Abstracts / Toxicology Letters 221S (2013) S31–S56 S45

as a readout of drug-induced neuronal activity.By mapping theneuronal substrates that change activity in response to sleep-and wake-inducing drugs, we hope to identify critical zebrafishsleep circuits. The function of these circuits in zebrafish sleepcan then be dissected by traditional genetics or with optogenetictechniques.

http://dx.doi.org/10.1016/j.toxlet.2013.06.161

W11-3The utility of the zebrafish for drug safetyassessment: an industry perspective

Matthew J. Winter, Jonathon Ball, Alex Goonesinghe, MalcolmHetheridge

AstraZeneca, Safety, Health & Environment, Brixham, Devon, UK

Recently the zebrafish has emerged as a credible, 3Rs-friendly,non-mammalian model for human drug safety assessment.In particular, the embryolarval form facilitates the use of lowamounts of test compound in a microplate; attributes that allowdevelopment of higher throughput assays for use in frontloading(i.e. conducting earlier) functional drug safety assessment.

The range of safety assessment disciplines for which promis-ing zebrafish based assays have been developed/proposed is wide:from developmental toxicity to cardiovascular function; and fromototoxicity to behavioral-based assays addressing adverse effectssuch as seizure and abuse liability. In our experience, however,relatively few validated assays are utilized routinely within phar-maceutical company safety assessment screening cascades.

Here,using our own experiences and examples from the liter-ature, we explore the reasons for the apparent underutilizationof this promising model by the pharmaceutical industry, withinthe context of the industry expectations for new models. Topicsfor discussion include: expected prediction of (pre)clinical out-come;desired levels of throughput and compound requirements;expected levels of automation; and the recurrent issues of rela-tive exposure concentrations, therapeutic versus assay effect levels,metabolic capabilityand clinical relevance.

In addition, we highlight applications that appear particularlypromising, for example gaps in current safety screening strategiesthat could be filled using this model. The overall aim is to pro-vide the audience witha better understanding of what is expectedof a zebrafishmodel/assay before it is suitable for application inan industrial setting, with the ultimate aim of aiding thegreaterexploitation of this highly promising model.

http://dx.doi.org/10.1016/j.toxlet.2013.06.162

W11-4Predicting drug-induced hepatotoxicity inzebrafish larvae

N. Mesens 1, A. Crawford 2, A. Menke 3, F. Van Goethem 1, C.Esguerra 2, A. Wolterbeek 3, P. De Witte 2, J. Van Gompel 1

1 Drug Safety Sciences, Janssen Pharmaceutical Companies ofJohnson&Johnson, Beerse, Belgium, 2 Laboratory of Pharmaceuticaland Biological Sciences, Katholic University of Leuven, Leuven,Belgium, 3 TNO Triskelion bv., Zeist, The Netherlands

Zebrafish larvae represent an attractive lower animal model tofill the gap between high throughput in vitro cellular assays andconventional preclinical animal testing. The model may, in partic-

ular, be useful in predicting human liver toxicity.Drug-induced liverinjury (DILI) is poorly predicted by single- cell- based assays, likely-due to the lack of physiological integrations with other cells withinthe liver. The whole liver found in the zebrafish could provide addedvalue in a screening strategy for DILI.

The aim of this study was to set up an assay for assessing DILI inzebrafish larvaeby assessing the expression of a liver specific pro-teinin the larval liver. Hereto, LFABP10 was chosen as a marker sincetissue- specific fatty acid binding proteins were recently suggestedas plasma markers for tissue injury (Pelsers et al., 2005). In addition,investigating proteomic biomarkers during hepatotoxicity in ratsshowed that LFABP proteins were diminished after acetaminophenexposure; suggesting downregulation during acute hepatocellularnecrosis (Yamamoto et al., 2006).

The effect of reference compounds on fabp10 expression wasthen investigated. Compounds were selected to induce the major-ity of hepatotoxic phenotypes in humans (cholestasis, steatosisand necrosis) with knowninsults such as inhibition of the bile saltexport pump, mitochondrial toxicity and reactive metabolite for-mation.

Hepatotoxic compounds were identified by asignificant changeinthe expression of the liver-specific protein fabp10. Differ-ent expression patterns were observed and compared with thehistopathological changes in the liver, both in adults and zebrafishlarvae. Whole genome micro-array analysis was included to eluci-date the mechanisms of DILI.

Reference

Pelsers, M.M., Hermens, W.T., Glatz, J.F., 2005. Fatty acid-binding proteins as plasmamarkers of tissue injury. Clin Chim Acta. 352 (Feb (1-2)), 15–35.

Yamamoto, T., Kikkawa, R., Yamada, H., Horii, I., 2006. Investigation of proteomicbiomarkers in in vivo hepatotoxicity study of rat liver: toxicity differentiationin hepatotoxicants. J Toxicol Sci. 31 (Feb (1)), 49–60.

http://dx.doi.org/10.1016/j.toxlet.2013.06.163

W11-5New approach to a predictive toxicityevaluation with a zebrafish assay

Ainhoa Alzualde

BBD BioPhenix S.L., San Sebastián–Donostia, Spain

Toxicological screening is important for the development ofnew drugs and for the extension of the therapeutic potential ofexisting molecules. As behavior reflects integration of the vari-ous functional components of the nervous system alterations inbehaviour could be indicative of toxicity. Changes in behaviourcould be also observed due to indirect effects of drugs on otherphysiological systems. Zebrafish has a high genetic homologywith humans and an important parallelism in organogenesis andfunctional mechanisms. Its small size, rapid development, highreproducibility and transparency make zebrafish an ideal model forscreening large number of compounds. Here we present an assaybased on locomotor activity evaluation of zebrafish larvae com-plemented with a general morphological screening. Biobide hasvalidated this assay in zebrafish with several compounds. Larvaewere treated with toxic, neuroactive and innocuous compoundsduring 2 days and their locomotor activity was evaluated at 6dpfunder two light-dark cycles. Afterwards, a general morphologi-cal screening was carried out. Preliminary results indicate thatzebrafish embryos show locomotorbehavior and/or morphologicalalterations in response to drug treatments allowing us to detectpotential toxicity of compounds. Zebrafish are a good model to beused as a complementary to in vivo models in the Drug Discovery

S46 Abstracts / Toxicology Letters 221S (2013) S31–S56

process providing pharmaceutical industry with new assays whichmight decrease the current huge gap between in vitro and in vivoassays.

http://dx.doi.org/10.1016/j.toxlet.2013.06.164

Workshop 12: Identifying, assessing and managing allergens infood

W12-1Trends in food allergy and impact on publichealth

Jonathan O’B Hourihane

Paediatrics, University College Cork, Ireland

Food allergy is a major focus of clinical/public health attention.Issues of regulatory importance such as the use of precautionarylabelingare the focus of EU and FDA attention and clinical decisionsremain difficult over who should have adrenaline autoinjectorsavailable and who should be offered immune-modulatory treat-ments. The avoidance of the most allergenic foods in infancy (milk,egg, peanut) does not appear to prevent the initiation of thesefood allergies at a population level. Conversely studies are explor-ing the effect of early introduction of such foods to promote theacquisition of oral tolerance to these foods, like other foods notknown for their allergenicity.The clinical management of estab-lished food allergy is changing rapidly at present, moving fromadvice about extreme vigilance and stringent, total avoidance ofallergens to cautious exposure to allergens whose antigenicity isattenuated by processing such as heating. This is not advised forall food allergens however, as peanut’s allergenicity is increasedby heating, unlike egg and milk,which are decreased. New foodallergies are constantly being described but their impact on pub-lic health cannot be accurately assessed on the basis of anecdotalreports. Europe-wide studies have allowed the characterization ofpaediatric food allergies in different regions of Europe, showingdistinct regional patterns that must be considered by EU and otherregulators. Such collaborative international studies are continuingand will soon enable to map how infant allergies resolve or persistas children grow up and whether new allergies develop in thesewell characterized populations.

http://dx.doi.org/10.1016/j.toxlet.2013.06.166

W12-2A regulator’s approach to risk assessment offood allergens

Sue Hattersley

Food Standards Agency, London, UK

In order to protect the health of food allergic consumers, regu-lators need to assess the risks posed by particular allergenic foodsin food products. In recent years, legislation has been introducedin many countries thatrequires clear labelling of certain allergenicingredients used in pre-packaged foods. However, such legislationgenerally does not include any thresholds for labelling of theseintentional ingredients, nor does it cover accidental presence ofan allergenic food as a result of cross contact at some point inthe food production process. Some legislation, such as that in theEuropean Union, does allow for certain highly processed ingredi-ents derived from the specified allergenic foods to be exempt fromallergen labelling requirements. However, there are no published

limitsfor maximum residual levels of allergenic proteins in suchingredientsupon which cases for exemption can be based.

The absence of internationally agreed allergen thresh-olds(action levels) leads toinconsistency in the approachestaken by food businesses to their decision-making on whetheror not to use allergen cross contact precautionary labelling ontheir products. It is also one possible reason for differences in theapproaches taken by different regulatory bodies when decidingon appropriate enforcement actions for foods where allergencross contact has been detected. This presentation will set out aregulatory approach to allergen risk assessment,including labellingof intentional ingredients, exemptions from statutory allergenlabelling requirements and ‘free-from’ claims, as well as assessingthe risk posed by possible allergen cross contact.

http://dx.doi.org/10.1016/j.toxlet.2013.06.167

W12-3Identification of new food allergens of publichealth relevance

Geert Houben

TNO, Zeist, The Netherlands

Identification of food allergens of public health relevance isimportant for various reasons. For existing known allergens, it mayhelp in the prioritization of allergens to be covered in labeling legis-lation: which allergenic ingredients or ingredients derived from anallergenic source should be labeled because of a potential allergenicrisk and which not? Identification of food allergens of public healthrelevance is also important in the allergenicity assessment of novelfood proteins and proteins from biotechnology/genetically mod-ified species and the subsequent safety assessment and decisionregarding market approval for foods or food ingredients containingthese proteins. Yet, today, there is no generally applicable quanti-tative hazard assessment approach for the identification of foodallergens of public health relevance.

TNO is developing a quantitative approach for the identifi-cation of new food allergens of public health relevance, basedon knowledge on existing known allergens. For such approach,a crucial step is designing a framework for scaling the (relative)allergenicity of food proteins. A scaling framework was developedin which allergenicity is represented by two independent andmeasurable dimensions: the prevalence of allergies for variousallergens and the potency for elicitation of allergic reactions inallergic individuals. This scaling approach was built in a stepwiseapproach for assessing allergenic risks of new food proteins.

A blue print of the approach under developmentwill be pre-sented and approaches considered for the further development willbe discussed.

http://dx.doi.org/10.1016/j.toxlet.2013.06.168

W12-4Experimental approaches to predict allergenicpotential of novel food

Charlotte B Madsen, Stine Kroghsbo, Katrine L Bøgh

Technical University of Denmark, Søborg, Denmark

There are many unanswered questions relating to food allergysensitization in humans. We don’t know under what circumstancessensitization takes place i.e. route (oral, dermal, respiratory), age,