Board A-01

Hsiaoling Wang, Alfred Del Grosso, Joan May, Laboratory of Analytical chemistry, Division of Manufacturing and Product Quality, CBER, FDA, Rockville, MD USA

Benthezonium chloride (BZC) serves the role ofas an anti-microbial agent preservative in the AAnthrax vaccine. Its content is currently determined by a non-specific, tedious and environmentally unfriendly titrimetrication method that is general for quaternary ammonium compounds. In this study a new sensitive and specific HPLC method has been developed for the quality control purposedetermination of BZC in this product of the vaccine. The isocratic HPLC analysis is rapid with a low limit of detection of 0.5ppm. The Unique unique chemical environment of BZC in the vaccine and sample pretreatment are discussed. along with Tahe comparison of analytical results obtained by thesis of new and old methods is also reported. The HPLC method has smallerdemonstrated an average RSD% of tion 1.6% compared to that of 3.3% that was obtained from the titrimetric method in for parallel experiments. The percent difference of two methods is less than 4%. Recovery of analyte by the HPLC procedure was 42.3% greater than the titrimetric method. The new method gives has been evaluated as yielding better precision with much less sample and time.

Board A-02

Z. Li, Ph.D.¹, S.A. Rubin, M.S.¹, M. Merchlinsky, Ph.D.², K.M. Carbone, M.D.¹, LPRVD¹ and LDV², DVP/OVRR/CBER, FDA, Bethesda, MD 20892

Universal vaccination with DryVax™, the only US FDA licensed smallpox vaccine, was associated with serious adverse events (approximately 1 in 5,000 vaccinees) and was stopped in 1971. Recent biowarfare/bioterrorism defense initiatives have stimulated the development of new smallpox vaccines. However, there are no validated pre-clinical virulence assays with which to test safety of these new vaccines prior to use in humans. Here we report the development of a small animal virulence assay for vaccinia based smallpox vaccines. Using newborn mice, we utilized the attenuated vaccine DryVax™ as a reference vaccine and the virulent WR strain of vaccinia virus as a positive control to develop a standardized virulence assay. The WR strain produced significantly greater and more rapid onset of mortality than the DryVax™ vaccine reference. Expression of virus antigen and infectious virus replication in the brain was also significantly different between the two strains. In addition, the appearance of high titer virus antibody correlated with the clearance of virus from brain. These data demonstrate the utility of this assay to provide a reference standard with which to perform pre-clinical virulence testing of new vaccinia-based smallpox vaccine strains.

Board A-04

R.M. Jacobs, ORA, San Francisco District Laboratory, FDA, Alameda, CA

While FDA's concern in terrorism has mostly involved biological agents, there are several other classes of toxic agents that could be effective in the food supply. These agents could also pose devastating health effects. Moreover, some of these "other" agents pose little risk to the terrorist, require little expertise for use, and are readily available. As a class of agents, toxic elements, i.e. "metals", (including alkylated forms) have a wide range of toxicity and health effects. The onset of the toxicity can be subtle and the effects can be irreversible. The necessary "tampering" could be done prior to the food entering U.S. commerce. To be practical and effective, either a food or a food ingredient vehicle would have to be chosen that can be easily tampered, consumed with a high frequency, in large quantities, or purchased in large quantities, e.g., infant formula, juice concentrate. That's the "bad news". The "good news" is that tools are available to readily detect most of the toxic elements that are practical agents. Portable XRF units are available to detect some of the toxic elements. In the lab, ICP-MS can be used to identify and quantify virtually all the elements that pose a practical hazard. FDA presently has a limited capability to prevent or respond to these circumstances.

Board A-05

Z. Li, S.A. Rubin, M. Merchlinsky, K.M. Carbone, DVP/OVRR/CBER, FDA, Rockville, MD 20892

Universal vaccination with DryVax™, the only US FDA licensed smallpox vaccine, was associated with serious adverse events (approximately 1 in 13,000 vaccinees) and was stopped in 1971. Recent biowarfare/bioterrorism defense initiatives have stimulated the development of new smallpox vaccines. However, there are no validated pre-clinical virulence assays with which to test safety of these new vaccines prior to use in humans. Here we report the development of a small animal virulence assay for vaccinia based smallpox vaccines. Using newborn mice, we utilized DryVax™ as the attenuated vaccine reference strain and the virulent WR strain of vaccinia virus as a positive control to develop a standardized virulence assay. The WR strain produced significantly greater and more rapid onset of mortality than the DryVax™ vaccine reference. Expression of virus antigen and infectious virus replication in the brain was also significantly different between the two strains. In addition, the appearance of high titer virus antibody correlated with the clearance of virus in the brain tissue. These data demonstrate the utility of this assay to provide a reference standard with which to perform pre-clinical virulence testing of new vaccinia-based smallpox vaccine strains.

Board A-07

F. S. Fry¹, L.E. Rodriguez-Saona², F.M. Khambaty¹, and E.M. Calvey¹, ¹CFSAN and ²JIFSAN, 5100 Paint Branch Parkway, College Park, MD 20740-3835

To address the need for a fast and sensitive method for the detection and classification of Bacillus spp., the use of FT-NIR spectroscopy and multivariate pattern recognition techniques was evaluated. The complex cellular composition of bacteria yields FT-NIR vibrational transitions, including overtone and combination bands, which might permit discrimination. . Multiple isolates of four Bacillus species, namely B. cereus, B. thuringiensis, B. subtilis, and B. amyloliquefaciens were studied, each evaluated through repeated trials. After growth, the bacterial cells were either resuspended in saline solution (0.9%) or in ethanol (70% v/v) to address safety concerns. The cells were then concentrated on an aluminum oxide membrane, yielding a thin, uniform film. This membrane filtration procedure generated reproducible FT-NIR spectra, indicating procedural robustness. Principal Component Analysis and Soft Independent Modeling of Class Analogy of transformed spectra in the 5100-4200 cm^-1 region exhibited clusters that permitted accurate species-level classification and some strain specific discrimination of most of the isolates. We conclude that the above FT-NIR technique shows promise for the rapid and accurate classification (with minimal sample manipulation) of Bacillus species of public health concern and the agriculturally beneficial strains that are otherwise very similar pathogens.

Board B-01

J.R. Hayes^1,2, S.D. McDermott¹, R. Reimschuessel¹, and D.D. Wagner¹, ¹CVM, FDA, Laurel, MD 20708, ²University of Maryland, College Park, MD 20742

Few studies have characterized the populations of bacteria from retail fish that can harbor important mobile antimicrobial resistance determinants that may be disseminated to complicate the treatment of human infectious disease. Enterococci are an important group of indicator organisms for fecal contamination that possess a special affinity to exchange such resistance elements. In an effort to preliminarily describe the populations of Enterococcus spp. and their associated resistance profiles, enterococci were isolated from retail salmon, Tilapia spp., catfish, and trout filets from the metropolitan D.C. area. A total of 59 isolates were recovered from 91 sampled filets. The incidence of Enterococcus spp. varied with the filet class, with detection rates of 72%, 75%, 50%, and 39% from salmon, Tilapia spp., catfish, and trout filets, respectively. E. faecalis was the most frequently isolated species, although differences in the ratios of E. faecalis to E. faecium were apparent among the filet classes. While no vancomycin resistance was detected, antimicrobial resistance profiles demonstrated that erythromycin and tetracycline resistance predominates among isolates of E. faecalis and resistance to high-level aminoglycosides predominates among E. faecium isolates. Interestingly, a single isolate of high-level gentamicin-resistant E. faecalis was isolated from a Tilapia spp. filet.

Board B-02

S Simjee^1*, D G White¹, P F McDermott¹, D D Wagner¹, M J Zervos², S M Donabedian², L L English¹ and R D Walker¹, ¹US FDA, CVM, Muirkirk Road, Laurel, MD 20708, ²William Beaumont Hospital, Royal Oak, MI 48073

Several studies have suggested that animal enterococci may be a potential reservoir of antibiotic resistance genes, but this has yet to be definitively proven. The transfer of resistance genes from human to animal enterococci has never been reported in the USA. Over a two-year period (1996-1998), 35 enterococcal isolates were recovered from dogs diagnosed with UTI's at the Michigan State University Veterinary Teaching Hospital. One E. faecium isolate displayed high-level resistance to vancomycin (MIC>16 µg/ml) and gentamicin (> 256 µg/ml). Initial molecular analysis revealed the presence of Tn1546 and aac6'-aph2", responsible for high-level vancomycin and gentamicin resistance, respectively. Both genes could be transferred by conjugation. Detailed molecular analysis of Tn1546 identified insertions of IS1216V in ORF1 and insertions of IS1251 between vanS and vanH. This particular form of Tn1546, with IS1216V and IS1251 insertions, has only been described in human clinical isolates of VRE found in the USA. Comparison, by PFGE, of the canine VRE isolate against over 300 human VRE isolates from Michigan, however, failed to yield any significant similarities. To our knowledge, this is the first report of a Tn1546 like element commonly found in human VRE to be isolated from a house pet in the USA. This data suggest that human flora may serve as a reservoir of transferable resistance genes to bacteria isolated from animals.

Board B-03

R.A. Miller, R. Reimschuessel, Division of Animal and Food Microbiology, Office of Research, FDA-CVM, Laurel, MD 20708

Currently there are no standard protocols for in vitro antimicrobial susceptibility testing (AST) of aquatic and marine microorganisms. Since many of these microorganisms grow under different environmental conditions than mammalian pathogens, it is necessary to develop methods for testing which will optimize the growth conditions.

Following the standard methods for disk diffusion susceptibility testing provided by the NCCLS, four ATCC® strains, Escherichia coli 25922, Aeromonas salmonicida ssp. salmonicida 33658, Listonella (Vibrio) anguillarum 19264, and Photobacterium damselae ssp. piscicida 51736 were tested against eight antimicrobials commonly used in worldwide aquaculture. Isolates were incubated on Mueller-Hinton agar (MHA) and MHA supplemented with 1.5% NaCl at four temperatures, 22 °C, 25 °C, 30 °C, and 35 °C. Results showed a slight decrease in zone diameter with increasing temperature on MHA for all organisms except P. damselae. L. anguillarum performed better on MHA with NaCl, and P. damselae did not perform well on either media. E. coli was the only isolate to perform well at all temperatures, and exhibit zone diameters in the acceptable range (18-40mm) for all drugs tested. A. salmonicida, a cooler temperature-tolerant organism, E. coli, and L. anguillarum, yielded the best results at 30°C, and have significant potential as QC organisms for in vitro AST of aquatic and marine pathogens.

Board B-06

Susceptibility of Planktonic and Biofilms of E. coli and Listeria innocua to Selected Disinfectants

Laila Ali, FDA, CFSAN, Washington, DC 20204

Biofilm formation is initiated when bacteria are deposited from an aqueous phase onto the attachment substratum. These bacteria are able to adhere to the surface by the adsorption of cell surface adhesive polymers, which may be polysaccharides produced by bacteria. Biofilms have been recognized as a potential source of contamination, which may lead to product spoilage and/or foodborne illness. In this research, the biofilm was artificially initiated to determine the effectiveness of different disinfectants on biofilm elimination and reduction of potential microbial contamination. Phenol, bleach, hydrogen peroxide, and quatericide were chosen as disinfectants. The results showed that the minimal concentration of disinfectants required to eradicate the biofilms were 25 ppm, 1%, and 50 ppm for bleach, phenol, and quatericide, respectively, for E. coli and Listeria innocua strains. The results showed that the planktonic E.coli strain proved to be significantly more resistant to bleach and quatericide at 12.5 ppm than the planktonic Listeria innocua.

Board B-07

H.G. Claycamp and C. M. Lathers, CVM, FDA Rockville, MD 20855

Antimicrobial resistance risk assessment (ARRA) is risk assessment focused on estimating adverse human health effects from the uses of antimicrobial drugs in humans or domesticated animals. ARRA differs from microbial risk assessment (MRA) by focusing on antimicrobial resistance genes (ARGs) as hazardous agents, rather than on only pathogenic microbes and toxins. Although ARRA and MRA overlap when the organism harboring ARGs is itself a pathogen, ARRA also considers resistance transfer to nonpathogenic bacteria, such as human commensal strains. Commensal bacteria are opportunistic pathogens in several exposure scenarios including, most notably, nosocomial exposures in hospitals. The ARRA process generally follows a classical risk assessment paradigm, including elements of hazard identification, dose-response assessment, exposure assessment and risk characterization. Alternatively, some advocate further compartmentalization of ARRA into "release" and "consequence" assessments. Subdivision of the paradigm might be useful to assess the risks from food animal applications of antimicrobial drugs, because release assessment captures the farm-to-market introduction of ARGs, while exposure and consequence assessment capture the human factors. The relative merits of the classical and the subdivided paradigms are discussed.

Publish Only (B)

James Wilson¹, Theresa To¹, Aaron Margolin², and Patrick Regan¹, ¹Winchester Engineering and Analytical Center, Winchester, MA, ²University of New Hampshire, Durham, NH

Sigma A expression between germicide-treated and untreated Mycobacterium bovis BCG cells was measured using RT-PCR and molecular beacons. Mycobacterium bovis (10⁷) was exposed to 2.5% glutaraldehyde for 5 and 90 minutes at 20°C. Sigma A expression was analyzed immediately following treatment (day 0) and again after a 10 day incubation in a 37°C 5% CO₂ incubator. Relative fluorescence units (RFU) were measured at 519 nm. Analysis for day 0 was 100 RFU for the 5-minute exposure and 97 RFU for the 90-minute exposure. The untreated control for day 0 was 150 RFU. Day 10 revealed an increase in the RFU's for the 5-minute exposure (112 RFU), and a decrease in the RFU's for the 90-minute exposure (33 RFU). Sigma A expression level for the untreated cells after a 10-day incubation was 220 RFU. The results indicate that cell viability can be determined by endpoint analysis and a molecular beacon reporter system. Further evaluation along with the need for a quantitative method will be determined. This technique could improve the current AOAC Tuberculocidal method. Experiments were performed in triplicate.

Board C-01

Xin Du, Ewa Marszal, and Andrew Shrake, Laboratory of Plasma Derivatives, Division of Hematology, Office of Blood Research and Review, CBER, FDA

The normal physiological role of alpha-1-proteinase inhibitor (A1-PI) is inhibition of neutrophil elastase. A1-PI, purified from human plasma, is a licensed biologic used to treat patients with mutant A1-PI, which polymerizes and accumulates in hepatocytes thereby reducing circulating levels causing progressive emphysema and, in a substantial portion of these patients, leading to severe liver disease.

Normal A1-PI can be induced to polymerize by partially unfolding it by treatment with a low concentration of denaturant or by heating at GT 37 deg C. Previously, our laboratory demonstrated that fully folded, purified disulfide-linked dimer, formed in 1.4 M guanidine-HCl (Gu) in the absence of reducing agent, spontaneously polymerizes at LT 37 deg C. To investigate the nature of the interactions involved in the formation of dimer and polymerization of it and monomer, we studied the effect of ethylene glycol on the polymerization of: (1) partially purified dimer in buffer at 4 and 25 deg C; (2) monomer in buffer heated at 45 and 60 deg C; and (3) monomer in 1.4 M Gu at 25 deg C.

Results suggest that the formation of the dimeric intermediate from monomeric intermediates involves hydrophobic interactions whereas, in the further polymerization of the dimer and monomer, such interactions may play a less significant role.

Board C-03

S. F. Ali, M. Oetinger, J. Skinner, W. Slikker, Jr. & S. Z. Imam. Neurochemistry Laboratory, Division of Neurotoxicology, National Center for Toxicological Research/FDA, Jefferson, AR, 72079.

Methamphetamine (METH) is a widely used drug of abuse. It causes long-lasting reductions in dopamine (DA) content, tyrosine hydroxylase activity and dopamine transporters in the striatum. It also produces oxidative stress by generating reactive oxygen and nitrogen species. METH has been reported to upregulate p53 and downregulate bcl-2 expression in striatum of mice after repeated doses. Caspase-3 is a downstream effector involved in the dissolution of the nucleus. Activation of Caspase-3 follows the onset of apoptosis. Here, we studied the role of caspase inhibition on METH-induced alterations in the expression of striatal p53 and bcl-2 in mice. Adult male C57 mice were treated with 4x10 mg/kg of METH at 2 h intervals. Another groups of mice also received a specific caspase-3 inhibitor (CIII, 0.5 mg/kg) or a general caspase inhibitor (GC, 0.5 mg/kg) along with METH or alone. Saline treated animals served as control. Mice were sacrificed 72 h after the last dose of METH. METH administration resulted in significant up-regulation of p53 and down-regulation of bcl-2 in mice striatum. GC failed to protect against this genetic alteration caused by METH but CIII significantly protected against METH-induced genetic changes in the striatum. METH also resulted in the significant depletion of DA and its metabolites DOPAC and HVA. CIII provided a significant attenuation, whereas GC failed to provide any protection against dopaminergic neurotoxicity. The present data demonstrate that METH-induced dopaminergic neurotoxicity caused genetic alterations, which leads to the onset of programmed cell death, however, it can be protected by specifically targeting the downstream effector action of Caspase-3.

Board D-02

T.B. Whitaker¹, M.W. Trucksess², F.S. Thomas¹, and A.B. Slate², ¹USDA, ARS, Raleigh, NC 27965, ²CFSAN, FDA, Washington, DC 20204

The objectives were to measure the variability of an acceptable Cry9C test procedure, determine the distributional characteristics among sample test results, and evaluate the performance of several sampling plans for corn meal processed from genetically modified (GM) StarLink corn. A sampling plan is defined by a test procedure and an accept/reject limit. The testing procedure consists of sampling and analytical steps. In our experiment, each bulk sample was spiked with 7 levels of GM StarLink Corn seeds (containing Cry9C protein) at 0.0, 0.01, 0.025, 0.05, 0.075, 0.10, and 0.125 %. The Cry9C protein in each bulk sample was determined using replicate analyses on replicate 2 g test samples with 2 different ELISA methods. The sampling and analytical variances associated with testing cornmeal was determined. The sampling step accounted for 97.3% of the total variability. The distribution among sample test results was normally distributed. Operating characteristic curve (OC) that predicts false positives (good lots rejected) and false negatives (bad lots accepted) associated with a sampling plan can be used to evaluate the performance of specific sampling designs. OC curve can be computed once the variability and distributional characteristics of the test procedure are known. OC curves showed that four 10 g samples are required to all test to be less than the accept/reject limit.

Board D-03

Mary W. Trucksess, CFSAN, FDA, Washington, DC 20204

The purpose of this interlaboratory study was to determine the accuracy, repeatability and reproducibility parameters of the EnviroLogix ELISA kit method for the determination of Cry9C protein, a protein produced by genetically-modified corn, StarLink, in 8 types of corn-based foods (starch, refined oil, soft tortillas, tortilla chips, corn flakes, corn puffs, corn muffins, and corn bread). Blind duplicates of control samples, spiked samples, and incurred samples were analyzed. Cry9C protein produced and purified from a bacterial host was used to prepare spiked test samples at 2.72 ng /g and 6.8 ng/g. Cry9C protein from StarLink corn flour was used to prepare spiked samples at 1.97 ng/g. Average recoveries for samples spiked with corn flour Cry9C protein at 1.97 ng/g ranged from 73% to 122%, within-laboratory relative standard deviations (RSD_r) ranged from 6% to 22%, and between laboratory standard derivations (RSD_R) ranged from 16% to 56%. The incurred test samples were found to contain Cry9C protein at levels ranging from 0.8 to 3187 ng/g depending on the product. The RSD_r ranged from 5 to 16% and RSD_R from 11 to 71% for the incurred samples. Results of statistical analysis indicate that this method is applicable to determine Cry9C protein in the 8 types of corn-based products containing Cry9C protein (from StarLink ) at levels of > 1.97 ng/g.

Board D-04

Sufian Al-Khaldi, Charles Warner, Magdi Mossoba, and Hatsuichi Mimata. CFSAN, FDA, Washington DC 20204.

Potassium bromate (KBrO₃) has been used as a food additive in the treatment of flour and barley and as a constituent in cold-wave hair solution. It is also formed as a by-product during the disinfection of water by ozonation. KBrO₃ is reportedly known to increase bacterial mutation, and to cause renal cell tumors and mesotheliomas of the peritoneum as follicular cell tumours of the thyroid in the rat. It has been suggested that KBrO₃ can oxidize guanine present in DNA, thus forming 8 OH-deoxyguanonsine (8-oxodG).

The objective was to study the reaction of glutathione with bromate with IR spectroscopy to determine if the spectroscopic data would permit identification of this substance. In addition, the reaction of bromate with guanine residues in DNA with activation by glutathione would be used to develop rapid confirmatory DNA detection method for the presence of bromate. Data indicates that (i) the oxidation of guanine may be monitored by infrared spectroscopy, and (ii) the oxidized guanine may exhibit fluorescence alteration in binding capability with Cy3-deoxycytosine compared with non-treated guanine. Characteristic infrared spectra collected as a function of reaction time will be discussed. DNA microarray data for hybridized cytosine with guanine compared to oxidized guanine will be presented.

Board E-02

G.C. Ziobro and S.M. Cichowicz, CFSAN, FDA, College Park, MD 20740

The past decade has seen a tremendous growth in the botanical dietary supplement industry. The starting point for any supplement ingredient is the accurate identification of the plant material(s) used to make the supplement. One common way to identify the material is to compare it to a preserved, accurately identified specimen in a herbarium. Traditional herbaria are primarily collections of the pressed, dried, flowering portions of plants from any plants. The FDA Herbarium is a specialty herbarium. We are collecting herbaria sheets, roots, stems, seeds, and/or barks used in botanical dietary supplements. These materials are used to assist the Agency in the identification of these materials used either individually or as part of mixtures in supplements. Currently the FDA collection consists of about 2000 specimens either collected ad hoc, reserve material from FDA research, materials donated by other herbaria or botanical gardens, or samples of commercially prepared consumer products

Board G-01

M.Pletnikov^1,2, S.Rubin¹,Y.Nishino^1,2,T.Moran², D. Dietz ^1,2, K. Carbone^1,2, LPRVD/DVP/OVRR/CBER, FDA, Bethesda, MD^1,, Johns Hopkins University, Baltimore, MD²

A hotly debated and controversial association between childhood vaccines and autism has jeopardized public safety and effectiveness of vaccination programs. However, improving benefit/risk communication to the public and providers is hindered by the poorly understood pathogenesis of autism and the mechanisms by which individual susceptibility to viruses may lead to expression of autism-like symptoms. We study autism-like neurodevelopmental damage using our rat animal model of autism based on neonatal Borna virus disease (BDV) infection. Multidisciplinary analysis of the neurobiological and genetic features of this model gives new insights into the issue of pediatric vaccine safety and suggests the utility of the model for better understanding risk factors associated with pediatric vaccinations.

Publish Only (G)

M.G. Paule

The need to use animal models for predicting drug effects on the nervous system has led to the development of automated systems for assessing specific behaviors--identical in both humans and lab animals--that represent aspects of specific 'cognitive' processes. At NCTR, a battery of tasks has been developed to model specific processes associated with motivation, color and position discrimination, time estimation, short-term memory, and learning. Maintenance of task continuity across species allows for the determination of interspecies similarities in brain function and aids in the extrapolation of data from animals to humans. Comparative data indicate, for example, that the performance of well-trained monkeys in this Operant Test Battery (OTB) is generally indistinguishable from that of children. The demonstration that human OTB performance correlates significantly with intelligence (IQ scores) highlights the relevance of these measures, as does their sensitivity to specific clinical entities such as attention deficit/hyperactivity disorder. Comparative drug studies show the monkey to be a good predictor of drug effects in humans. Recent studies have shown that rodent performance in some of these complex tasks is also very similar to that of both monkeys and children. Thus, tools such as the NCTR OTB may provide the opportunity for extensive interspecies comparisons of cognitive processes, and provide the means for predicting the effects of psychoactive agents in humans--including children--using relevant endpoints from a variety of animal models.

Publish Only (G)

R. Johann-Liang, S.J. Singer, R. Roca, and R. Albrecht. CDER, FDA, Rockville, MD

The QL antimicrobials are not approved for pediatric patients except for ciprofloxacin in anthrax. However, the drugs are widely used in children. With exclusivity extensions under the 1997 Food and Drug Administration Modernization Act, industry is increasingly studying QLs in children. We reviewed all spontaneous postmarketing QL-associated pediatric reports from the FDA's Adverse Event Reporting System (AERS) database up to May 2001. The Warnings, Precautions, and Adverse Reactions sections of all currently marketed QL package inserts were also reviewed to see if the reported adverse events were listed. A total of 115 nonduplicated reports, involving 147 adverse events, were found in children < 13 years with the following suspect QLs: ciprofloxacin 52, nalidixic acid 26, alatro/trovafloxacin 9, norfloxacin 9, ofloxacin 9, levofloxacin 5, cinoxacin 3, gatifloxacin 1, lomefloxacin 1. The most frequently reported events, without regard to causality or route of administration were: CNS events (total 41 with reports of generalized convulsions 9, anxiety/emotional lability 5); gastrointestinal events (29 with nausea/vomiting/diarrhea 14, increased LFTs 6, hepatic failure 2); skin/hypersensitivity (24 with rash 9); renal/urogenital (10 with acute renal failure 4); musculoskeletal (8 with arthralgia 3). No previously unknown adverse events were reported in this pediatric group, and all events reported more than once are listed in the safety sections of the QL labeling. This information can help guide safety monitoring for children undergoing clinical trials with QL antimicrobials.

Board H-01

J.C. Bowers, M.O. Walderhaug, and M.D. Miliotis*, CFSAN, FDA, College Park, MD 20740, and Washington, DC 20204*

CFSAN is sponsoring a study where twenty volunteers will be fed raw oysters containing V. cholerae non-O1 to determine the effect of a food matrix on dose-response. To reduce the risk to the volunteers and to enhance the probability of obtaining useful data, we propose to use the con-tinual reassessment method (CRM) as the experimental protocol. The CRM is a Bayesian dose allocation design in which sequential groups of subjects are administered the a posteriori esti-mate of the dose corresponding to a prespecified response probability. The method was originally developed for estimating the maximum tolerated dose (MTD) in Phase I clinical drug trials, where the MTD is typically defined as the dose resulting in 20% probability of adverse response. For the purpose of microbial risk assessment, a higher targeted response level is necessary since it enhances the probability of identifying or estimating the curve's location and shape parameters, and is appropriate where the response (illness) is not grave. We used a computer-based simula-tion to determine how the probability of identifying the dose-response and precision of parameter estimates vary across targeted response levels of 25%, 50%, and 75%. We show that while a prespecified response level of 25% is inadequate, a response level of 50% and 75% provide adequate dose-response information. The study will provide an estimate of the food matrix effect for V. cholerae and may also be applicable to food matrix effects on other foodborne pathogens.

Board H-02

Helen S. Barold, MD and Carole C. Carey, RN, MEng, CDRH, FDA, Rockville MD 20850

Sudden cardiac death accounts for over 50% of all cardiac deaths in the US and is the cause of an estimated 300,000 deaths per year. The age-adjusted death rates are higher in men than women (410.6 versus 274.6 per 100,000), however women were more likely to have unwitnessed out of hospital death than men. In a large retrospective cohort study from Seattle and King County, there were 10879 out-of-hospital cardiac arrests of which 7069 (64.98%) were in men and 3810 (35.02%) were in women. We examined the data from six clinical studies submitted to the FDA for premarket approval of an implantable cardioverter defibrillator (ICD). These devices are designed to prevent or treat sudden cardiac death due to a ventricular arrhythmia to determine what percentage of women were enrolled. Out of the 1343 total patients enrolled in the clinical trials, only 222 (16.53%) were women. The percentage of women who completed clinical trials for devices designed to prevent or treat SCD is much less than the percentage of women who are at risk for sudden cardiac death. The FDA should encourage sponsors to enroll more women in their clinical trials in addition to encouraging physicians to be aware that more women may benefit from ICDs.

Board H-03

Syed R. Ahmad, Parivash Nourjah, Anne Trontell, FDA/CDER/OPDRA, HFD-430, Rm 15B-08, 5600 Fishers Lane, Rockville, MD, 20857

Background: When FDA approves a new drug not much is known about its teratogenic potential. Despite the lack of human data on medication safety in pregnancy, pregnant women are routinely prescribed drugs. Currently, the FDA risk classification system for describing and interpreting the teratogenic risk of drugs uses single letters A, B, C, D or X. Pregnancy category X drugs are contraindicated in women who are or may become pregnant. Objectives: The objective of this study was to describe drug use in pregnancy and to determine if category X drugs can be captured in two national surveys. Methods: We analyzed data from two surveys conducted by the National Ambulatory Medical Care Survey (NAMCS) and the outpatient component (OPD) of the National Hospital Ambulatory Medical Care Survey (NHAMCS). These surveys capture up to 6 medications. Results: In NAMCS, the most common classes of drugs mentioned were vitamins/minerals (73%), hematologic agents (25.5%), and antimicrobial agents (7.8%). In NHAMCS, the most common classes of drugs were vitamins/minerals (73.3%), hematologic agents (18.3%), and antimicrobial agents (9.7%). In both surveys, category X drugs were mentioned in about 1% of the visits. Conclusion: Drug use in pregnancy is extensive. These surveys captured a very small sample of category X drugs and cannot be used to estimate utilization of these drugs by pregnant women accurately.

Board H-06

R.R. Wei and W.G. Wamer CFSAN, FDA, Washington, DC 20204

Several naturally occurring anthraquinones are present in botanical ingredients added to cosmetics. We investigated the phototoxicity of two prominent anthraquinones: chrysophanic acid (CA) and emodin (EM). Human skin fibroblasts were incubated overnight with medium containing CA or EM. Fibroblasts were then irradiated with ultraviolet A (UVA) or visible light. Phototoxicity was assessed by measuring the ability of fibroblasts to form colonies after irradiation. We found that treatment with 10 µM CA or 20 µM EM, combined with 2.9 J/cm² UVA light, inhibited colony formation by 50%. Treatment with 20 µM CA or 40 µM EM, combined with 2.1 J/cm² visible light, inhibited colony formation by 50%. These results justify additional studies investigating the phototoxic potential of cosmetics containing naturally occurring anthraquinones.

Board H-08

H.S. Ko and J. K. Wilkin, CDER, FDA, Rockville MD 20857

Objective: To review the clinical studies involving chemopreventive agents submitted to the Division of Dermatologic and Dental Drug Products. Method: Search in the database for drug applications and examine the design of the clinical investigations in the applications. Results: There are 11 INDs in the Division that involve chemoprevention for dermatologic conditions. The indications are primarily skin cancers. Seven of them are under the NCI, three from independent investigators, and one from a commercial sponsor. The products include retinoids, botanical, and nonsteroidal antiinflammatory agents. Three studies involve topical agents and the remainder use systemic products. The study subjects vary from apparently healthy individuals to transplant recipients, and patients with actinic keratosis or previous surgery for carcinoma or melanoma. Apart from one study on the pharmacokinetics of the drug, the other trials look at the reduction of risk or regression of a presumed "premalignant" lesion. The duration of study is between a few months to 5 years. Conclusions: Chemoprevention studies need sufficient power and duration in relation to the risk of the population being studied. Use of high risk patients may help to reduce sample size and duration of study. Skin lesions are easily observable and facilitate study on the potential of drugs for chemoprevention. Surrogates for skin cancers need to be validated.

Board H-09

K. Kaidbey¹, B. M.. Sutherland², P.V. Bennett², D.A. Dennis³, W.G. Wamer³, C. Barton³ and A. Kornhauser³, ¹Ivy Laboratories, Philadelphia, PA 19104; ²Brookhaven National Lab, Upton, NY 11973; ³Food and Drug Administration, Washington, DC 20204

Glycolic acid (GA) is used widely in a large number of cosmetics products for daily use over long periods of time. Its application has dramatically increased in the last decade. This study was conducted to investigate the effects of GA, applied in a cosmetic formulation, in modifying UV-sensitivity, and to determine whether the effect was reversible after discontinuing GA applications. 29 healthy subjects of both sexes (skin type I-III) were recruited. The subjects were divided into two groups: in group 1 (n=16), photosensitivity was evaluated by determination of sunburn cells (SBC's) and the minimal erythema dose (MED); in group 2, (n=13) cyclobutyl-pyrimidine dimers (CPD's) were evaluated. The test material consisted of 10% GA, pH 3.5, in a cosmetic formulation (vehicle). The material and vehicle were applied to rectangular areas over the mid-back region daily (6x per week), for 4 consecutive weeks. An additional area served as an untreated control. At the end of treatment, a single dose of 1.5 MED of UVB was delivered to treated and untreated sites on all subjects. Shave biopsies were obtained 24 hrs after UV exposure in group 1 for enumeration of SBC's, and immediately after UV exposure in group 2 for determination of CPD's. In addition, in group one, at the end of the 4 week treatment period, and again one week later, the MED was determined in each treated and control area. Treatment with GA resulted in a 18% reduction in MED, as compared to the vehicle treated sites, and an increase in number of SBC's by a factor of 1.9. These changes induced by GA were statistically significant. The CPD's were elevated but not to a statistically significant level. The MED and the number of SBC's returned to control values after one week of termination of treatments. In conclusion, topical application of GA sensitizes the skin to the damaging effects of UVB and this process is reversible.

Publish Only (H)

S.D. Dubey, CDER, FDA Rockville, MD 20857

In many scientific investigations, the percent change index to measure the effectiveness of a new product relative to an active control is considered appropriate. Because this index involves a ratio of two random functions, it presents challenging problems in addressing important inferential issues pertinent to bias, p-value, lower and/or upper bound of a confidence interval for true percent change index and related matters. In this paper, the author has mathematically derived many insightful results which have profound implications in designing research studies optimally, analyzing research data carefully, and evaluating the strength of scientific evidence more accurately. Because of the space constraint, the following selected results are stated here. 1. Bias associated with the usual estimate of the true percent change index will never be negative. 2. Covariance between the ratio of the study mean of treatment response(x) and the study mean of the control response(y), x/y and y will always be negative. 3. Bias is a product of correlation(x/y,y), standard error(x/y), and coefficient of variation(y). 4. Bias will disappear when correlation(x/y,y) is zero. These results show that we can minimize bias by choosing the best available control group and reducing the variability associated with x/y. More insightful results are derived when we have stochastic independence between x/y and y.

Board I-01

Dianne E. Godar¹, Fredrick Urbach², Francis P. Gasparro³ and Jan C. van der Leun⁴.¹US Food and Drug Administration, Center for Devices and Radiological Health, Rockville, MD 20852; ²Temple University School of Medicine, Department of Dermatology, Philadelphia, PA 19085; ³Thomas Jefferson University, Department of Dermatology, Philadelphia, PA 19107; ⁴Ecofys, Utrecht, The Netherlands, NL_3526KL

Since 1986, people were told they got about 80% of their lifetime UV dose by the age of 18. This myth originated from a paper that concluded diligent use of sunscreen (SPF 15 or higher) during the first 18 years of life would reduce the lifetime incidence of non-melanoma skin cancers by 78%. This conclusion, combined with the fact that squamous cell carcinoma (SCC) is dependent on the cumulative dose, mistakenly led others to believe people get about 80% of their lifetime UV dose by the age of 18. However, analysis of actual exposure data shows that people get less than 25% of their lifetime dose by the age of 18. Thus, people of all ages should protect themselves from being exposed to too much UV radiation.

Board I-02

C. D. Ellis, CDRH, FDA, Rockville, MD 20852

Recent studies suggest that most American women do not recognize heart disease as their number one health threat. While 36% of American women will die from heart disease, a recent American Heart Association survey found that only eight percent fear heart disease as their leading cause of death.

More women die of heart disease than men, even though it has long been thought of as a man's disease.  Heart disease presents itself very differently in men and women, making it even more crucial for women to have a better understanding of the symptoms and causes of heart disease which can differ greatly from those commonly associated with the disease.

The television story will feature prominent heart disease experts such as Dr. Elizabeth Ofili, the first African-American female President of the National Association of Black Cardiologists, Martha Hill, past president of the American Heart Association, Dr. Lori Mosca, Director of Preventive Cardiology at the Columbia University College of Physicians and Surgeons, and heart attack survivors.

Board J-01

A.C. Batac, L.Y. Marshall, M.M. Nickerson, T.C. Smith and P.D. Schreuders, Biological Resources Engineering Department, University of Maryland, College Park, MD 20742

The current method for injection of glucagon (a hormone that raises blood sugar) into an unconscious diabetic involves many steps and can be confusing to inject. The proposed design involves three steps and will be easier for inexperienced caretakers to use. For the GlucaGun to be successful, there must be three distinct functions. The first function is separation. Since glucagon is unstable as a liquid, the crystallized glucagon must remain separate from the diluting solution. Therefore, two distinct chambers must exist to house the glucagon and the diluting solution. The next function is the mixing of the two components. When the GlucaGun is needed, the glucagon and diluting solution must be fully mixed together. The last function is injection. The GlucaGun must be able to deliver 1cc of glucagon and diluting fluid to the unconscious individual.

The proposed design was a hand-held auto-injection unit containing a mixing chamber. The mixing chamber contains conduits with bends to force mixing. The chamber also contains a separation barrier that keeps the solution and glucagon separate until the GlucaGun is activated. A prototype of the GlucaGun was manufactured and tested against all design specifications. The prototype met all design specifications and is considered successful.

Board J-02

J.C. Hutter, H.M.D. Luu, and L.W. Schroeder, CDRH, FDA, Rockville MD 20852

We developed a physiological model of intraocular gas transfer in vitreoretinal surgery. The model was calibrated using published results for the rabbit. We validated the model by comparing the predicted and experimental results. The model was scaled up to humans, and predictions of gas expansion, half-life, and intraocular pressure were found to correlate very well with clinical results. Mass transfer in the eye was controlled by diffusion of the gases through plasma and membranes. Intraocular pressure depended on several complicating factors such as the physiological condition of the eye as well as the medications being used. The transport and thermodynamic properties of the gases injected that favor elevations in intraocular pressure were identified. For gases such as perfluoropropane, elevations in intraocular pressure are possible following an increase in volume and/or purity of the injected gas. For gases that diffuse faster than perfluoropropane, there are minimal effects on intraocular pressure due to these changes.

Board K-01

S. J. Chirtel, M. S. Boyer, K. R. O'Neil and R. J. Blodgett OSAS, CFSAN, FDA

Several FDA Centers collect adverse event reports as an integral component of their post-market surveillance programs. The proposed method estimates the expected number of adverse events per product class (E) by assuming independence of product classes and adverse event classes. The expected proportion of total adverse events that fall in each cell is the product of marginal proportions of the product class and adverse event class. The number of observed events per cell is (N). The quantity log (N/E) follows a distribution at each N that is roughly Normal, but with a slight right skew. The mean and variance of log (N/E) are calculated for each N. These parameters are then fitted with a smooth function over the range of N, and finally, standard deviation bands are calculated for the distribution. Observations five or six standard deviations from the mean are considered signals. Advantages of the new method are transparency and minimal computing requirements for any dataset size. The signals from this method are compared to the Empirical Bayes Geometric Means (EBGM) which are signals processed by the Bayesian-based Gamma-Poisson method of DuMouchelle.

Board K-03

G.A. Pennello, T.Z. Irony

Bayesian analyses are part of an increasing number of premarket submissions to FDA of experimental medical devices. Bayesian analyses take advantage of prior information on safety and effectiveness parameters by formally combining it with clinical data via Bayes Theorem. Bayesian analyses are particularly helpful because good prior information is often available from studies of the same or earlier-generation devices, and sample sizes for device trials are typically small, making prior information a crucial component of the analysis. In addition, Bayesian statistics are also helpful because they are more flexible than classical statistics with respect to, interim looks, modifications of trials, and missing data. This poster discusses the design, conduct, and analysis of Bayesian device trials from a regulatory perspective.

Board K-04

Lilly Yue, CDRH, FDA, Rockville MD 20850

In this presentation, some frequently encountered design and analysis issues in non-inferiority medical device trials will be discussed from a statistical reviewer's perspective. These issues mainly include the purpose of a non-inferiority trial, the formulation of hypothesis testing, the choice of an active control, the choice of a non-inferiority margin, the estimation of required sample size, and the analysis strategy. The presentation, augmented with examples, will focus on the current practice of non-inferiority studies in medical device submissions.

Board K-05

Yi Tsong, Meiyu Shen, QMRS, OB/CDER

In order to assure that the manufacturing procedure is yielding drug product in capsule or dosage form equivalent to that which formed the basis of NDA (New Drug Application) approval, dissolution testing is required by regulatory agency. It is required to confirm that the dissolution compliance of the product with the declared dosage. In United States, the USP dissolution test with the acceptance sampling plan is provided as a standard to determine compliance with the dissolution specification. There are different acceptance sampling plans provided in Japan and Europe. These different plans are constructed without specifying the objective in terms of the parameters of interest. We propose the parameter requirement and compare the operating characteristics of the plans under the same requirements. Statistical based procedures are also proposed to address the requirements.

Board K-07

S.Y.Zhou¹, N.T. Lao¹ and R.A. Borders², ¹CDRH, FDA, Rockville, MD 20850, ² Kimberly-Clark Corp., Roswell, GA 30076

In an inter-laboratory study for Ethylene Oxide (EO) residual determination using the automated headspace gas chromatographic (HSA-GC) method, a Matrix Factor (MF) was introduced to quantify the calibration factors for a medical device polymer (Ref. Poster No. 219). To define a MF appropriately, it was necessary to evaluate the dose-response standard curves measuring from the PA charts of various concentrations of the EO solution. This study addressed the problem of lacking a statistically valid method of developing a standard curve for calibrating values of EO residual. The typical linear standard curves were found inadequate because of variance associated with Peak Area (PA) exhibits heterogeneity across EO concentration levels. Therefore, other models for calibration were considered. This study analyzed the MFs for two polymers: plasticized polyvinyl chloride (PVC) and high-density polyethylene (HDPE). The statistical properties of the standard curve and their impact on MF values were investigated and discussed using data from twelve participated laboratories.

Board K-08

Velasco C., Delongchamp R., Chen J., Kodell R., NCTR, FDA, Jefferson AR 72079

Array data is generally normalized before evaluating differential expression. Normalization typically is a scaling factor that is applied to all genes on the array. The presence of splotch and/or saturation effects in array data limit the effectiveness of the array-wide normalization and a local adjustment seems to work better under such circumstances. The consequence of local adjustment on the evaluation of differentially expressed genes is assessed through simulation. A previously proposed model for generating intensity data is used and the effects of various normalization procedures are explored.

The median adjustment outperforms the mean adjustment under the model and conditions simulated. However, the gain in type I and type II error rates by the use of the median adjustment is modest. Modeling of array data is difficult due in part to its noisy nature, but important insights on testing for differentially expressed genes in the presence of splotches and saturation nuisances are given.

Board K-09

Yi Tsong, Sue Jane Wang, H.M. James Hung^, Office of Biostatistics/CDER

In practice, "non-inferiority" active controlled trials have been designed for three different objectives: establishing evidence of efficacy over placebo, preserving a given percentage of the effect size of the active control and establishing evidence of no much inferior to active control. All three different objectives can be represented by the same set of hypotheses with the parameters defined differently. The various designs and statistical analysis procedures for active controlled trials proposed in the literature can be summarized into two basic approaches; the historical controlled trial approach, and the cross-study comparison approach. Each approach has its limitations. The two approaches may lead to consistent and scientific conclusions when the constancy assumptions hold. Alternative approaches may be considered when the constancy assumptions are invalid.

Board K-10

Yi Tsong, Wen Jen Chen and Chi Wan Chen, CDER

Shelf life determination has been conventionally considered a statistical modeling and prediction issue. Under this framework, an ANCOVA modeling approach is used to identify one of the many possible models to describe the linear changes of the chemical attributes under study. However, the role of regulatory review of shelf life determination is to evaluate the data collected from the stability study and decide whether the proposed shelf life can be supported. For a single factor study, the objective is to test against the null hypothesis that some of the batches have shelf life shorter than the proposed shelf life. When all individual batches have estimated shelf life longer than the proposed shelf life in a single-factor study the null hypothesis is rejected and there is no need to perform the pooling tests or to identify the best model. This approach can be generalized to multifactor design in order to simplify the data analysis of a complicated multiple-factor stability study.

Board K-11

Satish C. Misra, Ph. D. and Peter A. Lachenbruch, Ph. D., Division of Biostatistics, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20852

This paper presents and discusses issues related to statistics in diagnostic imaging and a CBER/FDA perspective. A general discussion of the objectives of an imaging/screening test, design and randomization issues, issue of gold standard, analysis, per patient versus per lesion analysis, intent-to-treat versus evaluable population , adjustments for population prevalence rates, blinded versus on-site readers, etc. are discussed here. Special emphasis is given to "Intent to Diagnose" (ITD) analysis. The imaging and diagnostic studies are usually open-label. There are no effective ways to include the results of the patients who dropout from the study after they have met the on-study eligibility criteria or the patients whose images are not available or interpretable. This paper explores various possibilities and problems associated with early dropouts and non-availability of interpretable scan results. Some problems CBER/FDA has faced in evaluating applications related to diagnostic imaging have been discussed.

Board K-12

Peng T. Liu, CFSAN, FDA, Washington DC 20204

The nonlinear programming technique is adopted to determine the best design strategy for treating a non-pairwise matching case-control study with constraints. The graphical solution technique is recommended for insight into the mechanisms of the optimization process. The optimization procedure can be divided into four steps: 1) graph the region of each constraint expressed by an inequality equation, 2) determine the feasible solution region satisfied by all the constraints, 3) graph a family of objective functions, and 4) determine the optimum solution by the intersection between the objective function and the boundary of the feasible solution region or the critical point.

First Place Poster - 2002 FDA Science Forum

Board K-13

L. Holness1, M. Knippen 2, L. Simmons 3 CBER, FDA, Rockville MD 20852

Introduction: Transfusion related acute lung injury (TRALI) is recognized as a life-threatening complication of blood component transfusion therapy. TRALI manifests as a clinical constellation of signs and symptoms characterized by dyspnea, hypotension, fever, and severe bilateral non-cardiogenic pulmonary edema that usually occurs within 1 to 6 hours post-transfusion. Blood components collected from previously transfused and/or multiparous donors are most often implicated in TRALI, and most have been found to contain HLA- or granulocyte-specific antibodies. We are aware that in about 75 percent of cases reported to us, testing confirmed that HLA and/or granulocyte-specific antibodies were passively transferred to the recipient.

Methods: CBER receives and monitors reports of transfusion-related fatalities and other serious outcomes reported by blood transfusion and collection facilities

Findings: Transfusion related fatality reports submitted to us over the past 4 years indicate a gradual increase in the number of deaths (6 out of 57 to 8 of 62) or (10.5 to 12.9%) attributable to TRALI. The fatality rate over the past 4 years has been in excess of 10 percent

Conclusion: Actual occurrences of TRALI may be are more frequent than either our data or the literature would suggest. It is important to raise awareness of this injury among the medical community so that affected patients may be treated promptly and appropriately. Also, strategies are needed to identify donors whose blood products are most likely to cause a TRALI reaction, so that preventative measures can be applied.

Publish Only (K)

Marina Kondratovich, James Reeves, and Radha Menon, CDRH, FDA, Rockville MD, 20850

We discuss the problem of comparing the two dichotomous tests and combinations of them with combining rules OR or AND for positive results when only those individuals who are positive on either tests are submitted to verification of true disease status (for example, digital rectal examination and prostate specific antigen test for the detection of prostate cancer; Papanicolaou test and human papillomavirus test for detection of cervical neoplasia). Unbiased estimates of the true positive rate (sensitivity) and true negative rate (specificity) cannot be estimated directly. However, unbiased estimates of the ratio of true positive rates, ratio of false positive rates, and positive predictive values may be obtained. Using the delta method, we constructed the 95% confidence intervals for these ratios (normal approximation). We discuss also estimates of negative predictive values and prevalence of disease.

Board L-01

S. Serfling, N. Schibblehut, J. Schrider, C. Gieseker, R. Reimschuessel, Division of Animal and Food Microbiology, FDA-CVM, Laurel, MD 20708

Aquaculture is becoming an increasingly important source of fish available for human consumption. As the number of aquaculture facilities grows, so does the need to develop safe and effective drugs for treating fish diseases. In response to this, the FDA's Center for Veterinary Medicine (CVM) Office of Research (OR) has greatly expanded its commitment to aquaculture research. CVM's newly renovated, state-of-the-art, aquaculture research facility is located in Laurel, MD. There are four main research rooms totaling approximately 4,600 sq. ft., including a radioisotope fish lab and an infectious studies area. Incoming water from either the well or municipal supply is processed in a primary filtration system with computerized temperature regulation to provide a wide range of aquatic life-support systems. Research at the facility will focus on both regulatory priorities and the needs of the aquaculture community. Species being studied include tilapia, rainbow trout, Atlantic salmon, channel catfish, large mouth bass, striped bass, toadfish and goldfish. Priorities include studying biodistribution, residue persistence, metabolism, efficacy, and environmental effects of drugs and other chemicals used in aquaculture. Collaborative projects and internships are in place with Universities, Veterinary Schools, Federal and State agencies

Board L-02

L. Cruz, K. Chin, E. Yu, and L. Lee, San Francisco District, FDA

A February 2001 consumer complaint of histamine-related illness from the Las Vegas, NV, Washoe County Health Department triggered FDA action. The SAN-DO laboratory analyzed Tasteless Smoke treated yellowfin tuna steaks. All 18 subs were found to be decomposed, with eight subs over FDA's Defect Action Level of 50 ppm of histamine. Levels ranged from 51-258 ppm.

Carbon monoxide-treated and "tasteless smoke"-treated tuna products typically appear highly desireable, usually a brighter watermelon-red in color rather than the usual burgundy color of fresh or fresh-frozen tuna. The appearance can hide decomposition including significant levels of histamine. Since February 2001 SAN-DO has analyzed 106 treated tuna products imported through California ports and found 28 samples with histamine levels above the current guideline. Seven of these had histamine levels greater than FDA's poisonous action level for tuna of 500 ppm, ranging up to 2060 ppm. The high levels of histamine in a product that visually appears to be of superior quality indicate a considerable risk to consumers of CO-treated tuna.

Board L-03

Rainer Buchalla, Kim M. Morehouse, and Timothy H. Begley, US Food and Drug Administration, Office of Food Additive Safety, 200 C Street SW, Washington DC 20204, USA

Estimating exposure to radiolysis products of polymers is an important part of the regulatory evaluation of packaging materials for use in food irradiation. In recent years, several groups have investigated some of the more widely used polymers using headspace or thermal desorption techniques coupled with gas chromatography-mass spectrometry (GC-MS). The coupling of liquid chromatography (LC) with mass spectrometry (MS) allows the characterization of heavier and more polar molecules, which cannot be analyzed by GC. Poly(ethylene terephthalate) (PET) is a polar polymer which already contains large amounts of polar low molecular-weight (low-MW) material. Thus, the ability to analyze polar non-volatile compounds is critical for an evaluation of radiation's effect on PET.

Preliminary results with an amorphous PET sheet irradiated with ⁶⁰Co-gamma-rays at 25 and 50 kGy confirm earlier results that were obtained by LC with UV-detection. That is, the concentrations of the major compounds that are already present in the non-irradiated PET do not change perceptibly. However, we find a small but significant increase in terephthalic acid (mono-) ethylester, from less than 1 ppm in the non-irradiated control to ca. 2 ppm after 50 kGy, which has not been described before. The finding is important because it gives an impression of the sensitivity of the analytical method. Additionally, it shows again that even very radiation-resistant polymers can form measurable amounts of low-MW radiolysis products.

We are currently trying to optimize our methods for the most polar compounds eluting near to the solvent front. We also intend to analyze other PET samples, such as crystalline PET films and PET irradiated under vacuum. Valid experimental methods for the analysis of polymers are needed in other areas regulated by the FDA as well, e.g., for the evaluation of recycled materials for food packaging, or for the "chemical characterization" of medical device materials.

Board L-04

Characterizing Bacterial Populations in Dinoflagellate Cultures

L. Vargas1, L. Ruiz2, B. Tall3, S. Conrad3, S. Hall3, 1HACU Program, 2Workforce Recruitment Program for College Students with Disabilities, 3CFSAN, FDA, Washington, DC 20204

For our research on seafood toxin we produce isolates of marine phytoplankton that are clonal with respect to the photoautotrophic alga but for which the accompanying bacterial population is uncharacterized. While axenic (bacteria free) cultures are of interest in some circumstances, they are difficult to produce, even harder to maintain, and generally not needed for our work. It is of great interest, however, to understand both the identity and the dynamics of the bacteria that do occur in our cultures. Preliminary studies have revealed some interesting patterns of bacterial type and growth dynamics.

Board L-05

M.M. Mossoba, F.M. Khambaty, and F.S. Fry, CFSAN, FDA, College Park, MD 20740

The two species Bacillus cereus and B. thuringiensis are close relatives of the spore-forming pathogen B. anthracis. B. thuringiensis is an important source of an insecticidal toxin that is sprayed on crops in the form of spore-containing preparations of crystalline protein toxins. On the other hand, B. cereus is a ubiquitous soil bacterium and a human pathogen that may cause contamination in the food industry. The only reported difference between B. cereus and B. thuringiensis are genes carried on plasmids, and that they could be regarded as essentially one species. In the present study, five strains of each of B. cereus and B. thuringiensis were characterized by Fourier transform infrared spectroscopy for subsequent classification by principal components analysis and hierarchical cluster analysis. For each of the ten strains analyzed, replicates were prepared and measured on ten different days to investigate the possible sources of variance in the sampling procedure. Preliminary analysis provided excellent discrimination between three strains of each of the B. cereus and B. thuringiensis species investigated. This study demonstrated the potential of this mid-infrared spectroscopic procedure for the rapid, repeatable, and precise classification of Bacillus spp., with minimum sample preparation. Extension of this rapid identification technique could easily be applied to other biologically important species of Bacillus.

Board L-06

K. H. Seo¹, R. E. Brackett¹, I. E. Valentin-Von¹, and P. S. Holt², ¹CFSAN, FDA, 200 C street SW, Washington, DC 20204, ²ARS, USDA, 934 College Station Road, Athens, GA 30605

Salmonella Enteritidis (SE) human outbreaks have been mainly associated with contaminated eggs or foods containing eggs. Homogenizing shell eggs using a stomacher, an electric blender, and hand massage have been adapted for SE detection methods in eggs in various studies. However, no study has been attempted to find the effect of the sampling methods on the growth of SE in raw eggs. Two experiments were conducted using two different inoculum levels, 10 cells or 100 cells/pool of ten eggs, respectively. Four different sampling methods, stomaching, electric blending, hand massaging, and whipping for 30 s or 60s, were used to homogenize egg pools. Egg contents were then incubated at 37C, and SE colonies were enumerated after 24 and 48 h incubation. After 48 h incubation, no growth of SE was observed in samples inoculated with less than 10 cells/pool and stomached or electrically blended whereas SE counts in eggs prepared by hand massaging or whipping reached approximately 1x10⁹ CFU/ml and 3x10⁸ CFU/ml, respectively. In trial 2, when eggs inoculated with 100 cell/pool, significant differences in SE counts between physical blending group (hand-massaging and whipping) and electric blending group (stomaching and machine blending) were observed after 24 h incubation

Board L-07

I.E.Valentín-Bon.¹; K.H. Seo¹, R.E. Brackett¹, T.S. Hammack¹, W. Andrews¹ , CFSAN, FDA, Washington, D.C. 20204

The effectiveness of 2 methods for the recovery of Salmonella Enteritidis (SE; phage types 4, 8, 13, 13A) from jumbo shell eggs was evaluated. The first method used in the comparison consisted in a pre-enrichment of the sample (1993 Poultry Science 72:1611-1614) and the second was developed by the Animal and Plant Health Inspection Service (APHIS). Three bulk samples of eggs containing 220 liquid whole eggs each were artificially inoculated with high (1x10³ CFU/ml), medium (100 CFU/ml) and low (10 CFU/ml) populations of SE cells. Twenty sub samples containing approximately 10 eggs each were withdrawn from each of the inoculated bulks samples and incubated for 4 days at room temperature (23°C). For the APHIS method, each pool was cultured by direct plating onto brilliant green agar, (BG), and xylose-lysine desoxycholate (XLD) agars. For the pre-enrichment method, 25 g portions, from each pool, were enriched in modified Tryptic soy broth with FeSO₄, selectively enriched in tetrathionate and Rappaport-Vassiliadis broths and streaked to BG, bismuth sulfite, and XLD plates. SE isolates were confirmed biochemically and serologically. The pre-enrichment method recovered significantly more SE isolates (p < 0.05), from all the phage types, than the APHIS method in all of the experiments. From a total of 478 test portions 332 were SE-positive by the pre-enrichment method and 160 were positive by the APHIS method. The pre-enrichment method provides greater sensitivity for the isolation of SE in contaminated egg slurries.

Board L-10

T. T. Tran, R. N. Matthews, C. R. Warner, and S. J. Chirtel. U.S. Food and Drug Administration, Washington, DC.

Test portions (25 g) of fresh-cut broccoli, celery, lettuce, mung bean sprouts, and scallions were treated with aqueous solutions of cetylpyridinium chloride (CPC, 0.1%) or 3 commercial vegetable wash preparations (VWP), namely F, SH and VW, with sonication. Indigenous microflora on the produce were enumerated by spiral plating on Standard Methods agar. Surviving organisms on CPC-, F-, SH-, and VW-treated produce at 25^oC for 5 min showed a log₁₀ differential of 0.4, 0.5, 0.4, and 0.6 compared to the control (untreated produce), respectively. A log₁₀ differential of 2.1, 1.7, 1.6, and 1.6 was realized with treatments at 50°C for 3 min, respectively. There were significant differences between treatments and time-temperature combinations (P < 0.05); but no interactions between time-temperature combinations and treatments, and no significant differences among the efficiencies of the disinfectants within each time-temperature combination. CPC is used as an antiseptic and disinfectant in commercial mouthwash preparations. VWP are claimed to remove pesticides, chemicals, waxes, as well as bacteria from the surfaces of fruits and vegetables. Results of this study suggest that aqueous solutions of CPC and VWP can be used in combination at 50°C as surfactants and disinfectants to effectively remove and reduce the viability of microbial contaminants from fresh produce.

Board L-11

T. T. Tran, J. I. Uwaleke, R. L. Thunberg, C. R. Warner, and S. J. Chirtel. U.S. Food and Drug Administration, Washington, D.C. 20204.

The effect of chloramine (80ppm), chlorine (200 and 2000 ppm), ethanol (10%), and ozone (2ppm) on the aerobic spoilage bacteria of broccoli, celery, lettuce, mung bean sprouts, parsley, and scallions was investigated. Test portions (25 g) were treated with aqueous solutions of these disinfectants, and then analyzed for aerobic plate counts (APC). The effectiveness of different sanitation regimens was estimated by the difference (D) between the APC (in log₁₀ cfu/g) of controls (C) and treated portions (logD). The mean C values ranged from 5.5 to 9 log₁₀ cfu/g. The overall effectiveness of ozone, chloramine, chlorine 200 and 2000 ppm, and ethanol were 0.2, 0.5, 1.2, 1.9, and 1.7 logD, respectively. Significant differences (p<0.05) in counts (logD) were seen in 1 out of 5, 4 of 5, 5 of 5, 5 of 5, and 5 of 5 produce categories tested with ozone, chlorine 200 and 2000 ppm, chloramine, and ethanol, respectively. Sonication, done in experiments with chloramine and chlorine, significantly (p<0.05) improved the overall effectiveness of these disinfectants by 0.2 logD. In practical terms, ozone was the least effective; and ethanol, the most effective and economical. Moreover, the results showed the limited effectiveness of these sanitation agents against the compact and resilient biofilms formed on surfaces and/or in crevices of vegetables.

Board L-12

R.A. Benner, Jr., P. Rogers, and W.F. Staruszkiewicz, CFSAN, FDA, Washington DC 20204

Putrescine, cadaverine, and indole were evaluated as chemical indicators of decomposition for wild and aquacultured Penaeid shrimp decomposed under controlled conditions at 0, 12, 24, and 36°C and in field verification studies with commercial shrimp samples collected and evaluated by FDA experts. Putrescine, cadaverine, and indole levels increased with time as the temperatures of decomposition increased. Putrescine, at a reject level of 3 ppm, confirmed sensory evidence of decomposition more frequently than did cadaverine or indole at reject levels of 3 ppm or 25 µg/100g, respectively. Based on this research, putrescine appears to be the most comprehensive chemical indicator for decomposition in fresh or frozen Penaeid shrimp studied to date.

Board L-13

A.P. Jacobson, M.L. Johnson, T.S. Hammack, and W.H. Andrews CFSAN, FDA, College Park, MD. 20740

The lowest detection level (LDL) of the U.S. Food and Drug Administration's Bacteriological Analytical Manual Shigella culture method was determined for 6 produce types (parsley, cilantro, celery, white onion, shredded carrots, and strawberries). With unstressed cells, LDLs were less than 10 cfu/25g on all produce types. With chill-stressed cells, LDLs were less than 10 cfu/25g, except with parsley (23 cfu/25g) and strawberries (17 cfu/25g) with one strain of S. sonnei. With freeze-stressed cells, LDLs with one strain were 11 cfu/25g and 52 cfu/25g for carrots and strawberries, respectively. With the other strain used, LDLs were less than 10 cfu/25g.

The efficiency of 6 reducing agents (Oxyrase®, thioglycollate, titanium (III) chloride, L-cysteine, L-cystine, and dithiothreitol) was compared for recovering chill-stressed and freeze-stressed S. sonnei cells during enrichment. Oxyrase®, at a concentration of 20 ul/mL in Shigella broth, consistently recovered the lowest numbers of chill-stressed and freeze-stressed S. sonnei cells and is a potential alternative to the more impractical GasPak anaerobic system.

Board L-14

Badar Shaikh, Nathan Rummel, Stanley Serfling, Christopher Middendorf and Renate Reimschuessel. Office of Research, CVM, FDA,, Laurel, MD 20708

Albendazole at 10 mg/kg body weight was incorporated into ~ 10x10 mm squares of fish food formulated in a gelatine base and fed as a single dose to six rainbow trouts. Each fish was held in a separate section of water tank set at the temperature of 12°C. Muscle plus skin tissue samples were collected at 8, 12,18, 24, 48, 72, and 96 hours post dose. These samples were homogenized in dry ice and subjected to extraction and cleanup procedures. The final extracts were analyzed for parent drug albendazole and its major metabolites, albendazole aminosulfone, albendazole sulfoxide and albendazole sulfone using high performance liquid chromatography and fluorescence detection. Albendazole was detected in only 8 and 12 hour post dose samples. Albendazole sulfoxide, a pharmacologically active metabolite, was detected in 8, 12, 18, 24 and 48 hour post dose samples and low levels of albendazole sulfone, an inactive metabolite, were detectable until 96 hours. A third metabolite, albendazole aminosulfone, was not detected.

Board L-15

S Simjee^1*, D G White¹, P F McDermott¹, and J J Maurer², ¹US FDA, CVM, Muirkirk Road, Laurel, MD 20708, ² University of Georgia, Dept Of Avian Medicine, Athens, GA 30602

Synercid, a streptogramin, was approved in the United States in 1999 for treatment of vancomycin resistant enterococci. Another streptogramin, virginiamycin, has been used in veterinary medicine for over two decades. Enterococci resistant to virginiamycin are cross-resistant to Synercid. A study was conducted to determine the prevalence of streptogramin resistance genes in enterococci and coagulase negative staphylococci isolated from poultry litter in 24 different farms across Georgia, USA. A total of 44 enterococci and 28 staphylococci isolates displayed MIC's > 4 µg/ml to Synercid and/or virginiamycin. These isolates were further analyzed for the presence of streptogramin associated resistance genes by PCR studies. The prevalence of known streptogramin resistance genes was: satG, 2.27% (n=1) of enterococci; satA, 84% (n= 37) of enterococci; vgaB, 67.85% (n=19) of staphylococci. The presence of vatA, vatB, vatC, vgaA, vgbA or vgbB was not detected, however, ermB was detected in 68% (n=30) of enterococci. Using sat degenerate primers 84% of enterococci (n=37) and 7.14% (n=2) of staphylococci were positive. This study suggests that poultry litter may serve as a potential source harboring enterococci and staphylococci carrying known and yet to be identified streptogramin resistance genes in enterococci.

Board L-18

R.M. Jacobs, ORA, San Francisco District Laboratory, FDA, Alameda, CA

The use of Pb-based and other metal-based inks for labeling candy wrappers has been a regulatory issue over the past decade. While the U.S. and the E.U. abandoned the use of metal-based inks in food wrappers some time ago, Pb and chromium (Cr) have been found in imported candy wrappers from Mexico and the Philippines. Understanding the physical nature and location of these inks in the wrapper is key to the regulatory process. FDA regulatory action is largely limited to those instances where the metal(s) can be shown to contaminate the candy. Conducting an analysis in such a way to illuminate both the locations of the ink and its ability to leach under simulated physiological conditions are key analytical aspects. Pb-based inks have been found on the exterior and interior surfaces, and in the matrices of wrappers. For some plastic wrappers, exterior and matrix Pb does not appear to pose an exposure hazard for children, providing the wrappers are not consumed or masticated. In those instances, considerations for regulatory action are being referred to CPSC. Where Pb can be shown either to migrate through the wrapper or physically transfer to the product, FDA regulatory action appears probable. However, the quantity of metal(s) is important. The use of hand-held XRF devices can be useful in identifying wrappers with metal-based pigments.

Board L-20

P.P. Eilers1, S.M. Conrad1, K.D. White1, C. Beaudry1, G. Langlois2, J. Matweyou3, G. Plumley3, S. Hall1, 1CFSAN, FDA, Washington DC 20204, 2CA Dept. Health Services, Berkeley CA 94704, 3Univ. of Alaska, Fairbanks AK 99701

Natural toxins from plankton can cause serious human illness when accumulated by shellfish. Dinoflagellates of the genus Alexandrium, which are the source of Paralytic Shellfish Poisons along both coasts of the USA, occur in populations with distinct toxin compositions. These differing compositions have implications for both toxin detection and regulatory policy. We are therefore culturing Alexandrium from various locations along the Pacific and Atlantic coasts and analyzing their toxin compositions so that we can plot the distributions of different compositional types.

Board L-21

S.M. Conrad1, S. Hall1, G. Langlois2, P. Anderson3, C. Dolan4, J.M. Hickey5, M. Shute 6, 1CFSAN, FDA, Washington DC 20204, 2CA Dept. Health Services, Berkeley CA 94704, 3ME Sea Grant Program, Univ. ME, Orono ME 04469, 4Univ. NH Cooperative Ext., Durham NH 08824, 5MA Div. Marine Fisheries, MA, 6CT Dept. Agriculture, Milford CT 06460

Volunteer plankton observer networks are proving to be a powerful leveraging tool for providing better food safety protection with limited FDA and State resources. Sightings of toxic plankton species by volunteer observers in the field provide early, detailed warnings of possible shellfish toxicity to state regulators, who can therefore focus their toxicity monitoring effort to the times and locations of greatest concern. The plankton observations furthermore indicate the type of toxin to be tested for. Experience with several of these programs over a 5-10 year period allows us to draw some conclusions about their performance, and to derive some insights about optimal program structure.

Board L-22

Ronald Pace, Susan Jackson and Yuan Hu, ORA, FDA, Jamaica, NY

Current culture methods are labor intensive and time consuming when handling many samples, and in order to overcome these problems we developed this research project. The objective of this project is to produce a rapid and sensitive detection method to analyze food commodities for multiple pathogens. We chose to approach this method by using PCR to detect Salmonella spp and shiga-toxigenic E. coli in the same PCR reaction. This method can be used to detect more than one pathogen at a time and reducing the time required for analysis would be beneficial to the FDA, the manufacturer and the consumer. We proposed a method to simultaneously detect shiga-toxigenic E. coli and Salmonella spp. in apple cider. Apple cider has been indicted in several foodborne outbreaks involving both E. coli O157:H7 and Salmonella spp. in the United States. In this method, we used a general enrichment step to support the growth of both shiga-toxigenic E. coli and Salmonella spp. After 24 hours incubation in the universal pre-enrichment broth, the DNA from the organisms was isolated. These steps were followed by PCR and gel electrophoresis for the detection of the PCR amplicons of interest. Our results proved this method is able to successfully detect Salmonella spp and shiga-toxigenic E. coli in less time than either of the current BAM methods used to individually detect E. coli 0157:H7 or Salmonella spp.. Rapid assays based on our nucleic acid amplification techniques have great potential for identifying different pathogen in the food.

Board L-23

A.M. Lando, DMS/OSAS/CFSAN/FDA, College Park, MD 20740

Proper storage of food in the home is an important practice for preventing foodborne illnesses. Data about consumers' self-reported refrigeration practices from the 1998 Food Safety Survey were analyzed descriptively and by logistic regression. Twelve percent of the 2001 respondents reported having trouble deciding whether to refrigerate a product in the past three months. Those most likely to have trouble deciding whether to refrigerate or not were those likely to be more attuned to food safety issues. They included: females, those with some college or higher education, the middle aged, people who look at many sources of food information, respondents who thought that a household member had a recent foodborne illness, and those who believe that it is a very common problem for people to get food poisoning from handling food at home. The most problematic foods were ones stored on a shelf when purchased that might need to be refrigerated after opening to maintain safety. Of the respondents who reported having trouble deciding whether to refrigerate, nine percent incorrectly decided not to refrigerate a product that needed refrigeration to maintain safety. These results suggest that additional education may be needed to reach less aware consumers about proper refrigeration. They also suggest that storage information on packages is particularly important for foods that can be stored at room temperature until opened and then need refrigeration.

Board L-24

W.F. Staruszkiewicz and P.L. Rogers, CFSAN, FDA, Washington, DC 20204

Consumer illnesses due to scombroid poisonings from decomposed seafood have been a continuing problem for many years. Research studies have shown that histamine is one of the indicators of the scombrotoxic condition in fish and that other amines such as cadaverine may also be involved. Guidance for the handling of fish on-board fishing vessels to prevent the formation of the toxic condition has been limited by a lack of data on changes which occur in fish from the water to delivery at dockside. To acquire the level of detail which is needed for the development of guidelines, studies were conducted at-sea to measure the changes in levels of histamine, cadaverine and putrescine in mahimahi and tuna which were captured and held in seawater at 26 or 35 C for up to 18 hours. Each of the fillets from the treated fish were analyzed for histamine, cadaverine and putrescine. The results show that at 26 C, 13 hours of incubation were required before a histamine level of 500 ppm was reached in mahimahi. At 35 C, a 500 ppm level was reached within 10 hours. Similiar results were found for skipjack and yellowfin tuna. Cadaverine concentrations were more sensitive to incubation conditions than were those of histamine. At both temperatures, an increase in the level of cadaverine preceded an increase in histamine. The study also demonstrated that histidine decarboxylase could be retained in frozen samples of fish and result in further increases in histamine upon thawing.

Publish Only (L)

C.P. Giri, D.B. Shah, R. B. Raybourne, and D. J. Kopecko, FDA-CFSAN/CBER, Washington, DC/Bethesda, MD.

S. Typhimurium DT104 strains have gained recent prominence in U.S. food borne infections, raising the possibility of novel DT104 virulence attributes. The focus of the present study was to identify specific DT104 virulence functions up-regulated during infection of human intestinal INT407 cells. Sau3A1-restricted and size-fractionated (0.4-1.6 kbp) fragments representing the entire DT104 chromosome were cloned into a multiple cloning site upstream from the promotor-less, GFP and CAT tandem gene reporter sequences, developed in a pUC bacterial expression vector. Recombinant plasmids were electroporated into a DT104 strain and the resultant library of ~40,000 Amp^R DT104 recombinants were used to infect 1-day old INT407 cells. Following a 1 hr infection period, remaining extracellular bacteria were killed by the addition of 100 ug/ml gentamicin and those intracellular bacteria expressing the CAT reporter gene were selected by incubation with 100 ug/ml chloramphenicol (Cm) for 8 hrs at 37^o C. This selection was serially repeated four times and the resulting Cm^R S. Typhimurium were also sorted by flow cytometry for intracellular GFP expression. Constitutive Cm^R isolates were distinguished from the desired inducible Cm^R recombinants on plate media. Sequence analyses of individual isolates and comparison with the S. Typhimurium genome are being used to identify affected DT104 genes which will be analyzed for novel virulence activity.

Publish Only (L)

K. H. Seo¹, R. E. Brackett¹, I. E. Valentin-Von¹, K. A. Lampel¹, and P. S. Holt², ¹CFSAN, FDA, 200 C street SW, Washington, DC 20204, ²ARS, USDA, 934 College Station Road, Athens, GA 30605

Although contamination of eggs with Salmonella Enteritidis (SE) occurs sporadically rather than endemically, many of the cases have nonetheless involved grade-A table eggs. An assay was developed for the specific detection of SE in eggs, using a novel application of the fluorogenic 5' nuclease assay (TaqMan). In this assay, a segment of the gene sef14 specific to Salmonella group D strains such as Salmonella Enteritidis and Salmonella Dublin was amplified. The amplification of the target gene products was monitored in real-time by incorporating fluorescent dye-labeled gene-specific probes in the PCR reaction. This method correctly detected and distinguished Salmonella Enteritidis from nearly 100 of non-group D Salmonella and other non-Salmonella strains. Detection of sef-14 gene was linear for DNA isolated from samples containing 10⁴ to 10⁹CFU per ml of a pure culture of Salmonella Enteritidis. When tested with DNA prepared from the enrichment broth using the developed PCR method, and compared with the results using a conventional culture method which takes up to 5 days, 100 % correlation was observed. The sensitivity of this assay was 1 CFU per 600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low levels of SE in egg samples.

Publish Only (L)

K. H. Seo¹, Holt, P. S.², R. E. Brackett¹, Stone, H. D.², Greene C. R.², and Gast, R. K², ¹CFSAN, FDA, 200 C street SW, Washington, DC 20204, ² ARS, USDA, 934 College Station Road, Athens, GA 30605

Salmonella Enteritidis (SE) has been a major cause of human salmonellosis related to the consumption of egg and poultry products. In chickens infected with SE, the IgA directed against SE antigens can be found in both bile and mucosal secretions. Because the crop is the first host gastrointestinal environment encountered by SE after infection, the presence of specific IgA at this location can influence the survival and persistence of an SE infection. The presence of specific IgA antibodies in crop has not previously been reported. In this report, we show that chickens, infected with SE by oral gavage, produce secretory IgAs (sIgAs) that specifically bind to numerous SE antigens. Chickens infected with SE showed strong sIgA response against flagella in both bile and crop. The O.D values of ELISA test in positive bile and crop were 1.17 and 0.38, respectively, and were significantly different from that of negative samples. Western immunoblotting revealed that ~13.5, ~56, ~62, ~80, and ~143 kDa polypeptides were immuno dominant proteins in bile while ~56, ~62, and ~80 kDa were found to be strong antigens in crop. Although all three immunogens in crop were detected strongly in bile, reactions against the ~56 kDa polypeptides were stronger in crop than in bile.

Publish Only (L)

S. A. McCarthy¹, J. G. Wilkes², R. Holland³, M. Holcomb², J. O. Lay, Jr.², and S. M. Musser⁴, FDA CFSAN, Dauphin Island, AL¹, NCTR, Jefferson, AR ², ORA, Jefferson, AR³, and CFSAN, Washington, DC⁴

The pandemic spread of Vibrio parahaemolyticus O3:K6 included two outbreaks in the U. S. Two mass spectrometric methods were examined for their ability to distinguish the O3:K6 outbreak strains from strains involved in a previous outbreak based on biochemical constituents. Whole bacterial cells were examined by pyrolysis mass spectrometry (PyMS) and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The two-dimensional canonical variate score plot for samples analyzed by PyMS distinguished V. parahaemolyticus O3:K6 from previous outbreak strains. Ions with positive relative weights by pattern recognition were associated with spectra for the O3:K6 outbreak strains; negative weight ions were associated with spectra for strains involved in a previous outbreak. The MALDI-TOFMS spectra of all V. parahaemolyticus strains were similar; however, the O3:K6 strains shared a "doublet" peak representing a unique protein not found in the other strains. The simple handling requirements, short analysis time (2.5 days), and distinctive spectra obtained demonstrate the potential use of PyMS and MALDI-TOFMS for identification of whole bacteria in epidemiological investigations.

Publish Only (L)

Michael D Wong, San-Do, 1431 Harbor Bay Parkway, Alameda CA 94502

MCPD, a manufacturing contaminate and potential carcinogen, created during the production of some soy sauces, is scheduled for regulation by the European Union in 2002 at less than 20mg/1000kg. A method for analysis is published in the AOAC International utilizing a mass spectrometer and a deuterated internal standard. San-Do, in partnership with the Food & Drug Lab Branch, Dept. of Health Service, State of California, has been evaluating the method.

Originally, before purchasing its own GC/MSD, San-Do would perform the extraction; the State Lab would quantitate on the MS. Both labs used the Hewlett Packard 5973 MSD, using the SIM (selective ion monitoring) mode. Calibration standards were prepared between 0-2.0 mg/ml; original regression data indicate the standards followed a quadratic curve. A few fortified soy sauces were prepared, spiked between 6.25-250 mg MCPD /1000 kg soy sauce. Results so far have been inconsistent: lower levels were generally non-detectable, higher levels were generally recoverable, but not at the fortified amount.

Publish Only (L)

K. H. Seo¹, R. E. Brackett¹, I. E. Valentin-Von¹, and P. S. Holt², ¹CFSAN, FDA, 200 C street SW, Washington, DC 20204, ²ARS, USDA, 934 College Station Road, Athens, GA 30605

The detection of Salmonella Enteritidis (SE) in eggs is limited by the infrequent occurrence of SE contaminated eggs, the very small numbers of SE in SE-positive eggs and the inhibitors naturally present in egg albumin. Recently, a commercially available rapid detection kit has been developed. The Reveal for Salmonella Enteritidis (Reveal-SE) test system is the only available commercial rapid detection kit for specifically identifying SE among other Salmonella serotypes. In all three trials, methods involving direct plating of nonselective broth or selective broth culture steps detected a significantly higher percentage of contaminated egg pools than did direct plating of straight egg cultures, but required 48 h longer to provide results. The Reveal-SE test system showed very close correlations with the results from the direct plating methods. In trial 3, when extremely low levels of SE (4 cell/220 eggs) were inoculated and analyzed by direct plating and the Reveal-SE, all enrichment steps were required to get the maximum number of SE-positive samples. The Reveal-SE test kit, when used following double step enrichment (pre and selective enrichment), detected more SE-positive samples than direct plating when 30-day old eggs were inoculated with 45 cell/220 eggs.

Board M-01

K L Thompson¹, M L Mirsky², E Kadyszewski², and F D Sistare¹, ¹CDER, FDA, Laurel, MD and ²Pfizer, Inc., Groton, CT

As part of an ILSI sponsored consortial evaluation of the application of genomic methodologies to mechanism-based risk assessment, the relationship between traditional toxicologic endpoints and gene expression responses to the nephrotoxic drugs was examined. For one study, 5 male Sprague Dawley rats received a single dose of 5 mg/kg cisplatin i.p. 6 days later, the animals were sacrificed, standard serum chemistry and urinalysis parameters were measured, and the kidneys processed for histopathology and RNA. Three animals had moderate renal tubular necrosis and regeneration, and highly elevated serum levels of BUN and creatinine. Two animals had mild or no evidence of tubular necrosis and regeneration. Gene expression profiles were evaluated on Phase-1 Rat(CT) cDNA microarrays using samples from individual animals, pooled controls, and pooled treated animals. The observed patterns of gene expression in this study were consistent with known mechanisms of cisplatin nephrotoxicity, highly reproducible, and reflective of the extent of renal injury observed from analysis of histopathology and clinical chemistry.

Board M-02

R. Duncan¹, P. Salotra², N. Akopyants³, S. Beverley³, H. Nakhasi¹, ¹Center for Biologics Evaluation and Research, FDA, Bethesda, MD; ²Molecular Biology Lab, Institute of Pathology (ICMR), New Delhi, India; ³Washington U., St. Louis, MO.

Leishmania donovani causes visceral disease in 500,000 new cases each year. The toxicity and cost of the treatment and new challenges such as emerging drug resistant strains, urge us to accelerate the pace of discovery of new genes that may play a role in pathogenesis. Consequently, we are developing a genomic microarray for L. donovani.

To insure maximal representation of genes required for pathogenesis, parasites were isolated from a kala-azar patient and cultured minimal passages before preparing DNA. The DNA was sheared by nebulization and 1.0-1.5kb fragments were ligated into the pZErO vector. Colonies were picked and grown in 96 well plates to produce plasmids for sequencing and PCR products for spotting onto microarrays. Agarose gels of plasmids to date indicated 92% of colonies picked were productive clones. Sequencing results to date indicated that 66% of the clones were homologous to L. donovani genes, L. major genes and L. major genomic sequences that have not been analysed in genbank, 32% are unknown and 2% are empty vectors.

The L. donovani microarray will be hybridized with cDNA probes from parasites induced to differentiate in culture. Each induced probe will be mixed with a common reference probe to identify genes that are increased or decreased in expression over the course of differentiation. Data from such an analysis will be discussed.

Board M-03

W. Tong², R. Perkins², J. Anson^{1 1}NCTR; ² Logicon ROW Sciences, Jefferson, AR 72079

Modern toxicology has focused on understanding biological mechanisms involved in the expression of toxicity. Advanced technologies such as genomics and proteomics offer new approaches for investigating disease and toxicity at the molecular level, and for discovery of corresponding biomarkers. Data from the new experimental platforms are both huge in number and noisy in content. Extraction of useful knowledge requires bioinformatics for data management and analysis. The NCTR Bioinformatics Laboratory (NBL) provides an informatics infrastructure and data analysis capability available within FDA. NBL expertise spans across computational chemistry, molecular modeling, simulation and scientific programming, bioinformatics, chemoinformatics, software engineering, scientific visualization, and Internet technology. The poster illustrates NBL capabilities by presenting several on-going projects: (1) microarray data management; (2) microarray data analysis, including normalization, significance analysis, clustering and classification; (3) protein gel image analysis and visualization; (4) predictive toxicological models using QSARs and other data mining tools; (5) web-based database development; (7) knowledge-base development. The NBL mission is to conduct collaborative research within FDA, and we invite you to contact us. NCTR contact person: Jeanne Anson at janson@nctr.fda.gov

Board M-04

J.C. Fuscoe, V.G. Desai, W.S. Branham, and C.L. Moland, NCTR, FDA, Jefferson, AR 72079

The new "-omics" technologies promise to revolutionize the understanding of biological systems, including toxicology. Toxicogenomics combines the emerging technologies of genomics, proteomics, and bioinformatics to identify and characterize mechanisms of action of known and suspected toxicants. This information will aid in the extrapolation of surrogate organism data to humans, as well as provide biomarkers for determination of risk. To efficiently utilize this new technology, the NCTR has developed a Center for Functional Genomics to provide a validated database by standardizing the printing and processing of DNA microarrays, and for the analysis of the large amounts of data generated. To accomplish this, the Center will standardize molecular, analytical, and informatic tools for the production of validated gene expression databases for a variety of surrogate organisms, as well as for humans. There are three crucial areas of expertise that are necessary for the optimal application of microarray data to toxicology. These are informatics, statistics, and biomarkers. The NCTR has a long history of combining these elements in toxicological research covering a wide range of endpoints (biomarkers). The Center for Functional Genomics will offer new tools for fundamental, hypothesis-driven investigations aimed at interpretation and/or revision of basic scientific concepts to address emerging public health needs.

Publish Only (M)

R. L. Bernstein, San Francisco District, U. S. Food and Drug Administration

Over 50 bacteria have had their genomes completely sequenced. In most cases the complete genomic DNA sequences can be readily accessed by researchers over the Internet, along with computer algorithmic tools to aid in data mining and analysis of genes and proteins. Many of the bacteria are pathogens of regulatory significance. Genomic comparisons of variant strains of the same species reveal genes and proteins specific to pathogens, such as toxins. The E. coli K12 and E. coli O157 genomes differ by over 900,000 basepairs, and further analysis shows the pathogenic O157 EHEC strain sports over 1000 genes not found in the nonpathogenic K12 strain.

Bioinformatics approaches include analyzing known protein exotoxins at the sequence and structure level. Clostridial neurotoxins form a closely related family. PSI-BLAST and HMM searches of protein databases reveal other members of toxin families in unrelated organisms. Toxin domain-specific searches sometimes find human proteins from the human genome databases that share distantly related common domains, implicating the toxin's mechanism of pathogenesis. Analysis of the three anthrax toxin proteins and their human cellular receptor promises new insight into vaccine development. Toxin genes make good PCR targets for detecting pathogens in foods.

The genomic DNA sequences and associated annotated protein databases provide an unprecedented opportunity for using bioinformatics approaches to enhance the regulatory mission.

Board N-01

S. Martin, J. Kanicki

Today, active-matrix liquid crystal displays (AMLCDs) with high-resolution formats with up to nine mega-pixels are available. When the color filters required for full-color are removed, the maximum luminance of the monochrome unit is increased by a factor of at least 2, surpassing the maximum luminance of diagnostic cathode-ray tubes (CRTs). AMLCDs have recently become candidate displays for high quality diagnostic imaging workstations. However, the characterization of the image quality of AMLCDs challenges the methods used for evaluating CRT monitors. In this paper, we report on image quality metrics and measurements for high-resolution medical imaging monochrome AMLCDs using methods described in the VESA Flat Panel Display Measurement Standard, and methods and test patterns proposed by the AAPM Task Group 18. We characterize the performance of the AMLCDs in combination with a diagnostic workstation regarding differential luminance response, pixel luminance and aperture ratio, contrast ratio with varying target size, electronic cross-talk, veiling glare, reflection coefficients and angular dependency of the luminance and contrast.

Board N-02

Nicholas N. Petrick¹, Heang-PingH.P. Chan², Berkman B. Sahiner², Mark M.A. Helvie², Lubomir L.M. Hadjiiski², ¹CDRH, FDA, Rockville MD 20857, ²Department of Radiology, University of Michigan, Ann Arbor, MI 48109

We have been developing a computer algorithm to detect breast masses on digitized screen/film mammograms. A digitized mammogram is first processed with an adaptive enhancement filter followed by local region growing. Morphological and texture feature classifications are then used to identify potential masses. . A digitized mammogram is first processed with an adaptive enhancement filter followed by local region growing. Morphological and texture feature classifications are then used to differentiate identify potential masses from normal tissue. We have evaluated our massthis mass detection algorithm on independent sets of mammograms collected at by the University of Michigan (UM) and the University of South Florida (USF). Each institution used a similar laser digitizer when acquiring cases but with different optical density ranges. The optical density ranges for the UM and USF digitizers were 0.0-4.0 O.D. and 0.0-3.6 O.D., respectively. Each institution used a different laser digitizer with different optical density characteristics for digitizing the images. For the malignant preoperative cases in the combine data set, the computer algorithm detected the cancer in 83% (130/156) of the cases at a marker rate of 1.0 per film. If the marker rate was lowered to 0.5 per film, the cancer detection rate fell slightly to 77% (120/156). In this presentation, we will describe the computer vision techniques used in the algorithm and compare the performance results obtained with cases from the individual institutions. The consistency of our mass detection algorithm with variations in case, institution and laser digitizer characteristics will be discussed.

Board N-03

G. Jacobs¹, R.F. Cullison² and J.N. Johannessen³, ¹Glenelg High School, Glenelg, MD; ²CVM & ³CFSAN, FDA, 8301 Muirkirk Road, Laurel, MD 20708

Whole shell eggs gradually lose moisture, leading to viscosity changes that can indicate relative age. Differences in MR parameters such as T2 relaxation should also occur. One dozen freshly laid, unfertilized eggs were collected from white leghorn chickens, allowed to equilibrate in the MRI lab overnight, then baseline mass and MR measurements, including T1, T2 and 3-D MRI scans were performed. Half were then kept at 4°C (FR group) and half at 25°C (RT group), and measurements were repeated at 2, 4 and 6 weeks.

The RT eggs showed a more rapid decrease in T2 and mass than the FR group. T2 changes proved a more sensitive measure of aging and storage condition than mass loss. A significant loss in mean mass from baseline was not seen until 4 weeks in the RT group and 6 weeks in the FR group. In contrast, the T2 values showed a highly significant drop relative to baseline values (P<.001) in both groups at 2, 4, and 6 weeks. Differences between FR and RT eggs were also more significant for T2 values than for mass. The mass difference between groups was not significant until 4 weeks (8% difference; .02<p<.05) and wasn't highly significant until 6 weeks (11% difference; p<.005). By 2 weeks there was a 14% difference in mean T2 between groups (p<.002), which increased to 21% then 27% (p<.0001) at 4 and 6 weeks, respectively.

Board N-05

George C. Ziobro, Alan R. Olsen and Patricia A. Valdes Biles

FDA analysts use optical microscopy in various regulatory procedures. What the analyst sees in focus is limited by the depth of field of the lens. By incrementally focusing through the specimen in the Z-direction the analyst builds a mental 3D image of the sample and the characteristics used to identify the sample. Recently developed off-the-shelf software allows the analyst to build a single, in-focus micrograph from the series of incremental images that the analyst observes through the microscope. The resulting image then can be printed and used for the training of other analysts and/or as a court exhibit. These micrographs are particularly important in demonstrating key characteristics of botanical or entomological specimens. Examples of recent research applications using digital imagery software illustrate the value of computer imaging in high priority FDA research and regulatory work involving disease vectors and botanicals.

Board O-01

Development of a Function-based Standard Methodology for Screening Medical Device materials with Potential to Activate Complement by the Classical Pathway

Grace Bushar, Daniel B. Lyle, and John J. Langone. Molecular Biology Branch, OST/CDRH/Food and Drug Administration, Rockville MD-20857.

This paper describes the development and characterization of an inexpensive, rapid, function-based standard methodology for screening materials for classical pathway complement activation. The assay detects hemolysis of sheep red blood cells treated with human complement and C4 deficient Guinea Pig complement, following exposure of the human complement to a medical device material. Complement is a group of blood proteins, which are part of the immune system. Inappropriate activation can have serious adverse health effects. Some medical device materials when in contact with patient's blood or body fluids are known to activate complement. Materials being considered for new medical devices need to be tested for complement activation.

Board O-02

G.M. Vodeiko, J. McInnis, and R.A. Levandowski, LPRVD, DVP, OVRR, CBER, Bethesda, MD

The low yield of some influenza B viruses can be a hindrance to the production of inactivated influenza vaccines in eggs. Influenza B/Beijing/184/93 (B93, a low growth [LG] strain in eggs) and B/Shangdong/7/97 (S97, a high growth [HG] strain) represent the two antigenically distinct hemagglutinin (HA) lineages circulating in human populations since 1988. Compared to B93, S97 produces 8 fold higher yield by HA titer and 100-1000 fold higher yield by EID50. The aim of this work was to generate HG reassortants between B93 and S97, and to analyze their genetic and phenotypic characteristics. We first determined the sequence of the 8 RNAs of S97 and discovered that it is a natural reassortant since its genome contains PB1, PB2, HA, NA, and NS genes from B/Victoria/2/87-like ancestors, and PA, NP, and M genes from B/Yamagata/16/88-like ancestors. Using S97 and B93 we produced an HG reassortant (HGR8), which acquired PB1, PA, NA, M, NS RNAs from S97, and other genes including HA, PB2 and NP from B93. Alignment of the deduced AA sequences of B93, S97, HGR8, and B/Yamagata/16/88 (an HG strain in the same HA lineage as LG B93) restricted the number of AAs that could be correlated with growth phenotype to one AA each in PB1, NB, BM2, and NS2 proteins; 2 AAs in the NS1 protein; and six AA in the NA protein. None of the six AAs of NA was in the highly conserved NA enzyme active site, but AA residue 248, which is adjacent to AA Ser 247 flanking the enzyme active site, is different for HG (Ile 248) and LG (Thr 248) strains. Although the results of AA alignment indicate that multiple genes potentially contribute to the growth properties of influenza B virus in eggs, the phenotypic characteristics of B93, S97, and R8 (plaque size, and kinetic of reproduction in eggs) support the major role of the NA.

Board P-01

Office of the Commissioner, FDA, 5600 Fishers Lane, Rockville, MD 20857

The Committee for the Advancement of FDA Science (CAFDAS) is an internal advisory committee to the Commissioner, Senior Advisor for Science, and the Senior Science Council. Designed to address FDA-wide science issues from a working-level scientist perspective independently of center or discipline, CAFDAS is composed of two representatives appointed from each Center and ORA to serve three-year terms. The primary objective of CAFDAS is to provide a forum for working level scientists to advise and assist the Commissioner in promoting an increase in the overall effectiveness and productivity of FDA science. Most importantly however, CAFDAS is a conduit for ideas and concerns from working-level scientists to senior FDA management concerning the state of science within the FDA. This includes providing constructive guidance and solutions to scientific challenges the agency currently faces as well as future workforce needs. In the past CAFDAS has addressed such topics as leveraging, debated the need for the implementation of sabbatical programs, and has been a strong advocate of quality-of-work life issues. Recently, CAFDAS was instrumental in the evaluation and selection of OSCC intra-agency research grants. The committee's focus for the coming year is to provide FDA scientists with information on intramural and extramural funding sources available to agency scientists and the resources necessary to compete for such funding.

⁺Current Committee Members: P.A. Orlandi, Chair; P.J. Faustino, Vice-Chair; N. Alderson, Sr. Advisor for Science; A.S. Khan; E.F. Petricoin; C.J. Rosebraugh; R.G. Kaczmarek; E. Jensen; P.F. McDermott; D. Momcilovic; T. Patterson, B. Coles; D.T. Heitkemper; E. Katsoudas; and C.F. Bové.

Board P-02

Laila Ali¹, Paul Schreuders², and Andrea Lomander², ¹FDA, CFSAN, Washington, DC 20204, ²Biological Resources Engineering Department, University of Maryland, College Park, MD 20742

Biofilms from a wild type E. coli strain were grown on steel billets in circulating starved medium for up to 48 hrs at 25°C. The billets were either polished or polished then scratched. This study investigated the percent coverage of living bacteria on the surfaces and the area and circularity of individual biofilm patches at different times of growth, with or without a sanitizing treatment. Deionized water, 200 ppm chlorine, or ultrasound was applied on the biofilm for 5 min and the biofilm was stained for viability. Images of the stainless steel at locations captured randomly over the surface, along scratched lines across the surface, and at the intersection of 2 scratches were compared. The biofilms that were treated with chlorine showed a significantly smaller percentage of surviving cells than biofilms treated with ultrasound or water. The circularity is different depending on whether the biofilm is grown on a smooth or scratched surface.

Board Q-01

Dorothy B. Abel¹, Hugh G. Beebe, M.D.², Mark M. Dedashtian³, Michael C. Morton⁴, Megan Moynahan¹, Louis J. Smith⁴, and Steven L. Weinberg⁵ (Workshop Steering Committee for the conference participants), ¹FDA/CDRH/ODE, 9200 Corporate Blvd., Rockville, MD 20850, ²Jobst Vascular Center, ³Edwards Lifesciences, Inc., ⁴W. L. Gore & Associates, ⁵Biomedical Device Consultants

Since their early introduction into controlled clinical trials within the United States, current endovascular aortic grafts have shown various types of problems. Although design and construction details vary among different endovascular grafts and the failure modes have had a variety of causes and clinical effects, it remains common to all endovascular grafts that the required pre-clinical testing did not predict these results. The recognized need for improved pre-clinical testing in an attempt to reduce unanticipated, clinical device failures resulted in an FDA-sponsored workshop on endovascular graft pre-clinical testing held in Rockville, Maryland, USA on July 31-August 1, 2001.

The workshop had international attendance by over 120 people, including 34 invited participants who represented device manufacturers, the medical community, FDA and testing facilities. A steering committee prepared a detailed worksheet specifying content areas that was distributed to conference participants in advance of the meeting. Each of the topical areas was introduced by a clinical expert presentation followed by moderated discussion among invited participants. Additional comments from interested public attendees were included. Workshop discussions were recorded and reviewed in transcript form by the steering committee. Summaries are provided for each topic. Additional results shown in table form, with copies and transcripts of presentations are available by Internet access at: http://www.fda.gov/cdrh/meetings/073101workshop.html

Board R-02

J.W. Karanian, D. Wray-Cahen, A.E. Ashby, S.L. Hilbert, P. Abii and W.F. Pritchard, OST, CDRH, FDA, Laurel MD 20708

Pre-clinical animal studies of medical devices are used to address safety, short-term effectiveness and surgical handling characteristics of medical devices prior to the initiation of clinical trials. In order to help meet the FDA's need for in-depth knowledge of animal models of human disease and device intervention, the CDRH Laboratory of Large Animal Research (LLAR) (i) conducts applied research in support of medical and hybrid device regulatory issues, (ii) provides education through courses and workshops and (iii) provides regulatory support through review of pre-clinical animal studies. The LLAR is establishing animal models of disease and investigates the safety and effectiveness of diagnostic and therapeutic devices. The LLAR is housed at the MODII animal research facility with animal management and veterinary support provided by the CVM Office of Research. The LLAR facility includes fluoroscopy and surgery suites, laboratories, pre- and post-operative animal holding, darkroom, and steam and gas sterilization. Specific capabilities include diagnostic ultrasound, interventional radiology, animal surgery, in vivo monitoring and hemodynamics, in vitro physiology and pharmacology, pathology and nutritional manipulation. LLAR represents a collaborative effort integrating the science and technology of multiple disciplines in evaluating product performance, developing new test methods, and performing applied research in support of current and evolving regulatory issues.

Board R-05

Theresa C. Smith and Victor Krauthamer, CDRH, FDA, Rockville, MD 20852

Electrical defibrillation and cardiac medications may interact and affect resuscitation outcome. Commonly, out-of-hospital defibrillation results in asystole or secondary arrhythmias, with poor patient survival. In this work we examined how the drug epinephrine (commonly given during cardiac resuscitation) interacts with defibrillators. We tested the hypotheses that epinephrine shortens the duration of arrest following a defibrillator shock, and that epinephrine increases the occurrence of a secondary arrhythmia (bigeminy). Isolated cardiomyocytes from 10-day-old chicken embryos were tissue cultured and in a shaker bath that did not allow the cells to settle. These cells formed myoballs or 3-d aggregates of cells. These myoballs were then plated in dishes and paced at 2 volts above threshold. The cells were shocked with a defibrillator for 1 msec at 100 volts. The arrest time following the shock was measured along with the occurrence of bigeminy. Following a defibrillator shock, epinephrine shortened the arrest time from 19.12±2.70 to 13.23± 1.91 seconds (N=50, P<0.05, one tailed t-test). Bigeminy occurred more frequently in the epinephrine treated subjects (37.5±13% vs. 24±12%), but not with a 95% confidence interval. We concluded that epinephrine has the effect of shortening the arrest (asystole) time following a shock, but we could not conclude that it increased the incidence of a secondary arrhythmia.

Board R-06

A. D. Lucas, S. A. Brown, V. Hitchins, S. McNamee, K. Merritt, T. Woods, CDRH, OST, FDA, Rockville MD

Ethylene oxide (EO) is commonly used to sterilize medical devices. Residual EO depends partly on the type and size of polymeric material. Many medical devices are resterilized with EO, which may affect the amount of residual EO. A major concern is the amount of residue that is bioavailable. Using the method developed by AAMI for headspace analysis of EO residues, we examined different polymers using various extraction conditions, vehicles, and various resterilization cycles. Different polymers desorb EO differently. PU 75D had much higher EO residue while PU 80A had barely detectable levels. Repeated extraction of material was necessary to obtain total EO residues. Different extraction vehicles influence the amount of EO detected. Media had some coeluting interferences, while water and oil did not. The oil had some significant reproducibility problems. Resterilization had little influence on the samples we studied. Care in establishing analytical conditions for EO is needed to obtain useful information.

Board R-07

A.A. Rodriguez, T.M. To, A.B. Margolin

AOAC Method 966.04 is used to assess the sporicidal activity of disinfectants. These products are used to reprocess reusable medical and dental devices. Several steps in the method lack standardization, causing it to yield inconsistent results. We have developed three modifications to the method in an effort to increase its reproducibility. These are a) standardization of the spore propagation media, b) establishment of a minimum spore load for the carriers and c) the use of stainless steel carriers. The present study compared the original and modified methods using Bacillus subtilis and Clostridium sporogenes. The aim of the study was to generate data to show that the modified method is more reproducible than the current one. The titers of carriers prepared by the AOAC Method varied by as much as 3.4 logs. Those prepared by the modified method fluctuated by no more than 0.9 logs. Thus carriers in the modified method provided a more uniform challenge to the disinfectant. All of the carriers tested passed the 2.5M HCl test and were sterilized by the test disinfectant. This shows that adoption of the method modifications did not alter the stringency of the test. The modified method was more controlled and reproducible.

First Place Group Award - 2002 FDA Science Forum

Board R-08

J.W. Karanian, D. Wray-Cahen, S.L. Hilbert, J Vossoughi, M. Laredo, W.F. Pritchard,

Laboratory of Large Animal Research, OST/CDRH/FDA, Laurel MD 20708

Large animal studies were designed to (1) develop models which evaluate devices per intended use and instructions for use and (2) define acute and long-term failure modes of both experimental and marketed significant risk devices. The histopathologic response from injury to healing was assessed for angioplasty balloons, stents, stent grafts and arteriovenous graft fistulas. Angioplasty balloon-injured swine coronaries develop acute thrombus (4h) followed by the development of neointimal hyperplasia (NH) by 30 days. Significant NH resulted in coronary stenosis and loss of vascular reactivity. Similarly, stents implanted in the carotid and coronary arteries of swine may develop mural thrombus and a foreign body response (inflammatory phase) followed by remodeling. Excessive proliferation (NH) leads to stenosis and loss of vascular patency. In addition, synthetic grafts (PTFE) anastomosed between the carotid artery and jugular vein (arterio-venous fistula) as a model of hemodialysis access grafts failed at the venous anastomosis due to significant NH. Thus thrombosis and NH led to compromised device performance in these models. These failure modes, that were assessed both radiographically and histopathologically, are comparable to those reported in humans. Preclinical studies of vascular interventions in swine provide information on potential failure modes and may be considered an accurate predictor of device safety and effectiveness.

Board R-09

Victor Krauthamer^* and Theresa Crosheck^{+ *}CDRH, FDA, Rockville MD 20852,⁺Marquette U., Milwaukee WI 53201

Many medical devices use high-rate electric currents to affect neural function. We examined stimulation rate versus action potential threshold and sustained firing rate for two model neurons: the rabbit myelinated fiber and the unmyelinated leech touch cell. Both models produced their characteristic action potentials. One effect of the high pulse rates was a net depolarization. This was confirmed experimentally in leech neurons when 500 Hz pulses relieved conduction block and caused irregular firing. Effects of stimulation rate on action potential firing rate were complex when stimulation rate exceeded the maximum firing rate. Many of these frequency-related effects upon firing are explained by the interaction of stimulus rate with the cell's refractory period. For the leech neuron, refractory period remained relatively constant, and the maximum firing rate was reached at several stimulation frequencies (in phase with the refractory period). For the myelinated axon, refractory period was frequency-dependent so that maximum firing decreased as stimulation frequency increased.

First Place Group Award - 2002 FDA Science Forum

Board R-11

J.W. Karanian, D. Wray-Cahen, S.L. Hilbert, J. Vossoughi, P. Abii, M. Laredo, W.F. Pritchard, Laboratory of Large Animal Research, OST/CDRH/FDA, Laurel MD 20708

This study was designed to (1) define the effects of gender on the histopathologic response of the coronary artery (LAD) to angioplasty balloon overinflation and (2) develop an animal model to adequately predict the effects of devices on the health of men and women. Balloon overinflation (20%) of the LAD was performed in sexually mature swine that were either intact males (M, n=14) and females (F, n=11) or castrates (MX, n=14; FX, n=14; respectively). The balloon injury generally disrupted the internal elastic lamina and induced neointimal hyperplasia (NH) consisting primarily of loosely organized smooth muscle cells interposed in fibrotic extracellular matrix. Both the injury index (II), a six-point injury score, and the proliferation index (PI), the ratio of neointima area/media area, were dependent on gender and hormonal state. II was 1.4-fold greater for M than F. II for MX was 64% lower than for M. Conversely, II for FX was 1.5-fold greater than for F. The greater II in the FX cohort correlated directly with the PI (NH). Similarly, the PI was greater in M relative to either F or MX. A marked NH was noted in M. The NH correlated with the hormonal state (i.e., intact or castrate) in both males and females. These data suggest that local or systemic hormone therapy may provide beneficial effects and thus enhance long-term device performance.

Board R-12

J. M. Al-qahtani MD*, I. W. McLean MD^¶, R. P. Weiblinger MPH^§ and M. N. Ediger PhD^§ *King Fahd Hospital of the University Alkhobar, Saudi Arabia, ^¶Department of Ophthalmic Pathology Armed Forces Institute of Pathology, Washington, DC, ^§Center for Devices and Radiological Health Food and Drug Administration, Rockville, MD

The study was designed to determine if moderate numbers of low-fluence, 193-nm excimer laser pulses modify or damage the corneal stroma. To address this research question the corneal epithelium of fresh bovine eyes was scraped-off and the exposed stroma was irradiated with 200 low-fluence, laser pulses from an argon fluoride excimer laser. This process was performed on 5 eyes each at two laser fluences – 10 mJ/cm² and 30 mJ/cm². The 10 irradiated and 3 control (unirradiated) corneas were sectioned and studied by electron microscopy. In addition, the maximum and minimum thickness of the anterior layer of randomly orientated collagen fibers was measured in electron micrographs. The result were that the mean maximum thickness of the anterior randomly oriented layer of collagen was 1.23 ± 0.45 µm in the control corneas, 0.67 ± 0.32 µm in the corneas irradiated at 10 mJ/cm² and 0.10 ± 0.12 µm in the corneas irradiated at 30 mJ/cm². A thin, electron-dense pseudomembrane was noticed at both fluences.

Thus we report the removal of bovine corneal stroma at 10 mJ/cm² - below the previously reported ablation threshold of 20 mJ/cm² : the mean thickness of corneal stroma removed is 0.7 and 1.1 microns at fluences of 10 and 30 mJ/cm², respectively, and finally, a thin pseudomembrane at the surface, observed at both fluences, is the only collateral damage.

Board R-14

A.B. Boam¹, M.B. Eydelman¹, F.C. Lum², P.M. Silverman¹, D.R. Lochner¹, D. J. Apple³, M. Escobar-Gomez³, L. Werner³, S.K. Pandey³, J. M. Schmidbauer³, S. Arthur³, ¹CDRH, FDA, Rockville MD, ²American Academy of Ophthalmology, San Francisco CA, ³Center for Research on Ocular Therapeutics and Biodevices, Storm Eye Institute, Medical University of South Carolina, Charleston SC

Safety and efficacy outcomes of IOL implantation in adults under 60 were compared to adults 60 years of age and older (the current indication for IOLs approved in the U.S.). Data from the FDA, Storm Eye Institute, and the AAO NEON databases were analyzed for short- and long-term outcomes of IOL implantation. Statistical analyses for significance were performed where appropriate. A comprehensive literature review was conducted in an effort to identify safety and efficacy outcomes of IOL implantation and their relationship to age at implantation. Statistical analyses of the FDA "Grid" and NEON database indicated that adults under 60 achieved equivalent or better outcomes when compared to adults over 60. Analysis of explanted IOLs and pseudophakic globes indicated similar rates and types of complications for the two groups. Examination of reports from FDA's MDR System revealed statistically equivalent rates for most categories of reportable events. The literature review revealed no additional safety or efficacy concerns specific to adults under 60 not identified in the FDA "Grid." Collaboration between the FDA, the AAO and the Storm Eye Institute together with a literature review resulted in the conclusion that the performance of IOLs in adults under 60 is comparable to those over 60.

Board R-15

T.O. Woods¹ , L.W. Grossman¹, A.A. Graham¹, & J.N. Johannessen², ¹CDRH, FDA, Rockville, MD, ²CFSAN, FDA, Laurel, MD

Recently, a 6 year old boy undergoing an MRI scan was killed by a portable oxygen cylinder that was pulled into the scanner's magnetic field. Investigation of the incident led to questions about the operation of oxygen regulators in large magnetic fields in and near MR imagers. We obtained approximately 20 oxygen regulators from several manufacturers, of types used with portable "E" gas cylinders. Some (intended for pediatric use) have maximum flow rates less than 5 L/min.; others (for adult use) have maximum flow rates of at least 15 L/min. Flow rates were measured outside the 5 gauss line, and at two worst case locations at the entrance to the bore of the FDA MOD-1 2T MRI scanner. Flow rates for some regulators were changed by the magnetic field, though the magnitudes of the changes may not be clinically significant.

Board R-16

S.C. Wood¹, D. Wray-Cahen², J.W. Karanian², W.F. Pritchard², D.B. Ly1e¹ and J.J. Langone ¹. Molecular Biology Branch (1), Laboratory of Large Animal Research (2). OST/CDRH/FDA, Rockville, MD

A porcine balloon injury model has been established using angioplasty with balloon overinflation of 20%. In this model, there is neointimal hyperplasia in the damaged vessel within 28-30 days. Damage to the vessel wall and the subsequent exposure of blood components to the extracellular matrix following the injury suggests that the activation of complement (C') is likely. Fixation of C'-reactive protein (CRP) in damaged tissues amplifies the deposition of C'. CRP and C' may also play a significant role in the proliferative responses of injured tissue and the overall rate of wound healing. To study these events in more detail, a porcine specific ELISA for the C3a component of C' has been established. Currently, C5b-9 and CRP ELISAs are being developed. Importantly, a protocol for the immunohistochemical localization of C3 on the endothelium is being developed which will be extended to C5b-9 and CRP. We plan to follow total C' levels, the generation of C' products, CRP and inflammatory cytokines such as IL-1, IL-6 and TNF from the time of balloon injury out to day 30. Additionally, immunohistochemical localization of C3, C5b-9 and CRP will be performed on the injured tissue. Development of porcine-specific ELISAs in conjunction with an immunohistochemical approach may delineate the role(s) that C' and CRP may play in amplifying inflammatory and wound healing responses that are manifest in the porcine arterial overstretch injury.

Board R-17

Loren A. Zaremba, CDRH, Rockville, MD 20850

Magnetic resonance imaging (MRI) is an extremely useful imaging technique. However, it can be also be used to obtain information regarding temperature. The proton resonant frequency changes by 0.01 ppm/°C. This phenomenon can be used to estimate temperature distribution using a technique called phase mapping. Preliminary tests have been conducted at the 2T MOD I MRI facility using a heater immersed in a phantom to produce an increase in temperature. Temperature data were acquired using a spin echo sequence with data acquisition displaced by 8 msec from the spin echo to produce a phase change of about .043 rad/°C. A comparison of temperature measurements using the phase mapping technique and a Luxtron fiberoptic probe suggests that an accuracy of 1°C can be achieved in vitro with this method. The technique can potentially be used to estimate the temperature distribution surrounding implanted medical devices such as brain stimulators and pacemakers during MRI procedures.

First Place Group Award - 2002 FDA Science Forum

Board R-18

D. Wray-Cahen¹, J.W. Karanian¹, K.B. Carmody¹, J. Vossoughi², P.O. Abii¹, and W.F. Pritchard¹, ¹Laboratory of Large Animal Research, OST, CDRH, FDA, Laurel, MD, ²Biomed Res. Foundation, Washington, DC

Recent reports have suggested that female patients emerge more quickly from anesthesia than males. Sex steroids may play a role in these differences. To determine the effect of gonads and gender on anesthesia recovery time, we observed four groups of sexually mature pigs (242±3lb body wt.): intact males (M, n=10), castrate males (MX, n=7), intact females (F, n=9), and ovariectomized females (FX, n=13). Anesthesia was maintained by isoflurane. Pigs were mechanically ventilated during a coronary angioplasty procedure. Times (in minutes) post-anesthesia to elicit responses to stimuli and motor control parameters were noted. Castration, but not ovariectomy, reduced the response times. MX were quicker than M to develop righting reflex (X ± SEM; 119±13 v 233±27), sit (108±13 v 256±26), and stand (120±19 v 250±29), respectively (P<0.05). M took more than 2-times longer than F to exhibit post-anesthesia responses: nasal pinch (103±21 v 24±6), leg pull (114±32 v 25±6), lift head (142±23 v 38±9), righting reflex (233±27 v 77±10), sit (256±26 v 88±14), and stand (250±29 v 102±18), respectively (P<0.05). These data suggest that pigs may be a useful model for studying gender differences in recovery from general anesthesia and that both gender and sex steroids play a role in the response.

First Place Group Award - 2002 FDA Science Forum

Board R-19

W.F. Pritchard, S.L. Hilbert, D. Wray-Cahen, M. Laredo, L. Watson, A.E. Ashby, J.W. Karanian, Laboratory of Large Animal Research, OST/CDRH/FDA, Laurel MD 20708

This study was designed to define the effects of estrogen replacement therapy (ERT) on the hemodynamic and pathologic response to coronary angioplasty balloon overinflation. Females underwent ovariectomy and either received daily ERT (8 µg/kg 17b-estradiol, im, n=10) or no treatment (FX, n=14). One cohort was left intact (F, n=11). ERT was initiated one month post-ovariectomy and continued for at least 4 weeks before balloon injury of a coronary artery (LAD). One month following injury, each animal was placed under general anesthesia, the heart was surgically exposed and a blood flow probe was placed around the LAD just distal to the injury site. Nitroprusside (vasodilator) and epinephrine (vasoconstrictor) were administered by direct intracoronary infusion just proximal to the injury site in the LAD. Dose-response curves for coronary blood flow and systemic pressure were defined (dose range: 0.01-6.0 µg/kg/min). All agonist response curves were modulated by ERT. Both the maximum response and sensitivity to nitroprusside for the ERT cohort was more than 2-fold greater than for FX and also higher than that observed in F. Estrogen replacement therapy or localized hormone delivery (e.g., estrogen eluting stents) may provide beneficial hemodynamic effects and thus enhance long-term device performance.

Publish Only (R)

Keith A. Wear, CDRH, FDA, Rockville, MD 20852.

Although ultrasonic attenuation from calcaneus (heel bone) is useful in the diagnosis of osteoporosis, the processes governing the interactions between ultrasound and bone are not well understood. Attenuation results from absorption and scattering. The purpose of this study was to investigate a theoretical model describing backscatter from calcaneus. Scattering is assumed to originate from the surfaces of trabeculae, which are modeled as long thin cylinders with radii small compared with the wavelength. For mediolateral insonification at diagnostic frequencies, the model predicts that scattering is approximately proportional to frequency cubed. Measurements from 16 human calcaneal samples in vitro were in good agreement with theory. Since attenuation is approximately proportional to frequency to the first power, these data suggest that absorption is likely to be a larger component of attenuation than scattering.

Funding from the FDA Office of Women's Health is gratefully acknowledged.

Publish Only (R)

Keith A. Wear,¹ and David W. Armstrong III², ¹CDRH, FDA, Rockville, MD 20852, ²National Naval Medical Center, Bethesda, MD, 20889.

This study was undertaken in order to investigate the use of calcaneal (heel bone) ultrasonic backscatter for the diagnosis of osteoporosis. Broadband ultrasonic attenuation (BUA), speed of sound (SOS), and average backscatter coefficient (ABC) were measured in calcanea in 47 women. All three ultrasound variables had comparable correlations with hip bone mineral density. BUA and SOS were rather highly correlated with each other. The logarithm of ABC was only moderately correlated with the other two. The three ultrasound parameters exhibited similar negative correlations with age. These results taken collectively suggest that ABC conveys important diagnostic information independent of that contained in BUA and SOS and therefore may be useful as an adjunct measurement in the diagnosis of osteoporosis.

Funding from the FDA Office of Women's Health is gratefully acknowledged.

Publish Only (R)

T.M. To, G.W. Varney, A.B. Margolin

One of the most important properties of the male condom is its ability to act as a barrier to the passage of sexually transmitted diseases (STDs) including human immunodeficiency virus (HIV) and hepatitis B virus (HBV). The current accepted method for demonstrating barrier properties of male condoms to virus penetration, required for 510(k) submission, has only been validated utilizing latex condoms. The test consists mainly of filling the condom with buffer containing virus and determining whether any viruses penetrate that barrier during submersion in a collection buffer. With a challenge virus titer of 1 x 10⁸ pfu/ml the test can detect virus penetration in latex condoms as low as 2 x 10^-6 ml and a hole size of 2 µm. Increases in the incidence of latex allergies have driven consumer demand for the use of non-latex materials in the manufacture of condoms. The water leak test routinely used to assure quality of non-latex condoms can not detect small holes that might allow passage of virus. The smallest defect that can be observed during the water leak test is larger than the HIV virus. Virus penetration was determined for polyurethane condoms containing holes of known sizes produced by laser drilling. The results indicate that this method can detect penetration of challenge virus suspension in the range of 1.3 - 2.7 x 10^-6 ml and a single hole 2 µm in diameter. The sensitivity of this test for polyurethane condoms is comparable to that for latex condoms.

Publish Only (R)

W. Boivin, L. Kerr, S. Mailhot, L. O'Malley, J. Teixeira

FDA regulates the effectiveness of medical gloves in protecting health care workers from infectious agents. A durability test method was developed during a research project which took place in FY 2001, Assessment of the Durability of Vinyl Medical Gloves. This method is comprised of both a donning test and a shaker-table abrasion test. Using the method, 100 vinyl gloves representing four different manufacturers resulted in a 42% defect rate. This defect rate agrees very well with the published defect rates for vinyl gloves in clinical use, 40% ± 10%. Latex examination gloves will be tested next. Latex gloves have a published defect rate of 7% ± 5% for clinical use. While the durability test method can establish the durability of examination and surgical gloves of a variety of materials, the FY 2001 research project assessed only the durability of vinyl medical exam gloves compared to latex medical exam gloves. The current project expands the scope of this previous research by including other glove materials and designs. The durability test method developed during the FY 2001 project will be adapted for the gloves selected for testing in the continuation project. Three sets of each type of glove will be tested. The first set of gloves will be conditioned using the durability test method. In order to verify that the method accurately simulates in-use conditions, a second set will be donned by members of this research group and conditioned through a regime of tasks simulating the stresses introduced during actual use. The third set of gloves will be a group of controls that will not be stressed. After conditioning, all gloves will be water-leak tested and the results recorded. This research will result in a reproducible method which manufacturers can use in the design validation of their products. Results are being presented to the ASTM standards committee and will be published in appropriate journals.

Board S-01

D. G. Hayward and P. M. Bolger, U. S. Food and Drug Administration, 200 C St SW, Washington, DC 20204

The US EPA and US FDA discovered g/kg levels of TCDD in an anti-caking agent used in the soybean component of chicken feed. Immediate intervention by US FDA stopped further distribution of contaminated soybean meal by manufacturers in early June of 1997. Eleven chicken containing baby foods were selected for dioxin analysis from a cross-section nationwide sampling in the last half of FY 97 to the first half of FY 98. Sampling resumed in FY 2000. TCDD was measured in 8 of 11 baby food samples from FY 97/98 (range = 0.025 to 0.28 ng/kg wet weight or 0.25 to 5 ng/kg fat). TCDD was not detected in six 1999 samples tested (LOD = 0.02 ng/kg wet wt). The mean FY 97/98 TCDD concentration was significantly higher than the mean FY/00 with ½ LODs substituted for non detects (Wilcoxon rank sum p-value <0.005). If the sampling of baby food in the US in 1997 was representative, then percentage of young chickens (94.6% of all slaughtered or 7,412,000,000 in 95/96) exposed to ball clay can be estimated to be between 2.6 and 7.2%. Using the mean contaminated young chicken TCDD value of 18 ng/kg lipid and the median of all baby food samples (FY/97-98) as 0.462 ng/kg lipid, then the total young chickens exposed is 2.6%, or using the mean (1.3 ng/kg lipid) 7.2%.

Board S-02

J.A. Casanova, F.J. Schenck and L.K. Gross, Southeastern Regional Laboratory, FDA, Atlanta, GA 30319<

Nitrate and nitrite are used as food preservatives. There are indications that carcinogenic nitrosamines may be formed from nitrates at the higher pH values found in baby stomachs. Nitrate and nitrite were extracted from fruit and vegetable baby foods and quantified using ion chromatography with post-column derivatization. Nitrite was directly determined using post-column addition of Griess type reagents to produce a dye detected at 535nm. Nitrate was reduced post column with vanadium (III) chloride to nitrite, which reacted with the Griess reagent. The ability to simultaneously determine both nitrite and nitrate in a single chromatographic run without destroying the colored dye formed in the final reaction has several significant advantages. First, the use of the toxic, carcinogenic, metal cadmium, used in the official AOAC method to reduce the nitrate to nitrite, was avoided. Second, the chromatographic resolution, sensitivity and reliability are considerably improved because the reduction occurs entirely in solution and does not require contact with a metal surface for reaction. Finally, no additional labor is required to determine both nitrate and nitrite in comparison to most official methods of analysis that are presently used for nitrate and nitrite analysis. Average recoveries of spiked nitrate residues ranged from 85-120%. The ion chromatography method and the official AOAC method yielded comparable results for baby food samples containing incurred nitrate residues.

Board S-03

F.J. Schenck¹ and G.E. Mercer², ¹Southeastern Regional Laboratory, FDA, Atlanta GA 30319, ²Pacific Regional Laboratory Northwest, FDA, Bothell WA 98021

A rapid multiresidue solid-phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of organochlorine and organophosphorus pesticide residues in fresh fruits and vegetables, which does not require the use of methylene chloride, is presented. Pesticide residues are extracted with acetone and water is removed from the extract by salting out with magnesium sulfate and sodium chloride. Cleanup of the dried acetone extract is performed on an aminopropyl SPE column. Average recoveries of 8 spiked pesticide residues (acephate, chlorpyriphos, p,p'-DDE, dimethoate, lindane, malathion, methamidophos and omethoate) ranged from 70-110%. The SPE method and the method of Luke et al. (1975) yielded comparable results for samples containing incurred pesticide residues. Recovery data will be presented on a number of organophosphorus, organochlorine and organonitrogen compounds using flame photometric and mass selective detectors.

Board S-04

S.B. Turnipseed and J.E. Roybal, A.P. Pfenning. ORA, FDA, Denver, CO 80225

A multiresidue procedure previously developed to confirm fluoroquinolone (FQ) residues in catfish tissue has been used to positively identify the same residues in salmon. Using a single quadrupole instrument with in-source collision induced dissociation, ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin residues were positively identified in salmon muscle fortified at 20-80 ppb. These residues were also confirmed in extracts from incurred salmon tissue with final drug concentrations ranging from 10-1000 ppb.

In addition, this method was adapted for use with an ion trap LC/MSⁿ instrument by collecting data dependent MS² and MS³ scans to yield structurally significant ions. Salmon control, fortified and incurred tissue were reanalyzed for confirmation of FQs using the same extraction and chromatographic conditions developed for the initial LC/MS method. A comparison of the data obtained with a single quadrupole and the ion trap instrument will be presented. The overall result of this project was to develop a validated regulatory confirmation method that is suitable for transfer to other laboratories and can be adapted for use by facilities with different types of mass spectrometers.

results of the analysis of fortified shrimp tissue using this multiresidue approach will be presented.

Board S-05

J.A. Casanova, F.J. Schenck and A.D. Williams, Southeastern Regional Laboratory, FDA, Atlanta, GA 30319

N-methyl carbamate insecticides (carbamates) comprise an important class of pesticides used for crop protection. Because of their toxicological effects and recent implication as endocrine disrupters there is a need to screen for carbamates in foods at trace levels. Since many of these compounds are polar and/or heat labile, they cannot be determined by gas liquid chromatography. The most common methods for the determination of carbamates entails using a reverse phase liquid chromatographic (LC) separation, followed by a postcolumn alkaline hydrolysis and derivatization to produce a fluorogenic derivative. Carbamates are typically confirmed by liquid chromatography with mass spectrometric (LC/MS) detection. This poster describes work on the evaluation of various instrumental parameters that will allow for the detection of trace levels of carbamates using LC/MS. The relative sensitivity of the LC/MS with two atmospheric pressure interfaces, electrospray (ESI) and atmospheric pressure chemical ionization (APCI) was evaluated. Some of the parameters studied were effect of various mobile phase modifiers/buffers and acquisition modes (SIM vs. MS/MS). MS tuning parameters (i.e. nebulizing gas flows and temperatures of the interfaces) were optimized for each analyte of interest prior to sample analysis. Limits of detection were determined for 14 carbamates in peach and green bean samples.

Board S-06

C.L. Flurer, M.B. Jones, B.S. Barnes, J.B. Crowe, J.R. Loeliger, D.F. Crockett, N. Ranieri, M.R. Witkowski, R.D. Satzger and K.A. Wolnik, Forensic Chemistry Center, FDA, Cincinnati, OH 45237

Within the past six months, various suspect tablets of a popular lifestyle drug, many of which were purchased over the Internet, have been analyzed by high-performance liquid chromatography (HPLC), headspace gas chromatography, inductively coupled plasma-atomic emission spectrometry (ICP-AES), Fourier-Transform infrared spectroscopy (FT-IR) and image analysis. Although all tablets analyzed to date have been potent, the HPLC impurity profiles and the residual solvents found indicate that the source(s) of the active ingredient in the suspect products are not consistent with the authentic. FT-IR and ICP-AES are used for formulation analysis, and image analysis is used to evaluate the morphology of the tablets. The information obtained from the various techniques is correlated to link sources of either the active ingredient or the tablets themselves, in order to provide assistance in on-going criminal investigations.

Board S-07

S.B. Clark¹, G.J. Nandrea¹, M.R. Madson¹, J.N. Sofos², and J.A. Hurlbut³, ¹FDA, ORA, Denver, CO, ²Colorado State University, Ft. Collins, CO, and, ³ Western Washington University, Bellingham, WA

Non-steroidal anti-inflammatory drugs (NSAID) are widely used in both human and veterinary medicine because they suppress or reduce inflammation, pain, swelling, heat, hyperemia, and loss of bodily function caused by various forms of arthritis. Prolonged use of NSAID is discouraged because possible side effects include gastric intestinal ulceration that can sometimes be accompanied by anemia and disturbances in platelet function. A 1992 survey of 2000 veterinarians whose practices were devoted to at least 50% dairy and beef cattle, revealed that approximately 88% (1,146/1,306) of the respondents prescribed NSAID in combination with antibiotics. Flunixin meglumine (FX) and phenylbutazone (PB) are two NSAID that are not permitted for lactating dairy cows. Our FY01 research produced a rapid screening method for flunixin meglumine (FX) and phenylbutazone (PB) residues in raw milk. For the test, raw milk is directly applied to Neogen® Corporation's FX and PB ELISA (enzyme-linked immuno-sorbent assay) kits. The kits are sensitive in the low part-per-billion range (0.5ppb FX and 5ppb PB) in milk.

Board S-08

Allen P. Pfenning, José E. Roybal, Sherri B. Turnipseed, Steve A. Gonzales, U.S. Food and Drug Administration, ADRC, Denver Federal Center, Denver, CO 80225-0087

A preliminary gas chromatographic (GC) method is described for determining residues of chloramphenicol (CAP), florfenicol (FF), florfenicol amine (FFa) and thiamphenicol (TAP) in bovine muscle and liver tissues. The tissues are extracted with basic ethyl acetate, centrifuged, evaporated, reconstituted in water, defatted and passed through a propyl sulfonic acid (PRS)/C₁₈ Solid Phase Extraction (SPE) column system. The C₁₈ column, retains CAP, FF, TAP and mCAP; the PRS column retains the FFa. An additional aliquot of internal standard is added to the PRS eluate. The eluates are individually derivatized with Sylon BFT (BSTFA:TMCS, 99:1), and the analytes determined by GC with electron capture detection (ECD).

Board S-10

P. J. Kijak¹, S.B. Turnipseed², W.Cui¹, and J.E. Roybal², ¹ CVM, FDA, Laurel, MD 20708, ² ORA, FDA, Denver, CO 80225

The development of better methods capable of screening aquaculture products for large numbers of drug residues from a single sample will provide the agency with tools needed to effectively monitor the Nation's food supply. By using generic extraction techniques, combined with the liquid chromatography ion trap mass spectrometry (LC/MSⁿ), methods can be developed that will screen a sample for the presence of most commonly used aquaculture drugs. Progress to date in the development of LC/MSⁿ method for the separation and identification of drug residues that are likely to be found in shrimp tissue is reported. Chromatographic and mass spectrometric conditions have been investigated and optimized for over twenty compounds. These compounds include quinolones, sulfonamides, phenicols, oxytetracycline, and therapeutic dyes.

Generic extraction methods for the isolation of drug residues from shrimp tissue are being evaluated. We are investigating the use of different types of solid phase extraction cartridges, specifically Oasis® reverse phase and various cation exchange columns, for extraction and clean-up of drug residues in shrimp. Preliminary results of the analysis of fortified shrimp tissue using this multiresidue approach will be presented.

Board S-11

Raafat Fahmy, Bill Staber, Bill Marnane, Dennis Bensley, Jack Zupan and R. Gary Hollenbeck

Purpose:
The objective of this project is to determine the effect of selected equipment and process changes on the physical characteristics and performance attributes of sulfamethazine boluses.

Experimental design:
A full-factorial experimental design was conducted to produce 144 sets of sulfamethazine boluses. Independent design variables were in three phases of the production process: Granulations were manufactured by two mixer types (low shear and high shear), with different binder levels (regular and +/- 5% of the amount of starch), with two different lubricant levels (regular and 2X increase) and two mixing times (3 and 15 minutes.) resulting in a total of 24 blends. Each blend was then compressed at three compression forces (30, 45, and 60 Kn), and two RPM's - (slow and fast) - to expose the granulations to different compaction dwell times.

Results:
For each bolus set, the thickness, weight, and breaking strength of 10 – 15 boluses were measured and correlated. Within sets, there was a very good correlation between tablet thickness and tablet weight. Also within sets, the correlation between tablet weight and tablet breaking strength was apparent. On average, tablet thickness declines as compression force increases, and tablet breaking strength increases as compression force increases.

The statistical analyses indicated that the changes in the experimental levels of compression force and final blend time account for about 15% change in tablet strength; a doubling of the lubricant level decreased tablet strength about 10%. The weakest factor in the experimental design was mixer type (high shear vs low shear).

Conclusion:
¯All of the design variables appear highly statistically significant for tablet strength except mixer type.

Board S-12

Louis Lue and Susan T. Hadman, 1NRL, ORA, FDA, 158-15 Liberty Avenue, Jamaica, New York 11433

Novobiocin (NOV) is widely used in treatment of urethritis in men, mastitis in dairy cattle, and poultry diseases. NOV is the choice for treatment, particularly, when the pathogens are resistant to common antibiotics such as penicillin, streptomycin or erythromycin. We developed an HPLC method for determination of NOV: isocratic elution (1.0 mL/min) of Bondapak C18 column (300 mm X 4.6 mm) with mobile phase consisting of acetonitrile : 10 mM phosphoric acid (66:34, v/v) was found to be appropriate for the analysis of NOV. The injection volume was 10 µL and the detector was set at 254 nm. This HPLC method has limit of detection (LOD) 3.5 ng/mL and limit of quantitation (LOQ) 35 ng/mL. The calibration curve for this method was linear from 35 ng/mL to 100 µg/mL. NOV and the internal standard, coumarin, had retention times 6.78 and 3.88 min, respectively. The resolution between coumarin and NOV was 9.0. This simple and rapid HPLC method was developed for determination of NOV in bulk. In comparison with the official USP microbial assay for NOV, this HPLC method is more selective, sensitive and offers positive identification.

Board S-15

G.M. Ware, A.D. Williams

Pure hypoglycin A (HG-A) is not commercially available for use in quantitative studies. This study reports on a simple and efficient method to isolate HG-A from ackee seeds. HG-A is extracted from ackee seeds with ethanol:water 80:20 and concentrated to dryness. The residue is dissolved in 1:1 dioxane:water and treated with triethylamine and, then, derivatized to N-t-Boc-HG-A-benzyl ester with N-t-butyloxycarbonyl (Boc) and benzyl bromide. The ester is isolated from the reaction mixture by chromatography on silica gel. The N-t-Boc-HG-A-benzyl ester was characterized by LCMS. Upon hydrolysis with 4M HCl, pure HG-A is obtained, free of leucine, in gram quantities.

Board S-16

Gary J. Lehr¹, Thomas L. Barry¹, John D. Franolic², Glenn Petzinger¹, and Peter Scheiner³, ¹FDA, ORA, Northeast Regional Laboratory, Jamaica, NY 11433, ² FDA, CDER, Rockville, MD 20855, ³ York College of the City University of NY, Jamaica, NY 11433

A gradient elution HPLC system was developed to separate methoxsalen from three of its known impurities: isopimpinellin, bergapten, and ammidin. The system consists of a methanol-6%THF(aq) mobile phase, phenyl column, and detection at 254 nm. The gradient HPLC procedure was applied to seven lots of methoxsalen from five different manufacturers. Six of the seven lots tested contained isopimpinellin as the major impurity at a concentration range of 0.2 to 2.5 %. Identification of the impurity as isopimpinellin was accomplished by a combination of HPLC, preparative chromatography, APCI LC/MS, and NMR.

Board S-18

José E. Roybal, Allen P. Pfenning, Sherri B. Turnipseed, Steve A. Gonzales, and Joseph M. Storey, FDA, Animal Drugs Research Center, Denver, CO 80225

A liquid chromatographic procedure for the quantitative determination of diminazene diaceturate (DD) in raw bovine milk is presented. DD is extracted from raw milk by chilled aqueous centrifugation. Isolated from milk components utilizing SPE-CN and eluted using a methanol-ion pairng reagent. LC is performed on a Phenomenex LUNA CN column with isocratic elution using an acetonitrile-buffered mobile phase with counter ion. The LC effluent is monitored at a detection wavelength of 370nm utilizing a D₂ lamp. Under these parameters, DD retention time is @ 6-7 minutes with a peak response of 4.8mAU/20ng. Recovery, detection limits, and critical factors affecting the analysis in milk is presented.

Board S-20

George M. Hanna, Food and Drug Administration, Northeast Regional Laboratory, 158-15 Liberty Avenue, Jamaica, New York 11433-1034, USA

Arisolochic acids (about 14 compounds) are found as constituents in plants from the family Arisolochiae. There are over 600 species in the family, found both in Asia and in the United States. Aristolochia species include Birthwort, Dutchman's pipe and Virginia snakeroot. All Aristolochic acids are considered antineoplastic agents with acute nephrotoxicity. Nephropathy, the progressive form of renal fibrosis was developed in many individuals who take weight-reducing pills containing Chinese herbs. The FDA is taking action against any product that is confirmed to contain any of aristolochic acids. ¹H NMR methodology for determination and characterization of the aristolochic acid components was developed utilizing a 400 MHz spectrometer without the need of pure reference standards. The developed methodology is able to differentiate between the compounds called aristolochic acids, assess chemical structure of these toxic compounds, and determine the relative ratio for an each component.

Board S-21

¹H NMR methodology is described for the determination and the characterization of S-adenosyl-L-methionine utilizing a 400 MHz spectrometer without the need of pure reference standards. The developed methodology is able to differentiate between the biologically-active (S)-S-diastereoisomer and the biologically-inactive (R)-S-diastereoisomer of S-adenosyl-L-methionine, assess chemical structure of these compounds, and determine the quantity of each isomer in the dietry suppliment formulation. The NMR methodology was found suitable to monitoring the stability of S-adenosyl-L-methionine in aqueous media, with the ability to detect degradation products adenine, homoserine lactone, S-pentosylmethionine and adenosine. The quantitative analysis was based on the integrals for the methyl proton of 2-methyl-2-propanol served as an internal standard at 1.24 ppm and the methine proton H'1 of the S-adenosyl-L-methionine ribose ring at 6.06 ppm. The accuracy was established by analyzing synthetic mixtures of the analyte and internal standard. Excellent agreement was verified between the assay results and the quantities of analyte in the mixture.

Board S-22

Adrian Weisz^a, Constance M. Murphy^a, Eugene P. Mazzola^a, Yoichiro Ito^b, ^aCenter for Food Safety and Applied Nutrition, FDA, Washington, DC 20204, ^bNHLBI, National Institutes of Health, Bethesda, MD 20892

Quinoline Yellow (QY, Color Index 47005) is a color additive widely used for foods in Europe (E-104) and for drugs and cosmetics in the USA (D&C Yellow No. 10) and Japan (Yellow 203). It is manufactured by condensing 2-methyl-quinoline with phthalic anhydride. The condensation product, 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione, is then sulfonated and the resulting products are isolated as sodium salts. The QY's degree of sulfonation determines its future uses. The QY that consists primarily of a mixture of the sodium salts of monosulfonic acid isomers (with up to 15% of the disodium salts of the disulfonated isomers) may be certified by the U.S. Food and Drug Administration (FDA) as D&C Yellow No. 10 for use in drugs and cosmetics. By contrast, QY that contains mostly di- and trisulfonated components is used for coloring food and/or drugs and cosmetics in other countries. This primarily di-and trisulfonated QY is not certifiable in the United States. For the development of analytical methods to be used for FDA batch certification of D&C Yellow No. 10, purified mono-, di- and trisulfonated components of QY are required as reference materials. These compounds are not commercially available. The present study describes the preparation of starting materials for the synthesis of QY monosulfonated in the indenedione moiety.

Board S-23

B.R. Petigara and A. Weisz, Office of Cosmetics and Colors, Food and Drug Administration, Washington, DC 20204

D&C Orange No. 5 (O5, Colour Index 45370:1) is a U.S.-certified color additive used in drugs and cosmetics. O5 consists of a mixture of 4',5'-dibromofluorescein and 2',4',5'-tribromofluorescein accompanied by smaller amounts of subsidiary colors (e.g., 2',4',5',7'-tetrabromofluorescein, 4'-bromofluorescein, fluorescein), intermediates and synthetic byproducts. O5 is batch-certified by the FDA to ensure compliance with specifications required by the Code of Federal Regulations. Currently, the analysis of the main components and the subsidiary colors of O5 involve separation on a 20 x 20 cm semipreparative TLC plate followed by spectrophotometric quantification. While this method is effective, it is tedious, time-consuming, prone to errors and produces appreciable quantities of solvent waste. The present study reports the development of a rapid, reliable and economic videodensitometric method that permits quantification of the main components and the subsidiary colors of multiple samples on the same 10 x 10 cm TLC plate. The method is applicable for use in routine batch-certification analyses.

Board S-24

MICHAEL E. STACK, U.S. Food & Drug Administration, USA

Ackee fruit is a popular food in Jamaica and other Caribbean countries. The arils of the ripe fruit can be eaten safely but consumption of other parts of the fruit or of the unripe arils causes death from hypoglycemia. Deaths from hypoglycemia have occurred this year in Haiti as a result of ackee consumption. Hypoglycin A, the toxic principle from ackee fruit is shown to contain two isomers, which can be separated by liquid chromatography. Baseline separation of the two isomers can be achieved using a C8 reverse phase column using water + acetonitrile + trifluoroacetic acid (950 + 50 +5) as the mobile phase. The two compounds are detected using post-column derivatization with o-phthalaldehyde (OPA) and fluorescence detection. Both compounds are molecular weight = 141. The liquid chromatographic separation and post-column detection can be used to determine hypoglycines in ackee fruit.

Board S-25

D. S. Jackson, D. F. Crockett, L.A. Kaine, D. T. Heitkemper, and K.J. Mulligan, Forensic Chemistry Center, FDA, Cincinnati, Ohio

Commercial bleach (sodium hypochlorite) has been indicated as the source of adulteration in a number of tampering investigations examined at the Forensic Chemistry Center (FCC). Commercial bleach is a cleaning and bleaching agent that contains 4-6% sodium hypochlorite, a strong oxidizer that can cause injury to eyes and skin and is harmful if swallowed. It is inexpensive, found in most households, and causes injury when used improperly. A primary function of the FCC is to provide laboratory support in product tampering investigations and to perform studies that allow FDA to better respond to alleged tamperings. A study was initiated at the FCC in which commercial bleach was added to selected beverages to determine the stability of the hypochlorite in the spiked beverages and to determine the effects of the bleach on the beverage matrix. This report describes the findings of our study.

Board S-26

Mary C. Carson, Cristina Nochetto, David N. Heller, Valerie B. Reeves, CVM, FDA, Laurel, MD 20708

Veterinary drugs are used in food animal production to improve animal health and promote growth. Generally, drug sponsors must supply methods capable of both quantifying and confirming residues in tissue or other edible products prior to the US FDA approving a New Animal Drug Application (NADA). However, NADA methods are almost always single analyte methods, and may only apply to one species. To maximize efficiency of regulatory monitoring, regulatory agencies need multiresidue and multiclass methods. Mass spectrometry (MS) is the preferred technique for confirmation. Ion trap MS is particularly well suited for the development of multiresidue methods. It provides full scan data, yielding a high certainty of identification. Its selectivity permits the use of cruder extracts in analysis, allowing "generic" sample preparation. Finally, MSⁿ capability is useful for the confirmation of several drugs which exhibit only nonspecific losses in the first fragmentation. This presentation will emphasize ion trap MS confirmation of aminoglycoside residues in tissue using data-dependent scan acquisition techniques. The utility of this approach in a regulatory monitoring program will be discussed.

Board S-27

R.D. Satzger, 6751 Steger Drive, Cincinnati, Ohio 45237-3097

The Forensic Chemistry Center serves as a national laboratory for rapid method development and research related to forensic issues involving foods, drugs and other FDA regulated products. The Forensic Center is staffed by a multidisciplinary team of scientists with an expertise in problem solving. The mission of the Forensic Center is to maintain the capability to respond immediately to incidents involving counterfeit or adulterated products and other high priority issues requiring rapid method development. Various recent cases involving adulterated or unapproved drugs, dietary supplements, and medical devices will be presented along with solutions using a coordinated analytical approach.

Publish Only (S)

J.L. Daft, R.E. Smith, D.F. Graham, D.L. Terrazas

Current trace findings of 2,4-D (a phenoxyacetic acid), dicamba (a methoxybenzoic acid), clopyralid (a pyridinecarboxylic acid), and quinclorac (a quinolinecarboxylic acid) in certain food indicate that the acid-herbicide methodology needs continual updating. The chlorophenoxy-acid method reported here, which methyl-esterfies the analytes by ion-pair alkylation, is being validated for several additional acid herbicides and other acid compounds, i.e., chlorophenols (antimicrobials and biocides) that come through the method satisfactorily. Further updates include 1) streamline the makeup of multiple-component reference solutions to reduce variation between the separately acquired acids and esters, 2) adjust the extractions and cleanups for maximum recovery of acid-herbicide types, e.g., by using a stronger PAM-1 mixed-ethers fraction during the Florisil cleanup step for improved recovery of certain analytes, and 3) minimize the background-matrix interference during the GC determinative step by using an XSD detector. These minor adjustments will give the analysis more accuracy, and expedite it.

Board T-01

J Zhang¹, E H Herman¹, D G Robertson², A Knapton¹, D P Chadwick¹, and F D Sistare¹, ¹CDER, FDA, Laurel, MD 20708; ²Ann Arbor Laboratories, Pfizer Inc., Ann Arbor, MI

The present study was initiated to compare the extent of vasculitis induced by SK&F 95654 (SKF) between SHR and WKY. Vascular injury (mesenteric small vessel vasculitis with accompanying mast cell degranulation) was far more severe while cardiac toxicity (necrosis, inflammation, edema) was slightly more severe in SHR than in WKY 24 h after a single s.c. dose of 100 or 200 mg/kg SKF. In contrast, at this time renal lesions (tubular hyaline droplets, protein casts, glomerular vacuolization) was far more severe and hepatic lesions (degeneration, inflammation) were found to be only slightly more severe in WKY than in SHR. Renal toxicity in WKY was associated with increases in serum levels of BUN, GGT, and creatinine. Cardiac toxicity in both strains was associated with increases in serum cardiac troponin T. No significant alterations in ALT, AST or Bi were associated with hepatic histopathology. Metabonomic analysis of urine samples showed treatment-related alterations in NMR spectral patterns that reflected the differences in toxicity profiles induced by SKF in SHR and WKY. These results indicate that contrasting changes in biofluids in SHR with WKY provides a strategy for identifying and evaluating biomarkers especially for PDE inhibitor-induced vasculitis.

Board T-02

S. Rubin ¹, D.Liu², J. McCullers³, M. Pletnikov⁴, Z.Ye, R. Levandowski¹ and K. Carbone ^1,4, ¹CBER/FDA, Bethesda, MD.; ² NIH/NIA, Baltimore, MD.; ³ St. Jude Children's Research Hospital, Memphis, TN; ⁴ Dept. Psychiatry, Johns Hopkins University, Baltimore, MD.

Influenza viruses have long been associated with a number of central nervous system (CNS) complications ranging from encephalopathy and encephalitis to neuropsychiatric disorders. Therefore, the risk of neurological adverse experiences following administration of replicating, live, attenuated influenza virus vaccines. Moreover, although these vaccines are administered intranasally, a potential direct pathway to the brain, no validated neurovirulence assay exists to evaluate their neurotoxicity. To address this concern, a rat model of influenza virus CNS infection was developed to evaluate the possible human neurovirulence potential of influenza vaccines. Newborn rats were inoculated intracranially with four influenza A virus strains representing a range of neurotoxicity. Two virus strains known to infect the CNS (a virus strain adapted to growth in rodent brain and a clinical isolate from a patient with influenza virus associated encephalitis) replicated to higher titers with a wider distribution in rat brain compared to two strains not associated with CNS infection in humans (a vaccine strain and a wild type respiratory isolate). Therefore this assay correctly identified the relative neurotoxicity of these virus strains and, thus, may prove useful in evaluating the neurovirulence potential of new live attenuated influenza virus vaccine strains.

Board T-03

P.A. Orlandi, D.-M. Chu, L. Carter, D. Antwi, and K.A. Lampel. CFSAN, FDA, Washington, D.C. 20204.

Many obstacles affect the success of conventional methods to detect and identify pathogenic microorganisms during the course of epidemiological investigations of food-borne outbreak, clinical diagnoses, and environmental surveillance studies. The detection of bacteria, protozoan parasites and viruses in these particular milieus presents a unique challenge. Whereas traditional microscopic and bacteriological methods that incorporate pre-enrichment can be employed for some pathogens, similar methods for the rapid isolation and detection of many others are either not currently available or not sufficiently selective. Additionally, the matrix from which these organisms must be isolated further complicates analysis. Few analyses can be performed in a timely and cost-effective manner. We have recently developed a rapid detection protocol that combines size-exclusion filtration with our previous work with FTA filter technology. This simple yet rapid and efficient method captures suspected microorganisms with an extraction-free, filter-based matrix to prepare DNA templates for use in the polymerase chain reaction (PCR). The advantage of filter-based technology is the elimination of cumbersome, costly, and time-consuming preparative steps to purify and concentrate isolates. In conjunction with other novel PCR applications, these protocols can provide a means for more rapid and sensitive detection and identification of many human pathogens associated with food- and waterborne illness and environmental contamination. Likewise, it could easily be adapted for the clinical diagnosis of emerging human pathogens for which cost-effective means may not be available.

Board T-04

J Zhang¹, E H Herman¹, G Holt², K T Blanchard³ , H V Ratajczak³, A Knapton¹ and F D Sistare¹, ¹CDER, FDA, Laurel, MD 20708; ²Oxford GlycoSciences, Montgomery Village, MD 20886; ³ Boehringer Ingelheim Pharmaceuticals, Inc. Ridgefield, CT 06877

The present study was initiated to determine whether changes in plasma levels of proteins could be detected using 2-d gel proteomic discovery technology in samples from animals exposed to drugs which cause vasculitis. Plasma samples were collected from Sprague-Dawley rats (SD) rats 1, 2, 4 and 24 hr after treatment with a single sc 100mg/kg dose of SK&F 95654. Morphological evaluation confirmed the presence of mesenteric vasculitis (associated with mast cell activation and neutrophil migration) in these rats. Two-d gel proteomic analyses detected increases in the plasma levels of apolipoprotein AI and a₂-macroglobulin. These proteins indicate that an acute phase response was involved. APPs are synthesized in the liver in response to the cytokines (IL-6, TNFa, and IL-1) released by activated macrophages, monocytes, and mast cells at sites of inflammation. The number APPs: C-reactive protein (CRP) and serum amyloid A (SAA) have been suggested as clinical indicators of arterial inflammation and enhanced risk for heart attack. Increases in haptoglobin levels have also been reported in patients with systemic idiopathic vasculitis. Samples of plasma were also obtained and analyzed from beagle dogs given two proprietary test compounds daily for 9 to 14 days. Significant elevations in plasma concentrations of CRP, SAA, and haptoglobin were observed in these animals, associated with pathologically confirmed venous vasculitis. These results indicate that APPs may serve as biomarkers of host-response to drug-induced vasculitis in rats and dogs.

Board T-06

Grisselle Martinez , Karlygash M. Yermukan, ORA, FDA, Alameda, CA

A fast and reliable method for the detection of live Salmonella cells in alfalfa sprouts using reverse transcriptase PCR was developed during this study. Total RNA was isolated from sprout samples spiked with Salmonella. The ST11 and ST15 primers, Aabo et al. were used to amplify a 429 bp-long fragment during RT-PCR. The resulting DNA fragment was isolated and sequenced. This method was also tested using sprouts homogenate spiked with Salmonella and naturally contaminated sprouted seeds cells. The detection limit was ~10³ - 10⁴ cells. The estimated time of completion for the rinse, RNA isolation, RT-PCR and gel electrophoresis was 6 hours for 5-10 samples. The advantages of this method over the currently used rapid method, are: a) the detection of live (vs. non-viable) cells, b) the procedure is completed in a significantly shorter period of time.

Board T-07

Daniel B. Lyle, Bethany M. Gaskill, and John J. Langone. Molecular Biology Branch, OST/CDRH/Food and Drug Administration, Rockville MD-20857.

Damage to endothelial cells, which line the interior of blood vessels, is involved in re-blockage (restenosis) of cardiac arteries opened by balloon angioplasty and/or stent placement, and may involve an increase in the concentration of intracellular calcium. We have developed a fluorescent plate reader methodology using 24-well culture plates and the calcium-sensitive dye Fluo-4 to quantify calcium signaling in human umbilical vein primary endothelial cell monolayers (HUVECs). Serum and substrate variables were investigated for optimal monolayer formation. Instrument parameters were optimized to detect both active uptake of Fluo-4 over time and increases in intracellular calcium. Calcium signaling in endothelial cells is an indicator of cell function, cell damage in apoptosis/necrosis, and complement-mediated immunotoxicity.

Board T-08

B.Z. Zmudzka1, T. Tadokoro2, N. Kobayashi2, S. Ito3, K. Wakamatsu3, V.J. Hearing2, J.Z. Beer1. 1Food and Drug Administration, Rockville, MD, 20857; 2National Cancer Institute, Bethesda MD 20892; 3Fujita Health University, Toyoake, Aichi 470-1192, Japan.

We are evaluating the usefulness of various methods for measuring UV responses in human skin, in particular DNA damage and repair and melanin content. These studies involve subjects representing various racial/ethnic groups and different UV sensitivities. The minimal erythema dose (MED) of each subject is determined to assess UV sensitivity. Shave biopsies are taken before exposure, immediately after exposure to approximately 1 MED of UV, and then 1 day and 7 days later. Induction of DNA damage (cyclobutane pyrimidine dimers, CPD) is detected in sections by indirect immunofluorescence using a monoclonal antibody (TDM-2) specific for CPD and the fluorescein isothiocyanate (FITC). Nuclear DNA is counterstained with propidium iodide (PI). DNA damage is expressed as the ratio of the intensities of the FITC fluorescence for CPD and that of the PI fluorescence for DNA. The available data show that DNA damage measured by CPD is most significant immediately following UV exposure, but is repaired more than 50 % within 1 day. However, melanin contents measured by Fontana-Masson staining

do not change consistently with UV exposure. Diffused reflectance (DR) spectra were taken in the exposed and unexposed skin areas using a Minolta spectrophotometer (CM-2002) to independently derive melanin content. In addition, concentrations of eumelanin and pheomelanin were determined by chemical analysis using HPLC. Melanin content derived from DR measurements correlated better with the eumelanin content determined by chemical analysis than with that determined by the Fontana-Masson staining. Also, the DR-derived and chemically determined melanin content increases were consistent with the expected effects of UV exposure. These studies make it possible to further analyze correlations between melanin content and DNA damage, repair efficiency, and the degree of photoprotection afforded in the skins of individuals from different racial/ethnic origins and phototypes.

Board T-10

B. Nhi Nguyen, Jogarao VS Gobburu, FDA, CDER, Rockville, MD 20852

Purpose To compare individual and population QT correction methods for drugs with different effects on heart rate (HR) and QT.

Methods Three models of QT correction were applied to dose, QT and HR data for drug A and B. The general equation for the models is QT = µa* RR^µb, where b = 0.5 for Bazett's model, 0.33 for Fridericia's model, and b is estimated for each subject for the Power model (individual correction). Estimation was performed using the naïve pooled (NP) and nonlinear mixed effects (NM) approaches. Potential covariate models were tested. Model selection was based on the standard log-likelihood ratio test (LRT).

Results The Power model estimated by NONMEM provided the best fit of the data.

Conclusions Individual correction of QT-HR data is better than population methods of QT correction.

Board T-11

Main components of UV-induced tan: pigmentation and erythema

B.Z. Zmudzka¹, G.N. Stamatas², S.A. Miller¹, N. Kollias², J.Z. Beer¹, ¹Center for Devices and Radiological Health, Food and Drug Administration, Rockville, MD, ²Methods and Models Development, Johnson & Johnson Consumer Products Worldwide, Skillman, NJ

We investigated the usefulness of diffuse reflectance (DR) measured with a Minolta spectrophotometer (CM-2002) to evaluate UV-induced erythema and pigmentation in individuals of different racial/ethnic origin. Small areas on the subjects' backs were exposed to different doses of UV radiation from FS lamps. Color changes in the exposed skin sites were assessed visually, and documented by photography and DR at days 1, 2, 8, and 16 after irradiation. The amounts of melanin, oxy- and deoxyhemoglobin were calculated from the DR measurements. The melanin pigment was evaluated from the absorbance spectrum in the range of 630-700 nm. The spectrum was then corrected for melanin absorption and the apparent concentrations of oxy- and deoxy-hemoglobin were calculated. At skin sites receiving 1 MED, the erythema was pronounced at day 1 while, at doses >2 MED, the erythema response was stronger at day 2. The erythema remained discernible 8 and 16 days after exposure. In dark complexioned individuals the increase in pigmentation at days 8 and 16 was pronounced even at 1 MED. In contrast, increase of pigmentation in fair skin required higher doses. DR can be used to measure erythema and pigmentation in human skin independently of the constitutive pigmentation. These two phenomena contribute to UV-induced change in skin color generally described as a tan. The relative contribution of pigmentation and erythema depends on constitutive pigmentation, post-exposure time, and UV dose. It may also depend on the emission spectrum of the UV source.

Board T-12

L. Bockstahler¹, Zhongming Li², K. Van Houten¹, N. Nguyen², J. Langone¹, M. Brennan² and S. Morris² , ¹CDRH, ²CBER, FDA Rockville, MD 20857

The emergence of drug resistant strains of Mycobacterium tuberculosis (MTB) is a serious public health problem. We are developing a PCR-PNA-ELISA as a diagnostic method using point mutations in genes associated with isoniazid and rifampin resistance in MTB. PCR with fluorescein-labeled primers is used to amplify specific regions of the cloned mycobacterial genes. Biotinylated PNAs bound to streptavidin-precoated microtiter wells are incubated with (denatured) PCR-amplified mutant or wild type gene sequences. After hybridization, color (405 nm) is developed following addition of an anti-fluorescein conjugate and appropriate substrate. Thus far we have established the hybridization temperatures (50-55^oC) and other experimental conditions suitable for detecting a number of clinically relevant point mutations in the katG and rpoB genes using 15-mer PNA probes. Hybridization of PCR-amplified MTB DNA sequences that contain these point mutations with mutant-specific complementary PNAs result in 6-10 fold increases in ELISA response compared to hybridization using MTB wild type-specific PNAs. Using the PCR-PNA-ELISA method with the cloned MTB gene system, drug resistant MTB sequences can be identified in less than 24 hours.

Board U-01

T. Joshua Pfefer, Ph.D. and Marwood N. Ediger, Ph.D., CDRH, FDA, Rockville MD 20857

A wide variety of fiberoptic probe designs have been used for in vivo fluorescence spectroscopy, yet the effect of probe geometry on spatial interrogation of tissue is not well understood. In this study, the influence of probe design parameters on light propagation were investigated though a series of Monte Carlo simulations. Four primary parameters were studied: numerical aperture, optical fiber diameter, source-detector fiber separation distance and fiber-tissue spacer thickness. Simulation results indicated that single fiber probes tend to target superficial layers and that the level of depthwise selectivity decreases with increasing fiber diameter. Multi-fiber probes were able to selectively target subsurface regions. Increased illumination-collection spacing produced deeper and more homogeneous tissue probing. Transparent spacers decreased the relative probing depth for multi-fiber probes while increasing the total fluorescence signal. This study provides theoretical evidence that probe design strongly affects fluorophore detection. Thus, customization of probe design for specific applications may lead to improvements in the clinical efficacy of fluorescence-based diagnostic devices.

Board U-02

D.N. Busick and D.J. Chwirut, CDRH, FDA, Rockville, MD 20852

Endovascular stents are structural scaffolds implanted into native or graft vasculature to maintain lumen patency after angioplasty or atherectomy. The use of these devices is expanding rapidly (estimated 500,000 cases per year in the U.S.), incorporating new technologies and new manufacturers. The ODE workload for these Class III products is extremely heavy. The goal of this project is to produce standardized, validated methodologies for the preclinical testing required for IDE approval. Such standardization will alleviate some of the burden on ODE and hopefully expedite new product approvals. The project is being coordinated with the activities of ASTM F04.30.06 (Interventional Cardiology Task Group) and the AdvaMed Interventional Cardiology Task Group.

Board U-03

D.M. Witters, J.P. Casamento, H.I. Bassen, and P.S. Ruggera, Center for Devices and Radiological Health, FDA, Rockville MD 20852

CDRH has received over 90 incident reports of ambulatory medical device malfunctions (e.g., implanted electrical stimulators for pain, cardiac pacemakers) due to electromagnetic interference (EMI) from electromagnetic (EM) security systems such as metal detectors and anti-theft systems. At present, medical device EMI is not considered to present a major public health hazard. However, the increasing use of electromagnetic security systems and analysis of the reports raises concerns about the potential rise in risks to users of electrically powered medical devices when exposed to emissions from these systems. FDA has joined with the Federal Aviation Administration (FAA) to develop solutions aimed to maintain airport security while minimizing the risks of medical device malfunctions due to EMI from metal detector security systems. Work includes developing standardized test methods for active medical device susceptibility to EMI from metal detectors, and emissions measurements from selected metal detectors. Information from this project is being used to help formulate consensus standards for both medical devices (e.g., the Association for the Advancement of Medical Instrumentation or AAMI) and the security industry (such as the American Society for Testing and Materials or ASTM). The poster presents information on the analysis of device EMI incidents, measurements of the emissions from metal detector systems, and medical device EMI test methods.

Board U-04

Ilko K. Ilev, Ronald W. Waynant, Kimberly R. Byrnes¹, Juanita J. Anders¹, V. Michelle Chenault, CDRH, FDA, Rockville, MD 20857, ¹USUHS, Bethesda, MD 20814

A novel and simple approach for on-off-switching laser radiation delivery into a precise tissue area using smart, tissue-activated optical fiber probes is demonstrated. These laser delivery systems are based on the use of a single delivery fiber with a specially shaped tip having an angled (porro prism) or a retroreflecting (corner cube) profile. The shaped fiber tip acts as a smart, tissue-activated probe because of the frustrated-total-internal-reflectance effect caused by the refractive-index change of the surrounding medium. The fiber tip provides a safe way for laser delivery with only two possible states of tissue illumination: the off-state (no tissue illumination), when the fiber tip is out of the tissue area and the laser emission is backreflected due to total-internal-reflection; and the on-state (maximum tissue illumination), when the fiber tip is touching the absorbing tissue and becomes "transparent", and thus the laser energy is coupled into the absorber. The fiber-probe-based system can be used for minimally invasive laser therapy and biosensing into a micro-scale tissue area with laser emission over a broadband spectral range that includes the visible, near- and mid-infrared.

Board V-02

J.L. Ferreira, S.J. Eliasberg, P. Edmonds, M.A. Harrison, 1,2ORA,FDA, Atlanta, GA 30309, 3Georgia Inst. of Tech., Atlanta, GA 30332, 4University of Georgia, Athens, GA 30602.

A food borne outbreak of type A botulism was linked to chili. Four samples of chili were examined for pre-formed type A botulinal toxin using two ELISA procedures and the mouse bioassay. One of the four samples was positive for type A botulinal toxin using all three methods. Three of the samples were negative for botulinal toxins using the three methods. The mouse bioassay indicated that type A toxin was present at 10,000 minimal lethal dose (MLD)/gram of product. The ELISA tests indicated a toxicity of either 7,650 with one method or 8,350 MLD/g with the second method. The sample toxicity determined by the ELISA was estimated by comparing samples to a standard curve generated with standard type A neurotoxin in casein buffer. The ELISA methods are more rapid than the mouse bioassay since the toxin type can be determined in one day. The mouse bioassay is more sensitive than the ELISA but usually requires multiple assays to obtain the toxin type and toxicity. Type A culture isolates from the sample were also verified using one of the ELISA methods.

Board V-03

G. L. Hartman, San Francisco District Laboratory, FDA, Alameda, CA 94502

The CDC has identified Norwalk-like viruses as the leading cause of foodborne illness in the U. S. Currently there are no official methods for the detection of Norwalk-like viruses in foods. A real-time PCR assay was develop a method for the routine screening of produces rinses for Norwalk-like viruses. The method is based on the TaqMan 5'-nucleaseassay. The Norwalk-like virus specific assay was tested in artificially contaminated produce rinses and found to be rapid and sensitive. It has the potential for use in the routine screening of produce rinses for Norwalk-like viruses.

Board V-04

T.S. Hammack, M.L. Johnson, A.P. Jacobson, and W.H. Andrews CFSAN, FDA, College Park, MD 20740

A comparison of the relative efficiencies of Universal Preenrichment (UP) broth and lactose broth for the recovery of a variety of Salmonella serovars from both pasteurized and unpasteurized apple cider, as well as pasteurized apple juice, was made. Juice was contaminated with single Salmonella serovars at high and low levels of 0.4 and 0.04 cfu/mL, respectively. The juice was aged for a minimum of 5 days at 2-5°C. On the day that analysis was initiated, each of 20 test portions (25 mL) of the contaminated juice were preenriched in both UP broth and lactose broth. The BAM Salmonella culture method was followed thereafter. For pasteurized apple cider, UP broth recovered significantly (p < 0.05) more Salmonella-positive test portions than did lactose broth (112 test portions and 75 test portions, respectively). For unpasteurized apple cider, UP broth recovered significantly more Salmonella-positive test portions than did lactose broth (326 test portions and 221 test portions, respectively). For pasteurized apple juice, UP broth recovered more Salmonella-positive test portions than did lactose broth (93 test portions and 81 test portions, respectively). These results indicate that UP broth should replace lactose broth for the analysis of pasteurized and unpasteurized apple cider, and pasteurized apple juice.

Board V-07

D. K. Lau, ORA, FDA, San Francisco District Laboratory, Alameda CA 94502

Most fresh produce is consumed raw without any type of microbiologically lethal processing. Low level, sporadic pathogenic contamination of produce may occur in the field and orchards where crops are grown. Due to the complex supply and distribution patterns, widespread outbreaks can occur. Because of the relatively short shelf life of the product, rapid analytical time is needed for regulatory decisions. Salmonella, an important food-borne pathogen, has been found in several produce commodities such as cantaloupe and cilantro. A fluorogenic 5' nuclease (TaqMan) assay has been developed to detect Salmonella PCR products directly by monitoring the increase in the fluorescence of a dye-labeled DNA probe. The purpose of this study is to determine the sensitivity of the kit coupled with a rapid DNA sample preparation step to detect Salmonella from several produce commodities. Results demonstrated a sensitivity level as low as 10 organisms in some of the produce tested (cantaloupe, green onions, parsley, and tomatoes). Detection time was less than 4 hours without a pre-enrichment step.

Second Place Poster - 2002 FDA Science Forum

Board V-08

Deborah A. Loveys, ORA, FDA, Atlanta, GA 30309

As a part of a food safety initiative, the FDA routinely samples high-risk field products such as cantaloupes, lettuce, tomatoes, strawberries, green onion, cilantro and celery. Due to difficulties in applying conventional enrichment-based culture methods to the detection of Shigella species, the polymerase chain reaction (PCR) has become the method for which regulatory action is taken for this organism. Though this method is effective for some forms of produce, the presence of product-based PCR inhibitors preclude its application to strawberries, raspberries and cilantro. Here, we report a rapid, cost-effective and efficient method for PCR detection of Shigella from these product rinses. This method can successfully detect S. dysenteriae at levels as low as 10²-10³ cfu (strawberry, raspberry) and 10³ cfu (cilantro) in sample rinses that cannot be evaluated by present methods. These detection levels are in accord with those obtained from PCR amplification of cellular pellets from serial dilutions of Shigella broth cultures. Inclusion of this extraction method into the existing microbiological method will provide for a more sensitive and effective measure of Shigella contamination in a wider range of high-risk produce.

Board V-09

Patrick McDermott. Center for Veterinary Medicine, Office of Research. Laurel, MD. 20708

The Division of Animal and Food Microbiology (DAFM) was established in 1998 within CVM's Office of Research. The mission of DAFM is to conduct basic and applied research to support regulatory decision-making by the Center for Veterinary Medicine. This research involves the isolation, identification, and characterization of microorganisms potentially harmful to animals and humans. DAFM's research explores the effects of antimicrobial use in food animal species including poultry, livestock, and aquaculture. In particular, research focuses on microbial use as it relates to: 1) efficacy against pathogens, 2) changes in the environmental microbial ecology, and 3) the development of antimicrobial resistance in pathogenic and commensal microorganisms.

Board V-10

A. Debrabant1, A. Padilla1, N. Lee1, D. Scott2, R. Noiva3, D.M. Dwyer4, H. Nakhasi1, 1 DETTD, CBER, FDA, Bethesda, MD, 2 U. Illinois, Urbana, IL, 3 U. S. Dakota, Vermillon, SD, 4 NIAID, NIH, Bethesda, MD

The protozoan parasite Leishmania donovani (Ld) is the causative agent of human visceral leishmaniasis that is responsible for hundred of thousands of death per year worldwide. In addition, U.S. army and travelers to endemic areas are at risk for leishmaniasis and deferred for blood donations. However, there is no effective vaccine against Leishmania to date. Our approach to develop novel live Leishmania vaccine is to attenuate parasites by disrupting their quality control of protein folding in the endoplasmic reticulum (ER) since it is a crucial check point for the release of secreted or membrane-bound proteins that are involved parasite virulence. We characterized two essential components of the parasite ER quality control machinery, the calcium binding protein calreticulin and protein disulfide isomerase. Ld mutant cell lines overexpressing modified sequences of these chaperones were made. Results showed that these mutant parasites have a defective secretory pathway and show reduced survival in human macrophages which are the target cells in an infected individual. The virulence of these attenuated parasites is currently being evaluated in animal models. Such attenuated strains of Leishmania have potential as novel live vaccine against human leishmaniasis.

Board V-11

Martha L. Gay; Frederick S. Fry, Jr.; Denis Andrzejewski; Steven M. Musser, CFSAN, FDA, College Park, MD 20740

MALDI mass spectra obtained from bacteria contain mass information which is characteristic of the bacteria. Initial investigations into the application of a variety of pattern recognition techniques to this problem indicated that the species of bacteria could be determined with minimal operator intervention. Further investigations using this approach made it clear that models constructed with these techniques are easily overwhelmed by chemical noise.

To address these problems, we constructed a mask which filtered out all but the most relevant peaks in a spectrum. This mask was constructed using a training set of spectra obtained from parahaemolyticus, V. vulnificus, E. coli, and S. aureus. Software was developed to aid in constructing masks and to construct data sets which use masks. Examination of a test set of spectra of V. parahaemolyticus and V. vulnificus demonstrated that these species can be separated using SIMCA or PCA models without the use of a mask. With the use of a mask the differences are more evident and it is possible to separate several serotypes.

Board V-12

S Simjee^1*, D G White¹, P F McDermott¹, D D Wagner¹, M J Zervos², S M Donabedian², L L English¹ and R D Walker¹, ¹US FDA, CVM, Muirkirk Road, Laurel, MD 20708, ²William Beaumont Hospital, Royal Oak, MI 48073

Several studies have suggested that animal enterococci may be a potential reservoir of antibiotic resistance genes, but this has yet to be definitively proven. The transfer of resistance genes from human to animal enterococci has never been reported in the USA. Over a two-year period (1996-1998), 35 enterococcal isolates were recovered from dogs diagnosed with UTI's at the Michigan State University Veterinary Teaching Hospital. One E. faecium isolate displayed high-level resistance to vancomycin (MIC>16 µg/ml) and gentamicin (> 256 µg/ml). Initial molecular analysis revealed the presence of Tn1546 and aac6'-aph2", responsible for high-level vancomycin and gentamicin resistance, respectively. Both genes could be transferred by conjugation. Detailed molecular analysis of Tn1546 identified insertions of IS1216V in ORF1 and insertions of IS1251 between vanS and vanH. This particular form of Tn1546, with IS1216V and IS1251 insertions, has only been described in human clinical isolates of VRE found in the USA. Comparison, by PFGE, of the canine VRE isolate against over 300 human VRE isolates from Michigan, however, failed to yield any significant similarities. To our knowledge, this is the first report of a Tn1546 like element commonly found in human VRE to be isolated from a house pet in the USA. This data suggest that human flora may serve as a reservoir of transferable resistance genes to bacteria isolated from animals.

Board V-13

S Simjee^1*, D G White¹, P F McDermott¹, and J J Maurer², ¹US FDA, CVM, Muirkirk Road, Laurel, MD 20708, ² University of Georgia, Dept Of Avian Medicine, Athens, GA 30602

Synercid, a streptogramin, was approved in the United States in 1999 for treatment of vancomycin resistant enterococci. Another streptogramin, rginiamycin, has been used in veterinary medicine for over two decades. Enterococci resistant to virginiamycin are cross-resistant to Synercid. A study was conducted to determine the prevalence of streptogramin resistance genes in enterococci and coagulase negative staphylococci isolated from poultry litter in 24 different farms across Georgia, USA. A total of 44 enterococci and 28 staphylococci isolates displayed MIC's > 4 µg/ml to Synercid and/or virginiamycin. These isolates were further analyzed for the presence of streptogramin associated resistance genes by PCR studies. The prevalence of known streptogramin resistance genes was: satG, 2.27% (n=1) of enterococci; satA, 84% (n= 37) of enterococci; vgaB, 67.85% (n=19) of staphylococci. The presence of vatA, vatB, vatC, vgaA, vgbA or vgbB was not detected, however, ermB was detected in 68% (n=30) of enterococci. Using sat degenerate primers 84% of enterococci (n=37) and 7.14% (n=2) of staphylococci were positive. This study suggests that poultry litter may serve as a potential source harboring enterococci and staphylococci carrying known and yet to be identified streptogramin resistance genes in enterococci.

Publish Only (V)

Y.C. Shieh, K.R. Calci, and J.W. Woods, FDA Gulf Coast Seafood Lab., Dauphin Island, AL.

Enteric viruses such as Norwalk-like viruses are major etiological agents of shellfish-borne gastrointestinal diseases in the US. We developed a sensitive method to detect viruses in contaminated shellfish (AEM 1999, 65: 4709) that removed PCR inhibitors, concentrated low levels of viruses, detected and confirmed viruses with Southern hybridization of PCR amplicon. We further modified the detection portion of the method to a simple and economical one-step procedure using 96-well fluorometer to read directly the hybridized amplicon in PCR tubes. This replaces conventional gel electrophoresis, Southern transfer, nucleic acid hybridization and colorimetric detection. This one-step detection system allowed the use of our original PCR conditions except that a probe was end-labeled with Cy3 fluorophore at the 5'-end and a Black Hole quencher at the 3'-end and was added at the beginning of PCR reaction. The detection efficiency by this method was comparable to Southern analysis at the level >0.27 pfu of poliovirus/PCR. For the reaction containing <0.27 pfu, the fluorometric measurements of the first round PCR viral amplicon were not as sensitive as Southern analysis; however, equivalent sensitivities were achieved with fluorogenic nested PCR. Concentrates of eleven oyster samples exposed to municipal sewage were tested for enteroviruses; the fluorogenic detection correlated 100% to Southern analysis. This fluorometric method requires less expensive equipment than is needed by real-time PCR and less time to complete than by Southern analysis.

Publish Only (V)

Chorng-Ming Cheng, Khan T. Van, Wen Lin and James Lin, PRL-SW, ORA, FDA, Los Angeles CA 90015

Salmonella is an important food and water-borne pathogen around the world. It causes acute gastrointestinal illness. The infective dose can be as low as 15-20 cells. A PCR method has been developed with custom designed primers to amplify a 420-bp fragment of Salmonella-specific invA gene. The system has been tested with 57 strains of field-isolated Salmonella spp. and non-Salmonella spp., as well 112 samples of various food matrixes. The results showed that the method had 100 % specificity and high sensitivity (0.7 CFU/g). This newly developed PCR method is much more rapid, sensitive, and specific for detection of Salmonella spp. than the traditional BAM method and the AOAC-approved VIDAS method.

Publish Only (V)

G. L. Hartman, San Francisco District Laboratory, FDA, Alameda, CA 94502

E. coli O157:H7 has an infectious dose of less than 10 organisms. The current method for detection of E. coli O157:H7 in produce rinses require the use of a 24 hr enrichment followed by plating of the enrichment and checking for typical colonies. In an effort to reduce the amount of time required to eliminate negative samples, a commercially available 5' fluorogenic assay (TaqMan O157:H7 Kit) was evaluated for use in spiked produce rinses. The assay was able to detect less than 10 organisms in spiked produce (>1 organism/ml) rinses after a 24 hour incubation in the BAM enrichment broth. The assay, if used for screening routine samples, can eliminate negative samples in 24 hours and allows for positive samples to be analyzed by the official methodology.

Board W-02

Yuan Hu¹, Azra Shahidi², Soon Park², Dennis Guilfoyle¹ and Irvin Hirshfield³, ¹U.S. FDA, Northeast Regional Laboratory, Jamaica, NY 11433; ²U.S. Department of Veterans Affairs, Medical Center, Bronx, NY 10468; ³St. John's University, Jamaica, NY 11439

Viral hepatitis is a major global health problem. Hepatitis C (HCV) is one of seven known viruses (A,B,C,D,E,G and TTV) that together account for the majority of cases of viral hepatitis. The life cycle of the HCV is poorly understood due to the lack of an efficient cell culture system. To address this need, we recently established a cell culture system for the replication of HCV by using the human T and B leukemia cell lines. These two cell lines were in vitro infected by hepatitis C positive pooled patient serum. HCV RNA was extracted from infected cell lines at different times after infection and a sequence of the virus 5' untranslated region was analyzed. Hepatitis C minus strand RNA was detected in the infected cell lines by highly strand-specific recombinant Thermus thermophilus DNA polymerase (rTth) based reverse transcription and a highly sensitive nested polymerase chain reaction (rTth RT-nested-PCR) method. PCR products were analyzed by direct DNA sequencing. These results indicate that hepatitis C virus can replicate in T and B-lymphocytes. This model should represent a valuable tool for the detailed study of initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.

Board W-03

A. Selvapandiyan¹, A. Debrabant¹, R. Duncan¹, S. Bertholet², P. Salotra³, and H.L. Nakhasi¹, ¹LBPUA/DETTD/CBER/FDA and ²LPD/NIAID/NIH, Bethesda, MD 20892, USA and ³Institute of Pathology/ ICMR, Delhi, India

The protozoan parasite, Leishmania donovani, causes visceral disease in humans by multiplying in the macrophages. We have isolated a complete copy of a gene encoding the L. donovani homologue of centrin, a centrosome associated protein responsible for cell division in other eukaryotes. We confirmed that Leishmania centrin is a calcium binding protein binds with higher affinity at its C-terminal putative Ca²⁺ binding domain. Immuno-fluorescence analysis localizes the protein in the area of basal body. Expression of centrin mRNA and protein correlated with the growth of the parasite, suggesting that LdCenp plays a functional role in parasite growth. This role is further supported by the observation that over-expression of N-terminal deleted LdCenp in the parasite resulted in reduction of the parasite growth rate. FACS analysis, on the slow growing parasites, showed an accumulation of cells in the G2 stage just prior to cell division compared to control cells. Thus, the expression of such a mutant centrin could have a negative dominant effect by disrupting the function of the endogenous centrin.

Board W-05

Nancy Lee¹, Sylvie Bertholet², Alain Debrabant¹, Jacqueline Muller³, Robert Duncan¹ and Hira L. Nakhasi¹*, ¹DETTD/OBRR/CBER/FDA, ²LPD/NIAID/NIH, ³DVP/OVRR/CBER,FDA, Bethesda, MD 20892

We have demonstrated the features characterizing programmed cell death (PCD) in the unicellular protozoan parasite Leishmania donovani, the causative agent of visceral leishmaniasis. We report that PCD is initiated in stationary phase cultures of promastigotes and both in actively growing cultures of axenic amastigotes and promastigotes upon treatment with antileishmanial drugs (Pentostam and amphotericin B). However, the two cell types respond to antileishmanial drugs differently. The features of PCD in L.donovani are nuclear condensation, nicked DNA in the nucleus, DNA ladder formation, decrease in the mitochondrial membrane potential and induction of a caspase-like activity. PCD in both stationary phase culture and upon induction by amphotericin B resulted first in the decrease of mitochondrial membrane potential followed by simultaneous change in plasma membrane permeability and induction of caspase-like activity. In addition, upon amphotericin B treatment, caspase-like activity was also induced in L. donovani and L. major parasites that were freshly isolated from infected animals. Our study demonstrates that the characteristic features of PCD exist in both laboratory adapted as well as freshly isolated unicellular protozoan Leishmania parasites.

Publish Only (W)

James Wilson¹, Theresa To¹, Aaron Margolin², and Patrick Regan¹, ¹Winchester Engineering and Analytical Center, Winchester, MA, ²University of New Hampshire, Durham, NH

The ability to determine the presence of viable mycobacterium post-disinfection is essential. We are developing a molecular-based method to detect the 85B mRNA of Mycobacterium bovis BCG. A simple and reproducible procedure was developed to extract RNA from mycobacterium cells. Serial 10-fold dilutions of Mycobacterium bovis BCG cells were sonicated for 10 seconds followed by an Instagene^® procedure to collect total nucleic acid. Samples were DNased. The presence of intact RNA was observed by ethidium bromide stained denaturing formaldehyde gel electrophoresis. Results showed that RT-PCR targeting the 85B mRNA of Mycobacterium bovis BCG could be detected to less than 20 organisms. This technique will be used to determine the difference between disinfected and non-disinfected mycobacterium after a period of culturing.

Publish Only (W)

Grisselle Martinez, ORA, FDA, Alameda, CA

Salmonella ATTC strains 9700, 12325, 29934, 9482, E. coli 25922, E. coli DH5a and Shigella sonnei 9290 were transformed with four different E. coli vectors carrying the Green Fluorescent Protein Gene, from the jellyfish Aqueorea victoria. All 28 strains were successfully transformed into the ampicillin resistant/GFP⁺ phenotype using the CaCl₂ incubation method. All transformed colonies fluoresced brightly under UV light illumination. The transformed strains appeared stable and fluorescence was maintained after repeated sub-culturing in media containing ampicillin. Expression levels and plasmid stability varied between strains.

Board X-01

T.L. Smith¹, H.E. Watson¹ and K.J. Mulligan², ¹Elemental Analysis Group, Total Diet Program, FDA, Lenexa, KS 66214, ²Forensic Chemistry Center, FDA, Cincinnati, OH 45237

The National Health and Nutrition Examination Survey [NHANES] serves to collect information about the health and diet of the people of the USA. A recent analysis of NHANES data (I and III) on Iodine suggests that the nutritional status of this essential element in women of child-bearing age is approaching levels at which WHO guidelines suggest that there is need for concern. Also, there are indications of an increased incidence of certain autoimmune disorders that have been associated with excess Iodine. Hence, there is a need to return to routine monitoring of Iodine as part of market basket analysis within the Total Diet Program. A former method utilized an alkaline dry ashing procedure for sample preparation. This was vulnerable to erratic recovery and that ultimately lead to the procedure being placed in abeyance. After Fischer et al. (J. Assoc. Off. Anal. Chem. 69(4), 687 – 689 (1986)), the method described here uses a ternary acid (HClO₄/HNO₃/H₂SO₄) digestion that is initially conducted under reflux conditions to retain volatile forms of iodine. Subsequent analysis monitors the catalytic effect of iodide on the reaction between As(III) and Ce(IV). The method has been implemented manually but can be readily automated. Quantitative recovery of Iodine from 13 market basket items (distributed across 6 food groups) and 4 NIST standard reference materials is demonstrated. This provides a basis for re-introducing of Iodine analysis into the Total Diet monitoring program.

Board X-03

W. Yip, E. P. Wolkow, M. Iorsh and M. R. Islam, ORA, FDA, Jamaica, NY 11433

Comparisons were made using two methods in the digestion of baby formula, to determine its mineral composition. One being the more traditional Dry Ashing Method (DA) and the other being the Microwave Digestion Method (MD). The predigestion step in MD was modified by running an open cell digestion program, followed by a more traditional closed cell digestion program. Inductively coupled Plasma Emission Spectroscopy (ICP) was used for mineral determination. Both methods gave analytically acceptable results. The MD procedure took 3 hours. The DA procedure took 3 days. MD gives slightly higher values of minerals than DA. The percent relative standard deviation was lower for MD than DA in the case of mineral compositions, standard reference materials and spiking determinations.

Board Z-01

C.D. Ellison, A.S. Hussain, and L.X. Yu., FDA/CDER/DPQR, Rockville, MD 20857

PURPOSE: To improve upon first-order one compartment drug absorption models by applying nonlinear methods to small intestinal transit, and to use this model for predicting the extent of intestinal absorption. METHODS: This nonlinear model uses a Weibull equation to describe small intestinal flow in the absence of drug absorption and dissolution. Weibull parameters were derived from literature data of small intestinal transit flow. With first-order absorption, the resulting mass balance model in the small intestine is described by the equation below, where M_i is amount of drug in the intestine and k_a is the absorption coefficient. Fraction of dose absorbed can be calculated by integration. RESULTS: Model parameters t and b were determined to be 217 min and 3.31 (dimensionless). The predicted fractions of dose absorbed for ketoprofen, naproxen, antipyrine, fluvastatin, and propranolol agree well with experimental values. Predicted values for metoprolol, atenolol, and terbutaline are in fair agreement with experimental values. The predicted value for furosemide is lower than the experimental value and the predicted value for enalaprilat is higher than the experimental value. CONCLUSIONS: The current one-compartment model reasonably predicts fraction of dose absorbed for passively absorbed drugs.

Board Z-02

HMD Luu, CS Kim and JC Hutter, CDRH, FDA, Rockville, MD 20852, CFSAN, FDA, Laurel, MD 20708

Bisphenol A (4,4'-isopropylidene-2-diphenol, BPA), polycarbonates and epoxy resins, is found in a variety of consumer products including food and drink packaging materials, baby feeding bottles, dental sealants, hemodialyzer casings, etc. BPA is a potent endocrine disruptor in animals. A comprehensive risk assessment of BPA exposure requires information about both the dosimetry of both free BPA as well as free endogenous estrogen. We have developed a physiologically based pharmacokinetic (PBPK) model to assess BPA bioavailability and its interaction with endogenous 17-ß estradiol (E₂) in rats and humans. In normal adult female, the model predicts no significant displacement of E₂ from sex hormone binding proteins in plasma following a single oral BPA exposure from five canned foods (30 µg/can x 5 =150 µg) or a dental sealant (1000 µg). However, the predicted free BPA in plasma increased compared to E₂ following repeated daily exposure to BPA (150 µg/day) in food for 7 days or more. Since BPA can cross the placenta and blood- brain barrier, exposure to BPA in the neonate and infant may cause developmental effects. Further modeling research is needed to assess the potential reproductive and developmental risks through out the life cycle of a woman, from fetus to mature adult.

Board Z-03

C.M. Lathers

Clinical pharmacologists must limit bacterial antimicrobial resistance (R) in humans to contain morbidity and mortality. Antibiotics (A)s as growth promoters influence resistance (R) prevalence in animal bacteria and is a risk factor for emergence of A R in human pathogens. R genes may transfer from bacteria of animals to pathogens in human intestinal flora via the food chain or contact. To prevent R in humans requires decreased use of As, good animal husbandry and hygienic measures to stop cross contamination. Poole (J Pharm Pharmacol 2001) targets 3 primary mechanisms of R, i.e., A inactivation, target site modification, and altered uptake via restricted entry and/or enhanced efflux, to develop new As. Bacterial R may be evaluated by using A pharmacokinetic (PK) values obtained from serum and MICs to reveal culture and sensitivity tests. Thomas et al (Antimicrobial Agents Chemotherap 1998) combined review of PK/PD and MICs values to examine data. Regulatory scientists must interpret scientific uncertainty, assess, communicate and manage risk, and convert science into regulatory policy to protect public health, food animals, and the food supply. This author opinion does not reflect FDA policy.

Board Z-04

C.M. Lathers ¹, P.L. Schraeder, ² and H.G. Claycamp¹, CVM, FDA Rockville MD 20855 ¹ and Albert Einstein Med Ctr and Jefferson Medical College ², Philadelphia, PA 19141

Clinical pharmacologists, neurologists, internists, and all health care givers must consider the efficacy, safety, and side effect profile of a given antiepileptic (AED) when determining which drug is best for a given patient. A number of new AEDs, including topiramate and lamotrigine, have been developed for chronic focal and secondarily generalized epileptic seizures. Efficacy of these drugs as anticonvulants does not seem to be superior to that of traditional anticonvulsants such as phenobarbital; however, the advantage of the new drugs is a different spectrum of possible adverse events. Newer AEDs may or may not induce sedation, and may minimize noncompliance by reducing side effects of lethargy and cognitive impairment. The difficulty in achieving therapeutic dosage because of side effects makes one consider whether these agents are "better" than the oldest and most side effect prone AED, phenobarbital. The new AEDs have less frequent interactions leading to improved tolerability with co-medication. This exercise compares two "new" AEDs, topiramate and lamotrigine, with phenobarbital by comparing efficacies and side effects using relative odds ratios, a method commonly used in drug development research. Development of new algorithms and/or new knowledge will bring beneficial tools to FDA reviewers of new animal or human drug applications. Opinions are those of the authors and do not represent opinions or policy of the FDA.

Board Z-05

O. A. Chiesa, J. von Bredow, P. Chamberlain, J. Peggins, R. Idowu, K. Moulton, B. Dominguez,. FDA, CVM, OR, Laurel, Maryland

Achieving and maintaining steady state drug concentrations are an important requirement for the accurate estimation of drug partitioning in pharmacokinetic studies. The long-term infusion of drug solution in conscious, free-moving large animals is very difficult and has only been achieved in a limited number of large animal surgical hospital centers. In these cases the animals are tethered to a long coiled tube used to provide the intravenous solution. The long tube system introduces an exceedingly large dead volume and the system requires constant supervision to be certain that tangles don't occur. A battery powered, self-contained ambulatory pump and drug solution for long-term intravenous infusion has been described for use in humans and in small animals. The short term use of ambulatory pumps for the administration of metabolic solutions has also been described in confined bovines, however, no reports are available which describe the chronic, long-term intravenous infusion of drug solutions using an ambulatory pump in the free-moving bovine. In a preliminary study designed to attain a seven day steady state plasma concentration of ciprofloxacin in a lactating dairy cow, the human use ambulatory intravenous infusion pump was attached to a custom fitted harness, designed for the free-moving bovine. Aseptic placement of left and right jugular catheters was made prior to intravenous infusion of the ciprofloxacin solution. The seven-day chronic intravenous infusion was interrupted for only a few minutes every twenty-four hours to change the drug solution container. Throughout this preliminary study, milk and plasma samples were collected to confirm that steady state plasma levels of ciprofloxacin were achieved and to measure the transfer of this antibiotic into the milk of the lactating dairy cow.

Board Z-06

P. Whittaker¹, V.C. Dunkel¹, H.E. Seifried², J.J. Clarke³, R.H.C. San³, ¹CFSAN, FDA, Washington, DC 20204, ²NCI, Bethesda, MD 20892, ³BioReliance, Rockville, MD 20850

We found that elemental and salt forms of Fe, including compounds used in dietary supplements and for food fortification, induced mutagenic responses in L5178Y mouse lymphoma cells. To study the mechanisms of the observed mutagenic effect and cellular toxicity, iron chelators were evaluated to select a compound that was not mutagenic and had limited toxicity. The iron chelators included those used clinically, those under development for clinical applications as well as those used in non-clinical applications. The mutagenic activity of the iron chelators was assessed in L5178Y mouse lymphoma cells. Eight of the 12 iron chelators induced mutagenic responses. Among those chelators used clinically or developed for clinical use, the only compound that did not induce a mutagenic response was a starch deferoxamine conjugate. In contrast, deferoxamine mesylate was mutagenic and showed the highest toxicity in this group of chemicals. The other three chelators that were not mutagenic were Na₂EDTA, phytic acid, and ferrozine. As reported previously, exposure of mouse lymphoma cells to Fe⁰, Fe⁺², and Fe⁺³ resulted in the induction of mutagenic responses with likely different mechanisms of iron metabolism. With the availability of nonmutagenic chelators with different specificities, we will be able to assess mechanisms of action.

Board Z-08

L.X. Yu¹ and C. W. Andrews², ¹FDA/CDER /DPQR, Rockville, MD 20857; ²GlaxoSmithKline, Research Triangle Park, NC 27709.

PURPOSE: This investigation evaluates a recent Quantitative Structure Bioavailability Relationship (QSBR) model (Andrews et al. Pharm Res. 17:639-643 (2000)) by using it to predict human absolute bioavailability of 20 recently approved drugs. METHODS: The QSBR model was revised to include the bioavailability of additional compounds in the training set as well as new descriptors. The revised model was used to predict the bioavailability of 20 drugs approved by the FDA from April 1999 to March 2001 and whose human absolute bioavailability is available. RESULTS: The training set contains 606 compounds with mean bioavailability of 56.4. The QSBR model obtained by stepwise regression had an R² of 0.83 containing 121 descriptors. Any descriptors that appear in one or two drugs were not included. It was found that the model could reasonably predict the bioavailability of 90% of the studied drugs: alosetron, dofetilide, entacapone, esomeprazole, gatifloxacin, letrozole, levetiracetam, linezolid, meloxicam, moxifloxacin, pantoprazole, pravastatin, rivastigmine, rizatriptan, rofecoxib, rosiglitazone, tolterodine, and zolmitriptan. The bioavailability of zaleplon was overpredicted and the bioavailability of terbinafine was underestimated. CONCLUSIONS: This modified QSBR model reasonably predicts human absolute bioavailability.

Board Z-10

O.A. Chiesa, P. Carter, J. von Bredow, J. Peggins, K.E. Moulton, R. D. Walker, FDA, CVM, OR, Laurel, Maryland

In an effort to evaluate the potential for the transfer of the antimicrobial agent ciprofloxacin to a nursing neonate during early lactation, ciprofloxacin was administered to a lactating Holstein cow. The dose the cow received was adjusted to approximate a dosing regimen similar to that used in human medicine. To accomplish this, ciprofloxacin was administered, as a continuous intravenous infusion for seven days, using an ambulatory pump. Serum and milk samples were collected prior to the administration of ciprofloxacin and then at 12 hours intervals for 9 days. In addition, to determine the effect the continuous intravenous delivery rate had on the cow's commensal flora, fecal samples were collected prior to treatment, once a day during treatment and 120 hours after the treatment was terminated. Escherichia coli was isolated from the fecal samples and identified using standard isolation and identification techniques. Antimicrobial susceptibility testing was performed in accordance with NCCLS standards. This study is designed to correlate systemic concentrations of ciprofloxacin with concentrations found in milk. It will provide insight as to the effect therapeutic doses of ciprofloxacin have on the susceptibility of intestinal commensals such as E. coli.

Board Z-11

S.H. Haidar, S. Doddapaneni and D. Udo. CDER, FDA, 5600 Fishers Lane, Rockville, MD 20857

Compartmental pharmacokinetic (PK) parameters were obtained for Drug x following single doses in patients with normal renal function and those with moderate and severe renal impairment. Drug x plasma levels were simulated for each group using multiple doses reflecting the proposed course of therapy. Simulations were used to evaluate alternate dosing regimens for patients with severe and moderate renal impairment. The effect of other patient demographic factors on Drug x clearance and exposure were also evaluated. The proposed dose in patients with moderate and severe renal impairment appears to result in unacceptably high plasma levels of Drug x. A dosing regimen of half the normal dose with a bolus of the normal dose appears to provide the best (near normal) exposure in this patient population.

Board AA-01

W. Howard Cyr¹ and Joanne Locke²

1. Former President of the FDA Chapter of Sigma Xi
2. FDA's Plain English Coordinator

Judging scientific posters is a difficult task, particularly when posters cover diverse disciplines. Criteria are established to give all judges a consistent approach to evaluating posters. Nevertheless, judging can still be subjective and therefore many excellent posters may go unrewarded. The FDA Chapter of Sigma Xi has judged posters at the many FDA Science Forums. Judges are chosen from Sigma Xi members and from winners of previous poster exhibits. The first author of this poster is one of the two people who started the first FDA - Sigma Xi poster exhibit and has been a judge at each event. The second author is the lead for FDA's Plain English initiative. We offer our criteria for a good poster, which must be clear, concise, important, relevant and eye-catching. The most common mistake is to present too much information, too much data, and too many words. One should be able to get a good grasp at the "bottom line" of a poster by reading the title and conclusions.

Board AB-01

Min C. Chen¹, Shiew-Mei Huang¹, Robert Mozersky², Julie Beitz¹ and Peter Honig¹. ¹Center for Drug Evaluation and Review (CDER), Rockville, MD, ²Center for Food Safety and Applied Nutrition (CFSAN), Washington DC

We evaluated in November 2001 a total of 548 adverse reaction reports submitted to CDER's Adverse Event Reporting System (AERS) and CFSAN's Adverse Event Monitoring System (AEMS) of St. John's Wort (SJW) used concomitantly with prescription drugs for possible cases of drug interaction. Published literature articles and the approved product labeling of interacting drugs were also reviewed. With the exception of forty case reports that indicated possible drug-SJW interaction, the majority of spontaneous reports did not implicate any role for SJW products in the reported adverse reactions. Based on the available information, there appeared to be pharmacokinetic interactions between SJW and drugs that are substrates of P450 CYP3A and/or p-glycoprotein, such as cyclosporin, levonorgestrel/estradiol, and sildenafil, resulting in drug level decrease, breakthrough bleeding/pregnancy and lack of effect, respectively. In addition, there appeared to be pharmacodynamic interactions between SJW and SSRI's (selective serotonin reuptake inhibitors), such as fluoxetine, fluvoxamine, and venlafaxine, or MAO inhibitors resulting in exaggerated hypertensive reactions and serotonin syndrome. Patients and health professionals need to be aware of these important possible interactions. Additional studies are needed to assess mechanistic issues and the full spectrum of clinical implications arising from these interactions.

Board AB-03

Syed R. Ahmad, Anne Trontell, Marilyn Pitts, Claudia Karwoski, Julie Beitz. FDA/CDER/OPDRA, HFD-430, Rm 15B-08, 5600 Fishers Lane, Rockville, MD, 20857

Background: The use of itraconazole and terbinafine have been associated with liver failure. We estimated the risk of liver failure with these antifungal drugs. Methods: A search in the FDA's AERS database identified cases of liver failure with both drugs. Reporting rates of liver failure were calculated based on the number of domestic cases reported to the FDA and the total number of prescriptions dispensed in the U.S. The reporting rates were compared with the background rate of liver failure which is 1 per million person years. Results: A search in AERS identified 6 and 5 domestic cases of liver failure in association with itraconazole and terbinafine respectively. The total number of prescriptions dispensed for itraconazole and oral terbinafine was 2.8 million and 6.6 million respectively. The estimated reporting rate of liver failure associated with itraconazole based on 6 domestic cases was 55 and 36 per million person-years of exposure assuming 14 and 21 days of therapy respectively. For terbinafine, the reporting rate was 6 per million person-years of exposure with 43 days of therapy. Conclusion: These data suggest that the reporting rates of liver failure among users of both drugs are higher than expected despite known underreporting of cases to the FDA. The risk of liver failure may be much greater than what has been reported. Physicians should be aware of liver failure risk with these drugs and consider stopping therapy if there is any evidence of liver toxicity.

Board AB-04

J.K. Zawadzki, L.Green, D.J.Graham, Food and Drug Administration, Rockville, MD

The thiazolidinedione (TZD) troglitazone was approved in the United States for the treatment of Type 2 diabetes mellitus in 1/97 and was withdrawn in 3/00 because of hepatotoxicity. To describe the potential hepatotoxicity of the other marketed TZDs, rosiglitazone and pioglitazone, we reviewed spontaneous adverse events (AEs) reported to FDA MEDWATCH during the initial 15 months of marketing for each drug in the US. We also reviewed AEs received during the first 15 months of marketing for two other antidiabetic agents, metformin and glyburide. Two distinct case definitions were applied: hepatitis and acute liver failure. Hepatitis was defined as elevation of alanine or aspartate aminotransferase above 3 times the upper limit of normal in temporal relation to drug use, in the absence of any other plausible etiologies. Acute liver failure was defined as severe hepatic dysfunction leading to encephalopathy, liver transplantation, or death, in the absence of other identified etiologies of hepatic failure. RESULTS: See Table below.

DISCUSSION: Underreporting of AEs is well recognized, with a majority of AEs not reported to the FDA. Knowledge about troglitazone withdrawal and recommendations for liver enzyme monitoring during TZD use may have augmented the spontaneous reporting rate of TZD-associated hepatotoxicity associated with rosiglitazone and pioglitazone. In the initial 15 months of marketing, with a similar number of prescriptions for each drug, fewer cases of hepatitis and acute liver failure were reported with the use of rosiglitazone and pioglitazone than with troglitazone use.

Board AB-05

D.J. Graham, L. Green, J.R. Senior, P. Nourjah. Office of Drug Safety/CDER

Troglitazone (TGZ), the first thiazolidindione agent developed for treatment of type II diabetes mellitus, caused cases of acute liver failure within 9 months of approval in 1997. Two "Dear Doctor" letters were sent in October and December 1997, advising physicians to test serum transaminase levels before and monthly during treatment with TGZ. The drug was withdrawn by the U.K in December 1997, but in the U.S it was widely used, and acute liver failure cases mounted, despite a third letter in July 1998 advising more monitoring. An Advisory Committee meeting in March 1999 recommended that TGZ should stay on the market but increased monitoring requirements yet again. Analysis of the 94 cases reported to FDA disclosed short time of progression from jaundice to encephalopathy, mean 31 days (median 24 days). There were 65 deaths and 10 transplants (2 died). In 19 cases, rapid progression occurred in less than a month, and less than the recommended monitoring interval. Biopsy, autopsy, or explant tissue in 27 cases showed hepatocellular necrosis, at times massive with liver collapse, with cholestasis less often described. We found that monitoring was seldom done as advised despite the three letters to physicians and the publicity surrounding the Advisory Committee meeting. In April and July 1999, two other thiazolidindione agents, rosiglitazone and pioglitazone, were approved for marketing, and it was hoped that they might be less likely to cause hepatotoxicity, which proved to be the case. TGZ (REZULIN) was withdrawn in March 2000.

Board AC-01

S.H. Stern, R.M. Gagne, H.H. Knox, M.P. Divine, R.J. Doyle, C.A. Finder, R.G. Kaczmarek, R.V. Kaczmarek, H.L. Rourk, T.B. Shope, Jr., D.C. Spelic, O.H. Suleiman, and S.A. Tucker, FDA, Rockville MD 20852

Since FDA's mandatory performance standard for x-ray CT equipment became effective in the mid-1980s, significant technological advances have been adopted in clinical practice. Current radiation-safety regulations have not kept pace with these developments and with the need to assure the lowest dose for the best image quality practically achievable. Involving approximately 58 million exams annually in the U.S., CT is a vitally important, beneficial modality whose radiation doses are relatively higher than those of other procedures. A group of CDRH and FDA Economics Staff is working to facilitate dose reduction through consideration of CT amendments in six technical areas: (1) standardization and definition of nomenclature of radiological variables, (2) display of an index of patient radiation dose that would be able to be automatically recorded within a facility's quality assurance system, (3) limitation of the x-ray field to that used for image formation, (4) "automatic exposure control" through modulation of x-ray tube output according to patient dimensions, (5) "CT fluoroscopy," and (6) cone-beam CT. We describe preliminary projections of technical feasibility, impact on clinical practice, impact on individual and population dose, and extent of harmonization with voluntary international standards.

Board AC-03

Meyer Katzper, CDER, FDA, Rockville MD 20852

Review activities in the agency involve specialties, as needed. By combining reviews we aim to obtain an integrated drug assessment. Actually we get a set of disjoint assessments. Another limitation of the review process involves the accumulation of experience and information. Individual reviewer experience is hard to transfer. Ideally, it would be possible to capture the knowledge obtained from the many NDAs that are reviewed. The proposed "Inclusive Medical Modeling" approach allows for incorporating cumulative knowledge and performing integrated assessments. Currently there are efforts to look at combined information, as in the Informatics and Computational Safety Analysis and Pharmacometrics Staffs. However, medical applications are not looked at in terms of the entire environment in which the medicine will be used. Application of computer-based modeling can incorporate a multiplicity of relevant factors. As an example consider a bacterial infection. Effective antibiotic therapy relies on a cooperative interaction of diverse physiological and pharmacological factors. An appreciation of these interrelationships is necessary to determine effective dosing strategies. In addition to the efficacy and toxicity of the drug, one should model the dynamics of bacterial growth and the development of resistivity. Toxicity and sources of infection must be considered along with host immune system and general health status. "Inclusive Medical Modeling " provides a paradigm for integrating relevant factors.

Board AC-04

H.R. Prasanna¹, R.C. Lyon¹, E.H. Jefferson¹, C.D. Ellison¹, T.W. Moore², J.A. Spencer², A.S. Hussain³, CDER, FDA, ¹Kensington, MD 20895; ²St. Louis, MO 63101; ³Rockville, MD 20852

Control of particle size is often necessary in pharmaceutical development and formulation. The particle size of pharmaceutical powders can influence performance (solubility, dissolution, bioavailability, stability, aerosol suspension) and manufacturing properties (flow, blend uniformity, tablet porosity and hardness). These factors can impact on safety and efficacy of the drug product. A meaningful particle size specification or acceptable size range needs to be linked to performance or a manufacturing property. We selected drug product dissolution as a performance standard. Nitrofurantoin powder was sifted into six fractions (20-32µ, 32-45µ, 45-63µ, 63-90µ, 90-150µ, 150-212µ). Each fraction was formulated into drug product (capsules) by dry blending of nitrofurantoin, lactose, cornstarch and talc. Dissolution profiles for each product were collected at pH 5.7 and modeled based on the Noyes-Whitney equation. This provided a direct linkage between dissolution and predicted particle size. The acceptance criteria established for dissolution could be translated to acceptance criteria for particle size distribution. The results from standard particle size methods (laser diffraction, light obscuration, image analysis) were compared with the size distributions predicted by modeling the dissolution results. By integrating these data with dissolution results, we have developed a performance-based approach to setting particle size specification.

Board AC-05

R.C. Lyon¹, E.H. Jefferson¹, L.X. Yu¹, A.S. Hussain², CDER, FDA, ¹Kensington, MD 20895; ²Rockville, MD 20852

This study explores the application of near-infrared (NIR) spectroscopic imaging for a pharmaceutical quality assurance problem - blend uniformity in the finished dosage form. A system based on array detector technology has recently been developed that can rapidly collect high contrast NIR images. This system bypasses the problems associated with single point mapping, and simultaneously reveals the type, distribution and relative abundance of chemical components within a particular system. Furosemide tablets were imaged to demonstrate the capabilities of this system. For comparison, these tablets were also analyzed by traditional NIR spectroscopy. Both approaches were used to evaluate drug product homogeneity. Four grades of blended tablets were evaluated: unblended, poorly blended, moderately blended and well blended (commercial-grade) tablets. The imaging system was capable of clearly differentiating between each grade and distinguishing between acceptable and unacceptable manufacturing processes. The distribution of active ingredients and excipients were rapidly visualized based on the spectral properties of these components. The NIR spectral imaging system provides a rapid approach for acquiring spatial and spectral information on pharmaceuticals. The technique has potential for a variety of applications in product quality assurance and could significantly enhance the manufacturing process.

Board AC-06

A.L. Ellis, CDER, FDA, Rockville MD 20857

Drug products intended for ototopical application should undergo testing for delayed contact hypersensitivity, dermal irritation and sensitization. Nonclinical pharmacokinetic data for a drug substance administered via the otic route may not be necessary if clinical administration is unlikely to result in measurable blood levels. It may, however, be useful to determine whether a drug penetrates an intact tympanic membrane or estimate the exposure to the middle and inner ears in an animal model when this barrier is not intact. State of the art ototoxicity testing frequently utilizes the guinea pig. A dosing catheter can be implanted through the tympanic bulla to allow drug to be administered directly to the middle ear, enhancing local exposure. Testing the auditory brainstem response (ABR) at an array of frequencies that ensures adequate coverage of the auditory range will identify functional hearing loss. Cochlear damage will be revealed through microscopic examination and enumeration of the hair cells. Gross and microscopic examination of the tissues of the middle ear, including mucosa, bone, tympanic membrane, and auditory ossicles will identify histopathologic changes indicative of irritation or injury. It is recommended that ototoxicity test groups include the product intended for clinical use, a vehicle control (especially if some components of the drug product have not been used in approved ototopical products) and a group that receives active drug at a concentration in excess of that to be used in the clinic to ensure that the animal model is being adequately challenged and provide an added margin of safety.

Board AC-07

G. J. Robbie, Ph.D.¹, H. Jeong², P. J. Marroum, Ph.D¹, J. Venitz, MD, Ph.D.³, M. Mehta, Ph.D.¹, L. Lesko, Ph.D¹. ¹CDER, FDA, Rockville, MD. ²UIC, IL. ³MCV, VA.

Purpose: To perform sensitivity analysis to identify factors influencing bioequivalence study outcomes in terms of parent drug and metabolite.

Methods: Monte Carlo simulations of parent drug and metabolite were generated using an one compartment model with first order absorption. Differing amounts of drug released from test and reference products, and different rate limitations such as, dissolution, absorption, and elimination rate limitations for both parent drug and metabolite were simulated at various levels of variability in inter-compartmental transfer rate constants. Each simulation scenario consisted of 1000 studies with 24 subjects in each study. The percentage of studies which failed the bioequivalence goalposts for AUC and C_max of parent drug and metabolite were calculated.

Results. Differential sensitivity of parent and metabolite Cmax and AUC to variability in different rate constants was observed. In all 38 scenarios, results based on either parent or metabolite bioequivalence metrics reflected true bioinequivalence, but not necessarily true bioequivalence of the test product.

Conclusion. Differential sensitivity of parent and metabolite Cmax and AUC might have direct implications in powering bioequivalence studies.

Board AC-08

J.S. Taylor¹, R.C. Lyon¹, H.R. Prasanna¹, A.S. Hussain², CDER, FDA, ¹Kensington, MD 20895; ²Rockville, MD 20852

The concern that expiration dating may markedly underestimate the actual shelf life of drug products has become an issue recently. For example, the AMA is currently evaluating the clinical and fiscal consequences associated with setting pharmaceutical expiration dates. Long term shelf life data from the Shelf Life Extension Program (SLEP) has been evaluated to address this issue. This program probably contains the most extensive source of stability data available. The FDA administers the SLEP for the U.S. Military. Drug products selected from the stockpiles are tested to determine if their shelf life can be extended past the labeled expiration date. This approach was not applied to dispensed drugs, but only to products stored in original sealed containers. This report summarizes extended stability profiles for 96 different drug products (1122 lots). 84% of the lots were extended for an average of 57 months past the original expiration date. Only 10 drug products were unstable beyond their expiration date; meaning that most of the lots for these few drug products failed initial extension. This data supports the assertion that many drug products can be extended past the expiration date, but this additional stability period is highly variable. Due to the lot-to-lot variability, the stability and quality of extended drug products can only be assured by continual testing and systematic evaluation of each lot.

Board AC-09

M.J. Shein, D.W. Feigal, Jr., L.J. Gill, T.M. Nearing, CDRH, FDA, Rockville MD 20850

A subcommittee of the Science Advisory Board recently completed an external review titled "Science at Work" at CDRH. Feedback on the process from the subcommittee presented here support its use as a model for other Centers interested in planning such a review. Foremost, the review was not limited to CDRH research programs, such as the laboratory, but i. Instead the review focused on the role of science as used by the Center in all of the CDRH's activities. A single product area, electrical ostimulation devices, was selected for in-depth review and as a pretext for selecting expert external reviewers. The review began with a one day orientation to the work and organization of CDRH, a subsequent 4-day site visit and a follow-up report writing day. During the site visit the review committee was asked to role play, in "CDRH's shoes" with real examples of early product development and post-marketing issues. Four detailed device case studies were presented, and the Committee was given the chance to put CDRH "on the spot" by picking any application, review, research project or employee to look at an issue in-depth. Management, staff, and device manufactures were interviewed. This poster presents a As shown here, subcommittee members regarding the usefulness of the elements of the review and its process to determine the suitability of this approach for others to use. The survey found that broadening opening of the scope of the review to include all Center activities was essential, and rated the staff interviews the most important single element. They also found the early kick-off meeting and background materials to be very important.

Board AC-10

C. M. Monroe, Detroit District Laboratory, Detroit, MI 48207

The Food and Drug Administration/Department of Defense Shelf Life Program began in 1985 as a pilot test with 58 drugs with 137 individual lots. The principle upon which the program is based is to artificially accelerate the degradation of the drug product using controlled conditions of temperature, humidity and time. An unstressed portion of a sample from a given lot of a drug product is tested and another portion of the same sample after it is stressed is tested. The amount of stress induced degradation is then measured using stability indicating methodology. Using that data, a computer model is used to extrapolate how long that lot can be stored under labeled storage conditions before it will reach the lower limit of its' established potency standard. Unique analytical problems encountered in this project and their solutions will also be described.

Board AC-12

M.A. Cheeseman, A.B. Bailey, N. C. Braier, R. F. Chanderbhan, E.J. Machuga, C.J. Sheu and M. L. Twaroski, CFSAN, FDA, College Park, MD 20740

Human exposures to substances used in food-contact materials are typically below 150 micrograms per person per day. At such low exposures the toxicological endpoint of most concern is carcinogenicity and safety data is often limited to genotoxicity data. A test data set of more than 600 compounds was used to evaluate the relative capabilities of structure activity analyses and genotoxicity testing to identify food contact substances of potential carcinogenic concern at low dietary exposures. Structure activity analyses and genotoxicity testing show comparable predictive capabilities and some complementarity. In addition, examples of the use of structure activity analysis to estimate the upperbound likely risk of food contact substances will be presented.

Board AC-14

Exchange of teaching materials via web-based technology supports sharing of innovative materials in clinical pharmacology. Professional associations, such as The American College of Clinical Pharmacology (ACCP), play a role by providing an exchange mechanism. ACCP offers the Educational Series of the J of Clin Pharmacology, Teaching Clinics, and Symposia, many of which may be placed on the ACCP web site. The Journal's educational series publishes articles based upon teaching programs designed for advanced students in medical/pharmacy/or other allied health programs. ACCP teaching symposia develop case histories illustrating pertinent principles in clinical pharmacology, including Clinical Pharmacology Problem Solving (CPPS) units. A web-based slide exchange for members allows sharing visual information for educational purposes. The Web is a chaotic, unorganized forum of almost limitless information and thus links in the teaching section of the ACCP web site could direct members to useful sites, such as those related to the development of new drugs, including the basic principles for developing and conducting a clinical trial. This becomes a potential role for the FDA Staff Colleges as they work to teach those within and outside the Agency about the drug development process using the world wide web. New web based knowledge will also bring beneficial tools such as those offered by ACCP to FDA reviewers of new animal or human drug applications. Opinions are those of the authors and do not represent opinions or policy of the FDA.

Board AD-01

W. Slikker, Jr.¹, G. Newport¹, J. Schnellman¹, Y. Itzhak², M. G. Paule¹, S. F. Ali¹ and J. DeGeorge³, .¹NCTR/FDA, Jefferson AR 72079, . .²Univ. of Miami, Miami FL 33136, ³CDER/FDA, Rockville MD 20857

In studies designed to determine the general toxicity and neurochemical and behavioral effects of the anorectic agent dfen, adult monkeys were dosed for 90 days with 5 mg/kg dfen (N=8) or vehicle (N=3) i.m., BID. Mean body weight loss during exposure was 9.5% ± 2.5. Peak plasma concentrations were 2.2 µmol (±0.4) and 0.9 µmol (±0.1) for dfen and the major active metabolite nor-dfen, respectively, and did not change with the length of treatment. Three of the 8 treated animals performed food-reinforced operant behavioral tasks. Significant behavioral effects of treatment were noted in both the learning and discrimination tasks. Rates of responding and associated endpoints (e.g., percent task completed; reinforcers earned) were the metrics most affected, whereas response accuracies generally were not. At the end of the 90 days of treatment, brain 5-HT concentrations were significantly decreased in frontal cortex and hippocampus and 5-HT transporter (Bmax) was decreased significantly in all brain regions examined. We conclude that with dosing at 5 mg/kg BID, peak dfen plasma levels can be maintained at 10-20x human therapeutic peak plasma levels. This 90-day dfen exposure results in significant body weight loss, neurochemical alterations and disruption in food-reinforced behavior.

Board AD-03

Cristina McLaughlin and Clark Nardinelli, FDA/CFSAN, Washington, DC

Risk assessments and cost-benefit analyses both require estimates of exposure to hazards, and estimates of exposure to food-borne hazards require estimates of food consumption. In this paper we describe the uses of various food consumption data sources in risk assessments of food-borne hazards and cost-benefit analyses of food safety policies. As more data on food consumption become available, risk assessors and economists need to be aware of and use all available data, partly because of legal requirements calling for more thorough analyses of risks and policies involving food safety. Many different databases on food consumption are available, including: Continuing Survey of Food Intakes by Individuals (CSFII), USDA's Food Consumption Prices and Expenditures (USDA-ERS), and IRI's Infoscan supermarket scanner data. Also, there are smaller, product-specific databases that can be used in risk assessments for particular hazards. We will discuss methods and guidelines for integrating more than one data source, according to its characteristics, into a risk assessment or cost-benefit analysis. We will include examples of how different databases can be combined to generate consumption estimates, and those estimates can then be used in risk assessments and cost-benefit analyses.

Board AD-04

A. D. Hitchins, CFSAN, FDA, Washington DC 20204-0001

Risk assessment (RA) of US foodborne listeriosis uses dated and foreign L. monocytogenes contamination data as well as modern domestic data. The possible bias due to data age and origin (GDP/capita) on % prevalence in the US was investigated. Downward (pasteurized or raw milks, hot dogs, raw seafood, soft cheese), upward (smoked & preserved seafoods, fresh vegetables) and no (raw seafood) normalization was indicated for older age data. Origin-wise, downward (raw or pasteurized milks, hot dogs, cheese, frozen dairy products, fresh vegetables, ready to eat meats), upward (cooked or preserved seafood) and no (smoked seafood) normalization was indicated for lower GDP/capita data. Such normalization is applicable to RA in any country.

Board AD-05

C.M. Lathers, Center Veterinary Med (CVM), FDA, Rockville, MD

One responsibility of veterinary medicine is to protect animal and human health. Food animal production uses antibiotics to enhance production. Regulators evaluate new production technology to assure animal safety and safe, edible products and to make public policy decisions by assessing risks/benefits. Risk assessment is the method of systematically identifying and assessing factors that influence the probability and consequences of a negative event occurring. CVM's first risk assessment addressed the potential human health impact of Campylobacter effects associated with use of fluoroquinolines in food-producing animals. CVM used the Monte Carlo method to estimate risk by probability distributions that reflect the uncertainty and variability in the data used for the assessment. Enterococci faecium is a species more likely to be resistant to antibiotics of last resort. Effective control of multiple-drug resistant enterococci will require a better understanding of the transfer of E. faecium from animals to humans and the interaction between E. faecium, the hospital environment, and humans; prudent antibiotic use; better contact isolation in hospitals; and better surveillance. CVM will model these factors in a second, more complex, risk assessment designed to examine the indirect transfer of resistance from animals to humans. Use of risk assessments allows researchers, the industry, regulatory authorities, and educators to make better policy decisions regarding antimicrobial use in food animals and humans and the development of resistance. Opinions are those of the author and do not reflect FDA or US government policy.

Board AD-06

K. Uhl¹, D. Kennedy¹, S. Kweder¹, CDER, FDA, Rockville, MD 20850

Drugs that carry a concern of teratogenicity are often contraindicated for use during pregnancy and are classified as Pregnancy Category X. A search of the electronic version of the 2001 Physicians' Desk Reference yielded 117 Category X drugs. The label/package insert for each drug was reviewed to identify risk management (RM) strategies. The majority of the labels included as the sole RM strategy either a black box warning and/or a contraindication for use in women who are or may become pregnant. Only 16 drugs contained specific RM strategies in the label directing the clinician and/or patient, e.g., number and type of contraception methods, frequency of pregnancy testing. Only 2 drugs (Accutane® and Thalomid®) had formal RM programs. This study demonstrates the varied RM approaches in labeling for Pregnancy C X drugs. There is a need for consistency in the classification of Category X drugs and the RM strategies utilized for them in labeling.

Board AD-07

Dianne E. Godar¹, Steven P. Wengraitis², Jack Shreffler³ and David H. Sliney²; ¹US Food and Drug Administration, Center for Devices and Radiological Health, Rockville, MD 20852, ²US Army, Center for Health Promotion and Preventive Medicine, Aberdeen Proving Ground, MD 21010, ³US Environmental Protection Agency (EPA), National Exposure Research Laboratory, Research Triangle Park, NC 27711

The UV doses of Americans were never measured, but are needed to assess the risks for UV-related health effects. We extracted the average Americans outdoor daylight-times from the EPA's NHAPS survey. Knowing this, and that people are exposed to UV about 30% of the time they are outdoors, we calculated the average Americans UV doses by multiplying by the available terrestrial UV readings. We find the average erythemal UV doses of Americans are about 25,000 J/m²/yr., 22,000 for females and 28,000 for males, or 33,000 J/m²/yr. including a conservative vacation in the continental U.S. (7,800 J/m²). Thus, we can now assess the risks of UV-related health effects for Americans.

Board AE-01

N. Wolins1, T. Mondoro1, J. Lozier1, T. Eggerman1, E. Jones1, R. Morgan2, E. Aguilar-Cordova3, and J. Vostal, 1Center for Biologics Evaluation and Research, FDA, Bethesda, MD, 2National Human Genome Research Institute, National Cancer Institute, NIH, Bethesda MD, 3Harvard University, Boston, MA

Adenovirus based vector are utilized in gene therapy. Unfortunately, these viral treatments cause complications including thrombocytopenia. We wanted to determine whether adenovirus decreased platelet counts by shortening the time platelets circulate or by hindering their replenishment by the bone marrow. To address this question, we measured platelet count and survival time in virally-treated monkeys. Adenovirus infusion decreased both the platelet counts and platelet survival times in a dose-dependent manner. In fact, a dose of 6 x 10¹² virus particles/kg lowered the number of circulating platelets from 374,000 to 52,000 and shortened the half-life from 111 h to 22.2h. The rapid loss of circulating platelets and the shortening of their circulation time is indicative of platelet destruction. Furthermore, viral treatment did not observably alter the megakaryocyte number in the bone marrow, and platelet counts started to rebound 96 hours after adenovirus treatment. These data suggesting that the bone marrow was functioning. Thus, adenovirus lowers platelet count by shortening the time platelets circulate.

Board AF-01

Daniel F. Molefe¹, Division of Biometry and Risk Assessment, 3900 NCTR Road, Jefferson, Arkansas, 72079
James J. Chen¹, Paul C. Howard^2,3, Donald P. Forbes⁴, Christopher P. Sambuco⁴ and Ralph L. Kodell¹, ¹Division of Biometry and Risk Assessment, NCTR, FDA. ²Division of Biochemical Toxicology, NCTR, FDA. ³National Toxicology Program Center for Phototoxicity, NCTR, FDA. ³Argus Research Laboratories, Charles River Companies, Horsham, PA

The single induction test procedure for modeling multiple tumor data by Kodell and Chen (2001) is extended to a test for tumor multiplicity in multiple induction experiments. The new test procedure can detect overall differences in the distribution of observed tumors between two groups as well as isolate whether the detected overall difference is due to the difference in the distribution of the number of induced tumors and/or the difference in the distribution of the to tumor observation. An illustration of the proposed test procedure was accomplished using data from a photocarcinogenicity experiment. The data set contained male and female mice exposed to no light or two doses of light following either no treatment or treatment with vehicle for 40 weeks, and the occurrence, size, and multiplicity were recorded weekly for each mouse. The results of the analysis when using the proposed test

were compared to the results coming from the logrank test, the negative binomial test and Dunson's test (2000). Monte Carlo simulation studies were used to show that the estimated parameters were fairly close to the true parameters. In addition, simulation studies of the type I error showed that the logrank test had a type I error fairly close to the desired nominal values while the proposed test was slightly anti-conservative and the negative binomial test was conservative.

Board AF-02

Z.K. Binienda, P.K. Kowalke, B.T. Thorn, M.A. Beaudoin, and W. Slikker, Jr. Div. Neurotoxicology, NCTR, FDA, Jefferson, AR 72079

Electroencephalography (EEG) is a noninvasive and a relatively inexpensive technique for assessing spontaneous electrocerebral activity using either scalp electrodes or electrodes implanted in specific brain regions. The objective of the present study was to assess the relationship between low frequency EEG changes and behavioral seizure type in rats exposed to domoic acid. Domoic acid (DOM), is of interest to the FDA because of its natural presence in some seafood, and its potential to produce neurotoxicity and death in humans. Here, electrocorticogram (ECoG) was monitored in adult, male Sprague-Dawley rats injected intraperitoneally with DOM at 2.2 and 4.4 mg/kg. The ECoG was recorded via a tether and swivel system at least one week after animal instrumentation (implantation of the EEG electrodes). The EEG frequency analysis based on the fast Fourier transform (FFT) technique revealed an associations between slow-wave EEG activity (delta; 1.25-4.50 Hz, and theta; 4.75-6.75 Hz) and type of behavioral seizures following DOM administration. Initial analysis suggests that EEG events precede motoric seizures.

Board AF-03

Z. Chen and R. Osterberg, CDER, FDA, Rockville, MD 20852

Ophthalmic drug products can be administered topically (as drops applied to the eye), intravitreally, intracamarally, systemically, as implants, and by some other ocular routes. Regardless of the mode of administration, ocular and systemic toxicity concerns should be addressed. To define the toxicity profile of ophthalmic drugs, general toxicology studies should be conducted using both ocular and systemic (e.g., oral) routes. Route specific studies, including pharmacokinetic and toxicity evaluations, should be performed with the proposed human clinical routes. The systemic bioavailability and ocular distribution following topical exposure should be defined early in the development process. Ocular toxicity should be assessed in at least 2 appropriate species. To support human trials, the dosing frequency, drug concentrations and study duration in nonclinical studies should be at least equal and preferably exceed the maximum frequency, concentration and duration of clinical human studies. The assessment of ocular toxicity should include slit lamp biomicroscopy, funduscopy, tonometry, histopathology and other measurements at appropriate times. If the drug binds to melanin, animals with pigmented eyes should be used.

Board AF-04

P.C. Howard¹ and W.G. Wamer², ¹NCTR, FDA, Jefferson, AR 72079, ²CFSAN, FDA, Washington, DC 20204

It has been estimated 3% of the population in the US has tattoos. Although a large number of both inorganic and organic pigments are currently used for dermal implantation in tattoos, there are currently no pigments approved by the FDA for this use. A collaborative project between CFSAN and NCTR has recently been established to develop methods for testing the safety of tattoo pigments. Studies being conducted at CFSAN and NCTR focus on in vitro and in vivo methods to assess tattoo pigment safety. Investigators in CFSAN are applying in vitro screening methods for identifying cytotoxic and photocytotoxic tattoo pigments. Preliminary results from in vitro screening tests suggest that a small number of pigments currently marketed for tattooing are phototoxic. Studies being conducted at NCTR focus on the chemical composition and metabolism of tattoo pigments, as well as development of animal models for assessing the acute and long-term safety of pigments used in tattooing. Additionally, studies at NCTR will examine the effects of UV light on biological responses to tattoo pigments. The methods being developed at CFSAN and NCTR should provide a foundation for establishing tests for the safety of tattoo pigments.

Board AF-05

ENHANCED HISTOPATHOLOGY WITH MORPHOMETRY, IMMUNO-AND IN-SITU STAINING IN IMMUNOTOXICITY STUDIES OF THE MYCOTOXINS, AFLATOXIN B1 (AFB1) AND DEOXNIVALENOL (DON).

DM Hinton¹, MJ Myers², A. Perlloni¹, F. Hines¹ ,R Raybourne¹, RE Sotomayor¹, J Shaddock³, A Warbritton³, M Chou³ , and J. J. Pestka⁴. ¹USFDA, CFSAN, Laurel, MD; ²USFDA, CVM, Laurel, MD; ³USFDA, NCTR, Jefferson, AR, and ⁴ Michigan State Univ., East Lansing, MI.

AFB1 is a known carcinogen and affects immune function in animals. DON has been shown to cause an IgA nephropathy in various murine strains. For AFB1, we were interested in the immunotoxic mechanisms relevant at low doses and in an intermittent exposure regimen. Fisher-344 male rats were fed for 40 weeks intermittently (4 weeks-on and 4 weeks-off) doses of 0.01, 0.04, 0.4 and 1.6 ppm of AFB1 and for 40 weeks continuously at the 1.6-ppm level. Groups of animals were sacrificed at 4-week intervals up to 20 weeks and then at 40 weeks. Lymphoid tissues were removed and processed for routine histopathology, immunoperoxidase staining for pan-T and pan-B lymphocytes (spleen), the proliferative antigens Ki67 and PCNA, and morphometric analysis, i.e. cortical/medullary ratio of the thymus, the area ratio of the periarteriolar lymphocyte sheath (PALS) in the spleen, and the relative nodular and germinal centers in the Peyer's patches. For comparison to structural changes observed, splenic cell suspensions were analyzed by flow cytometry for % T (CD3) and % B (CD45R) cells and % helper (CD4) and % suppressor (CD8). Portions of the suspension were stimulated in culture for analysis of the productive capacity for cytokines IL-2 (Con-A), IL-1 and IL-6 [LPS, LPS + (interferon (INF) and (INF + NGMA, a nitric oxide synthetase inhibitor]. Complex changes were seen in the various T- and B-cell areas of the lymphoid organs in this study design, and concomitant affects on cell numbers and type of cells were usually comparable to functional changes, but not always, e.g. increases in T-cell numbers and decreased IL-2 productive capacity as a function of AFB1 dose. For DON, various murine strains were used for comparison to the known effects seen in the B6C3F1strain. IL6 deficient knock-out (IL6KO), the wild type strain (IL6W) and B6C3F1 mice were fed diets with 10 ppm DON and diets without DON. Morphometric analysis revealed greater changes in the Peyer's patches for IgA production as well as the IL6 and IL2 mRNA determined by in-situ hybridization. Although, work on these studies is on-going, results obtained strongly support the use of enhanced histopathology in immunotoxicity studies since these methods are powerful tools for evaluating dosing and mechanistic effects.

Board AF-06

P. Vath¹, W.G. Wamer² and D.E. Falvey¹, ¹University of Maryland, College Park, MD 20742, ²CFSAN, FDA, Washington, DC 20204

Aloe emodin (AE) is a naturally occurring anthraquinone present in aloe vera. A recent report suggests that AE can enhance photocarcinogenesis in an animal model. We have examined the photochemical and photobiological mechanisms underlying the photoactivity of AE. Using laser flash photolysis, we found that irradiation of AE with UV light efficiently produces the excited triplet state of AE. Subsequently, these excited state species interact with molecular oxygen to produce singlet oxygen, a potent oxidizing agent. To investigate the role of singlet oxygen in the photocytotoxicity elicited by AE, we compared inhibition of cellular growth photosensitized by AE in buffers prepared in water (H₂O) or deuterated water (D₂O). Because singlet oxygen has a longer lifetime in D₂O than in H₂O, this experimental approach yields information on the importance of singlet oxygen in cellular damage. We found that the photocytotoxicity of AE in buffers prepared from D₂O is dramatically higher than the photocytotoxicity elicited by AE in buffers prepared in H₂O. We conclude that oxidative damage, involving the formation of singlet oxygen, plays a significant role in the photocytotoxicity associated with AE.

Board AF-07

T.J. Flynn and C. Madubata, CFSAN, MOD-1 Laboratories, Laurel, MD 20708.

The intestine has recently been identified as a key organ mediating drug-drug and drug-food interactions. A human colon adenocarcinoma cell line (Caco-2) retains many of the functions of normal intestinal epithelium. Testosterone is known to be hydro-xylated by several different cytochromes P450 (CYP) in both the liver and intestine. Caco-2 cells were exposed for 18 hr to model drugs and food components (3-100 µg/mL). Then, 200 µM tes-tosterone was added on the apical surface. After 5 hr at room temperature, unreacted testosterone and hydroxylated metabolites in the basolateral medium were quantitated by LC. Viability and cell monolayer integrity were assessed by measuring trans-epithelial electrical resistance. Both increases and decreases of various metabolites were observed suggesting test compound-specific induction or inhibition of one or more CYP and, therefore, the potential for adverse interactions with drugs.

Board AG-01

Ron Honchel¹, Ph.D., Carol S. Trempus², Ph.D. and Frank D. Sistare¹, Ph.D., ¹CDER, FDA, Laurel, MD 20708, ²LECM, NIEHS, Research Triangle Park, NC 27709

Transgenic Tg.AC mice develop large numbers of papillomas within 26 weeks in response to dermal application of certain tumorigens. We performed a solar-simulated light (SSL) dose-response study in order to determine the threshold levels of SSL that are tumorigenic to Tg.AC and FVB/N mice (the parental strain of Tg.AC). Groups of 7 male and female Tg.AC mice were then exposed to a fraction of the minimal erythemal dose (MED) of SSL (mice received 0, 0.1, 0.2, 0.3, 0.4, or 0.6 MED of SSL). Groups of 7 male and female FVB/N mice were exposed to 0, 0.1, 0.3, or 0.4 MED of SSL. Mice were exposed to SSL 5 days per week for 26 weeks. Because no tumors developed, all mice were then observed for an additional 12 weeks with no SSL exposure. During this 12 weeks, 14/26 of the Tg.AC mice and 2/14 of the FVB/N mice receiving 0.4 MED or greater of SSL developed large nonpapilloma skin tumors. In situ hybridization for transgene expression and p53 immunohistochemical staining was performed on all SSL-induced tumors. Unlike typical tumorigen-induced tumors in Tg.AC mice, transgene expression was not detected in the Tg.AC UV-induced tumors (0/13). However, all tumors, including FVB/N, stained positive for p53 expression (15/15). These results indicate that Tg.AC transgenic mice, surprisingly, do not respond in a transgene dependent manner to genotoxic doses of UV light delivered from chronic suberythemal SSL exposure.

Board AG-02

Karl K. Lin and Feng Zhou, CDER/FDA, 5600 Fishers Lane, Rockville, MD 20857

The model described in Dunson, et al. (2000) was applied to the data of studies submitted by drug sponsors to test if the dosing of a drug affect the probability of developing skin papillomas on the test animals. However, we encountered problems in using the SAS NLMIXED procedure in the fitting the above 5-parameter model to the data. The first encountered problem is that the iterative method used in the SAS procedure could not converge. The second encountered problem, which is more serious and dangerous in our opinion, is that the SAS procedure, when it went through, gave two very different sets of estimation and test results when two different sets of initial values were used.

The reason for above problems is the sparsity of tumor data of the mouse study. Under this circumstance, the use of the SAS NLMIXED procedure to fit the full 5-parameter model won't be able to give stable, reliable, and even meaningful results. A simpler version of the above model was actually applied to the data. This is the model derived from the above 5-parameter full model by dropping the two parameters measuring the drug effect on multiplicity of papillomas and the factor for the variability in expression of the transgene of individual animals.

Board AH-02

A.S. Carlin, A.S. Hussain, and L.X. Yu,. FDA/CDER/DPQR, Rockville, MD 20857

PURPOSE: To determine the effect of sodium lauryl sulfate (SLS) on solubility and intrinsic dissolution rate of cyclosporine. METHODS: Solubility was measured by shake flask method with 24hr equilibration. The intrinsic dissolution apparatus (Wood apparatus, VanKel type^TM) and USP dissolution apparatus (w/900mL vessel) were used to determine the intrinsic dissolution rate of cyclosporine. Compression force, dissolution medium volume, die position, and rotation speed used in the study were 2000psi, 900mL, 0.5in (from vessel bottom), and 100 rpm. RESULTS: See table. Plots of solubility and intrinsic dissolution vs. %SLS were linear from 0 to 1.5% SLS. CONCLUSIONS: Aqueous solubility and intrinsic dissolution rate as functions of SLS concentration were determined. Solubility and intrinsic dissolution are well correlated.

Board AH-03

P.J. Faustino¹, AB. Ciavarella¹, RC. Lyon¹ and A.S. Hussain², CDER, FDA, ¹Kensington, MD 20895; ²Rockville, MD 20852

FDA's Guidance for Industry, "Bioanalytical Methods Validation" provides assistance to sponsors and applicants of IND's, NDA's, ANDA's in developing bioanalytical method validation information used in human bioavailability and bioequivalence (BE) studies requiring pharmacokinetic information. FDA conducted and analyzed samples from a cross-over BE study (n=18 subjects, n=508 samples) with the model drug metoprolol. This study provided the opportunity to effectively overview the guidance "approach" and "processes" that defines bioanalytical regulatory methods validation. Application and review of the guidance processes: reference standard preparation; pre-study validation and in study validation, were undertaken. Reference standard preparation includes spectroscopic (NMR, IR, UV) and chromatographic identification. Pre-study validation includes (a) stability of drug substance, dosage form and biological matrix and (b) accuracy, linearity, recovery, specificity, precision, quality control (QC) and acceptance criteria data. In study validation includes calibration, QC and acceptance criteria data. This validated FDA intramural BE study meets or exceeds all defined specification criteria defined by the guidance and provides a step-by-step review of the guidance with supportive validation information.

Board AH-04

Michael A. McLaughlin*, Benjamin J. Cañas and Samuel W. Page, Food & Drug Administration, Center for Food Safety and Applied Nutrition, Office of Plant, Dairy Foods and Beverages, Division of Natural Products, HFS-347, 5100 Paint Branch Parkway, College Park, MD 20740-3835

Submitting Laboratory: Michael A. McLaughlin*, Benjamin J. Cañas and Samuel W. Page, Food & Drug Administration, Center for Food Safety and Applied Nutrition, Office of Plant, Dairy Foods and Beverages, Division of Natural Products, HFS-347, 5100 Paint Branch Parkway, College Park, MD 20740-3835, * Tel.: 301-436-1958, Fax: 301-436-2644, E-mail: mam@cfsan.fda.gov

Method Author: Nicholas Low, Department of Applied Microbiology and Food Science, University of Saskatchewan, 51 Campus Dr., Saskatoon, Saskatchewan S7N 5A8, Canada, Tel: 1-306-966-5037, Fax: 1-306-966-8898, E-mail: nicholas.low@usask.ca
(*) - to whom correspondence should be addressed

Collaborators: R. Ardenghi, N. Barda, C. Barry, A. Brause, B. Canas, E. Coppola, T. Eisele, M. French, V. Giller, D. Hammond, L. Hegeman, D. Kruger, S. Lewis, N. Low, R. Lyons, G. Martin, H. Nivens, R. Roderick, M. Siguvin, P. Stober, S.Tefera

Fifteen collaborating laboratories analyzed five sets of five blind duplicate samples of apple juice concentrate for the presence of added hydrolyzed inulin syrup (HIS) and high fructose corn syrup (HFCS). The procedure involved determining the Brix value of the apple juice or apple juice concentrate and preparing a dilution to 5.5° Brix. 100 µL of the 5.5° Brix dilution were then freeze-dried in a gas chromatograph (GC) autosampler vial. The sugars in the freeze-dried residue were converted to their trimethylsilyl derivatives by the addition of a silylation reagent and heated at 75° C for 30 minutes. After derivatization, the solution was analyzed by gas chromatography equipped with a flame ionization detector (FID). The five sets of test portions used in the study consisted of commercial apple juice concentrate and commercial apple juice concentrate with added HIS (6.9% or 16.0%) or HFCS (8.1% or 17.0%). Detection of the presence of the added syrups was ascertained by the identification of unique marker peaks derived from carbohydrate by-products (retrograde sugars) present in the two sugar syrups. The sensitivity (p₊) of the method to detect the two syrups in the apple juice concentrate ranged from 0.9733 – 0.9867. The high level of sensitivity was complemented by the low false negative rates (pf_-) (0.0133 – 0.0267) demonstrated in the study. The results from the authentic apple juice concentrate tests produced a specificity (p_-) value of 0.9733 with a false positive rate (pf₊) of 0.0267. Study results indicate that the method was highly selective and reliable in detecting the presence of added HIS (6.9% - 16.0%) or HFCS (8.1% - 17.0%) in apple juice.

Board AH-05

L.X. Yu¹, J.T. Wang¹, P.J. Faustino¹, C.D. Ellison¹, S.E. Tabibi², R.I. Poust³, J.M. Jordan³, W. Wilson³, R. Song⁴, and A.S. Hussain¹, ¹FDA/CDER, Rockville, MD 20857. ²NCI, Bethesda, MD 20852, ³University of Iowa, Iowa City, IA52242, ⁴CDC, Cincinnati, OH 45226

PURPOSE: To identify the critical formulation and process variables of direct compression tablets of the model BCS low permeability/low solubility drug furosemide using a factorial design approach. METHODS: The formulation and process variables studied include the amount of lactose, microcrystalline cellulose, croscarmellose sodium, magnesium stearate, and tablet hardness. 24 lots of tablets of 80 mg furosemide with total weight of 320 mg were manufactured based on a full factorial design. Dissolution testing was conducted in pHs 4.5, 5.8, and 7.4 buffers using USP apparatus II with 5, 10, 15, 30, 45, and 60 min sampling times. The results were analyzed by ANOVA. RESULTS: The in vitro dissolution at low pH is more discriminating than that at high pH. Hardness has no significant effect on dissolution at any of tested pHs. The effect of lactose or microcrystalline cellulose is significant after 5 min (p<0.05). Croscarmellose sodium and magnesium stearate have significant effects at low pH and early time points but not at high pH or later time points. CONCLUSIONS: The identification of these critical variables allows us to design desired formulations for clinical testing.

Board AH-06

S.H. Stern, R.V. Kaczmarek, D.C. Spelic, and O.H. Suleiman, CDRH, FDA, Rockville MD 20850

To estimate patient radiation dose from x-ray CT examinations, specially trained inspectors from 39 States surveyed 263 CT facilities across the U.S. in randomly selected samples proportional to State populations. Surveyors measured x-ray exposure (air kerma) centrally and peripherally in a standard head dosimetry phantom as well as free in air on the scanner axis of rotation. Facilities provided metrics of patient irradiation technique, estimated frequencies of various head and body examinations, and gave quality-assurance information. Our most important observation is that on average for any given examination of the head or body, effective doses E are significantly smaller with helical-scanning techniques than with axial-scanning techniques. For the most frequent routine examinations of adult patients E is 2 mSv (S.D. = 1 mSv, n = 45) for head exams; 12 mSv (S.D. = 7 mSv, n = 21) for abdomen-pelvis exams; 6 mSv (S.D. = 4 mSv, n = 21) for chest exams; 6 mSv (S.D. = 4 mSv, n = 19) for abdomen exams; 15 mSv (S.D. = 10 mSv, n = 18) for chest-abdomen-pelvis exams; and 6 mSv (S.D. = 4 mSv, n = 15) for pelvis exams. From facility reports of examination workload, we estimate that there were 58 ± 9 million CT examinations and procedures in the U.S. during the survey year 2000-01. The preliminary findings suggest that advances in CT scanner technology since the previous survey in 1990 have been rapidly adopted in clinical practice and have thereby significantly affected population dose.

Board AH-07

E. Kage, DFS, ORA, FDA, Rockville MD 20857

The Office of Regulatory Affairs, Division of Field Science (DFS), is responsible for managing the National Check Sample Program (NCSP), which is a critical component of the ORA Quality Assurance Program. The NCSP is an interlaboratory testing scheme designed to demonstrate uniformity of results among participating laboratories. It provides a means of evaluating ORA field laboratories' analytical capabilities and quality of evidence documentation with respect to regulatory actions. In addition, the NCSP can aid in the identification and resolution of problems that the program may uncover. Proficiency testing has been identified as one of the required steps towards laboratory accreditation for all Center and ORA regulatory testing and calibration laboratories. This is the only nationally organized and managed proficiency testing program for ORA field laboratories. Each field laboratory analyzes selected samples from major program areas using established regulatory methods. The program areas include pesticides, microbiology, mycotoxins, drugs, filth, decomposition, and metals. DFS coordinates the selection and distribution of samples with the assistance of an Ad Hoc Committee composed of field representatives.

Board AH-08

C. R. Clavet¹, A. B. Margolin² and P. M. Regan^{1 1}WEAC, ORA, FDA, Winchester, MA, ²University of New Hampshire, Durham, New Hampshire

Monoclonal antibody H1206 anti-HSV-2 gG-2 bound to tosylactivated magnetic dynabeads® (Dynal®) has been used to isolate recombinant G-2 from solution as well as native gG-2 from solubilized HSV-2 infected cell extracts. The immunoaffinity purified recombinant and native protein reacted strongly to monoclonal antibody H1206 and human serum samples when immunoblotted. The immunopurifed gG-2 was unreactive to monoclonal antibody H1379 anti-HSV-1 gG-1, goat anti-serum infected with HSV-1 strain F and a human serum previously determined to have no detectable antibodies to HSV by ELISA. In addition, a number of human and rabbit HSV positive antisera were obtained from John Stewart M.D. at the Centers for Disease Control (CDC), Atlanta, GA. Immunoblotting of the rabbit and a limited number of the human HSV positive samples were in agreement with the CDC HSV serological designation. Sera characterized by Western blot and serological reactivity to the immunopurifed gG-2 could be used to determine the accuracy of clinical serological immunoassays used to determine HSV-2 seropositivity.

Third Place Poster - 2002 FDA Science Forum

Board AH-11

Baker, Karen H.* and McCullagh, Linda**, *Center for Devices and Radiological Health and **National Institutes of Health

Gloves in the health care workplace exist to fit many different uses and activities. Choosing the best glove for a specific task is often confusing. Gloves are made from many different materials, both natural and synthetic, powdered and non-powdered, sterile or non-sterile. Health care professionals need information to help select the appropriate glove barrier as protection from potential hazards. Hazards are grouped into three categories: biological, pathogens found in blood and body fluids; chemical, pharmaceuticals, therapeutic agents and disinfecting agents; and, physical, punctures, needle sticks, blades, and burns. The U.S. Food and Drug Administration regulates medical gloves as Class I medical devices, requiring premarket notification. In addition, FDA required compliance with several ASTM standards.

There are several requirements for glove labeling, including device name, country of origin, material of construction and donning powder identification and directions for use among others.

Also included is information regarding latex allergy and appropriate web site addresses for additional information.

Publish Only (AH)

J.L. Daft

The results from testing the gas chromatographic halogen specific detector (XSD) under real-analysis conditions are as follows: 1) the detector responded similar to the electrolytic-conductivity detector (ELCD) for the organochlorine compounds tested, half as much for the organobromines, and one-tenth or less as much for the organofluorines and an organoiodine; 2) for the 64 compounds tested at the specified trace conditions, its best linear range was from 0 to 125% chart-scale deflection, no autoscale; 3) it showed less co-extracted and derivatized background and matrix effects than the electron-capture detector (ECD) and ELCD; and 4) it worked well with the cyanopropylphenyl-phase columns, yielding altered retention patterns for conclusive residue confirmations. For the repeated 5-µL auto-injections of 57 compounds, 1) the detector yielded a one-system variation (CV) of 3%, and 2) it held up for weeks at a time under the repeated injections of rigorous food samples. In conclusion, the GC-XSD system determines specific foodborne organohalogens effectively.