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 diff