Abstract
100 SERIES: METHODOLOGY
P100 Qualification of Pharmacodynamic Markers by Flow Cytometry
E. Moisan1, M.S. Piché1, C. Dumont1, A. Bassiri2, and L. LeSauteur1
1Immunology, Charles River Laboratories, Preclinical Services Montreal Inc., Montreal, Canada
2GlaxoSmithKline
P101 Evaluation of an Alternative Anesthetic for ERG Recording in Dogs
C. Li and M. Vézina,
Charles River Laboratories Montreal
A novel anesthetic combination is described for electroretinograms (ERGs) in the dog. Ketamine/Xylazine, a commonly used anesthetic combination, is known to have a limited impact on ERGs; however, recovery times vary between individual animals, can be lengthy, require prolonged monitoring, and in rare occasions, results in tremors, convulsions and/or mortality. The alternative presented reduces recovery time, allows for the use of a reversal agent thus reducing need for invasive procedures and the risk of anesthetic-related mortality.
Two male and 2 female beagle dogs were assigned to a single group and were subjected to 3 different anesthetic combinations on 3 different occasions for ERG assessments. On the first occasion, dogs were anesthetized with Ketamine (5 mg/kg) and Dexmedetomidine (0.02 mg/kg) by intramuscular (IM) injection (0.9 mL/kg). Following ERGs, the Dexmedetomidine was reversed with Atipamezole (0.2 mg/kg) by IM injection (0.02 mL/kg). On the second occasion, animals were anesthetized with Ketamine (10 mg/kg) and Dexmedetomidine (0.01 mg/kg) by IM injection (0.12 mL/kg). Following ERGs, the Dexmedetomidine effect was reversed for 2/4 animals with Atipamezole (0.1 mg/kg) by IM injection (0.02 mL/kg). The remaining 2/4 animals recovered prior to Atipamezole administration. On the third occasion, animals were anesthetized with Ketamine (35 mg/kg) and Xylazine (2 mg/kg) by IM (0.45 mL/kg) as a comparator and were allowed to recover without intervention.
ERG data indicated the 10 mg/kg Ketamine/0.01 mg/kg Dexmedetomidine combination produced generally comparable ERG data compared to the Ketamine/Xylazine combination. This combination of Ketamine/Dexmedetomidine had the added benefits of a sufficient anesthetic effect and duration for ERGs, a controlled and reduced recovery time using the reversal agent Atipamezole and fewer side effects than the higher dose (0.02 mg/kg) of Dexmedetomidine.
P102 A Labor-Efficient (Single-Handed) Jugular Venipuncture Method in the Rat
R. Falvo, C. Li, and A. Adamou
Charles River Laboratories Montreal
A labor-efficient, single-handed technique for the collection of blood via the jugular vein is described for the rat. The value of this technique is that blood samples can be obtained from rats using fewer technical resources and without the complicating factors of restraining devices or anesthesia.
A total of 36 males and 36 females were ordered and received at 3 different age and weight groups. Twelve (12) male and 12 female rats at each of 7, 11 or 16/17 weeks old were assigned to 1 of 3 groups based on age and weight, and were subjected to one of 2 blood sampling procedures at multiple timepoints. The youngest rats (Group 1) weighed 175 to 260g, the intermediate-aged rats (Group 2) weighed 251 to 439g, and the oldest rats (Group 3) weighed 293 to 547g. Six (6) rats/sex/group were subject to the standard jugular blood collection (use of limb/head restraint) procedure or the single-handed bleeding technique.
Observed clinical signs suggest that the standard method is slightly more stressful and is likely due to the use of limb/head restraints, while there are no restraints used for the single-hand procedure (which is the same to the method of restraint for dose administration). For both techniques, non-adverse increases in white cell parameters were noted with a slightly greater effect in the smaller/younger animals. Additional effects and considerations noted in the smaller/younger animals using the single-hand technique included increases in creatinine kinase, and the importance of proper positioning and holding, as well as the size of the animal.
P104 Enhanced Pre- and Postnatal Developmental Toxicity Validation Study in Socially Housed Cynomolgus Monkeys
S. Henwood1, J. McCulloh1, A. Mitchell1, A. Fuchs2, and G. Weinbauer2
1Covance-Madison 2Covance-Münster
A study was designed to validate procedures for enhanced pre- and postnatal developmental (ePPND) toxicity studies in nonhuman primates by replicating Covance-Münster key processes at Covance-Madison and evaluating and comparing outcomes from the Covance- Münster and Madison facilities. Females were paired with sexually mature males for a maximum of 48 hours. Daily vaginal swabs and ultrasound examinations were performed on Days 18, 19, and 20 post coitum to confirm pregnancy. Upon confirmation of pregnancy, maternal animals were housed in social-style pens. Twenty four pregnant females received 0.9% saline at a dose volume of 5 mL/kg via intravenous injection in the cephalic vein once weekly beginning on Gestation Day 20 until delivery. Maternal animals were observed daily for mortality, and detailed observations and body weights were recorded weekly. Vaginal swabs were obtained daily and pregnancy was monitored by ultrasonography, including embryo fetal measurements in utero. Following delivery, infant evaluations, including morphological measurements, neurobehavioral assessment, grip strength evaluation, ophthalmic and electrocardiographic examination, and skeletal assessment by X-ray, were conducted. Replication of procedures across two laboratory sites for pen housing, mating, ultrasounds, and infant evaluation yielded comparable results, confirming that ePPND toxicity studies can be successfully performed at the Covance-Madison facility.
P105 Single Dermal-Dose Subcutaneous Microdialysis Study in Gottingen Mini-Pigs Collected by an Automated Sampling System
Bradley Gien1, William Jo2, Susan Lucas2, and David Hopper1
1BASi
2Dow Pharmaceutical Sciences
Microdialysis of tissue depots can be utilized to determine local concentrations of molecular compounds in live, non-terminal animals. In this study, the baseline and post-administration concentrations of a dermally-applied endogenous small molecule were determined in subcutaneous microdialysis samples from mini-pigs. Four Gottingen mini-pigs, previously treated with test article daily for 2 weeks, were anesthetized and held in slings during the procedure. Two 10 mm microdialysis loop probes were inserted subcutaneously (2 probes/animal) under the dose site, lateral to the dorsal midline. The incoming line to each probe was perfused with Ringers Solution by a Bee syringe pump, and the outgoing line was connected to a Culex Fraction Collector sample collection unit. When all animals were prepared, and the microdialysis probe flow established, a baseline microdialysis sample was collected. Test article was applied topically to a proscribed area of skin over the probes, and 8 additional samples were collected over a period of 4 hours. All samples consisted of 30-minute cumulative collections at a flow rate of 1.0 µL/minute for a total volume of 30 µL/sample. Samples were frozen and shipped to Dow Pharmaceutical Sciences for LC/MS/MS analysis.
The endogenous test substance was detected in all pre-dose dialysate samples. Concentrations increased slightly in the first 30-minute post-dose interval samples from 5 of 8 sampling sites. Concentrations decreased over time and were generally below baseline levels in the second and subsequent post-dose interval samples. The Culex automated sample collection process was successfully used to automatically collect subcutaneous microdialysis samples from anesthetized mini-pigs over a 4½-hour collection period. All mini-pigs were returned to their home runs with no adverse effects of anesthesia or microdialysis procedures. This model has potential for in-life assessment of subcutaneous levels of dermally-applied drugs in mini-pigs.
P106 A New Noise-Removal Tool Increases Information Available from ECG Recordings Catherine
Catherine Kelly1, Mutsumi Miyamoto1, Marina Brockway2, and Brian Brockway2
1Huntingdon Life Sciences, E. Millstone, NJ
2VivaQuant, St. Paul, MN
Henriques et. al. (2010) demonstrated that noise can render up to 90% of 24-hour telemetered preclinical ECG recordings from freely moving non-human primates uninterpretable. This is a frequent and significant problem in drug safety studies. Some investigators have gone to the extent of placing leads on the epicardium while others have proposed the use of intravenous leads in order to reduce uninterpretable data in ECG recordings. These approaches have demonstrated success, documenting a decrease in uninterpretable data to between 5 and 20% (Henriques et. al., Authier et. al.). However, these techniques require more invasive procedure and can increase the study cost significantly. We tested a new technology, Multi-Domain Signal ProcessingTM (MDSP denoising), (Brockway and Hamlin, 2011) to determine whether it can reduce the incidence of uninterpretable data and to evaluate whether it has any impact on the accuracy of interval measurements obtained from the recordings. Five 24-hour recordings obtained using DSI D70-PCTs and Dataquest were analyzed by an experienced operator using EMKA ECGauto before and after removing noise with MDSP. Removing noise with MDSP before measuring intervals reduced the portion of recordings rejected by the EMKA system to about 10%, comparable to the improvement achieved by intravenous and epicardial ECG sensing leads. No significant difference was found in the interval estimates obtained with or without noise removal. In addition, removing noise reduced operator time required to analyze a 24-hour recording by about 50%. This new technology shows considerable promise in preclinical drug safety studies.
P108 Developmental Toxicity Evaluation in Socially Housed Cynomolgus Monkeys (Macaca fasciularis)
C. Marc Luetjens, and Gerhard F. Weinbauer
Covance Laboratories GmbH, Münster, Germany
Recent developments in regulations governing the use of nonhuman primates (NHPs) demand social housing whenever possible and have become mandatory in many countries and this poses a specific challenge for study conduct since NHPs with timed pregnancies cannot be obtained commercially. The female cynomolgus monkey fertility rate is 35-45% per mating cycle and 60% per female animal upon repeated mating trials. Special approaches are required for mating under social housing until a cage is populated by pregnant animals. Group-related effects such as rank order fights, incompatibility, pronounced effects on ovarian cyclicity, or other adverse issues may interfere with the maintenance of gestation in pregnant animals and could have a negative impact on developmental toxicity studies. Otherwise socially housed animals may profit from experiencing birth events and handling of neonates by observing cage mates. Under these settings, neonates/infants interact and develop with cage mates of comparable age. For the present investigation, maternal and infant body weights, and pre- and postnatal losses were compared for single and social housing. Qualitative assessment in several hundred pregnant NHPs under social housing indicates good compatibility with little aggression towards each other. Body weight development was advanced in maternal control animals housed socially (> 100 animals) vs housed singly (> 200 animals). For pre-and postnatal losses, social housing appear superior with a higher number of live infants at birth and at study termination with a 5-10% reduction of pre- and postnatal loss rates and an approx. 50% reduction in stillbirth rate. In conclusion, social housing is feasible for developmental toxicity studies and apparently provides distinct advantages over single housing in the context of developmental toxicity evaluation.
P109 Toxicokinetic Comparison Using Perforated Dried Blood Spots (PDBS), Traditional DBS and Plasma in Rats Given an Oral Gavage Dose of Acetaminophen
Stephen Ploch, Christie Scheurell, Magdy Abdelhameed, Marni Peterson, Glenn Hanson, Jon Winkler, and Fumin Li
Covance Laboratories, Madison, WI
The use of accurate microsampling has been proposed as a method to address the confounding impact of variable hematocrit levels observed in dried blood spot toxicokinetic analysis. The purpose of this study was to evaluate and compare the toxicokinetics of acetaminophen by traditional dried blood spot (DBS) sampling methodology; to compare the utility of perforated dried blood spot (PDBS) accurate microsampling (i.e. 5 µL) and traditional DBS sampling (∼20 µL samples); as well as tail vein and jugular vein sample collections in rats. Acetaminophen was administered as a single 150 mg/kg dose by oral gavage to five male rats. Blood samples were collected from all animals predose and through 24 hours postdose. At each timepoint, whole blood samples were collected from a tail vein using a capillary tube (DBS) or a Drummond incremental pipette (PDBS) and applied to PDBS cards (Grade 222 paper) and two types of DBS cards (Grade 222 paper and DMPK-C cards). At each timepoint, whole blood samples were also collected from a jugular vein. A portion was applied to a DMPK-C card (DBS), a portion saved as whole blood, and another harvested to plasma. The samples were assayed for acetaminophen using validated LC-MS/MS methods. Exposure (Cmax and AUC0-24) to acetaminophen was similar using the whole blood, plasma, and traditional DBS methods; when comparing samples collected via the tail vein and jugular vein; between the Grade 222 cards and DMPK-C cards; and between the Drummond pipette (PDBS) and capillary pipette (DBS). In conclusion, DBS methods using sample collections from the tail vein, the 5 µL Drummond pipette (PDBS), and the Grade 222 card produced similar toxicokinetic profiles and should be considered for use in nonclinical toxicology studies.
P110 Evaluation of the Effects of Dark Cycle Shortening on CD-1 Mice in Chronic Toxicology Studies
Debra Farrell1, Raluca Kubaszky1, Roy Forster2
1CiToxLAB North America, Canada,
2CiToxLAB France
The standard lighting regimen for regulatory toxicology studies in rodents considered suitable for normal animal activity, neuroendocrine regulation and for decreased risk of possible retinal damage due to prolonged exposure to light, is typically 12 hours light and 12 hours dark with occasional interruptions for infrequent activities (i.e. toxicokinetic sampling). The purpose of the current assessment was to evaluate or compare GLP data collected from one carcinogenicity study with a shortened dark cycle (10 hours dark, from 9 pm to 7 am, and 14 hours light, from 7 am to 9 pm, due to twice daily dose administration or toxicokinetic blood collection) against a 13-week study performed under standard lighting conditions, with 12/12 hours light/dark and once daily dosing. All the other conditions of animal housing, care and use were similar between the two studies. Parameters monitored included clinical observations, body weight, body weight gain and food intake, measured weekly for the first 13 weeks and monthly thereafter. In the carcinogenicity study mice, palpable masses were examined after Week 26 and ophthalmological examination and white cell differential counts assessed at 12 months and beyond. At the completion of each experiment, macroscopic and microscopic pathology examinations were performed. Evaluation of food intake, body weight progression, clinical observations, differential white cell counts and ophthalmological examination results following 12 months on the reduced dark cycle revealed generally minor differences from mice exposed to a standard 12-hour cycle. There were no clinical observations or gross or microscopic findings considered related to the 2-hour reduction in the dark cycle in the carcinogenicity control animals.
P111 Seizure Liability: Characterization of Epileptic Events and Automated Seizure Detection in Telemetered Non-Human Primates
Simon Authier1,3, Mylene Pouliot1, Sylvain Bernasconi2, Thibaut de Geyer d’Orth2, Eric Troncy3, and Raluca Kubaszky1
1CIToxLAB North America, Laval, QC, Canada
2NOTOCORD®, Paris, France
3Faculty of Veterinary Medicine, University of Montreal
Early identification of compounds with seizure liability represents an important objective in drug development and a major safety concern given potentially life threatening consequences to patients. EEG and EMG monitoring by telemetry is a recognized methodology to assess pharmacological effects of test articles on the central nervous system of various species of laboratory animals, including non human primates. Continuous telemetry monitoring generates a considerable amount of data which translates into analysis challenges when using manual review. A new software application for seizure detection was developed in non-human primates (NHP). The prototype evaluates temporal and spectral features and compares them to features extracted from a sliding reference window. Detected seizures are validated based on a decision tree and artifact rejection algorithms. The automated prototype was used to evaluate periods of up to 68 hours of continuous EEG data and all detections were subject to a visual review. All animals presented at least one epileptic event. A detection sensitivity of 86.4% with 2.3 false positives per hour of signal was achieved using the default parameters of the prototype. Detection sensitivity increased however to 100% when selecting optimal settings for each individual. The processing time was approximately 5 minutes for a 24-hour EEG telemetry signal. These promising results reflect progress in data analysis. Further improvements are investigated to maintain a detection sensitivity of 100% essential with the model, while keeping the number of false detections low to increase data analysis efficiency.
P112 Comparison of Different Skin Models to the 3T3 NRU PT for Chemically-Induced Phototoxicity
L. Pratt, G. DeGeorge, and D. Cerven
MB Research, Spinnerstown, PA
The 3T3 neutral red uptake assay for phototoxicity (3T3 NRU PT) is currently the only in vitro assay approved for the screening of potentially phototoxic compounds. However, the fact that the model uses a mouse fibroblast monolayer instead of human-derived cells or cell lines raises questions as to its predictivity for human exposure to phototoxins in the environment and from medicinal and cosmetic products. While the OECD guidelines (432) are based on the 3T3 results generated by the ZEBET group and the intra-laboratory validation, it is stated that other cells can be used with the same test procedure, if equivalency is demonstrated (paragraph 9). These studies report a side-by-side comparison of the 3T3 results generated in our laboratory to those obtained from three human skin-derived cell-based systems. We compare the HaCaT keratinocyte cell line, primary adult human epidermal keratinocytes (HEKa) and a 3D human skin model using the same panel of chemicals to determine if a human-based system is as predictive as the 3T3 test model. The chemicals tested are a subset of those used to validate the 3T3 system. The 3T3 negative chemicals (Hexachlorophene, SDS and L-histidine) show similar negativity in the three human-derived skin systems. Of the 3T3 positives, Chlorpromazine and Norfloxacin were determined to be equal in responsiveness in the monolayer human cell lines to the 3T3 system, while the 3D human skin model was less sensitive by 30 to 100 fold in effective chemical concentration, but had photo-irritation factors (PIF) within the “probable phototoxin” range defined in the 3T3 assay. All EC50 concentration values for the 3D skin model are much higher than the monolayer cell systems, but are comparable in PIF value ranges to those of the 3T3 system. Amiodarone, a weak positive in the 3T3 system, is also very weakly positive for the HaCaT cells and 3D skin model, but fails to be positive for the HEKa. Necessary modifications to the 3T3 protocol for the specific culture conditions of each alternative model with their respective positive and negative aspects are described.
P113 IVSA: In Vitro Sensitization Assay
M. Troese, G. DeGeorge, L. F. Pratt, and D. Cerven,
MB Research Laboratories, Spinnerstown, PA 18968
Regulatory agencies worldwide are seeking non-animal or in vitro testing methods that embrace the 3Rs philosophy. Strong support now exists in the consumer products, pharmaceutical and cosmetics industries to develop and validate alternative assays for use in safety testing programs. Our group has adapted the Sens-it-iv in vitro sensitization NCTC IL-18 response assay of Corsini, et al. (Toxicol In Vitro 23 (5):789-796, 2009) to use in the MatTek EpiDerm™ model. This Sens-it-iv testing paradigm was from a monolayer culture system to a 3D tissue with IL-18 secretion into the media to identify dermal sensitizers. In intra-lab validation tests, IL-18 release was measured after a 24-hour topical exposure of twelve sensitizers and five irritants/non-sensitizers. Either distilled water or EtOH was used as solvent vehicles. A Stimulation Index (SI) was calculated relative to the solvent vehicle for each test material. An SI > 2.0 result was considered biologically significant and best fit the data for identification of a positive sensitizer using a 2x2 contingency table and Cooper statistics. The overall accuracy of the assay was 94%, with no false positives. The sensitivity, specificity, positive and negative predictivity were 100%, 83%, 92% and 100%, respectively. IVSA produced a dose-response with dinitrochlorobenzene and 4-nitrobutylbromide. In addition, IVSA correctly identified two photosensitizers (+UVA): chlorpromazine and olaquindox. Thus, IVSA is a useful and highly predictive tool for identifying dermal sensitizers without the use of animals.
P114 Use of Confocal Microscopy to Examine Ultra-Mild Ocular Irritation in Cultured Porcine Corneas
M. Piehl, M. Carathers, G. DeGeorge, and D. Cerven
MB Research Laboratories, Spinnerstown, PA 18968
Confocal microscopy allows for “optical histological” sectioning of a living tissue such as the cornea to determine tissue viability within the corneal epithelium without the lengthy process of traditional histology. We have developed a novel assay, PorFocal, which uses cultured waste porcine corneas from the meat industry to assay individual corneal cell death with high sensitivity due to a confocal microscopy endpoint. In PorFocal, test substances are placed directly onto living corneal tissue in culture; therefore, solubility of the test substance is irrelevant. PorFocal cultured corneas are maintained in a living state for 7 days with daily application of the test substance. This multiple-exposure dosing schedule allows for quantification of extremely mild ocular cell death with additive effects over time. These additive effects are then measured by quantification of individual stained dead cells within the corneal tissue by confocal microscopy. Corneal tissue is imaged in an “optical histological” manner where a series of image “slices” are acquired at increasing depths into the corneal tissue. The images can then be digitally reconstructed to display the entire corneal tissue volume imaged. Six cultured corneas per test material were treated with phosphate buffered saline (PBS, negative control), or 10-fold dilutions of benzalkonium chloride (BAC; 0.1%, 0.01%, and 0.001%) or sodium dodecyl sulfate (SDS; 0.5%, 0.05%, and 0.005%). Corneas were dosed topically with 50-μL of test substance daily for 7 days. On day 8, corneas were stained with dead cell stain and imaged using confocal microscopy. All dead cells were counted for each tissue field and statistical analysis was performed using ANOVA. Test substances dosed at 10-fold dilutions were statistically significant (p<0.001) in a dose dependant manner. These data demonstrate the potential sensitivity of the PorFocal assay.
P115 Glucose Tolerance Test and Clinical Chemistry in Male Beagle Dogs
Y. Wang, Z. Qiu, Y. Wu, E. He, D. Zeng, J. Shen, Y. Lu, G. Zhang, Q. Zhu, S. Li, X. Tian, X. Gong, X. Miao, X. Li, J. Schumacher, M. Moldenhauer, M. Tate, and L. Lin
Covance Pharmaceutical R&D, Shanghai, 201318, China; Covance Laboratories, Inc. Madison 53704, USA
Large animal models are required for diabetic drug discovery in addition to rodent models. There are limited data available worldwide especially in China. The oral glucose tolerance test (OGTT) is extensively used to screen new diabetic drugs. To establish this screen method in laboratory dogs, nine naïve male beagle dogs from a pool of eleven were selected and assigned to three groups (3/group). Following overnight food-fast, Group 1, 2, and 3 animals received glucose by oral gavage at 1, 2, and 2.5 g/kg, respectively. Their glucose levels in serum, plasma and whole blood (by glucometer) were measured at 0, 30, 60 and 120 minutes post-dose. In addition, a full panel of clinical pathology parameters was tested from fasted and non-fasted dogs in plasma and serum for profiling. OGTT demonstrated that all dogs were well tolerated with oral glucose administration. Their blood glucose levels were comparable between serum, plasma and whole blood with highest read-out in plasma and lowest in whole blood by glucometer. The dogs receiving 1 g/kg glucose showed higher blood-glucose spike at 30 minute time points when compared with other glucose level dogs. The clinical pathology data showed that serum triglyceride (32 mg/dL) and BUN (13 mg/dL) in fasted dogs were lower than that in non fasted dogs (triglyceride 55 mg/dL and BUN 21 mg/dL), while creatinine and bilirubin were higher in the non-fasted dogs. In conclusion, beagle dogs can be utilized in diabetic research and model establishment.
P116 Predictivity of Non-Clinical Repolarization Assay Data for Clinical TQT Data in FDA Database
J. Koerner1, J.P. Valentin2, J Willard1, E.J. Park1, D. Bi1, W.T. Link1, M. Fiszman1, D. Kozeli1, M. Skinner2, H.M. Vargas3, L. Cantilena4, G. Gintant5, T. Wisialowski6, and S. Pettit7
1FDA, 2AstraZeneca, 3Amgen, 4Uniformed Services University of the Health Sciences, 5Abbott, 6Pfizer, 7HESI on behalf of the Cardiac Safety Pro-Arrhythmia Working Group
Drug-induced QT interval prolongation and Torsades de Pointes remain serious public health issues when new pharmaceuticals are brought to the market place. Under the auspices of ILSI Health and Environmental Sciences Institute (HESI), a consortium involving representatives from pharmaceutical companies and regulatory agencies has been initiated. One objective is to assess the predictivity of non-clinical repolarization assays in relation to clinical measures of QT interval prolongation, i.e. establish a quantitative risk assessment for individual compounds. To this end, the consortium conducted a retrospective analysis of non-clinical and clinical data housed in the U.S. Food and Drug Administration database. This presentation will provide a final analysis of the predictivity of data from Thorough clinical QT (TQT) studies and nonclinical studies submitted to support marketing approval of >150 new pharmaceuticals. Nonclinical studies analyzed include in vitro ion channel assays for effects on hERG current, action potential duration (APD) in isolated cardiac tissue, and in vivo ECG studies (QTc). Normalized concentration response data is compiled for each assay and compared across studies to determine the predictivity of nonclinical to clinical data. This analysis will be shared along with estimation of sensitivity and specificity of these assays at various nonclinical to clinical exposure multiples. The results suggest that a robust non-clinical risk assessment provides reasonable predictivity when the clinical QT signal is of sufficient magnitude (>10ms).
P117 Enhanced Delivery of Intranasally-Administered Nucleoside Drugs to the Central Nervous System
Mansi Krishan1, Gary Gudelsky2, Pankaj Desai2, and Mary Beth Genter1
1Environmental Health and 2 College of Pharmacy, University of Cincinnati
Delivery of therapeutics to the brain to treat neurological diseases is a challenge due to impenetrable nature of the blood brain barrier (BBB). Intranasal (IN) drug administration is a non-invasive approach for rapid direct delivery from the nose to the central nervous system (CNS), thereby minimizing systemic exposure. The current study focuses on the use of the IN route for delivery of the nucleoside drug gemcitabine (GEM). In order to enhance drug delivery to the CNS, we used papaverine (PV), which has been demonstrated to transiently increase the permeability of the brain-tumor barrier and BBB after systemic administration. The BBB and nasal epithelial tight junctions (TJs) share many proteins in common. We hypothesize that by transiently increasing the permeability of nasal epithelial tight junctions using PV, we will increase the concentration of GEM in the brain extracellular fluid (BECF) following IN delivery, with the goal of delivering therapeutic concentrations of nucleoside drugs to the CNS. Experimental methods include IN administration of fluorescein isothiocyanate-dextran beads (FD4) to determine IN drug distribution, in-vitro GEM recovery, in vivo brain microdialysis for BECF collection, HPLC analysis to measure GEM concentrations in BECF, histopathology, and western blot analysis to study the time course of altered levels of tight junction (TJ) proteins in the olfactory epithelium (OE). A non-toxic dose of PV, which enhanced delivery of GEM to BECF, was determined. BECF pharmacokinetics of GEM shows AUC=5.55±0.84 μg.h/ml for PV (1.4%) + GEM (50mg/kg) treated animals (n=4), compared to 1.5±0.29 μg.h/ml without PV treatment (n=4). The IN drug concentration in BECF was comparable to the AUC values when GEM and another BBB permeabilizer were administered intravenously. Studies with FD4 beads showed significant deposition in the OE, suggesting drug uptake through OE. Western blot analysis showed that IN PV treatment increases permeability through OE TJs by transiently decreasing the levels of TJ protein occludin. Thus, it appears that transient permeabilization of nasal epithelial TJs provides a non-invasive means to enhance delivery of nucleoside drugs to the CNS.
P118 Qualification of 96-Well High Throughput In Vitro Micronucleus and Comet Assays
L.F. Stankowski1, Jr., K. Pant1, T.E. Lawlor1, and M.J. Aardema1,2
1BioReliance by SAFC, Rockville, MD USA
2Marilyn Aardema Consulting, LLC Fairfield, OH USA
We recently qualified 96-well high throughput versions of the in vitro micronucleus (MN) assay in CHO cells, and the Comet assay in TK6 cells. The MN assay evaluated the ten reference compounds in OECD TG 487 using MicroFlowTM kits (Litron Laboratories; 10 independent trials), while the Comet assay evaluated six reference compounds using the Japanese Center for Validation of Alternative Methods (JaCVAM) Comet validation protocol (version 6.2; four independent trials). All trials were performed, using 7-10 concentrations in duplicate cultures with and without exogenous metabolic activation (S9), to assess inter- and intra-experimental variability, as well as sensitivity and specificity. Consistently positive responses were observed in the MN assay for the model genotoxins mitomycin C and cytosine arabinoside –S9, and benzo(a)pyrene and cyclophosphamide +S9 (all with a clastogenic signature); and for colchicine and vinblastine with and/or without S9 (both with an aneugenic signature). In agreement with the expected results, nongenotoxins di(2-ethylhexyl)phthalate, nalidixic acid, pyrene and sodium chloride were reproducibly negative in all MN trials at all dose levels ±S9. Reproducible positive results were observed in the Comet assay for the genotoxins 2-aminoanthracene, 9-aminoacridine, ethyl methanesulfonate and methyl methanesulfonate, while reproducible negative results were observed for the nongenotoxins cycloheximide and triton-X. These results support the use of these high throughput assays for genotoxicity screening in general and are being employed in the US EPA ToxCast program.
P119 Development of the Replacement Ocular Battery Tier 1 – Chorioallantoic Membrane Vascular Assay
D.R. Cerven, D. Hall, G.L. DeGeorge
MB Research Laboratories, Spinnerstown, PA
As the initial tier of the Replacement Ocular Battery (ROBATT) – a tiered testing strategy for regulatory classification of ocular irritation without the use of live animals – the Chorioallantoic Membrane Vascular Assay (CAMVA) was used to screen the ocular irritation potential of 52 chemicals with known levels of severity ranging from non-irritant to corrosive. Changes to the vasculature of the Chorioallantoic Membrane (CAM) were evaluated and a reference value (RC50) computed for each test chemical. Individual animal data from the ECETOC database were available for 37 of the 52 chemicals. Fifteen chemicals were selected after consultation with representatives of the EPA and FDA. Basic EPA classification information was available for these materials but individual animal data were not.
Based on the results of the CAMVA screen, 30 chemicals with irritating potential will advance for further evaluation in the Bovine Cornea Opacity and Permeability Assay (BCOP). These chemicals are expected to be in the moderate to corrosive range of ocular irritancy. Twenty-two chemicals with slight to non-irritating potential will be evaluated in the Porcine Confocal Assay (PorFocal). These chemicals are expected to be in the non-irritating to slightly irritating range.
Initial comparison of the CAMVA screen results with ECETOC and FIFRA data indicated evidence of over-prediction (5 of 30) for moderate - corrosive chemicals and under-prediction (3 of 22) for slight - nonirritating chemicals. Since the ROBATT is a multi-tiered test system, all test chemicals will be evaluated in at least two models before being classified into an appropriate regulatory corrosive category.
ROBatt is a two-year research grant funded by the NIH and FDA to develop a tiered testing strategy of alternatives to replace the need for using live rabbits in ocular irritation classifications.
P120 Development of the Replacement Ocular Battery (Robatt) Tier 2 – BCOP
D. R. Cerven, D. Wolfinger, and G. DeGeorge
MB Research Laboratories, Spinnerstown, PA
The Replacement Ocular Battery (ROBatt) is being developed as a Tiered Testing Strategy to replace Draize in vivo ocular irritation testing. It comprises (1) Chorioallantoic Membrane Vascular Assay (CAMVA), mimicking the vascular reaction of the conjunctiva; (2) Bovine Cornea Opacity/Permeability Test (BCOP), to assess mild to moderate corneal damage; (3) Porcine Corneal Opacity/Reversibility Assay (PorCORA), to discriminate severe irritants from ocular corrosives; and (4) PorFocal Assay, to separate non-irritants/slight irritants.
Initially, CAMVA segregated 52 chemicals with known levels of ocular irritation into two groups. The 30 most irritating were tested by BCOP, which measures reduction in corneal light transmission following insult. Fluorescein permeability detects corneal changes that may not be associated with opacity. Corneal damage was calculated as In Vitro Score (IVS) = Opacity Score + (15 x Permeability Score). A BCOP IVS threshold of 12.0 was chosen to assign 17 chemicals as severe/corrosive to be tested in PorCORA, and 13 chemicals as moderate irritant category (HMIS 2, GHS 2A and EPA III). ROBatt categorizations were compared with those obtained from the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) database and information provided by the NIH and FDA. Of the 13 materials in the moderate category, 5 classified Category III (USEPA FIFRA guidelines), 5 classified Category IV, and 3 were Category II. Results indicate a need to establish an IVS range below which the test material would be evaluated by PorCORA to establish Category IV classification. ROBatt is a 2-year research grant funded by the NIH and FDA.
P121 Linking Chemical Categories to Adverse Outcome Pathways for Chronic Toxicity
M.T.D. Cronin1, S.J. Enoch1, M.D. Nelms1, J.C. Madden1, A-N Richarz1, C. Yang2
1Liverpool John Moores University, UK
2Altamira LLC, Columbus OH
The formation of chemical categories allows for read-across to be performed to predict the toxicity of chemicals. Increasingly, category formation and read-across are being seen as one of the few reliable solutions to predict chronic (organ level) toxicity. Category formation relies on the use of appropriate methods to identify similar compounds. In this investigation, to develop categories for chronic toxicity, the anchor point was the consideration of appropriate Adverse Outcome Pathway (AOPs) relating to human organ level toxicity. AOPs record information relating to the perturbation of biochemical pathways which may result in an adverse effect. Rational in vitro or other testing can identify areas of chemical space associated with an AOP, which can then be used to define chemical categories associated with the pathway. Focusing on liver toxicity, the use of reactive fragments associated with known mechanisms of toxicity e.g. reactive hepatotoxicity was evaluated. Structural rules, fragments and properties, associated with these mechanisms of toxic action at the organ level have been identified and coded (e.g. as SMARTS strings) into a KNIME Workflow. However, reactivity alone has poor sensitivity and specificity. As a result categories were sought for other AOPs relevant to liver toxicity. Key challenges for this approach are the identification and verification of mechanisms of action. The research leading to these results has received funding from the European Community’s 7th Framework Program (FP7/2007-2013) under grant agreement n° 266835 and from Cosmetics Europe and from the European Community’s 7th Framework Programme Innovative Medicines Initiative Joint Undertaking (IMI-JU) eTox Project (grant agreement n° 115002).
P122 Health Risk Ranking Framework for the Life Cycle of Nanomaterial-Containing Products: Comparison of Industrial Versus Consumer Application Settings
A.K. Madl1, K. Unice2, M. Kreider2, M. Kovochich1, and I.G. Bebenek1 and M.M. Abramson2
1ChemRisk, LLC, Aliso Viejo, CA,
2ChemRisk, LLC, Pittsburgh, PA
Engineered nanoscale materials have provided significant advancements in a wide range of industries. Because of their unique combination of size, structure, morphology, physical/ chemical characteristics, and quantum properties, understanding the potential hazards and health risks of engineered nanomaterials and the products that utilize these materials present unique challenges. Multiple formulations and a range of applications add further complexity to the array of different scenarios for potential exposure to nanomaterials. With the diverse use of nanomaterials in occupational, consumer and environmental settings, there is a significant need for risk assessment tools for hazard and health risk evaluations within industry and government. Thus, a hierarchical risk ranking framework was developed as a methodological approach to identify and prioritize hazards and health risk scenarios involving nanomaterials. This conceptual risk ranking framework was applied to a case study of two types of nanostructured materials in an industrial and consumer setting. A nanomaterial hazard classification scheme was established based on 1) the location of the nanoscale structure in the system, matrix or material, and 2) the intrinsic nanomaterial physical and chemical properties. Utilizing a control banding framework, a numerical risk ranking was derived based on a customized hierarchical scheme of product and nanomaterial characteristics, use and exposure patterns, and toxicological information. The sensitivity of the risk ranking to the assumptions used in the analysis and the use of a numerical score for risk ranking were also assessed. Through the development of this hierarchical framework, several parameters were apparent as primary drivers for influencing the risk ranking estimates, including releasibility from the system or matrix, exposure pathway and intensity, bioavailability, biopersistence, and severity of health effects. There were evident differences in hazard ranking between nanostructured materials of similar elemental composition but different morphologies. Furthermore, risk ranking scores differed for nanomaterials handled in an industrial setting as compared to a consumer application setting. Although additional health-based research would reduce uncertainties to identify materials of greatest potential risk, the existing approach provides a basic structure to classify and prioritize potential risks of existing and newly developed nanomaterials or nano-enabled products. This framework offers a novel approach that applies hazard/risk ranking taking into account information about the parent product and substructure, use and exposure propensity, and aspects of physicochemisty that influence toxicity criteria throughout the nanomaterial lifecycle.
P123 A Study in Telemetry Implanted Cynomolgus Monkeys to Evaluate Select Cardiovascular Parameters in Animals Restrained in Procedure Cage or Restraint Chair (Pole and Collar)
N. Lalayeva1, N. Bailey1, J. Kenfield1, N. Makori1, T. Beck1, and R. Nagata2
1SNBL USA, Ltd., Everett, WA, USA
2Shin Nippon Biomedical Laboratories, Ltd., Kagoshima, Japan
The nonhuman primate is a well established animal model for nonclinical toxicology studies. For many decades, standard preclinical practice has involved the use of either chemical restraint, typically by ketamine or manual restraint methods, such as hand-catching or pole and collar systems. Restraint-associated stress can influence values of many study parameters including hemodynamic and cardiovascular measurements as well as clinical chemistry, hematology, and behavioral assessment. The Procedure Cage is a patented animal restraint apparatus designed in Japan by Dr. Ryoichi Nagata. The Procedure Cage allows the primary animal enclosure to remain a safe area for the animal by separating all procedure activities from the home cage. The USDA mandates research facilities to evaluate alternatives utilizing the principles of the 3 R’s. The Procedure Cage is a refinement in restraint technique. This study was conducted to compare cardiovascular parameters for restraint in the Restraint Chair (RC) vs. Procedure Cage (PC). Six telemetry implanted animals were monitored and data analyzed for heart rate (HR) and mean arterial blood pressure (BP) using a validated Notocord Telemetry System. On non-restraint days data were captured for 15 min every 3 hrs and on restraint days, data were captured from 2 hrs prior to 1 hr post restraint. In a series of procedures performed, the average HR for RC and PC animals was 176±19 bpm and 151±12 bpm, respectively. The average BP was 105±3 and 93±3 for RC and PC animals, respectively. The RC animals returned to baseline HR and BP levels only 22 and 33% of the time, beginning at 15 and 30 min post procedure, respectively. Whereas, the PC animals returned to baseline HR and BP levels 50 and 91% of the time beginning at 5 and 10 min post procedure, respectively. In conclusion, the procedure cage animals’ HR and BP values were closer to baseline levels throughout testing were less variable, and returned to baseline with a shorter latency, and at a greater rate than in animals tested using the restraint chair.
P124 Drug Discrimination Testing and Substitution Assessments in Adult Male SD Rats of Morphine Prepared in Multiple Vehicles
Jonathan D. Toot, Matt R. Bennett, Melissa J. Beck, Tim A. Pringle, Jacob Perl, Liz C. Lendrum, Donald G. Stump
WIL Research Laboratories, 1407 George Road, Ashland, OH, 44805-8946
The objective of this study was to assess the drug discrimination testing paradigm utilized at WIL Research Laboratories using morphine as the reference compound with test drug substitutions of morphine prepared in a variety of commonly used and specialty vehicles in the rat. The animals used for this study consisted of adult Sprague Dawley males, with 10-15 rats/group. Rats were first trained by food pellet reinforcement under a fixed ratio 20 schedule to discriminate between the compound associated levers when administered the reference compound, morphine (5.6 mg/kg), or the vehicle control (saline) via IP injection approximately 30 minutes prior to each session. Discrimination testing was conducted following compound substitution of morphine at 0, 0.3, 3.0, 5.6, and 9.0 mg/kg prepared in saline, 60% PEG 400 in water, 70% PEG 400 in water, 50% DMSO in saline, 35% Tween 80 in saline, and 32% ethanol in saline. Using this paradigm, the first 20 lever presses were reported as the relative percent of RC lever selection. The results of this study showed RC lever selection (>80%) for substitution with morphine (5.6 and 9.0 mg/kg) in saline, 60% PEG, 70% PEG, 35% Tween 80 and ethanol, with 50% DMSO only showing partial substitution (>30%). However, the 0 and 0.3 mg/kg morphine prepared in 70% PEG partially substituted for morphine, which failed to substitute in the other vehicles tested. Behavioral observations following dose administration attributed to the vehicles consisted of uncoordinated use of hindlimbs, circling, walking on tiptoes, and hunched posture. Therefore, the DD testing paradigm was able to characterize morphine in various vehicles for a comparison of potential discriminative properties.
P125 Self-Administration Assessment and Bioanalytical Analysis of Morphine in Adult Male Rats
Jonathan D. Toot, Michelle L. Hackman, Tim A. Pringle, Seth R. Bell, Ashley N. DePizzo, Alison E. Johnson, Sara M. Loris, Melissa J. Beck, Donald G. Stump
WIL Research Laboratories, 1407 George Road, Ashland, OH, 44805
The objectives of this study were the following: 1) assess the self-administration testing paradigm utilizing a Latin square study design, 2) demonstrate the positive reinforcing effects in this model with the test drug morphine, 3) assess plasma morphine concentration levels in low/mid/high responders following testing and 4) demonstrate the ability of morphine to be self-administered in the rat within the scope of the European Medicines Agency guidelines. The animals used for this study consisted of adult Sprague Dawley male rats, at approximately 16 weeks of age at the initiation of training. Rats were trained to press the active lever in order to receive an IV infusion (reinforcer) of the training/reference compound, morphine, during a maximum 2 hour test session on a fixed ratio schedule. This was followed by alternating between extinguishing that behavior with saline and substitution sessions with morphine (0.05, 0.2, and 0.6 mg/kg/infusion). Using this paradigm, animal responses were measured as the average number of infusions per session. During testing, morphine at 0.2 mg/kg/infusion reliably maintained self-administration behavior under baseline conditions. Saline, 0.05, and 0.6 mg/kg/infusion substitution for the reference compound resulted in an extinction and/or decrease in self-administration behavior. Following the completion of testing, rats were bled under isoflurane anesthesia, at the following time points: 0 hr (end of test session), 1 hr, 2 hrs, 6 hrs and 22 hrs (prior to the next session). Plasma morphine concentrations corresponded to the identified low/mid/high responders ranging from 7 to 239 ng/mL at 0 hr, with levels at or below the LOQ within 6 hrs. The self-administration Latin square testing paradigm, as well as the reference compound training and substitution levels were able to successfully establish the positive reinforcing potential of morphine consistent with its known abuse liability. Therefore, these results further validate the self-administration methodologies described at WIL Research.
200 SERIES: TEST SYSTEMS
P201 Pharmacodynamic Glycemia Effects From Rapid and Long-Acting Insulins Administered at Mealtime to Alloxan-Induced Diabetic Miniature Yucatan Swine
L.D. Brown, T.J. Madsen, E.C. Blair, B.C. Hanks, K.P. Horlen, J. Hiemstra, A. Stricker-Krongrad, J.J. Liu, and G.F. Bouchard
Sinclair Research Center, LLC and Sinclair BioResources, LLC
Pharmacodynamic effects from various insulins with known properties in humans were studied in the Yucatan miniature swine for comparative purposes. Yucatan miniature swine (Sus scrofa, at least 3 months, 20-60 kg) were made diabetic with intravenous alloxan and regulated on insulin. Animals were considered diabetic if they became hyperglycemic (≥150 mg/dL) within 2-5 days following induction. All procedures were on overnight fasted animals (no feed or insulin for 18 hrs). Our diabetic miniswine average baseline bG of 429 ± 84.5 (SD) mg/dL (N=148 measurements) while non-diabetics average 58.7 ± 8.2 (SD) mg/dL (N=238 measurements). For this study, well-known prototypical marketed insulins (Apidra™, Humalog™, Lantus™; 0.25 or 0.45 U/kg s.c.) were administered at mealtime, then blood glucose profiles recorded using handheld glucometer devices (One Touch Ultra®, Lifescan) over the next 8 hrs (rapid-acting) or 24 hrs (long-acting). Venous blood samples were collected from VAPs for bG readings. Blood glucose profile data in the diabetic Yucatan generally compared well to published human glucodynamic data for glycemia effects following insulin. These data suggest the Yucatan diabetic model has similar pharmacodynamic responses to the presentation of exogenous insulins for onset and peak effects but not duration for the rapid-acting insulins. The long-acting insulin (Lantus™) peaked in swine where no peak (same action throughout day) is normally reported in humans. The cited differences could be due to either the duration of the fasting plus relative high doses of insulin administered, the use of a small number of animals in this study, or a biological difference in the glucodynamic profiles of rapid-acting insulins between minipigs and humans.
P202 Miniature Swine Model of Phototoxicity Testing
H. Leigh1, P.D. Forbes2, C. Lawson1, D.Y. Kim3, D. White1, L.D. Brown1, D.R. Wehmeier1, J. Liu1, J. Hiemstra1, and G.F. Bouchard1
1Sinclair Research Center, LLC, Auxvasse, MO, USA
2Aclairo PDG, Inc., Vienna, VA, USA
3Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO, USA
This study determined the threshold doses for “solar erythema” and for phototoxic responses to 8-methoxypsoralen (8-MOP) in white skin Hanford and grey skin Yucatan miniature swine. For threshold erythema determinations, the UVR exposures included both UVA (315 – 400 nm) and UVB (290 – 315 nm) radiation by positioning one fluorescent ‘sunlamp’ among 10 ‘PUVA’ lamps. With this configuration the UVR exposures ranged from 0.5 - 2.8 times the “instrumental MED” (MEDi) for Hanford and from 1.0 - 5.6 times the MEDi for Yucatan. For phototoxicity determinations (i.e., with and without topically-applied graduated concentrations of 8-MOP), the UVB component was minimized by extinguishing the sunlamp, thus permitting higher UVA exposures. The Hanford had the lower UV erythema dose threshold (1.0-1.4 times the MEDi) and the erythema that developed was readily observable. The exposure doses for the phototoxicity test were 5J/cm2 of UVA in 35 minutes or 10 J/cm2 in 70 minutes. The phototoxic (vascular) response to 8-MOP was observed in the two highest concentrations (0.01% and 0.1%) in Hanford pigs, in a dose-related manner. Microscopic evidence of a dose-related response was also observed as the concentration of 8-MOP increased. This verified that the Hanford miniature swine is the preferable strain for phototoxic effects. In contrast, UVR exposure of the Yucatan pig skin produced tanning rather than erythema, confirming that the Yucatan is the more appropriate strain for studying the melanization response. Thus, Hanford and Yucatan miniature swine have cutaneous photobiological responses that reflect their respective strain differences regarding skin melanization.
P203 A T-Cell Dependent Antibody Response (TDAR) Validation Study in Cynomolgus Monkeys
Richard Early, Hong Yu, Fei Zhou, Dandan Lin, and Ying Jiang
WuXiAppTec (Suzhou) Co. Ltd., Suzhou, Jiangsu, PR China
Immunogenic responses to a single intramuscular (IM) injection of keyhole limpet hemocyanin (KLH; approximately 2.5 mg/kg) were assessed in cynomolgus monkeys (3 males and 3 females per treatment) over a 28 day period. Three treatments consisted of daily oral water + a single IM saline injection (
There were no clinical signs clearly related to KLH or CTX. CTX decreased white blood cells, total lymphocyte and lymphocyte subsets (pan-T, T-helper, T cytotoxic/suppressor, natural killer and B cells), reticulocytes and circulating red cell mass relative to pretest levels and other groups. There were no findings KLH- or CTX-related in the serum chemistry data. KLH induced a marked anti-KLH IgG and IgM antibody response beginning on Day 8. Anti-KLH IgG and IgM antibodies were similar to or greater in the males receiving
P205 A New Approach for Genotoxicity Testing of Dermal Exposures: The 3D Human Skin Micronucleus Assay
Marilyn Aardema1, Shambhu Roy2, Anna Szkudlinska2, Keyan Wang2, Jing Shi,2 Scott Hickman2, and Wannie Madraymootoo2
1Aardema Consulting, LLC
2BioReliance Corporation, Rockville, MD
Since skin is the site of the highest exposure to many chemicals, drugs and other products like cosmetics, an assay for the assessment of genotoxicity in skin can be a valuable addition to a testing approach. This is especially applicable to cosmetic ingredients that can no longer be tested in in vivo genotoxicity assays due to the 2009 EU 7th Amendment ban. To meet these needs, the 3D reconstructed human skin micronucleus assay (RSMN) in EpiDermTM was developed. This assay offers a more biologically relevant in vitro approach to assess genotoxicity compared to standard 2D in vitro genotoxicity assays since it provides a functional stratum corneum that takes into account permeability, appears to have normal dermal metabolic capability and is expected to have more normal DNA repair and cell cycle control unlike p53 deficient cell lines used in most standard genotoxicity assays. To meet the increasing interest in this assay, we have developed a GLP RSMN assay that also incorporates CREST labeling of micronuclei for identification of aneugenic versus clastogenic mechanisms. Qualification studies with chemicals of various modes of action were conducted including mitomycin C (cross-linker), vinblastine sulphate (aneugen), methylmethane sulfonate (clastogen). Results show dose-dependent increases of MN and demonstrated good reproducibility and comparability to previously published results. Non-carcinogens, such as 4-nitrophenol, are negative in the assay. Chemicals like vinblastine sulphate induce a large increase in micronuclei with positive CREST labeling indicative of aneugenicity. The GLP RSMN assay is a promising new in vitro assay for genotoxicity testing.
P206 Minipigs Display No Gender Bias in Local Reactions in Dermal Toxicity Studies
N.C. Ganderup
Ellegaard Göttingen Minipigs, Denmark
The pig/minipig has established itself as the model of choice for dermal toxicity studies, and is furthermore mentioned in the nonbinding recommendations for wound healing models given by the US FDA. Using pharm/tox reviews from NDAs (New Drug Application, available from Drugs@FDA) the author investigated whether there was a gender bias in local reactions in dermal studies.
More than 40 NDAs where the minipig was used as the non-rodent species were identified. Dermal testing was involved in 24 of the NDAs, and 36 dermal studies were described. No pattern of gender bias in local reactions was identified. The studies had wide ranges in a series of parameters: group size (1-6 animals/sex/group), duration (10 days to 52 weeks), exposure (15min/day to 23hrs/day), dosing method (occluded, non-occluded, or patch).
Only two cases (in separate NDAs) of gender specific differences in local reactions were identified: (1) one instance dermatitis was observed in all groups except low-dose females, and in another case (2) dermatitis appeared more frequently in females than in males. In five cases, differences between male and female local reactions were observed in a single animal of one or both sexes in a given study, i.e. not a group effect.
This investigation is unlikely to have uncovered all available literature on this matter so any generalisation should be done with care. However, it seems reasonable to conclude that gender based differences in local reactions in dermal studies are not a general phenomenon in the minipig.
P207 Body Temperature Monitoring in Anesthetized Beagle Dogs, to Establish Acceptable Conditions for Conduct of Pharmacokinetics or Safety Assessments of Thermo-Sensitive Compounds
D. Maynard1, C. Copeman1, Y. Trudel1, C.E. Swenson2
1Charles River Laboratories Montreal
2Celsion Corporation
Investigations were performed to determine whether the body temperature of beagle dogs (two males and two females) can be maintained within a range of 36.5 to 37.5ºC while under general anesthesia over a two hour period. The body temperature of these dogs was also monitored for up to 4 hours following the end of anesthesia. The ability to maintain a tight core temperature range was investigated in order to establish appropriate conditions to support future pre-clinical studies for temperature-sensitive compounds.
The body temperature monitoring was performed using two approaches to allow comparative data recording through the use of a common (calibrated) rectal thermometer and oximeter thermometer probe placed in the esophagus of the dogs. Recordings were performed before, during and up to 4 hours after the end of the anesthesia.
The investigations confirmed that body temperature of beagle dogs can be maintained within a range of 36.5 to 37.5ºC while under general anesthesia but required the presence of heating devices (e.g., Bair Huggers and/or heating pumps) to prevent fluctuations outside of the targeted range. In general, the temperature of the animals returned to pre-anesthesia ranges 1.5 to 2 hours after the end of the anesthesia. As such the dog, maintained under these conditions, presents an acceptable model for preclinical assessments of thermo-sensitive compounds.
P208 Diet Acclimation of NZW (Crl:KBL[NZ10]) Rabbits for Use in Embryo-Fetal Development Studies
A.I. Martin, M. Freke, A. Hoberman, E. Lewis, R. Perras, I. Primakova, and K. Robinson
Charles River, Montreal; Charles River Canada, Pennsylvania, St Constant
Poor food intake in pregnant rabbits can lead to adverse outcomes including abortion. Initial validation studies with a line of New Zealand White (NZW) rabbits, showed a high incidence of inappetence and adverse pregnancy outcomes. Several studies were conducted to optimize diet acclimation procedures at the breeding facility to match the certified diet used at the testing facility. Once the diet acclimation was optimized, a study was conducted that met the ICH guideline for evaluation of embryo-fetal development (EFD). Naturally bred adult female NZW (Crl:KBL[NZW]) rabbits were received (22 rabbits) from Charles River, Canada on Day 0 or 1 postcoitum (pc). For the premating diet acclimation the does were fed increasing percentages of an irradiated version of PMI 5322 for 10 days before being given this diet alone at 150 or 180g/day for a further 10 days. The animals were fed 180g/day of PMI 5322 during the study. The does were dosed with standard vehicles from Days 7 to 19 pc by oral gavage, intravenous injection, or inhalation; in addition there was a room control. Food consumption was measured daily and body weights were collected twice weekly. Clinical observations and any signs of abortion or premature delivery were recorded twice daily. Does were bled for laboratory investigations on Day 20 pc. Terminal examinations on Day 29 pc included a necropsy, corpora lutea counts and evaluation of the uterine contents. Live fetuses were weighed and examined for external, visceral and skeletal abnormalities. The does showed a high pregnancy rate and a low abortion rate. The occurrence of periods of inappetence during gestation was negligible. Ovarian, uterine and fetal parameters were comparable to other lines of NZW rabbits, with slightly higher litter sizes and consequently slightly lower fetal weights noted. In conclusion, the diet acclimation procedure decreased the inappetence that was seen in previous rabbit reproductive toxicology studies and that this line of NZW rabbit is suitable for use on EFD studies.
P209 Evaluation of Background Data From CD1 Mice Used in Carcinogenicty Studies
P. Mansell, A. Adamou, G. Hennig, L. Kangas
Charles River Laboratories Preclinical Services, Montreal, Quebec, Canada
The CD1 mouse has been used in conventional rodent bioassays for the assessment of carcinogenic potential in drug development for several decades.
To establish the continuing relevance of the CD1 mouse as an appropriate and stable model for carcinogenicity testing, data collected over a twenty year period from Crl:CD1 mice housed in a conventional non-barrier facility was evaluated. Primary end points chosen included survival, body weight and body weight gain, food consumption and incidence of common neoplastic and non-neoplastic lesions. Dosage routes included oral gavage, dermal, dietary and nose only inhalation.
In the majority of studies, mice received a standard rodent diet containing 20% protein fed ad libitum, and there was no evidence of any changes in food consumption patterns over the period of investigation when comparing recent studies to studies from 1990’s. Similarly, body weights changes over time were not apparent and this correlated with the previously observed stability in food consumption.
There was no evidence of major trends in changes of survival rates over time and introduction of social housing appears to have no influence on survival. Overall survivability at 104 weeks in males was 52% (ranged from 38 to 86%) and 46% (30 to 84%) in females.
Neoplastic lesions were commonly observed in several tissue types including adrenal glands, hemolymphoreticular tissue, liver and lung. These tumor types are typical of what has been reported in the literature and indicate a level of uniformity across the strain and there was no evidence of significant increases in any tumor type.
It is concluded that the CD1 mouse is still a relevant model for assessment of carcinogenic potential of pharmaceuticals. The uniformity of the model has been demonstrated over a prolonged time course for carcinogenicity testing and successful drug registration.
P210 A Comparative Assessment of Vena Cava Diameter in Rats, Dogs and Non-Human Primates
J. Forget and C. Copeman
Charles River Laboratories Montréal
Rats, dogs and non-human primates commonly used laboratory species for conduct of intravenous infusion studies have remarkably different vessel sizes which can influence responses at the site of delivery (vena cava), in particular for formulations with irritant potential. The vessel and catheter size, and rate of delivery will influence the blood volume/dilution capacity at the site of delivery, and will contribute to the extent of turbulence at the catheter tip; factors that can further exacerbate local irritancy. The relevance of the rodent versus the non-rodent models within a safety assessment program regarding the predictability of a local response may therefore arise. Comparative assessments of vena cava diameters between Sprague-Dawley rats, beagle dogs and cynomolgus monkeys were performed using fluoroscopy and by direct measurement (using callipers) prior to euthanasia, to determine diameter of the vena cava, at the age and weight ranges of these commonly used species, to compare to that reported for humans, and serve as guidance for likelihood of model predictability. The correlation of the vessel diameter in relation to body weight was also determined.
Minor expected variability were seen between the 2 methods of measurement, however, comparative differences to that reported for humans, could be evaluated. Vena cava of rats was on average 0.15X, cynomolgus monkeys 0.30X and beagle dogs 0.55X that of humans. Vena cava diameter did not show correlation to weight in rats while closer correlation was noted in dogs and primates, in the age range evaluated. With these measurements and correlations considered, studies conducted in Beagle dogs, using 9-12 Kg dogs, may serve as a better model for assessment of irritancy potential within a central vein, when this represents the planned administration site for the clinical scenario.
P211 A High Throughput Metabolite-Based Biomarker Approach to Predict Developmental Toxicity Using Human Embryonic Stem Cells
P.R. West, A.M. Smith, K.R. Conard, J.A. Palmer, L.A. Egnash, R.E. Burrier, and E.L.R. Donley
Stemina Biomarker Discovery, Inc., 504 S. Rosa Rd., Suite 150, Madison, WI 53719
Birth defects are the largest cause of infant morbidity and mortality in the United States. Teratogens, defined as substances that cause one or more fetal abnormalities during development, are responsible for 5-10% of all birth defects. More predictive developmental toxicity screens would reduce the prevalence of birth defects and increase pharmaceutical and chemical safety. Current rodent models for developmental toxicity testing do not adequately correlate to human response, resulting in only 62% concordance to humans. Human embryonic stem (hES) cell technology is an innovative and robust alternative to predict developmental toxicity of chemicals. We have developed a targeted, more rapid and highly predictive assay based on specific biomarker metabolites identified in our original high resolution LC/MS metabolomics based developmental toxicity screen: devTOX. The biomarkers were identified in the devTOX assay after analysis of metabolomics data obtained from hES cells exposed to 23 known human teratogens and non-teratogens and are representative of an all-human hES cell based test system. The biomarkers represent different metabolic pathways and show high individual predictivity (70-88%), while combinations result in even more highly predictive results (88-93%). This new targeted biomarker approach continues to utilize the power of LC/MS methods and allows for much higher throughput at significantly lower cost. This assay is an attractive new model for high throughput, early assessment of the developmental toxicity potential of a broad range of chemicals in an all human derived system.
P213 Detection of Idiosyncratic Hepatotoxicity Potential with Metabolite Profiling
W. Mattes1, B. van Ravenzwaay2, N. Moeller3, T. Walk3, P. Ruiz Noppinger3, and H. Kamp2
1PharmPoint Consulting, Poolesville, MD
2BASF SE, Ludwigshafen, Germany
3metanomics health GmbH, Berlin, Germany
Idiosyncratic drug-induced liver injury (iDILI), where hepatotoxicity occurs in a few individuals treated with a drug that shows no liver injury in routine animal toxicology studies, is a serious concern for both the pharmaceutical industry and regulatory agencies. The question remains whether the potential for iDILI might have been detected preclinically if new measures had been added to the usual complement of clinical and histo-pathology. One promising measure is metabolite profiling (metabonomics/metabolomics). To address the question of whether iDILI could be detected with this approach, 15 compounds were selected: phenytoin, flutamide, propyl-thiouracil, methotrexate, captopril, nefazodone, nevirapine, valproic acid, and zidovudine as positive for iDILI potential, with atropine, lamivudine, mannitol, neomycin, streptomycin, and vancomycin as negative. Classifications were taken from public sources. Metabolite profiles were taken from MetaMap®Tox, a unique database of metabolite profiles from rat plasma obtained during 28 day rat studies of over 500 substances. This database, developed by metanomics GmbH and BASF SE, was also used to create “mechanism of action” (MoA) metabolite signatures (“toxicity patterns”) for a number of know toxicities. Eight of the nine compounds with iDILI potential had metabolite profiles that matched with patterns of various liver toxicities, including cholestasis, oxidative stress, acetaminophen-type toxicity and peroxisome proliferation. Only methotrexate showed no such matches. By contrast, only two of the six non-DILI compounds showed weak matches with liver enzyme induction (atropine and neomycin) or general liver toxicity (atropine). These results suggest that metabolite profiling may indeed detect compounds with iDILI potential.
P214 Comparison of Historical Control Data in Two Strains of Rat Used in Carcinogenicty Studies
R. Massarelli, A. Adamou, G. Henning, L. Kangas
Charles River Laboratories
Comparative data collected over a 20-year period from Sprague Dawley IGS and Wistar (Hannover) rats housed in a conventional non-barrier facility was assessed. Selected parameters of interest included survival, body weight and body weight gain, and incidence of common neoplastic and non-neoplastic lesions. Dosage routes included oral gavage, subcutaneous injection, and nose-only inhalation.
Notable differences among the two strains included lower mean body weights that were associated with increased survival rates and a lower incidence of spontaneously occurring tumors over a two-year period in the Wistar (Hannover) rats. In recent studies, early indications of marginally improved survivability in Sprague Dawley rats can be attributed to a reduction in protein content of the diet from 20% to 14% at this laboratory.
Similar types of neoplastic and non-neoplastic lesions were seen in both strains, however at a greater incidence in Sprague Dawley rats. Spontaneously occurring tumor types were seen in many tissues including pituitary, adrenal, and mammary gland. There was no evidence of significant changes in tumor types in the data evaluated.
It is concluded that collective data over 20 years demonstrate that both Sprague Dawley and Wistar (Hannover) rats remain appropriate models for use in carcinogenicity studies. However, given the above-mentioned benefits (increased survival, and lower body weights and incidences of spontaneous tumor types), a trend towards the use of Wistar (Hannover) rats in North America has been observed. Prolonged effects of the reduction in protein limits in the diet on survival and body weights changes will continue to be evaluated.
P215 Evaluation of a Novel Telemetry System for Use with Group-Housed Primates in Toxicology Studies
Shannon A. Stutler, and David M. Cox,
Research Support Division, US Army Medical Research Institute of Chemical Defense
Although the Animal Welfare Regulations and the Guide for the Care and Use of Laboratory Animals emphasize meeting social needs of nonhuman primate species known to exist in social groups, both documents provide for single housing of animals for scientific, medical and social incompatibility reasons. Historically, nonhuman primates assigned to research protocols requiring telemetric monitoring have been singly housed because existing technology did not reliably allow individual data collection from socially housed animals. We evaluated the use of a novel telemetry system under various housing conditions. Using physostigmine to simulate a nerve agent exposure, we also assessed the system’s ability to record data of interest in our toxicology studies. Four female rhesus macaques were surgically implanted with telemetry devices that monitored blood pressure, a biopotential, temperature and physical activity. After surgical recovery and baseline data collection, they were anesthetized and administered a single dose of physostigmine intravenously. Approximately 4 min after onset of clinical signs, animals received an intravenous antidote. Blood was collected at various times post-physostigmine to assess acetylcholinesterase levels. Additionally, telemetry data were continuously recorded while animals were singly, pair- or group-housed. Individual data streams were discernible under all housing conditions. Physiologic values changed significantly shortly after physostigmine administration, but all returned to baseline within 8 h. Physical activity decreased initially, but returned to normal by 24 h post-physostigmine. Since all animals were treated, data were confounded by handling effects during compound administration. Although data analysis would have benefited from handled controls, our study clearly demonstrated this system’s utility in monitoring nonhuman primates under social housing conditions and in collecting the complex physiologic data required for toxicology studies at our institute. These results foreshadow enhanced animal welfare through social housing while meeting scientific needs.
P216 In Vitro Evaluation of Organ Specific Toxicity in a Panel of Five Human Primary Cell Lines
J. Chesnut-Speelman, K.F. Marcoe, U. Warrior, P. Chiu, and J. Baumgartner
Ricerca Biosciences, Bothell, USA
Development and validation of in vitro models for predictive risk assessment of organ-specific toxicities is now a priority for drug discovery. In vitro cell-based screening assays utilizing organ/tissue-specific cell lines combined with sensitive injury detecting biomarkers to evaluate hepatic-, cardiac-, dermal-, and nephro-toxicities would be important tools to improve early identification and de-selection of compounds with potential liabilities. In this study a diverse panel of five human primary cell lines including primary human hepatocytes (HPH), Normal Human Dermal Fibroblasts (NHDF-neo), Human Renal Proximal Tubular Epithelial cells (HRPTEpiC), Human Umbilical Vein Endothelial cells (HUVEC), and Human Umbilical Mesenchymal Stem cells (HUMSC) were evaluated using compounds with known organ/tissue toxicities to investigate the utility of this cell panel to accurately assess cytotoxicity and predict organ-specific toxicity. High content screening (HCS) with automated fluorescence microscopy and image analysis based technology was used to determine general toxicity based on cell viability. Experiments were conducted at 24 and 48hrs post compound exposure. Organ-specific drug induced injury was further evaluated by measuring levels of KIM-1(Kidney Injury Molecule 1) protein, a well accepted marker of renal tubular injury and the heat shock protein HSP27, a marker of oxidative and chemical stress. KIM-1 and HSP27 levels were measured in the cells after 48hrs of compound exposure. Compounds were tested at a top final assay concentration of no more than 100µM. In terms of general cytotoxicity the majority of the compounds tested had a similar effect (IC50 value) in all cell lines. Organ-specific differential toxicity responses were demonstrated with KIM-1. Only the HRPTEpiC cell line demonstrated significant KIM-1 expression post compound treatment. On the other hand, drug- induced HSP27 expression was found to be compound dependent, ubiquitous among the cell lines tested and elevated at molar concentrations that coincided with IC50 values of cell viability. Our results indicate that cell viability data from different primary cell lines has relatively equal value in assessing general cytotoxicity and identifying selective toxicity with in vitro cell-based assays requires more complex evaluation with relevant organ/tissue specific cellular injury biomarkers.
P217 Development of Improved In Silico Models for Predicting Rodent Carcinogenicity
Lidiya Stavitskaya1, Naomi L. Kruhlak1, Kevin P. Cross2, Barbara L. Minnier1, David A. Bower2, Suman Chakravarti3, Roustem D. Saiakhov3, R. Daniel Benz1
1FDA Center for Drug Evaluation and Research (CDER), Silver Spring, MD
2Leadscope, Inc., Columbus, OH
3MultiCASE, Inc., Beachwood, OH
The two-year rodent bioassay is conducted to evaluate the carcinogenic potential of new pharmaceuticals intended to be dosed for six months or longer. Given the length and cost of these studies, early evaluation of rodent carcinogenicity using predictive models with high sensitivity and negative predictivity would be of great benefit. The first generation rodent carcinogenicity predictive models were built in 1998 with high specificity (87 - 90%) but low sensitivity (38 - 48%). In an effort to improve their sensitivity, new models for female mouse, male mouse, female rat, male rat, mouse combined, and rat combined were constructed using CASE Ultra and Leadscope Enterprise. The non-proprietary training database was enhanced with up-to-date data harvested from FDA CDER approval packages and published literature to give a total of 1534 structures. The cross-validation performance statistics for the new carcinogenicity models range from 43% to 62% in sensitivity and 60% to 73% negative predictivity. Additionally, the performance of the newly developed carcinogenicity models, as well as Derek for Windows structural alerts, was assessed using an external validation set comprised of 710 chemicals. Overall performance statistics ranged from 60% to 80% in sensitivity and 68% to 84% negative predictivity. The predictive performance was further improved by combining predictions across all three software programs, justifying a positive overall call with two independent positive predictions. The improved predictive properties of the new (Q)SAR models will provide faster and more effective evaluation of potential drug candidates.
P218 Integrated In Silico Modeling for the Prediction of Chronic Toxicity
A-N. Richarz1, M.R. Berthold2, E. Fioravanzo3, D. Neagu4, C. Yang5, J-M. Zaldívar-Comenges6, M.T.D. Cronin1
1Liverpool John Moores University, UK
2KNIME.com AG, Switzerland
3Soluzioni Informatiche srl, Italy
4University of Bradford, UK
5Altamira LLC, Columbus OH
6EC JRC, Italy
The COSMOS (Integrated In Silico Models for the Prediction of Human Repeated Dose Toxicity of COSMetics to Optimise Safety) Project addresses the safety assessment needs of the cosmetics industry, without the use of animals. The main aim of the COSMOS Project (www.cosmostox.eu) is to develop freely available tools and workflows to predict safety to humans following the use of cosmetic ingredients. The integrated suite of computational workflows being developed includes models based on the threshold of toxicological concern (TTC) approach, innovative chemistry such as chemical categories, read-across and quantitative structure-activity relationships (QSARs) related to key events in adverse outcome pathways (AOP), and multi-scale modeling based on physiologically-based pharmacokinetics (PBPK). Initial results include the development of the COSMOS database for curated repeat dose toxicity data as well as information on skin permeability, development of a robust inventory of materials used in cosmetics products and their associated chemical structures, datasets for better TTC analysis and the extension of the current TTC approach to cosmetics, as well as a KNIME platform including workflows to identify structural rules, fragments and properties associated with particular mechanisms of toxicity. In addition, a multi-scale modeling approach to predict target organ concentrations and extrapolate from in vitro to in vivo exposure scenarios has been established. The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7/2007-2013) under grant agreement n° 266835 and from Cosmetics Europe.
P219 Relative Sensitivity of Preclinical Phototoxicity Test Systems to UVR Exposure and the Effect of Phototoxins on this Response
D. Learn, M. Dougherty, S. Jackman, M. Arocena, and A. Hoberman
Preclinical Services, Charles River Laboratories, Horsham PA
Preclinical evaluation of in vivo preclinical phototoxicity requires a thorough understanding of the response of the test system chosen, along with the cutaneous reactions of known phototoxins in response to ultraviolet radiation (UVR) exposure. Equally important is understanding how to translate those findings to clinical phototoxicity and thus establish phototoxicity risk assessment. The cutaneous response of the albino hairless mouse, pigmented albino mouse, Long-Evans rat, Sprague-Dawley rat, albino hairless rat and albino guinea pig to UVR exposure was evaluated using a compact xenon arc solar simulator filtered to mimic noonday summer solstice sunlight in a design modified from the Sun Protection Factor (SPF) testing method. The cutaneous endpoint evaluated was the observational minimal erythema or edema dose (MED). The hairless mouse showed a cutaneous MED response very similar to lightly pigmented human skin, as described in the literature. The other Test Systems showed variations from the mouse, with up to 10 times the UVR dose required to elicit a MED. Pigmentation did not appear to substantially affect the MED response of the or hairless mouse or between the lightly and darkly- pigmented skin of the Long-Evans rat. There was little gender difference in any test system. However, the response to the known phototoxin 8-methoxypsoralen in the Long-Evan rat is similar to that of the hairless mouse with the same UVR dose. These results indicate that most test systems used in preclinical phototoxicity evaluations are more resistant to UVR exposure alone than humans, but the UVR dose required to elicit a phototoxin-induced skin response reduces this difference.
P220 Comparison of Daily Intravenous Infusion in Different Rat Strains
W. Fritz, R. Hall, J. Eggers, L. Hudgens, A. Brooks, and A. Alexander
Covance Laboratories Inc., Madison, WI
Inherent differences in rat strains, particularly related to animal size and feasibility of surgically implanting intravenous catheters and maintaining catheter patency, need to be considered when designing a typical infusion study as these differences may influence the nature of the data obtained. This study compared the feasibility of daily intermittent infusion to Wistar and Sprague Dawley rats.
Surgically-catheterized Wistar [RccHan:WIST and Crl:WI(Han)] and Sprague Dawley [Hsd:Sprague Dawley®™ SD®™ and Crl:CD(SD)] rats were infused with 10 mL/kg sterile saline (2 hours/day) over 28 days. Mortality, catheter patency, clinical signs, body weights, food consumption, ophthalmic and macroscopic necropsy findings, and effects on clinical pathology parameters were monitored
One Crl:CD(SD) male was found dead on Day -7 but no cause of death was determined, and one Hsd:Sprague Dawley®™ SD®™ female was sacrificed on Day -5 due to a retracted catheter. Clinical signs, most commonly discolored haircoat and sores/scabs, were generally noted in more Hsd:Sprague Dawley®™ SD®™ animals, but with low frequency and of limited duration. One RccHan:WIST and one male and female Crl:WI(Han) had scabs or swelling while nothing was noted in Crl:CD(SD) animals. Catheter patency was generally comparable between strains. Body weights increased comparably in all strains, although most dramatically in Crl:CD(SD) animals; similar effects were noted on food consumption. Few obvious differences were noted for clinical pathology parameters between the different strains. The presence of an indwelling venous catheter was more apt to result in a secondary inflammatory condition and Sprague Dawley rats generally had higher inorganic phosphorus than Wistar rats. At necropsy, local inflammation at the infusion site and skin/subcutis were considered most likely due to the implanted intravenous catheter. Thickened infusion site and/or abnormal contents surrounding the infusion site was observed in one Crl:WI(Han) male while a thoracic scab; abdomen or inguinal sore; or thickened dorsal thorax, were noted macroscopically in two Hsd:Sprague Dawley®™ SD®™ males and one RccHan:WIST female.
In conclusion, rats from both strains were able to be successfully dosed via daily intravenous infusion for 28 days. Clinical signs and pathology findings typically associated with chronically catheterized animal models were observed with similar frequency in both strains, none of which would have prevented rats from either strain from being used for a typical 4 week daily infusion study.
300 SERIES: GENERAL TOXICOLOGY
P300 Continuous Ambulatory Intrathecal Infusion Study in the Cynomolgus Monkey: The Outcome of Unexpected Adverse Effects in Vehicle (L-Arginine) Treated Control Animals
S. Korte, and B. Niggemann
Covance Laboratories GmbH, Münster, Germany
Intrathecal infusion via the lumbar route is a specialized clinical route for drug delivery in humans. Unknown interactions of new vehicle components in the cerebrospinal fluid (CSF) of non-human primates can be a risk for timely and successful pre-clinical drug development for the increasing number of compound classes. Twenty-two surgically implanted female cynomolgus monkeys (Macaca fascicularis) were continuously administered with a flow rate of 0.02 mL/kg/hour. Animals were dosed either the vehicle or vehicle with the test item at different dose levels. Eight animals (incl. 2 control monkeys) were noted with severe pareses of the lower extremities. To investigate whether this finding was induced by the vehicle (vehicle per mL: 3.12 mg sodium dihydrogen phosphate dihydrate 20 mM, 0.20 mg polysorbate 80, 5.31 mg sodium chloride, 10 mg L-arginine free base, adjust to pH 6.0 and fill up to 1.0 mL with water for injection) twenty-two male animals were used for the following study design. Each time 6 male animals were allocated to a control (NaCl) and vehicle (without L-arginine) dose group and each time 5 animals were allocated to a vehicle (including L-arginine) and test item (with L-arginine as part of the vehicle) dose group. Assessment of toxic effects was based on evaluation of clinical signs, neurological and behavioral assessment, clinical pathology results (including bacteriological, viral and CSF screening), organ weights, macroscopic and microscopic changes, as well as magnet tomography. Each of the 3 animals in the vehicle group or the test item with L-arginine group showed paresis. Beside these six animals no other animals were seen with adverse clinical effects. In summary 14 out of 32 animals receiving L-arginine showed paralysis of the lower extremities following 5 to 25 days of continuous intrathecal administration. All diagnostics performed could help to describe secondary effects and clinical signs but could not explain the reason for the paresis. In conclusion, the below described L-arginine vehicle induced severe paresis of the lower part of the cynomolgus body when administered by continuous intrathecal infusion for 5 days or more.
P301 Changes in Drug Metabolizing Enzyme and Transporter Gene Expression in Liver and Kidney During Postnatal Development of the Rat
LeeAnne McLean1, M. Youreneff1, S. Juelch1, F. Nanna1, D. Beckman1, F. Bolognani2, P. Moulin2, S-D Chibout2
1Novartis Institutes for Biomedical Research (NIBR), East Hanover, NJ
2NIBR, Basel, Switzerland
There are substantial changes in expression of drug metab-olizing enzymes that occur during maturation that can have a profound impact on drug pharmacokinetics and pharmacodynamics in the juvenile compared to adult. To further understand these changes, we collected liver and kidney from juvenile rats at different stages of postnatal development to evaluate changes in expression using Affymetrix whole genome microarrays (Rat230 2.0). Tissues were collected from untreated animals at postnatal days 7, 14, 22, 29 and 36, using 5 males and 5 females per group. Tissues from adult animals at 14 weeks of age were used for comparison. Analysis was primarily focused upon the Phase I and Phase II drug metabolizing enzymes as well as Phase III drug transporters. In the liver, there was a significant age-dependent increase in a number of metabolizing enzymes, including the rat homologs to human CYP2D6, CYP2C19 and CYP1A2 which were up-regulated in both males and females and the CYP2C19 homolog which was up-regulated over time in males only. Changes in expression were also noted in the liver for a number of common drug transporters such as MDR1, OCT1 and OAT2. In the kidney, there was a significant increase in most of the tubule-expressed metabolizing enzymes and transporters as nephrogenesis progressed over time. As with the liver, there were several genes that were regulated in a gender-dependent fashion, showing selective increased or decreased expression over time. This sexual dimorphism, which is rodent-specific, could be clearly visualized by principle component analysis, with divergence of the sexes occurring between postnatal days 22 and 29 in both liver and kidney. Results from this study support a better understanding of the effects of age on drug PK/PD which have implications in juvenile toxicity studies as well as potential impact for pediatric indications.
P302 Differential Expression of Cannabinoid Type 2 (Cb2) Receptors by Human B Cells, T Cells, and Monocytes
Julie T. Castaneda1,2, Sylvia M. Kiertscher2, Airi Harui2, Michael D. Roth2
1Interdepartmental Program in Molecular Toxicology
2The Division of Pulmonary and Critical Care Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1690 USA
Cannabinoids, which represent the primary bioactive constituents of marijuana, are known to activate cannabinoid receptors CB1 and CB2 and induce complex downstream signaling events. Their activation by marijuana smoke can have wide-ranging health effects. While these receptors are expressed in many organ systems, their distribution and function within the human immune system is poorly understood. Using a monoclonal antibody against the C-terminal portion of CB2, we reproducibly detected CB2 on the surface of fresh circulating B cells from non-smoking subjects. In contrast, CB2 was not detected on the surface of T cells or monocytes. However, all three leukocyte subsets demonstrated high intracellular CB2 protein expression (after permeabilization). Quantitative real-time RT-PCR demonstrated similar CB2 mRNA expression by all three cell types and correlated with total protein expression rather than extracellular CB2 levels. We hypothesize that receptor internalization and signaling through intracellular CB2 might play an important role in mediating the biologic effects of cannabinoids. In order to study this hypothesis, we developed a model trafficking assay using human A549 lung tumor cells expressing low levels of CB2 (A549-CB2) and human embryonic kidney cells expressing high levels of CB2 (293T-CB2). As in human B cells, these cell lines exhibit both extracellular and intracellular CB2, and mRNA expression correlated with total cellular expression. When exposed to THC, cell surface CB2 expression rapidly declined in a concentration- and time-dependent manner. Flow-based fluorescent imaging (ImageStream Cytometer, Amnis Corp) confirmed translocation of labeled receptor from the surface to intracellular locations when exposed to THC. These tools can now be applied to investigate the interrelationships between CB2 receptor location, ligand binding, internalization, signaling, and the role of cannabinoid-based drugs and endogenous cannabinoid ligands in regulating human immune function.
400 SERIES: SPECIAL STUDIES
P401 Differential Gene Expression Responses by Hepatic Lobe in Fischer Rats Treated with Carcinogen Diethylnitrosamine
M.W. Trimble1, B. Li2, D. Henderson2, S. Stepaniants2, M.L. Parrish2, K. Billings1, A.K. Sharma1, and G.A. Boorman3
1Covance Laboratories Inc., Madison, WI
2Covance Genomics Laboratory, Seattle, WA
3Covance Laboratories Inc., Chantilly, VA
The liver is usually treated as a single organ for safety assessment in rodent studies; however, biological endpoints may not be uniform across liver lobes in response to exogenous compounds. In order to elucidate lobe-specific differences, groups of male Fischer rats (8 animals/group/interval) were given 0 or 7.5 mg/kg/day of the genotoxic hepatic carcinogen, diethylnitrosamine (DEN), by daily oral gavage for two and four weeks. Samples of the right lateral, right median, and left lateral hepatic lobes were obtained for gene expression analysis and morphological assessment. Animals given DEN had minimal to slight apoptosis/necrosis of individual hepatocytes, usually around central veins. An infiltrate of macrophages/lymphocytes was often associated with the presence of necrosis. Additionally, the number of mitotic figures was minimally increased in the hepatocytes of a few animals given DEN when compared with the controls. Lobe differences were not identified microscopically; however, the gene expression data indicated that the left lateral lobe is functionally different from the right lateral and right median. Gene expression profiles were assessed for three animals/group/interval using whole genome microarrays. In the rats treated with only the vehicle, 397 genes (FDR p-value < 0.01) are differentially expressed (DE) between the left lateral lobe and the other lobes. Upon DEN treatment, 94 (FDR p-value < 0.01) genes showed differential expressions between the left lateral lobes and the other two lobes. While some of the DE genes in vehicle-treated animals remained unchanged, differential expression of most genes were attenuated. In contrast, a different set of DE genes was induced upon DEN treatment. The gene expression profile differences may provide clues as to the mechanism of lobular differences in response to DEN treatment.
P402 Early Hematological Effects of Hexahydro-1-Nitroso-3,5-Dinitro-1,3,5-Triazine (MNX), Environmental Degradation Product of RDX, Persist to Cause Delayed Myelosuppression in Rats
S. Jaligama1, V.M. Kale2, M.S. Wilbanks3, E.J. Perkins3 and S.A. Meyer1
1Dept of Toxicology, Univ of Louisiana at Monroe, Monroe, LA 71209
2College of Pharmacy, Roseman Univ of Health Sci, South Jordan, UT 84095
3US Army Engineer Research and Development Center, Vicksburg, MS 39180
MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), an environmental nitro reduced product of munitions RDX, contaminates military sites. Our previous studies identified bone marrow (BM) and spleen as hematological targets of acute oral MNX in rats. Splenic hemosiderosis and loss of blood granulocytes and BM Granulocyte Macrophage Colony Forming Cells (GM-CFCs) occurred at 14d post exposure (NOAELs 47 mg/kg). To address whether late effects of MNX are due to persistence of early hematological effects or are late-onset due to required expression period, female Sprague-Dawley rats were orally gavaged with MNX from 0 to 94 mg/kg and hematology, organ weights and BM GM-CFCs were evaluated at 2, 7, 10, 12, and 14d. Relative spleen weight decrease at 2d; splenic hemosiderosis at (≥ 47 mg/kg) 2d, 7d and 14d; and increased macrophage activity in splenic red pulp at 2d (≥ 47 mg/kg) were effects that persisted for 14 days. Blood granulocytes and inflammatory cytokine RANTES were significantly increased at 2d. Immunohistochemistry (IHC) of MNX (94 mg/kg) treated rat iliums (24h, 48h and 10d) and flowcytometric analysis of BM cells (48h and 10d) showed increased macrophage infiltration. Gene expression analysis showed activation of NFkB signaling pathway in BM cells at 10d. GM-CFCs were unaffected at 2 and 7d, but decreased at 10–14d by 47 mg/kg MNX. In addition, direct MNX exposure at concentrations higher than blood levels did not suppress GM-CFC colony formation/differentiation in culture. Collectively, these data show that while splenic effects are early onset and persist for at least 14d, myelosuppression is delayed presumably because of time required for development of inhibitory effects of preceding BM inflammation on myelopoiesis.
P403 Comparative Neurotoxic Effects of Dexmedetomidine and Ketamine in Fetal Monkey Brains
Edward Koo1, and Timi Oshodi2
1Research and Development - Preclinical, Hospira, Inc., Lake Forest, IL, USA
2Maccine Pte Ltd, Singapore
Previous studies have demonstrated that ketamine induced apoptosis and degeneration in developing monkey brains. Dexmedetomidine (Dex) is a general anesthetic with a different mechanism of action from ketamine. The present study compared the neurotoxic effects of the two drugs in fetal Cynomolgus monkeys. Twenty pregnant monkeys at approximate gestation day 120 were divided into 4 groups. Group 1 animals were cage controls and did not receive any treatment. Group 2 animals were dosed with ketamine at 20mg/kg IM followed by a 12-hour infusion at 20-50mg/kg/hr. Group 3 animals received Dex at 3µg/kg IV over 10 minutes followed by a 12-hour infusion at 3µg/kg/hr (HED). Group 4 animals received Dex at 30µg/kg IV over 10 minutes followed by a 12-hour infusion at 30µg/kg/hr (10X HED). Six hours following end of infusion, all animals were anesthetized and each fetus was removed by C-section. Blood samples from both the dams and fetuses were measured for the concentration of Dex. The perfusion-fixed brains were removed from all fetuses and processed for paraffin sections. Serial sections were cut through the frontal cortex and were stained to detect for apoptosis (Caspase 3 and TUNEL) and neurodegeneration (silver stain). There were no significant neuroapoptotic lesions present in untreated fetal brains. In-utero treatment with ketamine resulted in marked apoptosis and degeneration in the frontal cortex. In contrast, fetal brains from animals treated with Dex showed none to minimal neuroapoptotic or neurodegenerative lesions at both the low- and high-dose levels; lesion incidence for both Dex groups were similar to untreated controls. PK samples confirmed systemic exposure of Dex in both dams and fetuses.
The current study showed that unlike ketamine, Dexmedetomidine at both the low-dose (HED) and at 10X HED; did not induce apoptosis in the developing brain of Cynomolgus monkey.
P404 Rapid Evaluation of Sub-Lethal Effects of Nanoparticles in Escherichia coli
Chitrada Kaweeteerawat1,4, Angela Ivask1, Heidi Fischer2, Patricia Holden2,3 and Hilary Godwin1,4
1University of California Center of Environmental Implication of Nanotechnology, University of California, Los Angeles
2Department of Biostatistics, University of California, Los Angeles
3Donald Bren School of Environmental Science and Management, University of California, Santa Barbara, California
4Molecular Toxicology Interdepartmental Program, School of Public Health, University of California, Los Angeles, California
Synthesis and use of nanoparticles has skyrocketed during the past decade. To ensure that nanotechnology is safely and sustainably developed, toxicity of nanoparticles need to be determined. Here, we report the application of a suite of sub-lethal assays as well as a growth inhibition assay to a series of silver and metal oxide nanoparticles in bacteria (Escherichia coli). Fluorescent assays such as PI/SYTO, XTT, DiBAC, and H2DCFDA were used to measure viability, respiration rate, membrane potential, and ROS production, respectively. These studies revealed that toxicity of the silver particles studied correlates with size and surface charge, with smaller particles and particles with a more positive surface charge being more toxic. By contrast, the toxicity of the metal oxide particles studied correlates most with the energy of the conduction band, although dissolution of some of the particles also plays an important role.
P405 Role of Cell Acidification in Zinc-Induced Toxicity of Olfactory Neurons
Heidi Hsieh1, Hassane Amlal2, and Mary Beth Genter1
1Department of Environmental Health, Division of Environmental Genetics and Molecular Toxicology
2Department of Internal Medicine, University of Cincinnati, Cincinnati, OH
Zinc has long been touted as a panacea for the common cold. However, there has been some controversy over whether an intranasal (IN) zinc gluconate gel (Zicam), purported to fight colds, causes anosmia, or the loss of the sense, of smell in humans. Previous evidence has shown that IN zinc sulfate solutions can cause anosmia in humans, as well as significant damage to the olfactory epithelium in rodents. However, more recent work has claimed to show that zinc gluconate is less toxic than zinc sulfate. Using an in vitro olfactory neuron model (the rat Odora cell line) to compare the toxicity of zinc sulfate and zinc gluconate on immature and mature rat olfactory sensory neurons, we found that the toxicity of both zinc salts was similar. Interestingly, toxicity to Odora cells occurred at significantly lower zinc concentrations (0.1 – 0.4 mM) than that found in Zicam nasal gel (35 mM), which adds credibility to the epidemiological link between IN zinc exposure and anosmia. We tested the hypothesis that zinc toxicity was caused by inhibition of the HVCN1 proton channel, leading to acidosis and apoptotic cell death. Olfactory sensory neurons in vitro are able to maintain their intracellular pH through a Na+/H+ exchanger, specifically NHE1, and a Cl−/HCO3− exchanger. Zinc sulfate, at non-toxic levels, had no impact on intracellular pH via proton transport either after acute exposure or after 24 hours incubation with the cells. In conclusion, zinc toxicity is not mediated through an acidification of intracellular pH in olfactory neurons in vitro.
500 SERIES: ENVIRONMENTAL TOXICOLOGY
P500 Endocrine Disruptor Screening Program (EDSP) Tier 1 In Vitro Assays Indicate Glyphosate Does Not Interact With Estrogen and Androgen Receptors nor Inhibit Steroidogenesis
E.G. Webb, D.A. Saltmiras, and S.L. Levine
Monsanto Company, 800 N. Lindbergh Blvd, Saint Louis, MON 63167
The USEPA’s Endocrine Disruptor Screening Program (EDSP) includes 11 validated Tier 1 screening assays that evaluate the potential for a chemical to interact with the estrogen, androgen and thyroid endocrine pathways. The first list of 67 compounds subject to the EDSP Tier 1 Screening Test Orders, which included glyphosate, were selected based on exposure potential and not based on known or suspected interaction with the endocrine system. Potential for glyphosate to interact with estrogen receptor, androgen receptor, and inhibit steroidogenesis was evaluated in five Tier 1 in vitro assays following EPA’s OCSPP 890 series test guidelines in accordance with Good Laboratory Practice. Estrogen receptor binding and estrogen receptor transcription activation assay results indicate that glyphosate is not an estrogen receptor agonist or antagonist. Additionally, results from the androgen receptor binding assay indicate that glyphosate is not an androgen receptor agonist or antagonist. Glyphosate was also evaluated in Phase 3 of the OECD validation of the H295R steroidogenesis assay. Results from the two independent laboratories demonstrated no potential for glyphosate to affect steroidogenesis (i.e., no impact on the production of testosterone or estradiol). Consistent with results from the H295R assay, glyphosate did not inhibit CYP19 in the human recombinant microsomal aromatase assay. These five validated in vitro endocrine screening assays provide unequivocal evidence that glyphosate does not interact with the estrogen receptor or androgen receptor, nor does glyphosate inhibit steroidogenesis.
P501 Hydrocarbon Oil Induced Autoimmune Diffuse Alveolar Hemorrhage (DAH)
Prasad Priti, Valera Isela, and Singh Ram Raj
Autoimmunity and Tolerance Laboratory, Department of Medicine and Pathology, UCLA, Los Angles, CA 90095
A community comparison study in people living in houses with higher levels of hydrocarbon oil [2,6,10,14-tetramethylpentadecane (TMPD)] in house dust near an oil field waste site in New Mexico found a higher prevalence of systemic lupus erythematosus (SLE) and related rheumatic diseases in exposed as compared to unexposed population. This suggests a possible role of exposure to environmental agents such as hydrocarbon oil (TMPD, also known as pristane) in inducing autoimmune diseases. Indeed, a single injection of TMPD induces a SLE-like disease in mice. Intriguingly, different strains of mice develop different manifestations of SLE. For example, C57BL/6 and C57BL/10 mice, but not other strains tested, develop diffuse alveolar hemorrhage (DAH), a serious pulmonary complication of SLE. DAH is fatal in over 50% of cases, and without any effective treatment. In order to investigate the mechanisms underlying DAH development, 0.5 ml TMPD was administered intraperitoneally once to C57BL/6 mice (controls were administered saline), and we analyzed different immune cells, with a focus on dendritic cells (DCs) and DC subsets. Studies thus far show that five of six TMPD injected mice, compared to none of controls, developed pulmonary hemorrhage within 2 weeks of injection. We found a profound cellular infiltration with monocytes, granulocytes, CD19+ B cells, CD4+ and CD8+ T cells in lungs of TMPD exposed mice. In addition, there were increased plasmacytoid DC (pDC) in lungs, but reduced frequency in the bone marrow of TMPD injected mice. Ongoing studies will determine whether a local expansion or preferential recruitment and migration from the bone marrow to lungs accounts for increased lung accumulation of pDCs that then elicit local inflammatory response.
P502 Effects of Asian Sand Dust on Lung Allergeic Inflammation in Mice Immunized Beforehand by Ovalbumin
Miao He1,2, Takamichi Ichinose1, Seiichi Yoshida1, Hirohisa Takano3, Masataka Nishikawa4, and Guifan Sun2
1Department of Health Sciences, Oita University of Nursing and Health Sciences, Oita, Japan
2Department of Environmental and Occupational Health, College of Public Health, China Medical University, Shenyang, China
3Environmental Health Division, Department of Environmental Engineering, Graduate School of Engineering, Kyoto University, Kyoto Daigaku-Katsura, Nishikyo-Ku, Kyoto, Japan
4Environmental Chemistry Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan,
Asian sand dust (ASD) event may result in a significant influence on an asthmatic patient. However, for obvious reasons, there is no experimental study in which asthmatic patients are exposed to ASD. This study was undertaken to clarify the effects of ASD on lung eosinophiliain mice immunized beforehand by ovalbumin (OVA). CD-1 mice were instilled intratracheally with OVA four times at 2-week intervals. Simultaneous intratracheal administration of OVA and ASD (OVA + ASD sim) at the last OVA treatment or intratracheal administration with ASD 1 day before (OVA + ASD pre) /after (OVA + ASD post) the last OVA treatment was performed to investigate the effects of OVA and ASD exposure timing. The three kinds of treatment (OVA + ASD pre; OVA + ASD sim; OVA + ASD post) aggravated allergic lung inflammation and proliferation of goblet cells in the airway epithelium in mice, as evidenced by the cellular profile of bronchoalveolar lavage fluid (BALF) and pathological examination. As an overall trend, these changes were paralleled with the expression of Th2-associated effecter molecules and eosinophil relevant cytokine chimokines in BALF as well as the production of OVA-specific IgG1 compared with OVA treatment alone. OVA + ASD sim aggravated lung eosinophilia remarkably compared with the other treatments. The order of the potency of the aggravation was OVA+ASD pre < OVA+ASD post <OVA+ASD sim. These results indicate that ASD has a potent effect in activating lung eosinopilia in mice immunized beforehand by OVA. The simultaneous exposure of asthmatic patients to ASD and its antigen may have serious consequences for such individuals.
600 SERIES: DATA SETS
P600 Reference Control Data for the Common Marmoset (Callithrix jacchus): A Comparison with Macaques
S. Korte and B. Niggemann
Covance Laboratories GmbH, Muenster, Germany
Preclinical safety assessments of pharmaceuticals using the common marmoset (Callitrix jacchus) have increased in number and complexity over the last decade. The marmoset monkey is the smallest nonhuman primate commonly used in biomedical research. This New World primate clearly differs from Old World primates in a variety of functions, including reproduction, endocrine signalling and immunology as well as histological findings. Due to the limited body weight and organ size and in comparison with macaques the sample volumes (e. g. blood, urine, and ejaculate) or diagnostic capabilities (e.g. ophthalmologic evaluation) are limited. Assuming 300 to 480 g body weight the marmoset has a circulating mean blood volume between 19.5 to 31.2 mL (65 mL/kg), whereas the cynomolgus monkey with 2.5 to 5.0 kg has a mean blood volume of 175 to 350 mL (70 mL/kg). In this work we present reference values and ranges for a large array of relevant diagnostic parameters for the normal male and female marmoset, and a comparison is made to macaque monkeys.
Twenty one clinical chemistry and 22 hematology parameters were compared, 488 marmosets underwent eye fundus diagnostics, as well as electrocardiogram evaluations. Respiratory rate as well as male fertility parameters (testosterone, testicular volume) were determined and brought into relation to data from 1711 macaques. Organ weight data from control animals were recorded at time of necropsy. In summary, the marmoset is an established laboratory species for preclinical studies with many physiological similarities to the macaque. Only a few significant differences have been described in marmosets such as low AP, G-GT and elevated reticulocytes. Heart and respiratory rate are increased in the marmoset due to markedly different body size.
P601 Historical Background Data of Extended Reproductive Parameter Assessments in Repeat Dose Toxicity Studies
R. Love1, N. Lalayeva1, R. Watson1, S. Oneda1, N. Makori1, P. Franklin1, T. Beck1, and R. Nagata2
1SNBL USA, Ltd., Everett, WA, USA
2Shin Nippon Biomedical Laboratories, Ltd., Kagoshima, Japan
Male and female reproductive parameters are routinely assessed in repeat dose toxicity studies using sexually mature cynomolgus monkeys (Macaca fascicularis: Mfl). The aim of this presentation is to provide comprehensive historical control background data in sexually mature male and female animals. The following parameters were evaluated in nonclinical studies: menstrual cycle (MC) length evaluated for abnormal cycle length; serum 17-beta estradiol (E2) and progesterone (P4) concentrations during normal and abnormal cycles; sperm count, motility, and concentration following semen collection by electrostimulation; testicular volume assessments; and microscopic sperm evaluations. Mean cycle length of individual cycles was 30.8 days (range 17 to 63 days) and 94% of MC were of normal length (22 to 38 days). Peak E2 and P4 concentrations were on menstrual days (MD) 13 and 23, respectively, in normal MC. Peak E2 and P4 were delayed in prolonged MC (MD34 and MD59, respectively). Intervals between E2 and P4 peaks and between E2 peak and next menses were extended in prolonged cycles while changes in the interval between P4 peak and next menses was minimal. Mean sperm count was 251.7x106/ejaculate (min-max: 5.1 – 1342.3x106/ejaculate); mean sperm motility was 75% (min-max: 9 – 97%) and mean sperm concentration was 1151.2x106/mL (min-max: 63.5 – 7302.8x106/mL). Mean testicular volume was 33.56 mL (min-max: 13.35 – 60.50 mL). The following abnormalities were noted during sperm morphology evaluations: head – absent, detached, tapered, small; neck – refracted, kinked; mid-piece – refracted, kinked, absent, thin; and tail – refracted, kinked, absent, shortened, coiled, detached, doubled. Historical background data are a vital reference tool in chronic toxicology studies using Mfl because of the large inter- and intra-animal variability normally encountered.
P602 Normal Data on Selected Lineages of Miniature Swine
L. Brown, J. Liu, D. Brocksmith, J. Trickey, A. Golightly, J. Hiemstra, and G.F. Bouchard
Sinclair BioResources, LLC & Sinclair Research Center, LLC, Auxvasse, MO USA
In an effort to further support biomedical research, we recently updated a comprehensive dataset on normal data (reference intervals or ranges) for our four lineages of miniature swine. Included are Yucatan, Hanford, Sinclair S-1, and Micro-Yucatan lineages. This effort collates and summarizes normal biological and physiological data collected over many years. Data categories include: uses in biomedical research, body measurements (biometrics), growth, coat colors, clinical pathology (hematology, chemistry, coagulation, urinalysis), organ weights, background histopathology findings, blood glucose, ocular, diet/feeding, cardiovascular, ECG, dermal, reproduction data, normal rectal temperatures, and references. Over 200 tables of data are presented. These data are offered to veterinarians, biomedical investigators, preclinical clients, and university staff to facilitate research when using our miniature swine animal models. This poster will outline the available data and present representative data tables. Access to the full pdf file is now on the Sinclair Bio Resources website and is available by request.
700 SERIES: PRECLINICAL SAFETY ASSESSMENT
P701 Seminal Excretion of Blosozumab and Assessment of Exposure Risk to the Developing Embryo/Fetus
William Breslin, Kim Hilbish, Todd Page, and David Coutant
Lilly Research Laboratories, Indianapolis, IN
Blosozumab (Bmab) is a humanized IgG4 monoclonal antibody targeted against sclerostin protein that is being developed as a bone anabolic agent for the treatment of osteoporosis. Given the increasing concern for potential fetal harm due to excretion of drugs into semen, studies were conducted in rabbits to assess the potential for Bmab excretion into semen after systemic administration, absorption from the vagina after intravaginal (IVG) administration, and transfer to the fetus after maternal intravenous (IV) injection. Bmab was administered by IV and/or IVG routes at 6 or 100 mg/kg to male and female rabbits. Seminal plasma and blood serum (male, female and fetal) were evaluated for Bmab concentration at various times post-dosing. Bmab was detected in the seminal plasma of male rabbits after IV administration at a maximum mean concentration of 1.8 ug/mL (6 mg/kg dose) and 12.2 ug/mL (100 mg/kg dose), which was approximately 100-370x lower than the concentration present in serum. The total dose of Bmab in 4 ejaculates collected over a 72 hour period post-dosing was 4-4.5 orders of magnitude lower than the total administered dose. In female rabbits, the serum concentrations of Bmab following IVG dosing were approximately 3.5 orders of magnitude lower than following IV administration. On gestation day 29 (GD29), eight days after the last maternal IV dose, fetal serum levels were approximately 1.5x maternal levels in both the 6 and 100 mg/kg groups. Considering the observed levels of excretion of Bmab into semen, absorption from the vagina and systemic placental transfer, the estimated GD29 fetal exposure to Bmab via semen from a male rabbit administered 100 mg/kg was extremely low (3.2*10−5 ug/mL) and 8 orders of magnitude (1.2*10−8) lower than placental exposure following the same IV dose to the female. In conclusion, these data indicate a negligible embryo-fetal exposure risk from the excretion of Bmab into semen.
P702 Testicular Toxicity in Mice Related to Testis Specific Serine Kinase Inhibition by a Selective Kinase Inhibitor
Rosana M. Magpantay1, Dinah Misner2, Kamran Ghoreishi1, Joseph R. Piccotti1, and Mike Breider1
1Exploratory Toxicology, Celgene Corporation, San Diego, CA
2Genentech, South San Francisco, CA
Protein kinases are important targets for the development of novel drugs due to their important role in signaling of various growth factors, cytokines, and hormones and their key function in the regulation of cell growth, apoptosis, and differentiation of hematopoietic cells. To characterize multi-dose tolerability and target organ toxicity, in vivo studies were conducted with two specific kinase inhibitors, CC0489731and CC0487971, in development for the treatment of autoimmune disorders. In two separate studies, the kinase inhibitors were administered to either C57BL/6 or CD1 male mice once daily for 4 days (doses of 100, 300, and 1000 mg/kg for CC0489731and doses of 100, 300, and 500 mg/kg for CC0487971). In CC0489731-treated C57BL/6 male mice, significant weight loss occurred at all doses. Animals treated with 1000 mg/kg/day did not tolerate the dosage and required an early sacrifice on Days 2 and 4. Test-article related increases in CK, AST, ALT, RBCs, hemoglobin and hematocrit occurred at doses ≥300 mg/kg/day. Histopathological changes included lymphoid depletion (thymus, spleen and mesenteric lymph node) at doses ≥100 mg/kg/day, and hepatocellular vacuolation/single cell necrosis, renal tubular degeneration, gastrointestinal mucosal attenuation and ulceration, and testicular seminiferous tubular degeneration at doses ≥300 mg/kg/day. In CC0487971-treated CD1 male mice, significant weight loss was observed only at 500 mg/kg/day and histopathological changes included thymic lymphoid depletion at doses ≥300 mg/kg/day. These compounds had similar kinase inhibition panels except for CC0489731having potent inhibition of testis specific serine kinase 1 (TSSK1; IC50 = 0.6109 µM), compared to no evident TSSK1 inhibition by CC0487971. These results support the role of TSSK1 inhibition in the pathogenesis of testicular seminiferous tubular degeneration in male mice.
P704 Safety Pharmacology Assessment NU100 (Interferon Beta-1b), NME for Treatment of Multiple Sclerosis (MS)
Christopher L. Douglas1, David W. Hobson2, and Vijay Tammara3
1Covance Laboratories Inc
2Lone Star PharmTox LLC
3Nuron Biotech
Relapsing-remitting MS (RRMS) is defined as MS in which patients have relapses of MS and stable periods between relapses which may include new or worsening symptoms that last more than 48 hours.
NU100 is a proprietary recombinant human interferon beta-1b developed as a monotherapy for treatment of RRMS. NU100 is undergoing Phase 3 clinical trials in Europe. Treatment with interferon beta is associated with a 30-40% prevalence of neutralizing antibodies after two years of treatment with interferon beta. By reducing or eliminating the immune response, NU100 is expected to prevent reduction in clinical efficacy and improve safety and tolerability for RRMS patients. The purpose of this study was to evaluate the potential acute cardiovascular, respiratory, and neurological effects of NU100 when administered by subcutaneous injection to male cynomolgus monkeys in a parallel dosing design. ECG, heart rate, blood pressure, and abdominal body temperature measures were made by radiotelemetry in undisturbed freely-behaving animals up to 19 hours postdose. Respiration rate was derived from the blood pressure waveform. Assessment of neurological function was made by veterinary clinical neurological examinations on a separate dosing day. The general health of monkeys was assessed based on mortality, clinical observations, food consumption, body weight, and intraabdominal body temperature.
NU100 generally well-tolerated, was not associated with any clinical or neurological signs, and had no apparent effect on ECG, hemodynamic parameters, or respiration rate up to 19 hours postdose. Administration of NU100 at 0.06 or 0.28 mg/kg was associated with mild body temperature increases (up to +0.9°C) for up to 6 hours postdose.
P705 Safety of Mavrilimumab, an Anti-Gmcsf Receptor Alpha Monoclonal Antibody, in Cynomolgus Monkeys: Relevance for Human Safety
Patricia Ryan, Joel Leininger, Matthew A Sleeman, Marlon Rebelatto, Bing Wang, Hong Lu, Xiaomin Chen, Chi-yuan Wu, Lorin Roskos, Heidi Towers, Kathleen McKeever, and Rakesh Dixit
MedImmune, LLC
Mavrilimumab (CAM-3001) is an investigational human IgG4 monoclonal antibody (MAb) targeting GM-CSF receptor alpha. GM-CSF plays a central role in the pathogenesis of rheumatoid arthritis (RA) through the activation, differentiation, and survival of macrophages and neutrophils. MedImmune is currently pursuing development of mavrilimumab for the treatment of RA. To support clinical development, the nonclinical safety of mavrilimumab was evaluated in several studies with cynomolgus monkeys as the pharmacologically relevant species. At therapeutically active doses, mavrilimumab had an acceptable safety profile following repeated IV or SC dosing with no meaningful changes in any of the toxicological endpoints, including microscopic findings in any tissues other than lung. Although exaggerated pharmacology-enabling high doses led to accumulation of foamy macrophages in lung, with adverse lung pathology observed in some animals, a clean no-observed-adverse-effect-level (NOAEL) without any foamy macrophages in lung was clearly established. Overall, the nonclinical safety results supported the continued clinical development of mavrilimumab. In clinical studies in RA patients, mavrilimumab has demonstrated good clinical activity with adequate safety to support further clinical development. A Phase 2b study of mavrilimumab in subjects with RA is in progress.
P706 Reproductive Effects of Lead Acetate Administered in Drinking Water to Wistar Rat on an Extended One Generation Toxicity Study
Raluca Kubaszky1,2, David J. Esdaile1, and Roy Forster3
1CiToxLAB, Hungary
2CiToxLAB North America, Canada
3CiToxLAB, France
A full, GLP study was conducted at CiToxLAB Hungary jointly with Syngenta according to the draft OECD 443 guideline on the Extended One-Generation Toxicity Study (EOGRTS). Lead acetate was provided in drinking water to the F0 adult Wistar rats at 0 (NaAc control), 100, 800 and 1700 ppm Pb, from 2 to 4 weeks pre-mating, gestation, and lactation, and to the F1 generation to adulthood. The F0 adults were mated and evaluated for standard in-life parameters, reproductive function and histopathology. Litters were assigned to 3 subgroups for clinical pathology/thyroid function/neurotoxicity; immunotoxicity; and reproductive toxicity assessments. Selected pups were evaluated for developmental milestones and growth, neurobehavioral assessment (FOB, grip strength, landing foot splay, automated monitoring of motor activity by a video tracking system), TDAR immunotoxicity (IgM response to sheep-RBC challenge) and thyroid hormones; standard and/or neuro-histopathology with in-situ perfusion and serial brain sectioning were performed. Decreases in sperm parameters were noted in both F0 and F1 males, including up to 17% lower than control sperm counts and reductions in motility or increased number of sperm with abnormal morphology at all the dose levels tested; however, the mating, fertility and gestation indices in the F0 animals were unaffected by treatment. At the high dose, effects on early postnatal survival, lower pup body weights throughout lactation, decreases in anogenital distance, and delayed vaginal opening (2 day delay in 90% positive rate) and preputial separation were observed. Slight peripheral neuropathy was seen in high dose adults only, without functional effects.
P707 Vanadium Trioxide: 14-Day Nose-Only Repeated Exposure Inhalation Toxicity Study in Rats
A. I. Nikiforov1, K. D. Ehman2, L. C. Fisher1, and W. D. Johnson3
1Toxicology Regulatory Services Inc., Charlottesville, VA, USA
2RTI International, Research Triangle Park, NC USA
3IIT Research Institute, Chicago, IL, USA
This nose-only repeated exposure inhalation study was conducted to determine the potential toxicity of aerosolized vanadium trioxide (V2O3) powder to groups of male and female Sprague-Dawley rats. Rats were exposed to V2O3 at mean measured concentrations of 0, 0.0022, 0.022 or 0.25 mg/L (0, 2.2, 22 or 250 mg/m3) for 6 hours per day, 5 days per week, over the course of 2 weeks. The corresponding overall mean mass median aerodynamic diameters (MMAD) of the test atmospheres were 1.92, 2.02 and 2.25 µm with the geometric standard deviation (GSD) ranges of 1.70-2.49, 1.67-2.85 and 1.53-2.38 µm. No test-substance related mortality or clinical signs of systemic toxicity were observed. Body weight, body weight gain and food consumption were statistically significantly decreased for males and females in the highest exposure group (250 mg/m3). Although differences were observed in some organ weights at the highest concentration only, the lung was identified as a target organ. Lung weights (absolute and relative to final body weight) were increased in all exposure groups with the increase achieving statistical significance in the mid and high exposure groups. Microscopic evaluations revealed mixed cell infiltrates in the alveolus and interstitium of the lung, dark pigmentation of the alveolar macrophages, hyperplasia of the bronchial and mediastinal lymph nodes and alveolar histiocytosis. Compared to controls, all test substance exposure groups showed an increase in bronchial alveolar lavage (BAL) parameters, with the exception of a statistically significant decrease in the number of lymphocytes. The effects on BAL parameters, lung weights and lung histopathology were considered to be adaptive responses to high concentration V2O3 exposure, perhaps suggestive of particle overload rather than an indication of toxicity. Based on reduced body weight at the highest exposure concentration (250 mg/m3), the no-observed-adverse-effect-level (NOAEL) for systemic toxicity in this study was 22 mg/m3.
P708 Vanadium Trioxide and Vanadium Pentoxide: Comparison of Short-Term Repeated Exposure Inhalation Toxicity Studies
A. I. Nikiforov1, K. D. Ehman2, L. C. Fisher1, and J. A. MacGregor3
1 Toxicology Regulatory Services Inc., Charlottesville, VA, USA
2RTI International, Research Triangle Park, NC USA
3Toxicology Consulting Services, Arnold, MD, USA
In a series of acute inhalation toxicity studies using both rats and mice, vanadium pentoxide (V2O5; oxidation state V) was determined to be more acutely toxic than other vanadium compounds, including vanadium trioxide (V2O3; oxidation state III). To determine if the apparent association between oxidation state and toxicity extends to repeated exposure, data from repeated-exposure inhalation studies were compared, i.e. two V2O5 16-day US National Toxicology Program (NTP 2002) whole-body inhalation studies and the current V2O3 14-day nose-only inhalation toxicity study. In the first NTP (2002) study, male and female F344/N rats were administered V2O5 at concentrations of 0, 2, 4, 8, 16 or 32 mg/m3 for 16 days. Based on the results of this study, a second study focusing on lung histopathology was conducted in female F344/N rats administered V2O5 at concentrations of 0, 1, 2 or 4 mg/m3 for 16 days. In a comparable study design, a 14-day nose-only inhalation toxicity study was conducted with a lower oxidation state vanadium compound (V2O3) in male and female Sprague Dawley rats at concentrations of 0, 2.2, 22 or 250 mg/m3. The V2O3 14-day inhalation study included a similar concentration and one exceeding the range (approximately 2 and 250 mg/m3, respectively) relative to both 16-day V2O5 studies and an intermediate concentration (22 mg/m3) within the concentration range of the first NTP study. Final body weights were decreased at the highest V2O3 concentration and at the two highest concentrations of V2O5 in the first NTP study. Lung histopathological findings for V2O3 were minimal or mild in almost all instances over the exposure range of 2.2 to 250 mg/m3, whereas for V2O5 at concentrations from 1 to 4 mg/m3, findings were severe and frequent. A comparison of the toxicological findings from the repeated-exposure inhalation studies will be presented and the results will be discussed in the context of oxidation state.
P710 In Vitro and In Vivo Safety Pharmacology Assessment of GLYX-13
E.A. Hanan1, A. Bruening-Wright2, D. Houck3, and D. Minnick3
1AVANZA Laboratories, Gaithersburg , MD
2ChanTest Corporation, Cleveland, OH
3Naurex, Inc., Evanston, IL
GLYX-13 is a novel and selective NMDAR partial agonist in development as an adjunctive therapy for patients with depression who do not respond adequately to current therapy. Preclinical data have demonstrated that GLYX-13 has robust antidepressant-like activity, rapid onset of effect, long-acting duration of effect, and no signs of CNS-related side effects. Cardiovascular safety of GLYX-13 was evaluated in Beagle dogs following a single bolus intravenous injection at 30, 100, and 500 mg/kg. Salivation occurred immediately following dosing at all dose levels. Additional effects seen at 500 mg/kg included emesis and red discoloration of the skin and eyes. All of these changes were transient and non-adverse. Cardiovascular profiling and body temperature parameters were evaluated using a sterile telemetry unit implanted in a subcutaneous pocket in the left flank of each animal. Data were retrieved from the telemetry system (DSI Ponemah) at the following intervals: 2, 5, 10, 20, and 60 minutes postdose, and 4 and 24 hours postdose. There were no test article effects on electrocardiograms (heart rate, RR, PR, QRS, QT, QTc intervals, or rhythm abnormalities), blood pressure, or body temperature at any dose level. The in vitro effect of GLYX-13 on the hERG channel current was not significantly different from control. The inhibition was 1.5 ± 0.6% compared to 0.5 ± 0.4% in control at a concentration of 5 mM. Therefore the IC50 for the inhibitory effect of GLYX-13 on hERG potassium current was estimated to be greater than 5 mM. The positive control (60 nM terfenadine) inhibited hERG potassium current by 84.2 ± 0.4%, confirming the sensitivity of the test system. Respiratory safety of GLYX-13 was evaluated in Sprague Dawley rats following a single bolus intravenous injection at 30, 100, and 415 mg/kg. Respiratory function parameters were measured in conscious animals using a plethysmograph chamber with an attached pneumotachometer. Data were retrieved from the telemetry system at the following intervals: predose, 2, 5, 20, 30, and 60 minutes postdose, and 4 hours postdose. There were no test article effects on respiratory safety parameters (tidal volume, minute volume, or respiratory rate) at any dose level.
P711 Safety Evaluation of GLYX-13 in Rats and Dogs Following Intravenous Dosing TWICE Weekly for 13 Weeks
C.S. Godin1, E.A. Hanan1, D. Houck2, and D. Minnick2
1AVANZA Laboratories, Gaithersburg, MD
2Naurex, Inc., Evanston, IL
GLYX-13 is a novel and selective NMDAR partial agonist in development as an adjunctive therapy for patients with depression who do not respond adequately to current therapy. GLYX-13 has robust antidepressant-like activity, rapid onset and long-acting duration of effect, and no signs of CNS-related side effects. Animals were randomized to four groups and dosed twice weekly for 13 weeks by intravenous injection at doses of 30, 90, and 300 mg/kg/dose (rats) and 20, 60, and 200 mg/kg/dose (dogs). Parameters evaluated in both studies included mortality, physical examinations, cageside observations, body weight, food consumption, ophthalmology, clinical pathology, gross pathology, absolute/relative organ weights, histopathology, and toxicokinetics; ECGs were evaluated as part of the dog study. There were no effects of GLYX-13 in rats except for a 20% decrease in body weight gain in females at 300 mg/kg/dose during the dosing phase. The decreased body weight gain was not adverse because there were no effects on food consumption, and there were no gross, clinical or anatomical pathology findings. In the dog, a test article-related and dose-dependent increase in incidence and frequency of salivation was noted for both sexes at 20, 60, and 200 mg/kg/dose during the dosing phase which was not considered adverse and did not persist into the recovery phase. There was an increase in total serum protein in males at 20 and 200 mg/kg/dose on SD 93 which was not considered adverse or toxicologically significant, and did not persist into recovery. Higher values for mean RBCs, hemoglobin, and hematocrit were also present in males at 200 mg/kg on SD 93. This and the higher serum protein suggested a slight degree of dehydration that was not considered adverse or toxicologically significant. In conclusion, GLYX-13 was well tolerated in both species and there were no apparent target organs. The no-observed-adverse-effect level was 300 mg/kg when given twice weekly to rats for 13 weeks and 200 mg/kg when given twice weekly to dogs for 13 weeks.
P712 Efinaconazole: Developmental and Reproductive Toxicity Potential
Jo W1, Glynn M1, Minowa K2, Sanada H2, Nejishima H2, Calvarese B1, Pillai R1, Mutter L1
1Dow Pharmaceutical Sciences, a Division of Valeant Pharmaceuticals North America, LLC, 1330 Redwood Way, Petaluma, CA 94954, U.S.A
2Kaken Pharmaceutical Co., Ltd., 301, Gensuke, Fujieda, Shizuoka, Japan
Efinaconazole is a triazole NCE in development for topical treatment of nail fungal infections. The developmental and reproductive toxicity potential of efinaconazole was characterized via subcutaneous injection in rats (fertility and early embryonic developmental, embryofetal, and peri/post-natal reproductive toxicity) and rabbits (embryofetal toxicity) in accordance with current ICH guidances. Toxicokinetics of efinaconazole and its metabolites in rat and rabbit plasma, and presence in rat milk were assessed. There were no male reproductive effects, even in the presence of paternal toxicity (local toxicity, extramedullary hematopoiesis), and the paternal reproductive NOAEL was 25 mg/kg/day. Maternal toxicity (local toxicity, transient decreased body weight gain during lactation), the basis of high dose selection, was noted in all studies and was comparable to non-gravid animals. Maternal reproductive toxicity (estrous cycle prolongation) was noted in the fertility study, and the NOAEL was 5 mg/kg/day. Embryofetal toxicity, characterized by placental effects (increased placental weight and size, vacuolated decidua cells) and/or death (increased embryofetal death, decreased liveborn litter size and perinatal survival), was noted in the rat embryofetal toxicity and peri/post-natal development studies at maternally toxic doses. Efinaconazole did not affect rabbit embryofetal development at any dose level. Embryofetal NOAEL ranged from 2 mg/kg/day (placental effects) to 5 mg/kg/day (lethality) in rats to >10 mg/kg/day in rabbits. The observed toxicities are not unexpected and are consistent with marketed azole antifungal drugs. Efinaconazole, unlike some other antifungals, was not teratogenic and did not impair fertility and/or parturition.
P713 Utilization of the Zucker Diabetic Fatty (ZDF) Rat Model for Investigating Hypoglycemia-Related Toxicities
JJ Horvath,* MA Kirby,§ BA Zinker,§ E Janovitz,∫ MA Tirmenstein,* L Patrone,* R Mangipudy,* TP Reilly*
*Drug Safety Evaluation, §Discovery Biology, ∫Discovery Toxicology, Bristol-Myers Squibb, New Brunswick, Hopewell and Princeton, New Jersey
Glucokinase catalyzes the initial step in glycolysis (formation of glucose-6-phosphate), which in pancreatic beta cells culminates in insulin release. Pharmacologic activation of glucokinase has potential as a treatment for type II diabetes. A proprietary glucokinase activator (BMS-GKa) allosterically activates glucokinase (in vitro EC50 = 39 nM in 12 mM glucose). In preclinical studies, BMS-GKa given orally once per day for 1 month to healthy, euglycemic Sprague Dawley (SD) rats and beagle dogs, caused significant and prolonged hypoglycemia (serum glucose <50 mg/dL [nadir of ∼ 30 mg/dL] for up to 8 hours post daily dosing). At the highest doses, clinical signs of hypoglycemia including ataxia, tremors, recumbency, and melena were observed in both species following the first dose or by Day 3. In dogs, melena was also observed at lower doses. In both species, degenerative changes were observed microscopically in the stomach (glandular mucosa) and sciatic nerve (axons), and in rats, heart (myocardium) and skeletal muscles. These changes occurred at exposures similar to those planned for early clinical studies. To investigate whether the adverse effects caused by BMS-GKa in these nonclinical studies were secondary to exaggerated pharmacology, i.e. prolonged hypoglycemia, BMS-GKa was given orally once per day for 1 month to markedly hyperglycemic, insulin-resistant male Zucker diabetic fatty (ZDF) rats at doses that achieved exposures similar to and exceeding (∼ 2 to 7x) those achieved in euglycemic SD rats and dogs. In contrast to the effects observed in euglycemic SD rats, only minimal reductions in serum glucose (all but 1 value above 100 mg/dL), and no clinical signs of hypoglycemia or pathologic changes in target organs were observed in ZDF rats. These findings demonstrated that the adverse effects observed with BMS-GKa in SD rats, and presumably dogs, were secondary to pharmacologically induced hypoglycemia and provided a compelling justification for proceeding to early clinical studies. The male ZDF rat was shown to be an effective model for risk assessment when toxicologic investigations in euglycemic animals are complicated by effects secondary to exaggerated pharmacology.
P714 Cardiovascular Assessment of a Monoclonal Antibody in a Repeat Dose Safety Pharmacology Study in Nonhuman Primates
Michael J. Santostefano1, Michael J. Engwall2, Nancy Everds3, Chris Sheckler4, Wei-Jian Pan4, and Hugo M. Vargas2
1Toxicology Sciences, Amgen Inc., Seattle, WA
2Toxicology Sciences, Amgen Inc.,Thousand Oaks, CA
3Pathology, Amgen Inc., Seattle, WA
4Pharmacokinetics and Drug Metabolism, Amgen Inc., Seattle, WA
We reported recently that acute administration of a monoclonal antibody (mAb), AMG X, induced platelet activation, thrombocytopenia, and loss of consciousness with hypotension in monkeys (Santostefano et al, 2012). In addition, we demonstrated that platelet-activation was not observed with other pharmacologically-related mAbs, including AMG Z, illustrating that the platelet activation of AMG X likely occurred through an off-target mechanism. Based on the paucity of published safety pharmacology data describing effects after repeat doses, especially for a mAb, and the hypotension risk observed with AMG X, we investigated the cardiovascular profile of AMG Z. Specifically, male mAb-naïve instrumented cynomolgus monkeys (N = 6/group) were administered 0 or 300 mg/kg AMG Z by subcutaneous injection once weekly for 4 weeks. Hemodynamic parameters and electrocardiographic assessment were recorded continuously for at least 3 days post weekly dosing based on the time to maximum serum concentration, followed by 1 hour recordings every 4 hours through 7 days post dose. No AMG Z effects on hemodynamic parameters or electrocardiographic intervals were noted, and there was no evidence of electrocardiographic waveform abnormalities or arrhythmias when monitored for up to 1 month post dosing. This case report illustrates a design for a repeat-dose safety pharmacology study for a mAb. The value for repeat-dose safety pharmacology study could be utilized to address a putative target liability, better understand non-target mediated pharmacological effects, or for issue resolution based on adverse functional effects (i.e., cardiac arrhythmia, blood pressure changes, CNS activity) observed in nonclinical or clinical studies.
P715 Preclinical Evaluation of Potential Effects of the Phosphatidylinositol-3-Kinase Inhibitors GDC-0941 and GDC-0980 on Electrocardiogram Parameters
Eric Morinello, Laurent Salphati, Joseph A. Ware, Lisa P. Wong, and Amy H. Kim
Genentech, South San Francisco, CA
GDC-0941, a pan-inhibitor of class I phosphatidylinositol-3-kinase (PI3K), and GDC-0980, a pan-inhibitor of class I PI3K and mammalian target of rapamycin, are small molecules being developed by Genentech for the treatment of cancer. A recent report described an increase in the action potential duration in canine ventricular myocytes exposed to the PI3K inhibitors BEZ235 and PI-103 in vitro, QTc prolongation in isolated hearts from p110α-deficient mice, and QTc prolongation in mice treated with a p110α-specific inhibitor and suggested these findings may represent a risk of QT prolongation and arrhythmias for molecules in this class. The potential for GDC-0941 and GDC-0980 to induce QT prolongation or other adverse effects on electrocardiogram (ECG) parameters was evaluated in preclinical studies. No significant inhibition of cardiac hERG channel current was observed at clinically relevant concentrations in vitro. Potential effects on ECG parameters were also evaluated in vivo in a dedicated GLP cardiovascular safety pharmacology study in telemetry instrument-implanted dogs and in a 4-week GLP repeat-dose dog toxicity study. Neither inhibitor induced QTc interval prolongation or other effects on ECG parameters in dogs at clinically relevant exposures. In summary, QTc prolongation is not anticipated to occur at therapeutic doses of GDC-0941 or GDC-0980. In addition, the studies with GDC-0941 and GDC-0980 demonstrated that the previously described effects of other PI3K inhibitors or PI3K ablation on action potential duration and QTc interval are not relevant to all PI3K inhibitors at clinically relevant exposures in vivo.
P716 Nonclinical Safety Assessment of Efinaconazole 10% Solution for Onychomycosis Treatment
Jo W1, Glynn M1, Nejishima H2, Sanada H2, Minowa K2, Calvarese B1, Pillai R1, Mutter L1
1Dow Pharmaceutical Sciences, a Division of Valeant Pharmaceuticals North America, LLC, 1330 Redwood Way, Petaluma, CA 94954, U.S.A
2Kaken Pharmaceutical Co., Ltd., 301, Gensuke, Fujieda, Shizuoka, Japan
Onychomycosis (tinea unguium) is a common, persistent and difficult to treat fungal nail infection. Several antifungals are approved for onychomycosis but a safe and effective treatment has remained elusive. Efinaconazole is a triazole antifungal NCE in development as a 10% solution for the topical treatment of onychomycosis. We evaluated the repeat dose toxicity, genotoxicity and carcinogenicity potential of efinaconazole/efinaconazole 10% solution in accordance with current FDA and ICH guidances. Efinaconazole and efinaconazole 10% solution were well tolerated with chronic dosing in rats (via subcutaneous injection) and minipigs (via dermal application), respectively; no target organs of systemic toxicity were identified. Efinaconazole 10% solution was also well tolerated in a 13-week dermal mouse study conducted as the dose range finder for the carcinogenicity study. Only minor vehicle-related skin toxicity (transient erythema, modest hyperkeratosis and mild microscopic inflammation) was noted in minipigs and mice; the NOAEL was the high dose (an enhanced strength, 30% efinaconazole). Repeated subcutaneous injections of a propylene glycol vehicle were not well tolerated in rat and local reactions appeared to be exacerbated by efinaconazole. Efinaconazole was not a mutagen in an in vitro bacterial mutation (Ames) assay, and was not a clastogen in an in vitro mammalian chromosomal aberration and in vivo mouse munucleus assays. Efinaconazole 10% solution was not carcinogenic in mice after daily dermal application for 2 years. The repeat dose toxicity and carcinogenicity studies established high margins of safety based on systemic exposure (plasma AUC); mean efinaconazole exposure at the nonclinical NOAELs ranged from 70- to 250-fold the highest individual exposure observed in a maximum use clinical PK study in onychomycosis patients. Based on low toxicity demonstrated in nonclinical models and very low systemic exposure associated with clinical treatment, the authors conclude that efinaconazole 10% solution is safe for chronic topical onychomycosis therapy.
P717 An In Vivo Subcutaneous Model in Rat to Evaluate Triclosan Dissipation from a Combination Product and a Triclosan Risk Assessment K
George, T. Builee, K. Chen, L. Johns, S. Savidge and J. Oldham
*All authors are affiliates of Ethicon
ETHICON Absorbable Plus Sutures are a line of combination products containing the antibacterial agent triclosan impregnated on Polyglactin 910, Poliglecaprone 25, and Polydioxanone material. To characterize patient exposure to triclosan from Plus Suture an in vivo rat model was developed to determine the rate of chemical dissipation.
Coiled Coated Plus Antibacterial Sutures of VICRYL™, Monocryl™, PDS™ and PDS™ Barbed and corresponding non-triclosan-containing controls were explanted after subcutaneous implantation in rats at various scheduled necropsy time points. The dissipation of triclosan was determined by HPLC/with UV detection and compared to baseline and controls to determine the amount transferred to surrounding tissue. A burst of triclosan dissipation occurred within the first 24hr for all groups. VICRYL™ Plus had two distinct dissipation intervals, a rapid loss of 80% within 24hr and a slower loss of the remaining content by 7.4 days, this loss was independent of suture size (2-0 and 5-0). The apparent t1/2 (half life) of triclosan in Monocryl™ Plus 2-0 and PDS™ Plus 2-0 were 5.2 and 2.7days.
Suture dimension had no impact on dissipation as evidenced by VICRYL™ Plus Sutures, similarly suture configuration had no impact on dissipation as evidenced by PDS™ Plus and PDS™ Plus Barbed sutures which had similar profiles of t1/2 of approximately 3 days. Maximum patient exposure to triclosan from suture occurs within the first day and can reach levels of up to 0.13mg/kg/day. Biocompatibility studies showed that Plus Suture is non-toxic, non irritating, and not a chemical pyrogen. Local tissue response from an implantation study shows it has comparable tissue reaction, healing response and absorption to controls. Triclosan does not cause developmental or reproductive toxicity, it is non-genotoxic and non carcinogenic and not a sensitizer in guinea pig. It is rapidly absorbed by oral and dermal exposure and rapidly removed by feces and urine. The authors conclude, triclosan has an acceptable margin of safety in Plus Suture.
P718 Medical Device Biocompatibility Evaluation of a Silicone Coated Tungsten-Rhenium Alloy Surgical Needle
K. George*, T. Builee*, T.A. Barbolt†, R.W. Hutchinson* and J.W. Oldham*
*Affiliates of Ethicon and †T.A.B. Consulting
A new surgical needle for cardiovascular surgery was developed to provide improved mechanical performance and reduced penetration force over current surgical needles. This needle was manufactured using a novel Tungsten-Rhenium (WRe) alloy with a new silicone-based lubricious coating. The biocompatibility of this needle was evaluated by conducting cytotoxicity, intracutaneous reactivity, sensitization, acute systemic toxicity, pyrogenicity, and hemocompatibility studies in accordance with ISO 10993, and FDA #G95-1 Guidelines. Headspace analysis using GC/MS was conducted to determine if residual organic solvents from the coating process remained on the needle. The results of this program of biocompatibility studies indicated that the silicone-coated WRe alloy surgical needle was non-cytotoxic, non-irritating, non sensitizing, non-acutely toxic, non-pyrogenic, and hemocompatible, and residual solvents were not detectable at levels of toxicological concern. Overall, this biocompatibility assessment demonstrates that the new coated surgical needle is biocompatible and the authors conclude there are no safety concerns to patient.
P719 9-Month Study of a Latanoprost-Containing Subconjunctival Device in Dutch-Belted Rabbits
Harjeet Kaur1, Bart Jessen2, Paul Miller3, Robert Leedle2, and Mark Evans2
1Covance Laboratories Inc., Madison, WI
2Pfizer Worldwide Research and Development, San Diego, CA
3Comparative Ophthalmic Research Laboratory, University of Wisconsin Veterinary Medical Teaching Hospital, Madison, WI
Latanoprost is used for the treatment of increased intraocular pressure to prevent the progression of glaucoma. Since the lack of compliance with topical ocular dosing may compromise efficacy, alternate methods of delivery are being sought. A 9-month study was conducted to assess the safety and tolerability of a latanoprost-containing biodegradable device. Dutch-belted rabbits were implanted subconjunctivally with up to 5 placebo or drug-containing devices containing from 50 to 190 μg of latanoprost per device. Study assessment consisted of irritation scoring, clinical signs, ophthalmic exams, electroretinography, and ocular histology of cohorts at 3 and 9 months post implantation. The implants were well tolerated, with the most severe clinical signs and irritation observed in the first few days of the study associated with the implantation procedure. Mild conjunctival congestion persisted through week 13 of the study and tended to correlate with the number of devices and presence of drug. Ophthalmic exams revealed no effects beyond the ocular surface irritation, including no effects on intraocular pressure, corneal thickness, or ERG parameters. Microscopically, implants at the 3-month necropsy were associated with cavities (containing the implants), fibrous encapsulation, and an infiltrate of macrophages, sometimes as multinucleate cells, into the implant cavity. Drug-containing implants were often associated with additional inflammatory cell infiltrates within the implant subconjunctival cavities and adjacent to the implant sites. At the 9-month necropsy, neutrophils were no longer common among the inflammatory cell infiltrates, most implants were fragmented and disintegrating, and fibrovascular proliferation was present within implant luminal remnants. Conclusion: Latanoprost-containing subconjunctival device were well-tolerated in rabbits.
P720 Safety Evaluation of a Maltogenic Amylase Enzyme from Bacillus licheniformis Intended for Use as a Food Processing Aid
Nicole Muntean, Vincent Sewalt, and Quang Q. Bui
Danisco US, Inc. (Dupont Industrial Biosciences Division), Palo Alto, CA 94304, USA
Amylase MAA is an enzyme used as a processing aid in bakery, grain and carbohydrate processing to convert starch into sugar and brewing. It is produced from a recombinant modified strain of Bacillus licheniformis MDT06-221 expressing a maltogenic amylase from G. stearothermophilus. A battery of toxicology studies was conducted to investigate its potential to cause adverse effects in humans using methods complying with OECD Guidelines, Principles of Good Laboratory Practice and all subsequent OECD consensus documents. Acutely, Amylase MAA is not an eye irritant, produces mild skin irritation and is considered as non hazardous by oral ingestion as substantiated by a LD50 greater than 2000 mg/kg bw. No evidence of mutagenic activity was shown in the Ames assay up to the highest recommended dose of 5000 μg/plate. Further, in the in vitro cytogenetic test using peripheral human lymphocytes cells, Amylase MAA did not induce structural and numerical chromosomal aberrations in the presence and absence of S-9 mix up to the highest recommended concentration (5000 μg/ml). In a 13-week oral gavage study conducted in Wistar rats, repeated daily oral administration of Amylase MAA at doses ranging from 0 to 80 mg TOS [total organic solid]/kg bw/day failed to induce systemic toxicity. There were no treatment-related changes in body weight, feed consumption, hematology and clinical chemistry at study termination. Further, no differences in the functional and clinical observations and no treatment-related macroscopic and histopathologic changes were detected. The NOAEL was established at the highest dose tested, 80 mg TOS /kg bw/day (55.6 mg total protein/kg bw/day). Under the worst-case scenario that Amylase MAA is applied at the maximum rate and the enzyme is not destroyed and/or removed during processing, the use of the enzyme is not expected to result in adverse effects to humans. Since the human daily cumulative exposure (0.203 mg TOS/kg bw) occupies only 25% of the pADI and the margin of safety was calculated to be approximately 400 the use of Amylase MAA as a food processing aid is not of human health concern. The authors conclude that Amylase MAA is safe for uses in foods.
800 SERIES: MOLECULAR MECHANISMS
P801 Disordered Copper (Cu) Transport Across the Blood-CSF Barrier Following Manganese Exposure: The Role Of Cu Transporters ATP7A and ATP7B
Xue Fu, Yanshu Zhang, Wendy Jiang, and Wei Zheng
Purdue University
Increased Cu levels in blood, saliva and brain were found in the Mn-exposed animals and humans. However, the underlying mechanism remains unknown. ATP7A and ATP7B function as the Cu exporters to transport Cu from the cytosol to membrane to maintain the intracellular Cu homeostasis. This study was designed to test the hypothesis that Mn exposure disrupted the Cu homeostasis at the blood-CSF barrier (BCB) by interfering Cu transport functions of ATP7A/B. Rats received i.p. injection of 6 mg Mn/kg as MnCl2 or saline, 5 d/week for 4 wks. Increased Cu and Mn levels in serum and CSF were detected following Mn exposure. An in situ ventriculo-cisternal perfusion by infusing [64Cu] and a space marker [14C]-sucrose was conducted to determine the Cu clearance function of BCB. Results showed that Mn exposure significantly increased the [64Cu] radioactivity by 2.6 fold, as compared with controls, in the CSF effluent collected from the cisternal magna, suggesting a reduced Cu clearance by the BCB. Confocal images of plexus tissue displayed ATP7A translocated from cytosol toward the apical membrane, while ATP7B re-localized oppositely from cytosol toward the basal membrane. ATP7A/B were observed located at perinuclear region in control Z310 cells. Treatment of the cells with 100 μM MnCl2 for 24 hrs led to a significantly decreased ATP7A/B fluorescent intensity. Both mRNA and protein expression levels of ATP7A/B in the tissue and Z310 cells were significantly reduced in Mn-exposed rats and Z310 cells. The two-chamber Transwell [64Cu] transport studies showed that Cu efflux was significantly reduced following Mn exposure, or when ATP7A expression was knocked down by siRNA. Collectively, these data suggest that subchronic Mn exposure distorts the Cu balance at BCB by impairing the Cu clearance function of BCB; the effect appears to be due mainly to the reductions and translocations of ATP7A from cytosol toward the apical membrane and ATP7B from cytosol toward the basal membrane. This may lead to an increased intracellular [64Cu] buildup and decreased Cu efflux. Consequently Mn exposure results in a high Cu level in brain. (Supported by NIH/RO1-ES008146)
P802 Boric Acid Induces ER Stress and the ATF4 and ATF6 Branches of the Unfolded Protein Response in DU-145 Prostate Cancer Cells
Sarah E. Kobylewski1, Kimberly A. Henderson1, Kristin E. Yamada1, and Curt D. Eckhert1,2
1Molecular Toxicology, UCLA
2
Environmental Health Sciences, School of Public Health, UCLA
Boric acid (BA) is a ubiquitous dietary component. Low levels reduce the incidence and mortality of prostate cancer. Our studies have shown that low doses of BA significantly inhibit proliferation of DU-145 prostate cancer cells without inducing apoptosis. At similar doses we see inhibition of calcium (Ca2+) release from the ryanodine receptor (RyR) in the endoplasmic reticulum (ER) in response to RyR agonists. Stored Ca2+ in DU-145 cells was reduced by 22% when treated with 10 μM BA. Altered cellular Ca2+ homeostasis can lead to acute ER stress and the unfolded protein response (UPR), a cellular coping mechanism which responds to the accumulation of unfolded or misfolded proteins in the ER. The objective of the current study is to identify if physiological levels of BA induce ER stress and the UPR in DU-145 cells. This response may be a key to understanding the mechanism by which BA slows the proliferation of these cells and BA’s chemopreventative nature. ER stress was indicated in BA-treated DU-145 cells with a dose-dependent increase in ER swelling and vacuolization. We also saw the accumulation of TIA-1 foci in the cytoplasm of DU-145 cells treated with low doses of BA, a stress granule marker, another indicator of ER stress. The UPR consists of three branches: PERK, ATF6, and IRE1. Analysis of the three UPR branches demonstrated that physiological doses of BA activated the ATF4 and ATF6 branches of the UPR, but not the IRE1 branch. There was also an inhibition in global protein translation, one of the first actions of the UPR. With the exception of CHOP, a pro-apoptotic protein, UPR gene and protein markers were significantly induced by BA treatment. Overall, our data indicates that physiological doses of BA induce ER stress and the ATF4 and ATF6 branches of the UPR in DU-145 prostate cancer cells.
P803 SMAD3 Mediates Cigarette Smoke Extract (CSE) Stimulation of Vascular Endothelial Growth Factor (VEGF) Release by Cultured Human Lung Fibroblasts
Maha Farid1,2, Yoko Gunji1, Joel Michalski1, JooHun Park1, Schunichiro Iwasawa1, Jun Ikari1, Xing Qi Wang1, Hesham Basma1, Amy J. Nelson1, Xiangde Liu1, and Stephen Rennard1
1Department of Internal Medicine, Pulmonary, Critical Care, Sleep and Allergy Division, University of Nebraska Medical Center, Omaha, NE, USA
2Department of Environmental, Agricultural and Occupational Health, Collage of Public Health, Omaha, NE, USA
RATIONALE: Cigarette smoke is the major cause of chronic obstructive pulmonary disease (COPD), yet pathogenic mechanisms are not fully understood. Vascular endothelial growth factor (VEGF) is one of the major regulators of endothelial cell survival and is believed to play a role in the pathogenesis of COPD. Fibroblasts are a significant source of VEGF in the lungs; however the effect of cigarette smoke extract (CSE) stimulation of VEGF release by fibroblasts is not fully understood. We hypothesized that CSE-induced disturbed VEGF release by human lung fibroblasts is a potential pathogenic mechanism that could contribute to COPD. METHODS: CSE was prepared by modification of the methods of Carp and Janoff (Am.Rev. Respir.Dis, 1978). Human fetal lung fibroblasts (HFL-1) were exposed to different concentrations of CSE (2.5, 5, 7.5 and 10% in serum free media) and for different durations (24, 48 and 72hours). VEGF release into the media was measured using ELISA. TGF-β1/Smad3 as a potential pathway for the observed effect was also investigated using Smad3 and TGF-β1 receptor siRNA. RESULTS: CSE induced VEGF release by HFL-1 was observed 48 hours after exposure and increased further after 72 hours. The increase in VEGF release by CSE exposed HFL-1 (79.8 ± 11.4pg/105 cells at 48hrs 10% CSE exposure and 169.8 ±19.3 pg/105 cells at 72hrs of 10% CSE) was significantly higher than that of control (38.38 ± 3.2pg/105 cells at 48hrs and 47 ± 6.5 pg/105 at 72hrs, both P<0.05. Silencing of Smad3 by siRNA not only eliminated the stimulatory effect of CSE on VEGF release (219.3 ± 35.8 pg/105 cells of the non-targeting control-siRNA vs 70 ±9.6 pg/105 cells of the Smad3-siRNA transfected cells, p<0.05), but also inhibited baseline VEGF production (84.1±0.75 pg/105 cells of control-siRNA vs 26.2 ± 4.4 pg/105 cells of Smad3-siRNA p<0.05). Suppression of TGF-β1 receptor by the pharmacological inhibitor (SB431542) markedly reduced VEGF release by HFL-1 in response to CSE and this effect was confirmed by TGF-β1 receptor siRNA. CSE also induced the phosphorylation of Smad3 in HFL-1 after 1hr of stimulation as shown by immunostainig. Nicotine inhibited VEGF release by HFL-1 in a dose and time dependent manner. CONCLUSIONS: Our data indicates that CSE stimulates VEGF release by lung fibroblasts and Smad3 modulates the stimulation of VEGF release. Nicotine is not the active agent responsible for this response in HFL-1. The ability of lung fibroblasts to produce VEGF may play a role in disease pathogenesis.
P804 Lead Exposure and Obesity are Comorbidity Factors for Osteoporosis by Inhibiting WNT/Beta-Catenin Signaling
E. Beier1, T. Sheu2, Y. Sun2, M. Kung1, R. Mooney2, and J.E. Puzas1, 2
Department of Environmental Medicine1, Department of Pathology2, University of Rochester
The obesity epidemic affects at least 14.6% of children in the US with an even higher proportion among the underprivileged. This latter population is also exposed to higher levels of environmental lead (Pb) due to its persistence in old homes and inner-city residences. Previous findings have independently identified both Pb and obesity as etiological factors in the development of osteoporosis. Interestingly, Pb-treated mice and those fed a high-fat diet share a number of other characteristics, including depression of bone formation and bone marrow steatosis. Increased adiposity of the bone marrow is itself associated with bone loss, and this is evident in cases of ovariectomy, immobilization, and glucocorticoid treatment, as well as in osteoporotic postmenopausal women. Additionally, we have found that Pb treatment up-regulates the expression of sclerostin, a potent inhibitor of Wnt/beta-catenin signaling, which is an important molecular switch governing bone homeostasis. From these findings we hypothesize that Pb exposure and obesity both act through depression of Wnt signaling in mesenchymal cells to promote their differentiation into adipocytes, subsequently decreasing number of osteoblasts, resulting in bone loss. Using concomitant exposure to Pb (50 ppm in the drinking water) and high-fat diet (60% kcal from fat) in mice we found that they additively reduce bone mass, increase bone marrow adiposity, and reduce beta-catenin and Runx2 levels in stromal cells. This correlated with reduced expression of the osteogenic transcription factor Runx2 and elevated expression of the adipogenic transcription factor Pparg. Pb and high-fat diet also act in vitro to promote adipogenic potential of mesenchymal cells by increasing lipid droplet formation, increasing activity of Pparg, and decreasing beta-catenin activity. These results provide mechanistic insight for targeted therapeutic intervention in bone diseases resulting from exposure to Pb, obesity, and a combination thereof.
P805 Telomerase Reactivation Reverses Telomere Shortening in Human Skin Keratinocytes: A Potential Mechanism of PCB Carcinogenesis
P.K. Senthilkumar, A. Klingelhutz, L.W. Robertson, and G. Ludewig
Graduate Program in Human Toxicology, University of Iowa, Iowa City, IA
Polychlorinated biphenyls (PCBs), an environmental pollutant, are probable human carcinogens. Activation of telomerase activity and lengthening of telomeres are key steps in carcinogenesis. In our study, immortal human skin keratinocytes were exposed to PCB126 at 5μM concentration for 90 days. Cells were re-seeded every 6th day with fresh medium and telomerase activity, telomere length, cMyc, hTERT, hTR, TRF1, TRF2, CYP1A1 mRNA, CYP1A1 activity and superoxide, hydroperoxide level were determined. PCB126 caused the prominent reduction of telomerase activity (50%), hTR and hTERT mRNA (10%), telomere length (40%) and cell growth, along with an increase in TRF1, TRF2, CYP1A1 mRNA (and activity), and in superoxide and hydroperoxide levels from day 6 to 48; treatment with PCB126 was continued until day 90. From day 54 on, an increase in cell growth, cMyc, hTERT, and hTR mRNA level (to 130%) along with re-activation of telomerase activity (to 100%) and re-elongation of telomere length (to 90%) was observed. From day 66, a decrease in TRF1 and TRF2 mRNA levels were observed, which allows for an increased access of telomerase enzyme to telomeres. This study shows for the first time that PCBs initially reduce telomerase activity, telomere length, and cell growth, with possible mechanistic connections to increased oxidative stress, but prolonged exposure lead to telomerase re-activation, telomere lengthening and increased cell growth, a hallmark in carcinogenesis.
900 SERIES: REGULATORY AND GUIDANCES
P900 Guidance on Extractables and Leachables Testing of Medical Devices in Combination with a Drug or Biologic
Brenda Faiola, Ph.D., D.A.B.T
Aptiv Solutions, Raleigh, NC
Combination products are therapeutic and diagnostic products that combine drugs, devices, and/or biological products. A combination product comprised of a medical device and either a drug and/or a biologic poses unique regulatory challenges in the pathway to gaining agency approval/clearance. An important issue that Sponsors of such combination products need to confront early on in development is the assessment of extractables and leachables from the device into the drug or biologic. However, the Office of Combination Products (OCP) within the FDA has not issued a comprehensive guidance document on extractable/leachable testing for combination products; therefore, many Sponsors must seek advice on this topic. The purpose of this poster is to provide important information regarding several aspects of extractables and leachables including: i) the impact of these chemicals with regard to assay and study design considerations, ii) the relevant guidance documents to refer to, iii) the toxicological evaluation of these chemicals, iv) the determination of an allowable limit for a leachable, and v) the use of the threshold of toxicological concern approach for risk assessment purposes. The number of combination products submitted for review to the FDA for fiscal years 2004 to 2010 ranged from approximately 250 to 350 each year according to the OCP annual reports. With advances in technology, the number of combination products will likely rise and therefore toxicologists will increasingly need to become more familiar with aspects of leachables and extractables in order to be effective members of these product development teams. Presenting this information in this poster will provide a useful resource for toxicologists involved in the development of combination products.