Abstract
100 Series—General Toxicology
P101: International Travel Grant: Single and Joint Action Toxicity Evaluation of Neonicotinoids Pesticides (Imidacloprid and Thiamethoxam) Against Brackish Water Shrimps (Macrobrachium malcolmsonii)
F. V. Doherty1 and I. A. Aneyo2
1Department of Biological Sciences, Yaba College of Technology, Lagos, Nigeria
2Department of Zoology, University of Lagos, Lagos, Nigeria
Neonicotinoids represent the largest selling class of pesticide, highly persistent and detected frequently in the environment. This study aimed at evaluating the joint action effects of 2 neonicotinoid pesticides used in commercial farming on Macrobrachium malcolmsoni. Macrobrachium malcolmsoni were exposed to 1/10th and /100th of lethal concentration (LC50) obtained for each chemical for 5 days to assess genotoxic effect using micronucleus and comet assay. The 96-hour LC50 obtained for imidacloprid and thiamethoxam were 13.08 µgL and 0.59 µgL, respectively, indicating thiamethoxam was more toxic. The joint lethal concentration was 3.01 µgL. DNA fragments in Macrobrachium malcolmsoni exposed to 1/10th LC50 were significantly higher compared to those exposed to 1/100th LC50. There was no significant difference (P > .05) between control shrimps and those exposed to 1/100th LC50 of imidacloprid. The number of micronucleated cells were significantly higher in shrimps exposed to 1/10th LC50 of the pesticides and was lowest in the control. The number of vacuolated cells were lowest in the shrimps exposed to 1/100th LC50 of the pesticides. There was no significant difference (P > .05) in the total number of cells counted in the control shrimps and those exposed to the combination of imidacloprid and thiamethoxam at 1/10th and 1/100th LC50. Comet DNA, tail DNA, and tail moment were significantly higher in the shrimps exposed to the combination of imidacloprid and thiamethoxam. This study confirmed that Comet and micronucleus assays are useful tools in determining the genotoxic effect of Neonicotinoid pesticide and can be used as bioindicators in the aquatic ecosystem.
P102: North American Graduate Fellowship 2019: Canonical NF-kB Signaling Mediates Mn-Induced YY1 Activation in Human H4 Astrocytes
A. Rizor1, E. Pajarillo1, M. Aschner2, and E. Lee1
1Department of Pharmaceutical Sciences, College of Pharmacy, Florida A&M University, Tallahassee, FL, USA
2Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA
Chronic exposure to manganese (Mn) causes a neurological disorder known as manganism. The mechanisms underlying Mn neurotoxicity are not well understood but include dysregulation of the astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2), leading to excess extracellular glutamate levels and subsequent excitotoxicity. Decreases in EAAT2 expression and activity are associated with pathogenesis in several neurological disorders, including Parkinson disease and manganism, and involve the repressive action of the transcription factor Yin Yang 1 (YY1). In the present study, we investigated the mechanisms of Mn-induced YY1 activation to determine whether Mn increases YY1 expression and activity via activation of the canonical nuclear factor (NF)-kB pathway, mediated by the upstream release of reactive oxygen species (ROS) and tumor necrosis factor (TNF)-α. We also tested if Mn activates YY1 via phosphorylation, leading to the transcriptional repression of EAAT2. Our results demonstrate that Mn exposure induces canonical NF-kB activation via release of ROS and TNF-α, leading to the phosphorylative activation of YY1 and corepressor histone deacetylases (HDACs). Manganese-induced YY1 activation results in increases in YY1 nuclear translocation, YY1/HDAC interaction, and EAAT2 repression. Accordingly, treatment with IKK16, an IKKB inhibitor, C-87, a TNF-α inhibitor, and antioxidants attenuated Mn-induced activation of NF-kB and YY1/HDACs and restored EAAT2 expression. Taken together, these results indicate that Mn-induced oxidative stress/inflammation upregulates YY1 via the canonical NF-kB pathway. Manganese also activates YY1/HDACs via phosphorylation, leading to EAAT2 reduction and subsequent excitotoxic neuronal injury.
P103: Feedlot-Derived Steroid Hormones in Surface Waters Adjacent to Concentrated Animal Feeding Operation (CAFO)
O. J. Ayodeji1 and P. Smith1
1Department of Environmental Toxicology, Texas Tech University, Lubbock, Texas, USA
Steroid hormones used as growth promoters in beef cattle feeding operations can contribute to pollution of surface waters. Transport of these compounds from concentrated animal feeding operations (CAFOs) into the environment has generally been associated with movement along the soil-water system, aerial deposition of dust, and direct application of wastes. Elevated concentrations of multiple agrochemical classes in fugitive particulate matter emissions, playa lake water samples, and wildflower samples collected near beef cattle feeding operations have been documented. Steroid hormones transported into the environment from CAFOs have potential to act as endocrine disrupting compounds. In this study, surface water samples collected from 10 retention ponds affected by CAFOs were examine for occurrence of estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), melengestrol acetate (MGA), and trenbolone acetate (TbA). Concentrations of steroids ranged from 2.29 to 6.33 ng/L for trendione, 31.95 to 3803.24 ng/L for β-TbA, 6.06 to 2497.55 ng/L for α-TbA, 9.27 to 156.60 ng/L for E1, 7.86 to 510 ng/L for α-E2, 5.10 to 461.58 ng/L for β-E2, and 1.10 to 63.08 ng/L for MGA. The most frequently detected were E1, β-E2, β-TbA, and α-TbA at 80%, 70%, 60%, and 60% of CAFOs, respectively. Results of the current study suggest that use of steroid growth promoters on CAFOs contributed to observed concentrations of these compounds in adjacent retention ponds.
P104: Histomorphological and Redox Delineations in the Testis and Epididymis of Albino Rats fed With Green-Synthesized Cellulose
C. Otuechere1, A. Adewuyi2, O. Adebayo1, E. Yawson3, O. Kabiawu1, and B. Okubio1
1Department of Biochemistry, Redeemer’s University, Ede Town, Osun State, Nigeria
2Department of Chemistry, Redeemer’s University, Ede Town, Osun State, Nigeria
3Department of Anatomy, Redeemer’s University, Ede Town, Osun State, Nigeria
It has also become increasingly necessary to diversify the production of cellulose for biomedical applications. In this study, cellulose was green synthesized from Sesamum indicum (GSC), an aphrodisiac medicinal plant in Nigeria. The impact of the oral administration of GSC on the antioxidant status and histomorphology of the testes and epididymis of rats was studied as follows: control (saline), 100, 200, and 400 mg/kg GSC for 14 consecutive days. Although there were no effects on organ weights and organosomatic indices, the albumin levels were markedly reduced in animals fed with GSC relative to the control. Exposure to GSC elicited differential effects on the redox systems of the testes and epididymis. GSC caused nonsignificant changes in superoxide dismutase, catalase, reduced glutathione, and nitric oxide levels, whereas it significantly decreased glutathione peroxidase and malondialdehyde levels in the testes of the treated rats. Contrariwise, GSC treatment significantly decreased superoxide dismutase and nitric oxide levels but caused a significant increase in glutathione peroxidase and reduced glutathione levels. Furthermore, the histomorphology of the testes and epididymis at the scale bar of 200 nm revealed extravasation of the germinal epithelium of the epididymis at doses of 200 and 400 mg/kg GSC. Conversely, at the scale bar of 400 nm, treatment-related aberrations such as sloughing of germinal cells and spermatogenic arrest (testes) and chromatolytic alterations (epididymis) were observed at the doses of 200 and 400 mg/kg GSC. Thus, GSC administration evoked pro-fertility effects in rats at lower doses, but its effects at higher doses calls for caution.
P105: International Travel Grant Neuroprotective Effects of Resveratrol Against Arsenic-Induced Cognitive Deficits via Alterations in Estrogen Receptor Signaling and ERK1/2 Pathway in Mouse Hippocampus
K. Mehta1, B. Kaur1, K. P. Kumar1, and P. Dhar1
1All India Institute of Medical Sciences, New Delhi, Delhi, India
Chronic inorganic arsenic (iAs) exposure is well associated with cognitive dysfunctions and alteration of several neurochemical signaling pathways in central nervous system. The correlation between neurotoxicity and oxidative stress induced by iAs exposure opens up an opportunity for identifying potential of natural polyphenolic compounds that could serve as beneficial therapeutic agents. We aimed to examine the neuroprotective role of Resveratrol (if any) against toxic effects of chronic iAs exposure in mouse hippocampus and exploring the mechanisms (cognitive functioning, oxidative stress, estrogen signaling, BDNF/NMDAR neurochemical signaling, and ERK1/2 pathway) underlying these neurotoxic effects. Adult female mice were exposed to As2O3 (2 and 4 mg/kg) alone, resveratrol (40 mg/kg) alone, or both via oral route (45 days). Assessment of cognitive functioning was carried out from day 33 to 46. On day 46, the animals were sacrificed and brain tissues, thus obtained were further processed (immunohistochemistry and Western blotting). Long-term exposure to iAs significantly enhanced the anxiety-related responses and impaired the cognitive functions. Arsenic-induced hippocampal oxidative damage was evident through alterations in Glutathione and nitric oxide levels. Inorganic arsenic alone exposed animals showed significant alteration in estrogen receptor signaling with a concomitant reduction of BDNF and NMDAR expression in mice hippocampus. Substantial recovery in all these altered mechanisms along with activation of ERK1/2 signaling was observed in the hippocampus of resveratrol supplemented animals. Our findings demonstrate that resveratrol supplementation attenuates arsenic-induced neurotoxicity in mouse hippocampus via activation of estrogen receptor expression and ERK1/2 signaling pathway. This study might provide therapeutic insight toward a treatment for arsenic-induced neurotoxicity.
P106: North American Graduate Fellowship 2020 Chlorpyrifos-Induced Transgenerational Modifications of Serotonin-Mediated Behaviors in Caenorhabditis elegans
C. McClure1 and P. Allard1
1University of California, Los Angeles, CA, USA
Chlorpyrifos (CPF) is an organophosphate pesticide in the United States despite evidence that it can cause life-long, persistent changes in the serotonergic system of mammals. However, there is little known about the transgenerational effects of behavior after exposure to this pesticide. We will evaluate this pesticide using a self-designed and standardized behavioral model in Caenorhabditis elegans. We hypothesize that CPF will alter serotonin-controlled behavior and that this will be transgenerational. We propose to design assays to monitor the activity of serotonin pathways which can then be used for chemical assessment. We have characterized the enhanced slowing responses (ESR) via food deprivation and basal slowing responses (BSR), unexposed, for both wild-type worms and a mutant strain. Wild-type worms display decreased speed in their ESR, as expected (fold change = 1.6, P < .0001, 1-way analysis of variance, 17-19 repeats) when compared with their BSR. A mutant strain, tph-1, does not accumulate serotonin. We expect worms to increase speed when compared to wild type. We found this to be true (fold change = 2, P value = .0153, Student t test, 6-7 replicates), validating this is as our mechanism of action. In addition, preliminary data indicate that there is a decrease in the hyper-roaming response after CPF exposure on wild-type worms (1 repeat) when compared control. This in-depth behavioral analysis and subsequent dose–response study is the first of its kind and will address a significant gap of knowledge into the transgenerational effects of serotonin modifying agents on behavior.
P107: International Travel Grant Evidence of Oxidative Damage, Endocrine Disruption, and Male Reproductive Dysfunction Accompanying Di-n-butyl Phthalate Exposure in Wistar Rats
J. Olugbodi1, R. Bello1, and G. Adebayo-Gege2
1Bingham University, Karu, Nasarawa, Nigeria
2Baze University, Abuja, Nigeria
Di-n-butyl phthalate (DnBP) is a widely reported chemical used in manufacturing household products—rocket fuel and paints—and has been noted to pose lot of danger to the body due to its toxicity, causing a great health challenge and concern. This study aimed at providing evidence regarding the DBP-induced reproductive dysfunction in male Wistar rats. Male Wistar (n = 28) was randomly divided into 4 groups (n = 7). The rats were orally administered with vehicle, DBP (100, 250 and 500 mg/kg) for 28 days. Thereafter, sperm kinematics and motility, selected biomarkers of oxidative damage, endocrine disruption, inflammation, and DNA damage were evaluated. DnBP-treated rats showed decrease in testis and epididymis weights. The cauda epididymal sperm total motility, progressive motility and epididymal sperm concentration, curvilinear velocity, straight line velocity, average path velocity, linearity, straightness, and beat/cross-frequency were significantly decreased (P < .05) in all treated groups. Increased levels of sperm abnormality, nonprogressive motility, wobble, and immotility was observed in all treated groups. The DnBP increases testicular and epididymal malondialdehyde level and hydrogen peroxide generation but significantly decreases (P < .05) catalase, superoxide dismutase, glutathione, glutathione peroxidase, and glutathione S-transferase activities in the testes and epididymis. Also, DnBP induced hormonal imbalance, markedly increased expressions of testicular cyclooxygenase-2, inducible nitric oxide synthase, and 8-hydroxy2-deoxyguanosine. Histological study revealed moderate congestion of vessels, edema in the testes, fibrotic connective tissues, and lack spermatozoa storage in the epididymis of DnBP-treated rats. Overall, DnBP induced reproductive dysfunctions, inflammation, and DNA damage in male rats.
P108: International Travel Grant Epidemiology Following 2-Methyl-4-Chlorophenoxyacetic Acid (MCPA) Poisoning
T. M. Wijerathna1, F. Mohamed2, I. B. Gawarammana3, and N. A. Buckley4
1South Asian Clinical Toxicology Research Collaboration, Faculty of Medicine, University of Peradeniya, Peradeniya, Central Province, Sri Lanka
2Department of Pharmacy, Faculty of Allied Health Science, University of Peradeniya, Peradeniya, Central Province, Sri Lanka
3Department of Medicine, Faculty of Medicine, University of Peradeniya, Peradeniya, Central Province, Sri Lanka
4Discipline of Pharmacology, Faculty of Medicine and Health, The University of Sydney, Clinical Pharmacology and Toxicology Research Group, Sydney, New South Wales, Australia
2-Methyl-4-chlorophenoxyacetic acid (MCPA) is a widely used chlorophenoxy herbicide. Self-poisoning with MCPA can be fatal. The objective of this study is to describe the epidemiology of MCPA self-poisoning in Sri Lanka. All consenting MCPA self-poisoning patients admitted to 9 general hospitals in Central Province, North Central Province, and Southern and North Western Province of Sri Lanka over a period of 14 years were considered. Epidemiologic, demographic, and clinical data were recorded on a study-specific datasheet. There were 1464 patients; 64% were male. The median time from ingestion to admission was 4 (interquartile range [IQR]: 2-6) hours, and 43% were transferred from peripheral hospitals. The overall case-fatality rate was 4.4% (95% CI: 3.4-5.6). The median age of the patients was 27 (IQR: 20-38) years. Patients who died were older (42 [IQR: 30-54] years for death vs 27 [IQR: 20-36] years for survivors). The median amount of MCPA ingested by patients who died was also greater (100 [IQR: 36-200] mL in death vs 55 [IQR: 16-140] mL in survivors). There was an increase in MCPA poisonings following the bans on paraquat in 2011 and glyphosate in 2014. The high proportion of men with MCPA poisoning likely reflects the availability of pesticides for male farmers. Older people ingested larger doses and were also more likely to die. Banning some pesticides causes an increase in other pesticide poisonings; however, the case-fatality of MCPA is not higher than that of paraquat or glyphosate.
P109: International Travel Grant Metal Mixture Exposure and Lifelong Consequences in Brain Cholinergic System and Spatial Learning and Memory of Rats
C. B. B. Davuljigari1, S. R. M1, and R. R. Gottipolu1
1Division of Environmental Health, Department of Zoology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India
In this study, we examined the effects of metal mixtures on neurochemical and behavioral functions and also investigated possible interactions among metals in rats. Male rats were lactationally exposed to 0.2% Pb in drinking water of the mother from postnatal day 1 (PND 1) to PND 21. Manganese (Mn) was separately given to PND 28 age-group rats at a low (2.5 mg/kg) dose through intraperitoneal injection for 4 weeks (3 alternative d/wk). To study the combined effect, Pb-exposed animals were exposed to Mn (2.5 mg/kg body weight) from PND 28 for 4 weeks. The results showed that synaptosomal AChE, mitochondrial aconitase, total superoxide dismutase (SOD), Cu/Zn-SOD, and thioredoxin reductase activities decreased, whereas ACh and malondialdehyde levels increased in the cortex, cerebellum, and hippocampus following individual and combined exposures to metals at PND 60, 4, 12, and 24 months age-group rats. While the activity of Mn-SOD marginally increased with Mn exposure and decreased with metal mixture exposure. These alterations were greater in the hippocampus with Pb exposure and in the cortex of Mn and Pb + Mn-exposed rats. Significant deficits were observed in spatial reference and working memory and exploratory behavior following exposure to both metals individually and together. In conclusion, our data indicate that exposure to the mixture of metals greatly increased the late-life impairments in the brain chemistry and behavioral functions of rats by altering the Pb deposition, whereas the late-life effects of Mn was very marginal suggesting neurotoxicity of Pb scenario should be considered within the context of metal mixture exposures.
North American Travel Grant Prenatal Exposure to an Environmentally Relevant Phthalate Mixture Accelerates the Aging of the Ovary in Multiple Generations of Female Mice
E. Brehm1, C. Zhou1, L. Gao1, and J.A. Flaws1
1Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana-Champaign, IL, USA
Phthalates are known endocrine-disrupting chemicals that have been shown to disrupt reproduction. Our laboratory found that prenatal exposure to a relevant phthalate mixture accelerated reproductive aging in multiple generations of mice. However, it was unknown if prenatal exposure to phthalates accelerates the aging of the ovary, which is defined as a decrease in the follicle pool and an increase in cysts. Thus, we tested the hypothesis that prenatal exposure to a phthalate mixture accelerates the aging of the ovary in multiple generations of mice. Pregnant mice were orally dosed with control or a phthalate mixture (20-500 mg/kg/d) daily from gestational day 10 to birth. Adult F1 females born to these dams were used to create the F2 and F3 generations by mating them with unexposed males. At 13 months, ovaries were collected for analysis. Prenatal exposure to the phthalate mixture (200 µg/kg/d) decreased the number of primordial follicles and increased the occurrence of cystic ovaries in the F1 generation. The mixture (20-200µg/kg/d) increased the percent of primary follicles, and it (200 mg/kg/d) decreased the percent of antral follicles in the F2 generation. Further, the phthalate mixture (20 and 500 mg/kg/d) increased the percent of primary follicles, and it (20-200 µg/kg/d) decreased the percent of preantral follicles in the F3 generation of female mice. Overall, these data suggest that prenatal exposure to a phthalate mixture may accelerate the aging of the ovary in a multi- and transgenerational manner in mice.
P111: North American Travel Grant Comparative Assessment of the Effects of Cypermethrin and Permethrin on Fetal Growth, Placental Morphology, and Neurodevelopment in Mice
B. A. Elser1, K. Kayali1, F. Rimi1, and H. E. Stevens1
1University of Iowa, Iowa City, IA, USA
Prenatal exposure to pyrethroid insecticides is associated with increased risk of neurodevelopmental delay and low birth weight in children. However, there is a significant gap in knowledge about the mechanisms by which pyrethroids alter fetal growth and neurodevelopment, and what the relative impact is of type-I versus type-II pyrethroids. To address these questions, pregnant CD1 dams (8 weeks old) were treated with the type-I pyrethroid permethrin (1.5, 15, and 50 mg/kg), the type-II pyrethroid α-cypermethrin (0.3, 3, and 10 mg/kg), or corn oil vehicle via oral gavage from gestational days 6 to 16 (n = 10 dams per treatment group). Placentas were evaluated for structural alterations and the presence of lesions. Fetal brains were assessed for cortical volume and microglia morphology. Cypermethrin had no effect on maternal weight gain at any dose; however, treatment with 1.5 mg/kg and 50 mg/kg permethrin increased maternal weight gain. Cypermethrin treatment at 10 mg/kg reduced fetal body weight; however, permethrin had no effect on fetal body weight. Neither treatment induced placental necrosis or hemorrhage; however, 10 mg/kg cypermethrin treatment reduced the size of the placental labyrinth zone. In comparison, permethrin did not affect placental layer morphology. Furthermore, cypermethrin treatment decreased cortical volume and induced a dose-dependent shift from ramified to ameboid microglia. Placental labyrinth zone thickness was significantly correlated to fetal body weight and degree of microglia ramification in the fetal brain, suggesting a potential link between cypermethrin-induced placental dysfunction and fetal development. In summary, these results demonstrate alterations in fetal growth, placental morphology, and neurodevelopment induced by the type-II pyrethroid cypermethrin.
P112: North American Graduate Fellowship 2020 Subchronic Diisononyl Phthalate Exposure Affects Colonic Health in Adult Female Mice
K. Chiu1 and J. A. Flaws2
1Division of Nutritional Sciences, College of Agricultural, Consumer and Environmental Sciences, Urbana, IL, USA
2Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA
Diisononyl phthalate (DiNP) is a common phthalate that is used to make polyvinyl chloride flexible. Diisononyl phthalate is used in many consumer products such as toys, clothing, food packaging, and building materials. Exposure to DiNP has been shown to aggravate immune responses. However, little is known about DiNP and its effects on the gastrointestinal tract. We hypothesized that subchronic exposure to DiNP alters histology, hormone levels, and gene expression related to inflammation and cell cycle regulation in the colon. To test this hypothesis, adult female mice were orally dosed with corn oil vehicle, 20 µg/kg, 200 µg/kg, 2 mg/kg, 20 mg/kg, or 200 mg/kg DiNP for 10 to 14 days and then euthanized during diestrus immediately after dosing. Colons were collected for histological examination, hormone analyses, and gene expression analyses. Histological analysis showed that DiNP exposure at environmentally relevant doses significantly increased colonic damage compared to control. Diisononyl phthalate exposure in treatment groups 200 µg/kg and above had significantly decreased estradiol concentrations in extracted lipids compared to control. Diisononyl phthalate did not significantly affect cytokine gene expression in the colon compared to control. However, DiNP exposure increased Ifng and Tnf expression in a dose-dependent manner compared to control. Further, 200 µg/kg DiNP significantly downregulated Ccnd2 compared to control. High levels of DiNP did not alter the expression of many of the cell cycle regulators compared to control. These data suggest that DiNP exposure causes colonic damage and hormonal changes and may interfere with cell growth and immune responses in the colon.
P113: A Comparison of Historical Control Data From Cynomolgus Macaques of Chinese, Cambodian, Vietnamese, and Mauritian Origin
M. J. Caruso1, Z. Lloyd1, C. Carter1, D. Patrick1, M. Muzyka1, E. Debien2, A. Wilcox3, C. Papagiannis3, S. Bulera3, and T. J. Baird1
1Charles River Laboratories, Mattawan, MI, USA
2Charles River Laboratories, Sherbrooke, Quebec, Canada
3Charles River Laboratories, Reno, Nevada, USA
Cynomolgus macaques, the most common nonhuman primate (NHP) species selected for nonclinical toxicology studies, are generally derived from purpose-bred sources in a variety of geographic locations including Southeast Asia (China, Cambodia, and Vietnam) and Mauritius. However, their importation remains a challenge due to a shortage in NHP availability. Importantly, considerable genetic and environmental variability associated with geographic origin could affect toxicology study results and interpretation. This study compiled historical control data from 2 to 9.4 year old, healthy control cynomolgus macaques sourced from China (n = 750 males/741 females), Cambodia (n = 282 males/271 females), Mauritius (n = 225 males/226 females), or Vietnam (n = 122 males/120 females). Data were used to evaluate the impact of geographic origin on standard nonclinical toxicology study end points. Parameters included clinical observations, body weight, ophthalmoscopic examinations, clinical pathology (hematology, coagulation, clinical chemistry, and urinalysis), and anatomic pathology (organ weights and gross/microscopic lesions). No toxicologically meaningful baseline differences were observed among cynomolgus macaques originating from China, Cambodia, and Vietnam for any parameter evaluated. When comparing similar parameters in Southeast Asian- and Mauritius-derived cynomolgus macaques, we identified numerous clinicopathologic differences (eg, smaller-sized erythrocytes, decreased lymphocyte counts and cholesterol concentration, increased globulin concentration). Findings suggest that Southeast Asian geographic origin has little impact on baseline variability in the evaluated parameters supporting interchangeable use of cynomolgus macaques from these specific subpopulations. Further studies are required to determine the impact of the baseline clinicopathologic differences between Southeast Asian and Mauritian cynomolgus macaques within the context of a nonclinical toxicology study.
P114: Spontaneous Incidence of Infrequent Neurological Clinical Signs Across Species: A Retrospective Analysis
C. Ballesteros1, A. Parenteau1, C. Li1, R. Tavcar1, and S. Authier1
1Charles River Laboratories Montreal ULC, Laval, Quebec, Canada
The identification of possible adverse effects is crucial in the development and safety assessment of drugs in toxicology and safety pharmacology studies. Potential background clinical observations in healthy animals can confound the interpretation of test item-related effects in toxicology and pharmacology studies. The purpose of this retrospective analysis was to determine the prevalence of incidental clinical observations which could be interpreted as neurologically related in control animals. We compiled clinical observations documented in control animals from 306 good laboratory practice–compliant toxicology studies which were conducted between 2009 and 2019 at Charles River Laboratories Montreal ULC. These studies consisted of a total of 6910 control animals (both males and females) with dosing phases ranging from 1 day to 39 weeks and recovery phases ranging from no recovery to 26 weeks. All control animals across these studies were administered an appropriate vehicle consistent with the drug formulation buffer and route of administration of their drug-treated counterparts. Species evaluated consisted of Beagle dogs, Sprague-Dawley rats, New Zealand White rabbits, Göttingen minipigs, Cynomolgus monkeys, and CD-1 mice. Among the clinical signs compiled which could be interpreted as neurologically related, we observed signs such as salivation, tremors, uncoordinated behavior, myoclonic jerks, muscle twitches, change in muscle tone, muscle atrophy, full muscle contraction, circling, clonic convulsion, tonic convulsion, hunched back, piloerection, and hypersensitivity. Here, we present the incidence proportion and rate of some of these spontaneous observations across the different control species, which could allow for a better interpretation of treatment-related data that is confounded by neurologically related incidental background findings.
P115: Gentamicin-Induced Ototoxicity in Hartley Guinea Pigs
J. A. Klein1, B. C. Cox2, L. Cox2, J. R. Manker2, M. Randle2, S. Tupal2, A. E. Turner2, S. E. Boley1, G. Bouchard1, and J. G. Turner2
1Sinclair Research Center, Auxvasse, MO, USA
2Turner Scientific, Jacksonville, IL, USA
Ototoxicity was investigated in male and female Hartley Guinea Pigs treated with 14 consecutive days of gentamicin at 40, 80, or 160 mg/kg given subcutaneously and compared to saline-treated controls. Guinea pigs are a common model of ototoxicity, but no sex differences have previously been reported. Baseline auditory brainstem response (ABR) tests were performed before the first dose and again 14 days after the last dose of gentamicin or saline. Cochlea were dissected using a whole mount preparation and immunofluorescent staining was performed using myosin VIIa and the nuclear dye, Hoeschst.
P116: Pexidartinib Impairs Liver Mitochondrial Functions Causing Hepatocyte Death
Q. Shi1, L. Ren1, K. Papineau1, L. Schnackenberg1, and W. Mattes1
1Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR, USA
Pexidartinib is a recently approved small molecule kinase inhibitor with a boxed warning for hepatotoxicity. As mitochondrial liability has been increasingly associated with drug toxicity, this study was designed to examine the potential role of mitochondrial injury in pexidartinib-induced hepatotoxicity. Freshly isolated rat liver mitochondria, submitochondrial fractions, and primary human hepatocytes were treated with pexidartinib at clinically relevant concentrations, and then mitochondrial functions and cell death were assessed. In isolated mitochondria, the state 3 oxygen consumption rates of glutamate-malate and succinate-driven respirations were both decreased by pexidartinib, with the former being affected more profoundly, while the state 4 oxygen consumption rates were barely affected. In rat liver submitochondrial fractions, the activities of respiratory chain complex I and V, particularly I, were significantly inhibited by pexidartinib; complexes II, III, and IV were not inhibited. In primary human hepatocytes, pexidartinib depleted adenosine triphosphate (ATP), increased reactive oxygen species, and caused cell death as measured by the cell counting kit-8. However, the caspase activities were unaffected. All the effects noted above occurred at concentrations of 1- to 2.5-fold human peak blood levels achieved at recommended therapeutic doses. Taken together, these data suggest that pexidartinib selectively inhibits mitochondrial respiratory chain complex I/V causing ATP shortage and oxidative stress which further lead to hepatocyte death, suggesting mitochondrial injury may contribute to the mechanism of pexidartinib-induced hepatotoxicity.
P117: Snuggle Wrap-Based Restraint Method for Continuous Infusion in Juvenile Nonhuman Primates
N. Lalayeva1, J. Reynolds1, G. Aylor1, J. Forget1, and T. Rogers1
1Altasciences Preclinical Seattle, LLC, Everett, WA, USA
With the increase of pediatric pharmaceuticals in preclinical studies, the ability to adapt to the use of a younger and smaller test system is necessary to assure process evolution and to reduce stress on the research animals. The routine method for infusion restraint at our facility includes the use of a procedure cage. This method would have been highly stressful for animals under a year of age and was not considered suitable for small primates. Alternative “snuggle wrap” method was developed to allow young animals to remain immobile while still feeling comforted by a hugging-like sensation. Given that social pairs needed to remain in proximity, the “snuggle board” was also developed. The boards hold 4 snuggled animals closely together which kept social pairs in visual and auditory contact during dose administration. Animals were visually monitored, provided treats, and manipulated throughout the dosing period. This method was successfully utilized in two 13-week studies with intravenous infusions in animals of: 6 to 9 months, 12 to 15 months, and 40 to 48 months of age. The data sets collected using the snuggle wrap or the procedure cage were pooled and utilized for comparison. Based on the data, there are no apparent differences between the 2 restraint methods. Therefore, both procedure cage and snuggle wrap methods can be successfully utilized based on the age and size of the animals with the snuggle wrap method being the most suitable for younger primates.
P118: Aerosol From a Heated Tobacco Product Causes Lower Induction of Hepatic Drug Metabolism and Reduced Drug Interaction Compared With Cigarette Smoke
D. Bovard1, K. Renggli1, D. Marescotti1, S. Majeed1, C. Pak1, A. Iskandar1, K. Luettich1, S. Frentzel1, J. Hoeng1, and M. C. Peitsch1
1PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland
The main mechanism behind drug interactions with cigarette smoke (CS) involves activation of hepatic cytochrome P450 (CYP) isoenzymes by the polycyclic aromatic hydrocarbons. This study aimed to evaluate the impact of exposure to aerosol generated from Tobacco Heating System 2.2 (THS 2.2; Philip Morris International) on human liver xenobiotic metabolism and subsequent drug metabolism, compared with the impact of exposure to CS. We exposed HepaRG spheroids to various concentrations of aqueous THS 2.2 aerosol or CS fractions for 48 and 96 hours and evaluated hepatotoxicity by measuring adenosine triphosphate levels. At 0.8 puffs/mL (∼100 times the nicotine concentrations in the plasma of IQOS users after 10 puffs), the THS 2.2 fraction was not hepatotoxic, while the CS fraction was highly hepatotoxic. We used a liquid chromatography–mass spectrometry approach to measure the activity of 8 CYP enzymes in the liver spheroids after exposure to subtoxic doses of the aqueous fractions of THS 2.2 aerosol and CS for 96 hours. The CS fraction induced a 2-fold and 3-fold increase in the activity of CYP1A1/1B1 and CYP1A2 enzymes, respectively, but inhibited CYP3A4 enzyme activity. At comparable concentrations, exposure to the THS 2.2 fraction resulted in smaller changes in CYP enzyme activities relative to those observed with CS. Compared with exposure to the aqueous fraction of CS, exposure to the aqueous fraction of THS 2.2 aerosol resulted in vitro in reduced hepatotoxicity, smaller changes in liver xenobiotic metabolism, and reduced alteration in the metabolism of clozapine, in which pharmacodynamics is known to be modified in smokers.
P119: The Göttingen Minipig as a Model for Skin Immunology
L. Allais1, N. Ravas1, and F. Condevaux1
1Charles River Laboratories, Saint-Germain-Nuelles, Rhone, France
The skin hosts a sophisticated immune system providing an immunological defense against pathogens and maintaining body’s homeostasis. When the barrier function of the skin is disrupted following injury or as a result of a disease, a cascade of events is triggered involving both innate and adaptive immune responses. Atopic dermatitis or eczema is one of the most common skin disorders and is characterized by a chronic inflammatory response mainly causing skin rash with different degrees of severity. In view of many similar properties shared between porcine and human skin in terms of structure, composition, and functions, we investigated a contact dermatitis model in Göttingen minipigs using 2,4-dinitrochlorobenzene (DNCB), a well-known sensitizer. Four minipigs were sensitized with 10% DNCB and then challenged with 1% DNCB 12 days later. Main skin immune cells were analyzed by flow cytometry and/or immunohistochemistry 24, 48, and 72 hours after DNCB challenge. Epidermal T cells increased 48 and 72 hours, whereas Langerhans and dermal dendritic cells decreased 48 and 72 hours after challenge. The latter changes suggested a potential migration of the dendritic cells from the skin before probably reaching the draining lymph nodes. There was also a time-dependent increase in dermal MHCIIlow cells, indicating infiltration of circulating inflammatory cells such as monocyte-derived macrophages. The DNCB-related changes obtained in our study were consistent with contact dermatitis responses and suggested that the Göttingen minipig skin could represent a relevant model for human skin immune pathologies, whereas skin immune properties could be more questionable in other laboratory animals.
P120: Establishing the “Normal” Immune Response in Cynomolgus Monkey and Rat for Use in Preclinical Immunotoxicology Safety Assessment
Z. F. Faraahi1, J. Munday1, K. Hunjan1, and A. Shaw1
1Covance Ltd, Harrogate, North Yorkshire, UK
Preclinical immunotoxicity assessments are part of repeat-dose toxicity studies recommended by regulators for improved prediction of potential adverse effects on the immune system. Evaluating T-cell-dependent antibody response (TDAR) using anti-keyhole limpet hemocyanin (KLH) antibody provides an overall measure of host immune function yet benefits from parallel immunophenotyping (IPT) and cytokine analysis for a more comprehensive immunotoxicology understanding. Limitations of these assays are high inter-animal variability, lack of established normal range, and ambiguity of toxicologically significant TDAR-inhibition levels in standard in vivo animal models. As such, there is a need to evaluate these issues and to establish standard criteria in rat and cynomolgus monkey for the classification of immunotoxicants. Healthy rats (n = 10) or cynomolgus monkeys (n = 37) were administered 300 µg/kg KLH subcutaneously at the start of the study to induce an initial immune response and were given a consecutive dose approximately 2 weeks later to enable secondary immune response. Serum anti-KLH IgG/IgM antibody titers were quantified by ELISA method using a validate cutoff antibody titer method. In the same animals, correlations between TDAR-KLH, pro-inflammatory serum cytokine concentrations measured using EMD Millipore MILLIPLEX MAP kits (interleukin [IL]-1β, IL-2, IL-4, IL-6, IL-10, monocyte chemoattractant protein-1, tumor necrosis factor-α, and interferon-γ), and peripheral blood IPT measured using flow cytometry (CD3+, CD4+, CD8+, CD14+, CD16+, and CD20+) were also assessed throughout the course of the toxicology study. In conclusion, our TDAR-KLH, IPT, and cytokine data provided a more accurate means to classifying changes outside of a “normal” humoral immune response in rat and cynomolgus monkey for use in preclinical immunotoxicology studies.
P121: Long-term Neurological Implications of Occupational Jet Fuel Exposure in the Air Force
T. D. Vincent1, W. J. Culpepper2, E. K. Dursa2, G. Wolff3, J. Escobar3, and A. Schneiderman2
1Department of Veterans Affairs, Environmental Health Program, Post-Deployment Health Services, WA, USA
2Department of Veterans Affairs, Epidemiology Program, Post-Deployment Health Services, WA, USA
3Epidemiology Consult Service, US Air Force School of Aerospace Medicine, Wright-Patterson Air Force Base, OH, USA
Military service introduces Servicemembers to numerous hazards associated with occupational activities. Exposure to jet fuels is one of the more commonly experienced hazards, regardless of branch. Although the acute effects of occupational jet fuel exposures have been well-documented, less is known about the potential long-term impact on health. The Department of Veterans Affairs and the US Air Force School of Aerospace Medicine conducted a pilot study to investigate the effects of jet fuel on long-term health outcomes using occupation codes as a surrogate measure of exposure. Study findings suggested that occupational exposure to jet fuels was associated with an increased risk (up to 22%) of receiving a diagnosis that reflects both acute and chronic effects on the nervous system when compared with AF personnel who were not likely exposed to jet fuels due to their normal duties. These effects appear to be a function of the intensity/frequency of exposure in an occupation, as well as the duration of occupation, but not deployment to a conflict. This relationship will be investigated further to better inform policy on the use of personal protective equipment and the need for routine monitoring of health conditions associated with occupational fuels exposure.
P122: A 6-Month Inhalation Toxicology Study in Apoe −/− Mice Demonstrates Substantially Lower Effects of E-Vapor Aerosols Compared With Cigarette Smoke in the Respiratory Tract
J. Szostak1, E. T. Wong2, B. Titz1, K. M. Lee3, A. Kumar3, Y. Xiang1, A. Kuczaj1, P. Vanscheeuwijck1, M. C. Peitsch1, and J. Hoeng1
1PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland
2Philip Morris International Research Laboratories Pte. Ltd, Singapore
3Altria Client Services LLC, Richmond, VA, USA
Cigarette smoke (CS) is the major cause of chronic obstructive pulmonary disease. Considerable attention has been given to the reduced harm potential of e-vapor products. We investigated the effects of exposure to e-vapor aerosols (nicotine and flavor) and mainstream CS on emphysematous changes, lung function, and molecular alterations on the respiratory tract of Apoe −/− mice. Mice (n = 10-12) were exposed daily (5 d/wk) for 6 months to fresh air (sham) or aerosols from 3 different e-vapor formulations—(1) carrier (propylene glycol and vegetable glycerol), (2) base (carrier and nicotine), or (3) test (base and flavors)—or to CS from 3R4F reference cigarettes. Aerosol concentrations in CS and base or test aerosols were matched at 35-µg nicotine/L. Exposure to CS impaired lung function and caused marked lung inflammation and emphysematous changes. Dysregulation of the lung transcriptome, lipidome, and proteome was also observed in response to CS exposure. These effects (functional, structural, and biochemical responses) were substantially reduced in response to all e-vapor aerosol exposures. In comparison to sham, the e-vapor aerosol exposures caused only a slight increase in lung inflammation and nasal olfactory epithelium irritation under the test conditions. In conclusion, the study demonstrated that, in comparison to CS, e-vapor aerosols demonstrated substantially lower biological responses in the respiratory tract.
P123: Critical Success Factors (CSFs) in the Safety Assessment of COVID-19 Vaccines
L. Bertrand1, P. Ancian2, J. DeLarichaudy1, P. Singh2, S. Gould1, and N. Truchot-Limonne1
1Charles River Laboratories, Saint-Germain-Nuelles, Rhone, France
2Charles River Laboratories, Evreux, Cedex, France
In 2020, the world has confronted the reality of living with the COVID-19 pandemic. Many are now asking, “What have the last few months taught us about critical success factors (CSFs) in the safety evaluation of COVID-19 vaccines?” We compiled what we have learned while conducting nonclinical safety studies on COVID-19 vaccines and describe drivers for species selection, end point evaluations, and determination of analytical methods. The CSFs we identified include experience, speed, and innovation that together are essential for translation of animal safety data into human vaccine trials. Species selection is usually based on the ability to generate an immune response and to enable the administration of the full human dose. Histopathology exams are performed on selected vital organs, injection sites, and lymphoid organs. Immune response is evaluated by an effective method to detect the antigen-specific serum IgG response. Biodistribution data may be required for viral vectored vaccines, and for mRNA vaccines, the concentration of mRNA encapsulated in lipid nanoparticle formulations can be quantified using specific analytical methods using the RiboGreen assay, which has been developed specifically for RNA vaccines. Reproductive and developmental data may also be required for license approvals. In conclusion, the safety assessment of COVID-19 vaccines is generally comparable to other prophylactic vaccines in terms of study design, end points, and reliance on analytical methods, but with access to nonclinical development experience, innovative analytical platforms and regulatory and scientific task forces to meet the critical timelines, an accelerated yet assured safety evaluation of COVID-19 vaccines can be achieved.
P124: Dietary Methyl Donor Restriction Alters Lipid Metabolism in a Colorectal Cancer Model
O. Aladelokun1, M. Hanley1, T. Moore2, J. Mu2, C. Giardina3, and D. Rosenberg4
1Center for Molecular Oncology, University of Connecticut Health Center, Farmington, CT, USA
2Statistical Consulting Services, University of Connecticut, Storrs, CT, USA
3Department of Molecular and Cellular Biology, University of Connecticut, Storrs, CT, USA
4The University of Connecticut School of Medicine, Farmington, CT, USA
We previously showed that depletion of dietary methyl donors (folate, choline, methionine, and vitamin B12) dramatically suppresses intestinal adenoma development in Apc-mutant mice, with associated changes in colonic cell turnover and inflammatory gene expression. A previous metabolomic analysis in ApcΔ14/+ mice found an unanticipated reduction in the total carnitine pool that mediates fatty acid oxidation associated with tumor reduction. In the present study, we performed a computational analysis of colonic metabolites using MetaboAnalyst 4.0 and found that the methyl donor deficient (MDD) diet caused significant alterations to long-chain fatty acids (LCFAs) β-oxidation and acetyl group transfer into the mitochondria. Changes in fatty acid metabolism were accompanied by lower expression of genes required for colonic fatty acid uptake, biosynthesis, and mitochondrial oxidation, including Cd36, Slc27A1, Acc, and Acadvl. The MDD diet also decreased the levels of LCFAs: palmitate, palmitoleate, and oleate; increased the levels of anti-inflammatory n-3 polyunsaturated fatty acids (PUFAs): docosahexaenoate and eicosapentaenoate. Similar to the colonic changes, a metabolomic analysis of the plasma revealed a systemic reduction in acylcarnitines and the LCFAs, arachidonate, and oleate, while increasing n-3 PUFAs. Linear regression analysis relating plasma metabolites and tumor number showed n-3 PUFA levels significantly correlated with fewer tumors, whereas the TCA cycle-derived “oncometabolite” 2-hydroxyglutarate correlated with a higher tumor number. Our data further highlight reductions in systemic LCFA levels, transport, and mitochondrial oxidation as metabolic pathways that are altered by MDD. These changes may impact tumor growth by limiting the proliferative capacity of colonocytes and suppressing systemic inflammatory responses.
P125: Assessment of Subcutaneous Radio-Telemetry Device Implantation in Cynomolgus Monkeys
J-C. Queudot1, J. Forget1, S. Nechev1, A. Celori1, B. Megrath1, and T. Hernandez1
1Altasciences Preclinical Seattle, LLC, Everett, WA, USA
The feasibility of 2 different methods, intra-abdominal versus subcutaneous, of telemetry device implantation in nonhuman primates (NHPs) were investigated, with the goal of potentially reducing the trauma caused by surgical implantation of telemetry devices without negatively impacting the quality of data. The standard route of telemetry device implantation in NHPs is intra-abdominal which is an invasive surgery and requires significant postoperative care, while the subcutaneous device implantation approach uses a less invasive surgery procedure with a shorter recovery. Cardiovascular and body temperature data, as well as rectal temperatures and clinical pathology (hematology, coagulation, and serum chemistry), were monitored over a 6-week period. No marked differences in cardiovascular readings were noted between both implants. Blood pressure, heart rate, and electrocardiogram were all found to be within normal ranges for both groups. There was a notable difference in body temperature between the 2 implant sites compared to rectal temperatures with 1.5 degrees lower for intra-abdominal and 3 to 4 degrees lower for subcutaneous implants. Minimal changes in clinical pathology observed during the postimplantation phase in both groups were consistent with low-grade inflammation. The feasibility of sustaining subcutaneous implant placement was limited by the animal size, due to the lack of a subcutaneous space that would allow the device to sit comfortably. Considering that the level of inflammation was comparable between the 2 methods and indicated no differences in cardiovascular readings, subcutaneous telemetry implants appear to be a suitable, and less invasive, alternative to intra-abdominal implants in NHPs of sufficient size.
P127: Determination of Normal: Flow Cytometry Analysis of Major Immune Cell Populations in Peripheral Blood of Naive Cynomolgus Monkeys
M. Patel1, A. S. Wong1, and M. Poirier1
1Altasciences Preclinical Seattle, LLC, Everett, WA, USA
Changes in immune cell populations in the peripheral blood of cynomolgus monkeys, as determined by flow cytometry, are routinely used as an immunological end point in toxicology studies. To determine what constitutes a “normal” value for immune cell populations in the peripheral blood, we analyzed large cohorts of naive adolescent (males: n = 84, females: n = 84) and infant (n = 33) cynomolgus monkeys. We determined relative percentage and absolute counts of immune cell populations, such as CD3+ total T cells, CD3+CD4+ T cells, CD3+CD8+ T cells, CD3-CD20+ B cells, CD3-CD16+ NK cells, and CD3-CD14+ monocytes. The number of total T cells was 3.06 ± 1.25, 3.24 ± 1.35, 2.88 ± 1.12, 4.97 ± 1.55 × 106 per mL, number of B cells were 1.34 ± 0.75, 1.45 ± 0.90, 1.23 ± 0.56, 1.85 ± 1.01 × 106 per mL, the number of NK cells was 7.59 ± 4.24, 8.56 ± 4.44, 6.63 ± 3.81, 4.48 ± 1.94 × 105 per mL, and the number of monocytes were 2.48 ± 1.09, 2.46 ± 1.22, 2.50 ± 0.94, 4.44 ± 2.08 × 105 per mL for total adolescent both sexes combined, adolescent male, adolescent female, and infant cynomolgus monkeys, respectively. Our results indicated significant differences based on age and sex. Infants had higher counts of total lymphocytes, monocytes, total T cells, CD4+ T cells, and B cells compared to adolescents (P < .005, Mann Whitney U test) and lower absolute counts of NK cells (P < .0001, Mann Whitney U test). We also observed sex-dependent differences where females had lower counts of CD3-CD16+ NK cells compared to males (P < .001, Mann Whitney U test). Overall, these reported values serve as a guideline for improved understanding of the various toxicology-related changes introduced by therapeutic treatments during drug development.
P128: Exploring Immunoconditioning Regimens for Cell Therapies in Murine Models for Toxicology Studies
C. Li1, K. Bujold1, C. Ballesteros1, A-M. Downey1, K. Wong1, and S. Authier1
1Charles River Laboratories Montreal ULC, Laval, Quebec, Canada
Selection of immunoconditioning regimens for cell therapy (CT) studies is a balancing act to ensure cell engraftment, minimal impact on toxicology end points, and/or clinical relevance. Immunoconditioning using radiation and/or chemotherapy can be included in toxicology studies to enable/maximize cell engraftment and to ensure clinical relevance. Fractionated radiation in combination with cyclophosphamide was evaluated for bone marrow transplant (BMT). For CT in NSG mice, single fraction radiation or chemotherapy alone was used. Bone marrow transplant recipient mice received cyclophosphamide (50-120 mg/kg, days 6 and 5) and total body irradiation (TBI; 3 daily fractions; 350-500 Gy) prior to BMT. Bone marrow cells from donor C57BL/6, C57BL/6 CD45.1, or Tg (CAG-EGFP) C57BL/6 CD45.2 mice were isolated by flushing bones (femur and tibia) with PBS 1X. Cells were filtered through a 40-µm cell strainer followed by centrifugation and cell pellet resuspension to reach 20 × 106 cells/mL. An automated viability cell count was performed on the cell formulation, prior to dosing. For BMT in immunocompetent mice, 3 daily fractions of 5 Gy combined with high-dose cyclophosphamide (120 mg/kg, twice) were associated with mortalities, while 3 daily fractions of 4 Gy with low-dose cyclophosphamide (50 mg/kg) was tolerated. The NSG mice used in CT studies tolerated a single γ radiation TBI dose of 2 Gy (1.6 Gy/min) with minimal clinical signs or effects on pathology end points. Clinical pathology parameters at 30 days after radiation were comparable to nonirradiated NSG mice. In conclusion, important differences exist for immunoconditioning/myeloablation and optimization is essential for each application.
P129: Evaluation of Evoked and Non-Evoked Pain End Points in the Complete Freund’s Adjuvant (CFA) Pain Model in Rats
H. Rashid1 and R. Samadfam1
1Chrales River Labs, Senneville, Quebec, Canada
Introduction: Persistent pain is a major hallmark of arthritis. The complete Freund’s adjuvant (CFA) model in rodents is often used to mimic human arthritic pain. In the present study, we have compared the thermal heat-evoked and the dynamic weight bearing non-evoked pain end points in the rat CFA model.
200 Series—Regulatory Toxicology
P201: North American Travel Grant Effect of PFOA on DNA Methylation and Tight Junctions in Mouse Colon Tissues
F. Rashid1 and J. Irudayaraj2
1Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA
2Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA
Perfluorooctanoic acid (PFOA) is a widespread contaminant in the environment, that is, implicated in several toxicities. The intestinal wall is first surface of interaction with any toxicant after exposure through the oral route. Previous studies have demonstrated: increased intestinal bioaccumulation of PFOA in different experimental animals, PFOA-induced changes in DNA methylation of several tissues, and alterations in expression of tight junction (TJ) genes due to epigenetics changes. Nevertheless, evidence on the toxicity of PFOA on intestinal epigenetics and TJ genes is limited. Thus, the present work investigates PFOA-induced changes in expression of epigenetic genes of the colon in relation to TJ genes alterations. CD-1 mice were orally dosed with 4 different concentrations of PFOA (1, 5, 10, or 20 mg/kg/d) for 10 consecutive days, and its bioaccumulation and induced changes in the expression of TJ and epigenetic genes in the colon were investigated. The PFOA was accumulated in very high concentrations in the colon tissue and decreased the expression levels of DNA methyltransferase genes (Dnmt3a and DNMT3b). However, no significant alterations were observed in expression levels of 10-11 translocation (TET1 and TET2) genes. We also found a significant reduction in the mRNA expression of TJ protein-1 (TJP1) at the highest concentration of PFOA. Furthermore, the mRNA expression levels were significantly increased for occludin (OCLN) and Claudins (CLDN2, CLDN3, CLDN8) genes at higher concentrations. Therefore, our study demonstrates that PFOA initiates DNA methylation alterations and changes the expression of genes necessary for preserving the integrity of the colon barrier.
P202: Permitted Daily Exposure Values—Application Considerations in Toxicological Risk Assessments
D. J. Ball1 and W. P. Beierschmitt2
1D&B ChemTox, LLC, Southbury, CT, USA
2D&B ChemTox, LLC, Waterford, CT, USA
Permitted daily exposure (PDE) values are used by some toxicologists to support the safety qualification of various types of impurities found in drug substances and drug products (DP). Permitted daily exposure values are important tools for the toxicologist, but one must be aware of their limitations to ensure they are used appropriately and effectively in the risk assessment process. Two impurity examples from DP clinical development programs, benzyl alcohol (BA) for neonatal exposure and maleic anhydride (MA) for inhalation delivery, are provided to highlight the importance of performing a comprehensive analysis of all available animal and human safety data for impurity qualification. For BA, a PDE for a parenteral DP for neonates must take into consideration an appropriate body weight adjustment (0.5 kg) to support safety at a dose below that known to elicit gasping baby syndrome (16 mg/d). For MA, a PDE calculated from a 2-year oral rat study (10 mg/d) does not provide an acceptable safety margin for respiratory tract effects, whereas one calculated from a 1-month inhalation rat study (0.0017 mg/d) provides a significantly lower dose to mitigate risk for an inhaled DP. These case studies highlight that in addition to repeat-dose animal toxicology data, the toxicologist must take into account specific risks for special populations (BA) and use of the appropriate animal study to assess route/target organ considerations (MA) to calculate PDE values to support human safety.
P203: Reference Values of Clinical Pathology Parameters in Juvenile Cynomolgus Monkey (Macaca Fascicularis) Aged Up to 6 Months
C. M. Luetjens1, A. Fuchs1, A. Baker2, P. Bushdid3, H. Flaherty3, and G. F. Weinbauer1
1Covance Preclinical Services GmbH, Münster, North Rhine–Westphalia, Germany
2Covance Laboratories Inc, Madison, WI, USA
3Covance Laboratories Inc, Greenfield, IN, USA
Clinical pathology parameters of nonhuman primates are important markers for physiology, pathology, and toxicology in preclinical research. However, reference data of infant cynomolgus monkeys are limited. In the present retrospective study, we established hematological and biochemical reference levels for infant animals between 1 and 6 months of age. We summarize data obtained from enhanced pre- and postnatal developmental studies at Covance sites in Madison and Münster. A total of 16 enhanced pre- and postnatal developmental studies were selected and analyzed, with data from up to 11 female and 24 male infants from Madison and from up to 64 females and 56 males from Münster, in order to examine reference levels of 18 hematological and 37 biochemical parameters. Although the infants were kept in very similar social housing conditions, the parameters evaluated at the 2 different sites have been analyzed separately. Reference intervals were separately established by age (weeks 4 to 26) and sex and analyzed by 2-way analysis of variance. Independently of the origin of the data and the sex of the infants, in some organ systems, infant developmental–related clinical pathology changes were found. The hematological system showed age-related adaptions for several parameters (eg, erythrocytes and lymphocytes) as well as for parameters of the humoral immune response system (immunoglobulin). Additionally, some parameters of the hepatic (eg, glutamate dehydrogenase, aspartate, and alanine aminotransferase) and nephritic system (urea) showed also age-related adaptions. In conclusion, a comprehensive and systematic reference of clinical pathology parameters for cynomolgus infants was established.
P204: BioCelerate Nonclinical Protocol and Report Harmonization Initiatives
S. Lopes1, S. Moesgaard2, G. Schulze3, P. Brinck2, M. Carfagna4, T. Fukushima5, J. Horvath3, and N. Vansell1
1Pfizer, Groton, CT, USA
2Novo Nordisk, Copenhagen, Copenhagen, Denmark
3Bristol Myers Squibb, New Brunswick, NJ, USA
4Eli Lilly, Indianapolis, IN, USA
5Shionogi & Co., Osaka, Osaka, Japan
BioCelerate is an industry consortium driving initiatives to increase efficiency in early-stage R&D. Our ongoing projects resulted in the release of a Common Protocol Template for repeat-dose toxicology studies in November 2019 and a targeted release of an associated study report template in December 2020. Advantages to using common templates for sponsors, CROs, and sites include (1) less time and effort to develop a protocol and the associated report for a study, (2) improved overall study quality by decreasing errors due to unfamiliarity with protocol/report layout, (3) optimization of time in managing multiple studies, and (4) optimization of time spent on critical data analysis and review. For both common templates, streamlined process instructions were defined where large variations in preferences existed. For the protocol template development, public feedback periods and webinars were held to collect stakeholder feedback. Poll questions indicated most stakeholders used multiple protocol templates and experienced problems associated with inconsistency, inefficiencies, and reduced study execution quality. To begin development of an associated common report template, the BioCelerate team collaborated with pharm-tox reviewers from the US Food and Drug Administration in January 2020 and subsequently hosted an open webinar to gather input from stakeholders. This template contains: (1) standard sections with common text that may be used with little to no editing; (2) major headings that are recommended to not be deleted; however, additional subheadings can be added as needed; and (3) a layout commonly used by biopharma/CROs. The common templates and supporting implementation materials can be downloaded from the BioCelerate website.
P205: Derivation of a Health-Based Exposure Limit for Cesium Using Nonclinical and Clinical Data
A. L. Kimzey1, B. T. Welsh1, A. R. Chesney1, C. Fonck2, C. O’Neill2, and M. L. Wood1
1ToxStrategies, Inc., Katy, TX, USA
2BioMarin Pharmaceutical Inc, Novato, CA, USA
Cesium chloride density gradients have been used for decades to purify DNA-containing and gene therapy products; however, residual cesium removal is critical. Cesium can mimic and compete with potassium in vivo, leading to adverse effects involving the cardiac ion channel, historic Ether-à-go-go-Related Gene. Cesium can induce QT prolongation, and because of this, it was used as a positive control in early nonclinical QT pharmacology models. Additionally, cesium was banned as an alternative medicine by the Food and Drug Administration in 2018 due to serious adverse events, including QT prolongation and death. A safe dose or permissible daily exposure (PDE) for cesium has not been identified in the ICH Q3D(R1) guideline for Elemental Impurities; therefore, a literature review of pharmacokinetics, nonclinical toxicology, and clinical safety data was performed. Data quality were evaluated and a dog safety pharmacology model that reported cardiac effects resulting from escalating intravenous doses of cesium (Hanich et al, 1988) was selected to derive a health-based exposure limit, preferable over human data obtained from overdose patients who had administered toxic or unknown cesium doses, or to the majority of other nonclinical studies performed at concentrations well above the no-observed-adverse-effect level (NOAEL). Based on Hanich (1998), a health-based exposure limit of 170 mg/kg for a single dose of residual cesium was derived per ICH Q3D(R1) from the dog cardiac study NOAEL. This PDE can be used to set release specifications for residual cesium as an impurity in a drug product. If necessary, this value could be validated using a modern good laboratory practice cardiac telemetry study in dogs.
P206: A Training and Validation Plan for Extended One Generation Reproductive Toxicology Studies
M. L. Foster1, M. Creamer-Hente1, D. A. Donahue1, D. Mahapatra1, P. Ovwigho1, and J. L. Henry1
1Integrated Laboratory Systems, Inc, Research Triangle Park, NC, USA
The special training and validation needed for conduct of developmental/reproductive toxicology studies is beyond the scope of normal toxicology studies. We present results of our training and validation plan used to meet current Organization for Economic Cooperation and Development (OECD) 443 Extended One Generation Reproductive Toxicology guidelines, including specific tests for reproductive toxicity, developmental neurotoxicity, and developmental immunotoxicity. Automated motor activity, acoustic startle, and sperm analysis systems were validated through conduct and documentation of performance scripts. Method development trials were performed for potassium ferricyanide staining of uterine implantation sites, in situ perfusion of juvenile rats for neuropathology, and preparation of sheep red blood cells for T-cell dependent antibody responses. Extensive staff training for performing functional observational battery (FOB) was initially conducted using formal classroom training followed by assessment of naive rats and rats treated with model neurotoxicants. Motor activity and FOB-positive control studies used Sprague Dawley rats (n = 15 rats/sex/treatment group) with saline, chlorpromazine (6 mg/kg), or d-amphetamine (4 mg/kg) for motor activity; corn oil, 4,4’-DDT (75 mg/kg) and carbaryl (100 mg/kg) for FOB. Expected motor activity and FOB effects were observed following treatment. Functional observational battery training resulted in high inter-rater reliability among the first 3 technicians trained. A subset of animals (n = 3 rats/sex/treatment group) were perfused following the FOB. Brain and cervical spinal cord histopathology and brain morphometry were performed. Perfusion produced high-quality specimens for evaluation although treatment-related neuropathology was not observed in any animals. Validation studies, although resource and time demanding, prepare laboratories for the conduct of these OECD studies.
P207: Implementation and Validation of a Skin Sensitization Protocol for In Silico Hazard Assessments
C. Johnson1, D. Bower1, K. P. Cross1, S. Miller1, and G. Myatt1
1Leadscope, Inc, Columbus, OH, USA
In silico assessments support hazard identification in cases where the synthesis and purification of impurities present challenges, such as with extractables and leachables. Recently published in silico toxicology protocols, that provide the user with database references, criteria for in silico tool use, and rules and principles that govern the weight-of-evidence assessment, were implemented into functional software. The implemented protocols provide an overall assessment, in addition to the reliability and/or confidence of the prediction. The user is also guided through an expert-review process according to the principles that are outlined in the published protocols. The skin sensitization protocol includes an assessment of the “skin sensitization in vitro” and “skin sensitization in rodents” sub-end points. An external validation using approximately 3000 structures was conducted. In cases where there was agreement between the “skin sensitization in vitro” and “skin sensitization in rodents” assessments, a sensitivity and specificity of 85% and 75% was obtained. A decrease in sensitivity (75%) and specificity (62%) was observed when only 1 sub-end point was responsible for the overall prediction. These results do not consider an expert review, which increases the accuracy and reliability of in silico predictions. In cases where the overall end point remains unassigned, the protocol provides information on the reaction domain or individual model predictions that could facilitate an expert-review and/or help identify the best approach to testing. The results obtained imply that with continued growth of the databases and models, the in silico approach used is a viable tool for the assessment of skin sensitization hazard.
P208: Optimization of an Indirect Ventricular Contractility Assessments: Heart Rate Correction of the QA Interval
R. Tavcar1, M. Accardi1, Y. Zhong1, E. Boulay1, and S. Authier1
1Charles River Laboratories Montreal ULC, Laval, Quebec, Canada
Cardiovascular effects, and especially heart rate (HR) changes, are among the most frequent unintended drug effect in drug development. Most cardiac parameters including the maximal rate of rise of left ventricular pressure (dP/dtmax) are correlated to HR, and correction methods can be used to enhance. dP/dtmax requires a transmural pressure catheter which is not compatible with telemetry added to toxicology studies. The time interval from the Q wave on the electrocardiogram to point A on the aortic pressure wave (QAI) may serve as an indirect index of cardiac contractility We assessed the HR to QAI correlation and HR to dP/dtmax correlation in rats; dogs, nonhuman primates. Higher HR was associated with shorter QAI. The dP/dtmax values were positively correlated to HR. The correlation between QAI and dP/dtmax suggests that in nonhuman primates and dogs, HR-related changes in QAI are nearly independent of dP/dtmax, whereas in rats a strong negative correlation exists between the 2 parameters (y = −704.1*× + 5.113). A HR correction applied to QAI reduced the slope of each respective trend line which enabled comparison of QAI changes in the presence of a drug effect on HR. Corrected QAI values were noted to be more consistently corrected to values closer to 0 than dP/dtmax suggesting that heart rate–corrected QAI values may present a more consistent heart rate dependence. Heart rate–corrected dP/dtmax and QAI can improve assay sensitivity to detect drug-induced inotropic effects. This observation maybe applicable in the context of the high prevalence of chronotropic effects in toxicology.
300 Series—Safety Evaluation Nonpharmaceuticals
P301: Dose-Response Analysis in GARD for Assessment of Skin Sensitizer Potency
J. Schmidt1, A. Forreryd2, R. Gradin2, and H. Johansson2
1SenzaGen, Inc, Raleigh, NC, USA
2SenzaGen AB, Lund, Sweden
The development of nonanimal methods for hazard identification of skin sensitizers has progressed actively, and several assays capable of attaining acceptable prediction performances are available. In contrast, the advancement of alternative methods for hazard characterization, that is, potency classification of skin sensitizers, is, though a coveted end point, still lacking. To this end, the application of the GARDskin assay for potency assessment by extension of the assay’s original protocols was evaluated. Whereas the default assay analyzes the induced gene expression levels in SenzaCells following exposure to a test substance at a single concentration, the gene-level effects were here evaluated in a dose-response manner. Specifically, a set of 31 chemicals of varying sensitizing potencies was used to assess the feasibility of such an approach. For each chemical and concentration, a decision value was generated by classification with the GARDskin support vector machine. Then, the lowest concentration where a chemical could establish a positive GARDskin prediction was estimated using linear interpolation. Thus, the chemicals’ end point results consisted of concentration estimates which were hypothesized to be reflective of their skin sensitizing potency. Indeed, the predicted GARDskin dose-response concentrations were compared to LLNA’s EC3 values and a statistically significant linear correlation of 0.74 (P = 4.1 × 10−4) was observed between the 2 measures. This suggest that a modified GARDskin protocol based on dose-response measurements facilitates assessment of skin sensitizing potency analogous to in vivo gold standards, enabling both improved prioritization and decision-making in stages of product development without resorting to animal experimentation.
P302: 3T3 Neutral Red Uptake Phototoxicity Test (3T3NRUPT) Vehicles: A Historical Comparison
L. F. Pratt1 and G. L. DeGeorge1
1MB Research Labs, Spinnerstown, PA, USA
The in vitro 3T3 NRU PT identifies potentially phototoxic test substances that are induced after exposure to light. Substances identified by this test are likely to be phototoxic in vivo. Depending on solubility, different solvents are used as vehicles to dilute the substance for the test. Test substances are diluted to 8 concentrations and applied to a 3T3 cell monolayer plated in two 96-well tissue culture plates. One plate is exposed to 5J/cm2 of solar simulated light (+SSL), while the other plate is placed in the dark (No SSL). The concentration at which the viability of the cells is 50% of untreated cells (IC50) is calculated for each condition; the photo irritation factor (PIF) is a ratio of the IC50 +SSL cells to the IC50 No SSL cells. The positive control chlorpromazine (CPZ) must have a PIF value greater than 5. We present 14 years of historical data +SSL, No SSL, and PIF values of CPZ in the vehicles of Hanks’ Balanced Salt Solution (HBSS), 1% dimethyl sulfoxide (DMSO) in HBSS, and 1% ethanol (EtOH) in HBSS comparing the effects of vehicle choice on CPZ. The average PIF values of chlorpromazine in the 3 vehicles are 38.2 (HBSS), 30.9 (1% DMSO), and 33.1 (1% EtOH); the average IC50 No SSL are 26.2, 26.2, and 25.3, and the average IC50 + SSL are 0.685, 0.847, and 0.764, respectively. The dilution vehicle used has potential to affect these values, but it is concluded that these vehicles are acceptable without compromising study results.
P303: Electronic Cigarettes and Vaping: The Impact of Sweet Flavorings in the PMTA Submission
E. Neyens1
1Elizabethtoxpath Consulting Inc, Vancouver, British Columbia, Canada
Electronic cigarettes contain e-liquids that are aerosolized with an electronic nicotine delivery system (ENDS) device upon consumption. The ENDS devices differ from combustible cigarettes in that they produce an aerosol by drawing and heating an e-liquid over a battery-powered coil. The e-liquid is typically composed of 1 single or various flavors, with or without nicotine, which are often diluted in a mixture of propylene glycol and/or vegetable glycerol. One of the major concerns that should be addressed in the Pre-Market Tobacco Application (PMTA) is the presence of newly aerosol-formed chemical constituents such as diacetyl (2,3-butanedione), and/or acetyl propionyl (2,3-pentanedione) which could be associated with the toxicity of fruit and sweet flavors. During an ENDS risk assessment, various e-liquids with and without flavors were analyzed for aerosol harmful and potentially harmful constituents. It became evident that the presence of appealing sweet, even GRAS-approved flavorings, was associated with extreme high concentrations of 2,3-butanedione and/or 2,3-pentanedione. Based on NIOSH proposed limits, it was decided to withdraw various sweet flavorings from the PMTA submission, as their aerosol concentrations would have certainly induced respiratory toxicity during the vaping experience. Therefore, we can highlight the importance of a well conducted aerosol risk assessment in addition to the ingredient risk assessment. Both investigations should demonstrate either a lack of toxicity or a reduced toxicity of the ENDS products compared with the classical combustible cigarette smoke for a determination of Appropriate for the Protection of Public Health.
P304: A Novel Secure Software Platform Developed to Conduct Risk Assessments of ENDS
L. Neilson1, S. Blackburn2, E. Minet1, Y. Wilding1, and A. J. Bernal3
1Broughton Nicotine Services, Earby, Lancashire, UK
2Broughton Software, Skipton, North Yorkshire, UK
3ToxCreative LLC, Laguna beach, CA, USA
E-liquids for electronic nicotine delivery systems (ENDS) are complex mixtures, which makes determining toxicological hazards and quantifying health risks an involved process, for product development and regulatory approval. Ingredients must be screened for toxicological hazards during ingredient selection, and formulations are often refined during product development to ensure exposures are maintained under toxicological thresholds. Software platforms with the flexibility to input product and consumer-specific variables are limited for ENDS and often not designed for efficient, documented, systematic assessment of chemical information, including toxicological reference values, in silico predictions, and literature. Here, a secure platform based on a robust decision-making algorithm firstly assigns hazard outcomes for ingoing ingredient screening or emissions output data to aid product development and then integrates product-specific consumer topography with a tier-based selection of toxicological reference values to determine maximum acceptable exposure across variable consumption scenarios. Practical examples will be presented, including the hazard screening determinations for carcinogenic, mutagenic, and reprotoxic (CMR) end points based on a range of authoritative sources and a hierarchical approach for an example e-liquid. In term of the base e-liquid ingredients, the results display a negative CMR outcome for propylene glycol (PG) and glycerol and identified the reprotoxic alert for nicotine. Furthermore, an aerosol emission data set will display risk estimates based on the selected reference value and consumption scenarios. For PG and glycerol, the rationale to use reference values from lower tiers is explained to provide insight to the approaches used to build the algorithm of the platform to support regulatory submissions.
400 Series—Toxicology Methods
P401: Determination of the Protective Effect of D-Methionine and N-Acetyl Cysteine in Ototoxicity Induced by Cisplatin in Zebrafish Embryos
A. Alday1, A. Muriana1, and C. Quevedo1
1Biobide, San Sebastian, Gipuzkoa, Spain
Death of mechanosensory hair cells is a common denominator in many forms of hearing impairment. Hair cell loss is most commonly due to environmental insults, including exposure to excessive noise or ototoxic drugs, or progressive loss due to aging. D-Methionine and N-Acetyl-Cysteine are known to be otoprotective agents against the ototoxicity effect produced by drugs like cisplatin, neomycin, or gentamicin. Screening of new compounds is essential for preventing this disorder. Zebrafish is a valuable model for studying hair cell development. Zebrafish have hair cells on their body surface organized into a sensory system called the lateral line. The hair cells are organized into small groups called neuromasts, making this system particularly convenient for hair cell death and protection screens. With the purpose to validate the use of zebrafish lateral line as a screening tool for the identification of potentially otoprotective drugs, we have evaluated the otoprotective effect of the D-Methionine and N-Acetyl-Cysteine in ototoxicity induced by 20-µM Cisplatin. First, larvae were treated with 8 concentrations of each of the agents to explore the maximum concentration at which lethality and malformations were not induced. Afterward, larvae were treated at 2 concentrations selected based on the previous phase and the number of neuromast were quantified as end point after proper staining with DASPEI. Results obtained demonstrate the otoprotective effect of the agents, showing the zebrafish to be a sensitive and cost-effective assay to screen potential otoprotective chemicals. Validation study with a larger reference compounds should be carried out to validate the assay.
P402: Next Generation Toxicology—Predicting Drug-Induced Liver Injury by Computable Biological Network Models
K. Renggli1, H. Yepiskoposyan1, U. Kogel1, V. Belcastro1, M. Talikka1, M. C. Peitsch1, and J. Hoeng1
1PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland
A fundamental understanding of the signaling cross talk is critical not only to the prediction of drug toxicity but also for the elucidation of pathophysiological mechanisms. Systems toxicology approaches with extensive molecular measurements provide a deeper view into the downstream signaling of drugs. In order to interpret these rich data sets, new methods have to be developed that allow for mechanistic understanding. We built a causal biological network model of drug-induced liver injury (DILI) to gather available liver-signaling specific knowledge into a structured graphical presentation and to facilitate high throughput data interpretation. All model relationships (network edge) were extracted from original literature, annotated with originating source (PMID) and biological context (ie, species, tissue/cell type, and disease state). The statements were scripted in Biological Expression Language, which facilitates computation, and the final model covers all the important signaling pathways involved in hepatic cell death, including the combination of MEK/ERK, mTOR, AKT, and p38 pathways. To interpret transcriptomic data, a second layer is used to infer the activity of the backbone nodes (inferable nodes, iNodes) from transcriptomic data, instead of overlaying gene expression changes onto the model. We have scored the DILI model with transcriptomic data from phenobarbital and phenylhydrazone-treated mouse liver. The model correctly classified phenobarbital as PXR activator and phenylhydrazone as hypoxia-inducing compound. This demonstrates that the DILI model can be used to investigate the impact of xenobiotics on biological systems of interest; it provides means for both quantitative impact assessment and mechanistic understanding of toxicant effects on the liver.
P403: Intratracheal (IT) Atomization of IT Liquid Delivery of a Test Article Results in Similar Pulmonary Distribution in a Preclinical Ferret Model
T. R. Yellowhair1, P. Kuehl1, and A. Werts1
1Lovelace Biomedical Research Institute, Albuquerque, NM, USA
Determining the most advantageous method to deliver inhaled therapeutics (AAVs, antibiotics, small molecules, small interfering RNAs, etc) in preclinical models and how this translates clinically presents many challenges. Preclinical ferret models are essential as they share comparable pulmonary physiology and clinical signs to humans when infected with many respiratory viruses. We asked whether intratracheal (IT) atomization has advantages in pulmonary test article (TA) delivery over liquid IT delivery. To test this, we used a clinical IT atomizer (MADgic Laryngo-Tracheal Mucosal Atomization Device; Teleflex) in vitro and in vivo, with or without the atomization tip intact. Both the intact and altered device were tested using a reporter TA (2% Evans blue dye in saline, commonly used to assess gross distribution and permeability) in vitro (open air or confined in artificial tracheas, 5-8 mm diameter), with dye distribution recorded. In vivo lung distribution was tested in ferret tracheas (n = 10) ranging from 3.5 to 5 mm. In vitro tests demonstrated that constricting the intact device in artificial tracheas reduced aerosol generation and increased liquid delivery by the device. In vivo TA delivery resulted in inconsistent and patchy delivery to the pulmonary parenchyma, independent of device modification status, and position of the animal at dosing. In summary, atomization in a confined space resulted in aerosol-airway wall interactions that precipitated the nebulized product into a liquid. Furthermore, IT delivery of dye by atomization or a liquid resulted in similar pulmonary distribution in ferrets, suggesting that IT atomization in small animals may not be advantageous in TA delivery over IT liquid administration.
P404: Drug Administration in Neonatal and Juvenile Rats: Challenges and Opportunities
B. Attalla1 and J. Younan1
1ITR Laboratories Canada Inc, Baie D’Urfe, Quebec, Canada
As regulatory agencies now require that the safety of drugs aimed at treating medical conditions affecting pediatric patients be assessed in juvenile animals, there is an increasing need for neonatal and juvenile animal models of relevant age to be used as test systems in nonclinical studies. The administration of test materials to neonatal and juvenile rats presents several challenges including, the size of the animal, its fragility, and the lack of acclimation to the procedures, such as dosing and handling. Our laboratory developed techniques to administer dose formulations intravenously, intramuscularly, or subcutaneously to rats as of 2 days of age, and as often as twice daily, and by oral gavage to rats as young as 4 days of age. Given the small size and the fragility of young rats, animals are identified using a unique tattoo system that employs indelible digit ink. For dosing, animal movement is usually restrained using wet ice, which also serves as anesthetic. Another major challenge is data interpretation since the biological systems of neonatal, juvenile, and adult populations are different. We compiled a database for clinical pathology parameters from naive rat pups at 4, 7, 14, and 21 days of age and concluded that red blood cell counts increase, while mean corpuscular volume decreases with age. Hemoglobin, hematocrit, platelet, and reticulocyte counts tend to remain stable over time, whereas increases in liver enzymes and decreases in urea levels were noted as animals age.
P405: How to Test Cynomolgus Monkeys (Macaca fascicularis) for Learning Ability in Their Home Cage? First Experience With a Touch Screen–Based Setup
D. Smieja1, A. Dütting1, C. M. Luetjens1, and S. Korte1
1Covance Preclinical Services GmbH, Muenster, North Rhine-Westphalia, Germany
Assessment of learning and memory is often performed for regulatory juvenile toxicity or enhanced pre- and postnatal development studies, for drugs targeting the central nervous system. There is currently no method in place for animals above 2 years of age. Basic visual discrimination tasks were offered to cynomolgus monkeys (Macaca fascicularis) using an automated software-controlled touch screen device, the Monkey-CANTAB Intellistation. It was placed in front of the home cage for time limited sessions daily for up to 15 sessions. Ten cynomolgus monkeys (2-9 years) housed singly or in groups were trained. During individual sessions, animals were freely moving inside the home cage compartment. Initial task was a touch training paradigm. Touches to colored squares on the screen were automatically food rewarded. After successful training, 2-alternative forced choice discrimination tasks (2 symbols differing in shape) were introduced. All monkeys responded positively to the offered behavioral tasks. All animals passed the touch training, that is, 40 correct touches, on average within 2 sessions. Acquisition of a first basic discrimination task (≥70% correct choices over ≥40 trials) took on average 4 sessions, and acquisition time for further tasks declined to on average 2 sessions. Touch performance was high, with up to about 160 trials per 15-minute session. The results confirm feasibility of home cage–based automated assessment of learning ability under study housing conditions, and this setup and paradigm is also applied to regulatory toxicity studies. Feasibility for juvenile animals remains to be assessed.
P406: Applicability of GARDskin for Accurate Assessment of Challenging Substances in the Context of Skin Sensitization Testing
J. Schmidt1, A. Forreryd2, H. Johansson2, J. Li2, and A. Johansson2
1SenzaGen, Inc., Raleigh, NC, USA
2SenzaGen AB, Lund, Sweden
Identifying chemicals with skin sensitizing properties is a key end point within hazard testing with several in vitro assays having been proposed. Understanding applicability domains and limitations of such assays is important to ensure reliable classifications and to highlight the chemical space where novel methods can contribute to fill important data gaps. A subset of the chemical space that remains challenging to accurately assess includes pre-/pro-haptens, surfactants, and hydrophobic substances. The GARDskin assay is a state-of-the-art testing strategy evaluating transcriptional patterns of a genomic biomarker signature in a human dendritic-like cell line following exposure, in order to provide machine learning–assisted classifications. The assay was recently subjected to a formal validation procedure (Organization for Economic Cooperation and Development [OECD] TGP 4.106) and reported predictive accuracy of 94%. In the current study, the potential of GARDskin to overcome issues facing currently validated assays was evaluated by testing challenging substances (n = 12) from the abovementioned chemical spaces. These were selected from a publication by Mehling et al (2019) and generated overlapping data to chemicals previously tested by OECD validated in vitro assays and a series of RHE-based assays. Novel GARDskin data were generated for this study (n = 3) or obtained from recent publications (n = 9), and results evaluated against available human data (n = 10). GARDskin exhibited predictive accuracy of 80%. Corresponding performances of RHE-based models ranged between 30% to 70%, while the ITS reported an accuracy of 40%. In conclusion, GARDskin is capable of filling data gaps where validated assays have shown technical limitations, thus contributing to an expanded applicability domain of nonanimal-based skin sensitization assays.
P407: Combining Liver Micronucleus Assay to Existing Multi-End Point Studies to Reduce Animal Usage
S. W. Bruce1, M. Young1, and D. Bruning1
1MilliporeSigma, Rockville, MD, USA
MilliporeSigma has been investigating the ability to combine the liver micronucleus assay with the existing peripheral blood micronucleus and liver comet assays as an ongoing effort to reduce animal usage in line with 3Rs. In this effort to reduce animal usage, we have thus far-tested 4 compounds combining peripheral blood micronucleus, liver micronucleus, and liver comet assays in a modified 7-day exposure regimen using a single set of animals. In these studies, 4 to 5 weeks old male and female rats were administered diethylnitrosamine (DEN), dimethylnitrosamine (DMN), cyclophosphamide monohydrate, or 1,2-Dimethylhydrazine-2HCl (1,2-DMH) for 3 consecutive days. On day 4, peripheral blood was collected to measure micronuclei. On day 7, animals were administered test article again just prior to euthanasia for the collection of liver to prepare agarose slides for alkaline comet and measurement of micronuclei by flow cytometry. We observed a statistically significant increase in the liver micronuclei frequency after 3 days of administration DEN, DMN, and 1,2-DMH. We also observed that only the frequency of liver micronuclei was increased but the blood micronuclei with DEN, DMN, and 1,2-DMH. We observed a statistically significant increase in % tail DNA in the liver after 4 administrations of DEN and DMN, and an equivocal response with 1,2-DMH. The simultaneous analysis of liver micronucleus, blood micronucleus, and liver comet demonstrates that the 3 assays can be combined into a multi-end point assay to study liver-specific genotoxicants.
P408: Qualification and Technology Transfer of Duplex Sequencing for In Vivo Mutant Analysis in Big Blue Fisher 344 Rats
R. R. Young1, C. Valentine2, F. Y. Lo2, T. LI2, L. Williams2, and J. Salk2
1MilliporeSigma, BioReliance Testing Services, Rockville, MD, USA
2TwinStrand Biosciences, Seattle, WA, USA
Duplex sequencing (DS) was used to directly measure background and chemically induced mutant frequency (MF) and mutational spectra in endogenous genomic regions in Fisher 344 Big Blue (BB) transgenic rats. Duplex sequencing permits measurement of MF and mutant spectra in any species, tissue, or DNA segment. The error rate is less than 1 × 10−7, below the background MF of mammalian genomic DNA. Here, we compared the MF and spectra in BB rats exposed to vehicle, ethylnitrosourea, and benzo(a)pyrene (BaP) for up to 28 days with tissues collected on day 31. We showed that DS produced low, reproducible background mutant frequencies and similar mutational spectra in liver, bone marrow, and germ cells from testis. Significant increase in MF and treatment-specific shifts in mutant spectra were seen with both mutagens in all tissues except for BaP in testis. Unsupervised clustering analysis of simple mutant spectra and trinucleotide spectra showed patterns unique to each treatment. Results of the 3 treatment conditions were similar to results separately reported for BB mice. Technology transfer of the DS kit and data analysis is underway. Preliminary results showed similar quality metrics in isolation, hybridization, and library preparation suggesting the technology is transferrable. Duplex sequencing is robust in detecting changes in MF and mutant spectra in endogenous DNA in rats. In summary, our results show that DS is an acceptable method for measurement of in vivo mutagenesis and is promising as a new nonclinical biomarker of human cancer risk.
P409: In Silico Strategy for Predicting Globally Harmonized System (GHS) Categories
G. Myatt1, D. Bower1, K. P. Cross1, C. Johnson1, and S. Miller1
1Leadscope Inc (Now part of Instem), Columbus, OH, USA
The Globally Harmonized System (GHS) classification for acute oral toxicity is extensively used in manufacturing and transportation. An alternative method to calculate a GHS category would support the 3Rs (reduction, refinement, and replacement of animal studies). This poster describes an in silico strategy employing multiple computational methodologies coupled with an expert review. The technology to perform such an assessment is presented including details on how a battery of computational models (both expert rule-based and statistical-based) are used within a decision tree to predict a GHS category. In addition, elements to consider as part of any expert review are presented. The analysis shows that multiple methodologies coupled with an expert review minimizes the number of misclassifications in which a chemical is predicted to be in a more toxic GHS category, such as predicted to be category 3 but experimentally determined to be category 2. Such an approach is fit-for-purpose since it avoids misclassification of highly potent chemicals that, if tested, would have a low LD50 value. This poster illustrates that based on this strategy, around 95% of chemicals (from a blind study of over 2000 chemicals) were either correctly predicted or were predicted with a more conservative category with the majority of these predictions being in the correct category or one category more toxic.
P410: Evaluation of the Liver-Chip to Recapitulate Signals of Clinical Hepatotoxicity Observed With a Novel Therapeutic
J. Vogt1, D. de Almeida Nagata1, L. M. Norona1, A. Fullerton1, and W. R. Proctor1
1Genentech Inc, South San Francisco, CA, USA
Drug-induced liver injury (DILI) remains one of the leading causes of drug attrition and post-market withdrawal. Assessment of hepatotoxicity risk in drug discovery is difficult given limitations related to in vitro translatability; complex interplays between metabolic, autocrine, and paracrine signaling pathways among different cell types leads to poor concordance between preclinical and clinical results. Microphysiological systems (MPS) are emerging technologies with the potential to recreate a liver microenvironment and recapitulate organ physiology and associated drug toxicities. While we believe this technology holds promise, implementation of these systems is challenging, due to limitations on biological thresholds and interpretation of results compared to other systems. We performed analogous experiments using donor-matched hepatocytes cultured in 2-dimensional static monolayer and an MPS Liver-Chip platform. Using confocal microscopy, enzyme-linked immunosorbent assay and real-time quantitative PCR, we identified that MPS improved cell morphology and viability, increased basal levels of albumin and urea, and induced higher expression of metabolic enzymes (CYP1A, CYP1A2, CYP2C8, and CYP3A4). An internal small molecule inhibitor (SMI) known to increase liver transaminases in clinical trials without inducing in vitro toxicity was used to evaluated sensitivity of this MPS model. Our SMI decreased levels of albumin, increased LDH secretion and downregulated transcription of transporters (BSEP, MDR3, and OST) when compared to vehicle, suggesting that MPS has increased sensitivity to identify DILI compared to simpler assays. Despite our favorable results, determination of appropriate end points, dosing strategies, and biological thresholds is still necessary to understand clinical translatability and integration of MPS in the prospective screening of novel molecules.
P411: Tolerability of Corn Oil as Vehicle in Prenatal Developmental Toxicity Studies in Rabbits After Optimization of Food Intake
M. Doomen-van den Hoven1, D. van den Oetelaar1, K. van Dycke-Hartman1, and M. Beekhuijzen1
1Charles River Laboratories, ‘s-Hertogenbosch, North Brabant, the Netherlands
Using corn oil as a vehicle in rabbit prenatal developmental toxicity studies via oral gavage is challenging since corn oil can cause disturbances itself (ie, stress, diarrhea, reduced, and fluctuating food consumption), which may result in preterm sacrifices. We evaluated whether optimization of food intake (restricted amount of pellets and fresh fruit/vegetables) during gestation resulted in a more consistent food intake after administration of corn oil via oral gavage in rabbits. Two groups of 6 time-mated New Zealand White rabbits received either corn oil or Elix water (2 mL/kg/d) via oral gavage from gestation day (GD) 7 to 28. Rabbits received a restricted amount of pellets (125 gr/d) and hay (daily) and fresh fruit/vegetables (twice weekly). Clinical signs, body weights, and food intake were collected. At C-section on GD 29, data were collected from females, litters, and fetuses. Rabbits receiving water showed a very consistent daily food intake during gestation, whereas rabbits receiving corn oil (despite improvement compared to historical control data) still showed a reduced, fluctuating food intake at several daily intervals. Additionally, developmental effects (ie, increased number of late resorptions and postimplantation loss and decreased fetal weight) were observed in corn oil–treated rabbits when compared to rabbits treated with water. Given that administration of corn oil (2 mL/kg/d) by oral gavage in pregnant rabbits may cause effects on food intake and developmental parameters, we do not recommend to use corn oil as a vehicle in rabbit prenatal developmental toxicity studies.
P412: Case Study With Chloroquine and Hydroxychloroquine by Using a Thermal Aerosolization Process Coupled to SUPER SESI-HRMS
T. Zivkovic Semren1, M. Fatarova, S. Majeed1, A. R. Kolli1, A. Mazurov1, F. Martin1, A. Kuczaj1, J. Hoeng1, M. C. Peitsch1, N. Ivanov1, and P. A. Guy1
1Philip Morris Products S.A., Neuchâtel, Switzerland
Severe acute respiratory syndrome coronavirus 2 was identified in January 2020 as the cause of the SARS-like atypical pneumonia called coronavirus disease 2019. Recent publications have brought attention to the possible benefit of chloroquine (CQ) and its analog hydroxychloroquine (HCQ) in treatment of patients infected by this coronavirus. Previous research has demonstrated a narrow margin between the therapeutic and toxic doses of CQ and HCQ. In this study, we investigated the feasibility of developing inhalable forms of the antiviral drugs CQ and HCQ. The drugs were solubilized in an appropriate carrier and subjected to thermal aerosolization to generate an aerosol. The liquid formulation was evaporated by heating and subsequently cooled, which triggered nucleation and condensation processes that lead to aerosol formation. The aerosol was generated and assessed by using a programmable dual syringe pump coupled to the aerosol generation device, which guaranteed active drawing of a specified volume of air. The chemicals were detected by using secondary electrospray ionization interfaced with a Q Exactive mass spectrometer (MS). Transfer rate was assessed by analyzing the chemicals present in aerosol particles trapped in a Cambridge filter pad, by using liquid chromatography coupled to MS detection. Both CQ and HCQ solutions showed good potential for thermal aerosolization. These formulations are currently undergoing further improvements in parallel with transfer rate optimization related to thermal aerosol generation and gas/liquid phase partitioning. The developed methods and protocol for thermal drug aerosolization could have potential applications in treatment of infected people.
P413: Strategies on Assessment of Receptor Occupancy via Flow Cytometry
A. S. Wong1, M. Patel1, and M. Poirier1
1Altasciences Preclinical Seattle, LLC, Everett, WA, USA
Flow cytometry has been the method of choice to assess receptor occupancy (RO) of antibody-based immunotherapies due to its ability to measure binding on multiple cell populations simultaneously. Receptor occupancy assessment at the preclinical phase not only informs on dosage in clinical trials, it also demonstrates that the relevant species has been selected for safety assessment through adequate target binding. Receptor occupancy assessment using flow cytometry involves the measurement of target receptors bound by the drug (bound receptor), target receptors not bound to the drug (free receptor), and total target receptors present (total receptor). The methods for measuring these parameters can be categorized into 2 approaches based on the detection reagents used. In one approach, fluorescently labeled competing and noncompeting antibodies to the drug are used to detect free and total receptors, respectively. Here, the competing antibody is often the labeled drug itself, and we have observed that in samples with antidrug antibodies present, this approach can lead to inaccurate levels of free receptor measured. The other approach utilizes a fluorescently labeled secondary antibody to detect both bound and total receptors. Staining with a single detection antibody means that direct comparison can be made between bound and total receptors, removing difficulties from different binding affinities between reagents. We have observed that identifying a suitable secondary antibody is crucial especially when the targeted populations consist granulocytes and monocytes. These technical considerations when incorporated early in the RO design will aid to determine the most suitable approach and avoid common pitfalls in assay designs.
P414: Skin Sensitization Potency of Fragrance Materials Assessed Using Kinetic Direct Peptide Reactivity Assay (kDPRA)
M. Na1 and A. M. Api1
1Research Institute for Fragrance Materials, Woodcliff Lake, NJ, USA
Currently, several validated nonanimal methods are available to assess the skin sensitization potential of chemicals. Each of these assays addresses a specific key event in the adverse outcome pathway (Organization for Economic Cooperation and Development [OECD], 2012). The direct peptide reactivity assay (DPRA) and amino acid derivative reactivity assay are validated to test the chemical’s ability to activate the molecular initiating event (OECD TG442C). When used in combination with other nonanimal methods, DPRA has been shown to be valuable in hazard identification of skin sensitizers. However, determining the potency of skin sensitizers using nonanimal methods remains a challenge. Recently, it was suggested that kinetic DPRA (kDPRA) could be utilized to assign the skin sensitization potency class of a chemical (Wareing et al, 2017). In this modification from the standard DPRA, the chemical’s reaction with a model peptide are measured at multiple concentrations and multiple time points. A rate constant for this reaction has been shown to be a good predictor of the skin sensitization potency (Natsch et al, 2020). Herein, we report data and analysis of 60 fragrance ingredients (49 sensitizers and 11 non-sensitizers) in the kDPRA method, in comparison with existing animal and human data. For this data set, 12 of the 49 sensitizers and 2 of the 11 non-sensitizers exhibited peptide reactivity. The 14 materials that have shown peptide reactivity were predicted as stronger sensitizers, when compared to the animal and human data for this data set.
P415: Downsizing Sinclair S-1 Miniature Swine Utilizing a Nutritional Approach—Physical Data
G. Bouchard1, M. K. Pannu2, and D. Brocksmith3
1Sinclair Research Center, Auxvasse, MO, USA
2Sinclair Research Center, LLC, Auxvasse, MO, USA
3Sinclair Bio Resources, LLC, Auxvasse, MO, USA
The objective was to evaluate different nutritional management approaches to reduce the size of the Sinclair miniature swine (SMS), while maintaining normal physiology and health status as part of a SMS downsizing program. The food regimen used for the SMS has not been optimized for growth management and allows full body weight (BW) development while maintaining 20% to 30% body fat, even during the acute growth phase. Two diets were evaluated: Sinclair S-9 and Purina 5081. Sinclair S-9 diet is used in the SMS production (19% protein, 3.7% fiber, 3.2 Kcal/g). Purina 5081 diet is commercially available across the United States (14% protein, 14% fiber, 2.4 Kcal/g). Groups were control (S-9; regular daily regimen amount, n = 7); G1 (S-9; 20% reduction, n = 7); G2 (5081, 25% reduction, n = 7). Physical exams, body condition scores (BCS), BW, and behavioral assessments were performed weekly. There has been a significant (P < .05) reduction in BW. Weights from control animals (Purina CU S-9; 26.5 ± 3.8 kg) were reduced in G1 and G2 animals by −29.1% (20.8 ± 2.6 kg) and −63.4% (15.2 ± 1.8 kg), respectively. The BCS for control, G1 and G2, were 3.3, 3.0, and 2.7, respectively.
500 Series—Safety Evaluation Pharmaceuticals
P501: North American Travel Grant Investigation of Genotoxic Potential of Pharmaceutical Agents in the Chicken Egg Model (CEM)
T. Kobets1, J-D. Duan1, K. Brunnemann1, E. Vock2, U. Deschl2, and G. Williams1
1New York Medical College, Valhalla, New York, USA
2Boehringer Ingelheim Pharma GmbH&Co. KG, Biberach an der Riss, Baden-Württemberg, Germany
DNA reactivity poses a major safety concern for pharmaceuticals. Thus, a set of 14 medicines with different pharmacological effects was evaluated for DNA damage in the Chicken Egg Model (CEM). Investigated pharmaceuticals were of the following types: antibiotic (ciprofloxacin), nonsteroidal anti-inflammatory (ibuprofen), antipyretic analgesics (phenacetin and its metabolite acetaminophen), sedative barbiturate (phenobarbital), antihypertensive vasodilator (hydralazine), antifungal (griseofulvin), local anesthetics (lidocaine and prilocaine), antineoplastic (teniposide, streptozotocin, cyclophosphamide, and mitomycin C), and estrogen modulator (tamoxifen). A genotoxin, quinoline, served as a positive comparator. DNA damage was evaluated in the livers of chicken embryo-fetuses, which on days 9 through 11 of incubation were administered 3 daily injections of selected pharmaceuticals at various dosages into the air sac and terminated 3 hours after the last dose. The 32P-nucleotide postlabeling (NPL) and comet assays were used to detect formation of DNA adducts and strand breaks, respectively. Ciprofloxacin, ibuprofen, phenacetin, acetaminophen, and phenobarbital yielded negative results, as expected based on their lack of or borderline genotoxic potential in vitro and in vivo. Nevertheless, low solubility limited dose range of several compounds tested in CEM. In contrast, other pharmaceuticals produced positive outcomes in at least one of the assays, congruently with the established genotoxic and/or carcinogenic potentials of these compounds in other systems. Moreover, chromatographic patterns of DNA adducts produced by lidocaine, prilocaine, and tamoxifen in CEM were similar to those previously detected by the NPL technique in rodent livers. Overall, CEM provided reliable detection of genotoxicity of several therapeutic agents and yielded no false positive results.
P502: Incidence of New Toxicities in Biologics and Small Molecules Upon Long-Term Dosing
H. Prior1, B. Labram1, and F. Sewell1
1National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), London, UK
A recent international consortium reviewed the use of 2 species in drug development, concluding that current ICHS6(R1) approaches for use of a single species for long-term toxicity studies could be used more widely for biologics and other modalities, including small molecules. The cross-company data set was examined to investigate how often new toxicities are identified in long-term toxicity studies in rodents and non-rodents and whether use of only one species would “miss” toxicities of concern for human safety. Paired studies (short-term: ≤6 week and long-term: 13-39 week) were available for 31, 33, or 29 molecules in rodent, non-rodent, or both species, respectively. Toxicities in different target organs (eg, hematology, immune system, etc) were compared between study durations and absence or presence of new effects in long-term studies were determined. New effects were identified in long-term studies for 7 small molecules and 4 biologics (rodent), and for 7 small molecules and 4 biologics (non-rodent). For 10 biologics using 2 species in short-term studies, new long-term toxicities may have been missed in 2 or 1, depending on whether the rodent or non-rodent were progressed, respectively. For 19 small molecules using 2 species in short-term studies, new long-term toxicities may have been missed in 7 (or a different 7) depending on whether the rodent or non-rodent were progressed. There are examples where the long-term studies in rodent, non-rodent, or both did not identify new toxicities for small molecules and biologics, leading to wider opportunities to run these studies in only a single species.
P503: A Humanized Mouse Model to Predict Immunogenicity of Impurities in Generic Peptide Drugs
J. M. Sung1, R. Becker1, A. Knapton1, E. Pang1, and K. Howard1
1US Food and Drug Administration, Silver Spring, MD, USA
There is potential for impurities in chemically synthesized generic peptide products to elicit unwanted immune responses. To address these regulatory challenges, we utilized bone marrow-liver-thymus immune humanized mice that develop a fully engrafted human immune system. Our goal was to determine the predictive value of the animal model compared to in silico and in vitro immunogenicity testing methods. Initially, we evaluated salmon calcitonin, known to induce antidrug antibodies in patients, and 2 of its isolated impurities which were predicted highly immunogenic in silico and in vitro. Teriparatide, which is not associated with immunogenicity in patients, was tested as a negative control to validate our animal model. Mice were dosed daily with salmon calcitonin, impurities, or teriparatide for 28 or 56 days at different doses and routes of application. Dose-response data from intranasal or subcutaneous drug administration will be presented. Histology showed intranasal salmon calcitonin caused more severe inflammation in the nasal cavity compared to saline control or impurities alone. Flow cytometric analysis showed salmon calcitonin increased immune responses in a dose-dependent manner and enhanced the number of cells producing class-switched antibody. Lower B-cell responses in teriparatide-treated mice support clinical observations of low immunogenicity to teriparatide. This model allowed us to investigate doses of peptide drugs and routes of exposure that have the potential to trigger immune responses from drug products expected to produce immunogenicity. This model will be used to further investigate humoral immune responses to generic peptide drugs and impurities from these generic products and originator products.
P504: Reproductive and Developmental Toxicity Studies With a Tropomyosin-Related Kinase Inhibitor, ASP7962, in Rats
D. Kigami1, A. Okada1, and H. Ito1
1Drug Discovery Research, Astellas Pharma Inc, Tsukuba, Ibaraki, Japan
ASP7962 is a small molecule inhibitor which inhibits tropomyosin-related kinases, TrkA, TrkB, and TrkC, with 50% inhibition concentration of 0.155, 1.41, and 1.09 µmol/L, respectively, and was under development for treatment of painful knee osteoarthritis. As these 3 Trks are well known to be involved in neuronal development in central and peripheral nervous systems, reproductive and developmental toxicity studies with ASP7962 were of interest to test the inhibitory effects on embryo–fetal and postnatal development. In the first study, ASP7962 was orally administered to pregnant SD rats from implantation until closure of the hard palate (from day 7 to 17 of gestation) or from implantation until before delivery (from day 7 to 21 of gestation). In a subsequent study, ASP7962 was orally administered to pregnant SD rats from implantation to weaning (from day 7 of gestation to day 20 after delivery). These studies showed corneal abnormalities (TrkA), reductions in the level of milk present in the stomach (TrkB), loss of pain responses (TrkA/B), and decreased reflexes (TrkB/C) in pups at the higher dose levels used in our studies. These deficiencies in corneal innervation as well as facial motor neuron, sensory neuron, and spinal motor neuron development are also seen in knockout mice lacking Trk A, B, or C, and thought to be attributed to the corresponding kinase.
P505: Species Used for Toxicity Assessments of Small Molecules and Monoclonal Antibodies Approved by EMA (2016-2019)
F. Sewell1, B. Labram1, and H. Prior1
1National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), London, UK
A recent NC3Rs-led international collaboration reviewed species use in pharmaceutical toxicology studies using a data set of 172 unmarketed drug candidates. Here, we present data reviewed within European Public Assessment Reports, investigating trends for 55 small molecules (SMs) and 23 monoclonal antibodies (mAbs) authorized by the EMA between 2016 and 2019. For SMs, short-term (1 month) toxicity was often assessed in 2 species, with rat/dog (16 SM) or rat/cynomolgus monkey (12 SM) most commonly used. However, a similar proportion (24 SMs) used 3 or more species, typically adding mouse studies, and 1 SM used published in vivo data instead of conducting new studies. For mAbs, 2 performed short-term toxicity studies in mouse only and another mAb included only in vitro toxicity data. Twenty mAbs used cynomolgus monkey, either alone (12 mAbs) or in combination with a rodent (5 mAbs) or a second non-rodent (3 mAbs). Opportunities to reduce the number of species used for long-term (3-9 month) toxicity studies are accepted within ICHS6(R1): Here, 6 mAbs reduced from 2 to 1 species, progressing the nonhuman primate in all cases. Small molecules often reduced from 3 or more to 1 rodent and non-rodent species (21 SM); reasons were not provided for any of these decisions. This and our unmarketed data set (and other publications) illustrate that variable approaches are being used, reflecting the diversity of drug modalities currently in development. These case-by-case considerations provide opportunities to apply the 3Rs while enabling generation of robust safety data.
P506: Developing Mechanism-Based Models for Complex Toxicology Study End Points Using Standardized Electronic Submission Data
D. P. Russo1, K. P. Cross2, and K. Snyder3
1Oak Ridge Institute for Science and Education, Oak Ridge, TN, USA
2Leadscope, Inc, Columbus, OH, USA
3US Food and Drug Administration, Center for Drug Evaluation and Research, Silver Spring, MD, USA
To ensure the safety of human subjects participating in clinical trials, the US Food and Drug Administration (FDA) Center for Drug Evaluation and Research requires submission of nonclinical pharmacology and toxicology studies for all new pharmaceuticals prior to initiation of clinical studies. While these data provide a valuable resource to inform the dosing, design, and safety monitoring of clinical studies, attrition during drug development due to unexpected clinical toxicity indicates the need for development of novel methods to improve the interpretation of these data. The FDA has begun to require the submission of Standard for Exchange of Nonclinical Data (SEND) data sets along with study reports for all repeat-dose toxicology studies initiated after December 17, 2017, providing a constant source of structured in vivo toxicology study data. The SEND data provide a unique data landscape for informatics-based approaches to identify safety signals and alerts from nonclinical data prior to initiation of clinical trials. In this work, we leveraged the wealth of animal toxicity data contained within > 1800 SEND data sets (submitted via Investigative New Drug Applications) to develop a workflow to train machine learning models from different SEND end points (eg, clinical chemistry lab tests) to predict various mechanisms of animal hepatotoxicity. These models were able to identify preclinical safety signals previously confirmed to be biomarkers for different animal hepatotoxicity mechanisms (eg, alanine/aspartate aminotransferases). This proof of concept suggests this workflow could be expanded to identify novel biomarkers for hepatotoxicity as well as be amended for other complex toxicity end points (eg, nephrotoxicity) and inform on their mechanisms.
P507: An Investigation of the Reinforcing Potential of Centanafadine in Rats Trained to Self-Administer Cocaine
D. Heal1, S. Smith1, H. Day2, Z. Turnbull2, and R. Elbekai3
1DevelRx Ltd, Nottingham, Nottinghamshire, UK
2RenaSci Ltd, Nottingham, Nottinghamshire, UK
3Otsuka Pharmaceutical Development & Commercialization, Inc, Rockville, MD, USA
Centanafadine is a novel, small-molecule, norepinephrine, dopamine, and 5-HT reuptake inhibitor being developed by Otsuka as treatment for attention deficit hyperactivity disorder (ADHD). Centanafadine’s reinforcing potential has been investigated by intravenous (IV) self-administration in cocaine-trained rats. Male, Sprague-Dawley rats were trained to IV self administer cocaine (0.36 mg/kg/inj) on a fixed-ratio (FR5) reinforcement schedule. After saline extinction, the reinforcing effect of centanafadine (0.003, 0.01, 0.03, and 0.1 mg/kg/inj), methylphenidate (0.01, 0.03, and 0.1 mg/kg/inj) or bupropion (0.03, 0.1, and 0.3 mg/kg/inj) were evaluated by FR5 IV self-administration. Results are mean ± SEM, n = 8 to 9 rats/group. Cocaine (0.36 mg/kg/inj) maintained self-administration at levels (units = inj/session) greater than saline (19.9 ± 0.1 vs 4.8 ± 0.2, P < .001). The 2 lowest centanafadine doses (0.003 and 0.01 mg/kg/inj) did not maintain significant levels of self-administration (7.2 ± 1.2 and 5.8 ± 1.3, respectively). The 2 highest doses (0.03 and 0.1 mg/kg/inj) maintained self-administration above saline (12.8 ± 2.4 and 15.2 ± 2.9, respectively; both P < .001). All methylphenidate doses (0.01, 0.03, and 0.1 mg/kg/inj) maintained self-administration above saline values (8.8 ± 2.2, P < .05; 18.8 ± 1.5, P < .001; 20.0 ± 0.0, P < .001, respectively). All bupropion doses (0.03, 0.1, and 0.3 mg/kg/inj) maintained self-administration above saline (10.4 ± 2.2; 20.0 ± 0.1; 20.0 ± 0.0, respectively; all P < .001). The rank order of potency for maintaining self-administration was methylphenidate > bupropion = centanafadine. Centanafadine served as a positive reinforcer at the 2 higher doses in cocaine-trained rats. When the results for centanafadine are compared against other ADHD drugs and those which have been reported to show efficacy in this disorder, they indicate centanafadine will pose a lower risk of abuse than methylphenidate (C-II) and a similar level of risk to bupropion (not scheduled).
P509: Applying Machine Learning Approaches for Early Prediction of Drug-Induced Phospholipidosis—Can We Replace Some of Our In Vitro Testing in the Near Future?
L. T. Anger1, K. C. Wu1, F. Melnikov1, and C. Hasselgren1
1Genentech Inc, South San Francisco, CA, USA
Drug-induced phospholipidosis (PLD) is a lipid storage disorder characterized by excessive accumulation of drug and polar phospholipids in acidic subcellular compartments, such as lysosomes. It is usually considered adaptive and reversible. Over 50 marketed drugs cause PLD; however, it may negatively impact labeling and market development and, as an example, Amiodarone has a black box warning for pulmonary toxicity. Distribution and trapping of compounds into lysosomes are well-understood key processes involved in PLD induction and these are mainly driven by their physicochemical properties (mainly LogP and pKa), which makes the end point particularly suitable for applying computational approaches to generate highly accurate predictive models. To reliably predict drug-induced PLD, we combined 3 orthogonal in silico approaches: A state-of-the-art Machine Learning model (Random Forest classification model developed in-house on in vitro data of Genentech small molecule drug candidates) together with 2 well-established methods published in the literature based on physicochemical properties and chemical substructure patterns. The consensus approach of these 3 approaches resulted in high accuracies during internal validation, achieving a correct classification rate of 75%. We furthermore implemented this methodology in an ongoing drug discovery project, where we demonstrate prospective validation through using PLD predictions as a prefilter for compound selection and prioritization of experimental in vitro testing. Finally, using selected examples of in-house and literature compounds, we investigated the translatability of PLD from in silico to in vitro and to in vivo achieving overall high concordances.
P510: Are Existing Environmental Assessment Approaches Appropriate for Novel Drug Products?
I. A. Bamgbose1, I. Mohar2, and T. Verslycke1
1Gradient, Boston, MA, USA
2Gradient, Seattle, WA, USA
Over the last 30 to 40 years, the potential environmental impacts associated with the use of human drugs has been of increasing concern. In 1998 and 2006, the US Food and Drug Administration and the European Medicines Agency, respectively, developed guidance for evaluating environmental safety as part of marketing authorization of new human drugs. These guidance documents present a framework for evaluating environmental risks associated with new drug approval using environmental fate, exposure, and ecotoxicity data. These guidance documents were developed at a time when most new human drugs were small molecules. However, recent years has seen a rapid increase in the development of nonclassical drugs. The aim of this analysis is to evaluate whether the existing framework for drug environmental safety assessment is appropriate for nonclassical drugs by considering a number of case examples, such as a drug containing a naturally occurring nucleic acid analog and an endocrine active drug. On the basis of these examples, a number of recommendations are put forth. For example, transformation and metabolism data and structural similarity can be used to determine whether a meaningful increase in environmental exposure for a naturally occurring substance is expected. Clinical safety data and tailored ecotoxicological testing can be used to evaluate endocrine active substances. Thus, while the existing testing frameworks may not always be appropriate for nonclassical drugs, they do provide an accepted framework against which fit-for-purpose environmental safety assessments can be developed and justified.
P511: Monoclonal Antibody Co-Dosing Strategy to Mitigate On-Target ADC Toxicity: A Case Study in Cynomolgus Monkeys
R. Yeager1, B. Bawa1, R. Sura1, C. Phipps2, I. Figueroa3, M. Anderson4, A. Oleksijew4, E. Boghaert4, and E. Reilly4
1AbbVie Preclinical Safety, North Chicago, IL, USA
2AbbVie DMPK/BA, North Chicago, IL, USA
3AbbVie DMPK/BA, San Francisco, CA, USA
4AbbVie Discovery Oncology, North Chicago, IL, USA
Antibody drug conjugates (ADCs) are an approved oncology therapeutic modality. Consistent with advancements in the field to develop new payloads, this case study compares a clinical-phase first-generation ADC (“ADC-1”) with a discovery-phase next-generation ADC (“ADC-2”), which includes a more potent payload with different mechanism of action. For both ADCs, the same c-Met targeting monoclonal antibody (mAb) was utilized. c-Met is a validated cancer target with some expression in normal tissues, including the gastrointestinal (GI) tract. In nonclinical and clinical studies, the ADC-1 safety profile was consistent with nonspecific antigen-independent toxicity; on-target GI toxicity was not observed. In contrast, in monkey studies with ADC-2, dose-limiting GI toxicity was observed. Therefore, a strategy for unconjugated c-Met mAb co-dosing was designed to evaluate potential mitigation of ADC-2 on-target toxicity. First, pharmacokinetic/pharmacodynamic (PK/PD) modeling in monkey and tumor-bearing mouse studies were conducted to determine whether mAb co-dosing may affect ADC-2 serum exposures and/or efficacy. The PK/PD modeling identified an optimal dose of mAb that saturates the normal target and increases ADC-2 serum exposures. Mouse efficacy studies indicated that mAb co-dosing did not affect ADC-2 tumor growth inhibition in 3 xenograft models with high expression of cMet. Subsequently, a monkey study was conducted; as predicted from the modeling, mAb co-dosing effectively mitigated the ADC-2 on-target lower GI toxicity, resulting in improved tolerability at higher projected serum exposures. In summary, this case study suggests a potential clinical strategy for improvement of the therapeutic index for ADCs with potent payloads through the mitigation of on-target toxicities.
P512: Overview of the Toxicology Studies Conducted to Support the Development of Cenobamate (YKP3089) for Epilepsy
S. M. Melnick1, H. W. Kim1, and K. J. Glenn1
1SK Life Science, Inc, Paramus, NJ, USA
Cenobamate is an anti-seizure medication approved in the United States for the treatment of partial-onset (focal) seizures in adults and appears to be working through sodium channel inhibition and γ-aminobutyric acid type-A positive allosteric modulation. In single-dose toxicology studies, cenobamate was orally administered to mice, rats, and monkeys. Multiple oral dose toxicology studies with toxicokinetic assessments were conducted in mice for up to 3 months, rats for up to 6 months, and monkeys for up to 1 year. Genotoxicity was evaluated with in vitro (Ames and mammalian cell gene mutation) and in vivo (mammalian erythrocyte micronucleus) assays. Oral-dose carcinogenicity studies were performed for up to 6 months in transgenic rasH2 mice and up to 2 years in rats. In single-dose toxicology studies, dose-dependent central nervous system (CNS)-related changes (ataxia, tremor, loss of righting reflex, immobility, decreased respiration) were observed in all species treated with cenobamate. In multiple-dose toxicology studies, CNS-related clinical signs occurred with cenobamate doses ≥60 mg/kg/d in mice, ≥24 mg/kg/d in rats, and ≥27 mg/kg/d in monkeys. In these studies, the no-observed-adverse-effect-levels were 30 mg/kg/d, 12 mg/kg/d, and 18 mg/kg/d, respectively, for the longest study duration. Cenobamate displayed a steep dose-response and CNS adverse events were associated with higher plasma concentrations. Cenobamate did not induce mutagenic/clastogenic potential. Cenobamate-treated transgenic mice and rats did not show increased incidences of neoplastic findings. The toxicologic package fully supported the development of cenobamate for the treatment of partial-onset seizures. Supported by SK Life Science, Inc.
P513: Effects of Sex, Age, Country of Origin, and Dosing Routes on Plasma Cytokine Profiles in Healthy Cynomolgus Monkeys (Macaca fascicularis)
L. Zhao1, A. Forrest1, and V. Patel1
1Charles River Laboratories, Reno, NV, USA
Circulating cytokines are important end points frequently assessed in toxicity studies to reflect immune system status. Cynomolgus monkey is a useful non-rodent species in preclinical testing; however, detailed reference levels for cytokines have not been reported in this species. The levels of interleukin (IL)-1β, IL-1RA, IL-2, IL 4, IL-5, IL-6, IL-8, IL 10, IL 12/23 (p40), IL 13, IL-17A, monocyte chemoattractant protein (MCP)-1, MIP-1β, interferon-γ, tumor necrosis factor (TNF)-α, G-CSF, and GM CSF were determined in plasma samples from 198 healthy cynomolgus monkeys from several toxicity studies using a validated multiplex assay. Most of the cytokines evaluated were below the lower limit of quantification before vehicle administration, with detectable levels of TNF-α, IL-8, and MCP-1 observed in a few animals. After IV bolus injection or IV infusion with inert vehicle, transient and low magnitude increases (2- to 5-fold) of IL-1RA, IL-6, IL-8, MCP-1, and TNF-a were observed between 1 and 8 hours postdose. Furthermore, the cytokine increases occurred earlier and were of a higher magnitude following IV infusion when compared to IV bolus injection (except for MCP-1), suggesting a relationship to any stress associated with dosing procedures. The results also revealed that male or sexually mature animals had higher plasma levels of IL-8 when compared to females or immature animals, respectively, while there were no differences in cytokine baselines for monkeys with countries of origin from China and Mauritius. This study indicates that variability in baseline cytokine levels, animal sex and age, and dosing routes should be considered when interpreting cytokine data from cynomolgus monkeys and evaluating potential test article–related changes.
P514: Toward Quantitative Potency Predictions in Safety Assessment: Robust Concentration-Response Analyses With Case Studies
F. Melnikov1, C. Hasselgren1, and L. T. Anger1
1Genentech, San Francisco, CA, USA
Predictive methods have gained wider acceptance in chemical safety assessment and drug development. However, several opportunities to improve activity predictions and model confidences through increased data quality data and consistent data processing remained. For example, in preclinical safety assessment, many compounds were screened in the 2-point format. Traditionally, these data were not analyzed in concentration-response format and did not have a quantitative potency estimate. Here, we developed a robust concentration-response inference routine that can reliably estimate chemical potency across experimental formats. We demonstrated its applicability using historic Ether-à-go-go-Related Gene (hERG) inhibition and cytotoxicity data sets. In 90% of all cases, the difference between 50% inhibition concentrations (IC50s) derived from 2-point and 6-point concentration-response curves was below 0.25 log units and did not exceed the expected assay reproducibility. We developed quantitative predictive models for hERG inhibition and cytotoxicity potency based on the resulting data sets with several thousand IC50 estimates. XGboost gbtree regression algorithm implemented in R statistical environment was chosen for the final model. Models’ performance was assessed with external validation. The models performed similarly to those built for categorical end points but allowed for improved uncertainty assessment and error propagation. Furthermore, the standardized concentration-response inference and quantitative modeling approaches enabled researchers to rank-order synthesized and virtual compounds based on potency estimates and thus helped analyze chemical trends.
P515: Utilizing CDISC SEND Data to Generate Historical Control Incidence From a Large Database of Toxicology Studies
J. Horvath1, M. Carfagna2, C. Eley3, T. Fukushima4, W. Houser1, C. Sloan1, T. Page2, J. Anderson5, K. Snyder5, and D. P. Russo5
1Bristol Myers Squibb, New Brunswick, NJ, USA
2Eli Lilly, Indianapolis, IN, USA
3Pfizer, Inc, Groton, CT, USA
4Shionogi & Co, Ltd, Osaka, Kansai, Japan
5CDER, US FDA, White Oak, MD, USA
BioCelerate is an industry consortium driving initiatives to increase efficiencies in early stage R&D. The implementation of the Standard for Exchange of Nonclinical Data (SEND) model represents an opportunity to apply large-scale data analytics to toxicology data. Before this opportunity can be realized, differences in SEND implementation that make it difficult to conduct cross-study analysis must be addressed. In partnership with Food and Drug Administration (FDA), 6 cross study analysis use-cases and areas in SEND data sets that are significant drivers of variability which negatively impact cross-study analysis were identified. Herein, the focus is on the background-control data use-case and approaches for improved data harmonization. The most relevant variables to extract from SEND data sets that allow SEND repositories to function as robust and easy-to-use historical control databases have been defined. The US FDA Center for Drug Evaluation and Research repository of >1500 SEND data sets was queried to gain insight into how SEND is being applied with respect to these parameters. Proposals for harmonization methods and test scripts to transform data were developed to allow consistent extraction of these variables across SEND data sets. A framework for the development of proposed solutions from which a user could assemble the results for specific end points for all control animals in a user-defined subset of studies is provided. Such a resource could be used to provide greater context into the significance of potential toxicologic findings observed in individual studies. By implementing these proposed approaches, stakeholders will be able to take advantage of the opportunity presented by SEND data sets to enable increased efficiency and productivity in early stage R&D.
P516: Nonclinical Safety Assessment of Repeated Administration and Biodistribution of a Novel Rabies Self-Amplifying mRNA Vaccine in Rats
A. Stokes1, J. B. G. Dubois2, N. B. Abdeljelil2, J. Haruna2, O. Binazon3, J. Young4, M. Ringenberg1, and A. Rodriguez3
1GlaxoSmithKline, Rockville, MD, USA
2Citoxlab (CRL), Laval, Quebec, Canada
3GlaxoSmithKline, Wavre, Belgium
4Stagebio, Mason, OH, USA
The novel self-amplifying mRNA (SAM) technology for vaccines consists of an engineered replication-deficient alphavirus genome encoding an RNA-dependent RNA polymerase and a gene of the target antigen. To validate the concept, rabies glycoprotein G was chosen as the antigen and cationic nanoemulsion (CNE) was selected as the delivery system. To characterize the local tolerance, potential systemic toxicity, and biodistribution of this vaccine, 2 nonclinical studies were performed. In the repeated dose toxicity study, the SAM vaccine was administered intramuscularly to rats on 4 occasions at 2-week intervals followed by a 4-week recovery period. Self-amplifying mRNA–related changes consisted of a transient increase in neutrophil count, α-2-macroglobulin, and fibrinogen levels. Transient aspartate transaminase and alanine aminotransferase increases were also noted in females only. At necropsy, observations related to the elicited inflammatory reaction, such as enlargement of the draining lymph nodes, were observed that were almost fully reversible by the end of the recovery period. In the biodistribution study, rats received a single intramuscular injection of the SAM Rabies vaccine, and the distribution of the material was characterized in various tissues including blood over 60 days. The RNA was found at injection sites and in draining lymph nodes 1 day after administration, then generally decreased in these tissues but remained detectable up to day 60. Rabies RNA was also transiently found in blood, lungs, spleen, and liver. These results characterized the SAM Rabies (CNE) vaccine as well-tolerated by the rats and supported initiation of the subsequent clinical trial.
P517: In Vitro Assessment of Farnesoid X Receptor Antagonism to Predict Drug-Induced Liver Injury Risk
L. M. Norona1, A. Fullerton1, C. Lawson1, L. Leung1, J. Brumm1, T. Kiyota1, J. Maher1, C. Khojasteh1, and W. R. Proctor1
1Genentech, Inc, South San Francisco, CA, USA
Given the importance of farnesoid X receptor (FXR) in bile acid homeostasis, drug-related FXR antagonism may be an important mechanism of drug-induced liver injury (DILI). However, assessment of this across drugs associated with DILI and predictivity to identify hepatotoxicants retrospectively is lacking. Here, we used an orthogonal approach comprised of a primary human hepatocyte target gene assay and commercially available reporter assay to evaluate potential FXR antagonist effects of 74 DILI positive and 85 DILI negative drugs. Following treatment, expression of FXR target genes or reporter activity was evaluated. Careful consideration for cytotoxicity was taken to mitigate false positive signals. We report for the first time that FXR antagonism is not highly prevalent across a diverse set of clinically relevant compounds. Accordingly, there was minimal to moderate predictivity to identify DILI retrospectively using FXR antagonism alone. While both assays were comparable in sensitivity (24%-27%) and specificity (87%-93%), the positive likelihood ratio for the reporter assay was 3.84, supporting a moderate predictive value. Given the multifactorial nature of DILI, we examined the value of FXR antagonism to predict DILI risk while enriching for high exposure and bile salt export pump inhibition as additive metrics. Based on these data, FXR antagonism was not a meaningful predictor of clinical DILI and did not add significant predictive value when combined with other risk factors explored here.
P518: An Investigation of the Discriminative Properties of Centanafadine in Rats Trained to Discriminate D-Amphetamine From Saline
R. Elbekai1, H. Rowley2, and D. Heal3
1Otsuka Pharmaceutical Development and Commercialization, Rockville, MD, USA
2Renasci Limited, BioCity, Nottingham, UK
3DevelRx Limited, BioCity, Nottingham, UK
Centanafadine is a novel, small-molecule, norepinephrine, dopamine, and 5-HT reuptake inhibitor being developed by Otsuka to treat attention deficit hyperactivity disorder (ADHD). Centanafadine’s discriminative properties have been investigated using rats trained to discriminate d-amphetamine from saline. Freely-fed, female, Lister hooded rats were trained using sweetened milk on a FR5 reward schedule to discriminate d-amphetamine (0.3 mg/kg intraperitoneal [IP]) from saline in a 2-choice lever-pressing task. Test sessions were 2.5 minutes (non-rewarded) plus 7.5 minutes (rewarded). Only results from the non-rewarded part were used to evaluate compounds. Generalization to d-amphetamine was set at >75%. Results are mean ± SD, n = 7 to 9 rats/group. Orally administered d-amphetamine (0.125-1.0 mg/kg) dose-dependently generalized to the IP-injected d-amphetamine cue, validating the test for detecting the effects of drugs administered by this route. Centanafadine did not generalize to d-amphetamine at 0.3, 3, 5, or 6 mg/kg, PO; only the highest dose (10 mg/kg, PO) generalized to d-amphetamine (76.3%). Methylphenidate (1-5 mg/kg, PO), phentermine (1-3mg/kg, PO), and bupropion (1-30 mg/kg, PO) all dose-dependently generalized to d-amphetamine. Rank order of potency was d-amphetamine > phentermine > methylphenidate > centanafadine > bupropion. Drug-discrimination testing revealed that moderate doses of centanafadine were not recognized as d-amphetamine-like, but centanafadine generalized to d-amphetamine at high dose. When these results are compared against other ADHD drugs and those which have been reported to show efficacy in this disorder, they predict that centanafadine will pose a lower risk for abuse than methylphenidate (C-II) or d-amphetamine (C-II) and a level of risk similar to bupropion (not scheduled).
P519: AAV8-Mediated Expression of Acid α-Glucosidase (GAA) in Nonhuman Primates Demonstrates Transgene-Specific Immune Responses and Toxicity
M. Eggers1, C. Vannoy1, J. Brassard2, C. Sadhu1, J. Huang1, H. Chavan3, C. Fonck1, and F. Mavilio1
1Audentes Therapeutics, San Francisco, CA, USA
2Jacqueline Brassard Toxicologic Pathology Consulting, Tustin, CA, USA
3Charles River Laboratories, Reno, NV, USA
Pompe disease is an autosomal recessive neuromuscular disorder caused by loss-of-function mutations in the acid α-glucosidase (GAA) gene, leading to glycogen accumulation in tissues, progressive muscle weakness, and cardiac insufficiency. Enzyme replacement therapy is currently the only available treatment. Systemic administration of AT845, an investigational AAV8 (Adeno-associated virus serotype 8) vector containing the human GAA gene under the control of a muscle-restricted promoter, to Macaca fascicularis monkeys resulted in an anti-GAA humoral immune response, cardiac and skeletal muscle inflammation, elevation of cardiac biomarkers, and echocardiogram abnormalities at the highest doses (2 × 1014 and 5 × 1014 vg/kg). A second, more limited study completed with the same vector containing the macaque GAA gene at the 2 × 1014 vg/kg dose, exhibited comparably increased levels of GAA expression in cardiac and skeletal muscle with no detectable immune response or tissue toxicity. This suggests that the toxicity observed with AT845 may be due to an anti-GAA xenogeneic immune response to the transgene product rather than to GAA protein overexpression. Western blot analysis of GAA from AT845-treated monkeys showed abnormal processing of the human GAA protein compared to the monkey GAA protein, which could in part explain the potential immunogenic nature of the human GAA. These studies illustrate some of the challenges of assessing the potential toxicity of gene therapy products when expressing human proteins in preclinical species. Overall, our data demonstrate AT845 is well tolerated and leads to robust, dose-dependent increases in GAA activity in skeletal muscle and the heart in primates.
P520: Toxicity Assessment of Pharmaceutical Dye Sunset Yellow FCF (aka FD&C Yellow No. 6)
J. Cohen1, C. Marsh1, and T. Lewandowski2
1Gradient, Boston, MA, USA
2Gradient, Seattle, WA, USA
Safety reviews of “inactive” pharmaceutical excipients have identified potential sources of human toxicity. In one case, amaranth (aka FD&C Red, and long considered one the safest colorants) was eventually removed from use over tumorigenicity concerns. FD&C Yellow is another certified dye approved for use in drug formulations (eg, Pepcid Complete) since 1986. Despite the widespread use, this chemical has not been thoroughly evaluated for safety even though studies in animals and humans suggest potential developmental and reproductive toxicity (DART) at high doses. We conducted PubMed and Google literature searches to evaluate carcinogenicity, mutagenicity (ICH M7 compliant), DART, consider potential mechanisms of action, and propose a permissible daily exposure. Study quality issues including sample size and material purity were noted. In vitro tests with FD&C Yellow 6 indicate the formulation is not mutagenic when manufactured to specification. Reproductive and target organ toxicity effects are inconsistently reported across several studies and lack a clear mechanism of action (effects on testes, sperm parameters, mammary glands, thymus weight). The most recent study conducted according to good laboratory practice concluded no male reproductive toxicity up to the highest dose tested (1475 mg/kg bw/d). A multigenerational study in rats reported potential developmental toxicity (decreased pup body weight), with a no observed adverse effect level of 375 mg/kg bw/d. Permissible daily exposures of 75, 15, and 5.25 mg/d were calculated for adults, children, and neonates, respectively, which included modifying factors for interspecies variability (5), intraspecies variability (10), and severity of effect (fetal toxicity without maternal toxicity, 10).
P521: Building a Seizure Testing Strategy in Toxicology: Considerations for Molecular Mechanisms
S. Authier1, Y. Zhong1, and M. Accardi1
1Charles River Laboratories Montreal ULC, Laval, Quebec, Canada
Understanding the mechanism(s) for drug-induced seizures (DIS) may help guide the structure analysis and computational chemistry to avoid this liability. A wide range of chemical entities have been previously associated with DIS (eg, antidepressants, antipsychotics, antibiotics, analgesics, etc). An understanding of the molecular mechanisms underpinning DIS is often challenging to achieve. The etiology of DIS can result of either a deficit in inhibitory (eg, γ aminobutyric acid) or elevation in excitatory (eg, glutamate) signaling, although other neurotransmitter systems are also known to play a role. The mechanism(s) underlying this altered neuronal signaling and how these changes interact with other non-brain receptor-driven DIS-associated changes such as metabolic disturbances, electrolyte imbalances, altered drug metabolism, and withdrawal effects are often not fully understood. We summarize molecular mechanisms which were reported in DIS relevant to drug development. With a better understanding of the DIS molecular mechanisms, targeted in vivo or in vitro strategies may be applied to characterize the DIS-risk. From a pragmatic standpoint, characterization of DIS manifestations in toxicology studies (ie, seizure onset, seizure duration, nature of the seizures, electroencephalogram, associated prodromal clinical signs) serves to build an assay panel to investigate this potential liability. Electroencephalogram data (surface electrodes or telemetry) show that most seizures occur when drug exposure is higher (as opposed to late-onset seizures) and susceptibility stratification based on GABA-related DIS presents species differences in the following order Beagle dogs > Sprague-Dawley rats > Cynomolgus monkeys > Göttingen minipigs with a more than 2-fold difference between canines and minipigs.
P522: Validation of the Use of a Telemetry Study to Measure Hemodynamic, Electrocardiographic, and Respiratory Parameters in the Conscious Dog
P. B. Senese1, M. K. Pannu2, S. E. Boley2, G. Bouchard2, M. D. Zammit1, J. Huang1, K. R. Doherty1, and M. R. Gralinski1
1CorDynamics, Inc, Chicago, IL, USA
2Sinclair Research Center, Auxvasse, MO, USA
Animal research remains a critical component to safety evaluation of candidate drugs, with telemetry providing insightful cardiorespiratory data. Safety pharmacology supports reducing animal numbers while optimizing study outcomes. Therefore, Beagle dogs were instrumented with Data Sciences International (DSI) implants (L11/L11 R), and a structured Latin Square crossover design was used to ascertain the discrete effects of verapamil, dofetilide, and doxapram. Hemodynamic, electrocardiographic, and body temperature data were captured using the DSI Ponemah v5 data capture telemetry system. EMKA electrocardiogram (ECG) Auto v3 was used to interrogate the ECG signals. Baseline values for all groups were as expected for the telemetered Beagle dog. Verapamil at 15 mg/kg significantly decreased blood pressure and caused a substantive increase in the PR interval over most of the study duration, with 2° atrioventricular block developing in 4 of 8 dogs. Dofetilide at 0.3 mg/kg significantly increased the PR interval during the 6-hour monitoring period, while 0.1 mg/kg and 0.3 mg/kg dofetilide significantly increased the corrected QTcH interval. Increases in both delayed and lost 1:1 atrioventricular conduction were noted in all dogs following 0.3 mg/kg dofetilide, while supraventricular ectopy increased in a dose-dependent fashion. Administration of 4 mg/kg doxapram elicited significant increases in blood pressures over the 4-hour monitoring period as well as a notable increase in respiratory rate, tidal volume, and minute volume shortly after administration. Thus, the expected effects of each test compound were accurately detected in Beagle dogs, validating the usage of an optimized safety pharmacology study to meet cardiorespiratory ICH guidelines.
P523: Consideration of Species Selection, Ocular Route of Administration, and Analytical Support for Ocular Safety Studies
H. Voute1, E. Drevon-Gaillot1, N. Truchot-Limonne1, L. Bertrand1, L. Allais1, P. Ancian2, and P. Singh2
1Charles River Laboratories, Lyon, Auvergne-Rhône-Alpes, France
2Charles River Laboratories, Evreux, Normandie, France
We have conducted over 50 ocular safety studies for a variety of disease therapies over the last 5 years in multiple species including rabbits, dogs, minipigs, and monkeys. Retrospective analysis indicated that drug properties and the type of test item (ie, gene therapy, Ab, biologics, small molecules) were associated with certain species and routes of ocular administration (instillation, intravitreal injection, subretinal). Study designs were reviewed by species/type of test item and trends were observed, particularly when the therapeutic candidate was a human protein, antibody, or gene therapy, due to specificity for a human target and possible lack of pharmacological activity in certain species. Ophthalmological examinations (slit lamp, tonometry, pachymetry, electroretinography, optical coherence tomography) were also reviewed by type of test item. Specific histopathology methods depended on the eye anatomy of the species. For viral vectored and gene therapy products, biodistribution studies and customized analytical methods were required. The type of analytical support needed is often critical for product development and safety testing to link the distribution of the test item to observed effects. The methods used for eye exams can differ based on anticipated effects in the anterior versus the posterior part of the eye. In conclusion, we determined that species selection, ocular route of administration and analytical support for ocular safety studies usually depends on the pharmaceutical or biologic properties and formulation of the therapeutic candidate.
P524: A Human Proximal Tubule Kidney Chip for Accelerated Therapeutic Development
S. S. F. Jeanty1, S. Jadalannagari1, J. Ronxhi1, D. Manatakis1, J. Sliz1, S. J. Kerns1, K-J. Jang1, L. Ewart1, and D. E. Ingber2
1Emulate Inc, Boston, MA, USA
2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USA
Drug-induced nephrotoxicity accounts for a majority of acute kidney injury cases and is a primary cause of clinical attrition for lead therapeutic candidates. The lack of predictive preclinical tools is a key factor in the failure to translate preclinical results into clinical tests that are sensitive enough to detect early biological markers of kidney injury. There is significant evidence that renal proximal tubule is the primary target for most nephrotoxic compounds. Therefore, there is a desperate need for more human-relevant proximal tubule models to evaluate early indicators of nephrotoxicological events. Here, we present an engineered Proximal Tubule Kidney-chip that more accurately captures in vivo phenomena by recreating the natural tubular-peritubular interface. The Human Proximal Tubule Kidney-Chip features 2 fluidic channels separated by a porous membrane that is coated with extracellular-matrix proteins, thereby creating a tubular epithelium and a vascular endothelium channel. The tubular epithelium is established with human primary renal proximal tubule cells, while the vascular endothelium consists of human primary renal microvascular endothelial cells cultured under continuous physiological flow to form the Proximal Tubule Kidney-Chip. We have shown that the proximal tubule epithelium expresses transporters that are key to proper kidney function in vivo, which are typically absent in conventional culture systems. Using well-known nephrotoxicants, including cisplatin and gentamicin, we have also demonstrated toxic responses at physiologically relevant concentrations. This Proximal Tubule Kidney-Chip recreates key physiological features of the human kidney and is a promising predictive tool to assess drug safety and efficacy.
P525: Historical Control Database of Cynomolgus Macaque Spermatozoa
A. Celori1, L. Baydak1, P. Ampo1, S. McGrath1, S. Kaur1, J. Hynek1, and J. Forget1
1Altasciences Preclinical Seattle, LLC, Everett, WA, USA
The assessment of sperm is useful to characterize potential adverse effects of pharmaceuticals on the reproductive system in males. Standard sperm assessments include motility, concentration, and morphology. Although the cynomolgus macaque is a common test species, in particular, for biotechnology-derived pharmaceuticals, ICH S5(R2) highlights that historical background data are lacking for nonhuman primates (NHP) in this area. Nonhuman primate semen is at this time evaluated based largely on the WHO Laboratory Manual for the Examination and Processing of Human Semen. Few publications exist describing normal cynomolgus macaque sperm, which coupled with the low numbers of animals typically used on studies, limit the translatability to humans of any potential effects observed in monkeys. Establishing robust criteria for normal NHP spermatozoa is crucial to accurately interpreting potential toxicological effects. To alleviate this gap, sperm concentration, motility, and morphology were collated from over 400 NHPs between 2007 and 2019 to generate an historical control database to assist in the interpretation of cynomolgus macaque sperm. All animals were either naive or had been dosed with a control/sham article. Sperm was collected via direct penile electrostimulation. Ejaculate volume was recorded, and sperm motility and concentration were assessed using the TOX IVOS or TOX IVOS II computerized assisted sperm analysis systems. Morphology was evaluated using light microscopy. Descriptive statistics (mean, SD, and quartile ranges) were generated to summarize the normal ranges for each parameter.