Abstract
100 Series—General Toxicology
P101: The α7-nAChRs and Heme Oxygenase-1 Pathway Are Key Mediators of Nicotine Protective Actions During Endotoxemia
A. M. Wedn1, S. M. El-Gowilly1, and M. M. El-Mas1
1Alexandria University, Alexandria, Egypt
The nicotinic/cholinergic anti-inflammatory pathway protects against kidney injury induced by endotoxemia. Here, we tested hypotheses that (1) nicotine guards against endotoxic manifestations of hemodynamic and renal dysfunction in rats and (2) the α7-nAChRs/heme oxygenase-1 (HO-1) cascade arbitrates lipopolysaccharide (LPS) nicotine interaction. The systolic blood pressure (SBP), inflammatory status, and renovascular reactivity to vasodilators and vasoconstrictors were assessed 6 hours after intraperitoneal administration of LPS (5 mg·kg−1). Lipopolysaccharide reduced SBP and elevated systemic and renal markers of inflammation along with mortality rate. The LPS-induced renal dysfunction was manifested as increase in serum urea and creatinine and decrease in renal reactivity to vasoconstrictors and vasodilators. These LPS effects were sex-related, as they were more prominent in male rats. The apparent female resistance to LPS effects was accompanied by intense induction of HO-1 renal expression compared with their male counterparts. The endotoxic manifestations were dose dependently reversed by nicotine. Such favorable actions of nicotine (1) disappeared in rats cotreated with methyllycaconitine (MLA, α7-nAChR blocker) or zinc protoporphyrin (HO-1 inhibitor) and (2) were accompanied with MLA-sensitive increases in renal HO-1 expression beyond levels achieved by LPS. We also report that anti-inflammatory and renal vasoconstrictor rectifying effects of nicotine were reproduced after treatment with hemin (HO-1 inducer) or tricarbonyldichlororuthenium (II) dimer (CORM-2, carbon monoxide–releasing molecule), but not bilirubin. Contrarily, only bilirubin replicated actions of nicotine on endotoxic mortality and renal vasodilator dysfunction. Together, current evidence suggests key roles for α7-nAChRs and HO-1 signaling in the nicotine counteraction of lethal, inflammatory, hemodynamic, and renal consequences of endotoxemia.
P102: Comparative Analysis of the Hepatotoxicity and Nephrotoxicity of a Commercial Energy Drink and a Local Kolanut Drink in Experimental Male Rats
A. A. Adebisi1, O. O. Ogunlabi2, and B. O. Adegbesan2
1Lagos State University College of Medicine, Ikeja, Lagos, Nigeria
2Olabisi Onabanjo University, Ago Iwoye, Ogun State, Nigeria
Emerging evidence has linked energy drink (ED) consumption with several negative health consequences. This study compares the impact of a commercial energy drink (CED) and a local Nigerian kola nut drink (KD) on hepatic and renal health of male rats. Twenty-eight male Wistar rats were randomly assigned to 4 equal groups. The groups—control, E1, E2, and E3—received daily oral treatment of placebo, CED (10 mg/kg caffeine), KD1 (10 mg/kg caffeine), and KD2 (20 mg/kg caffeine), respectively, for 28 days after which serum lipid parameters, glucose, insulin, electrolyte (Na+, K+, Cl−, HCO3 −), urea, creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were assessed. Our results show that CED significantly (P < 0.05) increased glucose (>200%), insulin (170%), total cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, and triacylglycerols and decreased high-density lipoprotein cholesterol. Urea, creatinine, AST, and ALT increased by 30% ± 6% and serum electrolyte levels were also altered. Animals treated with KD2 had marginal increases in urea and sodium within the group, while all the measured indices for KD1-treated rats were similar to control group. The results indicate that intake of CED at the test dose (about 2-3 cans of EDs) is hepatotoxic, nephrotoxic, hyperglycemic, and insulin intolerance inducing. Conversely, intake of KD1 and KD2 did not flag any noticeable metabolic threat, probably due to other beneficial phytochemicals acting synergistically. Moreover, the caloric content of the KD drinks would be much lower than the CED since no artificial sugars were added. However, further toxicity and histological inquest is necessary to establish safe dosage of the Nigerian KDs.
P103: Mechanisms of YY1 Activation via NF-κB Signaling in Manganese-Induced Impairment of Astrocytic Glutamate Transporters
A. Rizor1, E. Pajarillo1, J. Johnson1, M. Aschner2, and E. Lee1
1Florida A&M University, Tallahassee, FL, USA
2Albert Einstein College of Medicine, Bronx, NY, USA
Chronic exposure to manganese (Mn) causes a neurological disorder known as manganism, which shares the pathological features of Parkinson disease (PD). However, the mechanisms of Mn toxicity are not well understood. Manganese induces the dysregulation of the astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2), which regulates synaptic levels of the neurotransmitter glutamate. Manganese-induced decreases in EAAT2 expression and function lead to excess synaptic glutamate and excitatory neurotoxicity and are associated with neuropathogenesis in PD and manganism. Manganese activates the transcription factor Yin-Yang 1 (YY1), which binds its consensus site on the EAAT2 promoter and represses EAAT2 expression. In the present study, we investigated the mechanisms of Mn-induced YY1 activation via nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in human astrocyte H4 cells. We hypothesized that Mn activates the NF-κB-YY1 pathway via oxidative stress and pro-inflammatory signaling. The results revealed that the initial target of Mn is inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), an upstream kinase that activates NF-kB. Accordingly, pharmacological inhibition of IKKβ abolished Mn-induced NF-kB activation and increases in YY1 expression—with concomitant restoration of EAAT2 messenger RNA/protein levels. Our findings also demonstrated that Mn-induced reactive oxygen species production mediated IKKβ activation, as pretreatment with the antioxidants N-acetylcysteine and α-tocopherol abolished Mn-induced IKKβ activation, inhibited NF-κB p65 nuclear translocation, and attenuated YY1 promoter activity and expression. These results indicate that Mn-induced oxidative stress and pro-inflammatory signaling may activate IKKβ, the upstream kinase of NF-κB, to upregulate YY1 and impair EAAT2.
P104: Prenatal Exposure to Cypermethrin Alters Placental Function and Neurodevelopment in Mice
B. A. Elser1, R. C. Dhakal1, K. Wang1, H. J. Lehmler1, and H. E. Stevens1
1University of Iowa, Iowa City, IA, USA
Prenatal exposure to pyrethroid insecticides has been identified as a risk factor for low birth weight and neurodevelopmental delay in children. Cypermethrin, a type II pyrethroid, has broad agricultural and household use. Previous work in our lab demonstrated that 10 mg/kg cypermethrin administered via oral gavage from gestational days 11 to 14 reduces fetal body weight and disrupts development of GABAergic progenitors in the embryonic mouse brain. To address the molecular mechanisms responsible for these effects, migrating GABAergic progenitors were isolated from fixed embryonic brain tissue via laser capture and were subsequently analyzed via RNA-seq. Pathway analysis of differentially expressed genes found that cypermethrin altered several upstream regulators with critical roles in the differentiation and migration of GABAergic progenitors, including SOX6, PPAR-alpha, and MAPK8. In addition, pathway analysis implicated alterations in the pentose phosphate pathway as a major contributor to cypermethrin’s effects, as well as several pathways involved in the synthesis and catabolism of amino acids. These changes suggest that cypermethrin may affect energy provision to embryonic brain, a process dependent on placental function. Interestingly, measurement of cypermethrin via gas chromatography–mass spectrometry found that levels in amniotic fluid were below the limit of detection, despite an average concentration of 500 ng/mL in maternal serum. These results suggest an indirect mechanism by which cypermethrin alters neurodevelopment. Consistent with this notion, cypermethrin upregulated genes responsive to oxidative stress in placental tissue, including Hif-1alpha, Sod1, and Txrnd1. As such, impacts of pyrethroid exposure on fetal growth and neurodevelopment may be mediated by placental toxicity, which should be further investigated.
P105: Microcystin-LR Induces Multi- and Transgenerational Reproductive Toxicity Potentially Through Changes in Chromatin Structure
C. E. Moore1, M. Blumenkrantz1, and P. Allard1
1University of California Los Angeles, Los Angeles, CA, USA
The public health risk from microcystin-producing harmful algal blooms continues to increase worldwide. With over 100 congeners, microcystin-LR (MCLR) is the most commonly studied and detected microcystin and is known to cause acute hepatotoxicity at high concentrations through inhibition of serine/threonine protein phosphatases. Recent studies highlight lower nonlethal exposure to MCLR causes reproductive toxicity, yet there are limited to no multi- and transgenerational data published on how MCLR may alter offspring reproductive health after parental in utero exposure. Using the highly conserved and well-established reproductive alternative model Caenorhabditis elegans, parental L4 C. elegans are exposed to MCLR (0.1-100 μg/L) for 48 hours and reproductive end points are evaluated in parents (P0) and first-generation (F1) offspring directly exposed to the toxin and in third-generation (F3) offspring, which is the first generation not directly exposed to the toxin after 48-hour P0 exposure. To evaluate whether MCLR desilences the normally transcriptionally silenced germ line, the C elegans strain NL2507 is utilized, with a GFP transgene expressed in somatic cells, but it is epigenetically silenced in the germ line via accumulation of histone repressive marks. Generations F1 and F3 ancestrally exposed to MCLR concentrations below World Health Organization guidelines for maximum MCs in drinking water increased germ line desilencing and germ line apoptosis. Studies in F3 suggest this may be due to impaired chromosomal organization during diakinesis, the stage at which oocytes mature before fertilization. These preliminary results suggest MCLR exposure poses multi- and transgenerational reproductive health risks.
P106: Withdrawn
P107: Behavioral, Molecular, and Histological Responses of Nanoparticle Formulation and Naked Vincristine in Mice
C. T. Bluette1, A. M. Shoieb2, Q. Peng3, B. Manickam2, W. Huang3, E. Shin4, W. Zhang4, Y.-H. Song4, and C.-N. Liu2
1Pfizer Inc, Cambridge, MA, USA
2Pfizer Inc, Groton, CT, USA
3Pfizer Inc, San Diego, CA, USA
4Pfizer Inc, Pearl River, NY, USA
The application of chemotherapeutic agent vincristine (VCR) is limited by chemotherapy-induced peripheral neuropathy (CiPN). A new formulation of VCR has been proposed and developed based on encapsulation of this drug in nanoparticles. We hypothesized that the nanoparticle drug might be less neurotoxic by differentiating absorption and distribution to the peripheral nerve compared with the unencapsulated drug. In this study, we assessed whether VCR encapsulation in nanoparticle alleviates CiPN using behavioral gait analysis (CatWalk), and histological and molecular biological (RT-qPCR) approaches. Adult C57BL/6 mice were assigned to 3 groups (empty nanoparticle, nano-VCR, free-VCR; each n = 8). After 16 days of dosing, the animals were euthanized for blood and sciatic nerve sample collection. It was shown that intraperitoneal injection of nano-VCR (0.15 mg/kg, once daily) and the empty nanoparticle did not result in the decrease in run speed and increase in step cycle and stance observed following injection of free-VCR (P < 0.05). Histology of the sciatic nerve samples failed to show a difference in incidence or severity of axonal degeneration between the nano-VCR-dosed and free-VCR-dosed animals. Likewise, a nervous tissue–enriched microRNA miR-183 in the blood did not show significant difference between the nano- and free-VCR groups (P > 0.05). Empty nanoparticle administration did not cause behavioral, microRNA, or structural changes. In conclusion, this study suggests that the nano-VCR may alleviate behavioral changes of CiPN but did not improve the structural changes of CiPN in the peripheral nerve. Nanoparticle properties, including drug load, release rate, or other factors, may be optimized to improve biological observations.
P108: Translatability of Nonhuman Primate Cytokine Data to In-Life Parameters in Nonclinical Toxicology Studies
C. Do1, V. Bunker1, J. Forget1, and T. Rogers1
1Altasciences Preclinical Seattle LLC, Everett, WA, USA
Cytokines are important immunoregulatory proteins that have gained focus in safety assessment. Interpreting cytokine data comes with challenges due to the variable nature of their stimuli and responses. Contributing factors to the variability in cytokine expression include species-specific reactions, individual variations, dose–response relationships, and unanticipated immunotoxicity. Therefore, evaluating cytokine measurements in conjunction with additional parameters such as clinical observations, and clinical pathology data, can be used to provide more definitive assessments in nonclinical safety studies. In several case studies, cytokines were evaluated for the presence of a dose–response relationship. Multiplex platforms such as Luminex or MSD were used in determining cytokine levels in nonhuman primates. In multiple studies, increases in interleukin 6 (IL-6), a pro-inflammatory cytokine, and monocyte chemoattractant protein-1, a monocyte chemoattractant, were consistently correlated with a rash, diarrhea, lethargy, or hunched posture. More specifically, increases in both analytes ranged from undetectable (at predose) to over 20,000 pg/mL (after dosing). In other instances, elevated IL-2 (>2,000 pg/mL) or IL-12 (>350 pg/mL) correlated with bruising, injury, or abnormal feces, which were not necessarily considered test article related. In cases of test article–related effects, animals becoming moribund also had elevated tumor necrosis factor α (TNF-α) and IL-6 (>20,000 pg/mL for both). Therefore, when interpreting cytokine variations for assessment of potential toxicity, other measurements such as clinical observations and clinical pathology parameters should also be considered in addition to the test article–related effects.
P109: Toxicity Profile of MV8, a Lipid-Based Vehicle Used in Nonclinical Safety Studies
C. L. Neilan1, X. Linghu1, A. Phimister2, and M. P. Dillon1
1IDEAYA Biosciences, South San Francisco, CA, USA
2SeaviewPharma, Kensington, CA, USA
Selection of an appropriate vehicle to utilize in nonclinical safety studies of poorly water-soluble compounds can be challenging due to the lack of detailed toxicity data for various excipients. Here, we report the identification and toxicity profile of a microemulsion formulation “MV8” (Cremophor RH40, Labrafil M2125, propylene glycol, Transcutol: 10:3:3:4), which was used to maximize the oral absorption of 2 lead candidates, compound 1 and compound 2. In 7-day repeat-dose toxicity studies of MV8 alone or compound 1 in MV8, dosing volumes of 3 mL/kg and 2 mL/kg were well tolerated when administered once daily to rats and dogs, respectively. In 7-day toxicity studies of compound 2 formulated in MV8, the dosing volume was increased in both species in order to maximize both the compound dose and exposure in animals. When administered at a dose volume of 5 mL/kg, MV8 did not cause any toxicity in the rat. However, MV8 administered once daily at 3 mL/kg in the dog produced neutrophilic inflammation in the submucosal gut-associated lymphoid tissue (GALT) of the small and large intestine. Neutrophilic or mixed inflammation unassociated with GALT inflammation was also observed in the mucosa and/or submucosa of the large intestines. These data provide evidence that MV8 vehicle can be successfully utilized in nonclinical studies of shorter duration in the rat up to 5 mL/kg, but dosing volumes should not exceed 2 mL/kg in the dog due to its toxicity and therefore potential to confound interpretation of test article–related gastrointestinal toxicity findings in this species.
P110: Manganese Exposure Induces the Release of Exosomes Containing Misfolded α-Synuclein by Impairing Endosomal Trafficking and Protein Degradation Machinery
D. D. Rokad1, D. Harischandra1, D. Luo1, S. Sarkar1, V. J. Lawana1, D. Kim1, H. Jin1, V. Anantharam1, An. Kanthasamy1, and Ar. Kanthasamy1
1Iowa State University, Ames, IA, USA
Environmental exposure to excessive manganese (Mn) increases risk of chronic neurological diseases, including Parkinson disease (PD). Aggregated α-synuclein (αSyn) is a pathophysiological characteristic of PD. α-Synuclein can be released from neurons by exosomes, facilitating the spread of misfolded proteins, which can trigger a neurotoxic response. We recently discovered that Mn enhances release of misfolded αSyn via exosomes, but the underlying mechanisms are unclear. To understand the Mn-induced exosomal αSyn release, we examined how Mn modulates endosomal trafficking and protein degradation. We exposed MN9D neuronal cells stably expressing human wild-type (WT) αSyn (MN9D-αSyn) to Mn (300 μM) for 24 hours. Manganese significantly suppressed the expression of endosomal recycling protein Rab11a, both at protein and messenger RNA (mRNA) levels, suggesting Mn downregulates endosomal recycling mechanisms, thus forcing late endosomes to mature into multivesicular bodies. Ectopic expression of WT Rab11a significantly mitigated exosome release in untreated and Mn-exposed MN9D-αSyn cells, whereas ectopic expression of mutant Rab11a (S25N) increased exosome release. Intriguingly, our analyses also revealed that Mn exposure upregulated mRNA and protein levels of Rab27a, an endosomal protein that mediates exosome release, suggesting Rab27a upregulation contributes to Mn-induced exosome release. Our analysis of MN9D-αSyn cells shows Mn upregulated the expression of the autophagosomal markers LC3-II and Beclin-1 but downregulated the lysosomal marker LAMP2, suggesting an impairment of autophagolysosome formation following Mn exposure. Results from other lysosome assays, LysoTracker staining, Cathepsin D activity assay, confirmed Mn-induced lysosomal dysfunction. Together, these novel findings demonstrate Mn compromises endosomal trafficking, leading to autophagic/lysosomal impairment, thereby promoting exosomal release of misfolded αSyn.
P111: USP Admissions Evaluation of Coptis Species Rhizome and Indian Barberry Stem: Cautionary Labeling for Drug Interactions
E. Madden1, H. Oketch-Rabah1, B. Gurley2, A. Roe2, and T. Low Dog2
1United States Pharmacopeia, Rockville, MD, USA
22015–2020 USP DSAE JS
3United States Pharmacopeia, Rockville, MD, USA
Coptis Species Rhizome (CSR) and Indian Barberry Stem (Berberis aristata; IBS) are ingredients in some dietary supplements (DS) in the United States. Prior to developing DS quality monographs, the US Pharmacopeia (USP) performs an admission evaluation that includes an assessment based on available information that an ingredient does not present a serious health risk. There is evidence that berberine, an isoquinoline alkaloid present in CSR and IBS, can interact with conventional medications. To assess their interaction potential, we evaluated available clinical, animal, metabolic, and pharmacokinetic data for CSR and IBS. No information on drug interactions was identified for CSR and IBS. In addition, no interactions were apparent in diabetic patients given IBS as an add-on therapy to hypoglycemic and lipid-lowering drugs; however, drug metabolism was not assessed. In the absence of interaction data, berberine and goldenseal (Hydrastis canadensis; another berberine-containing plant) were evaluated. Available clinical data show that berberine and goldenseal can significantly inhibit CYP3A4 and CYP2D6 activity and affect drug clearance. Based on findings for berberine and goldenseal, the USP DSAE Joint Standards-Setting Subcommittee (JS3) determined that a potential for drug interactions with CSR and IBS exists. Thus, in the interest of protecting public health, the USP DSAE JS3 directed that a cautionary labeling statement be included in USP monographs for CSR and IBS as follows: Dosage forms prepared with this article should bear the following statement: “Coptis Species Rhizome or Indian Barberry Stem contain berberine which may interact with medications. Consult your healthcare provider before using.”
P112: In Vitro Whole Blood Clotting Evaluation of Hemostasis Altering Compounds in Rat and Human Using the TEG 5000 Thrombelastograph System
A. Pinçon1, J. E. McCartney1, and F. M. Poitout-Belissent1
1Charles River Laboratories Montreal ULC, Senneville, Quebec, Canada
Thromboelastography (TEG) is a global assay evaluating whole blood clotting for the detection of thrombotic or hemorrhagic events. It is sensitive to coagulation and fibrinolytic factors concentrations and altered platelet counts and function. The purpose of this study was to evaluate the potential use of TEG for in vitro testing of hemostasis-altering compounds. Citrated human and rat whole blood were spiked with heparin, tissue plasminogen activator (tpA), or platelet GIIbIIa inhibitor. Samples were analyzed by TEG after addition of CaCl2 with or without Kaolin activator. On the TEG 5000 System, the spiked blood is placed in an oscillating cup, as whole blood begins to clot, its viscosity increases; a moving pin placed in the cup provides an overview of coagulation processes. The different TEG parameters reflect the reaction time, clot kinetics and strength, and fibrinolysis. With similar CaCl2 treatment, rat samples were hypercoagulable compared with human samples, and kaolin-induced activation was not necessary for rat samples. Heparin-spiked human and rat samples showed increased reaction time, decreased clot kinetics, and decreased clot strength compared with control samples, consistent with decreased coagulability. The tpA-spiked human samples showed decreased clot kinetics and strength consistent with increased fibrinolysis. Platelet GIIbIIa inhibitor–spiked human samples showed increased reaction time and decreased clot kinetics consistent with decreased platelet function. In vitro TEG testing is valuable to help characterize clotting and clot lysis changes in human and rat citrated whole blood samples and to provide information about the potential effect of drug candidates.
P113: Toxicological Effects of Neonicotinoid Pesticide on Developmental Stages of Drosophila melanogaster (Fruit Fly) F1–F3 Generations
F. Doherty1, I. H. Obe1, O. Alafia2, and A. Muoghara1
1Yaba College of Technology, Lagos, Nigeria
2Lagos State University, Lagos, Nigeria
Fruit flies (Drosophila melanogaster) have been employed as classical model for malformation investigations due to their short reproduction cycles and high fecundity. The aim of the study was to evaluate the effects of neonicotinoid pesticide on 3 generations of D melanogaster. Drosophila melanogaster obtained from the wild were crossed and cultured in media containing different concentrations of imidacloprid insecticide (control, 0.1%, 1%, 2%, 5%, and 10%). The flies were cultured for 3 filial generations (F1–F3). The effect on the developmental stages of the flies was assessed by recording the number of eggs, larva, pupa, and adult and larva–pupa emergence period. The conversion rate of egg–larva and larva–pupa was analyzed and the chromosomes preparations were made using the salivary glands of the third instar larvae and incidence of inversions counted and recorded for the 2L, 2R, 3L, and 3R chromosome arms. The emergence time increased, number of progenies decreased, and number of eggs observed reduced with increasing concentrations. The 10% F3 generation produced the most severe effects on fecundity and viability. Chromosome analysis indicated an increase in the number of inversions with higher concentrations. These effects are possibly caused by hormonal disturbances, interference with vitellogenin synthesis, and an egg-laying nonpreference for treated medium. All these effects have the potential of being passed from generation to generation, as observed from the genotoxic effects on the chromosomes. Neonicotinoid pesticides have been proven to possess negative effects on the fecundity and viability of D melanogaster over 3 generations.
P114: Altered Mental Status With Increased Osmolar Gap
H. R. Ahmad1, E. Chang1, C. Odinkemere1, C. Anyanwoke1, A. Chima1, P. Tanksley1, I. Alinnor1, and M. Crawford1
1Morehouse School of Medicine, Atlanta, GA, USA
Isopropyl alcohol is found in common household items and can be easily abused. We present a case of a patient with altered mental status secondary to isopropyl alcohol ingestion.
P115: Withdrawn
P116: Ziram, a Pesticide Associated With Parkinson’s Disease, Increases Excitability and Synaptic Vesicle Release in Aminergic and Glutamatergic Neurons
J. Harrigan1, D. Brambila1, D. E. Krantz1, and F. E. Schweizer1
1University of California, Los Angeles, CA, USA
Exposure to environmental toxins, including the dithiocarbamate fungicide ziram, is linked to an increased risk of Parkinson disease (PD). We hypothesize that molecular targets of ziram could serve as novel entry points into disease-associated pathways. Using calcium indicators, we previously observed that ziram increases excitability in aminergic processes at the Drosophila neuromuscular junction (NMJ). We now extend these findings to show that ziram also increases calcium signals at the level of abdominal ganglion aminergic cell bodies. However, the cells do not exhibit the ziram-triggered calcium response when the processes are severed, indicating that ziram targets distal portions of aminergic processes. Using electrophysiological measurements, we find that ziram affects glutamatergic neurons through 2 distinct mechanisms. First, it increases vesicle release probability at the NMJ, and second, it increases excitability of glutamatergic and aminergic cells. It was previously reported that disruption of protein ubiquitination increases vesicle release in mammalian neurons. Since ziram inhibits the first step of this pathway, we tested ubiquitination inhibitors at the NMJ. Pharmacological disruption of this pathway phenocopied ziram and increased vesicle release probability, indicating that ziram may act through this pathway. Interestingly, these compounds did not change excitability. To investigate ziram’s excitability targets, we used channel mutants to show that the ether-a-go-go potassium channel phenocopies ziram induced excitability but does not exhibit increased release probability. Ziram thus increases both vesicle release and excitability via separate molecular mechanisms in multiple cell types. We are currently investigating how either pathway might contribute to idiopathic PD.
P117: Repeated Ultrasound-Guided Liver Biopsies in Nonhuman Primates
S. Mason1, J. Forget1, D. Benedict1, C. Cruzen1, B. Megrath1, A. Celori1, and K. Watson1
1Altasciences Preclinical Seattle LLC, Everett, WA, USA
Despite several improvements in serology, the need to collect a sample directly from an organ remains a valuable option in nonclinical research. The liver biopsy is an invasive procedure with potential risk for complications. Nonetheless, the procedure has been refined to become a rapid, precise, and safe collection in nonhuman primates (NHPs), with a relatively short recovery period. In order to mitigate the risk associated with liver biopsies, a 2-D ultrasound was used to identify major structures in and around the liver, to allow for the safe insertion of the biopsy needle (14 or 16 G) and to monitor any potential bleeding postretraction of the needle. Ultrasound-guided nonterminal single liver collections have been successfully performed on 150 NHPs. Repeated nonterminal liver collections were performed on 541 NHPs with 2 or 3 biopsies (4-7 days apart) and on 237 NHPs with 6 to 9 monthly collections. Minor complications such as prolonged recovery from sedation, second incision needed, or difficulty penetrating the hepatic capsule were noted for a handful of animals without affecting the health of the animals. A monthly frequency has been shown to be successful for long-term studies requiring up to 9 liver biopsies per animal and no less than 4 to 7 days between occasions for up to 3 biopsies per animal. Based on these observations, ultrasound-guided liver biopsies have been proven to be minimally invasive, safe, and well tolerated in NHPs for nonterminal repeated collections.
P118: Development of an IL-31-Induced Pruritus Model in Cynomolgus Monkeys
J. Forget1
1Altasciences Preclinical Seattle LLC, Everett, WA, USA
Itching and scratching has evolved as an important protection mechanism against various threats to the skin. Pruritus is a severe itching condition most commonly associated with skin disorders such as atopic dermatitis (AD) or psoriasis. Recent clinical trials have demonstrated significant improvement of pruritus in patients with AD after administration of an anti-interleukin-31 (IL-31) receptor antibody. In order to evaluate the efficacy of therapies intended to treat pruritic skin diseases, an IL-31 pruritus cynomolgus monkey model has been established. Cynomolgus monkeys were administered subcutaneous (SC), intradermal (ID), or intravenous (IV) injection(s) of cIL-31, ranging from 0.3 to 24 µg/kg. Pharmacological activity was monitored based on the number of scratching and self-grooming events over a 24-hour period. Intradermal injections of cIL-31 in cynomolgus monkeys at doses of 6, 12, 19.5, and 24 µg/kg resulted in a consistent and robust systemic pharmacological response, as evidenced by a 3- to 6-fold increase from baseline. The pharmacological response was more pronounced between 0.5 and 1.5 hours postdose and appeared to restore nearly to baseline levels by 24 hours postdose. The IV route elicited a comparable scratching response to the highest ID levels tested but with smaller dose levels (0.25-10 µg/kg); SC provided the least useful data. Based on these observations, ID or IV injections of cIL-31 in cynomolgus monkey resulted in a consistent and robust model for future research and development of treatment strategies for AD and other pruritic skin diseases.
P119: The Possible Neuroprotective Role of Resveratrol Supplementation on Arsenic Trioxide–Induced Neurotoxicity in Female Mice Hippocampus
J. Mehta1, B. Kaur1, K. K. Pandey1, and P. Dhar1
1All India Institute of Medical Sciences, New Delhi, Delhi, India
Exposure to arsenic through consumption of contaminated drinking water is associated with affliction of various organ systems. The reports of cognitive deficits following exposure to iAs are suggestive of targeted influence of arsenic on the central nervous system. Hence, the need of the hour is to identify safe therapeutic approaches for amelioration of arsenic-induced toxicity. The present study focused on the role of resveratrol in amelioration of arsenic trioxide (As2O3)-induced alterations on cognition, oxidative stress, and estrogen signaling in female mice hippocampus. Adult female mice were exposed to As2O3 (2 and 4 mg/kg body weight [bw]) alone, resveratrol (40 mg/kg bw) alone, or both by oral route for 45 days. From day 33 to 46 of the experimental period, behavioral tests (OFT, EPM, and MWM) were carried out. On day 46, the animals were sacrificed either by perfusion fixation or by euthanasia, and the brain tissues thus obtained were processed for immunohistochemistry and molecular study, respectively. Behavioral tests revealed enhanced anxiety levels and impairment of cognitive functions in As2O3 alone exposed groups. Oxidative stress markers (GSH and NO) in hippocampal tissue of As2O3 (2 mg/kg bw) alone exposed animals were significantly altered, suggesting As2O3-induced oxidative stress. Arsenic trioxide alone exposed animals also showed apparent reduction in estradiol levels and estrogen receptor β expression in the hippocampus. On the contrary, all these parameters were found to be restored in resveratrol-supplemented animals. These observations add a new perspective to the neuroprotective role of resveratrol on cognition, oxidative stress, and estrogen signaling in the hippocampus of mice following As2O3 exposure.
P120: Generation and Use of Glutathione S-transferase (GST) Humanized Mice to Predict Toxicity of GST Substrates in Humans
K. A. Behrens1, A. Sherry1, R. Dye1, J. P. Parker1, L. A. Jania1, J. N. Snouwaert1, Z. Xiang1, M. S. Nguyen1, and B. H. Koller1
1University of North Carolina at Chapel Hill, NC, USA
Exposure to toxic electrophiles, which can occur through metabolic intermediates or environmental exposures, results in damage to endogenous nucleophiles such as DNA, proteins, and lipids. Protection against this toxicity occurs through the conjugation of these electrophiles to glutathione, which is mediated by the glutathione S-transferase (GST) enzymes. To date, experimental animal models have been used to further our mechanistic understanding of GSTs, but interpreting these results for a human population is complicated by interspecies differences in the number and structure of these genes. To address this problem, we generated a panel of mice in which mouse GST genes were deleted and populated with their syntenic GST Pi, GST Mu, and GST Theta human loci and thus express the human GST isoforms. These GST families were selected for humanization because they harbor common polymorphisms in the human population that are associated with altered disease risk and response to treatment regimes. We used the model substrate benzo[a]pyrene (B[a]P) because of its well-documented toxicity and the role of GSTs in inactivating its toxic phase I metabolites. We demonstrated a critical role for GSTs in B[a] P metabolism and protection against organ damage and assigned this protection to specific human and mouse GST gene families. The humanized mice are therefore a useful model for testing the toxicity of compounds that are detoxified by GSTs for phase II metabolism. Furthermore, these humanized mice can be used for future individualized risk assessments in people carrying common disease-associated GST polymorphisms, such as the null GSTT1 and GSTM1 alleles.
P121: Rutin Attenuates Manganese-Induced Hippocampal and Striatal Toxicity via Inhibition of Iron Depletion, Oxidative Stress, Inflammation, and Suppressing the NF-κB Signaling Pathway
K. W. Nkpaa1, G. I. Onyeso2, and K. Z. Kponee3
1University of Port Harcourt, Port Harcourt, Rivers, Nigeria
2Rivers State University, Port Harcourt, Rivers, Nigeria
3Harvard T.H. Chan School of Public Health, Boston, MA, USA
Excess exposure to manganese (Mn) promotes oxidative stress and neuroinflammation. Rutin (RUT) has been found to exhibit both antioxidative stress and anti-inflammatory properties. This study aimed to investigate the effects of RUT on Mn accumulation, endogenous iron (Fe) depletion, oxidative stress, inflammation, and nuclear factor kappa B (NF-κB) signaling pathways in the hippocampus and striatum of Mn-induced rats. Rats were treated with 30 mg/kg Mn body weight alone or orally cotreated by gavage with RUT at 50 and at 100 mg/kg body weight for 35 consecutive days. Thereafter, we investigated Mn and endogenous Fe levels, acetylcholinesterase activity, oxidative stress markers, pro-inflammatory cytokines, and NF-κB in the hippocampus and striatum of rats. The results indicate that Mn induced Mn accumulation, Fe depletion, oxidative stress, inflammation, and the activation of acetylcholinesterase activity and NF-κB signaling pathways in the hippocampus and striatum of the rats. However, RUT attenuated Fe depletion, oxidative stress, and inflammation and suppressed acetylcholinesterase activity and NF-κB pathway via downstream regulations of tumor necrosis factor α, interleukin Iβ, and interleukin 6. Taken together, our present study demonstrates that RUT abrogates Mn-induced striatal and hippocampal toxicity via inhibition of Fe depletion, oxidative stress, neuroinflammation, and suppressing the NF-κB signaling pathways. Our results indicate that RUT may be of use as a neuroprotective agent against Mn-induced neuronal toxicity.
P122: MicroRNA as Regulators of 1,3-Butadiene-Induced Strain- and Tissue-Specific Effects in Mice
L. Lewis1, G. Chappell1, J. Blazier1, K. Konganti1, J. Israel2, J. Simon2, K. Eklund2, T.S. Furrey2, and I. Rusyn1
1Texas A&M University, College Station, TX, USA
2University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
1,3-Butadiene is a known human carcinogen that is both an occupational and an environmental health hazard. Genotoxicity is an established mechanism of 1,3-butadiene carcinogenicity; however, it does not explain the tissue-specific tumor development observed in mice. Our previous work demonstrated strain- and tissue-specific alterations in epigenetic effects in response to 1,3-butadiene exposure, which may contribute to tissue-specific toxicity. MicroRNA (miRNA) represents another epigenetic mechanism for regulating gene expression posttranscriptionally and has been implicated in carcinogenesis. In this study, we tested the hypothesis that miRNA regulate strain- and tissue-dependent transcriptional and epigenetic responses to 1,3-butadiene exposure in CAST/EiJ and C57BL/6J mice. These mice were exposed to 0 or 625 ppm 1,3-butadiene (6 hr/d, 5 d/wk) for 2 weeks. We evaluated changes in messenger RNA (mRNA) and miRNA expression by sequencing the lung, liver, and kidney tissues. We observed strain- and tissue-specific mRNA and miRNA expression profiles in response to 1,3-butadiene exposure. The miRhub algorithm was used to predict miRNAs that are master regulators of 1,3-butadiene-induced mRNA expression. We identified 9 miRNA as significant candidates across tissues and strains. In the lung of 1,3-butadiene-exposed C57BL/6J mice, mir-142-5p was significantly decreased and predicted to target significantly upregulated genes involved in chromatin remodeling such as KDM1B, a lysine demethylase for H3K4, a marker of transcription. In addition, mir-142-5p was predicted to target genes involved in DNA damage repair such as CtIP. The results of this study indicate that miRNA may mediate strain- and tissue-dependent variability of 1,3-butadiene-induced epigenetic effects and potentially greater tissue susceptibility to carcinogenesis.
P123: The Placenta: A Mediator of Thyroid Hormone Disruption Following Gestational Exposure to Flame Retardants
M. T. Ruis1, and H. M. Stapleton1
1Duke University, Durham, NC, USA
The placenta is an ephemeral organ composed of both maternally and fetally derived tissues separated by a semipermeable membrane that facilitates the exchange of nutrients, gases, hormones, and waste between the mother and fetus. Numerous studies have shown that this barrier is permeable to environmental pollutants such as flame retardants (FRs), which are ubiquitously detected in human serum, breast milk, and cord blood. Flame retardants have also been shown to disrupt thyroid hormone homeostasis. In this study, Wistar rats were orally exposed to a mixture of FRs for 10 days during gestation. Thyroid hormone levels in dam serum, fetuses, and their paired placenta were quantified. The placental tissue was microdissected to separate fetal from maternal placental tissue to better understand the how thyroid hormone levels differ within specific placental tissues. We observed a significant dose effect of FRs on T3 levels in dam serum. Our data suggested reduced T3 levels in the maternal portion of the placenta while increased T3 levels in fetuses, signifying that the placenta may be a mediator of thyroid hormone disruption during pregnancy. Our data indicated sex-specific differences in T3 levels in the maternal portion of the placenta. Additionally, we observed a significant difference of T3 in the maternal portion of the placenta based on gestational age of the fetus. These data suggest it is critical for future studies to consider the sex, placental tissue type, and gestational age of the fetus when drawing conclusions on the impact of a xenobiotic on thyroid hormone homeostasis.
P124: Changes in the Renal Function Biomarker, Symmetric Dimethylarginine (SDMA), Following Gentamicin-Induced Proximal Tubular Injury in the Rat
D. M. Hamlin1, A. E. Schultze1, D. J. McCrann III2, R. Mack2, C. Drake2, R. E. Murphy2, J. A. Cross2, M. Strong-Townsend2, and M. J. Coyne2
1Eli Lilly and Company, Indianapolis, IN, USA
2IDEXX Laboratories, Inc., Westbrook, ME, USA
Techniques for measuring renal function in rodent renal disease models include direct measurement of glomerular filtration rate (GFR) or indirect methods such as evaluating serum creatinine (sCr), blood urea nitrogen, or the presence of proteinuria. Symmetric dimethylarginine (SDMA) is a dimethylated derivative of arginine which is eliminated primarily by renal clearance and shown to be an accurate and precise biomarker for determining estimated GFR. The purpose of the study was to evaluate SDMA in rats following gentamicin-induced renal tubular injury. Eighty male Sprague Dawley CD IGS rats were assigned to one of 4 dosage groups (0, 20, 50, and 100 mg/kg gentamicin sulfate). Rats were dosed for 4 or 10 days and creatinine clearance was used to determine estimated GFR for each rat at the end of the dosing period. Kidney histopathology and concentrations of sCr, serum SDMA, and cystatin C, along with the urine biomarkers, µAlbumin, clusterin, cystatin C, KIM1, NGAL, and osteopontin, were measured. In the 4 treatment dose groups, there was a slight, nonsignificant decrease in creatinine clearance as the dose of gentamicin increased but a significant decrease in creatinine clearance with the 10-day dosage regimen (P < 0.001). A significant (P < 0.01) negative correlation was found between creatinine clearance and SCr and SDMA. No correlation was found between creatinine clearance and serum cystatin. Significant (P < 0.01) positive correlations were found between SDMA and sCr, urinary clusterin, urinary µALB, and kidney pathology scores. Symmetric dimethylarginine was found to be a good estimator of changes in GFR in rats with gentamicin-induced tubular injury.
P125: Changes in Renal Function Biomarkers, Including Symmetric Dimethylarginine (SDMA), Following the Induction of Passive Heymann Nephritis (PHN) in the Rat
D. M. Hamlin1, A. E. Schultze1, D. J. McCrann III2, R. Mack2, C. Drake2, R. E. Murphy2, J. A. Cross2, M. Strong-Townsend2, M. J. Coyne2, and K. Riley2
1Eli Lilly and Company, Indianapolis, IN, USA
2IDEXX Laboratories, Inc., Westbrook, ME, USA
Symmetric dimethylarginine (SDMA) is a dimethylated derivative of arginine which is eliminated by renal clearance and shown to be an accurate and precise biomarker for determining estimated glomerular filtration rate (eGFR). The purpose of this study was to evaluate SDMA in rats following induction of passive Heymann nephritis. Ninety-six male Sprague Dawley CD IGS rats were used and assigned to one of 2 groups (control and treatment). Passive Heymann nephritis was induced using commercial sheep anti-Fx1A serum; 0.9% sodium chloride for injection was used as vehicle control. Rats were dosed once at 7.5 mL/kg intravenously, and 12 rats were sacrificed on day 9, 16, 21, and 28 after treatment. Creatinine clearance was used to determine eGFR for each rat 1 day prior to sample day. Kidney histopathologic examination and concentrations of serum creatinine (sCr), SDMA, and cystatin C, along with the urine biomarkers, µAlbumin, clusterin, cystatin C, KIM1, NGAL, and osteopontin, were measured. There were no significant changes in creatinine clearance, SDMA, sCr, or cystatin C concentrations between the 2 groups for the duration of the study. Time progressive increases in urinary albumin and clusterin concentrations and increases in urinary cystatin C, NGAL, and KIM1 concentrations that were not progressive over time were observed. No changes in urine osteopontin concentrations occurred at any time. The lack of change in SDMA, sCr, and serum cystatin C levels indicates that minimal loss of kidney function occurred in this model over the dose and time period chosen for the study.
P126: Neuromuscular and Cardiac Deficits in Acid α-Glucosidase (GAA)-Deficient Mouse Model of Pompe Disease
M. Eggers1, M. Harter2, D. Gauvin2, R. Pussinen3, J. Brassard4, and J. J. Cunningham1
1Audentes Therapeutics, San Francisco, CA, USA
2Charles River Laboratories, Mattawan, MI, USA
3Charles River Laboratories, Kuopio, Finland
4Jacqueline Brassard Toxicologic Pathology Consulting, Tustin, CA, USA
Pompe disease is a genetic neuromuscular disorder caused by the loss of acid α-glucosidase (GAA), an enzyme critical for glycogen metabolism. This results in glycogen accumulation predominantly in skeletal and cardiac muscle. Mice deficient for GAA are currently used as a model for Pompe disease. While the histopathology in GAA−/− mice closely mimics the human disease, the neuromuscular and cardiac functional deficits are poorly characterized. The neuromuscular behavior was evaluated by accelerating rotarod and inverted screen test together with cardiac function by implanted telemetry electrocardiogram (ECG) in male and female GAA−/− mice at 3 months of age and followed for 3 months. During rotarod evaluation, animals were placed on a rotating rod set to accelerate from 0 to 40 rpm over 3 minutes. For grip response, animals were placed on an 8 × 12 cm inverted wire screen and time to fall was measured. At baseline, GAA−/− mice showed an ∼15% deficit in fall time compared with wild-type littermate controls for both the rotarod and inverted screen test. Six weeks later, mice showed similar differences compared with controls. Quantitative and qualitative ECG data were assessed at baseline (3 months) and 12 weeks later (6 months). Functional deficits (rotarod, grip response, and ECG) correlated with histopathologic data that showed glycogen accumulation in the skeletal and cardiac muscles of these mice, despite high interanimal variance. These data show that studies must be powered to see differences. These data show that GAA−/− mice exhibit both biochemical and functional deficits similar to human Pompe disease.
P127: An Acute Intravitreal Toxicity and Toxicokinetic Study of Nanolipoprotein Conjugated Fab (RabFab-NLP) in New Zealand White Rabbits
N. G. Tassew1, S. T. Laing1, K. Loyet1, S. Crowell1, C. Schuetz1, C. Blanchette1, and W. Shatz1
1Genentech, Inc, South San Francisco, CA, USA
The objective of this study was to evaluate the ocular toxicity and toxicokinetics of RabFab-NLP administered intravitreally to male New Zealand white rabbits. RabFab-NLP is a novel conjugate designed to prolong ocular half-life through an increase in hydrodynamic radius compared with Fab alone (∼12 vs ∼3 nm). Assessment of toxicity was based on mortality, clinical signs, body weight, ophthalmic evaluation, and microscopic assessment of ocular tissues. RabFab-NLP-related vitreous opacity and lens opacity were observed as early as day 3 during slit-lamp examination. Microscopic findings were present in the lens, vitreous cavity, and/or optic nerve head. In the lens, findings included cortical liquefaction, subcapsular lens epithelium proliferation, and/or cortical vacuoles in the posterior cortex. Very slight peripheral fibrillar material and mononuclear cell infiltrate were present in the vitreous cavity, and mononuclear cell infiltrate was present in the optic nerve head. Toxicokinetic behavior was consistent with a prolonged ocular half-life of RabFab-NLP (serum t½ = 5.5 days) compared with RabFab alone (serum t½ = 3.1 days), and ∼1.75-fold prolongation in half-life for RabFab-NLP compared with RabFab alone is consistent with what was expected based on the increase in hydrodynamic radius. However, the observed toxicokinetic data were highly variable between individual animals before and after the appearance of antidrug antibodies in all RabFab-NLP-treated animals and none of the RabFab-treated animals. Based on the ocular toxicity observed, it was concluded that a single intravitreal dose of RabFab-NLP administered at 1.3 mg/eye exceeded the maximum tolerated dose in rabbits.
P128: Impact of Hepatic CYP-Mediated Metabolism on the Tissue Distribution and Persistence of Inhaled Naphthalene
N. Kovalchuk1, L. Van Winkle2, and X. Ding1
1University of Arizona, Tucson, AZ, USA
2University of California at Davis, Davis, CA, USA
The in vivo persistence of lipid-soluble toxicants, such as naphthalene, can be affected by the body mass and by the rate of its biotransformation to water-soluble metabolites. Naphthalene bioactivation by P450 enzymes in the lung is the major mechanism for naphthalene-induced airway damage. Here, we aimed to identify factors that modulate the level or duration of lung exposure to naphthalene, which will likely influence the extent of lung toxicity. We examined naphthalene levels in various tissues following the termination of a 4-hour inhalation exposure to naphthalene at 10 ppm, comparing between wild-type (WT) mice on a regular diet and those on a high-fat diet to induce obesity, and between WT and liver-Cpr-null (LCN; deficient in hepatic naphthalene metabolism) mice that were fed a regular diet. At 1 to 20 hours after termination of naphthalene inhalation, naphthalene levels were the highest in white adipose tissues, compared with liver, lung, and kidney, in all mice. Tissue levels of naphthalene (ng/g tissue) were not increased in obese, compared to nonobese, WT mice, though there was a small increase in the lung at 1 hour. However, naphthalene levels were generally much higher in LCN than WT mice in all organs tested at various postexposure times (7- to 17-fold higher at 1 hour). These results indicate that for naphthalene, rate of hepatic metabolism is more important than capacity of tissue storage in determining persistence of exposure in the lung and suggest that a combination of low hepatic metabolism and obesity may further enhance lung toxicity. (Supported in part by NIEHS grant ES020867.)
P129: Environmental Concentrations of Triclosan and Triclocarban: Genotoxic, Embryotoxic, and Histological Effects in Various Life Stages of the African Sharptooth Catfish
R. O. Jimoh1, and T. O. Sogbanmu1
1University of Lagos, Lagos, Nigeria
Antimicrobial additives in personal care products (PCPs) such as triclosan (TCS) and triclocarban (TCC) are of significant environmental concern due to their potential toxicity to nontarget aquatic organisms. We investigated the genotoxic, embryotoxic, and histological effects of environmentally relevant concentrations (sublethal) of TCS and TCC in the African sharptooth catfish (Clarias gariepinus). The methods utilized were relative acute toxicity studies in C gariepinus embryos and fingerlings; micronucleus assay (genotoxicity studies); acute toxicity, hatching success, number of heart beats per minute, and developmental abnormalities in C gariepinus embryos from 0 to 72 hours postfertilization (embryotoxicity studies); and gill and liver histological evaluations. All studies were conducted according to standard methods. The acute toxicity studies revealed that the 24-hour LC50 and 26 hour EC50 (nonhatching) values for C gariepinus embryos were 16.48 and 11.07 mg/L, respectively for TCS; 46.08 and 41.93 mg/L, respectively, for TCC. The 96-hour LC50 of TCS and TCC against C gariepinus fingerlings was 16.04 and 41.56 mg/L, respectively. There was a significant dose-dependent increase (P < 0.05) in micronuclei and binucleated cells in the erythrocytes of exposed fishes compared with the control. Hatching success, number of heart beats per minute, and percentage abnormalities in the treated embryos decreased statistically (P < 0.05) compared with control. Mild to severe lamellar necrosis was observed in the gills of treated C gariepinus over the period of 28 days. The study demonstrates the need for regulatory measures and monitoring of the use of TCS and TCC in PCPs in order to mitigate potential adverse effects to aquatic organisms.
P130: Hematology and Biochemistry Reference Data in Juvenile Rats at 4, 7, and 21 Days of Age
B. Attalla1, A. Fotovati1, and J. Younan1
1ITR Laboratories Canada Inc., Baie-D’Urfé, Quebec, Canada
Due to physiologic differences between neonates, children, and adults, juvenile animal models of relevant age must be used as test systems in nonclinical studies in order to evaluate the safety of drugs targeting pediatric human patients. Inherent differences exist between mature and immature biological systems. As such, certain markers of toxicity, such as the clinical pathology parameters, of juvenile animals differ from those of their adult counterparts. Therefore, for the interpretation of results observed in juvenile toxicology studies, age-specific sets of standard clinical pathology parameters are needed for reference. In order to obtain reference hematology and clinical chemistry data from naive Sprague Dawley rat pups, our laboratory collected, processed, and analyzed samples from a total of 391 male and female animals at postnatal days 4, 7, and 21. The standard hematology and clinical chemistry parameters used in general toxicology studies were measured from rat pups at each of the critical stages mentioned above. Each parameter was statistically analyzed and compared with the data sets available from adult Sprague Dawley rats. The data analyses showed important differences in the early stages of development when compared with data obtained from adult rats. In addition, the clinical pathology data obtained from rat pups at different stages of their development can be used as a reference range for juvenile toxicology studies.
P131: Effects of Intravenous or Subcutaneous Polysorbate 80 (Tween 80) in Telemetered Beagle Dogs
R. Tavcar1, C. Li1, E. Boulay1, S. Maghezzi1, I. Reeves2, M. Demers2, E. Gazaille2, and S. Authier1
1Citoxlab, a Charles River Company, Laval, Quebec, Canada
2Université de Montréal, St-Hyacinthe, Quebec, Canada
Polysorbate 80 is a nonionic surfactant and emulsifier commonly used as a vehicle in preclinical formulations. Polysorbate 80 is associated with immunoglobulin E-independent anaphylactoid reactions in various species, including humans, and with dogs recognized as the most sensitive species. Doses of 0, 0.3, and 1 mg/kg were given as an intravenous (IV) bolus or subcutaneously (SC) to beagle dogs following a Latin square with continuous telemetry cardiovascular monitoring for 24 hours. A separate dose of 3 mg/kg was administered as a 15-minute IV infusion with and without diphenhydramine (1 mg/kg, SC) pretreatment with histamine concentration measurements and cardiovascular monitoring by telemetry. Doses of 0, 0.3, and 1 mg/kg SC were not associated with any significant effects, while one out of 4 dogs showed transient facial edema at 1 mg/kg IV but with minimal cardiovascular changes. At 3 mg/kg, body temperature was noted to decline with a −2°C nadir achieved at 45 minutes postdose and recovery to baseline comparable values by 105 minutes postdose. Parallel to the cardiovascular changes, plasma histamine concentrations peaked at 20-minute postdosing at 3 mg/kg and diphenhydramine were associated with a 3-fold reduction in histamine release with a significant attenuation of cardiovascular effects. In conclusion, polysorbate 80 was not associated with any significant effect at 1 mg/kg SC or at 0.3 mg/kg IV.
P132: Development of Exosome-Based Biomarkers for Manganese Neurotoxicity
S. Manne1, N. Kondru1, H. Jin1, E. Y. Lee2, M. Lewis2, V. Anantharam1, X. Huang2, A. Kanthasamy1, and A. Kanthasamy1
1Iowa State University, Ames, IA, USA
2Penn State Milton S. Hershey Medical Center, Hershey, PA, USA
Long-term exposure to manganese (Mn) is known to cause neurological, parkinsonian-like symptoms in exposed individuals by affecting the extrapyramidal motor control system. Occupationally exposed individuals like welders are at high risk due to constant exposure to Mn-rich welding fumes. No blood-based biomarkers are available for Mn poisoning in humans except for expensive imaging techniques, like brain magnetic resonance imaging. In the present study, we developed a rapid, sensitive quantification method that detects α-synuclein aggregation by a real-time quaking-induced conversion (RT-QuIC) assay in Mn-exposed individuals. We generated a high-quality recombinant human wild-type α-synuclein protein as a substrate for this assay and optimized the RT-QuIC assay conditions to quantify the α-synuclein-seeded aggregation. Moreover, we determined the diagnostic utility of the α-synuclein RT-QuIC assay using exosomes isolated from a blinded cohort of serum and plasma samples from a population exposed to welding fumes and age-matched controls. We could differentiate welders from controls with >95% sensitivity and specificity with the RT-QuIC assay, suggesting that exosomal α-synuclein aggregates may serve as a circulating biomarker for Mn neurotoxicity. Furthermore, next-generation sequencing of serum exosomes showed changes in several small RNAs, such as microRNAs, Piwi-interacting RNAs, and transfer RNAs, that may potentially contribute to Mn neurotoxicity and thus could also evolve as potential biomarkers. Collectively, our findings demonstrate the use of a sensitive, high-throughput RT-QuIC assay for discovering α-synuclein-based biomarkers and identify potential exosome-associated small RNA biomarkers. (Acknowledgements—ES 026892, Lloyd and Armbrust endowments.)
P133: Obesity and Prostate Cancer Progression: Role of the Chemokine CXCL12
S. Ahn1, A. Saha1, J. DiGiovanni1, and M. Kolonin2
1University of Texas at Austin, TX, USA
2The University of Texas Health Sciences Center at Houston, TX, USA
Obesity is a significant risk factor for several cancers in both men and women. A number of studies have shown that obesity is associated with increased prostate cancer (PCa) progression and higher mortality. However, the mechanism(s) for obesity-driven PCa remain unclear. Previously, we showed that obesity enhances PCa progression by changing the surrounding tumor microenvironment, especially the white adipose tissue (WAT). The WAT consists of many cells, including inflammatory cells, mature adipocytes, and adipose stromal cells (ASCs). Here, we further investigated the role of ASCs and ASC-derived CXCL12 in obesity-driven PCa progression. In HMVP2 cells, expressing high levels of CXCR4, we observed activation of MAPKs, AKT, STAT3, and NFkB following treatment with CXCL12. These CXCL12-induced signaling pathways were inhibited by treating with AMD3100, a CXCR4 antagonist, and also by CXCR4 knockdown. Our data demonstrate that obesity promotes EMT in tumor cells and increases invasion of surrounding WAT into the tumor stoma. Treatment of mice with AMD3100 significantly inhibited the growth of HMVP2 tumors and suppressed EMT in obese mice. Furthermore, D-CAN, a hunter-killer peptide developed for targeting ASCs, also suppressed obesity-induced EMT and tumor growth in HMVP2 tumor allografts. Lastly, AMD3100 in combination with D-CAN showed a significant reduction in PCa growth in obese mice compared with either agent used alone. These results provide evidence that ASCs activate the CXCL12/CXCR4 signaling as well as other pathways playing an important role in obesity-driven PCa progression. Our data suggest this pathway as a novel therapeutic target for inhibiting obesity-driven PCa growth and progression.
P134: Comparison of Organ Weight (Absolute and Relative to Brain) in Wistar Han Rats at Different Ages
S. McPherson1, and E. Wang1
1WuXi AppTec (Suzhou) Co Ltd, Suzhou, Wuzhong, China
Organ weights are routinely assessed during the conduct of toxicology studies. Differences in organ weight both relative and absolute in Wistar Han rats at 3 different ages (11-12, 20-21, and 33-34 weeks) were summarized. The data sets were analyzed using graphs of organ weight to body weight and organ weight to brain weights and further analyzed looking at the Y-intercepts, slopes, correlation coefficient, and coefficients of determination. The analysis showed: organs with highest relative growth rates: thyroid > liver > heart > kidneys > lungs > spleen > pituitary. Decreases in relative growth rates in absolute weights in males and/or females: thymus > adrenal glands. Male organs with highest relative growth rates in absolute weights: prostate > epididymis > testes. Female organs with highest relative growth rates in absolute weights: uterus > ovaries. Based on these results, it is suggested to interpret organ weight data based on absolute weights and ratios to body weight for those with relatively higher correlation index with body weight and based on absolute body weights for those with little or no correlations with either body/brain weights. The age ranges investigated have generally covered that of rats used on repeat-dose toxicology studies ranging from 2 to 26 weeks. Therefore, these background data are used in distinguishing changes due to treatment and variation; also identified were the more sensitive organs in high/low relative growth rates and sex differences, which can be used as scientific justification when reviewing trends in organ weights in long-term studies.
P135: Alterations of Expression of RNA Modification Regulators by Carcinogens in the Alternative Chicken Egg Model
T. Kobets1, J.-D. Duan1, K. D. Brunnemann1, D. A. Iacobas2, S. Iacobas1, E. Vock3, U. Deschl3, and G. M. Williams1
1New York Medical College, Valhalla, NY, USA
2Prairie View A&M University, Prairie View, TX, USA
3Boehringer Ingelheim Pharma GmbH&Co. KG, Biberach an der Riss, Baden-Württemberg, Germany
The role of RNA modifications in the development of pathologic conditions, including chemical carcinogenesis, remains unclear. The dynamic nature of the epitranscriptome modifications is regulated by enzymes, such as methyltransferases, demethylases, and pseudoU-synthetases. The current study utilized microarray platform to investigate the presence and expression of genes that encode for the aforementioned enzymes in the alternative to animal chicken egg model (CEM) after 3 daily injections with 10 established genotoxic and epigenetic carcinogens and their comparators, including dialkylnitrosamines, aromatic amines, polycyclic aromatic hydrocarbons, aflatoxins, clofibric acid, and phenobarbital. The CEM uses metabolically active livers from embryo–fetal chicken organisms, which by definition are not yet subjected to regulation for animals, collected 3 hours after the last dosing on incubation day 11. Chemical-specific deregulation of 21 genes that encode for several RNA modification enzymes in various species, including humans, was observed in the CEM. Genotoxic hepatocarcinogens dialkylnitrosamines produced most significant changes. Specifically, upregulation of demethylases ALKBH5, FTO, and ALKBH3 and downregulation of pseudoU-synthase genes PUS1, PUS10, PUS7, and PUSL1 were observed, among other changes. The majority of genotoxic hepatocarcinogens also altered the expression of several small nucleolar RNAs, including SNORD17, SNORA62, and SNORA81, which guide methylation and pseudouridylation of ribosomal RNA and transfer RNA. Other genes that can be potentially involved in the epitranscriptome alterations were detected. These findings lead to the hypothesis that chemicals can produce epitranscriptome modifications, which in combination with established genotoxic and/or epigenetic effects contribute to carcinogenesis. Based on our findings, CEM is an appropriate model to investigate this hypothesis.
P136: Changes in Mouse Lung and Blood Transcriptomes Upon Pulmonary Exposure to Multi-Walled Carbon Nanotubes
T. O. Khaliullin1, M. S. Newman1, L. M. Fatkhutdinova2, N. Yanamala3, E. R. Kisin3, and A. A. Shvedova3
1West Virginia University, Morgantown, WV, USA
2Kazan State Medical University, Kazan, Republic of Tatarstan, The Russian Federation
3National Institute for Occupational Safety and Health, Morgantown, WV, USA
Pulmonary exposure to multiwalled carbon nanotubes (MWCNT) has been shown to cause local inflammation, fibroproliferation, and immunotoxicity in small animal studies. However, the search for representative biomarkers of exposure is an ongoing endeavor. Whole blood gene expression profiling is a promising new approach to identification of novel biomarkers over traditional, invasive biopsy. The goal of this study was to look for correlates between messenger RNA (mRNA) expressions in lung tissue and in whole blood after MWCNT exposure. Mice were administered 40 µg of MWCNT or US Pharmacopeia (USP)-grade phosphate-buffered saline via pharyngeal aspiration and sacrificed 56 days after treatment. RNA microarray studies were performed alongside histopathology of lung sections. Ingenuity pathway analysis indicated inflammatory response and connective tissue organization markers, with an emphasis on immune cell trafficking, phagocytosis, and adaptive immune responses. Hematopoiesis and activation of cancer/tumor development pathways had the opposite activation status in lungs and blood. The only common disorder between lungs and blood was “hypersensitivity.” There were several common, upregulated genes between whole blood and lungs, important for the adaptive immune responses: cxcr1, cd72, sharpin, and slc11a1. Trim24, important for TH2 cell effector function, was downregulated in both data sets. Hla-dqa1 (MHC-II) mRNA was upregulated in the lungs and downregulated in the blood, as was lilrb4, which controls the immune response reactivity. In conclusion, we have shown that transcriptome changes occurring in the lungs of MWCNT-exposed mice do not necessarily produce a replicable pattern in the whole blood; however, specific systemic responses may be shared between transcriptomic profiles.
200 Series—Regulatory Toxicology
P201: Evaluation of the Utility of ToxCast HTS and High-Throughput Toxicokinetic Data for Food Chemical Safety Risk Assessment via Comparison With In Vivo Animal Data
A. E. Turley1, J. Zang1, K. Paul Friedman2, R. S. Judson2, and S. C. Fitzpatrick1
1US FDA, College Park, MD, USA
2US EPA, Research Triangle Park, NC, USA
New approach methodologies (NAMs) are currently being developed and evaluated for use in chemical safety risk assessment, including chemicals used in food. New approach methodologies include in vitro high-throughput screening (HTS) assays, such as the ToxCast and Tox21 assays. The ToxCast/Tox21 assays have been run for thousands of compounds, including hundreds of compounds used in food. However, the relationship of these NAM data with traditional in vivo animal data, and the utility of NAMs for risk assessment, remains under evaluation. The goal of the present study is to evaluate the utility of ToxCast/Tox21 HTS data in food safety risk assessment. To do this, bioactive concentrations of a subset of food use compounds in ToxCast were converted to oral equivalent doses (OEDs) via in vitro to in vivo extrapolation using either in vitro– or in silico–based toxicokinetic parameters for a subset of food use compounds. These OEDs were then compared with doses demonstrated to cause effects in in vivo animal tests (using data compiled by US Environmental Protection Agency [EPA] and US Food and Drug Administration). Initial comparisons demonstrated great variability in the correlation between ToxCast and in vivo data, so steps are being taken to further refine the toxicokinetic information, chemical groups, and in vivo end points in an effort to identify additional information and conditions necessary to utilize HTS data for preliminary food safety assessment. This work does not reflect the official policy of the US EPA.
P202: Application of Read-Across and Low-Hazard Approaches in the Human Health Risk Assessment of Existing Chemicals Under the Chemicals Management Program (CMP): A Case Study on Nonane, 2,2,4,4,6,8,8-Heptamethyl (HMN)
A. Luu1, C. Campbell1, S. Mohr1, A. Zidek1, V. Bergeron1, and S. Kuchta1
1Health Canada, Ottawa, Ontario, Canada
In 2016, the Government of Canada launched the third phase of the Chemicals Management Program. This phase is addressing the remaining 1,550 priority chemicals of the original 4,300 chemicals identified as priorities for assessment. To improve efficiencies and to address challenges for substances that lack sufficient data on potential health effects, Science Approach Documents (SciADs) and read-across approaches have been developed. Depending on the nature of the substance, some of these approaches may be combined in a human health risk assessment context. In 2017, the Government of Canada published a Science Approach Document for Substances With Low Human Health Hazard Potential, which represents a hazard-based evaluation framework for substances that have inherently low toxicity. For these substances, a qualitative approach to risk characterization may be considered, thereby streamlining the human health risk assessment. The utilization of this approach is informed either by substance-specific data or by data acquired through read-across. Read-across represents another approach whereby available information from a similar chemical (analogue) is extrapolated/interpolated to a chemical lacking data on one or more hazard end points (target chemical). The read-across process involves a series of steps, the principal ones being problem formulation, profiling the target chemical, identification/characterization of analogues, data gap filling, and uncertainty assessment. A case study on nonane, 2,2,4,4,6,8,8-heptamethyl (also referred to as heptamethylnonane) is presented, whereby substance-specific information and data acquired from read-across provided the foundation for the proposed use of a “low-hazard” approach, as outlined in the Substances With Low Human Health Hazard Potential SciAD.
P203: BioCelerate Protocol Harmonization Initiative
M. A. Carfagna1, P. R. Brinck2, K. Matsuyama3, T. Fukushima3, G. E. Schulze4, C. Clayton4, and T. Page1
1Eli Lilly and Company, Indianapolis, IN, USA
2Novo Nordisk, Copenhagen, Byen, Denmark
3Shionogi & Co, Ltd, Osaka, Kansai, Japan
4Bristol-Meyers Squibb, Jersey City, NJ, USA
BioCelerate, a subsidiary of TransCelerate BioPharma, Inc, is a preclinical industry consortium driving initiatives to increase efficiency and productivity in R&D. The objective of the common templates for nonclinical studies initiative is to drive efficiency in the preclinical space by decreasing the time to develop a protocol and thus start a study. An additional objective of the initiative is to improve overall study quality by decreasing errors due to unfamiliarity with protocol formatting. The initial focus is on developing a protocol template for FIH-enabling repeat-dose general toxicology studies. BioCelerate is collaborating with various stakeholders (contract research organizations, health authorities, biopharma, software vendors, and standard setting bodies) to evaluate options and potential recommendations for protocol content and structure. Input from these stakeholders will ensure that the protocol template will achieve our stated objectives of improving toxicology study efficiency and quality, while also being implementable by a wide range of stakeholders. To prepare a framework for engagement, the consortium has created an initial protocol template for stakeholder input. The process to develop the initial protocol template utilized the OECD Good Laboratory Practice as a starting point and excluded process instructions where large variations in preferences exist. A webinar was held on February 26, 2019, to introduce the initiative and collect feedback from stakeholders. Responses to poll questions indicated that a majority of stakeholders currently use multiple protocol templates and experience problems associated with protocol inconsistency, including process/time inefficiencies and reduced quality of study execution and a mixed response regarding the inclusion of SEND information in the protocol.
P204: Classification of EPA Ocular Irritants and Nonirritants by the OptiSafe Test Method
P. Vij1, S. J. Lebrun2, and G. L. DeGeorge1
1MB Research Laboratories, Spinnerstown, PA, USA
2Lebrun Labs LLC, Anaheim, CA, USA
The Environmental Protection Agency (EPA) eye irritation classification system is routinely used to categorize ocular toxicity. This system classifies chemicals that damage the eye after 24 hours (category III, II, or I) and those that do not cause damage (category IV). This latter classification is aligned with the standard definition of an “ocular nonirritant.” The OptiSafe test is a novel, shelf-stable, in chemico method that can be used to discriminate ocular irritants/corrosives from nonirritants without use of animal tissues or cells. OptiSafe measures ocular damage via a proxy for the corneal stroma (water-soluble molecules), damage to phospholipid bilayers, and the potential to induce pH extremes in a system (pH buffering system of the eye). Chemicals in this study were selected based on a wide range of EPA classifications, chemical and physical properties, high-quality in vivo reference data, and chemical stability. Selected chemicals (37) including surfactants not previously tested were aliquoted into coded vials and tested blind in triplicate. The coded vials were tested, and results were reported as either EPA category IV (nonirritant) or not (EPA category III, II, or I). The OptiSafe test method applied to these 37 test chemicals achieved a sensitivity of 100% (26/26), specificity was 81.8% (9/11), and overall accuracy was 94.6% (35/37). These results suggest that OptiSafe may be an important tool in the complete classification of eye hazards, especially for surfactants, as well as cosmetics and other substances applied to or around the eye.
P205: Identifying Eye Irritants (GHS Category 2) Using Validated, Nonanimal Tests
B. J. Varsho1, P. Vij1, D. Sergeyev1, and G. L. DeGeorge1
1MB Research Laboratories, Spinnerstown, PA, USA
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. By using a dual-assay/approach system—the combination of the EIT and BCOP test—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 category 2A and 2B cannot be made. After testing 42 chemicals, we correctly identified 93% of the category 2A/B 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 using this testing strategy would greatly reduce reliance on Draize Rabbit Eye Tests.
P206: QSAR Models for Identifying Pesticides Exhibiting High, Moderate, and Low Toxicity in Honey Bees
M. Girireddy1, and R. Saiakhov1
1MultiCASE Inc, Beachwood, OH, USA
Pesticides are important for crop production, but sometimes they harm species like honey bees (Apis mellifera), which are of immense ecological and economical importance since they contribute to crop pollination and honey production. Several regulatory guidelines were introduced by ECHA (OCSPP 850.3020, Chapter R7b: R.7.11), EFSA, US Environmental Protection Agency (EPA), and OECD (213, 214) to protect these species. In the past, various QSAR models were built with regard to these guidelines. However, they have training sets below 250 compounds and are limited to specific classes of chemicals and modes of action. To overcome these limitations, we have built QSAR models to predict acute toxicity (LD50) of chemicals toward honey bees in line with OECD and US EPA guidelines. The toxicity data were collected from various public sources. Lethal dose of compounds ranged from 0.0029 μg/bee to 1 000 001 μg/bee. Binary labels were assigned in accordance with US EPA specifications (eg, <2 μg/bee as highly toxic, 2 to <11 μg/bee as moderately toxic, 11 to <100 μg/bee as slightly toxic, and ≥100 μg/bee as not acutely toxic). The training data set size ranged from 298 to 719 compounds. All models demonstrated external set validation performance in the range of 43% to 67% sensitivity, 60% to 100% specificity, 74% to 100% positive accuracy, 9% to 94% negative accuracy, and 58% to 73% coverage. With bootstrap cross validation, models exhibited 62% to 79% sensitivity, 65% to 93% specificity, 68% to 82% positive accuracy, 52% to 96% negative accuracy, and 63% to 74% coverage. Thus, we successfully built interpretable QSAR models for predicting high, moderate, and low toxicity of pesticides toward honey bees.
P207: Confirming the Safety of Ingredients Used in Cosmetic Products Available in Canada—A Review of Current and Proposed Approaches
I. Dobre1
1Health Canada, Ottawa, Ontario, Canada
Cosmetics are one of the largest classes of products used by consumers worldwide and subject to close public, media, and regulatory scrutiny. Phthalates, parabens, triclosan, and coal tar dyes are only a few of the cosmetic ingredients showcased in recent years for their potential to cause adverse health effects. The ever-growing diversity of cosmetic products on the market is appealing to consumers but also raises important questions for regulators regarding the safety of these products and their respective ingredients. Access to product formulation data, along with robust regulations, has positioned Health Canada as a leader in promoting cosmetic product safety. With its mandatory cosmetic notification process, Health Canada is able to screen cosmetic products for compliance with conditions set out by the Food and Drugs Act and Cosmetic Regulations. To identify ingredients that may pose a human health concern, the notified product formulations are cross-referenced with the Cosmetic Ingredient Hotlist (Hotlist)—an administrative list of over 600 ingredients that are either prohibited or restricted in cosmetic products. The Hotlist also serves as a valuable source of ingredient safety information for various stakeholders, including industry, general public, and other parts of the federal government. Recognizing the increasingly diverse cosmetic product landscape, maintaining a scientifically supportable Hotlist is paramount to confirming the safety of current and future cosmetic products.
P208: 28-Eight-Day Somatic Gene Mutation Study of 1-Bromopropane in Female Big Blue Mice via Whole-Body Inhalation: Support for a Carcinogenic Threshold
J. T. Weinberg1, R. R. Young2, and M.E. Stelljes3
1Charles River Laboratories, Ashland, OH, USA
2MilliporeSigma, BioReliance Testing Services, Rockville, MD, USA
3SLR International Corporation, Martinez, CA, USA
A 2-year inhalation rat and mouse cancer study by the National Toxicology Program (NTP) on 1-bromopropane (1-BP), a brominated solvent most commonly used as a vapor degreaser, showed significant increase in tumors in the lung of female mice and in the large intestine of male and female rats. The most sensitive end point was lung tumor in female mice. Mice of both sexes had hyperplasia and inflammation of the nose and showed regeneration of lung tissue. The NTP assumed that these tumors were due to genotoxic effects and that a linear dose–response relationship was appropriate. It is plausible that, similar to chloroform, hyperplasia and inflammation are required as initial events for tumor development. If true, then a threshold-based model may be more appropriate for 1-BP. To test this hypothesis, a 28-day repeat-dose inhalation Big Blue assay was conducted using female transgenic B6C3F1 mice. The target exposure concentrations and the exposure regimen were identical to those used by the NTP. Results demonstrated no elevation in mutant frequency of the cII transgene in lung, colon, or liver. Positive controls produced statistically significant increases in mutant frequencies across all tested tissues. These results demonstrate that 1-BP does not induce cII mutants in lungs, colon, or liver under the testing conditions. These data have important ramifications in the quantitative evaluation of tumor results for this chemical and support a mechanism of action where a threshold for carcinogenicity is plausible.
P209: Good Laboratory Practice (GLP) in the Academic Environment
J. L. Zahorsky-Reeves1, and D. M. Breazeal1
1University of California Los Angeles, Los Angeles, CA, USA
Good Laboratory Practice (GLP) is a quality system concerned with the organizational process and the conditions under which nonclinical health and environmental safety studies are planned, performed, monitored, recorded, archived, and reported under the US Food and Drug Administration (FDA) Code of Federal Regulations 21 Part 58, which was first enacted in the United States in 1978. These studies are generally conducted at contract research organizations, where dedicated employees commit full-time efforts. However, this can prove to be too cost prohibitive to university faculty members without substantial financing, typically from “Big Pharma” partners. In an attempt to assist our investigators with the US FDA approval process, our animal care services division embarked on setting up a GLP-compliant testing facility at a major university. This undertaking posed a complicated set of trials and tribulations to get this highly regulated program off the ground in an academic setting. While it was successful to a degree, we will present the challenges associated with organizing and training the right personnel; developing sufficient SOPs and associated forms; preparing the facilities, including monitoring of the environment; writing a comprehensive study protocol; certifying and maintaining equipment; handling of test and control articles; properly archiving all data and study materials; and preserving a relationship between the sponsor, the QA unit, and animal services as the testing facility. Each academic institution should decide whether GLP-compliant studies are something into which they are willing to invest their employees’ and their facilities’ considerable time and effort.
P210: Model Characterization: Gamma Irradiation for Immunoconditioning Cell Therapy Toxicology and Tumorigenicity Studies in NOD/SCID/IL2Rγnull (NSG) Mice
F.- H. Paradis1, K. Wong1, C. Li1, R. Tavcar1, and S. Authier1
1Citoxlab, a Charles River Company, Laval, Quebec, Canada
The NOD/SCID/IL2Rγnull mouse is a relevant model for in vivo toxicology and tumorigenicity studies evaluating human cell therapies due to their severe deficiency in adaptive and innate immune response, enabling cell engraftment. Data were compiled from control NOD/SCID/IL2Rγnull (NSG) mice exposed to gamma irradiation at 200 cGy (160 cGy/min) (N = 41 males, 68 females) or 0 cGy (N = 10 males, 10 females) and had then been dosed with vehicle or positive control via a single intravenous injection. Retrospective data evaluation included mortality, clinical observations, body weights, hematology, and external and internal macroscopic observations. There was no mortality in any of the 129 control (irradiated and nonirradiated) mice up to the 20-week observation period. Although the irradiated group showed a minor decrease in body weight (−5.6% and −2.7% in males and females, respectively), the body weight changes at 21 days postirradiation were similar to that of nonirradiated controls. Clinical signs in irradiated mice were low in incidence and severity and did not differ significantly from nonirradiated mice. Hematology, including white blood cell and neutrophil counts, from irradiated mice at weeks 13 and 20 revealed comparable values to nonirradiated animals, confirming bone marrow recovery. In contrast, irradiated mice treated with a positive control (HL-60) were euthanized prior to week 13. Finally, there were no irradiation-related differences in macroscopic observations with lymphoid atrophy identified comparably in irradiated and nonirradiated groups. These results support the use of gamma irradiation for immunoconditioning prior to cell engraftment in NSG mice in the context of regulatory toxicology and tumorigenicity studies.
P211: Use of Dog Studies in Evaluating the Safety of Food and Color Additive Petitions Submitted to the US FDA
N. Anyangwe1, S. C. Fitzpatrick2, B. Flannery3, A. Mattia1, H. Schaefer3, T. Tyler1, and C. Whiteside4
1Office of Food Additive Safety, CFSAN/US FDA, College Park, MD, USA
2Office of the Center Director, CFSAN/US FDA, College Park, MD, USA
3Office of Analytics and Outreach, CFSAN/US FDA, College Park, MD, USA
4Retired US FDA, College Park, MD, USA
There has been a decline in the number of dog studies submitted to the US Food and Drug Administration (FDA) since the year 2000. This observation, coupled with the US FDA goal of modernizing toxicology, prompted our analysis of the role of dog studies in food ingredient safety assessments. CFSAN reviewed its Food Applications Regulatory Management database to identify and determine the impact of dog studies on decisions regarding the safe use of food and color additives. We identified food additive petitions (FAPs) and color additive petitions (CAPs) that included one or more dog studies. These dog studies were classified as having a decisive impact on safety decisions for less than 50% of FAPs and CAPs. They were further characterized based on the following criteria: addressed a safety concern, used to calculate acceptable daily intakes, used to conclude safety in toxicology memoranda, provided a reason for withdrawal of a petition, or the dog was the most sensitive species tested. These findings may enable CFSAN to determine when a toxicity study in dogs could provide toxicological information not otherwise available using other experimental model systems and may help to identify end points for potential evaluation using modern in vitro or in silico techniques.
300 Series—Safety Evaluation Nonpharmaceuticals
P301: Medical Devices Extraction: Solvent Selection Challenges and Solutions
A. Luniwal1
1North American Science Associates (NAMSA), Inc, Northwood, OH, USA
General principles of toxicology play an important role in evaluation of biological safety and risk assessment of extractable and leachable exposure from clinical use of medical devices. The regulatory landscape for medical devices’ biological safety evaluation is ever changing. Recent publication of revised ISO 10993-1:2018, Biological Evaluation of Medical Devices—Part 1: Evaluation and Testing within a Risk Management Process, has brought material and chemical characterization of medical products to forefront of the risk-based approach for evaluation of biological safety. To that effect, chemical characterization of appropriate device extracts has become the predominant approach. Selection of the appropriate extraction vehicle for chemical characterization plays an important role in effective chemical testing strategy design. A review of % nonvolatile extractable (also known as NVR) limits provided in the Code of Federal Regulations Title 21—Food and Drugs, Part 177: Indirect Food Additives: Polymers, is performed to develop solvent selection criteria for common types of polymers used in medical products. An aggressive solvent is expected to generate higher % of NVR than a more compatible solvent. Based on these limits, extraction vehicle can be categorized as aggressive (e.g., incompatible) or compatible.
P302: Reproductive Disorders in Male Rats with High-Fructose Diet Consumption Starting from Juvenile Age to Puberty
G. M. Shayakhmetova1, O. Y. Tkachenko1, A. V. Matvienko1, I. S. Blazhchuk1, and V. V. Kovalenko1
1SI (Institute of Pharmacology & Toxicology NAMS of Ukraine), Kyiv, Ukraine
There is compelling evidence that hypercaloric, high-fructose diet can induce a whole range of metabolic alterations and supposedly metabolic syndrome, which merits special attention in boys because of androgen deficiency development. However, evidences concerning effects of high-fructose diet in the juvenile period on future male reproductive function are insufficient. The study aimed to analyze effects of high-fructose diet consumption starting from juvenile age to puberty on reproductive function in male rats. Juvenile male Wistar rats were divided into 2 groups: control and high-fructose diet (replacement of drinking water with 10% fructose starting from postnatal day 23 up to 83). Pronounced decrease in serum testosterone (28% as compared with control) accompanied with 2-fold increase in luteinizing hormone and follicle-stimulating hormone levels was apparent in males with high-fructose intake. Absolute and relative weight and volume of testes, as well as absolute weight of epididymis, reduced in these rats. Such results are consistent with those on 26% sperm count decrease. High fructose intake was accompanied with destructive changes in spermatogenic epithelium: spermatogenic index, number of spermatogonia, and cells at XII stage of spermatogenesis decreased. Simultaneously epithelium exfoliation into the lumen of seminiferous tubules increased. In part of animals consuming fructose solution detected dystrophically altered epithelial cells in epididymis tubules. Fertilizing capacity of these males remained unchanged, but fertility rate of intact females fertilized by them decreased. In addition, level of preimplantational embryonic death raised 2.2-fold. Results indicate that high fructose consumption starting from juvenile age up to puberty causes reproductive impairment in male rats.
P303: Demonstrating a Lack of Skin Sensitization Hazard Potential for Alpha-Glycosyl Isoquercitrin (AGIQ) Utilizing the Local Lymph Node Assay
D. A. Donahue1, P. Vij2, D. Sergeyev2, K. P. Burke1, S.-M. Hayashi3, M. Koyanagi3, R. R. Maronpot4, B. J. Varsho2, G. L. DeGeorge2, and L. Recio1
1Integrated Laboratory Systems, Inc., Research Triangle Park, NC, USA
2MB Research Laboratories, Spinnerstown, PA, USA
3San-Ei Gen F.F.I., Inc., Osaka, Japan
4Toxicologic Pathology Resources, Raleigh, NC, USA
Skin sensitization is an important aspect of safety assessment and is a key end point in the toxicological evaluation of chemicals. The compound under study, alpha-glycosyl isoquercitrin (AGIQ), is used in Japan as a food additive and was granted generally recognized as safe status by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) in 2005 and US Food and Drug Administration (US FDA) in 2007. Alpha-glycosyl isoquercitrin is highly absorbable and has been shown to possess antioxidative properties. The safety and toxicity information for AGIQ is still sparse, and there is paucity of data for AGIQ’s potential to cause skin sensitization. The Local Lymph Node Assay (LLNA) has been used for more than two decades to assess a substance’s potential to induce an allergic response and is considered the gold standard of skin sensitization hazard identification. The LLNA was used, in accordance with OECD test guideline 442B, to assess AGIQ’s potential to cause a type IV allergic response. Formulations were analyzed for concentration and homogeneity before dosing and results were ± 10% of target. No excessive irritation was observed after the irritation screen (ear swelling <25% and erythema score <3) when AGIQ was tested at 5%, 10%, and 25%. Based on the lack of irritation, AGIQ was evaluated at 10%, 25%, and 50% (in N, N-dimethylformamide) in the main test, resulting in stimulation indices of less than the positive threshold of 1.6; 1.2, 1.4, and 1.2, respectively. Therefore, AGIQ is not a dermal sensitizer in the LLNA.
P304: Withdrawn
P305: Utilization of Physiologically Based Pharmacokinetic Modeling to Assess Dermal Uptake of Bisphenol A (BPA) and Its Skin and Systemic Exposure in Humans
B. Wiśniowska1, S. Linke2, R. Pirow2, A. Luch2, Z. Tylutki1, and S. Polak3
1Jagiellonian University Medical College, Kraków, Malopolska, Poland
2German Federal Institute for Risk Assessment (BfR), Berlin, Germany
3Certara UK (Simcyp Division), Sheffield, South Yorkshire, United Kingdom
Physiologically based pharmacokinetic (PBPK) modeling plays an increasing role in the pharmaceutical and chemical industries (Laroche, 2018). The present study aimed to develop a PBPK model able to describe the fate of free and conjugated bisphenol A (BPA) after dermal exposure. Simcyp Simulator V18 (Certara, United Kingdom) was used to develop PBPK models. The simulator provides the parametrization for the physiological parameters for various populations. Substance-specific parameters (eg, pKa, logP, skin layers diffusion, and partition coefficients) were taken from the literature or predicted with the use of QSAR models. Literature available in in vitro ADME parameters were scaled to the whole organ level. Various dermally applied doses (50-600 μg) and application areas (10 80 cm2) were used to simulate BPA exposure resulting from thermal papers receipts handling (Liu, 2017). The simulated average total BPA concentration was below experimental LOD (0.015 ng/mL) up to 450 μg (the estimated highest amount of dermal load of BPA, based on Biedermann, 2010); however, there were individuals with Cmax higher than LOD with the dose of ∼100 μg (1/60 individual) and 200 μg (7/60 individuals). These results are consistent with the results of Liu’s study in which only for one participant (after repeated exposure) plasma concentration was above LOD. The PBPK modeling is an appropriate way to predict internal exposure to consumer-relevant chemicals such as BPA. It can be a powerful tool to simulate various exposure scenarios (eg, exposure time, dose, levels, and routes). The influence of the variability within a population and uncertainties of input parameters can be addressed.
400 Series—Toxicology Methods
P401: Detection of Human Cytokines in Humanized Mice
P. Cherukuri1, R. Qualy1, J. Munday2, A. M. Frantz3, B. E. McIntosh3, B. Wilkinson1, and A. L. Rowse1
1Covance Laboratories Inc, Greenfield, IN, USA
2Covance Laboratories Ltd, Harrogate, North Yorkshire, UK
3Covance Laboratories Inc, Madison, WI, USA
Cytokine measurement is an important end point in immunotoxicology safety assessment, and the ability to measure cytokines within humanized mice provides an additional safety end point as well as mechanistic insight within the model. To determine whether human cytokines (interleukin 2 [IL-2], IL-6 IL-10, interferon γ [IFNγ] and tumor necrosis factor α [TNFα]) can be detected in murine matrices and human cytokines (recombinant and ex vivo stimulated) were spiked into serum or tissue lysates (brain and liver) from naive NOD-scid mice. No matrix interference was detected for all analytes for all matrices, with the exception of minimal matrix interference of IL-10 in tissue lysates. The specificity of the human reagents to selectively detect human cytokines was confirmed by the insignificant detection of murine cytokines (both endogenous and recombinant) using human reagents in comparison with the positive detection observed using mouse specific reagents. To confirm use within a humanized mouse model, serum and tissue samples were collected for cytokine assessment from NSG mice engrafted with CD34+ cells (Hu-CD34-NSG) or peripheral blood mononuclear cells (PBMCs; Hu-PBMC-NSH) and nonengrafted NSG mice (controls). Human cytokines were detected in serum above nonengrafted NSG cytokine levels in one or both of the engrafted models. In brain lysates, no cytokines were detected in nonengrafted mice, with detection of only IFNγ, TNFα, and IL-10 in at least one of the engrafted models. In liver lysates, all 5 cytokines were detected but cytokine levels were comparable among engrafted and nonengrafted mice. These data demonstrate an acceptable assay to detect human cytokines within humanized mouse models to be used for preclinical assessment of therapeutic agents.
P402: Characterization of Longitudinal Quality Controls for Immunophenotyping in Cynomolgus Monkey
M. P. Brandt1, J. A. Hollenbaugh1, M. P. Shea2, A. M. Frantz2, and A. L. Rowse1
1Covance Laboratories Inc, Greenfield, IN, USA
2Covance Laboratories Inc, Madison, WI, USA
Immunophenotyping (IPT) of peripheral whole blood is a standard immunotoxicology end point for preclinical studies in primate species. Due to limited stability of whole blood, samples are processed at discrete time points over the course of a study. Here, the use of lyophilized peripheral blood mononuclear cells (Lyo-PBMCs) as a longitudinal control for blood IPT was investigated. The consistency of IPT data is commonly assessed using lymphocyte summation (LSUM) and T-cell summation (TSUM), which evaluate the relationship of T-cell, B-cell, and natural killer cell (TBNK) populations. However, LSUM and TSUM analysis is limited to standard TBNK phenotypes and is generally focused on data analysis. Therefore, a current limitation of IPT assays is the lack of a quality control that can be processed with each batch of samples. Custom Lyo-PBMCs were generated using pooled whole blood from cynomolgus macaques, acting to provide a reagent with COA and expiration date documentation. Reconstitution of cells was optimized for staining consistency, and staining procedures were developed to allow parallel processing of Lyo-PBMCs with whole blood IPT. Surface and intracellular markers for the identification of TBNK populations, memory T cells, and proliferating (Ki-67+) cells were successfully detected using Lyo-PBMCs. The TBNK phenotypes were assessed using replicate measurements and found to produce consistent data through time and using multiple analysts. In conclusion, Lyo-PBMCs are easy to use, are suitable as longitudinal quality controls, and may be validated for use with individual IPT panels.
P403: Development of a Zebrafish Larvae Screening Assay to Identify Compounds With Immunotoxicity and Anti-Inflammatory Activity
I. Iturria1, O. Jaka1, C. Martí1, A. Muriana1, A. Altuna1, and M. J. Mazón1
1Biobide, San Sebastian, Gipuzkoa, Spain
The zebrafish is a unique model for pharmacological manipulation of the innate and adaptive immune response. There are several transgenic lines available to visualize cells from the innate and adaptive immune systems. Taking advantage of zebrafish embryo transparency, we can test immunotoxic compounds by quantifying these cell populations. We have developed an assay in zebrafish larvae to detect compounds with specific toxicity for the innate immune system and to screen and identify new anti-inflammatory drugs. Two transgenic lines have been used: neutrophil-specific Tg (mpx: GFP)i114 and macrophage specific Tg (mpeg: mcherry). Different reference compounds with a known effect on the immune system were chosen and their doses selected after carrying out a maximum tolerated concentration assay. To determine compounds’ toxicity at the innate immune system, embryos were exposed from 72 high-power field and the population of neutrophils and macrophages was quantified by fluorescence microscopy. Inflammation was induced by sterile injury of the tail fin and neutrophil recruitment to the wound site, in the presence of reference compounds, was assessed. The ability of the compounds to suppress the expression of inflammatory genes (il1b, tnf-a) was also evaluated by quantitative polymerase chain reaction. To determine compounds’ toxicity at the adaptive immune system, embryos from the transgenic line Tg (cd4-1: mcherry) were exposed to the test compounds from 4 dpf. The effect in the formation of the thymus was evaluated by fluorescence microscopy at 6 dpf. These zebrafish assays show to be a cost-effective assay over mammalian models for the identification of new anti-inflammatory drugs and the evaluation of immunotoxicity.
P404: Withdrawn
P405: Withdrawn
P406: Development of Large Animal Models of Transtympanic Injection into the Middle Ear and Ototoxicity
A. Marie1, L. Fontaine1, S. Raghunathan1, M. Williams-Fritze2, R. Abutin2, R. Tzafriri2, and C. Sage1
1CILcare, Lexington, MA, USA
2CBset, Lexington, MA, USA
Though rodents are widely used in auditory studies, including ototoxicity and inner ear pharmacokinetics (PK), some studies require larger species with middle and inner ear anatomy more similar to humans. Safety evaluation guidance ask that inner ear exposure and auditory brain stem response (ABR) should be determined for otic test articles. We tested if adult swine and sheep could be used for auditory studies. Investigation of the acoustic ABR threshold in swine and sheep using a purpose-built multichannel ABR system revealed insensitivity to anesthesia protocols across all frequencies tested and confirmed that our system is sensitive enough to be used with a minimally invasive subdermal electrode versus a brain implant. We determined that medical device implantation for safety evaluation into both species could utilize the same middle ear surgical approach. Further examination of the external auditory canal (EAC) anatomy through the tympanic membrane (TM) showed that sheep were better suited for transtympanic (TT) injection with an EAC/TM approximating human architecture than swine (EAC and TM at 90° angle). The feasibility of using sheep in PK studies was demonstrated through a TT injection of compound X to rats and sheep. Inner ear fluid was collected 6, 24, and 72 hours postinjection. Bioanalysis detected the compound in both species at 6 hours, 24 hours (rat only), but not at 72 hours in both species. In summary, these studies demonstrate that a large animal species may be successfully utilized in standard safety auditory evaluation, including TT injections, a surgical approach reaching the middle ear, and ABR assessments.
P407: Withdrawn
P408: Investigating the Distribution of an Antitumor Compound in the Brain of a Glioblastoma Mouse Model by Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry (MALDI IMS)
F. Xie1, H. Tran1, and S. Castellino1
1GlaxoSmithKline, Collegeville, PA, USA
GSK-A is an antitumor compound under development for the treatment of glioblastoma. In this study, quantitative matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) was employed to investigate the brain distribution of GSK-A in the intracranial PDX mouse tumor model (G12). Starting 4 days after intracranial tumor implantation, mice received an oral administration of GSK-A at 100 mg/kg twice daily or a vehicle-only dose. Following 2 days of dosing, in the treated brain tissues, IMS demonstrated GSK-A was localized in the glioblastoma tumor region and the ventricles. After 15 days of GSK-A dosing, in contrast to the vehicle treatment group, brain tissues did not display typical glioblastoma tumors; rather, parenchyma disruption was identified in the brain. Ion images of GSK-A were shown to be localized in the disrupted regions as well as the ventricles. To assess the concentration differences of GSK-A in the tumor/disrupted regions and the rest of the brain parenchyma, a 2-stage hurdle model was built to estimate ion intensity and signal detection probability surfaces. Summary measures from the surfaces were then used in a mixed-effects model to test for differences between regions while adjusting for intermouse variation using random effects. Significant GSK-A concentration differences between the tumor/disrupted regions and the rest of the brain parenchyma region were demonstrated, supporting specificity of GSK-A to localize to tumor-associated regions. Furthermore, several lipids, including cholesterol esters and a sphingomyelin, were identified by IMS in the brain tissues as potential molecular markers of tumor pathogenesis and/or GSK-A pharmacology in this glioblastoma model.
P409: Alternative Vehicles Allow the LuSens Test Method to Predict Dermal Sensitization of Mixtures
M. Cicchini1, L. F. Pratt1, P. Vij1, B. J. Varsho1, and G. L. DeGeorge1
1MB Research Laboratories, Spinnerstown, PA, USA
Predicting dermal sensitization is an important component of the acute toxicity testing battery. The second key event (KE2) in the dermal sensitization adverse outcome pathway is characterized by keratinocyte activation. The OECD 442D guideline details the in vitro keratinocyte ARE-Nrf2 Luciferase-based LuSens Test, herein referred to as the LuSens Test, to predict UN GHS sensitizers (category 1) or nonsensitizers. We performed a series of LuSens Tests using our validated protocol under the OECD 442D guideline. One limitation of this methodology for testing chemicals and mixtures is the limited solubility of potential test substances in the guideline-approved vehicles, dimethyl sulfoxide (DMSO), media, and water. To expand its applicability domain, we sought to validate the use of additional vehicles in the LuSens Test. We demonstrated 8 additional vehicles to be compatible in the LuSens Test, as determined by their ability to appropriately promote positive and negative control luciferase induction responses. Using DMSO or propylene glycol, the LuSens Test was able to predict the dermal sensitization potential of 9 commercially available mixtures. Dermal sensitization predictions agreed in 6 of 6 mixtures tested where safety data sheets were available. For 2 of 2 lotion mixtures tested, which were not expected to induce a sensitization response, no induction was seen. Lastly, a shampoo mixture had a positive prediction in both the LuSens Test and the KE3 guideline-based assay. Together, these data demonstrate that the LuSens Test can be performed on mixtures using an expanded list of intralaboratory validated alternative vehicles.
P410: Withdrawn
P411: Withdrawn
P412: Comparison of Microscopy-Based, Conventional Flow Cytometry and Imaging Flow Cytometry Methods for In Vitro Micronucleus Test in TK6 Cells
H. Xie1, M. A. Rodrigues2, and R. Kulkarni1
1BioReliance Toxicology Testing Services by MilliporeSigma, Rockville, MD, USA
2Amnis Flow Cytometry, Luminex Corporation, Seattle, WA, USA
The in vitro micronucleus (MN) assay is a commonly used test for the evaluation of clastogenicity and aneugenicity of drugs, chemicals, cosmetics, food additives, and medical device extracts. A reliable and accurate high-throughput assay system for detecting MN cells has long been desired. Both microscopy and conventional flow cytometry methods have been developed and validated, and both are routinely used to perform the MN assay. However, these methods have several limitations, such as long turnaround time and scorer variability with microscopy, as well as lack of visual confirmation of events and potential interference of apoptosis with flow cytometry, especially in p53 proficient cell lines. In this study, we conducted a comparative evaluation of MN frequencies determined by conventional flow cytometry, multispectral imaging flow cytometry, and microscopy-based scoring. The MN frequencies of known apoptosis inducers with and without gentotoxicity were tested in TK6 human lymphoblastoid cells. The cells were exposed to 5 to 10 concentrations of the compounds for 4 hours in the presence of metabolic activation and 27 hours in the absence of metabolic activation. After exposure, the cells were processed for manual microscopic scoring, conventional flow cytometry, and imaging cytometry analysis. The %MN and % apoptosis measurement appeared higher using the conventional flow method. Imaging flow and microscopic scoring gave similar apoptosis measurement. The results provide information to better assess the potential correlation of high proportions of apoptotic cells and false-positive MN results using conventional flow cytometry.
P413: hTERT Immortalized Kidney Models for Drug Screening and Toxicity Assays
K. Grady1, L. Rodriguez1, and J. Huuskonen1
1ATCC, Gaithersburg, MD, USA
Kidney is a major organ in the body responsible for the elimination of many xenobiotics and prescription drugs and, having relevant models for drug interaction and toxicity studies, is thus a necessity. Primary cells and continuous cell lines have traditionally been used in these studies. We have generated human telomerase reverse transcriptase (hTERT) immortalized renal proximal tubule epithelial cells (hTERT-RPTEC) that can overcome the limitations of donor variability and senescence of primary cells yet show key primary cell functionality. These cells have the expected expression of E-cadherin and CD31 and display typical dome formation morphology. One limitation of primary RPTEC is that the expression of key solute carrier (SLC) transporters OAT1, OCT2, and OAT3 is lost during culture and expansion of these cells. To overcome this, we have stably overexpressed each of these carriers in the hTERT-RPTEC background. These cells maintain correct marker expression (both overexpressed SLC and endogenous RPTEC markers) and growth characteristics. Functional uptake studies using fluorescently labeled 6-carboxyfluorescein and ethanaminium iodide, as well as inhibition of the uptake by novobiocin and cimetidine, demonstrate the functionality of the overexpressed SLCs. These cells have also been used to study the mechanism of drug toxicity (eg, antiviral drugs tenofovir, tenofovir DF, cidofovir) using cell viability assays. In aggregate, all these data demonstrate that the immortalized hTERT-RPTEC and the SLC-expressing stable variants (hTERT-RPTEC-OAT1, -OCT2, and -OAT3) are reliable kidney models that can be used for drug screening and toxicity assays.
P414: hTERT Immortalized Adult Dermal Melanocytes as an In Vitro Model to Study Melanogenesis
L. Rodriguez1, K. Grady1, and J. Huuskonen1
1ATCC, Gaithersburg, MD, USA
Skin pigmentation is a complex process within melanocytes, and mutations in the multiple genes that regulate it are characteristic in numerous skin disorders, including hyperpigmentation, hypopigmentation, and mixed hyper-/hypopigmentation. Melanin expression in adult melanocytes is also influenced by additional extrinsic and intrinsic factors, such as hormonal changes, inflammation, age, and exposure to UV light. In order to better understand the melanocyte biology, there is a need for relevant biological models. Human telomerase reverse transcriptase (hTERT) immortalized melanocytes described here are a very robust model for studying melanocyte function by providing primary melanocyte functionality but exhibiting “immortalized” characteristics for more than 40 population doublings (PDL) without signs of replicative senescence. These dermal melanocytes maintain a consistent expression of the melanocyte-specific marker tyrosinase-related protein 1 but lack the expression of a fibroblast-specific TE7 marker. Melanin production is maintained through the life cycle, declining only after PDL 45. Functionality similar to primary melanocytes was demonstrated by a concentration-dependent melanin production by melanogenesis enhancers latanoprost and stem cell factor, and a reduction in melanin production in response to hydroquinone. Last, incorporation of these melanocytes into an organotypic coculture with fibroblasts and keratinocytes enhances the growth and formation of the culture, as well as shows enhanced pigment production. All these data together demonstrate that the hTERT immortalized melanocytes display form and function very similar to primary melanocytes and offer a viable solution for building reliable and complex dermal models.
P415: TargetTri: Safety Assessment and Derisking of Novel Drug Targets
J. Venhorst1, G. J. Kalkman1, T. Rouhani-Rankouhi1, E. P. van Someren1, and S. Folkertsma1
1TNO, Zeist, NH, the Netherlands
Assessing safety liabilities of (exploratory) drug targets in the early preclinical phase of development is an important aspect of managing the drug discovery pipeline. On the one hand, toxicity information is imperative for the derisking of therapeutic targets that are progressing through the development pipeline. On the other hand, safety considerations can aid in the prioritization and selection of exploratory drug targets. Both contribute to the reduction of attrition rates that have hampered the launch of new drugs in recent years. TNO, a nonprofit research organization, is developing the in silico target triaging platform “TargetTri” together with pharmaceutical partners. By combining data mining, text mining, and systems biology/network analysis, this web-based platform offers insight in various aspects of target-related toxicities in separate views. These views provide, for example, information on diseases, biological (adverse) effects, single-nucleotide polymorphism, transgenic models, pharmacological tools, clinical data, expression profiles, and subsequent risk mitigation strategies. The TargetTri platform has been applied to analyze safety risks of Oncostatin M, identified as a potential therapeutic target for cardiovascular disease by the CarTarDis consortium. Identified major risks concerned anemia and increased susceptibility to infection based on clinical and preclinical data. Reduction of myocardial remodeling and angiogenesis may also be of concern for the atherosclerotic patient based mainly on preclinical observations. The occurrence of such effects needs to be evaluated in subsequent derisking assays supported by TargetTri. This research is supported by the Dutch Ministry of Economic Affairs.
P416: Refinements to Intravenous Infusion Systems in Sprague Dawley Rats Aid in the Successful Outcome of a Complex Study Design
T. R. Gleason1, A. J. Evans1, J. L. Shaffer1, and J. D. Bultman1
1Charles River Laboratories, Ashland, OH, USA
The Infusion Department at Charles River Ashland was given a challenge: administer a 15-second intravenous dose to rats every 10 minutes for 1 hour on study days 86 and 149. A surgically implanted catheter was used to allow for intravenous infusion via ambulatory tethered system. Due to the study design, several areas of concern were noted, including the extended duration of the study warranting concerns for patency, the condition of the port, and social housing for animal welfare. An exteriorized port using magnetic connections was determined to be the best option with consideration given to animal growth, catheter length and tip style, and security of the catheter to the port, while also allowing for social housing. The study initiated with 116 male and 116 female rats (Toxicology: 60/sex; Pharmacokinetic: 24/sex; Cardiovascular Telemetry: 32/sex). The animals were checked for patency and locked with 20 IU/mL heparinized saline every other week. Two animals were electively euthanized due to a compromised port. Of the surviving animals, 96% of the toxicology animals remained bidirectionally patent until scheduled main and recovery necropsy on study days 87 and 100, respectively, and 94% of the pharmacokinetic and cardiovascular remained bidirectionally patent until scheduled necropsy on study day 151. System refinements (catheter length, tip style, and security) facilitated the exceptional patency rates and the successful dosing of the study and allowed for social housing for most of the study.
Expanded Solvent Selection for In Vitro Sensitization Testing Using the GARD Assay Platform
J. Schmidt1, O. Larne2, and U. Mattson2
1SenzaGen, Inc, Maplewood, MN, USA
2SenzaGen, AB, Lund, Sweden
With increased use of chemicals, allergic reactions have grown as well. Some substances, known as sensitizers, induce allergic contact dermatitis and can severely affect the quality of life. To prevent harmful exposure, chemicals are typically required to be tested for safety. Historically, testing has been performed on animals, but recent regulations as well as economic and public interests demand replacement with animal-free methods. The GARD platform is an in vitro, state-of-the-art assay developed for predicting sensitizers. Development of the GARD platform included use of 2 solvents: dimethyl sulfoxide and water. However, due to varying chemical properties, many substances or mixtures are not soluble in either of these solvents. Therefore, there is high interest in increasing the array of usable solvents compatible with the GARD assay. To do this, several solvents with varying polar or nonpolar dissolving capabilities were selected. After identifying concentrations not cytotoxic or causing changes in cell phenotypes, we analyzed these solvents by the GARD method to demonstrate assay compatibility. To demonstrate bioavailability of the solvents to the cells, and show correct classification, a known sensitizer was dissolved in each solvent and used as a positive control.
We identified both polar and nonpolar solvents that did not affect the cells, were predicted as nonsensitizers, and correctly predicted the positive control. We demonstrate that GARD is compatible with a wider range of solvents, thus extending the applicability domain to include test items with low aqueous solubility, opening the door for testing difficult-to-test substances such as UVCBs and other mixtures.
P418: Optimizing Intracecal Cannulation for Direct Administration in Rats
G. Iacono1, J. Douville1, B. Richard2, F. Emond1, C. Foucault1, M. Halle1, and N. Hébert1
1Charles River Laboratories Montreal ULC, Senneville, Quebec, Canada
2Progenity, Inc, San Diego, CA, USA
Gastrointestinal diseases affect a large population across the world. Current treatment options include anti-inflammatory drugs, immune system suppressors, and, where ineffective, surgical removal of the entire colon and rectum. Herein, we evaluated the feasibility of direct delivery via an intracecal catheter and access port for repeated daily administration. Rats had a catheter surgically implanted at the apex of the cecum, and the catheter was connected to a vascular access button (VAB) in the interscapular region. Following an appropriate postoperative period, a patency check was performed prior to the start of dosing to ensure integrity of the system. A fixed dose volume of 1 mL followed by a 0.1 mL flush of 0.9% sodium chloride for injection US Pharmacopeia was administered daily. Most animals received their dose for approximately 28 consecutive days. There were no clinical signs or significant body weight changes related to the daily administration. Complications due to the size of the VAB were encountered in a few animals and consisted in moderate to severe swelling with discharge around the VAB site. For animals surviving until study completion, the placement of the catheter was confirmed to be adequate. Microscopic findings were considered minimal and expected following abdominal and intracecal catheter surgery. Optimization of the model was subsequently pursued and involved the implantation of a smaller VAB. In conclusion, the repeat administration by direct delivery in the cecum using surgical cannulation is a feasible model in the rat and can be used in the preclinical development of drugs intended for gastrointestinal diseases.
P419: Development of a Large Animal Model for Combined Seizure and Cardiovascular Liability Assessment
N. Sadekova1, K. Norton1, J. Sessenwein1, M. Metea2, F. Emond1, C. Copeman1, and E. Wachman1
1Charles River Laboratories Montreal ULC, Senneville, Quebec, Canada
2Preclinical Electrophysiology Consulting, LLC, Boston, MA, USA
Evaluation of central nervous system (CNS) liabilities is traditionally limited to behavioral assessments. However, due to the growth in compounds targeting CNS indications and novel approaches for delivering drugs to the CNS, the need for more sensitive CNS assessments has arisen in recent years. Regulatory agencies are now routinely requesting that electroencephalography (EEG) studies be conducted. These assessments have predominantly been conducted using analogue telemetry, which is limited to 2 EEG leads. However, advances in digital technology can improve sensitivity and expand assessments that can be conducted within a single model. The objective of this study was to examine the feasibility of a dog model implanted with both an easyTel+L_EEETA and an easyTel+L_EPPTA telemetry unit for concurrent assessment of EEG, electrocardiograph (ECG), and blood pressure (BP). Six Beagle dogs were implanted with 3 EEG leads (frontal, parietal, and occipital), an electromyography lead, lead II ECG, and femoral BP catheter. Animals were dosed with pentylenetetrazol to determine the sensitivity of the model, with video monitoring, EEG and ECG waveforms, and BP collected continuously for a minimum of 24 hours. Pentylenetetrazol administration induced location-specific prodromal EEG and frank seizures and the direct impact on ECG morphology and blood pressure could be assessed concurrently. These results demonstrate that EEG and ECG/BP assessments can be conducted in parallel, reducing the need for multiple studies, while the addition of a third EEG lead increases the sensitivity of the model to detect seizures. This model may provide application to distinguish suspected hypoxia driven syncope from a true seizure.
P420: Refinement of Techniques for Cardiovascular Assessments on Repeat-Dose Toxicology Studies
K. Norton1 and N. Sadekova1
1Charles River Laboratories Montreal ULC, Senneville, Quebec, Canada
Over the past decade, jacketed telemetry has become the gold standard for assessing cardiovascular assessments on repeat-dose toxicology studies. However, the method itself is not without issues. The jacketing of animals requires several days of acclimation to reduce stress-induced heart rate elevations. Inconsistent lead placement across monitoring occasions results in variable electrocardiograph (ECG) interval durations, as such reducing the model sensitivity, and muscle artifact impacts signal quality. Additionally, loss of data can be encountered due to displacement and disconnection of leads due to animal movement. Advances in telemetry implants have seen considerable reduction in device weight and size, allowing them to be adapted for use on toxicology studies. To determine the feasibility of utilizing surgical telemetry on repeat-dose studies, we investigated implant functionality, ECG waveform morphology quality, blood pressure drift, and ECG interval duration consistency in dogs instrumented with DSI-M11 implants. Data were evaluated over multiple studies up to 7 months in duration. All implants remained functional over the 7-month period. Resting heart rates and blood pressures were comparable throughout the study, with no blood pressure drift noted over time. The ECG morphology was of a higher resolution than surface leads, with less muscle and movement artifact, allowing for a greater number of cycles to be assessed in a given monitoring period. This study confirms that surgical telemetry can be readily incorporated into repeat-dose studies, providing a higher-quality stable ECG and blood pressure waveform while removing monitoring limitations and stress induced by jacketing animals.
P421: Withdrawn
P422: Microsampling Blood Collection Technique in Juvenile Rodent Studies
M. Felx1, B. Heroux1, and K. Robinson1
1Charles River Laboratories Montreal ULC, Senneville, Quebec, Canada
Toxicokinetic (TK) assessments are important components of juvenile toxicology studies to allow appropriate interpretation of results as required in International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, US Food and Drug Administration, and European Medicines Agency guidance. Age-matched rodent models (rats and mice) are frequently the species of choice for juvenile toxicology studies. The total blood volume present in this size/age of animals presents challenges when using traditional blood sampling techniques and often requires that sampling be performed as terminal procedure or the pooling of samples from several pups to provide sufficient volume of blood for analysis. Advances in bioanalytical technology have allowed the potential to use smaller blood sample volumes. Microsamples are defined as a volume of up to 50 µL. To decrease animal use and improve the quality of data, a nonterminal microsampling technique permitting sampling over multiple occasions from the same mouse or rat pups was developed. CD-1 mice and Crl: CD(SD) Sprague Dawley rats were sampled using a butterfly connected to a syringe. The butterfly was premarked to identify target volume and blood withdrawn from the jugular vein. The microsampling technique using conscious, nonterminal blood sampling was successfully validated in mouse pups at day 7 postpartum (pp) and rat pups at day 4 pp. Repeat sampling was also possible when volumes remained within allowable limits. These techniques allowed the reduction of the animal usage by up to 50% by removing satellite TK animals, provided a complete TK profile from a single animal, and allowed 2 of the 3 R objectives to be directly implemented on routine studies: reduction and refinement.
P423: Minimizing the Unpredictability of Flow Cytometry Gating Modifications on Lymphocyte and T-Cell Summation Acceptance Criteria for Canine Whole Blood Immunophenotyping
M. P. Shea1, A. L. Rowse2, and A. M. Frantz1
1Covance Laboratories Inc, Madison, WI, USA
2Covance Laboratories Inc, Greenfield, IN, USA
Flow cytometry is a preferred method when performing immunophenotyping for safety assessment and clinical diagnostics. Due to the multivariable nature of flow cytometric analysis, appropriate quality checks are often used as acceptance criteria to confirm the reliability of data. Lymphocyte summation (LSUM) and T-cell summation (TSUM) are 2 common examples of criteria that display the respective proportion of identified lymphocytes and T cells within a sample. Many experimental factors (eg, antibody/clone selectivity, lyse/wash conditions) govern the robustness and accuracy of these 2 acceptance criteria. Additionally, the final gating strategy utilized for flow cytometry data analysis is a critical component in determining LSUM and TSUM values. Using data acquired from a newly validated in-house canine immunophenotyping method that delineated T- and B-cell subsets using antibodies against CD45, CD3, CD4, CD8, and CD21, we calculated LSUMs and TSUMs comparing a variety of different plausible gating strategies. We found that LSUM values were particularly sensitive to small changes in gate shape when lymphocytes were gated on a CD45 versus side-scatter plot. Conversely, LSUMs were less sensitive to small gating changes when lymphocytes were gated using forward versus side-scatter characteristics. T-cell summation values were relatively unchanged through all gating strategies tested. Our findings suggest that careful consideration of gating strategy is critical to achieve robust and repeatable results, as monitored with LSUM and TSUM acceptance criteria, when performing immunophenotyping analysis.
P424: Detection of Peripheral Blood Foxp3+ Regulatory T Cell Population in a Preclinical Setting
M. Patel1, A. Wong1, and J. Lindsay1
1Altasciences Preclinical Seattle LLC, Everett, WA, USA
Research focused on T regulatory (Treg) cells has seen an increase for immunotherapy treatments designed to treat various malignancies. For instance, studies in humans and animal models have shown that by suppressing tumor immunity, Tregs can prevent the host immune system from detecting and controlling tumor growth. As a result, interest in targeted depletion of Treg cells has also increased in preclinical and clinical settings. However, it is difficult to quantify and characterize the naturally occurring Treg population characterized as CD3+CD4+ CD25+Foxp3+ using flow cytometry in peripheral blood. This is due to difficulties involved in Foxp3 staining and relatively rare presence of Treg cells in peripheral blood. To alleviate these concerns, we developed a method that can reproducibly detect a large number of Treg cells in 250 µl of whole peripheral blood of cynomolgus monkeys; this allows effective evaluation of therapies designed to characterize or deplete Treg cells. In animals analyzed using our developed method, the Foxp3+ Treg population ranged from 2.1% to as low as 0.2% of total lymphocyte population. Nonetheless, our method was reliably able to detect up to 21,500 Foxp3+ Treg cells in peripheral blood. The results were confirmed using an extracellular method of detecting Treg population characterized as CD3+CD4+CD25+127low. Our method provides a reproducible way of detecting Foxp3+ Treg cell population in cynomolgus monkeys and presents a unique opportunity of studying modulation of the Treg cell population in vivo.
P425: Urine Quantitative Analysis of 26 Novel Fentanyl Analogs and Metabolites by LC-MS/MS and Its Clinical Significance
M. Akhtar1, M. Ficurilli1, L. Pham1, and M. B. Homme1
1Acutis Biosciences, East Northport, NY, USA
Fatalities caused by opioid overdose continue to rise in the United States without a sign of abating. An increasingly growing portion of these deaths involves synthetic opioids, especially fentanyl and its structural analogs. In 2017, they were responsible for more than 28,000 deaths, surpassing the number of deaths due to heroin and prescription opioids. Synthetic opioids are an increasingly major public health hazard requiring attention from many fields, including clinical toxicology laboratories. Our laboratory has, in response, developed and validated an analytical method for the analysis of the 26 novel fentanyl analogs in urine. Chromatographic separation and detection were achieved using an Agilent Technologies 1290 liquid chromatograph coupled with an Agilent 6460-triple-quadrapole mass spectrometer with a jetstream electrospray source. Linearity for all analytes was established along the range of 0.01 to 20 ng/mL. The limit of detection for all analytes in urine ranged from 0.01 to 0.025 ng/mL. This method has shown to be selective and specific, providing no evidence of carryover or interference concerns. Finally, approximately 500 clinical samples were submitted for analysis. Of those samples, 97 samples were positive for 11 distinct fentanyl analogs that would have avoided detection in routine clinical analyses. It is worth emphasizing that carfentanil and its major metabolite, norcarfentanil, were detected in one clinical sample. Incorporating this sensitive and highly specific method into the workflow will allow clinical toxicology laboratories to more effectively assist clinicians in mitigating the public health hazard due to fentanyl and its analogs.
P426: Intra-articular Injection: Compartmental Dosing and Species Selection
T. McKinnon1, J. Douville1, J. Sweet2, M. M. Laffins3, A. Varela1, and N. Doyle1
1Charles River Laboratories Montreal ULC, Senneville, Quebec, Canada
2Charles River Laboratories, Mattawan, MI, USA
3Charles River Laboratories, Reno, NV, USA
Intra-articular injections into the knee joint of preclinical models for assessment of drug candidates to evaluate local efficacy or safety risks present challenges due to lesser compartment size in these animal models than encountered clinically. For safety assessment, multiples of clinical dose also need to be considered, presenting further challenges. To this effect, tolerance of injected volumes in the knee joint was evaluated in different species (mouse, rat, rabbit, cynomolgus monkey, beagle dog, sheep). Intra-articular injections of a radiolucent material were performed under anesthesia to evaluate radiologically the acceptable dose volume without extra-articular leakage. Methods for collection of synovial fluid (via lavage) were also established. The following volumes were considered acceptable for intra-articular injection for the tested species: 0.006 mL in mice, 0.1 mL in rats, 1 mL in rabbits, 0.6 mL in monkeys, 2 mL in dogs, and 4 mL in sheep. Similar volumes of lavage solution can be administered to ease synovial fluid collection. In general, the volume collected after lavage is slightly less than 50% of the volume injected. These data allow a good estimation of the knee joint space in the different species. These volumes were also considered acceptable from a histopathological perspective with generally slight changes consistent with injection procedures seen. Overall these data can be used for comparison with human joint space and provide a good range of acceptable animal species to be considered for dose level and species selections for use in safety assessment of therapeutic agents for intra-articular administration.
P427: Use of Thromboelastography (TEG) in Preclinical Studies
N. Lalayeva1 and K. Thrall1
1Altasciences Preclinical Seattle LLC, Everett, WA, USA
Preclinical drug development focused on coagulation will typically include frequent analyses of hematology and coagulation. In addition, thromboelastography (TEG) is useful to monitor clot strength and fibrinolysis and to help to diagnose platelet dysfunction and hypercoagulability. Thromboelastography must be initiated shortly following blood collection; thus, it is essentially a point-of-care technique. In practice, whole blood is collected in sodium citrate and an aliquot of citrated blood is added to a vial containing kaolin. The kaolin mixture is then pipetted into a disposable TEG cup and analyzed on a calibrated machine. The resulting TEG tracing provides data on the rate of clot formation (alpha-angle), time to achieve a certain clot strength (K), time to initial fibrin formation (R), overall stability of the clot (MA), and decrease in stability of the clot at 30 or 60 minutes (LY30 or LY60). Thromboelastography analysis has been utilized in preclinical studies focused on bleeding disorders and platelet dysfunction in multiple large animal models and correlated well with conventional hematology data. For example, a TEG tracing illustrating long R and K times and low alpha-angle and MA suggestive of thrombocytopenia directly correlated with decreased platelet counts. The long R time suggestive of a deficiency in clotting factors was compared to measured levels of individual coagulation factors. Data indicate that coagulation factors VIII, XI, XII, and XIII were markedly altered during the critical point of thrombocytopenia. These data support the continued use of multiple approaches to evaluate the coagulation cascade to provide the most meaningful interpretation.
P428: Withdrawn
P429: Using Pathology Findings to Improve Animal Welfare and Toxicology Study Quality
R. R. Hukkanen1, K. G. Nelson2, N. Dybdal3, and P. V. Turner4
1Eisai, Inc, Cambridge, MA, USA
2Charles River Laboratories, Mattawan, MI, USA
3Genentech, South San Francisco, CA, USA
4Charles River Laboratories, Wilmington, MA, USA
Responsibility for promoting and enhancing the 3Rs (replacement, reduction, and refinement) in toxicology research rests with all individuals working with animals. The study pathologist can be a powerful advocate for animal welfare by working collaboratively with animal care personnel at all levels. Veterinary clinical and anatomic pathologists contribute to key discussions during toxicology study planning but also can provide important recommendations for refinements to toxicology SOPs and institutional practices based on day-to-day observations in their work. Veterinary pathologists are ideally positioned to recognize opportunities for improvement in husbandry practices and study designs. A series of case examples are provided to demonstrate how veterinary pathology findings can be used to improve toxicology study quality through animal welfare refinements. Examples include potentially confounding study lesions in mice related to ulcerative dermatitis, pododermatitis in dogs held on raised floors, potential test article immunosuppression concerns in nonhuman primates, intranasal epithelial ulceration in rats, dosing irregularities in rodents, and refinement of intramuscular dosing in minipigs. In all cases, further investigations, discussions, and practice refinements permitted veterinary pathologists to tease out, reduce, or eliminate potentially inaccurate or confounding study results. Encouraging open discussions about challenging pathology issues that arise periodically in the course of toxicology research can ultimately improve animal welfare and toxicology study quality.
P430: Ongoing Validation of the Liver Micronucleus Assay
R. Kulkarni1, S. Bruce1, M. Young1, and S. Roy1
1BioReliance Toxicology Testing Services by MilliporeSigma, Rockville, MD, USA
The in vivo micronucleus (MN) assay is performed to assess genotoxic potential in rodents. Since liver is the major site of metabolism and many genotoxicants may act as hepatocarcinogens, detecting genotoxicity in the liver is an effective approach to predict hepatocarcinogenicity. Although MN assessment in other tissues has been attempted, it was limited by either complicated methods or lack of integration into other in vivo genetox assays. BioReliance Toxicology Testing Services is participating in a ring trial with Litron Labs to develop a high content flow cytometry–based rat river MN assay. Under this ring trial, BioReliance has thus far tested 2 compounds, diethylnitrosamine (DEN), a liver specific genotoxicant, and 1,2-dimethylhydrazine-2HCl (1,2-DMH), a known carcinogen. In these studies, 4 to 5 weeks old rats were administered DEN or 1, 2-DMH for 3 consecutive days. On day 4, peripheral blood was collected to measure MN in reticulocytes. On day 7, animals were euthanized to collect left lateral liver lobes. The liver lobes were processed to prepare single cell suspensions and MN were labeled for flow cytometric analysis. A fraction of the liver cells was used to prepare slides for comparative manual scoring. We observed a statistically significant increase in the liver MN frequency in both DEN and 1,2-DMH. We also observed that only the frequency of liver MN was increased but not for blood MN. The simultaneous analysis of liver and blood MN could be a valuable tool to study liver specific genotoxicants that may be missed when analyzing micronucleus in the blood reticulocytes only.
P431: Intratracheal Administration in Juvenile Sprague Dawley Rats
J. Laporte1, H. Mirsadeghi1, A. Fotovati1, K. McInally1, and P. Aughton1
1ITR Laboratories Canada Inc, Baie-D’Urfé, Quebec, Canada
Data generated from juvenile toxicity studies can contribute to the assessment of potential drug toxicity in the pediatric population that is not adequately assessed in reproductive toxicity risk assessments and standard toxicology studies. Direct intratracheal administration is a highly effective pulmonary delivery route bypassing first-pass metabolism and permitting a direct and local delivery of a small amount of drug to the lungs. The objective was to establish a single and repeat intratracheal administration of saline in juvenile Sprague Dawley rats at 4, 10, and 14 days of age. Prior to intratracheal administration of saline, each juvenile rat was anesthetized by inhalation of an isoflurane/oxygen mixture. The tracheal opening was located in rats using the transillumination method. Saline (≤1 mL/kg) was delivered using a Penn-Century MicroSprayer or a feeding needle (24G × 1 cm). To confirm the intratracheal dosing procedure, some rats were dosed with Evan’s blue dye mixed in saline and determined in lungs at necropsy. Rats were dosed once or daily with saline for 5 consecutive days. Mortality, clinical signs, and body weight were monitored after dosing. Rats were euthanized upon completion of dosing and blood was collected via cardiac puncture. Lung weight was measured and bronchoalveolar lavage was collected. Rats survived a single and repeated dose of saline using the transillumination method with a Penn-Century MicroSprayer or feeding needle administration with minimal clinical signs. In addition, dose volume, body weight, and blood and bronchoalveolar volumes will be used as a reference for future juvenile toxicology studies.
P432: Characterization of a Proinflammatory Response in Cynomolgus Monkeys Using a 17 Cytokine Multiplex Assay
A.- M. Downey1, R. Genard1, R. Riffon1, C. Li1, R. Tavcar1, I. Boulay1, and S. Authier1
1Citoxlab, a Charles River Company, Laval, Quebec, Canada
Cytokine profiling has become common in toxicology studies involving immunomodulating agents. Sample volume requirements often represent a challenge in laboratory animal species including nonhuman primates owing to competing assays, including toxicokinetics, clinical chemistry, hematology, and occasionally antidrug antibodies. The cytokine response to lipopolysaccharide (20 µg/kg, IP, Escherichia coli O127: B8) was characterized by monitoring 17 cytokines at baseline, 1, 2, 6, and 24 hours postchallenge in telemetered cynomolgus monkeys (n = 8) using a Luminex multiplex assay. Plasma samples, control and standard were incubated for 2 hours at room temperature in a 96-well plate with precoated magnetic beads with either one of the antianalyte antibodies. Samples were then incubated with a biotinylated detection antibody for 1 hour before adding streptavidin-phycoerythrin conjugate for 30 minutes. After washing steps, using magnetic plate separator, samples were resuspended in Drive Fluid and read using MAGPIX system. Median fluorescent intensity data were analyzed using a 4-Parameter Logistic curve fit for interpolating each analyte concentrations in the samples. The minimum volume required per sample was 20 µL for analysis in duplicate using a minimum required dilution of 1/3. Significant increases were noted for all cytokines but with differences in time to peak levels for granulocyte-macrophage colony-stimulating factor (GM-CSF; 2 hours), G-CSF (2 hours), interferon (IFN; 2 hours), interleukin 2 (IL-2; 6 hours), IL-10 (1 hours), IL-17A (1 hours), IL-1ra (2 hours), IL-13 (6 hours), IL-1b (2 hours), IL-4 (6 hours), IL-5 (1 hours), IL-6 (2 hours), IL-8 (2 hours), monocyte chemoattractant protein-1 (MCP-1; 2 hours), tumor necrosis factor α (TNF α; 1 hours), macrophage inflammatory protein 1β (MIP-1b; 1 hours), and IL-12(p40) (6 hours). This assay appeared suitable for cytokine profiling in toxicology studies involving cynomolgus monkeys.
P433: Effect of Housing on Stress-Related Parameters in NHPs
T. Rogers1, V. Bunker1, B. Darimont2, R. Burke2, A. Tenney1, K. Kraszewski1, and S. Nechev1
1Altasciences Preclinical Seattle LLC, Everett, WA, USA
2Avidity Biosciences, La Jolla, CA, USA
In preclinical studies, it is important to reduce animal stress, both for animal welfare reasons and to minimize the impact on toxicologic parameters. Effects of stress on in-life parameters (body weight, food consumption), clinical pathology (white blood cell and differential counts), organ weights (endocrine, immune, reproductive), and histopathology (lymphoid, gastrointestinal tract) are well documented. Myostatin (MSTN), a member of the transforming growth factor β superfamily, is a negative regulator of muscle deposition and has been shown to increase in response to psychological stress in mice; increased MSTN correlated directly with decreased muscle mass. Plasma levels of MSTN were measured in 15 young adult male cynomolgus monkeys during a 2-week period of housing in standard 4 packs (pairs and trios) and after transfer into gang housing enclosures (3 animals per cage) that meet or exceed European Union standards. Average MSTN levels in standard housing ranged from approximately 3,900 to 7,100 pg/mL, and in gang housing from 1,700 to 5,000 pg/mL. On an individual animal basis, average MSTN levels decreased by 10% to 32%, suggesting potential stress reduction in the gang housing setting. The significance of this change in MSTN levels is unknown. Potential correlation with other stress-related parameters, including body weights, clinical pathology, and behavioral evaluations, was assessed. The specific benefits of gang versus standard housing were examined to better understand the manifestations of stress in a laboratory environment that are relevant to toxicology study outcomes.
P434: Withdrawn
500 Series—Safety Evaluation Pharmaceuticals
P501: Withdrawn
P502: Assessment of the Sensitivity of a Newly Developed Cynomolgus Monkey Cytotoxic T Lymphocyte Activity Assay to Various Immunomodulatory Agents and Mechanisms In Vitro
B. Frank1, H. Lebrec1, and X. Wang1
1Amgen Inc, South San Francisco, CA, USA
The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of studies and end points and, if deemed necessary based on causes for concern, additional functional assays. CTL activity impairment by immunomodulatory agents is recognized to be a contributor to decreased antiviral defense and increased cancer risk. We developed a BiTE-molecule-mediated CD8+ CTL activity assay that is applicable to both in vitro and ex vivo experimentation in nonhuman primates in the context of toxicology studies. While an ex vivo analysis of long-term studies focuses on the chronic impact on CTL function, the in vitro analysis captures an acute effect in the presence of the drug. Here, we describe the sensitivity of the CTL activity assay to various immunomodulatory therapeutics targeting multiple pathways and mechanisms in vitro. The assay demonstrated immunosuppression with corticosteroids, and inhibitors of Janus kinases (JAK), mTOR, phosphodiesterase, purine and pyrimidine synthesis, and calcineurin. Conversely, the immune checkpoint inhibitors tested enhanced the BiTE-molecule-mediated CTL activity. The agents that did not acutely influence CTL activity at all concentrations evaluated were inhibitors of TNF, IL-6R, IL-12/IL-23p40, CD28-CD80/86 costimulation, sphingosine-1-phosphate (S1P) receptors, and alpha-4 integrins. Since the BiTE-molecule-mediated T-cell activation acts independently of traditional MHC-I antigen presentation and requires less of costimulatory molecules, this unique mechanism of action is likely to prove insensitive to certain pathways. Taken together, our in vitro results demonstrate this assay to be sensitive to some immunomodulatory agents in a mechanism-specific manner.
P503: Juvenile Animal Study in Rats With the Aromatase Inhibitor Letrozole Administered for 9 Weeks, Including Recovery and Reproductive Phases
C. J. Murphy1, M. A. Dorato1, S. Y. Goodnight1, A. Arepalli1, and J. W. Henck2
1BASi Gaithersburg, Gaithersburg, MD, USA
2Toxicology Solutions Consulting LLC, Leeds, UT, USA
The aromatase inhibitor letrozole produces adverse effects on growth, skeletal development, sexual maturation, behavior, and reproductive anatomical and functional parameters when administered to rats during postnatal maturation, primarily through antiestrogenic properties. Letrozole was utilized as a positive control to support validation of a juvenile animal toxicology program. Sprague Dawley rats from untreated dams were administered vehicle (0.5% methylcellulose) or 0.05, 0.3, or 2.0 mg letrozole/kg once daily via oral gavage from postnatal days (PND) 4 through 70. They were evaluated for alterations of general toxicology/core end points, developmental landmarks, clinical and anatomic pathology, reproduction, and neurological development. The most prominent treatment-related effects were attributed to letrozole pharmacology. Body weight and weight gain of letrozole-treated animals were affected in all dose groups, being significantly lower in males and higher in females than concurrent controls. Sexual maturation and reproduction were also affected in all letrozole groups. Vaginal opening was delayed by 3 to 4 days, and irregular estrous cycles occurred in 30% to 100% of females. Following cohabitation on PND 90 with males from the same dose group, all control females mated and were confirmed pregnant, while in females administered letrozole, mating indices were 0%, 11%, and 0% at 0.05, 0.3, and 2.0 mg/kg/d, respectively. Mean uterus weights were decreased at all letrozole doses, consistent with the microscopic finding of mild or marked uterine atrophy. Reproductive end points were within normal limits for males. Results of this study demonstrated juvenile toxicity consistent with known effects of letrozole.
P504: Nonclinical and Clinical Safety of Modified Virus Ankara (MVA)–Based Immunotherapy Products Targeting Cancer and Infectious Diseases
C. Spring Giusti1, G. Lacoste1, A. Tavernaro1, L. Laruelle1, and E. Quemeneur1
1Transgene S.A, Illkirch-Graffenstaden, France
Transgene is developing specific modified virus ankara (MVA)-based immunotherapy products targeting the tumor-associated MUC-1 antigen (TG4010) and human papillomavirus (HPV)16 E6-E7 oncoproteins (TG4001) in cancer indications. A therapeutic MVA vaccine expressing hepatitis C antigens (TG4040) was stopped after a phase II. Taken together, transgene has a very strong safety record in animal and human mainly after local administration of MVA for long-term treatment periods. The toxicity profiles of TG4010, TG4001, and TG4040 were first investigated in more than 500 animals (mice, rats, rabbits, and primates) following single and/or repeated administrations. All MVA constructs were well tolerated. Hematology and histopathology did not reveal any signs of potential immunotoxicity, alteration in immune system organs (eg, thymus, spleen, lymph nodes, and bone marrow), or sign of amyloidosis. Minor local reactions were observed at the injection sites. In addition, TG4001 did not show any maternal toxicity or adverse effects on embryo-fetal or pre- and postnatal development. To date, 800 patients received at least one dose of MVA-based product, regardless of the indication and/or the route of administration. Tolerance of MVA immunotherapeutics was good and side effects mainly consisted of injection site pain and influenza-like symptoms. The well-established tolerance of recombinant MVA both in animal and human along with the scientific rationale to combine MVA-based immunotherapeutics with anti-PD1 or anti-PDL1 supported the ongoing evaluation of TG4010 in combination with Nivolumab and TG4001 in combination with Avelumab. Modified virus ankara could also be assessed as a potential safe platform for patient specific neoantigen directed immunotherapy.
P505: The Use of Emerging Safety Biomarkers in Nonclinical and Clinical Safety Assessment—The Current and Future State: An IQ DruSafe Industry Survey
D. A. Dalmas1, J. Burkhardt2, J.- C. Gautier3, J. Harding4, M. Singer5, L. Ramaiah6, W. J. Reagan2, A. E. Schultze7, J. Smith8, and T. Zabka9
1GlaxoSmithKline, Collegeville, PA, USA
2Pfizer, Groton, CT, USA
3Sanofi, Paris, Vitry-sur-Seine, France
4AstraZeneca, Hertfordshire, Stevenage, United Kingdom
5Janssen, San Diego, CA, USA
6Bristol-Meyers Squibb, New Brunswick, NJ, USA
7Eli Lilly and Company, Indianapolis, IN, USA
8Boehringer Ingelheim, Ridgefield, CT, USA
9Genentech Inc, South San Francisco, CA, USA
Successful implementation of emerging safety biomarkers (ESBs) can potentially accelerate drug development (DD). Because translatable biomarker utilization by pharmaceutical/biotech companies is often undisclosed, the true impact on DD remains unclear. The IQ DruSafe Biomarker Working Group conducted a survey of large, midsized, and small companies to assess the current and future state of ESBs found in biofluids (ie, protein-, activity-, metabolites-, miRNA/ mRNA-biomarkers). Sixty-seven questions were designed to determine current use in the nonclinical and clinical space, identify opportunities and gaps or barriers to greater implementation, understand impact on advancement of DD, and benchmark perspective on regulatory acceptance. There was general agreement (regardless of company size) regarding percent of companies indicating current use of ESBs in the nonclinical and clinical spaces, use in non–Good Laboratory Practice (GLP) and GLP studies, and use for forward and reverse translation; however, frequency of use varied. Inclusion of ESBs in investigational new drug applications, although limited, was similar across companies. For the future state, there is agreement of a need for broader application in both nonclinical and clinical phases of DD and a need for more robust science balanced with practical approaches to evaluate strengths and weaknesses of ESBs. Additionally, there is a need for increased cross-industry collaborations to develop well-validated assays and decrease the time/resources required for ESB development. For increased biomarker acceptance, value of clear written criteria by health authorities was identified. Overall, the survey highlights similarities and differences across companies and offers suggestions to improve use and implementation of ESBs for the advancement of DD.
P506: Safety Profile of Calicheamicin and Calicheamicin ThioMab Antibody Conjugate in Rats
F. Zhong1, M. Schutten1, T. Lin1, B. Vollmar1, T. Pillow1, M. McDowell1, and C. Frantz1
1Genentech, Inc, South San Francisco, CA, USA
Calicheamicin is a class of antitumor antibiotic cytotoxin that is 4,000× more potent than doxorubicin, a common antitumor cytotoxin used in cancer therapy. Calicheamicins target DNA and cause strand scission. We evaluated the potential for use of calicheamicin as part of an antibody drug conjugate (ADC) for the treatment of cancer. Currently, gemtuzumab ozogamicin (GO), a calicheamicin ADC to anti-CD33, and ainotuzumab ozogamicin (IO), a calicheamicin ADC to anti-CD22, have been approved for relapsed or refractory myeloid leukemia and B-cell precursor acute lymphoblastic leukemia, respectively. Main toxicities of GO/IO in the clinic are hepatotoxicity with veno-occlusive disease, increases in liver enzymes, thrombocytopenia, and neutropenia. Here, we describe the use of THIOMAB antibody technology (TDC) to try to improve the nonclinical safety of ADCs and to more fully understand the mechanism of the cytotoxic payloads action to allow for better optimization. In our study, we tested free calicheamicin or anti-CD22-calicheamicin using the THIOMAB conjugation in rats. Rats were dosed with single dose of free calicheamcin up to 0.1 mg/kg, or anti-CD22-calicheamcin-TDC up to 10 mg/kg weekly for 2 doses. A single dose of calicheamicin was not tolerated at 0.1 mg/kg, with the main target organs being the bone marrow, gastrointestinal, and lymphoid tissues. Repeat dose of anti-CD22-calicheamicin was well tolerated up to 10 mg/kg weekly for 2 doses. The major target organs of toxicity were the bone marrow and lymphoid tissues. In conclusion, calicheamicin antibody conjugate was better tolerated than that of free calicheamicin at equivalent payload dose levels.
P507: In Vivo Assessment of Antibody-Dependent Enhancement of Influenza B Infection
G. K. Rao1, R. A. Prell1, S. T. Laing1, S. C. M. Burleson2, A. Nguyen1, J. M. McBride1, C. Zhang1, D. Sheinson1, and W. G. Halpern1
1Genentech, Inc, South San Francisco, CA, USA
2Burleson Research Technologies, Inc, Morrisville, NC, USA
A theoretical safety concern proposed in the influenza literature is that therapeutic antiviral antibodies could have the potential for antibody-dependent enhancement (ADE) of infection and disease. Antibody-dependent enhancement may occur when virus-specific antibodies at subtherapeutic, non-neutralizing concentrations facilitate virus uptake and, in some cases, enhance replication, which can lead to an exacerbation of virus-mediated disease. Alternatively, ADE may occur due to antibody-dependent complement activation exacerbating virus-mediated disease in the absence of increased replication. As a result of this theoretical safety concern, safety assessment of anti-influenza antibodies may include an in vivo evaluation of ADE of infection and/or disease. The current studies were conducted to investigate the potential of MHAB5553A, a broadly specific, neutralizing therapeutic anti-influenza B antibody, to elicit ADE of infection and disease in mouse models of influenza B infection. In parallel studies, female DBA/2J mice were infected with either influenza B/Victoria/504/2000 or influenza B/Brisbane/60/2008 representing distinct lineages. Assessment of ADE was based on an integration of results from multiple end points, including infectious lung viral titers and genomes, body weight, mortality, lung weight, and histopathology. In these studies, the high dose of 15 mg/kg MHAB5553A resulted in substantial attenuation of influenza pneumonia, with more modest effects at 1.5 mg/kg, whereas MHAB5553A treatment at 0.15 or 0.015 mg/kg was generally comparable to vehicle-treated controls. Our results demonstrate that MHAB5553A across a broad range of doses did not enhance primary influenza B infection or disease in this model and represent a nonclinical derisking of the ADE potential with this antibody.
P508: Methods for Safety Assessment of a Topical Wound Therapy Ointment as a Medical Device
M. T. Finneran1, B. Yasso2, S. C. Gad3, B. J. Varsho2, and G. L. DeGeorge2
1Mark Thomas Finneran, MD, Inc, Wooster, OH, USA
2MB Research Laboratories, Spinnerstown, PA, USA
3Gad Consulting Services, Raleigh, NC, USA
The US Food and Drug Administration Center for Devices and Radiological Health (CDRH) has precedent for classifying topical wound therapies as class II medical devices. As a result of classification of this topical wound therapy ointment as a blood path indirect, external communicating medical device, CDRH required a material-mediated pyrogenicity test and a study to evaluate effects after subcutaneous implantation. Because administration of a viscous ointment intravenously to rabbits was not appropriate for pyrogenicity testing, an atypical extraction scheme was employed. The ointment was spread in a thin layer over the bottom of sterile borosilicate glass petri dishes and covered with extraction solvent (physiological saline [PS]). The ratio of PS to area was based on the circulating blood and lymph volume to the total body surface area of an Hra: NZW SPF rabbit. Petri dishes were agitated and maintained at 37°C for 72 hours. Rabbits were treated with the PS extract following ISO international standard 10993-12:2012(E). For the subcutaneous implantation study, sterile Silastic tubing (1.5 mm ID) was filled with the ointment. Once filled, the tubing was cut into 1 cm lengths for implantation. For the safety evaluation, each of 10 Crl: CD(SD) rats was administered one ointment-filled tubing length in a surgically prepared 1 × 1 cm subcutaneous pocket on the left or right flank, just off the dorsal midline. Empty tubing was administered in the same manner to a control group of 10 rats. The implantation period was 14 days.
P509: Investigation of the Nrf2 Stress Response at Cellular Level to Detect Compounds Inducing Oxidative and Electrophilic Stress
H. Gerets1, K. Tilmant1, F. Atienzar1, and J.- P. Valentin1
1UCB BioPharma SPRL, Braine L’Alleud, Waals Brabant, Belgium
Drug-induced liver injury remains a significant reason for drug attrition. Drug-induced liver injury can be caused by several mechanisms; one of these is the formation of reactive metabolites (RM) leading to oxidative and electrophilic stress. Since such event activates NF-E2-related factor-2 (Nrf2), it seems relevant to measure the Nrf2 activation. In the present study, Nrf2 activation was evaluated using 2 approaches. Firstly, 3 human hepatocytes donors and HepG2 cells were exposed to 5 compounds known to produce RM and 4 non-RM-forming compounds. The expression of 7 genes under the regulation of Nrf2 was measured, enabling the determination of an index considering the results of the 7 genes. Subsequently, the mean index for the RM-forming and the non-RM-forming compounds was calculated for each donor, and the midpoint between these 2 values was determined, which represents the donor-specific threshold, and the compounds were classified accordingly. All 3 donors obtained a sensitivity of 80% and a specificity between 75% and 100%. HepG2 cells obtained 80% sensitivity but very poor specificity of 0%, which makes them less suitable, compared with human hepatocytes donors, for screening of electrophilic stress. Secondly, the Antioxidant-Response-Element-Reporter-Cell Line, AREc32, was exposed to the same set of compounds in addition to 13 RM-forming and 5 non-RM-forming compounds. Using an arbitrary cutoff of 1.5, a sensitivity of 78% and a specificity of 88% were obtained. Comparing both approaches led to the conclusion that acceptable predictive values and easiness of use make the AREc32 assay more suited to investigate Nrf2 activation compared with gene expression approach.
P510: A Comparison of Environmental Assessment Requirements of New Human Drugs in the US and the EU
I. Mohar1, T. D. Lunsman1, and T. Verslycke2
1Gradient, Seattle, WA, USA
2Gradient, Cambridge, MA, USA
Market authorization of new human drugs in both the United States and the European Union (EU) requires an assessment of potential environmental impacts. In the United States, drug approval is overseen by the US Food and Drug Administration (FDA), and an environmental assessment (EA) is required pursuant to the National Environmental Policy Act. In the EU, market authorization is overseen by the European Medicines Agency (EMA), and an EA is required under Directive 2001/83/EC. Both agencies have published guidance documents outlining EA requirements. While the EA requirements of veterinary drugs are largely harmonized, the framework for conducting EAs of human drugs is much less harmonized. The objective of our analysis was to compare and contrast the EA framework in the United States and EU. We reviewed current guidance and incorporated recent EA experience. We identified some general similarities such as the prioritization of endocrine active drugs, the use of tiered testing frameworks, and the preference for guideline studies conducted in accordance with GLPs. However, we identified many important differences in the US FDA and EMA frameworks, including the production and use criteria that trigger EAs, the points at which physicochemical properties are considered, and the specific tests needed to satisfy EA requirements. For example, in the United States , the predicted annual production volume may qualify a drug for EA exemption, while in the EU, both the indicated dosage and certain physicochemical properties must be considered for potential EA exemption. Our analysis illustrates the need for careful and early planning by pharmaceutical companies to ensure global EA compliance.
P511: Safety Evaluation of High Potency Mutagenic Impurities: A Timely Case Study
J. Cohen1, W. Liu2, and M. Jordi2
1Gradient, Cambridge, MA, USA
2Jordi Labs LLC, Mansfield, MA, USA
Recently, hundreds of lots of angiotensin II receptor blockers have been recalled due to the presence of high potency mutagenic impurities (nitrosamines) found in these products as a result of a manufacturing process change. Chemical analysis of such high potency mutagenic impurities can be challenging due to the high instrument sensitivity and low background noise necessary to identify these structures, and the very low detection limits necessary for quantitation. In addition, limitations on the commercial availability of chemical standards can require alternative quantitation strategies. We present here a case study chemical analysis and toxicological evaluation of amino-drug intermediates prepared via the reduction of aromatic nitro groups to the corresponding amines. The resulting amino-drug intermediates were characterized using Quadrupole Time of Flight Gas and Liquid Chromatography Mass Spectrometry (QTOF-GCMS and QTOF-LCMS), facilitated using differential analysis software to identify low-level impurities. Structure confirmation by nuclear magnetic resonance and Fourier-transform infrared spectroscopy was conducted on isolated impurities. Toxicological review of the resulting nitroso- and azoxy-impurities identified no compound-specific data. Both the nitroso- and azoxy-compounds fall within the “cohort of concern” as defined by International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M7 guidance, for which the standard Threshold of Toxicological Concern may not be sufficiently health protective. Carcinogenicity data for similarly structured compounds were therefore used to establish compound-specific safety limits. This case study illustrates some of the potential challenges and strategies for chemical analysis and toxicological qualification of pharmaceutical impurities.
P512: Withdrawn
P513: Optimized Monitoring of Peripheral Neuropathy on Preclinical Studies
N. Sadekova1, J. Douville1, K. Norton1, P.- A. Robillard1, G. Muhire1, F. Goulet1, and L. Chouinard1
1Charles River Laboratories Montreal ULC, Senneville, Quebec, Canada
Peripheral neuropathy is defined by a damage to the autonomic, motor, and/or sensory nerves. The etiology can be varied: diabetes, hereditary disorders, inflammatory infections, autoimmune diseases, or poor nutrition, but may also be an important side effect of certain classes of drugs, especially anticancer agents. Given the translatable nature and clinical significance of this condition, it is critical to characterize nerve function during the preclinical phase. Nerve conduction velocity (NCV) has been previously shown to be a sensitive and valid electrophysiological index of induced neuropathies. In this study, the objective was to validate the sensitivity of the NCV assessments in rats with drug-induced neuropathy and to correlate any effects with histopathological findings. Rats were treated with cisplatin, vincristine, and pyridoxine following regimens known to induce nerve damage. Caudal and digital NCV as well as tibial motor conduction were measured following treatment. Results showed a deficit in the caudal sensory nerve following administration of cisplatin, vincristine, and pyridoxine and a severe loss of function for the digital sensory nerve following administration of vincristine and pyridoxine. A slight prolongation of the onset latency of the tibial motor nerve was also noted following cisplatin and pyridoxine treatment. Histopathological evaluation of paraffin and resin embedded peripheral and central nervous tissues confirmed the results of NCV measurements. In conclusion, the results of this study show that NCV assessments in the rat provide a reliable and sensitive assessment of the onset of changes associated with drug-induced neuropathy.
P514: A Nonclinical Animal Model to Predict Therapeutic Protein and Biological Drug Product Immunogenicity
J. M. Sung1, C. L. San Emeterio1, S. Hosain1, A. D. Knapton1, and K. E. Howard1
1US FDA, Silver Spring, MD, USA
Biological drugs can elicit unwanted immune responses affecting patient safety and drug efficacy, and animal models are needed to understand adverse human responses. Humanized mice have been shown capable of modeling immune toxicity, but some models show inadequate adaptive immune responses, specifically lacking class-switched, antigen-specific antibody formation. The goal of this study was to evaluate humanized mouse models for T-cell-dependent antibody responses. We compared strain-specific antibody responses using bone marrow-liver-thymus (BLT) immune humanized mice made from 4 unique strains of immune compromised mice (NOG, NOG/hGM-CSF/hIL-3, NOG/hIL-6, and NCG). Following humanization, BLT humanized mice were immunized either keyhole limpet hemocyanin, tetanus toxoid, Hepatitis A vaccine, or saline to test antigen-specific stimulation. Following immunizations, antibody class-switching and specificity were evaluated using ELISA and flow cytometry. Results show that NOG/hGM-CSF/hIL-3 and NOG/hIL-6 strains support improved B-cell production and maturation compared with other strains. Further, in contrast to previously published data, NOG/hGM-CSF/hIL-3 and NOG/hIL-6 strains show development of humoral immunity, supported by the presence of typical B-cell subsets (switched memory B cells and plasma cells) and enhanced isotype-switched, antigen-specific immunoglobulin G response to immunization. In conclusion, we show an improved preclinical humanized mouse model with more complete humoral immune function. Future studies will use these models to investigate humoral immune response to biological drug products to better understand immunogenicity and adverse events in patients.
P515: Sildenafil versus Ambrisentan in a Rat Model of Pulmonary Arterial Hypertension
P. B. Senese1, T. Martinez1, K. R. Doherty1, J. Bi1, H. Wang1, M. Zammit1, and M. R. Gralinski1
1CorDynamics, Inc, Chicago, IL, USA
We have previously demonstrated the development of increased pulmonary artery pressure (PAP) and hypertrophy of pulmonary vascular smooth muscle akin to pulmonary arterial hypertension (PAH) after a single dose of semaxanib and hypoxia. Further, partial reversal of PAH symptoms has been shown following treatment with sildenafil. This study was undertaken to compare the effectiveness of sildenafil versus the selective type-A endothelin receptor antagonist ambrisentan in this model. Male Sprague Dawley rats (223-300 g) received a single dose of semaxanib (200 mg/kg SC) on day 1 and then were placed in a low oxygen (13%) chamber from day 1 to 21. Rats were housed in normoxic conditions from day 21 to 56. Test compounds were orally dosed from day 28 to 56, with sildenafil given at 60 mg/kg/day (twice a day) and ambrisentan given at 10 mg/kg/d (once a day). Pulmonary artery pressure, arterial pressure, and thoracic organ weights were measured on day 56. Relative to vehicle, PAP decreased more with ambrisentan than with sildenafil. Systolic PAP decreased 11% following sildenafil and 29% following ambrisentan (diastolic PAP: −33% and 33%, mean PAP: 24% and −31%, respectively). Arterial blood pressures and heart rate were similar to vehicle-treated animals. Necropsy findings showed a decrease in right ventricle (RV) weight and Fulton’s Index in animals given either sildenafil or ambrisentan. Right ventricle weight decreased by 22% and 30% while Fulton’s Index decreased by 17% and 25%, respectively. Under the conditions of this study, ambrisentan appears to be a reliable positive control in this model of PAH.
P516: Investigation of Repeated Oromucosal Application of Triamcinolone Acetonide-Lidocaine Gel in Sprague Dawley Rats
K. Handattu1, S. Biswas1, and B. Mahdevan1
1Abbott Healthcare Pvt. Ltd., Mumbai, Maharashtra, India
Triamcinolone acetonide belongs to the class of synthetic glucocorticoids with potential anti-inflammatory activity. Lignocaine is a well-established local anesthetic of the amide type that has been used for many decades. Both compounds have been studied as monotherapy products in nonclinical models and are approved for use in patients. However, there is limited nonclinical evidence to support the safety of triamcinolone acetonide and lignocaine for use in combination. Therefore, to understand the safety of the combination product, a 28-day repeated-dose oromucosal toxicity study was conducted in Sprague Dawley rats through once daily application of 0.1% triamcinolone acetonide and 2% lidocaine gel. Three different dose levels were studied based on the previous nonclinical toxicology information on the monotherapy products and on the proposed clinical therapeutic dose. Results from the study did not reveal any additional toxicity or local reactions due to simultaneous exposure of triamcinolone acetonide and lignocaine in the gel form. However, characteristic effects of glucocorticoids such as reduced body weight and decrease in weights of spleen and thymus accompanied with the microscopic lymphoid depletion and reduction in peripheral blood leukocytes were evident. In addition, lymphoid tissues like axillary and mesenteric lymph nodes, mandibular lymph nodes, and gut associated lymphoid tissue showed lymphoid depletion. All these effects were reversible in nature and were consistent with the distinctive effects of glucocorticoids indicative of their immunosuppressive property. Thus, the results of the present study revealed the safety of 0.1% triamcinolone acetonide and 2% lidocaine gel, providing further insights for clinical development.
P517: Identification of Safety Margins for Polysorbate 80 Exposure in Canines
K. Holbrook1, D. Andrews1, W. Sutherland1, A. Coppi1, S. Barry1, M. Escamilla1, and J. Bussiere1
1Amgen, Thousand Oaks, CA, USA
Polysorbate 80 (PS80; Tween 80) functions as a dispersing agent, solubilizer, or stabilizer in many pharmaceuticals. Topical or parenteral administration of PS80 has been associated with allergic reactions, including local injection site reactions as well as systemic effects consistent with anaphylactoid-type reactions. These reactions are characterized by the direct release of histamine from mast cells (degranulation). Nonclinical safety assessments of PS80 in vivo have mainly focused on canine model systems, a species well established to be particularly sensitive to PS80. However, there are conflicting data about the dose of PS80 required to elicit an anaphylactoid reaction in this model system. Therefore, systematic studies in anesthetized dogs were conducted to better define this dose using multiple routes of administration, based on a combination of cardiovascular data, clinical signs, and biomarkers of mast cell degranulation. An intravenous (IV) bolus of 1 mg/kg PS80 caused a positive anaphylactoid reaction, including increased heart rate, hypotension, and clinical signs associated with allergic reactions (eg, reddened muzzle). However, a subcutaneous injection of PS80 up to 1 mg/kg did not lead to any adverse effect or anaphylactoid reaction. Likewise, IV infusion of PS80 up to 0.5 mg/kg and IV bolus injection of up to 0.3 mg/kg did not elicit an anaphylactoid reaction in dogs. These studies will enable a better understanding of the safety multiples associated with this common excipient in many commercial pharmaceuticals.
P518: C3d/C4d Deposition on Platelet Correlates With Thrombocytopenia in Monkeys With 2′-O-methoxyethyl Antisense Oligonucleotide (2′-MOE ASO) Treatment
L. Shen1, P. Narayanan1, J. Schroeder1, A. Wong2, S. Oneda2, B. R. Curtis3, J. P. Nolan4, A. Frelinger5, J. A. Engelhardt1, and S. P. Henry1
1Ionis Pharmaceuticals Inc, Carlsbad, CA, USA
2Altasciences Preclinical Seattle LLC, Everett, WA, USA
3Blood Research Institute Blood Center Wisconsin, Milwaukee, WI, USA
4Cellarcus Technologies, La Jolla, CA, USA
5Boston Children’s Hospital, Boston, MA, USA
Sporadic severe thrombocytopenia (TCP; Platelet <50 K/μL) was observed with a low incidence (2%-4% at doses > 5 mg/kg) in Asian-sourced cynomolgus monkeys given 2′-MOE ASOs. The potential mechanism(s) involved was studied in one source of Mauritian monkeys that has shown to be more sensitive to ASO-induced TCP, along with the Asian-sourced monkeys (Cambodian origin). ISIS 405879, a 2′-MOE ASO, induced severe TCP (Platelet < 50 K/μL) and mean platelet volume increase in 7/9 Mauritian monkeys but not in the Cambodian monkeys after 16 weeks of treatment at a dose of 40 mg/kg/week. Mild increases in monocyte chemoattractant protein-1 and Von Willebrand factor were observed in monkeys from both sources. Increases in total serum immunoglobulin M (IgM), along with antiplatelet IgM (and, to a lesser extent, antiplatelet IgG) and antiplatelet factor 4 IgM levels, were observed in both sources but were more pronounced in the Mauritian monkeys. Increased platelet-bound C3d and C4d levels were detected in all thrombocytopenic Mauritian monkeys but not in the unaffected Mauritian or Cambodian monkeys. Plasma Bb and C3a increases were seen in all monkeys from both sources and no C4a increase in the fluid phase, suggesting a local complement pathway activation on the platelet, possibly secondary to IgM antibody deposition or greater complement receptor interaction in those thrombocytopenic Mauritian monkeys. Overall, these data suggest an enhanced innate immune response induced by ISIS 405879 treatment, which leads to anti-PLT antibody generation (likely IgMs), complement fixation on PLT surface, and ultimately an increased PLT clearance in the circulation, resulting in TCP.
P519: Development of a Challenge Model to Study the CD8-Specific Response in Mauritius Cynomolgus Monkey
C. Dumont1, R. Graveline1, M. Haida1, D. Poulin1, R. Samadfam1, S. Kronenberg2, F. Regenass2, and M.- S. Piche1
1Charles River Laboratories, Senneville, Quebec, Canada
2F. Hoffmann-La Roche Ltd., Basel, Switzerland
Although CD8 T cells play a crucial role in cancer immunity, no model is currently available in nonhuman primates (NHP) to monitor this response. Since cancer immunotherapy agents are often tested in nontumor bearing NHP for safety assessment, the animal models currently available do not allow monitoring of potential exaggerated pharmacology and efficacy of these new drug candidates. The goal of this study was to develop an NHP challenge model that specifically elicits a CTL response, in order to evaluate the efficacy of compounds modulating this response. Nonhuman primates were therefore immunized with 3 replication incompetent recombinant adenovirus vectors, each containing the coding sequence for 3 SIV proteins. Moreover, 2 out of the 3 groups of animals received 2 compounds triggering different checkpoint inhibitors known to modulate the T-cell response, allowing monitoring of the T-cell activation in lieu of a tumor or live virus model. Animals receiving both compounds presented an increase in the activation profile of their T cells. This response was characterized by a proliferation profile, the expression of surface activation markers, an upregulation of several checkpoint molecules, and antigen specific responses of the T cells. Increases in the distribution of CD8 T cells and regulatory T cells as well as an increase in the cytolytic activity of the CD8 T cells were also observed. Taken all together, the results suggest that this adenovirus challenge model can be useful to evaluate the efficacy and potential exaggerated pharmacology of new drug candidates targeting CD8 T cells.
P520: Predicting Contractility Risk of Cancer Agents With Adult Human Primary Cardiomyocytes
N. Abi-Gerges1, T. Indersmitten1, W. Nguyen1, P. Ratchada1, K. Truong1, G. Page1, P. E. Miller1, and A. Ghetti1
1AnaBios Corporation, San Diego, CA, USA
Tyrosine kinase inhibitors (TKIs) provide effective cancer treatments, but they are often associated with cardiotoxicity. To enable the development of a new generation of safer TKIs, it is critical to establish novel strategies that can help rank the cardiotoxicity of TKIs early in discovery. We have recently shown that adult human primary cardiomyocytes from donor hearts can be used for in vitro cardiac safety studies. In the present work, we investigated the ability of noninvasive measurement of cardiomyocyte contractility to predict the cardiac safety risk associated with 8 TKIs. We selected both known noncardiotoxic (Afatinib, Dasatinib, Erlotinib, Gefitinib) and cardiotoxic (AZD7762, Imatinib, Sorafenib, Vandetanib) drugs. Our data demonstrate that isolated adult human primary cardiomyocytes are differentially affected by safe and cardiotoxic TKIs. While afatinib, dasatinib, erlotinib, and gefitinib had no effects on contractility up to concentrations equivalent to 30-fold the Cmax at therapeutic dose, AZD7762, imatinib, sorafenib, and vandetanib inhibited contractility with IC50 values of 0.8 μM, 44 μM, 1.2 μM, and 4.6 μM, respectively, which closely match the therapeutic plasma concentrations (Cmax). With these data, we generated a “safety index” (IC50/Cmax) to reflect the cardiotoxicity of these 8 TKIs. We found that TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. Thus, adult human primary cardiomyocytes can provide a useful strategy for the early assessment of cardiac risk associated with anticancer TKIs. Furthermore, this study resulted in the unexpected finding that human cardiomyocyte contractility can be affected by toxic TKIs, irrespective of the effects on the vasculature and chronic cardiac remodeling.
P521: Pharmacological Inhibition of RORg in Wild-Type Mice Does Not Reproduce the High Incidence of Thymic Lymphomas Observed in Knockout (KO) Mice
P. Parris1, M. Liljevald2, J. Jirholt2, M. Söderberg2, L. Kärrberg2, R. Pehrson2, U. Andersson2, J. Hornberg2, and P. Åberg2
1Astrazeneca, Melbourn, Cambridge, United Kingdom
2AstraZeneca, Mölndal, Gothenburg, Sweden
Mice deficient in RORg/gt protein have a high incidence of T-cell lymphomas. Over 50% of knockout (KO) mice die of thymic lymphomas within the first 4 months (Ueda et al, 2002) and conditional KOs show similar phenotype (Liljevald et al, 2016). However, there is no evidence to date of lymphoma risk in humans carrying RORg loss-of-function mutations; therefore, potential translation to larger species is unclear. Mice are recognized as sensitive to T-cell lymphoma development, and in addition, it is unclear whether pharmacological inhibition would differentiate from full ablation of RORg/gt. Though thymic alterations upon RORg inhibition have been documented in rat (Guntermann et al, 2017) and monkey, no subsequent tumor development has been reported. Therefore, we conducted an investigative 6-month mouse study during the nonclinical safety program of AZD0284 (a small molecule RORg inhibitor for the treatment of psoriasis), in addition to 1-month rat and dog toxicology studies. The chronic mouse study was conducted to characterize the histopathological, cellular, and molecular effects of AZD0284 in thymus and blood; the 1-month studies included a general assessment of thymus phenotype. After 6 months, 1 mouse of 60 (1.67%) presented with thymic lymphoma. There were no effects or preneoplastic lesions in the thymus of remaining animals. This demonstrates that pharmacological inhibition of RORg does not reproduce the cumulatively increasing incidence of lymphoma reported in the KO mice (>70% at 6 months). Chronic toxicology and carcinogenicity studies with AZD0284 will further address thymus phenotype development in nonclinical species and contribute to the integrated risk assessment.
P522: Comparison of Single, Paired, and Group Housing on Cardiovascular Parameters and Body Temperature in Telemetered Cynomolgus Monkeys
P. Singh1, S. Loriot1, F. El Amrani-Callens1, M.- E. Duclos1, R. Forster1, and A.- I. El Amrani1
1Charles River Laboratories, Evreux, France
In the present investigation, we compared 3 housing conditions in 4 well-acclimated male telemeter-implanted cynomolgus monkeys. Body temperature (BT) and cardiovascular parameters, including heart rate (HR), arterial blood pressure (systolic [SAP], diastolic [DAP], and mean arterial pressure [MAP]), and ECG parameters (PQ interval, QRS complex, and QT interval) were continuously recorded by telemetry over a period of 19 hours. The animals were housed in ETS-123 compliant cages and data were recorded first under group housing and then under single and paired housing conditions using a cross-over design. When compared with single housing conditions, paired housing had no significant effect on cardiovascular parameters, but group housing led to significant decreases in HR (106 ± 9 vs 139 ± 4 beats/min, P < 0.001), DAP (64 ± 3 vs 87 ± 6 mm Hg, P < 0.001), MAP (83 ± 3 vs 106 ± 7 mm Hg, P < 0.001), and increases in QT interval (297 ± 15 vs 247 ± 3 ms, P < 0.01). There were no statistically significant changes between single- and group-housed animals in SAP or QT corrected for changes in HR according to the Bazett (QTcB) formula or the individual QT correction method (QTca). In group-housed animals, there was a statistically significant increase in BT (38.8 ± 0 vs 38.2 ± 0°C, P < 0.01). Based on quantitative cardiovascular parameters, the present preliminary findings suggest a benefit of group housing conditions over single or paired housing in cynomolgus monkeys.
P523: Withdrawn
P524: Interspecies Comparison of Embryo-Fetal Data Among Control Groups of Sprague Dawley Rats, New Zealand White Rabbits, and Göttingen Minipigs
A. Parenteau1, F.- H. Paradis1, A.- M. Downey1, S. Ellemann-Laursen2, K. Hill3, P. Singh4, J. Hargitai5, R. Forster4, R. Tavcar1, and S. Authier1
1Citoxlab, a Charles River Company, Laval, Quebec, Canada
2Citoxlab, a Charles River Company, Ejby, Koge, Denmark
3Citoxlab, a Charles River Company, Kansas City, KS, USA
4Citoxlab, a Charles River Company, Evreux, Normandy, France
5Citoxlab, a Charles River Company, Veszprem, Hungary
Species-dependent differences in relative incidence of spontaneous variations and malformations can be considered in the assessment of the translational value of reproductive and developmental safety assessments. 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 = 824), rabbits (n = 540), and minipigs (n = 70) from vehicle control groups were included in the analysis equating to 10,749 rat, 5,073 rabbit, and 378 pig fetuses collected at term by caesarean section. Preimplantation loss was more frequent than postimplantation loss in the rat and rabbit, whereas the opposite was observed in the minipig. Several external and visceral malformations and variations such as domed head, bent tail, abdominal edema, and anal atresia were observed in all 3 species. Visceral malformations of the heart and major blood vessels were remarkably more frequent in the minipig and rabbit, respectively; ventricular and atrium septum defects were observed in 1.9% and 2.1%, respectively, for the minipig fetuses, whereas they were observed in equal or less than 0.02% in the rat and rabbit fetuses evaluated in this study. Characterization of species-dependent differences in spontaneous variations and malformations can be useful during interpretation of embryo-fetal development study results. The current analysis identified relevant differences between commonly used species in reproductive toxicology with potential implications during data assessment.
P525: Calculating Safety Margins in Toxicity Studies Using Total Systemic Exposure Versus Unbound Systemic Exposure
S. K. Mukherjee1 and J. B. Ferry1
1Merck & Co, Inc, West Point, PA, USA
Safety margin, a key aspect of nonclinical toxicity studies, is calculated by dividing the systemic exposure (AUC) at NOAEL in nonclinical studies by the AUC required for clinical efficacy. Generally, the total (bound + unbound) plasma concentration (fTp) is used to calculate an AUC. However, the validity of using fTp has been questioned, as it is the unbound plasma concentration (fup) that elicits a pharmacological effect. Plasma protein binding (in nonclinical species and humans) and target organ toxicity data were collected for discontinued Merck compounds to determine whether there would be a meaningful change in safety margin with fup instead of fTp. These compounds had been discontinued from development either due to nonclinical toxicity or due to clinical adverse effects. The data set comprised 118 small molecule compounds. A >3-fold difference in fup was selected as a meaningful difference in plasma protein binding between nonclinical species and humans. In rats, dogs, and nonhuman primates, approximately 4%, 5%, and 3% of the compounds, respectively, had a >3-fold difference in fup when compared with humans. Following an assessment of the toxicity profile for the compounds with a >3-fold difference in fup between humans and nonclinical species, it was concluded that calculation of safety margins using fup would have still led to the discontinuation of these compounds from development. Therefore, although fup can be used for calculation of safety margin, experience shows that no difference in drug discontinuation decisions would have been made whether risk assessment was based on fup or fTp.
P526: Save Your MTD: How to Confirm Spinal Cord Infarction After Lumbar Intrathecal Bolus Administration of Oligonucleotides in Mature and Juvenile Cynomolgus Monkeys
P. Korytko1, A. von Keutz2, F. Runge2, L. Mecklenburg2, S. Zander2, B. Pajaziti2, J. Luft2, F. Ludwig2, M. Wozniak3, and S. Korte2
1Preclinical GPS, Bainbridge Island, WA, USA
2Covance Preclinical Services GmbH, Münster, NRW, Germany
3Medical University of Lublin, Lublin, Poland
Intrathecal (IT) injection is a common dose route in juvenile nonhuman primate (NHP) toxicology studies for antisense oligonucleotides under development for neurodegenerative diseases. Although rare, IT dosing procedures can result in non-drug-related spinal cord infarction (0.1% from >30 NHP toxicology studies; >1,000 animals; >4,500 doses). We present diagnostic features of rare non-drug-related spinal cord infarction in a juvenile NHP (1.6 kg, L2/L3) dosed with 0.75 mL drug solution + 0.25 mL artificial cerebrospinal fluid (CSF) by lumbar puncture. The spinal cord infarction was diagnosed as non-drug-related based on the following findings: (1) low incidence in group or study; (2) high amounts of blood in extracted CSF prior to dosing; (3) delayed hind-limb paralysis/lameness that begins hours after dosing instead of at time of dosing; (4) reduced muscle tone postdose and loss of patellar and foot grip reflexes at 2 to 4 hours postdose instead of immediately after dosing; (5) magnetic resonance imaging evidence of infarction (43-46 mm spinal cord edema from level Th11 to lower endplate of L3 with L2 spinal canal stenosis); (6) macroscopic evidence of infarction; and (7) histopathological evidence (day 3) of necrosis within the spinal cord resulting in dorsal spinal cord malacia, as well as focal axonal degeneration, foamy macrophages, and multifocal vacuolation with axonal degeneration of spinal nerve roots. Although spinal cord infarction is a rare procedure-related finding, this finding may be seen with the increased use of IT dosing in juvenile NHP. This pattern of findings characterizes a non-drug-related spinal cord infarction in juvenile NHP.
P527: Range of Neurological Signs in Cynomolgus Monkeys After Intrathecal Bolus Administration of Oligonucleotides
S. Korte1, J. Luft1, A. von Keutz1, F. Runge1, T. Voss1, M. Wozniak2, S. Zander1, F. Ludwig1, and L. Mecklenburg1
1Covance Preclinical Services GmbH, Münster, NRW, Germany
2Medical University of Lublin, Lublin, Poland
Intrathecal (IT) dosing is a common route of administration in cynomolgus monkey preclinical safety studies conducted for oligonucleotides that target central nervous system diseases. Herein we report on neurological signs that have been observed in 30 IT studies conducted in 1,496 cynomolgus monkeys: (1) Neurological findings occurring under dosing/end of sedation. In 6 animals, severe muscle tremor (rhythmic contraction and relaxation) or muscle spasticity (tightness) was observed under injection of the test article. The dosing procedure was discontinued and intravenous treatment with diazepam was initiated, which improved the symptoms in 2 cases. The other 4 animals were euthanized. (2) A transient absence of lower spinal reflexes. This observation occurs in up to 2% of animals injected with artificial cerebrospinal fluid (control) and is observed in 20% to 80% of test article dosed animals, with the incidence increasing with dose. The transient loss of lower spinal reflexes is considered test article related, but it is not considered adverse. (3) Neurological findings occurring between 30 minutes and 4 hours after dosing. Those findings are characterized by any adverse combination of ataxia, paresis or paralysis of the hind limbs, nystagmus, urinary incontinence, or muscle tremor. (4) Neurological findings due to spinal cord hemorrhage. In 3 animals, neurological findings were characterized by paralysis of hind limbs, absence of patellar reflexes, and grip reaction within 2 to 4 hours after dosing. These symptoms are compatible with a spinal cord ischemic lesion (confirmed by magnetic resonance imaging). Those findings result from a hemorrhage that is created during the injection procedure.
P528: Evaluation of Silicone Rubber as a Potential Impurity in a Drug Product
T. A. Lewandowski1, S. Pacheco-Shubin1, and I. Mohar1
1Gradient, Seattle, WA, USA
Silicone components used in pharmaceutical production subjected to high heat or solvents can degrade over time. We evaluated a drug product suspected to be contaminated with small amounts of silicone polymer particulate. In addition to siloxane monomers and oligomers, silicone rubber also contains additives including antioxidants and plasticizers. Our risk assessment addressed 3 questions: (1) To what extent will constituents of silicone rubber be absorbed in the gastrointestinal (GI) tract? (2) What is the toxicity of the siloxane monomers/oligomers? (3) What is the toxicity of potential additives in silicone rubber? We concluded that only very limited GI absorption of the constituents of silicone rubber will occur because the parent material is resistant to degradation by stomach acid. Animal studies of various siloxanes (linear, cyclic, with variable substitution) indicate minimal adverse effects associated with subchronic or chronic oral exposure. Studies of additives in silicones, performed in the context of food safety evaluation, also indicate potentially extractable concentrations of additives are likely well below established regulatory guidelines. Assuming a 50 kg individual ingested drug product contaminated with the equivalent of 0.25 cm3/day of silicone rubber and the silicone additives are extracted in the GI tract with similar efficiency as reported in food safety studies, additive exposure would be approximately 0.025 μg. This is far below the Threshold of Toxicological Concern or relevant reference doses. Overall, we concluded that the presence of particles of silicone rubber as an unintended impurity in a drug product did not pose a significant risk to patients.
P529: Establishing an In Vitro Proximal Tubular Epithelial Cell (PTEC) Model to Address Kidney Toxicity In Vivo
T. Jiang1, J. Funk2, and M. Gubler2
1Roche Innovation Center Shanghai, Shanghai, China
2Roche Innovation Center Basel, Basel, Switzerland
The kidneys are essential organs that fulfill many vital functions, such as filtration of blood to eliminate metabolic waste products. The proximal tubular epithelial cells (PTECs) are especially susceptible to both small molecule and large molecule drugs because of well-developed transport systems for reabsorption of ions, organic solutes, and peptides from the primary urine. In order to develop safe medicines devoid of kidney liabilities, predictive animal and human-derived PTEC models were developed to assess nephrotoxicity in vitro with cytotoxicity readouts including measurement of intracellular ATP, quantification of stress-induced release of cellular injury markers, and so on. Several small molecule compounds that had been found nephrotoxic in rats were tested in the in vitro PTEC model and showed moderate cytotoxicity to rat PTECs. It was further determined that the cytotoxicity was not driven by toxic metabolites but rather moderate intracellular accumulation of the parent compounds. The results were correlated nicely with proximal tubule degeneration/necrosis in rats, which suggests that this model can serve as an effective tool to address kidney toxicity in vivo.