Abstract
P1: A 28-Day Intrathecal Toxicity Study With Trastuzumab in Cynomolgus Monkeys
Angélique P.J.M. Braena, Josée Perronb, Pierre Tellierb, Anthony R. Catalaa, Gerry Kolaitisa, and Wanping Genga
a Hoffmann – La Roche, Nutley, NJ, USA; b Charles River Laboratories Preclinical Services Montreal, Senneville, Quebec, Canada
Trastuzumab is indicated for the treatment of patients with breast cancer whose tumors overexpress the HER2 protein. Brain metastases have been observed in breast cancer patients, being the highest in women with HER2-overexpressing tumors. The blood-brain barrier and blood cerebrospinal fluid (CSF) barrier, however, may prevent trastuzumab from reaching appropriate concentrations in the brain and CSF following standard intravenous administration. Case studies have been reported in which trastuzumab was intrathecally administered for the treatment of metastatic brain cancer in humans. However, no preclinical safety evaluation has been performed for intrathecal application of trastuzumab. In order to evaluate the potential of local effects, a 28-day toxicology study with weekly intrathecal administration of trastuzumab was performed in cynomolgus monkeys, a trastuzumab cross-reactive species. Five male and female monkeys were evaluated per dose group, of which two monkeys/sex/group were evaluated after a 4-week recovery period. Animals were assigned to 3 groups which received 4 weekly doses of 0, 3 or 15 mg trastuzumab (equivalent to 0, 0.051, or 0.24 mg/g brain weight). No test article-related in-life, neurological, clinical pathology, or anatomic pathology findings were noted. The concentrations of trastuzumab in the CSF and serum were determined. At the high dose of 15 mg, maximum CSF concentrations (Cmax) of 403 µg/mL were attained. The applied doses and CSF concentrations achieved in the current study greatly exceeded those reported in patients. Combined with the results of clinical case studies, the results of this 4-week toxicology study in monkeys support future studies to evaluate intrathecal application of trastuzumab in patients with brain metastasis in HER2-positive breast cancer.
P2: Combination Toxicity Evaluation of Direct Acting Anti-Hepatitis C Virus (HCV) Agents With Pegylated Interferon-α (pIfnα) and Ribavirin (RBV)
T.W. Salcedo, R. Lange, J. Moehlenkamp, H. Janke, D. Walker, R. Diters, J. Woicke, K. Cimon, M. Abbott, H.G. Haggerty, T.P. Reilly, G.E. Schulze, and M.H. Davies.
Drug Safety Evaluation, Bristol-Myers Squibb Co.
HCV infection remains a significant health concern worldwide, with over 170 million individuals infected. Chronic infection may progress to cirrhosis, end-stage liver disease, and/or hepatocellular carcinoma. The current approved treatment for chronic HCV infection is a combination of pIFNα and RBV. While this therapy can result in sustained virologic responses with reduced risk for progressive disease, it is not effective in all patients and is limited by adverse events requiring dose reduction or discontinuation. New classes of antiviral medications are being developed as adjuncts or replacements to pIFNα and/or RBV therapy and include direct-acting antiviral agents (DAAs) that target specific viral components. To evaluate the non-clinical toxicity profile of novel DAA small molecules when combined with pIFNα/RBV, we administered individual BMS-DAAs to cynomolgus monkeys by daily oral gavage for up to 1-month. The same groups of monkeys also received marketed formulations of pIFNα (subcutaneously every other day) and RBV (oral solution daily). DAA doses were chosen to generate clinically relevant systemic exposures and pIFNα/RBV doses were multiples of approved clinical doses. Additional groups of monkeys received either BMS-DAA vehicle alone and/or BMS–DAA vehicle with pIFNα/RBV and served as control groups. Standard toxicokinetic, toxicologic, and pathologic endpoints were assessed along with immunogenicity against pIFNα. As validation of the experimental system, changes consistent with known effects of RBV on red cell turnover, IFNα inhibition of erythropoiesis, and anti-IFNα antibody formation were evident in groups receiving these agents. The addition of BMS-DAAs to the pIFNα/RBV regimen did not result in new toxicities relative to the BMS–DAA or pIFN/RBV treatments alone. This approach provides a useful model in which the non-clinical safety of novel anti–HCV agents may be evaluated for toxicokinetic and/or toxicologic interactions with pIFNs/RBV.
P3: Withdrawn
P4: Withdrawn
P5: A 26-Week Repeat-Dose Toxicity Study in Cynomolgus Monkeys With Xoma 052, a Novel Monoclonal Antibody Targeting IL-1 Beta
Kathleen Meyer1, Kenneth Der1, Jeremy Ma1, Yero Espinoza1, Liching Cao1, Carolyn Gasper1, and Charles Bechtel2
XOMA (US) LLC1, Berkeley, California and Charles River Laboratories2, Reno, Nevada.
XOMA 052 is an ultra-high affinity (300 fM) humanized IgG2 monoclonal antibody that specifically binds to IL-1 beta (IL-1β) and inhibits activation of the IL-1 receptor. This activity is expected to prevent the cellular signaling events that produce inflammation. IL-1β is a proinflammatory cytokine involved in the development of many diseases including Type 2 diabetes, rheumatoid arthritis, gout. XOMA 052 has been evaluated in two Phase 1 clinical studies in Type 2 diabetes designed to assess safety, pharmacokinetics and measures of glycemic control and systemic inflammation. It was well tolerated and demonstrated clinically meaningful biologic activity in diabetes and systemic inflammatory assessments. The cynomolgus monkey was selected for safety evaluation due to similar binding affinity and in vitro functional activity between human and monkey IL-1β with XOMA 052. XOMA 052 was administered by subcutaneous injection weekly for 27 weeks at doses up to 90 mg/kg, with a recovery period of 3 months. The nonclinical safety evaluation included daily clinical observations, food consumption, body weight, hematology, clinical chemistry, coagulation parameters, flow cytometry, urinalysis, ophthalmic examinations, electrocardiography (ECG), heart rate/blood pressure, T cell-dependent antibody response (TDAR), toxicokinetics, immunogenicity, as well as macroscopic and microscopic pathology. The results showed no toxicologically significant findings and the no-observed-adverse-effect level (NOAEL) was considered to be 90 mg/kg. This study supports the phase 2 clinical evaluation of XOMA 052 in patients with Type 2 diabetes as well as other chronic indications.
P6: Preclinical Safety of Recombinant Human Hyaluronidase (rHuPH20)
RC Liu1, M Zepeda1, KC Crowder2, N Lalayeva2, N Makori2, M Bauman2, BJ Sugarman1, GI Frost1, WH Bee1.
1Halozyme Inc, San Diego, CA. 2SNBL USA, Everett, WA.
rHuPH20 is a novel drug delivery enzyme that is locally degrading hyaluronan to transiently increase bulk fluid flow. rHuPH20 improves the subcutaneous (SC) absorption profiles of fluids and co-injected drugs and biologics. Formulation with rHuPH20 permits delivery of volumes that substantially exceed the typically tolerable SC limits and achieves bioavailability of protein therapeutics that approximate that of IV administration. In support of this novel drug delivery permeation enhancer and to characterize the preclinical safety profile, rHuPH20 was evaluated for acute, subacute and chronic toxicity in the Cynomolgus monkey.
IV or SC injections of 30 mg/kg (3,600,000 U/kg) rHuPH20 dosed acutely, and 5 mg/kg (600,000 U/kg) dosed once daily for 7 days, elicited no adverse findings, as anticipated from the short t½, low systemic exposure and bioavailability of ≤5% following SC dosing. A 9-month study in monkeys dosed SC once weekly at 0.02, 0.2, and 2 mg/kg/dose (2,400, 24,000, 240,000 U/kg/dose) found no systemic toxicity. Minimal perivascular lymphoplasmacytic infiltration was observed at the injection sites in the mid- and high-dose, showing substantial improvement after a 4-week recovery. The finding was likely a local response of monkeys to the injection of a human protein and therefore considered non-adverse. The NOAEL was 2 mg/kg. Very low levels of plasma hyaluronidase activity were detectable at the high-dose only (for ≤6 months in males, for 9 months in females). Loss of plasma hyaluronidase activity correlated with increased levels of hyaluronidase neutralizing activity. Collectively, these studies establish that high SC doses of rHuPH20 resulted in very low systemic exposure and no systemic toxicity, which is consistent with rHuPH20’s activity as a locally-acting, transiently-active, fully reversible permeation-enhancing excipient.
P7: Embryo-Fetal Development Study of Pertuzumab Administered By Intravenous Injection To Pregnant Cynomolgus Monkeys
Ortega S*1, Arima A*2, Chihaya Y2, Allison D1, Braen A3, Lauriault V4, Dybdal N1.
1Development Sciences, Genentech Research and Early Development, SSF, CA; 2SNBL Ltd., Kagoshima, Japan; 3Hoffmann-La Roche, Nutley NJ; 4formerly Genentech, currently private consultant, SSF, CA.
Pertuzumab, a recombinant humanized monoclonal antibody directed against the extracellular domain of the human epidermal growth factor receptor, type 2 (HER-2), was administered IV twice weekly for nine doses [loading dose of 0, 30, 100, or 150 mg/kg on Gestation Day (GD) 19, and maintenance dose of 0, 10, 33.3, or 100 mg/kg on GD 26, 29, 33, 36, 40, 43, 47, and 50] to 12 pregnant cynomolgus monkeys per group during the period of fetal organogenesis (GD 20 to 50). Between GD 25 and 70, abortion or embryo-fetal death was confirmed in 20 of 36 animals receiving test article. The decrease in fetal survival was dose related (0/12, 4/12, 6/12 and 10/12, respectively). On GD 100, at scheduled cesarean section, significantly low, or tendencies toward low values in amniotic fluid volume (oligohydramnios), fetal weight, and absolute and relative lung and kidney weights were noted in all test article groups when compared with the control group. In addition, hypoplasia (delayed development) of several kidney components was observed microscopically, with dose dependent severity, in all fetuses from all test article groups. Some external, visceral and skeletal abnormalities also noted were considered related to oligohydramnios and/or delayed fetal development. No test article-related changes were noted in the number of vertebral centrum, or microscopically, in the heart or placenta in any group. Systemic Pertuzumab exposure in dams and fetuses was confirmed. In conclusion, administration of Pertuzumab to pregnant cynomolgus monkeys between GD 19 and 50 was associated with high fetal lethality and oligohydramnios accompanied by delayed development of the fetal kidneys, and some external, visceral and skeletal abnormalities. The results of this study indicate that dosing pregnant cynomolgus monkeys with a monoclonal antibody from GD 20 to 50 is sufficient to cover the critical period of fetal organogenesis and to provide relevant reproductive safety information.
*Equal authorship
P8: Embryo/Fetal Development in Cynomolgus Monkeys Exposed to Rituximab
A. Vaidyanathan1, K.McKeever1, H. Tsusaki2, S. Eppler1, S.Ortega1, J. Beyer1
1Genentech, South San Francisco, CA, USA; 2Shin Nippon Biomedical Laboratories, Ltd., Kagoshima, Japan
Rituximab is a chimeric murine/human engineered IgG1 anti-CD20 monoclonal antibody, which selectively depletes CD20-expressing cells in peripheral blood and lymphoid tissues. As part of the registration-enabling program in rheumatoid arthritis as well as other potential non-oncology indications, a cynomolgus monkey embryo-fetal development (EFD) toxicology study was performed. Importantly, critical events in fetal cynomolgus monkey immune system development, such as T and B-cell development occur during the second trimester of gestation. Although maternal dosing was ended during the first trimester at gestation day (GD) 50, maternal and fetal exposure was confirmed at all doses of rituximab during the second trimester at GD 100. Consequently, both dams and fetuses demonstrated B-cell depletion in either peripheral blood (dams; as judged by lymphocyte counts) or lymphoid tissues (fetuses). In this study, female cynomolgus monkeys were administered rituximab intravenously at doses of 0/0, 15/20, 37.5/50 and 75/100 mg/kg (loading dose/study dose) from GD 20 to 50. Pregnancies were terminated on GD 99-102 by cesarean sections on all dams. There were no test article-related abnormalities in fetal body weight, placental weight, external measurements, or in visceral and skeletal findings in the any of the rituximab groups. Noteworthy findings in dams were limited to a significant decrease in lymphocyte count, which resulted from the pharmacologic effect of B-cell depletion. There was a trend toward B cell recovery in dams on GD 103 as compared to GD 50. Noteworthy findings in fetuses included significantly lower absolute and relative spleen weight as compared to study controls. Immunohistochemistry (IHC) on lymphoid tissues revealed rituximab-related B-cell depletion in sections of the spleen and lymph node, consistent with expected pharmacology. Although reversibility of this finding was not demonstrated in this study, follow-up studies have confirmed the reversibility of B-cell depletion following fetal and neonatal exposure. In conclusion, under the conditions of this study, rituximab had no teratogenic effects on the developing fetus. Rituximab administration to maternal animals during GD 20-50 resulted in placental transfer of rituximab to the fetus and resulted in B-cell depletion in fetal lymphoid tissue.
P9: Seven Day Repeat Dose Exploratory Toxicity Study of a Targeted Hepatitis C Drug
Douglas Fuhrer1, Lin Pan1, Brad Buckman1, Steve Ammons1, and Scott Seiwert1
1Research and Development Division, InterMune, Inc, 3280 Bayshore Blvd., Brisbane, CA
Principals of medicinal chemistry were applied to a promising novel scaffold structure to specifically target HCV polyprotein processing with optimal drug-like properties. In order to evaluate preliminary TK and drug safety characteristics a 7-day repeat dose toxicity study was performed with gene expression analysis at doses of 0, 50, 100, and 200 mg/kg in both sexes of Sprague-Dawley Rats on a drug candidate. No significant dose dependent effects on body weights, food consumption, blood coagulation, clinical observations, organ weights, or hematology were found. C max and area under the concentration-time curves (AUC) were comparable between Day 1 and Day 7 indicating that exposure was not altered with repeat dosing although exposure was not linear with dose. The highest plasma C max seen was with the medium dose (100 mg/kg) suggesting that the high dose toxicity seen was not C max driven. Clinical chemistry analysis of plasma showed a statistically significant increase in the high dose treated group for bilirubin (females), BUN (females) and phosphorous (males) with similar trends in both sexes. At necropsy minimal hepatocyte single cell necrosis, minimal hepatocyte increased mitosis (5/12 animals), and minimal hepatocyte single cell necrosis (1/6 males), and eosinophilic degeneration (1/6 females) was noted in addition to abnormal gastrointestinal (GI) contents found in many of the 200 mg/kg dosed animals. Liver Gene Expression ToxFX analyses were negative for all 29 toxicity profiles measured. Acute rat and NHP studies at higher exposure levels support GI effects as the major target for dose limiting toxicity.
P10: Three-Month Toxicity Study With Cremophor RH 40/Polyethylene Glycol 400 or With Solutol HS 15/Polyethylene Glycol 400 In Rats
Alan Stokes, Chris Merrill, and Holly Jordan
Safety Assessment, GlaxoSmithKline, Research Triangle Park, NC
GlaxoSmithKline (GSK) Safety Assessment only considers non-traditional vehicle formulations when conventional formulations do not provide adequate systemic exposure to assess toxicity. One challenge with this strategy is the absence of toxicology information on these formulations. This study was conducted to collect toxicity data with two non-traditional vehicle formulations. Groups of rats were given water, 30:70 Cremophor RH 40/polyethylene glycol (PEG) 400, or 30:70 Solutol HS 15/PEG 400 once daily for 91 days by oral gavage. Administration of the Cremophor formulation was associated with minimal, focal/multifocal hepatocellular necrosis that was not considered adverse based on the minimal severity and the absence of increases in serum hepatobiliary markers. Both PEG-containing vehicles were associated with increased relative kidney weights in males. There were no correlating macroscopic or microscopic findings in the kidney or bladder, though both formulations were associated with increased urine specific gravity and minimally altered urine electrolytes in both genders. Males given the Cremophor formulation also had mildly increased serum urea and females had markedly increased urine calcium/creatinine ratio. Urine phosphorus/creatinine ratio was also increased in males and females given the Cremophor formulation. Males and females given the Solutol formulation had decreased urine glucose parameters and increased urine calcium/creatinine; males had increased urine volume. Males in both groups had minimally decreased serum electrolytes and females had minimally increased serum cholesterol. Administration of either formulation was also associated with decreased thymic weight with no concurrent microscopic findings. Neither formulation produced overt toxicity in 91 days of dosing but the Solutol formulation produced fewer effects in rats.
P11: Thyroid Gland Pathology and Evidence of Hypothyroidism in Sprague-Dawley and Long-Evans Rats Treated with Amiodarone for Seven Days
Richard A. Peterson, David L. Krull, Mary J. Keener, Sundeep A. Chandra.
GlaxoSmithKline, Safety Assessment, Research Triangle Park, NC 27709
Amiodarone is a cationic-amphiphilic, anti-arrhythmic drug associated with drug-induced phospholipidosis in several cell types of humans and preclinical species. Amiodarone is associated with hypothyroidism in humans, although characterization of amiodarone-induced changes in rodent thyroid glands has not been reported. Amiodarone causes hypothyroidism by inhibiting 5’-deiodinase resulting in preferential conversion of triiodothyronine (T3) to inactive reverse-T3, and follicular cell cytotoxicity. Male Sprague-Dawley (n = 5) and Long-Evans (n = 5) rats were treated orally with Amiodarone at 300 mg/kg/day for 7 days. All animals survived to terminal necropsy. Treated animals lost up to 15% body weight compared to vehicle controls. Follicular cells exhibited prominent vacuolation and showed evidence of degeneration/necrosis and decreased colloid. Immunohistochemistry (IHC) for thyroglobulin showed differences when compared with vehicle controls, there was decreased immunoreactivity for thyroglobulin within follicular cell cytoplasm and within the follicular colloid, confirming depletion of colloid. Transmission electron microscopy showed prominent multilamellar bodies within lysosomal structures of follicular cells (phospholipidosis), blunting and fusion of follicular cell microvilli, decreased colloid and follicular cell degeneration/necrosis. Vacuolated, hypertrophic pituitary cells in the adenohypophysis were prominent in treated rats from both strains. IHC for thyrotropin stimulating hormone (TSH) showed that the vacuolated hypertrophic pituitary cells were strongly immunoreactive, indicating that the cells were thyrotropes. The pituitary change suggests hypothyroidism, due to a lack of negative feedback of active T3, subsequent increased TSH expression and secretion, and accumulation of TSH within hypertrophic thyrotropes. A limited clinical chemistry profile showed up to a 2.4X (mild to moderate) increase in serum cholesterol in treated rats when compared with controls and increased hepatocellular lipid by TEM, which hints at a hyperlipidemic state as would be expected in hypothyroidism. Extra serum from the study was not available to evaluate T3, T4, and TSH levels. This study characterizes the morphologic and ultrastructural aspects of thyroid gland changes associated with amiodarone treatment in the rat.
P12: From Phenotyping to Host Resistance Models: A Comprehensive Immunotoxicologic Investigation of a Gamma Secretase Inhibitor in Rats
WJ Freebern1, FG Burleson2, SQ Wells1, M Slade1, B Grubor1, GD Pilcher1, TP Sanderson1, HG Haggerty1
1Bristol-Myers Squibb Co., 2Burleson Research Technologies
An immunologic investigation of BMS-708163, a gamma-secretase inhibitor in development for Alzheimer’s disease, was initiated after peripheral blood (PB) and splenic lymphocyte depletion occurred in female rats treated daily for 1-month with BMS-708163 (AUC ≥ 32900 ng·h/mL, ~4X clinically active exposure). In investigative studies designed to characterize these findings, an array of specialty endpoints were performed including flow-cytometric based PB and splenic T-cell, B-cell, and IgM phenotyping; immunofluorescence analyses of splenic T-cell, B-cell and IgM; and T-cell dependent antibody response (TDAR). In female rats administered BMS-708163 for 1 month, exposure-dependent decreases in IgM expression per circulating B-cell occurred at AUC ≥ 3000 ng·h/mL. At AUC ≥ 4670 ng·h/mL, decreased number of IgM+ B-cells in the splenic marginal zone (MZ) was observed. At higher exposures (≥ 22800 ng·h/mL), decreases in PB T- and B-cells and splenic T-cells were observed. These findings were associated with ≥ 97% suppression of TDAR to keyhole limpet hemocyanin. In these studies and in 3- and 6-month rat toxicology studies, no opportunistic infections occurred supporting that the observed changes in immune parameters do not compromise rat net immune health. To investigate the potential effect of BMS-708163 on host immunity, two host resistance models in aged (10-12 months old) females were utilized to explore the relationship between the affected immune parameters and net immune health: 1) a streptococcal model in which aged rats were challenged IV with Streptococcus pneumoniae, a blood-borne pathogen infection that reportedly requires splenic MZ activity for clearance and 2) an intranasal influenza A model to evaluate the complex interactions of cell-mediated and humoral immune functions. Although PB and splenic lymphoid drug-related changes occurred in both models and the IgM-specific responses to streptococcus and influenza were impaired, there were no drug-related effects on survival or impairment of pathogen clearance supporting that the complexity and redundancy of the immune system provided sufficient net immune health in the presence of gamma secretase inhibition.
P13: Safety Profiles of Cruciferin-Rich (Puratein) And Napin-Rich (Supertein) Canola Protein Isolates Following Subchronic Dietary Toxicity Studies in Sprague Dawley Rats
L.A. Mejiaa, C. K. Korgaonkarb, M. Schweizerc, C. Chengelisb, G. Maritb, M. Novillab, E. Ziemerb, P. Hughesa, R. Grabiela and M. Empiea
aArcher Daniels Midland Company, Decatur, IL; bWIL Research Laboratories, LLC, Ashland, OH; cBurcon NutraScience Corporation, Winnipeg, Canada.
Ninety day subchronic dietary toxicity studies of a napin-rich canola protein isolate (Supertein) and a cruciferin-rich canola protein isolate (Puratein) were conducted separately using Sprague-Dawley rats. Supertein and Puratein were obtained from canola using a proprietary process. Besides inherent nutritional value, these isolates possess unique functional properties such as emulsifying/binding/transparent characteristics for use in processed foods. Rats were fed for 90 days an AIN-93 G based protein-free diet containing respectively 5%, 10% and 20% (w/w) Supertein or Puratein (test articles). Vitamin-free casein (20% w/w) was used as control. Body weights, food consumption, locomotor activity, behavioral and clinical pathology parameters were recorded at various study intervals, with organ weight measurements, macroscopic and microscopic examination at study termination. There were no test article related effects on clinical observations, ophthalmic examinations, functional observational battery parameters, motor activity, clinical pathology parameters, macroscopic or microscopic findings for Supertein or Puratein. Lower body weight gains, correlating with lower food consumption, were noted in the 10% Supertein-treated males and 20% Supertein-treated males and females. Lower food consumption, most notable during the first week of the study, suggested lesser palatability of Supertein containing diets. A slightly higher thyroid/parathyroid weight ratio was noted in the 20% Puratein-treated males and females without a histopathological correlate. Based on these results and comparison with relevant WIL historical control reference range values, the no observed adverse effect level (NOAEL) for both protein isolates was the highest fed level of 20% which as a mean daily intake for 90 days was equivalent to 12.5 g/Kg BW/day for males and 15.0 g/Kg BW/day for females for Supertein and to 11.2 g/KgBW for males and 14.1 g/Kg BW/day for females for Puratein. In conclusion, Supertein and Puratein were considered safe under the conditions of the study.
P14: Soft Tissue Mineralization Induced by Mek Inhibitor G-573 in Rats
D. Diaz, K. P. Allamneni, H. Hiraragi, N. La, J. Tarrant, E. F. Choo, and D. M. Dambach
Genentech, Inc.
The RAS/RAF/MEK/ERK signaling pathway represents an important focus in oncology research, and MEK, a kinase downstream of Ras and Raf oncogenes, constitutes a high priority target. Several small molecule MEK inhibitors are currently in clinical trials for oncology and autoimmune indications. G-573, a selective MEK inhibitor which shows efficacy in xenograft models, caused soft tissue mineralization in the rat, secondary to serum inorganic phosphorous elevation. Male rats were given G-573 at 0, 3, 10 and 20 mg/kg/day via oral gavage for 14 days. Animals were euthanized on day 15 (terminal necropsy) and day 21 (recovery necropsy), with additional blood sampling at day 3 and day 7 for haematology and serum chemistry evaluation. G-573 caused tissue mineralization that was dose-dependent and related to systemic levels of inorganic phosphorous. Sensitivity of tissues for mineralization varied with the stomach affected at lower elevations of serum phosphorous concentrations when compared to baseline, whereas other tissues such as heart and vasculature were only affected at relatively higher phosphorous concentrations. The rat appears to be particularly sensitive to this type of toxicity, although the reason for this is unknown as MEK seems to have a highly conserved role in systemic phosphorous regulation. This finding is consistent with previously described mineralization caused by structurally-distinct MEK inhibitors, suggesting that soft tissue mineralization might be potentially on-target. We provide a plausible mechanistic rationale to explain the hyperphosphatemia-associated soft tissue mineralization as a direct effect of MEK inhibition, involving MEK-driven FGF-23/Klotho signaling in the kidney.
P15: Developmental and Pre-/Postnatal Reproductive Toxicity of rHuPH20
S Nadjsombati1, A Hoberman2, D Newcomb2, BJ Sugarman1, GI Frost1 and WH Bee1.
1Halozyme Inc, San Diego, CA, and 2CRL, Horsham, PA.
rHuPH20 is a novel drug delivery enzyme that is locally degrading hyaluronan to transiently increase bulk fluid flow. rHuPH20 improves the subcutaneous (SC) absorption profiles of fluids and co-injected drugs and biologics. Formulation with rHuPH20 permits delivery of volumes that substantially exceed the typically tolerable SC limits and achieves bioavailability of protein therapeutics that approximate that of IV administration. In support of this novel drug delivery permeation enhancer, rHuPH20 was evaluated for developmental and pre-/postnatal toxicity in pregnant mice.
In the range-finding and developmental toxicity study, pregnant mice were injected SC with rHuPH20 QD on DGs 6-15 at doses of 0, 1, 3, 10 and 30 mg/kg and 0, 3, 9 and 18 mg/kg, respectively. Caesarean-section and necropsy occurred on DG 18. 1/8 dam in the 10 mg/kg-group had a litter that was 69% resorbed while 4/8 dams in the 30 mg/kg-group had litters that were 100% resorbed. The litter size of the 30 mg/kg group was reduced as compared to the controls. Fetal body weights were significantly reduced in the 9 and 18 mg/kg groups compared to the vehicle control group. The number of late resorptions was increased in the 9 and 18 mg/kg groups and was considered test article related. Cmax and AUC(0-24) values generally increased on day 15 as compared to those on day 6. In the prenatal/postnatal study, pregnant mice were dosed QD 6 to DL 20 at doses of 0, 3, 6 and 9 mg/kg. All natural delivery and litter observations were unaffected by dosages of rHuPH20 as high as 9 mg/kg/day. The NOAEL of rHuPH20 were: maternal 18 mg/kg, developmental 3 mg/kg and prenatal/postnatal 9 mg/kg. rHuPH20 proved to be embryo-fetotoxic but did not show an overt dysmorphogenic potential. NOAEL’s established in these studies provide wide safety margins for proposed human use.
P16: Why Use Miniature Swine in Dermal Research
Liu, J.1; Brown, L.1,2; Madsen, T.1; Lawson, C.1; Blair, E.1; Bouchard, G. F.1,2
1Sinclair Research Center, LLC, Auxvasse, MO, USA; 2Sinclair BioResources, LLC, Auxvasse, MO, USA
Swine have been used extensively in dermal research because of the similarities of the integument to humans. Miniature swine offer many distinct advantages for dermal research. Skin researchers require efficient animal models which are predictive of human responses. Pig skin is anatomically, physiologically, biochemically and immunologically similar to human skin, and the skin is ‘fixed skin’ like humans. Pig skin mirrors human skin in having a sparse haircoat, a relatively thick epidermis, similar epidermal turnover kinetics, lipid composition and carbohydrate biochemistry, lipid biophysical properties, and a similar arrangement of dermal collagen and elastic fibers. Porcine or miniature swine dermal models offer significant advantages and have a record of predicting treatment modalities in humans over models with loose skin. Included are models for wound healing (including delayed diabetic model), phototoxicity, dermal toxicology, dermal PK/TK, iontophoresis, dermal irritation, burns, hypertrophic scarring, contact allergic dermatitis, depigmentation and chemical vesication. These dermal study types will be briefly described and aspects of these studies in miniature swine will be discussed. Miniature swine models provide useful safety and efficacy data for novel cutaneous therapy product development. Miniature swine offer researchers unique tools in dermal research.
P17: Göttingen Minipigs As The Nonrodent Species in the IND For KNS-760704
SW Frantz,1 DR Demady,2 JA Dalton,1 EJ Popke,1 EJ Tasker,1 E Ingersoll,2 and VK Gribkoff21
MPI Research Inc., Mattawan, MI and 2 Knopp Neurosciences, Pittsburgh, PA
Although the beagle dog remains a pharmaceutical industry standard for many nonrodent toxicology studies, adverse pharmacodynamic responses can necessitate the use of an alternative nonrodent species. During pre-IND studies with KNS–760704, a CNS drug in Phase II clinical trials for the treatment of ALS, single doses of KNS-760704 administered to dogs produced significant exposures after gavage administration of 2.5, 7.5, or 25 mg/kg but also produced dose-limiting toxicity (emesis) immediately following dosing; the single-dose no-observed-adverse-effect-level (NOAEL) in dogs was 2.5 mg/kg. A no-observed-effect-level (NOEL) could not be identified due to emesis at all dosages, suggesting that dogs were not an acceptable nonrodent species. In minipigs, the single-dose NOAEL was 25 mg/kg (AUC0-t of 18.5 μg·hr/mL), with little emesis noted, while 7.5 mg/kg (AUC0-t of 3.0 µg·hr/mL) was the NOEL. A lower incidence and severity of clinical signs following single doses of KNS–760704 supported minipig selection for further toxicology testing and 75 mg/kg was selected as the high dose level for a 2-week IND-enabling toxicity study.
The definitive minipig studies employed a combined acute (MTD, single doses up to 300 mg/kg) and 2-week multiple dose (doses of 0, 7.5, 25, or 75 mg/kg/day) study designs with a 2-week recovery period. KNS–760704 was well tolerated in pigs, with only moderate clinical signs (decreased activity, inappetence, salivation, and mild emesis) that were not present during the recovery phase and no adverse treatment-related anatomic or clinical pathology findings. The NOAEL was considered ≥75 mg/kg/day (Day 13 AUC0-24 of 128.6-197.7 µg·hr/mL); 7.5 mg/kg/day (Day 13 AUC0-24 of 9.0-9.9 µg·hr/mL) was the NOEL in this 2-week study.
Finally, a cardiovascular study in Göttingen minipigs (3/sex) at 0, 7.5, 25, or 75 mg/kg showed only minor effects, with a NOAEL of 75 mg/kg. Thus, multiple toxicology studies using Göttingen minipigs have successfully supported first-in-human and ongoing Phase II clinical trials for KNS–760704.
P18: The Utility of the Minipig in P38 Map Kinase Inhibitor Testing
J. Schützsack1, A. Gibbs2, J. Parish2, K. Gill2
1 LEO Pharma A/S, 55 Industriparken, DK-2750, Ballerup, Denmark; 2Covance Laboratories Ltd, Otley Road, Harrogate HG3 1PY, UK
P38 map kinase inhibitors are currently investigated as a target for many types of inflammatory diseases due to their inhibitory action on pro inflammatory cytokines such as TNFα and IL-1β and other inflammatory mediators. The systemic repeat 28-day dose toxicity of a P38 MAPKi has been evaluated in rats and minipigs. The minipig was chosen as the non-rodent species as there were no major qualitative or quantitative interspecies differences between man and minipig in an in vitro comparative metabolic profiling study. Also, the minipig was shown to be a reasonable predictor of human AUC0-24 and Cmax concentrations. Signs of toxicity included lymphoid proliferation in rats and GI inflammation in minipigs as indicated by increases in WBC and acute phase reactants and confirmed histologically. A number of target related toxicities have previously been reported clinically and preclinically e.g. hepatic, cardiac, CNS and skin toxicities but were not observed in these studies. Rats were much less sensitive towards the adverse effects of the compound and tolerated doses 10-100-fold higher than minipigs. The reason why rats and minipigs show this difference in sensitivity to the adverse effects of the compound and the different toxicities is not known. Previously J.W. Davis (SOT 2008) reported that species-specific toxicities have been observed with kinase inhibitors and the dog is uniquely sensitive to p38 MAPK toxicity due to over-expression of p38 in B lymphocytes resulting in acute lymphoid necrosis and colonic haemorrhage. Consequently, the NHP has traditionally been selected as the second species. Even though the minipig was more sensitive than the rat it is considered of much less sensitivity than the dog and comparable to the NHP and human. These data demonstrate that the minipig is an excellent alternative to the primate for the toxicological evaluation of p38 MAPK inhibitors and thus nicely in line with the new proposed directive on animal experimentation (86/609/EEC) with provisions to reduce the number of NHP to an absolute minimum. The minipig does not share the problems encountered with the use of the dog due to over-expression of p38 and demonstrated excellent concordance with the human in this respect.
P19: Normal Organ Weight Data for the Yucatan Miniature Swine
Liu, J.1; Brown, L.1,2; Madsen, T.1; Lawson, C.1; Blair, E.1; Bouchard, G. F.1,2
1Sinclair Research Center, LLC, Auxvasse, MO, USA, 2Sinclair BioResources, LLC, Auxvasse, MO, USA
Swine have been used extensively in research because of correlated similarities to humans. Miniature swine offer many distinct advantages for researchers but reference normal data is sometimes lacking for miniswine. In an effort to generate a database on baseline information for Yucatan miniature swine, we are reporting quality controlled organ weight data from naïve juvenile (~1 month) and young adult (5-8 month) Standard Yucatan miniature swine of both genders. Absolute organ weights (g), organ to body weight ratio, and organ to brain weight ratio (%) are offered. Number of animals per gender (N), Mean, Standard Deviation, and Range for each parameter are presented in Tabular form. Weight data on brain, adrenals, epididymis, heart, kidneys, liver, lungs, ovary, pituitary, prostate, seminal vesicle, spleen, testis, thymus, thyroid w/parathyroid, body weight and the appropriate ratios are presented for the two age categories of Yucatan.
P20: Application of Animal Models for AN0128 Topical Formulation Development
C-W. Chen, D. Imbert, and S. Chanda
Anacor Pharmaceuticals, CA
AN0128 is a novel borinic acid ester small molecule with good antibacterial activity against Propionibacterium acnes (P. acnes) and moderate anti-inflammatory activity. The selection of topical formulations of AN0128 for the treatment of acne was based on the criteria of optimization of skin penetration, anti-inflammatory activity in selected in vivo models and minimization of skin irritation. The animal models used in support of AN0128 formulation development included: 1) phorbol myristate acetate (PMA)-induced mouse ear edema model of inflammation, 2) hairless guinea pig model of skin irritation, and 3) minipig model of skin irritation. Eight formulation prototypes with greater flux (3 to 5 fold enhancement compared to previously tested cream formulation) through human cadaver skin were selected for entering animal studies. Six of eight prototypes showed greater inhibition of PMA-induced ear swelling compared to cream formulation, and three of six prototypes showed significant inhibition (P< 0.05) compared to the corresponding vehicle control. In the hairless guinea pig study, non-occluded dermal application of AN0128 and vehicle formulations once daily for 5 days resulted in slight to moderate dermal findings, and all formulations and vehicles were classified as non-irritants. Two formulations were selected for the further evaluation of potential skin irritation in a minipig model. No systemic or dermal toxicity was observed after twice daily (6 hours apart) non-occluded dermal applications for 7 consecutive days, and no systemic exposure of AN0128 was evident, therefore, these two formulations were considered as clinical formulation candidates. In summary, the use of multiple application sites (6-8 sites per animal) in the hairless guinea pig model is considered as a useful screening tool for the early phase of topical formulation development. The application of efficacy and skin irritation models to topical drug development aids selection of optimal clinical formulations.
P21: Sporatic Occurrence of Low Fertility Rates in Control CD Rats
Hentz, KL†; Neal, BH†; Tyl, RW‡; Hoberman, AM*; and Wilson, D.#
†Exponent, 1800 Diagonal Road, #300, Alexandria, VA 22314, ‡RTI International, 3040 Cornwallis Road, Research Triangle Park, NC 27709-2194, *Charles River Preclinical Services, 905 Sheehy Dr. Bldg. A, Horsham, PA 19044, #sanofi-aventis US, Inc. 9 Great Valley Parkway, Malvern, PA 19355
In recent reproductive/developmental studies fertility rates for control CD rats were observed to be considerably lower than expected (<80%) based on historical control rates. We have explored the factors that were a possible cause of the reduced fertility. Various environmental and study-specific factors were considered and excluded. Compared to historical controls or the F0 generation data, several observations in the F1 generation control group of a recent two generation reproductive study suggest the observed reduction in fertility may be inherent in the test animal model as used. The F1 observations included: increased body weight of F1 animals, an increased number of dams in prolonged or persistent estrus, decreased mating success, and increased litter size. Similar findings were observed in a different laboratory using animals from the same animal supplier. The observations in these studies are compared to historical control data and the findings considered in our evaluation of reduced fertility are presented.
P22: Comparison of Sprague Dawley IGS and Wistar (Hannover) Strains of Rat Used in Carcinogenicty Studies
P Mansell, S Y Smith, R L Gregson, L Kangas
Charles River Preclinical Services, Montreal, Quebec, Canada.
The choice of rat strain for two year carcinogenicity studies is influenced by several factors. It is advisable that drug development programs should be undertaken using the same strain throughout for consistency. Sponsor preferences can dictate strain and selection is largely influenced by historical data and experience with a given strain.
Data collected over a ten year period from Sprague Dawley IGS and Wistar (Hannover) rats from a conventional non-barrier facility, were compared. Primary end points included survival, body weight and body weight gain, onset, progression and incidence of common neoplastic and non-neoplastic lesions.
Restricted feeding regimes were found to influence growth rates, body weight, survival, and tumor progression in the Sprague Dawley rat. Housing regimens appeared to have less influence on these parameters. In contrast Wistar rats growth and survival allowed for ad libitum feeding and terminal body weights were lower than the Sprague Dawley. Associated with the lower bodyweights were increased survival rates and a lower incidence of spontaneously occurring tumors over a two year period.
The type of lesions seen in these strains differed principally in incidence with no one lesion being peculiar to either strain. Spontaneously occurring tumors were broadly similar in both strains and included neoplasms of pituitary, adrenal, and mammary tissue amongst the most common recorded.
In conclusion, strain selection for carcinogenicity studies should be considered by at least the 13 week range finding stage with reference to previous work performed with the drug class. Since there are no major differences in spontaneously occurring tumors between Sprague Dawley IGS and Wistar (Hannover) rats, both strains have been used for carcinogenicity testing and successful drug registration.
P23: Withdrawn
P24: Feasibility of Continuous Intravenous Infusion in Long-Evans and Brown Norway Rats
A. Prefontaine, Y. Trudel, S. Caron, C. Copeman
Charles River Preclinical Services, Montreal, Quebec, Canada
An increase number of new therapeutics have the potential to either elicit ocular effects or interact with the melanogenesis process. These new therapeutics may be better evaluated in a strain with pigmentation in order to assess accordingly the toxicity. The Sprague Dawley rat is frequently the species of choice for intravenous infusion toxicity study and has been used in our laboratories for this route for over 25 years.
This study was conducted to assess the feasibility of using two such strains of pigmented rats such as; the Long-Evans and Brown Norway rats for the conduct of continuous intravenous infusion for toxicology testing. The animals were catheterized and infused at a rate of 0.4 mL/h for 10 consecutive days with 0.9% Sodium Chloride for injection USP. The parameters that were evaluated consisted of daily detailed examination, weekly body weights and food consumption, ophthalmology, hematology, biochemistry, urinalysis, serial blood collection to mimic toxicokinetic assessments, macroscopic observations at necropsy, organ weights and histopathology.
Continuous intravenous infusion for 10 days had no significant effects on clinical observations, body weights, food consumption, ophthalmology and macroscopic evaluations. There were no unexpected events encountered during serial blood collection. Minor changes observed for clinical pathology and histopathology for Long-Evans and Brown Norway rats were comparable with those seen in infusion studies conducted in Sprague Dawley rats. At the same age, Brown Norway rats had significant lower mean body weight and food consumption when compared to the Long-Evans and Sprague Dawley rats. As the body weight range and growth rate of Brown Norway are relatively lower than the other two strains at a similar age, and therefore there should be consideration for use of slightly heavier (older) rats at time of cannulation.
This study demonstrates that the Long-Evans and Brown Norway rats provide an appropriate pigmented animal rodent model for use in toxicology studies.
P25: Evaluation of a Chronic Bile Duct Cannulation Rat Model
Yifan Luo, Guy B. Mulder, Yiying Luo, and Terrance F. Fisher
Charles River, Wilmington, MA
In the development of novel pharmaceutical compounds, pharmacokinetic parameters, such as the extent of biliary excretion, are often characterized. Rats are frequently the first species used to characterize drug clearance and bioavailability, and because rats lack gallbladders they provide an ideal model for examining biliary drug disposition. This study evaluated bile flow rate and liver function in rats after ligation of the bile duct and shunting of bile via catheter into the duodenum. Eight adult male CD rats (Charles River, Raleigh, NC) weighing between 225 and 250 grams were surgically implanted with bile duct catheters. Bile flow was assessed by collecting two 15 minutes AM and PM samples. Blood samples were taken every other day during the 14 days study for liver function analysis. The average bile flow rates were 1.81 ml/hr and 1.69 ml/hr for the AM sampling and PM sampling respectively. The values of Albumin (ALB), Total protein (TPR) Alanine transaminase (ALT) and Alkaline Phosphatase (ALK) remained consistent during the study period. The values of Aspartate transaminase (AST) and Total bilirubin (TBIL) were consistent during the first 8 days post surgery; after which values increased sharply for the duration of the study. The results of this study suggest that the rat bile loop model produce an average bile flow of 1.75 ml/h. Considering the change liver function parameters, this model is best suited for studies of approximately one week in duration.
P26: Refinement of Non-Human Primate Use in Toxicology: Evaluation of Genetic Diversity in Three Geographical Sources of Cynomolgus Monkey (Macaca fascicularis)
Donald Waller1, Jean Dubach2, Ken Draper3, Philippe Baneux1, Thomas Welsh1
1Prelabs, Oak Park IL, 2Loyola University Medical Center, Maywood IL, 3Draper Consulting, Reno NV
Recent developments of novel therapeutic platforms (e.g., siRNA and various gene therapies) have exploited the expanding field of human genetics. DNA sequencing of targeted genetic loci has revealed genetic variation and similarities that underlie observed idiosyncratic and generalized trends of response to xenobiotics and lend efficiency to the pursuit of therapies for intractable human diseases. Due to genetic similarity between humans and non-human primates, the non-human primate model has become important for evaluation of safety and efficacy using many types of therapeutic biologics. Breeding programs to standardize laboratory rodent genotypes resulted in better predictability and reproducibility of toxicology data in those species, and it is reasonable to assume that understanding the genetic variability of sourced non-human primates will ultimately provide further refinements to the use of this important animal model. As a first step in defining the genetic diversity in laboratory-sourced non-human primates, we evaluated genetic variation between Indonesian, Vietnamese (atb), and Mauritius cynomolgus monkeys (M. fascicularis) at defined microsatellite loci. Blood samples were collected from individuals of each animal geographic source. Loci were amplified and sized using a capillary gel electrophoretic system. Observed population diversity indices (eg: Ho, A, R, FIS, FST) within and among groups will be presented.
P27: Seizure Liability Assessment in Zebrafish
Demian Park, Louis J. D’Amico, Wen Lin Seng, and Patricia McGrath
Phylonix Pharmaceuticals, Inc., Cambridge, Massachusetts, USA. Email: pat@phylonix.com
Numerous marketed drugs and potential drug candidates in various classes, including antiepileptics, analgesics, antidepressants, antiphychotics, anaesthetics, antiarrthymics, and antibiotics, have been associated with increased risk of seizure and epilepsy, and the FDA has included this endpoint in S7A, Guidance for Industry. We recently examined patterns of locomotor activity in 6 day zebrafish using an infrared camera that acquires images continuously. We tested 7 drugs known to induce seizures in mammals: pentylenetetrazole (PTZ), 4-aminopyridine, amoxapine, methoxychlorate, aminophylline hydrate, cefazolin sodium and bicuculline methoidide, and 3 negative control drugs, acetaminophen, verapamil and dexamathasone. We screened multiple (20-42) animals simultaneously and recorded activity of each zebrafish for one hour. Average distance travelled during high speed (>20mm/sec) movement was measured in one minute intervals and we observed a dose-dependent increase in zebrafish treated with seizure-inducing drugs. Similar to PTZ, used routinely as a seizure-inducing drug in mammalian models, positive drugs increased high speed movement, however, the time course of drug effects varied. Advantages of this comparatively high throughput animal model include, drug delivery directly to fish water, small amount of drug required, and statistically significant number of animals per experiment.
P28: Whole Zebrafish ROS Assay for Drug Toxicity Screening
Chun-Qi Li, Yingli Duan, and Patricia McGrath
Phylonix Pharmaceutical, Cambridge, MA 02139
In this study, using CM-H2DCFA, a ROS specific fluorogenic dye, we developed a quantitative microplate-based whole zebrafish assay to rapidly screen drug candidates. Initially, we confirmed ROS assay specificity using positive control compounds, hydrogen peroxide (H2O2) and 4β-phorbol 12-myristate 13-acetate (PMA). Next, we established a linear relationship between fluorescence intensity and number of zebrafish per microwell. Signal/noise ratio was > 2 using 3 zebrafish per microwell. 3 day post fertilization zebrafish were selected as the optimum stage for assessing drug effects. We further validated the whole zebrafish microplate assay using 7 additional known mammalian ROS inducers. ROS production increased in zebrafish treated with Cisplatin and DCA (Dichloroacetate) at 1000 µM (p < 0.05 and p < 0.01, respectively), PMA at 1 and 10 µM (p < 0.001), TSA (Trichostatin A) at 25 and 50 µM (p < 0.001), Menadione at 100 and 500 µM (p < 0.01 and p < 0.001), TBHP (tert-Butyl hydroperoxide) at 2500 and 5000 µM (p < 0.05 and p < 0.01), and Ethanol at 2.5% and 5% (p < 0.001). Negative control compound NAC (N-Acetyl-L-cysteine) did not induce ROS at test concentrations up to 1000 µM. Whole zebrafish microplate ROS results were further confirmed visually using live zebrafish fluorescence ROS staining. Compared to results in mammalian systems, overall prediction success rate for assessing ROS production in zebrafish was 100% (
P29: Design, Conduct and Completion of Validation Studies for a New Toxicology Facility
Andy P. Mould, Mingyi W. Trimble, Jay C. Albretsen, and Steve M. Glaza
Covance Laboratories Inc., Chandler, AZ
Following the construction of a new toxicology facility, validation studies are routinely completed to demonstrate the successful ability to conduct animal studies. However, a literature search did not reveal a common approach or design for such studies. Therefore, 1-month studies in 4 common laboratory animal species (rat, mouse, dog, monkey), conducted in compliance with GLP regulations were designed and included many procedures commonly used in toxicology studies. Commonly used vehicles (HPMC, NaCl, ground diet), or loratadine, were administered daily via oral gavage or capsule, or twice weekly via intravenous or subcutaneous injection. Observations within the studies included clinical signs, body weights, food consumption, physical, ophthalmic, ECG, and neurological examinations, vital signs, blood pressure determinations, and clinical and anatomic pathology. Blood samples were also collected for bioanalytical analysis (all studies) and serology (rat study only). Dose formulations were accurately prepared, and dosing was successfully completed in all studies. Only minor clinical signs were observed, and no marked or adverse findings were seen in any of the other study parameters, although minor, procedure-related microscopic findings were found in the SC and/or IV groups of all species. Bioanalytical results in all studies demonstrated successful and expected exposure to loratadine. In the rat study, all serology results were negative. None of the dosing routes or study procedures had any effect on the health of the animals. Overall, the study designs were considered sufficiently inclusive and robust for the validation of new, or existing, toxicology facilities involved in conducting GLP studies.
P30: Validation of Procedures and Evaluation Methods for Assessment of Embryofetal Toxicity, Using Retinoic Acid, in Sprague Dawley Rats
Sukhdeep Sahambi, Annie LeBlanc, Cedric Gordon, and Glenn Washer
LAB Research Inc. 445 Armand-Frappier Blvd., Laval, (QC), Canada
Mammalian development is an extremely sensitive process and susceptible to a myriad of disruptions during various stages of embryonic and fetal development. Retinoic acid (RA), a variant of Vitamin A, is an essential component in normal mammalian development as well as adult growth and fluctuations in its levels during critical stages can result in a multitude of teratogenic/embryotoxic/toxic outcomes. An established teratogen, RA has been identified to affect neural tube and limb development. As a part of the developing reproductive-embryofetal toxicology facility at LAB Research Inc., timed-pregnant Sprague Dawley (SD) rats were orally administered (10 mL/kg) with 0, 5, 10, 20, 30 mg/kg RA in dimethylsulfoxide (DMSO; doses selected based on literature as well as a pilot study in non-pregnant adult female SD rats) from gestation day (GD) 6 to 17. Maternal body weights (BW) and food consumption (FC) were monitored throughout the gestation period. Pups were delivered by cesarean on GD21 and an external as well as internal exam was performed. RA treatment at 30 mg/kg resulted in the death of 1 dam on GD 14. The treated dams showed abnormal and uncoordinated behavior with salivation, decreased activity with partially closed eyes. RA treatment reduced maternal FC and consequently the BW between GD 7 to 12. BWs remained lower in RA treated animals throughout the remainder of gestation. At 30 mg/kg, RA resulted in a complete resorption of conceptus during early pregnancy. In the remaining treatment groups, up to 60-fold increase in the number of resorptions and 62-fold decrease in the number of live fetuses was noticed. The pups in the RA treated dams were relatively smaller in size and exhibited external malformations such as exencephaly, along with skeletal malformations in the rib cage and vertebral column. The above results were consistent with results published by other researchers and thus demonstrated that the laboratory’s procedures and evaluation methods were suitable for identification of RA induced effects on embryofetal development and maternal toxicity in Sprague Dawley rats.
P31: Validation of Immunohistochemical Staining
Mylène Valin1, Kate Lillard-Wetherell1, Thomas Lemarchand2, Palate Bernard2, and Roy Forster2
1Aperio, San Diego, CA, USA, 2CIT, Evreux, France
In our facility, antibody reagents and the corresponding immunohistochemical staining procedures are subjected to a validation process prior to bringing into use. Full validation consists of the determination of specificity, optimal concentration [C], repetitivity, reproducibility, linear range and dynamic range (from concentration without background noise to extinction of signal). Full validation is performed on antibody reagents for tissue cross reactivity studies and is also proposed for use on GLP studies with antibodies in development for human therapeutic use. The validation process is less exhaustive when dealing with antibodies for research use only. Each new staining technique is optimized for performance manually and by automated instrument (IHC Discovery XT, Ventana) on frozen and fixed tissues. For frozen tissues, optimization includes assays of post-fixation. The most critical step is determination of the optimal concentration. This is performed using image analysis software to provide quantification of staining (stained surface area or number of stained cells) as a function of antibody concentration; the optimal concentration and linear range are derived from this dose-response curve. In some cases intra and inter-batch variability can be substantial and in these cases qualitative validation only may be possible. High technical standards, reproducible section thickness, the use of digital slide acquisition and new approaches in image analysis (as provided by Genie software) can significantly improve intra- and inter-batch variability.
P32: Validation of Developmental Immunotoxicology Assays in Infant Cynomolgus Monkeys
Satterwhite, Christina M, Auyeung-Kim, Diana, Fraser, Stephanie, Dumont, Carolyne, LeSauteur, Lynne and Chellman, Gary
Charles River Laboratories, Preclinical Services
The cynomolgus monkey has emerged as the primary nonclinical reproductive toxicology model for biotherapeutics. Biotherapeutics are a class of drug that may have increased potential to intentionally modulate the immune system due to the mechanism of action of the drug or have unanticipated adverse affects on the immune system. The regulatory agencies require immunotoxicology in order to evaluate immune system function using a weight of evidence approach. Assays used to assess impact on immune function such as T–cell dependent antibody response (TDAR), immunophenotyping via flow cytometry, natural killer (NK) cell cytolytic activity, and the measurement of total immunoglobulins are used on a case by case basis. These assessments are characterized in adult cynomolgus monkeys in the preclinical toxicology setting; however in the developing postnatal cynomolgus monkeys, data using the same immune function tests are lacking. The aforementioned immune function tests were evaluated in six (3 male, 3 female) postnatal cynomolgus monkeys from approximately 6 months to 12 months of age. Primary and recall immune responses to immunization with KLH were observed at 6, 9 and 12 months monitored by serum levels of anti-KLH IgM and IgG antibodies. Immunophenotyping, Immunoglobulins, and NK cell activity were evaluated monthly. Peripheral blood lymphocyte subsets were evaluated via marker sets for monocytes, T, B and NK cells. Lymphocyte subset percentages in individual infant absolute count values did not significantly change with age. Total IgM values monitored by serum levels were constant between the ages of 6 to 12 months; it was observed that females had higher levels of total IgM than males. In contrast, total IgG increased steadily for both males and females. Immunoglobulin E was measurable over the course of the study and there was an absence of measurable IgA. NK cell cytolytic activity increased with age. This data serves as a reference for future developmental immunotoxicity studies in infant cynomolgus monkeys.
P33: The use of the Tg.RasH2 Mouse in Carcinogenicity Studies
Joseph Younan BSc, Dipl.1, Lev Kolodzieyski, DVM, PhD2
1Ecotoxicology, Director, Toxicology; 2Director, Pathology, ITR Laboratories Canada Inc.
The objective of the study was to evaluate the response of the Tg.rasH2 mouse test system to two known carcinogens and to demonstrate its utility as a quicker and more cost-effective alternative to the conventional two-year mouse bioassay.
Fifteen (15) male and 15 female Tg.rasH2 mice were assigned to each of 3 dose groups. Animals of Group 1 received purified water by oral gavage once daily for 26 consecutive weeks. Animals of Group 2 received a total of 3 intraperitoneal injections of Urethane (1000 mg/kg each) at 2-day intervals (i.e., on Day 1, 3 and 5), while animals of Group 3 received a single intraperitoneal injection of MNU (75 mg/kg). Parameters monitored during the study included mortality, clinical observations, including examinations for the presence of palpable masses, body weights and food consumption. Upon completion of the 26-week treatment/holding period (Groups 2 and 3), all surviving animals were euthanized and subjected to a necropsy examination. Subsequently, tissues collected from all animals were examined histopathologically.
A total of 2 Control, 28 Urethane-treated and 23 MNU-treated animals died or were preterminally euthanized due to poor clinical condition during the study. Bronchiolo-alveolar carcinoma of the lungs and hemangiosarcoma of the spleen were considered to be the cause of mortality/morbidity in the 2 Control females. Bronchiolo-alveolar adenoma and/or carcinoma and hemangiosarcoma were considered to be the cause of mortality/morbidity in the majority (26/28) of the Urethane-treated animals, whereas malignant lymphoma of the hemolymphoreticular system was the cause of death/morbidity in the in the majority (17/23) of the MNU-treated animals.
Thus, the intraperitoneal administration of 2 known carcinogens (Urethane and MNU) to Tg.rasH2 mice followed by a 26-week holding period produced clearly higher incidences of benign/malignant tumors and tumor bearers resulting in significantly higher mortality rates among the animals treated with the carcinogens in comparison to Controls.
P34: Self-Administration Testing and Amphetamine Substitution in Adult Female Rats
Jonathan D. Toot, Melissa J. Beck, Donald G. Stump, Julie S. Varsho, and Mark D. Nemec
WIL Research Laboratories, LLC. 1407 George Road, Ashland, OH, 44805-8946
The objective of this study was to 1) assess the self-administration (SA) testing paradigm utilized at WIL Research Laboratories, LLC, 2) demonstrate positive reinforcing effects in this model with amphetamine and 3) demonstrate the ability of cocaine to be self administered (or maintain SA) in the rat within the scope of the European Medicines Agency guidelines. The animals used for this study consisted of adult Sprague Dawley females, at approximately 8 weeks of age at the initiation of training. Rats were trained to press the active lever in order to receive an infusion (reinforcer) of the training/reference compound (RC), cocaine. This was followed by extinguishing that behavior with saline and ultimately testing with the test article compound, d-amphetamine (AMPH), during substitution sessions. Using this paradigm, animal responses were measured as the average number of infusions administered per session. During testing, 0.6 mg/kg/infusion RC reliably maintained SA under baseline conditions. Saline substitution for RC resulted in extinction of this response (p<0.001). Rats were then re-exposed to RC following successful extinction and were shown to reacquire the SA behavior (p<0.001). Once baseline performance was stable, subjects were assigned to a substitution test with individual doses of AMPH (0.0125, 0.025 or 0.05 mg/kg/infusion). The average number of infusions at the 0.025 and 0.05 mg/kg/infusion dose levels (p<0.001) and at the 0.0125 mg/kg/infusion dose level (p<0.01) were significantly greater than infusions during saline sessions. The SA testing paradigm as well as the training and testing compounds, were able to successfully establish the positive reinforcing potential of AMPH consistent with its known abuse liability. Therefore, these results validate that the SA methodologies described are in accordance with the EMEA guidelines.
P35: Drug Discrimination Testing and Substitution With Cocaine, Amphetamine, Desipramine and Morphine in Adult Female Rats
Jonathan D. Toot, Melissa J. Beck, Donald G. Stump, Julie S. Varsho, and Mark D. Nemec
WIL Research Laboratories, LLC. 1407 George Road, Ashland, OH, 44805-8946
The objective of this study was to assess the drug discrimination (DD) testing paradigm utilized at WIL Research Laboratories, LLC, with cocaine, amphetamine, desipramine and morphine in the rat within the scope of the European Medicines Agency guidelines. The animals used for this study consisted of adult Sprague Dawley females (n=10-12), approximately 8 weeks of age at initiation of testing. Rats were trained by food pellet reinforcement under a fixed ratio 20 schedule to discriminate between the compound-associated levers when administered the reference compound (RC), cocaine (10 mg/kg), or the vehicle control (saline) via intraperitoneal injection approximately 15 minutes prior to the drug discrimination session. Discrimination testing was conducted with substitution of the following compounds: cocaine hydrochloride (COCAINE) at 1.25, 2.5, 5 and 10 mg/kg; d-amphetamine sulfate (AMPH) at 0.025, 0.1, 0.5 and 1.0 mg/kg; desipramine hydrochloride (DESP) at 0.1, 1.0, 5 and 10 mg/kg; and morphine hydrochloride (MORPH) at 0.25, 0.5, 1.0, 2.0 and 4.0 mg/kg. Using this paradigm, the first 20 lever presses were reported as the relative percent of RC lever selection. The results of this study showed RC lever selection (>80%) for subsitution with COCAINE and AMPH at dose levels above 2.5 mg/kg and 0.5 mg/kg, respectively. As expected, DESP resulted in intermediate levels of substitution over the dose levels administered, with RC lever selection between 10 and 60%. Also, MORPH did not substitute over the dose levels administered, with RC lever selection below 25%. The DD testing paradigm was able to characterize compounds selected for their known discriminative potential. Therefore, these results validate that the methodologies described are in accordance with the EMEA guidelines.
P36: Endocrine Disruptor Screening Program: Demonstrating Laboratory Proficiency in Conducting Tier I Mammalian In Vivo Assays
Susan Borghoff, Jeff Davis, Theleria Hackett, Pamela Sproul, Glenda Moser
Integrated Laboratory Systems, Inc. Durham, NC, 27713
The Endocrine Disruptor Screening Program has been developed to identify environmental contaminants for their potential to affect the estrogenic, androgenic and/or thyroid hormone systems of humans and wildlife. There are a number of proposed Tier I screening assays which include in vitro (e.g. AR and ER binding, steroidogenesis, and aromatase assays), amphibian metamorphosis and fish reproductive screening assays, along with 4 in vivo mammalian assays. The mammalian assays include the Uterotrophic and Hershberger bioassays which screen for estrogenic and androgenic/antiandrogenic activity, respectively. The gender-specific pubertal assays screen for multiple modes-of-action in male (e.g. antithyroid, androgens/antiandrogens, and steroid enzyme mediated) and female (e.g. estrogenic/antiestrogenic and thyroid mediated) rats. OECD guidelines exist for both the Uterotrophic (440) and Hershberger (draft) assays and the EPA is currently finalizing study protocols for the pubertal assays. Laboratory Performance Criteria has been established for all 4 assays. The Uterotrophic assay was conducted using the OECD guideline immature rat protocol with the administration of 4 doses of ethinyl estradiol. Uterine wet and blotted weights were compared to historical data showing the laboratory’s ability to carry out this assay. The Hershberger assay was conducted using testosterone propionate (3 doses) to evaluate agonist responses and 0.4 mg/kg testosterone propionate co-administered with flutamide (3 doses) to evaluate antagonist responses. Performance criteria including coefficient of variation limits on specific endpoints and comparison to historical values successfully meet the OECD guideline criteria. Meeting these criteria is essential to demonstrate the ability of an individual laboratory to conduct these assays.
P37: From Concept to Operation: The Launch of a New GLP Toxicology Facility
Mingyi W. Trimble, Andrew P. Mould, Jay C. Albretsen, Steve M. Glaza, and Monique Heiser-Wong
Covance Laboratories Inc., Chandler, Arizona
The timeline, project management tool, and key events for the launching a new toxicology facility operating under Good Laboratory Practices (GLP) will be presented. It is not an easy task going from 77 acres of undeveloped desert land to a fully operational, state-of-the-art, 288,000 square foot facility designed for general toxicology and supporting services. A multi-disciplinary organizing committee with representatives from operations, science, quality assurance, and administration was formed to steer decisions going from design concept to ground breaking to certificate of occupancy to competent functionality. The committee worked closely with internal and external stakeholders and served as a central information exchange throughout the project. Managing the initial move in, study initiation, and staffing took extensive planning and coordination between builders and suppliers, as well as temporary and permanent staff. Major facility, IT infrastructure, and equipment validation soon followed, and a business continuity plan was established. To further demonstrate the newly built facility was ready to conduct animal studies, four 1-month GLP studies were successfully performed in four common laboratory animal species (i.e., rat, mouse, dog, and monkey). The site qualification process involved logistics, scientific, and regulatory expertise, while accommodating potential client, local and federal agency auditors, and applicable accreditation agencies. The utilization of a multi-faceted organizing committee in establishing a new GLP toxicology facility was shown to be an invaluable tool for similar future endeavors. The specific functions of the organizing committee and the lessons learned throughout the process will be discussed.
P38: A New Option for Group Housing Cynomolgus Primates in Toxicology Studies
Jay C. Albretsen, Mingyi W. Trimble, Andrew P. Mould, and Steve M. Glaza
Covance Laboratories Inc., Chandler, Arizona
The social, physical health and psychological benefits of housing primates in groups has been recommended for several years. However, in many U.S. facilities, primates have traditionally been housed singly for toxicology studies due to limited space, the need to keep experimental variables to a minimum, and for the ease of collecting data. In addition, doorway and hallway size at facilities generally make it difficult to use mobile cages of sufficient size to allow several monkeys to be housed as a group. Group housing of primates for toxicology studies has been successful in Europe; however, the cages used are often permanently installed into the rooms and are considerably larger than traditional mobile cages. Unfortunately, installing caging of that size in many U.S. laboratories is not practical. In order to gain the benefits known to occur with group housed primates and provide a mobile cage useful in most facilities; a custom built mobile housing unit was developed to allow group housing of up to four cynomolgus monkeys. This unit was designed as a bank of four cages, two on top and two on the bottom. Dividers open between all individual cages to allow movement of animals throughout the entire space. This group housing layout is maintained continuously once compatible groupings are achieved, but each primate can be singly housed if necessary. In our experience, additional time is required to achieve group compatibility within a housing unit, particularly with larger, more aggressive males. While it was not always possible to group-house four primates, in general cynomolgus monkeys group housed in these units showed less aggression, were easier to handle, and had fewer observations of fecal abnormalities indicating the animals were under less stress. Our results demonstrate the benefits of group housing cynomolgus monkeys in mobile cages that could be used in other toxicology laboratories.
P39: How to Integrate Safety Pharmacology End-Points During Toxicological Studies in Non Human Primates For Biologics?
H. Ficheux, G. Froget and J.J Legrand
CIT, Evreux, France
Existing guidance ICH M-3; S6; S7A; S7B, provide requisites for the conduct of appropriate safety pharmacology and toxicology assessment. However, inherent differences between biologics and small molecular entities support different approaches in non clinical safety assessment. As a result, opportunities exist for alternative strategies for the integration of safety pharmacology end-points in general toxicological studies. Based on our own experience, we describe an approach in non human primates to explore a new biologic that matches not only toxicological criteria, but also meets guideline criteria. Requirements from the ICH S7A core battery were met by the following: Respiratory function is evaluated by the use of a facial mask. Respiratory volumes, flows and bronchoconstriction index are recorded on different occasions; Cardiovascular functions are assessed by measurements of blood pressure using tail/leg cuff while the ECGs are recorded on separate occasions by external telemetry; and, finally, some aspects of CNS are investigated during the study, including detailed clinical observation, social behavior and body temperature - TK measurements are also made at the most appropriate time-points. The sequence of evaluation of these different parameters is a critical issue in order to avoid interferences between the different measurements. Since ECG is considered as the most critical end-point, it has been decided to perform it on days when no other parameters are to be assessed. Blood pressure, respiratory function as well as body temperature are monitored every other day under light anesthesia and can be associated with TK samplings. Such a design while fulfilling most regulatory compliances presents not only the additional benefit of reducing quantities of compound, time of development and cost but also meets the ethical constraints of reducing the number of animals used.
P40: Biological Validation of an External Telemetry System in Conscious Non-Human Primates
Philip Atterson, John Setser, Tonya Cobb, Laurie Shellhammer, John Yohe
WIL Research Laboratories, LLC, Ashland, OH
Collection of electrocardiographic recordings from conscious animals during the conduct of toxicology studies usually requires the use of chemical or physical restraint which may lead to stress and/or tachycardia that could confound data interpretation. In addition, the recordings are limited and are restricted to being collected two or three times during a one month study. This restricts analysis to gross changes in waveform morphology and does not allow for a detailed assessment of potential arryhthmogenic events. There has been an increased acceptance of the use of non-implantable, non-invasive telemetric systems such as the Jacketed External Telemetry (JET) as a tool to allow for the assessment of cardiac safety issues in freely moving animals on toxicology studies. This system allows for continuous data collection from canines and non-human primates over extended periods of time providing valuable data regarding the cardiac safety of drugs at high doses. The purpose of this validation study was to assess the sensitivity of the JET system to detect ECG changes in conscious, freely moving non-human primates when compared to data simultaneously acquired from the same animals via surgically implanted telemetry devices. In order to fully assess the performance of the JET system, the animals were treated with moxifloxacin, a broad spectrum fluroquinolone antibiotic, a compound that evokes measurable QT interval prolongation. The invasive and non-invasive systems produced comparable ECG signals and both displayed the ability to allow detection of QT interval changes following treatment with moxifloxacin. The results indicate that the JET system may be used on toxicology studies as a credible alternative to traditional methods.
P41: A New Impedance-Based Method for Combined Cardio-Pulmonary Data Acquisition in Freely Moving Telemetered Animals
Philip Atterson, Ken Kearney, Teresa Gleason, Tim Edwards, Gene Kopp
WIL Research Laboratories, LLC, Ashland, OH
The Safety Pharmacology ICH S7A guidelines mandate that effects on respiratory function be evaluated. The majority of these evaluations are currently conducted in rodents as measurement of respiratory parameters in large animals is notoriously complicated. Current systems for large animal respiratory measurement usually require some level of restraint (e.g use of chairs or sling systems) leading to stress-induced artifacts that confound data interpretation. Current systems also require extensive training of the animals to accept the jacketed systems. However, recent advances in implantable technology, specifically implantable impedance telemetry technology, allows for the accurate acquisition of respiratory frequency and tidal volume in an ambulatory large animal model. The device is an adaptation of the DSI TL11M2 D70 telemetry implant. The addition of impedance leads allows for concurrent acquisition of cardiovascular data (blood pressure, ECG), body temperature, and respiratory data (respiratory frequency, tidal volume). The device is implanted such that the impedance leads are located between the sternum and spine at the mid-thorax level. Direct comparison to the pneumotach model illustrates high concordance and accuracy of respiratory endpoints in conscious beagle dogs. The use of the implantable impedance device also eliminates the confounding agents associated with animal restraint, thereby providing cleaner respiratory data.
P42: Electroretinography (ERG) Of C57/B and NIH III Mice
Leigh, H.1; Blair, E.1; Liu, J.1; Brown, L.1,2; Bouchard, G. F.1,2
1Sinclair Research Center, LLC, Auxvasse, MO, USA, 2Sinclair BioResources, LLC, Auxvasse, MO, USA
Electroretinography (ERG) is a unique tool for measuring the electrical potentials of the retina and evaluating in-development drug products for potential ocular effects, such as induced retinal degeneration. The mouse has become a popular model for the study of retinal degeneration, and new generation electrodes have been used successfully to obtain electroretinograms from mice as young as 14 days. The C57/B mouse (female, 7-8 weeks, 13-17g) and NIH III strain of nude mouse (male, aged 6-8 weeks, 19-28g) were used in this baseline study of niave animals. We measured ERGs of 5 C57/B and 59 NIH III untreated mice using the Diagnosys LLC, Espion2 ERG machine. All C57/B mice had acceptable ERG recordings while 12 (20%) of the NIH III mice had flat-line recordings, which might be a result of retinal degeneration. Mean values for retinal ERG potentials of the NIH III and C57 are presented below. These data offer baseline ERG measurements for the NIH III and C57 strains of mice.
P43: A Method Development Study to Assess the Effectiveness of Measures to Prevent Cross Contamination During Topical Application of a Test Article to the Göttingen Minipig
Will Ruddock1 and Ovidiu Jumanca2
1Toxicology Clinical Veterinarian 2ITR Laboratories Canada Inc
Although the rabbit is commonly used for the assessment of primary dermal irritation, pigs have generally been considered to be a better model for the more sophisticated study of dermal permeability and toxicity. Many studies have shown the close resemblance of human and minipig skin in terms of architectural structure/morphology, histology and physiological characteristics. As a model in toxicology, the minipig has been used extensively in dermal/topical application studies. One of the most significant issues for dermal studies is the extensive measures required to prevent cross-contamination of blood and tissue samples, taken to monitor local and systemic exposure to the test article. This is more difficult with the minipig simply because of it’s size and temperament.
ITR has completed a method development topical application study utilizing the Göttingen minipig. The study objective was to assess the effectiveness of control processes designed to preclude contamination of Control animals/samples with test substance. In the study, test animals were treated by non-occluded topical application of Lidocaine 2.5% cream (EMLA) for 4 hours/day over 7 consecutive days. Control animals were similarly treated with Glycerin USP. Specific standard procedures were identified in the study protocol to highlight the areas of concern for the technical groups. This included those working with the animals, processing blood samples to plasma and the analytical department. Blood samples were collected at various time points during the treatment phase, processed and analyzed. Results from the analysis of blood samples collected during the study revealed that there were no measurable levels of drug in the Control animals at any time point. Treated animals, however, showed appropriate levels of drug at all time points. It can be concluded therefore, that the measures employed on the study were effective in preventing contamination of Control animals and samples with drug.
P44: Intravaginal Dose Delivery in Rabbits
G. Glazier, Y. Trudel, and S.Y. Smith
Charles River, Preclinical Services Montreal, Quebec, Canada
The non–clinical testing of drugs targeting the female population requires a technique for delivery that will mimic the intended clinical use. To meet this demand, we have developed techniques using the New Zealand White (NZW) rabbit for the intravaginal administration of various compounds in the form of creams, gels, suppositories and capsules for indications such as hormone replacement therapy, contraception, infection and infertility. The vaginal route for drug delivery has many advantages such as insensitivity to pain, rich vascularisation and elimination of the hepatic first-pass effect which means lower doses can be administered. The reproductive tract of the female NZW is convenient for drug administration and favours retention of vaginal formulations depending on their consistency. Their lack of an estrous cycle results in minimal variability in the permeability of the vaginal membrane and ensures consistency in histological, biochemical and physiological properties. Consequently, this makes them an excellent animal model for pharmacokinetic studies in addition to their body weights (3.0 to 3.5 kg) which allows for ample blood sampling. Preparation of the test system for administration requires proper acclimation to the dosing techniques including digital priming of the clitoris. In addition to the standard toxicity study endpoints, including blood collection for assessment of drug exposure, daily expulsion/backflow assessments are recorded, and a qualitative assessment of vaginal tissue irritation is performed concomitant with the weekly detailed examination. Surgical implantation of a capsule into the vaginal wall was performed on 44 and 33 females while under anesthesia. Successful retention rates varied from 62% to 100% depending on the length of the in-life phase which varied from 7 to 90 days. Vaginal administration of a cream to 52 females, daily for 90 days showed negligible to slight irritation associated with the dosing technique and backflow rates ranged from 0.8% to 8.2% depending on the consistency of the cream formulation. Daily digital vaginal suppository administration to 35 females for a period of 5 days generally resulted in well-defined erythema and/or slight edema and backflow observations were noted for at least one animal per group. These studies support the use of the NZW rabbit for single and repeat vaginal administration of creams/gels and highlight the experimental limitations associated with suppository and capsule use.
P45: An Evaluation of Patency in Chronic Venous Catheters
Yifan Luo, Guy B. Mulder, Yiying Luo, and Terrance F. Fisher
Charles River, Wilmington, MA
Pharmacokinetic studies in rats are most effectively and humanely performed with a chronically implanted catheter system that allows for repeated blood sampling from an animal with minimal restraint. Once a catheter is surgically implanted, the amount of time that it reliably remains patent will set practical limits on its use. The patency life can be affected by many factors including flushing regimen and frequency as well as type of locking solution. This study was designed to compare the patency life affected by (1) catheter flushing manipulation, ie, flushing vs. non-flushing and (2) type of lock solution, ie, Taurolidine citrate solution (TCS) and 50% heparinized dextrose. Forty adult male CD rats (Charles River, Raleigh, NC) weighing between 245 and 275 grams were surgically implanted with jugular vein catheters that were locked with either TCS or 50% heparinized dextrose. The rats were randomly allocated into eight groups with five animals per group. While one group of animals with each locking solution were flushed every 7 days, catheters in the other groups were not manipulated in anyway until on day 14, day 21 and day 28 when catheter patency was assessed. Patency was classified as: Fully Patent (successful blood withdrawal on first attempt); Patent on Flush (successful blood withdrawal after infusion of saline); Partially Patent (unsuccessful blood withdrawal but patent for infusion) and Non-patent (unsuccessful blood withdrawal and infusion). On day 7, 90% of catheters remained fully patent in the flushing groups. The fully patent rates dropped to 80% for both flushing and non-flushing groups on day 14. On day 21, the patent rates were 20% higher in non-flushed catheters than those flushed every 7 days. On day 28, the numbers of patent catheters were 2.5 times greater in non-flushed catheters than flushed catheters. Overall, the patent rates were 3% higher in catheters locked with Dextrose than those locked with TCS. The results of this study suggest that non-manipulated jugular vein catheters maintained longer patency life than catheters flushed once weekly. Catheters locked with 50% heparinized dextrose showed a slightly improved patency rate than catheters locked with TCS solution, although the differences were not statistically significant.
P46: Ocular Damage Reversibility of Three Commercially Available Personal Care Products Using the PorCORA
Piehl, M1, Donahue, DA2, Avalos, J2, and Cerven, D1
1-MB Research Laboratories, Spinnerstown, PA, 2-Kao Brands Company, Cincinnati, OH
To ensure consumer safety, ocular irritation testing is routinely performed on personal care products. Two ocular assays, the Chorioallantoic Membrane Vascular Assay (CAMVA) and Bovine Corneal Opacity and Permeability Assay (BCOP), are widely used in the cosmetic industry since they do not require the use of animals. These assays provide reliable data predicting ocular irritation and are inexpensive to conduct. To complement the CAMVA/BCOP assays, the Porcine Corneal Opacity Reversibility Assay (PorCORA) was developed using an ex vivo model to predict reversibility of ocular irritants. In the current study, three commercially available consumer products (a shampoo, a hair color glaze, and a 12% hydrogen peroxide product) were tested in the PorCORA for ocular damage reversibility. The PorCORA indicates that under the exaggerated in vitro study conditions the surfactant-based shampoo may cause irreversible ocular damage: histological changes occurred in the squamous-cell layer of the corneas and mild to moderate changes in the basal-cell layer. However, literature does indicate that reversibility of ocular damage occur in vivo following exposure to shampoo. Furthermore, the PorCORA predicts that under the same study conditions used for the shampoo, ocular damage caused by a hair color glaze and a 12% hydrogen peroxide paroduct are fully reversible with histology reporting only minimal or mild microscopic effects to the superficial squamous-cell layer. Like the shampoo, literature also indicates that reversibility of ocular damage occur in vivo following exposure to hydrogen peroxide. The PorCORA assay, in conjunction with other ocular irritation assays can be used to predict the extent of ocular damage and reversibility that products may cause following consumer eye exposure.
P47: Chorioallantoic Membrane Vascular Assay (CAMVA) and Bovine Corneal Opacity and Permeability Assay (BCOP) Historical Data on Personal Care Products Over Fourteen Years
Newhard, W1*, Donahue, DA2*, Kaufman, L3, Avalos, J2, and Cerven, D1
1-MB Research Laboratories, Spinnerstown, PA, 2-Kao Brands Company, Cincinnati, OH, 3-Scripterra Scientific, Wooster, OH, *-co-first authors
The Chorioallantoic Membrane Vascular Assay (CAMVA) and Bovine Corneal Opacity and Permeability Assay (BCOP) are two common assays used to determine ocular irritation for consumer-use products. These assays do not require the use of live animals, provide reliable predictive data, provide results similar to in vivo models and are rapid and inexpensive to conduct. Data from 321 studies performed from 1995 to 2009 (a total of 345 test materials assessed by CAMVA and/or BCOP) were compiled to determine the feasibility of predicting ocular irritation for various formulations. Review of the data from both assays found that hair shampoos, skin cleansers, and hair styling sprays (containing ethanol) were repeatedly predicted to be ocular irritants. In contrast skin lotions/moisturizers were repeatedly predicted not to be ocular irritants. Based on the findings for these product types, future ocular irritation testing (i.e., CAMVA/BCOP) can be nearly eliminated as long as formulations are compared to those previously tested. For example, skin cleanser irritation appears to be solely dependent on surfactant species and level in these formulations.
For other product types (e.g., deodorants, make-up removers, hair styling, body sprays) it was concluded that these products should continue to be tested in CAMVA/BCOP for ocular irritation potential because either significant variability exists in the historical data (non-spray hair stylers) or the historical sample size is too small to permit definitive conclusions (deodorants, make-up removers, massage oils, facial masks, body sprays, and hair styling products).
P48: A Robust In Vitro assay For Predicting and Ranking Clinical Neutropenia Caused by Tyrosine Kinase Inhibitors
Gary Dos Santos & Emer Clarke
ReachBio LLC, Seattle WA, USA
Tyrosine Kinase Inhibitors (TKIs) represent a new class of rationally designed drugs. The success of Imatinib, targeting the ABL tyrosine kinase in CML, has prompted the development of other TKIs for the treatment of various cancers and inflammation. Although more successful than conventional therapies, myelotoxicity/neutropenia is often a dose-limiting toxicity of TKIs. As intended applications of TKIs extend beyond oncology to less life-threatening indications such as inflammation, the tolerance for myelotoxicity will decrease and understanding the relationship between efficacy and myelotoxicity will become increasingly important earlier in the drug development process.
Using hematopoietic progenitor colony forming cell (CFC) assays as an in vitro model for hematopoiesis and myelotoxicity, we determined the IC50 values of 6 currently marketed therapeutic TKIs (Imatinib, Lapatinib, Erlotinib, Dasatinib, Sorafenib and Sunitinib) and compared them to the degree of clinical myelotoxicity (neutropenia) reported for these drugs in the literature. Normal human bone marrow cells from three different donors (each evaluated separately) and the various individual TKIs were mixed with methylcellulose-based media, plated in 35 mm dishes in triplicate and the progenitor cell colonies were microscopically enumerated on day 14. Not only was the rank order of the TKIs' myletoxic potential determined in vitro identical to that reported for their potential to induce clinical neutropenia, but there was also a direct correlation (R2 = 0.81) between clinical myelotoxicity and the IC50 values derived from the in vitro CFC assays, with lower IC50 values associated with increased neutropenia (IC50 range: 0.008 ug/mL (Dasatinib) -> 100 ug/mL (Lapatinib). These results demonstrate that when performed by skilled personnel, the in vitro human CFC assay is highly reliable and useful for accurately predicting and ranking the myelotoxic potential of TKIs.
P49: Withdrawn
P50: Withdrawn
P51: Role of Epigenetics in Dioxin-Induced Differential Regulation of the Human CYP1A1 and CYP1B1 Genes
Sudheer R Beedanagari* and Oliver Hankinson
Molecular Toxicology Interdepartmental Program, UCLA
The Aryl Hydrocarbon Receptor/ARNT heterodimer mediates carcinogenesis by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) and certain polycyclic aromatic hydrocarbons (PAHs) by activating gene transcription. Metabolism of PAHs by CYP1A1 and CYP1B1 plays a major role in carcinogenesis mediated by these compounds. The overall goal of this study is to understand the mechanisms involved in differential regulation of the human CYP1A1 and CYP1B1 genes. We are studying the regulation of these genes in three different human cell lines (MCF-7, MDA-MB 231, and HepG2) as the endogenous expression and induction levels of these genes upon treatment with TCDD differ significantly between these cell lines. To elucidate the mechanisms involved in the differential regulation of these genes, we studied the recruitment of AHR and RNA PolII at their enhancers and promoters, respectively. The data indicate that RNA PolII, but not AHR recruitment, correlates with CYP1A1 and CYP1B1 expression. Subsequently, we characterized the different dioxin- inducible chromatin modifications (i.e acetylation, methylation, and phosphorylation) that occur across the CYP1A1 and CYP1B1 enhancer and promoter regions using the chromatin immunoprecipitation (ChIP) assay. The results show H3K9Ac, H3K14Ac, H3K4me3 and H4Ac chromatin modifications at the CYP1A1 and CYP1B1 promoter regions strongly correlate with the TCDD-induction of the CYP1A1 and CYP1B1 genes in different cell lines. These modifications, at least in part are likely to contribute to the differential induction of these genes upon treatment with TCDD. We also studied the role of DNA methylation in silencing the CYP1B1 gene in HepG2 cells. The results from our bisulfite DNA sequencing demonstrated strong methylation of CYP1B1 promoter region in HepG2 cells, but not in MCF-7 cells. Furthermore there the enhancer region of the CYP1B1 gene was only partially methylated in both cell lines. This clearly complements our initial ChIP data, demonstrating AhR recruitment of the CYP1B1 enhancer region in both cell lines is due to partial methylation, where as PolII recruitment at the CYP1B1 promoter, only in the MCF-7 cells, but not in the HepG2 cells is due to complete unmethylated, and methylated nature of the region respectively. In conclusion our data clearly demonstrate the potential role of both epigenetic mechanisms; DNA methylation and histone modifications in silencing the CYP1B1 gene expression in the HepG2 cell line.
Acknowledgements: S.R.B was supported by the UC Toxic Substances Research & Teaching Program (TSRTP) and NIH grant CA28868.
P52: Gene Expression Profiling of Reconstituted Human Skin Exposed to Chlorpromazine Points to a Mechanism of Action Involving Stress Damage
M.J. Cunningham1, S. Magnuson2, M. Falduto2, L. Pratt3 and G. DeGeorge3
1Nanomics Biosciences, Inc., 2GenUs Biosystems, Inc., 3MB Research Laboratories.
Chlorpromazine (CPZ) is a known phototoxin but the cellular and genetic mechanism by which this toxicity occurs is still unknown. Reconstituted human skin samples, an in vitro system closely modeling human skin in vivo, were exposed to CPZ at a dose correlating with the EC10. The skin samples were treated with CPZ in the dark as well as exposed to UV light and gene activity was investigated with mRNA expression microarrays at 1, 6, and 20 hr. Samples not treated with CPZ and kept in the dark or exposed to UV light were used as controls.
cRNA targets were reverse-transcribed from total RNA isolated from each of the treatment samples. The labeled cRNA were hybridized onto whole human genome mRNA expression microarrays. The microarrays contained 41,000 probes corresponding to unique genes. The data was analyzed using a tiered approach. Coefficients of variation (CV) from all the probes passing quality measures or a total of 10,299 probes for each biological sample within the treatment groups ranged from 5.3 to 19.2%. The least variability was observed with the principal components analysis (PCA) for the skin samples not treated to CPZ under either dark or light conditions. The profiles for each treatment group were more similar by time point rather than by light/dark exposure. The numbers of down-regulated genes ranged from 10 to 52 and the numbers of up-regulated genes ranged from 96 to 236. Noteworthy genes which were down-regulated included genes involved in differentiation and normally expressed in epithelial tissue, such as CXCL14 and DAPL1. VEGF-A and several genes (e.g. HSPA6, ERO1L and ANGPTL4) related to oxidative stress and hypoxia were up-regulated. Funding was provided by NIEHS Grant No. 5-R44-ES-11927-02.
P53: Dersalazine Sodium is not Genotoxic in an Extended Test Battery
Janer G, Nadal T, Merlos M, and Balsa D
Pharmacology & Toxicology, Palau Pharma S.A, Spain
Dersalazine sodium is a new chemical entity result of linking a potent PAF antagonist with anti-TNFα properties (UR-12715) and 5-aminosalicylic acid (5-ASA) by an azo bond. Dersalazine sodium is carried unaltered through the upper gastrointestinal tract until azo-reduction by the colonic bacteria splits dersalazine sodium into the two metabolites. The compound is currently in Phase II clinical development for ulcerative colitis. Due to some particularities of this molecule, e.g., its low systemic exposure, its high colonic exposure, the presence of an azo-bond, and its azoreduction to UR-12715 and 5-ASA in the colon, the genotoxicity battery recommended by current ICH guidelines (S2B Genotoxicity) might have not been sufficient to assess its mutagenic potential. This work presents the results of the extended battery that we used to evaluate the genotoxic potential of dersalazine. The standard battery recommended by the guidelines was performed not only with dersalazine sodium, but also with its main metabolite UR-12715. Thus, two in vitro studies (Ames and chromosomal aberration test) and an in vivo study (mouse bone marrow micronucleus test) were performed with both compounds. In addition, an Ames test in metabolic reductive conditions was carried out with dersalazine to mimic dersalazine metabolism in the colon. Finally in order to evaluate dersalazine genotoxicity at the site of highest exposure, a Comet assay in the colon of rats treated orally with high doses of dersalazine was performed. The results of all the assays were negative, indicating that dersalazine and UR-12715 are not genotoxic.
P54: Alpha- and Gamma-Tocopherol Quinone are not Mutagenic in a Variety of In Vitro Genotoxicity Assays
LF Stankowski, Jr1, H Murli1, A Hawi2, S Paisley2, and R Stoll3
1Covance Laboratories, Inc., Vienna, VA 22182, 2Penwest Pharmaceuticals, Co., Patterson, NY 12563, 3Stoll and Associates, LLC, Storrs-Mansfield, CT 06268
Quinones are common constituents of biologically active molecules and are involved in a variety of biological processes. Those derived from vitamin E falls into 2 classes: fully substituted alpha-tocopherol quinone (a-TQ), and partially substituted quinones including beta-, gamma- and delta-quinones. Partially substituted para-quinones are considered structural alerts for mutagenicity due to their potential to form Michael adducts with cellular nucleophiles. In particular, gamma-tocopherol quinone (g-TQ) has been reported to be an arylating agent and highly mutagenic in AS52 cells. In contrast, a-TQ lacks these characteristics as it is fully substituted. In these studies, we have examined the potential genotoxicity of a-TQ and g-TQ in a battery of in vitro genotoxicity tests performed in compliance with ICH and OECD guidances and GLP regulations. a-TQ and g-TQ were uniformly negative in a 5-strain bacterial reverse mutation (Ames) assay up to 5000 µg/plate ±S9. Negative results also were observed for both in a chromosome aberration assay in purified human peripheral blood lymphocytes, treated for three hours ±S9 and 24 hours –S9, up to toxic limits. Finally, both were evaluated in the AS52/XPRT assay, up to cytotoxic or solubility limits, and under significantly more rigorous conditions, and both were negative ±S9. Thus, a-TQ and g-TQ have been demonstrated to be negative in a variety of in vitro genotoxicity assays, including the lone test system previously purported to have produced a positive response. An in vivo mouse micronucleus assay currently is in progress and those results also will be reported.
P55: Inorganic Calcium (Ca2+) Induces DNA Damage In Vivo
Carol Beevers1, Morten Fjordholt2, Jørgen Schützsack2, David Kirkland1, David Tweats3 and Iain Stuart2
1Covance Laboratories Ltd, Otley Road, Harrogate HG3 1PY, UK (email: Carol.Beevers@covance.com); 2LEO Pharma, 55 Industriparken, DK-2750, Ballerup, Denmark; 3The School of Medicine, University of Swansea, Swansea, SA2 8PP, UK
Our current research has focussed on the toxicological implications of manipulating intracellular Ca2+ with the most recent and remarkable finding being the potential of inorganic calcium to induce DNA damage in vivo. The hypothesis put forward here is that by allosterically modulating the calcium sensing receptor (CaSR) present on the cell surface of the cells in the rat stomach, high doses of calcium carbonate cause a constitutive activation of the receptor due to the high local concentration in the stomach.
The intracellular signaling pathway is thus over-stimulated, causing an extreme rise in intracellular calcium concentration and possibly other signaling pathways ultimately leading to DNA strand breaks. Several publications demonstrate that disruption of the content and movement of various cations, including Ca2+, can lead to inhibition of DNA synthesis, production of reactive oxygen species, DNA damage and ultimately apoptosis (Dopp et al. 2004). A clear link between activation of the CaSR and DNA strand breaks has been shown by Wu et al. in vitro by “rescue” of cells without a functional CaSR (Wu et al. 2005). In order to investigate this possibility, stomach cells harvested from rats dosed orally with CaCO3 were assessed for statistical changes to both Tail Intensity and Moment relative to vehicle/control in Comet assays. A significant increase in both Tail Intensity and Moment over a 40 fold concentration range (50-2000 mg/kg/day) was observed after three doses at 0, 24 and 45 hours with necropsy at 48 hours. There was no evidence of histopathological changes in the stomach tissue or formation of reactive oxygen species. These results suggest that care should be taken when interpreting comet data from experiments using high doses of Ca2+-regulating agents that act on Ca2+–dependent signal transduction. Furthermore, studies with calcimimetic compounds are planned to confirm the proposed molecular action in vivo.
P56: Withdrawn
P57: Lysosome Impairment, Phagocytosis Inhibition and Retinopathy
Shuyan Lu1, Sharon A Sokolowski2, Eileen Blasi1, Bart Jessen1
1DSRD La Jolla, PGRD, Pfizer Inc., 2DSRD Groton, PGRD, Pfizer Inc
Retina is a highly compact neural structure composed of distinct layers. The retinal pigmented epithelium (RPE) is the pigmented cell layer just outside the neurosensory and serves a number of crucial metabolic functions including the phagocytosis and removal of shed photoreceptor cell outer segments. Drugs with a basic moiety can accumulate in the lysososme and disturb lysosome function including phagocytosis, which will retard the clearance of outer segments and ultimately induce retinopathy. To test this hypothesis we chose a compound that contains a basic moiety and induced retinopathy in long term in vivo studies in rats In vitro assays including viability, LC-3 staining, and phagocytosis were conducted using the human retinal pigmented epithelium cell line ARPE-19. These studies showed that this compound caused cellular vacuolation and dose dependent cytotoxicity that was related to the initial cell number and dosing volume, indicating an accumulation effect. This compound also decreased phagocytosis function of ARPE-19, and induced accumulation of LC-3, suggesting a blockage of the autophagy process. EM analysis of eyes taken from an in vivo study showed the presence of large phagolysosomes, supporting the hypothesis of impairment of lysosome function as a mechanism for retinal toxicity. Twenty three proprietary compounds for which the in vivo effects on the retina were known were tested in a phagocytosis assay to examine the prevalence of this cellular mechanism in retinal toxicity. A correlation amongst the physiochemical properties, in vitro lysosomal pathway effects, and in vivo retinal effects was established. This data reveals the importance of physiochemical properties and lysosome accumulation as a common mechanism for drug induced retinopathy and demonstrates the potential usefulness of in vitro screening in predicting this liability.
P58: Evaluting the Potential Endocrine Distrupting Activity of DE-71 in the Intact Adult Male Rat Screening Assay
Jeff Davis, Glenda Moser, Bill Iverson, and Susan Borghoff
Integrated Laboratory Systems, Inc. Durham, NC. 27713.
The Intact Male Rat Screening Assay is designed as a mode-of-action screening assay for evaluating endocrine activity. The primary purpose of this assay is to detect androgen and thyroid active compounds, and substances that can inhibit steroid biosynthesis. To assess the potential endocrine activity of DE-71, a polybrominated diphenyl ether mixture, adult male Sprague Dawley rats were dosed orally for 15 consecutive days with DE-71 (0, 3, 30, 60 mg/kg) or allyl alcohol (AA) (0, 10, 30, 40 mg/kg) which served as a negative control. The liver, thyroid, and reproductive organs (testes, epididymides, prostate, and seminal vesicles) were weighed, and the testis, epididymis, and thyroid evaluated histopathologically. Serum hormone levels (testosterone, estradiol, DHT, LH, FSH, prolactin, T3, T4, and TSH) were measured in all animals following 15 days of dose administration. Absolute liver weight and liver:body weight increased with no change in thyroid weight in animals treated with DE-71. Histopathological evaluation of testis and epididymis from rats treated with DE-71 (60 mg/kg) or AA (40 mg/kg) revealed no treatment-related changes, however, the thyroid from animals administered 30 or 60 mg/kg DE-71 revealed treatment-related changes consisting of follicular cell hypertrophy and hyperplasia, smaller follicles, less colloid and less eosinophilic colloid compared to the vehicle control or other treatment groups. An increase in serum TSH concentration with a corresponding decrease in T4 was observed with treatment of DE-71 with no changes in these levels measured in animals administered AA. The results from this study will be part of the validation database for use in assessing the relevance and reliability of this screening method. (This study was funded by the American Chemistry Council.)
P59: Possible Protective Role of Propolis Against the Hazardous Effect of Triphenyltin on Reproductive Performance and Seminal Plasma Biochemistry of Male Rabbits
Mokhtar I. Yousef1*, Kamel I. Kamel2, Mervat S. Hassan3, Ahmed M. A. El-Morsy3
1Department of Home Economic, Faculty of Specific Education, Alexandria University, 14 Mohamed Amin Shohaib Street, Moustafa Kamel, P.O.Box. Roushdi Alexandria 21529, Egypt; 2Animal Production Research Institute, Dokki, Giza, Egypt; 3Central Lab. for Food and Feed (CLFF), Agriculture Research Center, Giza, Cairo, Egypt
Triphenyltin (TPT) is known to cause endocrine disruption, reproductive toxicity and a decrease in testosterone production. It is involved in the production of reactive oxygen species. Propolis has been reported to be an important antioxidant. Therefore, the present investigation aimed to elucidate the possible protective effects of propolis in alleviating the toxicity of triphenyltin chloride (TPTCl) on reproductive performance, testosterone levels, lipid peroxidation and enzyme activities in seminal plasma of male New-Zealand white rabbits. Six rabbits per group were assigned to 1 of 4 treatment groups: 0 mg propolis and 0 mg TPTCl/kg bw (control); 50 mg propolis/kg bw; 0.5 mg TPTCl/kg bw; 0.5 mg TPTCl plus 50 mg propolis/kg bw, respectively. Rabbits were orally administered their respective doses every day for 12 weeks. Results showed that semen quality was deteriorated following treatment with TPT. It significantly (P<0.05) decreased libido (by increasing the reaction time), ejaculate volume, sperm concentration, total sperm output, sperm motility (%), total motile sperm per ejaculate (TMS), packed sperm volume (PSV), total functional sperm fraction (TFSF), normal and live sperm and semen initial fructose. While, initial hydrogen ion concentration (pH), and dead sperm were increased. Also, testosterone levels, body weight (BW), relative weights of testes (RTW) and epididymis (REW) were decreased. Levels of thiobarbituric acid-reactive substances and lactate dehydrogenase were increased, while the activities of glutathione S-transferase, transaminases and phosphatases were decreased in seminal plasma of rabbits treated with TPTCl compared to control. Propolis alone significantly increased testosterone levels, BW, RTW, REW, semen characteristics and seminal plasma enzymes, and decreased the levels of free radicals and lactate dehydrogenase. Furthermore, the presence of propolis with TPTCl alleviates its toxic effects. Therefore, the present study concluded that propolis can be effective in the protection of TPTCl-induced reproductive toxicity.
P60: Dopaminergic Toxicity During Development Alters Locomotor Activity in Larval Zebrafish
Terra D. Irons1,3, Robert MacPhail2, Deborah L. Hunter1, Beth Padnos1 and Stephanie Padilla1.
1Integrated Systems Toxicology Division; 2Toxicology Assessment Division, U.S. Environmental Protection Agency, Research Triangle Park, NC, USA; 3Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC
Exposure to pesticides is thought to be implicated in the etiology of parkinsonism. In an effort to develop a rapid in vivo screen for pesticides that are dopaminergic poisons, we are characterizing the phenotypes associated with dopaminergic poisoning of zebrafish (Danio rerio) larvae. The neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) acts on dopaminergic neurons, causing parkinsonism in mammals. In this study, zebrafish were exposed to MPTP during development for more complete characterization of the dose-response function. Zebrafish were exposed to 0.08-50 µM MPTP in a 96-well microtiter plate from 5- hours to 5-days post fertilization, followed by one depuration day. On the sixth day, larvae were evaluated for structural alterations, and locomotor activity was assessed. Developmental exposure to the highest dose of MPTP (50 µM) produced teratogenic changes, as well as hypoactivity, consistent with previous studies. In contrast, lower, non-teratogenic doses (3.1, 6.3, and 12.5 µM) produced hyperactivity. Larvae that were exposed to high doses of MPTP during development showed similar decreases in motor activity when compared to mammals and previous studies in zebrafish; however, the unique finding in the current study is that those exposed to lower doses of MPTP manifested hyperactivity.
This abstract does not necessarily reflect Agency policy.
P61: DDA, a Biomarker for Determination of DDT Exposure in Wildlife Specimens
Zhenshan Chen1,2, Li Cui2, Kyle Aratani2, Arthur Joseph2, Ohimai Unoje2, Weiguo Song2,3 and Robert I. Krieger1,2
1Environmental Toxicology Graduate Program; 2Personal Chemical Exposure Program, Dept of Entomology, Univ. of California, Riverside, CA 92521; 3Shanghai Academy of Agricultural Sciences. 2901 Beidi Road, Shanghai, China 201106
DDA, [bis(p-chlorophenyl)acetic acid, CAS# 83-05-6], is a water-soluble metabolite of DDT but not DDE. Controversies over DDT and its major metabolite DDE have become public concerns since the publication of Rachel Carson’s Silent Spring. Recently attention has been directed toward their possible endocrine disrupting effects in wildlife and humans. Although the two persistent chemicals occur together their toxicologies differ. Measurement of rapid DDA excretion provides a means to clarify the extent of actual concurrent DDT exposure. An analytical method for the determination of DDA in chicken or bird excreta was developed. DDA in excreta was acid hydrolyzed, extracted with hexane and derivatized using pentafluorobenzyl bromide and diisopropylethyl amine at room temperature for 1h. DDTs (DDT/DDD/DDE) and DDA were detected using GC-MS in SIM mode. The method limit of detection (LOD) is 1 ppb in feces. A 4 d feeding study with chickens was done using non-toxic levels of DDT/DDD (10, 100 and 1000 ppm in diet). Chicken excreta were collected daily. About 1% of DDT ingested was excreted daily as DDA during active DDT administration. Less than 0.01% was excreted as DDE. DDA excretion decreased rapidly (within 2 days) after DDT feeding stopped. The rapid excretion of DDA in excreta may be a useful biomarker of DDT exposure in the wildlife.
P62: Toxicity of Chloropicrin Vapor to Plants and Seeds
J. Weinberg1, M. Amato1, D. Voltz2, J. Butala3
1WIL Research Laboratories, LLC, Ashland, OH; 2The Ohio State University, Wooster, OH; 3Toxicology Consultants, Gibsonia, PA
The objective of this study was to evaluate potential adverse effects of chloropicrin vapor on seed germination and young plant growth when administered for 6-hours on 2 consecutive days. Seeds and young plants of 6 representative monocotyledon and dicotyledon species were exposed to target concentrations of 0, 200, 1,000 and 10,000 ppb chloropicrin. Each test or control group consisted of 10 replicate pots containing seeds or young plants. A post-exposure observation period of 14-days (seeds) or 21-days (plants) was employed. Plants were observed daily for mortality and/or signs of phytotoxicity throughout the 14- or 21-day post-exposure period. The modified Horsfall and Barratt rating scale was used to assess plant damage or phytotoxicity. There were no test article-related effects on seedling emergence, survival or wet or dry biomass for 5 of the 6 species. There was a statistically-significant decrease in wet biomass for ryegrass seedlings at the 10,000 ppb exposure level. Exposure of young plants to 10,000 ppb produced chlorosis and mortality in cucumbers and resulted in a statistically-significant reduction in wet and dry biomass for all other species tested, except for corn. The no-observed-effect concentration (NOEC) for chloropicrin vapor exposure of 6 plant species for 2 consecutive days was 1000 ppb (1 ppm).
P63 Withdrawn
P64: Cytotoxicity and Necrotic Cell Death in Human Bronchoalveolar A549 Cells Following Exposure to Fine Particulate Matter (PM2.5) Collected in Southeastern Louisiana
Brian Bourgeois, Viswanathan Rangan, and John Owens
Southern University; PhD Program in Environmental Toxicology; Baton Rouge, LA, 70813
The cytotoxicity of PM2.5 on A549 human bronchoalveolar carcinoma cells was studied, utilizing a Promega Cell Titer 96 Aqueous assay. Cell viability curves were generated in triplicate for three different batches of PM 2.5 suspensions. Batch 1 was utilized at concentrations of 600μg/mL, 300μg/mL, 200μg/mL, 100μg/mL, and 50μg/mL; batch 2 was utilized at concentrations of 250μg/mL, 200μg/mL, 100μg/mL, 50μg/mL, and 10μg/mL; batch 3 was utilized at concentrations of 181.8μg/mL, 145.44μg/mL, 109.05μg/mL, 72.7μg/mL, 36.3μg/mL, and 18.15μg/mL. Batch 1 showed an IC50 of 238µg/ml; batch 2 showed an IC50 of 59µg/ml; and batch III showed an IC50 of 130µg/ml. The presence of transition metals in PM2.5 particulates is believed to be the major cause of toxicity. As a result, cytotoxicity was also measured in the presence of deferoxamine mesylate, a well known transition metal chelating agent, at deferoxamine concentrations of 0.75mM, 0.50mM, and 0.0375mM. Tests showed a remarkable reduction in PM2.5 cytotoxicity. The reduction in cytotoxicity suggests that deferoxamine mesylate provides protection to A549 cells, and, that transition metals are indeed involved in mediating PM2.5 toxicity. To decipher the mechanism of cell death by conducting experiments to differentiate apoptosis from necrosis for the highest concentration of PM2.5 that was used on A549 cells for cytotoxicity studies. At the highest concentration used (600 ug/ml) the three different PM 2.5 extracts that was tested showed 50 ± 16%, 45 ± 14%, 65 ± 19% and 70 ± 20 %, 62 ± 25%, 60 ± 19% cells undergoing necrosis by Annexin-V FITC/ Propidium Iodide and Acridine Orange/Ethidium bromide dual staining respectively.
P65: Withdrawn
P66: Osmotically Active Constituents in Moist Smokeless Tobacco Extracts
Jeff Edmiston, Don Farthing, Rob McKallip, Catherine Lombard1, Chris Tucker and Marc Fariss
Altria Client Services, Richmond, Virginia; 1RemX Specialty Staffing c/o Altria Client Services, Richmond, Virginia.
Leukoplakia, a reversible oral mucosal injury, has been noted in some smokeless tobacco users. The specific properties of moist smokeless tobacco (MST) related to this injury are unknown. One possibility is the induction of hyperosmolarity at the site of smokeless tobacco use. In the present study, we investigated the source of hyperosmolarity in extracts of smokeless tobacco as well as the possible role of hyperosmolarity in smokeless tobacco-induced oral injury using an in vitro model. The hyperosmolarity observed in MST extracts appears to be attributable to the presence of sodium, calcium and potassium salts, and to a lesser extent acetic acid. These materials were used to create a solution with similar osmotic properties as the MST extracts. This synthetic solution was compared to MST extracts with similar osmolarity in an in vitro inflammatory cell viability model using the MM6 cell line. Using this model system, approximately 60% of the loss of cell viability induced by the MST extract could be attributed to hyperosmolarity, indicating hyperosmolarity may be playing an important role in MST extract induced MM6 cell death in this in vitro model.
P67: Measuring Pesticide Availability And Transferability Using Rubber Latex Gloves of Strawberry Harvesters as a Potential Dosimeter
Gayatri Sankaran1,2, Yanhong Li2, Zhenshan Chen1,2, Terry Lopez1,2, Li Cui2, Song Wei-guo2,3, Helen Vega2, Robert I. Krieger1,2.
1Environmental Toxicology Graduate Program; 2Personal Chemical Exposure Program, Dept of Entomology, Univ. of California, Riverside, CA 92521; 3Shanghai Academy of Agricultural Sciences. 2901 Beidi Road, Shanghai, China 201106
Pesticides are used in modern organic and conventional agriculture when growers chose to produce berries for paying customers rather than giving them away to pests. Low level pesticide exposures occur primarily due to hand contact with foliage residues. Strawberry harvesters frequently use light rubber latex gloves to reduce pesticide exposure, to cosmetically protect the skin from accumulation of dirt and juice, and as a food safety measure.
Potential harvester exposure (µg/harvester-h) may be expressed as the product of dislodgeable foliar residues (DFR, µg/cm2), an empirical transfer coefficient (cm2/h) specific to strawberry harvesting, and the working period (h). However, only a portion of the DFR is transferred to harvesters who contact leaf surfaces during harvest. Our overall goal is to develop a simple, clear and reliable indicator of strawberry harvester exposure. The residues transferred from leaf surfaces to harvester gloves may be used to predict external potential harvester insecticide, fungicide and miticide exposure.
Latex gloves have been randomly collected since 2005 as they are discarded by harvesters at strawberry farms in Santa Maria, CA. The highest average glove residues occur at the PHI (Pre-Harvest Interval). Residues from insecticides, fungicides and miticides have been detected in ethyl acetate extracts of gloves worn for 2 to 2.5 h work periods. Residues on gloves range from 19.7 to 0.001 mg/pair. Up to 8 residues may be detected in extracts. When leaf malathion DFRs were 0.025 ug/cm2 to 0.16 ug/cm2, the corresponding glove residues were about 1.67 mg/pair. The available residue will be a portion of the total glove residue during any work period. Since hands contribute 50% to 90% of total strawberry harvester pesticide exposure, glove residues may represent an indirect means to predict potential long-term, low-level harvester exposures.
P68: Withdrawn
P69: Does Diexpoxybutane (DEB) Increase Cancer Stem Cells and Allow Resistance to Anti-Tumor Therapy?
Lea A. Dixon2, Eduardo Martinez-Ceballos1, 2
Department of Biological Sciences1 and Environmental Toxicology Program2 Southern University and A&M College Baton Rouge, LA, 70813
1,3-butadiene (BD) has been deemed as an environmental pollutant and is also a volatile gas used in the production of rubber, plastics, insulation, acrylics, and other polymers. Its ubiquitous occurrence in environmental and industrial areas has been shown to increase the risks of respiratory illnesses. In addition, BD is a known mutagen and human carcinogen, and possesses multi-organ systems toxicity that includes bone marrow depletion, spleen, and thymus atrophy. After entering the body, BD is metabolized to its most toxic metabolite, Diepoxybutane (DEB). Recent studies suggest that some environmental pollutants may increase the stem cell population in several human cancers and that these cancer stem cells can acquire resistance to anticancer drug treatments. In order to elucidate the cellular and molecular mechanisms mediating the acquired resistance to carcinogens in cells, we investigated the effect of DEB on the prevalence of cancer stem cells among the human prostate cancer cell line DU145. We observed that DEB (1µM) increases the incidence of cancer stem cells within the DU145 cell population. By employing specific cell pathway inhibitors, we determined the role played by various cell survival pathways during the DEB-induced prevalence of cancer stem cells in DU145 cells.
P70: Clinical Trials Outside of the United States
CL Kruger1 and AW Hayes1,2
1Spherix Consulting and 2Harvard School of Public Health
Despite introduction of a number of agents for treatment of Type 2 diabetes, glucose control in this population remains unsatisfactory because average HbA1c’s are well above 8%. D-Tagatose, an epimer of D-fructose, is proposed for use as an oral antidiabetic agent in Type 2 diabetics under diet and exercise control. An ongoing single-blinded study being conducted in the U. S. and India is designed to establish the minimum dose of D-tagatose capable of producing a significant reduction in HbA1c. With an estimated 40 million people suffering from diabetes in India, placement of diabetes trials in this country is growing. Conduct of a clinical trial in India requires experience with and knowledge of the regulations, ethics, good clinical practice and skills for clinical trial management. For this trial, D-tagatose was administered orally with meals, three times daily (TID) at 2.5, 5.0, and 7.5 g. After 6 months on drug, patients in the 7.5 g group experienced an average reduction of 0.3% in HbA1c from the HbA1c of the 2.5 g group. At that same 6-month point, the 5.0 g group averaged a reduction in HbA1c of 0.05% from the 2.5 g group. These data, combined with the fact that D-tagatose is a naturally occurring compound with no known contraindications to current Type 2 diabetes treatments, suggest a place for D-tagatose in the treatment regime as either a stand-alone or an adjunct therapy.