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Office of Science and Technology Annual Report Fiscal Year 1999

October 1, 1998 – September 30, 1999

May 2000

Food and Drug Administration
Center for Devices and Radiological Health

Table of Contents

PREFACE

OFFICE OF SCIENCE AND TECHNOLOGY STAFF

INTRODUCTION

TECHNICAL CONSULTATIONS

FORENSIC ANALYSES

RESEARCH

BIOEFFECTS

Cellulose Acetate

Developing a PCR-PNA-ELISA Procedure for Rapid Detection of Point Mutations in Mycobacterium Tuberculosis Genes Associated with Drug Resistance

Effects of Implant Coatings on Heart Cell Function

Safe Use of Electrostimulators in Acupuncture

Safety of High Frequency Electrical Stimulation of the Nervous System

Laboratory Testing of Radiofrequency Cardiac Ablation Devices

In-Vitro Safety Studies of Cardiac Electrical Defibrillation Devices

Production of Autoantibodies in Response to Silicone Gel/Oil

Large Animal Cardiovascular Research Program

BIOEFFECTS-RADIATION

Quantitative, Biologically Relevant Parameters for Testing and Standardizing Skin Response to UV

Full Spectrum Apoptosis

UVA1 Radiation Triggers Two Different Final Apoptotic Pathways

Potential Therapeutic Uses of Magnetic Fields to Mitigate Reperfusion Injury

ELECTROMAGNETIC INTERFERENCE

Electronic Article Surveillance Systems (EASS) and Metal Detectors as Sources of Interference with Implanted Medical Devices

IMAGING

Mammography

RF Safety of Patients with Metallic Implants in Magnetic Resonance Imaging 

Maximizing Velocity Measurements with Digital Particle Image Velocimetry 

Tissue Characterization 

IMMUNOTOXICOLOGY/TOXICOLOGY 

Effects of Particulates on Immunologic Function – In Vitro Methods 

Immunotoxic and Morphologic Effects of Biomaterial Particles In Vivo 

Natural Rubber Latex-associated Allergies: Identification and Quantitation of NRL Proteins 

Molecular Biomarkers for New Methods for Safety Assessment 

INFECTION CONTROL 

Decontaminating Particles Exposed to Bacterial Endotoxin 

Transmissible Spongiform Encephalopathies (TSE’s) and FDA-Regulated Products 

Lack of Latex Porosity: A Review of Virus Barrier Tests 

Calculation of Virus Transmission Through Synthetic Barriers Under Realistic Use Conditions 

Oven-Aged Latex Gloves Do Not Become Porous 

Reuse of Single-Use Devices 

Long-Term Survival of Pseudomonas Aureginosa in Diluted Liquid Disinfectants 

Using Fluorescent Microspheres to Evaluate Barrier Integrity 

Virus Adsorption Varies with Barrier Material and with Latex Formulation 

LASERS 

Fibers and Waveguides Used in Minimally Invasive Surgery 

Optical Phantoms in the Evaluation of Therapeutic Laser Medical Devices 

MATERIAL DEGRADATION 

Effects of Flex-Fatigue and Abrasion on Glove Barrier Integrity 

Pharmacokinetic Modeling of 4,4’-Methylenedianiline Released from Reused Polyurethane Dialyzer Potting Materials 

Validation of Proposed Revision to USP Physical Test 881 - Tensile Strength of Surgical Sutures 

OPTICS 

Intraocular Lenses

Noninvasive Detection of Diabetes 

Excimer Laser Ablation Studies 

Operation Microscopes, Endoilluminators, and Ophthalmic Instruments 

Pulsed Xenon Lamps

RISK ASSESSMENT 

Microbial Risk Assessment 

Development and Evaluation of a Swine Model to Assess the Preclinical Safety of Mechanical Heart Valves 

Risk Assessment of Dioxin in Tampons 

Average Annual Solar UV Dose of Continental US Citizens 

STANDARDS DEVELOPMENT 

Apnea Monitor Physiologic Waveform Test Method Development 

ASTM MR Safety and Compatibility Test Methods Development 

ASTM Committee F-04 Tissue Engineered Medical Products (TEMPS) 

Endovascular Stent Standards Development 

Modification of ASTM Standard Test Method F1671 for Detection of Small Tear Latex 

Development of a Standard Practice for Testing for Alternative Pathway Complement Activation in Serum by Solid Materials 

Radiation Safety Standard for Personnel Security Screening Systems 

Sterilization, Reuse, and Package Integrity 

Standard Methods for Evaluating the Blood Damage Potential of Materials Used in Medical Devices 

Evaluation of the Standard Tests for Neurological Shunts 

Examination of Mechanical Prosthetic Heart Valve for an Acoustic Signature of Cavitation 

Sterility Assurance Levels

International Standards Development 

Tissue Engineering 

Ophthalmic Standards Support 

Guideline for the Detection of Mycobacterium Tuberculosis and other Pathogenic Mycobacteria by the Polymerase Chain Reaction Procedure 

Alginate in Tissue-Engineered Medical Products 

TECHNICAL SUPPORT

Book of Hazards 

Proposed New Wireless Medical Telemetry System to Minimize Risks from Electromagnetic Interference (EMI) 

Development of an In Vitro p53 Gene Mutation Assay for Cancer Risk Studies 

FDA-Wide Genetox Network 

Noncoherent Optical Radiation Program 

Laser Field Compliance Program 

Laser Product Evaluations 

Laser Standards Support 

Sunlamps: FDA Publishes an Advanced Notice of Proposed Rulemaking (ANPRM) 

Surgeon and Patient Examination Gloves 

Guidance on the Implementation of the Biomaterials Access Assurance Act of 1998 

X-ray Calibration Laboratory 

X-ray Calibration Laboratory Computer System 

Microwave Oven Instrument Calibrations 

ULTRASOUND 

Improved Ultrasound Contrast-Detail Phantom

Medical-Use Hydrophone Calibration Via Time-Delay Spectrometry 

Ultrasound Bone Densitometry

APPENDIX A - OST Publications 

APPENDIX B - OST Presentations 

APPENDIX C - Academic Affiliations of OST Staff 

APPENDIX D - OST PATENTS

APPENDIX E - OST-Sponsored Research Seminars 

APPENDIX F - Research Contracts and Interagency Agreements 

APPENDIX G - STANDARDS ORGANIZATIONS 

A. NATIONAL STANDARDS ORGANIZATIONS

B. INTERNATIONAL STANDARDS ORGANIZATIONS

APPENDIX H - Abbreviations and Acronmyms 

 

The Office of Science and Technology (OST) is the laboratory of the Center for Devices and Radiological Health (CDRH) of the Food and Drug Administration.

The Office of Science and Technology (OST) supports the scientific basis for the Agency’s regulatory policies through development of independent laboratory information for regulatory and other public health activities of the Center for Devices and Radiological Health (CDRH). OST accomplishes this mission by managing, developing, and supporting standards used for regulatory assessments; performing laboratory evaluations and analyses in support of CDRH premarket and postmarket activities; developing data needed for current and future regulatory problems; and performing research, anticipating the impact of technology on the safety, effectiveness, and use of regulated products.

Specifically, OST develops and conducts research and testing programs in the areas of physical, life, and engineering sciences related to the human health effects of radiation and medical device technologies. It provides expertise and analyses for health-risk assessments. The Office also develops new or improved measurement methods, techniques, instruments, and analytical procedures for evaluating product performance and reliability. OST provides innovative solutions to public health problems through the development of generic techniques to enhance product safety and effectiveness. The laboratory activities of the Office have four major focus areas: characterization of the constituents or components of products; measurement of product performance; bioeffects which derive from human exposure to radiation or medical devices; and radiation metrology in support of Agency regulation of radiation-emitting products.

The purpose of the OST Annual Report is to update our readers about OST's organization, staffing, and intramural science activities; provide a summary of our direct lab support for pre-market review and compliance cases; and provide a bibliography of scientific publications, presentations, contracts, patents, and research seminars of the Office for 1998. The Annual Report is an overview rather than a comprehensive accounting. For additional information, please contact us. The Report might also be viewed as a source of information regarding areas in which Cooperative Research and Development Agreements (CRADAs) can be initiated with interested institutions. Comments are welcome on the programs described in this report. We hope you find this report useful and informative, and we invite any comments you might want to offer.

October 1, 1998 – September 30, 1999

Organization Chart [PDF]

The Office of Science and Technology (OST) is the laboratory of the Center for Devices and Radiological Health (CDRH) of the Food and Drug Administration. OST supports the scientific basis for the Agency’s regulatory decision making through development of independent laboratory information for regulatory and other public health activities of the Center for Devices and Radiological Health (CDRH). OST accomplishes this mission by working in four areas: managing, developing, and supporting standards used for regulatory assessments; providing technical consultations and performing laboratory evaluations and analyses in support of CDRH pre-market and post-market activities; developing forensic data needed for current and future regulatory problems; and performing research, anticipating the impact of technology on the safety, effectiveness, and use of regulated products. In this section of the OST Annual Report these activities will be summarized and a few highlighted.

The organization of OST is structured along technical department lines, similar to the organization of university departments of expertise, in contrast to the organization of the Offices of Device Evaluation (ODE) and Compliance (OC), which are organized along business lines. This enables the other Offices of the Center to identify and use specific expertise solving problems or consulting on the various regulatory functions of the Agency. These "bread-and-butter" activities of the Office also serve OST staff by placing them in everyday contact with an evolving industry and with the "use-problems" that occur with devices. Table 1 shows the relative changes in these interactions in the pre-market review area over recent years. One of the areas in which OST can contribute most effectively to the Center’s regulatory programs is through contribution to the development of guidance documents, such as guidance to reviewers and manufacturers related to the approval requirements of a specific product. Table 2 is an illustrative list of these types of consultations for 1999. On occasion, the OST contribution is limited to developing and validating an appropriate test method for determining a specific piece of information needed for a particular device. Table 3 is a list of these types of efforts in OST this year. OST receives many requests to provide expertise and information which cannot be characterized into any of the above tables. Table 4 is a summary of the requests from the Center, and Table 5 is a summary of requests from non-CDRH sources. Finally, OST contributes to the Center’s active postmarket surveillance activities. Table 6 is a summary of those activities performed in fiscal year 1999.

Table 1 – Premarket Review – Technical Consultations

Table 2 – Premarket Review – Significant Contribution as an Author of New or Revised Guidance

Table 3 – Premarket Review – Guidance Test Method Development or Evaluation

Shelf Life Testing of Medical Gloves

Table 4 – Premarket Review – Miscellaneous Contributions

Table 5 – Technical Consultations for Non-CDRH Clients

Table 6 – Ad Hoc Postmarket Surveillance Activities

Failure of a medical device in-service is seldom without significant adverse consequences to the patient on whom the device is being used at the time. Analyzing the failures and identifying the siblings of the failed device often require understanding the possible mechanisms of failure and deciding on an experimental approach while working with very little data. Table 7 is an example of the forensic analyses performed this year. OST also provides several calibration services in direct support of CDRH, ORA, and state inspectors of radiation-emitting products. Table 8 is a list of those calibration services and the numbers of devices from all calibrated sources.

Table 7 – Forensic Analyses

Laser Pointers (10 samples)
Laser Rangefinder (2 samples)
Contamination of hemodialysis machine blood lines
Sunlamps
Ferris Wound Dressings

Table 8 - Instrument Calibrations

Diagnostic X-ray Probes – 1350
Mammography X-ray Probes – 278
KVp Meter - 136
Digaphots - 108
Microwave Probes - 25
Laser Probes – 22
Beam splitter - 1
C-series Calorimeter - 2

RESEARCH

Key words: neutrophils, cellulose acetate, hemodialysis, oxidative response, biocompatibility

Following an adverse clinical event involving seven patients undergoing renal dialysis using 12-year-old cellulose acetate hemodialyzers, this in vitro study was proposed in an effort to characterize the inflammatory response to the constituent cellulose acetate (CA) fiber materials. Chemiluminescence (CL) and apoptosis assays were used to determine if human neutrophils were activated by cellulose acetate fiber materials and/or are sensitive to degradation/alteration of these fibers over time. For the CL assays, 60-minute exposure was followed by secondary stimulation with n-formyl-met-leu-phe or phorbol-12-myristate-13-acetate. The inflammatory response as measured by the respiratory burst of neutrophils was stimulated (p £ 0.05) by CA fiber exposure significantly over control. There was a trend toward an increased response with exposure to older fibers. Apoptosis was increased 12% with exposure to the more aged fibers versus 2% with the new fibers. More work is needed to determine the specific nature of the interaction of inflammatory cells with CA materials, but early evidence does suggest that neutrophils are activated by CA and display an altered response to more aged fibers.

Key words: polymerase chain reaction, PCR, peptide nucleic acid, PNA, in vitro diagnostics, Mycobacterium tuberculosis.

Tuberculosis remains a global health problem, and the emergence of antibiotic drug resistant strains of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, threatens public health. Recent molecular biological studies have shown that many of the specific genetic mutations that cause resistance in MTB are single-base change ("point") mutations. Classical techniques for detecting drug-resistant mutant strains of MTB are slow and tedious. New methods for quick diagnosis of drug-resistant MTB strains are needed. An OST molecular biologist and collaborators from FDA’s Center for Biologics Evaluation and Research (CBER), are developing a PCR (polymerase chain reaction)–PNA (peptide nucleic acid)–ELISA (enzyme-linked immunosorbent assay) procedure for rapid and convenient detection and identification of single-base change ("point") mutations in drug-resistant genes of MTB.

A model system has been developed for this work, and it involves the MTB katG gene, which is associated with resistance to the commonly used MTB antibiotic "isoniazid." The katG gene was cloned by the CBER collaborators into a plasmid vector using techniques of recombinant DNA. The PCR method with fluorescein-labeled primers is used to amplify specific regions of the cloned mutant katG gene. Short biotinylated PNAs that attach to avidin-precoated ELISA plates are then synthesized and used as probes for detecting mutant katG gene DNA sequences. PNA molecules are chemical strands that contain DNA bases attached to a protein-like (peptide) structural "backbone," and they hybridize, or bind, with single-stranded DNA molecules. PNAs are useful probes for detecting mutant nucleic acids because they have high thermal stability, strong binding capacity, and high binding specificity. The PNA probes used in this study are designed so that they will hybridize specifically with DNA sequences of the (denatured) PCR-amplified mutant katG gene (but not DNA sequences of the wild-type katG gene or any non-specific mutant gene). Non-specific DNA sequences are dehybridized by washing at 50-55oC. PNA molecules are chemical strands that contain DNA bases attached to a protein-like (peptide) structural "backbone." PNA probes are useful for detecting mutant nucleic acids because they have high thermal stability, strong binding capacity, and high binding specificity. After hybridization, color is developed following addition of an anti-fluorescein conjugate and appropriate substrate (ELISA procedure), and the amount of color formation is quantified using a spectrophotometer.

Thus far, the hybridization and other experimental conditions suitable for the successful detection (within 24 hours) of mutations in two different positions of the katG gene have been established using 15-mer PNA probes. The detection of further mutations in the katG gene will be investigated by the FDA investigators, and similar studies using an additional model drug-resistant MTB gene are planned. In addition to development of this in vitro diagnostic procedure, the laboratory experience gained from the project should also enable the investigators to make informed premarket evaluations of commercial in vitro diagnostic devices used for the detection of wild-type and mutant strains of MTB.

Key words: cardiac, biocompatibility, surface modification, calcium, implant

Chemical surface modification techniques are being applied to implant materials to achieve better biocompatibility and permit functional interaction between implant and tissue. Modified surfaces are being testing in a number of cardiac devices, including implant fabrics, heart valves, artificial blood vessels, and pacemaker electrodes. One type of surface modification that has gained recent prevalence is the use of self-assembled monolayers (SAMs) of biocompatible molecules. In anticipation of the use of such surface modification for cardiac devices, scientists in OST have collaborated with the chemistry department at the George Washington University to test the effects of various SAM coatings on heart cell function.

Dissociated cells from chicken embryo hearts were plated onto two different modified surfaces. One SAM was a hydrophilic triamine; the other was a hydrophobic perfluorinated compound. Both anatomical and functional characteristics of individual heart cells were measured on each of these surfaces. Quantitative morphology showed no significant differences in either the size or shape of the cells. Intracellular electrical recording demonstrated normal electrical activity of the cells on both surfaces. Optical measurements of intracellular calcium changes during the cardiac action potential showed abnormal calcium signals from cells grown on the perfluorinated surfaces. The calcium signal in these cells was smaller and had a missing component. Calcium in heart cells is related to contractility, cellular regulation and signaling. The abnormal calcium changes of cells grown on this surface would imply functional impairment. The fact that important functional differences were demonstrated with no significant difference in morphology underscores the need for physiological testing of medical device materials.

Key words: electroacupuncture, stimulator calibration standards

OST has been involved with issues of infection control regarding acupuncture needles; however, the next issue to be confronted by CDRH with medical devices associated with acupuncture will be electrostimulators. The concern with these devices is whether they have the potential to do serious harm through electric shock or excessive current. An electrical engineering mentorship student from Marquette University performed an analysis of representative devices used for electroacupuncture, specifically the electrostimulators used in conjunction with inserted acupuncture needles. The approach consisted of comparing the claimed outputs of a number of marketed electrostimulators with electronic evaluations (via oscilloscope) and combining that information with actual use parameters obtained from electroacupuncture clinical trial publications, as well as with safety considerations provided in performance standards (ASTM) and FDA safety concerns. The measurements indicated that the output from each of the representative stimulators was significantly different from the manufacturer’s values. Thus, each stimulator should be individually calibrated. Determinations of the unbalanced current resulting from the pulse waveforms indicated that at high pulse frequencies (100 Hz) there may be localized injury from the excess current. Thus, the users should be adequately trained to minimize risk from using the electrostimulators in the clinical setting.

Key words: excitotoxicity, nerve, deep brain stimulation, spinal cord, cochlea, stimulation

Electrical stimulation is used to treat a number of nervous system disorders as well as in the replacement of damaged neural elements. Treatments generally involve stimulating nerves at a rate no higher than the maximum impulse rate of nerve cells, typically 300 pulses per second. Recent clinical evidence, however, suggests that high-frequency stimulation may increase the therapeutic value of nerve stimulation devices. Examples include the improved relief of pain via spinal cord stimulation, better sound with cochlear implants, and the treatment of Parkinson's disease symptoms with deep brain stimulation. However, the safety of these forms of high-frequency electrical stimulation has not been systematically investigated. OST scientists are performing neurophysiological experiments combined with theoretical analyses designed to determine the deleterious effects of high-frequency electrical stimulation on neural tissue.

Theory predicts that high-frequency current pulses will depolarize nerve cells, lower the threshold of stimulation, and increase excitability. These occur with an increase in sodium ion permeability. OST experiments involving intracellular microelectrode recording from single-nerve cells demonstrated that depolarization does, in fact, occur. These experiments also show that high-frequency stimulation increases excitability and relieves action potential conduction block. The resulting safety concern is that prolonged stimulation will cause an ion imbalance due to intracellular sodium or calcium ion accumulation. This imbalance could overload the metabolic machinery of the nerve cell and cause a toxic effect (excitotoxicity). Preliminary experimental evidence has demonstrated that short periods of high frequency stimulation cause the activation of a metabolic pump for extruding sodium ions from cells. This metabolic pump activation causes an eventual depression in excitability. Future studies will investigate the safety relevance of the excitability changes and the conditions under which neural damage could occur from high-frequency stimulation.

Key words: cardiac, ablation, arrhythmias, radiofrequency, RF, thrombus

Radiofrequency (RF) catheter ablation is a commonly used procedure for the management of cardiac arrhythmias. The procedure uses RF electrical energy delivered via catheter electrode(s) to generate thermal lesions, which disrupt the aberrant electrical pathways that cause potentially lethal cardiac arrhythmias. One of the difficulties in researching the heat-transfer mechanisms is that much of the available data regarding heat-transfer issues have been conducted in animal studies. The variances in the numbers from study to study make the interpretation of mechanism difficult to determine. Several courses of study have, therefore, been undertaken in the past year to address this issue and include 1) development of durable simulated human tissue materials (phantoms), and 2) development of an advanced convective heat transfer test cell.

A significant effort has been applied towards the development of phantom materials over the past several decades. The majority of this work has been directed towards microwave and ultrasound applications. Nearly all phantom materials used at ablation frequencies (480-550 kHz) are water-based salt solutions, since the mode of heating is primarily resistive heating. These materials are effective in evaluating electric field strength and thermal fields in homogeneous systems. However, these methods are not effective in evaluating heterogeneous systems that involve materials that have several different electrical properties. One technique for examining these heterogeneous systems is to use a bi-phasic system where a liquid and a solid each represent different materials (e.g. blood and heart muscle). However, because both materials are typically water-based, ion exchange occurs between the materials and each material is degraded significantly after only a few minutes. OST scientists have developed several new phantom materials that are not salt-based and are not, therefore, susceptible to this ion exchange. Used in conjunction with existing phantom materials, these materials will extend the ability to examine more complex systems.

Researchers also directed their efforts during FY 1999 toward developing a heat transfer test cell to examine heat dissipation issues, both at the surface of a phantom and in the near electric field of an ablation electrode. A sophisticated test cell containing these phantom materials has been developed  that produces a series of holographic projections of the thermal washout from ablation electrodes exposed to a flow field. The goal of this effort is to refine understanding of heat transfer phenomena where significant blood flow is involved in order to evaluate the potential for adverse effects downstream from the ablation site.

Key words: cardiac, stimulator, defibrillator, dysfunction, safety

OST performs safety studies on medical devices for electrical defibrillation of the heart. This work applies to a number of devices, including implanted and external cardiac defibrillators. The work concentrates on the possible deleterious effects of these devices on live cells and how these harmful effects can be avoided. In FY 1999, OST scientists continued their work on the safety of electroshock from cardiac defibrillators and focused on the mechanism by which strong electric shocks can induce cardiac dysfunction.

Both automatic implanted cardioverter defibrillators (ICD) and the recent pediatric use of automatic external defibrillators (AED) can generate local electric fields in the heart that are capable of inducing cellular dysfunction. Previous OST studies have demonstrated that electroshock-induced dysfunction involves a prolonged calcium ion elevation in heart cells. The calcium elevation is associated with a period of refractoriness to pacing. Such cellular dysfunction is related to the production of secondary arrhythmias following defibrillator shocks. For this calcium-related dysfunction, OST scientists determined its threshold, relationship to shock strength, relationship to shock waveform, and the cell locale of the effect. A drug that blocks the calcium channel blocked much of the calcium increase following a strong shock but not the refractoriness to pacing.

Experiments performed on heart cells with different channel characteristics showed that the calcium elevation is due to contributions through nonspecific membrane pores, through calcium channels, and from cytosolic stores. Strong shocks are hypothesized to cause an initial poration of the cell membrane, entry of sodium of calcium ions, cell depolarization, opening of voltage-sensitive calcium channels and the release of cytosolic calcium stores. This hypothesis suggests how drugs and metabolic state can modulate defibrillator safety and effectiveness.

Key words: autoantibodies, silicone gel

Studies have been conducted using a female rat model to assess the production of autoantibodies in response to the injection of silicone gel/oil (taken from a breast implant) mixed with connective tissue proteins. Results of this study demonstrated the presence of anti-collagen antibodies in the serum of rats injected with only oil or only gel/oil. To confirm the results of these studies, the 21-month experiments are being repeated using normal Sprague-Dawley rats and autoimmune susceptible Dark Agouti rats. In addition to the test emulsions used in the initial experiment, scientists included oil and gel from a second source.

Key words: cardiovascular, animal models, biomechanics, interventional radiology

CDRH has established a large animal cardiovascular research program to develop and study models of cardiovascular disease, vascular injury, and long-term vascular implant performance. This research brings together an interdisciplinary group of both government and non-government scientists and clinicians with expertise in cardiovascular physiology and pharmacology; radiology; pathology; cardiology; animal science (swine); tissue biomechanics; and the molecular biology of vascular disease. The laboratory, which is located at the FDA’s Center for Veterinary Medicine in Laurel, Maryland, can be used for animals ranging from rodents to large swine. The space includes a small animal procedural lab, wet lab, gross pathology lab, in vitro physiology lab, pre- and postoperative holding areas, and an interventional radiology/surgery suite with a supporting darkroom and film processor. The interventional radiology suite allows performance of diagnostic angiography as well as interventional procedures (e.g., balloon angioplasty, stent placement, and selective catheterization) under sterile conditions with general anesthesia. The laboratory also utilizes the long-term holding facilities of the FDA/CVM complex at Greenbelt, Maryland.

OST scientists in collaboration with ODE are using the laboratory to study the effects of gender and hormonal state on the function and mechanical properties of coronary arteries and on the healing response of arteries to balloon injury. The motivation for the study is the observed greater incidence of cardiovascular death in postmenopausal women and in men of all ages compared to premenopausal women. There is epidemiological evidence that estrogen replacement therapy in postmenopausal women provides some protection against coronary artery disease. The proliferative response of an artery following angioplasty, a major cause of restenosis following this treatment, may also be reduced by estrogen therapy.

In this study, the effects of balloon injury of the coronary artery are evaluated in a swine model representing permutations of gender and hormonal state. The four cohorts are normal controls (mature and hormonally intact males and females), castrated males, and ovariectomized females (a model of female menopause) with and without estrogen replacement. The left anterior descending coronary artery is intentionally injured by balloon dilation, the same technique used in the treatment of coronary artery disease. This results in a proliferative healing response by the vessel wall. One month later, the heart and coronary arteries are surgically isolated, and coronary blood flow is directly measured and evaluated for changes in response to directly infusing vasoactive drugs directly into the coronary artery. Samples of the coronary artery are collected for in vitro measurement of biomechanical and biomolecular properties and smooth muscle contractility. Researchers then perform a complete histopathological study of the injured site. The scientists also collect samples to study differences in gene expression relevant to normal and abnormal function of the vascular wall.

The project addresses gender and hormonal influences on hemodynamic, biomechanical, pathological, and molecular parameters of normal vascular function as well as the responses of the vasculature to injury. The results should shed light on gender and hormonal differences in the progression and treatment of cardiovascular disease in humans and allow for better definition of the preclinical (animal) testing which should be conducted prior to testing cardiovascular devices in humans.

Key words: UV sensitivity, ultraviolet radiation, sunlamps

FDA regulates a variety of ultraviolet radiation (UV)-emitting or –transmitting products including sunlamps, sunscreens, cosmetics, and photosensitizing drugs. The number of people who use such products is very high.

Acute and delayed adverse effects of exposure to ultraviolet radiation (UV) are widely recognized. To improve public health policies in this area, FDA is actively involved in revising or developing national and international standards (e.g., the FDA Sunlamp Performance Standard; IEC 60 335-2-27, UV and IR Appliances; UL 482, Sun/Heat Lamps; CIE TC6-48, Typical Minimal Erythema Doses. Also, FDA currently develops or revises guidelines for different UV-related products. However, the current knowledge provides inadequate scientific basis for such standards, guidelines, and policies. This applies to all users of the UV-related products. The gaps in our knowledge are particularly severe for non-Caucasians. In fact, it is not clear how to test or predict human UV responses and general UV sensitivity.

For these reasons, the Human Photosciences Research Facility has been established at CDRH. It includes a UV exposure room, a biopsy room, a biopsy processing lab, photographic documentation studio, a measurement section housing three optical instruments (Minolta spectrophotometer, DiaStron Erythema/Melanin Meter, and an Optical Coherence Tomograph obtained from Philips Research, The Netherlands). Also included are two mechanical instruments (BTC-2000 Suction Device and Diastron torsional ballistometer), high-frequency ultrasound (Taberna Pro Medicum DUB-20 Plus) equipment, and a reception/conference room. This facility is used by a multidisciplinary team, including investigators from OST and OSB as well as those from CFSAN, NCI/NIH, Washington Hospital Center, and Philips Research Laboratories. The team investigates the usefulness of novel physical parameters and biomarkers for testing and standardizing human skin response to UV. This study is conducted on 110 volunteers divided into 11 groups on the basis of their skin type (Fitzpatrick, I-VI) and their racial/ethnic origin (OMB classification 0990-0208: American Indian or Alaska Native; Black or African American; Asian; Hispanic or Latino; Native Hawaiian or Other Pacific Islander; White.) Small (2x2 cm) areas are exposed to different UV doses. Then, at different times, the erythema (skin reddening), pigmentation (tan) and edema are evaluated by eye and using several instruments. Epidermal elasticity and viscoelasticity are measured. Structural changes are examined using optical coherence tomography and the ultrasound technique. Skin biopsies from the exposed and unexposed areas are collected and analyzed for cellular and molecular changes.

It is critical to the outcome of this study that the optical radiation dosimetry be of the utmost integrity (e.g., traceable to NIST). Accurate spectral measurements of UV radiation are required, not only to determine the exact exposure schedule for each individual - which varies with presumed skin type - but also to protect ancillary personnel from inadvertent exposure to UV.

Figure 1 shows the emission spectrum of the exposure source used in the CDRH human study. External filtration (Kodacel cellulose acetate film) is required to remove wavelengths neither found in natural sunlight or used in tanning devices.

As mentioned above, this study afforded OST scientists the opportunity to use and evaluate an Optical Coherence Tomograph (OCT) (from Philips Research Laboratories). It was hoped that this device might be useful in determining the structure of the upper layers of the skin and aid in predicting skin UV sensitivity by revealing UVB-related effects not measurable by other means. The device employs a 670-nm wavelength laser diode to interrogate the top 1 mm of skin surface. OST scientists used the image data to examine the optical attenuation of UV-exposed and unexposed skin. Early results indicate that a correlation exists between the attenuation of 670-nm photons through the top 0.6-0.8 mm of unexposed skin and the experimentally determined MED. Data for eleven subjects (each displayed in a different color) are shown in Figure 2.

The study is ongoing at this time, and the data have been collected on 38 subjects. The results confirm substantial variation of Minimal Erythema Doses (MED) within conventional skin type categories. The data indicate that racial/ethnic origin is not a primary factor determining individual UV sensitivity. They also confirm induction of extensive DNA damage following exposure to 1 MED.

The results of this study will facilitate safety and efficacy analyses of many FDA-regulated products and should help to select parameters for clinical trials of FDA-regulated products. They should provide support for modernizing public health policies in the area and help consumers to protect themselves from skin cancers and premature skin aging.

Key words: apoptosis, cell death, apoptotic pathway, megapore

Many wavelengths of radiation cause apoptosis. Apoptosis, or Sunburn Cell, is a term that only describes the morphological changes a cell undergoes during this mode of cytotoxic cell death. The terms immediate, intermediate, and delayed apoptosis segregate the different apoptotic mechanisms into three kinetic categories, while the terms pre-programmed cell death (prePCD) and programmed cell death (PCD) describe the underlying mechanisms. Immediate apoptosis (T£ 15 min post exposure) triggered by photodynamic therapy (PDT) or UVA1 singlet-oxygen damage to mitochondrial membranes depolarizes the inner transmembrane potential, which opens the mitochondrial megapore at the "S" site (sulfhydryl sensitive). It is a prePCD mechanism of apoptosis because all the necessary components are constitutively synthesized and only need to be activated and/or released. Intermediate apoptosis (T>15 min£ 4h) initiated by PDT, UVA, high-dose UVB or UVC damage to receptors and/or cytoplasmic components leads to opening the megapore at the "P" site (pyrimidine dinucleotide sensitive). It is also a prePCD mechanism. Delayed apoptosis (T>4h) induced by PDT, UVA, PUVA, UVB, UVC and X-ray damages to DNA also eventually leads to opening the megapore at the P site. However, it is a PCD mechanism of apoptosis because transcription and/or translation are required. Each mechanism activates one of two final apoptotic pathways. PDT and UVA can trigger immediate apoptosis by causing the cyclosporin A-sensitive S site of the megapore to release apoptosis-initiating factor, which activates a final apoptotic pathway that is primarily caspase-independent. Whereas, PDT, UVA, PUVA, UVB, UVC, and X-rays initiate intermediate and/or induce delayed apoptosis by causing the cyclosporin A-insensitive P site of the megapore to release cytochrome c, which activates another final apoptotic pathway that is primarily caspase-dependent. The apoptotic mechanism(s) which is (are) initiated depend(s) somewhat on the cell type; but primarily depends on the wavelength and dose of radiation, as well as the type, concentration, and intracellular location of the photosensitizer or chromophore.

Key words: apoptosis, cyclosporin A, pre-programmed cell death, mitochondria, radiation

Because UVA1 radiation is used therapeutically, this in vitro study addressed the question, "how does it work?" To begin addressing this question, UVA1 radiation was first established to reduce the survival of transformed T and B lymphocytes in a linear, dose-dependent manner using clonogenic assays, transmission electron microscopy, Annexin V, and flow cytometry. The primary mechanism was determined to be immediate (<20 min) preprogrammed cell death (pre-PCD), an apoptotic mechanism that does not require protein synthesis post insult, by quantifying the apoptotic cells over time in the absence or presence of a translation inhibitor.

To explore how UVA1 radiation triggers immediate pre-PCD apoptosis, reactive oxygen species (ROS) and mitochondrial function were altered during exposure using a variety of agents, while a specific fluorescent probe, JC-1 (5,5’, 6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide), was used to examine mitochondrial transmembrane depolarization. To show which UVA1-mediated ROS damages mitochondrial membranes, the following established ROS generating systems were used: 1) singlet oxygen, rose bengal or delta-aminolevulinic acid (increases endogenous protoporphyrin IX) with visible light; 2) superoxide anions, vitamin K₃; 3) hydroxyl radicals, X-rays; 4) mixed ROS, high-dose UVB; and 5) none, anti-Fas cross-linking antibody and blocking antibody. Cyclosporin A was used along with these systems to distinguish between the two final pathways because it inhibits mitochondrial permeability transition (PT) or megapore opening at the "S" site (sulfhydryl sensitive) but not at the "P" site (pyrimidine dinucleotide sensitive).

The collective results show UVA1 radiation triggers both final apoptotic pathways. It primarily mediates singlet-oxygen damage to the cyclosporin A-sensitive S site of the mitochondrial megapore, triggering immediate pre-PCD apoptosis by depolarizing the inner transmembrane potential causing PT. However, it also mediates superoxide-anion damage to the cyclosporin A-insensitive P site of the megapore. The S site releases apoptosis-initiating factor, which primarily triggers a caspase-independent final apoptotic pathway, while the P site releases cytochrome c, which primarily activates a caspase-dependent final apoptotic pathway.

Key words: magnetic fields, coronary reperfusion injury, ischemic stress

Continuing from previous investigations on the potential adverse effects of magnetic fields, OST investigated one of several proposed therapeutic effects of magnetic fields. Following a myocardial infarction, perfusion of the myocardium may be re-established by therapeutic interventions including thrombolytic agents and/or percutaneous angioplasty. Reperfusion of the hypoxic myocardium results in the formation of reactive oxygen intermediates which have deleterious effects, including further necrosis of cardiac tissue. Other investigators have reported that applying weak magnetic fields to avian embryos prior to re-perfusion prevents the toxic effects of re-oxygenation in this model. It has been postulated that this protective effect is due to the activation of heat shock factors by magnetic field exposure. These investigators believe that this avian model suggests the possible therapeutic use of weak alternating magnetic fields prior to thrombolytic therapy or angioplasty in patients being treated for myocardial infarction. Since the study was funded by commercial interests and a patent has been awarded for a medical device based on the reported effects, it seems likely that an IDE will be submitted to CDRH for human studies.

In order to position the Center to evaluate submissions involving this use of magnetic fields to treat coronary artery disease, OST attempted to verify the investigations reported in the avian embryo. Chicken embryos were exposed by "windowing" the eggs and the embryos made anoxic using an argon atmosphere. Embryos pre-exposed to 60-Hz 8 m T magnetic fields for 20 minutes were compared to unexposed control embryos. Observers blinded to the treatment scored the embryos at 30-minute intervals checking for the presence or absence of heartbeat. When the percentage of surviving control embryos had fallen to 30-50%, the embryos were re-oxygenated, and myocardial toxicity was monitored by observing the return of a heartbeat. Experiments using a total of 182 control embryos and 169 magnetic field exposed embryos showed a slight trend toward higher recovery in exposed embryos (70% vs. 65%), but the difference was not statistically significant. OST concludes that it is unlikely that weak magnetic field exposure protects myocardium against reperfusion injury and that any proposed clinical trials of this treatment be supported by data from a mammalian model.

Key words: magnetic field, electronic article surveillance systems, EASS, electromagnetic interference, implanted devices, metal detectors

OST engineers have completed magnetic and electric field mapping of eight electronic article surveillance systems (EASS). The results of these tests were published in the

September-October issue of Compliance Engineering. These data were also presented at the 1999 annual Association for the Advancement of Medical Instrumentation (AAMI) symposium. Table 9 and figure 3 provide summaries of these electromagnetic field-strength measurement data. A discussion of the EASS issue was presented at a public meeting of the CDRH Technical Electronic Product Safety Standards Committee (TEPRSSC) in conjunction with a review of the risks associated with exposures to magnetic fields of patients wearing certain medical devices. These risks included interference with the proper operation of implanted medical devices, such as cardiac pacemakers and defibrillators, and spinal cord stimulators. Comparisons of the electromagnetic field measurements made in the laboratory with performance requirements with the European Standard EN 50061/A1 -1995 (Safety of Implantable Cardiac Pacemakers) are shown in figure 4.

A walk-through metal detector was obtained from the Federal Aviation Administration for use in the medical device interference study. OST's three-dimensional electromagnetic field-strength mapping apparatus was relocated to a new laboratory. The optimal location space was identified via comparative measurements in the laboratory location vs. measurements in the OST/EPB outdoor test facility (figure 5). The required support structure was designed and constructed using non-magnetic components.

Key words: mammography, phantom, dosimetry, thermoluminescent dosimetry

Work has continued in the area of mammography on the optimized mammography system (OMS). The film-screen combination used with the system for the past several years consisted of a Kodak Min-R Medium screen and Kodak Min-R H film. Both of these products have been discontinued, so it was necessary to find a suitable replacement with the high sensitivity (speed) required by the OMS. Suggestions were solicited from manufacturers, and Kodak and Fuji responded. The film-screen combinations recommended by both firms were tested with the OMS at the NIH Clinical Center. Both have sufficient sensitivity. A series of phantom images was made with each combination, and scored by the radiologists working on the project. A final choice of the combination to be used for the clinical trial of the OMS will be based on analysis of the phantom scores.

OST continued the project to provide experimental verification of the exposure-to-dose conversion factors used in the American College of Radiology Mammography Accreditation Program. The measurements with the conventional source configuration (Mo-anode tube, Mo filter) were successfully concluded, showing agreement with previous experiments and theoretical calculations that is within the limits of experimental error. The graduate student working on the project received a Master's degree from Georgetown University in April 1999. While this work validates the method used, the work of verifying the conversion factors for new, dose-reducing anode and filter choices remains to be completed.

OST also continued work on the project evaluating the potential for reading the mammographic phantom images by machine. The two packages obtained from outside sources and the OST-developed software were compared quantitatively on a range of phantom images. The results were presented at a Mammography Symposium jointly sponsored by the University of Virginia and the American Association of Physicists in Medicine, and was held in Charlottesville, Virginia, on September 24-25, 1999.

Key words: MRI safety, Implants, specific absorption rate, magnetic fields

Magnetic Resonance (MR) imaging has become a widely used medical procedure. Manufacturers of implant medical devices are routinely submitting claims that their devices are safe and effective in an MR environment. As a result, OST scientists have been studying patient heating due to the interaction of metallic implants with the strong radio frequency (RF) magnetic field produced by MR devices.

MR devices produce an RF magnetic field that results in RF energy absorption by patient tissues. The absorbed RF energy, denoted by the parameter Specific Absorption Rate (SAR), usually occurs in non-uniform patterns within the patient's body. A metallic implant can interact with the RF magnetic field of the MR device resulting in further concentration of local RF heating in tissues near the implant.

Computer modeling was performed of the specific absorption rate (SAR) distribution in a realistic model of the human body containing a metallic implant (24-cm long wire). This model was exposed to circularly polarized fields from a model of an MR birdcage body coil (64 MHz, 1.5 Tesla). The results were compared with those obtained from a human-shaped phantom composed of only muscle tissue and a rectangular (39x25x55 cm) phantom model filled with either muscle tissue or cerebro-spinal fluid (CSF). The results of the calculations indicated rather different patterns of SAR values in the phantom models. These differences in SAR patterns may be caused by several factors, including differences in geometry and dielectric properties between the models.

The heating pattern near the metallic implant concentrates at the tip of the metallic wire. Although different SAR distributions are observed in the realistic human model--the muscle human model and the muscle rectangular model--the heating at the tips of the metallic wires in all three models are evident. However, the RF field penetration into the interior of the saline (CSF fluid) rectangular model is low compared to the rest of the models, resulting in very little heating at the tip of the metallic wire. The result of this study indicates the need to use the proper model and the proper MR coil source to represent the realistic SAR patterns of patients with metallic implants exposed to MR RF magnetic fields.

Virtually all manufacturers of implantable devices that claim MRI compatibility utilize experimental models of the human body with simple, nonrealistic configurations. The OST computer modeling indicates that this can cause an underestimate of the induced SAR and resulting heating caused by implants used in patients undergoing an MRI exam .

Key words: flow visualization, DPIV validation model, standard test method

Digital particle image velocimetry (DPIV) is a flow visualization tool that provides a quantitative two-dimensional velocity vector map of flow patterns in clear plastic models. This measuring technique can identify zones of flow stagnation or high shear stress, either of which can lead to adverse effects in blood flow devices, such as artificial heart valves. The previous DPIV flow visualization system utilized a video camera, room lighting, and a dual-pulsed strobe-lighting source to measure velocities up to 1 m/s. Since peak blood velocities in the human aorta and through heart valves can reach several m/s, some improvements and new developments were required.

These improvements included new instrumentation consisting of a digital PIV camera and frame grabber, an acousto-optic laser modulator (AOM), a digital delay/pulse generator, a fiber optic link, and new version of the DPIV software to integrate this new equipment. The digital camera is operated in the triggered dual exposure mode, and the camera and frame grabber are integrated for real-time image capture and storage on a DVD recorder with a new version of Visiflow DPIV software (Ver 6.14). The CW argon laser is modulated by applying a dual-pulse TTL signal to the input of the acousto-optic modulator, which produces a modulated laser beam with a repetition rate of 30 Hz. The characteristics of the driving pulses to the AOM are adjustable and include the pulse width, time between pulses (D t), and the duty factor for the pulses (on time/off time).

Scientists measured a dry powder linear and rotary model, along with a Poiseuille fluid flow model. Schematics of the setups for these tests are shown in figure 6 (linear/rotary motion model) and figure 7 (Poiseuille fluid flow model). Preliminary results demonstrated correct direction vectors but large velocity magnitude errors for the linear/rotary models as well as for the Poiseuille fluid flow model. This error is attributed to the large duty factor (longer laser pulses) required to properly illuminate the models at the higher velocities. This result is similar to those obtained by others and indicates that high duty factors limit the accuracy in measuring high fluid velocities.

OST is currently in the process of characterizing the maximum useful measurable velocities for all three validation models (linear, rotary, and Poiseuille fluid flow) using various combinations of duty factors and time between images. In order for DPIV to accurately measure velocity, the illuminated sequential images must show the particles as particles and not streaks. The longer laser pulses cause this streaking. However, lower duty factors may not provide enough illumination for proper analysis. Possible solutions to this laser power problem will be investigated in FY 2000.

Key words: ultrasound, pattern recognition, liver disease, prostate cancer, research

OST scientists, in collaboration with their colleagues from Georgetown University (GU), George Washington University (GWU), and the University of Vermont (UVM), have begun a study titled "Combining Clinical, Sonographic, and Elastographic Features to Improve the Detection of Prostate Cancer," supported by funding from the US Army. This 2.5-year effort will apply and evaluate the use of ultrasonic tissue characterization, elastography, and pattern recognition methods that OST scientists and their collaborators have developed to detect prostate cancer. The efforts this year were focused on the relocation of the data-acquisition portion of the investigation to the UVM laboratory. Ultrasound (US) data are now being acquired from about one to two in vitro prostatectomy samples per week; data from about 30 samples have been acquired thus far. There have been advances in the last year in the US data-acquisition process as well as the data-analysis process. OST has also been working with the pathology department at UVM to better align the pathology data with the US data. Additionally, an OST-sponsored doctoral student at GWU defended his dissertation on "Classification Performance and Reproducibility of New Parameters for Quantitative Ultrasound Tissue Characterization."

Key words: particles, cytokines, wear and degradation, macrophages, standards

Wear and corrosion of implanted medical devices, such as dental and orthopedic prostheses, may produce particles, which may lead to acute and chronic inflammatory responses in the body. In order to evaluate the inflammatory potential of these particles, OST scientists have continued to refine and develop an in vitro assay using a macrophage cell line that has been incorporated into an ASTM standard (F04.16.01: Practices for Testing for Biological Responses to Particles In Vitro). In this assay, murine macrophage cells are exposed to particles or chemicals and then evaluated for cytotoxicity, production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), both inflammatory cytokines, and production of nitric oxide (NO).

In the past, OST scientists have used this assay to study the inflammatory potential of various types of particles, such as polymethylmethacrylate, titanium oxide, hydroxyapatite, silica, diamond, cadmium oxide, and aluminum hydroxide. A recent interest in evaluating polyethylene (PE) particles required developing special techniques to get the PE particles that float on the culture medium surface to be in contact with the macrophages. The method was refined for floating particles by growing the macrophages on glass coverslips and then inverting the coverslip ‘cells-side-down’ onto the particles. High density PE (HDPE) and ultra high molecular weight PE (UHMWPE) gave differing results. HDPE in the presence of bacterial LPS, a known macrophage activator, had little effect on TNF-alpha production compared to levels induced by HDPE alone, while UHMWPE plus LPS decreased TNF-alpha production from levels induced by UHMWPE alone. HDPE plus LPS tended to increase NO and IL-6 production, while UHMWPE with LPS tended to decrease NO and IL-6 production.

Thus far, these studies using the in vitro macrophage assay have demonstrated that 1) particles generated from different medical device materials can produce specific and unique TNF-alpha, IL-6, and NO responses; 2) some particles stimulate TNF-alpha production and some do not; and 3) some particles enhance, some inhibit, and some have no effect on the induction of TNF-alpha and NO production by murine macrophages in response to LPS added to the particles.

Key words: particles, spleen, in vivo, immunotoxicity

Particles from medical devices may be generated in place within tissue surrounding an implant or may be deliberately implanted in humans, and can cause adverse biological responses. OST scientists from several disciplines have collaborated on a long-term project to examine the effect of chemical composition and size of particles on immune system responses in the mouse to medical device associated materials.

Polystyrene (PS), polyethylene (PE) and/or polymethylmethacrylate (PMMA) particles of various diameters were injected into mice and various parameters were assessed at various time intervals post-injection. The parameters examined included distribution of the particles in peritoneal exudate cells, distribution of the particles within the tissues of the mouse, changes in the morphology and weights of internal organs affected by particle deposits, and changes in several immunologic markers. Preliminary studies showed that chemical composition was the major factor that determines the nature of the response. Particle size was less significant, although it was apparent that some particles were too large to be readily phagocytized. Tissue distribution varied depending on the particle; PMMA particles were transported to the spleen, presumably activating immune responses, while the PS particles remained in the tissue comprising the peritoneal cavity and promoted formation of tissue adhesions. The activation of macrophages measured by the release of nitric oxide (NO) was most evident with the large PS particles, which were not readily phagocytized by single cells. Analysis of the proliferative activity of the spleen cells revealed enhancement of the Con A-responsive population and suppression of the LPS-responsive population by some particles.

Further studies are being conducted to evaluate effects on the specific functions of immune cells in relation to cellular and/or antibody responses. This research was presented at the FDA Science Forum in February 2000 and will be presented at the American Association of Immunologists meetings in summer 2000. The results of these in vivo studies will be compared to the results of OST in vitro studies in order to validate in vitro approaches to biocompatibility testing and ultimately reduce the number of animals needed for extensive testing of biomaterials.

Key words: natural rubber latex, allergies, contributing factors, protein measurement

Natural rubber latex (NRL) in medical devices induces a Type 1 allergy, which may be life-threatening in individuals highly sensitized to NRL-containing proteins. Although awareness of allergy to NRL proteins has increased in the last several years, the prevalence of sensitization in health care providers and in the general population is still significant.

OST scientists are undertaking research projects that are focused on identifying the specific NRL proteins that are allergenic, and they are developing appropriate reagents to be used as standards for the protein and/or allergen quantitation tests. The clinical relevance of various in vitro methods for measurement of potentially allergenic proteins has not been established, and the identity of all latex allergens has not been determined yet. The OST studies revealed a) the existence of a number of major allergenic proteins present in various latex products, and b) the specificity of allergenic response for each sensitized individual depends on the type of product and the pattern of exposure. Based on these findings, it became evident that full and complete evaluation of the allergenic potential of finished NRL products will not be possible until all potential allergens are identified. Through the comparison of NRL proteins from various sources, OST scientists concluded that the total protein level might be a reliable indirect measure of the potential allergenicity of NRL products. They developed a protocol for an ELISA-based immunoassay for protein quantitation based on a rabbit antiserum to NRL proteins. The method was compared with other methods that measure total protein (Modified Lowry and HPLC) and a method that measures specific allergens (RAST inhibition assay). The best correlation of the ELISA inhibition assay thus far has been observed with amino acid analysis by HPLC. Further comparisons will be performed with skin testing as a gold standard for evaluation of allergenicity.

OST scientists have also established an animal model of latex allergy by prolonged exposure of Balb/C mice to NRL proteins, and they also developed an assay for NRL-specific mouse IgE antibodies as a measure of the allergenic response. This assay is providing OST scientists the possibility to study mechanisms, kinetics, and dose-response relationships of allergy induction. This model is also being used in collaboration with industry to evaluate the effects of some environmental factors on the development and intensity of sensitization. The preliminary results indicate that bacterial endotoxin and some disinfectants used in hospitals may, depending on the dose, either enhance or suppress development of NRL allergy. Researchers are continuing efforts to evaluate the potential factors contributing to the development of NRL allergy and to continue further developing methods to reliably predict the potential allergenicity of NRL-containing devices.

Key words: stress proteins, heat shock proteins, pre-clinical test method development,

bisphenol A, endocrine disruption, estrogen,, uterus, liver

OST scientists are developing, refining, and validating more sensitive and predictive pre-clinical methods for improved safety assessments, standards, and risk assessments. New molecular biomarkers of exposure and toxicity must be carefully validated with traditional assays and standards for use in preclinical safety evaluation and in risk assessment activities. The rationale for assessing molecular biomarkers is that such targets are usually the first responses induced by potentially hazardous materials and chemicals. In order to be effective and useful, a biomarker should be detectable prior to the onset of overt tissue damage. OST scientists are evaluating enhanced expression of "stress" proteins (sometimes called heat shock proteins) as a method that will more reliably predict potential adverse effects of device materials and other chemicals in two major target systems in the body: the endocrine system and the liver.

CDRH is concerned with the potential for certain medical device materials or other leachable substances used in device manufacture to mimic or interfere with functions of endogenous hormones and disrupt endocrine homeostasis. For example, bisphenol A (Bis-A), a plasticizer found in some medical devices, has the potential to act as a xenoestrogen. OST scientists are collaborating with researchers at the Department of Biology, George Washington University, on characterizing the estrogenic potential of Bis-A and developing the stress protein response as a molecular biomarker of exposure to estrogenic compounds in general using well known estrogens. The utility of the heat shock protein response to reliably predict the endocrine disruption potential of materials and leachates is being evaluated using side-by-side comparisons with the traditional uterotrophic (uterine hypertrophy, i.e., increased tissue wet weight) assay.

In studies using mice, OST scientists have shown that such changes can be detected in a number of stress proteins (specific heat shock proteins) in the mouse uterus in response to the administration of estradiol, a potent model estrogenic compound. Three endpoints - uterine hypertrophy, histology, and heat shock protein expression - were used to examine the estrogenic response of Bis-A. The changes in expression of heat shock proteins were correlated with the estradiol effects on uterine hypertrophy, the standard assay. In a study comparing the protein induction biomarker response in three strains of mice commonly used in safety testing, one strain was not as sensitive to the effects of estradiol. The major conclusions thus far are that heat shock protein induction is a more sensitive indicator of estrogenic effect than is uterine hypertrophy. Results of the estrogen studies were presented at the 1999 and 2000 Annual Meetings of the Society of Toxicology and form the basis of a graduate student thesis at the George Washington University.

OST scientists, in collaboration with researchers from the University of Arizona, Department of Pharmacology and Toxicology, are also studying the stress protein induction response as a biomarker of exposure and toxicity for chemicals that damage the liver. The study involved the administration of cadmium, a well-known hepatotoxic metal that OST scientists use as a positive control compound, to two strains of laboratory rats that are commonly used in safety testing. The results showed that induction of specific stress proteins occurred prior to the detection of standard clinical indicators of liver damage in one of the rat strains. However, the results also demonstrated significant differences between the two rat strains, i.e., the stress protein response was not induced prior to the onset of severe hepatotoxicity in the second rat strain tested. Results of these studies were presented at the 1999 and 2000 Annual Meetings of the Society of Toxicology.

These studies of new biomarkers for substances that interfere with the endocrine system and for hepatotoxic chemicals illustrate the need to fully understand the expression of particular biomarkers in various model systems (species, strains, cell types and tissues). It is also important to understand the genetic differences that may produce varied results, especially if these new biotechnologies are to be incorporated as surrogates for or adjuncts to traditional standard methods for safety assessment.

Key words: particles, lipopolysaccharide, nitric oxide, macrophage

Medical device implants that remain contaminated with bacteria or endotoxin after sterilization procedures can produce fever in the patient. In their submissions to FDA, manufacturers must provide evidence that their sterilized implantable devices are at or below a given endotoxin level. Medical device particles that prove to be sterile by some standard methods may still be contaminated with the endotoxin lipopolysaccharide (LPS), which is not destroyed by autoclaving. Thus, in order to detect macrophage responses to medical device particles that are not influenced by endotoxin contamination using the new ASTM standard (F04.16.01 - Practices for Testing for Biological Responses to Particles In Vitro), it is important that the particles to be tested be sterilized and LPS-free (or barely detectable LPS levels). OST scientists discovered that particles intentionally contaminated with small amounts of LPS stimulate the production of nitric oxide by macrophages in vitro. This method of testing for endotoxin contamination may be more sensitive than the standard limulus amebocyte assay (LAL). OST scientists demonstrated that 70% ethanol can inactivate LPS and may constitute an appropriate method to remove LPS from particles that are being tested for inflammatory potential. This work was published in the Journal of Biomedical Materials Research 46: 434-437 (1999).

Key words: Transmissible Spongiform Encephalopathies (TSEs), product safety, infection control, TSE risks

As a key member of the FDA InterCenter TSE Working Group (FDA TSE WG) and Chair of the CDRH TSE Working Group, the Center Coordinator for Biotechnology continues to demonstrate leadership and consistent and productive outstanding scientific expertise and efforts in Agency initiatives to resolve numerous cross-cutting scientific and regulatory issues regarding TSEs. These TSEs impact FDA-regulated products with broad implications for public health. Significant initiatives of the FDA TSE Working Group taken to protect public health and to alert industry to appropriate safeguards measures have included 1) letters to the industry, 2) recommendations and guidance, and 3) organizing meetings of the FDA TSE Advisory Committee (TSEAC) to allow for public input into proposed Agency actions. These actions will increase confidence that FDA-regulated products are free of potentially infectious material for TSEs.

The September 1999 International Workshop on Clearance of TSE Agents from Blood Products and Implanted Tissues sponsored by FDA’s Center for Biologics Evaluation and Research (CBER) and CDRH was convened to address the needs of scientists and regulators for biological reference standards and for a harmonized approach to evaluating process-validation data for TSEs. The workshop was a follow-up initiative of the June 1998 Joint Institute for Food Safety and Nutrition International Workshop on TSE Risks that identified the need to discuss standardization of clearance methods for TSE agents and of process validation for those methods on an international level. A summary report of the ATSE Clearance Workshop has been prepared for publication in Developments in Biologic Standardization, and the workshop transcripts have been edited for the FDA web site. The report summarizes the current status and agreement on the need for standardized clearance procedures for products and product components. The World Health Organization (WHO) effort on developing biological reference materials for TSEs is a step in that direction, with FDA input being provided by CBER and CDRH members of the FDA TSE WG.

Additionally, the Center Coordinator for Biotechnology has been key in developing the agenda/issues for the semiannual TSEAC meetings which have included, among others, presentation of the Draft Guidance for the Preparation of a Premarket Notification Application for Processed Human Dura Mater (which has been completed and published) and recommendations on material sourcing for FDA-regulated products from sheep in BSE/scrapie countries. Participation in several national and international forums such as the Cambridge Healthtech Institute Conference on TSEs continues to educate and communicate Agency actions on TSEs and FDA products to the greater scientific community.

Key words: latex, porosity, virus barriers

OST scientists have reviewed evidence regarding whether latex films, as found in condoms and medical gloves, are effective barriers to virus passage together with new data from additional tests. The primary focus was to determine whether latex films are porous, as opposed to having occasional manufacture-induced defects. The published and new evidence from studies using viruses are consistent only with the presence of occasional defects and are not consistent with porosity sufficient enough to allow virus passage. However, quality control of manufactured products based on acceptable quality levels using standardized tests does not guarantee that every sample is perfect. The risk of a specific product is related to the defect rate, the use situation, and the disease of interest, in particular, the quantity of virus-carrying fluid that is needed to constitute an "infectious dose." The possibility of latex film hydration leading to porosity and ultimately to virus passage was also found to be unlikely and not supported by data.

Key words: virus transmission, computational fluid dynamics, barrier evaluation, transport modeling

Scientists often perform tests on the effectiveness of synthetic barriers (gloves, condoms, instrument sheaths, etc.) to virus transmission under conditions that do not reflect actual use. For example, static test conditions are typically employed while, in reality, considerable motion is associated with use of the barrier. In order to extend laboratory results to more realistic conditions, OST has developed a mathematical model for simulating virus transport through synthetic barriers. The model was recently used to estimate the amount of virus that would be transmitted through a defect in a condom during coitus.

Input into the model was the pressure difference across the condom surface, which was previously measured by OST scientists during coital simulations. The pressure waveform was periodic (a period of 2 seconds was used), with a maximum pressure difference across of approximately 60 millimeters of mercury and a minimum of approximately minus-20 millimeters of mercury. Defects of various cross-sectional shapes were considered, from circles to wide ellipses. The rate constants characterizing the interaction force between the virus and latex were obtaine