Mouse bioassay detection method

It is obvious from the provisional risk assessment values for microcystins, and, being of the same order of magnitude of mammalian toxicity, similar values may be calculated for the cyanobacterial neurotoxins, that sensitive detection methods are required to detect these low concentrations of toxins. Of the biological methods of detection discussed earlier, the mouse and invertebrate bioassays are not sensitive enough without concentration of water samples, in that they are only able to detect mg of microcystins per litre. Only the immunoassays (ng-/rg 1 and the protein phosphatase inhibition assays (ng O... 

Some Chemical Considerations Relevant to the Mouse Bioassay. Net toxicity, determined by mouse bioassay, has served as a traditional measure of toxin quantity and, despite the development of HPLC and other detection methods for the saxi-toxins, continues to be used. In this assay, as in most others, the molar specific potencies of the various saxitoxins differ, thus, net toxicity of a toxin sample with an undefined mixture of the saxitoxins can provide only a rough approximation of the net molar concentration. Still, to the extent that limits can be placed on variation in toxin composition, the mouse assay can in principle provide useful data on trends in net toxin concentration. However, the somewhat protean chemistry of the saxitoxins makes it difficult to define conditions under which the composition of a mixture of toxins will remain constant thus, attaining a reproducible level of mouse bioassay toxicity is difficult. It is therefore useful to review briefly some of the chemical factors that should be considered when employing the mouse bioassay for the saxitoxins or when interpreting results. Similar concepts will apply to other assays. 

Mouse Bioassay. The mouse is the traditional animal of choice for detecting biological activity due to STX and TTX. Mice receive an intraperitoneal injection of sample and are observed for symptoms of intoxication, i.e., dypsnea, convulsions, and death. This method is effective for detecting biological activity of STX and TTX in numerous samples. For the standard STX assay, one mouse unit is defined as that quantity of STX injected i.p. in 1 ml solution that will... 

Experimental evidence indicates that many marine bacteria produce TTXs. However, TTX production by some bacteria has not been validated since TTX and anhydro-like TTX are described as "difficult to detect" by using HPLC and GC-MS methods, and show no activity in the mouse bioassay. 

An improved high pressure liquid chromatographic (HPLC) procedure Tor the PSP toxins is described. The method involves separation of the toxins on a polystyrene divinylbenzene resin colunn (Hamilton, PRP-1) in the reversed phase mode using heptane and hexane sulfonic acids as ion-pairing reagents. Detection of the toxins is by fluorescence following post-column alkaline periodate oxidation. The sensitivity of the HPLC method is better than the standard mouse bioassay by at least a factor of four for each of the individual toxins. 

European regulations set the limit for azaspiracid toxins content in shellfish that is to be destined to human consumption in 160 pg of azaspiracid eqnwalents/kg in the whole body or any part edible separately (EU 2002). This limit has a higher valne than the NOEL to allow for detection by mouse bioassay, the most commonly nsed method due to the lack of standards for chemical analysis. 

A series of mouse bioassay procedures (biological methods), differing in the test portion (hepat-opancreas or whole body) and in the solvents used for the extraction and purification steps, can be used for detection of the toxins DSR Sensitivity and selectivity depend on the choice of the solvents used for the extraction and purification steps, and this should be taken into account when making a decision on the method to be used, in order to cover the full range of toxins. 

Mouse bioassay is the current detection method for most of the marine toxins, with the exception of domoic acid. The reason for this is that it has been protecting consumers for the past decades, ever since monitoring systems were implemented in many countries. Therefore, food safety authorities are reluctant to allow replacement of the monse bioassay, unless a solid evidence is provided to sustain current levels of consumer protection. This evidence is legally sustained in Europe by the requirement of an internationally recognized validation study for any method that could be an alternative to the bioassay. " A recent ontbreak of mice toxicity in bivalves took place in Arcachone, France, and the source of this toxicity was unknown. This was a very good example of universal... 

The fluorimetric method of Bates and Rapoport [8], based on the oxidation of PSP toxins in alkaline conditions to form fluorescent derivatives, was incorporated into a detection method with the PSP toxins separated in a chromatographic column by Buckley et al. [17]. This method set the basis for the development of a high pressure liquid chromatography with postcolumn reaction system that was subsequently improved to achieve a better toxin separation and adequate sensitivity [18]. Sullivan et al. [ 19] evaluated its applicability to shellfish toxicity monitoring, by comparing the results obtained by the HPLC method and the standard Association of Official Analytical Chemists (AOAC) mouse bioassay. They found, in general, a good correlation between the two methods. However, Cl and C2 toxins could not be separated and individually quantified. Further improvements and modifications... 

Implementation of new methods in regulatory work faces several critical difficulties. Among them, lack of certified reference materials, QC/QA protocol application and pending validation studies hinder the transformation of good ideas into official methods for practical regulatory work. For example, Inami et al. recently compared two immunoassays and a 5 h neuroblastoma assay with the MBA for presence/absence detection of saxitoxins in shellfish. The study concluded that a reduction in the number of mouse bioassays in the state of California could be achieved, provided that the alternative assays were applied as a screening tool. 

Consequently, a new ofQcial procedme for analyzing seafood that would be capable of separating YTXs and DSP-toxins in distinct layers (Figure 13.9) was set up. In fact, the old ofQcial scheme did not allow any separation between YTXs and DSP-toxins therefore the mouse bioassay—the reference method in Europe to detect toxicity in seafood—could not lead to any confident assessment of the toxin(s) involved. The new method, however, presents some weak points in particular, based on the difference of solubility between YTXs and OA, it fails when the extract to be analyzed contains less hydrophilic YTXs such as their desulfo derivatives, which are being recovered in the OA-containing fraction. 

On the basis of the scientific literature, the use of the mouse bioassay as a method of reference for marine biotoxins is no longer considered appropriate. Relevant international expert publications have reported the reliability of in vitro detection methods, which can be used as alternatives to animal experiments. Also, the European Commission has recognized the needs of the analytical community to develop methods alternative to animal testing. " ... 

In recent years a number of biochemical and chenucal detection methods alternative to mouse bioassay have been developed. Although the biochemical assays are excellent for screening out... 

Owing to the difficulties in distinguishing between DSP toxins and pectenotoxins by the traditional mouse bioassay, several chemical analyses have been developed. These methods are based on liquid chromatography (LC) with detection by ultraviolet (UV) absorption [4-7,20,25,26], fluorescence (FL) [6,7,9-11,25-27], andmass spectrometry (MS) [5-8,10-21,23,24,28-36]. Amajor drawback of these LC techniques is the lack of analytical standards. In the present chapter, chemistry, metabolism, and chemical detection methods of pectenotoxins are described. Pharmacology and toxicology of pectenotoxins will be discussed in other chapters. 

Garthwaite, L, et al., Comparison of immunoassay, cellular, and classical mouse bioassay methods for detection ofneuro toxic shellfish toxins, m Immunoassays for Residue Analysis, Beier, R. and Stanker, L., Eds., American Chemical Society, Washington DC, 19196, pp. 404 12. 

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