2004 FDA Science Forum

Poster Abstracts by Category and Board Number

CATEGORY A: ANALYTICAL CHEMISTRY: METHODS DEVELOPMENT AND APPLICATION
CATEGORY B: BIOLOGICAL ENDPOINTS
CATEGORY C: MICROBIOLOGY & VIROLOGY
CATEGORY D: PHARMACOLOGY, TOXICOLOGY, AND PHARMACOKINETICS
CATEGORY E: BIOINFORMATICS/"OMICS"
CATEGORY F: ENGINEERING AND PHYSICS
CATEGORY G: NUTRITION AND OBESITY
CATEGORY H: FOOD SAFETY INITIATIVE
CATEGORY I: COUNTERTERRORISM
CATEGORY J: REGULATORY SCIENCES
CATEGORY K: MODELING & POPULATION-BASED STUDIES
CATEGORY L: VALIDATION, TESTING, STANDARDIZATION, AND QUALITY ASSURANCE
CATEGORY M: SCIENCE COMMUNICATION/POLICY/LEVERAGING/OUTREACH

CATEGORY A: ANALYTICAL CHEMISTRY: METHODS DEVELOPMENT AND APPLICATION
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A-01

ICP-MS Method Development and Determination of Mercury in Canned Tuna
L. D. Athey, D. J. Edge, FDA, Atlanta GA

The presence of toxic elements such as mercury is a concern of the United States Food and Drug Administration, FDA. FDA labs routinely analyze for this element in food. The method currently being used involves a digestion using a mixture of nitric and sulfuric acid which convert mercury compounds to the mercuric (Hg++) form. The mercuric ions in the solution are converted to elemental mercury by reduction with stannous chloride and determined by cold vapor AAS. This study proposes the determination of mercury by ICP-MS using a microwave digestion on canned tuna products. This method for trace metals provides a rapid, specific and easily controlled assay for the analysis of heavy metals with minimal sample preparation and acid usage.

A-03

Modification of a method for isolation of teucrin A and other diterpenoids from authenticated Teucrium Chamaedrys L.
P. R. Sundaresan¹, S. Slavoff², E. Grundel¹, K. White¹, J. I. Rader¹, ¹FDA, ²JIFSAN

Neoclerodane diterpenoids are present in Teucrium species, commonly known as germander. Germander has been used in folk medicine as a stimulant, diaphoretic and a diuretic. It has been marketed in European countries as an adjuvant to weight control. Severe hepatotoxicity resulted from its use and preparations containing germander have been prohibited in France since 1992. Previous reports from this laboratory have shown that teucrin A and other diterpenoids (e.g. teucrin 5) can be isolated from the aerial parts of authenticated T. chamaedrys L. by HPLC and identified by LC/MS. For the purpose of isolation and characterization of teucrium diterpenoids on a preparative scale, we adopted a solid phase extraction (SPE) method for concentrating large volumes of teucrin A and teucrin 5 fractions obtained after semi-preparative HPLC of methanol extracts. For SPE of teucrin A, we employed a 500 mg styrene divinylbenzene tube (Phenomenex, Torrence, CA). The average recovery of teucrin A standard was 100.9 % in two separate experiments. For SPE of teucrin 5, we used Strata -X sorbent tube and the average recovery was 101.2%. The semi-preparative HPLC of the methanol extract of aerial parts of teucrium plant powder was carried out using a 250x 10 mm Synergi 4 Max-RP 80 A column (Phenomenex, Torrence, CA). The column was monitored at 220 nm with a diode array detector and fractions were collected with a Beckman SC-100 fraction collector. These procedures improved the efficiency of the isolation of in-house standards for diterpenoids.

A-05

Problems and Solutions for Quantitative Assessment of OVI's in Ipratropium Bromide by GC-Headspace R. L. Campbell, R. Needham, S. Jansen-Varnum, PDL, FDA, Philadelphia, PA

Ipratropium bromide is a bronchodilator, in the form of an inhalation aerosol, for the treatment of bronchial asthma, bronchitis, emphysema, and chronic obstructive pulmonary disease (COPD). It is also approved as a nasal spray (Atrovent®, Boehringer Ingelheim) for treatment of allergic and non-allergic rhinorrhea for children as young as six years. More recent studies indicate that Ipratropium bromide is an effective treatment for emergency situations in infants as young as six weeks. As a drug used on membranes, and administered as an aerosol, the question of organic volatile impurities in the raw material is of great importance. Most methods used to evaluate volatile impurities involve headspace GC applications. Typical methods may not be sufficiently quantitative to provide real safety data. Accuracy ranges are 80-140% with precision limits up to 15% rsd even using internal standards. These difficulties in obtaining reliable quantitative data are due to the complexities of the experiment. The representation of species in the headspace gases is strongly dependent on the effects of temperature and pressure on phase equilibria. The presence of water even in trace amounts affects the apparent volatility. Contamination on the order of 1-2% can lead to the formation of azeotropic moieties with unique volatilities. Addition of a drying agent will not disrupt the azeotrope once formed. The experimental design employs temperatures that are extremely relative to the phase equilibrium. We will show how a headspace GC method development benefits from considerations of the phase equilibrium, system and chemical control and present an improved method for Ipratropium bromide.

A-06

Determination of Phthalic Acid in the Color Additives D&C Red Nos. 21 and 22 (Eosin Y) Using High Performance Liquid Chromatography
A. Weisz, M. B. Meyers, Office of Cosmetics and Colors, CFSAN, FDA, Chantilly VA 20151

D&C Red No. 21 (R21, C.I. 45380:2, mainly 2',4',5',7'-tetrabromofluorescein), its disodium salt D&C Red No. 22 (R22, C.I. 45380, eosin Y), and their lakes are U.S. certified color additives used in drugs and cosmetics. These color additives are batch-certified by the US Food and Drug Administration (FDA) to ensure compliance with specifications described in the Code of Federal Regulations (CFR). R21 is manufactured by condensing phthalic anhydride (or acid) with two equivalents of resorcinol, partially purifying the resulting fluorescein and then brominating it. Hydrolyzing R21 with sodium hydroxide yields R22. The unreacted starting material phthalic acid (PhthAc) is specified in the CFR as "not more than 1%." The current method for the analysis of PhthAc for batch certification involves separation on a cellulose column using gravity flow elution, followed by spectrophotometric determination, which is not automated and is time consuming. Further, it may not separate PhthAc from other components present. The current work describes the development and application of an automated HPLC method for the determination of PhthAc in R21/R22 that provides better separations and quantification, and is more time efficient for routine certification analyses.

A-08

Development of Methods for Qualitative and Semi-Quantitative analyses of Protective Antigen in Final Product of Human Anthrax Vaccine
H. Wang, J. C. May, FDA

Protective antigen (PA) is considered the most important component in the human anthrax vaccine. It stimulates a protective immune response against infection with Bacillus anthracis. PA content in the vaccine varies from lot to lot due to the nature of the fermentation procedure. Thought PA structure is determined in 1977 and it is easily detected during its manufacturing in its bulk condition (using aliquots of the fermentation), the qualitative and quantitative analyses of PA in final product don't exist. The difficulty is caused by interactions between the PA and adjuvant-Alhydrogel and many impurities in the final product of the vaccine. This study is focused on investigating effective desorption solution to release PA from the adjuvant based on the proposed mechanism of interactions and developing qualitative and semi-quantitative analyses methods of PA after desorption.

A-09

Evaluation of the QuEChERS Method for Veterinary Drug Residues and Other Residues
S. E. McMullen, V. A. Vega, F. J. Schenck, SRL, ORA, FDA, Atlanta, GA

A fast and inexpensive method for the analysis of pesticide residues in fruits and vegetables, QuEChERS, was developed at a USDA-ARS Laboratory and has been evaluated extensively. The QuEChERS method results in significant reductions in solvent usage, hazardous waste and analytical time. In this study, the QuEChERS method was extended for the first time to the analysis of veterinary drugs in fish tissue and anti-oxidants in dog food. The basic procedure entails vortexing an aliquot of product with acetonitrile, and effecting a phase separation by salting out with MgSO₄ and NaCl. Cleanup of the acetonitrile extract was performed using dispersive SPE (vortexing with SPE sorbent and MgSO₄). Gas chromatography or liquid chromatography was then utilized to perform quantitative analysis of the residues.

A-10

Improved HPLC Method for The Determination of Impurities in Pyridostigmine Bromide Tablets.
L. J. Rossi, D. E. Saudarg, S. Varnum, S. N. Ali, FDA/PHI-DO/Science Branch, Philadelphia, PA 19106

"Pyridostigmine bromide (PB) is an FDA-approved drug for two civilian uses: treatment of myasthenia gravis, a neuro-muscular disorder, and for reversing the effects of some anesthetic drugs. However, PB is considered to be an investigational, not experimental, drug when used as a pre-treatment against chemical warfare agents. Evidence of the effectiveness of PB as the pre-treatment for Soman was based solely on animal studies because it is unethical to intentionally expose people to lethal nerve agents in order to test the effectiveness of a drug." (http://www.defenselink.mil/news) The current USP monograph of Pyridostigmine bromide tablets does not require testing for its impurities. However, there exists a firm's method to measure impurities in this product but this method requires the use of Ro1-5237 reference standard; one of the major known impurities of pyridostigmine bromide. Unfortunately, this is not a Pharmacopeial reference standard and is available only from its supplier in Austria. In this work we present an improved stability indicating method for the qualification of pyridostigmine bromide and impurities using only Pyridostigmine Br standard. This method has been completely validated. Data from recovery studies, robustness and ruggedness indicate that this method is superior to the current methods of analysis.

A-11

Rapid aflatoxin extraction from creamy and crunchy peanut butter.
V.A. Vega, FDA/SRL/CHEM II

A rapid extraction technique for the isolation and subsequent liquid chromatographic (LC) determination of aflatoxins B₁, B₂, G₁, and G₂ in creamy and crunchy peanut butter is described. Peanut butter samples were extracted with a methanol 15% sodium chloride (7+3) solution followed by a second extraction with methanol. The extract was subjected to a cleanup using a Vicam Aflatest^® immunoaffinity columns (IAC). Control samples for both smooth and crunchy peanut butter were fortified at four different levels for each aflatoxin B₁, B₂, G₁, and G₂ . The average aflatoxin B₁, B₂, G₁, and G₂recoveries of smooth peanut butter were 95.2, 89.9, 94.1, and 62.4% respectively, and for the crunchy peanut butter were 92.4, 84.3, 85.5, and 53.7% respectively. This extraction method and the official AOAC International method 991.31 produced comparable results for peanut butter samples (1). This method provides a rapid, specific and easily controlled assay for the analysis of aflatoxins in peanut butter with minimal solvent usage. Organic solvent consumption was decreased by 85% and hazardous waste production was decreased by 80% in comparison with the AOAC method. Along with the decrease in solvent consumption, significant saving in time was observed.

A-12

Modifications and Adaptations of a Rapid Antibody Assay for Chloramphenicol in Honey
S. E. McMullen, J. A. Lansden, F. J. Schenck, SRL, ORA, FDA, Atlanta, GA

A screening method for the presence of chloramphenicol (CAP) in honey with sensitivity to 0.3 ppb is evaluated. This screening method is a simple rapid antibody assay using [³H]chloramphenicol and a binding reagent. Analysis of different types of honey showed considerable differences. Honey can be liquid, crystallized (creamed), or partially crystallized and is classified by the USDA into seven color categories (water white, extra white, white, extra light amber, light amber, amber, and dark amber). Fortified and non-fortified liquid amber honey tested appropriately with the negative control provided with the Charm II unit. However, approximately 70% of creamed honey samples fortified at 0.6 ppb did not test positive for the presence of chloramphenicol using the provided negative control. Different matrix quenching effects were evaluated and measures were taken to account for these effects by establishing different assays for different honey types.

A-13

Validation of an in vitro Method for the Determination of Cyanide Release from Prussian Blue
Y. Yang¹, C. R. Brownell¹, N. Sadrieh², J. C. May³, A. V. Del Grosso³, R. C. Lyon¹, P. J. Faustino¹, ¹DPQR, OPS, FDA, Silver Spring, MD, ²OPS, FDA, Rockville, MD, ³LAC, OVRR, FDA, Kensington, MD

Prussian Blue (PB) was approved by the FDA in October 2003 for the treatment of radioactive metal contamination resulting from a terrorism incident. However, cyanide can be released from decomposition of PB, and therefore raises a concern for the safety of the product. The objective of this study was to measure the extent of cyanide relese over a wide pH range (from 1 to 12). Cyanide was assayed using the Spectroquant cyanide test kit (Merck). Since this photometric method requires that the measurement of solution be within pH 5.5 to 6.0, therefore samples and standards need to be adjusted to this pH range. The process of pH adjustment significantly affected the photometric measurement of cyanide. Consequently, the analysis method was optimized and validated for every pH value. The validation parameters evaluated included accuracy, precision, quantitation limit and linearity, range and specificity. The intra-day precision (CV%) ranged from 2.4 to 8.1 for the measurement solutions under pH 1, 2, 3, 5, 7, 9 and 12, respectively. The intra-day accuracy (%) was from 94 to 104 for the solutions at pH 1, 2, 3, 5, 7, 9 and 12, respectively. The linear range was 0.05 to 0.5 ppm (mg/L). The R² ranged from 0.9955 to 0.9998. The validated method was successfully implemented to determine cyanide release from PB under various pH conditions and at different time-points (1 to 48 hrs).

A-14

Methodology for particle size analysis of modified cyclosporine preparations
A. M. Wokovich, W. H. Doub, FDA/CDER/OPS/OTR/DPA

Cyclosporine is an immunosuppressive drug used in the prevention and treatment of transplant rejection. Currently there is a proposed USP monograph for particle size testing of cyclosporine modified oral solutions using a z-average size calculation. Additionally, different methodologies and reporting formats are found in literature reports of cyclosporine particle size measurement which makes it difficult to compare products. Here we report particle size measurement of four cyclosporine modified oral solutions and four cyclosporine modified capsules using a single technique, photon correlation spectroscopy (PCS). Products were found to vary in their polydispersity index. If the polydispersity index indicates that the sample is monodispersed, then z-average size is used; if the polydispersity index indicates that the sample is polydispersed, then volume distribution is used. Therefore, the z-average size calculation in the proposed USP monograph may not be the best mathematical calculation to apply to all cyclosporine modified solutions.

A-PO-15

Evaluation of a Surface Rinse Protocol for the Detection of Hepatitis A from Green Onions
D.A. Loveys, Southeast Regional Laboratory, Atlanta GA

A recent report of more than 550 illnesses and three deaths from a Hepatitis A (HAV) foodborne outbreak at a restaurant in Monaca, PA, demonstrates the substantial impact that HAV contamination has on public health. CDC and FDA investigation has since causally linked this outbreak to the consumption of uncooked or minimally heated fresh green onions present in several products purchased at a popular restaurant. We report here the results of a preliminary comparative evaluation of a surface rinse protocol capable of detecting HAV contamination by reverse-transcription PCR.

Glycine (Gly), Threonine (Threo) and Tris buffer rinse solutions containing Tween-80 were evaluated for their effectiveness in removing HAV strain HM175 from the surface of green onions inoculated with 10, 100, 1000 and 10,000 pfu/onion bunch (~120g). HAV particles were collected and concentrated by PEG precipitation and total RNA was purified using commercially available RNA isolation columns. Subsequent reverse transcription and HAV-specific primary and nested PCR were performed, essentially as described and published by the Centers for Food Safety and Nutrition, FDA.

We have found that a single primary HAV-specific PCR was sufficient to detect HAV from onions inoculated at a level of 1000 pfu using all buffer solutions tested. In contrast, we were able to detect HAV from onion samples inoculated at a lower level of 100 pfu when Threo and Tris buffers were used but detection at this level was dependent on secondary PCR amplification.

The protocol and data presented provide a simplified, rugged and feasible template for a field adaptable method suitable of detecting HAV surface contamination on high risk products. Additional studies aimed at optimizing the reverse transcription and PCR reactions are ongoing and should serve to further define a reproducible lower limit of detection and defendable performance characteristics.

A-16

Maintaining High Quality Data in Surveys of U.S. Food Products for Acrylamide
J. A. Roach, S. M. Musser, D. Andrzewjewski, M. L. Gay, D. Nortrup, OSAS, FDA, College Park

Recent data from many international food agencies demonstrate that acrylamide is produced by high temperature cooking. As a result, it is found in a number of common food products. We devised a specific, sensitive method for the quantitation of acrylamide in a wide variety of food matrices in order to survey 35 U.S. food product types for acrylamide. The analysis entails aqueous room temperature extraction, SPE cleanup and analysis by ESI LC-MS/MS. The method has been fully validated. It was used to survey more than 450 different food products. Results of this survey show significant variability in analyte levels in certain food types, suggesting that it may be possible to reduce acrylamide levels in those foods. Additionally, consumer preferences for both appearance and consistency of food produced in the home were found to cause significant variation in acrylamide levels.

A-17

Effects of consumer food preparation techniques on acrylamide formation
L. S. Jackson¹, F. Al-Taher², J. E. Jablonski¹, G. Fleischman¹, T. Bowden², ¹FDA/NCFST, ²IIT/NCFST

Acrylamide forms in high-carbohydrate foods during high temperature processes such as frying, baking, roasting and extrusion. Although acrylamide is known to form during industrial processing of food, high levels of the chemical have been found in home-cooked foods, mainly potato- and grain-based products. This poster will focus on 1) the effects of cooking conditions (e.g. time/temperature) on acrylamide formation in consumer-prepared foods, 2) the use of surface color (browning) as a indicator of acrylamide levels in some foods, and 3) methods for reducing acrylamide levels in home-prepared foods. As with commercially processed foods, acrylamide levels in home-prepared foods tend to increase with cooking time and temperature. In experiments conducted at the NCFST, we found that acrylamide levels in cooked food depended greatly on the cooking conditions and the degree of "doneness", as measured by the level of surface browning. For example, French fries fried at 180-190ºC for up to 5 min had acrylamide levels of 55 to 2130 ng/g (wet weight), with the highest levels in the most processed (highest frying times/temperatures) and the most highly browned fries. Similarly, more acrylamide was formed in "dark" toasted bread (white, wheat, rye, potato, multigrain, English muffins) slices (43.7-610.7 ng acrylamide/g wet weight), than "light" (8.27-217.5 ng acrylamide/g) or "medium" (10.9-213.7 ng acrylamide/g) toasted slices. Analysis of the surface color by colorimetry indicated that some components of surface color ("a" and "L" values) correlated highly with acrylamide levels. This indicates that color could be used as an indicator of acrylamide formation during cooking. Methods that could be used by consumers to reduce acrylamide levels include proper storage of the foods before cooking, food pretreatments (i.e. acid dips, washing treatments, etc.), recipe changes, and careful control of cooking conditions. More work is needed to develop practical methods for reducing acrylamide formation in home-prepared foods without changing the acceptability of these foods.

A-18

HPLC Enforcement Methods for the Separation and Identification of Certifiable Color Additives
B.R. Petigara, OCAC, FDA, College Park, MD

The FDA District labs need primary and confirmatory analytical methods for the enforcement of CFR listing regulations for color additives in imported and domestic food, drug, and cosmetic products. Qualitative methods were developed for the separation and identification of certifiable color additives, using high performance liquid chromatography (HPLC) and photodiode array (PDA) detection. The first method describes the separation and identification of color additives used in food products, including FD&C Yellow No. 5, FD&C Yellow No. 6, FD&C Blue No. 1, FD&C Blue No. 2, FD&C Green No. 3, FD&C Red No. 40, and FD&C Red No. 3. These color additives were separated using an Xterra RP18 column (2.1 x 150mm) and gradient elution with aqueous ammonium acetate and methanol. The second method describes the separation and identification of color additives used in drug and cosmetic products, including D&C Yellow No. 10, D&C Red No. 33, D&C Red No. 21, D&C Red No. 27, D&C Red No. 17, D&C Red No. 7, D&C Orange No. 5, D&C Orange No. 4, FD&C Yellow No. 5, FD&C Yellow No. 6 and FD&C Blue No. 1. Two formerly certifiable colors, D&C Red No. 19 and D&C Red No. 8, were also included in the study. These color additives were separated using a larger length and diameter Xterra RP18 column (4.6 x 250mm) and gradient elution with aqueous ammonium acetate and methanol. Both methods will be expanded to allow the identification of selected non-permitted color additives that have been found in adulterated food, drug, and cosmetic products during FDA District analyses.

A-19

Determination of Cholesterol in Food Products making a Declaration of Low Cholesterol
C.L. Redding, FDA, Atlanta, GA

The Food and Drug Administration regulates food products claiming no or low cholesterol on the label. Research was conducted to develop a capillary gas chromatographic method for the determination of cholesterol in food products claiming low cholesterol, less than 20 mg/serving, and cholesterol free, less than 2.0 mg/serving. The proposed method uses saponification, followed by neutralization of base, dilution with water, concentration of cholesterol on to a solid phase extract (SPE) column, fractionation to remove extraneous material and elution of cholesterol, TMS derivatization and determination by capillary GC using a flame ionization detector (FID). The SPE purified extract was derivatized with 1% Power Sil-Prep/hexane. The TMS-cholesterol was separated on a 30 m DP-5 capillary. The capillary column provided complete separation from other plant sterols. Calibration and system suitability data indicate the TMS-cholesterol formation is quantitative and stable. The correlation coefficient was r=0.999 and the limits of detection and quantitation were 2.64 and 9.64 microgram/mL, respectively. The method was validated for several food products claiming low or no cholesterol. Recovery data for added cholesterol at levels of 2 to 50 mg per serving size will be presented. Cholesterol was confirmed by GC/MS.

A-20

Development of Methods for Characterization of Hypericum perforatum L.
F.B. Williams, OFAS, CFSAN, FDA, Washington, DC

Hypericum perforatum L. is a widely used herbaceous perennial plant commonly known as St. John's Wort. This botanical has been used in the treatment of depression and as an anti-inflammatory agent. St. John's Wort is prepared and sold in multiple forms: tinctures, teas, tablets, capsules and dried plant. Because there is an enormous demand for dietary supplements, it is possible that other materials can be substituted when the authentic material is in short supply. Comparison of liquid chromatographic "fingerprints" of extracts of authentic botanicals with finished products may provide useful indications of product composition.

The analysis of dietary supplements can present a significant challenge to the analyst. Botanical extracts are often complex, necessitating the use of high resolution analytical techniques such as HPLC and LC/MS. Improved analytical methods can increase precision, accuracy of measurements, improve quality control of products (potentially) and permit verification of product claims.

A-21

Analysis of Germander (Teucrium chamaedrys L.) Diterpenoids by High Performance Thin-Layer Chromatography (HPTLC)
R. J. Michelson¹, P. R. Sundaresan², J. I. Rader², ¹JIFSAN, ²FDA

Germander (T. chamaedrys L.) has been identified as an adulterant in several skullcap (Scutellaria lateriflora L.) herbal preparations and is known to be hepatotoxic. Several important diterpenoids have been isolated and identified from this species (Teucrins1-9). No reference standards are commercially available and therefore in-house standards must be developed for these compounds. In this study, we utilized a standard TLC solvent system. This will be followed by High Performance Flash Chromatography (HPFC) and HPLC procedures in the isolation and identification of in-house standards. TLC analysis was performed on a methanol extract of the powdered aerial parts of authenticated T. chamaedrys L. Varying concentrations (10-50 µL) of the methanol extract were spotted (Linomat V sample applicator) on 10x10 cm silica gel 60 F₂₅₄HPTLC plates. Plates were developed in a solvent system of methylene chloride:methanol (19:1) for ~25 min, sprayed with Ehrlich's reagent, exposed to concentrated HCl vapor for 30 min., then heated for 10 minutes at 100°C. This procedure yielded poor band separation. However, a solvent system of 10:10:4 hexane:ethyl acetate:methanol provided good separation, indicating 8-10 well defined bands. Pre-washing the plates in methanol and drying thoroughly before spotting also improved the separations. Standards of Teucrin compounds (Teucrin 1, 5, 6, 7) were gifts from the NCNPR, University of Mississippi. Different standards were developed alongside the methanol extract of Germander and Rf values were compared. We will correlate the Rf values found in the HPTLC analysis to column volumes for the HPFC method to further improve the efficiency of preparing in-house standards.

A-24

Application of Novel Analytical Methodologies to Compliance Related Activities
B. J. Westenberger¹, D. Y. Toler¹, T. W. Moore¹, A. M. Wokovich¹, A. P. Smith¹, T. G. Lipe¹, K. D. Story¹, A. S. Fussner¹, R. E. Kolinski¹, L. K. Revelle¹, S. Jenney², C. D. Ellison³, ¹DPA/OTR/OPS, St. Louis, MO, ²DPA/OTR/OPS, White Oak, MD, ³DPQR/OTR/OPS, White Oak, MD

Because FDA/CDER is responsible for assuring the safety and efficacy of human drugs, the tremendous increase in the availability of pharmaceutical products via the internet poses a serious problem for the Agency with regard to the safety and efficacy of these unapproved products. Recently the Division of Pharmaceutical Analysis (DPA) was involved in a small survey to assess the quality of five selected drugs (Fluoxetine, Warfarin, Metformin, Levothyroxine, and Phenytoin) purchased from foreign internet sites. Four of these drugs require individualized titration of the dose and therefore could pose a life-threatening situation if the potency of the product is insufficient. Several techniques were used in assessing the quality of these products including: Near-Infrared Imaging, Near Infrared Spectroscopy, Thermal Gravimetric Analysis, drug release rate by Dissolution and stability indicating HPLC analysis. In addition, physical characteristics such as weight variation, shape, color and dimensions were recorded as well as observations regarding the packaging in which the products arrived. Results will be presented with an emphasis on how these newer techniques complement each other.

A-25

Survey of the Multi-Stable Isotopic Composition of Naproxen
J. P. Jasper¹, J. A. Spencer², B. J. Westenberger², A. M. Wokovich², L. F. Buhse², ¹Molecular Isotope Technologies, ²FDA/CDER/OPS/OTR/DPA

A survey of twenty-six samples of Naproxen from six manufacturers located in four countries was performed using multi-stable isotopic composition to study the potential of this technique in determining the isotopic provenance of Active Pharmaceutical Ingredients (APIs). Naproxen samples from Italy, India, Ireland, and the United States of America were analyzed for three isotope ratios (¹³C/¹²C, ¹⁸O/¹⁶O, and D/H) by either elemental analyzer/isotope ratio mass spectrometry [EA/IRMS: carbon (d¹³C)] or by thermal conversion-EA/IRMS [TCEA/IRMS; hydrogen (dD) and oxygen (¹⁸O)]. Stable isotopic ratios are reported in the standard d-notation, which represents parts-per-thousand (‰) variations from the isotopic ratios of international standards. Bivariate and trivariate isotope graphs show marked clustering of the data for five out of the six manufacturers. A plot of the carbon versus oxygen isotopic compositions of this sample suite shows point groupings that reveal differences in the isotopic provenance of their sources. Isotopic variation may result from differences in raw materials originating at different geographical locations ("thermodynamic fractionation"). Different synthetic pathways (e.g., precipitation vs. distillation) can also alter stable isotope ratios ("kinetic fractionation") and are country independent. Different combinations of the two fractionation processes accrue for each manufacturer's product rendering drug materials from different sources distinguishable.

A-26

Physico-Chemical Characterization of Meningococcal Group A Polysaccharide Vaccines and Chemically Activated Meningococcal Group A Capsular Polysaccharides
S. E. Norris, D. I. Freedberg, DBPAP,CBER,FDA,Bethesda MD

Two crucial factors dictate Meningococcal Group A CPS (capsular polysaccharide) vaccine immunogenicity: 1. molecular size, and 2. degree of O-acetylation. We have developed and now report physico-chemical methods to accurately measure these quantities. We use a combination of MALLS (multiple-angle laser light scattering) and ¹H NMR (nuclear magnetic resonance) to characterize Meningococcal Group A CPS. Meningococcal Group A CPSs have varying levels of O-acetylation on multiple sites of the repeat unit.¹H NMR assignments allow us to differentiate H-2 of O-Acetylated residues from H-2 of non-O-Acetylated residues. This facilitates assessment of immunogenicity. Further, when the CPS is activated by periodate oxidation, its size and O-acetylation state may change. With MALLS analysis, we are able to quantify any change in molecular size. We will present methods that can be used to characterize the size and degree of O-acetylation of activated CPSs, which are later, conjugated to immunogenic protein carriers. These techniques allow for better characterization of CPS vaccines.

A-27

Determination of 17-alpha-Methyltestosterone in Tilapia
M. Lopez, P. Chu, R. Reimshuessel, S. Serfling, C. Geiseker, FDA

The synthetic androgen, 17a-methyltestosterone (MT) is commonly used in tilapia fry for inducing sex reversal. When the proper withdrawal time is observed following the sex reversal therapy, residues are expected to be diluted through biotransformation and growth of fish. Failure to observe the proper withdrawal time, however, could result in residues being found in edible tissues. There also exists a potential for misuse as a growth promoter. Analytical methods capable of measuring sub-part-per-billion (ppb) levels are, therefore, needed to monitor for MT residues in tilapia. In this poster, we present an LC/MS/MS method for the quantitation of MT in tilapia tissues at 0.8 ppb. The sample cleanup involves two solid-phase extractions and two solvent partitions. The results of the method validation are presented.

A-28

Differentiation of Enterobacter sakasakii Strains Using Protein Expression Profiles Generated by LC/MS
T. L. Williams, S. G. Edelson-Mammel, R. L. Buchanan, S. M. Musser, FDA

Background:Enterobacter akazakii is an emerging foodborne pathogen that can cause sepsis, meningitis, or necrotizing enterocolitis in infants, particularly premature neonates. The presence of this gram-negative bacterium has been detected at low levels in powdered infant formulas and its presence in these non-sterile infant formulas has been associated with several outbreaks in neonatal intensive care units worldwide. There have been reports that E. sakazakii is more thermotolerant than many other Enterobacteriaceae although there is substantial diversity in thermal resistance among E. sakazakii strains.
Methods:We have developed a method for generating bacterial protein profiles from the LC/MS chromatogram of bacterial cell lysates. The method translates the chromatographic and multiply-charged protein information into one comprehensive mass versus intensity spectrum. After the data is converted, a number of software tools can be used to compare bacterial protein profiles and monitor changes in protein expression that may be linked to pathogenicity, or, in this case, thermal tolerance. When significant changes in protein expression profiles are identified, the proteins of interest can be purified, sequenced, and examined for alterations in primary sequence composition and/or posttranslational modifications.
Results:This work describes the LC/MS analysis of 12 strains of E. sakasakii and the identification of protein biomarkers used to differentiate strains. The protein biomarkers were sequenced to provide insight into why certain strains were more thermal tolerant than others.

A-29

Analysis of Black Cohosh and Blue Cohosh rhizomes for Triterpene Glycosides
S. Satchithanandam¹, K. D. White¹, P. Delmonte², J. I. Rader¹, ¹FDA, College Park, MD, ²Visiting Scientist, FDA, College Park, MD.

Caulophyllum thalictroides (Blue Cohosh) and Aceta racemosa (Black Cohosh) are perennial North American plants that range from Southern Ontario, Canada, to Georgia, USA. The dried roots were used by Native American women for menstrual and menopausal discomforts. Both have been actively promoted as alternatives to estrogen, since they were believed to have estrogen-like benefits without the unpleasant or harmful side effects. However, recent studies indicate that some of the components of these plants are toxic to humans. The purpose of this work is to identify some of the active components in Black Cohosh and Blue Coshosh. The rhizomes of authenticated Black Cohosh and Blue Cohosh were obtained from Botanical Liaisons, Boulder, CO. They were powdered in a blender and sieved through a # 60 sieve. Ten grams of each powder was extracted overnight with 50 mL methanol. The mixtures were centrifuged and the supernatants were analyzed by HPLC, using a J'sphere ODS-M80 column (250 x 4.6 mm), with a 0.1% aqueous formic acid-acetonitrile gradient as a mobile phase and an evaporative light scattering detector. A reference standard triterpene glycoside mixture (ChromaDex, Santa Ana, CA.) was used to establish reference conditions for HPLC. Seventeen triterpene glycoside peaks in Black Cohosh and five in Blue Cohosh were identified based on published relative retention times. We are currently in the process of collecting fractions of individual peaks using HPLC trapping for the identification of components.

A-30

Evaluation of Commercial Chemometric Software used to Determine Content Uniformity of Acetaminophen Tablets
E. H. Jefferson¹, R. C. Lyon¹, C. D. Ellison¹, J. A. Spencer², W. N. Worsley¹, A. S. Hussain³, H. Wu³, ¹DPQR, FDA, Silver Spring, MD, ²DPA, FDA, St. Louis, MO, ³OPS, FDA, Rockville, MD

Validation of chemometric software and models is an important componant of Process Analytical Technology. The objective of this study was to compare the preformance of commercial chemometric software packages for modeling and prediction of results. Six software packages (GRAMS/AI 7.00, Pirouette 3.02, PLS Toolbox 3.0, SIMCA-P 10.0, The Unscrambler 7.8, and VISION 2.51) were compared by using them to perform partial least squares analyses on the same set of near-infrared (NIR) spectroscopic results. The same model parameters were used for each software package: size of validation set (N = 140) and prediction set (N = 130), number of principal components (PC=5), NIR spectral range (1114-2300 nm), NIR spectral increment (2 nm), preprocessing (Savitzky Golay 2nd derivative, 15 point smoothing, 2nd order polynominal), and cross validation approach (full cross validation, "leave-one-out"). The data set consisted on NIR spectra from acetaminophen tablets (27 total lots) prepared by direct compression with microcrystalline cellulose at 9 different dose strengths (60-120 mg) and 3 different compressions (600, 900, 1200 kg). NIR spectra of 10 tablets per lot were collected using a FOSS NIRSystems 6500 spectrometer. The acetaminophen content value for each tablet was determined by HPLC. Based on a common set of statistical parameters (PRESS, Bias, RMSEC, RMSEP) defined by the study, the PLS models and the predicted results generated by the software packages were nearly identical.

A-33

Determination of Tetracycline Residues in Shrimp and Whole Milk by HPLC
W. C. Andersen, J. E. Roybal, S. A. Gonzales, S. B. Turnipseed, A. P. Pfenning, L. R. Kuck, FDA

Tetracyclines are broad spectrum antibiotics that have been used worldwide in both veterinary medicine and in aquaculture. Extensive use has raised concerns over increased resistivity of microorganisms to these drugs. Moreover, the presence of tetracyclines in food products of animal origin may cause allergic reactions in some individuals. Methodology is necessary to monitor food products for drug residues to protect the public health. We have recently developed two rapid quantitative methods for the simultaneous determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) in shrimp and in whole milk. These methods are particularly well-suited for routine regulatory analysis because they avoid the use of complex buffers and extraction procedures, and can be performed quickly. The methods are also compatible with mass spectrometric confirmation. Both methods rely on a simple extraction of the shrimp or milk matrix with succinic acid followed by isolation on an Oasis HLB solid phase extraction column. Isocratic liquid chromatographic analysis of the eluate with UV detection resulted in the detection of all three tetracycline residues from shrimp and milk samples fortified at 50, 100, 200, 300, and 400 ng/mL (ppb) with overall recoveries from 77 to 93 % and RSDs of less than 10 %.

A-34

Analysis of Avermectin Residues in Milk by LC-MS using An Atmospheric Pressure Chemical Ionization /Atmospheric Pressure Photoionization Source
S. B. Turnipseed, J. E. Roybal, W. C. Andersen, L. R. Kuck, FDA

The avermectins, ivermectin (IVR), doramectin (DOR), eprinomectin (EPR), and moxidectin (MOX), are anthelmintic compounds that may be administered to cattle. Different ionization techniques, including atmospheric pressure photoionization (APPI), were evaluated for the detection of avermectin residues in milk. Using APPI by itself to ionize these compounds was compared to using APPI together with APCI. It was found that the relative response of these drugs using different ionization protocols varied depending on the compound and the mobile phase. When monitoring negative ions, the use of the UV lamp increased the MS response. However, the best response for these compounds was obtained using the APCI/APPI source in the positive ion mode without any discharge current applied to the corona needle, whether the UV lamp was on or not. Using this unusual mode of ionization, an MS/MS method was established to monitor the product ion scans of the sodiated molecular ions with an ion trap instrument. A simple solid phase extraction method was used to isolate the residues from whole milk. Milk fortified with these residues (0.5-20 ng/g) and milk samples from dosed animals were analyzed using this method. EPR, DOR and IVR were confirmed in all of the extracts analyzed. MOX was confirmed in all samples fortified at 5 ng/g or higher. Acceptable recoveries (> 60%) and relative standard deviations (RSDs < 20%) were observed for the residues at the following levels: EPR and IVR (1 ng/g); DOR and MOX (5 ng/g).

A-35

Detection of Mycobacterium tuberculosis (MTB) Drug Resistant Strains using Microarrays and Allele-Specific PCR
X. Tang¹, J. J. Langone¹, S. L. Morris², L. E. Bockstahler¹, ¹FDA, White Oak, MD, ²FDA, Bethesda, MD

Point mutations in the MTB genes rRNA polymerase subunit B (rpoB), catalase-peroxidase G (katG), and ribosomal protein S12 (rpsL) are associated with the resistance of MTB strains to rifampin, isoniazid, and streptomycin, respectively. We have developed an allele specific PCR protocol combined with microarrays for rapid detection of the point mutations in these genes. In this protocol a pair of allele-specific primers for each detected position was designed to generate dye-labeled type (wild or mutant) specific DNA fragments (target). Each of the paired primers carries a DNA oligo-nucleotide tag with a distinct sequence. The tags hybridize to their complementary anti-tag probes spotted on a glass slide with a robotic microarrayer. Ten pairs of allele specific primers have been designed for detecting ten point mutations in these three MTB genes. We have applied this method to nine clinical isolates. Three of them are wild types with no mutations found in those ten positions that we are currently examining. Two of the isolates were found to have mutations in the katG gene and are isoniazid resistant. Three of the isolates had mutations in both the katG and rpoB genes and are resistant to isoniazid and rifampin. One of the isolates had mutations in all three genes and is resistant to isoniazid, rifampin and streptomycin. All of the data obtained had strong and specific signals and very low background noise. The results demonstrate that these techniques can be used to specifically detect drug resistant strains of MTB. The methodology may have broad applications for point mutation detection.

A-36

Gene Expression in Human Embryonic Stem Cell Lines: Unique Molecular Signature
B. Bhattacharya¹, T. Miura², R. Brandenberger³, J. Mejido¹, Y. Luo⁴, A. X. Yang¹, B. H. Joshi¹, I. Ginis⁵, S. R. Thies⁴, M. Amit⁶, I. Lyons⁷, B. G. Condie⁸, J. Iskovitz-Eldor⁶, M. S. Rao⁵, R. K. Puri¹, ¹Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, OCTGT, CBER, Bethesda, MD, ²c, ³Geron Corporation, 230 Constitution Drive, Menlo Park CA, ⁴3Geron Corporation, 230 Constitution Drive, Menlo Park CA 94025. 4BresaGen Inc. 111 River Bend Rd, Athens, GA 30605, ⁵Laboratory of Neuroscience, National Insititute on Aging, Baltimore, MD, ⁶Department of Obstetrics and Gynecology, The Rambam Medical Center and Faculty of Medicine, Technion, Haifa 31096, Israel, ⁷BresaGen Inc. 111 River Bend Rd, Athens, GA, ⁸Department of Genetics, University of Georgia, Athens GA

There is a huge scientific and public interest in the development of novel cellular therapies derived from human embryonic stem (huES) cell lines. These cells have the ability to differentiate into a variety of cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. However, little is known about the mechanism of differentiation of huES cells and factors regulating cell development. Understanding the molecular identity of these cells will help FDA reviewers to develop appropriate guidance for sponsors when cellular therapies derived from huES cells will be tested in the clinic. To address these issues, we have produced high quality microarrays containing 16,659 seventy base pair oligonucleotides to examine gene expression in six of the eleven approved huES cells lines. Gene expression was compared against pooled RNA from multiple tissues (universal RNA) and genes enriched in huES cells were identified. All six-cell lines expressed multiple markers of the undifferentiated state and shared significant homology in gene expression (overall similarity coefficient >0.85). A common subset of 92 genes was identified that included Nanog, GTCM-1, connexin 43 (GJA1), oct-4 and TDGF1 (cripto). Gene expression was validated by a variety of techniques including comparison with databases, RT-PCR, focused cDNA microarrays, EST enumeration and immunocytochemistry. Comparison with published "stemness" genes revealed a limited overlap suggesting little similarity with other stem cell populations. Several novel ES cell specific expressed sequence tags (ESTs) were identified and mapped to the human genome. These results represent the first detailed characterization of undifferentiated huES cells and provide a unique set of markers to profile and better understand the biology of huES cells. This information will be critical in the development of cellular therapies for a variety of conditions using differentiated cells derived from huES cell lines.

A-38

Rapid Bacterial Identification using Infrared Spectroscopy: Meeting Real World Challenges using a Dedicated Expert System
J. Dubois¹, C. Keys², E. M. Calvey³, F. S. Fry¹, ¹OSAS, FDA, College Park MD, ²OPDFB, FDA, College Park MD, ³OSC, FDA, College Park MD

Bacteria identification using spectroscopic methods has been the focus of much research during the last two decades. Examples of successful identification or discrimination systems are numerous. However, important limitations have been observed for the infrared-and Raman-based identification systems arising from the fact that the chemical composition of bacteria is quite uniform and advanced chemometrics are needed to sort through the information and extract the small differences. Calibrated identification systems have shown promising results, but impose restrictions, most important of which is the fact that an organism must be in the calibration set for it to be reliably recognized by the system. This creates the need to gather very large databases, further complicating the chemometrics needed to sort through the closely-resembling spectra.

Our approach to solve this problem is to reduce the size of the database needed for reliable identification by using an expert system that utilizes input from the user about the isolation and microbial enrichment process to target a particular subset of bacteria which may be the contaminant of interest. By eliminating a large number of organisms prior to using the infrared spectra for specific identification, a much smaller subset, or database, can be used. Results presented will demonstrate the improvement of the performance of the infrared spectrum-based identification method using this strategy. To our knowledge, it is the first time that classical microbiological data and infrared spectral information are combined in an expert system to identify bacteria.

A-39

Preparative Separation for Biologically Active Compounds by High-Speed Countercurrent Chromatography
Y. Ito¹, L. L. Ng², ¹NIH, Bethesda, MD, ²CDER, FDA, Rockville, MD

Purification of drug substances has been an art since pharmaceuticals were first sold. Currently, it ranges from simple solvent precipitation techniques to chromatographic separations. The established preparative chromatographic techniques: reversed-phase, normal-phase and chiral separations, use a solid stationary phase and a liquid mobile phase for separation. A less known chromatographic technique but one with a great potential is high-speed countercurrent chromatography (HSCCC). This technique has advantages over the established techniques including a great saving of processing time for complex mixtures.

Countercurrent chromatography was discovered by Dr. Yoichiro Ito in the 1970s. HSCCC separates using a liquid stationary phase and a liquid mobile phase resulting in highly efficient separations in a few hours. Chromatographic separations by HSCCC have been established for inorganic elements, dyes, vitamins, peptides, proteins, lipids, sugars, alkaloids, antibiotics, botanicals, etc. For this poster presentation, the theory and application of HSCCC for the purification of pharmaceutical drug substances will be discussed. Discussion will also include the pros and cons of HSCCC.

A-40

Validationof a High Performance Liquid Chromatographic (HPLC) Bioanalytical Method for Furosemide
P. J. Faustino, C. R. Brownell, E. B. Asafu-Adjaye, A. B. Ciavarella, Y. S. Yang, R. C. Lyon, DPQR, OPS, FDA, Silver Spring, MD

A simple and highly specific gradient high-performance liquid chromatographic method is reported for the determination of furosemide (a diuretic) in plasma from patients given a solid oral dosage form. The method was validated according to the FDA's Guidance for Industry, "Bioanalytical Method Validation" for clinical pharmacokinetic (PK) studies. Furosemide and internal standard (IS) naproxen were extracted from plasma by an improved liquid-liquid extraction method. The extracted analytes were dried under vacuum and re-constituted in 250 µL of mobile phase and placed in light protected vials. Chromatographic separation was achieved on a C₁₈ column 250 X 4.6 mm using an acetonitrile (ACN)-phosphate buffer (pH=2.1) gradient elution of 38-45% ACN over 20 minutes. The flow rate was 1.0 mL/min and the injection volume was 75 µL. Detection was by fluorescence (EX 270, EM 390). All validation acceptance criteria as defined in the guidance were met. Furosemide and IS were well resolved from co-eluting species. The average extraction efficiency was >85% from plasma. The lower limit of quantification (LOQ) was 25 ng/mL for plasma. The linear range was 25 to 1500 ng/mL. The r² ranged from 0.991 to 0.999. Intra-day and Inter-day precision ranged from 4 to 10% and 5.0 to 9.2% respectively. This method was utilized routinely for normal volunteers receiving therapeutic doses of furosemide to evaluate PK differences from formulations that had undergone major manufacturing changes.

A-41

Determination of 2,4,6-Tribromoaniline in the Color Additives D&C Red Nos. 21 and 22 (Eosin Y) Using Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry
A. Weisz¹, D. Andrzejewski², I. Rasooly¹, ¹Office of Cosmetics and Colors, CFSAN, FDA, Chantilly, VA 20151, ²Office of Scientific Analysis and Support, CFSAN, USFDA, College Park, MD 20740

D&C Red No. 21 (R21, C.I. 45380:2, mainly 2',4',5',7'-tetrabromofluorescein), its disodium salt D&C Red No. 22 (R22, C.I. 45380, eosin Y), and their lakes are U.S. certified color additives used in drugs and cosmetics. These color additives are batch-certified by the US Food and Drug Administration (FDA) to ensure compliance with specifications described in the Code of Federal Regulations (CFR). In a prior study in our lab, 2,4,6-tribromoaniline (TBA), a previously unknown contaminant and potential carcinogen, was found to be present in R22. In order to assess the need for limiting specifications in the CFR, information was needed on the presence of TBA in related color additives. This work describes the development of a solid-phase microextraction/GC-MS method for the determination of TBA in R21, R22 and their lakes. The method is being applied to the determination of TBA in 23 lots of these color additives from both domestic and foreign manufacturers that were certified during the past two years.

A-42

The Use of Acetic Acid to Moderate the Matrix-Induced Effects Encountered in the Gas Chromatographic (GC) Analysis of Organophosphorus Pesticides.
F. J. Schenck¹, Y. S. Johnson², ¹Southeast Regional Laboratory, ORA, FDA, Atlanta, GA, ²Minnesota Department of Agriculture, St. Paul, MN

Matrix-induced enhancement is a phenomenon commonly encountered in the gas chromatographic analysis of certain pesticides in foods. When standards are dissolved in organic solvent and injected into the GC, analyte losses and peak tailing may result due to active sites in the GC inlet and column. Significant improvements in the chromatography may result when sample matrix components are present, resulting in an enhanced response for analytes in sample matrix extracts as compared to analytes in pure organic solvent. The accepted explanation is that the matrix components mask the active sites. While preparation of standards in blank matrix extract can be used to compensate for matrix-induced effects, this approach cannot be used by FDA for enforcement purposes. We have found that the addition of minute quantities of acetic acid, as little as 1- µL/mL, to pesticide standard solutions in organic solvent can greatly reduce or eliminate matrix-induced effects. The effect of acetic acid on matrix-induced effects for over 80 organophosphorus pesticides was studied. The organophosphorus pesticide acephate, which has been widely misused on foods and is extremely susceptible to matrix effects, was studied in detail.

A-43

Variability Associated with Sampling, Sample Preparation and Analysis for the Determination of Peanuts in Energy Bars and Milk Chocolate
M. W. Trucksess¹, T. B. Whitaker², A. B. Slate³, K. M. Williams⁴, V. A. Brewer⁵, P. Whittaker⁶, J. T. Heeres⁷, ¹CFSAN/ONPLDS, FDA, College Park, MD, ²US Department of Agriculture, ARS, North Carolina State University, Raleigh, NC 27695, ³US Department of Agriculture, ARS, North Carolina State University, Raleigh, NC, 27695, ⁴CFSAN/OARSA, FDA, Laurel, MD 20708, ⁵CFSAN/OPDF, FDA, College Park, MD 20740, ⁶CFSAN/ONPLDS, FDA, College Park, MD 20740, ⁷CFSAN/FDA/University of Maryland, College Park, MD 20740

Studies were conducted to determine the recovery of an ELISA method applied to determining peanuts in energy bars and milk chocolate, and determine the sampling, subsampling and analytical variances associated with testing energy bars and milk chocolate for peanut proteins. Peanut contaminated energy bars, non-contaminated energy bars, incurred milk chocolate containing known levels of peanut and peanut free milk chocolate were analyzed. A commercially available ELISA kit was used for analysis. The sampling, sample preparation, and analytical variances associated with each step of the test procedure were determined for energy bars. The sample preparation and analytical variances were also determined for milk chocolate. Variances were found to be functions of peanut concentration. Sampling, subsampling and analytical variability associated with measuring peanuts in energy bars accounted for 53.1%, 43.5% and 3.4% of the total testing variability respectively. Subsampling and analytical variability associated with measuring peanuts in powdered milk chocolate accounted for 61% and 39% of the total testing variability respectively. For energy bars the effect of increasing sample size from one to four bars, subsample size from five to 20 g, and number of aliquots quantified from one to two on reducing the sampling, sample preparation, and analytical variances was demonstrated. For powdered milk chocolate the effect of increasing subsample size from 5 to 20 g and number of aliquots quantified from one to two on reducing sample preparation and analytical variances were demonstrated. This study serves as a template for application to other foods, and extrapolation to different sizes of samples and subsamples as well as numbers of analyses.

Clear Science Communication Award - 2004 FDA Science Forum

A-44

Hand-held microfluidics multi-channel immunosensor for detection of multiple microbial toxins simultaneously.
I. Treisel¹, M. Manion¹, N. Sergeev², B. Shapiro¹, A. Rasooly², ¹JIFSAN - University of Maryland, Silver Spring, MD, ²OST, FDA, Silver Spring, MD

Immunosensors are a specialized type of biosensor, which utilize antibodies for detection. The goal of this project is to develop a small, simple and inexpensive hand-held immunosensor for detecting multiple microbial toxins simultaneously, incorporating microfluidics and lab-on-a-chip technology. Microfluidics allows miniaturization of the device and quicker detection. The device utilizes a "sandwich antibody" approach with colorimetric detection based on peroxidase. The potential sensitivity is similar to conventional ELISA, while using smaller amounts of sample and reagents.

The base of the device is a nitrocellulose membrane which is used to immobilize the primary antibodies for capturing the antigen. A vinyl layer with the micro-channels and chambers for the different antibodies is placed on the nitrocellulose membrane, supported by plexiglass and tape layers.

To demonstrate the instrument capabilities, we used Staphylococcal Toxic Shock Syndrome Toxin (TSST-1), which causes Toxic Shock Syndrome, and staphylococcal enterotoxin B (SEB), a food poisoning agent. The device currently is capable of detecting up to ten pathogens or toxins simultaneously, although this number can be increased. Other main advantages of the device are that it does not require any external equipment or electrical power for operation and the device production is rapid and inexpensive.

The results suggest that our portable hand-held immunosensor has potential value for field clinical and food safety analyses and for food and homeland security, as well as for on-site and personal diagnostics that may improve public health.

A-45

Perfluoro chemicals: Potential migration from food packaging
T. H. Begley¹, R. Neches², R. A. Walker², ¹FDA, ²Univ. of Maryland

Recent epidemiologic studies show perfluoroctanesulfonate and perfluorooctanoic acid to be in human blood. It is thought that these chemicals may possibly be bioaccumulating and many studies are investigating possible sources of exposure. These chemicals and other perfluoro chemicals are widely used for the manufacture and processing of food packaging, which are therefore potential sources of exposure to these chemicals. Some of the uses of perfluoro chemicals in food packaging include non-stick coatings (polytetrafluoroethylene) for cookware and paper coatings for oil and moisture resistance.

There is very little information, if any, on the types of perfluoro chemicals that actually migrate from these types of coatings into food. This is because these types of chemicals are not easily measured by routine conventional analytical techniques like GC-MS or LC-UV. Today liquid chromatography-mass spectrometry (LC-MS) has become a more routine analytical technique, therefore potential migrants from perfluoro coatings can be more easily characterized. Data will be presented on the types of perfluoro chemicals that are used in food packaging. Additional data will be presented on the potential for migration of these chemicals into foods or food simulating liquids.

A-46

A receptor binding assay for the saxotoxins: Development and implementation of an alternative to the mouse bioassay for Paralytic Shellfish Poisons
S. Hall¹, G. R. Strichartz², E. Moczydlowski³, E. P. Mazzola⁴, Y. F. Lam⁵, L. Rutledge⁶, F. Van Dolah⁷, S. M. Etheridge¹, J. Deeds¹, S. M. Conrad¹, ¹WSL, DSAT, OS, CFSAN, FDA, Laurel, MD, ²Anaesthesia Research, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, ³Biology Department, Clarkson University, PO Box 5805, Potsdam, N.Y. 13699-5805, ⁴Instrumentation and Biotechnology Branch, DGSS, OSAS, OO, ⁵Department of Chemistry, University of Maryland, ⁶American Radiolabeled Chemicals, ⁷NOAA

Paralytic shellfish poisoning (PSP) is a potentially lethal human illness caused by the saxitoxins, which are produced by marine plankton and accumulate in seafood. The growth of plankton, including the species that produce the saxitoxins, is patchy and ephemeral; the occurrence of the toxins is therefore correspondingly so, and requires constant monitoring to assure consumer safety. While toxicity monitoring alone cannot provide cost-effective management, it is an inescapable requirement. Traditionally, toxicity monitoring for PSP has been conducted with the mouse bioassay. Though quite effective for rapidly detecting toxicity at the regulatory limit, the mouse assay is frequently criticized for its use of live animals and limited sensitivity. Because of the number of saxitoxin congeners, their differing potencies, and the variations in toxin composition that occur, the most practical way to design a non-animal assay is to employ the selectivity of the native receptor site on the voltage-activated sodium channel. The practicality and fundamental validity of this approach was demonstrated in 1984 by Hall and Strichartz. The primary impediment to implementation of this assay has been the availability of a suitably labeled toxin, both of the exchange-tritiated saxitoxin originally used, and of improved labeled toxins free of its limitations. A very productive collaboration between the FDA, the International Atomic Energy Agency, NOAA, and a private company (American Radiolabeled Chemicals) has succeeded in re-establishing the supply of exchange-tritiated saxitoxin. Efforts at the Washington Seafood Laboratory will now focus on developing improved labels and implementation of the assay as a regulatory tool.

A-49

Diffusion of Limonene in Polyethylene
W. Limm¹, T. H. Begley¹, T. Lickly², S. G. Hentges³, ¹FDA, College Park, MD, ²Dow, Midland, MI, ³American Plastics Council

Diffusion coefficients of limonene in various linear low-density polyethylene (LLDPE) and low-density polyethylene (LDPE) resins have been determined from sorption data using a thermogravimetric method. From these data, one can determine whether polymer synthesis parameters such as choice of catalytic process or comonomer result in substantial differences in polymer barrier properties.

For example, LLDPE is currently manufactured using either one of two distinct catalytic processes: Ziegler-Natta (ZN) and metallocene, a single site catalyst. ZN catalysis is a heterogeneous process that has proven itself over the last half-century. It involves a transition metal compound containing a metal-carbon bond that can handle repeated insertion of olefin units. In contrast, metallocene catalysis has fewer than 20 years of history, but has generated much interest due to its ability to produce highly stereospecific polymers in very high yield. In addition to high stereospecificity, metallocene-catalyzed polymers are significantly lower in polydispersity than traditional ZN counterparts.

Absorption and desorption testing of heat-pressed films made from LLDPE and LDPE resins of varying processing parameters indicates that diffusion coefficients of limonene in these resins do not change substantially enough to warrant a change in the way FDA accesses LLDPE barrier properties.

A-50

ICP-MS Method Development and Determination of Lead and Other Toxic Elements From Food
L.D. Athey, FDA, Atlanta GA

The presence of toxic elements such as lead, cadmium and arsenic are a concern of the United States Food and Drug Administration, FDA. FDA labs routinely analyze these elements in food. The methods currently being used involve conventional wet digestion and dry ashing techniques for sample preparation. This study proposes the determination of trace metals by ICP-MS using a microwave digestion on a wide variety of products. The study will focus on the applicability at the violative level (0.25 ppm of lead in food sample and various levels for other elements). The method for trace metals provides a rapid, specific and easily controlled assay for the analysis of heavy metals with minimal sample preparation and acid usage.

A-51

Materials Testing of Recalled Particle-Contaminated Blood Bags
J. C. Hutter¹, D. C. Richardson¹, M. K. McDermott¹, E. T. Chen¹, J. Crowe², F. Platek², M. Witkowski², B. Poindexter³, J. Vostal³, ¹CDRH, FDA, Rockville MD, ²FCC, FDA, Cincinnati OH, ³CBER, FDA, Bethesda MD

On January 31, 2003 it was reported that an American Red Cross (ARC) worker noticed mysterious white particles suspended in bags of blood collected at an Atlanta blood bank. An immediate inspection of the available inventory of blood at the Atlanta blood center indicated that numerous bags contained unknown particles (Halbfinger, NY Times). The ARC initiated a voluntary recall of multiple lots of a particular type of collection bag (10,000 units); and reported the observations to both the Centers for Disease Control and to the FDA's Center for Biological Evaluation and Research (CBER). Samples of suspect blood bags and control lots of blood bags were submitted to CBER, the FDA's Center for Devices and Radiological Health and the FDA's Forensic Chemistry Center for materials analysis. A variety of analytical techniques were employed in the analysis of the blood bags and the components used to manufacture the blood bags. Micro analytical techniques were used to analyze the small particles from the affected bags. No defects or degradation of the bag materials were observed in the course of analysis. The anticoagulation solutions in unused bags from affected lots were found to be consistent with the labeled contents. Isolation of the unknown particles and micro analysis found them to be consistent with fatty acids and proteins. This poster will describe the types of techniques and methods used to analyze the suspect blood bags as well as the particles isolated from them.

A-52

Confirmation of sulfamethazine, sulfathiazole, and sulfadimethoxine residues in condensed milk and soft-cheese products by liquid chromatography tandem mass spectrometry
S. B. Clark¹, S. B. Turnipseed¹, M. R. Madson¹, J. A. Hurlbut², L. R. Kuck³, J. N. Sofos⁴, ¹FDA, ²Western Washington University, Bellingham, WA, ³University of Colorado, Boulder, CO, ⁴Colorado State University, Ft. Collins, CO

A liquid chromatographic tandem mass spectrometry method is described for the simultaneous detection of three sulfonamide drug residues at 1.25 ppb in condensed milk and soft-cheese products. The three sulfonamide drugs of interest are: sulfathiazole (STZ), sulfamethazine (SMZ), and sulfadimethoxine (SDM). The method includes extraction of the product with phosphate buffer, centrifugation of the diluted product, followed by application of a portion of the extract onto a Water's Oasis HLB 3cc (60mg) extraction cartridge. Confirmation was completed on a liquid chromatography tandem mass spectrometry (LC/MS/MS) system. Validation was performed with control and fortified-control condensed bovine milk at 2.5, 5 and 10 ppb sulfonamides. In addition, this method was applied to imported flavored and unflavored condensed milk and cream cheese bars, and the presence of STZ and SMZ residues were confirmed in three out of six products.

A-53

Penicillins in Active Pharmaceutical Ingredients and Formulations of Cephalosporins
L. P. Lue¹, A. Vancura², ¹NRL, ORA, FDA, Jamaica, NY 11433, ²St. John University, Jamaica, NY 11439

Cephalosporins (CEF) are b-lactam antibiotics with cephalosporinic nucleus, produced by a fungus Cephalosporium acremonium and modified chemically.Chemical synthesis involves conversion of penicillin nucleus into a cephalosporinic nucleus. Therefore, it is very common to find the contaminant of penicillin in API's and dosage forms of cephalosporins. The penicillin contamination in cephalosporins is not acceptable in medicinal practice because some patients are allergic to penicillin. The HPLC on C18 reversed phase columns with UV or fluorescence (FD) detection has been utilized to detect penicillin in API's and dosage forms of cephalosporins. FD enhanced the detection level ten times. The low levels of penicillins in cephalosporins (ppb) after derivatization with HgCl₂ or 4-fluoro-7-nitro-benzofuran were detected by FD.

A-54

Simultaneous screening and confirmation of multi-class antibiotic residues in shrimp by a LC-MSn method
H. Li, W. Cui, P. J. Kijak, FDA

The indiscriminate use of antibiotics in shrimp aquaculture can lead to illegal drug residues in shrimp sold to US consumers. The abuse of antibiotics in shrimp production may cause health, environmental, and drug-resistance problems. As most shrimp consumed in the US is imported, the FDA needs methods to screen for illegal drug residues to protect US consumers. A multi-class, multi-residue LC-tandem-MS method has been developed to quickly screen and confirm a wide variety of antibiotic residues in shrimp Presently, 18 compounds, representing 6 classes of drugs, i.e., tetracyclines, sulfonamides, quinolones, fluoroquinolones, (leuco)dyes, and triazine-triones, are included in the screening procedure. They are either unapproved or banned by US FDA for use in shrimp aquaculture. An extraction with 5% trichloroacetic acid, followed by SPE clean-up, provides clean sample extracts for analysis. Ion-trap mass spectrometry provides highly specific and sensitive monitoring for the residues. The target concentrations for method validation are 200 ng/g for oxytetracycline (one tenth current action level), 100 ng/g for toltrazuril sulfone, 25 ng/g for sulfaquinoxaline, and 10 ng/g or lower for the other 15 drugs. Method validation was conducted using both fortified and incurred shrimp, and several commonly raised shrimp species. Combined with a quantitative method to be developed in a future project, FDA will have a wide spectrum, high throughput method to detect illegal drug residues in shrimp.

A-55

A GC-MS Study of the Addition Reaction of Aryl Amines with Acrylic Monomers
M. Farahani¹, J. M. Antonucci², ¹OGD, CDER, FDA, Rockville, MD, ²Polymer Division, NIST, Gaithersburg, MD.

The interaction of carboxylic acid groups with the amine functionalities of aryl amines, can lead to the free-radical polymerization of acrylic monomers. In this study, the Michael addition reaction of primary and secondary aryl amines with acrylic monomers such as acrylic acid (AA) was investigated. Equivalent amounts of either p-toluidine (PT) or N-phenylglycine (NPG) and AA were combined in polar solvents such as ethanol. The reactions were conducted at ambient (23° C) or near-ambient (37-60° C) temperatures. Samples (about 3-5mg) of these products were then trimethylsilylated with a solution consisting of 0.4 ml of bis (trimethylsilyltrifluoroacetamide (BSTFA) and 0.4 ml of acetonitrile by heating for 30 min at 140° C under N₂. These derivatives were characterized by gas chromatography-mass spectrometry (GC-MS). The GC-MS analyses suggest that one mol of the primary amine PT had reacted with two mol of AA to yield the expected N-p-tolyliminodipropionic acid. Similarly, the secondary amine NPG added to 1 mol of AA yielded the corresponding mixed iminodiacid, N-phenyliminoacetic-propionic acid. It would appear that the Michael reaction of primary and secondary amines with acrylic monomers might offer a general, facile synthetic route to a variety of tertiary amines. Aryl amino acids of the type synthesized in this study may find use in a number of dental applications, e.g., as surface-active adhesive agents and as polymerization initiators or activators.

A-56

The Systematical Optimization of Liquid Chromatography Tandem Mass Spectroscopy (LC-MS-MS) Analytical Methods
S. Wang, A. Thompson, Northeast Regional Laboratory, ORA, FDA, Jamaica, NY 11433

Systematic optimization of LC-MS-MS method is the process of finding the best set of operational parameters by both rational and experimental approaches. Rational approach is used 1) to determine a few most likely qualified sets of operational parameters from an infinite number of candidates, and 2) to design experiments which will produce the best set from the few predetermined sets.

A complete optimization covers all LC-MS-MS steps. Useful optimization for any given step will be achieved only if optimizations for all preceding steps are done. Thus, optimization must follow the same pathway as analytes: separation, ionization, ion-transmissions, ion-breakdown, and ion-detection.

Although the signal to noise ratio ( S/N) is more important than sensitivity, in mass spectrometry manuals discussions on tune involve total sensitivity only, but not S/N due to its complexity and difficulty.

In this poster we will illustrate 1) how to systematically optimize LC-MS-MS analysis of nitrofuran in shrimp not only for sensitivity, but also for S/N, and 2) how to approach the complete optimization step by step for analytes in mobile phase, then in method blank, and finally in shrimp sample. Using this method we were able to obtain: 1) better than 10 pg on column sensitivity for nitrofuran and 2) confirm nitrofuran in contaminated shrimp at sub-ppb level by increasing S/N or reducing/limiting interference from the matrix.

A-57

Evaluation of the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) Approach to Pesticide Residue Analysis for USDA Pesticide Data Program (PDP) Samples.
A. Brown¹, F. J. Schenck², M. Reliford¹, ¹Florida Department of Agriculture and Consumer Services, Tallahassee, FL, ²Southeast Regional Laboratory, ORA, FDA, Atlanta, GA

The USDA pesticide data program (PDP) is a federally funded program that collects data on pesticide residues in agricultural commodities for purposes of risk assessment. there is an emphasis on those commodities consumed by infants and children. low levels of detection, in many cases at part per billion levels, are required. the program allows the epa to make necessary risk assessments and regulatory decisions based on the actual exposure data. analytical services are provided by 9 state and 2 federal laboratories. typically the methods used for the analysis of pesticide residues in foods have tended to be labor intensive and entailed using large volumes of solvent that resulted in large volumes of hazardous waste. recently, a rapid and inexpensive approach to the analysis of pesticide residues in fruits and vegetables, named quechers has been reported. over 50 pear samples were analyzed for 137 pesticide residues for the pdp program. spike recoveries ranged from 72% to 120%. loqs ranged from 1 ppb to 125 ppb. using the quechers method resulted in a 65% reduction in solvent usage, when compared to the traditional multiresidue method previously used.

A-58

LC/MS Analysis of Eiconsanoid Expression in Biological Tissue: Method Validation and Application
S. A. Varnum¹, L. Rossi¹, H. Yue², M. R. Borenstein², M. F. Barbe², A. E. Barr², K. I. Strauss³, ¹FDA, ²Temple University, ³University of Cincinnati

Work-related usculoskeletal disorders (MSD) are the result of prolonged repetitive, forceful, or awkward movements. In recent studies, we have shown that performance of highly repetitive tasks is associated with an increase in serum levels of IL1a and widespread increases in activated macrophages and inflammatory cytokine in musculoskeletal tissues. Prostaglandins (PGs) are important mediators of pain and tissue inflammation. Some of Hydroxyeicosatetraenoic acids (HETEs), dihydorxyeicosatetraenoic acids (DiHETrEs) and epoxyeicosatrienoic acids (EETs) are reported to have anti-inflammatory properties. PGs, HETEs, DiHETrEs and EETs are bioactive eicosanoids. Thus, a correlation of IL1a expression with eicosanoid expression will be helpful to understand the inflammatory process, and this is enabled by a series of LC/MS analysis of over 20 eicosanoids. A selective solid phase extraction method, reverse-phase HPLC method and mass spectrometric detection under negative selected ion monitoring mode with atmospheric electrospray ionization source are completely described for all eicosanoid species. PGF2a, PGE2, PGD2, PGJ2, 14,15-DiHETrE, 11,12-DiHETrE, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 9-HETE, 8-HETE, 5-HETE, 11,12-EET, 5,6-EET, 14,15-EET, 8,9-EET, arachidonic acid are the eicosanoids of interest. Deuterium labeled 15(s)-HETE-d8, 14,15-EET-d8, 11,12-EET-d8, 8,9-EET-d8, and AA-d8 at 5, 6, 8, 9, 11, 12, 14, 15-position are used as internal standards. This method has been applied to tissue samples from several biological matrices.

A-59

A Case of Self-Induced Ion Suppression in Electrospray Liquid Chromatography Mass Spectrometry
J. C. Reepmeyer, H. Ye, DPA, CDER, FDA, St. Louis, MO

Ion suppression is a phenomenon in liquid chromatography (LC) mass spectrometry (MS) that may occur when an analyte co-elutes with a substance that hinders its ionization, and thus, reduces its signal response. A stable isotope-labeled internal standard is considered to be an ideal internal standard for LC-MS because the compound co-elutes with the analyte and experiences the same solvent effects, matrix-mediated ion suppression effects, and any transient changes in MS sensitivity. During the analysis of equine conjugated estrogens by LC negative ion atmospheric pressure electrospray ionization (ESI) MS, we observed that the signal response of estrone sulfate-d₄ (ES-d₄) and other deuterated estrogen sulfates in an internal standard solution decreased markedly when this internal standard solution was used to prepare a solution of natural source conjugated estrogens. This signal reduction persisted after sample clean up by solid phase extraction, suggesting that it was caused by something other than the sample matrix. Solutions of estrone sulfate (ES) and ES-d₄ were analyzed by LC-ESI-MS separately and at various combinations of concentrations of 0.06-60 ng/µL in aqueous organic mobile phases containing ammonium acetate buffer, acetic acid, or ammonia. Electrospray responses from mixtures of solutions of ES and ES-d₄ showed that each compound suppressed the signal of each other. A compound at low concentration in the presence of its analog at high concentrations showed the greatest degree of ion suppression, sometimes higher than 75%. ES experienced a self-induced ion suppression, which resulted in a continuous non-linear response curve over a concentration range of 1000.

A-60

Determination of Ginsenosides, Metals, Pesticides and VOCs in Herbal Remedies
R. E. Smith, R. Luchtefeld, M. M. Fleming-Jones, D. Eide, A. Abdalla, D. Foran, N. Adams, S. Nickols, S. Ryan, H. Watson, FDA

The concentrations of ginsenosides in ginseng have been determined by LC-MS. The amounts found varied considerably in different dosage forms. A liquid extract, tea bags, instant tea and capsules all contained ginsenosides a, b and c. It was found that more could be extracted from tea bags if they were steeped for a longer time than recommended on the label, and if they were used more than once. One tea bag that was labeled as a mix of green tea, licorice and ginseng, had no detectable ginsenosides. Also, bilobalide, amentoflavone and ginkgolides were determined in ginkgo. These and other herbs were analyzed for metals, pesticides and volatile organic compounds (VOCs). As much as 0.16 ppm cadmium and 0.65 ppm lead were found in one sample, using graphite furnace AA. Other metals, including sodium, potassium, phosphorus, calcium, magnesium, nickel, copper, cobalt, chromium, iron, aluminum, manganese, strontium, titanium, vanadium and zinc were determined by inductively coupled plasma (ICP) spectrometry. The pesticide biphenthrin was found in another sample using GC with electron capture detection and GC-MS. Several volatile compounds (VOCs) were found using purge and trap GC-MS. This included 1296 ppm benzene in an herbal tea, 469 ppm benzene and 789 ppm styrene in star anise, 959 ppm toluene in valerian, and sulfur dioxide in another valerian sample. Also, NMR was used to determine the complex carbohydrates that were present in different herbal remedies.

A-61

Isolation and Quantitative Analysis of Phosphopeptides in a Neurotoxicity Model using Phosphoprotein Isotope-coded Solid-phase Tags (PhIST)
D. Cawthon¹, M. B. Goshe², Z. A. Xu¹, H. M. Duhart¹, W. Slikker Jr.¹, S. F. Ali¹, ¹Div. of Neurotoxicology, NCTR/FDA, Jefferson, AR, ²Dept. of Molecular and Structural Biochemistry, N.C. State University, Raleigh NC

A primary biological mechanism of action underlying the toxicity of products regulated by the FDA involves the reversible phosphorylation of numerous cellular proteins. Our understanding of various models of neurotoxicity will be greatly enhanced by the development of high throughput techniques that allow quantitative mapping of phosphoproteins and their respective sites of phosphorylation following a toxic insult. We are currently developing a Phosphoprotein Isotope-coded Solid-phase Tag (PhIST) approach. In this approach, sites of phosphorylation are derivatized with a chemically reactive group. The solid-phase reagent captures and labels the reactive peptides with either light or heavy stable isotope-coded tags that are used to differentiate control from treated samples. The labeled peptides are released from the solid-phase support by UV photocleavage and analyzed by mass spectrometry. This technique provides important improvements over previous methods because it allows the quantification of the relative phosphorylation of a peptide between treated and control samples; facilitates the identification of the exact sites of multiple phosphorylation events, and enables labeled phosphopeptides to be recovered in high yield. A dopaminergic cell line (PC12) was treated with the neurotoxicant N-methyl-4-phenylpyridinium (MPP⁺) and the extracted phosphoproteome was mapped. Using this technique, several hundred phosphopeptides were isolated and quantified.

Outstanding Poster Award - 2004 FDA Science Forum

A-62

Solid-phase immunosensor for quantitative rapid detection of antibiotic residues in raw unprocessed milk
N. A. Anis¹, J. C. Gotthardt¹, M. S. Khalil², L. H. Stanker³, D. E. Menking⁴, J. J. Valdes⁴, J. Park⁴, ¹FDA, CVM, Rockville, MD 20855, ²University of Maryland, Baltimore, MD 21201, ³USDA,, Albany, CA 94710, ⁴U.S. Army, ABG, Aberdeen, MD 21010

Contamination of milk by drug residues represents a major public heath hazard. Current methods for residue detection are either very slow (microbial growth assay) or laborious and time consuming (e.g., HPLC, GC, or GC-MS). A simple detection system that is cost effective and user-friendly is needed for rapid screening of milk samples for drug residues. A solid-phase competitive fluoroimmunoassay using a 12 channel fully automated flow fluorometer adopted for rapid screening of ceftiofur in raw unprocessed milk was developed. The fluorescent signal was generated by the binding of fluorescein-labeled secondary antibodies (anti-mouse IgG-FITC) to a protein complex, consisting of BSA-conjugated ceftiofur and monoclonal anti-ceftiofur antibodies, immobilized on the surface of polymethyl-methacrylate (PMMA) beads. Binding of the anti-ceftiofur antibodies to the BSA-ceftiofur-coated beads was inhibited by the free ceftiofur in milk samples in a dose dependent manner. The binding of the mAb anti-ceftiofur to the BSA-ceftiofur-coated beads was specific as the mAb anti-ceftiofur was not bound to beads coated with non-specific BSA-antigen or beads coated with non-specific proteins such as casein, BSA, or IgG. The Limit of Quantitation (LOQ) of the assay was 0.3 ppb. The dynamic range of the assay was 1 ppb to 50 ppb. The assay discriminated effectively between ceftiofur which was readily detected at 5 ppb and other commonly used antibiotics which were not detected at 100 ppb. The assay provided direct, simple, rapid, and specific screening of milk samples without sample preparation that is usually needed for residue detection by instrumental analysis.

A-63

Separation of Equilin Sulfate and 8,9-Dehydroestrone Sulfate by Reversed-Phase HPLC

J. C. Reepmeyer, J. F. Brower, H. Ye, DPA, CDER, FDA, St. Louis, MO

Equilin-3-sulfate (EqS) and 8,9-dehydroestrone sulfate (DHES) are two isomers found in equine conjugated estrogens that are reported to have estrogenic activity. These geometric isomers, differing only by the position of a double bond in the steroid B-ring, were not resolved on a C-18 column during the analysis of conjugated estrogens by liquid chromatography-mass spectrometry (LC-MS) using acetonitrile-ammonium acetate buffer as the mobile phase. The purpose of this work was to evaluate various types of columns, mobile phases, and chromatographic conditions suitable for the separation of these compounds by reversed-phase chromatography using a mobile phase that is compatible with LC-MS. A conjugated estrogen possesses a nonpolar steroid ring convalently bonded to a polar sulfate or glucuronide moiety which makes it possible to use a selected amine counter ion to assist in the separation by paired ion reversed-phase chromatography. Separations were attempted using octadecyl (C-18), phenyl, diphenyl, pentafluorophenyl, phenyl-hexyl, and triacontanyl (C-30) silica-based columns, a C-18 polyvinyl alcohol column, a carbon-coated zirconia-based column (Zr-CARB), and a porous graphitic carbon column (Hypercarb).Chromatographic separation was unsuccessful using columns that depend primarily on hydrophobicity, such as octadecylsilane columns. Partial and full resolution was achieved on phenyl and diphenyl columns presumably due to a stronger p - p interaction between the phenyl ring of the stationary phase and the conjugated phenyl ring of DHES than that of the unconjugated phenyl ring of EqS. Excellent separation of these geometric isomers, better than any separation on a silica-based column, was achieved on two carbonaceous columns: Zr-CARB and Hypercarb.

A-64

Analytical Method Development for the Determination of Kava in Functional Foods
L. S. de Jager, G. A. Perfetti, G. W. Diachenko, OFAS, CFSAN, FDA, College Park, MD

The use of natural therapies and herbal dietary supplements as alternatives to western drugs is increasing in the United States. It is estimated that herbal supplement sales exceeded $4 billion in 2000. Since the passage of the Dietary Supplement Health and Education Act of 1994, marketing of supplements containing herbal ingredients has increased. Over the past few years, several food products containing botanical ingredients have appeared on the market. These "functional foods" occupy a tenuous position between dietary supplement and food. Most botanical dietary supplement ingredients have not been determined by the FDA to be Generally Recognized as Safe (GRAS) nor approved as food additives and may potentially be subject to regulatory action if GRAS self-determination is not sustainable under FDA regulations. Although methods have been developed to detect marker compounds from botanicals, little work has been done for the detection of these compounds in complex food matrices. Analytical methodology has been developed for the detection of six kava lactones, the characteristic bioactive compounds of kava (Piper methysticum), in functional food and drink products. Various sample preparation techniques were used in conjunction with LC-UV and LC-MS for the detection and quantification of these marker compounds. Food and beverage products claiming to contain kava were obtained and analyzed for the marker compounds. Concentrations of kava lactones in commercially available "functional" beverages varied greatly, ranging between 35 and 1040 µg per 240 mL serving. Recovery studies for the solid phase extraction method were completed resulting in average analyte recovery of 80%. Analysis of chocolate and drink mix products yielded between 12 and 135 mg kava lactones per serving and analysis of tea products showed between 4.7 and 11 mg per serving. The results of the food and beverage analyses will be presented.

A-65

Determination of Egg Proteins in Food Products
M. W. Trucksess¹, K. M. Williams², V. Velez-Vega³, ¹CFSAN/ONPLDS, FDA, College Park, MD, ²CFSAN/OARSA, FDA, Laurel, MD 20705, ³University of Puerto Rico

Egg is one of the five major allergens that are responsible for more than three-quarters of food allergies in children. Food allergic responses can be controlled by avoidance of the allergenic foods. The applicability of a commercial enzyme-linked immunosorbent assay kit (ELISA) for the detection of egg proteins in food products such as cookies, crackers, salad dressings and raw and cooked noodles was evaluated. A National Institute of Standards & Technology (NIST) whole dried egg powder reference material, SRM 8415, was used as a standard. A homogeneous and stable aqueous egg suspension was prepared for the evaluation of the performance of the Veratox for Egg Allergen Test (Neogen Corp., Lansing, MI). This test does not detect egg yolk proteins. Each gram of the aqueous dried egg suspension contained 643 µg whole dried egg (219 µg dried egg white), 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. When the various foods were spiked at level of 24 mg/kg of the SRM 8415 equivalent to 8.2 mg/kg egg white, recoveries for whole egg averaged about 30%. Recoveries calculated based only on egg white content was about 80%. The limit of detection averaged about 1 mg/kg. All foods containing egg as indicated on the ingredient label were found to be positive. No false positives occurred in samples that did not contain eggs. Cooked noodles contained less than 1% of the egg found in uncooked noodles. The boiling of the noodle could have reduced the immunoreactivity of the egg proteins to the antibodies used in the kit or rendered the egg proteins non-extractable.

A-68

DNA microarray for food safety analysis.
N. Sergeev^1,2, M. Distler³, S. Courtney³, S. F. Al-khaldi², D. Volokhov⁴, V. Chizhikov⁴, A. Rasooly^1,2,5, ¹FDA Center for Devices and Radiological Health, ²FDA Center for Food Safety and Applied Nutrition, ³JIFSAN-University of Maryland, ⁴FDA Center for Biologics Evaluation and Research, ⁵NIH-National Cancer Institute.

The large and diverse number of food microbial pathogens and their virulence factors has fueled interest in technologies capable of detecting multiple pathogens and multiple virulence factors simultaneously. The primary aim of this project was to develop a multipathogen oligonucleotide microarray (FDA-1) for simultaneous analysis of S. aureus enterotoxin genes, Listeria spp., Campylobacter spp. and Clostridium perfringens toxin genes using a single chip. Our two-step method require an initial PCR amplification step, followed by identification of the fluorescently labeled amplicons by hybridization to a DNA microarray. DNA microarrays are small, solid supports onto which large numbers of gene-specific DNA sequences are attached in an orderly arrangement at high density.

For FDA regulatory use, three elements were incorporated to increase confidence in this system: multiple genes for microbial identification, multiple oligonucleotide probes (oligoprobes) for each specific gene, and quality control oligoprobes to monitor array spotting and DNA hybridization. The combination of these elements enhances the reliability of detection and reduces the chance of erroneous results due to the genetic variability of microbes or technical problems with the microarray.

The method described here was used successfully for identification of S. aureus enterotoxin genes, Listeria spp., Campylobacter spp. and Clostridium perfringens. The results presented demonstrate the potential of oligonucleotide microarrays for detection of microbial pathogens relevant to food safety and biodefense.

A-69

Formulation Of Sulfamethazine Boluses With Varying Release Rates And Assessment Of The Predictive Potential Of NIR On Drug Release.
A. Tatavarti¹, R. M. Fahmy², L. X. Yu Zou¹, M. William², B. Dennis², H. Gary¹, H. Steven¹, ¹University Of Maryland, ²CVM.FDA, Rockville, MD)

Purpose To develop sulfamethazine bolus formulations, with three different release rates (slow, medium and fast) using a cornstarch based wet granulation process, and assess the potential of NIR to predict drug release. Methods A simple formulation containing sulfamethazine, cornstarch and magnesium stearate was employed. Sulfamethazine and cornstarch were weighed and mixed in a 'high shear granulator'. A 10% starch paste was slowly added to the mixer bowl while mixing at high speed. The wet mass was passed through an 8-mesh screen and dried for 1 hour at 60^°C in a convection tray dryer. The dried granulation was passed through a 10-mesh screen and appropriate amounts of extragranular starch and magnesium stearate were weighed and mixed with the granulation in a twin-shell blender; the lubricant (Mg stearate) was added last and mixed for two minutes. The tablets were compressed on a Stokes B2 rotary tablet press running at 30 rpm. Each sample was scanned in reflectance mode by NIR. The data was analyzed using the Vision^® software. Dissolution was conducted on a USP II apparatus operating at 100 rpm. Results: Drug release increased as the amount of starch paste (intragranular) increased and the amount of extragranular starch decreased,. Also, as the compression force increased, drug release increased. Thus, modulation of these three parameters lead to different release rates with a cumulative release of 30, 55 and 100% release over a two-hour period for the slow, medium and fast formulations. A PLS model with three factors was built to predict drug dissolution from the three formulations. A calibration correlation of 0.9798 with a standard error of calibration (SEC) of 5.7% and a validation correlation of 0.9736 with an SEC of 6.5% were obtained. Conclusions Sulfamethazine formulations with distinctly different release rates were successfully developed based on a simple starch based formulation. The release rates from the three formulations were predicted with good accuracy based on a PLS model.

A-70

Assessment Of NIR Spectroscopy For Process Analytical Testing Of Sulfamethazine Boluses Using Different Statistical Models.
R. M. Fahmy¹, H. WU², A. Tatavarti³, L. Y. Zou³, W. Marnane¹, D. M. Bensley¹, A. Hussian², G. Hollenbeck³, S. Hoag³, ¹CVM, FDA, Rockville, MD, ²PDS, FDA, Rockville, MD, ³School of Pharmacy, UMAB

Purpose: The goal of this study is to assess the utility of NIR spectrometry for the determination of content uniformity, tablet hardness and dissolution rate in sulfamethazine veterinary bolus dosage forms.

Methods: Eight formulations containing sulfamethazine, corn starch and magnesium stearate were manufactured and scanned. The formulations were wet granulated with a 10% (w/w) starch paste in a high shear granulator and dried 60°C in a convection tray dryer. The tablets were compressed on a Stokes B2 rotary tablet press running at 30 rpm. Each sample was scanned in reflectance mode by NIR. The data were analyzed using the Vision^® and Unscrambler^® software.

Results and Conclusions: The simple linear regression model was able to predict content uniformity with a correlation coefficient of 0.986 at 1658nm; the use of Partial Least Squares (PLS) gave similar results. However, for tablet crushing strength and dissolution simple linear regression was inadequate, but PLS calibration models successfully correlated NIR spectra with crushing strength and dissolution. This was probably due to the fact that crushing strength and dissolution are more dependent upon the tablet's physical microstructure than content uniformity which is solely dependent upon tablet composition. Principle Component Analysis (PCA) of the different formulations and the raw materials indicated that the models were highly dependent upon physical attributes.

A-73

COMFREY I: Simultaneous analysis of hepatotoxic pyrrolizidine alkaloids and N-oxides in comfrey root by LC-ion trap mass spectrometry
J.C.A. Wuilloud, S.R. Gratz, B.M. Gamble and K.A. Wolnik, U.S. Food and Drug, Administration, Forensic Chemistry Center, Cincinnati, Ohio 45237

Pyrrolizidine alkaloids (PAs) are a large group of structurally similar compounds with wide geographical and botanical distribution. The experimental study of PAs has been primarily driven by the combination of their potential toxicity and their documented presence in the popular herb, comfrey. The roots and leaves of comfrey have been traditionally used to treat a broad spectrum of diseases and ailments. In the modern era, comfrey is used as a primary ingredient in many dietary supplements. The purpose of the current study was to develop an electrospray LC-MS method for the analysis of pyrrolizidine alkaloids (PAs) in comfrey. Published data offers an extensive list of PAs and their N-oxides, however, standards are not commercially available for any of the PAs typically found in comfrey. This method allows the simultaneous analysis of pyrrolizidine alkaloids and their corresponding N-oxides, the predominant form, without the requirement of a tedious reduction step. In addition, the specific fragmentation pathways observed in MS-MS experiments permits differentiation and identification of more than twenty pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides without standards. Chromatographically, the use of a Zorbax-Aq C₁₈ column allowed mobile phase composition in excess of 97% water, which was necessary to resolve the many PA isomers present in comfrey. Those PAs that are not stereoisomers were readily resolved on the Aq column using a water-acetonitrile gradient as the mobile phase.

A-74

COMFREY II: Investigation of pyrrolizidine alkaloids and their N-oxides in commercial comfrey-containing products and botanical materials by LC-ESI-MS
J.C.A. Wuilloud, S.R. Gratz and K.A. Wolnik, U.S. Food and Drug Administration, Forensic Chemistry Center, Cincinnati, Ohio 45237

Pyrrolizidine alkaloids (PAs) and their N-oxides are a class of compounds found in several plant families, most notably comfrey. Some PAs are potentially toxic to the liver and/or lungs in humans and may cause acute liver failure, cirrhosis, pneumonitis and pulmonary hypertension. PAs may also be carcinogenic to animals, and have been linked to the development of hepatocellular and skin squamous cell carcinomas as well as liver angiosarcomas. According to some experimental studies, the quantity of PAs present in herbal teas and dietary supplements is sufficient to be carcinogenic in exposed individuals. An optimized method for the extraction and analysis of PAs and their N-oxides in botanical materials and commercial comfrey containing products has been developed using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The identification is based on MS-MS experiments conducted in our previous research (COMFREY I). Extraction conditions were optimized and the method was applied to the characterization of two different species of authenticated comfrey root and ten herbal remedies. Chromatographic profiles were generated for each commercial product and compared to the profiles generated for authentic root samples. Each product that was labeled to contain comfrey revealed measurable quantities of pyrrolizidine alkaloids. Notably, some of the chromatographic profiles of products labeled as extracts from one comfrey species, Symphytum officinale, were more consistent with the chromatographic profile of the comfrey species Symphytum uplandicum.

A-PO-01

Real-Time PCR Method Identifies Salmonella in Environmental Samples following Six Hour Enrichment
D. Bark¹, E. Analaul¹, K. Pride², J. Hu², ¹FDA, Bothell, WA, ²WSPHL, Bothell, WA

Surveillance for salmonella represents a major effort in the examination of food for the retail market by the food and drug administration (fda). the conventional fda Salmonella method requires 5 days for identification, followed by serologic testing. The rapid serologic method (Vidas) shortens identification by 2 days but requires conventional confirmation and serologic testing. The purpose of this study is to develop a rapid real-time PCR (RT-PCR) screening assay using Smart Cycler (Cephied) for detecting Salmonella spp. in foods. GyrB gene, which encodes the subunit B protein of DNA gyrase (topoisomerase type II) has gained more attention recently because of its higher level of sequence variation that allow differentiation of closely related bacterial species. This assay utilizes a set of Salmonella -specific primers and fluorogenic probe that targeted on a polymorphic region of gyrB gene. The specificity of the assay was confirmed by testing 51 Salmonella serotypes and 14 non-Salmonella enteric bacterial species or strains. Preliminary tests of this RT-PCR method in recovering Salmonella spp. directly from spiked potato mixed with powdered milk provided a sensitivity of 5x10² CFU/g. Analysis of naturally contaminated Oyster samples following 6-hour enrichment revealed positive RT-PCR results. Interestingly, the RT-PCR method failed to detect Salmonella spp. from the samples after 18 hour enrichment. Examination of the PCR inhibitory factor(s) in the 18-hour enrichment samples is in the process. In conclusion, this real-time PCR could be used as a sensitive screening method for specific detection of Salmonella spp. in environmental and retail food samples following short enrichment.

A-PO-02

Ion Chromatographic Determination of Nitrate and Nitrite in Vegetable and Fruit Baby Foods using Suppressed Conductivity Detection
S. E. McMullen, J. A. Casanova, L. K. Gross, F. J. Schenck, SRL, ORA, FDA, Atlanta, GA

An ion chromatographic method for the determination of nitrate and nitrite in vegetable and fruit baby foods was developed. The introduction of nitrate or nitrite to food may be natural or artificial as a preservative. Due to the higher pH found in baby's stomachs, nitrate can act as a reservoir for the production of nitrite. Exposure to nitrite by infants can result in methemoglobinemia (blue baby syndrome). There are also indications that carcinogenic nitrosamines can be formed from nitrites at the higher pH. In this method, nitrate and nitrite were separated on a hydroxide-selective anion exchange column using on-line electrolytically generated high-purity hydroxide eluent and detected using suppressed conductivity detection. Average recoveries of spiked nitrite residue ranged from 91-104 % and spiked nitrate residue ranged from 87-104 %. This method and the official AOAC method yield comparable results for samples containing incurred nitrate residue. This method eliminates the hazardous waste associated with the use of cadmium found in the official AOAC method.

A-PO-03

NMR Regulatory Analysis: Enantiomeric Purity Determination for Desoxyephedrine and Methamphetamine
G.M. Hanna, NRL,ORA,FDA, Jamaica NY

Direct enantiomeric purity determination methodologies for desoxyephedrine and methamphetamine were developed utilizing a 400 MHz¹H NMR spectroscopy. Efficient enantiomeric differentiation was obtained by using a diamagnetic chiral solvating agent to dissimilarly perturb the NMR spectra of the enantiomeric solutes. Nonequivalence behavior was studied in terms of all variables that affect population and intrinsic spectra of the fast diastereomeric solvates. Resolved enantiomeric resonance signals suitable for quantification were obtained by optimization of experimental conditions. Enantiomeric purity was determined on the basis of the relative intensities of the corresponding enantiomeric proton signals; assignment of enantiomer configuration was based on the relative field position. Results of the analysis of synthetic enantiomeric mixtures by the proposed methods demonstrated excellent agreements with the known values of the enantiomers present. The developed methodology represents a powerful and rapid tool for regulatory enantiomeric purity determination for (S)-(+)-methamphetamine, the widely abused, DEA schedule II, controlled substance, and (R)-(-)-desoxyephedrine, over-the-counter nasal decongestant.

A-PO-04

Total Mercury Analysis of Tuna by Microwave Assisted Digestion and Dedicated CVAAS
R. M. Jacobs, J. Hosting, M. S. Leung, ORA, Alameda , CA

The presence of mercury (Hg) in fish continues to be a public health issue. While Hg in the form of methylHg (MeHg) is the primary Hg form of concern, analyses for MeHg are expensive, time consuming and require high-level analyst expertise. Analyses for total Hg can be more rigorously controlled and are more cost effective. Since the predominate form of Hg in most fish species is MeHg, an analysis for total Hg is essentially equivalent to performing an analysis for MeHg. FDA is currently collecting information regarding the level of Hg in several species of fish. While both the digestion technique (closed-vessel microwave assisted digestion) and the determination technique, Cold Vapor Atomic Absorption Spectroscopy (CVAAS), are well established techniques for elemental determinations, they have not been previously validated and employed by FDA as the sole means for estimating the level of mercury in fish. The results shown here are those from an assignment issued by the Center for Food Safety and Nutrition. While the assignment called for 300 samples of canned tuna, only those samples (N=151) analyzed by SAN-DO Lab are discussed here.

The method utilized a MARS-5 digestion system equipped with Omnivessels (CEM Corp., NC, USA) and a CETAC model M6000 Mercury Analyzer (CETAC Corp., NE, USA). While the digestion conditions (9ml nitric acid) proved adequate for weights >1g of fish, the typical portion of tuna analyzed was ~1g. Omnivessels are designed to release combustion gases at ~350psi. The use of ~1g of fish, using a ramp to a temperature of 200⁰C after 15 min., with an additional 10 hold at 200⁰C, did not typically yield visual evidence of venting. The CETAC model M6000 Mercury Analyzer is a dedicated Hg AAS. A full range of QC parameters and materials were employed to demonstrate both precision and accuracy: method blanks (MBK), MBK spikes (MBKSP), sample replicates, sample spikes, reference materials (RM-50), independent check solution (ICV, NIST, SRM 1641d) and CCV. All samples, RMs, etc. were run in duplicate. While the M6000 allows Hg to be quantified at sub ppb levels, the instrumental conditions, sample size and sample dilution were set to be able to quantify fish containing 0.01 mg/kg.

The samples (12 x ~6oz. cans/pouches) from the same lot code were collected by 4 districts over a 3 week period. The12 subs were combined, drained, and blended into a fine paste. The analyses were conducted in 4 individual runs. In general, QC parameters fell within a range of 95-105% recovery. Sample precision for duplicate portions ranged from 0 - 12.5%, average 2.17%. The Hg for all the tuna type ranged from < 0.01 - 0.664 mg/kg, average 0.253 mg/kg. Solid white Albacore (N=67) and chunk white Albacore (N=22) had similar levels of Hg, range 0.169 - 0.664 mg/kg, average 0.359 mg/kg, and range 0.188 - 0.564 mg/kg, ave. 0.336 mg/kg, respectively. The range and ave. of Hg in solid light (N=9), chunk light (N=47), and chunk Tongol (N=6), were 0.048 -0.533 mg/kg, ave. 0.262; <0.01 - 0.344 mg/kg, ave. 0.086; and 0.051 -0.181 mg/kg, ave. 0.095, respectively.

The method was able to quantitatively convert incurred MeHg to total (inorganic) Hg. The method allows same-day-as- received analysis and has high throughput potential. Since most samples needed to be diluted 495-fold for analysis by the CETAC M6000, the LOQ could have been set much lower than 0.01 mg/kg and still retained both a high level of accuracy and precision. Prior validation studies by SAN-DO Lab, this work, and additional validation studies by the CFSAN will result in a new Elemental Analysis Manual method, EAM 4.5.

A-PO-05

Effects of cell-free hemoglobin on hypoxia inducible factor (HIF) and heme oxygenase expression in response to ischemia in flow-adapted endothelial cells
L. P. Yeh, A. I. Alayash, OBRR, FDA, Bethesda, MD

Oxidative and cell signaling events associated with the infusion of cell-free hemoglobin (Hb)-based oxygen therapeutics have been ascribed to redox reactions between Hb and biological peroxides (i.e. ₂O₂, LOOH,and ONOO^-). We investigated the interplay between Hb, oxidative and signaling mechanisms of endothelial cells grown in hypoxic (static) (PO₂ = 2 mmHg) and in a laminar-flow chamber followed by abrupt cessation (stimulated ischemia followed by oxidative burst). Co-incubation of the oxy form (HbFe²⁺) of diaspirin cross-linked Hb (DBBF-Hb), a blood substitute, with hypoxic BAECs increased the expression of HIF-1a in a manner that corresponded linearly with the decay of HbFe²⁺ and accumulation of the ferric form (HbFe³⁺).Inclusion of HbFe³⁺with hypoxic BAECs produced twice as much expression in the HIF-1a and HO-1 proteins as apposed to HbFe²⁺alone, and HbFe²⁺ plus hypoxia. In addition, higher and more persistent levels of the ferryl form (HbFe⁴⁺) (due to the consumption of endogenous peroxides) were found in the hypoxic media containing Hb. Using RT-PCR we measured the levels of HIF-1 mRNA in cells exposed to shear stress of 22 dynes/cm²for 2 hours followed by cessation for 30 min in the presence or absence of DBBF-Hb. In the presence of Hb, HIF-1 mRNA expression increased two-fold compared to that of shear alone (with Hb), or shear/cessation (w/o Hb). Moreover, under shear/cessation conditions the levels of mRNA expression were as high as that seen under hypoxic conditions. DBBF-Hb creates an oxidative milieu and modulates key cell-signaling pathways by competing with peroxides required for the de-activation of HIF-1a, which may result in alterations of important physiological mediators.

CATEGORY B: BIOLOGICAL ENDPOINTS
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B-01

Laboratory of Preclinical Studies Program Overview
J. W. Karanian, D. Wray-Cahen, A. E. Ashby, S. L. Hilbert, W. F. Pritchard, Laboratory of Preclinical Studies, OST, CDRH, FDA, Laurel, MD

The Laboratory is investigating the safety and effectiveness of a range of interventional therapeutics, including cardiovascular and minimally invasive devices and related adjunctive therapeutics and their interaction with the body in both normal and health-compromised swine models. As part of this effort, we identify, evaluate and develop more predictive preclinical models of device use and related failure modes as well as evaluate, retrospectively, the data from animal models that have been used to support market applications. Together, these models and the study of the models address the problems of identification and assessment of regulatory science issues associated with novel interventional and combination therapeutics and delivery technology. The swine models of disease include those with vasculopathy induced by diet (atherogenic high fat/high cholesterol diets), mechanical manipulation (balloon angioplasty, stenting), hormonal manipulation (castration, hormone replacement therapy, streptozotocin-induced diabetes), hemodynamic alterations (vascular ligation, fistulas) and/or immune stress. These studies contribute to the identification of critical scientific and safety issues for current and emerging technologies based on an analysis of failure modes and clinical outcomes. The results of all of these studies have a direct impact on the effectiveness and consistency of the Center's preclinical review of device applications.

B-02

Impact of gender, diet and regional hemodynamics on arterial endothelial cell gene expression profiles in a swine model of vascular disease
W. F. Pritchard¹, A. G. Passerini², C. Shi², N. M. Francesco², E. Manduchi², G. R. Grant², J. W. Karanian¹, D. Wray-Cahen¹, P. Chuan², A. E. Ashby¹, C. J. Stoeckert², P. F. Davies², ¹Laboratory of Preclinical Studies, OST, CDRH, FDA, Laurel, MD, ²University of Pennsylvania, Philadelphia, PA

Atherosclerosis preferentially develops in susceptible areas of the arterial system that are defined by the regional vascular hemodynamics. Regions of disturbed flow (DF) are susceptible to atherogenesis whereas regions of undisturbed laminar flow (UF) appear protected. Coordinated regulation of gene expression by endothelial cells in these regions results in differing regional phenotypes. Identification of the differences in gene expression in susceptible and protected areas may lead to targets for therapeutic intervention. Previous work in adult castrate male pigs revealed the coexistence of pro- and anti-atherosclerotic gene expression profiles in susceptible regions. We hypothesize that these profiles may be modified by gender or diet to impact lesion development. In this study of sexually mature intact pigs, linearly amplified RNA from freshly isolated endothelial cells of DF (inner aortic arch) and UF (descending thoracic aorta) regions of arteries in females and males on a normal diet and males on a diet high in fat and cholesterol was used to profile differential gene expression. Using human cDNA arrays, differentially expressed genes were identified and selected genes were validated by quantitative real-time polymerase chain reaction and/or immunostaining. Analysis of the gene transcription profiles revealed themes in differential expression across multiple classes of biological pathways. This study of regional endothelial cell gene expression in sexually mature intact males and females on a normal diet and males on an atherogenic diet provides new insights into the development of atherosclerosis and the role of hemodynamics in athero-susceptibility.

B-03

High Cholesterol-High Fat Diet in Swine Leads to Atherosclerotic Lesions and a Delayed Healing Response Following Coronary Interventions
J. W. Karanian¹, D. Wray-Cahen¹, A. E. Ashby¹, R. Virmani², F. Kologie², S. L. Hilbert¹, W. F. Pritchard¹, ¹Laboratory of Preclinical Studies, OST, CDRH, FDA, Laurel, MD, ²Armed Forces Institute of Pathology, Washington, DC

The predictive value of existing animal models for establishing safety and effectiveness of interventional cardiovascular devices is limited. Experimental studies are typically performed in normal healthy blood vessels of juvenile female and castrate male swine as models for diseased vessels in humans. This study was designed to (i) define the effects of a high fat-high cholesterol diet (20-24 weeks; fed juveniles through sexual maturity) on development of coronary atherosclerosis in male and female swine, and (ii) evaluate vascular healing and restenosis across gender. After 16-20 weeks on the diet, the coronary arteries underwent angioplasty, stenting or no intervention. Mean blood cholesterol was 268mg% at that time (vs 79mg% in animals receiving a normal diet). Vessels were harvested 8 weeks later for histopathologic analysis. In general, de novo fatty streaks were noted with macrophages or foam cells in vessels with and without interventions. These lesions were especially prevalent in the proximal coronary arteries and most marked in the right coronary (no intervention) as compared to the left anterior descending and left circumflex. The most advanced restenosis lesions were in the intact males as compared to the females. Healing rates were delayed post-stenting, evidenced by proteoglycan-rich/fibromuscular-poor hyperplasia and inflammation around the stent struts. The impact of gender and experimental atherosclerosis on healing and restenosis after coronary interventions will be defined histomorphometrically and histomorphologically. This interim analysis supports continued development and potential use of an in vivo animal model of atherosclerotic disease to more predictively model human disease and interventional cardiovascular device performance.

B-04

Response of Reactive Nitrogen Components of the Nitric Oxide Cascade to Angioplasty Balloon Injury in Coronary Arteries is Gender- and Hormone-Dependent in a Swine Model
D. Wray-Cahen¹, T. H. Elsasser², B. Wilkins², S. L. Hilbert¹, W. F. Pritchard¹, A. E. Ashby¹, J. W. Karanian¹, ¹FDA/CDRH/OST, Laurel MD 20708, ²USDA/ARS/ANRI/GBL, Beltsville, MD 20705

We previously demonstrated that gender and hormone status affects neointimal hyperplasia (NH) response to balloon angioplasty-induced injury (PTCA). Reaction products of vascular nitric oxide have the potent capacity to nitrate cellular proteins to form nitrotyrosine (NT); nitration impairs cell function. In this study, we determined if changes in abundance of phosphorylation-activated endothelial nitric oxide synthase (p-eNOS) or NT correlate with gender-related differences in healing response to PTCA and identified affected vascular cell types. Changes in NT and p-eNOS in response to PTCA in a coronary artery (LAD) were compared in intact male (M; n=4), intact female (F; n=4), and ovariectomized female (Fx; n=4) sexually mature swine. Thirty days after PTCA, injured and non-injured distal sections of LAD were harvested; quantitative immunohistochemistry was used to localize and measure NT and p-eNOS content. PTCA induced significant NH in M and Fx, with proliferative responses more than 5-fold greater in M and Fx than in F (P<0.05). Protein nitration was more intense in injured sections displaying significant NH compared to non-injured sections within the same vessel. In M, differences in p-eNOS staining were greatest in NH of injured sections, compared to the uninjured sections. Cells in NH and adventitia (P<0.05) in M and Fx had 95 and 70% more NT-specific pixilation, respectively, than that quantified in F. Nitration of vascular cell proteins can occur as a consequence of PTCA and is influenced by gender and hormone status. Nitration may underlie elements of stenosis (i.e., NH) and related adverse outcomes following therapeutic vascular interventions.

Runner-up Poster Award - 2004 FDA Science Forum

B-05

Local Delivery of Estrogen Inhibits Stenosis Following Angioplasty Balloon Injury in a Swine Model: A Preliminary Report on the Safety and Effectiveness of a Trans-Vascular Needle Injection Catheter
J. W. Karanian¹, R. Virmani², D. Wray-Cahen¹, A. E. Ashby¹, S. L. Hilbert¹, W. F. Pritchard¹, ¹Laboratory of Preclinical Studies, OST, CDRH, FDA, Laurel, MD, ²Armed Forces Institute of Pathology, Washington, DC

We have previously shown that coronary arteries in swine develop neointimal hyperplasia (NH) by 30 days after balloon angioplasty. This NH leads to stenosis, a failure mode associated with balloon angioplasty and stenting. In this earlier study, intact females had less NH than ovariectomized females, with a lesser degree of stenosis. However, chronic systemic estrogen replacement therapy (ERT) did not significantly reduce NH or stenosis in ovariectomized females. Recent reports analyzing the risks/benefits of ERT have focused on chronic systemic administration, not localized single dose delivery to the interventional site. The current study was designed to assess the safety and effectiveness of local peri-vascular administration of 17 a-Estradiol (100 µg in 350 µl vehicle) vs. vehicle alone to inhibit NH and stenosis after angioplasty. We have shown that an endoluminally delivered needle injection catheter reliably delivered 17 a-estradiol or vehicle alone (control) immediately following balloon angioplasty (peri-vascular drug delivery successful in 47 of 50 attempts). Angiographic evaluation demonstrated circumferential and longitudinal distribution in the peri-vascular space around the intervention site with no extravasation noted. Gross tissue analysis at explant (30 days post-intervention) showed no hemorrhage or petechia. Interim analysis showed little or no NH after balloon angioplasty with estrogen as compared to angioplasty with vehicle. The pharmacodynamic results combined with the pharmacokinetic analysis of drug levels will be presented. These preliminary data are consistent with the proposition that local drug delivery, via needle injection catheter, may provide a reliable, safe and efficacious therapy for the treatment of vascular disease and restenosis.

B-06

Measurement of mouse p53 codon 270 CGT to TGT mutant fraction as a potential biomarker for estimating the co-carcinogenicity of sunlight and FDA-regulated products
T. L. Verkler¹, L. H. Couch², B. J. Miller², P. C. Howard², B. L. Parsons¹, ¹DGRT, NCTR, FDA, Jefferson, AR, ²DBT, NCTR, FDA, Jefferson, AR

Biomarkers of skin tumor development are needed to investigate the co-carcinogenicity of sunlight and chemical exposure. The mouse p53 codon 270 CGT to TGT mutation is a dominant negative mutation detected by DNA sequencing in 38% of mouse squamous cell carcinomas (SSCs) induced by simulated solar light (SSL). To use this mutation as an early biomarker of skin tumor development, it must be possible to detect the mutation in a large excess of normal DNA. Therefore, Allele-specific Competitive Blocker-PCR (ACB-PCR), a sensitive, DNA-based method for the detection of rare point mutations, was developed for the mouse p53 codon 270 CGT to TGT mutation. The ACB-PCR assay that was developed can quantify the p53 mutation within a 10,000-fold excess of wild-type allele. To characterize the utility of the assay and characterize the prevalence of this mutation as sub-populations within skin tumors, eight SSL-induced SSCs were analyzed for p53 codon 270 CGT to TGT mutation. In three independent ACB-PCR experiments, the p53 mutant fraction in each sample was measured relative to a standard curve constructed from mutant fraction standards. The p53 mutation was detected as a sub-population in each of the eight tumor DNAs analyzed. Specifically, three SSCs had p53 mutant fractions between 10^-4 and 10^-3. Five SSCs had p53 mutant fractions between 10^-3 and 10^-2. These results demonstrate that ACB-PCR can quantify p53 codon 270 CGT to TGT mutation and this mutation is likely to be a useful biomarker for studying tumor induction and progression in response to SSL.

B-07

Nitric Oxide Detection in Cell Types Involved in Vascular Restenosis.
J. C. Shallcross¹, D. B. Lyle¹, V. M. Hitchins², J. J. Langone¹, ¹Molecular Biology Branch, OST/CDRH, FDA, Rockville MD, ²Health Sciences Branch, OST/CDRH, FDA, Rockville MD

Macrophages, neutrophils, and other cell types when activated produce cytokines that can stimulate smooth muscle cell proliferation, a major mode of restenosis. One of the toxic substances produced by activated macrophages is nitric oxide (NO), which can be used as an indicator of activation. Robust NO production by the murine macrophage cell line RAW 264.7 may serve as a means to determine the ability of polymeric materials used in resorbable stents and coatings to interfere with restenosis-suppressing eluting drugs or stimulate "rebound" restenosis. Fluorescent dyes were used to determine, down to nanomolar concentrations, NO elaboration (DAF-FM [4-amino-5-methylamino-2,7-difluorofluorescein]) or NO-produced nitrites (DAN [2,3-diaminonaphthalene] ) from RAW cells stimulated with E. coli O26:B6 lipopolysaccharide (LPS). By 24 hour exposure to LPS, a strong dose-response (into the micromolar range) of nitrite production by RAW cells was detected using DAN. A parallel response was detected using DAF-FM, indicating that the nitrite detection was due to nitric oxide production and oxidation. The degree of nitrite production by RAW cells paralleled gross morphological changes indicative of differentiation (cell photos taken at same magnification). Using the same nitrite-responsive detection system, NO production was observed in the nanomolar range from a porcine endothelial cell line (PECSV.46) exposed to acetylcholine. Using DAN, significant nitrite production could be detected from RAW cells as early as 12 hours post-exposure to 5.0 ng/ml LPS. In this model system, rapamycin (used in drug-eluting stents to inhibit restenosis) did not affect LPS-stimulated nitrite production.

B-08

An In-Bred Rat Strain (the FDA "Asthmatic Rat") for Predicting the Allergic Potential of Proteins and for Evaluating the Efficacy of Drugs and Dietary Supplements in Respiratory (Asthmatic) and Cardiovascular Studies
D. M. Hinton¹, M. Lorenzo¹, S. B. Harper¹, S. Francke-Carroll², R. K. O'Neill², S. J. Chirtel², M. S. Calvo¹, ¹OARSA, FDA, Laurel, MD, ²OSS, FDA, College Park, MD

A colony of an in-bred Sprague-Dawley rat strain has been maintained at CFSAN for testing of the allergenicity of food proteins. It was used in the 1990s in collaborative CDER/CFSAN studies for evaluating cardiovascular effects of ß-agonist drugs. Recently (2004, FDA Office of Women's Health) we proposed testing the efficacy of soy dietary supplements in ovariectomized female asthmatic rats mimicking the post-menopausal status in women. The in-bred Sprague-Dawley rat was first reported by Holme and Piechuta (Immunology, 42, 1981) for testing efficacy of anti-asthmatic drugs after challenge with egg albumin (OA). We have evaluated respiratory responses and histopathology after sensitization and challenge to various food proteins, i.e. purified ara-H2 from peanuts, glycinin and ß-conglycinin from soy, tropomyosin (TROPO) from chicken muscle, and OVA. Also, we have compared the respiratory responses of the Asthmatic rat to the normal out-bred Sprague-Dawley using a non-invasive whole body plethysmograph. Asthmatic female rats were more sensitive than males. Using a time/incremental dose protocol (one to ten g), greater responses were seen for OVA than with ara-H2, the soy proteins, and tropomyosin with female rats at the one µg dose. Tentative ranking at the low doses would be OVA>ara-H2>SOY>>TROPO. At the 10 µg dose with males, a tentative ranking would be ara-H2>OVA>SOY>>TROPO. The tropomyosin was chosen as a non-allergen protein control. No responses were seen with this protein. In conclusion, this animal model has been shown to be predictive of allergenic potential of proteins and should allow for evaluation of the allergenicity of genetically modified foods.

B-09

Role of Dlk, a stromal cell factor, in development and function of lymphocytes.
R. Raghunandan, P. S. Riggins, E. Rudikoff, S. R. Bauer, FDA

Stem cell based therapies hold great potential for development of novel therapies to repair, replace, restore and regenerate diseased or damaged cells, tissues and organs. It is important to understand pathways and molecules that regulate expansion and differentiation of stem cells to be able to use them in novel cellular therapies. Our lab uses tissue culture and animal models to investigate the role of Dlk in differentiation of stem cells. Dlk belongs to the Notch/Delta family of molecules, which are involved in the regulation of hematopoietic stem cell and T and B cell development and differentiation. Dlk knockout mice were generated to understand the in vivo and in vitro role of Dlk in B and T cell development. We observed multiple changes in B and T cell development and function in these mice by in vivo and in vitro investigations. In vivo changes in the B cells included increased numbers of immature B cells and marginal zone cells in primary lymphoid follicles of the spleen in Dlk KO mice. As a result, functional properties of B cells were altered as seen in perturbed serum IgG levels and immune response to DNP-KLH antigen. In vitro experiments indicate there was increased ability to grow without IL7 in the KO Pre-B cells. T cells had decreased response upon CD3 stimulation. These studies show how molecules in the Notch-Delta family can have profound impact on stem cell development, which manifest as perturbations in both development and function of B and T cells.

B-12

Notch signaling in organ development and regeneration
B. McCright, M. Kraman, E. Barak, DCGT There is a great deal of commercial and public interest in developing cell therapy applications that use stem cell or progenitor cell populations to treat degenerative diseases. The main obstacles impeding the development of effective cell therapies are the identification and characterization of useful progenitor cell populations. Therefore the study of signaling pathways known to regulate tissue development is highly relevant to the understanding and regulation of novel cell therapies. Notch signaling is required for the development and regeneration of almost every mammalian organ system studied thus far. In our lab we analyze the role of Notch2 signaling in the repair and development of mammalian organs using two approaches. The first approach is to study the effects of targeted mutations in Notch2 using the mouse as our model system. We have made mice that contain hypomorphic, null, conditional null, and conditionally active alleles of Notch2. Mice that are homozygous for a hypomorphic allele of Notch2 have embryonic developmental defects in the kidney, liver, and heart. Epithelial tissues such as kidney podocytes, and intrahepatic bile ducts require Notch2 for their development. By using the Cre-lox recombination system we have specifically inactivated Notch2 in specific cell types demonstrating the cell autonomous nature of the Notch2 phenotypes. The second approach we use is to genetically modify embryonic stem cells and test their ability to differentiate into specific cell types in vitro. Knowledge gained from studying the requirements for Notch signaling during tissue repair will enable the evaluation and development of new cell therapies.

B-13

Increased Apoptosis of Erythroid Cells during Anemia Recovery in Prion Protein Deficient (Prnp-/-) Mice.
J. H. Zivny¹, K. Holada², J. Simak¹, J. G. Vostal¹, ¹DH, OBRR, FDA, Bethesda MD, ²Charles University, Prague, Czech Republic

The cellular prion protein (PrPc) is involved in the pathogenesis of prion diseases but its normal function is not known. PrPc is expressed on hematopoietic cells and red cells. We investigated the role of PrPc in erythropoiesis using a mouse erythroleukemia cell line (MEL) and a mouse model of accelerated erythropoiesis induced by acute anemia. Erythroid differentiation of MEL induced prion protein gene (Prnp) mRNA expression and up-regulated cell surface PrPc from 11,700 ± 1,400 to 20,400 ± 2,700 molecules/cell. Severe anemia, induced with phenylhydrazine, increased Prnp mRNA expression in mouse bone marrow and spleen, increased erythroid cell-specific antigen (Ter119) expression in bone marrow to 214 ± 19% (p<0.01) and was associated with 274 ± 17% (p<0.01) increase of PrPc expression, compared to non-treated mice. We compared erythroid recovery in PrPc knock out mice (Prnp-/-) and control mice of the same genetic background. Induction of anemia resulted in significantly higher number of reticulocytes in wild type (WT) mice than Prnp-/- mice, 52.8 ± 1.8% vs. 38.6 ± 1.8%, respectively, without significant differences in bone marrow Ter119 expression. Before anemia apoptosis of bone marrow erythroid cells was equivalent between Prnp-/- and WT mice, as measured by annexin-V-FITC staining. With anemia induction the apoptosis of erythroid Ter119+ cells in bone marrow was significantly higher in Prnp-/- mice, 285±55 MFI, compared to WT mice, 129 ± 40 MFI (p=0.008). These data suggest that up-regulation of PrPc during erythroid differentiation may represent a protective mechanism that diverts erythroid Ter119+ cells away from apoptosis.

B-14

A New Method for Rapid Genome Analysis of Sabin Poliovirus Vaccine Strains Directly from Stool Specimens by a Combination of full-length PCR and Oligonucleotide Microarray Hybridization
M. Laassri, K. Chumakov, CBER, FDA, Rockville, MD

Recent outbreaks of poliomyelitis caused by vaccine-derived virus have raised concerns that vaccine-derived poliovirus may continue to circulate after eradication and revert to virulence. Therefore analysis of genetic stability of OPV both in cell cultures and in the organisms of vaccine recipients is a high priority. Here we report the use of full length PCR (FL-PCR) combined to oligonucleotide microarray hybridization for rapid amplification and analysis of genomes of excreted Sabin poliovirus strains in stool samples of vaccinees following administration of the trivalent oral poliovirus vaccine.

The FL-PCR assay detected approximately 50 TCID₅₀ of each poliovirus in stool samples. Although PCR inhibitors are often present in stool specimens, we found that 1: 6 RNA dilution consistently allowed us to amplify full-length cDNA directly from stool samples. For the analysis of stool specimens collected from vaccinees we developed oligonucleotide microarrays for detection and quantification the revertants in the viral 5' UTR. We found high levels of revertants in the 5' NCR of OPV after one week of OPV administration. And for mutation screening in VP1 we used the MARSH assay that we developed before to screen the emerging point mutation. We found a sample with two mutations, one in codon 35 of VP1 (C 2590àU, SeràPro), and another in codon 99 (A 2783àC, ThràLys). The latest mutation is located in the region coding for antigenic determinant of the virus, suggesting that it may have emerged under immune pressure.

In addition to an enhanced sensitivity for rapid detection and characterization of poliovirus genome, this PCR method combined with oligonucleotide microarray hybridization, permits direct characterization of virus in stool specimens without further passage in culture, preventing selection of genetic variants that may not accurately reflect the virus composition in the original specimen

Clear Science Communication Award - 2004 FDA Science Forum

B-15

Adverse events (AEs) among patients receiving autologous cultured chondrocytes
M. A. Malek, A. K. Mohan, J. Polder, M. M. Braun, T. R. Cote, FDA

Background: Carticel is an autologous chondrocyte product used to repair cartilaginous femoral condyle defects.
Methods: We reviewed patient demographics and anatomic site for AEs submitted to FDA
Manufacturers must report AEs, however, reporting by clinicians and others is voluntary; therefore, AE reporting likely underestimates event occurrences. Moreover, AEs may be causally or only coincidentally related to the product.
Results: A total of 301 AEs among patients receiving Carticel were reported to the FDA from 1996-2003. Patients had a median age of 38 (range:13-60); 62% were male. Among the 277 reports that noted the anatomic site, 95% (264) were femoral condyles, 4% (11) were other knee sites, and 1% (2) were outside the knee. More than one AE was reported for 152 (50%) patients. AEs reported included: graft failure (25%), delamination (22%), tissue hypertrophy (18%), chondromalacia (13%), adhesions (12%), loose body (9%), meniscal tears (9%), infection (7%), and other miscellaneous complications (22% in aggregate, none exceeding 4%). There were three deaths reported: two following motor vehicle accidents and one from intracranial hemorrhage.
During 8/22/97-8/22/03, Carticel was implanted in 7724 patients. During that same period, FDA received AE reports for 157 patients. Therefore, at least 2% of all Carticel treatments were followed by an AE report. Conclusions: Most AEs described structural defects in the graft or other mechanical failures. Post marketing AE reports were similar to recognized AE's that are currently included in the product label. Given underreporting and AEs that have not yet occurred, this proportion likely underestimates event occurrences.

B-16

The Detection of Gluten Using Commercial ELISA-based Assays
J. X. Nguyen¹, F. S. Thomas², S. P. Amato¹, V. A. Brewer², E. A. Garber², ¹Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, MD 20742, ²DNP, OPDF, CFSAN, US FDA, College Park, MD 20740

Cereals, besides being consumed in prepared foods directly, are also used as binders/extenders to increase water and fat content, to improve texture, taste, and cost of production. It is therefore critical for individuals allergic to gluten, or suffering from celiac disease, that all foods containing gluten be properly labeled. In 1991 the AOAC published an ELISA-based method (991.19, 32.1.24 First Action 1991, Final Action 2001) for the detection of gluten using reagents supplied by Medical Innovations Limited (Australia) and Cortecs Diagnostics (France). This method was validated for the identification of gluten in wheat flours, cookies, baking mixes, starch, soup, and cooked ground beef. The assay was validated for commodities containing 160 ppm gluten; however, the limit of detection using gluten standards was less than 5 ppm. The current program is to evaluate and validate test kit methods for the detection of food allergens in foods and the confirmation of whether or not they are allergen-free for specific allergens, such as gluten. The test kits can provide a quick and reliable method for the industry to detect the presence of the specific allergenic food that is not labeled as containing wheat for example, and can more effectively prevent these products from reaching the consumer. Therefore, in order to meet the need for an assay sufficiently sensitive to detect gluten at concentrations known to cause allergic reactions, a survey was conducted of commercially available gluten test kits and preliminary evaluations were conducted using spiked samples and commodities known to contain gluten.

B-17

Determination of Optimal Conditions for Detecting Nitric Oxide Involved in Adverse Reactions to Cardiovascular Devices.
J. C. Shallcross, D. B. Lyle, J. J. Langone, Molecular Biology Branch, OST/CDRH, FDA, Rockville MD

Restenosis of unblocked cardiac arteries involves migration and/or proliferation of smooth muscle cells, which may be inhibited by nitric oxide from regenerated endothelial cells. Cardiovascular disease, smoking, diabetes, obesity, and ageing are associated with a decrease in endothelial cell NO production. Determining the optimal conditions for measuring nitric oxide (NO) at nanomolar concentrations (where endothelial cells elaborate NO) may facilitate in vitro study of vascular injury by cardiovascular devices, in particular drug-eluting stents. We have compared two modes of detection for quantifying NO in the nanomolar range: the fluorescent dyes DAF-FM (for NO) [4-amino-5-methylamino-2,7-difluorofluorescein] and DAN (for the NO product nitrite). DAF-FM was excited at 495 nm, emission measured at 515; and DAN was excited at 375, emission measured at 415. We found that the ability of DAF-FM to detect NO generated from the nitric oxide-donor DEANO is dependent on the type of medium used, concentration of DAF-FM in the medium, and amount of fetal calf serum (FCS) present. Optimal conditions for detecting NO by DAF-FM in the nanomolar range were: PBS with no FCS present, using 0.1 uM extra-cellular DAF-FM. Optimal conditions for detection of nitrites by DAN in the nanomolar range were when conversion of nitrates to nitrites by enzymatic reaction was not done. The presence of nitrate reductase raised the background into the micromolar range. Detection can be accomplished using 96-well plates, with a simultaneous standard curve of NO generated by DEANO, producing mean and standard deviations for statistically significant determination of responses.

B-20

Genetics of the Murine Immune Response to Human Coagulation Factor IX
J. N. Lozier, N. Tayebi, OBRR, FDA, Rockville, MD
We used the mouse as a model system to test the hypothesis that the antibody response to factor IX is controlled in part by genetic factors, especially histocompatibility antigens. Six mouse strains were tested for antibodies to human factor IX following protein injections, or a single injection of an adenovirus vector that expresses human factor IX. A/J mice had the highest response and C57BL/6J mice had the lowest response to human factor IX after injection with protein or adenovirus vector. We used the adenovirus factor IX vector to immunize ten AXB and twelve BXA recombinant inbred mouse strains and observed highly significant LOD scores (>4.5) for the D17Mit62 marker, which is 1 centimorgan from the mouse MHC locus (H2). Experiments in mice with chimeric major histocompatibility complex genes indicate that class IaK and/or class II H2 genes are the key MHC H2 genes, but other gene(s) contribute to the antibody response. Markers from chromosomes 10 within 0.5 centimorgans of the interferon gamma gene show linkage (LOD scores of ~2.5-2.9) to the factor IX antibody response. This study suggests the hypothesis that histocompatibility (and other) genes may influence factor IX inhibitor development in humans with hemophilia B.

B-21
Development and Validation of a Gentamicin-Induced Subclinical Renal Injury Model in Rats.
E. F. Madden, R. P. Brown, P. L. Goering, Division of Life Sciences, FDA/CDRH White Oak, Silver Spring, MD 20903

Patients with pre-existing risk factors are prone to developing acute renal failure following exposure to certain drugs or nephrotoxicants. Recognition of this increased sensitivity has led to a proposal to use animal models of renal failure as an adjunct to standard safety assessment studies. However, there are disadvantages associated with existing renal failure models, including use of survival surgery and protracted development time. Also, animals in overt renal failure may be insensitive to low doses of nephrotoxicants. The goal of this project was to develop a model of subclinical renal injury (SRI), validate it against other renal failure models, and assess its sensitivity to a challenge nephrotoxicant. To develop the SRI model, male S-D rats were dosed daily with 250 mg/kg gentamicin (s.c.) for 3 days. BUN, blood creatinine, and urinary protein/creatinine ratio values in gentamicin-treated rats were similar to controls, but urinary NAG levels increased by 5-fold. SRI persisted for up to 7 days. The sensitivity of our SRI model was validated with a NOAEL dose of HgCl₂. Gentamicin-treated rats challenged with 0.25 mg Hg/kg (i.v.) exhibited a 2-fold increase in BUN, blood creatinine, urinary NAG and urinary protein/creatinine levels after 24 hours. However, two models of renal failure -5/6 nephrectomy and a 40 mg/kg/day x 10-day gentamicin dosing regimen- did not demonstrate increased sensitivity of the kidney to HgCl₂. In conclusion, our SRI model avoids disadvantages associated with existing renal failure models and demonstrates increased sensitivity to Hg compared to healthy animals. [Supported in part by FDA OSHC.]
B-22
Accelerated scrapie pathogenesis following intraspinal administration: analysis of abnormal prion protein distribution
O. Maximova, R. Taffs, P. Piccardo, K. Pomeroy, D. McMahon, D. Asher, Laboratory of Bacterial Parasitic and Unconventional Agents, DETTD, OBRR, CBER, FDA

The pathogenesis of transmissible spongiform encephalopathies (TSEs) is not yet completely understood despite numerous studies. TSE agents are thought to spread from site of entry to the central nervous system (CNS) by two pathways: lymphoreticular and neural. The purpose of this study was to reproduce part of the neural stage of scrapie by inoculating the agent into the mid-thoracic spinal cord and then analyzing the appearance and distribution of the abnormal prion protein (PrP^Sc) within the CNS. Hamsters were inoculated with the 263K strain of scrapie agent by either the intracerebral (IC) or intraspinal (IS) route, and patterns of PrP^Sc deposition were analyzed by immunohistochemistry (IHC). Results confirmed previous observations of faster onset of symptomatic disease following IS inoculation compared with the direct IC route. IHC analysis of PrP^Scdistribution within CNS revealed different pathogenesis following IS or IC administration. Following IC inoculation, PrP^Sc accumulated first throughout the cerebral white matter with only slight involvement of areas adjacent to the ventricles. After IS inoculation there was a different pattern of accumulation, suggesting the primary spread of scrapie agent with the flow of interstitial and cerebrospinal fluids. Dissemination of PrP^Sc by rostral glial transmission along the nerve fibers may be a secondary pathway. We describe the first IHC study of PrP^Sc distribution following IS infection and the advantages that this route of infection offers, both to investigate TSE pathogenesis and for faster assays of infectivity.

B-24
Production of IL-6, IL-1 beta, TNF-alpha, and nitric oxide by murine macrophages, murine bone cells, and human bone cells exposed to alginates of different MG ratios and different purities.
V. M. Hitchins, K. Merritt, FDA

Alginates are being studied as possible scaffolds in tissue engineered medical products and as encapsulation material for living cells. In addition, alginates are being used in wound dressings to support tissue repair and regeneration. Studies by various investigators on the effects of alginates on the immune system and inflammatory responses have shown variable results. It is generally proposed that the ratio of mannuronic acid to guluronic acid in the alginates will influence the biological response. In this study, alginates of various M/G ratios were used to stimulate murine macrophages with the production of IL-1 beta, IL-6, nitric oxide (NO) and TNF-alpha evaluated. The results of this study indicated that many of these preparations were contaminated with bacterial endotoxin (LPS) which is a potent producer of all of the cytokines studied. The level of LPS is far more important than the M/G ratio. Detailed reports on the biological response to alginates in the literature seldom define the source of the alginate and almost never state the LPS concentration. In order to compare responses in various studies comparing alginates, the LPS level must be reported. Determining the level of LPS in alginates requires special methods.
B-25
Chronic exposure to NMDA receptor and sodium channel blockers during development: long-term effects on cognitive function in monkeys and rats.
M. G. Paule¹, C. M. Fogle¹, R. R. Allen², E. C. Pearson³, T. G. Hammond⁴, E. J. Popke⁵, ¹Div Neurotox, FDA, Jefferson, AR, ²Peak Statistical Services, Evergreen, CO, ³AstraZeneca R&D Charnwood, Loughborough, Leics, UK, ⁴AstraZeneca R&D, Loughborough, Leics, UK, ⁵Wyeth Research, Chazy, NY

The effects of chronic administration of MK-801 and remacemide on the acquisition and performance of several complex behaviors were assessed in juvenile monkeys. LO and HI doses of both drugs were given daily per os for 18 months. HI doses of both agents delayed visual discrimination acquisition whereas HI doses of remacemide greatly decreased learning task acquisition, an effect which lasted months after treatment. Thus, chronic blockade of fast sodium (Na) channels by remacemide (perhaps in conjunction with NMDA receptor blockade) has long-term consequences in monkeys but MK-801 was well tolerated. To explore the relative contributions of NMDA receptor and Na channel blockade to these effects, MK-801, phenytoin (Na channel blocker) or combinations were given to rats performing tasks similar to those used in monkeys. LO and HI doses of both drugs and LO and HI drug combinations were given daily for nine months beginning at weaning. HI MK-801 and HI COMB impaired performance in multiple tasks: response rate decreases indicated decreased motivation and/or motoric abilities; acquisition was decreased for both groups for audio/visual discrimination, but only for the HI MK-801 group in a learning task. The rat data indicate that blockade of fast Na channels (phenytoin) is well tolerated but that blockade of NMDA receptors alone (MK-801) has long-lasting adverse consequences. These data contrast markedly with those obtained in the monkey demonstrating that, at least in some cases, the rat is not a good predictor of drug effects in primates. (Supported by AstraZeneca and the National Center for Toxicological Research).
B-26
Multicolor flow cytometry (MCFCM) analysis of prefursor states in chronic lymphocytic leukemia (CLL): B Cell Monoclonal Lymphocytosis (BCML).
T. Schleinitz¹, W. Telford², S. Perfetto³, N. Caporaso², M. Stetler-Stevenson², F. Abbasi¹, V. Zenger¹, G. Marti¹, ¹Rockville, MD, ²NCI, ³NIAID

Seven-color flow cytometry was used to investigate normal donor (ND), familial CLL donors and unaffected first degree relatives (FDR) using CD45, CD14, CD3, CD4, CD8, CD16+CD56, CD19, CD20, CD5, CD23, kappa, lambda, IgD, IgM, CD79b, CD27, CD38, and CD69 antibodies. Conventional lymphocyte subsets (T, B, NK, CD4 and CD8 T cells) were easily identified and served as internal positive controls. On the basis of CD20 mean fluorescence intensity, we were able to define six B cell subsets in ND whole blood (N=7): two dim CD20 and four CD20 bright subpopulations. CLL intraclonal heterogeneity was not apparent even though pattern variation between patients (N=5) clearly suggested immunophenotypic heterogeneity. MCFCM also permitted the analysis of normal remaining B cells in CLL (RNBC). These remaining remnant polyclonal B cells were enriched for CD27+, IgD-IgM-, IgDdimIgM+ and IgD-IgM+ cells. MCFCM study was performed in seven FDR from four CLL families. The existence and prevalence of a CD5+ and CD5- BCML was confirmed as found in a previous study (Clinical Cytometry 2003;52B:1-12) suggesting that BCML may be a precursor state chronic B lymphoproliferative disease. As predicted, several patterns were noted. Polyclonal remnant B cells were identified. In one pattern, the BCML was identical to CLL: CD20dim, CD5+, CD23+, lambda dim, IgD+/dim, IgM dim, CD27 dim, CD38-, CD69+/dim. In a second pattern, two distinct subsets could be identified within the clone. A third pattern consisted of CD19, CD20 IgM-IgD+ lambda light chain restricted B cells.
B-28
Evaluation of Commercial Immunology-based Diagnostic Assays for the Detection of Egg in Food
E. A. Garber¹, V. A. Brewer¹, S. P. Amato², J. X. Nguyen², ¹DNP, OPDF, CFSAN, US FDA, College Park, MD 20740, ²Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, MD 20742

It has been estimated that 1.5 % of adults and 6% of children are allergic to food. Of these an estimated 125 die each year due to severe reactions. In order to address the need for validated methods that can be used to test food products for the presence of allergens, the Food and Drug Administration has initiated studies to evaluate commercial immunology based diagnostic assays for the detection of food allergens. Four commercial ELISA-based diagnostic assays for the detection of egg proteins were evaluated with six different matrices representative of the various forms of processing routinely employed in the preparation of egg containing products. The matrices examined included bread with the egg applied as a surface glaze and baked for 10 min at 178 ºC, muffins with the egg in the batter and baked for 10 min at 218 ºC, pasta with the egg absorbed into the pasta and analyzed before and after boiling (uncooked and cooked) for 10 min, vanilla ice cream with the egg mixed into the ice cream and subjected to 5 min at 80 ºC, salad dressing with the egg mixed in with no further processing, and dissolved in PBS buffer. Six levels of egg were spiked into the various matrices ranging from 0 through 100 mg NIST whole egg standard (SRM 8415) per gram commodity. Cooking had a significant effect on the sensitivity of the various assays. A survey of commodities labeled as containing egg products also showed a reduced sensitivity towards baked goods.
B-29
Performance Tested Method Validation Study of ELISA-based Diagnostic Assays for the Detection of Peanuts in Food
D. L. Park¹, S. Coates², V. A. Brewer¹, E. A. Garber¹, M. Abouzied³, K. Johnson⁴, B. Ritter⁵, D. McKenzie², ¹DNP, OPDF, CFSAN, US FDA, College Park, MD 20740, ²AOAC Research Institute, Gaithersburg, MD 20877, ³Neogen Corporation, Lansing, MI 48912, ⁴R-Biopharm, Inc., Marshall, MI 49068, ⁵ELISA Technologies, Inc., Gainesville, FL 32609

Performance Tested Method^SM validations for the detection of peanut protein in four different food matrices were conducted under the auspices of the AOAC Research Institute. In this blind study, commercially available immuno-diagnostic kits were validated. The three commercially available kits evaluated were Neogen Veratox for Peanut, R-Biopharm RIDASCREEN® FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrices employed were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut.

Analysis of the samples were conducted by laboratories representing industry, international and U.S. governmental agencies. All three commercial kits successfully detected all spiked samples. Inasmuch as a limited number of samples were evaluated by only three laboratories, the maximum confidence level for each kit is 80%. Hence, the combination of two of the test kits yields a confidence level of 95%. It is therefore recommended that all field samples be analyzed with at least two of the validated kits.
B-30
Evaluation of a Commercial ELISA-based Milk Allergen Quantitative Test Kit
S. P. Amato¹, E. A. Garber², ¹Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, MD 20742, ²DNP, OPDF, CFSAN, US FDA, College Park, MD 20740

The American Academy of Allergy, Asthma, and Immunology estimates over 40 million Americans suffer annually from allergies. Several immunology-based detection kits have become commercially available for the detection of food allergens. The Veratox Quantitative Milk Allergen Test Kit manufactured by the Neogen Corporation (Lansing, MI) was evaluated for the detection of milk in four different food commodities-margarine, lemon sorbet, orange juice, and infant formula. Samples from all four foods were spiked with milk at concentrations of 0.0, 2.5, 10.0 and 25ppm. At all concentrations containing milk, the test kit was able to detect the milk. The test kit was also able to quantify the amount of milk protein at 10.0 and 25ppm. A large variance in the response of the assay was observed with samples spiked at 2.5ppm Incubation of spiked food samples up to 72 hours at 4°C had no effect on the detection of milk protein. Lemon sorbet had an average fractional recovery of 0.5 and showed the lowest recovery of the four foods. Both margarine and orange juice had fractional recoveries of 1. Infant formula had fractional recoveries of 0.9.
B-34
Development of recombinant Brucella abortus strain RB51 as a carrier for HIV-1 pol protein expression vector: an approach for development of a vaccine for AIDS
Y. Ophir¹, A.T.M. S. Hoque¹, S. Khurana¹, W. Wang¹, A. C. Brown¹, R.Vemulapalli², G. G. Schurig³, B. K. Felber⁴, H. Golding¹, Basil Golding¹. ¹ US FDA, CBER, Bethesda, MD 20892, ²Purdue University, West Lafayette, IN 47907, ³Virginia Tech, Blacksburg, VA 24061, ⁴NCI, NIH, Fredrick, MD 21701

Previously, we have shown that Brucella abortus (BA) can act as an efficient carrier and adjuvant for HIV peptides or proteins to induce CTL and antibody responses. Based on these finding we wished to express Human immunodeficiency virus (HIV) genes in BA. HIV gene expression is tightly regulated at several levels, which is an obstacle for vaccine development. One of other obstacles to an effective plasmid DNA based vaccine is the low level of expression of HIV genes in mammalian cells due to the instability of HIV mRNAs. To overcome this obstacle, bacteria expressing high levels of HIV protein may be useful as a vaccine candidate for AIDS. Here, we report use of BA strain RB51 as a carrier of HIV-1 pol genes to maximize HIV-1 pol protein expression in the bacterial strain. We have constructed plasmid expression vector encoding HIV-1 pol sequences under the control of Brucella SOD promoter sequence. The construction of the expression vector was confirmed by PCR and sequencing. Thereafter, the expression plasmid DNA was transformed into the BA RB51 strain by electroporation. The bacterial cells carrying the expression plasmid were grown in TSB supplemented with antibiotic to OD₆₀₀ 1-1.5 by shaking at 37°C. The whole cell lysate was prepared and analyzed for expression of pol proteins. The protein was separated by SDS-PAGE and electroblotted onto PVDF membranes, and pol protein was detected using pol specific polyclonal antibodies. Our experiment results demonstrated that use of BA might be valuable tool as a carrier of HIV-1 protein for vaccination against HIV.
B-35
Evaluation of Expression and Toxicity of Hydrodynamic Gene Transfer in Mice
A.T.M. S. Hoque¹, R. H. Smith², B. Golding¹, ¹DH, CBER, US FDA, Bethesda, MD 20892; ²NHLBI, NIH, Bethesda, MD 20892

We are considering use of non-viral plasmid DNA gene transfer technology for gene expression in vitro and in vivo. We have constructed an expression vector, encoding the luciferase reporter gene, using adeno-associated virus serotype 2 backbone including internal terminal repeats and p5 promoter. First, we evaluated the ability of several transfectants i.e saline, Hanks' Balanced Salt solution (HBSS), Lipofectamine 2000, Polyethylenimine (PEI; 9:1) in 20 mM HEPES, PEI (9:1), PEI (6:1) or PEI (9:1) and Lipofectamine 2000 in 5% glucose solution for the delivery of DNA vector into 293 cells in vitro, and measured luciferase protein expression. Our results show that the transfection complexes containing Lipofectamine 2000-DNA, and PEI (9:1)-Lipofectamine 2000-DNA in 5% glucose results in increased luciferase expression in the cells compared to PEI (6:1 or 9:1)-DNA, saline-DNA (naked), or HEPES- DNA complexes. Secondly, we evaluated the ability of DNA-complexed with above transfectants to transfect liver cells in vivo after hydrodynamic tail vein injection in mice. Our in vivo results show that the naked-DNA (in 5% glucose solution) gene transfer resulted in higher luciferase protein expression in the liver until 15 days compared to DNA-complexed with other transfectants. Thirdly, we evaluated the toxicity after the gene transfer. Our results show that PEI-DNA and PEI-Lipofectamine 2000-DNA complexes produce higher systemic and liver toxicity in mice compared to naked-DNA or Lipofectamine-DNA complexes. The toxicity and liver necrosis are severe until day 3 of post-injection. However, mice recover by day 15. We conclude that the hydrodynamic injection with naked-DNA is preferred as a liver transfectant in terms of expression and safety compared to DNA-complexed with other delivery systems.
B-PO-01
UVA1-Mediated Receptor and Cytokine Changes of Transformed Lymphocytes
D. E. Godar, A. D. Lucas, OST, FDA, WhiteOak, MD

UVA1 radiation (340-400 nm) causes singlet-oxygen damage that depolarizes mitochondrial membranes triggering immediate apoptosis (T<10 min), while it also causes oxidative damage to DNA inducing delayed apoptosis (T>24 h). In this study, we examined some potential therapeutic endpoints associated with UVA1-mediated immediate and delayed apoptosis, such as receptor and cytokine changes. We quantified the number of membrane-bound CD3 receptors on transformed T lymphocytes (Jurkat) and the number of membrane-bound CD19 receptors on transformed B lymphocytes (Daudi) using flow cytometry. We also quantified the release of the cytokines IFN-g and IL-2 using enzyme-linked immunosorbent assays. Out of the entire population of cells, only the apoptotic Daudi cells immediately decreased CD19 expression via capping, while only the apoptotic Jurkat cells increased CD3 receptor expression 24 h after exposure. Both receptor changes occurred in a UVA1 dose-dependent manner. We also examined other T-cell receptors, such as CD4, CD25 and CD69, but they did not change for up to 24 h following exposure. During UVA1-triggered immediate apoptosis of Jurkat T cells, IFN-g levels were found to increase in a dose-dependent manner at 4 h, but returned to base-line levels at 24 h following exposure; whereas, there was no significant change in IL-2 at 4 or 24 h following exposure. Thus, UVA1-triggered immediate apoptosis causes a rapid decrease in the number of CD19 receptors on Daudi B cells and release of IFN-g from Jurkat T cells at 4 h, and UVA1-mediated delayed apoptosis causes an increase in the number of CD3 receptors on Jurkat T cells.
B-PO-02
Identification of IL-13 Receptor alpha2 as a Molecular Target by Microarray analysis in Adrenomedullin transfected or treated Human Tumor Cells: Mechanism of Target Regulation
B. H. Joshi¹, P. Leland¹, A. Calvo², L. Montuenga², J. E. Green³, R. K. Puri¹, ¹LMTB, DCGT, OCTGT, CBER, FDA, Bethesda, MD, ²Dept. Histology and Pathology, University of Navarra, Pamplona, Spain, ³Laboratory of Cell Regulation and Carcinogenesis, NCI, NIH, Bethesda, MD

Previously, we have identified a tumor-associated target in the form of Interleukin-13 receptors (IL-13R) on a variety of human tumor cell lines. IL-13Ra2 chain, the predominant IL-13 binding protein of the IL-13R complex serves as a novel target for immunotherapy of cancer by a chimeric fusion protein consisting of IL-13 and a truncated form of Pseudomonas exotoxin (IL-13PE). By microarray analysis of adrenomedullin (AM) transfected human prostate tumor cell lines, we have also identified upregulation of IL-13Ra2 gene by AM. We demonstrate here that IL-13Ra2 mRNA and protein is also up regulated in two human breast tumors (MCF-7 and MDA-MB-231) and one prostate carcinoma (PC-3) cell line after exogeous AM treatment. In order to understand the mechanism of up-regulation, we performed actinomycin-D treatment studies, which showed that IL-13Ra2 and AM genes are transcribed at a higher rate in AM treated tumor cells compared to control cells. Similarly, cycloheximide treatment studies demonstrated the half-life of IL-13Ra2 gene is enhanced by AM treatment. These abilities of AM were exploited for receptor targeted cancer therapy by using IL-13PE. This molecule was highly cytotoxic to AM treated tumor cells compared to untreated cells. Our results suggest that IL-13Ra2 chain can be up regulated by AM and this property can be exploited for breast and prostate cancer therapy. These studies provide unique insight into the mechanism of action of tumor targeted agents and understanding of novel technology in the context of the development of oncology products including gene therapy and tumor vaccines.

CATEGORY C: MICROBIOLOGY & VIROLOGY
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C-04
Improved Method for Enrichment of E. coli O157:H7 is More Effective Than Six Standard U.S. and International Methods
M.A. Grant, FDA, ORA, Pacific Regional Laboratory Northwest, Bothell, WA

An improved procedure for detection of E. coli O157:H7 and other Shiga toxin-containing E. coli (STEC) was recently developed. This method was compared to five standard methods, including FDA, USDA, Canadian, British and ISO, as well as to Buffered Peptone Water (BPW). When pure cultures of STEC were tested, the new procedure was superior to all other methods by factors ranging from 2 to 60-fold. When STEC cultures were tested under conditions representing real food samples, the acid enrichment method was even more effective, with detection levels as much as 1000-fold higher than some standard methods.

The new protocol can also be used for rapid detection by PCR and real time PCR. By conventional PCR the new method yielded strong bands (stx1/stx2) after electrophoresis. In contrast, gene products could not be detected by 4 methods and 2 methods produced bands distinctly weaker than the acid method. When samples were analyzed by real time PCR, the new method was also superior; stx1 and stx2 gene products being detected with Ct values of 19.44 and 17.50, respectively. In contrast, the Ct values for all other methods, including FDA, ranged from 24.44 to undetectable.

The new enrichment method can be universally applied to numerous detection methods including PCR, ELISA, cultural, and IMS. It therefore holds considerable potential to improve the ability of public health agencies worldwide in their efforts to detect E. coli O157:H7 and other Shiga toxin-containing E. coli.
C-06
Preliminary study: Effectiveness of Ferrioxiamine E as supplement to isolate Salmonella Enteritidis from shell eggs
I. E. Valentin-Bon¹, K. H. Seo¹, R. E. Brackett², R. Mehta³, ¹OPDF, DDES, DA, CFSAN, College Park, MD, ²OD, CFSAN, FDA, College Park, MD, ³JIFSAN Program, University of Maryland

The use and effectiveness of the hydroxamate siderophore, Ferrioxamine E as supplement to isolate Salmonella Enteritidis from white shell eggs was tested. Ferrioxamine E is a siderophoric compound biotechnologically produced by Streptomyces pilosus. As a growth factor of S. Enterica serotypes, FE can reduce the pre-enrichment and detection time in less than 20 hours; compared with conventional methods that require a pre-enrichment of 24 hours to isolate Salmonella spp. To determine the minimum concentration of FE needed to enhance SE growth, egg samples were pre-enriched with different FE concentrations (0, 2, 200, 2000, 20,000 ng/g), and inoculated with low levels of SE (0.2 CFU/g). Samples were incubated at 37°C for 24 hours. The optimum concentration of FE was determined to be 200 ng/g of liquid egg. Growth curves were performed with 200 ng/g of FE, and without 0 ng/g of FE to calculate the lag phase and the exponential growth rates of SE in eggs. After 24 hours of incubation at 37°C, the SE population increased markedly from 2 log CFU/g to 8 log CFU/g from eggs enriched with 0 and 200 ng/g of FE, respectively. Using a linear growth model the lag phase for SE with 0 ng/g of FE was 12.743 versus 1.371 hours with 200 ng/g of FE. The exponential growth rate for the control SE with 0 ng/g of FE was 0.20642 log CFU/hour versus 0.40279 for 200 ng/g. Ferrioxamine E improved the efficiency to detect SE in eggs by accelerating the growth of the bacteria that is usually present in low number in shell eggs.

C-08
Prevalence and Antibiotic Susceptibility Profiles of Salmonella and Escherichia coli Recovered from Plant Based Feed Commodities - Results of a National Survey
P. J. Carter¹, S. D. McDermott¹, P. Cullen¹, A. Glenn¹, S. Ayers¹, J. Paige², D. D. Wagner¹, ¹FDA, Laurel MD, ²FDA, Rockville MD

To better understand the role that animal feeds play in the dissemination of foodborne pathogens and in antibiotic resistant phenotypes, the FDA has conducted surveys of commodities used to manufacture animal feeds. This 2003 survey was intended to provide data on the distribution of antibiotic resistance determinants associated with Salmonella and E. coli in plant based commodities. A total of 79 samples were collected and analyzed.The Salmonella (4 isolates) recovered were serotyped and antibiotic susceptibility testing showed susceptibility to 16 antibiotics. A comparison to data resulting from a survey of rendered animal by-products completed in fiscal year 2002 showed forty two (34%) of rendered animal by-products were positive for Salmonella compared to four (5%) of the plant based feed commodities. The salmonellae recovered from the rendered by-products were also uniformly susceptible to all antibiotics. Forty three percent of the plant based feed commodities were positive for E. coli. Susceptibility tests of the E. coli isolates showed resistance to tetracycline in 9% of the 54 isolates while 13% were resistant to cephalothin. Similar susceptibility profiles were seen in isolates collected from animal by-products during 2002. The data indicates that Salmonella is far more prevalent in rendered animal by-products than in processed plant based feed commodities. E. coli is found at approximately equal prevalence in both. Also, the presence of antibiotic resistant phenotypes is not common in either commodity. Thermal processes commonly used in feed manufacturing would likely reduce the number of positive samples in complete feeds to even lower levels.

C-09
Simple and Reliable Method for Genetic Characterization of the Influenza B Virus Reassortants
V. Y. Lugovtsev, C. Strupczewski, G. M. Vodeiko, R. A. Levandowski, FDA, Bethesda MD

The ability of influenza viruses to reassort can be used to understand viral evolution and to create viruses with specified phenotypes. The theoretical maximum number of possible gene combinations in random reassortment between two influenza viruses is 256 (2 to the 8^th power). Therefore, even with a selection scheme screening for progeny viruses with particular gene segment constellations is laborious, time-consuming and expensive. To overcome these limitations in screening large numbers of influenza B virus reassortants we adapted a method based on single-strand conformation polymorphisms (SSCP) to examine RT-PCR products of viral gene segments. This method provided an unambiguous identification of the origin of each of the viral genes and permitted us to optimize the selection of reassortant clones with desirable genomes. The high specificity of SSCP allows discrimination of single nucleotide differences. Therefore, this method can be used to assess the purity of selected viruses and to detect mixed viral populations. This method was tested on numerous "B/Beijing/184/93 x B/Shangdong/7/97" reassortants and was reproducible, simple, fast and cost-effective. Once established the method requires no special instruments and expensive chemical compounds and can be performed in a general laboratory setting.

C-10
Evaluating the Effects of Oxytetracycline on the Microflora of a Recirculating Aquaculture System
S. Serfling, C. Gieseker, R. Miller, E. Squibb, T. Brady, R. Reimschuessel, Center for Veterinary Medicine, Office of Research, Division of Animal Research

According to the United Nation's Food and Agriculture Organization, aquaculture is the fastest growing food producing industry in the world. Because of this rapid development, and the shift towards more intensive farming methods, the aquaculture industry has encountered many viral and bacterial diseases, and the need for treatments to control and prevent disease. As a result, public health concerns have arisen about chemical residues and the development of antibiotic resistance in fish tissue. In the United States there are two antibiotics approved by the Food and Drug Administration for aquaculture, oxytetracycline - Terramycin ®, and sulfadimethoxine:ormetoprim - Romet-30 ®. These antibiotics are added to feed during the manufacturing process. Any food that is not eaten by fish, and the drug residues excreted in fish feces, will eventually reach pond sediments, or be captured in bio-filters used by recirculating aquaculture systems. Antibiotic residues are adsorbed in these sediments, which can create selective pressure on the microorganisms found in this environment. The purpose of this study is to survey bacteria found in recirculating aquaculture systems and to find a potential indicator organism. In addition to defining the bacterial isolation and identification methods, the isolated organisms will be tested for susceptibility to oxytetracycline following antibiotic susceptibility methods developed at CVM for aquatic organisms. Data from this study will help identify potential microorganisms to monitor for changes in antimicrobial susceptibility patterns following antibiotic use in recirculating aquaculture systems.
C-11
Severe pulmonary pathology after intravenous administration of adenovirus vectors in cirrhotic rats
J. S. Smith¹, J. Tian¹, J. N. Lozier², A. P. Byrnes¹, ¹DCGT, FDA, Bethesda MD, ²DH, FDA, Bethesda MD

Normally, when adenoviral vectors (AdVs) are administered by intravascular injection, the majority of the vector is rapidly cleared from the circulation by the reticuloendothelial system (RES), primarily by Kupffer cells (KCs), macrophages that reside in liver sinusoids. This results in a rapid innate immune response and subsequent liver pathology. In cirrhotic animals, however, we have previously shown that the biodistribution of intravenously administered AdVs is altered due to the presence of pulmonary intravascular macrophages (PIMs), which cause a shift in RES vector uptake from the liver to the lungs. Because macrophages respond to AdVs by releasing pro-inflammatory cytokines, including IL-6 and TNF-a we speculated that uptake of vector by PIMs might cause pulmonary damage in cirrhotic rats. We therefore compared the innate response to AdVs in normal and cirrhotic rats. Cirrhosis was induced in rats by bile duct ligation for four weeks, AdV was injected into the tail vein, and animals were sacrificed six hours later. We found that cirrhotic rats which received high doses of AdV developed severe pulmonary hemorrhagic edema and, when compared to normal rats, reacted to vector with enormous increases in TNF-a and IL-6. Severe coagulopathy was also seen, marked by hemolysis and prolongation of PT and aPTT. Because a similar shift in RES uptake from the liver to the lungs is known to occur in humans during cirrhosis and other diseases, there is the potential that intravascular administration of adenovirus vectors might cause lung pathology in such patients.
C-12
Isolation and Characterization of a Non-Phospholipase D Hemolysin from Photobacterium damselae subspecies damselae.
J. Trinidad-Prerez¹, M. H. Kothary¹, S. R. Monday², P. Whittaker², G. Modderman¹, I. Agba¹, A. Nyarko¹, R. P. Gonzalez-Nieves¹, M. Marrero-Moran¹, T. DeSiano¹, S. Taherkhani¹, N. C. Flores¹, J. Arnold³, B. D. Tall¹, ¹FDA, Laurel, MD, ²FDA, College Park, MD, ³Nat. Aqaurium, Baltimore, MD

Photobacterium damselae subspecies damselae (Pdd) has a wide host species range and causes illness in seafood species and humans. Nine isolates were identified using the API20E and MIDI identification systems and by a duplex PCR assay targeting concomitantly, a species-specific 16s rRNA gene sequence and the phospholipase D (damselysin) hemolysin gene. All of the isolates were positive for the 16s rRNA gene; none of the isolates were positive for the damselysin gene; yet 7 of the 9 isolates were hemolytic on sheep blood agar. The present study describes the isolation and characterization of this hemolysin. First, the effect of growth conditions on expression of the hemolysin was determined for cultures grown in CYE broth containing either 0%, 3% or 6% NaCl. The growth temperature for these cultures were either 20°, 30°, or 37° C. Culture supernatants were sampled over time using a hemolysin assay with sheep erythrocytes. Results showed that 30°C and 0% NaCl favored the expression of the hemolysin and maximum expression occurred within 6 hours, whereas no hemolytic activity was observed in overnight culture supernatants. The Pdd hemolysin was purified by ammonium sulfate precipitation and sequential hydrophobic interaction chromatography. The hemolysin is heat labile, and has a molecular weight of 64,000 daltons by SDS-PAGE and an isoelectric point of ca. 4.7. In summary, these results demonstrate that P. damselae subspecies damselae produces a second hemolysin that is different than the damselysin previously described and that environmental cues such as temperature and NaCl affect its expression.

C-13

Analysis of Bile Acid-Mediated Aminoglycoside Sensitivity in Lactobacillus
C. A. Elkins, L. B. Mullis, NCTR, FDA, Jefferson AR

Few studies have been conducted on antimicrobial resistance in lactobacilli presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 strains representing 14 species using agar disc diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i.) they were more susceptible (up to 256-fold in some cases) to deconjugated bile acid, cholic acid, then to conjugates, taurocholic or taurodeoxycholic acid, and (ii.) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain, L. plantarum WCFS1, at increasing concentrations of ox gall displayed corresponding decreases in resistance to all tested aminoglycosides and ethidium bromide. This effect was dramatic and potentially clinically relevant with the gentamicin MIC, decreasing from over 1 mg/ml to 4 µg/ml in just 3.8 mg/ml ox gall. In uptake studies with [³H(G)]-gentamicin at pH 6.5, bile acids and reserpine, but not CCCP, increased gentamicin accumulation over control levels. The effect was dramatic particularly with cholic acid, increasing up to 18-fold, whereas only modest levels of uptake could be achieved with taurocholic acid or ox gall. Since L. plantarum and particularly WCFS1 is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that cholic acid, but not taurocholic acid, effectively permeabilizes the membrane. However, our data also indicate that there is a modest contribution of a potential aminoglycoside efflux mechanism at pHs that approach the intestinal lumen.

C-PO-15

Interference of Food Matrix Components on the Detection of Escherichia coli O157:H7 in Produce using Real Time Quantitative PCR
P. Gedalanga¹, D. M. Williams-Hill², B. H. Olson¹, ¹University of California, Irvine, ²FDA-PRL-SW

Escherichia coli O157:H7 (EHEC) has been associated with outbreaks of human gastroenteritis due to the consumption of contaminated produce. Real-time PCR allows for detection and confirmation of EHEC to be completed in 48 hours or less. However, interference by matrix components on PCR efficiency is a concern, especially for low-level pathogen detection amidst high background. In this study, we begin to quantitate the effects of inhibition and interference associated with detecting EHEC from various produce matrices (lettuce, green onions, sprouts, broccoli, and celery) both as rinses and as minces of the product. The latter is important as EHEC has been shown to travel through the root system of plants into the internal veins. For analysis of real-time PCR efficiency of produce rinses, products are spiked with EHEC and rinsed with PBS solution (1:1, w/v). For minces, produce products are fed EHEC-spiked water over a period of five days. The products are rinsed thoroughly, and then minced in a 1:1 (w/v) of PBS. DNA will be extracted from both rinse and mince solutions and screened for five EHEC specific genes: stx1, stx2, eaeA, rfbE, and fliC using the RotorGene 3000 and dual-labeled Taqman probes. Recovery is quantitated by comparing the cycle threshold of the samples against a standard curve generated from an EHECcontrol strain (ATCC # 43895). These results will contribute to understanding the role of matrix interference in quantifying bacterial pathogens in environmental samples.

Outstanding Poster Award - 2004 FDA Science Forum

C-16

Microarray analysis of Bacillus cereus group virulence factors.
N. Sergeev¹, M. Distler², V. Chizhikov³, A. Rasooly¹, ¹OST, FDA, Silver Spring, MD, ²JIFSAN - University of Maryland, Silver Spring, MD, ³FDA, Silver Spring, MD

Members of the Bacillus cereus group bacteria are environmental organisms which encompass at least four closely related species: B. anthracis, B.cereus, B. thuringiensis, and B. mycoides. B. anthracis causes anthrax and B. cereus causes food-borne disease. B. thuringiensis has economic significance because it is an insect pathogen.

The taxonomy of this group is complex. It has been suggested that these closely related species should all be grouped as members of B. cereus. Most of the primary differences between B. cereus, anthracis and thuringiensis are the presence or absence of several plasmid-borne virulence factor genes.

The primary aim of this project was to develop an oligonucleotide microarray for the detection of the major B. cereus virulence factor genes. We postulated that each species may contain a different combination of virulence factors that can be used for distinguishing between these Bacillus species. In addition, the profile of virulence factors may indicate the potential virulence of a strain.

Our method requires an initial PCR amplification step, followed by identification of the PCR amplicons by hybridization of the fluorescently labeled single-stranded DNAs to an oligonucleotide microarray. We used ninteen Bacillus virulence factors genes (nheA, hneC, sph, plc, hblD, hneB, piplc, hblA, hblC, entFM, cytK, bceT, cryA, spaB, lef, cya, pa, cap and capB) divided into four groups for multiplex PCR amplification and labeling. Our oligonucleotide microarray contains DNA sequences representing all nineteen genes.

In our preliminary results, the B. cereus group virulence factor microarray successfully detected the virulence factor genes in the tested strains and was able to identify several B. cereus group species. The results presented here demonstrate the potential of oligonucleotide microarrays for food safety and biodefense.

C-19

Evaluation of the Real Time PCR Assay for Rapid, Specific Detection and Enumeration of Enterobacter sakazakii in Infant Formula
K. H. Seo¹, G. Thammasuvimol¹, M. L. Kotewicz², R. E. Brackett¹, ¹OPDF, FDA, College Park, MD, ²OARSA, FDA, Laurel , MD

Enterobacter sakazakii (E. sakazakii) is a rare cause of invasive infection with high mortality rates in neonates. Powdered milk-based infant formulas have been attributed to the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, an assay was developed for the specific detection of E. sakazakii in infant formula using an application of the fluorogenic 5' nuclease assay (TaqMan). A set of primers and probe was designed using E. sakazakii partial macromolecular synthesis (MMS) operon; rpsU gene, 3' end, and primase (dnaG) gene, 5' end. The specificity of the assay was evaluated using nearly 100 genus Enterobacter and 60 non-Enterobacter strains including Enterobacter cloacae. The newly developed assay enables us to detect 10² CFU/ml in pure culture and in reconstituted infant formula in 50 cycles of PCR reaction without enrichment procedure. Also, the assay was specific to discriminate E. sakazakii from all other Enterobacter and non-Enterobacter strains tested. The developed real-time PCR assay could save up to five days and eliminate the need for plating samples on selective/diagnostic agars and biochemical confirmation steps. The real-time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.

C-PO-20

E. coli O157:H7:- Long-term Survival in Culture Media and Production of Small Colony Variants
M.O. Paul, NRL, FDA, Jamaica, NY

E. coli O157:H7 (EHEC) is a significant cause of foodborne illness in the US, with roughly 3% mortality among infected persons. As a potential agent of bioterrorism, it is important to evaluate survival characteristics of this organism under different conditions. Brain Heart Infusion (BHI) and Lauryl Sulfate (LS) broths were inoculated with E. coli O157:H7 (ATCC #43888). After overnight incubation, aliquots of each medium were stored at room temperature (RT, ~25° C), refrigerator temperature (FT, 4°C), and frozen (FZ, -20°C). At various intervals CFU/ml were estimated in duplicate on Tryptic Soy Agar with Yeast Extract (TSAYE) plates. After a 100-day period, from an initial mean CFU/ml of 5.9 x 10⁸ at day 0, CFU/ml for BHI cultures, were 2.3 x 10⁸, 2.5 x 10⁷ and 9.2 x 10³ at day 100, for storage at RT, FT and FZ, respectively; for LS cultures, CFU/ml were from 1.4 x 10⁸ initially, to 4.0 x 10⁵, <1, and <1, for storage at FT, RT and FZ, respectively. During storage, small colony variants (< 1 mm in diameter, vs. > 10 mm), not previously described for EHEC, were observed on TSAYE plates used for CFU/ml determinations. Overall, during the 100-day period, with respect to culture and storage conditions, EHEC survived equally well in BHI cultures kept at RT and at FT, and substantively at FZ; whereas, survival in LS was restricted to cultures kept at FT. Small colony variants, induced in other bacteria after exposure to antibiotics, are probably of survival value for EHEC, and their pathogenic significance is being investigated.

C-PO-22

Salmonella Serotype Trend Analysis Using a Microsoft Access Database
D. M. Williams-Hill¹, D. Farmer², D. R. Solis¹, ¹FDA, PRL-SW, ²FDA, ARL

In light of bioterroist threats to public health, it is important to isolate all Salmonella serotypes for epidemiological trace-back. An initial review of 278 worksheets from Salmonella positive seafoods isolated at PRL-SW showed that 64% of the O-antigen serotypes were isolated from RV and TT enrichment media. The remaining 36% of serotypes were isolated from either RV or TT, indicating efficient isolation of Salmonella requires the use of two enrichment media. The study has been expanded to include data from the ARL serotyping laboratory where O and H antigen serotyping is performed on Salmonella isolates from FDA laboratories nationwide. A critical component has been the design and implementation of a database to accumulate this data and generate the appropriate statistical analysis. We have created a relational database in Microsoft Access that records and trends the biochemical reactions of Salmonella analysis. The database details enrichment mediums, biochemical profiles and serological characteristics as well as information on food types and country of origin and conducts trend analysis that can be presented in a variety of report formats. We created a GUI (Graphic User Interface) and made it user friendly with Menu Bars and Toolbars. Microsoft Access is readily available and can be used in FDA labs nationwide to normalize data storage formats, to facilitate information exchange in a secured encrypted format (Access Encryption) and to create reports that are customized to personal preferences. Here we present the use of this database in assessing whether the isolation of Salmonella serotypes is enrichment-media dependent.

C-26

Real-Time PCR assay for the detection of Salmonella in Foods
C. M. Cheng, K. T. Van, W. S. Lin, N. N. Tran, L. C. Phan, PRL-SW, Irvine, CA

The genus Salmonella is an important food and water-borne pathogen around the world. It causes acute gastrointestinal illness. The infective dose can be as low as 15-20 cells. Conventional methods for detection of Salmonella spp. in foods are generally time-consuming and labor-intensive. A real-time PCR method has been developed by FDA, PRL-SW with custom designed primers to amplify a 262-bp fragment of Salmonella-specific invA gene. The system has been tested with 255 strains of field-isolated Salmonella spp. and non-Salmonella spp., as well 312 samples of various food matrices. The results showed that the method had 100 % specificity and high sensitivity (0.1-0.7 CFU/g). This newly developed real-time PCR method is much more rapid, sensitive, and specific for detection of Salmonella spp. than the traditional BAM method and the AOAC-approved VIDAS method.

C-27

Development of methods for detection of egg antibodies (IgY)
B.D. Paul, FDA

Several reports have shown that chickens can develop antibodies to antigens from human pathogens. Such antibodies are transferred from the blood stream to the eggs yolk of laying hens and stored as IgY, the yolk immunoglobulin. IgY is slightly bigger that IgG; it has a longer Fc region increasing the molecular weight from 150 kDa to 165 kDa, but the Fab antigen binding moieties remain the same. Antibody production in eggs is an economically attractive venture since large quantities may be produced at relatively low cost. However, there are no commercially available tests for assessing the presence of such antibodies. At the Northeast Regional Laboratory we used a vaccine preparation of Candida albicans to develop an Enzyme Linked Immunosorbent Assay (ELISA) for detecting the Candida antibody, and worked with a commercial manufacturer to alter a Micro Immune Fluorescence (MIF) assay for the detection of Chlamydia pneumonia antibody in chicken egg yolks. These tests were used to determine the presence and quantities of the two antibodies in egg samples from chickens whose eggs yolks were claimed to prevent or cure candiadiasis and pneumonia. Of thirty eight egg yolk samples tested for Candida IgY antibodies only one showed a significantly higher antibody level than the shop-bought control egg. Antibody levels of twenty-nine samples tested for Chlamydia antibody were as follows: 18 < 1:8, the lowest dilution, 9, 1:8 to 1: 32 and one sample fluoresced non-specifically. Similar results were obtained for the control eggs, therefore, the antibody claim could not be substantiated.

C-PO-29

Improved Rapid Procedure for Shigella Detection in Fresh Produce
W. S. Lin, C. M. Cheng, K. T. Van, M. S. Esteves, PRL-SW, FDA, Irvine CA

With recent increasing demand of fresh produce, there is the greater potential of food borne illness associated with these products. Shigella is one of the food pathogens associated with fresh produce. The infectious dose is estimated to be about 10 cells. The traditional BAM method for Shigella spp. detection is time consuming and labor intensive. The PCR method developed by CFSAN may not be sensitive enough to detect a low number of Shigella cells in fresh produce due to low cell recovery rate and the lack of an enrichment procedure, hence increasing the health risk for shigellosis. The FDA Pacific Regional Laboratory - Southwest has developed a two-step enrichment procedure that can boost cell number 10⁹ fold prior to PCR amplification. Shigella cells were first resuscitated in universal preenrichment broth at 42°C for four hours following by aerobic incubation in shigella broth with novobiocin for additional eighteen hours at the same temperature. The DNA templates prepared from this enrichment are used to amplify a 181-bp fragment of Shigella specific ipaH gene by a newly developed real-time PCR method with custom designed primers and probe.
The system has been tested with twelve varieties of fresh produce spiked with four Shigella species. The results showed that the method had 100% specificity and high sensitivity (1 CFU/100 ml) for all four Shigella species. The whole procedure can be completed within 24 hrs and has great potential for screening Shigella spp. in fresh produce.

C-30

Prevalence and Antimicrobial Susceptibility Profiles of Enterococcus spp. Isolated from Plant Based Feed Commodities - A National Survey
P. Cullen¹, S. D. McDermott¹, P. J. Carter¹, J. C. Paige², D. D. Wagner¹, ¹CVM, FDA, Laurel MD 20708, ²CVM, FDA, Rockville MD 20855

The food animal production environment has been a focal point in assessing human and animal health risks associated with antimicrobial use and in developing intervention and mitigation strategies to limit the spread of antibiotic resistance at the farm level. This study was part of an ongoing survey to investigate the role various animal feed components may have in the dissemination of antibiotic resistance in the environment. A total of seventy-nine samples of plant based feed commodities were collected from across the United States and cultured for Enterococcus. Ninety-one percent (72/79) of the samples were positive for Enterococcus. This high prevalence of enterococci was comparable to that previously reported in association with rendered animal by-products (84%). In the plant based commodities, E. faecium was the most often recovered species (90.7%) followed by E. faecalis, E. hirae, and E. mundtii, E. durans, and E. casseliflavus. A total of 162 enterococci were recovered and tested for susceptibility to 17 antimicrobials. Resistance to tetracycline (9.9%), erythromycin (9.3%), and ciprofloxacin (6.2%) was detected. High-level kanamycin and streptomycin resistance (>1024 µg/ml) was detected in (5.5%) and (1.2%) of the isolates, respectively. No resistance was detected to quinupristin/dalfopristin (Synercid ^TM) in any of the non-faecalis species. All isolates were susceptible to chloramphenicol, penicillin, vancomycin, and linezolid. The high prevalence of enterococci found in various plant based feed ingredients, and the antibiotic resistances associated with them, demonstrate the potential for animal feed to serve as a reservoir for resistance genes.

C-31

Growth of Salmonella during Sprouting of Naturally Contaminated Alfalfa Seeds as Affected by Sprouting Conditions
T. Fu¹, O. M. VanPelt², K. F. Reineke¹, ¹CFSAN/NCFST, FDA, Summit-Argo, IL, ²NCFST, Illinois Institute of Technology, Summit-Argo, IL

Alfalfa sprouts contaminated with Salmonella have been linked to a number of foodborne disease outbreaks in recent years. The source of contamination is frequently the seeds used for sprouting. Many studies have examined the growth of Salmonella during sprouting of contaminated seeds and shown that the pathogen proliferates rapidly and reaches high numbers during sprouting. Most of these growth studies, however, were conducted using lab-scale sprouting systems under conditions very different from those used in commercial operations. Whether similar growth kinetics will be observed during commercial sprouting operations remains to be determined. We have built a mini-drum sprouter equipped with an automatic irrigation system similar to those used in commercial operations. The growth of Salmonella during sprouting of naturally contaminated alfalfa seeds in this mini-drum sprouter was compared with growth observed during sprouting in jars under conditions commonly used for home sprouting. The level of Salmonella, while increased by as much as 4 logs after 48 h of sprouting in jars, remained constant during the entire sprouting period in the mini-drum sprouter. The effect of sprouting temperature and irrigation frequency on Salmonella growth was examined. Decreasing the irrigation frequency from every 20 min to every 2 h resulted in an approx. 2-log increase in Salmonella counts, and increasing the sprouting temperature from 20°C to 30°C increased the Salmonella counts by as much as 3 logs. Finally, the effect of chemical treatment of seeds on Salmonella growth was examined. Salmonella grew to a slightly higher level during sprouting of seeds treated with 20,000 ppm calcium hypochlorite compared to levels observed with un-treated seeds.

C-32

Risk Assessment Development through Inter-Center Collaboration: Development of a Microbial Food Safety Guidance for the Center Veterinary Medicine
J. W. Punderson, H. G. Claycamp, W. T. Flynn, J. M. Gilbert, B. Hooberman, K. R. Lampe, L. Tollefson, R. D. Walker, S. S. Yan, J. Powers, A. Sheldon, J. Mulinde, FDA

FDA's Center for Veterinary Medicine published Guidance for Industry #152: "Evaluating the Safety of Antimicrobial New Animal Drugs with Regard to Their Microbial Effects on Bacteria of Human Health Concern", on October 23, 2003. The guidance was developed in a collaborative process involving FDA's Center for Drug Evaluation and Research, their Anti-Infective Drug Medical Advisory Committee, open public meetings, and comment dockets allowing input from all interested parties.

Bacterial resistance to antimicrobial agents is a complex problem with potential human health impact that may be attributable, in part, to the use of antimicrobial drugs in food-producing animals. Food-producing animals may be the recipients of the same, or closely related, antimicrobial drugs as those administered to humans, to promote animal health and productivity. Once exposed to an antimicrobial agent the treated animals can serve as reservoirs of resistant commensal or pathogenic bacteria which may infect humans through consumption of contaminated food products.

The guidance outlines a qualitative risk assessment (QRA) process as one possible mechanism for evaluating the microbial food safety of antimicrobial new animal drugs. The factors considered in the ranking process included both broad factors (efficacy and development of antimicrobial resistance), and specific factors (utility in therapy of food-borne infection and availability of therapeutic alternatives).

This document will serve as a template for both the pharmaceutical industry and for the Center for Veterinary Medicine in assessing the importance of antimicrobial agents for use in animals raised for food production relative to the importance of those drugs in human medicine.

C-33

Screening of gene-combinational variants of influenza B reassortants by DNA microarray technique.
G. M. Vodeiko, A. V. Ivshina, C. M. Strupczewski, K. M. Chumakov, R. A. Levandowski, FDA, Rockville, MD

Success of annual Influenza vaccine production depends on timely preparation of high growth (HG) virus seeds. Reassortment of eight RNA segments, traditionally used for influenza A, has not been practical for influenza B in part because the HG donor virus strain has not been established and genetic determinants of influenza B HG were not identified. To estimate the growth potential of different combinations of RNA segment we analyzed 200 virus isolates selected after reassorting of two (HG and LG, low growth) influenza B viruses. For genotyping of genome segments we used oligonucleotide microarray (OM) technique, which allowed us to analyze 1600 samples in relatively short time. OM revealed that some reassortants contain copies of both parental RNA for one or two segments. The results also showed that relative proportion of patterns the RNA segment combination in isolates was different from calculated, what would occurred if reassorting of RNA segments was at random. The addition of immunoselection against hemagglutinine (HA) of HG parent resulted in gene constellation with over-presentation of PB2 RNA of LG parent, which suggests the linkage of PB2 with HA. Phenotypic study indicated, that HG progeny viruses correlated best with the presence of HA, NA and NS from the HG parent. The results suggest that gene constellations in reassortment with and without selective pressures are not random, and HG property can be transfer by reassortment.

C-34

Characterization of a Putative Metacaspase Involved in the Programmed Cell Death Pathway of the Human Pathogen Leishmania.
N. Lee, A. Selvapandiyan, H. L. Nakhasi, A. Debrabant, LBPUA, DETTD, OBRR, Bethesda MD

The protozoan parasite Leishmania donovani is the causative agent of human visceral leishmaniasis that is responsible for hundred of thousands of deaths per year worldwide. In addition, U.S. Military personnel and travelers to endemic areas are at risk for leishmaniasis and are also deferred for blood donations. To date, there is no effective vaccine against Leishmania and parasites show increasing resistance to current anti-leishmanial drugs. Programmed cell death (PCD) is an essential part of cell biology and is thought to have evolved not only to regulate growth and development in multicellular organisms but also to have a functional role in the biology of unicellular organisms. In higher eukaryotes caspases are key effector molecules of PCD. We have previously described characteristic features of PCD in L. donovani. Such features include activation of a caspase-like activity in dying parasites present in stationary phase cultures and in parasites treated with the anti-leishmanial drug amphotericin B. Recently we identified a putative metacaspase gene (LdMC) in L. donovani. We showed that the transcription of LdMC was increased in stationary phase parasites when other features of PCD are also observed. Further, overexpression of LdMC in Leishmania renders the parasites more sensitive to amphotericin B induced cell death, suggesting that LdMC is probably involved in the Leishmania PCD pathway. Since metacaspases are not present in the human host, the LdMC represent a new target that could be exploited for the development of novel anti-leishmanial drugs that would trigger the parasite PCD pathway.

C-35

Mutational Pattern in High-Growth Influenza B Viruses Adapted for Replication in Eggs
V. Y. Lugovtsev, G. M. Vodeiko, R. A. Levandowski, FDA, Bethesda, MD

Epidemiologically important influenza B viruses exhibit variable growth capabilities in embryonated chicken eggs (CE). Serial passaging in CE remains the main way to obtain viruses with high growth characteristics, but not all strains are easily adaptable. Reverse genetics is a promising strategy for introducing genes that can contribute to CE-adaptation, but the molecular basis for the high growth phenotype of influenza B viruses remains unclear. We analyzed the mutational pattern of influenza virus B/Victoria/504/2000 adapted to high-growth phenotype by multiple serial passages in CE. Mutations resulting in amino acid (AA) substitutions were found only in HA gene of the two virus variants selected because of their excellent growth in CE. AA substitutions at position 162 and 196 were common for both of them, but one virus variant was also altered at AA position 141. By analogous alignment of the sequences of the HAs of the selected influenza B viruses with sequences of influenza A viruses having known 3D structure of HA, all 3 AA changes were predicted to be near the rim of the receptor-binding pocket of HA. Both of the selected viruses showed high yield in CE, but the virus with AA substitution at the position 141 demonstrated considerable antigenic divergence from the original reference virus. Our results indicate that adaptive mutations in HA contribute to higher growth capabilities, but some of them would not be desirable for vaccine production.

C-38

COMPARISON OF THE HEPARIN-BINDING AFFINITIES OF SUBGROUP B VIRULENT AND ATTENUATED RESPIRATORY SYNCYTIAL VIRUSES.
Y. Q. Li¹, P. Budge², R. L. Crim³, J. A. Beeler⁴, ¹DVP, FDA, Bethesda , MD, ²VRC, NIH, Bethesda, MD, ³DVP, FDA, Bethsda, MD, ⁴DVP, FDA, Bethesda, MD

The aim of our investigation is to evaluate and compare affinity of heparin-binding of a known attenuated RSV subgroup B strains cp52 with a wild type, subgroup B virulent respiratory syncytial virus (RSV) B1 isolate to determine if heparin-binding affinity correlates with virulence in vivo. We used heparin agarose affinity chromatography (HAAC) to evaluate binding affinity of the two RSV surface glycoproteins (F and G). F and G proteins eluted from HAAC using NaCl step gradients were detected in Western blot. The results demonstrate that multiple glycosylated forms of RSV subgroup B attenuated cp-52 fusion protein bound heparin with high affinity. In contrast, a subgroup B virulent virus B1 bound heparin with lower affinity. These data indicate that heparin-binding affinity may prove to be a useful, rapid in vitro marker of attenuation that will facilitate characterization of attenuated strains for testing in young infants and provide new information about RSV virulence.

C-39

Development of improved assays for evaluating vaccines against West Nile Virus and other emerging viral pathogens.
J. Soto, R. Levis, J. Weir, OVRR, FDA, Bethesda, MD

Several types of vaccines to protect against West Nile virus infection are currently in development. These include live, attenuated viral vaccines, subunit vaccines and DNA vaccines. A critical parameter for successful vaccine development is the ability to measure virus specific neutralizing antibody after immunization. The current tests available for measuring antibody have potentially high levels of background due to existing antibodies to other flaviviurses, are time consuming and require special facilities to conduct the testing. We have begun studies to develop improved assays for measuring vaccine efficacy that will enhance existing methodologies. Current tests to measure immune response to West Nile virus measure envelope protein specific IgM and IgG in acute and convalescent sera, respectively. These tests are ELISA based and are very sensitive, but can have background signal if an individual has had previous exposure, either vaccination or naturally occurring, to other flaviviurses such as dengue, yellow fever or Japanese encephalitis virus. In addition, the need to conduct secondary testing to confirm positive results makes the test lengthy and requires the use of high level containment facilities. Published reports have shown that vaccination or infection with flaviviurses elicits antibodies against other flaviviral proteins. We are working to improve the current assays by developing an antibody capture assay that will measure antibodies to the West Nile nonstructural viral proteins and establishing a system to measure West Nile neutralizing antibodies. These assays will be useful both for evaluation of vaccine response and as assays for determination of vaccine potency.

C-40

HIV-1 entry is governed by thiol/disulfide interchange in gp120 during conformational changes required for fusion
I. Markovic¹, T. S. Stantchev², K. H. Fields¹, L. J. Tiffany¹, M. Tomiæ³, C. D. Weiss¹, C. C. Broder², K. Strebel³, K. A. Clouse¹, ¹FDA, ²USUHS, ³NIH

HIV-1 gp120 envelope (Env) interaction with its primary receptor, CD4, triggers structural rearrangement in gp120 that facilitates binding to an appropriate co-receptor. Gp120/co-receptor engagement induces other conformational changes in the envelope that drive fusion between the virion and the target cell membrane. Protein disulfide isomerase (PDI) is a catalyst with redox-isomerase activity, which facilitates this Env conversion from its inactive to the fusion-competent conformation. Anti-PDI agents block Env-mediated fusion, whereas exogenously added PDI restores fusion to levels observed in untreated control cells. We postulate that the window of opportunity for PDI action is downstream from exposure of CD4-inducible epitopes (e.g., 17b, 48d) on gp120, but prior to assuming the fusogenic six-helix bundle. The present study shows that gp120 induces assembly of PDI, CD4 and CXCR4 into a tetramolecular protein complex at the virus-cell contact site. This suggests a new role for gp120, which appears to involve recruitment of the other three components into a protein complex that serves as a portal for HIV-1 entry. Our findings support the concept that Env conformational change depends on the well-co-ordinated action of a tripartite system where PDI works in concert with the receptor and co-receptor to allow HIV-1 to penetrate the cell-membrane barrier.

C-PO-01

Absence of Chlamydia pneumoniae DNA in Normal Blood Donors from the United States
G. Gao¹, G. Le Parc², G. Nemo³, E. Tabor¹, ¹FDA, Rockville, MD, ²Florida Blood Services, St. Petersburg, FL, ³National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

Chlamydia pneumoniae, an occasional cause of pneumonia, bronchitis, sinusitis, and pharyngitis, has been found in the cholesterol plaques of patients with atherosclerosis and in the peripheral blood of persons with cardiovascular diseases. C. pneumoniae DNA has been reported to be detectable in peripheral blood mononuclear cells of 59% of 101 coronary heart disease patients in Sweden and in circulating leukocytes from 20% of 60 patients with symptomatic carotid artery stenosis, peripheral arterial occlusive disease, or aortic aneurysms in Germany. It has also been reported that C. pneumoniae DNA can be detected in circulating PBMCs of 8.9% of 237 U.S. blood donors, 26% of 19 U.S. blood donors, 13% of 51 German blood donors, and 46% of 52 Swedish blood donors. The present study was conducted to determine whether the reported prevalence of Chlamydia pneumoniae DNA in U.S. blood donors could be confirmed. Methods: Buffy coat DNA was isolated from 211 normal blood donors. These included 100 blood donors from the Washington, DC, metropolitan area (kindly provided by the Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD) (42 females, 58 males; median age = 46.0 years) and 111 blood donors collected as part of the Transfusion Safety Study, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD (56 from Los Angeles, CA, 29 from San Francisco, CA and 26 from New York City, NY) (14 females, 97 males; median age = 41.4 years). C. pneumoniae DNA was assayed by touchdown PCR targeting the 16S ribosomal RNA (rRNA) of C. pneumoniae and real-time quantitative PCR targeting the C. pneumonia pmp4 gene. Results: None of the 211 normal blood donors had detectable C. pneumoniae DNA in their buffy coat DNA when assayed with a limit of detection of 100gEq/pg DNA. Validatio ntests on donor buffy coat DNA preparations, performed by spiking each of 10 samples with 1000 gEq/pg of C. pneumoniae DNA, were all positive, thus ruling out the possibility that inhibitory substances might be present in the buffy coat DNA preparations. Conclusions: C. pneumoniae DNA could not be detected in any of 211 buffy coat samples from normal blood donors in the U.S. A few previous small studies had reported the detection of C. pnemoniae DNA in 8.9-26% of U.S. blood donors. The absence of detectable C. pneumoniae DNA in the donors in the present study could reflect different prevalences in different donor populations. Another potential explanation could be the fact that the DNA in the present study was derived from buffy coats rather than from peripheral blood mononuclear cells, since the concentration of the C. pneumoniae DNA in buffy coats may be lower than that in concentrated preparations of monocytes. To address this issue, additional testing is in progress on purified monocytes from normal blood donors.

C-PO-03

Rapid Detection of Shigella using Real Time PCR
D. K. Lau, J. Kwan, FDA, SANDO, Alameda, CA

Shigella are highly infectious agents that are transmitted by the fecal oral route. Each year an estimated 448,240 cases of shigellosis occur in the United States, mostly due to Shigella sonnei. Shigellosis is particularly common and causes recurrent problems in settings where hygiene is poor and can sometimes sweep through entire communities. The disease is caused when virulent Shigella attach to, and penetrate epithelial cells of the intestinal mucosa. After invasion they multiply intracellularly and spread to contiguous epithelial cells resulting in tissue destruction. The FDA conventional method for Shigella isolation is dependent upon traditional enrichment and biochemical identification. The development of a rapid detection method for Shigella would greatly benefit the FDA's regulatory response to shigellosis. The purpose of this project is to develop a method using real-time PCR or 5'-nuclease assay to detect Shigella in various food matrices in less than 8 hours.

C-PO-04

Examination of Seafood Decomposition by the Microbiological Detection of Histamine-Forming Organisms
D. K. Lau, T. T. Huynh, FDA, SANDO, Alameda, CA

Histamine is a chemical health hazard that causes scombroid poisoning. Certain bacteria such as Morganella morganii, Enterobacter aerogenes, and Klebsiella pneumoniae produce a decarboxylase enzyme that converts the free histidine in the muscle of the fish to histamine. Organoleptic examination and chemical assay are the traditional methods used by FDA to detect decomposed fish. This research seeks a different method to screen decomposed fish through microbiological testing. This project involves the testing of the microbiological detection and enumeration method for Morganella morganii, Enterobacter aerogenes, and Klebsiella pneumoniae.. The detection method will utilize biochemical tests while the enumeration method will be MPN based. The detection and enumeration method of the histamine-forming organisms will be also tested on spiked frozen tuna samples at different time periods. The project will provide an alternative method for the detection of decomposition in seafood.

C-PO-05

Comparison of the VIDAS and TECRA Campylobacter Assay with the Traditional BAM Method.
D. K. Lau, G. Hall, FDA, SANDO, Alameda, CA

Campylobacter jejuni and Campylobacter coli are recognized as the leading causes of bacterial foodborne diarrhoeal disease. In the United States, Campylobacter species are isolated from about 5% of the patients with diarrhea and the annual incidence of infection is estimated to be 50 per 100,000 population while salmonellosis is only 16 per 100,000 population. The current FDA BAM method for detection of Campylobacter requires specialized media, microaerophilic conditions, and a minimum of five days for analysis and a maximum of two weeks for confirmation. The FDA method is dependent upon traditional enrichment and biochemical identification. The limitations of the current analytical method include the amount of labor required by the analyst to conduct the analysis and the time required performing the analysis. ELISA assays are quite sensitive and specific. The FDA currently uses Salmonella VIDAS, Listeria VIDAS, and the TECRA Staph enterotoxin kit for screening. The use of ELISA assays could shorten the time for analysis by screening out negative samples and identifying presumptive positives for further analysis. TECRA Campylobacter kit and the VIDAS Campylobacter kit will be able to detect Campylobacter in a short amount of time. Since both kits are immuno-based, specificity and sensitivity may be comparable to each other. Some food matrices may inhibit the ELISA tests.

C-PO-06

Development of a Real Time PCR Method for the Detection of Campylobacter jejuni and Campylobacter coli.
D. K. Lau, J. Huang, FDA, SANDO, Alameda, CA

Campylobacter sp. is a group of spiral-shaped bacteria that is recognized as the leading bacterial cause of diarrhea and gastroenteritis. The symptoms include diarrhea, cramping, abdominal pain and fever. The habitat of Campylobacter is in the intestinal tract of a wide variety of wild and domestic animals, especially birds. Campylobacter jejuni grows best at the body temperature of a bird without causing illness of its carrier. Food and water contaminated with untreated animal and human waste are the causes of campylobacteriosis. The foods include meats, poultry, fruits, shellfish and unpasteurized milk. The microaerophilic and fastidious nature of Campylobacter makes conventional methods cumbersome. Currently, the FDA BAM method requires a minimum of 4 days for isolation and more than one week for biochemical identification. Recent molecular technology involving the 5'-nuclease assay or Real-Time PCR can shorten the detection of pathogens to hours instead of days. This project will involve the development of the primers and probes for C. jejuni and C. coli and consequent testing for their specificity and sensitivity. This method will be applied in several food matrices including shellfish, dairy and produce. The results will expedite regulatory actions and thus achieve FDA's commitment in consumer protection.

C-PO-07

Development of a Real Time PCR Method for the Detection of Enterobacter sakazakii
D. K. Lau, J. W. Rotton, FDA, SANDO, Alameda, CA

Enterobacter sakazakii is a Gram-negative organism that was only recognized as a unique species in 1980, before which it was simply referred to as a 'yellow-pigmented Enterobacter cloacae'. In recent years, however, the emerging pathogen has become associated with powdered infant formula in isolated cases of illness ranging from bacteremia and necrotizing entercolitis to neonatal meningitis. The current FDA method for the analysis of E. sakazakii in powdered infant formula-which relies on traditional enrichment, plating, and biochemical identification-takes 5 days and still does not distinguish between E. sakazakii and other types of enterobacteria. Recent technology advances in PCR could allow for much faster and more reliable detection time. Therefore, we attempt to design a method using the Real-Time PCR technology (5' nuclease assay). The first step in such a procedure necessarily involves primers and probe design for unique portions of the E. sakazakii genome. After the primers and probe are designed, they will be tested against other members of the Enterobacteriacae family to insure specificity. The eventual development of a more rapid PCR-based method for this pathogen could expedite regulatory actions and thus better protect neonates and babies from potentially life-threatening illnesses.

C-PO-08

Real Time PCR Detection of Listeria monocytogenes
D. K. Lau, C. T. Peters, FDA, SANDO, Alameda, CA

Listeria monocytogenes is a Gram-positive organism that is ubiquitous in soil and water and is associated with foodborne illness. It is easily transmitted to agricultural food products from soil and animal vectors. Listeria has been associated with adulteration of raw food products such as, unpasteurized milk, meats, vegetables, and processed, ready-to-eat foods that become adulterated after processing. The clinical manifestations of mild listeriosis may include nausea, fever, vomiting, diarrhea to more severe clinical manifestations of septicemia, encephalitis, and meningitis. Listeriosis can lead to cervical infections inducing spontaneous abortions and stillbirths in pregnant women in the first six months of pregnancy. Currently, the FDA method requires a minimum of five days for analysis and a maximum of two weeks for confirmation. The FDA method depends upon traditional enrichment, biochemical identification, and serological classification. Recent technology in molecular biology involving the 5'-nuclease assay or real-time PCR can shorten the detection of pathogens to hours instead of days. The development of the method begins with the primer and probe design for the pathogen. This project will involve the development of the primers and probes for Listeria monocytogenes and consequently testing them for specificity and sensitivity for the pathogen. Development of a rapid method that can detect Listeria monocytogenes in less than a day will expedite regulatory actions and thus protect the consumer from adulterated products.

C-PO-09

Development of a Rapid Screening Method for Hepatitis A Virus on Green Onions
G.L. Hartman, SAN-DO, FDA, Alameda, CA

A simple and rapid screening method for the detection of Hepatitis A virus (HAV) in green onions was developed. HAV is detected by a one step, real-time reverse transcriptase-PCR (rt RT-PCR) that incorporates SYBR Green detection of amplified RT-PCR products. This method was developed as a result of an outbreak of Hepatitis A linked to green onions served at a national chain restaurant that involved more than 600 infections and 3 deaths. The HAV is isolated from green onions using a high pH glycine buffer to wash the virus from the green onions and a polyethylene glycol precipitation to concentrate the virus. The viral RNA is extracted from the PEG precipitation by a TRI Reagent extraction and the RNA is amplified in a single-tube one-step real-time RT-PCR detection system which incorporates SYBR green to rapidly screen the produce. A first derivative melt curve was used to verify HAV amplification and to rule out the presence of primer dimers and non-specific amplification. The Hepatitis A virus assay was tested in artificially contaminated green onions and found to be rapid and more sensitive than a previously published two-step RT-PCR . Multiple samples can be screened in 6-8 hours. The method is being further refined to include a TaqMan assay. The dual labeled TaqMan probes will be used for confirmation of samples found positive by the SYBR green assay, replacing the need for tissue culture as a confirmation step.

CATEGORY D: PHARMACOLOGY, TOXICOLOGY, AND PHARMACOKINETICS
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D-01

Photochemical decomposition of the dichlorobenzidine-based tattoo pigment, Pigment Orange 13, in simulated solar light
P. C. Howard¹, N. V. Gopee², Y. Cui², F. E. Evans³, L. H. Couch², M. I. Churchwell², D. R. Doerge², ¹NTP Center for Phototoxicology, NCTR, FDA, Jefferson, AR, ²Div. Biochemical Toxicology, NCTR, FDA, Jefferson, AR, ³Div. Chemistry, NCTR, FDA, Jefferson, AR

The art of tattooing has experienced a renaissance over the past several decades, with an increase in popularity especially among youth, and migration from the use of inorganic metals (e.g. iron oxides, HgS) to the use of organic pigments. Many of the yellow, orange, or red pigments reported to be used in tattoo inks are derivatives of 3,3'-dichlorobenzidine. We have used Pigment Orange 13 (PO13; CI 21110), a bisazo pigment of 3,3'-dichlorobenzidine and 1-phenyl-3-methyl-5-pyrazolone (1,3,5-PMP) as a model bisazo pigment for examining photochemical stability of this class of compounds. PO13 was dissolved in tetrahydrofuran, deoxygenated using argon gas, and exposed in 2 mm cuvettes to simulated solar light using a 6.5 kWatt xenon arc solar simulator, with radiation equivalent to terrestrial summer sunlight. The PO13 solution decolorized, indicating photochemical decomposition. The products were analyzed by HPLC, isolated using a combination of column chromatography, TLC, and HPLC, and analyzed individually by NMR and MS. The initial photochemical reduction of PO13 involves hetero- or homonuclear cleavage of one of the hydrazone groups to yield, respectively, the 1,3,5-PMP derivative of 3,3'-dichlorobenzidine or the 1,3,5-PMP derivative of 3,3'-dichloro-4-aminobiphenyl. 3,3'-Dichlorobenzidine and 3,3'-dichloro-4-aminobiphenyl were also identified, presumably formed by sequential photochemical reduction. The identification of other photochemical products is in progress to elucidate further the pathways for photodecomposition of PO13. These data demonstrate that photochemical decomposition of a representative 3,3'-dichlorobenzidine-based tattoo pigment using simulated solar light in an aprotic solvent results in the release of potentially carcinogenic dichlorobenzidine and dichlorobiphenyl derivatives.

D-02

Response of SKH-1 mouse skin following the acute injury of tattooing
N. V. Gopee¹, Y. Cui¹, G. Olson², A. Warbritton², B. J. Miller¹, L. H. Couch¹, W. G. Wamer³, P. C. Howard⁴, ¹Div. Biochemical Toxicology, NCTR, FDA, Jefferson, AR, ²Charles River Companies, Jefferson, AR, ³Office of Cosmetics and Colors, CFSAN, FDA, College Park, MD, ⁴NTP Center for Phototoxicology, NCTR, FDA, Jefferson, AR

Tattooing is a rapidly increasing practice involving as many as 20 million U.S. citizens. The toxicological and photocarcinogenic safety of the pigments used in tattoo inks have not been reported. As an initial step to assess their toxicological properties, we have studied the cutaneous impact of tattooing SKH-1 hairless mice, using commercial 14-pt needles and commercially available tattoo inks or inks containing water and CdS or Fe₃O₄. The dorsal tattoos were ~ 2x25 mm, and the mice were sacrificed at 0.5, 1, 3, 4, 7, or 13 days following tattooing. Histological evaluation of the skin revealed dermal hemorrhaging at 0.5 and 1 day, followed by dermal necrosis at 3 days that decreased to 17% of the original amount by day 13. Dermal inflammation was present from days 3 through 13. Epidermal necrosis and inflammation were immediately apparent (0.5 day) and decreased in occurrence and frequency to 0% by day13. External inguinal and axillary lymph nodes contained tattoo pigments, the inguinal being most reactive as evidenced by hyperplasia at all timepoints. Ornithine decarboxylase (ODC), cyclooxygenase-1 (COX-1), and COX-2 protein level in total skin increased significantly at 0.5-4 days in the skin and decreased to control levels by day 13, and COX-1 and COX-2 results were corroborated by immunohistochemical staining. The increase was greatest in the tattooed skin compared to the intra-tattooed skin, and in some cases the protein levels were elevated in nearby normal skin. ODC and COX-2 protein levels were elevated in the inguinal lymph node. Interleukin-1(beta) (IL-1b) and IL-10 protein levels were dramatically increased at all time points in the inguinal lymph nodes but suppressed in the tattoo and intra-tattooed skin, with maximal suppression between 0.5-4 days. Cutaneous nuclear protein concentrations of nuclear factor-kappa B (NF-kB), the transcription factor for a number of these inflammatory mediators, were elevated between 0.5-4 days. These data demonstrate that: 1) tattooing skin induces an inflammatory response similar to acute injury; 2) inflammatory cells respond to the presence of the tattoo ink and remove the ink compounds to the regional lymph node; 3) the skin in general recovers from the insult within 13 days.

D-03

Metabolism of a tattoo pigment, Pigment Yellow 74, using rat liver microsomal protein or xanthine oxidase
Y. Cui¹, N. V. Gopee¹, F. E. Evans², L. H. Couch¹, M. I. Churchwell¹, D. R. Doerge¹, P. C. Howard³, ¹Div. Biochemical Toxicology, NCTR, FDA, Jefferson, AR, ²Div. Chemistry, NCTR, FDA, Jefferson, AR, ³NTP Center for Phototoxicology, NCTR, FDA, Jefferson, AR

Tattooing has increased in popularity in the past decade and now is considered a common form of personal artistic expression. Recent publications point out the increase in tattoo popularity has been accompanied by a change in the pigments used in tattoo inks from inorganic salts to organic-based pigments. Very little is known about the chemical compositions of these pigments or their biological availability, metabolism, or toxicity. In these initial studies, we examined the metabolism of Pigment Yellow 74 (PY74; CI 11741), a common monoazo pigment in yellow tattoo inks. Oxidative metabolism of PY74 was determined following aerobic incubation of PY74 with liver microsomes isolated from 3-methylcholanthrene-treated male F344 rats. PY74 solubility in the incubations was increased by inclusion of bovine serum albumin to 10 mg/mL. Consumption of PY74 and formation of metabolites was linear for up to 30 min at 0.5 mg/mL microsomal protein. The predominant metabolite was isolated by HPLC and identified as a ring hydroxylation product (4-hydroxylation on the 2-methoxyaniline ring) using NMR and MS methods. The identification of the second metabolite and determination of the CYP450 specificity of PY74 metabolism are in progress. PY74 contains a nitro group on the 2-methoxy-4-nitroaniline group that should be available for nitroreduction. PY74 was incubated with 0.2 U/mL xanthine oxidase under anaerobic conditions and the products extracted. The nitroreduction of PY74 to the amino-derivative of PY74(NH₂-PY74) was detected by comigration on HPLC (UV-VIS and fluorescence detection) with authentic NH₂-PY74, and additionally confirmed by MS analysis. These results demonstrate that PY74 can be metabolized by both aerobic oxidative and anaerobic nitroreductive pathways, and that these monoazo tattoo pigments should not be considered as inert chemicals.

D-04

Photodecomposition of Pigment Yellow 74, a pigment in tattoo inks
Y. Cui¹, L. H. Couch¹, A. Spann¹, N. V. Gopee¹, F. E. Evans², M. I. Churchwell¹, L. D. Williams¹, D. R. Doerge¹, P. C. Howard³, ¹Div. Biochemical Toxicology, NCTR, FDA, Jefferson, AR, ²Div. Chemistry, NCTR, FDA, Jefferson, AR, ³NTP Center for Phototoxicology, NCTR, FDA, Jefferson, AR

Tattooing has become a popular recreational practice in the past decade. Very little is known concerning the pigments used or the toxicology, phototoxicology, or photochemistry of the pigments. Yellow tattoo inks were obtained from commercial sources and the component responsible for the color was extracted and quantitatively determined by HPLC with UV-VIS and MS detection. The monoazo Pigment Yellow 74 (PY74; CI 11741) was the major pigment found in seven yellow tattoo inks. PY74 was dissolved in tetrahydrofuran, deoxygenated using argon gas, and exposed in 2 mm cuvettes to simulated solar light using a 6.5 kWatt xenon arc solar simulator, with radiation equivalent to terrestrial summer sunlight. UV-VIS and HPLC analyses indicated PY74 photodecomposed after exposure to the simulated solar light. Multiple products were isolated using a combination of column chromatography, TLC, and HPLC. Three of the major products were identified by NMR and MS as N-(2-methoxyphenyl)-3-oxobutanamide and its 2-hydrazine derivative, and N,N'-bis(2-methoxyphenyl)urea. These products are indicative of photochemical reduction of the hydrazone double bond in PY74. The rate of formation of these products is solvent dependent and inhibited by the presence of oxygen. These results suggest that the photochemical decomposition of PY74 can result in the release of aromatic amine products.

D-05

An In Vitro Assay for Identifying Photoactive Tattoo Inks
W. G. Wamer¹, P. C. Howard², ¹CFSAN, College Park, MD, ²NCTR, Jefferson, AR

Tattoo inks, used for general body tattooing or permanent make-up, contain a wide range of organic pigments, inorganic pigments and diluents. Little is known about the toxicological properties of pigments used in tattoo inks. While there have been reports of phototoxicity associated with tattoo pigments such as cadmium sulfide, there have been no systematic studies designed to identify photoactive tattoo inks. We have developed an in vitro assay that allows screening commercial tattoo inks for their photoactivity. Thirty-three commercially available tattoo inks, marketed by three vendors for use in permanent make-up, were screened for photoactivity. Human skin fibroblasts were treated for 18 hr with up to 100 µg/cm² of each product. The cultures of skin fibroblasts were then washed to remove culture media, and exposed to a filtered metal halide lamp emitting 10 J/cm² long wavelength ultraviolet (i.e. 320 - 400 nm) and 49 J/cm² visible light. After irradiation, fibroblasts were re-plated at a low cell density (800 cells/60 mm dish) and incubated for 10 to 14 days to allow the growth of cell colonies. Cell colonies were then fixed in methanol, stained with Giemsa stain, and counted. A tattoo ink was identified as photoactive if treatment with ink, followed by exposure to light, resulted in inhibition of colony growth. A significant number of the tattoo inks were found to be photoactive. Although the clinical significance of this photoactivity is unclear, our results suggest that additional studies to evaluate the in vivo phototoxicity of tattoo inks are warranted.

D-06

Effects of Androstenedione on In Utero Development in Rats
R. L. Sprando¹, T. F. Collins¹, T. N. Black¹, N. Olejnik¹, E. Grundel¹, D. I. Ruggles², ¹Division of Toxicology, ²Division of Mathematics

This study was conducted to characterize the effect of androstenedione on estrous cyclicity, mating behavior and fetal development. Thirty-day old rats received corn oil alone or androstenedione (in corn oil) at one of four concentrations (0, 1.0, 5.0, 10.0 or 30.0 mg/kg body weight) by gavage for two weeks prior to mating, during the mating period and throughout gestation. Dose related increases in serum androstenedione, estradiol and estrone were observed in all androstenedione treated animals at gestation day 20. A statistically significant increase in serum testosterone concentration was observed in the 30 mg/kg dose group. Feed and fluid consumption were not affected by androstenedione treatment during the pre-mating or gestational periods, however a statistically significant decrease in the number of females with regular estrous cycles was observed in the 10.0 and 30.0 mg/kg dose groups. Exposure to androstenedione did not affect mean body weight gain during pre-mating or gestation. Slight not statistically significant reductions in the number of implants, number of viable fetuses and number of viable male fetuses were observed in the 30.0 mg/kg androstenedione group. Reductions were not observed in the number of corpora lutea. Fetal growth in terms of fetal weight, crown-rump length, anogenital distance and the number of external abnormalities was not affected. Androstenedione had no specific effect on the development of individual bones, including sternebrae or the development of soft tissues. Organ weights (expressed per gram of body weight or per gram of brain weight) were not affected by androstenedione treatment.

D-07

Hepatotoxicity of Androstenedione in Pregnant Rats
S. C. Sahu, P. P. Sapienza, R. L. Sprando, I. A. Ross, C. S. Kim, T. J. Flynn, P. L. Wiesenfeld, T. F. Collins, M. W. O'Donnell, W. D. Johnson, OARSA, FDA, Laurel, MD 20708

Androstenedione, a naturally occurring steroid hormone, is a dietary supplement used to enhance athletic performance. Little is known, however, about the safety of its use by young adults including women of child bearing age. To test the possible adverse effects of hepatic failure with androstenedione treatment, this study was undertaken to examine its hepatotoxic potential using a pregnant rat model. Pregnant rats (6 rats/dose) were exposed to androstenedione in corn oil by gastric intubation with increasing doses (0, 5, 30 and 60 mg/kg/day) beginning 2 weeks before mating and continuing through gestation day 19. On gestation day 20, blood and livers were collected from the dams for analysis of hepatotoxicity endpoints. Serum ALT, AST, GST, LDH, GSH, microsomal P450 and nuclear DNA damage and lipid peroxidation were used as biomarkers of hepatotoxicity. Statistical significance was assessed by Analysis of Variance. Under these experimental conditions, no significant differences were found in any of these biomarkers over the concentration range examined.

D-08

Effects of Oral Androstenedione on Steroid Metabolism in Liver of Pregnant Female Rats
T. J. Flynn, P. P. Sapienza, P. W. Wiesenfeld, R. L. Sprando, S. Sahu, I. A. Ross, C. S. Kim, M. W. O'Donnell, W. D. Johnson, T. F. Collins, FDA, Laurel, MD

Alteration of steroid hormone metabolism in liver is a potential mechanism of endocrine disruption. Steroid hormones are oxidized by numerous cytochrome P450s (CYPs) in liver. Androstenedione, the in vivo metabolic precursor of both androgens and estrogens, is also a dietary supplement used to enhance athletic performance. It is not known if dietary androstenedione can modulate normal hormone levels by altering liver enzyme activities. Alteration of hormone levels could be especially devastating during pregnancy. In a teratology study, mature female rats were gavaged with 0, 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Livers were removed from dams concurrent with Caesarian sections on gestation day 20. Liver microsomes were incubated with 200 µM testosterone. The reaction products were isolated and analyzed by LC. There were no significant effects on the formation of 15a-, 16 a-, or 2a-hydroxytestosterone. Formation of 16a-, 15b-, 7a-, 16b-, and 2b-hydroxytestosterone was increased significantly vs. control at the highest dose level only. Formation of 6b-hydroxytestosterone increased significantly at both the 30 and 60 mg/kg/d dose levels. Endogenous formation of androstenedione decreased significantly at 5 mg/kg/d but was not significantly different from control at higher doses. The data suggest that high dietary levels of androstenedione induce some maternal liver CYPs that metabolize steroid hormones. Until data acquisition from the teratology segment of this study is completed, it is unknown whether these alterations in steroid metabolism are associated with adverse effects on either the dams or their offspring.

D-09

Oral Androstenedione Decreases Doscosahexaeonic Acid in Livers of Pregnant Female Rats
P. W. Wiesenfeld¹, P. P. Sapienza², T. J. Flynn², S. Sahu², C. S. Kim², I. A. Ross², W. D. Johnson², R. L. Sprando², T. F. Collins², ¹OARSA, FDA, Laurel MD, ²OARSA, FDA, Laurel, MD

Anabolic steroid hormones alter sex hormone and lipid metabolism. Androstenedione, a naturally occurring steroid hormone, is widely used as a dietary supplement. Because androstenedione is metabolized to androgens it is used to increase muscle mass. It is typically taken by athletes, and possibly by women of child bearing age. In vitro studies in our laboratory using hepatocytes exposed to androstenedione (> 25µg/ml), suggested that it may also increase intracellular lipid (hepatosteatosis). Since changes in steroid hormone production and lipid metabolism might alter pregnancy outcome, the present study was conducted to address this concern. In a teratology study, mature female rats were gavaged with vehicle (corn oil), 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Livers were removed from dams on gestation day 20. No dose effects on animal and liver weight were observed. Total fat, cholesterol, protein and fatty acids profiles were determined in liver homogenates. As the dose of androstenedione increased (0 to 5 mg/kg) there was a small decrease in total fat, but there were no notable changes in the amount of total liver cholesterol or protein. As dietary androstenedione increased there was a noteworthy decrease in liver docosahexaenoic acid (DHA, C22:6) from 12.4% to 10.9% of total fatty acids. DHA is an essential fatty acid for fetal neural and visual development. The possible adverse effects of androstenedione on neural or visual development remain to be determined.

D-11

Characterization of the Effect of Deoxynivalenol on Selected Male Reproductive Endpoints
R. L. Sprando¹, T. F. Collins¹, T. N. Black¹, N. Olejnik¹, D. I. Ruggles², ¹Division of Toxicology, ²Division of Mathematics

The effect of deoxynivalenol (DON) exposure on selected male reproductive endpoints was examined. Adult male rats received vehicle alone (distilled water) or DON (in distilled water) at one of four concentrations (0.5, 1.0. 2.5 or 5.0 mg/kg body weight) by gavage for 28 days. Body weight gain and final body weight of animals in the 5.0 mg/kg dose group and feed consumption in animals in the 2.5 mg/kg and 5.0 mg/kg dose groups were reduced compared to controls. Fluid consumption was not affected in any of the treated groups. Epididymal and seminal vesicle weights expressed per gram of body weight and per gram of brain weight were significantly lower than controls in the 2.5 and 5.0 mg/kg dose groups. Prostate weight expressed per gram of brain weight and per gram of body weight was significantly lower than controls in the 5.0 mg/kg dose group. Dose related decreases in homogenization resistant testicular spermatid numbers and cauda epididymal sperm numbers per gram of cauda epididymis were observed in the 5.0 mg/kg DON treatment group. Dose related changes in sperm motility were not observed across treatment groups however swimming speed was increased in the 2.5 mg/kg dose group. Serum FSH and LH concentrations were increased in a dose dependent manner across all treated groups while serum testosterone concentrations were decreased in a dose related manner across all dose groups. A slight increase in germ cell degeneration, sperm retention and abnormal nuclear morphology was observed in the 2.5 mg/kg and 5.0 mg/kg dose groups.

D-12

The Radioactive Drug Research Committee - Microdosing
O. H. Suleiman, R. Fejka, M. Walsh, R. Farkas, FDA

Since 1975 the Food and Drug Administration (FDA) has allowed basic clinical research involving radiolabeled drugs at microdose, or sub pharmacologic levels, to be conducted without an FDA Investigative New Drug (IND) application. Although the concept of "microdosing" has only been introduced recently, this program, codified in 21 Code of Federal Regulations (CFR) 361.1 established a process by which a medical institution can conduct such research under an FDA approved Radioactive Drug Research Committee (RDRC). The program's intent was to encourage preliminary human research. Currently there are 83 committees overseeing such research in the United States.

RDRC research is considered basic science research. This research is intended to obtain basic information regarding the metabolism (including kinetics, distribution, dosimetry, and localization) of a radioactive drug. It is not intended for therapeutic, diagnostic, or preventive benefit to the research subject, and is not intended to determine the safety and effectiveness of the drug. These type of investigations would require an IND. RDRC research must be conducted in a facility licensed for the use of radioactive materials in humans and conducted by qualified study investigators. The research protocols must be approved by the associated Institutional Review Board (IRB). Research subjects must be properly selected, informed, and any adverse events must be reported to the FDA. Quality assurance testing of the radioactive drugs must be performed. Both the pharmacologic dose of the radioactive drug to be administered must be low enough to not cause any clinically detectable pharmacologic effect in humans, and specified organ and whole body radiation dose limits must not be exceeded.

D-13

Fish Drug Analysis - A Searchable Database of Residues and Pharmacokinetics Data in Fish.
R. Reimschuessel¹, L. Stewart², E. E. Squibb³, K. Hirokawa⁴, T. Brady⁵, D. H. Brooks⁶, B. Shaikh⁷, C. Hodsdon⁸, ¹CVM, FDA, Laurel, MD 20708, ²Northwestern U., Evanston, IL 60201, ³U. Maryland, Columbia, MD 21044, ⁴St. George's University, P.O. Box 7, Grenada, West Indies, ⁵U. Maryland, College Park, MD 20770, ⁶CVM, Rockville, MD 20855, ⁷CVM, Laurel, MD 20708, ⁸Independent Computing, Morris Plains, NJ 07950

There are currently very few FDA approved drugs for use in fish, primarily due to the high cost of conducting the research to obtain the data required for drug approval. One strategy recommended to reduce costs is to define species groups for particular studies, such as the human food safety evaluation. A large amount of supportive data, however, must be compiled in order to demonstrate that information from a model species will accurately predict the responses of all the members of a group. Compared to mammals, there is a general lack of the literature describing drug depletion or pharmacokinetic studies in fish. Certainly there are a few fish species, channel catfish and salmonids, which dominate the literature. As part of CVM/FDA's commitment to streamlining the drug approval process for minor species, we have begun to develop a database of literature detailing drug metabolism, depuration and pharmacokinetics in multiple fish species. This current database consists of over 150 articles which include data from 50 species (40 genera) of fish. Data fields include genus species, water temperatures, the average animal weight, sample types analyzed, drug name, dosage, route of administration, metabolites identified, protein binding, clearance, volume of distribution in a central compartment (Vc) or volume of distribution at steady state (Vd) and drug half-lives (t ½). Additional fields list the citation, authors, title, and internet links. This database will be a valuable resource to investigators of drug metabolism in aquatic species as well as government and private organizations involved in the drug approval process for aquatic species.

D-14

Pre-Clinical Testing of Plant-Produced a-Galactosidase A in a Mouse Model of Fabry Disease
M. P. Gelderman¹, K. L. Oliver², A. T. Yazdani³, G. J. Murray³, S. J. Garger², R. B. Holtz², R. O. Brady³, ¹LCH, DH, CBER, Bethesda MD 20892, ²LSBC, Corp., Vacaville CA 95688, ³DMNB, NINDS, NIH, Bethesda MD 20892

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of the lysosomal enzyme, alpha-galactosidase A (a-gal A). This results in an accumulation of the glycosphingolipid globotriaosylceramide (Gb₃) in vascular endothelial cells and mainly affects the skin, kidneys, heart and brain. Enzyme replacement therapy is currently under intensive investigation in patients with this disease. The present study examined the therapeutic potential of plant-produced a-gal A in a-gal A knockout (KO) mice, a murine model of Fabry disease.

Groups of KO mice (n=8) received a total of eight intravenous injections of a-gal A at varying doses (0.05 - 0.5 mg/kg body weight) every two weeks over a period of 16 weeks. All of the treated mice remained healthy throughout the studies although they produced enzyme-specific antibodies as determined by ELISA. This humoral response is similar to that observed in many Fabry patients who received a-gal A replacement therapies. The saline-injected animals (control group) did not produce any detectable anti-a-gal A antibodies.

Analysis of the IgG isotypes, revealed only IgG₁ antibodies in the treated mice. There was no indication of neutralizing effect of the exogenous a-gal A. Analysis of multiple tissues of the mice five days after the last administration of a-gal A at 0.5 mg/kg body weight showed the following reductions of tissue Gb₃: liver 84±2 %; heart 65±3 %; spleen 70±8 % and 33±5 % in the kidneys. No significant reduction was seen in the lungs, small intestine or brain. Overall, this study provides evidence for a therapeutic potential of plant-produced a-gal A.

D-15

Assessment of Skin Absorption and Metabolism of Arachidonic Acid & Glyceryl Arachidonate Using In Vitro Diffusion Cell Techniques
A. R. Eppler¹, M. E. Kraeling¹, R. R. Wickett², R. L. Bronaugh¹, ¹Office of Cosmetics and Colors, USFDA, Laurel, MD 20708, ²University of Cincinnati, Cincinnati, OH 45267

Arachidonic acid (AA) has been used in cosmetics as a surfactant-cleansing and -emulsifying agent. Glyceryl arachidonate (GA), a skin conditioning agent and emollient, may be hydrolyzed in skin to AA, whose metabolites are pro-inflammatory mediators of irritation. In vitro percutaneous absorption/metabolism studies were initiated because of the lack of AA dermal absorption data observed by the Cosmetic Ingredient Review Panel. AA and GA were applied in an oil in water emulsion (2mg/cm²) for 24hr to 200um thick skin samples in flow-through diffusion cells. Skin penetration, expressed as a percent of the applied dose, was analyzed on viable fuzzy rat and human skin with [¹⁴C]AA, while [³H]GA was applied to viable and cadaver human skin. To assess for skin metabolism, Epiderm was similarly dosed with [³H]GA, then analyzed by HPLC. Absorption of AA through rat skin was 19.8 ± 2.5% (mean ± SEM) compared to only 1.3 ± 0.1% through human skin. Total AA penetration (receptor fluid plus skin levels) in rat and human skin was 50.3 ± 3.1% and 18.4 ± 2.0%, respectively. Total GA penetration of viable skin was 21.4 ± 1.1% with only 3.2 ± 0.2% absorbed through the skin. In cadaver skin, 2.2 ± 0.3% GA was absorbed through skin with a total penetration of 14.8 ± 0.9%. Epiderm receptor fluid analysis found ~50% absorption and 3.0 ± 2.1% GA conversion to AA. In conclusion, AA and GA penetration was observed through human and/or rat skin while GA absorbed through Epiderm exhibited metabolism to AA.

D-16

Effect of chronic azathioprine treatment on germ-line transmission of Hprt mutations in C57BL/6 mice
S. V. Bendre, J. G. Shaddock, V. N. Dobrovolsky, R. H. Heflich, DGRT, FDA/NCTR, Jefferson, AR

Chronic treatment with the immunosuppressant drug azathioprine (Aza) can result in large increases in HPRT lymphocyte mutant frequency (MF) in both humans and mice (e.g., 1 x 10^-1vs. normal MF of 1-10 x 10^-6). These increases are not due to mutation induction, but to the selection and amplification of pre-existing HPRT mutant lymphocytes. A similar selection in germ cells could result in an increased frequency of offspring with the Lesch-Nyhan syndrome. To test this possibility, 55 male C57BL/6 mice were treated with 10 mg/kg Aza for 24 weeks; 10 control mice were treated with the vehicle. Each of these animals then was bred with untreated females for a total of 8 weeks. The resulting female offspring were sacrificed at 28 days of age to measure lymphocyte Hprt MFs. Since the Hprt gene is on the X-chromosome, female mice with a germ-line Hprt mutation should have Hprt MFs of 1-5 x 10^-1. After completing the breeding, all the surviving Aza-treated mice also were sacrificed and their Hprt MFs determined. Twelve of 53 Aza-treated breeders had highly elevated Hprt MFs (0.001-2.4 x 10^-1), indicating that the Aza treatment had selected for somatic cell mutants. None of 371 female offspring (331 from treated fathers), however, had Hprt lymphocyte MFs consistent with germ-line transmission; 364 had MFs of 0-6 x 10^-6(normal for mice of this age), and 7 had slightly elevated MFs of 16-55 x 10^-6. The results indicate that Aza therapy does not promote high levels of germ-line Hprt mutation transmission.

D-17

In Vitro Percutaneous Absorption and Metabolism of 2-Nitro-p-Phenylenediamine in Human and Fuzzy Rat Skin
J. J. Yourick, R. L. Bronaugh, Office of Cosmetics and Colors, USFDA, Laurel, MD

2-Nitro-p-phenylenediamine (2NPPD) is a "coal-tar" dye used in semipermanent and permanent (tinting color) hair dye products. We previously reported on the percutaneous absorption and metabolism of 2NPPD in fuzzy rat skin and intestinal tissue (Toxicologist, 36:188, 1997). These studies were expanded to include absorption and metabolism in human skin from ethanol and a semipermanent formulation, and in fuzzy rat skin from ethanol and a permanent formulation. Dosing vehicles were applied to skin for 30 min and absorption was measured over 24 hr using flow-through diffusion cells (0.64 cm²). 2NPPD metabolites were determined by HPLC. In human skin, the percentages of total applied dose absorbed (receptor fluid + skin) over 24 hr were 9.2 ± 5.7 (mean ± SD) and 9.5 ± 3.2 for the ethanol and semipermanent vehicles, respectively, with approximately 3% remaining in skin. In rat skin, the percentages of total applied dose absorbed over 24 hr were 9.3 ± 1.2 (mean ± SEM) and 4.2 ± 0.1 for the ethanol and permanent formulation vehicles, respectively, with approximately 3% remaining in skin. 2NPPD rapidly penetrated both human and fuzzy rat skin with 47% and 64%, respectively, of the total absorbed dose found in the receptor fluid within 3 hr. In human and rat skin, 2NPPD was metabolized to triaminobenzene and N4-acetyl-2NPPD. 2NPPD was also metabolized to a sulfated 2NPPD metabolite in rat skin, but not in human skin. In summary, 2NPPD is rapidly absorbed and extensively metabolized in both human and rat skin. 2NPPD metabolism upon absorption appears to be species-dependent.

D-18

An Assay of Eiconsanoid Expression in Brain Tissue Following Injury and Treatment with a COX-2 Inhibitor
S. A. Varnum¹, L. Rossi¹, H. Yue², M. R. Borenstein², M. F. Barbe², A. E. Barr², K. I. Strauss³, ¹FDA, ²Temple University, ³University of Cincinnati

An LC/MS method to simultaneously identify and quantitate a wide array of bioactive eicosanoids derived from rat brain has been developed, validated and applied to aid in understanding expression mechanisms associated with injury, inflammation and treatment. Prostaglandins (PGs), hydroxyeicosatetraenoic acids (HETEs), dihydorxyeicosatetraenoic acids (DiHETrEs) and epoxyeicosatrienoic acids (EETs) expression occur as a result of oxidation of arachadonic acid. Their expression in tissue is followed by reverse-phase HPLC separation and detection under negative selected ion monitoring mode of quadrupole mass spectrometer (MSD) with atmospheric electrospray ionization source. PGF2a, PGE2, PGD2, PGJ2, 14,15-DiHETrE, 11,12-DiHETrE, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 9-HETE, 8-HETE, 5-HETE, 11,12-EET, 5,6-EET, 14,15-EET, 8,9-EET, arachidonic acid were the eicosanoids analyzed in this study. Deuterium labeled 15(s)-HETE-d8, 14,15-EET-d8, 11,12-EET-d8, 8,9-EET-d8, and AA-d8 at 5, 6, 8, 9, 11, 12, 14, 15-position were used as internal standards for quantitation. 24 eicosanoids were separated by LC/MS and quantified using selected ion monitoring (SIM) mode. The LOQs and LODs were estimated to be 1ng/ml and 0.1 attog/ml individually by flow injection analyses. This method has been applied to the injured rat brain treated with a cyclooxygenase-2 inhibitor, DFU. Significant variations in eiconsanoid expression are noted in injured rats and in rats treated with the COX-2 inhibitor. Variations in expression were also noted with treatment protocols.

D-19

Clinical Pharmacology and Biopharmaceutics Issues for Regulatory Submissions Involving Transdermal Delivery Systems
D. J. Chatterjee, A. Parekh, J. Hunt, H. Malinowski, OCPB, CDER, FDA, Rockville, MD

Purpose: Transdermal delivery systems (TDS) are currently the subject of an increasing number of regulatory submissions. Regulatory research within the Agency is directed towards optimizing critical Clinical Pharmacology and Biopharmaceutics (CPB) information during drug development, regulatory review process and labeling decisions.

Methods: Review experience with current NDAs/INDs and previous regulatory submissions involving TDS are being compiled to evaluate value of CPB studies for product performance and labeling information.

Results: The following emerge as significant regulatory CPB issues with TDS: (i) Comparability on exposure-response - claims of altered PK/PD profiles with TDS is evaluated after comparison with conventional dosage form; (ii) Patch adhesion - dependent on patch design, adhesion is assessed best during phase 3 clinical trials; (iii) Site of application - site 'switch-ability' may be justified with adequate PK data; (iv) Environmental/weather conditions - recommended that the effect of heat, humidity, sun-burning, elevation of body temperature, shaving, showering and health-club conditions be addressed; (v) In vitro release - decision on choice of time points for specifications be made earlier than pre-NDA stage; correlation of in vitro dissolution data to in vitro skin flux across human cadaver skin may provide valuable IVIVC information (vi) In vivo release - labeled delivered dose is generally calculated from relative bioavailability and assay for drug remaining after patch use (vii) Drug transfer on contact - recommended that potential for drug transfer to others be addressed (viii) Intrinsic Factors - issues related to age/race/gender/body weight may be addressed by enrolling adequate number and variety of patients (ix) Disposal - since many drugs considered for TDS have abuse potential, amount remaining in patch after use is a critical determinant of abuse potential and adequate disposal techniques.

Conclusions: Information from CPB studies is critical to ensure optimal product quality and use. Inclusion of CPB information in product labels will exceedingly benefit health care providers and patients.

D-20

In Vitro Percutaneous Absorption of Acrylamide and Styrene in Human Skin From Cosmetic Vehicles
M. E. Kraeling, R. L. Bronaugh, Office of Cosmetics and Colors, USFDA, Laurel, MD

Acrylamide (ACR) and styrene (STY) are residual monomers of safety concern that remain as an impurity in polymers used in hair, nail and skin care products. These residual monomers may be substantially absorbed through skin. Therefore, we conducted studies to measure the extent of ACR and STY absorption in human skin relevant to exposures from consumer products. [¹⁴C]ACR (specific activity of 5 mCi/mmol) was applied to skin in flow through diffusion cells for 24 h using an oil-in-water (O/W) emulsion at doses of 0.2, 2.2 and 13.3 µg/cm². ACR was also applied in a 2% polyacrylamide gel cream at a dose of 0.3 µg/cm². The amount of ACR absorbed in skin layers and receptor fluid was measured and expressed as the percent of the applied dose absorbed. ACR was rapidly absorbed from both formulations into receptor fluid at 33 - 53%, with peak absorption occurring at 6 h. Only 4 - 7% ACR remained in the skin. [¹⁴C]STY (20 mCi/mmol) was applied to skin in the O/W emulsion at a dose of 4.1 µg/cm². STY was also rapidly absorbed with peak absorption occurring at 6 h. Only 1.2% of the applied dose of STY was absorbed into the receptor fluid with 0.12% remaining in the skin. The low absorption of STY may be due predominately to the volatility of this monomer.

D-21

Gill Histopathology Induced by the Mid-Atlantic, Bloom-Forming Dinoflagellate Karlodinium micrum.
J. R. Deeds¹, R. Reimschuessel², A. R. Place³, ¹US FDA Washington Seafood Laboratory, Laurel, MD 20708, ²US FDA Center for Veterinary Medicine, Laurel MD 20708, ³University of Maryland Biotechnology Institute, Center of Marine Biotechnology, Baltimore, MD 21202

Karlodinium micrum is a common dinoflagellate in US Mid-Atlantic waters that has been associated with fish kills in other parts of the world. K. micrum occasionally forms large blooms in shallow, poorly flushed estuarine tributaries typical of this geographic region. K. micrum is often found in association with dinoflagellates of the genus Pfiesteria but all dinoflagellates that co-occur with Pfiesteria were previously believed to be non-toxic in US waters. We have recently shown that US east coast isolates of K. micrum produce a suite of ichthyotoxins which we believe to be responsible for several Maryland fish kills dating back to 1996.

In this study we examined the effects of KmTx 2, isolated from a 2002 South Carolina fish kill, on zebrafish (Danio rerio). We have previously shown KmTx 2 to cause cellular toxicity by non-specifically increasing permeability to vertebrate plasma membranes. Here we found KmTx 2 to be acutely toxic (100% in <1 hr) to zebrafish at concentrations > 0.5 µg/ml. At concentrations >0.1 µg/ml some toxicity occurred (40%) with gills appearing to be the main organ affected. Environmental concentrations of 0.1-0.8 µg/ml KmTx 2 have been measured during fish kills. Primary gill alterations included a loss of secondary structure in gill lamellae including: occlusion of interlamellar spacing, edema, and mild necrosis. At concentrations > 0.5 µg/ml severe necrosis of all gill epithelia was observed. In fish surviving the 4 hour treatment, TEM analysis of gills showed alterations to gill chloride cells.

This study provides a mechanistic explanation for the association between blooms of K. micrum and fish kills.

D-22

Preclinical Development of New Drugs That Exhibit Side Effects of Enhancing Thyroid Hormone Metabolism by Induction of Hepatic UDPGT Enzymes: Inadequacy of Using Rat as an Animal Model for Predicting Human Risks of an IND and NDA
KUEI-MENG. WU, DAVDP, ODEIV, FDA, Rockville, MD

New drugs that enhance glucuronidation of thyroid hormones in rats often trigger a sequence of toxicity events during chronic administration: reduction of T4/T3, elevation of TSH levels and thyroid gland hyperfunction/growth. Hepatocellular hypertrophy and thyroid follicular hyperplasia are often observed with increased liver and thyroid organ weights. The thyroid may develop goiter, adnomas and carcinomas over long-term drug administration. This unique toxicity profile appears to be species-specific because thyroxine in rodents is metabolized rapidly, without thyroid hormone binding globulin that serves as a reserve, as in humans. Thus, elevations of TSH were not reported in humans for UDPGT inducers such as delavirdine, nicardipine, phenobarbital and spironolactone, all of which produce thyroid tumors in rats. Further, the human thyroid is less sensitive to prolonged TSH stimulation than that of the rat (e.g., endemic goiter patients with high TSH due to iodine deficiency do not develop thyroid cancer). In view of the species difference in sensitivity of the thyroid between rodents and humans, using the rat as an model to explore target organs of toxicity for a new drug that significantly enhances T4/T3 glucuronidation and increases TSH levels would not be adequate. In this case, the rat exhibits a compromised and dysfunctional hypothalamo-pituitary-thyroid system that, together with the secondary effects resulting from thyroid disorders, would confound the toxicity profile explored in preclinical toxicity testing and render the model an inadequate risk predictor for the new drug in humans. Under such conditions, IND and NDA sponsors of drugs exhibiting this activity profile should be encouraged to use alternative animal species for toxicity exploration to provide a more meaningful human risk prediction.

D-23

An experimental PDE IV inhibitor-induced vascular injury (VI) associated with increased mast cell degranulation and elevated serum levels of acute-phase proteins in Sprague-Dawley rats.
J. Zhang¹, R. Honchel¹, J. L. Weaver¹, A. Knapton¹, E. H. Herman¹, F. M. Goodsaid², J. W. Davis², I. Y. Rosenblum², F. D. Sistare¹, ¹OTR, FDA, White Oak MD, ²Schering-Plough Research Institute, Lafayette, NJ

Evaluation of drug-induced VI has been hampered by a lack of early clinical signs and accessible biomarkers. As part of an ongoing effort to establish a rat model of drug-induced VI, rats were gavaged with an experimental PDE IV inhibitor at doses of 3 to 160 mg/kg/day (mkd) for 1 to 9 days and sacrificed 24 hr after the last dose. Doses of 6 mkd and higher resulted in systemic toxicity. Vascular lesion development appeared to be both time- and dose-dependent. A semiquantitative scale (0 to 5+) was used to grade mesenteric vascular lesions. Lesions increased in both incidence and severity from 0.6+ to 4.7+ following three doses of 3, 6, or 40 mkd. Similar lesions and scores were observed following 9 doses of 3 mkd (1.2+) or 7 doses of 9 mkd (3.0+). In mesenteric vasculature, the compound induced arteriolar fibrinoid necrosis, arterial hemorrhage and necrosis, and perivascular inflammation, accompanied by endothelial cell (EC) activation, mast cell degranulation, increased nitrotyrosine formation on EC, and apoptosis of EC's and smooth muscle cells. VI correlated well with the extent of neutrophilia, lymphopenia, and increased serum levels of haptoglobin and a1-acid glycoprotein at all time points. Increased serum IL-6 was seen only at early time points. The pathologic findings suggest that mechanisms responsible for the VI might involve peroxynitrite-mediated damage to EC and a concurrent activation of certain immune system components. Further work is required to refine the model and elucidate the pathogenesis of VI. We are evaluating additional blood-based biomarkers to identify drug-induced VI and differentiate it from inflammation. A more specific biomarker panel will be critical to expand our capabilities in safety monitoring of preclinical or clinical studies with PDE inhibitors.

D-24

Enhanced Sensitivity of Female Rats to Azoxymethane following Dietary Soy Isoflavone Supplementation
B. Magnuson¹, K. Daly¹, M. Malik¹, T. Wang², S. Francke-Carroll³, ¹Dept. of Nutrition and Food Science, University of Maryland, College Park, MD, ²Phytonutrients Laboratory, USDA, Beltsville, MD, ³Pathology Branch, OSAS, US FDA, College Park, MD

Soy isoflavones are becoming popular supplements among middle-aged women based on their potential to protect against breast cancer and their alternative use as hormone replacement therapy. The numerous published animal feeding studies with soy isoflavones used mainly young male rats or mice. We report here observations made in female rats during a preliminary study conducted to investigate the effects of dietary soy supplementation and age on colon cancer development using the well known, azoxymethane (AOM)-xenobiotic to induces colon cancer. Young (1 mo), mature (11 mo) and old (22 mo) female Fisher 344 rats were fed either the control diet (AIN-93G) or the AIN-93G diet containing 0.4% isoflavone supplement for 1 week. Rats were then injected with 20 mg/kg AOM and maintained on the diets for another 12 weeks. In all age groups, isoflavone-supplemented rats had greater weight loss and a slower recovery compared to rats fed the control diet. Weight loss due to AOM and time needed to regain weight increased with age. Five of the 21 rats fed the isoflavone-supplemented diet died prematurely, whereas all control diet rats survived. Uterine weights, serum estradiol and serum isoflavone levels were measured. Evaluation of the expression of the P-450 enzyme and CYP2E1 is in progress. The increased sensitivity of older female rats to the AOM / soy isoflavone-diet combination needs further examination because women, and particularly older women, are the prime target population for consumption of soy supplements.

D-25

Pulmonary Targeting of Sustained Release Formulation of Budesonide in Neonatal Rats
V. Arya¹, I. Coowanitwong², B. Brugos², W. S. Kim², R. Singh², G. Hochhaus², ¹CDER/FDA/Rockville, MD, ²University of Florida

The high incidence of adverse effects in preterm infants after systemic corticosteroid administration has provided an impetus for exploration of alternate routes of corticosteroid delivery. The use of the inhalation route for delivering corticosteroids has been widely recognized but has surprisingly met with modest success. We hypothesize that the pulmonary administration of sustained release formulations of inhaled corticosteroids to preterm infants will result in a higher benefit/risk ratio as compared to inhalation therapy with the free (non-sustained) drug. To test our hypothesis, ex vivo receptor binding studies were performed to monitor the cumulative lung, liver and brain receptor occupancies as a function of time in neonatal (10-11 days old) rats after intratracheal instillation of either uncoated budesonide or poly (l-lactic acid) (PLA) coated budesonide. The average receptor occupancies in the lung, liver and brain after administration of uncoated budesonide were 58.4 ± 12.9 %, 56.4 ± 6.8 % and 38.3 ± 6.7 % respectively. However, after administration of PLA coated budesonide, the average AUC estimates in the lung, liver and brain were 75.8 ± 3.7 %, 46.6 ± 14.5 % and 29 ± 7 % respectively. The results from our study suggest sustained receptor occupancy in the lungs of neonatal rats after administration of PLA coated budesonide that results in lower systemic exposure (as indicated by low liver receptor occupancy). The data strongly underscore the urgent need to develop sustained release pulmonary-targeted delivery systems of corticosteroids for the treatment of CLD in preterm infants. The administration of inhaled corticosteroids using pulmonary targeted drug-delivery systems will potentially result in higher local effects and reduced side effects.

Note: This work was performed at the University of Florida as part of Dr. Arya's Doctoral Dissertation.

D-26

Do P-Glycoprotein Transporters Modulate the Brain Permeability of Inhaled Steroids in Preterm Infants?
V. Arya¹, M. Issar², V. DeMarco², S. Shrestha², G. Hochhaus², ¹CDER/FDA/Rockville, MD, ²University of Florida

Poor development of vital organs predispose the preterm infant to pulmonary disorders such as chronic lung diseases (CLD). Corticosteroids prevent lung inflammation and help to reduce the incidence of CLD. However, administration of systemic corticosteroids in preterm infants is often associated with neurological complications such as cerebral palsy etc. We hypothesize that the poor development of the p-gp transporters in preterm infants (due to immaturity of the blood brain barrier) results in higher brain concentrations of the corticosteroid. TAP was administered intra-tracheally to neonatal (25 and 50 µg/kg) and adult rats (100 µg/kg). The % receptors occupied in the brain and liver at 1hr were determined using an ex vivo receptor binding assay. To assess the involvement of P-gp transporters, TAP was administered intravenously (100 µg/kg) to wild type mice and mdr1a knock out mice (mice lack expression of p-gp). The % free receptors in the local (brain) and systemic (liver) organs were monitored using an ex vivo receptor binding assay. AUC_0-6hrwas estimated from the % free receptors vs time profiles. The neonatal rats showed significantly higher brain receptor occupancy (44.5 ±13 % and 50.4 ± 25.7 % after 25 and 50 µg/kg respectively) than adult rats (11 ± 1.7%). The average hepatic receptor occupancies were similar in wild type and knockout mice (34.9 % vs 37.8 %). However, the average brain receptor occupancies were significantly higher in knockout mice (47.5 %) as compared to wild type mice (11.5%). The data strongly suggest the pivotal role played by P-gp transporters in modulating the permeability of steroids across the blood brain barrier.

Note: This work was performed at the University of Florida as part of Dr. Arya's Doctoral Dissertation.

D-27

Effect of Defibrillator-Drug Interactions on Cytosolic Calcium in Cultured Cardiac Myocytes.
R. Renukananda, V. Krauthamer, CDRH, FDA, Rockville, MD

The number of people receiving electrical defibrillation is constantly growing due to increasing prescription of these devices. This increases the risk for dangerous interactions between these defibrillator shocks and cardiovascular medications taken by patients. This project examines defibrillator-drug interactions and gives us preliminary results on the impact of interaction between medication and electrical defibrillation. From previous results, we hypothesize that following a defibrillator shock the beta adrenergic agonist isoproterenol, will improve intracellular calcium dynamics and shorten the duration of the arrest time which is the period of time following a shock prior to the restoration of normal heart rhythm. Isoproterenol is a medication used to treat shortness of breath, emphysema, asthma and other lung diseases. On the other hand, the L-type calcium channel blocker verapamil will increase the duration of arrest time. Verapamil is a drug used to control arrhythmias, high blood pressure and angina. Dissociated heart cells from 10 day old chicken embryos were tissue cultured and incubated in a shaker bath. The heart cells adhered to each other to form beating spherical aggregates which were later plated in Petri dishes. These cardiac myocytes were then stained with fura-2AM, a calcium specific dye for fluorescence recording. In the presence of isoproterenol (1 M), the duration of arrest time was decreased by 20-30%, and both the amplitude and duration (from peak to ½ amplitude) of the calcium action potential increased. These effects were reversed after the drug was wash-out. Preliminary results suggest that in the presence of verapamil (2 M), following a defibrillator shock decreases the calcium concentration during the arrest, increases the duration of the arrest, and decreases both the amplitude and duration of the calcium action potential. This demonstrated cellular interaction of pharmacologic agents with electric defibrillator shocks suggests a need for clinical investigations of such effects.

D-28

Phototoxicity, Photochemistry and Photophysics of 2,3-Diaminophenazine - a Common Contaminant in Hair Dyes.
P. K. Fu¹, C. Turro², ¹FDA, ²The Ohio State University

Aromatic amines, such as o-phenylenediamine (OPD), have been used extensively in commercial hair dyes and in the synthesis of agricultural pesticides. Air oxidation of OPD results in the formation of 2,3-diaminophenazine (DAP). Although DAP has been shown to be mutagenic in both prokaryotic and eukaryotic systems, its phototoxicity remains largely unexplored. The present work focuses on the pH-dependent photophysical properties of DAP, and demonstrates that DAP photoinduces DNA damage in pUC19 plasmid in vitro, as well as photocytotoxicity in human skin fibroblasts. DAP exhibited weak intercalative binding to double stranded DNA (K_b = 2.8 x 10³M^-1), and was able to nick plasmid DNA in the presence of oxygen upon irradiation with visible light. The concentration of DAP which resulted in 50% cell death (LC₅₀) was 172 µM in the dark and 13 µM following irradiation of the DAP-containing cell cultures with visible light (400 - 700 nm, 30 min, 5 J/cm²). The 13-fold increase in toxicity upon exposure to visible light shows the need for further study of the photocytotoxicity of contaminants such as DAP.

D-29

Database Consolidation of Significant Toxicological Data from Genetic Toxicity Studies Submitted to CFSAN/OFAS and CDER
J. N. Mayer¹, O. Lo¹, M. A. Cheeseman¹, C. P. Nelson¹, R. D. Benz², E. J. Matthews², N. L. Kruhlak², J. F. Contrera², C. Yang³, M. L. Twaroski¹, ¹OFAS, FDA, College Park, MD, ²OPS/ICSAS, FDA, Rockville, MD, ³LeadScope, Inc., Columbus, OH 43212

Structure searchable electronic databases are a valuable new tool that will assist the FDA in its mission to promptly and efficiently review incoming submissions. CFSAN/OFAS, in collaboration with LeadScope, Inc., is consolidating genetic toxicity data submitted in petitions from the 1960's to the present day. CDER/OPS/ICSAS is separately gathering similar information. Presently this data is distributed in various locations as paper files, microfiche, and non-standardized toxicology memoranda. The organization of the data into a consistent, searchable format should reduce paperwork, expedite the toxicology review process, and provide valuable information to industry that is currently available only to the FDA. Furthermore, by incorporating chemical structures with the genetic toxicity information, biologically active moieties can be identified and used to develop QSAR modeling and testing guidelines. Additionally, chemicals devoid of data can be compared to known structures, allowing for improved safety review through the identification of analogs. Four database frameworks have been created: bacterial mutagenesis, in vitro chromosome aberration, in vitro mammalian mutagenesis, and in vitro micronucleus. Controlled vocabulary for these databases has been established. The separate databases are all tied together into an overall genetic toxicity database for standardization and easy accessibility. Beyond the genetic toxicity databases described, additional databases for subchronic, chronic, and teratogenicity studies are in preparation.

D-30

Loratadine and Terfenadine Interaction with Nefazodone: QT Implications
S. H. Haidar¹, C. Rosebraugh², E. O. Fadiran³, R. J. Meyer², ¹OGD, FDA, Rockville, MD, ²OND, FDA, Rockville, MD, ³OCPB, FDA, Rockville, MD

Loratadine (Claritin®) is an anti-histamine used widely in the treatment of symptoms of hay fever and other allergies. Terfenadine is another anti-histamine which has been withdrawn from the market due to cases of Torsades de Pointes associated with the drug's prolongation of the QT interval. Both drugs are substrates of cytochrome P450 3A (CYP3A). Nefazodone is an anti-depressant and a well known inhibitor of CYP3A. A drug interaction study was conducted to evaluate the effect of co-administration of nefazodone on the pharmacokinetics as well as pharmacodynamics (QT prolongation) of loratadine and terfenadine. This was a randomized, double-blind, multiple-dose study in sixty seven healthy subjects (males and females) divided into five treatment groups: Terfenadine + placebo (n = 13), terfenadine + nefazodone, (n = 14), loratadine + placebo (n = 13), loratadine + nefazodone (n = 14), and placebo + nefazodone (n = 13). Blood samples and ECG data were collected on days 7 and 15 of the study. The results of this study were published by Abernethy et al., in 2001. Subsequently, the data were submitted to the FDA for re-evaluation of the QT analysis using an approach different from the one used by the authors'. The FDA evaluated changes in the QT interval, while the authors presented actual QT values. Results suggest Fridericia's method provided the least bias in heart rate correction. Additionally, FDA analysis showed that co-administration of nefazodone resulted in pronounced increases in the QT interval for terfenadine, and little effect on loratadine pharmacodynamics.

D-31

Ceramide Mediated Release of Membrane Microparticles from Endothelial Cells is Controlled by Caspase 1 Activity and in Contrast to Executive Apoptotic Blebbing is Kinase Independent
J. Simak, J. Novak, Laboratory of Cellular Hematology, Division of Hematology, OBRR, CBER, FDA, Rockville, MD 20852

Flow cytometry analysis of endothelial cell membrane microparticles (EMP) in blood is a valuable diagnostic tool for studying vascular injury and may be used for testing in vivo adverse effects of biologics, drugs, and devices. Little is known, however, about the mechanism of EMP release. We studied the role of cytoskeleton, ceramide pathways, and caspases in EMP release from cultured human umbilical endothelial cells (HUVEC) induced by an antineoplastic drug camptothecin (CPT). EMP of 0.2-1.5µm in size stained with annexin V-FITC were counted. Myosine ATPase inhibition by 5 nM 2,3-butanedione monoxime markedly enhanced CPT-induced EMP release (23,100±1,600 EMP/10³cells/4hrs) as compared to CPT treatment alone (13,700±1,500 EMP/10³cells/4 hrs). Destabilization of actin with 100 nM cytochalasin D had only a slight inhibitory effect. A general kinase inhibitor staurosporin (ST, 1 µM ) alone induced a release of very high counts of EMP (42,300±3,000 EMP/10³ cells/ 4 hrs). Treatment of HUVEC with 50 µM C₂-ceramide alone, as well as accumulation of the cell's own ceramide by an inhibitor of acid ceramidase (50 M N-oleoyl ethanolamine), caused a significant EMP release. Preincubation of HUVEC with 200 µM caspase 1 inhibitor Z-YVAD-FMK resulted in a 47±16% inhibition of C₂-ceramide-induced EMP release, while 200 M caspase 3 inhibitor Z-DEVD-FMK caused 5±11 % inhibition only. Our results indicate that a CPT-induced EMP release in HUVEC is kinase and myosine ATPase independent and is not affected by actin destabilization. In addition, a ceramide mediated EMP release seems to be controlled by caspase 1 but not caspase 3 activity, suggesting that it may occur before the execution phase of apoptosis.

D-32

Gentamicin drug residue levels in kidney biopsy samples obtained from live, standing holstein steers
O. A. Chiesa, R. Cullison, K. Moulton, D. Heller, C. Nochetto, M. Smith, M. Thomas, OR, FDA, Laurel, MD

Bovine treated with gentamicin are known to harbor kidney drug residues for many months following the last dose. Due to the high cost of maintaining a large number of gentamicin dosed steers or cows, the detailed gentamicin residue depletion has not been defined. The objective of this study is to reduce the total number of steers required to perform a gentamicin drug residue depletion study and to reduce the inter-animal variability by obtaining multiple, sequential kidney biopsy samples from each animal.

Eight healthy adult Holstein steers were dosed intramuscularly with three doses of gentamicin sulfate (4 mg/Kg). The kidneys of each animal were biopsied three times over a period of 100 to 180 days following the last dose of gentamicin The concentrations of gentamicin in the kidney biopsy samples were determined with a LC-MS-MS procedure with a sensitivity of 25 ppb. At 3 months following the last dose of gentamicin the kidney level averaged 250 ppb which depleted an additional 40% during the following 3 months.

The minimally invasive surgical procedure was successful and provided three biopsy samples in all of the animals. Each biopsy yielded a sample of 50 to 100 mg of kidney cortical tissue, sufficient to perform the analytical procedure. The minimally invasive surgical procedure provides multiple sequential biopsy samples from the living animal. The analytical evaluation of drug residue in these samples provides multiple time-related drug residue depletion data from the same live animal. The procedure allows titration of drug residue levels in each animal to required end points without slaughter.

D-PO-01

Underlying age-related liver pathology increases azoxymethane toxicity in F344 female rats in an aging study on colon cancer chemoprevention with dietary soy.
S. Francke-Carroll¹, K. Daly², T. Wang³, B. Magnuson², ¹OSAS, US FDA, College Park, MD, ²UMD, College Park, MD, ³USDA, Beltsville, MD

The influence of age and nutrition on the development of cancer is of growing interest to the public health. Aging studies in rodents, however, hold many challenges. Established cancer models, such as the azoxymethane (AOM) colon cancer model in rats routinely require a standardized dose of AOM for all test groups. Applied to comparative aging studies, animals that differ significantly in age may receive the same standardized dose of AOM based on body weight, although older animals are potentially more sensitive.

We report here inadvertent acute AOM-induced toxicity in female F344 rats due to underlying age-related liver pathology. The rats were part of a study investigating the effect of dietary soy and aging on the development of AOM-induced aberrant crypt foci (ACF). Control and test animals of three age groups (1, 11 and 21 month old), with seven animals per group received one dose of 20 mg/kg AOM s.c., to induce colonic ACF. Three 21-month animals and one 11-month animal developed fatal toxic signs within 96 hrs. post injection. Histopathological evaluation revealed acute liver-, adrenal gland- and gastro-intestinal-toxicity. Pre-existing age- related changes including hepatocellular adenomas, were also noted in the livers of all four animals. We further discuss how underlying, age-related liver pathology may influence the outcome of aging research in laboratory rodents.

CATEGORY E: BIOINFORMATICS/"OMICS"
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E-01

Survey of Genetic Variations Among 356 Human Genes That Are Established Pharmaceutical Intervention Targets
R. Jiang, K. Nandabalan, R. Judson, J. C. Stephens, C. Xu, Genaissance Pharmaceuticals,Inc., New Haven, CT 06511

We have discovered and compiled a database of single nucleotide polymorphisms (SNPs) and haplotypes of 356 pharmaceutically important human genes by re-sequencing specific regions, including exons, exon-intron boundaries, untranslated regions and 5' and 3' flanking regions, using a panel of 79 unrelated individual DNAs: 20 African-Americans, 19 East Asians, 20 European-Americans, 17 Hispanic-Latinos and 3 Native Americans. The level and distribution of variability in these genes, and the specific nucleotide changes involved in each polymorphism were examined across different ethnic groups. The biochemical properties of the SNPs were compared using the Grantham matrix to evaluate the functional impact of the nonsynonymous substitutions. We found that 62% of the SNPs identified occurred at a frequency of 1% or greater, and 30% of these SNPs overlapped with major public SNP databases. On average, we identified ~5.1 SNPs/kb and the promoter/upstream regions appeared to be the most polymorphic (~5.8 SNPs/kb). Our analysis showed that pharmaceutically relevant genes have a substantial amount of genetic variability in both coding and potential regulatory regions. This finding clearly indicates the need for a pharmacogenomics strategy from the very beginning of the drug discovery and development process. It also advocates for the ultimate individualization of therapies in terms of selection of medicine and dosing scheme based on the individual genetic profiles.

E-04

Natural Genetic Variation as a Tool in Understanding the Role of CETP in Lipid Levels and Disease
J. Thompson, M. Lira, K. Durham, A. Seymore, P. Milos, Pfizer Global Research and Development, Groton, CT, USA

Cardiovascular disease continues to be the major cause of death in developed countries despite the great strides made in prevention resulting from dietary changes, smoking cessation, and statin treatment of at-risk individuals. While improved compliance with these treatments will undoubtedly further reduce the rate of cardiovascular disease, its continued high prevalence make additional avenues for lowering risk necessary. Elevated HDL cholesterol (HDL-C) has been shown in numerous studies to have a protective effect on disease; but, at present, there are no marketed, well-tolerated pharmacological treatments for significantly raising HDL-C. One potential target for therapeutic intervention is inhibition of cholesteryl ester transfer protein (CETP). Despite our knowledge of CETP's role in lipid transfer, understanding its role in disease has been hindered by uncertainties surrounding the different possible mechanisms of HDL's protective effects and the lack of good animal models to adequately test the its role in disease processes. To circumvent these problems, much effort has been directed at examining genetic variation in CETP as a tool for more directly understanding its role in humans. We have more thoroughly characterized the genetic variation and used a variety of techniques to determine its association with HDL-C. Individual genotyping of CETP and other genes potentially involved in modulating HDL-C via fluorescence polarization revealed that CETP is more highly associated with HDL-C than endothelial lipase, LXRa, or LXRb. A partial genomic scan of 17,129,893 bp encompassing 290 genes using pooled DNAs from individuals with extreme HDL-C on high density arrays corroborated these results.

E-05

Pharmacogenetic analysis of polymorphisms in the chemokine receptors CCR5 and CCR2 in the clinical development of a CCR5 antagonist (UK-427, 857) for the treatment of HIV/AIDS
M. Penny¹, S. Myrand², C. Lin², M. Boucher¹, M. Man², I. James¹, ¹Pfizer Global Research & Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent, UK, ²Pfizer Global Research & Development, Ann Arbour Laboratories, Plymouth Road, Ann Arbor, MI 48105 USA

Both viral and host factors have a role in HIV pathogenesis; the best-characterised host genetic factor is the CCR5 receptor. Polymorphisms in CCR5 have been associated with resistance to infection and slowing of disease progression. UK-427, 857 is a CCR5 antagonist currently in development at Pfizer for HIV/AIDS, it is well tolerated and over 300 healthy volunteers have received single or multiple doses of the compound. As CCR5 antagonists function by binding to the host receptor to block HIV infection it is possible that an individuals response to UK-427, 857 may vary depending on their CCR5 genotype. A total of 16 CCR2/CCR5 SNPs were genotyped in a total of 165 healthy subjects recruited from four phase I studies for initial, exploratory, pharmacogenetic studies. Genotypes were correlated with clinical endpoints including CCR5 expression. Allele frequencies were not significantly different from published frequencies and there were no significant (p<0.05) deviations from HWE observed. CCR5 promoter haplotypes were determined using the EM algorithm and DNA sequencing. A novel CCR5 promoter haplotype was identified in two subjects. There was evidence that the CCR5-D32 polymorphism correlates with CCR5 expression, however no significant associations between CCR5 polymorphism and any safety and toleration clinical endpoints tested were observed. This study underlies the development of a pharmacogenetic strategy to assess the impact of genetic variation on response to UK-427, 857. Future pharmacogenetic studies will assess the impact of common CCR5 variants on safety, toleration and antiretroviral activity in response to UK 427, 857 in HIV infected patients.

E-06

Association of Serotonin Transporter Polymorphism with Early Response to SSRIs in Patients with Major Depressive Disorder
K. Durham¹, S. Webb¹, P. Chappell¹, C. Clary², J. Dunn², E. Giller¹, A. Seymour¹, H. Sakul¹, ¹Pfizer, Groton/NL, ²Pfizer, New York

A common polymorphism (5HTTLPR) within the promoter region of the serotonin transporter gene (SLC6A4) has been shown to influence response time to paroxetine in elderly depressed and overall response to fluoxetine in younger subjects with major depressive disorder (Pollock et al., 2000; Smeraldi et al., 1998). Based on these findings we hypothesized that a similar effect in response time to sertraline, fluoxetine, and paroxetine would be observed in subjects homozygous for the Long allele (LL) at 5HTTLPR in our clinical trials for Major Depressive Disorder (MDD). Results presented here encompass six independent studies (one with sertraline, and 2 and 3 studies respectively with fluoxetine and paroxetine as active comparators). Study 1 targeted an elderly population with MDD while studies 2-6 had a target population of age 18 and older. Primary efficacy variable reported here is HAM-D across all six studies. In all studies, except study 6, the LL group tended to respond to SSRIs sooner than subjects homozygous (SS) or heterozygous (LS) for the Short allele. In study 1, percentages of HAM-D responders at week 2 were 20.8 and 14.3% for the LL and SS/LS groups, respectively. For studies 2 through 6 combined, these percentages were 30.6 and 18.4% for the LL and SS/LS groups, respectively. These results complement evidence to previous reports suggesting that genetic variation in the serotonin transporter gene confers significant effect on the response time to SSRIs.

E-08

DNA Microarray Analysis of Bacterial Gene Expression in MMR-defective Salmonella enterica
S. A. Jackson¹, K. Dudley¹, S. Porwollik², M. McClelland², J. E. LeClerc¹, T. A. Cebula¹, ¹OARSA, FDA, Laurel MD, ²Sidney Kimmey Cancer Center, San Diego CA

The high incidence of mutators found among natural populations of E. coli and Salmonella can help to explain the emergence of antibiotic resistance and the penetrance of virulence genes within the prokaryotic community. Among these mutators, many are defective in methyl-directed mismatch repair (MMR), which is responsible for maintaining the integrity of the genome by correcting mismatches that occur during DNA replication and by acting as a barrier to genetic exchange between species. Defects in MMR result in a mutator phenotype characterized by a high incidence of spontaneous mutations and increased genetic exchange through homeologous recombination. In an effort to understand possible benefits of the mutator phenotype during infection by natural pathogens, our lab previously tested the effects of MMR defects on survival of the human pathogen Salmonella enterica serovar Enteritidis in a mouse hepatocyte cell line. We obtained the surprising result that these mutators survived and multiplied preferentially within the cultured cells. In order to further characterize this property, we analyzed the global gene expression profiles of mutator and non-mutator strains of S. Enteritidis, using a novel non-redundant microarray that represents ~98% of the genes found in S. Typhimurium and S. Enteritidis. From these experiments, we demonstrated that the expression of many of the invasion genes that flank the mutS gene are strongly down-regulated in the mutS strain. Control of flagellar biosynthesis, however, is positively influenced by the mutS defect. The implications of these findings are currently being investigated.

E-09

Potential Role of Toxicogenomics in Mechanistic and Predictive Toxicology
J. T. Auman¹ , H. K. Hamadeh² , P. R. Bushel¹ , C. J. Tucker¹ , K. Blanchard³ , S. Jayadev³ , R. Stoll³ , C. A. Afshari² , R. Tennant¹ , R. S. Paules¹ , ¹National Center for Toxicogenomics, NIEHS, Research Triangle Park, NC 27709, ²Amgen Inc, Thousand Oaks, CA 91320, ³Boehringer-Ingelheim Pharmaceuticals Inc, Ridgefield, CT 06877

Traditional toxicology deals with measuring endpoints of toxicity, evaluating one compound at a time in a very time consuming manner. The classic parameters of toxicity evaluated in such efforts are dose and time dependent and rarely allow for predictions of toxicity. One of the main challenges for toxicologists in the 21^st century is the identification of highly sensitive and accurate predictive biomarkers for exposure, pharmacological effect and toxicity of environmental hazards. A promising tool to aid in this effort is the genome-wide analysis of gene expression, from which scientists may extrapolate predictive signatures of exposure and effects. This would allow scientists to rapidly classify unknown compounds as being similar to groups of known compounds, thus potentially predicting their beneficial and adverse effects. In order to accomplish this goal we are creating a knowledge base of gene expression patterns following exposure to various hepatotoxicants. By phenotypically anchoring changes in gene expression to conventional parameters of toxicity, such as histopathology and clinical chemistry, we aim to identify gene expression signatures that can predict the propensity of environmental hazards to damage the liver (Toxicol Pathol 2002 30: p470). To demonstrate the utility of this technology for predictive toxicology, we have been able to predict the class identity of blinded samples based on their gene expression profile (Toxicol Sci 2002 67: p219 & p232). Eventually, we hope to achieve the goal of developing signatures that can serve as predictive biomarkers in the screening process of unknown compounds for hepatotoxicity.

E-11

Design of a Bead-Based High-Throughput Gene Expression Assay to Determine Estrogenic Potential
J. M. Naciff¹ , B. D. Richardson¹ , K. G. Oliver² , M. L. Jump¹ , S. M. Torontali¹ , K. D. Juhlin¹ , G. J. Carr¹ , J. R. Paine¹ , J. P. Tiesman¹ , G. P. Daston¹ , ¹The Procter & Gamble Company Miami Valley Labs: Cincinnati, Ohio, ²Radix BioSolutions: Georgetown, Texas

In previous studies, using high density oligonucleotide arrays, we have shown that the exposure to various estrogen receptor agonists induced a characteristic gene expression profile in the developing reproductive system of the female rat (Naciff et al., 2002, 2003). From this set of genes, we have chosen a group that would show the strongest prediction of estrogen activity in order to develop a high-throughput gene expression assay. In this assay, based on the Luminex xMAP system, carefully selected oligonucleotides were covalently linked to fluorescently-coded microspheres, in order to generate a rapid multiplexed assay platform that quantifies 20 different gene transcripts of interest in the same sample. However, this assay can be multiplexed to evaluate up to 100 distinct analytes simultaneously in a single sample, in a 96 well plate format. We have made several improvements on existing bead-based assays, mostly designed to evaluate protein levels, resulting in an assay highly correlated in sensitivity and precision to the GeneChip(TM) microarray technology. We have achieved detection levels down to 1 attomole per analyte, which allows the detection of rare messages in complex cRNA samples, using as little as 2.5 µg of cRNA. This assay offers increased throughput with decreased costs compared to existing gene expression technologies. Our data clearly shows that this approach can be used to create a high-throughput screening test to identify potential gene expression changes induced by a given treatment. Its multiplexing capability offers the opportunity to screen large numbers of chemicals to determine their potential therapeutic and toxic properties, in a cost- and animal-effective manner.

E-12

The utility of gene expression profile analysis for elucidating genotoxic mechanisms
D. Dickenson¹ , T. Hu² , M. Aardema² , J. Aubrecht¹ , ¹Pfizer Global Research and Development, Groton, CT 06355, ²Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH. 45253

During the safety evaluation process of new drugs, a battery of in vitro genotoxicity tests is conducted. Positive results are not uncommon and their biological relevance needs to be determined. Without this, the risk assessment of genotoxic agents is generally based on linear extrapolation methods, though there is substantial evidence that some chemicals may exhibit a clear thresholded dose-response. Hence, gaining an insight into genotoxic mechanisms, i.e., differentiation of DNA-reactive vs. DNA non-reactive, is essential for risk assessment to humans. This results in laborious and time consuming follow-up strategies. Genotoxic stress triggers a variety of biological responses including the transcriptional activation of genes regulating DNA repair, cell survival and cell death. Thus, we evaluated the utility of the gene expression profile analysis in L5178Y cells for gaining an insight into genotoxic mechanisms using a set of model agents. The gene expression changes were compared with micronucleus induction and direct DNA damage. First, we investigated whether gene expression profiles can differentiate between DNA reactive and DNA non-reactive mechanisms of genotoxicity associated with general toxic stress. Furthermore, using a set of model compounds we have developed a set of specific genes that distinguished genotoxic mechanisms and evaluated its usefulness using data from multiple laboratories. Although, more experimental work including evaluation of a large number of compounds and stresses is necessary to refine and enhance our understanding of gene expression profiles and toxic pathways, our results suggest the potential utility of gene expression profile analysis for evaluating molecular mechanisms of action of genotoxicants.

E-15

Liver Cancer Analysis Using Cross Species Mapping Based on Rat Gene Expression Profiling
H. Fang¹ , W. Tong¹ , S. H. Yim² , J. M. Ward² , R. Perkins¹ , Y. P. Dragan¹ , ¹NCTR, FDA, 3900 NCTR Rd Jefferson, AR 72079, ²Center for Cancer Research, National Cancer Institute

Liver cancer is a public health concern in many parts of the world. In order to develop methods for the analysis of microarrays of cancer tissue, microarray studies were performed on tumor samples obtained from albumin-SV40 transgenic rats. These transgenic rats develop hepatic neoplasms. Gene expression profiles were generated from 2 control, 2 adenoma and 2 carcinoma samples with NCI Incyte mouse chip containing 9984 using a dye flip approach. Data from microarray analysis of liver tumors from these animals demonstrated an altered gene expression. Our analysis found that 2223 genes were differentially expressed across the sample sets using an F test with P<0.05. Genes related to cell cycle control, cell proliferation, apoptosis, transcriptional regulation, and protein metabolism were altered. A novel visualization tool that permits the efficient analysis of gene expression in the context of chromosomal alterations based on cross species gene expression mapping was developed to assist in the analysis of this type of data. Using this tool and bioinformatics approaches, gene expression in regions of previously identified chromosomal aberrations associated with early hepatic neoplasms in this transgenic rat model were examined. These results indicate that the altered gene expression associated with rat liver tumor development may be useful in the analysis of human liver cancer.

E-16

Gene arrays, cell culture process changes, and comparability
M. Hanson¹ , K. Brorson² , S. Lute² , A. Moreira¹ , ¹University of Maryland, Baltimore County, ²FDA (OBP/DMA)

Changes in biopharmaceutical processes (media, chromatography support, formulation, etc.) have, in some cases, resulted in modifications of the final products. However, in many of these cases, the mechanism by which these modifications occur is unclear. Recently we have explored using global gene expression data to elucidate the molecular mechanism by which these changes at the cell culture level affect biological products. Here we will present preliminary data demonstrating the effect that sodium butyrate, a common enhancer of protein production, has on global gene expression at the transcript level as well its effect on monoclonal antibody comparability. Ultimately, we hope to establish the possibility of using global gene expression data for product comparability studies.

E-21

Analysis of Microarray Expression Signals Generated by RNA Labeled with Fluorescent Molecules Compared with Resonance Light Scattering Particles
D. G. Ranamukhaarachchi¹ , J. Z. Langone¹ , F. Chen² , A. D. nandanie¹ , M. Schneider³ , R. K. Elespuru¹ , ¹CDRH/FDA, ²Northwestern University, ³Butler University

Generation of gene expression signals by commonly used fluorescent molecules (e.g. Cy3 and Cy5) in microarrays demands quantities of total RNA that cannot be usually obtained in clinical samples. This requires either invasive sampling or RNA amplification strategies that may affect the accuracy of signals. Sensitivity of signal detection becomes more critical when low abundance transcripts are considered. We compared a novel non-fluorescent nanometer-sized metal colloidal particle-based signaling method (Resonance Light Scattering [RLS] particles) with fluorescence-based signal generation under a variety of microarray fabrication conditions using a broad range of starting total RNA amounts. The results indicated a vastly improved sensitivity in detection of signals by RLS particle labeling compared with fluorescence. RLS particles generated signals from as little as 0.5 µg of total RNA, while maintaining the dynamic range and linearity of signal intensity. This compares with a minimum of 20 µg total RNA detected using fluorescence. Microarrays fabricated on poly-L-lysine coated glass slides showed poor performance with increased background by RLS particles and need to be further evaluated. The impact of labeling method on analytical performance of microarray devices and improved sensitivity by nanometer particles is illustrated.

E-22

Gene Expression Profiling in a Rat Cardiac Ischemia Model
P. Koza-Taylor¹ , B. Lu¹ , M. Wenfang² , S. Eustis³ , X. Li³ , M. Lawton¹ , ¹Molecular and Investigative Toxicology, Groton, CT, USA, ²Comparative Medicine, Pfizer, Groton, CT, USA, ³Pathology, Pfizer, Groton, CT, USA

Cardiotoxicity in preclinical animal species is a common finding during development of vasoactive or cardiotonic drugs. There are multiple mechanisms of cardiotoxicity, with cardiac ischemia being the most common cause. We have utilized a rodent model of cardiac ischemia for biomarker identification and mechanistic insight. In this model, male Sprague-Dawley rats were anesthetized with isoflurane, and a left thoracotomy performed, exposing the heart and the left coronary artery. For ligation, the left anterior coronary artery was tied to occlude the blood flow. For sham control, the needle/suture were passed through under the artery, but not tied. Heart and whole blood samples were collected for gene expression profiling at 3, 6, 9, 24 and 48 hours after coronary artery ligation or sham surgery. Moderate acute cardiomyocyte necrosis at 3-9 hours and necrosis with mixed inflammatory cell infiltration at 24-48 hours were observed in the left free ventricular wall after ligation. Principal component analysis (PCA) of gene expression data showed clear separation between the sham and treated groups. An immediate-early stress response was observed starting the early time points and increasing at the later time points. The genes differentially expressed in the ischemia-induced animals fell into distinct clusters: the down-regulated genes included metabolism, glycolysis and catalase pathway members whilst up-regulated genes included acute phase and inflammatory response, heme oxidation pathway members, heat shock proteins, matrix metalloproteinases and fibronectin. The transcriptional profile in the hearts of ischemic rats was similar to catecholamine- and minoxidil-like responses, suggesting common mechanisms of ischemic cardiac injury.

E-24

Gene Expression Profiling of Type I Latex allergy by Fluorescent Differential Display PCR
D. G. Ranamukhaarachchi¹ , M. Tinsworth² , A. D. Nandanie¹ , J. Z. Langone¹ , R. K. Elespuru¹ , ¹CDRH/FDA, ²Thomas Wooten High School

Type I allergic responses to proteins in natural rubber latex (NRL) gloves pose a significant problem among health care workers. Type I hypersensitivity is an IgE-mediated response regulated by the balance of Type 1 (Th1) and Type 2 (Th2) CD4+ T helper cells. Molecular diagnosis and new therapeutic approaches to Type I allergy could be targeted to differentially regulated genes as a response to NRL-allergens. We developed a gene expression profiling approach for latex allergy using fluorescent differential display PCR, based on the hypothesis that T helper cells in peripheral blood lymphocytes (PBL) from allergic and non-allergic individuals challenged in vitro with NRL-allergens would show differential gene expression responses. The long-term goal is to develop a diagnostic microarray for latex allergy using differentially expressed genes. We used 17 primer combinations and excised 184 bands representing differences in expression levels between allergic/non-allergic subjects as well as different times (12, 24, 48 hrs.) post allergen treatment within the PBLs from the same subject. Approximately 35% percent of excised bands corresponded to genes expressed moderate to strong with >4-fold difference in expression level. Less than five percent of the bands were detected either only in allergic or non-allergic (uniquely expressed genes). Gene expression analysis post allergen treatment indicated a time dependent regulation of responding genes within 24 hrs. and then decline of the response. We sequenced these bands and searched their sequence similarities in the GenBank^TM database using BLAST program. The significance of these genes in Type I allergy is currently being studied.

E-26

Using correlation analysis and hierarchical clustering to discriminate genes associated with hepatic vasculitis from subsequent hepatic inflammation
B. Lu¹ , L. Nelms¹ , G. Floyd² , M. Lawton¹ , ¹Molecular and Investigative Toxicology, Groton, CT, USA, ²Pathology, Pfizer, Groton, CT, USA

In pre-clinical studies, compound X induced hepatic vasculitis in rats. In a subsequent investigative study, groups of male Sprague-Dawley rats were given daily oral doses of vehicle, 10 mg/kg or 1000 mg/kg of compound X and animals were euthanized 24, 48, 144 and 240 hours after first dose. Liver samples were harvested for gene expression profiling with RG_U34A Genechip and serum was used for proteomics analyses. Clinical chemistry, hematology and histopathology data were also collected from the study. At 24 hours, histological vascular changes induced by high-dose compound X were consistent with endothelial activation, and frank vascular inflammation that sometimes extended secondarily into the hepatic parenchyma was evident at 48 hours. Clinical chemistry and hematology data both reflected the presence of inflammation at 48 hours. We then focused on genes that were differentially regulated at 24 or 48 hours to look for potential biomarkers for liver vasculitis. We used statistical methods to calculate and identify genes that are highly correlated with severity of the vascular and perivascular inflammation. We have compared the hepatic gene expression profile induced by compound X to other compounds known to induce a hepatic acute phase response secondary to inflammation at sites other than the liver. Using data from Gene Logic's ToxExpress database, we also subtracted genes activated by hepatic inflammation from the compound X response to select candidate genes that are involved in the hepatic vascular inflammatory response rather than in the hepatic acute phase response that occurs secondary to inflammation.

E-27

Using P-Value Plots and ROC-Like Curves to Assess Statistical Significance of cDNA Array Data on Gene Expression
R. R. Delongchamp¹ , J. F. Bowyer² , J. J. Chen¹ , R. L. Kodell¹ , ¹Division of Biometry and Risk Assessment, ²Division of Neurotoxicology, NCTR

A cDNA array assays expression levels in tissue samples for hundreds to tens-of-thousands of genes. In studies evaluating treatment-induced changes, selecting a significance level for by-gene hypothesis tests requires dealing with the multitude of treatment contrasts. P-values from these tests order the genes such that a p-value cutoff divides the interrogated genes into two sets. The set of genes selected as affected will have false positives while the set selected as unaffected will contain false negatives. The large number of p-values provides information for estimating the number of true null hypotheses (truly unaffected genes), m₀. A p-value plot can be used to develop computational methods for estimating m₀, as well as a visual aid for analysis and interpretation of cDNA array experiments. With a reliable estimate of m₀, the false-positive and false-negative rates associated with any p-value cutoff can be estimated. The optimal cutoff depends upon the relative costs of misclassification. Here, a method analogous to methods developed for ROC curves is proposed for selecting the cutoff. The large number of p-values gives good resolution to the ROC-like curves. The false discovery rate and the false non-discovery rate associated with the selected cutoff are important error rates that can be estimated a posteriori. A functional genomics study is used for illustration.

E-28

The use of gene expression profiling for identifying potential biomarkers of reproductive toxicology
L. Nelms¹ , R. Chapin² , B. Lu¹ , S. Curry² , M. Wilhelms³ , M. Elwell³ , D. Pelletier¹ , M. Lawton¹ , ¹Molecular and Investigative Toxicology, Groton, CT, USA, ²Investigative Developmental Toxicology, Groton, CT, USA, ³Pathology, Pfizer, Groton, CT, USA

More sensitive clinical markers of damage to the male reproductive system are needed. Testicular toxicity in rodents and other preclinical species is currently determined by a histologic evaluation of tissues from treated animals. In humans, an ejaculated sperm count has been the gold standard for many years. However, sperm count has some drawbacks: it is highly variable across individuals and from one sample to another within a single individual, and it reflects damage that happened weeks or months previously. We are using emerging "omics" technologies to identify clinically relevant, "leading" biomarkers of testis toxicity. To find candidate biomarkers, rats were treated with well-described toxicants to produce varying degrees of testis damage. Serum was profiled by 2D gels (proteomics), urine by NMR (metabonomics), and various tissues (testis, prostate, liver, and whole blood) by gene expression profiling (genomics). For this communication, male Sprague Dawley rats were dosed with the well-known testicular toxicant, 1,3-dinitrobenzene (DNB) at 0, 2, 5, or 10 mg/kg/day, and samples collected after 2, 4, 7, and 10 days of treatment. The histologic lesions were as described previously: Sertoli vacuolization, and apoptosis of round spermatids and spermatocytes, followed by sloughing of dead and dying germ cells. The degree of these effects varied with dose and duration. Gene expression analysis using Affymetrix GeneChips also revealed changes that varied with time and dose. Experimental validation of candidate markers using a variety of techniques (in situ hybridization, RT-PCR, regulation by other compounds, etc.) will be described.

E-30

Gender differences in expression of liver genes: Statistical design and analysis
R. R. Delongchamp¹ , S. Dial² , A. J. Harris³ , ¹Division of Biometry and Risk Assessment, ²Center for Hepatotoxicity, NCTR, ³Center for Toxicology and Environmental Health, LLC, 615 W. Markham, Little Rock, AR 72201

Gene expression levels in liver samples from 9 males and 9 females were analyzed for gender differences. The cDNA arrays interrogated 31,110 genes using an experimental design that adjusted for sources of variation associated with arrays, hybridizations and processing conditions. All of the data were analyzed for gender differences. However, the genes were partitioned into 2,800 that were clearly expressed (expressed genes) and 28,310 that had expression levels in the background range (not expressed genes) in order to better manage false positive rates associated with a large number of comparisons. The distribution of p-values from the 'not expressed' group was consistent with no gender differences. The distribution of p-values from the 'expressed' group indicated that about 8 % of the genes had a gender difference. Unfortunately, estimated false discovery rates exceed 80 % for sets of genes selected based upon their p-values essentially making it impossible to identify specific genes with a gender difference. Among expressed genes, the estimated fold-changes (expression in males / expression in females) were small. The largest observed fold-change was 1.55. The 95 % confidence bounds on the estimated fold-change are less than 1.4 fold for 79.3 %, and few (1.1 %) exceed 2-fold.

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How many replicates are needed to identify a fraction of the unknown number of truly altered genes in microarray experiments
S. Wang¹ , J. Chen² , ¹FDA, Rockville, MD, ²FDA, Little Rock, Arkansas

Microarray technology allows simultaneous comparison of expression levels of thousands of genes under each experimental condition. This research concerns sample size calculation to identify differentially expressed genes under two conditions (e.g., treated versus control samples, diseased versus normal samples). In a typical experiment, only a fraction of genes is expected to be differentially expressed between two samples. Sample size determination depends on a number of factors including the specified significance level (a), the desired statistical power (1 - b), the fraction (h) of truly altered genes out of the total g genes studied, and the effect sizes (D) for the altered genes. This paper proposes a method to calculate the number of arrays required to detect at least 100 l% (where 0 < l < 1) of the truly altered genes under the model of an equal effect size for all altered genes. Based on the proposed approach, to identify up to 90% of truly altered genes among the unknown number of truly altered genes, the estimated numbers of arrays needed appear to be manageable. The proposed method offers a simple and practical way to determine the number of arrays needed in microarray experiments. An example data set is used to illustrate the use of the proposed approach to plan microarray experiments.

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Liver Cancer Analysis Using Cross Species Mapping Based on Rat Gene Expression Profiling
H. FANG¹ , W. TONG¹ , S. H. Yim² , J. M. Ward² , R. Perkins¹ , Y. P. Dragon¹ , ¹FDA, ²

NCI Liver cancer is a public health concern in many parts of the world. In order to develop methods for the analysis of microarrays of cancer tissue, microarray studies were performed on tumor samples obtained from albumin-SV40 transgenic rats. These transgenic rats develop hepatic neoplasms. Gene expression profiles were generated from 2 control, 2 adenoma and 2 carcinoma samples with NCI Incyte mouse chip containing 9984 using a dye flip approach. Data from microarray analysis of liver tumors from these animals demonstrated an altered gene expression. Our analysis found that 2223 genes were differentially expressed across the sample sets using an F test with P<0.05. Genes related to cell cycle control, cell proliferation, apoptosis, transcriptional regulation, and protein metabolism were altered. A novel visualization tool that permits the efficient analysis of gene expression in the context of chromosomal alterations based on cross species gene expression mapping was developed to assist in the analysis of this type of data. Using this tool and bioinformatics approaches, gene expression in regions of previously identified chromosomal aberrations associated with early hepatic neoplasms in this transgenic rat model were examined. These results indicate that the altered gene expression associated with rat liver tumor development may be useful in the analysis of human liver cancer.

E-34

Evaluation of six analytic strategies for a typical cDNA gene expression data
J. Zhang¹ , Y. Tsong¹ , L. Zhao² , ¹FDA, ²Fred Hutchinson Cancer Research Center

Microarray technology provides a global overview of thousands of different genes simultaneously and it has been commonly used for drug development as well as for much broader biomedical researches. OTR/CDER/FDA conducted a two color based cDNA microarray experiment including six microarry chips. Each chip includes treated and controlled samples, which are from kidney tissues of rats. The primary objective is to identify genes that are differentially expressed between treated and controlled groups. Despite the seemingly simplicity, there are numerous methods for analyzing such data in a statistically rigorous manner. Here we focus on six different analytic strategies, each of which is derived under a set of reasonable assumptions. We use GenePlus software to analyze the data set of the experiment carried out at OTR/CDER/FDA.

E-35

Gene Selection for Sample Classifications in Microarray Experiments
C. Tsai, J. Chen, NCTR,FDA,Jefferson, AR

DNA microarray technology provides useful tools for profiling global gene expression patterns in different cell/tissue samples. One major challenge is the large number of genes relative to the number of samples. The use of all genes can suppress or reduce the performance of a classification rule due to the noise of none discriminatory genes. Selection of an optimal subset from the original gene set becomes an important pre-step in sample classification. Selection approach of an optimal subset involves two steps : calculating a discriminatory index for ranking genes with differential expressions and determining a cutoff from the ranked scores. We consider several statistical discrimination indices based on the Receiver Operating Characteristic curve, and use both the family-wise error (FWE) rate and false discovery rate (FDR) as criterions for gene selection. A public colon cancer dataset is used to evaluate the performance of selected gene sets for sample classification using two well-established classification algorithms: k-nearest neighbors (KNN) and support vector machine (SVM). The predicted accuracies are assessed by performing the internal and external cross-validations. Using the SVM classification, the overall accuracies of classification using 10 genes can reach more than 90% for internal cross-validation, and above 85% for the external cross-validation. The selected gene sets appear to perform better than or comparable to several results reported in literatures using univariate or multivariate discriminant analysis. Our approach uses the univariate analysis without performing multivariate search.

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Formulation of RNA Performance Standards for Regulatory Toxicogenomics Studies
B. A. Rosenzweig¹ , P. S. Pine¹ , J. C. Fuscoe² , K. L. Thompson¹ , ¹CDER, FDA, Silver Spring, MD, ²NCTR, FDA, Jefferson, AR

Genome-wide measurement of gene expression has the potential to enhance non-clinical assessments of drug safety and action, but there is acknowledged variability among users and between platforms due to differences in probe sequence, protocol non-uniformity, and other technical issues. To enable regulatory reviewers to objectively evaluate the quality and content of gene expression data, it is important to identify performance characteristics associated with well-conducted microarray experiments. A collaborative research project was initiated to test the feasibility and value of using a set of mixed tissue samples to evaluate the ability of different laboratories and microarray platforms to detect designed-in differences in gene expression between two complex biological samples. Total RNA was isolated from four tissues (brain, liver, kidney, and testis), pooled across sets of 8 male Sprague Dawley rats for each of 3 independent sets of animals, and assayed on Affymetrix RAE230A arrays. Tissue-selective probes were identified and mapped to Agilent, Amersham, and NCTR microarray platforms using sequence identifiers. Two mixed tissue samples composed of different defined proportions of total RNA from the four tissues were prepared. Tissue-selective probes demonstrating consistent ratios of expression between the two mixtures in well-conducted assays are candidate markers of assay performance. The selection of these probes will be evaluated by testing the samples at multiple sites and on multiple array formats. Incorporation of measures of assay performance, such as a mixed tissue standard, into toxicogenomic data submissions could help assure regulators that high quality data is being submitted to support a regulatory review.

E-39

Infrastructure For Reporting Toxicogenomics Data - Toward The Harmonization
S. Sansone¹ , M. D. Waters² , J. Fostel² , S. D. Pettit³ , W. Tong⁴ , A. Brazma¹ , P. R. Bushel² , R. S. Paules² , W. Pennie⁵ , U. Sarkans¹ , P. Rocca-Serra¹ , R. W. Tennant² , ¹European Molecular Biology Laboratory, The European Bioinformatics Institute (EMBL-EBI), Cambridge, UK, ²NIEHS National Center for Toxicogenomics (NCT), Research Triangle Park, NC, ³Health and Environmental Sciences Institute (HESI) Committee on Genomics, ⁴NCTR, Center for Toxicoinformatics, FDA, Jefferson, AR, ⁵Pfizer, Kalamazoo, MI

To fully realise the potential of this emerging interdisciplinary field we believe that it is necessary to move toward the establishment of common infrastructure for exchanging toxicogenomics (and similarly pharmacogenomics) data¹. As part of a collaborative undertaking, the HESI Committee on Genomics, the NIH NIEHS-NCT, the FDA NCTR Center for Toxicoinformatics, and the EMBL-EBI have initiated an harmonization process for reporting array-based toxicogenomics data in their respective databases/knowledge bases, using common descriptors, standard data storage and exchange format and harmonized nomenclature. This collaboration has developed in the establishment of a Toxicogenomics Working Group (TWG) within the MGED Society². The TWG provides a public forum for the creation of an internationally compatible informatics platform exploiting use of MGED accomplishments (MIAME, MAGEand the MGED Ontology). Recently, the MGED TWG has established links with the Pharmacogenomics Standards Group, joint project of HL7, CDISC and I3C, aiming to define the requirements for submission to FDA and clarify data formats and standards. Discussions are ongoing to exploit any possible form of integration and harmonization of the efforts. Ultimately the creation of internationally compatible informatic systems will enhance the impact of the individual datasets and provide the scientific and regulatory community with easy access to integrated data in a structured standard format, facilitating data exchange, comparison and analysis.

Mattes WB et al. (2004). Database development in toxicogenomics: issues and efforts. Environ Health Perspect Toxicogenomics: doi:10.1289/txg.6697
MGED Society: http://www.mged.org

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Report from the Non-Clinical Pharmacogenomics Subcommittee of the Pharmacology Toxicology Coordinating Committee on the assessment and review of pharmacogenomics data.
J. K. Leighton¹ , K. Thompson¹ , S. Biade¹ , P. Brown¹ , R. Duncan² , A. Ellis¹ , P. Harlow¹ , W. Harrouk¹ , S. Pine¹ , M. Rivera¹ , T. Robison¹ , L. Rosario¹ , A. Worobec¹ , K. Zhang¹ , ¹CDER, FDA, Rockville MD, ²CBER, FDA, Rockville MD

Over the past several years, both FDA and the pharmaceutical industry have recognized the potential importance of pharmacogenomics to drug development. To gain both expertise in this field and a regulatory framework for reviewing these data, pharm/tox reviewers and researchers within CDER and CBER formed a subcommittee to address these issues. The charter of this subcommittee incorporates the following goals: 1) to develop a guidance on the submission of nonclinical pharmacogenomics data; 2) to develop internal consensus on the best means of interpreting these data and the regulatory implications on drug development; 3) to gain Agency expertise in this field; and 4) to develop mechanisms to communicate with sponsors how reviewers will use pharmacogenomics data in product assessments. Over the past year, the subcommittee has 1) evaluated several voluntary submissions of genomic and ancillary toxicology data from pharmaceutical companies on compounds not undergoing regulatory review and 2) gained experience in the use of a reference toxicogenomics database to interpret genomic data. We report on our preliminary evaluation of data content and quality control metrics useful for evaluating nonclinical pharmacogenomic submissions. Voluntary genomics submissions from industry have been and will continue to be useful tools that help both researchers and regulators gain experience in reviewing and analyzing toxicogenomics data and developing guidelines for the submission and review of these data.

E-41

Issues Associated with a Mock Submission Using Microarray Data
L. Reid¹ , R. Cohn¹ , W. Jones¹ , T. Goralski¹ , F. Goodsaid² , ¹Expression Analysis, Inc., Durham, NC, ²Schering-Plough, Lafayette, NJ

We conducted a test submission using microarray-based data as a collaborative exercise between FDA and industry. The data files were derived from a toxicogenomics study with eighteen rat samples that had been hybridized to Affymetrix U34A GeneChips. The objectives were to: (1) contribute to building and refining a process in which microarray data may be incorporated into submissions to FDA; (2) provide a suitable framework in which to develop recommendations; and (3) contribute to the development of consensus around the specific elements of applicable recommendations. The scope of the submission was defined to address: (a) laboratory infrastructure (i.e. hybridization platform, protocol, chip reader); (b) informatics infrastructure (including data management, IT, statistical system infrastructure and associated quality assurance/quality control measures); (c) study-specific sample processing and array performance characteristics (i.e. the use and interpretation of RNA standards and controls, quality control metrics, data quality, and array performance characteristics); (d) detection and mitigation of biases related to processing; (e) statistical analysis methods and related considerations; (f) toxicology results independent of toxicogenomic data; and (g) interpretation (i.e. toxicogenomics discussed in the context of the toxicology data to provide an overall interpretation of experimental results). We summarize issues encountered and lessons learned in each of these areas, as well as the value gained by all participants.

E-43

Multiplexed Kinase Substrate and Signal Transduction Profiling of Human Ovarian Cancer: Towards Patient Tailored Therapeutics
J. D. Wulfkuhle¹ , J. A. Aquino² , L. Young³ , C. Liang⁴ , V. S. Calvert⁵ , D. Fishman⁶ , G. Coukos⁷ , P. J. Munson⁸ , L. A. Liotta⁹ , E. F. Petricoin⁵ , ¹NCI-FDA Clinical Proteomics Program, NCI, Bethesda, MD, ²NCI-FDA Clinical Proteomics Program, NCI, Clinical Proteomics Program, Bethesda, MD, ³CIT, NIH, Bethesda, MD, ⁴Vigenetech Inc., N. Billerica, MA, ⁵NCI-FDA Clinical Proteomics Program, FDA, Bethesda, ⁶Northwestern University School of Medicine, Chicago, IL, ⁷University of Pennsylvania School of Medicine, Philadelphia, PA, ⁸CIT, NIH, Bethesday, MD, ⁹NCI-FDA Clinical Proteomics Program, NCI. Bethesda, MD

Cancer can be classified as both a genomic and a proteomic disease in which gene mutations result in aberrant protein activity within the cell. Defects in cell signaling pathways play a central role in cancer cell growth, survival, invasion and metastasis. Profiling the activity of signaling pathways in the context of the disease process can highlight disease-related alterations that represent the patient-tailored drug targets of the future. We are working to characterize the rewiring of protein signaling networks that occurs during the progression of invasive ovarian cancer. In this study, we used reverse-phase protein array technology coupled with Laser Capture Microdissection and phosphospecific antibodies to examine the activation status of several key molecular "gates" involved in cell survival and proliferation signaling in human ovarian tumor tissue. Populations of 25,000-30,000 epithelial cells were microdissected from a large number of invasive ovarian carcinomas and lesions representing a variety of benign, epithelial-related and unrelated lesions. These cells were lysed in volumes of approximately 50 uL, and 100 nL of each lysate arrayed on nitrocellulose-coated glass slides. These arrays were analyzed for the expression and activation status of over 40 valdiated key phospho-specific endpoints that act as surrogates for kinase activity function from multiple signaling pathways. Arrays were probed with antibodies specific for a variety of molecules involved in pathways such EGF receptor signaling, cell survival and apoptosis control, and cell proliferation. Overall, global signaling profile analysis suggests that patterns in signal pathway activation in ovarian tumors may be patient-specific rather than histotype or stage specific. Bayesian clustering analysis performed with a preliminary dataset 38 tumors and 23 signaling endpoints classified the tumors into two large groups, one containing 7/8 endometrioid tumors in the study set and this group exhibited generally higher levels of phosphorylated MARCKS, IRS-1 and ErbB2 than the second group. Signaling profiles will ultimately be correlated with a variety of clinical parameters including treatment response and patient outcome. We expect that profiling the status of signaling pathways in individual ovarian cancer tissues will move us closer to the reality of patient-tailored therapy where appropriate treatments are chosen and monitored based on the proteomic signature of a tumor before, during and after therapy.

E-45

Differentiating Benign States from Non Small Cell Lung Cancer Using Serum Proteomic Profiling
C. D. Ching¹ , P. Mosca¹ , V. Fusaro² , V. Rajapakse² , S. Ross² , T. Veenstra³ , T. Conrads³ , E. Petricoin² , L. Liotta⁴ , H. K. Lyerly¹ , D. Harpole¹ , ¹Duke University Medical Center, Durham NC, ²NCI/FDA Clinical Proteomics Program, Bethesda, MD, ³SAIC/NCI, Frederick, MD, ⁴NIH/NCI, Bethesda, MD

In 2004, 173,770 new cases and 160,440 deaths due to lung cancer are estimated in the US. Its lethality is partially attributed to advanced stages of the disease at diagnosis. Helical computed tomography scans have a reported sensitivity for stage I tumors of 70-85% but also high false-positive rates. Subsequent evaluation of suspicious lung nodules, involving bronchoscopy, percutaneous needle biopsy, and/or surgery can be associated with morbidity/expenses that often outweigh the benefits of the original screening exam. An adjuvant serum test to distinguish patients with benign disease from those with malignant disease would be of great clinical value. In this study, we used a high-resolution time-of-flight mass spectrometer to generate discriminatory serum proteomic profiles with the goal of differentiating benign and malignant states. Serum was collected from 153 patients, 86 with pathologically verified non-small cell lung cancer and 67 with smoking histories but no known lung cancer. Samples were applied to WCX2 SELDI chips by a Biomek 2000 robotic liquid handler. Low molecular weight (<12 kd) proteomic patterns were generated with an API QSTAR Pulsar LC/MS/MS System. Proteomic patterns were analyzed utilizing three independent bioinformatics systems employing diverse pattern recognition algorithms. Each algorithm was applied to a training set and separate blinded validation set. For all algorithms, blinded validation yielded sensitivities of 70-100% and specificities of 60-94% for distinguishing benign states from non-small cell lung cancer. If confirmed in larger clinical studies, this technology can potentially reduce the number of invasive procedures for patients with CT diagnosed noncalcified lung nodules.

E-46

A Proteomic Approach to Understanding Host Cell Protein Impurities and the Immunoassays Used to Measure Them
K. Champion¹ , S. Frie² , M. Vanderlaan¹ , ¹Dept. of Analytical Chemistry, Genentech, Inc., South San Francisco, CA 94080, ²Dept. of Analytical Operations, Genentech, Inc., South San Francisco, CA 94080

Residual host cell protein impurities are normally quantified in immunoassays using antibodies raised to a crude cell lysate. The host cell protein value obtained in the ELISA is actually a measure of the immunoreactive content of the sample. We have developed multi-product ELISAs using antibodies raised against a single crude cell lysate. As long as the antigen content of the immunogen is similar to the antigen content of samples, the value obtained in the ELISA is a reasonable measure of host cell protein mass. In order to better understand the composition of host cell protein populations, we used 2-D electrophoresis to analyze an E. coli cell lysate to which antibodies were raised, as well as cell lysates from several other E. coli strains and culture conditions (Champion et al., 1999, Proteomics, 1, 1133-1148). Since these proteome profiles represent cells that are not expressing a recombinant protein, we also compared the proteome of recombinant human growth hormone-expressing cells with the corresponding non-producing cells (Champion et al., 2003, Proteomics, 3 (7), 1365-1373). These 2-D proteomes were compared on a qualitative basis. The results revealed a high degree of similarity for the 'antigen population'. Most of the observable protein differences occur in proteins of low abundance near the limit of detection. To further our investigations, we performed immunofractionation and 2D immunoblot analyses and demonstrated that hundreds of E. coli proteins (covering a broad range of mass and charge) are immunoreactive. Not surprisingly, our results suggested that some of the proteins were immunodominant. This was confirmed for a pair of E. coli proteins when they were analyzed in the ELISA either alone or with the cell lysate.

E-47

Protein Microarrays: Monitoring Patient Response to Biologic Therapies
V. Espina¹ , S. Cowherd¹ , E. F. Petricoin² , L. A. Liotta¹ , ¹National Cancer Institute, Center for Cancer Research, Laboratory of Pathology, FDA-NCI Clinical Proteomics Group, Bethesda, MD 20892, ²Food and Drug Administration, Center for Biologics Evaluation and Research, Office of Cellular and Gene Therapy, Bethesda, MD 20892

Proteomics has the potential to revolutionize diagnosis and disease management. Protein microarrays are emerging as a novel approach for characterization of protein signaling networks. This type of molecular profiling allows real time monitoring of the cellular proteome information content, providing diagnostic and prognostic windows for guiding patient management. The reverse phase protein microarray format allows monitoring of the in vivo states of normal, pre-malignant and malignant cell populations and is showing utility in understanding deranged cell signaling networks.

Our protein microarray utilizes an immobilized heterogeneous protein mixture as a bait molecule, and an antibody directed against the immobilized protein of interest as a capture molecule. Pure cell populations from heterogeneous biopsy material are procured with laser capture microdissection (LCM). Solubilzation of the captured cells produces a lysate containing the entire cellular proteome. These whole cell lysates are immobilized on a nitrocellulose coated glass slide. The lysate is printed in a mini dilution curve ensuring that the antigen antibody interactions are in the linear dynamic range for each antigen-antibody pair and protein concentration.Immunostaining of the protein microarrays allows relative quantitation of phosphorylated and non-phosphorylated forms of the cell's key signaling proteins. In this way, protein signal pathways can be mapped and thus become the starting point for individualized therapy. This protein microarray technology is currently in use at the National Institutes of Health in Phase II clinical trials of metastatic breast and ovarian cancer. Cell survival and apoptotic protein pathways are monitored as biological markers of disease progression in these clinical trials.

E-PO-48

Genomics, Proteomics and Metabonomics analysis of acetaminophen-induced hepatotoxicity in the mouse
F. Pognan¹ , M. Coen² , S. U. Ruepp*³ , R. P. Tonge⁴ , J. C. Lindon² , J. K. Nicholson² , E. M. Lenz⁵ , I. D. Wilson⁵ , ¹Safety Assessment, AstraZeneca Pharmaceuticals, Wilmington, DE 19850 USA, ²Biological Chemistry, Faculty of Medicine, Imperial College London, South Kensington, London SW7 2AZ, UK, ³Safety Assessment, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG UK, ⁴Enabling Science & Technology, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG UK, ⁵Drug Metabolism and Pharmacokinetics, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG UK (*Present address: Non-Clinical Drug Safety, F. Hoffmann-La Roche Ltd, 4051 Basel, Switzerland)

Acetaminophen has been administered at pharmacological, sub-toxic and toxic doses to overnight fasted mice. Animals have been sacrificed at different time points from 15 minutes to 4 hours post-injection. Genomics analysis in liver has been performed using nylon filter arrays, Affymetrix chips and RTQ-PCR. Proteomics on liver mitochondrial subfractions has been run using the quantitative fluorescent 2D-DIGE method and metabonomics ¹H NMR spectra from liver tissue, tissue extracts and plasma from mice dosed with acetaminophen have been analyzed to identify biochemical changes arising from hepatotoxicity. As soon as 15 minutes post-injection, centrilobular hepatocyte mitochondria were already slightly enlarged and GSH total content decreased by a third at the top dose. mRNA changes were observed from 30 minutes onward and most of the protein changes in mitochondria were present at 15 minutes post-injection, thus preceding most of the gene expression changes. The principal metabonomic changes comprised a decrease in hepatic glucose and glycogen in intact tissue, coupled with an increase in lipid content, plus increases in the levels of glucose, pyruvate, acetate and lactate in plasma, and increases in alanine and lactate in the aqueous tissue extracts. These three sets of data have then been interpreted together in terms of common metabolic pathways to show a shift in energy metabolism. The results show that these three technology platforms together offer a complementary view into cellular responses to toxic processes, providing new insight into the toxic consequences, even for a therapeutic as well-studied as acetaminophen.

E-50

Quantum Dots-A New Detection System For Reverse Phase Protein Microarrays
D. Geho¹ , N. Lahar¹ , V. Espina¹ , P. Herrmann¹ , E. Petricoin¹ , L. Liotta¹ , K. Rosenblatt² , ¹FDA (CBER)-NCI Clinical Proteomics Program, Bethesda, MD, ²UT Southwestern Medical Center, Dallas, TX

Reverse-phase protein microarrays enable the quantitative analysis of cell signaling pathways within clinical specimens using limited amounts of microdissected cancer and normal cells. A conventional approach to protein microarrays utilizes a chromogenic detection system and linear amplification using catalyzed reporter deposition. While this method has been very successful, limitations of the colorimetric methodology include its limited dynamic range and one target-at-a-time approach. Quantum Dots (Quantum Dot Corporation, Hayward, CA), nanoparticle semiconductor crystals with fluorescent properties, are now being used as reporter agents for biological systems. These reagents have improved optical properties with large extinction coefficients, broader absorbance ranges, narrow emission bandwidths, and higher quantum yields. Because of these properties, they yield a much brighter signal than organic fluorescent reporter molecules. We are investigating the integration of reverse-phase microarrays with Quantum Dot reporter technology for the molecular profiling of signaling pathways in cancer cells. Because Quantum Dots resist photobleaching, the signals can be time-averaged, potentially offering a sensitive assay with a wide dynamic range. Additionally, because catalyzed reporter deposition is not required and numerous different reporter wavelengths are available, protein microarrays can theoretically be probed in a multiplex fashion. Quantum Dot® microarray analysis of an intracellular signaling protein has shown that catalyzed reporter deposition unnecessary. Integration of Quantum Dot® technology into protein microarray platforms may provide a means for subtle measurements of intracellular signaling pathways while surveying multiple molecular targets simultaneously through multiplexing.

E-51

Signal pathway profiles of inherited and sporadic renal cell carcinoma using reverse phase protein microarrays
R. L. Grubb III¹ , J. Yeamans² , V. S. Calvert³ , J. D. Wulfkuhle³ , W. M. Linehan⁴ , M. M. Walther⁴ , L. L. Liotta³ , E. F. Petricoin³ , ¹Urologic Oncology Branch, National Cancer Institute, Bethesda, MD & FDA/NCI Clinical Proteomics Program, Bethesda, MD, ²Department of Urology, Georgetown University, Washington, DC, ³FDA/NCI Clinical Proteomics Program, Bethesda, MD, ⁴Urologic Oncology Branch, National Cancer Institute, Bethesda, MD

Deregulation of cell signaling pathways underpins carcinogenesis. Reverse phase protein arrays are a new proteomics tool that couple Laser Capture Microdissection (LCM) with protein microarrays to analyze changes in signaling pathway activation. We have applied reverse phase protein array technology to examine the status of signaling involved in pro-survival and apoptotic pathways in renal tissue in hereditary and sporadic clear cell renal cell carcinomas. Frozen specimens of renal cell carcinoma and benign tissue were stained with a modified hematoxylin protocol. Pure specimens of renal epithelium were obtained by LCM. 4,000-6,000 LCM shots were pooled and lysed in buffer containing SDS sample buffer and Tissue Protein Extraction Reagent. After cell lysis, samples were boiled for 10 minutes at 100°C and 3 nL of the lysate were arrayed onto noncharged nitrocellulose slides with a glass backing. Staining was performed with an automated stainer using a biotinyl-linked peroxidase catalyzed signal amplification system. Commercially available phospho-specific and total antibodies against checkpoints in signaling pathways were used. Using reverse phase protein arrays coupled with LCM-obtained whole cell protein lysates, the status of signaling pathways between benign and malignant renal tissue will be studied. The states of phospho-specific checkpoints will be examined to determine changes in signaling events between benign and malignant renal tissue and between patients. Analyzing these specimens with multiplexed reverse phase protein arrays, the states of signaling changes between patient-matched benign and malignant renal tissue can be determined. Patterns in cellular signaling events between sporadic and hereditary clear cell renal cell carcinomas will be examined.

E-53

Tailoring Material Surfaces To Function As Harvesting Agents For Serum Proteome Analysis
D. Geho¹ , L. Lahar¹ , M. Lowenthal¹ , D. Johann¹ , P. Herrmann¹ , A. Mehta² , E. Petricoin¹ , L. Liotta¹ , ¹FDA (CBER)-NCI Clinical Proteomics Program, Bethesda, MD, ²FDA (CBER)-NCI Clinical Proteomics Program, Boston, MA

Low molecular weight molecules shed as a result of turnover and degradation of proteins from physiological and pathophysiological processes throughout the body provide a potential individualized portrait of a patient's state of health. Within the blood stream, low molecular weight molecules are rapidly cleared as they pass through the kidney unless they are bound to protecting, or carrier, molecules. These molecules effectively amplify the population of low molecular weight molecules by protecting them from size-mediated renal clearance mechanisms. One current challenge for clinical proteomics is to devise agents that selectively harvest carrier molecules from the blood so that the low molecular weight cargo they carry can be recovered and analyzed in a systematic manner. We are investigating whether material surfaces, such as derivatized silica, can be used as platforms for isolating carrier proteins and their low molecular weight cargo. To this end, silane-coated silica surfaces with controlled pore sizes have been activated with glutaraldehyde for covalent protein binding. One iteration involves the transformation of the silica surfaces into affinity capture agents in order to fractionate serum carrier proteins for individual study. The low molecular weight biomarker cargo can then be eluted from the carrier proteins for further proteomic study. Once defined, in the short term, these harvesting agents could be applied for systematic and reproducible ex vivo handling of patient blood samples as we study proteomic signatures of disease. In the future, harvesting agents designed using similar techniques could be infused as in vivo harvesting agents.

E-56

Software Tools for Clinical Proteomics
D. J. Johann¹ , M. D. McGuigan² , S. Tomov² , G. R. Whiteley³ , L. A. Liotta¹ , E. F. Petricoin⁴ , ¹NIH, ²BNL, ³SAIC, ⁴FDA

Regulatory science is now encountering advanced computer-aided diagnostic systems that are preparing to deploy a new paradigm involving the integration of medical imaging with high throughput molecular medicine tests. A synergistic test composed of a targeted imaging study correlated with genomic or proteomic test(s), offers the potential of a tremendous medical advancement, especially in difficult clinical scenarios involving the analysis and characterization of tumors. Software toolkits are a crucial component of these systems, both for the management of these large and complex data streams, and the novel algorithms used in the formation of a multiplexed diagnostic. Open source provides a mechanism for leveraging existing toolkits, sharing expertise, accelerating development, and furthering both software and systems science in new complex multi-disciplinary fields.

New types of proteomic biomarker technologies that correlate with clinically meaningful outcomes are active research areas in the Surrogate Biomarker Endpoint (SBE) scientific field. Clinical trials incorporating advanced biomarker techniques are scheduled to begin in the near future at the NCI, Center for Cancer Research. The first trial will be to clinically evaluate a multiplexed biomarker "pattern" obtained from mass spectrometer analysis of the low molecular weight proteome present in blood for monitoring the recurrence of ovarian cancer. The pattern is comprised of ions whose combined relative intensities serve as a discriminator for a disease phenotype. New SBE methods will therefore involve FDA filing of innovative software tools for the management and analysis of this challenging data.

E-59

Using Decision Forest to Classify Prostate Cancer Samples Based on SELDI-TOF MS Data - Assessing Chance Correlation and Prediction Confidence
W. Tong, Q. Xie, H. Hong, L. Shi, R. Perkins, E. Petricoin, FDA

Class prediction using omics data is playing an increasing role in diagnosis, prognosis and risk assessment. Omics data are usually noisy and represented by relatively few samples and a very large number of predictor variables (e.g., genes of DNA microarray data or m/z peaks of mass spectrometry data). These characteristics pose a significant challenge for most classification approaches in assessing a model's potential random correlation and overfitting of noise. This article proposes use of a novel pattern recognition method, Decision Forest, which combines the results of multiple heterogeneous but comparable Decision Tree models to produce a consensus prediction. The degree of chance correlation and prediction confidence for samples not used in fitting are rigorously assessed by simultaneously integrating Decision Forest with an extensive cross-validation and randomization testing. A Decision Forest model was developed to predict presence of prostate cancer using a proteomic dataset generated from surface enhanced laser deposition/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Comparison of model prediction with imposed random correlation demonstrated biological relevance of the model and the reduction of overfitting in Decision Forest. The model achieved 99.2% sensitivity and 98.2% specificity for the ~80% of samples during cross-validation with high confidence. Importantly, a list of model-selected peaks could be useful for biomarker identification. Decision Forest should be equally applicable to other omics data, such as gene expression data or metabonomic data. The Decision Forest algorithm is available upon request.

E-61

The domain analysis of C. botulinum types A neurotoxin and its relationship with other botulinum serotypes
S. K. Sharma¹ , H. D. Shulka² , ¹FDA, CFSAN, College Park, MD, ²Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202

Botulinum neurotoxins (BoNTs) are highly potent poisons that are produced by seven serotypes of Clostridium botulinum. The BoNT molecule is post-translationally proteolyzed to form a 1,285 amino acid di-chain molecule in which the two chains, 50 and 100 kDa, remain linked by a disulfide bond at N-terminal end of a heavy chain. The 150 kDa neurotoxin dimer is further complexed with other associated proteins known as "Neurotoxin Complex". The mechanism of neurotoxin action is a multistep process which leads to the cleavage one of three different SNARE proteins essential for synaptic vesicle fusion and transmission of the nerve signals to muscles: synaptobrevin, syntaxin, or SNAP-25. In order to understand the precise mechanism of neurotoxin in a host, the domain structure of the neurotoxin was analyzed among different serotypes of C. botulinum. The results indicate that neurotoxins type A, C, D, E and F contains a coiled-coil domain and types B and type G neurotoxin do not. Interestingly, the phylogenetic analysis based on neurotoxin sequences has further confirmed that serotypes B and G closely related. These results suggest that neurotoxin has multi domain structure and coiled coil domain plays an important role in oligomerization of neurotoxin in host. The domain analysis may help to identify effective antibodies to treat botulinum toxin intoxication. A detailed understanding of the architecture, catalysis, and recognition properties of these domains will also help to reveal how the toxins achieve their functional diversity.

E-63

Dextromethorphan to dextrorphan urinary metabolic ratio not sensitive enough to assess modest changes in CYP2D6 activity
J. C. Gorski¹ , S. Huang² , L. Li¹ , M. A. Hamman¹ , S. D. Hall¹ , ¹Division of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis, IN, ²Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, MD

The ability of the dextromethorphan (DTM) to dextrorphan (DT) urinary metabolic ratio (UMR) to detect subtle changes in CYP2D6 activity is not well defined. 10 extensive metabolizers and 1 poor metabolizer were studied on two separate occasions. Serum and urine were collected over 24 hours after DTM (30 mg) oral dosing. DTM and DT were quantitated by LC-MS (serum) and HPLC-fluorescence (urine). The DTM oral clearance (CL_PO), DTM/DT AUC ratio, and DTM/DT UMR were determined. DTM CL_PO was significantly correlated with the DTM/DT AUC ratio (r = 0.98) and DTM/DT UMR (n= 22; r = 0.765). Exclusion of the poor metabolizer resulted in loss of correlation between DTM CL_PO and DTM/DT UMR (r < 0.001) but not DTM/DT AUC ratio(r = 0.84). Intra and inter subject variability for DTM CL_PO, DTM/DT AUC ratio, and DTM/DT UMR was estimated to be 15%, 26%, 35% and 32%, 68%, 100%, respectively. Assuming an effect size of 30%, type I error rate at 5%, 80% power and a crossover design requires a sample size of 14, 34 or 56 patients when the primary outcome measure is DTM CL_PO, DTM/DT AUC ratio, DTM/DT UMR, respectively. If 14 subjects are used in a crossover study design and the DTM/DT UMR is used, the effect size would have to be 395% to maintain 80% power. Cross-sectional studies are similarly affected when the DTM/DT UMR is the principle outcome. The limitations of the DTM/DT UMR should be taken into account when modest effect sizes are expected.

Supported by NIH Grants M01RR00750 and FDT001756.

E-PO-01

Listeria innocua, indicator of Listeria monocytogenes
R.L. Bernstein, FDA San Francisco District, 1431 Harbor Bay Parkway, Alameda, CA 94502

Bioinformatics analysis of the available complete genomic DNA sequences of Listeria monocytogenes and Listeria innocua, supported by extensive field and laboratory sampling experience, strongly suggest that L. innocua can act as an effective indicator organism for L. monocytogenes. L. monocytogenes is pathogenic, responsible for approximately 500 deaths annually from foodborne infections in the U. S. L. innocua is non-pathogenic. Neither bacterium occurs naturally in foods, and the presence of either usually indicates human contamination. L. monocytogenes is a highly regulated organism in foods. Sampling of foods and processing material may sometimes recover L. innocua more easily than L. monocytogenes when both are present. Genome sizes are comparable: though L. innocua's genome is larger, L. monocytogenes has critical virulence genes. Gene-by-gene comparison strongly indicates conservation of metabolic and physiologic capacities between these highly related organisms, apart from the added virulence factors of L. monocytogenes. Correspondingly, growth conditions and laboratory tests involving biochemical parameters are virtually identical. Based on these observations, L. innocua ought to attain status as an indicator of L. monocytogenes.

CATEGORY F: ENGINEERING AND PHYSICS
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F-01

Abrasion Occurring During Emergency Response Can Hasten Loss of Glove Integrity
M. R. Schwerin, D. C. Richardson, CDRH, FDA, Rockville, MD 20852

Emergency Medical Technicians (EMTs) and fire and rescue personnel are often the first to respond to any mass casualty event to administer aid to the injured. This study was designed to address a scenario in which the possibility of a partially collapsed structure and/or a biohazard threat is assumed. Glove selection was made by surveying the rescue communities in the field to determine which brand/models were widely used. Using protocols reported previously, natural rubber latex glove specimens were first abraded and then flexed until ionic permeation was confirmed. Data were plotted as elapsed abrasion cycles versus cycles to fatigue failure. We concluded that extremely low levels of abrasion, similar to occupationally-caused abrasive wear (not detectable by casual inspection), led to significant reduction in the anticipated protection time afforded by latex gloves usually worn during duty. The measured reduction in time to perforation does not correlate to the manufacturing variation in glove thickness by one brand/model in the same lot. In the most extreme abrasive exposure we tested - at 75% of the minimum cycle count needed for perforation - the time to barrier failure was reduced an average of 40.6% (range: 12.2% to 77.9%).

F-02

Characterization of Material Properties of Natural Materials Using a Cell-Based Test Method
D. S. Kaplan¹, T. Vegella², R. Malinauskas¹, D. Schaefer³, Y. Deng⁴, J. Krause⁵, ¹OST, FDA, Rockville, MD 20850, ²OSM, FDA, Rockville, MD 20850, ³Dept. Physics, Astronomy and Geosciences, Towson Univ., Baltimore, MD 21252, ⁴Material Science and Engineering Lab, National Institute of Standards and Technology, Gaithersburg, MD 20899, ⁵Dept. Biology, Univ. Maryland, College Park, MD 20742

The biomedical and pharmaceutical industries are continually testing materials that may serve as scaffold materials for tissue engineered medical products (TEMPs). One such class of materials that is showing great promise is natural materials, which include materials such as alginate, chitosan, collagen and hyaluronan. These materials appear to have excellent biocompatibility and immunogenicity properties when properly purified. However, many of the natural materials used in TEMPs have little or no materials characterization as to their chemical, physical or mechanical properties and levels of contaminants (i.e., endotoxin levels). This has often resulted in variability in the products produced from these starting materials. To ensure the functionality of these biopolymers in an application, they must be fully characterized as to their physical, chemical and mechanical properties.

Our laboratory is developing a series of techniques aimed at characterizing both the natural materials and the cells employed in the TEMPs constructs. Physical characterization is performed through quantitative histomorphometric techniques, including Atomic Force Microscopy (AFM). Cell characterization of phenotypic stability is performed through cell-based assays for viability, adhesion and histomorphometry. This poster will describe the set of analytical techniques which we are employing to characterize the material properties of natural materials.

F-04

Lubricants Used by Consumers of Latex Gloves and Condoms Significantly Decrease Tensile Strength of Latex Gloves
D.L. Walsh, FDA/CDRH/OST, Rockville, MD

Medical glove and condom manufacturers have received multiple inquiries from consumers regarding whether or not certain lubricants are compatible with their products. The American Society for Testing and Materials (ASTM) formed Task Group D11.40.07 to address this issue. The Task Group developed a Round Robin test protocol for evaluating the compatibility of various consumer lubricants with natural rubber latex ("latex") gloves and condoms. This poster presents the results of the FDA/CDRH/OST laboratory as a participant in this Round Robin study. Three lubricants commonly used by medical glove and/or condom users (Keri® Lotion, Astroglide® personal lubricant, and Monistat® suppositories), as well as a positive control (Vaseline®), were tested for compatibility with latex. The lubricants were applied to latex glove specimens, conditioned for 60 minutes, and removed from the specimens prior to tensile testing. As expected, the positive control (Vaseline®) had a significant detrimental effect on tensile strength when compared to an untreated control group. Two of the test lubricants (Keri® and Monistat®) also had significant detrimental effects on tensile strength, while the third lubricant (Astroglide®) had no effect. No significant effect in elongation at break was expected for any of the test or control groups, yet a decrease was noted for the Monistat® group. These results will be pooled with those of other Round Robin participant laboratories and together will form the basis of the precision and bias statement of a new ASTM standard test method for evaluating the compatibility of consumer lubricants with latex barrier products.

F-05

In Vitro Steady Flow Evaluation of Clot Capture Efficiency and Clot Location for Five Commercial Vena Cava Filters
R. A. Robinson¹, R. A. Malinauskas¹, S. A. Yazdani², S. S. Das¹, J. K. Wozniczka³, D. Yabut³, H. F. Bushar⁴, S. F. Stewart¹, L. W. Grossman¹, ¹OST, FDA, Rockville, MD, ²Texas A&M, College Station, TX, ³Marquette University, Milwaukee, WI, ⁴OSB, FDA, Rockville, MD

Deep vein thrombosis and pulmonary embolism (PE) occur in over 600,000 U.S. patients annually, with approximately 50,000 deaths annually. Over 90,000 of these patients cannot be anticoagulated, and are implanted with vena cava filters to prevent PE. Recently, a number of serious cases of thrombosis, vena cava occlusion, and obstructed filters were reported to the FDA. This study was designed to identify potential thrombus forming mechanisms. Five commercial filters were evaluated in an in vitro physiological model of the vena cava, by injecting 2850 synthetic polymer gel clots. Clot capture efficiency was assessed as a function of filter type, vena cava diameter (small 20 mm and large 28 mm), vena cava orientation (horizontal or vertical), and clot diameter and length. Binary logistic regression was used to model the data. Clots were more likely to be captured in the small than in the large vena cava (p < 0.0001), and more likely to capture clots in the vertical orientation than in the horizontal orientation (p < 0.0001). Large long clots were more likely to be captured than small short clots (p < 0.0001). Filter ranking for all data in descending order of capture efficiency was: Simon Nitinol, Trapease, Vena Tech, Titanium Greenfield, and Bird's Nest filters (P < 0 .0001). However, the clinical relevance of these findings is not fully understood. Clot capture location was also dependent on filter design. Future studies will use flow visualization and clot location information to identify aberrant flows, which may contribute to thrombo-emboli and device occlusion. These studies will be useful in developing standardized testing protocols for CDRH guidance documents, to facilitate submissions and reviews and to help bring safer devices to market.

F-07

Integrating Design of Experiment and Multivariate Data Analysis as a PAT Tool to Extract Critical Process Knowledge from NIR Spectra and Explore the Causal Link Between Tablet Processing Conditions and Drug Dissolution Rate
H. Wu¹, R. C. Lyon², A. S. Hussain¹, C. D. Ellison², E. H. Jefferson², J. K. Drennen III³, D. DasGupta³, R. J. Voytilla³, L. Bejic³, ¹OPS, FDA, Rockville, MD 20852, ²DPQR, OPS, FDA, Silver Spring, MD 20993, ³Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Dequesne University, Pittsburgh, PA 15282

Near-infrared (NIR) spectroscopy has become an essential method of Process Analytical Technology (PAT) in the pharmaceutical industry. While the NIR spectra of solid pharmaceutical dosage forms are rich in both chemical and physical information, it is often difficult to extract essential process knowledge from spectral data. Multivariate data analysis techniques, such as principal component analysis (PCA) and partial-least squares regression (PLS), are mainstay techniques for the analysis of NIR spectral data. The objective of this work is to apply PCA and NIR spectroscopy to gain critical knowledge of a pharmaceutical manufacturing process. Two independent data sets for theophylline and furosemide tablets were generated according to a predefined experimental design (DOE) protocols. The theophylline tablets (immediate-release, and controlled-release formulations) were manufactured by direct compression, wet granulation, and direct compression-film coating. The furosemide tablets were manufactured through direct compression and wet granulation. Tablets were selected from each batch, scanned using NIR reflectance spectroscopy, and subjected to hardness and dissolution testing. A PCA model of the NIR data was created and used to analyze the effect of processing changes on tablet spectra. It was observed that the first and second principal components were indicators of tablet hardness and chemical composition. Furthermore, using the PCA model, a causal link between coating level and drug dissolution was identified. These results confirm the utility of NIR spectroscopy and multivariate data analysis as PAT tools, and illustrate their potential for investigating the causal link between pharmaceutical processing parameters, and performance characteristics of the subsequent solid dosage forms.

F-08

Computational Analysis of Fluorescence Spectroscopy Devices with Angled Delivery and Collection
J. Pfefer¹, A. Agrawal¹, R. Drezek², ¹OST, FDA, Rockville, MD, ²Rice University, Houston, TX

Optimization of illumination-collection parameters may lead to an improvement in the efficacy of fluorescence spectroscopy devices for minimally invasive detection of diseases such as cervical neoplasia. While device-tissue interface geometry has been shown to have a strong influence on the origin of detected fluorescence, prior studies have not provided significant information on devices which deliver and collect light at non-normal angles relative to the tissue surface. Optical fibers with beveled surfaces enable the delivery and/or collection of light at a range of angles. This feature may make it possible to interrogate subsurface tissue regions with a greater degree of spatial selectivity. In this study, simulations were performed using a Monte Carlo model of fluorescent light propagation in homogeneous tissue so as to estimate the optical behavior of the probes. Specifically, the effect of tissue optical properties, illumination angle, collection angle and illumination-collection fiber separation distance were investigated. While the advantage of an angled approach was greatest for tissue with lower levels of attenuation, the relative sensitivity at a specific targeted depth doubled in several cases as the illumination or collection angle was increased from 0 (flat) to 45 degrees. In conclusion, our model indicates that angled fiber probes may significantly improve a device's ability to selectively interrogate specific tissue regions or layers.

F-09

Toward Understanding Intracellular Light-Tissue Interaction Mechanisms Using Optical Nanobiosensors and Nanoimaging
I. K. Ilev¹, R. W. Waynant¹, E. Gorman¹,R. Weiblinger¹, K. Byrnes², T. Romanczyk², J. Anders², R. Claus³, A. Gandjbakhche⁴, ¹US FDA, CDRH, Rockville, MD 20850, ²USUHS, Bethesda, MD 20814, ³Virginia Tech, Blacksburg, VA 24061, ⁴NIH, Bethesda, MD 20892

Minimally invasive photonic biosensor and imaging techniques are potential alternatives to conventional medical methods for diagnosis of diseases because these techniques offer an effective, highly sensitive, fast and painless way for sensing and monitoring of various biomedical quantities. However, in order to optimize effectiveness and ensure safety of optical biosensing and imaging devices as they progress from concept to consumer, fundamental mechanisms of light-tissue interactions and photochemical processes need to be understood at cellular and intracellular levels. Here, we present a systematic study of basic intracellular light-tissue interaction mechanisms using fiber-optic nanobiosensor probes and highly sensitive imaging in the subwavelength nanoscale range. The nanobiosensors are utilized for direct probing and chemical analysis within individual cells. In this way we can detect small concentrations of target molecules or intracellular analytes such as reactive oxygen species, calcium, and glucose. In addition, the study includes comparable experiments based on an in vivo bulk animal model. Using smart tissue-activated fiber-optic probes, we investigate optical properties and light transmission characteristics of single and multiple tissue layers and blood utilizing either coherent or incoherent broadband light sources in the visible and near-infrared. The results of these studies are critical for understanding basic medical processes including optical diagnostics, photodynamic cancer cell killing, low power laser therapy, effects of light-activated oxygen, generation of beneficial chemicals and cellular repair. The findings of these studies will be incorporated into guidance documents, thus providing a solid foundation for future regulatory decisions on optical biosensing and imaging devices.

F-10

The Role of Young's Modulus in the Development and Performance of Laser Ablation Phantoms
D.D. Royston, FDA OST Rockville, MD

The laser ablation efficiency of tissue has been shown to depend upon a tissue's mechanical properties, such as its Young's modulus. This study was undertaken to determine the Young's modulus of some commonly used gel phantoms and compare those values to those of tissue. The role of Young's modulus in determining a gel's suitability as an ablative phantom is also discussed. The Young's modulus of polyacrylamide and agar gels was determined using a simple laboratory balance that was modified to measure the elastic modulus of small tissue samples. The apparatus was calibrated with plastisol cylinders of known Young's modulus. Polyacrylamide gels of 95, 90, 86, 80, and 75% water concentration and a 2% agar gel were constructed and their Young's modulus determined.
The Young's modulus of the polyacrylamide gels was found to vary linearly with the water concentration (R=0.996). The range of Young's modulus values for these gels was from 2.2 to 67.99 kPa. These values disagree with modulus values (31 to 866 kPa) found in a previous ablation study of similar gels. However, other literature values were found to agree with those found in this study. The Young's modulus found for the agar gel, 60.9 kPa, also agrees with other literature values. A 2% agar gel has a Young's modulus of 61 kPa. A 20% polyacrylamide gel has a Young's modulus of 50 kPa. These values are similar to literature values for in vivo ventricular myocardium.

F-12

Mechanical Characterization of Calcaneus Bone
N. C. Dermody¹, J. H. Graham², T. O. Woods², ¹Department of Biomedical Engineering, Marquette University, Milwaukee, WI 53201, ²Division of Mechanics & Materials Science, CDRH, FDA, Rockville, MD 20850

This project is part of an ongoing effort to develop methods for using ultrasound in the diagnosis of osteoporosis. In previous work, human calcaneus bone samples were thoroughly characterized using ultrasound (backscatter, attenuation, and sound speed measurements) and micro computed tomography (measurements of bone morphology). The objective of this work is to relate the previous measurements to the mechanical properties of the bones. Protocols and test fixtures were developed to mechanically test and evaluate bone samples in compression. Fifty-five test specimens were prepared from 33 bone samples. Compressive strength, compressive modulus, apparent density and volume fraction of bone were determined for each sample. Strength and modulus showed good power-law correlations with apparent density (r=0.66 and 0.56, respectively), and a very strong correlation with each other (r=0.94).

CATEGORY G: NUTRITION AND OBESITY
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G-01

Nutrition Information in Restaurant Menus: An Online Survey
C.F. McLaughlin, FDA, College Park, MD

The purpose of this study is to examine the current state of nutrition information in restaurant menus by examining the menus available on the Internet. The type of nutritional information, when available online, includes calories, calories from fat, total fat, sat fat, sodium, along with other nutritional information. The survey also shows that in recent months, a growing number of restaurant websites are presenting nutrition information as an interactive tool, such as a meal builder or a printable version or both.

Preliminary observations show that while some websites provide very complete nutrition information, this practice is not yet characteristic of the industry. Moreover, except for a very few websites, it is not possible to determine, if the nutrition information available online, is available on the premises as well. The nutrition information, when provided, is generally a separate file from the online menu. A few online menus are available in a format that probably resembles the actual restaurant menu (on premises) but none of these menus showed information on calories or fat.

The study was conducted by reviewing the top 100 firms that used "Eating Places" as their designated primary function in the D& B Market Identifiers (Dialog File 516) limited by records that contained restaurant sales information with annual sales of $30 million or above. The type of restaurant chains sampled included casual dining, fast food and other (upscale, pizza delivery, buffets, etc).

G-03

A Social Sciences Approach to Public Policy with Special Emphasis on Consumer Information, Food Choices, and Weight Management
R. Williams, P. Vardon, FDA, College Park, MD

The social sciences provide a systematic approach to obtaining knowledge about human behavior. Humans, equipped with the capacity to think, alter their behavior under changing conditions, not least when they obtain new information. Success in implementing public policy must be judged pragmatically to determine whether a policy actually solves human problems. To evaluate the success of a policy, social scientists use theory and observation of human responses through an array of research tools to derive an inference that is a tentatively accepted generalization. Scientific progress is made through research that expands generalizations and improves confidence in the validity and the accuracy of predictions.

With new nutritional information, people react, or not in ways that can be predicted. Using the disciplines of social psychology and economics, social scientists predict how consumers adjust their consumption and how manufacturers change the foods they produce. Thus, there is a symbiotic relationship between the life science of nutrition and the social sciences. The nutritionists tell us what information we need, and the social sciences tell us how to use the information to accomplish the common goal of better health.

Examples of the social science contribution to the obesity problem are given in this series of posters, by determining a) how consumers react to changes in food labels on packaged food and in restaurants; b) how manufacturers react to labeling requirements with new products and product reformulation and c) what the current retail environment is now for nutrition labeling. .

G-04

FDA's Food Safety Survey 2001: Use, Understanding and Perception of Food Labels by Food-Allergic People
K. A. Vierk, K. M. Koehler, S. B. Fein, D. A. Street, CFSAN, FDA, College Park, MD

The Food Safety Survey (FSS) is a national random digit-dial survey of American consumers conducted periodically by the Food and Drug Administration. One purpose of FDA's 2001 FSS was to describe opinions about various characteristics of food labels among adults with self-reported food allergy.

A total of 4,482 persons responded to the survey and of these individuals, 471 (9%) self-reported having a food allergy. Among the persons with food allergies, 306 persons stated that they read food labels to avoid allergenic foods. The following percentage of the 306 persons responded that these labeling issues were serious or very serious for managing an allergy: 1) 36%, words on some ingredient lists are too technical or hard to understand; 2) 42%, different words for the food a person is allergic to are used on different food products; 3) 29%, some ingredient lists are so long that it is hard to find the ingredient that the person is concerned about; 4) 41%, food labels do not always alert people that new ingredients have been added to a food; and 5) 44%, some ingredient lists give a general name for an ingredient without specifying the source. Additional information from the FSS will be presented; for example, responses on labeling issues will be compared for those who reported having doctor-diagnosed food allergies and those whose reported symptoms correspond with symptoms for IgE-mediated allergies.

Clear Science Communication Award - 2004 FDA Science Forum

G-05

Consumer's Use of Food and Restaurant Labeling: Focus Group Results
A. M. Lando, J. M. Labiner, R. A. Williams, CFSAN, FDA, College Park, MD 20740

A series of eight focus groups were conducted in four locations around the U.S. to explore how consumers use the Nutrition Facts Panel on food labels and consumer interest in nutrition labeling in fast food restaurants. Focus groups are guided discussions of about 8-10 people and are often used to explore consumer reactions and thoughts to different topics or stimuli. They are useful for getting the range of opinions that consumers may have about a specific topic and for that reason are often done prior to surveys or experimental studies. The groups were segregated by gender and education. All participants were 18 years or older, regular food shoppers, and consumers of fast food. Results indicate that participants, especially women, cared about nutrition and reported using the Nutrition Facts Panel. At the same time, however, many also said that they do not always consider nutrition when deciding what to eat. Taste, convenience, price, and what their family will eat were often at odds with healthy eating. Many participants read the Nutrition Facts Panel for a specific nutrient. Overall, participants cared about calories and many nutrients including saturated fat, total fat, cholesterol, carbohydrates, and sodium. In general, participants thought it was misleading to label a product usually consumed in one sitting, such as a 20 oz soda or a large muffin, as multi-serving. Participants expressed interest in having nutrition information posted in fast food restaurants, but said they might not always use this information.

G-06

Determination of Iodine in Food: A Comparison between Manual Analysis and Automated Analysis
T. L. Smith¹, R. Luchtefeld², K. J. Mulligan³, R. E. Smith⁴, ¹Elemental Analysis Group, Total Diet Program, US FDA, Lenexa, KS 66214, ²Total Diet Pesticide Research Center, US FDA, Lenexa, KS 66214, ³Forensic Chemistry Center, US FDA, Cincinnati, OH 45237, ⁴UMKC Dept. of Pharmacology, Kansas City, MO 64110

As part of the Total Diet Study, the US FDA monitors the concentrations of toxic and nutrient elements. Iodine analysis is being reintroduced in the Total Diet Study. The analysis described uses a ternary acid digestion (nitric, perchloric, sulfuric) which is carefully controlled by conducting the reaction under reflux using a condensing cold finger. The subsequent analysis is based on the catalysis by iodine of the oxidation-reduction reaction between Ce⁺⁴ and As⁺³. This analysis was first implemented manually. 4 NIST Standard Reference Materials (SRMs) and 13 items from the 2001-02 Market Basket were successfully analyzed. We reported on this work at the 2002 FDA Science Forum. In the interest of efficiency, an autoanalyzer has been acquired. This instrument is the QuikChem^® FIA+ 8000 Series by Lachat, a Hach Company brand. After some minor modifications to a method which was provided by Lachat, we have successfully analyzed 3 NIST SRMs and 13 similar items from the 2003-04 Market Basket. This poster compares and contrasts the two modes of evaluation and demonstrates that the automation of the determinative step has made the analysis of Iodine a practical part of the Total Diet Study.

G-07

Nutrition and Health: Preliminary Findings from the 2002 FDA Health and Diet Survey
C.-T.J. Lin, FDA

Knowledge of what consumers say about nutrition and health is useful to the agency in deliberating and developing effective communication strategies and other actions to help consumers make informed diet choices. This poster will report preliminary findings from the 2002 FDA Health and Diet Survey about what consumers say about (1) the relationships between diet and certain chronic illnesses, (2) fats and cholesterol, (3) dietary management practices, and (4) use of food labels. The findings suggest many consumers know about the general relationships between diet and certain chronic illnesses. But many of them are also uncertain about fats and cholesterol. They say they use food labels for a variety of purposes.

G-08

2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PHIP)-induced tandem double CC->TT mutations in the mitochondrial DNA of rats: effects of strenuous exercise.
R. K. Panguluri, M. G. Manjanatha, S. A. Ferguson, M. E. Bishop, L. E. Lyn-Cook, A. Aidoo, FDA

CCàTT mutations known to occur in cancer-related genes are associated with reactive oxygen species (ROS) which are also induced by food-borne heterocyclic amines (HCAs). Due to the continuous exposure of HCAs in the diet and the importance of mutations in disease processes and aging, we have exposed rats to PhIP and have used a modified non-radioactive, PCR-RFLP-SSCP method to screen mitochondria for this genetic aberration. Male and female rats subjected to a treadmill exercise and their respective controls were continuously exposed to 50 mg/kg PhIP for 10 days. The treadmill exercise continued for 5 weeks after PhIP treatment. At sacrifice, colon, kidney, liver and muscle were processed for the isolation of mitochondrial DNA. Using PCR primers, 841 bp region of NADH subunit 6/tRNA-Glu/cytochrome b of the mitochondria was amplified. The PCR products were digested and separated on Mutation Detection Enhancement gels. The variant bands on the gels were then isolated and sequenced. DNA sequence analysis revealed CCàTT tandem transition mutations in the region of cytochrome b of mitochondrial DNA (14135-14136). None of the tissues from PhIP-treated and exercised male rats showed CCàTT mutations in the mitochondrial DNA; however, among the non-exercised male group, the frequency markedly increased from 12% - 50%. In contrast, PhIP exposed female rats displayed high frequency of CCàTT mutations in both exercised (17% - 37%) and non-exercised groups (14% -57%). Also, PhIP-treated male and female rats in the exercised group exhibited a decrease in the frequency of CCàTT mutations. Tandem mutation was not found in the liver of male, non-exercised rats. These results indicate that CCàTT tandem mutations occur in the mitochondrial DNA of PhIP-treated rats and suggest that this novel mutation could be indicative of exacerbated ROS production in the face of PhIP exposure. Our results also suggest the benefits of exercise in human health.

G-09

Psammomys obesus (Fat Sand Rat) as a Model of Nutritionally Induced "Diabesity": Understanding Food Consumption, Composition and Behavior
A. A. Cotterell¹, J. C. Anglin², V. M. Chenault¹, ¹FDA, ²Howard University

The prevalence of obesity has increased over thepast few decades and is one of the leading public health issues in the U.S.. Obesity is associated with a number of adversemedical conditions - diabetes, cardiovascular disease, hypertension, cancer, arthritis and pulmonary dysfunction. Diabetes is also associated with a number of degenerating and life threatening diseases such blindness, kidney and heart disease. Medical problems, previously associated with adulthood, are more prevalent in children. Overweight adolescents have an increased risk of becoming obese adults, which increases the risk for chronic diseases. The sand rat is an excellent model for the study of nutritionally induced diabetes, insulin resistance, and obesity. These rodents eat a low calorie, high fiber, electrolyte rich plant (saltbush) in the wild. In captivity, when fed standard rat chow, obesity results with all the concomitant complications. Understanding the consumption and composition of rodent chows in an animal model of "diabesity" will help us understand obesity and glucose homeostasis in humans. This study consists of the commercial development and evaluation of a high fiber/low energy diet based on a "saltbush comparable" formulation and a second diet at least 5% higher in fiber and 2% lower in protein. It was proposed that these diets were healthier. Body weight, food intake, behavior, blood parameters and clinical symptoms were compared and contrasted. The results were surprising in that Test Diet 1 was not well tolerated and Test Diet 2 produced some changes in glucose homeostasis. Additional analyses are underway to elucidate the results obtained.

CATEGORY H: FOOD SAFETY INITIATIVE
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H-01

Investigating Sources of Paralytic Shellfish Poisoning Toxins in a Novel Vector: Abalone
S. M. Etheridge¹, R. Benner¹, G. C. Pitcher², C. S. Roesler³, S. Hall¹, K. White⁴, ¹US FDA, Washington Seafood Laboratory, Laurel, MD, ²Marine and Coastal Management, Cape Town, South Africa, ³Bigelow Laboratory for Ocean Sciences, W. Boothbay Harbor, ME, ⁴US FDA, Instrumentation and Biophysics Branch, College Park, MD

Dinoflagellates of the genus Alexandrium are the most common sources of paralytic shellfish poisoning (PSP) toxins, and suspension-feeding bivalves are the usual vectors. However, in 1999 PSP was detected in the abalone Haliotis midae off the coast of South Africa. As a result, harvesting was temporarily suspended on the West Coast where the highest abalone toxicity was observed. This is noteworthy since, unlike the common vectors of the saxitoxins, abalone are neither filter feeders nor carnivores. Instead they feed on seaweed and scour benthic organisms from rock surfaces. The toxin profile in these gastropods differed from that of the local Alexandrium population, a potential toxin source; although, this difference may be attributed to biotransformation by the abalone. Additionally, the lack of seasonal trends in abalone toxicity has made it difficult to ascertain the toxin source. Thus, the goal of our research was to identify the source of abalone toxins. To date PSP toxins have been detected in Alexandrium sp., the kelp Eklonia maxima (the in situ food source for abalone), and bacteria isolated from abalone. While the toxin source remains unclear, the need for including abalone and other non-traditional vectors in monitoring programs for PSP is clearly evident. Though PSP toxins have not been reported in US abalone, determining the source of toxins in South African abalone may identify potential threats of toxic abalone in the US. Our current and future investigations address 1) whether/how Alexandrium toxins may be transferred to abalone, 2) toxin production by kelp versus toxic organisms growing on kelp, and 3) autonomous toxin production by bacteria.

H-03

Printing Microarrays of Bacteria for Identification by Infrared Microspectroscopy
M. M. Mossoba¹, S. F. Al-Khaldi², J. P. Kirkwood³, J. Sedman³, A. A. Ismail³, F. S. Fry¹, ¹OSAS/FDA, College Park, MD, ²DMS/FDA, College Park, ³McGill University, Canada

Infrared (IR) microspectroscopy is a non-destructive and label-free analytical technique that has been used for the identification of bacteria. In general, bacterial samples are manually deposited onto a substrate for IR measurement. The possibility of applying an automated bacterial sampling step based on Microarray technology was explored. In the present study, the feasibility of applying contact deposition technology to print intact bacterial cells as arrayed spots (150 µm diameter) on optical substrates and agar slides was demonstrated. Protocols for printing microarrays of whole-cells were evaluated and optimized by IR microspectroscopy and imaging. Parameters that were refined included pin capacity, deposition mode, and spatial distribution of microarrays. Using a contact micro-spotting robotic system, representative foodborne bacteria (Yersinia enterocolitica 289, Staphylococcus aureus ATCC 19075, Salmonella typhimurium DT104 (serotype 104 B), Listeria monocytogenes, Enterobacter cloacae, Citrobacter freundii, Klebsiella pneumoniae KP73, and Escherichia coli) were arrayed on zinc selenide and agar slides. In the latter case, arrayed bacterial spots were allowed to grow on a BHI agar slide, stamped onto an infrared slide, and in both cases analyzed by IR and chemometric data analysis. Dendrograms generated by hierarchical cluster analysis indicated clustering of the descendants into their respective branches. Potential applications and advantages, such as high throughput printing and IR imaging, of using microarray technology over manual sampling procedures for bacteria will be discussed.

H-04

Prevalence and Antimicrobial Resistance of Campylobacter spp. Isolated from Retail Foods of Animal Origin in the United States D. G. White, L. English, P. Carter, S. Bodeis, T. Proescholdt, K. Lee, P. F. Mcdermott, R. Singh, D. Melka, R. D. Walker, CVM, Office Of Research

Campylobacter jejuni is one of the principal causes of acute bacterial gastroenteritis in the U.S. In an attempt to determine the incidence of Campylobacter contamination of retail meats in the U.S., 814 samples of retail raw meats (chicken, turkey, pork and beef) were randomly obtained from 209 grocery stores in the state of Iowa from June 2001 to June 2002, and cultured for Campylobacter. Campylobacter isolates were further identified to species and tested for susceptibility to 4 antimicrobial agents by the NCCLS recommended agar dilution method. A total of 192 Campylobacter isolates were recovered from the samples with contamination rates ranging from 75% of chicken breasts to 0% of ground beef samples. Overall, C. jejuni was the predominant species recovered (59%), followed by C. coli (41%). C. jejuni was also the predominant species recovered from chicken (63%), where as C. coli predominated from ground turkey (73%). Of the 114 C. jejuni strains susceptibility tested by agar dilution, 18% had an MIC > than 4 µg/mL for ciprofloxacin, whereas 5% exhibited MICs > 8 µg/mL for erythromycin. Among the 78 C. coli isolates tested, 29% of strains displayed MICs > than 4 µg/mL for ciprofloxacin, and 37% of strains exhibited MICs > 8 µg/mL for erythromycin. C. coli also tended to exhibit reduced susceptibility to doxycycline as compared to C. jejuni strains (55% vs. 28%, respectively). These findings merit sustained surveillance of bacteria recovered from throughout the food production continuum to detect emerging antimicrobial resistance phenotypes.

H-05

Antimicrobial susceptibility profiles among Salmonella spp. recovered from retail foods of animal origin, NARMS 2002
D. G. White¹, S. Ayers¹, A. Glenn¹, E. Hall-Robinson¹, S. Zhao¹, P. F. Mcdermott¹, S. Friedman¹, A. Walker¹, T. Proescholdt¹, R. D. Walker¹, J. E. Stevenson², ¹CVM, Office Of Research, ²centers For Disease Control And Prevention The National Antimicrobial Resistance Monitoring System (NARMS) retail food arm is a collaborative effort between CDC, FoodNet sites and the U.S. Food and Drug Administration (FDA). Salmonella were recovered from a convenience sample of chicken breast, ground turkey, ground beef and pork chops purchased from grocery stores in the participating 6 FoodNet states (CT, GA, MD, MN, OR, TN). Minimum inhibitory concentrations were determined via broth micro-dilution methods using the Sensititre^TM susceptibility system and interpreted according to the NCCLS standards. One-hundred and sixty-nine of 2513 retail meat samples (7%) were contaminated with Salmonella. After microbiological analysis, 153 Salmonella were tested for antimicrobial susceptibility. The majority of Salmonella isolates were susceptible to the antimicrobials tested (43%), however, resistance was observed to tetracycline (46 %), streptomycin (35 %), sulfamethoxazole (23 %), ampicillin (19 %), and kanamycin (12 %). Twenty-one Salmonella isolates were resistant to at least one antimicrobial, whereas 15 isolates displayed resistance to 7 or more antimicrobials. Multi-drug resistant S. Newport was recovered from ground beef, ground turkey, and pork chop samples. All isolates were susceptible to ceftriaxone, however, 11% were considered resistant to the veterinary extended-spectrum cephalosporin, ceftiofur. All isolates were also susceptible to ciprofloxacin, however, 5% of isolates were resistant to nalidixic acid. The nalidixic acid resistant Salmonella were all recovered from ground turkey samples. This study demonstrated that multiple antimicrobial-resistant Salmonella isolates were present in retail food of animal origin and may reflect a reservoir of resistance in animals that can be transmitted through the food processing chain.

H-06

Food Additive Petition Expedited Review
A. D. Laumbach, J. C. Wallwork, R. L. Martin, FDA

Reports of foodborne illnesses caused by microbial contamination have increased over the past few years. While America's food supply is among the safest in the world, every year millions of Americans become ill and some deaths occur as a result of infections caused by foodborne pathogens. The Food and Drug Administration is concerned about this increase in foodborne illness, and has undertaken several actions in an attempt to address this issue. One such action has been to give priority to the review of petitions for food additives that are intended to decrease the incidence of foodborne illness through their antimicrobial action against human pathogens that might be present in food. The process and requirements for considering petitions for designated expedited review will be presented.

H-07

ICP-MS Analysis of Dietary Supplements for Toxic Heavy Metals
J. Wong, K. Shippey, C. Castro, L. Lau, SAN-DO, Alameda, CA

The dietary supplement market in the US is large and continues to grow due to demographic trends as well as by the unsubstantiated perception that the supplements can and will prevent diseases and enhance health. The supplement industry, unlike the drug industry, is not rigorously regulated by FDA. Lack of regulation means less reliability in the quality of the products, domestic and imported. SAN-DO lab has analyzed several popular supplements in the past few months and found, not surprisingly, that unacceptable high levels of toxic metals were present in two supplements. The lack of regulation may be apparent when different lots of the same product have been analyzed. For example, analysis of several lots of one popular dietary supplement gave results from 0.5 mg/kg to 8 mg/kg of Lead This project uses ICP-MS for the analysis of toxic metals in popular supplements which include Echinacea/Goldenseal, Saw Palmetto, Black Cohosh, Glucosamine/Chondroitin, SAM-e (S-adenosylmethionine), Ginko Biloba, Salmon Oil, Flaxseed Oil, Cod Liver Oil and Ginseng. The ICP-MS was selected for its high sensitivity, multi-element capability, and fewer matrix interferences.

H-09

Effects of Gamma Irradiation on Color Additives
V. Komolprasert¹, T. Diel², G. Sadler², ¹FDA, Summit-Argo, IL, ²NCFST, Summit-Argo, IL

The effects of irradiation on color additives have not been studied. In this study, the two colorants used were chromophtal yellow 2RLTS and Irgalite Blue GBP. Each colorant was blended with all-purpose grade PS pellets at concentrations of 0.1, 0.5 and 1.0% (w/w) before extrusion to produce PS sheets. The PS sheets were cut into PS test specimens, and placed in 20 ml glass vials or 250 ml glass jars for subsequent gamma radiation at 10 and 20 kGy doses at ambient temperature. After irradiation, the test specimens were analyzed with FTIR, static headspace (HS) with GC/MSD, total polymer dissolution, and migration studies using 10% and 50% ethanol solution at 40°C for up to 10 days, in comparison with the non-irradiated test specimens.

Qualitatively, gamma radiation did not affect the IR spectra of pure colorant and PS test specimens measured in a range of 600-4000 wavenumbers. The qualitative HS/GC/MSD results suggest that irradiation did not generate new chemicals. Volatiles detected in the colorant specimens were not detected in the colored PS sheet specimens. Total polymer dissolution and analysis of the extracts by HPLC-PDA yielded the extraction of PS residues including phenol, phenyl ethanol, benzaldehyde, acetophenone and styrene. Irradiation increased the concentrations of these chemicals but had no effect on styrene. Amounts of PS solids migrating from PS test specimens into 10% and 50% ethanol food simulating solvents were in a range of 0.0035 - 0.013% (w/w) based on polymer weight. The average global migration of these solids was in a range of 0.07 - 0.25 mg/dm^2.

H-11

Identification of Significant Co-resistance among Quinupristin-Dalfopristin-Resistant Enterococcus faecium Isolated from Retail Meats
J. R. Hayes¹, D. D. Wagner¹, L. L. English¹, P. J. Carter¹, T. Proescholdt², K. Y. Lee², D. G. White¹, ¹OR, CVM, FDA, Laurel, MD, ²OSC, CVM, FDA, Rockville, MD

The long-term use of the streptogramin analogue, virginiamycin, in food animal production has led to a reservoir of Q-D-resistant E. faecium (QDREF) isolates from animal products. This study was undertaken to determine the magnitude of association between resistance to Q-D and other antimicrobial agents among isolates of E. faecium from retail foods of animal origin.

From a collection of E. faecium isolated from retail meats from Iowa, Q-D susceptible (587) and resistant populations (238) were statistically compared to determine the degree of association between Q-D resistance and other tested antimicrobials, measured by the prevalence of resistance and described by the odds ratio (OR).

Among the QDREF isolated from the four meat sources, significant co-resistance phenotypes were observed with tetracycline (57-93%, OR 2.3-18) and lincomycin (88-98%, OR 4.7-39) among isolates from poultry and ground beef. Q-D resistance was associated with resistance to erythromycin among ground turkey (60%, OR 1.8) and chicken (35%, OR 3.0) isolates whereas co-resistance to tylosin was significant among isolates from chicken (32%, OR 3.9) and ground beef (46%, OR 12). Co-resistance was observed with high-level streptomycin resistance among chicken isolates (53%, OR 7.6) and to nitrofurantoin (67%, OR 2.3) and high-level kanamycin resistance (74%, OR 72) among isolates from ground beef.

The finding of significant co-resistance phenotypes among QDREF recovered from different retail meats suggests that there are differences in the antimicrobial selection pressures exerted among the tested food animal production environments and that other antimicrobial agents may co-select for QDREF within these environments.

H-12

Establishing a Food Chain Link Between Pyrodinium bahamense (Dinophyceae) and Paralytic Shellfish Toxin (PST) Poisoning Due to Consumption of Southern Puffer Fish (Sphoeroides nephelus) from Florida Waters.
J. Deeds¹, L. Flewelling², B. Richardson², S. Etheridge¹, K. White³, S. Conrad¹, J. Landsberg², S. Hall¹, K. Steidinger², ¹US FDA Washington Seafood Laboratory, Laurel MD, 20708, ²Florida Fish and Wildlife Conservation Commission, St. Petersburg FL, 33701, ³US FDA Instrumentation and Biophysics Branch, College Park MD, 20740

In 2002, 19 unexpected cases of Puffer Fish Poisoning were reported in the United States (14 from Florida, 3 from New Jersey, and 2 from Virginia). After all cases were linked to consumed puffer fish originating from the Indian River Lagoon (IRL), near Titusville on Florida's east coast, puffer fish harvesting was banned in the IRL and public health warnings were issued for consumption of puffer fish caught from the Titusville area. Coordinated studies between the Florida Fish and Wildlife Conservation Commission, the FDA's Washington and Gulf Coast Seafood Laboratories, Canada's Institute for Marine Science, and the NOAA Marine Biotoxins Laboratory determined that PST's (saxitoxin and saxitoxin-congeners) were the causative agents in human intoxication and that the dinoflagellate Pyrodinium bahamense was the putative toxin source. These were the first confirmed reports in the US of puffer fish PST poisoning and PST producing Pyrodinium bahamense in Florida waters.

In 2004, the FDA WSL, in collaboration with the Florida Marine Research Institute, will evaluate a hypothesized food chain link between PST producing dinoflagellates and southern puffer fish (Sphoeroides nephelus). This will be accomplished through mass culturing of PST producing dinoflagellate species, such as P. bahamense, experimental contamination of small shellfish species endemic to the IRL, followed by feeding to southern puffer fish. Future studies will also compare various puffer fish species, including northern puffers (Sphoeroides maculates) which are harvested commercially on the US east coast. Our goals are to evaluate the potential human health risks that puffer fish pose as a new vector for PST poisoning in the US and to evaluate P. bahamense as a new source of PST's in Florida waters.

H-13

The impact of prolonged virginiamycin feeding in chickens on streptogramin susceptibility in native Enterococcus faecium
P. Cullen, P. F. McDermott, S. D. McDermott, S. K. Hubert, D. D. Wagner, CVM, Office Of Research, Laurel, MD

Background. The use of antimicrobial agents in food animals and the potential impact on resistance in human infections is an area of intense public health concern. The streptogramin virginiamycin has been used for nearly 30 years in food animal production to prevent disease and promote weight gain. Virginiamycin induces complete cross-resistance to quinupristin/dalfopristin (Q/D). As part of a quantitative risk assessment being developed by the FDA, the effects of virginiamycin on the development of Q/D resistance of Enterococcus in a poultry model production ecosystem was examined. Methods. We conducted four consecutive experiments using broiler chickens raised on common litter. In each replicate, one group of birds was fed virginiamycin continuously in feed at 20 g/ton and a second group served as the non-treated control. Fecal samples were collected and cultured for Enterococcus one, two, three and seven weeks after beginning medication. Results. E. faecium with Q/D MICs above the resistance breakpoint (>4 µg/mL) appeared at Week 7 in the first replicate, and at Week 1 in the second and third replicates. No strains displayed MICs >32 µg/mL. PCR failed to detect vatD, vatE, vgaA, vgbA or ermB in any of the isolates. The msrC gene was detected in 97% of resistant E. faecium. Approximately 2 weeks passed between flocks, during which time most litter-derived E. faecium maintained MICs above the resistant breakpoint. Virginiamycin was removed from the feed for the fourth replicate, and all strains displayed pre-treatment MICs by Week 7. Conclusions. We examined the temporal relationship of resistance development to antibiotic exposure, MIC levels, and persistence of the resistance once the selective pressure of the antibiotic was removed from the environment. Our findings show that, in a experimental setting, resistance required continuous streptogramin exposure to be maintained in the broiler gut. This suggests that management of litter and controlled use of streptogramin antimicrobials could help limit the dissemination of Q/D-resistant Enterococcus out of the poultry production environment.

H-15

Antibiotic Resistance Patterns of Foodborne Bacterial Pathogens Isolated from Retail Meats: NARMS Retail Meat Surveillance
D. G. White¹, S. Ayers¹, L. English¹, A. Glenn¹, S. Gaines¹, S. Zhao¹, J. Abbott¹, S. Qaiyumi¹, S. Friedman¹, P. Carter¹, P. Cullen¹, S. Mcdermott¹, P. F. Mcdermott¹, R. Singh¹, D. Melka¹, E. Hall-Robinson¹, T. Proescholdt¹, A. Walker¹, R. D. Walker¹, J. E. Stevenson², ¹CVM, Office Of Research, ²centers For Disease Control And Prevention

The NARMS Retail Food Study is a collaborative effort between CDC, ten FoodNet sites (California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York, Oregon and Tennessee) and the U.S. Food and Drug Administration (FDA). The NARMS Retail Food Study uses a standardized method to monitor the prevalence of antimicrobial resistance among Campylobacter, E. coli, Salmonella and enterococci isolated from a convenience sample of meat and poultry from selected grocery stores. Data collection began January 1, 2002. Each site visits at least one grocery store per month, not returning to the same store for at least two months, and purchase packages of meat or poultry including 10 packages of chicken breasts, 10 packages of pork chops, 10 packages of ground turkey and 10 packages of ground beef. Each site cultures the rinse from each sample for the presence of Salmonella and Campylobacter using isolation procedures that have been adapted from the FDA's Bacteriological Analytical Manual. In addition, Georgia, Maryland and Tennessee culture the rinse for E. coli and enterococci. Isolates are forwarded to FDA for confirmation of species identification, antimicrobial susceptibility testing, and PFGE analysis. This collaborative surveillance project will provide data on the prevalence of antimicrobial resistance in enteric bacteria contaminating retail meats. By examining the prevalence of antimicrobial-resistant enteric bacteria we will better understand the extent of antimicrobial resistance in the food supply and will be better equipped to implement mitigation strategies to reduce the incidence of zoonotic foodborne illness by multidrug resistant pathogens.

H-16

What makes the standardization of detection methods for food allergen so troublesome?
C. D. Westphal, M. R. Pereira, K. M. Williams, R. B. Raybourne, FDA, Laurel, MD

The detection of food allergens is based on the analysis of multiple proteins, allergens and non-allergens, by polyclonal antibody-based immunoassay. There are "non-universal" commercial kits in the market that differ in their ability to detect allergens for the following reasons. Food allergens are proteins but not all of the proteins are allergens. Proteins are compounds with a complex structure. A single antibody binds a few amino acids from the target sequence (epitope). If this target structure is affected by processing or other environmental factors, the detection of that allergen will be altered as well. Moreover, food products may contain more than one allergen with very different structure and stability characteristics. Polyclonal antibodies are found to be adequate for the detection of allergens in food because they are able to bind multiple epitopes of different proteins. Regarding aspects related to detection methods, it is known that the type of standards (processed vs. non-processed food, purified proteins vs. extract) used to raise antibodies or to be used as calibrators is one explanation for differences between commercial kits. In addition to this, we have found that pH and ionic strength of sample extraction buffers have a critical qualitative and quantitative impact in the final results because of the different ability of buffers to extract proteins from the sample. This is an additional factor explaining the existing differences among commercial kits, and should be also considered in the standardization process along with standardization of calibrators.

H-17

Detection of Irradiated Foods Containing Sugars Using ESR Spectroscopy: A preliminary study 8
M. Miyahara¹, T. Mashimizu², Y. Kobayashi³, and T. Maitani¹, National Institute of Health Sciences (NIHS)¹, Setagaya, Tokyo; Japan Electron Optics Laboratory (JEOL)², Akishima, Tokyo; Japan Atomic Energy Research Institute (JAERI)³, Takasaki, Gunma

After extensive studies on irradiated food for over 50 years, food irradiation technology has been recognized as a safety measure for food hygiene. Some detection methods for irradiated foods are international methods of the General Codex Methods, but development of new detection method are still required for international labeling regulation. In this study we discuss detection method for irradiated dry fruits using ESR. Those dry fruits contain fine crystalline glucose, fructose, and/or sucrose on the surfaces. Those crystals retain radicals, which are induced by irradiation. ESR is a radical monitor. Samples were prepared by modified procedures suggested by EN. Samples were dried over silica gel in vacuum for 2 days. ESR can detect those radicals in the fruits (figs, raisins, apples). Some of the signals were detectable at 1kGy at initial stage of storage. Those results were sufficient for dry fruits which are irradiated for insect disinfestations (the recommended dose is 1kGy for dry fruits). The shapes of ESR spectra for irradiated dry fruits were different from sample to sample. This results may caused from difference of sugar component ratios in sample. Sucrose can be added to most samples in food process, therefore those ESR gave typical spectra of sucrose.

H-PO-01

Selective Isolation of Salmonella Enteritidis from Mixed Cultures in Eggs Using Ferrioxamine E Supplementation
K. H. Seo, I. E. Valentin-Bon, R. E. Brackett, OPDF, FDA, College Park, MD

Direct plating method for detection of Salmonella Enteritidis (SE) in raw eggs has been hindered due to the antimicrobial and iron-restricting compounds in albumen. Studies show that utilization of ferrioxamine E as sole source of iron distinguishes Salmonella enterica serotypes Typhimurium and Enteritidis from a number of related species, including Escherichia coli (E. coli). However, the samples used for these studies were artificially contaminated with exclusively SE, so no interference with a number of related species could have taken place. The effectiveness of ferrioxamine E as a selective supplement was evaluated by inoculating whole eggs or egg white with proportionally mixed cultures of Salmonella Enteritidis phage type 13a (0.1 ml of 10² CFU/ml) and E. coli K12 (0.1 ml of 10⁹ - 10² CFU/ml each). After 24 h of incubation at 37C, the SE populations recovered from whole eggs supplemented with ferrioxamine E were 10³- 10⁷ CFU/ml. However, 0 - 10³ CFU/ml SE were isolated for samples without ferrioxamine E. In egg white, the SE population for samples supplemented with ferrioxamine E was 10³-10⁶CFU/ml, whereas no SE was detected for samples without supplementation. The growth of E. coli in whole egg supplemented with ferrioxamine E was inhibited when the proportion of the mixed culture was under 1:100 (SE versus E. coli). In egg white, whether the samples were supplemented or not, the growth of E. coli was not significant. Our data indicate that ferrioxamine E could be used in direct plating method for detection of Salmonella Enteritidis in raw eggs to increase SE growth rate while selectivity is maintained.

CATEGORY I: COUNTERTERRORISM
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I-01

Determination of Cesium Binding to Prussian Blue (A Treatment for Internal Radioactive Metal Contamination)
P. J. Faustino¹, Y. Yang¹, J. J. Progar², C. R. Brownell¹, N. Sadrieh³, J. C. May², D. A. Place⁴, E. Leutzinger⁴, F. Houn⁵, S. A. Loewke⁵, M. M. Nasr⁴, R. C. Lyon¹, ¹DPQR, OPS, FDA, Silver Spring, MD, ²LAC, OVRR, FDA, Kensington, MD, ³OPS, FDA, Rockville, MD, ⁴ONDC, OPS, FDA, Rockville, MD, ⁵ODE3, OND, FDA, Rockville, MD

Prussian Blue (PB) was approved by the FDA in October 2003 for the treatment of radioactive metal contamination resulting from a terrorism incident. PB (ferric-hexacyanoferrate (II)), a metal ligand is able to enhance the excretion of radioactive isotopes of cesium and thallium from the body by a mechanism of ion exchange and/or physical adsorption on the large molecular surface. The study objective was to determine if chemical and physical factors such as pH, particle size, and drying procedure have any influence on cesium binding to PB. The experimental pH conditions of the assay ranged from 1 to 9, to cover all possible pH levels in the gastrointestinal tract in humans. Measurements of cesium binding were made between 1 and 24 hours, to cover gastric and intestinal tract residence time using a validated Atomic Emission Spectroscopy (AES) method. The results indicated that pH and incubation time affect cesium binding of both the Active Pharmaceutical Ingredient (API) and drug product. The lowest cesium binding was at pH 1 and 2 (100 mg/g), whereas maximal cesium binding was at pH 7.5 (250 mg/g) at 24 hours. Drying of API and PB Drug product reduced cesium binding capacity at all timepoints. This highlights the importance of the moisture content in product performance. Currently, we are studying the effect of particle size on both cesium binding and cyanide release, as a potential factor that may be used in predicting product quality.

I-02

Ethical Issues in Clinical Studies on Biodefense Products
H.S. Ko, CBER, FDA, Rockville, MD

Background. In clinical studies on products to be used for biodefense, there may be situations where prompt administration can be life-saving. Obtaining informed consent and institutional review board (IRB) approval can be challenging under emergency conditions.

Methods. The protocols for clinical studies in fourteen investigational new drug applications for immune globulin products of potential use in biodefense were reviewed. Information on IRB approval and informed consent was specifically sought. This included any departure from requirements under FDA regulations.

Results. In three of the studies, the original protocol suggested that the attending physician could sign informed consent for the patient in emergency. The protocols were revised to allow for consent only by the patient, parent/guardian, or an authorized person. In four of the studies, waiver for IRB approval was requested. Subsequently the sponsor agreed to obtain protocol approval by a central IRB instead of multiple local IRBs.

Conclusions. These findings illustrate the difficult ethical issues which may be encountered in clinical trials for the study of products for biodefense. Such issues may need to be resolved on a case-to-case basis for optimal protection of human subjects in emergency situations.

I-04

Identification of Critical Amino Acid Residues on Ebola Virus Glycoprotein Required for Cell Entry
O. Mpanju, B. Fraser, C. Wilson, CBER, FDA, Bethesda, MD 20892

The glycoprotein (GP) of the filoviruses Ebola and Marburg, made up of disulfide-linked surface GP1 and transmembrane GP2 subunits, is known to facilitate receptor binding and cell entry. The objective of this study was to identify GP residues that mediate Ebola entry into susceptible cells by using site-directed mutagenesis of the GP1 cDNA, targeting codons for amino acid residues that are conserved among nine strains of Ebola GP.
We used retroviral vector pseudotypes bearing the Ebola (Zaire) (wild-type or mutant) envelope GP and the lac-z reporter gene to infect cell lines of either human (HEK 293 and HeLa) or monkey (Vero) origin. The number of cells expressing lac-z was used to estimate the level of infection in target cells.
We have so far mutated and analyzed ten of fifteen conserved GP1 residues originally targeted for mutation to alanine. Mutating four of the residues - Pro34, Asp49, Pro126 or Arg130 - did not affect the ability of Ebola GP to mediate infectivity in test cell lines. However, mutating Lys84, Arg85, Phe88, Tyr109, Phe159 or Phe160 rendered the GP incapable of mediating infectivity. Immunoblot analysis of vector producing cell lysates or of purified virions demonstrated that the mutant GP was correctly expressed and assembled into all four retroviral pseudotypes exhibiting infectious phenotype, and five of six mutant envelopes with a non-infectious phenotype.
We are currently assessing whether Lys84, Arg85 and Phe88 make up a linear epitope critical for infectivity by studying the antiviral activity of peptides spanning this region of Ebola GP1.

I-11

Identification of Foodborne Bacteria Pathogens and Aerobic Endospore-Forming Bacilli Using Fatty Acid Profiles
P. Whittaker, M. M. Mossoba, S. Al-Khaldi, B. D. Tall, F. S. Fry, M. P. Yurawecz, V. C. Dunkel, CFSAN, FDA, College Park, MD

A gas chromatographic (GC) analysis method was used 1) to determine the cellular fatty acid profiles of various foodborne microbial pathogens, and 2) to compare the fatty acid profiles of spores and vegetative cells of the same endospore-forming bacilli. Fifteen different bacteria, representing 8 genera (Staphylococcus, Listeria, Bacillus, Yersinia, Salmonella, Shigella, Escherichia, and Vibrio) and 11 species, were used to prepare the bacterial fatty acid methyl esters (FAMEs). The endospore-forming bacilli were processed to obtain pure spores and whole cell FAMEs for GC analysis. The cells were cultured on brain heart infusion agar at 35°EC for 24 hours, harvested and then subjected to saponification, methylation and extraction into hexane:methyl tert-butyl ether. The bacterial FAMEs were then measured by GC. A database for each bacterial agent was prepared using fatty acid profiles from 5 replicates prepared on different days. The results showed that these fatty acid intensity profiles were unique for each of the 11 species and that they could be used as a fingerprint for the organisms. The cellular fatty acid profiles for B. anthracis and B. cereus show that there are two fatty acids, iso 17:1 w10c and 17:1 anteiso, that are unique in these species. Iso 17:1 w10c is present in B. cereus vegetative cells and spores but is not observed in B. anthracis. The 17:1 anteiso fatty acid is present in B. anthracis cells but not in B. cereus cells. Fatty acids 16:0 2OH and 17:0 iso 3OH are present in B. anthracis and B. cereus spores but not in the vegetative cells. In summary, this analytical method provides a procedure for the identification of bacteria and spores based on their unique fatty acids.

I-12

Utilization of Lateral Flow Devices for the Detection of Ricin in Food Matrices
M. A. McLaughlin, E. A. Garber, DNP, OPDF, FDA, College Park, MD

Ricin, the toxin derived from the castor bean (Ricinus communis), is a potential threat to the security of the US food supply. The ability to rapidly and reliably detect the presence of this select agent in foods is essential to the FDA's food security mission. This project involved the adaptation of lateral flow devices (LFDs) originally designed for environmental samples to use in the more complicated matrices found in foods. Several types of LFDs were evaluated with one commercial product and one government source device adopted for the trials. Proper sample manipulation allowed for the establishment of lower limits of detection (LOD). Both devices were able to detect ricin in both solid and liquid matrices at levels well below those known to pose a health risk. The methods were then incorporated into protocols designed for distribution to field personnel. These protocols provide the first level of a two-tiered system involving an LFD initial screen followed by a commercial ELISA test for positive and borderline results.

I-13

Generation and Characterization of Bovine and Human Anthrax Immune Globulins in Conventional and Transchromosomal Cattle
J. Riemenschnneider¹, K. Stabler², T. Sathiyaseelan², K. Haffer², M. Bramstedt², J. Robl², B. Golding¹, ¹CBER, FDA, Bethesda, MD, ²Hematech L.L.C. Sioux Falls, SD

Passive immunization with polyclonal immune globulins (IG) may be an important treatment for life-threatening anthrax infections. There is evidence in animals that it may be effective in treating disease, especially if combined with antibiotic therapy. Polyclonal IG for passive immunization have certain advantages over other therapeutics, such as their relative ease to produce in animals and have an expansive antigenic specificity range. Cloned Transchromosomal (Tc) bovines that are capable of producing human polyclonal antibodies were developed. This process involved introducing a human artificial chromosome (HAC) that carries the entire sequences of human IG heavy and light chain loci into bovine fibroblasts and producing Tc cattle by somatic cell cloning technology. Adult conventional and Tc cattle were immunized with various formulations of anthrax antigens including recombinant protective antigen (PA), edema factor (EF) and lethal factor (LF). Serum was collected at several time points after immunization. Antibody titers were measured against PA, LF, and EF using ELISA tests. All vaccinated animals showed a robust antibody response after immunization. A macrophage-based toxin neutralization assay (TNA) was performed and revealed that bovine antibodies generated in the immunized conventional and Tc cattle have high neutralizing activity. Mice challenged with Sterne strain anthrax spores were treated after exposure with purified bovine IG produced from the immunized cows. Protection levels from this lethal challenge was 90-100% when the bovine IG preparation was given by passive transfer, even if not given until 24 hours after challenge. Additional mouse challenge/passive transfer experiments are underway and will be discussed.

I-15

Determination of Cyanide Release from Prussian Blue (A Treatment for Internal Radioactive Metal Contamination)
Y. Yang¹, C. R. Brownell¹, N. Sadrieh², J. C. May³, V. A. Del Grosso³, D. A. Place⁴, E. P. Duffy⁴, F. Houn⁵, M. M. Nasr⁴, R. C. Lyon¹, P. J. Faustino¹, ¹DPQR, OPS, FDA, Silver Spring, MD, ²OPS, FDA, Rockville, MD, ³LAC, OVRR, FDA, Kensington, MD, ⁴ONDC, OPS, FDA, Rockville, MD, ⁵ODE3, OND, FDA, Rockville, MD

Prussian Blue (PB) was approved by the FDA in October 2003 for the treatment of radioactive metal contamination resulting from a terrorism incident. PB (ferric-hexacyanoferrate (II)) is used to enhance the excretion of radioactive isotopes of cesium and thallium from the body. However, PB can decompose under certain pH conditions in the gastrointestinal tract and lead to release of cyanide ions. The purpose of this study was to determine the extent cyanide release from 5 lots of PB Active Pharmaceutical Ingredients (API) and 3 lots of PB drug products under different pH conditions (pH 1 to 12), over a range of incubation times (1 to 48 hr) by using a validated in vitro method. Cyanide-release from both API and drug product showed a pH-dependent profile. Cyanide-release reached the highest concentration at pH 1, and declined gradually as pH increased to pH 7. The lowest concentration was observed at pH 7, but cyanide ion release increased again as the pH rose to 12. The highest level of cyanide-release observed was 414 µg/g, after 48 hr incubation at pH 1. The variation of cyanide-release from batch to batch was not significant among the APIs, but was remarkable among the drug products. Considering that the average fatal dose of hydrogen cyanide in humans is 50 to 60 mg, even at the highest oral daily dose of 10 g, the amount of cyanide released from PB does not present a safety concern.

I-16

The Detection Of Ricin In Food Matrices Using ELISA Technology
E. A. Garber, M. A. McLaughlin, DNP, OPDF, CFSAN, US FDA, College Park, MD 20740

Ricin is a potent ribosome inactivating protein (RIP-2) present in beans of the castor plant, Ricinus communis. Due to recent events, the detection of ricin has become a serious concern to the FDA. As part of an effort to develop protocols for the detection of ricin in food products, a commercially available ELISA assay was evaluated and compared with commercially available lateral flow devices (LFDs) in regards to specificity and sensitivity. Limits of detection (LOD) were determined for ricin added to solid and liquid food matrices. In all cases the ELISA was significantly more sensitive than the LFDs. Both the ELISA and the LFDs were capable of detecting ricin at levels below and equivalent to concentrations known to present a health risk. A two tier procedure is proposed for the screening of food samples in which the LFDs are employed as an initial screen followed by ELISA analysis of those samples that elicit either a positive or borderline response.

I-18

Anthrax Lethal Toxin Alters the Inflammatory Response of Endothelial Cells
A. D. Steele, J. M. Warfel, F. D'Agnillo, OBRR, FDA, Bethesda MD

Bacillus anthracis lethal toxin (LTx) plays a major role in the pathogenesis of anthrax. Vascular endothelium is a potential target for LTx particularly during the systemic phase of anthrax infection. This study examines the effect of LTx on adhesion molecule expression, chemokine production, and permeability. Primary human microvascular endothelial cells were treated with anthrax lethal factor (LF), protective antigen (PA) or both (LTx) with or without costimulation by TNF-a or IL-1b. Surface expression of adhesion molecules was analyzed by flow cytometry and immunofluorescence microscopy. Soluble adhesion molecules and IL-8 were measured by ELISA. Endothelial cell permeability was assessed by a fluorescent-labeled albumin assay. Proinflammatory cytokines elevated permeability and increased the expression of E-selectin, ICAM-1, VCAM-1, and IL-8. LTx enhanced TNF-a- or IL-1b-induced VCAM-1 expression after 12 hours. PA or LF alone or the combination of PA and LF_E687C, an inactive LF mutant, had no effect on cytokine-induced VCAM-1 expression. LTx enhanced cytokine-induced ICAM-1 expression, but did not alter E-selectin expression. LTx alone increased ICAM-1 expression at 24 hours, but did not significantly increase E-selectin or VCAM-1. LTx also dramatically inhibited cytokine-induced IL-8 release by 6 hours. Furthermore, TNF-a-induced endothelial monolayer permeability was exacerbated by LTx. Overall, LTx inhibited cytokine-induced IL-8 release while enhancing cytokine-induced adhesion molecule expression and permeability. These findings suggest that LTx may contribute to the progressive stages of anthrax infection by altering the normal inflammatory responses of endothelium. A better understanding of anthrax pathogenesis will enhance the FDA's ability to provide effective science-based regulation of anthrax-directed therapies.

I-19

Development of Drug Medical Countermeasures for Threat Agents when Ethics Prohibit the Use of Human Subjects: A Regulatory Perspective
T. C. MacGill, L. K. Schrager, M. V. Mathis, F. R. Pelsor, B. Leissa, M. Purucker, M. D. Murphy, OCTAP, CDER, FDA, Rockville MD

With the increased threat of biological, chemical, radiological or nuclear attacks, there is a critical need to develop medical countermeasures to threat agents. New drug approvals by the U.S. Food and Drug Administration (FDA) require substantial evidence of efficacy for the specified indication and demonstrated safety for the use in humans, a standard that is usually met through the conduct of adequate and well controlled clinical trials. As it would be unethical to intentionally expose human volunteers to extremely toxic agents and because naturally occurring exposures are generally rare, it is infeasible to conduct clinical studies for many threat agents. For this reason, the FDA promulgated a new regulation -- 21 CFR 314 Subpart I for drugs; 21 CFR 601 Subpart H for biologics - commonly referred to as the "Animal Rule," in 2002. Under this rule, acceptance of animal data as primary evidence of efficacy, in conjunction with human safety data, may be utilized as the basis of approval given that specific criteria are met. Validated animal models of human disease and the ability to interpret and extrapolate animal pharmacokinetics and pharmacodynamics data to an effective dose in humans will be crucial in the development of drug medical countermeasures to these threat agents. This presentation will examine the Animal Rule and current strategies for using this important new regulatory tool to facilitate and expedite the approval of counterterrorism drug products.

I-20

CpG protects newborn mice against lethal infection with Tacaribe, a prototypical New World Arenavirus
J. A. Pedras-Vasconcelos, D. I. Verthelhyi, DTP, FDA, Bethesda MD

Stimulation of the innate immune system by determinants expressed by infectious microorganisms serves to limit the early spread of a pathogen while promoting the development of antigen-specific immunity. Unmethylated CpG motifs present at high frequency in bacterial but not vertebrate DNA trigger an immune cascade resulting in improved antigen uptake/presentation by APC, and the secretion of polyreactive Ig, chemokines and cytokines by B cells, NK cells, DCs, and monocytes. Synthetic oligodeoxynucleotides (ODN) expressing CpG motifs have been shown to induce protection in a variety of animal models against parasitic, bacterial and viral infections including Hemorrhagic fever-causing agents such as Ebola Zaire and Marburg. Their ability to induce protection against a variety of pathogens make CpG ODN potential candidates as "universal" treatments that would improve the host's ability to survive an infection -regardless of the pathogen involved- allowing for time for more specific protective measures (vaccines or antimicrobial agents) to be set in place. While the protective effect of CpG ODN in adults is known, their effectiveness in neonates, which have reduced number of DC, B and T cells and inefficient production of IFN-gamma by T cells is unclear.

New World Arenaviruses (Tacaribe serocomplex) not only cause significant morbidity and mortality in South America, but have recently been classified as Category A pathogens due to their potential for weaponization as bioterrorism agents. In neonatal C57BL/6 and Balb/c mice, Tacaribe virus (TCRV) induces a lethal meningoencephalitis within 15 days of intraperitoneal (IP) or intracerebral (IC) challenge. Administration of CpG ODN at the time of infection resulted in 40-60% survival for IP infections and 20-30% survival for IC infections. Protection is partly explained by a significant reduction in viral load in the brain, and can be achieved even if the ODN are administered 3 days after challenge. T cells are known to play an important role in the pathogenesis, but which subsets are involved is unclear. Indeed, 25% of IL-10/12 DKO mice survive infection. Administration of CpG ODN to these mice results in improved survival despite brain viral loads similar to those observed in WT mice. In contrast, mice that lacked IL-4 (IL10/IL-4 DKO) failed to be protected by the ODN. This suggests that aTh1 response may be deleterious, while a Th2 response may be protective.

In summary, CpG ODN can activate the immature immune system of neonates to induce protection from an otherwise lethal Arena virus infection.

I-22

Safety and efficacy of CpG ODN as vaccine adjuvants and immunoprotective agents in healthy and immunocompromised subjects
V. W. Wang, D. I. Verthelyi, DTP, FDA, Bethesda, MD

One of the major recent advances in the field of immunology has been the identification of germline encoded Toll-like receptors (TLR) which recognize conserved microbial determinants that activate the cells of the innate immune system to limit the early spread of a pathogen while promoting the development of antigen-specific immunity. Unmethylated CpG motifs present at high frequency in bacterial but not vertebrate DNA are recognized by Toll-like receptor 9 (TLR 9) expressed by B cells and plasmacytoid dendritic cells (pDC). The interaction of TLR 9 with DNA bearing CpG motifs triggers an immune cascade resulting in improved antigen uptake/presentation by APC, and the secretion of polyreactive Ig, chemokines and cytokines by B cells, NK cells, dendritic cells, and monocytes. Oligodeoxynucleotides (ODN) containing CpG motifs mimic the ability of microbial DNA to activate the innate immune system and have been shown in mice to provide protection against a wide range of bioterror and emerging infections. Due to evolutionary divergence, the CpG motifs that are active in mice are weak in primates and vice versa dictating the need to assess their safety and efficacy in relevant animal models.

In primates, distinct types of CpG ODN have been identified: "D" type ODN trigger pDC to secrete IFNa, monocytes to mature into functionally active DC, and NK cells to secrete IFNl, whereas the "K" type ODN primarily stimulate B cells and monocytes to proliferate and secrete IgM, IL-10 and IL-6. Using a primate model we show that activation of the innate immune system using local or systemic CpG ODN type D protects macaques from an infectious challenge with L.major. In addition, when used as vaccine adjuvants CpG ODN improved the response to vaccines against Hepatitis B (HBV) and L. major in male and females macaques.

Immunocompromised subjects are particularly at risk of infection. PBMC from HIV-infected patients respond to CpG ODN stimulation in vitro, although the magnitude of this response is lower in hosts with advanced disease. In a primate model, Local administration of D ODN to SIV-infected macaques significantly reduced the severity of the lesions resulting from a cutaneous L. major challenge. Further, SIV infected macaques, which are unable to respond to the HBV vaccine, produce a significant antibody response when the CpG-HBV vaccine is administered to animals with viral loads <10⁷ copies/ml. These findings support the development of clinical studies to assess the use of CpG ODN as a novel strategy to respond to undefined and emerging microbial threats.

I-24

Rapid response to unrecognized and uncharacterized viral agents attacks
Y. Hu¹, I. Hirshfield², ¹U.S. Food and Drug Administration, Northeast Regional Laboratory, Jamaica, NY 11433, ²St. John's University, Jamaica, NY 11439

For defense against bioterrorist attacks, rapid identification and characterization of the responsible unknown agent are crucial. Once the relationship between a new agent to other known organisms is established, appropriate culture conditions, serologic tests, nucleic acid detection and even therapeutic strategies can be rapidly developed. The most recent technologies for detecting and identifying previously unrecognized pathogens are differential display (DD), representational difference analysis (RDA), subtractive hybridization, expression library screening, broad-range polymerase chain reaction and serial analysis of gene expression (SAGE). But they are all time-consuming and not very reproducible approaches. In this project, we used hepatitis C virus as a hypothetical unrecognized virus and "discovered" it in the sample. Also, we developed a new subtraction hybridization method to suite our need. The method was first to remove most of the common RNA/DNA by nested-PCR-based subtractive hybridization with a single-strand deletion technology to reduce the number of false positives. Following subtraction, a cDNA library was constructed and displayed by dot blot hybridization assay. This new genomic subtraction method will be suited to a rapid response to an attack by an unrecognized viral agent.

CATEGORY J: REGULATORY SCIENCES
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J-01

The use of a Biliary Stent Clearance Database in the Review of Metallic Biliary Stents for Malignant Neoplasms
G. Gonzalez, J. C. Nipper, A. S. Yustein, K. M. Olvey, L. L. Dart, J. W. Cooper, K. A. Straughn, C. Y. Neuland, ODE, FDA, Rockville, MD 20850

In recent years, the Gastro-Renal Devices Branch (GRDB) has received large numbers of 510(k)s for metallic, expandable stents indicated for the palliation of malignant neoplasms in the biliary tree. Since 1999, over 125 such submissions have been reviewed, many of them as Special 510(k)s under CDRH's "New 510(k) Paradigm." As documented in the clinical literature and in MDRs received into FDA, many of these devices are being used off-label as vascular stents, a use for which limited safety data are available. With so many submissions, especially those subject to Special 510(k)s' 30-day review timeframes, it became difficult for reviewers to keep track of the types of devices receiving clearance, in terms of their design, dimensions, materials and labeling claims. To resolve these problems, GRDB created a database of cleared biliary stents. This database lists all the biliary stent clearances since 1995 and includes information, such as: stent dimensions, materials of construction, design characteristics (e.g., balloon expandable or self-expanding), and delivery catheter parameters. The data were manually collected from CDRH's records (IMAGE) by GRDB's student intern (KS). The database, which is an Excel document, offers search capabilities, so that reviewers may find individual predicate documents or perform global searches focusing on one or more device parameters. The use of the database has enabled GRDB reviewers to quickly identify devices featuring novel technological features, such as a new material being used, or a stent length or diameter previously not considered. It has also helped with reviews of Office of Compliance (OC) actions.

J-02

Genotoxic Impurities and Degradants in the Drugs: How do we regulate them?
R. S. Harapanhalli, D. Mellon, HFD-170, CDER, FDA, Rockville, MD

At a molecular level, mutagenicity can be caused by chemicals that interact with the DNA and lead to an adduct formation such that the inherent repair mechanism fails and the DNA replication with incorrect base insertion due to such a lesion leads ultimately to complete replication with base substitution or base deletion. The adduct formation includes covalent bond formation on the DNA bases such as alkylation, cyclization, intercalation and minor-groove binding. The DNA bases, deoxyguanosine, deoxyadenosine, deoxycytidine, and deoxythymidine are so electron rich as to also act as nucleophiles and potentially interact with electron affinic chemicals containing certain structural alert moieties. Some of such structural alerts are epoxides, arylamines, aryl nitro compounds, primary halides, alkylsulfonate esters, aldehydes, and a,b-enones that are Michael-reactive acceptors. Many such structural alert containing chemicals have been shown to be genotoxic and carcinogenic in several in vitro and in vivo experimental models. A critical aspect of the multi-disciplinary drug review process at the Agency is to identify and to appropriately limit such structural alert-containing impurities and degradants in the drugs. Although the International Conference on Harmonization (ICH) has recommended thresholds for reporting, identifying, and qualifying the impurities and the degradants, they have stressed that certain impurities with specific safety concerns such as genotoxicity should be limited to levels far below those recommended for ordinary drug-related impurities. For mutagenic chemicals, in reality there is no threshold since it only takes one molecule at the right place and right time to inflict a mutagenic event that may lead to carcinogenicity. This presents the Agency with a challenge to identify safe thresholds for such chemicals in the review process. The current regulatory approaches followed in the Division of Anesthetic, Critical Care and Addiction Drug Products of CDER will be presented in identifying structural alerts in various stages of drug development and in limiting them to certain levels deemed safe from the point of mutagenic risk to the public versus the benefit of the drugs. Additionally, the importance of the review process in identifying potential mutagens early in the drug development will be highlighted.

J-03

Biopharmaceutical Classification of Representative Fluoroquinolone Drugs
D. A. Volpe, R. L. Hunt, FDA, CDER, DPQR, Silver Spring, MD

Representative fluoroquinolones (ciprofloxacin, levofloxacin, lomefloxacin, ofloxacin) were categorized according to solubility, permeability and dissolution characteristics based upon the methods outlined in FDA/CDER's biopharmaceutic classification system (BCS) guidance for industry. The pH-solubility profile of the drugs was determined using a traditional shake-flask method in aqueous buffers. The solubility class was established by calculating the aqueous volume sufficient to dissolve the highest dose strength. Permeability was measured in an in vitro Caco-2 assay with demonstrated method suitability. The permeability class and efflux potential were ascertained by comparing test drug results to standard compounds. Dissolution testing was carried out in a USP Apparatus II at 50 rpm in 0.1 N HCl, pH 4.5 buffer, and pH 6.8 buffer. The dissolution class was determined by calculating the percent dissolved at 30 minutes. The solubility experiments determined that the fluoroquinolone drugs were deemed to be highly soluble at pH 1, 3, 4, 5 and 7.5, with the exception of ofloxacin at pH 7.5. Ciprofloxacin was classified as low permeability in the Caco-2 assay whereas ofloxacin, lomefloxacin and levofloxacin were classified as highly permeable drugs, correlating to their respective known human bioavailabilities. The four drugs were subject to efflux and active transport in varying degrees. Lomefloxacin and ofloxacin drug products were rapidly dissolving, as >85% dissolved within 30 minutes in the three buffers. Based upon our experiments, levofloxacin and lomefloxacin are class I drugs, whereas ofloxacin and ciprofloxacin are class II and III, respectively.

J-04

Scientific Review of Chemical Sterilization Indicator Devices
P. A. Fox, C. A. Hawthorne, C. Lin, CDRH

A chemical indicator is a medical device intended for use in health care facilities such as hospitals, doctor's offices, or rehabilitation centers. At these facilities, users include a chemical indicator in each sterilization cycle with products that need to be sterilized.

Management of sterilization technologies in health care facilities requires the use of monitoring procedures as part of each facility's quality systems program. Chemical indicators are an integral part of monitoring sterilization. Chemical indicators can help guard against problems associated with user error, sterilizer malfunction, inadequate air removal, and inadequate sterilant penetration. Effective use and performance of sterilizers in health care facilities is important in preventing nosocomial infections. Nosocomial infections are directly attributable to 60 - 80 thousand deaths and an estimated cost of approximately 5 billion dollars per year.

In the guidance entitled, Premarket Notification [510(k)] Submissions for Chemical Indicators, FDA describes the measures we recommend to address the performance characteristics of these devices. These measures, which include performance testing and conformance with FDA-recognized voluntary standards, are intended to mitigate the associated risks to health. Together with FDA's general regulatory controls, quality systems requirement for device design and manufacturing, labeling, and post market oversight, the measures described in the guidance optimize the safety and effectiveness of devices while minimizing redundant regulatory requirements.

J-06

Quality assessment and effects of Cremophor in the paclitaxel formulation
L. S. Hsieh, J. M. Jee, H. B. Patel, J. E. Simmons, ONDC, CDER, FDA, Rockville

The non-ionic surfactant Cremophor served as an inactive excipient in the formulations used as drug delivery vehicle for anticancer agents: paclitaxel and docetaxel. An example of such a product, Taxol® is formulated in a 1:1 combination of polyoxyethylated castor oil (Cremophor® EL) and dehydrated ethanol. After reconstitution for intravenous injection, Cremophor forms a large polar micelle to transport and deliver the entrapped drug to the target. Recent studies indicated that the presence of Cremophor also exhibits biological and pharmacological activity and exerts intrinsic adverse effects even though inert. It has been observed that increasing the amount of Cremophor unexpectedly decreases the absorption of paclitaxel. One of the explanations may be the physical-chemical character, such as Critical Micelle Concentration or presence of acid and base stabilizers, however, such factors affecting drug loading and deposition has never been fully evaluated. Therefore, we examined the hypothesis of possible chemical-physical factors affecting the micelle-drug stability of Cremophor containing formulations involving paclitaxel. The results indicated that paclitaxel remains stable in the presence of Cremophor, but the loading efficiency, the disposition and membrane affinity appear to be affected by the quality of Cremophor. It is proposed that proper chemical-physical evaluation of excipients is needed to provide an integral component for the drug safety and quality, as well as to enable a more rational approach in the development of formulation for poorly water-soluble agents. Hopefully, the knowledge gained from our study could also be valuable when updating Agency regulatory guidance.

J-07

From Controlled Trials to Clinical Practice: Monitoring Transmyocardial Revascularization Use and Outcomes
E. D. Peterson¹, P. Kaul², R. G. Kaczmarek³, B. G. Hammill¹, P. W. Armstrong², C. R. Bridges⁴, T. B. Ferguson, Jr.⁵, ¹Duke Clinical Research Institute, Durham, NC, ²Univ. of Alberta, Edmonton, Canada, ³CDRH, FDA, Rockville, MD, ⁴Univ. of Pennsylvania, Philadelphia, PA, ⁵Louisiana State Univ. Health Sciences Center, Ne w Orleans, Louisiana

We sought to examine trends in the use and outcomes of transmyocardial revascularization (TMR) in community practice. We also identified important risk factors for TMR and compared outcomes of TMR combined with coronary artery bypass graft surgery (TMR + CABG) versus bypass alone in patients receiving incomplete revascularization. Baseline characteristics and outcomes in patients who received TMR at 173 hospitals participating in the Society of Thoracic Surgeons (STS) National Cardiac Database were compared with those from six published TMR trials. Multivariate logistic regression was used to identify clinical risk factors for mortality with TMR. Risk-adjusted mortality was also compared for TMR + CABG relative to CABG only in patients not amenable to complete traditional revascularization. Between January 1998 and December 2001, the number of STS hospitals and total procedural counts increased markedly, driven predominately by more TMR and CABG cases. Overall mortality rates for TMR alone and TMR + CABG were 6.4% and 4.2%, respectively. Operative risks were significantly higher in those patients with recent myocardial infarction, unstable angina, and depressed ventricular function. Among patients receiving incomplete revascularization, TMR + CABG was not associated with decreased mortality risk compared with CABG alone, adjusted odds ratio 1.11 (95% confidence interval 0.74 to 1.67). The use of TMR, and in particular TMR + CABG, is expanding in community practice. The results of this study point out the potential for a reduction in TMR morbidity and mortality through optimization of the timing of the procedure.

J-08

From Concept to Reality: FDA's Recommendations for the Design, Manufacturing, Clinical Evaluation, and Postmarket Surveillance of Class III Implantable Middle Ear Hearing Devices
T. Cygnarowicz¹, K. Baker¹, V. Malshet¹, J. Kane¹, W. Regnault², S. Jaffee¹, S. Weininger², J. Jorgens², M. Wollerton³, G. Koustenis⁴, M. Hoban⁵, B. Beard², T. Woods², J. Casamento², E. Mann¹, ¹Office of Device Evaluation, ²Office of Science and Technology, ³Office of Health and Industry Programs, ⁴Office of Surveillance and Biometrics, ⁵Office of Compliance, CDRH, FDA, Rockville, MD

Implantable middle ear hearing devices (IMEHD) are a new approach to help patients with hearing loss. The IMEHD is a class III device, which requires a premarket approval application (PMA). The IMEHD combines external and implanted components to achieve amplified sound perception.

After being issued for public comment, a draft guidance was presented at an open meeting of the FDA Ear Nose and Throat Devices Advisory Panel to help ensure adequate public participation. Public comments and recommendations of the Panel were considered and the guidance revised accordingly before being issued as final in 2003.

The guidance entitled, Implantable Middle Ear Hearing Device describes FDA's recommendations for performance characteristics, testing and design, voluntary standards and clinical studies for the IMEHD. The guidance document was a multidisciplinary collaborative effort involving experts in audiology, biocompatibility, toxicology, mechanical, electrical, and biomedical engineering, material science, clinical and surgical otolaryngology, and statistics from CDRH's Office of Device Evaluation, Office of Compliance, Office of Science and Technology, Office of Surveillance and Biometrics, and Office of Health and Industry Programs.

J-09

CFSAN's Risk Management Framework
L. Carson, S. Choudhuri, S. Dennis, P. Klein, FDA

CFSAN conducted a retrospective study to survey, evaluate, and define risk management decisions and processes. The center has been using the principles of risk management (RM) in nutrition, food and cosmetics safety continuously but had not fully examined the general principles and corporate philosophy embedded within our mission, decision-making, and culture. The project set forward to glean the framework, including best practices used by CFSAN leadership in grappling with difficult, diverse and complex risks of the food and cosmetics supply in providing the appropriate level of protection for the U.S. consumer. The RM framework developed from this retrospective look is simple/ flexible, iterative, systematic, science-based, and transparent /open. We identified seven components for successful risk management decision-making within the RM framework:

Triggers/ inputs. These are indicators of issues or concerns that may require a RM decision.
Prioritization. Weighing the relative importance, the capacity to accomplish and the timeframe for action.
Process. A means to gather and communicate data and information from both internal and external resources.
Decision. Identification and selection of option(s) to mitigate, reduce, or eliminate the risk in a practical, cost effective manner.
Implementation. Acting on the decision; mobilize program resources through established or new procedures/ policy/ testing/ investigation.
Outcome. The impact of the implementation of the decision.
Monitor/evaluate/modify. Active measurement and assessment of the outcome and determination if changes are needed.

J-10

The Regulatory Impetus for Developing Condition-of-Use Drug Product Photostability Studies.
W.C. Timmer, Division of New Drug Chemistry I, OPS, CDER, FDA, Rockville MD

At present there is no uniform policy and/or test procedure(s) for performing photostability studies under normal conditions-of-use. In particular, the ICH Q1B Guidance on the Photostability Testing of New Drug Substances and Products addresses the photostability of drug products within their marketing package. The proposed condition-of-use criteria would determine the photochemical behavior of a drug product, over a suitable period of time (e.g., 8 to 12 hours), and under simulated or actual in-use conditions. Appropriate measures could be taken (e.g., mitigation measures) once the photochemical behavior of a drug product is known. Naturally, condition-of-use studies primarily apply to topical drug products, where their application to the body involves areas that are exposed to light radiation (i.e., arms, face, scalp, etc.). The rationale for performing condition-of-use studies with non-topical drug products will be examined. Data from several representative condition-of-use studies will be presented. Finally, a brief discussion of experimental methods, as well as their advantages and limitations, will be presented.

J-11

ORA Laboratories and Research Centers Program Capabilities
H. M. Jones, C. A. Montalvo, FDA/ORA/ORO/DFS

The Office of Regulatory Affairs (ORA) has 13 laboratories located throughout the United States of America and Puerto Rico. FDA's primary regulatory analytical activities are conducted in these 13 laboratories. The ORA laboratories are organized into five Regional Labs, four District Labs, and four Specialty Labs. The Northeast, Southeast, Arkansas, Pacific Southwest and Pacific Northwest Regional Labs are large general servicing laboratories providing service in most major analytical programs. Kansas City, Denver, Detroit, and San Francisco District Labs provide services in several major programs and have specialties in specific areas of analysis. The Philadelphia and San Juan District Labs, Winchester Engineering and Analytical Center and the Forensic Chemistry Center are specialty labs engaged in specific areas of laboratory service. There are three Research Centers located in three of the ORA Laboratories. These are the Animal Drug Research Center located in the Denver District Laboratory, the Total Diet and Pesticide Research Center located in the Kansas City District Laboratory, and the Seafood Products Research Center located in the Pacific Regional Laboratory Northwest.

The primary role of our scientists is the analysis of FDA-regulated products. Research, method validation, technical training, inspections, and public outreach are also important activities within ORA labs. Our labs are equipped with the full range of sophisticated instrumentation and modern techniques appropriate for their specific scientific programs.

J-12

Connectivity Standards Utilizing Computers for Analyzing Data from Blood Glucose Monitors and Insulin Delivery Systems
H. Sauberman, E. Hausman, J. Callaghan, FDA , Rockville MD

One of the key steps in the development of future devices for managing the diabetic patient is the development and application of reliable automated systems. To this end, one must be able to identify and/or develop appropriate connectivity standards for the analysis and interpretation of data from glucose detection and monitoring systems, and from insulin delivery systems. Important parameters in the design of a total product monitoring system include: proof of underlying scientific and medical principles; software development under accepted life-cycle processes; device compatibility issues; human factors considerations; and the ability to retrieve and utilize data for interface with a patient's stored medical record for appropriate disease management. All of these identified processes need to be developed within the constraints of a rigorous quality assurance and quality control program.

J-13

Modeling the Effects of Food Handling Practices on the Incidence of Foodborne Illness
D. Kendall¹, A. Ritzert², C. Viator¹, S. Karns¹, B. Durocher¹, ¹RTI International, NC, ²FDA, College Park, MD

Each year millions of cases of foodborne illness occur in the United States. Antecedent to each case of foodborne illness is contamination of food by pathogens and failure to destroy or sufficiently control these pathogens in retail food establishments or in households. One of FDA's roles as a food safety institution is to research the causes of foodborne illnesses and provide guidance and regulation to industry and consumers on ways to better control or eliminate foodborne pathogens.

In response to FDA's mission in food safety, we have developed a quantitative model to estimate the effects of food handling practices in retail establishments and households on the incidence of foodborne illness. This stochastic simulation model uses data from scientific literature, foodborne disease outbreak studies, county-level retail establishment sanitation inspection reports, federal and state foodborne disease surveillance systems, and expert elicitation to estimate expected reductions in foodborne illnesses if changes in food handling practices occur in retail food establishments and households.

This model will be a useful tool for FDA economists, analysts, and scientists to use in estimating how the incidence of foodborne illness may be reduced by changing regulations or issuing guidance that affect food handling practices or food manufacturing practices.

J-14

Prioritizing sources of variability in genomic profiling data for standards and guidance development
R. K. Elespuru¹, J. C. Fuscoe², R. K. Puri³, D. G. Ranamukhaarachchi¹, G. A. Pennello⁴, J. Roayaei⁴, S. M. Morris², T. Martinsky⁵, D. Mendrick⁶, J. K. Leighton⁷, J. J. Chen⁸, ¹OST, FDA, Silver Spring MD, ²DDR, FDA, Jefferson AR, ³OCTGT, FDA, Bethesda MD, ⁴OSB, FDA, Rockville MD, ⁵Telechem, Sunnyvale, CA, ⁶GeneLogic, Gaithersburg MD, ⁷OND, Rockville MD

FDA bears the responsibility for approving microarray-based genomic diagnostic devices and for evaluating genomic data submitted as evidence of safety or efficacy of therapeutic products. Standards for submission and/or evaluation of genomic data are a source of concern for both FDA and industry. The goal of this inter-center project is to prioritize sources of variability in microarray data; this could aid in focusing additional experimental queries, guidance development and experimental standards. The project also provides a mechanism for inter-center collaboration, team building among researchers and reviewers, and integration of industry experience. The outcome should be an enhanced capability to address standards development and accept new technologies as they arise. Building on recent studies, we are implementing an inter-lab validation exercise to assess variables affecting microarray data that appear to be significant and controllable. The study design addresses microarray issues in an FDA framework. For example, the expectation would be that protocols would be controlled, array platforms could differ significantly, and array and sample stability would be important independent of platform. Effort will focus on microarray fabrication, different array platforms, sample preparation, sample (RNA) stability, array stability, surface chemistry, target or probe labeling, and microarray processing. A fractional factorial experimental design has been developed to study effects of the factors with a feasible number of slides. Supported by the FDA Office of Science, Health and Communication (OSHC).

J-15

Transient bulging fontanelle after vaccinations: Reports to the U.S.Vaccine Adverse Event Reporting System
S. B. Freedman¹, J. Reed², R. P. Wise², D. R. Burwen², A. Weiss³, R. Ball⁴, ¹Div. of Pediatric Emergency Medicine, Children's Memorial Hospital, Northwestern University Feinberg School of Medicine, Chicago, IL, ²OBE, FDA, Rockville, MD, ³Dept. of Pediatrics, Children's Memorial Hospital, Northwestern University Feinberg School of Medicine, Chicago, IL, ⁴OBE, CBER, FDA, Rockville, MD

Published case reports of transient bulging fontanelle (TBF) after vaccinations in the U.S. include 2 positive rechallenges, but most antedate current vaccination schedules and lack neuroimaging. The Vaccine Adverse Event Reporting System (VAERS) monitors vaccine safety through "passive surveillance." Limitations include underreporting and uncertainty whether vaccinations caused the reported events in most cases, but VAERS can detect new risks and monitor known side effects. We describe recent cases in VAERS of TBF after vaccinations. We searched for reports with bulging fontanelle. We contacted sources for reports received between1/1997 and 6/2002. A definite TBF case had bulging fontanelle(s); normal neuroimaging study and cerebrospinal fluid (CSF); without depressed consciousness, focal neurologic findings, or identified etiology. Follow-up confirmed normal neurologic development. Probable cases lacked lumbar puncture or neuroimaging but met the other critera. After excluding 26 reports for infections, abnormal neuroimaging, or other inconsistencies with case definitions, 7 definite and 11 probable TBF case ages ranged from 2.7-6.5 months. Intervals from vaccination to symptoms varied from 5 hours to 4 days. Bulging fontanelles resolved in 1-7 days. Patients received various vaccines: Hib (14), DTaP (10), DTP (8), Hep B (8), pneumococcal conjugate (7), IPV (5), OPV (4), and rotavirus (1). 15 (83%) children were febrile. None had positive rechallenge. No probable case had neuroimaging; 6 had normal CSF. These cases suggest that benign TBF rarely follows routine infant vaccinations, but, without further research and additional reports, we cannot conclude that vaccines cause TBF or ICH.

J-16

Topical Drug Classification
L. F. Buhse¹, R. E. Kolinski¹, B. J. Westenberger¹, A. M. Wokovich¹, J. A. Spencer¹, C. W. Chen², S. Turujman², M. Gautam-Basak², G. J. Kang³, A. Kibbe⁴, ¹DPA/OTR/OPS, St. Louis, MO, ²ONDC/OPS, Rockville, MD, ³OGD/OPS, Rockville, MD, ⁴School of Pharmacy, Wilkes University, Wilkes Barre, PA

The existing classification of dosage forms for topical drugs needs to be re-examined to ensure that definitions for different dosage forms are based on consistent principles and that dosage forms can be distinguished from one another. Current definitions of lotions, gels, creams and ointments vary depending on literature source, market history, traditional use or application type. The purpose of this study is to obtain a scientifically based, systematic classification of dosage forms for topical drugs.

A variety of prescription and over-the-counter lotions, gels, creams, and ointments are evaluated using a variety of techniques including rheology, loss on drying (LOD), thermal behavior, appearance and composition. Rheology is the most discriminating property separating creams and lotions. LOD and composition separate ointments and creams. Composition and thermal behavior separate gels from the other dosage forms. New definitions and a decision tree are presented to assist in the determination of the appropriate nomenclature for a topical dosage form.

J-17

Blend Uniformity and In-process Dosage Unit Content Uniformity Testing of the Highly Potent and Narrow Therapeutic Range Drugs
R. S. Harapanhalli, E. P. Duffy, ONDC, OPS, CDER, FDA, Rockville, MD

Recently the Agency released a draft guidance entitled "Powder blends and finished dosage units- a stratified in-process dosage unit sampling and assessment." The Guidance is based on the recommendations of the PQRI working group on blend uniformity analysis. The Guidance states that the methods described are not intended to be the only methods for meeting Agency requirements to demonstrate the adequacy of powder mix. It states further that the traditional powder blend sampling and testing can be used as well. The Guidance also states that the formulations with extremely low dose and/or high potency may call for more rigorous sampling than is described in assessing the uniformity of powder blends or the uniformity of content of the finished dosage units. This poster presentation focuses on specific issues and concerns associated with highly potent drugs that often have narrow therapeutic range and are formulated at very low strengths. Redacted case studies are presented highlighting the blend uniformity problems identified during the review process and the recommendations made by the CDER review division on the blend and in-process content uniformity analysis. The case studies highlight the fact that the blend uniformity is related not only to the particle size distribution but also to the morphic form of the drug substance and their agglomerates since the morphic forms may differ in the density and powder flow properties and hence may contribute to the non-uniformity of the blend. The studies reveal that overmilling of the drug substances due to empirically driven scale up operations often results in the formation of agglomerates that may have to be removed thorough a screening process before formulation into the drug products. The studies also reveal that for highly potent drugs with narrow therapeutic range, formulated at very low doses, both blend uniformity analysis and in-process dosage unit content uniformity are critical to the assurance of overall quality and hence one may not replace the other. Finally, the importance of proactive interaction of the reviewers with the drug applicants during the beginning stages of drug development on the subject of blend uniformity and in-process unit dosage content uniformity will also be highlighted.

J-18

Experience Evaluating QT Prolongation Data
L. Kenna, A. Parekh, D. J. Chatterjee, H. Sun, M. J. Kim, S. Ortiz, J. Hunt, H. Malinowski, DPEII, OCPB, CDER, FDA, 5600 Fishers Lane, Rockville, MD 20857

Purpose. A preliminary concept paper under development at FDA advises on QT assessment study design. This paper brings to the forefront common challenges in such studies as assessed by the comparison of NDAs reviewed in our division. Methods. Eight NDAs with QT studies reviewed during the past 2 years were compared with respect to design, data analysis and regulatory outcome. These critical issues are presented for consideration. Results. Critical issues include (i) EXPOSURES TESTED: supratherapeutic versus normal doses: Doses tested often cover extreme exposure expected in a clinical setting; (ii) SAMPLING: Duration: measurements around and beyond Tmax captures maximal or delayed effects, Intensity: no standard number of replicates; (iii) DATA ANALYSIS: QT Correction Methods: Fridericia most common; multiple corrections in a given submission, Baseline: No standard duration of measurement or number of replicates; no standard approach to assessing baseline effect, Drug Effect: Various mean analyses, standard outlier analyses, PK/PD analyses should focus on individual response; (iv) CONTROLS: Placebo and positive controls recommended, but involve ethical considerations; some use of Active Controls; (v) STUDY POPULATION: ethical considerations; (vi) REGULATORY DECISION: outcomes include labeling and recommendations to perform additional studies. Conclusions. Our experience suggests that QT prolongation assessment is a regulatory challenge involving the interplay of many factors. Well-controlled studies lead to the most successful outcome.

J-19

Identifying and visualizing strong signals in an Adverse Event database using a polynomial response surface model
S. J. Chirtel, R. J. Blodgett, M. S. Boyer, K. R. O'Neill, FDA, CFSAN, OSAS, College Park, MD

Several FDA centers collect adverse event reports as an integral component of their post-market surveillance programs. Separating genuine signals from background "noise" involves creating a two-way table of compounds (or foods) and symptoms. The expected number (E) of reports for an adverse event for any food or compound in any cell of the table is estimated by multiplying the marginal proportion of the compound class by the marginal proportion of the symptom class and multiplying the result by the total number of reports in the data base. The actual number of symptom reports observed (O) for each compound (food) in each cell is divided by E and then log transformed. In a presentation at the 2002 FDA Science Forum, the authors showed that when the log (O/E) is plotted against O, the distribution of log (O/E) is slightly skewed at each O, with decreasing mean and variance as O increases (Adverse Event Funnel), and that statistical outliers from this pattern may be easily identified using standard methods. We have extended our earlier findings by examining the distribution of the quantity log (O/E) as a function of E. The E's of all the cells in the table are ranked in increasing value and placed in approximately 15 groups. On plotting log (O/E) vs. the E groups, a curved band with a negative slope and roughly constant variance is evident. Strong signals are identified as outliers from this distribution. Finally, the quantity log (O/E) may be graphed simultaneously vs. the O and E parameters in a 3-D plot, where the O's are ranked and grouped in a similar fashion to the E's. A curved saddle-like surface (Adverse Event Surface) is evident, with a few points visible far above the surface. When the quantity log (O/E) is regressed against O, E and their higher order terms and cross products, a response surface model is generated. Ordinary studentized deleted residuals from the regression model are roughly normally distributed and may be used to identify the model "outliers" or adverse event signals. This visualization procedure may be useful in describing the behavior of other signal detection procedures such as the Bayesian-based Gamma-Poisson and neural network methods.

J-20

Radiation Dose and Risk Assessment
O. H. Suleiman, R. Fejka, M. Walsh, R. Farkas, FDA, Rockville, MD

In FDA many disciplines deal with assessing radiation risk, and although the medical benefit usually outweighs the radiation risk, public sensitivity to radiation continues. It is important for FDA staff without radiation expertise to understand the fundamental relationship between radiation dose and risk, and radiation doses associated with typical medical and non-medical sources of radiation.

In the U.S. annual radiation dose limits exist for members of the general public (1 mSv), occupational workers (50mSv) , mammography (3 mGy) and fluoroscopy (10 R/m) equipment, and nuclear medicine research subjects under the authority of a Radioactive Drug Research Committee, RDRC, (30 - 50 mSv for adults, and 3 - 5 mSv for children).

Radiation dose limits do not exist for routine medical examinations and other clinical research.

Clinical and research doses range widely, with high doses exceeding the 3 mSv an individual will receive from natural background sources and approaching several years' worth of natural background radiation. The increasing complexity of new imaging procedures and emerging technologies has increased the need to better understand the radiation doses patients and subjects receive. A patient undergoing a fused positron emission tomography (PET)/computed tomography (CT) procedure will receive significant doses from both the internal radioactive drug and the external x-ray based CT procedure. Radiation doses from these various medical procedures will be compared, along with a discussion of radiation terminology. The concept of organ dose will be presented along with the International Council on Radiation Protection (ICRP) term "effective dose", which allows partial body irradiations to be compared with whole body irradiations. Current ICRP, National Council on Radiation Protection (NCRP), Nuclear Regulatory Commission (NRC), and FDA dose standards will be presented.

J-21

CFSAN Food Additives Regulatory Management (FARM) System
J. Ziyad¹, G. A. Brindza², B. G. Girmay¹, A. M. Rulis¹, L. M. Tarantino¹, ¹CFSAN/OFAS, FDA, College Park, MD 20704, ²CFSAN/OMS, FDA, College Park, MD 20704

Pre-market safety review and approval of regulated food ingredients by the Center for Food Safety and Applied Nutrition (CFSAN) is an important responsibility for the Center's Office of Food Additive Safety (OFAS).The FARM Project's electronic information management system is designed to support electronic processing, review, maintenance, and reporting for food ingredient submissions: food and color additive petitions, Food Contact Notifications (FCNs), Generally Recognized as Safe Notices (GRNs) and Biotechnology Consultations. FARM provides reviewers and managers with state-of-the art analytical and search tools to support safety reviews, evaluations, and decisions and they spend less time searching for, processing, and sharing paper- based information. FARM currently supports industry electronic submissions, Freedom of Information (FOI) requests and correspondence further improving efficiencies for industry and FDA. Users access the FARM system database, run queries, and generate real-time reports on submissions, decisions, time intervals, and general trends just to name a few possibilities. FARM users are trained based on their roles. Recently, an interactive Computer-Based Training (CBT) module was developed to help improve training and to save the cost of contractor facilitated training. The FARM Project is used in other agency initiatives such as EFOI and Correspondence Management Systems.

The FARM System architecture and its code have been shared with the National Cancer Institute Center for Bioinformatics Initiative, National Institutes of Health and FDA's Office of Regulatory Affairs. The FARM System currently serves as a model for other systems within CFSAN and certain modules may be expanded to other centers throughout the Agency.

J-PO-01

Time course of cII gene mutant manifestation in the liver, spleen and bone marrow of N-ethyl-N-nitrosourea-treated Big Blue transgenic mice
J. Wang, X. Liu, M. Moore, R. Heflich, T. Chen, NCTR, FDA, Jefferson, AR 72079

The time between the treatment and sampling (mutant manifestation time) is a critical variable in in vivo transgenic mutation assays. There are, however, limited data describing the optimal sampling time for detecting mutations in various tissues from mutagen-treated animals. In this study, we investigated the cII gene mutant frequencies (MFs) in the liver, spleen and bone marrow of Big Blue transgenic mice. Six-month old mice were treated i.p. with a single dose (125 mg/kg) of N-ethyl-N-nitrosourea (ENU) and the animals were sacrificed and assayed for cII MFs 1, 3, 7, 15, 30 and 120 days later. The MFs in the liver cII gene of ENU-treated mice were 73, 71, 87, 106, 224 and 382x10^-6 at the respective sampling times. Compared with that of concurrent controls [(73±8)x10^-6], MFs were significantly increased since day 15. The MFs in the spleen cII gene of ENU-treated mice were 63, 67, 150, 210, 289 and 268x10^-6 respectively, while the MF was (54±5)x10^-6 in the concurrent controls. The MFs were increased significantly at day 7 and reached a plateau at day 30. In bone marrow, compared with that of concurrent controls [(34 ±14)x10^-6], all the MFs of ENU-treated groups (253, 374, 278, 236, 238 and 190x10^-6) were increased significantly with the maximum at day 3. The results confirmed that the time required to reach the maximum MF is tissue specific, with the approximate time for maximum cII MF response being: bone marrow, 3 days; spleen, 30 days; and liver, more than 30 days.

CATEGORY K: MODELING & POPULATION-BASED STUDIES
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K-01

Testing superiority and non-inferiority hypotheses in active controlled clinical trials
Y. Tsong¹, J. J. Zhang², ¹HFD-705, CDER, FDA. Rockville, MD, ²HFD-705, CDER, FDA, Rockville, MD

Switching between testing for the hypothesis of superiority and the hypothesis of non-inferiority has been an important statistical issue in the design and analysis of active controlled clinical trials. In practice, a two-stage testing procedure is often used. It has been shown that there is no type I error rate adjustment required to switch to testing for the hypothesis of non-inferiority once the data fail to support the superiority claim. Neither is the adjustment required to switch to testing the hypothesis of superiority once the null hypothesis of non-inferiority is rejected. This has been shown to be the case in trials with a properly pre-specified non-inferiority margin in a generalized historical control approach. However, when using a cross-trial comparison approach for non-inferiority testing, controlling the type I error rate may become an issue with the conventional two-stage procedure. We proposed to adopt a single-stage simultaneous testing concept as proposed by Ng (2003) for testing both hypotheses of non-inferiority and superiority simultaneously. The Fieller confidence interval procedure as proposed by Hauschke et al (1999) is used in the proposed approach.

K-02

Is Cross-Trial Comparison a Preferred Answer for Non-Inferiority Testing
Y. Tsong¹, W. Chen², ¹HFD-705, CDER, FDA, Rockville, MD, ²HFD-715, CDER, FDA. Rockville, MD

The procedures of non-inferiority testing can be catgorized into two types of approaches: the generalized historical control approach and the cross-trial comparison approach. The cross-trial comparison approach was originally proposed for testing a non-inferiority null hypothesis that the efficacy of the test treatment (i.e. mean difference between the test and placebo treatments) is no larger than a pre-specified proportion of the efficacy of the active control treatment (i.e. mean difference between the active control and placebo treatments) assuming there is a placebo arm in the current active control treatment. In this presentation, we will discuss the difference between cross-trial comparison and generalized historical control approaches in assumptions, independence, switching between superiority testing and non-inferiority testing, group sequential analysis, data transformation and multi-center trial setting.

K-03

Data Transformation with Non-inferiority or Equivalence Tests
Y. Tsong, J. J. Zhang, L. Chen, HFD-705, CDER, FDA. Rockville, MD

When sample sizes are small, the assumptions of normality and homoscedasticity of variance are important for using parametric methods such as t-tests, analysis of variance and analysis of covariance for the analysis of continuous endpoints of a clinical trial. Proper transformation of data is often considered as an option by statisticians to enhance normality and reduce heterogeneity. Though the literature is rich in data transformation, almost all articles were focused on testing the hypothesis of superiority. Additional difficulties may be encountered in testing the hypothesis of non-inferiority when one needs to transform X + d instead of X. Depending on the hypothesis statof P-gp-mediated efflux on drug absorption were done at low drug concentrations far below the saturation concentration (Km). At these concentrations, the large ratio of CL_eff-ABsuggests that P-gp-mediated efflux has prominent clinical implications. This conclusion, however can mislead as to the importance of P-gp transport to clinical practice. In our model, no significant role of P-gp in oral bioavailability was observed at the concentrations obtained from normal clinical doses. Conclusions: Although numerous in vitro or in vivo experiments with animals have indicated a substantial role of P-gp on oral bioavailability, the efflux by P-gp may not be significant at higher concentrations. Therefore, an experimental design that includes the concentration range found in clinical application needs to be considered to apply the data from in vitro or in vivo animal studies to humans.

K-04

A Statistical Reviewer's Perspective on the Application of Propensity Score Analysis to Non-randomized Medical Device Clinical Studies
Lilly. Yue, CDRH, FDA, Rockville MD

Propensity score analysis is a versatile statistical method used in observational studies, mainly for making valid comparison of treatments by controlling for many confounding covariates. Recently, there is an increased interest in applying this method to non-randomized medical device studies. For this presentation in particular, the advantages and limitations of this method will be discussed and illustrated from a regulatory statistical reviewer's perspective.

K-05

Putting a Risk Assessment Model to Work: Listeria monocytogenes 'What If' Scenarios
C. Carrington, S. Dennis, R. Whiting, R. Buchanan, FDA

In October 2003, FDA released a quantitative assessment of the relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods (see www.foodsafety.gov/~dms/lmr2-toc.html). The risk assessment examined systematically the available scientific information and data to predict the relative risks of serious illness and death associated with consumption of 23 types of ready-to-eat foods that may be contaminated with Listeria. The risk predictions described in the risk assessment document are based on a complex model and provide our best estimate of the current situation in the U.S. However, the model input parameters can be changed and the resulting change in the model outputs collected. This process, referred to as conducting 'what-if' scenarios, can be used in many applications. These scenarios compared with the baseline estimations of risk illustrate the impact of storage time, storage temperature, and contamination level on the risks per serving. The models may also be used to evaluate the expected public health impact of preventive controls such as storage limits, sanitation improvements, or new processing technologies. The impacts of hypothetical changes in a process, such as limits on storage time or temperature, also provide insight to how the different components of the model interact. The 'what-if' scenarios modeled in this risk assessment provide insight to the impact on public health of limiting storage times, reformulating a food so L. monocytogenes can't grow, avoiding high temperature refrigeration storage, and reducing contamination levels.

K-06

Data Feasibility Evaluation of 'Major' CFSAN Risk Assessments
S. Dennis, M. Miliotis, J. Hicks, D. Carlson, V. Bunning, FDA

Risk assessment is a valuable tool to enhance the scientific basis of regulatory decisions and solve high priority public health problems. Risk assessments conducted by CFSAN are identified and selected using a decision-based approach that considers appropriate use of the Center's resources. CFSAN's process for identifying and selecting a 'major' (cross-cutting, resource intensive) risk assessment is divided into four phases: concept generation, problem identification, data feasibility determination and disposition. Candidate risk assessment nominations are first solicited from CFSAN offices (CONCEPT GENERATION). CFSAN senior managers (the leadership team) recommend which risk assessments to pursue according to CFSAN's regulatory needs (PROBLEM IDENTIFICATION). One possible recommendation is to conduct a DATA FEASIBILITY EVALUATION. A data feasibility evaluation determines whether sufficient data are available to conduct the specific risk assessment. Subject matter experts review the available peer-reviewed data and literature and evaluate the quality of the information gathered. The evaluation provides a description of the scope of the problem and identifies risk management questions, risk assessment questions, a conceptual model, and data gaps. The conclusions of the data feasibility evaluation should include recommendations for the type of risk assessment, if any, that is warranted. Ultimately, the leadership team decides whether to conduct a risk assessment and identifies resources to accomplish the project (DISPOSITION PHASE).

K-07

Adverse Events Reported to the Center for Devices and Radiological Health on Local Anesthetic Infusion Pump Systems.
S. L. Brown, A. E. Morrison, OSB, FDA, Rockville, MD

The purpose of this report is to characterize adverse event reports on continuous direct local anesthetic infusion into surgical wounds using infusion pump systems devised for this purpose. These pump systems typically consist of disposable, non-electric pumps, or electromechanical pumps, that deliver a continuous infusion at controlled rates for a specified length of time. Postoperative pain may be managed by continuous direct infusion of anesthetic into a surgical wound. This technique is reportedly used in a variety of surgeries. For instance, inguinal hernia repair, upper abdominal surgery, laparoscopic nephrectomy, cholecystectomy, knee arthroplasty, podiatric and ob-gyn procedures. Adverse events during direct local anesthetic infusion into surgical wounds, with an infusion pump system, have been reported to CDRH through the Medical Device Reporting (MDR) system. These reports involve adverse events reported for surgeries performed at a variety of surgical sites including orthopedic, gastrointestinal, podiatric, and others. Complications encountered with these infusion pump systems include tissue necrosis, surgical wound infection, and cellulites. We summarize 40 injuries that occurred using direct local anesthetic infusion pump systems. These reports may represent sentinel events, that is an early warning that is representative of a problem that is widespread, or alternatively, these may be isolated incidents.

K-08

Dynamics of Bisphenol A Distribution in the Neuroendocrine Tissues
J. C. Hutter¹, M. D. Luu¹, C. S. Kim², ¹CDRH, FDA, Rockville MD, ²CFSAN, FDA, Laurel MD

Bisphenol A (BPA) is a known xenoestrogen with similar properties to 17b-estradiol. BPA is a hydrophobic compound, and this affects the pharmacokinetics both compounds in mammals. In a previous study we measured the distribution of BPA in female F344 rats exposed to oral doses of 0.1, 10, and 100 mg/kg. The results indicated distribution to target neuroendocrine organs (pituitary, brain stem, cerebellum, hippocampus, hypothalamus, frontal cortex, caudate nucleus) at all doses tested. Using these results, we developed a pharmacokinetic model to predict the dynamic uptake and excretion of bisphenol A by various routes of exposure (po, iv, sc, ip). The model was able to simulate the entire time course (48 h) following exposure in rats over the dose range tested. The model indicated that the observed initial rapid uptake was due to the slow kinetics of plasma protein binding. With the exception of the GI tract, the highest uptakes were found in the liver and kidney. BPA was also found to persist in neuroendocrine organs. Binding in plasma and tissues influenced uptake and retention of BPA in target tissues.

K-09

Prediction of Mutagenic Response in Salmonella typhimurium Using MultiCASE Quantitative Structure Activity Relationship Modules
R. D. Benz¹, E. J. Matthews¹, N. L. Kruhlak¹, R. D. Saiakhov², L. Fisher², G. Klopman², J. F. Contrera¹, ¹CDER, FDA, Rockville, MD 20857, ²MultiCASE, Inc., Beachwood, OH 44122

We have developed MCASE/MC4PC quantitative structure activity relationship modules and used them with a human expert analysis methodology developed by the FDA/CDER Informatics and Computational Safety Analysis Staff (ICSAS) to predict the mutagenic response of test chemicals in Salmonella typhimurium. This system produces mutagenicity predictions with high precision by basing positive determinations only on significant, structurally similar mutagenicity alerts that have been detected by two or more modules. We prepared a total of sixteen different MCASE/MC4PC modules, five using data from tests done with no exogenous metabolic activation using a liver S9 fraction, five with rat liver S9 present, five with hamster liver S9 present, and one composite that includes data from all testing conditions. Considered were Salmonella strains TA100, TA1535, TA1537, TA97, and TA98. Tests of the predictive performance of the program demonstrated that the modules exhibited good coverage for FDA regulated direct food additives (87-96%) and food contact substances (86-92%), but not as good for marketed pharmaceuticals (56-87%) or new/unmarketed drugs (44-82%). An experiment using 289 test compounds not included in the training data set showed that the new MCASE/MC4PC mutagenicity modules using the FDA analysis methodology produce high predictive value (88.9%), specificity (98.3%), and low false positives (11.1%) for known mutagens, but a relatively high rate of false negatives (39.6%) resulting from too few examples of mutagenic pharmaceuticals in FDA's records. We discuss experimental factors which influence the program's predictive performance and potential regulatory and lead compound selection applications.

K-10

Sample Size Determination for Validation of a Screening Method
F. D. McClure, Q. F. Graves, US FDA, CFSAN \ OSAS, College Park, MD 20740

A screening method is usually validated by applying it to a group of test samples each of which is known to contain or not to contain some level of analyte. In designing a validation scheme to test a screening method that generates quantitative values, a sample size was needed to ensure, with a stated level of confidence, that the test met predefined performance specifications with respect to its sensitivity (i.e., probability of correctly classifying "true" positive test samples) and false positive rate (probability of incorrectly classifying "true" negative test samples), where positives and negatives were to be determined by the dichotomization of analyte values assumed to be normally distributed. In determining the desired sample size, we adopted and extended the Greenhouse and Mantel procedure that minimizes the cost of sampling and testing.

K-11

A Comparison of Microbial Dose-Response Models Fitted to Human Data
H. Moon¹, R. L. Kodell¹, J. J. Chen¹, D. W. Gaylor², ¹NCTR, FDA, Jefferson, AR 72079, ²Gaylor and Associates, LLC, Eureka Springs, AR 72631

A study of mathematical two-parameter and three-parameter dose-response models for microbial risk assessment is conducted using infectivity and illness data on a variety of microbial pathogens from published studies with human volunteers for gastro-intestinal pathogens. The purpose is to evaluate the degree of model uncertainty exhibited by the models in order to determine whether two-parameter models might suffice for most microbial dose-response data or whether three-parameter models should generally be fitted. Model variability is measured in terms of estimated ED₀₁'s and ED₁₀'s, with the view that these effective dose levels correspond to the lower and upper limits of the 1% to 10% risk range generally recommended for establishing benchmark doses in risk assessment. Based on the ranks of the ED₀₁ and ED₁₀ values among eight mathematical models, we investigate model uncertainty among the two-parameter models and the three-parameter models for the data examined. A further evaluation of the two-parameter models is conducted to investigate if any one of the two-parameter models might be favored over the others.

K-12

Benchmark Dose Modeling of Mercury-Induced Acute Renal Failure in Sprague-Dawley Rats with Renal Insufficiency Compared to Healthy Controls.
R. P. Brown, E. F. Madden, P. L. Goering, Division of Life Sciences, FDA/CDRH White Oak, Silver Spring, MD 20903

Experimentally induced renal insufficiency (RI) increases the sensitivity of rodents to the adverse effects of a subsequently administered nephrotoxic agent. Since patients with renal insufficiency are at increased risk of developing acute renal failure (ARF) following exposure to nephrotoxic compounds, compared to healthy individuals, the use of animal models of renal insufficiency improves the clinical relevance of toxicity test results used for the risk assessment of nephrotoxic compounds released from medical device materials. Benchmark dose modeling was used to quantify the magnitude of the increased sensitivity in male Sprague-Dawley rats, compared to healthy controls, following IV injection of mercuric chloride (HgCl₂). RI was induced by 3 daily SC injections of gentamicin (250 mg/kg). Healthy control animals received SC saline x 3 days. HgCl₂ (0.025 to 0.5 mg Hg/kg) was administered on Day 4 and blood was collected for analysis 24 hours after HgCl₂ injection. The dose-response relationship is based on the number of animals in ARF, defined as 2x the mean value of BUN in healthy, non-mercury exposed animals. BMD50[control]/BMDL50[RI] ratios for mercury-induced ARF were in the range of 3-14, with only one dose ratio > 10. Assuming a similar increased sensitivity exists for patients with RI exposed to mercury or other nephrotoxic agents, compared to healthy persons, these results suggest that the default uncertainty factor of 10 used in noncancer risk assessment to account for interindividual variability in a population response to a given dose of a compound is adequately protective in most cases.

K-14

Gout Attack Modeling and Simulation
M. Katzper, CDER, FDA, Rockville MD

Gout may occur when subjects have elevated uric acid and deposit urate crystals in joints, soft tissue and urinary tract. However, individuals who are hyperuricaemic may never experience an attack. Under-excretion of urate is found in about 90 per cent of patients with gout. The underlying reasons for under- excretion of urate are unknown. A low purine diet can decrease serum urate levels by up to 15 per cent. Allopurinol decreases the production of uric acid by inhibiting the necessary enzyme xanthine oxidase. Because of the self limiting nature of gout attacks and the great variability in their occurrence there may be difficulty in assessing the efficacy of a preventive drug. A model that captures the behavioral characteristics of gout attacks can be used to generate simulated data representing a clinical trial to evaluate the efficacy of a preventive drug. Varying the factors incorporated in the model indicates how they affect the frequency and duration of attacks. The model is designed to study what reduction in acute gout attacks over a specified period is needed to show significance.

K-15

Breast Pump Adverse Events: Medical Device Reports to Center for Devices and Radiological Health
S. L. Brown¹, R. A. Bright¹, D. E. Dwyer¹, B. Foxman², ¹OSB, FDA, Rockville, MD, ²University of Michigan School of Public Health, Ann Arbor, MI

Breast milk is widely acknowledged to be the most complete form of nutrition for infants, with a range of benefits for infants' health, growth, immunity, and development. Breast pumps are medical devices used by nursing mothers to maintain their milk supply while away from their infant and to collect milk for their infant when they are separated. Success in expressing and collecting milk may be a determining factor in a woman's ability to successfully breast feed her infant, especially for mothers with premature infants who are unable to nurse at birth or for working mothers who are unable to nurse their infants during working hours. We searched the CDRH Manufacturer and User Facility Device Experience (MAUDE) database for adverse events reported with the use of breast pumps. There were 37 adverse event reports, 30 for electric or battery operated pumps and 7 for manual pumps. We characterize manual and electric breast pump adverse events reported to the CDRH.

K-16

Can mold allergy be triggered via the oral route?
S. Luccioli¹, J. Malka-Rais², T. M. Nsouli², L. Chiazze², J. A. Bellanti², ¹CFSAN/ FDA, ²Georgetown University, Washington, DC

It is generally accepted that mold allergy involves sensitization by inhalation of mold spores through the respiratory tract. However, there is recent evidence to suggest that sensitization through the GI tract may be an alternate pathogenetic route. Despite these observations, only a limited number of studies have been performed to evaluate whether oral ingestion of molds may provoke hypersensitivity reactions in mold allergic patients. Twenty-four adult patients with histories of various allergic disorders, including asthma, were initially selected based on skin test positivity to mold(s) or other inhalants following which all subjects underwent a single blind, placebo-controlled oral challenge to increasing doses of a mixed preparation of molds (mold mix) commonly found in foods, [kindly provided by Allergen Laboratories, Liberty, MO], (Alternaria, Cladosporium, Aspergillus, Mucor, Fusarium, Epicoccum, Pulullaria and Penicillium species). Of these patients, 16 were found to be skin test positive by skin-prick K-test (SPT) and/or intradermal test (ID) to the mold mix preparation, and 8 allergic patients were negative to both. All subjects were challenged orally with increasing doses of the mold mix (from 100 ng to 1 mg of individual molds) representative of mold concentrations that commonly contaminate foods. Symptoms were assessed at 15 minute intervals during the challenge period and by telephone follow-up at 24 hours using a modified scoring system (MSS), for which 5 points were assigned for each symptom, within a specific organ system, elicited by the challenge. Based upon the skin testing methodology, it was possible to differentiate the subjects into three groups based on mold sensitivity through skin testing, the provocation challenge (MSS score) and the average dose provoking concentrations (ADPC). In contrast to the positive relationship of skin sensitivity to MSS score an inverse relationship was seen between ADPC and dermal reactivity to molds within the 3 groups. The results of these preliminary studies suggest that ingestion of molds, at doses commonly found in foods, may provoke hypersensitivity reactions in mold allergic patients and may thus be a contributing factor to allergic symptoms in mold sensitive patients.

K-17

Surgical Stapler Adverse Events Reported to the Center for Devices and Radiological Health
S. L. Brown, E. K. Woo, OSB, FDA, Rockville, MD

The use of stapling devices has become standard practice in many operations, and these devices have many applications including ligation and division, resection, anastomosis, and fascial closure. The Food and Drug Administration regulates surgical staplers as a medical device. Manufacturers and health care providers report adverse events occurring during the use of surgical staplers to the FDA. Two FDA adverse event databases, the Manufacturer and User Facility Device Experience (MAUDE) database and the Alternative Summary Reporting (ASR) database were searched for adverse events related to the use of surgical staplers. A CDRH database, Oracle System Center Automated Retrieval (OSCAR), that includes information on recalls was searched for surgical stapler recalls and the reason for these recalls. We characterize adverse events from 112 death, 2,180 injury, and 22,804 malfunction reports from FDA adverse event databases. We describe 22 recalls for these products that are described in an FDA database. A majority of these recalls are related to manufacturing or design problems. The overall incidence of these events remains unknown, however, because these products are used so frequently, even uncommon adverse events may affect many patients. It is important for health care providers to report adverse events to manufacturers so that they may work to improve the design of these devices and reduce use errors that contribute to the events.

K-PO-18

Estimating the Safe Starting Dose in Clinical Trials and the No Effect Level for Humans Using Chemical Structure Activity Modeling of the Human Maximum Recommended Daily Dose (MRDD) of Pharmaceuticals
J. F. Contrera, E. J. Matthews, N. J. Kruhlak, R. D. Benz, OPS, FDA, Rockville, MD

The estimation of the maximum safe starting dose in clinical trials in healthy adult volunteers for pharmaceuticals and the no effect level (NOEL) dose for chemicals is currently based on an extrapolation of the results of animal toxicity studies. This process requires the use of multiple safety factors to compensate for the uncertainty underlying applying animal toxicity results to humans. The objective of this research was to model human MRDD data for pharmaceuticals and to develop quantitative structure activity relationship (QSAR) software to predict the human MRDD. The predicted MRDD can be used as the basis for estimating the maximum safe starting dose in clinical trials and the no adverse effect level (NOEL). A database of the MRDD for over 1300 pharmaceuticals was modeled using MDL QSAR software and E-state and connectivity molecular topological descriptors. Our predictive models performed well, with 74 -78% of predicted MRDD values for 120 internal and 160 external validation compounds falling within a range of 0.1-10 times the actual MRDD value (±1-log-fold). MRDD models derived from human data should provide a more accurate and specific estimate of MRDD and NOEL than current risk assessment models that extrapolate from animals to humans and may have broad drug development and regulatory applications.

K-20

Some Statistical Fallacies in Analyzing the Data Collected from the Adverse Events Reporting System
P. T. Liu, D. A. Street, FDA

Due to the nature of the multiple-symtom multiple-exposure Adverse Events Reporting System (AERS), neither the compound categories nor the symptom categories of the two-way AERS table are mutually-exclusive. The entries among different cells are dependent. The use of the conventional contingency table approach for analyzing the AERS table is thus inapproppriate.

The purpose of this article is to examine the statistical fallacies that arise from mistreating the AERS table as a contingency table in analysis. The simplest "two-compound two-symptom" model is given as an example for illustrating "proper" and "improper" statistical analyses.

K-21

Nonspecific musculoskeletal disorders in pediatric patients: analysis of ambulatory care visits from the National Ambulatory Medical Care Survey, a national representative database
E. O. Ibia, S. Singer, P. Nourjah, E. E. Navarro, CDER, FDA, Rockville, MD

The evaluation of drug-associated pediatric musculoskeletal adverse events (DAPMAE) is hampered by scant information on the frequency of nonspecific musculoskeletal disorders (NMD) in children. To estimate the proportion of NMD among pediatric ambulatory care visits in the US, we used ICD-9-CM codes to query the National Ambulatory Medical Care Survey (NAMCS), 1993 - 2000. We defined NMD excluding ICD9 codes for known musculoskeletal disorders and refined the definition through consultation with a panel of pediatric rheumatologists. We queried the three principal visit diagnoses to exclude injury- or infection-related visits, and also excluded follow-up visits. Finally, we reviewed DAPMAE reports in FDA's Adverse Event Reporting System (AERS) database for selected antimicrobials, 1989 - 2002, to understand the types of DAPMAE being reported to AERS.

Approximately 60% of ICD-9-defined NMD had a diagnosis of an injury or infection, which were excluded from further analysis. Of 146,448,900 annual visits in children < 17 years, 1,032,800 (7/1000) were for NMD. Over 90% of NMD visits were prompted by pain/discomfort in weight-bearing limbs or axial skeleton. The proportions of NMD visits/1000 visits were 4.1, 7.4, 12, and 9.6 for children <4, 5-9, 10-14, and 15-16 years, respectively. Of children visiting for NMD, mean age was 8.9 years with both sexes equally represented. Similarly, joint/limb pain and swelling were the most frequently reported DAPMAE in AERS. These reported DAPMAE were sometimes confounded by concomitant therapy and other underlying conditions.

In this exploratory study, a minority of pediatric ambulatory care visits were for NMD. NMD defined by ICD codes were confounded by infection and injury. Nonetheless, if validated by other methods, this screening strategy as a prelude to the determination of NMD rates may find utility in evaluating drug safety.

K-23

A Clinical Trial Simulation (CTS) Approach for Designing Studies Investigating the Similarity in Pharmacokinetic (PK) - Pharmacodynamic (PD) Relationships Between Different Patient Populations
N. S. Berry, Y. Wang, H. Sun, P. Lee, OCPB, FDA, Rockville, MD

Section 115 of FDAMA provides adult to pediatric bridging studies to extrapolate effectiveness and safety information, given the two populations share a similar PK-PD relationship. This project was conducted to develop a quantitative methodology to optimize the design of such a bridging study. The main objective of the project was to simulate trial designs where a bridging study would adequately describe the PK-PD relationship (as determined by clinical and/or statistical significance). The trial simulation consisted of 3 components: (1) patient characteristics, (2) drug model, and (3) study design. Studies with different patient distributions of age, body weight, and gender were simulated. In addition, the potential effects of age and body weight on drug pharmacokinetics and therapeutic response were considered in the simulation. Study design factors, such as number of subjects and sampling scheme, were examined for optimal study performance. Various combinations of patient characteristics, drug response, and study design were evaluated for study power. Both univariate and multivariate equivalence statistical approaches were evaluated for scenarios on an observational level. Preliminary results indicate CTS may help optimize the clinical trial design of adult to pediatric bridging studies and may further establish confidence intervals for concluding a PK-PD relationship is similar.

K-24

Construction of a Human Adverse Effects Database for Modeling Quantitative Structure-Activity Relationships (QSARs)
N. L. Kruhlak¹, E. J. Matthews¹, J. L. Weaver², R. D. Benz¹, J. F. Contrera¹, ¹FDA/CDER/OPS/ICSAS, Rockville, MD, ²FDA/CDER/OTR/DAPR, Silver Spring, MD

DA/CDER's Informatics and Computational Safety Analysis Staff (ICSAS) is conducting a pilot study to determine whether computational toxicology methods can be developed to predict the potential human adverse effects of pharmaceuticals based on post-marketing report data. The adverse effect data chosen for this investigation were extracted from FDA/CDER's Spontaneous Reporting System (SRS) database and included over 1.6 million adverse drug reaction (ADR) reports for 8620 drugs and biologics listed for 1191 Coding Symbols for Thesaurus of Adverse Reaction (COSTAR) terms. The ICSAS Adverse Effects database contains the generic names and molecular structures for a subset of 1515 organic chemicals that are suitable for modeling with commercially available QSAR software packages. ADR reports from the SRS database were pooled for the first five years that a pharmaceutical was marketed. To estimate patient exposure during this period, shipping units for each pharmaceutical were used as a denominator, resulting in the creation of the Adverse Effect Index (AEI), where AEI = (# ADR reports/# shipping units) × 1,000,000. A pharmaceutical defined as active for a single COSTAR endpoint is characterized by 4 ADR reports, >20,000 shipping units, and an AEI 4.0 during the first five years of marketing. Furthermore, test chemicals evaluated as active must contain a statistically significant structural alert at two or more toxicologically related endpoints. We also report the use of a combination QSAR module which pools observations from two or more toxicologically related COSTAR term endpoints to provide signal enhancement for poorly represented adverse effects.

K-25

The Odds-Risk Inequality and Odds-Risk Conversion
P. T. Liu, D. A. Street, FDA

The odds ratio calculated from a retrospective study can be used for estimating the relative risk when the disease incidence is rare. However, the accuracy of such an estimation is determined not only by the incidence rate, but also by the exposure rate and relative risk.
The pourposes of this article are (1) to develop a deterministic model for describing the functional relation between the odds ratio and relative risk, (2) to define and prove the odds-risk inequalities, (3) to determine the upper or lower limit of odds ratio estimates, (4) to generate the odds-risk ratio tables necessary for evaluating the impact of those variables affecting the odds-risk discrepancies, and (5) to demonstrate how to convert an odds ratio to a relative risk

K-26

Evaluation of six QT correction formula including a new nonlinear model based on continuous QT measurement from Holter recording
J. Zhang, S. Machado, S. Haidar, FDA

Several formulae have been proposed in the literatures to adjust the QT interval for heart rate. The most common approaches are still Bazett's or Fridericia's corrections, which adjust the QT interval by dividing by the square root or cube root of the RR interval, respectively. Because both formulae do not work well at low and high heart rates, other approaches have been developed. We will evaluate five existing correction formulae: Bazett's, Fridericia's, linear (Framingham), exponential (Sarma), log-linear (Eli-Lilly). We will also propose a new approach using a modified nonlinear logistic model to capture the curvature in the relationship between the QT and RR interval. Comparisons of performance among the different methods will be provided. The data were collected based on 24-hour Holter monitoring for a group of 45 male patients with ischemic heart disease who were on drugs known not to prolong the QT intervals. For each subject, ECG data were continuously collected for up to 72 hours over a large range of heart rates.

K-28

Can we recruit additional subjects for a failed study and still maintain the type one error rate?
T-H. Ng, FDA, Rockville, MD

When a study fails to meet the study objective but comes close, it is tempting to recruit additional subjects in the hope that enlarging the study will allow achievement of a statistically significant result. Such an action is operationally similar to performing an unplanned interim analysis; in general, it would lead to an inflation of the type I error rate and therefore, would be unacceptable. This problem always exists when evaluating continuous endpoints because the nominal a level is attained by the statistical tests. However, for discrete outcomes such as the proportion of successes the nominal a level is frequently not attained by the statistical tests. In such cases, the "leftover" a may be "spent" by the second analysis with additional subjects and the prespecified type I error rate is maintained.

K-30

A Note on Quan-Zhang's Method for Estimating the Standard Deviation for a log-Transformed variable
L. Chen, Y. Tsong, FDA, Rockville

Quan and Zhang (2003) proposed several confidence interval estimators for the standard deviation of a log-transformed variable using the sample mean and sample standard deviation in original scale. These methodologies are all based on asymptotic theorem. Note that the convergence rate of the sampling distribution of the proposed variance estimator under log scale is highly related to the skewness of the distribution under the original scale. When the skewness is large, the Quan-Zhang' Method will have very poor performance. It may greatly underestimate the standard deviation, so as the power of the test.

K-PO-01

Determination of Sample size for setting tolerance limits in clinical trials with dichotomous outcomes
B.G. Zaslavsky, OBE/DB, Rockville, MD

Statistical evaluation and planning of clinical trials frequently utilizes the notion of confidence intervals. The definition of confidence intervals is well known and is broadly accepted by biomedical researchers. In some clinical trials the notion of tolerance interval is utilized. This notion is applicable to the clinical trials that evaluate products based on their performance in a part of the population. The tolerance intervals are rarely mentioned in clinical protocols, even though they are used often in some clinical studies. When the outcomes of a trial are continuous, the tolerance intervals are used in validation and process control of medical products. Tolerance intervals for normally distributed variables can be calculated using available statistical tables. For dichotomous outcomes, the definition of a tolerance interval is complicated, and no tables are available to calculate it. This research provides an algorithm for calculating sample size in the clinical trials with binary outcomes, given the level of confidence and tolerance. The sample size may be calculated using StatXact.

Key words: confidence, tolerance, StatXact, binomial distribution, negative binomial distribution

CATEGORY L: VALIDATION, TESTING, STANDARDIZATION, AND QUALITY ASSURANCE
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L-01

Evaluation of the BAX System for the Detection of Salmonella spp. in Spice Samples X. Lai, M. J. Palmieri, NRL, FDA, Jamaica, NY

This study was undertaken to validate the reliability of BAX system to detect Salmonella spp. in naturally contaminated spice samples. The BAX system and VIDAS-SLM were evaluated in parallel to screen for Salmonella spp. in 257 spice samples, representing more than 38 kinds of spices from 30 countries. The BAX system identified 14 samples as positive while the VIDAS-SLM identified 30 samples as positive. All 14 samples identified as positive by the BAX system were among the 30 samples identified as positive by the VIDAS-SLM and all these 14 samples were confirmed as true positive by BAM (Bacteriological Analytical Manual) method. Of the 30 samples identified as positive by the VIDAS-SLM, 23 were confirmed as true positive and 7 as false positive by BAM method. These results indicate that about 8.9% (23/257) of spices were naturally contaminated with Salmonella spp. The BAX System identified 60.8% (14/23) of Salmonella spp. contaminated spices whereas the VIDAS-SLM identified all the contaminated spices (23/23) but also reported 23.3% (7/30) false positive results. Based upon these results, the use of the VIDAS-SLM would be better to be used to safeguard the release of contaminated spices by FDA.

L-03

Effects of cleaning on removal of peanut allergens from food-contact surfaces
L. S. Jackson¹, J. E. Schlesser¹, T. Bowden², T. J. Fu¹, S. M. Gendel¹, M. Moorman³, ¹FDA/NCFST, ²IIT/NCFST, ³Kellogg Co.

Undeclared allergens can be inadvertently introduced into food due to cross-contamination during manufacture. Inadequate cleanining of shared processing equipment is believed to be a major source of cross-contamination. Little information exists on the adequacy of cleaning regimens for the removal of allergenic protein(s) from food-contact surfaces and the extent of allergen carry-over due to use of shared processing equipment. The objectives of this work were to 1) determine the efficacy of cleaning protocols for removing peanut allergens from food-contact surfaces and 2) measure allergen cross-contamination caused by the use of the same food-contact surface. For washing experiments, plates of different food-contact materials (stainless steel, teflon, polyethylene, urethane, polycarbonate) were contaminated with a known amount of peanut butter, and then washed [water, chlorinated alkali detergent (CAD), acid detergent (AD), each at room temperature and 62.8ºC] for 30 min. After cleaning, the plates were swabbed and peanut residues measured with an ELISA peanut assay. For cross-contamination experiments, peanut butter cookie dough was applied to the surface of the plates. The dough was removed and replaced with an equal amount of sugar cookie dough. Peanut transferred to the sugar cookie dough was measured by ELISA. The cleaning protocols differed in their ability to remove peanut butter from the food-contact surfaces. Room temperature water was not effective at removing peanut butter from any of the materials while hot (62.8ºC) water was not effective for cleaning urethane and teflon plates. Hot CAD and AD solutions were successful at cleaning all five materials. Room temperature CAD was not effective in removing peanut butter from some of the plates. Cross-contamination experiments showed high levels of transfer of peanut from food-contact surfaces to sugar cookie dough. These results demonstrate that detergents need to be used as directed by the manufacturer to be effective at cleaning equipment surfaces. Food processors need to evaluate the efficacy of cleaning protocols for each food-contact surface, piece of equipment and processing line.

L-04

Virus-Retentive Filter Nomenclature: Characterization of the coliphage PR772 recommended for virus filter performance testing
S. C. Lute¹, H. Aranha², D. Tremblay³, D. Liang⁴, H. W. Ackermann³, B. Chu⁴, S. Moineau³, K. Brorson¹, ¹OBP/DMA, ²Pall Corp., ³Université Laval, ⁴SUNY, Stony Brook

Virus filtration is a key clearance unit operation in the manufacture of recombinant protein, monoclonal antibody and plasma-derived biopharmaceuticals. Recently, a consensus has developed among filter manufacturers and end-users about the desirability of a common nomenclature and a standardized test for classifying and identifying viral-retentive filters. Because of its reported size, the coliphage PR772 (Tectiviridae family) has been used as a surrogate for mammalian viruses sized around 50-60 nm. The PDA virus filter task force has proposed the use of PR772 as a potential model bacteriophage to standardize nomenclature for 'large pore size' virus retentive filters (filters designed to retain viruses larger than 50-60 nm in size). In this study, we analyze specific properties of the PR772 critical to support its use for the standardization of virus filters. The complete genomic sequence of the virulent phage PR772 was determined and analyzed. Its genome contains 14,946 bp with an overall GC content of 48.3 mol% G+C. Sequence analysis revealed 32 open reading frames of at least 40 codons. Comparative analysis of PR772 nucleotide sequence with the genome of the Tectiviridae prototype phage PRD1 revealed a 97.2% identity at the DNA level. Dimensions measured on electron micrographs estimated the size of PR772 at 73 nm ± 3 nm. Using dynamic light scattering, its hydrated, natural-state, hydrodynamic diameter was estimated at 82 ± 6 nm, appropriate for use to test large viral-retentive filters. Finally, dynamic light scattering of PR772 preparations purified on CsCl gradients reveals that the phage preparations appear to be largely monodispersed. In summary, PR772 appears to be an appropriate model bacteriophage for standardization of nomenclature for larger pore size viral retentive filters.

L-05

Issues in Using Databases of Pre-Recorded Physiological Signals to Test Medical Devices
C. Ho, S. B. Kurtzman, FDA

Bench testing with databases of pre-recorded physiological signals is a common step in the verification and validation of a medical device. For example, in the case of a new ECG monitor, the physiological signals are ECG tracings that have previously been recorded from patients using other ECG monitors. Human overreaders then annotate the tracings to determine the occurrence of significant clinical events such as ventricular tachycardia. These annotations can be used to determine the sensitivity and specificity of the new ECG monitor.

This article aims to highlight some issues associated with such testing, such as the way the pre-recorded signals are presented to the new monitor, the adequacy of single channel versus dual channel versus multi-channel testing in the bench testing, and the need for the human overreaders and the new monitor to use the same set of rules for determining the occurrence of an event. These issues are also applicable to other monitors such as the apnea monitor. Some suggestions for addressing the issues are also presented.

* This paper represents the professional opinion of the authors and is not an official document, guidance or policy of the U.S. Government, the Department of Health and Human Services, or the Food and Drug Administration, nor should any official endorsement be inferred.

Note: This abstract is part of an article previously published under: * Charles Ho and Steven B. Kurtzman, "Issues in using databases of pre-recorded physiological signals to test medical devices", Biomedical Sciences Instrumentation 39 (2003) 169-174.

L-06

Evaluation of Devices for the Surgical Treatment of Presbyopia.
B.A. Drum, CDRH, FDA, Rockville, MD 20850

Presbyopia is the loss of ocular accommodation (ability to focus the eyes to see clearly at close range) with increasing age. All people eventually become presbyopic, usually between the ages of 45 and 55 years, and need optical assistance to see clearly at near distances. Nonsurgical remedies for presbyopia have been available for many years in the form of reading and multifocal (bifocal, trifocal or progressive) spectacles. More recently, refractive surgical device manufacturers have undertaken the development of surgical remedies. While some of these proposed remedies are also multifocal in nature (e.g., monovision LASIK treatments, multifocal intraocular lens [IOL] implants), others (e.g., accommodating IOL implants, scleral expansion procedures) actually aim to restore the accommodative ability of the eye.

Most accommodation restoration methods are invasive, high-risk procedures, and some assume controversial theories of accommodation that appear inconsistent with known changes in the aging crystalline lens. It is crucial that the effectiveness of these methods be evaluated with accurate and unbiased tests of accommodative range. Unfortunately, clinical measurements of accommodative range still rely on either the subjective "push-up" method or dynamic retinoscopy, both of which are susceptible to biases that can lead to selective overestimates of postoperative accommodation. Automatic refractometers and aberrometers are potentially capable of objective measurements of accommodative range, but most are not equipped to make these measurements without modification. New instruments designed explicitly for the objective measurement of accommodative range are needed to adequately assess the effectiveness of devices that claim to restore accommodative capability to presbyopic eyes.

L-07

Evaluation of Diagnostic Devices: Analytical Limits at Low Levels
M. Kondratovich¹, K. Linnet², ¹Division of Biostatistics, CDRH, FDA, Rockville, MD 20850, U.S.A., ²Laboratory of Clinical Biochemistry, Psychiatric University Hospital, DK-8240 Risskov, Denmark

Characteristics of the analytical performance of quantitative methods at low levels are very important in the evaluation of in vitro diagnostic devices. Two such critical performance characteristics at low levels are the limit of detection (the smallest amount that the method can reliably detect to determine presence or absence of an analyte) and the limit of quantitation (the smallest amount that the method can reliably measure quantitatevely). According to recent ISO standards, the limit of detection of the method should be estimated taking both type I and II errors into account. The authors present the concepts of the limit of blank, the limit of detection, the limit of quantitation and the relationships among these limits. Other concepts such as analytical sensitivity and functional sensitivity are discussed. The connection between the limit of detection for quantitative methods and the 95% interval around the cutoff for qualitative methods are also discussed. The authors consider various schemes for reporting results. These schemes depend on the relationship between the observed results of the quantitative method and the analytical limits of the method at low levels.

L-08

An international collaborative evaluation of a new mumps virus vaccine neurovirulence safety test
S. Rubin¹, M. Afzal², J. Johannessen³, H. Hsu¹, R. Taffs¹, K. Carbone¹, ¹FDA, Rockville MD, ²NIBSC, UK, ³FDA, Rockville, MD

Prior to the widespread use of mumps vaccines, mumps virus was the leading cause of viral meningitis and encephalitis in developed countries. Due to the highly neurotropic properties of wild type mumps viruses, neurovirulence testing of virus seeds used in the manufacture of mumps vaccines is required. Such testing has historically been performed in monkeys, as prescribed by regulatory authorities. However, results obtained from these tests have raised questions as to whether this assay can reliably discriminate neurovirulent from non-neurovirulent mumps virus strains. For example, despite passing neurovirulence safety test scores, the risk of aseptic meningitis following administration of the Urabe vaccine strain (used in Japan, Latin America and Europe) is approximately 1/10,000 and that of the Leningrad-3 vaccine strain (used in Russia, Croatia, India and other countries) is approximately 1/1,000. This has resulted in withdrawal of mumps vaccines, public resistance to vaccination, and, in some countries, complete cessation of national vaccination programs. Thus, as new strains of mumps vaccine continue to be licensed for clinical use, development of a validated mumps virus neurovirulence test with relevance to human disease remains an important public health objective. To address this issue we have recently developed a rat model that has successfully been used in our laboratory to assess the human neurovirulence potential of several mumps virus strains. To better evaluate the performance of the rat neurovirulence safety test, an interlaboratory collaborative study was initiated. Results of this study are presented and demonstrate that the test is reproducible, robust, and predictive of human neurovirulence.

L-10

Stability to Digestion and Processing Conditions as Criteria for Protein Allergenicity Assessment
T. Fu¹, C. Hatzos², ¹CFSAN/NCFST, FDA, Summit-Argo, IL, ²NCFST, Illinois Institute of Technology, Summit-Argo, IL

Advances in biotechnology have resulted in an increased number of foods containing proteins of non-food origin. The allergenic potential of these new proteins needs to be assessed to ensure food safety. Digestive stability and resistance to food processing conditions (e.g., heating and acid hydrolysis) are among the criteria used by the agricultural biotechnology industry to assess the allergenic potential of novel proteins. The utility of digestive and processing stabilities as predictive tools for protein allergenicity assessment, however, has not been thoroughly validated. We have evaluated the predictive value of digestive and acid stabilities by comparing the relative resistance of food allergens and non-allergens to digestion in standard simulated gastric and intestinal fluids and to hydrolysis in acidic solutions at different pHs, and determined whether a ranking order can be established relating a protein's allergenicity to its digestive and acid stabilities. The results showed that the correlation between the digestive and acid stabilities of a protein measured in vitro and allergenic potential is not always present. In addition, it was found that protein digestibility as measured by in vitro assays is greatly influenced by the assay conditions used. A protein's relative digestibility and thus its perceived allergenic potential are largely determined by how digestion assay results are interpreted. Standardized criteria relating measured digestibility to allergenic potential are needed. To facilitate the establishment of such criteria, information regarding the range of digestibility exhibited by food allergens in defined conditions is needed. We have developed a database consisting of the digestibility of 21 known food allergens in simulated gastric and intestinal fluids at various enzyme to test protein ratios.

L-13

Evaluation of Statistical Methods for Mouse Lymphoma Assay (MLA) Data
M. M. Moore¹, J. Clements², R. Delongchamp¹, A. Thakur³, B. Myhr³, ¹FDA, Jefferson, AR, ²Covance, Harrogate, United Kingdom, ³Covance, Vienna, VA

The MLA Workgroup of the International Workshop on Genotoxicity Tests (IWGT) has developed a new recommendation for data analysis. To evaluate the performance of statistical methods the group utilized 398 experimental data sets from 10 laboratories. Twenty-seven statistical methods were applied. When p<0.05 was used to determine whether an experiment was positive or negative, there was agreement among the various statistical methods for 40% of the data sets. For the other 60%, the methods gave different positive/negative calls. Graphical representations of the 398 experiments were used to classify the data into various curve shapes. A small subset (about 10%) of the data gave curve shapes that clearly defined positive responses (large dose-related mutant frequency increases). The rest of the data fell into categories that showed small dose-related increases, no dose-related increase or where it was difficult to visually discern a positive trend. It was this latter set of experiments where the statistical methods gave the most divergent determinations. In these situations the choice of statistical method would determine whether a response was positive or negative. Thus the Workgroup could not recommend a specific statistical method. However, because a uniform method is needed to analyze MLA data, the Workgroup proposes an approach that combines statistical analysis with a global evaluation factor based on the inter-laboratory variability observed for the solvent/negative control mutant frequency.

L-14

Development of an HIV-1 subtype panel for standardization of HIV NAT assays
J. Hu¹, S. Lee², R. Taffs², O. Wood², A. Machuca³, A. Vallejo², I. Hewlett², ¹CBER/FDA, Bethesda, MD, ²CBER/FDA Bethesda, MD, ³CBER/FDABethesda, MD

Current available Nucleic acid technolgies (NAT) assays for HIV are based on different technologies. To both standardize and to evaluate the performance of NAT assays for detection of multiple HIV subtypes (A-G) , CBER developed panels based on cultured virus isolates spiked in negative human plasma. A panel of specimens consiting of dilutions of virus stocks was sent to 5 independaet laboratories to accurately determine copy numbers. A consensus value for HIV RNA copy number was assigned to each member based on statistical analysis of results from each of these five laboratories. These panels can be used to validate and standardize qualittative and quantitative assays used to screen the blood supply or to monitor patients on antiretroviral therapy. The panels also serve as reference materials for manufacturers to comply with sensitivity requirements and to harmonize NAT in the international setting.

L-15

Development of West Nile virus standards to correlate infectivity and RNA copy number in blood
M. Rios, K. Srinivasan, O. Wood, S. Daniel, K. Tomioka, A. Williams, I. Hewlett, CBER/FDA, Bethesda, MD

West Nile virus (WNV) first appeared in the US in 1999 with 62 reported human cases and 6 deaths. In the year 2002 there were 4156 cases reported and 288 deaths. Investigations carried out by CDC identified human to human transmission through organ transplants, and 21 cases associated with blood transfusion. Nucleic acid tests (NAT) have been developed for blood donor screening in order to prevent transmission. Currently, there are no quantified standards for WNV and viral load has been determined by infection of Vero cells in culture and expressed in plaque forming units (PFU). The objective of this work was to generate standards with known correlation of PFU and viral copies to standardize and evaluate the sensitivity of different WNV NAT assays.Viral copy numbers and PFU values of a WNV virus stock was tested in a collaborative study in multiple laboratories and it was found that RNA copies were approximately 2 logs higher than PFU. This information allows better definition of viremia in WNV disease. The virus stocks are being formulated into a dilution series for a second larger collaboratiove study. Results will be analyzed statistically and used to assign values to panel members. The panel will be useful in licensure of WNV NAT assays and for lot release surveillance of future licensed WNV NAT assays.

L-16

Evaluation of Quality Attributes of Prussian Blue using Chemical Imaging and Particle Size Distribution
P. J. Faustino¹, C. D. Ellison¹, Y. Yang¹, C. R. Brownell¹, N. Sadrieh², E. P. Duffy³, F. Houn⁴, S. A. Loewke⁵, M. M. Nasr³, R. C. Lyon¹, ¹DPQR, OPS, FDA, Silver Spring, MD, ²OPS, FDA, Rockville, MD, ³ONDC, OPS, FDA, Rockville, MD, ⁴ODE3, OND, FDA, Rockville, ⁵ODE3, OND, FDA, Rockville, MD

Prussian Blue (PB) was approved by the FDA in October 2003 for the treatment of radioactive contamination resulting from a terrorism incident. PB or Fe₄[Fe(CN)₆]₃ is synthesized by reacting excess iron chloride with potassium ferricyanate. The resulting precipitate is filtered and dried at temperatures greater than 100°C. Processing may generate vast differences in particle size and shape for the PB Active Pharmaceutical Ingredient (API). These studies were done to indicate whether particle size might be a potential measure of product quality. Particle size determination of API was done using ultrasonic sifting whereas chemical imaging (CI) was used for PB drug product particle sizing. Results indicate statistically significant differences in the particle size distribution of five commercial API lots. Similarly, differences in particle size of all five PB drug product lots were also noted. To compare lots, the linear function of the geometric mean mass of the particle size distribution was calculated. The d50 (distribution at 50%) for API lot 1-5 was 229, 160, 165, 170 and 38 microns, respectively. Using CI, the uniformity of distribution of the API and the major excipient (microcrystalline cellulose) within PB drug product capsules was evaluated. A partial least squares chemometric analysis of compositional histograms indicated only 1 lot had a uniform distribution of PB while two lots had "bimodal" distributions in the final drug product. Further work is being done to correlate particle size with both cyanide release and cesium binding.

L-17

Detection of Avian and Mammalian Retroviruses using a Single-tube Fluorescent Product-Enhanced Reverse Transcriptase (STF-PERT)Assay.
Y. K. Ma, A. S. Khan, CBER, FDA, Bethesda, MD

We previously developed a TaqMan fluorescent probe-based product-enhanced reverse transcriptase assay in which the reverse transcriptase and polymerase chain reaction steps are set-up in a single tube, in two compartments separated by AmpliwaxÔ (designated as STF-PERT; J. F. Sears and A. S. Khan, 2003, J. Virol. Methods, 108; 139 - 142). This simplification of the original two-step PERT method resulted in increased assay reproducibility and handling efficiency while maintaining the sensitivity (<10 virions equivalent RT activity). The PERT assay is currently recommended for retrovirus testing in vaccine development. A critical step in the manufacture of a safe biological product is selection of a cell substrate that has been demonstrated to be free of adventitious agents. Retroviruses are a particular safety concern in biologics since they integrate and persist stably in the normal DNA as a critical part of their replication cycle. Furthermore, all species contain retroviral sequences, of which, some may generate infectious virus. To evaluate potential endogenous retrovirus contamination of vaccine cell substrates, we have evaluated the sensitivity of the STF-PERT assay for avian and mammalian retroviruses using AMV and MLV RTs, respectively, and relevant titered virus stocks. The results indicated similar sensitivity of detection of AMV and MLV RTs (<10 virions equivalent RT) and detection of one infectious particle of endogenous avian retrovirus (RAV-0) and endogenous murine retrovirus (AKR-L1).

The broad and sensitive detection of retroviruses by the STF-PERT assay can help in early selection of "clean" cell substrates for production of safe vaccines and other biological products.

L-18

A Standardized MIC Testing Method at 22°C and 28°C Approved by the NCCLS Aquaculture Working Group
R. A. Miller, R. D. Walker, R. Reimschuessel, OR, FDA, Laurel MD

To accurately assess the susceptibility of aquatic bacterial pathogens to antimicrobial agents, it is first necessary to develop appropriate standardized antimicrobial susceptibility testing (AST) methods. To facilitate this, the National Committee for Clinical Laboratory Standards (NCCLS) established an Aquaculture Working Group (AWG), charged with developing standardized AST methods for aquatic bacterial pathogens. The AWG has published an NCCLS report M42-R providing a standardized agar disk diffusion testing method for aquatic isolates. A second publication describing newly standardized methods for determining minimum inhibitory concentrations (MICs) will be published in 2004.

In an effort to standardize a broth microdilution AST method and establish quality control (QC) ranges, members of the AWG coordinated a 10 laboratory multi-national collaborative study. Following procedures in the NCCLS documents M31-A2 and M37-A2, this study was conducted in accordance with NCCLS recommendations. Using two previously characterized QC strains, Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658, in cation-adjusted Mueller-Hinton broth incubated at 22°C for 24-28 hours and 44-48 hours, 28°C for 24-28 hours, and 35°C (E. coli for QC) for 16-20 hours, ten antimicrobial agents of interest to global aquaculture (enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, ormetoprim-sulfadimethoxine, oxolinic acid, gentamicin, and trimethoprim-sulfamethoxazole) were tested in dried 96-well panels manufactured by TREK Diagnostic Systems. All of the proposed MIC QC ranges generated from this study were approved by the NCCLS Veterinary AST Subcommittee for testing at 22°C, 28°C, and 35°C in a dried format. This represents the first standardized reference method for MIC testing of aquatic isolates.

L-19

Development of an alternative, ELISA-based potency test for licensed rabies vaccines.
A. Kumar, J. Muller, M. Wright, C. Anderson, R. Levis, OVRR, FDA, Bethesda, MD

We have previously presented data on the development of an alternative potency test for rabies virus vaccine products licensed in the United States. The current test, the NIH potency test, is an animal-based immunogenicity assay. The alternative test we are developing is a capture ELISA that measures the level of rabies antigen in the vaccine. A critical quality standard that the ELISA-based potency test must meet is the ability to measure a subpotent vaccine. To validate the ability of this new ELISA-based test to measure the potency and stability of vaccine product over time, we have exposed the test vaccine to a variety of different conditions designed to degrade its potency. These include modification of storage conditions, temperature changes, and adverse chemical conditions. We will present data comparing the ELISA-measured potency of these vaccines to the traditional animal-based immunogenicity assay. Electron microscopic analysis of virus particles in treated vaccine samples may show the structural changes in the virus that correlate with a loss of potency. The studies described above will be expanded to all rabies vaccines licensed in the United States.

CATEGORY M: SCIENCE COMMUNICATION/POLICY/LEVERAGING/OUTREACH
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M-01

UV Doses Worldwide and Beyond
D.E. Godar, OST, FDA, WhiteOak, MD

UV radiation affects human health. The only beneficial health effect is the formation of pre-vitamin D in the skin; whereas, there are many detrimental effects: sunburn, ocular damage, photoaging, immune suppression, DNA damage, and skin cancer. Skin cancer is the most diagnosed of all the cancers. In 2002 in the US, there were over one million new cases of skin cancer. That means 1 out of every 285 people got skin cancer and, it is estimated that 1 out of 67 people born in 2003 will get melanoma sometime during their life. Skin cancer is increasing at an alarming rate (4-6%/yr) around the world and has now reached so-called "pandemic" proportions. Thus, it is important to know what UV doses people get throughout their lives around the world. This review covers how the outdoor UV doses are weighted for different biological effects, the most common measuring devices for terrestrial and personal UV doses, the natural effects on terrestrial and personal UV doses, the time people spend outdoors, their percent personal ambients, and the terrestrial and personal UV doses of adult outdoor and indoor workers as well as children and adolescents around the world. Overall, outdoor working adults get about 10%, while indoor workers and children only get about 3% (2-4%) of the total available annual UV. People's UV doses increase with decreasing latitude; most indoor working adult Europeans get 10,000-20,000, American's get 20,000-30,000, and Australian's are estimated to get 20,000-50,000 J/m²/yr (excluding vacation, which can increase the dose by 30%).

M-02

CBER Severe Acute Respiratory Syndrome (SARS) Research Working Group
D.R. Taylor, CBER SARS Research Working Group, FDA, Bethesda, MD

The CBER SARS Research Working Group was formed in response to the recent pandemic of 2003. The group shares multiple goals including communication across offices, information sharing, discussion of current topics related to SARS research and regulatory issues, the fostering of collaborations and sharing of reagents and resources and the development of group proposals. Projects have been undertaken by CBER researchers to help expand the knowledge of this emerging virus to better enable good regulatory decisions and recommendations. Our projects are focused at understanding the SARS virus so that we can help to ensure vaccine safety and efficacy, safety of the blood supply and the development and analysis of diagnostic reagents. We have established collaborations with the Laboratory of Infectious Diseases, NIAID, the SARS Team at the CDC, and with researchers at several universities in order to obtain reagents and exchange ideas. Several projects have been initiated by CBER researchers including; 1) Virus inactivation studies for killed vaccine applications, blood and blood products, and laboratory use outside BSL3, 2) Development of virus neutralization and ELISA assays for the characterization of the humoral response and to test vaccine candidates, 3) Understanding the potential for Antibody-Dependent Enhancement in vitro and in animal models, 4) Detection of virus in asymptomatic patient samples to help ensure the safety of the blood supply, 5) Developing SARS-specific diagnostics through analysis of novel antigens, 6) Expression assays to elucidate the role of these proteins in the pathogenesis of the virus that may be important for the development of antiviral drugs, 7) Expression of S1 spike protein as a virus-like particle to enhance vaccine potency, 8) Identification of SARS-specific epitopes and development of specific antibodies for passive immunotherapy to prevent infection and treat disease related to SARS coronavirus.

Clear Science Communication Award - 2004 FDA Science Forum

M-04

Study Pregnant Women? Yes, You Can!
K. Uhl¹, M. A. Miller², D. L. Kennedy¹, ¹CDER, OND, FDA, Rockville, MD, ²OC, FDA, Rockville, MD

The DHHS regulations governing the inclusion of pregnant women in clinical research were first promulgated in 1978 and are found in 45 CFR 46 Subpart B. The original Subpart B was criticized for being overprotective, possessing language that was exclusionary to pregnant women. In 1994, a PHS Human Subject Regulation Drafting Committee was convened to evaluate possible revisions to Subpart B. Subpart B was subsequently revised and finalized November 13, 2001 (60 FR 56775). In the revised regulation, the presumption of exclusion of pregnant women, i.e., "no pregnant women may be involved unless...", was changed to a presumption of inclusion, i.e., "pregnant womenmay be involved in research if". This important change is not well known by many including experts in the field of ethics. Many people are likely to resist the idea that pregnant women may now be enrolled in drug trials. This is unfortunate, as considerable research is needed on health problems that occur or worsen during pregnancy. It is imperative that all investigators, study auditors/inspectors, institutional review boards, and FDA reviewers, among others, are knowledgeable of the current Subpart B regulations so that information on maternal safety and efficacy and fetal safety can be collected in well-designed research studies. Without such an understanding, undue obstacles may hinder or even stop studies involving pregnant women.

M-05

The United States Regulatory Agencies Unified Biotechnology Website and the Database of Completed Regulatory Reviews: Development and Use
R. I. Merker, K. Ricker, FDA, College Park, MD

Under a policy developed in 1986 (51 FR 23302), biotechnology-derived products are regulated under a "coordinated framework" of existing laws and regulations administered by the U. S. Environmental Protection Agency (EPA), the U.S. Department of Agriculture (USDA), and the Food and Drug Administration (FDA). Products are regulated according to their intended use; the evaluation of different aspects of a single product may come under the purview of more than one agency. To respond to requests by various domestic and international groups, these regulatory agencies have created a unified Interagency Website that provides information on U.S. government review of bioengineered crop plants. The centerpiece of the website, a database entitled U.S. Database of Completed Regulatory Agency Reviews, contains descriptive information and hypertext links to agency documents on bioengineered crop plants intended for food and feed that have completed all recommended or required reviews for food, feed, or planting use in the United States. We provide a brief overview of this joint interagency project and examples of the uses of the database.

M-06

The Pediatric Research Equity Act, The Best Pharmaceuticals for Children Act, Emergency Research, and Informed Consent: Uncharted Waters or Rough Seas
S. K. McCune¹, S. F. Goldkind², ¹CDER, FDA, Rockville, MD, ²OC, FDA, Rockville, MD

The Pediatric Research Equity Act (PREA), signed into law on December 3, 2003, requires pediatric studies of safety and efficacy for certain pharmaceutical products that have potential pediatric use. The Best Pharmaceutics for Children Act (BPCA), enacted on January 4, 2002, provides the impetus to study off-patent drugs and it reauthorizes the pediatric exclusivity incentive program. The latter legislation has resulted in label changes in 63 products. There will be an increase in the number of pediatric trials in the future driven by PREA and BPCA, and some of these trials will involve emergency research. These complex situations mandate thoughtful solutions to protect the rights of the individual patients while obtaining the scientific data to promote optimal drug therapy for the entire pediatric population. A number of physicians, ethicists and regulators at FDA, NIH and OHRP have discussed the role of informed consent in pediatric emergency research and the following questions have emerged:

Is it possible to conduct emergency research in pediatrics given an especially vulnerable population with distraught parents?
Can permission from parents be obtained in a timely and effective manner given the stressfulness of the situation?
How can permission best be obtained in an emergency setting with a narrow therapeutic intervention window?
Are the considerations different for a pediatric population?
Is the exception from informed consent (CFR 50.24) truly applicable in pediatrics?
Would parents have a different set of considerations for themselves and for their children regarding the exception to informed consent?

M-07

Patient Perspective in the Product Review and Approval Process
M. L. Covington¹, J. M. Minor¹, R. M. Klein², ¹OSHI, FDA, Rockville, MD, ²OSHI, FDA Rockville, MD

The development of a Food and Drug Administration (FDA) regulated product benefits from the inclusion of input from the end user before it is approved for use in the general population. Patients, the ultimate consumer of drugs, biologics and medical devices, are involved in the clinical trial a sponsor conducts to determine the safety and effectiveness of a product. Patients are in a unique position to advise the agency about their concerns and the risks they are willing to take relevant to a specific product. Consequently, the FDA includes patients in the product review and approval process of products for serious and life-threatening diseases such as HIV/AIDS and cancer.

Since the inception of the Patient Representative Program in 1991, more than 100 Patient Representatives (PR) have participated in advisory committee deliberations on products for the treatment of HIV/AIDS, cancer, diabetes, cardiovascular disease, etc. More recently, 25 Patient Consultants (PC) have participated in end of Phase 2 discussions between FDA and sponsors to address issues related to cancer clinical trial designs, expanded access of investigational drugs, and labeling. PRs and PCs receive training about FDA and the drug review process. Part of their commitment to these programs is to take back what they have learned about FDA to their advocacy communities.

This presentation will discuss the history of how patient advocates, once critical and distrustful of the agency, have become an important part of the product review and approval process through the Patient Representative and Patient Consultant Programs, and played an essential role in the evolution of the regulatory review process.

M-08

Development of Policies to Manage Risks Associated with Increasing Use of Medical Devices in the Home
P. J. Phillips¹, S. Gardner², H. Albersheim³, S. Berman⁴, H. J. Duggirala³, M. Eakle², P. Jahnes⁵, P. Jones⁶, M. Mendelson³, A. Pinkos⁷, M. Warner², M. A. Wollerton³, M. Weick Brady², A. A. Ciarkowski¹, ¹ODE, ²OSB, ³OHIP, ⁴OSM, ⁵OC, ⁶OST, ⁷OIVD

The CDRH Home Health Care Committee (HHCC) is addressing the rapid increase of medical devices that are used in the home. In general, the medical device industry is experiencing exponential growth that is producing an abundance of devices that with diverse options and often easier to use (e.g., automatic external defibrillator). Over the past decade, however, medical devices that were designed for medical professionals are now in common use by patients.

Concurrent with this trend is an increasing number of reports indicating new risks associated with the use of medical devices in the home such as:
* Family members making changes to the device settings
* Patients lack operating instructions for the device resulting in errors
* Device malfuntion through lack of maintenance
* Complex devices incorrectly used
* Some patients/caregivers lack skills to properly operate the device

To consider policy options for managing these risks, the HHCC conducted:
* Literature review
* Focus Group Meetings
* Rapid Response Surveys
* Open public discussion with other governments and nongovernment organizations

The following options are under consideration to manage these risks:
* Certification of home care medical devices by Third Parties
* Collaboration with nongovernmental organizations to address education and training requirements
* Leverage CDRH efforts with Standard Development Organizations to promulgate human factor standards for these devices
* Develop an FDA web site for labeling and instructions for use
* Write brochures to assist users in properly using devices in the home

M-10

An FDA perspective on dermatologic drug development: The FDA Reviewer and Dermatology Advisory Committee Meetings
M. C. Luke, MD PhD, J. K. Wilkin, MD, DDDDP, ODE V, Rockville, MD

The FDA medical reviewer's role is to evaluate information submitted to INDs and NDAs and to review conclusions from other disciplines for relevance. The FDA dermatologist reviewer is crucial to the evaluation of dermatologic drug products. The FDA reviewer is responsible for identifying issues, preparing and presenting the material related to the clinical safety and efficacy of the drug under review. The dermatologist reviewer evaluates the content of the NDA, literature, recommendations from the Advisory Committee, expert consults and other relevant information and recommends either approval or non-approval of dermatologic drug product marketing applications. FDA Advisory Committees make recommendations on issues related to FDA's regulatory responsibilities. The issues that have been brought to Advisory Commitees are many and varied. A retrospective review of the topics brought to the FDA Dermatology Advisory Committee meetings over the past 20 years gives insight and perspective on the issues for which FDA dermatology medical reviewers evaluate and make regulatory recommendations. A descriptive analysis was conducted with regard to INDs and NDAs reviewed by the reviewers in the Division. The topics from Dermatology Advisory Committees were summarized with regard to indications and presented for discussion. The descriptive results are presented.

M-11

Committee for the Advancement of FDA Science (CAFDAS): The FDA Scientist's Liaison to the Commissioner's Office
N. Alderson¹, S. N. Ali², A. Debrabant³, C. A. Elkins⁴, R. Fahmy⁵, P. J. Faustino⁶, M. A. Grant², J. Johannessen¹, A. S. Khan³, M. Kulka⁷, A. D. Lucas⁸, M. Manjanatha⁴, S. H. Stern⁸, H. Trinh¹, J. L. Ward⁵, G. E. Wood⁷, ¹OC, FDA, ²ORA, FDA, ³CBER,, FDA, ⁴NCTR,, FDA, ⁵CVM, FDA, ⁶CDER, FDA, ⁷CFSAN, FDA, ⁸CDRH, FDA

The Committee for the Advancement of FDA Science (CAFDAS) serves as an internal advisory committee to the Commissioner, Associate Commissioner for Science, and the Senior Science Council, addressing FDA-wide science issues from a scientist's perspective, functioning independently of center or discipline. The primary objective of CAFDAS is to aid in enhancing the agency's science infrastructure by: serving as a conduit for ideas and concerns from scientists to senior FDA management regarding the state of science in FDA; providing comments to the Commissioner on science policy questions and planning, from the perspective of the working scientist; and providing practical suggestions and constructive solutions to achieve Agency-wide scientific excellence.

Some selected CAFDAS activities include: review of OSHC inter-center collaborative science projects; promotion of leveraging information on competitive research funding sources and mechanisms; discussion of a mini-sabbatical program for FDA scientists; and implementation of a Science Seminar Series for the Commissioner's Office.

CAFDAS is composed of two representatives appointed from each Center and ORA to serve a three-year term. CAFDAS members represent research, review and compliance aspects of the FDA mission.

More information regarding CAFDAS' history, Meeting Minutes and Leveraging Activities is available at: http://first.fda.gov/cafdas.

M-12

Committee for the Advancement of FDA Science (CAFDAS): Leveraging Activities
N. Alderson¹, S. N. Ali², A. Debrabant³, C. A. Elkins⁴, R. Fahmy⁵, P. J. Faustino⁶, M. A. Grant², J. Johannessen¹, A. S. Khan³, M. Kulka⁷, A. D. Lucas⁸, M. Manjanatha⁴, S. H. Stern⁸, H. Trinh¹, J. L. Ward⁵, G. E. Wood⁷, ¹OC, FDA, ²ORA, FDA, ³CBER, FDA, ⁴NCTR, FDA, ⁵CVM, FDA, ⁶CDER, FDA, ⁷CFSAN, FDA, ⁸CDRH, FDA

A principal focus of CAFDAS efforts has been to provide FDA scientists with information on available funding sources and the resources necessary to compete for such funding. To effectively serve FDA scientists in identifying and pursuing leveraging options, CAFDAS has compiled documents (Series on Leveraging Techniques) that identify resources and procedures to use within FDA's Leveraging Program (http://www.fda.gov/oc/leveraging). These resources provide the FDA scientist with information about leveraging activities, mechanisms to seek out collaborative research partners and research projects, experiences of others who have succeeded in leveraging resources to develop projects, and some of the pitfalls in establishing such collaborative efforts. This information resource is designed to give all FDA scientists, both research and regulatory, a starting point for pursuing external resources and partnerships including Memoranda of Understanding (MOUs), Cooperative Research and Development Agreements (CRADAs), Interagency Agreements (IAGs), Contracts, and Material Transfer Agreements (MTAs).

The CAFDAS Leveraging Activities including the Series on Leveraging Techniques, and a compilation of FDA and other Leveraging Resources are available at: http://first.fda.gov/cafdas.

M-PO-01

Pediatric Information in Package Inserts of HIV Anti-retroviral Drugs: How much pediatric pharmacokinetic (pK), dosing, safety and efficacy information are currently available from the electronic Physician's Desk Reference (ePDR)?
R. Johann-Liang, D. M. Sillivan, J. S. Murray, D. B. Birnkrant, DADVP, FDA, Rockville, MD

Appropriate drug labeling for children continues to be part of a critical agenda for the FDA and the pediatric community. There are now 21different anti-retroviral drugs (ARVD) approved to treat adult HIV infection. This study examined what labeling was available for pediatrics. We accessed the ePDR for labels of each of the approved ARVD and queried the words "pediatric" and "child". We found 24 separate labels (7 nucleoside analogs with 2 fixed-dose combination and 1enteric-coated dosage forms, 3 non-nucleoside analogs, 1 nucleotide analog, 8 protease inhibitors with 1 separate dosage form, and 1 fusion inhibitor) in the ePDR (updated 12/03). Ten of 23 (43%) labels for oral use had dosage form (solution, suspension, or powder for reconstitution) appropriate for infants or young children. Pediatrics was mentioned in the following sections of the label: Clinical Pharmacology 21/24, Indications and Usage - Clinical Studies 5/24, Warnings 6/24, Precautions 24/24, Adverse Events (AE) 9/24, Dosage and Administration 16/24, and Patient Package Insert 7/11. Usable pK and dosing information were available for 14/24 (58%) with 7 having dosing for infants. Safety information was also available for 14/24 (58%) with 4 adding significant information for children (pancreatitis, rash, nephrolithiasis, granulocytopenia AEs were higher in children than adults). Efficacy information was available for 5/24 (21%) and 4 more labels had "evaluation is on-going" statements. Although the collective effort for pediatric ARVD labeling has been commendable, continued diligence is needed to adequately label ARVD as well as all needed drugs for infants and children.

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Last updated on 2004-AUG-11 by frf