Paralytic shellfish poisoning (PSP) is a serious and sometimes fatal outcome of the consumption of seafood contaminated with saxitoxin and its congeners, which are produced by marine dinoflagellates of the genera Alexandrium
and by several genera of freshwater cyanobacteria [1
]. The geographic distribution of PSP-inducing organisms is increasing, and on a global scale, around 2000 cases of PSP are reported each year, with a mortality rate of 15% [3
For many years, evaluation of the safety of seafood for human consumption has been based on a mouse bioassay (MBA), which involves intraperitoneal injection of an extract of the seafood in mice, with death as the endpoint. This assay has been approved as a reference method for paralytic shellfish toxins by the Association of Official Analytical Chemists [4
]. Such an assay is, however, deemed by many to be ethically unacceptable and, further, its validity is questionable since it involves intraperitoneal injection rather than the oral route through which humans are exposed to the PSP toxins. The use of the MBA is now being phased out in several countries, and alternative chemical and functional assays for the paralytic shellfish toxins have been subjected to interlaboratory validations and approved by AOAC following review. These include two HPLC fluorescence methods [5
], one using pre-column oxidation (AOAC 2005.06) and the other using post-column oxidation (AOAC 2011.02). Both of these methods allow quantitation of individual saxitoxin analogues present in a sample. A receptor binding assay has also been validated and approved (AOAC 2011.27) which determines a composite measure of sample toxicity based on the ability of sample extracts to compete with radiolabeled saxitoxin for binding to voltage-gated sodium channels [7
As of 2010, more than 50 analogues of saxitoxin had been identified [8
]. Instrumental methods for the quantitation of saxitoxin and many of its congeners in seafood are now available. Such methods permit the assessment of the concentration of the individual toxins in a seafood sample and this, together with knowledge of the relative toxicity of the various compounds, permits the overall toxicity of the sample to be determined, enabling assessment of the potential risk to human health.
The relative toxicities of saxitoxin congeners are expressed as “Toxicity Equivalence Factors” (TEFs), which define the toxicities of these substances as a ratio of that of saxitoxin itself. Again, an MBA has been used for the estimation of TEFs for saxitoxin congeners. An assay for saxitoxin itself was developed by Sommer and Meyer in the 1930s [9
], based on the relationship between the dose of pure saxitoxin administered to mice by intraperitoneal injection and the time to death of the animals. The amount of saxitoxin in the sample injected, expressed as “Mouse Units”, was determined from the table of death-times established by these authors. Although validated only for saxitoxin itself, this MBA has more recently been applied to saxitoxin congeners, and TEFs for such congeners have been estimated from this data [10
The validity of this approach is questionable. The assay depends upon intraperitoneal injection which negates the role the digestive system may play in either detoxifying some compounds, or in some cases, increasing their toxicological effect. Furthermore, the MBA is a bioassay, not a toxicological parameter, and it has been shown that TEFs derived from this method do not correlate with those derived from median lethal doses determined by approved toxicological methods [11
]. The use of the MBA also assumes that the dose death-time relationships for saxitoxin congeners are the same as that for saxitoxin itself. This too has been shown to be untrue [11
]. The inadequacy of the present TEFs for risk assessment was noted in the Scientific Opinion of the European Food Safety Authority Panel on Contaminants in the Food Chain, which indicated the need for establishing robust TEFs based on the relative oral toxicities of the saxitoxin congeners [10
]. In a recent Expert Panel review of TEFs [12
], it was agreed that the most relevant parameter for their determination was relative toxicity by oral administration and the Expert Panel recommended revisions to the presently used TEFs for certain saxitoxin congeners. Oral toxicity data are now available for neosaxitoxin, decarbamoyl saxitoxin, gonyautoxins 1&4 and gonyautoxins 2&3 [11
]. As a continuation of these studies, we now report the acute toxicities of gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6), decarbamoyl gonyautoxin 2&3 (dcGTX2&3), decarbamoyl neosaxitoxin (dcNeoSTX), N
-sulfocarbamoyl gonyautoxin 2&3 (C1&2) and N
-sulfocarbamoyl gonyautoxin 1&4 (C3&4) by two methods of oral administration and a comparison of these data with the acute toxicities of these substances by intraperitoneal injection. The objective of this study is to add to the list of published TEFs for saxitoxin congeners based on oral administration in order to provide more robust TEF data applicable to the way in which humans are usually exposed to the major saxitoxin congeners found in seafood.
As expected, the acute toxicities of the saxitoxin congeners by gavage were lower than those by intraperitoneal injection, most likely due to slower absorption via the oral route. Materials injected intraperitoneally are generally rapidly and extensively absorbed, leading to high tissue levels and toxicity. Slower absorption via oral administration may allow more time for detoxification and/or excretion of the test material before toxic levels are reached. It should be noted, however, that there were wide variations in the ratios between the toxicities by the two routes of administration. This difference was most pronounced with dcNeoSTX, which showed one of the lowest toxicities by injection, but the highest by gavage and by feeding.
It has been argued that administration by feeding, rather than by gavage, is the most relevant route for toxicity determinations in rodents, since the semi-solid content of the stomach of these animals does not permit mixing of the material given by gavage, which may flow around the stomach contents and rapidly enter the duodenum. When given by feeding, however, the test material becomes mixed with the stomach contents of rodents in the same way that substances are distributed throughout the liquid contents of the human stomach, leading to relatively slow release into the absorptive areas of the gastrointestinal tract [13
]. This is consistent with the observation that the absolute values of the acute toxicities of the saxitoxin derivatives were lower by feeding than by gavage. The ratio between the toxicity by feeding and that by gavage ranged from 2.1 to 2.6 for C1&2, GTX5 and dcNeoSTX, which is consistent with results with other saxitoxin congeners [11
]. The ratios for dcGTX2&3 and GTX6 were higher, however (4.1 and >6, respectively). The reason for this disparity is not presently known. Possibilities include the conversion of these compounds into less toxic substances during the relatively long residence time in the stomach of the animals or the inhibition of stomach contraction or of the opening of the pyloric sphincter, leading to slower release into the duodenum.
For accurate risk assessment, it is essential that relevant and accurate TEFs for saxitoxin and its congeners are available. At present, the relative risk to human health of saxitoxin derivatives is largely based on TEFs calculated from the specific activities of these substances determined in the MBA. As shown previously [11
], the relative acute toxicities of a number of saxitoxin congeners by intraperitoneal injection do not correlate with their relative specific activities in the MBA. This is consistent with the observation that the death time-dose curves for the saxitoxin derivatives are not the same as that for saxitoxin itself [11
In the present study, the acute toxicities of GTX5, GTX6, dcGTX2&3, dcNeoSTX, C1&2 and C-3&4 were determined. MBA data are available for GTX5, GTX6 and dcGTX2&3 [10
]. No MBA data on epimeric mixtures of C1&2 or C3&4 are available. Also, the MBA figure given by European Food Safety Authority (EFSA) for dcNeoSTX [10
] is regarded as incorrect. The figure given is that from Sullivan et al. [14
], though these authors did not determine the specific activity of dcNeoSTX, but assumed that it was the same as that of decarbamoyl saxitoxin. In order to facilitate comparison, we determined the specific activities of the C-toxin equilibrium mixtures and that of dcNeoSTX. It should be noted that the equilibrium mixtures of the epimers of dcGTX2&3, C1&2 and C3&4 were evaluated in these studies, rather than the individual epimers, since the latter substances are never found in isolation in seafood, but invariably as equilibrium mixtures.
A comparison of the TEFs derived from the MBA, acute toxicity by intraperitoneal injection and by oral administration of the above toxins is shown in Table 4
. Again, there was no correlation between the TEFs derived by the MBA and those from acute toxicity by intraperitoneal injection. The TEFs based on the MBA were similar to those based on oral toxicity for GTX5 and C3&4, but were higher for GTX6, dcGTX2&3 and C1&2 and lower for dcNeoSTX. The TEFs based on toxicity by feeding were ~40% lower than those proposed by EFSA for GTX5 and dcNeoSTX, and more than five times lower for GTX6.
The results of the present study suggest that the currently used TEFs for some of the above compounds should be revised based on the available oral toxicity data, and this has been recommended in a recent Expert Panel review [12
]. In this way, appropriate regulatory limits can be set that are not so high as to endanger human health and not so low that they cause unnecessary loss to the seafood industry through destruction of product or closure of harvesting areas.