American College of Toxicology 2016 Annual Meeting Poster Abstracts

Note: Poster abstracts are indicated with letters preceding the poster number: Poster (P), Student Travel Award (STP), and International Travel Grant (IGP).

100 Series—General Toxicology

P101 - qEEG as a Biomarker of Animal Welfare: Assessing the Influence of Different Caging Environments and Ventilation Protocols in Cynomolgus Macaques (Macaca fascicularis)

S. Authier^1,2, E. Boulay^1,2, M. Pouliot¹, M. Accardi¹, and R. Forster¹

¹CiToxLAB, Laval, Québec, Canada

²Faculty of Veterinary Medicine, University of Montréal, Saint-Hyacinthe, Québec, Canada

Environment has an impact on animal welfare, but limited tools are available to quantify animal well-being. Monitoring of cardiovascular parameters could not identify beneficial effects of social housing in cynomolgus macaque (Macaca fascicularis). This study evaluated the effect of caging configuration and ventilation on biomarkers of anxiety in cynomolgus macaques (n = 6). Stress levels were monitored by means of electroencephalographic (EEG) parameters, while each macaque was either single- or pair-housed with a familiar or an unfamiliar cage mate in either an AAALAC compliant (12 days) or ETS-123-compliant cage (12 days). On occasion, extraneous stressors were introduced (eg, an unfamiliar staff) to determine the response to spontaneous stressors. Ventilation in the room was adjusted from 6 to 12 air changes per hour with continuous monitoring of air quality (ammonium, CO₂, and total particle). Quantitative electro-encephalography (qEEG) suggested that single-housed animals displayed higher levels of anxiety biomarkers throughout the experiment with progressive adaptation to the isolated state. Pair-housed animals showed signs of decreased stress/anxiety with the presence of a familiar cage mate. An unfamiliar cage mate was associated with variable results, as some animals showed an initial increase in biomarkers of anxiety and stress, while others presented reduced stress with an unknown cage mate. Our results suggest that qEEG can be used to evaluate well-being in cynomolgus monkeys.

P102 - Adaptive Response in Mouse Bone Marrow Stromal Cells Exposed to 900 MHz Radiofrequency Fields: Impact of Poly ADP-Ribose Polymerase (PARP)

Y. Cao¹, J. Tong¹, M. Geng¹, Q. He¹, L. Zong¹, Y. Sun¹, and J. Li¹

¹Soochow University, Suzhou, Jiangsu Province, China

This study examined whether nonionizing radiofrequency field (RF) exposure is capable of inducing poly ADP-ribose polymerase (PARP) in bone marrow stromal cells (BMSCs) and whether it plays a role in RF-induced adaptive response (AR). Bone marrow stromal cells (BMSCs) were exposed to 900 MHz RF at 120 μW/cm² power intensity for 3 hours/day for 5 days and then challenged with a genotoxic dose of 1.5 Gy gamma-radiation (GR). Some cells were also treated with 3-aminobenzamide (3AB, 2 mM final concentration), a potent inhibitor of PARP. Unexposed and sham (SH)-exposed control cells as well as positive control cells exposed to gamma radiation (GR) were included in the experiments. The expression of PARP mRNA and its protein levels as well as single strand breaks in the DNA and the kinetics of their repair were evaluated several times after exposures. The results indicated the following: (a) cells exposed to RF alone showed significantly increased PARP mRNA expression and its protein levels compared with those exposed to SH and GR alone. (b) Treatment of RF-exposed cells with 3AB had diminished such increase in PARP. (c) Cells exposed to RF + GR showed significantly decreased genetic damage as well as faster kinetics of repair compared with those exposed to GR alone. (d) Cells exposed to RF + 3AB + GR showed no such decrease in genetic damage. Thus, the overall data suggested that nonionizing RF exposure was capable of inducing PARP, which has a role in RF-induced AR.

P103 - Mitochondrial Neurotoxic Pesticides Promote Epigenetic Dysregulation by Histone H3 and H4 Hyperacetylation in Dopaminergic Neuronal Model of Parkinson Disease

A. Charli¹, D. Luo¹, M. Langely¹, M. Ay¹, H. Jin¹, A. Vellareddy¹, A. Kanthasamy¹, and A. Kanthasamy¹

¹Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, IA, USA

Growing evidence implicates continual exposure to environmental pesticides in the etiopathogenesis of Parkinson disease (PD). Previously, we demonstrated that dysregulation of histone acetylation homeostasis induced by environmental pesticide exposure increases the susceptibility of dopaminergic neurons to degenerative processes. Using confocal microscopy and Seahorse bioanalyzer, we recently showed that exposing N27 dopaminergic neuronal cells to the greenhouse pesticide tebufenpyrad induced significant neurotoxicity by affecting mitochondrial structure and function. These observations promoted us to examine whether tebufenpyrad-induced mitochondrial dysfunction influences epigenetic histone acetylation. Western blot analysis revealed that exposure of N27 cells to tebufenpyrad (3 μM) drastically increased histone (specifically H3 and H4) acetylation. The classic mitochondrial complex-1 inhibitor rotenone also induced histone H3/H4 acetylation. Immunocytochemical analysis revealed that tebufenpyrad and rotenone exposure preferentially acetylate lysine sites K23 and K5 on H3 and H4, respectively. 3-D reconstruction of confocal microscopy images performed using IMARIS demonstrated vivid architectural changes in nuclear morphology. Further analysis by means of mitochondria-defective N27 dopaminergic stable cells obtained by CRISPR/Cas9-based knockdown of TFAM protein also revealed hyperacetylation of histone H3/H4 compared to the vector control stable cells. Similar results of histone H3/H4 hyperacetylation were observed in the transgenic MitoPark mouse model of PD that is deficient of mitochondrial function. Importantly, western blotting and immunohistochemical analysis unraveled hyperacetylation of histone H3/H4 in the nigral dopaminergic neurons from PD brains. Collectively, our data reveal a novel interplay between mitochondrial dysfunction and epigenetic dysregulation of core histone acetylation, which together may play a critical role in the etiology of environmentally linked PD.

P106 - Evaluation of the Effect on Heart Rate When Providing Supplemental Treats After Oral Dose Administration in the Minipig

J. Sentz¹, M. Miyamoto¹, J. Sheehan¹, A. Camacho¹, and C. Dziak¹

¹Envigo, Somerset, NJ, USA

In cardiovascular (CV) safety pharmacology minipig studies, it has been observed that feeding can cause an increase in heart rate (HR), which may remain elevated for several hours. To limit the impact of feeding on HR, and to ensure the collection of high quality CV data, the use of treats such as apples or pears following dose administration must be restricted. However, treats have been shown to provide positive reinforcement to dosing procedures and to improve animal welfare. So, the suitability of apple juice (AJ) as a post-dose treat on minipig studies was investigated. To assess the effect of AJ on HR, 2 tests were performed. In each test, data were collected continuously for 26 hours. After 2 hours of data collection had elapsed, AJ was presented to 4 minipigs for ∼15 seconds. In the first test, AJ was presented in the absence of restraint and dose administration. In the second test, AJ was presented following restrained oral gavage administration of tap water. The data acquired from these tests were compared to 2 data sets previously collected from the same minipigs in which AJ was not presented, restraint occurred, and an oral gavage dose of vehicle control was either administered or not. Review of the data in its entirety revealed that the presentation of AJ has a minimal effect on HR in the minipig compared to restraint and associated dosing procedures. It would appear then that AJ is a viable candidate for positive reinforcement on CV safety pharmacology minipig studies.

P107 - Toxicity Study of Mycosynthesized Silver Nanoparticles on Pseudomonas Putida.

I. Gupta^1,2, A. Anderson³, and M. Rai^1,4

¹Nanobiotechnology Laboratory, Department of Biotechnology, S.G.B. Amravati University, Amravati, Maharashtra, India

²Department of Biotechnology, Institute of Science, Aurangabad, Maharashtra, India

³Department of Biology, Utah State University, Logan, UT, USA

⁴Laboratório de Química Biológica, Instituto de Química, UNICAMP, Cidade Universitária “Zefferino Vaz,” Barao Geraldo, SP, Brazil

Silver nanoparticles have unique optical, electrical, and thermal properties as compared to their bulk counterparts. They are being applied for various purposes ranging from photovoltaic systems and chemical sensors to biological systems. Specifically in biological systems they are mostly used as antibacterials. But with rise in their use, there is risk of development of hazardous effects to humans and the environment. With reference to many studies performed in vitro and in vivo, there are various toxicological effects of silver nanoparticles on eukaryotes and prokaryotes. Therefore, there is an urgent need to study the toxicological aspects of silver nanoparticles to microbes that are helpful for maintaining the environment. In the present study, we made an attempt to assess the toxicity of biologically synthesized silver nanoparticles on Pseudomonas putida, a beneficial soil microbe. For this study, we synthesized silver nanoparticles by using various fungi, followed by their characterization through UV-vis spectrophotometry, FTIR, X-ray diffraction, nanosight LM20—a particle-size distribution analyzer—and TEM. By using soil bacterial biosensors generated in P. putida, we investigated the toxicity of silver nanoparticles. The results suggest inhibition of silver nanoparticle-exposed P. putida, thereby showing the hazardous effect on soil environment.

P108 - Complementarity of In Vitro and In Vivo models for the Evaluation of the Effects of Pharmacological Substances on Gastric Tissues

S. Goineau¹, S. Bezivin¹, L. Barrais¹, C. Legrand¹, L. Lecouflet¹, E. Hayes¹, and V. Castagne¹

¹Porsolt, Le Genest-Saint-Isles, France

Gastric mucosa is frequently exposed to various gastric irritants, and there is a continuing requirement to develop new gastroprotective agents. The present study compares the effects of sucralfate and rebamipide in in vitro and in vivo indomethacin-induced gastric damage models. For the in vitro approach, rat gastric mucosal cells were incubated with vehicle, test, or reagent substances. The release of lactate dehydrogenase (LDH) and mitochondrial function were measured. For the in vivo approach, rats were administered with the vehicle or test substance before an acute indomethacin dose. Subsequent gastric lesions were macroscopically examined. In vitro, indomethacin induced gastric cell injury (decrease in mitochondrial function and LDH release) in a concentration-dependent manner, starting from 3 mM. Indomethacin-induced cell damage was inhibited by 3 and 5 mg/mL sucralfate (LDH release) and by 0.3 and 1 mM rebamipide (mitochondrial function and LDH release). In contrast, sucralfate accentuated indomethacin-induced decrease in mitochondrial function. In vivo, indomethacin (30 mg/kg) caused the occurrence of gastric ulcers 6 hours post-dosing. Sucralfate and rebamipide (300 mg/kg) exhibited gastroprotective effects against indomethacin-induced gastric lesions (decreased number of gastric ulcers: −50% P < 0.05 and −22% NS and reduced length of gastric lesions: −62% P < 0.05 and −29% NS). These findings suggest that cultured gastric cells offer a promising primary tool for evaluating the cytotoxic or protective effects of compounds. However, data from models are still needed to confirm in vitro data. Using both approaches provides a more comprehensive insight into the effects of test compounds on the gastric mucosa.

P110 - Emesis in Toxicity Studies in Canines: Impact of Choice of the Vehicle and Route of Administration

P. Limaye¹, J. Flavin¹, R. Thiher¹, and D. Dandekar¹

¹Xenometrics LLC, Stilwell, KS, USA

Canines are used as a nonrodent species for toxicity studies. Emesis is a common clinical finding in dogs and can be a confounding factor in determination of toxicity. In the present study, we evaluated the correlation between the incidences of emesis and growth rate as well as clinical pathology parameters in beagle dogs. Data were collected from a total of 22 commonly used nonclinical vehicles covering 24 subchronic canine toxicity studies conducted in our laboratory via various dosing routes, including oral (gavage, dietary, or capsule), intravenous (IV) bolus, or infusion. The data indicated that emesis occurred independent of any alterations triggered by the route of dosing. In general, sporadic incidences of emesis were seen in all the 24 studies, and the percent incidence of emesis ranged from 3% to 262% in males and 0% to 228% in females. No correlation was found between emesis incidence and body weight gain or food consumption. There were no changes in the hematology and serum chemistry profiles. In conclusion, our data clearly indicate that, although there were differences in the emesis incidence across the various vehicles utilized, the most important factor for selecting a dosing formulation remains pharmacokinetics (exposure) of the test article using a specific vehicle. This is the first database, to the best of our knowledge, that provides a direct correlation between the nonclinical vehicles and associated incidences of emesis and its comparison with the general health status of the beagle dogs during the course of repeat-dose toxicity studies.

P111 - Developmental Toxicology Validation Program Evaluating the Teratogenic Effects Produced When 6-Aminonicotinamide or Acetylsalicylic Acid Are Administered to Pregnant New Zealand White Rabbits or Sprague-Dawley Rats, Respectively, on Specific Gestation Days

C. Murphy¹, M. Dorato¹, S. Goodnight¹, and S. Harris²

¹Smithers Avanza Toxicology Services, Gaithersburg, MD, USA

²Stephen B. Harris Group, San Diego, CA, USA

Known teratogens have been utilized to generate fetal abnormalities to support developmental toxicology training programs. These technical validation studies investigated the teratogenicity of 6-aminonicotinamide (6-AN) in New Zealand white rabbits and acetylsalicyclic acid (ASA) in Sprague-Dawley rats. Time-mated rabbits were dosed orally once on gestational day (GD) 9 or 10 with 8 mg/kg 6-AN (controls: 40 litters (L), 345 fetuses (F); GD 9: 12 L, 49 F; GD 10: 11 L, 51 F). Time-mated rats were dosed once orally on GD 9 or 10 with 500 or 625 mg/kg ASA, respectively (controls: 64 L, 755 F; GD 9: 11 L, 123 F; GD 10: 10 L, 115 F). A laparohysterectomy was performed on GD 21 (rats) and GD 29 (rabbits). Fetuses were weighed, and external, visceral, and skeletal examinations were performed. Fetal weights decreased and postimplantation loss increased in dosed groups. Primary external abnormalities in rabbits were craniofacial, limb, and tail defects. Kidney and lung visceral abnormalities were present, but cardiovascular defects (persistent truncus arteriosis) were most prevalent. Rabbits dosed on GD 9 showed a higher incidence of skeletal malformations than GD 10 (absent and/or fused limb bones, ribs, and vertebrae). ASA-dosed rats presented externally with an increased frequency of neural tube, abdominal wall, tail, and craniofacial defects. Visceral malformations were mainly cardiovascular (absent aortic arch and truncus arteriosus). Skeletal abnormalities included fused and/or absent rib, skull, and vertebrae. In conclusion, 6-AN or ASA administered to pregnant rabbits or rats, respectively, during organogenesis resulted in malformations consistent with published literature and support their continued use as positive controls.

P112 - Characterization of Proliferating Human Hepatocytes As a Model System for Drug Interaction Studies and Toxicity Screenings

A. Noerenberg¹, L. Tolosa², M. José Gómez-Lechón², and R. Jover²

¹upcyte technologies GmbH, Hamburg, Germany

²Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe, Valencia, Spain

Liver toxicity represents a major cause of adverse effects and drug withdrawal. To efficiently predict the safety profile of a drug candidate, there is a constant need for reliable preclinical test systems. While primary human hepatocytes (pHH) remain the gold standard for predicting drug responses, restricted availability and a rapid loss of the hepatic phenotype limit their use. We previously reported the generation of proliferating “upcyte” hepatocytes, maintaining a differentiated phenotype. To evaluate their suitability for preclinical compound testing, we compared upcyte hepatocytes generated from different donors with HepG2 cells and pHH. Expression analysis revealed that the mRNA profile of upcyte hepatocytes comes closer to pHH than HepG2 cells. The cells maintained important hepatic functions including ureogenesis, glycogen storage, lipid accumulation, and albumin secretion. All donors were sensitive to prototypical CYP inducers such as PB and RIF, similar to pHH. Consistent with these findings, they exhibited measurable activity of several phase I and phase II enzymes (eg, CYP2B6, CYP2A6, CYP3A4, and UGT1A1). Importantly, upcyte hepatocytes predicted acute and long-term hepatotoxicity, as determined by a multiparametric HCS approach using established model compounds. Affected parameters included overall viability, apoptosis, mitochondrial membrane potential, oxidative stress, and intracellular Ca²⁺ levels. In addition, known steatosis-inducing agents provoked lipid accumulation and downregulation of the steatosis biomarker FOXA1. In conclusion, our data indicate that upcyte hepatocytes are more comparable to pHH than HepG2 cells. Given the additional advantage of virtually unlimited cell access, upcytes represent a promising hepatic model in drug development and preclinical toxicological safety assessments.

P113 - Use of Proliferating and Differentiated Upcyte Hepatocytes to Predict Toxicological Outcomes

A. Noerenberg¹, G. Levy², and Y. Nahmias²

¹upcyte technologies GmbH, Hamburg, Germany

²The Hebrew University of Jerusalem, Jerusalem, Israel

Primary human hepatocytes (pHH) have limitations for drug testing because of the low availability of liver tissue. We have developed a technique that causes pHH to proliferate while retaining a metabolic competent polarized phenotype (upcyte hepatocyte). To assess the utility of upcyte hepatocytes for toxicity screening, we used a library of known hepatotoxins and similarly structured control compounds. First, we compared the 24-hour acute TC50 toxicity profile of 6 compounds in differentiated upcyte hepatocytes, pHH and HepG2 cells. A permutation test showed that the TC50 toxicity profile of upcyte hepatocytes for these 6 compounds was not significantly different from that of pHH (P = 0.466; n = 4), whereas the HepG2 profiles were significantly different (P = 0.03; n = 4). We then measured TC50 values of 23 additional compounds and found an excellent correlation between all donors of upcyte hepatocytes and pHH (R² = 0.99). Second, we compared the toxicological profile of proliferating and differentiated upcyte hepatocytes. The TC50 of 9 known hepatotoxic drugs clustered into 2 groups. One group (aflatoxin B1, acetylsalicylic acid, and acetaminophen) showed no difference between both. The second group, which included 6 of the 9 hepatotoxic compounds, showed significantly higher toxicity in differentiated than in proliferating cells, suggesting that metabolic activation was required for the toxic effect. Our results demonstrate that upcyte hepatocytes from multiple genotypes show toxicity profiles similar to those of pHH. Moreover, proliferating and differentiated upcyte hepatocytes from the same donor can be used to discriminate mechanism of toxicity.

P114 - Evaluation of Growth and Plasma Membrane H⁺-ATPase Inhibition in Candida albicans by Selected Diselenide Analogs of Ebselen

N. Orie¹, M. Pietka-Ottlik², and B. Billack¹

¹St. John’s University, Jamaica, NY, USA

²Wrocław University of Technology, Wrocław, Poland

Ebselen (EB) exerts its antifungal activity, in part, by inhibiting the yeast plasma membrane, H⁺-ATPase (Pma1p). The present study investigated the antimycotic activity of a panel of diselenide analogs of EB: EB-double, and EB-1Se through EB-6Se. Suspensions of a fluconazole (FLU)-resistant strain, S2, were prepared by broth microdilution and incubated for 48 hours with each test compound. No diselenide exhibited growth inhibitory activity comparable to EB. Studies using another FLU-resistant strain, ATCC 96901, demonstrated results similar to that in S2. Next and in order to determine whether or not the diselenides could inhibit the activity of Pma1p, the effects of EB-double on medium acidification by intact S2 cells and of EB-6Se on isolated Pma1p enzyme were studied. Although EB-double demonstrated slight inhibition after a 10-minute interval, no efficacy at inhibiting medium acidification in intact yeast cells after 30 minutes was noted; the IC50MA for both time points was greater than the maximum test concentration. Despite minimal effects on growth and activity of the Pma1p in intact yeast cells, ATPase inhibitory studies from the partially purified S2 membrane preparation showed that inhibition by EB-6Se was both similar to EB and concentration dependent. This observation suggests that bulky diselenides may be less efficacious than EB because of size and a limited accumulation in yeast cells. More work will be needed to resolve this issue. To our knowledge, the data presented here represent the first time diselenide analogs of EB have been characterized in a FLU-resistant strain of C. albicans.

P115 - Localization of Lactate Transporters in Rat and Rabbit Placentae

C. Picut¹, N. Moore², J. Charlap³, T. McGiver¹, and E. Parker¹

¹Charles River Laboratories Hillsborough, LLC, Hillsborough, NC, USA

²Ubrs GmbH, Postfach, Basel, Switzerland

³Charles River Laboratories, Inc, Horsham, PA, USA

This study characterizes the distribution of monocarboxylate transporters (MCT) in the placenta of rat and rabbit, 2 species commonly used in developmental and reproduction toxicity studies. MCT isoforms 1 and 4 mediate plasmalemmal transport of lactate and pyruvic acid and generally are expressed on either the maternal or fetal side of the syncytiotrophoblasts. The polarity of MCT isoforms are completely opposite in human and mice, yet nothing is known of the MCT isoform polarity in rat or rabbit. Placentae from pregnant Sprague-Dawley rats at gestation days (GD) 11, 14, 18, or 20 and from pregnant New Zealand white rabbits at GD 13, 18, and 28 were subjected to immunohistochemical detection of MCT1 and MCT4. In the rat, MCT1 was located on the syncytiotrophoblast adjacent to maternal blood, while MCT4 was expressed by the syncytiotrophoblast apposed to fetal blood, similar to the polarity observed in mice. In the rabbit, MCT1 was expressed on the portion of cell membrane adjacent to fetal blood, while MCT4 was expressed on that part of the syncytiotrophoblast adjacent to maternal blood in a fashion similar to human placenta. In both rat and rabbit, MCT1 decreased during gestation, but MCT4 was consistently strong, indicating perhaps its important role in ridding the fetal blood from lactic and pyruvic acid buildup. The results suggest that the rabbit placenta may handle energy metabolism and substrate transport in a manner similar to humans and may be a preferable species over rat to use in certain investigations.

P116 - Minipig Jacket Design Enabling Simultaneous Electrocardiogram Collections and Dermal Dosing

C. Savidge¹, C. Artis¹, M. Miyamoto¹, A. Camacho¹, and T. Ramani¹

¹Envigo, Somerset, NJ, USA

The minipig is quickly becoming a widely accepted scientific model in toxicology studies with a cardiac component because of the similarity between the pig and the human heart. Because of this similarity, the ability to collect continuous electrocardiogram parameters on a variety of minipig study designs is critical to the scientific community. In the past, these critical data parameters have been avoided on minipig dermal toxicity studies, because the design of the jacket used to secure the electrodes in place for electrocardiogram collections would interfere with the procedure and location for dose application. The most common dose site used for dermal application on the minipig is across the dorsal area and onto the flanks of the animal. It totals approximately 10% of the total body surface area based on bodyweight. This dose site does not interfere with the placement of the electrodes for electrocardiogram collections; however, the jacket commonly used to secure the electrodes and to house the transmitter covers the entire trunk of the animal, thus creating the challenge of performing both dermal dosing and electrocardiogram tracings simultaneously. By modifying the jacket design to have an opening large enough to allow for the entire dermal dose site to be unaffected during the collection process but secure enough that the electrodes stay in place, we can successfully add electrocardiogram collections on any minipig dermal toxicity study.

P117 - Oligohydramnios in Cynomolgus Macaques

A. Rozner¹, J. McCulloh¹, and S. Henwood¹

¹Covance, Madison, WI, USA

Amniotic fluid provides fetal protection and space for normal fetal movement and limb development and contributes to the development of the fetal lungs, kidneys, and urogenital tract. Amniotic fluid is initially produced by the placenta but by the second half of pregnancy is primarily supplied by lung secretions and urine production of the fetus. Low amniotic fluid, or oligohydramnios, has been correlated to adverse outcomes including intrauterine growth restriction, malformations, fetal mortality, early term delivery, and delivery complications in humans; however, the direct effect of oligohydramnios is debated. No published data is available for oligohydramnios in nonhuman primates. This study provided a subset of cynomolgus macaques with low amniotic fluid noted qualitatively by ultrasound as early as gestation day (GD) 75 (normal gestation length of 165 days). Animals were monitored throughout gestation, delivery, and lactation. High-water-content supplements, including fruits and Gatorade, were offered to maternal animals starting around GD 145 until delivery in an attempt to increase water consumption and amniotic fluid levels, a common therapy in humans. The effect of oligohydramnios on fetal heart rate, delivery, postnatal infant body weight, respiration rate, and abnormalities was evaluated. After receiving high-water-content supplements, amniotic fluid levels appeared to increase in some animals. The results of this study suggest that increasing water consumption through supplementation may be a treatment option for oligohydramnios in the nonhuman primate.

P118 - Validation of 6- and 24-Well Plate Bacterial Mutagenicity Assay

S. Sawant¹, A. Brianna Smith¹, and L. Xu¹

¹Covance Inc, Greenfield, IN, USA

Testing for genotoxic activity of new chemical entities is used as an early surrogate for carcinogenic potential. Testing for mutagenicity is required by regulatory agencies to conduct first in human trials and/or pharmaceutical marketing. To reduce mutagenicity-related late stage attrition, early screening is beneficial to maximize hazard identification with minimal resources. The 6- and 24-well plate formats are miniaturized versions of the standard plate Ames assay that require significantly less compound and are similar to the plate incorporation method. The purpose of this validation study is to demonstrate a comparable sensitivity of the miniaturized versions to the full plate assay. Ten known Ames negative (anthracene, 6-mercaptopurine, l-methionine) and positive (2-aminoanthracene, benzo[a]pyrene, 7,12-dimethylbenz[a] anthracene, ICR-191, 2-nitrofluorine, 4-nitroquinoline-N-oxide, sodium azide) compounds and 2 pharmaceutical-related starting materials/impurities (1-H-pyrazole-4-boronic acid and 4-methoxycarbonyl-phenylboronic acid) were used in the validation. Studies were conducted simultaneously using full, 6-well, and 24-well plates for each validation compound, with and without metabolic activation system. Overall study conclusions were compared between the miniaturized formats and the full plate assay. Additionally, sensitivity of the miniaturized assays was evaluated by comparing corresponding test compound concentrations between 6- and 24-well plate formats with the full plate assay results. Comparison of the studies demonstrates that both miniaturized formats correctly predict the mutagenicity outcome of all test compounds in the full plate assay and with a high (∼95%) concordance. The results show that the miniaturized formats provide an opportunity to conduct mutagenicity evaluation with less compound and at an earlier stage in discovery.

P119 - Keyhole Limpet Hemocyanin Effects on Immunotoxicology Parameters in Nonclinical Toxicology Studies

J. Setser¹, R. Tadagavadi¹, J. England¹, L. Manson¹, J. Hyde¹, V. Peachee¹, and N. Makori¹

¹Charles River Laboratories Ashland LLC, Ashland, OH, USA

Keyhole limpet hemocyanin (KLH) is commonly used for nonclinical T cell-dependent antibody response (TDAR) studies; however, its effect on frequently assessed immune parameters is largely unknown. Here, we examined blood, spleen, thymus, and bone marrow leukocytes in KLH (300 μg IM injection)-administered female rats treated via oral gavage with vehicle (distilled water) or cyclophosphamide (CPS, 15 mg/kg/day). Parameters evaluated include differential counts of leukocyte subsets by flow cytometry on peripheral blood (pretest, days 8 and 22) and at necropsy (day 22) on bone marrow, thymus, and spleen. Histopathology of lymphoid organs was also performed. KLH administration had no impact on CD4T cells, CD8T cells, and B cells in blood, spleen, thymus, and bone marrow. Rats treated with CPS + KLH presented statistically significant decreases in blood lymphocytes that correlated with lower absolute total T cells (CD3+, −71%) and subsets (CD3+CD4+, −75%; CD3+CD8+, −65%) and B cells (CD3− CD45RA+, −99%) at 8 days following dosing compared to vehicle control. Similar changes were noted in the CPS-only group at day 8 and persisted (CPS only and CPS + KLH cohorts) through day 22. Spleen and thymus of rats treated with CPS or CPS + KLH exhibited lower, although not statistically significant, T and B lymphocyte counts. Administration of CPS, with or without KLH, resulted in lymphoid depletion in the spleen and lower thymus weight. In conclusion, KLH use as an immunogen for assessment of TDAR does not appear to influence routinely assessed immunological parameters.

P120 - Bile Acids as Biomarkers to Assess Liver Impairment in Polycystic Kidney Disease

J. Slizgi¹, M. Su², W. Jai², K. Brouwer¹, and B. Brock³

¹University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

²University of Hawaii Cancer Center, Honolulu, HI, USA

³Brock Scientific Consulting, LLC, Montgomery Village, MD, USA

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of kidney cysts and declining renal function with frequent development of cysts in other organs including the liver. Polycystic kidney disease (PCK) rats, a rodent model of ADPKD, have been used to understand hepatorenal disease progression and evaluate pharmacotherapeutic interventions. Biomarkers that describe the progression, degree of liver impairment, and/or hepatic cyst burden in ADPKD would be useful. In the present study, hepatic cyst volume was measured in PCK rats at 12, 16, and 20 weeks. After 20 weeks, wild-type (n = 4) and PCK (n = 4) rats were sacrificed and 42 bile acids were analyzed in the liver, bile, serum, and urine by LC-MS/MS. Metabolomic analysis revealed significant increases in total bile acids in the liver (13.2-fold) and serum (5.5-fold) in PCK rats, with modest increases in the urine (3.0-fold). Bile acids associated with hepatotoxicity were increased. Total serum bile acids were associated with markers of liver impairment (ie, liver weight, total liver bile acids, total hepatotoxic liver bile acids, and cystic volume) (r² > 0.85; P < 0.05). A multiple analysis of variance revealed a statistically significant effect (P < 0.001), indicating that markers of liver impairment independently associated with serum bile acids. These results indicate that bile acids are increased in PCK rats, which may contribute to disease progression and/or potential susceptibility to bile acid-mediated hepatotoxicity. Therefore, serum bile acids may be useful biomarkers of liver impairment in ADPKD.

P122 - Role of p53 on Mediation of Energy Metabolism in Cell Malignant Transformation Induced by Radon

J. Tong¹, M. Geng¹, Y. Cao¹, J. Li¹, and X. Liu¹

¹Department of Toxicology, School of Public Health, Medical College of Soochow University, Suzhou, Jiangsu, China

Radon has long been recognized as a human carcinogen leading to lung cancer, but the underlying mechanisms remain obscure. The aim of the present study was to explore the role of p53 in mediation of energy metabolism in malignant transformation of human bronchial epithelial (BEAS-2B) cells induced by radon. A p53 knockout BEAS-2B cell model was set up to observe the adverse effects induced by long-term exposure of radon. Alterations in cell proliferation, energy metabolism, and expression of p53 target genes were detected by biochemical and molecular biological assays. The results showed that malignant transformation occurred in cells exposed to radon at 15 passages of cell culturing, along with increased levels of lactate and lactate dehydrogenase (LDH) and ratio of NAD+/NADH, as well as upregulation of p53 target gene synthesis of cytochrome c oxidase 2 (SCO₂), TP53-induced glycolysis, and apoptosis regulator (TIGAR). The study provided the first evidence that p53 plays an important role in radon-induced cell transformation by regulation on cellular energy metabolism.

P124 - Options for Enhanced Analysis of Spermatogenesis in the Nonhuman Primate Model

G. Weinbauer¹, G. Hoffmann², and M. Luetjens¹

¹Covance, Münster, Germany

²Covance, Greenfield, IN, USA

Spermatogenesis is a highly complex process comprising multiple cell divisions and special cellular differentiation. This process is highly organized, with germ cell generations topographically arranged into so-called spermatogenic stages. Unlike in rodents, spermatogenic stages in macaque and human are less segmentalized, rendering qualitative and quantitative analyses comparatively challenging. This work investigates options for enhanced spermatogenesis analysis in the cynomolgus monkey. Retrospective evaluation of 15 studies comprising over 200 animals with specific testicular analyses was conducted. Analyses included detailed spermatogenic evaluation (modified Davidson’s fixative, hematoxylin-eosin, and/or PAS staining) of up to all 12 spermatogenic stages. In 4 studies, cells from a small testicular fraction and stained with propidium iodide were quantified by flow cytometry (10 000 cells/sample). From over 90 animals, semen samples were analyzed for sperm number and motility. In 2 shorter-term studies (<8 weeks), stage-specific analysis revealed stage-specific effects that were not apparent during routine histological analysis. Results from quantitative flow cytometry analyses coincided with histological observations. Sperm numbers were highly variable, with poor statistical power requiring group sizes of more than 50 animals. In conclusion, additional stage-related analysis is recommended to characterize specific germ cell effects over 1.5 spermatogenic cycles (approximately 8 weeks) but not for routine histopathology. Seminal sperm number determination has a low sensitivity and may be useful only under particular circumstances. Flow cytometry appears useful if quantitative information on testicular cell populations is required.

P125 - Light-Induced Retinal Degeneration in Sprague- Dawley Rats in a Drug Safety Study at a Nonclinical Research Laboratory

L. Zwick¹, J. Bartoe¹, D. Cooper¹, J. Albretsen¹, and D. Patrick¹

¹MPI Research, Mattawan, MI, USA

Retinal degeneration from intense or prolonged exposure to light can occur in albino rats and has the potential to confound drug safety evaluation. During a 26-week oral toxicity study of a pharmaceutical test article in Sprague-Dawley rats, ophthalmoscopic examination prior to termination revealed bilateral retinal atrophy in 4 rats treated with the test article and 2 with the water vehicle. All affected rats were housed in the uppermost cages. Histopathologic examination of the eyes was limited to rats in the control and high-dose groups. Microscopic lesions consistent with retinal phototoxicity were present in the eyes of 9 rats in the control group and 2 rats in the high-dose group, including the 3 rats in these groups that had ophthalmoscopic lesions. All but one of the affected animals was housed in the top row of cages, indicating a relationship of the retinal lesions to the intensity of the light exposure. Degeneration was of minimal to moderate severity, typically bilateral, and involved the central retina bordering the optic disc. Affected portions of the retina were hypocellular and exhibited variable loss of rod and cone photoreceptor processes, disorganization of the inner and outer nuclear layers, and narrowing or absence of the plexiform layers. Light-induced retinal degeneration is uncommon at this facility and was most likely due to the maximal light intensity exceeding the recommended range. Additional in house studies are currently investigating the pathophysiology of light-induced retinal lesions in rats and evaluating the efficacy of multiple preventative measures.

*STP126 - Antioxidant Status of the Brain and Hematological Evaluations of Subacute Toxicity to Sodium Selenite

D. Babayemi¹, A. Esther¹, A. Victoria¹, M. Aminat¹, A. Fola², and A. Oladipo¹

¹Biochemistry Department, Federal University of Agriculture, Abeokuta, Ogun, Nigeria

²Chemistry Department, Federal University of Agriculture, Abeokuta, Ogun, Nigeria

Selenium is an antioxidant and an important micronutrient affecting multiple physiologic functions. In order to investigate the effects of sodium selenite exposure on some hematological parameters and antioxidant status of the brain, rats were exposed to sodium selenite (16 ppm, 32 ppm, and 64 ppm) in their drinking water for 4 weeks. Control animals received distilled water for 4 weeks. Activities of catalase (CAT), glutathione peroxidase (GPx), xanthine oxidase (XO), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH), the concentration of reduced glutathione (GSH) and vitamin C (Vit C), and the levels of lipid peroxidation (MDA) and protein carbonylation (PC) were assayed for in the plasma and brain using the spectrophotometric method. Hematological results showed a 78% increase in the white blood cells count of 64 ppm and a 0.92% increase in neutrophils count when compared to the control (P < 0.05). In the plasma, there were significant increases (P < 0.05) in the activities of XO and GPx of the sodium selenite-exposed groups compared with the control, though these were not dose-dependent. A negative dose-dependent relationship was observed in the effects of sodium selenite on GSH and Vit C. The relative organ (brain) to body weight showed significant increases (P < 0.05) in 16 ppm (300%) and 32 ppm (300%) groups compared to the control. There were significant decreases (P < 0.05) in the brain Vit C and activity of CAT; these were also dose-dependent. Our findings suggest that sodium selenite is a possible pro-oxidant at higher concentrations.

*STP127 - Differential CB2 Expression Patterns Are Dependent on Activation in Human B Cells

J. Castaneda¹, A. Harui¹, S. Kiertscher¹, and M. Roth¹

¹University of California, Los Angeles, Los Angeles, CA, USA

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 significance of differential CB2 expression patterns and the toxicity of cannabinoids on immune function remain unknown. In preliminary studies, tonsillar naive (IgD+/CD38+) and quiescent memory (IgD−/CD38−) B cells were found to express surface CB2, but activated B cells (IgD−/CD38+) expressed low to no surface CB2. Interestingly, all 3 subsets express intracellular CB2. We hypothesize that B cell activation downregulates surface CB2 and plays an active role in differential CB2 expression patterns. We designed an in vitro model to mimic activation and isotype switching in naive B cells by cross-linkage of the B cell receptor with costimulatory molecules. In vitro experiments demonstrate that naive B cells exposed to activation conditions downregulated surface CB2, and cells expressing IgD did not downregulate surface CB2. Isotype-switched cells also demonstrate a downregulation of surface CB2. Our findings suggest that CB2 is differentially regulated in naive versus activated/isotype switched B cells. Furthermore, confocal microscopy was used to locate intracellular CB2, but CB2 was not found to colocalize in mitochondria, lysosomes, or nucleus. The intracellular location of CB2 and its role in intracellular signaling remains unknown. These experiments establish a model for examining the interaction between CB2 receptors and intracellular signaling events that lead to B cell activation, differentiation, and isotype switching. Ultimately, investigating the immunoregulatory effects of cannabinoids will help understand the exploitation of the CB2 pathway for therapeutic purposes.

*STP128 - Variation in cyp1a Detected in 3 Genetically Divergent Strains of Zebrafish

L. Holden¹ and K. Brown¹

¹Portland State University, Portland, OR, USA

Zebrafish (Danio rerio) are a widely utilized model system for toxicological studies, but exact lab strains are often not reported. Genetically divergent strains—AB, TU, and WIK—have extensive genomic copy number variation (CNV) covering an average of 1,445 genes. To assess effects of CNV in a toxicological system, we examined 2 genes important in the aryl hydrocarbon exposure pathway. Using publicly available microarray data (GEO Accession: GSE28328), we evaluated aryl hydrocarbon receptor 2 (ahr2) and cytochrome P450 subtype 1A (cyp1a) genes—a receptor and metabolic enzyme, respectively—for evidence of CNV. No CNV were observed across ahr2, but the data indicate a novel CNV across cyp1a in WIK as compared to AB and TU strains. qPCR of cyp1a narrowed the CNV region to exon 5 (of 7), and resulted in a 15-fold relative reduction of cyp1a in WIK versus AB and TU strains. Interestingly, cyp1a exon 5 amplicons did not separate by size, but rather showed a decrease in intensity. This suggests that the cyp1a exon 5 CNV may be a single copy loss, yet still present in WIK. Furthermore, we exposed adult zebrafish from the same 3 strains to 131 ppb PCB126 for up to 48 hours and quantified cyp1a mRNA expression using reverse transcription (RT)-qPCR. RT-qPCR results indicate a 14-fold relative increase in cyp1a mRNA expression in WIK versus AB and TU after 9 hours of exposure, indicating aberrant regulation of cyp1a in WIK. Our results highlight the importance of understanding within-species genomic variation in toxicological assessments.

*STP129 - Neurotoxicological Effects and the Impairment of Spatial Recognition Memory in Rats Caused by Subchronic Exposure to Silica Nanoparticles

S. Husain Mustafa Rizvi¹, A. Parveen¹, F. Mahdi², and A. Ali Mahdi¹

¹King George’s Medical University, Lucknow, UP, India

²Era’s Lucknow Medical College and Hospital, Lucknow, UP, India

Silica nanoparticles (SiNPs) have attracted extensive interest because of their easy surface modifications and highly hydrophilic properties and potential applications in biomedical and industrial fields. The present study was planned to examine the effect of subchronic SiNPs exposure on the dopaminergic system, and also to explore the novel mechanisms involved in cognitive impairment and neuronal apoptosis in the corpus striatum (CS) of male Wistar rats. Male Wistar rats were divided into 2 groups (control and experimental) of 6 animals each. Intranasal exposure of SiNPs (80 nm) at a dose of 0.75 mg/kg was given to experimental rats for 90 days. The results demonstrated impairment in neurobehavioral indices and a significant increase in lipid peroxide levels (LPO), hydrogen peroxide (H₂O₂), superoxide (O₂ ⁻), and protein carbonyl content, whereas there was a significant decrease in the activities of the enzymes, manganese superoxide dismutase (Mn SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione (GSH) content, suggesting an impaired antioxidant defense system. Protein and mRNA expression of cytochrome c, Bcl-2, Bax, p53, caspase-3, caspase 12, and CHOP/Gadd153 suggested that alteration in the levels of these proteins was involved in oxidative stress-mediated apoptosis in treated rat brain CS. Moreover, electron microscopic studies clearly showed mitochondrial and endoplasmic reticulum dysfunction. The result of the study suggested that subchronic SiNPs exposure has the potential to alter behavioral activity and to bring about changes in biochemical, neurochemical, and ultrastructural profiles in the CS region of rat brain.

*STP130 - Phthalate Mixture Exposure Reduces Antral Follicle Growth and Hormone Production in Mouse Ovaries

C. Zhou¹ and J. Flaws¹

¹Univeristy of Illinois, Urbana, IL, USA

Phthalates are used in building materials, medical devices, and personal care products. Most studies on phthalates have focused on single phthalates, but it is important to study mixtures of phthalates because humans are exposed to such mixtures daily. Thus, we tested the hypothesis that exposure to an environmentally relevant phthalate mixture decreases ovarian antral follicle growth and hormone production. Antral follicles from adult CD-1 mice were cultured with vehicle control or phthalate mixture (1-500 μg/mL; n = 6 cultures, 6-12 follicles/treatment/culture) for 96 hours. The mixture was based on the composition of phthalates detected in women and included 35% diethyl phthalate, 21% di(2-ethylhexyl) phthalate, 15% dibutyl phthalate, 15% diisononyl phthalate, 8% diisobutyl phthalate, and 5% benzylbutyl phthalate. During culture, antral follicle diameters were measured every 24 hours to monitor growth. After culture, media and follicles were subjected to measurements of sex steroid hormones and atresia, respectively. The phthalate mixture (100 and 500 μg/mL) decreased antral follicle growth and androstenedione (10, 100, and 500 μg/mL), testosterone (10 and 500 μg/mL), estradiol (10, 100, and 500 μg/mL), and estrone (100 and 500 μg/mL) levels compared to control (P < 0.01). The mixture (10, 100, and 500 μg/mL) also induced oocyte fragmentation. These data suggest that exposure to a phthalate mixture significantly inhibits antral follicle growth, reduces several sex steroid hormone levels, and induces oocyte fragmentation. Further, these data suggest that the phthalate mixture adversely affects female reproduction. NIH P01 ES022848, EPA RD-83459301, and an Environmental Toxicology Fellowship.

*IGP131 - Updates in Nickel Allergy: In Vitro Regulation of Interleukin-12 Cytokine Family in Human Dendritic Cells and Identification of Naive T-cells Specific to Nickel

R. Bechara^1,2, D. Antonios², M. Azoury¹, H. Azouri², and M. Pallardy¹

¹INSERM UMR996, Paris-Sud University, Paris-Saclay University, Chatenay-Malabry, Iles de France, France

²Saint Joseph University, Laboratory of Toxicology, Beirut, Lebanon

Allergic contact dermatitis (ACD) is a major cause of occupational skin disease, and nickel is among the most prevalent contact allergen. Dendritic cells play an important role in the type of ensuing immune response through differential cytokine production. In particular, the IL-12 cytokine family plays a major role in the generation of allergen-specific T cell responses. Moreover, the manifestation of nickel-induced ACD has been ascribed to T cell activation, demonstrating the presence of nickel-specific T cells. In this work, we studied the effect of nickel on the IL-12 cytokine family and evaluated the frequency of human naive T cells specific to nickel in peripheral blood. Immature human monocyte-derived dendritic cells (Mo-DC) were differentiated from monocyte CD14+, and at day 5, they were stimulated with NiSO₄. Moreover, naive CD4+ T cells were stimulated once a week with human autologous dendritic cells loaded with NiSO₄. Activation of nickel-specific T cells was detected using interferon-γ EliSpot. First, we showed that nickel induced the production of IL-12p40, IL-23, and IL-27 in Mo-DC, and its effect on these cytokines correlated with the expression of their subunits. In the second part, naive CD4+ T cells specific to nickel were detected in 6 of 7 of the tested donors. Frequency of nickel-specific naive CD4+ T cells varied from 0 to 2.03 nickel-specific naive CD4+ T cells per million naive CD4+ T cells, with a mean value of 0.80. Finally, our results describe a novel effect of nickel on the IL-12 cytokine family and show, for the first time, a quantification of pre-existing CD4+ T cells specific for nickel.

*IGP132 - Activation of PERK-eIF2α Signaling and Downregulation of M2-mAChR Causes Cholinergic Dysfunction in Silica Nanoparticles-Exposed Rats

A. Parveen¹, S. Husain Mustafa Rizvi¹, F. Mahdi², and A. Ali Mahdi¹

¹King George’s Medical University, Lucknow, UP, India

²Era’s Lucknow Medical College and Hospital, Lucknow, UP, India

With the increase in use and wide application of engineered nanomaterials, the safety issues associated with them are being questioned by the scientific community all over the world. Among the various nanoparticles, silica nanoparticles (SiNPs) have profound uses in numerous consumer products, including food industry products, cosmetics, and medical products. The present study was planned to examine the effect of subchronic exposure of SiNPs on the cholinergic system of male Wistar rats. The male Wistar rats (wt: 150-200 gm) were divided into 2 groups (control and experimental) of 6 animals each. Intranasal exposure of SiNPs (10 nm) at a dose of 0.75 mg/kg was given to experimental rats for 90 days, after post treatment of 90 days. The results showed that SiNPs promote learning and memory impairment, which may be associated with reactive oxygen species overproduction and oxidative damage in the frontal cortex (FC) and hippocampus (HC) of rats. We also studied expression of p53, Bax, Bcl2, and Cyt c together with endoplasmic reticulum stress-related proteins like CHOP, PERK, p-PERK, eIF2α, GRP-78, and caspase 12 in FC and HP. We observed a decrease in the binding of muscarinic-cholinergic receptors in FC and HC associated with reduced CHRM2 mRNA levels, acetylcholinesterase activity, and expression of ChAT in treated rats. The results of the study suggested that subchronic SiNPs exposure has the potential to alter the PERK-EIF2α signaling together with downregulation of M2 mAChR, which may play a critical role in cognitive impairment observed in SiNPs-treated rats.

*IGP133 - Developmental Toxicity in Wistar Rats Treated with Ethanolic Seed Extract of Moringa Oleifera

A. Njan¹, B. Ejimkonye¹, O. Afolabi¹, and O. Olorundare¹

¹University of Ilorin, Ilorin, Kwara, Nigeria

Moringa oleifera is widely sought after for its medicinal and nutritional properties. Pregnant women use Moringa as food supplement thus, are also referred to as ‘mother’s best friend’ due to its perceived milk production property. Despite medicinal and nutritional function of Moringa, paucity of information on its safety remains an important challenge. The present study aimed to determine developmental toxicity following oral administration of ethanolic seed extract of Moringa oleifera on gravid Wistar rats. Eight weeks old female Wistar rats were separated into 4 groups (n = 10). The control group received water ad libitum while the treatment groups received 50, 200 and 800 mg/kg bwt (body weight) of extract, respectively, from gestation days 1-19. Body weight, food, and water consumption was observed daily in the pregnant rats. Necropsies were performed 1 day before the expected parturition on half of the animals and parameters (implantations, uterine weight, foetal resorptions, death, viability and body weight, placenta weight, crown-rump length, tail length, and fetal number) were determined. The remaining pregnant rats were allowed to give birth and foetal parameters were assessed. No cesarean-sectioning or litter parameters were affected by the test article at doses of 50 and 200 mg/kg body weight. However, incidence of fetal alterations was high at 800 mg/kg. This suggests that the extract may be safe for use at these doses by female during organogenic period of pregnancy, whereas the extract dose at 800 mg/kg body weight portends mild toxicity in pregnancy.

*GFP134 - Beneficial Effect of Carnosine on Motor Function in the Thy1-aSyn Mouse Model of Parkinson Disease

M. Bermudez¹ and M.B. Genter¹

¹University of Cincinnati, Cincinnati, OH, USA

Parkinson disease (PD) is the second leading neurodegenerative disease, affecting millions of people worldwide. No cure exists for this devastating disease. PD is characterized by several motor and prior onset nonmotor deficits, including gait instability and decreased olfactory function. Molecular hallmarks of PD include protein aggregates and oxidative stress. Our studies evaluated a novel mechanism-based treatment for PD, using the Thy1-aSyn mouse model of PD. Carnosine, an endogenous dipeptide abundant in muscle, brain, and the olfactory system, declines with age and pathological conditions. Recent in vivo studies indicate that carnosine reduces protein aggregation and protects against oxidative stress, 2 features of PD. Therefore, we hypothesized that intranasal (IN) administration of carnosine would significantly reduce disease progression in the Thyl-aSyn mouse model of PD. Wild-type and Thy1-aSyn mice were treated IN with 2 mg/day of carnosine or sterile water (as control) for 2 months. Immunohistochemistry, buried food pellet, and the challenging beam traversal (CBT) tests were used to evaluate tissue structure and sensorimotor functions at the beginning and end of treatment. Olfactory function and structure were preserved, and alpha-synuclein (aSyn)-positive inclusions were notably lower in the olfactory epithelium of carnosine-treated Thy1-aSyn mice compared to controls. Strikingly, in the CBT test, the number of errors per step was lower in the carnosine-treated Thy1-aSyn group compared to the untreated Thy1-aSyn group (0.5 vs 0.7, P < 0.05). Our novel findings suggest that carnosine prevents the progression of motor deficits and aSyn aggregation in the Thy1-aSyn model of PD.

*GFP135 - Proteasome Activity is Modulated by Parkinson in Dopamine Axon Terminals Following Neurotoxic Insult With MPTP

T. Lansdell¹, K. Lookingland¹, and J. Goudreau¹

¹Michigan State University, East Lansing, MI, USA

The motor symptoms of Parkinson disease are the result of chronic degeneration of nigrostriatal dopamine (NSDA) neurons. While NSDA neurons are susceptible, tuberoinfundibular dopamine (TIDA) neurons are spared. This pattern of susceptibility can be recapitulated with exposure to the selective DA neurotoxicant MPTP. In this model, the recovery of TIDA neurons is concurrent with increased parkin expression. Parkin, an E3 ligase, is able to enhance proteasome activity in cellular assays. We hypothesized that increased parkin expression in TIDA neurons acts to protect these neurons through maintenance of proteasome activity. MPP+, but not MPTP, decreased proteasome activity in vitro, and this was attenuated in the presence of purified parkin. Proteasome activity was decreased in striatal tissue and synaptosomes derived from Park2−/− mice compared to those from WT mice. MPTP caused a decrease in striatal parkin expression and proteasome activity in both WT and Park2−/− mice. In contrast, proteasome activity was impaired in the median eminence of Park2−/− mice, but not in WT mice. Overexpression of parkin in the substantia nigra resulted in increased proteasome activity in ST synaptosomes. Further studies mediating the overexpression of parkin in the substantia nigra will determine if parkin overexpression in NSDA neurons will protect axon terminals from loss of proteasome activity. Overexpression of parkin in the arcuate nucleus of Park2−/− mice will determine if parkin expression is sufficient to restore proteasome activity in the ME. These results indicate that parkin plays a role in maintaining proteasome activity following acute neurotoxicant injury in DA neurons.

*GFP136 - Thyroid Hormone Disruption Alters Oligodendrocyte Development in Larval Zebrafish

K. Walter¹, X. Chen¹, B. Puschner¹, and P. Lein¹

¹University of California Davis, Davis, CA, USA

Thyroid hormones (THs) are critical for proper neurodevelopment and can influence multiple neurodevelopmental processes in different brain regions, including the differentiation and maturation of oligodendrocytes and subsequent myelination. Many environmental contaminants disrupt TH signaling, which is widely postulated to be a convergent mechanism of developmental neurotoxicity (DNT) across diverse chemical classes. However, testing this hypothesis has been limited by the identification of specific neurodevelopmental endpoints directly affected by THs. To address this gap, we determined whether disrupting TH signaling alters oligodendrocyte maturation in larval zebrafish, coincident with altered swimming behavior. To modulate TH signaling, larval zebrafish were treated with exogenous T4 or T3 beginning at 6 hours post fertilization or were injected as 1-cell embryos with a morpholino to knock down the TH transporter MCT8. The Tg(Sox10: RFP; Mbp: GFP) zebrafish line was used to visualize and count mature oligodendrocytes in both the brain and spinal cord using an automated imaging and analysis system. Treatment with T4 and T3 significantly increased the number of mature oligodendrocytes in the spinal cord at concentrations that also caused hyperactivity in larval swimming behavior tests, whereas preliminary data suggest that MCT8 knockdown decreases larval swimming activity and may decrease the number of mature oligodendrocytes. This characterization of how modulating TH signaling influences oligodendrocyte maturation in larval zebrafish will facilitate further mechanistic studies of TH’s role during neurodevelopment, which will be critical for developing screening tests to assess TH-disrupting chemicals for DNT risk. Supported by US EPA (grant R83550 to PJL) and the NIEHS (T32 ES007059 predoctoral fellowship to KMW).

200 Series—Regulatory Toxicology

P201 - Cardiac Contractility: Correction Strategies Applied to Telemetry Data From an HESI-Sponsored Consortium

S. Authier^1,2, E. Boulay¹, V. Jacquemet^3,4, A. Vinet^3,4, J. Doyle⁵, J. Pierson⁶, and M. Pugsley⁷

¹CiToxLAB, Laval, Québec, Canada

²Faculty of Veterinary Medicine, University of Montréal, Saint-Hyacinthe, Québec, Canada

³Département de Physiologie Moléculaire et Intégrative, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada

⁴Centre de Recherche, Hôpital du Sacré-Coeur, Montréal, Québec, Canada

⁵Data Science International, St. Paul, MN, USA

⁶HESI, Washington, DC, USA

⁷Department of Toxicology, Purdue Pharma L.P., Cranbury, NJ, USA

While the QT has a long history of heart rate (HR) correction, limited investigations have been undertaken to assess the impact of cardiovascular parameters on left ventricular (LV) contractility. Cardiac contractility is affected by preload (Cyon-Frank-Starling), afterload (Anrep), and HR (Bowditch). Modeling was completed using contractility data from chronically instrumented beagle dogs in a HESI consortium in either a single or double 4 × 4 Latin square study (n = 4 or 8/site). Forty-four correction models (11 fitting formulas × 2 modeling approaches [universal and individualized]) × 2 correction approaches (linear or proportional) were evaluated. 3-D/2-D cloud analysis of the beat-to-beat data for the negative control and positive controls (pimobendan, and itraconazole or atenolol) were used to evaluate correlations between the various parameters and derive an optimal correction method. Results: dP/dtmax was best correlated to HR and the LV systolic pressure with a correlation coefficient of 0.8. In decreasing order, dP/dtmin, arterial blood pressure (systolic, mean, and diastolic), arterial pulse pressure, and LV end-diastolic pressure showed reduced correlation to dP/dtmax. Subject-specific models improved the correction by up to 14% when compared to universal correction models. The non-linear correction model was superior to the linear model. Results suggest that the optimal correction formula for dP/dtmax would be subject-specific, nonlinear, and would include HR and LV systolic pressure. Correcting contractility for HR and LV systolic pressure may enhance data interpretation in nonclinical drug safety assessments. Similar analysis could be considered for other species used in safety pharmacology.

P202 - Strategies for EEG Interpretation in Preclinical Studies: Chasing Biomarkers of Seizure Activity

S. Authier^1,2, D. Paquette¹, M. Pouliot¹, and R. Forster¹

¹CiToxLAB, Laval, Québec, Canada

²Faculty of Veterinary Medicine, University of Montréal, Saint-Hyacinthe, Québec, Canada

Drug-induced seizures are a concern in preclinical development given the life-threatening consequences. Telemetry studies generate a significant amount of data that require analysis to identify seizure activity biomarkers. EEG traces were obtained in cynomolgus monkeys, beagle dogs, and Sprague-Dawley rats using telemetry with implanted electrodes from the 10 to 20 system (Cz-Oz, C3-O1, and C4-O2). Automated and manual analysis methods were compared to identify EEG biomarkers of seizure. Automated seizure detection tools identified spike trains but could not detect more complex biomarkers of increased susceptibility to seizure such as increased synchrony, isolated sharp waves and isolated spikes, or isolated spike-and-waves patterns. In large animals, EEG traces obtained in a Cz-Oz derivation presented the lowest artifact level that was optimal for automated seizure detection. Abnormal EEG often occurred when clinical signs (eg, autonomic signs or muscular activity such as myoclonic jerks) were present and review of traces when abnormal behavioral signs were presented increased sensitivity. Abnormal EEG activity without any clinical sign (including absence of seizure) was most frequently observed around T_max, and a manual review of traces around this period was a valuable strategy to increase sensitivity of the review. Handling/ restraining activities triggered seizure in some cases. Tremors were rarely associated with abnormal EEG activity, but this remains a regulatory concern. Automated EEG analysis alone is insufficient to evaluate traces for biomarkers of seizure activity but can serve as a first line tool to identify areas of interest. Manual review of selected time points around Tmax was the most sensitive strategy to identify seizure biomarkers.

P203 - Overcoming Data Integration Challenges to Produce SEND Data Sets

M. Bausman¹, J. Fohey¹, J. Pitzer¹, and D. Sullivan¹

¹Covance Inc, Madison, WI, USA

Nonclinical toxicology studies that include phases run at multiple test sites (aka multisite studies) are commonplace in the industry and can involve 1 or more organizations. With the FDA requirement for including SEND (Standard for Exchange of Nonclinical Data) data sets in NDA submissions taking effect on December 17, 2016, integrating data generated from the test facility and multiple test sites into 1 submission-ready and compliant SEND data set is challenging. Assorted data types may be generated on various instruments and software and even paper. Ensuring compatibility of these sources is a shared responsibility between the organizations involved in study conduct. Covance assembled a multidisciplinary team to develop a validated process to integrate bioanalytical and toxicokinetic endpoints from disparate source systems into the SEND dataset. The project involved (1) investigation of software solution details, such as versions, configurations, settings, limitations; (2) proposed software use proof of concept; (3) validation of the final process; and (4) operational rollout and control plans. The resulting process for data generated within Covance is over 80% automated, implemented globally at Covance, and compliant with SEND Implementation Guide 3.0 specifications. Subsequently, we addressed this challenge within our organization while also gaining the added benefit of developing specification of data transfer requirements when the sponsor or a third-party vendor holds responsibility for generating these and other data types.

P204 - Reduction and Refinement of Rodent Juvenile Toxicity Studies: The Simple Approach to Cross-Fostering

E. Richmond¹, S. Clode¹, and M. Blackwell¹

¹Sequani Ltd, Ledbury, United Kingdom

The need for juvenile rodent toxicity studies to support pediatric clinical testing of pharmaceutical products has become increasingly commonplace, with appropriately designed nonclinical juvenile studies providing safety data, predictive of toxicity in the pediatric population. While study designs vary hugely because of their tailored approach, almost exclusively these are large and complex studies, with animal usage exceeding any other study in the nonclinical program. It is vital therefore that all options to reduce and refine these studies are explored. There are opportunities in basic study design, but the simple process of how animals are selected for study is often overlooked, despite the significant impact this can have on total animal usage and the robustness of data. The commonly adopted pup selection method only allocates to study a small number of the dam’s natural offspring per litter, because of concern over maternal genetic bias. The cross-fostering approach has often been considered a superior method, whereby the offspring are distributed to multiple litters, removing any genetic bias or culling, and allowing all offspring to be selected for use. This reduces animal usage, provides better-quality data and greater study flexibility. However, because of practical feasibility, crossfostering has been traditionally discounted as idealist. This poster aims to raise awareness of a unique cross-fostering approach, developed in a joint collaboration with our animal supplier. It involves cross-fostering to specific requirements by the supplier before arrival at the test laboratory and has been shown to reduce total rat usage, typically by >65%.

P205 - Characterizing Compound Classes by Rodent Carcinogenicity Tumor Severity and Type

D. Guo¹, N. Kruhlak¹, L. Stavitskaya¹, K. Cross², and D. Bower²

¹US FDA, Silver Spring, MD, USA

²Leadscope, Inc., Columbus, OH, USA

Characterizing carcinogenic classes by tumor type and severity is crucial for understanding the structure–activity relationships for rodent carcinogenic potential and for accurately developing (Q)SAR models predicting rodent carcinogens. To identify characteristics and severity of tumors between classes, we developed a scoring system considering the number of tumor types in a given study group and whether a particular tumor type is conserved across sexes and species. We classified 1,186 compounds in the FDA rodent carcinogenicity database using 92 Leadscope structural alerts and characterized the classes by tumor type and severity. The alert classes covered 82% of 979 carcinogens; 51% were non-genotoxic, including steroids, statins, dialkyl phenols, phenylureas, thiophenes, and urethanes. Each class consisted of both single-site and multisite carcinogens. While nitrosamines, polycyclic aromatic systems, and aliphatic N-nitros had high tumor severity scores with multisite tumors, they were predominately single-species tumors. Some classes with high scores had predominately single-site tumors, including quinones, diphenyl-ketones, and azepines. Classes with high tumor severity scores and multi-species tumors included polycyclic-aromatic systems, polycyclic aromatic hydrocarbons, trichloro(fluoro)ethylenes, and hexachlorohexanes. Ninety-five percent of the 979 carcinogens caused tumors in both sexes. Classes characterizing single-sex tumors were not identified in this study. Some carcinogenic classes had only moderate tumor severity scores and single-species tumors, including hydrazines, aziridines, pyrrolizidine alkaloids, quinolines, benzsufonic esters, and methyl sulfonic esters. The classification of rodent carcinogens by tumor type and severity provides a more detailed approach to characterize rodent carcinogens and their structure–activity relationships, leading to more accurate (Q)SAR modeling of rodent carcinogenicity through the identification of more precise model features.

P206 - Enhanced Imaging Has Proven to Be a Safety Net During the Conduct of Regulatory Studies With Nonhuman Primates: The Value of Hard Data Before Necropsy

S. Korte¹, M. Wozniak², F. Runge¹, T. Voß¹, F. Ludwig¹, and A. von Keutz¹

¹Covance Preclinical Services GmbH, Münster, Germany

²Department of Pediatric Radiology, Medical University of Lublin, Lublin, Poland

The cynomolgus monkey represents an important primate model for preclinical safety evaluation, as it has shown to predict toxicity and pharmacodynamics of many pharmaceuticals under development. The present work examined the use of multislice spiral computed tomography (CT) and magnet resonance (MRI) for in vivo imaging during the conduct of regulatory toxicity studies. CT produces detailed cross-sectional images. Post-processing of raw data enables the generation of 3-dimensional (3-D) images of organs. MRI is typically used for viewing axial, coronal, and sagittal sections of bones, ligaments, and the brain. Two geriatric monkeys (above 10 years, 4.0 and 6.7 kg) for CT and 1 monkey (2 years) were subjected to in vivo imaging during the course of a regulatory toxicity study (CT) or directly after quarantine (MRI). For MRI, an Excite Echospeed SR120 GE scanner performing coronal T1 and T2 weighted sections and axial T1 and T2 weighted sections was used, with and without contrast agent. CT-examined monkeys showed severe degenerative changes in multiple joints with the appearance of arthritis. MRI (T2 weighted) with axial images revealed a unilateral enlargement of the posterior cornus of the left lateral ventricle and thin cerebral tissue in this area, typical for a hydrocephalus internus. The implementation of in vivo imaging techniques allows detection of spontaneous background findings in nonhuman primates. This would be useful for excluding specific animals from being assigned to regulatory toxicity studies, and could also be used for efficacy testing in the nonhuman primates.

P207 - Comparison of the Biological Effects of Long-Term Exposure of Human Bronchial Epithelial Cells to Total Particulate Matter From 3R4F Cigarette Smoke or Aerosol From the Candidate Modified Risk Tobacco Product (cMRTP) THS2.2

M. Van der Toorn¹, D. Marescotti¹, S. Johne¹, K. Baumer¹, D. Bornand¹, R. Dulize¹, C. Merg¹, A. Sewer¹, J. Hoeng¹, and K. Luettich¹

¹Philip Morris International R&D (part of Philip Morris International group of companies), Neuchâtel, Switzerland

Chronic cigarette smoke exposure is responsible for airway epithelial changes underlying lung tumorigenesis, although the molecular events are not fully elucidated. We mimicked chronic exposure in smokers’ airways by continuously exposing BEAS-2B cells to TPM from 3R4F cigarette smoke or aerosol of the Tobacco Heating System 2.2 (THS2.2.), a cMRTP, for 12 weeks. Several endpoints, including proliferation, DNA damage, oxidative stress, epithelial-mesenchymal transition (EMT), and wound repair were assessed monthly. Soft-agar assays were performed after 12 weeks, and resulting clones were tested for invasion. Cells were also collected regularly for systems toxicological analysis. Increased oxidative stress and DNA damage were noticeable within the first 2 weeks of 3R4F TPM treatment but subsided thereafter, indicating adaptation. Four-week 3R4F TPM treatment resulted in crisis and EMT. By week 8, cells regained E-cadherin expression, suggesting EMT was reversible. Increased MMP levels and decreased wound repair were noted in 3R4F TPM-treated cells at week 12. However, these changes were not observed following prolonged treatment of BEAS-2B cells with the same or a 5-fold higher concentration of THS2.2 TPM, while a 20-fold higher concentration also increased oxidative burden, DNA damage, and caused reversible EMT. In addition, anchorage-independent growth was observed in 3R4F and THS2.2 TPM-treated BEAS-2B cells. 3R4F TPM-derived clones were invasive, while THS2.2 TPM clones were not. Systems toxicological analysis indicated an overall smaller biological impact of THS2.2 compared with 3R4F TPM, confirming a differential effect of the 2 TPMs. Further analysis by DNA sequencing and in vitro xenograft assay is currently underway.

P208 - Comparison of Rodent Liver Tumor Mode of Actions and Relevance to Humans

A.M. Lynch¹ and J.E. Klaunig²

¹ToxPlus Consulting, LLC, Haymarket, VA, USA

²Indiana University, Bloomington, IN, USA

The liver is a frequent target of tumor formation following chronic exposure of rodents to chemical agents. Our understanding of liver carcinogenesis in rodents and humans has advanced to allow us to define those mechanisms and pathways involved and relate the findings in rodents to potential human risk. Risk assessment models depend on the similarity of rodents to humans when utilizing the data generated using these rodent models. However, limitations of the rodent model exist and include species specific responses as well as extrapolation of high-dose effects to low-dose exposures. To address these limitations, a human relevance framework concept was developed by the International Programme on Chemical Safety (IPCS), which incorporates the identification of a mode of action (MOA). Several MOAs have been identified that can be categorized into genotoxicity (DNA reactive) and nongenotoxicity effects. Nongenotoxic MOAs include receptor-mediated (CAR, PPAR, AHR, hormonal, and PXR) cytotoxicity, inflammation, infection, oxidative stress, and mitogenesis. In addition to the key events, modulating and associated events were identified for each mode of action. Examples of agents that function through each MOA and the relevance of that MOA to humans were reviewed and compared. In the application of rodent MOA findings to human relevance, the importance of dose response and comparative biological responses need to be considered in the setting of regulatory limits and determining the safety of drugs and chemicals.

P209 - Withdrawn

P210 - Vacuolation of Tubular Epithelial Cells in the Kidneys of CByB6F1 Mice in 28-Day Dose Range Finding (DRF) Studies

M. Paranjpe¹, J. Belich¹, M. McKeon¹, and C. Brown¹

¹BioReliance, Rockville, MD, USA

CByB6F1-Tg(HRAS)2Jic (−/− homozygous c-Ha-ras; wildtype littermates of the cross between C57BL/6JJic-Tg[HRAS]2Jic hemizygous male mice with BALB/cByJJic female mice) are commonly used in the DRF studies conducted prior to 6-month carcinogenicity studies conducted in Tg.rasH2 mice. We have been conducting these studies since 2003; however, only recently a unique finding was seen in the kidneys in which there was vacuolation of tubular epithelium in mice treated with vehicle with pH of ≤4.0. This finding was noted in studies conducted between 2014 and 2016. There were a few studies conducted before 2014 that had pH in the vehicle ≤4.0, but we had not seen any similar findings prior to 2014. In these recent studies, in which the pH of the vehicle was ≤4.0, there was clear vacuolation of tubular epithelial cells in the tubules of the cortex extending toward the cortico-medullary interface. This vacuolation was generally of minimal to mild degree. Such vacuoles were clear, the cells were rounded, and the nuclei were centrally placed. Such tubules with vacuolation of epithelium can be easily distinguished from the ones without the vacuoles. In the test article-treated mice of the same studies, if the pH of the formulations remained below ≤4.0, then often the incidence and/or severity of such vacuoles increased with possible exacerbation by the test article. The males were affected more than the females. This poster will also demonstrate photomicrographs of tubules with and without vacuolation of tubular epithelium in the vehicle-treated groups of both sexes.

P211 - The Use of Open Field Testing as Part of a Withdrawal Study on Risperidone in Juvenile, Adolescent, and Adult Rats

A. Rasmussen¹ and K. Puton¹

¹H. Lundbeck A/S, Valby, Denmark

The purpose of this study was to perform an evaluation of the withdrawal effects of risperidone in rats. Animals were treated twice daily for 21 days by oral gavage followed by a 7-day off treatment period. Animals of 3 different age groups were used: juvenile animals, dosing start at day 21 of age—corresponding to a child 2 years of age; adolescent animals, dosing start at day 45 of age—corresponding to a child 12 years of age; adult animals, dosing start at day 90+ of age—corresponding to an adult (16+ years of age). Effects of withdrawal from risperidone were noted in clinical signs, and these were different between the age groups tested. Although not part of a standard withdrawal study, we included an open-field assessment, evaluating the animals during treatment, at the end of treatment, and during the off-treatment period. While it was found that adult and adolescent animals generally demonstrated the same patterns of behavior, they differed somewhat from the juvenile animals with respect to parameters such as mean velocity, distances traveled, and movements with the inner and outer fields. The possible use of open-field assessments as part of a standard withdrawal study is discussed.

P213 - Developing a 2-Component QSAR System to Predict In Vivo Micronucleus Induction

R. Saiakhov¹ and S. Chakravarti¹

¹MultiCASE, Inc, Beachwood, OH, USA

In vivo micronucleus induction assay is an essential part of the regulatory required genotoxicity test battery. As previously reported, a statistical-based QSAR model for evaluation of in vivo micronucleus induction in mice was developed and demonstrated good performance. In this study, we have developed an approach that allows taking advantage of previously available and newly acquired data utilizing statistical and rule-based methodologies. The new system includes: (1) a statistical model, (2) a newly developed rule-based in vivo micronucleus model, and (3) Micronucleus Konsolidator (an expert knowledge tool based on a database of experimental data on in vivo micronucleus tests performed on mice and rats). The Micronucleus Konsolidator also incorporates an analysis of all available supporting information, which helps to resolve “out of domain” and “inconclusive” calls. External validation demonstrates a performance of the rulebased model, exceeding the statistical model in sensitivity by 4% and in negative predictivity by 3%. In addition, the Konsolidator demonstrates improved coverage by 12%, while its performance accuracy lies between those of individual statistical and expert MNT models. We conclude that using both statistical and rulebased QSAR systems coupled with Konsolidator can improve the predictive performance of the in vivo micronucleus suite of models. The effect of adding new data provided by NIEHS Japan is also discussed.

P214 - Drug-Induced Neurotoxicity Assessments: Validation of a Neurological and Functional Observation Battery in the Sinclair Miniature Swine

A. Stricker-Krongrad¹, M. Zhong¹, A. Fuller¹, D. Brocksmith², J. Liu¹, and G. Bouchard^1,2

¹Sinclair Research Center, Columbia, MO, USA

²Sinclair BioResources, Columbia, MO, USA

The central nervous system, along with cardiovascular and respiratory systems, has been listed in the safety pharmacology core battery of the harmonized test guidelines for preclinical testing of human pharmaceuticals (ICH S7A, 2000). A functional observational battery (FOB) has been recommended in the guideline as the first-tier neurotoxicity screening that encompasses motor activity, behavioral changes, coordination, sensor/motor reflex responses, and body temperature. The miniature swine is an appropriate species for neurological and behavioral studies. Its central nervous system and, specifically, the brain are similar to those of human with respect to tissue composition, gyrencephalic structure, and developmental growth and myelination patterns. In addition, behavioral studies, as well as modeling of neurological human diseases, have been increasingly performed in minipigs. Because the miniature swine has increasingly been used as an alternative to dog or nonhuman primate in regulatory toxicology studies, we wanted to validate an FOB for the safety pharmacology assessment of pharmaceutical products intended for human use. A swine FOB protocol was developed with multiple observation parameters to monitor effects on 6 major categories related to sensory and motor functions, autonomic and voluntary functions, and behavior. A crossover study was performed to capture the neurological effects of CNS acting agents administered to the Sinclair minipigs via intramuscular route. Amphetamine, ketamine, and diazepam were demonstrated to positively or negatively dose-dependently affect different parameters related to autonomic functions, excitability, behavior, motor activities, gait, and reflexes.

P215 - Feasibility of Capillary Microsampling for Toxicokinetic Profile Analysis in the Juvenile Rat From Postnatal Day (PND) 4 Onward

D. Perks¹, J. Burnett¹, and G. Weinbauer²

¹Covance Laboratories Ltd, Harrogate, United Kingdom

²Covance Preclinical Services GmbH, Münster, Germany

Microsampling has received considerable interest during recent years within the pharmaceutical industry as this approach provides many scientific, ethical, and cost improvements over existing practices. The objective of the current research was to validate a method for capillary microsampling in the juvenile rat from postnatal day 4 (PND 4) in order to generate toxicokinetic (TK) data. In parallel, the welfare implications of repeated microsampling in juvenile rats were explored. The study was conducted under the authority of the UK Home Office license. Wistar (Han) rats were housed under controlled environmental conditions. Four groups of up to 14 Crl: WI(Han) rats were allocated as follows: group 1 (control), group 2 (no-dose microsampling), group 3 (no in-life bleeds), and group 4 (capillary microsampling). Group 5 comprised 54 animals with traditional TK sampling. Rats (groups 3, 4, and 5) were dosed by the oral (gavage) route with pioglitazone; the control group received vehicle (gelatine 0.6% (w/w) and carboxymethylcellulose sodium 0.9% [w/w] in purified water). Group 2 received no treatment. Clinical signs, body weights, and hematology data were collected during the study. The results showed that changes to body weights and hematology were statistically insignificant between the microsampled groups (groups 2 and 4) and the control groups (groups 1 and 3). These parameters are considered important markers of the erythropoetic effect and useful to highlight any adverse effects of repeated blood sampling in the juvenile animals. The toxicokinetic data generated by the microblood sampling technique were in concordance with the data generated by the standard blood sampling method.

*GFP216 - Changes in Biomarkers Related to λ-Acehalothrine and Chlorpyrifos Exposure Across the Application Season in Adolescents

B. Okeke¹, D. Rohlman¹, A. Ismail^1,3, G. Abdel-Rasoul³, J. Olson², and K. Wang¹

¹University of Iowa, Iowa City, Iowa, USA

²University of Buffalo, Buffalo, New York, USA

³Minoufiya University, Egypt

Organophosphates (OP) and pyrethroid (PYR) compounds are the most widely used insecticides, worldwide. Although there is evidence of OP’s impact on neurobehavioral performance and health outcomes, there is a paucity of data examining the impact of PYR exposure. Furthermore, fewer studies have examined the impact of repeated exposures across the application season or the impact of coexposures. Adolescents’ varied metabolism, rates of growth, and developmental state makes them a unique group due to their increased vulnerability. The goal of this study is to examine the changes over time in PYR metabolite levels in adolescent pesticide applicators and nonapplicators. Urine samples were collected over 5 weeks from the adolescents’ employed (n = 33; times 1-6) and not employed (n = 24; time 1) by the Ministry of Agriculture before, during, and after the PYR application portion of the season. This provided an accurate account of the exposure changes within applicators and versus nonapplicators. These samples were analyzed for Cyhal Acid (μCA), a specific metabolite for λ-Acehalothrine and 3-phenoxybenzoic acid (3-PBA), a nonspecific PYR metabolite. Mean metabolite levels for the applicators were 3.17 μg/creatinine at T1 and 2.82 μg/creatinine for non-applicators showing a difference. Preliminary analysis on the applicators group demonstrated an increase in μCA before (0.12 μg/creatinine; T1) and during application season (0.65 μg/creatinine; T3) with a decrease at the end never fully recovering (0.40 μg/creatinine; T6). Future research will be to understand the association of these metabolite levels with neurobehavioral function and to examine the synergistic effect of the co-exposure to chlorpyrifos and λ-cyhalothrin.

300 Series—Safety Evaluation Nonpharmaceuticals

P301 - AlphaTRAK and ReliOn Confirm Glucometers for Use in Nonclinical Laboratory Testing: Anticoagulant Use and Sample Site Analysis

A. Chesney¹, B. Mancl¹, J. Smith¹, L. Quarberg¹, and M. Schroeder¹

¹Covance Laboratories Inc, Madison, WI, USA

The AlphaTRAK (Zoetis) and the ReliOn Confirm (ARKRAY) glucometers are designed for use in animal models, but their reliability for use with anticoagulants during nonclinical safety studies has not been evaluated. Additionally, the use of alternative, less stressful sample collection sites in monkeys has not been evaluated for the ReliOn Confirm glucometer. This study compared blood glucose values using the AlphaTRAK (in dogs) and the ReliOn Confirm (in nonhuman primates) glucometers with a standard validated clinical chemistry analyzer. Blood was collected from the jugular vein in dogs, and the femoral vein, finger, or tail stick in monkeys. Blood glucose values achieved from AlphaTRAK glucometers were significantly higher than the chemistry analyzer in dogs, regardless of anticoagulant used. In monkeys, average blood glucose values were not significantly different from the chemistry analyzer with added K2EDTA; however, all other anticoagulants had significantly higher levels when obtained from ReliOn Confirm glucometers compared to the chemistry analyzer. There were significant differences in blood glucose values from finger stick and femoral vein collection sites in monkeys. Blood glucose values from tail sticks had the largest variance, suggesting a lack of precision compared to other collection sites. Alternative sample collection sites do not appear to have the precision (tail) or accuracy (finger) necessary to replace the femoral vein. However, these data support being able to use finger or tail prick interchangeably. In comparing studies where anticoagulants or alternative collection sites are used, only studies using the same site or anticoagulant should be compared.

P302 - In Silico Prediction of Acute and Chronic Effects

C. Hasselgren¹, G. Myatt¹, and K. Cross¹

¹Leadscope, Inc, Columbus, OH, USA

There are many situations in which complete toxicological studies are not possible because there is either not enough time to perform the experiment or it is not possible because of cost restrictions or other impediments. To support a rapid response to hazard and risk assessment, new in silico methods based on different (Q)SAR methodologies have been developed to predict acute and chronic toxicity as well as specific organ effects. Multiple (Q)SAR methodologies were developed to predict toxicity categories, such as acute toxicity ranges based on the Globally Harmonized System of Classification and Labeling of Chemicals classifications, eg less than 5 mg/kg, 5 to 50 mg/kg, 50 to 300 mg/kg, 300 to 2,000 mg/kg, 2,000 to 5,000 mg/kg, and greater than 5,000 mg/kg. Training and reference sets were created from existing Leadscope databases that include over 250,000 studies for over 150,000 unique chemicals with acute toxicity data. The toxicity studies were categorized according to their target organ effects and used to identify structural alerts. This poster will present how structural alerts based on specific organ effects are related to study duration. The poster will also present a validation of the categorical prediction models for acute and chronic toxicity ranges.

P303 - Safety Assessment of the Flavoring Agent Perillaldehyde: Genotoxicity Battery

S. Hayashi¹, C. Hobbs², M. Lloyd³, R. Bowen³, L. Lillford³, S. Taylor⁴, and R. Maronpot⁵

¹Japan Flavor and Fragrance Materials Association, Tokyo, Japan

²Integrated Laboratory Systems, Inc, Research Triangle Park, NC, USA

³Covance Laboratories Ltd., Harrogate, United Kingdom

⁴International Organization of the Flavor Industry, Brussels, Belgium

⁵Maronpot Consulting LLC, Raleigh, NC, USA

Perillaldehyde, a natural monocyclic terpenoid found most abundantly in the herb perilla, has a long history of use as a flavoring ingredient to add spiciness and citrus taste to foods. Perillaldehyde is designated by the Ministry of Health, Labor, and Welfare in Japan as unlikely to harm human health, is affirmed as “generally recognized as safe” by the US Flavor and Extract Manufacturers Association, and was judged to be safe by the Food and Agriculture Organization of the United Nations/World Health Organization Joint Expert Committee on Food Additives. In response to a request for more data by the European Food Safety Authority, a genotoxicity battery consisting of a bacterial reverse mutation assay (Ames assay), an in vitro micronucleus assay in human lymphocytes, an HPRT assay in mouse lymphoma cells, and a micronucleus/comet assay in Wistar Han rats was conducted. Although previously reported Salmonella typhimurium strain TA98 assays were negative, and perillaldehyde induced mutation in the absence of metabolic activation in TA98. The comet assay was negative for duodenum; a weakly positive response in liver was measured only at a hepatotoxic dose of perillaldehyde and fell within laboratory historical data. All other genotoxicity assays were negative. In conclusion, these data do not provide indication of any genotoxic potential for perillaldehyde, and they provide the primary basis for recent scientific opinions regarding perillaldehyde genotoxicity announced by several international organizations responsible for safety assessment of food additives and flavorings.

P305 - Can Nonclinical Repolarization Assays Predict the Results of Clinical Thorough QT Studies? A HESI-FDA Retrospective Analysis

E. Park¹, G. Gintant², D. Bi¹, D. Kozeli¹, S. Pettit³, J. Pierson³, M. Skinner⁴, J. Willard¹, T. Wisialowski⁵, J. Koerner¹, and J. Valentin⁶

¹US FDA, Center for Drug Evaluation and Research, Silver Spring, MD, USA

²AbbVie, Integrative Pharmacology, North Chicago, IL, USA

³HESI, Washington, DC, USA

⁴AstraZeneca, Drug Safety and Metabolism, Macclesfield, Cheshire, United Kingdom

⁵Pfizer, Drug Safety Research and Development, Groton, CT, USA

⁶UCB-Biopharma SPRL, Non-Clinical Development, Braine l’Alleud, Belgium

Avoidance of drug-induced QT interval prolongation remains a significant hurdle in drug development. An HESI consortium involving representatives from industry, academia, and government convened the Cardiac Safety Proarrhythmia Working Group to better characterize the utility of nonclinical assays to detect and predict clinical QT interval prolongation utilizing data from submissions to US FDA from 2006 to 2012. This communication summarizes the analysis and discusses strengths and limitations of current nonclinical approaches. A retrospective analysis of an anonymized HESI-FDA database consisting of 150 drugs was conducted. Results from 3 nonclinical assays (functional IKr current block [hERG], cardiac action potential duration (APD), and QTc interval in animals (in vivo QTc) were quantitatively assessed for concordance to clinical thorough QT (TQT) study outcomes based on free drug exposures. Assay sensitivity, specificity, positive and negative predictive values, likelihood ratios, and nonclinical test accuracies (receiver operator curves, ROC) were calculated. All 3 assays demonstrated robust specificity but poor sensitivity at low to intermediate multiples (ie, ≤10×) of the free clinical reference concentration (CRC). The in vivo QTc assay provided the most robust positive and negative predictive values. An integrated analysis demonstrated that sensitivity increased from 1× to 30× the CRC, whereas specificity in the same range was relatively high and the overall concordance achieved a maximum of 0.77 at 30× the CRC. This analysis highlights the strengths and limitations of these nonclinical assays to detect and predict clinical QT prolongation and the need for a more comprehensive mechanism-driven approach focused on proarrhythmic risk.

P306 - Determining the Dermal Absorption of [14C]-Amitraz Using the Triple Pack Approach

J. Thomas¹, N. Pineiro Costas⁷, G. Burin², J. Gerhart³, J. Walraven⁴, C. Parks⁵, and I. Villard⁶

¹Charles River Laboratory, Ashland, OH, USA

²Technology Sciences Group, Inc, Washington, DC, USA

³Merial, Inc, a Sanofi Company, Duluth, GA, USA

⁴Formerly Merial, Inc, a Sanofi Company, Duluth, GA, USA

⁵Virbac Animal Health, Ft. Worth, TX, USA

⁶Véto-pharma, Villebon-sur-Yvette, France

⁷Charles River Laboratory, S Hertogenbosch, the Netherlands

Human dermal absorption is often overestimated by in vivo experiments in rats. Rat in vivo dermal absorption (TG 427) data can be corrected to better estimate human absorption based on in vitro skin absorption assays using rat and human skin (TG 428, the “triple pack”). Human dermal absorption of [¹⁴C]-Amitraz was evaluated using the triple pack approach. Dermal doses equivalent to 20, 60, and 100 μg/cm² of [¹⁴C]-Amitraz were applied to a 10 cm² area; after a 6.5-hour exposure period, radioactivity slowly penetrated the skin of male rats. After washing, a mean of 37% of the applied dose was considered to be absorbed or absorbable among all dose groups. Absorption pathways did not appear to be saturated. During in vitro experiments, dermal doses equivalent to 20, 60, and 100 μg/cm² were applied to a 1 cm² area for 6 hours. The in vitro dermal absorption of Amitraz in corn oil was 1% ± 0.3%, 0.9% ± 0.4%, and 0.8% ± 0.2% through human skin and 4% ± 2%, 6% ± 3%, and 7% ± 3% through rat skin and results in a human/rat absorption ratio of approximately 1:4, 1:6.7, and 1:8.8 at concentrations of 2, 6, and 10 mg Amitraz/mL, respectively. The human skin absorption values are 9.25% (37%/4) at 2 mg/mL, 5.4% (36%/6.7) at 6 mg/mL, and 4.3% (38%/8.8) at 10 mg/mL. The triple pack approach to estimating human dermal absorption has the advantage of addressing differences in absorption between rats and humans without requiring the use of human subjects.

*STP307 - Biomarkers of Fibrosis in Workers Exposed to Multi-Walled Carbon Nanotubes

T. Khaliullin^1,2, L. Fatkhutdinova³, O. Vasilyeva³, R. Zalyalov³, I. Mustafin³, E. Kisin², M. Eileen Birch⁴, N. Yanamala², and A. Shvedova^1,2

¹West Virginia University, Morgantown, WV, USA

²National Institute for Occupational Safety and Health, Morgantown, WV, USA

³Kazan State Medical University, Kazan, Russia

⁴National Institute for Occupational Safety and Health, Cincinnati, OH, USA

Distinctive physicochemical properties of multi-walled carbon nanotubes (MWCNT) offer numerous technological advantages. At the same time, their large-scale manufacturing and broadened applications in the fields of composite materials, energy production, consumer products, and biomedicine raises the likelihood of human exposure to these materials, increasing concerns about their potential adverse health effects. This necessitates the assessment of their potential health effects in humans. The current study was carried out at NanotechCenter Ltd Enterprise (Tambov, Russia), where large-scale manufacturing of MWCNT was established. The goal of this small cross-sectional study was to evaluate the applicability of potential biomarkers of occupational exposure to MWCNT. Air samples were collected at the workplaces using filter-based devices to quantitate amounts of elemental carbon and perform particle analysis by TEM. Biological samples of nasal lavage, induced sputum, and blood serum were obtained from MWCNT-exposed and non-exposed workers for assessment of inflammatory and fibrotic markers. It was found that exposure to MWCNTs caused a significant increase in IL-1β, IL-6, and TNF-α inflammatory cytokines and KL-6, a serological biomarker for interstitial lung disease, in collected sputum samples. Moreover, we found an exposure-related increase of serum TGF-β1 levels in younger workers. Overall, the study has revealed accumulation of inflammatory and fibrotic biomarkers in biofluids of workers handling MWCNTs at the manufacturing facility. The obtained results should be useful for planning the health surveillance programs in nanoindustry. More epidemiological studies involving a greater number of workers and a longer latency period, however, are required to substantiate our findings.

*IGP308 - A Comparative Study on the Neuromuscular Activity and Cognitive Ability of Mice Exposed to Sugar and Stevia

F. Aigbe¹, J. Akinde¹, F. Osuntokun¹, O. Oyebiyi¹, M. Chijioke¹, O. Afolayan¹, and O. Adeyemi¹

¹College of Medicine, University of Lagos, Surulere, Lagos, Nigeria

Stevia has been regarded as a safer sugar substitute with therapeutic benefits. The aim of this study is to compare the effects of sugar and organic stevia (within acceptable dose limits) on neuromuscular activity as well as cognitive functions of mice. Refined table sugar (880-4,048.4 mg/kg) and organic stevia (29.6-148 mg/kg) were orally administered to mice daily for 90 days. During this period, the neuromuscular activity of a set of these mice was assessed weekly using muscle grip, traction, and rota rod tests, while the cognitive ability of another set of treated mice was assessed weekly using Y maze test and, after 90 days, via Morris water maze test. At the expiration of the exposure period, the mice were humanely sacrificed and the frontal lobes and cerebellum of their brain tissues were excised for assessment of oxidative stress biomarkers and histology. In the rota rod test, stevia reduced neuromuscular activity (significant for the first 6 weeks) throughout the exposure period, while sugar reduced (significant for the first week) in the first 5 weeks. Stevia also, more significantly (P < 0.05) than sugar, reduced alternations in Y maze test and increased escape latency in the Morris water maze test. No significant effect on brain weight was observed with stevia or sugar. Stevia significantly, at P < 0.05 and P < 0.01, increased frontal lobes’ malondialdehyde levels when compared to control and sugar-treated mice, respectively. These findings show that stevia induces undesirable changes in neuromuscular and cognitive functions and should, like sugar, be used with caution.

*IGP309 - Tabernaemontana Elegans Decreases Proliferation of HepG2 Hepatocarcinoma Cells with Subsequent Necrotic Cell Death

W. Cordier¹ and V. Steenkamp¹

¹University of Pretoria, Pretoria, Gauteng, South Africa

Herb-induced hepatotoxicity is an increasing phenomenon. Preclinical assessment of herbs is imperative to reduce the occurrence of hepatotoxicity. Tabernaemontana elegans is used ethnomedicinally for the treatment of pulmonary disorders. The aim of the study was to assess the hepatotoxic potential of different extract preparations of the root using an in vitro HepG2 hepatocarcinoma model. After 72 hours exposure to the plant extracts, cell density, mitochondrial membrane potential, oxidative stress, fatty acid accumulation, caspase-3 activation, and ATP levels were determined. Cell death and cellular kinetics was assessed 24 and 72 hours after exposure. The hot water extract was determined not to be cytotoxic below 100 μg/mL, and induced negligible oxidative stress and minimal alteration to mitochondrial parameters. This extract, at 100 μg/mL, induced a G2/M-block (20.93%) after 24 hours, with necrosis (23.79%) after 72 hours The methanol extract was found to be highly cytotoxic (IC50 = 3.07 μg/mL), which was paralleled by mitochondrial depolarization (0.68-fold), reactive oxygen species generation (1.85-fold), fatty acid accumulation (5.80-fold), and caspase-3 activation (50.80-fold). The methanol extract also induced a G2/M-block (9.24%) and necrosis (13.92%), albeit less than the hot water extract. Mechanistically, the extracts resulted in altered bioenergetics (as reflected by fatty acid accumulation and ATP reductions) due to mitochondrial toxicity. Caspase-3 activation would suggest apoptosis, but necrosis was preferred, possibly due to an interruption of pro-apoptotic mediation related to ATP depletion. Necrotic effects may predispose inflammatory hepatitis; thus, caution must be exercised to ensure safety, especially in light of the risks observed with hot water-induced cytotoxicity.

*GFP310 - Cannabinoid Toxicity Versus Antiepileptic Potential Using Zebrafish

D. Carty¹, C. Thornton¹, S. Cutler¹, and K. Willett¹

¹University of Mississippi, University, MS, USA

Dravet syndrome is a pharmaco-resistant form of epilepsy afflicting 1 out of every 21,000 children born in the United States. While cannabidiol (CBD) has shown anecdotal efficacy in reducing seizure frequency, little is known concerning the potential adverse side effects of CBD on brain development and subsequent adult disease. The goal of this project is to compare the relative developmental toxicities of CBD and Δ9-tetrahydrocannabinol (THC) exposure to the effectiveness of cannabinoids as potential anticonvulsants. We measured mortality at 24 and 96 hours postfertilization (hpf), touch response suppression, yolk sac and pericardial edemas, axis mutations, fin deformities, and gene expression of 9 mRNAs critical in morpho-neurogenesis. Additionally, 120 hpf zebrafish were exposed to pentylenetetrazol (PTZ) and select antiepileptic drugs then subsequently assessed using the ViewPoint ZebraBox locomotor tracking system. THC and CBD shared similar adverse morphologic and behavioral outcomes, but lacked similarity in terms of differential gene expression in key morphogenesis/neurogenesis genes. Additionally, CBD proved to be upward of 6 times for potent than THC. Diazepam (50 μM), but not CBD (1.6 μM), significantly reduced PTZ-mediated locomotion compared to control. This comparative approach is highly relevant due to the known adverse effects THC exposure has on humans including brain development deficits and social disorders, while the developmental toxicity of CBD is unknown. Supported by Grant Number P20GM104932 from the National Institute of General Medical Sciences (NIGMS), a component of the National Institutes of Health (NIH).

400 Series—Toxicology Methods

P401 - Predose Phase Echocardiography Assessments for Canine Toxicology Studies: Are 2 Better Than 1?

T.M. Bartko¹, K. Ashcroft-Hawley², N.D. Carneal-Frazer¹, R.M. Harrison¹, J.J. Kremer¹, N.B. Olivier³, and M.A. Osinski¹

¹Covance Laboratories, Madison, WI, USA

²Covance Laboratories, Harrogate, North Yorkshire, United Kingdom

³Michigan State University, College of Veterinary Medicine, East Lansing, MI, USA

Echocardiography is used in nonclinical studies as a noninvasive technique to evaluate heart size and function. Two frequent questions arise: (1) Is there a need for multiple echocardiograph measurements during the predose phase? (2) If so, which collection is more representative of future collections? Data from 4 toxicology studies (2012-2015) were used. Echocardiographs were collected on lightly sedated beagles once daily on 2 consecutive days during the predose phase (up to 208 animals) and once in the dosing phase (36 to 40 days later in up to 51 control animals). Echocardiography parameters of heart size and ventricular function were analyzed from single heartbeats. To assess consistency between the 2 predose collections and between each predose collection and the first on-study collection, a paired t test (alpha : 0.05) was performed to generate 95% confidence limits. Data were generally comparable between the predose collections, with the exception of heart rate when the first collection was higher (21 BPM, males [16-26]; 21 BPM, females [15-26]). Trivial but statistically significant mean differences were also observed in ejection fraction (1.7%, males [0.3-3.0, 95% confidence intervals]; 2.1%, females [0.7-3.4]), stroke volume (1.1 mL, males [0.6-1.6]; 0.5 mL, females [0.1-1.0]), and systolic left ventricular area (−0.11 cm², males [0.21-0.01]; −0.15 cm², females [0.22-0.08]). All other measurements were not statistically different. Despite similarities between predose collections, data from the first predose phase collection showed a higher correlation to the first on-study collection. These data support that a single predose echocardiography assessment is sufficient for most nonclinical canine toxicology studies.

P402 - Immunophenotyping of HPBMC by Multicolored Flow Cytometry Analysis of Cell Surface and Intracellular Markers—Comparison of Fresh Versus Cryopreserved

F. Lauer¹ and S. Burchiel¹

¹University of New Mexico, Albuquerque, NM, USA

Multicolored or multi-fluorescence flow cytometry is a tool by which changes in immune status can be described and monitored in terms of phenotype and function of white blood cells. A change in immune status from the population as a whole demonstrates immune system distress such as illness, infection, or decreased function. We have developed a method using 2 tubes and 11 colors (17 different markers) to describe the immunological phenotypes of the white blood cells found in human peripheral blood mononuclear cells (HPBMC), as well as T cell subsets, which describes the functional aspect of the T cell. This poster describes procedures that allow HPBMC to be purified from whole blood, cryopreserved so that samples can be held for weeks or months, and assayed in groups coming from remote or distant research labs or sites. HPBMC were isolated and cryopreserved followed by analysis of cell surface markers (CSM; T cells, B cells, monocytes, and NK cells) and intracellular staining (ICS) of T cell subsets (Th1, Th2, Th17, and Treg). To validate our procedures, we obtained freshly isolated HPBMC from 5 healthy donors. HPBMC were assayed for CSM and ICS, and the remainder of the cells were cryopreserved. The cryopreserved cells were thawed at various times and assayed for CSM and ICS, and the results were compared with those from the fresh samples. Results demonstrated that multi-fluorescence flow cytometry for CSM and ICS of previously cryopreserved samples is an alternative to the analysis of fresh samples. Supported by NIH RO1 ES-019968.

P403 - Further Evaluation of Chemicals and Mixtures for Skin Sensitization Potential and Potency Using a Reconstructed Human Epithelium (3-D) Tissue Model and the IVSA

M. Troese¹, B. Varsho¹, D. Weisensee², and G. DeGeorge¹

¹MB Research Laboratories, Spinnerstown, PA, USA

²Cell Systems Biotechnologie Vertrieb GmbH, Troisdorf, Germany

Human skin models release IL-18 in response to a wide range of dermal sensitizing chemicals. Using a 3-D skin model (epiCS) in the in vitro sensitization assay (IVSA), we measured IL-18 secretion as a biomarker of sensitization. In this test model, we are able to achieve 90% accuracy when testing 20 pure test chemicals (11 sensitizers, 7 non-sensitizing irritants, and 2 nontoxic materials). Further analysis of the data revealed that test chemical concentrations that induced a 2-fold increase in IL-18 secretion (stimulation index: SI-2) allowed for derivation of potency categorizations. The IL-18 SI-2 was directly proportional to the potency of the sensitizer. A limitation of all other OECD-validated in vitro sensitization assays is that test substances that are mixtures are often not compatible with the test system. An advantage of the epiCS test system is that the 3-D tissues can be topically dosed, like skin, with a wide variety of substances, such as liquids, gels, powders, and waxes. Next, we explored the feasibility of expanding IVSA’s applicability domain. We obtained sensitizing and non-sensitizing mixtures (as listed on their SDS documents) from several commercial sources used in different industries for assessment. These included hair dyes, caulking material, adhesives, antimicrobial fuel additive, and propolis (dietary supplement). A positive response (SI ≥ 2) was detected for all the known sensitizing mixtures. In summary, the IVSA was able to correctly distinguish pure sensitizing chemicals and mixtures from non-sensitizing materials with high accuracy and sensitivity.

P404 - Vaginal Administration of Lactobacilli and Microbiome Analysis in the Gottingen Minipig

C. Günther¹, N. Thomas¹, and K. Gutberlet¹

¹Bayer Pharma AG, Berlin, Germany

A local and systemic toxicity study was conducted in Gottingen minipigs with twice-daily intravaginal administration of hydroxypropylmethylcellulose (HPMC) capsules filled with a probiotic Lactobacillus lyophilisate (LBL). Three groups of 6 females were treated for 4 weeks at doses of 0 (placebo capsules filled with lactose/cellulose: 2 g/animal), 0.8, and 2 g LBL/animal. A further control group remained untreated. Study included a post-treatment observation period of 14 days. The complete set of standard clinical parameters was conducted. Additionally, intravaginal examination, measurement of intravaginal pH, and immunological investigation were performed. The vaginal microbiome was investigated using microbiological analysis and bacterial 16S mRNA PCR. Twice-daily intravaginal dosing of Lactobacillus lyophilisate in HPMC capsules exhibited neither any local irritation in the vagina nor specific pathogenicity due to the LBL used. However, 5 of 18 treated animals (3 verum and 2 placebo treated) showed signs of severe intrauterine infection. Histopathological examination revealed correlating endometritis and metritis, ascending urinary infection, and, in the animals with marked ulcerative endometritis, also degeneration of the liver, acute myocardial degeneration, and lymphoid depletion in thymus, spleen, and lymph nodes. Microbiologic investigation showed that the Lactobacillus strain used neither colonized the vagina nor the uterus. In addition, pH in the vagina stayed around neutral. Microbiologic and PCR analysis showed growth of commonly found facultative pathogen vaginal bacteria (eg, E coli and S aureus) in the intrauterine fluid of investigated infected animals. All other uteri did not show any bacterial growth.

P405 - Effects of Social Housing on Cannibalism in Mice and Rats

H. Hsieh¹, M. Simons¹, J. Hartman¹, and H. Dale¹

¹Covance Laboratories, Inc, Madison, WI, USA

With the increasing focus on animal welfare and 3Rs, there has been a move toward socially housing animals. We investigated whether social housing led to an increase in cannibalism in rats and mice depending on gender, route of administration, and length of study. From January 2013 to May 2016, there were 263 studies with socially housed mice and 989 studies with socially housed rats at Covance Madison. Out of the 263 mice studies, there were 9 studies with 23 instances of cannibalism. The 9 studies could be broken down into 2 acute studies, 2 chronic studies, and 5 carcinogenicity studies. Male mice are not socially housed, so all instances of cannibalism in mice involved females. The majority of instances of cannibalism in mice occurred within the first 20 days (19/23), with the majority involving a single study where the TA was administered intracranially (13/23). From the 989 rat studies, there were 35 studies and 83 cannibalism incidents (46 males, 37 females), with the majority of studies being carcinogenicity studies (24/35) that utilized oral gavage (19/24). In the rats, 14 instances of cannibalism occurred within the first 20 days of the study; however, since there were many longer studies, the majority of instances of cannibalism were from week 30 to the 2-year mark. In conclusion, the move to socially housing rodents resulted in a cannibalism incidence rate of less than 0.1% in rats and female mice.

P406 - Development of an In Vitro Method for the Generation of Nonhuman Primate Cytokines as an Aid to the Establishment of Cytokine Multiplex Assays Used in Support of Preclinical Safety Assessment

K. Hunjan¹, C. Fielding¹, C. Cooper¹, S. Kirk¹, and J. Gaminde¹

¹Covance Laboratories Ltd, Harrogate, United Kingdom

Evaluation of circulating cytokine levels in the nonhuman primate (NHP) provides vital information on the animals’ immune status, particularly during safety evaluation studies for novel biotech products where increased, adverse cytokine production is suspected. Multiplex assays, measuring multiple cytokines, provide a powerful screening tool while requiring limited blood volume. Establishment of a multiplex cytokine kit requires positive controls for validation and, as a source of controls, for monitoring kit performance. Circulating levels of many cytokines in naive NHPs are often below the kit’s lowest detection limits, making it difficult to assess kit suitability. We attempted to generate positive samples of NHP cytokines by taking cynomolgus whole blood (EDTA) and generating peripheral blood mononuclear cells (PBMCs). Subaliquots of PBMCs were cultured with various stimulants (LPS, PWM, PHAN, and PMA) to initiate cytokine release. Sampling at various timepoints during culture established optimum timepoints when each cytokine level was maximal. Cytokine production was evaluated using Merck Millipore Kit Cat# PCYTMG-40K-PX23 (designed specifically for NHP cytokines). Results showed marked increase of certain cytokines (IL-1b; IL-2; IL4; IL-6; IL10; IL13; IFN-γ; TNF-α; IL-8; MCP-1; MIP1-α; MIP1-β, G-CSF), which were maximal at 24 hours. Cytokine levels varied depending on the stimulant used, with IFN-γ production markedly increased in response to PMA, PWM, and PHA but not LPS, whereas IL-1β increased markedly to LPS, PWM, and PHA but not PMA. Minimal or no increase was observed with IL5, IL15, IL12/23 (p40), IL-17a, IL-18, and TGF-α, indicating that alternate stimulants are required to obtain maximal yield.

P407 - Genomic Analysis of L5178y Tk+/− Cells and Their Induced Tk−/− Mutant Colonies

J.N.D. Battey¹, D. Smart¹, N. Sierro¹, D. McHugh¹, P. Vanscheeuwijck¹, M.C. Peitsch¹, and N.V. Ivanov¹

¹PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland

The mouse lymphoma assay (MLA) is a standard method for screening compounds for genotoxicity. The assay measures the induction of trifluorothymidine resistance by loss of thymidine kinase 1 function in L5178Y Tk+/− cells. The cells have only 1 functional allele at the Tk1 locus at the distal end of chromosome 11, and loss of Tk1 function is caused either by loss of this portion of the chromosome, thus resulting in loss of heterozygosity (LOH), or by loss of function in the remaining allele due to local mutations in the gene. The genomic mechanisms by which this occurs are only partially understood, so we here explore the use of next generation sequencing to deepen our understanding. The genome of the L5178Y tk+/− (clone 3.7.2C; IVGT) cell line was resequenced, allowing polymorphic sites relative to the mouse reference genome to be identified and compared to those observed in various mouse species that have been sequenced to date. The polymorphisms were analyzed with respect to their likely functional effects and the mutations possibly explaining L5178Y mutagen sensitivity. Finally, several TFT-resistant mutant colonies, which had been induced by mutagens methyl methanesulfonate and 7,12-dimethylbenz[a]anthracene, were sequenced. It is shown how LOH in TFT-resistant clones can be mapped by surveying known polymorphic sites on chromosome 11.

P408 - Differential Effects of DNA Methylation in Malignant Transformation of BEAS-2B Cells Induced by Radon and Cigarette Smoking

J. Li¹, H. Huang¹, Y. Ji¹, Y. Cao¹, M. Geng¹, and J. Tong¹

¹School of Public Health, Medical College of Soochow University, Suzhou, Jiangsu, China

To investigate the mechanism of lung cancer caused by radon and cigarette smoking, immortalized human bronchial epithelial cells (BEAS-2B) were directly exposed to radon and cigarette smoking on the transwell membrane. After the exposure, cells were cultured until to 15 passages exposure and further growth to 20 passages. Biological characteristics of malignant transformation were detected. Appropriate exposure of radon was 20,000 Bq/m³ at 30 minutes, and exposure of cigarette smoking was 20% and 10 minutes. A series of sequential steps emerged among cells that, exposed to radon and cigarette smoking for 15 to 20 passages, showed similar characteristics of malignant cells. The apoptosis was significantly lower in cells after exposure. The capacity of colony formation rate was significantly lower in the combined group compared to the control group and was up to 24.00% ± 0.31% in soft agar after exposure for 15 passages and generation for 20 passages (P < 0.05). The genome-wide methylation level was higher in the control group than the exposure group. Using the Illumina 450k methylation chip, we screened abnormal methylation-specific genes. CDK5RAP1 genes were much lower in the combined group; SPDEF genes were highly expressed in the single group; ABCG1 genes were highly expressed in the radon group; PTPRM genes were much lower in the cigarette-smoking group. A model of malignant transformation of human cells in vitro induced by radon and cigarette smoking was established. Abnormal methylation genes may play a different role during the malignant transformation of BEAS-2B cells induced by radon and cigarette smoking in vitro.

P409 - Flow Cytometry and Microscopy Method Comparison for In Vivo Micronucleus Studies in Rat Bone Marrow and Peripheral Blood

J. Godin-Ethier¹, F. Merah¹, G. Marceau¹, M. Charlebois¹, S. Thébaud¹, and A. Nelson¹

¹ITR Laboratories Canada, Inc, Montréal, QC, Canada

We compared the relative strengths and limitations of microscopic and flow cytometric methods for micronuclei scoring in rat bone marrow and peripheral blood. Five animal replicates were treated independently at 24-hour intervals. In each replicate, 1 group was treated with cyclophosphamide at 20 mg/kg via intraperitoneal (IP) injection, and a second group was either treated IP with 0.9% sterile saline solution or with purified water via oral gavage as vehicle controls. Bone marrow was collected 24 hours post treatment and evaluated by fluorescence microscopy. Peripheral blood was collected 48 hours post treatment and evaluated by fluorescence microscopy using supravital staining and by flow cytometry. At least 2,000 erythrocytes were evaluated by manual method and over 10,000 by flow cytometry. Overall, the results were as expected with all 3 methods. There were low micronuclei frequencies with the vehicle controls and a significant increase in micronuclei frequencies in cyclophosphamide-treated groups. However, the results percentages obtained with the bone marrow scored manually and peripheral blood scored automatically were closer: (0.13%, 1.99%), (0.13%, 1.57%), with vehicle and cyclophosphamide, respectively. The results obtained with peripheral blood stained supravitally were less evident (0.08%, 0.67%). When using the supravital method, it was difficult to select enough young immature erythrocytes for scoring. The flow cytometry method is clearly superior for analysis of peripheral blood, while the standard fluorescent microscopy remains a reliable method for analyzing bone marrow preparations.

P410 - Flow Cytometry for In Vitro Assessment of Genotoxic Substances

F. Merah¹, G. Marceau¹, S. Lavallée¹, F. Leroux¹, and A. Nelson¹

¹ITR Laboratories Canada, Inc, Montréal, QC, Canada

Flow cytometric analysis offers new possibilities in genetic toxicology. Here, we detail the implementation of the micronucleus assay by flow cytometry. CHO-K1 cells were exposed to vehicle or genotoxins (cyclophosphamide, CPA; colchicine, CLC; and mitomycin C, MMC) in 2 modes of exposure: short (approx 4 hours with additional 24 hours in normal media) and extended (approx 26 hours). Dead cells were tagged with ethidium monoazide and then lysed and labeled with a DNA dye. DNA fluorescence intensity was acquired on a flow cytometer for the quantitation of haploid/diploid nuclei, hypodiploid nuclei, and micronuclei. The relative population doubling of each sample was calculated with the help of fluorescent beads. In short exposure, the relative amount of hypodiploid nuclei and micronuclei for the vehicle were 0.2% and 1.3%, respectively. With CLC (0.1-0.3 ng/mL), hypodiploid nuclei were 0.2% to 3.0% and micronuclei 1.5% to 6.1%. In short exposure with metabolic activation, hypodiploid nuclei and micronuclei for the vehicle control were 0.4% to 2.9%, respectively. With CPA (1-3 ng/mL), hypodiploid nuclei were 0.6% to 0.7% and micronuclei 6.0% to 13.5%. In extended exposure, hypodiploid nuclei and micronuclei for the vehicle were 0.1% to 1.2%, respectively. With MMC (0.05-0.25 ng/ mL), the relative amounts of hypodiploid nuclei were from 0.4% to 0.5% and micronuclei from 5.2% to 11.6%. Exposure with CPA and MMC induced dose-dependent increases in micronuclei in the short and extended protocols. Exposure with CLC induced dose-dependent increases in both hypodiploid and micronuclei. The analysis of in vitro nuclei by flow cytometry provides an alternative to microscopic slides reading, with capacity for high throughput.

P411 - Effects of Sedation by Acepromazine on Routine Clinical Pathology Parameters and Intraocular Pressure in Rabbits

J. Miller¹, T. Arndt¹, S. Stake¹, K. Perronne¹, S. Schumacher¹, and A. Kamholz¹

¹Covance Laboratories Inc, Madison, WI, USA

We evaluated the effect of acepromazine (ACE) on clinical pathology (CP) parameters and intraocular pressure (IOP) when administered via subcutaneous injection to 2 rabbit strains (New Zealand White [NZW] and Dutch Belted [DB]): male and female NZWs (2 groups, 5 animals/sex/group) and male DBs (2 groups, 3 animals/group). Animals were dosed with saline (control) or ACE. IOPs were obtained and CP samples collected before and approximately 30 minutes after dosing. Compared with predose and control values, minor differences in post-dose CP results included lower red cell mass and total and absolute differential white blood cell counts; minor electrolyte alterations were noted, but no clear effect was identified. Postdose covariate-adjusted mean IOPs of NZWs were not affected by ACE for males, but were significantly lower for females administered ACE. Post-dose covariate-adjusted mean IOPs of ACE-treated DBs were significantly lower compared with control DBs; however, this was due to an increase in IOP (approximately 3.5 mm Hg) relative to predose for the control group. IOPs of the ACE-treated DB group were comparable with predose values. Increased IOPs for the control DBs was considered due to stress from handling, restraint, and increased room activity for IOP measurement and CP sample collection. In conclusion, the use of ACE as a presample collection sedative for either rabbit strain had no consistent impact on IOPs, though may have resulted in a decrease for NZW females. Minor differences in routine CP tests were small in magnitude and not considered adverse or markers of adversity and therefore considered acceptable.

P412 - Impact of Social Housing on Time-Mated Rabbits in an Embryo-Fetal Development Study Design

M. O’Hara², P. Knepley¹, A. Fetter¹, P. Parsons¹, and K. Derfler¹

¹Covance Research Products, Inc, Denver, PA, USA

²Covance Laboratories, Greenfield, IN, USA

Sixty female NZW rabbits housed in a commercial production facility were randomized into a group of 30 single-housed rabbits and a group of 30 pair-housed rabbits (15 pairs). Every effort was made to eliminate other variables between the 2 groups: same caging design, location within building, caretakers, feed program, microenvironment, etc. From age 15 weeks until breeding at age 25 weeks, all pair-housed rabbits remained compatible but exhibited a 20% incidence of minor cosmetic defects (hair barbering, skin nicks) associated with normal social interactions. Upon breeding, 14 of the 15 pairs rapidly became socially incompatible. Effort to achieve 100% conception rates in the socially housed rabbits was double that of individually housed rabbits. Social housing was therefore found to be incompatible with the embryo-fetal development study design.

P413 - Human Pluripotent Stem Cell-Based Assay Predicts Developmental Toxicity Potential of ToxCast Chemicals

J. Palmer¹, T. Knudsen², L. Egnash¹, P. Kothiya², K. Houck², and E. Donley¹

¹Stemina Biomarker Discovery, Inc, Madison, WI, USA

²National Center for Computational Toxicology, US EPA, Research Triangle Park, NC, USA

Worldwide initiatives to screen for toxicity potential among the thousands of chemicals currently in use require inexpensive and high-throughput in vitro models to meet their goals. The devTOX quickPredict platform is an in vitro human pluripotent stem cell-based assay used to assess a wide range of chemicals (ie, pharmaceutical, environmental, and industrial compounds) for potential developmental toxicity affecting differing developmental lineages. The assay is being used by the United States Environmental Protection Agency (EPA) to screen the ToxCast chemical library in support of Tox21. A 2-tier testing strategy was employed to screen a total of 1,066 chemicals in human embryonic stem (hES) cells, guided by the AC50 (half maximal activity concentration) across multiple cytotoxicity assays in ToxCast. To date, 347 chemicals were tested in an 8 concentration dose-response in this assay. Spent media was collected to measure changes in biomarkers of developmental toxicity (ornithine and cystine) together with cell viability measurements. A preliminary analysis revealed a signal in 15% to 18% of all ToxCast chemicals tested based on a default threshold biomarker ratio (ORN/CYSS) <0.88. In most of these cases the concentration producing an effect in the biomarker ratio fell below the AC50 for cell viability. Model performance (28-compound training set) showed a balanced accuracy of 82% (sensitivity 0.71, specificity 1.0). The data presented here demonstrate the utility of the assay in screening and prioritizing compounds for further testing. (Disclaimer: this abstract does not reflect EPA policy.)

P414 - Communications on Nonclinical Studies in the Electronic Era

L. Pan¹, X. Ruan¹, H. Lv¹, and S. McPherson¹

¹WuXi AppTec (Suzhou) Co. Ltd, Suzhou, China

During conduct of nonclinical studies, study monitors need to monitor studies. This can be achieved by onsite monitoring of the ongoing studies or, more often, due to geography and travel constraints rely on the study director’s (SD’s) routine verbal or written updates. For all studies, data transfer is important, the nature and frequency of the transfers being dependent on the type of study and responses observed. With many CROs collecting data electronically, these transfers are simple. Yet to maintain protection, these transfers can be cumbersome in that the files need to be password protected and the sponsor is reliant on the SD extracting the data files. To overcome this, some CROs/pharmas have developed their own portals whereby the SD uploads the study data and documents. The advantage of these systems is that there is no need to password-protect the files. One step above are systems that allow the client to view the data in real time. The systems are secure in that the client can only view the studies and data that were assigned by the system administrator and the SD. Study data are available in table or graph form and can also be exported for local storage. The systems can also be used for transferring other study files, especially those of large volume. By using such secure systems, the study monitor is able to access the electronic data set directly and monitor the studies more closely, no matter their geographic location.

P415 - Acute and Repeated Effects of the Intra-Articular Administration in Rats and Dogs

M. Pecoraro¹, I. Sartori¹, P. Griffini¹, A. Ferrara¹, and I. Faustinelli¹

¹Aptuit, Verona, Italy

Osteoarthritis is a chronic degenerative disease, slowly progressive and irreversible, affecting the adult population and causing pain that reduces quality of life. Pharmacological treatments mainly consisted of NSAIDs and corticosteroids. Adverse effects of systemic therapies are well known; therefore, intra-articular injection can provide a useful alternative given the local effect on their site of action. In order to support clinical studies, regulatory agencies require preclinical toxicology studies in rodent and nonrodents. The amount of information available on the use of intra-articular injection in rats and dogs are limited. The aim of these studies was to assess the feasibility and tolerability of the repeated intra-articular injections in rats and dogs. Animals were treated by intra-articular injection (fixed volume) once a week for 5 weeks in the right femorotibial joint and euthanized approximately 24 hours or 15 days after the last administration. The following endpoint/parameters were evaluated: body weight, food consumption, clinical observations, clinical pathology, organ weights, macroscopic observations, and microscopic observations of the injection site. No changes in body weight, clinical pathology, or organ weight were observed. Slight bleeding of the injection site and/or slight to moderate dark or red area around the injection site were observed immediately after dose administration in a few animals. Microscopically, inflammation of the synovial membrane, occasionally associated with hemorrhage of the subcutaneous tissue surrounding the femorotibial joint, was observed that generally correlated with red areas observed macroscopically. Full recovery of microscopic findings was observed after 14-day recovery period.

P416 - Prenatal and Early Postnatal Development of the Thyroid Gland in the Rat: Histologic and Morphometric Endpoints

C. Picut¹, C. Swanson¹, T. McGiver¹, M. Acciani¹, and E. Parker¹

¹Charles River Laboratories Hillsborough, LLC, Hillsborough, NC, USA

The late fetal and early postnatal period in the rat is purported to be a critical time point for evaluation of thyroid endpoints in order to detect goitrogens because after GD 18, the animal is no longer dependent on maternal thyroid hormone. In order to establish the baseline values for histopathology and morphometry endpoints over this time period, we collected thyroid glands from normal rats GD 15 through PND 50 (2-3 males and 2-3 females per day), and stained serial sections with H&E, PAS, and Ki67 (immunohistochemistry). The slides were scanned using a Hamamatsu nanozoomer, and the height of follicular epithelial cells, the relative amounts of colloid, and the proliferation index were evaluated with Visiopharm software. The results show that epithelial height is maximum at GD 18, dramatically declines through PND 4, and thereafter plateaus until PND 40 when height returns to its maximum. Colloid is first observable at GD 18, increases to 35% of the gland mass by PND 4, and reaches a stable maximum by PND 30. Proliferation index is maximum at GD18, declines precipitously by PND 5, and thereafter stabilizes except for a brief spike at PND 25. Sexual dimorphism manifests with females having consistently higher epithelial height from PND 4 to PND 40, males having higher height after PND 40, and females having more colloid after PND 30. Histopathology and morphometry are relatively simple endpoints, compared to thyroid weight or serum hormone levels, and may be routinely incorporated in safety assessment studies designed to identify endocrine disruptors.

P418 - Genotoxicity Assessments in Metabolically Competent Human HepaRG Cells: MN and CometChip

L. Recio¹, J. Winters¹, K. Ketner¹, C. Yauk², J. Buick², L. Ngo³, B. Engleward³, and C. Swartz¹

¹ILS, RTP, NC, USA

²Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON Canada

³Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

Human hepatoma-derived HepaRGTM cells retain many of the metabolic capabilities of primary human hepatocytes and represent a readily available model of metabolically competent cells for use in genetic toxicity testing. Integrated Laboratory Systems (ILS) is developing a model for assessing genotoxicity using HepaRGTM cells, with a goal toward developing medium throughput assays for micronucleus induction and DNA damage without the need for addition of exogenous S9. In collaboration with researchers at MIT and Health Canada, ILS has successfully demonstrated the ability of HepaRGTM cells to detect genetic damage. HepaRGTM cells seeded into collagen-coated plates were allowed to recover metabolic functions for 7 days, then exposed to test compounds daily for 3 days. Using Litron’s In Vitro Microflow kit, chromosomal damage was detected with 100% specificity and 100% sensitivity following exposure to 6 non-genotoxins and 8 known genotoxins, including 1 non-DNA reactive genotoxin and 3 genotoxins requiring metabolic activation. In addition, expression analysis of several metabolism-related genes evaluated in cyclophosphamide-exposed HepaRGTM cells demonstrated dose-related increases in expression of CYP2B6, CYP3A4, and UGT1A1, indicating appropriate metabolic functionality of the exposed cells. In a side-by-side comparison of CometChip (Trevigen) and the comet assay using standard 2-well Trevigen slides, exposure to EMS, MMS, cyclophosphamide, and benzo[a]pyrene resulted in similar response patterns between the 2 formats. The same exposures performed in 96-well culture plates allowed direct well-to-well transfer to the CometChip, thus increasing assay efficiency. Assays incorporating HepaRGTM cells can provide potential follow-up to positive results from S9-based in vitro assays used for regulatory testing.

P420 - Analytical Validation for Peripheral Blood Immunophenotyping of Myeloid, T, and B Cell Populations in Cynomolgus Macaques

A. Rowse¹, J. Puchalski¹, H. Hu², J. Holroyd³, and A. Frantz¹

¹Covance Laboratories Inc, Madison, WI, USA

²Covance Laboratories Inc, Shanghai, China

³Covance Laboratories Inc, Harrogate, United Kingdom

The purpose of this study was to validate myeloid, T regulatory (Treg), T cell, and B cell antibody panels for immunophenotyping evaluation of peripheral blood in cynomolgus macaques. The myeloid panel identifies plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), and stem cells and includes lineage markers (CD3, CD14, CD20), DC markers (HLA-DR, CD123, BDCA-2, CD11c), and the stem cell marker CD34. The Treg panel includes T cell lineage markers (CD3, CD4, CD8) as well as activation markers (CD95, CD25) and the intracellular Treg marker Foxp3. The T cell panel allows identification of central and effector memory cells and includes T cell lineage markers (CD3, CD4, CD8), activation/ memory markers (CD95, CD28, CCR7), and the proliferation marker Ki67. The B cell panel includes B cell lineage markers (CD19, CD20, CD40), differentiation markers (IgD, CD27), and the proliferation marker Ki67. We validated a total of 76 phenotypes, including 3 myeloid populations, 26 Treg phenotypes, 29 T cell phenotypes, and 18 B cell phenotypes, representing an exhaustive list of potential populations of interest. Furthermore, we assessed intra-assay precision and interassay precision, as well as interanalyst precision for each of the 4 antibody panels. For precision measurements, we were able to meet the acceptance criteria with CV of <25% for phenotypes comprising >5% of the parent population (lymphocytes or, for the myeloid panel, lymphocytes and monocytes). Phenotypes that comprised <5% of the parent population had a greater variation with the CV usually >25%.

P421 - Metabolites, Lipids, N-Glycans, and Histology on the Same Biopsy

L. Liang¹, R. Drake², H. Tao², and D. Troyer³

¹University of Alberta, Edmonton, Alberta, Canada

²Medical University of South Carolina, Charleston, SC, USA

³Eastern Virginia Medical School, Norfolk, VA, USA

Deriving more information from tissue samples deepens the toxicologic, diagnostic, and prognostic information obtained from sometimes-scarce biopsies. We use a histology workflow, which allows upstream detection of lipids and metabolites and downstream glycoprotein profiling. Histopathology is a standard procedure that consumes tissues and requires intact architecture. Traditionally, tissues are fixed in formaldehyde and processed into paraffin to preserve architecture for morphological studies. Powerful genomic and proteomic assays are used for the assay of macromolecules in paraffin-embedded tissues. These methods require extraction and disruption of tissue architecture and therefore consume tissue. We have changed the workflow by using alcohol as a primary tissue fixative, followed by secondary fixation in formalin and embedding in paraffin. When alcohol is used as a primary tissue fixative, metabolites and lipids are extracted into solution while tissue architecture is preserved. Liquid chromatography-mass spectroscopy (LC-MS) is used to assay lipids and metabolites in the alcohol. About 2,500 metabolites have been positively or putatively identified in small tissue biopsies (5 mg). Metabolite normalization and quantitation are enabled using a universal metabolome standard combined with chemical labeling (dansylation). Sphingomyelin, phosphatidylcholine, and over 216 lipid peaks were detected. Furthermore, N-glycan profiles can also be obtained from histologic sections of the same paraffin-embedded tissues using MALDI imaging spectrometry, yielding more information downstream. The upstream metabolite/lipid data and the downstream glycoprotein profiling are obtained without disrupting tissue architecture. Thus, existing methods that use paraffin-embedded tissues can be performed on the exact same tissue sample.

P422 - The h-CLAT for Assessment of Dermal Sensitization Potency of Commercially Available Mixtures and the OECD Proficiency Chemicals

M. Carathers¹, B. Varsho¹, P. Vij¹, and G. DeGeorge¹

¹MB Research Laboratories, Spinnerstown, PA, USA

In order to explore the applicability domain for more pure chemicals and mixtures (including commercial products), we performed a series of studies using the Human Cell Line Activation Test (h-CLAT) protocol under the latest OECD Test Guideline. THP-1 cell expression of CD86 and CD54 from a new, expanded set of validation chemicals and several complex mixtures were measured. A variety of products from the petroleum, agrochemical, food, beauty, and chemical industries were obtained via retail outlets and evaluated. Known positive (via safety data sheets) mixtures were assessed along with similar nonsensitizing mixtures. These included non-PPD-containing hair dye, propolis extract, diesel fuel additive, a pesticide, and commercial acrylate-based sealants. In addition to mixtures, we evaluated the OECD proficiency test chemicals, which includes DNCB, phenylenediamine, nickel sulfate, 2-mercaptobenzothiazole, R(+) limonene, imidazolidinyl urea, and the nonsensitizers, lactic acid, isopropanol, glycerol, and 4-aminobenzoic acid. All chemicals were able to be exposed at a low or nonirritating concentration, yielding a CV75 or higher viability, as determined by flow cytometry. Sensitizer potency was measured by the concentration of test chemical that induced a relative fluorescence intensity (RFI) that was a threshold-positive response (CD86 = 200%, CD54 = 150%) of control. Two sets of draft OECD guidelines proficiency chemicals were tested for a total of 16 pure chemicals (6 non-sensitizers and 9 sensitizers). The h-CLAT correctly predicted 9 of 9 sensitizing and 5 of 6 non-sensitizing chemicals, for an overall accuracy of 93.7%.

P423 - Insulin Deficiency Induction in Male Beagle Dogs to Establish a Reliable Disease Model for the Serial Evaluation of Peripheral Blood Glucose Levels Following Insulin Treatment

K. Voss¹, J. Kieper¹, N. Adkins¹, J. Bova¹, and B. Roche¹

¹Charles River Laboratories Ashland, LLC, Ashland, OH, USA

Millions of people are afflicted with diabetes, a chronic disease in which the body either does not produce enough insulin or effectively utilize the insulin it does produce. Disease management is pivotal to survivability and quality of life; therefore, there is a large focus to increase adherence to a patient’s disease management plans. New drugs are being developed and marketed to increase end-user compliance, which requires a reliable preclinical disease model to effectively evaluate these innovative compounds. This study established a colony of 6 insulin-deficient dogs as a model for evaluating blood glucose levels following novel insulin treatments. Induction of insulin deficiency was accomplished by an intravenous injection of alloxan (50 mg/kg at 200 mg/mL) and streptozotocin (30 mg/kg at 120 mg/mL) in 0.05 M citrate buffer, pH 4.5 ± 0.1. Veterinary care was implemented prior to and during chemical induction of the disease to ensure animal safety. Blood glucose levels were evaluated and maintained with subcutaneous injections of insulin every 12 hours and following feeding. Animals were maintained in this disease state for up to 3 months with clinical pathology screenings, body weights, and daily blood glucose levels measured for evaluation of animal health. Serial oral glucose tolerance tests (OGTTs) were conducted after induction of insulin deficiency. Consistency of results from each dog’s repeated OGTT confirmed the disease state through quantification of each area under the curve. In conclusion, chemical induction of insulin deficiency in these dogs resulted in a reliable model of disease for evaluation of novel insulin treatments.

P424 - Comparison of Clinical Pathology Data From Pen/Group-Housed and Individually Housed Cynomolgus Monkeys

C. Voyer¹, A. Provencher¹, and N. Hebert¹

¹Charles River Laboratories, Sherbrooke, Québec, Canada

In alignment with the 3Rs, a higher demand for group-housed nonhuman primates (NHPs) has been seen over the historical individually housed paradigm. In support of these housing changes and overall 3R initiatives, our objective was to evaluate potential differences related to housing conditions on standard clinical pathology parameters by comparing baseline data from pen/group-housed animals versus individually housed animals. Clinical pathology data from 93 male and 92 female pen/group-housed NHPs were compared with data from 94 and 64 female individually housed NHPs. All animals were experimentally naive, healthy, adult (2 to 6 years old) cynomolgus monkeys, fed with the same diet and maintained under comparable room environmental conditions. Clinical pathology evaluations were performed with the following analyzers: Siemens Advia 120 (hematology), Stago STAR 4 (coagulation), and Roche P800 (biochemistry). Statistically significant changes were seen in hematology, coagulation, and biochemistry. Taking into account expected biological variability for the species and assay methodology, noteworthy changes were 71% to 73% decreased neutrophil, 37% to 46% increased lymphocyte, and 85% to 62% increased eosinophils counts, possibly indicative of lower stress levels in the pen/group-housed animals. Clinical chemistry changes consisted of 20% to 27% increased urea, 86% to 89% decreased phosphorus, 5% to 5% decreased total protein, and 8% to 11% decreased globulin. As comparison to baseline and to concurrent controls data determines potential experimental compound effect on clinical pathology, the observed differences were not considered an influence on overall outcome and interpretation for pen/group-housed NHPs within a given nonclinical study, but housing should be considered if/when assessing data across studies for a given species.

*GFP425 - Heritable Environmental Effects Mediated by Germline Chromatin Alterations

J. Camacho¹, N. Gentry¹, G. Gutierrez¹, L. Hosohama¹, A. Clark¹, and P. Allard¹

¹UCLA, Los Angeles, CA, USA

Dynamic remodeling of chromatin imparts an epigenetic regulatory role in several biological processes. Our goal is to examine the influence of environmental chemicals on the epigenetic remodeling and chromatin silencing of germ cells. Germ cells are an especially important context in examining alterations in histone modification patterns, as they undergo periods of profound DNA demethylation and consequently have a high reliance on histone marks to ensure genomic stability. However, these events take place in mammals early during embryogenesis, which creates issues of accessibility to the cells and ease of study. Here, we utilize the powerful genetic system Caenorhabditis elegans. Our approach uses a reporter strain, where GFP is epigenetically silenced in the germline, to examine epigenetic regulation after chemical exposure. Results indicate that de-silencing effects after exposure to the environmental compound bisphenol-A (BPA) last for at least 3 generations (up to 3-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. Additionally, we have linked possible toxic effects in C. elegans caused by chemical disruption of chromatin through environmental exposure. We found a significant increase (**P ≤ 0.01) in embryonic lethality in offspring of worms exposed to BPA, and these effects can be reversed after subsequent exposure to drugs that promote the maintenance of repressive marks. Together, our latest work hopes to establish the epigenetic pathway requirements for transgenerational environmental effects.

*GFP426 - Expression of Pulmonary Inflammatory Cytokines: Influence of Excessive Ethanol Exposure on BEAS-2B Cells and Antimicrobial Peptides, Ll-37

O. Ogunsakin¹, T. Hottor¹, and M. McCaskill¹

¹Tulane University, New Orleans, LA, USA

Pulmonary inflammatory cytokines’ expression can be influenced by chronic exposure to excessive ethanol concentration. Fewer research projects have been carried out to elucidate mechanisms of ethanol’s adverse effect and the association between pulmonary cells, vitamin D, cathelicidin/LL-37 (antimicrobial peptides) and pulmonary inflammatory cytokines. Depletion of vitamin D levels through pathway disruption and/or altered expression of inflammatory cytokines may result in potential pulmonary toxicity. The objective of this project was to understand toxicological mechanisms through which excessive ethanol exposure influences antimicrobial peptides in pulmonary epithelium. BEAS-2B cells, immortalized broncho-epithelial cells, were grown and cultured according to manufacturer’s protocol. The cultured cells were treated with varying concentrations of 1 μM diallyl disulfide (DADS), 80 mM ethanol (EtOH), and varying concentrations of calcidiol for 10 hours. The resulting homogenate were assayed using commercial cathelicidin/LL-37 ELISA kit, inactive vitamin D Immunodiagnostic Systems ELISA kit, and Multi-Analyte ELISArray Kit (MEH-004A). Excessive ethanol exposure reduced levels of vitamin D by 40% in the treated BEAS-2B cells. At varying concentration, cells treated with calcidiol had higher levels of cathelicidin/LL-37 when compared to a control group. However, increasing levels of LL-37 protein peaked at 50 ng/mL, because at 100 ng/mL, a statistically significant reduction (*P < 0.05) in levels of LL-37 protein was observed. In addition, significant expression of IL1α, IL1β, IL8, and GM-CSF, *P < 0.05, were noticed. Vitamin D and LL-37 are actively involved in immune functions against respiratory infections. Chronic ethanol exposure among vulnerable populations can alter their functions and expression of inflammatory cytokines.

500 Series—Safety Evaluation Pharmaceuticals

P501 - SPARCL: Use of a Novel Technology in Validation of a Nonhuman Primate and Rat Cardiac Troponin-I Assay in Serum

I. Boulay¹, J. Derek Ng Yan Hing¹, K. Dumaresq-Doiron¹, J. Mercier¹, R. Riffon¹, C. Chadwick², and S. Authier^1,3

¹CiToxLAB, Laval, Québec, Canada

²Life Diagnostics, West Chester, PA, USA

³Faculty of Veterinary Medicine, University of Montréal, Saint-Hyacinthe, Québec, Canada

Cardiac troponin-I (cTnI) is released following cardiac muscle injury. Historically quantified by ELISA, an alternative assay methodology was explored for analysis of cTnI in toxicology studies. The SPARCL (Spatial Proximity Analyte Reagent Capture Luminescence) assay lies on proximity assay in homogenous phase. Two affinity purified cTnI-specific antibodies were used, one conjugated to HRP and one to acridan, a chemiluminescent substrate. When these 2 antibodies are brought into close proximity due to specific binding and when a solution containing hydrogen peroxide is added, HRP catalyzes oxidation of proximal acridan molecules, causing a chemiluminescent reaction, which is captured by a plate-based luminometer. To characterize assay performance, samples were obtained after coronary ligature, which increased the cTnI levels over 8 hours. Samples were used to define precision, ruggedness, parallelism, stability, and minimum required dilution. An acceptable standard curve fit was obtained between 0.1 and 5.0 ng/mL. Parallelism was demonstrated using serum samples from different NHPs and rats to span the widest range of dilution folds. Samples at low and high concentrations were used to demonstrate stability after 3 freeze-thaws, overnight at room temperature and after long-term storage at −70°C. In conclusion, a SPARCL assay for the quantification of cTnI in NHP and rat serum was validated. The assay presented short assay run time, and allowed a high throughput for method validation and a sample analysis with an analytical range that covered relevant concentrations upon cardiac injury in NHP and rat.

P502 - Comparison of Multi-Ion Channel Inhibition Profiles

H. Huang¹, M. Pugsley², R. Forster¹, and S. Authier^1,3

¹CiToxLAB, Laval, Québec, Canada

²Purdue Pharma L.P., Cranbury, NJ, USA

³Faculty of Veterinary Medicine, University of Montréal, Saint-Hyacinthe, Québec, Canada

hERG/IKr inhibition has been considered a major risk factor for arrhythmia in drug development for nearly 2 decades with known limitations. A mechanistic understanding of ion channel interaction during cardiac action potential has emerged as a strategy to identify proarrhythmic risk, beyond a simple measure of hERG/IKr inhibition. Currently, multiple ion channel inhibition screening is proposed to evaluate early and late repolarization effects. The purpose of this analysis was to evaluate a quantitative method to compare multiion channel inhibition profiles. HEK 293 cells with stable expression were used in manual whole cell configuration. IC50 values for ion channels (Nav1. 5, Kv4. 3, Cav1. 2, hERG, and Kir2. 1) were normalized for IKr inhibition, and the resulting ratios were subjected to statistical comparison. Cisapride showed a dose-dependent inhibition on multiple channels with IC50 values (μM) of 0.0138 for hERG, 0.81 for Cav1.2, 20.2 for Nav1.5, 15.4 for Kv4.3, and >10 μM for Kir 2.1. The IC50 values (μM) obtained from terfenadine were 0.018 (hERG), 0.71 (Cav1.2), 4.8 (Nav1.5), 3.26 (Kv4.3), and >10 (Kir 2.1). For verapamil, IC50 values (μM) were 0.53 (hERG), 16.7 (Cav1.2), 11.9 (Nav1.5), >10 (Kv4.3), and >100 (Kir 2.1), respectively. After normalizing to hERG IC50, cisapride ratios were significantly different from verapamil (P = .04). Ratios for terfenadine versus cisapride were not significantly different. We concluded that multiple ion channel inhibition can help evaluate potential effects on the cardiac action potential and estimate the proarrhythmic risk in patients. This analysis evaluated a simple quantitative strategy to compare multiple ion channel inhibition profiles.

P503 - Seizure Liability Assessments Using Hippocampal Brain Slice: Comparison of Multiple Preclinical Species

M. Accardi¹, R. Forster¹, and S. Authier^1,2

¹CiToxLAB, Laval, Québec, Canada

²Faculty of Veterinary Medicine, University of Montréal, Saint-Hyacinthe, Québec, Canada

Hippocampal brain slice can be used as an in vitro assay to characterize seizure liability early in drug development. Traditionally, this assay has relied upon the rat hippocampal slice, which sometime possesses limited translational value. Species differences are often noted during in vivo seizure liability assessments, and drug safety testing may benefit from early ranking of the various animal models. Transverse hippocampal brain slices were isolated from animal models (eg, rat, minipig, dog, nonhuman primate). Population spikes (PS) were evoked through Schaffer collateral pathway stimulation via a concentric bipolar stimulating electrode and recorded using conventional in vitro electrophysiological techniques via an extracellular electrode placed into the CA1 cell body layer. Preclinical animal hippocampal slices displayed a concentration-dependent increase in PS area and number in the presence of the proconvulsant pentylenetetrazol (PTZ; 0.1-10 mM). However, nuanced and distinct differences were observed in certain species with respect to their sensitivity to PTZ. For instance, PS area reached significance at lower thresholds in nonhuman primates compared to rats (1 vs 10 mM), whereas rats demonstrated greater evoked PS numbers when compared to monkeys (2.3 vs 1.8). Our results suggest that the nuanced differences between hippocampal slices isolated from different preclinical animal models could influence the design of nonclinical seizure liability studies and their associated data interpretation. Hippocampal brain slices from multiple preclinical animal models can be a valuable approach to complement seizure risk assessment.

P504 - Comparison of Selected Densitometry Endpoints in Single and Socially Housed Sprague-Dawley Female Rats

M. Boyer¹, J. Jouniaux¹, A. Varela¹, and S.Y. Smith¹

¹Charles River Laboratories Montréal ULC, Senneville, Québec, Canada

In view of the current requirements to socially house animals in support of 3R initiatives, selected densitometry parameters from singly and socially housed ovariectomized (OVX) and non-OVX control female Sprague-Dawley rats were evaluated in order to determine if social housing could have an impact on bone density, area, and mineral content. Single- housed and socially housed animals were kept in mesh-bottom cages of appropriate size and provided with comparable diet during the evaluation periods. Whole body, femur, and vertebrae were evaluated using dual energy X-ray absorptiometry (DXA). The regions of interest included whole body, spine, and femur, and the selected parameters included bone mineral density (BMD), bone mineral content (BMC), and area. The age of the animals was taken into consideration during the evaluations. There were no clear trends observed that would be indicative of a difference related to the housing conditions of the animals in any of the parameters evaluated. In conclusion, there are no meaningful differences in area, BMC, and BMD values noted between single- and socially housed OVX and non-OVX female Sprague-Dawley rats, respectively.

P505 - Weight Loss and Decreased Food Consumption in Diet-Induced Obese Mice Administered Celastrol Were Likely Due to Toxicity Instead of Leptin Sensitization

A. Brown¹, J. Gao¹, V. Beaulieu¹, D. Panza¹, M. Chen¹, and J. Boisclair¹

¹Novartis Institutes for Biomedical Research, Cambridge, MA, USA

Obesity is associated with resistance to the adipocyte-derived hormone leptin, which provides input to the hypothalamus regarding peripheral energy stores. Celastrol, a natural product from the thunder god vine, was recently reported to decrease food consumption and produce weight loss in diet-induced obese (DIO) mice, but not ob/ob mice (which lack functional leptin), presumably by reversing leptin resistance. A study was conducted to evaluate whether celastrol’s proposed pharmacology occurs independently from toxicity. Male DIO C57BL and ob/ob mice received either vehicle alone, 0.1 or 1 mg/kg celastrol (N = 8/group) by intraperitoneal (IP) injection prior to the dark cycle once daily for either 8 (DIO) or 9 (ob/ob) days. Decreased food consumption and body weight loss occurred at 0.1 mg/kg in DIO mice and at 1 mg/kg in both strains. Celastrol produced peritonitis and transmural inflammation in the GI tract to a greater extent in DIO mice compared to ob/ob mice. Decreased serum albumin and increased globulin occurred to a greater extent in DIO mice, consistent with the increased inflammatory response observed microscopically. In a separate study, celastrol plasma exposures were equivalent between DIO and ob/ob mice following a single 0.1 mg/kg IP dose. Celastrol is highly lipophilic (cLogP = 7.1) and was nonselective in a secondary pharmacology screen. In conclusion, decreased food consumption and weight loss in DIO mice were likely due to celastrol toxicity instead of leptin sensitization. It is critical to dissociate purported pharmacologic effects on food consumption from responses confounded by toxicity.

P506 - In Vitro Potentiation of gH2AX Responses by Small Molecule DNA Repair Inhibitors as a Means to Characterize Genotoxic Mode of Action—Proof of Concept with the PARP Inhibitor Olaparib

D. Bernacki¹, S. Bryce¹, J. Bemis¹, and S. Dertinger¹

¹Litron Labs, Rochester, NY, USA

Previous work has shown that a multiplexed in vitro flow cytometric assay known as MultiFlow reliably distinguishes clastogens, aneugens, and nongenotoxicants. In this assay nuclei are released with a nonionic, DNA is stained, and several nuclear epitopes are simultaneously labeled with fluorescent antibodies (gH2AX, phospho-H3, and p53). The current work was conducted to evaluate whether this platform could provide additional information about clastogenic mode of action (MoA). For these experiments, TK6 cells were treated with 0 or 1μM of the PARP inhibitor olaparib. Cells were then treated with each of 22 diverse clastogens over a range of concentrations. At several time points gH2AX, phospho-H3, and p53 readings were taken. Of the endpoints studied, gH2AX responses were affected the most, generally at the earliest time points (1-2 hours). Proast software was used to calculate the concentration at which gH2AX responses were 2× higher than solvent control (“doubling dose”). A potentiation factor (PF2X) was calculated by dividing the doubling dose without olaparib by the doubling dose in the presence of olaparib. PARP inhibition was observed to potentiate the genotoxicity of small alkylators, ROS generators, and topoisomerase I inhibitors. The greatest effects were observed for methyl methanesulfonate and 1-methyl-3-nitro-1-nitrosoguanidine (PF2X = 7 and 4, respectively). Little to no potentiation was observed for anti-metabolites or topoisomerase II inhibitors (PF2X ≤ 1.19). Taken together, these data are encouraging, as they suggest that the influence that cotreatment with DNA repair inhibitor(s) has on gH2AX or other biomarkers may provide an efficient means of characterizing genotoxicants’ MoA.

P507 - A QSAR System to Predict Xenobiotic Metabolites and Their Toxicological Properties

S. Chakravarti¹, A. Sedykh², and R. Saiakhov¹

¹MultiCASE, Inc, Beachwood, OH, USA

²Former employee of MultiCASE, Inc, Beachwood, OH, USA

We report here an algorithm to predict and/or search possible human metabolites of xenobiotics and calculate their toxicity properties within 1 software system. The algorithm is a combination of 4 components working together: (1) a collection of highly predictive QSAR models for predicting potential sites of metabolism for different phase I and phase II biotransformations; (2) a collection of QSPR models to calculate physicochemical properties of metabolites, eg, LogP, water solubility, etc; (3) a comprehensive database of known human metabolites of a large number of xenobiotics; (4) a collection of CASE Ultra models and databases to predict/search for various toxicological endpoints, (eg, mutagenicity, carcinogenicity, human adverse effects, etc) We have built QSAR models for various phase I and phase II biotransformation reactions (oxidative, reductive, hydrolytic, and conjugation) to calculate the probability of various sites of metabolism (SOMs) on the query chemical helping to identify major and minor metabolites. The accompanying database contains ∼15,000 known parent-metabolite pairs in humans, literature references, and biotransformation details. The CASE Ultra toxicity models help in predicting toxicity potential of every predicted metabolite. The software application also contains a real-time SOM prediction and display during structure sketching to assist in drug design. This report will provide details of the software, QSAR models, and examples to illustrate the system.

P508 - Derisking Chronic Neprilysin Inhibition and Tumor Progression

A. Del Rio Espinola¹, O. Grenet¹, J. Moggs¹, C. Buono², O. Turner³, and H. Schoenfeld³

¹Novartis Institute of Biomedical Research, Basel, Switzerland

²Novartis Institute of Biomedical Research, Cambridge, MA, USA

³Novartis Institute of Biomedical Research, East Hanover, NJ, USA

Sacubitril/valsartan (Entresto) is the first angiotensin receptor neprilysin (NEP) inhibitor approved by the FDA and EMA to reduce cardiovascular mortality and hospitalization for worsening heart failure. NEP is expressed in several organs (eg, kidney, lung, and brain), with highest levels in the renal proximal tubule. NEP cleaves several physiologically relevant substrates, including natriuretic peptides, and inactivates several signaling peptides (eg, endothelin-1 and fibroblast growth factor-2) with a physiological role in cell migration and neovascularization. We assessed the theoretical risk that NEP inhibition could contribute to tumor progression in silico by evaluating potential associations between variants in the gene encoding neprilysin (MME) and tumor phenotypes in public Genome Wide Association Studies (GWAS), germline (eg, OMIM, dbSNP), and somatic mutation databases (eg, TCGA, COSMIC). Germline variants in MME have not been reported to be associated with tumor phenotypes. Moreover, the somatic mutation pattern of MME does not resemble the classical tumor suppressor gene (eg, PTEN, p53) and is shared with surrounding genes. Most tumor copy number alterations observed in MME are copy number gains, and most mutations are missense mutations without evidence of any hotspot, in contrast to tumor suppressor genes characterized by copy number losses and truncating mutations. These results complement rodent studies showing no evidence of carcinogenicity when sacubitril was orally administered to mice (doses up to 1,200 mg/kg/day) and rats (doses up to 400 mg/kg/day) for 2 years. In conclusion, in silico genetic analyses do not support a theoretical risk of neprilysin inhibition contributing to neoplastic progression.

P509 - Development of an Approach for Cerebrospinal Administration in the Gottingen Minipig

J. Douville¹, F. Emond¹, C. Foucault¹, G. Iacono¹, J. F. Lafond¹, R.S. Jacques¹, and C. Copeman¹

¹Charles River Laboratories, Montréal, Québec, Canada

Minipigs have been used extensively in toxicology studies involving the dermal route of administration. However, growing interest has been observed over the past years in using the minipig as an alternative nonrodent species on various types of safety studies. When considering nontraditional routes of administration, the particular morphology of the minipig may present challenges. Given that many neurodegenerative diseases represent a large pool of unmet needs, and that an increasing number of therapeutic molecules being developed are biologics that cannot cross the blood-brain barrier, we sought to develop a suitable surgery-based approach for cerebrospinal delivery of therapeutic molecules. Based on previous experience of our laboratory in other species, we opted for the implantation of a catheter in the intrathecal space at the lumbar level. In order to be able to follow pharmacodynamic and pharmacokinetic parameters, we also optimized a technique for collection of cerebrospinal fluid (CSF) by direct puncture in the cisterna magna. The intrathecal catheter was implanted via a microlaminectomy at the L5 vertebra followed by a durotomy, and the catheter was inserted intrathecally. The catheter was secured and connected to a PinPort access port exteriorized in the interscapular region. Animals were administered saline by intrathecal injection twice weekly over 14 days. Endpoints evaluated included body weights, clinical observations, hematology, coagulation, clinical biochemistry, gross observations at necropsy, and histopathology. The results obtained are comparable to historical data, and, as such, feasibility of intrathecal administration and CSF collection in the Gottingen minipig was confirmed.

P510 - Putamen and Lateral Ventricle Administration in the Cynomolgus Monkey

J. Douville¹, F. Emond¹, C. Foucault¹, G. Iacono¹, J. F. Lafond¹, R.S. Jacques¹, and C. Copeman¹

¹Charles River Laboratories, Montréal, Québec, Canada

The putamen, along with the caudate nucleus, forms the dorsal striatum of the brain. It plays a key role in movement coordination and in various types of learning. It is involved in many degenerative neurological disorders, like Parkinson and Huntington diseases. Novel therapies, such as large molecules, have low penetration potential due to the blood-brain barrier or to a short half-life. Therefore, delivery of drugs directly into the target brain compartments may be indicated. In large animals, accurate delivery into a specific brain structure represents a greater challenge. In monkeys, because of marked variability in the size and shape of the head, documented coordinates are considered approximations. To support the development of drugs intended for intracerebral administration, feasibility, reproducibility, and tolerability of targeted putamen and lateral ventricle injections were evaluated. Once the surgical approach was established, a range of volumes were injected bilaterally at different rates to evaluate diffusion and reflux using ink. Following recovery from surgery, the animals were kept for an observation period of at least 14 days. Clinical signs, body weights, food intake, neurological evaluations, clinical pathology parameters, and histopathology of the brain were evaluated. The animals showed no clinical signs related to the injections. There was no effect on body weight, food intake, hematology, coagulation, or biochemistry. Neurological evaluations showed normal behavior, gait, postural reactions, cranial, and spinal nerve functions. The histopathology evaluation demonstrated that the targeted brain structures were reached with minimal background changes. Overall, the putamen and lateral ventricle administration was successfully established.

P511 - Comprehensive In Vivo Nonclinical Safety Assessment of NSI-189, a Small Molecule New Chemical Entity for the Treatment of Major Depressive Disorder

G. Furman¹, R. Christopher², and K. Johe³

¹Paracelsus, Inc, Leucadia, CA, USA

²CPC, LLC, San Diego, CA, USA

³Neuralstem, Inc, Germantown, MD, USA

NSI-189, a benzylpiperizine-aminiopyridine, is a small molecule new chemical entity that stimulates neurogenesis of human hippocampus-derived neural stem cells in vitro and stimulates neurogenesis in mouse hippocampus in vivo. In the safety pharmacology program, no test article-related neurobehavioral effects were noted in mice administered ≤500 mg/kg NSI- 189; decreased body temperature was noted at 500 mg/kg. In telemeterized dogs, slightly increased heart rate; associated shortening of the RR, PR, and QT intervals; and a slightly increased respiratory frequency were noted at 250 and 400 mg/kg; minute volume was slightly increased at 400 mg/kg. In 28-day toxicity studies, the NOAELs for NSI-189 in mice and dogs were 250 and 100 mg/kg/day, respectively. In mice, adverse neurobehavioral observations and mortality were observed at 500 mg/kg/day. In dogs, adverse neurobehavioral observations were noted at doses above the NOAEL. In a 26-week toxicity study in mice, the NOAEL for NSI-189 was 250 mg/kg/day. Adverse neurobehavioral effects, transient reduction in body weight, and non-adverse adaptive increases in liver weights were observed at 500 mg/kg/ day. In a 13-week toxicity study in dogs, a NOAEL of <50 mg/kg/ day was identified. During the dosing period, compound-related neurobehavioral observations, salivation, emesis, paleness, cool to touch, fecal changes, and thin appearance were noted in 1 or more animals across all NSI-189 treatment groups. No target organs were identified histopathologically in either species when NSI-189 was administered for up to 3 (dogs) or 6 (mice) months.

P512 - Urethane-Induced Carcinogenesis in Tg.rasH2 Mice: A Comparative Study With Literature

G. Krishna¹, G. Gopalakrishnan¹, S. Ganiger², H. Krishnappa², and K. Kamala²

¹Supernus Pharmaceuticals, Inc, Rockville, MD, USA

²Advinus Therapeutics Ltd, Bengaluru, India

Tg.rasH2 mice constitute a validated mouse bioassay system for evaluation of carcinogenic potential, confirmed to be sensitive to both genotoxic and nongenotoxic carcinogens (Long et al, 2010; MacDonald et al, 2004). Besides, this is one of the models specified in ICH Guidance, enabling 6-month carcinogenicity studies as an alternative to traditional 2-year bioassays. The objective of the present study was to demonstrate the induction of lung neoplasia in Tg.rasH2 mice using urethane (positive control) and to compare the observations with existing literature. Tg.rasH2 mice (Taconic Farms, Hudson, NY), 20/gender, were dosed at 1,000 mg/kg/day urethane in saline (IP) on study days 1, 3, and 5 with appropriate controls. Standard toxicological observations were made per carcinogenicity assessment protocol, and noteworthy findings were compared with existing literature and controls. In the urethane group, mortality was noted beginning day 57, and all remaining urethane-treated mice were sacrificed on day 116 due to 50% mortality. Grossly, urethane caused tumors in lung, spleen, and stomach, and these were confirmed microscopically as bronchiolo-alveolar adenoma and adenocarcinoma (100% mice), hemangioma and hemangiosarcoma (85% mice), and squamous cell carcinoma of non-glandular stomach (25% mice), respectively. Urethane is known to induce lung and spleen tumors in mice (Shah et al, 2012), and the observations reported in the current study were comparable; thus, the test system was considered validated. In addition, the stomach nonglandular-type tumors observed in this study seem to be unique and, as such, may serve as an additional target organ for urethane-induced tumor assessment in such routine carcinogenicity studies.

P513 - An Intradermal Toxicity Study of a Therapeutic Nanoparticle Cancer Vaccine in Sprague-Dawley Rats

S. Godin¹, S. Stewart², and S. Fuller²

¹Smithers Avanza, Gaithersburg, MD, USA

²Panacea Pharmaceuticals, Gaithersburg, MD, USA

HAAH is prevalent on tumor cells but is not recognized by the immune system. A vaccine containing an N-terminal portion of the HAAH protein as a fusion protein with the gpD antigen on the surface of bacteriophage lambda is being developed as a therapeutic cancer vaccine. The purpose of this study was to evaluate toxicity and local tolerance when administered by intradermal injection and to evaluate reversibility of any treatment-related toxicity following 2-week and 4-week recovery periods. Four groups of rats received 3 injections of either the control article or the vaccine at 3 different doses once every 3 weeks. Treatment with the vaccine had no effect on mortality, physical examinations, dermal Draize observations, body weights, food consumption, body temperature, ophthalmologic observations, gross pathology, organ weights, hematology, or clinical chemistry. Levels of antibody in groups receiving vaccine showed measurable antibody to both recombinant HAAH and to the recombinant bacteriophage construct, which was both dose- and dose number-dependent. A slightly prolonged but nonadverse increase in prothrombin time was present in both sexes 2 weeks following the last dose, which resolved in males by 4 weeks following the last dose but remained minimally prolonged in females given 3 × 1,011 particles. Microscopic findings were present at the injection site in animals given >1 × 1,011 particles, and consisted of mild or moderate mononuclear or mixed inflammatory cell infiltrates in the dermis and/or subcutis. These findings were considered non-adverse and resolved during recovery.

P515 - Observation of an Apparent Species-Specific Effect of Anti-miR-122 Inhibitors on Body Weight Gain in Sprague-Dawley Rats

C. Heinlein¹, C. Berman², K. Liu¹, S. Neben¹, K. Kersjes¹, C. Manalo¹, and J. Grundy¹

¹Regulus, San Diego, CA, USA

²Berman Consulting, Wayland, MA, USA

RG-101 is a GalNAc-conjugated anti-miR-122 oligonucleotide currently under clinical development for treatment of chronic hepatitis C virus (HCV) infection. Pharmacological activity biomarkers of miR-122 inhibition common to all evaluated species (mice, rats, monkeys, and humans, when feasible to evaluate) typically include moderate effects on aldolase A mRNA derepression in liver, reductions in serum cholesterol, and increased serum alkaline phosphatase. In pharmacology studies with RG-101 and/or various unconjugated anti-miR-122 inhibitors in mice, rats, and monkeys, a finding apparently unique to rats was dose-related attenuation of body weight gain (BWG), with significant reductions seen at ≥10 mg/kg for RG-101. Repeat-dose toxicity studies of RG-101 in mice (45-450 mg/kg), rats (0.1-88.2 mg/kg; evaluating 10-fold dose increases), and cynomolgus monkeys (15-150 mg/kg) demonstrated no-observed-adverse effect levels (NOAELs) of 450, 8.8, and 45 mg/kg (given once monthly for a total of 4 doses). In general, toxicities seen across species were similar, with the liver identified as the target organ of toxicity, which is consistent with the high and persistent concentrations of RG-101-related oligonucleotides observed in this tissue. Adverse effects, when seen, were typically limited to expected toxicities such as increased liver weights and minimal single-cell hepatocellular necrosis. In addition, consistent with the pharmacology studies, mean dose-dependent decreases (ranging from ∼14%-61%) in BWG relative to controls were also observed in rats only (females and males at doses ≥0.1 and ≥8.8 mg/kg, respectively). The mechanism for this BWG effect in rats has not yet been determined, but is considered unlikely to be clinically relevant.

P516 - Immune Humanized Mouse Model: Autoimmunity Induced by Nivolumab

K. Howard¹, L. Zadrozny¹, and J. Weaver¹

¹US Food and Drug Administration, Silver Spring, MD, USA

Checkpoint inhibitors represent a new class of therapeutics in the treatment of cancer that have demonstrated remarkable clinical effectiveness. However, some patients have experienced serious adverse autoimmune effects including pneumonitis, hepatitis, colitis, nephritis, dermatitis, encephalitis, and adrenal or pituitary insufficiency. These adverse events were not predicted by nonclinical studies in nonhuman primates. To determine if the bone marrow-liver-thymus (BLT) immune humanized mouse model could model these autoimmune effects, we studied the effect of nivolumab on immune humanized NOG mice. Mice were treated with 2.5, 5.0, or 10.0 mg/kg nivolumab or saline intraperitoneally twice weekly for 4 weeks. Mouse body weights were monitored twice weekly, and observations of behavior and appearance were noted daily. Blood was evaluated pre-study, at 14 days, and at necropsy. Necropsy was completed at 28 days or sooner if weight loss >20% or morbidity was noted. Survival was reduced in a dose-dependent manner, and limited ALT elevations were noted. Similar to the adverse reactions reported in humans, histopathologic findings showed that most mice in all dose groups developed pneumonitis and hepatitis, with nephritis, dermatitis, and adrenalitis also noted in some individuals. Additional histopathologic findings including pancreatic atrophy, myositis, and osteomyelitis were seen in only a few individuals from the 2.5 and 10 mg/kg dose groups. Flow cytometric analysis showed increased T cell activation with concomitant loss of PD-1 expression. These findings show that this fully engrafted humanized mouse model can demonstrate autoimmune adverse effects of anti-PD1 therapy.

P517 - Ex Vivo Evaluation of Immune Enhancement in the Cynomolgus Monkey in Response to In Vivo Exposure to Several Checkpoint Inhibitors

S. Kirk¹, A. Head¹, C. Cooper¹, C. Clarkson¹, A. Iqbal¹, and K. Troth¹

¹Covance Laboratories Ltd, Harrogate, United Kingdom

Recent years have seen an increase in “checkpoint inhibitors” as potential therapies for a variety of malignancies. Checkpoint inhibitor binding by their respective ligands can induce apoptosis in activated T cells, and this mechanism can be exploited by certain malignancies evading immune surveillance. However, blocking this interaction offers a mechanism for some anticancer therapies. During preclinical development, it is desirable to measure exposure and activity of such therapies ex vivo. In this study, we have evaluated a suite of assays to confirm activity of 2 novel checkpoint inhibitors (mab A and mab B) throughout the course of a preclinical toxicology study. Assays utilized in this study were receptor occupation (by flow cytometry) to evaluate free receptor levels on PBMCs, lymphocyte activity upon stimulation with Staphylococcal enterotoxin B (SEB), and lymphocyte activation when cocultured with a target cell line (K562). Initial experiments using a conjugated (APC-Cy7) form of mab A have established optimum conditions to determine free receptor levels (mab B work is ongoing). Results from SEB stimulation (using IL-2 production to indicate activation) showed increased production in the presence of mab A and B when compared to controls. Coculture of lymphocytes with K562 cells, in the presence of an anti-PD-1 control antibody, showed a dose-dependent increase in IFN-γ production. Work is ongoing with regard to evaluating lymphocyte activation in the presence of mab A and B. In conclusion, initial experiments show that our suite of assays have the potential to monitor the presence and activity of checkpoint inhibitors ex vivo.

P518 - Validation Study of Neuropharmacologic Screening in Rats Administered Oral d, l-Methylphenidate HCl or Chlorpromazine HCl

P.J. Kruzich¹, N.E. Suttles¹, A.M. Hermann¹, and L. D. Hopper¹

¹BASi, Inc, Mt Vernon, IN, USA

The International Conference on Harmonization (ICH) 7A regulations were developed to protect clinical trial participants receiving investigational test articles from adverse pharmacodynamic effects. The Safety Pharmacology Core Battery of ICH7A seeks to investigate cardiovascular, respiratory, and central nervous system (CNS) functions potentially impacted by investigational test articles. The goal of this study was to validate a simplified modified Irwin assay where behavioral observations in treated groups are compared to control animals across time points to aid in determining possible CNS and autonomic nervous system (ANS) effects of administered drugs. Groups of 10 male Sprague-Dawley rats were administered vehicle, 20 mg/kg chlorpromazine (CPZ), an antipsychotic drug (APD), or 50 mg/kg methylphenidate (MPH), a psychostimulant, orally by gavage (5 mL/kg). Animals were observed prior to administration and at 0.5, 1, 2, 4, 8, and 24 hours following administration. Evaluations included home cage observations, hand-held observations, open field observations, elicited behaviors, and rectal temperatures. Administration of CPZ and MPH was associated with time-dependent changes in activity and several other CNS and ANS observations associated with CPZ and MPH administration. Our results suggest this simplified format complies with ICH7A guidelines and is valid for CNS and ANS screening of novel compounds in support of investigational new drug-enabling studies.

P519 - A Compound Lead Optimization Strategy to Establish a Correlation Between In Vitro Mast Cell Activation and In Vivo Cmax to Identify Maximum Tolerated Dose in Rat Species

D. Lee¹, T. Nguyen¹, N. Valle¹, S. Ubhayakar¹, X. Liang¹, Y. Chen¹, C. Heise¹, and M. Koehler¹

¹Genentech, South San Francisco, CA, USA

Drug-induced anaphylaxis-like responses have been observed shortly after dosing with certain types of chemical compounds in preclinical and clinical studies. These types of compounds are commonly described as a cationic amphiphilic class of molecules that possess physical chemical attributes of positive charge and both hydrophilic and lipophilic properties. Examples of such compounds include the neuropeptide substance P and the antibiotics vancomycin and polymyxin. Previous investigative in vitro and in vivo studies have found an association with this type of acute toxic reaction to the mechanistic action of histamine that is released due to mast cell degranulation in an IgE-independent fashion. In our study, we describe a tractable method to explore and characterize the potential of novel compounds in an antibiotic drug discovery program to induce histamine release from rodent mast cells in vitro. And furthermore, we show good in vivo translation of the in vitro results to that of rodent tolerability when select compounds are dosed via intravenous infusion and are ranked based on C_max exposure and systemic histamine concentration. This method was used to move forward compounds of desired potency and pharmacokinetic properties that demonstrated the potential for higher safety margins for acute histamine toxicity into repeat dose safety studies. The methodology outlined provides an impactful compound safety derisking strategy that is amenable to the lead optimization process in a small molecule discovery program and incorporates the 3Rs principle for animal use in research.

P520 - Food Restriction and Administration of Immunogens: Impact on Parameters Routinely Monitored in Nonclinical Studies

N. Makori¹, J. Setser¹, J. England¹, J. Hyde¹, R. Tadagavadi¹, and V. Peachee¹

¹Charles River Laboratories Ashland LLC, Ashland, OH, USA

It is established that decreased feed consumption in toxicology studies correlates with decreased body weights. There is, however, limited information relating reduced food consumption to hematology, peripheral blood, lymphoid tissue, T and B cells and subsets, organ weights, and histopathology. In this study, parameters were evaluated in food-restricted female rats. Five rats each were assigned to 4 cohorts: water (DI) alone, DI and keyhole limpet hemocyanin (KLH), restricted food, restricted food and KLH. Another cohort of 10 rats were administered sheep red blood cells (sRBC, 1 × 108), with 1 group of 5 administered DI and the second group cyclophosphamide (CPS) once at 15 mg/kg/day. Results showed no changes in hematology or in blood and tissue lymphocytes measured by flow cytometry in the food restriction group and in the food restriction with sRBC or KLH groups. Cumulative body weights were lower in CPS (−7 grams) and sRBC (−10 grams) cohorts, but not in animals administered DI and sRBC or KLH. Lower spleen (absolute, 41.7% and relative 38.5%) and thymus (absolute, 75.5%, and relative, 75%) weights were noted in CPS and sRBC (or KLH) groups but not in DI and sRBC or KLH groups. These changes correlated with lymphoid organ cell depletion. Since none of these immunotoxicology effects were seen in food-restricted animals, including those administered KLH, we conclude that sRBC, KLH, or food restriction is unlikely to cause effects that can interfere with data interpretation in some of the routinely monitored parameters in toxicology studies in rats.

P521 - Characterization of the Carcinogenicity Risk of SPD602

K. Yip¹, N. Pierson¹, and J. McNulty¹

¹Shire, Lexington, MA, USA

SPD602 (deferitazole) is a novel iron chelator being investigated by Shire for the treatment of chronic iron overload in patients with hereditary and acquired transfusion-dependent anemias. In a 2-year rat carcinogenicity study, male rats developed renal adenomas and carcinomas, as well as atypical hyperplasia and degenerative/regenerative nonneoplastic changes in the kidney. In addition, a high level of chronic progressive nephropathy was observed in male rats, which, in combination with the nonneoplastic kidney findings, are thought to progress to neoplastic lesions. In order to progress the clinical program, 2 nonclinical safety studies were conducted to understand the risk associated with the findings seen in the male rats. In the first study, transgenic mice (Tg rasH2) were administered SPD602 for 6 months to assess the carcinogenic risk. In the second study, male rats were fed a basal diet or 1 high in iron and administered SPD602 for 13 weeks in order to assess the effects on the kidney. We hypothesized that the high levels of iron, intended to mimic the disease setting, would have protective effects on the kidney. In that study, urinary kidney biomarkers were assessed and kidneys were examined for cellular proliferation (PCNA, Ki-67) along with histologic assessment. Overall, no evidence of SHP602 carcinogenicity was seen in the transgenic mouse study, and in the 13-week study there was a reduction in the severity of histologic changes in the kidney along with a delay and reduction in biomarker changes seen in animals fed a high-iron diet.

P522 - Continuous Assessment of Blood Pressure, Heart Rate, and ECG Interval Durations in Juvenile Beagle Dogs Using Telemetry

K. Norton¹ and A. Kirouac¹

¹Charles River Laboratories, Senneville, Québec, Canada

To meet regulatory expectations from US FDA and EMA for testing of pediatric drugs, there may be a need to study the function of the cardiovascular system in juvenile animals. However, there is a lack of historical baseline data and positive reference data in juvenile animals. Surgically instrumented telemetry provides an integrated cardiovascular assessment, and the feasibility of using this model in juvenile dogs was examined. Weaned pups (2/sex) were instrumented with DSI PhysioTel Digital L11 transmitter implants at 9 weeks of age. The patency of the telemetry implantation over time was demonstrated by assessing baseline ECG and blood pressure data for 24-hour periods from 9 to 30 weeks of age. Cardiovascular changes were evaluated following the oral administration of 10 mg/kg of L-NAME at 10, 20, and 30 weeks of age. Baseline changes, secondary to age, were as expected, with decreased heart rate and increased blood pressure observed from weeks 9 to 30. L-NAME induced expected increases in blood pressures and corresponding decreases in heart rate with secondary increases in PR and QT interval duration. Telemetry implant patency was demonstrated up to 30 weeks of age, with a single exception of a blood pressure catheter patency compromised by 20 weeks of age due to damaged (obstructed) vessel. In conclusion, this evaluation demonstrated the feasibility of conducting surgically instrumented telemetric ECG and blood pressure measurements in juvenile dogs. This model provides a comprehensive evaluation to assess the potential effects of a drug on the cardiovascular system of juvenile dogs.

P523 - Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) as a Biomarker for Cell Viability and Cardiac Function

C. Obejero-Paz¹, L. Ellison¹, J. Kramer¹, and A. Bruening-Wright¹

¹Charles River Laboratories, Cleveland, OH, USA

The simultaneous measurement of impedance and field potential signals from 2D hiPSC-CM cultures is a powerful tool to investigate drug effects on cardiac cell viability and excitation contraction coupling. Impedance signals are proportional to the number of cells growing on top of the recording electrodes; we show that cardiotoxic drugs like doxorubicin decrease the basal impedance in a concentration- and time-dependent manner. In addition, the impedance signal changes transiently due to cell contraction. The transient component of the impedance signal is complex, showing multiple peaks. Here, we dissected pharmacologically the twitch components using modifiers of sarcomere activity, including the myosin II inhibitor blebbistatin, the sarcomere ATPase activator omecamtiv mecarbil, and the calcium sensitizer pimobendan and the calcium channel agonist FPL64176 as activator of the excitation-contraction coupling machinery. Impedance recordings of hiPSC-CMs were measured at 35°C using a cardioECR instrument (ACEA Biosciences) pacing the cells at 0.67 Hz. Activators and inhibitors of sarcomere activity increased and decreased the positive peak amplitude and contraction and relaxation areas, respectively, but had only minor effects on the negative peak amplitude, suggesting that the peaks underlie different mechanisms. Notably, the negative peak was increased in a concentration-dependent manner by FPL64176, suggesting the involvement of calcium entry. The data suggest that (1) the basal impedance is a valuable tool to assess cell viability, (2) the positive peak of the twitch impedance is a valuable surrogate of the sarcomere activity in hiPSC-CMs, and (3) the initial negative peak changes are a surrogate for calcium entry.

P524 - Juvenile Mouse Study of Albiglutide, a GLP-1 Protein for Type II Diabetes

L. Posobiec¹, K. Brannen², D. Fisher³, C. Maier¹, S. Fernandez¹, and S. Laffan¹

¹GlaxoSmithKline, King of Prussia, PA, USA

²Merck, West Point, PA, USA

³Charles River, Horsham, PA, USA

Albiglutide (GSK716155) is a human glucagon-like peptide (GLP)-1 albumin fusion protein approved for adult type II diabetes. In response to a regulatory request in support of pediatric development, a juvenile toxicity study was conducted, with a focus on determining whether albiglutide accelerated sexual maturation. Mice were subcutaneously administered vehicle or albiglutide up to 50/100 mg/kg/day once daily using dose escalation from postnatal day (PND) 14 to alleviate body weight effects, and twice daily from PND 28 to 35 to maintain systemic exposure, followed by 9 weeks off-dose. An interim necropsy was performed at an age when 20% of the mice were expected to have physical hallmarks of sexual maturity, and hormones were assessed at an age just prior to anticipated sexual maturation. There were dose-dependent transient body weight gain reductions at all doses from PND 14 to 21, then increased body weight gain from PND 22 to 35; both expected pharmacological effects. There were transient food consumption reductions at 50/100 mg/kg/day, resolving after PND 32. Reversible non-adverse aspartate and alanine aminotransferase increases occurred in males at 50/100 mg/kg/day without microscopic correlates. There were no effects on behavior, the first estrous age or estrous cyclicity, sexual maturation physical hallmarks, hormone levels, or sexual maturity parameters observed histologically in either sex; therefore, accelerated sexual maturation was not observed. Antidrug antibodies were not associated with altered toxicokinetics. The no-observed-adverse-effect level for growth, development, sexual maturation, and toxicity in juvenile mice from PND 14 was 50/100 mg/kg/day, the highest dose tested.

P525 - Small Molecule BTK Inhibitors Exacerbate Spontaneous Background Pancreatic Changes in Sprague-Dawley Rats

A. Prefontaine¹, J. Bai¹, E. Polack¹, and E. Tien¹

¹Biogen, Inc, Cambridge, USA

Bruton’s tyrosine kinase (BTK) is a member of the TEC family of cytoplasmic tyrosine kinases involved in B cell and myeloid cell signaling. Inhibitors of BTK are being developed to treat a variety of immunologic disorders including hematological neoplasia and autoimmune diseases. Administration of structurally diverse BTK inhibitors for ≥7 days have been shown to produce rat-specific pancreatic changes. BIO-0556375, a reversible BTK inhibitor, produced minimal/mild hemorrhage, fibrin and/or pigment deposition, and fibrosis within islets as well as hemorrhage, chronic inflammation, and acinar atrophy in exocrine regions of the pancreas in Sprague-Dawley (SD) rats after 14 days at doses ≥100 mg/kg/day. A 14-day investigative study in SD rats with BIO 0556375 was performed to further characterize the pancreatic lesions and their progression. SD rats received 150 mg/kg/dose of BIO-055637 BID for up to 14 days. Clinical pathology and histopathology evaluation was performed following 3, 7, and 14 days of dosing along with general in-life monitoring. Transient minimal increases in lipase and cholesterol were observed at 4 hours post dose on days 3, 7, and 14 with no evident increase in severity over time and no changes in amylase and glucose levels. The pancreatic histopathology seen in this study are similar to those observed with other BTK inhibitors in rats and suggestive of an exacerbation of spontaneous background findings seen in this species. Additional investigation is warranted to further understand the mechanism of BTK inhibitors to cause these pancreatic changes.

P526 - Design of an In Vivo Phototoxicity Study

A. Rasmussen¹ and C. Longobardi²

¹H. Lundbeck A/S, Valby, Denmark

²Research Toxicology Centre S.p.A., Pomezia, Italy

The assessment of phototoxicity of small molecules is now a standard part of nonclinical development. Several guidance documents are available advising companies on how to examine their compounds. This poster reports how the FDA guidance was used for designing an in vivo phototoxicity study in rats using lomefloxacin, a known phototoxicant, as the test compound. In our study, nude RH/rnu rats were exposed to radiation in the form of UV-A (40 Joules/cm²) and UV-B (0. 8 Joule/cm²) light. Some animals were administered a single dose of lomefloxacin (50 mg/kg) 1 hour prior to UV irradiation, while other animals were administered vehicle. Similar groups were maintained without UV irradiation. After termination of UV irradiation, all animals were subjected to detailed examinations for up to 72 hours, including dermal skin observations and eye assessments, followed by a histopathological assessment of ears and skin. The validity of the study design and the choice of test compound were demonstrated as (1) all irradiated animals displayed dermal signs of photoirritation, while the nonirradiated animals showed no signs of photoirritation, (2) only low incidences of acute dermal inflammation were seen in the non-irradiated animals, while a range of histopathological findings were made in the irradiated animals, and (3) treated animals exhibited the strongest signs of erythema, demonstrating that the UV-irradiation contained enough energy to photo-activate lomefloxacin, causing dermal reaction in the non UV-exposed body areas. We consider this study design adequate for assessment of phototoxicity of small molecules.

P527 - Renal Complications in Chronic Diabetic Minipigs

S. Roth¹, J. Liu¹, G. Bouchard¹, and A. Stricker-Krongrad¹

¹Sinclair Research Center, Columbia, MO, USA

Objective: Assess kidney pathophysiology in chronic insulin-dependent diabetic Yucatan miniature swine. Methods: Type I diabetes was induced in Yucatan miniature swine, and animals were maintained on a daily insulin regimen for more than 3.5 years. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured in conscious diabetic and normal control animals. Results: BUN ranged from 19 to 23 mg/dL, and creatinine ranged from 1.10 to 1.47 mg/dL; no significant abnormalities were noted in albuminuria. RBF was remarkably increased in the diabetic minipig when compared to normal animals: 240.9 ± 59.9 versus 103.7 ± 8.6 mL/min, respectively. GFR was moderately increased: 36.3 ± 5.0 versus 17.6 ± 1.7 mL/min, respectively. Gross kidney findings ranged from normal (majority of animals) to moderately shrunken, lobulated with white foci (in 1 animal). Histopathologic findings ranged from little to mild aberrations (majority of animals) to severe interstitial fibrosis, severe glomerular sclerosis, and tubular nephrosis in 1 animal. Conclusions: Chronic diabetes in Yucatan miniature swine manifests with a renal pathophysiological tableau corresponding to the hyperfiltration phase of early diabetic kidney disease (increased glomerular hydrostatic pressure and increased glomerular filtration rate in absence of increased albumin secretion rate). This corresponds well to the 50% elevation in GFR seen early in type 1 human diabetes and in some type 2 diabetic patients. This indicates that the diabetic mini swine could provide a good model to test preventative approaches for progressive kidney therapies, using GFR hyperfiltration as a marker of early renal damage.

P528 - Validation of Neutral Red Uptake Test (NRU-test) for Cytotoxicity Assessment in CHO-WBL Cells

S. Roy¹, S. Bruce¹, Y. Xu¹, and R. Kulkarni¹

¹BioReliance, Rockville, MD, USA

In vitro cytotoxicity tests such as neutral red update (NRU) assay can be used as alternative toxicity tests to reduce the total number of animals needed for acute oral toxicity tests. However, NRU assay is also one of the most used cytotoxicity tests for chemicals, pharmaceutics, cigarette smoke condensate, and plant and medical devise extracts. This test is generally performed in BALB/c 3T3 cells. The modal number of chromosomes in BALB/c 3T3 cells is 78 with a range of 62 to 109. The stem line number is hypotetraploid, which makes this a karyotypically unstable cell line. In contrast, the CHO-WBL cells are well characterized and karyotypically stable (2N = 20 ± 2), and can be obtained from European Collection of Authenticated Cell Cultures. This cell line is also used in genetic toxicology assays for regulatory submission. We explored the possibility to using CHO-WBL cells in NRU assay, as this can provide relevant information of cytotoxicity and can help in dose level selection in genetic toxicology study as well. We tested 12 chemicals listed in the NTP database with known different mode of action, using a 96-well plate as per the ICCVAM protocol. The positive control selected was sodium lauryl sulfate (SLS). The IC20, IC50, and IC80 values were calculated for each chemical. Our validation results indicate that CHO-WBL cell is also suitable to use in the NRU assay for cytotoxicity assessment.

P529 - De-Risking Chronic Neprilysin Inhibition and Age-Related Macular Degeneration

K. Mansfield¹, Y. Gao¹, C. Buono¹, V. Sasseville¹, I. Pruimboom², D. Theil², and H. Schoenfeld³

¹Novartis Institute of Biomedical Research, Cambridge, MA, USA

²Novartis Institute of Biomedical Research, Basel, Switzerland

³Novartis Institute of Biomedical Research, East Hanover, NJ, USA

Sacubitril/valsartan (Entresto) is the first angiotensin receptor neprilysin (NEP) inhibitor approved by the FDA and EMA to reduce cardiovascular mortality and hospitalization for worsening heart failure. NEP is expressed in several organs, including kidney, lung, and brain, with the renal proximal tubule displaying the highest levels. NEP cleaves a number of physiologically relevant beneficial substrates, including atrial natriuretic peptide, C-type natriuretic peptide, and, to a lesser degree, B-type natriuretic peptide. Emerging evidence indicates that amyloid-beta (Aβ), a NEP substrate, is elevated and may be a factor in the pathology of late-stage age-related macular degeneration (AMD). The current studies were undertaken to address the theoretical risk that chronic NEP inhibition by sacubitril/valsartan could contribute to ocular Aβ elevations. Target localization of NEP and other Aβ-degrading enzymes was performed in normal ocular tissue from rodents, cynomolgus monkeys, and humans using immunohistochemical (IHC) and/or in situ hybridization (ISH) methodologies. NEP protein was detected in endothelial cells of the choroid in normal mice and rats. However, neither NEP protein nor mRNA was detected in normal cynomolgus monkey or normal human neuronal retina, retinal pigmented epithelium, and choroid. No microscopic ocular findings were observed in hematoxylin and eosin-stained sections from chronic preclinical studies conducted with either sacubitril/valsartan or sacubitril in rodents and cynomolgus monkeys. These identified species differences suggest that NEP inhibition is unlikely to result in ocular Aβ increases in primates or humans. In conclusion, NEP inhibition by sacubitril/valsartan is unlikely to precipitate the development of AMD in humans.

P530 - Development of Hepatic/Immune Humanized Mice

K. Semple¹ and K. Howard¹

¹US Food and Drug Administration, Silver Spring, MD, USA

Preclinical testing of therapeutics is confounded by species differences in hepatic metabolism and immune system function. The ability to accurately predict drug-induced toxicity and adverse immune responses in large and small molecule therapeutics is limited by the lack of appropriate animal models. Immune compromised mice reconstituted with human hepatocytes are a promising model for the assessment of drug-induced liver toxicities. However, some drug toxicities are believed to be caused by interaction of the immune system with the drug and hepatocytes. Hence, the goal of this study was to develop a dual humanized mouse that has human hepatic and immune function. TK-NOG mice were treated with ganciclovir to ablate murine hepatocytes. Mouse serum liver enzymes were assessed to determine the magnitude of mouse liver damage and to predict mouse liver ablation rate. The mice were then transplanted with human hepatocytes intrasplenically and with human thymus and liver tissue under the renal capsule. Three weeks following surgery, mice were given human hematopoietic stem cells intravenously. Thereafter, human serum albumin ELISA assay and flow cytometry were used to determine the level of hepatic and immune engraftment, respectively. Results show TK-NOG mice had serum albumin levels ≥5 mg/ml over several months, indicating stable, long-term engraftment of human hepatocytes. TK-NOG mice engrafted an immune system containing >500/μL human leukocytes in peripheral blood. T- and B-lymphocytes and monocytes were present in PBMC, spleen, and bone marrow. A dual-humanized model can improve our ability to understand drug-induced liver injury and enhance pharmaceutical safety in preclinical and post-marketing phases of development.

P531 - A Current Industry Perspective on Assessing Hepatotoxicity Risk in Drug Discovery

J. Vogt¹, C. Lawson¹, D. Misner¹, D. Dambach¹, and W. Proctor¹

¹Genentech, South San Francisco, CA, USA

Drug-induced liver injury (DILI) is a leading cause of attrition, blackbox warnings, and post-market withdrawal. Extensive efforts are made to assess hepatotoxicity risk in drug discovery, which is difficult as there is poor concordance of preclinical species to identify human hepatotoxicants. Like others, Genentech employs a panel of in vitro models to investigate potential DILI risk, including cytotoxicity assays using primary human hepatocytes and human spheroid liver microtissues, mitochondrial toxicity assays, and transporter inhibition studies with additional consideration for projected dose/exposure. However, it is difficult to use human in vitro data to make decisions on compound progression in the absence of preclinical in vivo signals of hepatotoxicity. To address this, we employ a weight-of-evidence approach considering parameters that have been identified clinically to characterize risk for DILI within our small molecule programs. In this situation, no individual assays/parameters are decisional or used in isolation, but considered as hazard identification to holistically capture risk for hepatotoxicity. Qualification and implementation of these in vitro models using a test set of known DILI positive and negative compounds will be described, as well as how these assays are positioned for compound lead optimization and investigative studies. Case studies describing how this multiparametric approach was leveraged to explore mechanisms of hepatotoxicity for small molecules associated with preclinical and/or clinical hepatotoxicity will be presented. In conclusion, we believe this approach captures DILI risk prior to candidate nomination in a way that enables project progression even in the presence of individual in vitro flags of DILI.

P532 - Development of a Screening Strategy for Prediction of Drug-Induced Vascular Injury (DIVI): Creation of a DIVI Database and Compound Screening to Evaluate Translation and Predictivity of In Vitro and Ex Vivo Assays

T. Wisialowski¹, A. Fensome², D. Fish¹, L. Gauthier³, C. Houle¹, W. Hu⁴, S. Jenkinson⁴, M. Lawton¹, D. Potter¹, T. Schroeter¹, D. Stedman¹, L. Warren¹, and G. Yanochko⁴

¹Pfizer, Inc, Groton, CT, USA

²Pfizer, Inc, Cambridge, MA, USA

³Pfizer, Inc, Andover, MA, USA

⁴Pfizer, Inc, La Jolla, CA, USA

The occurrence of drug-induced vascular injury (DIVI) in nonclinical toxicology studies often has a significant impact on compound development based on safety margins, and typically results in compound termination or a delay in development often with a conservative risk management approach for clinical testing. However, there is relatively poor understanding regarding the incidence of this finding in nonclinical toxicology studies. We have reviewed results from internal studies and generated a “DIVI Database” of small molecule compounds with study-related metadata to better understand and characterize DIVI. We identified >70 small molecule compounds from a variety of primary pharmacological targets and therapeutic areas with DIVI across species (rat, dog, and monkey), and revealed that the overall incidence rate for DIVI is ∼8% of compounds and studies. When DIVI is observed, it tends to occur in specific vascular beds within the heart (ie, dog) or mesentery (ie, rat), although it can occur in a variety of vessels. We are currently testing DIVI and non-DIVI compounds in a suite of in vitro (eg, rat smooth muscle and endothelial cells—cytotoxicity) and ex vivo (eg, rat aortic rings—vascular reactivity) assays to explore mechanistic and pharmacological pathways involved in the development of DIVI and to determine the assays’ translation and predictive capacity. Preliminary results suggest that we should be able to develop a prospective DIVI screening strategy to provide early hazard identification and risk assessment for project teams prior to advancement of compounds into early in vivo safety studies.

P533 - Application of Small RNA Sequencing to Identify MicroRNAs in Drug-Induced Cardiac Toxicity Using iPSC Cardiomyocytes

X. Yang¹, L. Ren¹, M. White¹, and W. Mattes¹

¹FDA/National Center for Toxicological Research, Jefferson, AR, USA

For many cancer patients, the benefits of novel life-saving anticancer agents, such as tyrosine kinase inhibitors (TKIs), are often offset by drug-related cardiotoxicity. Previous clinical studies in cardiac diseases, including myocardial infarction and heart failure, fuel the notion that circulating miRNAs may serve as sensitive and specific biomarkers for heart injury. In the current study, we treated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes with a panel of TKIs up to 24 hours, and conducted small RNA sequencing to generate a profile of miRNA expression after drug treatment. From the altered miRNAs that were differentially expressed (>2-fold, P < 0.05), we chose to further investigate miR-1/133b, which are muscle-enriched, but not cardiac-specific; and miR-208b, which is highly specific to the heart. Using qRT-PCR, we confirmed the dose-, time-dependent increased expression of these candidate miRNAs in the cell-free medium. In addition, extracellular miR-1 increased as early as 1 hour post-treatment. Therefore, miRNAs have a potential to complement the existing cardiac troponin biomarker. These results suggest that miR-1, miR-133b, and miR-208b may be important miRNAs to predict TKI-induced cardiac injury before irreversible heart damage. As more TKIs are developed, early recognition and management of drug-induced structural cardiotoxicity will be important to reduce their associated morbidity and mortality.

P534 - Investigating Nontarget-Mediated Toxicity and ADC Uptake

L. Yu¹, A. Cercillieux¹, N. Corr¹, Y. Zhu¹, D. Sheinson¹, N. Stagg¹, H. Wang¹, D. Misner¹, and K. Staflin¹

¹Safety Assessment, Genentech, Inc, South San Francisco, CA, USA

Antibody-drug conjugates (ADCs) are a class of therapeutics intended to deliver highly potent drugs to tumors and spare normal tissue. However, non-target mediated toxicity has been observed in both preclinical and clinical settings. The mechanism of action (MOA) of nontarget mediated accumulation and uptake is largely unknown. One example of a nontarget mediated toxicity observed clinically with ADCs containing microtubule inhibitors such as maytansinoids is peripheral neuropathy, which in some instances is not readily identified in preclinical studies. We have developed an in vitro model validated with chemotherapeutic agents known to induce clinical neuropathy, in an effort to capture the neurotoxicity effects seen clinically. This method utilizes a multiplexed, kinetic, and high-throughput imaging system to assess compounds for their ability to effect neurite dynamics and cytotoxicity in human induced pluripotent stem cell (iPSC)-derived neurons. The characterization of this in vitro cell model suggests suitability to test chemotherapeutic-dependent changes in neurite dynamics as a mimicry of neuropathy. This model was used to investigate the contribution of antigen independent uptake mechanisms that may lead to nontargeted toxicity, such as Fc gamma receptor (FcgR), macropinocytosis, and mannose receptor dependency. These ongoing efforts aim to get a better understanding of ADC mediated neurotoxicity and target-independent uptake contributing to accumulation in nontargeted cells.

*GFP535 - Using Biopharmaceutics Drug Disposition Classification System (BDDCS) Classification to Evaluate the DILI Causative Potential of Bile Salt Export Pump (BSEP) Inhibition

R. Chan¹ and L. Benet¹

¹University of California San Francisco, San Francisco, CA, USA

Drug-induced liver injury (DILI) is a leading cause of drug failure in clinical trials and a major reason for drug withdrawals from the market. The present study examined the clinical impact of the BDDCS in evaluating the severity of DILI warning in drug labels approved by the FDA, the withdrawal status due to adverse drug reactions (ADRs), and the role of BSEP inhibition. FDA drug labels for 182 registered drugs were assessed for their BSEP inhibition. Next, the distribution of BSEP inhibition in each FDA hepatic liability category, DILI severity assignment, and BDDCS class were evaluated, as was the correlation of all sources of FDA hepatic liability and BDDCS class. Results were analyzed using chi-square tests for trend in proportions. When BSEP inhibition data were correlated with FDA drug labels of registered drugs, we observed no discernible pattern between BSEP inhibition and DILI severity assessment categories (P = NS). Yet when FDA hepatic adverse reactions were correlated with BDDCS class there was a highly significant result (P < 0.05). Our results show that the majority of BSEP inhibitors are BDDCS class 2 drugs (84.6%, n = 33/39), with concomitant decreases in the percentages of BDDCS class 1 and 3 drugs as BSEP inhibition increases. It appears that an apparent correlation of BSEP inhibitors with DILI is not related to the BSEP inhibition process, but due to the fact that the great majority of BSEP inhibitors are BDDCS class 2 compounds, which are highly correlated with DILI independent of the BSEP process.

*GFP536 - Generation of Homogeneous Site-Specific Ligation of Small Molecule to Monoclonal Immunoglobulins Resulting in Improved Toxicities Profile

S. Feng¹, D. Lac¹, G. Bhardwaj¹, and K. Lam¹

¹UC Davis School of Medicine, Sacramento, CA, USA

To develop a versatile and reliable site-specific ligation chemistry to create immunoglobulin conjugates for therapeutic use has remained a challenge. One of these applications is for the development of antibody drug conjugates (ADC) for cancer therapy. Non-specific ligation methods have been traditionally used to chemically modify immunoglobulins. However, these modifications often lead to heterogeneous products, making optimization of the biological, physical, and pharmacological properties of ADCs very challenging. Higher frequency of off-target toxicity is associated with non-specific ligation ADCs. Site-specific ligation chemistries have also been developed to ensure high-quality clinical products, but most require reengineered monoclonal antibodies with several reactive residues for conjugation. Here, we propose a site-specific affinity linker that exploits the highly conserved nucleotide-binding pocket (NBP) located within the Fab region of immunoglobulins. This linker serves as an anchor for covalent derivatization of the NBP via “proximity covalent ligation chemistry.” Several characterization data have confirmed that the ligation only occurs at Fab fragments, and the generated ADC through our platform has demonstrated better antitumor growth and improved target cell recognition ability compared to conventional antibody therapeutics in the cellular level and tumor animal model. Therefore, we propose to fully characterize the efficiency and specificity of the affinity element and the toxicity profiles of the resulting immunoconjugates.

P537 - Identification of Specific Biomarkers for Liver Toxicity in Rats Using Multiplexed Mass Spectrometry

P.J. Sherratt¹, K. Ghoreishi², C. Drew², and M. Myers¹

¹Nonclinical Development, Celgene Summit, NJ, USA

²Nonclinical Development, Celgene, San Diego, CA, USA

Hepatotoxicity is a major clinical concern during drug development. Any increases in the serum enzyme activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in a patient can lead to the clinician discontinuing treatment due to concerns of liver toxicity. Unfortunately, these biomarkers although sensitive are not necessarily specific to liver injury. Both transaminases can be elevated due to damage in other tissues, such as muscle, or due to metabolic adaptation. A broader panel of biomarkers are needed to confirm if changes in these biomarkers are due to true liver injury or not. For novel biomarker discovery, there is often an absence of an assay to test a new biomarker. One approach that minimizes this the initial investment in biomarker assay development is the use of mass spectrometry. Here, we describe the evaluation of a panel of potential hepatotoxicity biomarkers using a multiplexed mass spectrometry approach to measure protein levels for multiple biomarkers in a single serum sample. This is part of work to support the Preclinical Safety Testing Consortium Hepatotoxicity Working Group. The biomarkers evaluated include glutathione S-transferase α, colony stimulating factor 1, colony stimulating factor 1 receptor, regucalcin, cytokeratin 18, 4-hydroxy phenyl pyruvate dioxygenase and factor 7. The performances of these biomarkers were evaluated in rats treated with model liver, kidney and muscle toxicants. The results demonstrate that regucalcin, cytokeratin 18 and 4-hydroxy phenyl pyruvate dioxygenase have the potential to be specific markers of hepatotoxicity.