Metallothioneins (MTs) are a family of proteins involved with regulating intracellular zinc concentration and compartmentalization. Blue-green algae (BGA) supplements are described as an antioxidant superfood and considered a treatment for metabolic and cardiovascular issues; however, there is. Family System Therapy Rachel Gunn, Hums 102 Ivy Tech Community College Abstract Family Therapy, sometimes called family focus therapy or family systems therapy is a style of psychological therapy that works to revolution the relationships within families to help them better cope with varies problems.

1. Structural Family Therapy Training
2. Structural Family Therapy Pdf

P101 - Comparison of Background Organ Weight Data in the Göttingen and Chinese Bama Minipig

The minipig is often used in regulatory preclinical toxicology studies as the choice among nonrodent species. It presents a favorable profile as a nonrodent toxicology model in terms of its similarity to humans and also in terms of its applicability to different study types (Bode et al 2010). Studies for general toxicology programs can be performed by oral, dermal, parenteral, and inhalation routes. Physiologically, the minipig also has advantages for use in reproductive and safety pharmacology studies. In the United States and Europe, the Göttingen minipig is often selected, while in China the Bama pig is usually the strain of choice. Organ weight data from control animals from toxicology studies using the Bama at Wuxi Apptec Suzhou were compared to published data for the Göttingen minipig. The data sets were compared to see if there were any notable differences between the two strains of animals. To correct for any differences in body weight, the relative organ weight data (relative to body weight) was used. Analysis of these data sets showed that overall the weights for the Göttingen minipig were generally toward the lower to medium range of that of the Bama pig of comparable age.

P102 - Bone Marrow Suppression Secondary to Argemone Mexicana Intoxication: Report of Three Cases

Epidemic dropsy is a cluster of manifestations resulting from adulteration of food with Argemone mexicana oil. We present three patients with pancytopenia secondary to argemone intoxication. Patient 1 presented with progressive paresthesias with an acute onset of a blanching rash and lower extremity swelling. Examination was significant for sinus tachycardia with a pulse of 110bpm. Mild pallor; non-pitting, tender, 2+ pedal edema; and a macular telangiectatic lower extremity rash were noted. CBC revealed pancytopenia (table 1) with mild anisopoikilocytosis on peripheral smear. Bone marrow aspiration and trephine revealed hypocellular marrow with proportionate reduction of all three cell lines. Viral serologies, including parvovirus, CMV, EBV, HIV, and hepatitis, were negative. Patient 2 had a similar clinical presentation in addition to fever for 20 days. Bone marrow biopsy revealed similar findings. Patient 3 had a brief onset of rash, paresthesias, and pedal edema. A bone marrow aspiration was declined by the family. A diagnosis was confirmed by history of use of cooking oil extracted from locally grown mustard seeds. Positive nitric acid test in the cooking oil confirmed the contamination.

After three weeks of supportive management, pancytopenia resolved completely.

In the classic study by P. Sen Gupta et. al, anemia was the most common hematological presentation. Pancytopenia with hypocellular marrow is a previously undescribed manifestation of dropsy, and we propose a maturation defect as the presumed pathogenesis of the same.

STP104 - Loss of Hepatic Aryl Hydrocarbon Receptor Alters the Thermogenic Properties of White Fat and Protects Against Diet-Induced Obesity

The aryl hydrocarbon receptor (AhR) is a cytosolic, ligand-activated transcription factor commonly known for its role in xenobiotic metabolism. However, the generation of AhR-null mice has revealed physiological roles for the AhR in lipid metabolism and energy expenditure. The AhR is highly expressed in the liver, which is a major endocrine regulator of both overall lipid and energy homeostasis. To investigate how AhR activity in the mouse liver affects lipid metabolism, we utilized adult female (8–10 weeks) AhR-floxed (AhRfx/fx controls) and liver-specific AhR-CKO (AhRfx/fx Alb-CreERT) mice. We discovered a novel phenotype wherein AhR-CKO mice exhibited significantly reduced body weight gains and increased expression of uncoupling protein 1 (UCP1) in gonadal white adipose tissue (GWAT) depots vs. controls while maintained on a normal chow diet. AhR-CKO mice brown adipose tissue (BAT) and GWAT also revealed significant increases in uncoupled mitochondrial respiration rates compared to controls, consistent with the increased expression of UCP1 the and multilocular morphology observed in H&E sections. These changes are indicative of browning of the white adipose tissue. We also placed mice on a 41 Kcal % high-fat diet (HFD) for three months, which revealed a resistance to increased weight gain and hepatic steatosis in AhR-CKO vs. controls. Our results demonstrate novel physiological roles for the AhR in the liver in modulating the thermogenic properties of white fat as well as protection against weight gain and fatty liver while on an obesogenic diet.

STP106 - Hepatic Overexpression of Annexin A1 and A2 Underlies the Heteroprotection by Thioacetamide Against a Lethal Dose of Acetaminophen in Mice

Compensatory tissue repair (CTR) in thioacetamide (TA)-primed rats protects them against acetaminophen (APAP)-induced lethality. This study was aimed at investigating the mechanisms of CTR-mediated heteroprotection in mice. Male Swiss Webster mice were primed with a low dose of TA (40 mg/kg-bw in 10 ml distilled water/kg-bw, intraperitoneally [i.p.]). TA-induced liver injury, CTR, and expression of annexin A1 and A2 (ANX1 and ANX2), the endogenous inhibitors of the death protein secretory phospholipase A2 (sPLA2), were measured over a time course of 84h after TA priming. Both centrilobular necrosis and CTR peaked at 36h after TA priming as indicated by significantly increased alanine transaminase (ALT) and aspartate transaminase (AST) activities, histological findings, and proliferating cell nuclear antigen immunostaining. TA priming resulted in overexpression of ANX1 and ANX2 at 36 to 84 hours and 1260 hours, respectively. A lethal dose of APAP (600 mg/kg-bw in 10 ml 0.45% NaCl/kg-bw, pH 8.2, i.p.) was given at 12, 24, or 36 hours after TA priming. TA-priming did not affect the rise in plasma ALT, AST, and sPLA2 levels seen 2 hours after the APAP overdose. Neither these biochemical markers of liver injury nor histology suggested any escalation of hepatic injury at later time points (12 and 24h after APAP overdose), consistent with 100% survival of the TA-primed mice as compared to nonprimed mice, which suffered 100% mortality. Inhibition of ANX1 and ANX2 biosynthesis using cycloheximide (40 mg/kg-bw in 5 ml distilled water/kg-bw, i.p.) abolished this heteroprotection. In conclusion, ANX1 and ANX2 overexpression abolishes APAP-induced expansion of liver injury.

STP107 - Role of Metallothionein in Zinc-Induced Anosmia: In Silico, In Vitro, and In Vivo Studies

Zinc is both an essential and potentially toxic metal. Oral zinc supplementation can reduce the duration and severity of the common cold; however, intranasal administration of zinc-containing gels causes olfactory dysfunction in some humans. Using RNAseq analyses of zinc exposure in the olfactory neuron cell line Odora, we identified pathways associated with oxidative stress, ATP synthesis, and metal response as being highly upregulated. Odora cells demonstrated elevated markers of oxidative stress, which returned to baseline after 24 hours; however, ATP levels continued to decline over time and did not return to baseline.

Metallothioneins (MTs) are a family of proteins involved with regulating intracellular zinc concentration and compartmentalization. MTs are polymorphic in humans, with variants of MT1 conferring enhanced sensitivity to cadmium. Mt1/2 shRNA knockdown Odora cells did not show a significant difference in zinc sensitivity; however, at baseline they exhibited increased hydrogen peroxide and a depleted level of reduced glutathione. We also administered zinc gluconate intranasally to MT1/2 knockout mice and assessed olfactory toxicity via behavioral assays and histology. WT mice recovered their sense of smell earlier and at a higher percentage by the end of study, while showing significantly improved tissue recovery (organization and thickness of the olfactory epithelium) at the end of the study (28 days), compared to the knockout mice. We hypothesize that MTs play an important role in the recovery of olfactory mucosa in vivo after zinc-induced tissue damage, which may be related to the reduced rate of cell proliferation observed in the double knockdown Odora cells.

P108 - Evaluation of MicroRNAs As Potential Biomarkers of Acute Pancreatic Injury in Rats

Pancreatic injury in rats is primarily detected through histopathological changes and conventional serum biomarkers such as amylase/lipase. However, amylase/lipase have a short half-life and are markers of pancreatic acinar, not islet cell injury. We investigated whether circulating microRNA (miR) levels that are enriched in acinar cells (miR-217) or islet cells (miR-375) could serve as markers of pancreatic injury. Rats were treated with a single ip dose of either vehicle, the pancreatic acinar cell toxicant—caerulein (0.05 mg/kg), or the cell toxicant—streptozotocin (STZ) (150 mg/kg). Rats were necropsied at 4, 24, and 48 hours; and pancreas, liver, heart, kidney, and skeletal muscle were collected and analyzed for histopathology. Blood was collected at necropsy and processed to serum for amylase/lipase enzymatic determinations and miR qPCR analysis. Caerulein induced degeneration/necrosis of acinar cells after 4 hours that persisted after 24 and 48 hours, and increased amylase/lipase levels at 4 hrs but not at 24 or 48 hours. Increases in miR-217 (100-1000x controls) persisted for 48 hours following caerulein and only occurred in rats with evidence of acinar cell damage. STZ induced islet cell necrosis after 4 hours followed by atrophy at 24 and 48 hours. STZ did not induce significant increases in either amylase or lipase but did induce increases in miR-375 levels at 4 (90x controls) and 24 hours (5x controls). No significant increases in miR-375 were observed in caerulein-treated or miR-217 in STZ-treated rats. Used together, miR-217 and miR-375 represent promising biomarkers of exocrine and endocrine pancreatic injury in rats.

P109 - Species, Strain, Age, and Gender Differences in Locomotor Activity and Exploration: Protocol Development for Neurobehavioral Testing in Rodents

Evaluation of locomotor activity is routinely conducted during neurotoxicity testing to screen for central nervous system dysfunction. Baseline motor activity parameters (distance traveled, time spent moving, beam breaks, thigmotaxis) were evaluated in untreated male and female Sprague-Dawley rats from two suppliers (Harlan [Hsd:SD] and Charles River [Crl:CD]) on PND 45 and 60 and in male CD-1 mice on PND 45. Rearing was also evaluated to assess exploration. Evaluations were conducted in an open field (16X16 in.) and/or a rectangular (7x15 in.) enclosure in an automated photocell-based activity chamber for 60 minutes. In the 16x16 frame, male CD-1 mice (n=4) moved and reared more than male Hsd:SD rats (n=4), indicating greater exploratory behavior in mice. Rats habituated faster than mice regardless of chamber size, suggesting that a shorter protocol (e.g., 35 minutes) could be used for rats. Larger group sizes are needed to confirm these preliminary findings. In the 7x15 frame, Hsd:SD females (n=20) moved more than Hsd:SD males (n=20) at PND 45 and 60, and more than Crl:CD females (n=7) at PND 60 within the first 5 minutes. Hsd:SD and Crl:CD males (n=7) had similar activity levels in the first 10 minutes, while the apparent more rapid habituation of Hsd:SD males may have resulted from small sample size variability. No difference in thigmotaxis, as measured by time spent in periphery, was observed between the two chamber sizes. Species, strain, gender, age, and frame size should be carefully considered when developing a motor activity protocol.

P111 - Development of a Model of Dermal Inflammation and Irritation (Urticaria) in the Miniature Swine

Urticaria is dermal edema that results from vascular dilation and leakage of fluid into the skin in response to molecules released from mast cells. Our objective was to develop a reproducible dermal urticaria model in miniature swine. Three Hanford minipigs (3 to 18 months old) were used in the study. The animals were pricked on their back skins with a skin test device (Lincoln Diagnostics Inc, Decatur, IN) that were loaded with vehicle or histamine. The irritation and wheal and flare responses of the dermis were evaluated with Draize scoring and with wheal size measurement. The reactions of the skin were assessed at 10, 20 or 25, and 45 minutes after test formulation application. Histamine dose-dependently induced skin irritation at both 10 and 20 minutes after treatment. The most prominent erythema and edema (Draize score) responses were observed at 10 minutes after treatment, which were slightly diminished at 20 minutes after treatment. Histamine also caused skin wheal that ranged between 4 and 7 mm in diameter. When urticaria was induced with 25 and 50 mg/mL histamine, the topical administration of Allegra as well as Cortaid inhibited both the dermal irritation and the wheal and flare responses. The inhibitory effects of Allegra and Cortaid were observed at 25 and 45 minutes post dose. In conclusion, a histamine-induced urticaria model has been established in the Hanford miniature swine and can be used for the testing of topical treatments for dermal irritation and inflammation.

STP112 - Aluminum Induces ER Stress-Mediated Neuro-Inflammation in Human Neuroblastoma SH-SY5Y Cells: Implication of Oxidative Stress and Apoptosis

The neurotoxic potential of aluminum (Al) is evidenced by studies indicating involvement of this metal in a variety of neurodegenerative diseases including Alzheimer’s disease. Inflammation and apoptosis are key hallmarks of aging and neuronal disorders. ER stress is known to elicit inflammation through the activation of unfolded protein response (UPR). In the present study, we aimed to investigate the role of Al in mediating ER stress-induced inflammatory responses in neuroblastoma cells. Lactate dehydrogenase release assay revealed that Al compromised the membrane integrity of neuroblastoma cells, probably due to membrane damage, as indicated by enhanced levels of lipid peroxidation. Elevation in reactive oxygen species (ROS) levels was associated with a weakened antioxidant defense system manifested by decreases in catalase activity and cellular glutathione and suppressed Nrf2 responses. Study of ER stress-mediated activation of UPR revealed that Al-enhanced protein levels of PERK, EIF2α, and inflammatory markers like NF-κB, NLRP3, HMGB1, and nitric oxide. Furthermore, Al-altered TNF α, IL1β, IL6, and IL10 mRNA levels. In addition, these changes were associated with initiation of apoptotic response as indicated by caspase activation and cytochrome c release. However, apoptosis induced by Al exposure was devoid of p53 induction as indicated by western blot and PCR analysis of p53. Study of epigenetic modifications revealed that miR-125b could be responsible for Al-induced p53 suppression. The overall findings indicated that Al induced UPR activation through ER stress, which resulted in induction of inflammatory pathway and apoptotic proteins in neuroblastoma cells independent of p53.

P114 - Take Your Child to Work Day in an Animal Research Facility? Improving Perceptions Using Three Es: Education, Engagement, and Enrichment

Children are regularly exposed to many views about animals in research, whether at school, through peers, or through media sources. These views impact their perceptions of animals in research. Unfortunately, many of these views have negative connotations. It is unusual for children to be exposed to positive views of animals in research. Participation in Take Your Child to Work Day (TYCTWD) allows animal research facilities to provide children with positive experiences. Many people in research are proud of their work and love the opportunity to share that work; however, a common problem faced is animal facility rules/regulations, which restrict or inhibit those opportunities. We established a program that allows our facility to participate in TYCTWD while upholding facility rules/regulations. Age eligibility, number of participants per workshop/session, and application procedures are clearly defined. Letters are sent to participants explaining the program, rules, and expectations for TYCTWD. The program consists of several hour-long workshops, including interactive education sessions focusing on animal use in research, equipment demonstrations, and vivarium tours. Employees volunteer to manage and facilitate each workshop, all participants follow facility SOPs, and the IACUC reviewed the program. We use TYCTWD as a form of youth outreach focusing on improving perceptions of animals in research using three E’s: Education (presentations, workshops, tours), Engagement (participants, employees), and Enrichment (human, nonhuman). Participants share their experience in this program with teachers and school peers, thus increasing our reach. Success of this program is illustrated by active participation for eight consecutive years with continued growth.

P116 - Site-Specific Covalent Chemical Ligation of Small Molecule to Monoclonal and Polyclonal Immunoglobulins at Conserved Nucleotide Binding Site

Unlike traditional chemotherapy, antibody drug conjugates (ADCs) have demonstrated a target-specific toxin delivery platform to eliminate cancers. Clinical-approved ADCs, produced by chemical nonspecific ligation via lysine or cysteine residues, are highly heterogeneous. It results in various populations of the ADCs of different drug-to-antibody ratios (DARs) and a distinct pharmacokinetics profile, which enhances the side-effect toxicity. Site-specific ligation of toxins or ligands to antibodies has demonstrated to be more target-oriented in biodistribution and less prone to aggregation. Current strategies for synthesis of the homogeneous ADCs include use of engineered antibodies, such as reactive cysteine residues, unnatural amino acids, aldehyde tags, or enzymatic transglutaminase onto the specific site of protein. In this present study, we took advantage of the reported nucleotide-binding pocket in F(ab) arms by developing indole-based OBOC peptide libraries, for the identification of affinity elements that can be used as site-specific derivatization agents against both commercially available monoclonal and polyclonal immunoglobulins. This allows one to generate ADCs much more efficiently and affordable financially. Immunoconjugates resulting from such affinity elements can lead to high therapeutic efficacy and low off-target toxicity, thereby improving the maximal tolerated dose.

P117 - Effect of Connexin32 on Hepatic Tumors in Mice

Connexin32 (Cx32) proteins are components of gap junctions (GJ) and are expressed in various organs and tissues, including the liver. Cx32 has an important role in the maintenance of tissue homeostasis through cell-to-cell communication and the control of cell growth, differentiation, and tumor formation. In order to investigate the effect of Cx32 on hepatic tumor development, we examined the incidence of hepatic tumors in Cx32 knockout mice (Cx32KO). Cx32KO mice and wild-type mice (C57BL/6) were bred for approximately 24 months. In the Cx32KO mice, a statistically significant increase in hepatocellular carcinoma was observed in the males, and there was a tendency toward an increase in hepatocellular adenoma in males and hepatocellular adenoma and hepatocellular carcinoma in females compared to wild-type mice. There was no apparent difference between Cx32KO mice and wild-type mice in the other organs. In order to investigate the expression of Cx32 in hepatic neoplastic and pre-neoplastic lesions, immunostaining of Cx32 was conducted in spontaneous hepatic lesions in a mice carcinogenicity study (C57BL6J, 24-month, po). The observed localization and expression patterns of Cx32 could be classified into three patterns: decrease in intensity (clear cell foci), increase in the number and spot size (foci and tumor stained with basophilic), decreased intensity in the intercellular area and increased intensity in the perisinusoidal area of hepatocytes (foci and tumor stained with eosinophilic). Based on the results of our observations, it was considered that Cx32 has a suppressive effect against hepatic tumorigenesis and affects the morphological character of hepatic tumors.

P119 - The Minipig As an Alternative Nonrodent Model in Nonclinical Research

The ICH Harmonized Tripartite Guideline Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals M3(R2) recognizes the use of a rodent and nonrodent species in nonclinical toxicology studies. The nonrodent species is not specified, and although traditionally canines and nonhuman primates have been used, the use of the minipig as an alternative has been recently highlighted by the 2010 RETHINK European FP6 project. While the minipig is being used in this laboratory in both our North American and European facilities for a wide range of safety studies including pharmacokinetic, safety pharmacology, and toxicity studies using various dose routes, namely oral (gavage and capsule), intravenous and subcutaneous injections or infusion, target tissue dosing, intranasal, dermal and ocular (instillation, intravitreal, and subretinal injections), and wound healing studies, based on the available data as presented below, there is no evidence of changes in the number of minipigs used for safety studies over the last five years that would suggest that the species is replacing either the dog or nonhuman primate in safety assessment studies. However, since the minipig has become recognized as a suitable alternate nonrodent species and because there is a sufficient body of historical data that enables unequivocal data interpretation, continued consideration should be given to use of the minipig as an alternate model of choice.

P123 - Reduced Effects of Aqueous Aerosol Extract from THS2.2, a Candidate MRTP, Compared with a Reference Cigarette on the Adhesion of Monocytic Cells to Human Coronary Arterial Endothelial Cells Evaluated Using In Vitro Systems Toxicology

Smoking is a major risk factor for the development of cardiovascular diseases. Modified risk tobacco products (MRTP) are designed to reduce smoking-related health risks. The present study aimed to evaluate the impact of THS2.2, a candidate heat-not-burn technology-based MRTP, compared with a reference cigarette (3R4F), on the adhesion of monocytic cells to human coronary arterial endothelial cells (HCAECs), a critical stage in atherosclerosis development, using a functional in vitro adhesion assay combined with systems toxicology. HCAECs were treated for 4 hours with conditioned media of human monocytic mono mac 6 (MM6) cells preincubated with low or high concentrations of aqueous extracts from THS2.2 aerosol or 3R4F smoke for 2 hours (indirect treatment), unconditioned media (direct treatment), or fresh aqueous extract (fresh direct treatment). Previous results showed that aqueous 3R4F smoke extract induced the adhesion of MM6 cells to HCAECs via distinct direct and indirect concentration-dependent mechanisms. Leveraging the same experimental and computational framework, significant reduced effects of aqueous THS2.2 aerosol extract on MM6 cell-HCAEC adhesion were measured, also supported by markedly diminished molecular changes such as gene expression in both endothelial and monocytic cells. A shift toward 10 and 20 times higher concentrations of aqueous THS2.2 aerosol extract was required to observe similar effects as the ones measured with 3R4F in both fresh direct and indirect exposure modes, respectively. In conclusion, our in vitro systems toxicology investigations revealed reduced effects of THS2.2, a candidate MRTP, on monocytic cell-endothelial cell adhesion compared with a reference cigarette.

P124 - Toxicological Evaluation of the Heat-Not-Burn THS2.2 Product in a 90-Day OECD Inhalation Study Complemented with Systems Toxicology

A 90-day rat inhalation study was performed in accordance with OECD test guideline 413 to characterize potential adverse effects caused by subchronic exposure to the Tobacco Heating System (THS) 2.2 aerosol, a heat-not-burn tobacco product, and to compare them to those induced by the smoke generated from the combustible reference cigarette 3R4F. In addition, a systems toxicology approach with additional animals was included to further characterize the exposure effects on the transcriptome and proteome of the lung.

Sprague-Dawley rats were exposed for a period of 13 weeks to filtered air, to three concentrations of mainstream 3R4F smoke (8, 15, 23μg/l nicotine), or to THS 2.2 aerosol (15, 23, 50μg/l nicotine). Exposure was confirmed by numerous biomarkers, well reflecting test atmosphere constituent concentrations. Histopathological evaluation of the respiratory tract from animals exposed to THS 2.2 aerosol showed a lower inhalation toxicity compared to animals exposed to 3R4F smoke. Lung inflammation in THS 2.2-exposed groups was dramatically lower (or absent) compared to 3R4F. This was consistent with gene and protein expression profiling data from the lung that revealed reduced perturbation in inflammatory process networks and reduced protein expression related to inflammation. Quantitative transcriptomics and proteomics analyses showed a clear reduction of the impact on toxicity pathways activated by cigarette smoke in the lungs. Overall, the data indicated that aerosol from the THS 2.2 caused significantly reduced systemic and respiratory tract toxicity in comparison to mainstream cigarette smoke.

P125 - Systems Toxicology Analysis of Cardiovascular and Respiratory Endpoints from ApoE-/- Mice Showed Similar Effects after Switching to a Candidate Modified Risk Tobacco Rroduct, THS 2.2, or to Smoking Cessation

Cigarette smoking is a risk factor for chronic obstructive pulmonary disease and cardiovascular disease (CVD). ApoE-deficient mice are prone to developing atherosclerosis and emphysema, making them an ideal model in which both pathologies can be assessed. We evaluated the effects of cigarette smoke (CS) from a conventional cigarette (3R4F) and aerosol from THS 2.2, a modified risk tobacco product (MRTP). ApoE-/- mice were exposed for up to 8 months to the test aerosol for 3 hours/day, 5 days/week, with a target nicotine concentration of 30 ╡g/l. After 2 months of exposure to CS, cessation and switching groups were further exposed for up to 6 months to fresh air or THS 2.2. A battery of markers of disease progression were investigated including atherosclerotic plaque formation, pulmonary inflammation, pulmonary function, and lung emphysema. Exposure to CS induced time-dependent molecular, physiological, and inflammatory pulmonary responses in ApoE-/- mice consistent with emphysematous changes. The area and volume of atherosclerotic plaques measured in the aortic arches was higher in CS-exposed animals compared to both sham and MRTP-exposed animals at all time points, evaluated using both stereometric measurements and micro-computed tomographic analysis. Significant changes in the lung transcriptome and proteome of ApoE-/- mice were observed in response to 3R4F exposure compared to sham controls. Smoking cessation and switching to THS2.2 resulted in lower activation levels compared to 3R4F. Both smoking cessation and switching to the MRTP aerosol halted the rate of disease development as assessed by inflammatory, histopathological, and molecular endpoints, typically within 1 to 3 months postexposure.

P126 - Historical Control Data for Developmental Toxicity Parameters in Harlan Sprague-Dawley Rats

The Sprague-Dawley rat (Hsd:SD; Harlan Laboratories) is being used for developmental toxicity studies by the National Toxicology Program. Since historical control data is limited for developmental parameters in the Hsd:SD rat, we have compiled control data from developmental toxicity studies conducted between February 2014 and March 2015. Vehicles used included corn oil, sterile water, and 0.5% aqueous methylcellulose. Rats were dosed by oral gavage once daily from gestation day (GD) 6 through 20. Embryo/fetal mortality, fetal weight, and fetal and placental morphology were evaluated on GD 21. Mean fetal weight ranged from 5.06 to 5.38 grams, and the mean number of fetuses per litter was 11.0–14.0 (N=20–23 litters/group from six studies). The most common observations (mean % affected fetuses/litter) included short and full supernumerary thoracolumbar rib (17–29% and 0.3–4%, respectively), supernumerary interparietal ossification site (0.9–8%), isolated ossification site in the frontonasal suture (0–2%), 4th cusp in the aortic and pulmonary semilunar valves (0.8–7% and 0–2%, respectively), absent and short innominate artery (1–3% and 0.4–4%, respectively), and dilated ureter (0–4%). Other selected observations occurring at lower incidence (1–2 fetuses) included meningocele, exencephaly, malrotated limb, omphalocele, ventricular septal defect, thick ventricle wall, absent subclavian artery, dilated pulmonary trunk, retina fold, fused lumbar centrum, or thoracic arch. Compared to the Crl:CD rat (Charles River Laboratories), the Hsd:SD rat displays a different pattern of fetal abnormalities, most notably a 4th cusp in the semilunar valves and a higher incidence of short supernumerary rib and short/absent innominate.

P127 - Characterization of Transdermal Pharmacokinetics of Ketoprofen in Sinclair and Göttingen Minipigs

Our objective was to characterize the transdermal pharmacokinetics of a ketoprofen cream formulation in the Sinclair and Göttingen breed of minipigs. Minipigs have a fixed skin tightly attached to the subcutaneous tissues similar to that in humans, which makes them a preferable model for dermal studies. Three minipigs per gender per strain (9.1–9.9 months old) were used in the study. Four mL of 2.5% ketoprofen cream (100 mg ketoprofen) was applied on an area on the back of each minipig that was approximately 3% of the total body surface area. Blood samples were collected at 4, 8, 12, 24, 28, 32, and 36 hours post-dose. The plasma samples were analyzed with API 4000 LC/MS/MS system with a stable d3-labeled ketoprofen used as the internal standard. The linear range of quantitation for ketoprofen was 50 to 25600 ng/mL. The PK parameters were calculated with PKSolver. Individual animal data were analyzed with the noncompartmental analysis. Transdermal pharmacokinetic parameters of ketoprofen were (N= 6; mean +/- SD; Sinclair versus Göttingen): t1/2 (h): 12.2 +/- 2.0 versus 11.7 +/- 3.9; Tmax (h): 8.0 +/- 2.5 versus 9.3 +/- 2.1 and Cmax (ng/m): 94.3 +/- 65.6 versus 173.1 +/- 71.8. Although ketoprofen was shown to be absorbed better in Göttingen than in Sinclair minipigs in the average plasma concentration time curves, no statistically significant difference was observed between all PK parameters. These data suggested that ketoprofen has a similar ADME process in both minipig lineages.

GFP128 - Regulation of CB2 Expression by In Vitro Activation and Isotype Switching in B Cells

Cannabinoids, the primary bioactive constituents of marijuana, activate cannabinoid receptor CB2, which is expressed on the surface of human B cells but not on T cells. The toxicity of cannabinoids on immune function remains to be determined. In preliminary studies, human tonsillar B cell subsets were analyzed for the expression of cell surface CB2 by flow cytometry. While naive (IgD+/CD38-/CD27-) and quiescent memory (IgD-/CD38-/CD27+) B cells expressed cell surface CB2, activated B cells (IgD-/CD38+/CD27dim) expressed low to no detectable surface CB2. We hypothesized that B-cell activation down-regulates surface CB2, and exposure to cannabinoids plays a biologic role in isotype switching. In vitro experiments demonstrated that naive tonsillar B cells exposed to activation conditions down-regulated surface CB2 and expressed activation marker CD27. Cells still expressing IgD did not demonstrate down-regulation of surface CB2. We have designed an in vitro model to mimic isotype switching in naive mature B cells from cord blood by cross-linkage of the B cell receptor with co-stimulatory molecules. We will examine differences in CB2 expression that occur before/after immunoglobulin (Ig) isotype selection. Our findings suggest that CB2 is differentially regulated in naive versus activated/isotype-switched B cells. Cells will be treated with selective CB2 agonists and antagonists to determine the impact on isotype switching and the resulting expression of Ig subtypes. These experiments will establish a model system for examining the two-way interaction between CB2 receptors and signaling events that lead to B cell activation and differentiation.

GFP129 - Evaluation of the Functional Toxicity of a Commercially Available Canine Blue-Green Algae Dietary Supplement

The dietary supplement industry is mainly self-regulated, with limited safeguards in place, yet over a fourth of surveyed households feed supplements to their dogs. Blue-green algae (BGA) supplements are described as an antioxidant superfood and considered a treatment for metabolic and cardiovascular issues; however, there is limited data to support these purported health benefits. A commonly sold species of BGA, Aphanizomenon flos aquae, is grown in Upper Klamath Lake, Oregon, and can be contaminated with naturally occurring algae toxins. Microcystins (MCs) are acute hepatotoxic BGA toxins with over 100 known variants. MCs inhibit serine/threonine protein phosphatases (PP) 1 and 2A. PP inhibition assays (PPIA) may be a useful screening tool to determine if PP inhibitors, such as MCs, are present in BGA- containing products. A previously developed PPIA was utilized to test the functional toxicity of a 100% certified organic Aphanizomenon flos aquae BGA supplement that had been given to an 11-year-old pug dog for 3.5 weeks at the recommended dose of <1g/day. The dog was hospitalized due to persistent lethargy, inappetence, high liver enzymes, and a coagulopathy. The PPIA found the BGA supplement to significantly inhibit PP1 activity (p<0.001), suggesting PP inhibition played a major role in the observed hepatopathy in the dog. Presence of MCs in the supplement were confirmed using liquid chromatography-mass spectrometry/mass spectrometry. Two out of four MC variants tested for (MC-LR, -LA, -RR, -YR) were present, 166ng/g MC-LR and 962 ng/g MC-LA, showcasing the potential major health risks associated with BGA supplements.

GFP130 - Investigation of Persistent Organic Pollutants As Environmental Risk Factors for Hyperthyroidism

Feline hyperthyroidism has increased consistently in frequency since its recognition in 1979 and is one of the leading causes of morbidity and mortality in cats over six years of age. The cause(s) of feline hyperthyroidism are still unknown and few studies have rigorously investigated the contribution of environmental factors such as exposure to polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs), which are known to disrupt thyroid hormone (TH) regulation and signaling through multiple mechanisms. This project aims to determine whether environmental exposure to PBDEs and/or PCBs is associated with the clinical diagnosis of hyperthyroidism in felines. Approximately 40 age- and sex-matched cats were enrolled into each diagnostic group, hyperthyroid or euthyroid controls. Sera were analyzed for 13 PBDE and 12 PCB congeners by gas chromatography/mass spectrometry (GC/MS). Logistic regression analysis shows that some PBDE and PCB congeners (BDE100, BDE47, PCB175, and PCB180) are found in significantly higher concentrations in serum of hyperthyroid compared to euthyroid cats. Data from owner surveys will be assessed to gain insight into environmental sources of exposure. PBDEs and PCBs are ubiquitous in the environment and bioaccumulate in human and animal tissues; thus, it is essential to develop a better understanding of their role in thyroid disruption and disease. Cats share their environment with humans but are exposed to higher levels of PBDEs and PCBs; therefore, they can also serve as a model for human risk to aid in the prevention of environmentally induced thyroidopathies.

GFP131 - Decreased Proteasome Activity in Brain Regions Containing Nigrostriatal Dopamine Neurons Occurs through a Parkin Independent Process

The motor symptoms of Parkinson’s disease are caused by loss of nigrostriatal dopamine (NSDA) neurons. While NSDA neurons are susceptible, tuberoinfundibular dopamine (TIDA) neurons are spared. Exposure to MPTP recapitulates this pattern of susceptibility. Following MPTP exposure, parkin expression is increased in the mediobasal hypothalamus while there is no increase in the ventral midbrain. Parkin is an E3 ligase that directly modulates the activity of the 26S proteasome. The role of parkin on proteasome activity was measured in tissue from the striatum and median eminence in WT mice treated with MPTP. CT-L activity in the striatum was increased 4 hours after MPTP exposure and subsequently decreased. The amount of 26S complex also increased and then declined over the remaining time. In contrast, the amount of 20S proteasome complex increased over the time of the experiment. CT-L activity in the median eminence was not significantly changed following MPTP exposure. The role of parkin in maintaining proteasome activity was assessed using Park2-/- mice. While MPTP exposure decreased activity in NSDA neuronal regions of both WT and Park2-/- mice, proteasome activity in TIDA neuronal regions was decreased only in the Park2-/- mice. This suggests a differential role for parkin in maintaining proteasome activity in NSDA and TIDA terminal regions following neurotoxicant exposure.

IGP132 - Xenobiotics Metabolism In Vitro: Implications for Chemical Risk Assessment in Biomedical Research

Protecting public health is the primary reason why risk assessment of xenobiotics, e.g. agrochemicals, is of utmost importance. Humans are inevitably exposed to agrochemicals via different routes, at different dose levels, and for varying periods of time. Exposure to pesticides is a global challenge to risk assessment. Risk assessment needs reliable scientific information, and one type of information is the metabolic fate and toxicokinetics of compounds. Toxicokinetics refers to the movement of a xenobiotic into, through, and out of the body and is divided into several processes including absorption, distribution, metabolism, and excretion. Metabolism is one of the most important factors that can affect the overall toxic profile of a xenobiotic. During metabolism, the chemical’s first reaction is typically catalyzed by phase I enzymes, usually by the cytochrome P450 (CYP) system, and then conjugated to a more soluble and excretable form by phaseII enzyme systems. It is possible to characterize metabolic routes, metabolic interactions, and to assign which CYP is involved in the metabolism of a certain compound by different in vitro approaches. In general, these enzymatic reactions are beneficial in that they help to eliminate foreign compounds. Sometimes, however, these enzymes transform an otherwise harmless substance into a reactive form—a phenomenon known as metabolic activation. The aim of our studies is to elucidate metabolic factors and interactions of the insecticide benfuracarb in human and animal in vitro hepatic models, to extrapolate in vitro data to in vivo situations, and to incorporate toxicokinetic data into human health risk assessment processes.

IGP133 - Antidepressant Effect of Taurine in Rats Exposed to Chlorpyrifos

Chlorpyrifos is an organophosphate insecticide that can induce depression in humans and animals. Taurine is a semi-essential amino acid and a potent antioxidant with antidepressant effect. The aim of this study was to evaluate the antidepressant effect of taurine in male Wistar rats exposed to chlorpyrifos. Fifty rats were distributed into five groups of ten rats each. The groups received the following treatments once daily by oral gavage for 16 weeks: DS (distilled water), SY (soya oil, 1 ml/kg), TR (taurine, 50 mg/kg), CH (chlorpyrifos, 4.25 mg/kg, 1/20th LD50) and TR+CH (taurine and chlorpyrifos sequentially). The forced swim test was used to assess the antidepressant effect of taurine at week 16. Additionally, brain malondialdehyde (MDA) concentration and the activities of brain antioxidant enzymes (superoxide dismutase [SOD] and catalase [CAT]) were evaluated in the rats at the end of the study. Significant reductions in the immobility time were recorded in the DS, TR, and TR+CH groups compared to the CH group, respectively. The brain MDA concentration of the rats in the DS, SY, TR, and TR+CH groups were significantly decreased compared to the CH group, respectively. Brain SOD activity was increased in the TR and TR+CH groups, while brain CAT activity was enhanced in the TR group compared to the CH group, respectively. The antidepressant property of taurine was manifested as a reduction in the immobility time of the rats. It is postulated that taurine elicited its antidepressant effect probably through its antioxidant effects in the brain.

P202 - A CALUX Assessment of Petrogenic PAH Contamination in Gulf of Mexico Seafood Following the Deepwater Horizon Oil Spill

Through a community-based participatory research (CBPR) approach, the GC-HARMS consortium conducted a four-year assessment of petrogenic polycyclic aromatic hydrocarbon (PAH) contamination in Gulf of Mexico seafood from Louisiana, Mississippi, and Alabama following the 2010 Deepwater Horizon (DWH) oil spill. Contamination was measured using the EPA-approved CALUX^®chemical-activated luciferase gene expression) bioassay, based on aryl hydrocarbon receptor responsiveness to PAHs. It was chosen under the notions that 1) petrogenic PAHs have similar toxicological manifestations as the well-studied pyrogenic PAHs, 2) other methods of quantification (GC-MS) impart less human health-relevant toxicological insight (relative toxic potency) on the PAH’s in vivo toxicology. We analyzed 432 PAH extracts using benzo[a]pyrene (BaP) as the reference standard. The results were disseminated to our community members in the form of conservative seafood consumption guidelines, based on the published cancer slope factor for BaP and exposure duration values for the DWH spill. We also evaluated PAH contamination between species and states and over time to identify any trends or contamination hotspots. We identified a significant difference in PAH levels between samples collected at the earliest time studied and those collected later in the study. We also found oysters to contain significantly higher levels of PAHs than crab, shrimp, and finfish. It is noteworthy that we identified high PAH levels in samples collected in a control location that was not directly oiled by the DWH spill. We conclude that our data suggest a ‘background level’ of PAH contamination in the Gulf of Mexico.

P203 - Strategic Approaches to the Use of SEND Controlled Terminology

Starting in December 2016, SEND (Standard for Exchange of Nonclinical Data) will become mandatory for nonclinical FDA submissions. One requirement of SEND is the mapping of specified study terms to CDISC SEND controlled terms (CT). However, the SEND model itself requires inclusion of original terms recorded by pathologists and other scientists, in addition to the mapping of these terms to SEND CT. This dual representation provides sponsors with strategic options. For example, it is possible to create glossaries within LIMS (Laboratory Information Management Systems) using SEND controlled terminology. With that option, the identical information will populate SEND variables for results as collected as well as results mapped to controlled terminology. In contrast, if sponsors prefer to retain their own terminologies, their original results will be captured in SEND in addition to being mapped to controlled terminology within SEND. Because SEND is a standard electronic format, it can be used to build data warehouses and repositories. The decision of which controlled terminology option to choose should therefore be driven by data analysis strategies (which terms are the most appropriate and which data to capture and use for searches).

P204 - Template, User Guide, and Examples for Nonclinical Study Data Reviewer’s Guide for SEND Submissions

According to FDA’s Study Data Technical Conformance Guide V2.2 (June 2015), preparation of a Study Data Reviewer’s Guide (SDRG) is recommended as an integral part of a CDISC standards-compliant study data submission. An SDRG template, completion guidelines, and examples for clinical studies have been available since May 2013. Recently, the PhUSE/FDA Nonclinical SDRG Working Group, with representation from pharma, CROs, and SEND solution vendors, has developed an SDRG for nonclinical studies with inputs and feedback from the FDA. These materials can be found at www.phusewiki.org/wiki/index.php?title=Nonclinical_SDRG_Template_and_Guide. The nonclinical SDRG should describe for each study any special considerations that may facilitate use of the dataset by FDA reviewers and data managers. These include clarification of relationships between the study report and SEND datasets; identification of SEND standards, controlled terminologies and versions used in the datasets; a summary of the included datasets; conformance observations relating to FDA SEND validator rules; and decisions related to data standards implementations, including deviations and errors where applicable. The SDRG should include a high-level summary of the process by which the SEND datasets were created from study data. Each SDRG should be specific to a particular study to enable effective use by FDA reviewers and data managers. Highlights of recommendations for authoring a nonclinical SDRG will form the basis of this poster presentation.

STP206 - Petrogenic PAH Contamination in Galveston, Texas, Seafood Species Before and After the 2014 Houston Ship Channel Bunker Oil Spill

Following the 2010 Deepwater Horizon oil spill, we developed a consortium to assess petrogenic PAH levels in Gulf of Mexico seafood using a community-based participatory research approach. Through this consortium we collected seafood samples from Galveston, Texas, which were used as control samples for the Deepwater Horizon spill. In March of 2014, the oil barge Kirby collided with the tanker M/V Summer Wind, causing the release of approximately 168,000 gallons of bunker oil in the Galveston Bay. Bunker oil differs from light crude because it is enriched for high molecular weight PAHs like fluorene, fluoranthene, and pyrenes. A recent study shows this oil contains 100 times more BaP than the DWH oil. As these large PAHs are the main toxins of concern to humans following an oil spill, this oil may contaminate local seafood and drive PAH toxicities in those who consume such contaminated seafood. Using previously developed community partnerships, we collected samples over the course of the 2014 Bunker oil spill and after cleanup. Using this longitudinal sample set, we generated PAH extracts and assessed PAH contamination using GC/MS (wt PAH/wt sample) and the EPA-approved in vitro CALUX bioassay (PAH toxic equivalency/wt sample). To our knowledge, we are the first to show that PAH levels in local seafood were significantly elevated following the HSC spill. Further, those levels remained above the baseline levels we identified in 2012 and 2013, for the entirety of the 1-year study period after the spill.

P207 - Cosmetic Safety Testing Roadmap to Regulatory Approval in North America, Europe, and Japan

The cosmetics industry is a robust business, achieving greater than $250 billion in worldwide sales in 2013. Although major recognizable brands constitute the largest fraction of this total, there are thousands of small companies that operate in the same markets. Enterprise architect 11 keygenguru 9. While the industry association International Cooperation on Cosmetic Regulation (ICCR) seeks long-term harmonization of testing requirements, currently unaligned governmental agencies in different countries make navigation of safety requirements daunting for major manufacturers and Kafkaesque for smaller firms. While also laudable, the development, validation, approval, and implementation of new non-animal safety assays often make the path to cosmetics approval seem more formidable than ever. To aid in navigation of the kaleidoscopic variables of cosmetics ingredients and final cosmetics formulation safety testing, the authors have developed a roadmap to safety assessment, focusing on requirements of the United States and the European Union.

P208 - Analysis of Bacterial Mutagenesis Tester Strains and Carcinogenicity Data by Chemical Class

The ICH M7 guidance on pharmaceutical impurities describes how to handle impurities that are DNA reactive, including performing bacterial mutation testing. The guidance allows for deviations from fully adequate protocols and compliance with Good Laboratory Practices for practical reasons whereby the selection of bacterial tester strains may be limited to those proven to be sensitive to the identified alert. Bacterial tester strains were analyzed for sensitivity to particular compound classes and structural alerts using a large database of over 10,000 compounds with genetic toxicity data. Almost half of the structural alerts were highly correlated with a specific tester strain (including consideration of metabolic activation). 69% of these correlating alerts were most sensitive to either Salmonella TA98 or TA100 and 14% to Salmonella TA102 or E. coli. For example, it was determined that 95% of mutagenic primary aromatic amines are positive in at least Salmonella TA98 and TA100 with and without metabolic activation, with TA98 with metabolic activation being the most sensitive. This analysis was extended to include analysis of carcinogenicity data, including specific lesion sites. The Leadscope Knowledge Sharing Program extended this analysis to include proprietary corporate information without revealing business-sensitive compounds or data. This presentation describes how this analysis was used to support the efficient adherence to the M7 guidance and a corresponding reduction in cost by testing fewer strains and requiring lower compound amounts.

P209 - Performance and Application of the MultiCASE Rule-Based Expert System

ICH M7 guidelines for in silico bacterial genotoxicity testing require the use of both statistical and expert rules systems. To better satisfy these regulations we have updated our rule-based system of mutagenicity, GT_EXPERT, which now consists of 180 structural alerts, representing general mechanisms and refining factors. Our updated expert system helps to reduce false positives in several important structural chemical classes, such as tertiary aromatic amines, hydrazines, and oximes. We have compared its performance on an independent dataset of 4,000 compounds screened by the Division of Genetics and Mutagenesis of the National Institute of Health Sciences of Japan. On this heavily skewed dataset (576 actives and 3,442 inactives), the updated rule-based system has sensitivity of 70% (+1%), specificity of 79% (+4%), positive predictive rate of 37% (+5%), and negative predictive rate of 94% (+1%), while prediction coverage (91%) was the same. We also demonstrate several testing scenarios, in which expert rules can be used to invalidate unreliable predictions from weak statistical alerts.

P210 - Microsampling Conscious Rat Jugular Technique for Serial Collection of Toxicokinetic Samples

The large blood volumes required for traditional toxicokinetic (TK) analysis on rat toxicity studies present animal welfare concerns, requiring TK satellite groups that typically consist of 3-4 animals/sex/group. Collecting 0.5 mL of blood at up to eight time points could significantly impact a main study animal and would likely exceed allowable volume limits. Recently improved bioanalytical sensitivity has resulted in decreased sample volume requirements, allowing development of a plasma microsampling strategy. Our goal was to use microsampling on GLP and non-GLP rat studies and ultimately incorporate TK sampling into main study animals and eliminate TK satellite groups. To this end, the Sarstedt Minivette^® device was evaluated for practicality, GLP compliance, and quantitative equivalence. The Minivette^® was used to collect 50 μμL whole blood samples from the jugular vein of conscious rats. Plasma (10 μL) was extracted and diluted with blank plasma prior to bioanalysis. This technique was validated against our traditional TK methods and provided quantitative equivalence to conventional samples while reducing total blood volume to 0.4 mL. This significant decrease in blood volume collected has allowed collection of TK samples directly from main study animals and elimination of TK satellite groups, thus providing a full TK profile for toxicity study animals. In our ongoing implementation of this study design, TK time point samples are serially collected from each of the first four rats/sex/group. This technique contributes to the 3Rs (refine, replace, reduce) by eliminating up to 32 animals per study while providing high-quality TK evaluations for toxicity studies.

P211 - The Applicability of QSAR Modeling for Impurities/Metabolite Profiling of Agrochemicals

The use of quantitative structureactivity relationship (QSAR) models is increasing, especially for toxicological potential profiling of impurities and metabolite found in the production and use of agrochemicals. Most important, the predictions made are becoming increasingly accepted by regulatory authorities worldwide. One such QSAR tool is DEREK Nexus. This is an expert knowledge-based system that predicts whether a chemical is toxic in humans, other mammals, and bacteria. The application is a high-throughput screen for multiple endpoints and uses expert knowledge rules derived from toxicological databases. Software applies the rules to make a qualitative assessment on the toxicity potential of any given compound. DEREK Nexus is an increasing model of choice in the pharmaceutical, crop protection, and chemical industries for assessing impurities and metabolites. Use of the model can in some instances preclude studies in animals. The use of and acceptance of DEREK predictions by worldwide regulatory authorities does depend on the presentation of the correct output and interpretation of that output. To facilitate the consideration of a QSAR model for regulatory purposes, it should be associated with the following information: 1. A defined endpoint; 2. An unambiguous algorithm; 3. A defined domain of applicability; 4. Appropriate measures of goodness-of-fit, robustness; and, predictively, 5. A mechanistic interpretation, if possible.

Presentation of this information will be discussed together with the limitations of the QSAR. How expert interpretation can add value to the DEREK output in the regulatory setting will be illustrated.

P213 - SEND Nonclinical Data Submissions: Topics to Focus on Now

On December 17, 2014, the FDA announced new binding guidance that defines requirements for standardized electronic data in regulatory submissions for certain studies that begin after December 17, 2016. For nonclinical data, this begins a new era for the submission of study data in the United States. An unprecedented level of implementation activity is now occurring throughout the industry. Through the collaborative work of contract research organizations (CROs), industry sponsor companies, vendors, and the FDA, the SEND (Standard for Exchange of Nonclinical Data) Implementation Guide (SENDIG) (www.cdisc.org/send) was developed. The SENDIG continues to evolve to include additional study and data types. Two industry organizations that collaborate regularly on the development and implementation of nonclinical standards are CDISC (Clinical Data Interchange Standards Consortium) (www.cdisc.org) and PhUSE (Pharmaceutical Users Software Exchange) (www.phusewiki.org/wiki/index.php?title=PhUSE_Wiki). This poster highlights some key topics that the industry is focusing on to comply with the SEND standard. The latest development activities and targeted rollout for new SEND versions will be described, including an updated version (v3.1) targeted for this year. The poster will also highlight important considerations for managing data that makes use of multiple versions of the standard and verifying the quality and completeness of SEND datasets; best practices for presenting metadata with submissions; specific sections of the nonbinding Technical Conformance Guide; dataset challenges recognized by nonclinical FDA reviewers to date; and understanding the full value of the Study Data Reviewers Guide (www.phusewiki.org/wiki/index.php?title=SDRG).

P214 - The Assessment of Single-Housed vs. Pair-Housed Rats on the Irwin Test

The purpose of safety pharmacology is to investigate effects of the test substance on vital functions. In this regard, cardiovascular, respiratory, and central nervous systems are usually considered the vital organ systems that should be studied in the core battery. As a result, the ICH S7A guideline (2001) specifies that the effects of test substance on motor activity, behavioral changes, coordination, sensory/motor reflex responses, and body temperature should be evaluated through the use of a modified Irwin or other appropriate test. In 2011, the Guide for the Care and Use of Laboratory Animals (8th Edition) stated that members of a social species should be socially housed whenever possible, including rodent species. WIL Research adopted this policy for rodent Irwin studies conducted to meet the ICH S7A guideline in 2014.

Recent studies have evaluated space needs and the effects of social housing, group size, density, age, and housing conditions for rodents, and have reported varying effects on behavior (such as aggression) and experimental outcomes. Based on this work, an effort was conducted at WIL Research to determine the differences in behavior (CNS effects) when comparing results from single-housed vs. pair-housed animals as outlined below: Did the background incidence of findings change between housing conditions?; Were baseline findings prior to dosing consistent regardless of housing?; and Were data more or less variable as a result of these housing changes?

In order to answer these questions, a comparison to historical control and internal positive control data were utilized.

P301 - Skin Sensitization Model by Quantitative Structure Toxocity Relationships (QSTR) Approach

In silico assessment of skin sensitization is increasingly needed owing to the problems concerning animal welfare, as well as excessive time consumed and cost involved in the development and testing of new chemicals. Previously, we developed a skin sensitization model from human and animal data, which was reported at 24JSAAE. Its accuracy was 61.2% (sensitivity 60.7%, specificity 62.8%) by external validation. This time we developed a skin sensitization QSTR model using only animal data (LLNA, 471 chemicals, ICCVAM Test Method Evaluation Report [NIH Publication Number 09-6439]) by using K-step Yard sampling (KY) methods (US Patent No. 7725413, 2010), one model KY method (US Patent Application), and ordinary discriminant function based on EC3 values to discriminate strong sensitizers from weak sensitizers. All data analysis was performed using ADMEWORKS/Model Builder (Fujitsu Kyushu Systems Limited Japan). The result was that a total of 320 compounds (212 positive sensitizers and 108 negative sensitizers) were used in this study. 288 compounds were used to train a QSTR model, and external validation studies were performed on a separate set of 32 compounds. The concordance of QSTR prediction for LLNA data was 71.9% (sensitivity 54.5%, specificity 81%). We concluded that the concordance was better than our previous model and indicates that the data from human and animal studies were qualitatively different from each other. Acknowledgment: This work was supported by JSPS KAKENHI Grant Number 25293148.

P302 - Aerosol from a Candidate Modified Risk Tobacco Product Has Reduced Effects on Chemotaxis and Transendothelial Migration Compared to Combustion of Conventional Cigarettes

Cigarette smoking increases the risk of cardiovascular disease. Reduction of harmful constituents by heating rather than combusting tobacco without modifying the amount of delivered nicotine could be a promising new approach to reduce harmful effects associated with cigarette smoking. In this study, we investigated the effect of extract from a new candidate modified risk tobacco product (MRTP), the tobacco heating system (THS) 2.2, on the migratory behavior of monocytes in comparison with extract of smoke from combustible 3R4F reference cigarettes. THP-1 cells, a monocytic cell line, and human coronary arterial endothelial cells (HCAECs) were used to analyze chemotaxis and transendothelial migration (TEM) of monocytes in response to C-X-C motif ligand 12 in conventional and impedance-based systems. To assess the influence of extracts from THS2.2 aerosol and 3R4F smoke on toxicity and inflammation, flow cytometry and ELISA assays were used. The results show that treatment of THP-1 cells with extract from 3R4F or THS2.2 induced a concentration-dependent increase in cytotoxicity and inflammation (IL-8 and TNF-α secretion). The inhibitory effects of THS2.2 extract for chemotaxis and TEM were at least ∼18 times less effective compared to 3R4F extract. Furthermore, we found decreases in the integrity of an HCAEC monolayer in a concentration-dependent manner. However, for all other examined endpoints, the extract from 3R4F showed more than one order of magnitude stronger effects than that from THS2.2 extract. These data indicate the potential of a heat-not-burn tobacco product to reduce the risk for cardiovascular disease compared to combustible cigarettes.

P304 - Evaluation of the Chronic Toxicity and Carcinogenicity Potential of Smokeless Tobacco in Wistar Han Rats

A two-year chronic oral toxicity/carcinogenicity study was conducted in Wistar Han rats with snus tobacco blend and an extract of that blend, according to good laboratory practices. Control groups consisted of feed without tobacco. Test article groups consisted of dosed feed with tobacco blend used in snus and dosed feed with an aqueous extract of that tobacco blend, administered at 0.2, 2, or 5 mg nicotine/kg body weight/day to Wistar Han rats. These dosages simulated potential exposure in humans ingesting smokeless tobacco or an extract of smokeless tobacco, to enable bridging these data to snus epidemiology data. There were no test article-related changes in survival, clinical observations, ophthalmic exams, clinical pathology, or organ weights. Treatment-related changes included dose-related increases in plasma nicotine and cotinine (indicating appropriate exposures), decreases in body weights, and some alterations in feed consumption. Histologically, a few uterine carcinomas and epididymal mesotheliomas were noted, and these exhibited statistically significant increasing trends in incidence; mammary gland adenomas (females), skin basal cell carcinomas (females), and thyroid follicular cell adenomas (males) exhibited decreasing trends. Tumor incidences were within the expected spontaneous range for historical controls for Wistar Han rats. Therefore, it was concluded that chronic exposure of Wistar Han rats to either a blend of tobacco used in snus or an extract of that blend does not lead to adverse toxicity or increased carcinogenicity.

P305 - Assessment of the Tobacco Heating System 2.2, a Candidate Modified Risk Tobacco Product, on Human Organotypic Bronchial and Nasal Epithelial Tissue Culture Using Systems Toxicology Approach

New tobacco products with the potential to reduce the risk associated with smoking are under development and require a careful safety assessment strategy. In line with the 21st century toxicology paradigm and with the use of human in vitro organotypic models as a substitute for animal testing, we exposed human nasal and bronchial epithelial tissue culture to an aerosol generated by a candidate modified risk tobacco product (cMRTP), named Tobacco Heating System 2.2 (THS2.2), versus air (sham control) or mainstream smoke from conventional cigarettes with the same nicotine content as THS2.2.

Different endpoints (cytotoxicity, cilia beating frequency, CYP1A1/1B1 enzyme activity, inflammatory markers release, morphological and transcriptomic changes) were analyzed at 4, 24, 48, and 72 hours after exposure to identify and compare the dose- and time-dependent effect of the different test item used.

By using systems toxicology-based risk assessment approaches combining computable biological network models and gene expression changes, we compared the molecular perturbations in both conventional product and cMRTP exposure conditions. A significant impact was quantified over different combustible cigarette smoke (CS) post-exposure time points in the networks representing cell death, inflammation, proliferation, and cellular stress; instead, the impact of THS2.2 (at a comparable dose) was closer to sham controls and detectable only shortly after exposure (4 hours). Pro-inflammatory markers release was also significantly reduced in THS2.2. Overall, these results highlight a reduced toxicity of THS2.2 acute exposure for both nasal and bronchial epithelial tissue culture when compared to CS.

P306 - A 14-Day Toxicity Repeat-Dose Study of PSOA By Oral Gavage in Rats

This study follows prior work assessing repeat-dose toxicity of peroxysulfonated oleic acid (PSOA). PSOA was dosed daily via gavage to Sprague-Dawley rats (10/group, 5M/5F) for 14 days at 0, 100, 300, and 1000 mg/kg/day. Endpoints evaluated were clinical signs, body weight, food/water consumption, clinical pathology, organ weight, gross pathology, and histopathology. Mortality occurred at mid (10%) and high (30%) doses. Clinical signs were noted at all PSOA doses, with dose-response of incidence and severity. Survivors in the control, low-, and mid-dose groups gained body weight. High-dose group survivors all gained weight, but loss was observed periodically. Decreased food/water consumption occurred at mid (one rat) and high doses (all rats). Clinical pathology endpoints were largely unaffected aside from select parameters at mid and high doses, which had questionable biological significance or appeared as adaptive responses. Organ weight effects were confined to high dose with some observations attributable to decreased body weights and/or nutritional status (females). Gross pathology was unremarkable, with all non-gastric findings considered unrelated to PSOA. Histopathological lesions, confined to the stomach, were observed in all high-dose rats and three of four surviving mid-dose females. No stomach lesions observed in mid-dose males or in rats at low-dose or control. Overall, effects were observed at all PSOA doses; however, the effects observed at the low-dose were clinical signs of minimal severity in a minority of rats, so a study NOAEL of 100 mg/kg/day was identified. Effects observed for PSOA are related to local gastric irritation, not the result of systemic toxicity.

P307 - Acute Inhalation Toxicity of Oleoresin-Capsicum-Containing Smoke Used for Crowd-Control Applications

Oleoresin Capsicum (OC) is an oily resin found in cayenne and other varieties of peppers used in law enforcement as a debilitating irritant and inflammatory agent. Although traditionally delivered as a liquid aerosol (pepper spray), combusted OC carried in smoke from pyrotechnic devices has also proven effective. The short-term inhalation toxicity of OC-containing smoke was assessed using whole-body inhalation exposure methodology. Five male and five female Crl:SD(CD) rats were exposed at a total smoke concentration of 19.4 mg/l for 5 minutes. Particle size of the smoke was determined using cascade impaction; the mass median aerodynamic diameter (MMAD) was 1.9 ╡m. The concentration of the total smoke was determined by gravimetric technique, and the corresponding OC concentration in air was calculated based on the fraction of the combusted product containing OC. Animals were observed throughout exposure and daily for 14 days afterward. Body weights were collected on study days 0, 7, and 14. Early in the exposure period, all rats displayed avoidant behaviors such as efforts to escape the enclosures within the exposure chamber. By the end of the exposure period, all animals were lethargic, but regained normal activity levels after removal from the chamber and being washed free of smoke residue. All rats displayed no adverse signs at three hours postexposure. All animals gained weight during the study, and survived to the scheduled necropsy.

P308 - Cyclodextrin Glucanotransferse: 90-Day Rat Toxicity Study and Genotoxicity Battery

Cyclodextrin glucanotransferase (CGTase) is used commercially in the manufacture of food, pharmaceuticals, and cosmetics. In anticipation of its use in production of alpha-glycosyl isoquercitrin, a water-soluble form of quercetin, microbiologically derived CGTase was evaluated for its toxic potential.

CGTase, produced by Bacillus pseudalcaliphilus DK-1139, was evaluated in a genotoxicity battery following OECD guidelines and included a bacterial reverse mutation assay, in vitro and in vivo micronucleus (MN) assays, and a comet assay using B6C3F1 male and female mouse tissues. These same genotoxicity assays were also conducted for sodium sulfate, a contaminant of CGTase preparation. In addition, CGTase was administered by gavage in water at daily doses of 0, 250, 500, and 1000 mg/kg/day in a 90-day toxicity study in Sprague-Dawley rats.

Results: CGTase did not induce mutations with or without metabolic activation in the bacterial reverse mutation assay. Micronuclei were not induced either in in vitro with or without metabolic activation or in male or female mice. Furthermore, there was no induction of DNA damage in male or female mouse liver, stomach, or duodenum in the comet assay. Sodium sulfate also tested negative in these same genotoxicity assays. In the 90-day repeated dose rat study there were no treatment-related adverse clinical or pathological findings.

Conclusion: The genotoxicity assays and repeated dose toxicity study support the safe use of CGTase in production of alpha-glycosyl isoquercitrin.

P309 - Cilia of Human MucilAir Are Less Impacted by the Exposure to the Aerosol of a Candidate Modified Risk Tobacco Product Than to Whole Smoke from Conventional Cigarettes In Vitro

Mucociliary clearance is an important defense mechanism that mediates removal of foreign particles and chemicals from the airways. Cilia beating thereby plays a key role that determines the rate of mucus clearance and thus constitutes a vital function of respiratory epithelia. Cigarette smoke has been reported to adversely impact cilia function in vitro and in vivo, by changing cilia beating frequency (CBF) or impairing ciliogenesis. To monitor CBF, semi-automated methods such as CiliaFA combine high-speed video recording with the ability to determine CBF. Using CiliaFA, we were able to confirm that the MucilAir CBF can be modulated in vitro by either 100 μM isoproterenol or a temperature shift to 4°°C. Moreover, in vitro exposure of MucilAir with whole smoke from conventional cigarettes (3R4F) caused a decrease in the total surface area of the culture showing active cilia beating. Cilia on the epithelial cell surface that were detected to be still active after 3R4F exposure showed variable beat frequencies, ranging from normal to decreased CBF. Compared to 3R4F cigarette smoke exposure, the effect of equivalent concentrations (based on nicotine) of a candidate modified risk tobacco product (THS2.2) aerosol was much less, judged from its effect on total surface area showing active cilia and from the determined cilia beating frequencies. Overall, this study clearly distinguished the effects of THS2.2 from the deleterious impact of 3R4F on cilia beating.

P310 - Dermal Exposure, Absorption, and Hazards from Use of Thymol, a Terpene-Based Skin Penetration Enhancer

Thymol (2-isopropyl-5-methylphenol) is a naturally occurring constituent of plants of the genus Thymus (including the culinary herb common thyme) and, as a monocyclic monoterpene, is a member of the ╘terpene╒ family of compounds that have been extensively studied by the pharmaceutical industry for their use as skin penetration enhancers (penetrants) to aid in the transdermal delivery of drugs across the stratum corneum. The safety of exposure associated with thymol was considered by the US FDA and HC for its use in other applications, but uncertainties remain with regard to MPLs of this compound in dermally applied drugs; considering that thymol at high concentrations is known to be irritating to the skin. The objectives of the present exercise were to (i) quantify the transdermal dose of thymol absorbed after a single use of a hypothetical topical medication containing ∼1% thymol, (ii) assess the MoS from a single use of such a drug, (iii) assess safety concerns related to human health from repeated, long-term use of the penetrant, and (iv) identify a provisional maximum permissible level of thymol in the dermally applied drug. A number of variables influence the extent to which any compound is absorbed across the skin, and to account for the uncertainty in quantifying the dermally absorbed dose of thymol we investigated and compared multiple methodologies to determine exposure. A safety evaluation was completed by combining the results of our exposure assessment with a provisional toxicological reference value we derived de novo utilizing traditional MoS approaches (i.e., NOAEL/Exp).

IGP311 - Toxicological Studies of Icacina trichantha Tuber Extract and Fractions

Icacina trichantha Oliv. (Icacinaceae) is a traditional herb used in Nigeria and West Africa, locally known as urumbia or eriagbo (referring to its emetic effect) among the Ibos of Nigeria, or gbegbe (meaning to cleanse) by the Yorubas of western Nigeria. The tuber is prescribed by herbalists to treat food poisoning, constipation, and malaria. The plant drug is also a household medicine for emergency uses; hence, virtually every household in Nigeria would keep a supply of the macerated tuber in alcohol as a first-aid. Recent studies on I. trichantha revealed its anti-hyperglycemic, antimicrobial, analgesic, and sedative effects. Previous phytochemical studies have shown the presence of diterpenes and diterpene alkaloids in three species of Icacina. This study was aimed at evaluating the toxicological effects of chronic administration of I. trichantha tuber extract in rats and also the in vitro toxicological effects of its fractions and isolates. Graded doses of the extract (A=0g/kg, B=0.25g/kg, C=0.5g/kg and D=1g/kg) were administered to rats in feed for 90 days. The effects on the liver, kidney, heart, and pancreas and also on body weight, clinical behavior, hematology, and serum biochemical parameters were investigated. In vitro toxicity studies were also carried out on human melanoma cells designated MDA-MB-435, human colon cancer cells (HT-29), and normal cells (3T3-L1). There were no treatment-related histopathologic lesions in the various organs studied. However, the crude extract and some of the fractions displayed remarkable cytotoxicity on the cell lines.

IGP312 - Application of Robust Cost-Effective In Vitro (Eco)Toxicity Screening Methods for the Initial Grouping of Engineered Nanomaterials in Respect of Their Hazard: EU FP7 Project NANOVALID

According to the Woodrow Wilson Database there are currently more than 1,600 nanoparticles-containing consumer products on the market. At the same time, toxicologists and the public are concerned about the potential environmental and human health risks associated with nanoparticles (NPs, with the size of 1-100 nm). Currently, there are numerous European Union (EU) research consortia dedicated to nanosafety, including the flagship FP7 project NANOVALID. The main aim of the NANOVALID project is to develop a set of reliable reference methods and materials for physico-chemical characterization and hazard identification of NPs, whereas our group focused on the development of (eco)toxicological in vitro toxicity assays. Altogether, 12 different test organisms and cell lines and 16 toxicity endpoints were tested for their potential to disclose the (eco)toxicity of 9 well-characterized NPs. The assays were based on a wide variety of ecologically relevant non-vertebrate organisms of different food-web levels, medically and hygienically important microbial strains and mammalian cells in vitro. As a rule, the toxicity pattern for NMs was quite similar, whatever the assay and organism. The toxicity decreased in the following order: Ag>ZnO>CuO>Au> MWCNTs >SiO2=TiO2. The results obtained allowed us to rank the NMs into different toxicity categories. Taking into account the test results, test cost, relevance, and variety in terms of biological organization of the test species, we suggest the following test battery for the cost-efficient nanotoxicity screening comprising three acute assays: bacteria Vibrio fischeri 30-min bioluminescence inhibition assay, Daphnia magna 48-h immobilization assay, and 48-h murine fibroblasts neutral red uptake test in vitro.

P401 - Standardized Neurological and Physical Diagnostics During Intrathecal Drug Delivery of Cynomolgus Monkeys: You Better Know the Background Findings

Detailed neurological and physical examination is an essential part of toxicity studies in Cynomolgus monkeys undergoing intrathecal drug delivery. The scientific literature, however, reveals little background data of such parameters. Hence, physical and neurological examinations were performed in unsedated animals (n≥240, on at least five occasions) during the predose phase and the dosing phase of toxicity studies (predose, 4 and 8 hours postdose). In case of findings that did not recover within 8 hours postdose, additional examinations were performed. Physical examinations included abdominal palpation, eye, ear, and nose examination, body temperature, heart, and lung auscultation. Neurological examinations included general sensomotory aspects, cerebral reflexes (pupillary, orbicularis oculi, cornea) and spinal reflexes (patellar, anal). Furthermore, the foot grip reflex (a rod was placed under the foot and the animal should grip this rod) was recorded. In healthy animals, the only background finding on multiple occasions predose was a decreased patellar tendon reflex present on several days for the same animal. Effects that were related to the intrathecal administration (procedure-related, as also occurring in control animals) were either a temporarily absent or reduced patellar reflex or the absence of the anal reflex. The foot reflex was never absent. In all cases observed the findings were of transient nature and recovered within 24 hours. No trend was observed for a procedural-related repeat of findings in the same animals. Individual physical examinations predose did not reveal any abnormality. Body temperature in healthy primates was 35.9 to 39.5°°C with a circadian and sex-specific pattern.

P402 - Method Enhancement for Intrathecal Lumbar Bolus Delivery and CSF Sampling in the Anesthetized Cynomolgus Monkey for the Safety Assessment of Oligonucleotides

Crossing the blood-brain barrier becomes an increasing need in terms of drug delivery for pharmaceutical development (Turner 2003). Guidance in literature using dosing techniques on anesthetized Cynomolgus monkeys is still limited. Lumbar intrathecal administration of monkeys (above 2.3kg) is feasible for up to 53 weeks (Felice 2011). A dose volume of 0.75mL-2.0mL of test item formulation, followed by 0.25mL of an artificial CSF can be used for each injection, if the approximate dosing volume of CSF is withdrawn in forefront. For best practice a micro incision of the skin is conducted using a 20G needle before introduction of the spinal needle. Dosing is elaborated with a Pencan Paed^® pencil-point needle (25G) for pediatric use. Intrathecal administration is performed via lumbar puncture at level L3-L4 by slow manual infusion over 1 min to fasted anesthetized animals (ketamine and medetomidin; atipamezole was used as antidote). It has to be confirmed that the position of the needle opening is pointing toward the head of the animal prior to dose administration. Additionally, the needle (with syringe) remains in the dosing site for at least 30 seconds after a CSF flush. Following the administration, the animal will carefully be returned in the transport box and placed in a lying position for 15 minutes before the antidote is applied. Review of toxicokinetic profiles of CSF versus tissue (brain sections, spinal cord sections) have confirmed a higher payload when applying the above, which may have broader implications for CNS treatment with biopharmaceuticals.

P403 - Application of a Labeled Stem Cell Animal Model for Investigative Safety Assessment of Stem Cell Populations In Situ

The advent of stem cell technologies has created new approaches for evaluating the toxic effects of drugs. While there is growing application of isolated stem cell culture systems in drug safety testing, new animal models have recently been developed where specific organotypic stem cells express fluorescence proteins, permitting easier tracking of these populations and their response to drugs in situ. To explore the utility of such models for in vivo investigative safety testing, we have evaluated a mouse model in which green fluorescence protein (GFP) expression is driven by the Lgr5 gene promoter. The Lgr5 gene is a well-characterized stem cell marker, expressed by basal intestinal crypt cells as well as by stem cells in other organs. By digesting sampled tissues to single cells and using flow cytometry, it is possible to quantitatively determine the effect of compounds on Lgr5-positive stem cell populations. We evaluated two compounds known for their ability to cause toxicity in the small intestine, and obtained comparative data on their effects on the Lgr5-positive stem cells in the basal crypts. Flow cytometry-based analysis was corroborated by histopathological assessment of these cells in the small intestine. In summary, we demonstrated the utility of a labeled stem cell animal model to obtain quantitative evidence of stem cell toxicity in vivo. The model can be applied to address specific mechanistic hypotheses related to stem cells in the toxicologic response of tissues to drugs.

P404 - The Systems Toxicology Challenge: Omics Data to Predict Exposure Response and Mechanisms of Toxicity

Risk assessment in the context of 21st century toxicology relies on the elucidation and understanding of mechanisms of toxicity. For that purpose, datasets generated by high-throughput technologies (e.g., high-throughput/content screening) combined with various omics data types are now generated in vitro to test a diverse set of chemicals (e.g. ToxCast). The development of relevant computational approaches for the analysis and integration of these big data remains challenging. The current scope of sbv IMPROVER (Industrial Methodology for Process Verification in Research; http://sbvimprover.com/) is the verification of methods and concepts in systems biology research via challenges opened to the scientific community. Previous challenges brought new insights on methods and their associated results that address questions about diagnostic signatures, the translatability of biological responses/processes across species, and the relevance of biological causal network models. A new sbv IMPROVER challenge will be introduced, aiming at evaluating methodologies for the identification of specific biomarkers of exposure when organisms are exposed to individual chemical molecules or mixtures. Participants will be provided with high-quality data sets to develop predictive models/classifiers. For this challenge, the integration of a priori biological knowledge in the development of computational approaches may be required to enable biological interpretability/understanding of the predictions. The results and post-challenge analyses will be shared with the scientific community, and will open new avenues in the field of systems toxicology.

P405 - Determinants of Ischemic Wound Healing in the Diabetic Yucatan Miniature Swine

Our objective was to evaluate the delay in healing in full-thickness ischemic wounds using a bipedicle flap model in diabetic miniswine. Full thickness paraspinous chronic ischemic wounds were created in three diabetic miniswine (duration of induced diabetes onset >10 months). The wounds were allocated to the three animals and were composed of a: six bipedicle ischemic flaps (5 x 15 cm: 75 cm2) with center punch biopsy (diameter: 0.8 cm) with corresponding six non-ischemic control full-thickness punch biopsies or b: two larger bipedicle ischemic flaps (9 x 24 cm: 216 cm2) with center punch biopsy (diameter: 5 cm) with corresponding two non-ischemic control full-thickness punch biopsies. Each of the flaps had silicone film underlying the flap. Time to complete healing was determined by photographic documentation of wound size. Although not statistically significant due to the small sample size (N=6), the 8-mm-diameter punch full-thickness wounds on 75 cm2 bipedicle flaps showed delayed healing at day 12 (0.15 +/- 0.06 versus 0.28 +/- 0.11 cm2, control versus ischemic, p<0.1) and were fully delayed and/or incompletely healed at day 21. The 5-cm-diameter full-thickness wounds on 216 cm2 bipedicle flaps did not heal because of apparent ischemic necrosis and the dehiscence of one of the flaps. Our data showed that the 8 mm ischemic wounds showed clear evidence of impaired wound healing in our diabetic Yucatan miniswines. The 5 cm full-thickness ischemic wound model showed evidence of impaired wound healing with associated necrosis.

P406 - A Method for Sampling Vitreous Humor and Performing Ocular Histopathology Evaluation in the Same Eye of Nonhuman Primates

In nonclinical intravitreal injection (IVT) studies, collection of vitreous at necropsy for assessment of drug concentration generally renders the eye unsuitable for histopathology due to distortion/disruption of ocular tissues from the morphological collapse of the globe. We have developed a method where vitreous is sampled and the globe is then re-expanded or bisected, then fixed for histopathology evaluation. After removal of aqueous humor, vitreous volumes of 0.2 mL-0.8 mL were collected from eight eyes by inserting a 1-cc needleless syringe into a small scleral incision. Equal volumes of 10% neutral-buffered formalin were then injected into the scleral incision in order to re-expand the globe and speed ocular fixation. The incision site was sealed for some eyes using tissue adhesive, and all eyes were fixed in formalin. Histopathological evaluation demonstrated that eyes with up to 0.8 mL of vitreous collected had similar findings to intact control eyes when tissue adhesive was used to seal the incision. In comparison, eyes without sealant were notably more collapsed in shape, with greater distortion and disruption of globe tissues. Additional eyes were bisected using a Thomas blade following collection of 0.4 mL vitreous, and half the globe was fixed in formalin, and microscopic examination revealed relatively intact eye tissues competent for follow-on assessments. In summary, increased insight can be derived from preclinical IVT studies by simultaneous collection of vitreous fluid and tissues from the same injected eye, while potentially reducing animal use.

P407 - Thermoprofiling: A Key Tool for the Toxicological Evaluation of Vapor Products

Vapor products (e.g., e-cigarettes, tanks) have gained popularity with consumers searching for alternate tobacco products. With increased popularity comes increased regulatory and public health interest. As a fairly new product class on the marketplace, a comprehensive toxicological evaluation strategy is needed to ensure these products are acceptable for intended consumer use. Vapor products consist of e-liquid-containing cartridges attached to power units, which heat the e-liquid and generate an aerosol. Toxicological scrutiny of the aerosol, e-liquids, and structural materials is an essential part of a robust evaluation strategy. Thermoprofiling studies assess the exposure temperatures of various cartridge components during aerosol generation. Such studies, with the use of thermocouples and thermal imagers, allow for temperature measurements of the heat source and other structural materials. When thermoprofiling studies are coupled with other studies including 1) thermogravimetric, 2) pyrolysis, 3) chromatographic profiling, and 4) quantitation of aerosolized toxicants of concern, a clearer picture emerges on the e-cigarette profile. An example decision tree is presented, illustrating how these types of data can be integrated in a robust risk assessment paradigm. Such an approach allows for responsible, data-driven decision-making. Following this framework, materials that have the required functionality and the most favorable toxicological profiles can be selected for use in marketed products.

P408 - Comprehensive Analysis of Tissue-Enriched Plasma MicroRNAs in Cynomolgus Monkeys

To identify the utility of circulating microRNAs (miRNAs) as novel biomarkers for risk assessment in nonclinical toxicology study, we collected the background data on plasma miRNAs enriched in the liver (miR-122 and miR-192), skeletal muscle (miR-133a and miR-206), and heart (miR-1, miR-208a, miR-208b, and miR-499) obtained from male (n = 25) and female (n = 25) healthy cynomolgus monkeys. Total RNA, including miRNA, was extracted from 200 μL of EDTA-plasma using miRNeasy Mini Kit. The absolute number of the miRNAs was quantified by Taqman microRNA assay specific for the target miRNAs. A synthetic cel-miR-238 was used as exogenous control for normalization. The original data were not normally distributed, so that a logarithmic transformation was performed for each miRNA. Then, the value for two standard deviations below and above the mean was defined as reference range. Six out of eight miRNAs: miR-122, miR-192, miR-133a, miR-206, miR-1, and miR-499, were detectable quantitatively in cynomolgus monkey plasma by RT-qPCR, either with or without preamplification. Regarding the difference between upper and lower limits of the reference range, miR-133a showed more than approximately 900-fold variability, which was the largest among miRNAs examined, while miR-192 showed the smallest variability (8-fold). No significant gender, inter-study, inter-, and intra-individual differences were detected. In conclusion, it is important to use a standardized protocol to compare the results from different studies. These historical data regarding the variation of circulating miRNAs in cynomolgus monkeys is valuable in interpreting the assay results from nonclinical toxicology studies.

P409 - Characterization of Intra-Articular Dose Administration in the Stifle Joint of Rabbits for Toxicity Testing

Intra-articular administration is a standard therapeutic option for patients with rheumatoid arthritis, and pharmaceutical companies are increasing the research and development of injectable treatments. Therefore, there is an unmet need for reliable dosing models in toxicity safety testing. The rabbit stifle joint is attractive for toxicology testing of injectable treatments for rheumatoid arthritis because it is the largest synovial joint in the animal’s body and is equivalent in structure and function to the human knee. Intra-articular dosing in rabbits is one model currently being used in general toxicology studies to assess the safety of injectable compounds. In this study, intra-articular dose administration was characterized in the stifle joints of New Zealand white rabbits. An anesthesia regimen was developed for the intra-articular injection procedure. Volumes of ≤400 ╡L were successfully injected into the stifle joint of rabbits. Injection success was confirmed by visual inspection of contrasting agent within the articular space, with minimal leakage and no rupturing of the synovial membrane observed. A post-procedural analgesia regimen was developed to alleviate pain and discomfort. Bilateral and multiple injections were also explored and determined feasible. In addition, successful synovial fluid recovery without flushing was demonstrated. Histopathology of the stifle joint demonstrated that the injection procedure caused minimal inflammation or effects on the stifle joint or articular cartilage. In summary, intra-articular dose administration by injection to the stifle joint(s) of rabbits is a successful procedure for use in preclinical safety evaluation.

P410 - Potency Ranking of Dermal Sensitizing Chemicals Using the IVSA and Epics^® Skin Tissues

Human 3D reconstructed skin epidermal equivalents have been shown to release IL-18 in response to a wide range of dermal sensitizing chemicals. The concentration of these chemicals that produce greater than a threshold positive response (stimulation index, SI≥2.0) is correlated to their potency or strength in an in vitro sensitization assay (IVSA). In our experiments, 4-nitrobenzyl bromide (NBB) and 2,4-dinitrochlorobenzene (DNCB) were strong inducers of IL-18 secretion into the culture medium (SI-2=0.02% and 0.03%, respectively). The strong sensitizer p-phenylenediamine (PPD) had an SI-2 of 0.13%. Cinnamaldehyde (CA) (SI-2=0.33%) and isoeugenol (IE) (SI-2=0.56%) were moderate sensitizers, while eugenol (EU) (SI-2=0.75), resorcinol (RES) (SI-2=2.9%) and hexylcinnamaldehyde (HCA) (SI-2=8.1%) were weak sensitizers. Sensitizer potency ranked using an SI-2 as follows: NBB > DNCB > PPD > CA > IE > EU > RES > HCA, with NBB, DNCB, and PPD classified as strong; CA, IE, and EU as moderate; and RES and HCA classified as weak sensitizers. Of the total of 18 chemicals tested, seven were irritants and two were non-sensitizers (glycerol and isopropanol); of these, only chlorobenzene (50%) was incorrectly predicted as a sensitizer. epiCS^® gave an accuracy of 89% and sensitivity of 89%, and all other Cooper statistics (specificity, and negative and positive predictivity) values were 89%. In summary, measuring IL-18 release from 3D tissues allows for highly accurate and sensitive identification of dermal sensitizers. Also, the ability to rank-order potency of these chemicals based on SI-2 values of IL-18 secretion is a powerful tool for further classification into potency categories.

P411 - Progress on the Society of Toxicology: Colgate Palmolive 2014 Grant for Alternative Research: In Vitro Co-Culture Assay for Identification of Dermal Sensitizers

To better predict dermal sensitization by immunotoxicants, we report results on a cell co-culture model consisting of 3D reconstituted human epidermis skin model (RHE) cultured above media suspension of Langerhans cell-like differentiated CD34+ progenitor cells (pDCs). Test chemicals were topically dosed directly onto the RHE stratum corneum, mimicking the dermal route of exposure. We hypothesized that this co-culture model of human skin-like tissues and Langerhans-like cells could allow a more mechanistic and predictive in vitro sensitization assay. We measured two markers of sensitization response: IL-18 secretion into culture media and CD86 expression on pDCs. Initially, we exposed RHE tissues to chemicals overnight then transferred the conditioned media to pDCs cultured for an additional overnight period. This sequential treatment was compared to a co-culture method, where RHEs were cultured above pDCs and exposed to test chemicals by topical application to the RHE. Using the sequential treatment method, the strong sensitizer 4-nitrobutylbromide (NBB) induced a 1.1-fold increase in CD86. In contrast, NBB induced a 33.7-fold increase in CD86+ pDCs using the co-culture method. We next tested sensitizing chemicals (isoeugenol, hexylcinnamaldehyde, dinitrochlorobenzene) and non-sensitizers (lactic acid, phenol) in the co-culture model. Topical treatment induced dose-dependent increases of IL-18 secretion by all sensitizers, but not by non-sensitizers. The sensitizers and the non-sensitizer Phenol also induced significant increases in CD86 expression on pDCs; lactic acid did not. These studies demonstrate that RHEs cultured with pDCs show promise in a co-culture in vitro sensitization assay, allowing cell-cell communication between two critical skin-resident cell types, critical for dermal sensitization.

P412 - A Continuous Intravenous Infusion Qualification Study in Juvenile Sprague-Dawley Rats

The ability to administer drugs intravenously to neonatal rats is critical for the assessment of nonclinical development of some pharmaceuticals. The objective of this study was to assess the tolerance of continuous intravenous infusion of saline via a surgically implanted catheter following surgical procedures to neonatal Sprague-Dawley rats starting on day 21 postpartum for 35 days. Animals underwent surgery on day 21 postpartum, and a medical-grade catheter was surgically implanted so the tip of the catheter was located in the vena cava at approximately the level of the kidneys. The catheter was secured in place and exteriorized at the nape of the neck through a subcutaneous tunnel from the inguinal area to the dorso-cervical area. Following recovery from surgery, the animals were placed into two groups of 10 animals per/sex with a third (non-operated) group consisting of five animals/sex used as controls. Animals received 0.9% saline at a rate of 0.2 or 0.4 mL/hour via an infusion pump. Data were collected on mortality, clinical signs, body weights, food intake, clinical pathology, and histopathology. Food consumption and body weight were reduced moderately for pups that underwent surgical procedures in comparison to the controls. Preliminary results obtained from this study provided sufficient evidence that the surgical implanted catheter to weanling rats on day 21 postpartum was well tolerated at both dose rates and did not result in any unexpected background changes normally observed with surgically implanted catheters.

P413 - Use of Microsampling Procedures to Reduce the Number of Rodents Needed for Toxicokinetic Evaluations

Limitations of blood volume in rats and mice have traditionally resulted in the requirement for a large number of satellite animals in toxicity studies in order to obtain the number of blood samples required to allow for appropriate toxicokinetic evaluations. Several laboratories have developed techniques for obtaining microsamples (≤100 ╡L) of blood to obtain plasma or serum samples that can be analyzed using modern analytical methods. This presentation describes procedures that have been developed in our laboratory and results of a study performed to document their effectiveness in obtaining microsamples (50 ╡L) of acceptable quality from rats (Han Wistar) and mice (CD-1 and TgRasH2) of varying age and size and to demonstrate the numbers of samples that can be obtained with no adverse impact on animal health and survival. Results demonstrated that at least eight microsamples could be obtained from Han Wistar rats in a 24-hour period with no adverse effects. For mice, two to five microsamples could be obtained over 24 hours, depending on animal size, with no adverse effects. Plasma of appropriate volume and quality for microanalysis was obtained.

P414 - Refinement of Mini/Microsampling Collection Methods: How Far Can We Go?

The overall objectives for the use of microsamples are to reduce the number of animals required for parameter assessment, reduce animal stress, and increase the amount of data collected per animal by allowing more parameters and full profiles to be evaluated in a single animal. In addition to optimizing bioanalytical evaluations to allow for the use of microsamples, biomarkers and T-dependent antibody response (TDAR) can also be analyzed with minimal blood volumes. Microsamples are generally defined as a volume of up to 50 μL. Alternatively, minisamples, representing volumes of up to 80 μL, can be used when a larger volume of blood is required. The use of standard mini/microsampling approaches were found to be challenging with inconsistent collection. Consequently, modifications to the mini/microsampling collection methods were evaluated to assess reproducibility of volume of collection for samples of up to 80 μL with minimal invasiveness. The use of a butterfly with different gauges of needles and capillary types was assessed for collections in mice, rats, rabbits, dogs, and NHPs at different collection sites within each species to obtain optimal collection techniques. A list of assays and parameters that can be analyzed using mini/microsamples was also collated. In conclusion, collection of samples of volumes of up to 80 μL to allow mini/microsample analysis was easily performed on several species and allowed analysis of a number of biomarkers and TDAR assessments in addition to bioanalysis for toxicokinetic assessments. These represent key refinements of blood sample collection, also allowing subsequent improvements in study designs.

P415 - GFP+ Human Stem Cell-Derived Neurons Amenable to High-Content and High-Throughput Assays

High-throughput (HTS) and high-content screening (HCS) assays often utilize primary or stem cell sources, which are not amenable to large-scale screening and can require extensive cell culture and processing prior to imaging. Our goal was to develop GFP+ human stem cell-derived neural progenitors to provide a scalable solution to the previously labor intensive, single-endpoint, and hence limited nature of discovery and toxicology assays, while preserving the breadth and quality of data. Human neural progenitors derived from embryonic stem cell (WiCell h9) were modified with a nonviral vector encoding a green fluorescent protein (GFP) reporter gene using a selectable piggyBac™ system to produce the ArunA/Transposagen genetically modified hNP1GFP+TM line. Upon differentiation and selection, MAP2 expression increased to >90%, resulting in more mature neurons that exhibit significantly longer neurite-length post-thaw. A cytotoxicity profile was generated for the hNP1GFP+™ human neural progenitors to ensure response to toxins, and detection and quantification under assay conditions. Cell viability and cell migration, two established neurotoxicity endpoints, were measured in cultures exposed to a panel of five compounds. A CellTiter96™ viability assay and a high-throughput cell migration/proliferation assay were used. hNP1GFP+™ cells exhibit detectable dose-dependent response to toxins tested (cytotoxicity and cell migration), providing a sensitive cell-based human neurotox assay platform. Positive and negative controls produced results as expected, and can thus be used to normalize multi-plate data in larger screens. Overall, we conclude that hNP1™ human neural progenitor cells can be genetically modified and expanded to produce migration-assay ready cells.

P416 - Correlation Between Nasal Epithelial Injury and In Vitro Cytotoxicity Data Using a Series of Small Molecule Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitors

Obesity is associated with leptin resistance, which may arise because of increased expression of PTP1B in the hypothalamus. PTP1B inhibitors (lipophilic molecules with phosphate mimetic) were investigated as agents for treating obesity with intranasal (IN) dosing as a means for direct nose-to-brain delivery. Although food consumption was decreased in diet-induced obese (DIO) male C57BL/6 mice following acute IN dosing (via pipet) of various PTP1B inhibitors, nasal discharge was observed. Studies were conducted to investigate local effects on the nasal airway, develop SAR, and explore potential mechanisms. PTP1B inhibitors produced dose-dependent injury to nasal epithelia (squamous, transitional, respiratory, olfactory) in mice following one or two doses (≥0.03 mg/day). Compounds with cLogP > 3.0 were the most toxic. Whereas a pharmacologically-inactive analog of a PTP1B inhibitor-produced nasal injury (along with decreased food consumption), ketorolac (a marketed IN drug) produced no lesions at the same dose (0.3 mg/day) and only minor changes at 3 mg/day. Rat fibroblast CLR1213 cells were exposed to various PTP1B inhibitors, ketorolac, paraquat, or Na dodecylbenzene sulfonate (NDS; detergent) for 24 hours followed by measures of cytotoxicity. The most potent PTP1B inhibitors were similar to NDS, whereas ketorolac was the least toxic compound. The in vitro ranking of cytotoxic potency was similar to the in vivo results. In conclusion, PTP1B inhibitors injured nasal epithelium through a mechanism independent from PTP1B inhibition, likely due to nonspecific cytotoxicity such as disruption of cell membranes. In vitro cytotoxicity screens should be considered for evaluating a series of molecules intended for IN delivery.

GFP417 - Examining Environmental Effects on the Germline Epigenome

Many factors challenge our ability to assess the safety of chemicals. Of particular importance is the potential effect of chemicals on the germline epigenome, which can alter biological processes over several generations. There is a great need to explore the influence of environmental compounds on the epigenome and to develop methods that allow us to quickly and efficiently examine this question. We have established the use of the genetic model system, the worm Caenorhabditis elegans, as a relevant model for epigenetic and reproductive toxicity assessment. By taking advantage of the C. elegans genetic tools, we propose to comprehensively identify chemicals for their ability to disrupt the germline chromatin. We are making use of a worm strain where GFP is specifically epigenetically silenced in the germline. We previously showed that valproic acid, a well-known mammalian histone deacetylase inhibitor, disrupts the germline epigenetic state leading to the de-silencing of the transgene in the germline. Our current experiments test this concept further by exposing the worms to environmental compounds. We tested vinclozolin and BPA to analyze a disruption in maintenance and/or establishment of epigenetic marks. Results indicate that de-silencing effects last for at least three generations (up to three-fold compared to our DMSO control). Furthermore, most worms showing de-silencing in the first, second and third generation originate from worms showing de-silencing in the parental exposed generation, indicating that the effect is inherited. Thus, we conclude that valproic acid, vinclozolin, and BPA disrupt the germline epigenome over several generations in a heritable fashion.

GFP418 - Inhibitory Effects of Alcohol on Lung Epithelial Cells (Nuli-1) via Reduction in Expression of Vitamin D Proteins and Activation of Cathelicidin/LL-37

In the lungs, alcohol has been found to have potential adverse impact on epithelial cells by interfering with synthesis of vitamin D proteins and pulmonary antimicrobial peptides. The goal of this study is to understand the mechanism of this inhibition on lung epithelial cells by characterizing the expression of active and inactive vitamin D and deactivation of cathelicidin (LL-37) using Nuli-1 cells, an hTERT-immortalized human airway epithelial (HAE) cell line.

Nuli-1cells were grown in a serum-free medium: airway epithelial cell basal medium (ATCC PCS-300-030) with bronchial epithelial cell growth kit (ATCC PCS-300-040) additives. After culture, the cells were dosed with different concentrations (10, 20, 50, 80, and 100uM) of alcohol and diallyl disulfide (DADS). Subsequently, the cells were lysed and the homogenate was prepared for assay using 25(OH) Vitamin D EIA immuno-diagnostic-systems (ids) and 1, 25(OH2) Vitamin D EIA immuno-diagnostic-systems (ids) for inactive and active vitamin D proteins, respectively. We used human cathelicidin/LL-37 (Hycult Biotech) to quantify LL-37 proteins.

The concentrations of 25(OH)D3, in the alcohol-exposed cells were statistically reduced by 60%. In 1, 25 (OH)2D3, the concentrations were much more reduced (40%). In addition, the levels of LL-37 were statistically reduced (p<0.05) at higher concentrations of the alcohol. The opposite effects were observed with the addition of DADS.

Our preliminary results showed exposure to excessive alcohol has potential adverse health outcomes on the lungs by impairing the immune functions of vitamin D and LL-37 antimicrobial peptides in the pulmonary system.

IGP419 - In Vitro Antimicrobial and Antifungal Activity of Phospholipases A2 isolated of Bothrops asper Snake Venom from Panama

The increasing emergence of resistant microorganisms both in clinical, veterinary, and agricultural sectors makes it necessary to develop new strategies to control these pathogens. Some strategies are more effective than others. One of the best alternatives contemplated the search for new antimicrobial and antifungal agents with biological activity, with effective action so that the microorganisms do not generate resistance easily, and which have significant scientific, economic, and social impact.

Phospholipases A2 (PLA2s) are a superfamily of ubiquitous proteins in nature. Some phospholipases A2 present in snake venom (svPLA2s) and peptides derivatives thereof, exhibit strong fungicidal and bactericidal activity that might have pharmacological and biotechnological applications.

The proteins were isolated from the venom of Bothrops asper from Panama, using ion exchange chromatography CM Sepharose (0.05 M NH4HCO3 buffer, pH 8.1). The bactericidal activity of MTX-I and-II was tested on strains of E. coli (ATCC 29648). MTX-I and-II showed an inhibitory effect in the growth of the strains in a dose-dependent manner, when incubated for 30 minutes at 37°C in PBS, pH 7.4, containing 1% peptone. In addition to its lethal effect on bacteria, MTX-I and-II were effective on cultures of C. albicans (ATCC 24433). The mechanism of action involved in the cytotoxic effect has been poorly investigated. However, the toxicity of MTX-I and-II on bacteria appears to involve disruption of the cell membrane through cationic residues and hydrophobic amino acids at the C-terminus of the protein. Additional tests are needed to study the mechanism of action of MTX-I and-II on bacteria and fungi.

P502 - Serum Trace Element Levels in Children Receiving Antiepileptic Drug Therapy: A Cross-Sectional Study

Conventional antiepileptic drugs (AEDs) have been shown to alter the homeostasis of trace elements—copper, selenium, and zinc—in epilepsy patients. The newer AEDs are considered to have a more acceptable safety profile, but this confidence is somewhat guarded in the absence of long-term data. This cross-sectional study evaluated the status of trace elements in epileptic children treated with conventional and newer AEDs and compared them with healthy controls. The study included 92 epileptic children and 28 healthy controls. The participant distribution was as follows, Group I: phenytoin (PHT) monotherapy (n=35), Group II: valproate (VPA) monotherapy (n=30), Group III: valproate plus levetiracetam (VPA+LEV)(n=27), Group IV: healthy controls (n=28). Serum levels of seven trace elements, e.g. zinc, copper, magnesium, manganese, iron, selenium, and strontium, were determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Phenytoin monotherapy was associated with increased copper (1568.8μg/L vs. 1053.6 μ g/L, p=0.002) and strontium (37.0 μ g/L vs. 30.7 μ g/L, p=0.014) concentrations. Valproate monotherapy-treated children had decreased serum zinc (1010.5 μ g/L vs. 1242.9 μ g/L, p=0.003) and selenium levels (67.0 μ g/L vs. 84.7 μ g/L, p=0.02) as compared to healthy controls. However, in the VPA+LEV group, no significant differences were observed in trace element profile as compared to healthy children. Concluding, a significant difference in trace element levels in VPA- and PHT-treated epileptic children as compared to controls suggests a possible association between AED therapy and trace element alterations. However, levetiracetam, when used in combination with valproate, was not associated with these alterations. These findings further support its favorable adverse effect profile as compared to conventional AEDs.

P503 - Evaluation of Investigational Anticancer Compound INC280 in the Rat for Auditory Liability

Many anticancer compounds, such as those that are platinum-based, have demonstrated clear ototoxicity. With the development of new and more innovative types of anticancer treatment outside these classes of heavy metals, the risk of ototoxicity is expected to be limited. INC280, a c-MET inhibitor, is currently being investigated in clinical trials for solid tumors with cMET dysregulation. A preclinical ototoxicity study was conducted to investigate the potential risk for hearing loss after exposure to INC280. The ototoxicity study was designed to capture the potential risk by assessing the auditory brainstem response (ABR), otic histopathology of relevant otic tissues, and cytocochleogram of the organ of Corti. Since the toxicity studies for INC280 were conducted in rats, the ototoxicity study was also conducted in the same species as opposed to the more standard species (such as guinea pig). INC280 treatment, at 60 and 90 mg/kg/day for 6 weeks, produced no adverse changes in ABR, or in cytocochleogram and otic histopathology at terminal necropsy or at the end of recovery, suggesting that this compound had no evidence of ototoxicity in this study; the positive control (gentamicin) produced signs of ototoxicity in the validation study and the concurrent study.

P504 - Use of the Electromechanical Window in Anesthetized Guinea-Pigs to Assess Pro-Arrhythmic Risk in Early Drug Development

It is recognized that QTc interval prolongation is an imperfect predictor of Torsade de Pointes (TdP) liability. The electromechanical window (EMw) was recently proposed as an alternative risk predictor of TdP in preclinical models. The goal of this study was to differentiate the effects of 26 reference agents on the EMw and assess the predictivity of the EMw as compared to hERG inhibition or QTc interval prolongation alone to predict arrhythmic risk. Changes in EMw and QTc interval durations by 26 well-characterized pro- and non-arrhythmic agents were evaluated in the ketamine/xylazine anesthetized guinea pig model and quantified by measures of EMw EC-10 and QTc5. Arrhythmogenic risk was assessed using the hERG, QTc, and EMw indexes (calculated by dividing the hERG IC50, QTc EC10, or the EMw EC-10 by their respective free therapeutic Cmax plasma concentration) and compared to the known arrhythmic outcome in the clinic. An EMw shortening concomitant to a QTc interval duration increase was observed with drugs known to have high TdP risk liability. Non-torsadogenic compounds did not cause EMw shortening although some prolonged the QTc interval. The predictive capacity of the EMw index (88%) is greater than using QTc or hERG indexes (73 and 69%, respectively) to identify torsadogenic drugs. When the directional response of the EMw components (QLVPend or QT interval) is considered, the predictive capacity is further improved (>95%). The EMw complements QTc interval measurements in early drug development and increases the translatability over existing preclinical tools in predicting clinical arrhythmia(s).

P505 - Evaluation of Hepatotoxic Potential of Acetominophen and Amiodarone on Zebrafish Embryos

Liver is an essential organ for the detoxification of ingested chemicals and plays a pivotal role in various biochemicals and physiological processes; hence, liver toxicity is one of the major causes for the withdrawal of newly discovered molecules. Peculiar characteristics, viz. high-fecundity rate, transparent eggs, less maintenance cost, and livelihood in E3 medium without external feeding, make zebrafish (Danio rerio) a suitable vertebral model for the hepatotoxicity assay. On 3 days post-fertilization (dpf), embryos were treated with various concentrations of acetaminophen and amiodarone for 72 hours. Embryos from both treatment and control groups were observed daily for mortality and other abnormalities. No mortality was observed in the acetaminophen-treated groups. At 25 μ M, 15 μ M and 10 μ M amiodarone concentration, 100%, 78.5%, and 64.2% mortality was observed, respectively, and captured images of embryos were analyzed through Image J software to evaluate liver necrosis on 6 dpf. Significantly decreased mean pixel intensity was observed at 1000 μ M, 2000 μ M, and 5000 μ M acetaminophen concentrations compared to the control group. Significantly decreased mean pixel intensity was observed at 5 μ M, 7 μ M, and 10 μ ╡M amiodarone concentrations compared to the control group. Based on the results, it is concluded that NOAEL for amiodarone and acetaminophen is 3 μ M and 500 μ M, respectively. Both molecules are potentially hepatotoxic for zebrafish embryos, and the results of this study support the use of zebrafish embryos to predict hepatotoxicity in vertebrates, as the results obtained are in line with the proven hepatotoxic effects of both drugs in humans.

P506 - Molecular Mechanisms Underlying Development of Pectoral Fin Malformation in Zebrafish Embryos Treated With Thalidomide

In previous zebrafish developmental studies, we observed that thalidomide, but not lenalidomide or pomalidomide treatment of zebrafish embryos, resulted in pectoral fin malformations. The crucial window for development of fin malformations by thalidomide exposure is between 2 and 14 hours post-fertilization (hpf). In order to characterize mechanisms underlying these developmental defects induced by thalidomide, but not lenalidomide or pomalidomide, we conducted a toxicogenomic study in which zebrafish embryos (2 hpf) were treated with each compound for 6 and 12 hours. RNA was collected and data were generated using zebrafish microarrays. Lists of genes uniquely perturbed by each treatment were analyzed using the ToxWiz systems toxicology application. Interrogation of signaling pathways identified sonic hedgehog and Wnt pathways as being enriched only by the thalidomide treatment. Within the sonic hedgehog pathway, mRNA for genes, including GSK3b, SHH, SMO, and GLI-1/-2, were increased following thalidomide treatment. In additiona, mRNA for Wnt signaling genes NFKB1, WIF1, BTRC, and Wnt1 were also increased. Analysis of gene ontology molecular mechanism clusters found enrichment by thalidomide for genes in the embryonic limb and skeletal system morphogenesis networks. Perturbed genes related to limb morphogenesis included developmental genes and transcription factors such as HOXD10, WNT5A, and TP63. By contrast, gene lists from zebrafish treated with either lenalidomide or pomalidomide showed only weak enrichment in signal transduction pathways that did not map to limb/skeletal gene ontologies. Increased transcript levels of these developmental genes by thalidomide demonstrates the major contributing pathways responsible for fin malformation in zebrafish embryos.

P507 - The Genotoxic Potential of Pacritinib, a Novel Non-Myelosuppressive JAK2-FLT3 Inhibitor

Pacritinib is a novel, orally active, dual JAK2-FLT3 inhibitor being developed for the treatment of life-threatening myeloproliferative diseases. Anti-tumor activity of pacritinib has been demonstrated in tumor models driven by JAK2 or FLT3 mutations. The genotoxic potential of pacritinib was evaluated using the standard battery of studies as per ICH guidelines. In a bacterial reverse mutation test (Ames assay), there were no biologically significant increases in the number of revertant colonies in any of the five S. typhimurium strains (TA98, TA100, TA1535, TA1537, and TA102) tested, with or without metabolic activation. In an in vitro chromosomal aberration study using human peripheral blood lymphocytes, pacritinib did not induce structural chromosomal aberrations in the presence or absence of metabolic activation. In an initial in vivo bone marrow micronucleus assay in male and female Balb/C mice (n=3-5/sex/group/timepoint), single oral doses of 1,000 or 2,000 mg/kg resulted in significant increases in mean micronuclei counts in males at 48 hours post-dose, as compared with controls. However, due to the lack of dose-proportional increases in micronuclei and the low statistical power associated with this evaluation, a second in vivo bone marrow micronucleus assay was conducted in male and female ICR mice (n=10/sex/group/timepoint). In this study, pacritinib did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes following a single oral administration of pacritinib at up to 1,500 mg/kg, the highest dose tested. Overall, these data indicate that pacritinib lacks genotoxic potential and supports the chronic use of this agent in myeloproliferative diseases.

P508 - Sphingosine-1-Phosphate Lyase Inhibition Causes a Decrease in Heart Rate

Sphingosine-1-phosphate (S1PR1) agonists, such as fingolimod, provide strong validation for the arrest of lymphocyte egress from secondary lymphoid organs as an immune-suppressive therapy for inflammatory diseases. Such agonists typically elicit bradycardia, atrio-ventricular block, and hypertension. Lymphocyte sequestration can also be achieved through blockade of S1P Lyase (S1PL), with resulting elevation of S1P and activation of its receptors in lymphoid tissues. However, the cardiovascular effects of S1PL antagonists have not been reported. The purpose of this study was to investigate potential cardiovascular effects of S1PL inhibition and compare it to the effects of an S1PR1 agonist after acute i.v. infusion in anesthetized rats and 5-day oral administration in telemeterized rats. i.v. infusion of fingolimod (0.1, 0.3, 1.0 mg/kg/30 min) decreased HR and increased PR interval with no effects on BP, similar to clinical effects with a single dose administration. In contrast, i.v. infusion of the S1PL antagonist #31 (from Weiler et al., 2014, J Med Chem) did not cause changes in HR, BP, or PR interval in the anesthetized rat. Similar to results in anesthetized rat, fingolimod caused a decrease in HR after oral dosing (3 mg/kg) but also caused an increase in BP after 2 days of dosing. S1PL inhibition also caused a decrease in HR after oral dosing. These data suggest that the S1PL inhibition carries the same risk for decreased HR that SIP agonists have demonstrated in the clinic and would not provide immunosuppression with an improved safety profile over fingolimod.

P509 - Optimization of a Flow Cytometric Assay for the Dectection of Polyfunctional CD8+ Cytotoxic T-lymphocytes in Cynomolgus Macaque Whole Blood Based on CD107a Degranulation and Cytokine Secretion

While methods such as the chromium release and CD107a degranulation assays exist to measure CD8+ cytotoixic T lymphocyte (CTL) function in humans and rodents, there are no established ex vivo assays that can directly characterize changes in CTL function in cynomolgus macaques. Herein, we present the optimization of a whole blood flow cytometry assay that permits the simultaneous measurement of CD107a degranulation and intracellular INFg and TNFα secretion, for the characterization of circulating polyfunctional CTL in macaques. Initial experiments performed to determine CD107a expression in whole blood stimulated with Staphylococcus aureus enterotoxin B did not produce a robust response in macaque CTLs. Subsequent experiments (n=8) demonstrated that cross-linking of anti-CD3 along with co-stimulation (anti-CD28/anti-CD49d mAb) more robustly enhanced surface expression of CD107a (7.1±5.9% positive cells). Boolean analysis of stimulated cynomolgus whole blood (n=8) demonstrated that the highest proportion of responding polyfunctional CTLs were dual positive for CD107a expression and TNFα production (24 ± 7.6% positive cells). The addition of cyclosporine A at 1 uM to whole blood samples resulted in the reduction of polyfunctional CTLs co-expressing CD107a with TNFα, IFNg, or both (72, 96, and 97% decreases, respectively). Cyclosporin A also caused the loss of cytokine expression within a population of CTLs preserving expression of CD107a. In summary, we have optimized stimulation conditions for characterizing polyfunctional CTL in cynomolgus macaque whole blood, and further characterization of the effects of various immunosuppressive compounds in this setting will help assessing the value of this assay for nonclinical safety assessments.

P510 - Comparison of Left Ventricular Contractility to RR Interval Relationship in Freely-Moving Rats, Dogs, and Monkeys

The QT-RR relationship has been extensively examined in nonclinical models, but a paucity of data exist to characterize the left ventricular pressure (LVP)-RR relationship across species. LVP was continuously monitored by telemetry in conscious rats, dogs, and cynomolgus monkeys following transmural telemetry catheterization. The circadian changes to the LVP-RR relationship were evaluated in each species. dP/dt+ max and contractility index as a function of heart rate showed circadian effects in all species. Only cynomolgus monkeys had statistically significant different (p<0.01) slopes during the two periods, which was expected because of the significant circadian cycle changes documented in this species. The highest linearity of the LVP-RR relationship was observed in the rats (R2 = 0.75). Also, the variability between individual animals was the lesser in this species when compared to large animals. Our results suggest that the conscious rat LVP model is associated with a high sensitivity owing to low variability observed in this species when compared to Beagle dogs and cynomolgus monkeys. Characterization of the LVP-RR relationship for each individual animal prior to drug administration can be valuable to enhance interpretation of ventricular contractility data.

P511 - Gastroinstestinal Motility: Motility and Motor Migrating Complex (MMC) Evaluations in Rats, Dogs, and NonHuman Primates

Drug-induced effects on gastrointestinal motility are observed with a number of approved drugs, but available nonclinical assays for assessing such effects are limited in drug development. Fluoroscopic video imaging was used to assess oesophageal and gastric motility in rats using a buccal iodixanol radio-opaque meal. In Beagle dogs and cynomolgus monkeys, this methodology was used to monitor gastric and intestinal motor migrating complexes and gastric emptying times using 10x3 mm radio-opaque beads and/or barium solution. In dogs, gastric pH was sequentially measured from fasted animals or after a wet food meal. In rats, baseline oesophageal transit time was 2.29 ± 0.74 sec, and a significant increase was induced by atropine (1 and 5 mg/kg) and morphine (5 and 20 mg/kg). Baseline gastric motor migrating complexes (MMC) in nonhuman primates during the day was 2.46 ± 0.61 contractions per minute with an average gastric emptying time of a semi-solid meal at 242 ±71 min. Morphine (2 mg/kg) significantly increased gastric emptying time in cynomolgus monkeys. In dogs, feeding was shown to significantly increase gastric emptying time compared to the fasted state but also significantly decreased gastric pH for a period of 5 hours. MMC present circadian cycle patterns and controlling for daytime is an important factor in functional evaluations. Large animals presented individual MMC patterns, and comparison of drug-treated sessions with a time-matched control period from the same animal yielded optimal sensitivity. In large animals, a crossover study design is preferable whenever possible.

P512 - Left Ventricular Pressure (LVP) and Contractility in Freely-Moving Rats: The Application of Marginal Distribution Analysis

Chronotropic effects are the most frequent cardiovascular change in drug development. Interpretation of left ventricular contractility requires correction for heart rate and circadian cycle effects. Left ventricular pressure (LVP) was measured in conscious rats by telemetry with a transmural catheter. Pharmacological agents known for their positive inotropic (pimobendan, morphine and amrinone) or negative inotropic (atenolol and itraconazole) properties were administered to rats, and cardiovascular function was monitored for at least 24 hours after each dosing. The results were analyzed by the application of a marginal distribution curve on the contractility index and heart rate. In the rat model, pimobendan (3 to 30 kg/kg, po), morphine (2 to 20 mg/kg, sc) and amrinone (10 to 100 mg/kg, capsule) induced dose-dependent increases in the contractility index compared to control. Atenolol (1 to 100 mg/kg, po) decreased the contractility index in a dose-dependent manner. Similar to previous reports, the negative inotropic effects of itraconazole (10 to 100 mg/kg, po) on LVP were not observed in the rat telemetry model. The application of marginal distribution analysis to telemetry data at Tmax increased sensitivity compared to analysis for the entire 24-hour period. We explored a new analysis strategy to dissociate true positive or negative inotropic pharmacological changes from physiological effect of heart rate on ventricular contractility. The application of the marginal distribution curve was considered a valuable tool in the overall interpretation of drug-induced changes in cardiac contractility.

P513 - Neurological Effects of Fulranumab, an Anti-NGF Monoclonal Antibody, Following 15 Weeks of Treatment in Sexually Mature Cynomolgus Monkeys

Fulranumab, a fully human IgG2 anti-nerve growth factor (NGF) monoclonal antibody, is in clinical development as a biotherapeutic for pain. Antagonism of NGF function prevents hyperalgesia and allodynia in animal models of neuropathic and chronic inflammatory pain. Extensive neurological evaluations were performed in cynomolgus monkeys given 10 or 50 mg/kg/week fulranumab SC for 15 weeks followed by a 12-week recovery period. Fulranumab treatment had no effect on the central nervous system, density of intraepidermal nerve fibers in footpads, the size or number of myelinated nerve fibers in the sural nerve, or the ganglion volume, neuron number, or average neuron size within the thoracic dorsal root ganglia. Increased density of satellite glial cells in one or more ganglia of the sympathetic nervous system (SNS) was observed histologically at both dosages in both sexes at the terminal and recovery necropsies. Additionally, decreases in ganglion volume, total calculated neuron number, and estimated neuron size in the superior cervical ganglion, were observed in stereological evaluations. The decreases in total calculated neuron number and estimated neuron size were not associated with any indications of neuronal cell necrosis or SNS dysfunction, suggesting the decreased total calculated neuron number was due to under-recognition during the stereology evaluation. The apparent increase in the density of satellite glial cells was attributed to the reduced size of the neurons. Complete/partial recovery of these effects was demonstrated following the 12-week recovery interval. Overall, these changes were not considered adverse because they were not associated with any signs of SNS dysfunction.

P514 - Effects on Neurohistopathology in Sexually Mature Cynomolgus Monkeys of Acute and Subacute Administration of Fulranumab, an Anti-NGF Monoclonal Antibody

Fulranumab, a fully human IgG2 anti-nerve growth factor (NGF) monoclonal antibody, is in clinical development as a biotherapeutic for pain. In a 15-week repeated-dose study of fulranumab in cynomolgus monkeys, an increase in glial cell density was observed histologically in one or more ganglia of the sympathetic nervous system (SNS). Stereological evaluation showed decreases in ganglion volume, total calculated neuron number, and estimated neuron size in the superior cervical ganglion, but no indications of neuronal cell necrosis. To further characterize the time course of neurohistopathology in the SNS, an acute/subacute study was performed in sexually mature cynomolgus monkeys. Fulranumab was administered SC either as a single-dose injection or as five weekly injections of 1, 10, or 50 mg/kg/dose (n = 3/sex/time point/group). Animals were necropsied three days after the last dose (on day 4 for single-dose or day 32 for five-dose animals). Decreased neuron size and an increased glial cell density were observed histologically after a single dose of 50 mg/kg in females and at all dosages in both sexes after five weeks. There was a trend toward a decrease in ganglion volume and total calculated neurons by stereological evaluation in all fulranumab-treated groups in both subsets. There was no evidence of death, degeneration, necrosis, or apoptosis of neurons in the SNS. Decreased average neuron size was interpreted to be at least partially, if not completely, responsible for the morphologic findings (small neurons, increased glial cell density) and the trends towards decreasing ganglion size and total calculated neuron counts.

P515 - Investigating Concordance of Nonclinical and Clinical Toxicity Through Integrated Analysis

Since 1938 and the passage of the Food, Drug, and Cosmetic Act (FD&C Act) the FDA has reviewed the safety of new drugs prior to their marketing, resulting in a wealth of safety information on a huge number of drugs. We describe an approach wherein for a drug or group of drugs the safety data for both clinical and nonclinical studies are extracted from regulatory submission data. Using standard allometric scaling tables based on body surface area (BSA), the dose data for nonclinical species are converted to human equivalent doses (HED), then normalized to the daily mg dose as given by the FDA label. This fraction (DoseFrac) of the total daily dosage allows direct comparison between nonclinical and clinical safety findings. Furthermore, the clinical relevance is immediately apparent: toxicities observed at a DoseFrac of 1 are those occurring at therapeutic levels. For certain classes of drugs, e.g. many antivirals, nonclinical signals of hepatotoxicity are seen at a DoseFrac value of 1, predictive of the observed clinical hepatotoxicity. For tyrosine kinase inhibitors, the analysis shows that liver toxicity is observed at therapeutic doses for sunitinib, but is seen in animal models only at higher equivalent doses. By contrast, sorafenib treatment results in liver toxicity in animal models at HEDs considerably lower than the therapeutic dose. This approach allows for identifying those drugs where the endpoints in nonclinical studies do or do not predict the clinical safety concerns, and hence areas for research in improving methods for safety assessment.

P516 - Renal Tumors in 26-Week Tg.rasH2 Mouse Carcinogenicity Studies

We describe observations of previously unreported renal tubular adenomas and a carcinoma in seven mice from four recent 26-week Tg.rasH2 mouse carcinogenicity studies using mice from Taconic Biosciences, Germantown, NY. From 2004 through 2013 we observed no renal tumors in Tg.rasH2 mice in our studies. In four studies conducted in 2014, seven mice out of 800 (both sexes) had multiple adenomas and one carcinoma, present in either the same kidney or both kidneys. Most of the tumors, in control and high-dose mice, males and females, were amphophilic, solid and lobular in nature, but some exhibited cystic and papillary characteristics (photomicrographs to be presented). One tumor classified as a carcinoma was present with multiple renal adenomas in the same mouse. All animals with renal tumors reached terminal sacrifice. There were no other remarkable lesions present in these individual animals. Although these tumors had some similarities with the amphophilic-vacuolar renal tumors noted in Sprague-Dawley and Fischer 344 rats, which are thought to be familial in nature, these mice with tumors were not linked to any particular set of parents or animal barrier. We believe that these tumors developed randomly. The environmental conditions at our animal facility, including the type of feed, bedding, cleaning detergents, the lighting cycle, humidity, or temperature ranges were the same for all studies conducted at BioReliance. These renal tumors have now been added to our Tg.rasH2 neoplastic historical control database.

P517 - Safety Evaluation of Chronic Antithrombin Silencing in Nonhuman Primate and Expanded Therapeutic Index in an Hemophilia A Mouse Model

ALN-AT3, a subcutaneously administered investigational RNAi therapeutic targeting antithrombin (AT), is currently in clinical development (NCT02035605) for the treatment of hemophilia and other rare bleeding disorders. Once-weekly dosing of ALN-AT3 results in potent, dose-dependent, and reversible silencing of plasma AT in multiple preclinical species. The objective of these studies was to evaluate the tolerability of ALN-AT3 when administered over a chronic dosing period to normal (nondiseased) cynomolgus monkeys at therapeutically relevant dose levels and transgenic hemophilia A (HA) mice at exaggerated dose multiples. ALN-AT3 was administered weekly to monkeys for 39 weeks at 0.15, 0.3, and 0.5 mg/kg (35, 60, and 70% steady-state AT inhibition, respectively). ALN-AT3 was administered weekly to HA mice (35/sex/grp) for 26 weeks at 10 and 30 mg/kg (20- and 60-fold the mouse ED80). Potential effects were evaluated by clinical signs, body weight, clinical pathology, plasma AT, organ weights, and histopathology. ALN-AT3 was well tolerated in monkeys with no adverse changes to clinical condition or clinical or anatomic pathology parameters. ALN-AT3 was also well tolerated in HA mice. HA mice are inherently fragile and vulnerable to procedural stress, and as such there was a prominent incidence of early mortality in control animals (40% overall). In contrast, mortality was significantly reduced in ALN-AT3-treated groups (6% overall). Chronic administration of ALN-AT3 was well tolerated in monkeys, a relevant preclinical model. Chronic dosing at exaggerated dose levels (>90% AT inhibition) in HA mice was also well tolerated and resulted in a survival benefit relative to saline-treated controls.

P518 - Assessment of Safety and Efficacy of Ofatumumab in Human Immune System Mice

Ofatumumab is an FDA-approved anti-CD20 monoclonal antibody (mAb) for secondary treatment of chronic lymphocytic leukemia. Several mechanisms of target cell killing occur through anti-CD20 therapeutics including complement-dependent cell lysis (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and apoptosis. It is well known that there is a lack of animal models expressing human-specific receptors for many biologic therapeutics. Therefore, we evaluated the ability of human immune system (HIS) mice to recapitulate the effector functions and potential toxicities of ofatumumab. HIS mice were administered a single treatment at one of three doses: 2 mg/kg (low), 8 mg/kg (middle), and 15 mg/kg (high), or control (saline). Ofatumumab was diluted in saline to concentrations of 0.2–0.75 mg/ml prior to i.v. administration to reduce the likelihood of immediate hypersensitivity reaction. Samples for flow cytometric assessment and serum were taken immediately prior to administration and then at 1, 2, 5, 8, 11, 16, and 21 days post-treatment. Mice were euthanized at days 8, 11, 16, and 21 post-administration to determine the completeness and durability of B-cell depletion in blood, spleen, and bone marrow. Results showed that HIS mice were fully depleted of B-cells in all tissues sampled, with the low-dose group reconstituted at day 11 with high- and middle-dose groups remaining depleted at study end. Only isolated adverse effects such as weight loss in high-dose mice were observed. As the primary effector mechanism of this product in humans is CDC, it suggests that human immune system mice can mediate this effector mechanism.

P519 - Contractility Assessment in Conscious and Anesthetized Crl:CD Rats Instrumented with Telemetry for Assessment of Left Ventricular Function

Structural Family Therapy Training

Cardiovascular safety of new chemical entities is a crucial component in the evaluation for advancing lead candidates. The trend is to conduct these evaluations earlier in the process to remove any compounds from the pipeline that may have a risk to affect cardiac function negatively. There are a host of assays that are conducted in this early space as tools to evaluate cardiac safety. The rat is a common specie evaluated early in development with a focus on toxicology. Although the rat does not possess the IKr channel for assessment of ventricular repolarization, it is a proven specie for the evaluation of hemodynamic effects of test compounds. This study evaluated the effects of a positive inotrope (pimobendan) under both conscious and anesthetized conditions in rats instrumented with left ventricular pressure catheters via telemetry. Pimobendan was administered as both an intravenous (2 mg/kg) and oral formulation (6 mg/kg). The IV response evaluated under anesthetized state for 1 hour post-dosing, and the oral response evaluated for 6 hours post-dosing and then anesthetized from hours 6 to 7 for comparison, all while telemetry data were being collected continuously. Both conditions (anesthetized and conscious) detected the pimobendan increase in dP/dtmax after IV and oral administration. The anesthetized preparation (isoflurane) for the oral dosing resulted in muted responses for heart rate and mean arterial pressure compared to IV dosing, adjusting for anticipated Cmax. A rat, instrumented with left ventricular pressure, displayed increases in contractility under both conscious and anesthetized conditions following IV or oral administration.

P520 - Analyzing OEL Values for the Pharmaceutical Industry: A Novel Approach for Hazard Characterization of Unstudied Molecules

Many pharmaceutical companies have adopted the use of health-hazard bands (HB) as a tool for risk assessment and management for protection of workers in R & D and manufacturing. Banding provides an assessment of the relative hazards of actives, intermediates, and other materials when there are limited data to provide guidance for appropriate handling practices and engineering controls. Typical datasets to establish a band include, at least, pilot test data. A significant challenge in pharmaceuticals is associated with the handling of many new development compounds for which little to no data are available prior to candidate selection, especially for those molecules of very high potency or pharmacologic activity. The purpose of this investigation was to develop a grouping scheme for small and large molecules in very early development to inform safe workplace practices prior to derivation of HBs or occupational exposure limits (OELs). OEL values for approximately 1750 pharmaceutical actives were collected and grouped based on those OELs vs. internal HB ranges. Materials were subsequently parsed based on therapeutic indication and mechanism of action for additional precision in the grouping scheme. The handling practices and industrial hygiene expectations of each early development compound (EDC) group correlate to an internal health hazard category (HHC) scheme. Due to the differences between small and large molecule pharmacology, separate grouping schemes were developed. Using the scientific basis of OELs, this EDC grouping strategy facilitates and informs appropriate and safe practices during discovery and very early drug development plus other operations that require health assessments.

P521 - Protective Effect of Topical Brimonidine Administration on UVR-Induced Skin Tumors, Epidermal Hyperplasia and Cell Proliferation in the Skin of Albino Hairless Mice

The anti-tumor protective effect of the α2A-agonist brimonidine tartrate on UVR-induced skin carcinogenesis in hairless mice was evaluated in a topical 52-week photocarcinogenesis assay, with standard tumor and clinical observation endpoints. The effects of topical brimonidine administration on keratinocyte hyperplasia in an acute model of UVB-induced proliferation of basal keratinocyte and hyperplasia, with exposure UVB either immediately before or 1 hour after topical brimonidine administration, repeated 23 hours and 46 hours after the UVB exposure in albino hairless mice, was also evaluated. Topical administration of 0.8%, 1%, or 2% brimonidine significantly reduced UVR-induced tumor development by all tumor criteria. The unbiased median week to tumor was increased, tumor yield and incidences were reduced, and tumor yield was reduced, as compared with mice exposed to UVR alone or the vehicle and UVR. In addition, incidences of UVR-induced erythema, flaking, wrinkling, and skin thickening were reduced in a brimonidine-dependent manner. Brimonidine at 2% administered before or after acute UVB exposure decreased epidermal hyperplasia by 23% and 32% and epithelial cell proliferation by 59% and 64%, respectively. None of these effects could be attributed to UVR absorption of brimonidine. This study demonstrated that topical brimonidine delays the appearance of skin tumors in mice and UVR-induced epidermal hyperplasia. This reduction by brimonidine may be explained by a pharmacological effect upon keratinocyte proliferation. These results suggest new therapeutic uses of brimonidine for chemoprevention of hyperproliferative epidermal disorders, of UVR-photodamage and/or predisposition to cutaneous actinic keratosis, or squamous cell carcinoma.

P522 - The Toxicology and Toxicokinetics of ALN-PCSsc, an Investigational RNAi Therapeutic Targeting PCSK9

ALN-PCSsc is an RNAi therapeutic consisting of a small interfering RNA (siRNA) that has a triantennary N-acetylgalactosamine (GalNAc) ligand covalently attached to the sense strand. It targets proprotein convertase subtilisin/kexin type 9 (PCSK9). GalNAc facilitates hepatocellular uptake via the asialoglycoprotein receptor. PCSK9 binds to low-density liproprotein receptors (LDLR), leading to their degradation. Reduction in PCSK9 results in increased numbers of hepatocellular LDLRs and a subsequent reduction of serum LDL cholesterol. Single subcutaneous (SC) doses of 6 and 10 mg/kg administered to cynomolgus monkeys resulted in reductions in plasma PCSK9 (-80% from baseline) and LDL (≥ -60% from baseline) for at least 40 days post-dose. ALN-PCSsc was not genotoxic in bacterial, human lymphocyte or rat bone marrow micronucleus assays. There were no ALN-PCSsc-related changes in ECGs, hemodynamics, respiration rate, or body temperature in monkeys at 250 mg/kg every other week (3 doses); therefore, the no observed effect level (NOEL) for cardiovascular/respiratory safety pharmacology was ≥ 250 mg/kg. One-month and 15-week repeat-dose rat and nonhuman primate toxicology studies were conducted with once-weekly, once-every-other-week, and once-monthly dosing schedules. There were no dose-limiting toxicities and only minor hepatocellular changes. The no observed adverse effect levels (NOAELs) in both species were ≥ 250 mg/kg. Because of receptor-mediated uptake by liver, plasma exposure to ALN-PCSsc was low and ALN-PCSsc primarily distributed to liver but was also detected in kidney (the primary organ of excretion). These results provide large dose and liver exposure multiples supporting ongoing clinical trials.

P523 - Cerebrospinal Administration Using the Intracerebroventricular Route in the Beagle Dog

Cerebrospinal administration is a very effective way to cross the blood-brain barrier and deliver therapeutic compounds to the central nervous system, especially large molecules. Various catheter and access port systems have been used, each with their own technical constraints and limitations including loss of patency and/or risks of infection. In this study, we present a novel method to allow for delivery in the lateral ventricles of the brain. Six animals had a needle guide combined with an access port that was surgically implanted on the cranium. The position of the cannula was determined using pre-defined coordinates, and the entry site was formed by drilling through the cranium. The duramater was delicately cut with the bevel of a needle and the access port was positioned and secured to the skull. Correct alignment with the lateral ventricles was confirmed in surgery by the insertion of a spinal needle with a stylet to a pre-defined depth; spontaneous rise of cerebrospinal fluid (CSF) in the hub indicated successful positioning. Recovery from surgery without indication of adverse sequelae was 100% despite the invasiveness of the procedure. Animals were administered 0.9% sodium chloride for injection, USP twice weekly for 14 days. Endpoints evaluated included body weights, clinical observations, hematology, coagulation, clinical biochemistry, neurological examination, and gross observations at necropsy, as well as histopathology. The results obtained are comparable to historical data and confirmed that the intracerebroventricular (ICV) administration route was a safe approach to cross the blood-brain barrier and deliver drug candidates to the central nervous system.

P524 - An Integrated Analysis of Genomic and miroRNAExpression Profiles to Characterize the Specific Toxicological Signatures for Drugs with Similar Chemical Structure

Amiodarone and benzbromarone share similar chemical structure, but exhibit different liver injuries in the clinical practice. Benzbromarone has been withdrawn due to the severe liver injury, while amiodarone is still widely prescribed in the clinical practice. Therefore, it is important to explore the novel parameters that are able to distinguish the difference of hepatotoxicity between the two drugs and help develop biomarkers to predict the outcome of liver injury at earlier stages of drug discovery and development. With this aim, we analyzed both miRNA and mRNA expression data of liver samples in rats treated with amiodarone and benzbromarone at multiple doses and timepoints. We did an integrated analysis of the differentially expressed miRNAs (DEMs) and genes (DEGs) and their corresponding biological pathways. We identified signatures of genomics and miRNA expression, which may be used to distinguish differences in liver injuries related to these two compounds. In addition, enriched toxicological functions indicated that there exist compound-specific biological functions and pathways involved in their mechanisms of hepatotoxicity. In that regard, our findings demonstrated that exposure of amiodarone and benzbromarone engage in diverse compound-specific toxicological functions by altering specific miRNAs and genes. We conclude that an integrated analysis of both miRNA and mRNA expression profiles can explain differences of liver injuries for drugs that are structurally similar.

P525 - A Compatibility Study of Socialization and Jacketed Telemetry (JET) in Cynomolgus Monkeys

The past few years have seen growing efforts to socialize laboratory animals to enhance the animal welfare during toxicology studies. Jacketed external telemetry (JET) offers a non- or minimally-invasive approach to collect continuous ECG recordings during toxicology studies in order to integrate the evaluation of key safety pharmacology parameters such as heart rate and PR, QRS, QT, and rate-corrected QT intervals. Continuous JET recordings allow for the real-time visualization of drug-induced effects, which is not feasible with the snapshot ECG collections typically employed in repeated-dose toxicology study designs. The current objective was to evaluate the reference effects of moxifloxacin on cardiovascular parameters of five socially housed cynomolgus monkeys using JET telemetry. As anticipated, moxifloxacin administration of 30 and 100 mg/kg resulted in a dose-dependent prolongation of ∼6 and 17 ms in the rate-corrected QT interval, respectively. The study demonstrates that high- quality cardiovascular data and detection of drug-related changes in ECG parameters can be obtained using JET in socially housed primates, thus enabling the incorporation of safety pharmacology endpoints on a repeat-dose toxicology study.

P526 - The Influence of Caging and Environment on Lymphocyte Subpopulations in Cynomolgus Monkey

Nonhuman primates (NHP) are routinely used as a nonrodent animal model during preclinical safety evaluation on the basis of proven suitability and comparability between NHP and humans. The cynomolgus monkey (Macaca fascicularis) is the preferred NHP model used in routine safety evaluation of biopharmaceuticals. There is an expectation when using the NHP that an evaluation of changes in lymphocyte subpopulations of both peripheral blood and lymphoid tissue using flow cytometry as well as other standard immunological endpoints be included in protocols. However, there is a high degree of inter- and intra-group variability in lymphocyte marker cell counts in NHP studies, and this is often attributed to ‘stress.’ Given the restricted supply and relatively small numbers of animals used in toxicology studies, it is critical to gain a clear understanding of the changes that occur in animals during the period before dosing commences in order to establish a clear baseline against which to identify possible effects of the test substance. In this study an assessment of immune phenotype marker baseline data from over 450 animals was undertaken, focusing on the type of caging and environmental factors that have been implicated as stressors for primates. Analysis of these data has indicated that the type of primate caging can influence the immune response. It is considered that the enriched environments of gang enclosures, including opportunities for foraging, climbing and swinging, and the provision of other objects to stimulate interest, create a reduced-stressed environment.

P527 - The eTOX Data-Sharing Project—From Vision to Reality

The eTOX project aims to collect, extract, and organize preclinical safety data from pharmaceutical industry legacy study reports and publically available toxicology data into a searchable database to facilitate data mining and the development of innovative in silico models and software tools to predict potential safety liabilities of small molecules. The eTOX-consortium consists of 13 pharmaceutical companies, 11 academic institutions, and 6 SMEs working together under the sponsorship of the Innovative Medicines Initiative (IMI) since 2010. The participating partners embrace expert knowledge in computational modeling, toxicology, pathology, and database design, liaising within the project in an integrative working environment.

After establishing an effective data sharing intellectual property (IP) protection within an ‘honest broker’ approach, the project was able to compile a unique, well-curated dataset of currently more than 6,000 study reports, corresponding to ca. 1,800 test compounds. Treatment-related findings have been classified within the database, reflecting the interpreted study outcome of every report. A suite of ontologies, built through OntoBrowser and now released by eTOX in the public domain, enable the user to directly compare observed effects or toxicities of chemically similar structures (read-across).

The newly developed in silico tool eTOXsys is a single-user interface, which manages search queries on the high-quality preclinical database and organizes requests to a steadily growing collection of independent prediction models. Aspects of IP rights for data sharing, definition of ontologies, design of database structure, development of in silico models, data analysis, validation, and sustainability, as well as exemplary use case examples, will be discussed.

P528 - Human Pluripotent Stem Cell-Based Assay Accurately Predicts Developmental Toxicity Potential of Compounds with Different Mechanisms of Toxicity

Structural Family Therapy Pdf

Current worldwide initiatives to screen thousands of chemicals currently in use for toxicity potential, such as REACH and Tox21, require inexpensive and high- throughput in vitro models to meet their goals. Development of innovative in vitro toxicity screening assays aimed at reducing or replacing the use of animal models in compound safety testing is critical to meet the safety requirements for multiple industries. We utilized a previously developed, predictive, in vitro human pluripotent stem cell-based assay to assess the developmental toxicity potential of a wide range of chemicals (i.e., pharmaceutical, environmental, and industrial compounds) that affect differing developmental lineages. The assay is currently in use by the United States Environmental Protection Agency (EPA) to screen the ToxCast chemical library in support of Tox21. Human pluripotent stem cells were exposed to eight or nine concentrations of over 75 compounds with known in vivo developmental toxicity outcomes. Spent media was collected to measure changes in biomarkers of developmental toxicity (ornithine and cystine), and cell viability was measured. The assay predicted the developmental toxicity potential across a diverse set of chemotypes with 83% accuracy when compared to known in vivo toxicity (79% sensitivity, 89% specificity). Additionally, the assay was able to predict the developmental toxicity potential of compounds adversely affecting all lineages of development (e.g. neural, cardiac, skeletal). The assay’s concordance with reported developmental toxicity in vivo demonstrates the utility of the assay for supporting initiatives, such as Tox21, in prioritizing compounds for further testing.

P529 - Susceptibility Factor for DILI: TAK-875 Alters Bile Acid Homeostasis In Vitro and In Vivo

TAK-875, a GPR40 agonist in development for Type 2 diabetes, was terminated in Phase 3. Nonclinical toxicology studies demonstrated liver effects in rat (clinical chemistry) and dog (TAK-875 crystal formation in biliary tract) at high exposures; the mechanistic relevance to humans is unclear. Additional in vitro and in vivo studies were performed to elucidate the liver signal. TAK-875 inhibited hepatobiliary transporters in vitro (BSEP, MRPs, NTCP) at low micromolar concentrations. In single- or repeat-dose studies, TAK-875 caused a dose-dependent increase in serum total bile acid (BA) in mouse, rat, and dog at exposures exceeding the clinical exposures. Analysis by LC/MS demonstrated an increase in conjugated and unconjugated individual BA. There were no microscopic findings in rodent studies, which suggests that the increase in BA may be due to transporter inhibition. Dog BA elevations preceded liver injury that was characterized by ALT, AST, and ALP increases and portal to periportal granulomatous inflammation with neutrophils and giant cells around intralesional crystalline. There was a trend of decreasing BA concentrations in dog bile, which may affect solubility thresholds of TAK-875. In summary, both in vitro and in vivo studies demonstrated that TAK-875 can affect hepatocyte transporters. It is unclear whether the BA increase observed in vivo is due to inhibition of efflux/influx transport. Transporter inhibition could be a susceptibility factor for TAK-875 liver injury by a direct mechanism leading to a buildup of toxic BA within the hepatocyte, or by an indirect mechanism where altered bile could lower TAK-875 solubility and crystallization threshold.

P530 - In Vitro Characterization of Immune Potential for Long-Acting Ophthalmologic Drug Delivery Carrier

The challenge with ophthalmologic treatment for diseases such as age-related macular degeneration (AMD) is the requirement for monthly or bi-monthly intravitreal (IVT) injections. To reduce the frequency of injections, biocompatible and polymer-based materials such as poly (lactic-co-glycolic acid) (PLGA) microspheres have been considered for long-acting drug delivery applications. However, these materials have the potential to trigger inflammation and induce foreign body reaction (FBR) in various tissues including the eye. To further investigate the soluble mediators of FBR and its mechanism of action, we have developed in vitro assays to characterize their immune potential. A total of 14 cytokines/chemokines were examined from human peripheral blood mononuclear cell (PBMC) cultures (N=3) with PLGA microsphere and other biomaterial, Polymer A. In addition, their potential for inducing macrophage fusion (in vitro mimicry of FBR) and cytokine/chemokine release from macrophages was assessed. Despite the slight differential immune profile of biomaterials, the temporal and quantal immune response suggested predominant innate immune responses, with upregulation of IL-1b, IL-8, TNFa, MCP-1, MIP-1a, MIP-1b, and RANTES. Furthermore, the in vitro data primarily correlated with in vivo histopathology and in situ hybridization results from the rabbit eye administered with these same biomaterials. Overall, the in vitro results suggest in vivo translatability and immune players associated with FBR in the eye. Ultimately these in vitro assays may be useful as potential screening tools for long-acting ophthalmologic drug delivery system and help improve current chemistry to minimize inflammatory responses.

P531 - Validation of a Multiplex Bead-Based Immunoassay (Luminex) for Analysis of Th1/Th2/Inflammatory Cytokines in Cynomolgus Monkey Plasma

A study was undertaken to validate a multiplex bead-based immunoassay using the xMAP technology for the quantitation of IL-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/23(p40), IL-13, IL-15, IL-17, IL-18, IFN-α, IFN-g, TNF-a, GM-CSF, G-CSF, MCP-1, and MIP-1β in monkey EDTA plasma. The standards and quality control samples were assayed using a custom multiplex bead-based immunoassay kit (Millipore). The validation parameters assessed included specificity, selectivity and sensitivity, linearity of dilution/parallelism, intra/inter assay precision, and accuracy and stability. To further evaluate sensitivity of the method, cytokine detection in plasma following administration of an anti-CD3 antibody to several monkeys was performed. Validation parameters assessed met the acceptance criteria for 19 of the 20 cytokines. To further evaluate the sensitivity of the method, plasma samples from stimulated cynomolgus monkeys, with an anti-CD3 monoclonal antibody, were analyzed and compared to plasma samples from unstimulated monkeys. Increases in IL-10, IL-1RA, IL-6, MIP-1β, IFN-γ, IL-2, TNF-α, IL-5, and G-CSF were observed following treatment with the anti-CD3 mAb. Because of differences in the scheme of dilution, MCP-1 analysis will be performed separately. The validation data demonstrated that this multiplex bead-based immunoassay is suitable to measure cytokine endpoints from preclinical studies. In addition, these results show that the method is sensitive enough to detect even subtle changes in cytokine profiles in cynomolgus monkeys that may occur not only in disease state, but also as a test article-related effect in a preclinical trial.

P532 - In Vitro Cytokine Release Assay in Risk Assessment: Comparison of Whole Blood and PBMC Methods

Cytokine release syndrome is a potential serious complication associated with the clinical use of monoclonal antibodies (mAbs). Here, we compared two common formats of cytokine release assays (CRA) using either whole blood (WB) or peripheral blood mononuclear cells (PBMCs) from the same donors in a soluble assay setup. In each format we exposed cells to several clinically relevant mAbs known to induce cytokines to varying degrees. Cytokine levels (IFNγ, IL-1β, IL-2, IL-6, IL-8, TNFα) were determined in supernatants using multiplex Luminex kits. The goal was to characterize the cytokine release response in each assay format using the same donors and note similarities and differences in response to the chosen antibodies, thus allowing a more informed decision when choosing a format for future studies. When comparing the clinical source of anti-CD52 (alemtuzumab) to the commercially available clone (YTH34.5), both sources of anti-CD52 demonstrated a similar response across donors in both assay formats. Exposure to anti-CD28 was more variable with IL-2 levels greater in WB than PBMCs, while TNFα and IL-8 levels were higher in PBMCs than WB. Our results indicate that the anti-CD52 clone, YTH34.5, is a potential substitute for alemtuzumab to use as a comparator in CRAs and that generally PBMCs react in a more sensitive fashion than WB to the selected mAbs. However, in choosing a CRA approach for novel molecules, it is important to consider the expected MOA, the cellular expression of the target, and the potential for differing cellular components to contribute to a response.