ACT 2021 Virtual Annual Meeting Poster Abstracts

The abstracts within this PDF were reviewed and approved by the ACT Program Committee. Every effort has been made to reproduce the abstracts as submitted. The author(s) of the abstracts are solely responsible for the data and content provided. The American College of Toxicology does not claim to validate, promote, or share in the representation of the content provided by the submitting author.

100s—General Toxicology

P101: North American Graduate Fellowship Recipient: Toxicity of Polychlorinated Biphenyls and their Human-relevant Metabolites in Astroglial Cells

N. Paranjape¹, B. Cagle¹, R. Tjalkens², H.-J. Lehmler¹, and J. Doorn¹

¹University of Iowa, Iowa City, IA, USA

²Colorado State University, Fort Collins, Colorado, USA

Exposure to polychlorinated biphenyls (PCBs) correlates with developmental neurotoxicity and, recently, with neurodegenerative disorders. However, the underlying mechanisms remain unknown. Because normal brain function is largely astrocyte dependent, we hypothesize that astrocytes play an important role in PCB-mediated neurotoxicity. We assessed the toxicity of four PCB congeners, 4-chlorobiphenyl (PCB3), 3,3’-dichlorobiphenyl (PCB11), 2,3’,4-trichlorobiphenyl (PCB25) and 2,2’,5,5’-tetrachlorobiphenyl (PCB52) and their corresponding human-relevant hydroxylated and sulfated metabolites, in C6 cells (rat glioma cell line) or primary glial cells (from C57BL/6 mice) exposed to test compounds at a concentration range of 0.5 to 50 μM for 24 h. The MTT assay was employed to determine cell viability, also in the absence and presence of 100 μM dopamine. Reactive oxygen species (ROS) generation was analyzed using fluorescent probes, MitoSox Red and CellRox Green in C6 cells. PCB52 and both its metabolites were the most toxic in C6 cells at LC₅₀ concentrations of 8.4 μM (PCB52), 2.2 μM (4-OH-PCB52) and 8.8 μM (4-PCB52-sulfate) thus, further studies were focused on these compounds. ROS was generated at concentrations of PCB 52 and its metabolites much below the LC₅₀ concentrations and was largely mitochondria-targeted. The presence of dopamine significantly increased cell viability in C6 cells exposed to 4-PCB52-sulfate but not other test compounds. PCB52 and 4-OH-PCB52 were similarly cytotoxic to primary glia as observed in C6 cells. These findings suggest that key cellular processes in astrocytes are impaired in a structure-dependent manner following PCB exposure. Future studies will assess the functional implications of these findings in PCB-mediated neurotoxicity.

P102: North American Graduate Fellowship Recipient: Alcohol-induced TFEB Dysregulation and Lysosome Impairment Lead to Hepatotoxicity in HIV-exposed Hepatocytes

M. O. New-Aaron¹, P. Thomes¹, M. Ganesan¹, R. S. Dagur¹, K. K. Kusum¹, L. Y. Poluektova¹, and N. A. Osna¹

¹University of Nebraska Medical Center, Omaha, Nebraska, USA

Background: Although etiologies of hepatotoxicity among alcohol-abusing HIV patients are multifactorial, alcohol remains the least explored “second hit” for HIV-related hepatotoxicity. We recently showed that ethanol (EtOH) metabolites impair lysosomal functions, which enhances HIV-induced hepatotoxicity. Objective: We investigated how the combination of EtOH metabolites and HIV affects lysosome functions. Methods: In this study, huh 7.5 cells stably transfected with CYP2E1 and designated as RLW cells were exposed to acetaldehyde generating system (AGS) for 24h and then infected with HIV-1_ADA. Lysosomal damage was assessed by galectin 3/LAMP1 colocalization and cathepsin leakage. Lysosome biogenesis –regulating factor, TFEB, was measured by RT-PCR (mRNA), Western-blot (proteins), and immunofluorescence (immunostaining). Calcineurin, ZKSCAN3, and STUB1 were analyzed by Western blot. Results: Combined exposure to AGS+HIV induced the highest levels of lysosome leakage and impaired lysosomal biogenesis corresponding to our previous report on the accumulation of HIV proteins and hepatocyte apoptosis. Retention of TFEB in the cytosol and its reduced translocation to nucleus was associated with AGS+HIV-suppression of proteasome activities, suppressed expression of E3 ligase STUB1, induction of 14-3-3 protein and ZKSCAN3, leading to attenuation of lysosomal biogenesis by TFEB. Conclusion: This implies that HIV and EtOH metabolites damage lysosomes and simultaneously prevent damaged lysosome repair, thereby contributing to HIV-induced hepatotoxicity.

P103: Tris(1,3-dichloro-2-propyl)phosphate Disrupts the Trajectory of DNA Methylation within Developing Zebrafish Embryos

S. Avila-Barnard¹, A. Reddam¹, J. L. Wiegand¹, D. C. Volz¹, V. Cheng¹, and S. Dasgupta¹

¹Department of Environmental Sciences, University of California, Riverside, Riverside, CA, USA

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is an organophosphate-based flame retardant used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post fertilization (hpf) reliably disrupts DNA methylation from cleavage (2 hpf) through early-gastrulation (6 hpf). Therefore, the objective of this study was to determine whether TDCIPP-induced effects on DNA methylation persist beyond 6 hpf. First, we exposed embryos to vehicle or TDCIPP from 0.75 hpf to 6, 24, or 48 hpf, and then conducted bisulfite amplicon sequencing of target loci using genomic DNA derived from whole embryos. Within both vehicle- and TDCIPP-treated embryos, CpG methylation was similar at 6 hpf and CHG/CHH methylation were similar at 24 and 48 hpf (relative to 6 hpf). However, relative to 6 hpf, CpG methylation was lower within vehicle-treated embryos at 48 hpf and TDCIPP-treated embryos at 24 and 48 hpf – an effect that was driven by acceleration of CpG hypomethylation. However, we found that, even at high µM concentrations, TDCIPP had no effect on key enzymes that regulate CpG methylation (DNA methyltransferase, ten-eleven translocation enzyme, demethylase, and thymine DNA-glycosylase), suggesting that TDCIPP-induced effects on CpG methylation are independent of direct enzymatic activation or inhibition. Finally, using 5-methylcytosine (5-mC)-specific whole-mount immunochemistry (IHC), we found that TDCIPP exposure from 0.75-6 hpf resulted in an increase in 5-mC abundance at 6 hpf, suggesting that TDCIPP-induced effects on CpG methylation vary by concentration and developmental stage. Therefore, we are using 5-mC-specific, whole-mount IHC to determine how TDCIPP disrupts the trajectory of CpG methylation during embryogenesis.

P104: Lipid Shelled PSMA-targeted Nanobubbles: Acute Toxicities in Male Rats

L. Bernard¹, R. Reams², J. Liu¹, E. Abenojar³, J. Basilion³, and A. Exner³

¹Frontage Laboratories, Inc., Concord TWP, OH, USA

²StageBio, Mason, OH, USA

³Case Western Reserve University, Cleveland, OH, USA

Lipid shelled octafluoropropane nanobubbles (NBs) targeted to the prostate specific membrane antigen (PSMA) are currently under development as an imaging agent for biomedical ultrasound. Acute toxicity of these agents was assessed in 10 male Sprague-Dawley rats treated with a single intravenous dose of vehicle (saline) or 70 mg/kg of NBs (mean diameter of ∼300 nm), the maximum feasible dose, followed by sacrifices on Day 2 (5 rats) and Day 8 (5 rats). There were no adverse effects on body weight, food consumption, hematology, clinical chemistry, behavior, or survival. Changes in cytokines included 3.5% lower mean IL-1β value on Day 2 and lower mean IL-1β, IL-4, and TNF-α values on Day 8 (-64, -11, and -47%, respectively). Other non-adverse effects included 29% lower mean white blood cell counts on Day 2, and increased spleen weights on Day 2 (19%) and Day 8 (27%). Increased spleen weights correlated microscopically with minimal to mild increased macrophage cellularity on Day 2 and minimal increased macrophage cellularity on Day 8. Increased macrophage cellularity, attributed to clearance of the nanobubbles, was considered of insufficient severity to impact splenic function. Follow up definitive toxicology studies are warranted to further advance the imaging agent as a viable formulation with a clinical use trajectory. 

P105 – North American Travel Grant Recipient: Carbon Nanodots and Their Biomedical Impact: Their Cytotoxicity and Effects on ProInflammatory Molecules through In Vitro and In Vivo Studies

S. Belperain¹, ZY. Kang¹, N. Chiu¹, and Z. Jia¹

¹University of North Carolina at Greensboro, Greensboro, NC, USA

Cardiovascular disease (CVD) is a term used to encompass a variety of diseases that involve the heart or blood vessels. One of the most common forms of CVD is atherosclerosis, and the formation of this disease is triggered by an influx of inflammatory molecules such as cytokines, chemokines, and adhesion molecules, leading to a dysfunctional endothelial lining. Carbon nanodots (CNDs) are a new wave of nanoparticles that have shown promising results in the field of biomedical research due to their small size, photoluminescent characteristics, drug delivery potential and green synthesis. The effect of CNDs on inflammatory molecules has not been largely researches; therefore, this study examined the impact of these nanoparticles on cell viability and tumor necrosis factor-alpha (TNF-α) induced expressions of interleukin (IL)-8, interleukin-1 beta (IL-1β), chemokine (C-C motif) ligand-2 (CCL2), chemokine (C-X-C motif) ligand-a (CXCL1) and intercellular adhesion molecule-1 (ICAM-1) in human endothelial cells (HMEC-1). Results showed that CNDs had no effect on cell viability and subdued TNF-α induced pro-inflammatory molecules in HMEC-1s. This prompted further exploration of CNDs through an in vivo study of C57BL/6 mice. Administration of CNDs showed a reduction of TNF-α as well as TNF-α induced expressions of specific inflammatory molecules CCL2 and CXCL1 while showing no change in animal body weight throughout the trials when comparing to control. Studying the cytotoxicity as well as the prospective anti-inflammatory effects of CNDs has only just begun; however, these results demonstrate the potential biomedical uses for these nanoparticles in the aid of treating atherosclerosis.

P106: North American Graduate Fellowship Recipient: Concordance Analysis of Human Nonalcoholic Steatohepatitis Patients to Identify a Rodent Model of Renal Transporter Expression

K. L. Frost¹, J. L. Jilek¹, E. L. Toth¹, M. J. Goedken², and N. J. Cherrington¹

¹University of Arizona, Tucson, Arizona, USA

²Rutgers University, Piscataway, New Jersey, USA

Alterations to renal elimination processes can lead to adverse drug reactions. Nonalcoholic steatohepatitis (NASH) is known to alter hepatic dtransport but may also affect renal transporters. This study investigates renal physiological changes in rodent and human NASH for identification of a model that recapitulates human alterations. Quantitative protein expression by surrogate peptide LC-MS/MS on renal biopsies from NASH patients were used for concordance analysis with rodent models including methionine and choline deficient (MCD), atherogenic (Athero) or control rats; Lepr ^db/db MCD (db/db); C57BL/6J fast food thioacetamide (FFDTH), American lifestyle induced obesity syndrome (ALIOS) or control diet. OAT3 showed an upward trend in all rodent models except the FFDTH (from 3.20 to 2.39 pmol/mg protein) making it the only model to represent human OAT3 changes. OAT5 significantly decreased in mice (from 4.59 to 0.45 db/db;1.59 FFDTH; 2.83 ALIOS, respectively) but a significantly increased in MCD rats (1.67 to 4.17 pmol/mg protein) suggesting the mice are appropriate to compare to human. MATE1 showed no change in human or rodent while MRP4 decreased in mice (from 0.87 to 0.34 db/db; 0.29 FFDTH; 0.18 ALIOS pmol/mg protein) recapitulating human expression. MRP3 decreased in human and rodents (from 2.71 to 1.53 MCD; 1.68 Athero, respectively; from 9.49 to 8.33 db/db; 8.82 FFDTH, 8.44 ALIOS pmol/mg protein, respectively). These data suggest variations in renal physiology are elicited by NASH and the FFDTH concordance analysis is significantly correlated to human expression. These models provide a valuable resource to identify human variability in renal elimination with various substrates. 

P107: International Travel Grant Recipient: Cytotoxicity and Activation of Stress-activated Receptors from Fish in a Battery of Cell-based Bioassays to Assess the Environmental Quality of Soil from Major e-waste Sites in West Africa

C. T. Eze¹, A. A. Otitoloju¹, O. O. Eze², A. M. Ali³, T. E. Ugochukwu⁴, J. L. Lyche⁵, A. Goksøyr⁶, and O. A. Karlsen⁶

¹University of Lagos, Akoka-Yaba, Lagos, Nigeria

²University of Nigeria Nsukka, Nsukka, Enugu, Nigeria

³Institute of Marine Research, Nordnes, Bergen, Norway

⁴Federal University Oye-ekiti, Oye, Ekiti State, Nigeria

⁵Norwegian University of Life Sciences, Oslo, Oslo, Norway

⁶University of Bergen, Bergen, Bergen, Norway

Specific e-waste associated chemicals are released into the environment during informal e-waste recycling activities. These chemicals could possibly impact ecosystems and consequently, human health. Therefore, cytotoxicity and induction of stress-activated receptors from fish was used as a battery of cell-based bioassays to assess the environmental quality of soil from major e-waste site in Nigeria, Benin and Ghana. The e-waste soils were analysed for a wide spectrum of organic contaminants of emerging concerns (CECs) and persistent organic pollutants (POPs) using combinations of chromatography and mass spectrometry. As expected, significantly higher concentrations of these chemicals were measured in the e-waste soils than in reference soils. The study revealed that e-waste soil-derived extracts (polar and non-polar) and elutriate (water soluble fractions) reduced COS-7 cell viability in a concentration-dependent manner and significantly activated the transcriptional activities of our xeno-sensing receptors. Overall, non polar e-waste-soil derived extracts were more cytotoxic compared to polar e-waste soil-derived extracts and elutriates. The highest fold induction of estrogen receptor alpha (15-fold), aryl hydrocarbon receptor 1-alpha (29-fold), aryl hydrocarbon receptor 2-alpha (39-fold), peroxisome proliferator-activated receptor alpha-1(3-fold) and peroxisome proliferator-activated receptor alpha-2 (28-fold) from Atlantic cod was observed with polar N4 (from Nigeria), non-polar B4 (from Benin), non-polar B4, non-polar N4 and non-polar B3 respectively. Conclusively, the combination of in vitro bioassays focusing on cytotoxicity and activation of Atlantic cod stress-activated receptors by e-waste soil-derived extracts and elutriates highlighted these e-waste sites as significantly polluted areas.

P108: North American Travel Grant Recipient: Oxidative Stress in Lens Cells Induces an Inflammatory Response and Triggers Immune Surveillance of Ocular Structures

B. Thompson¹, Y. Chen¹, D. J. Orlicky², D. C. Thompson³, and V. Vasiliou¹

¹Department of Environmental Health Sciences, Yale School of Public Health, Yale University, New Haven, Connecticut, USA

²Department of Pathology, Anschutz School of Medicine, University of Colorado, Aurora, Colorado, USA

³Department of Clinical Pharmacy, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, Denver, Colorado, USA

Purpose: Recent studies have indicated that the lens is not an immune privileged site; lens dysmorphogenesis or damage to the ocular surface (by corneal scratch) can trigger immune surveillance of the lens and other ocular structures. However, additional factors that can trigger inflammation of the lens and recruit immune cells to ocular structures remain to be identified. Methods: Glutamate-cysteine ligase catalytic subunit (Gclc) was conditionally deleted from ocular tissues to deplete GSH and induce ROS in the lens, termed KO. RNA-seq was conducted on postnatal day (P) 1 lens from KO and CON mice and bioinformatic analysis conducted. BSO (GCL inhibitor) and BSO/N-acetylcysteine (NAC, antioxidant) co-treatment were administered to cultured lens epithelial cells (21EM15), cytokine expression was analyzed by RT-qPCR and ROS detected by DHR probe. Cytokine expression was analyzed by RT-qPCR, ROS detected by DHR probe and immune cells identified in ocular structures by immunohistochemical staining in the lenses and eyes of KO mice aged P21. Results: Gene ontology analysis of upregulated genes in the lenses of KO mice aged P1 revealed enrichment in terms associated with the immune system. Impairment of Gclc in 21EM15 cells upregulated cytokine expression and promoted ROS accumulation, which was rescued by NAC co-treatment. Innate immune cells surveilled multiple ocular structures of KO mice aged P20, and was associated with increased lenticular expression of cytokines and ROS levels. Conclusions: Our results provide the first evidence that oxidative stress can induce inflammation in lens cells and promotes recruitment of ocular tissues of GSH-deficient mice.

P109: International Travel Grant Recipient: Abrogation of Cadmium-induced Hepatic Damage in Albino Rats by Vanillin Conjugated Zinc-Iron Nanoparticles

C. A. Otuechere¹, A. Adewuyi¹, A. Ekozin¹, S. Eromosele¹, and O. Ibekwe¹

¹Redeemer’s University, Osun State, Osun, Nigeria

Cadmium (Cd) has been reported to elicit hepatic oxidative stress and injury. Emerging reports of vanillin as a therapeutic molecule highlight its potential biomedical applications, while the magnetic nanoparticles could serve as drug molecule carriers. This study was designed to evaluate the synergistic effects of vanillin, conjugated with bimetallic Zinc-Iron magnetic nanoparticles (VMN), on sero-clinical indices, oxidative stress parameters, and histology in the liver of Cd-treated rats. VMN was synthesized via a simple chemical route and characterized using X-ray diffraction, thermogravimetric analysis, scanning electron microscope, and particle size distribution. Rats were treated with 1 mg/kg Cd bodyweight alone or co-treated per os, with VMN at 100 and 200 mg/kg body weight for five consecutive days. The results indicate that Cd significantly depleted the levels of albumin, globulin, albumin/globulin ratio, superoxide dismutase, glutathione, glutathione S-transferase, and escalated the levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, catalase, glutathione peroxidase, and lipid peroxidation. However, VMN, especially at the 200 mg/kg dose, attenuated these Cd-induced deviations. Furthermore, treatment with VMN ameliorated the presence of centrilobular and necrotic hepatocytes observed in the liver after exposure to Cd. Overall, our data suggest that VMN may be used as a hepatoprotective molecule in Cd-induced hepatic injury.

P110 – North American Graduate Fellowship Recipient: The Discovery of DiNP-Degrading Microbes and the Negative Impacts DiNP has on Colon Health in Female Mice

K. Chiu¹, ST. Bashir¹, A. Abdel-Hamid¹, I. Cann¹, R. Nowak¹, and J. Flaws¹

¹University of Illinois at Urbana-Champaign, Urbana, IL, USA

Di-isononyl phthalate (DiNP) is a common plasticizer found in polyvinyl chloride, toys, and faux leather. The most common exposure to DiNP is through ingestion; however, inhalation, dermal absorption, and intravenous absorption can also occur. Little was known about the effects of DiNP on the colon. Thus, study tested the hypothesis that exposure to DiNP alters colon histology, hormone levels, gene expression, and protein levels related to inflammation and cell cycle regulation and that the colon contains microbes that degrade DiNP. To test this hypothesis, adult female CD-1 mice were orally dosed with corn oil vehicle or DiNP (0.02 – 200 mg/kg) for 10 days. Distal colons were collected for histological examination, hormone analyses, and gene expression and protein analyses. Colonic contents were collected to identify bacteria that degrade DiNP. Histological analysis showed that DiNP exposure significantly increased colonic damage compared to control. DiNP exposure also significantly altered the gene expression of several cell cycle regulators (Ccnb1, Aifm1, and Bcl2l10) compared to control. Further, DiNP exposure altered gene expression and/or protein levels of several cytokines, tight junctions, and goblet cells compared to control (p<0.05). Lipid extractions from the distal colon revealed that DiNP exposure significantly decreased estradiol concentrations compared to control. Finally, bacteria isolated from colon contents show that they can use DiNP as a carbon source. These data suggest that DiNP exposure causes colonic damage and interferes with the colonic immune and endocrine microenvironment. The changes in the colon may be partly mediated by DiNP-degrading bacteria.

P111: The Effect on Bodyweight of Rats after Inhalation Administration Compared to Oral Administration

S. Moore¹, N. Ettridge¹, and J. Damiano²

¹Labcorp Drug Development, Huntingdon, Cambridgeshire, UK

²Labcorp Drug Development, Somerset, NJ, USA

Oral and inhalation administration are common dose routes of non-clinical development for assessing chemical safety. The standard approach to ensure compliance of inhalation studies is to restrain rats for a period of 6 hrs/day for 5 days/week. By comparison, oral administration only takes a few minutes per animal. A comparison was conducted over a period of a 13 week study using the same rat strain (RccHan WIST) to assess the effect on bodyweight due to inhalation restraint. Data was meaned from the control groups from at least 6 random selected studies per route of administration. The results showed that there a gradual difference in bodyweight gain from week 4 of the in-life part of the study. By week 13 of the dosing period, the mean male and female oral animals had a bodyweight of 415g and 240g respectively. This is compared with male and female inhalation animals having a bodyweight of 349g and 220g respectively. The difference in bodyweights for the male animals was statistically significant from week 9 of the dosing period. The difference in bodyweights for the female animals was not statistically significant. In conclusion, there was a statistically significant reduced bodyweight gain for male inhalation animals that were restrained for a 6hrs/day 5days/week when compared to the male oral administration animals of the same strain over the same dosing period. This reduced bodyweight gain effect was not observed for the female animals over the same dosing period.

P112: The Role of Peroxiredoxin IV (Prx4) in Intestinal Inflammation and Disease Progression

P. Thapa¹, H. Jiang¹, N. Ding¹, Y. Hao¹, A. Alshahrani¹, J. Fujii², and Q. Wei¹

¹University of Kentucky, Lexington, Kentucky, USA

²Yamagata University, Yamagata, Yamagata, Japan

Commonly found in pesticides, textile dyes, fuels for rockets, hydrazine is a classified environmental hazard agent metabolized to toxic azoxymethane (AOM) to cause mutagenic effect. Administration of dextran sodium sulfate (DSS) in drinking water to mouse leading to intestinal inflammation is a widely appreciated model of inflammatory bowel disease (IBD) in human. We hypothesize that Prx4 plays a role in intestinal inflammation through the change of local environment. Our goal is to determine how the presence of Prx4 contributes to inflammation, redox homeostasis, and inflammatory disease progression. We have established Prx4 knockout and Srx-Prx4 double knockout mice. These mice were treated with DSS or AOM/DSS and tissues were collected. Our results indicate that loss of the genes Prx4 and Srx alters the mutagenic effect of these agents in mouse by providing resistance against the development of colon and rectum tumors. Ongoing studies include the evaluation of DSS-induced inflammation in mice with different genetic backgrounds, and the disease progression from IBD, hyperproliferation, aberrant foci formation to intestinal adenocarcinoma.

This work was partially supported by the National Institutes of Health R01CA222596, National Institute of Environmental Health Sciences T32ES07266, Department of Defense W81XWH-16-1-0203, American Cancer Society RSG-16-213-01-TBE.

P113: Cross-study Analysis of SEND Datasets Using an R Package: sendigR

K. Snyder¹, M. Carfagna², W. Houser³, B. Larsen⁴, B. Paisley², D. Russo⁵, and Y. Ali⁶

¹US Food & Drug Administration, Silver Spring, Maryland, USA

²Eli Lilly and Company, Indianapolis, IN, USA

³Bristol Myers Squibb, New Brunswick, New Jersey, USA

⁴NovoNordisk, Copenhagen, Copenhagen, Denmark

⁵Rutgers University, Piscataway, New Jersey, USA

⁶Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, USA

The CDISC-SEND data standard has created new opportunities for collaborative development of open-source software solutions to facilitate cross-study analyses of toxicology study data. A public private partnership between BioCelerate and FDA/CDER was established in part to develop and publicize novel methods of extracting value from SEND datasets. As part of this work in collaboration with PHUSE, an R package (sendigR) has been developed to enable end users to easily construct a relational database from any collection of SEND datasets and then query that database to perform cross-study analyses. The package includes an R Shiny application with a graphical user interface, allowing users who are not familiar with the R programming language to perform cross-study analysis. Experienced R programmers, on the other hand, will be able to integrate the package functions into their own custom scripts/packages and potentially contribute improvements to the functionality of sendigR.

P114: A Decade of Plant Exposures and Toxicities: 2009-2019

M. DeMent¹, M. Ellis¹, and E. Ehrenpreis¹

¹Advocate Lutheran General Hospital, Park Ridge, IL, USA

Introduction. The National Poison Data System (NPDS) reports more than 2.2 million toxic exposures annually on average, with 13,642 cases related to plant toxicities. Of these plant exposures, 74% involve children <12 years of age. Currently, there is only one comprehensive review of plant toxicities evaluating cases from 1983 to 2009 (Krenzelok & Mrvos, 2011). Materials and Methods. We evaluated single-substance, human subject, plant exposures from 2009 to 2019 using deidentified data from the NPDS. Results. We found the annual incidence of plant versus total NPDS exposures decreased from 0.71% (2009) to 0.61% (2019). There were 213 plant species reported, although 50,828 cases were due to an unknown species. The most common species involved, Capsicum annuum and Phytolacca americana, changed compared to the previous decade. The pediatric population remained most affected by plant toxicities, with 65% of cases occurring in children <5 years old. Most cases were unintentional (91%) and either not followed due to low probability for toxicity (60%) or had no effects or minor effects (33%). The male to female ratio in moderate or major outcomes was 2:1 versus a 1:1 male to female ratio in mild cases. Of the 18 reported fatalities from 2009-2019, all occurred in adults >20 years of age. The majority were intentional (56%) and primarily due to cardiac glycosides (67%). Conclusions. The current study presents useful data for physicians, poison control centers, public health officials, and parents in identifying risks and enabling early recognition of relevant toxidromes requiring higher levels of care.

P115: Triphenyl Phosphate-induced Pericardial Edema is Associated with Elevated Ionocytes within Zebrafish Embryos

J. L. Wiegand¹, V. Cheng¹, A. Reddam¹, S. Avila-Barnard¹, and D. C. Volz¹

¹University of California Riverside, Riverside, CA, USA

Triphenyl phosphate (TPHP) is an organophosphate-based flame retardant used worldwide. TPHP blocks cardiac looping during zebrafish embryogenesis – an effect that is dependent on pericardial edema. Within zebrafish embryos, epidermal ionocytes express ion transporters responsible for maintaining an ionic/osmotic balance between the external aqueous and internal embryonic environment. Therefore, the objective of this study was to identify the potential role of ionocytes in mediating TPHP-induced pericardial edema. First, TPHP exposure from 24 to 72 h post fertilization (hpf) resulted in a significant increase in pericardial edema and ionocyte abundance at 72 hpf relative to vehicle-treated embryos. In addition, co-exposure of embryos to mannitol, an osmotic diuretic that increases the osmolarity of surrounding water, from 24-72 hpf blocked TPHP-induced effects, suggesting that elevated ionocytes are strongly associated with pericardial edema. However, initiation of exposure at 30 hpf (vs. 24 hpf) mitigated TPHP-induced effects on ionocyte abundance at 72 hpf even though the severity of pericardial edema was similar following exposure from 24-72 hpf and 30-72 hpf, suggesting that 1) 24-30 hpf represents a critical window of exposure for TPHP-induced effects on ionocyte abundance and 2) an increase in ionocyte abundance may not be required for pericardial edema. Therefore, we are using morpholino knockdown and mRNA-sequencing to determine whether the absence of ionocytes alters TPHP-induced pericardial edema and corresponding effects on the embryonic transcriptome. Overall, our findings to date suggest that TPHP may have different mechanisms of toxicity leading to an increase in ionocyte abundance and pericardial edema within developing zebrafish embryos.

P116: Body Weight and Organ Weight Analysis in Cambodian, Chinese, Vietnamese, and Mauritian Longtailed Macaques (Macaca fascicularis) – Recommendations for Separate Reference Databases

K. Ashcroft-Hawley¹, G. Chadalapaka², Z. Faraahi¹, M. Meindel², A. Newell¹, R. Rose³, A. Shaw¹, S. Templeton¹, and G. Weinbauer⁴

¹Labcorp Early Development Laboratories Limited, Harrogate, England, UK

²Labcorp Drug Development, Madison, Wisconsin, USA

³Labcorp Early Development Laboratories Limited, Huntingdon, England, UK

⁴Labcorp Drug Development, Munster, Nordrhine-Westfalia, Germany

Longtailed macaques are the predominant (>90%) primate species for biopharmaceutical drug development. This demand, combined with COVID-19-related transport bans, led to explore whether animal origin matters for safety assessment. This work evaluates body weight and organ weight data (absolute and adjusted) in up to 1310 animals of Cambodian, Chinese, or Vietnamese (Asia mainland), or Mauritian origin for ages 2-3, 3-4, 4-5 and 5-6 years. Asia mainland origins are considered a specific genetic cluster being quite divergent from the Mauritian cluster, and it is common practice to maintain separate reference databases for island versus mainland origins. Age-adjusted body weights were highest in Mauritian compared to other origins (p < 0.05). Differences were significant (p < 0.05) for both sexes and absolute differences attained 300-500 grams for females and 700-1200 grams for males. Mauritian males had larger absolute reproductive organ weights, most evident for testis weight (p < 0.05), supporting an earlier sexual maturity attainment compared to Asia mainland. Other absolute organ weight differences were higher weights for adrenal, kidney, liver, pituitary, spleen, and thymus in Mauritian origin. Differences were also apparent within Asia mainland origins with lowest body weight in Chinese animals vs other Asian origins. Absolute organ weights differences were also found for adrenal, heart, kidney, liver, ovary, pituitary, and uterus. Based on the latter findings, it is recommended to also maintain separate reference databases for each Asia mainland origin. Separate databases are expected to reduce variability and yield more accurate reference data.

P117: Iodoacetic Acid Exposure Alters Immune and Tight Junction Genes in the Colon of Adult Female Mice

J. Chiu¹, K. Chiu¹, A. Gonsioroski¹, and J. Flaws¹

¹University of Illinois at Urbana-Champaign, Urbana, IL, USA

Iodoacetic acid (IAA) is an unregulated iodinated water disinfection byproduct (DBP) that poses a significant health hazard to human health. In vitro studies have indicated that IAA is cytotoxic, mutagenic, genotoxic, and tumorigenic in mammalian cells. Initial exposure to IAA in drinking water occurs through the gastrointestinal (GI) tract. Yet, no studies have investigated the impact of IAA exposure on the GI tract. Thus, this study tested the hypothesis that IAA exposure alters the morphology and gene expression of immune and cell health factors in the colon compared to control. To test this hypothesis, adult female mice (n = 8 mice/group) were orally dosed with water or IAA (0.5, 10, 100, or 500 mg/L) for 35 days. Mice were euthanized in diestrus. Distal colons were collected for morphological evaluation and gene expression analysis. Morphological analysis revealed that 0.5 mg/L IAA exposure significantly decreased colon weight and length compared to control (p<0.05). IAA exposure (10 mg/L) also decreased expression of tight junction (Zo-2) and proinflammatory immune (Il6) markers compared to control (p<0.05). Exposure to IAA did not significantly alter expression levels of several cell cycle regulators (Ccna2, Ccnb1, Ccnd2, Ccne, and Cdk4), cell cycle inhibitor (Cdkn1a), immune factors (Il17, Tlr4, and Tlr5), and tight junctions (Zo-1, Zo-3, and Ocln) compared to control. These findings suggest that IAA exposure causes colonic damage by altering morphology, decreasing the integrity of gut barriers, and decreasing immune responses in the colonic immune microenvironment. Supported by NIH R21 ES028963 and NIH T32 ES007326. 

P118: Analysis of Inflammation Lipid Mediators Following Acute Sarin Surrogate Exposure and Novel Oxime Therapy

C. Y. Price¹, M. K. Ross¹, B. S. Backer¹, and J. E. Chambers¹

¹Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA

The sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP), a potent organophosphorus compound (OP), causes acute neurotoxicity by inhibiting acetylcholinesterase (AChE) in the brain. Acute OP exposure mediates toxic cholinergic signs and exacerbates mechanisms of neuroinflammation, as well as brain damage leading to long-term neurologic and cognitive deficits. Neuroinflammation is regulated by lipid mediators derived from fatty acid metabolism. Our lab has designed a series of oxime AChE reactivators (US Patent 9,227,937) that enter the brain, reduce time to cessation of seizure-like activities, and prevent neuropathology after NIMP exposure. In this study, critical areas of seizure initiation (piriform cortex and hippocampus) were examined for pro- and anti-inflammatory lipid mediators in an atropinized rat model of an acute lethal NIMP (0.6 mg/kg, s.c.) exposure and novel oxime therapy (146 μmol/kg, i.m.) at 1- and 4-days. Following NIMP exposure alone, the pro-inflammatory mediators (e.g., prostaglandins PGD2 and PGE2) were decreased at day 1 when compared to controls but had increased by day 4, while the anti-inflammatory mediator 14,15-Epoxyeicosatrienoic acid (14,15-EET) was increased at day 1 and was progressively higher at day 4. Increases in pro-inflammatory mediators were reduced at day 4 but not at day 1 in those animals given the novel oxime therapy. These results, observed in both the piriform cortex and hippocampus, suggest that NIMP can induce neuroinflammatory lipids at day 4 and the novel oxime therapy could attenuate this effect. Thus, reducing OP-induced neuroinflammation might contribute to the neuroprotection observed with these novel oximes. (Supported by NIH U01NS107127).

P119: International Travel Grant Recipient: Neuroinflammatory Mechanisms in Manganese-induced Noradrenergic Axons Degeneration in the Rat Cerebral Cortex and Hippocampus

C. B. Davuljigari¹, A. Yenukolu¹, P. Kutagolla¹, and S. R. Motireddy¹

¹Department of Zoology, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India

Manganese (Mn) is an essential nutrient metal required for the development of the brain. Chronic exposure to Mn can result in different neurodegenerative diseases, whose precise molecular mechanism remain unclear. Our previous studies demonstrated that Mn-induced neurotoxicity is associated with mitochondrial damage by impairment in the activity of the aconitase enzyme. This study aims to examine whether Mn-induced mitochondrial defects promote neuroinflammation and its association with noradrenergic axons degeneration in the cerebral cortex and hippocampus regions of the rat. Male pups were lactationally exposed to Mn (6mg/Kg body weight) through intraperitoneal injection for two weeks (5days/week) from PND 15 to PND 28. We examined the density of noradrenergic axons in the frontal cortex (M1 & M2) and hippocampus (DG, CA1, and CA3) using immunohistochemical staining at PND 29- and 3-months age group rats. The density was significantly lower in the selected frontal cortex and hippocampus regions in response to Mn-exposure. The expression of noradrenergic transporter (NET) was also significantly decreased following exposure to Mn. We found that Mn exposure substantially augmented NLRP3 abundance, caspase-1, inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor α (TNF- α) gene expression in both cortex and hippocampus regions however the observed impairments in the density of noradrenergic axons and neuroinflammatory markers were more pronounced in the cortex region than the hippocampus. Together, our findings demonstrated that Mn exposure induces noradrenergic axons degeneration through neuroinflammatory mechanisms suggest that NLRP3 activation plays an important role in Mn-induced neurotoxicity. Supported by SERB Grant No. YSS/2015/000289.

P120: The Effect of Novel Oxime Acetylcholinesterase Reactivator on Sarin Surrogate-Induced Changes of Microglial Activation Biomarker IBA-1

D. C. Stanford¹ and J. E. Chambers¹

¹Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA

Exposure to organophosphates (OPs) may induce neurological deficits resulting from long-term damage. To investigate the neuroprotection of oxime therapy for OP poisoning, ionized calcium-binding adapter molecule 1 (IBA-1) was measured as a neuropathology biomarker. IBA-1 is a protein indicating activated microglia, recruited during microgliosis. Comparisons were made between currently approved oxime treatment 2-PAM and Oxime 20, a novel oxime from a platform of substituted phenoxyalkyl pyridinium oximes (US Patent 9,227,937), both of which reactivate OP-inhibited acetylcholinesterase. Oxime 20 has displayed efficacy within the brain in in vivo tests where 2-PAM has not. The OP tested was sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP). Rats were administered a lethal dose SC of NIMP (0.6 mg/kg) or vehicle. After one hour, IM injection of oxime (2-PAM, Oxime 20; 146 µmoles/kg) was administered. Three animals per treatment group (vehicle, NIMP, NIMP+2-PAM, and NIMP+Oxime 20) were sampled four days post-treatment. Brain sections containing the CA1 region of the hippocampus were sectioned and immunohistochemically stained for IBA-1. Slides were imaged to quantify the total number of immunostained cells per area of the CA1 region in each image. A one-way ANOVA test showed no significant difference among test groups. However, a trend of increased IBA-1 staining in the NIMP group was seen compared to the vehicle group. Activated microglia in NIMP groups are indicative of brain insult from the OP. Additionally, a trend of neuroprotection with less IBA-1 staining was seen in both NIMP+2-PAM and NIMP+Oxime 20 groups compared to NIMP alone. (Supported by NIH U01NS107127).

P121: An Inter-Laboratory Case Study to harmonize Zebrafish Light-Dark Transition Test to predict Developmental Neurotoxicity for the OECD Guidance Document

A. Alzualde¹, J-H. Hsieh², L. Ellis³, V. Schiavone⁴, L. Truong⁵, J. Legradi⁶, D. Rubbini⁴, C. Woodland⁷, N. Kluver⁸, K. Ryan², M. Behl², J. Terriente⁴, A. Muriana¹, R. Tanguay⁵, M. Sachana⁹, B. Hill¹⁰, S. Padilla¹¹, T. Shafer¹², and E. Hessel¹²

¹Biobide, San Sebastián, Guipúzcoa, Spain

²Division of the National Toxicology Program, National Institute of Environmental Health Sciences, North Carolina, North Carolina, USA

³National Research Council of Canada, Nova Scotia, Nova Scotia, Canada

⁴ZeClinics SL, Barcelona, Barcelona, Spain

⁵Department of Environmental and Molecular Toxicology, Sinnhuber Aquatic Research Laboratory, Oregon State University, Corvallis, Oregon, USA

⁶Environment & Health, VU University Amsterdam, Amsterdam, The Netherlands, The Netherlands

⁷Innovation and Science Integration Unit, New Substances Assessment and Control Bureau, Healthy Environments and Consumer Safety Branch, Health Canada / Government of Canada., Canada, Canada, Canada

⁸Department of Bioanalytical Ecotoxicology, Helmholtz Centre for Environmental Research GmbH - UFZ, Leipzig, Leipzig, Germany

⁹Organisation for Economic Co-operation and Development (OECD), Paris, Paris, France

¹⁰European Commission -DG Joint Research Centre (JRC), Ispra, Ispra, Italy

¹¹Center for Computational Toxicology and Exposure, Biomolecular and Computational Toxicology Division, U.S. Environmental Protection Agency, North Carolina, North Carolina, USA

¹²Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, Utrecht, The Netherlands

Developmental neurotoxicity (DNT) is a key complex area in toxicology. The predictivity of some OECD guidelines using animal models such as TG 426 and TG 443, may be limited and they are not fully used in common practice. This implies a need to establish human-relevant in vitro models to assess the DNT potential of chemicals. OECD is building a guidance document containing a testing battery of in vitro tests that encompasses the critical processes in brain development. Since zebrafish are not considered an experimental animal model until 120 hpf, this whole-organism model presents an added value to study DNT. To assess the impact of adding the zebrafish behavioral model into the guidance, a group of experts agreed on a protocol for the light-dark transition test. Zebrafish are chemically exposed in a 96-well plate from 6-120 hpf and immediately tested following the light-dark transition test. Twenty-eight known DNT compounds were tested in 5 different laboratories. Data analysis was based on recording the locomotor activity of the larvae during the testing period. Some functional features such as the presence of an inflated swim bladder highlighted their importance on behavior. Consequently, the group agreed on modifications in the harmonized protocol to improve the performance and avoid swim bladder issues. Upon experimental repetition, a benchmark dose analysis will be performed to determine the critical effect dose of each compound for comparison of results across laboratories. The future goal is to use this zebrafish model to predict DNT and add it to the OECD guidance.

P122 – North American Graduate Fellowship Recipient: Transgenerational Impact of Pesticides on Behavior and the Epigenome

C. McClure¹, M. Blumenkrantz¹, and P. Allard¹

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

Chlorpyrifos (CPF) is a ubiquitously used pesticide which can facilitate effective farming practices by way of insect control. Its direct and intergenerational exposure has been associated with an increased prevalence of mood disorders such as anxiety and depression. However, whether CPF can also elicit behavioral alterations in a transgenerational manner is not known. Based on its ability to alter behaviors but also epigenetic marks, we hypothesize that CPF may elicit heritable epigenomic changes that will lead to transgenerational behavioral endpoints. To investigate this, we utilized the genetic model organism C. elegans to screen for behavioral changes in response to CPF, in directly exposed (P0) and transgenerational (F3) generations. Using the WormLab, which is able to track and analyze behavioral patterns in C. elegans, we have found that exposure to CPF alters several behaviors in both the P0 and F3 generations. Our higher concentration (15μM) led to a significant decrease in both wavelength and amplitude when compared with our lower concentration (2.5μM) (two independent trials, 120 worms, one-way ANOVA, P <0.0001). Our high dose-treated worms also performed significantly less reversals when compared to our DMSO control (P <0.0001). We conclude that our high dose is having a significant effect on behavior transgenerationally. Following these clearly demonstrated transgenerational effects, we will employ targeted epigenomic approaches to investigate alterations in the epigenome that may be mediating this impairment. This novel assessment is important to public health as it demonstrates the need to evaluate toxicants in our environment that can have long-lasting generational effects.

P123: Preclinical Safety Evaluation of Chronic LRRK2 Inhibition with DNL201 in Cynomolgus Monkeys

R. Meisner¹, J. A. Bravo¹, W. H. Jordan², N. Sharma³, W. A. Meier³, A. Sharma³, D. Diaz¹, and J. C. Sasaki¹

¹Denali Therapeutics, South San Francisco, California, USA

²StageBio, Mount Jackson, VA, USA

³Covance Laboratories, Inc., Madison, WI, USA

One of the most common genetic risk factors for Parkinson’s disease (PD) are mutations in leucine-rich repeat kinase 2 (LRRK2). Increased LRRK2 activity impairs lysosomal function and is thought to contribute to the pathogenesis of PD; thus, inhibition of LRRK2 is a potential disease-modifying therapeutic approach for PD. DNL201 is a first-in-class, potent, selective, small-molecule, ATP-competitive investigational LRRK2 kinase inhibitor. Previously reported toxicology evaluation of LRRK2 inhibitors includes repeat-dose studies of  28 days. To support long-term clinical trials, DNL201 was administered po, BID to cynomolgus monkeys at 0 (vehicle), 8, 16, or 32 mg/kg/day for 39-weeks, followed by a 12-week recovery period. Toxicity evaluations included clinical, ophthalmic, physical, respiratory, ECG and neurological examinations, clinical pathology, urinary biomarkers, anatomic pathology, electron microscopy (EM) evaluation of the lungs, and toxicokinetic assessment. DNL201 treatment was associated with non-adverse minimal to slight microscopic lung changes (vacuolated pneumocytes, increased lamellar bodies by EM) at ≥ 16 mg/kg/day. Changes were not associated with cellular injury nor inflammation, and did not progress following 9 months of treatment, when compared with studies of shorter duration. In the kidneys, non-adverse microscopic minimally or slightly increased pigment in renal tubular epithelial cells was observed. No DNL201-related effect on renal function was observed. Anatomic changes in lung and kidney were fully reversible following the 12-week recovery period. These data support the safety of chronic DNL201 administration for potential treatment of PD and indicate that LRRK2 inhibitors are unlikely to affect respiratory or renal function in humans in the therapeutic range.

P124: In silico Prediction of Thyroid Receptor Binding Affinity to Detect Potential EDCs

M. Girireddy¹, R. Saiakhov¹, and S. Chakravarti¹

¹MultiCASE Inc., Beachwood, Ohio, USA

The ability to identify endocrine disrupting chemicals (EDCs) is important due to their potential interferences with the endocrine system, resulting in various developmental, reproductive, and neurological adverse effects. In addition to androgen and estrogen receptor binding, thyroid receptor binding also helps to identify potential EDCs. In this regard, we built a QSAR model on thyroid receptor antagonism with rat in vitro data (Cell-based GH3-TRE-Luc assay) utilizing the structural fragments of compounds. This model identifies compounds that inhibit thyroid receptors, thereby may detect some of the EDCs that are not caught by androgen, estrogen, and aryl hydrocarbon receptor models. It successfully predicts most known EDCs which work via thyroid antagonism, e.g., bisphenols, pesticides, pharmaceutical agents, etc. Bootstrap validation metrics of the model are 89.9% sensitivity, 85.6% specificity, and 0.943 ROC-AUC. External validations are 80.17% sensitivity, 87.35% specificity and 0.859 ROC-AUC. Our model contains 566 alerts with positive and negative effects, mined automatically from training data, contributing to the interpretability of the results. The usefulness of our model lies in its ability to identify structural alerts within a query compound that are statistically related to the thyroid receptor inhibition. Thus, the combination of high interpretability and higher accuracy makes this QSAR model potentially valuable for identifying EDCs acting via thyroid receptor binding mechanism.

P125: SEND Data Analyses to Enable the Comparison of Multiple Studies

M. Carfagna¹, K. Snyder², W. Houser³, B. Paisley¹, Y. Ali⁴, C. Eley⁵, T. Fukushima⁶, S. Leuenroth-Quinn², R. Thompson⁷, J. Anderson², G. Ullmann⁸, and B. Larsen⁸

¹Eli Lilly and Company, Indianapolis, IN, USA

²US Food & Drug Administration, CDER, White Oak, Maryland, USA

³Bristol Myers Squibb, New Brunswick, New Jersey, USA

⁴Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, USA

⁵Pfizer, Groton, Connecticut, USA

⁶Shionogi & Co. Ltd., Osaka, Japan, Japan

⁷Johnson & Johnson, Springhouse, Pennsylvania, USA

⁸NovoNordisk, Copenhagen, Copenhagen, Denmark

Since the SEND requirement became effective in 2016, organizations have collected SEND data on multiple compounds intended for the same target and for a single compound tested across multiple study durations and species. The availability of these types of SEND data provides the opportunity for cross study comparison of toxicity profiles. The objective of this work, a collaborative effort between BioCelerate and FDA, is to develop data harmonization/transformation strategies and apply analytic techniques to enable an understanding of the similarities and differences between two or more SEND data sets. Example use cases include understanding a single compound’s toxicity profile across all studies performed or evaluating on- versus off-target toxicity for multiple compounds intended for the same pharmacological target. SEND data sets were transformed to integrate categorical microscopic histological findings with numerical body weight data. Similar body weight changes were identified using clustering (e.g., dose groups with body weight loss greater than 15%) of data from multiple studies. Incidence and severity of microscopic data at the organ and finding level were clustered by dose groups with similar changes. An analysis of the data components are planned and visualizations are being developed to allow for the comparison of the clustered body weight and microscopic data across multiple studies. Cross-study comparisons using SEND data are expected to improve the identification of unique findings related to the intended target and duration of dosing.

P126: Supporting Efficiencies in Nonclinical Toxicology Studies Through Protocol and Report Templates

S. Lopes¹, N. Vansell¹, P. Lynch², B. Gery², M. Carfagna³, T. Fukushima⁴, B. Emde⁵, S. Moesgaard⁶, P. Brinck⁶, J. Ingram-Ross⁷, and A. Kusnierz⁷

¹Pfizer, Groton, Connecticut, USA

²Bristol Myers Squibb, New Brunswick, New Jersey, USA

³Eli Lilly and Company, Indianapolis, IN, USA

⁴Shionogi & Co. Ltd., Osaka, Japan, Japan

⁵Boehringer-Ingelheim Pharma GmbH & Co.KG, Biberach, Germany, Germany

⁶NovoNordisk, Copenhagen, Copenhagen, Denmark

⁷Johnson & Johnson, Springhouse, Pennsylvania, USA

BioCelerate, an industry consortium driving initiatives to increase efficiency in early-stage R&D, has an ongoing project that resulted in the release of a nonclinical common protocol template for repeat-dose toxicology studies and an associated nonclinical common report template. These common templates and associated supporting materials can be downloaded from the BioCelerate website. For the protocol template development, public feedback periods and open webinars with CROs were held to collect feedback. Feedback indicated that the variety and complexity of nonclinical template formats across organizations can result in inefficient protocol/report authoring and numerous review cycles by all stakeholders. The companion report template was developed with feedback from US FDA OCS pharm tox, and an open webinar was held to gather additional input. Advantages to using these common templates for all stakeholders include (1) less time and effort to develop a protocol and the associated report for a study, (2) improved overall study quality by reducing the risk of errors due to unfamiliarity with protocol/report layout, (3) optimization of time in managing multiple studies, and (4) optimization of time spent on critical data analysis and review. Utilizing the publicly available versions of both templates, the team undertook a two-part project with participating companies to assess the value and functionality of the BioCelerate templates. Learnings from this experience will be shared to assist in the broader understanding and potential adoption of the templates by industry.www.biocelerate.org.

P127: Harmonization of SEND Implementation to Enable Historical Control Data Analysis: Recommendations for Exchanging Vehicle Details Using SENDIG v3.1

W. Houser¹, M. Carfagna², and G. Sato³

¹Bristol Myers Squibb, New Brunswick, New Jersey, USA

²Eli Lilly and Company, Indianapolis, IN, USA

³Eisai Co., Ltd, Tsukuba, Ibaraki, Japan

A collaborative team of both PHUSE and BioCelerate members was established with the goal of recommending ways to harmonize SEND information within areas with a high variability to enable common historical control data analysis. One such area in CDISC SENDIG v3.1 is vehicle description. When planning toxicology studies, researchers choose appropriate vehicles to deliver test compounds to animals requiring a selection of components compatible with the test articles. The concentration of each component should typically be within a well-tolerated range for the species of animals and not expected to exacerbate treatment-related toxicity or interfere with study interpretation. Additionally, pH, temperature, viscosity, and other properties of mixtures are important to consider for compatibility with delivery route, method, species. Selection of an appropriate vehicle can be improved by considering previous experiences with various components in toxicology studies. Toxicologists, having recognized this potential, have previously manually collated this information; a slow, laborious task. SEND datasets have the potential to simplify this analysis through an automated data analysis process if we develop better algorithms and improve the standardization in SEND, as current versions (3.0/3.1) allow for free-text fields for treatment vehicles. This project, has evaluated various approaches to address this and sought input from CDISC and the community of SEND users to determine an effective solution with relatively low effort. The conclusion is summarized here but further details are to be published in a paper later this year.

P128: Pre-clinical Strategies in Rodents Studies Using Volumetric Absorptive Microsampling (VAMs)

N. Lalayeva¹, J. Forget¹, and J. Plomley²

¹Altasciences Preclinical Seattle LLC, Everett, Washington, USA

²Altasciences, Laval, Quebec, Canada

The adoption of blood microsampling as the preferred collection technique for the entirety of a drug development program has increased, particularly for those indications requiring pediatric or vulnerable and critically-ill study populations, wherein low-volume patient-centric sampling offers enormous benefit. Blood microsampling in the nonclinical space affords both, a reduction and refinement in experimental rodent use, through the collection of small volume from less disruptive locations, ability to correlate toxicological effects with exposure in the same individuals, as well as circumventing the hematocrit (HCT) effect due to sampling volume. Nonetheless, microsampling in rodents has its limitations, especially in mice, where blood volume is the greatest challenge. Volumetric Absorptive Microsampling (VAMs) technology, sampled onto a Mitra^TM microsampling device, is a suitable method for non-clinical studies. Using VAMs (10 μL/sample), a pharmacokinetic (PK) rat study can successfully be completed with 3 animals/dose level (serial), and a PK mouse study, with 8 animals/dose level (split in 2 subsets - sparse); representing a reduction in population by 50% and 60%, respectively, compared to study designs using a standard sample volume of 0.5 mL/sample. Further refinement of study designs for regulated rodent studies includes a full or partial consolidation of satellite toxicokinetic and main study groups which results in significant improvement in animal use. Although drug development processes and regulations are very conservative, various microsampling strategies are now available and proven successful for drug development program, including studies to support clinical trials, with significant ethical benefits.

P129: Intracerebroventricular Injection in the hTau.P301S Mouse: Development of a Mouse Model for the Evaluation of Potential Drug Candidates for Neurodegenerative Disorders

G. Quesseveur¹, S. Nesme², P. Vignand¹, P. Sibilia², and L. Allais¹

¹Charles River, Safety Assessment, Lyon, Rhone-Alpes, France, ²Charles River, Research Models and Services, Lyon, Rhone-Alpes, France

Alzheimer’s disease (AD) is an age-associated neurodegenerative disorder clinically characterized by a decline in cognitive function and pathologically defined by the presence of senile plaques, neurofibrillary tangles (NFTs), and neuronal loss. Over 100 genetically engineered mouse lines recapitulating some aspects of AD clinicopathology have been developed during the past quarter-century. The hTau.P301S (PS19) mouse model is one of the most common tauopathy transgenic lines in which human 4R tau with P301S mutation is controlled by the mouse prion promoter. The blood-brain-barrier (BBB) limits access to the central nervous system (CNS) which complicates the development of potential therapeutics for neurodegenerative disorders (NDs). In order to circumvent the BBB, injection paradigms are being developed to directly deliver potential therapeutics into the brain. One of these methods is intracerebroventricular (ICV) injection which consists of injecting material into the cerebral lateral ventricles (brain cannulae), resulting in diffusion of the injected material into the CNS through the cerebrospinal fluid. An 18-week exploratory safety and PK study was conducted in this mouse model to test multiple ICV injection paradigms with an oligonucleotide to identify a technique for future testing of potential drug candidates for NDs. Brain, kidney and blood samples were collected at selected timepoints from 12 to 24 weeks old mice dosed with the compound following one or multiple ICV injections. Standard toxicological endpoints were evaluated during the study. Preliminary results demonstrate feasibility of repeated ICV administration in this model with high success rate and low to no occurrence of administration-related clinical signs.

P130: Recording and Analysis of Physiological Signals in Adult Male Rats using a Bluetooth-Based, Non-Invasive External Telemetry System (DECRO® Jacket)

J. Vial¹, S. Baudet¹, C. Bory¹, M. Ravaz¹, V. Roger¹, R. Fares², and T. Flenet²

¹Charles River, Safety Assessment, Lyon, Rhone-Alpes, France

²Etisense SAS, Lyon, Rhone-Alpes, France

Non-invasive measurements of vital functions, such as ECG, respiration and activity via jacketed external telemetry systems, are common in non-rodent toxicology studies. The availability of such a solution in rodent toxicology studies would be highly beneficial, not only because additional safety endpoints could be collected but also because of the 3Rs potential. In this context, the DECRO® solution was developed and successfully applied for rats in efficacy studies and academic research. We aimed to expand DECRO® use under the same experimental conditions as in a 4-week toxicology study, in a C.R.O. environment. Usability of DECRO® to record and analyze vital signs was tested in eight, pair-housed, male Wistar rats. Baclofen (respiratory depressant and sedative) or vehicle (n=4 per group) was administered orally, once in Weeks 1, 2 and 4. ECG was recorded with patch electrodes, respiration with inductance bands, and activity by accelerometry. Signals were recorded from -2h to 22h versus dosing time and analyzed using proprietary software, yielding heart rate, activity, and several respiratory parameters. Main results showed that: 1) the jackets remained functional despite pair housing; 2) >90% of ECG and >60% of respiratory signals were analyzable; 3) baclofen induced expected marked respiratory depressant, sedative and tachycardic effects compared to vehicle. Effects lasted for 2 to 6 hours postdose and their magnitude attenuated with aging of the rats. Integration of non-invasive monitoring of vital functions in pair-housed rats using the DECRO® jacketed external telemetry system for 4 weeks was shown to be technically feasible and ethically advantageous. 

P131: Evaluation of the NCG Mouse for Cell Therapy Toxicology Assessments

V. Smutova¹, C. Para¹, J. Rowe², S. Festin², M. Stoute¹, C. Li¹, and S. Authier¹

¹Charles River Laboratories, Laval, Quebec, Canada

²Charles River Laboratories Research Models and Services, Wilmington, Massachusetts, USA

NCG triple immunodeficient mice on a NOD/Nju background lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function. The objective of this study was to qualify the NCG mouse model for toxicity, engraftment and tumorigenicity assessments of cell therapies, using CD34+ hHSPCs adult mobilized cells with various myeloablation regimens. Mice were irradiated with 1.4 to 2 Gy or administered Busulfan to assess conditioning and injected 0.1 - 1.0 x 10⁶ cells/mouse CD34+ hHSPCs or PBS (control). The positive control animals received a tumor cell line (i.e. 2 x 10⁶ HL-60 cells/mouse). hCD34+ cell donors were stimulated with either Mozobil® (MOZ), G-CSF (Neupogen®) or a combination of both (MOZ/G-CSF). Blood samples were collected to assess engraftment rates by flow cytometry, body weights were recorded periodically up to 20 – 30 weeks post-dose. No significant clinical signs or body weight changes were observed. On week 18 post-injection, the percentage of human CD45+ cells of total lymphocytes was 48.4 ± 29 (MOZ), 56.4 ± 11.1 (MOZ/G-CSF) and 17.9 ± 12.6 (G-CSF) in comparison with 84.5 ± 9.84 when donor cells were obtained from cord blood. In a positive control group administrated with HL-60 cells, the expected mortalities due to tumorigenesis were observed between days 27 and 40 post-dose. Conditioning regimens that were tested did not significantly affect body weight or survival and engraftment rates were adequate. The NCG mouse model was considered suitable for use in cell therapy toxicology studies.

P132 – International Travel Grant Recipient: Chronic Arsenic Exposure Alters the Cognitive Behaviour and Estrogen Signaling in Female Mouse Hippocampus

K. Mehta¹, B. Kaur¹, K. K. Pandey¹, and S. Kaler¹

¹All India Institute of Medical Sciences, New Delhi, Delhi, India

Exposure to endocrine disrupting compounds (EDC) induces behavioral abnormalities associated with reproduction. Inorganic arsenic (iAs) is identified as a potent EDC, to which humans are routinely exposed deliberately or inadvertently. We therefore examined the effects of chronic iAs exposure on cognitive behaviour and estrogen receptor (ER) signaling in CA1 region (hippocampus) of adult female mice. Mice were randomly divided into control and experimental groups receiving distilled water and arsenic trioxide (2 and 4 mg/kg bw As₂O₃) respectively via oral route for 45 days. A behavioural study (open field, elevated plus maze and Morris water maze) was conducted from day 33 to 45 to evaluate status of anxiety, locomotion and learning and memory abilities. On day 46, brain tissues were procured either by perfusion fixation or by euthanasia and were processed further for immunohistochemistry, ELISA, western blotting and atomic absorption spectrophotometry. Compared to controls, mice exposed to 2 and 4 mg As₂O₃ exhibited locomotor deficits, high anxiety levels and deficits in spatial learning and memory. As₂O₃ exposure resulted in downregulation of ERα levels but no change in ER β levels, thereby suggesting differential effect of iAs on ER regulation. Additionally, the brain estradiol (E2) levels were found to be decreased after As₂O₃ exposure compared to controls. Brain tissues of experimental mice receiving 2 and 4 mg As₂O₃ showed iAs accumulation, thereby indicating that iAs crosses the blood brain barrier. Our findings suggest that chronic iAs exposure in mouse hippocampus induces long-lasting adverse effects on cognitive behaviour via modifications in ER signaling.

P133: Inhibition of Rho-associated Protein Kinase (ROCK) Induces Acute Hypotension in Dogs That Correlates with Plasma Pharmacokinetics and in vitro Potency

C. K. Choi¹, E. Kadakia¹, S. Gyoneva¹, A. Shah¹, P. Li¹, M. Kirkland¹, F. Zheng¹, and R. Gilfillan¹

¹Biogen, Cambridge, MA, USA

ROCK is a critical downstream effector of the small GTPase RhoA, which regulates the cytoskeletal dynamics and organization for numerous cellular functions. In blood vessels, ROCK can alter endothelial permeability by modulating myosin-mediated cellular contractility and cell-cell adhesion. Thus, the kinase is a key target for treating conditions of altered vascular permeability. When developing a potent small molecule ROCK inhibitor, however, reduced contractility in vascular smooth muscle cells could pose a serious risk of reduced blood pressure (BP). To investigate whether effective ROCK inhibition could cause significant reduction in BP, we conducted step-wise in vitro and in vivo studies. First, the human endothelial cells hCMEC/D3 were treated with various concentration of published ROCK inhibitors, and the resultant myosin light chain phosphorylation (pMLC) was measured to identify cellular IC₅₀. Linear correlation between cellular and biochemical IC₅₀ demonstrated the accuracy of the pMLC assay. Next, we selected one potent ROCK1/2 inhibitor (7.6 nM cellular IC₅₀) and conducted pharmacokinetics studies in dogs to determine the in vivo doses for ROCK1/2 IC₅₀, IC₉₀, and 3x IC₉₀, followed by a canine cardiovascular (CV) study. Interestingly, we detected acute and significant BP decrease (20 to 50 mmHg mean arterial), starting at ∼50 % predicted ROCK1/2 inhibition. The dynamics and magnitude of BP effects correlated with the drug plasma concentrations, indicating that hypotension was compound-mediated. Taken together, our findings demonstrate that potentially efficacious levels of ROCK1/2 inhibition induce acute hypotension that would require evaluations of orthostatic hypotension and related CV safety in humans.

P134: Withdrawn

200s—Regulatory Toxicology

P201: A Decision Tree Supporting the Use of Regulatory Frameworks in Food Ingredient Safety: An Industry Perspective

C. M. Crincoli¹, J. L. van de Ligt², A. T. Pavel³, and A. K. Eapen¹

¹Cargill, Inc., Wayzata, MN, USA

²University of Minnesota, St. Paul, MN, USA

³Cargill, Inc., Washington, DC, USA

In the United States, the regulatory framework for food ingredient approval allows for some flexibility, which can be challenging to navigate. The Food and Drug Administration (FDA) published their Final Rule on substances generally recognized as safe (GRAS) for their intended use in food in 2017. While the Final Rule didn’t deviate substantially from the proposed rule under which FDA had been operating the GRAS Program, there were some definitive margins put around best regulatory practices. With the burden of proof of safety being identical to a Food Additive Petition (FAP), if not more robust in the GRAS program, industry has proportionally chosen this route over an FAP where sufficient published data is available to support a GRAS conclusion due to the time for review by the FDA and subsequent conclusion to be reached. GRAS gives industry the opportunity to assure the safety of new ingredients while providing our customers, and ultimately the consumers, with nutritious, high-quality, and safe ingredients for use in our food supply. As an ingredient supplier, we have the responsibility to provide our customers with ingredients that are compliant and safe for use at their intended levels. Given the constant shifting demands of the consumer, research and development opportunities require nimble approaches and agility in application. We have developed a decision tree for individual ingredients and applications which help direct the flow of research and time spent in ensuring our ingredients meet the high safety and quality standards, while allowing for speed to shelf.

P202 – North American Travel Grant Recipient: Effect of Subacute Exposure to PFAS Compounds on Gut Microbial Functions and Liver Metabolome in Mice

F. Rashid¹, V. Dubinkina¹, S. Maslov¹, and J. Irudayaraj¹

¹University of Illinois at Urbana Champaign, Urbana, Illinois, USA

The animal gut microbiota consist of 100 trillion microbes which drives remarkable metabolic activities in the gut. The alteration of gut microbiota due to xenobiotic toxicity can lead to severe health consequences. Due to high levels of perfluoroalkyl and polyfluoroalkyl substances contamination in environment, we conducted this study to determine key changes induced by Perfluorooctane sulfonate (PFOS) and GenX exposure in the gut microbial functions and liver metabolism. Six groups (n=3) of male mice were treated with 5, 10, and 20mg/kg of PFOS and 10, 20, and 100 mg/kg of GenX. Two separate control groups (n=3) of mice were treated with the vehicle control. 16S rRNA sequencing and untargeted metabolome analysis were used to analyze the gut microbiome and liver changes respectively in exposed mice. PFOS was found to have more profound impact in mice by inducing amino acid biosynthesis pathways in small intestine and various fermentation pathways in colon. Whereas, GenX mainly induced the biosynthesis pathways of amino acids, purine and vitamin B12 in small intestine and in colon it induced vitamin B12 and B1 synthesis pathways. Metabolomics analysis revealed a total of 491 significantly altered compounds between treatment groups in liver (p<0.01). These metabolites are associated with host metabolic pathways mainly involved in lipid synthesis, steroidogenesis, and metabolism of amino acids, nitrogen, and bile acids. In conclusion, PFOS and GenX exposure lead to key alterations in the gut microbial functions in both small intestine and colon, as well as significant alterations in liver metabolome leading to host metabolic disorders.

P203: Determining Occupational Hazard Control Categories for Biocatalytic Enzymes

E. Fung¹, A. Buerger², J. Parker¹, C. Boles², M. Masuda-Herrera³, A. Trejo-Martin³, A. Maier², and J. Bercu³

¹Cardno ChemRisk, Aliso Viejo, CA, USA, ²Cardno ChemRisk, Cincinnati, OH, USA, ³Gilead Sciences, Foster City, CA, USA

The use of biocatalytic enzymes, which are potential inhalation sensitization hazards, in the pharmaceutical industry is increasing. Occupational controls of these enzymes are often based on exposure thresholds for Subtilisin, while enzyme-specific thresholds are typically not available. A framework for assigning biocatalytic enzymes to occupational hazard control categories was developed based on review of literature and benchmarking with biopharmaceutical sector practices. Possible outcomes, which are based on the weight-of-evidence for each enzyme, include: 1) deriving an enzyme-specific occupational safety limit; and 2) assigning to pre-defined occupational hazard categories. First, if enzyme-specific data are available, an enzyme-specific safety limit may be derived. In the absence of such data, a moderate potency category is assigned if the enzyme contains human peptide sequences. Next, screening classification with a relevant in silico prediction tool is conducted to determine whether the enzyme degrades structural macromolecules and/or is identified as an allergen. If either outcome is “yes,” the enzyme is assigned to the high potency category; if both outcomes are “no,” the enzyme is assigned to the moderate potency category. This category assignment may be amended as new enzyme-specific data, including human use experience with quantitative exposure data and empirical data from in vitro and in vivo studies, become available. In applying the framework, we found that most biocatalytic enzymes lack specific data, and while available non-clinical assays provide some evidence, further refinement and validation of such assays is needed. Nonetheless, the framework provides a pragmatic tool for relative categorization of biocatalytic enzymes.

P204: Volume Impact of Blood Withdrawal on Selected Hematology Parameters in the Longtailed Macaque (Cynomolgus Monkey, Macaca fascicularis)

J. Tripp¹ and G. Weinbauer¹

¹Labcorp Drug Development, Muenster, North Rhine-Westphalia, Germany

Longtailed macaques are the predominant primate (>90%) species for development of biopharmaceuticals. Blood collections are indispensable in regulatory safety assessment studies but balancing between required and standard recommendations for blood volumes can be challenging. Current blood volume withdrawal recommendations for macaques mostly refer to good practice guides whilst experimental data-based recommendations are scarce. This retrospective work investigates reticulocytes (RC), red blood cells (RBC), hemoglobin (HB) and packed cell volume (PCV) relative to collected blood volume from 16 dose identification studies conducted between 1993–2006. Since recommended limits for blood volume have become more tightly regulated the current limits were applied to the recent studies. Studies comprised 150 animals (87 females, 63 males, 2.5–7.5 years old, 2.5–7.7 kg body weight) with median study duration 22 days (range 5–112). Within-study blood parameter alterations were assessed from baseline 95 and 99 percentiles. RC values increased (p<0.05) and RBC, HB and PCV values decreased (p<0.05) in 12 of 16 studies. In 9/12 studies volumes (mL/kg) were within limits and in 3/12 studies exceeded current limits up to 30% over the study period (p < 0.05). In 4/12 studies without hematology changes, collected blood volumes were similar to those 9 studies with hematology changes. No relationship (p>0.05) was seen regarding frequency of exceeding limits versus frequency of blood parameter change irrespective of applying a 95 or 99 percentile reference range. In summary, the selected hematology parameters frequently changed from baseline but without a direct relation to the collected blood volumes suggesting a more complex relationship.

P205: Factors Contributing to Significant Toxicokinetic Differences for Small Molecule Active Pharmaceutical Ingredients in Pregnant Versus Non-Pregnant Rats and Rabbits

Z. Liu¹, J. L. Valentine¹, S. Altman-Hamamdzic¹, and S. K. Mukherjee¹

¹Safety Assessment and Laboratory Animal Resources, Merck & Co, Inc, West Point, PA, USA

Toxicokinetic (TK) data from the rat and rabbit toxicology studies for 39 small molecule drug candidate compounds from year 1999 to present were analyzed to determine whether it is possible to predict systemic exposure (AUC and C_max) in pregnant animals from similar data in non-pregnant animals. To reduce TK variability, only datasets that were matched exactly for dose levels, formulation and vehicle types, dose regimens, and closely for study duration were critically analyzed. The initial analysis demonstrated that 35 of 39 compounds had concordant (within ±2-fold) exposures regardless of pregnancy; 4 of 39 compounds had significant (above ±2-fold) TK differences in pregnant vs. non-pregnant rats; 1 of these 4 identified compounds also had significant TK differences in pregnant vs. non-pregnant rabbits. Further analysis showed that these 4 compounds had low solubility, pH-dependent solubility with very low solubility below pH=7, high lipophilicity, and low oral bioavailability; a combination of these properties with pregnancy-related physiological changes including higher food consumption, lower gastrointestinal pH, higher intestinal lymphatic absorption and longer gastric transit time especially during the third trimester of pregnancy might lead to significant TK changes in pregnant animals. It was concluded that approximately 90% of compounds had concordant exposures between pregnant and non-pregnant animals, and that it is possible to predict TK in pregnant animals using non-pregnant animal data for most compounds. However, for a subset of compounds that share chemical properties identified above, caution should be taken when extrapolation is made about TK in pregnant animals based on non-pregnant TK data.

P206: Derivation of the No Expected Sensitization Induction Level (NESIL) for dermal Quantitative Risk Assessment (QRA) by the Research Institute for Fragrance Materials

I. G. Lee¹, M. Na¹, M. Lavelle¹, and AM. Api¹

¹Research Institute for Fragrance Materials, Inc., Woodcliff Lake, NJ, USA

Some fragrance ingredients may have the potential to induce skin sensitization in humans but can still be safely formulated into consumer products. Quantitative risk assessment (QRA) for dermal sensitization is required to determine safe levels at which potential skin sensitizers can be incorporated into consumer products. The no expected sensitization induction level or NESIL is the point of departure for the dermal sensitization QRA. Sensitization assessment factors (SAFs) are applied to the NESIL to determine acceptable exposure levels at which no skin sensitization induction would be expected in the general population. We elucidate the key steps involved in the derivation of the NESIL for use in the QRA of fragrance ingredients. Generally, this process involves, collating and analyzing all available historical data on a given fragrance ingredient, determining the chemical’s potency, and finally deriving a weight of evidence (WoE) NESIL. When limited or no information is available on a chemical, a read-across may be used to fill the data gap or the dermal sensitization threshold (DST) approach may be applied where appropriate before further testing is considered. We provide case studies illustrating how the WoE approach is applied to generate a NESIL for fragrance ingredients. These case studies highlight the importance of using a WoE approach to set a NESIL. We outline the process as it currently stands at the Research Institute for Fragrance Materials (RIFM), but it is dynamic and is bound to change with evolving science as new approach methodologies (NAMs) are actively incorporated.

P207: Addressing Critical Aspects in Design and Conduct of Extended One-Generation Reproductive Toxicity (EOGRT, OECD443) Studies under REACH Following Regulatory Advice and the Subsequent Impact of Changes in Study Design for Ongoing and Future Studies

S. D. Renaut¹, P. B. Bushdid², D. Myers¹, S. Hershberger², and N. Barraclough³

¹Labcorp drug development, Eye, Suffolk, United Kingdom

²Labcorp Early Development Laboratories Inc., Greenfield, Indiana, USA

³Labcorp Early Development Laboratories Limited, Harrogate, England, United Kingdom

The Extended One Generation Reproductive Toxicity Study (EOGRTS, EU B.56, OECD TG 443) has been the information requirement for reproductive toxicity under REACH (Annexes IX and X, Section 8.7.3.) since March 2015. In July 2021 an evaluation of OECD 443 studies led by the European Chemicals Agency (ECHA) revealed critical issues with study designs that had the potential to compromise data analysis and interpretation. Resulting regulatory advice had an immediate impact upon the design of ongoing and planned OECD 443 studies. Recommendations emphasized demonstrating the highest possible dose level showing Reproductive effects without severe suffering or deaths in P0 animals and provided clarity on expectations for histopathology of Cohort 1B animals and intermediate and low dose level groups. Required investigations for F2 offspring and Immunotoxicity assessment in adults and offspring were clarified along with expectations for adequate documentation of methodology. A significant recommendation was the need for 60 offspring/sex/group to be evaluated for attainment of sexual maturation. For many study design variants this requires an additional cohort of animals on study, having a significant impact upon resource and cost. Retrospective analysis of studies conducted at Labcorp compares anonymized sexual maturation data to assess the relative merits of increasing the number of animals evaluated. These recommendations have resulted in the necessity to amend the design of ongoing and planned studies ultimately affecting resource, time and cost on a study which was already complex and costly. The relative merits and consequences of these recommendations are discussed from a practical perspective.

P208: Development of a Structure-Activity Relationship Profiler to Predict Mechanism-Based Inhibition of a Metabolite on CYP Enzymes

A. Bassan¹, R. Selvam², D. Bower³, K. P. Cross³, L. Stavitskaya², X. Yang⁴, D. A. Volpe², A. Amberg⁵, and G. J. Myatt³

¹Innovatune, Padova, PD, Italy

²Division of Applied Regulatory Science, Office of Clinical Pharmacology, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA

³Instem, Columbus, Ohio, USA

⁴Guidance and Policy Team, Office of Clinical Pharmacology, Center for Drug Evaluation and Research, Food and Drug Administration, Ellicott City, Maryland, USA

⁵Sanofi, R&D Preclinical Safety, Frankfurt, Frankfurt am Main, Germany

The newly finalized FDA In Vitro Drug Interaction Studies — Cytochrome P450 (CYP) Enzyme- and Transporter-Mediated Drug Interactions guidance states that in vivo drug-drug interactions caused by metabolites may be possible even if the in vitro studies suggest that the parent drug alone will not inhibit any major CYP enzymes. Structural alerts of metabolites associated with mechanism-based inhibition (MBI) represent an important element in the decision tree underlying the selection of in vitro CYP enzyme inhibition studies (Yu, H., Tweedie, D., 2013. Drug Metab. Dispos. 41, 536–540). Consequently, an extensive literature search was performed to identify alerts for MBI of CYP enzymes. These were collected from 14 key publications and associated with formation of reactive metabolites including bioactivation that results in MBI. This information was encoded within a structure-activity relationship profiler that included 734 unique substructure rules encoded within about 70 structural alerts. A proprietary database of CYP3A4 enzyme time-dependent inhibition (n = 320; 220 inhibitors) was used to qualify 20 alerts as predictive and 50 alerts as indeterminate based on available evidence (i.e., number of examples and precision of the alert). The newly developed profiler will provide a faster and more effective evaluation of potential drug-drug interaction caused by metabolites.

300s—Safety Evaluation Nonpharmaceuticals

P301: International Travel Grant Recipient: In vitro Anticancer Capacity and Ameliorative Effects of Methanol Leaf Extract of Picralima nitida on Cisplatin-induced Toxicities in Rats

A. A. Adedapo¹, A. A. Yusuf¹, O. O. Falayi¹, I. O. Ogunmiluyi¹, B. S. Ogunpolu¹, T. O. Omobowale¹, A. A. Oyagbemi¹, O. A. Adejumobi¹, O. O. Oguntibeju², and M. A. Yakubu³

¹University of Ibadan, Ibadan, Oyo, Nigeria

²Cape Peninsula University of Technology, Bellsville, Western Cape, South Africa

³Texas Southern University, Houston, Texas, USA

Objectives: The anti-proliferative effect of the methanol leaf extract of Picralima nitida on Human Colorectal Adenocarcinoma Cell lines (HT-29) was investigated using the Cell Titer 96 MTT assay. The extract was also evaluated in cisplatin-induced toxicities to determine its potent herbal properties. Methods: The in vivo studies were carried out in forty laboratory animals divided into four groups of 10 rats per group. On day 9 blood pressure measurement of all groups was taken and the changes in body weight were also determined. On day 10 blood samples were taken for serum chemistry and thereafter the animals were sacrificed and tissues such as kidney, liver and heart were harvested. Serum assayed, histopathology and immunohistochemistry were thereafter evaluated. Results: In vitro anticancer study showed that the extract at all doses caused cytotoxicity of the HT29 cells. In vivo study showed that the plant extract also improved weight changes caused by cisplatin and reversed the toxic changes produced by cisplatin on serum chemistry, oxidative stress, histopathology and in immunohistochemistry the extract caused significant decrease in the levels of nuclear factor kappa beta, cardiac troponin and mineralocorticoid receptor, but increase in the protein expression of Nrf2 when compared to the toxicant group. Conclusion: The extract thus has anti-cancer, anti-inflammatory, antioxidant and anti-renin properties.

P302: Medical Device Thaumaturgy: The Chemistry and How it Can Affect Your Toxicological Risk Assessment

S. Belperain¹, A. Van Cott¹, B. Dagger¹, S. Mimche¹, D. A. Donahue¹, D. Gilbert¹, L. Yu¹, D. Nazarenko¹, D. W. Eaker¹, and A. Hendricker¹

¹Becton Dickinson, RTP, North Carolina, USA

The International Organization for Standards, (ISO) 10993 includes guidelines that establish a universal standard on chemical characterization and biological testing for medical devices. Chemical characterization studies have become a focal point, allowing for some, if not all, potentially bypassing biological testing once a toxicological risk assessment is performed on the identified compounds. ISO-10993-12 outlines recommendations on how the extraction of devices should be completed; however, even with these recommendations, selecting the correct extraction environment is challenging. With inappropriate extraction methods, degradation of the product occurs, with detection of substances that may represent the materials of construction but may not represent substances that a patient may be exposed to in a clinical setting. Using over 100 medical devices, we considered the impact of several variables on the resulting extraction profile. Results indicated: 1) different medical device sterilization techniques can result in different extraction profiles; 2) an extraction ratio using device surface area/solvent volume vs a ratio using device weight/solvent volume had minimal impact on the extraction profile; and 3) the different materials present in medical devices (metals, polymers, etc.) require tailoring the extraction solvent to the device material to arrive at a clinically relevant extraction profile. A carefully designed extraction and chemical characterization study for medical devices may adequately identify and qualify clinically relevant hazards. Subsequent hazard and exposure assessments of the leachable substances are an integral part of an ISO 10993 based risk assessment that may adequately indicate overall biological safety for medicals device without the need for animal testing.

P303: To Stain, or Not to Stain, That is the Question: Determining the Applicability of Staining in ISO 10993-5 Cell Cytotoxicity Studies

C. L. Crocker¹, M. Dauterman¹, D. W. Eaker¹, and K. Crapnell¹

¹Becton Dickinson, RTP, North Carolina, USA

The cytotoxicity assay is an in vitro assay based on the cellular response secondary to exposure to product/material extracts (or direct contact) with confluent layers of appropriate cell lines. Known cell lines include the normal human lung fibroblast cell line Wistar Instar fetus 38 (WI-38) or the murine fibroblast-like NCTC clone 929 strain L (L929); and both are established cell lines per ISO 10993-5. The goal of this study was to demonstrate the equivalence of these two cell lines with and without staining in the cytotoxicity assay. The assay followed ISO 10993 Part 5 and Part 12 standards, cells were seeded into 48-well plates and treated with serial dilutions of Triton X-100 and allowed to reach confluency of greater than 80% to observe a graded cytotoxic response. Cells were then imaged unstained. Cells were then fixed, stained with crystal violet or trypan blue and were graded for cytotoxic effects using the USP/ISO grading system. For both cell lines, the severity of the cytotoxic response was dose responsive. The higher the concentration of the positive control (Triton X-100), the more extensive the cell growth inhibition, cell morphological changes, and cell lysis, regardless of stain. Staining with crystal violet or trypan blue did not inhibit the ability to grade the cytotoxic response Although, L929 cells are widely used in cytotoxicity testing, this study also demonstrates that WI-38 cells are comparable and appropriate for use in the cell cytotoxicity assay and that staining allows for ease in determining subtleties between the various grades.

P304: Pick and Choose: Are MTT Assays Right for Medical Device Testing?

S. Belperain¹, K. Crapnell¹, H. G. Brooks¹, D. A. Donahue¹, A. Van Cott¹, D. Nazarenko¹, and D. W. Eaker¹

¹Becton Dickinson, RTP, North Carolina, USA

The MTT assay (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is an established cytotoxicity assay for medical device testing per ISO 10993-5. It is a colorimetric assay that quantitatively measures cell viability based upon the cell’s capability to actively metabolize MTT into formazan. This viability assay has been widely used across numerous research fields; therefore, its limitations are very well established. It is known that MTT itself has cytotoxic tendencies and some research has suggested certain antioxidants can produce false positive results. The goal of this study was to determine the accuracy of the MTT assay when used for medical devices. An MTT assay was conducted in parallel with a commonly used cytotoxicity assay known as an elution assay, which is an ISO-10993-5 approved study that is not only used to measure cell lysis and death but also changes in cell morphology. The MTT assay followed the manufacturer recommendations. For the elution cytotoxicity assay, the procedure followed ISO standards. For this initial experiment, a total of 26 runs with 5 different medical device components was conducted. Our expectations were to see similar results between the two assays; however, we found this to not be the case. With the MTT showing 46% of the results as cytotoxic and the elution assay only 19%, it leads to the question on the accuracy for the MTT assay. Despite the MTT assay being well-established, the inconsistency in these initial results indicates the potential for false-positive readings, and further testing is needed to determine MTT limitations with medical devices.

P305: Skin Sensitization Potency Assessments of Fragrance Materials using the GARDskin Dose-Response Assay

M. Na¹, U. Mattson², R. Gradin², H. Johansson², A. Forreryd², and AM. Api¹

¹Research Institute for Fragrance Materials, Inc., Woodcliff Lake, NJ, USA

²SenzaGen AB, Lund, Lund, Sweden

Several New Approach Methods for hazard identification of skin sensitizers have been developed and incorporated as OECD Test Guidelines. However, the methods for potency assessment are still lacking. GARDskin (OECD TGP 4.106) was initially developed to identify skin sensitizers by monitoring transcriptional patterns of a biomarker signature in a dendritic-like cell line. The predictive capacity of GARDskin has been demonstrated previously, with 95.8% accuracy, 91.7% positive predictive value, and 100.0% negative predictive value (1 false positive, n = 24) (Johansson et al., 2019). To derive potency information, a strategy based on dose-response measurements in GARDskin, referred to as the GARDskin Dose-Response assay, has recently been proposed. The readout of the assay corresponds to the lowest concentration required to exceed the binary classification threshold in GARDskin. This concentration correlates with LLNA EC3 and human NOEL values and linear regression models have been established to exploit these relationships for potency predictions. In this blinded study, 12 fragrance materials (10 very weak sensitizers and 2 weak sensitizers) were evaluated in GARDskin Dose-Response. Results were evaluated by comparing predicted values to the reference potency categories. Three of the very weak sensitizers were predicted as non-sensitizers by GARDskin Dose-Response. For the remaining nine materials which were predicted as sensitizers, the predicted EC3 and NOEL values closely approximated the reference data for most materials. Based on results from this dataset, GARDskin Dose-Response appears useful for potency assessment for weak sensitizers and may constitute a promising strategy for deriving a point-of-departure for quantitative risk assessments.

P306: The Safety of Bael Tree Fruit as a Dietary Supplement Ingredient

E. Madden¹, H. Oketch-Rabah¹, N-C. Kim¹, S. Jordan², and T. Low Dog²

¹United States Pharmacopeia, Rockville, MD, USA ²2015-2020 USP DSAE JS3, United States Pharmacopeia, Rockville, MD, USA

Bael tree fruit derived ingredients are used in some dietary supplements (DS). Prior to developing quality monographs, US Pharmacopeia (USP) performs an Admission Evaluation (AE) that includes an assessment of available information to determine whether or not a dietary ingredient presents a serious health risk. Available clinical data show no serious effects associated with Bael tree fruit powder at intakes of 2–6 g/day for up to 60 days, exceeding intakes of 50–3900 mg/day in currently marketed DSs. One repeat-dose study reported liver histopathology findings in rats fed Bael tree fruit extract at an estimated human equivalent dose of 5,676 mg/day, exceeding intakes of 500–1000 mg/day in DSs. However, histopathology data in control animals was not reported, thus limiting the interpretation of results. Egeline (syn. aegeline), one of the constituents of Bael tree fruit, was used as an ingredient in reformulated OxyELITE Pro supplements, which were linked to an outbreak of nonviral hepatitis in 2013. However, etiologic factor(s) responsible for the outbreak remains unknown. Furthermore, the egeline in OxyLITE Pro was suspected to have been synthetically produced. Proposed USP monographs for Bael tree fruit derived ingredients have an acceptance criterion of NMT 0.05% of egeline. Applying this criterion, DSs containing Bael tree fruit derived ingredients would provide low amounts of egeline (0.05% × 50–3900 mg/day = 0.025–1.95 mg/day) in order to meet USP standards. Bael tree fruit derived ingredients were considered by the USP DSAE Joint Standards-Setting Subcommittee (DSAE JS3) and admitted for USP‐NF monograph development.

P307: DNA Methylation Changes in Genes Related to Parkinson’s Disease in Mice Treated with Intranasal Carnosine

J. T. Toebbe¹ and M. B. Genter¹

¹University of Cincinnati, Cincinnati, OH, USA

Parkinson’s disease (PD) is an increasingly prevalent disease in our society. With the increase in cases, awareness and research efforts get stronger. One of the suspected causes is aggregation of α-synuclein (aSyn), a common protein in the central nervous system. While aSyn normally aids neurotransmission in pre-synaptic neurons, the protein can aggregate under several conditions: oxidative stress, Ser129 phosphorylation, and overexpression. aSyn aggregation in the olfactory epithelium (OE) may extend into deeper brain regions such as the substantia nigra (SN), where dopaminergic neuron loss is a key feature of PD. Previous work from our lab showed that a PD mouse model (Thy1-aSyn) treated with intranasal (IN) carnosine showed mitigation of motor dysfunction, increased mitochondrial activity, and decreased accumulation of aSyn in the OE and SN. To understand the basis for these beneficial effects, analysis of DNA methylation of key genes in PD was undertaken. DNA was isolated from the OE of mice treated with IN vehicle or carnosine and used for reduced representation bisulfite sequencing. The resulting data set included over 30,000 restriction fragments, and data analysis showed differential methylation of CpG islands in fragments located in or near promoter regions. The promoter and first exon methylation status of aSyn was not affected by carnosine treatment. This preliminary analysis to find changes in DNA methylation will provide us with data needed to proceed onto RT-qPCR for validation of expression changes in genes that are associated with the beneficial effects of IN carnosine treatment in the Thy1-aSyn mouse model of PD.

P308: Immunophenotypical Characterisation of Immune Checkpoint Receptors Expression in Cynomolgus Monkeys and Human Healthy Volunteers in Resting and in T Cell Stimulatory Conditions In vitro

M. Cocco¹, J. Hollenbaugh², A. Rowse³, C. Cooper¹, Z. Faraahi¹, K. Cadwallader⁴, J. Munday¹, D. Craig-Meyer⁴, S. Morgado⁴, K. McGee¹, and E. Perkins¹

¹Labcorp Early Development Laboratories Limited, Harrogate, England, United Kingdom

²Labcorp Early Development Laboratories Inc., Greenfield, Indiana, USA

³Labcorp Drug Development Inc., Ann Arbor, Michigan, USA

⁴Labcorp Early Development Laboratories Limited, Huntingdon, England, United Kingdom

Immunotherapeutics targeting immune checkpoint receptors or their ligands (i.e., immune checkpoint inhibitors), have been groundbreaking in the field of oncology, radically changing the approach to treatment and improving the clinical outcomes of an ever-expanding list of solid tumors and hematological malignancies. However, as with other successful therapeutics before them, immune checkpoint inhibitors are not devoid of side effects, collectively regarded as immune-related adverse events (irAEs), that are not easily uncovered in preclinical immunotoxicological investigations, often due to the very low expression of their targets in immunologically unchallenged relevant species. As a contributing partner of imSAVAR (immune safety avatar: https://imsavar.eu/), an Innovative Medicines Initiative (IMI) funded project aiming to improve the efficacy and safety of immunomodulatory therapies, we characterized the expression of a broad range of immune checkpoint receptors in peripheral blood mononuclear cell subpopulations (PBMCs) derived from cynomolgus monkeys (Macaca fascicularis) and human healthy volunteers, in resting and T-cell stimulatory conditions using flow cytometry and cross-reactive antibody panels. We defined groups of checkpoint receptors with shared similarities and key differences in the two species, which included expression under resting conditions and selective response to IL-2 alone or in combination with antibodies anti-CD3 and anti-CD28 across main lymphocytes subsets. We anticipate the results of our investigation will help in supporting data driven decisions in the design and careful consideration in the interpretation of preclinical studies involving immunotherapeutics directed against these targets.

400s—Toxicology Methods

P401 – North American Travel Grant Recipient: A Validated Method for the Quantitation of Desmosine in Plasma, Urine, and Formalin-Fixed Autopsied Lung Tissue Specimens by LC–MS-MS

M. Fagiola¹, J. Avella², G. Gu¹, and J. Cantor¹

¹St. John's University, Department of Pharmaceutical Sciences, Queens, New York, USA

²Nassau County Medical Examiner, East Meadow, New York, USA

Desmosine is an elastin-specific crosslink that can be isolated and quantified to determine the progression of elastic fiber damage in chronic obstructive pulmonary disease (COPD) and other disorders. The current paper describes a method for measuring desmosine in plasma, urine, and formalin-fixed autopsied lung tissue specimens using LC–MS-MS to further determine its validity as both a biomarker for elastin degradation and a means of assessing respiratory status during routine medicolegal investigations. One calibration curve using calibrators prepared in solid tissue, ranging from 40 to 2000 ng/mL (ng/g), was developed. Quality control specimens were prepared in solid tissue as well as plasma and urine to verify proper calibration of desmosine concentration. The method was evaluated for the following parameters using ANSI/ASB standards: linearity/calibration model, limits of detection, quantification, and upper limit of linearity, bias and precision, ionization suppression/enhancement, interference studies, dilution integrity, sample stability, and carryover. This method is suitable for use in forensic laboratories because validation was performed on instrumentation routinely used in this setting. Additionally, in cases where no apparent gross lung pathology may be present at autopsy, desmosine concentrations in formalin fixed tissues may provide insight into the potential contribution of undiagnosed COPD to the loss of life. In addition to potentially serving as a screening procedure for COPD, the method provides a real-time measure of COPD drug efficacy and may therefore supersede the use of less sensitive tests such as pulmonary function studies and computed tomography.

P402: Evaluation of Escherichia coli as a Microbial Tool in Residue Monitoring of Enrofloxacin in Milk

G. Sarathchandra¹ and M. Bothinathan¹

¹Tamilnadu Veterinary and Animal Sciences University, Chennai, Tamilnadu, India

Antimicrobials are used in dairy cattle to treat disease and to improve feed efficiency. The widespread use of antimicrobials has created potential residue problems in dairy products. The problems associated with the antimicrobial residues in milk includes the risk of allergic reactions after consumption and transfer of antimicrobial resistant bacteria from livestock to human through contaminated food products. This study aims to monitor Enrofloxacin antimicrobial drug residue in milk by a simple microbial screening method. Two hundred milk samples comprising of 100 cow and 100 buffalo milk were collected from organized dairy farms. Milk samples were extracted for the antibiotic Enrofloxacin and were examined for the presence of Enrofloxacin antibiotic residues by microbial tube test method using the indicator organism Escherichia coli. The results of this study revealed 5 positive buffalo milk samples (5%) for enrofloxacin antimicrobial drug residues. This study explains that microbial screening method has a unique advantage of a high-throughput, economical and simple tool that can be used to screen the existence of antibiotic residues in foods of animal origin.

P403: Withdrawn

P404: Dual-Telemetry Implant Model for Simultaneous Cardiovascular and Electroencephalographic Evaluation in Dogs

C. L. Douglas¹, G. Friedrichs², H. Drey¹, H-M. Tang², and M. A. Osinski¹

¹Labcorp Early Development Laboratories Inc., Madison, WI, USA, ²Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA

ECG/hemodynamic recording and EEG are widely used tools for assessment of in vivo cardiovascular (CV) and brain (CNS) function, respectively, and these techniques directly correlate with clinical assessments. However, these tools have not previously been routinely combined into a single conscious animal. Simultaneous, continuous assessment of the CV and CNS systems, along with behavioral observations via time-synchronized video recording, would allow rapid assessment of the interplay of EEG perturbations and CV endpoints in cases of untoward findings or unexplained observations. Furthermore, data extracted from these models could elucidate strategies to advance programs from various therapeutic areas that may require a better understanding of the downstream consequences of the combined perturbations, as failure of either system often causes collapse of the other. Here we present a conscious canine model that combines two commonly-used DSI PhysioTel digital telemetry devices in a single animal: (L03 for EEG, body temperature, and activity, plus M11 for ECG, blood pressure, body temperature, and activity). Positive control articles were used to evaluate sensitivity of CV (0.3 mg/kg dofetilide) and CNS (6, 30, and 60 mg/kg pentylenetetrazole) parameters. The combined model is robust and sensitive to CV and EEG effects, including prolongation of the QT interval and induction of abnormal EEG, in agreement with historical and published data obtained using single telemetry implants. The ability to evaluate the two organ systems simultaneously reveals new insights into the interplay between the CV and CNS systems, including simultaneous quantification of CV and EEG measures in relation to sleep state.

P405: Validation of the ToxProfiler Reporter Assay for Toxicological Profiling and Determination of the Underlying Mode of Action

B. ter Braak¹, L. Wolters¹, T. Osterlund¹, B. van de Water², and G. Hendriks¹

¹Toxys, Oegstgeest, ZH, The Netherlands, ²Leiden University, Leiden, ZH, The Netherlands

Classical in vitro chemical safety evaluation often relies on simple cytotoxicity endpoints. However, the underlying mechanisms are usually poorly understood from these general assays. In order to gain further understanding of cell stress mechanisms we have developed the ToxProfiler assay, a unique new approach method (NAM). ToxProfiler can be applied to accurately quantify chemical-induced cellular stress response pathways to reveal the toxicological Mode of Action (MOA) of novel medicines, (agro)chemicals, cosmetics and food ingredients. ToxProfiler consists of a collection of 7 unique genetically engineered human liver HepG2 cell lines. Each cell line contains a green fluorescent protein reporter that reflects a specific cellular stress response pathway, oxidative stress, genetic stress, ER stress, ion stress, protein stress, autophagy and inflammation. To visualize activation of the different cellular stress response reporters, we developed an automated live cell confocal imaging and data analysis pipeline to derive a unique quantitative toxicity fingerprint of chemicals. A point of departure (POD; lowest concentration at which a significant response was observed) is determined for each endpoint in order to directly compare the potency from one chemical to another. Validation of ToxProfiler was performed using numerous compounds all showing the expected profiles of toxicities. The compounds were then clustered according to the tox-profiles to provide evidences of read-across. To summarize, we developed and validated a new mechanism-based in vitro reporter assay, ToxProfiler, that can provide mechanistic toxicity information for early chemical safety testing, read-across, adverse outcome pathways (AOP) and weight-of-evidence (WoE) approaches.

P406: Neurobehavioral Testing of Juvenile Miniature Swine

S. Boley¹, J. Klein¹, M. Haney¹, and C. Selby¹

¹Sinclair Research Center, Auxvasse, MO, USA

Nonclinical juvenile studies are an essential method for assessing potential safety hazards for products intended to be administered to pediatric patients, and there has been a steady increase in the number of these studies conducted in the past decade. One of the concerns of using novel therapeutics in pediatrics is due to the continued development of the brain and nervous system in children. The use of neurobehavioral testing in nonclinical juvenile toxicity studies allows for the understanding and mitigation of some of the concerns related to potential test article-related toxicity for the brain and nervous system. We have developed a testing system and scoring rubric that allows for juvenile minipigs, as young as 1-month old, to be tested for neurobehavioral changes that may be due to test article administration. This age in the minipig is consistent with the development stage of a human at 2 years of age. Although the approach is not novel in adult pigs, it has not been previously described in juvenile animals. This assessment includes mental status, standing/posture, gait/mobility, tremors/convulsions, respiration character, respiration rate, strabismus, nystagmus, ear reflex, snout reflex, response to handling, muscle tone, dorsal pressure reaction, positioning reaction, hopping reaction, and vocalization. The use of this assessment has been employed successfully in GLP studies and was accepted by the regulatory agency to move a product forward into pediatric patients.

P407: Development of Long-Term Cecal Catheterization for Repeated Infusions in Miniature Swine

K. McGinley¹, E. Callahan¹, and M. Haney¹

¹Sinclair Research Center, Auxvasse, MO, USA

Small intestinal catheter implantation has been described in multiple species. However, literature is limited on catheter placement in the cecum of swine. This study aimed to allow liquid drug delivery directly to the cecum for repeated, sedation-free dosing in order to investigate the GI toxicity of a test article seen following oral administration. Yucatan swine (n=6), sedated and maintained on isoflurane, had surgical implantation of an intestinal burp valve catheter into the cecum. Through a paramedian incision, the catheter was inserted into the cecum and secured and was tunneled to a subcutaneous pocket on the lateral neck. A port head was attached and secured to underlying muscle. Incisions were closed and ports were aseptically flushed with sterile saline at least monthly. Catheter placement was successful in all animals. Duration of infusions were up to 6 hours up to every 4 days. Complications included port infections (n=2), resulting in euthanasia at 65 and 125 days post-surgery. Other complications (n=1) involved catheter separation from the port. Catheters remained functional at least 125 days in the surviving 3 pigs. In conclusion, catheter implantation is a viable option for repeated dosing into the cecum. This presents a refinement to more invasive methods by delivering consistent, targeted dosing without sedation. Though alternative methods for sample collection or capsule dosing are required, cecal catheters provide a sustainable route for long-term fluid administration. Thus, cecal catheters facilitate a reduction in animal numbers and improvement in data quality by enabling repeated dosing without systemic effects of sedation.

500s—Safety Evaluation Pharmaceuticals

P501: Pharmacology/Toxicology Review of Extractables and Leachables (E/Ls) in Generic Drug Products: Common Deficiencies and Successful Practices

C. Gupta¹, A. Chaudhary¹, S. Rayavarapu¹, S. T. King¹, and R. T. Dorsam¹

¹Division of Clinical Review, Office of Bioequivalence, Office of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA

Extractables and leachables (E/Ls) are chemical entities that might be extracted from and/or migrate from the manufacturing components or the container closure system of a drug product. The presence of E/Ls can affect the safety of a drug product. FDA’s Office of Generic Drugs- Pharmacology/Toxicology (Pharm/Tox) assessors evaluate the safety of E/Ls in Abbreviated New Drug Applications (ANDAs) to ensure that generic drugs have similar safety profile as their respective reference listed drugs (RLDs). The authors surveyed 114 Pharm/Tox assessments of E/Ls in generic drug products. The survey identified deficiencies in 70% of E/L studies and applicant’s safety justification reports in the first Pharm/Tox review cycle. The commonly identified deficiencies were: (a) inappropriate analytical methods; (b) inappropriate threshold values, maximum daily dose (MDD) or maximum daily exposure (MDE); and (c) inappropriate toxicological justification (e.g., use of in silico methods or Cramer classification for general toxicity justification, lack of consideration of context of use of drug product). These deficiencies warranted additional information from the applicants and led to multiple review cycles. Successful E/L safety justifications included appropriate safety concern threshold and MDD to calculate the analytical evaluation threshold of E/L studies, appropriate calculation of MDEs, and included toxicological assessments (genetic, systemic and/or local toxicity) that considered proposed context of use (i.e., dose, route of administration, duration of use, and patient population) in the safety justification. Overall, this abstract highlights common deficiencies in E/L submissions and identifies successful practices to improve ANDA submission quality.

P502: Comparative Toxicity of Free and Liposomal Drugs to Human iPSC-derived Cardiomyocytes: A Mitochondrial Respiration Study

M. C. Fortin¹, Q. Wang¹, and T. Grammatopoulos²

¹Jazz Pharmaceuticals, Inc., Philadelphia, PA, USA

²BioEnergetics, LLC., Boston, MA, USA

Even though cancer patients might benefit from continuing anthracycline-based chemotherapy, their use is constrained by clinically-determined lifetime exposure limits due to cardiotoxicity. A liposomal formulation of doxorubicin has demonstrated mitigation of some of these effects in patients. The purpose of this study was to 1) develop a model that could recapitulate this benefit in vitro and 2) apply this model to assess the relative cardiotoxicity of CPX-351, a liposomal formula containing daunorubicin:cytarabine in a 1:5 molar ratio, vs free daunorubicin+cytarabine. Specifically, hiPSC-derived cardiomyocytes (∼15,000 cells/well) were treated with free or liposomal drug(s) at concentrations ranging from 0 to 1000 ng/mL, for one, two, or three, bidiurnal 24-h treatment periods. The oxygen consumption rate (OCR) was measured using the Seahorse XF96 platform on day two, four, six, and eight, and the basal, ATP-linked, maximal, and reserve capacity parameters were calculated. This design allowed evaluation of the dose-response and cumulative-exposure-response. Vehicle-control-treated hiPSC-derived cardiomyocytes displayed relatively stable OCR values over the duration of the study. Free doxorubicin caused significant time- and dose-dependent decreases in OCR values (p<0.05), while liposomal doxorubicin caused minimal changes. This finding is consistent with the clinical observation that liposomal doxorubicin is less cardiotoxic than free doxorubicin. Similarly, CPX-351 showed minimal effects on OCR values, while the free daunorubicin+cytarabine drug combination caused significant time- and dose-dependent decreases in OCR parameters (p<0.05). In this preclinical model, CPX-351 appears less cardiotoxic than the free daunorubicin+cytarabine drug combination. Additional studies will be required to further characterize the cardiotoxicity profile of CPX-351.

P503: AAV2-hAADC (Eladocagene Exuparvovec) Vector Biodistribution and Transgene Expression of Human Aromatic Amino Acid Decarboxylase (hAADC) cDNA following Administration in Monkeys by Multiple Routes: Superiority of Intraputaminal versus Intracerebroventricular and Intrathecal Routes

D. R. Compton¹, S. J. DeMarco¹, and P. Yalamanchili¹

¹PTC Therapeutics, Inc., South Plainfield, New Jersey, USA

AADC deficiency is a genetic disorder of enzyme loss with decreased neurotransmitter synthesis. Potential therapies include CNS delivery of an adeno-associated virus (AAV) packaging the human enzyme (hAADC) cDNA. A biodistribution/expression study was conducted in monkeys to evaluate AAV2-hAADC following intraputaminal (iPut), intracerebroventricular (ICV), or intrathecal (IT) administration to determine the acceptability of less invasive (ICV/IT) routes for possible infant dosing. The ICV/IT dose was 10-times that of the iPut route to compensate for lower bioavailability via CSF delivery. Results show all three routes produced comparable CSF vector levels. The iPut route yielded the highest levels of transgene-derived expression in the putamen, globus pallidus, and caudate, while that in the spinal cord and dorsal root ganglia (DRG) were undetectable. Unlike ICV/IT, the iPut route produced no vector in blood. The highest vector levels in ICV/IT groups were in the spinal cord and DRG. In conclusion, an ICV/IT dose 10-times that of the iPut dose was appropriate, given all routes produced comparable CSF transgene levels. Regarding efficacy, only iPut was acceptable, as ICV/IT routes produced insufficient expression in the caudate/putamen and globus pallidus. Regarding safety, all routes proved well tolerated while only the iPut route had no detectable vector in blood, suggesting less likelihood of off-target toxicities. Intraputaminal dosing also resulted in the lowest ADA levels. For iPut dosing only, biodistribution to DRG (a target of special toxicological concern), did not occur. These data demonstrate that, based on safety and efficacy, iPut administration of eladocagene exuparvovec is superior to ICV/IT administration.

P504: Nonclinical Safety Profile of PN-943, an Orally Peptide-Based Molecule, Supports Its Safe Use in Ulcerative Colitis Patients

J. Zhao¹, L. Wang¹, Y. Pu¹, N. Modi¹, J. Tovera¹, G. Zemede¹, G. Ledet¹, M. Masjedizadeh¹, and D. Liu¹

¹Protagonist Therapeutics, Newark, CA, USA

PN-943, a specific α4β7 integrin inhibitor, is an oral, gastrointestinal-restricted, peptide-based drug candidate currently in a Phase 2 trial in moderate to severe ulcerative colitis (UC). The toxicity profile of PN-943 was evaluated in a comprehensive program of GLP-compliant studies. PN-943 did not elicit any treatment-related adverse effects in any of the rat and cynomolgus monkey 28-day, 13-week, 6-month (rat), or 9-month (monkey) repeat-dose GLP toxicology studies. Both dose-range finding and definitive embryo-fetal development (EFD) studies in rats and rabbits were also completed. In definitive EFD studies, both maternal and developmental no-observed-effect-level (NOEL) was 1000 mg/kg/day in rats and 75 mg/kg/day in rabbits. All in vitro and in vivo genetic toxicology studies were negative, indicating a low genotoxic potential of PN-943. No PN-943 treatment related effects were observed in the safety pharmacology studies for cardiovascular, respiratory, and central nervous systems. Margins-of-safety (MOS) were thus calculated to be minimally 4.7x and 26.4x from the steady-state area-under-curve (AUC) values obtained from the 6-month rat and 9-month monkey toxicity studies, respectively. The observed steady-state human exposure of PN-943 following oral dosing of multiple doses of 450 mg twice daily (BID) (900 mg daily) in healthy volunteers was used for MOS comparisons. Furthermore, the highest dose tested in a 4-week rat limit dose toxicity study,1000 mg/kg/day, was considered as NOEL, resulting in MOS of 26.4x over 450 mg BID (900 mg daily) in humans. In summary, the available toxicology results support the continued development of PN-943 as a potential human therapeutic for UC.

P505: HUMIMIC–InHALES — Human Aerosol Test Platform for Assessing Respiratory Toxicity and Systemic Human Effects of Inhaled Aerosols

K. Renggli¹, S. Steiner¹, F. Rambo², A. Sandoz¹, D. Bovard¹, H. Erfurth², K. Schimek², B. Ataç², U. Marx², and J. Hoeng¹

¹Philip Morris International, Neuchâtel, Neuchâtel, Switzerland

²TissUse GmbH, Berlin, Berlin, Germany

Philip Morris International and TissUse GmbH present the first specifically designed in vitro system for systemic risk and efficacy assessment of aerosols. The logical combination of aerosol delivery testing for advanced cell culture systems and cutting-edge microfluidic microphysiological systems (MPS) enables the system to physically mimic cell type interconnections and systemic aerosols delivery. The Independent Holistic Air–Liquid Interface Exposure System (InHALES) was designed as a mechanical replica of the whole human respiratory tract; that is, cell cultures can be exposed at the air–liquid interface in a model of each relevant respiratory tract compartment. The existing InHALES was engineered to implement TissUse’s proprietary commercial HUMIMIC microfluidic platform. The HUMIMIC MPS technology is capable of maintaining and culturing miniaturized organs that emulate the biological function of their respective full-size counterparts over long periods. The technology allows flexible and customized combination of different tissue constructs or organ equivalents on a disposable chip-based MPS. The resulting HUMIMIC–InHALES system supports the development of several type of systemic substance assays for aerosol exposure, e.g., acute/chronic toxicity and long-term disease treatment (efficacy) assays. For this purpose, we can leverage our knowledge of existing advanced in vitro models and their application in toxicity, metabolism, and disease modelling. Available organ models include the liver, intestine, skin, vasculature, neuronal and cardiac tissue, cartilage, pancreas, kidney, fat, tumors, and bone marrow. Furthermore, the system provides a means to understand the underlying systemic interplay and crosstalk between these biological models in their true physiological context, which conventional assays often neglect.

P506: Toxicology-based Exposure Limits for Residual HEK-293T Cell DNA and Protein

I. Mohar¹ and T. A. Lewandowski¹

¹Gradient Corporation, Seattle, WA, USA

Immortalized mammalian cell lines are useful tools to generate biological drugs, including antibodies, gene-therapy vectors, and vaccines. As living cells, however, these manufacturing tools can introduce host cell components, such as DNA and protein, as manufacturing impurities in the final drug product. We reviewed the publicly available toxicology data for a commonly used cell line, HEK-293T, in order to 1) determine if the WHO limit of 10 ng of residual host cell DNA per dose can be applied to the HEK-293T cell lineage and 2) identify a toxicology-based limit for residual HEK-293T cell protein. Drug regulatory agencies consider HEK-293T to be a weakly tumorigenic continuous cell line, which therefore poses the theoretical risk of introducing "immortalization factors" into a drug product. For residual DNA, we found no published data to support concluding that residual HEK-293T DNA can transform a normal human cell. While the xenogeneic DNA (e.g., Adenovirus serotype-5 (Ad5) E1A, Ad5E1B, and SV40 large T antigen [SV40LTA]) present in HEK-293T cells may pose a theoretical oncogenicity hazard, standard manufacturing processes that involve nuclease- or chemical-mediated DNA fragmentation and purification can reasonably be expected to reduce this hazard to a tolerable risk. For residual protein, SV40 LTA was concluded to not pose a meaningful risk; instead, innate immune activation was identified as the most relevant hazard, and a limit based on high-mobility group box protein-1 (HMGB1) was proposed. Collectively, the available toxicology data for HEK-293T cells provide reassurance that residuals can be controlled at amounts where patient risk is negligible.

P507: Assessing the Carcinogenicity of Vadadustat, an Oral Hypoxia-Inducible Factor (HIF) Prolyl Hydroxylase Inhibitor, in Rodents

H. L. Conboy¹, D. J. Hoivik¹, and M. Rabinowitz ¹

¹Akebia Therapeutics, Cambridge, Massachusetts, USA

Vadadustat is an investigational oral hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) being developed for the treatment of anemia due to chronic kidney disease in adult patients who are dialysis-dependent (DD) or non-dialysis-dependent (NDD). The purpose of this study was to evaluate the potential carcinogenicity of vadadustat in mice and rats. In CByB6F1/Tg-rasH2 hemizygous (transgenic) mice, 5, 15, and 50 mg/kg/day of vadadustat was administered by oral gavage for 6 months. In Sprague-Dawley rats, 2, 7, and 20 mg/kg/day was administered for 2 years by oral gavage. In both studies, vadadustat did not impact survival or body weight. There was a mild increase in RBC parameters in the high dose groups, which was indicative that pharmacologically relevant exposures were reached. There was no statistically significant incidence of tumors in either sex that was outside published incidences in control animals. In mice, a dose of 50 mg/kg/day was associated with AUC exposures of 94700 ng·hr/mL in male mice and 167000 ng·hr/mL in female mice. The exposure achieved in males is approximately 0.15-fold and 0.21-fold the dose achieved in NDD and DD subjects respectively and in females is approximately 0.27-fold and 0.40-fold projected exposures in NDD and DD subjects, respectively. In rats, a dose of 20 mg/kg/day was associated with a vadadustat AUC (combined sexes) of 216000 ng·h/mL, which is approximately 0.36- and 0.52-fold projected exposures in NDD and DD subjects, respectively. In conclusion, there was no carcinogenic effect attributed to vadadustat in mice or rats under the conditions of these studies.

P508: In vitro Evaluation of Compound-related Changes on Rabbit Hemostasis by Thrombin Generation Assay

A. Pinçon¹, S. Woaye Koi¹, and F. Poitout-Belissent¹

¹Charles River Laboratories, Senneville, Québec, Canada

The thrombin generation assay (TGA) is a functional test evaluating the global clotting potential of plasma. It is sensitive to different coagulation factors concentrations and can detect hypocoagulable and hypercoagulable conditions. Aims: Rabbits are common animal models used to study compounds that can impact hemostasis. The purpose of this study was to evaluate the in vitro effects of Heparin®, Tissue Factor (Dade® Innovin®) and Rivaroxaban on rabbit TGA, using a calibrated automated thrombogram (CAT). Methods: Accuracy, Linearity and Sensitivity to Heparin (Leo Pharma), Tissue-Factor (Innovin, Siemens) or Rivaroxaban (Cayman Chemical) on TGA were evaluated using citrated rabbit plasmas. All TGA parameters were tested: lag time, time to peak, start tail, peak and endogenous thrombin potential (ETP). Results: Intra and Inter-assay precision of neat rabbit plasma samples was <20% for all TGA parameters. Linearity of dilution was not conclusive as ETP and Peak increased with dilution and were not proportional to the dilution factor, suggesting an inhibition of anticoagulant factors. Thrombin generation in spiked rabbit plasma samples was completely inhibited with Heparin at 0.4 UI/L and inhibited down to 90% with Rivaroxaban at 300 ng/mL (decreased peak and ETP, and increased times), and it was accelerated up to 27.4% (higher peak) with Innovin (1:3000, dilution), compared to control samples. Conclusion: TGA is a sensitive assay for in vitro evaluation of compounds anticoagulant or procoagulant activity in rabbits. Plasma samples cannot be diluted for TGA evaluation.

P509: Challenges and Gaps in Immunosafety Assessments of Pharmaceutical Drugs: An Industry Survey

M. Collinge¹, S. Akella², B. Fogal³, K. Fraser⁴, C. L. Fuller⁵, K. S. Harper⁶, J. Jabbour⁷, C. C. Maier⁸, L. Malherbe⁹, N. B. Marshall⁸, K. Raman¹⁰, G. K. Rao¹¹, H. S. Skaggs¹², F. Weber¹³, and H. Neff-LaFord¹⁴

¹Pfizer, Inc, Groton, CT, USA

²AbbVie, Redwood City, CA, USA

³Boehringer-Ingelheim, Ridgefield, CT, USA

⁴Takeda, Cambridge, MA, USA

⁵Merck, West Point, PA, USA

⁶UCB, Slough, Berkshire, United Kingdom

⁷Gilead Sciences, Foster City, CA, USA

⁸GlaxoSmithKline, Collegeville, PA, USA

⁹Eli Lilly and Company, Indianapolis, IN, USA

¹⁰Amgen, South San Francisco, CA, USA

¹¹Genentech Inc, South San Francisco, CA, USA

¹²Incyte Corporation, Wilmington, DE, USA

¹³F. Hoffmann La- Roche Ltd., CH-4070, Basel, Switzerland

¹⁴Seagen, Bothell, WA, USA

Immunotoxicology/immunosafety science is rapidly evolving, with novel modalities and immuno-oncology among the primary drivers of new tools and technologies. One challenge with new technologies is understanding the uptake across industry and impact/acceptance by regulatory authorities. As such, the Immunosafety Working Group of IQ/DruSafe set out to better understand some of the key challenges in immunosafety evaluation, gaps in the science, and current limitations in methods and data interpretation. To this end, a survey was developed with the aim of providing a baseline understanding of the needs and challenges faced in immunosafety assessments, the tools currently being applied across the industry, and impact of feedback received from regulatory agencies. Leveraging industry experience is critical for understanding the current state of immunosafety evaluation in drug discovery and development and will help to focus future efforts across the industry. In addition to identifying some of the broader challenges and gaps, this initial survey focused on current practices and challenges in two key areas, the T-dependent antibody response (TDAR) and the cytokine release assay (CRA), with particular emphasis on feedback from regulatory agencies. Among some of the key challenges identified were translation of immune-related safety findings observed in nonclinical species to human, understanding the relevance and safety implications of exaggerated pharmacology, and the lack of appropriate preclinical models. Additional key outcomes from the survey with respect to the TDAR and CRA will be presented.

P510: Toxicity and Toxicokinetic Assessment of an Anti-Tubercular Drug Pretomanid in Cynomolgus Monkeys

R. S. Bruning-Barry¹, J. L. Ambroso¹, J. Dillberger², and T. J. Yang³

¹RTI International - Research Triangle Park, NC, Research Triangle Park, North Carolina, USA

²J. Dillberger LLC, Nashville, Indiana, USA

³Global Alliance for TB Drug Development, New York, New York, USA

Pretomanid is a nitroimidazooxazine antimycobacterial drug recently approved in the United States, Europe, and India as part of a three-drug, all oral regimen, consisting of bedaquiline, pretomanid, and linezolid (BPaL) for 6-months treatment of adults with pulmonary extensively drug-resistant tuberculosis (XDR-TB) or with complicated forms of multidrug-resistant tuberculosis (MDR-TB). To support clinical development and approval, the Sponsor conducted a series of repeat-dose oral toxicity studies up to 39 weeks’ long in cynomolgus monkeys. Target organs in monkeys were the heart (decreased heart rate, QT prolongation), eyes (cataracts), central nervous system (ataxia, tremor, convulsion), and liver (hepatocellular hypertrophy). NOAELs were identified for both sexes in all studies. The NOAEL decreased with increasing study duration; however, systemic exposures to pretomanid at all NOAELs equaled or exceeded exposure in patients at the approved dose (200 mg), except for females in the 39-week study. Adverse effects involving the heart, eyes, and central nervous system were not seen in human subjects during clinical trials, possibly because systemic exposure remained below the threshold for these effects.

P511: Non-Human Primate (NHP) Husbandry and Impact on NHP Health: Outcomes from an IQ DruSafe/3RS Industrial Benchmark Survey

S. Salian-Mehta¹, J. Poling², J. Birkebak¹, S. Rensing³, C. Carosino⁴, R. Santos⁵, W. West⁶, K. Adams⁷, H. Klein⁷, and D. Fillman-Holliday⁸

¹Gilead Sciences, Foster city, California, USA

²Eli Lilly and Company, Indianapolis, IN, USA

³Abbvie, North Chicago, Illinois, USA

⁴Seagen, Bothell, WA, USA

⁵Pfizer, New York, NY, USA

⁶Boehringer Ingelheim, Ridgefield, CT, USA

⁷Incyte Research Institute, Wilmington, DE, USA

⁸Glaxosmithkline Pharmaceuticals, Philadelphia, PA, USA

Health issues (abnormal feces, failure to thrive, stress-induced abnormal behavior) in Non-Human Primates (NHPs) can confound toxicity study interpretation or lead to animal exclusion contributing to the NHP shortage. A working group cosponsored by DruSafe and 3Rs Translational and Predictive Sciences Leadership Groups of the IQ consortium conducted a survey to benchmark pre-study screening and veterinary care practices and their impact on NHP health and to identify opportunities to improve quality and consistency of NHP husbandry. Improvements to animal welfare can reduce health issues that can confound toxicity study interpretation or lead to animal exclusion. Eleven companies participated providing separate responses for studies conducted in-house and at CROs from 3 regions (North America [NA], Europe and Asia) for an aggregate of 33 responses. A majority conducted studies at NA CROs (39%) or in-house (36%) using Chinese (33%) or Cambodian (27%) origin NHPs. Forty-Five percent of respondents had pre-study health issues (fecal, etc.) on ≥ 1 studies with the highest incidence observed in Vietnam origin NHPs (80%). Variable pre-screening and quarantine practices across facilities did not provide clear connection to lowering health issues. Husbandry practices including behavioral assessments, environmental enrichment and consistent diets were associated with a lower incidence of health issues. Survey also captured approaches used to diagnose and manage abnormal feces in NHPs. The survey highlighted opportunities for harmonizing screening criteria across industry and for improved tracking and sharing of health screening results, leading to further refinement of NHP veterinary care practices, higher quality studies, and reduced NHP use.

P512: Development and Cross-Species Comparison of Cytokine Release Assays

A. McDermott¹, Z. Matthews¹, A. Bonow¹, A. Lucchini¹, and A. Frantz¹

¹Labcorp Drug Development, Madison, Wisconsin, USA

Cytokine release assays (CRAs) are essential in vitro assays for the de-risking of monoclonal antibody therapeutics prior to first in human studies. However, the identification and selection of appropriate controls for cytokine release assays across different formats remains a challenge to assay design and interpretation. In the current study, we sought to compare the performance of multiple commonly used controls across the most popular CRA formats, whole blood and peripheral blood mononuclear cells (PBMCs), in both human and non-human primates, an important pre-clinical safety species. Whole blood or PBMCs isolated from at least 3 donors were incubated for approximately 24 hours with either anti-CD3, phytohemagglutinin, PMA and Ionomycin, or all four in combination in the liquid phase. After incubation, plasma or cell culture supernatant was collected and assessed for levels of IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFα using the V-PLEX Proinflammatory Panel 1 Human Kit or IFNγ, IL-1β, IL-2, IL-6, IL-8, and IL-10 using the Proinflammatory Panel 1 NHP Kit on the Meso Scale Discovery platform. Overall, PBMCs produced more cytokines than whole blood when stimulated by the same concentration of the same mitogen, including IL-2 and TNFα production in response to PHA. Likewise, stimulation conditions containing PMA and Ionomycin consistently produced the highest level of cytokines as compared to other stimulation conditions in the same format, most notably seen in IL-2 production. Collectively, these data represent a formal assessment of candidate control compounds in the most popular CRA formats.

P513: Efficient Applied Toxicology during COVID-19: A Preclinical Study that Advanced Clinical Use of Ampion

L. Goldberg¹, H. Cherevka¹, and P. Kuehl²

¹Ampio Pharmaceuticals, Englewood, CO, USA

²Lovelace Biomedical, Albuquerque, NM, USA

Background: The effective application of preclinical toxicology studies for therapeutics is essential and critical during COVID-19. Traditional nonclinical studies require multiple animal species in large groups, often taking a year to complete. Ampion is a biologic drug containing a blood-derived cyclized peptide and small molecules that target the innate immune response responsible for inflammatory disease to provide a treatment for COVID-19 patients. The FDA identified a need to assess safety in animals with Ampion via inhalation before first-in-human trials. A right-sized study was designed with FDA feedback to assess the local toxicity in the lung and other respiratory tissues at a range of inhalation doses. Methods: Rats were exposed to multiple doses of nebulized Ampion delivered via nose-only inhalation daily over 5 days. Aerosol delivery was optimized to ensure exposure at a maximum feasible dose at least 10x higher than the planned human dose. Results: Ampion was well tolerated for all exposure groups with no abnormal clinical signs, changes in lung function, hematology/chemistry, and no adverse findings during gross/detailed pathology. The No Observed Adverse Effect Level (NOAEL) daily dose is equal to 15x higher than the proposed human adult nominal dose. Based on the results, the FDA awarded an active Investigational New Drug (ClinicalTrials.gov: NCT04606784). Discussion: This program is an example of efficient application of preclinical toxicology that is responsive to the COVID-19 pandemic and FDA requirements.

P514: A Six-Month Pre-clinical Safety and Biodistribution Study of cyno-AT845, a Muscle-Directed AAV-based Gene Replacement Therapy for Pompe Disease

M. Eggers¹, J. Brassard², A. Moss¹, P. Purushothaman¹, N. Fitch³, and C. Fonck¹

¹Audentes Therapeutics, San Francisco, CA, USA

²Jacqueline Brassard Toxicologic Pathology Consulting, Tustin, CA, USA

³Charles River Laboratories, Reno, NV, USA

Pompe disease is a rare autosomal recessive neuromuscular disorder caused by loss-of-function mutations in the acid α-glucosidase (GAA) gene. These mutations prevent GAA mediated hydrolysis of cellular glycogen, leading to glycogen accumulation in tissues. The clinical phenotypes include progressive muscle weakness and cardiac insufficiency, which can vary in age of onset as well as rates of progression and severity. Enzyme replacement therapy is currently the only available treatment for Pompe. AT845 is an investigational Adeno-Associated Virus Serotype 8 (AAV8) vector containing the human GAA gene under the control of a muscle-restricted promoter designed to express human GAA synthesis and restore activity directly in skeletal muscles and heart after a single intravenous dose. To determine biodistribution and safety, a six-month study was conducted in Macaca fascicularis monkeys using a similar construct containing the cynomolgus macaque GAA gene (cyno-AT845) dosed at 6 x 10¹³, 1 x 10¹⁴, and 2 x 10¹⁴ vg/kg. Vector-derived GAA protein was noted in the heart and quadriceps muscle, while dose-related tissue activity of GAA was increased by administration of cyno-AT845 in diaphragm, heart and quadriceps muscle. Distribution to cardiac tissue was dose-dependent and demonstrated a direct relationship between vector copy number and GAA protein expression. Collectively, the pathology findings in the heart, skeletal muscle, and lumbar and thoracic dorsal root ganglia were limited to non-adverse mononuclear and mixed cell infiltrates. Overall, cyno-AT845 is well-tolerated and led to dose-dependent increases in GAA activity in skeletal muscle and the heart of Macaca fascicularis monkeys.

P515: Case Studies Demonstrating the Process and Challenges of Deriving Exposure-Based Limits for Impurities in Pharmaceutical Drug Products Administered Directly to the CNS

A. L. Kimzey¹, B. Welsh¹, and M. Wood¹

¹ToxStrategies, Katy, TX, USA

To ensure the safety of pharmaceuticals, the entire drug formulation must be characterized adequately for potential toxicity, including an evaluation of product- and process-related impurities. Initial evaluations of these impurities are based on nonclinical and clinical data that are evaluated to characterize their safety. Data are identified by conducting a systematic search and comprehensive review of sources that include primary literature databases, regulatory authority websites, and other industry or government literature. A summary of this information is used to develop a risk assessment or toxicology monograph, with the aim of deriving a data-based exposure limit, such as a permissible daily exposure (PDE), or ascertaining key data gaps that may require additional toxicity testing to be consistent with regulatory guidance (e.g., ICH Q3). The monograph can also be used to set manufacturing specifications during process development or to justify specification limits to a regulatory authority within the context of a specific disease indication and route of administration. Compounds formulated for direct-to-brain routes, including intrathecal (IT) or intracerebroventricular (ICV) administration, can be particularly difficult to assess, because these routes are less common, and the available impurity safety information is often limited. The literature review and risk assessment process will be presented, including derivation of an exposure limit during three challenging case studies of impurities (a salt, an alcohol and an endogenous molecule) present in pharmaceutical drug products that are administered directly to the central nervous system.

P516: Mechanistic Investigation of Drug-induced Liver Toxicity Using Cellular Causality Assays in Human 3D InSight^TM Liver Microtissues

B. Filippi¹, R. Kostadinova¹, K. Sanchez¹, A. Pajak¹, N. Zapiórkowska-Blumer¹, and A. Wolf¹

¹InSphero AG, Schlieren, N/A, Switzerland

Human 3D liver microtissues are more predictive than 2D hepatocyte cultures to screen for clinical drug-induced liver injury (DILI) (Archives of Toxicology, vol. 91, 2849-2863, 2017). This new study shows that Human 3D liver microtissues can also be used for investigation toxicology. Using specific treatment of the microtissues, the mechanisms of toxicity of Acetaminophen (APAP), Aflatoxin B1 and Trovafloxacin are successfully dissected in vitro. Acetaminophen (APAP) produces a toxic phase I metabolite subsequently detoxified by GSH in vivo. Correspondingly, APAP toxicity is potentiated by the GSH synthesis inhibitor L-Buthionine Sulfoximine in 3D liver microtissues. Trovafloxacin induces toxicity in inflamed liver in vivo. Correspondingly, co-treatment of 3D microtissues with lipopolysaccharides potentiates the hepatotoxicity of trovafloxacin. Finally, metabolism of Aflatoxin B1 by cytochrome P450 (CYP) 3A4 produces reactive epoxides in vivo. Correspondingly, inhibition of CYP3A4 by the pan-specific CYP inhibitor 1-aminobenzotriazole decreases Aflatoxin B1-mediated cytotoxicity in Human 3D liver microtissues, which suggests the formation of a reactive drug-metabolite. In summary, the physiological relevance of Human 3D liver microtissues makes them effective tools to study the toxicological mechanisms of clinical drug-induced liver injury in vitro. The scalability of InSphero’s Human 3D liver microtissues offers the opportunity for DILI screening of specific mechanisms of toxicity in medium to high throughput format (96-well and 384-well plate format).

P517: Withdrawn

P518: One Month Repeat Dose Intracameral Ocular Toxicity Assessment of TTHX1114 in Rabbits and Dogs

G. Wang¹, B. Short², D. Eveleth³, and P. S. Terse⁴

¹Lovelace Biomedical Research Institute, Albuquerque, NM, USA

²Atheln, Inc., Laguna Beach, CA, USA

³Trefoil Therapeutics, Inc., San Diego, CA, USA

⁴National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA

Ocular and systemic safety following 5 once weekly intracameral injection of TTHX1114, a human fibroblast growth factor-1 derivative, were evaluated in rabbits and dogs to support clinical development for treatment of Fuchs Endothelial Corneal Dystrophy. Groups of animals (N=4-6/sex) were administered 0 (vehicle), or 88, 265 or 882 ng/eye of TTHX1114 into one eye (10 µL/eye), followed by in-life and terminal evaluation 1 day following the last injection and a 28-day recovery phase. There were no TTHX1114 related systemic effects in clinical observations, body and organ weight, clinical pathology, or systemic microscopic findings in either species, or safety pharmacology effects in respiratory function, blood pressure or electrocardiography in dogs. No TTHX1114-related ocular effects were identified based on ophthalmic examination, intraocular pressure, electroretinography, corneal thickness and corneal endothelial cell density. Draize scoring detected injection procedure-related minimal-mild conjunctival redness and/or iritis that was slightly exacerbated at ≥265 ng/eye in rabbits and at 882 ng/eye in dogs. The only TTHX1114-related microscopic effect was iris posterior epithelial cytoplasmic vacuolation that was minimal-mild, partially reversible at ≥ 265 ng/eye in rabbits and minimal-moderate at 882 ng/eye in dogs with persistence at recovery and this finding was considered nonadverse based on minor severity in most animals and lack of related ophthalmic examination findings. There was no dose-related increase in systemic TTHX1114 concentrations and no minimal plasma anti-drug antibody response. The no observed adverse effect level of TTHX1114 was considered to be 882 ng/eye in both rabbits and dogs. (Supported by NCATS under TRND Program Contract No. HHSN271201700017I)

P519: Evaluation of the Anti-Keyhole Limpet Hemocyanin (KLH) T Cell-Dependent Antibody Response (TDAR) Across Multiple Pre-Clinical Mammalian Species

E. Bruder¹, A. McDermott¹, A. Repic¹, A. Lucchini¹, and A. Frantz¹

¹Labcorp Drug Development, Madison, Wisconsin, USA

The T cell-dependent antibody response (TDAR) is dependent on multiple functional immunological processes that interact over a period of days and months. The production of antigen-specific immunoglobulins is well-described and plays a role in adaptive immunity. Pre-clinical safety assessment of a test article’s influence on the TDAR is essential, and having the ability to do so across preclinical species is both scientifically relevant and cost-effective. To establish a TDAR model, optimal doses of KLH (i.e., doses eliciting maximum antibody production without pathology) were administered to non-human primates (NHP), canines, rats, and minipigs, and anti-KLH IgG and IgM titers were assessed. Serum was collected pre-dose and over multiple days out to and including Day 29. Anti-KLH IgG and IgM were measured using an electrochemiluminescent immunoassay, with signal captured using a MSD S600 Sector Imager. For all species except the minipig, the temporal relationship between IgG and IgM was exhibited (i.e., IgM response precedes IgG response). In addition, the minipig was the only species that did not exhibit a sustained increase in IgG titers over time (out to Day 29). When examining the magnitude of the IgG and IgM responses across species, the data show that the largest increase in response occurred with rat IgG. This multi-species study suggests that the KLH-TDAR can be assessed, with confidence, using species-specific antibodies towards IgG and IgM. The data provide insight into assay feasibility as a part of safety assessment and immunogenicity studies, and have shown that responses across species are uniform and comparable.

P520: Withdrawn

P521: A Comparison of Microelectrode Array and Neurite Outgrowth in Neurotoxicity Assessment

C. Strock¹, S. Qin¹, Y. Feng¹, and J. Bradley¹

¹Cyprotex, Watertown, MA, USA

Domoic acid is a neurotoxin first associated with the poisoning of 107 individuals and three subsequent deaths on Prince Edward Island in 1987 after these individuals consumed mussels containing this toxin. In our lab, we use domoic acid as a tool compound when testing neurons on a microelectrode array (MEA) platform. Domoic acid completely eliminates all spontaneous spike activity when treating neurons at concentrations ≥1 µM. When tested in a neurite outgrowth assay, using a high content imager, domoic acid does not affect cell health or neurite outgrowth when tested up to 25 µM for 72 hours. The chemotherapeutic paclitaxel did not demonstrate seizurogenic liabilities > 10 µM on the MEA but had an IC50 of <0.05 µM for neurite outgrowth when tested in an HCS assay. GABA_A antagonist, picrotoxin, had a seizurogenic MEA profile as low as 1 µM but did not affect cell health and neurite outgrowth at concentrations up to 50 µM. With the results from domoic acid, paclitaxel, picrotoxin and 10 additional control compounds, we have determined that the MEA platform is more sensitive for detecting electrophysiological CNS liabilities than the neurite outgrowth assay. Alternatively, compounds which affect neurons by non-electrophysiological cytotoxic effects can be detected more sensitively with the HCS neurite outgrowth assay than the acute MEA assay. Therefore, an effective overall strategy would be to test compounds for safety in both assays.

P522: Real-time MRI for Intraparenchymal Convection-enhanced Delivery (CED) Infusion in NHPs with Target Coverage Quantification Using 7T MRI Masks

J. Stare¹, M. V. Accardi¹, K. Bullock², E. Larson¹, S. Maghezzi¹, K. Wong¹, R. Kubaszky³, C. Li¹, and S. Authier¹

¹Charles River Laboratories, Laval, Quebec, Canada, ²Kelly Bullock Art, Mitchell, Ontario, Canada, ³ToXcel, Gainesville, Virginia, USA

Advances in targeted delivery of novel therapies directly into the brain using convection-enhanced delivery (CED) have prompted the implementation of clinical methodologies in non-clinical models. Real-time intraoperative MRI was developed using the ClearPoint stereotactic drug delivery system in non-human primates (NHPs) for safety and efficacy studies. Given the current availability of lighter (1.8-2.2 kg) NHPs, and the prevalence of gene therapies targeting the pediatric population, a custom MRI atlas was developed to improve post-hoc image analysis of target structure coverage. NHPs (2.0 - 4.0 kg) were anaesthetized and scanned using a 7T MRI scanner. Using a combination of MP2RAGE, SPACE, and Multi-Echo images, custom bilateral masks outlining target subcortical structures, including the putamen, globus pallidus, thalamus, and substantia nigra, were manually defined using 3D visualization software. Anatomical accuracy was verified against existing NHP atlases. Masks were then overlaid on T1-weighted images of intraparenchymal gadolinium infusions (10-250 µL dose volume delivered at 1-5 µL/min) into targeted structures. The resultant segmentation was used to quantify the percent coverage of gadolinium-containing infusates in the targeted subcortical structures. By using custom masks that better reflect the size and positioning of the target structures within our NHP population, we present a clinically relevant approach whereby dose volumes and rates maximize tissue coverage whilst minimizing off-target leakage in smaller NHPs that are used for regulatory toxicology. By adapting an existing clinical approach, translational considerations are more readily met in the preclinical environment and better reflect the expected clinical context.

P523: Assessing Abuse Liability Using Read-across and Structural Alerts

G. J. Myatt¹, D. Bower¹, K. P. Cross¹, L. Stavitskaya², and S. Miller¹

¹Instem, Columbus, Ohio, USA

²Division of Applied Regulatory Science, Office of Clinical Pharmacology, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA

The ability to predict abuse liability of a drug candidate based on a computational assessment of its chemical structure has the potential to aid in the rapid identification of less addictive compounds by prioritizing chemicals within early discovery programs and formulating targeted testing strategies. In the present study, an in silico method was developed using a newly compiled database containing 4,178 chemicals with known abuse liability from sources such as US Drug Enforcement Administration’s (DEA) schedule list, as well as 3,106 chemicals lacking abuse potential. This database can be used to support assessment of abuse potential using a read-across methodology. In addition, 69 structural alerts have been compiled from both an analysis of the literature and identified directly from the new database. These structural alerts have been annotated with summarized information on the chemicals matching the alerts, including their DEA schedules, pharmacological action and in vivo findings. The matching database examples alongside the alert annotation will support a thorough expert review of the material. A preliminary assessment of the performance of these alerts over the reference database shows a balanced accuracy of 89%, sensitivity of 94% and negative predictivity of 99%. This poster will review how a combination of read-across, available data, application of the alerts and an expert review can be used to assess abuse liability.

P524: Using Metabolically Similar Analogs in Read-Across to Establish Dialkyl-N-Nitrosamine Potency

K. P. Cross¹ and G. J. Myatt¹

¹Instem, Columbus, Ohio, USA

Management of N-Nitrosamine impurity levels in pharmaceutical drug substances and products is guided by ICH M7 where they are defined as Cohorts of Concern. Regulatory agencies have suggested using read-across of rodent carcinogenicity TD₅₀ values for structurally-similar compounds to assess the potency of various N-nitrosamines. They have set provisional acceptable daily intake limits for several common N-nitrosamines based upon experimentally measured TD₅₀ values of close structural analogs. However, the TD₅₀ values for N-nitrosamines span 4 orders of magnitude with the potency of dialkyl-N-nitrosamines dependent upon the phase I metabolism mechanisms (alpha-carbon hydroxylation, beta-carbon hydroxylation, denitrosation) and the degree to which a mechanism is inhibited or competes with another. Solubility, phase II liver metabolism (glucuronidation, sulfation, glutathione conjugation), the stability of metabolites and their size, distribution, and DNA targets, also affects N-nitrosamine potency. To address these scientific issues, an ad hoc workgroup of over 20 companies and universities was established. Our analysis has shown that small dialkyl-N-nitrosamines, such as those having only methyl or ethyl groups, represents a small group of highly potent compounds that is distinctly different in mechanism (or rate) than large, feature-rich compounds. While the former undergo unfettered alpha-carbon hydroxylation, the latter are affected by steric bulk, steric hinderance and hydrogen availability at the alpha-carbon site as well as electronic effects such as the proximity, and strength of electron-withdrawing groups, to the alpha-carbon. We illustrate how read-across assessments of dialkyl-N-nitrosamines can be better supported by identifying mechanistically-relevant analogs as part of selecting structural similar analogs for consideration.