S40 Abstracts / Toxicology Letters 229S (2014) S40–S252

P1: Alternative animal models

P-1.1Utility of the HRNTM (hepatic cytochrome P450reductase null) mice for investigatingmechanisms of liver toxicity ofcarboxylic-acid-containing drugs

James Akingbasote 1,∗, Alison Foster 2, Sunil Sarda 3, Huw Jones 4,Ian Wilson 5, Gerry Kenna 6

1 Institute of Translational Medicine, University of Liverpool,Liverpool, Merseyside, UK, 2 Translational Safety, Drug Safety andMetabolism, AstraZeneca, Alderley Park, Cheshire, UK, 3 DiscoverySciences, AstraZeneca, Alderley Park, Cheshire, UK, 4 PathologicalSciences, Drug Safety and Metabolism, AstraZeneca, Alderley Park,Cheshire, UK, 5 Department of Surgery and Cancer, Imperial College,London, UK, 6 Safety Science Consultant, Cheshire, UK

Many carboxylic-acid-containing drugs cause liver injury inhumans. Examples include fenclozic acid, which was withdrawndue to jaundice observed in clinical trials, and diclofenac, whichremains widely prescribed despite being associated with liverdamage. To explore whether metabolic bioactivation mediatedby cytochrome P450 (CYP) or UDP-dependent gluronyltransferase(UGT) enzymes could play a role in these toxicities, covalentbinding (CVB) assays were performed using liver microsomal incu-bations from wild-type and hepatic cytochrome P450 reductasenull (HRNTM) mice, which are deficient in hepatic CYP activity.Additionally, wild-type and HRNTM mice were administered fen-clozic acid and diclofenac orally for 7 days and the effect on clinicalchemistry biomarkers and liver pathology investigated. High levelsof CYP-mediated CVB of [14C]-fenclozic acid and [14C]-diclofenacwere observed in wild-type microsomes, but not in HRNTM micro-somes. No UDPGA-mediated CVB was detected in microsomesincubated with [14C]-fenclozic acid. Exposure to fenclozic acid(100 mg/kg) for 7 days resulted in a significant (p < 0.05) increase inplasma alanine amino transferase (ALT) in wild-type but not HRNTM

mice. In HRNTM mice liver histopathology changes and markedlyelevated ALT, glutamate dehydrogenase and alkaline phosphataselevels were evident. Treatment with diclofenac and fenclozic acid“normalised” the liver clinical chemistry parameters. These datademonstrate that fenclozic acid undergoes CYP but not UGT medi-ated bioactivation and that HRNTM mice can provide a valuableinsight into the role of hepatic CYPs in drug metabolism. However,HRNTM mice are not well suited to investigations of liver toxicity,due to impaired background liver function.

http://dx.doi.org/10.1016/j.toxlet.2014.06.180

P-1.2Screening of compounds that alter sleep–wakestate in zebrafish

Olaia Holgado 1, Arantza Muriana 1, Anne Dekeyne 2, CarolineLouis 2, Ainhoa Alzualde 1,∗

1 BBD BioPhenix SL, Donostia, San Sebastian, Spain, 2 Unité deRecherche de Découverte en Neurosciences, Institut de RecherchesServier, Croissy-sur-Seine, France

The zebrafish is emerging as an alternative to mammaliananimal models. Although it is more phylogenetically distantfrom humans than mammals, zebrafish are vertebrate thatdisplay other advantageous characteristics including embryo trans-

parency, which allows direct visualization and evaluation ofinternal organs; or small size, which allows to manipulate themeasily and to use 96-well plates to array embryos. The model isideal to be used as a predictive tool in Drug Discovery, being able ina high extent to compare the results with those obtained in mam-mals. Increasing knowledge in zebrafish gives more support to thisalternative model.

Physiological, biochemical and behavioral daily rhythms areregulated by circadian clock. Apart from having many biologicaladvantages, zebrafish are diurnal and present highly conserved cir-cadian and sleep regulation systems, so they have been postulatedalso as a good model for circadian rhythm studies. Behavioral profil-ing in zebrafish reveals a conserved vertebrate neuropharmacologyand identifies regulators of rest/wake states. For screening pur-poses, working with zebrafish larvae has many advantages in termsof rapidity and throughput.

The main objective of this study was to set up a protocol to detectcompounds with potential to alter rest/wake states in zebrafish. Forthis purpose, zebrafish larvae were exposed to Melatonin and otherreference drugs as Barbiturates and Benzodiazepines. Diurnal andnocturnal locomotor activity and responsiveness of larvae are themain endpoints related to sleep states. This study supports the useof zebrafish in order to detect compounds that alter the sleep–wakestates.

http://dx.doi.org/10.1016/j.toxlet.2014.06.181

P-1.3Analysis of the influence of vehicles on thesensitizing potential of 3 allergenic compounds

Marie-Pierre Berrada-Gomez 1,∗, Hanna Dalhuchyts 1, SandyRattier 1, Catherine Bidan 1, Hervé Groux 2, Pierre-Jacques Ferret 1

1 Pierre Fabre Dermo Cosmetique, Toulouse, France,2 IMMUNOSEARCH, Grasse, France

Purpose: Cutaneous sensitization results from the induction ofan immune response following the exposure of the skin to a sub-stance. A compound sensitizing potential depends on its capacityto penetrate the skin. This potential could be reduced or increasedby the bioavailability of the substance. Thus, the use of an appro-priate vehicle could play a leading role in the bioavailability of thecompound in order to decrease the sensitizing risk.

Method: The aim of this study was to determine the influenceof 5 cosmetic forms (oil-in-water emulsion, cleansing water, oil,water-in-oil emulsion, microemulsion) on the sensitizing potentialof 3 allergens. Cinnamaldehyde, isoeugenol and hydroxycitronel-lal were tested at several concentrations 10%, 5%, 2%, 1%, 0.1% onthe Sens-is model. This assay assesses the ability of the test item tospecifically induce the gene expression of irritation and sensitiza-tion biomarkers in an in vitro 3D model of reconstructed skin.

Results and conclusion: The 3 compounds sensitizing potentialhas been significantly reduced by the microemulsion while it hasbeen significantly increased by the cleansing water. These obser-vations have been less important with the emulsions.

On the other hand the oil vehicle has not presented any influenceon this potential.

Moreover it would be interesting to carry out a study with com-plex mixtures as perfumes used in cosmetic products and comparethe results to those obtained with the Human Repeated Insult PatchTest. The Sens-is model could also provide relevant data to assessthe safety of hypoallergenic products.

http://dx.doi.org/10.1016/j.toxlet.2014.06.182