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CFSAN Regulatory Codes: VI.C.
CFSAN Program Priority Codes:
Start Date: 00    Completion Date: 00

Statement of Research Problem:
While plankton are, in general, a vital component of the marine biosphere, some species produce potent toxins that accumulate in seafood and put human consumers at risk. Existing management programs have dealt moderately well with the problem in the past, but are challenged by novel toxins and changing temporal and special patterns of occurrence. At present, the publicity given Pfiesteria has aroused concerns that the toxins it produces might accumulate in seafood. Despite the lack of any evidence of a public health risk, solid data are needed to properly address the situation. Aside from this, there is a general need for a better understanding of the various kinds of organisms and toxins known to cause human illness, better detection methods for them, and development of novel management strategies that will provide better detection at lower cost

Specifically, goals will focus on characterizing biotoxins, such as the known toxins of paralytic shellfish poison (PSP), neurotoxic shellfish poison (NSP toxins, primarily brevetoxin and metabolites=PbTx), diarrhetic shell fish poison (DSP), etc., ciguatera fish poisoning toxin (CFP), and totally unknown toxins such as those recently evident in Pfiesteria piscicida and buffalo fish that may be present in seafood. Levels of contamination likely to pose human health hazards will be assessed and the means/tools needed to control such biotoxins developed. These are multidisciplinary studies involving chemistry (including elucidation of structure as a basis for developing quantitative tests), toxicology and molecular biology.

Statement of Project Objective(s):
Provide tools for establishing control procedures, critical control points and critical limits (i.e. action levels) for all biotoxins affecting consumers of seafood. Develop management tools and strategies to address the problem of seafood contamination by natural toxins from plankton. Provide improved detection methods. Develop more cost-effective, reliable programs for scientifically based marine biotoxin management and ensure that existing programs deal effectively with new threats.

Anticipated Impact on FDA Regulatory Program:
Effect the establishment of control procedures, critical control points and critical limits (i.e. action levels) for biotoxins affecting consumers of seafood.

Project Priority Changes During FY2000: none

Administrative Liaison(s): George P. Hoskin: 202/418-3172

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
Dickey, R.	OS/DSATOS	1.0, 1.0, 1.0	1-5
Plakas, S.	OS/DSATOS	1.0, 1.0, 1.0	2
Granade, R.	OS/DSATOS	1.0, 1.0, 1.0	1-3
El Said, K.	OS/DSATOS	1.0, 1.0, 1.0	2
Jester, E.	OS/DSATOS	1.0, 1.0, 1.0	1-3
Mowdy, D.	OS/DSATOS	1.0, 1.0, 1.0	1-3
	Total FTE:	6.0, 6.0, 6.0

Other Personnel:


Name	Office/Division	FTE [00,01,02]	Component
Johannessen, J.	OARSA/DTR	0.4, 0.4, 0.4	1-4
Jackson, R.	OARSA/DTR	0.2, 0.2, 0.2	1-4
Rabbani, I.	OARSA/DTR	0.4, 0.4, 0.4	1-4
	Total FTE:	1.0, 1.0, 1.0

Collaborators: Dr. S. Musser (CFSAN); Dr. M. Poli (USAMRIID); Dr. K. Shea (UC-Irvine); Dr. G. Martin (Pharmacia-Upjohn); Dr. D.J. Faulkner (Scripps Instit. Oceanogr.)

Component 1 Objectives:


1. Develop and evaluate in vitro methods as alternatives to official mouse bioassay.
   
2. Convert existing PbTx radioimmunoassay to enzyme-linked immunosorbant assay (ELISA) format and evaluate as alternative to mouse bioassay
Component 1 FY 2000 Deliverables:

1. Adapt anti-brevetoxin antibodies to ELISA format.
   
2. Test performance fidelity against standard brevetoxins and NSP shellfish matrices. Recommend continuation or discontinuation of NSP ELISA development.
Component 1 FY 2000 Progress:
Component 1, deliverable 1 is incomplete. The anti-brevetoxin antibodies have been formatted for microplate ELISA assay, however, some loss in affinity for standard brevetoxin was observed. NSP shellfish matrices have not been tested against this format. It will be necessary to synthesize new ligand and optimize conjugation chemistry for binding of ligand to microplate substrate. Deliverable will be extended into FY01
Technical Barriers to Meeting Component 1 Objectives or Deliverables: none listed
Component 1 FY 2001 Deliverables:

1. Complete FY2000 deliverable.
   
2. Reduce complexity of NSP-ELISA to suit field application of immunoassay (e.g. develop dip-stick test) and test against NSP shellfish matrices. Recommend use, limitations for use, or rejection.
Component 1 FY 2002 Deliverables:
Explore feasibility of developing molecular imprintable polymer (MIP: a rugged "artificial" antibody) assay for NSP as a less expensive and durable alternative to biological antibody-based immunoassay. Recommend development or rejection of NSP-MIP assay concept.

Component 2 Objectives:


1. Investigate NSP biotoxin/metabolite in vivo dose-response by intraperitoneal and peroral routes of administration.
   
2. Identify relationship to naturally incurred NSP biotoxin/metabolite residues, and assess validity of current NSP guidance level.
   
3. Investigate brevetoxin absorption, metabolism and elimination in shellfish by exposure to radiolabelled toxin under controlled laboratory conditions.
Component 2 Deliverables:

1. Describe chemical identification of brevetoxin metabolites from eastern oyster.
   
2. Determine toxicological significance of parent brevetoxins and brevetoxin metabolites from NSP contaminated shellfish.
   
3. Complete study of radiolabelled brevetoxin absorption, metabolism and elimination in the eastern oyster.
Component 2 FY2000 Progress:

1. Component 2, deliverable 1 is complete: the molluscan metabolites of brevetoxin have been identified.
   
2. Component 2, deliverable 2 is complete: however, this deliverable is to be expanded to include a more thorough examination of parent toxin and molluscan metabolite contributions to composite toxicity.
   
3. Component 2, deliverable 3 is incomplete: in vitro characterization of molluscan absorption, metabolism and elimination of brevetoxins has made excellent progress against significant experimental obstacles and gaps in the knowledge base. The in vitro study has reproduced toxin metabolism profiles observed in situ red tide episodes and is tracking well toward completion in FY2001.
Technical Barriers to Meeting Component 2 Objectives or Deliverables:
Component 2, deliverable 3: difficulties were encountered with regard to radiochemical purity and stability of commercially obtained 3H-brevetoxin.
FY 2001 Deliverables:

1. Complete study of radiolabelled brevetoxin absorption, metabolism and elimination in the eastern oyster
   
2. Report on brevetoxin absorption, metabolism and elimination in eastern oyster (Crassostrea virginica). Propose criteria for regulatory applications.
   
3. Report on NSP-mouse bioassay toxicological assessment.
FY 2002 Deliverables:

1. Prepare report in-house addressing relevance of study to current NSP guidance level.
   
2. Assess the need for extension of NSP-brevetoxin metabolism studies in other aquatic food species (e.g. gastropods, finfish, crustaceans). If insufficient basis for support is found this component will be concluded.
   

Component 3 Objectives:
Isolate/purify ciguatoxins from toxic finfish collected from ciguatera endemic regions. Refine in vitro and instrumental methods for the determination of CFP in finfish.
Component 3 Deliverables:


1. Isolate and characterize ciguatoxin standards for use in methods development and toxicological assessment.
   
2. Describe comparison of cytotoxicity with LC/MS methods for determination of ciguatoxins in finfish. Recommend use, limitations for use, or rejection.
   
3. Compare other methods against cytotoxicity and LC/MS for the determination of ciguatoxins in finfish.
Component 3 FY2000 Progress:
Component 3, deliverable 1 is complete. Caribbean ciguatoxin C-CTX-1 has been isolated and is currently used as a standard in ciguatera outbreak analyses. Additional ciguatoxin standards are near final stages of chromatographic purification and will be characterized by mass spectral and NMR when final products are obtained.
Component 3, deliverable 2 is complete. In vitro cytotoxicity assays and benchtop LC-MS have been combined with some success in ciguatera outbreak investigations. The combination of methods provides semi-quantitative sub-ppb screening of fish for sodium channel specific toxicity and confirmation of ciguatoxin (CTX) by LC-MS.
Component 3, deliverable 3 is incomplete. The comparison of in vivo mouse bioassay, in vitro cytotoxicity, LC/MS, immunoassay and native receptor binding has been delayed pending completion of other component deliverables. In vivo, in vitro and immunoassays have been compared and an interim report has been submitted to management. The study awaits matching LC/MS and native receptor binding data for completion.
Technical Barriers to Meeting Component 3 Objectives or Deliverables: none listed
FY 2001 Deliverables:

1. Continue Isolation and characterization of ciguatoxin standards for use in methods development and toxicological assessment.
   
2. Describe comparison of in vitro with LC/MS methods for determination of ciguatoxins in finfish. Recommend use, limitations for use, or rejection.
   
FY 2002 Deliverables:

1. Continue Isolation and characterization of ciguatoxin standards for use in methods development and toxicological assessment.
   
2. Explore feasibility of developing immunochemical assay for CFP. Recommend development or rejection of CFP immunoassay concept.

Component 4 Objectives:
Develop in vivo dose-response model for incurred ciguatoxin residues in finfish; determine correlations with in vitro and instrumental methods of analysis (incl. case/outbreak analyses where possible); and propose critical levels/limits for control of CFP seafood hazard.
Component 4 Deliverables:


1. Design study protocol and perform range-finding experiments for mouse bioassay dose-response for purified and naturally incurred ciguatoxins. Determine correlation with in vitro and instrumental methods.
Component 4 FY2000 Progress:
Component 4, deliverable 1 is incomplete and will be rescheduled for FY2001.
Technical Barriers to Meeting Component 2 Objectives or Deliverables: none listed
FY 2001 Deliverables:

1. Design study protocol and perform range-finding experiments for mouse bioassay dose-response for purified and naturally incurred ciguatoxins. Determine correlation with in vitro and instrumental methods.
   
FY 2002 Deliverables:

1. Complete study of CFP mouse bioassay dose-response. Propose regulatory guidance level for CFP.

Component 5 Objectives:
Prepare large-scale cultures of okadaic acid producing algae, isolate and purify okadaic acid standards. Characterize okadaic acid absorption, metabolism and elimination in shellfish under controlled laboratory conditions. Isolate molluscan metabolites of DSP toxins and elucidate chemical structures.
Component 5 Deliverables:


1. Prepare large-scale cultures; isolate and prepare okadaic acid standards for use in shellfish exposure studies.
   
2. Design study protocol and perform range-finding experiments for okadaic acid absorption, metabolism and elimination in selected shellfish species.
Component 5 FY2000 Progress:
Component 5, deliverable 1 is complete. Okadaic acid standard has been produced and is available for use in shellfish exposure studies.
Component 5, deliverable 2 is incomplete. Design of study protocol and range-finding experiments will not begin until completion of brevetoxin metabolism study. Deliverable will be rescheduled for FY2001.
Technical Barriers to Meeting Component 5 Objectives or Deliverables: none listed
FY 2001 Deliverables:

1. Design study protocol and perform range-finding experiments for okadaic acid absorption, metabolism and elimination in selected shellfish species.
FY 2002 Deliverables:

1. Complete study of okadaic acid absorption, metabolism and elimination in selected shellfish species.

Dickey R, Jester E, Granade R, Mowdy D, Moncreiff C, Rebarchik D, Robl M, Musser S (1999) Monitoring brevetoxins during a Gymnodinium breve red tide: comparison of cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts. Natural Toxins 7(4):157-165.

MA, Musser SM, Dickey RW, Eilers PP, Hall S (2000) Neurotoxic shellfish poisoning and breve toxin metabolites: a case study from Florida. Toxicon 38(7):981-993.

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Hypertext updated by dav 2001-OCT-02