Poster Categories:

Third Place Poster - 2001 FDA Science Forum

Board A01

Prion hypothesis postulates that the conformantionally changed isoform of prion, PrPsc, is the TSE infectious agent which propagates by serving as a template to convert host prion protein (PrPc) to additional molecules of PrPsc. It has been shown that 2/3 of PrPc in blood is present in plasma. Interestingly, also portion of TSE infectivity present in blood has been shown to reside in plasma. The source of plasma PrPc and TSE infectivity is not known. We used monoclonal antibodies 1562 and 6H4 for study of PrPc expression on cultured endothelial cells (EC). Flow cyto-metry demonstrated that human umbilical vein ECs (HUVECs) and bovine aorta ECs are clearly positive for PrPc. We measured the number of PrPc molecules on HUVECs during growth in culture. The expression of PrPc peaked significantly (p<0.01) after 48 hours, when HUVECs were reaching confluence. Treatment of HUVECs with calcium ionophore led to shedding of PrPc positive microparticles. PrPc positive microparticles were also found in cell culture supernatant of non-stimulated HUVECs. Our results demo-nstrate that vascular ECs express PrPc and should be considered as a possible source of plasma PrPc and TSE infectivity.

Board A02

O-R-polyHbA0, an intra- and intermolecularly cross-linked derivative of human HbA₀, was found to have lower oxygen affinity, a reduced Bohr effect and no measurable cooperativity (n_max=1). In addition, we found that the enthalpy change (DH) associated with oxygen binding is reduced by a factor of two in the modified protein. The kinetics of O₂ and CO binding to the proteins were examined by rapid mixing techniques. In unmodified HbA₀, inositol hexaphosphate (IHP) causes an increase in oxygen dissociation rate (k_off) and a decrease in CO association rate (k_on). Neither of these allosteric effects were seen in parallel studies with O-R-polyHbA₀. The apparent lack of T®R transition seen with O-R-polyHbA₀ was confirmed by circular dichrosim (CD) studies, as reflected by absence of changes in the Soret region of the CD spectrum (~420 nm) upon the addition of IHP. Our results also indicate that locking HbA₀ in the T-conformation with O-raffinose may compromise its ability to effectively deliver oxygen as blood substitute and increase its susceptibility to oxidative damage.

Board A04

About 15 % of patients homozygous with Z mutant a₁-PI develop liver disease. The disorder is linked to accumulation of high molecular weight a₁-PI polymers in hepatocytes. Understanding the mechanism of polymerization is crucial for the rational design of therapeutics and for assessing the safety of a₁-PI products. We suggest a new model of polymerization, which is at odds with a previously proposed loop-sheet (i.e. head-to-tail) monomer polymerization model. In contrast, we propose that a₁-PI dimer (perhaps head-to-head) is the polymerizing unit. Our hypothesis results from an observation that a₁-PI polymers, formed in 1.4 M guanidine-HCl, after folding by dilution, polymerize further. Dimers, isolated from such solutions and from polymer solutions generated by heating, form polymers in buffer at ambient temperature. The rate of polymerization is temperature and concentration dependent. Until now, both denaturant- and heat-induced polymerization studies have been used to support the loop-sheet polymerization model. However, polymers formed from dimers produced by these two methods migrate differently by native PAGE. This suggests that the dimers are different and, thus, the polymerization mechanism(s) may be different. Further work is needed to determine if either of these mechanisms is physiologically relevant. However, the ability of both types of dimers alone to polymerize in vitro suggests that dimer may also be the relevant polymerizing unit in hepatocytes.

Board A05

In rodent model of transmissible spongiform encephalopathy (TSE), infectivity in plasma was mostly absent in preclinical phase but appeared during symptomatic stage of disease. Prion protein (PrPc), expressed on many cell types, is believed to play an essential role in TSE infectivity propagation. We found PrPc expression on human umbilical vein endothelial cells (HUVEC) and investigated if PrPc could be released from HUVEC in the form of membrane microparticles (MP). Using flow cytometry, MP were identified by size 0.3 - 3.0 mm and by annexin V binding. MP count increased overnight in medium of unstimulated cells, but this was not enhanced by TNFa (1000 U/mL). However, when cells were activated for 25 hrs with TNFain the presence of cycloheximide (50mg/mL) to promote apoptosis, a dramatic increase in MP count was observed (83 000 MP/10³ cells), as compared to TNFa (15 000 MP/10³ cells) or cycloheximide (13 000 MP/10³ cells) treatment alone. Apoptosis of HUVEC was studied after 5mM camptothecin treatment. Release of MP after camptothecin treatment could be partly inhibited (25 - 30 %) by 50 mM Z-DEVD-fluoromethyl ketone, a caspase 3 inhibitor. Endothelial MP expressed PrPc, antigens in abundance on MP were CD147 and CD31, while CD51/61 was found in low levels. PrPc positive MP released by apoptotic endothelial cells may play an important role in spreading infectivity in blood during symptomatic stage of disease.

Board A06

The quantification and identification of xenobiotic reactive intermediates is difficult in the absence of highly radiolabeled drug. These intermediates can be detected, however, by measuring the formation of adducts to intracellularly generated radiolabeled glutathione (GSH). Freshly isolated adherent rat hepatocytes were incubated overnight in thiol-free medium. They were then exposed to 100 mM methionine and ³⁵S-labeled methionine in otherwise thiol-free medium to replete cellular GSH pools with intracellularly generated ³⁵S-labeled GSH. After 3 hours the test substance was added and the cells were incubated for an additional 24 hours. The medium was then processed using C₁₈ solid phase extraction and chromatographed by C₁₈ HPLC with radiochemical detection. Adduct peaks were assumed to be those not present in solvent controls. The cells from these incubations were then disrupted and GSH was quantified by reaction with monobromo-bimane followed by HPLC analysis with fluorescence and radiochemical detection to obtain values for the cellular concentration of GSH and the specific activity of the GSH pool. Adduct formation with acetaminophen was used for a positive control. Application of the method was demonstrated for other known hepatotoxins including the drug troglitazone and felbamate metabolites.

Board B02

Like the evaluation of average equivalence of two drugs, for quantitative measurements, the assessment of equivalence in terms of regression line is often conducted for diagnostic medical devices, with the objective of demonstrating that the slope is close to 1 and the intercept is close to 0. And, for qualitative measurements, the evaluation of equivalence with respect to sensitivity and specificity is frequently performed, with the objective of showing that the difference between sensitivities (specificities) of two devices is close to 0. In practice, in order to claim equivalence, one current exercise is to demonstrate that a confidence interval on slope (intercept, sensitivity or specificity) includes 1(0), while another one is to show that the confidence interval falls completely within a pre-specified clinical meaningful relevant range on slope (intercept, sensitivity or specificity). The two practices correspond to methodologies known as the power approach and the interval hypothesis procedure (i.e., two one-sided tests procedure), respectively. The two procedures will be compared with examples.

Group Award for Leveraging Activities - 2001 FDA Science Forum

Board B03

We are evaluating the usefulness of non-invasive and biomarker methods for measuring UV responses in human skin. The observations will involve 110 subjects representing different UV sensitivities within six racial/ethnic groups. The non-invasive techniques include two reflectance spectroscopy methods, optical coherence tomography, ultrasound imaging, and two mechanical methods: ballistometry and a suction technique. Shave biopsies are analyzed for DNA damage and repair, and for melanin production and distribution. At the time of writing, data have been collected on 52 subjects. They indicate that, in addition to the visible erythema and pigmentation, UV causes skin changes that are discernible with almost all the techniques examined. Induction of DNA damage following exposure to 1 Minimal Erythema Dose (MED) was readily detectable. Data confirm substantial MED variation within conventional skin types and indicate that racial/ethnic origin is not a significant factor determining individual UV sensitivity.

Group Award for Leveraging Activities - 2001 FDA Science Forum

Board B04

The role of racial/ethnic origin in determining individual UV sensitivity remains unclear. We are evaluating usefulness of various biomarker methods for measuring UV responses in human skin, and we are analyzing UV-induced DNA damage and repair within different racial/ethnic groups. This study will involve 110 subjects representing different UV sensitivities with six racial/ethnic groups. Minimal Erythema Dose (MED) of each individual was determined to assess the sensitivity for UV irradiation before biopsies. Shave biopsies are taken before exposure, immediately after exposure to approximately 1 MED of UV, and then 1 day and 7 days later. Induction of DNA damage (cyclobutane pyrimidine dimers, CPD) is detected in sections by indirect immunofluorescence using a monoclonal antibody (TDM-2) specific for CPD and the fluorescein isothiocyanate (FITC). Nuclear DNA is counterstained with propidium iodide (PI). DNA damage is expressed as the ratio of the intensities of the FITC fluorescence for CPD and that of the PI fluorescence for DNA. The available data show that DNA damage measured by CPD is most significant immediately following UV exposure, but is repaired more than 50 % within 1 day. However, melanin contents measured by Fontana-Masson staining do not change consistently with UV exposure.

This study will eventually compare the immediate DNA damage, the repair efficiency and the degree of photoprotection afforded in the skins of individuals from different racial/ethnic origins and phototypes 1-6.

Group Award for Leveraging Activities - 2001 FDA Science Forum

Board B05

We are studying UV-induced erythema in a cohort of 110 volunteers divided into 11 groups on the basis of their skin type (Fitzpatrick, 1-6) and their racial/ethnic origin (OMB classification 0990-0208: American Indian or Alaska Native; Black or African American; Asian; Hispanic or Latino; Native Hawaiian or Other Pacific Islander; White.) Small areas on the backs of the subjects are exposed to different doses of UV radiation from FS lamps. After 1, 2, 8, and 16 days, skin color changes are evaluated visually and instrumentally with a Minolta spectrophotometer and a DiaStron Erythema/Melanin Meter. The first instrument takes measurements at 10-nm intervals (400–700 nm), the second one at 546, 632, and 905 nm. Until now, the data have been collected on 52 subjects. Administered doses ranged from 72 to 3333 J/m² (CIE Erythema Action Spectrum weighted at 298 nm). The sensitivity of the visual and instrumental observations is similar, although in some cases erythema can be measured instrumentally in the exposed areas that are not easy to evaluate visually. Results confirm wide variation of the Minimal Erythema Doses (MED) within skin types. For example, for the subjects with a skin type 2/2.5, and 3/3.5 the MED ranged from 171-400 and 150-709 J/m², indicating significant overlap.

Board B06

Clinical efficacy endpoints can be designated as primary or secondary in many ways. Some secondary endpoints taken alone or together can potentially be primary if so designated in the protocol, while others only play a supportive role. To handle endpoints, there are two classes of widely used analysis strategies. In one class, each endpoint is allocated some proportion of the desired total alpha for its individual hypothesis to be tested. In the other, some closed test procedures are performed sequentially from the aggregate of the associated pre-specified hypotheses to the individual hypotheses. Which of these competing strategies is optimal is generally unknown, depending on the true state of nature about the alternative hypotheses. In practice the true state of nature, particularly about which endpoint(s) will show a more favorable effect size, is unknown and extremely difficult to predict in advance. Consequently, many fixed-sample-size trials fail if the strategy is chosen wrongly. In this presentation, we explore some adaptive strategies which use the results of interim analyses to adjust the final analysis of multiple endpoints and select the primary endpoint(s). The adaptive strategies preserve type I error rate and can maintain the desired power level. Some caveats about the operational aspect of the strategies are given. We also explore some other strategies to aid interpretation of statistical evidence.

Board B07b

Three paragraphs in 21 CFR 201.57 (f)(8) describe requirements for the content and format of labeling human prescription drugs for nursing mothers. If a drug is absorbed systemically, the labeling should contain information about excretion of the drugs in human milk and effects on the nursing infant, and describe adverse effects in animal offspring. The purpose of this project was to review statements regarding nursing in labeling extracted from the current PDR. Among the labels with nursing information, 353 contained only a general statement and 52 contained a general statement applying to drug class (for example, estrogen or thiazides). Only 126 labels mentioned that the drug was found in human milk, and 89 labels provided a drug level in human milk. The conditions on which the level was measured (for instance, steady state, single dose, etc.) were usually not described. Adverse events in nursing babies were described in 31 labels. One hundred forty seven labels only provided information on nursing animals and their offspring without correlation to human circumstance. Results show that labeling of human prescription drugs for nursing mothers needs more clinically relevant information. FDA's Pregnancy Labeling Team is currently considering a new labeling regulation to provide better information about the use of drugs during lactation.

Board B08

Pulmonary artery catheters (PAC), also known as Swan-Ganz catheters were introduced into critical care practice in the 1970's. PAC's provide clinicians with critical hemodynamic data not available from other clinical resources. Despite recent controversies in risk-benefit ratios in use of the device, approximately 1.5 million catheters are sold domestically per annum. Pulmonary artery (PA) rupture is the most acute, and often fatal, complication associated with use of PAC, with a reported incidence between 0.016% and 1.0%, and mortality well over 50%. A review of the Food and Drug Administration's (FDA) Manufacturer and User Facility Device Experience (MAUDE) database revealed 714 total medical device adverse event reports (death, injury and malfunction, associated with use of PAC) between June, 1993 and June 1999. Among 61 reported PA rupture patients were within the age range of 52-89 years. This analysis of catheter-related PA rupture yields the following profile of susceptibility: female gender, age and presence of pre-existing cardiovascular disease. Among these reported risk factors, the most notable finding was the frequency of occurrence of PA rupture in postmenopausal females. Case report review and analysis indicate a need to further investigate the relationship between factors related to PAC use and PA rupture. Further study to clarify measurable outcomes to delineate what relationship, if any, exists between gender, age and PA rupture in use of PAC is recommended.

Board B09

A provision of the 1998 Demographic Final Rule requires that sponsors of investigational new drug (IND) applications provide in their annual reports the total number of subjects initially planned for inclusion in the study and the number entered into the study to date, tabulated by race, age group, and gender (Federal Register Vol. 63 No. 28). The objective of this project is to assess whether participation in clinical trials of these demographic subgroups was documented and reported in annual reports submitted during the first quarter of 1999. Demographic information was extracted from clinical trial protocols described in 413 IND annual reports received by CBER and CDER between January 1, 1999, and March 31, 1999. Analysis of data from approximately 1,250 protocols is ongoing.

Board C01a

Cataract surgery with subsequent implantation of an intraocular lens (IOL) has become one of the most successful and widely performed surgical procedures in this country. Many surgeons are turning to flexible IOLs such as those made from silicone as they can be implanted through a smaller incision than the conventional acrylic lenses. A smaller incision decreases scarring and distortion of the cornea. Silicone IOLs pose special problems for the manufacturer during optical testing. Due to the index of refraction of silicone, the IOLs must have a high surface curvature. These higher curvatures result in much more spherical aberration when the lens is tested in isolation in air. If the effects of spherical aberration can be minimized while leaving the other optical properties under test the same, in principle, silicone lenses could be tested against the more demanding criteria applied to conventional acrylic lenses. One method is to use an additional lens during the testing of the silicone IOL that would undo, or null, the effects of spherical aberration while leaving the other optical properties under test the same. A two element null lens, optimized for the modulation transfer function (MTF) performance of a 20 D silicone IOL, was designed and tested. The performance of the null lens was examined for silicone IOLs with in situ powers from 16 D to 24 D. Improvements in resolution, resolution efficiency and MTF were obtained over the tested power range.

Board C02

Lubricants such as PEG and spermicides such as N9 are applied to PU condoms before packaging. This may lower the condom strength due to plastization of the PU, leading to breakage of the condom during use, resulting in an undesired pregnancy and/or transmission of disease. It was found that after soaking PU condoms in N9 and PEG, mass uptake and mechanical properties increased and the glass transition temperature decreased with increased N9 concentration in PEG. The loss in properties will need to be weighed against the benefit provided by a given concentration of N9 in the lubricant. Most of the changes occurred in the first 20 hours of soaking, so the effect of N9 and PEG on the long term shelf life of the condom is likely not an issue. In addition, mechanical properties of the film used to make the condom varied with direction because of molecular orientation which occurred during manufacturing. This suggests that the condom should be cut to optimize this property-orientation relationship.

Group Award for Leveraging Activities - 2001 FDA Science Forum

Board C03

MRI is rapidly emerging as an essential diagnostic tool for the evaluation of toxicology, pathophysiology and pathology at the pre-clinical levels. MRI techniques have distinct characteristics that complement more traditional toxicological/pathological evaluations of experimental animals. In addition to animal studies, MRI can provide a non-invasive method for gathering data on the internal texture, chemical composition, pH, temperature, function or structure of a wide range of materials, fixed tissues, and foods. MRI data has the potential to contribute significantly to the resolution of regulatory issues. Heretofore, an in-house MRI capability has not been available to FDA scientists.

The FDA has recently established a 2-Tesla research MRI facility at MOD I. The magnet and hardware were obtained from the NIH. Space for the MRI facility was identified at MOD I. CFSAN provided funds for transport of the 7,000-pound superconducting magnet and for building modifications needed to accommodate the magnet and instrumentation. CFSAN also provided funds to purchase and install a new console and data system. Installation of the new console was accomplished in August, 2000.

A number of non-animal projects are already under way or in the planning stages. The veterinary infrastructure necessary for animal studies is now under development. When complete, the facility will be capable of imaging animals ranging in size from rats to dogs and primates. Other technical improvements and pulse sequence implementation is also progressing. Sample images and data generated at the MRI facility will be presented.

Board C04

Suspected tampering cases have included both accidental and deliberate holes or punctures in product packaging. Identification of the object(s) used to make the holes has been demonstrated to reduce the initial analysis time and determine if the hole was consistent with known objects. With emphasis placed upon items that could be used to make a suitable hole and/or inject or remove liquids, thirty-eight (38) puncturing objects were used to produce simulated tampering holes in plastic bottles. Stereoscopic light microscopy (SLM), polarized light microscopy (PLM) and scanning electron microscopy (SEM) were all used to determine unique puncture hole characteristics. SLM was valuable in performing initial hole analysis and observing the presence of raised "halos" around the puncture site. PLM showed substrate compression and sites of transmitted light extinction. SEM analysis provided internal hole edge characteristic information or "tool marks". The eruption surface around the inside edges of the holes provided characteristic shapes usually dependent on the cross-sectional profile of the penetrating object. The preliminary results of this study show that there are a number of unique and identifiable characteristics in the eruption surfaces of holes in plastic bottles made by penetrating objects. Continuing research at the FCC will include developing a complete method for hole classification and the preparation of a guide for the identification of puncture holes.

Board C05

Behavioral measures of frequency selectivity, e.g., psychophysical tuning curve (PTC), are time-intensive and require cooperation from subjects. In contrast, measures ofcochlear frequency selectivity based on the distortion-product (2f₁-f₂) otoacoustic-emission ratio function (DPRF), i.e., f₂/f₁ ratio paradigm, are objective, passively obtained and time efficient. Animal data has shown that the frequency of the maximum-amplitude 2f₁-f₂ distortion-product (f_d*) in the f₂/f₁ ratio function is the same as the "tip-tail" slope-change frequency (f_z) in the neural-frequency tuning curve (FTC) for f₂; however, no comparable human data are available. Based on the analogous relationship PTCs have to FTCs, the current study evaluated the relationship between the "tip-tail" slope-change frequency (F_xb) in 2 kHz and 4 kHz PTCs, and the f₁ frequency in DPRFs (2 kHz and 4kHz) when 2f₁-f₂ was at maximum amplitude. Results showed that the two frequencies (F_xb & f₁) were essentially the same in both functions (PTC & DPRF) when f_d* occurred.

Board C06

Introduction. Space program technology results in direct benefits for humans living on Earth (J Clin Pharmacol 30:223, 1990). Biomedical spin-offs include a: cardiac ventricular-assis pump; clinical fetal heart monitor; electrocardiogram and electrogastrogram monitor; and devices to engineer human tissue and to detect changes in the blood supply of cancer tumors. Methods. A literature search was conducted. Results. The DeBakey VAD device, a miniaturized ventricular-assist pump, provides a bridge to the availability of an actual heart organ. Problems were friction damaged blood cells caused by turbulent flows in many pump parts and stagnant regions in the pump resulting in blood clots. Supercomputer simulation of fluid flow in rocket engines designed the VAD to move cardiac blood efficiently with less red blood cell damage and clot formation. Flexible material employed for wing surface measurements developed portable technology to provide fetal heart monitoring. The monitor documents and stores fetal heart rate data non-invasively, resulting in decreased fetal mortality. The BioLog, a portable electronic unit, stores 48-hours of myoelectric activity of the stomach and heart to study gastric function and autonomic activity associated with space motion sickness. On Earth it monitors patients unable or unwilling to work with stationary monitors. The BioScan improves cancer treatment by detecting treatment-induced changes in cancerous lesions of breast and skin. It monitors antiangiogenesis factors, to limit cancer growth by inhibiting blood supply. The sensor locates hot spots during fires and search and rescue missions, spots faulty welds, observes volcanoes and detects radiation severity in the Van Allen Belt. Conclusions. The benefit of scientific investments in the US space program are summarized. Author opinions do not reflect policy of the US federal government.

Board D01

The U.S. Food and Drug Administration (FDA), has been successful in responding to the unique challenges presented to the agency by the AIDS epidemic. Since AIDS was observed and recognized by scientists at the Centers for Disease Control and around the world in 1981, the agency has continually made the fight against this dread disease a top priority. In order to keep pace with the critical needs and issues surrounding a disease for which there is still no known cure, FDA took a proactive approach to identify and implement change where necessary, while keeping within its statutory requirements under the Federal Food Drug and Cosmetic Act. The three major areas for change that the agency focused on were 1) The drug approval process; 2) Including the patient's perspective on advisory committees; and 3) Creating educational tools to combat the barrage of fraudulent products and treatments targeted toward Persons Living with AIDS (PLWAs).

Board D02

In an active controlled clinical trial without a placebo arm, the study objective is increasingly more often to show that a new treatment is no less effective than the active control within some non-inferiority range. Two issues behind this objective are that of whether the new treatment is efficacious relative to standard of care or a putative placebo and whether the new treatment preserves a certain fraction of control effect. Two types of statistical analysis methods are employed in recent pharmaceutical applications to address these issues. One requires that non-inferiority margin be set in relation to preservation of a target portion of control effect prior to commencement of the active control trial (ref: CBER/FDA). The other aims at directly testing for the effect of the new treatment relative to the nonconcurrent placebo or for preservation of some fraction of control effect (ref: Hasselblad and Kong, Fisher, a version of Simon, etc.). These methods carry some key assumptions. The effect of the active control is often estimated from a collection of historical control trials using random effect modeling of DerSimonian and Laird. In this presentation we show that statistical validity of the latter methods rests highly upon the assumption that the control effect does not change from historical trials to the concurrent active controlled trial and the conditions under which normal approximation is appropriate for the statistics in the random-effect modeling. The former methods are generally quite insensitive to these conditions though they can be ultra conservative when the assumptions are met.

Board D03

A key quantity in the evaluation of monitoring devices is precision of measurement. If monitoring devices cannot measure true values precisely, then they are of limited utility in distinguishing normal patients from abnormal patients by trends in these values over time. An example of such devices are bone densitometers, which are being used to diagnose osteoporosis by monitoring bone mineral density. To assess the utility of a monitoring device, the average precision is less helpful than the precision for individuals. Therefore, we give prediction and tolerance intervals for the precision of repeated measurements on an individual. For bone densitometers, we also consider the minimal time needed between measurements to detect with reliability an accelerated trend that suggests osteoporosis. The minimal time calculation adjusts for the normal trend and for the effects of any drugs that the patient might be taking.

Board D05a

The "full compliance" model is introduced for estimating the "best" inspection interval, for quantifying the inspection Impact and for determining the optimal inspection scheme. The model can also be used for verify the following general inspection principles quantitatively: 1) The increment of inspection has a "diminishing effect", 2) The inspection of a high risk plant has a greater impact than the inspection of a low risk plant and 3) The maximum inspection impact can be achieved by equal inspection intervals.

Board D05b

The multiple-screening procedures have been adopted to determine the sample size for the "classification" problem, such as that of labeling a compound as either an "irritant" or "nonirritant". The procedures can be summarized as follows: Construct a mixture binomial model for describing the performance of the Draize test,Estimate the parameters (sensitivity and specificity) of the model, Examine all feasible regulatory decision strategies,Evaluate the error rates (i.e., false positive and false negative rates) associated with each feasible strategy, and Select the optimal decision strategy. This study intends both (a) to justify theclaim that the two- or three-rabbit test suffices for making irritancy decisions and (b) to harmonize the international regulations for eye irritation by comparing the potential error rates associated with the existing regulations adopted by different agencies.

Board D06

Both of the Bayesian approach with the assumption of a uniform prior from 0 to 1 and the classical sampling theory approach produce the identical confidence interval for the "zero" positive rate. Therefore, when we impose any restriction to reduce the sample space of a uniform prior, such a procedure could effectively shorten the confidence interval. This article discusses the reasons to impose some restrictions in the uniform prior to reduce the required sample size in regulatory policy decisions.

Board D07

Methodology of Removing the Effect of Confounding Variables in Receiver Operating Characteristic (ROC) Analysis
M.Kondratovich CDRH, FDA, Rockville MD 20850

The area under an ROC curve (AUC) is often used to summarize the ability of a diagnostic test to discriminate between two groups. In many clinical studies, confounding factors (e.g., age) influence the distributions of the test variable and can affect the performance of the diagnostic test. For example, the mean value of the ultrasound test for the bone status becomes lower with increasing of age. The diseased population is generally older than non-diseased population, therefore, the age distribution of diseased and non-diseased groups in the retrospective studies are very different and the ability of the diagnostic test to discriminate these populations, which is measured by AUC, is usually overstated because some portion of the difference in the test distributions is due to the difference in age. The author considered not-age-matched and age-matched ROC analysis and showed that only age-matched ROC analysis yields correct estimates of the diagnostic capability of the medical device. A weighted ROC analysis is offered by the author which allows age-matched ROC analysis.

Board D08

Since 1996, through August 29, 2000, CDRH has received 1372 reports of serious injuries and 24 reports of death associated with the use of hemostasis devices, used to stop bleeding from the femoral artery following cardiac catheterization for diagnostic or interventional purposes. Despite the fact that 1996 NCHS data indicate that 63.3% of these procedures are performed on males (1,156 thousand of 1,827 thousand), 51.7% of serious injuries (983 of 1903) and 62.5% of deaths (15 of 24) have been reported in females (p<.0001 and p=.01, respectively). Studies which have assessed the risks of hemostasis device use compared to no device use (i.e., use of manual compression to stop bleeding) have produced mixed results, and we could not find any studies which assessed gender differences in risk. Despite the issuance of a Dear Doctor Letter in 1999, reports of serious injuries and deaths associated with the use of these devices appear to be increasing.

A concept paper that we submitted to the Office of Women's Health to study this issue was one of ten that were accepted for further consideration, with a request to submit a full proposal to them. The proposal that we submitted is an epidemiologic study that will assess the safety of these devices and the gender differential in risk associated with them. This will be accomplished by ascertaining incidence and relative and attributable risks for various serious injuries and death associated with the use of these devices, in women as compared with men.

This presentation looks at estimated rates of voluntary and mandatory medical device adverse event reports involving hemostasis devices received through our Medical Device Reporting (MDR) system, by year of report, type of adverse event, and gender.

Group Award for Leveraging Activities - 2001 FDA Science Forum

Board D09a

Human Illness from Seafood Toxins: Outbreak Investigation
S. Hall¹, P.P. Eilers¹, S.M. Conrad¹, S.M. Musser¹, M. Poli², ¹CFSAN, FDA, Washington DC 20204, ²USAMRIID, Frederick MD 21702

Human consumers can and do become seriously ill when they consume seafood contaminated with natural toxins. While we strive to avoid all human illnesses due to such seafood toxins, those illnesses that occur provide important opportunities to learn and improve our regulatory programs. This poster describes some of our work on outbreaks of paralytic shellfish poisoning (PSP) in Washington state and neurotoxic shellfish poisoning (NSP) in Florida.

Board D10

In reviewing premarket notification (510(k)) submissions for pulse oximeters in non-motion and motion status, several statistical issues have been raised in determining whether a new device is ‘substantially equivalent (SE)' to a legally marketed predicate. For the oximeters with non-motion status, there are fundamental problems regarding the study design, success criterion, and data analysis, that remain to be solved. On the other hand, claims of being resistant to motion artifacts are new, and our review experience with such submissions is limited. Since the statistical hypotheses have not been clearly stated, the examination of distribution assumptions for the summary statistics are often ignored, and the statistical modeling may not be appropriate, there may be some degree of subjectivity in the final SE recommendation. To help resolve the fundamental problems in determining SE, we recommend some relevant statistical hypotheses and the associated power/sample size calculation. We propose to perform appropriate data analyses for repeated calibration measurements based on established methods. The impact of violations in model assumptions for different scenarios will also be explored.

Board D11

Total serum homocysteine (tHcy), is an indicator of folate status and a possible risk factor for vascular disease. The purpose of this cross-sectional study was to examine the association between tHcy, folate intake from food and supplements, and alcohol consumption in the elderly. We studied 278 subjects, 66-94 y, in 1993. Total folate intake from food frequencies was negatively associated with tHcy in linear regression models adjusted for age, sex, creatinine and albumin. We found an interaction between food folate intake and supplement use. Food folate intake had an inverse dose-response relationship with tHcy that was limited to non-users of supplements. For users of supplements containing folate and vit. B-12, predicted tHcy was 1.5 umol/L lower than for non-users, and independent of food folate. We found a positive association with tHcy for alcohol intake >=60 drinks/mo compared with low intake and an interaction of alcohol use with folate intake and supplement use. Compared with alcohol users, non-users had higher predicted tHcy and a diminished inverse dose-response relationship of food folate intake with tHcy. Due to the prevalence of vascular disease in the elderly, identifying modifiable factors related to tHcy is especially important.

Board D12

Clinical trials are often designed with more than one test treatment with the objective to show the superiority over the control treatment of one or both of the test treatments. In a group sequential setting, the test treatment arm may be terminated at any interim look once it is shown to be superior to the control arm. The test treatment may also be dropped with a low conditional probability of rejecting the null hypothesis. In both situations, the unused sample size of the dropped test treatment arm may be redistributed to the remaining arms in order to improve the power of rejecting the null hypothesis. The strategy is proposed to maintain the type I error rate, improve the power and minimize the error rate of dropping an effective arm. The properties of the proposed strategy are studied using a simulation plan.

Board D14

At a minimum, intensities recorded on cDNA arrays require adjustment for non-specific binding and arbitrary scaling before comparisons across arrays. The typical procedure is to subtract an estimate of background intensity within arrays and scale across arrays based upon housekeeping genes. In general, neither adjustment is straightforward to implement. For example, subtracting background estimates can generate negative estimates for the level of gene expression, and these cases preclude the use of variance-stabilizing transformations such as logarithms. Housekeeping genes, incorporated into arrays as internal standards, also can be problematic when used to adjust for the arbitrary scale. They often exhibit within-array differences that contradict assumptions necessary for valid scale adjustments. Graphical diagnostics and alternative adjustment strategies are presented that can be applied to the intensities from a set of arrays whenever the treatments do not affect the level of gene expression for the majority of genes.

After adjustment, remaining differences in intensities between two arrays are frequently evaluated by the fold change. An implicit assumption is that genes with larger fold changes are more likely to reflect true differences in levels of expression than those with smaller changes. That is, the fold change is presumed to order genes by the evidence of a treatment effect. Some problems with this assumption and ways to deal with these problems are discussed. In addition, the fold-change criterion is generalized to comparisons involving several arrays.

rdelongchamp@nctr.fda.gov

Board D15

Usually, in applying market approval of a new drug, more than one clinical trials are conducted to support efficacy or safety claims. No appropriate statistical approach exists that can evaluate evidence from many studies collectively. In this research, we propose a new multiplicity adjustment approach to evaluate several independently conducted studies collectively. This multiplicity approach is flexible in selecting decision rules so that it is possible to find a decision rule which achieves the optimal power for a specified alternatives and a certain decision error. To control the decision error, a concept of overall hypotheses is proposed which are the combinations of individual study's hypotheses. As the number of studies increases, we could have many choices of overall hypotheses. Using this new approach, the maximum type I error can be derived under any overall hypotheses. A couple of examples are presented to illustrate the application of the method in evaluating several independently conducted studies.

Board D16

In alcohol treatment clinical trials, conventional evaluation of efficacy outcomes often focuses on the time-to-first-event, where the event may be "any drinking" or "heavy drinking", in addition to multiple outcomes such as frequency of drinking episodes, percent of abstinence, etc. after treatment. For each subject, when all events can be considered as a body of evidence, there may be a more comprehensive measure of treatment effect on a subject or patient level. In excessive alcohol users/alcoholics, treatment may be considered effective when treatment effect can be distinguished on recurrent events statistically in some manner but not necessarily on time-to-first-drinking. In addition, gap time between events and frequency of drinking episodes are embedded in the pattern of favorable treatment effect. The potential utility of time-to-recurrent-event analysis methods will be explored via a real example for evaluation of alcohol treatments on the basis of multiple episodes.

*The views expressed in this abstract do not necessarily represent those of the U.S. Food and Drug Administration.

Board D17a

Serial dilution experiments to find the concentration of a target microbe may involve a confirmation test. The use ofa confirmation test implies another microbe can be confused with the target microbe. Statistical models can account for this possible confusion.

Board D18

Introduction. FDA approved etanercept for use in rheumatoid arthritis (RA) in 1998; infliximab was approved for Crohn'sdisease in 1998 and RA in 1999. Both agents antagonize the effects of tumor necrosis factor (TNF). Since TNF plays a role in host defenses, therapeutic agents that block TNF might increase risk for serious infections. Methods. FDA's Adverse Event Reporting System (AERS) receives spontaneous reports of suspected side effects from domestic and foreign sources. Most AERS reports are submitted by health care providers and do not necessarily imply that the product caused the event. We evaluated post-licensure reports of infection associated with use of etanercept and infliximab through October 1, 2000. Results: Infections accounted for 2,782 (21%) of 13,000 reports associated with use of etanercept and 226 reports (20%) of 1,100 reports for infliximab. Commonly reported infections included pneumonia, sepsis, cellulitis, septic arthritis, pyelonephritis and osteomyelitis. Lower numbers of reports were seen with etanercept for some for opportunistic infections relative to infliximab. There were 17 cases of tuberculosis (11 foreign, 6 U.S.), 6 cases of Pneumocystis carini pneumonia (PCP), 3 cases of histoplasmosis, and 3 cases of listeriosis with infliximab. This compared to 3 reports of TB, 3 reports of PCP, no report of histoplasmosis and no report of listeriosis for etanercept. Many infliximab TB cases had unusual presentations, including 6 with miliary patterns and 8 with other extra-pulmonary involvement; 2 of these patients died. Conclusion. Substantial proportions of adverse event reports for both products describe infections, consistent with the package inserts, which prominently warn of risks for serious infections. Reports raise concern of risks for tuberculosis and other opportunistic infections with TNF-antagonists. Differences between these products (biologic mechanism of action, dosing schedules, concomitant medications including use of immunosuppressants, precise indications, and geographic distribution) could explain the observed discrepancy in case numbers, such as the larger number of reports of TB with infliximab. Clinicians should be alert to screening, prevention and treatment of TB in patients treated with TNF antagonists. .

While investigations continue, we urge physicians to be alert for and report (MEDWATCH, 800-332-1088) adverse events, especially opportunistic infections and TB.

Board D19

ArrayTrack is in-house software for management and analysis of filter array experiment data through an interactive user-friendly interface. A relational database (ORACLE) is used to store all experiment data, including target sample information (species, treatment protocol, cell lines and etc) and array information (gene ID and position and array image), as well as raw and analyzed expression data. In order to facilitate data mining, three visualization and analysis functions are available for users to explore information in data. The ScatterPlot Viewer displays multiple X-Y plots for pairwise comparison of expression data from different experiments based on the same array. Any point (associated with a particular gene in the array) in the plots can be selected to directly link to a variety of public databases for research, such as GenBank, UniGene, Genome Database, OMIM, LocusLink, GeneCards and SWISS-PROT. The ArrayImage Viewer graphically displays changes in gene expression for a single experiment on a constructed microarray image. Users can select, index and search genes in the image by defined criteria, such as expression fold range, and the identified genes can also be researched by directly linking to the public databases. The BarChart Viewer presents the gene expression behavior for a selected gene in bar chart form across different experiments that may or may not use the same arrays. The system is extensible to other microarray data and is ready for web deployment.

Publish Only (D)

We have previously shown that an active transport of glutamic acid (glu) operates at the choroid plexus, a CSF-blood barrier. There is also high metabolic activity converting glu to non-toxic glutamine (gln) in the miniswine choroid plexus (Kim et al., Brain Res. 709:59-64, 1996). The glu uptake and its metabolism were further investigated with the choroid plexi from the rat brain in vitro. Tissues were incubated with 100 mM glu for 30 min in artificial cerebrospinal fluid (CSF). Analyses of glu and gln were performed by HPLC. Glu uptake was very pronounced after only 30 second. The changing pattern of gln concentration in the choroid plexus tends to follow that of glu. Glu uptake and its metabolism were challenged with ouabain, sodium azide, domoic acid, 3-nitropropionic acid (3-NPA) or lead nitrate during incubation for 10 min. Uptake was significantly inhibited, ranging from 62% to 83% (ANOVA, Dunnett's t, P<0.05, n=10), with the individual test compound. However, the concentration of gln was significantly reduced by 1 mM sodium azide (63%), 100mM 3-NPA (45%), and 10 mM lead nitrate (44%), respectively, (Dunnett's t, P<0.05, n=10). These results indicated that the metabolism of glu to gln, in addition to an active transport system in the choroid plexus, may also serve to regulate glu concentration in CSF. Possible involvement of 3-NPA and lead nitrate in the regulation of cellular metabolism of the glu or other cellular function in the choroid plexus remains to be determined.

Board E01

Brand name retail cuts of beef, chicken and turkey were purchased from supermarkets in the Maryland suburbs of Washington DC to assess the carriage rate and the antibiotic resistant phenotypes of enterococci present on the meat. Random sampling took place over the course of one year (8/99 – 8/00). Samples were aseptically transferred from commercial packaging into sterile plastic bags and rinsed in buffered peptone water. Ten ml of these rinses were stored frozen until cultured for enterococci. One-ml aliquots were transferred to enterococcosel broth and incubated for up to 48 hrs at 45°C. Esculin positive tubes were further cultured for isolation and identification of enterococcal strains to species. All isolates were assayed for susceptibility to 29 antibiotics using Sensititre™(TREK diagnostics). Six of these are drugs used as growth promotants in livestock and poultry. Enterococcus positive rates were 29/43 (chicken), 11/32 (turkey) and, 16/24 (beef). No Enterococcus faecium were isolated from the beef samples. There were obvious differences in the resistance profiles among isolates from the different meat types especially in the case of beta-lactams, aminoglycosides, fluoroquinolones, macrolides, and streptogramins. In general, isolates from beef were far more susceptible to the antibiotics tested than were isolates from the avian species. Results suggest that the resistance profiles of enterococci isolated from retail meat reflect the antibiotic selective effect associated with antibiotic usage in the various production classes.

Board E02a

Two different semi-rigid amorphous copolyester (PET) materials were irradiated at 5, 25, and 50 kGy at ambient temperature with Co⁶⁰ radiator or electron beam accelerator, and subjected to migration experiments using 10% ethanol and 100% n-heptane food simulating solvents, maintained at 40°C for up to 10 days. The HPLC results indicate that no new chemicals are generated by irradiation. A 5 kGy dose did not significantly affect either PET, but at 25 and 50 kGy doses a slightly different effect between gamma and e-beam irradiation on low MW oligomers was observed in the PETs. However, these differences did not significantly affect the migration data. Based on the migration data, an estimated maximum dietary exposure for each migrant was 0.10 µg/kg.

Board E03a

Safe food handling labels on raw meat and poultry products are designed to provide consumers with information on safe food handling practices to reduce foodborne illness. This study uses the FDA 1998 Food Safety Survey to examine the prevalence of consumer awareness of the label four years after it was put on raw meat and poultry packages and consumer characteristics related to the awareness. It is found that two thirds of consumers have seen the label and three quarters of them could remember safe food handling advice on the label. Those who have seen the label tend to perceive higher risk of foodborne illness at home, know a larger number of foodborne pathogens, perceive higher risk in unsafe handling practices, have safer hand and cutting-board washing practices, and be aware of recent foodborne incidents. The findings will be investigated for implications on consumer food safety education and on the use of handling labels on foods regulated by FDA.

Board E03b

The capability of the Alpha-MOS Fox 3000 system to discriminate between samples of acceptable and decomposed canned albacore, yellowfin, and skipjack tuna was determined using packs of fish that had been classified by sensory analysis prior to canning. The MOS (metal oxide sensor) responses were analyzed using Principle Component Analysis (PCA) and/or Discriminate Function Analysis (DFA). There was discrimination between acceptable and decomposed albacore when albacore samples were run as a single group. Discrimination between acceptable and decomposed samples of skipjack, yellowfin, or a mixture of the two species was achieved as a single group. Results will be compared to time/temperature abuse conditions and putrescine & cadaverine levels. This technique has potential for use as a screening technique to discriminate between acceptable and decomposed processed fish when sensory expertise is not available.

Board E04a

Dried pig ear dog treats have been implicated in human salmonellosis cases in Canada. To determine whether similar pet treats available in the U.S. were also contaminated, one-hundred and fifty-eight, randomly sampled, imported dog treats made from dried pig ears and other animal parts, were assayed for Salmonella. Forty-nine percent (n=78) of dog treat samples were positive for Salmonella. Twenty-seven different Salmonella serotypes were recovered including Anatum (n=10), Typhimurium (n=7), and Infantis (n=7). The majority of Salmonella isolates were susceptible to the 17 antimicrobials tested, however, resistance was observed to tetracycline (26 %), streptomycin (23 %), sulfamethoxazole (19 %), and chloramphenicol (8 %). Twenty-eight Salmonella isolates were resistant to at least one antimicrobial, whereas 10 isolates displayed resistance to 4 antimicrobials.Two S. Typhimurium DT104 isolates displayed the characteristic penta-resistance phenotype (ACSSuT). One isolate (S. Brandenburg) was resistant to 8 antimicrobials including ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, cephalothin, gentamicin, and apramycin. Salmonella isolates were further screened for the presence of class 1 integrons via PCR. While the majority of Salmonella isolates assayed did not contain class 1 integrons, three Salmonella isolates displaying resistance patterns of ACSSuT or KACSSuT possessed two chromosomal integrons of 1 and 1.2 kb. In conclusion, this study indicates that animal derived dog treats available in the U.S. are a potential source of animal and human salmonella infections.

Board E04b

Dietary exposure to Vibrio vulnificus occurs through consumption of raw shellfish. Systemic infection with V. vulnificus is fatal in over 60% of cases. Eleven different strains of V. vulnificus with a range of virulence (as determined in the iron-overloaded mouse model) were tested for sensitivity to the soy isoflavone, genistein. Bacteria (approximately 10⁶ cfu/mL initially) were incubated 24 hours at 30°C in APS broth containing genistein (0-600 mM). Survival and growth were monitored continuously by resazurin dye reduction. Lag phase duration (LPD) and maximum growth rate (MGR) were determined by fitting the bacterial growth curves to a Gompertz function. LPD increased and MGR decreased in a concentration-dependent manner for all strains of V. vulnificus with total growth inhibition in all strains at 500 mM.

Board E05

Staphylococcal enterotoxins (SET) are a family of nine major serological types of heat stable enterotoxins. SET a leading cause of gastroenteritis resulting from consumption of contaminated food, are analyzed using toxin specific antibodies. DNA microarray chips are miniature arrays of gene-specific DNA targets immobilized on a chip surface which are used for identification of DNA sequences. The staphylococcal DNA microarray being developed at CFSAN, is a chip containing DNA sequences of SET and other virulence genes. This chip can be used for analysis of SET and staphylococcal virulence factors. The strategy used here is to design universal PCR primers for amplification and labeling of enterotoxin genes with Cy5 fluorescent dye. These labeled probes could be used for hybridization with the staphylococcus gene chip. The chip will include all the enterotoxin genes as well as other Staphylococcal virulence factors. Potential use of this model is to screen other food microbial pathogen.

Board E07

The use of antimicrobials in the animal production environment is coming under increasing scrutiny as contributing to the development of resistance in human bacterial pathogens. Synercid?, a streptogramin antimicrobial, was approved in the United States in 1999 for treatment of vancomycin resistant enterococci. However, another streptogramin, virginiamycin, has been used in veterinary medicine for over two decades. Enterococci resistant to virginiamycin are also cross-resistant to Synercid. A study was conducted to determine the prevalence of streptogramin resistance genes in E. faecium isolated from retail poultry in the greater Washington DC area. To date, 10 chicken and 12 turkey retail samples have been analyzed. Enterococcus faecium was isolated from every retail meat sample. All isolates displayed MIC's > 4 mg/ml to Synercid and/or virginiamycin. These isolates were further analyzed using PCR for the presence of streptogramin associated resistance genes. The vgbA gene was identified in 58.3% (n=7) of turkey and 40% (n=4) of chicken E. faecium isolates. The vatD (satA) and vatE (satG) genes were not detected among any of the isolates. However, degenerate acetyltransferase PCR primers amplified a product of approximately 150 bp for E. faecium isolated from both chickens (30%, n=3) and turkeys (8.3%, n=1). The presence of vatA, vatB, vatC, vgaA, vgaB or vgbB was not detected, however, ermB was detected in 75% (n=9) of turkey and 50% (n=5) of chicken E. faecium isolates. This study suggests that retail poultry are a potential source of known, and yet to be identified, streptogramin resistance genes in E. faecium.

Board E08

The recent approval in the US of irradiation for red meat has led to a renewed interest in the effects of ionizing radiation on plastic packaging materials. At present, little is known on one important aspect of packaging irradiation, i. e., the formation of low-molecular weight (‘volatile') degradation products, and the extent to which these compounds might migrate into food.

A variety of radiolysis products has been identified in different single plastic films and also in a few multilayer materials by several groups using dynamic headspace or thermal desorption (TDS) techniques. However, the available data on the effects of desorption temperature suggest that, in some cases, the products or a fraction of them might be analytical artifacts, i. e., thermal degradation products of some (radiation-induced) precursors.

We are currently refining dissolution-precipitation and other extraction methods and developing GC and HPLC methods to determine the extent to which some characteristic "target products" are genuine. Preliminary results with polyamide-6 (nylon) irradiated at 25 kGy indicate that pentanamide, which was observed in relatively high concentrations (ca 200 mg/kg) in TDS-experiments, might indeed be an analytical artifact. However, many radiolysis products of polystyrene (PS) such as acetophenone, benzaldehyde, or 1-phenylethanol appear to be genuine.

Consistent with earlier TDS-results, the acetophenone level in PS increases 5 to 6-fold after a 25 kGy dose, the concentrations of the various radiolytic compounds appear to be in the low ppm (mg/kg) range, and the chromatograms support the earlier conclusion that it is possible to differentiate analytically between radiation-sterilized and non-irradiated polystyrene ("irradiation detection").

In the case of irradiated plastics, there is clearly a need to confirm the results obtained by headspace and thermal desorption techniques (the methods of choice to identify low-molecular weight components in polymers). However, quantifying trace levels of small organic molecules in polymer matrices by "conventional" techniques is still an analytical challenge. Currently under way are experiments to "upscale" the initial extraction steps, and to extend the approach to other polymers. Ultimately, the aim is a comprehensive analysis of radiolysis products / potential migrants at a level of 100-200 ppb (mg/kg) in the polymers.

Board E09

Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are recognized as a major cause of human gastroenteritis worldwide. Subtyping has been widely used in the characterization of C. jejuni and C. coli isolates from different sources and plays an essential role in epidemiological studies. Genotypic methods, such as pulsed-field gel electrophoresis (PFGE) and ribotyping, are useful tools for subtyping bacterial pathogens. In this study, a total of 115 Campylobacter (71 C. jejuni and 44 C. coli) isolated from 47 poultry meat samples from retail stores in suburban Maryland were analyzed by PFGE with SmaI and ribotyping with PstI. PFGE and ribotyping both gave distinct groups of the organism at the species level. They also were able to differentiate isolates under species. The 71 C. jejuni were grouped into 32 PFGE patterns and 19 RiboGroups, whereas 44 C. coli to 18 PFGE patterns and 7 RiboGroups. A total of 50 PFGE patterns and 26 RiboGroups were identified among the 115 Campylobacter isolates. The combination of RiboGroups and PFGE patterns gave 68 genotypes, of which 54 had multiple PFGE patterns within one RiboGroup, and 31 had multiple RiboGroups within one PFGE pattern. Several identical genotypes were observed in isolates obtained from different supermarket chains at different sampling times. The results showed that PFGE had a higher discriminatory power than ribotyping, and that the two methods were complementary in typing Campylobacter. More than one typing method is helpful, in some cases necessary, to differentiate microbial pathogens in epidemiological investigations.

Board E10

Detection of Staphylococcal Enterotoxins by a Superantigen-Induced Cytotoxicity Assay
Tim Hawryluk FDA NRL, Jamaica NY 11433

Food poisoning due to ingestion of toxins elaborated by certain strains of Staphylococcus aureus is an area of intense regulatory concern to the FDA. The toxins themselves are capable of causing severe gastrointestinal illness at very low concentrations. Presented here is the development of a bioassay that detects staphylococcal enterotoxins A, B, C, and D by exploiting their ability to induce a superantigenic cytotoxic T-cell response. The detection system used is a tissue culture based colorimetric cytotoxicity bioassay that has detected picomolar (10^-12 mol/liter) concentrations of staphylococcal enterotoxin A (SEA) in a food matrix. This method has also detected heat-treated SEA.

Group Award for Leveraging Activities - 2001 FDA Science Forum

Board E11

FDA is responsible for seafood safety. In the case of natural toxins in shellfish, which can be a significant hazard to consumers, the risk has generally been managed by monitoring the shellfish for toxicity. While this is necessary, it has fundamental limits when used alone. We are investigating the use of volunteer observer networks to gather information that the state shellfish programs can use to focus toxicity monitoring on the times, locations, and toxins of greatest concern, thus making the overall program more effective and less expensive. Such networks are currently operating in several states on the Pacific and Atlantic coasts. Through the participation of citizen volunteer observers we are leveraging the limited resources of the FDA and states to assure a higher level of seafood safety than would otherwise be possible.

First Place Poster - 2001 FDA Science Forum

Board E12

Flies were collected at caged-layer poultry houses that had produced eggs that were implicated as the food vehicle in recent outbreaks of Salmonella Enteritidis infections. The flies were grouped into pools that were tested for Salmonella spp. A total of 15 pools of house flies, Musca domestica L. and 7 pools of dump flies, Hydrotaea aenescens (Wiedemann) (Diptera: Muscidae) were tested. Salmonella Enteritidis was isolated from 2 of the 15 pools of house flies. Other isolates included: Salmonella Infantis from one pool of house flies and one pool of dump flies, and Salmonella Heidelberg from one pool of houseflies. These results are a strong indication that house flies and other disease-carrying flies should be considered a potential risk factor in the prevention of outbreaks of Salmonella Enteritidis involving eggs.

Honorable Mention Poster - 2001 FDA Science Forum

Board E13

During the two two-week long sessions at International Summer Language School, from June 21^st to July 4^th, 2000, sixteen fifth- and sixth-graders and from July 6^th to 19^th, 2000, eighteen fifth- and sixth-graders participated in a Food Safety Program at "Borovoe" in Novosibirsk, Russia. This summer school has been operational since 1995. The program is organized by Linguistic Gymnasia #164 and the Department of Education of the city of Zelenogorsk. Students were from the Linguistic Gymnasia and other schools of Zelenogorsk in Russia. Many materials were used to increase their knowledge. At the end of each session, each group gave their own play/presentation along with a Food Safety song. Their play/presentation demonstrated what they had learned from their two-week Food Safety program. Each student in these two groups was awarded a Food Safety certificate along with a Fight Bac! Seal on it for their participation in this program.

Board E14

The effect of chlorine on spoilage aerobic bacteria of broccoli, celery, lettuce, mung bean sprouts, and scallions was investigated. Test portions were treated with aqueous chlorine solutions, with or without sonication, and analyzed for aerobic plate counts (APC). The reduction (R) in APC in log₁₀cfu/g (logR) as compared to controls (APC_C) was used to evaluate the efficiency of chlorine. The mean APC_C ranged from 5.5 to 8 log₁₀ cfu/g. The overall mean logR values were 0.9 and 1.4 at 200 and 2000 ppm chlorine, respectively. There were significant differences (p< 0.05) in logR values of 4 out of 5 vegetables, except broccoli. Overall, the efficiency of chlorine was significantly (p< 0.05) improved by only 0.2 logR with sonication. This suggests the compact nature of biofilms formed on raw produce. However, the limited usefulness of chlorine realized may represent a final but significant step that could be achieved in the home to minimize the risk of possible contamination of ready-to-eat vegetables with microorganisms of public health significance.

Board E15

This study evaluated the survival of Campylobacter jejuni in cilantro, green pepper, and romaine lettuce. Test portions (25 g) were spiked with C. jejuni JH93, packaged under normal air, vacuum, and a modified atmosphere. C. jejuni counts (CjC) and aerobic plate counts (APC) were done after 0, 3, 6, 9,13, and 15 days storage at 4°C. Enrichment recovery of C. jejuni was also performed. The APC increased about 1 log₁₀ cfu/g from the initial counts and remained unchanged throughout the experiments. CjC decreased linearly (r = -0.89) during storage at 4°C, with an overall D value of 3 days. The die-off rate of C. jejuni in modified atmosphere was slightly slower compared to normal air or vacuum. The blood-free enrichment system did not significantly improve the recovery of C. jejuni compared to direct plating procedure. Results indicate that at refrigeration temperatures, C. jejuni can survive long enough in cilantro, green pepper and lettuce to pose a risk for the consumer, especially when such products are packaged under modified atmosphere.

Board E17

Worldwide, Vibrio parahaemolyticus (Vp) is the leading cause of seafood-related gastroenteritis and the expression of a thermostable direct hemolysin (TDH) is important in disease. TDH is cytotoxic to a variety of host cell types. The mechanism of TDH-mediated pathogenesis of Vp is unknown. In this study, the interaction of TDH with cell membranes was investigated using electron spin resonance (ESR) techniques. TDH added to membranes containing 10% cholesterol caused a significant decrease in the mobility of lipids and the collision frequency of these lipids with O₂ compared to membranes without cholesterol suggesting that TDH binds to cholesterol and was inserted through the membrane bilayer leading to changes in membrane function analogous to cell lysis. In summary, these results demonstrate that the insertion of TDH into membranes is cholesterol-mediated and may explain the diversity of cell types that are affected by TDH .

Board E18

Modified Atmosphere Packaging (MAP) extends the shelf life of produce, and becomes an important technology to maintain a high quality product for consumer acceptance. However, extension of product shelf life also invites the potential proliferation of organisms that may threaten consumer health. The impact of MAP on the growth of S. aureus was evaluated on cilantro, lettuce, and green peppers, inoculated with 10³-10⁴ CFU'g, and incubated at three different temperatures (4°, 15°, and 25°C) under normal atmosphere, vacuum, and modified gas atmosphere (2% O₂, 18% CO₂, balance N₂). Samples were analyzed for S. aureus on Baird-Parker agar, and for APC on Plate Count agar. Data showed that S. aureus decreased by 1 log CFU/g in cilantro, and 1-2 log CFU/g in green peppers and lettuce following 9 days incubation. Survival of S. aureus was favored by modified gas atmosphere mwith all produce studied. Conversely, the APC increased by 1-2 log CFU/g, and was favored by air. The sensory quality of the produce remained acceptable at 4°C regardless of storage temperature. The competitive natural flora of these produce prevented the proliferation of S. aureus and subsequent production of enterotoxin.

Board E19

Use of antimicrobials in the animal production environment is coming under increasing scrutiny as contributing to the development of resistance in human bacterial pathogens. Synercid^TM, a streptogramin antimicrobial, was approved in the United States in 1999 for treatment of vancomycin resistant enterococci. Another streptogramin, virginiamycin, has been used in animal production, including poultry, for over two decades. Enterococci resistant to virginiamycin are cross-resistant to Synercid. A study was conducted to determine the prevalence of streptogramin resistance genes in enterococci and coagulase negative staphylococci isolated from poultry litter in 24 different farms across Georgia, USA. One-hundred and ten coagulase negative staphylococci and 90 enterococcal isolates were identified and speciated. The most common enterococcal species identified included, hirae (n=41), and faecium (n=24), whereas sciuri (n=42), lentus (n=23), and xylosus (n=15) were the most common staphylococcal species. A total of 44 enterococci and 28 staphylococci isolates displayed MIC's > 4 µg/ml to Synercid and/or virginiamycin. The satA gene was identified in 84% (n=37) of synercid resistant enterococci, whereas satG was identified in only 1 isolate (E. faecium). Interestingly the satA E. faecium was also positive for satG and ermB. Sixty-eight percent of staphylococci were positive for vgaB (n=19). vatA, vatB, vatC, vgaA, vgbA or vgbB were not detected in any enterococcal or staphylococcal isolates, however, ermB was detected in 68% (n=30) of enterococci. Using sat degenerate primers, amplified PCR products were produced in 84% of enterococci (n=37) and 7.14% (n=2) of staphylococci. This study suggests that enterococci and staphylococci in poultry litter serves as a reservoir for known and yet to be characterized streptogramin resistance genes.

Publish Only (E)

An oil spill usually prompts the immediate closure of the affected fishing waters. An important criterion for reopening is taint-free seafood from those waters. To set chemical criteria for reopening, the constituents of the petrochemical taint must be known. By the use of dynamic headspace gas chromatography/mass spectrometry, we determined over 50 volatile compounds in oyster, lobsters and salmon artificially tainted by tank exposure to crude oil and diesel oil contaminated water. Most of these compounds were aromatic hydrocarbons: alkyl homologues of benzene, naphthalene and tetrahydronaphthalene. The presence of taint was determined by conventional sensory assessment. An electronic nose was able to detect petrochemical taint in seafood at the human sensory threshold (low ppm) level.

Publish Only (E)

Analysis of extracts from shellfish exposed to Gymnodinium breve red tides show that the brevetoxins are quickly accumulated and metabolized by shellfish. The metabolites include diastereomeric mixtures of the C50 cysteine adduct, their sulfoxidation products and a thioglycolic acid adduct. The metabolites are eliminated by shellfish slowly (>60 days) and retain toxicity in mammalian in vitro and in vivo test systems. The metabolites are not recovered by the official NSP diethyl ether extraction protocol. Substituting acetone extraction, in vitro screening and LC/MS has improved the recovery of composite toxicity and achieved ppb level sensitivity for parent brevetoxins and metabolites from shellfish matrices. These methods are currently used in the study of brevetoxin dispositon, metabolism and elimination in shellfish, and can be further refined to monitor shellfish toxicity resulting from G. breve red tide exposure.

Publish Only (E)

Recent outbreaks of Vibrio parahaemolyticus infections in North America were due to consumption of raw oysters. All clinical isolates possessed the tdh gene common to pathogenic strains. Although gene detection in pure cultures by PCR is very efficient, inhibitors present in oysters interfere with the PCR reaction and prevent detection of low numbers of pathogenic V. parahaemolyticus. In this study, the detection limits of different methods (boiling, boiling/Chelex, phenol:chloroform:isoamyl alcohol, CTAB/NaCl, GITC/chloroform, GuSCN/diatomaceous earth, and PERFECTg DNA filters) for extraction of DNA from enrichments of oyster homogenates were compared using PCR. Freshly harvested and retail market oysters (Crassostrea virginica) were homogenized (1:10 oyster:APW), inoculated with 0 to 10³ CFU/g of tdh+ V. parahaemolyticus, and enriched overnight at 35°C. The CTAB/NaCl method was the most efficient for extraction of DNA from fresh oyster enrichments. Detection limits for pathogenic V. parahaemolyticus were 10⁰ to 10¹ CFU/g in initial inocula (10⁴ to 10⁸ CFU/g after enrichment). The same numbers of cells (10⁰to 10¹ CFU/g) were detectable in only 20% of inoculated market oysters, perhaps due to overgrowth of the pathogenic strain by inherent microflora or the presence of other PCR inhibitors.

Publish Only (E)

Analysis of tissue extracts from shellfish exposed in the laboratory to pure brevetoxins (PbTx-2 and -3) indicated rapid accumulation and, for PbTx-2, metabolism. After 72-hr waterborne exposure to PbTx-3, parent PbTx-3 concentrations in whole oyster were ten-fold higher than initial water concentrations. While oysters minimally metabolized PbTx-3, they rapidly metabolized PbTx-2 with a metabolic profile similar to that observed after a Gymnodinium breve red tide event. Parent PbTx-2 was not detectable (recoverable) in exposed oyster tissues. Major metabolites of PbTx-2 found in exposure water and oyster tissues by LC/MS included a cysteine adduct and the reduction product PbTx-3. Metabolites of PbTx-2 were toxic by in vitro assay, whereas only one peak of activity associated with the parent compound occurred in PbTx-3 exposed oysters. Metabolites of PbTx-2 were detectable throughout an 8-week depuration period, and are likely targets in monitoring shellfish toxicity.

Publish Only (E)

Caliciviridae such as Norwalk-like virus (NLV) are major viral etiological agents of shellfish-borne gastrointestinal disease in US. Bivalve molluscan shellfish accumulate microorganisms, including human viruses, present in their growing waters. To detect viruses in oysters, we developed a method that removed PCR inhibitors and concentrated low levels of seeded viruses from oyster tissue. To further evaluate the method, we surveyed oysters that were depurated (2 weeks) and then suspended in racks (2 weeks) in close proximity to a municipal sewage out-fall for NLV genogroups. Multiple sets of PCR primers corresponding to NLV polymerase and capsid genomic regions were utilized and evaluated for their detection patterns of NLV in contaminated oysters. Using Southern transfer/hybridization and nucleotide sequencing of amplicon, we identified NLV G2 in 60% of the relocated oyster samples during a 9-month survey. NLV in oysters was associated with high titers of coliphages in oysters (³ 3 log/100 g) and surrounding water (³ 2 log/100 ml)during cooler months. The accumulation of NLV in shellfish was not associated with bacterial indicators, fecal coliform and E. coli. The methodology developed allows for NLV identification in environmentally contaminated oysters, and provides a tool for the evaluation of current and alternative indicators of NLV in shellfish and shellfish growing water.

Board F03

Unmethylated CpG motifs present in bacterial DNA stimulate a rapid innate immune response and condition the adaptive immune response. Bacterial DNA or synthetic oligonucleotides (ODN) containing CpG motifs induce lymphocyte proliferation and the secretion of cytokines and chemokines. Mice treated with CpG DNA become resistant to subsequent infection by a variety of bacterial, viral and parasitic pathogens. Further, CpG ODN act as strong immune adjuvants, improving the immunogenicity of soluble proteins and DNA vaccines.

Transition to clinical use required the identification of motifs that are optimally stimulatory in humans. By synthesizing and analyzing hundreds of novel ODN and testing them on PBMC from a large number of donors (N=30), we identified two structurally and functionally distinct "clusters" of CpG ODN that activate human PBMC in vitro. The "K" cluster consists of short (12 bases) phosphorothioate ODN containing a TCGTT or TCGTA stimulatory motif. These ODN stimulated a 20 fold increase in cell proliferation (p< 0.0001), and IL-6 production (p<0.01) and a 100 fold increase in the production of IgM (p<0.0001). "K" ODN act primarily on B cells and monocytes/dendritic cells.

In contrast, "D" ODN are longer (18 bases), phosphorodiester, contain a palindromic PuPy CpG PuPy stimulatory motif and a poly G tail. These ODN primarily stimulated NK cells to secrete IFNgamma (p<0.05), induced lower levels of IL-6 secretion and no IgM. Both types of ODN induce the up-regulation of the expression of MHC class II and various co-stimulatory molecules (including CD40, CD80, and CD86) indicating that they induce dendritic cell maturation in vitro.

Studies conducted using Rhesus monkeys (which respond to the same CpG ODN clusters as human PBMC) indicate that CpG ODN magnify the in vivo response to protein antigens and provide protection against infectious agents. Indeed administration of CpG DNA together with ovalbumin significantly increased the titers of antigen-specific antibodies. In addition, administration of CpG ODN prevented the development of cutaneous lesions when monkeys were challenged by Leishmania amazonensis. These findings support the development of CpG ODN as vaccine adjuvants and immunoprotective agents, and suggest that selective use of "K" vs "D" ODN may facilitate the induction of immune responses designed to protect against specific infectious agents.

Board F08

Synthetic overlapping peptides derived from the F protein amino acid sequence were used to identify a linear heparin binding domain (HBD) within the F protein. Three peptides, (F15, F16 and F78), each representing various overlaps of a linear HBD within the F protein specifically bound to heparin agarose. In addition all 3 peptides bound to Vero and A549 cells, and this binding was inhibited by soluble low molecular weight heparin, bovine lung heparin and purified RSV A2. Only one of the three peptides, peptide F16, (K₁₃₁ ® F₁₄₀), showed modest inhibition of RSV infectivity. Taken together, these data suggest that a linear HBD is located within the F2 portion of RSV-F and that this domain plays a functionally significant role in RSV infectivity.

Board G01

LLAR is housed in MODII, a complex of offices, laboratories, animal facilities, and pastures. The CVM Office of Research provides the laboratory with management and veterinary support. LLAR contains an interventional radiology lab and surgery suites, in vitro physiology lab, wet lab, necropsy room, pre- and post-operative animal holding, darkroom, and steam and gas sterilization. We are establishing animal models of human disease and investigate the safety and effectiveness of diagnostic and therapeutic devices. The Laboratory program includes (i) applied research in support of medical and hybrid device regulatory issues, (ii) education through Staff College-sponsored courses and a center-sponsored Workshop Series and (iii) pre-clinical review support of animal protocols and data for medical and hybrid device 510k, IDE and PMA applications.

Board G02

Our laboratory is evaluating safety and effectiveness (S&E) issues associated with percutaneous coronary balloon angioplasty. The study is designed to (i) define the effects of gender and hormone replacement therapy on the response of the cardiovascular system to interventional devices regulated by the FDA and investigated in human clinical trials and (ii) develop an animal model to adequately predict the effects of new cardiovascular devices on the health of men and women. This work provides the basis for assessing S&E differences between genders in the vascular responses to interventional devices and is directly addressing the mechanisms by which ovarian hormones influence cardiovasular disease. Angiographic, morphometric and hemodynamic data will be presented.

Board H01a

Various types of nuts are used by the food industry. Processing destroys the botanical characteristics used to identify them. Accidental cross contamination of nut meats in food manufacturing plants has resulted in severe allergenic reactions and even deaths when the products were consumed. Extracting the water-soluble proteins from isolated nut meats and analyzing them by SDS-polyacrylamide gel electrophoresis, a distinctive banding pattern or "fingerprint" is obtained. Using this method it is possible to identify nut meat fragments as small as 5 mg. The method is able to distinguish very closely related nuts from English walnuts, black walnuts, and pecans. The identity of an unknown nut meat fragment can be determined in less than 2 hours.

Board H01b

Many medical devices are designated by the original equipment manufacturer to be for single use only. In the interest of cost containment, many healthcare institutions are reprocessing these devices in their own facility or contracting with third party reprocessors. Some reprocessors will add an oil based lubricant to the reprocessed device to aid in insertion or slipperiness. The purpose of this study was to determine if the addition of the oil based lubricant would make cleaning more difficult. The lubricants used in the study were glycerol and various oils: cotton seed, mineral, and silicone. Studies with 96 UV well plates and a plate reader indicated that the lubricants did not affect the binding or removal of proteins. Removal of the oil could be assessed using a wavelength of 215nm and required additional steps beyond that to remove protein. Cottonseed oil was the most difficult to remove. Procedures using 0.5M NaOH and an ultrasonic cleaner were needed to remove the oil.

Board H02

Several high pressure liquid chromatography (HPLC) methods for the analysis of vitamin A in foods and feeds have been previously reported but these have not been validated in non-food matrices. A validated HPLC method is needed for the determination of vitamin A and b-carotene in the various matrices presented by dietary supplements. The performance of a reverse-phase method with methanol-isopropanol gradient elution was evaluated with standard retinyl derivatives and b-carotene. The reverse-phase method is capable of separating retinol from other retinyl derivatives and b-carotene. Two types of extraction were employed to extract the analytes from the dietary supplements: 1) a hexane-methylene chloride extraction for soft gel capsules containing b-carotene supplement in soft-gel capsule indicated 92% of total b-carotene was in the all-trans form. Most of the vitamin A and b-carotene in the products examined (n=4) was also in the all-trans form. In conclusion, the reverse-phase method is suitable for simultaneous determination of retinyl derivatives and b-carotene in dietary supplements.

Board H03a

The Food Quality protection Act establishes more stringent standards for pesticides in foods and relies on analytical results from regulatory agencies to provide data on the presence of trace levels of pesticide residues in foods for risk assessment purposes. These trace level analyses will require better sample cleanup procedures than are in common use currently. This poster will describe the results of a study in which sample extracts will be subjected to cleanup with various solid phase extraction (SPE) cartridges. SPE cleanups using graphitized carbon black, C-18, strong anion exchange, aminopropyl and primary secondary amine SPE cartridges will be evaluated using liquid chromatography with UV and fluorescence detection. Further, the development of a regulatory confirmatory procedure employing liquid chromatography/mass spectrometry will be described.

Board H03b

High performance capillary electrophoresis and HPLC were evaluated as techniques for regulatory analysis of Nystatin in bulk drug substances and finished dosage forms. A capillary electrophoresis method has been developed which has demonstrated to be rugged with reproducible results from one column to another. Because Nystatin is rapidly decomposed in the presence of methanol, used as a modifier, a preservative (BHT) is added to the solution of analyte dissolved in dimethylsulfoxide, to retard the decomposition. To increase the precision of the assay an internal standard, Novobiocin, is added to the methanol modifier, improving the precision to 0.86%RSD and 0.88%RSD on two separate columns. The column used is a fused silica hollow capillary tube 72 cm. in length, 50 cm. to detector and with a 50m internal diameter. Nystatin is an all-trans-tetraene system with an amino sugar, mycosamine, attached which can easily be complexed with a borate buffer.

Board H05

There is currently no official method for the analysis of fatty acids in infant formulas. AOAC Official Method 996.01 for Fat Analysis in Cereal Products was extended to the analysis of milk based infant formula Standard Reference Material (SRM) 1846 to determine its applicability to analyze infant formulas. Samples were hydrolyzed with 8N HCl and extracted with ethyl and petroleum ethers. Fatty acid methyl esters (FAMEs) were analyzed by gas chromatography. Amounts of individual fatty acids were calculated from peak areas. Total fat was calculated as sum of individual fatty acids expressed as triglycerides. Measured values for individual fatty acids were highly reproducible. The coefficients of variation (SD/Mean x 100) were <5% for the major fatty acids indicating good precision. Mean measured values for fatty acids were +1 SD of the certificate values. The measured value for total fat as triglycerides (Mean + SD) 26.27+ 0.25% was in agreement with the certificate value for SRM 1846(27.1+ 0.59%). After analysis, 12 different powdered and 13 different liquid infant formulas were measured by the same method. In powdered formulas, saturated fatty acid (SFA) content was 41.05 + 4.0%, monounsaturated fatty acid (MUFA) content was 36.97 + 3.4 %, polyunsaturated fatty acid (PUFA) content was 20.17 + 3.1 % and total trans fatty acid content was 1.30 + 1.3%. In liquid formulas, SFA content was 42.29 + 3.0%, MUFA content was 36.05 + 2.5%, PUFA content was 20.65 + 2.4% and total trans fatty acid content was 0.88 + 0.54%. In both powdered and liquid formulas, fat and linoleic acid contents fell within specifications in CFR (1997) 21 § 107.100a (i.e., minimum linoleic acid content, 300 mg/100kcal and fat content, 3.3 - 6.0 g/100kcal).

Board H06

This collection of related procedures determines the amount of oxytetracycline (OTC) present in trout tissue (muscle with skin attached), biofilter sand, sediment, and tank water from a recycle aquaculture tank. OTC is extracted from the matrices using different techniques, depending on complexity of the matrix and desired OTC detection level in that matrix. Listed in order of complexity, OTC is extracted from tank water by dilution with acidic buffer containing ethylenediaminetetraacetic acid (EDTA); from biofilter sand by shaking with 0.1N HCl; from sediment by homogenization and shaking with buffer/EDTA; from ground trout by homogenization and shaking with buffer/EDTA (twice), with further cleanup and concentration of the extract on a polymeric solid-phase extraction (SPE) cartridge. The four procedures all use the same reversed-phase gradient chromatography on a polymeric column with UV detection at 350 nm. The lower limit of detection (estimated) and upper limit of validation for each of these four matrices are 0.04 to 4.0 µg/g (ppm) (trout), 0.03 to 20 ppm (biofilter sand), 1 to 6,000 ppm (sediment), and 0.003 to 10 ppm (water). Recoveries range from 82% to 108%, with RSD <20% over the applicable concentration ranges. These procedures were used to monitor OTC residues derived from medicated feed use in a recirculating aquaculture system.

Board H07

During our method development for Staphylococcus aureus enterotoxins we found it difficult to obtain stable electrospray conditions using the standard instrument configurations. Replacement of stainless steel needles with fused silica capillaries to prevent the binding of proteins onto the stainless steel well known and some instrument manufacturers provide this option with their electrospray interfaces. Applying high voltage to the solution becomes problematic and is usually accomplished at the tip of the capillary in some way such as plating a conductive metal onto the fs tip. Obtaining a stable spray was difficult, inconsistent and dependent on many factors, such as, solvent composition, relative distances of the various hardware components and nebulizer gas flow rate. It was recently reported that remote location of the high voltage attachment (Fales modification) during infusion experiments resulted in improved ion detection. The Fales modification involved using a fused silica capillary for the electrospray needle and connecting the HV lead to a position upstream from the tip, such as the syringe needle of a syringe pump, which essentially applied the potential directly to the mobile phase. ¹ Our application of this method was to insert a capillary LC column and attach the HV to the column end fitting. This investigation reports the results of the changes that were necessary to stabilize the spray at low flow rates when using capillary LC/ES/MS.

Board H08a

Water miscible solvents such as acetone and acetonitrile will effectively extract both polar and nonpolar pesticide residues from nonfatty foods. The addition of sodium chloride to the resulting acetonitrile-water or acetone-water extracts (salting out) will result in the separation of the water from the organic solvent. Since the organic solvent may still contain some residual water, which would interfere with the later gas chromatographic determination, drying agents, such as sodium sulfate or magnesium sulfate are used to remove the water from the organic extracts. In the present study, we used NMR to study the composition of the phases resulting from salting out and the relative effectiveness of sodium sulfate vs. magnesium sulfate as drying agents. The study showed that considerable amounts of water remained in the organic phase after phase separation. Sodium sulfate was a relatively ineffective drying agent, removing little or no residual water from the organic solvent. Magnesium sulfate proved to be a much more effective drying agent.

Board H08b

The Food Quality Protection Act establishes more stringent standards for pesticides in foods and relies on analytical results from regulatory agencies to provide data on the presence of trace levels of pesticide residues in foods for risk assessment purposes. These trace level analyses will require better sample cleanup procedures than are in common use currently. Sample extracts of apples, plums, green peppers and collard greens, obtained using the FDA (acetone extraction) and Canadian Pest Management Regulatory Agency (acetonitrile extraction) methods for pesticides were subjected to cleanup with solid phase extraction (SPE) cartridges. Graphitized carbon black (carbon), C-18, strong anion exchange (SAX), aminopropyl (amino) and primary secondary amine (PSA) SPE cartridges were evaluated. The relative sample cleanup provided by these SPE columns was evaluated using gas chromatography with electron capture, flame photometric and mass spectrometric detection. The amino and PSA cartridges were found to provide the most effective cleanup, removing the greatest number of sample matrix interferences. The carbon cartridges removed most of the visible plant pigment in the extracts, but did little to eliminate matrix interferences "seen" by the detectors. Likewise, the C-18 and SAX cartridges removed some plant pigment along with minor amounts of sample matrix coextractants.

Board H09

Nineteen lots of hydrochlorothiazide drug substance from five different manufacturers, and four different countries of origin (USA, Italy, Hungary, and Croatia), were analyzed for the presence of impurities. A gradient elution chromatographic system, using acetonitrile/water as the mobile phase, was developed, which separated two known impurities of hydrochlorothiazide, 4-amino-6-chloro-1,3-benzenedisulfonamide and chlorothiazide, as well as a late-eluting, unknown, recurring impurity. The late-eluting, unknown impurity was isolated by preparative liquid chromatography followed by preparative thin-layer chromatography. It was characterized by Electrospray Ionization LC/MS as a 2:1 hydrochlorothiazide-formaldehyde adduct of the parent drug substance. The dimer is believed to form through the double condensation reaction of hydrochlorothiazide with excess formaldehyde during the parent compound's synthesis. The concentration of this impurity ranged from 0.02% to 1.1% (area %), and was above the 0.1% USP impurity threshold in 16 of the 19 lots examined.

Board H11

Amphotericin B (AmB) is one of the most potent antifungal agents and the drug of choice in treatment of serious fungal infections. An HPLC method was developed to determine AmB in bulk, and formulations, for injection, tissue culture, cream and lotion. This HPLC method applied a C18 reversed phase column, 3.9 x 300.mm, with a mobile phase consisting of acetonitrile, water, and acetic acid, (40: 54: 6 v/v), flow rate, 1.8 mL/min, detected with wavelength at 405 nm. The comparison made between results of HPLC determination and that of microbial assay showed good agreement in corresponding dosages. This HPLC method enables FDA personnel to perform drug assay in a more selective, sensitive and time efficient manner than the current outdated time consuming microbial assay.

Board H12

The recent FDA Biopharmaceutics Classification System (BCS) guidance allows for the use of an acid-base titration method, providing justification is given to support this method to predict equilibrium solubility of the test drug. To evaluate an acid-base titration method compared to the traditional shake-flask method, 30 drugs were selected from the World Health Organization List of Essential Drugs and solubility-pH profiles were determined. The acid-base titration method relies on a characteristic shift in the mid-buffer region of the titration curve when precipitate is present. The shake-flask method consists of 24 hr shaking of saturated solutions, filtration and assaying by UV detection. The drug compounds consisted of acids, bases and ampholytes, with pK_a values ranging from 2.0 to 12.0 and log P values ranging from -1.0 to 5.5. Intrinsic solubility values ranging from 0.5 to 20,000 mg/ml were determined by running acid-base titrations over the pH range 1.7 to 12.2. The acid-base titration method proves to be comparable to the shake-flask method for determining solubility-pH profiles and intrinsic solubility values for test drugs.

Board H13

Inorganic forms of arsenic have been linked to an increased risk of cancer, while some organic forms of arsenic are considerably less toxic or even nontoxic. By far seafood accounts for most of the arsenic found in the average diet. The major form of arsenic found in fish and shellfish is arsenobetaine, a nontoxic species. In edible seaweed, arsenic-containing ribofuranosides (arsenosugars) have been identified and often account for the majority of the arsenic found. However, there have been reports of some seaweed samples containing high levels µg/g) of inorganic arsenic. Additionally, recent studies have demonstrated that dimethylarsinic acid (DMA) is the primary arsenosugar metabolite found in the urine after ingestion. DMA is thought to be a cancer promoter. In this presentation, the determination of both total arsenic and speciated arsenic in several seaweed samples will be described. Total arsenic is measured in the samples by ICP-AES after microwave digestion. The identification and determination of individual arsenosugar species is accomplished through the use of HPLC-ICP-MS and HPLC-MS using electrospray ionization.

Board H14

D&C Red No. 21 (R21, Colour Index 45380:2) and D&C Red No. 22 (R22, CI 45380) are U.S.-certified color additives listed for use in drugs and cosmetics. R21 and R22 consist of a mixture of mainly 2',4',5',7'-tetrabromofluorescein (Br₄F) and its disodium salt, respectively, accompanied by smaller amounts of subsidiary colors (e.g., lower halogenated subsidiary colors, the ethyl ester of Br₄F and fluorescein). R21 and R22 are batch-certified by the FDA to ensure compliance with specifications required by the Code of Federal Regulations. Currently the main component and the subsidiary colors of R21 and R22 are analyzed by a multi-step procedure that includes use of a 25x25 cm semipreparative TLC plate for each sample, followed by spectrophotometric quantification. For the analysis of the ethyl ester, liquid-liquid chromatography is presently used with celite as support followed by spectrophotometric quantification. While these indirect methods are effective and reproducible, they are tedious, time-consuming and produce appreciable quantities of solvent waste. The present study reports the development of a simple, rapid and reliable videodensitometric method that permits quantification of all the subsidiary colors (specified for R21 or R22) of multiple samples on the same 10x10 cm TLC plate. The method is applicable for use in routine batch-certification analyses.

Board H16

Chiral recognition was achieved using fast diastereomeric interactions with a diamagnetic chiral solvating agent and a paramagnetic chiral lanthanide shift chelate. Each chiral probe was able to translate the spatial environment of tramadol nuclei into different magnetic environments measurable by both high- and low-field NMR spectrometers. The required resolution of enantiomeric spectral lines was obtained by optimizing the experimental conditions in terms of populations of solutes and chiral probe and temperature. Two ¹H NMR methodologies for the direct determination of enantiomeric purity of the analgesic chiral drug tramadol hydrochloride were developed. The tramadol methoxy protons provided remarkable enantiomeric resolution in both approaches. Chiral lanthanide shift chelate induced much larger nonequivalence magnitudes, an important feature when using low-field NMR Enantiomeric purities were determined on the basis of the relative intensities of the corresponding enantiomeric proton resonance; the assignment of enantiomeric configurations was based on the relative field position of these resonances. Based on the analysis of synthetic enantiomeric mixtures using the two approaches the results agreed with the known values of each enantiomer present in the mixture samples. The optically pure enantiomers were used to establish the minimum sensitivity of the ¹H NMR spectroscopic chiral analysis.

Board H17

Determinations of chiral impurities of two anesthetics were developed utilizing a 400 MHz ¹H NMR spectroscopy. Chiral selectivity was obtained by using a chiral solvating agent that has two important characteristics: (a) the presence of functional groups that are complimentary to those of the chiral drug for significant interaction to occur; and (b) it has a group of high diamagnetic anisotropy near its stereogenic center for translating spatial environment of chiral drug nuclei into different magnetic environments measurable by NMR spectroscopy. Two sets of resolved enantiomeric resonance signals suitable for quantitative usage were obtained by optimization of experimental conditions in terms of populations of solutes and chiral solvating agent and temperature. Enantiomeric purity was determined on the basis of the relative intensities of the corresponding enantiomeric proton resonance; the assignment of enantiomeric configuration was based on the relative field position. Results of the analysis of synthetic enantiomeric mixtures by the proposed method demonstrated an excellent agreement with the known values of the enantiomers present.

Board H18

The ICH Q6A guidance has suggested that it is technically difficult to measure solid state forms in the drug product. Diffuse reflectance near infrared (NIR) spectroscopy has been evaluated as a method for quantifying the anhydrous and monohydrate forms of the active ingredient in a finished dosage form. Nitrofurantoin was selected for this study because commercial dosage forms contain anhydrous (A) nitrofurantoin alone or as a mixture with the monohydrate (M). The state of hydration affects product performance. XRD, DSC and TGA were used to characterize A and M and validate the NIR results. The NIR spectrum of A was different from that of M. A was characterized by a strong R-NH band at 2150 nm. M was characterized by OH bands of water at 1420 nm and 1920 nm. Physical mixtures of M (0.5% - 80% w/w ) were prepared either with A or with commercial products. Linear relationships were observed between the intensity of the 1420 nm peak of M and its concentration. The limit of detection of M was 1% in mixtures of M and A and 2% in mixtures of M and commercial products. NIR spectroscopy enabled the detection and quantification of anhydrous and monohydrate forms of nitrofurantoin in formulations. This may serve as a method to monitor drug product stability based on changes in the state of hydration.

Board H19

A liquid chromatographic method with fluorescence detection is described for determining residues of the pesticide azamethiphos in salmon tissue. The sample is extracted with ethyl acetate, centrifuged, dehydrated, evaporated, reconstituted in water and defatted with hexane; aqueous phase is passed through a C₁₈ Solid Phase Extraction column. The column is eluted with methanol, the eluate evaporated to dryness, and taken up in 10% acetonitrile in water. The LC/FLD system employs a C₁₈ column, ACN:H₂O (32:68) mobile phase and 230 nm excitation, 345 nm emission wavelengths. Composited salmon tissues were fortified with AZA at 5, 10, 21, 42 and 83 ng/g, or ppb (the target level = 10 ng/g). Overall recoveries were 86%, with between day variability of 5.3%. The method detection limit was calculated as 1.2 ppb AZA. The LOQ as determined empirically by this method is the lower limit of the standard curve, approximately 5 ppb.

Board H20

A rapid qualitative method for the screening and confirmation of flunixin meglumine (FX) and phenylbutazone (PB) residues in kidney tissue is described. Bovine kidneys were swabbed in federally inspected slaughterhouses and the swabs were shipped to the Denver District FDA Laboratory for screening. The sample swabs were extracted in phosphate buffer and analyzed using Neogen® Corporation's Flunixin and Phenylbutazone ELISA (enzyme-linked immuno-sorbent assay) kits. The kits were sensitive in the low part-per-billion range (³ 32ppb FX and 106 ppb PB) in kidney tissue. Confirmation of the presumptive-positive swab extracts was performed on a single quadrupole mass spectrometer coupled to a liquid chromatograph with an electrospray interface. In addition, a LC/MS method was developed to confirm PB residues in a more representative bovine kidney tissue sample. Using this procedure, PB could be confirmed in 5 g of kidney tissue at 50 ppb. In an initial survey, approximately 16% (48/295) of the kidney swabs received for screening by ELISA were found presumptive-positive for either FX or PB or both drugs. Additional analyses of kidney swabs and tissue collected from slaughterhouses located throughout the country are currently underway. A description and evaluation of the methodology used, as well as results from these surveys will be presented.

Board H21

A chemical reaction initiated by light (photolysis), commonly known as a photochemical reaction (PR), is a variation of on-line, pre- or post-column derivatization. Certain advantages to the PR are: on-line continuous reaction, reagent-less real-time derivatization, easily reproducible reaction, requires no post-column dilution of sample or additional pumps, less hazardous reagents are required, no secondary cooling are necessary, a large number of reactions are possible and it is compatible with most LC detector types. We have applied PR to alter the detectability of various veterinary drugs. The use of a PR post-column reaction for the fluorimetric detection of several anthelmintics of the avermectins (AVR) family, the electrochemical detection of chloramphenicol (CAP) and meta-CAP, the conductivity detection of several "phenicols" and the detection of other veterinary drugs are presented.

Board H23

The development of an in vitro dissolution test for a bolus presents several challenges: selection or design of an appropriate dissolution apparatus and identification or formulation of a dissolution medium capable of providing sink conditions and sufficient buffer capacity. Potentially discriminating in vitro dissolution testing of veterinary boluses containing sulfa drugs with dosages up to 5 g can be accomplished using USP Apparatus II with conventional volumes and stirring rates in an aqueous medium specially designed to provide and maintain sink conditions. The design of an appropriate buffer system to be used as the dissolution medium for weakly acidic or weakly basic drugs can be accomplished by using standard theoretical relationships fitted to real solubility and buffer data.

Publish Only (H)

Carbon (graphitized carbon black) and amino (aminopropyl, primary secondary amine, or trimethyl amino) solid phase extraction (SPE) cartridges are widely used for the isolation of pesticide residues from fresh fruits and vegetables. The surfaces of both the carbon and amino sorbents will interact strongly with acidic plant pigments to the extent that strong organic solvents such as acetone and acetonitrile are incapable of desorbing them. When organic solvent extracts of food samples are eluted through tandem carbon-amino SPE cartridges, the pigmented sample matrix coextractants will be retained on the SPE cartridges while the pesticides will be eluted through the SPE cartridges. Most published methods use an acetonitrile/toluene (3/1, v/v) elution solvent mixture, since nonpolar aromatic pesticides which are strongly adsorbed on the graphitic carbon surface can be desorbed using solvent mixtures containing toluene. Unfortunately, fairly large volumes (25-30 mL) of elution solvent are required. We have found acetone-toluene (3/1, v/v) to be a much more efficient elution solvent. Ten pesticides that had been identified in the literature as being strongly retained on either carbon or amino SPE cartridges were studied. Nine of the pesticides studied could be quantitatively recovered with less than seven mL of acetone/toluene. While the pesticide hexachlorobenzene could not be quantitatively recovered with 25-mL of either solvent mixture, recoveries were much higher with the acetone/toluene elution solvent.

Publish Only (H)

A multiresidue solid phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of nonpolar organochlorine and polar organophosphorus pesticide residues in milk has been developed. The method uses an acetonitrile extraction followed by an SPE cleanup using C-18 and aminopropyl SPE cartridges. Organophosphorus pesticides are determined by gas chromatography with flame photometric detection. After further cleanup of the extract with Florisil SPE cartridges, organochlorine pesticides are determined by gas chromatography with electron capture detection. Recoveries ranging from 60-120% and limits of quantitation of £1.0 ppb were obtained for the pesticides chlorpyriphos, p,p'-DDE, dimethoate, lindane, methamidophos and malathion.

Board K01

Damage to endothelial cells, which line the interior of blood vessels, is involved in re-blockage of cardiac arteries opened by balloon angioplasty and/or stent placement. Complement may play a role in this process, through activation by catheter and stent materials, or by damaged cells or exposed tissue matrix. Inappropriate activation of blood complement by either the antibody-dependent "classical" pathway or the antibody-independent "alternative" pathway can directly damage cells and/or attract/activate white blood cells which are known to participate in restenosis. We have developed flow cytometric techniques using the calcium-sensitive dye Fluo-4 plus propidium iodide that quantify cell lysis of porcine and human endothelial cells exposed to complement-competent or complement-inactivated porcine or human serum.

Board K02a

A major problem with cornstarch as a donning powder is adsorption of NRL proteins resulting in the aerosolization of allergen during glove use. The propensity of NRL protein binding to donning powder and its extractability has not been evaluated quantitatively. The ASTM ELISA (enzyme-linked immuosorbant assay) for antigenic NRL proteins was used to estimate the amount of antigenic latex associated with glove powder. A series of fortification studies was conducted using virgin glove powder and proteins from ammoniated latex to evaluate recovery and extraction efficiency. The overall recovery of ammoniated latex protein from glove powder was on the average above 75%. The residual amount of protein "associated" with the glove powder (following overnight incubation of the powder and the NRL protein)) was less than 25%, but the remaining amount of NRL protein "bound" to the glove powder (less than 1%) was not amenable to extractable using aqueous solutions. The measurement of glove extracts with and without donning powder indicates that some amount of residual protein is still associated with the glove powder of some gloves, even after extraction. The results indicate that the ELSIA may be a good method to quantitate the antigenic protein on donning powder.

Board K02b

This research investigated the effectiveness of low power laser irradiation (LPLI) with either a 632.8nm or 810nm laser in promoting regeneration of acutely transected corticospinal tract (CST) axons. Laser treatment was applied for 14 days, beginning immediately after CST lesion at vertebral level T9. At five weeks post-lesion, a fluorescent, anterograde tracer was injected into the motor cortex to visualize CST axons. Caudal to the lesion, non-irradiated control rats had significantly fewer axons (p<0.001) which regrew a significantly shorter distance (p<0.001) than rats treated with the 632.8nm or 810nm lasers. The 632.8nm laser treatment group had an average of 48.3 axons which regrew an average 9 mm past the lesion, while the 810nm laser treatment group had a significantly greater number of axons (138.7, p<0.05) which regrew 13.7 mm caudal to the lesion. Immunohistochemistry was used to show an increase in the number of cells labeling positively for ED1 (a marker for macrophages and activated microglia) and RP3 (a marker for neutrophils) in tissue treated with the 810nm laser vs. both the 632.8nm laser and non-irradiated tissue. Therefore, we found that LPLI with an 810nm laser was most effective in promoting axonal regrowth and promoting inflammatory cell invasion following CST lesion. This adds support to the possible use of LPLI as a non-invasive therapy for acute spinal cord injury.

Board K03

In situ polymers are used by mixing two or more compounds which are then placed immediately and directly in tissues to form a unique product. These reactions can generate heat, reactive oxygen species, free radicals, and other by-products of unknown toxicities; but the final polymer is biocompatible. Guidelines written by standards organizations test polymers in a final form prior to use as a medical device. To better estimate the cytotoxicity of these in situ polymers, various means of introducing the reacting material to cells in culture were developed. Coating the material on a sterile glass cover slip then adding the cover slip to the in vitro test system immediately provided reasonable cytotoxicity data that reflected actual use conditions. For in situ polymeric devices which are more viscous, such as dental materials and bone cements, a mold was used which was placed directly into cell culture. Details of the methods developed to test in situ polymers are presented with the goal of obtaining in vitro toxicity data that reflects the actual use of the material.

Board K05

Drug photocarcinogenicity is the ability of a drug to enhance the carcinogenic effect of UV light. Tg.AC mice have been shown to develop large numbers of papillomas within 26 weeks in response to dermal application to carcinogens. This propensity to develop skin tumors in response to dermal carcinogen exposure may make Tg.AC mice an ideal alternative short-term model for photocarcinogenicity testing. Since UV light alone is a carcinogen, our first objective was to determine the threshold levels of solar simulated light (SSL) that are tumorigenic to Tg.AC mice. These results would then guide the dose of SSL to be used in future Tg.AC photocarcinogenesis studies. The acute minimal erythemal dose (MED) was determined in both Tg.AC mice and FVB/N mice (the parental strain of Tg.AC mice). Groups of 7 male and female Tg.AC mice were then exposed to a fraction of the MED of SSL (mice received 0, 0.1, 0.2, 0.3, 0.4, or 0.6 MED of SSL). Groups of 7 male and female FVB/N mice were exposed to 0, 0.1, 0.3, or 0.4 MED of SSL. Mice were exposed to SSL 5 days per week for 26 weeks, then observed for an additional 12 weeks with no SSL exposure. No significant papilloma incidence was observed with any group after 26 weeks of treatment with SSL or after 12 additional weeks of observation. However, most of the Tg.AC mice receiving the highest doses of SSL developed large nonpapilloma skin tumors between week 26 and 38. The absence of papillomas in all SSL treatment groups indicates that "background" incidence to week 26 will be low. The nonpapilloma tumorigenic response seen after 26 weeks in high SSL dose groups is encouraging of further studies evaluating the Tg.AC mouse response to photocarcinogens.

Board K06

Particles from orthopedic implants have been shown to induce inflammatory responses in the host. Ti6Al4V particles were tested alone and with LPS in RAW 264.7 cells to determine their effects on the induction of TNF-a, IL-1b, IL-6 and NO.TNF-a was measured using the L929 cell cytotoxicity assay. NO was measured with the Griess reagent. IL-1b and IL-6 were measured using immunoassay kits. LPS alone significantly stimulatesTNF-a. Ti6Al4V alone stimulates TNF-a slightly above controls. Ti6Al4V particles and LPS produced an additive effect. IL-1bwas not stimulated by LPS, Ti6Al4V or a combination of Ti6Al4V and LPS. LPS alone induced IL-6, while Ti6Al4V had no effect. LPS alone induced NO while Ti6Al4V did not. Increasing concentrations of Ti6Al4V with LPS decreased the NO response by LPS alone. Thus our results show that Ti6Al4V is not very inflammatory in terms ofTNF-a, IL-6, IL-1b and NO.

Board K07

We have studied the ability of some beta-adrenergic antagonists, analgesics and quinolone antibiotics to block the function of Pgp in Pgp-expressing NIH3T3 and MDCK/MDR1 cells. Both cell lines expressed Pgp on the plasma membrane as detected by MRK16 mAb and by flow cytometric assays. As fluorescent substrates, rhodamine123 and daunorubicin were used. In Pgp expressing NIH3T3 and MDCK/MDR1 cells, propranolol, pindolol and labetolol inhibited Pgp, but at in vitro concentrations higher than therapeutic levels. The analgesic drugs, propoxyphene and loperamide block the function of Pgp at or close to the therapeutic levels. The quinoline antibiotics tested did not block Pgp, even at levels higher than therapeutic levels. These findings suggest that some drugs inhibit the Pgp-mediated drug efflux and therefore, can act clinically as modulators of the multidrug resistance phenotyped cancer cells. In addition, these drugs, if coadministered with other Pgp substrates may increase the absorption through the intestine or the penetration through the blood brain barrier.

Board K08

RAW 264.7 (ATCC TIB 71) cells can be stimulated to produce NO by the addition of low amounts (1 to 5 ng/ml) of lipopolysaccharide (LPS) from E. coli 0127:B8 and Ps. aeruginosa. RAW cells do not produce detectable NO levels after the addition of up to 100 ng/ml lipoteichoic acid (LTA) derived from either Strep. pyrogenes or S. aureus. NO production was measured using the Griess reagent to detect nitrite in the media 48 and 72 hrs after LPS was added to the cells. Earlier studies showed that NO detection may be as, or more, sensitive than the Limulus amebocyte lysate test to detect the presence of LPS. Cells were responsive to LTA since both LTA and LPS can stimulate tumor necrosis factor-a production. Approximately 10 times more LTA than LPS is needed to stimulate similar TNF-a levels in RAW cells. Thus measuring NO stimulation with RAW cells may be another method to detect the presence of low levels of LPS in water or on device materials.

Board K09

When dealing with drugs, which contain opiates, we are concerned with potential physical dependence and abuse. Withdrawal symptoms are an indication of physical dependence. In a pain reliever submission the sponsor was asked about possible withdrawal effects of their drug. They used the WOW scale (Weak Opiate Withdrawal) in a clinical trial to measure the withdrawal symptoms of their slow release drug versus the immediate release compound and placebo. Our analysis of the data has included looking at the overall scores and also grouping the raw data into major withdrawal categories. Within the limitations of the data WOW scores for the slow release product are generally higher than placebo, but lower than the immediate release product. Scores are available for four sequential days. Changes in the aggregate data over the sequential days are small, making it difficult to draw any clear inference with respect to differential withdrawal. The averages for the physiological and psychological categories present a complex pattern. For the trial we analyzed a large number of our categories showed the slow release drug having its highest withdrawal score in the second day. However, what is troublesome both regarding the aggregated score and the score for the categories is the low percentage with respect to the total possible score that is achieved. This seems to indicate that the WOW scale is a poor measure of withdrawal symptoms for weak opiates.

Board K10

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

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

Board K11

This study investigated the improvement of cutaneous wound healing after low power laser irradiation in diabetic and non-diabetic Fat Sand Rats (Psammomys obesus). Bilateral, full thickness, circular wounds were made in the dorso-lumbar skin of Fat Sand Rats. Laser treatment with a 632.8 nm HeNe laser (output power of 16mW, energy density of 4J/cm²) was applied to the left wound of treated animals for 4 days beginning immediately after injury. At days 1,2, 3, 4, and 10 post-injury, the wound sizes were measured using electronic calipers. At 10 days post-injury, the animals were euthanized and the wound tissue was removed, post-fixed, and sectioned. Sections were then stained with hematoxylin and eosin for histological analysis. Histological scoring showed a significant increase in score in non-diabetic animals that were treated with the low power laser. An increase in percentage of wound closure was measured, using calibrated electronic calipers, in both non-diabetic and diabetic Fat Sand Rats that received treatment with the low power laser at 4 days post-injury versus those animals that received no laser irradiation. These results suggest that low power laser irradiation may be a beneficial treatment for wound healing, particularly when healing is impaired, such as in Type II diabetes.

Board L01

Imaging systems which "look" at a large area with a small detector using a optical lenses are commercially available. These imaging systems can be quite inefficient depending on the characteristics of the optical coupling. This inefficiency raises radiation safety and protection issues. We have implemented an optically coupled imaging system which includes a digital detector, i.e., a CCD camera, in our laboratory. The laboratory system has provided the ability to investigate when a system of this configuration starts to develop a "secondary quantum sink" as a result of poor optical coupling. Representative measures of imaging performance such as detective quantum efficiency (DQE) were obtained using the laboratory system. Experimental measurements have been made of the large area gray-scale transfer, resolution and noise properties of the laboratory system. The observed DQE at low spatial frequency is quite low at 6 percent and possesses a limited bandwidth. Given the commercial availability of optically-coupled imaging systems, it is important to recognize the system parameters and characteristics which can lead to the emergence of inefficient optical coupling and to the presence of a "secondary quantum sink." Interestingly, this investigation re-visits actions in the radiological health past in that optically coupled imaging systems were used in chest screening programs in the 60s and 70s. These older generation imaging systems were also inefficient in their optical coupling and led to patient exposure concerns at that time.

Board L02a

A previous investigation in our laboratory linked cellulose acetate (CA) membrane degradation with adverse health effects in hemodialysis patients. A population molecular model was developed to track CA degradation of a dialyzer membrane during storage. The model used a random number to select individual polymer molecules out of a population, and then to select a site on the molecule for the degradation reaction to occur. The resulting molecular fragments were then redistributed into the population. Molecular weight averages and acetyl content were recalculated as the reaction simulation proceeded. The model was validated with experimental measurements on dialyzers stored up to 13.3 years. It was found that the degradation reactions can be accurately modeled as random events and that the reaction events occur at constant rates. The shelf life of these devices was estimated using the model predictions and animal test results.

Publish Only (L)

Magnetic field strength measurements were made around 8 hand-held and 10 walk-through metal detectors. Measurements were collected at use sites including airport terminals, federal and state government buildings, and a local high school. Walk-through metal detectors had considerably higher magnetic field strengths (up to 3741 mG p-p) than hand-held metal detectors (up to 76 mG p-p) The frequency of the magnetic field signal for walk-through detectors was 0.1 to 3.5 kHz while that for hand-held detectors was 89 to 133 kHz. Waveforms for all hand-held metal detectors were sinusoidal; those for walk-through metal detectors were varied with most being saw-toothed and pulsed. Due to their higher field strengths and the pulsed nature of their magnetic fields, walk-through metal detectors may pose a higher risk for medical device electromagnetic interference (EMI) than would hand-held units.

Second Place Poster - 2001 FDA Science Forum

Board M01a

StarLink^TM is corn which has been genetically modified by insertion of the cry9C gene to allow expression of the insecticidal Cry9C protein. This product has been approved by US EPA for animal and non-food use, but not for use in human food. The presence of either the protein or the gene in the human food supply would render the commodity adulterated under FDA regulations. Independent laboratories initially reported the presence of cry9C in taco shells, leading to a voluntary recall of the product by the manufacturer. With assistance from Aventis Crop Sciences, the developer of StarLink^TM corn, CFSAN scientists developed a method for determination of the presence of StarLink^TM using PCR analysis for the cry9C DNA in corn flour, corn meal, and tacos shells. The method has been applied to a number of processed corn products including cereals, baby foods, and chips. Extraction methods were developed to increase detection of the target gene in food products and the method transferred to ORA field laboratories for use in assignments to analyze corn flour, corn meal, and finished food products for the presence of this adulteration.

Board M01b

For implementation of CDER's Pediatric Final Rule, information on inpatient pediatric use of marketed drugs is needed to determine how substantial is their use in pediatric patients. Two pilot studies compared pediatric drug use (1997-1998) in 139 general hospitals and 19 children's hospitals for eleven drugs: albuterol (inhaled), buspirone, ciprofloxacin, cisapride, dopamine, enalapril, metformin, nifedipine, omeprazole, paclitaxel, and pamidronate. Parameters evaluated were the numbers of discharges mentioning these products, age and gender of patients, and primary discharge diagnosis. The number of discharges mentioning these 11 drugs was much higher in the children's hospitals than in the general hospitals. Demographic and diagnostic patterns of use were similar between children's hospitals and general, teaching hospitals. Differences in use in the general, non-teaching hospitals were seen primarily in the 12-16 year age group. These studies suggest that significant pediatric inpatient drug use occurs in children's hospitals, especially for more severe or rare illnesses. Nationally representative inpatient drug use information from a larger sample of multiple pediatric care arenas is needed to address CDER's mission concerning drug safety and effectiveness in pediatrics.

Board M02

A sensory panel was designed to determine the ability of the consumer to see hair, a filth analyte, in food. The survey was conducted in two phases. In Phase IA 48 people filled out a demographic survey and observed ten cards. Each card was divided into nine squares whose background color was randomly chosen from nine predetermined colors to mimic the color of mushrooms. To each square was applied a single hair. Five hair lengths (0, 3, 5, 7, 10 mm), and 5 hair types (human black, human brown, human white, cat/dog, and rat/mouse) were used. Two separate cards were also prepared and used for training the panelists. Results from Phase IA indicated that there was no significant difference observed for several of the background colors and between lengths 7 and 10 mm. The panelists were asked to view a second set of fourteen cards (Phase IB) prepared from 6 background colors, 5 hair lengths (0, 1, 3, 5, 10 mm), and 4 hair types (human black and brown were combined). In Phase IB there was no significant difference observed between lengths 3 and 5 mm. Based on the results of Phase IB, the panelists were presented with a series of petri dishes containing canned sliced mushrooms with 0-4 hairs of 4 hair lengths and 4 hair types. A positive result was if the panelist saw a hair in a quadrant in the petri dish.

Board M03

Herpes simplex viruses (HSV) are the most common cause of genital ulceration worldwide. Enzyme-linked immunosorbent assays (ELISA) are routinely used confirm the presence of the virus. Kit manufacturers generate their own quality control test challenge. There is a need to develop a standard viral challenge preparation protocol to best evaluate HSV test kits. Research includes the use of fundamental virus propagation and titration principals. Here we outline work on the propagation and titration of HSV-2 for use in evaluating a HSV antigen detection ELISA kit. Three virus preparations, freeze thaw liberated virus, supernate without host-cell liberation, and sonication-liberated virus from concentrated infected cells, were tested with an ELISA kit. Results with a commercially available antigen detection kit showed that although each virus challenge was adjusted to have the same TCID₅₀, there was a measurable difference in the absorbencies with the antigen kit. The sonicated cells that contained the highest concentration of host cell material gave the highest absorbance readings. The supernate that contained the lowest amount of host cell material gave the lowest absorbance readings. This supports the hypothesis that an increase in cellular material would directly influence the test result independent of the virus titer. This work emphasizes the need to standardize the virus preparation procedure to better evaluate the HSV antigen ELISA test kits.

Board M04

Through 1991 FDA received reports of 22 cases of hemorrhagic stroke (HS) associated with phenylpropanolamine (PPA) used as an appetite suppressant (AS,73%) and nasal decongestant (ND,27%), primarily in young females (95%) and first-day users (55%). FDA hypothesized that PPA was associated with an increased risk of HS, particularly with first-day use. Yale conducted a study from 1994 to 1999 to test this hypothesis and showed a statistically significant increase in risk of HS among female AS users and first-day ND users of PPA. From 1991-2000, FDA received additional 22 reports of PPA-associated HS. An OTC advisory committee reviewed these data and concluded that PPA could not be considered safe, leading to a public health advisory against PPA use and its withdrawal from the market.

Board M05

Measuring NO production by RAW 264.7 (ATCC TIB 71) murine macrophage cells may be a sensitive method to detect the presence of LPS in the water reservoirs of table top steam sterilizers. Several water samples from the reservoirs or water spiked with crude endotoxin from R. picketti or E. coli were added to cells. Cell supernatants were harvested after 48 and 72 hr after addition of water or LPS. NO production was measured as nitrite using the Greiss reagent. The LPS-spiked and some unspiked water samples from reservoirs of sterilizers stimulated RAW cells. Steam sterilizers may not completely inactivate LPS. Heating water with high concentrations of E. coli LPS to135°C for 3 or 10 min did not completely inactivate LPS as measured by NO production. Water samples with bacteriostatic agents containing nitrite gave false positive results. NO production by stimulated RAW cells may be a sensitive method to detect LPS in water.

Board M07

Due to extensive serological cross-reactivity commercially available enzyme immunoassays have failed to distinguish between herpes simplex type 1 (HSV-1) and herpes simplex type 2 (HSV-2) infections. Manufacturers will continue to use whole prototype HSV antigens in the production of their immunoassay test kits until the next generation of HSV kits manufactured with type-specific glycoproteins are tested and accepted for general use. Consequently, the extensive serological cross-reactivity between the two viruses will continue to give misleading or inaccurate results with respect to the correct herpes virus type. The purpose of this project is to develop a serum panel with well-defined reactivity to HSV types 1 and 2 utilizing the Western blot technique, purified native HSV type specific and recombinant glycoproteins. Helix pomatia lectin affinity (HPA) chromatography purified glycoprotein reacted strongly with HSV-2 gG-2 monoclonal antibody (H1206) and a serum that has a high reactivity to recombinant gG-2. A limited number of serum samples that reacted to the recombinant gG-1 did not react to the HPA purified HSV-2 glycoprotein or the recombinant HSV-2 glycoprotein when immunoblotted. Sera characterized in this manner are available in large volumes and could be used to determine the accuracy of both the old type test kits and the next generation of kits manufactured with HSV 1 and HSV-2 type specific glycoproteins.

Board N01

As part of our efforts to apply MALDI/TOF to the characterization of food borne pathogens, we have investigated the application of a variety of pattern recognition programs to the identification of bacteria based on their spectra. MALDI spectra were obtained on samples of V. fluvialis, V. vulnificus, and L. monocytogenes. For each sample, selected spectra were combined, smoothed, and baseline subtracted. To fill gaps in this mass/intensity list, masses for which no value was recorded are assigned an intensity of 0. To ensure that variables are correctly compared, spectra were further smoothed using a binning technique. Factor analysis and cluster analysis modules from two vendors performed comparably. Using all non-zero variables, we could easily distinguish the three groups. In addition, V. fluvialis and V. parahaemolyticus were more similar to each other than to L. monocytogenes. Preliminary experiments with the Statistica Neural Net indicated that it was possible to train a network to identify these bacteria based on their spectra, but the sample size was too small to permit us to eliminate the possibility of overtraining. Best results were obtained when the 25 or 50 most intense peaks were used with a bin width of 10 or 20. These conditions eliminated noise while preserving unique features of the spectra.

Publish Only (O)

A method, which involves an enzymatic hot start nested PCR in a single closed tube, was developed for the sensitive detection of Shigella spp in Northeast Regional Laboratory FDA. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from Shigella spp. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of Shigella spp, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. In the standard nested PCR, the second-round PCR is set up in the presence of first-round PCR amplification products, which increases the risks of assay contamination by product DNA. Contamination is a widely recognized problem with PCR. The introduction of this standard nested PCR into routine testing in FDA laboratories was hampered by its frequent tendency to create false-positive results because of carry-over contamination. This problem can be solved by our one-tube nested PCR and uracil-N-glycosylase (UNG) anti-contamination technology. This method achieved better results than any other PCR technique. Retaining high sensitivity of the conventional nested PCR, our one-tube nested PCR reduced greatly the risk of carry-over contamination. It is also cost and time saving. We believe that one-tube nested PCR plus UNG offers a useful method for detecting Shigella spp at extremely low levels in food.

Board O02

Salmonella ATTC strains 9700, 12325, 29934, 9482, E. coli 25922, E. coli DH5α and Shigella sonnei 9290 were transformed with E. coli vectors carrying the Green Fluorescent Protein Gene, from the jellyfish Aqueorea victoria. All strains were successfully transformed into the ampicillin resistant/GFP⁺ phenotype using the CaCl₂ incubation method. All transformed colonies fluoresced brightly under UV light illumination (366nm). The transformed strains appeared stable and fluorescence was maintained after repeated sub-culturing in media containing ampicillin. Expression levels varied between strains.

Board O03

The unicellular protozoan parasite, Leishmania donovani, causes visceral disease in humans. Two major developmental stages, flagellated promastigote that resides in the gut of sandfly and aflagellated amastigote that survives in animal macrophages, are involved in the parasite's life cycle. In order to identify genes that control the process of growth/differentiation, we have isolated a complete copy of a gene encoding Ca⁺⁺ binding centrosome associated ‘centrin' homologue, which has been shown to be involved in cell growth in higher organisms. Leishmania centrin is a single copy gene and codes for an ~17 kDa protein. The E. coli expressed Leishmania centrin protein cross-reacts with the antibody to human centrin2 (H-cen2) but not with that of either Chlamydomonas or human centrin3. Antibodies to H-cen2 and Leishmania centrin cross-react with a similar size protein in Leishmania cell lysate suggesting its function is similar to H-cen2. Both centrin mRNA and protein levels are significantly reduced during the stationary phase of the cell culture, suggesting a regulatory role in cell growth.

Board O04

The emergence of resistance to antimicrobial agents in bacterial pathogens is a worldwide problem that has been associated with inappropriate use in human and veterinary medicine, and animal husbandry. Recently, plasmid-mediated b-lactamases with extended resistance spectra, such as cephamycinase, CMY-2, has emerged. In this study, 69 E. coli and 21 Salmonella isolates recovered from cattle, poultry, swine and retail meats that exhibited decreased susceptibilities to ceftiofur and ceftriaxone, were examined for the presence of bla_CMY genes using PCR. PCR analysis revealed that fifty-four E. coli isolates and all of the Salmonella possessed a bla_CMY gene. DNA sequence analyses of 9 bla_CMY PCR products (4 from E. coli and 5 from Salmonella) indicated that identified bla_CMY genes were 95% to 99% homologous to previously reported bla_CMY-2 genes in Klebsiella pneumonia and Salmonella senftenberg. The bla_CMY gene from an E. coli strain isolated from retail meats was successfully transferred to two strains of E. coli O157:H7 by conjugation. Both transconjugates demonstrated resistance to ceftiofur, ceftriaxone, cefoxitin, cephalothin, ampicillin, and amoxicillin/clavulanic acid. Subsequently, the bla_CMY gene was cloned into expression vector pET34b+, which was transformed into E. coli BL21(DE3)LysS. The clone displayed decreased susceptibilities to the six antimicrobials but at different levels compared to the wild type strain. Further sequence analyses of the cloned E. coli bla_CMY gene revealed 100% homology to a previously reported bla_CMY-4 gene from an E. coli strain isolated from leukemia patients. Our results indicate that bla_CMY is commonly present in ceftiofur- and ceftriaxone-resistant E. coli and Salmonella of animal origin isolates, and that this plasmid-mediated resistance is readily transferred among bacteria through conjugation. The emergence and dissemination of ceftiofur- and ceftriaxone-resistant E. coli and Salmonella may present significant therapeutic challenges in animal and human health care.

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Board P01

Iron deficiency anemia (IDA) is a major public health problem in Mexico, and affects approximately 1.5 billion individuals worldwide. Fortification is regarded as a cost-effective, long-term method for preventing IDA. An in vitro simulated digestion method was adapted as a preliminary screening procedure to assess the bioavailability of elemental Fe when used for fortification in Mexican and US tortillas. Mexican tortillas were prepared by the National Institute of Nutrition and the US tortillas were commercially prepared. The tortillas were made with corn masa flour and were not fortified with Fe. A total of 22 Fe compounds were obtained from 14 companies, and included 15 samples of elemental Fe (carbonyl, electrolytic, reduced, and atomized), which were then added to the tortillas. Electrolytic Fe had the highest % dialyzable Fe in both the Mexican (3%) and US (1.5%) tortillas. The values for carbonyl Fe were 1.9 and 0.7% for Mexican and US tortillas, respectively, and 1.8 and 0.6% for reduced Fe. Ferrous sulfate and ferrous fumarate were approximately 5% for the Mexican tortillas and 2% for the US tortillas. Overall, the US tortillas had lower dialyzable Fe values than the Mexican tortillas. In the human bioavailability study of Forbes et al. (1989) values of 3.4% and 4.5% were obtained for electrolytic Fe and FeSO₄, respectively. In the present study, using the in vitro procedure with Mexican tortillas, similar values were obtained, that is, 3% for electrolytic Fe and 5% for FeSO₄. The carbonyl Fe results we obtained were also similar to results reported in two studies by Hallberg et al. (1986) of approximately 1% for carbonyl Fe and 5.5% for FeSO₄. The data from this study indicate that the in vitro method provides a simple screening tool for assessing elemental Fe bioavailability and can reduce the number of animals used in determining Fe bioavailability by the AOAC method.

Board P02

Fortification of cereal-grain products have contributed to increased dietary iron intake and reduction in iron deficiency anemia, and to increased folate intake of women of child-bearing age and reduction in the risk of neural tube birth defects. With recent increases in fortification, there is concern in the US that excess intake of iron and folic acid may result in toxic manifestations. Our objective was to measure iron and total folate in breakfast cereals and compare assay to label values for % Daily Value. We also determined the amount of a ready-to-eat breakfast cereal adults would eat and compared this to the labeled serving size. Twenty-nine cereals were analyzed for iron content and 28 cereals were analyzed for total folate. When the labeled value was compared to the assayed value for iron content, 21 of the 29 cereals were 120% or more of the label value and 8 were 150% or more of the label value. Analyzed values for iron ranged from 80 to 190% of label values. Analyzed values for folate ranged from 98 to 320% of label values. Of the 28 cereals analyzed for folate, 14 had values exceeding label declarations by more than 150%. Serving size quantities were estimated in 72 adults who regularly ate cereal. The median analyzed serving size was 47 g for females, 61 g for males with a combined median of 56 g as compared to the label value of 30 g. For adults, the amount of cereal actually consumed was approximately 200% of the labeled serving size. When the levels of iron and folate are higher than labeled and the quantity of cereal consumed is more than the indicated serving size, the intake of both will be significantly greater than the label value. It will be important to continue monitoring serum ferritin and folate levels in the fourth National Health and Nutrition Examination Survey (NHANES IV).

Publish Only (P)

Lutein is a major carotenoid in human blood. Reports suggest that lutein may have specific functions in protecting against age-related macular degeneration and cancer. Some dietary supplements contain a mixture of lutein, natural tocopherols and other components. Limited data are available on the impact of dietary supplements containing such mixtures of nutrients. We determined the effect of graded levels of Flora GLO ¬FG|, a dietary supplement containing lutein derived from marigold flowers, on tissue levels of retinoids in rats. Concurrent with the decrease in plasma retinol was an increase in liver retinyl esters in rats fed the three highest dietary levels of FG (p<0.05). The ingestion of a supplement containing a carotenoid that does not have vitamin A activity had an impact on the storage of retinoids in the liver and on the transport of retinol in plasma. These results indicate that some component/components in the dietary supplement modulated tissue concentration of retinoids.

Publish Only (P)

High doses of alpha-tocopherol affect vitamin K (vitK)-dependent coagulation factors. There is limited information on the effects of tocopherols on tissue concentrations of vitK ¬phylloquinone (K₁) and menaquinone-4 (MK-4)|. Research suggests a unique role of these vitK compounds at the tissue level. Gamma-tocopherol is the major naturally occurring isomer of vitamin E in foods and is a component of mixed tocopherol supplements. We measured tissue concentrations of vitK in rats fed diets containing reduced and adequate levels of alpha-tocopherol and supplemented with gamma-tocopherol. Gamma-tocopherol accumulated in tissues of all supplemented rats. Concentrations of K₁ in heart and spleen of rats fed adequate alpha-tocopherol were lower than rats fed reduced alpha-tocopherol. MK-4 increased in the heart, testes and spleen of rats fed diets supplemented with gamma-tocopherol. These data suggest that alpha-tocopherol and gamma-tocopherol affect tissue concentrations of vitK differently and may affect vitK function.

Honorable Mention Poster - 2001 FDA Science Forum

Board Q01

Reducing errors and noncompliance in using medicines was explored using focus groups of limited or non English speaking /reading Chinese, Vietnamese, Native Hawaiian, Samoan, Korean, and Cambodian consumers, and pharmacists serving those communities. Los Angeles District Public Affairs Specialist (PAS) partnered with UCLA School of Public Health to conduct the focus groups. Pharmacists were questioned about how they interacted with non-English speaking Asian and Pacific Islander patients, and how the pharmacists assessed patient understanding. Pharmacists also identified what information was needed to help them to assist their customers. Consumer focus groups were asked about how they obtained information about medications and their informational needs. Traditional practices and linguistic and literacy limitations were also discussed with both audiences. PAS also conducted educational workshops to present the Take Time to Care medication safety messages (Read the Label, Ask Questions, Avoid Problems, Keep a Record). The messages were illustrated with stories from the communities about problems that arose from lack of compliance and /or understanding of these messages. Translated brochures of the Take Time to Care messages were provided to the workshop audiences. Workshop participants and focus groups shared a wealth of stories and suggestions. Based on the focus group results and workshops, common label directions will be translated and the Power Point workshop presentation will be adapted to each community. A set of recommendations and references for cultural practices will also be completed.

Honorable Mention Poster - 2001 FDA Science Forum

Board R01

A new animal model has been developed using the Holstein cow and her own calf to monitor offspring exposures to drugs in utero and/or through consumption of mother's milk. Two drugs used in human medicine, phenylbutazone and ivermectin, were tested as prototype drugs in the cow-calf model. Both drugs have long elimination half-lives in the human and the bovine, and both are secreted into milk at low levels, such that the resulting estimated dose to the nursing human infant is 5% of the maternal dose per day for phenylbutazone and 2% of the maternal dose per day for ivermectin. The literature suggests that the use of these drugs is compatible with breast-feeding. In the cow-calf model, lactational exposure to 1% of the maternal dose of phenylbutazone twice daily resulted in appreciable accumulation of the drug in the calf's plasma after a 7-day exposure period. A lactational exposure to 3% of the maternal dose twice daily, for 7-days, maintained a steady-state plasma level in a calf that was also exposed to phenylbutazone in utero. For ivermectin, a lactational exposure to 0.5% of the maternal dose of ivermectin twice daily resulted in plasma drug accumulation in calves approximating 50% of the adult therapeutic blood level after 7-days exposure. Thus the cow-calf model dramatically demonstrates that exposures of newborn to low levels of phenylbutazone or ivermectin, or to drugs with similar physico chemical characteristics, in breast milk may not be considered safe due to the potential for these compounds to accumulate in the newborn.

Board R02

The fungus Cunninghamella elegans was used as a microbial model of mammalian metabolism to biotransform the tricyclic antidepressant drug mirtazepine. Mirtazepine has a unique action in that it blocks presynaptic a₂-adrenoreceptors as well as postsynaptic 5-HT₂ and 5-HT₃ receptors. Cultures of C. elegans were grown in Sabouraud dextrose broth at 28°C for 32 hr, then dosed with mirtazepine. After 96 hr of incubation, the metabolites were extracted with ethyl acetate, separated by reverse-phase HPLC, and identified by mass and ¹H NMR spectrometry. The fungus produced three human metabolites, 8-hydroxymirtazepine, N-desmethylmirtazepine, and mirtazepine N-oxide. It also produced the mouse metabolite, 13-hydroxymirtazepine, and a novel metabolite, 12-hydroxymirtazepine.

Board R08

Drug ‘A' is currently used for the acute relief of an attack and prophylaxis of angina pectoris due to coronary artery disease. The new formulation of A is not bioequivalent to the marketed. The impact of this pharmacokinetic (PK) bioinequivalence on the pharmacodynamic (PD) vasodilatory effect is unknown. Objective: To establish a PK/PD model for the comparison of PD effects of a new and marketed formulation of drug ‘A'. Methods & Results: Plasma data were obtained from the single dose, open-label, randomized, 3-way crossover study, where the equal doses of drug ‘A' were administered as marketed reference (treatment 1), and new formulation (treatments 2 and 3). PD effect on the digital pressure waveform was measured during the period of expected maximal antianginal effect of the drug. The PD end-point was real time systolic BP: diastolic BP ratio. PK/PD analysis was conducted using NONMEM. Drug ‘A' PK was best described by a one-compartmental model with first order absorption fitted simultaneously for all three treatments: CL (SE, %) 10.0 (10.30) L/kg, V 86.9 (18.41) L, ka 0.906 (16.56) hr-1. Relative systemic availability of drug ‘A' for each treatment was estimated as 1:1.25:1.36, respectively. Individual predicted plasma concentrations were used as an input to the linear PD model, and model was fitted to each treatment data separately, results for each treatment were very similar. Finally, the vasodilatory effect vs drug concentration data from all three treatments were combined and fitted simultaneously to the linear model to obtain general parameter estimates and individual predictions of effect at Cmax. Conclusion: Although the new formulation has not met the bioequivalence criteria with reference for both Cmax and AUC, this inequivalence in PK does not seem to result in significant differences in the pharmacodynamics.

Board R09

Veterinary public health is another frontier in the fight against human disease. The veterinary public health scope includes the control and eradication of zoonoses, diseases that are naturally transmitted between vertebrate animals and man. These diseases pose a continuous hazard to the health and welfare of the public. Zoonoses include more than 100 diseases, including salmonellosis. For example, approximately 20% of U.S. broiler chickens are contaminated with Salmonella, while more that 80% are contaminated with Campylobacter. The veterinary public health scope, in addition to the control and eradication of zoonoses, also includes the development and supervision of food hygiene practices, laboratory and research activities, and education of the public. It is important to understand how antibiotics are used in humans and in food animals and how their use affects the evolution of antibacterial resistance. An understanding of the epidemiology of antimicrobial resistance allows development of preventive strategies to limit existing resistance and to avoid emergence of new strains of resistant bacteria (Curr Opin Microbiol 2: 494, 1999). Food animal production in the United States currently uses large amounts of subtherapeutic doses of antibiotics for disease control. Aarestrup and Wegener (Microbes Infect 8:639,1999) conclude that the use of large amounts of antibiotics for disease control in food animal production results in the spread and persistence of antimicrobial-resistant zoonotic bacteria. A heavy antimicrobial drug selective pressure in overcrowded populations of production animals creates favorable environments both for the emergence and the spread of antibiotic resistance genes (Acta Vet Scand Suppl 92: 23, 1999). Pedersen et al (Vet Rec 145: 50, 1999) emphasized that appropriate use of antibiotics for food animals will preserve the long-term efficacy of existing antibiotics, support animal health and welfare, and limit the risk of transfer of antibiotic resistance to humans.

Board R10

This article is a documentary of the history of the Am Coll Clin Pharmacol (ACCP) and the J Clin Pharmacol, summarized by leaders who played key roles in the growth of the profession of Clinical Pharmacology (CP). Together, the college, the journal, and all clinical pharmacologists working in academia, industry, CROs, or government in many different subspecialty areas of the discipline contribute to the advancement of CP, the development of new drugs, and to an improved quality of life for mankind. Achieving leadership in health care in an era of change requires actions to be dynamic and flexible. Leaders must be capable of self-development and self-education. Leaders must examine challenges from top to bottom and build on the leadership foundations of vision, courage, and knowledge. Strong leaders are needed for the future of CP to address the rapidly changing environment for health care givers and the challenges faced by those working in drug development or training new leaders. One lesson from the past, from the professional life of Harry Gold, is that it is very important to convey the excitement of the field of CP and to pass on this excitement and knowledge base to those currently leading the educational process necessary to keep clinical pharmacologists in the forefront of the medical arena of today and tomorrow. The college became an instrument for change in the field of CP. Forward thinking efforts of the college did not allow stagnation. All founding leaders of the college were possessed of a dream of "what could be and what should be." Two points should be emphasized: the importance of teaching teachers how to teach and the importance of forming national networks, such as the college, to concentrate on the role of teaching students. Today's students are the leaders of tomorrow. The training of students by qualified mentors in the field of CP continues over many years. The ACCP continues to prepare for the changing demands of a new millennium.

Board R11

Background: St. John's wort (SJW) is a popular over-the-counter herbal remedy that has been implicated in drug interactions with substrates of several cytochrome P450s (CYPs). The effect of SJW on CYP activity in vivo was examined using a probe drug cocktail.

Methods: A three period, open-label, fixed schedule study design was employed. Tolbutamide (CYP2C9), caffeine (CYP1A2), dextromethorphan (CYP2D6), oral midazolam (intestinal wall and hepatic CYP3A) and intravenous midazolam (hepatic CYP3A) were administered before, with acute SJW dosing, and after two weeks of SJW intake to determine CYP activities.

Results: Acute administration of SJW had no effect on CYP activities. Chronic SJW administration caused a significant (p<0.05) increase in oral clearance of midazolam from 121.8±70.7 to 254.5±127.8 and a corresponding significant decline in oral bioavailability from 0.28±0.15 to 0.17±0.06. In contrast to the over 50% decrease in AUC when midazolam administered orally, chronic SJW administration caused a non-significant 20% decrease in AUC when midazolam was given intravenously. There was no change in CYP1A2, CYP2C9, and CYP2D6 activities as a result of SJW administration.

Conclusion: SJW administration resulted in a significant and selective induction of CYP3A activity in the intestinal wall. SJW did not alter the CYP2C9, CYP1A2, and CYP2D6 activities.

Acknowledgement: FDA grant#FD-T-001756-01; the authors acknowledge the contribution of Drs. Jerry Collins and Peter Honig

Board R13

Six lactating Holstein cows were administered an intravenous bolus dose of 500 mg of furosemide (Lasix Injection 5%) on three consecutive days. Milk samples were collected approximately twelve hours after the last treatment and at twelve-hour intervals thereafter up to 72 hours postdose (six milking total). After defating and deproteination, the milk samples were analyzed for furosemide concentrations by HPLC using a reversed phase column, a mobile phase of acetonitrile/phosphate buffer (pH 3) and a fluorescence detector. The results showed that the average concentration of furosemide in milk collected from six cows 24 hours after the last treatment was within the safe level (10 ppb). No furosemide was detected in 36 hours postdose milk.

Board R14

Fluticasone propionate (FP), a novel potent corticosteroid with low systemic bioavailability, has a significant therapeutic efficacy in the treatment of asthma. The main adverse effect - suppression of adrenal function - has been assessed by the measurement of plasma cortisol level. For the prediction of pharmacodynamic effect based on the dose of drug, modeling is applied. Modeling of cortisol (CT) suppression has the additional complexities due to nonstationary secretion of cortisol (circadian rhythm) and down-regulation effect. Previous models describe cortisol secretion as a cosine function (Jusko, 1993) or as sum of linear functions (Derendorf, 1995). We applied a superposition of 2 Bateman functions in order to characterize pulsatile circadian rhythm of cortisol in plasma. CT and FP levels were measured over 24 hrs in 12 healthy subjects after inhalation of 0.5, 1, and 2 mg of FP, and placebo. NONMEM was used to model PK/PD. PK of FP was best characterized by a two-compartment model with first order absorption (CL/F 158.8 L/hr, V/F 2800 L, Ka 7.1 hr-1). CT plasma levels were described by two pulsatile Bateman functions over 24 hrs. The effect of FP was modeled using the Hill equation with inhibition of cortisol secretion. Cortisol peaks were estimated to occur in pulses at 3:30 a.m. (large sharp) and 1:30 p.m. (small shallow) with an amplitude ratio of 7:1. The IC₅₀ of FP was 350 pcg/mL, which is consistent with predicted values for receptor affinity. Cortisol suppression (adverse effect) will have negligible values at the therapeutic doses (50 mcg).

Board R16

The FDA focused attention of practitioners and pharmaceutical manufacturers on whether use of anticonvulsant drugs contributes to or prevents sudden unexpected death in epileptic persons. Consideration of the possibility of sudden unexpected death in epileptic persons when developing new anticonvulsant drugs was emphasized by the FDA convened panel of scientists assigned to review data on the risk of sudden unexpected death in epileptic persons in patients taking lamotrigine (LTG). The sudden unexpected death rate in epileptic persons in this conhort of patients was comparable to that expected in young, poorly controlled epileptics (Epilepsia 38-47, 1997). Estimated sudden unexpected death rates in patients receiving any of these newer anticonvulsant drugs are similar to those observed in patients receiving other anticonvulsant drugs. Cumulative evidence suggested sudden unexpected death rates in epileptic persons reflect population rates and not a specific drug effect. In summary, four new anticonvulsant drugs, LTG, gabapentin, topiramate, and tigabine have FDA required warning labels with data on the risk of sudden unexpected death in epileptic persons in association with use of each drug. None of these four newer anticonvulsant drugs showed an associated increased risk of sudden unexpected death nor did their use show a decreased risk. The above represents the opinion of the authors and does not reflect policy of the FDA or the U.S. government.

Board R17

Endocrine disruptors provide a unique challenge to both toxicologists and to clinical pharmacologists. Perhaps the biggest challenge is to resolve the controversy of whether endocrine disruptors do or do not negatively impact on human health. Carefully designed human studies will be necessary to answer this question. The endocrine disruptors cause dose-dependent toxic effects for most of the responses they induce. The inverted U-shape curve for hazard and risk assessment of estrogenic compounds and other endocrine disruptors needs to be studied. There appears to be an age-related (utero or early postnatal exposure) toxicity effect of endocrine disruptors associated with a critical window of exposure. Clinical pharmacologists and toxicologists need to work together to ascertain why the effect of endocrine disruptors in adult populations differ from the utero or early postnatal exposure. The role of dietary, lifestyle, and genetic factors as they influence hormonal responses needs to be deciphered. Indeed, government regulators and scientists from all disciplines must work together to determine the next steps global society must take to limit the unwanted effects of endocrine disruptors. Safe et al. (CAST July 16, 2000) noted that several government agencies and the U.S. Congress worked with the National Research Council, an agency of the National Academy of Sciences, to evaluate the existing scientific data relevant to endocrine disruptors to provide advice. Clinical pharmacologists must also work with these agencies and their scientists to evaluate the scientific data and to provide advice to governmental agencies. Clinical pharmacologists must work hand in hand with veterinary clinical pharmacologists to definitively establish whether endocrine disruptors have a negative effect on human health and to determine how the doses at which this occurs effect wildlife and the environment.

Board R18

Lack of gravity during space flight causes inner ear malfunction due to mismatched canal and statolith afferent stimuli, leading to an illusionary tilt which may result in motion sickness. Bagain reported phenergan abates symptoms of space motion sickness during space flight. Observations in fish are analygous to the clinical observation that maintenance of the ability to observe the horizon diminishes the chance of motion sickness in persons susceptible to developing symptoms. Data from 2 carp flown on an 8-day mission of Spacelab-J provided evidence for sensory-motor disorder and readjustment during the early phase of microgravity. We propose to develop a new goldfish model to predict pharmacodynamic and/or pharmacokinetic (PD/PK) effects of drugs used to treat sickness administered in differing gravity. Ground-based studies will provide baseline data for comparison obtained during parabolic flights (1, 2, and 0-g) and space flight (0-g). Goldfish will be immersed in solution containing phenergan (Griezerstein and Smith, 1976). A trained observer will observe, record, and photograph the percentage swimming and reflex behavioral movements exhibited. A better understanding of the mechanism of the neurophysical and neurovestibular changes associated with altered gravity loads will permit targeted selection of pharmacologic agents to prevent or lessen neurovestibular dysfunction and associated motion sickness.

Honorable Mention Poster - 2001 FDA Science Forum

Board R19

Background: St. John's wort (SJW) is a popular over-the-counter herbal remedy that has been implicated in drug interactions with several substrates of p-glycoprotein. The effect of SJW on p-glycoprotein activity in vivo was examined using fexofenadine as selective probe drug.

Methods: A three period, open-label, fixed schedule study design was employed. Fexofenadine, 60 mg, was administered orally before SJW dosing, with acute SJW dosing (900 mg), and after two weeks of SJW (300 mg tid) intake to determine p-glycoprotein activity.

Results: Acute administration of SJW significantly increased the Cmax and AUC of fexofenadine by 1.4- and 1.3-fold, respectively but did not alter the half-life or renal clearance. Chronic SJW administration caused a nonsignificant 0.85-fold decrease in fexofenadine AUC and Cmax relative to the untreated phase with no change in half-life or renal clearance of fexofenadine. When compared to the acute treatment phase, chronic SJW caused a significant decreases in fexofenadine AUC and Cmax of 0.66- and 0.62-fold and respectively.

Conclusion: Acute dosing of SJW administration resulted in an inhibition of intestinal p-glycoprotein and an elevated AUC of fexofenadine. Chronic SJW treatment reversed the inhibition seen on acute dosing and resulted in enhancement of intestinal p-glycoprotein activity and reduced AUC of fexofenadine.

Acknowledgement: FDA grant#FD-T-001756-01; the authors acknowledge the contribution of Drs. Jerry Collins and Peter Honig

Board R20

Cutaneous absorption of a topical corticosteroid product may be sufficient to result in systemic effects. Adrenal function may be suppressed by systemic exposure to the absorbed corticosteroid even in the absence of measurable plasma levels. Thus, the pharmacodynamic evaluation of adrenal suppression is potentially a more sensitive indicator than pharmacokinetic parameters of clinically relevant systemic exposure.

A review of the New Drug Applications (NDAs) for topical corticosteroids shows that different approaches have been used for testing adrenal suppression. The studies for testing two low-potency, two medium-potency and two high-potency topical corticosteroids available in the U.S. have been reviewed. These studies show the following differences in design: enrollment criteria, amount, area and duration of study drug application, time points for testing, use of controls, methodology and criteria for determining suppression, withdrawal criteria, and planning for data analysis. The conclusions drawn from these studies are dependent on the above factors in the study design.

It is important that testing for adrenal suppression be performed after maximal exposure of the lesions to the drug product commensurate with labeling conditions. Challenge using Cortrosyn is preferable to simple measurements of plasma or urine levels of corticosteroids. However, the optimal dose of Cortrosyn and the optimal time for testing remain to be determined.

Board R21

Three lots (A, B, C) of OTC soft-gel ginseng capsule product were evaluated for their solubilization characteristics. Capsules from each lot in 10 mL of water, 0.1% or 0.5% Triton-X at 37°C, were shaken and sampled after 3 and 5 days. The extent of solubilization, as measured by the total amount of ginsenosides in each sample by HPLC, seems to indicate lot to lot variability in the solubilization of the product within a medium, and also from medium to medium. For each lot, Triton-X100^® in the solubilizing medium appeared to improve overall solubilization. For lot A, the total amounts of ginsenosides were 411.4, 684.6 and 666.3 g/mL in water, 0.1% Triton-X100, and 0.5% Triton-X100 respectively for day-3. For lot B, the day-3 amounts were 578.3, 452.0 and 721.1 µg/mL in water, 0.1% and 0.5% Triton-X100 while lot C yielded 408., 702.7 and 820.5 g/mL ginsenosides in water, 0.1% and 0.5% Triton-X100 respectively. Day-5 produced 171.1, 626.5 and 675.5 g/mL for lot A, 425.8, 568.2 and 18.9 g/mL for B, and 398.3, 660.5, 683.7 µg/mL for lot C respectively in water, 0.1% and 0.5% Triton-100X. Solubility studies at different pHs with ginsenosides R_b1 and R_e indicated that they were stable at slightly acidic to neutral pHs but were completely degraded at very low pH. Under the study's conditions, there was complete dissolution of the ginsenosides within an hour.

Board R22

Providing scientific evidence underpinning the biological and toxicological activity of natural products being used in proposed or current clinical trials as chemopreventive agents is critical in providing guidance in determining the safety and efficacy of these agents. Chemoprevention, the intervention with specific agents to prevent, inhibit, or reverse carcinogenesis, is an important strategy for potentially controlling the occurrence and spread of cancer. Chemoprevention is relatively new and the development and evaluation of chemopreventive agents is a largely uncharted process. Additional mechanistic data are needed. Because of the lack of early diagnosis and poor therapeutic responsiveness of pancreatic cancer, survival beyond five years is rare and the median survival in most patients are less than six months. Currently, very few biochemical or molecular targets have been identified in pancreatic cancer. We have recently examined the chemopreventive potential of several dietary agents against pancreatic cancer. A number of these agents are natural constituents of the human diet. The public is increasingly consuming large amounts of these agents without proper information regarding their interaction with other approved drugs, or whether these agents respond differently in normal, diseased or cancer cells. In our in vitro cellular model, phytoestrogens and metabolites were found to strongly inhibit female pancreatic tumor cell growth; however, coumestrol and equol stimulated the growth of male pancreatic tumor cells. Genistein also demonstrated gender differences in the inhibition of growth of pancreatic tumor cells. Black and green tea extracts and components of tea extracts, such as the polyphenols and mixed theaflavins, were strongly inhibitory in both female and male pancreatic tumor cells. These agents have also shown positive effects on several biomarkers such as downregulation of K-ras, NF-kB, cyclooxygenase–2 (Cox-2), and kallikrein; upregulation of maspsin and MnSOD was shown in these studies. Changes in enzymes involved in activation and detoxification pathways were also noted. Modulation of the various genes demonstrated that these agents exert possible mechanistic action in pancreatic tumor cells through inhibition of oncogene expression, restoration of tumor suppressor function, inhibition of angiogenesis, and inhibition of basement membrane degradation, which is critical for the spread of cancer to other organs. We have recently proposed studies to further establish mechanistic and efficacy data on chemopreventive agents currently being used in clinical trials or those currently approved by FDA for pancreatic cancer. Studies will be carried out with these drugs alone or in combination with a number of proposed naturally-occurring chemopreventive agents which are currently being consumed in high amounts by the public.

Publish Only (R)

A high performance liquid chromatographic (HPLC) method using a thermal energy analyzer (TEA) for detection is described for the determination of diethanolamine (DEA) in shampoo products containing fatty acid diethanolamides. Shampoo products were dissolved in 6 N glacial acetic acid and mixed with sodium nitrite for one hour. The reaction mixture was then dried and redissolved in acetone for N-nitrosodiethanolamine (NDELA) determination by HPLC/TEA. For method validation, recovery of DEA from two shampoo products at fortification levels of 25, 250, and 1000 ppm ranged from 76 to 104%. The method was used to analyze twenty shampoo products containing fatty acid diethanolamides. Nineteen products were found to contain DEA at levels ranging from 140 to 4,060 ppm.

Board S02

The FDA Center for Devices and Radiological Health (CDRH) collaborates with the Conference of Radiation Control Program Directors in a unique federal-state partnership to characterize the radiation doses patients receive and to document the state of the practice of diagnostic radiology. Each year the Nationwide Evaluation of X-ray Trends (NEXT) survey program selects a particular radiological examination for study and captures radiation exposure data from a nationally representative sample of U.S. clinical facilities. Approximately 45 states provide radiation control personnel to conduct the surveys. CDRH staff compiles, analyzes, and publishes survey results on population exposure, radiographic and fluoroscopic technique factors, diagnostic image quality, and film processing quality. Surveys are repeated periodically to track trends as technology and clinical practices change. Since 1973 NEXT has been conducting surveys on examinations related to the adult chest, abdomen, lumbosacral spine, upper gastrointestinal fluoroscopy, mammography, computed tomography, dental radiography, and pediatric chest radiography. This exhibit will highlight the scope of the NEXT program and provide recent survey results and observations.

Board S03

The Center for Devices and Radiological Health is proposing nine changes to the U.S. x-ray equipment-performance standard that will reduce unnecessary radiation emitted during fluoroscopy. Principal radiation risks to patients are the potential for skin burns and the long-term possibility for cancer induction. To assess the impact of this proposal, we estimate the numbers of radiation burns that would be avoided, years of life that would be spared cancer mortality, and their respective pecuniary correlates. This analysis applies to upper gastrointestinal fluoroscopic examinations, percutaneous cardiac interventions, and the following proposed equipment requirements: (1) increased minimum half-value layer promoting more penetrating, skin-sparing x-ray energies, (2) limitation of x-ray beam area primarily to the region of clinical interest, and (3) displays of radiation emission rate and cumulative skin-entrance dose values to the operator during actual fluoroscopic examinations. Using demographic, exposure, dose, and risk data from various sources, we infer that the benefits of the amendments would exceed their estimated costs.

Board S04a

Although data-derived UFs are preferred over default values when establishing a Tolerable Intake (TI) value for compounds released from medical devices (ISO/DIS 10993-17), pharmacokinetic and pharmacodynamic data are rarely available to derive such values for a specific patient population. In the absence of the necessary data, a default UF of 10 is typically used to account for interindividual variability in the exposed population. Although this default UF is intended to account for variability in the response of the general population (including sensitive subgroups) to a toxic compound, it is not clear whether the default UF is adequately protective for critically ill or injured patients, since injury and illness can alter pharmacodynamic responses. To address this question, a literature search was conducted to identify the mean dose associated with a discrete pharmacodynamic endpoint (i.e., loss of righting reflex, seizure threshold, ED50, LD50) in healthy vs. diseased/injured experimental animals. The ratio of the mean doses associated with an effect in healthy animals:diseased/injured ranged from less than one to 250; however, the overwhelming majority of the ratios were <3 and most were <2, suggesting that the default UF of 10 is probably adequate to protect critically ill and injured patients from the adverse effects of compounds released from devices when they are exposed at doses equivalent to or less than the TI.

Board S04b

Long-term systemic toxicity testing is recommended for some implanted and externally communicating medical devices under the ISO 10993-1 standard. However, such tests are not only costly, but can also use a large number of animals, relative to other biocompatibility tests. Therefore, employing short-term screening techniques would be useful in determining when long-term, in vivo toxicity testing is appropriate for medical devices. CDRH is exploring a two-step process involving a "threshold of toxicological concern" and cytotoxicity data to improve this decision-making process. Although cytotoxicity data are currently used in a qualitative fashion in the biological evaluation of medical devices, recent analyses have suggested that cytotoxicity data can be used in a more quantitative manner to determine when in vivo testing is required. Linear regression of quantitative cytotoxicity (IC50) data and LOAEL values from long-term toxicity studies reveal a good correlation between these variables. As a result, it is possible to estimate IC50 values that correspond to the proposed threshold of toxicological concern of 0.1 mg/day. Although there are many details to be worked out before such a screening approach can be used in a meaningful way for regulatory decision-making (e.g., cell type, duration of test, endpoint), it appears that quantitative cytotoxicity data could be useful in helping to determine when long-term systemic toxicity testing would be appropriate for medical devices.

Board S05

According to 21 CFR 320.22, FDA may waive the Bioavailability/Bioequivalence requirement for oral solution dosage forms (e.g., elixirs, syrups, and other solubilized dosage forms). Sorbitol, a widely used sweetener in oral solution dosage forms (and food products) may reduce bioavailability of certain low intestinal permeability drugs by increasing the osmotic pressure of intestinal fluids. A replicate, two-way crossover clinical study was conducted in 20 normal healthy human volunteers to evaluate the relative bioavailability of ranitidine (a model low intestinal permeability drug) from solutions containing 5 grams of sorbitol or sucrose. This study was approved by RIHSC. Plasma samples were analyzed by a validated HPLC method. Peak ranitidine concentration in plasma (Cmax) and the area under the drug concentration – time curve (AUC) were reduced by about 50% for the sorbitol solution compared to sucrose solution. Data from this study suggests that a significant risk of bioinequivalence exist between sucrose and sorbitol containing oral syrup products for low permeability drugs.

Board S06a

Foodborne Listeria monocytogenes concentration data are sparse but they are generally accompanied by prevalence (presence/absence) data. In contrast, prevalence data are relatively frequent but are generally not accompanied by concentration data. This situation complicates the modeling of concentration frequency distributions in exposure estimations since the distribution shape(s) are unknown. Pooling data for all foods, while possibly less than ideal, was investigated as a potential stop gap simplification pending accumulation of more data. The logarithm percentage of samples with contamination ³100 cfu/g was directly proportional to the corresponding log % prevalence (r² = 0.68). The relationship accounted for 1 log (90%) of the 2-log variability range of the % ³ 100-cfu/g levels of contamination. The remaining 1 log variation was only partly due to the food category. Thus, log % prevalence values can be transformed to predict log ± 0.5 log % concentration values ³100 cfu/g. Also, log % concentration values ³ 10 cfu/g can be predicted. These observations provide an empirically based way to optimize the use of prevalence data which are unaccompanied by concentration data.

Board T01a

Three compounds that modify the effects of serotonin (d-fenfluramine HCl (DFEN), fluoxetine HCl (FLUX) and p-chloroamphetamine HCl (PCA)) were examined in male and female rats to compare their effects on perceptual and attentional processes. The acoustic startle response (ASR) and pre-pulse inhibition (PPI) of the ASR was measured before, during, and up to two weeks after drug administration. Male rats showed a larger startle response than females during drug administration and two weeks after drug cessation. During drug administration, DFEN (but not FLUX or PCA) significantly suppressed ASR in male and female rats. PCA and DFEN (but not FLUX) significantly reduced PPI in female rats only. Two weeks post-drug, PCA and DFEN increased PPI in female rats only. In addition, the suppression of ASR by DFEN was retained over the two week interval in females only. Further, PCA increased ASR in both sexes after the recovery interval, although it had no effect on ASR at dosing. Effects of serotonergic compounds on ASR (a measure of responsiveness) were similar in both sexes, although DFEN effects were only retained in females. However, effects of these drugs on PPI (a measure of information processing) are highly dependent on the gender of the animal. Imaging and histology are being used to correlate changes in behavior with observable changes in the brain caused by these drugs.

Group Award for Leveraging Activities - 2001 FDA Science Forum

Board T02

Seafood Toxins: Characterizing the Source and Nature of Toxins Accumulated by Shellfish
P.P. Eilers¹, S.M. Conrad¹, S. Hall¹, G. Langlois², J. Matweyou³, G. Plumley³, ¹CFSAN, FDA, Washington DC 20204, ²CA Dept. Health Services, Berkeley CA 94704, ³Univ. of Alaska, Fairbanks AK 99701

Natural toxins from plankton can cause serious human illness when accumulated by shellfish. As part of our mandate to assure seafood safety we need to characterize these toxins and the organisms that produce them. This poster describes some of our recent work in this area including studies of toxin composition in Alexandrium spp. and production of domoic acid by different populations of Pseudo-nitzschia.

Board T04

We investigated the role of oxidative damage in the biological effects photosensitized by aloe emodine (AE), a naturally occurring component of aloe vera. Skin fibroblasts were incubated with 20 µM AE for 18 hr. Fibroblasts were then exposed to UVA radiation (0 to 2.5 J/cm² ). Inhibition of colony formation was used to assess cytotoxicity. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoG) were used as markers for oxidative damage in cellular DNA and RNA, respectively. Treatment with 20 µM AE and 1.7 J/cm² UVA radiation inhibited the growth of fibroblasts by 50%. Similarly treated fibroblasts contained significant levels of oxidative damage in RNA ( 6.7 ± 1.7 8-oxoG/10⁵ G). Treatment with 20 µM AE and 2.5 J/cm² UVA radiation resulted in higher levels of cytotoxicity (85%) and oxidative damage in both RNA and DNA± 0.7 8-oxoG/10⁵ G and 1.5 ± 0.2 8-oxodG/10⁵ dG, respectively). Therefore, photodynamic activity of AE, resulting in oxidative damage to cellular components, may play a role in the photobiology of AE.

Board T05a

Phloxine B is a halogenated xanthene dye. The ability of phloxine B to induce transformation and mutation has not been determined. In this study we investigated cellular transformation photosensitized by phloxine B using C3H 10T1/2 mouse embryo cells treated with phloxine B plus visible light. Cells were incubated with 75 µM phloxine B in medium for 2 hours. After three washings with PBS, the cells, in PBS, were irradiated with various doses of visible light (0-3 J/cm²). The cells were then detached and replated in new 60 mm dishes. Positive controls were cells treated with 8-methoxypsoralen plus UVA radiation. Cell transformation was determined after incubation for six weeks by examining type II and III foci and calculating the transformation frequency. The results showed that the transformation frequency was 0.000157 ± 0.000073 and 0.011 ± 0.0047 at visible light doses resulting in 50% and 90% cell cytotoxicity, respectively. This preliminary study indicates phloxine B plus visible light treatment can induce transformation in C3H 10 T1/2 cells.

Board T05b

Better systems for evaluating the carcinogenic potential of new drugs, medical devices and food additives are needed. A large body of data indicates that mutations in the p53 gene are the most common genetic alteration in human cancer. We are working to develop an in vitro p53 mutagenicity screening system, using a strain of Saccharomyces cerevisiae constructed by Moshinsky & Wogan (Environmental & Molecular Mutagenesis 35: 31-38, 2000). This strain contains a plasmid with a human p53 cDNA insert. Cells containing mutations in the p53 gene can be detected via two reporter genes acting in concert to allow detection and selection of mutants. We have worked to optimize the generation and recovery of cells containing mutant p53. Variables include: the physiological state of cells during treatment, mutant expression time, mutant colony development conditions, conditions affecting differential survival of mutant and wild-type cells. We have also developed tests to differentiate true p53 mutants from other cells surviving the selection system, particularly those that have lost the p53 plasmid. Future work will include 1) characterization of the spectrum of p53 mutations that are recovered in the system; 2) comparison with the IARC data base of p53 mutations found in human tumors⁵; and 3) test of the mutagenic response of model carcinogens.

Board T06a

The TgrasH2 transgenic mouse, is a short-term carcinogenesis model, currently being used for pharmaceutical safety testing. Such alternative models are acceptable substitutes for one species in the conventional 2-year cancer bioassay. Confidence and widespread use of this model will require proof of transgene structural and functional stability over successive generations of breeding. This model was created by microinjection of the transgene into developing mouse zygotes. We have shown previously that transgenic models created this way may be prone to genetic instability if inverted repeats of injected DNA sequence are formed. Our labs have rigorously assessed transgene orientation in the TgrasH2 hemizygous mouse, which reportedly contains 5-6 copies of the transgene, a fragment of human hybrid DNA derived from normal, non-mutant, c-Ha-ras. A careful selection of DNA probes and traditional Southern blotting is necessary to perform a complete assessment of transgene orientation. We caution suppliers against relying solely upon PCR for quality control of their breeder colonies. We have analyzed the TgrasH2 mouse transgene sequence for the presence of potentially unstable head-to-head or tail-to-tail inverted repeats and have convincing evidence that none are, or ever were present.

Board T06b

Rapid advances in computer technology have allowed expansion of quantitative electroencephalography (EEG) techniques in neurotoxicology. EEG is applied to assess spontaneous electrocerebral activity using either scalp electrodes or electrodes implanted in specific brain regions. Ibogaine (IBO) and domoic acid (DOM) are both of interest to FDA because of their potential to produce neurotoxicity. Here, cortical EEG (ECoG) was monitored in adult, male Sprague-Dawley rats injected with IBO, cocaine and IBO or DOM. Frequency analysis based on the fast Fourier transform (FFT) technique revealed that IBO at a high dose decreased threshold for cocaine induced seizures. Rats treated with DOM had the seizures along with a significant increase in the low frequency ECoG. The results indicate that noninvasive, spectral EEG analysis may represent a useful approach in neurotoxicity assessment.

Board T07

Environmental "endocrine disruptor" chemicals may either modulate or mimic the actions of endogenoushormones on the normal course of brain development. We have studied exposures to the chemical intermediate Nonylphenol (NON), the contraceptive agent Ethinylestradiol (EE), and the phytoestrogen Genistein (GEN) (exogenous mimics of 17-b-estradiol), as well as the fungicide vinclozolin (VIN, an anti-androgen). Dams and their pups were dosed orally by adding either NON, GEN, EE or VIN to their feed from GD7 through weaning and until sacrifice at PND50. The sexually dimorphic central nucleus of the medial preoptic area (MPOC) of the hypothalamus, often utilized as a biomarker of estrogenic actions on the rat brain, was rendered in 3-D for computation of its volume. None of the compounds altered MPOC volume in female offspring. However, in males, moderate doses of NON, GEN, or EE each reduced the volume of the MPOC. There were no effects of VIN on MPOC size in either males or females. Following adjustment for variations in uterotrophic potency, it was observed that a similar dose range of each compound was associated with decreases in MPOC volume. This finding suggests that common properties of these endocrine disrupters may underlie their effects on both uterine and hypothalamic growth.

Board T08

High level soy trypsin inhibitor (TI) was fed to male swine from 1-39 wk of age. TI caused moderately lower feed consumption and reversible decrease in growth from 2 to 5 wk of age but not thereafter. All swine were active and had normal appearance and behavior. Gross and histopathological analysis of major organs except the spleen were within normal limits. TI increased extramedullary hematopoiesis in the spleen at 6 but not 39 wk, consistent with mild physiological macrocytic anemia. TI had no adverse effect on the pancreas. At 6 wk of age, TI increased pancreatic protein and amylase activity but not trypsin or chymotrypsin activity. Clinical chemistry, hematology, and plasma cholecystokinin were determined at 6, 18, 30, and 39 wk of age.

Board T09

Understanding the strengths and limitations of alternative models, like the Tg.AC assay, for evaluating the potential carcinogenicity of pharmaceuticals requires assessment of assay specificity through studies that specifically target biologically active compounds. To select drugs that might produce potential false positive results in the Tg.AC assay, we screened 100 noncarcinogenic drugs for in vitro induction of the gadd153 promoter, shown previously as correlative with Tg.AC activity. From this screening, amiloride, dipyridamole, and pyrimethamine were selected for testing in a skin paint study at MTDs or MFDs. No skin papillomas were observed in Tg.AC mice treated for 26 weeks with any of the 3 drugs, adding further favorable evidence of appropriate specificity for this model. Administration of the positive control compound in a 2.4% DMSO vehicle was shown to have a strong inhibitory effect on papillomagenesis by TPA.

Honorable Mention Poster - 2001 FDA Science Forum

Board T10

Women are exposed daily to D₄ because it is used in numerous personal care products including hair sprays, lotions, antiperspirants and breast implants. The estimated average daily intake of D₄ for a woman who used multiple products except breast implants is 0.158 mg/kg/day (11.1 mg/day). A physiologically- based pharmacokinetic (PBPK) model was developed to determine the bioavailability of D₄ leached from saline filled silicone breast implants and after repeated inhalation exposures of D₄ from personal products. The model was calibrated using previously published ¬¹⁴ C| D₄ distribution studies as reported by Kirkpatrick et al (1996). The results were extrapolated to simulate multiple exposures of D₄ in women by inhalation and breast implantation. The model was validated using published inhalation data in rats and humans. The kinetic results indicated accumulation of D₄ in women more than men in various target tissues such as fat, liver and kidneys following single and repeated exposures by IV, inhalation and implant exposure. Due to its high lipid solubility (Log P _oct/water = 5.1), D₄ persisted in fat with a half- life of 11.1 days following inhalation and 18.2 days following breast implant exposure. For women exposed to both personal care products and breast implants, the accumulation of D₄ in fatty tissues should play an important role in the risk assessment of breast implants.

Board T11