Abstract
100 Series—General Toxicology
P101: Protein Malnutrition Alters Mitochondrial Electron Transport Chain and Antioxidant Enzymes in the Cortex and Cerebellum: Modulatory Roles of Selenium and Zinc
O. L. Adebayo1, G. A. Adenuga2, and R. Sandhir3
1Redeemer’s University, Ede, Nigeria
2Olabisi Onabanjo University, Ago-Iwoye, Nigeria
3Panjab University, Chandigarh, India
Protein malnutrition is a major problem in the tropic contributing to high morbidity and mortality rates especially among children. Evidence suggest that protein malnutrition disturbs the functions of mitochondrial electron transport chain (mETC) and antioxidant enzymes, but little is known on the modulatory roles of Se and Zn on these parameters. Hence, the study was carried out to investigate the roles of Se and Zn on mETC and antioxidant enzymes in the cortex and cerebellum of protein malnourished rats. Rats were randomly assigned to 6 groups. Adequate protein diet and protein malnourished (PM) groups were provided with 16% and 5% casein, respectively, for 10 weeks. At the end of week 10, Se and Zn were supplemented at a concentration of 0.15 and 227 mg/L, respectively, in drinking water for 3 weeks, after which the rats were sacrificed under mild ether anesthesia by decapitation and the brain regions were taken. The data suggested reductions in the mETC enzymes, catalase, and Mn-superoxide dismutase activities and in the levels of glutathione (GSH), GSH/GSSG ratio, and MTT reduction in both the cortex and cerebellum of PM-fed rats. Moreover, there were significant (P < 0.05) increases in lipid peroxidation, reactive oxygen species generation, oxidized glutathione (GSSG) levels, and mitochondrial swelling following ingestion of PM diet in these brain regions. Se or Zn supplementation, however, restored the alterations in all the parameters analyzed, suggesting their efficacy in protecting against protein malnutrition-induced oxidative stress.
P102: The Genotoxicity of the Aqueous Extract of Ingestible Camphor
F. R. Aigbe1, A. A. Ajasa1, B. T. Omowunmi1, U-. O. Akpan1, M. C. Chijioke1, and O. O. Adeyemi1
1University of Lagos, Lagos, Nigeria
A form of camphor has been identified in some quarters as ingestible and its aqueous extract is commonly used as a home remedy for various ailments. A Nigerian survey once revealed that 50% of patients with colorectal cancer had ingested this form of camphor at least in early childhood. This study aims to investigate the genotoxicity of the aqueous extract of ingestible camphor (AEIC). The study was performed using Allium cepa root assay as well as micronucleus and comet assays using mice. In the A cepa test, onion roots were grown in tubes containing AEIC at 0.02 to 4 mg/mL, respectively, for 3 days. For micronucleus and comet assays, 5 groups of 6 mice each were orally administered distilled water (10 mL/kg), AEIC (3.53, 17.65, and 88.25 mg/kg), and cyclophosphamide (10 mg/kg), respectively, for 21 days. Following exposure, A cepa root as well as mice femoral bone and blood samples were collected and appropriately processed for relevant data. For A cepa assay, significant (P < 0.05) reductions in root length and mitotic index (with 50% reduction at 4 mg/mL by the third day) were observed. Chromosomal aberrations, including bridged and laggard types, were observed in meristematic cells of A cepa roots. Significant (P < 0.05) increase in micronuclei of polychromatic erythrocytes was noted for AEIC (3.53 and 17.65 mg/kg)-treated mice. In the comet assay, AEIC increased comet area and comet deoxyribonucleic acid. These findings reveal that AEIC is genotoxic; appropriate guidelines are required for its therapeutic application.
P104: Behavioral, Molecular, and Physiological Responses of Embryo-Larval Zebrafish Exposed to Types I and II Pyrethroids
O. M. Awoyemi1 and J. Crago1
1Texas Tech University, Lubbock, TX, USA
Pyrethroid pesticides have become increasingly relevant in the environment due to their broad-scale agricultural and urban applications. However, there are limited studies comparing the developmental toxicities of types I and II pyrethroids in nontarget species at environmentally relevant concentrations. The goal of this study was to compare the behavioral, molecular, and physiological effects of pyrethroid exposures in larval zebrafish (Danio rerio). Zebrafish embryos were exposed to type I (bifenthrin, permethrin) and type II (deltamethrin, λ-cyhalothrin, fenvalerate, esfenvalerate) pyrethroids at 1,000, 10, 0.1, 0.01 µg/L starting at 5 hours postfertilization through 5 days postfertilization (dpf). Swimming behavior (distance traveled and velocity) of zebrafish larvae was monitored on DanioVision observation chamber at 5 dpf. The stability of the pyrethroids across the 5 days was analyzed using ultra performance liquid chromatography with tandem mass spectrometer. Gene expression was analyzed using real-time qualitative polymerase chain reaction; total reactive oxygen species (ROS), total protein content, and enzymatic activities were determined using —UV-Vis spectrophotometer. The initial concentrations of all 6 pyrethroids were significantly (P < 0.05) reduced at days 3 and 5 of the exposure time line. However, both the types I and II pyrethroids had significant (P < 0.05) effects on the behavior of the zebrafish larvae, as compared to control. The types I and II pyrethroids had significant (P < 0.05) effects on the total ROS production, as well as the expressions of certain genes, including Nrf2a, Cas9, and p53, which were indicative of oxidative stress in the zebrafish embryo-larval development. Further studies are being carried out to assess the enzymatic activities in zebrafish exposed to these pyrethroids.
P105: Characterization of Focal Chorioretinal Atrophy in Sprague Dawley Rats as a New Model of Human Geographic Atrophy, the Late Stage of Dry Age-Related Macular Degeneration
J. T. Bartoe1, M. J. Leahy1, R. F. Boyd1, K. G. Nelson1, C. S. Nutting1, and T. S. Vihtelic1
1Charles River, Mattawan, MI, USA
P106: Identification of Immunophenotyping Biomarkers and Characterization of Peripheral Blood Lymphocytes Populations Distribution in Minipigs
M. Zhong1, C. Shoemake1, D. White1, D. Brocksmith2, J. Liu1, G. Bouchard1, and A. Stricker-Krongrad1
1Sinclair Research Center, LLC, Auxvasse, MO, USA
2Sinclair Bio Resources Inc, Auxvasse, MO, USA
We developed a complete panel of flow cytometry biomarkers for characterization of the major populations of porcine peripheral blood mononuclear cells (PBMCs). We subsequently analyzed distributions of these immune cells in blood samples of 4 breeds of minipigs. There were a number of common features associated with distribution of the major PBMCs in blood samples of different breeds of minipigs (Hanford, Yucatan, Sinclair, and Gottingen). T lymphocytes were the predominant population of PBMCs (around 70% of total PBMCs with drops to 61% in 8-month-old male Sinclair and to 56% in 8-month-old female Yucatan). Natural killer (NK) cells were composed of 3 populations of PBMCs that were CD16+CD8−, CD16+CD8+, and CD16-CD8+, respectively. CD8+ lymphocytes represented the majority body of NK cells, with CD16+CD8− lymphocytes to be the least fraction (no more than 8% of total NK cells). Although αβ as well as γδ T-cell receptors were identified in NKT cells, αβ T cells comprised no less than 75% of NKT cells. In summary, a novel flow cytometry approach has been developed to characterize the major porcine PBMCs in one single assay. This assay will facilitate immune investigations during nonclinical pharmacology and toxicology studies.
P107: In Silico Toxicological Evaluation of New Antileishmanial Derivatives
L. A. Miceli1,2, M-. L. F. Lira2, R. K. F. Marra2, J. C. Borges3, A. P. Gomes2, M. L. Bello1, C. R. Rodrigues1, A. M. R. Bernardino2, V. F. Amaral1, and H. C. Castro4
1Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
2Universidade Federal Fluminense, Niterói, Brazil
3Instituto Federal do Rio de Janeiro, Rio de Janeiro, Brazil
4Postgraduate Program in Science and Biotechnology, Federal Fluminense University, Niterói, Brazil
Leishmaniasis is a neglected disease with few treatment options with a high toxicity level. In this work, we evaluated the in silico toxicity profile of a new series of pyrazolyl benzenesulfonamide derivatives with significant antileishmanial activity. Therefore, 3a-h, 3i, and 3j toxicity profiles were determined according to the rule-of-five developed by Lipinski and Veber filter, also comparing them with reference drugs glucantime and ketoconazole. According to our Lipinski’s rule-of-five analysis, the compounds presented favorable parameters, which, together with Veber filter, pointed to a good availability and oral absorption. We also performed a virtual high-throughput screening to evaluate the presence of toxicophores, reactive groups, and/or toxic interferences in biological assays also known as Pan-Assay Interference Compounds (PAINS). However, no PAIN was detected in any structure of this series in contrast to the reference drug ketoconazole. These data pointed this series as promising to further in vivo and in vitro assays.
P108: Subchronic Inhalation Toxicity Study of Methylchloroisothiazolinone/Methylisothiazolinone Mixture in Wistar Rats
S-. J. Choi1, Y. Heo1, S-. H. Bae1, G. Seo1, and K. Lee1,2
1Korea Institute of Toxicology, Jeongeup, Republic of Korea
2University of Science & Technology, Daejeon, Republic of Korea
Methylchloroisothiazolinone/methylisothiazolinone (CMIT/MIT) mixture has been used for various biocidal products, including the humidifier disinfectant in Korea. Inhalation of humidifier disinfectant containing CMIT/MIT mixture as the active ingredient was considered one of the causes of adverse health effects like pulmonary diseases. This study was investigated to the toxicity of CMIT/MIT mixture by a 90-day repeated inhalation exposure in accordance with Good Laboratory Practice guideline. Male and female Wistar rats were exposed to a commercial humidified disinfectant (1.124% CMIT and 0.368% MIT) at a mass concentration of 0, 1.8, 7.8, or 15.5 mg/m3 for 6 h/d, 7 d/wk, for 13 weeks. Mortality, clinical signs, body weights, and food consumption were observed during exposure periods. Clinical chemistry, hematology, gross findings, organ weights, and histopathology were also determined. Biochemical analyses in bronchoalveolar lavage fluid (BALF) or lung tissue were conducted. No toxicological effects were observed including histopathological examination. However, pulmonary inflammation and injury biomarker levels in BALF were significantly changed in male rats of 7.8 or 15.5 mg/m3 exposure group. Biomarkers of pulmonary fibrosis, such as collagen, were significantly increased in male rats of 7.8 or 15.5 mg/m3 exposure group. Although the results of biochemical analyses did not correlate with histopathological changes, these results suggest that CMIT/MIT mixture induced to be the possible cause of pulmonary diseases by repeated inhalation exposure. Based on the results, the no observed adverse effect levels for both sexes was considered to be 15.5 mg/m3 in rats.
P109: The Challenges of Bioanalysis for Dermal Toxicokinetic Studies: A Case Study Measuring Lidocaine and 2,6-Dimethylaniline in Minipig Plasma, Skin, and Dermal Tapes
Q. I. Li1, T. Magers1, B. King1, B. J. Engel1, R. Bakhtiar2, C. Green2, D. Dehler1, and R. Shoup1
1AIT Bioscience, Indianapolis, IN, USA
2Teva Branded Pharmaceutical Products R&D, Inc, West Chester, PA, USA
Dermal drug delivery can have desirable toxicokinetic properties such as controlled and sustained drug release over time, avoidance of first-pass metabolism, and improved patient compliance. A comprehensive dermal TK package to support these programs creates unique challenges for bioanalysis, including the measurements of skin penetration rates, systemic exposure, and local exposure. Typically, dermal delivery seeks one of 2 outcomes: high local concentration with minimal systemic exposure or maximal skin penetration and systemic exposure with minimal residual concentrations. Often, it is necessary to develop wide-range or dual-range assays capable of measuring the extremes of the expected concentrations. Herein, we feature the bioanalysis of lidocaine and its key metabolite, 2,6-DMA, for 5 dermal formulations tested in minipigs. The challenges of wide detection ranges, blank matrix generation, and tissue and tape extractions are elucidated and addressed. Sensitive liquid chromatography with tandem mass spectrometry methods were developed to measure lidocaine in minipig plasma, tissue biopsies, and dermal tapes with lower quantitation limits of 25 pg/mL in plasma, 15 ng/g tissue, and 5 ng/tape. The 2,6-DMA was measured in plasma and skin tissue homogenates by ultrafiltration and (for tissue) by further derivatization with 4-methoxybenzoyl chloride to form the corresponding benzamide derivative, which extended the lower limits of quantitation to 200 pg/mL for each. Quantitation limits were approximately 7-fold lower than previously reported for lidocaine and 3-fold lower for 2,6-DMA.
P110: Combined Administration of α-Cypermethrin and Prenatal Stress Alters Embryonic Brain Development
B. A. Elser1, H. J. Lehmler1, and H. E. Stevens1
1University of Iowa, Iowa City, IA, USA
Prenatal exposure to α-cypermethrin is a risk factor for adverse neurodevelopmental outcomes in children. The prenatal maternal environment plays a critical role in determining how toxicants reach the developing fetus. Psychological stress has been shown to cause significant changes to physiology and may influence chemical toxicity to the embryo through alterations in maternal xenobiotic metabolism and placental function. As such, the present study examined effects of prenatal exposure to α-cypermethrin on mouse embryonic brain development, alone and in combination with maternal stress. CD1 dams were administered either α-cypermethrin (10 mg/kg body weight) or corn oil vehicle via oral gavage from embryonic days 11 to 14 (E11-E14). In addition, half the mice from each treatment were subjected to a widely used repetitive stress protocol of restraint under bright light (3 × 45 min/d) from E12 to E13. Neurodevelopmental outcomes were assessed by immunohistochemistry in E14 brain tissue. Restraint stress in both vehicle- and cypermethrin-treated dams reduced maternal weight gain relative to litter size from E11 to E14. In contrast, cypermethrin treatment reduced embryonic body weights in both control and stressed dams. Only combined administration of cypermethrin and restraint stress resulted in delay of GABAergic progenitor migration into the cortical plate, which depends on molecular mechanisms influenced by cypermethrin. Embryonic forebrain volume was unaffected by any treatment; however, combined cypermethrin and stress administration increased ganglionic eminence proliferative zone volume. In conclusion, these findings suggest that prenatal exposure to cypermethrin and maternal stress may act together to disrupt GABAergic circuit development through multiple mechanisms.
P111: The Involvement of the Aryl Hydrocarbon Receptor in the Toxicity of Polychlorinated Biphenyls
N. A. Eti1, S. Flor1, V. E. Klenov1, K. Iqbal2, J. Watt3, W. H. Watson4, A. F. Keating5, K. N. Gibson-Corley1, M. J. Ronis3, M. J. Soares2, G. Ludewig1, and L. W. Robertson1
1University of Iowa, Iowa City, IA, USA
2University of Kansas Medical Center, Kansas City, KS, USA
3LSUHSC, New Orleans, LA, USA
4University of Louisville, Louisville, KY, USA
5Iowa State University, Ames, IA, USA
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in the regulation of biological responses to planar aromatic hydrocarbons and also regulates xenobiotic-metabolizing enzymes such as cytochrome P-450. We have previously described the sequence of events following exposure to dioxin-like polychlorinated biphenyl (PCB) congeners like PCB126 that bind avidly to the AhR. Our hypothesis is that toxic manifestations following exposure to PCB126 are mediated through the AhR. To test this, we created an AhR knockout (AhR KO) model using CRISPR/Cas9. Comparison was made to wild-type (WT) male and female Holtzman Sprague Dawley rats. Rats were injected by a single intraperitoneal dose of corn oil vehicle or PCB126 in corn oil. After 28 days, a necropsy was performed to collect organs and to analyze the expression of genes and changes leading to histopathology. As a result, PCB126-exposed WT rats showed significant weight loss compared to AhR-KO rats. Similarly, relative thymus weights were significantly lower and relative liver weights were significantly higher than AhR-KO rats exposed to PCB126. The expression of genes encoding enzymes related to xenobiotic and intermediary metabolism was unaffected in the AhR-KO rats following PCB126 exposure as opposed to WT where expression was altered. The WT rats had decreased serum glucose, while KO rats showed no significant changes. Therefore, all adverse manifestations were observed in WT rats, while none were detected in AhR-KO rats, indicating the direct involvement of the AhR in the mediation of toxicity due to PCB126 exposure.
P112: Comparisons of Historical Control Clinical Pathology Data From Sprague Dawley Rats and Wistar Han Rats of Similar Ages
J. Ford Jr1, J. Albretsen1, L. Cregar1, G. Gibson1, and S. Denham1
1MPI Research, A Charles River Company, Mattawan, MI, USA
Sprague Dawley (SD) rats have been traditionally used in toxicology studies; however, the use of Wistar Han (WH) rats in toxicology studies is increasing due to their smaller size and increased longevity. Historical control data may be used to aid interpretation of clinical pathology effects noted during toxicology studies, particularly in the absence of control groups, or to identify vehicle effects or outlier data. Selecting an appropriate historical control data set is important due to differences in rat strain, sex, collection site, and age. Clinical pathology historical control data from SD and WH rats of varying ages and collection sites were generated and compared. Hematology, coagulation, clinical chemistry, and urinalysis data were collected over a 4- to 6-year period from SD and WH control rats. Following appropriate statistical analysis and review, tables were generated based on strain (SD or WH), sex, age (6-9 weeks old, 10-17 weeks old, 18-25 weeks old, 26-37 weeks old, and 38 weeks and older), and site of collection (sublingual or vena cava). Overall, WH rats had lower total leukocyte, neutrophil, lymphocyte, and platelet counts in both sexes across all ages and sites of collection; lower cholesterol concentrations in females across all ages and sites of collection; lower alkaline phosphatase activity in both sexes up to 17 weeks old; and lower urine volume in both sexes aged 38 weeks or older. All other differences identified in the data were either minimal or sporadic and of no biologic significance.
P113: Evaluation of Liver Metabolism Following Phenobarbital Treatment in the Göttingen Minipig
R. Forster1, J. B. Nielsen2, C. Bansard1, J. Decorde1, C-. A. Erratico1, M. Fonsi1, P. Ancian1, C. Parmentier3, M. Untrau3, L. Richert3, and P. Singh1
1Citoxlab France, Évreux, France
2Citoxlab Denmark, Lille Skensved, Denmark
3KaLy-Cell, Plobsheim, France
Phenobarbital (PB) is well known as an inducer of hepatic metabolizing enzymes in the rat with characteristic toxicological outcomes following long-term exposures, but relatively little is known about the consequences of phenobarbital treatment in Göttingen minipigs. In the present study, we have characterized the hepatic effects of an acute treatment with PB, and particular attention was given to the effects on xenobiotic metabolizing enzymes. Male Göttingen minipigs (4-5 months of age) received PB by the oral route for 6 consecutive days at a dose level of 15 mg/kg/d, while control animals received vehicle only. Following sacrifice on day 7, a panel of phase I and II metabolizing enzymes was investigated by messenger RNA (mRNA) expression and by enzyme activity measurements. Differential gene expression in the liver was investigated on Affymetrix porcine whole-genome microarrays. Liver weights were increased by 46% (absolute) and 42% (relative), correlating with histopathology findings of mild diffuse hepatocellular hypertrophy. The PB treatment resulted in isoform-specific increases in CYP enzymes and UGT enzymes, as measured by mRNA expression and specific activities. These findings were broadly consistent with the upregulation of CYP isotypes identified by microarray analysis. This study demonstrates that similar hepatic changes are seen in PB-treated minipigs to those seen in PB-treated rats. Further study is warranted to determine the toxicological and Adverse Outcome Pathway potential following longer term exposure in this model. It is concluded that the hepatic effects of PB treatment translate to the Göttingen minipig model.
P114: Impact of Serum on Calcium Transient Assays Performed on Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes
G. Froget1,2, M. Petit2, M. Paquet2, L. Paulhac2, and P. Kitchener2
1Porsolt, Le Genest-Saint-Isle, France
2Fluofarma, Pessac, France
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as a preclinical tool for detecting cardiotoxicity. These cells express physiologically relevant cardiomyocyte channels and, through integrated measurements, allow for the assessment of compounds which may simultaneously affect multiple channels or have an indirect effect on other signaling pathways. icell Cardiomyocytes2 (Cellular Dynamics International [CDI]) were used in this work. The CDI recommends using serum supplemented growth medium for calcium assays; however, serum can potentially bind molecules and affect their potency. Therefore, the aim of this study was to evaluate the effects of serum on the contractile activity of hiPSC-CMs after treatment with cisapride, quinidine, and nifedipine. icell Cardiomyocytes 2 were plated and maintained, according to the manufacturer’s instructions, in serum-containing media. Seven days after plating, hiPSC-CMs were loaded with a calcium indicator and calcium transients were monitored from spontaneously beating cells either in Tyrode, without serum, or in Maintenance Medium with serum. Basal beat frequency was similar in both assay media. One-hour treatment with cisapride and quinidine resulted in dose-dependent decreases in beat frequency in Maintenance Medium. In Tyrode, the same treatments had no effects on beat frequency. Treatment with nifedipine resulted in a dose-dependent increase in beat frequency in both assay media, with a steeper curve in serum-containing Maintenance Medium than in Tyrode. These results highlight the importance of careful consideration when choosing assay medium to perform toxicologically relevant calcium transient assays on icell Cardiomyocytes 2 and their potential influence on the results.
P115: Exposure to a Naturally Occurring Polycyclic Aromatic Hydrocarbon Mixture Triggers an Aggressive Phenotype in Estrogen Receptor–Positive Breast Cancer Cells
L. M. Gearhart-Serna1, J. B. Davis1, N. Jayasundara1, M. K. Jolly2, S. J. Sauer1, H. Levine2, R. T. Di Giulio1, and G.R. Devi1
1Duke University, Durham, NC, USA
2Rice University, Houston, TX, USA
Exposure to environmental chemicals has recently been linked to the promotion of rapidly proliferating and aggressive breast cancer subtypes. Although the association of environmental endocrine-disrupting chemicals (EDCs) in breast cancer risk is well-documented, there is a lack of studies elaborating on associations or the mechanism by which EDCs may contribute to cancer progression. In this study, we used a naturally occurring complex mixture of polycyclic aromatic hydrocarbon (PAH) EDCs derived from a contaminated Superfund site. We evaluated the ability of low-dose PAH mixture exposure to affect traditional toxicant signaling—upregulation of the aryl hydrocarbon receptor (AhR) and cytochrome p450 (CYP1A1)—in estrogen receptor–positive and triple negative breast cancer as well as normal mammary cell lines. Viability, 2-D proliferation, 3-D organoid growth, and levels of antioxidant superoxide dismutase (SOD1) and antiapoptotic X-linked inhibitor of apoptosis protein (XIAP) were also investigated. We further utilized the CDC NHANES database to calculate mean US adult intake exposure for select PAHs to validate physiological relevance of our PAH mixture doses. ER+ breast cancer cells treated with low-dose PAH mixture relevant to US adult exposure exhibited upregulation of toxicant (AhR, CYP1A1) signaling as expected, as well as increased 2-D proliferation and adaptive stress response signaling (SOD1, XIAP). The 3-D organoid growth of triple negative cells was also observed, all markers of a hyperproliferative and aggressive tumor phenotype. Collectively, these results suggest that exposure to low-dose EDC mixtures such as PAHs can play a promoting role in breast tumor progression.
P118: Cyp2b-Null Male Mice Are Susceptible to High-Fat Diet-Induced Obesity Due to Changes in PUFA Metabolism and Response to Hepatic Lipids as Measured by RNAseq
M. M. Heintz1, R. Kumar1, and W. S. Baldwin1
1Clemson University, Clemson, SC, USA
CYP2B6 metabolizes xenobiotic and endobiotic compounds, including drugs, pesticides, plasticizers, steroids, and poly-unsaturated fatty acids (PUFAs). Recent studies using the cytochrome P450 oxidoreductase knockout mouse revealed steatosis coupled with induced Cyp2b10. Data from our laboratory using RNAi-mediated Cyp2b knockdown mice indicate age-onset obesity. To better investigate the role of CYP2B in lipid metabolism, a Cyp2b triple knockout mouse lacking Cyp2b9, Cyp2b10, and Cyp2b13 was developed. Wild-type (WT) and Cyp2b-null mice were fed a normal diet (ND) or a 60% high-fat diet (HFD) for 10 weeks. Male but not female Cyp2b-
P119: Saccharomyces Cerevisiae (Yeast) Helping Our Understanding About Mn-Induced Neurotoxicity
R. B. Hernandez1, H. Moteshareie2, D. Burnside2, B. McKay2, and A. Golshani2
1Unifesp, Diadema, Brazil
2Carleton University, Ottawa, Canada
Manganese (Mn) is an essential element; but in humans, chronic and/or acute exposure to this metal can lead to neurotoxicity and neurodegenerative disorders including Parkinsonism by unclear mechanisms. To better understand the effects of Mn exposure on the biology of a cell, we exposed the yeast, Saccharomyces cerevisiae nonessential gene deletion collection to a subinhibitory concentration of Mn and followed by targeted functional analyses. This screen produced a set of 68 sensitive gene deletion mutants and following the functional enrichment analysis of these genes revealed the process of protein biosynthesis as a possible target for Mn-induced toxicity. Consistent with this, we identified that Mn reduced the total levels of yeast ribosomal RNA molecules and the expression of a β-galactosidase reporter gene, in a dose-dependent manner. This was subsequently supported by analysis of ribosome profiles, whose results suggested that Mn-induced toxicity was associated with a reduction in active polysomes. Finally, using Comparative Toxicogenomic Database, we revealed that Mn shared certain similarities in toxicological mechanism with neurodegenerative disorders, including Alzheimer, Parkinson, and Huntington diseases. Altogether, these findings contribute to the current understanding of the mechanism of Mn neurotoxicity and neurodegeneration.
P120: Sharing Study Details and Data in INTERVALS From the Assessment of the Impact of Aerosol From the Carbon-Heated Tobacco Product 1.2, a Potential Modified Risk Tobacco Product, on Human Organotypic Gingival Cultures
J. Hoeng1, S. Boue1, F. Zanetti1, and M. C. Peitsch1
1PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland
The US Food and Drug Administration defines modified risk tobacco products (MRTP) as any tobacco product sold or distributed to reduce harm or the risk of tobacco-related disease associated with commercially marketed tobacco products. Establishing a product’s potential as an MRTP requires scientific substantiation, including toxicity studies and measures of disease risk relative to that of cigarette smoking. As part of a 21st-century toxicology assessment framework, human gingival epithelia organotypic cultures were exposed repeatedly to nicotine-matching concentrations of carbon-heated tobacco product (CHTP) 1.2 aerosol or 3R4F cigarette smoke (CS), as well as a nondiluted (100%) CHTP 1.2 aerosol, and subsequently characterized. The results demonstrated the absence of cytotoxicity and reduction in pathophysiological alterations, toxicological marker proteins, and inflammatory mediators following exposure to CHTP 1.2 aerosol as compared with 3R4F CS. All relevant data files from this assessment study were deposited in INTERVALS (www.intervals.science), an online platform developed by PMI to enable independent, third-party collaboration and data analysis by proactively sharing protocols, tools, and data from assessment studies. Data files are accompanied by the relevant information to foster reproducible research and by high-level summaries of obtained results. This platform can serve as an invaluable source of scientific assessment data, able to host all results from various studies, including in vivo inhalation studies, in vitro studies, and clinical studies. INTERVALS aim to enable the necessary dialogue between industry, independent reviewers, the public health community, and regulatory agencies that can validate the harm reduction potential of these products.
P121: A Catalogue of Somatic NRF2 Gain-of-Function Mutations in Cancer
M. J. Kerins1 and A. Ooi1
1University of Arizona, Tucson, AZ, USA
Identification and characterization of somatic mutations in cancer have important prognostication and treatment implications. Genes encoding the nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor and its negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), are frequently mutated in cancer. These mutations drive constitutive NRF2 activation and correlate with poor prognosis due to increased clearance of chemotherapeutics in NRF2-activated tumors. Despite its apparent significance, a comprehensive catalog of somatic NRF2 mutations across different tumor types is still lacking. Here, we catalog NRF2 mutations in The Cancer Genome Atlas database. A total of 226 unique NRF2-mutant tumors were identified from 10,364 cases. The NRF2 mutations were found in 21 out of the 33 tumor types. A total of 11 hotspots were identified. Of these, mutation to the R34 position was most frequent. Notably, R34 and D29 mutations were overrepresented in bladder, lung, and uterine cancers. Analyses of corresponding RNA sequencing data using a de novo derived gene expression classifier showed that the R34 mutations drive constitutive NRF2 activation with a selection pressure biased against the formation of R34L. Of all R34 mutants, R34L conferred the least degree of protein stabilization, suggesting a protumor NRF2 half-life threshold. Our findings offer a comprehensive catalogue of NRF2 mutations in cancer that can help prognostication and NRF2 research.
P122: Comparative Cytotoxicity of Respirable Surface-Treated/Untreated Rock Dust Particles in THP-1 Macrophages
T. O. Khaliullin1,2, E. R. Kisin2, N. Yanamala2, S. Guppi2, M. Harper3, T. Lee4, and A. A. Shvedova1,2
1West Virginia University, Morgantown, WV, USA
2CDC/NIOSH/HELD, Morgantown, WV, USA
3Zefon International, Ocala, FL, USA
4CDC/NIOSH/PMRD, Pittsburgh, PA, USA
Calcium carbonate rock dust (RD) is used in mining to reduce the explosibility of coal dust, but the potential for human exposure raises health concerns. To improve the RD aerosolization, several types of anticaking surface treatments exist. The aim of the study was to evaluate cytotoxicity of 4 respirable RD samples: untreated/treated limestone (L/TL), untreated/treated marble (M/TM), with crystalline silica (SiO2) as a positive control in a THP-1 human macrophage model. The coating on TM was stearate based, while the TL coating was based on silicone. Respirable fractions were generated and collected using FSP10 high flow-rate cyclone samplers. After the PMA differentiation, cells were exposed for 24 hours to RD and SiO2 at concentrations ranging from 0 to 0.06 mg/cm2. At 24 hours, there was significant dose-dependent lactate dehydrogenase, inflammatory cytokines, and chemokines release as well as increased caspase-1 activity in SiO2- and TM-exposed cells, but not in other RD. To test whether the increased toxicity of TM was uptake related, THP-1 cells were treated with phagocytosis inhibitor cytochalasin D (CytD) or inhibitor of V-ATPase bafilomycin A (BafA), followed by exposure to RD or SiO2 for 6 hours. The CytD treatment blocked the uptake and mitigated or significantly decreased cytotoxic effects in all samples. The BafA completely prevented caspase-1 activation and partially rescued SiO2- but not TM-exposed cells, suggesting different mechanisms of toxicity. Overall, stearate-treated particles were able to induce inflammatory response in THP-1 cells; however, it was much less pronounced compared to SiO2.
P123: Impact of Obesity on DNA Structure-Induced Genetic Instability in Mice
P. Kompella1, G. Wang1, J. DiGiovanni1, and K. M. Vasquez1
1The University of Texas at Austin, Austin, TX, USA
Many studies have reported an elevated risk and poor prognosis for a variety of cancers in people with high body mass index. Obesity is thought to stimulate oxidative stress that can cause DNA double-strand breaks (DSBs) and reduced DNA repair capacity. Double-strand breaks result in gene translocations consequently disrupting oncogenes in cancers such as Burkitt lymphoma (BL). The c-MYC oncogene in BL harbors vulnerable breakpoint “hotspots” enriched in repetitive DNA sequences that can adopt intrinsically mutagenic alternate DNA structures (ie, non-B DNA; eg, H-DNA). Such structures may increase the accessibility of DNA to oxidative damage and impact DNA repair efficiency and accuracy. We hypothesize that obesity increases DNA structure-induced genetic instability in vivo, contributing to mutation “hotspots” in cancer. Groups (n = 4-5) of transgenic mice harboring a human H-DNA-forming sequence (mapping to a breakpoint hotspot in BL) within a mutation reporter were put on control, calorie-restricted, or diet-inducing obesity (DIO) for 13 weeks. Mutagenesis assay results indicate that the mutagenicity of H-DNA-forming sequences was significantly elevated in obese mice compared to normal-weight mice. Mutation spectra results revealed deletions, point mutations, and transversion events. Detection of a breakpoint hotspot in obese mice indicates the influence of DIO on the formation of H-DNA-induced DSBs. Next-generation sequencing data showed elevated mutation events in and around H-DNA-forming sequences in obese mice DNA. These results can help us define the molecular mechanisms underlying the effects of obesity on endogenous mutational “hotspots” and also aid in the development of strategies to prevent and/or attenuate the obesity–cancer link.
P125: High-Throughput and Sensitive Quantification of α-Synuclein Protein Aggregation in Welder’s Serum: Relevance to Development of Circulating Biomarkers for Manganese Neurotoxicity
S. Manne1
1Iowa State University, Ames, IA, USA
Chronic exposure to Mn is known to affect the extrapyramidal motor control system, resulting in Parkinsonian-like neurological symptoms. Welders exposed to Mn-rich welding fumes prone to Mn neurotoxicity. Recently, we demonstrated that Mn interacts with α-synuclein (αSyn) protein and promotes its aggregation in cell culture and animal models of Mn neurotoxicity. Although a brain magnetic resonance imaging is traditionally used to diagnosis Mn neurotoxicity in humans, no reliable blood-based biomarker is available. In this study, we developed a sensitive quantification method that detects the effect of Mn exposure on αSyn aggregation by a real-time quaking-induced conversion (RT-QuIC) assay. First, we generated a high-quality recombinant human wild-type αSyn substrate and synthesized the αSyn filaments, or pre-formed fibrils (PFFs), from this substrate. The PFFs generated were tested vigorously for internal quality control using defined criteria set by the Michael J. Fox Foundation. Next, we optimized RT-QuIC assay conditions to further quantify αSyn aggregation in cell cultures and slice culture models. Importantly, we also examined the utility of RT-QuIC assay in detecting αSyn aggregation in welder serum samples. Serum exosomes from 29 welders exposed to welding fumes and 16 age-matched control participants were tested for αSyn seeding activity in a blinded RT-QuIC assay. We could differentiate the welders from controls with >95% sensitivity and specificity using this assay, suggesting that exosomal αSyn aggregates can serve as a circulating biomarker for Mn neurotoxicity. Collectively, our findings demonstrate the efficacy of using a highly sensitive, high-throughput RT-QuIC diagnostic assay for detecting Mn-induced αSyn aggregation in occupationally exposed individuals.
P128: Derangement of Lipid Profiles and Antioxidant Status in the Testes of Rats Subchronically Fed Sucrose Diet
C. A. Otuechere1, P. O. Adeniji2, M. M. Adeyanju3, and B. A. Salau1
1Department of Chemical Sciences, College of Natural Sciences, Redeemer’s University, Ede, Osun State, Nigeria
2Transport and Tourism Studies, College of Management Sciences, Redeemer’s University, Ede, Osun State, Nigeria
3Department of Biochemistry, Faculty of Basic Medical Sciences, Obafemi Awolowo College of Health Science, Olabisi Onabanjo University, Ogun State, Nigeria
Sucrose has been implicated in the etiology of many diseases with pronounced effects on major organs such as the liver, kidney, and brain. However, there is paucity of information on the effect of its long-term exposure in the testis. This study, therefore, investigated the subchronic effect of sucrose diet in testicular organ of rats. Twenty-four animals were grouped into 4 and fed varying concentrations of sucrose diet (SUD) for 6 months as follows: SUD 0%, SUD10%, SUD20%, and SUD 30%, respectively. Sucrose diet elicited a significant and dose-dependent increase in triglyceride and total, low-density lipoprotein and very-low-density lipoprotein cholesterols levels with a concomitant decrease in high-density lipoprotein cholesterol level, when compared to control groups. A similar trend was observed in oxidative stress markers as high SUD significantly increased (P < 0.05) testicular malondialdehyde level with a corresponding depletion of reduced glutathione. Additionally, subchronic ingestion of high-dose SUD altered testicular antioxidant status by significantly reducing glutathione S-transferase, superoxide dismutase, and catalase activities in comparison to controls. In conclusion, we opine that testicular function may be impaired in rats exposed to long-term SUD via the combined mechanisms of lipid profiles derangement and oxidative stress.
P129: Comparison of Tris (2-Ethylhexyl) Phosphate and Di (2-Ethylhexyl) Phosphoric Acid Toxicities in a Rat 28-Day Oral Exposure Study
G. Pelletier1, M. Rigden1, G-. S. Wang2, D. Caldwell2, S. Siddique3, K. Leingartner1, I. Kosarac4, and C. Kubwabo3
1Hazard Identification Division, HECSB, Health Canada, Ottawa, Ontario, Canada
2Scientific Service Division, HPFB, Health Canada, Ottawa, Ontario, Canada
3Exposure and Biomonitoring Division, HECSB, Health Canada, Ottawa, Ontario, Canada
4Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario, Canada
Flame retardants phased-out over health concerns are sometimes replaced by alternative compounds whose toxicities may not be as well studied. In order to address data gaps, the toxicities of the flame-retardant tris (2-ethylhexyl) phosphate (TEHP, CAS# 78-42-2) and related di (2-ethylhexyl) phosphoric acid (DEHPA, CAS# 298-07-7) were assessed according to Organisation for Economic Co-operation and Development Test Guidelines 407. Briefly, adult male and female rats were administered TEHP (300, 1,000, and 3,000 mg/kg body weight) or DEHPA (20, 60, and 180 mg/kg body weight) by gavage for 28 consecutive days. Rat health and weight were monitored, urine and feces were collected, and tissues were harvested at the terminal necropsy. Quantification of TEHP and DEHPA in urine hinted at the enzymatic hydrolysis of TEHP to DEHPA in males, at higher DEHPA excretion rates in females, and at the presence of a saturable binding site or renal reabsorption mechanism for TEHP. Although impaired body weight gains were observed only in TEHP-treated males, administration of TEHP and DEHPA often resulted in similar alterations of hepatic functions, hematology, and serum chemistry, in both sexes. Squamous epithelial hyperplasia and hyperkeratosis observed in the nonglandular forestomach of DEHPA-treated rats was most likely caused by the slightly corrosive nature of this chemical. The etiology and toxicological significance of the periodic acid–Schiff stained crystals observed in the testis interstitial cells of TEHP-treated males remains to be determined and warrants further investigation. Better characterization of the toxicity of flame retardants will ultimately improve human health risk assessments and offer informed substitution choices.
P130: Impact of Thermally Abused Oil on Gut Inflammation and Colon Tumorigenesis in Mice Fed a Standard Diet or the Total Western Diet
S. Phatak1, K. Campbell1, R. Boomen1, E. Speas1, K. Hintze1, W. Helferich2, and A. Benninghoff1
1Utah State University, Logan, UT, USA
2University of Illinois at Urbana–Champaign, IL, USA
The average American diet is characterized by highly processed foods that are energy dense, yet nutrient poor. Deep frying these foods contributes to the formation of several toxic compounds known to negatively influence health outcome, including increased colorectal cancer (CRC) risk. Fifty million adults and one-third of all children consume fast food daily in the United States. Previous work showed oxidized lipids were absorbed, damaging cellular proteins in vitro, and they intensified circulation of inflammatory cytokines in vivo. The objective of this study was to determine the impact of the total Western diet (TWD) for rodents and thermally abused oil (TAO) on health end points associated with chronic inflammation and CRC. The AOM + DSS model of inflammation-associated CRC was employed with a 2 × 2 experimental design, where C57BL/6J mice were fed standard AIN93G or TWD, each prepared with either fresh oil or TAO. End points assessed included colon tumors, symptoms of colitis, food consumption, body composition, and organ weights. AIN93G + TAO did not significantly impact any of the measured parameters as compared to AIN93G + fresh oil. Energy intake in mice provided TWD + TAO was significantly greater than observed in mice-fed TAO + fresh oil; however, this change did not translate to increased body weight or fat mass gain. Also, exposure to TAO did not enhance colitis symptoms or colon tumorigenesis in mice fed either basal diet. Overall, these data suggest that exposure to TAO via either a standard diet or a Western-type diet had little impact on health status in mice.
P131: Inhibition of Human Liver Carboxylesterase by Organophosphate Flame Retardants and Plasticizers: Implications for Pharmacotherapy
A. L. Phillips1 and H. M. Stapleton1
1Duke University, Durham, NC, USA
Isopropylated triarylphosphate esters (ITPs) and tertbutylated triarylphosphate esters (TBPPs) are chemicals used as plasticizers and as flame retardants. Despite widespread human exposure in the US population, there is currently little information regarding their potential effects in the human body. In this study, ITPs, TBPPs, and related commercial flame-retardant mixtures were evaluated individually at a range of concentrations (0.1 nM-100 µM) to investigate their potential in vitro inhibition of purified human hepatic carboxylesterase (hCE1). Triphenyl phosphate (TPHP), 4-tertbutyl phenyl diphenyl phosphate (4tBPDPP), and house dust extracts (n = 20; 10 and 100 µg/mL doses) were also tested for their effect on the activation of imidapril, an angiotensin-converting enzyme inhibitor prodrug known to be metabolized to its bioactive form (imidaprilat) by hCE1. IC50 values for the test chemicals ranged from 17.6 to >90,400 nM, with 4 commercial mixtures and 4 individual chemicals having IC50 values lower than 100 nM. Both TPHP and 4tBPDPP were potent inhibitors of imidapril activation (IC50 = 57.7 and 15.8 nM, respectively.) Kinetic experiments suggested that TPHP and 4tBPDPP were noncompetitive inhibitors of hCE1 (Ki = 49.0 and 17.1 nM, respectively). While limited hCE1b inhibition was observed by dust extracts at 10 µg/mL, significant inhibition (reductions of up to 33%) was observed for 10 out of 20 extracts at 100 µg/mL. Taken together, these results highlight the potential for hCE1 inhibition by ITP and TBPP isomers at environmentally relevant concentrations and demonstrate that these chemicals may compromise the efficacy of pharmacotherapy strategies that rely on prodrug activation by hCE1.
P132: Neuroinflammation: Combined Administration of P7C3-A20 and Ibuprofen Protects Rat Spiral Ganglion Neurons After Aminoglycoside Deafening
M. T. Rahman1, B. Peng2, C. Kane1, B. M. Gansemer1, J. H. Parker1, A. A. Pieper3, and S. H. Green1
1The University of Iowa, Iowa City, IA, USA
2University of South Carolina, Columbia, SC, USA
3Case Western Reserve University, Cleveland, OH, USA
P133: Acute Poisoning in Children—Facts From Egypt
N. Ramadan1
1Kasr Al Ainy Faculty of Medicine, Cairo University, Cairo, Egypt
P134: Inhibitory Effect of Arsenicals on Protein Tyrosine Phosphatase 1B
S. H. M. Rizvi1, A. Londhe1, S. R. Sagabala1, P. C. Lin2, C. Huang3, and B. Boivin1
1SUNY Polytechnic Institute, Colleges of Nanoscale Sciences and Engineering, Albany, NY, USA
2Center for Cancer Research, National Cancer Institute, Frederick, MD, USA
3Department of Environmental Medicine, New York University School of Medicine, New York, NY, USA
Inorganic arsenic (As) and methylated metabolites formed during its detoxification, such as monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), are thought to be cardiotoxic. Adverse effects of arsenicals occur through acute and prolonged perturbation of phospho-dependent cell signaling pathways through generation of reactive oxygen species (ROS) or direct binding to biomolecules. Protein tyrosine phosphatases (PTPs) superfamily involved in serine, threonine, and tyrosine dephosphorylation in vivo are transiently inactivated by ROS under physiological conditions and irreversibly inactivated in pathological conditions. Here, we investigated the role of As and its metabolites in PTP regulation. Interestingly, using the cysteinyl-labeling assay (CLA) to assess oxidation of the catalytic cysteine of PTPs in cells, PTP1B reversible oxidation was found to occur in cardiomyocytes exposed to MMA(III), but not As (III) or DMA(III). In order to understand MMA-mediated inactivation of PTP1B, we exposed PTP1B to arsenicals with or without reducing agents and observed that MMA(III) and DMA(III) inhibited PTP1B activity by ∼60% and 20%, respectively, while As minimally inhibited the phosphatase even at high concentrations. Importantly, MMA inhibition of PTP1B was reversible by the addition of reducing agents, suggesting that direct MMA-mediated inactivation of PTP1B is measured by the CLA. Exposing cardiomyocytes and mice to As inactivated PTP1B and caused increased phosphorylation of PTP1B substrates involved in cardiac hypertrophy. Finally, detection of arsenicals in the heart of exposed mice support that metabolites of As directly inhibit PTP1B activity to affect signaling pathways involved in cardiac hypertrophy, independently of vascular dysfunction.
P135: Tissue-Specific Accumulation of PBDEs in the Placenta and Effects on Thyroid Regulation
M. T. Ruis1, K. R. Baldwin2, S. M. Hall1, B. Horman2, H. Patisaul2, and H. M. Stapleton1
1Duke University, Durham, NC, USA
2North Carolina State University, Raleigh, NC, USA
The placenta is an ephemeral organ that plays many critical roles during fetal development. It is derived from both maternal and fetal tissue, which is separated by the placental barrier. The placental barrier is responsible for the transfer of nutrients, gases, and hormones and the protection of the fetus from pathogens, the maternal immune system, and xenobiotics. Recent studies have shown that there are numerous persistent organic pollutants (POP) that accumulate in cord blood, indicating that the placental barrier is permeable to environmental contaminants; however, the mechanism is unclear. In this study, we have microdissected term human placenta as well as placenta from Wistar rats orally dosed with a flame-retardant mixture and quantified tissue-specific POP accumulation. We observed significantly higher concentrations of POPs (∼2×) in the fetal portion of the placenta relative to the maternal tissues of the placenta, despite no differences in lipid content. This challenges the previously accepted hypothesis that POPs cross the placental barrier passively. We also observed sex-specific differences in thyroid hormone transporter gene expression in the placenta following in utero exposure to POPs using the Wistar rat as a model. This research could help explain several adverse pregnancy outcomes, such as intrauterine growth restriction and preeclampsia, which are believed to be a result of placenta dysfunction. This research can also be used to elucidate mechanisms of maternal transfer of harmful chemicals and causes of thyroid hormone disruption, which has been shown to lead to adverse neurodevelopmental outcomes.
P136: Nonclinical Safety Profile of Anti-HER2/CD3 T-Cell-Dependent Bispecific (TDB) Antibodies
K. Staflin1, L. Schutt1, T. Junttila1, C. De Zafra1, P. Katavolos1, N. Dybdal1, K. Totpal1, J. Li1, M. Hristopoulos1, R. Clark1, R. Ybarra1, M. Junttila1, D. Slaga1, J. Johnston1, M. Dennis1, M. Mathieu1, X. Chen1, D. Ellerman1, F. Zhong1, V. Clark1, A. Shelton1, and R. Prell1
1Genentech, South San Francisco, CA, USA
Cancer immunotherapy using bispecific antibodies that engage and induce T-cell-mediated tumor killing have shown promise in the clinic mainly in hematologic malignancies. One advantage of redirecting T cells to bind and kill tumor cells using this technology is that no preexisting antitumor immunity is required. We have developed a full-length T-cell-dependent bispecific (TDB) antibody targeting human epidermal growth factor receptor 2 (HER2) and cluster of differentiation 3 (CD3). Efficacy studies show a robust antitumor response in vitro as well as in xenograft studies in mice, potently killing HER2-positive target cells in a dose-dependent manner. Since HER2 expression is widely distributed in normal tissues, characterizing the safety of the HER2/CD3 TDB was a key driver in the development strategy. Three molecules with differing affinity for either the HER2 Fab or the CD3 Fab were produced and assessed in toxicity studies in cynomolgus monkeys. The purpose of this study was to elucidate how the different affinities to either target affected the safety profiles of HER2/CD3 TDB antibodies. In general, HER2/CD3 TDB-related findings were associated with transient T-cell activation, redistribution, and cytokine release with histopathological findings present in HER2-expressing epithelial tissues. Increasing the affinity of either the HER2 or the CD3 arm of the antibody affected tolerability and demonstrated that customizing the affinities of both targets is important to balance safety with efficacy.
P137: Laser-Induced Choroidal Neovascularization (CNV) as a Model of Age-Related Macular Degeneration (AMD) in the Minipig
A. Stricker Krongrad1, H. Leigh1, R. Harvey1, M. Jones1, S. Tellez1, J. Wicks1, and G. Bouchard1
1Sinclair Research Center, LLC, Auxvasse, MO, USA
Choroidal neovascularization (CNV) is a known cause of age-related macular degeneration (AMD). Lucentis ( ranibizumab) is a marketed treatment for AMD. Ocular similarities between humans and miniature swine make it a useful large animal model ocular therapy, and this study aimed to validate the miniature swine model of laser-induced CNV. In this study, CNV was generated in Sinclair minipig using retinal photocoagulation. After laser induction of CNV, animals received a single 25 µL intravitreal injection of either Lucentis (n = 4), or vehicle (n = 4). Eyes examinations and fluorescein angiography were performed to assess hemorrhaging and formation of CNV lesions. Choroidal neovascularization lesions developed as expected, as hypofluorescent spots and hemorrhages caused by laser tissue damage were observed during weeks 1 and 2. Grade 1 through 3 CNV lesions were present in weeks 1 and 2. Ocular opacities prevented fluorescein imaging in 2/4 vehicle-treated animals compared to 1/4 Lucentis-treated animals. At week 6, all hypofluorescent spots and hemorrhages had resolved and clinically significant grade 4 CNV lesions were present, defined by bright hyperfluorescence at the mid-time point and late leakage beyond the treated area during fluorescein angiography. Grade 4 lesions were present at a lower prevalence (25% of observed lesions) in Lucentis-treated animals compared to vehicle-treated animals (33% of observed lesions). Based on vitreous opacities, and anterior segment inflammation, vehicle-treated animals developed more severe ocular changes than Lucentis-treated animals. Lucentis treatment demonstrated an antiangiogenic effect, validating the Sinclair minipig as a model of wet AMD.
P138: A 6-Month Inhalation Study in ApoE
−/−
Mice to Investigate Cardiovascular and Respiratory Exposure Effects of E-Vapor Aerosols Compared With Cigarette Smoke
E. T. Wong1, J. Szostak2, T. Lee1, S. K. C. Wong1, G. J. Loh1, P. Leroy2, G. Vuillaume2, M. Lee3, M. C. Peitsch2, P. Vanscheeuwijck2, and J. Hoeng2
1PMI Research Laboratory, Singapore, Singapore
2PMI R&D, Philip Morris Products SA, Neuchâtel, Switzerland
3Altria Client Services LLC, Richmond, VA, USA
Chronic exposure to cigarette smoke is a risk factor for the development and progression of cardiovascular disease and chronic obstructive pulmonary disease. Considerable attention has been given to the potential reduced harm of e-cigarettes (e-cigs). Here, ApoE−/− mice were used to evaluate lung inflammation, atherosclerosis development, and the underlying molecular changes upon 6-month exposure to mainstream cigarette smoke (CS) from a 3R4F reference cigarette or to e-cig aerosols. Capillary aerosol generators were used to generate e-vapor products containing various e-liquids (“CARRIER” containing humectants alone, “BASE” containing humectants and 4% nicotine, and “TESTMIX” containing humectants, 4% nicotine, and flavors). ApoE−/− mice were exposed at matched nicotine concentration, 36 µg/L, to CS and the e-cig aerosols (“BASE” and “TESTMIX”). Aerosol particle sizes were within the respirable range, from 0.71 to 0.90 µm for 3R4F CS and 0.74 to 1.28 µm for e-cig aerosols. Pulmonary inflammation and atherosclerotic plaque areas, as well as cholesterol concentrations in serum or lipoprotein fraction, were quantified at months 3 and 6. In contrast to CS, exposure to e-cig aerosols resulted in no increase in leukocyte counts, serum cholesterol concentration, aortic plaque formation, and lung matrix metalloproteinase activity compared with exposure to fresh air. Furthermore, no differences were observed among the exposures to CARRIER, BASE, and TESTMIX aerosols. In conclusion, none of these e-liquid formulations gave rise to the disease mechanisms related to atherosclerosis and lung inflammation that were elicited by CS in the ApoE−/− mouse model.
P139: Interspecies Comparison of Embryo–Fetal Data Among Control Groups of Sprague Dawley Rats, New Zealand White Rabbits, and Göttingen Minipigs
F-. H. Paradis1, R. Tavcar1, F. Beaudry1, R. Kubaszky1, K. Kaaber2, K. Hill3, R. Gilmore3, P. Singh4, J. Hargitai5, D. Dandekar3, F. Spézia4, R. Forster4, and S. Authier1
1Citoxlab North America, Laval, Canada
2Citoxlab Denmark, Ejby, Denmark
3Xenometrics LLC, Stilwell, KS, USA
4Citoxlab France, Évreux, France
5Citoxlab Hungary, Veszprem, Hungary
Understanding species-dependent differences in relative incidence of spontaneous variations and malformations is important for reproductive and developmental safety assessment. The objective of this evaluation was to compare litter parameters and external, visceral, and skeletal malformations and variations across species in the Sprague Dawley rat, New Zealand white rabbit, and Göttingen minipig. Pregnant female rats (n = 871), rabbits (n = 465), and minipigs (n = 70) from vehicle control groups were included in the analysis equating to 11,460 rat, 4,486 rabbit, and 378 pig fetuses collected at term by cesarean birth. Preimplantation loss was more frequent than postimplantation loss in the rat and rabbit, whereas the opposite was observed in the minipig (rat: 10.9%, 4.7%; rabbit: 13.8%, 8.1%; minipig: 7.6%, 13.0%). Several external and visceral malformations and variations, such as domed head, skin discoloration, cleft palate, abdominal edema, and anal atresia, were observed in all 3 species. Visceral malformations of the heart and the major blood vessels were remarkably more frequent in the minipig. Ventricular and atrium septum defects were observed in 1.9% and 2.1% of the minipig and 0.02% and 0% in the rabbit, whereas they were not observed in any rat fetuses evaluated in this study. Our results indicate that the minipig presents a higher spontaneous incidence of heart malformations consistent with humans, as congenital heart defects are the most common types of birth defects in humans (approximately 1% of births). A thorough understanding of the similarities and differences in spontaneous malformations in different species is important to interpretation of embryo–fetal development studies.
P140: CYP2A6, CYP2B6, and CYP2E1 Atropselectively Metabolize Polychlorinated Biphenyls to Hydroxylated Metabolites
E. Uwimana1, P. Ruiz2, X. Li1, and H-.J. Lehmler1
1University of Iowa, Iowa City, IA, USA
2Division of Toxicology and Environmental Medicine, Computational Toxicology and Methods Development Lab, Agency for Toxic Substances and Disease Registry, Atlanta, GA, USA
Exposure to chiral polychlorinated biphenyls (PCBs) has been associated with neurodevelopmental disorders. Their hydroxylated metabolites (OH-PCBs) are also chiral and potentially neurotoxic; however, the formation of OH-PCBs by human cytochrome P450 (P450) isoforms is poorly investigated. We hypothesized that the biotransformation of PCB 91, PCB 95, PCB 132, and PCB 136 is mediated by different P450 isoforms. ADMET Predictor and MetaDrug software were used to predict P450 isoforms involved in the metabolism of chiral PCBs in silico. These predictions suggested a role of CYP1A2, CYP2A6, CYP2B6, CYP2E1, and CYP3A4 in the metabolism of chiral PCBs. Subsequent metabolism studies with recombinant human enzymes demonstrated that CYP2A6 and CYP2B6 oxidized PCB 91 and PCB 132 in meta position and that CYP2A6 oxidized PCB 95 and PCB 136 in para position. CYP2B6 played only a minor role in the metabolism of PCB 95 and PCB 136 and formed meta hydroxylated metabolites. Traces of para hydroxylated PCB metabolites were detected in incubations with CYP2E1, whereas no hydroxylated metabolites were detected in incubations with CYP1A2 or CYP3A4. These findings suggest that CYP2A6 and CYP2B6 play an important role in the oxidation of neurotoxic PCBs to chiral OH-PCBs in humans. Further studies are needed to characterize the enantioselectivity of the oxidation of PCBs by both P450 isoforms and assess the toxicity of the resulting OH-PCBs. The findings and conclusions in this presentation have not been formally disseminated by the CDC/ATSDR and should not be construed to represent any agency determination or policy.
P141: Comparison of Routine Clinical Pathology Parameters in Wistar Han Rats at Different Ages
Z. Wang1 and S. McPherson1
1WuXi AppTec, Suzhou, China
Clinical pathology parameters are routinely assessed during the conduct of toxicology studies. This study was conducted to see whether there were any differences in hematology and serum chemistry parameters in Wistar Han rats at 3 different ages: 11 to 12 weeks, 20 to 21 weeks, and 33 to 34 weeks. Rats were sourced from BioLasco Taiwan. The results from the 3 data sets were analyzed using the rats aging 11 to 21 weeks as a reference control point. Analysis showed overall while most of the parameters were comparable among the 3 age groups, some parameters did change over time. Analysis of the hematology data showed that slightly lower white blood cell count, lymphocytes (absolute and percentage), and increased neutrophils (percentage) in both sexes aging 33 to 34 weeks as well as males aging 20 to 21 weeks increased neutrophils count in males only and decreased reticulocytes amount in both sexes at both ages. Differences in serum chemistry included decreased alkaline phosphatase and inorganic phosphorus, increased glucose in both sexes at both ages, increased total cholesterol and triglyceride in both sexes aging 33 to 34 weeks as well as male rats aging 20 to 21 weeks, and increased alanine aminotransferase in both sexes aging 33 to 34 weeks. The tendency of change was consistent with the published reference for this species. These background data collected from this study can serve as a tool to help the toxicologist evaluate study data and put potential findings in context when compared to both the concurrent controls and these data sets.
P142: Epidemiology, Toxicokinetics, and Renal Injury of Self-Poisoning With Gloriosa superba
T. M. Wijerathna1,2, I. B. Gawarammana1,3, F. Mohamed1,4,5,6, D. M. Dissanayake2, P. I. Dargon7, and N. A. Buckley1,6
1South Asian Clinical Toxicology Research Collaboration, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka
2Department of Pathology, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka
3Department of Medicine, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka
4Department of Pharmacy, Faculty of Allied Health Science, University of Peradeniya, Peradeniya, Sri Lanka
5Department of Nephrology, Prince of Wales Hospital and Clinical School, University of New South Wales, Sydney, Australia
6Department of Pharmacology, Sydney Medical School, University of Sydney, Sydney, Australia
7Faculty of Life Sciences and Medicine, King’s College London, London, United Kingdom
Gloriosa superba is a flowering plant that contains colchicine. It is responsible for 44% of plant poisonings in Sri Lanka (case fatality rate 15%). Despite it being a common clinical problem, its epidemiology, toxicokinetics, and effects on the kidney have not been described. The objective of this study was to describe epidemiology, toxicokinetics, and renal injury through a large multicenter cohort study. The study consisted of 3 parts: reporting of epidemiologic data (n = 297), study of toxicokinetics (n = 72), and evaluation of acute kidney injury (AKI) and renal biomarkers (n = 45). Plasma colchicine levels were measured by high-performance liquid chromatography method. Acute kidney injury was studied using serum biomarkers: creatinine (sCr), cystatin C (sCysC), and creatine kinase (CK) and urinary biomarkers; creatinine, kidney injury molecule-1, clusterin, albumin, β-2-microglobulin, cystatin C, neutrophil gelatinase-associated lipocalin, osteopontin, and trefoil factors. Of 297, 29 died; 54% were males. Median concentration of colchicine was high in both elderly (4.7 [1.7-6.6] ng/mL) and deceased categories (7.8 [5.8-18.7] ng/mL). The area under the receiver operating characteristic curve (AUC-ROC) for uncorrected admission colchicine level was highly predictive of a fatal outcome, and this improved even further with 2 methods we developed to correct for the expected change with time. The best method had an AUC-ROC of 0.98 (95% confidence interval, 0.94-1.00) in predicting death, with 100% sensitivity and 96% specificity. Elimination exhibited first-order kinetics. sCr, sCysC, and CK were mildly elevated. Ingestion of G superba caused mild AKI and mild rhabdomyolysis.
P144: Clock1a Gene Mediates Bisphenol A-Induced Neurodevelopmental Toxicity in Zebrafish Embryo
J. Zhang1, Y. Chen1, and R. Yan1
1Soochow University, Suzhou, China
Bisphenol A (BPA) is one of the most common environmental pollutants with neurotoxicity. The aim of this study was to determine whether clock1a, an important circadian clock gene, was able to regulate the BPA-induced neurodevelopmental toxicity in zebrafish embryo. Using the zebrafish (wild-type, WT and clock1a gene knockout, clock1a−/−) embryo as a model system, 1, 5, or 25 μM of BPA was given and behavioral and locomotor tests were conducted. Cellular death/apoptosis and the clock1a gene expression were also detected to investigate the effect of clock1a on BPA exposure. The observations indicated that BPA treatment resulted in embryo malformations, shortened movement distance and movement times, increased apoptosis, and decreased survival rate accompanied by upregulated expression of Nrf2 and neurogenesis-related genes, Shha and Ngn1. The BPA treatment also promoted the binding of estrogen receptor to the estrogen-response elements of clock1a promoter. Since all these changes were more pronounced in clock1a−/− than in WT zebrafish, clock1a plays an important role in BPA-induced neurodevelopmental toxicity.
200 Series—Regulatory Toxicology
P201: Characterization of EEG Morphologies During Drug-Induced Seizures and Peri-Ictal Changes in Nonclinical Species
S. Authier1,2, M. Pouliot1, M. Accardi1, M. Dubuc-Mageau2, W. Tan2, A. Sanfacon2, V. Badalone1, and E. Boulay1,2
1CiToxLAB, Laval, Quebec, Canada
2Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canada
Regulatory agencies require that the most susceptible species be used for seizure liability assessment during drug development. A broad range of automated and manual methodologies are available to detect or identify ictal and interictal events in nonclinical electroencephalogram (EEG) obtained by telemetry. The strategy to detect seizure events is often based on EEG morphology and defining those events is essential to ensure appropriate algorithm selection. The current study aimed to analyze EEG morphologies observed during drug-induced seizures but also interictal events. The EEG traces from rats, dogs, minipigs, and nonhuman primates were evaluated for spike frequency, duration, patterns, and premonitory and postictal changes. Most drug-induced seizure events were self-limiting often with crescendo decrescendo spike trains. In all species, postictal EEG changes were observed over a period of a few minutes only after a seizure episode with resolution prior to clinical signs. Characteristic profiles were noted for individual animals but also with each drug (ie, similar patterns noted for a given individual or drug). Most drug-induced seizures were noted at times of maximal plasma exposure, but delayed seizure onset was noted in rare cases. Seizure onsets were associated with stimulation in some cases as reported with nondrug-related seizures. Quantitative EEG analysis generally revealed a progressive increase in most spectral frequencies above 4 Hz prior to ictus. Overall, this analysis reveals a rich diversity in EEG morphologies associated with drug-induced seizures with implications when selecting detection algorithms, EEG derivations, or during manual morphology review.
P202: Are All Ames Strains in the OECD Mutagenicity Test Guideline 471 Useful and Necessary? An Analysis of Large Mutagenicity Data Sets for the IWGT
K. P. Cross1, D. M. DeMarini2, L. F. Stankowski Jr3, E. Zeiger4, and R. Williams5
1Leadscope, Inc, Columbus, OH, USA
2US EPA, Research Triangle Park, NC, USA
3Charles River Laboratories, Inc, Skokie, IL, USA
4Errol Zeiger Consulting, Chapel Hill, NC, USA
5Lhasa, LTD, Leeds, United Kingdom
The International Workshop on Genetic Toxicology (IWGT) meets every 4 years to reach consensus recommendations on difficult or conflicting approaches to genotoxicity testing based upon practical experience and newly available data and analysis techniques. The IWGT met in Tokyo, November 2017, with one working group assessing the sensitivity and selectivity of the standard strains in the Ames test as specified in Organisation for Economic Co-operation and Development Test Guideline 471 and to make recommendations for a minimum viable strain profile. The discussions, based partially on bacterial mutation data in multiple strains from large (>10,000 compound) Leadscope and Lhasa databases, included: (1) defining criteria for determining significant selective responses across different strains; (2) identifying compounds producing selective responses using reported author calls; (3) confirming selective responses by examining dose–response data including metabolic activation and experimental conditions; (4) using statistical methods to objectively verify response differences; and (5) determining the frequency and chemotypes of compounds producing selective responses. Results indicated that Salmonella TA1535 added little information to a battery including TA100, and TA97/TA97a detected more unique mutagens than TA1537. Preliminary results suggest Escherichia coli WP2 uvrA pKM101 is more sensitive than TA102 or E coli WP2 uvrA sans plasmid. Preliminary results from additional direct comparison testing of 10 chemicals failed to confirm previously reported selectivity between related tester strains. For example, altertoxin I was previously reported to be positive in TA1537 and negative in TA97 but gave clearly positive responses in both tester strains when tested concurrently. [Abstract does not reflect policies of the US EPA.]
P203: Conducting a US FDA CVM Animal Drug Safety Study in a GLP Environment
R. Early1, S. McPherson2, B. Zhou2, and W. Salminen3
1RJEarly Consulting, Irvine, CA, USA
2WuXiAppTec (Suzhou) Co Ltd, Suzhou, China
3Camargo Pharmaceuticals, Cincinnati, OH, USA
Target animal safety (TAS) studies conducted for investigational veterinary medicines with the US Food and Drug Administration (US FDA) Center for Veterinary Medicine (CVM) require additional precautions beyond more routine Good Laboratory Practice (GLP) toxicology studies. These TAS studies can be viewed as a hybrid of a GLP toxicology study and a phase 1 clinical trial but conducted in the species for which the investigational veterinary medicine is indicated. These additional CVM-mandated requirements are to ensure no bias or influence on the results. These measures can at times be in conflict with testing facility SOPs and GLP and animal welfare regulations. Notable differences from nonclinical safety studies include anonymous drug administration and data collection; masking procedures for study personnel; adjustments in procedures for randomizing animals and drug administration; adjustments in data collection and processing procedures to prevent identification of treatments; limited access to previously collection data; masking of treatment groups on housing cages; and random placement of animals throughout the room. Another key difference from GLP toxicology studies is that GMP-produced clinical formulations are used and doses are often multiples of the clinical dose and may not reach a maximum tolerated dose. Statistical analyses are more complicated and include models up to repeated-measures analysis of covariance incorporating baseline as a covariate and treatment, sex, and time as fixed effects. In conclusion, TAS studies on animal drugs are a hybrid of GLP toxicology and phase 1 human clinical trials with increased emphasis on anonymous dose administration and data collection and more extensive statistical analyses.
P205: Definition and Implementation of a Skin Sensitization In Silico Protocol
C. Johnson1, D. Bower1, K. P. Cross1, and G. Myatt1
1Leadscope, Inc, Columbus, OH, USA
The contribution of in silico approaches to toxicological assessments is well recognized. However, there is a need to develop standardized procedures and guidelines to conduct in silico toxicological assessments. Toward this end, a cross-industry consortium comprising over 50 organizations proposed a general strategy for the development of protocols that allows for consistent, transparent, and reproducible evaluation of major toxicological end points using in silico methods. The skin sensitization end point is of particular interest. The guiding principles of animal research, the “3Rs” (replacement, reduction, and refinement), and the implementation of regulatory guidelines have encouraged the development of alternative (nonanimal) methods, such as, in silico methods in concert with new and existing in vitro and in chemico methods. Within the consortium, a framework was developed for hazard assessment of skin sensitization that comprised of effects and mechanisms associated with key adverse outcome pathway (AOP) events plus historical testing strategies and sources of information. The framework is flexible and can accommodate single and integrated approaches to testing and assessment. The procedure for deriving an overall assessment for skin sensitization using in silico approaches is presented along with the software implementing the framework. Several examples will be presented demonstrating (Q)SAR predictions of skin sensitization and how confidence scores are assigned and propagated to the overall hazard assessment.
P206: Social Housing and Pregnancy Success in the Nonhuman Primate (NHP): An Appraisal
C. M. Luetjens1, H. Grossmann2, and G. F. Weinbauer1
1Covance Preclinical Services GmbH, Münster, Germany
2Institut für Mathematische Stochastik, Otto-von-Guericke-Universität, Magdeburg, Germany
Enhanced pre- and postnatal developmental (ePPND) studies for biologics with pregnant nonhuman primates (NHPs) have become the gold standard if NHP is the relevant species. Spontaneous pregnancy losses and infant deaths are highly variable in long-tailed macaques (cynomolgus monkey), making it potentially difficult to ascertain test item-related effects; given this variability, it is important to assess and benchmark pregnancy outcome data relative to test facility historical control data, as described by Jarvis et al. The group size-related normograms of pregnancy outcome and early infant survival were comprised of data from embryo–fetal development and PPND studies. Since the publication of ICH M3(R2)(2009) and ICH S6(R1)(2011), these 2 study types have been replaced by ePPND studies for biologics. Importantly, the data reported in Jarvis are derived from singly housed animals. Since then, studies in pregnant NHPs have been conducted in socially housed animals including delivery and raising infants. This work compares the pre- and postnatal loss rates and infant development in (e)PPND studies under single and social housing. Qualitative assessment under social housing indicates good compatibility of maternal animals. It appears that under social housing pre- and postnatal losses tend to be reduced compared to single housing. Conceivably, the delivery process and managing the newborn is facilitated by social maternal housing. Interestingly, body weight development is clearly enhanced in infants raised under social versus single housing. Overall, social housing is strongly recommended for developmental toxicity studies in NHPs.
P207: Can We Expand the Use of One Species for Post-“First-in-Human” Studies, Within and Beyond ICHS6?
H. Prior1, N. Gellatly1, F. Sewell1, and I. Kimber2
1National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs), London, United Kingdom
2University of Manchester, Manchester, United Kingdom
An NC3Rs/Association of the British Pharmaceutical Industry international working group is reviewing the need for 2 species in regulatory toxicology studies. Within ICHS6 guidelines, one species may be used for longer term studies if toxicity profiles are “similar” in 2 species in short-term studies. We collected data on 172 compounds from 18 organizations to determine the incidence of similar target organ toxicities between species in short-term (First-in-Human or “FIH”) studies (2-13 weeks), to determine whether other guidelines could also adopt this principle. For compounds with additional longer term (“post-FIH”) data (13-39 week), FIH data were reviewed for a prospective exercise: comparing the blinded decision to conduct the post-FIH studies in one or 2 species, with the actual post-FIH study outcomes. Two species were used in FIH studies by 77 small molecules, 12 monoclonal antibodies (mAbs), 11 recombinant proteins (RP), 12 synthetic peptides (SP), and 3 antibody–drug conjugates (ADCs). Toxicities were similar in both species for 32%, 83%, 36%, 42%, and 0% per molecule type, respectively. For 11 compounds following ICHS6 that progressed to post-FIH studies, 4 mAbs, 1 RP, and 1 SP had similar toxicities, yet only 2 mAbs reduced to one species. The prospective exercise decisions to reduce to one species or not were 100% accurate for mAbs, RP, and SP, and 70% accurate for small molecules. We propose that when toxicities are similar in 2 species at the FIH stage, subsequent studies could proceed in a single species with low risk to human safety.
P208: Trends Toward a Reduction in the Use of Recovery Animals for First-in-Human Studies From 2013 to 2017
F. Sewell1, N. Gellatly1, and H. Prior1
1National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs), London, United Kingdom
It is a regulatory requirement that recovery is assessed during pharmaceutical development, but flexibility as to how, where, or even if recovery animals are included. In 2014, a data sharing initiative identified opportunities to reduce recovery animal use by inclusion later in development, and in fewer studies or dose groups. A recent NC3Rs/Association of the British Pharmaceutical Industry international working group reviewing 2 species use within toxicology studies has also collected data on recovery animals, providing an insight into current trends. Data from 157 studies (started 2014 onward) to support First-in-Human (FIH) clinical trials were compared with 242 studies (the pre-2014 data set). There was a reduction in the proportion of studies including recovery animals compared with the earlier data set (small molecules 49% vs 68%; monoclonal antibodies [mAbs] 55% vs 81%). When recovery groups were included, the number of small molecule studies with control and high dose only was similar to previous data (77% vs 75%), but mAbs showed an increase in studies with control and high dose only (50% vs 33%), away from recovery in all groups. If considering species, there was a similar reduction in the proportion of studies including recovery animals for monkeys, dogs, and rats compared with the earlier data set (57% vs 85%, 41% vs 61%, and 49% vs 72%, respectively). Although these data suggest trends toward reduced and case-specific approaches, there remain opportunities to review and further reduce recovery animal use in FIH packages without impacting human safety.
P209: Reevaluation of Discordant Results in Related OECD TG471 Tester Strains
L. F. Stankowski Jr1, B. Lallier1, C. Tydrick1, R. Baba1, S. Tincher1, J. Callupe1, and V. Y. Kwok1
1Charles River Laboratories, Inc, Skokie, IL, USA
The International Workshop on Genetic Toxicology recently met with one group assessing the sensitivity/selectivity of tester strains used for Organisation for Economic Co-operation and Development (OECD) TG471 (Bacterial Reverse Mutation (Ames) test). Results from large (>10,000 compounds) databases were analyzed to assess relative responses between related tester strains: TA100/TA1535, TA97/TA1537, and TA102/WP2uvrA/WP2uvrA (pKM101). TA100/TA1535 comparisons were relatively straightforward, since both are required by OECD TG471 and comparison data typically were generated concurrently. Since only one strain from other pairs/triplets is required, comparison data generally were from different laboratories. Discordant results were often observed, especially when using a “2- (or 3-) fold” criteria. Ten chemicals producing discordant results were reevaluated concurrently: altertoxin I, chlorambucil, CI basic red, retrorsine, and 2-methylpropanenitrile in TA97/TA1537; and N,N-diethylnitrosamine, folpet, acrylonitrile, NiCl2, and p-toluenesulfonyl hydrazide in TA102/WP2uvrA/WP2uvrA (pKM101). Our GLP-compliant studies did not confirm previous discrepancies. For example, altertoxin I was reported to be TA1537 positive and TA97 negative, but concurrent testing gave a higher response (revertants per µg/plate) in TA97. Similarly, folpet gave conflicting results in TA102/WP2 uvrA with and/or without S9 but was uniformly positive here in TA102/WP2uvrA/WP2uvrA (pKM101) ±S9. Chlorambucil, previously reported to be TA1537 positive and TA97 negative (+S9 only), was negative here in both. Results of the database analyses, and these, suggest that TA1535 adds little value to a set containing TA100, TA97 detects more unique mutagens than TA1537, and WP2uvrA (pKM101) is more sensitive than TA102 or WP2uvrA.
300 Series—Safety Evaluation Nonpharmaceuticals
P301: Biochemical and Toxicological Implications of Ethylacetate Fraction of the Methanolic Extract of Plumbago zeylanica (Linn) Root
G. O. Ajayi1, O. Ademuyiwa2, J. A. Olagunju1, and F. A. Faduyile1
1Lagos State University College of Medicine, Ikeja-Lagos, Nigeria
2Federal University of Agriculture, Abeokuta, Nigeria
Plumbago zeylanica, Linn, is an important plant, commonly found in Southwest Nigeria, with numerous medicinal values. This study was aimed to evaluate the biochemical and toxicological effects of the administration of the ethylacetate fraction of the methanolic extract of Plumbago zeylnica root (EAME). In subchronic study, extract doses of 100, 200, and 400 mg/kg body weight were administered orally in rats for 28 days. In the plasma, subchronic administration of EAME at the highest dose increased the concentrations of protein by 11%, albumin 32%, glucose 153%, direct bilirubin 151%, total bilirubin 656%, creatinine 35%, and uric acid 29%. Activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and γ- glutamyltransferase were also increased by 6%, 39%, 76%, and 46%, respectively. The concentrations of total cholesterol, triglycerides, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and very-low-density lipoprotein-cholesterol were not significantly different (P > 0.05) compared with the control. The EAME induced histopathological alterations in the cellular architecture of the liver and lungs. Mild congestion in sinusoids and bile duct proliferation were observed in the liver, while the lung showed necrosis, edema, and hemorrhage. The alterations were more prominent in the lung of rats treated with 200 and 400 mg/kg body weight doses. No histopathological alteration was observed in the kidney. These findings indicate that EAME of P zeylanica root contains bioactive compounds which are toxic to the organism, but the action was organ-specific.
P306: Follow-Up Photosafety Testing for a Subset of Fragrance Materials
G. Ritacco1 and A. M. Api1
1Research Institute for Fragrance Materials, Woodcliff Lake, NJ, USA
Chemicals with significant UV/Vis absorbance have the potential to cause phototoxic effects. The UV/Vis absorbance spectra were obtained for nearly 2,000 fragrance raw materials; approximately 93% did not demonstrate significant UV/Vis absorbance. For materials that do absorb, further testing is warranted in order to ensure photosafety. Our photosafety testing is conducted in a stepwise manner, moving from hazard-based to risk-based in vitro assays, and confirming no-effect levels in humans. In the 3T3-neutral red uptake (NRU) phototoxicity test, Balb/c 3T3 mouse fibroblasts are exposed to serial dilutions of the fragrance materials. Phototoxic and cytotoxic potential is measured by test material-induced inhibition of NRU in the cells in both the presence and absence of UVA exposure. The materials we present were predicted to have phototoxic potential in the 3T3-NRU assay. The materials were then evaluated in human keratinocytes using the EpiDerm multi-dose phototoxicity assay (MatTek Corporation) in both the presence and absence of UVA. Each fragrance material was tested at 3 concentrations. The tissues were subjected to UVA for 60 minutes (6 J/cm2). In this assay, a test material that induces a decrease in viability of 30% or more, in the presence of UVA compared to viability in the absence of UVA, predicts phototoxicity. As a final step, human phototoxicity studies were conducted to confirm no-effect levels. The studies presented demonstrate a stepwise method to assess phototoxic potential of fragrance raw materials and arrive at a no-effect level for phototoxicity.
P307: Selection of Cancer and Noncancer Toxicity Values for Quantitative Risk Assessment of Harmful and Potentially Harmful Constituents in Combustible Tobacco Products
S. G. Sawant1, A. Amantana1, W. Xie1, S. Liu1, K. M. Marano1, and D. T. Szabo1
1RAI Services Company, Winston-Salem, NC, USA
The US Food and Drug Administration (US FDA) established a list of 93 harmful and potentially harmful constituents (HPHCs) in tobacco products/smoke. These are classified by the US FDA as respiratory, cardiovascular, reproductive developmental toxicants, and/or carcinogens. In order to quantitatively estimate human health risks of HPHCs from cigarette smoke exposure, cancer and noncancer toxicity values are preferred. The US Environmental Protection Agency (US EPA) has developed a hierarchical risk assessment approach to identify toxicity values for chemicals found at Superfund sites. However, several toxicity values have not been updated for decades and may no longer be scientifically defensible. Toxicity values are also available for several HPHCs from other regulatory/public health agencies. In the absence of pertinent US FDA guidance, our aim was to identify the most scientifically defensible toxicity values for HPHCs. A comprehensive review of US federal and state and international databases was conducted to identify chronic inhalation cancer and noncancer toxicity values for HPHCs identified by US FDA for reporting in cigarette smoke. The results indicate that study quality, date of the study, and the toxicity value derivation approach are critical factors in selecting a public health protective toxicity value. Specific to noncancer toxicity values derived using a critical effect other than a US FDA-identified toxicity end point(s) (respiratory, cardiovascular, and/or reproductive-developmental), scientific justification should describe the public health protective nature of the selected value. A similar selection approach can also be considered for other exposure routes to conduct a scientifically defensible quantitative risk assessment of exposure to tobacco-related HPHCs.
P308: The Porcine Corneal Opacity and Reversibility Assay and Assessment of the Drivers of Classification With Regard to Ocular Damage
P. Vij1, D. Sergeyev1, M. Carathers1, E. Delacruz1, B. J. Varsho1, and G. L. DeGeorge1
1MB Research Laboratories, Spinnerstown, PA, USA
The Draize Rabbit Eye test assesses damage to ocular structures, which are scored and weighted based on toxicological importance. The structures are cornea (CO), conjunctiva (Conj), and iris (IR). Corneal irritation is assessed by opacities on the cornea, Conj by impact on the vasculature, and IR damage by alteration of the function of the iris (constriction or dilation) and deepening of the rugae. Score weighting is according to CO damage accounting for 80 of the 110-point Draize scale, and conjunctival effects for 20 points, with IR for 10 points. Since CO scores have the heaviest weight, and most often are the drivers of eye irritation, we developed an ex vivo corneal model to assess these effects. PorCORA is an ex vivo ocular assay that distinguishes between a substances’ potential to cause severe (reversible) versus corrosive (irreversible) damage. We tested 56 chemicals ranging from corrosive (GHS category 1) to nonirritating (GHS not categorized) using Cooper statistics. We obtained an accuracy of 88% with a positive and negative predictivity of 91% (category 1) and 85% (not category 1), respectively. Reexamination of our data using the in vivo drivers of classification concept of Barroso et al and its database and methodology, we found 8 chemicals that had invalid tests (animals euthanized prior to day 21) or GHS classification not driven by corneal opacities. When these chemicals were curated in our data set, the accuracy improved to 90%, with a major change in sensitivity, which increased from 80% to 87%.
400 Series—Toxicology Methods
P401: Toxicological Characterization and Efficacy of an Inhaled Therapeutic Platform for Optimization of Protein Therapies
A. Aslam1, K. McInally1, and O. Blaschuk2
1ITR Laboratories Canada Inc, Baie-d’Urfé, Canada
2McGill University Health Centre, Montreal, Canada
As oral delivery of therapeutic proteins is often viewed as attractive for supporting patient compliance, the principal drawback is that these macromolecules are too large to pass through the epithelial and endothelial barriers without an absorption enhancer. An alternative to support protein therapy is by introducing aerosolized macromolecule delivery into the lungs. The extensive surface area of the lungs, its unique cellular properties of airway epithelium, and lower concentrations of drug metabolizing enzymes allow for rapid absorption of macromolecules into the systemic circulation that are less likely to be degraded. A preliminary experiment was performed to determine the toxicity and efficacy of a therapeutic platform when administered by inhalation exposure once daily for 5 consecutive days to Sprague Dawley rats. The proposed use of the therapeutic platform would introduce an absorption enhancer to optimize protein therapy through pulmonary drug delivery in tandem with a variety of inhaled macromolecules. The results of this experiment demonstrated that dose levels up to 26.0 mg/kg/d did not produce any adverse test article-related findings. The absorption-enhancing molecule was well tolerated, and there were no adverse clinical observations or systemic effects detected by assessment of clinical pathology parameters or following histopathological examination of all major organs. The respiratory tracts were examined histologically with no indications of local toxicity. The stability of the absorption enhancer in dose formulation solutions up to 50 mg/mL was determined in vitro by assessing the biological activity on epithelial cell–cell adhesion. The absorption enhancer was capable of disrupting adhesion of confluent epithelial cells.
P402: Human Cell Line Activation Test: Pitfalls and Considerations
G. L. DeGeorge1, P. Vij1, D. Sergeyev1, and B. J. Varsho1
1MB Research Laboratories, Spinnerstown, PA, USA
Skin sensitization resulting from exposure to a chemical is an important concern. The human cell line activation test (h-CLAT) was developed based on the role of Langerhans cells (LC) activation during the induction phase of skin sensitization and uses THP-1 cell line as LC surrogate. This in vitro test measures the augmentation of CD86 and CD54 expression in THP-1 cells (a human monocytic leukemia cell line) following a 24-hour exposure to a test substance (TS). Herein, we address some pitfalls, issues, and remediation in the h-CLAT procedure. The reactivity check, performed 2 weeks after thawing a new batch of cells, requires the viability of vehicle- and lactic acid (LA)-treated cells ≥90%. Both 2,4-dinitrochlorobenzene (DNCB) and nickel sulfate (NiSO4) should produce a positive response in CD86 and CD54 expression, and LA should produce negative response. Otherwise, remedial steps should be taken, including thawing a new batch of cells. Proactive measures should be made to bank frozen cell stocks at various passages. An appropriate solvent (saline or dimethyl sulfoxide) should completely dissolve the TS. If not, we present alternative vehicles (chosen with scientific justification) to broaden the applicability domain. A reliable CV75 should be derived from 2 independent screens. If it is difficult to attain, we present options for consideration. FACS buffer and 1% globulin preparation should be at 4°C. High maintenance of the flow cytometer is essential for reliable data acquisition. In addition, historical data on the doubling time and passage number should be maintained and monitored.
P403: Application of Inducible Pluripotent Stem Cells to Evaluate Chemical Effects on Human Adult Cardiomyocyte Differentiation, Cytotoxicity, and Function
K. Dreher1, V. Long III2, K. Das1, and C. Lau1
1National Health and Environmental Effects Research Laboratory, Office of Research and Development, US EPA, Durham, NC, USA
2Oak Ridge Institute for Science and Education, Oak Ridge, TN, USA
Adult human stem cell-derived cardiomyocytes (hSC-CM) were employed to evaluate the effects of chemicals on their cytotoxicity, differentiation, and function. The hSC-CM were exposed separately to saline or dimethyl sulfoxide vehicle, 10 nm Ag nanoparticles (NP, 3 or 6 µg/mL), perfluoroalkyl substances (PFNA, PFOS, 25-100 µM), or triclosan (TCS, 2.5-10 µM) and evaluated viability and function over 48 hours of exposure. No effect on hSC-CM cytotoxicity was observed. Ten nanometer Ag NP induced a 15% and 30% decrease in beat rate (BR) and beat amplitude (BAMP) at 3 µg/mL, respectively. The PFNA induced an increased beat rate irregularity index (BRI) up to 24 hours of exposure. The PFOS induced an increase in BRI over the 48-hour exposure period. The TCS induced an increase in BR and decreased BAMP over 48 hours of exposure. An human cardiac progenitor cell (hCPC) assay evaluated the effect of chemical exposures on their viability, differentiation, and function. The hCPCs were exposed to PFNA, TCS, or bisphenol A (BPA, 0.12-12 µM), as described previously, and evaluated 14 days postexposure. The hCPC cytotoxicity and differentiation were determined by fluorescent analysis using Red Dot1 nuclear and troponin T antibody in-cell Western staining, respectively. The BPA was not cytotoxic to hCPCs and had no effect on CM function. The PFNA decreased both hCPC viability at 100 µM and differentiation, BR, and BAMP at 25 µM. The TCS was cytotoxic to hCPCs at 10 µM. Results demonstrate hSC-CMs and CPCs are valuable models to evaluate chemical effects on adult cardiac developmental biology and toxicity. (This abstract does not represent US EPA policy.)
P404: Intravenous Dosing of Neonatal/Juvenile Rats as a Test System for Toxicology and Safety Evaluation of Drugs Targeting Pediatric Patients
A. Fotovati1, A. Dadkhah1, and W. Ruddock1
1ITR Laboratories Canada Inc, Baie-d’Urfé, Quebec, Canada
Administration of potential therapeutics to neonatal rodents is technically challenging. However, considering the metabolic and physiologic difference between adult and neonates, in order to evaluate the efficacy or safety of the drugs targeting pediatric patients, establishing a neonatal/juvenile test system is essential. We have developed a repeat intravenous injection dosing system for neonatal rats. A total of 20 neonates (10 of each sex) from 2 dams were divided into test and control groups. In test group, animals were anesthetized by either hypothermia or isoflurane inhalation. The temporal vein was located using transluminating light under magnifier or dissecting microscope, and following a gentle manual restrain, animals were injected with 50 µL of phosphate-buffered saline using an insulin syringe with a 31-G needle. Successful dosing was evidenced by blanching of the vascular network following injection. Furthermore, infusion of 1% Evans Blue confirmed successful dosing as it was evidenced by immediate blue discoloration of the injected pup. Animals in the control group were also anesthetized similarly but were not injected. Dosing was performed once every 24 hours and pups were monitored daily for any abnormality. Our results showed that the neonatal rats could be successfully dosed daily until at least postnatal day 4 and depending on the body size up to day 5. We suggest that our model can be used for efficacy studies or safety evaluation of pediatric drugs as single or up to 5 daily doses. Furthermore, to continue intravenous dosing, temporal vein could be replaced by jugular vein till adulthood.
P407: Determination of Optimal Neurological Evaluation During a 14-Day Oral Gavage Study of NBI-1 in the Rat
A. J. Jabbour1, G. S. Liang1, G. R. Loewen1, and H. P. Bozigian1
1Neurocrine Biosciences, San Diego, CA, USA
Sprague Dawley rats were administered vehicle and NBI-1 at 100 and 300 mg/kg/d via oral gavage for 14 consecutive days. Study parameters included functional (days 3, 7, and 14), electrophysiological (day 15), and microscopic (day 15) evaluations. No effects were noted at 100 mg/kg/d. At 300 mg/kg/d, functional assays showed limited usage of the hind limbs and paws, decreased locomotor activity, and a decrease in forelimb and hindlimb grip strength, with the latter more affected. Nerve conduction velocity measurements showed a severe deficit of the mixed caudal nerve and a milder effect on the pure sensory response recorded in the digital nerve at 300 mg/kg/d. In addition, a prolongation of the onset latency of muscle contraction following stimulation of the tibial motor nerve was noted. The relative deficit for the digital sensory and tibial motor nerves was similar. No effects were noted with the Hargreaves assessment. Microscopically, an increase in the incidence of nerve fiber degeneration was noted at 300 mg/kg/d. Nerve fiber degeneration was characterized by axonal fragments and myelin ovoids. Resin-embedded sections revealed the presence of fully demyelinated axons, not readily recognized in the sections of paraffin-embedded samples, and better demonstrated thinly myelinated (remyelinating) fibers as compared to paraffin sections. In conclusion, functional, electrophysiological, and neuropathological examinations were comparable in identifying test article-related effects at 300 mg/kg/ d. Resin sections allowed better characterization of the nature of nerve damage.
P408: A Study on the Calibration Characteristics of Toxic Compounds With the Solvent Effects
Y-.J. An1, Y-.H. Kim2,3, S-.J. Choi2, and K. Lee2,3
1Konyang University, Daejeon, Republic of Korea
2Korea Institute of Toxicology, Jeongeup, Republic of Korea
3University of Science & Technology, Daejeon, Republic of Korea
When assessing the toxicity of diverse substances, a first step is accurate quantitative analysis. The analytical methods can be selected by the physicochemical properties of target substances. In case of the complex mixture of substances, one needs an instrumental analysis such as chromatography. In order to accurately quantify analytes, the solvent effect, which can affect their quantitative analysis, should be evaluated. In this study, the solvent effects were assessed using 4 different solvents (methanol, hexane, phosphate-buffered saline [PBS], and dimethyl sulfoxide [DMSO]) and 3 toxic compounds (benzene, toluene, and methylisothiazolinone). The working standards containing 3 toxic compounds were prepared by the dilution of each solvent and analyzed by the gas chromatography–mass spectrometry system. The response factor values of target analytes were different depending on the solvent types. Benzene and toluene had highest response factor values in the hexane solvent (33,674/ng benzene and 78,604/ng toluene). In contrast, the response factor of methylisothiazolinone was the highest at 9,067/ng in the PBS solvent. Methanol and DMSO solvents had fairly good values of R 2 > 0.99 and relative standard deviation (RSD) <10%. The R 2 and RSD values of methylisothiazolinone in the hexane solvent recorded the low of 0.0562% and 10.6%, respectively. Benzene and toluene had low R 2 and RSD values in the PBS solvent (mean R 2 = 0.9892 ± 0.0146 and mean RSD = 13.3% ± 4.1%). The results and the reliability of the quantitative analysis are different depending on the analyte and solvent types. Therefore, the quantitative analysis should be conducted by considering the solvent.
P409: Assessing Mitochondrial Toxicities by Live-Cell Imaging Using a Novel ATP Sensor
T. Kiyota1, H. Irimagawa1, C. Schramm2, M. Bowe2, J. Shean2, C. Lawson1, D. Appledorn2, and W. R. Proctor1
1Investigative Toxicology, Department of Safety Assessment, Genentech Inc, South San Francisco, CA, USA
2Department of Biology, Essen BioScience Inc, Ann Arbor, MI, USA
Drug-induced mitochondrial toxicity is associated broadly with different organ toxicities. As such, compound-related mitochondrial effects are commonly assessed for lead optimization. The glucose/galactose switch assay (Glu/Gal assay) is traditionally employed but suffers from technical challenges and false-negative signals due to intrinsic cytotoxicity that often limits its utility for identifying clinically relevant toxicants. Here, we subjected a test set of 12 compounds selected based on known mitochondrial liabilities including 2 negative controls to a novel adenosine triphosphate (ATP) sensor-expressing cell-based Glu/Gal assay with a cytosolic ATP sensor and IncuCyte S3. Images were kinetically captured every 15 minutes for the first 3 hours, followed by every 1 hour for 21 hours. Results from the assay were compared across a traditional end point CellTiter-Glo (CTG) Glu/Gal assay at 24 hours and a cell-based mitochondrial respiration Seahorse assay. Of the 12, 9 were properly identified as mitochondrial toxicants or controls appropriately. However, the remaining 3 of 12 showed their different mitochondrial toxicity profiles in the 3 assays examined. Detailed description of these effects in each system and the overall kinetics of this novel tool will be presented. In general, the ATP sensor-based assay better identified compounds associated with mitochondrial toxicities than the CTG end point alone due to the ability to identify meaningful changes between glucose- and galactose-cultured cells prior to overlapping cytotoxicity. Taken together, these results suggest that the ATP sensor-based assay can provide meaningful data for mitochondrial risk assessment in drug discovery. Further studies are underway to continue to evaluate this novel technology.
P410: Comparison Between the Standard and the 6-Well Ames Assay
R. Kulkarni1, E. Dakoulas1, J. Nicolette2, and A. Patel1
1MilliporeSigma, BioReliance Toxicology Services, Rockville, MD, USA
2Abbvie Inc, North Chicago, IL, USA
The bacterial reverse mutation (Ames) assay is the cornerstone of assays employed in a battery of tests for many regulations conducted as a part of the safety assessment. The Organisation for Economic Co-operation and Development (OECD) Test Guideline 471 describes the conduct of the standard Ames assay for regulatory submissions. After the release of the International Conference on Harmonisation guideline for impurities (ICH M7), regulatory agencies are accepting the 6-well “mini Ames” assay, especially when the test articles are hard to synthesize. As such, 2 platforms of the Ames test were compared: the 6-well plate and the standard 100 mm plate test at AbbVie, Inc and MilliporeSigma, BioReliance Toxicology Services. The 6-well test uses the same plating procedure and evaluation methods as the standard test but requires 20% of the reagents and test chemical. Additionally, the 6-well version uses a limit concentration of 1,000 µg/well versus 5,000 µg/plate. Benzo (a) pyrene (BaP), N-ethyl-N-nitrosourea (ENU),
P411: Predicting Drug-Induced Liver Injury In Vitro
S. Lamore1, C. Rowbottom1, S. Kapadnis1, A. Easter1, E. Tien1, J. Fikes1, K. Brouwer2, and J. Jackson2
1Biogen, Cambridge, MA, USA
2BioIVT, Durham, NC, USA
Drug-induced liver injury (DILI) is a common reason for late-stage drug failure and remains difficult to predict. The C-DILI assay uses Sandwich-Cultured Transporter Certified Human Hepatocytes (SCHH) cultured under physiologically relevant concentrations of bile acids and lipids, allowing for determination of alterations in bile acid disposition and hepatotoxicity. In this study, the hepatotoxicity potential and mechanism (cholestatic vs general) was assessed using the C-DILI assay for 4 preclinical candidates (BIO1-BIO4) found to be hepatotoxic in repeated-dose rat studies as indicated by histopathology and/or clinical chemistry changes. All 4 compounds were inactive in a BSEP inhibition assay at concentrations up to 100 µM (unless limited by solubility). The SCHH were exposed to test article under standard conditions or bile acid sensitization conditions and mechanism was determined based upon the lactate dehydrogenase (LDH) leakage profile across the 2 media conditions. Concentration-dependent increases in LDH were observed in SCHH treated with BIO1, BIO2, and BIO3 under both media conditions, suggesting that these 3 test articles have the potential to cause hepatotoxicity through a general toxicity mechanism rather than impairing bile acid homeostasis. In contrast, LDH leakage was 13× greater under sensitization media versus standard conditions following BIO4 treatment, suggesting that BIO4 has the potential to disrupt bile acid homeostasis in hepatocytes leading to bile acid-dependent or cholestatic hepatotoxicity. The C-DILI assay results for general hepatotoxicity were consistent with rodent findings; however, based on this assay, only BIO4 would have the potential to cause cholestatic hepatotoxicity in humans.
P412: Historical Data: Histopathology Lesions Observed in the Eyes of Control Rabbits
N. E. Langevin1, K. A. Schafer2, O. C. Turner3, B. J. McPherson4, and R. E. Rose4
1Novartis, Fort Worth, TX, USA
2Vet Path Services, Inc, Mason, OH, USA
3Novartis Institutes for BioMedical Research, East Hannover, NJ, USA
4Alcon Research, Ltd, Fort Worth, TX, USA
Information on background changes in the ocular tissues of laboratory rabbits (Oryctolagus cuniculus), a common species employed in ophthalmic toxicology studies, is sparse. This complicates the interpretation of changes in the face of the small sample sizes on any single toxicology study. The purpose of this work is to document the interstudy incidence of spontaneous or iatrogenic changes occurring in the eyes of control rabbits. Photomicrographs of select lesions were captured. The data set covers an 11-year period and includes pigmented and albino New Zealand rabbits, untreated eyes and eyes subjected to treatment with innocuous control articles, and studies featuring topical ocular and contact lens routes of administration. Changes in the anterior and adnexal tissues were more common than in posterior tissues. Mononuclear cell infiltration was the most common finding. The retina was the posterior tissue most commonly observed with spontaneous changes. There were no noteworthy differences across the routes of administration considered. Retinal changes were more common in albino as compared to pigmented rabbits. Understanding the incidence of spontaneous histopathology lesions should be an aid in accurate data interpretation. Characterization of these findings via photography and use of standard terminology following International Harmonization of Nomenclature and Diagnostic guidance is meant to encourage consistent diagnosis and descriptions among toxicologists and pathologists.
P413: A 6-Month Subcutaneous Infusion Qualification Study in Beagle Dogs
E. Langlois-Forget1, R. Tavcar1, M. S. Maghezzi1, J. Haruna1, R. Forster2, and S. Authier1
1Citoxlab North America, Laval, Canada
2Citoxlab France, Évreux, France
The ability to continuously administer drugs subcutaneously is critical to attain appropriate exposure of some pharmaceuticals, which offer advantages over traditional administration routes and translate to the intended clinical route. The objective of this study was to assess the tolerance and background observations following continuous subcutaneous infusion of saline via a surgically implanted catheter in beagle dogs for up to 6 months. Five male and five female beagle dogs underwent surgical implantation of a medical grade subcutaneous catheter. The tip of the catheter was positioned in the lumbar region and the catheter was exteriorized at the dorsocervical region through a subcutaneous tunnel via a cath-in-cath/port system. Normal saline was continuously infused at a rate of 0.1 mL/h generally for 1 month (3/sex) or between 4 and 6 months (2/sex). Animals were evaluated for clinical signs, body weight, limited clinical pathology, and histopathology. The dorsolumbar area facilitated clinical evaluation of the dosing site and the infusion rate was achieved with dosing accountability values generally within acceptance criteria (±5%). The animals showed normal body weight gain and limited clinical pathology changes. Results obtained from this study provided sufficient evidence that the surgically implanted catheter was well tolerated and did not result in any unexpected background changes other than those normally observed with surgically implanted catheters. Specifically, a mixed cellular inflammatory reaction was observed surrounding the subcutaneous catheter infusion site. There was no difference in the inflammatory reaction following saline infusion over a period of 4, 5, or 6 months.
P414: Electrolyte Quantification in the Aqueous and Vitreous Humor of Common Preclinical Species
C. Li1, L. F. Negro Silva1, P. J. Seadi Pereira1, R. Forster2, S. Authier1, W. Tan3, M. Dubuc-Mageau3, and A. Sanfacon3
1Citoxlab North America, Laval, Quebec, Canada
2Citoxlab France, Évreux Cedex, France
3Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canada
The assessment of ocular health in support of standard preclinical ocular examinations has generated interest in ocular toxicology testing. Few studies have quantified the clinical chemistry contents of aqueous and vitreous humors across species. The goal of this study was to quantify the electrolyte composition of aqueous and vitreous humors across 4 standard preclinical species. Aqueous and vitreous humors were collected from rabbits (n = 10), dogs (n = 10), monkeys (n = 10), and minipigs (n = 7). Intraspecies age and body weight ranges were comparable. Vitreous and aqueous humor electrolyte content were analyzed by a Cobas 6,000 module c501 set to plasma/serum mode. Parameters are expressed in mmol/L. Electrolyte concentrations in both aqueous and vitreous humors were comparable between species. Electrolyte concentrations in vitreous humor were phosphorus (0.31 ± 0.07 to 0.33 ± 0.19), calcium (1.41 ± 0.18 to 2.34 ± 0.33), sodium (132.80 ± 16.52 to 157.00 ± 17.58), potassium (5.15 ± 0.57 to 5.30 ± 0.36), and chloride (99.40 ± 14.53 to 116.18 ± 5.48). Electrolyte concentrations in aqueous humor were phosphorus (0.72 ± 0.05 to 0.90 ± 0.08), calcium (1.25 ± 0.10 to 2.18 ± 0.04), sodium (142.00 ± 6.11 to 153.67 ± 4.58), potassium (3.86 ± 0.06 to 5.26 ± 0.99), and chloride (97.80 ± 4.07 to 116.40 ± 2.97). Vitreous and aqueous humor concentrations of sodium, potassium, and chloride were generally similar to species-specific serum levels; however, phosphorus and calcium concentrations were slightly below serum levels. This study provided a baseline/reference of vitreous and aqueous humor electrolytes. Electrolyte measurement from aqueous and vitreous humors could be a useful parameter in the assessment of ocular toxicity in toxicology studies. The electrolyte composition of these anatomical segments may also have implications relative to test compound chemistry following intraocular delivery.
P415: DNA Methylation Aberrant in Radon and Cigarette Smoke Exposure
J. Li1 and Y. Chen1
1Soochow University, Suzhou, China
It is well known that cigarette smoking (CS) and/or radon (Rn) induce malignant transformation in lung cells. To investigate the mechanisms underlying lung carcinogenesis induced by CS, Rn, or Rn followed by CS using BEAS-2B cell line derived from human bronchial epithelial cells. BEAS-2B cells were exposed to R (20,000 Bq/m3) for 30 minutes, CS (20%) for 10 minutes, or Rn followed by CS for 40 minutes. Global and gene-specific DNA methylation modifications were measured by microarray and methylation-specific polymerase chain reaction. Cell cycle and apoptosis were determined by flow cytometry, while soft agar colony formation was conducted to assess the characteristics of malignant transformation. Data demonstrated global hypomethylation as well as gene-specific DNA methylation alterations in all treatment groups compared to unexposed control cells. In addition, Rn and CS produced DNA hypermethylation of protein tyrosine phosphatase receptor type M (PTPRM) and ectodysplasin A2 receptor (EDA2R), 2 genes related to malignant transformation. In all treatment conditions, cell proliferation and survival of malignant cells were increased, while apoptosis was initially first passage elevated but decreased at passages 5 to 15. Our results indicate that aberrant DNA methylation plays an important role in Rn- and/or CS-induced malignant transformation. In addition, BEAS-2B cell line may be used as an in vitro model to investigate mechanisms underlying malignant transformation induced by ambient environmental contaminants.
P416: Effects of WSCM, eVCM, and Nicotine on Monocyte Adhesion to Human Aortic Endothelial Cells
O. Makwana1, G. Smith2, H. Flockton2, G. Watters2, F. Lowe3, D. Breheny3, and W. Fields1
1RAI Services Company, Winston-Salem, NC, USA
2Covance Laboratories Ltd, Harrogate, United Kingdom
3British American Tobacco, Southampton, United Kingdom
Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 human monocyte adhesion to human aortic endothelial cells (HAECs). Here, we evaluated the effects of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapor conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen), and nicotine (NIC). The HAEC monolayers were grown in microfluidic channels and exposed to 0 to 3,000 ng/mL nicotine or 0 to 3,000 ng/mL nicotine equivalence of WSCM or eVCM for 24 hours. Activated THP-1 cells were perfused through the channels and left to adhere under static conditions. The perfusion, adhesion period, and wash cycle were performed 4 times with increasing adhesion periods (10, 20, 30, and 40 minutes). THP-1 cell numbers were quantified by counting adherent cells. Both WSCM and eVCM induced dose-dependent increases in THP-1 cell adhesion of up to 3-fold and 2-fold, respectively, compared to control. Adhesion regulation was linked to increases in ICAM-1 protein expression. Colocalization staining of ICAM-1 in HAECs and cd11b (MAC-in THP-1 cells) demonstrated adhesion molecule interaction in BioFlux plates. We conclude that the BioFlux system is able to model human monocyte adhesion to human endothelial cells in vitro and that WSCM drove the greatest increases in both adhesion and endothelial expression of ICAM-1.
P418: Integrated Analysis of Transcriptomics Data With the AOP Framework for Adverse Outcome Identification and Risk Assessment of Chemicals: Piperonyl Butoxide Case Study
N. O. Oki1, L. Farcal2, A. Abdelaziz2, O. Florean2, F. Hollinger1, T. Exner2, P. Kohonen3, R. Grafström3, and B. Hardy2
1Douglas Connect GmbH, Research Triangle Park, NC, USA
2Douglas Connect GmbH, Basel, Switzerland
3Karolinska Institutet, Stockholm, Sweden
European Union regulations limiting the use of new animal testing in cosmetics has made the existing information more valuable. Scientists need to mine as much useful information as possible from the existing knowledge while integrating in vitro and in silico approaches to support the risk assessment of chemicals. We have built upon a “Safety Evaluation Ultimately Replacing Animal Testing” 1 project framework for deriving in vivo toxicity responses from in vitro data. Piperonyl butoxide was selected as a compound of interest for understanding the effects of an unknown compound on the liver and for the identification of knowledge and methodology gaps which may improve our understanding of these mechanisms. In the course of this investigation, we integrated biological pathway data, disease information, and human in vitro transcriptomics data collected from a carcinogenicity study. Combining pathway enrichment, association analysis, and other modeling approaches with adverse outcome pathway information (AOPs), we identified areas of concern to support an evidence-driven risk assessment of the compound. The resulting pathways, chemical analogs, and disease associations were then combined with key events from specific liver AOPs to generate an AOP network of liver-related end points. The results show evidence supporting a case for PBO as a liver toxicant. They also show that when applied to relevant AOPs, this workflow can be used to fill knowledge gaps in AOPs and for verification across specific AOPs. Upon further refinement, this approach could support chemical risk assessment and identify data gaps where additional testing may be needed.
P419: Lipidomics Analysis of Metabolic Dysregulation in Diet-Induced Obese B6C3F1 Mice
L. K. Schnackenberg1, E. E. Jones1, J. Sun1, R. D. Beger1, W. B. Mattes1, and V. Varma1
1National Center for Toxicological Research, US FDA, Jefferson, AR, USA
A survey of health conditions and risk factors by the Center for Disease Control reported that in 2013 to 2014, 37.9% of adults older than 20 years were considered obese. Obesity has the potential to impact drug pharmacokinetics and pharmacodynamics as well as cause pathophysiological changes that will also alter the absorption, distribution, metabolism, and excretion of a drug. In order to understand the impact of obesity on drug toxicity and efficacy, it is first necessary to evaluate the effects of obesity in the model system. A diet-induced obesity model has been developed using B6C3F1 mice. As part of the evaluation of the obese phenotype, lipids in the liver and kidney have been evaluated using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) and the Lipidyzer platform. Lipids represent a major class of biomolecules that play essential roles in human physiology. Furthermore, the ectopic accumulation of lipids in key tissues, such as the liver and kidney, that can occur under conditions of obesity can impair organ function. MALDI IMS provided the spatial distribution of lipids throughout the tissue slices, while the Lipidyzer platform allowed for the quantitation of lipids from 13 different classes. The altered lipid profiles of the different lipid classes and their potential differential distribution in obese versus lean mice may provide novel insights into the mechanisms underlying lipid metabolic dysregulation in obesity.
P420: Validation of the In Vitro Reconstructed Skin Micronucleus Assay as a Potential Animal Alternative for Fragrance Materials
Y. Thakkar1, K. Joshi1, and A. M. Api1
1Research Institute for Fragrance Materials Inc, Woodcliff Lake, NJ, USA
The in vitro reconstructed skin micronucleus assay is a skin-based assay that measures the clastogenicity and aneugenicity potential. Reconstructed skin models are prepared from primary human cells and are expected to have DNA repair and cell-cycle control mechanisms similar to in vivo systems. They are also expected to exhibit a human metabolic capability that is more biologically relevant than the exogenous rodent metabolizing enzymes currently used in standard in vitro genotoxicity assays. The skin is an effective barrier to chemicals and has enzymes actively involved in metabolism which may prohibit materials applied to the skin from entering systemic circulation or allow detoxification to occur prior to systemic circulation. The major route of exposure to fragrances is via the dermal route, making the in vitro skin test system of greater relevance as an animal alternative follow-up study to the regulatory approved in vitro micronucleus assay for fragrance materials. A set of 13 fragrance materials were selected. Materials selected produced adverse results in the traditional in vitro micronucleus assay; however, negative responses were observed in the in vivo micronucleus assay. All 13 materials resulted in negative results in the in vitro reconstructed skin micronucleus assay. There was 100% concordance when compared to the results in the in vivo micronucleus assay. These findings strengthen the use of the in vitro reconstructed skin micronucleus assay as an assay to evaluate the clastogenicity and aneugenicity and as a potential follow-up animal alternative tool to support the safety of fragrance materials.
P421: Prediction of Placental Transfer in Rats for Small Molecules
J. L. Valentine1, A. R. Kaczor1, R. P. Sheridan2, M. L. Green1, A. G. Aslamkhan1, B. G. Redfern1, S. K. Mukherjee1, S. Altman-Hamamdzic1, and F. D. Sistare1
1Merck & Co, Inc, West Point, PA, USA
2Merck & Co, Inc, Kenilworth, NJ, USA
In vitro developmental toxicity assays can be vital in early development efforts to distinguish teratogenic potential of drugs. The mouse transcriptional embryonic stem cell test and rat whole embryo culture assay are utilized to identify most probable rat teratogens. With an integrated model, the sensitivity and specificity for teratogens and/or embryo-lethal drugs is 89% and 54%, respectively. The low specificity presents a gap in model predictivity, which suggests that there are a high number of false positives in both assays. Fetal plasma concentrations derived from placental transfer studies were examined for these compounds and it was determined that some false positives may be due to limited distribution across the placenta ultimately impacting fetal exposure. Hence, the in vitro results did not recapitulate the in vivo outcome. Thus, it is important to consider likely fetal exposure in the interpretation of in vitro studies that assess reproductive toxicity. However, at the time when the in vitro assays are performed in early drug development, fetal exposure data do not generally exist in nonclinical species, and only nonpregnant female or male exposure data are available. To aid early exposure assessment, our historical database (1999-2017) was examined and demonstrated that (1) the toxicokinetics of ∼80% of small molecule compounds had concordant exposures (±2-fold threshold) regardless of pregnancy status in rats and rabbits and (2) that an in silico model based upon quantitative structural activity relationship methods well predicted placental transfer in rats for ∼30 compounds examined.
P422: Identifying Eye Irritants (GHS Category 2) Using Validated, Nonanimal Tests
B. J. Varsho1, B. Yasso1, M. Troese1, E. Delacruz1, and G. L. DeGeorge1
1MB Research Laboratories, Spinnerstown, PA, USA
Organisation for Economic Co-operation and Development (OECD) Test Guideline 405 prescribes a weight of evidence (WOE) analysis and sequential testing strategy for the classification of acute eye hazards, including “perform validated and accepted in vitro or ex vivo ocular test(s).” Four tests qualify: 3 designed to identify severe eye irritation/corrosion (GHS category 1) and 1 to identify nonirritants (GHS no category). GHS category 2 (eye irritant) classification is impossible using any single test. The EpiOcular eye irritation test (EIT; OECD 492) classifies a substance to be no category, or contrarily causes (uncategorizable) eye effects. Conversely, the Bovine Corneal Opacity and Permeability (BCOP) test (OECD 437) is used for ruling in or ruling out category 1 effects. Using a dual-assay/approach system—the combination of the EIT and BCOP tests—we have determined, with a high degree of accuracy, GHS acute eye hazard category 2 chemicals that cause reversible eye irritation. When a BCOP test rules out GHS category 1 and the EIT rules out GHS no category, analysis of these results indicates the only other possible designation—category 2. Per GHS, category 2 classification defaults to category 2A, because differentiation between categories 2A and 2B cannot be made. After testing 42 chemicals, we correctly identified 93% of the category 2A/2B chemicals as category 2. The potential of the BCOP EIT dual-assay system, coupled with WOE evaluation, to correctly classify substances into GHS category 1, category 2A, and no category is encouraging. We predict this testing strategy would greatly reduce reliance on Draize tests.
P423: Cerebrospinal Administration in the C57Bl6 Mouse Using the Intracerebroventricular Route
C. Voyer1, J. Douville1, F. Émond1, C. Foucault1, M. Halle1, and J-.F. Lafond1
1Charles River Laboratories, Senneville, Montreal, Canada
An increasing number of molecules targeted to address neurological diseases and deficiencies are in development. As these are often larger molecules, failure to reach their target due to low penetration of the blood–brain barrier and/or short half-life in circulation, targeted administration of these drugs directly into the central nervous system might be indicated. In addition to allowing central nervous system exposure, this also reduces systemic exposure, which, at times, may result in unwanted toxicities. Administration into the intrathecal space is more difficult than routine approaches regardless of species, but these challenges are heightened when attempting these in smaller species such as mice. The mouse presents unique challenges given its size and delicate anatomy, and as such there are technical limitations for intrathecal administration. In order to circumvent these difficulties, we established capabilities to conduct intracerebroventricular injection in mice (C57Bl6) in our laboratories. Anesthetized mice were restrained, and dosing was guided using stereotaxic table and predetermined coordinates to target the right ventricular area. During trials, a portion of mice were used for confirmation of successful injection. Ink was injected, and accuracy evaluated visually following euthanasia and extraction of the brain from the skull and performing a coronal cut. Once the surgical approach was well established, an additional subset of mice was kept for 3 weeks to assess any potential delayed adverse effects following the procedure. This subset was then euthanized, the brain was collected, and histopathological evaluation was performed in order to characterize accuracy of the injection and any procedure-related findings.
P425: The Needleless Injection Site Swabbable Connector (NISSC): A Practical and Reliable Tool That Permits Dogs to be Exercised on Continuous Intravenous Infusion Studies
A. Zakaryan1, J. Younan1, D. Bennamane1, and A. Bocan1
1ITR Laboratories Canada Inc, Baie-d’Urfé, Montreal, Canada
Allowing dogs assigned to subchronic and chronic toxicity studies to get out of their home cage and exercise a few times a week is a standard practice at ITR that provides enrichment and is well in line with the recommendations of most Institutional Animal Care Committees. However, historically dogs on continuous infusion studies could not be exercised since they were surgically implanted with a permanent intravenous catheter. ITR has managed to identify a suitable quick release connector that allows the dogs to be disconnected from the infusion system for exercise. We have recently completed a 4-week method development study using the needleless injection site swabbable connector (NISSC) in which dogs were surgically catheterized in the usual way but NISSC was used to hook them up to the infusion pumps. During the study, the dogs were disconnected from the pumps for 5×/week and allowed to exercise freely for 20 min/d before being reconnected to the pump. There were no abnormal clinical observations, macroscopic signs of infection at the femoral infusion site, or changes in body weights, food consumption, and behavior that were considered to be due to the use of the NISSC. There were no incidents during the 20-minute daily exercise, and no difficulties were encountered during the disconnection and reconnection of the infusion system prior to and after dog exercise. Therefore, the NISSC was considered to be a safe, viable, low-cost, and very practical tool in permitting dog exercise on continuous infusion studies.
500 Series—Safety Evaluation Pharmaceuticals
P501: Isoproterenol Cardiotoxicity: Relating Hemodynamic Effects to Cardiac Troponin Release and Microscopic Tissue Analysis
M. Abernathy1, D. Best1, E. Wang1, D. Leishman1, and A. Schultze1
1Lilly, Indianapolis, IN, USA
Isoproterenol generates stress on the myocardium producing lesions in multiple species. A definitive study has not been conducted evaluating the hemodynamic and microscopic tissue effects of isoproterenol. Isoproterenol was administered subcutaneously at 0, 0.003, 0.01, and 0.03 mg/kg to rats instrumented with hemodynamic telemetry transmitters as well as naive age-matched animals. Peak change from baseline and area under the curve (AUC) analysis was performed on the hemodynamic and cardiac troponin I (cTnI) data. Dose and time-related effects on heart rate were elicited with average peak increases from baseline of 185, 194, and 192 beats/min at 0.003, 0.01, and 0.03 mg/kg, respectively. Mean arterial pressure (MAP) decreases were observed with dose and time dependence reaching average peak decreases of 22, 26, and 36 mm Hg. The largest average cTnI change from baseline values and microscopic severity was observed in the 0.03 mg/kg dose group with increased cTnI concentrations correlating with greater pathology severity in the 0.003 and 0.01 mg/kg dose groups. Peak change analysis yielded poor correlations between heart rate and cTnI and/or MAP and cTnI. The AUC-based linear regression analysis demonstrated a hysteresis of effect where in early heart rate increases correlated with later cTnI concentrations for the 0.003 and 0.01 mg/kg groups with early MAP decreases correlating with later cTnI concentrations for 0.03 mg/kg. These data support that hemodynamic effects in rats correlate with cardiac histopathology when analyzed using an AUC-based analysis versus peak change.
P502: Effects of Common Confounders in Toxicology Studies on JTp and Tpe as Novel Proarrhythmia Biomarkers
S. Authier1,2, A. Menard1, M. Dubuc-Mageau2, A. Sanfacon2, W. Tan2, and E. Boulay1,2
1CiToxLAB, Laval, Quebec, Canada
2Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canada
QTc was identified as a central proarrhythmic risk biomarker in ICH S7A, but recent clinical data have revealed limitations when using QTc mostly related to false positives. Drugs with balanced multi-ion channel inhibition were shown to prolong QTc without increased proarrhythmic risks. Research led by the US Food and Drug Administration has identified JTp as an alternate electrocardiography (ECG) biomarker to evaluate drug-induced proarrhythmic risk in clinical trials and HESI showed similar effects in canines and nonhuman primates. In humans or nonclinical species, proarrhythmic drugs are associated with an increase in QT but also JTp. The current study evaluated the effects of common ECG confounders on QTca, JTpca, and Tpeca. Beagle dogs were anesthetized and subjected to progressive hyperthermia (42°C), hypothermia (33°C), vagal nerve pacing, or epinephrine intravenous injections (0.03 mg/kg). All ECG parameters (QTca, JTpca, and Tpeca) were subjected to individual rate correction. QTca (slope −9.2 msec/°C) and JTpca (−8.7 msec/°C) durations were negatively correlated with core body temperature, but Tpeca was minimally affected. Epinephrine was associated with QTca and JTpca shortening, which could be related to undercorrection in the presence of tachycardia but minimal effects on Tpeca. As expected, vagal pacing was associated not only with QTca but also with JTpca prolongation, but with minimal effects on Tpeca. These results highlight the importance of potential confounders not only on QTc but also on JTpca and Tpeca. These potential confounding effects need to be considered in the interpretation of ECG biomarkers during proarrhythmic risk assessment in nonclinical drug development.
P503: Current and Future Trends in Nonclinical Drug Safety Testing: An Industry Survey
W. J. Brock1, P. McGovern2, W. Halpern3, M. Pugsley4, D. Jones5, T. McGovern6, and S. Authier7
1Brock Scientific Consulting, Montgomery Village, MD, USA
2USDA, Washington, DC, USA
3Genentech, South San Francisco, CA, USA
4Consultant, Fairfield, CT, USA
5Medicines and Healthcare Products Regulatory Agency, London, United Kingdom
6US FDA, Silver Spring, MD, USA, 7CiToxLAB, Laval, Quebec, Canada
The growth in drug development has been extraordinary during the past 20+ years, reflecting significant advancements in the basic sciences and a greater understanding of molecular pathways of disease. Toxicologists, pathologists, pharmacologists, and other safety assessment scientists are continuously challenged to ensure safety study designs reflect current standards of scientific and regulatory practice. Benchmarking industry practices are important to enable critical reflection of pharmaceutical testing, and the outcome of past industry surveys has influenced the evolution of best practices applied to drug safety testing. A broad survey of 37 questions was provided to the full membership of the American College of Toxicology and the Societies of Safety Pharmacology and Toxicologic Pathology. From the 2,550 possible participants, the response rate was ∼24%. Although survey participation was global, the majority of respondents (∼75%) were from the United States and Europe. The survey encompassed topics encountered in nonclinical safety assessment, including use of biomarkers of inflammation, cardiac/vascular, renal, and hepatic toxicity; common practices used to assess endocrine perturbations; carcinogenicity and genotoxicity; and juvenile and male and female reproductive toxicity. The survey also investigated the impact of regulatory meetings on program design, application of the 3Rs, and reasons for program delays. Results highlighted common toxicities (hepatic, hematopoietic, and gastrointestinal) and identified histopathology findings that frequently impact the no observed adverse effect level. Overall, the survey results provide a broad perspective of current practices of nonclinical safety assessment based on current experience in the scientific community.
P504: Nonclinical Correlates to Drug-Induced Liver Injury Produced by IDH305, an Isocitrate Dehydrogenase-1 Inhibitor Investigated for the Treatment of Cancer
A. P. Brown1, T. Heimbach2, S. Spence1, and W. Kluwe1
1Novartis Institutes for Biomedical Research, Cambridge, MA, USA
2Novartis Institutes for Biomedical Research, East Hanover, NJ, USA
Mutations in isocitrate dehydrogenase (isoforms IDH1 and IDH2) can result in altered catalytic function and aberrant production of the oncometabolite 2-hydroxyglutarate, which can result in cellular transformation and neoplasia. IDH305 is a highly selective allosteric inhibitor of the mutant IDH1R132 enzyme and studied in a phase 1 trial in patients with advanced malignancies. Hyperbilirubinemia and elevated aminotransaminases were observed and considered drug-related hepatic adverse events. In a 4-week oral toxicity study in dogs, increases in serum total bilirubin (conjugated and unconjugated), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyltransferase (GGT) occurred at ≥30 mg/kg, with total bile acids increased at 60 mg/kg. These serum chemistry changes were generally reversible. Increased ALT/AST values correlated with hepatocyte degeneration/necrosis, whereas increased bilirubin, bile acids, ALP, and GGT correlated with bile duct hyperplasia and bile stasis. Hepatic fibrosis, chronic-active inflammation, and biliary hyperplasia were seen in one female at 60 mg/kg in the recovery group. In vitro studies demonstrated that IDH305 inhibits proteins involved with biliary function. IDH305 inhibited the human bile salt export protein with an IC50 of 9.1 µM, uridine diphosphate glucuronyltransferase (UGT1A1) with IC50 of 0.13 µM, and organic anion transport proteins OATP1B1 and OATP1B3 with IC50 values of 1.7 and 2.4 µM, respectively. IDH305 undergoes hepatic metabolism and is a time-dependent inhibitor of CYP3A4/5. In conclusion, the liver and biliary systems are targets for IDH305 toxicity and the nonclinical data provided correlates with adverse events observed clinically.
P505: Intranasal Nilotinib in the Thy1-aSyn Mouse Model of Parkinson Disease
J. M. Brown1, S. A. Moore1, K. B. Seroogy1, and M. B. Genter1
1University of Cincinnati, Cincinnati, OH, USA
Parkinson disease (PD) is a common, debilitating neurodegenerative disease. Early symptoms include motor dysfunction and decreased olfaction. Aggregation of α-synuclein (αSyn) in the brain is a hallmark of PD and correlates with motor and nonmotor impairments in patients. Thy1-αSyn transgenic mice exhibit these and other features of PD observed in humans, making this a suitable model for studying drug intervention in PD. Nilotinib is US Food and Drug Administration approved to treat chronic myelogenous leukemia and a phase 1 clinical trial showed oral treatment improved symptoms in patients with PD, but serious side effects were reported. With the goal of decreasing drug-induced systemic toxicity and delivering nilotinib directly to the brain, we are investigating novel use of the intranasal route of administration for nilotinib in the Thy1-αSyn model of PD. After baseline neurobehavioral assessments, wild-type and Thy1-αSyn mice received vehicle or 0.5 or 0.25 mg/d nilotinib treatment (N = 18-26). After 8 weeks of dosing, behavioral assessments were repeated; brains and nasal cavities were collected for histology, αSyn immunohistochemistry, and stereological analysis. In the motor test, Thy1-αSyn mice treated with 0.25 mg/d nilotinib did not show increased errors/step after treatment compared with the vehicle and 0.5 mg/d dose (P values ≤0.002). For olfactory behavior, Thy1-αSyn mice given 0.5 mg/d nilotinib had increased latency to retrieve the buried pellet (P-value = 0.004). These data suggest in the Thy1-αSyn mouse model of PD, progressive motor dysfunction was prevented with 0.25 mg/d intranasal nilotinib, while 0.5 mg/d resulted in olfactory toxicity.
P506: CTP 692: Selective Deuterium Modification of d -Serine Markedly Decreases Renal Toxicity in Preclinical Testing
C. L. Brummel1, S. Vedananda1, D. Doller1, D. Wong1, R. Gallegos1, J. F. Liu1, S. Nguyen1, N. Uddin1, R. Tung1, and J. V. Cassella1
1Concert Pharmaceuticals, Lexington, MA, USA
CTP 692, a deuterated version of the naturally occurring amino acid
P507: Comparison of Subretinal Injection of iPSC-Derived RPE Cell Suspension or Subretinal Implantation of iPSC-Derived RPE on PLGA Scaffold in RNU Nude Rats
E. Budzynski1, P. Sonnentag1, M. T. Nork2,3, P. E. Miller2,3, S. D. Sorden1, C. A. Rasmussen1,2, K. E. Sampson1, A. Maminishkis4, V. Khristov4, B. S. Jha4, and K. Bharti4
1Covance Laboratories Inc, Madison, WI, USA
2University of Wisconsin, Madison, WI, USA
3OSOD LLC, Madison, WI, USA
4National Institute of Health, National Eye Institute, Bethesda, MD, USA
Dysfunction of retinal pigment epithelial (RPE) cells is a key factor in development of age-related macular degeneration. An induced pluripotent stem cell (iPSC)-derived RPE implant cultured on a biocompatible poly (lactic-co-glycolic acid or PLGA) scaffold was developed as an RPE replacement therapy. This study evaluated 9-month toxicity and tumorigenicity of iPSC-derived RPE when delivered into the subretinal space of RNU nude rats as a cell and scaffold suspension, equivalent to anticipated clinical dose, or as a cell implant on PLGA scaffold at a dose appropriately scaled for the rat eye. Suspension or implant was associated with white subretinal infiltrates on ophthalmic examinations correlating with hyper-reflective material (HRM) on optical coherence tomography (OCT) and microscopically RPE hyperplasia/choroidal fibrosis. Human cell marker immunolabeling in foci of RPE hyperplasia/choroidal fibrosis confirmed the presence of iPSC-derived RPE in eyes up to 9 months after dosing. In some eyes, transplanted cells integrated within the host RPE. In addition, suspension was associated with HRM in vitreous on OCT, correlating with pigmented cells (sometimes positive for human cell markers) in the vitreous. Outer retinal tubulations were present on OCT and correlated microscopically with the presence of pigmented cell rosettes centered around an amorphous eosinophilic or mineralized material. Suspension was associated with the presence of cells in the vitreous, while cells delivered on implants were generally localized to the subretinal space or host RPE only.
P508: Derivation of a PEL for Antibody-Conjugated Iron Dextran Nanoparticles
J. M. Cohen1, I. Mohar2, and T. A. Lewandowski2
1Gradient, Cambridge, MA, USA
2Gradient, Seattle, WA, USA
Antibody-conjugated iron dextran nanoparticles (antibody-IDNPs) are commonly used in research to efficiently enrich or deplete specific cell populations. In the context of cell-based therapies, antibody-IDNPs have been used to select specific cell populations to generate personalized treatments. Following manufacturing, low levels of these nanoparticles may be present as residual manufacturing impurities. The purpose of this assessment was to identify the potential hazards associated with anti-CD8 antibody-conjugated IDNPs (anti-CD8 IDNPs) being introduced into the patient and to propose a safe exposure level for this potential process-related impurity. In the absence of comprehensive toxicology data for anti-CD8 IDNPs, this assessment was supported by publicly available data for a similar antibody-IDNP approved for clinical use and individual components of the anti-CD8 IDNP. Based on a review of available information for anti-CD8 IDNPs and supporting data, we identified no evidence suggesting human exposure to residual anti-CD8 IDNPs would pose significant acute or chronic toxicity hazard. Immunogenicity against the conjugated mouse anti-human CD8 antibody is identified as the end point that may limit repeated exposure. Five possible approaches to deriving a permissible exposure limit are proposed based on human data with anti-CD34 IDNPs, human data with monoclonal mouse anti-CD3 antibody, human data with humanized anti-CD4 antibody, rodent data with unconjugated IDNPs, and nonhuman primate data with monoclonal mouse anti-CD8 antibody. The collective data indicate that limited exposure to residual anti-CD8 IDNPs is unlikely to pose a significant risk to a patient.
P509: Evaluation of PEG (Polyethylene Glycol) Accumulation and Vacuolation in Nonclinical Chronic Toxicology Studies of Pegvaliase (PEGylated Recombinant Phenylalanine Ammonia Lyase)
C. R. Corado1, T. Oppeneer1, A. Gershman1, S. Chandra1, M. J. Tomlinson2, C. O’Neill1, and L. Sullivan1
1BioMarin, Novato, CA, USA
2Nova Pathology, Bellingham, WA, USA
Intracytoplasmic vacuoles occur in multiple tissues in animals following repeated administration of PEG-conjugated molecules. Pegvaliase is a bacterial enzyme, phenylalanine ammonia lyase, which has been pegylated to reduce immunogenicity and increase the half-life of the enzyme. Pegvaliase is an enzyme substitution therapy intended for the reduction of blood phenylalanine levels in adults with phenylketonuria. We conducted a 26-week toxicity study in rats with a 17-week interim euthanasia. Additionally, we conducted a 36-week toxicity study in cynomolgus monkeys with a 13-week recovery. Animals in both studies were dosed twice weekly with control article or pegvaliase, by subcutaneous injection. No vacuoles were observed in any tissues in cynomolgus monkeys. Dose-dependent cytoplasmic vacuoles (predominantly in the tissue macrophages) were detected in the rat in adrenal gland, spleen, testis, liver, lymph nodes, and kidney (renal tubular epithelium), and no vacuoles were observed in the choroid plexus or the ependymal cells. PEG accumulation in rat tissues was semiquantitatively evaluated by immunohistochemistry using an anti-PEG antibody. Staining intensity was similar at 17 and 26 weeks, indicating that PEG accumulation reached steady state. There was complete reversibility of PEG-associated vacuoles during the 12-week recovery period in all tissues except the renal tubular epithelium. Vacuolation was not associated with adverse effects on organ function in any tissue and represents a normal elimination pathway for PEG. Clinical and laboratory monitoring in the pegvaliase clinical program has not revealed any evidence of PEG-related target organ damage.
P510: Early Characterization of Hematopoietic Toxicity Utilizing a Multilineage Differentiation Assay to Identify Potential Dose-Limiting Toxicities and Minimize Risk for Drug Attrition in Clinical Development
N. Corr1 and A. M. Fullerton1
1Genentech, South San Francisco, CA, USA
During drug discovery, high-throughput in vitro models are used to establish candidate profiles before progressing in vivo where it becomes prohibitive to explore more than a handful of lead molecules. Often, shortcomings in the in vitro screening paradigm are only brought to light in clinical trials and may result in costly late-stage attrition. In particular, oncology drug candidates have historically proven susceptible to poorly characterized hematopoietic toxicity that manifests in clinical trials as dose-limiting neutropenia, thrombocytopenia, and anemia. To better characterize these liabilities in the preclinical space, we describe a model utilizing simultaneous differentiation of CD34+ human stem cells into multiple hematopoietic lineages. This in vitro assay is well suited to first-tier screening of drug candidates for impact on multilineage differentiation using a novel 96-well format 14-color-flow cytometry-based methodology. In addition, the multilineage nature of this approach facilitates second-tier assessments such as the evaluation of target expression and mechanism of toxicity to further inform on differential sensitivities of specific lineages. Using this approach, we report the hematopoietic toxicity profiles of a variety of oncology drug classes, including epigenetic remodelers, cyclin-dependent kinases, and apoptosis regulators, and show clear concordance with the observed hematologic findings in clinical trials. These results demonstrate utility of this assay to quickly and thoroughly characterize hematopoietic liability in early discovery and lead optimization stages of drug development, providing a valuable tool to explore mechanism of action, inform on patient safety, and limit late-stage attrition due to dose-limiting hematopoietic toxicity.
P511: Cytokine-Mediated Suppression of CYP Enzymes by the Toll-Like Receptor 9 Agonist, IMO-2125, in Cultured Human Hepatocytes
P. Tarantino1, T. Sullivan1, B. Ogilvie2, and M. Czerwinski2
1Idera Pharmaceuticals, Cambridge, MA, USA
2Sekisui XenoTech LLC, Kansas City, KS, USA
The cytokine response to some drugs may suppress xenobiotic metabolizing CYP enzymes and affect the pharmacokinetics of coadministered small molecules. In this study, the potential of IMO 2125 for direct and cytokine-mediated effects on CYP activity and messenger RNA (mRNA) expression was investigated. Human blood was exposed to saline, IMO-2125 (10 or 100 µg/mL), or lipopolysaccharides (LPS) for 24 hours, followed by isolation of plasma and measurement of a panel of cytokines. Treatment of blood with LPS increased levels of pro-inflammatory cytokines, and treatment of whole blood with IMO-2125 caused >2-fold increases in IP-10, MIP-1α, tumor necrosis factor-α, and interferon (IFN)-α2a. Cultured hepatocytes from 3 donors were treated for 3 days with vehicle controls (dimethyl sulfoxide, saline), IMO-2125 (10, 30, or 100 µg/mL), omeprazole, phenobarbital, rifampin, or interleukin-6. Treatment of cultured hepatocytes with up to 100 µg/mL IMO-2125 had minimal effects on CYP1A2, CYP2B6, and CYP3A4(/5) activity and mRNA levels. Hepatocytes were also treated with pooled plasma (40%, vol/vol) from blood treated with saline, IMO-2125, LPS, or untreated control. Treatment of hepatocytes with plasma from IMO-2125-treated blood reduced CYP2B6 enzyme activity by up to 87% and mRNA level by up to 37%, as compared to saline-treated control. We observed that IMO-2125 caused release of cytokines unaffected by LPS, namely, MCP-1, MIP-1α, and INF-α2a. The results of this study indicate that cytokines other that those typically associated with inflammation may modulate CYP activity and mRNA expression.
P512: Metabolite Profiling and Identification in Dog Plasma and Tissues of Edasalonexent (CAT-1004), a Conjugate of Docosahexaenoic Acid and Salicylic Acid
R. Dagher1, M. Fonsi2, C-.A. Erratico2, H. Liu1, R. Forster2, and A. Nichols1
1Catabasis Pharmaceuticals, Inc, Cambridge, MA, USA
2Citoxlab, Évreux, France
Edasalonexent (CAT-1004) is an orally administered small molecule candidate drug that inhibits nuclear factor-κB, a transcription factor that drives inflammation, fibrosis, and muscle degeneration and is activated in Duchenne muscular dystrophy (DMD). Edasalonexent is currently in clinical development in 4- to 7-year-old boys with DMD, regardless of mutation. The present study was conducted to identify edasalonexent metabolites formed in beagle dogs in vivo and to determine their presence in the plasma, heart, liver, and skeletal muscle using liquid chromatography–high-resolution mass spectrometry. Beagle dogs were treated with 1,000 mg/kg/d for 39 consecutive weeks (this treatment was well tolerated, and no treatment-related signs of toxicity were recorded). Edasalonexent was biotransformed into 13 (males) and 10 (females) metabolites. Biotransformation observed included phase I (oxidation and hydrolysis) and phase II (mainly glucuro-conjugation) pathways with large overlaps between the 2 genders. The liver showed the presence of the highest number and levels of edasalonexent metabolites. In the heart, liver, and skeletal muscle of both male and female dogs, DHA represented between 80% and 99% of the total drug-related peak area. Consistent with the known metabolism of unsaturated fatty acids, oxidative metabolites in the DHA moiety were also identified. These results suggest that after administration of edasalonexent to beagle dogs, DHA is preferentially retained by heart and skeletal muscle, 2 affected tissues in DMD disease, more than the parent compound and/or any other metabolite detected in the same tissues. The DHA was also one of the major circulating metabolites observed in dogs.
P513: A 14-Day Intravenous Infusion Toxicity and Toxicokinetic Study of DUR-928, a Novel, First-in-Class, Investigational Therapeutic in Sprague Dawley Rats
L. R. DePass1, A. Miksztal1, E. Bui1, H. Wu1, C. Gordon2, and W. Q. Lin1
1Durect Corporation, Cupertino, CA, USA
2CiToxLAB, Laval, Quebec, Canada
DUR-928 is an endogenous sulfated oxysterol that has been demonstrated to play a key regulatory role in mammalian lipid metabolism, inflammatory responses, and cell survival. DUR-928 was administered as a daily 2-hour intravenous infusion at doses of 15, 50, and 150 mg/kg/d to 12 rats/sex/dose groups. Controls received the aqueous vehicle containing hydroxypropyl-β-cyclodextrin and sodium phosphate buffer. At the onset of dosing, the animals were 8 to 9 weeks old. DUR-928 was well tolerated, and there were no drug-related deaths during the course of the study. There was a slight drug-related reduction in body weight gain in males at the high dose. Vehicle-related, nonadverse, microscopic changes consisted of renal tubular vacuolation and pulmonary histiocytosis. DUR-928 was quickly eliminated from the plasma with half-lives ranging from 0.5 to 1.6 hours. On days 1 and 13, the systemic exposure to DUR-928 generally followed nonlinear kinetics (not dose proportional) over the entire dose range, suggesting possible saturable drug elimination processes. Predose (24-hour) plasma samples were free of DUR-928, consistent with a short half-life. AUClast and Cmax were generally similar on day 13 when compared to day 1, indicating the absence of plasma accumulation. In summary, DUR-928 was considered well tolerated. Based on the above results, the no observed adverse effect level (NOAEL) was the high dose of 150 mg/kg/d. At the NOAEL, the mean Cmax was 23,150 ng/mL and the mean AUClast was 58,225 h·ng/mL, averaged over both sexes and time intervals.
P514: Assessing the Cardiac, Thrombopoietic, and Neuronal Safety Liabilities of a Selective Immunoproteasome Inhibitor
E. Dere1, A. M. Fullerton1, J. Heidmann1, L. Yu1, E. Ladi1, C. Everett1, C. Eidenschenk1, P. Katavolos1, R. Pappu1, and K. Rajapaksa1
1Genentech, South San Francisco, CA, USA
The proteasome is the central proteolytic machinery of the ubiquitin proteasome system that is integral in clearing misfolded proteins and maintaining endoplasmic reticulum homeostasis. The immunoproteasome is derived from the constitutive proteasome and is abundantly expressed in immune cells. Bortezomib is a proteasome inhibitor approved for the treatment of multiple myeloma that has accompanied safety liabilities, including peripheral neuropathy, cardiovascular toxicities, and thrombocytopenia. These concerns are likely due to bortezomib’s indiscriminate inhibition of both the constitutive and immuno-specific β5 subunits (β5c and β5i, respectively) with comparable affinities. In this study, we investigated whether bortezomib-associated toxicities could be avoided by specifically inhibiting the immunoproteasome. Using compound X that possesses 3× greater selectivity for β5i over β5c (based on enzymatic activity), its toxicity in in vitro neurite outgrowth, cardiomyocyte, colony-forming unit-megakaryocyte, and platelet generation assays were assessed and compared to the responses elicited by bortezomib. The small selectivity increases in compound X provided a 10-fold improvement in the safety margins for thrombopoietic and neuronal toxicities. However, assays using human-induced pluripotent stem cell-derived cardiomyocytes found only minimal improvements in the safety margins for cardiovascular-related toxicities. These results indicate that this pathway should be further explored to understand if the safety liabilities can be mitigated with a selective immunoproteasome inhibitor.
P515: Chronic Intrathecal Administration in the Sprague Dawley Rat
J. Douville1, F. Émond1, C. Foucault1, and M. Halle1
1Charles River Laboratories, Montreal, Canada
In the context of a single patient IND application, we designed a study consisting in 2 doses administered 1 week apart in the intrathecal space of Sprague Dawley rats with takedowns at different time points to evaluate potential toxicity and safe starting dose of the test formulation. Following delayed onset of clinical signs during the observation period following second dose, regulators (US Food and Drug Administration) requested additional doses be given to remaining animals. Herein, we present the approach that allowed us to successfully give 7 intrathecal injections over a period of 144 days. Animals had a catheter implanted at the lumbar level. Once secured, the formulation was injected, and the catheter was heat-sealed using a hot clamp. The catheter was left in place and the skin was sutured closed. For each subsequent dose administration, the animals were anesthetized and the skin over the lumbar area was reopened. The catheter was retrieved, the previously sealed tip was cut off, and the dose was administered. The catheter was heat-sealed following dosing, and skin sutured. Doses were administered in this manner on days 1, 8, 71, 85, 99, 113, and 127. Technical issues were encountered in less than 10% of the animals. Animals were euthanized on day 144 based on toxicity indications as opposed to technical limitations. The procedure caused no clinical signs or effects on body weight, food intake, or neurological evaluation; only minimal to mild inflammation, compression, and/or nerve fibers degeneration were noted at the intrathecal injection site and lumbar spinal cord.
P516: Historical Immunophenotyping Data in Preclinical Species
C. Dumont1, G. Desilets1, and M-. S. Piche1
1Charles River Laboratories, Senneville, Quebec, Canada
Flow cytometry allows multiparametric analysis of thousands of particles per second and helps to adequately identify or functionally characterize complex cell populations of interest. It is often used in basic research, discovery, preclinical, and clinical trials. During the preclinical phase development of new drugs, flow cytometry has routinely been used for assessing the immunotoxic effects of a candidate drug by evaluating the immunophenotype of various cell populations in whole blood, tissues, or other matrices. Despite the fact that it is being used by multiple different laboratories, combined database presentation and comparison of preclinical immunophenotyping data in various animals, strains, and tissues/whole blood has been limited. The objective of this study was to compile relevant preclinical immunophenotyping data from monkeys (cynomolgus, rhesus), dogs (beagle), rats (Sprague Dawley, Wistar, Fisher), and mice (various strains) from different matrices. The database from a single site was extracted from control data generated from over 15 years of work. Specie, strain, age, cell populations, and sample types were grouped to provide a representation of expected cell population distribution profiles.
P517: Comparison of FGF Receptor 1 Klotho β-Dependent Agonist in CD1 and C57BL/6J DIO Mice
M. Felx1, A. Varela1, M. Guillot1, S. Rajan2, M. Schutten2, A. Adedeji2, D. W. Stevens2, and T. Gelzleichter2
1Charles River Laboratories, Montreal ULC, Senneville, Canada
2Genentech, Inc, South San Francisco, CA, USA
BFKB8488A is a bispecific antibody designed to mimic the metabolic hormone FGF21 through a klotho β-dependent activation of FGF receptor 1, being developed to treat metabolic disorders. BFKB8488A was administered weekly to 24-week-old CD1 mice and to C57BL/6J DIO (diet-induced obese) mice, a model of prediabetic type II diabetes and obesity. After 13 weeks, marked increases in food (up to 77%) and water consumption (up to 152%) were noted for CD1 mice, with minimal effect on body weight, consistent with a moderate decrease in fat mass. Clinical chemistry changes were limited to metabolic findings. Decreases in whole body area, bone mineral content (BMC), and bone mineral density were also observed, with no effect on bone length and/or bone width. Although similar increases in food and water consumption were noted for DIO mice, body weights of these mice were significantly decreased (down to 33%), mainly due to a loss of fat mass. Similar metabolic findings as those observed in CD1 mice were noted. DIO mice presented decreases in whole body area and BMC, with no changes in bone size. At terminal necropsy, liver weight changes, and microscopic findings in the adipose tissue, liver, kidney, and pancreas in DIO mice were considered secondary to pharmacologic effects of BFKB8488A. These data demonstrated that BFKB8488A treatment induced metabolic pharmacological effects with a greater amplitude in DIO mice. These results support the potential use of BFKB8488A as a new treatment for metabolic disorders.
P518: Further Development of TargetTri: A Toxicity-Based Triaging System for Novel Drug Targets
S. Folkertsma1, J. Venhorst1, E. van Someren1, F. van de Brug1, and G. Kalkman1
1TNO, Zeist, the Netherlands
TNO is developing TargetTri, a web-based system to aid in the triaging of novel drug targets. TargetTri can extract and visualize data on the fly for any of the 20k reviewed human proteins deposited in Uniprot, allowing highly efficient toxicological assessments and triaging of drug targets. TargetTri extracts information from (1) relevant databases (covering, eg, protein, compound, and pathway information); (2) in-house curated data on known toxicities of proteins; and (3) text-based resources using TNO’s text-mining tool ERIS. A customized ontology has been developed in-house to structure results from the latter based on toxicity and target organ. To allow intuitive and efficient analysis of the available data, various web views have been developed, including an interactive network view that links targets and their immediate biological network to diseases and toxicities, and a powerful heat-map view that shows the toxicity effects per organ. Other views display summary data, ligand information, and clinical trial data. Last year, we presented the prototype of TargetTri (validated with the data-rich target HSP90), which is currently further developed in a consortium of TNO and 3 pharmaceutical companies. Here, we present the progress, mainly based on comparison of TargetTri and in-house pharma safety assessments, made on (1) improvements of the text mining algorithm and the development of a dynamic ontology and (2) the addition of 2 new modules: risk mitigation (to de-risk identified safety liabilities) and off-target safety (based on ligand-binding profiles). This research is supported by the Dutch Ministry of Economic Affairs.
P519: Toxicity of Low Environmentally Relevant Concentrations of Cytostatic Drug 5-Fluorouracil in Cultured Human Cells and Zebrafish (Danio rerio)
G. Gajski1, M. Geric1, B. Zegura2, M. Novak2, J. Nunic2, D. Bajrektarevic2, R. Kovacs3, Z. Csenki3, B. Urbanyi3, A. Horvath3, N. Negreira4, M. Lopez de Alda4, D. Barcelo4, E. Heath5, T. Kosjek5, I. Zajc2, S. Baebler2, A. Rotter2, Z. Ramsak2, M. Filipic2, and V. Garaj-Vrhovac1
1Institute for Medical Research and Occupational Health, Zagreb, Croatia
2National Institute of Biology, Ljubljana, Slovenia
3Szent Istvan University, Godollo, Hungary
4Institute of Environmental Assessment and Water Research, Barcelona, Spain
5Jozef Stefan Institute, Ljubljana, Slovenia
We conducted a comparative in vitro toxicological characterization of 5-fluorouracil (5-FU) toward human lymphocytes (HPBLs), human hepatoma (HepG2) cells, zebrafish liver (ZFL) cells, and zebrafish (Danio rerio) in vivo using a 2-generation toxicity study. The 5-FU induced time- and dose-dependent decreases in cell viability in all 3 cell models. The sensitivity of ZFL and HepG2 cells toward the cytotoxicity of 5-FU was comparable while HPBLs were the least sensitive. Genotoxicity was determined by the comet and micronucleus assays. The ZFL cells were the most sensitive, and HPBLs were the least sensitive. The higher sensitivity of ZFL cells to 5-FU raises the question of the impact of such compound in aquatic ecosystem. Hence, for in vivo assessment, zebrafish exposure to 5-FU (0.01, 1.0, and 100 μg/L) was initiated with adult zebrafish (F0) and continued through the F1 and F2 generations. The exposure did not affect survival, growth, and reproduction of the zebrafish; however, histopathological changes were observed in the liver and kidney, along with genotoxic effects, at all 5-FU concentrations. Whole-genome transcriptomic analysis of liver samples of F1 generation zebrafish revealed dose-dependent increases in the number of differentially expressed genes. Although this chronic exposure did not affect the reproduction of the exposed zebrafish, it cannot be excluded that 5-FU can lead to degenerative changes, including cancers. Overall, the obtained data contribute to a better understanding of the potential adverse effects of cytostatic drugs in both humans and aquatic animals.
P520: PfCSP Self-Assembling Protein Nanoparticle (SAPN) Malaria Vaccine in Combination With Army Liposomal Adjuvant Containing Monophosphoryl Lipid A, QS21 (ALFQ), ALFQ, and Alhydrogel (ALFQA): Local and Systemic Toxicity Study Following 4 Intramuscular Injections to Male and Female New Zealand White Rabbits With a 28-Day Recovery Period
S. Godin1, D. Lanar2, and G. Matyas2
1Smithers Avanza, Gaithersburg, MD, USA
2Walter Reed Army Institute of Research, Silver Spring, MD, USA
Malaria causes serious illness and deaths every year, but an effective vaccine is not available. The purpose of this study was to evaluate the potential toxicity of a vaccine directed against the circumsporozoite surface protein of Plasmodium falciparum (PfCSP) formulated with adjuvants ALFQ and ALFQA in rabbits. Treatment had no effect on mortality, physical examinations, cageside observations, dermal Draize observations, body weights, body weight changes, food consumption, body temperatures, ocular examinations, gross pathology findings, and absolute and relative organ weights. All rabbits receiving the vaccine in combination with the adjuvants developed antibody titers to 2 specific vaccine antigens (NANP repeat and Pf16 C-term peptides). Alterations in clinical pathology consisted of increased serum globulin, increased neutrophil count, increased fibrinogen, and increased C-reactive protein, which were expected responses to immune stimulation and/or inflammation at the injection site(s) and most resolved during recovery. Microscopic findings consisted of minimal to moderate subcutaneous and/or skeletal muscle infiltration of inflammatory cells and minimal subcutaneous and/or skeletal muscle hemorrhage at injection sites which mostly resolved during recovery. Minimal or mild lymphoid hyperplasia occurred in iliac lymph nodes. These changes were expected responses to immune stimulation and/or injection trauma and had no deleterious effect on the overall health of the animals; therefore, all microscopic findings were considered nonadverse. In conclusion, the vaccine when formulated with ALFQ or ALFQA was well tolerated. The vaccine was immunogenic, and all observations were considered nonadverse and were consistent with expected inflammatory response and immune stimulation following vaccine administration.
P523: Real-Time Monitoring of Human Primary Cell Panel for Evaluating On/Off-Target Toxicity of Bispecific T-Cell Engagers
C. Jin1 and Y. Abassi1
1ACEA Biosciences, Inc, San Diego, CA, USA
Cancer immunotherapy empowers the patients’ own immune system to specifically recognize and destroy cancerous cells. Though designed to specifically kill cancer cells, side effects such as cytokine storms and on/off-target toxicity toward normal tissue/organs remain as considerable safety concerns that can be difficult to predict using animal models. Bispecific T-cell engagers (BiTEs) are engineered antibodies where the single-chain heavy fragments (scFvs) of 2 monoclonal antibodies are artificially fused together. One scFv is specific for target antigen expressed on cancer cells and the opposing scFv targets the CD3 receptor on T cells. These BiTE antibodies serve to facilitate the interaction of the immune system with the tumor cells. In order to explore potential on/off-target immune-mediated toxicity of BiTE antibodies, a panel of human primary cells representing different tissues were exposed to EpCAM-CD3 BiTE in the presence of peripheral blood mononuclear cells (PBMCs) and monitored in real time using the xCELLigence RTCA system. Our data show that EpCAM BiTE did mediate PBMC-mediated cytotoxicity of different cell types in a manner that correlated with the expression level of EpCAM. Importantly, an irrelevant BiTE (CD19) whose target is not expressed in the primary cells did not induce significant PBMC-mediated cytotoxicity. The extent of PBMC toxicity mediated through EpCAM-CD3 varied among different media conditions. Overall, our data suggest in vitro RTCA assays utilizing human primary cells can be used to evaluate the on/off-target toxicity of reagents used for cancer immunotherapy.
P524: Evaluation of Primary Hemostasis Using Bleeding Time Tests: Anatomical Sites and Species Comparison
C. Gross1, R. Kubaszky1, C. Li1, R. Tavcar1, and S. Authier1,2
1CiToxLAB, Laval, Quebec, Canada
2Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canada
Drug effects on hemostasis may represent intended pharmacology or can be an observed adverse effect. Bleeding time tests (BTT) are used for diagnosis in patients, but also to assess platelet function in nonclinical drug safety assessments. The goal of the study was to assess and compare anatomical sites for BTT in species commonly used for nonclinical drug development. Beagle dogs, cynomolgus monkeys, New Zealand white rabbits, and Sprague Dawley rats were used to assess BTTs at several anatomical regions (ie, gums, cranium, upper lip, pinna, inner cheek). A single use Surgicutt bleeding time device for consistency in incision length and depth (1 mm depth by 5 mm length) was employed. Cranium presented low interoccasion variability in all species, most likely due to cutaneous attachment to the cranial structures that enables stable application of Surgicutt devices. Gums and pinna (evaluated in dogs, monkeys, and rabbits only) presented intermediate variability. Lips showed greater variability, possibly due to differences in tissue immobilization and/or saliva levels. Between species, monkey lips (lower lip 10.9 minutes and upper lip 7.5 minutes) presented the highest interoccasion variability, but cranium (5.4 minutes), pinna (5.4 minutes), and inner cheek (4.7 minutes) showed values within expected ranges. These results support the use of BTT assessments in duplicate or triplicate at each time point to increase sensitivity of this assay in the context of toxicology assessments.
P525: Assessing the Predictive Ability of an In Silico Tool for Developmental Toxicity
S. Pacheco Shubin1 and T. A. Lewandowski1
1Gradient, Seattle, WA, USA
We conduct risk assessments to establish workplace exposure limits for developmental and reproductive toxicants (DART). Not all chemicals that may be present in the pharmaceutical workspace (eg, new drugs, process chemicals, impurities) may have data to determine their DART potential. In silico tools may be one way to fill in this data gap in cases where data are lacking. We evaluated the predictive ability of one in silico tool, the US Environmental Protection Agency’s Toxicity Estimation Software Tool (TEST) for 301 chemicals we have previously investigated for DART potential using in vivo data (primarily guideline animal studies). Our analysis excluded chemicals reported to be primarily male reproductive toxicants because this effect is not predicted by TEST. We compared the program’s predictions made with the US Food and Drug Administration (US FDA) and Consensus decision methods with the results of our in vivo based evaluations. Using the US FDA decision method, 57% of TEST predicted DART agents were also demonstrated selective DART agents in vivo (ie, true positives); using the Consensus method, the agreement was 74%. The US FDA decision method resulted in 40% false negatives (ie, predicting a chemical was not a DART agent when it is so in vivo), while the Consensus method had a better result, 23%. Our results show that programs such as TEST may be useful for initial screening but have a fairly high potential to incorrectly estimate DART risk.
P526: Evaluation of Liver Protein Biomarkers Induced by the Known Hepatotoxicants Acetaminophen, Buspirone, Finasteride, Nefazodone, and Flutamide Using a High-Throughput Proteomic Profiling LC-MS/MS Assay
A. Licollari1, D. Fronda1, A. Quaile1, J. Fanaras1, and R. Bagshaw1
1Nucro-Technics, Scarborough, Canada
P527: Determination of Curcumin in Dog Whole Blood for a Multidrug Cocktail by LC-MS/MS
M. X. Luna1, X. S. Yan1, K. Jackson1, Y. Liu1, G. Zaid2, C. West2, and L. Goodwin1
1KCAS Bioanalytical & Biomarker Services, Shawnee, KS, USA
2Genzada Pharmaceuticals, Sterling, KS, USA
A liquid chromatography with tandem mass spectrometer method was established for curcumin as part of an experimental antitumor cocktail in dog whole blood. The assay was validated over a range of 2.00 to 100 ng/mL and met all regulatory compliance. The precision (% CV) and accuracy (% Bias) of the method ranged from 2.0% to 8.6% and −4.8% to 8.4%, respectively, and determined stable for 6 hours on wet ice and 35 days at −70°C. Analysis of blood samples, following oral administration of the formulation, did not reveal significant levels of curcumin in dog. Additional investigations were conducted confirming the absence of measurable levels of curcumin other species. The sulfate and glucuronides of curcumin were subsequently determined as present at significant systemic levels in murine plasma, but not detected in murine brain homogenates. Methods have since been fully validated to support clinical investigations to measure curcumin, as well as the sulfate and glucuronide forms.
P528: Transient Aminotransferase Increases Following Acetaminophen Treatment in Rats
W. B. Mattes1, A. Regev2, P. Watkins3, R. J. Church3, M. Avigan4, D. Mendrick5, J. Greenhaw1, and Q. Shi1
1National Center for Toxicological Research, US FDA, Jefferson, AR, USA
2Eli Lilly and Company, Indianapolis IN, USA
3UNC Institute for Drug Safety Sciences, Chapel Hill, NC, USA
4Center for Drug Evaluation and Research, US FDA, Silver Spring, MD, USA
5National Center for Toxicological Research, US FDA, Silver Spring, MD, USA
A persistent concern in pharmaceutical development is the appropriate response to serum aminotransferase (AT) increases seen in patients given a new drug in clinical trials. While AT increases can portend serious liver injury, it is also well known that for certain drugs, such increases in clinical settings reverse with continued treatment, with no further evidence of liver injury. This well-documented phenomenon is often referred to as adaptation. A means to distinguish those AT increases that will resolve from those that portend serious liver injury is needed. We report here a pilot animal model of such transient AT increases. Sprague Dawley rats were given daily oral doses of 1.0, 1.5, and 2.0 g/kg acetaminophen and sacrificed after 8 days of treatment. Tail bleeds were conducted at days 1, 3, 6, and 7 of treatment. Alanine aminotransferase (ALT) increases were seen at day 3. Strikingly, (1) these increases were not seen in all animals and the extent varied greatly from animal to animal; (2) all ALT increases returned to baseline by day 6; (3) the increases were not dose dependent; (4) by contrast, no increases in total bilirubin were seen; and (5) while minimal to mild centrilobular necrosis was observed at day 8 for most of the treated animals, its grade was not related to the extent of ALT increase. This represents the first report of transient AT increases in a rodent model and offers the possibility for discovery of biomarkers of adaptation.
P530: Nerve Conduction Assessments of Disease Progression in Preclinical Rodent Models of Chronic Metabolic and Chemotherapy-Induced Peripheral Neuropathy
M. R. Metea1, G. D. Lyng2, and J. D. Mena2
1Preclinical Electrophysiology Consulting, LLC, Watertown, MA, USA
2Biomodels, LLC, Watertown, MA, USA
Peripheral neuropathies (PN) are common and debilitating disorders of various origin (metabolic: eg, diabetes, toxic: eg, chemotherapy, or genetic). Although acute rodent models of PN are widely used for the assessment of safety and efficacy of investigational compounds, there is a recognized need for establishing models of chronic disease. Electrophysiological measures of nerve impairment (or neurography) are noninvasive tools considered the “gold standard” for determining early changes in sensory and motor nerve function. We characterized biomarkers of PN such as changes in nerve conduction velocity (NCV) in spontaneously diabetic db/db mice (a model of type 2 diabetes); streptozotocin (STZ)-injected Sprague Dawley (SD) rats (a model of type 1 diabetes); and Balb/c mice treated chronically with the chemotherapeutic compound cisplatin (a model of toxic PN). Diabetes was induced in SD rats via a single 70 mg/kg STZ intravenous injection. The PN was induced in BALB/c mice by administration of cisplatin (4 mg/kg) over up to 4 weeks. Neurography was conducted at ∼4 and ∼8 weeks after treatment or confirmation of disease. Statistically significant decreased conduction velocity of the caudal, sciatic/tibial, or sural nerves were noted during and at the end of the study and are described in addition to recommendations for stimulation and recording. The results demonstrate the utility of electrophysiological tools in the assessment of drug effects on peripheral nerve function using murine model of peripheral neuropathy. Recommendations are made for including NCV measurements in toxicological studies and applicability to drug efficacy or safety projects.
P531: Drug-Induced Genome Instability Test (DiGIT): A Novel Strategy to Assess Cancer Risk of Human Therapeutics
S. Minocherhomji1, D. Murali1, and M. Fielden1
1Amgen Inc, South San Francisco, CA, USA
Cancer is a complex disease that constantly evolves and adapts to conditions of genome instability and increased metabolic, cellular, and replication stress (RS). Activation of mutagenic DNA repair pathways following persistent drug-induced RS can result in cellular damage or uncontrolled proliferation of a subset of cells, albeit at a cost of increased genome instability. The current battery of genotoxicity tests does not fully account for the repertoire of DNA repair pathways, including mutagenic DNA repair pathways that impact nuclear genome integrity. Therefore, further studies are required to assess the putative impact therapeutics can have in promoting genome instability as early indicators of tumorigenesis. Here, we show that small molecule inhibitors previously attributed as being nonclastogenic and/or nonmutagenic activate mutagenic DNA damage response (DDR) pathways associated with 53BP1 foci formation in cells from phenotypically normal individuals. 53BP1 is a known mediator of the mutagenic Nonhomologous End-Joining (NHEJ) DNA repair pathway that promotes the formation of oncogenic chromosomal rearrangements. Recruitment of 53BP1 to chromatin was increased in a dose-dependent and tissue-specific manner in single cells following treatment with test compounds known to induce genomic instability (low-dose aphidicolin, duvelisib, idelalisib, and hydralazine) and compounds suspected of causing genomic instability based on carcinogenicity outcomes (amiodarone and pravastatin). Nongenotoxic compounds not suspected of causing cancer based on rodent data did not induce significant effects on 53BP1 recruitment (
P532: Considerations for Qualifying VCN in Cell-Based Therapies
Mohar1 and T. A. Lewandowski1
1Gradient, Seattle, WA, USA
Recombinant lentiviral vectors (RLVs) are engineered replication-deficient viruses that have been used to deliver transgenic material for gene and cell-based therapies. Vector integration into the host cell genome is necessary for RLV-delivered transgene expression, but it can possibly contribute to cellular transformation. Presently, regulatory guidance documents state that vector integration and the risk of cellular transformation is poorly understood. Because of the lack of understanding, reducing target cell vector copy number (VCN) has been proposed as the only way to reduce the probability of cellular transformation. The US Food and Drug Administration (US FDA) has proposed that VCN should not exceed 5. This assessment reviews the publicly available regulatory guidance and safety data on RLV VCN with the objective of identifying (1) the basis of the US FDA limit and (2) a potential alternative VCN limit. Based on the available regulatory guidance and clinical, in vivo, and in vitro experimental data, we found limited support for the US FDA value, or any generic limit. Instead, the data indicate that a target VCN should be experimentally and empirically qualified for any permutation in the vector design, target cell, and dosing regimen, as these may individually and collectively impact the probability of transformation at the clonal and/or population levels. Further, the data suggest that simply reducing the VCN could alter or reduce treatment efficacy, and potentially place replicative stress on a subset of the RLV-modified cells.
P533: Retinal Effects in Albino Rats Exposed to Pramipexole and Constant Light
A. D. Rasmussen1 and K. C. Leere2
1H. Lundbeck A/S, Valby, Denmark
2Copenhagen University, Copenhagen, Denmark
Pramipexole is a dopamine agonist that has been on the market for 2 decades for the treatment of Parkinson disease. Pathological changes in the retina have been recorded in rats administered pramipexole in long-term studies, and while a 2-year clinical trial on retinal deterioration was unable to detect statistically significant effects in patients with Parkinson disease, up to 10% of general patients experience visual disturbances. Although the potential significance of rat retinal findings to the human situation has not been clearly established, they can thus not be disregarded. Mechanistic studies have suggested that constant light (24 hours) exposure conditions while receiving daily administration of pramipexole will accelerate retinal findings in nonpigmented rats and that these findings become apparent within 13 weeks. We investigated this further by performing a 13-week study where 2 groups of nonpigmented Wistar Han male rats were administered vehicle (water) and 2 groups were administered pramipexole (25 mg/kg/d). One group of each treatment scheme was kept under normal light conditions (12-hour light:12-hour dark) while the other groups were housed in constant light. Following 13 weeks, clear retinal damage in the form of atrophy was recorded in all animals exposed to constant light conditions, while no findings were recorded in animals housed under normal light conditions. Interestingly, we did not record any retinal effects of pramipexole. Specifically, administration of pramipexole caused no exacerbation of the light-induced retinal atrophy or caused any additional retinal findings. Consequently, our results do not corroborate previously published findings.
P534: Assessing the Genotoxicity of Drug Product Impurities by Combining In Silico Predictions With Read-Across for Analogs Having Experimental Data
J. S. Rathman1,2, C. Yang3, N. Skoulis2, A. Mostrag-Szlichtyng2, and O. Sacher3
1Ohio State University, Columbus, OH, USA
2MN-AM, Columbus, OH, USA
3MN-AM, Nürnberg, Germany
The ICH M7 guidance for assessment of mutagenic impurities in pharmaceuticals stipulate that, in certain circumstances, computational (in silico) toxicity assessment can be sufficient to conclude that an impurity is of no mutagenic concern. We present an approach that enhances conventional quantitative structure–activity relationship (QSAR) methods (expert rule-based and statistical models) with a rigorous and quantitative read-across approach. Read-across is used to evaluate the potential toxicity of an impurity based on experimental data available for analogs, compounds that are similar to the target molecule in a way that is relevant to the particular toxicity endpoint of interest. The decision theory approach used here, adapted from Dempster Shafer theory, takes into account the uncertainty associated with each evidence source (eg, accuracies of the expert rules and QSAR models, experimental study reliability, analog quality) and provides a systematic way to combine all available evidence sources to obtain a weight-of-evidence decision. Analog quality is determined based on both structure- and property-based similarity to the target impurity. An example is presented for a metaxalone impurity to illustrate how analogs are identified, qualified, and assessed, and how read-across on experimental data for analogs reduces uncertainty in the final weight-of-evidence decision. Additional examples are presented, including cases in which the read-across result conflicts with the in silico prediction.
P535: Comparison Between Toxicity Studies in the Juvenile Rat or Mouse—How Different Are They?
E. S. Richmond1, S-. A. Reynolds1, and M. Blackwell1
1Sequani Limited, Ledbury, United Kingdom
There are well-known differences between children and adults with respect to drug handling and the risk of adverse effects. Despite this, just 1 to 2 decades ago, most medicines were developed for adults and then prescribed to children without the safety support of clinical trials in pediatric populations, or even nonclinical safety data from appropriately aged animals that had been directly exposed during postnatal development. This realization has driven legislative and regulatory changes in both Europe and the United States, making it now mandatory to include pediatric assessments of all new drugs or line extensions for a patented product, unless they can be specifically excluded. The outcome has been a huge increase in the number of toxicity studies conducted in juvenile animals to support pediatric clinical trials, and a large body of work has been performed to understand the ontogeny of developing organ systems and the human significance. As rats have been the species of choice in most circumstances, the knowledge around this species has grown, but inevitably there are occasions when mice are of more human relevance. This has posed new challenges, with little industry understanding of background organ development or the practicalities of dosing juvenile mice. We sought to fill these knowledge gaps by conducting validation studies in the juvenile mouse, concluding that juvenile mice have some unique considerations, with discernible differences to rats. This poster documents the experience gained in performing regulatory toxicity studies in the juvenile mouse and highlights the main differences compared with rats.
P536: In Silico Toxicity Prediction of Roflumilast Degradation Products and Their Metabolites
M. S. Pinheiro1, G. M. Viana2, B. A. Abrahim-Vieira2, A. M. T. Souza2, L. M. Cabral2, H. C. Castro3, R. C. E. E. Marins2, V. P. Sousa2, and C. R. Rodrigues2
1University Federal of Rio de Janeiro, Rio de Janeiro, Brazil
2University Federal of Rio de Janeiro, Rio de Janeiro, Brazil
3University Federal Fluminense, Niteroi, Brazil
The present study reports the toxicity availability of roflumilast (RFL) degradation products (DPs). Eleven DPs of RFL were submitted to in silico studies of their toxicity properties using ADMET Predictor in qualitative and quantitative models. Hepatotoxicity parameters were specifically studied using relevant biomarkers and mutagenicity was predicted in 5 individual strains of Salmonella typhimurium. Acute toxicity (LD50) was also measured. All metabolites generated in silico, based on phase I and phase II for each DP, were evaluated for risk potential. The results showed that 4 DPs (DP-1, DP-4, DP-5, and DP-7) were identified as potential hepatotoxic compounds. The DP-1 and DP-7 also showed predisposition for respiratory sensitization. Mutagenic potential was observed for the DP-4 and DP-10. The metabolites produced for each DP, excluding DP-9, exhibited an important indicator of undesirable toxicity properties. All DPs metabolites from DP-1, DP-3, DP-4, DP-5, DP-7, DP-8, and DP-11 also displayed hepatotoxic potential. Mutagenicity was predicted for MI-3 (metabolite of DP-1), MIII-2 (metabolite of DP-3), MIV-3 (metabolite of DP-4), and MVII (metabolite of DP-7). All DPs showed higher LD50 values than RFL (389 mg/kg), considered not acutely toxic in rats. This value is classified as slightly toxic according to the Hodge and Sterner toxicity classification scale. Therefore, in silico studies from DPs of RFL showed relevant toxicity with efficient and robust results.
P537: New QSAR Models for Predicting Drug-Induced Liver Injury With Enhanced Sensitivity
R. D. Saiakhov1, N. L. Kruhlak2, L. Stavitskaya2, M. T. Kim2, and S. Chakravarti1
1MultiCASE Inc, Beachwood, NJ, USA
2US FDA Center for Drug Evaluation and Research, Silver Spring, MD, USA
Drug-induced liver injury (DILI) is one of the most common drug-induced adverse events (AEs) leading to life-threatening conditions such as acute liver failure. It is also the second most common cause of postmarket withdrawals or warnings. An earlier generation of quantitative structure–activity relationship (QSAR) models was developed for DILI endpoints based on predominantly negative training data collected from the US Food and Drug Administration adverse event reporting system (FAERS), which contains postmarket reports that depict the morbidity of AEs. The data sets contained only 10% to 50% positives, yielding models with high specificity and negative predictivity, but low sensitivity. In this study, we present a revised set of DILI QSAR models built using a previously reported downsampling method to improve sensitivity while maintaining structural coverage. The set of models covers the endpoints of liver damage, liver function, bile duct disorders, cholestasis, and gallbladder disorders. All models demonstrated bootstrap cross-validation performance in the range of 64% to 76% sensitivity, 56% to 67% specificity, 58% to 71% positive predictivity, and 62% to 69% negative predictivity, with an improvement in sensitivity of more than 30% over the earlier generation of unbalanced models. An assessment of the domain of applicability yielded coverage in the range of 70% to 80% through the implementation of a new algorithm that utilizes downsampled negative data in combination with predictions when an unknown structural fragment is identified in the test chemical. Collectively, these models provide an enhanced, high-throughput method for predicting DILI based on chemical structure for early screening of pharmaceutical candidates for hepatotoxic potential, as well as supporting regulatory safety decision making.
P538: In Vivo to In Vitro Detection of miR216-5p in Models of Acute Exocrine Pancreas Toxicity
M. R. Saunders1, A. Rabinowitz1, V. S. Carreira1, J. Y. Ma1, E. Wang2, V. Barlow3, J. Kanerva1, W. E. Glaab2, S. Snook1, and J.E. McDuffie1
1Janssen Research & Development, LLC, San Diego, CA, USA
2Merck & Co, West Point, PA, USA
3Janssen Research & Development, LLC, Spring House, PA, USA
Cell-based models that mimic biological responses of drug-induced injury are promising tools for early screening strategies. Diagnosis of drug-induced acute pancreatitis is challenging due to the lack of sensitive and specific biomarkers. Utilizing biochemical, molecular, and morphological studies, we monitored the kinetic activity of amylase and lipase and expression levels of pancreas-enriched microRNAs (miR216a-5p, miR216b-5p, miR217-5p, and miR375-3p) in the serum and pancreas of Sprague Dawley, Wistar Han, and Wistar Kyoto rats and conditioned medium and cells of the amphicrine cell line, ARIP, relative to controls. Following exposure to toxicants, Caerulein and para-Chlorophenylalanine, acute increases in amylase and lipase proteins were observed at 2 and 6 hours, while the pancreas-enriched miRNAs detected in the serum were significantly increased at 6 hours posttreatment in rats. Amylase activity and miR216b-5p were significantly increased in the conditioned medium of ARIP 2-D and 3-D models at 2, 6, 24, and 48 hours following treatment. The pattern of temporal changes in cellular amylase in vitro was comparable to in vivo findings. Microscopic findings consisted of acinar cell degeneration, necrosis, and inflammation in the rat pancreas. ViewRNA approaches verified the loss of miRNAs within the acinar cells. The detection of miR216b-5p as a candidate biomarker in the conditioned medium of ARIP cell models is predictive of exocrine cell toxicity. To our knowledge, this research provides the first example of in vivo to in vitro translation for pancreas-specific miR216b-5p, further supporting its use as a biomarker for assessing risk in preclinical drug discovery.
P539: Kinetic Analysis of Intratumoral Protein Production Following Intratumoral Injection of LNP-Formulated mRNA, and Comparison to Formulation-Invoked Cytokine Response In Vivo
Y. E. Timsit1, M. Pietras2, S. Wen3, M. Grondine3, N. Johnson2, A. Desai4, N. Derbyshire5, S. Bickerton6, S. Bates6, H. Barthlow1, C. Betts6, M. Fellows7, and C. Scott1
1Discovery Safety, Drug Safety and Metabolism, AstraZeneca, Waltham, MA, USA
2Lab Animal Sciences, Drug Safety and Metabolism, AstraZeneca, Waltham, MA, USA
3Oncology iMED, AstraZeneca, Waltham, MA, USA
4Advanced Drug Delivery, Pharmaceutical Sciences, AstraZeneca, Cambridge Science Park, Cambridge, United Kingdom
5Pathology, Drug Safety and Metabolism, AstraZeneca, Alderly Park, Macclesfield, United Kingdom
6Pathology, Drug Safety and Metabolism, AstraZeneca, Cambridge Science Park, Cambridge, United Kingdom
7New Modalities, Drug Safety and Metabolism, AstraZeneca, Cambridge Science Park, Cambridge, United Kingdom
Effective intracellular delivery of therapeutic messenger RNAs (mRNAs) requires nanoparticle carriers. While the mRNA contains modifications designed to avoid immune detection, a nanoparticle carrier that avoids immune detection has yet to be developed. Understanding mechanisms of nanoparticle carrier-elicited inflammation response will inform development of new nanoparticle carriers that do not invoke such a response. A key question is whether a relationship exists between the immune response and protein production from therapeutic mRNA. To this end, 2 lipid nanoparticle (LNP) formulations giving different levels of mRNA-directed protein expression were used. A luciferase mRNA was used to allow whole-body bioluminescent imaging (BLI) for measuring intratumoral (iTu) protein production kinetics. Serial plasma and tumor sampling were performed to measure inflammatory cytokines. After iTu dosing, BLI confirmed the difference in protein expression kinetics between the 2 LNPs (2.5-fold difference in area under the). Interleukin-6 response was similar for both LNPs, while KC response was lower for the LNP giving higher protein expression. With repeat dosing, attenuated cytokine response was observed, or as seen with plasma MCP-1 and KC, peak levels were reached sooner but did not exceed levels observed after first dose. This attenuated response with repeat dosing suggests immune tolerance, but further investigation is needed. Our results show that increased iTu protein production did not translate into greater cytokine response, suggesting other factors as inflammation drivers. Further characterization of iTu protein production and inflammation response will allow development of PBPK models to enable translational prediction of iTu protein expression and immune response.
P541: Continued Evaluation of Spheroid Human Liver Microtissues for Hepatotoxicity Risk Assessment in Drug Discovery
Vogt1, C. Lawson1, T. Kiyota1, A. M. Fullerton1, and W. R. Proctor1
1Genentech, South San Francisco, CA, USA
Drug-induced liver injury (DILI) is a leading cause of attrition, black-box warnings, and postmarket withdrawal. Assessment of hepatotoxicity risk in drug discovery is difficult due to poor concordance of preclinical species in identification of human hepatotoxicants. Like others, Genentech employs a panel of in vitro models to investigate potential DILI risk. One of these models is compound-induced cytotoxicity using 3-dimensional spheroid liver microtissues (LiMTs), which we have demonstrated as having increased sensitivity with comparable specificity to primary hepatocytes in identifying known hepatotoxicants. This initial work focused on a 110-compound set, of which 60% are DILI positive, that were heavily weighted with reactive metabolite formation as a proposed mechanism of toxicity. However, compound-related factors (mitochondrial effects, bile-acid transport inhibition, physicochemical properties, etc) associated with the increased sensitivity observed in this model remained unclear. We expanded the test set by an additional 60 compounds and profiled DILI risk factors to identify covariates of cytotoxicity in LiMTs and refine predictivity of this model. Data reanalysis of the expanded compound set resulted in decreased specificity of the assay by approximately 10% when LiMT cytotoxicity IC50 values were 10-fold or less than clinical Cmax. Compound attributes associated with cytotoxicity in LiMTs will be presented, focusing on both true and false DILI positive compounds. We present case studies highlighting utilization of this model during lead optimization and describe how LiMT cytotoxicity and other in vitro hazards of DILI are weighed prior to candidate nomination.
P539: Evaluation of Sex Differences in Diazepam and Amphetamine Conditioned Place Preference in Sprague Dawley Rats
K. Walker1, E. Sable1, V. Castagne1, N. Madar-Balakirski2, H. Kedar2, and C. Froger-Colleaux1
1Porsolt, Le Genest-Saint-Isle, France
2Teva Pharmaceuticals, Netanya, Israel
The US Food and Drug Administration guideline (January 2017) related to the assessment of abuse potential of drugs included the recommendation for a New Chemical Entity to be evaluated in both male and female rodents. Nevertheless, despite evidence for an impact of sex difference in humans, the corresponding impact in rodents needs further evaluation. The aim of this study was to evaluate the impact of sex on diazepam and amphetamine-induced conditioned place preference (CPP) in rats using a 2-chambered CPP apparatus and a biased procedure. In male rats, diazepam 2.5 mg/kg intraperitoneally (ip) did not affect the percent of time spent in the drug-paired compartment (% time D-P) compared with vehicle controls (+7%, NS). Amphetamine 2 mg/kg ip significantly increased the % time D-P (+27%, P < 0.01) with a similar trend at 1 mg/kg ip (+18%, P = 0.0675). In female rats, diazepam 2.5 mg/kg ip was also devoid of effects, whereas amphetamine 1 and 2 mg/kg ip significantly increased the % time D-P (+35%, P < 0.001 and +27%, P < 0.001, respectively). These data indicate the presence of reinforcing properties for amphetamine at 2 mg/kg ip in male and female rats and at 1 mg/kg ip in female rats, suggesting no marked sex difference in drug-induced CPP in the present experimental conditions. Further studies evaluating additional drugs would be useful to support the recommendation concerning the sex of animals in the new guideline, taking into account the 3Rs principle in preclinical research.
P543: Pancreatic Toxicity of Valosin-Containing Protein (VCP) Inhibitors in Rodents
F. S. Wolenski1, K. Enos1, J. Gavin1, S. Harrison1, A. Berger1, S. Gould1, and W. Mallender1
1Takeda Pharmaceuticals, Cambridge, MA, USA
The valosin-containing protein (VCP; p97) is an ATPase that helps segregate proteins from large cellular structures, such as the endoplasmic reticulum. A series of small molecule VCP inhibitors (VCPinhibs) were developed as a potential oncology therapy. The pancreas was identified as a target of VCPinhib toxicity after weight loss and excessive urination was observed in xenograft mice. Degeneration of cells in the islets of Langerhans was specific to the insulin-producing β cells. There was also a correlation between islet cell degeneration and blood glucose, consistent with pancreatic dysfunction. To determine whether this was a mouse-only effect, Sprague Dawley rats received 0.05 or 0.1 mg/kg of a VCPinhib either by a subcutaneous or intravenous administration. Single doses of 0.1 mg/kg VCPinhib were not tolerated due to body weight loss. These animals had a significant decrease in insulin with a correlative increase in glucose. Rats tolerated 2 weekly doses of the 0.05 mg/ kg VCPinhib without weight loss. Microscopic evaluation of pancreases from both dose groups revealed islet cell degeneration in most animals with the additional finding of acinar cell atrophy in all rats. VCPinhib toxicity was observed in other endocrine and exocrine tissues and may have contributed to the overall poor tolerability. These findings prompted the discontinuation of this series of VCP small molecule inhibitors.
P544: Renal Pathology Findings in Cynomolgus Macaques Administered Antibody Drug Conjugates (ADCs): Evaluating the Contribution of Antibody and Payload Attributes
R. L. Yeager1, M. Ramos1, T. Kambara1, R. Kikuchi2, G. Foley1, L. Gallenberg1, S. Ralston1, L. Loberg1, and K. Barnhart1
1AbbVie Preclinical Safety, North Chicago, IL, USA
2AbbVie DMPK-BA, North Chicago, IL, USA
Renal pathology findings were characterized for cynomolgus macaques administered antibody drug conjugates (ADCs) with “targeting” or “nontargeting” antibodies and payloads (same mechanism of action) of “moderate” or “high” permeability. All ADCs were produced by site-directed conjugation for a drug–antibody ratio (DAR) of 2 and utilized cleavable linker technology. The comparator ADC (“C-ADC”) was comprised of a targeting antibody with payload of moderate permeability. Three alternate ADCs (“A-ADC”) were produced: 2 with different nontargeting antibodies conjugated to the moderate permeable payload and 1with the targeting antibody conjugated to the high-permeable payload. Targeting ADCs had toxicokinetic profiles suggestive of target-mediated disposition, compared to nontargeting ADCs against antigens with restricted expression in normal tissues. Doses of 5 and 10 mg/kg (single-dose or 2× Q2W) of the C-ADC resulted in poor clinical condition. Changes in clinical pathology parameters attributed to altered proximal tubule function included decreased urine pH, increased urine glucose, protein and albumin, and increased serum blood urea nitrogen and creatinine. Minimal to mild changes in these parameters were evident prior to changes in clinical condition and correlated with dilated proximal tubules; progressive increases indicating greater severity correlated with proximal tubular epithelial degeneration/necrosis and hyaline casts. The C-ADC-associated renal findings were also observed to varying degrees with the A-ADCs. Both payloads showed significant accumulation in the kidney with exposure related to permeability. In conclusion, this analysis highlights that payload mechanism of action is the primary driver of the renal pathology findings observed across a set of diverse ADCs.
P545: Renal Toxicity in Sprague Dawley Rats Associated With Cyclopropylindoline (CBI)-Containing Antibody Drug Conjugates
F. Zhong1, M. Schutten1, P. Katavolos1, B. Latifi1, D. Leipold1, I. Amenabar1, O. Saad1, S. Hyung1, C. Sioson1, H. Wang1, and D. Lee1
1Genentech Inc, South San Francisco, CA, USA
CBIs are potent alkylating agents of the cyclopropylindoline class that target the minor groove of DNA. CBI antibody drug conjugates (ADCs) were developed to combat multidrug resistance with tubulin inhibitors. The main toxicity in the clinic related to adzelesin, a CBI class analog, is myelosuppression. In nonclinical studies using rodents and monkeys, the main toxicities associated with CBI-ADCs were myelosuppression and elevated liver enzymes. In addition, renal pathology characterized by tubular degeneration, regeneration, and interstitial fibrosis was observed in monkeys administered a single dose of a CBI-ADC after an extended drug-free recovery period. This kidney finding was not observed in rats. To investigate the mechanism of renal toxicity observed in monkeys, we sought to develop a rat model to investigate whether the renal toxicity can be recapitulated. Rats were dosed at 5 mg/kg weekly for 5 doses with a terminal necropsy or a 6-week recovery necropsy. Animals did not tolerate the full dosing regimen well and were euthanized ahead of scheduled necropsy after the fourth dose. During dosing, excessive urine output was observed consistent with polyuria. In conclusion, rat appears to be a less sensitive species to CBI-ADC-related renal degeneration and fibrosis, but still developed clinical signs consistent with renal function impairment (polyuria). The etiology of the renal functional impairment in rats is unknown and further investigations are pending.
600 Series—Late-Breaking
P601: Toxicity Value Selection for Quantitative Risk Assessment of Harmful and Potentially Harmful Constituents in Smokeless Tobacco Products
A. Amantana1, S. G. Sawant1, W. Xie1, S. Liu1, K. M. Marano1, and D. T. Szabo1
1RAI Services Company, Winston-Salem, NC, USA
The US Food and Drug Administration (US FDA) established a list of 93 harmful and potentially harmful constituents (HPHCs) in tobacco products and tobacco smoke, as required by the Family Smoking Prevention and Tobacco Control Act. The US FDA classifies these constituents as carcinogens, respiratory toxicants, cardiovascular toxicants, and/or reproductive/developmental toxicants. The abbreviated list of HPHCs for reporting in smokeless tobacco products includes acetaldehyde, arsenic, benzo[a]pyrene, cadmium, crotonaldehyde, formaldehyde, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and n-nitrosonornicotine (NNN). The US Environmental Protection Agency published a hierarchical risk assessment approach to identifying toxicity values for chemicals at Superfund sites. However, several toxicity values have not been updated for decades and may no longer be scientifically defensible. To assess risk associated with smokeless tobacco product consumption, a search of publicly available US federal and state databases and international databases for noncancer and cancer oral toxicity values was conducted. Search results included multiple values reported by different agencies. In the absence of US FDA guidance for selecting a toxicity value for QRA, we report a selection approach supported by the most current scientific methods that include study quality, date of study, and toxicity value derivation approach. For noncancer toxicity values derived from a critical effect other than the endpoint(s) published by US FDA, scientific justification was included to support the public health protective nature of the toxicity value(s) selected. This approach can be used to justify the selection of toxicity values for HPHCs identified by US FDA in smokeless tobacco and other products for which the oral route of exposure is relevant.
P602: A Systems Model of Liver Physiology Provides Mechanistic Insights From Toxicogenomics Data
T. Sharma1, P. Chandarana1, S. Priyadarsini1, A. Bhat1, K. Subramanian1, and S. Das1
1Syngene International Ltd, Bangalore, India
To understand the mechanisms of drug-induced liver injury, we have combined genomic data with a systems model of liver physiology to simulate the impact of the change in genes at different drug exposure levels. A gene expression array data set (GSE2303) for valproic acid (VPA) has been analyzed by 2 methods. In method 1, we used the differentially expressed genes and their neighbors from a natural language processing-derived network (34 genes at 4 hours, 167 at 24 hours, 300 at 48 hours posttreatment) as model inputs, while in method 2, 11 pathways found by gene set enrichment analysis were used as model inputs. In either case, the inputs were applied as a perturbation on the state of the model and simulations performed. Approach 1 predicted accumulation of intracellular triglycerides (TG) leading to steatosis due to changes in fat and carbohydrate metabolism enzymes including glucokinase, CPT1, FAS, and ACC and targets of transcription factors SREBP, CREB, and BTG2. The adaptive upregulation of fatty acid entry points to the direct inhibition of fatty-acid β-oxidation by VPA. The increase in TG was opposed by inhibition of de novo fatty acid synthesis, which could only partially mitigate the TG increase. Approach 2 also showed TG increase but it was minor. In addition, we could neither infer the direct target of VPA nor the adaptive regulations. Based upon this, we propose that method 1, differentially expressed genes and network neighborhoods along with simulation, provides an interesting semiquantitative approach to understand the toxicological impact of drugs on the liver.
P603: ReproTracker, a Human Stem Cell-Based Reporter Assay for In Vitro DART Assessment
P. I. Racz1, I. Brandsma1, T. Zwetsloot1, and G. Hendriks1
1Toxys BV, Leiden, the Netherlands
Testing for developmental and reproductive toxicology (DART) is a crucial part of the toxicological risk assessment. The DART mostly relies on animal testing because the current alternative in vitro assays such as embryonic stem cells tests often lack mechanistic insight and are difficult to translate to human risk due to interspecies differences. To improve in vitro identification of developmental toxicants, we developed a human-induced pluripotent stem cell (hiPSC)-based assay. To quantitatively assess the major developmental processes, we identified 43 potential biomarkers, marking different developmental stages from stem cells to mature tissues. To test whether compounds affect development, we optimized the differentiation protocols for hiPSC toward cardiomyocytes, hepatocytes, and neuronal cells and confirmed expression of various biomarker genes by real-time qualitative polymerase chain reaction. During differentiation, expression of the stem cell marker OCT4 decreased, while expression increased for MYH6 in cardiomyocytes, ALB and AFP in hepatocytes, and SOX1 and PAX6 in neural rosettes. Next, we investigated disruption of stem cell differentiation using 15 well-known teratogenic and nonteratogenic compounds from the ECVAM compound library. Exposure to the teratogens 5-fluorouracil, thalidomide, retinoic acid, and carbendazim resulted in a marked reduction of functional cardiomyocytes and led to downregulation of the specific biomarker genes for cardiomyocytes, hepatocytes, and neuronal cells. In contrast, the nonteratogenic compounds like Penicillin G did not affect proper differentiation of the hiPSCs. In conclusion, following the differentiation program by biomarker genes is a suitable tool for high-throughput, quantitative measurement of differentiation for DART screening.
P604: Assessment of Reproductive and Repeated-Dose Toxicity for Cyclopentyl Methyl Ether as a Residual Solvent in Pharmaceuticals
K. Inoue1, N. Otsuki2, and A. Hirose1
1National Institute of Health Sciences, Kawasaki, Kanagawa, Japan
2Nippon Zeon Co, Ltd, Tokyo, Japan
Cyclopentyl methyl ether (CPME; CAS 5614-37-9) is used as an alternative to other ethereal solvents in pharmaceutical chemical process development. Although data for the repeated-dose toxicity of CPME were available, detailed reproductive/developmental toxicity information has not been fully reported. Therefore, we performed a reproductive/developmental toxicity screening study and reevaluated the full report of the repeated-dose toxicity study, allowing detailed hazard characterization of CPME to support determining a permitted daily exposure level for the ICH guideline on impurities in pharmaceuticals. The studies were performed according to the Organisation for Economic Co-operation and Development Testing Guidelines 421 and 407. Sprague Dawley rats were treated with CPME by gavage at the doses of 50, 150, or 450 mg/kg/d in the reproductive/developmental toxicity study, or at doses of 15, 150, and 700 mg/kg/d in the 28-day study. In the reproductive/developmental toxicity study, lower body weight in males and longer gestation length were observed in F0 animals at 450 mg/kg/d. In F1 animals, lower body weight gain during days 1 to 7 of age was detected in the 450 mg/kg/d group of both sexes. In the 28-day study, salivation was observed in the male 150 and 700 mg/kg/d groups and the female 700 mg/kg/d group. However, the frequency was not continuous, but sporadic. In the female 700 mg/kg/d group, body weight gain became progressively lower than that of the control with statistical significance. Based on these findings, the no observed adverse effect levels for both the reproductive/developmental and 28-day repeated-dose toxicity studies were judged to be 150 mg/kg/d.
P605: Identifying Epigenetic Modifications of Harmful Algal Bloom Toxins Which Contribute to Transgenerational Reproductive Toxicity
C. E. Moore1 and P. Allard1
1University of California Los Angeles, Los Angeles, CA, USA
With changing climates, warmer water, and increased nutrient pollution runoff, the public health risk of microcystin-producing harmful algal blooms continues to rise. Of the over 100 microcysin (MC) congeners, MC-LR is the most commonly detected and studied. Well-established acute hepatotoxins, MCs inhibit serine/threonine protein phosphatases (PP) and alter cell signaling. Recent studies have highlighted the reproductive system as an additional target organ through oxidative stress, PP inhibition, and possible endocrine disruption. Furthermore, multigenerational studies in mammals and aquatic animals looking at the offspring of exposed parents have demonstrated changes in the F1 generation, including altered neurodevelopment, growth, and oxidative stress markers. In the alternative model, Caenorhabditis elegans MCs induce germline apoptosis and decrease brood size at P0, making it an ideal model to study the epigenetic impact of MCs on reproduction over several generations. To evaluate whether MCs de-silence the normally transcriptionally silenced germline, the C elegans strain NL2507 carrying an integrated low-complexity, highly repetitive array composed of a transgene coding for a fusion product between nuclear-localized LET-858 and GFP (pkIs1582[let-858:: GFP; rol-6(su1006)]) is utilized. This transgene is expressed in somatic cells, but it is epigenetically silenced in the germline via accumulation of the repressive marks H3K9me3 and H3K27me3. Prior exposure studies using bisphenol A have established this method to evaluate transgenerational toxicity (F3 and beyond). A range of environmentally relevant concentrations of MC-LR and MC-LF will be studied to understand epigenetic modifications occurring through several generations by measuring the germline H3K9 regulator H3S10p using immunofluorescence after MC exposure.
P606: Assessment of Neurotoxic Potential of 90 Blinded Compounds Using Zebrafish Embryos
C. Quevedo1, A. Muriana1, K. Ryan2, R. S. Paules2, M. Behl2, and A. Alzualde1
1Biobide, San Sebastian, Spain
2NIEHS-NIH, Research Triangle Park, NC, USA
Developmental toxicity and neurotoxicity of 90 blinded compounds containing known neurotoxicants (DNT/NT) and compounds with undetermined neurotoxic potential (eg, flame retardants, bisphenol A analogs) were assessed using zebrafish embryos. To evaluate developmental toxicity, chemicals were tested at 5 to 8 concentrations, and a teratogenic index was then calculated as the ratio between LC50 (mortality) and EC50 (based on morphological alterations). For neurotoxicity evaluation, embryos were treated at 3 days postfertilization with 5 doses, with the lowest concentration where morphological effects appeared selected as the highest concentration, and after 48 hours of exposure, locomotor activity was analyzed as indicative of neurotoxicity. Larvae from the developmental toxicity assay were analyzed for internal compound concentrations to determine the real concentration at which toxic effects were induced. Moreover, chemical concentration in the medium was also determined. After compound unblinding, within the 38 DNT/NT compounds, 19 (50%) were detected as neurotoxic. An additional 7 compounds were classified as toxic. The remaining 12 compounds did not induce any toxicity. However, the bioavailability data showed that in 11 of the 12 cases, the internal concentration did not reach 100 µM because of the limited uptake, instability, or precipitation. In addition, 9 of 43 (21%) compounds with “unknown” DNT/NT were classified as neurotoxic and other 24 were classified as toxic. This study demonstrates the utility of zebrafish embryos as a prioritization tool for further testing and underscores the importance of evaluating internal compound concentrations to better compare and contrast findings between zebrafish and other models.
P607: Identification of Circulating RNA Biomarkers of Testicular Toxicity in Dogs
J. C. Shing1, K. Schaefer2, T. Sharapova1, M. Rao1, S. E. Grosskurth1, K. Bodié2, K. Hempel2, E. A. Blomme1, W. R. Buck1
1AbbVie, Inc, North Chicago, IL, USA
2AbbVie Deutschland GmbH & Co KG, Ludwigshafen, Germany
Predictive indicators of testicular toxicity (TT) could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating small RNAs could serve as TT biomarkers in dogs. First, we conducted a dose range finding study using the testicular toxicant ethylene glycol monomethyl ether (EGME) in beagle dogs. Doses of 100 or 300 mg/kg/d for 1 week (n = 3/group) were not tolerated. In addition to testicular germ cell degeneration, significant decreases in lymphocytes and loss of the intestinal barrier associated with sepsis were observed in a dose-dependent manner. In a second study, a lower dose of 50 mg/kg/d was selected to avoid systemic toxicity for microRNA profiling using next-generation RNA sequencing. Two groups of dogs (castrated, noncastrated) were treated with EGME for 14 to 28 days (n = 2/group). Because EGME can cause toxicity to organ systems in addition to the testes, comparison to the castrated control group allows identification of biomarkers specific for TT. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Blood was collected daily during the dosing phase, followed by recovery for 29 to 43 days with less frequent sampling. Preliminary bioinformatics data analysis using linear modeling demonstrated that 5 microRNAs were elevated in noncastrated animals compared with castrated animals during the dosing period. Two microRNAs were elevated in noncastrated animals compared with castrated animals during recovery. Future studies will confirm potential biomarkers by real-time qualitative polymerase chain reaction. These data suggest that small RNA profiling is a promising approach for identifying novel biomarkers.
P608: Novel Extensions of the ToxTracker Genotoxicity Assay Provide Insight Into the Mode of Action of Aneugenic and Clastogenic Chemicals
I. Brandsma1, R. Derr1, N. Moelijker1, P. van Rossum1, and G. Hendriks1
1Toxys BV, Leiden, the Netherlands
ToxTracker is a mammalian stem cell-based reporter assay that detects activation of specific cellular signaling pathways upon chemical exposure. The assay consists of 6 different GFP-tagged reporters that can discriminate between induction of DNA damage, oxidative stress, and protein damage. Genotoxicity is detected by the Bscl2-GFP reporter for promutagenic DNA lesions and DNA replication stress, and the Rtkn-GFP reporter for DNA double-stranded breaks. Here, we investigated the ability to discriminate clastogens from aneugens and identify tubulin poisons and kinase inhibitors. We, therefore, included a DNA stain in the assay for cell-cycle analyses and polyploidy identification. The DNA cross linker cisplatin (clastogen) clearly induced both DNA damage reporters. There was a delay in cell-cycle progression with an accumulation of cells in S phase. However, no polyploidy was observed. In contrast, the tubulin poison taxol (aneugen) selectively activated the Rtkn-GFP DNA double-stranded break reporter but not the Bscl2-GFP reporter for DNA replication stress. The cell-cycle analysis showed a clear accumulation of cells in G2/M and an increase in polyploid cells. The aurora kinase inhibitor AMG900 (aneugen) did not activate the genotoxicity but induced a cell-cycle block in G2/M and a strong induction of polyploidy. Together, the differential activation of the genotoxicity reporters, in combination with the cell-cycle analysis and polyploidy detection, allows for rapid identification of clastogens and antigens and can further discriminate between tubulin poisons and kinase inhibitors.