American College of Toxicology 30th Annual Meeting

Poster Abstracts

P1: A 28-Day Intrathecal Toxicity Study With Trastuzumab in Cynomolgus Monkeys Ange´lique P.J.M. Braena, Jose´e 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 mg/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 HER2positive breast cancer.

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International Journal of Toxicology 29(1) 88-111 ª The Author(s) 2010 Reprints and permission: http://www. sagepub.com/journalsPermissions.nav DOI: 10.1177/1091581809357326 http://ijt.sagepub.com

P2: Combination Toxicity Evaluation of Direct Acting Anti-Hepatitis C Virus (HCV) Agents With Pegylated Interferon-␣ (pIfn␣) 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 pIFNa 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 pIFNa 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 pIFNa/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 pIFNa (subcutaneously every other day) and RBV (oral solution daily). DAA doses were chosen to generate clinically relevant systemic exposures and pIFNa/RBV doses were multiples of approved clinical doses. Additional groups of monkeys received either BMSDAA vehicle alone and/or BMS–DAA vehicle with pIFNa/ RBV and served as control groups. Standard toxicokinetic, toxicologic, and pathologic endpoints were assessed along with immunogenicity against pIFNa. As validation of the experimental system, changes consistent with known effects of RBV on red cell turnover, IFNa inhibition of erythropoiesis, and anti-IFNa antibody formation were evident in groups receiving these agents. The addition of BMS-DAAs to the pIFNa/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. Index Terms: Combination Toxicity, Interferon, Hepatitis C Virus

Poster Abstracts

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-1b) and inhibits activation of the IL-1 receptor. This activity is expected to prevent the cellular signaling events that produce inflammation. IL-1b 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-1b 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. Index terms: Monoclonal Antibody, XOMA 052, IL-1 beta

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. 1

Halozyme 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

89 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 permeationenhancing excipient. Index Terms: Hyaluronidase, Drug Delivery, Preclinical Safety

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. 1

Development Sciences, Genentech Research and Early Development, SSF, CA; 2SNBL Ltd., Kagoshima, Japan; 3 Hoffmann-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 89

90 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 Index Terms: Monoclonal Antibody, Fetal Toxicity, Amniotic Fluid

International Journal of Toxicology 29(1) 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. Index Terms: Cynomolgus Monkey, Anti-CD20, Embryo/ Fetal Development

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 1

P8: Embryo/Fetal Development in Cynomolgus Monkeys Exposed to Rituximab A. Vaidyanathan1, K.McKeever1, H. Tsusaki2, S. Eppler1, S.Ortega1, J. Beyer1 1

Genentech, 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

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Research 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. Cmax 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 Cmax seen was with the medium dose (100 mg/kg) suggesting that the high dose toxicity seen was not Cmax 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. Index Terms: Exploratory Toxicology, Expression Profiling, Targeted Hepatitis C Drug

Poster Abstracts

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. Index Terms: Non-traditional vehicles, Cremophor RH 40, Solutol HS 15

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

91 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 ¼ 5) and LongEvans (n ¼ 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. Index Terms: Phospholipidosis, Thyroid Gland, Hypothyroidism

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 1

Bristol-Myers Squibb Co., 2Burleson Research Technologies

An immunologic investigation of BMS-708163, a gammasecretase 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 ngh/mL, *4X clinically active 91

92 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 ngh/mL. At AUC  4670 ngh/mL, decreased number of IgMþ B-cells in the splenic marginal zone (MZ) was observed. At higher exposures ( 22800 ngh/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 BMS708163 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 bloodborne 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 IgMspecific 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. Index Terms: Immunotoxicology, Gamma Secretase Inhibitor, Host Resistance Models

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 a

Archer Daniels Midland Company, Decatur, IL; bWIL Research Laboratories, LLC, Ashland, OH; cBurcon NutraScience Corporation, Winnipeg, Canada. Ninety day subchronic dietary toxicity studies of a napinrich 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

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International Journal of Toxicology 29(1) 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. Index terms: Sub-chronic toxicity, Cruciferin-rich canola protein isolate (Puratein), Napin-rich canola protein isolate (Supertein)

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

Poster Abstracts 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. Index Terms: Mineralization, MEK, Phosphorous

P15: Developmental and Pre-/Postnatal Reproductive Toxicity of rHuPH20 S Nadjsombati1, A Hoberman2, D Newcomb2, BJ Sugarman1, GI Frost1 and WH Bee1. 1

Halozyme 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. Index Terms: Hyaluronidase, Drug Delivery, Reproductive Toxicology

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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 1

Sinclair 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. Index Terms: Dermal Model, Miniature Swine, Cutaneous Model

P17: Go¨ttingen 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 doselimiting 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 93

94 18.5 mghr/mL), with little emesis noted, while 7.5 mg/kg (AUC0-t of 3.0 mghr/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 2week 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 2week 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 mghr/mL); 7.5 mg/kg/day (Day 13 AUC0-24 of 9.0-9.9 mghr/mL) was the NOEL in this 2-week study. Finally, a cardiovascular study in Go¨ttingen 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 Go¨ttingen minipigs have successfully supported first-in-human and ongoing Phase II clinical trials for KNS–760704. Index Terms: Go¨ttingen Minipigs, KNS–760704, Nonrodent Toxicity

P18: The Utility of the Minipig in P38 Map Kinase Inhibitor Testing J. Schu¨tzsack1, A. Gibbs2, J. Parish2, K. Gill2 1

LEO Pharma A/S, 55 Industriparken, DK-2750, Ballerup, Denmark; Covance Laboratories Ltd, Otley Road, Harrogate HG3 1PY, UK

2

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 TNFa and IL-1b 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 94

International Journal of Toxicology 29(1) 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. Index terms: MAP kinase inhibitor, Minipig, Toxicity testing

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 1

Sinclair 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 naı¨ve 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. Index Terms: Organ Weights, Miniature Swine, 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

Poster Abstracts 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. Index Term: Topical Formulation, Animal Model, Skin Irritation

P21: Sporatic Occurrence of Low Fertility Rates in Control CD Rats Hentz, KLy; Neal, BHy; Tyl, RWz; Hoberman, AM*; and Wilson, D.# y

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 z

In recent reproductive/developmental studies fertility rates for control CD rats were observed to be considerably lower than expected (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. Index Terms: Zebrafish, Seizure, Behavior

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 4b-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 mM (p < 0.05 and p < 0.01, respectively), PMA at 1 and 10 mM (p < 0.001), TSA (Trichostatin A) at 25 and 50 mM (p < 0.001), Menadione at 100 and

97 500 mM (p < 0.01 and p < 0.001), TBHP (tert-Butyl hydroperoxide) at 2500 and 5000 mM (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 mM. 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% (7/7). These data underscore the high degree of conservation of drug toxicity pathways among species and suggest that whole zebrafish microplate ROS assay represents a predictive, reproducible animal model for screening ROS-inducing compounds. Index Terms: Zebrafish, ROS, Toxicity

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. Index Terms: Validation, Toxicology Studies, Facility 97

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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 reproductiveembryofetal toxicology facility at LAB Research Inc., timedpregnant 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. Index Terms: Sprague Dawley Rats, Retinoic Acid, Embryofetal Development

P31: Validation of Immunohistochemical Staining Myle`ne Valin1, Kate Lillard-Wetherell1, Thomas Lemarchand2, Palate Bernard2, and Roy Forster2 1

Aperio, 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 98

International Journal of Toxicology 29(1) 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. Index Terms: Immunohistochemistry, GLP, Antibodies

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

Poster Abstracts 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 1

Ecotoxicology, 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 MNUtreated 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 Urethanetreated 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. Index Terms: Transgenic, ras.H2, Carcinogenicity

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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 selfadministration (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