The Laboratory Mouse in Routine Food Safety Testing for Marine Algal Biotoxins and Harmful Algal

17 Pages·2014·0.58 MB·English

356 Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014
SPECIAL GUEST EDITOR SECTION
The Laboratory Mouse in Routine Food Safety Testing for
Marine Algal Biotoxins and Harmful Algal Bloom Toxin
Research: Past, Present and Future
Ian Stewart
South Australian Research and Development Institute (SARDI), Gate 2b, Hartley Grove, Urrbrae, SA 5064, Australia;
The Australian Seafood Cooperative Research Centre, Laffer Drive, Bedford Park, SA 5042, Australia; The University of
Queensland, National Research Centre for Environmental Toxicology, 39 Kessels Rd, Coopers Plains, QLD 4108, Australia
CatherIne MCLeod
South Australian Research and Development Institute, Gate 2b, Hartley Grove, Urrbrae, SA 5064, Australia; Seafood Safety
Assessment, PO Box 7125, Adelaide, SA 5000, Australia
Mouse bioassays have been a mainstay for detecting development of the source microalgae occurs under opportune
harmful concentrations of marine algal toxins in environmental conditions. These so-called blooms of microalgae
shellfish for over 70 years. Routine monitoring represent a public health hazard when their toxins are present in
involves intraperitoneal injection of shellfish extracts seafood at concentrations that can cause illness and death when
into mice; shellfish contaminated with algal toxins the product is consumed (1–4). Some seafood-related poisonings
are thus identified by mortality in exposed mice. With subsequently shown to be caused by marine microalgal toxins
the advent of alternative test methods to detect and have a long history: Native American Indians in Alaska were
quantify specific algal toxins has come increasing apparently aware that an infrequent luminescence in coastal
criticism of enduring use of mouse bioassays for waters was temporally linked to poisonous shellfish, taking
shellfish safety testing. However, the complete steps to monitor for such events (5). Captain George Vancouver
replacement of shellfish safety mouse bioassays wrote of an outbreak of fulminant neurological signs and
by chemical, antibody-based, and functional assays symptoms in a group of his sailors who had consumed mussels
has been and will continue to be a gradual process from an inlet in British Columbia they were exploring in 1793.
One of the mariners died within hours, in what would now be
for various reasons, including skills availability
described as a typical case of paralytic shellfish poisoning (6).
and instrument costs for chromatography-based
Likewise, ciguatera fish poisoning was described by seafarers in
toxin monitoring. Mouse bioassays for shellfish
the South Pacific from the early 17th century through to Captain
safety testing do not comply with modern standards
Cook’s expeditions in the 18th century (7, 8). By contrast, other
for laboratory animal welfare, specifically the
harmful algal bloom (HAB) intoxication syndromes are more
requirement in published official methods for
recently understood phenomena. Amnesic shellfish poisoning,
death as a test outcome. Mouse bioassays for algal
caused by the glutamate analog domoic acid that is produced
biotoxins in shellfish, as well as fundamental algal
by the pennate diatom Pseudonitzschia, was first reported
toxin research endeavors using in vivo models, are
after a mass neurointoxication outbreak in 1987 caused by
amenable to revision and refinement from a humane
contaminated mussels from Prince Edward Island, Canada (9).
endpoints perspective. Regulated hypothermia
Diarrhetic shellfish poisoning (DSP) was first described in
may be a useful and easily acquired nonlethal
detail in 1978, following outbreaks of gastrointestinal illness in
toxicological endpoint; objective determination of
Japan involving mussels and scallops (10, 11).
neuromuscular blockade may allow algal neurotoxin
This paper will examine the history and current use of the
testing and research to enter the domain of humane
mouse bioassay (MBA) as a reference method for detection and
endpoints evaluation. Relinquishing reliance on
quantification of harmful algal toxins from the saxitoxin (STX)
subjective test endpoints, including death, will
family in shellfish and lipophilic toxins in shellfish that include
likely also deliver collateral improvements in assay
toxins responsible for diarrhetic shellfish poisoning. STXs are
variability and sensitivity.
the causative agents of paralytic shellfish poisoning (PSP) and
are synonymous with paralytic shellfish toxins (PSTs). Toxins
of the okadaic acid (OA) group are the causative agents of DSP;
Marine microalgae can produce a range of structurally OA and its derivatives are synonymous with diarrhetic shellfish
diverse toxic compounds that bioaccumulate and in toxins (DSTs). However, the DSTs are classified through
some cases biomagnify in edible fish and shellfish. the predicates of chemical analysis within a larger group of
These toxins contaminate various types of bivalves, gastropod compounds that are readily separable by chromatographic
and cephalopod molluscs, crustaceans and finfish when mass methods, the so-called lipophilic toxins. Lipophilic toxins
include some structurally and functionally unrelated groups,
Guest edited as a special report on “Marine Toxins” by
some of which can co-occur with DSTs in shellfish tissues but
James Hungerford and Ana Gago-Martínez.
Corresponding author’s e-mail: [email protected] are not diarrheagenic as a principal feature of their activity, e.g.,
DOI: 10.5740/jaoacint.SGEStewart pectenotoxins and yessotoxins.
Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014 357
Regulatory detection of harmful concentrations of brevetoxins, toxic in the summer months, although with variable intensity.
the causative agents of neurotoxic shellfish poisoning, is That work was initiated in response to a large outbreak of PSP
also realized by an MBA (12). Brevetoxins have periodically in 1927, with 102 cases, including six fatalities, following
contaminated shellfish, mainly from U.S. Gulf states (13). consumption of mussels harvested from the Northern California
Historical and contemporary aspects of brevetoxin analysis, coastline (21). These same authors then described in more detail
including initial use of MBAs and subsequent development of the method for their PSP MBA (22) with further refinements
alternative analytical methods, mirror the approach to PST and by Medcof et al. (23). An interim proposal for a standardized
lipophilic toxin analysis presented in this paper. The challenges bioassay was released in 1956 (24) when purified STX became
of brevetoxin analysis, including discussion of specific available (25). The MBA for PSTs was subsequently adopted as
limitations of the MBA, have been reviewed by others (14, 15). an official AOAC method (26). The AOAC method published
We will not include further discussion of brevetoxin MBAs in 1959 remains current today (27). The MBA for PSP toxins
here, but the conclusions and recommendations regarding played a pivotal role in the research understanding of the
revision and refinement of MBAs used for routine testing of STX family of neurotoxins. Ed Schantz, the pioneering U.S.
shellfish for PSTs and lipophilic toxins are pertinent to the topic biochemist who first isolated and elucidated the structure of
of brevetoxin detection in shellfish in cases where and when STX, remarked that:
MBAs are used for that purpose. “We used the mouse assay to quantitate the in vivo response
The MBA for detection and quantification of ciguatera toxins in practically all of our work on the isolation, purification,
in finfish will be examined, as there are features of the test and and chemical and physical characterization of the various
the physiological responses of mice to these toxins that add to paralytic poisons in mussels, clams, scallops, and cultures of
the discussion. The use of PST and lipophilic toxin MBAs for [Alexandrium] species.” (28).
routine safety testing of commercial shellfish products will be Likewise, the mouse has been an invaluable research tool for
compared and contrasted with the application of the laboratory the study of most, if not all, of the structurally and functionally
mouse as a research tool in the field of harmful algal toxin diverse array of compounds that are classified as HAB toxins,
research, with particular attention to recent developments in the including the cyanobacteria toxins. To give just a few examples,
ethical use of laboratory rodents for scientific purposes. the laboratory mouse has been employed for investigation of
the mechanistic toxicity of freshwater and marine cyanotoxins
History of the Mouse Bioassay (29–32) and eukaryotic marine toxins such as domoic acid (33),
azaspiracids (34), OA (35), ciguatoxins (36), palytoxin (37),
Mice and other small vertebrates have had a role in biomedical and brevetoxins (38). The laboratory mouse continues to be a
research since the early 16th century. William Harvey used frontline model for assessing the acute toxicity of novel and
mice for his experiments on reproduction and the circulatory emerging HAB toxins (39, 40).
system. Selective breeding and genetic experiments began in
the 19th century (16). The turn of the 19th to the 20th century The MBA for Shellfish Safety Testing
saw investigation of spontaneous and transplantable tumors in
mice (17). The use of mice for experimental induction of cancers The two MBA methods currently in routine and widespread
was first reported in 1918, when Tsutsui in Japan demonstrated use for monitoring of commercial shellfish for food safety
that coal tar preparations applied to the skin produced lesions purposes are AOAC 959.08 for PSTs (27), and an MBA for
similar to those that had previously been demonstrated on rabbit lipophilic toxins, including the so-called DSTs. A reference
ear skin (18). method for the lipophilic toxins MBA was published by a
Premarket safety testing of drugs began in earnest following consortium of European national reference laboratories (41). A
a notorious mass poisoning in 1937, when 34 children and validated MBA is also used for detection of ciguatera toxins in
71 adults died of acute renal failure after ingesting a proprietary finfish (42), but application of that particular assay differs in
sulfa antibiotic formulation that contained a toxic solvent, one very important respect compared to those used for PSTs and
diethylene glycol. This event, centred on Tulsa, Oklahoma lipophilic toxins insofar as the ciguatoxin MBA is not routinely
and known as the Elixir Sulfanilamide Disaster, heralded used for food safety monitoring of fish prior to distribution or
the proclamation by the U.S. Congress of the 1938 Federal sale. However, some issues surrounding the development and
Food, Drug, and Cosmetic Act. Preceding legislation from use of the ciguatoxin MBA are pertinent to the topic of MBAs
1906 was aimed at preventing the distribution of adulterated for shellfish safety testing, so they will be discussed in this
food and mislabelled patent medicines. The U.S. Food and review.
Drug Administration (FDA) at the time was essentially a
policing agency that responded retroactively to food and MBA for PSTs
drug infringements; the 1938 Act for the first time required
pharmaceutical manufacturers to demonstrate premarket As outlined above, the MBA for testing shellfish contaminated
product safety (19). Animal models then became the mainstay by STXs was first developed in the United States in the 1920s;
for testing new food and drug ingredients (20). the assay was validated and adopted as a reference method in
The laboratory mouse has an 85-year history of contributing 1959. Regulatory oversight of commercial shellfish operations,
to the understanding of harmful algal toxin contamination in and associated interventions informed by mouse toxicity testing,
shellfish. Sommer and Meyer (21) presented a 7-year “poison was reported from the United States since the 1920s (5, 43) and
curve,” derived from mouse lethality tests that commenced Canada from the early 1940s (23). The STXs are a highly water-
in 1927, demonstrating seasonal toxicity spikes in San soluble group of compounds; early workers noted that toxic
Francisco-area mussels. Shellfish were consistently seen to be properties were degraded in alkaline or neutral solutions (5), so
358 Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014
the assay method was developed—and remains to date—using used in some countries for postconsumption confirmation of
acidic extracts injected parenterally (26, 27). ciguatoxin contamination, (e.g., 53, 54). However, confirmatory
detection and quantification of ciguatoxins in finfish by
MBA for Lipophilic Shellfish Toxins chemical or combined chemical and in vitro bioassay testing
has been adopted by other countries for their epidemiological
Occurrences of shellfish-associated gastrointestinal illnesses surveillance (55, 56). The development and adoption by food
unlike those known to be caused by enteric pathogens were first safety agencies across the world of different assay techniques
reported from The Netherlands in 1961 and Norway in 1968, with for detecting ciguatera toxins parallels the various current
further outbreaks—again from The Netherlands—in 1971 and approaches to testing shellfish for safe levels of paralytic and
1976 (44). A severe gastrointestinal mass intoxication from Chile lipophilic toxins.
in 1970 was retrospectively linked to a bloom of the dinoflagellate
Dinophysis acuminata (45). Following outbreaks in Japan in 1976 Alternatives to the MBA for Marine Algal Toxin Food
and 1977, Yasumoto and colleagues identified a lipid soluble Safety Testing
toxin from Dinophysis, which they named dinophysistoxin. This
compound had identical chemical properties to those of a toxin It is beyond the scope of this paper to present a detailed
extracted from mussels, so the term DSP was coined to describe review of the range of chemical, immunological and biological
this form of intoxication (10). Dinophysistoxin was subsequently tests that have been presented—either as research applications
shown to be an OA analog (46). Yasumoto et al. (11) described or more thoroughly assessed as working assays—as alternatives
an MBA method for OA toxins in which acetone extracts are to the MBA for PSTs, lipophilic toxins and ciguatera toxins and
reconstituted in a polysorbate emulsifier as an injectable vehicle. their advantages and disadvantages compared to the MBAs.
A modified procedure to partition acetone-soluble extracts Alternatives to the MBA and their relative merits have been, and
between diethyl ether or dichloromethane and water forms the continue to be, widely discussed in the literature—see examples
basis for the currently operating MBA for lipophilic toxins, the in review format (57–61). Rather, this review will critique
European Union (EU) harmonized standard operating procedure the current use of the MBA by comparing and contrasting
for detection of lipophilic toxins by MBA (41). The death of two the advantages and disadvantages of the technique with those
of three mice within 24 h is the endpoint for a positive toxicity of the various alternative approaches (chemical, physical,
result. immunological and biological) that have been recruited for
shellfish and finfish safety testing, or are—or were—in the
MBA for Ciguatera Toxins process of development to that end.
Table 1 presents a nonexhaustive summary of analytical
There is a long history of adopting a wide range of animal alternatives to the MBA, their classification as functional tests
models for detecting and quantifying ciguatoxins, including or otherwise, and their application as either routinely adopted
mongoose, cat, chicken, and mouse assays, as well as techniques or methods that are either still in development or
invertebrate assays based on mosquitoes, dipteran larvae and did not proceed to commercialization. As revealed in Table 1, a
brine shrimp (47–49). An MBA validated for quantitation remarkable and diverse range of approaches have been applied
of ciguatera toxins in fish flesh was reported in 1993 (50) to the detection and measurement of the three toxin groups
and subsequently described in detail in Intergovernmental found in shellfish and finfish for which the MBA is currently a
Oceanographic Commission/United Nations Educational reference method: PSTs, lipophilic toxins and ciguatera toxins.
Scientific and Cultural Organization (IOC/UNESCO) manuals The history of seafood safety testing for all three toxin groups
on harmful marine microalgae (42, 51). While the MBA has has progressed along broadly similar trajectories: from initial
been widely used as a research model, it is not applied in the reports of illnesses—often fatal or severely debilitating—
same way as the PST and lipophilic shellfish toxin bioassays grew the understanding that particular seafood products were
are used for prospective food safety testing. Because fish are hazardous to consume in some circumstances, with seasonality
not sessile and because precursor toxins undergo a complex and and geography often noted as contributory factors. Early
lengthy biotransformation to their final chemical configuration interventions to address basic, often qualitative, questions
as ciguatoxins in apex predator fish, routine testing of finfish of toxicity and food safety were informed by various whole
for presale or preconsumption confirmation of the absence of animal models; the history of implementation of a wide variety
ciguatoxins would need to be done on an individual specimen of bioassays—including informal predictive testing on humans
basis; batch testing is arguably not feasible. A 2-year research in some South Pacific island communities—for ciguatera fish
program to screen a single high-risk fish species, Seriola toxins is particularly instructive here. Useful bioassays were
dumerili, was conducted in Hawaii between 1979 and 1981 then refined and modified to serve as quantitative tools. More
using a radioimmunoassay technique. Ciguatoxin-negative sophisticated bioassays, as well as chemical and antibody-
fish were released for sale the morning after testing; the assay based tests, were the natural consequence of advances in the
was reported to be economically viable for fish weighing understanding of the molecular toxicology, biochemistry and
more than 9 kg (52). To our knowledge, there are no currently analytical chemistry of these toxins.
operative premarket screening programs for ciguatoxins in
finfish by any method, although there is a long standing but Advantages and Disadvantages of Alternatives to the
still unmet demand for a low cost, simple and reliable test. The MBA
principal food safety application of the MBA for ciguatoxins
is in retrospective testing of suspect fish for epidemiological The various approaches to seafood safety testing presented
surveillance. The MBA is, or was in recent years, routinely in Table 1 may be further summarized for the purposes
Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014 359
Table 1. Alternatives to the MBA for detection and quantification of STX, lipophilic and ciguatera toxin groups
Application
Toxin family Test (research, industry) Advantages and requirements References
STXs LC-FD precolumn Official AOAC method Ethical concerns: none, non-biological assay; (62–64)
oxidationa toxins can be characterized and quantitated
Technical competencies need to be more broadly available, business
models need to address capital and running costs
LC-FD postcolumn Official AOAC method Ethical concerns: none, non-biological assay; (65–68)
oxidation toxins can be characterized and quantitated
Technical competencies need to be more broadly available, business
models need to address capital and running costs
LC/MS Research Ethical concerns: none, non-biological assay; (69–71)
toxins can be characterized and quantitated
Technical competencies need to be more broadly available, business
models need to address capital and running costs
Capillary electrophoresis Research Ethical concerns: none, non-biological assay; (72, 73)
toxins can be characterized and quantitated
Technical competencies need to be more broadly available, business
models need to address capital and running costs
Ion-exchange Research Ethical concerns: none, non-biological assay; (74, 75)
chromatography toxins can be characterized and quantitated
Technical competencies need to be more broadly available, business
models need to address capital and running costs
Quantitative NMR Research Ethical concerns: none, non-biological assay; (76)
spectrometry toxins can be characterized and quantitated
Technical competencies need to be more broadly available, business
models need to address capital and running costs
Lateral flow Used as a screening test in Ethical concerns: none, non-biological assay; (77–80)
immunoassay some commercial shellfish rapid, low-cost, low-tech test
monitoring programs Activity correlates poorly with toxicity in samples with different
congener profiles; high false positives detected in some PST profiles
ELISA and other Research; some ELISA kits Ethical concerns: none, non-biological assay; (81–83)
immunoassays commercially available high sensitivity, potential for use as screening assays
Activity across congeners may not correlate with toxicity
Surface plasmon Research–promising potential Ethical concerns: none, non-biological assay; (84–86)
resonance assay for replacement of MBA using potential for rapid throughput
(optical biosensor) multiplexed antibodies Activity correlates poorly with toxicity
using single-antibody technique
Receptor binding assay Official AOAC method Ethical concerns: ex vivo functional assay (87–90)
(i.e., no live animal toxicity testing)
Labeled STX supply needs to be secured and assured, OH&Sb
and regulatory approvals regarding use and disposal of tritiated STX
need to be addressed in some countries
Saxiphilin competitive Research Ethical concerns: functional assay. Supply of receptor proteins needs (91, 92)
binding assay considering, but no live animal toxicity testing is required
More R&D requiredc
Membrane potential in Research Ethical concerns: in vitro or ex vivo functional assays; (93–100)
vitro assays no live animal toxicity testing required
Technical competencies need to be more broadly available, business
models need to address capital and running costs.
Some methods currently require lengthy incubation times
Membrane biosensor Research Ethical concerns: ex vivo functional assay (101, 102)
(frog bladder membrane) More R&D required
Insect bioassay Research Ethical concerns: invertebrate bioassays (103–105)
More R&D required
Lipophilic LC/MS Official EU method Ethical concerns: none, non-biological assay; (106–109)
toxins toxins can be characterized and quantitated
Technical competencies need to be more broadly available, business
models need to address capital and running costs
LC-FD Research Ethical concerns: none, non-biological assay; (110–112)
toxins can be characterized and quantitated
Technical competencies need to be more broadly available, business
models need to address capital and running costs
360 Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014
Table 1. (continued)
Application
Toxin family Test (research, industry) Advantages and requirements References
Capillary electrophoresis/ Research Ethical concerns: none, non-biological assay; (113, 114)
electrochromatography toxins can be characterized and quantified
Technical competencies need to be more broadly available, business
models need to address capital and running costs
ELISA and other Research Ethical concerns: none, non-biological assay; high sensitivity, (115–120)
immunoassays potential for use as screening assays
Activity across congeners may not correlate with toxicity; screening for
structurally unrelated toxins within the lipophilic toxin class will require
use of multiple ELISAs
Surface plasmon reso- Research Ethical concerns: none, non-biological assay; potential for rapid (121)
nance assay throughput
(optical biosensor)
Cell-based assays Research Ethical concerns: in vitro or ex vivo functional assays; (122–124)
no live animal toxicity testing required
Technical competencies need to be more broadly available, business
models need to address capital and running costs. Some methods
currently require lengthy incubation times
Protein phosphatase Research; mature technique Ethical concerns: enzyme-based functional assay, (125–127)
inhibition assay awaiting regulatory ac- no requirement for laboratory animal use
ceptance Suitable only for OA-group toxins
Rat bioassay Official method in Ethical concerns: in vivo non-lethal endpoint test (128, 129)
The Netherlands (examination of fecal consistency)
Semiquantitative, subjective endpoints;
suitable only for diarrhetic toxins
Ciguatoxins LC/MS Research Ethical concerns: none, non-biological assay; (56, 130,
toxins can be characterized and quantitated 131)
Availability of analytical standards is a current constraint; sample
extraction and cleanup is complex and labor intensive, therefore
unsuitable for screening
Radioimmunoassay Research, Reported success as a single-species screening tool, (52, 132)
now obsolete method Hawaii 1979–81 historical use;
requirement for radiation safety facilities and protocols
Immunobead assay Kits have been marketed Ethical concerns: antibody-based test; no requirement for (133–138)
in the past but none whole-animal testing. Marketed as a rapid, low cost,
currently available preconsumption safety test
Performance has been questioned. Production and sale of the most
recent product, Cigua-Check®, has ceased
ELISA Research Ethical concerns: antibody-based test; no requirement for whole-animal (139, 140)
testing
More R&D required to develop and validate a reliable kit
Receptor binding assay Research Ethical concerns: ex vivo test (141–143)
Tritiated brevetoxin supply needs to be secured and assured, OH&S
considerations and regulatory constraints to establish facilities for
handling radioisotopes need to be addressed
Mouse neuroblastoma Available as a screening test Ethical concerns: in vitro test using an immortalized cell line (55, 144,
assay at some facilities for research (i.e., no ethical constraints) 145)
and epidemiological Technical competencies to maintain cell lines and run assays
surveillance need to be more broadly available, business models
need to address capital and running costs.
Microplate fluorometry Research Ethical concerns: in vitro test (146)
More R&D required
Insect bioassay Calliphorid assay: research Ethical concerns: invertebrate assays (47, 147)
only; culicid assay now More R&D required
superseded by MBA
Other bioassays, e.g., Mongoose bioassay was Ethical concerns: plentiful, to the extent that village-scale and home (49, 148)
cat, mongoose, chicken, utilized in Hawaii; domestic bioassays using relatives and companion animals are extant.
guinea pig atrium, brine cat as a backyard assay in Use of backyard bioassays highlights an unmet demand for reliable,
shrimp, village elder, Australia low cost and simple analytical tests
mother-in-law assay
a FD = Fluorescence detection.
b OH&S = Occupational Health and Safety Administration.
c R&D = Research and development.
Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014 361
of comparing and contrasting with the MBA. It should be sample extraction and cleanup is also a necessary component
understood, however, that grouping analytical methodologies of analytical strategies to address matrix-related signal
that operate with disparate fundamental principles is an artificial suppression or enhancement (152). Another important challenge
construct, and it will fail to consider marginal benefits (such for a chemical methods approach is that these techniques can
as economies of scale) or marginal deficiencies (e.g., supply/ only detect and measure compounds for which analytical
demand misalignment of essential component/s) by which any standards (or, in the absence of reference materials, specific
particular method or method type may succeed or fail over the and sophisticated analytical techniques) are available. The issue
long term. of toxicity equivalence factors for the various STX congeners
Broadly speaking, chemical methods have an advantage over and lipophilic toxins (and, to a lesser extent, the ciguatoxin
nonspecific bioassays insofar as the chemical components of family) is also pertinent to the discussion of the relative
assayed samples can be accurately identified and quantified with merits of chemical analysis. Broadly speaking, proponents of
reference to known analytical standards. Matrix interferences, bioassay-based approaches point out that functional assays,
which are more-or-less inherent to particular methodologies— particularly whole-animal assays, offer considerable advantages
more so in the case of MS techniques—may be addressed by for preconsumption shellfish safety testing insofar as the
use of matrix-certified reference materials. The availability outcomes are not required to address or target specific congener
of analytical standards has been cited as a constraint to the profiles within the toxin group being analyzed. Provided the
development and application of chemical assays for algal extraction of shellfish samples is directed towards the toxin
biotoxins in seafood (103, 149, 150), but it should be noted that group of interest, i.e., acidified aqueous extracts for STXs and
such constraints are economic, not knowledge based. All of the emulsified extracts using various organic solvents for lipophilic
known STX, lipophilic toxin and ciguatoxin congeners can either toxins or ciguatoxins, the bioassay will return both qualitative
be chemically synthesized, produced by laboratory biosynthesis, information on the toxicity of the sample extract and quantitative
or isolated and purified through wild-harvest excursions. Supply estimates of total toxicity when the assay is standardized with
bottlenecks are essentially functions of resource availability, even reference to purified standard (PST assay; 27) or fish extracts
though access to particular analytical standards is currently very of known toxicity (ciguatoxin assay; 42). Chemical methods,
difficult due to separation and purification inefficiencies, with by contrast, can only provide toxicological information on
attendant cost implications. The ciguatoxins are arguably subject analyzed samples by way of quantifying known congeners for
to the most serious limitations in this regard, with only microgram which toxicity equivalence factors—or alternative measures of
quantities able to be extracted and purified from kilograms of fish relative toxicity—have been determined.
viscera using sophisticated and time-consuming techniques, let
alone the costs involved in capturing, transporting and storing Antibody-Based Methods: Advantages and
source tissues (130, 151). Disadvantages
Chemical Methods: Advantages and Disadvantages Immunoassays have been developed for both paralytic and
lipophilic toxins; several are commercially available, others are
Chemical methods currently in routine use for shellfish safety in various stages of precommercialization development (see
testing to detect and measure concentrations of paralytic and Table 1 for references). Immunoassays for detecting ciguatera
lipophilic toxins mainly use LC separation techniques, with toxins have been largely unsuccessful: several ELISAs,
fluorescence detection most commonly applied to STX analysis immunobead assays, and radioimmunoassay tests developed
and MS/MS generally applied to the analysis of the structurally over past decades are not currently available to address the
diverse range of compounds grouped in the lipophilic toxins class unmet global demand for a reliable, simple and low cost
(see Table 1 for references). Advantages of chemical techniques screening test for ciguatoxins in finfish [reviewed in (49)]. Two
are that individual toxins can be unequivocally identified and solid-phase immunobead tests had been commercially available:
quantified; these methods thus deliver a highly specific result, the Ciguatect assay and later the Cigua-Check kit; both were
usually with excellent sensitivity. The unequivocal specificity subject to unfavorable critical evaluation and subsequently
of HPLC-based techniques is particularly apposite for the withdrawn from the market (133, 134, 136). However, research
retrospective analysis of fish suspected to have caused ciguatera into ciguatoxin antibodies continues, with one group working
poisoning; such applications are very useful for epidemiological to develop a hapten from a synthetic partial ciguatoxin structure
surveillance, helping to support or exclude the diagnosis (8). (140, 153).
Other advantages of chemical methods are the capacity for high Immunoassays offer the general advantages of high sensitivity
throughput analysis achieved through utilization of autosamplers and ease of use; reliable execution of ELISAs and particularly
and other automated processes. Chemical methods of analysis the stick-type assays does not require highly sophisticated skill
do not require use of biological tissues or substrates; therefore sets and training. As such, antibody-based tests are relatively
they are unconstrained by ethical considerations. cost effective compared to sophisticated LC-based techniques.
Disadvantages of chemical methods are the requirements for One practical shortcoming of the application of immunoassays
sophisticated and expensive facilities, equipment—in terms of to many harmful algal toxins is congener cross-reactivity.
initial capital outlay, consumables and depreciation costs in Most HAB toxins, including the STXs, lipophilic toxins, and
light of rapidly advancing capabilities—and skill sets, as well ciguatoxins that are the focus of this review, belong to “families”
as the cost and availability of analytical standards, both purified of toxins. Each group or family comprises compounds related
reference standards and matrix-certified materials. The latter by core chemical structure that differ from each other in the
are particularly useful for MS methods, which are prone to presence and location of various functional groups. Thus the
matrix interferences; careful (read time-consuming and costly) STXs are a family of tricyclic tetrahydropurine alkaloids;
362 Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014
at least 57 variants are known to date (154). The ciguatoxins have the advantages of detecting toxicologically relevant
encompass more than 30 variants, all featuring a core structure endpoints and ease of use, but the impediments of poor
of 13-14 ether-linked rings (155). The lipophilic toxins are a specificity, rising costs and increasing ethical constraints. These
more disparate group of compounds, loosely categorized within topics will now be discussed, with particular focus on animal
a single entity. But within the structurally and functionally welfare considerations that are brought to bear on the use of
unrelated group of lipophilic toxins are yet more families of these tests in the second decade of the 21st century.
structurally related compounds, for example, the azaspiracids
with some 30 variants known, and more than 90 yessotoxin The MBA and Toxicologically Relevant Endpoints
analogs (156). The problem with congener cross-reactions,
where epitopes are presumably located on core structures, is that The MBA for monitoring the presence of STXs through
toxicity does not always correlate with cross-reactivity. Acute shellfish safety programs has a long history of protecting public
toxicity across the various analogs within a toxin “family” can health; many statements to that effect can be found in the
vary by several orders of magnitude in some cases. And different literature (e.g., 164–169). An important factor here—and indeed
toxin families that share some structural similarities can also be for any other type of assay used for food safety testing—is an
subject to cross-reactivity; an earlier ciguatoxin immunoassay absolute requirement for a low level of false-negative returns.
was shown to significantly cross-react with OA—both toxin This pertains to the sensitivity of the test. The MBA for PST
groups being cyclic polyethers—meaning that the assay could screening apparently meets this requirement; reports of PSP
not effectively discriminate between ciguatoxin and OA (157). illness outbreaks caused by consumption of shellfish harvested
In practice, the problem of poor analytical specificity for from commercial fisheries subject to regulatory oversight
particular toxin congeners presented by many immunoassays where the MBA is or was used for monitoring appear to be
manifests in hazard overestimation. For example, immunoassays infrequent in the literature. One such example is that of two
for PSTs are typically based on antibodies raised to STX, the U.S. outbreaks in 1972, in Washington and New England (170).
most toxic congener in the family of aquatic STXs. But STX- Washington State established its shellfish monitoring program
producing dinoflagellates can synthesize a range of STX for PSTs in 1957 (171). However, despite the apparent dearth
congeners—again, some of these analogs will be of considerably of similar publications, it is possible that mild cases of PSP
lower toxicity than that of STX—and some microalgae produce may go unreported, under-elicited or misdiagnosed. A form of
STX profiles in which low-toxicity variants are found in the publication bias may also operate in some instances in some
highest concentrations (158, 159). PSP immunoassays can, countries, whereby information on cases or outbreaks of PSP is
therefore, present an economic burden to seafood industries not readily accessible by the broader scientific community from
when these tests identify the presence of STX congeners at the peer-reviewed literature.
concentrations that do not represent the same risks to human The apparent low failure rate of the MBA for PST shellfish
health as would equimolar-range concentrations of STX alone. monitoring might have less to do with the inherent level of
Toxins can also be transformed to higher or lower toxicity protection provided by the assay itself, and may be more a
analogs by various biological, chemical or physical processes reflection of the wide margin of safety afforded by the test.
(158, 160–162); some analogs for which acute toxicity However, expert opinion remains somewhat divided; some
information is not available can sometimes be seen in shellfish suggest that the long history of use of the MBA, with scant
at high concentrations. In the latter case, food safety regulators evidence of failure to prevent outbreaks of intoxication in
tend to adopt a precautionary approach to risk management, by humans caused by consumption of product from monitored
which congeners of unknown toxicity are allocated a toxicity fisheries, is an indication that the public health policy and risk
equivalence factor corresponding to that of the analog with the management settings are about right (165, 168, 172). However,
highest known toxicity. In all these cases, immunoassays can other agencies (169, 173) have drawn on information from the
present the same problems of congener cross-reactivity that may clinical epidemiology of PSP and estimates of ingested doses
not relate to toxicity. to derive lowest-observable-adverse-effect levels (LOAEL)
The critique above does not, however, present a complete and, with applied safety factors, no-observable-adverse-effect
picture. Congener cross-reactivity is not universally levels (NOAEL). These latter approaches, applying formal
disadvantageous; on the contrary, this could be an important health risk assessment processes, have recommended acute
benefit in situations where cross-reactivity correlates well with reference doses that translate to guidance levels in shellfish
toxicity. And in practice, cross-reactivity between toxin congeners that are considerably lower than the current guidance level of
detected by any single assay kit can be highly variable. Low 800 µg STX eq./kg shellfish meat (75 µg STX eq./kg shellfish
cross-reactivity between the target analog and any other congener by the European Food Safety Authority (EFSA) approach;
may be useful where the secondary congener is of low toxicity, 110–170 µg STX eq./kg shellfish by the Food and Agriculture
but much less so in the case of congeners having very similar Organization/World Health Organization/Intergovernmental
toxicities [e.g., STX, neosaxitoxin, and gonyautoxin (GTX)- Oceanographic Commission (FAO/WHO/IOC; 169, 173). The
1]. For further consideration of these topics, see the review of 800 µg/kg regulatory level has been in place for many decades;
Humpage et al. (57). Contradictory conclusions regarding the it was first applied in the 1920s through the efforts of the early
proficiency of STX immunoassays can be found in the literature, PSP researchers from California, and doses in mouse units were
with reports of effective (163) and inadequate (80) performance. subsequently converted to STX and STX equivalents following
the isolation and purification of STX and determination of the
MBAs: Advantages and Disadvantages relative toxicities of the main STX congeners. Wekell et al.
suggest that the level of 80 µg STX/100 g shellfish tissue was
MBAs for food safety testing and research on harmful algae probably accepted as a compromise between the low sensitivity
Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014 363
of the MBA, at around 40 µg STX/100 g shellfish and the Next, we present an analysis of some key aspects of the
LOAEL of 200 µg STX/100 g shellfish reported from early MBA. We begin with an examination of death as a test endpoint.
studies (168).
Other authors have commented on the low sensitivity of the Laboratory Animal Welfare: the Movement Towards
PSP MBA (172, 174). Trace metals are known to interfere with Humane Endpoints Testing
the test (43). Turner et al. reported on the potential for the MBA
to underestimate PST toxicity, with a considerable decrease Animal experiments that are designed to conclude with
in the sensitivity of the assay due to interactions with trace death as the endpoint have long been the focus of criticism by
metals—particularly zinc—in oyster tissues (175). individuals and organizations with an interest in animal welfare
issues. The move away from lethal endpoints testing is gaining
Lipophilic Toxins MBA momentum in the 21st century, facilitated by the growing
subdiscipline of humane endpoints research, and by regulatory
The MBA for lipophilic toxins would appear to be a less imperatives in some countries that prohibit or severely restrict
sensitive safety test than the PST MBA, insofar as more the use of lethal endpoint experiments such as the median lethal
reports of human illness following consumption of shellfish dose (LD ), for example, the EU (193), Australia’s National
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sourced from monitored fisheries can be found in the literature Health and Medical Research Council (194), and the Australian
(e.g., 176–178). False-positive tests have also occurred, State of Queensland (195). Regulatory agencies in the United
representing unnecessary economic disruption for affected States are assessing humane endpoints models (196). Humane
shellfish fisheries (179, 180). Aside from criticism on ethical endpoints are one or more objectively determinable and pre-
grounds of this and other MBAs used for seafood safety established physiological and/or behavioral signs that prescribe
monitoring, which we discuss below, the lipophilic toxin the point at which a laboratory animal has delivered the required
MBA has been subject to pointed scrutiny regarding fitness- experimental outcome and can then be humanely withdrawn
for-purpose. Hess (181) suggests that the assay’s sensitivity from the experiment.
is too low to detect OA-group toxins at the regulatory level, The OECD published a report in 2000 on the use of humane
and that it cannot adequately detect azaspiracid and OA-group endpoints for laboratory animals used in product safety
toxins in processed (heat-treated) product. The EU has adopted testing (197). Humane endpoints testing is the focus of a
an LC-MS/MS technique as the reference method for toxins growing body of literature, featuring detailed discussion on a
in the OA, pectenotoxin, yessotoxin and azaspiracid families, wide range of specific neurological or immunological outcomes
with a transition period until the end of 2014 for member states and biomarkers of effect. For in vivo toxicology research,
to discontinue use of the MBA for routine detection of these authors describe methods for systematic determination of the
compounds in seafood (109, 182). moribund state (198) and checklists for detailed observation of
clinical signs that attempt to minimize subjective interpretation
Published Censure of MBAs for Routine Seafood of toxicological endpoints (199). But probably the largest
Safety Testing—Animal Welfare Issues contribution to an admittedly still rather small literature base on
nonlethal experimental toxicology concerns the measurement
As well as criticism on technical grounds, as discussed above, of regulated hypothermia in rodent models, which we now
continuing use of MBAs for seafood safety testing has been explore in the context of harmful algal toxin research and food
challenged by scientists working within the fields of harmful safety testing.
algae and seafood safety, and by authors with a primary interest
in animal welfare issues. Many HAB workers recognize the Regulated Hypothermia as a Toxicological Endpoint
ethical shortcomings of these tests, and they acknowledge that
these concerns have in the past and continue to be one of the Hypothermia has long been known to accompany
principal motivations for the move towards alternative testing experimental poisoning of animals; for example, Metzler
methods (e.g., 183–187). Campbell et al. note that these tests are in 1891 noted that a dog dosed with acetone lowered its
“widely considered to be antiquated, cruel methods…” (188). temperature to 34.3°C. More recently, Gordon and colleagues
Researchers and policy workers with a primary interest in found that laboratory rodents lower their body temperature in
laboratory animal welfare, who publish in journals indexed response to a range of toxic agents, including pesticides, toxic
by bibliographic databases commonly accessed by experts in metals, and organic solvents (200). Gordon established that
the biological and biomedical sciences (e.g., PubMed, Web of hypothermia in mice as a response to various toxic challenges is
Science), have forcefully expressed disapproval of MBAs for a regulated phenomenon, and that affected animals will not seek
shellfish safety testing (172, 189–191). These papers, together warmer ambient temperatures in an attempt to return to thermal
with an authoritative review by the Federal Institute of Risk equilibrium (201).
Assessment in Germany (192), represent an additional burden A hypothermia-inducing dose, the so-called HID , was
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of criticism directed towards routine shellfish safety testing presented as “…a more objective, reproducible, and earlier
that has potential to reflect unfavorably on the legitimate endpoint than that used in conventional LD estimation” (202).
50
use of well-designed in vivo studies for algal toxin research. The HID test measured the effects of pathogenic bacteria
50
Justifiably negative appraisal of the current use of MBAs for injected into mice; the authors suggested that HID has
50
shellfish safety testing from both HAB toxin experts and animal potential for acute toxicity testing.
welfare advocates arguably makes things incrementally more Hypothermia in laboratory rodents can be monitored by
difficult for scientists seeking to conduct research utilizing continuous radiotelemetry, whereby a small transmitter is
modern, exemplary ethical standards. implanted into the peritoneal space by a simple surgical
364 Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014
procedure; mice are allowed to recover for a week or so from of the two to modify into a nonlethal endpoints test, as the
the implant procedure, after which time data is continuously time between exposure and endpoint—currently death—
generated without further disruption to the animals’ behavior is very short; the official method requires the assay to be
and activities (203, 204). These systems are suitable only standardized such that death occurs 5–7 min after injection of
for research applications, as the equipment and operating test material (27). But that understanding arguably should not
costs are considerable. For high-throughput work such as preclude a modest research endeavor to assess the capacity of
shellfish safety testing, intermittent rectal thermometry is a hypothermia to serve as a reliable, predeath indicator of PST
potentially reliable, low-cost technique requiring a mouse intoxication. The greatest potential for refinement of these lethal
rectal probe, (a Type T thermocouple) and a battery-powered endpoint tests arguably lies with the lipophilic toxins assay,
digital thermometer (205). The procedure is simple to conduct currently conducted with death occurring some hours after
and well tolerated (particularly in mouse strains exhibiting exposure to toxicologically relevant doses. In the event that a
relatively low levels of handling stress, e.g., Balb/c), and core replicable pattern of measurable core hypothermia, maintained
temperature data are rapidly obtained. Minimally invasive over a consecutive series of readings in mice injected with
techniques for measuring regulated hypothermia in mice may lethal doses (i.e., shellfish harvesting-intervention doses) of
be achievable: tympanic membrane infrared thermometry has lipophilic toxins can be demonstrated—and contingent on the
been investigated experimentally on rodents such as rabbits same pattern failing to be observed with sublethal doses—then
and guinea pigs (206, 207); mouse skin temperature appears to this technique might be considered as a means of revising and
correlate well with rectal core temperature (208). refining the lipophilic toxin MBA.
The ciguatera toxin bioassay has considerable potential to
Hypothermia in Rodents in Response to Algal and be modified as a nonlethal test, given that this toxin is already
Cyanobacterial Toxins known to provoke hypothermia in mice (210–212). Indeed,
hypothermia measured by rectal thermometry is suggested as
Some microalgal and cyanobacterial toxins can initiate a adjunct data in the published ciguatera MBA (42). We have no
hypothermia response in laboratory rodents. Wheeler et al. information or estimates regarding current levels of use of the
in 1942 (32) showed that mice injected with toxic Microcystis ciguatera bioassay, and chemical methods are now available
become hypothermic, although early experiments with crude with quantification limits that meet or approach the guideline
cyanobacterial preparations need to be interpreted with caution in level of 0.01 ppb P-CTX-1 recently established by the FDA
this regard, as cyanobacteria contain lipopolysaccharides (LPSs) (56, 130, 220). A modest research program to investigate
in their cell walls. LPSs from heterotrophic bacteria, particularly the utility of modifying the ciguatera MBA as a nonlethal
the Enterobacteriaceae, are classical pyrogens; regulated endpoints test could be conducted if warranted by current and
hypothermia in small rodents in response to pyrogen exposure anticipated future usage levels. For the STX and lipophilic
is caused by immunological mechanisms analogous to those that toxins shellfish safety bioassays, similar considerations would
cause pyrexia in larger mammals such as humans (201). Purified need to apply with respect to hypothermic responses of
cyanobacterial LPSs will initiate hypothermia in parenterally exposure to the respective solvents. We discuss below some
dosed mice (205). The cyanotoxin cylindrospermopsin also particular aspects of the acidified aqueous vehicle used for
triggers core hypothermia in mice (209). the PST MBA concerning the potential for localized irritant
Several types of marine microalgal toxins are known to effects of the acidic vehicle. The dispersant vehicle (1%
cause hypothermia in laboratory rodents. Separate research Tween 60) used in the lipophilic toxins bioassay (41) might
groups have shown that ciguatera toxins will initiate a rapid be considered unlikely to initiate hypothermia in parenterally
and profound hypothermia in mice (210–213). As with exposed mice; if shown to be correct, the most important ethical
ciguatoxin, the structurally related cyclic polyethers maitotoxin benefits and the least methodological challenges might accrue
and brevetoxin also initiate hypothermia in mice (214); to the lipophilic toxins bioassay, should any or all of the three
parenteral exposure to brevetoxin provokes core and peripheral seafood safety bioassays be assessed for their potential to be
hypothermia in rats (215). modified as nonlethal endpoints tests. As for the current and
With regard to the four currently sanctioned MBAs for anticipated future usage levels of both the PST and lipophilic
seafood safety testing (brevetoxins, ciguatoxins, lipophilic toxin toxins assays, our suggestion that these tests be subject to
group, and STXs), brevetoxins and ciguatera toxins are known research investigation to determine the viability and reliability
to initiate rodent hypothermia, as noted above. Toxins of the of converting these assays to non-lethal endpoints tests using
STX family have not, to our knowledge, been investigated for regulated hypothermia and/or other humane outcome measures
their capacity to cause hypothermia in dosed mice. However, could be subject to challenge. Grounds for such challenge might
STX and the functionally-related biotoxin tetrodotoxin were be along the lines that these assays have limited future demand
shown in experiments from the 1960s and 1970s to cause because of the increasing acceptance of alternative chemical,
hypothermia in cats (216–218). A qualitative estimate of antibody and ex vivo bioassays by regulatory agencies in a
hypothermia, along with other nonlethal endpoints, has been growing number of countries. However, a strong case for the
adopted by Food Standards Agency Scotland for its diarrhetic continuing use of bioassays rests on the knowledge that these
shellfish toxin MBA (219). functional assays provide a measure of total sample toxicity,
Should toxins in the lipophilic and/or STX groups be shown whereas chemical and antibody-based tests do not. This is an
capable of initiating measurable and sustained hypothermia unresolved problem for non-bioassay methods, particularly
in mice at toxicologically relevant doses, this response has where several congeners with a broad range of toxicities are
the potential to be exploited as a nonlethal endpoint for these present in particular samples, or where uncertainties exist
bioassays. The PST bioassay would be the most difficult because toxicity equivalence factors are either not known or
Stewart & McLeod: JournaL of aoac InternatIonaL VoL. 97, no. 2, 2014 365
were not derived from oral exposure studies (221, 222). Some rectal thermometry (228). The assay thus modified by dermal
food safety agencies therefore argue that there will continue temperature measurement has subsequently been validated,
to be a need to monitor shellfish and other seafood by use with the recommendation that the revised assay is suitable as
of functional assays like the MBA. Ex vivo assays such as an interim test until such time as a robust and reliable in vitro
the receptor binding assay, recently adopted by AOAC as a assay can be adopted (229). Regulatory agencies are divided with
reference method for PSTs (89), research and development into regard to acceptance of a hypothermia endpoint in the MBA for
invertebrate bioassays (103–105), and in vitro assays such as pertussis vaccine safety: U.S. and European authorities insist on a
the mouse neuroblastoma assay may offer solutions here. But lethal endpoint test, while Japanese regulators and WHO accept a
where the MBA continues to be used for reasons including hypothermia endpoint (230).
familiarity, track record of safety, or convenience, there are
grounds to argue that this in vivo test should be refined in order The Laboratory Mouse in Harmful Algal Toxin
to comply with modern animal welfare standards, particularly Research
with regard to humane endpoints.
A recent estimate suggests some 400 000 mice are used While the principal focus of this discussion has been on
annually for marine biotoxin testing in Europe (223). This MBAs for marine biotoxin shellfish safety testing, the laboratory
number should decrease substantially in Europe over coming mouse has been, and continues to be, widely utilized for research
years as the lipophilic toxins bioassay is phased out by end of investigations into harmful algal and cyanobacterial toxins. The
2014 (182). Canada until recently used more than 40 000 mice mouse has been an invaluable catalyst for discovery in this field.
annually for PST monitoring (224). New Zealand’s response Bioassay-guided fractionation has been a fundamental tool
following its initial shellfish biotoxin event in 1993 used facilitating the isolation and purification of toxic compounds
an estimated 80 000 mice annually; however, this number from crude extracts (231). The discovery of the freshwater
declined considerably over ensuing years with the introduction cyanobacteria toxin cylindrospermopsin serves as an example
of phytoplankton monitoring and chemical methods for toxin here. A drinking water-associated toxin was thought responsible
detection (225). Both Canada (224) and the United Kingdom for an acute hepato-enteritis outbreak in North Queensland,
(226) have now discontinued MBA testing for shellfish safety. We Australia, in 1979 (232, 233). Extracts of a common tropical
have no information or estimates regarding the number of mice cyanobacterium, Cylindrospermopsis raciborskii, were found
used by other countries for shellfish safety testing; presumably to be toxic to mice (31). The toxic alkaloid cylindrospermopsin
those combined numbers are large, considering the estimates was purified from cyanobacterial extracts through bioassay-
from Europe and Canada. Demand for access to the MBA for guided testing of chromatographic fractions (234). The
shellfish safety testing appears likely to continue, in some mouse has also been integral to understanding a wide range
countries more so than others, for reasons including convenience, of biological responses to exposure to many, if not all,
cost, regulatory inertia and the irregular global availability HAB toxins, from toxicokinetics to immunotoxicology,
of analytical chemistry skills and infrastructure. Therefore, neurotoxicology, mechanistic toxicology and determination of
arguments that no further revision or refinement of MBAs can metabolic pathways. The acute toxicity of purified HAB toxins
be justified on the grounds that they face imminent and absolute and extracts of source microalgae and cyanobacteria has long
substitution by alternative chemical, antibody and ex vivo assays been determined experimentally by mouse intraperitoneal LD
50
may be flawed. Considering that the MBA is likely to be used for or oral toxicity studies. This practice, with death as an endpoint,
shellfish testing for some years to come, investigating the utility continues into the present day (e.g., 39, 40, 222, 234–236).
of modifying the tests to incorporate regulated hypothermia and/
or other nonlethal endpoints is warranted. If successful, flow-on Other Humane Endpoints for MBAs
benefits would accrue to the use of laboratory mice for HAB
research endeavors, as discussed below. The discussion above has focused on the potential utility
of regulated hypothermia as a rapidly acquired, objectively
Modification of a Vaccine Safety MBA to a Nonlethal measurable endpoint of acute intoxication caused by exposure
Endpoints Test to a structurally diverse range of harmful algal toxins. However,
even if a HID test is found to be a useful model for acute
50
In an endeavor that parallels the themes outlined in this paper intoxication by microalgal and cyanobacterial toxins, the
for revision of seafood safety testing MBAs, an MBA for QC movement toward improved ethical standards will continue.
testing has been subject to modification and refinement in order It is likely that the molecular and behavioral responses to
to move from a lethal endpoints test to a hypothermia endpoint acute intoxication, involving a cascade of pro-inflammatory
for detection of a toxic contaminant. Testing of acellular pertussis cytokines, regulated hypothermia and sickness behavior
vaccines for the presence of residual pertussis toxin is conducted manifesting in social isolation, immobility, anorexia and
with MBAs, originally designed—and still required by some adipsia are experienced by laboratory rodents as malaise, just as
regulatory agencies—as a lethal endpoints test. Japanese workers humans experience the symptoms of influenza caused by similar
took the lead in modifying the assay. Using hypothermia by rectal molecular mechanisms (238–241). So use of nonlethal endpoints
thermometry as an endpoint, the precision and sensitivity of the such as hypothermia or systematically determined moribundity
test was considerably enhanced compared to that of the lethal will not affect the severity of suffering, only its duration.
endpoint assay (227). Further refinement of the test, again from Experimental investigations into non-recovery anesthesia for
Japan with animal welfare considerations in mind, showed that the PST MBA showed that time to death is increased two-fold,
dermal temperature measured by IR thermometry on abdominal presumably because of anesthesia-associated hemodynamic
skin was equally as efficacious as core temperature acquired by decrements. The method shows promise, although assays using