Abstract
100 Series—General Toxicology
P102 - Absence of MRP4 Impairs Compensatory Liver Proliferation and Causes Hepatic Steatosis Following Two-Third Partial Hepatectomy
O. Aladelokun1, J. Manautou1, A. C. Donepud1, K. Flores1, F. Carvalho1, Y. Lee1, and G. Smith2
1University of Connecticut, Storrs, CT, USA
2University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Liver has remarkable capacity to regenerate, which is needed for survival during toxin-induced liver injury. Multidrug resistance-associated protein 4 (MRP4), an efflux transporter of the ATP-binding cassette (ABC) family, plays a crucial role in regulating both hepatotoxicity and adaptive changes during liver injury in mice. Previous works in our laboratory show the upregulation of hepatic MRP4 after acetaminophen (APAP) treatment. In mice, the absence of MRP4 increased susceptibility to the APAP and valproic acid-induced hepatotoxicity. We hypothesized that the loss of MRP4 will compromise the capacity of the liver to regenerate after partial hepatectomy (PHx). To study the role of MRP4 in liver regeneration, we performed two-third PHx on wildtype (WT) and Mrp4 knock out (KO) mice, and sacrificed them at 24-, 48-, and 72-hour time points. Liver regeneration and hepatic steatosis were evaluated using histological and gene expression analyses. A transient increase in serum ALT levels and hepatic Ki-67 levels, a marker for cell proliferation, was observed in both WT and KO mice with no apparent significant changes. Interestingly, histological evaluation using H&E and Oil Red O staining indicated microvessicular steatosis, a known phenomenon of PHx, in both WT and KO mice. However, KO mice have significantly more hepatic steatosis compared to WT mice. Although no significant changes were observed in liver mass gain, a remarkable increase in hepatic lipid accumulation was observed in the KO mice. Our finding indicates MRP4 as a novel genetic determinant for hepatic lipid homeostasis during liver regeneration.
P103 - Assessing the Efficacy of Aqueous Garlic Extract Against Sublethal Cyanide Toxicity Using Righting Reflex Recovery Paradigm in Mice
M. Avais1, S. Ali1, and A. Z. Durrani1
1University of Veterinary and Animal Sciences, Lahore, Punjab, Pakista
This study was carried out to assess the efficacy of aqueous garlic extract (AGE) as antidote against sublethal cyanide toxicity using righting reflex recovery paradigm in mice. Male mice were divided into 30 treatment groups. The righting reflex recovery paradigm was based upon signs of acute cyanide toxicity including disturbance of neuromuscular coordination viz the righting reflex. Using this model, AGE at different doses was tested precyanide and postcyanide either singly or in adjunction with sodium nitrite (SN) or sodium thiosulfate (STS). The efficacy was measured based upon reduction in righting reflex recovery time. When AGE was given singly at 250, 500, or 750 mg/kg IP or orally, precyanide or postcyanide, righting reflex recovery time was significantly reduced (P < 0.05) at increasing dose of AGE. When SN was used at 20 mg/kg IP, precyanide and postcyanide, righting reflex recovery time was 44.16 ± 0.477 minutes and 49.33 ± 0.494 minutes, respectively. STS at 600 mg/kg precyanide and postcyanide reduced the righting reflex recovery time to 17.33 ± 0.333 minutes and 25.5 ± 0.341 minutes, respectively. AGE (at 750 mg/kg) in combination with SN, the righting reflex recovery times were reduced to 15.50 ± 0.447 minutes and 21.66 ± 1.032 minutes, precyanide and postcyanide, respectively. Combination of AGE and STS precyanide and postcyanide decreased the righting reflex recovery time to 8.5 ± 0.42 minutes and 18.5 ± 0.494 minutes, respectively. On comparing the righting reflex recovery time of mice treated with AGE + SN or AGE + STS, it was significantly reduced (P < 0.05) when used AGE + STS. In conclusion, AGE either singly or in combination with STS is efficacious to treat sublethal cyanide toxicity in mice.
P104 - Chronic Effects of Low Toxicological Doses of Organic Arsenic Exposure on Some Enzymes of Energy Metabolism and Antioxidant System in the Kidney
D. Babayemi1, E. Adeiyi1, M. Abdulsalam1, A. Tijani1, H. Adubiagbe1, O. Dosumu1, T. Akinhanmi2, and O. Ademuyiwa1
1Biochemistry Department, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria
2Chemistry Department, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria
In order to investigate the chronic effects of low toxicological doses of organic arsenic exposure on some enzymes of energy metabolism and antioxidant system in the kidney, rats were exposed to cacodylic acids (20 and 40 ppm) in their drinking water for 15 weeks. Control animals received distilled water. Activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), NADase, and xanthine oxidase (XO) and the levels of nitric oxide (NO), reduced glutathione (GSH), and lipid peroxidation (MDA) were assayed for in the renal cytosol, while lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and respiratory chain enzymes activities were determined in the mitochondrial fraction using the spectrophotometric method. There were significant (P < 0.05) decreases in all measured parameters at the 40 ppm except for succinate dehydrogenase and cytochrome c oxidase compared to the control. The activities of renal LDH, MDH, and NADH dehydrogenase were significantly (P < 0.05) inhibited by 70%, 15%, and 17% respectively. A negative dose-dependent relationship was observed in the effects of cacodylic acid on activities of SOD and GPx. Our findings confirm that organic arsenic interferes with energy metabolism in etiology of renal toxicity. Keywords: cacodylic acid, antioxidant system, kidney, respiratory chain enzymes, chronic toxicity.
P106 - Beneficial Effects of Intranasal Carnosine on Olfactory, Motor, and Mitochondrial Function in a Mouse Model of Parkinson Disease
M. L. Bermudez1, M. B. Genter1, and K. Seroogy1
University of Cincinnati, Cincinnati, OH, USA
Parkinson disease (PD) is a common and debilitating neurodegenerative disorder with no known cure. PD is characterized by motor and nonmotor deficits, including gait instability and decreased olfactory function. Molecular hallmarks of PD include protein aggregates and oxidative stress. Recent in vivo studies indicate that the dipeptide carnosine reduces protein aggregation and protects against oxidative stress, 2 features of PD. Therefore, we hypothesize that intranasal (IN) administration of carnosine will significantly reduce disease progression in the Thyl-aSyn mouse model of PD. Wildtype and Thy1-aSyn mice were treated IN with 2 mg/d of carnosine or sterile water (as control) for 2 months, a regimen demonstrated to increase the concentration of carnosine in the brain. Immunohistochemistry, buried food pellet, and the challenging beam traversal (CBT) tests were used to evaluate alpha-synuclein (aSyn) aggregation and sensorimotor functions at the beginning and end of treatment. Olfactory function and structure were preserved, and aSyn-positive inclusions were notably lower in the olfactory epithelium of carnosine-treated Thy1-aSyn mice compared to control Thy1-aSyn mice. In the CBT test, the number of errors per step was significantly lower in the carnosine-treated Thy1-aSyn group compared to the vehicle-treated Thy1-aSyn group (P < 0.05; n = 6/group). Additionally, the maximal respiratory capacity of the mitochondria isolated from the striatum significantly increased in the carnosine-treated Thy1-aSyn compared to control Thy1-aSyn mice (P = 0.0005). Our findings suggest that carnosine improves mitochondria function and prevents the progression of the motor deficits and aSyn aggregation in the Thy1-aSyn model of PD. Supported in part by P30-ES006096.
P107 - Multigenerational Assessment of Cannabidiol and Δ9-Tetrahydrocannabinol Toxicity Using Zebra Fish
D. Carty1, C. Thornton1, and K. Willett1
1Department of BioMolecular Sciences, University of Mississippi, Oxford, MS, USA
In pharmacoresistant epilepsy, cannabidiol (CBD) shows promise in reducing seizure frequency when administered in conjunction with first-line anticonvulsants, but empirical evidence of CBD’s safety profile or potential developmental and multigenerational adverse effects are still unknown. The goal of this project is to compare multigenerational adverse outcomes of Δ9-tetrahydrocannabinol (THC) and CBD exposure in the highly relevant zebra fish model. Zebra fish (F0) were exposed to sub-LOAEL concentrations of CBD, THC, or DMSO beginning at the blastula through the larval stage, then allowed to mature under normal rearing conditions, and mated at 6 months to produce unexposed offspring (F1). Throughout early zebra fish development, gene expression markers for neurogenesis, neuroactivity, and germline fitness namely bdnf, c-fos, and dazl, respectively, were measured along with reproductive fitness (F0), morphology (F0 and F1), and anxiolytic behavior (F0 and F1). Following THC and CBD exposure, 6-month-old adults had reduced offspring production compared to DMSO. There were no obvious dysmorphologies in the F1 generation at 96 hpf, but parental exposure of CBD (0.03 mg/L) or THC (0.12 mg/L) significantly reduced or stimulated anxiolytic behavior in F1 larvae, respectively. Differential gene expression of bdnf, c-fos, and dazl was widespread in 96 hpf F0 fish, but changes in F1 gene expression were less affected. This work supports the potential for persistent reproductive impacts of developmental cannabinoid exposure, while more studies are needed to assess multigenerational impacts. Supported by P20GM104932 and R21DA044473-01.
P110 - Aerosol From Tobacco Heating System 2.2 Has a Limited Impact on DNA Methylation of Candidate Enhancers in Mouse Lung Compared With Cigarette Smoke
M. A. Choukrallah1, N. Sierro1, F. Martin1, K. Baumer1, J. Thomas1, S. Ouadi1, J. Hoeng1, M. C. Peitsch1, and N. V. Ivanov1
1PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland
Exposure to cigarette smoke (CS) is a major long-term risk factor for various diseases even decades after cessation. Epigenetic alterations, including changes in DNA methylation, have been proposed to create a long-term memory of CS exposure. Previous reports based on methylation arrays showed that CS exposure correlates with changes in DNA methylation; however, methylation arrays are limited to annotated loci and poorly cover distal regulatory elements. In this study, we used whole-genome bisulfite sequencing to assess the effects of CS extract (reference cigarette) and aerosol from the Tobacco Heating System 2.2 (THS2.2), a candidate modified risk tobacco product (MRTP), on DNA methylation in lung and liver tissues from apolipoprotein E-deficient mice (ApoE−/−) during an 8-month period of exposure. We found that in lung tissue, CS mainly induced hypermethylation of enhancers at late time points while promoters were less affected. This effect was strongly reduced upon cessation or switching to THS2.2. By contrast, chronic exposure to aerosol from THS2.2 had a limited effect on DNA methylation at both promoters and enhancers. We also identified members of the E-26 and forkhead families of transcription factors as potential players in the epigenetic response to CS exposure in lung tissue. In contrast to the lung, DNA methylation in the liver was largely insensitive to all investigated exposures. In summary, our investigations indicate that CS-related epigenetic alterations are tissue-specific and occur mainly at enhancers. Exposure to an aerosol from THS2.2 in the same system had only a limited effect.
P111 - Vehicle Toxicity: The Effects of Commonly Used Vehicles in a Rabbit Embryo–Fetal Development Study
D. A. Donahue1, C. A. Schroeder1, H. L. Jordan1, K. Quigley1, T. J. Sterrett1, H. A. Staudner1, C. K. John1, A. Y. Akinola1, A. K. Groeller1, T. Gust1, T. Reichter1, C. D. Qualls1, and G. F. Weinbauer2
1Covance Laboratories Inc, Greenfield, IN, USA
2Covance Preclinical Services GmbH, Münster, Germany
Five vehicles were chosen due to their frequent use in rabbit studies and their tendency to elicit overt vehicle toxicity. Methylcellulose, polysorbate 80, soybean oil (SO), polyethylene glycol 400 (PEG 400; 2 and 4 mL/kg), and propylene glycol (PG; 2 and 4 mL/kg) were administered to 8 time-mated female rabbits/vehicle from gestation day (GD) 7 to 19. Blood samples were collected for clinical pathology parameters on GD 7, 19, and 29. On GD 29 maternal macroscopic observations and fetal visceral and skeletal examinations were conducted. Several rabbits administered SO, PEG 400 (4 mL/kg), and PG (4 mL/kg) were euthanized in extremis due to poor general health. Treatment-related macroscopic observations were limited to animals administered SO, PEG (4 mL/kg), and PG (4 mL/kg). Dose administration of SO, PEG 400, and PG resulted in adverse effects on clinical pathology parameters in moribund animals; minor nonadverse changes were noted in surviving animals on GD 19 with no differences noted on GD 29. Effects on cesarean delivery parameters were limited to increases in percent postimplantation loss in animals administered SO, polysorbate 80, and PG (4 mL/kg) and decreases in fetal weights at 4 mL/kg PEG 400. Treatment-related fetal skeletal variations and malformations were limited to PEG 400 (4 mL/kg), PG (2 and 4 mL/kg), and SO fetuses. Based upon the severe decreases in maternal body weights and food consumption, decreased fetal weights and fetal skeletal abnormalities, the following are not recommended as vehicles in rabbit embryo–fetal developmental studies: SO, PEG 400, and PG.
P112 - Maternal Uterine Artery Ad.VEGF-DΔNΔC Gene Therapy for Placental Insufficiency Shows No Evidence of Harm in a Rabbit Model
P. Singh1, C. Dauzat1, C. Bansard1, A. Decheix-Nguyen1, S. Bentz1, F. Spezia1, R. Forster1, M. Lees2, T. Heikura4, and A. David3
1CiToxLAB France, Evreux, France
2Magnus Growth Ltd, London, United Kingdom
3University of Eastern Finland, Kuopio, Finland
4UCL Institute for Women’s Health, London, United Kingdom
P114 - Variability in Ovarian Follicle Count Data Collected Over 2 Decades
R. Gilmore1, K. Gothard1, K. Gothard2, N. Robben2, C. Atterson2, M. Gilmore2, and D. Dandekar1
1Xenometrics, LLC, Stilwell, KS, USA
2Xenometrics, LLC–Intern, Stilwell, KS, USA
Ovarian follicle counts (OFC), ovary weights, and corpora lutea counts are useful tools for the assessment of compound-related adverse reproductive effects in toxicology studies as set forward by regulatory agencies since 1998. High variability in OFC and impact of variability in OFC count on assessment of reproductive toxicity are highlighted in the STP position paper on ovarian follicular counting (Regan KR et al, 2005). For abovementioned reasons, robust laboratory-generated historical control database (HCD) remains extremely useful. OFC and ovarian organ weight data collected at our laboratory from 21 completed 2-generation reproductive toxicity studies conducted in Wistar Han rats since 1998 are presented in this article. Stepwise sections (levels 1-5) of paired ovaries collected from females that had viable litters were counted (n = up to 20 rats/study). Follicles were classified as (1) primordial follicle (PF)/preantral follicles consists of “primordial,” “primary,” and “secondary” follicles and (2) antral follicles (AF) consists of “tertiary” and “mature” follicles. Corpus luteum (CL) were counted from each ovarian section. Ovarian weights were recorded at necropsy and normalized with terminal body weights. Combined follicular data presented as mean and ± standard deviation from 1998 to 2012 contained PF = 71.60 ± 23.0, AF = 41.30 ± 16.6, and CL = 34.9±9.7. HCD over 5-year period: 1998 to 2002, 2003 to 2007, or 2008 to 2012 showed similar results. As expected, the ovary/body weight ratio data remained fairly consistent during this period. Overall, consistency in FC counts at our laboratory indicates that with established standard operating procedures variability in FC can be minimized.
P117 - 5-Fluorouracil- and Irinotecan-Induced Intestinal Mucositis in the Mouse
S. Goineau1, E. Gascoin1, L. Lecouflet1, L. Paulhac2, E. Hayes1, and V. Castagne1
1PORSOLT SAS, Le Genest Saint Isle, France
2FLUOFARMA, Pessac, France
Chemotherapy-induced mucositis is a common severe side effect experienced by colorectal cancer patients during treatment. We aimed to investigate the gastrointestinal toxicity of 5-fluorouracil and irinotecan in mice. Balb/C mice (n = 8/group) were injected with saline, 5-fluorouracil (30 mg/kg, IP) for 5 consecutive days (males and females) or irinotecan (75 mg/kg, IP) for 4 consecutive days (males only). Body weight and diarrhea were assessed over the test period and inflammatory and histopathological responses were investigated 7 days after the first injection. 5-Fluorouracil and irinotecan increased the diarrhea score as compared to controls (5-fluorouracil: +2.3 and +2.4, P < 0.001 for males and females and irinotecan: +0.9, P < .001). 5-Fluorouracil, but not irinotecan, tended to increase the myeloperoxidase activity in both males and females. 5-Fluorouracil and irinotecan increased ileum IL-1β levels (5-fluorouracil: +36%, NS, and +131%, P < 0.01 for males and females and irinotecan: +93%, P < 0.05) and plasma TNF-α levels (5-fluorouracil: +700% and +800%, P < 0.001 for males and females and irinotecan: +825%, P < 0.05) without modifying ileum TNF-α levels. Plasma IL-1β levels were below the limit of quantification in all groups. Both treatments affected the intestinal architecture and decreased the villus/crypt ratio (5-fluorouracil: −45% and −33%, p<0.001 for males and females and irinotecan: −23%, P < 0.01). These findings suggest that 5-fluorouracil- and irinotecan- induced gastrointestinal toxicity in mice presents similarities with the clinical intestinal mucositis manifestations. Therefore, these mouse experimental mucositis models offer a promising tool for evaluating the side effects of novel chemotherapeutic agents or the efficacy of potential treatments against chemotherapy-induced mucositis.
P118 - Characterization of a Novel Tobacco Vapor Device Using an In Vivo Inhalation Exposure System
S. Hirata1, H. Suzuki1, S. Onami1, and T. Sekine2
1Scientific Product Assessment Center, R&D Group, Japan Tobacco Inc, Yokohama, Japan
2Reduced-Risk Products Science, Emerging Products, JT International SA, Geneva, Switzerland
Devices that heat, rather than combust, tobacco have been reported to deliver vapors with less complex chemical compositions, and reduced biological effects, compared to cigarette smoke. In this study, we used a nose-only inhalation system to reveal, in rats, the acute effects of vapor from a novel tobacco vapor device (NTV), compared with that of the reference cigarette (3R4F) smoke. The NTV vapor and 3R4F smoke were generated under the modified Canadian intense puffing regimen and wet total particulate matter (WTPM), particle size, and selected constituents (propylene glycol [PG], glycerol, nicotine, carbon monoxide [CO], carbonyls, and 1,3-butadiene) were measured at the nose port. Aerodynamic particle sizes were measured at 0.80 (NTV) and 0.89 μm (3R4F). CO, carbonyls, or 1,3-butadiene weren’t detected in NTV vapor. Nicotine yield was lower, and PG was higher, in NTV vapor compared to 3R4F smoke. Ten male rats were subsequently exposed to air (control), NTV vapor (200, 600 and 1,000 μg/L WTPM), or 3R4F smoke (1,000 μg/L WTPM) for 1 h/d for 3 days. Following treatment, plasma nicotine and markers of exposure to CO, carbonyls, and 1,3-butadiene were found to be substantially lower in rats exposed to NTV vapor compared to 3R4F smoke. Histologically, only minimal changes in respiratory tract (nose and larynx) lesions were observed in the NTV groups compared to control, while the 3R4F group showed higher incidence. In summary, NTV vapor elicited substantially reduced acute biological effects on the rat respiratory system compared to cigarette smoke.
P119 - FH Inactivation Is Synthetic Lethal With Ferroptosis Inducers in Hereditary Leiomyomatosis and Renal Cell Cancer
M. Kerins1, A. Vashisht2, J. Wohlschlegel2, and A. Ooi1
1University of Arizona, Tucson, AZ, USA
2University of California–Los Angeles, Los Angeles, CA, USA
Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome characterized by inactivation of the Krebs cycle enzyme fumarate hydratase (FH). Following loss of heterozygosity of FH, HLRCC patients can develop kidney cancer of type 2 papillary morphology that is refractory to current radiotherapy, immunotherapy, and chemotherapy. Identification of targeted therapies against FH-inactivated tumors that limit toxicity to healthy cells remains a critical barrier to HLRCC treatment. In this study, we show that FH inactivation proves synthetic lethal with inducers of ferroptosis, an iron-dependent and nonapoptotic form of cell death. Specifically, we show that reconstitution of FH in an FH-inactivated cell line, UOK262, abrogates ferroptotic cell death induced by erasing, glutamate, and RSL3 drugs. Similarly, knockout of FH in Caki-2 kidney cancer cells sensitized cells to RSL3-induced toxicity. Mechanistically, we show that fumarate accumulation, which arises following FH inactivation, covalently modifies the enzyme glutathione peroxidase 4 (GPX4). Moreover, fumarate accumulation inhibits the capacity of GPX4 to eliminate toxic lipid peroxides, sensitizing the cells to further GPX4 inhibition by ferroptosis inducers. Induction of ferroptosis in FH-inactivated tumors represents a novel opportunity for synthetic lethality in cancer that limits toxicity to healthy tissues.
P120 - Impact of Diet-Induced Obesity on DNA Structure-Induced Mutagenesis in Mice
P. Kompella1, G. Wang1, J. DiGiovanni1, and K. Vasquez1
1The University of Texas at Austin, Austin, TX, USA
Obesity is an increasing global concern with several studies reporting a significantly elevated risk and poor prognosis of various cancers in people with body mass index > 30 kg/m2. Obesity stimulates oxidative stress that can cause DNA double-strand breaks (DSBs) and reduced DNA repair capacity. If rejoined improperly, DSBs result in gene translocations, a common cause of blood cancers such as Burkitt lymphoma (BL). Many BL patients carry chromosomal breakage and translocations at genomic “hot spots.” These “hot spots” are enriched in repetitive DNA sequences that can adopt alternate (non-B DNA) structures, such as H-DNA. Our laboratory has found H-DNA-forming sequences to be intrinsically mutagenic in mammalian cells and mice. Thus, we hypothesize that obesity increases DNA structure-induced genetic instability in vivo. Groups of transgenic mice containing H-DNA-forming sequence (mapping to a breakpoint hot spot in BL) were put on control diet, diet inducing obesity (DIO), or calorie restricted (CR) for 13 weeks. Mutation frequency data (MF) shows that DIO increases the mutagenic potential of H-DNA-forming sequences in obese mice (MF-7.88) compared to mice on control (MF-0.52) and CR diets (MF-0.66). Additionally, detection of a breakpoint hot spot in DIO mice DNA elucidates that obesity influences the formation of H-DNA-induced DSBs leading to large deletions (300-900 bp). The relevance of results to public health is significant as obesity is a known contributing factor to increased cancer risk, and defining the molecular mechanisms underlying the effects of obesity on endogenous mutational “hot spot” sequences will assist us in developing strategies to prevent and/or attenuate the obesity–cancer link.
P121 - THIOMAB Antibody Antibiotic Conjugate (TAC): Blue Discoloration in Rats Seen in Preclinical Safety Assessment Studies
N. Ljumanovic1, N. Stagg1, W. Halpern1, P. Katavolos1, A. Kamath1, M. Carrasco Triguero1, and D. Dambach1
1Genentech, South San Francisco, CA, USA
Staphylococcus aureus (S aureus) is one of the leading causes of bacterial infections in humans with serious complications such as pneumonia, endocarditis, and sepsis. Therapeutic options are becoming limited because S aureus can survive inside cellular organelles not exposed to effective antibiotic levels. Therapeutic approaches targeting intracellular persistence may provide novel treatment options for invasive S aureus infections. Our drug is a THIOMAB antibody antibiotic conjugate (TAC) that consists of a human immunoglobulin (Ig) G1 monoclonal antibody and a potent antibiotic (dmDNA31) against S aureus that is linked through a ValCit linker. In preclinical studies, we evaluated the toxicity of TAC in mice and rats following repeated doses of up to 500 mg/kg. Systemic exposure of TAC was confirmed at all dose levels. Although there was no clear evidence of toxicity in rats and mice, there was transient blue discoloration of serum and plasma at dose levels ≥50 mg/kg and blue discoloration of skin (limbs and snout) at dose levels ≥200 mg/kg that generally resolved within 24 hours. This was attributed to the antibiotic portion of the molecule, which is blue in color. Similar effects have been seen with rifampicin and other compounds in this class (red discoloration of tears, urine, etc) that are monitorable and not associated with adverse clinical effects. The purpose of this poster is to present the preclinical data with TAC and the transient nature of this blue discoloration in mice and rats.
P122 - Tolerability and Efficacy of a Novel HBV Ribonuclease H Inhibitor in Liver Humanized FRG Knockout Mice
K. Long1, E. Lomonosova2, Q. Li2, N. Ponzar2, J. Villa2, E. Touchette1, S. Rapp1, R. M. Liley1, R. M. Buller2, E. Wilson3, J. Bial3, J. Sagartz1, and J. Tavis2
1Seventh Wave Laboratories LLC, Maryland Heights, MO, USA
2Department of Molecular Microbiology and Immunology, The Saint Louis University Liver Center, Saint Louis University School of Medicine, St Louis, MO, USA
3Yecuris Corporation, Tualatin, OR, USA
Although a highly effective vaccine exists, at least 240 million people are chronically infected with hepatitis B virus (HBV), and about 2 billion people worldwide have been infected with the virus. Chronic HBV infection is a major cause of end-stage liver disease, including cirrhosis, liver failure, and hepatocellular carcinoma. Standard therapies inhibit viral replication but rarely eradicate the virus and, therefore, chronic treatment is required. As a result, novel treatments are urgently needed. The liver humanized FRG knockout (huFRG KO) mouse model, in which healthy human hepatocytes are transplanted into immune deficient mice with controllable liver disease, can support chronic infection with HBV, in addition to other hepatotropic infectious pathogens, and is an excellent model for evaluating novel therapies. The advantages of the liver humanized FRG KO mouse model will be discussed in addition to the in vivo testing funnel utilized for evaluating a novel HBV ribonuclease H inhibitor (compound #208). Prior to assessing efficacy in the chronically HBV-infected huFRG KO mouse model, pharmacokinetics, and dosing route selection were evaluated followed by acute and repeat dose tolerability. Once the appropriate dose was determined, efficacy in a chronically infected HBV huFRG KO mouse model was conducted. Treatment with compound #208 led to significant reductions in plasma viremia without affecting plasma HBV surface or envelope antigen levels, and viral titers rebounded following treatment cessation. Entecavir, an HBV polymerase inhibitor, was also tested as a positive control.
P123 - Assessment of Dietary Melamine and Cyanuric Acid in Lactating Dairy Cows: Relevance to Cow Health, Human Health, and Levels in Biological Specimens
C. E. Moore1, B. Puschner1, K. M. Bauer3, H. Knych1,2, L. W. Woods2, S. S. Stump2, and E. J. DePeters3
1Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA
2California Animal Health and Food Safety Laboratory System, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA
3Department of Animal Science, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA
Nephrotoxic melamine and cyanuric acid are used as a nitrogen-boosting contaminates in food products. Outbreaks of nephrolithiasis and acute kidney failure in children have been linked to melamine-contaminated infant formula. Melamine and cyanuric acid-contaminated pet foods resulted in acute renal failure and deaths in dogs and cats. To investigate these toxins’ potential to enter the food chain, the fate of melamine and cyanuric acid and their effects on animal health were studied in lactating cows, orally administered melamine or cyanuric acid at 30 mg/kg, then 300 mg/kg, for 10 days each. After dosing, serum, urine, and milk toxin concentrations were highly correlated with intake for both toxins individually. Dosing with 30 mg/kg of melamine resulted in detectable cyanuric acid concentrations in urine and milk, but not serum. Dosing with cyanuric acid resulted in detectable concentrations of melamine in urine, but not serum or milk. Pharmacokinetic modelling determined the C max, T max, and t 1/2λ of melamine at 30 and 300 mg/kg to be 6,200 and 38,000 ng/mL, 0.969 and 0.167 days, and 2.57 and 2.79 days, respectively, and cyanuric acid at 300 mg/kg to be 22,000 ng/mL, 0.167, days and 12.6 days. There were no demonstrable health effects caused by either toxin, and no pathological changes were identified at necropsy. Melamine and cyanuric acid were not detected in muscle tissues at 30 days postdosing. The results show that cows with no clinical signs can have melamine and cyanuric acid-contaminated milk and in vivo metabolism of both toxins occurs.
P124 - Background Incidence of Spontaneous Arrhythmia in Naive Beagle Dogs
K. Norton1, G. Boudreau1, and N. Sadekova1
1Charles River Laboratories, Montreal, Québec, Canada
Although Jacketed External Telemetry (JET) is widely utilized for continuous quantitative ECG evaluations on repeat-dose toxicology studies, arrhythmia detection is still limited to snapshot assessments. However, with advances in automated arrhythmia detection modules, it is now feasible to conduct comprehensive qualitative ECG review on continuously recorded data sets. One limitation that still remains, however, is a lack of historical data sets on the prevalence of spontaneous arrhythmia in beagle dogs, against which to compare the arrhythmogenic effect of novel compounds. The purpose of this study was to assess the types and incidence of spontaneous arrhythmia in naive dogs. Using JET, with a lead II configuration, ECG evaluations were conducted on 70 beagle dogs over a period of 24 hours. Additionally, repeat assessments were conducted on 20 animals to look for individual variance over time. Arrhythmia assessments were conducted using Data Sciences International Data Insights analysis module. All animals assessed had arrhythmias detected, with 7 types of waveform abnormalities and arrhythmia identified. Junctional escape complexes and premature atrial complexes (PAC) were the most common finding, with 2° atrioventricular block and premature ventricular complexes also observed at a frequent incidence. Arrhythmia levels were highly variable across animals, with some animals noted as having around 300 occurrences of PAC whereas other displayed none. The data set contained in this study confirms ability to continuously evaluate arrhythmias, and demonstrates the need for a solid historical database in order to account for normal animal variability when assessing the arrhythmogenic effect of novel compounds.
P125 - Modulatory Effects of Pterocarpus mildbraedii Extract on Inflammatory Transcription Factors in the Liver of Propanil-Treated Albino Rats
C. Otuechere1,2 and O. Farombi2
1Redeemer’s University, Ede, Osun State, Nigeria
2University of Ibadan, Ibadan, Oyo State, Nigeria
Propanil (PRP) is a highly selective post-emergent contact herbicide used widely in rice production. In light of the evidence that Pterocarpus mildbraedii, a leafy vegetable, possesses antioxidant and anti-inflammatory properties, we investigated the protective effects of a dichloromethane: methanol extract of Pterocarpus mildbraedii (PME) on PRP-induced inflammation in the liver of rats. Four groups of rats containing 6 rats/group received olive oil (control), PRP (200 mg/kg), PME (200 mg/kg), and PME (200 mg/kg) + PRP (200 mg/kg) orally, once daily, for 7 days, respectively. Expressions of phospho-nuclear factor-κB (pNF-κB) p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were measured by immunohistochemistry. Levels of myeloperoxidase (MPO), nitric oxide (NO), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), signal transducer and activator of transcription (STAT-3), p38 mitogen-activated protein kinase (MAPK), inhibitory-κB-α, and NF-κB p65, were also determined using ELISA kits. The results showed that PME significantly reduced PRP-induced increases in MPO, NO, p38 (MAPK), and STAT-3. Immunohistochemical studies also confirmed that PME significantly decreased the expressions of NFκB, iNOS, and COX-2 when compared with the group treated exclusively with PRP. Based on these findings, it is concluded that PME could contribute to reduction of inflammatory responses in PRP-intoxicated rats through modulation of transcription factors.
P126 - Comparison of Routine Toxicology Parameters Between the Göttingen and the Chinese Bama Minipig
L. Pan1, S. McPherson1, R. Wu1, M. Chen1, and T. Zhou1
1Wuxi AppTec, Suzhou, China
The minipig is often used in regulatory preclinical toxicology studies as the choice of the nonrodent species. In the United States and Europe, the Göttingen minipig is often selected, while in China the Bama pig is usually the strain of choice, both being accepted strains by the US FDA. Hemodynamic and electrocardiographic parameters, clinical pathology, and organ weight (adjusted to body weight) data of control animals from toxicology studies using the Bama at Wuxi AppTec Suzhou were compared to published data for the Göttingen minipig of similar age. The results from the data sets were analyzed using the Göttingen minipig as reference control point. Analysis of the hematology data showed slightly higher white blood cell count (WBC), neutrophils, basophils, eosinophils, and increased platelet counts for the Bama pig when compared to the Göttingen minipig. Differences in clinical chemistry were also evaluated and included an increased total protein/globulin, creatinine, reduced urea, and total cholesterol when compared to the Göttingen minipig. There were some differences in mean systolic and diastolic pressures with the Bama pig being slightly higher than Gottingen, which also had a higher baseline heart rate and minor differences in quantitative ECG parameters; organ weight data analysis showed that the overall weights for the data Göttingen minipig were generally toward the lower to medium range of that of the Bama pig of comparable age. These observed differences between the 2 strains of minipig enforce the need to be consistent in the strain of animal for a toxicology development program.
P127 - Spleen, Thymus, and Mandibular and Mesenteric Lymph Node Immunophenotyping in Sprague Dawley Rats
A. Phadnis-Moghe1, A. Rowse1, P. Brewer1, J. Olsen1, J. Puchalski1, and A. Frantz1
1Covance, Madison, WI, USA
Tissue immunophenotyping (IPT) by flow cytometry is routinely used for assessing immune-modulatory effects of test articles in toxicology studies. Methods for tissue IPT in rats were re-evaluated to include not only an assessment of immune cell populations and measurements of accuracy, precision, and stability but also CD4:CD8 ratio, sum of the relative percentages of the T cells (TSUM) and the sum of lymphocytes (LSUM), to provide possible acceptance criteria. IPT was conducted using a heterogeneous lymphocyte gating strategy along with the incorporation of a viability dye. Precision measurements included comparisons of cranial and caudal sections of the spleen, left and right aspects of the thymus, bilateral mandibular lymph nodes, and 2 sections of mesenteric lymph nodes. The inter-animal variability was acceptable using automated and manual methods for tissue dissociation and the IPT values were comparable. Slight differences were noted in the cellularity between the caudal and cranial sections of the spleen, left and right section of the thymus. The overall assay precision was acceptable with CV ≤10%, with higher variability noted as expected in the less frequent NK cells in the lymph nodes and early T cells in the thymus. Stability up to 72 hours for fixed samples and up to 24 hours for single-cell suspensions was noted for all tissues. In conclusion, a method was derived for measurement of lymphocyte subsets in rat tissue and acceptance criteria based on LSUM and TSUM that provides a robust method to evaluate changes in immune cell populations in rat tissues.
P128 - Multigenerational Consumption of a Western Diet for Rodents Promotes Colitis-Associated Colorectal Cancer in Third-Generation Offspring
S. Phatak1, K. Hintze1, R. Ward1, and A. Benninghoff1
1Utah State University, Logan, UT, USA
Colon cancer is the second leading cause of cancer-related death in the United States, claiming nearly 50,000 victims per year. Despite 70% to 90% of all incidence being attributed directly to diet, Americans routinely consume highly processed foods that are energy dense and nutrient-poor. The primary objective of this study was to determine the impact of transgenerational or multigenerational consumption of the total western diet (TWD), a Western-style diet formulated for rodents using human US nutrient intake data, in a murine model of inflammation-associated colorectal carcinogenesis. The hypotheses tested were (1) transgenerationally feeding TWD will promote colitis-associated colorectal cancer (CAC) in F3 offspring; and (2) cumulatively feeding TWD would further exacerbate disease development. C57BL/6J mice were bred for 3 generations, during which they were fed either a standard diet (AIN93G) or TWD during the F0 generation only, for the duration of F0 through F3 generations, or the F3 generation only. The azoxymethane and dextran sodium sulfate model of CAC was employed in F3 offspring, necropsied at 24 weeks. Notably, tumor incidence was increased by transgenerational exposure to TWD (92%) when compared to consecutive AIN93G exposure only (56%). Moreover, successive exposure to TWD markedly increased tumor burden (>3-fold increase) when compared to direct TWD exposure. In summary, ancestrally feeding TWD increased CAC incidence and disease severity in third generation offspring that were not directly fed this diet, and continuously feeding TWD for 3 generations distinctly exacerbated disease outcome in the third generation offspring.
P129 - Inhibition of Carboxylesterase Enzymes by Isopropylated Triarylphosphate Ester Flame Retardants: Implications for Pharmacotherapy and Cardiovascular Disease
A. Phillips1 and H. Stapleton1
1Duke University, Durham, NC, USA
Despite widespread human exposure and documented neuro- and developmental toxicity, there is currently little information regarding the fate of isopropylated triarylphosphate esters (ITPs) in the human body (Hammel et al, 2016; Behl et al, 2015). ITPs are used as plasticizers and flame retardants in residential couch cushioning (McGee et al, 2013; Phillips et al, 2016). Due to their bioaccumulative and potentially toxic properties, the EPA prioritized their risk assessment under the Frank R. Lautenberg Chemical Safety for the 21st Century Act, with a deadline for action of June 22, 2019 (US EPA, 2016). We have shown that rats orally dosed with 1,000 µg/d of an ITP-containing flame retardant mixture had significantly reduced hepatic carboxylesterase (Ces) activity (62% decrease) compared to controls. Importantly, triphenyl phosphate (TPHP), a structural analog of ITPs, irreversibly binds to the catalytic serine of Ces enzymes, rendering them permanently inactive (Morris et al, 2014). In the present study, in vitro Ces inhibition by individual ITP isomers present in commercial flame retardant mixtures found in common household products is assessed. Because hepatic Ces activity is known to be crucial for the bioactivation of imidapril, an angiotensin-converting enzyme inhibitor (ACE inhibitor) used in the treatment of chronic heart failure, the in vitro metabolism of imidapril is investigated in the presence and absence of various ITP isomers. Imidapril has a responder rate of approximately 50%, affected by polymorphisms of the CES1 gene (Huang et al, 2001). As such, Ces inhibition by ITPs could have a profound impact imidapril pharmacotherapy.
P132 - Suboptimal KLH T-Dependent Antibody Response Assay Dose Evaluation in Cynomolgus Monkey and Sprague Dawley Rats
A. Repic1, M. Leitermann1, J. Olsen1, J. Puchalski1, and A. Frantz1
1Covance, Madison, WI, USA
The TDAR assay has become a standard approach to assess immune function. The successful TDAR in any species is dependent on multiple functional immune processes. An “optimal” dose determined to maximize antibody production yet avoid unwanted effects on standard toxicology study endpoints has been used to monitor immunosuppression. Selection of a “suboptimal” dose for monitoring of test articles that might enhance immune response was desired, and monitoring of endotoxin levels in the KLH reagent was also determined. Eighty total SD rats (10/sex/group) were administered KLH at 0, 30, 100, or 300 μg/animal and 32 cynomolgus macaques (4/sex/group) administered 0, 0.5, 2.5, and 10 mg/animal twice, 29 days apart. Endotoxin levels in the doses administered ranged from levels below detection to near the recommended limit of 5 EU/kg, depending on the dose concentration. Administration of the high dose in both species was considered to have generated an ideal and optimal anti-KLH IgM and IgG primary and secondary response. The mid and low doses produced robust but dose-dependently and quantifiably lesser anti-KLH IgM and IgG response, and a delay in the kinetics of the response noted in rats especially when administered the low dose of 30 μg/animal/dose. Limited individual variability in anti-KLH IgM and IgG primary and secondary response was considered of potential relation to the endotoxin levels of the KLH reagent used, and highlights the importance of monitoring this variable when employing the TDAR. A suboptimal dose capable of detecting enhanced immune response resulting from immunostimulatory test articles was identified in both species.
P133 - Development of a Fixable Flow Cytometry-Based Natural Killer Cell Cytotoxicity Assay
A. Rowse1, J. Mutschler1, A. Phadnis-Moghe1, H. Chu1, J. Olsen1, J. Puchalski1, and A. Frantz1
1Covance, Madison, WI, USA
The natural killer cell (NKc) assay is an important tool for innate immune system functional assessment that has been commonly performed in support of standard toxicology studies by Chromium-release assay for more than 30 years. To remove radioactivity and enhance assay assessment capabilities, we developed a fixable flow cytometry-based assay that measures NK cytotoxicity using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques and fluorescently-labelled K562 as MHC I-deficient target cells. Due to the known biological variability of primates, especially with regard to NK activity, multiple effector: target (E: T) ratio and coculture lengths were evaluated. We determined that with 4 E:T ratios (50:1, 25:1, 12.5:1, and 6.25:1) and a short (2 hours) coculture, the viability of K562 target cells (assessed at each E: T using either propidium iodide (PI) or a fixable viability dye to identify dead cells) was sufficiently for robust generation of lytic unit calculations as have been used in the chromium-release assay. The samples stained with PI were acquired immediately (live) and those stained with a fixable viability dye were fixed with paraformaldehyde before acquisition on the cytometer. We found that the traditional live assay and the fixed assay produced comparable percent cytotoxicity as well as lytic unit at which 50% cytotoxicity is observed (LU50). Fixation of this coculture assay is important as it allows for increased numbers of samples to be assayed and will allow for future addition of assay components such as NK activation and degranulation markers to partner with the traditional NKc functional assay.
P135 - Development of an Intracameral Injection Technique and Ocular Examination for Beagle Dogs
J. Laporte1, R. Verhoeven2, and W. Ruddock1
1ITR Laboratories Canada, Montreal, Québec, Canada
2Envisia Therapeutics, Durham, NC, USA
The objective of the study was to set up an intracameral administration method along with ocular examination procedures and aqueous humor collection in beagle dogs. Prior to the injection, animals were sedated using intramuscular medetomidine and isoflurane. A reversal agent was provided after the injection. The eyes were flushed with povidone and saline solution prior to the injection. The needle was inserted into the cornea at 1 mm from the limbus and aimed toward the central anterior chamber space. A dose volume of 20 mL of saline was administered in each eye. Slit lamp and indirect ophthalmoscopy examination, pupillometry, tonometry, pachymetry, and specular microscopy of the right eyes were measured post-dose and an aqueous humor collection was performed 3 days after the injection from the left eye. The intracameral injection was well tolerated with minor findings noted related to the injection procedure. The intraocular pressure was 23.0 ± 2.1 mm Hg, the pupil diameter was 9.4 ± 0.71 mm, and the mean corneal thickness was 618 ± 51 mm measured 3 to 15 days after the injection. The cell density of the central corneal was 2,574 ± 70.0 cell/mm2 with 73% ± 0.7% of hexagonal cells and the cell density of the mean central inferior cornea was 2,606 ± 163 cell/mm2 with 69% ± 3.5% of hexagonal cells. An aqueous humor sample was successfully collected. In conclusion, a single intracameral administration, ocular examinations, and aqueous humor collection were successfully performed and well tolerated in beagle dogs.
P137 - FGFR Signaling During Human Hepatic Stellate Cell Activation
J. Schumacher1, B. Kong1, J. Wu1, J. Shen1, and G. Guo1
1Rutgers University, Piscataway, NJ, USA
Regulation of hepatic stellate cell (HSC) phenotype and proliferation plays a key role in the prevention and development of liver fibrosis. Our laboratory has previously shown that deficiency of fibroblast growth factor 15 (FGF15; FGF19—human homologue), an FXR-regulated endocrine FGF produced in the ileum, was protective against liver fibrosis in a high fat diet (HFD)-induced nonalcoholic steatohepatitis (NASH) mouse model. Despite the observed decrease in fibrosis in FGF15-deficient mice with NASH, there was severe steatosis and inflammation indicating that FGF15/19 may directly activate HSCs. Therefore, it was the aim of this study to investigate the mechanisms by which FGF15/19 affects HSC phenotype and proliferation. A human HSC line, LX-2, was treated with recombinant FGF19 protein and effects on activation of downstream signaling pathways and HSC phenotype were determined. LX-2 cells were also serially passaged to allow for transdifferentiation into a more activated phenotype. Expression of FGFR4, FGFR1, and β-KLOTHO, the receptors for FGF15/19, were determined in unpassaged and passaged cells. We found that FGFR1, FGFR4, and β-Klotho are all upregulated in the activated, passaged cells.
P138 - Does Balanopreputial Separation Have Effects on Fertility and Embryo–Fetal Development?
H. L. Jordon1, D. A. Donahue1, M. D. Haas1, A. C. Sellers1, W. B. Lawrence1, C. D. Qualls1, and H. A. Staudner1
1Covance Laboratories Inc, Greenfield, IN, USA
Current literature indicates there is a background incidence of female rats that fail vaginal opening and therefore may have difficulty or an inability to successfully mate. Balanopreputial separation is considered complete when the entire circumference of the prepuce can be retracted. If a line or an attachment is still present on the ventral surface, the animal is considered to fail balanopreputial separation. No data exist for determining whether males that fail balanopreputial separation can successfully mate or sire offspring or whether a failure in balanopreputial separation is congenital. Therefore, this pilot study investigated these effects on males that failed balanopreputial separation when mated to naive females. Three males that did and did not pass balanopreputial separation, each, were paired twice. Females in pairing 1 were allowed to litter out, F1 pups were culled to males only on PND 4 and starting on PND 40 F1 males were examined for completion of balanopreputial separation. Females in pairing 2 were euthanized and c-sectioned on gestation day (GD) 21 to evaluate macroscopic effects, uterine and cesarean section parameters, and fetal weights and observations. All males were euthanized with collection of reproductive tissues for histopathology. Results indicated that utilization of males that failed balanopreputial separation did not result in any adverse effects on fertility or fecundity in either pairing trial (final F1 balanopreputial separation and pathology-pending).
P140 - Time Course of Chlorpyrifos-Induced Neurogenic Inflammation and Airway Hyperreactivity in Rats
F. Shaffo1, A. C. Grodzki1, E. Schelegle1, and P. Lein1
1University of California, Davis, Davis, CA, USA
Occupational and environmental exposures to organophosphorus pesticides (OP) are associated with increased asthma incidence. Although the canonical mechanism of OP neurotoxicity is acetylcholinesterase (AChE) inhibition, we previously demonstrated that the OP chlorpyrifos (CPF) causes airway hyperreactivity (AHR) in adult rats 24 hours after exposure to levels that do not significantly inhibit AChE. We further investigated the persistence of this effect and whether neurogenic inflammation may be involved. Eight-week old male Sprague Dawley rats were administered 30 mg/kg CPF subcutaneously, and AHR was assessed at 24, 48, 72 hours, or 7 days postexposure using a Flexivent mechanical ventilator during electrical stimulation of the vagus nerves. Dorsal root ganglion (DRG) collected at the conclusion of physiological assessments were immunostained for inflammatory neuropeptides substance-P (SP) and calcitonin gene- related peptide (CGRP). Lungs, cerebella, and peripheral blood were collected from a separate cohort and assayed for AChE activity. CPF significantly increased airway resistance at 24 hours and 7 days postexposure, but not at 48 or 72 hours. SP and CGRP expression was elevated at all time points postexposure. AChE inhibition was significant only in the lung at 24 hours. These data indicate that CPF-induced AHR may occur through different mechanisms, with AChE inhibition driving AHR at early time points and neurogenic inflammation driving delayed AHR at 7 days postexposure. These findings may have significant implications for assessing the risk to human health and safety of CPF, and potentially other OPs. This work was supported by the NIH (grants R01 ES017592 and T32 HL07013).
P142 - Modulation of Ethanol Negative Impacts on Testes and Reproductive Function of Alcoholic Rats Treated With Antituberculosis Drugs
G. Shayakhmetova1, L. Bondarenko1, V. Rushchak2, and V. Kovalenko1
1SI Institute of Pharmacology & Toxicology NAMS of Ukraine, Kyiv, Ukraine
2SI Institute of Molecular Biology & Genetics NAS of Ukraine, Kyiv, Ukraine
Association between alcoholism and tuberculosis has long been known. Taking into account that both alcohol and antituberculosis drugs (ATD) could have negative impacts on male gonads, increase in adverse effects is probable. This study aimed to determine the combine effects of long-term alcohol consumption and ATD administration on rat testes parameters, male reproductive capacity, and antenatal development of their posterity. Wistar male rats were divided into groups: (1) control (intact), (2) chronic alcoholism, and (3) chronic alcoholism + ATD (ethambutol, isoniazid, rifampin, and pyrazinamide) administration. CYP2E1 mRNA and protein expression in alcoholic and alcoholic ATD-treated rats’ testes increased 3.6- to 5.9- and 1.2- to 1.4-folds as compared with control. In testes of control group, only 2 fractions of DNA fragments were present, while under alcohol administration their number increased to 4. In testes of alcoholic ATD-treated rats, 6 fractions of DNA fragments with weights over 1,000, 500, 300, 200, and 150 bp were present. The revealed disorders at testicular genome and proteome levels have had negative implications on spermatogenic epithelium parameters, sperm count, male fertility, and surviving of their offspring. In alcoholic ATD-treated rats, sperm number was lower than in control and alcoholics (2.4- and 1.6-folds, respectively). Fertility indexes in control and alcoholic groups were 93% and 71%, but in ethanol with ATD-treated group only 29%. The total embryonal/fetal death in alcoholic group increased 22% in comparison with control, in alcoholic with ATD coadministration group it was 100%. Thus, ATD administration modulated the impact of long-term alcohol consumption on testes and aggravated paternal-mediated negative effects on posterity.
P143 - Drug-Induced Vascular Degeneration and Intimal Proliferation in Sprague Dawley Rats: Histopathology and Mechanistic Investigation
M. Sonee1, B. Singh1, B. Damiano1, V. Urmaliya1,2, H. K. Lim1, I. R. Jennifer1, M. Duvall1, M. Kelly1, C. Louden1, and R. Mamidi1
1Janssen Research and Development LLC, Spring House, PA, USA
2Janssen Research & Development, Beerse, Belgium
The purpose of this study was to assess the potential toxicity of an investigational anticancer therapeutic agent when administered orally to male Sprague Dawley rats at 0, 10, 30, 50, and 70 mg/kg/d (vehicle, 20% hydroxypropyl-β-cyclodextrin) for up to 14 days. The following parameters were evaluated: clinical observations, body, and organ weights, toxicokinetics, clinical pathology, and macroscopic and microscopic pathology. Increases in exposure were dose-proportional from 10 to 50 mg/kg/d and less than dose-proportional from 50 to 70 mg/kg/d, and mean brain concentrations were higher than the respective plasma concentration. The 70 mg/kg/d rats were euthanized on day 8 due to CNS signs (dragging of hind limbs and posterior paralysis). Decreased grip strength was noted at 50 and 70 mg/kg/d. Macroscopically at 70 mg/kg/d, edema was noted in the hind limb. Microscopically at 30 to 70 mg/kg/d, degeneration/necrosis of the muscular wall, perivascular infiltration of mononuclear cells, and enlarged endothelial cells were observed in small arteries of several tissues and were attributed to hypertension. At 50 and 70 mg/kg/d, degeneration of small arterial wall and demyelination were present in the sciatic nerve, and demyelination was considered secondary to the vascular lesions. Separate safety pharmacology studies revealed off-target NPY1 agonist activity in vitro, and slight elevation of mean arterial blood pressure in conscious telemetered rats at 70 mg/kg/d. Thus, the compound’s secondary vascular pharmacology, potentially mediated by NPY1 activation, vasoconstriction, and blood pressure increase, may provide a mechanistic basis for the vascular findings at high doses.
P144 - Identifying the Human Cytochrome P450 Isoforms Involved in the Enantioselective Metabolism of 2,2′,3,4′,6-Pentachlorobiphenyl (PCB 91)
E. Uwiman1 and H. J. Lehmler1
1Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA, USA
Exposure to chiral polychlorinated biphenyls (PCBs) has been associated with neurodevelopmental disorders. Their hydroxylated metabolites (OH-PCBs) are also chiral and potentially toxic to the developing brain. Because the formation of OH-PCBs by human P450 isoforms is poorly investigated, we investigated the hypothesis that biotransformation of PCB 91 by human liver microsomes is enantioselective and mediated by different CYP isoforms. Racemic PCB 91 was incubated with pooled (pHLM), individual donor (iHLM) human liver microsomes or recombinant human CYP1A2, CYP2A6, CYP2B6, CYP2E1, or CYP3A4 at 37°C. Incubations with all HLM preparations resulted in the formation of 2,2′,3,4′,6-pentachlorobiphenyl-5-ol (5-91), 2,2′,3,4′,6-pentachlorobiphenyl-4-ol (4-91), 2,2′,4,4′,6-pentachlorobiphenyl-3-ol (3-100; 1,2 shift product) and 4,5-dihydroxy-2,2′,3,4′,6-pentachlorobiphenyl (4,5-91) from racemic PCB 91, with a rank order of 3-100 > 5-91 >> 4-91 >> 4,5-91. The first-eluting atropisomer of PCB 91 was enriched at low PCB concentration. The formation of the first-eluting atropisomer of 3-100 was nearly enantiospecific. The second-eluting atropisomer of 5-91 and the first-eluting atropisomer of 4-91 were enriched in all microsomal preparations investigated. CYP2A6 and CYP2B6 were the major CYPs involved in PCB 91 metabolism. CYP2A6 mainly formed 3-100, while 5-91 and 4-91 were minor metabolites. CYP2B6 almost exclusively formed 5-91, with 4-91 being a minor metabolite. As with HLMs, the formation of OH-PCBs by CYP2A6 and CYP2B6 was enantioselective. These findings suggest that there are inter-individual differences in the enantioselective biotransformation of PCB 91 to the corresponding OH- PCBs in humans, possibly due to differences in levels of CYP2B6 versus CYP2A6.
P145 - Bioanalytical Analysis of Sunitinib Microparticles for Ocular Administration
J. Vance1, B. King1, R. Lappin1, C. Crean2, W. Peterson2, J. Cleland2, and R. Shoup1
1AIT Bioscience, Indianapolis, IN, USA
2Graybug Vision, Redwood City, CA, USA
P146 - Enhanced Histone Acetylation Upregulates MDR1 and BCRP Transporters in Human Blood–Brain Barrier Cells
D. You1, X. Wen2,3, A. Morris1, J. R. Richardson4, and L. M. Aleksunes2,3
1Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ, USA
2Environmental and Occupational Health Sciences Institute, Rutgers University, Piscataway, NJ, USA
3Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ, USA
4Northeast Ohio Medical University, Rootstown, OH, USA
Multidrug resistance protein 1 (MDR1, ABCB1) and the breast cancer resistance protein (BCRP, ABCG2) expressed at the blood–brain barrier (BBB) are key efflux transporters that regulate the efficacy and/or toxicity of chemicals in the brain. Prior studies in cancer cells have pointed to the ability of histone deacetylase (HDAC) inhibitors to modulate the expression and function of MDR1 and BCRP. However, whether such regulation occurs at the BBB is unknown. In this study, we sought to test whether HDAC inhibitors could alter expression and function of MDR1 and BCRP at the BBB. To test this, we treated immortalized human brain capillary endothelial (hCMEC/D3) cells, a model of the BBB, with 6 HDAC inhibitors, valproic acid (VPA), sodium butyrate, romidepsin, apicidin, suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA), and assessed for expression and function of MDR1 and BCRP. HDAC inhibition following treatment was confirmed by increased levels of acetylated histone H3 protein. At 12 hours, VPA, apicidin, SAHA, and TSA upregulated MDR1 mRNA levels up to 200%. All 6 HDAC inhibitors significantly induced BCRP mRNA levels between 100% and 270%. Similarly, the protein expression of MDR1 and BCRP transporters was upregulated about 2-fold at 24 hours. Enhanced MDR1 expression corresponded with reduced intracellular accumulation of the substrate rhodamine 123. Collectively, these results demonstrate that HDAC inhibitors upregulate MDR1 and BCRP transporters at the BBB by histone modification. The clinical use of HDAC inhibitors may enhance efflux transporter activity at the BBB and restrict access of xenobiotics to the brain.
P147 - Oral Cyclosporine A Immunosuppressive Therapy Leads to Systemic Toxicity in the Royal College of Surgeon Rats
L. Zhang2, Y. S. Andrews-Swilling1, D. Bates1, T. Madsen2, Y. H. Ann-Fu3, J. R. Wicks3, D. White2, C. Hanks2, A. Stricker-Krongrad2, G. Bouchard2, and J. Liu2
1SanBio, Inc., Mountain View, CA, USA
2KCAS LLC, Shawnee, KS, USA
3Alizée Pathology, Thurmont, MD, USA
Twenty-four male and 24 female RCS rats were randomized into 2 groups with or without cyclosporine A (CsA) in drinking water. At 28, 56, 91, and 118 days, 6 rats per gender were euthanized. Growth, clinical pathology parameters, along with organ weight and histopathology evaluations were performed at the termination. Blood CsA concentrations were analyzed by LC-MS/MS at termination. Cyclosporine A was offered in drinking water at 210 mg/L, an immunosuppressive therapeutic level recommended by published literature. Blood CsA concentrations ranged from 1,670 to 6,310 ng/mL. This range was much higher than the values reported in the literature. Systemic CsA toxicities included decreased body weight, increased ALP, and BUN, and nephropathy.
P148 - Investigating Dose Fractionation Regimen to Mitigate Toxicity Associated With PBD-Containing Antibody Drug Conjugates in Rats
F. Zhong1, M. Schutten1, B. Latifi1, D. Leipold1, T. Lin1, J. Corpuz1, H. Wang1, and N. Stagg1
1Genentech, South San Francisco, CA, USA
PBD (pyrrolobenzodiazepine) is an alkylating agent that has been tested in the clinic for oncology indications. The main drug-related adverse events in patients treated with free PBD dimers or PBD-containing antibody drug conjugates (ADCs) are vascular leak syndrome and reversible hepatotoxicity (Hartley, 2011). In nonclinical PBD-ADC toxicology rat studies, the dose-limiting toxicity was body weight loss, bone marrow hypocellularity, and vascular leak-like syndrome (VLS), characterized by face or limb swelling. To investigate whether PBD-ADC-associated toxicities can be potentially mitigated by reducing C max while maintaining AUC, a dose fractionation study was conducted in rats following intravenous administration of anti-Her2-SG3203. Anti-Her2-SG3203 is an ADC synthesized through a cleavable linker to a potent PBD dimer. Animals were split into 2 groups, one given a single dose of 6 mg/kg to elicit VLS and the other given a fractionated dose of 1.5 mg/kg weekly for 4 doses. Toxicokinetic results showed that the fractionated dose reduced C max by 75%, while achieving overall exposure equivalent to that of a single 6 mg/kg dose. A single dose of 6 mg/kg was not tolerated; animals developed VLS and were euthanized early. Weekly dose of 1.5 mg/kg was associated with delayed onset of body weight loss, overt clinical signs, and VLS; however, it also resulted in mortality or early euthanasia. The affected target organs and the severity of the findings were comparable in both groups. In conclusion, under the conditions of this study, the dose fractionation regimen did not improve the overall safety profile compare to the single-dose administration.
200 Series—Regulatory Toxicology
P201 - In Silico Cardiomyocyte Action Potential Modeling Using the O’Hara-Rudy Model With GLP Manual Patch Clamp Inhibition Profiles From CiPA Ion Channels
S. Authier1,2, H. Huang1, M. Pugsley3, M. Accardi1, V. Jacquemet4, and R. Forster1
1CiToxLAB, Laval, Québec, Canada
2Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Québec, Canada
3Department of Toxicology, Purdue Pharma L.P., Stamford, CT, USA
4Département de Physiologie Moléculaire et Intégrative, Faculté de Médecine, Université de Montréal, Montreal, Québec, Canada
P203 - SEND in Your Submission
M. Bausman1, G. Hawkins2, and C. Sawczak2
1Covance Inc, Madison, WI, USA
2Covance Inc, Princeton, NJ, USA
Now that clearing the initial hurdle of implementing SEND (Standard for the Exchange of Nonclinical Data) is behind us, the focus is now on overcoming the next hurdle—the eCTD submission details for submission to the US FDA via the ESG (electronic submissions gateway). Today, only general toxicology and carcinogenicity studies conducted after specific dates have the requirement to have data for submissions presented in both the SEND format and the study report format. Because the requirement applies only to select studies, each submission will have a unique blend of legacy, in-scope and out-of-scope studies with their corresponding files types that must reside in the appropriate folder of the submission. Getting this right is paramount to avoiding delays and/or technical rejection. Guidance on where to place particular files is available in section 7 of the Study Data Technical Conformance Guide. With the looming requirement to include SEND data sets in IND submissions, and following this guide, Covance has prepared a trial submission representative of a typical IND submission that establishes our recommendation in order to achieve the optimal arrangement of files from various studies.
P204 - Revised Draft EMA Guidance on Early Clinical Trials: Potential Impacts and Industry Response
L. Butler1,2, P. Guzzie-Peck2,3, J. Hartke2,4, M. Bogdanffy2,5, Y. Will1,2, D. Diaz6,11, E. Mortimer-Cassen2,7, M. Derzi2,8, N. Greene2,9, J. DeGeorge2,10, T. Monticello2,11, and V. Kadambi2,12
1Pfizer Worldwide Research and Development, Groton, CT, USA
2International Consortium for Innovation and Quality in Pharmaceutical Development/DruSafe, Washington, DC, USA
3Janssen Research & Development, Springhouse, PA, USA
4Celgene Corporation, Nonclinical Development, San Diego, CA, USA
5Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, CT, USA
6Denali Therapeutics, San Francisco, CA, USA
7Astra-Zeneca Innovation Medicines and Early Development, Hertfordshire, United Kingdom
8Pfizer Worldwide Research and Development, Cambridge, MA, USA
9Astra-Zeneca Innovation Medicines and Early Development, Waltham, MA, USA
10Merck & Co, Inc MRL, West Point, PA, USA
11Comparative Biology and Safety Sciences, Amgen, Thousand Oaks, CA, USA
12Nonclinical Development Sciences, Blueprint Medicines, Cambridge, MA, USA
In November 2016, the EMA released draft guidance for sponsors planning early clinical trials with investigational pharmaceuticals, in response to a recent clinical study in which healthy volunteers experienced unexpected serious neurologic toxicity, including death (Kerbrat et al, 2016). In the first half of 2017, sponsors have received queries on EU Clinical Trial Applications that align with the revised draft guideline. Through the International Consortium for Innovation and Quality (IQ) and an EU pharmaceutical trade organization (EFPIA), industry has collaborated with EMA regulators, providing comprehensive, science-based written comments on the draft guideline. Further, a face-to-face workshop provided a forum for joint discussion of potential impacts on pharmaceutical development, and risk-based guideline revisions were proposed. In parallel, IQ/DruSafe member companies recently published a manuscript on their nonclinical approaches to progress small molecules to early clinical trials, concluding that following current nonclinical practices support clinical trial participant safety while advancing in the development of important new medicines (Butler et al, 2017). This conclusion is reinforced by an IQ/DruSafe nonclinical to clinical translational database indicating that absence of toxicity in animal studies predicts similar clinical outcomes (manuscript in preparation). All stakeholders agree that in cases where there is higher uncertainty regarding nonclinical to clinical translation, a more conservative approach to clinical trial design is warranted. Given the rapid evolution of pharmaceutical safety evaluation, health authorities and industry must continue to jointly engage to ensure that testing paradigms and regulatory guidance evolve in line with emerging science without impeding development of new medicines.
P205 - Derivation of a Maximum Concentration Limit (MCL) for Grain and Flour Dust
M. Chappel1, K. Haines1, L. Neil2, and F. DiGiovanni2
1NovaTox Inc., Guelph, Ontario, Canada
2AirZone One Inc, Mississauga, Ontario, Canada
Dust arising from grain and flour can be an important inhalation hazard in facilities that handle those bulk substances, and various agencies have derived occupational exposure limits (OELs) to protect worker health following inhalation of grain and flour dusts. Less well characterized is the potential health hazards to members of the general public that live in proximity to grain and flour handling facilities. To date, most jurisdictions appear to have taken the position that ambient air quality guidelines for particulate matter (PM2.5 and PM10) are sufficient to be applied directly to grain and flour dusts, however, these guidelines are based on different sources (eg, combustion by-products) and the endpoints may differ (eg, lung cancer). Generic PM guidelines may potentially be insufficiently or overly protective, as there are important physical, chemical, microbiological, and toxicological differences between dusts derived from organic sources compared to particulates derived from traditional PM sources. An endpoint of concern remains immediate- and delayed type 1 immunological responses (through IgE). A “maximum concentration limit” (MCL) to ensure protection of the general population was derived that could be applied to both grain and flour dusts; the approach proposed accounts for the various size fractions that may be emitted from these facilities and for which members of the general population may be exposed to.
P206 - ToxBox: A New Approach for Evaluating Anthropogenic Trace Substances in Drinking Water
A. Eckhardt1, T. Grummt1, M. Kramer2, T. Braunbeck3, and H. Hollert4
1German Environment Agency, Bad Elster, Germany
2RheinEnergie, Cologne, Germany
3Center for Organismal Studies, University of Heidelberg, Heidelberg, Germany
4Institute for Environmental Research, Aachen University, Aachen, Germany
Due to improvements in analytics, increasing numbers of substances were found in drinking water. The project ToxBox aimed at developing a test battery, allowing a rapid evaluation of substances in water. Attention was focused on genotoxic, neurotoxic, and endocrine effects, which are considered to be of most concern to the consumer. By the end of the project, guidelines are published describing the analytical methods. ToxBox was based on a theoretical concept, called “health-related indicator value,” which was developed by the German Environment Agency (UBA) for assessing substances with incomplete toxicological data. During the years, increasing amounts of substances were found in drinking water, requiring an evaluation offering rapid assessment of chemicals for which no data are available. In ToxBox, this was provided for genotoxicity, neurotoxicity, and endocrine effects. A hierarchic strategy is applied that enables a first assessment via relatively simple assays and only when these tests hint toward an effect, more tests are applied. Ames, Umu, and micronucleus assay form the panel for genotoxicity testing. Neurotoxicity will be assessed by comparing cytotoxic effects and the development of reactive oxygen species in human nervous versus liver cells. Additionally, neuron specific assays such as the neurite outgrowth test are performed. This is complemented by measuring the activity of acetyl choline esterase and the development of the side line organ in zebra fish. The test battery for endocrine effects consists of hormone specific reporter gene assays in addition to the H295R assay. During the project, some 60 substances were evaluated, allowing for a reliable test strategy.
P207 - Immunotoxicological Assessments in Juvenile Rats
J. Godin-Ethier1, J. Leiva1, M. Toneva1, F. Leroux1, I. Shaikh1, and A. Nelson1
1ITR Laboratories Canada; Baie d’Urfe, Montreal, Québec, Canada
The emergence of novel immunomodulatory therapeutics has brought more attention to the evaluation of potential adverse effects on the immune system, including the developing immune system. The T-dependent antibody response (TDAR) is considered as the main functional test to address immunotoxicity in nonclinical setting; however, assay designs need optimization for use in younger animals. In this study, keyhole limpet hemocyanin (KLH), selected as the T-cell-dependent antigen, was used to immunize juvenile rats. Cyclophosphamide (CPA) served as a positive control for immunotoxicity evaluation and demonstration of assay design appropriateness. Twenty-two days old rats were injected for 42 days with CPA at 0, 2, 4, or 6 mg/kg/d. Primary and secondary KLH immunizations were given on days 7 and 35. Anti-KLH antibody response was evaluated by ELISA from serum samples collected prior and following each immunization. Complementarily, immunophenotyping was performed on terminal blood samples to evaluate immunotoxic effects on lymphocyte subsets. An anti-KLH IgG response was detected following both immunizations, although the secondary challenge produced a more robust response. CPA treatment at 6 mg/kg/d significantly inhibited the anti-KLH response for both sexes on all occasions. Inhibition was also achieved at 4 mg/kg/d only for the secondary response, suggesting that the secondary immunization provides a more sensitive assessment of immunosuppression by CPA. Reduction in T lymphocytes was achieved with CPA at 2 mg/kg/d, and at 4 mg/kg/d for B lymphocytes and NK cells. The identification of a well-tolerated dose of CPA which induced significant immunosuppression will support future immunotoxicology studies in juvenile rats.
P208 - BioCelerate Toxicology Data Sharing Initiative: Development of a Centralized, Searchable Preclinical Data Repository for the Biopharmaceutical Industry
M. Graziano1, E. Vock2, G. Kahlbaugh3, W. Houser4, C. Fish5, T. Page6, T. Bjerregaard7, K. Mera8, T. Fukushima8, M. Kuzumoto8, and S. Frahm9
1Bristol-Myers Squibb, Princeton, NJ, USA
2Boehringer Ingelheim Pharma GmbH & Co KG, Biberach an der Riss, Germany
3Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, CT, USA
4Bristol-Myers Squibb, New Brunswick, NJ, USA
5GlaxoSmithKline, Ware, United Kingdom
6Eli Lilly and Company, Indianapolis, IN, USA
7Novo Nordisk, Copenhagen, Denmark
8Shionogi & Co, Osaka, Japan
9BioCelerate, Conshohocken, PA, USA
BioCelerate, a subsidiary of TransCelerate, was formed in 2015 as a preclinical industry consortium that could identify and implement initiatives to drive efficiency and productivity in early stage R&D. Toxicology Data Sharing (TDS) is the first initiative launched under BioCelerate and involves creation of a centralized, searchable toxicology data repository that will enable participants to make more informed decisions on compound progression based on increased understanding of on-target and off-target toxicity. The project has also been extended to include a repository of background control data. Moreover, the initiative has implemented a framework for successful and secure collaboration and data sharing within a biopharmaceutical industry consortium. Motivated in part by the US FDA’s 2011 Strategic Plan for Regulatory Science and binding guidance requiring CDISC SEND standard format for nonclinical data, the TDS initiative partnered with a technology vendor to design, develop, host, and maintain a cloud-based data lake to facilitate sharing and analysis of de-identified unstructured (eg, PDF) and structured (eg, SEND) data sets. The platform was designed for flexibility and modularity, built in line with our future-state vision, to enable voluntary data sharing and linking across the entire drug development continuum, from preclinical discovery through late phase clinical studies. Presented here are the core capabilities, use cases and value story driving the TDS initiative, the processes and system architecture for the core data sharing platform, and the principles guiding data sharing and collaboration among BioCelerate participants.
P209 - Risk From Pesticide Residues on Medical Marijuana: Myclobutanil as a Case Study
M. Chappel1 and K. Haines1
1NovaTox Inc, Guelph, Ontario, Canada
There are potential health risks associated with the use of pesticides on medical marijuana (MM) and related products. Pesticides used on food crops can also be used to control pests on MM, but the ultimate pathway of human exposure to residues differs (ie, inhalation of a combusted residue vs ingestion with first-pass metabolism); and existing regulatory levels that have been developed for pesticide residues on food (referred to as tolerances [United States] and maximum residue limits [Canada and Europe] developed based exclusively on oral toxicity reference values) may not be applicable for MM. The objective of this work was to quantitatively characterize the potential health risks associated with the fungicide myclobutanil (“Systhane”). Myclobutanil has been banned by most jurisdictions in which MM is legal; on the basis that it releases hydrogen cyanide following combustion. Despite the restrictions, some MM growers have been reported to be illegally continuing to use it to increase MM yields. Quantitative estimates of risk associated with inhaling combusted myclobutanil residue from MM use could not be obtained in the scientific literature. This case study identifies important points to consider in deriving tolerances and MRLs for any pesticide proposed for use on MM. Potential risk management and regulatory issues will likely become more important as growers/suppliers face increased economic pressure to maximize crop yields and cost-effectively satisfy the additional demand for MM and potentially recreational marijuana as its legalization becomes more widespread.
P211 - Managing Immune-Mediated Infusion Reactions in Nonclinical Studies
K. Mease1, A. Kimzey1, and J. Lansita1
1ToxStrategies, Katy, TX, USA
The observation of an infusion reaction (IR) in a nonclinical study can cause concern among investigators and regulators in the development of biotherapeutics. IRs can encompass a broad range of adverse events that may result in dose holidays or early euthanasia. Based on an examination of US FDA pharmacology reviews for US-approved biotherapeutics between 2004 and 2017, 19 out of 59 (32%) approved products were identified as producing nonclinical IRs; of these 19 approved products with nonclinical IRs, 16 resulted from delayed ADA-mediated reactions, 4 were first-dose reactions, and 1 biotherapeutic reported both types of reactions. Biomarkers such as cytokines (IL-6, TNFa, IFNg, IL1-b, IL-2), complement (sc5b-9, CH50, C3a, C5a, Bb), toxicokinetics, pharmacodynamic markers, antidrug antibodies, and immunohistochemistry can be informative to determine whether the reactions are immune-mediated or test-article related and if there is a potential risk to human subjects. Through savvy nonclinical study management that includes (1) a consistent and time matched biomarker sampling plan for control, and treated animals with and without IRs, (2) detailed IR incident tracking, (3) IR mitigation strategies, and (4) a refined necropsy plan; an evidence-based approach can be developed using peripheral blood and histopathology biomarkers to move a biotherapeutic with immune-mediated infusion reactions into clinical development, without the need for additional investigative in vivo nonclinical studies. In summary, this poster reviews US FDA approved biotherapeutics with nonclinical IRs, the biomarkers evaluated, and provides detailed guidance on study design and conduct.
P212 - In Silico Toxicology Protocols and Software Platforms
D. Bower1, K. Cross1, C. Hasselgren1, S. Miller1, G. Myatt1, and D. Quigley1
1Leadscope, Columbus, OH, USA
In silico toxicology is an important alternative approach to in vivo testing that provides a fast and inexpensive prediction of toxicity. Although running the models is fast, the whole process of making predictions, including selecting and acquiring the models, interpreting the results, performing an expert review, and documenting the results can be time-consuming and difficult to repeat. It is also challenging to defend the results, primarily due to a lack of published procedures for performing an in silico assessment. To support the development of such protocols, a 52-member consortium has been assembled and includes representatives from international regulatory agencies and government research laboratories in the United States, Canada, Japan, and Europe, as well as companies from major industrial sectors (eg, pharmaceuticals, cosmetics, food), academic groups and other stakeholders. The protocols will ensure any in silico assessments are performed in a consistent, repeatable, and well-documented manner to support wider uptake and acceptance of the approaches. A new software platform has been built that will make toxicity assessments based on these protocols. It includes a series of high-quality specialized curated databases and updated models integrated to be compliant with the published protocols. This poster will describe the general conclusions of the consortium concerning data and model relevance and reliability and overall confidence based on the weight of the evidence. This poster will outline the development of in silico toxicology protocols for major toxicological endpoints (eg, genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity).
P213 - Changes in Biomarkers in Adolescent Applicators and Nonapplicators Over Time Related to Organophosphate and Pyrethroid Insecticide Exposures
B. C. Okeke1, D. S. Rohlman1, J. R. Olson2, A. A. Ismali1,3, M. R. Bonner2, O. Hendy3, G. A. Rasoul3, and K. Wang1
1University of Iowa, Iowa City, IA, USA
2University at Buffalo, Buffalo, NY, USA
3Menoufia University, Al Minufya, Egypt
Agricultural practices commonly use multiple pesticides throughout a season for maximum effectiveness in minimizing infestation; however, research seldom examines the impact of these coexposures. Previous studies have hypothesized potential organophosphate (OP)-mediated pyrethroid (PYR) inhibition mechanism, as a result of OPs competing for the carboxylesterases responsible for hydrolyzing PYRs causing bioaccumulation. The goal of this study were to (1) identify changes in PYR/OP metabolite levels during the application season among pesticide applicators and nonapplicators; and (2) understand the interaction between the PYR and OP metabolite levels. As part of a 10-month longitudinal study examining adolescent pesticide application before, during, and after the application season, 3 pesticides were selected to the examine changes in metabolite levels. The pesticides were: λ-cyhalothrin, chlorpyrifos, and α-cypermethrin. Urine samples were analyzed from the adolescents employed (n = 33) and not employed (n = 24) by the Ministry of Agriculture in Egypt. Negative ion chemical ionization gas chromatography mass spectrometry was used to analyze the samples for 5 metabolites: 3,5,6-trichloro-2-pyridinol (TCPy), λ-cyhalothric acid (λ-cyhal), cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylycyclopropanecarboxylic acid (cis/trans-DCCA), and 3-phenoxybenzoic acid (3-PBA). TCPy and applicator status (P < 0.05; P < 0.01) increased the amounts of cis/trans-dcca and 3-PBA. Between the pre-PYR spray and during-PYR spray, there was an (P < 0.01) increase in the amount of cis/trans-DCCA and 3-PBA. Time (P < 0.01) impacted the amount of cis-dcca and λ-cyhal. Applicator status (P < 0.01) impacted the amount of 3-PBA and λ-cyhal. There was a positive association (P < 0.01) between TCPy levels, cis/trans-dcca and 3-PBA during the PYR spray period.
P214 - Background Incidence of Nuclear Cataract in Juvenile Sprague Dawley Rats
W. Roosen1, M. Van heerden1, G. Bailey1, and A. Ndifor2
1Janssen Research & Development, Beerse, Belgium
2Janssen Research & Development, La Jolla, CA, USA
During the development of a new molecular entity (NME) at Janssen Preclinical Development & Safety, fetal examinations in a rat embryo–fetal developmental study revealed unusually high incidences of granular foci within the lens in rats dosed with the NME. In a subsequent study, the incidence of fetal ocular findings developing in utero was investigated together with any subsequent recovery postnatally. The postnatal recovery was evaluated by performing ophthalmic evaluation on rat Sprague Dawley pups at 19 to 21 and 35 to 40 days of age, followed by histopathological evaluation. Ophthalmology of control pups showed unilateral focal nuclear cataract in 4.5% of the pups at days 19 to 21 and 12.2% on days 35 to 40, as well as bilateral nuclear cataract in 0.4% of the pups on days 19 to 21 and 0.8% on days 35 to 40. When day 21 fetuses were examined histologically, swelling of primary lens fibers was seen in 18% with 0.4% of the fetuses showing focal degeneration. When eyes of day 35- to 40-day-old-pups were examined histologically, swelling or degeneration of the primary lens fibers were absent. There was no histological correlation for nuclear cataracts diagnosed ophthalmologically. Since secondary lens fibers are formed throughout life, these compress the primary lens fibers of the nucleus, resulting in a relatively smaller and denser lens nucleus in adult rats. This is thought to have contributed to the insensitivity of histology to identify the nuclear cataracts.
P215 - US and EU Regulatory Initiatives to Expedite Drug Development and Registration
K. Spencer1 and M. MacNair1
1Shire, Lexington, MA, USA
It takes approximately 10 years from the discovery of a molecule to a medicinal product entering the marketplace. Clinical development averages 5 to 7 years and 2.6 billion dollars, yet the probability of clinical success is estimated to be less than 12%. Despite major scientific breakthroughs, regulatory authorities need to identify ways to expedite development, while providing evidence of safety and efficacy for severe diseases with high unmet medical need. The US FDA and EMA have developed initiatives to facilitate early and expanded engagement with regulatory authorities and provide additional support to promising product development strategies. These expedited regulatory pathways are intended to identify breakthrough drugs and streamline processes, supporting earlier patient access to important therapies. To qualify for these expedited programs the drug must be intended to treat a serious condition and address an unmet medical need. There are 4 CDER expedited review programs: Fast Track Designation, Accelerated Approval, Priority Review, and Breakthrough Therapy. Each has variations in eligibility criteria, but all serve to speed the timeframe of development and review. The EMA PRIority MEdicines (PRIME) initiative provides early and enhanced support to optimize development and speed MAA evaluation for medicines that demonstrate a potential to address an unmet medical need or provide therapeutic advantage. The benefit of these programs has been evidenced, as over half of CDER’s 2015 novel drug approvals received some form of expedited review. This is a positive step for drug companies, establishing relationships with regulatory authorities, and ultimately driving drug development for patients in need.
P216 - Developmental Thyroid Toxicity Study of Asulam Sodium Technical
D. Ujawane1, Q. Bui2, K. Hadiya1, M. Poshiya1, J. Rabadia1, F. Parikh1, J. Mistry1, A. Shinde1, D. Olson2, and M. Patel1
1Jai Research Foundation, Valvada, Gujarat, India
2United Phosphorus, Inc, Prussia, PA, USA
There are no validated and approved guidelines yet to investigate developmental thyroid toxicity. Asulam sodium technical (ASU) was administered by oral gavage to parental female Wistar rats at 30, 45, 67.5 mg/kg bw/d from GD-6 to LD-21. Pups were treated with ASU through oral gavage from PND 8 to PND 21. Water (vehicle control) and 6-propyl-2-thiouracil (PTU) were administered to parent and pups for similar duration. Pooled blood (per litter) was collected from fetuses on GD-20 and pups on PND-4. Blood was collected from dams on GD-20 and LD-22. Analysis of T3, T4, and TSH was carried out by ELISA. Microscopic examination of thyroid (with parathyroid) was conducted for GD-20 fetuses, PND-4 and PND-22 pups and GD-20 and LD-22 dams. No treatment-related effects were observed in the ASU-treated groups in body weight, organ weight, serum hormone levels, and microscopic examination in fetuses, pups, and dams. Whereas in the PTU treated group, a significant decrease in serum T4 and T3 and the corresponding increase in TSH were noted in parental animals, in fetuses of GD-20, and in pups of PND-4 and PND-22. PTU treatment also caused significant decrease in body weight and increase in thyroid (with parathyroid) weight in dams, fetuses, and pups and produced histopathological changes. Based on the results, it is concluded that the ASU is not a developmental thyroid toxicant. The results observed in the positive control group (PTU) confirm that the procedures used in this study met the criteria of a developmental thyroid study.
P217 - Trends in (Q)SAR and Bacterial Reverse Mutation (Ames) Assay Submitted in Generic Drug Applications to US FDA
H. Wang1, S. Rayavarapu1, M. Mueller1, S. Goel1, I. Surh1, S. King1, R. Dorsam1, M. Ritter1, and D. Shetty1
1US Food and Drug Administration, Silver Spring, MD, USA
Genotoxicity testing is essential for identifying and controlling genotoxic impurities. According to ICH M7, quantitative structure–activity relationship [(Q)SAR] assessment and Ames assay are pivotal to characterize the genotoxicity of impurities in drug products. This study is used to identify successful approaches and common deficiencies in (Q)SAR assessments and Ames assays in generic drug applications. The OGD pharmacology/toxicology team evaluated 29 (Q)SAR assessments and 47 Ames assays in generic drug applications submitted to OGD from March 2014 to September 2016. This evaluation is based on ICH M7 recommendations and OECD 471 protocols. Fourteen (48%) out of 29 (Q)SAR assessments failed to follow in silico methods described in ICH M7 guidance. Deficiencies included not using both expert rule-based and statistical-based methodologies (8 cases), using not appropriately validated software models (3 cases), and use of invalid (Q)SAR analysis in literature (3 cases). Seventeen (36%) out of 47 Ames tests did not appropriately characterize the bacterial mutagenicity of the tested impurities. Deficiencies included use of impurity-enriched test article rather than impurity alone (5 cases), uninterpretable results due to excessive impurity-related cytotoxicity (7 cases), absence of or inappropriate positive controls (3 cases), use of only 1 bacterial strain, rather than the recommended 5 (1 case), and testing only 4 doses (one case). The current evaluation identified several common deficiencies in (Q)SAR and Ames assays. It is hoped that this effort will be informative for future submissions and may ultimately improve the quality of genotoxicity justifications for impurities in generic drug submissions.
P218 - Modeling Cytokine Release Syndrome Using BLT Immune Humanized Mice Treated With Anti-CD28
H. Yan1, A. Knapton1, J. L. Weaver1, and K. E. Howard1
1US Food and Drug Administration, Silver Spring, MD, USA
Cytokine release syndrome (CRS) is a serious and potentially life-threatening complication typically associated with biological drug products. CRS represents an exaggerated immune response involving the release of inflammatory mediators and a recruitment and activation of immune cells. Preclinical testing in vitro and in vivo studies using nonhuman primates often fail to predict CRS. In order to determine whether bone marrow–thymus–liver (BLT) humanized mice or a CD34+ humanized mouse model could predict CRS, we tested an anti-CD28 monoclonal antibody (mAb) similar to TGN1412. Adalimumab, an antitumor necrosis factor-α-antibody not known to cause CRS, served as an active control. We evaluated immune cell activation and cytokine expression in 3 independent experiments. Mice were pre-bled and divided into 4 to 6 mice per treatment group with subsets of mice bled at 2, 4, 8, and 12 hours. Animals were euthanized at 24 hours posttreatment with blood and spleen collected. We used flow cytometry to assess immune activation and a custom 10-plex assay to assess cytokine levels. In BLT humanized mice, significant increases in levels of 10 human cytokines were identified in animals treated with anti-CD28 mAb. CD28+ cell detection was strongly reduced in anti-CD28 mAb treated group and increased T cell activation, identified with increased CD25, CD45RO, and CD69, was also noted. The CD34+ humanized mice demonstrated minimal ability to respond to anti-CD28. These results suggest that BLT immune humanized mice can demonstrate CRS when it is expected and may represent a model to identify CRS in preclinical testing.
P219 - Tumorigenic Assessment of JZP-110, a Dopamine and Norepinephrine Reuptake Inhibitor, in 2-Year Rodent Carcinogenicity Studies
J. Zhao1, K. Zomorodi1, and K. Allamneni1
1Jazz Pharmaceuticals, Palo Alto, CA, USA
(R)-2-Amino-3-phenylpropylcarbamate hydrochloride (JZP-110) is a selective dopamine and norepinephrine reuptake inhibitor with wake-promoting effects currently in late-stage clinical development for treating excessive sleepiness in adult patients with narcolepsy or obstructive sleep apnea. In 2 rodent carcinogenicity studies, JZP-110 was evaluated for a possible change in lifetime incidence of tumor formation. JZP-110 was administered orally once daily for up to 104 weeks at doses of 0 (vehicle), 20, 65, or 200 mg/kg in CD-1 mice, and at doses of 0 (vehicle), 35, 80, or 200 mg/kg in Sprague Dawley rats. There were no JZP-110-related effects on survival, food consumption, gross pathology, or histopathology up to the highest dose tested in either mice or rats. Overall, trends in JZP-110 dose-related decreases in animal body weight occurred in both sexes of mice and rats. In addition, JZP-110 dose-related hyperactivity was observed in rats. The body weight and clinical sign changes were consistent with results from previous repeat-dose rodent toxicity studies and were expected based on the JZP-110 pharmacologic properties. There was no JZP-110-related statistically significant increase in the incidence of any tumor type (neoplastic or nonneoplastic) in any tissue for either sex in mice or rats. At the highest, nontumorigenic dose of 200 mg/ kg in rodents, the steady-state plasma exposure is 3.9-fold (mice) to 10.4-fold (rats) higher than the exposure in humans at the highest recommended dose (300 mg/d). In conclusion, JZP-110 does not appear to increase tumorigenicity risk in humans. Supported by Jazz Pharmaceuticals.
300 Series—Safety Evaluation Nonpharmaceuticals
P301 - d -Arginine as a Novel Excipient Resulted in Retinal Degeneration Following Intravitreal Administration to Male New Zealand White Rabbits
Andaya1, V. Bantseev1, H. Booler1, C. Sioson1, and E. Thackaberry1
1Genentech, Inc, South San Francisco, CA, USA
High viscosity solutions are common problems in ophthalmology intravitreal (ITV) drug development. With a majority of drug excipients excluded from ITV ophthalmic drugs because of safety concerns unique to the eye, investigation into novel excipients for ITV drug administration is warranted. This study aimed to characterize the toxicity of
P302 - Sharing and Verifying Systems Toxicology Methods and Data via the INTERVALS and sbv IMPROVER Platforms
S. Boué1, A. K. Kuczaj1, D. Page1, L. Cannesson1, M. Seijo1, V. Belcastro1, E. Scotti1, F. Bonjour1, J. Hoeng1, and M. Peitsch1
1PMI R&D, Philip Morris Products S.A. (Part of Philip Morris International Group of Companies), Neuchâtel, Switzerland
International toxicology programs (eg, Tox21, ToxCast, EUToxRisk, SEURAT, and TG-GATEs) generate large and complex data sets. Moreover, important streams of data, software, and state-of-the-art methods are created via industrial R&D programs which are often not publicly shared. Therefore, sharing these industry-owned data sets represents a great opportunity to push forward frontiers of knowledge for the scientific community as a whole. A proof-of-concept database and website (INTERVALS) has been developed to share protocols, software, data, and results from inhalation studies and in vitro studies conducted by Philip Morris International R&D that assess potential modified risk tobacco products (MRTP). The data modeling for INTERVALS took into account the latest standards in terms of data sharing and reproducible research. Tools developed by the industry and/or academic groups may also be shared through such a platform. For example, AeroSolved, a powerful OpenFOAM-based software for CFD simulations of aerosol-related flow and deposition problems, has been made available through this platform. Our goal is to grow this initiative to establish a public global repository for 21st century preclinical systems toxicology MRTP assessment data. Additionally, in order to maintain scrutiny in data analysis and interpretation, we have developed and applied the sbv IMPROVER methodology to verify the output of research processes in industry. Whereas computational methods are benchmarked using computational challenges, a verification program engaging panels of independent experts confirms the excellence of the scientific methods used and the integrity of the results shared.
P303 - Can You Measure Calcitonin in Rat, Mouse, or Monkey Serum? Validation of an ECL Method for Calcitonin Quantitation
P. Pitsikas1, M. O. Pepin1, C. Ronacher1, P. Khatcherian1, S. Croteau1, G. Robin1, M. Pelletier1, F. Christ1, V. Klypa1, M. Djefel1, S. Cotton1, and S. Lavallée1
1Charles River, Montreal, Québec, Canada
Calcitonin can be used as a marker for various diseases and physical conditions. This hormone is involved in the reduction of blood calcium, counteracting the effects of PTH. The MSD platform was used to develop a more sensitive assay compared to commercially available kits. Multiple antibody pairs were tested for each species in order to identify the best pair and reduce the signal to noise ratio. Analytical validation parameters, including precision, relative accuracy, selectivity, parallelism, and stability assessments were performed. Sera from obese rats and mice treated with Ca gluconate were analyzed and detectable calcitonin endogenous levels were observed. Six levels of QCs were used to define the optimal dynamic range of the assay by conducting precision and accuracy assessments. The LLOQ and ULOQ for the rat assay (25.43 and 2604.17 pg/mL, respectively) met acceptance criteria, thus showing a wide dynamic range. Similar curve ranges were validated for both mouse and NHP. Parallelism assessments for calcitonin proved successful between undiluted samples and samples diluted 8-fold in rat serum between undiluted samples and samples diluted 3-fold in mouse serum and between undiluted samples and samples diluted 2-fold in monkey serum. Selectivity assessment demonstrated no matrix interference for all species. Short-term stability at ambient room temperature and in a refrigerator set to maintain 4°C were assessed. Freeze-thaw and long-term stability were also proven. Those methods are suitable to measure the most subtle changes in calcitonin in less than 130 µL of rat, mouse, or monkey serum.
P305 - Variance Analysis of Circulating miR-122 Levels in Humans
J. Vogt1, H. Irimagawa1, D. Sheinson1, M. Tseng1, P. Katavolos1, K. Rajapaksa1, and W. Proctor1
1Genentech, Inc, South San Francisco, CA, USA
miR-122 is a promising exploratory biomarker for detecting liver injury in preclinical and clinical studies. Elevations in serum or plasma have been associated with viral and autoimmune hepatitis, NASH, hepatocellular carcinoma, and drug-induced liver injury. These associations were primarily determined based upon population differences between the disease state and the controls. However, little is known about the variability and subsequent variance components of circulating miR-122 in humans, which affects the practical use of the biomarker in the clinic. Accordingly, we aimed to explore the observed inter- and intra-donor variability of this miRNA in healthy human volunteers. We developed and qualified a fit-for-purpose qRT-PCR assay to detect miR-122 and other circulating miRNAs in human serum. miR-122 relative expression was assessed via normalization to the average of several endogenous miRNAs using an adaptive algorithm. Preliminary results in serum from healthy volunteers (N = 91) demonstrated high variability with 175-fold dynamic range (95% reference interval). Thus, a longitudinal study of miR-122 expression was initiated. Six serum draws were performed over 6 to 10 weeks in 42 volunteers. Using variance analysis in this cohort, the intra- and inter-donor variability was evenly split with an overall total variance of 133×. Additionally, miR-122 was highly variable compared to other measured miRNAs: the next variable miRNA demonstrated a 44-fold reference interval. In contrast, ALT levels in this population exhibited a 5-fold total variance, with 80% of this variance due to inter-donor sources. In conclusion, the high variability of this biomarker limits its potential for use as a prospective clinical monitoring tool.
P306 - Use of Pharmacokinetic Data for Adjustment Factor Derivation for Benzoate Preservatives
D. Pizzurro1, K. Zu1, T. Lewandowski2, and J. Goodman1
1Gradient, Cambridge, MA, USA
2Gradient, Seattle, WA, USA
Data-derived adjustment factors are increasingly used in place of default uncertainty factors (UFs) by scientific and regulatory bodies tasked with deriving health-based safety criteria, such as acceptable daily exposures (ADEs) for substances in pharmaceuticals, foods, and the environment. Human health risk assessments for the common preservative sodium benzoate have not yet used this approach or considered existing clinical data on its use as a therapeutic agent. Based on comparisons of chemical-specific pharmacokinetic data (eg, areas under the curve) in rodents and humans, we derived a chemical-specific adjustment factor for the pharmacokinetic component of the interspecies UF (ie, PKinterCSAF) for benzoates in food. Our analysis indicated that (1) default interspecies extrapolation methods are overly conservative; (2) the metabolic capacity for benzoates is greater in humans than in rats, as demonstrated by comparisons of maximum rates of biotransformation in vitro; and (3) a conservative PKinterCSAF of 2 to replace the WHO/IPCS default of 4 for interspecies pharmacokinetic differences for acceptable daily intake (ADI) derivation is justified. This is supported by a comprehensive qualitative comparison of the absorption, distribution, metabolism, and excretion across species and the general safety observed at high exposures in clinical studies. In the case of sodium benzoate as a food preservative, this analysis indicates that the total current UF should be reduced from 100 to 25, which results in an increase in the ADI from 5 to 10 mg/kg bw/d. This analysis illustrates how chemical-specific pharmacokinetic data can reduce uncertainty in safety evaluations and ADE derivations.
P307 - Skin Sensitization Studies by Quantitative Structure–Toxicity Relationships (QSTRs): A Computational Approach
K. Sato1, Y. Kusaka1, and J. M. Ciloy2
1Department of Environmental Health, School of Medicine, University of Fukui, Fukui, Japan
2Fujitsu Kyushu Systems Limited, Fukuoka, Japan
P308 - Biocompatibility Evaluation of a Contact Lens Soaked in a Solution Containing ε-Polylysine
E. Si1 and C. Smejkal1
1CooperVision Inc, Pleasanton, CA, USA
ε-Polylysine (e-PL), a natural, biologically derived compound, is a broad-spectrum antimicrobial effective against both Gram+ and Gram− bacteria. To reduce bacterial contamination introduced during lens insertion, stenfilcon A, a silicone hydrogel contact lens, was packaged in solution containing 100 µg/mL e-PL. To assess biocompatibility of this lens product, cytotoxicity, acute ocular irritation, acute systemic toxicity, sensitization, and a 22-day contact lens wearing study in rabbits were conducted in accordance with ISO 10993 and 9394. An additional 1-day, multiple dose study of solutions with e-PL concentrations ranging from 50 to 500 ppm was conducted to evaluate ocular safety. Specifically, 50 μL test solution was instilled hourly into rabbit eyes for 8 doses. Contralateral eyes were instilled with vehicle. Both macroscopic and slit lamp examinations were employed to monitor rabbit ocular health. Biocompatibility study results indicated that 100 ppm e-PL-packaged lenses were noncytotoxic, nonirritating, nonsensitizing, and systemically nontoxic. Lenses worn for 22 days did not elicit any significant ocular irritation. Microscopic examination of various ocular tissue sections did not reveal any histomorphological changes caused by lens wear. The 1-day multiple dose study did not reveal any significant ocular irritation at any e-PL concentration (50, 150, and 500 ppm). Sporadic conjunctival redness and discharge (scores of 1) were detected but were not dose-related and thus considered incidental. Overall, the evaluations demonstrate that the lens prototype is biocompatible.
P309 - Expert Review of In Silico Predictions for Skin Sensitization Potency
J. Cohen1 and T. Lewandowski2
1Gradient, Cambridge, MA, USA
2Gradient, Seattle, WA, USA
Skin sensitization thresholds are important for establishing acceptable exposure limits (eg, in pharmaceutical manufacturing), and are typically determined based on data from the local lymph node assay (LLNA). Such data are not available for many chemicals that may require evaluation within restrictive budgetary and time constraints. For these situations, alternative methods such as predictive in silico programs may be considered. However, in silico estimates often require an additional level of expert scrutiny. We evaluated the impact of applying expert review to the predicted skin sensitization thresholds (EC3 values) from the in silico program Derek Nexus (Lhasa Limited, United Kingdom) for a set of 20 dermal sensitizers (target chemicals). This involved examining the similarity score and EC3 for each source chemical used to calculate an EC3 value for the respective target chemical, and removing outliers (eg, <50% similar). Some EC3 predictions, such as those for which all source chemicals were <50% similar, were considered “unreliable.” We then compared the predicted EC3 against potency classifications determined based on readily available guinea pig or human data. We found the Derek Nexus potency estimates were concordant with study data in 35% of cases (7 out of 20 chemicals). In 15% of cases (3 out of 20 chemicals), in silico estimates were health protective (ie, potentially overestimating hazard). However, in the remaining 50% of cases, in silico estimates were judged unreliable due to poor surrogate availability. Our results highlight the importance of expert review in the interpretation and application of in silico estimates for skin sensitization potency.
400 Series—Toxicology Methods
P401 - Evaluating In Silico and In Vitro Approaches for Identifying Developmental and Reproductive Toxicity in the Workplace
T. Lewandowski1, S. Pacheco Shubin1, I. Mohar1, and J. Cohen2
1Gradient, Seattle, WA, USA
2Gradient, Cambridge, MA, USA
We conduct risk assessments to establish workplace exposure limits for developmental and reproductive toxins. For some chemicals (eg, drug production intermediates) lack of animal data may be a challenge. To evaluate some available approaches for addressing data gaps, we compared zebrafish ova assay data and the predictions of US EPA’s TEST structure–activity relationship (SAR) program to in vivo data for 186 chemicals we previously investigated for in vivo DART effects. In our analysis, 38% of zebrafish positive chemicals were also in vivo animal assay selective DART agents (ie, true positives), 36% were not in vivo selective DART agents (ie, false positives). An analysis of the zebrafish false positives indicated that most (75%) produced DART effects only at maternally toxic doses, which cannot be discerned in the in vitro assay. The zebrafish false negative rate was 62%; all of these chemicals were suspected as DART agents in vivo based on end points not investigated in the in vitro assay (eg, male reproductive toxicity). SAR predictions were very conservative; 90% of SAR-positive chemicals were also positive in vivo (ie, true positives) but 92 chemicals that were also SAR positive were negative in vivo (80% false positive rate). These results suggest caution is needed when applying the in vitro zebrafish ova assay to fill DART data gaps; in vitro assays addressing multiple DART end points need to be considered. The SAR program we used may be useful for precautionary screening but has poor accuracy, leading to potential over-prediction of risk.
P402 - The Combination of Expert-Rule- and Statistical-Based In Silico Systems Significantly Improves Sensitivity in the Prediction of the Ames Assay Outcome
A. Brigo1 and W. Muster1
1Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, Basel, Switzerland
The International Conference of Harmonization (ICH) M7 guideline on the assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals mandates the initial assessment of their potential mutagenicity using computational tools. The computational evaluation should include 2 different methodologies: expert rule-based and statistical-based. In order to comply with such requirements, additional commercial in silico tools and models are made available to the scientific community. In the present study, we evaluated the performance of all combinations of 3 expert rule-systems (Derek Nexus, ToxTree, and Leadscope Expert Alert) and 3 statistical systems (Sarah, Leadscope Model Applier, and CASE Ultra). The validation was carried out using Roche Ames mutagenicity data sets comprising over 2,600 compounds. We previously described the performance of individual systems using the same proprietary data sets, which showed consistently low sensitivity values (between 8% and 54%) and relatively high specificity (69% to 95%). The combinations of expert rule-based and statistical-based systems consistently improved the sensitivity, up to 71%, with modest reduction in specificity. Low-molecular-weight subsets, containing an enriched genotoxic impurities chemical space, showed remarkable improvements in sensitivity, up to over 90% with a more substantial decrease in specificity (approximately 30%). At the same time, the positive predictive value (PPV) showed a limited increase (up to 61%), whereas the negative predictive value (NPV) remained consistently high (above 85%). Our study provides a thorough evaluation of the most commonly used commercial in silico systems for mutagenicity prediction and may guide the identification of suitable tools to comply with the ICH M7 requirements.
P404 - Frontloading Nonclinical Kidney Safety Evaluation in Staphylococcus aureus Minipig Surgical Infection Model Using Novel Biomarkers
Y. Chen1, J. Fernandez2, P. Acton2, C. McLahan2, L. Lee2, J. Wilson2, J. Hastings2, C. Cotto2, P. King2, C. Amuzie2, L. Varacallo2, H. Sanders3, G. van den Dobbelsteen3, J. Poolman3, and J. McDuffie1
1Janssen Research and Development, LLC, La Jolla, CA, USA
2Janssen Research and Development, LLC, Spring House, PA, USA
3Janssen Research and Development, LLC, Leiden, the Netherlands
Poorly translatable pharmacology models lend to significant failure of anti-Staphylococcus aureus vaccines in clinical trials. To increase the success rate of candidate vaccines, we developed a minipig surgical wound infection model, which has an immune system that is similar to humans. Several studies have described surgical infection models in rodents that were characterized by infection-induced glomerulointerstitial and tubulointerstitial nephritis. We developed a minipig model of surgical wound infection that is anticipated to closely resemble human disease. Various plasma-, serum- and urine-based biomarkers of infection, inflammation, and nephrotoxicity were assessed. Infection in the deep thigh muscle of minipigs correlated with increase in signal intensities detected using PET imaging. Elevations in circulating fibrinogen and haptoglobin as biomarkers of systemic inflammation. Elevations in urinary neutrophil gelatinase associated lipocalin (NGAL) as a biomarker of kidney tubular injury in the absence of changes in serum/plasma NGAL, sCr, or BUN. This new, more relevant animal model will enable frontloading kidney safety evaluation in during preclinical studies.
P405 - Feasibility and Optimization of Intrathecal Cannulated Surgical Model in Rats
K. DeSouza1, J. Herman1, N. West1, M. Kuhur1, M. Rauschenberger1, D. Ealy1, M. Molland1, and J. Andreoli1
1Covance Laboratories, Madison, WI, USA
Based on an increased interest in the delivery of compounds to the intrathecal space, this study was conducted to evaluate the feasibility and optimization of the intrathecal cannulated surgical model in rats. Male and female Sprague-Dawley rats were surgically implanted with lumbar intrathecal catheters and the catheters were accessible via either access plugs, PinPorts, or Vascular Access Buttons. Divalent free PBS (Dulbecco) was infused at a rate of up to 100 μL/h for approximately 1 hour, once daily for 5 days a week for at least 2 weeks. The dose was administered via an external pump with animals restrained in plexiglass devices during dose administration. Evaluations consisted of ease of connectivity (dosing), clinical evaluations, functional observational battery testing, cerebral spinal fluid, and standard clinical pathology evaluations, and histopathology evaluations of the catheter sites and spinal cord. Dose administration was successfully performed through each of the access routes; however, the button was found to be the superior approach based on a low occlusion rate, ease of managing and accessing the port, and the ability to socially house animals. No adverse changes in clinical condition or body weights were observed throughout the dosing phase. Background histological changes associated with the presence of the indwelling catheters were documented and catheter tips were confirmed to be within the lumbar space. In conclusion, intrathecal dose administration at a rate up to 100 μL/h via a vascular access button for up to 4 weeks has been validated for use on safety assessment studies.
P406 - Optimization of Complement Functional Assays for the Testing of Complement Inhibitory Therapeutics
A. Frazer-Abel1, T. Ton1, D. El Mehdi2, and P. Deschatelets2
1Exsera BioLabs, Aurora, CO, USA
2Apellis Pharmaceutical, Crestwood, KY, USA
P407 - Characterization of Maduramicin-Induced Toxicity Mechanism in Primary Chicken Myocardial Cells
X. G. Gao1, L. Peng1, H. Ji1, X. C. Ruan1, D. W. Guo1, and S. X. Jiang1
1Nanjing Agricultural University, Nanjing, Jiangsu, China
Maduramicin, a commercially important polyether ionophore, is extensively used for coccidiosis treatment in poultry. Previous reports showed that maduramicin has a relative narrow safety range of medication-induced intoxication in farm animals, with heart and skeletal muscle to be the major target toxic organs in chicken. To date, the toxicological mechanisms of maduramicin in chicken have not yet been systematic investigated. In this study, differential transcriptional analysis in primary chicken myocardial cells during a toxic dose of maduramicin were proceeded. A total of 1,442 differential expressed genes were identified, among which 810 genes were upregulated and 632 genes were downregulated. Transcriptomic analysis indicated that the maduramicin-induced toxicity mechanisms were linked to several important pathways, such as the cytokine-cytokine receptor interaction, apoptosis, ion channel regulation, and cytoplasmic vacuoles, which was further confirmed by RT-qPCR, western blot, ELISA, flow cytometry analysis, light, and transmission electron microscope (TEM) observation. ELISA results exhibited that pro-inflammatory factors TNF-α and IL-8 increased during a toxic dose of maduramicin. Flow cytometry and western blot analysis revealed the apoptosis rate of chicken myocardial cells was elevated significantly. Simultaneously, intracellular calcium level and Ca2+-ATPase activity were also increased. Morphology and TEM analysis found that massive vacuoles generated in cytoplasm of maduramicin-treated chicken myocardial cells. In conclusion, our results provide a systematic analysis on toxicological mechanisms of maduramicin in primary chicken myocardial cells, which are associated with multiple molecular mechanisms including the cytokine-cytokine receptor interaction, apoptosis, ion channel regulation, and cytoplasmic vacuoles.
P410 - Utility and Limitations of a Dual Cholate Shunt Assay in Preclinical Species
V. R. More1, F. D. Sistare1, R. J. Gonzalez1, Y. Li1, L. LaFranco1, E. Messina1, J. Lebron1, and K. Pearson1
1Merck & Co, Inc, West Point, PA, USA
Quantitative liver function tests utilize chemical tracers that are excellent substrates for hepatic clearance through uptake, metabolism, and excretion. A “dual cholate shunt” is a method utilized for assessment of hepatic function in patients with fibrosis in clinical trials for hepatitis C interventions. The assay uses oral (PO) and intravenous (IV) administration of stable labeled cholic acid tracers monitored over period of 3 hours. In this study, we describe the results from translating the assay in a rodent model of hepatic fibrosis, and in healthy dogs, and describe utility and the limitations of the assay in preclinical species. Four-week treatment of rats with 1 mL/kg CCl4 resulted in advanced liver fibrosis. During multiple stages of fibrosis development and following termination of treatment during a recovery period, the dual cholate shunt assay was performed in rats using modified protocol for rats. After 1 week of CCl4 treatment, severe hepatocellular injury was induced with 10-fold elevation in serum aminotransferases, however, no fibrosis was evident. The plasma concentration–time curves for both PO and IV cholate were significantly altered in CCl4-treated groups, with much higher area under the curve (AUC) compared to the vehicle group. However, the shunt values remained unchanged. After 4-week CCl4 treatment, hepatocellular injury was accompanied by advanced fibrosis. AUC values increased for both PO and IV cholate in CCl4-treated groups, however, shunt values remained unchanged. One week after CCl4 treatment was stopped, aminotransferase levels returned to normal, however, fibrosis scores remained severe. Interestingly, AUC change in PO cholate was higher that IV cholate at this time, and shunt value increased by >60% in fibrotic rodents. In healthy dogs, plasma concentration–time curves for both PO and IV cholates appeared similar to clinical data, and shunt value obtained were between 10% and 15%. Overall, we demonstrated that a “dual cholate shunt” assay can be translated to preclinical species. Hepatic fibrosis increased shunt values in rats, however acute injury diminished shunt changes. Further, this assay can be translated to dogs and plasma concentration–time curves for dogs align better with clinical data compared to rats.
P411 - ETS123 Group Housing of Mature Cynomolgus Moneys Undergoing 24/7 Continuous Intravenous Infusion for up to 8 Weeks: Applying the 3 R’s
S. Korte1, M. O. Niehoff1, T. F. Ludwig1, A. von Keutz1, F. Runge1, T. Voss1, and T. Pfaff2
1Covance Preclinical Services GmbH, Münster, Germany
2AiCuris Anti-Infective Cures GmbH, Wuppertal, Germany
Until today, continuous intravenous (IV) infusion studies in cynomolgus monkeys required single housing, because animals would manipulate backpacks and implanted catheters. Fourteen animals were pair housed (ETS 123 (2007)) in groups of 2 or 3. They were 4 to 7 years old and weighed 4 to 7 kg. Jacket training was conducted with all animals for up to 4 weeks, gradually increasing the weight of the backpack to a maximum of 20% of the body weight. The animals were free-ranging (5.68 m3). Animals were inspected daily and social interaction was also recorded. Then 10 animals were implanted with ports and catheters. After 3 weeks of recovery from surgery, animals again underwent training for 3 days. Thereafter, continuous infusion with 0.9% NaCl was started, lasting for 28 days. Over the course of the training period, jacket size and tightness of cable ties were individually adjusted. Infusion pumps and fluid bags were protected with a customized plastic cover. Canines were shortened as a precautionary measurement and the backpacks were equipped with additional straps for optimizing comfort. After 28 days of continuous infusion, individual infusion times were calculated (targeted infusion time–interruptions). The 10 animals had reached individual infusion times between 89.7% and 100% of nominal dosing. This study represents a successful application of the 3Rs principles, and it is concluded that with the described methodology it is possible to conduct 24 hours IV infusion studies over at least 28 days in free-ranging mature cynomolgus monkeys.
P412 - Procedural Refinement of Peripheral Infusion via a Tail Vein in Nonhuman Primates
J. Laliberte1, B. Heroux1, C. Brochu1, A. Provencher1, M. Cote1, and N. Hebert1
1Charles River Laboratories, Sherbrooke, Québec, Canada
The intravenous (IV) injection is a commonly used parenteral route of administration (ROA) in nonclinical studies for large molecules for pharmacokinetic studies as well as exposure comparability studies. For larger IV volumes, a temporary indwelling catheter is recommended (ie, peripheral infusion) for the administration. In our laboratory, we have recently adapted a method of peripheral infusion in nonhuman primates (NHP) via the tail vein using a modified restraint chair; the chair allows NHPs to be restrained in a more natural position, while the tail is easily available and secured to the back of the chair. In order to demonstrate the advantages of the tail vein infusion versus peripheral limb vein infusion, we have performed both methods in NHP populations (total of 8 animals) temporarily restrained on a chair for up to 0.5 hours and compared behavioral clinical observations, environment interaction, and stress-related physiologic parameters. Animals were healthy, 2 to 6 years old cynomolgus monkeys, fed with the same diet and maintained in comparable room environmental conditions. The refined infusion technique via a tail vein appears to be less stressful for the animals. The anatomy of the tail vein vs peripheral veins offers the advantage of being a larger and straighter blood vessel, with easier and rapid access for catheter placement, reducing risks of procedural complications such as phlebitis and hematomas. Given the dorsal placement, NHPs are less prompt to remove the catheter, reducing the risk of catheter replacements and allowing availability of the peripheral limb veins as alternate venipuncture sites.
P413 - Speckle Tracking for Noninvasive Semi-Automated Cardiac Contractility Assessments in Dogs, Minipigs, and Nonhuman Primates
N. Gendron-Parra1,2, E. Boulay1,2, C. St-Jean1,2, R. Forster2, E. Troncy2, and S. Authier1,2
1Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Québec, Canada
2CiToxLAB, Laval, Québec, Canada
M-Mode which was historically used for cardiac contractility assessment with echocardiography presents significantly limitations owing to the unidimensional rectilinear approach with high risks for variations. Speckle tracking imaging enables semi-automated contractility evaluation of the entire ventricular circumference with segmental ventricular analysis as well as global integrated contractility assessment. The goal of this study was evaluate of this noninvasive methodology in dogs, mini-pigs, and nonhuman primates in the context of toxicology studies. Echocardiography with speckle tracking imaging was performed on beagle dogs, Göttingens minipigs, and cynomolgus monkeys at baseline and after pimobendan and itraconazole administration. Dogs and minipigs were evaluated with the right parasternal short- and long-axis views. Cynomolgus monkeys were evaluated in apical and parasternal views. 2-D and 3-D video was acquired with electrocardiogram taken for systole and diastole marking for calculation of strain rate, displacement, and velocity (peak and time to peak). In dogs, endocardiac circumference strain per segment for the middle anterior, middle inferolateral, and middle inferoseptal segment were the most stable at baseline (SD < 11%). Pimobendan and itraconazole were associated with increased and decreased circumferential endocardial strain rate with peak effects noted at 3 to 10 hours post-dose. Standard deviation values were animal dependent. The linear proportional correlation for paired standard deviation values between 2 observers was R 2 = 0.948 supporting a strong animal dependence for variability. In conclusion, Speckle tracking with strain measurements could be considered to assess global or segmental ventricular contractility in nonrodents in toxicology.
P416 - Effects of WSCM, eVCM, and Nicotine on Monocyte Adhesion to Human Aortic Endothelial Cells
O. Makwana1, N. Cockcroft2, F. Lowe2, D. Breheny2, G. Smith3, H. Flockton3, G. Watters3, W. Fields1, and B. Bombick1
1RAI Services Company, Winston-Salem, NC, USA
2British American Tobacco (Investments) Ltd, Southampton, United Kingdom
3Covance Laboratories Ltd, Genetic and Molecular Toxicology, Harrogate, United Kingdom
Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology and associated devices to investigate THP-1 human monocyte adhesion to human aortic endothelial cells (HAECs). The aim of the study was to determine the effects of whole smoke conditioned media (WSCM; from University of Kentucky reference cigarettes 3R4F), electronic cigarette vapor conditioned media (eVCM; from a commercially available e-cigarette), and nicotine on monocyte adhesion to endothelial cells in the BioFlux. HAEC monolayers were grown in microfluidic channels and exposed to 0 to 3000 ng/mL nicotine, or 0 to 3000 ng/mL nicotine equivalence of WSCM or eVCM for 24 hours under gravity flow. Activated THP-1 cells were perfused through the microfluidic channels at low shear and left to adhere under static conditions. The perfusion, adhesion period and wash cycle was performed 4 times with increasing adhesion periods (10, 20, 30, and 40 minutes). THP-1 cell numbers were quantified by counting of phase contrast images from 3 fields of view. WSCM and eVCM induced dose-dependent increases in THP-1 cell adhesion of up to 3-fold and 2-fold, respectively, compared to control. Nicotine had no effect across all doses. Furthermore, adhesion regulation was linked to increases in endothelial ICAM-1 expression after WSCM exposure but not after eVCM and nicotine exposures. This test system effectively models monocyte-endothelial cell adhesion and is a useful tool for the future assessment of novel tobacco and nicotine delivery devices.
P417 - Transferability of the GARDskin Assay to 2 Independent Laboratories
A. Johansson1, M. Agemark1, R. Gradin1, O. Larne1, A. Malmborg Hager1, A. Edwards2, V. Hoepflinger3, F. Burleson2, H. Gehrke3, E. Roggen1, and H. J ohansson1
1SenzaGen, Lund, Sweden
2Burleson Research Technologies, Morrisville, NC, USA
3Eurofins, Munich, Germany
Chemical handling and chemicals in consumer products can cause undesired health effects. To protect workers and consumers, chemicals have to be safety tested. Traditional testing strategies for determination of a chemical’s sensitization potential have included animals, but today authorities, public and economic interests require animal-free models. The genomic allergen rapid detection skin (GARDskin) assay is an in vitro test that addresses this requirement, and is currently in a ring trial to confirm its validity (OECD TGP 4.106). The initiation of skin sensitization in vivo includes activation of dendritic cells. The GARDskin assay relies on test substance stimulation in vitro of SenzaCells; a human myeloid cell line with dendritic properties. Following incubation, a gene expression analysis of 200 predictive biomarkers is performed. A machine learning technique is used to analyze the high dimensional data and to perform the binary classification. In this study, we present data from the initial phase of the study including the laboratory transferability. Two laboratories, independent from SenzaGen and each other, performed the GARDskin assay to test 5 blinded chemicals. The test was repeated 3 times in each laboratory. The 2 laboratories were able to classify the 5 substances with 100% accuracy (5/5) in all 3 transfer studies, confirming that GARDskin is transferable to external laboratories. This paves the way for a successful final validation where the 2 external laboratories and the in-house laboratory at SenzaGen will test 28 blinded substances by the GARDskin assay.
P418 - The Distinct Molecular Signature and Functional Characteristics of Developing Neural Progenitor Cells from Human, Mouse, and Rat
S. Masjosthusmann1, M. Barenys1, J. Baumann1, D. Becker1, R. Deenen2, U. Hübenthal1, J. Klose1, K. Köhrer2, B. Petzuch1, C. Siebert1, and E. Fritsche1
1Institute for Environmental Medicine, Düsseldorf, Germany
2Heinrich-Heine-University Düsseldorf, Biological and Medical Research Center, Düsseldorf, Germany
Given the high vulnerability of the developing brain, there is concern that many compounds that have not been tested for their developmental neurotoxic potential place our children at risk. Therefore, testing for developmental neurotoxicity (DNT) represents an emerging issue in future risk assessment. One approach to handle this issue is the use of alternative methods that if combined in a testing battery allow reliable, faster, and cheap compound screening and prioritization for regulatory purposes. An in vitro methods that could be part a testing battery are aggregated primary neural progenitor cells (NPC) growing as neurospheres. This alternative method functionally mimic progenitor cell proliferation, migration, neuronal, and glial differentiation and therefore represents major neurodevelopmental key events in vitro. We performed a molecular and functional characterization of developing human and rodent NPC. Transcriptome analyses revealed that gene ontology (GO) biological processes involved in organ and tissue development and regulation of major neurodevelopmental processes are overrepresented in developing NPC. We found that despite conservation of GO categories across species, transcriptome changes are highly species-specific with only ∼10% overlap of regulated genes. Based on overrepresented neurodevelopmental processes enriched with gene–protein interactions data, we identified some important key regulators guiding neurodevelopment. Some of these regulators were experimentally validated in the functional end points of developing NPC across species. This pathway-to-function analysis provides information on the application domain of NPC and will contribute to higher confidence in NPC for DNT in vitro testing.
P419 - Baseline Parameters for Learning and Spatial Memory in the Morris Water Maze: Protocol Development for Neurobehavioral Testing in Rats
L. Miller1
1Southern Research, Birmingham, AL, USA
Evaluation of learning and memory is routinely conducted during neurotoxicity testing to screen for central nervous system dysfunction. Baseline Morris Water Maze parameters (latency to platform/% success, cumulative distance to platform, total distance moved, Gallagher’s proximity, time spent in slow swimming, % thigmotaxis time, and heading) were evaluated in untreated adult male (N = 6) Sprague Dawley rats (Charles River). EthoVision XT (Noldus, Inc) was used for the automated capture of rodent activity in the water maze. The following protocol was used: cued learning trials on day 1 (3 trials/animal), acquisition trials on days 2 to 8 (4 trials/animal/day), probe trials on day 11 (1 trial/animal), and reversal trials on days 13 to 15 (4 trials/animal/day). During the acquisition phase, animals required 3 days to learn the location of platform as evidenced by decreases in all parameters, and increased % success. During the reversal phase, 100% of the animals learned the new platform location by trial 4 on the first day. These preliminary data demonstrate the establishment of a robust learning paradigm for rats. Additional data are being generated to build a historical control database for neurobehavioral parameters.
P420 - Induced Clinical Pathology Changes Associated With Saline Intravenous Infusion Administration in Cynomolgus Monkeys
J. T. Mortensen1, W. Harvey2, S. Horbach1, and P. Parren1
1Genmab BV, Utrecht, the Netherlands
2Charles River Laboratories, Tranent, Scotland, United Kingdom
This experiment was conducted in order to investigate whether the routine handling and restraint procedures used for intravenous infusion of test compounds to cynomolgus monkeys in toxicology and pharmacokinetic studies could affect the clinical pathology measurements performed in toxicology studies, and which could be confounded with test compound-induced effects. Eight cynomolgus monkeys were given an intravenous infusion of sterile 0.9% saline according to the standard procedures of the laboratory. A battery of clinical pathology plasma enzymes and biomarkers were measured before infusion and at 4, 24, and 48 hours postinfusion. Small, transient variations were seen in many of the parameters measured after the infusion procedure. Most notable were transient increases seen between 4 and 24 hours postinfusion in the following parameters: aspartate aminotransferase (ALT), lactate dehydrogenase (LDH), total creatine kinase (CK), including CK isozyme CK-MM (mainly skeletal muscle) but not CK-MB (mainly myocardium) or CK-BB (mainly brain), as well as C-reactive protein (CRP). This investigation suggests that the procedures associated with intravenous infusion in cynomolgus monkeys, including handling, fixation, insertion of intravenous catheter, and the strained/restricted position, while in the restraint chair, may be stressful for the animal and may produce transient increases in plasma biomarkers, mainly related to the musculoskeletal system that could be mistaken for treatment-related reactions. Much care should be taken to limit forceful handling of the cynomolgus monkeys during the infusion process and the importance of including relevant control groups in toxicology studies is emphasized.
P421 - Illustration of Toxicological Methods Used in US EPA STAR Grants
K. Oo1, A. Page1, and M. Bonner1
1NCER/ORD/US EPA, Washington, DC, USA
Nationally, alternative toxicity testing methods and strategies have ushered in a next generation of toxicity testing, for example, high-throughput testing and next generation in vitro studies, to reduce, refine, and replace animals in research. This poster will highlight research projects from various US EPA funded Science to Achieve Results (STAR) grants to illustrate how research methodologies have changed over time and how these innovations are leading to improvements in our ability to predict and assess toxicity.
P422 - Micro Blood Sampling From the Marginal Ear Vein of the Göttingen Minipig for Nonclinical Safety studies
S. A. Reynolds1, S. Hackford1, and H. Blunt1
1Sequani Limited, Ledbury, United Kingdom
Over recent years, the use of the minipig for nonclinical toxicology studies has expanded, leading to the species becoming a viable nonrodent alternative, accepted by regulatory authorities. Although the minipig is a useful model due to possible extrapolation to man, the increased demands for blood sampling in nonclinical studies is considered a drawback. Traditional blood sampling routes, such as the cranial vena cava and jugular vein, can result in excessive stress over repeated occasions and, although alternatives exist (eg, vascular access ports), surgery is typically required, highlighting the need for an alternative approach. We have developed a technique for collection of blood samples from the minipig on repeated occasions, using a microsampling technique from the marginal ear vein. Blood samples of up to 50 µL were collected by capillary action after venepuncture on 6 occasions per sampling day; sufficient for a typical toxicokinetic profile in a regulatory toxicology study. Samples were collected successfully, with minimal restraint compared with existing techniques and a notable reduction in distress to the animals. Slight bruising to the ear was noted, but recovered rapidly and did not impair further sample collection. We describe how this technique was evaluated and subsequently used for obtaining repeated toxicokinetic blood samples during 2 regulatory dermal toxicity studies and show anonymized examples of the toxicokinetic profile obtained. Guidance to ensure successful use of the technique is provided.
P423 - Making Hematology Go Further: Dilution of Samples
K. Ridge1, H. Blunt1, D. Chuchu1, S. A. Reynolds1, and B. Finney1
1Sequani Limited, Ledbury, United Kingdom
Nonclinical toxicology studies are increasingly complex, commonly with addition of multiple biomarkers and repeated blood sampling. This creates particular challenges in small species (mice and juvenile rats) where it is difficult to collect the volume and quantity of samples required, without having to resort to numerous satellite animals. The advantages of microsampling techniques are not confined to small animals and can be significant in minipig studies. Considering the 3Rs, we tested the effects of dilution on minipig, mouse, and juvenile rat hematology samples obtained as follows: juvenile Crl: CD(SD) rat (n = 10) and adult Crl: CD-1 (ICR) mice (n = 20)—from the vena cava after death; adult Göttingen mini-pigs (n = 6)—from a temporary catheter inserted into the external jugular vein. Analysis was performed on an Advia 120. Samples were analyzed neat and diluted as follows: rats and minipigs—1 in 2, 3, 4, or 5 in Advia sheath fluid; mice—1 in 2, 3, or 4 in Advia sheath fluid or PBS. Results were calculated as percent change from neat sample value, and difference of ≤20% was considered acceptable. Typically, dilution of samples for hematology at ratios of 1:2 or 1:3 were acceptable, regardless of species. As variation was lowest in the 1 in 2 dilution, this was preferred, the diluent with the lowest variability was Advia sheath fluid. Although dilution may render some changes to WBC differentials, the benefits of being able to analyze basic hematology and other biomarkers, such as immunophenotype, in the same sample may outweigh disadvantages.
P424 - A Qualification/Feasibility Oral Gavage Study in Neonatal Sprague Dawley Rats and CD-1 Mice
C. Gordon1, M. Stoute1, M. Araujo Abrahim1, and R. Tavcar1
1CiToxLAB, Laval, Québec, Canada
The ability to administer drugs by oral gavage to neonatal rats is critical for the assessment of nonclinical development of many pharmaceuticals. The objective of these studies was to assess the tolerance of the procedures administering reverse osmosis water (ROW) to neonatal Sprague Dawley rats starting as early as postpartum days 4, 7, or 10 to day 20 and to neonatal CD-1 mice starting on postpartum days 7 to 21. Three pregnant rats and mice were received and each set of pups was divided into groups containing at least 5 pups/sex in the control group and 8 pups/sex in the treated group(s). Animals in dosed groups were administered ROW daily at a dose volume of 10 mL/kg and control animals were not dosed. The data collected included mortality, clinical signs, body weights, and macroscopic examination. The results obtained from these studies provided sufficient evidence that oral gavage to male and female neonatal mice daily, from postpartum day 7 to 21 and neonatal rats daily, from postpartum days 4, 7, and 10 to postpartum 20, was well tolerated even though there were 2 accidental deaths (mice) and a slight reduction in body weights and body weight changes observed prior to the start of dosing until termination of the dosed animals compared to the naïve controls. Macroscopic findings were considered background, spontaneous or related to experimental procedures.
P425 - Morphometric Assessment of GATA4 Expression in the Rat Testis During Postnatal Development
K. Takayama1, N. Li1, J. Goad1, C. Picut1, A. Remick1, and D. Brown1
1Charles River Labs, Durham, NC, USA
Histopathological evaluation of immature rat testes is a routine component of juvenile toxicity studies; however, the detection of abnormalities is complicated by the rapid development of organs in postnatal animals. A comprehensive description of normal testicular maturation with respect to the morphology and relative abundance of somatic and germ cells will be a useful tool in distinguishing toxicity-induced irregularities from intrinsic biological variation. The transcription factor, GATA4, is an established marker for Sertoli and Leydig somatic cells in seminiferous tubules. Previous investigations of GATA4 expression in the testes suggest a gradual decrease in protein and transcript abundance as juvenile rats approach adulthood. However, this effect has not been explored using computerized image analysis of whole-tissue sections. To this end, testes were harvested at various time points between postnatal days 3 to 70, fixed in modified Davidson solution, embedded in paraffin, and sectioned transversely (3 μm) before immunolabeling with GATA4. Slides were scanned at 20× and the tissue was analyzed using an algorithm to detect positively and negatively labeled nuclei. The results show a significant, time-dependent reduction in the relative number of GATA4-positive nuclei at postnatal days 3, 16, 27, and 46 (88%, 70%, 24%, and 10%, respectively). These data will guide the development of robust quantitative end points for evaluation of rat testicular development which may serve as the foundation for future models of endocrine dysfunction related to maternal and postnatal toxicities.
500 Series—Safety Evaluation Pharmaceuticals
P501 - Selection of Positive Control Cells for Use in Cell Therapy Tumorigenicity Assessment in NOD/SCID/IL2Rγnull (NSG) Mice Subjected to Immunoablative Whole-Body Co-60 Irradiation
I. Boulay1, K. Wong1, W. Fodor2, M. Chabicovsky3, A. Günzl3, R. Goetz3, S. H. Yee Hung1, A. Larivee1, J. Haruna1, K. Bujold1, W. Wierzbicki1, and S. Authier1,4
1CiToxLAB, Laval, Québec, Canada
2Cell Therapy Group, Bellingham, CT, USA
3MC Toxicology Consulting GmbH, Vienna, Austria
4University of Montreal, St-Hyacinthe, Québec, Canada
Tumorigenicity assessment is a key preclinical safety test in gene and cell therapies. The NOD-SCID IL2rγnull (NSG) is the most immunodeficient mouse allowing for cell engraftment. Given high radio-sensitivity of lymphoid lineages, total body irradiation (TBI) is a potent immunoablative conditioning procedure. To confirm validity of this mouse model for testing engraftment/tumorigenicity of human cells, a positive control cell line was tested. NSG mice receiving TBI were treated IV with cultured human promyelocytic leukemia cells (HL-60) at doses of either 0.2 × 106 or 2.0 × 106 with a 43-day observation period. Ionization chambers confirmed Co-60 exposures (195.3-196.8 cGy). All control NSG mice receiving TBI had no clinical signs with unremarkable necropsy results. Clinical signs in HL-60 treated NSG mice started on day 23, progressed more rapidly in mice receiving the high HL-60 cell dose. Blood smear morphology was normal in control mice, while atypical cells were noted in mice treated with HL-60 cells. Flow cytometry on spleen and bone marrow identified (P < 0.05) hCD45/hCD33 positive cells at days 25 and 36. HL-60 high dose was associated with higher mortality and greater number of masses/tumors than the low dose. Masses/tumors were noted in the subcutis, mesentery, abdomen, kidneys, prostate, vagina, urinary bladder, ovaries, testes, and salivary glands confirming a wide biodistribution. NSG mice exposed to TBI immunoablation identified tumorigenicity following human promyelocytic leukemia cell administration and thus, the cell line qualifies for use as positive control in standard tumorigenicity studies for human gene and cell therapy candidates.
P502 - Evaluation of EEG Seizure Detection Software Platforms to Optimize Sensitivity and Specificity of Ictal Event Detection
M. Accardi1 and S. Authier1,2
1CiToxLAB, Laval, Québec, Canada
2Faculty of Veterinary Medicine, St-Hyacinthe, Québec, Canada
The ability to detect epileptiform electroencephalographic (EEG) abnormalities in safety pharmacology assessments is an essential component of any prospective CNS safety program. However, EEG artifacts and electromyographic (EMG) contamination can complicate EEG analysis and hinder one’s ability to robustly and accurately identify epileptiform abnormalities. Accordingly, robust EEG detection software is an essential requirement for rapid and accurate analysis of EEG recordings. However, the diverseness of software and algorithms available to perform seizure and spike detections can potentially address a range of applications. Given this, we report a comparison between the seizure detection modules among leading commercially available EEG analysis software. Paired with continuous video monitoring to aid in the identification of clinical signs, showing a progression into convulsive activity (ie, tremors, twitches, myoclonic jerks, convulsions, etc), ictal EEG traces (induced by pentylenetetrazol, PTZ) were detected with high fidelity across software platforms (ie NeuroScore, EMKA seizure detection module). Our results highlight the value of more algorithmic options that could be used to help distinguish real abnormal EEG ictal events from artifacts or EMG contamination. Our results also highlighted the increased specificity of strategies that enabled higher complexity algorithm. No software could be used to identify isolated biomarkers of increased susceptibility to seizure (eg, isolated spikes and isolated sharp waves) and manual EEG review remains a major component of interpretation. Taken together, high-fidelity seizure detection software suites are available to aid in the detection of ictal EEG abnormalities within safety pharmacology programs.
P503 - Ex Vivo Assessment of Drug Effects on Platelet Function in Nonhuman Primate and Human Whole Blood
C. St-Jean1,2, E. Boulay1,2, N. Gendron-Parra1,2, R. Kubazsky2, R. Forster2, E. Troncy2, S. Authier1,2, and F. Nego-Silva1
1Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Québec, Canada
2CiToxLAB, Laval, Québec, Canada
High sensitivity assays to identify pro or antithrombotic effects are critical in preclinical safety testing considering the life-threatening consequences of drug-induced thrombosis or hemorrhage. This study evaluated the sensitivity of a coagulation assessment platform, to detect alterations in platelet response, clot formation, and thrombus lysis ex vivo. Blood was obtained from healthy cynomolgus monkey and human normal volunteers. Positive control agents including heparin and acetylsalicylic acid, and platelet activator, collagen, were added to the blood samples, ex vivo. Viscoelastic properties of the clot formation were assessed by thromboelastometry. In cynomolgus monkeys, mean coagulation time (CT) increased by 17%, 2 hours postcollection, whereas clot formation time (CFT) increased by 51% and maximum clot firmness (MCF) decreased by 9% in the same interval. Results were presented as % of change from an untreated baseline. With rising collagen concentrations (0, 1, 2, and 5 μg/mL), the CT was on an average shortened by 23%, 31%, and 36% with collagen concentrations of 1, 2, and 5 μg/mL, respectively. Signs of effect saturation were observed at higher concentrations. Heparin at relevant concentrations completely inhibited clot formation at 1 units/mL or above. However, at a lower concentration of 0.1 units/ mL, CT was extended by 148%, CFT was prolonged by 454%, while MCF was reduced by 32%. As previously reported, acetylsalicylic acid did not significantly affect rotational thromboelastometry. This study reports ex vivo platelet response characterization using rotational thromboelastometry as a strategy for screening of toxicological evaluations of drug effects using nonhuman primates and human blood.
P504 - Validation of a Nonhuman Primate Model of Ferric Chloride-Induced Venous Thrombosis
P. Gayheart-Walsten1, J. Baublits1, P. Atterson1, and S. Love1
1Xenometrics, Stilwell, KS, USA
Venous thromboembolism is a common disease comprising deep vein thrombosis and pulmonary embolism which can cause death or serious morbidity. It is a frequent complication among surgical patients and acutely ill patients hospitalized for conditions including congestive heart failure, acute respiratory insufficiency, rheumatologic disorders, and acute infection. The intent of the following study was to validate a model of antithrombotic efficacy in an acute anesthetized cynomolgus monkey model. End points of the study included thrombus weight, hematology, and coagulation parameters. The animals (n = 28) were anesthetized and the femoral veins isolated from the femoral artery and nerve. Parafilm was placed under the vein after isolating the vessel to protect surrounding tissue from the FeCl3. The vessels were allowed to rest for 5 minutes following isolation. Prior to thrombus induction, the animals were administered either 0.9% saline, heparin (250 IU/kg), or apixaban (0.1 and 1 mg/kg) as a 5-minute intravenous infusion at t = 0. A piece of filter paper presoaked in 5% or 10% FeCl3 solution was placed circumferentially onto the femoral veins above the parafilm at t = +10 minutes for 5 minutes. The FeCl3 solution concentration was alternated between left and right veins for each subsequent animal. Sutures were placed around the veins for removal and the thrombus was dissected, dried, and weighed. When comparing heparin and apixaban dosed animals with saline-dosed animals, statistical significance was observed in all end points confirming a valid model for future research.
P505 - Combination Toxicology Studies: Industry Perspective From IQ Consortium DruSafe Survey
J. Birkebak1, L. Buckley2, D. Dambach3, E. Musvasva4, K. Price5, S. Ralston6, and A. Sacaan7
1Gilead Sciences Inc, Foster City, CA, USA
2Eli Lilly & Co, Indianapolis, IN, USA
3Genentech Inc, South San Francisco, CA, USA
4Boehringer Ingelheim Pharmaceuticals Inc, Ridgefield, CT, USA
5Bristol-Myers Squibb Co, Princeton, NJ, USA
6AbbVie Inc, North Chicago, IL, USA
7Pfizer Inc, San Diego, CA, USA
There is increasing interest within the pharmaceutical industry to develop combination products to overcome drug resistance and treat complex diseases. Although regulatory guidance provides frameworks for nonclinical strategies to support therapeutic combinations, the need for, design of, and contribution of nonclinical studies is situational. The IQ DruSafe Leadership Group surveyed member companies to evaluate industry practice with combination toxicology strategies. Survey questions focused on general information about combination product programs and rationales for conducting (or not) combination toxicology studies. When a study was conducted, additional questions probed timing relative to clinical development, prior clinical experience, and perceived value from the study. Responses from 20-member companies represented a total of 79 programs across anti-infective, metabolic, and oncology therapeutic areas. Most (75%) combination products were small molecules. Fixed-dose formulations and individual drugs labeled for course were most common. When conducted, combination toxicology studies were performed based on scientific rationale, regulatory agency request, or expectation of regulatory request to avoid program delays. Companies perceived the studies to be of value when they provided increased confidence of no new or exaggerated toxicity, fulfilled a regulatory requirement, avoided developmental delays, or when new or exaggerated toxicity or pharmacokinetic interactions were identified. When studies were considered of low value, it was because no new or unpredicted toxicities were identified. Only 12% of the studies actually impacted clinical trial plans. Overall, the survey suggested that combination toxicology studies should be conducted when there is cause for concern or potential impact on the clinical program.
P506 - Vacuolation in Rat, Dog, and Human Hepatocytes: A Species Difference for a Mechanism of Toxicity?
W. Brock1, C. Strock2, G. Koelsch3, and D. Norton4
1Brock Scientific Consulting, Montgomery Village, MD, USA
2Cyprotex, Watertown, MA, USA
3CoMentis, South San Francisco, CA, USA
4Calvert Laboartories, Scott Township, PA, USA
Therapies targeting the α7 nAChR are being developed for a variety of CNS indications including schizophrenia and Alzheimer disease. In rat subchronic studies with PTC1318, a dose-related increase in hepatic vacuolation at ≥20 mg/kg were observed. At doses >100 mg/kg, increases in hepatic enzymes and changes in lipid parameters occurred. Hepatic findings in the liver were not observed in the dog, even at systemic exposures >50-fold than observed in the rat. An increase in ORO staining was observed in rat cryopreserved hepatocytes, with micro- and macrovesiculation vacuolation noted at ≥50 mg/kg. A trend toward reversibility was noted. A mechanistic in vitro vacuolation study evaluated the potential of the parent drug and a potential rodent-specific metabolite of PTC1318 (PTC1463) to induce vacuolation in rat, dog, and human hepatocytes. Hepatocytes were plated in collagen I-coated plates and incubated with test compounds followed by fixation and staining for microscopic imaging. PTC1318 and PTC1463 caused cytotoxicity in hepatocytes from all 3 species. IC50 values ranged from 72 to 85 and 114 to 253 µM, respectively. PTC1318 appeared to cause the formation of small vacuoles/vesicles in the rat hepatocytes up to 50 µM. PTC1318 also caused textural changes in the rat hepatocytes with punctate-type staining of the cytoplasm. No vacuolation or textural changes were observed in human and dog hepatocytes or with the PTC1463 in hepatocytes of any species. These data indicate that a different mechanism unrelated to a rodent-specific metabolite may be involved in the vacuole formation in the rat compared to dog and humans.
P507 - Intranasal Nilotinib in the Thy1-aSyn Mouse Model of Parkinson Disease
J. Brown1, S. Moore1, K. Seroogy2, and M. B. Genter1
1University of Cincinnati, Department of Environmental Health, Cincinnati, OH, USA
2University of Cincinnati, Department of Neurology, Cincinnati, OH, USA
Parkinson disease (PD) is a common and debilitating motor disorder. Early symptoms include motor dysfunction and decreased olfactory function and gastrointestinal motility. Aggregation of α-synuclein (aSyn) in the brain correlates with motor and nonmotor impairments in PD patients. Thy1-aSyn mice exhibit these and other features of PD observed in humans, making this a suitable model for studying drug intervention in PD. Nilotinib is an inhibitor of the tyrosine kinase c-Abl and is approved for treatment of Philadelphia chromosome-positive chronic myelogenous leukemia. A phase 1 clinical trial found motor improvement in PD patients treated with oral nilotinib, but serious side effects were reported. With the goal of decreasing drug-induced systemic toxicity and delivering nilotinib directly to the brain, we are investigating the novel use of the intranasal (IN) route of administration for nilotinib in Thy1-aSyn mice. Dose-finding studies with 1.0 mg/d IN nilotinib (n = 3) resulted in acute toxicity indicated by weight loss, anosmia, and olfactory epithelial damage, while 0.5 mg/d showed no histological damage. After baseline neurobehavioral assessments, wild-type and Thy1-aSyn mice received IN vehicle, 0.25 or 0.5 mg/d nilotinib treatment for 8 weeks. After 8-week dosing, behavioral assessments were repeated, and brains, nasal cavities, and lungs were collected for histology, aSyn immunohistochemistry, and stereological analysis. Behavioral results found the Thy1-aSyn mice treated with 0.5 mg/d nilotinib have less net increase in errors/step (P value = 0.038) compared with the controls, suggesting that IN nilotinib decreases the progression of motor dysfunction in the Thy1-aSyn PD mouse model.
P508 - QSAR Modeling for Japan NIHS QSAR International Collaborative Study to Predict Ames Mutagenicity
S. Chakravarti1 and R. Saiakhov1
1MultiCASE Inc, Beachwood, OH, USA
ICH-M7 is the first guideline to address the use of QSAR in place of an actual experiment for assessing carcinogenic risk of impurities in pharmaceuticals. Therefore, robust and highly predictive QSAR models are now a necessity for the prediction of Ames mutagenicity. The Division of Genetics and Mutagenesis, National Institute of Health Sciences, Japan, is collaborating with 12 QSAR builders around the world by providing high quality Ames mutagenicity data as training sets to improve QSAR models. MultiCASE Inc is a participant and we are reporting various aspects of model building and prediction using NIHS data. So far 3 batches of data have been released, and after combining with our existing training data we were able to build models with very high prediction accuracy, for example, we obtained 82% and 88% sensitivity and specificity, respectively, in predicting phase III chemicals. We will also discuss the importance of classification thresholds, ROC plots, and effect of adding the new data to our existing training sets on prediction accuracy.
P509 - Examining the Role of Mrp3, Mrp4, and Bsep Inhibition Individually and Synergistically and Their Relationship With DILI
R. Chan1 and L. Benet1
1University of California, San Francisco, San Francisco, CA, USA
DILI encompasses a spectrum from mild biochemical abnormalities to acute liver failure. Although, the underlying pathophysiological mechanism of DILI is still poorly understood, there is increasing evidence that cholestatic forms of DILI result from a drug- or metabolite-mediated inhibition of hepatobiliary transporter systems. In this study, we sought to evaluate individual and synergistic effects of in vitro inhibition of BSEP, multidrug resistance protein 4 (MRP4), and multidrug resistance protein 3 (MRP3) and their relationship with type of liver damage, US FDA drug labels, and the biopharmaceutics drug disposition classification system (BDDCS). The proportions of each of these transporter’s inhibitory individual and synergistic effects on marketed drugs were tabulated and statistically analyzed. Among all of the US FDA hepatic liabilities, the “warning and precautions” label section displayed the majority of drugs exhibiting BSEP, MRP3, and MRP4 inhibition, with MPR4 and BSEP inhibition showing a synergetic effect. When correlated with BDDCS, the overwhelming majority of inhibitors are BDDCS Class 1 and 2. In terms of the type of injury presented, BSEP, MRP3, and MRP4 inhibition were best associated with cholestatic injury, with MRP4 inhibition exhibiting the greatest effect. MRP3 and BSEP inhibition displayed synergy with hepatocellular injury. It appears that BSEP, MRP3, and MRP4 inhibition increase the risk of DILI. The field currently believes that DILI association is strongest with BSEP. Our analysis shows that drugs that tend to be BDDCS class 2 compounds are potentially BSEP, MRP3, and MRP4 inhibitors. However, BDDCS class and inhibition may be confounded as causing cholestatic and hepatocellular injury.
P510 - Triple Cannulation Approach for Biodistribution Studies of Neurologically Active Compounds in the Nonhuman Primate
J. Douville1, G. Iacono1, C. Copeman1, and T. Magee2
1Charles River Laboratories, Senneville, Québec, Canada
2Biogen Inc, Cambridge, MA, USA
In recent years, there has been increased interest in the development of novel therapies for the treatment of neurological diseases. When compounds are unable to cross the blood–brain barrier, direct delivery in the central nervous system (CNS) may be required. In this study, we present an optimized study design to evaluate biodistribution of an intrathecally administered compound in nonhuman primates involving triple cannulation into the CNS and extensive nervous tissue sampling. Animals had a lumbar intrathecal catheter surgically implanted for dose administration and cerebrospinal fluid (CSF) collection. A second catheter was implanted in the cisterna magna for CSF collection. Both catheters were connected to subcutaneous access ports. Finally, a needle guide port was implanted using a stereotaxic approach to access the lateral ventricle for CSF collection. Animals were distributed into 4 subgroups to allow for sparse sampling of blood and CSF from the 3 locations to assess distribution. At study initiation, all 12 animals were patent for dosing and CSF collection at the lumbar site, 9 of 12 remained patent at the cisterna magna site, and 8 of 12 remained patent at the ventricle site 4 weeks following surgical implantation. Despite loss of patency at a few sites, the primary end points of the study were maintained by evenly distributing the animals across time points and sampling based on the patency. To further evaluate biodistribution, tissues were obtained at the terminal time point from each animal. Based on the postsurgical recovery and sampling success, the triple cannulation method is a suitable approach to determine biodistribution within the CNS.
P511 - Characterization of the Göttingen Minipig for Cerebrospinal Administration and CSF Collection
J. Douville1, F. Emond1, C. Foucault1, G. Iacono1, J. F. Lafond1, and C. Copeman1
1Charles River Laboratories, Senneville, Québec, Canada
The minipig is considered an excellent surrogate nonrodent model for use on safety assessment studies given its many similarities with humans. It has been used extensively with the dermal, ocular, intravenous, and oral routes of administration, as well as on safety pharmacology studies. However, less work has been conducted via the cerebrospinal route. Given that many neurodegenerative diseases represent a large pool of unmet needs, and that an increasing number of therapeutic molecules being developed are biologics that cannot cross the blood–brain barrier, we investigated the suitability of the swine for neurological safety assessment studies. Following the development of a surgical implantation approach of lumbar intrathecal catheter, we mimicked dose administration twice weekly for 14 days using saline and monitored clinical signs, body weight, food intake, clinical pathology and histopathology of the spinal cord and brain. Cerebrospinal fluid (CSF) was also obtained from conscious animals via the catheter system. Results showed that the procedures had no effects on any of the parameters evaluated. Histopathology revealed background findings comparable to those observed using a similar implantation approach in other species. Additionally, we investigated the feasibility of intrathecal administration and CSF collection by direct percutaneous puncture at the lumbar and cisternal levels under anesthesia. Needle placement for injection/collection was successful at both locations; CSF was analyzed for clinical pathology parameters and compared with other species. The results obtained are comparable to historical data and as such feasibility of intrathecal administration and CSF collection in the Göttingen minipig was confirmed.
P512 - Challenges Associated With the Development and Validation of Flow Cytometry Assays
C. Dumont1, E. Moisan1, M. H. Côté1, M. Poirier1, and M. S. Piché1
1Charles River Laboratories, Inc., Senneville, Québec, Canada
Flow cytometry is a technology allowing multiparametric analysis of thousands of particles per second and helps to adequately identify or functionally characterize complex cell populations of interest. It is often used in basic research, discovery, preclinical, and clinical trials. With the increasing proportion of biologics in the pipeline, flow cytometry has proven itself to be an indispensable tool to assess safety, receptor occupancy or pharmacodynamics. Flow cytometry-based methods can be challenging to develop and validate, given the cellular measurement involved, the lack of standardized cellular reference materials and that these assays are often used for multiple different purposes. There are currently no guidelines for the validation of flow cytometry methods to be used in the context of preclinical studies. Some initiatives have been taken by various working committees in the writing of guidance documents describing flow cytometry method validation. However, these recommendations have not yet been integrated in an official document released by the regulatory agencies as it has been done for other analytical methodologies. Case studies of various flow cytometry methodology validations, custom build to overcome various challenges, will be presented. Validation design will be presented, which were adapted to address challenges such as sample stability limitations for shipment, inherent variability of functional end points, and low frequency populations. A one size fit for all validation design is not applicable to flow cytometry assay and a fit for purpose approach is required for each type of assay.
P514 - In Vitro Assessment of Erythrocyte Differentiation Following Treatment With Small Molecule CHK1 Inhibitors
S. Feng1, M. Holdren2, A. Fullerton2, and W. Proctor2
1University of California, Davis, Davis, CA, USA
2Genentech, South San Francisco, CA, USA
Bone marrow toxicity is one of the most common dose limiting adverse effects observed during oncology drug development. Mechanisms of bone marrow toxicity may include DNA damage, direct cytotoxicity, inhibition of cell cycle progression, or interruption of signaling pathways that govern HSC differentiation and proliferation. Chk1 is an important regulator of DNA damage checkpoints and plays an important role in normal DNA replication. Chk1 is a potential drug target for anticancer therapies in combination with DNA damaging agents. Chk1 is also highly expressed in hematopoietic stem cells (HSCs), T-cells, and erythroid progenitors as compared to other cell types. We have implemented an in vitro model, utilizing lineage specific differentiation of HSCs into subsequent erythroid progenitors which was used to characterize the effects of 2 small molecule CHK1 inhibitors on proliferation, viability, and differentiation of erythrocytes at various stages of the hematopoietic process. We investigated the role of DNA damage occurring subsequent to CHK1 inhibitor treatment at early and late stages of erythrocyte differentiation in an effort to further understand the mechanism by which these inhibitors elicit their effects on erythropoiesis and the potential safety concerns associated with those mechanisms of action. The results have shown Chk1 inhibitors alone contribute to DNA damage and differentiation of erythrocyte progenitors. We found that the late differentiation cells (CD71+ and CD235a+) and mature cells (CD71− and CD235a+) are more sensitive to Chk1 inhibitor treatment with higher Annexin V and Gamma-H2AX dose-dependent induction.
P515 - TargetTri: A Toxicity-Based Triaging System for Novel Drug Targets
J. Venhorst1, E. van Someren1, F. van de Brug1, G. Kalkman1, S. Folkertsma1, and C. Krul1
1TNO, Zeist, The Netherlands
To aid in the triaging of novel drug targets, TNO is developing a web-based system that builds upon a previously designed target safety assessment workflow. This system extracts information from (1) relevant databases (covering, eg, protein, compound, and pathway information), (2) in-house curated data on known toxicities of proteins, and (3) text-based resources using TNO’s text-mining tool ERIS. A customized ontology has been developed in-house to structure results from the latter based on toxicity and target organ. To allow intuitive and efficient analysis of the available data, various web-views have been developed. The interactive network view links targets and their immediate biological network to diseases and toxicities. The toxicity effects view displays identified effects per organ in a heat-map, and allows further analysis of the text-mining results by digging down to the source data. Other views display summary data, ligand information and clinical trial data. Validation against an in-house pharma safety report on the data-rich target HSP90 demonstrated that both the major toxicities (QTc prolongation, liver necrosis, and renal failure) and minor effects (visual disturbances) could be identified with TargetTri. Mined data from both curated databases and text (ERIS) further indicated a major role of HSP90 in cancer, in accordance with its therapeutic application. TargetTri can extract and visualize data on-the fly for any of the 20k reviewed human proteins deposited in UniProt, allowing highly efficient toxicological assessments and triaging of drug targets. This research is supported by the Dutch Ministry of Economic Affairs.
P516 - How Low Can You Go? Generation of Stable Dry Powder Aerosols at Less Than 10 Micrograms per Liter
M. Freke1, S. Oag2, M. F. Tellier1, C. Durie3, E. Moreau1, and A. Viau1
1Charles River Laboratories, Montreal, Québec, Canada
2AstraZeneca Drug Safety & Metabolism, Mölndal, Sweden
3Charles River Laboratories, Edinburgh, United Kingdom
Potent test compounds are increasingly driving down the target dose levels needed for inhalation safety assessment (iSA) toxicity testing. A new powder generation system, a joint venture between Charles River and AstraZeneca, has been developed with the objective of generating stable dry powder aerosols between 5 and 10 µg/L pure API (determined chemically using validated HPLC methods) using free-flowing powder to meet this need. In this experiment compound A, a micronized dry powder, was used. Compound A had been used previously in inhalation studies conducted by Charles River Laboratories, where by the mode of aerosolization required that the test substance was compressed into a pellet (Wright dust feeder) or a column (brush generator). Physchem profiling of compound A illustrated a poor flowability profile with moderate to high cohesiveness, which is not ideal if compaction is required for aerosolization. These inherent powder characteristics make this test substance ideal for this prototype validation of a free-flowing dry powder generator. Compound A was loaded into 1 of 2 prototypes, with aerosols generated into “flow-through” and then “flow-past” rodent chambers for at least 6 hours over several days at a target aerosol concentration of 6 µg/L. The spatial and temporal homogeneity of the generated aerosols were determined. Following modifications, both devices met their design requirement, reliably producing stable aerosols (%CV of <20) at concentrations ranging from 5 to 7 µg/L active API on multiple occasions. The next step will be to mass produce these devices at Charles River Montreal for use on upcoming SA studies.
P517 - Assessment of the Electrophysiological Effects of Doxorubicin in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes
S. Goineau1, M. Hunault1, C. Legrand1, E. Hayes1, G. Froget1, and V. Castagne1
1Porsolt SAS, Le Genest Saint Isle, France
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an efficient in vitro system to investigate potential cardiotoxic effects of drugs. The aim of this study was to investigate the electrophysiological effects of doxorubicin in hiPSC-CMs using multielectrode array (MEA) technology. Cardiomyocytes (Cor 4U Axiogenesis) were cultured in monolayer on 6-well MEA plates and were exposed to doxorubicin (1 and 10 µM) for 24 hours. At 24 hours, field potential was recorded from spontaneously beating cardiomyocytes on the MEA-2100 system (MultiChannel Systems) and lactate dehydrogenase (LDH) leakage was measured in the culture medium. Total spike amplitude (SA, µV), field potential duration (FPD, ms), and spontaneous beat rate (BR, bpm) were recorded. The FPD was subsequently corrected (FPDc) using Fridericia formula (FPD/RR0.33, where RR = inter-spike interval). The electrophysiological data are expressed in percent change from baseline and compared to a control group (n = 4-5 wells/group) using unpaired student t test. At 1 µM, doxorubicin had no effect on FPDc or LDH release, but decreased SA (−59% vs +1%, P < 0.001) and increased BR (−2% vs −14%, P < 0.05) compared to the control group. At 10 µM, doxorubicin decreased SA (−80% vs +1%, P < 0.001) and FPDc (+3% vs +12%, P < 0.001) and increased BR (+32% vs −14%, P < 0.001) compared to the control group, it also increased the LDH release (+47%, P < 0.001). These findings suggest that hiPSC-CMs evaluated using a MEA platform is a promising preclinical in vitro tool for predicting the cardiotoxicity induced by chemotherapeutic agents.
P518 - Nonrodent Isolated Mature Ventricular Cardiomyocytes as an Integrated Multi-Ion Channel Model for Proarrhythmia Screening
H. Huang1, M. Accardi1, M. Pugsley2, and S. Authier1,3
1CiToxLAB, Laval, Québec, Canada
2Department of Toxicology, Purdue Pharma L.P., Stamford, CT, USA
3Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Québec, Canada
Multiple ion channel inhibition screening on stable expression cell lines is used to assess the effects on action potential repolarization but primary cardiomyocytes may be relevant. HEK 293 cells with stable expression were used in manual whole-cell configuration. IC50 values for ion channels (Nav1.5, Kv4.3, Cav1.2, hERG, KvLQT1, and Kir2.1) were tested with various ion-channel blockers. Cardiomyocytes were isolated from minipigs, dogs, and nonhuman primates (NHP) ventricle. The action potential duration (APD) was significantly prolonged by the potent hERG blockers E-4031 (IC50 = 2.3 µM in HEK 293 cells) and dofetilide (IC50 = 4.8 µM) in NHP cardiomyocytes. The ADP90 was increased by 80% ± 12% at 20 µM for E-4031 and 54% ± 16% at 2 µM for dofetilide, respectively, in NHP cardiomycytes. ADP90 was decreased by 56% ± 9% for Cav1.2 blocker nifedipine at 5 µM (IC50 = 7.8 µM in HEK293) in NHP cardiomyocytes. Similar changes were observed with the same agents in minipig and dog isolated mature ventricular cardiomyocytes. The isolated cardiomyocyte dV m/dt max and action potential amplitude (APA) in all animal models tested were significantly reduced by Nav1.5 channel blocker TTx (IC50 = 5.4 µM in HEK293). Multiple ion-channel inhibition profiles are considered under the comprehensive in vitro proarrhythmia (CiPA) to discriminate proarrhythmic risk in the presence of QT prolongation. Isolated ventricular cardiomyocytes from nonrodents may be considered as a tool in proarrhythmic risk assessment.
P519 - In Vitro Stimulation of Lymphocytes Using Staphylococcal Enterotoxin B (SEB): A Comparison of Whole Blood Versus PBMC Methods
C. Cooper1, K. Troth1, A. Iqbal1, J. Holroyd1, J. Gaminde1, and S. Kirk1
1Covance Laboratories Ltd, Harrogate, North Yorkshire, United Kingdom
Better understanding the effects of exaggerated pharmacology during preclinical safety evaluation of biotechnology-derived immunomodulators, often requires a combination of in vivo studies, in an appropriate animal species, complemented by in vitro or ex vivo functional assays. A useful assay, when assessing (un)intended immunostimulation of a potential therapeutic, is the production of IL-2 by circulating leukocytes after stimulation with the superantigen SEB. Traditionally this is performed by in vitro stimulation of PBMC’s with SEB in the presence of a known concentration of therapeutic. However, to ascertain systemic exposure to the therapeutic in preclinical species, it is desirable to perform this assay ex vivo. Preparation of PBMC’s involves wash steps, which can remove the therapeutic from serum, therefore it is advantageous to perform evaluations in whole blood instead. In this study, we compared the performance of SEB stimulation, using PBMC’s versus whole blood. Whole blood and PBMC samples from 3 cynomolgus monkeys were cultured (37°C/5% CO2) with various concentrations of SEB for 24 and 72 hours, and IL-2 production measured as an indication of stimulation. At all concentrations of SEB (after 24 and 72 hours), IL-2 concentrations from whole blood were comparable or above those detected in PBMCs. The addition of an anti-PD-1 antagonist control also enhanced IL-2 production in whole blood samples cocultured with SEB. In conclusion, the utilization of whole blood instead of PBMCs provides a robust method that allows for the ex vivo measurement of immunomodulators in serum, while allowing for a more rapid experimental setup.
P520 - A Preclinical Safety Testing Algorithm for Antibody Drug Conjugate (ADC) Lead Optimization and Candidate Selection
D. W. Lee1, N. Stagg1, B. Vuillemenot1, H. Booler1, J. Bravo1, F. Zhong1, T. Nguyen1, M. Schutten1, and H. Wang1
1Genentech, South San Francisco, CA, USA
In the development of a new class of ADCs, we adopted a 4-tiered approach to generate robust data sets, while minimizing nonhuman primate usage: (1) in vitro stability and potency data across species to select drug-linkers for the target antibody, (2) in vivo efficacy in various mouse models to select 2 to 4 promising ADC candidates, (3) a single-dose rat tolerability assessment to select 1 to 2 candidates with satisfactory therapeutic index (TI), and (4) a repeat-dose toxicity study in cynomolgus monkeys to select lead candidate for further development. A single-sex (female), single-dose rat study was considered sufficient to evaluate tolerability, if no gender differences in toxicity profiles were expected or noted in previous studies. To initiate the studies, 2 doses were selected based on mouse efficacy data, incorporating a 10-fold margin over the minimum efficacious dose. If necessary, based on in-life tolerability, doses were then increased or decreased in subsequent cohorts to determine the maximum tolerated dose (MTD). Candidates that demonstrated satisfactory TI following tier 3 were progressed into a toxicity study in cynomolgus monkeys. Studies were typically conducted in 2 phases: a dose escalation phase with 1 to 2 animals/cohort to determine the MTD, followed by an expansion phase with up to 4 animals/cohort to further characterize toxicity with repeat dosing. Overall, we believe that these refinements in experimental design enabled robust optimization of specific linker drugs to each target antibody to maximize TI and candidate selection, and characterization of safety profiles to inform GLP toxicology study design, while reducing the number of animals used.
P522 - Individual Serum Bile Acid Profiling in Rats Aids in Human Risk Assessment of Drug-Induced Liver Injury due to BSEP Inhibition
J. Maher1, D. Potter4, L. Wong1, L. Schutt1, J. Tarrant5, J. Pang1, X Zhang1, R. Andaya1, L. Salphati1, Y. Ran1, L. An1, R. Morgan2, and S. Cepa3
1Genentech, South San Francisco, CA, USA
2Amgen, Thousand Oaks, CA, USA
3AbbVie, North Chicago, IL, USA
4Pfizer, Groton, CT, USA
5Gilead, Foster City, CA, USA
Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50 years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to predict in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine whether serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50 mg/kg troglitazone. Minimal serum transaminase elevations (approximately 2-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2 hours after dosing and returned to baseline values before 24 hours. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be missed by traditional toxicity end points in rats.
P523 - Characterization of Cox-1 Inhibitors Effect on Platelets by In Vitro Platelet Aggregation Assays
F. Poitout-Belissant1, L. Huard1, and J. McCartney1
1Charles River Laboratories, Senneville, Québec, Canada
Cox-1 inhibitors such as aspirin strongly inhibit platelet function induced by arachidonic acid and limit the production of serum thromboxane A2. Cox-1 inhibition effects on platelet function may be characterized in vivo on human or cynomolgus monkey platelets but can be easily determined by in vitro methodology. For the characterization of Cox-1 inhibitors, arachidonic acid is used as an agonist added to freshly prepared platelet rich plasma (PRP), before measurement of platelet aggregation by optical methodology. A positive control of platelet inhibition consists of acetylsalicylic acid into dimethyl sulfoxide (DMSO) and saline is used as negative control. Serial concentrations of compound are tested on PRP samples from different donors in order to determine the optimal concentration for which 50% of the platelets are aggregated (IC50). The IC50 obtained is then compared to the expected maximal concentration of the compound (C max) in order to predict its potential effects on platelets. Effects of compound on platelets are also tested with other agonists such as ADP or convulxin, the platelet aggregation induced by non-Cox-1 dependent activity is usually not altered or only slightly limited by Cox-1 inhibitors despite complete suppression of the Cox-1 activity. Evaluation of drug–drug interactions on platelets can be assessed by combining a Cox-1 inhibitor with a compound and evaluating the potential combined effect on platelet aggregation with one or more agonists depending on the nature of the compounds tested.
P524 - Predictivity of 3 In Vitro Assays for In Vivo Developmental Toxicity: A Status Report From the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) DruSafe Working Group 2
M. E. McNerney1, K. Augustine2, P. Barrow3, B. Beyer4, S. Engel5, B. Enright6, A. Fullerton7, M. Green8, K. W. Hew9, K. Hilbish10, J. Hui11, C. McGinnis3, G. Moffat12, W. Nowland5, N. Powles-Glover13, M. Schneidkraut14, D. Stanislaus15, K. Turner16, J. M. Vergis17, and M. Graziano18
1Bristol-Myers Squibb, New Brunswick, NJ, USA
2Bristol-Myers Squibb, Hopewell, NJ, USA
3Roche, Basel, Switzerland
4Sanofi, US, Bridgewater, NJ, USA
5Pfizer, Groton, CT, USA
6AbbVie, North Chicago, IL, USA
7Genentech, South San Francisco, CA, USA
8Merck, West Point, PA, USA
9Takeda, US, Deerfield, IL, USA
10Lilly, Indianapolis, IN, USA
11Celgene, Summit, NJ, USA
12Amgen, Thousand Oaks, CA, USA
13AstraZeneca, Macclesfield, Cheshire, United Kingdom
14Astellas, Northbrook, IL, USA
15GlaxoSmithKline, King of Prussia, PA, USA
16Janssen, Spring Hill, PA, USA
17Drinker Biddle and Reath, Washington, DC, USA
18Bristol-Myers Squibb, Lawrenceville, NJ, USA
For decades, investigators working in developmental toxicology have sought to identify in vitro or nonmammalian assays that would predict findings in conventional embryo–fetal development studies, traditionally conducted in rodents and rabbits. Reasons for this interest include regional mandates for 3Rs, pharmaceutical companies’ interest in early teratogenicity screens; and, recently, potential to defer or replace regulatory in vivo work with one or more assays. DruSafe, the preclinical safety leadership group within IQ, established a working group to evaluate the concordance of 3 such alternative assays (rodent whole embryo culture, zebrafish embryo culture, and embryonic stem cells) with findings reported from in vivo studies of developmental toxicity in rodents and rabbits. To date, data for 32 individual compounds have been entered into a new proprietary database. Additional contributions are sought. Criteria for compound inclusion are limited to provision of data from at least 1 alternative assay and 1 in vivo study, using test systems previously validated by individual sponsors. Sample queries include whether predictivity correlates with specific assays, whether it is related to broad pharmacological targets, whether one or more specific assays have better concordance, and whether lack of predictivity is associated with disparities between in vitro and in vivo concentrations.
P525 - Comet Assay in 8 Target Organs and Tissues Collected From Sprague Dawley Rats
F. Merah1, G. Marceau1, M. Ghoussoub1, S. Lavallee1, N. Boudjadja1, and A. Nelson1
1ITR Laboratories, Inc, Montreal, Québec, Canada
This work is about comet assay in organs collected from Sprague Dawley rats. This assay detects DNA damage and it is used as part of genetic toxicology testing. Six independent replicates were treated at 24 hours intervals and each replicate had 2 groups. The first group was treated twice by oral gavage with EMS at 200 mg/kg and the second group was treated with water by the same route. Tissues were collected (liver, stomach, kidney, and skin in the first 3 replicates; and brain, spleen, lung, and bladder in the last 3 replicates) in mincing buffer. Single-cell suspensions were prepared, embedded in agarose on glass slides, lysed, and subjected to alkaline unwinding and electrophoresis. The slides were then randomized and stained with ethidium bromide. The % DNA intensity was measured and compared between groups within each replicate. The mean percentage values of DNA intensity obtained in this study were significantly increased (P < 0.01) between negative controls (from 6.2%– to 11.9%) and EMS-treated tissues (from 33.2%– to 38.9%). Specifically, DNA intensity increased in liver from 7.1% to 33.2%, in lung from 6.2% to 36.0%, in stomach from 7.6% to 33.4%, in kidney from 6.6% to 33.3%, in brain from 7.6% to 35.5%, in skin from 11.9% to 38.9%, in bladder from 8.1% to 36.1%, and in spleen from 7.6% to 35.0%. The procedures used in this study demonstrated that EMS at 200 mg/kg reached the targeted organs which could be the first site of contact dependently of the test substance dosing method.
P526 - Detection of DNA Damage in Sprague Dawley Rat Skin by Using Comet Assay
F. Merah1, G. Marceau1, M. Ghoussoub1, S. Lavallee1, N. Boudjadja1, and A. Nelson1
1ITR Laboratories Inc., Montreal, Québec, Canada
Comet assay is used in genetic toxicology to detect DNA damage. In this study, we report on the reproducibility of this assay in rat skin and building a historical control database. Five independent experiments were performed with Sprague Dawley rats and each experiment had 2 groups. One group was treated with water (negative control) and the second one was treated twice with a positive control, ethyl methanesulfonate (200 mg/kg and 10 mL/kg), at 24 and 3 hours before euthanasia. At termination, a section (3 × 3 cm) of dorsal skin was shaved and removed. The skin was sliced into strips, immersed in a cold EDTA/PBS, transferred into a trypsin/HBSS overnight at 4°C, and then rinsed again in cold PBS. The epidermis was peeled from the dermis and transferred into a cold RPMI with 10% FBS solution. The epidermal strips were gently stirred to prepare single-cell suspensions, which were then passed through cell strainers. The isolated cells were processed for comet assay and % DNA intensity was compared between groups within each experiment. The results indicate that there is significant increase in DNA migration between the negative and the positive controls (P < 0.01); and the results are reproducible between the 5 experiments. The mean values of the negative and positive controls were (16.89, 41.94) and (13.66, 35.21), (7.37, 33.22), (12.49, 44.26), and (15.75, 39.33) in the 5 experiments. In summary, this technique can be used to prepare single-cell population to perform Comet assay on skin for dermal studies in rats.
P527 - Evaluation of the Effects of Nucleoside Analogs on Mitochondrial Function Using In Vitro Cell Systems
D. Misner1, D. Simic2, M. Otieno2, M. Kijanska3, J. Deval1, A. Standeven1, and S. Chanda1
1Alios BioPharma, South San Francisco, CA, USA
2Janssen Pharmaceuticals Inc, Spring House, PA, USA
3InSphero, Brunswick, ME, USA
Nucleoside analogs play an important role in treating viral diseases. However, many of these compounds have shown toxicities that have either halted clinical development or resulted in withdrawal from the market. Most of the nucleoside toxicities have demonstrated involve effects on mitochondrial function, which are believed to be a potential mechanism for the adverse effects observed (Feng et al, Antimicrob Agents Chem., 2016; Jin et al, Antiviral Res, 2017). In this study, we examine the effects of a range of nucleoside analogs (including balapiravir, zalcitabine, and INX-08189) on both the general cell viability and mitochondrial function (oxygen consumption rate [OCR] and extracellular acidification rate [ECAR]) using the Seahorse instrument. Nucleoside analogs with reported toxicities were evaluated, along with marketed compounds with no known mitochondrial liabilities as negative controls. Two different cell systems were used to assess these nucleoside analogs: PC3 human prostate cells and 3D human liver microtissues (hLiMTs) provided by InSphero. Cytotoxicity and mitochondrial function end points (OCR and ECAR) were assessed following 3 days of incubation with compounds for PC3 cells and 7 days for hLiMTs. In this report, we outline effects of selected nucleoside analogs using the 2 different cell systems, compare the sensitivity of each system to detect effects on mitochondrial function, and assess predictivity to known toxicity (or lack thereof) observed clinically. Differences in cytotoxicity measured by ATP and mitochondrial effects by Seahorse were readily detected. For zalcitabine and balapiravir, cytotoxicity IC50s were > 100 μM, while mitochondrial toxicity was noted at much lower concentrations using PC3 cells.
P528 - Electroretinography Evaluation in Minipigs: A Useful Animal Model for Ocular Preclinical Safety Assessment
L. F. Negro Silva1, C. Li1, R. Tavcar1, R. Forster2, and S. Authier1
1CiToxLAB North America, Laval, Québec, Canada
2CiToxLAB France, Evreux Cedex, France
Minipig eye shares many similarities to the human eye, they are a relevant choice of test system in ocular toxicology testing. Electroretinography (ERG) is a common end point employed to evaluate retinal toxicity following systemic, ocular instillation or intravitreal (IVT) administration. In this study, ERGs were recorded using a computerized system with a Ganzfeld dome and contact lens electrodes referenced to a subpalpebral electrode and a ground electrode placed at Cz (based on the 10-20 system). The ERG protocol included a scotopic luminance–response curve analysis (−4.09 log cds/m2 to 0.90 log cds/m2) with an average of three responses for each light intensity except for oscillatory potentials (OPs), where a single flash was also used at 0.41 log cds/m2. Photopic evaluations included a single flash (averaged response) followed by a flicker response at 30.3 Hz at 0.41 log cds/m2. The dose volume was 100 µL/eye. Three different reference agents were assessed across 3 groups: gentamicin (5 mg/left eye, 3 males), indocyanine green (0.25 mg/left eye, 3 males), and glycine (5 mg/left eye, 2 males and 2 females). OPs were reduced in all treated eyes. The magnitude of the reduction was greater in the gentamicin and glycine treated eyes and only slightly reduced in the animals given indocyanine green. Log K, which is representative of retina sensitivity, was reduced in the gentamicin and indocyanine green, treated eyes, but increased in glycine dosed eyes when compared with the contralateral untreated control eyes. The pharmacological response of the minipig to IVT appeared comparable to other laboratory animal species.
P529 - Electrolyte Quantification in the Aqueous and Vitreous Humor of Minipigs
L. F. Negro Silva1, R. Tavcar1, C. Li1, R. Forster2, and S. Authier1
1CiToxLAB North America, Laval, Québec, Canada
2CiToxLAB France, Evreux Cedex, France
The chemical properties of the physiological environment are important for intraocular dosing as it may affect the stability or solubility of the test article. The aqueous or vitreous humor of the minipigs is not well characterized. The goal of this study was to determine and quantify the electrolyte composition of aqueous and vitreous humor in the Göttingen minipig. Electrolyte content of vitreous and aqueous humor was analyzed using a Cobas 6000 module c501 set to plasma/serum mode. All parameters are expressed in mmol/L. The following electrolyte concentrations were obtained from vitreous humor: phosphorus 0.31 ± 0.07, calcium 1.41 ± 0.18, sodium 132.80 ± 16.52, potassium 5.23 ± 0.48, and chloride 99.40 ± 14.53. Electrolyte concentrations in aqueous humor were phosphorus 0.90 ± 0.08, calcium 1.25 ± 0.10, sodium 142.00 ± 6.11, potassium 5.26 ± 0.99, and chloride 97.80 ± 4.07. This study provided a baseline/reference for electrolyte content in minipig eyes.
P530 - Development of a Targeted Biomarker Assay to Predict Cardiotoxicity Potential Using Metabolomics and Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes
J. Palmer1, A. Smith1, B. Smart1, M. Colwell1, P. West1, V. Gryshkova2, J. P. Valentin2, A. Nogueira da Costa2, A. Delaunois2, E. Donley1, F. Kirchner1, and R. Burrier1
1Stemina Biomarker Discovery, Inc, Madison, WI, USA
2UCB Biopharma SPRL, Braine-l’Alleud, Belgium
Cardiac safety is one of the leading causes of late-stage compound attrition in the pharmaceutical industry and accounts for 28% of the safety related withdrawals of US FDA-approved drugs from the market. Current cardiac safety preclinical evaluations are heavily focused on approximately 3 to 7 main ion channels involved in maintaining the cardiac action potential; however, over 70 different types of ion channels are expressed in the heart and participate in the overall cardiac current. These safety testing methods overemphasize electrophysiological assessment of cardiotoxicity and fail to evaluate cardiomyopathy and other forms of structural cardiotoxicity. Metabolic perturbations are one of the primary mechanisms underlying the cardiotoxicity elicited by pharmaceuticals. Stemina has developed a biomarker-based assay for evaluating the cardiotoxicity potential of compounds based on changes in the metabolic signature of human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. In this study, we exposed hiPSC-derived cardiomyocytes to 60 compounds and analyzed the spent media using UPLC-HRMS-based metabolomics. The training set consisted of 20 noncardiotoxic compounds and 40 cardiotoxic compounds that elicit a broad range of outcomes, including structural and functional cardiotoxicity. Analysis of metabolomics data identified a set of biomarkers that represent different metabolic pathways. When combined with viability data, different combinations of these biomarkers were highly predictive of cardiotoxic potential (>80% accuracy). This assay is an attractive new model that can identify both structural and functional cardiotoxic compounds that could be used in conjunction with CiPA and other end points to provide a more comprehensive evaluation of a compound’s cardiotoxicity potential.
P531 - A Retrospective Analysis of In Vitro Hemolysis Studies for Pharmaceuticals
L. Pan1, Z. Wang1, H. Huang1, and S. McPherson1
1WuXi AppTec (Suzhou) Co. Ltd., Suzhou, Jiangsu Province, China
In vitro hemolysis study is required for parenteral drugs (especially injections) when submitting INDs to CFDA (Chinese FDA). Although not a pivotal study in the IND package for US FDA filing, this study is sometimes also required when there is a concern of the hemolytic potential of the drug under development. Currently, there are no regulatory guidelines or standard methods that were followed by all testing laboratories and the sponsors. A specific testing method using rabbit blood and visual inspection was previously recommended by the CFDA technical guidelines for testing of chemical drugs. This has been adopted to test other types of drugs including biologics as well and represent the majority of in vitro hemolysis studies performed at WuXi AppTec (Suzhou) Co Ltd. In addition to this, another method using human blood which measures the plasma hemoglobin and potassium concentrations and red blood cell count was also used on some programs. A retrospective analysis was performed on approximately 30 in vitro hemolysis studies completed so far. The results were summarized for and compared between these 2 testing methods. The analysis focused on the interpretation of results and the correlation of findings with in vivo animal studies. The results of the analysis showed that the rabbit blood and visual inspection method is more likely to be influenced by physical characteristics of the test article or the vehicle and may generate ambiguous results difficult to interpret.
P532 - Renal and Hematologic Comparative Effects of Dissociated Agonist of the Glucocorticoid Receptor (DAGR) and Prednisone in Dogs With and Without Food Restriction
Z. Radi1, M. Vogel2, J. Dybowski2, M. Peraza2, S. Portugal3, and D. Lettiere3
1Pfizer Worldwide R&D, Andover, MA, USA
2Pfizer Worldwide R&D, Cambridge, MA, USA
3Pfizer Worldwide R&D, Groton, CT, USA
Glomerulopathy and body weight gain were noted after chronic administration of a novel nonsteroidal dissociated agonist of the glucocorticoid receptor (DAGR) compound, fosdagrocorat, to beagle dogs fed an ad libitum diet. To further investigate the role of diet and treatment with either fosdagrocorat or the glucocorticoid comparator, prednisone, on renal safety, a 13-week investigative study was conducted in beagle dogs. Renal histopathology, clinical chemistry, urinalysis, glomerular filtration rate (GFR), body weight, heart rate, blood pressure, and hematology were investigated in restricted and ad libitum fed dogs administered either prednisone (2.2 mg/kg/d), fosdagrocorat (5 mg/kg/d), or vehicle for 13 weeks. Glomerulopathy was generally observed in fosdagrocorat- and prednisone-treated ad libitum, but not in feed-restricted or ad libitum vehicle-treated dogs. Kidneys from prednisone treated ad libitum dogs had more severe tubular degenerative changes when compared with fosdagrocorat. Increased urine volume and decreased urine specific gravity were generally present in prednisone- and fosdagrocorat-treated dogs, regardless of diet. These changes were not, however, associated with consistent changes in GFR. Fosdagrocorat or prednisone treatment to ad libitum dogs resulted in increased body weight as well as increased systolic blood pressure. Only prednisone treated ad libitum dogs experienced increased heart rate. Significant reductions in serum cortisol and absolute eosinophils were noted in both ad libitum and restriction fed prednisone- and fosdagrocorat-treated dogs. In conclusion, prednisone-dosed dogs fed ad libitum had greater overall glucocorticoid-induced renal effects than those dosed with fosdagrocorat.
P533 - Nonclinical Evaluation of a Disproportionate Human Metabolite of Venetoclax
J. Rhodes1, X. Wan1, A. Souers1, D. Hill1, C. Frantz2, E. Choo2, and S. Ralston1
1AbbVie, Inc, North Chicago, IL, USA
2Genentech/Roche, South San Francisco, CA, USA
M27 is a major human metabolite of the oncology drug, Venetoclax, an orally bioavailable and selective Bcl-2 inhibitor. In a phase 1 Venetoclax clinical study, M27 exposures at steady state (ss) averaged 33.8% as a percentage of the total plasma AUC (M27 + Venetoclax). In contrast, ss plasma levels of M27 in mice and dogs were ≤5.1% of human exposure, making M27 a disproportionate human metabolite. Although extensive nonclinical safety testing of metabolites is generally not required for advanced cancer (ICH S9), we are preparing for oncology indications that may fall outside the scope of ICH S9. Because of the low levels of M27 formed in animals, M27 was synthetized for nonclinical testing. As with Venetoclax, no evidence of genotoxicity was found (Ames and chromosome aberration assays). M27 has at least 172-fold less cell killing activity against a Bcl-2 dependent tumor cell line (RS4;11) than venetoclax, and in vitro secondary pharmacology assays predicted low off-target toxicity. Finally, M27 was qualified in a GLP-compliant 4-week toxicology study conducted in transgenic wildtype mice to support a potential future carcinogenicity study. Effects similar to those with Venetoclax were observed (decreases in peripheral blood lymphocyte counts and in hemoglobin) but were milder, consistent with the low in vitro potency of M27. The basis for this presentation (study designs and conduct, interpretation of data, review, financial support, and approval) was provided by AbbVie and Genentech/Roche, who are jointly developing Venetoclax. J. W. Rhodes, X. Wan, A. J. Souers, D. R. Hill, and S. L. Ralston are AbbVie employees and may own AbbVie stock/stock options. C. E. Frantz and E. F. Choo are employees of Genentech/Roche.
P534 - Multiparametric Validation of Emerging Translational Pancreatic Injury Biomarkers
M. Saunders1, J. Y. Ma1, B. Zimmerman2, Y. Chen1, J. Vegas1, and J. E. McDuffie1
1Janssen Research and Development, LLC, San Diego, CA, USA
2Janssen Research and Development, Spring House, PA, USA
Biomarkers serve as measurable indicators to predict the pharmacological effects of discovery compounds. In recent years, increased incidences of drug-induced pancreatic injury (DIPI) have been observed in preclinical toxicology studies in rats. The onset of acute DIPI appears as clinically mild to severe inflammation encompassing exocrine and endocrine cells, diffuse and mixed cell infiltration, and single-cell necrosis. Early diagnosis is hampered by the use of nonspecific and insensitive biomarkers, including serum amylase or lipase activity. MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that are well-conserved across different species. Due to their tissue specificity and relative stability, miRNAs hold promise as novel, translatable DIPI biomarkers. We have investigated pancreas-enriched miRNAs as circulating biomarkers of DIPI in serum samples from male Wistar Kyoto, Han Wistar, and Sprague Dawley rats, following a single subcutaneous injection with a known pancreas toxicant. These miRNAs were first characterized as highly enriched in the rat pancreas based on RNASeq and qRT-PCR data. We observed significantly increased serum levels of miR 217, miR 375, miR-216a, and miR-216b corresponding to microscopic findings of injury in the pancreas. Select miRNAs exhibited diagnostic sensitivity, demonstrating utility as candidate biomarkers to enable early prediction of DIPI in rats. The sensitivity and reproducibility of these findings were confirmed using Taqman quantitative PCR. Current efforts employing fluorescent in situ hybridization, branched DNA signal amplification, and flow cytometry will reveal the dynamic kinetic changes associated with protein and miRNA expression patterns among heterogeneous cell populations and single-cell changes in response to acute DIPI.
P535 - Bone Marrow Stem Cell Toxicity Assessment of GS-5801: A Prodrug of a Lysine Demethylase 5 Inhibitor Under Development for Chronic Hepatitis B
C. A. Snyder1, J. F. Kruplak2, I. N. Rich2, L. A. Burns-Naas1, and C. H. Tay1
1Gilead Sciences Inc, Foster City, CA, USA
2Hemogenix Inc, Colorado Springs, CO, USA
The landscape for targeting epigenetic mechanisms in drug development is still rapidly evolving. Currently, all approved epigenetic modulators are anticancer agents with well-described safety concerns. A common problem across the class is adverse, dose-limiting hematological toxicity (Butler et al, 2015). GS-5801, currently under development for treatment of chronic hepatitis B, is a prodrug of GS- 080, a small molecule inhibitor of lysine demethylase 5 (KDM5). KDM5 is an “epigenetic eraser” that catalyzes the demethylation of lysine 4 on histone 3 (H3K4). Development of GS-5801 for a nononcology indication necessitates an improved hematotoxicity profile compared to other marketed epigenetic modulators. In this study, we compare the toxicity profiles of GS-5801 and GS-080 in 7 primary human bone marrow stem cell populations (consisting of hematopoietic stem cells or granulocyte, macrophage, erythropoietic, megakaryopoietic, and T- or B cell progenitors) to vorinostat and azacytidine, marketed histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors, respectively. In this in vitro platform (HemoGenix HALO-384 HT), proliferation of each population was measured. GS-5801 and GS-080 were significantly less toxic when compared to vorinostat and azacytidine in all stem cell populations tested. For example, IC50 values in HPP-SP primitive stem cells for vorinostat, azacytidine, GS-5801, and GS-080 were 1, 1, >10 and >10 μM, respectively. The IC values provided wider safety margins over a clinically relevant free Cmax for GS-5801 and GS-080 when compared to the marketed epigenetic modulators. Taken together these results suggest that specific epigenetic alterations, such as histone methylation, are potentially less hematotoxic than DNA methylation and histone acetylation modifications.
P536 - Preclinical Case Studies of Biological Therapeutics: A Multi-Faceted Strategy of Testing Immunotoxic Potential and Pharmacodynamic Properties of Immuno-Oncology Monoclonal Antibodies
D. Szatkowski1, J. Wheeler1, B. Wang1, C. Thompson1, J. Crona1, C. Newsome1, A. Thekkumthala1, C. Gleason1, J. Sathish1, R. Burgeois2, C. DuMont2, M. S. Piche2, T. Bunch1, H. Haggerty1, and W. Freebern1
1Bristol-Myers Squibb, New Brunswick, NJ, USA
2Charles River Laboratories, Montreal, Québec, Canada
As new generations of biological therapeutics are being developed, including immuno-oncology (I-O) monoclonal antibody (mAb) therapies that target immune checkpoints and costimulatory molecules, the methodology to test both pharmacodynamic (PD) properties of these compounds and immunotoxic potential has evolved. In lieu of a 1-size fits all approach, the specific assays used to characterize the potential risks and appropriate doses of I-O mAbs have been tailored on a study-to-study basis. The strategies used to characterize the immunotoxic and PD activity of 3 I-O mAbs in investigational new drug (IND)-enabling studies in cynomolgus monkeys and results are described. Further, in an effort to fully assess the impact of I-O mAbs on immune system function in the absence of a tumor model in cynomolgus monkeys, the importance of antigen challenge in these studies is discussed. Important lessons learned from these studies include (1) careful consideration of both I-O mAb and antigenic dose-selection to encompass both PD end points and safety margins, (2) utilizing existing assays to assess PD, (3) leveraging data from early exploratory studies to assess kinetics and utility of assessments, and (4) identifying risk factors for immunotoxic events and employing strategic sample collection for follow-up analyses. Results from these IND-enabling studies continue to aid in our goal to ensure both efficacy and safety of our biologic therapeutics for patients.
P537 - The Process and Challenges of Deriving Exposure-Based Limits for Components and Impurities Present in Pharmaceutical Drug Products With Case Studies for an Excipient, a Genotoxic Impurity, and an Immunomodulator
B. Welsh1, A. Kimzey1, and J. Lansita1
1ToxStrategies, Inc, Katy, TX, USA
To ensure the safety of drug products in the pharmaceutical industry (eg, small molecules, biologics, cell therapies, and gene therapy), the components used during manufacturing or present in the drug formulation must be adequately characterized for potential toxicity. Such components may include product- and process-related impurities, degradants, solvents, or excipients, which are novel and/or formulated for a new administration route and/or disease indication. It is essential that nonclinical and clinical data are evaluated to characterize the safety of the components. These data are identified by conducting a systematic and comprehensive review of primary literature databases, regulatory authority websites, and other industry or government literature sources. Critical information includes (1) physical and chemical properties, (2) pharmacokinetic/ADME, (3) toxicology, (4) clinical, and (5) regulatory limits/guidance to support a thorough nonclinical and clinical safety evaluation. This information is then summarized in a risk assessment or toxicology monograph to derive a health-based exposure limit such as the permissible daily exposure (PDE), or to ascertain key data gaps that may require additional toxicity testing consistent with regulatory guidance (ICH Q3). The monograph can then be used to set manufacturing specifications during process development or to justify specification limits to a regulatory authority. The literature review and risk assessment process will be presented, including derivation of an exposure limit for 3 challenging case studies from therapeutic drug products: an approved excipient for a novel route of administration, a genotoxic impurity, and a potentially immunomodulatory impurity.
P538 - Determination of Free and Total Gemcitabine and 2′,2′-Difluoro-2-,2′-Deoxyuridine (DFDU) in Minipig Bladder TissuesX
X. S. Yan1, M. Luna1, J. Showalter1, and Y. Liu1
1KCAS, Shawnee, KS, USA
A highly selective assay was developed for the determination of gemcitabine and DFDU in pig bladder tissue by LC-MS. Methods were established for the free (nonphosphorylated) and total (free + mono, di and triphosphates) analyte forms. For the free method, finely minced pig bladder (0.4-0.6 g) was place into tared Precellys homogenization tube containing ceramic beads and 5 mL of homogenization buffer (20% methanol) per gram tissue added. Following homogenization and sonication, the samples were centrifuged and aliquots (150 mcL) of supernatants placed on 96-well plate (Phenomenex phospholipid removal) for extraction. The eluates were analyzed on an API-5000 using valve switching with Aquasil C18 columns (Thermo) in positive mode. For the total method, samples were similarly processed, with the addition of a digestion step for the extracts (overnight at RT using shrimp alkaline phosphatase solution in 25 mM Tris buffer, pH 7.5, containing 1 mM MgCl2 and 0.5 M ZnCl2). Authentic phosphorylated standards were not commercially available, so only gemcitabine diphosphate was used to confirm hydrolysis completion under enzyme. The precision (% CV) of the free and total methods were <12.9% and the accuracy (% bias) was <7%. The stability of the analytes in pig bladder homogenates was confirmed up to 4 hours at room temperature.
P539 - Characterization of Renal Pathology Findings in Cynomolgus Macaques Administered Antibody Drug Conjugates (ADCs)
R. Yeager1, T. Van Vleet1, B. Philip1, M. Ramos1, S. Mantena1, K. Barnhart1, M. Logan1, K. Whitney1, S. Ralston1, L. Loberg1, and B. Bawa1
1AbbVie Preclinical Safety, North Chicago, IL, USA
For oncology, antibody drug conjugates (ADCs) have the potential to provide targeted delivery of potent payloads (eg, cytotoxics) to cancer cells. The present analysis will focus on characterizing renal pathology findings in cynomolgus macaques administered various ADCs. With regard to monitoring for renal toxicity, important nonclinical study design considerations include timing of interim clinical pathology sample collections, sensitivity and specificity of urinary biomarkers, and post-dose recovery/observation duration. For discovery of oncology ADC programs of various antibody-linker/payload combinations, we have characterized 3 distinct sets of renal findings in monkey toxicity studies of 1 to 3 months duration. The histopathology findings are (1) acute tubular epithelial degeneration/necrosis with altered tubular function and metabolic changes (hyperglycemia), (2) increase in glomerular matrix with progression to tubular injury, and (3) degeneration of tubules and collecting ducts, associated with urothelial changes. For some of these findings, there appears to be a progression over time that may lead to fibrosis, with potential for delayed clinical presentation. Despite longitudinal clinical pathology sample collections, no premonitory serum chemistry/urinary signals for the histopathology findings were detected. A key consideration for nonclinical safety evaluation of ADCs is characterizing antigen-dependent versus independent toxicities. Some degree of nonantigen-mediated uptake of antibodies, and thus ADCs, is anticipated in various tissues including kidney. For each of the renal histopathology findings described in this study, there are on-going efforts to understand contribution of nonantigen-mediated uptake and pathogenesis associated with ADC payload mechanism of action.
P540 - In Vitro Mechanistic Studies of Nontarget-Mediated Toxicity and ADC Uptake
L. Yu1, N. Corr1, A. Cercillieux1, J. Vogt1, W. Proctor1, A. Fullerton1, H. Wang1, N. Stagg1, and K. Staflin1
1Genentech Inc., South San Francisco, CA, USA
Antibody drug conjugates (ADC) are designed to deliver highly potent drugs specifically to tumor cells and minimize exposure to normal tissues. However, nontarget-mediated ADC toxicity has been observed clinically (and preclinically) in a variety of organs and tissues. We investigated the role of nontarget-mediated uptake using fluorescent-labeled nontargeting antibody (Ab) treatment of cell types associated with off-target toxicity. We were able to detect nontarget-mediated uptake in neurons, endothelial cells, bone marrow cells, and kidney epithelial cells. Ab uptake across cell types was assessed using high-content imaging and confirmed by confocal microscopy. Potential mechanisms including mannose receptor (MR) and Fc gamma receptor (FcγR)-mediated uptake, and macropinocytosis were investigated. Although, pinocytosis is likely the dominant mechanism in most cells, these studies utilizing FcγR and MR-blocking reagents suggest that MR, FcγR, or combination of both significantly contribute to uptake in specific cell types. Our ongoing efforts aim to further understand the mechanism of actions of nontarget-mediated uptake, inform design and development of ADCs and reduce off-target toxicity.
P542 - Assessment Strategy for Mutagenicity of Extractable Components of Medical Device by QSAR Analyses, Published Data, and Bacterial Reverse Mutation Assay
V. Kostrubsky1, J. Kiernan-Lewis2, and R. Saiakhov3
1Johnson & Johnson Vision Care, Jacksonville, FL, USA
2University of Florida, Gainesville, FL, USA
3Multicase Inc, Beachwood, OH, USA
To reduce testing for genotoxicity of finished medical devices, a risk assessment of individually identified extractable chemicals was conducted according to principles of ICH M7 guideline. Genotoxic potential of 16 extractable components of a medical device was evaluated using quantitative structure–activity relationship (QSAR) models, published data, and results of bacterial mutation assay. Components represented hydrophilic and hydrophobic methacrylate esters including those containing siloxane, 2-hydroxyphenyl benzotriazole, and anthracenesulfonic acid derivatives, N, N-dimethylacrylamide, and a bis(benzoyl) phosphine oxide derivatives. A MultiCASE “case ultra” validated suite was applied using 2 QSAR modules: ICH M7 (statistical and expert models) and additional GenTox statistical model covering chromosomal aberration and micronucleus assays. Only anthracenesulfonic acid derivative was identified as ICH M7 positive and classified as class 3. Subsequent results of mutation assay demonstrated no mutagenicity (class 5). However, this extractable is still controlled below TTC of 1.5 μg/d due to GenTox alerts and expert analyses. Negative predictions were supported by published data with exception of no information for methacrylate esters of siloxane. Further expert analysis supported a nongenotoxic nature of these compounds since their siloxane and methacrylate functional groups do not contribute to genotoxicity in vivo. The overall approach in genotoxicity risk assessment of extractable device components using specific examples is presented.