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CFSAN Regulatory Codes: IV. A
CFSAN Program Priority Codes: 2.16a, 2.16b
Start Date: 10/1/99    Completion Date: 9/30/02

Research Problem:
Mycotoxins produced by molds are common contaminants of many important food crops, including wheat, corn, tree nuts, and peanuts. Some mycotoxins are found in fruits and vegetables. These contaminants have a broad range of toxic effects on animals, including carcinogenicity, immunotoxicity, neurotoxicity, and reproductive and developmental toxicity. They could have potential human toxicity. Regulatory limits, including international standards, have tremendous economic impact and should be developed on science-based risk assessments to have validity. The purpose of this research is to generate the information about exposure and toxicity data necessary for risk assessment, and to develop analytical methods to support control programs. Studies are designed to evaluate the effects of processing on mycotoxin levels in human foods and to assess various types of toxicity.

Project Objective(s):
This project will develop information needed for hazard identification (identification and production of mycotoxins), risk assessment (occurrence and toxicity) and risk management (development and validation of rapid analytical methods for monitoring to support regulatory actions).

Anticipated Impact on FDA Regulatory Program:
Project will provide the Agency with necessary data for setting and enforcing FDA guidelines on mycotoxins.

Project Priority Changes During FY2000:
No changes were necessary.

Administrative Liaison(s): Mary W. Trucksess: 202/205-4429

Research Personnel:


Name	Office/Division	FTE [00, 01, 02]	Component
Robert E. Eppley	OPDFB/DNP	1.0, 1.0, 1.0	1,2,3
Michael E. Stack	OPDFB/DNP	1.0, 1.0, 1.0	1,2,3
Frederick Thomas	OPDFB/DNP	1.0, 1.0, 1.0	3
Valerie Tournas	OPDFB/DNP	1.0, 1.0, 1.0	1,2
Mary W. Trucksess	OPDFB/DNP	0.5, 0.5, 0.5	3
Kathryn Young	OPDFB/DNP	1.0, 1.0, 1.0	1,3
Lauren S. Jackson	OPDFB/DFPP	0.5, 0.5, 0.5	4
Thomas Black	OARSA/DTR	0.5, 0.5, 0.5	5,6,7
Thomas Collins	OARSA/DTR	0.5, 0.5, 0.5	5,6,7
Larry Garthoff	OARSA/DTR	1.0, 1.0, 1.0	5,6,7
Dennis Hinton	OARSA/DTR	0.5, 0.5, 0.5	5,6,8
Stuart Long	OARSA/DTR	0.4, 0.4, 0.4	5,6,7
Nicholas Olejnik	OARSA/DTR	0.6, 0.6, 0.6	5,6,7
Dorothy Quander	OARSA/DTR	0.4, 0.4, 0.4	5,6,7
Roscoe Brown	OARSA/DTR	0.3,0.3, 0.3	5,6,7
James Rorie	OARSA/DTR	0.6, 0.6, 0.6	5,6,7
Michael Scott	OARSA/DTR	0.5, 0.5, 0.5	5,6,8
Matthew Smith	OARSA/DTR	0.4, 0.4, 0.4	5,6,7
Thomas Sobotka	OARSA/DTR	0.4, 0.4, 0.4	5,6,7
Rene Sotomayor	OARSA/DTR	1.0, 1.0, 1.0	6,8
Robert Sprando	OARSA/DTR	0.6, 0.6, 0.6	5,6,7
Melissa Washington	OARSA/DTR	1.0, 1.0, 1.0	6,8
	Total FTE	14.7,14.7,14.7

Collaborators: Dr. James Pestka, Michigan State University (#5). Dr. Fred Hines, Pathology Branch, OSS, CFSAN (#5,6,7,8). See also component descriptions and FY 2000 publications.

Component 1 Objectives:
Isolate, purify and identify suspect mycotoxins from Fusarium species, and other fungi identified as frequently occurring in foods. Produce selected mycotoxins by culturing the mold on grains or other suitable natural substrates. Isolate suspect mycotoxins for toxicological evaluations and for analytical reference standards.
Component 1 FY 2000 Deliverables:
Produce cyclopiazonic acid and isolate sufficient quantities for preliminary toxicological evaluation.
Component 1 FY 2000 Progress:
Literature search for production of cyclopiazonic acid (CPA) completed. CPA could be produced in liquid medium inoculated with Aspergillus flavus. Liquid chromatographic technique could be used for isolation and identification of the toxin. Support was provided to quality assurance of mycotoxin toxicological studies.
Technical Barriers to Meeting Component 1 Objectives or Deliverables: none listed
Component 1 FY 2001 Deliverables:


1. Isolated sufficient amount of Fusarium toxins for long term toxicological studies.
   
2. Develop procedure to isolate and identify toxins produced by molds found in foods including fresh produce.
Component 1 FY 2002 Deliverables:

1. Continue preparation of Fusarium mycotoxins.
   
2. Isolate and purify sufficient quantities of mycotoxins produced by molds found in foods and fresh produce for use in preliminary toxicological evaluation.

Component 2 Objectives:


1. To isolate and identify potentially toxigenic molds from fresh and minimally processed produce (excluding juice).
   
2. To test these isolates for toxin production.
Component 2 FY2000 Deliverables:

1. Conducted a limited survey on mold contamination of sprouts, ready-to-eat salads and pretreated (surface disinfected) fruits.
   
2. Start toxicity screening of isolated molds.
Component 2 FY2000 Progress:

1. About 200 samples of surface-disinfected (sodium hypochloride) fruits and produce (sprouts, ready-to-eat salads and pretreated fruits such as grapes, blueberries, strawberries, raspberries, blackberries, oranges, tangerines, lemons and limes) have been analyzed for mold contamination. Mold isolates have been identified.
   
2. Screened for toxicity of metabolites from molds isolated from disinfected fruits has been started. The brine shrimp and Bacillus megaterium bioassays have been used for the toxicity screening.
Technical Barriers to Meeting Component 2 Objectives or Deliverables:
Lack of an appropriate microscope-video camera-monitor system has slowed down the mold identification process.
FY 2001 Deliverables:
1. Continue testing FY2000 isolated molds for toxicity using the brine shrimp and Bacillus megaterium bioassays.
   
FY 2002 Deliverables:

1. Optimize growth conditions for molds that have been identified as toxic metabolite producers.
   
2. Maintain culture for toxin production and cooperate with Component 1 deliverables.

Component 3 Objectives
Develop and validate improved methods for rapid testing, investigate chromatographic methods, immunochemical methods, and emerging analytical techniques.
Component 3 FY 2000 Deliverables


1. Continue study of method for trichothecenes.
   
2. Report results of patulin in fruit juice study.
   
3. Develop method for ochratoxin A in grape juice and wine.
   
4. Validate a rapid method for fumonisins in corn and sorghum by conducting an international collaborative study.
   
5. Investigate microwave extractor for the analysis of mycotoxins in foods.
Component 3 FY 2000 Progress

1. Unexpected problems were encountered using a new gas chromatograph for trichothecene method development.
   
2. A method has been evaluated and will be applied to the analysis of wine and beer for ochratoxin A.
   
3. An immunoaffinity column cleanup and solution fluorometric method for fumonisins has been evaluated. The method performance is not yet suitable for a collaborative study.
   
4. Preliminary results of using microwave extractor for extraction of mycotoxins from foods showed little advantages over the conventional extraction.
   
Technical Barriers to Meeting Component 3 Objectives or Deliverables: none listed
Component 3 FY 2001 Deliverables

1. Develop analytical method for moniliformin in grains and prepare report.
   
2. Continue study of methods for trichothecenes.
   
3. Prepare report for ochratoxin A in wine, beer and grape juice.
   
4. Develop rapid liquid chromatographic/mass spectrometric (LC/MS) method for suspect mycotoxins in foods implicated in poisonings.
   
5. Continue investigation the use of microwave extractor for mycotoxin analysis.
Component 3 FY 2002 Deliverables

1. Continue development of rapid LC/MS methods for mycotoxins implicated in food poisonings.
   
2. Develop rapid methods for mycotoxins using biosensor.
   
3. Investigate the use of immunochemical methods for mycotoxins and other food contaminants (such as food allergens if Project 10 includes other food contaminants).

Component 4 Objectives:
Determine the effects of processing/chemical treatments on mycotoxins in foods.
Component 4 FY 2000 Deliverables


1. Report on the effects of electron beam irradiation with and without chemical treatments on a mycotoxin in corn.
   
2. Report on the effect of thermal processing in one selected mycotoxin produced by Fusarium species.
Component 4 FY 2000 Progress:

1. Use of electron beam irradiation on mycotoxin in corn has not been investigated.
   
2. A manuscript has been prepared to report the effects of thermal processing on fumonisins in foods.
Technical Barriers to Meeting Component 4 Objectives of Deliverables: none listed
Component 4 FY 2001 Deliverables:
Report on the effects of processing/chemical treatments on a mycotoxin produced by a selected Fusarium species.
Component 4 FY 2002 Deliverables:

1. Determine the effects of thermal processing and chemical treatments on moniliformin, zearalenone and other mycotoxins.
   
2. Study the effects of electron beam irradiation in combination with peroxide treatments on aflatoxin levels in contaminated grain.

Toxicological investigation of the effects of sub-chronic exposure to dietary, deoxynivalenol [DON], in IL-6 Deficient Mice. Collaborative study with Michigan State University.

Objectives:
Compare the known toxicological effect of DON on the immune (IgA) mediated nephropathy in kidneys in various mice strains in order to evaluate the possible immune mechanisms.
Evaluate the potential effects of DON on other target organs such as male reproduction and neurology (e.g., brain).
Component 5 FY 2000 Deliverables:


1. Complete the pathology evaluation and imaging analysis of the lymphoid tissues.
   
2. Complete neurochemical and histological analyses of sections of brain tissue to assess treatment related changes in neurocellular protein profiles and histochemical signs of neurodegeneration, microglial proliferation, and altered brain barrier permeability.
   
Component 5 FY 2000 Progress:.

1. The pathology report from EPL was submitted to FDA in June, 00. In addition, digital image acquisition of tissues has also been completed. The "morphometric" analysis is also near completion. Special immunostains (i.e. IgG and IgA) and in-situ hyrbridization staining for IL-6 and IL-2 gene expression is in progress.
   
2. In collaboration with NCTR, histological evaluations of brain sections revealed no treatment related neuropathological findings including neuronal degeneration, microglial proliferation, or changes in blood brain barrier permeability. Brain weights were also unaffected by treatment. In the absence of positive findings to guide selection of target brain areas, the plan for subsequent neurochemical analysis may be reconsidered.
Technical Barriers to Meeting Component 5 Objectives or Deliverables: none listed
Component 5 FY 2001 Deliverables:

1. Collaborate with Michigan State University on completion of manuscript for the immunotoxicity study.
   
2. Prepare final report/manuscript of work on the neurotoxicity of DON in IL-6 study.
Component 5 FY 2002 Deliverables:

1. Although all work in this component should be completed FY 2001, additional collaborative work with Michigan State University is being considered.

Selected toxins will be administered to rodents (rats) to evaluate their potential developmental, molecular (DNA damage), male reproductive, immuno- and neurotoxicity.

Objectives: Conduct comprehensive, multidisciplinary studies on selected mycotoxins to provide information which can be used in development of risk assessment guidelines and establishing "action levels". Specifically: (1) evaluate the effects of DON on both, female (in-utero exposure) and male reproductive functions in short tem dosing studies; (2) evaluate a new rat model which shows promise of being more sensitive immunologically (and neurologically) in toxicological studies of the various mycotoxins (e.g. DON); and (3) evaluate the clastogenic effects of DON in short tem dosing studies with rats.
Component 6 FY 2000 Deliverables:


1. Complete DON developmental toxicity final study. Begin final report.
   
2. Complete DON male reproduction study.
   
3. Run zearalenone developmental toxicity preliminary study and develop final protocol.
   
4. Develop protocol with OPDFB to investigate neurotoxicity of repeated DON exposure in rats.
   
5. Initiate in-vitro/in-vivo biochemical/immunotoxicity rat studies of DON, after OPDFB consult.
Component 6 FY2000 Progress:

1. The final developmental toxicity study on DON was completed.
   
2. A study to characterize the effect of DON on the male reproductive function was concluded. The protocol was written and approved. The live portion of the study was completed.
   
3. Safety issues that delayed a preliminary zearalenone study have recently been resolved. A protocol for the preliminary study has been submitted to the Safety Office.
   
4. DTR personnel met with the Director, Division of Risk Assessment, OPDFB, with regard to the various planned studies in this component (immunologic, neurotoxic, reproduction). A protocol plan designed to evaluate the effects of DON using the immunologically hypersensitive rat met with approval. This protocol is under development.
   
5. An OPDFB approved protocol to evaluate the clastogenicity/immunotoxicity of DON was approved by line management and the Safety Office. IACUC approval is anticipated so that studies can begin in FY4/00.
Technical Barriers to Meeting Component 6 Objectives or Deliverables:
The safety questions on zearalenone provided barriers to progress.
Component 6 FY 2001 Deliverables:

1. Complete DON final report and manuscript.
   
2. Run zearalenone final study and prepare final report.
   
3. Complete protocol and Initiate multidisciplinary investigation of DON with hypersensitive rat.
   
4. Compete biochemical and immunotoxicity studies. Initiate preparation of the final report(s).
   
5. Develop reproductive toxicity preliminary protocol to evaluate DON/Zearalenone combinations.
Component 6 FY 2002 Deliverables:

1. Complete DON manuscript (Developmental Toxicity Study).
   
2. Complete DON manuscript (Male Reproductive Toxicity Study).
   
3. Continue assessing utility of the hypersensitive rat model for determining the toxicity of DON.
   
4. Initiate/complete manusript(s) of biochemical and immunotoxicity studies dependent on progress in FY 2001.

Determine the toxic effect of fumonisins on both male and female rats.

FY 2000 Component 7 Deliverables:


1. Complete the final report of totally hydrolyzed fumonisin B₁ developmental toxicity study.
   
2. Complete report of neurobehavioral effects of fumonisin B₁ in rats.
Component 7 Progress:

1. The data from the study of hydrolyzed fumonisin have been retrospectively entered into the SAS system and tables are being developed for statistical analysis. The final report will not be completed by the end of the year because of work on other projects.
   
2. The preparation of a final report for the GLP protocol BFQ-392-T, The Utility of the Adult Rodent as a Model for Fumonisin (FB1) Neurobehavioral Toxicity (PI: T. J. Sobotka), has been completed as scheduled. This final report has been submitted to the Quality Assurance Unit for review and audit.
   
Technical Barriers to Meeting Component 7 Objectives or Deliverables: none listed
FY 2001 Component 7 Deliverables:

1. Prepare manuscript for the development toxicity of totally hydrolyzed fumonisin B₁.
   
2. Prepare manuscript of neurobehavioral effects of fumonisin B₁.
FY 2002 Component 7 Deliverables:
Further work on fumonisin that may be needed will be assessed in FY 2001.

Determine DNA/RNA adduct formation and detoxification enzymes in select tissues, as well as changes in lymphocyte cell subsets and function in lymphoid tissue as a function of time and cumulative dose in a study of continuous vs. intermittent dosing of aflatoxin B₁ (AFB₁) to assess the effects of interactions of AFB₁ with fumonisin B₁, and with cyclopiazonic acid (CPA) on biochemical endpoints, e.g. DNA and RNA adduct formation, and immunologic endpoints.

Component 8 FY 2000 Deliverables:


1. Complete manuscript on adduct formation in rat liver.
   
2. Initiate manuscript preparation on comparison of adduct formation in the rat kidney and liver.
   
3. Complete a second manuscript comparing functional and ex-vivo analysis of immunologic endpoints with the histopathology results.
   
4. Develop protocol(s) to evaluate biochemical and immunologic toxicity associated with combinations of AFB₁ and fumonisin B₁ (in-vivo and in-vitro studies) using the rat and/or rat hepatocytes and lymphocytes.
   
5. Use human hepatocytes, lymphocytes, and/or explants for the above study.
Component 8 FY 2000 Progress:

1. The study on DNA and RNA adduct formation after continuous and intermittent exposure to AFB1 has been completed. Results were presented at the annual meeting of the Society of Toxicology March 19-23, 2000.
   
2. A manuscript on a comparison between the liver and kidney is in progress.
   
3. The immunologic effects of AFB1 as well as the effects seen in the morphometric (imaging) analysis of the lymphoid tissues were presented at the annual meeting of the Society of Toxicology March 19-23, 2000. The final pathology report on the effects was received from EPL. New methods for quantitative assessment of the effects on lymphoid organs are nearing completion for all of the various tissues. Various tissues are being evaluated by special immunostaining for various cell types and proliferative biomarkers, e.g., PCNA, Ki67 and H3 histone mRNA. We anticipate this will be the most extensive immunologic evaluation of AFB1 conducted to date. This work has resulted in the elucidation of the possible mechanism of the carcinogenic properties of AFB1.
   
4. The Director, Risk Assessment Division, OPDFB, has stated that although mixtures of mycotoxins are the "real" world situation, there is currently no way of handling the data for establishing guidelines. No protocols have been developed for studying combinations of AFB1 and fumonisin. However, USDA is now soliciting grant proposals for studying these combinations.
   
5. No work has been done on the in-vitro correlates. Refocusing in FY 2000 is anticipated when meetings with FSI and OPDFB staff are convened.
Technical Barriers to Meeting Component 8 Objectives or Deliverables: none listed
Component 8 FY 2001 Deliverables:

1. Initiate and/or continue studies on the interactions of AFB₁ and fumonisin B₁.
   
2. Develop protocol(s) to evaluate biochemical and immunologic toxicity associated with combinations of AFB₁ and cyclopiazonic acid (in-vivo and in-vitro studies) using the rat and/or rat hepatocytes and lymphocytes.
   
3. Use human hepatocytes, lymphocytes and/or explants for the above study.
Component 8 FY 2002 Deliverables:

1. Refocusing in FY 2000 is anticipated when meetings with FSI and OPDFB staff are convened.

Roach, J.A.G., White, K.D., Trucksess, M.W., and Thomas, F.S. (2000) "Capillary gas chromatography/mass spectrometry with chemical ionization and negative ion detection for confirmation of identity of patulin in apple juice. J. AOACI, 83, 104-112.

Sahu CS, Chou MW, Sotomayor RE, Hinton DM, Barton CN, O'Donnell MW (2000). Effects of intermittent exposure of aflatoxin B1 on hepatic and testicular glutathione S-transferase in rats. J. Appl. Toxicol. 20:215-219

Sotomayor, R.E., Sahu, S., Washington, M., Hinton, D.M., and Chou, M (1999). "Temporal Patterns of DNA Adduct Formation and Glutathione S-Transferase Activity in the Testes of Rats Fed Aflatoxin B1: A Comparison with Patterns in the Liver," Environ. and Molecular Mutagenesis, 33: 293-302

Sprando, R.L., Pestka, J., Collins, T.F. X., Rorie, J., O'Donnell, M., Hinton, D.M., and Chirtel, S. (1999). "The Effect of Vomitoxin (Deoxynivalenol) on Testicular Morphology, Testicular Spermatid Counts and Epididymal Sperm Counts in IL-6KO [B6129-IL6 <tmlKopf > (IL-6 gene deficient)] and, WT [B6129F2 (wild type to B6129-IL6 with an intact IL-6 gene)] mice," Fd. Chem. Toxicol., 37: 1073-1079

Trucksess, M.W. (2000) "Mycotoxins" J. AOACI, 83: 442-448.

Trucksess, M.W., Cho, T.H. and Ready, D.E. (2000) "Liquid chromatographic method for fumonisin B1 in sorghum syrup and corn-based breakfast cereals", Food Addit. Contamin., 17, 161-166.

Trucksess, M.W. and Tang Y. (1999) "Solid-phase extraction method for patulin in apple juice and unfiltered apple juice" J. AOACI, 82: 1109-1113.

Smith, G.W., Constable, P.D., Eppley, R.M., Tumbleson, M.E., Gumprecht, L.A., and Haschek-Hock., W.M. (2000) "Purified fumonisins B1 decreases cardiovascular function but does not alter pulmonary capillary permeability in swine" Toxicological Sciences, 56: 240-249.

Hinton, D.M.¹, Myers, M.J.², Raybourne, R.¹, Sotomayor, R.E.¹, Shaddock, J.³, Warbritton, A.³, and M Chou³ (¹USFDA, CFSAN, Laurel, MD; ²USFDA, CVM, Laurel, MD; ³USFDA, NCTR, Jefferson, AR.) "Dynamics of the immune system of the rat in an intermittent dosing study with aflatoxin B1 (AFB1)." 2000 FDA Science Forum, FDA and the Science of Safety: New Perspectives, Feb. 14-15, 2000, Washington, DC.

Stack, M. E. (2000) Aflatoxins and other mycotoxins: "Thin layer chromatography", in Encyclopedia of Separation Science. Wilson, I.D., Adlard, E.R., Poole, C.F. and Cooke, M. Editors. Academic Press Ltd., London.

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Hypertext updated by dav 2001-OCT-02