Standards Agency (FSA) and the Home Office both recognise that the PSP bioassay is of particular concern relative to animal welfare and therefore aim to replace this method with one which minimises animal suffering. In the absence of a validated chemical test, a refinement of the current mouse bioassay was investigated in which test animals were anaesthetised prior to being injected with toxic sample.

An initial pilot study was performed, prior to this research project, to identify a suitable method of anaesthesia. This identified that the combination of fentanyl/ fluanisone/ midazolam, via the intraperitoneal route, was the preferred method of anaesthesia to investigate the aim of this project, which is whether PSP monitoring in shellfish on anaesthetised mice was as effective as the current testing scheme, which employs conscious (unanaesthetised) mice.

The project was split into two parts

a) The Calibration study. Known concentrations of saxitoxin (this is a positive reference standard for PSP and one of the components of the PSP toxin complex) were administered to both unanaesthetised and anaesthetised mice to investigate whether the response was altered by the anaesthesia. Calibration curves, relating time of death with toxicity (concentration of PSP toxin), were constructed for both unanaesthetised and anaesthetised mice. This study allowed comparison between three different methods:

1. The time of death of an unanaesthetised mouse was translated into toxin concentration using Sommers Table (current method);
2. The time of death of an unanaesthetised mouse was translated into toxin concentration using the newly developed calibration curve for unanaesthetised mice (calibration method):
3. The time of death of an anaesthetised mouse was translated into toxin concentration using the newly developed calibration curve for anaesthetised mice (anaesthesia method).

b) The monitoring sample study. Routine shellfish samples from FSA Scotland’s shellfish monitoring programme were tested on anaesthetised and unanaesthetised mice to evaluate whether the anaesthesia method could be used for routine monitoring of shellfish samples. The majority of the shellfish samples were also analysed by high performance liquid chromatography (HPLC) to provide further information on the PSP toxin profiles of the samples tested.

1.2. General Conclusions

a) Conclusions from the calibration study

• Anaesthesia caused a delay in the time of death.
• For unanaesthetised mice, not all mice died, even at high toxin concentrations.
• The time of death shows a decrease with higher toxin concentrations. The relationship between time of death and toxin concentration is different for unanaesthetised and anaesthetised mice. Therefore, two calibration curves were developed; one for unanaesthetised and one for anaesthetised mice.
• The validity of the current method in unanaesthetised mice was investigated by comparing results from the new calibration curve against those of the current method. It was found that, for high PSP concentrations, the current method used in the FSA Scotland monitoring programme could underestimate toxicity by up to 50%.
• The predicted toxin concentration was variable for all three methods (current method, calibration method and anaesthesia method). e.g. when a saxitoxin concentration of 80mg/100g shellfish was tested, the estimated toxicity ranged from 40mg/100g to 150mg/100g (where 40 is only just above the limit of detection, 80mg/100g is closure limit, and 150mg/100g is close to the toxin concentration that would cause illness in consumers). This variation was similar for all three methods.

b) Conclusions from the monitoring sample study.

• Although sufficient numbers of field samples were collected to allow for full assessment of the performance of anaesthesia in non-toxic and low-toxic samples, unfortunately, there were too few PSP positive samples in Scotland in 2002-2003 to allow a full statistical validation of anaesthesia in positive samples in this study.
• It was difficult to evaluate the suitability of using anaesthesia in PSP monitoring by comparison to the method currently in use, due to the inherent variability in the mouse bioassay.
• The current method and HPLC method tend to give similar monitoring toxicity estimates.
• The estimated toxicities from the calibration method and anaesthesia method tend to be higher than from the current method. Toxicities estimated from the anaesthesia method also exceed those based on the HPLC method.
• The calibration method (unanaesthetised mice) and anaesthesia method tend to give similar monitoring toxicity estimates, suggesting that use of anaesthetised mice is comparable with use of unanaesthetised mice.
• The calibration curves developed in the present study (calibration method and anaesthesia method) are likely to give more accurate toxicity estimates than the current method. This is because the newly developed calibration curves reflect current practice with respect to laboratory conditions and mouse breed, whereas the current method is based on practices that were in place almost 70 years ago.
• When the anaesthesia method is compared with the current method: when the two methods disagree on a monitoring open or monitoring closed decision, the anaesthesia method always results in monitoring closure. Results to date therefore suggest that the use of the anaesthesia method would protect the safety of the consumer.

Project Code: S02012