Relationship of Volatile Hydrocarbons to Lipids in Irradiated Meats
Kim M. Morehouse1 and Irwin A. Taub2, 1Food and Drug Administration, Center for Food Safety and Applied Nutrition, 200 C Street, SW, Washington, DC 20204, 2US Army Soldier Center, Natick, MA 01760
Hydrocarbons that are produced by ionizing radiation in fat-containing foods were analyzed. This analysis demonstrates that the types and relative amounts of these radiolytically-generated products are formed in a predictable manner. The data further show that the radiolytically-generated products from various meat products are all comparable. The use of the commonality and predictability of radiolysis products for the review of the safety of irradiated foods by the FDA will be discussed.
Detection and Classification of Bacteria Strains by Fourier Transform Near-Infrared Spectroscopy
L.E. Rodriguez-Saona1, F.M. Khambaty2, M.M. Mossoba2, F.S. Fry2 and E.M. Calvey2, 1JIFSAN-UMD,
2 CFSAN, Washington, DC 20204
The potential uses of Fourier-transform near infrared (FT-NIR) spectroscopy and multivariate techniques for rapid detection of bacterial contamination in solutions were evaluated. The complex cellular composition of bacteria yields FT-NIR vibrational transitions (overtone and combination bands) that can be used for classification and identification applications. Bacterial suspensions (E. coli HB101, E. coli O157:H7 ATCC43888 or Pseudomonas aeruginosa) were filtered through fiberglass or membrane filters (0.2 µm pore size) to concentrate the cells and eliminate the matrix which has a strong NIR signal. FT-NIR measurements were done using a diffuse reflection integrating sphere. Principal component analysis showed tight clustering of the bacterial strains at the information-rich spectral region of 5300-4000 cm-1. Preliminary ATR-FTIR (1800-900 cm-1) measurements of bacteria on the disposable filters gave spectral patterns typical of bacteria in the mid-infrared region. This methodology allows for the rapid and safe evaluation of potential bacterial contamination in liquids.
Determination of Polychlorinated Dibenzo-p-dioxins and Polychlorinated Dibenzofurans in Fish and Dairy Products by Quadrupole Ion Storage Mass Spectrometry/Mass Spectrometry
Tameka A. Bailey, R. Michael Glidden, E. Mark Harris, Jim R. Holcomb, Virginia L. Spencer and M. Paige Wilson, FDA, 3900 NCTR Rd. Jefferson, AR 72079
The 2,3,7,8-substituted PCDD/Fs have been classified as human carcinogens, with 2,3,7,8-TCDD being the most toxic. The analysis for PCDD/Fs in the US food supply has been ongoing within the FDA for several years now. The determinative step in the analysis has typically been made using HRMS. Recently the agency has utilized QISMS/MS as the determinative step. In the past year, FDAs Office of Regulatory Affairs collected and analyzed 144 samples of food for Dioxin with the QISMS/MS used in the determination step 131 times. 15 PCDD/Fs were measured using QISMS/MS and replicate analysis by HRMS produced comparable results.
1H NMR Direct Enantiomeric Purity Determination of the Chiral Sympathomimetic Drug, Ephedrine
G.M. Hanna, NRL, ORA, FDA, Brooklyn, NY 11232
Optical purity of ephedrine was obtained by fast diastereomeric interactions with a chiral solvating agent. Assignment of absolute configuration was based on relative field position of the resolved enantiomeric signals. Optimization of experimental conditions provided four significant resolved signals for quantitative use. Utilizing the relative intensities of the resolved enantiomeric signals, the analysis of synthetic mixtures of the enantiomers by the proposed NMR method resulted in assay values which agreed closely with the known quantities of each enantiomer in the tested mixtures. The optically pure enantiomers were used to establish the minimum amount detected by the proposed NMR spectroscopic method.
Nuclear Magnetic Resonance Simultaneous Analysis of Propantheline Bromide Structurally Related Impurities
G.M. Hanna, NRL, ORA, FDA, Brooklyn, NY 11232
A rapid, specific, and precise 1H-NMR spectroscopic method is described for the simultaneous analysis of propantheline bromide structurally related impurities, xanthene-9-carboxylic acid, xanthen-9-one, and 9-hydroxyxanthene. The impurities were calculated on the bases of relative intensities of corresponding signals at levels (mole/mole) of about 0.1%. NMR spectroscopy is potentially an exceptional technique for detection of impurities because it is sensitive to subtle changes in molecular structure, there can be equal sensitivity for all compounds (per nuclei), and there is no need for reference standards.
Direct Analysis of Trans Fatty Acids as Their FAMES by Gas Chromatography
A.P. Reid and D. Cantellops, Food and Drug Administration, 60 8th St. NE, Atlanta, GA 30309
A method is described for direct extraction and methylation of fatty acid methyl esters (FAMES) with an emphasis on the trans fatty acids. FAMES were produced by addition of sodium methoxide to the extraction solvent. After heating, the FAMES were separated by capillary GC (100m SP2560 column), quantitated with an FID detector and identified by GC-MS. A standard curve of trans FAMES over a concentration range of 0.01 mg/mL - 1.0 mg/ mL were analyzed (r>0.9990). Reproducibility was constant at each fatty acid level in the mixture. Analysis of a commercial margarine (n=10) gave a mean of 10.6g for total fat and 1.94g for the trans fat with %RSD of 2.27 and 2.08 respectively. SRM 1846 (milk based infant formula) was analyzed and the results closely compared to the certified fat and fatty acid values, (n=10) with a mean of 26.9 g for the total fat and 4.34g for the trans fat with %RSD of 1.57 and 1.50 respectively.
Stability of Ergot Alkaloids in Solution
George Ware, Mike Chasey and Paul Rader, FDA, 60 8th Street, Atlanta, GA 30309
The stability of 5 ergot alkaloids was studied for 8 weeks in different three solvent systems: methanol + water (1+1), acetonitrile + water (1+1), and an ergot alkaloid stabilizing solution (EASS) consisting of 100 g ethylene glycol, 100 g 1.2-propanediol and 1.0 g tartaric acid diluted to a liter with ethanol + water (25 +75). The stability of ergot alkaloids in each solvent system was studied at storage temperatures -18°, 25° and 40° C. Standards were analyzed by reversed phase HPLC with fluorescence detection. Decomposition of ergot alkaloid was evident in methanol + water and acetonitrile + water. The stability of ergot alkaloid decreased with increased storage temperature for both methanol and acetonitrile solution. The ergot alkaloid standards were stable for 8 weeks when stored in EASS at -18 ° C, and for about two weeks when stored at 25° C. Rate constants and half-life for ergot alkaloids standard will be discussed.
Identification of Oleandrin and Oleandrigenin in Oleander Extract by LC/MS and LC/MS/MS
Walden H. Lee and Kin S. Chiu, Pacific Regional Laboratory-Southwest, FDA, Los Angeles, CA 90015
Nerium Oleander is an ornamental shrub found in the sun belt areas of the United States. The plant contains "digitalis-like" cardioglycosides that are highly toxic to animals, including humans. Oleander has been reported to have therapeutic effects in cell proliferation disease and a patent exists for the production of an extract. Due to the unsubstantiated medical claim, the toxicity of the plant and its potential use as an herbal drug, an identification method for two toxic components, oleandrin and oleandrigenin, was developed. For regulatory purposes, the method uses reversed phase liquid chromatography (LC) with electrospray ionization/mass spectrometry (MS) and MS/MS detection.
Mycotoxins in Incaparina (Protein Supplement)
M.W. Trucksess, V.H. Tournas and K.D. White, CFSAN, FDA, Washington, DC 20204
Incaparina is mainly a mixture of cottonseed and corn flours. It is given to 80 percent of Guatemalan children in their first year of age to combat protein deficiency. The aflatoxins and fumonisins producing fungi such as Aspergillus flavus and Fusarium moniliforme are commonly found in corn and cottonseed. It is possible that one or more of these toxins may be found in the Incaparina. Five samples of Incaparina collected from different importers were examined for fungal contamination and analyzed for the toxins. A. flavus was the predominant fungus in all the samples whereas F. moniliforme was found in one of the samples. All samples contained aflatoxins ranging from 57-244 ng/g and fumonisins from 182-1256 ng/g. The identities of the aflatoxins were confirmed using a chemical derivatization method and LC/MS/MS analysis. The identities of the fumonisins were confirmed using LC/MS/MS analysis. Appropriate regulatory action was recommended for the import of Incaparina.
Aromatic Isocyanates: Convenient Derivatization Reagents for Chiral HPLC Analysis
C.A. Brunner1, W.M. Adams2, F.N. Russel3, R.L. Hunt1 and T.D. Doyle1, FDA, 1CDER, OTR, DPQR 2ONDC, Rockville MD, 3ORA, Atlanta, GA
Many chiral drugs and chiral food additive substances are difficult to resolve directly, may contain low-level enantiomeric impurities, or are present at low concentrations in biological fluids. The known facile conversion of primary and secondary amines to aromatic ureide derivatives, by rapid and quantitative reaction with the corresponding isocyanates, has been successfully applied in our laboratories to the stereo-chemical analysis of a wide variety of such problems. Alcohols are converted to carbamates, sometimes with remarkable procedural facility, as is illustrated by the analysis of racemic terfenadine. Examples include the resolution of racemic amphetamine, the detection of (S)-desoxyephedrine (methamphetamine) as a contaminant in the (R)-enantiomer, and the resolution and analysis of norephedrine at picogram levels in biological fluids. Food supplements based on amino acids, such as selenomethionine, will also be discussed.
Detection of Chromium (Cr) and Chromium Picolinate (CrP) in Dietary Supplements by Flame AAS and by LC-UV
Jeffrey A. Hurlbut, Susan B. Clark, W. D. Rowe and Sherri B. Turnipseed, FDA, PO Box 25087, DFC, Denver, CO 80225-0087
Two methods for analysis of Cr in dietary supplements are presented: a flame AAS analysis for Cr and an LC-UV analysis for CrP. Products containing CrP are water extracted, sonicated, filtered and used for either AAS or LC analysis. AAS analysis requires Na2SO4 as a masking agent, and the analysis is performed at 357.9 nm with a normal acetylene-air flame. LC analysis involves separation on a C18 column with 1:1 MeOH:H2O and detection at 266 nm. Nine supplements, with Cr levels ranging from 100 to 500 ug per dose, were analyzed. Recoveries based on label claims and RSD values were 103 % and 2.5 % by both methods. Spike recoveries ranged from 113 to 88 %. The levels of quantitation in ug Cr per dose were 50 for the AAS procedure and 0.6 for the LC method. The CrP used was synthesized, and the structure was confirmed by LC-MS.
Concurrent Determination of Four Fluoroquinolones; Ciprofloxacin, Enrofloxacin, Sarafloxacin and Difloxacin in Catfish, Shrimp and Salmon by LC with Fluorescence Detection
José E. Roybal, Calvin C. Walker, Allen P. Pfenning, Sherri B. Turnipseed, Steve A. Gonzales and Jeffrey A. Hurlbut, FDA, ADRC, Denver Federal Center, Denver, CO 80225-0087
An LC method is presented for the concurrent analysis of ENRO, CIPRO, SARA and DIFLX in catfish, shrimp and salmon. The procedure consists of extraction of tissue with acidified ethanol, isolation/retention on a cation exchange SPE column, elution with basic methanol and LC analysis with fluorescence detection. LC analysis is performed utilizing ACN/2%Acetic Acid (16+84) mobile phase with PLRP-S column and fluorescence detection, EX-278nm and EM-450nm. Data from the analysis of fortified and fluoroquinolone-incurred catfish, shrimp and salmon are presented.
LC/MS Confirmation of Ionophores in Animal Feeds
S.B. Turnipseed, J.E. Roybal, A.P. Pfenning and S.A. Gonzales, Animal Drugs Research Center, ORA/FDA, Denver, CO 80225
An LC/MS electrospray confirmation method has been developed to confirm four ionophores (monensin , lasalocid, salinomycin, and narasin) in a variety of animal feed using a single quadrupole mass spectrometer. The sodium ions of these compounds are dominant in the electrospray mass spectrum. Using optimized "in-source" collision induced dissociation, characteristic fragment ions seen previously using MS/MS can be observed. The drugs were extracted from the feed matrix using hexane/ethyl acetate and isolated using a silica solid phase extraction cartridge. These four ionophores were confirmed in both medicated feeds and non-medicated feeds fortified with these drugs at the 1-50 ppm level.
The Use of Differential Scanning Calorimetry and Thermogravimetric Analysis for the Drug Profiling of Pharmaceutical Finished Dosage Forms
George Pyramides1, Michael Levin2 and Louis P. Lue2,1Philadelphia Drug Lab, Custom House, 2nd & Chestnut Streets, Philadelphia, PA, 2 Northeast Regional Laboratory, 850 Third Avenue, Brooklyn, NY
The Northeast Regional Laboratorys Drug Profile Group has been using Thermal Analysis as one of the techniques for the Profile Analysis of Generic and Innovator Finished Dosage Forms. The analysis has been fine-tuned over the course of several years and a more uniform approach has been developed. This approach will be described using Ticlopine Tablets as an example. The analysis is multifaceted with the primary focus being the detection of substitution of one product for another and the batch formulation confirmation. Thermogravimetric Analysis was used to differentiate the Innovator from Generic products and gave a preliminary qualitative identification of the active ingredient and several excipients. Differential Scanning Calorimetry was used to further confirm individual components. Method of addition techniques, which consisted of individual components in varying amounts added to the powder from ground tablets, were used to probe the content of the tablets. The spiking experiments pointed to relative amounts of individual components and allowed for an estimate of the content. The resulting thermal curves were used to map out the effects of differing amounts of individual components on the thermal traces. This analytical procedure has proven useful in detecting product substitution.
Detection of Peanut Agglutinin in Chocolate and Cookies by Enzyme-Linked Immunosorbent Assay
T.Y. Yi, A.W. Smallwood, T.W. Brueggemeyer and R.D. Satzger, Forensic Chemistry Center, ORA, FDA, Cincinnati, OH 45237
Undeclared peanuts in foods often cause severe allergic reactions among sensitive individuals. In this report, peanut agglutinin (PNA) was used as a marker for peanuts in foods. An Enzyme-Linked Immunosorbent Assay (ELISA) was developed to determine the presence of PNA in chocolate and cookies. Affinity purified anti-peanut agglutinin (anti-PNA) and enzyme labeled anti-PNA were applied to 96-well microplates to capture and detect PNA. PNA was extracted from homogenized samples by phosphate buffered saline (PBS) and the extract was applied to the microplate for the ELISA. Control samples containing various nuts were analyzed as a check against false positives. The reported method reliably detected PNA at levels greater than 1 ng/ml. No false positive results were obtained at or above the detection level. A few samples showed possible cross reactivity with anti-PNA.
Rapid Screening for Organochlorine and Organophosphorus Pesticide Residues in Fruits and Vegetables Using a Sold Phase Extraction Cleanup
F.J. Schenck and Michael K. Hennessy, Southeastern Regional Laboratory, FDA, Atlanta, GA 30319
A rapid multiresidue solid-phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of pesticide residues in fruits and vegetables has been developed. The method is a modification of published methods developed by regulatory agencies in Canada and New York State. Pesticides are extracted from fruit and vegetable samples with acetonitrile and water is separated from the extract by salting out. Matrix coextractants are removed from the extract using tandem graphitized carbon black (GCB) and aminopropyl SPE columns. Acetone/toluene (3:1) was used as an elution solvent. By optimizing the SPE elution solvent and size of the GCB SPE column, we were able to significantly reduce the volume of solvent required for the SPE cleanup step compared to previously published SPE methods. Recovery studies were performed using samples containing both fortified and incurred pesticide residues.
Determination of Organochlorine and Organophosphorus Pesticide Residues in Eggs Using a Solid Phase Extraction Cleanup
F.J. Schenck1, D. Donoghue2 and H.Righter2, 1Southeastern Regional Laboratory, FDA, Atlanta, GA 30319, 2Center for Veterinary Medicine, FDA, Laurel, MD 20708
The AOAC method for the determination of pesticides in eggs will not recover the polar organophosphorus pesticides. A multiresidue solid-phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of nonpolar organochlorine and polar organophosphorus pesticide residues in eggs is described. The method uses an acetonitrile extraction followed by an SPE cleanup using graphitized carbon black and aminopropyl SPE columns. Organophosphorus pesticides are determined by gas chromatography with flame photometric detection. After further cleanup of the extract using Florisil SPE columns, organochlorine pesticides are determined by gas chromatography with electron capture detection. Studies were performed using eggs containing both fortified and incurred pesticide residues.
Differentiation between Real and Fake Shark Fin
George C. Ziobro1 and Luis A. Solorzano2, 1CFSAN, Microanalytical Branch, Washington, DC 20204, 2San Francisco District Laboratory, Alameda, CA 94502
A method has between developed to distinguish between real and fake shark fin. Shark fin is used in the cuisine of many cultures from the Pacific Rim, and it has gained popular medicinal usage as a cure for cancer. Shark fin currently markets for approximately $40.00 per pound. FDA labs encounter shark fin and there can be economic incentive to counterfeit real shark fin. Because no method exists to differentiate real from fake shark fin, we have developed a methodology to authenticate real from fake shark fin. Microscopic examination of fake product shows little internal detail and transverse striations when compared to authentic shark fin, which has a defined internal structures and no striations. Carrageenan [seaweed extracts] and sodium alginates are used in the food industry as thickening agents. This method relies on the principle that the gelling of carrageenan is a reversible process. Fake shark fin dissolves in sodium citrate and EDTA, while real shark fin that is composed of protein fibers will not.
Detection of Erythromycin-Resistant Determinant Genes in Human Clinical Staphylococcus aureus Strains by PCR
Khan, M. S. Nawaz, A. A. Khan and C. E. Cerniglia. Division of Microbiology, NCTR, FDA, Jefferson, AR 72079
A polymerase chain reaction method was used to detect the presence of erythromycin rRNA methylase genes ermA, ermB and ermC and the multicomponent efflux pumps msrA and msrB in twenty-five multidrug-resistant human clinical Staphylococcus aureus strains. All the strains possessed the multicomponent efflux pump, 88% (22) had the ermA, 72% (18) had the ermB and 4% (1) had the ermC gene. The ermC gene was found on a 2.5-Kb plasmid and the other genes were located on the chromosome. The presence of ermB gene in clinical isolates was surprising because the gene has been reported to be present mostly in the isolates of animal origin. Further, the lower occurrence of ermC over the ermA gene was also significantly different from the earlier observations where the ermC is believed to be predominantly present in erythromycin-resistant organisms. The restriction fragment length polymorphic (RFLP) analysis revealed four different EcoRI RFLP patterns (21.0, 10.5, 6.2 and 5.0-kb) of the ermA gene. Two-three copies of the ermA gene were found integrated into the chromosome.
Detection of Shigella by the Polymerase Chain Reaction: Culturing and Template DNA Preparation
Walter E. Hill1 and Keith A. Lampel2, 1ORA, FDA, Bothell, WA 98021, 2CFSAN, FDA, Washington, DC 20204
Shigella species can cause foodborne gastroenteritis and may have an infectious dose as low as 10 cells. Therefore, sensitive methods are needed to detect pathogenic strains in foods. We have used a PCR- assay targeting the ipaH gene. Microbial flora from lettuce samples was seeded with Kanr Shigella and after 6 hours incubation in Shigella Broth, initial levels as low as 5 cells/ml could be detected. Growth of Shigella in novobiocin, as recommended in the Bacteriological Analytical Manual, often decreased test sensitivity, as did incubation with kanamycin or overnight culturing. Studies with Percoll® gradients to separate foodborne PCR inhibitors were unsuccessful, possibly due to the strong inhibitory effect of Percoll® on PCR. Assay sensitivity was increased when washed cells were added directly to PCR tubes when compared with boiled cells. This project was supported by funds from OS/CAFDAS.
Predicting the Neurovirulence Potential of Viral Vaccines for the Human CNS: The Use of Mumps Virus for Model Development
S. A. Rubin, M. Pletnikov, R. Taffs, P. Snoy and K. Carbone
Development of an in vivo system for evaluating viral neurovirulence would be of enormous benefit in vaccine development as well as in the study of host-specific mechanisms involved in virus-induced nervous system disease. Here, mumps virus strains representing a wide range of neurovirulence in humans was used for the development of a model of viral neurovirulence using neonatal rats, a host shown to be highly sensitive to perturbations by perinatal virus infection. The relative neurovirulence of each mumps virus strain was assessed based upon histological examination of the brains for neuroanatomical abnormalities. Results demonstrated that the presence and severity of hydrocephalus was proportional to the severity of the strains known neurovirulence in humans. In addition, significant cerebellar developmental brain damage was noted in virus strains considered highly neurovirulent. If these findings can be reliably obtained in independent testing in other laboratories, there would be compelling evidence for the replacement of the poorly predictive monkey neurovirulence safety test for mumps virus vaccines with the more predictive neonatal rat model. Further, the neonatal rat model will be of great utility in elucidating the molecular pathogenesis of neurovirulence by mumps and other viruses.
Detection of Infectious Cryptosporidium Parvum Oocysts Following Disinfectant Treatment by Cell Culture-PCR
Patrick M. Regan1, James A. Wilson1, Charles R. Clavet1 and Aaron B. Margolin2, 1FDA, WEAC, Winchester, MA 01890, 2University of New Hampshire, Durham, NH 03824
Cryptosporidium parvum is a coccidian parasite that is excreted in the fecal material of an infected individual. Studies have shown that this organism is resistant to glutaraldehyde-based disinfectants. In immunocomopromised individuals it can be life threatening. Reusable endoscopes may become contaminated with C. parvum. Therefore, the ability to determine the efficacy of a disinfectant to inactivate C. parvum is important. Here we report on a cell culture nucleic acid amplification system to determine the oocysts infectivity after disinfectant treatment. It was found that with an oocyst challenge of 104 oocysts, infectivity could be detected by cell culture-PCR following a 90-min treatment in 2.5% glutaraldehyde. The use of a molecular-based technique to determine the infectivity of germicide treated C. parvum will provide information on the efficacy of liquid chemical disinfectants to inactivate C. parvum.
Multidisciplinary Assessment of Conventional and Innovative Approaches of Determining Adverse Effects of Known Neurotoxic Agents
D. S. Lester, H. Davis, P.S. Pine and J. P. Hanig, FDA/CDER, Division of Applied Pharmacologic Research, Neural and Cellular Research Program
Current approaches to neurotoxicity assessment include routine neurological and behavioral testing and/or conventional histological procedures. New imaging technologies may provide a more sensitive way to detect and identify potentially adverse neuronal effects. These innovative methodologies have yet to be compared with more commonly used methods. Subjects are treated with one of three pharmaceuticals, whose effects are known to range from no effect to neurotoxic. Neurotoxic effects range from destruction of cell layers to diffuse lesions. The effects of the agents currently under study range from no effect to neurotoxic. The approaches compared in these studies can be divided into three distinct categories: 1) Behavioral testing (acoustic startle, locomotor activity). 2) Histochemical staining (Nissl, silver stain, Fluorojade, GFAP). 3) Imaging approaches (autoradiographic biomarkers, magnetic resonance imaging microscopy, midinfrared spectral imaging). The comparison of data from each of these approaches using the same treated animals will provide an opportunity to evaluate the capabilities of each of these techniques and their potential contribution in the identification of potential agents that may cause adverse nervous system effects.
Effect of Dietary Restriction on Lymphocyte Hprt Mutant Frequency in Aging Rats
A. Aidoo, M.E. Bishop, R.A, Mittelstaedt, L.E. Lyn-Cook, P.H. Duffy and R. Heflich, NCTR, FDA, Jefferson, AR 72079
Dietary restriction (DR) reduces tumor incidence and retards aging in experimental animals. In the present study, we investigated whether or not DR affects the accumulation of somatic cell mutations in aging animals. Starting at 1.5 months of age, male CD rats were placed on different levels of dietary intake, ad libitum feeding, and 90%, 75% and 60% of the total calories consumed by ad libitum animals. Six, 12 and 24 months later, mutant frequencies were measured in the Hprt gene of spleen lymphocytes isolated from the rats. Mutant frequencies increased with age and were significantly higher in ad libitum rats compared with 60% DR rats: 4.5x10-6 vs. 3.3x10-6 (p<0.05) in 6-month DR rats; 10.3x10-6 vs. 7.1x10-6 (p<<0.05) in 12-month DR rats; and 17.4x10-6 vs. 7.8x10-6 (p<0.01) in 24-month DR rats. In addition, 24-month DR rats given 75% of the calories of the ad libitum diet had a mutant frequency, 10.7x10-6, between 60% and ad libitum rats. Rats in the 90% DR group had a mutant frequency, 18.2x10-6, similar to the ad libitum rats. Multiplex PCR of the Hprt gene in mutant clones from 12 and 24 month rats revealed deletions in 19% of mutants from ad libitum rats and 42% of mutants from 60% DR rats. However, because of the difference in Hprt mutant frequency in the tow groups, the estimated mutant frequency associated with deletions in DR rats (3.2x10-6) was very similar to the deletion mutant frequency in ad libitum rats (2.5x10-6). The results suggest that dietary factors are involved in spontaneous somatic cell mutation as well as in age-associated disorders such as cancer and that the excess mutant frequency found in ad libitum rats mainly is due to point mutation.
Developing a PCR-PNA-ELISA Procedure for Rapid Detection of Point Mutations in Drug Resistant Mycobacterium Tuberculosis Genes
L.E. Bockstahler1, Zhongming Li3, K.A. Van Houten1, N.Y. Nguyen2, J.J. Langone1, S.L. Morris2 and M.J. Brennan2, 1CDRH, 2CBER, FDA, 3Currently at American Red Cross, Rockville, MD 120857
The emergence of drug resistant strains of Mycobacterium tuberculosis (DR-MTB) remains a serious public health problem. New methods for the rapid diagnosis of DR-MTB are needed. We are developing a PCR-peptide nucleic acid (PNA)-based ELISA as a diagnostic using point mutations in katG, a gene associated with isoniazid resistance in MTB. Thus far we have established the hybridization temperatures (50-55oC) and other experimental conditions suitable for detection of two clinically relevant point mutations in the katG gene using 15-mer PNA probes. Hybridization of PCR-amplified MTB DNA sequences that contain these point mutations with mutant-specific, complementary PNAs result in ~ 6-9 fold increases in ELISA response compared to hybridization using MTB wild type-specific PNAs. Conversely, wild type MTB sequences hybridize more efficiently at 50-55oC with wild type PNAs than with the mutant-specific PNAs. Using this method, the DR-MTB strains can be identified in 24 hrs.
Loss of Palindromic Transgene Symmetry Generates a "Nonresponder" Phenotype in Tg.AC Transgenic Mice
B.A. Rosenzweig1, R. Honchel1, K.L. Thompson1, C. Strupczewski1, K.T. Blanchard2, S.M. Furst2, R.E. Stoll2 and F.D. Sistare1, 1CDER, FDA, Laurel, MD 20708, 2Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877
A subset of Tg.AC mice used for carcinogenicity testing of pharmaceuticals was found to be refractory to carcinogens. Both responders and nonresponders have ~40 copies of transgene but nonresponders had lost a 2 kb BamHI fragment hypothesized to result from palindromic transgene sequence. Restriction enzyme experiments revealed two subsets of nonresponder Tg.AC mice in which the 2 kb BamHI fragment had been reduced in size to 0.9 kb or 1.9 kb. We cloned and sequenced a PCR product derived across the putative "head-to-head" junction of the 1.9 kb BamHI fragment of nonresponder DNA. Our results confirm the palindromic orientation of the transgene in Tg.AC mice and show that loss of "head-to-head" symmetry in the structure, even by a deletion as small as 75 bp, results in loss of transgene expression.
Selection of Drugs to Test the Specificity of the Tg.AC Assay by Screening for Induction of the gadd153 Promoter In Vitro
K.L. Thompson, D.D. Broud and F.D. Sistare, CDER, FDA, Laurel, MD 20708
To assist in selecting pharmaceuticals to test the limits of specificity of the Tg.AC short-term carcinogenicity assay, a rapid throughput in vitro assay was developed that is predictive of in vivo activity in the Tg.AC assay. This assay measures activation of a hamster gadd153 promoter construct that is stably expressed in HepG2 cells. Of the 100 noncarcinogenic pharmaceuticals that were screened in the gadd153 assay, ~20% induced the gadd153 promoter. Several criteria were used to select the 3 best candidates for subsequent in vivo testing in a Tg.AC assay: whether drug solubility in acetone or ethanol was sufficient to elicit systemic toxicity, the level of promoter induction, in vitro potency, and cost. Testing whether noncarcinogenic pharmaceuticals that activate select stress response pathways in vitro can elicit papillomas in the Tg.AC skin paint assay will accelerate the evaluation of the specificity of the Tg.AC assay and assist in defining its appropriate usage in the safety testing of pharmaceuticals.
Development of a Standard Methodology for Screening Medical Device Materials for Alternative Pathway Complement Activation
Daniel B. Lyle, Grace S. Bushar and John J. Langone, Molecular Biology Branch, OST/CDRH/Food and Drug Administration, Rockville, MD 20857
Complement is a group of blood proteins that are part of the immune system. Inappropriate activation either by the antibody-dependent "classical" pathway or the antibody-independent "alternative" pathway can have serious adverse health effects. Some medical device materials when in contact with patient blood are known to activate complement by the alternative pathway. Materials being considered for new medical devices need to be tested for alternative-pathway complement activation. This paper describes the development of an inexpensive, rapid, function-based standard methodology for screening materials for alternative pathway complement activation based on rabbit RBC hemolysis. Results are validated by immunoassay for C4d and Bb complement pathway-specific components.
Quantitative, Biologically Relevant Parameters for Testing and Standardization of Skin Response to UV
The FDA Photosciences Network with contributors in, CDER, Laurel, MD 20708, CDRH, Rockville, MD 20852, CFSAN, Washington, DC 20204, NCTR, Jefferson, AR 72079, NCI/NIH, Bethesda, MD 20892
This study evaluates the utility of novel (both non-invasive and biomarker) methods in assessing skin responses to ultraviolet radiation (UV). Coordinated observations are carried out on humans in CDRH and NCI, guinea pigs and rats in CFSAN, transgenic mice in CDER, and hairless mice in NCTR. The use of comparable methodologies for humans and for animal models makes it possible to assess the validity of cross-species extrapolations related to UV-induced skin damage. This, in turn, should allow us to propose new approaches to the evaluation of the safety and efficacy of UV exposure-related products. Such FDA-regulated products include drugs, sunscreens, cosmetics, and sunlamps. The FDA is currently revising a number of public health policies in this area.
How to Measure UV Response in Human Skin?
J.Z. Beer, H.F. Bushar, D.J. Chwirut, M.N. Ediger, W. Lee, S. Matchette, S.A. Miller, H. Lopez, S. Weininger and B.Z. Zmudzka, CDRH, Rockville, MD 20852, E. Toombs, CFSAN, Washington, DC 20204, H. Jorgensen, Washington Hospital Center, Washington, DC 20010, K.S. Korossy, Kensington, MD 20895, V.J. Hearing, N. Kobayashi, T. Tadokoro, NCI/NIH, Bethesda, MD, 20892, E. Lenderink, Philips Research, Eindhoven, The Netherlands
A range of non-invasive and biomarker methods for measuring UV responses in human skin is being assessed. The observations are being carried out on a cohort of 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. Biopsies are analyzed for DNA damage and repair, and for melanin production and distribution. Preliminary data indicate that, in addition to the visible erythema and pigmentation, UV causes skin changes that are discernible with almost all the techniques examined.
Response of SKH-1 Mouse Skin to Simulated Solar and UVB Radiation
P.C. Howard, R.L. Sams, II, B.J. Miller, W.T. Allaben, NCTR, Jefferson, AR 72079, C. Okerberg, T.J. Bucci, Pathology Associates International, Jefferson, AR 72079, W.G. Wamer, CFSAN, Washington, DC 20204, J.Z. Beer, CDRH, Rockville, MD 20852, J.R. Bucher, NIEHS, Research Triangle Park, NC 27709
The FDA/NIEHS Phototoxicology Research and Testing Laboratory (PRTL) has been established at the NCTR in response to research and testing needs within both agencies. The facility has the capacity to expose large numbers of mice to simulated solar or fluorescent lamp generated (UVB) radiation. The response of the skin in SKH-1 mice to simulated solar and UVB radiation under conditions that will be used in upcoming tumor studies with SKH-1 mice is being evaluated. Markers for the response of the skin include epidermal thickness, epidermal basal cell proliferation rates, sunburn cell formation, and DNA damage. The data that are generated in these preliminary studies will be compared with the data from the UV/skin studies at the other FDA Centers, and will serve as the basis for quantifying the response of skin in unrestrained SKH-1 mice to electromagnetic radiation.
Biological Markers for Assessing the Effects of Cosmetics on Sensitivity to Ultraviolet Radiation
W.G. Wamer, R.R. Wei and A. Kornhauser, CFSAN, FDA, Washington, DC 20204
Recently, concerns have arisen about a new class of photosensitizing cosmetic ingredients. These "indirect" photosensitizers do not absorb UV radiation (UVR) yet increase UV-induced damage by altering the skin's optical properties or biochemical responses to UVR. We are using the guinea pig as an animal model to identify this type of photosensitizing cosmetic ingredient. Our recently completed pilot studies demonstrate that measurements of the minimal erythema dose, UV-induced thymine dimer formation and sunburn cell formation are sensitive markers for UVR-induced damage in the guinea pig. Using these endpoints, it is possible to assess changes in sensitivity to UVR equivalent to 20% of the minimal erythema dose (95 mJ/cm2 in the guinea pig). Additionally, these markers provide information on the mechanism(s) underlying increased sensitivity to UVR. This mechanistic information will aid in predicting the long term risks associated with increased sensitivity to UVR elicited by cosmetic ingredients.
Noninvasive Characterization of Diabetic Cataracts in Psammomys Obesus (Sand Rat)
V.M. Chenault, M.N. Ediger, FDA, Rockville, MD, C. DiCarlo, Uniformed Services University, Bethesda, MD
Psammomys obesus is a wild rodent in the subfamily Gerbillinae that inhabits the desert areas of the Middle East and Africa. This animal is unique in that it develops mild to moderate obesity, hyperglycemia and the complications of diabetes such as cataracts and vision loss when it consumes a high caloric diet. The cataracts produced will be characterized using conventional ophthalmic; dynamic light scattering and histologic techniques in conjunction with the biochemical blood parameters used in the assessment of diabetes.
Characterization and Creation of Defects in Condoms
L.N. Kerr, M.P. Chaput, S.M. Boyd, E.A. Galevi and P.A. Millward, WEAC, FDA, Winchester, MA 01890
Condom defects were characterized and compared with defects made using various creation techniques. Eighty-five percent of the condom defects examined were classified as either a hole (void in material) or a slit (puncture). Laser drilling and puncturing with a 160 um diameter acupuncture needle artificially introduced similar types of defects. Microscopic examination of the created defects, before and after water leak testing, showed that using the FDA water leak method does not increase the size of pre-existing microscopic defects. Examination also showed that these creation techniques produce reproducible defects within a condom type, with the size of acupuncture needle defects varying less than laser drilled defects. Results of water leak testing showed that the leakage characteristics of defects are affected by: the material type, the condom shape and size, the type of defect and the defect size, and the presence of lubricant.
Characterization of Radiated and Conducted Electromagnetic (EM) Energy from Fluorescent Lights
W.S. Boivin, S.M. Boyd, J.N. Coletta and L.N. Kerr, WEAC, ORA, FDA, Winchester, MA 01890
Concerns over radiated and conducted EM disturbances, and public exposure to EM fields from compact fluorescent lamps (CFLs) and fluorescent light (FL) fixtures demonstrate a need for FDA to investigate these sources. Radiated and conducted emissions were measured around 11 CFLs and 5 FL fixture configurations. Changing to energy efficient lighting reduced lower frequency EM fields and increased higher frequency fields. Spectrum analysis showed that FL fixtures and CFLs are broadband sources of EM radiation. Conducted disturbances were detectable from CFLs but very small compared to standards and not detectable from FL fixtures. All measurements were below standards limits for human exposure. When investigating device EMI, hospitals should be aware that converting to energy efficient lighting changes the EM environment.
Determination of Optimal Locations for Measuring the Maximum Rotational and Translational Forces Experienced by Metallic Patient Implants in MRI Systems
W.S. Boivin, S.M. Boyd, J.N. Coletta and L.N. Kerr, WEAC, ORA, FDA, Winchester, MA 01890
In a magnetic resonance imaging (MRI) unit, the strong static magnetic field and its spatial gradient may cause implantable metallic medical devices to experience rotational and translational forces. Four different MRI systems were mapped to identify the location of the maximum static magnetic field, and to calculate locations of the maximum field gradient and gradient product using a 3-axis Teslameter, thereby identifying the optimal locations for MR compatibility testing of metallic implants. For the 1.5 tesla (1.5T) MRI units, the optimal test location was 30 to 40 cm inside the face of the bore and near the walls of the bore. Translational forces would found to be higher in shielded than in unshielded units. The optimal test location for rotational force was at the isocenter for all of the 1.5T units.
The Evaluation of Various Papers for Usefulness in Condom Water Leak Testing
William S. Boivin, Louis G. OMalley and Vincent J.B. Pierdominici, Jr., WEAC, ORA, FDA, Winchester, MA 01890
Twelve different absorbent papers were evaluated for usefulness in condom water leak testing. One analyst placed 1 µl drops of water onto a 3x3 grid marked on each paper. Another analyst examined the paper, outlining any visible drops with a pen. Numerical scores were given to plot size and to comments about visibility and contrast. The sum of these two scores was used to rank the papers for usefulness in detecting water drops during condom leak testing. High contrast was the most important factor in determining the optimum paper. Brown blotter paper and brown paper hand towels tied for the highest score. Brown blotter paper was preferred over paper towels by the researchers, but only slightly. Brown paper hand towels cost much less than blotter paper and are therefore recommended for all sample analysis and research applications. Consistency can be improved by holding a container of brown paper hand towels aside exclusively for sample analysis.
A Simple Validation Model for Quantifying 2-D Flows in Medical Devices
R.A. Robinson and R.A. Malinauskas, CDRH, FDA, Rockville, MD 20852
A simple method was developed to validate and quantify any flow visualization system using digital particle image velocimetry (DPIV). It is important to assess accurately the flow characteristics of some medical devices (heart valves, blood pumps, vascular grafts, catheters, etc.), because aberrant flow can contribute to thrombus production and hemolysis, and ultimately to device failure or harm to the patient. This method uses a replaceable sanding belt, driven by a D.C. servomotor, to move actual or computer-generated particles in a well-defined linear motion. The linear speed of the belt is calibrated to an accuracy of 0.1% using a NIST traceable hand tachometer. This model provides for analyzing steady as well as pulsatile flows as found in human blood flow. The analysis of our test system demonstrated that a DPIV velocity measurement error of 1-3% was achievable for test velocities of 70 mm/s to 1000 mm/s. Accurate assessment of medical device flow characteristics can contribute to device longevity and an improvement in medical device safety.
Biaxial Flex-Fatigue and Viral Permeability of Oven-Aged Natural Rubber Latex Gloves
M.R. Schwerin1, D.L. Walsh1, D.C. Richardson1, C.D. Lytle2, R.W. Kisielewski1, L.B. Routson3 and R.M. Kotz1, 1CDRH, FDA, Rockville, MD, 2Formerly of CDRH, FDA, 3Visiting Scientist, PATH, Seattle, WA
Latex gloves, artificially aged at 70:C, were subjected to flexing and viral challenge to assess barrier integrity. A unique biaxial fatigue apparatus utilized an electrical signal to indicate a change in barrier properties, while viral permeability was determined using a modified version of ASTM F1671-97a. Results indicate that unaged powdered and powder-free gloves had significantly longer fatigue lives than unaged chlorinated gloves from the same manufacturer. Aging caused a significant change in the mechanical properties of the chlorinated gloves, but only a marginal increase for non-chlorinated gloves. When a change in the barrier properties was indicated by the electrical signal, viral passage was generally found. Viral permeability results for gloves that were not flexed showed no degradation in barrier integrity due to oven-aging alone.
Adverse Events Associated with Hemostasis Devices
B.A. Gallauresi, CDRH, FDA, Rockville, MD 20850
The Food and Drug Administration (FDA) has received numerous death and serious injury reports associated with the use of hemostasis devices since 1996. Hemostasis devices are used to seal off the femoral artery puncture site which is accessed for diagnostic and interventional cardiac procedures. Three types of hemostasis devices have been approved by FDA and are currently marketed: (1) Extravascular, a collagen plug placed a the femoral artery puncture site; (2) Intravascular, a collagen plug in combination with a biodegradable anchor placed inside the femoral artery; and (3) Surgical Suture, arterial closure is accomplished by deployment of sutures around the arterial entry site. The major problem reported with the use of these devices is bleeding from the femoral artery, which may result in death by exsanguination. Possible factors contributing to bleeding include patient selection, user training, and insertion techniques. The deaths and serious injuries reported to FDA have raised concerns about the role device user interface plays in adverse outcomes associated with hemostasis devices. As a result, FDA issued a notification to users recommending that all manufacturer warnings and instructions regarding patient selection and device use be carefully followed.
Modeling Rupture Rates of Silicone-Gel Breast Implants.
G. Penello1, S. L. Brown1, R. Natarajan2 and D. Feigal, CDRH, FDA, Rockville, 1CDRH, FDA, Rockville, MD 20850, 2U. Florida, Dept. of Statistics, Gainesville, FL 32611
In a recent study conducted by the FDA, 344 women underwent MRI examination to assess the rupture status of their silicone-gel breast implants. These women were among 907 women who completed an interview in which they were asked about the rupture status of any explanted implants. The MRI data show a peak and then a decline in rupture rates as a function of implant age (time since implantation), which suggests heterogeneity in the risk of rupture among women and/or implants. In this paper, we analyzed the interview data and the MRI data together, and modeled heterogeneity in the risk of rupture through random effects (frailties). Self-reported ruptures of explanted implants were verified by surgical records, when available. We modeled the data with parametric and semi-parametric survival models and compared their fits on the basis of deviance. We estimated odds ratios of rupture for implant age, implant type (single or double lumen), implant location (submuscular or subglandular), and implant manufacturer, and estimated implant age for a given probability of rupture. An important feature of the data is that implant age at time of rupture was never known, but interval censored according to the time of discovery of rupture.
Gender-Related "Higher-Than-Expected" Drug-Event Combinations in Spontaneous Adverse Drug Event Reports
Ana Szarfman, M.D., Ph.D., QMR, Office of Biostatistics, CDER, FDA
The large Food and Drug Administration (FDA) Spontaneous Reporting System (SRS) database is the most important of its kind in the U.S. Its primary objective is for the study of adverse drug effects and it is used to identify and document rare events not detected during randomized controlled clinical trials. It contains over 1.8 million records collected between 1968 to the present. We used the full SRS database to apply a new empirical Bayesian (EB) data mining method developed by William DuMouchel (The American Statistician, August 1999). We detect Gender-Related Signal Scores (SS) by computing the EB estimates separately for each gender and then comparing them visually by gender, drug, classes of drugs, events, and groups of events. Our studies show that women have higher SS than men in association with severe adverse drug events, including "torsades de pointes", "QT prolongation", "agranulocytosis", "bleeding", "renal events", "pancreatitis", and "liver events" with a higher number of drugs. This higher association is independent of drug use by gender. In contrast, "anaphylaxis", Stevens Johnson Syndrome", or "toxic epidermal necrolysis" do not show that higher SS are associated with a specific gender. Our results support the existence of higher SS in women in association with severe adverse drug events with old and newly marketed drugs. *Supported with a grant from OWH, OC, FDA.
The Medical Device Surveillance Network (MeDSuN)
S. Gardner and M. Flack, CDRH, FDA, Rockville, MD 20852
The increasing complexity of medical technology increases the potential for unanticipated and unintended consequences. The 1997 Food and Drug Administration Modernization Act provides the FDA with the opportunity to design and implement a national surveillance network, composed of well-trained clinical facilities, to provide high-quality data on medical devices in clinical use (MeDSuN). This program will enable FDA to improve its ability to detect and analyze medical device-related problems. To the extent that product failures can be identified before patients are injured, FDA can join with manufacturers and health care professionals in creating a safer health-care environment. It is important that this system will give FDA ready access to a network of clinical facilities that could potentially offer clinical insight into investigation of device problems and participate in specific research and educational efforts. FDA is now in the Phase 2 pilot for the MeDSuN program.
Developing Calibration Standards: The MQSA Calibration Facility
O.H. Suleiman1, D.C. Spelic1, S. Belella and M.S. West2, 1CDRH, FDA, Rockville, MD 20850, 2Mentor Technologies, Inc.
The Food and Drug Administrations Center for Devices and Radiological Health (CDRH) annually calibrates 300 sets of field equipment, specifically sensitometers and densitometers, which are used by the FDA inspection force to annually assure that approximately 10,000 clinical mammography facilities, certified by FDA, are in compliance with film quality standards under the Mammography Quality Standards Act (MQSA). Although national standards for densitometry exist and a primary standard is available by the National Institute of Standards and Technology (NIST), such standards are still undergoing development for light sensitometry. The development of a standard reference light sensitometer and how this unique FDA research is fulfilling a fundamental need in supporting FDAs regulatory mission will be presented.
SAS/AF Applications as Review Tools
Ted Guo, Ph.D., Mathematical Statistician, HFD-715, Rm. 10B-06, 5600 Fishers Lane, Rockville, MD 20857
Since 1996, a number of SAS/AF applications have emerged as software tools in assisting statistical reviews for CDER's statistical reviewers. These applications function under Windows platform, expand SAS's power and functionality, and handle some of the most frequent tasks in statistical reviews. These applications include 1. Carcin (for the carcinogenicity-study review); 2. Quickinfo99 (for the assessment of carcinogenicity-data-format conformity to the FDA's Guidance Document); 3. AnalystPro (for data exploration and management); 4. Stable97 (for the stability analysis). These applications are available to licensed SAS users at CDER and are downloadable from CDER's web site.
Testing Requirements for Automated External Defibrillators (AEDs)
C.C. Carey and W.Y. Saperstein, ODE, CDRH, FDA, Rockville MD 20850
The major breakthroughs in defibrillation current waveform together with the technical sophistication permitting extensive automation in function fueled development of a new generation of external defibrillators. These automated external defibrillators analyze heart rhythm to detect ventricular fibrillation and deliver the appropriate electrical energy to convert the pattern to normal rhythm. Public interest in these "smart defibrillators" has increased substantially, as well as support to implement the Chain of Survival concept in emergency cardiac care. AEDs are now being deployed in environs other than the traditional clinical setting or in ambulances. "First Responders" who are medically authorized using these new devices can now participate more effectively in treating cardiac arrest situations. This presentation will examine the technical issues and define the pre-market testing requirements needed to ensure that advances in AEDs are both safe and effective for the treatment of sudden cardiac arrest in this enlarging domain of use.
The Nationwide Evaluation of X-ray Trends 1998 Survey of Pediatric Radiography
A.E. Moyal, O.H. Suleiman, D.C. Spelic, R.V. Kaczmarek and R.J. Slayton
The Nationwide Evaluation of X-ray Trends (NEXT) program is a cooperative effort of FDAs Center for Devices and Radiological Health (CDRH) and the various radiation control programs to collect information regarding patient exposure and image quality. Each year surveys are performed on a single diagnostic x-ray modality. The survey collects information related to radiation dose and image quality. Past and future surveys include computed tomography (CT) (1990, 2000), dental radiography (1993, 1999), adult chest radiography (1984, 1986, 1994), abdomen and LS spine examinations (1987, 1989, 1995), and fluoroscopy (1991, 1996). In 1998, a first time survey of facilities performing pediatric chest radiography was conducted.
Measurement-Methods Comparisons for Medical Devices
L. Yue, G. Gray and S. Zhou, CDRH, FDA, Rockville, MD 20850
In an equivalence study for a new diagnostic device, the performance (e.g., accuracy and precision) is evaluated by comparing measurements from the new device to those obtained from a reference. In an ideal situation, a perfect standard (a so-called "gold" standard) exists and can be used as the reference. Therefore, the comparison can be made through calibration. In practice, however, the use of a gold standard may be too expensive or laborious, thus it is replaced by an imperfect standard. In this case, the measurements obtained from both the new device and the imperfect standard are subject to random measurement error. Ignoring the effect of the measurement error in the imperfect standard leads to bias in parameter estimates and inferences. A less well known problem is overcorrection for measurement error in the imperfect standard by misuse of orthogonal regression. We show the correct calibration and the effects of ignoring and overcorrecting for measurement error in imperfect standards. Examples will be given to illustrate the concepts and to promote more correct ways of accounting for measurement error.
Effect of Reprocessing on Hemodialyzer Performance: How Premarket Data Can be Used to Revise an Existing FDA Guidance Document
M. C. Provost1 and T.C. Lu2, CDRH, FDA, 1ODE, 2OSB, Rockville, MD 20850
Beginning in February 1997, FDA has required hemodialyzer manufacturers to submit 510(k)s seeking approval to relabel their devices for multiple use. The manufacturers collected data on the effects of each recommended reprocessing method on key hemodialyzer performance parameters, including ultrafiltration coefficient and clearance of standard small and large molecules. A database was constructed that contained all of the bench and clinical data submitted in fourteen 510(k) submissions from 12 different manufacturers. Statistical tools were used to analyze the data for significant findings. The results permitted a critical evaluation of the testing requirements described in an existing guidance for multiple use dialyzers. Revisions to the guidance document can then be made that are based on sound science. By limiting the requirements to only the most relevant tests, FDA can minimize review resources and industry can use the least-burdensome path to marketing clearance.
Analysis of Mined Clay Products for PCDDs/PCDFs By High-Resolution Mass Spectrometry (HRGC/HRMS) and Quadrupole Ion Storage Mass Spectrometry/Mass Spectrometry (QISMS/MS)
Jim Holcomb, Joseph Ferrario and Christian Byrne, FDA, Arkansas Regional Laboratory, 3900 NCTR Road, Jefferson, AR 72079, *EPA, OPPTS, Environmental Chemistry Laboratory, Stennis Space Center, MS 39529
A recent preliminary survey by the Center for Veterinary Medicine was conducted to determine the PCDDs/PCDFs concentrations in mined clay products used in manufacturing of animal feed ingredients. This limited survey includes collecting samples of clays, as well as, any other mined product that may be used in the processing of plant protein meal and finished feed. EPA extracted and analyzed a set of sixteen samples by HRGC/HRMS. The remainder of the extracts were then shipped to the FDA laboratory for analysis by QISMS/MS. The purpose of this study is to demonstrate that the quadrupole ion storage mass spectrometer can be used as a possible alternative determination or, supplemental technique in the analysis of PCDDs/PCDFs.
Group Sequential Testing for Superiority and Non-Inferiority Hypotheses in Active Controlled Clinical Trials
Sue-Jane Wang1, H.M. James Hung2, Yi Tsong3, Lu Cui2 , 1DBII/OB/CDER/FDA, Rockville, MD 20857, 2DB I, 3QMR
In a group sequential active controlled clinical trial, the study hypothesis may be a superiority or a non-inferiority hypothesis. When it is necessary to plan for testing the superiority and the non-inferiority hypotheses, we propose an adaptive group sequential closed test strategy by which the sample size is planned for testing superiority and is to be increased for showing non-inferiority given that it is deemed more plausible than superiority based on the observed sample path during the course of the trial. The proposed adaptive test strategy is valid in terms of having the Type I error probability maintained at the targeted a level for both superiority and non-inferiority. It has power advantage or sample size saving over the traditional group sequential test designed either for testing superiority only or for testing non-inferiority only.
Development of an in-vitro IgE test for Ethylene Oxide Hypersensitivity: A CRADA Study and Technology Transfer Agreement with Diagnostic Product Corporation, Inc., National Institutes of Health and Centers for Disease Control and Prevention
Kearby Fugate, Ph.D.1, S. Vadlamudi, D.V.M., Ph.D.1, Paul Turkeltaub, M.D.2and Charles Noah3, 1 CDRH, FDA, Rockville, MD 20850, 2 CBER, FDA, Bethesda, MD 20892, 3 ORA/FDA Denver, CO 80225
This Cooperative Research and Development Agreement (CRADA) is an extramural development of technology transfer with Diagnostic Products Corporation, Los Angeles, California 900454.
The purpose of the research is to develop and assess the effectiveness of a diagnostic product used to detect immunoglobulin E (IgE) mediated hypersensitivity in patients exposed to ethylene oxide sterilized medical devices (e.g., renal dialysis equipment).
Interest in ethylene oxide (EO) stems from its continuing importance as a sterilant of implantable devices, whose surfaces come in contact with body internal tissues, including blood. There have been reports of adverse reactions to EO in the scientific literature; nevertheless, currently available information is not amenable to provide a basis for projecting the incidence of hypersensitivity of EO in the U.S. population.
The Diagnostic Products Corporation provided sufficient RAST/ELISA test kits for testing sera for ethylene oxide hypersensitivity in the study populations. The sera for the study were generously provided from stored frozen sera banks by NIH and CDC investigators. These stored sera were collected from patient populations known to have been exposed to medical devices sterilized with EO.
The results from this study will be presented and discussed.
Development of a Guidance Document for a First-of-a-Kind Implantable Middle Ear Hearing Device
T. Cygnarowicz, M.A. and K. Baker, MSN, Ear, Nose and Throat Devices Branch, CDRH, FDA, Rockville, MD, 20850
Several clinical trials are currently ongoing for implantable middle ear hearing devices, an emerging hearing technology. As FDA anticipates submission of its first Premarket Approval Application (PMA), it concurrently is developing a guidance document for the arrangement and content of a PMA for this new device area. The document will guide regulated industry and FDA review staff regarding the processing, content and evaluation of regulatory submissions. Development of such guidance requires input from individuals from a variety of disciplines, including areas of biomedical research, academia, and industry. This approach offers FDA the opportunity to gain an interdisciplinary perspective for a first-of-a-kind device. In addition, current knowledge of existing hearing technologies, published literature, and input that was obtained during a recent ENT Devices Advisory Panel Meeting will result in a useful and applicable guidance for this emerging technology.
Utilities of the P-Value Distribution in Medical Research
H.M. James Hung1 and Robert T. ONeill2 1DBI, OB, CDER, FDA, Rockville, MD 20852, 2OB, CDER, FDA, Rockville, MD 20857
P-value is a widely used measure of strength of statistical evidence in medical research. In clinical trials, the P-value is often cited in rendering a decision of whether to reject a null hypothesis and accept an alternative hypothesis of interest. It is well known that under the null hypothesis, the distribution of the P-value based on a continuous test statistic is uniform over the interval [0, 1], regardless of the sample size of the experiment. In contrast, under the alternative hypothesis, the distribution of the P-value is a function of both sample size and the true value of the tested parameter. This presentation will explore potential utilities of P-value distribution in the design, analysis, and interpretation of results of clinical trials. Other related subjects to be considered include issues of replicability of statistical evidence in clinical trial settings. *The contents of this talk are partially based on the work of Hung, ONeill, Bauer and Köhne (1997, Biometrics, pp. 11-22).
Cefotetan-Induced Hemolytic Anemia: Review of 85 Cases
R.Viraraghavan M.D., M.P.H., DTM&H,1 A. Chakravarty Ph.D. ,2A. Szarfman M.D., Ph.D.3 and J. Soreth M.D.1, 1DAIDP, 2Division of Biometrics III, 3OPDRA, FDA, Rockville,MD 20850
Cefotetan use and reports of hemolytic anemia have prompted an FDA review of the overall number, severity, and causality of such cases. Concurrently, Zeneca Pharmaceuticals has submitted a request to strengthen the labeling to further promote awareness of this adverse event. A search of the Spontaneous Reporting System (SRS) and the World Health Organizations database revealed 85 cases of hemolytic anemia since Cefotetans approval in 1985, 14 of them fatal. Moderate degree of hemolysis was reflected in a drop in hemoglobin level by 5.3 mg/dL (n=13) and final hemoglobin level of 5.2 mg/dL (n=53). Transfusion was required in 48 patients (56%). New onset renal dysfunction was noted in 7 patients (8%). Direct antiglobulin test was positive in 50 patients (59%), and serologic studies revealed antibodies to cefotetan in 30 patients (35%). These data suggest that treatment with Cefotetan may induce a potentially severe autoimmune hemolytic anemia.
A Statistical Reviewers Perspective on Non-Inferiority Medical Device Clinical Trials with the PDP Process
Lilly Q. Yue and R. Lakshmi Vishnuvajjala, CDRH, FDA, Rockville, MD 20850
In this presentation, as statistical reviewers, we will discuss some frequently encountered problems with the Product Development Protocol (PDP) process in designing non-inferiority medical device clinical trials. The problems mainly consist of the specification of maximum tolerable difference to claim non-inferiority of study device, the specification of population parameters used in sample size calculations, the reality and sensitivity of a significance test, statistical methodology proposed in data analysis, and the method of handling missing values. Some of the problems seem trivial to statisticians, but are often overlooked. However, these issues are critical to the success of a clinical trial due to the special requirements of the PDP process, but not always clear to device company management. Examples are given to illustrate some of the issues.
The Importance of Predefined Differences in the Analysis of Adverse Treatment Effects
V. Freidlin, R. Srinivasan and H.S. Ko, CDER, FDA, Rockville, MD 20857
When a safety problem is anticipated for a drug, and a study is conducted to support a specific safety claim, relevant evidence similar to that for efficacy claims should be collected. An equivalence margin delta should be prespecified in the protocol and the 95% confidence interval approach used.
A number of examples will be presented to illustrate the point. In one example, reduction in ejaculate volume was anticipated for usage of a new drug. A specialized safety study, purported to demonstrate no difference between test drug and placebo, prespecified a delta of 10% as being clinically acceptable. However, the study had insufficient sample size and the confidence interval for the treatment difference fell outside the prespecified +/- delta. In spite of this, the sponsor concluded that there was no difference between test drug and placebo, because the treatment difference was non-significant (p=0.9). The examples will underscore the importance of defining appropriate criteria and implementing them in the analysis of adverse treatment differences. It is essential to note that if a safety study is not adequately powered, the trial may fail to show equivalence despite a small difference between the treatments.
Identification of Virus Genotypes Using Microarray Analysis
V. E. Chizhikov and K .M. Chumakov, CBER, FDA, Rockville, MD 20852
The main goal of the present study was to design oligonucleotide microchips for the rapid and precise discrimination of genetically related viruses. As an experimental model we used well-characterized genomes of three poliovirus serotypes. Oligonucleotides complementary to several serotype-specific regions of the VP1 coding sequence were synthesized and attached to the surface of glass microscope slides. The efficiency of hybridization of each oligonucleotide with Cy5-labeled poliovirus DNA probes was quantified using a laser fluorescence scanner. We found that the designed oligonucleotide microchip yielded a unique pattern of spots specific for each poliovirus genotype. Wild-type and attenuated polioviruses could be easily differentiated, opening the prospect of using this technology to control safety of live viral vaccines. In some cases one nucleotide mismatch was sufficient to differentiate between poliovirus serotypes. The use of this approach for the identification of other viruses will also be presented.
B Lymphocytes Serve as a Possible Extrahepatic Site for HCV Replication
Yuan Hu, MD, FDA, Northeast Reginal Laboratory, Brooklyn, NY 11232
Hepatitis C, a single-stranded RNA virus, is the etiologic agent in most cases of posttransfusion hepatitis in the Unite State. During the long-term study of hepatitis C virus (HCV), we failed to observe 100% correlation between the HCV replication and serious liver damage. To study possible extrahepatic sites for the replication of hepatitis C virus, the T and B-lymphocyte cell line in vitro infected by hepatitis C RNA positive serum was performed. We extracted the HCV RNA from infected, noninfected cell line and final cell wash supernatant. Nested RT-PCR and quantitation PCR amplified hepatitis C RNA was performed. Direct localization of hepatitis C RNA in the infected cell by in-situ PCR was also studied. We accurately analysed nested RT-PCR products by southern blot hybridization, quantitation PCR products by liquid hybridization, and in-situ PCR products by in-situ hybridization. All of three different hybridization used HCV specific biotinylated oligonucleotide probe. The PCR was sensitive and specific in identifying HCV in this study. Hepatitis C RNA detected only in the infected B-lymphocyte cell line but none of the noninfected cell line and T-lymphocyte cell line. The results suggect that B-lymphocyte may also allow hepatitic C viral replication. The detection of HCV RNA in B-lymphocyte is believed to be a sensitive and reliable indicator of infectivity. Further understanding of the role of hepatitis C viral replication in peripheral blood mononuclear leukocytes may lead to the ability to control hepatitis C infection. (This research work was done in the Bronx Lebanon Hospital Center)
Detection and Characterization of Erythromycin Resistant Methylase Genes in Gram-Positive Bacteria Isolated from Poultry Litter
M.S. Nawaz, S.A. Khan, A.A. Khan, R.S. Steele, J.V. Pothuluri and C.E. Cerniglia, Division of Microbiology, The National Center for Toxicological Research, Jefferson, AR 72079
The epidemiology of 4 different erythromycin-resistant methylase (erm) genes, ermA, ermB, ermC and msrA genes were determined in erythromycin-resistant staphylococci, enterococci and streptococci isolated from poultry litter. Most of these isolates were resistant to multiple antibiotics. Southern hybridization indicated that 4 of the 25 staphylococci contained ermC gene on various sized plasmids. Twenty one of the 25 staphylococci harbored the ermA gene exclusively on the chromosome. The ermA was found as a double chromosomal insert on a 8.0 and 6.2 kb EcoRI fragment in all ermA-positive staphylococci. Six of the staphylococci harbored the msrA gene. Dot-blot analysis indicated that all enterococci and streptococci hybridized with the biotinylated ermB gene probe. Southern hybridization analysis indicated that only 2 of the 19 erythromycin-resistant enterococci contained the ermB gene on the plasmid. The gene was localized on a 1.8 kb and 1.4 kb plasmid respectively in two E. faecium isolates. Our results indicate that the use of erythromycin for the treatment of staphylococcosis can select for erythromycin-resistance in Gram-positive bacteria in poultry litter.
Regional Expression of Heat Shock Protein-72 (hsp72) Induced in Rat Liver by the Hepatotoxicant Cadmium (CdCl2)
P.L. Goering1, R.R. Dugyala1, B.R. Fisher2 and R.J. Marler2, 1CDRH, FDA, Rockville, MD, 2Covance Laboratories, Vienna, VA
Many toxic agents enhance the expression of hsps, a cellular response that may be a useful biomarker of toxicity in pre-clinical test batteries. CdCl2 is lab tool we use that is known to produce necrosis across the liver. Determining the cell-specific localization of markers such as hsp72 will allow us to understand their roles in cell injury. Rats received single, sub-necrotic, iv dosages of CdCl2. No treatment-related histopathological changes were observed. No immunostaining signal specific for hsp72 was observed in untreated controls. The most intense signal was observed 16 hr after exposure to 2 mg Cd/kg. Hsp72 accumulation was predominantly localized in the cytoplasm of centrolobular hepatocytes, primarily around venules. These results demonstrated that hsp72 expression in response to acute Cd exposure occurs in hepatocytes located in centrolobular areas and adjacent to larger intrahepatic venules prior to the onset of overt cytotoxicity.
Phosphodiesterase Type III (PDE III) Inhibitor Induces Vasculitis Preceded by Apoptosis in Mesenteric Vessels and Lymphoid Tissues of Rats
J. Zhang1, E.H. Herman1, J.L. Weaver1, D.P. Chadwick1, D. Broud1, B.A. Rosenzweig1, A.D. Knapton1, V.E. Whitehurst2 and F.D. Sistare1, 1CDER, FDA, Laurel MD 20708, 2CDER, FDA, Rockville, MD 20857
Vasculitis has been seen in nonclinical studies associated with a number of PDE inhibitors but the relevance of these findings in determining human dosing and evaluating human risk is uncertain. Greater mechanistic understanding and accessible biomarkers predictive of this insidious and potentially life threatening toxicity are needed. In the present study we assessed the time course and a greater definition of the earliest manifestations of pathology following a single s.c. injection of 100 mg/kg of the PDE III inhibitor SK&F 95654. Our findings indicate that apoptotic cell death of EC and SMC may play an important role in the pathogenesis of SK&F 95654-induced vascular toxicity in rats and that this response is also observed in peripheral blood leukocytes.
Felbamate Metabolism to Potentially Toxic Metabolite(s) by Human Liver Tissue In Vitro
NR Hartman1, IM Kapetanovic2, C Lu3, CDTorchin2, AP Li3, HJ Kupferberg2 and JM Strong1,
1
Aplastic anemia and hepatic failure have been reported with use of the anticonvulsant drug Felbamate (FBM). The major FBM urinary metabolite identified in humans is 3-carbamoyl-2-phenylpropionic acid (CPPA) produced by oxidation of the mono-carbamoylated metabolite (MCF). Reactive intermediary metabolites during the oxidation of MCF to CPPA were proposed including an aldehyde (CMBA) which could undergo reversible cyclization to a more stable compound (CCMF) or b-elimination to yield a proposed toxic metabolite, a, b-unsaturated aldehyde (ATPAL). Our study demonstrated the metabolic conversion of MCF to CCMF and formation of the reactive metabolite ATPAL by human liver tissue including isolated hepatocytes (fresh and cryopreserved) in vitro. Hepatocyte viability, measured by the tetrazolium dye MTT, was significantly compromised when incubated with the postulated toxin, ATPAL.
FDA Inter-Center Neurotoxicity Working Group
T. Sobotka, B. Wilcox and W. Slikker
The FDA is a science-based regulatory agency with broad responsibilities for promoting and protecting the public health. The credibility and confidence in the FDA's regulatory decisions are based, in part, on the Agency's ability to apply sound science in its review and regulatory process. Efforts are needed to ensure that regulators have the appropriate expertise and necessary information in various scientific disciplines for making appropriate decisions based on high quality science. To assist the FDA in addressing regulatory concerns related to neurotoxicity, personnel from the Centers within the Agency, who are actively engaged in neuroscience-related research or regulatory activities, have organized an Inter-Center Neurotoxicity Working Group. Primary objectives of the group include promoting dialogue between regulatory and research scientists across the FDA, identifying and acting on current and significant issues of Agency relevance in the neurosciences, establishing collaborative research projects, and serving as an Agency resource for neuroscience expertise and information. The efforts of this Group to foster scientific communication within the FDA will enhance the ability of the Agency to address existing regulatory issues and anticipate future problems in the areas of neurobiology, neuropharmacology and neurotoxicology, to keep pace with rapidly emerging and complex technologies, and to respond in a timely, flexible and competent manner to new regulatory needs.
Differential Cytokine mRNA Expression in Swine Whole Blood and PBMC Cultures
H.F. Yancya.b, S.L. Ayresa and M.J. Myersa, aCVM, Laurel, MD 20708, bHoward University, Washington DC 20017
The kinetics of IL-2, IL-6, IL-8, and IL-10 gene expression were examined in concanavalin A (Con A)-activated whole blood (WB) and peripheral blood mononuclear cell (PBMC) cultures using RT-PCR. WB cultures reached maximal levels of production 24 hrs after addition of Con A, while PBMC IL-2 mRNA levels reached peak levels at 12 hrs. Peak WB IL-6 mRNA was achieved 12 hrs after stimulation, whereas PBMC IL-6 mRNA expression peaked at 24 hrs. WB cultures had peak levels of IL-8 6 hrs post Con A, while PBMC cultures did not achieve maximal production until 12 hrs after stimulation. Peak levels of IL-10 mRNA in WB cultures were observed 12-24 hrs post Con A stimulation, whereas peak levels in the PBMC cultures were observed 6 hrs post stimulation. This method results in a culture systems with reduced cost and time compared to traditional cell culture and isolation methods and overcomes the lack of assays for measuring cytokine protein levels.
Mutational Analysis of Avidity and Fine Specificity of Anti-Polysaccharide Antibodies
Kurt Brorson, Cynthia Thompson, George Wei, Michael Krasnokutsky and Kathryn E. Stein, Division of Monoclonal Antibodies, CBER/FDA
Using the polyfructose, bacterial levan (BL), as a model polysaccharide (PS), we analyzed how variable (V) regions affect binding in anti-PS monoclonal antibodies (mAb). Previously, panels of mAb were constructed from BL-immunized BALB/c and CBA/Ca mice (Boswell and Stein, J. Immunol. 157:1996, 1996). The BALB/c mAb were mostly germline VHJ606:Vk11, and a subset contained presumed somatic mutations in the CDR regions which correlated with increases in avidity for the b(2->1) inulin linkage of levan. The CBA/Ca mAb were more heterogeneous in V gene usage, but a subset of inulin-non-reactive mAb were VHJ606:Vl and had VH sequence differences in the CDR regions from the VHJ606 regions of the BALB/c mAb. In this report, VHJ606 antibodies containing various combinations of specifically mutated heavy and light chains were produced by engineered transfectants and tested for inulin avidity and levan binding. Two presumed somatic mutations seen in CDRs of the BALB/c hybridomas were shown to directly cause marked increases in avidity for inulin (VH N53H, 9 fold; VL N53I, 20 fold; together, 46 fold) but not for
b(2->6) levan. Exchange of either positions 50 or 53 in VH or the H3 loop between the BALB/c and CBA/Ca mAb resulted in either fine specificity shift or total loss of BL binding. 3-D models of the V regions suggested that residues that affect binding to inulin alone are near the edge of the CDR surface, while residues involved with binding both forms of levan and affect fine specificity are in the VH:VL junctional area.
Detection of Multidrug-Resistant Salmonella Typhimurium DT104 by Multiplex Polymerase Chain Reaction
Ashraf A. Khan, M. S. Nawaz, S. A. Khan and Carl E. Cerniglia, Division of Microbiology, Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079
Salmonella typhimurium definitive type DT104 is a pathogen for humans and animals with many strains having multiple drug resistance characteristics. The organism typically carries resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT-resistant). A multiplex PCR method was developed to simultaneously amplify four genes, florfenicol (flost), virulence (spvC), invasion (invA), and integron (int) from S. typhimurium DT104 (ACSSuT-type). Twenty two ACSSuT-resistant DT104 isolates in our collection gave 100% positive reactions to this PCR assay by amplifying 584-, 392-, 321- and 265-bp PCR products, using primers specific to the respective target genes. One Salmonella strain DT23, ACSSuT-resistant, phage type771 failed to amplify the 584-bp fragment, indicating that this method is specific for DT104 type ACSSuT resistant S. typhimurium strains. One clinical and one bovine ASSuT resistant strains that were sensitive to chloramphenicol and florfenicol did not yield a 584-bp fragment, indicating the absence of the flost gene. The method developed can identify ACSSuT-type DT104 strains from clinical, food environmental samples.
Update on the Recovery of Cold-Injured Campylobacter Jejuni from Inoculated Foods with the Blood-Free Enrichment System
T. T. Tran, FDA, Washington, DC 20204
The lowest recovery levels of cold-stressed Campylobacter jejuni in modified blood-free enrichment broth (mBFEB) and enriched cultures of crabmeat, mushrooms, raw milk, and raw oysters were determined. C. jejuni cells were cold-stressed before inoculation into mBFEB containing 10% food rinses. After a 24 and 48 h enrichment under ambient atmosphere, C. jejuni were isolated on modified CCDA-Preston agar. The overall mean recovery limit 50% end point (RL50) values, irrespective of food types and enrichment time, for the 5-, 10-, 20-, and 30-day stress procedure were 0.2, 1.3, 5.5 and 10.9 cfu / 5 g test portion, respectively. The effectiveness of the mBFEB decreased as injury time increased from 5 to 30 days. Further, with the increase of injury time, greater variations in recovery rates within and between foods were observed. Increase of enrichment time from 24 to 48 h did not appear to significantly improve (P>0.05) the recovery limits. This study indicates that C. jejuni cells survived well up to 30 days at 4-50 C and that the mBFEB system was effective in recovering cold-injured C. jejuni from the four foods studied at a contamination level of 1-20 cfu /5g food.
Microbiological Profile of Fresh Produce
T.T. Tran, R.L. Thunberg, R.N. Matthews and R.W. Bennett, FDA, Washington, DC 20204
The microbiological quality of fresh produce obtained at retail outlets (DP) as well as imported produce (IP) was investigated. Test portions were blended, filtered, and analyzed for aerobic (APC), Gram-negative (GNC), coliform (CC), and yeast and mold (YMC) counts. The overall arithmetic mean APC, GNC, CC, and YMC values were, respectively, 7.7, 7.3, 5.4, and 4.9 log10 cfu / g for the DP; and 8.1, 7.7, 6.4, and 6.3 for the IP. The CC represented approximately 74-83% of the GNC which, in turn, represented 95% of the APC. Yeasts accounted for 95% of the YMC. Of 191 samples (19 produce categories) tested, all were negative for E.coli O157:H7, Campylobacter and Salmonella. E. coli and/or Listeria spp., including L. monocytogenes, were found in 37 samples. The findings of E. coli and L. monocytogenes raised concerns over the risk of cross contamination among produce, especially at farmers markets and imported wholesalers. In the preparation of foods in the home, decontamination by washing with tap water and/or tap plus chlorinated water, and complete separation of ready-to-eat vegetables from other food products, especially raw meat, would reduce the risk of cross-contamination and ensure a much safer product before consumption.
Antibiotic Resistance Integrons in Shiga Toxin-Producing Escherichia Coli (STEC)
1
S. Zhao1, D. White1, S.Ayers1, S. Friedman1, B. Ge2, J. Meng2, L. English1, D. Wagner1 and S.Gaines1,
Integrons are mobile DNA elements with a specific structure consisting of two conserved segments flanking a central region (cassettes) that often encodes antibiotic resistance. This novel system has been identified in several Gram-negative pathogens. A total of 59 antibiotic resistant Shiga toxin-producing Escherichia coli (STEC) strains were analyzed, including 30 E. coli O157:H7 and 29 other STEC. One E. coli O157:H7 and 10 other STEC isolates displayed class 1 integrons of 1 and 2 kb in size respectively using PCR. Sequence analysis showed that the integron of E. coli O157:H7 exhibited ca. 80% homology with a previously described integron in the GenBank database, whereas the integron of an E. coli O111:H8 demonstrate 100% homology with the same GenBank sequence. The E. coli O157:H7 integron was transferable by conjugation. Southern hybridization analysis also indicated that the integron was carried on both plasmid and chromosome. The study suggests that integrons may contribute to acquisition and dissemination of antibiotic resistance genes in Gram-negative bacterial pathogens.
Campylobacter Jejuni 81-176 Specifically Associates with Microtubules and Dynein During Controlled Invasion into Human Intestinal Cells.
L. Hu and D. J. Kopecko, FDA-CBER, Bethesda, MD 20892
C. jejuni 81-176 enters cultured human intestinal cells via a process that requires microtubules (MTs), but is unaffected by depolymerization of microfilaments. Direct evidence of MT involvement in C. jejuni internalization was obtained by time-course immunofluorescence microscopic analyses. At early times during bacterial invasion, C. jejuni interacted with finger-like extensions of the cell membrane containing 1 or a few bundled MTs, which may represent an early host cytoskeletal response to a diffusible bacterial signal. C. jejuni were observed to align in parallel with MTs during entry, were found to co-localize specifically with MTs, and moved over 4 hr. to the perinuclear region. Orthovanadate, which inhibits dynein activity, markedly reduced C. jejuni 81-176 entry, and invading C. jejuni colocalized with dynein, suggesting that this molecular motor is involved in both C. jejuni entry and endosome-trafficking during bacterial internalization. We hypothesize that C. jejuni interact with a receptor in membrane caveolae, activating membrane-bound dynein, whose movement along MTs aids in the membrane invagination event that results in bacterial entry.
Rappaport-Vassiliadis Medium for the Recovery of Salmonella from Foods with a Low Microbial Load: Collaborative Study
T. S. Hammack, R. M. Amaguaña and W. H. Andrews, FDA, CFSAN, Washington, DC 20204
Twenty-three laboratories participated in a collaborative study to compare the relative effectiveness of Rappaport-Vassiliadis (RV) medium incubated at 42oC, selenite cystine (SC) broth (35oC) and tetrathionate (TT) broth (35 and 43oC) for the recovery of Salmonella from foods with a low microbial load. These foods included dried egg yolk, dry active yeast, ground black pepper, guar gum, and instant nonfat dry milk. For dry active yeast, lauryl tryptose (LT) broth, incubated at 35oC, was used instead of SC broth. All of the low microbial load foods were inoculated with high and low target levels of 0.4 and 0.04 colony forming units/g food, respectively. RVmedium was comparable or even more effective than the other selective enrichments for the recovery of Salmonella from all of the foods except guar gum. It is recommended that RV (42o) and TT (35o) be used that have foods with a low microbial load, except for guar gum. For the analysis of guar gum, the use of SC (35oC) and TT (35oC) is recommended.
Evaluation of Potential Surrogate Microorganisms for Validating Pathogen Reduction Treatments for Fruit Juices
S. Edelson-Mammel and R.L. Buchanan, CFSAN, FDA-200 C. St, SW, Washington, DC 20204
Validation of the effectiveness of potential treatments for reducing the level of pathogenic bacteria in fruit juice require "inoculated pack" studies under commercial conditions. This requires that surrogates microorganisms are available for use by food processors. Four potential surrogate organisms were compared to Escherichia coli O157:H7 and Salmonella Hartford HO610 for their use in orange juice production. The four organism selected were non-pathogenic E. coli biotype 1, Enterobacter aerogenes, Lactobacillus fermentum, and Klebsiella pneumonia. Four types of resistance were evaluated: thermal, sodium hypochlorite, acid, and alkaline resistance. Results show that the organism(s) chosen as a surrogate depends on the target pathogen and the treatment being tested.
Pragmatic Approach to Detecting Potential Clinical Strains among Foodborne Isolates of Listeria Monocytogenes
L. Evsen2, A.I. Yansaneh2, T.K. DeValroger2 and A.D. Hitchins1, 1FDA, CFSAN, Washington, DC 20204, 2University of Maryland, College Park, MD 20742
Foodborne (F) and clinical (C) isolates of Listeria monocytogenes (Lmo) are similarly pathogenic in laboratory tests. Epidemiological and prevalence data suggest that F and C differ in field conditions. An evolving approach to detecting the clinical potential of foodborne isolates is the use of conventional identification tests under adverse or extreme growth conditions. Mean growth rates were similar for F and C in selective medium at 30°C. Their ability to grow or not in non-selective medium near the 45° C maximum was similar. Their ability to grow in rhamnose medium at 45°C was slightly different (p=0.1). Abilities to grow in motility medium, at either 5 or 45°C, were different (p=0.1). Abilities to grow under 1 or more of these three conditions were significant (p<0.05). The approach deserves further study.
An Atypical Hemolytic Strain of Listeria
J. Johnson1, K. Jinneman1, G. Stelma2, B.G. Smith2, D. Lye2, J. Messer2, J. Ulaszek3, L. Evsen4, S. Gendel5, R. W. Bennett5 and A. D. Hitchins5, 1FDA, Seattle, WA 98041, 2EPA, Cincinatti, OH 45268, 3NCFST, Summit-Argo, IL 60501, 4University of Maryland, College Park, MD 20742, 5FDA, CFSAN, Washington, DC 20204
An unusual hemolytic (Hly) Listeria strain was isolated from a sample of imported boiled baby clam meat. Classical tests suggested it is either a rhamnose (Rha) minus L. monocytogenes (Lmo) or a xylose (Xyl) minus L. seeligeri strain. Lmo specific PCR analysis showed it is hly positive and iap minus. Also, it is phospholipase C (Plc) positive and a-methyl-D-mannoside positive. The serotype is 4 (5) with no factors 6-9. It is DIM positive and Lmo rRNA probe negative. The ribotype does not closely correspond to known ribotypes of Lmo or L. innocua (Rha variable, Xyl minus). It is apathogenic to immunocompromised mice. Apart from Hly and Plc, the strain must be deficient in other known or unknown virulence factor(s). The available data preclude a definitive assignment of the strain to a Listeria species or hybrid.
A Preliminary Evaluation of Two Chromogenic Agar Media for Detection of EnteroheMorrhagic Escherichia coli.
G. R. Garcia1, E. Sloan1, C. Ramirez1, S. M. Ramsey1 and J. N. Sofos1,2, 1Food and Drug Administration, Denver, CO, 2Colorado State University, Fort Collins, CO
Chromogenic agar media are reported to produce colonies with colors specific for different Escherichia coli serotypes. The objective of the chromogenic ingredients is to improve the selection and differentiation of enterohemorrhagic Escherichia coli (EHEC) isolates. A study was conducted to provide a preliminary evaluation of two chromogenic agar media (i.e., BCMTM O157:H7 (+) agar and Rainbow® Agar O157) without and with added novobiocin, in comparison with Sorbitol-MacConkey (SMAC) agar, SMAC with tellurite and cefixime (TC-SMAC) and Tryptic Soy Agar (TSA) with added 0.6% yeast extract, for their efficiency and ease of recovery of pure cultures of EHEC and other microorganisms. The results indicated that the chromogenic agar media tested might offer improved detection of the pathogen, compared to agar media currently used.
Detection of Poliovirus in Artificially Contaminated Oysters Magnetic Bead Capture and RT-PCR
Gary L. Hartman1 and Y.S. Carol Shieh2, 1San Francisco District Laboratory, Alameda, CA 94502, 2CFSAN, OS, GCSL, Dauphin Island, AL 36528
A simple and rapid method for the detection of enteric viruses in oysters has been developed. This method incorporates a viral concentration step using polyethylene glycol and the novel use of paramagnetic beads to capture the viral nucleic acid. The target genome is then amplified by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and detected by agarose gel electrophoresis and Southern hybridization. Presently, the classification of shellfish growing water is based on fecal coliform bacteria. However, the correlation between bacterial and viral contamination is not constant. Poliovirus, through the use of the Sabin strain for immunization, is found at a low level in human waste, providing a possible viral marker for fecal contamination of shellfish. This method has the potential to provide FDA a rapid way to detect poliovirus as an indicator of other enteric viruses, such as Norwalk and Norwalk-like viruses, in shellfish.
Antibiotic Susceptibility Profiles of Enterococcus Isolates from the Poultry Production Environment
L.L. English1, J.R. Hayes2, D.G. White1, S.W. Joseph2, L.E. Carr2 and D.D. Wagner1, 1OR, CVM, FDA, Laurel, MD 20708, 2 University of Maryland, College Park, MD 20742
Isolates of Enterococcus spp. have been cultured from litter samples and swabs taken from poultry transport containers and subjected to antibiotic susceptibility testing. The isolates have been screened against 33 antibiotics in a microtiter format utilizing Sensititer (Trek Diagnostics, Inc). A total of 269 isolates from 541 thus far collected have been tested. E. faecalis (61%), E. faecium (19%), and E. gallinarum (2.4%) are the species most frequently isolated. High-level streptomycin resistance is evident in about 30% of all isolates tested. There have been no vancomycin resistant isolates detected. Of particular interest, resistance to the streptogramin, quinupristin-dalfopristin (Synercid), is present in 70% of the E. faeceum isolates tested. Resistance to Synercid has been detected in 73% of all non E. faecalis isolates thus far tested.
Characterization of Fluoroquinolone Resistant Campylobacter spp. Isolated from Poultry
M.S. Nawaz1, A.A. Khan1, S.A. Khan1, R.S. Steele1, C.E. Cerniglia1 and R.A. Jones2, Division of Microbiology, 1The National Center for Toxicological Research, Jefferson, AR 72079, 2Center for Veterinary Medicine, FDA, Washington, DC
Campylobacteriosis is caused by Campylobacter jejuni and Campylobacter coli. Widespread use of the fluoroquinolones in veterinary medicine has enhanced the prevalence of fluoroquinolone-resistant Campylobacter spp. We have isolated more than 14 fluoroquinolone-resistant Campylobacter spp. from contaminated poultry samples. Morphological and biochemical characteristics indicated that these bacteria were Gram-negative, spiral shaped, hydrolyzed sodium hippurate and DNA. A majority of the strain were alkaline phosphatase and aryl sulfatase positive. All fourteen fluoroquinolone-resistant Campylobacter spp were resistant to multiple antibiotics. However, they were all sensitive to streptomycin. Plasmids (ca. 12 kb) were present in some strains of Campylobacter spp. One strain of Campylobacter spp. contained a plasmid measuring ca. 6.0 kb.
Extraction of Cryptosporidium parvum Oocysts from Foods
D.K. Lau1 and R.L. Bernstein Ph.D.2, 1ORA, FDA, Alameda, CA 94502, 2 San Francisco State University, San Francisco, CA 94132
Cryptosporidium parvum has recently been involved in significant foodborne outbreaks. The parasite can be transmitted through water into food in an oocyst form that is resistant to many environmental stresses and water treatment practices. While the numbers of oocysts in the environment are generally very low, the minimum infectious dose is small. In food, the limited size of the samples, the difficulties encountered in the concentration procedure, and the lack of an equivalent to the bacterial enrichment for Cryptosporidium spp., increase the need for sensitive detection methods. Immunomagnetic separation in conjunction with nested polymerase chain reaction has been used to detect the oocysts in lettuce extract. For more complex food matrices, an extraction procedure is required to isolate the oocysts. The aim of this study is to develop a rapid and sensitive method to extract and detect C. parvum oocysts in foods with future application to routine monitoring of foods.
Sensitivity of a Direct Polymerase Chain Reaction (PCR) Method for Shigella Species Inoculated in Produce Rinses
C. Ramirez1, J. N. Sofos1,2 and E. R. Singleton1, 1Denver District Laboratory, Denver, CO, 2Colorado State University, Fort Collins, CO
Methods for detection of Shigella species in food products need improvement, particularly for its isolation from raw vegetables. Current methods involve enrichment of product rinses, but competing bacteria may outgrow Shigella and interfere with detection. This research has applied a polymerase chain reaction (PCR) screening method that could be applicable universally in the analysis of vegetable products for Shigella. In this study we have evaluated the PCR method (developed by Dr. Keith A. Lampel) for detection of Shigella in produce rinses, without enrichment, and for determination of sensitivity (level of recovery), when viable cells were inoculated in broccoli, parsley, strawberries, and celery rinses. Detection was consistent at inoculum levels of ³ 102 CFU/ml of rinse. In general, the PCR method was successful in detecting Shigella spp. at levels of ³102 CFU/ml from non-enriched produce rinses.
Comparison of Resistance and Multi-Drug Resistance Patterns in Salmonella Enterica Serotype Heidelberg Isolated from Humans and Broiler Chickens
K. Hollinger1, L. Silvers1, P. Fedorka-Cray2, N. Marano3, F. Angulo3, K. Stamey 3 and L. Tollefson 1, 1FDA Ctr. for Vet. Med., Rockville, MD, 2USDA-ARS, Athens, GA, 3CDC, Atlanta, GA
Comparing salmonella isolates obtained from people ill with salmonellosis and divided into Heidelberg and non-Heidelberg groups indicated similar prevalence of resistance for all 17 antibiotics tested except for: chloramphenicol, gentamicin, kanamycin, and streptomycin. The largest difference between human groups was observed for gentamicin where 16.7% of the Heidelberg and 1.8% of the Non-Heidelberg groups were resistant. Comparison of human Heidelberg and broiler carcass Heidelberg isolate groups indicated similarity in prevalence of resistance for all antibiotics except ampicillin, gentamicin, kanamycin, sulfamethoxazole, tetracycline and ticarcillin. Evaluating multi-drug resistance patterns in S. Heidelberg, the top five ranked human patterns (seven patterns in all, because three patterns shared the same rank), included four of the top five broiler isolate patterns. Comparisons of percent resistance and multi-drug resistance patterns in human S. Heidelberg isolates appear to reflect those identified in broilers. Selection pressure due to antimicrobial drug use is the most consistently identified risk factor for antimicrobial drug resistance in humans, animals and the environment. Antimicrobials such as gentamicin are important in the treatment of disease in human and animal medicine and should be used prudently and judiciously. Further study is warranted to identify the impact of gentamicin use in the poultry industry and better characterize transmission of resistance through the food chain.
Development of a Simple, Rapid Pre-Screen for the Detection of E. coli in Foods
M. Jasmine Thompson, FDA/PRL-SW, Los Angeles, CA 90015
The public is still threatened by the presence of foodborne pathogens despite diligent efforts made by the agency. Screening food commodities are performed to limit food related illnesses. Conventional screening procedures of food commodities are inefficient as well as labor and cost intensive. The emergence of PCR provides laboratories with a rapid and sensitive strategy to screen for food pathogens. However, potential problems still exist ranging from laboratory contamination to inhibition of the PCR reaction by food matrices. Field laboratories need a reliable and rapid method that screens food for pathogens. The proposed study addresses these needs by providing: a universal method to eliminate inhibitory compounds by hybridizing target DNA sequences to specific, biotinylated PCR primers fixed to avidin coated microtiter plates followed by washing a universal method that can be adapted to rapidly and accurately enumerate target organisms. This study will lay the foundation for future work involving several different organisms.
Human Respiratory Syncytial Virus (RSV) Glycoproteins F, G and SH Can Form an Oligomeric Complex
S.A. Feldman and J.A. Beeler, Laboratory of Respiratory Viruses and Pediatrics, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20852
RSV interactions with heparin were evaluated using heparin agarose affinity chromatography (HAAC) and either RSV-infected cell lysates or lysates produced from vaccinia recombinant viruses expressing F, G or SH (vF, vG, vSH). When RSV-infected cell lysates were tested, F, G and SH were eluted. Similarly, vF and vG bound to heparin, whereas vSH did not bind. Likewise, when RSV-vaccinia lysates were mixed prior to HAAC, only F and G bound heparin. In contrast, following coinfection with vF, vG and vSH all three proteins were detected subsequent to HAAC suggesting an association of SH with F or G. Immunoprecipitation with aF, G or SH antibody also provided evidence of a high molecular weight F/G/SH complex. Using linear salt gradients, RSV F/G/SH-complexes eluted at higher salt concentrations than either purified F or G protein indicating a relatively higher affinity of the complex for heparin.
Identification of Estrogenic Endocrine Disruptors Using an Integrated Computational Approach
Weida Tong1, Roger Perkins1, Jie Wu1, Leming Shi1, Meihua Tu1, Hong Fang2, Robert Blair2, William Branham2and Daniel M Sheehan2, 1R.O.W. Science, 2FDA's National Center for Toxicological Research (NCTR), Jefferson, AR 72079
Considerable scientific, regulatory and popular press attention has been devoted to the Endocrine Disrupting Chemicals (EDCs), of which estrogenic chemicals figure prominently. A larger number of potential estrogenic EDCs are of interest to the FDA, including plastics chemicals, phytoestrogens, pharmaceutical chemicals, cosmetic ingredients and etc. There is a crucial need to identify potential estrogenic EDCs for subsequent experimental screening and testing. Here we report an integrated computational approach to priority setting of potential estrogenic EDCs. This approach rationally integrates different predictive computational models into a "Four-Phase" scheme so that it can effectively identify and prioritize ER-mediated EDCs. We anticipate that the same integrated scheme will be equally applicable to endpoints of other endocrine disrupting mechanisms, e.g., androgen receptor binding.
Forensic Method for Evaluating Contamination from Disease-Carrying Flies
Alan R. Olsen and Sherry A. Knight
The role of disease-carrying flies as passive vectors of foodborne pathogens is firmly established by scientific research. Recent scientific studies have identified flies as a potential risk factor in outbreaks of foodborne illness from Salmonella Enteriditis in eggs and Escherichia coli O157:H7 in beef and produce. Only certain species of flies present a reasonable likelihood of transmitting foodborne pathogens. A new forensic method was developed and validated to identify the limited number of fly species that are of regulatory concern as contributing factors to the transmission of foodborne pathogens. The method uses a combination of ecological, behavioral and morphological attributes that are unique to disease-carrying flies to evaluate observations of fly contamination. The results of this research will be published and distributed as portable fact sheets to be used by laboratory analysts, forensic scientists, field investigators, sanitarians and other food sanitation control officials. The results are the science base for proposed revisions to FDA guidance and international guidance (Codex Alimentarius) for evaluating contamination from disease-carrying flies in the areas of HACCP, egg safety and general food hygiene.
Developing a BAM Selective Enrichment Based Enumeration Method for Listeria Monocytogenes in Fresh Produce
T.K. DeValroger2, A.I. Yansaneh2, F.U. Ibebuchi2, R.E. Duvall1 and A.D. Hitchins1,
1FDA, CFSAN, Washington, DC 20204, 2University of Maryland, College Park, MD 20742
Risk assessment of foodborne listeriosis is constrained by sparse quantitative contamination data for Listeria monocytogenes (Lmo). Enumerating contamination is typically done, if at all, only when foods screen positive for listeria by selective enrichment. A hybrid version of these two steps was tested. Low numbers of Lmo were selectively enriched (24 h at 30°C) and the proliferated cells were enumerated by colony counts. The initial contamination level was calculated using an empirically derived mean growth rate. The back calculated and known initial numbers corresponded well with some kinds of sample matrices. An empirical correction was necessary for other matrices, such as fresh produce, where inhibition due to plant microbiota or constituents seems to occur. In practice, an isolates growth parameter will not known, so the mean growth rate was estimated for foodborne strains in general.
Chronic Bioassays for Liver Tumors in B6C3F1 Mice Using Idealized Weight Curves Produced by Controlled Feeding
J.E. Seng, W.T. Allaben and J.E.A. Leakey, NCTR, Jefferson, AR 72079
Dietary control (DC) to manipulate growth in B6C3F1 mice was used in an ongoing chronic bioassay. An idealized weight curve for male B6C3F1 mice, which predicts a terminal background liver tumor incid-ence of 15-20%, was constructed and tested using a known mouse hepatocarcinogen. This approach successfully maintained cohorts of mice at weights approximating their idealized target weights throughout the 2-year study. The mice exhibited less individual body weight variation than did their ad libitum-fed (AL) counterparts. This was associated with decreased variation in liver: body weight ratios, which allowed the demonstration of a significant dose response to the test chemical in the DC, but not the AL test groups. The test chemical, in AL and DC diet groups, increased terminal liver tumor incidence, but a significant dose-response was observed only in the DC mice. Furthermore, this dose response positively correlated with markers of peroxisomal proliferation in the DC mice only. Thus, DC not only improves terminal survival and decreases inter-assay variation, but can also increase assay sensitivity by decreasing intra-assay variation.
Developing Methodology for Using QSAR Programs to Estimate Toxic Potency
Mitchell A. Cheeseman
In order to use quantitative structure activity relationship (QSAR) software in regulatory decision-making it is necessary to develop a reliable methodology for relating the output of various QSAR programs to exposure. CFSAN/OPA, in cooperation with CDER/OTR, is developing methodologies to use QSAR software to estimate the likely carcinogenic potency of chemicals suspected of being carcinogens. A test set of chemicals of known carcinogenic potencies were used to calibrate three QSAR programs, Multicase, Topkat, and Oncologic. The output from these three QSAR systems in relation to the test data set has been used to develop parameters for estimating potency of unknown substances. In addition, QSAR modules have been developed integrating information on the carcinogenic potency of chemicals in existing modules and integrating maximum tolerated dose of chemicals to enable prediction of quantitative estimates of potency.
Ionizing Radiation Risk Assessment in Diagnostic and Interventional Radiology: Tissue Dose Handbooks
S.H. Stern and O.H. Suleiman, CDRH, FDA, Rockville, MD 20850
The principal risks associated with x-ray radiation absorbed by a patient undergoing a radiological procedure are the lifetime probability of cancer morbidity and mortality, the chance for transmitting a genetically heritable defect, and the possibility for acute skin injury. Assessment of any of these risks begins with estimation of absorbed dose incurred by each radiosensitive tissue. In an ongoing program unique in the U.S., CDRH develops and distributes handbooks of reference values of normalized tissue doses for representative radiological examinations. Handbooks published so far include those for general diagnostic radiography, pediatric radiology, mammography, upper-gastrointestinal and coronary-artery fluoroscopy. Work currently underway on a computed tomography handbook exemplifies the challenges in representing rapidly evolving clinical practice, scanner technology, and medical- and radiation-physics developments in a single, user-friendly compendium.
Special Populations: Testing and Labeling of New Drugs
D. B. Banks and T.A. Toigo, OSHI, FDA, Rockville, MD 20857
The FDAMA Section 115 working group recommended that the agency continue to gather and evaluate demographic data for product applications. The purpose of the project was to assess participation in testing, availability of clinical data, and labeling instructions for demographic subgroups for 81 new drugs approved by FDA in 1995-96. Results demonstrate that aged, female, and African American patients participated in clinical trials for nearly all drugs. Participation by other minority groups was low. Clinical data were available for more than half of the drugs for aged and female patients but for fewer than one fourth of the drugs for racial/ethnic minority groups. Labeling information was available for 24 drugs (aged), 13 drugs (females), 5 drugs (minorities). Further analyses of these data and review of more recent applications (1997-1999) are necessary to evaluate whether there are issues that regulatory guidance could help address.
Evaluation of the TOPKAT Computer-Based System for Predicting Carcinogenicity of Chemicals
M.J. Prival, CFSAN, FDA, Washington, DC 20204
The ability of the TOPKAT computer-based system (version 5.01) to predict the carcinogenicity of chemicals was evaluated. The predictions of the TOPKAT "NTP Rodent Carcinogenicity" module were compared to the results of bioassays conducted by the National Toxicology Program that had not been used in the learning set of the TOPKAT software. Positive and negative predictivity and overall concordance figures ranged from 40% to 64%. It would thus seem, from the analysis performed here, that TOPKAT in its present form is not useful in predicting the carcinogenicity of chemicals. Chemicals that have no apparent relationship to each other with regard to potential mechanisms of carcinogenic activity were often identified as similar by TOPKAT. Perhaps future versions of TOPKAT, utilizing larger data bases and more refined decision rules, will be more reliable in predicting carcinogenicity than the version currently available.
Fluoroquinolone Resistance in Chickens and Human Health Impact; A Quantitative Risk Assessment
K. Hollinger, D. Vose, M. Bartholomew, M. Miller, S. Thompson and L. Tollefson
Emerging antimicrobial resistance, due to use of antimicrobials, is a growing public health concern in human and animal medicine worldwide. The Center for Veterinary Medicine (CVM) has developed a risk assessment model that evaluates the risk to human health from resistant food borne pathogens attributed to the use of antimicrobials in food producing animals. A mathematical model was developed to relate the prevalence of resistant Campylobacter infections in humans due to consumption of chickens, to the prevalence of resistant Campylobacter in chickens. The model uses data from recently initiated surveillance systems (FoodNet and the National Antimicrobial Resistance Monitoring Program, [NARMS]) that monitor the actual incidence of foodborne disease and prevalence of fluoroquinolone resistance in humans and chickens, as well as from the published literature. This model may be updated annually to show changes in the level of resistance and incidence of campylobacteriosis. These changes may reflect the implementation of changes in food animal production, processing and indicate changes in the susceptibility of the human population allowing an annual reassessment of the magnitude of human health impact.
Dynamics of the Immune System of the Rat in an Intermittent Dosing Study with Aflatoxin B1 (AFB1)
D.M. Hinton1, M.J. Myers2, R. Raybourne1, R.E. Sotomayor1, J. Shaddock3, A. Warbritton3 and M Chou3, 1CFSAN, Laurel, MD, 2CVM, Laurel, MD, 3NCTR, Jefferson, AR
AFB1 is a known carcinogen and affects immune function in animals. Fisher-344 male rats were fed for 40 weeks intermittently (4 weeks-on and 4 weeks-off) doses of 0.01, 0.04, 0.4 and 1.6 ppm of AFB1 and for 40 weeks continuously at the 1.6 ppm level. Groups of animals were sacrificed at 4-week intervals up to 20 weeks and then at 40 weeks. Splenic cell suspensions were analyzed by flow cytometry. Portions of the suspension were stimulated in culture for analysis of the productive capacity for cytokines IL-2 (Con-A), IL-1 and IL-6 [LPS, LPS + gamma interferon (INF) and gamma INF + NGMA, a nitric oxide synthetase inhibitor]. Hematologic and other endpoints were measured also. Animals were 6 weeks of age at the start of dosing. Dynamic changes were seen in % lymphocyte T- and B-cells and cytokines for controls during the growth phase. A 7-fold increase for IL-2, e.g., was seen at 18 weeks of age. The effects of AFB1 were seen at the lowest doses tested and were not totally reversed after the "off-diet" cycle. Changes in cytokine profiles suggest a pro-inflammatory response. The significance of these changes in relation to functional responses and carcinogenicity need to be determined.
Rhabdomyolysis Associated with Lovastatin and Grapefruit Juice Interaction
M.H. Parks1, R. Marcus2, X. Wei1 and L. Green1, 1CDER, FDA, Rockville, MD 20857, 21 Medical Center Dr., Upland, PA 19013
Increases in drug levels attributed to drug-grapefruit juice interaction have been described for a number of CYP3A4 substrates such as dihydropyridine calcium antagonists and terfenadine. Similar interactions have been observed with the HMG-CoA reductase inhibitors metabolized by CYP3A4 but clinical adverse events have never been described. We describe a MedWatch report of a 60 year-old male with a history of heart disease, diabetes, and chronic renal insufficiency treated with Lovastatin 40 mg bid and Gemfibrozil 600 mg bid for > 5 years duration who switched from his routine daily consumption of orange juice to 8 oz. grapefruit juice in early October 1998. On October 13, 1998 the patient experienced diffuse muscle pains in the absence of any strenuous activity. The patient was seen in the emergency room 5 days later with rhabdomyolysis (CPK 44,860 U/L) and acute on chronic renal failure requiring hemodialysis. Plasma concentration of total HMG-CoA reductase inhibitors obtained 14 hrs after Lovastatin dosing was elevated at 109 ngEq/ml. The potential role of grapefruit juice in precipitating rhabdomyolysis in this patient on chronic treatment with Lovastatin and Gemfibrozil will be discussed.
What Have We Learned from the Recent Market Withdrawal of Terfenadine, Mibefradil and Astemizole? Shiew-Mei Huang, Brian Booth, Emmanuel Fadiran, Ramana S. Uppoor, Suresh Doddapaneni, Min Chen, Funmilayo Ajayi, Terry Martin and Lawrence J. Lesko, CDER, FDA, Rockville, MD
A Quality Assurance Working Group was formed in the Office of Clinical Pharmacology and Biopharmaceutics to determine "lessons learned" from the recent U.S. market withdrawal because of serious drug-drug interactions (D-DI). The goal of this initiative was to assess how new technology, scientific data and regulatory assessment and communication of risk could lead to improved paradigms for regulatory review of D-DI in the future. The results of this study demonstrate the technological evolution that has occurred over the past 15 years to more accurately forecast potential for serious D-DI. The conclusions of this study were that industry and regulatory authorities should work together to 1) assign a relative level of risk to D-DI in the product label, 2) use prominent warnings in the initial product label for serious D-DI and 3) develop effective ways to communicate key information in product labels to practitioners and patients about managing D-DI.
Use of Dietary Exposure Estimates to Determine the Equivalency of Veterinary Drug Maximum Residue Levels
S.C. Fitzpatrick1 and S.D. Brynes 2, 1 ORA, FDA, Rockville, MD 20857, 2 CVM, FDA, Rockville, MD 20855
Harmonization of standards for establishing veterinary drug maximum residue levels (MRLs) is a major goal of the international veterinary drug community. However, harmonized standards don't automatically lead to harmonized MRLs. Discrepancies can result from different, but equally scientifically valid, conclusions about the same data set, such as the choice of safety factors, food consumption factors, or the analyte used in monitoring programs or from different use levels in different geographical regions. Trade problems can occur when differences between MRLs are interpreted as a difference in the country's human food safety assessment of the residues. However, if use of a different MRL does not result in dietary exposure to residues above a country's established acceptable daily intake for that compound, even using very conservative estimates of exposure, that MRL should be considered equivalent. In conclusion, although harmonizing testing standards is worthwhile, more emphasis needs to be placed upon delineating guidelines for determining the equivalence of differing MRLs for the same compound.
Effects of Increasing Iron Supplementation
P. Whittaker and V.C. Dunkel, CFSAN, FDA, Washington, DC 20204
The effects of increasing levels of Fe on serum fatty acids, cholesterol, triglycerides, liver, heart, and pancreas were examined in male Sprague Dawley rats fed either Fe-deficient or carbonyl Fe-supplemented diets with 35 (control), 350, 3500 and 20,000 µg Fe/g for 12 weeks. As intake of Fe increased, total serum phospholipid fatty acids increased from 609 to 1,292 mg/l. Serum total cholesterol increased from 78 mg/dl in controls to 201 mg/dl at the highest level of dietary Fe. A dose-related increase in liver nonheme Fe from 18 to 3,500 µg/g correlated with increases in lipid peroxidation (r=0.87), measured by the lipid-conjugated diene assay. This oxidative change in the liver may have affected alterations in sterol synthesis, leading to increased serum cholesterol levels with concurrent increases in serum phospholipids and changes in the ratios of their saturated/unsaturated fatty acids. Increasing dietary Fe depletes antioxidants, causes lipid peroxidation, and oxidation of LDL-cholesterol that has been shown to be involved in atherosclerosis. An evaluation of animals with heart damage showed myocardial degeneration and cardiomyopathy with hemosiderin in interstitial macrophages or in myocardial fibers. The toxic effects of Fe overload include cellular apoptosis or necrosis in heart and pancreas and, when coupled with the findings on lipid peroxidation, suggests that oxidative stress is involved in the pathogenesis of the lesions.
Food Handling of Infant Caregivers as Risk Factors for Diarrhea
J.F. Guthrie, S.B. Fein and C.D. Falci, DMS/OSAS/CFSAN/FDA, Washington, DC 20204
Because infants are a high-risk group for foodborne illness, it is important to identify the food handling practices of their caregivers that may increase risk. Using data on 1,000 infants collected at ages 2-7 and 9 months, we conducted a series of multivariate analyses to examine the relation between infant diarrhea and handling practices for infant formula and solid foods. Many mothers did not follow recommendations, such as sterilizing bottles and water used for formula and discarding leftover solid food in a serving dish or jar. However, only holding prepared formula at room temperature was associated with higher rates of diarrhea, and only for older infants (5 - 7 months). No category of education, income, or mothers age was systematically related to the safety of handling practices of formula or solid foods. For infants in day care, the risk of diarrhea decreased when mothers provided the food themselves. These results will help to target risk communication information for infant caregivers.
The Effect of Different Levels of Dietary Restriction on Survival in the Sprague-Dawley Rat: Implications for Chronic Studies
P.H. Duffy and R.J. Feuers, NCTR, FDA, Jefferson, AR 72079
To resolve the problem of declining rodent survival in the bioassay, survival, growth and reproductive variables were monitored in a chronic study in which male Sprague Dawley (SD) rats (NCTR colony) were fed ad libitum (AL), or fed dietary restricted (DR) rations. The two-year survival rate for the AL and 10%, 25% and 40% DR groups was 63.4%, 87.5%, 87.5% and 97.5%, respectively. Rats from the NCTR colony were found to be ideal for chronic studies because they were less obese and much longer-lived than other SD strains. Although the survival rate for small AL rats exceeds the FDAs "Redbook" guideline (> 50%), the use of DR is advocated because it reduces individual variability in numerous variables, increases longevity and synchronizes the circadian rhythms among the test groups, thereby increasing the statistical power and accuracy of the bioassay. An evaluation of growth, reproductive and energy-intake parameters suggests that 10% DR may be the optimal diet to increase the survival rate of rodents in chronic studies without altering the baseline sensitivity to carcinogenesis and drug toxicity.
Interaction of Herbal Preparations with Drug Metabolizing Enzymes
J.E.A.Leakey, F.W. Lee and T.I.Sergeeva, NCTR, Jefferson, AR 72079
The US herbal dietary supplement market is experiencing unprecedented growth, and consumers do not always inform their physicians. This can lead to potential adverse interactions with prescribed drugs. We investigated interactions of extracts of five common herbs (St Johns wort, aloe vera gel, ginkgo biloba, echinacea and ginseng) with the hepatic UDP-glucuronosyltransferase (UGT) system. We demonstrated that lipophilic extracts of echinacea, St John's wort and ginseng all contained compounds that were UGT substrates. Additional UGT substrates appeared to be released from aqueous extracts of ginkgo, echinacea and ginseng by hydrolysis with ß-glucosidase. The echinacea extract also appeared to contain a potent non-competitive inhibitor of human hepatic UGT activity towards estriol. This study suggests that the concurrent use of herbal products such as echinacea may alter the pharmacokinetics and elimination of pharmaceuticals that are conjugated by hepatic UGT.
Silicone Gel Breast Implant Rupture Prevalence by Magnetic Resonance Imaging (MR) in a Population of Women in Birmingham, Alabama
S.L. Brown1, M.S. Middleton2, W.A. Berg3, M.S. Soo4 and G. Pennello1, 1CDRH, FDA, Rockville, MD 20850, 2University of California at San Diego School of Medicine, San Diego, CA 92103, 3University of Maryland School of Medicine, Baltimore, MD 21201, 4 Duke University Medical Center, Durham, NC 27710
Silicone gel breast implants have been available since the early 1960's and it has been estimated that between 1-2 million American women had breast implants by 1992. Breast implant rupture has been reported to the Food and Drug Administration in over one quarter of the 94,120 adverse event reports on silicone gel breast implants received between 1984 and 1995. In the current study, an unreferred population of women from Birmingham, AL with silicone gel breast implants were invited to have a MR examination of their breasts to assess the status of their breast implants. There were 344 women with silicone gel breast implants in the study. The status of their implants will be discussed.
Replacement Surgery and Silicone Gel Breast Implant Rupture: Self-Report by Women after Mammoplasty
S.L. Brown and G. Pennello, CDRH, FDA, Rockville, MD 20850
The purpose of this study was to examine the prevalence of revision surgery and of silicone gel breast implant rupture reported after explantation in a cohort of women from Birmingham, Alabama. Data were collected by a computer assisted telephone interview of 1,247 women previously identified in a larger National Cancer Institute cohort study of women with breast implants. Women who reported breast surgeries subsequent to their index mammoplasty were asked to consent to retrieval of the surgical records describing the surgery. Of the 1,247 eligible women, 907 responded to the interview. Excluding deceased (7) and unlocated (115) women, the response rate was 75%. Surgery in which an implant was removed or replaced was reported by 33.4% of the 907 women. The most common reason (103/303 surgeries) for surgery was problems with the implant that affected the breast such as suspected implant rupture or capsular contracture. Self-reported implant rupture will be compared to surgical record reports.
Health Status in Women with Silicone Gel Breast Implant Rupture or Extracapsular Silicone
S.L. Brown1, G. Pennello1, W.A. Berg2 , M.S. Soo3 and M.S. Middleton4, 1CDRH, FDA, Rockville, MD 20850, 2University of Maryland School of Medicine, Baltimore, MD 21201, 3 Duke University Medical Center, Durham, NC 27710, 4University of California at San Diego School of Medicine, San Diego, CA 92103
The objective of this study was to assess whether breast implant rupture or extracapsular silicone are associated with selected symptoms of connective tissue disease or with self-reported physician diagnosed disease. Women with silicone gel breast implants responded to a questionnaire which included questions on health status, satisfaction with implants, symptoms of connective tissue disease, and physician diagnosed disease. These women then had a magnetic resonance imaging (MR) examination of their breasts to determine the status of their implants with respect to rupture and extracapsular silicone. The self-report of symptoms and diagnosed connective disease was compared in women with implant rupture or extracapsular silicone to other women in the study.
Statistical Considerations in the Estimation of the Replication-Competent Retrovirus Stock Titer through Serial Dilutions
Tie-Hua Ng and Carolyn Wilson, CBER, FDA, Rockville, MD 20852
Replication-defective retroviral vectors are currently being tested in human gene therapy clinical trials. Replication-competent retrovirus (RCR) may be generated during the production of retroviral vectors. To facilitate the development of assays for RCR detection, the American Type Culture Collection in collaboration with FDA scientists produced a stock of RCR derived from a molecular clone. This RCR stock was assayed by exposing PG-4 cells to serial dilutions of virus and then enumerating the number of plaques formed. Each plaque represents an infectious particle. At dilution 10-k (k=5, 6, 7, 8, 9), the titer of the RCR stock may be estimated by the average number of infectious particles over several replications times 10k. A naive method of averaging these estimates over the dilutions does not take into account the correlation induced by the serial dilutions. This paper discusses an alternative method that accounts for the fact that the samples tested at a given dilution and the samples used to prepare the next dilution are from the same source.
Receiver Operating Characteristic (ROC) Analysis in the Multivariate Case
M. Kondratovich, R. Kotz, 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. The observation vector is usually not only the value of diagnostic test but other variables which are associated with one another (e.g., the measurement of speed of ultrasound in the evaluation of bones status and age of the patient are not independent factors for the prediction of fracture). In the case when it is necessary to estimate the discriminatory ability of each variable separately, the calculation of the AUC for one variable gives overstated values unless the distribution of the second variable is the same for the two groups. The authors developed weighted ROC analysis method which allows one to calculate the AUC of the variable in question without the effect of other potentially confounding variables, and, thus, to estimate correctly the diagnostic capability of the medical device.
Adaptive Testing with Multiple Sample Size Adjustment
Lu Cui, Division of Biometrics I, OB, CDER, FDA, Rockville, MD 20852
Sample size determination is an important but often difficult issue in planning clinical trials. Due to lack of prior knowledge of the treatment effect of interest in a clinical trial, the sample size needed to achieve a desired statistical power may be underestimated, leading to an insignificant trial result. One way to solve this problem is to make the trial more flexible so that sample size reestimation and modification can be made at an interim stage of a clinical trial. In this study, a new adaptive statistical testing procedure is proposed. This new procedure allows one to adjust sample size at each of a sequence of analyses based on the information accumulated up to that analysis. The new procedure is easy to use and guarantees a controlled type I error rate. With the increase of sample size, the power of the new procedure increases. The new test can be viewed as an extension of a two-stage test and an extension of a group sequential test with a fixed sample size.
Statistical Issues in Monitoring Postmarketing Adverse Event Reporting
Yi Tsong, Ph.D., CDER, FDA, Rockville, MD 20857
The FDA receives over 200,000 adverse drug reaction reports annually, and currently has over 1.6 million reports in its computerized database. It is one of the richest database available for detecting the signals of uncommonly frequent adverse events of any FDA approved drug. There were many statistical methods proposed for the various aspect of signal monitoring. Due to the voluntary reporting nature of the system, there are many factors that may influent the reporting. In this presentation, we will discuss some of the signaling methods proposed with adjustment for the factors. The data set of ADR reports of triazolam and temazepam received between 1981 and 1991 previously published at the 1992 FDA Advisory Committee Meeting is used to illustrate the approaches.
Comparison of Two Correlated Proportions: Bayesian and Frequentist Approaches
Telba Z. Irony, CDRH - FDA, Carlos A. B. Pereira, University of Sao Paulo, Brazil, Ram C. Tiwari, University of North Carolina
Suppose that a group of individuals is evaluated in order to assess the presence of a certain condition (present = yes, absent = no). After a treatment is administered, these individuals are evaluated again. The objective is to analyze whether or not there was a change - swing - in their state. Due to the longitudinal nature of data, only tests of hypothesis are usually performed - for instance the McNemar test. The estimation of the parameters of interest is usually more complicated. In this poster we suggest Bayesian solutions that make the estimation problem straightforward.
Thresholds in Nonlinear Models for Repeated Measurements
M. J. Bartholomew1 and C. Gennings2, 1CVM, FDA, Rockville, MD 20855, 2Department of Biostatistics, Medical College of Virginia, VCU
The literature contains numerous applications of threshold models in the linear or generalized linear model context for the analysis of cross-sectional data. Thresholds in nonlinear models for cross-sectional data are less prevalent in the literature. We extend nonlinear models with thresholds to the analysis of repeated measurement data. We obtain estimates for parameters for these new models using the Nelder-Mead simplex algorithm, an efficient searching algorithm. This method does not require the evaluation of derivatives of the nonlinear function in order to obtain the estimates for the mean model parameters themselves, although derivatives are required to estimate the variances of the parameters. An application of the procedure to fit a population-averaged model to a toxicological study in mice and an application of the procedure to fit a subject-specific model to specific activity of a labelled compound in test subjects are provided.
Rational Approaches for Choosing the True and False Positive Rates
S. D. Dubey CDER, FDA, Rockvill, MD 20857
In this paper, a Correct Decision Expectation Rate (CDER) Index is defined, developed, and described, which involves a function of the true and false positive rates with a sensible flexiblity factor. Similarly, a Trial Success Expectation Rate (TSER) Index is defined, developed, and described, which involves a valid and reliable measure of independent evidence, and the true and false positive rates. Various properties of these indices are derived and their consequences are discussed. Then a Trial Size Index is derived as a function of the CDER Index, the TSER Index, and the Expected Effect Size. Several insightful interactions among the CDER Index, the TSER Index, and the Trial Size Index are derived and their consequences are discussed. Finally, a wonderful world of interactive decision strategies involving true and false positive rates is produced with a view to assuring robust statistical evidence. The results of this paper are applicable under a spectrum of situations.
An Example of Analyzing Ordinal Categories of Response Using Continuation-Ratio Models
Henry S.H. Hsu, Ph.D., MPH; Peter A. Lachenbruch, Ph.D; Rolf. E. Taffs, Ph.D.
1401 Rockville Pike, HFM-215, Rockville, MD 20854
When the categories of response probabilities X1, X2, ..., Xk are ordered, the cumulative response probabilities, K1 = X1, K2 = X1+ X2, ... , Kk = X1+X2+...+Xk = 1, provide a basis to use the logit link function in the linear logistic regression. The parallel-lines regression model has the form:
log{Kj(x) / ¬1!Kj(x)|} = Ij + JTx, where J is the vector of slope parameters and x is the covariate vector. This proportional-odds model is commonly used in analysis of polychotomous data. However, there are situations such as toxicity or adverse event studies whose ordinal categories of response are formed as the sequence of conditional factors according to a nested or hierarchical scale. The probability of a positive response is Xj/(1!Kj-1), and the probability of a negative response is (1!Kj)/(1!Kj-1). Thus, the log odds corresponding to category j in a continuous-ratio model are log{Xj/(1!Kj)} which use the individual probabilities, rather than their cumulative distribution probabilities. In this presentation, we have used a computational procedure proposed by Berridge and Whitehead (1991) to analyze the neurovirulence test data of oral poliomyelitis vaccine, in which the nature of lesion severity was in ordinal hierarchical categories. We also compared the results based on the continuation-odds model and the proportional-odds model.
The Effect of Repeated Ethylene Oxide Sterilization on the Mechanical Strength of Synthetic Absorbable Sutures
T.O. Woods1, S.A. Brown1, K. Merritt2 and V.M. Hitchins2, 1Division of Mechanics and Materials Science, 2Division of Life Sciences, CDRH, FDA, Rockville, MD 20850
Sutures that are opened but not used are commonly reprocessed for reuse, though they are labeled for single use. The effect of repeated ethylene oxide (EO) sterilization on the knot strength of three types of absorbable sutures was tested. Suture inner packs were repacked and EO sterilized using a clinical protocol. Mean knot strength was measured out of package and after 1 and 2 Re-EO cycles. As is true for other devices, it is not possible to make general conclusions. Suture strength was not affected for some sutures; others increased or decreased in strength. Seals on some inner packs were destroyed during reprocessing, exposing the absorbable sutures to ambient humidity. While seal loss might not cause an initial strength loss, exposure to increased humidity for an extended time will cause suture degradation and loss of strength.
Reprocessing Single Use Biopsy Forceps for Reuse
K.Merritt, V.M. Hitchins, S.A. Brown and T.O. Woods, Division of Life Sciences, Division of Mechanics and Material Science, CDRH, FDA, Rockville, MD 20852
Economic considerations in the delivery of health care are enticing some facilities to reuse single use devices. Biopsy forceps, used together with an endoscope in gastrointestinal procedures, are among the devices that some entities are reprocessing. If these forceps are to be reused on another patient, they must be adequately cleaned and sterilized. We have been examining 3 types of single use GI biopsy forceps. These have an external polymer sheath covering the spring that operates either jaws or a snare. The snares have an open lumen. The jaw forceps appear sealed but actually there is an open lumen. Cleaning of these devices with a sequence of bleach, ultrasonic bath with detergent and enzyme, and water rinse appears to remove residual debris. However, drying the lumens of these devices is very difficult. Residual water may decrease the effectiveness of sterilization.
Effects of Different Sterilization Methods on Materials Used for Single Use Devices (SUDs)
S. A. Brown1, K. Merritt2, T. O. Woods1 and V. M. Hitchins2, 1Division of Mechanics and Materials Science, 2Division of Life Sciences, CDRH/ FDA, Rockville, MD 20852
Driven by economic and time constraints, some medical centers and third parties are resterilizing SUDs for reuse. The steam autoclave is quick, but most plastics used in SUDs can not survive the temperature. Thus, a number of new methods are being introduced on the market. To date, this program has studied the effects of five: EtO, peracetic acid + peroxide (Steris), high temp formaldehyde, (Chemiclave), low temp peroxide gas plasma - (Sterrad), and low temp peracetic acid gas plasma (Abtox). Tensile strength testing has shown that silicone elastomer is unaffected, whereas the strength of nylon, polyethyelene and latex was reduced by some of the methods. Depending on the formulation the strength of polyurethane either increased or decreased. The results demonstrate that the effect of sterilization depends on the method and the materials used in the device.
Effects of Use and Reprocessing on Single Use Coronary Catheters
S. A. Brown1, K. Merritt2, V. M. Hitchins2 and T. O. Woods1, 1Division of Mechanics and Materials Science, 2Division of Life Sciences, CDRH/ FDA, Rockville, MD 20852
Although sold for single use only, some medical devices, such as coronary catheters, are being processed for reuse. Over 400 PTCA and 300 EP catheters have been retrieved after single patient use at Walter Reed Army Hospital. After disinfection and cleaning, a variety of performance characteristics were determined, and then some were subjected to ETO sterilization and simulated reuse. The results demonstrated that cleaning was not a trivial problem. The balloon compliance data demonstrated model specific changes. Some catheters became more sticky making insertion more difficult. Some models of EP's were non-lumen, whereas others had hollow cores sometimes contaminated with blood. Damage to electrode seals exposed the lumens as well as copper wires connected to the electrodes. Unbeknownst to the user, subtle changes in device appearance may be associated with major changes in the performance of a used or reused device.
The Effect of Reprocessing on Single Use Electrophysiology Catheters
T.O. Woods1, S.A. Brown1, K. Merritt2 and V.M. Hitchins2, 1Division of Mechanics and Materials Science, 2Division of Life Sciences, CDRH, FDA, Rockville, MD 20850
Electrophysiology catheters (EPs) are one of the single use devices that are most often reported to be reprocessed and reused. Once it has been established that a used device can be cleaned and resterilized, it is necessary to show that its mechanical behavior has not been adversely affected. Torque and trackability, two clinically relevant mechanical properties of EPs, will be determined for a solid and hollow configuration of one model of EP catheter. The two types reflect a manufacturing change that was made without a change in model name. The two properties will be determined for new, unused catheters; for catheters after use in a single patient; and for used catheters subjected to a number of cycles of ethylene oxide sterilization, simulated reuse and reprocessing cycles. Results for the two catheter types will be compared.
Identifying and Characterizing Toxic Degradation Products in Cellulose Acetate Dialyzers
A.D. Lucas, J.A. Kalson, J.C. Hutter and R.R. Wallis, CDRH, FDA, Rockville, MD 20852
In September 1996, seven patients at Hospital A suffered headache, vision and hearing loss 7-24 h after hemodialysis treatment. Twelve-year-old dialysis modules were identified as a common link between these patients, with degradation postulated as the probable cause of this incident. Cellulose acetate (CA) dialysis membranes were retrieved and tested for degradation products. To verify the suspected cause of this incident, a series of in vitro, chemical and in vivo tests of various CA degradation products was conducted. Based on the toxicity of the material preparations to the cells, animal tests were performed. Rabbits displayed "red eye" when injected with degradation products. Chemical characterization indicated the toxic agent generated from degraded CA is water soluble, acidic and less than 20000 mw. The blood chemistry and eye evaluation of the rabbits strongly indicated that oxidative stress of CA at some point created degradation products that can reproduce some of the symptoms identified at Hospital A.
EVALUATION OF ANTIGENS IN THE ELISA INHIBITION ASSAY FOR NATURAL RUBBER LATEX PROTEINS
V. J. Tomazic-Jezic, A. D. Lucas, FDA/CDRH, Rockville
In the process of development of a ELISA Inhibition assay for the quantitation of the natural rubber latex (NRL) proteins, we investigated properties of proteins from ammoniated (AL) and non-ammoniated (NAL) raw latex, as coating antigens, immunizing antigens and reference antigens. The anti-NRL sera were produced by immunizing rabbits with AL, NAL and a mix of both antigens. Determination of affinity of rabbit antisera for binding to these antigens indicated strong preference of both anti-AL and anti-NAL antibodies to bind the immunizing antigen, while anti-Mix antiserum bound similarly to both antigens. Evaluation of the three sera in the ELISA inhibition assay with AL and NAL proteins as coating and reference antigens, further confirmed this finding. Anti-AL serum demonstrated a strong preference in inhibiting and binding to AL antigen, as compared to either NAL antigen or mix. In the case where NAL was a coating antigen, standard curves for both inhibition antigens (AL and NAL) with all three sera were more linear and uniform. Next we investigated the capacity of different anti-sera to inhibit proteins in glove extracts, comparing two coating antigens, AL and NAL. We observed a significant difference in the appearance of the inhibition curves of glove extracts with these two coat antigens. When NAL was used as a coat antigen, the inhibition curves by all three sera were similar. The results indicate that the selection of an appropriate combination of anti-serum, inhibiting and coating antigen is critical for the accurate measurements of the NRL proteins in the finished products.
Fitting Considerations for Nonlinear Pharmacometrics
M. Katzper, CDER, FDA, Rockville, MD 20857
When rich data sets are available for pharmacokinetic data, the standard two-stage approach is used to estimate population characteristics. This involves fitting of individual data followed by averaging over the parameters of all the individuals. The reason given for not using a one-stage naïve pooled data approach is that grouping and fitting the averaged data does not distinguishing the variability within and between individuals. Using scenarios without any intra-individual variability it can be demonstrated that the one-stage pooled data approach, when used for non-linear models, is inherently flawed in defining a population average irrespective of variability. An example is constructed using exponential decay for a number of individuals with differing clearances and perfect data with no variability. Such a group has a clearly defined parametric average. A transformed linearized fit can be used to calculate the average, which coincides with the parametric average. A non-linear least-squares fit of the untransformed data does not yield the parametric average.