Linz 2007 Abstracts of All Lectures and Posters · control (absolute ethanol) and irritants...

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Abstracts of All Lectures and PostersIn alphabetical order of the first authors – alphabetisch nach Erstautor/inn/en geordnet

In vitro models of pharmacosensitive andpharmacoresistant epileptiform activityhave been developed on the basis oforganotypic slice cultures of hippocam-pus. Slice cultures (on average 25/ani-mal) are prepared from 6 to 10-day oldrats and cultured under 5% CO2. Singleneuron and neuron population activitiesas well as extracellular K+ concentrationsare recorded with microelectrodes andion sensitive electrodes, respectively. Thedrugs tested were carbamazepine, pheny-toin, sodium valproate, phenobarbitalsodium, diazepam, clonazepam, gaba-pentin and ethosuximide. Drug concen-trations in vitro corresponded to uppertherapeutic-low toxic ranges in the brainextracellular fluid of patients treated withthe respective antiepileptic drugs. Highfrequency stimulation of Schaffer collat-erals in CA1 of hippocampus induces a

primary afterdischarge (PAD) in the hip-pocampus. The PAD consists of a tonicand clonic component, lasts between10–30 s and can be interpreted as an elec-trographic correlate of the maximal elec-troshock seizure (MES) test in animals.The pharmacosensitivity of the PAD issimilar to that of the MES-Test: the PADis inhibited by phenytoin and carba-mazepin and not by ethosuximide. 1,4-benzodiazepines and sodium valproatecause a partial suppression of the PADonly. Low magnesium or blockade of K+-channels reliably induces recurrent tonic-clonic seizure like activity in thehippocampus and the dentate gyrus. Inmore than 94% of 120 slice culturesinvestigated so far the studied antiepilep-tic drugs failed to block induction ofepileptiform activities even when toxicconcentrations were applied. The phar-

macoresistance persisted over the time invitro explored so far (2 months). We pre-sent here two simple to establish in vitromodels, one of pharmacosensitive, theother of pharmacoresistant epileptiformactivity of which the latter in comparisonto respective animal models is a prioripharmacoresistent. After further valida-tion the in vitro models might be wellsuited for inclusion in the panel of testsused for the identification of newantiepileptic compounds thus reducingthe number of animals currently used inanticonvulsive drug screening and testingand eventually replacing the respectiveanimal models.

Supported by set (Stiftung zur Er-forschung von Ersatz- und Ergänzungs-methoden zur Einschränkung von Tier-versuchen).

Lecture: free communications

In vitro alternatives to animal models for screeningand evaluation of anticonvulsive compoundsKlaus Albus, Abdul Wahab and Uwe Heinemann Institut für Neurophysiologie CCM, Charité-Universitätsmedizin Berlin (Berlin) (DE)e-mail: [email protected]

Keywords: hippocampal slice culture, seizure-like activity, antiepileptic drugs, pharmacosensitive, pharmacoresistant

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The bovine corneal opacity and permeabil-ity assay (BCOP) has been endorsed bythe ECVAM Scientific AdvisoryCommittee as an alternative method to invivo rabbit eye irritation test. Since porcinecorneas are more similar to humans thanthose of bovine and fresh porcine corneasare available locally, this study aims atusing porcine corneas for conducting theporcine corneal opacity and permeabilityassay (PCOP). As porcine eyes have dif-ferent size and shape from bovine eyes andporcine corneal holders are not availablecommercially, a set of new corneal holdersis needed to hold the porcine cornea in anatural manner during experiment. Newcorneal holders were constructed withinformation from non-contact laser scan-

ning of the anterior surface of porcineeyes. Modifications were made until theholders could hold the porcine corneawithout wrinkles and there was no leakageof culture medium from inside. The hold-ers also fit in a stand suitable for measure-ment with a UV-visible spectrophotometerin order to measure the opacity of thecorneas. A set of glass windows located atboth ends of the holders was checked toensure minimal absorbance at the wave-length used. The suitability of thesecorneal holders for eye irritation testingwas validated by studies regarding cornealmorphology and histology after mountingin the holder with and without irritants(absolute ethanol, acetone and 1% benz-alkonium chloride). The holders were uti-

lized in PCOP experiments with a few irri-tants using the National ToxicologyProgram Interagency Center for theEvaluation of Alternative ToxicologicalMethods (NICEATM) protocol for BCOP.The in vitro irritation scores of negativecontrol (normal saline solution), positivecontrol (absolute ethanol) and irritants(acetone and 1% benzalkonium chloride)were 0.5, 29.4, 39.7 and 28.9, respectively.Therefore, preliminary PCOP experimentsusing these corneal holders could distin-guish the non-irritant from the irritants.The new corneal holders could retain freshporcine corneas in natural manner duringexperiments without damaging corneal tis-sue and were suitable to use in PCOPassay.

Poster: free communications

Fabrication and validation of corneal holders forporcine corneal opacity and permeability assayPanida Asavapichayont, Suluck Ukong, Patamawan Phuagphong and Duangdeun MeksuriyenFaculty of Pharmacy, Silpakorn University (Nakornpathom) (TH)e-mail: [email protected]

Keywords: PCOP, corneal holders, eye irritation testing alternative, porcine

The next generation search engines areintelligent assistants. With the help ofbackground knowledge Transinsight’ssearch technologies speeds up the search(for answers) significantly and gives anoverview over large query results. Whenpeople search, they have questions inmind. For example if writing a report on1) chemical testing, 2) animal welfare or3) animal use alternatives, relevantinformation about the keywords in the

right context are needed in order to com-pile a comprehensive report. Today’ssearch technologies are by fare not elab-orated enough to cope with the complex-ity of such topics. Although texts holdanswers to most questions it is very dif-ficult to obtain them with classicalsearch engines, as they merely presentpossibly long lists of search results andleave it up to the user to find the answerto his/her question. To find answers

rather than to just search for them, ournext-generation search engines are intel-ligent and use background knowledgeand boost search to a next level of intel-ligence. The biomedical Web 2.0 searchengine www.GoPubMed.org is nowonline. In the talk we give examples ofhow knowledge can improve searchingsignificantly.

Keywords: animal use alternatives, search engines, GoPubMed ontology, semantic web, background knowledge, mesh, gene ontology

Lecture: computer assisted procedures

Knowledge based search – a new search machinefor knowledge intense domainsMichael R. AlversTransinsight GmbH (Dresden) (DE)e-mail: [email protected]

Keywords: plasmacytoid dendritic cells, skin sensitization and allergy, CD86 expression, REACH

Keywords: EpiVaginal, organotypic vaginal tissue model, irritation, women’s care products

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An in vitro predictive test system forassessing the allergenicity potential ofsubstances will have utility throughoutindustry to monitor products for contactallergenicity. Development of such non-animal alternative assay systems for skin sensitization hazard assessment iswithin the provisions of the EuropeanUnion chemicals policy known asREACH (Registration, Evaluation, andAuthorization of Chemicals). We investi-gated whether phenotypic and functionalchanges to subset of dendritic cells (DC),plasmacytoid DC (pDC), could be used to

identify allergens. To achieve this goal,normal human DC were generated fromCD34+ progenitor cells and cryopre-served. Frozen DC were thawed and thepDC fraction (CD123+/CD11c-) was har-vested using FACS sorting. The pDCwere cultured, expanded, and pulsed withchemical allergens (n=13) or irritants(n=7). Sub-toxic concentrations of eachchemical were determined using FACSanalysis of propidium iodide stainedcells. Results showed that exposure ofpDC (n=2-5 donors) to allergens induceda 1.5 fold expression of CD86 for 12 of

13 allergens tested. On the other hand, 7of 7 non-allergens did not result inincreased CD86 expression. Based onthese results, a preliminary predictionmodel was developed to identify chemi-cal allergens (sensitivity = 91-93% andspecificity = 93-100%). In conclusion,CD86 expression in pDC appears to be asensitive and specific predictor of aller-genicity of chemicals. When comparedwith existing animal models, the assay isadvantageous because high throughputscreening of chemicals using cells ofhuman origin is possible at low cost.

Poster: 7th cosmetics amendment

Allergenicity testing using plasmacytoid dendritic cells Seyoum Ayehunie, Maurine Snell, Helena Kandarova and Mitchell Klausner MatTek Corporation (Ashland, Massachusetts) (USA)e-mail: [email protected]

The utility of an organotypic vaginal-ectocervical (VEC) tissue model, com-posed on normal human cells, to test theirritation potential of vaginally-appliedchemicals and their formulations wasexamined. Histologically, the VEC tis-sues have nucleated basal and parabasalcell layer and glycogenated intermediateand superficial layers. To insure tissuereproducibility, standardized quality con-trol (QC) tests utilized the MTT assay todetermine the exposure time necessary todecrease the tissue viability to 50%(ET50). ET50 results for the positive con-trol (1% Triton X-100) showed the tis-sues to be highly reproducible; the

average intra-lot coefficient of variation(CV) was less than 10% and ET50s aver-aged 1.40 h ± 0.26 (n=55 lots). End-points including MTT ET50, histology,RT-PCR, and cytokine release patternswere used to evaluate 20 commerciallyavailable test articles. Upon exposure totest articles, the tissue model was able to discriminate between the mildness oftest articles. The ET50 values rangedbetween 3.5-7.0 h for contraceptives,between 6.9- >18 h for anti-itch creams,and between 1.7-2.7 h for femininewashes. Released cytokines and geneexpression levels showed that IL-1α, IL-1β, IL-6, and IL-8 were associated with

toxicity of test materials. In conclusion,the VEC tissue model will serve as a use-ful, highly reproducible, non-animal testmethod to assess the irritation potentialof vaginally applied chemicals and theirformulations. Models such as the epi-vaginal tissue which mimic the vaginalmicroenvironment could provide essen-tial data set for early identification of thebiological/toxicological property ofchemicals/products intended for vaginaluse. Development of such in vitro testmodel for risk-assessment and manage-ment of chemicals is in line with the newEuropean Union chemicals policy knownas REACH.


Irritation screening of vaginal-care products using EpiVaginal™, a human vaginal-ectocervical tissue modelSeyoum Ayehunie, Chris Cannon, Jeff Gimondo, Mitchell Klausner and Helena KandarovaMatTek Corporation (Ashland, Massachusetts) (USA)e-mail: [email protected]

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To guarantee non-hazardous application,the detoxification of vaccine againstWhooping Cough (Pertussis) has to bemonitored for residual Pertussis Toxin (PT)activity under standardized conditions. Thetraditional procedure to determine toxicityis done by animal testing, with the involve-ment of high resource (at least 15 mice pertest) and personnel costs. Due to that analternative testing method has to be devel-oped. The literature describes the activationof monocytes by PT. This effect wasattempted to utilise by applying theMonocyte Activation Test (MAT). For thisreason, human whole blood was incubatedwith PT and monocyte activation is mea-

sured by ELISA. PT is produced by theGram-negative bacterium Bordetella per-tussis and, therefore, it is naturally contam-inated with Endotoxin (Lipopoly-saccharide, LPS). The latter represents themost potent monocyte stimulator. We coulddetect LPS in each PT preparation testedwithout having any chance to remove it dueto the high affinity of LPS to PT.Furthermore, blocking of CD14 as the LPSreceptor on monocyte’s surface by mono-clonal antibodies abolished monocyte acti-vation by PT. In consequence, MAT is notsuited for the estimation of PT activity.Therefore, a new principle of detection hasto be found. To achieve this, the fluores-

cence characteristics of etheno-NAD(1,N6-Ethenonicotinamide adenine dinu-cleotide) are thought to be exploited.Etheno-NAD represents the fluorescenceanalogon of the natural substrate NAD(Nicotinamide adenine dinucleotide) of PT.In our new test, PT hydrolyses etheno-NADinto Nicotinamide and etheno-ADP-Ribose(1,N6-Ethenoadenosine 5’-diphosphori-bose). The cleavage of etheno-NAD leadsto an increase of fluorescence. An alterna-tive approach to assess PT activity is tomeasure these increasing fluorescence sig-nals. The data obtained with this new alter-native test principle for the estimation ofactive PT are discussed in the poster.


Alternative in vitro method for detection of pertussistoxin in vaccines to replace the mandatory animal testChristina Bache, Ingo Spreitzer, Bjoern Becker, Bettina Loeschner, Michael Schwanig and Thomas MontagPaul-Ehrlich-Institut (Federal Agency for Sera and Vaccines) (Langen) (DE)e-mail: [email protected]

Keywords: drug safety, alternative methods, pertussis toxin, monocyte activation test, fluorescence assay

Angiogenesis, the sprouting of new ves-sels from pre-existing ones and its inhi-bition, so-called anti-angiogenesis, arein the focus of modern medicine. In par-ticular, a promising strategy of cancertreatment combines chemo-, radio- andanti-angiogenic therapy. Identificationand evaluation of angiogenic and anti-angiogenic factors are still carried out inanimal models like the cornea model ordorsal skinfold chamber where sub-stances are tested in rabbits, mice andhamsters. The aim of our studies is thereplacement of these animal models byin vitro models of angiogenesis and anti-

angiogenesis. We have established invitro models of angiogenesis based oncultivated microvascular endothelialcells of different species and tissues. Inthe bovine model a method for quantita-tion of angiogenesis and anti-angiogene-sis was developed which should beadapted to different human endothelialcell cultures (from heart, lung and twoforeskins). This method is based on thestaging of the angiogenic cascade instrictly defined stages and thus allowsquantitation of all phases of angiogene-sis up to the development of capillary-like structures. Stimulated by a selective

medium the bovine cultures run throughall defined stages of angiogenesis within60 days. A similar run of angiogenesiscould be observed in one of the foreskinand the heart derived human cultures. Inthe other foreskin derived cells angio-genesis remained at a certain stage after30 days of culturing. In endothelial cellsof the lung angiogenesis began runningbackwards from day 30 onward. Im-munolocalization of endothelial expres-sion of collagen type IV, an essentialcomponent of the basal lamina, showeda slow but continuous expression in thebovine cultures over the entire cultiva-

Lecture: good cell culture practice

Establishment of realistic in vitro models ofangiogenesis: How to make the right choiceMahtab Bahramsoltani and Johanna PlendlInstitute of Veterinary Anatomy, Freie Universität Berlin (Berlin) (DE)e-mail: [email protected]

tion period. These observations wereconfirmed in the “angiogenic” heart andforeskin derived cultures which also ranthrough all stages of angiogenesis. Inopposite, in the “non angiogenic” cul-tures of lung and foreskin collagen typeIV was expressed very moderately

resulting in only a fragmentary extracel-lular collagenous network. Our resultsshow that only particular microvascularendothelial cells can be stimulated to runthrough all stages of angiogenesis invitro. Therefore only specific culturesmay be used for the establishment of

realistic in vitro models of angiogenesis.One potential reason for this may be theability of the cells to express collagentype IV. Further investigations are on theway to identify the expression profile ofendothelial cells to be chosen for in vitromodels of angiogenesis.

Matthew “Hiasl” Pan is a chimp, whowas abducted from West Africa in 1982to Vienna in Austria to be used forexperiments on AIDS and Hepatitis.Since his abduction was illegal, he wastaken in by customs and handed over toan animal shelter. A caretaker took himhome and raised him like a child in hisfamily. The company Immuno, who hadinstigated Matthew’s abduction, wonthe legal procedures to get him back.However, activists prevented the chimpbeing handed over. Later, chimp experi-ments were ended and Matthew was for-mally sold to the animal shelter.However, in 2007 the shelter wentbankrupt. That meant Matthew could besold and deported. As someone, whowas abducted as a small child, withmost likely his mother being killed inthe process in front of his eyes, who wasbrought to an alien environment andwho spent most of his life locked up,

and hence cannot look after himself andsafeguard his interests, and as someone,who is in imminent danger of beingdeported, he is the paradigmatic exam-ple of a person needing a legal guardian.If, indeed, he is a person. Hence, inFebruary 2007, I did an application tothe district court in Mödling (Austria)for a legal guardian for Matthew Pan.We argued that he is a person accordingto Austrian law. Firstly, that is becausethe law says that every human is a per-son. The only possible definition of ahuman within the law is the biologicaldefinition of genus. Scientists arguebiologically that chimps belong to thegenus homo and hence Matthew Pan is ahuman and a person according to thelaw. Secondly, the law clearly differen-tiates between human and person. Allhumans are persons but not all personsare humans. The definition of person-hood must be sought in the philosophy

of the enlightening era and is best scien-tifically described by what is referred toas “theory of mind”, the ability torecognise intentional states – and hencepersonhood – in others. Persons are allthose beings, who recognise person-hood in each other. Scientists say thatchimps have a “theory of mind”,Matthew has himself passed the mirrorself-recognition MSR test. Hence he is aperson and deserves a legal guardian. Inpractice, that would mean he is notsomeone else’s property but belongs tohimself. He can receive his own dona-tions and safeguard his own future bylegally fighting deportation procedures.Only as a person represented through alegal guardian will his interests berecognised and represented in a court oflaw. Only then is justice possible.Eventually, that means he could suethose responsible for his abduction andhis misery for damages.

Lecture: ethical and legal aspects in animal experimentation

Trial for personhood of a chimp Martin Balluch Verein Gegen Tierfabriken (Vienna) (AT)e-mail: [email protected]

Keywords: personhood, animal rights, chimp, legal guardian

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Keywords: in vitro model, angiogenesis, endothelial cells, collagen type IV

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The aim of the present publication is tocollect possible alternative methods in thefield of radiopharmaceutical research anddevelopment that could serve the idea ofthe 3Rs, namely replacing the animal testsor, if this is not possible, reducing thenumber of them and refining the tech-niques. In vitro cell-radiopharmaceuticalbinding assays (receptor-binding assay,immune-binding assay and non-specificbinding), the superfine, specific and sensi-tive nanoSPECT/CT (Bioscan/MedisoLtd) imaging method and the possible useof spontaneously occurring veterinarypatients will be considered as availablealternative methods in the field. In vitrocell binding assays proved to be replacing

the in vivo rodent xenograft models in thepreselection of possible radiopharmaceuti-cal candidates. The nanoSPECT/CThybrid (fusion) imaging method allowshigh-resolution pictures including correctanatomical structures and quantified func-tional content at different times after radio-pharmaceutical application using a single,anaesthetized animal that need not be sac-rificed at the end of the process. Re-use oflaboratory animals allowed us to comparethe characteristics of different agents on the very same biological model.Spontaneously occurring canine, felineand egzotic animal diseases are a constantsource of animal models for radiopharma-cists as well. Osteosarcoma, mammary

gland carcinoma, thyroid carcinoma, braintumours and a lot of others in dogs andcats might be the best known animal mod-els of the appropriate human diseases.Diagnosing and then treating them withthe most promising human methods pro-vides a chance for the suffering animalsand produces useful data for human oncol-ogists and biomedical researchers. Thiswork was conducted with the financialsupport of Mediso and Bioscan Ltd, Medi-Radiopharma Ltd, and under the umbrellaof different international scientific pro-grammes (EMIL-NoE 503569-2.2, IAEACRP No E1.30.33 and Indo-HungarianIntergovernmental Programme No2003/07).


3Rs in the field of radiopharmaceutical research and developmentLajos Balogh0, Domokos Máthé1, Gábor Andócs1, Réka Király1, András Polyák1, Julinanna Thuróczy1, Pradip Chaudhari1

0 NRIRRGyözö Jánoki National “F.J.C.” Research Institute for Radiobiology and Radiohygiene (Budapest) (HU); 1 Gyözö Jánoki National “F.J.C.” Research Institute for Radiobiology and Radiohygiene (Budapest) (HU)e-mail: [email protected]

Keywords: radiopharmaceuticals, nanoSPECT/CT, spontaneous diseases

A novel organization would like to intro-duce itself for the international partnersworking in the same field. The HungarianConsensus Platform on Alternatives(hucopa) was grounded by 12 foundingmembers in Budapest and has been start-ing to work. Hucopa is a full-right mem-ber of the European mother organization(European Consensus Platform onAlternatives – ecopa) and similarly con-sists of the 4 relevant platforms as: theAcademy (research and education), theIndustry (pharmaceutical, chemical andother companies), the Animal Welfare

(animal welfare and right organizations)and the Authority (government people).The main goal of hucopa is to increase theknowledge about alternative methods inthe public and specialists by the theory of3Rs recommendations. Performing theabove listed activities hucopa organizes 2meetings in a year (one for the 4 platformspecialists and another one for the public),platform-leaders and other key-personalsparticipate as many as possible othernational and international meetings,where they provide information alsoabout the activities of hucopa, take a close

co-operation with the European motherorganization (ecopa) and constantly com-municate with the prominents of the 4platforms and the medias. Hucopa wantsto open and work-together also with othernational 3Rs organizations. This workwas conducted by the financial support ofMediso and Bioscan Ltd, Medi-Radiopharma Ltd, and under the umbrellaof different international scientific pro-grammes (EMIL-NoE 503569-2.2, IAEACRP No E1.30.33 and Indo-HungarianIntergovernmental Programme No2003/07) (www.hucopa.com).


Hungarian consensus platform on alternatives – here we go!Lajos Balogh, Éva Hercsuth, Tibor Bartha, Zsuzsa Somfai and László Pallós Hungarian Consensus Platform on Alternatives (Budapest) (HU)e-mail: [email protected]

Keywords: new organization for alternative methods

Animal experiments are often used todetermine allergenic or irritating poten-tial of substances. To reduce, refine andreplace in vivo systems, new methodssuch as characterisations of surface anti-gen expression are currently under de-velopment. In the present study, weevaluated the maturation of bovine skin-derived dendritic cells (DC) to given hap-ten/irritants by means of surface antigenexpression. In contrast to in vitro analysisof DC derived by other methods, ourapproach takes advantage of a physiolog-ical skin barrier, which has to be pene-trated by a given substance before areaction occurs, thus more likely mimick-ing the in vivo situation. To analyse the

With the advent of nanotechnology, theprospects of manufactured nanomaterialsin many applications have progressedrapidly. The potential health effects ofthese nanoparticles (diameter less than0.1 µm) associated with human exposureare unknown. In order to avoid and toreplace toxicity studies with animalswhich are time consuming and stressfulto the animals we have established a

triple cell co-culture system composed ofepithelial cells, macrophages and den-dritic cells which simulates the mostimportant barrier functions of the epithe-lial airway: the surface active phospho-lipids, a tight epithelium, and cells of thedefence system. The exposition of cellcultures at the air-liquid interface withpolystyrene nanoparticles (0.05 µm) wasdone using a hand-held microsprayer.

Quantification of particles and intracellu-lar localisation of the particles was stud-ied by laser scanning and transmissionelectron microscopy. The visualizationand the quantification of the polystyreneparticles by laser scanning microscopyrevealed that the particles entered all celltypes, however, to a different extent.Using transmission electron microscopywe could show that the particles were

expression of different surface markers,dermatomized bovine udder skin (thick-ness 100 µm) was topically treated withknown haptens and irritants. Skin sam-ples were cultured in medium, incubatedfor 48 hours, emigrated DC were har-vested and analysed by flow-cytometryfor expression of CD14, CD80, CD86und MHC II. TDI and DNCB in concen-trations of 0.05% and 0.5% were used asstrong sensitizing haptens, whereas SDSin concentrations of 0.1%, 0.5% and1.0% was used as a strong irritant. Theseconcentrations were chosen based on ourunpublished observation that higher con-centrations induced cell-death. Exposureof DC to TDI and DNCB in tested con-

centrations led to an up-regulation of theactivation markers CD80, CD86 andMHC II, while CD14 (a monocytemarker) was dose dependently down-regulated. In contrast, exposure of DC toSDS up-regulated CD80/86 expressionbut down-regulated MHC II expression.Our results indicate the potential use of flow cytometric analysis to discrimi-nate between cellular reactions to aller-gens/haptens and irritants. As the bovineudder has no commercial value and isaccessible in large amounts, the proposedsystem may provide a useful approach totest such substances.

This study is supported by ZEBET(WK 3-1328-175).


Use of skin derived bovine dendritic cells todiscriminate between potential haptens and irritantsHelen Becker0, Dirk Werling1, Niall MacHugh2, Manfred Kietzmann3 and Wolfgang Bäumer 3

0 University of Veterinary Medicine Hannover (Hannover) (DE); 1 Department of Pathology & Infectious Diseases, RoyalVeterinary College (Hatfiled) (GB); 2 The Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies(Edinburgh) (GB); 3 Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover,Foundation (Hannover) (DE)e-mail: [email protected]

Keywords: dendritic cells, cell culture, contact sensitivty, activation makers

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Lecture: nanotoxicology

A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticlesChristina Brandenberger, Fabian Blank, Christian Mühlfeld, Peter Gehr and Barbara Rothen-RutishauserInstitute of Anatomy, Division of Histology, University of Bern (Bern) (CH)e-mail: [email protected]

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Three dimensional cell systems havebeen already established for the corneaand the skin. These models are – due totheir organ-specific properties – suitablefor the analysis of different substances

with regard to their biocompatibility andtoxicology which up to now could beexamined only by animal experiments.The airway represents also an importantentry gate for many different pathogenic

agents and environmental impacts as forexample nanoparticles. A three dimen-sional model of the trachea becomes alsomore and more important as an alterna-tive for animal experiments because it

within the cells either in membrane-bound agglomerates or as single particlesfree in the cytoplasm, hence having direct access to cytoplasmic proteins.Penetration of nanoparticles into mito-chondria and the nucleus could be shown.Our in vitro triple cell co-culture modelof the epithelial airway barrier offers a

great tool to study particle-lung cell inter-actions at the nanostructural level. Usingthe model and advanced microscopictechniques we have shown that nanopar-ticles (i.e. polystyrene particles) enteredthe epithelial cells as well as the cells ofthe defence system. Nanoparticles werefound intracellularly membrane-bound or

free in the cytoplasm as well as inorganelles, like in mitochondria and inthe nucleus. This points to their enormoustoxic and carcinogenic potential by inter-fering with the respiratory chain in themitochondria and with the DNA in thenucleus and perhaps also in the mito-chondria.

Poster: nanotoxicology

In vitro three dimensional trachea modelMartina Brenner, Jan Hansmann and Heike Mertsching Fraunhofer Institute for Interfacial Engineering and Biotechnology Fh-IGB (Stuttgart) (DE)e-mail: [email protected]

Keywords: epithelial airway model, nanoparticles, toxicity, laser scanning microscopy, transmission electron microscopy

Magnesium alloys are a promising newmaterial for the production of biodegrad-able implants in biomedical technology.While their technical properties are wellcharacterised, there is only limited infor-mation available concerning their degra-dation behaviour in biological matricesand their effect on human or animalorganisms. In the present studies a varietyof in vitro examinations were conductedto establish a test facility that enables thepre-selection of suitable candidates forfurther alloy development.

In cell culture investigations alloydegradation was examined by scanningelectron microscopy (SEM) and induc-

tively coupled plasma (ICP) measure-ments. The influence of the alloy elementson the viability and proliferation of murinekeratinocytes (MSC-P5) and murinefibroblasts (L929) was examined by sup-plementing the culture medium with saltsand by co-cultivation of the cells withalloy samples using membrane inserts.Using the in vitro model of the isolatedperfused bovine udder, acute effects ofalloys on skin, connective tissue and teatmucosa were investigated after subcuta-neous and intracisternal (teat) incubationof samples. Degradation and tissue distri-bution of the alloy elements were investi-gated by microdialysis and microscopy. As

parameters for tissue irritation the viabilityand the release of pro-inflammatory medi-ators (prostaglandin E2, tumour necrosisfactor alpha) was determined.

As a great variety of chemical elementsmay be alloyed with magnesium, a highnumber of different materials have to beevaluated concerning their biocompatibil-ity. By using the presented in vitro mod-els to pre-select promising candidates forin vivo trials, the use of laboratory ani-mals can be significantly reduced.

These studies are supported by theDeutsche Forschungsgemeinschaft (DFG)as part of the collaborative research cen-tre (SFB) 599: “Biomedical Technology”.


In vitro testing of the biocompatibility of degradablemagnesium alloys for use in biomedical technologyMichael Braun0, Christian Krause1, Burkhard Schwab2, Thomas Lenarz2, Friedrich-Wilhelm Bach2 and Manfred Kietzmann0

0 Institute of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Foundation (Hannover) (DE);1 Institute of Materials Science, Leibniz University of Hannover (Hannover) (DE); 2 Department of Otolaryngology, MedicalUniversity of Hannover (Hannover) (DE)e-mail: [email protected]

Keywords: biocompatibility, implants

shows similarities in structure and func-tion of the human trachea. This modelcould be used as a test system for theanalysis of different substances and itcould be also used as a biological graft.For the construction of a three-dimen-sional trachea model, we isolate porcinecells with an enzymatic treatment andseed them in preliminary tests on 24-wellinserts with a pore size of 1 µm. For fur-ther tests, the colonisation also occurs inan air liquid interface in a specialbiomembrane reactor with a pulsatile

flow of the optimized cell culture mediumand regulate breathing rate. The colonisa-tion occurs with Airway Epithelial CellGrowth Medium and by way of compari-son in DMEM Medium with differentsupplements. The cell characterizationoccurs via histological and immuno-his-tological methods and measurement ofthe ciliary beat frequency. In preliminarytests porcine respiratory cells were culti-vated on inserts and they show a longtermbut no metachrone ciliary activity. Thecells were also cultivated on an acellular-

ized scaffold in the bioreactor. Most ofthese cells are vital and show a high rateof proliferation and also longterm ciliaryactivity. There are ongoing experimentswith a co-culture of fibroblast cells andciliated epithelium cells and tests to initi-ate the metachrone ciliary beat in thebioreactor system. Our aim is to developa functional tubular trachea test system ina bioreactor in which the different cells ofthe trachea are cultivated as a co-cultureunder physiological conditions and simu-lation of the respiration.

Keywords: trachea, three-dimensional test system, primary cells, nanotechnology

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The measurement of cellular reactionsunder controlled in vitro conditions incell monitoring systems (CMS®) hasbeen established in our group in 1992.CMS® permit for non-invasive measure-ments of different parameters such asacidification, respiration and adhesionby the use of micro-sensors. Multi-elec-trode arrays (MEAs) can be applied todetect the electric activity in neuronalnetworks. Both features are combinedon a silicon neuro-chip allowing for par-allel measurements of metabolic andelectrical network parameters. The chipallows for multi-parametric monitoringof neurotoxic effects in cellular systemsfor hours and up to days. These proper-ties are key features in drug develop-ment, high-content screening, and

quantitative safety classifications in neu-rotoxicity as well as developmental-neu-rotoxicity studies. This may also beimportant for the industry when approx-imately 30,000 existing chemicals willbe evaluated for their toxicological prop-erties under the REACH program of theEuropean Commission. We work on thereduction of primary cell use by estab-lishing differentiated stem cell lines onour chips. Appropriate culture protocolsfor the promising D3-cell line (ATCC),guaranteeing the viability and the differ-entiation of the cells into neuronal net-works are currently developed. Newprotocols aim at the development ofspontaneous electrical activity in thenetwork. The metabolic activity of thedifferentiating cells is detected by the

Bionas® 2500 analyzing system with sixparallel SC1000-chip modules that weresolely designed for metabolic measure-ments. This system allows for the evalu-ation of three additional potentialend-points (cell adhesion, acidificationand oxygen consumption) in addition tothe electrical network activity. The dose-dependent responses for different testsubstances were analyzed with specialattention to effects at the different devel-opmental stages of the neuronal cell sys-tems. In addition, the prototype of a newglass neuro-chip has been developed,that is compatible to our silicon-chipsystem (see poster of Koester et al.). Theglass neuro-chip ensures microscopicobservability in future experiments.

Poster: EU-chemicals policy (REACH)

Bio-analytic silicon chips for the detection ofdevelopmental-neurotoxic effects of chemicals and drugs in the context of theEuropean REACH programSebastian Moritz Buehler, Philipp Julian Koester, Carsten Tautorat, Helene Altrichter, WernerBaumann and Jan GimsaUniversity of Rostock, Institute of Biology, Chair of Biophysics (Rostock) (DE)e-mail: [email protected]

Keywords: lab-on-chip, biochip, REACH, multi-electrode array, neuro-sensorchip, glass chip, neuronal networks, developmental neurotoxicology, DNT, neurotoxicity, action potential detection

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The determination of drug absorptionafter aerosol deposition is an importantstep in the development of new aerosolmedicines. Because of the complexity ofthe inhalation process mainly animalexperiments are used to predict the invivo drug absorption rate. Even if differ-ent cell lines like Calu-3 and A549 arewidely used, an in vivo realistic deposi-tion of medical aerosol particles on cellmodels is not yet realized. In our studywe investigated the influence of deposi-tion conditions like deposition device,fluid volume, particle size, and particlewettability on the absorption rate of phar-maceutical aerosols across cell models.We applied drug solutions/suspension viapipetting, or dry aerosol particles via aninsufflator syringe or with the aid of amulti stage cascade impinger on dry cell

monolayers and measured subsequentlythe drug absorption rate. Commercialavailable salbutamol and budesonideaerosols with different solubility and par-ticle sizes were used. In summary, airinterface deposition on dry epithelial sur-face resulted in increased absorption ratescompared with liquid interface applica-tion. The fluid volume in the apical com-partment controls at first the drug releaserate from particles. Thereafter, the fluidvolume influences the concentration gra-dient, which is according to Fick’s firstlaw the motor of passive transport pro-cesses. The limitations of the air interfacedeposition with the relatively simpleinsufflator syringe, however, becomeapparent in cases of drug formulationswhere aerodynamic properties of theaerosolized powder particles get critical.

To address also aerodynamic propertiesmore sophisticated setups like some mod-ified multi stage liquid impinger can beused. Air interface deposition on pul-monary cell culture models offers a wayto simulate the most important peculiarityof aerosol drug delivery: absorption of arelatively high metered dose after deposi-tion on a slightly wetted epithelial sur-face. Therefore, this approach is anattractive alternative to inhalation experi-ments on laboratory animals and mayprovide important additional informationabout pulmonary permeability of activepharmacological ingredients, as well asabout safety and efficacy of new excipi-ents (e.g. polymers, surfactants etc.) andformulations (micro- and nanoparticles,liposomes etc.).


Pulmonary cell culture models to study safety and efficacy of innovative aerosol medicines Michael Bur, Andreas Henning, Marc Schneider and Claus-Michael LehrSaarland University (Saarbruecken) (DE)e-mail: [email protected]

Keywords: alveolar epithelium, drug absorption, mucus, in vitro

The assessment of teratogenic effects ofchemicals is generally performed usingin vivo assays in rats and rabbits.Following the 3R concept and publicexpectations, the use of alternativemethods is promoted to reduce the num-ber of animal tests. From this perspec-tive, we have developed an in vitro testwith the zebrafish Danio rerio embryotest (DarT) combined with an exogenous

mammalian metabolic activation system(MAS), able to biotransform proterato-genic compounds. Cyclophosphamideand Benzo[a]pyrene were used as pro-teratogens to test the efficiency of thisassay. Briefly, the zebrafish embryoswere co-cultured at 2 hpf (hours postfecundation) with varying concentra-tions of the test materials, induced malerat liver microsomes and NADPH for 60

min at 32°C under moderate agitation inTris-buffer. The negative control (testmaterial alone) and the vehicle control(MAS alone) were incubated in parallel.For each parameter, 20 eggs were usedfor statistical robustness. Afterwards fishembryos were transferred individuallyinto 24-well plates filled with fishmedium for the next 48 hours at 26°Cwith a 12 hour-light cycle. Terato-

Poster: ecotoxicology

Development of a screening assay for teratogenicity: combination of DarT with a mammalian metabolic activation systemFrançois Busquet 0, Roland Nagel1, Friedrich von Landenberg0, Stefan O. Mueller 0 and Thomas H. Broschard 0

0 Institute of Toxicology, Merck KgaA (Darmstadt) (DE); 1 Institute of Hydrobiology, TU Dresden (Dresden) (DE)e-mail: [email protected]

genicity was scored using morphologi-cal endpoints. No teratogenic effectswere observed in fish embryos exposedto the proteratogens alone, i.e. withoutmetabolic activation. In contrast, Cy-

The aim of the present study was theestablishment of an in vitro test system toreveal the potential risk to human healthof nanoparticles at the workplace. The

essential advantage of in vitro investiga-tions is to be non-invasive, the employeesdon’t have to be bothered and the workroutine doesn’t have to be intercepted. At

occupational settings test cells onTranswell® inserts were exposed to theworkplace atmosphere or to particle fil-tered air for 1 to 3 hrs using a CULTEX®

clophosphamide and Benzo[a]pyreneinduced teratogenic effects in fishembryos in the presence of themetabolic activation system. The sever-ity of malformations increased with the

proteratogen concentration. We con-clude that the application of the MASwill improve and refine DarT as a pre-dictive and valuable alternative methodto screen teratogenic compounds.


Toxicity of TBTC and Arsenic on adult and fetal blood progenitors Cristina Croera, Mary Carfi and Laura Gribaldo ECVAM, Institute for Health and Consumer Protection, Joint Research Center (Ispra) (IT)e-mail: [email protected]


Toxicological investigation of nanoparticles – effectson human cells Letizia Farmer0, Alexander Graff 1, Sandra Szameit 2, Eva Valic3 and Helga Tuschl0

0 Toxicology, Austrian Research Centers GmbH – ARC (Seibersdorf) (AT); 1 Österreichische Staub- undSilikosebekämpfungsstelle (Leoben) (AT); 2 Molecular Diagnostics, Austrian Research Centers GmbH – ARC (Seibersdorf) (AT);3 Austrian Worker’s Compensation Board (AUVA) (Vienna) (AT)e-mail: [email protected]

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Many environmental pollutants target thehematopoietic system, aging directly onadult tissue (bone marrow) or, indirectly,on the fetal progenitors (umbilical cordblood) by crossing the placental barrier.Tributyltin Chloride (TBTC) and Arsenic(Sodium Arsenite) are widely present inthe environment as agricultural pesticidesor contaminants, and in fewer amounts, inplastic, glass or ceramic industry. Both thecompounds are toxic in vivo on differenthematopoietic compartments, indeedTBTC causes thymus atrophy in rodents,depletion of lymphocytes in spleen andlymph nodes and alteration of serumimmunoglobulin levels, while Arsenicinduces immunotoxicity, in addition toskin and lung cancer and cardiovascularlesions. The aim of this study was to eval-

uate the in vitro toxicity of these com-pounds on human blood progenitors, com-paring their effect on the capability toclone (GM-CFU) of bone marrow (BMC)and cord blood mononuclear cells (CBC).Our results indicated that both the com-pounds have a relevant toxic effect both onadult and fetal tissue, being TBTC muchmore toxic than Arsenic. BM resultedmore sensitive than CBC, being IC50 ofTBTC in this tissue (0.03 µM) two timeslower than in CBC (0.07 µM), and IC50 ofArsenic (0.34 µM) even 10 times lowerthan in CBC (1.22 µM). Moreover, at verylow concentrations (0.006 µM), TBCTinduced a strong significant increase ofclonogenicity of BM progenitors, differ-ently than in CBC. Our data indicate thathuman hematopoietic progenitors are

strongly affected by the toxic effect ofArsenic and TBTC. Bone marrow GM-CFU resulted more sensitive than cordblood, in particular after Arsenic treat-ment. Most colony forming cells are foundin CD34+ fraction, which is higher in BMthan CB. Although CB contains a lowernumber of CD34+, they have a highercapacity to form colonies. This couldexplain CB less sensitivity to the toxiceffect of these chemicals. Further investi-gations will be needed to check thishypothesis or to identify different mecha-nisms of action exerted on the two cellpopulations. Taking into account that thesexenobiotics pass through the placental bar-rier, the effects of their exposure are ofgreat concern for health on both adults andnext generations.

Keywords: TBTC, arsenic, blood progenitors

Keywords: zebrafish, embryo, MAS, cyclophosphamide, benzo[a]pyrene

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System. Two types of co-cultures weretested: In the first type differentiatedmacrophages were exposed and post-incubated with human lung epithelialcells. In the second type differentiatedmacrophages were seeded on human lungepithelial cells and the co-culture wasexposed. As endpoints for particle expo-sure cell viability (WST-1 assay), oxida-

tive stress (DHR-Assay) and pro-inflam-matory cytokines (BDTM CBA-Assay)were evaluated. Cell viability testingshowed a negative effect at high expo-sure. In cells exposed to the workplaceatmosphere an increased oxidative burstwas detected compared to cells exposedto particle filtered air. Exposure of co-cul-tures resulted in significantly enhanced

TNF-α, IL-6, IL-1β and IL-8 levels. Wecould show that our in vitro exposure sys-tem is very well adapted for the assess-ment of adverse effects of nanoparticlesat the workplace. Our results indicate thatnanoparticles involve an occupationalrisk and further experiments will be per-formed to analyse additional endpoints.


In tube evaluation of the effect of cobalt, nickel andalbendazol upon rumen fermentation Sandor G. Fekete0, Tamas Veresegyhazy 1, Hedwig Febel 2 and Emese Andrasofszky 1

0 SzIU Faculty of Veterinary Science Budapest (Budapest) (HU); 1 SzIU Faculty of Veterinary Science Budapest1 (Budapest) (HU); 2 Research Institute of Animal Breeding and Nutrition, Herceghalom (Budapest) (HU)e-mail: [email protected]

Keywords: in vitro test, co-culture, nanoparticles, workplace

In vivo investigations have been carriedout in order to determine the effect ofalbendazol, cobalt and molybdenum on

microbial fermentation in the rumen. Fouradult sheep were used in the tests. Therumen cannulated wethers had an average

weight of 75 kg. Their basal diet consistedof 2 kg meadow hay and 0.3 kg corn meal.In the in vitro trial (Phase 1) one experi-

Poster: good cell culture practice

Possibility to assess the harmfulness of minerals using cell culture Sandor G. Fekete and Peter GalfiSzIU Faculty of Veterinary Science Budapest (Budapest) (HU)e-mail: [email protected]

Keywords: cell lines, cadmium, nickel, cobalt, molybdenum, IC50, reference compound

The usefulness of cell cultures in traceelements research will be illustrated bythe presented experiences. The cell typesused in the present study were supplied byICN Flow (UK): NBL-1 (MDBK)-bovinekidney; HEp-2-human carcinoma of lar-ynx; J-111-human monocytic leukaemia;Chang liver, human liver; HeLaS3-cloneof the human HeLa; MMT 060562-mouse mammary tumour; MG-63-humanosteogenic sarcoma and A-549-humanlung carcinoma. The culture medium wassupplemented with the salts dissolved inPBS. After 72-hour incubation the propor-tion of live cells was determined. Theprinciple of the measurement is the bio-logical oxidation of cells: the assay con-tains tetrasolium salt (MTS) and electron

uncoupling compound (PMS). The MTSis reduced to formazan by the live cells.The coloured formazan produced dis-solves in the culture medium and can bemeasured directly. The quantity of arisingformazan is proportional to the living cellcount. The 50% cell multiplicationinhibitory concentration (IC50) can be cal-culated by regression analysis. Low cad-mium sulphate concentrations abruptlyinhibit cell multiplication without anytransition. The IC50-values of the differentcell lines are in the same range (0.025 to0.082 mmol/l). Based on these data, cad-mium can be treated as a general cell tox-icant. The IC50-value of ammoniummolibdate was between 0.21 and 1.03, fornickel ammonium sulphate between 0.09

and 0.42 and for cobalt sulphate between0.17 and 0.53 mmol/l. Thus the inhibitoryconcentrations of these three nutritivecompounds are in the same range. Basedon this series of trials, and comparing theIC50-values of Cd and the Ni, Mo, and Cosalts, one can state that the toxic or nutri-tive effect of a given trace element can beevaluated only in comparison with theIC50 of a biologically known chemicalcompound. Since the inhibitory concen-trations of cadmium sulphate are ten- tofifty-fold (in the range of 0.025 to 0.082mmol/l) lower than those of the essentialtrace elements (in the range of 0.09 to1.03 mmol/l), cadmium sulphate can beused as a reference compound in this kindof testing.

mental group was formed by 4 tubes foreach treatment. One of them served as thepositive control, containing only untreatedruminal fluid from the sheep (but receiv-ing added starch and fibre as energysource), the others were supplementedwith albendazol, cobalt + molybdenum oralbendazol + cobalt + molybdenum con-taining salt solutions. The concentrationsused were as follows: albendazol 10,cobalt (as CoCl2) 15 and molybdenum (asammonium molybdenate) 18 mg/litre. Invivo trial (Phase 1): During 14 days afterthe first sampling (to obtain ruminal fluidfor the in vitro study), the animals

received salt solution containing albenda-zol, cobalt + molybdenum and albendazol+ cobalt + molybdenum, daily into theirrumen through the cannula. The quantitiesof salts effected the same concentrationsas in case of the in vitro trial. On day 14,“treated” (adapted) rumen fluid was col-lected from each animal, and the in vitrotrial (Phase 2) was carried out in the sameway as described previously. When alben-dazol was given to the untreated rumenfluid, the degradation of organic matterincreased by 25 to 30% and that of proteinby 40 to 100%, in case of both the alfalfaand corn substrate. In contrast, when salts

were given to an adapted rumen fluid invitro, the degradation of alfalfa proteindecreased. Simultaneous application ofthe albendazol and the two salts tountreated rumen fluid increased the degra-dation of corn protein. In contrast, thesame substances, given to an adaptedrumen fluid in vitro diminished organicmatter degradation both in the alfalfa andthe corn substrate. Data suggest that theanthelminthic albendazol may enhanceprotein degradation, while the cobalt +molybdenum or their combinations withthe albendazol tend to decrease it.

Keywords: in vitro, rumen fluid, cobalt, nickel, molybdenum, fermentation, degradation, protein, organic matter

Sera are obtained from whole blood fromadult, calf or foetal animals. After thenatural clotting process, which may takeseveral hours at 4°C, the blood consists ofserum and a blood clot containing of 95%red blood cells, 5% platelets and less than1% fibrin strands. In comparison when aPRP (Platelet-rich plasma) clots, the clotcontains 4% red blood cells, 95%platelets and 1% white cells. The cellgrowth promoting components are theplatelet derived growth factor (PDGF)and mostly the transforming growth fac-tor Beta (TGF β). Fibronectin and vit-ronectin are also present. They are thecell adhesion molecules found in plasmaand fibrin itself. The experiments pre-sented were aimed to isolate, characteriseand test in vitro on different cell cultures

the growth promoting material from thecalf blood clot. The blood was collectedand allowed to clot. The whole contentwas centrifuged at 2500 rpm for 20 min-utes and the supernatant (=serum) wasaspirated off. The clot was quicklywashed with the physiological solution(20 minutes); the content was centrifugedat 2500 rpm for 20 minutes. The super-natant was collected and stored at -20°C(Fraction I). To the remaining clot sterile0.4 M PBS, pH=5.8, was added for 2hours. After centrifugation at 2500 rpmfor 20 minutes, the supernatant was col-lected and stored at -20°C (Fraction II).To the remaining clot PBS-Glucose,pH=7.3, was added for 24 hours. Aftercentrifugation at 2500 rpm for 20 min-utes, the supernatant was stored at -20°C

(Fraction III) All fractions were sterilisedby 0.2 membrane filtration, and analysedby SDS-PAGE. The cell growth promo-tion/inhibition activity in comparison toSR-2.0552P (Serum replacement basedon porcine ocular fluid) and FCS (foetalcalf serum) was tested on: chicken intesti-nal epithelial cell line, WISH, PLA-2 andCaco-2 cell line. The results of the exper-iments were: (1) Fractions I - III showedgrowth promoting activity that differedaccording to the cells tested. (2) Theactivity was strongest on the transformedcells (Chicken intestinal epithelial cellline, CaCo-2). (3) The optimal concentra-tion was 6-8% in Eagle’s medium. (4) Inthis range up to 80% of SR-2.0552P orFCS activity could be obtained.


Cell-growth promoting fractions originated from calf blood clotBratko Filipic0, Srecko Sladoljev 1, Lidija Gradisnik2, Tanja Botic’ 2, Eva Ruzic’-Sabljic’ 0, Avrelija Cencic 2 and Srecko Koren0

0 Microbiology and Immunology, University of Ljubljana (Ljubljana) (SI), 1 Institute of Immunology (Zagreb) (HR); 2 Faculty of Agriculture, University of Maribor (Maribor) (SI)e-mail: [email protected]

Keywords: calf, blood clot, growth factors, cell growth in vitro

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Avian influenza (AI) H5N1 virus, theextremely contagious strain, can bedeadly to domestic poultry. Without“jumping” the species barrier it can sel-dom cause infections in humans. Such aninfection coincided with devastating epi-demics in poultry farms in Asian coun-tries, with the reported mortalityapproaching 100%. Closely related to thishighly pathogenic avian influenza (HPAI)is strain H5N2, being antigenically simi-lar to H5N1 although distinguishablefrom an influenza H5N1 virus isolatedfrom a human in Hong Kong.Nevertheless the phylogenetic analysis ofthe haemagglutinin (HA) genes showed

that the highly pathogenic H5N2 clus-tered with the human H5N1 virus isolatedin Hong Kong, even though up to nowthere are no reports of the transmission ofthis virus from bird to human. The pre-sented experiments were aimed to findout if low doses of HuIFN-Alpha N3 andPoIFN-Beta affect the replication of theAI (H5N2) virus in the embryonatedchicken egg and through this the survivalof the infected embryo. The obtainedresults were evaluated on the basis of theEID50 value: 10 - 4,249/0.1ml, and werethe following: virus (10-4) + IFNs givensimultaneously shows the effectivenessof low doses (150 I.U.) of IFN, resulting

in 100% embryo survival and 100% HAinhibition. Virus (10-4) + IFNs givenafter 2h: only the high doses (15.000) arein some way effective (33,3 % survival).IFNs + virus (10-4) after 2 h are probablymore important, because this pre-treat-ment with low doses and infection after 2hours resulted in a decrease of HA as wellas 66,7% survival. This is probably con-nected with the neuraminidase (NA) inac-tivation. Further experiments will showhow to optimize the effectiveness ofHuIFN-Alpha N3 and PoIFN-Betatoward the resistance of AI(H5N2) viruswith different effectors like different l-amino acids.


HuIFN-Alpha N3 and PoIFN-Beta affect thereplication of Avian Influenza (H5N2) virus in theembryonated chicken eggBratko Filipic 0, Irena Ciglar Grozdanic’1, Zeljko Gottstein1, Tatjana Sindik-Milo 2, Srecko Sladoljev 2, Hrvoje Mazija 1, Srecko Koren 0 and Eugen Soos 1

0 Institute of Microbiology and Immunology, University of Ljubljana (Ljubljana) (SI); 1 Veterinarian Faculty, Dep. of AvianDiseases with Clinics (Zagreb) (HR); 2 Institute of Immunology (Zagreb) (HR)e-mail: [email protected]


News from the neurosphere front Ellen Fritsche, Michaela Moors, Kathrin Gassmann, Thomas Rockel, Jessica Heinrichs, Jason E. Cline and Josef AbelInstitut für umweltmedizinische Forschung gGmbH (Düsseldorf) (DE)e-mail: [email protected]

Keywords: Avian Influenza (H5N1), Avian Influenza (H5N2), HuIFN-Alpha N3, PoIFN-Beta, Chicken embryo, Haemaglutination,Embryo survival

Because developmental neurotoxicitytesting (DNT) requires large amounts ofanimals, alternative methods are neededto reduce animal consumption. There-fore, we have established a human invitro model for DNT based on normalhuman neural progenitor (NHNP) cells,which are cultured as proliferating neuro-spheres. On appropriate extracellularmatrices, NHNP cells migrate radiallyout of the sphere and thereby differentiateinto the major neural cell types of thebrain. During brain development, cell

proliferation, apoptosis, differentiationand migration are fundamental processes.Therefore, we developed in vitro meth-ods, which can detect effects of chemi-cals on these endpoints. NHNP cellproliferation can be measured using theCell Titer blue assay (Promega) in a highthroughput 96-well format. This testdetermines mitochondrial activity of liv-ing cells. The same assay is used to testfor cytotoxicity of chemicals on NHNPcells and can be multiplexed with a cas-pase-3/-7 assay which assesses apoptosis-

induced effector-caspase acitvity. Inimmunocytochemical investigations, celldifferentiation analyses revealed thatcAMP and retinoic acid induce neuronaldifferentiation, while methylmercurycauses an increased formation of astro-cytes in NHNP cells. Oligodendrocytedevelopment was promoted by thyroidhormone and inhibited by exposure tolead in these cultures indicating that weare able to influence cell determination ofNHNP cells. Measurements of the trackthat NHNP cells wander during their dif-

In Sri Lanka, a herbal decoction (DC)comprised of Nigella sative (seeds),Hemidesmus indicus or its very closerelative Cryptolepis buchananii (root),and Smilax glabra (rhizome) has beenused for many years by a particular fam-ily of indigenous medical practitioners(personal communication, Ayurvedicphysician, Dr. N. Jayathilake) for thetreatment of cancer, despite the lack ofscientific evidence to prove or disproveits therapeutic efficacy. Recent in vivoinvestigations have demonstrated thatthe above decoction can offer significantprotection against diethylnitrosamineinduced hepatocarcinogenic changes inrats. The aim of the present study was to investigate how changes in liver cellintegrity could contribute to the anti-hepatocarcinogenic actions of the decoc-tion comprised of Nigella sativa,Cryptolepis buchananii and Smilaxglabra. For this purpose, an in vitrostudy was conducted using human hep-atoma HepG2 cells. For experimental

purpose, HepG2 cells were cultured inT25 plates (1x106 cells/plate) in DMEMsupplemented with 10% foetal bovineserum. Cells in test plates were exposedto different doses of the decoction (10 µg/ml, 80 µg/ml, and 160 µg/ml) fora total of 72 h. Cells in control plateswere maintained in the same mannerwithout exposure to the decoction.Cellular integrity in both test and controlwas assessed by (a) microscopic exami-nation of cell morphology, (b) evalua-tion of lactate dehydrogenase (LDH)release from cells, cellular ATPase activ-ity, and intracellular reduced glutathione(GSH) status, at the end of 6 h, 24 h, 48 h and 72 h incubation with or withoutthe decoction, respectively. Resultsdemonstrate that compared to the con-trol the decoction even at a dose of 10µg/ml induced a 12.99% increase in theleakage of cellular LDH along with14.79% and -8.94% decreases in cellularATPase and GSH, respectively at the endof 72 h incubation. Reduction in cell

growth was microscopically apparenteven as early as 6h post-incubation. Itwas interesting to note that the cellularGSH in test cells was marginally higherthan the corresponding control at eachtime point. The overall outcome of thisstudy indicates a potent cytotoxicitytowards human liver cancer cells onceexposed to the decoction of Nigellasative (seeds), Cryptolepis buchananii(root), and Smilax glabra (rhizome).This direct effect on cells helps confirmthe results of recent in vivo investiga-tions, which demonstrated a protectionagainst chemically induced hepatocar-cinogenesis in rat liver. The marginalincrease in GSH subsequent to decoc-tion exposure is supportive for a possibleanti-oxidant role of the decoction, whichis essential for the protection againstchemically induced cancers. It may beconcluded that cytotoxicity and antioxi-dant activity may be two mechanismsthrough which the DC mediates its anti-hepatocarcinogenic actions.

Poster: evidence-based toxicology

Effects of a decoction of Nigella sativa, Cryptolepisbuchananii and Smilax glabraon human hepatoma HepG2 cell integrityBandula Galhena 0, M. I. Thabrew 0, F. D. Paul Solomon1 and V. A. Hanna Rachel1

0 Department of Biochemistry and Clinical Chemistry, Faculty of Medicine, University of Kelaniya, Sri Lanka (Ragama) (LK); 1 Department of Human Genetics, Sri Ramachandra Medical College & Research Institute, Chennai, India (Chennai) (IN)e-mail: [email protected]

Keywords: Nigella sativa, Cryptolepis buchananii, Smilax glabra, cytotoxicity, HepG2 cell line, hepatoma

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Keywords: DNT, neurosphere, in vitro method

ferentiation revealed that exposure tomercury (HgCl2 or CH3HgC), ethanol orcarbaryl led to an inhibition of NHNPcell migration. Investigations of theunderlying mechanisms indicated thatNHNP cell migration is regulated via the

MAP kinase ERK1/2-dependent andERK1/2-independent pathways, whichare regulated by PKC, EGFR and srcfamily kinases. Therefore, interference ofchemicals with these target moleculesmay cause disturbed cell migration and

impair brain development in humans. Insummary, we set up NHNP cells as ahuman in vitro model for DNT testing byestablishing endpoints which detect alter-ations in basic processes involved in braindevelopment.

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Current methods of body fat evaluationuse either isolated tissue samples or invivo techniques using instruments.However, only a few in vivo instrumentscan distinguish between different bodyfat tissues, yet produce only numericaldata. Consequently, determination ofthe ratio of subcutaneous (SCAT) andvisceral fat (VAT) becomes a manualprocess. Visualisation of the shape andsite of adipose tissues inside a living

animal was also limited. In this presen-tation, we focus on an alternativeapproach, which overcomes these issuesin rodents. The Computed TomographyScanner LaTheta™, Aloka Inc., Japan,includes a fast and exact in vivo body fatevaluation method for rodents with avisual display. We show an example ofan easy to use screening procedure forbody fat measurement in mice: Imagedata giving a visual impression is cap-

tured in less than a minute per scan.SCAT and VAT ratio or weights areevaluated immediately with just onemouse click. This method has beendeveloped especially for long-termstudies in mice and other small labora-tory animals. CT is also suitable forother complementary methods such asfast tumour monitoring or bone parame-ter evaluation, saving animal use withinthese research areas.

Poster: computer assisted procedures

Bounded animal use through in vivo body fat screening with the laboratory CT LaTheta™ Adam Glowalla and Wiebke Zinnser-NoethenZinsser Analytic GmbH (Frankfurt) (DE)e-mail: [email protected]

Keywords: small animals, rodent, mouse, rat, in vivo, body fat, screening, long-term study, CT, functional imaging

Scientists are obliged by animal welfarelegislation not to conduct animal experi-ments, if alternative methods are reason-ably and practicably available to obtainthe information. Scientists should consultliterature and other relevant informationsources on alternatives prior to any exper-imental study using laboratory animals.Over the years scientists have pointed outthe difficulties in identifying informationon alternatives among the increasingamount of scientific publications stored

in electronic-data retrieval systems. Thecomprehensive search guide, initiatedand financed by ECVAM, aims at provid-ing a step-by-step approach for data-retrieval procedures for untraineddatabase users. The project has beenstarted in 2007 and will be finished in2008. The guide will consist of five sec-tions: (1) Legislation Requirements of theEU, (2) Main Problems during DataRetrieval, (3) Information RetrievalSystems, (4) Basic Search Principles, and

(4) Search Terms, Key Words and theirUse. Each section will be represented bydata sheets that inform about its maincharacteristics. The data sheet’s format isthe very reason to create a kind of a “file-card box”. The “ECVAM Guide” will bemade available via the Internet by theECVAM Database service on AlternativeMethods to animal experimentation (DB-ALM) currently accessible at the address:http://ecvam-dbalm.jrc.ec.europa.eu.


Best search practice for animal alternatives – theECVAM guide for untrained database usersBarbara Grune0, Annett J. Roi 1, Amrei Schnock0, Florian Spiegel 0, Sebastian Spiegel 0, Antje Döhrendahl 0, Susanne Skolik0 and Horst Spielmann 0

0 Center for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET) (Berlin) (DE); 1 European Centre for the Validation of Alternative Methods (ECVAM) (Ispra) (IT)e-mail: [email protected]

Keywords: animal welfare, animal testing alternatives, legislation requirements, information retrieval, searching strategies,search principles, search terms

Poster: computer assisted procedures

Partition and diffusion – the experimental basis for in silico modeling of skin absorption Steffi Hansen0, Claus-Michael Lehr 0, Ulrich Schaefer 0, Arne Naegel 1, Dirk Feuchter1, Michael Heisig1, Gabriel Wittum 1 and Dirk Neumann 2

0 Saarland University, Biopharmaceutics/Pharmaceutical Technology (Saarbruecken) (DE); 1 University of Heidelberg, Simulationin Technology (Heidelberg) (DE); 2 Saarland University, Centre for Bioinformatics Saar (Saarbruecken) (DE)e-mail: [email protected]

Keywords: in silico modeling, skin absorption, diffusion, partition

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Pharmaceutical and cosmetic industriesas well as governmental institutions havediverse interest in in vitro skin investiga-tion such as bioavailability studies, riskassessment and consumer protection(EU-chemicals policy REACH). The highdemand conflicts with an insufficientavailability of human skin. Animal andbioengineered skin is used alternativelyhowever; large interspecies variabilitiesand insufficient barrier formation limittheir significance for the situation in man.In addition the use of animal skin for cos-metic applications will largely berestricted due to the 7th cosmeticsamendment from March 2009. Therefore

computer simulations may be a suitablealternative. Skin absorption may bedescribed as partition between donor andsuperficial stratum corneum lipids, inter-cellular lipids and corneocytes, stratumcorneum and viable epidermis and diffu-sion through lipids, corneocytes andviable skin layers. Based on these parti-tion and diffusion coefficients Heisig etal. (1996) developed a mathematicalmodel that is able to follow non-steady-state drug penetration into the stratumcorneum. Our aims were first to deter-mine these partition and diffusion coeffi-cients experimentally in order to limit thenumber of unknowns and thus increase

the predictive power of the model.Second, concentration-depth profiles andpermeability data were needed to validatepredictions. Experimental and simulateddata will be presented for two exemplarycompounds: flufenamic acid (lipophilic,ionizable) and caffeine (hydrophilic, non-ionizable).

ReferencesHeisig, M., Lieckfeldt, R., Wittum, G. et

al. (1996). Non steady-state descrip-tions of drug permeation through stratum corneum. I. The biphasic brick-and-mortar model. Pharm. Res. 13,421-6.


The use of a human in vitro airway epithelium tissuemodel (EpiAirway) for metabolism, toxicity screeningand drug delivery applicationsPatrick Hayden, Robert Jackson, Helena Kandarova and Mitchell KlausnerMatTek Corporation (Ashland, Massachusetts) (USA)e-mail: [email protected]

In vitro airway models are urgentlyneeded as alternatives to animal testingfor compliance with REACH legislation.The current poster describes a highly dif-ferentiated in vitro model of human tra-cheal/bronchial epithelium (EpiAirway).The model is produced by culturing nor-mal human tracheal/bronchial epithelialcells on microporous membrane inserts atthe air-liquid interface. Here we presentphysical and biochemical characteriza-

tion of EpiAirway morphology, barrierfunction and drug metabolizing capabil-ity. Histological cross-sections ofEpiAirway cultures show an in vivo-like,pseudo-stratified structure with numerousapical cilia. The useful lifespan of thecultures is at least 1 month. Dot blot anal-ysis demonstrates apical mucin secretion.Transmission electron microscopyreveals ultrastructural detail of cilia andtight junctions between cells. Trans-

epithelial electrical resistance (TEER) of300-500 ohms x cm2 demonstrates func-tionality of tight junctions. RT-PCR geneexpression experiments were conductedto evaluate baseline and inducible expres-sion of CYP isoforms in EpiAirway cul-tures derived from 4 individual donors.CYP1A1 (weak), CYP1B1, CYP2A6,CYP2B6 (weak), CYP2C8 (weak),CYP2C19, CYP2D6, CYP2E1 andCYP3A5 are constitutively expressed,

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Pulmonary administration of novel micro-and nano-scaled drug and gene deliverysystems is a suitable alternative to injec-

tion, even though the drugs must over-come numerous barriers in the lung toreach the target site. Our aim was to inves-

tigate the in vitro toxicity of low molecu-lar weight polyethylenimine (PEI)towards specific cells of the human distal

In 1986 Degussa’s Krefeld site becamethe world’s first large-scale manufacturerof super absorbing polymers (SAP). Theproduct FAVOR® is a cross linked, par-tially neutralized sodium polyacrylateand its primary function is to absorb liq-uids and keep them retained, even underpressure. Depending on the type of liq-uid, FAVOR® can absorb 30–500 timesits own volume. SAP is an essential partof modern hygiene articles like baby diapers, and adult care specialities. Tomeet todays and future needs of thosehygiene articles, FAVOR® is continu-ously improved by our research anddevelopment. We demand high safety

requirements from all improved FAVOR®

products. Despite strict control of the rawmaterials we additionally use a battery ofin vitro screening tests to guarantee thesafety of FAVOR®.

Here we demonstrate how in vitro toxi-cological test methods are capable to meetthe strict demands of the world’s leadingsupplier of SAPs. The first safety assess-ments of FAVOR® were performed on thebasis of the required in vivo test methodsaccording to international guidelines.However, results from valid in vitro testmethods have always been generated inparallel and today these in vitro data serveas our standard. Using the respective in

vitro test battery, each modification andimprovement of the product now has tocomply with our well established refer-ence in vitro data. In the present studyextracts and gels derived from modifiedSAPs have been tested to detect possibleeffects on the human skin using in vitroreconstructed human skin models.Additionally, the data have been amendedby BALB/c 3T3 NRU-Assays and HET-CAM Tests, all performed under GLP con-ditions. From our results we conclude thatthe battery of these in vitro tests is suitableto confirm a consistent and sustainablecutaneous tolerance towards each of thechemically modified SAPs.


In vitro toxicity of polyethyleneimine nanoparticleson human distal lung cells in mono- and co-culture M. Iris Hermanns 0, Peter Dubruel 1, Chiara Uboldi 0, Etienne Schacht 1 and James Kirkpatrick 0

0 Johannes Gutenberg University of Mainz, Institute for Pathology (Mainz) (DE); 1 Ghent University, Polymer Chemistry &Biomaterials Research Group (Ghent) (BE)e-mail: [email protected]

Keywords: in vitro screening, safety assessment, HET-CAM, BALB/c 3T3 NRU, reconstructed skin, cutaneous tolerance

while CYP3A4 and CYP3A7 were notdetected. 3-Methylcholanthrene (3MC)strongly increased expression ofCYP1A1 and slightly increased CYP2B6and CYP2C8 expression. Thus, CYPexpression in EpiAirway shows a high

concordance with CYP expression of invivo human bronchial epithelium. TotalGST activity in EpiAirway was demon-strated by measuring conjugation of glu-tathione with 1-chloro-2,4-dinitroben-zene. The results demonstrate that the

EpiAirway model possesses in vivo-likephysical and biochemical attributes forevaluating airway irritation, toxicitypotential, metabolism and delivery ofdrugs and environmental/occupationalchemicals.


When in vitro toxicology meets a global player:research and development of Degussa’s superabsorbing polymers are accompanied by alternativemethods in toxicologyEckhard Heisler0, S. Weimans0, F. Furno1, H. Schmidt1 and A. Schnurstein0

0 Laboratorium für Toxikologie und Ökologie; Stockhausen GmbH (Krefeld) (DE); 1 Degussa BU Superabsorber; Stockhausen GmbH (Krefeld) (DE)e-mail: [email protected]

Keywords: EpiAirway, REACH, CYP expression, TEER, metabolism, delivery of drugs

lung barrier. Therefore, we studied theuptake and influence of PEI on a humanlung epithelial cell line with characteris-tics of alveolar type II and Clara cells(NCI H441) and on the microvascularendothelial cell line (ISO-HAS-1) in an invitro co-culture model of the distal lung.This co-culture model mimics the twobarriers a therapeutic drug has to pass toreach the systemic circulation. Differentconcentrations of unlabelled and OregonGreen 488 (OG)-conjugated 25 kDa PEI(0.14–2.23 µg/cm2) in serum-free mediumwere used for cellular uptake studies overa 2 hr incubation period by fluorescentreading and confocal laser scanningmicroscopy (CLSM). Possible mitochon-drial damage after exposure to PEI wasstudied using the MTS assay. In addition,

viability of the cells was assayed by stain-ing for Ki67. In the in vitro co-culturemodel, the influence of PEI-nanoparticleson barrier properties was investigated bymeasuring trans-bilayer electrical resis-tance (TER). The uptake of OG-conju-gated PEI in the serum-free mediumdepended on the incubation time and onthe nanoparticle concentration. PEInanoparticles were taken up by bothmicrovascular endothelial cells and distallung epithelial cells, whereas the lattertake up OG-conjugated PEI much faster.TER-values of the PEI exposed co-cul-tures stayed similar compared to the con-trols without PEI during incubation up to2 hrs. Additionally, the expression of Ki67remained similar compared to cells with-out PEI. A concentration of 1.12 µg/cm2

OG-conjugated and unloaded 25 kDa PEIwas biocompatible with the cells, as it didneither affect the mitochondrial activity(MTS assay) nor the cell viability in a 24 hr mitogenic assay. Incubation with1.12 µg/cm2 OG-conjugated PEI nanopar-ticles did not affect the TER-values of theco-culture model resulting in a sustainedfunctional barrier after 24 hrs. Our systemwill therefore be a suitable model toinvestigate the targeting of drugs or genesto lung epithelium and endothelium viacoupling to OG-conjugated PEI-nanopar-ticles. The in vitro co-culture model willbe useful to further systematically studythe uptake and trafficking of new drugcarrier systems, delivering drugs to deeplung epithelial cells and across a distallung barrier.

Keywords: pulmonary drug delivery, nanoparticles, distal lung, alveolar type II, Clara cells, microvascular endothelial cells

Modern risk assessment, pharmacotoxi-cology and dermatology research impliesthe development of in vitro models withhigh reproducibility and confidentialityto the native situation. In this study wepresent data obtained by the use of“Epidermal Skin Test 1000” (EST-1000,CellSystems Biotechnologie VertriebGmbH, St. Katharinen, Germany). EST-1000 is a reconstructed human epidermiswhich shows a high comparability tonormal human in vivo skin and which

has already shown its abilities in the fieldof in vitro skin corrosion and irritationtesting. The major focus of this work wasto compare the toxicity properties of sev-eral substances, e.g. nerol and chlorpro-mazin, in the absence or presence of UVradiation (UVR) light. Without UVRmost of the tested substances have pro-voked none or weak effects to EST-1000while in the presence of UVR EST-1000they have shown increased histologicaldamages (H & E staining) and a signifi-

cant reduce of cell viability (conversionof MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)).Furthermore the tissue reacts with anincreased release of Lactate Dehydro-genase (LDH) as determined by standardELISA assay. We conclude from theseresults, that EST-1000 is a useful tool fora correct classification of substancesconcerning their phototoxic properties.


Determination of skin phototoxicity properties of different compounds using epidermalskin test 1000 (EST-1000)Jens Hoffmann0, Eckhard Heisler1, Astrid Thiemann 1, Sabine Weimans1 and Horst W. Fuch0

0 CellSystems Biotechnologie Vertrieb GmbH (St. Katharinen) (DE); 1 Laboratorium für Toxikologie und Ökologie, DegussaStandortservice Essen/Krefeld (Essen/Krefeld) (DE)e-mail: [email protected]

Keywords: reconstructed skin, epidermis, phototoxicology, evidence-based toxicology

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The Bionas®2500 analyzing system provides acell based in vitro monitoring of dynamic cellbehaviour influenced by test compounds. It is avery useful tool for the analysis of cytotoxic anddose dependent effects as well as for the inves-tigation of regeneration effects after treatmentand removal of test compounds. Investigationsof adherent cells are already established.Currently in focus of European MedicinesAgency (EMEA) is the study of immunotoxic-ity, defined as unintended immunosuppressionor enhancement. EMEA recommends the eval-uation of potential adverse effects of pharma-ceuticals on the immune system. On cellularlevel this could be achieved by examination ofdynamic immune cell behaviour in reliance onpotential immunotoxic compounds. One pre-requisite for such studies is the analysis of sus-

pension cells because immune cells commonlygrow non-adherent. By the Bionas®2500 ana-lyzing system a continuous monitoring of phys-iological parameters of suspension cells is nowpossible. Our silicon chip based analyzing sys-tem is able to observe 2 different physiologicalparameters (acidification, respiration) per chip.Six chips can be measured in parallel. Changesof acidification activity and respiration ratescaused by test compound are hints of the effecton the cell physiology and presumably thecytotoxic property of the compound used.Usually changes in cell physiology, especiallyon immune cell physiology are indices forimmunotoxicity. Generally we have successfulestablished the using of suspension cell for theonline monitoring of oxygen consumption andacidification behaviour. An important advan-

tage of our Bionas®2500 is the continuousdetection of all parameters from a few hours toseveral days contrary to the so called end pointtest like MTT or LDH release. Our analyzingsystem is able to record in real time all com-pound actions on the living cells until the end-point of treatment and moreover subsequentlythe recovering effects after removing the com-pounds. First experiments revealed effects ofdoxorubicin on T-lymphocyte Jurkat cells.Doxorubicin (Dox) is known as an anticancerdrug and also as an immunosuppressive agent.Different concentrations of Dox were appliedand a dose-dependent decrease of acidificationactivity and respiration rates could be observed.Regeneration of Jurkat cells after removingDoxorubicin from the medium could not beobserved.


Online monitoring of suspension cells in the focus of in vitro immunotoxicity-studiesBritta Jehle0, Sabine Drechsler 0, Axel Kob0, Marcus Wego 0, Miriam Nickel 0, Ingo Freund1, Ralf Ehret 0 and Elke Thedinga 0

0 Bionas GmbH (Rostock-Warnemünde) (DE); 1 Biophysics Institute, Bioscience Department, University Rostock (Rostock) (DE)e-mail: [email protected]

Keywords: cell metabolism, suspension cells, immunotoxicity, in vitro method, label-free, non-invasive

Increasingly concerns about limitations ofregulatory toxicological methodologies, suchas an insufficient test method assessment orthe lack of transparency and consistency indata integration and interpretation, areexpressed. We identified the concept of evi-dence-based medicine (EBM) as a promisingstarting point to remedy these issues as clini-cal medicine faces a similar problem of com-bining state-of-the-art scientific and traditionalapproaches. Evidence-based medicine strivesfor making decisions regarding patients, e.g.by providing sound scientific bases via sys-tematic reviews of available evidence.

Systematic reviews, being scarce in toxicol-ogy, could be a valuable tool for mapping thestate of today’s toxicology in a consistentmanner. Moreover, obvious fundamental par-allels between diagnostic and toxicologicaltests suggest that the available methodologyfor an evidence-based diagnostic test assess-ment might be transferable to toxicology. Inaddition, quality assurance aspects, qualityassessment tools and elaborated experimentaldesigns of EBM might constitute approachesrelevant for toxicology. Along with Guzelianet al. (2005), we propose an evidence-basedtoxicology (EBT) (2006). To discuss this pro-

posal with toxicologist and other interestedscientists, the first international forum towardan evidence-based toxicology will be held inComo, Italy, October, 15th-18th, 2007(www.ebtox.org).

ReferencesGuzelian, P. S., Victoroff, M. S., Halmes, N.

C. et al. (2005). Evidence-based toxicol-ogy: a comprehensive framework for cau-sation. Hum. Exp. Toxicol. 24, 161-201.

Hoffmann, S. and Hartung, T. (2006). Towardan evidence-based toxicology. Hum. Exp.Toxicol. 25, 497-513.

Keywords: evidence-based, transparency, test assessment, decision making

Lecture: evidence-based toxicology

Toward an evidence-based toxicology Sebastian Hoffmann European Commission, DG JRC (Ispra) (IT)e-mail: [email protected]

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Keywords: microarray, nephrotoxicity

Keywords: InterNICHE, Humane Education Award, funding, replacement, education, training

As technology develops and we enter therealm of omics, standardisation of in vitrocell culture techniques has become moreimportant than ever. In the case of DNAmicroarrays where it is possible to mea-sure the transcriptional activity of over47,000 transcripts, every effort must betaken to limit the effect of unknowns in agiven experiment. Although this technol-ogy is still relatively expensive the cost in

time for analysis is the major factor.Therefore, engaging this technology within vitro cell culture requires a consider-able amount of foresight in an attempt topre-empt possible costly (financial andtime) experimental design errors. In the6th Framework project, PREDIC-TOMICS “Short-term in vitro assays forlong-term toxicity”, we attempted to per-form an interlaboratory comparison of

microarray data from four European lab-oratories. In each laboratory, the humanproximal tubule cell line HK-2, wastreated with Cyclosporine A and RNAwas isolated. At three different centresmicroarray processing was conductedusing Affymetrix HGU-133 Plus 2 genechips. The approach, experiences, resultsand whether or not we were successfulwill be discussed.


Standardisation of cell culture techniques: a prerequisite to effective multi laboratory comparison of microarray data Paul Jennings Innsbruck Medical University (Innsbruck) (AT)e-mail: [email protected]

The InterNICHE Humane EducationAward is an annual grant program estab-lished to support ethical and effectiveeducation and training within biologicalscience, medicine and veterinarymedicine. Supported by Dutch organisa-tion Proefdiervrij, the Award has offeredan annual 20,000 Euro (US$ 27,000)since 2002. The Award was initiallyfocused on one region or country, such asIndia, and has been global since 2005.Applicants may be teachers, students,campaigners or other individuals com-

mitted to best practice education andtraining. Proposals are assessed primarilyaccording to their potential to replaceharmful animal use, based on number ofanimals and/or severity of procedures,potential pedagogic effectiveness, andinnovation, resourcefulness, and overallethical design. Funds are split betweenthe successful applicants, who number onaverage 8 per year. Examples of projectsthat have been funded include the devel-opment and implementation of freeware,videos, models and mannekins; purchase

and use of existing alternatives, includingan advanced surgery training device;establishment of a student-based self-experimentation program to replace ani-mals in physiology practical classes; andestablishment of a client donation pro-gram to secure ethically sourced animalcadavers for replacement of animals usedfor anatomy and surgery training.Specific projects will be described in thepresentation, and the impact of support-ing multi-local humane education initia-tives assessed.

Poster: vocational training

InterNICHE Humane Education Award: assessing the international impactNick Jukes 0 and Siri Martinsen 1

0 InterNICHE (Leicester) (GB); 1 InterNICHE Norway (Oslo) (NO)e-mail: [email protected]

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The EpiOcular™ tissue model (OCL-200) is an organotypic model of thehuman corneal epithelium (HCE) cul-tured from normal human keratinocytes.Paraffin embedded, histological cross-sections show the structure ofEpiOcular™ closely parallels that of the HCE. Quality control of weeklyEpiOcular™ batches is performed usingthe MTT assay, which historically hasbeen the in vitro endpoint of choice forEuropean and US regulators. The expo-sure time needed to reduce the viability to50% (ET50) for 0.3% Triton X-100 isdetermined. Since 1997, yearly average

ET50 values have ranged from 20.6 min-utes to 25.0 minutes with average coeffi-cients of variation under 7%. Thus,EpiOcular™ has demonstrated long termreproducibility over the past 10 years.This is important since regulators requirethat test methods provide consistent dataduring and after the validation process.EpiOcular™ has been increasingly usedby many personal care and householdproduct companies to determine the ocu-lar irritancy of their products withoutusing animals. In particular, EpiOcular™has been very useful for evaluating watersoluble and low irritancy materials.

Currently, a new protocol to handleindustrial chemicals spanning a range ofchemical categories and irritancies hasbeen developed. 41 materials includingalcohols, hydrocarbons, amines, esters,and ketones have been evaluated. Using astraightforward MTT tissue viability protocol, a prediction model for discrim-inating between ocular irritants and non-irritants was developed which resulted in100% sensitivity and 63% specificity.This new protocol will prove very useful in fulfilling the provisions ofREACH (Registration, Evaluation, andAuthorization of Chemicals) in the EU.


Expanded applicability domain for assessing ocularirritation using EpiOcular™ human cornel tissuemodelYulia Kaluzhny, Helena Kandarova, Laurence Thornton, Patrick Hayden and Mitchell KlausnerMatTek Corporation (Ashland, Massachusetts) (USA)e-mail: [email protected]

Keywords: EpiOcular™, ocular irritation, REACH, MTT

Safety assessment of new products forhuman use requires genotoxicity testingto insure their non-carcinogenicity.Current assays are based on non-humancell systems and result in low specificitydue to the lack of human-like metabolismand low relevance to the target organs.Dermally exposed substances require atest which determines skin-specific geno-toxicity. Starting in 2009, manufacturersof cosmetics products will need to assessgenotoxicity using non-animal methods.Thus, industrial organizations such asCOLIPA are actively pursuing develop-ment of micronucleus assay (MNA) for

dermally exposed chemicals. EpiDerm™forms a 3D skin-like tissue that is highlyreproducible and contains epidermis-likebarrier. Also, EpiDerm™ possesseshuman in vivo-like biotransformationcapabilities including CYP450, GST, andUDP enzymatic activity. The EpiDerm™MNA protocol utilizes two 10 µl topicaldoses of test material given 24 hoursapart in the presence of 3 µg/ml ofCytochalasin-B in the medium. Cells areharvested from the tissue 24 hours afterthe last dose. This protocol results in thegeneration of a reproducible populationof binucleated cells (43.9% +/-7.6) with a

low background frequency of micronu-cleated cells (0.08% +/-0.08). We haveshown dose-related, statistically signifi-cant increases in micronuclei inductionfor 3 model genotoxins and for 5 out of 6 rodent skin genotoxins. In addition, 4 non-genotoxic chemicals have beenevaluated and shown to be negative.Finally, 3 genotoxins that require meta-bolic activation were also positive in theMNA. In conclusion, the MNA that uti-lizes EpiDerm™ with inherent metabolicactivity holds excellent promise to predictgenotoxicity while avoiding animal wel-fare issues.

Lecture: 7th cosmetics amendment

Genotoxicity screening using the EpiDerm™ human3D model skin specific micronucleus assayYulia Kaluzhny, Patrick Hayden, Viktor Karetsky, Helena Kandarova and Mitchell KlausnerMatTek Corporation (Ashland, Massachusetts) (USA)e-mail: [email protected]

Keywords: EpiDerm™, genotoxicity, 7th cosmetics amendment, micronucleus assay

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During 2001-2003, refined EPISKIN andEpiDerm™ in vitro skin irritation proto-cols were developed (Cotovio et al.,2005; Kandárová et al., 2005), whichresulted good correlation between in vivoand in vitro data. Both methods werebased on the idea of a common protocol,comprised of 15 min application and 42 hpost-exposure period, and measuring thereduction of tissue viability using MTTendpoint. In 2004, the two skin modelsproceeded into the ECVAM validationstudy with the aim of replacing acute skinirritation tests performed in rabbits. Thestudy revealed that the EPISKIN assay(based on MTT endpoint) showed suffi-cient sensitivity and specificity to beendorsed as a replacement of the in vivotest. The EpiDerm™ model was recog-nized as a validated constituent withinOECD testing strategy. Faller andBracher (2002), Schaefer-Korting et al.(2007) and others demonstrated that dif-ferences in the barrier properties of thereconstructed human epidermal modelsexist. We hypothesized that the “falsenegative” outcomes observed forEpiDerm™ using the common protocolwere likely due to EpiDerm™’s enhancedbarrier. Therefore, we modified the com-mon protocol by extending the exposuretime from 15 min to 60 min. Using thismodified protocol, we re-tested 60 readilyavailable chemicals from pre-validationand validation studies and obtained sig-nificant increased sensitivity, without lossof specificity, using EpiDerm™. Themodified assay provided a very balancedoutcome, resulting in total accuracy of80%. An inter-laboratory study will beperformed with chemicals endorsed by

ESAC to evaluate the reproducibility ofthe improved performance of EpiDerm™method. Taking into account evidenceabout the variability (Gilman et al., 1978;Weil and Scala, 1971; Worth and Cronin,2001) and borderline predictive capaci-ties of the Draize test in terms of humanhealth effects (Calvin, 1992; Campbelland Bruce, 1981; Robinson et al., 2000;Basketter et al., 2004), it can be con-cluded that both EpiDerm™ skin irrita-tion protocols provided sufficient levelsof sensitivity and specificity. The presen-tation will summarize results obtainedwith the two EpiDerm™ protocols com-pared to in vivo rabbit data, existing andrecently performed human patch study(Jírová et al., 2007).

RererencesBasketter, D. A., York, M., McFadden, J.

P. and Robinson, M. K. (2004).Determination of skin irritation poten-tial in the human 4-h patch test.Contact Dermatitis 51, 1–4.

Calvin, G. (1992). New approaches to theassessment of eye and skin irritation.Toxicol. Lett. 64, 157–164.

Campbell, R. L. and Bruce, R. D. (1981).Comparative toxicology I. Direct com-parison of rabbit and human primaryskin irritation responses to isopropy-lmyristate. Toxicol. Appl. Pharmacol.59, 555–563.

Cotovio, J., Grandidier, M. H., Portes etal. (2005). The in vitro skin irritation ofchemicals: optimisation of theEPISKIN prediction model within theframework of the ECVAM validationprocess. Altern. Lab. Anim. 33 (4),329–349.

Faller, C. and Bracher, M. (2002).Reconstructed skin kits: reproducibil-ity of cutaneous irritancy testing. SkinPharmacol. Appl. Skin Physiol. 15(Suppl. 1), 74–91.

Gilman, M. R., Evans, R. A. and DeSalva, S. J. (1978). The influence ofconcentration, exposure duration, andpatch occlusivity upon rabbit primarydermal irritation indices. Drug Chem.Toxicol. 1 (4), 391–400.

Jírová, D., Basketter, D., Bendová, H. etal. (2007). Human Skin Irritation StudySupports Results of 3D Human SkinModel In Vitro. OEESC 2007. Golden,Colorado. Downloadable at http://www.mines.edu/outreach/cont_ed/oeesc/P9.pdf

Kandárová, H., Liebsch, M., Gerner, I. etal. (2005). EpiDerm skin irritation testprotocol – Assessment of the perfor-mance of the optimised test. Altern.Lab. Anim. 33 (4), 351–367

Robinson, M. K., Osborne, R. andPerkins, M. A. (2000). In vitro andhuman testing strategies for skin irrita-tion. Ann. NY Acad. Sci. 919, 192–204.

Schaefer Korting, M., Bock, U.,Diembeck, W. et al. (2007). Recon-structed Human Epidermis for SkinAbsorption Testing: Results of theValidation Study. Altern. Lab. Anim.Accepted for publication

Weil, C. S. and Scala, R. A. (1971). Studyof intra- and interlaboratory variabilityin the results of rabbit eye and skin irri-tation tests. Toxicol. Appl. Pharmacol.19 (2), 276–360.

Worth, A. P. and Cronin, T. D. (2001). Theuse of bootstrap resampling to assess thevariability of Draize tissue scores.Altern. Lab. Anim. 29, 557–573.

Lecture: EU-chemicals policy (REACH)

Increased sensitivity of the EpiDerm™ skin irritationprotocol evaluated in the ECVAM skin irritation validation study Helena Kandarova, Patrick Hayden, Erin Spiller, Mitchell Klausner, Joseph Kubilus and John Sheasgreen MatTek Corporation (Ashland, Massachusetts) (USA)e-mail: [email protected]

Keywords: EpiDerm™, skin irritation, validation, in vitro methods, ECVAM

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The potential for chemicals to cause skineffects such as corrosion is a concern ofindustrial toxicologists in their assess-ments of possible worker and consumersafety issues. Moreover, the U.S.Department of Transportation (DOT), thenew regulation on the Registration,Evaluation and Authorization ofChemicals (REACH), and other interna-tional and national regulatory agenciesrequire that substances should be labeledas to skin corrosivity potential. In the past, skin corrosion assessments werebased on tests involving topical applica-tion of test substances to the skin of rab-bits. However, based on two ECVAMValidation studies performed during1996-2000 (Fentem et al., 1998; Liebschet al., 2000) with two reconstructedhuman skin models (EpiDerm™ andEPISKIN), the OECD approved use ofskin models as regulatory accepted meth-

ods (OECD TG 431) replacing the in vivotest. In the present study, the EpiDerm™skin corrosion test was repeated with thecommercially available test substancespreviously used in both ECVAM valida-tion studies. The aim was to demonstratethe long term reproducibility and reliabil-ity of the EpiDerm™ model and themethod, as required by regulators (Rispinet al., 2006). The data obtained show verygood correlation over a period of 7 years.We also conducted a formalized evalua-tion of a procedural modification pro-posed several years ago to correctlypredict the corrosive potential of materialsthat interfere with the MTT endpoint nor-mally used in the method. An attempt wasmade to optimize this method and demon-strate with several chemicals (sometimesreported to be false negatives) the predic-tive capacity of the EpiDerm™ cor-rosivity assay when this procedural modi-

fication is included. The presentation willsummarize data from the above men-tioned studies and describe optimizedmethods for assessing materials whichinterfere with the MTT endpoint.

ReferencesFentem, J. H., Archer, G. E. B., Balls, M.

et al. (1998). The ECVAM InternationalValidation Study on In Vitro Tests forSkin Corrosivity. 2. Results andEvaluation by the Management Team.Toxicology in Vitro 12, 483-524.

Liebsch, M., Traue, D., Barrabas, C. et al.(2000). The ECVAM PrevalidationStudy on the Use of EpiDerm for SkinCorrosivity Testing. ATLA 28, 371-401

Rispin, A., Stitzel, K., Harbell, J. andKlausner, M. (2006). Ensuring qualityof in vitro alternative test methods:Current practice. Regul. Toxicol.Pharmacol. 45 (2), 97-103.


In vitro skin corrosion test: reproducibility over timeand optimized methodology for testing chemicals interfering with the MTT endpointHelena Kandarova0, Hans Raabe1, Gregory Chua0, Mitchell Klausner 0, Joseph Kubilus0, Patrick Hayden0, Seyoum Ayehunie 0, Yulia Kaluzhny 0, Rodger Curren 1 and John Sheasgreen0

0 MatTek Corporation (Ashland, Massachusetts) (USA); 1 Institute for In Vitro Science, Inc. (Gaithersburg, Maryland) (USA)e-mail: [email protected]

Keywords: EpiDerm™, skin corrosion, REACH, MTT endpoint, regulatory toxicology

Validated in vitro methods are urgentlyneeded as alternatives to animal testingfor compliance with REACH and EUCosmetics Directive legislation. Some ofthe in vitro assays/tissue models havebeen, or are being, formally validated for

use in testing for corrosivity, irritation ortoxicity. However, regulatory agenciesand other users require assurance thatmodels will provide consistent, qualitydata over time, not just during validation(Rispin et al., 2004). Recommended

guidelines for good cell/tissue culturepractice include e.g. full characterizationof cells or tissues, sampling of each lotfor performance, and regular use of con-trols and benchmark chemicals to pro-vide assurance of the consistency in


Long term reproducibility of in vitro reconstructed skin models for regulatory testing purposes – Evidence of good tissue culture practiceHelena Kandarova, Mitchell Klausner, Joseph Kubilus, Paul Kearney, Patrick Hayden, Yulia Kaluzhny and John SheasgreenMatTek Corporation (Ashland, Massachusetts) (USA)e-mail: [email protected]

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performance of the models and assays(Cooper-Hannan et al., 1999; Coecke etal., 2005; Rispin et al., 2006). Onedemonstration of good cell/tissue culturepractice for commercially manufacturedskin models is their long-term repro-ducibility and consistence performancein validated test assays. In compliancewith GMP and ISO standards, MatTekCorporation evaluates the quality of eachlot of commercially manufactured prod-uct. The release criteria include: viabilitydetermination, proof of product sterility,screening of histology and evaluation ofthe barrier properties of the model usingthe MTT ET50 assay with the benchmarkchemical Triton X-100. Acceptance andrejection criteria have been establishedfor each in vitro product and are strictlyadhered to. The presented poster summarizes data showing long-termreproducibility of EpiDerm™ and

Antimicrobial/biocidal properties of TiO2,ZnO and CuO have found use in waterpurification, dentistry, wood preservation,antimicrobial textiles etc. Currently, nano-size formulations of TiO2 and ZnO aregrowingly used in consumer products (cos-metics, sunscreens, etc.), mostly as UV-protection ingredients. However, theirpotential harmful properties are poorlystudied. Due to their small size, nanoparti-cles gain new properties (optical, physical,etc.) and thus also new biological proper-ties are expected. Thus, nanosize metaloxides (as well as all types of nanoparti-cles) need to be tested for toxicity even ifthe bulk materials (like ZnO) have beentested and allowed to used in consumerproducts. The aim of this study was to elu-

cidate how the size of ZnO, CuO and TiO2

particles influences their toxic properties.For that, nano and bulk formulations ofthese metal oxides were studied for theirgrowth inhibitory effects towardsSaccharomyces cerevisiae – a widely usedeukaryotic model. All the studied metaloxides were purchased. The sizes ofnanoscale metal oxides were as follows:ZnO (50-70 nm), CuO (~30 nm) and TiO2

(25-70 nm). S. cerevisiae S288C wasgrown batch-wise on orbital shaker (200rpm) at 30°C in malt extract broth (pH 5.4)that was supplemented with various con-centrations of metal oxides. The metaloxide suspensions in growth medium werecharacterized for nanoparticles size distri-bution using Nanosight LM10™ system.

The growth of yeasts was evaluated byviable cell counts at 4, 8, 24 and 48 hours.The toxic effect of metal-oxides on thegrowth of S. cerevisiae was characterizedand quantified from the respective dose-effect curves as IC50 and IC20 (reduction inthe specific growth rate by 50% or 20%,respectively). Our data showed that bothZnO formulations were of analogous toxi-city (8-h IC50 about 300 mg/l). However,nano CuO (8-h IC50 about 60 mg/l) andnano TiO2 (IC50 about 2500 mg/l) wasabout 10-20 fold more toxic than respec-tive bulk formulations. This is the firstcomparison of the potential adverse effectsof nanosize and bulk ZnO, CuO and TiO2

on yeast Saccharomyces cerevisiae.

EpiOcular™ reconstructed tissue mod-els. Quality controls for each tissue lot ofEpiDerm™ and EpiOcular™ were per-formed and the yearly averages havebeen calculated. Since 1997, EpiOcular’syearly average ET50 values ranged from 20.6 minutes to 25.0 minutes.Coefficients of variation for negativecontrol tissue have averaged under 6%for the entire historical data set. Theyearly average CV for all tissues neverexceeded 6.5%. For EpiDerm™, theyearly average ET50 ranged from 6.0 to7.5 h. The CV for the negative controltissue averaged under 7.5% for everyyear since 1996. Results of quality con-trol data for commercially availableproducts, manufactured over the past 10years have address regulatory concernsregarding long-term reproducibility andhave fulfilled of the standards for goodcell/tissue culture practice.

ReferencesCoecke, S., Balls, M., Bowe, G. et al.

(2005). Guidance on Good Cell CulturePractice: A Report of the SecondECVAM Task Force on Good CellCulture Practice. ATLA 33, 261-287.

Cooper-Hannan, R., Harbell, J. W.,Coecke, S. et al. (1999). The principlesof Good Laboratory Practice: applica-tion to in vitro toxicology studies. Thereport and recommendations of ECVAMworkshop 37. ATLA 27, 539-577.

Rispin, A., Harbell, J. W., Klausner, M. etal. (2004). Quality Assurance for In VitroAlternative Test Methods: QualityControl Issues in Test Kit Production.ATLA 32, Suppl. 1, 725-729.

Rispin, A., Stitzel, K., Harbell, J. et al.(2006). Ensuring quality of in vitro alter-native test methods: Current practice.Regul. Tox. Pharmacol. 45, 97-103.s


Effect of ZnO, TiO2 and CuO particle size on thegrowth of Saccharomyces cerevisiaeKaja Kasemets, Marina Romet and Anne KahruNational Institute of Chemical Physics and Biophysics (Tallinn) (EE)e-mail: [email protected]

Keywords: REACH, EpiDerm™, EpiOcular™, long-term reproducibility, good cell/tissue culture practice

Keywords: nanoscale metal oxides, toxicity, Saccharomyces cerevisiae

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Animal use resulting in harm or death hashistorically played an integral role in vet-erinary education, in disciplines such assurgery, physiology, biochemistry,anatomy, pharmacology, and parasitology.However, many non-harmful alternativesnow exist, including computer simulations,high quality videos, “ethically-sourcedcadavers” such as from animals euthanizedfor medical reasons, preserved specimens,models and surgical simulators, non-inva-sive self-experimentation and supervisedclinical experiences. Veterinary studentsseeking to use such methods often facestrong opposition by faculty members,who usually cite concerns about theirteaching efficacy. Consequently, studies ofveterinary students were reviewed compar-

ing learning outcomes generated by non-harmful teaching methods with thoseachieved by harmful animal use. Of elevenpublished from 1989 to 2006, nineassessed surgical training – historically thediscipline involving greatest harmful ani-mal use. 45.5% (5/11) demonstrated supe-rior learning outcomes using more humanealternatives. Another 45.5% (5/11) demon-strated equivalent learning outcomes and9.1% (1/11) demonstrated inferior learningoutcomes. Twenty one studies of non-vet-erinary students in related academic disci-plines were also published from 1968 to2004. 38.1% (8/21) demonstrated superior,52.4% (11/21) demonstrated equivalent,and 9.5% (2/21) demonstrated inferiorlearning outcomes using humane alterna-

tives. Twenty nine papers in which com-parison with harmful animal use did notoccur illustrated additional benefits ofhumane teaching methods in veterinaryeducation, including: time and cost sav-ings, enhanced potential for customisationand repeatability of the learning exercise,increased student confidence and satisfac-tion, increased compliance with animal uselegislation, elimination of objections to theuse of purpose-killed animals, and integra-tion of clinical perspectives and ethicsearly in the curriculum. The evidencedemonstrates that veterinary educators canbest serve their students and animals, whileminimising financial and time burdens, byintroducing well-designed teaching meth-ods not reliant on harmful animal use.

Keywords: alternative, animal experiment, education, training, veterinarian, veterinary surgery


Educational studies establish the efficacy of humaneteaching methods in veterinary educationAndrew KnightAnimal Consultants International (London) (GB)e-mail: [email protected]

Biocompatibility testing of dental materialsis an essential requirement for their applica-tion in clinical practice. The objective of thepresent study is to investigate the in vitrocytotoxic and membrane toxic potential ofsix glass ionomer cements (Ionofil® Molar,Argion® Molar, KetacTM Molar Aplicap,Ketac™ Silver Aplicap, Alpha® Fil, Alpha®

Silver), one silicophosphate cement (CuproDur® N), one compomer (F2000) and onedenture resin (Kallocryl®). The test samplesare prepared in practically relevant sizesaccording to the manufacturer’s instruc-tions. The cytotoxicity is determined usingthe XTT tetrazolium reduction assay which

bases on the ability of living cells to reducethe colourless tetrazolium salt XTT to anorange water-soluble formazan product.The membrane toxicity is examined bymeans of the [3H]arachidonic acid releaseassay. The U937 permanent cell line is cho-sen because of its human origin and theability to perform many monocytic func-tions. Biocompatibility is comparativelyevaluated on the base of CD50 (doserequired to kill 50% of the cells) and MTF(membrane toxicity factor) values. Theresults show CD50s between 43 and 107mg/0.2 ml for glass ionomer cements and154 mg/0.2 ml for the compomer F2000

after 1-hour exposure. Kallocryl® did notexert any cytotoxicity within the concentra-tion range tested. The cytotoxicity increaseswith prolonged exposure: CD50 values werefound to be 7 to 41 mg/0.2 ml for glassionomer cements after 24-hour and 0.23 to31 mg/0.2 ml after 48-hour exposure. TheCD50s of F2000 were 73 and 52 mg/0.2 mland of Kallocryl® 149 and 32 mg/0.2 mlafter 24-hour and 48-hour exposure, respec-tively. The membrane toxicity of the sam-ples proved to be generally low. After1-hour exposure most of the tested dentalmaterials showed no membrane toxicity(MTF).


In vitro assessment of cytotoxic and membrane toxic potential of dental materialsHans-Peter Klöcking, Heiko Wagner and Renate KlöckingInstitute of Pharmacology and Toxicology/Working Group Erfurt, University of Jena (Erfurt) (DE)e-mail: [email protected]

Keywords: dental materials, cytotoxicity, membrane toxicity, XTT tetrazolium reduction assay, [3H]arachidonic acid assay

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The acquisition of electrophysiologicalparameters from cultivated stem cellsduring and after their differentiation intonerve or heart muscle cells is a novelapproach with the potential to reduce ani-mal experiments. A new modular glasschip system (MOGS) has been devel-oped, with a periphery compatible to our

existing silicon neuro-sensorchip system.The glass chip (www.gesim.de) ensuresmicroscopic observability. It features amulti-electrode array (MEA) with 52electrodes for detecting the electric activ-ity, e.g. in neuronal networks or car-diomyocytes. An integrated interdigitatedelectrode structure (IDES) in the cell-cul-

ture trough allows for impedance mea-surements to evaluate cell adhesion as anindicator for the viability of adherentlygrowing cells. The trough also contains atemperature sensor in direct proximity tothe cells. In the system, cells can be cul-tured for longer periods, from days up tomonths, as evidenced by electrically


Modular glass chip system for the acquisition of the electric activity and physiological parametersof differentiated stem cellsPhilipp Julian Köster, Sebastian Moritz Buehler, Jan Sakowski, Carsten Tautorat, HeleneAltrichter, Werner Baumann and Jan GimsaUniversity of Rostock, Institute of Biology, Chair of Biophysics (Rostock) (DE)e-mail: [email protected]

The assumption that animal models arereasonably predictive of human outcomesprovides the basis for their widespread usein toxicity testing and in biomedicalresearch aimed at developing cures forhuman diseases. To investigate the validityof this assumption, the comprehensive“Scopus” bibliographic biomedical data-bases were searched for published system-atic reviews of the human clinical ortoxicological utility of animal experiments.Of 27 reviews retrieved, the authors con-cluded that the animal models were sub-stantially consistent with or useful inadvancing clinical outcomes in only twocases, and the conclusion in one case wascontentious. Included were reviews of theclinical utility of experiments expected byethics committees to lead to medicaladvances, of highly-cited experiments pub-lished in major journals, and of chim-

panzee experiments – the species mostlikely to be predictive of human outcomes.Seven reviews failed to clearly demonstrateutility in predicting human toxicologicaloutcomes such as carcinogenicity and ter-atogenicity. Consequently, animal datamay not generally be assumed to be sub-stantially useful for these purposes.Possible causes include interspecies differ-ences, the distortion of experimental outcomes arising from experimental envi-ronments and protocols, and the poormethodological quality of many animalexperiments evident in at least 11 reviews.No reviews existed in which a majority ofanimal experiments were of good quality.While the latter problems might be min-imised with concerted effort, given theirwidespread nature, the interspecies limita-tions are likely to be technically and theo-retically impossible to overcome. Yet,

unlike non-animal models, animal modelsare not normally subjected to formal scien-tific validation. Instead of simply assumingthey are predictive of human outcomes, theconsistent application of formal validationstudies to all test models is clearly war-ranted, regardless of their animal, non-ani-mal, historical, contemporary or possiblefuture status. Expected benefits wouldinclude greater selection of models trulypredictive for human outcomes, increasedefficiency during the development ofhuman pharmaceuticals, and decreasedwastage of animal, personnel and financialresources. The poor human clinical andtoxicological utility of most animal modelsfor which data exists, in conjunction withtheir generally substantial animal welfareand economic costs, justify a ban on ani-mal models lacking scientific data estab-lishing their human predictivity or utility.


Animal experiments scrutinised: systematic reviews demonstrate poor human clinical and toxicological utility Andrew Knight Animal Consultants International (London) (GB)e-mail: [email protected]

Keywords: animal experiment, animal study, clinical trial, human outcome, systematic review

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active neural networks formed from pri-mary cells. With approximately 250,000primary cells needed per chip, one sacri-ficed laboratory mouse supplies cells forseveral hundred chips. This allows for areduction in the number of animals usedfor certain tests. Nevertheless, our aim isto replace primary cells by stem celllines. The new stem cell systems will be

interesting for research into the therapyof neurodegenerative diseases, e.g.Morbus Parkinson as well as for thedevelopment of pharmaceutical com-pounds and the testing of neurotoxic anddevelopmental neurotoxic effects. Themicroscopic observability of MOGSallows to correlate network structure tonetwork activity and to test regenerative

techniques in degenerative disease cellmodels. Other applications are in thefields of substance development for fertil-izers, food, household chemicals andconsumer goods or in the clinical, envi-ronment, food and military sectors. Theuse of MOGS as an alternative method toanimal experimentation should be vali-dated by ECVAM in the future.

Keywords: lab-on-chip, biochip, REACH, multi-electrode array, neuro-sensorchip, glass chip, neuronal networks, developmentalneurotoxicology, DNT, neurotoxicity, action potential detection

In February 2006, the German AnimalWelfare Federation started its 3rd surveysince the establishment of ethics commit-tees for animal experiments according to §15 of the German Animal Welfare Act inGermany in 1987. The survey of 1988focused on general parameters within thelicensing process. In 1995, changesregarding the previous survey and alsospecific developments after the Germanreunification were analysed. As animalwelfare has been included as a “state goal”in the German constitution in 2002, thepresent survey aims at an analysis of spe-cific changes within the licensing process.

In this context, the increase in value of ani-mal welfare as a consequence of its inclu-sion into the constitution was of majorinterest. According to law experts the newlegal situation would have to result inhigher requirements concerning the ethicaljustifiability within applications for animalexperiments. The question to which extentthe above mentioned increase in value haspenetrated into the work of ethics commit-tees and licensing authorities is thereforean important aspect of the survey. Otherimportant aspects include changes in thework of ethics committees and licensingauthorities since the last survey as well as

suggestions for improvement that havebeen regarded useful from the point ofview of members of ethics committees andlicensing authorities. The survey is basedon questionnaires that have been sent tolicensing authorities and members ofethics committees in February 2006. Oneof the main results is that the importanceof animal welfare within the licensing pro-cess, after inclusion of animal welfare intothe German constitution, has not changedor changed only to a little extent. It alsobecomes clear that ethical parameters arestill of minor importance when animalexperiments are licensed.


Survey of the German Animal Welfare Federationconcerning the work of ethics committees andlicensing authorities for animal experimentsRoman Kolar, Irmela Ruhdel Deutscher Tierschutzbund / German Animal Welfare Federation (Neubiberg) (DE)e-mail: [email protected]

Keywords: animal experiments, ethical committee, authorisation procedure

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The chorioallantoic membrane test sys-tem (CAM) represents a borderline sys-tem between in vitro and in vivo systems.In the last years the CAM has become thestandard model system for investigationsin angiogenesis. Because of the immun-odeficiency of the chick embryos in thefirst half of breeding, the membrane canaccept inoculation of various tumor cellsor tissues. In the last couple of years we have established the chorioallantoicmembrane system for research in tumorbiology especially as a test system fortumor chemosensitivity. For this purposewe used different methods for the appli-cation of test substances. After topicalapplication onto the membrane we couldshow the time-course of plasma concen-trations in the embryo circulation. If theembryonic development is taken intoaccount, the model is very reproducible.In a recent study we could show, that the

effect of the substances topically appliedis not caused by direct uptake of thetumor cells like in cell culture. Wedropped the substance onto the mem-brane in a different area clearly separatedfrom the tumor cells. For analysis of theplasma-level kinetics, blood of was col-lected from the chorioallantoic veins atdifferent time points. The effects seen inthese tumor cells were comparable withthe effects seen in xenotransplanted miceafter intraperitoneal application. In otherstudies, the substances were administeredintravenously into the CAM vessels.Using fluorescent markers, we couldshow the redistribution in the membraneand the absorption in the tumor cells.Recently, Taizi et al. reported about theCAM as a novel in vivo system for testingtherapeutics on human leukemias. In thisreport, three days after injection ofhuman leukemia cells in the CAM veins a

single dose of doxorubicin was adminis-tered i.v. The strengths of this model fortesting new pharmacologically activesubstances are several-fold. First, it savesmice, further it is quick to perform, eco-nomic and it requires only very smallquantities of precious novel compounds.Upon successful pretesting in this model,additional experiments are required toconfirm the pharmacotherapeutical corre-lation between the xenotransplantationmodels in mice and fertilized chickeneggs. In our presentation we will give areview of the current application methodsin CAM found in the literature.

ReferenceTaizi, M., Deutsch, V., Leitner, A. et al.

(2006). A novel and rapid in vivo sys-tem for testing therapeutics on humanleukemias. Experimental Hematology,Vol. 34, 12, 1698-1708.


Refinement of tissue biopsy for PCR basedgenotyping of GM mice by using hair samples Dirk Korthaus, Lill Andersen, Simone Müller, Caroline Lassnig and Thomas Rülicke Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna (Vienna) (AT)e-mail: [email protected]


Chorioallantoic membrane model system – differentapplication methods for test substances Karin Kunzi-Rapp Felicitas Genze Institute for Laser Technologies in Medicine and Metrology, University of Ulm (Ulm) (DE)e-mail: [email protected]

The efficient and reliable genotyping is oneof the most important aspects for the gen-eration and breeding of GM rodents. Toisolate genomic DNA from mice differenttechniques of invasive and non-invasive tis-sue biopsies are known. Tail biopsy is stillthe routinely used but also most invasivemethod. Therefore, hair follicles for thepreparation of chromosomal DNA are dis-cussed as possible source and several stud-

ies have been published to demonstrate thefeasibility. The use of hair samples couldreduce distress and impairment of the ani-mal’s well-being. However, efficiency andreliability of hair sampling as routinemethod remains to be certified. The presentstudy was conducted to investigate severalparameters that might have an effect ongenotyping results, to be more confident inthe use of hair follicles for DNA isolation.

Tested parameters are hair colour, age ofmice (hair cycle stages), sampling methodas well as storage of the biopsies.Moreover, the risk of cross contaminationwas proved by micro satellite analyses. ForDNA preparation different protocols wereused to meet the requirements for DNApurification either for detecting a knockoutallele by standard PCR or transgene inte-gration by real-time PCR.

Keywords: mouse, genotyping, hair, PCR, DNA purification

Keywords: chorioallantoic membrane, CAM, hen’s egg test, application methods

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Derek for Windows: conforming to the OECDprinciples for (Q)SAR validation Kate Langton and Carol Marchant Lhasa Limited (Leeds) (GB)e-mail: [email protected]

The OECD Principles for (Q)SAR Validationwere established in 2004 and are intended toidentify the types of information considereduseful for the regulatory review of (Q)SARmodels and their predictions. The five princi-ples have been applied to Derek for Windows(DfW), a knowledge-based expert systemwhich predicts the toxicity of a compoundfrom its chemical structure. Principle 1 (adefined endpoint): DfW predicts for toxico-logical endpoints such as mutagenicity, car-cinogenicity and skin sensitisation.Information on specific assays associatedwith an endpoint is included in the support-ing evidence provided with each prediction.Principle 2 (an unambiguous algorithm): Thereasoning methodology used by DfW hasbeen described in the public domain.

Predictions are based on alerts, examples andrules, which are supported by comments andreferences describing their basis. This sup-porting evidence can be explored for individ-ual predictions within the program, or theknowledge base as a whole can be viewed inthe accompanying editor. Principle 3 (adefined domain of applicability): At present,the applicability domain for an alert isdefined in terms of the chemical environmentit describes. Where sufficient evidence exists,this may be refined by consideration ofphysicochemical properties such as molecu-lar weight and log P. Principle 4 (appropriatemeasures of goodness-of-fit, robustness andpredictivity): Alerts in the DfW knowledgebase have historically been supported by theinclusion of four or five supporting examples,

with additional evidence appearing in com-ments and supporting references. Potentialsources of unseen data suitable for the pur-poses of external validation include that con-tributed to Vitic restricted data groups. Theseprovide an opportunity for industry to sharenon-commercially-sensitive data, not cur-rently in the public domain, for mutual bene-fit. Principle 5 (a mechanistic interpretation,if possible): Where evidence is available, themechanistic basis of an alert or rule isdetailed in the associated comments and sup-porting references. Examples will be pre-sented to illustrate the existing high degree ofconformity of DfW to the OECD Principlesfor (Q)SAR Validation, and the potential forfurther enhanced compliance will be dis-cussed.

Poster: revision of Directive 86/609/EEC

Next of Kin: the use of primates in experiments in the EU Gill Langley and Katy Taylor BUAV (British Union for the Abolition of Vivisection) (London) (GB)e-mail: [email protected]

Keywords: in silico, toxicity prediction, regulatory, quantitative structure-activity relationship

The Next of Kin project constitutes anextensive review of the public, scientific,legislative and ethical issues regarding theuse of non-human primates in scientificresearch and testing in the EU. The finalreport highlights a discrepancy betweenpublic unease about the use of primates andtheir treatment in the laboratory. 10,392 pri-mates were included in the EU statistics in2002, a 14% increase on previous years,although many more are held in laborato-ries. The largest users of primates areFrance, UK and Germany. The majority areused within the pharmaceutical industry forsafety and efficacy assessment (e.g. 726 pri-

mates were poisoned in LD50 tests). UK sur-veys of public concern about the use of pri-mates in research consistently show that themajority of the public does not support theiruse, even for the study of serious medicalproblems. This may in part reflect ourincreased understanding of the capacity ofprimates to reason, use tools, communicatesemantically, count, develop cultures andpractise deception; suggesting that they arenot only self aware but are aware of others.The potential for primates to suffer withinthe laboratory environment is therefore con-siderable and is reflected in stereotypies,suppressed immune function and physiolog-

ical disturbances which may confoundresearch findings. In particular, the transportof old world monkeys from non-EU coun-tries (78% of these) causes significant dis-tress. Despite the use of primates in thepharmaceutical industry, the current failurerate from entry to Phase 1 clinical trials tomarket is over 90%, largely attributed toinability to predict human ADME, toxicityand efficacy. Although alternative technolo-gies, such as in vitro, are being developed,increased funding of the development ofnon-animal methods is urgently requiredand should be incorporated into the currentrevision of EC Directive 86/609.

Keywords: primate, alternatives, EU legislation, animal experiments

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In the fields of neurotoxicity and devel-opmental neurotoxicity, there is a lack ofsuitable human experimental systems.Most of the available cells are neuro-blastoma cells with a transformed pheno-type. Especially for experiments withdopaminergic neurons, mostly primarycells are required. We developed an alter-native culture model based on LUHMEScells, a human neuronal precursor thatwas conditionally immortalized by aswitchable myc gene. We established amodel based on the toxicity of the

endogenous dopamine of the cells. Thecells died slowly upon exposure tomethamphetamine and iron, and this wasfound to be due to oxidative stress trig-gered by the dopamine released fromvesicles to the cytosol. As a consequenceof the oxidative stress, the cytoskeletonwas damaged, stress kinases were acti-vated and apoptosis was triggered. Wepresume that this sequence of eventsreproduces those that may occur duringParkinson’s disease and methampheta-mine-induced neurotoxicity. Since the

most frequently applied neurotoxin inprimate and murine models ofParkinson’s disease is 1-methyl-4-phenylpyridinium, we also examined theeffect of this compound. Concentrationsas low as 5 micromolar depleted glu-tathione, reduced the cellular energycharge, and eventually killed the cells.This is usually only observed in primaryneurons, but not in a cell line. Thus thenew LUHMES cell model may have thepotential to substitute primary cells formany experiments.

Keywords: Parkinson’s disease, neurodegeneration, methamphetamine, neurotoxicity, 1-methyl-4-phenylpyridinium

Microglia cells are of large pharmacolog-ical interest as they appear to be involvedin chronic pain mechanisms and arealways found to be activated in chronicneurodegenerative diseases. Microgliaare derived from the bone marrow andare more related to macrophages than tothe other neural cells derived from neu-roectoderm. They have a limited prolifer-ative capacity in culture and requiretherefore continuous re-isolation formbrain tissue to be available for experi-mentation. As an alternative to primarytissue, various microglia cell lines have

been generated and one of the most-fre-quently used ones is the BALB/c basedBV-2 cell. We present here initial experi-ments describing the suitability and limi-tations of BV-2 cells as surrogate modelfor primary microglia. For this approach,the inflammatory potential of the cell linewas compared to that of primarymicroglia from mice. The transcriptionalprofile was compared on two differentarray platforms and by direct measure-ment of various cytokines. In addition,the in vitro data were compared to the invivo reaction of microglia. The overall

result was, that BV-2 cells do not showresponses, not seen in primary cells, butthat they also do not mount the full spec-trum of activities observed in primarymicroglia. The genes induced in vivo,were also identified in the in vitro sys-tems. However, the cell cultures showedregulations, not detected in vivo, which isa point requiring attention. In conclusion,the BV-2 cells appear to be a good modelsystem for certain screen purposes, butbasic mechanistic research may requirethe use of additional experimental sys-tems.


The value of the BV-2 cell line as model for primary microgliaMarcel Leist 1,2, Lund Sören2, Andre Schrattenholz 3 and Peter Pörzgen 2

1 University of Konstanz (Konstanz) (DE); 2 H. Lundbeck A/S, Valby (DK); 3 Proteosys GmbH, Mainz (DE)e-mail: [email protected]


A human neuronal model system with a potential to substitute primary culturesMarcel Leist 1, Stefan Schildknecht 1, Alberto Salvo-Vargas1 and Julie Lotharius 2

1 University of Konstanz (Konstanz) (DE); 2 H. Lundbeck A/S, Valby (DK)e-mail: [email protected]

Keywords: inflammation, neurodegeneration, cell line standardisation, cell line characterisation, primary cells

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Alternative methods (AM) and their successin many areas of research have a relativelylow visibility outside the field. This is par-tially due to the fact, that there is no cleardefinition of what an AM is, and which valueit generates. The latter point is of majorimportance, not only for the public supportof this research area, but also as a judgmentbasis for financing and granting systems.This presentation attempts to highlightimportant issues linked to the visibility andjudgment of AM, and points out potentialways forward. A constructive dialog on thistopic can broaden the field of AM, sharpen

its cutting edges, and give it a profile clearlyrecognizable from outside. In the core areaof regulatory toxicology of chemicals, cos-metics and agricultural products, AM are rel-atively clearly defined. Here, some of theburning questions are, whether 1:1 substitu-tions are possible for safety areas differentfrom acute toxicity, how one deals with theconcept of severity of stress in view of ben-efit calculations, and how far AM have avalue as filters, in addition to substitutinganimal based methods. Especially the latterpoint is of major importance in the field ofpharmaceutical research and development.

Here the use and value of AM seems to bevastly underestimated, if one judges theirimpact based on the available statistics andpublications. Most of the AM data, e.g. theuse of in vitro metabolism systems, Amesassays, hERG assays and acute cytotoxicityassays never appear in statistics or publica-tions, although they are an integral part ofmost drug discovery screening plans. Thus,it appears that the application and impact ofAM should not be measured indirectly,based on the animal numbers in the officialstatistics on experimental animals. Moredirect and active measures will be suggested.


Judging the value of alternative methodsMarcel LeistUniversity of Konstanz (Konstanz) (DE)e-mail: [email protected]


Proinflammatory response in human alveolarepithelial cells exposed to ultrafine zinc oxide (ZnO)particles at the air-liquid interface (ALI)Anke-Gabriele Lenz, Ingrid Beck-Speier, Ellen Bitterle, Helga Hinze-Heyn, Erwin Karg, Bernd Lentner, Holger Schulz and Konrad MaierInstitute for Inhalation Biology, GSF-Research Center for Environment and Health (Neuherberg) (DE)e-mail: [email protected]

Keywords: animal statistics, toxicological domains, severity of stress, test strategies, visibility, judgement

Keywords: epithelial cells, air-liquid interface, nanoparticles, zinc oxide

We have recently developed a novel exposuresystem which allows a dose-controlled expo-sure of bronchiolar or alveolar epithelial cellsto airborne nanoparticles at the ALI. The sys-tem allows for a homogeneous and quantifi-able deposition of nanosized particles in astagnation point aerosol flow (Tippe et al.,2002; Bitterle et al., 2006). We intend to applythis system for exposures of cellular models tonanoparticles for dose titrations and identifica-tion of toxicological endpoints in order toreduce animal studies. Using the ALI system,we studied toxic effects of nanosized ZnOparticles. Human alveolar epithelial type IIcells (A549), chosen as biological target, werecultured on Anodisc membranes to confluentmonolayers (1x106 cells/cm2) and placed inspecially designed chambers. Commercially

available ZnO particles (0.024–0.071 µm;AlfaAesar) were dry-dispersed using a dustgenerator (TOPAS) and diluted with humidi-fied filtered air to an aerosol concentration of8.6x105 particles/cm3, with a count medianparticle diameter of 136 nm. Exposure of thecells for 3 h resulted in a total deposition of393 ng/cm2. After 3 h exposure to airborneZnO, cell viability remained unchanged,while transcription of the proinflammatorycytokine IL-8 increased 4-fold. Subsequentincubation of the cells in medium for another2 h led to a further increase of the IL-8 tran-scripts up to 23-fold over controls. In addition,ZnO exposure upregulated the expression ofcyclooxygenase-2 and enhanced the release ofprostaglandin E2. Analysis of IL-8 transcrip-tion assessed in submerged exposures using a

particle concentration of 10 µg ZnO/106cells/ml led to comparative results. In conclu-sion, airborne ZnO provoked a proinflamma-tory response. However, the concomitantupregulation of cyclooxygenase-2 and releaseof prostaglandin E2 suggests the activation ofa counteractive pathway. The particle doseattained at the ALI is well defined in contrastto exposures under submerged conditions.

ReferencesBitterle, E., Karg, E., Schroeppel, A. et al.

(2006). Dose-controlled exposure of A549epithelial cells at the air-liquid interface toairborne ultrafine carbonaceous particles.Chemosphere 65, 1784-1790.

Tippe, A., Heinzmann, U., Roth C. (2002).Deposition of fine and ultrafine aerosol par-ticles during exposure at the air/cell inter-face. J. Aerosol Sci. 33, 207-218.

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Significant progress has been made in Russiaduring 2005-2007 concerning awareness andimplementation of alternatives in education.Several major outreach visits involving pre-sentations and demonstrations of alternativeshave been made by a team of InterNICHEcampaigners, bringing the concept ofhumane education to large audiences. Mediainterest in these new approaches has resultedin nationwide and international coverage.Outreach letters have been sent direct toteachers across the country to promote the

resources that have been developed. Printedresources and on-line information have beencomplemented by the distribution of free-ware physiology and pharmacology alterna-tives – all translated into Russian. A librarygives hands-on access to alternatives nation-ally, and the donation of computers and alter-natives to some institutes has establishedexemplary multimedia laboratories. Thismulti-pronged strategy has now led to agree-ments being made between InterNICHE andseveral institutes to abandon animal experi-

ments for teaching purposes at the depart-mental and faculty level. Implementation ofalternative tools has been achieved at severalinstitutes, with multimedia directly replacingthe annual use of several thousand animals.The sharing between teachers of the experi-ence of implementation has now begun.There have also been isolated cases of deter-mined student conscientious objection, andthe small number of Russian campaigners isslowly growing, empowered by the suc-cesses of recent years.


Outreach, alternatives awareness and replacement in RussiaLena Maroueva 0 and Nick Jukes1

0 InterNICHE-Russia & VITA (Moscow) (RU); 1 InterNICHE (Leicester) (GB)e-mail: [email protected]

Keywords: InterNICHE, Russia, replacement, education, freeware

Photochemical damage to retinal pigmentepithelial (RPE) cells and photoreceptorsis involved in the pathogenesis of age-related macular degeneration (AMD).Numerous studies have suggested thatphotochemical damage includes oxidativeevents by which retinal cells die by apop-tosis in response to photooxidative injury.In the absence of a suitable in vitro model,many mechanistic examination of lightinduced photooxidative injury were exam-ined by animal experiments. ARPE-19, anon-transformed human diploid retinalpigment epithelial cell line that displaysmany of differentiated properties typical tothe RPE in vivo, exhibit a number of mor-phological and biochemical indications ofdifferentiation but only when cultures areallowed to grow to post-confluent densi-

ties for several weeks. At this stage,ARPE-19 cells express RLBP1 andRPE65, both of which are synthesized bydifferentiated RPE in vivo. In vitro differ-entiated ARPE-19 cells may therefore be asuitable cell model for mechanistic exam-inations of light induced cell damages.With the aid of in vitro differentiatedARPE-19 cells we examined the cytopro-tective role of mitogen-activated proteinkinase phosphatase-1 (MKP-1) of lightdamaged cells. Analysis of gene expres-sion showed, that the expression of proteinphosphates 1 and 8 was significantlyinduced (8- and 5-fold). The results ofmicroarray analysis were verified by west-ern blot analysis using a light dose of 1- 4J/cm2. Low light doses (2 J/cm2) inducedsignificantly MKP-1 expression and inac-

tivated JNK. Phosphorylation of p38 andERK were not altered upon treatment.High light doses (3 and 4 J/cm2) decreasedthe expression of MKP-1 which resulted inactivation of SAPK/JNK. After 24 hrecovery time the high light dose treatmentinduced apoptosis (PARP-cleavage) andincreased cell death. By means of in vitrodifferentiated ARPE-19 cells we coulddemonstrate for the first time that theMAPK phosphatase MKP-1 is capable ofinactivating the stress-inducible proteinkinase (JNK). Inactivation of the JNKpathway inhibits apoptosis and amelio-rated cell viability. The cytoprotectiveproperties of MKP-1 do not appear to bemediated by the p38 or the ERK pathwaybut rather by the JNK pathway in differen-tiated ARPE-19 cells.


Differentiated ARPE-19 can serve an in vitro modelfor the study of light damage to the eyeMohammad Reza Lornejad-Schaefer 0, Hans Konrad Biesalski1, Harald Schoeffl 0 and Juergen Frank0

0 zet – Centre for Alternative and Complementary Methods to Animal Testing (Linz) (AT); 1 University of Hohenheim, Inst. ofBiological Chemistry and Nutrition (Stuttgart) (DE)e-mail: [email protected]

Keywords: cell line, age-related macular degeneration, light damage, cell differentiation

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This paper describes “ethically sourced”animal cadavers and tissue, as defined bythe InterNICHE Policy, and addresses theimportance of using cadavers and tissueonly from these sources when material isneeded for the purpose of education andtraining. The attitudes developed by stu-dents and trainees using ethically sourced

material and conventional sources arecompared and discussed. Examples aregiven where the use of ethically sourcedcadavers and tissue has been successfullyimplemented in practical classes foranatomy and surgery. Potential use forresearch and testing purposes is alsobriefly evaluated. The paper outlines the

potential practical problems of suchcadaver use and offers examples of howthey may be overcome. The impact onveterinary colleges and society of “clientdonation programs” for sourcing animalcadavers is also addressed.

The small intestine is the place of diges-tion. Oral applied substances are takenup over the intestine epithelium into theblood vessel system. The evaluation of

different substances regarding their toxi-city and absorption is mainly still madeby the LD50 via the use of animal exper-iments. Our aim is their limitation by the

development of a 3D physiologicalintestinal test system. The matrix used inour intestine module consists of aporcine jejunal segment with an obtained


Ethically sourced animal cadavers and tissue:considerations for education and trainingSiri Martinsen0 and Nick Jukes1

0 InterNICHE Norway (Oslo) (NO); 1 InterNICHE (Leicester) (GB)e-mail: [email protected]

Keywords: InterNICHE, ethically sourced, cadavers, tissue, Policy, education, training, replacement

The InterNICHE Policy on the Use ofAnimals and Alternatives in Education isa comprehensive document in 10 sec-tions that addresses all aspects of workwith animals and alternatives in life sci-ence education and training. The Policypresents guidelines to ensure effectiveand fully ethical acquisition of knowl-edge and skills. It includes a definition ofalternatives in education and of harm,

and presents individual policies on dis-section, the sourcing of animal cadaversand tissue, work with live animals forclinical skills and surgery training, andfield studies. As well as addressing non-animal alternatives, therefore, it has asignificant focus on the ethical use of,and work with, animals and animal tis-sue. It also addresses the use of animalsfor the production of alternatives them-

selves. The Policy demonstrates the pos-sibilities for full replacement of harmfulanimal use in education and training.Examples from across the world of prac-tical classes that accord with the Policywill be given. Recommendations willalso be made for ethics committees, foruniversity policy towards student choice,and for legislation.


From Policy to Practice: illustrating the viability of full replacementSiri Martinsen0 and Nick Jukes1

0 InterNICHE Norway (Oslo) (NO); 1 InterNICHE (Leicester) (GB)e-mail: [email protected]


Development of a physiological three-dimensionalintestinal test systemJacqueline MichaelisFraunhofer Institute for Interfacial Engineering and Biotechnology (Stuttgart) (DE)e-mail: [email protected]

Keywords: InterNICHE, Policy, guidelines, education, training, implementation, replacement

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Keywords: intestinal test system, drug absorption, vascularised matrices

Keywords: non-invasive imaging, fMRI, heat, rat, image processing

vascular system. The vascular system isreseeded with primary microvascularendothelial cells and the lumen of thematrix with primary enterocytes or cellswith similar characteristics (e.g. Caco-2cells). The intestine model is cultivatedin a bioreactor system under arterial per-fusion with growth medium simulatingphysiological conditions. The absorptionof different substances into the bloodvessel system can be experimentallydetermined through the descending

venous branch. The cell characterisationand the proof of epithelial and endothe-lial cell monolayers takes place by histo-logical and immunohistological analysis.The evaluation of the epithelial barrierfunction and other qualification parame-ters are to be established in the future bythe use of different substances viaHPLC, ELISA and photometric methods.In preliminary tests Caco-2 cells andendo-thelial cells could be successfullyco-cultivated during a 14-day period on

the matrix. First absorption studies ofpeptides on a native small intestine seg-ment were compared to our system. Ouraim is the establishment of a physiologi-cal 3D intestinal in vitro test system. Bythe complexity of our test system anadjustment to the human body over phar-macokinetic models is quite conceivable.Oral bioavailability of drugs and activeingredients could be tested due to thegiven blood vessel structures in our testsystem.


New animal model for objective pain research: non-invasive functional imaging in anesthetized animals by BOLD fMRINicole Jennifer Motzkus0, Marina Sergejeva 1, Lubos Budinsky0, Kay Brune0 and Andreas Hess1

0 Doerenkamp Professorship, Innovations in Animal and Consumer Protection, FAU Erlangen-Nuremberg (Erlangen) (DE); 1 Institute of Pharmacology, FAU Erlangen-Nuremberg (Erlangen) (DE)e-mail: [email protected]

The experience of acute pain is an elementary sensation necessary for main-taining individual integrity and well-being in interaction with theenvironment. However, repetitive nox-ious input can induce chronic pain stateswithout any biological advantage.Chronic pain is getting a health problemof increasing importance (prevalence ofchronic low back pain, migraine) nowa-days. Traditional behavioural pain exam-inations in animals are highly stressfuland subjective. Contributing to the 3Rsnon-invasive imaging approaches likefMRI in anesthetized animals would sig-nificantly reduce the stress for animalsand simultaneously refine and improveobjective measurements of pain withoutthe need of a behavioural readout. Having

established a model for acute mild pain infully anesthetized animals we now couldshow, that this model can be extended toinvestigate repetitive measurements.Moreover, optimized data-analysis strate-gies help to reduce the number of experi-ments needed to obtain statisticallysignificant activation maps. We couldshow that reliable and quantifiable BOLDresponses in pain related brain areas (e.g.thalamus, somatosensory cortex, cingu-late cortex, hypothalamus, entorhinal cor-tex, hippocampus, amygdala) nicelycomparable to human studies (Valet et al.,2006) , were activated during the differ-ent sessions. In conclusion, our computercontrolled topical repetitive heat stimula-tion of the rat hind paw is a robust stimu-lation paradigm leading to reliable BOLD

activation of sensory and especially painrelated pathways. It is well suited forrepetitive stimulations. The results can bequantified with respect to brain area, size,and intensity in relation to the tempera-ture applied. Therefore, this non-invasiveanimal pain model at minimal animalstress level due to the anaesthesia of theanimal is highly objective and well quali-fied for studying chronification of painresponses.

ReferenceValet, M., Sprenger, T., Boecker, H. et al.

(2006). Repetetive pain exposure:Neuronal correlates in the humanbrain. Proceedings of the 11th WorldCongress on Pain, 431-438.

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Since the year 2000, there has been a strongawakening to the concept of the 3Rs amongstacademic and bureaucratic circles responsi-ble for curricula preparation and revisions insecondary/graduate education in life sciencesand in the fields of pharmacology, medicineand veterinary education in India. In a coun-try as vast as India, with thousands of educa-tional institutions, every policy change,effectively saves the lives of millions of ani-mals. In the year 2000-01 the Indian SchoolCertificate Board of Education and CentralBoard of Secondary Education, deleted allanimal dissection in higher secondary educa-tion. In 2003, the Pharmacy Council of India,

decided to include computer aided learninginstead of all pharmacological applicationsusing live animals in about 500 pharmacycolleges across India. In 2006, the UniversityGrants Commission, of the Government ofIndia, issued a directive to all universitiesteaching zoology in graduate and post gradu-ate courses, to implement available alterna-tives and other meaningful exercises toreplace animal dissection. In 2007, theMedical Council of India has proposed acomplete ban in dissection in the teaching ofClinical Pharmacology in medical schools inIndia, which will become effective by theend of this year. The changes have been rela-

tively slower in veterinary education.However, there have been significantchanges in that the Veterinary Council OfIndia has directed that lesser number of ani-mals should be used in anatomy teaching andsupplemented by computer aided learning.Clinical cases now replace the use of experi-mental animals in veterinary surgery teach-ing. These changes over the years have beenlargely due to the efforts of People forAnimals (PFA), Committee for the Purposeof Control & Supervision of Experiments onAnimals (CPCSEA) and the InternationalCenter for Alternatives in Research &Education (ICARE).

Not only after the introduction of the EUchemicals policy REACH, the investiga-tion of dermal and transdermal drugabsorption is of great interest for the cos-metic and pharmaceutical industries, aswell as for governmental institutions. Thenagain, a reduction of the number of in vitroand in vivo experiments is desirable fromthe point of view of animal protection anddue to economic and legislative con-straints. This work addresses this issue andpresents a framework for the simulation ofdiffusion through human stratum corneum:The mathematical model is based on abrick and mortar geometry for modellingthe stratum corneum, which is extended byan additional compartment representing

the more permeable deeper skin layers.Transport through this skin model isdescribed by time-dependent diffusionequations. Together with transmission con-ditions, the interfaces and appropriateboundary and initial conditions, thisdescribes a partial differential equation,which is solved numerically. The secondclosely interwoven component is theexperimental setup, which allows the deter-mination of input parameters for the math-ematical model. A database of theseparameters was collected for one lipophilicand one hydrophilic substance, namelyflufenamic acid and caffeine. Two differentexperimental setups were considered.While the first is an a priori approach, the

latter one is based on an a posteriori anal-ysis. The validation of the model is provenby tape stripping experiments, which areperformed following standard procedures.This allows a resolution of the diffusionprocess in space and time. The study is oneof the first studies to present a comparisonbetween simulation and in vitro diffusionexperiment. The diffusion coefficient in thecorneocytes is shown to be a criticalparameter in the model. The simulatedconcentration depth profiles obtained usingexperimental input data show good agree-ments with the experimental ones. Thefuture work will include enlarging the setof substances as well as studying the effectof keratin binding.


Alternatives changing the face of education in IndiaM. Iris Hermanns 0, Peter Dubruel 1, Chiara Uboldi 0, Etienne Schacht 1 and James Kirkpatrick 0

I-Care (Chennai) (IN)e-mail: [email protected]

Keywords: alternatives, education, India

Keywords: stratum corneum, skin, tape stripping, numerical simulation

Poster: free communication

In silico model of skin penetration based onexperimentally determined input parametersArne Naegel 0, Steffi Hansen1, Dirk Neumann1, Claus-Michael Lehr 1, Ulrich Schäfer 1, Gabriel Wittum0 and Michael Heisig 0

0 University of Heidelberg (Heidelberg) (DE); 1 Saarland University (Saarbruecken) (DE)e-mail: [email protected]

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In the application under article 8 paragraph 1Tierschutzgesetz (TierSchG) the ethical justi-fication must set out scientific evidently. Thisdemand is written down in article 7 para-graph 3 TierSchG. Attempts to refuse exper-iments on animals on the basis of ethicalreasons failed until 2002 because the respon-sible authority was only entitled to review theexplanation of the applicant with regard toplausibility. This opinion has been declaredclearly by the Administrative Court of Berlinin connection with the refused experimentson monkeys in the year 1994. The experi-ments had to be approved. Finally, in the year2002 animal welfare could be established asaim of state in article 20a of the constitution.From now on parliamentary questions andinquiries of animal welfare organizationsreached the competent authority at regular

intervals. They want to know, why the estab-lishment of animal welfare in the constitutiondoes not result in dramatically reduced ani-mal experiments or reported animals used inprocedures, respectively. Other frequentlyasked questions are how the application pro-cedure changed since then and whether moreexperiments on animals are rejected now.Seeing that especially in Berlin the yearlyreported numbers of animals increase and theanswers to the Parliamentary questionsremained unsatisfactory the Parliament ofBerlin passed the resolution to extent thecommission under article 15 TierSchG (com-mission which assist the responsible author-ity whether to authorize experiments onanimals) by including moral philosopher.This request was translated into action inSeptember 2005. Three proven moral

philosopher specialized in animal ethic areappointed to the commission as additionalconsultant on the recommendation of the ani-mal welfare organizations. Since then thecommission has seven regular members eachwith two deputies. The first experience hasshown that the particular knowledge of thephilosophers is only needed in individualcases. This could be shown exemplarily inconnection with an application concerningexperiments on monkeys, which was rejectedon the basis of ethical reasons in 2006 provenin detail through the moral philosophers. Theapplicant abstained from his possibility toinstitute proceedings against the responsibleauthority. A lot of questions remained openlike the ethical limits of action and the abso-lute limit of suffering for animals.

Poster: ethical and legal aspects in animal experimentation

Ethical justification concerning the application under article 8 paragraph 1 of the German animal welfare act (TierSchG) after including animalwelfare in the constitutionHeidemarie RatschLandesamt für Gesundheit und Soziales Berlin (Berlin) (DE)e-mail: [email protected]

Keywords: animal welfare, ethical justification, application

In 1991, the European Community devel-oped a regulatory framework, Directive91/414/EEC defining strict rules for theauthorisation of plant protection products(PPPs). The Directive requires very exten-sive risk assessments for effects on healthand environment to be carried out, before aPPP can be placed on the market and used.The use of pesticides is recognised as posingthreats both to human health and the envi-

ronment. In order to address these concerns,the European Commission adopted a newstrategy aimed at improving the way pesti-cides are used across the EU.

On 12 July 2006 the Commission pub-lished a proposal for a Regulation on PPPs(COM(2006) 388 Final). Although the pro-posal requires producers to share data fromanimal tests in order to avoid duplicate animaltesting, much more than that is necessary to

ensure that animal testing under thisRegulation is only undertaken as a last resort.There is no mention of a need or objective tominimise animal testing, ensuring access torelevant information to avoid duplicate ani-mal testing, or the promotion and use of non-animal test methods and intelligent testingstrategies. It is important that when theRegulation and its annexes are finally adoptedthat these objectives are fully integrated.

Lecture: revision of agrochemicals directive

Animal welfare and the revision of EU legislation on plant protection productsKirsty ReidEurogroup for Animals (Brussels) (BE)e-mail: [email protected]

Keywords: animal welfare, plant protection products, data sharing, alternative methods

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Since the adoption of the Directive86/609/EEC in 1986 important progresshas been made in science and new tech-niques. Additionally, the demand of thepublic for a better protection of laboratoryanimals increased as shown by a recentinternet consultation of the EuropeanCommission. Therefore, a revision of the

legislation is urgently needed. Since 2002the work to revise the text began and thefirst draft is expected in autumn 2007. Totruly meet the goal to protect laboratoryanimals essential improvements are neces-sary. The scope of the Directive should beextended to cover all scientific procedurescovering basic research and education as

well as to include certain invertebrates andembryonic forms. Due to ethical reasonsthe use of non-human primates should beprohibited. An effective authorisation pro-cedure for all procedures using animalsshould be established. This should includea harm/benefit assessment and a retrospec-tive analysis of all animal experimental

Lecture: revision of directive 86/609/EEC

Revision of directive 86/609: demands from theanimal welfare point of view Irmela Ruhdel and Roman KolarDeutscher Tierschutzbund / German Animal Welfare Federation (Neubiberg) (DE)e-mail: [email protected]

The START-UP project is concerned withthe identification and proposals to abolishbottlenecks in the 3Rs approach in phar-maceutical discovery and development.The goal of the project is the organisationof 3 workshops in order to determine a)the state of the art of each of the 3Rs inthe EU, b) to assess European strengthand gaps in 3Rs and c) the identificationof rate limiting steps on the political, sci-entific, technological level. As a result, aConsensus Paper containing the conceptsand suggestions for a Roadmap for futureresearch will be produced. Stakeholders(among them European PharmaceuticalIndustries (EPI)) have identified bottle-necks in drug development and in theintegration of in vitro methods. Earlyidentification of wrong candidates forfurther development and avoiding effortsfor under-performing candidates, areessential for the competitiveness of

European Industry. Identification of bot-tlenecks in the implementation of reduc-tion, refinement and replacement ofanimal experimentation in drug R&D,should assist in identifying the best invitro and in vivo systems, and to speed upthe drug development process. Existinghurdles in the scientific, technological,political and environmental level (includ-ing regulatory), play a substantial roleand are rate-limiting in developing newdrugs, including biological entities(almost 50% of the currently developedproducts). ecopa (the quadripartiteumbrella NGO for alternatives) structureswith its VUB partner this support actionaround 3 major workshops which will bepreceded by 2 Expert Meetings redefin-ing and prioritising current bottlenecks in3Rs methodology; with EPI, drug discov-ery and development. Each phase has itsown specific needs, and analysing the

present limitations and gaps needs to beaddressed, e.g., many cell systems do notyet have the required stability forgenomics, proteomics or metabonomicsanalysis; many current in vitro cell sys-tems lack crucial bioactivation capability.Consequently, the status of satisfactory“predictive” pharmacology and toxicol-ogy in vitro has not yet been reached. Interms of politics and ethical concerns,considerable differences in regard to theuse and development of transgenic ani-mals, human tissues and stem cells createan atmosphere of insecurity for an effec-tive academia and industry cooperation.The final goal of this action is aConsensus Document that analyses pre-sent status and possible solutions. Basedthereon, a Road Map to implement thestrategy for a better integration of 3Rs inthe EU drug discovery and developmentstrategy will be proposed.

Keywords: 3R methology, bottlenecks, politics, ethical concerns, consensus document


ecopa's EU FP7 project START-UP: Scientific and technological issues in 3Rs alternatives research in the process of drugdevelopment and Union politics Vera Rogiers, Bernward Garthoff and Jose Castell European Consensus Platform for 3R-Alternatives (ecopa), Belgium. e-mail: [email protected]

Keywords: EU legislation, animal experiments, scope, primates, authorisation procedure, 3Rs

Keywords: human hepatocytes, acetaminophen, electron microscopy, glycogen, rough ER, smooth ER

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projects. There should be a system oflocal, regional and/or national ethics com-mittees in which animal welfare specialistsand lay persons have to be incorporated.Procedures causing severe suffering to ani-mals should not be permitted. Greatertransparency on animal use is essential. Amain focus should be put on the impor-tance of implementing replacement andreduction methods to the fullest extent.

Current standards of housing and careshould completely and immediately bereplaced with those agreed in Appendix Aof the European Convention ETS 123 aslegally binding minimum standards.Nevertheless, the German Animal WelfareFederation is of the opinion that a signifi-cant reduction of the numbers of animalexperiments performed in the EuropeanUnion can only be obtained if the revision

of Directive 86/609 is taken as an opportu-nity to implement a paradigm shift sanc-tioning animal experiments. There shouldbe a general ban on inflicting pain, suffer-ing or damage to animals in the sector ofanimal experimentation. Exceptions fromsuch a ban could then be regulated accord-ing to the specific provisions of a revisedDirective 86/609.

Human hepatocytes are the in vitro sys-tem of choice to study drug-induced pro-cesses in man. We have developed andvalidated HEPAC2: a serum-free long-term culture system for human hepato-cytes. Cellular viability and liverfunctions (urea and albumin production)were monitored daily. These functionsremained relatively constant for up to 3weeks. We used HEPAC2 to study theeffects of repetitive drug treatments onhepatocellular functions and morphology.Acetaminophen (APAP) was used as amodel substance. Hepatocytes wereexposed to 18.6 mM APAP for 24 h.

Subsequently, culture medium wasreplaced by medium without APAP andthe same exposure scenario was repeatedevery 4 days. During APAP treatmenturea and albumin secretion werereversible reduced by 15-30% and 70-80%. Cytochrome P450 2E1 and 1A2were active for at least 3 weeks, since cel-lular response to APAP did not changeduring the first 4-5 cycles of exposure toAPAP. Electron microscopy revealed thatAPAP led to a complete replacement ofrough ER by smooth ER and degradationof glycogen. After removal of APAP, hep-atocytes refilled their glycogen stores

within 1 day, while it took about 2-3 daysfor complete regeneration of rough ER.Following the exposure protocol, thesame ultrastructural changes were alsofound after the 3rd and 5th treatment ofhuman hepatocytes with 18.6 mM APAP.Metabolism of APAP via glucuronidationand sulfation was analyzed by HPLC.Our data demonstrate the suitability ofHEPAC2 to serve as a tool for repetitivescreening of drug-mediated changes onhepatocyte functions. Furthermore, itmay help to overcome the sparse avail-ability of human hepatocytes for testingdrug-mediated responses in man.


A human hepatocyte long-term culture system servesas a drug testing alternative to animal models:results of metabolic assays and electron microscopy Dieter Runge0, Donna Beer Stolz 1, Ewa C. Ellis 2, Stephen C. Strom2, George K. Michalopoulos2,Christine Berg0, Jan G. Hengstler 3 and Anett Ullrich0

0 PRIMACYT GmbH (Schwerin) (DE); 1 Dept. of Cell Biology, University of Pittsburgh (Pittsburgh) (US); 2 Dept. of Pathology,University of Pittsburgh (Pittsburgh) (US); 3 4Leibniz Research Center for Working Environment and Human Factors (Dortmund) (DE)e-mail: [email protected]

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The aim of this initiative is to understandhow metabolism of ingredients in in vitromodels compares with metabolism inhuman skin, in order to develop appropri-ate alternative testing strategies for topically applied cosmetic/toiletry ingre-dients. New in vitro models for assessingskin sensitisation and genotoxicity poten-tial should be characterised metabolicallyin order that any data generated can beinterpreted in the context of human riskassessment. Two key questions are

addressed here: Qu 1 “Is a chemicalingredient metabolised within the skinupon exposure?” Ingredients could beapplied to ex vivo human skin, or wecould use “simpler” in vitro models (e.g.3D skin model or keratinocytes) tometabolically profile the ingredient. Suchapproaches would provide “mechanismof action” data for risk assessment. Qu 2“Are in vitro models (used in predictiveassays) metabolically competent in anappropriate way to allow an accurate pre-

diction of potential toxicity?” Here, itwill be necessary to determine themetabolic capacity of in vitro models toassess if they are sufficiently representa-tive of metabolism in human skin.Enzyme expression (protein) profiles andfunctional activity (i.e. whether enzymesactively biotransform chemicals) arebeing investigated in a range of models.

Work funded by the EuropeanCosmetics, Toiletries and PerfumeriesAssociation (COLIPA)


COLIPA skin metabolism project towards the inclusionof appropriate metabolism in in vitro alternatives forskin sensitisation and genotoxicityKarsten Ruwiedel0, Camilla K. Pease1, Ellen Fritsche0, Robert J. Edwards 2, Paul Carmichael 1, Pierre Aeby3 and Carsten Goebel 4

0 Department of Molecular Toxicology, Institute for Environmental Health Research (IUF gGmbH), Heinrich-Heine-UniversityDuesseldorf (Duesseldorf) (DE); 1 Unilever, Safety & Environmental Assurance Centre (Sharnbrook) (GB); 2 Imperial CollegeLondon, Hammersmith Campus (London) (GB); 3 The Procter and Gamble Company, Wella-Cosmital (Marly) (CH); 4 The Procter andGamble Company, Wella Service GmbH (Darmstadt) (DE)e-mail: [email protected]

Keywords: skin, metabolism, in vitro, sensitisation, genotoxicity

There is an increased awareness thatmarine biotoxins can have a seriousadverse effect on public health and theeconomy. Many of these biotoxins areproduced by phytoplankton that are sub-sequently ingested and thus accumulatedby shellfish. As a result there is a regula-tory requirement for routine testing. TheEU aims to replace the existing biologicaltest method, the in vivo mouse bioassay,with alternative methods. Our work hasfocused on the marine biotoxin aza-

spiracid-1 (AZA-1) and the developmentof an in vitro assay to assess AZA-1 tox-icity. AZA-1 is found in shellfish and isresponsible for gastrointestinal distur-bances in humans. Therefore, in order tomimic the in vivo situation the humancolonic cell line Caco-2 was cultured onmicroporous membrane supports. Thisenabled us to examine the impact ofAZA-1 on barrier function, as measuredby transepithelial electrical resistance(TER). TER is a useful index of the func-

tion of these cells in maintaining thetransport of solutes and water. Significantreductions in TER were detected at 5, 10and 100 nM AZA-1 at all time-pointsexamined. This decrease in TER corre-sponded with increased permeability ofthe epithelial barrier. This resultprompted us to examine the componentsof the tight junction, namely members ofthe claudin family. Alterations in claudinfamily members have been reported inaffecting the level of tight junction


Development of a functional in vitrobioassay for the marine biotoxin azaspiracid (AZA)using human colonic epithelial cellsGavin E. Ryan, Kevin Cunningham and Michael P. RyanUniversity College Dublin (Dublin) (IE)e-mail: [email protected]

H4IIE hepatoma cells exposed toanisoosmotic media are an alternative toanimal testing studying osmotically regulated hepatic gene expression. Theuse of cultured H4IIE cells exposed toanisoosmotic media is an experimentalalternative for animal experiments tostudy the dependence of hepatic geneexpression and metabolism on cell hydration. The in vitro H4IIE modelavoids confounding variables possiblycaused by secondary effects of high saltanimal treatment. Betaine-HomocysteineS-Methyltransferase (BHMT, EC 2.1.1.5)catalyzes a methyl transfer from betaineto homocysteine to form dimethylglycineand methionine. BHMT is expressed athigh levels in livers of humans, rats andguinea pigs, and its activity is largely reg-ulated at the expression level. BHMTactivity becomes essential when folate-dependent remethylation is impaired due

to nutritional deficiencies or geneticdefects. In mouse models of homocystin-uria, hyperhomocysteinemia and betainedepletion were associated with fatty liver,which was preventable by betaine supple-mentation. BHMT gene expression isseverely decreased in rat liver cirrhosis,providing more evidence for an associa-tion between disrupted betainemetabolism and liver disease. Betainealso is an osmoprotectant that accumu-lates intracellular under conditions ofhigh extracellular osmolarity. Cell hydra-tion changes induced by anisoosmolaritycritically affect liver metabolism andgene expression. The BHMT expressionand activity were decreased in liver ofNaCl-loaded guinea pigs (Delgado-Reyes, 2005). In the course of geneexpression studies using nylon cDNA-arrays we found that hyperosmolarity(405 mosmol/l) suppressed the Bhmt

mRNA expression in H4IIE rat hepatomacells. This was confirmed by Northernblot analysis, which in addition unraveleda pronounced induction of Bhmt mRNAexpression by hypoosmotic (205mosmol/l) swelling. Osmotic regulationof Bhmt mRNA expression was largelyparalleled at the levels of BHMT proteinand enzymatic activity. It may be part ofa cell volume-regulatory response andadditionally lead to metabolic alterationsthat depend on the availability of betaine-derived methyl groups.

ReferenceDelgado-Reyes, C. V., Garrow, T. A.

(2005). High sodium chloride intakedecreases betaine-homocysteine S-methyltransferase expression in guineapig liver and kidney. Am. J. Physiol.Regul. Integr. Comp. Physiol. 288(1),R182-187.


H4IIE hepatoma cells exposed to anisoosmotic mediaare an alternative to animal testing studyingosmotically regulated hepatic gene expression Christine Schäfer 0, Mohammad Reza Lornejad-Schäfer 0, Jürgen Frank 0, Harald Schöffl 0, Lars Hoffmann 1, Thor Gehrmann 2, Dieter Häussinger 2, Ertan Mayatepek1,Bernd Schwahn 1 and Freimut Schliess 2

0 zet – Centre for Alternative and Complementary Methods to Animal Testing (Linz) (AT); 1 Heinrich-Heine-University, Clinic for General Pediatrics (Düsseldorf) (DE); 2 Heinrich-Heine-University, Clinic for Gastroenterology, Hepatology, andInfectiology (Düsseldorf) (DE)e-mail: [email protected]

Keywords: BHMT, hepatoma cell, cell volume, osmoregulation

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integrity and thus barrier permeability.Western blotting analysis showed thatAZA-1 induced a significant dose-depen-dent increase in claudin-2 expression inboth cytosolic and membrane fractions,indicating an alteration in intracellularprotein localisation. Another member ofthe claudin family, claudin-4, increasedin the cytosolic fraction in response to

AZA-1. In contrast, claudin-3 proteinlevels were decreased in the membranefraction. This alteration of claudin levelswas confirmed to be in part due to sig-nalling via the MAPK pathway, ERK 1/2.The p38 and JNK/SAPK signalling path-ways were also activated although no rolein the regulation of barrier permeabilitywas observed. These results suggest that

AZA-1 is capable of disrupting theintestinal barrier via alterations in tightjunction protein expression. This assayhas proved to be sensitive for detection ofAZA-1 and could prove to be a usefultool in detecting known and unknownmarine biotoxins.

Keywords: azaspiracid, bioassay, claudin, tight junction

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Keywords: fish cell lines, fish embryo, regulatory testing, ecotoxicology, fish tests

The liver is the largest internal organ re-sponsible for the metabolism. Therefore,it represents an interesting analysis sys-tem for substances and active agents inpharmacy, chemistry and cosmetics. Ex-isting in vitro liver test systems are veryartificial and no real alternative to animalexperiments. The used cells show only asmall part of their biological reactions invivo. Essential for hepatocyte (HC) vital-ity and function in in vitro longterm cul-ture are 1) a vascularisation to guaranteetransport of nutrients, metabolites andgases, 2) extracellular matrix (ECM) con-tact and 3) the co-culture with endothelialcells (EC). ECs of the liver build up a fil-tration barrier between blood and HC,and are involved in the control of impor-

tant metabolic processes. We created avascularised matrix for longterm co-cul-ture of HC and EC. Basis is a porcine je-junal segment with an obtained vascularsystem, which is chemically acellu-larised. Hepatocytes and endothelial pro-genitors are isolated from porcine biop-sies. Firstly the vascular system of thematrix is reseeded with progenitor cells.In the second step hepatocytes in colla-gengel suspension are seeded on the ma-trix lumen. During the cultivation periodthe matrix is perfused with medium overthe artery in a bioreactor system simulat-ing physiological blood flow. The cultureof hepatocytes on the matrix shows goodresults for cell growth and conservationof liver specific functions. The hepato-

cytes synthesise albumin and urea duringthe whole cultivation period. Phase I andII metabolism of dextrometorphan couldbe shown over 21 days. Immunohisto-chemical stainings demonstrate HC pro-liferation, receipt of differentiation andthe formation of tight and adhering junc-tions between the cells. EC precursorcells were seeded successfully in the vas-cular bed of the matrix. After 3 weeks ofculture the cells express endothelial spe-cific markers. Our liver module can beused to analyse substances and activeagents and to examine regeneration pro-cesses or cell to cell interactions. At theconclusion of the work, the goal is thetransfer of the results to a human model.


A vascularised liver cell module as an alternative to animal experimentsJohanna Schanz, Kirstin Linke and Heike MertschingFraunhofer Institute for Interfacial Engineering and Biotechnolgy (Stuttgart) (DE)e-mail: [email protected]

Keywords: liver module, metabolism analyses, vascularisation

Ecotoxicological risk assessment relies,to a significant part, on the evaluation ofchemicals and effluents in fish. Examplesof regulatory tests include the fish acutetoxicity test (OECD 203) and – as achronic test – the early life stage toxicitytest (OECD 210). In fact, fish are themost widely used vertebrates in environ-mental risk assessment. Therefore,according to the 3Rs, a refinement orreplacement of fish tests is highly desir-able. In light of this, we work toward theadvancement of fish cell line assays as

well as fish embryo tests so that they canbe used instead of fish. Two strategies arebeing followed. The first is to systemati-cally explore an array of fish cell lines fortheir functional abilities and most sensi-tive set-up to indicate acute toxicity tofish. One very promising cell line is therainbow trout gill model, RTgill-W1,which has previously been shown capableof predicting the toxicity of paper milleffluents to fish. The second strategy is toexplore the expression of genes inembryos of zebrafish as predictors of pro-

longed or chronic toxic effects. Focushere is on robust genes that are expressedupon chemical exposure in embryos andlater life stages and on the functionalcharacterisation of the role of such genesin the expression or prevention of toxic-ity. An example is the induction of thegenes that code for the metabolic enzymecytochrome CYP1A and the stress pro-tein HMOX-1. These genes were found tobe induced in the embryo in the samerange of concentrations that elicit toxiceffects in the Early Life Stage Test.

Poster:Lecture: ecotoxicology

Towards replacing or refining fish tests in chemicalregulation and effluent testing – strategies toadvance fish cell lines and embryos as alternativesKristin Schirmer, Katrin Tanneberger, Doris Völker and Stefan ScholzHelmholtz Centre for Environmental Research – UFZ, Department of Cell Toxicology (Leipzig) (DE)e-mail: [email protected]

A new report (2006) from FAO (Food andAgriculture Organization of the UnitedNations) states that livestock production is oneof the major causes of the world's most press-ing environmental problems, including globalwarming, land degradation, air and water pol-lution, and loss of biodiversity. Regarding cli-mate change, FAO estimates that livestock areresponsible for 18% of greenhouse gas emis-sions, a bigger share than that of transport. Theconsensus report (2007) from IPCC’s(Intergovernmental Panel on Climate Change)confirms that global emissions must start tofall within the next 15 years and then be cut toaround half of 1990 levels by 2050 if the worldis to have a fair chance of preventing irre-versible and possibly catastrophic globalchanges. In the same time global production ofmeat is projected to more than double from

229 million tonnes in 1999/2001 to 465 mil-lion tonnes in 2050. The environmental impactper unit of livestock production would have tobe cut down by three quarters to reach IPCC’sgoal. FAO proposes promoting research andextension of cutting edge technology.Improved genetics, for example, could greatlyreduce emissions of gases (carbon dioxide,methane, etc.) and of nutrients per unit of out-put. It is foreseeable that these measures willlead to animal welfare problems in research aswell as in production itself. Traditional meatproduction is based on growing whole organ-isms. The main share of input is lost for main-tenance, heat increment, travel, etc. Only 10%to 25% of metabolizeable energy input pro-duces animal biomass. And depending onspecies only 51% to 78% of animal biomass isuseable for human nutrition. Producing meat

directly through cell cultures could be muchmore efficient. Tissue engineered meat couldhave dramatic environmental advantages.Meat with healthier compositions of fatty acidscould be produced. The biggest usage of ani-mals, which affects an annual number of 50billion individuals worldwide, disregardingwater animals, could be reduced and replaced.The market potential is huge: meat has anannual turnover of 250 billions USD. But inspite of existing research and an existing Dutchpatent, cultured meat is still a visionary topic.The Future-Food-project (www.futurefood.orgwas published in July 2007) aims to accelerateresearch into and development of culturedmeat. It is in contact with various researchersand NGOs and can provide their contactaddresses as well as potential funding contacts(e.g. in the food-industry).


Cultured meat; advantages of manufacturing meatproducts through “tissue-engineering” technologyKurt Schmidinger and Harald BalluchVerein gegen Tierfabriken (Vienna) (AT)e-mail: [email protected]

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Keywords: in vitro, skin sensitization, prediction, gene expression

Up to now, the skin sensitizing capacity ofchemicals has been investigated using invivo animal tests. Due to the ethical and eco-nomical burden associated with research onanimals, the urge for a validated in vitroalternative is very high. The aim of the pre-sent study was to identify genetic alterationsin human dendritic cells in response to (non-)sensitizing chemicals and to extractrelevant markers for predicting skin sensiti-zation. In animal studies, Langerhans cells

have been identified as potent antigen-pre-senting cells that play a crucial role in thedevelopment of allergic contact dermatitis.We used CD34+ stem cell-derived dendriticcells, isolated from cord blood, as an in vitroalternative for Langerhans cells. The cellswere exposed to chemicals with known(non-) sensitizing properties (nickel sul-phate, dinitrochlorobenzene, oxazolone,eugenol, sodium dodecyl sulphate and ben-zalkonium chloride) for different time peri-

ods. Using microarray analysis, we revealeda set of genes with an expression profile thatis strongly associated with the sensitizingcharacter of the exposure compound. For anumber of genes, this result was confirmedby real-time RT-PCR. The genes selectedhere are valuable candidates to predict thesensitizing potential of different classes ofchemicals and deserve further validationusing a more extended set of (non-)allergicsubstances.


Gene expression profiling in dendritic cells to predict skin sensitizationElke Schoeters0, Nathalie Lambrechts0, Karen Hollanders0, Geert Verheyen0, Inge Nelissen0, An Van Rompay0, Jef Hooyberghs0, Rosette Van Den Heuvel0, Hilda Witters0, Vigor Van Tendeloo1, Zwi Berneman1 and Greet Schoeters0

0 Flemish Institute for Technological Research (VITO), Centre of Expertise in Environmental Toxicology (Mol) (BE); 1 Antwerp University Hospital (UZA), Laboratory of Experimental Hematology (Edegem) (BE)e-mail: [email protected]

Keywords: tissue-engineering, livestock, climate change, pollutio

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Precision cut lung slices (PCLS) offer thedistinctive opportunity to gain insightinto lung morphology and physiologyunder in vitro cell culture conditions.Objective of this project is the develop-ment of an in vitro technique for the

assessment of immune modulating (aller-gic) substances while avoiding animaltesting. This study is part of the EuropeanUnion project SENS-IT-IV. Extraction oflung tissue (mouse) was performeddirectly post mortem to conserve vitality

of the tissue. Lungs were filled and cutwith a special microtome. After prepara-tion of PCLS with a thickness of approx-imately 220 µm slices were cultivated at37°C under cell culture conditions. PCLSwere exposed to respiratory (e.g. ammo-


Mouse precision cut lung slices (PCLS) as alternative for in vivo respiratory toxicology testing:focus on immune modulating LPS and allergensKatherina Sewald, Simone Switalla, Maja Henjakovic, Tibor Veres, Norbert Krug and Armin BraunFraunhofer Institute of Toxicology and Experimental Medicine, Department of Immunology, Allergology and Immunotoxicology(Hannover) (DE)e-mail: [email protected]

Given the significant potential of chemi-cals and drugs to interfere with develop-ment of the nervous system, regulatory testguidelines have been adopted for the pre-diction and assessment of developmentalneurotoxicity (U.S.EPA OPPTS 870.6300and OECD TG 426). However, current invivo test methods are laborious, costly andnecessitate use of high numbers of labora-tory animals. Around 1,000 pups have to be handled in an in vivo DNT study andat least 140-mated dams are used to produce enough pups from different litters available to the tests. Moreover, thestudy design is complex and clear recom-mendations for optimal methodologicalapproaches in DNT studies are lacking. Inaddition, under the REACH program of

the European Commission it is planned toevaluate approximately 30,000 existingchemicals for their toxicological proper-ties. Prediction of developmental neuro-toxic effects is a key feature in thetoxicological profile of a compound. Thissituation will considerably increase thenumber of laboratory animals. Validatedalternative methods for developmentalneurotoxicity testing are not available.Thus, standardized, predictive screens forthe evaluation of developmental neurotox-icity need to be available with the ultimategoal of increased efficiency in terms ofreduced animal use and higher throughputcompared to whole-animal testing usingthe existing guidelines. In a newly estab-lished joint project funded by the German

Ministry for Research and Education ourfinal goal is to develop standardized pre-dictive cell-based in vitro assays for devel-opmental neurotoxicity testing. Differentcomplementary cell models which repre-sent selected developmental stages of thedeveloping brain in vivo will be investi-gated to predict developmental neurotoxi-city in vivo from in vitro data. These cellmodels are (1) embryonic stem cells, (2)human neural progenitor cells, (3) humanteratocarcinoma cells, (4) neuro-sensor-chips. To assess neural developmentmolecular and mechanistic endpoints likedifferentiation, migration, proliferation,apoptosis and analysis of electrophysio-logical data will be established.


The new German BMBF joint project on thedevelopment of predictive in vitro tests for develop-mental neurotoxicity testingAndrea Seiler0, Werner Baumann1, Gerd Bicker2, Ellen Fritsche3, Elke Genschow0, Jan Gimsa1,Katrin Hayess0, Wolfgang Kaufmann4, Martina Klemm5 and Andre Schrattenholz5

0 Federal Institute for Risk Assessment (Berlin) (DE); 1 Universität Rostock, Institut für Biowissenschaften (Rostock) (DE); 2 Stiftung Tierärztliche Hochschule Hannover, Abt. Zellbiologie, Physiologisches Institut (Hannover) (DE); 3 Institut fürumweltmedizinische Forschung (IUF), an der Heinrich-Heine-Universität gGmbH (Duesseldorf) (DE); 4 BASF AG, BASFAktiengesellschaft, GV/TD – Z470 (Ludwigshafen) (DE); 5 ProteoSys AG (Mainz) (DE)e-mail: [email protected]

Keywords: developmental neurotoxicity, in vitro, embryonic stem cells, human neural progenitor cells, human teratocarcinomacells, neuro-sensorchips

Alveolar epithelial cells reside in an airliquid interphase and are responsible formaintaining an efficient gas exchangebetween the blood and alveolar space.Many diseases arise due to the breakdownin this function often involving a severehypoxic component. As with many reduc-tionist approaches designed to study alve-olar dysfunction, technical difficultiesarise when attempts are made to model theprecise effects of altered oxygen tensionon pulmonary cell function when using

standard cell culture techniques. Duemainly to the inherent nature of static cellculture cellular oxygen consumption dur-ing respiration creates oxygen gradientsfrom atmospheric tensions through the liq-uid environment of the culture medium tomuch reduced levels at the surface of thecell. As multiple factors such as cell num-ber and time of culture can influence oxy-gen tension, such reductionist cell culturetechniques are inherently inappropriate forthe study of altered oxygen tension on cel-

lular function. At best experiments con-ducted under static liquid culture arepoorly controllable and highly variable, atworst artifactual. Therefore we have devel-oped a system which can mimic the nor-mal environment in the lung and can alsobe used to alter oxygen tensions. The sys-tem is described in detail here. We antici-pate this system to be useful in hypoxiaregulation studies and in investigations ofoxygen tension on alveolar epithelial celldifferentiation.

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A new approach to study hypoxia in vitroSara Signorelli0, Paul Jennings0, Martin O. Leonard1, Thomas Lechleitner0, Sonia Aydin0 and Walter Pfaller0

0 Institute of Physiology, Dept. of Physiology and Medical Physics, Innsbruck Medical University (Innsbruck) (AT); 1 School ofMedicine and Medical Science, UCD Conway Institute, University College Dublin (Dublin) (IE)e-mail: [email protected]

niumhexachloroplatinate, AHCP), andcontact (e.g. cinnamaldehyde) allergens,to positive (LPS) and negative controls(salicylic acid). Vitality of the lung sliceswas either controlled by measurement ofLDH enzyme activity or live/dead stain-ing using confocal laser scanningmicroscopy (CLSM). Cytokines andchemokines were detected with Luminextechnology. Dendritic cell markersCD11c, MHC class II, CD40 and CD86were investigated in living mouse PCLSin situ using CLSM. Ex vivo incubationof PCLS with LPS induced both changes

in cytokine profile and in expression ofcell surface markers. LPS induced anddexamethasone prevented LPS inducedrelease of cytokines and chemokines suchas interleukin (IL)-5 (180% for LPS to0% for LPS/dexamethasone), IL-1(1550% to 325%), TNF-α (5340% to470%), IL-12 (723% to 170%) andRantes (1060% to 120%) in PCLS.Expression of MHC class II, CD40 andCD11c but not CD86 could be observedin naive untreated PCLS. After incuba-tion of PCLS with LPS exclusively astrong enhancement of MHC class II was

found. Treatment to AHCP increased IL-10 (220%), TNF-α and IL-1 up to 160%and MIP-1β up to 70%. Eotaxin, G-CSFand IL-12 showed increasing amounts upto 75%. Stimulation with cinnamalde-hyde resulted in a significant decrease ofMIP-1β up to 50%, but showed nochanges in expression of other cytokines.Salicylic acid showed compared to AHCPan increase of TNF-α-expression, but e.g.no increase of IL-1α. Thus, the method ofPCLS might provide an in vitro techniqueto predict immune modulating potenciesof inhaled substances.

Keywords: PCLS, LPS, allergens

Keywords: lung, air liquid interphase culture, hypoxia

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The embryonic stem cell test (EST) is ascientifically validated in vitro assay thatwas established to classify compoundswith respect to their embryotoxic poten-tial. This assay assesses the embryotoxicproperties of chemicals by the evaluationof inhibitory effects on differentiation ofcontracting myocardial cells compared tocytotoxic effects on undifferentiatedmurine embryonic stem cells and differ-entiated 3T3 fibroblasts. On the basis oftest data of 27 compounds a biostatistical

prediction model (PM) was developedwhich enables the EST to predict theembryotoxic potential of test chemicalswith an accuracy of 78%. In order to con-tinually improve the predictivity of theEST, we intended to enlarge the data baseof the validated EST by testing additionalchemicals including pharmaceutical sub-stances. In this study, we have testedeight different substances: benzoic acid,d-penicillamine, methyl azoxymethanolacetate, Y-27632, mometasone furoate,

and three pharmaceutical inhouse sub-stances. Different molecular and func-tional endpoints were assessed: detectionof myosin heavy chain expression byflow cytometry analysis, microscopicevaluation of contracting cardiomyocytesand analysis of cytotoxic effects onembryonic stem cells and fibroblasts. Onthe basis of these in vitro endpoints wewere able to correctly predict the embry-otoxic potential in vivo.


Enlargement of the database of the validatedembryonic stem cell test (EST): testing of eightsubstancesBirgitta Slawik, Katrin Hayess, Roland Buesen, Elke Genschow, Anke Visan, Katharina Schlechter, Horst Spielmann and Andrea SeilerFederal Institute for Risk Assessment (BfR) National Center for Documentation and Evaluation of Alternative Methods to AnimalExperiments (ZEBET) (Berlin) (DE)e-mail: [email protected]

Keywords: in vitro, embryotoxicity, embryonic stem cell test, data base enlargement

The Paul-Ehrlich-Institute (PEI) is theFederal Agency for Sera, Vaccines andBlood Products in Germany. During thelast 10 years we have tested and optimiseddifferent Monocyte activation tests(MATs) for the replacement of the rabbitpyrogen test with emphasis on humanwhole blood as monocyte source. Whereason a research lab scale the use of freshlydrawn human whole blood was conve-nient, for routine application (industry,agencies) there are severe obstacles like:safety (blood-born infectious diseases),

standardisation (donor-to-donor varia-tion), availability and legal issues. Basedon our experiences we developed a simplemethod to cryopreserve human wholeblood at -80°C (-80°C freezer). Weachieved storage stability at -80°C up totwo years. Storage in the vapor of liquidnitrogen (-140°C) is possible, and resultsin even longer shelf life. The donatedblood is tested for infectious markersaccording to transfusion regulations.Routinely we pool the blood of 10 donorsbefore cryopreservation in order to aver-

age the donor-to-donor variation. The sus-ceptibility for non-endotoxin pyrogens isdonor-dependent; therefore, we stronglyrecommend the pooling of monocytes.The frozen blood aliquots can be shippedsafe, easy and economic at -80°C on dryice. The cryopreservation procedure couldbe applied successfully on human aphere-sis monocytes, too. The -80°C cryopreser-vation technique offers an easy andconvenient methodology to guarantee asafe, stable and reliable source of mono-cytes for MATs.


Cryopreserved human whole blood (-80°C):development and optimisation of a stable monocytesource for alternative pyrogen tests (Monocyteactivation tests)Ingo Spreitzer, Bettina Löschner, Christina Bache, Christian Schneider, Kay Hanschmann andThomas MontagPaul-Ehrlich-Institut (Federal Agency for Sera and Vaccines) (Langen) (DE)e-mail: [email protected]

Keywords: alternative pyrogen test, monocyte activation test, cryopreserved blood, cryocopreserved apheresis monocytes

In vitro permeation experiments areimportant alternative methods to reducein vivo animal tests for toxicological riskassessment and pharmacological researchexperiments. Although experiments onanimal skin are accepted models, manyunknown influences on permeation ratescomplicate a prediction of permeationthrough human skin based on in vitroexperiments in other species. Therefore,we investigated the epidermal lipid com-position and histological parameters (e.g.

morphology and density of hair follicles,stratum corneum thickness) in dogs, cat-tle, pigs, and rats. To elucidate the influ-ence of these parameters on transdermaldrug transport we studied percutaneouspermeation rates in the four species usingtest substances chosen based on differentlipophilicity and molecular weight (flufe-namic acid, ibuprofen, indomethacine,and salicylic acid). Permeation parame-ters were obtained in Franz diffusion cellswith split skin (thickness 500 µm) over a

period of 30 hours. Epidermal lipids werequantified after extraction with chloro-form-methanol using high-performancethin-layer chromatography. Furthermore,histological parameters were obtainedfrom 8 µm thick vertical and horizontalslices (haematoxylin-eosin staining). Thepermeation experiments showed an equalranking of permeabilities for all sub-stances (rat > cattle > dog > pig).Significant differences were found incholesterol, cholesterol ester, cholesterol


Establishment of a three-dimensional cell culturesystem of the canine endometriumKatharina Stadler 0, Johannes Handler1, Susanne Schönkypl2 and Ingrid Walter0

0 Department for Pathobiology, Veterinary University Vienna (Vienna) (AT); 1 Clinic for Horses, Ludwig-Maximilians-University(Munich) (DE); 2 Small Animal Practice (Vienna) (AT)e-mail: [email protected]


Comparison of percutaneous permeation withepidermal lipid composition and histologicalparameters in four different species Jessica Stahl, Frank Niedor and Frank KietzmannInstitute of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Foundation (Hannover) (DE)e-mail: [email protected]

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Frequently, severe pathological disordersin the adult bitch concern the uterine tract,in particular the uterine glands. Ex-periments on living animals that help to elucidate their pathogenesis should besubstituted by in vitro systems due to ethical reasons and practicability.Conventional monolayer cell culture sys-tems are not appropriate to study this pur-pose as the cells show dedifferentiationand unnatural behaviour. Therefore theaim of our study was to develop a three-dimensional in vitro cell culture systemthat reflects the dynamic processes of thein vivo endometrium relating to morphol-ogy, epithelial cell polarity, secretion pat-terns, proliferation, and expression ofsteroid hormone receptors. Endometrialglands of uteri obtained after routine ovar-iohysterectomy of anestrous dogs (n=24)

were isolated by collagenase-digestionlasting eight hours. Subsequently, theywere cultivated on various extracellularmatrix components (stromal and base-ment membrane) to maintain differenti-ated uterine glands. The cultivation ofuterine gland explants on each matrix wasextended over four days and repeated twotimes. Samples were taken every day for immunohistochemical (cytokeratin,vimentin, laminin, Ki-67, progesteronereceptor, estrogen receptor) and lectin his-tochemical stainings and for electronmicroscopic analysis. The immunohisto-chemical detection of cytokeratin verifiedthe epithelial nature of the explantedstructures, vimentin was used for exclu-sion. Laminin immunostaining showed avarious amount of basement membranecomponents. Using electron microscopy

we could confirm the polarized differenti-ation of the epithelial cells. Ki-67immunostaining showed a low mitoticactivity of the epithelial cells according toanestrous uterine specimens. For the anal-ysis of the secretion patterns of theextracted glands lectin histochemistry wasperformed and demonstrated high confor-mity with the original uterine tissue. Ahigh number of oestrogen receptors wasfound in endometrial and cultured glands,whereas progesterone receptors were low.With the establishment of this three-dimensional cell culture system we cre-ated an analytical tool to mimic thecomplex endometrial architecture. Itshould be possible to transfer this systemto other tissues and species and thereforerepresents an important contribution toavoid animal experiments.

Keywords: endometrium, in vitro, dog

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A cell culture model for the detection of chemical or physical agents with potential pro- or antioxidative properties using 8-hydroxy-2’-deoxyguanosine in cellular DNA as toxicologicalendpointAlois Strasser0, Harald Kühnel0, Günter Marik1 and Ivo Schmerold1

0 Institute of Physiology, Veterinary University of Vienna (Vienna) (AT); 1 Institute of Pharmacology and Toxicology, VeterinaryUniversity of Vienna (Vienna) (AT)e-mail: [email protected]

Oxidative DNA damage is widelyaccepted to be linked to mutation andmalignant transformation in eukaryoticcells. Although in vitro as well as in vivotest systems are in use for the identifica-tion of oxidants there is still a vital needfor feasible in vitro screening systems. Inorder to test their suitability to detectoxidative cytotoxicity we exposed lym-phoblasts to a chemical and a physicalagent both with known pro-oxidativeactivity mediated by the generation ofreactive oxygen species (ROS) and by theformation of 8-hydroxy-2’-deoxyguano-sine (8-OH-dG) in cellular DNA.Commercially available murine lym-phoma cells (YAC-1) were exposed tohydrogen peroxide (H2O2, 1 mM) and

FeCl2 (50 µM) or UV-C-irradiation (peakwavelength 253.7 nm; dose: 6 mJ/min xcm2) for distinct periods of time. In sepa-rate experiments, the free radical scav-enging compound TEMPO (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl)was added (1 mM) in order to demon-strate its protective activity. A HPLC system equipped with UV and electro-chemical detection was employed tomeasure the levels of 8-OH-dG in iso-lated cellular DNA. DNA of untreatedYAC-1 cells contained 5.95 ± 3.06 µmol8-OH-dG/mol guanine. Following expo-sure to H2O2/FeCl2 for 10, 25, 60 or 180min levels of 8-OH-dG increased time-dependently to more than threefold.Exposure to UV-C light also caused a

time-dependent rise in 8-OH-dG by a fac-tor of 40 (60 min) to 100 (360 min). Inboth cases, the addition of TEMPOalmost completely inhibited the increasein concentrations of 8-OH-dG. Based onthese results, YAC-1 cells represent apromising and feasible in vitro system for the detection of DNA oxidants interms of an acute generation of 8-OH-dG.Ongoing investigations aim at the possi-ble oxidative effects of nitroheterocycliccompounds of the nitroimidazole andnitrofuran type and the identification offurther compounds with anti-oxidativeactivity. Also the capability of YAC-1cells to repair oxidative DNA-lesions ispresently under investigation.

Keywords: percutaneous permeation, epidermal lipids, histology, epidermis, stratum corneum, hair follicles, comparative study

Keywords: 8-hydroxy-2’-deoxyguanosine, pro-oxidative, DNA damage, antioxidative, in vitro

sulfate, ceramide 3, galactosylcerebro-sides, and phospholipids. Ceramide 4could only be found in pig skin.Moreover, the histology showed a com-plex variety of results. From the large

number of factors possibly influencingpercutaneous permeability a correlationwas only found to stratum corneum thick-ness (positively correlated with the per-meation rate) and total epidermal lipid

content (inversely correlated with the per-meation rate). As the latter result is con-tradicting to other investigations, furtherstudies will be required to clarify speciesrelated influences on skin permeability.

Transgenic animals, mostly transgenicmice, are important models to study(human) gene function and disease inbiomedical basic research. With the rapidadvances in sequencing the whole humangenome, the great challenge today is toascribe the function of the thousands ofgenes in normal physiology, develop-ment, and disease. One powerfulapproach to decipher the functions ofgenes is the manipulation of genes inintact animals. The production of trans-genic animals that enable to transmit thegenetic modification (gene knock-out,knock-in, or mutation) to their offspringrequires germline transformation. The

most commonly used techniques are: (1)microinjection of the transgene DNA intothe pronucleus of fertilized eggs, (2)embryonic stem (ES) cell manipulation,and (3) the cre-lox technology for tar-geted homologous recombination. Allmethodologies have success rates below10%. Thus, the process of transgenesis iswasteful and involves the infliction ofpain, distress and suffering of the geneti-cally modified (GM) animals. Besidesthat, the subsequent establishment ofhomozygous transgenic strains fromfounder animals is also a wasteful pro-cess, generating a large number of sur-plus animals, for which the strange term

“waste animals” is used in the literature.A careful mangement of breedingcolonies is mandatory in order to reducethese numbers (Robinson et al., 2003).The number of laboratories and compa-nies generating knock-out mice hassteadily increased since invention of thistechnique in the 1980s. At present, about3,000 knock-out strains are available, andthe numbers are growing exponentially(Knight and Abbot., 2002). In the tablebelow, the numbers of transgenic animalsused in basic research in Germany,Switzerland and the UK are listed. Whilethe use of genetically modified animalsincreased dramatically in the last decade,

The aim of our present research is theestablishment of an in vitro test system toreveal the sensitizing potential of chemi-cals. Therefore, we developed the ARCimmune toxicity chip, a DNA microarraycontaining 65 immune genes in addition toa series of housekeeping genes, negativecontrols and normalization controls.Contact sensitizers (nickel, Bandrowski’sbase, sodium-2,4-dinitrobenzenesulfonate,1-chloro-2,4-dinitrobenzene, alpha-hexyl-cinnamaldehyde, hydroquinone, eugenol)and irritants (sodium dodecyl sulphate,methyl salicylate, TritonX-100) wereapplied to immature dendritic cells (iDC)

cultured from human peripheral bloodmonocytes. Gene expression in chemical-treated cells was compared to gene expres-sion in solvent-treated samples. Takinginto account the problem of donor vari-ability, cells from 3 or 4 donors wereexposed to each chemical to identifyimmune genes differentially expressed inall biological replicates. In addition, tran-scriptional changes in dendritic cellsexposed to selected chemicals were ana-lyzed on whole genome microarrays toinvestigate effects on genes not repre-sented on our chip. Real-time PCRs wereperformed to confirm treatment-related

differences in gene expression detected onmicroarrays. Employing the ARC immunetoxicity chip, we could identify severalimmune-relevant genes that were up- ordown-regulated after exposure of iDC tosensitizers, while no significant effect wasdetected after application of irritants.Allergens well-known as strong sensitizersinduced differential expression of a highernumber of genes than treatment with mod-erate and weak allergens. Our results indi-cate that our system could be used as an invitro alternative to animal tests, providedmarker genes not affected by donor vari-ability are employed.


Discrimination of contact allergens and irritantsemploying the ARC immune toxicity chipSandra Szameit0, Klemens Vierlinger0, Letizia Farmer1, Helga Tuschl1 and Christa Nöhammer0

0 Austrian Research Centers GmbH – ARC, Molecular Diagnostics Unit (Seibersdorf) (AT); 1 Austrian Research Centers GmbH –ARC, Toxicology Unit (Seibersdorf) (AT)e-mail: [email protected]


Dramatic increase in numbers of transgenic mice –we must take action now! Tina Tauss0, Harald Schöffl0, Helmut Appl0, Walter Pfaller1 and Gerhard Gstraunthaler2

0 zet – Centre for Alternative and Complementary Methods to Animal Testing (Linz) (AT); 1 zet – Centre for Alternative andComplementary Methods to Animal Testing (Innsbruck) (AT); 2 Department of Physiology and Medical Physics, InnsbruckMedical University (Innsbruck) (AT)e-mail: [email protected]

Keywords: DNA microarrays, dendritic cells, immunotoxicological testing, contact sensitizer

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there was a general decline in the overallnumber of experimental animals, whichis mainly due to following the 3R princi-ples and the invention of alternativemethods. Thus, there is an urgent need toreduce at least the dramatic increase inthe numbers of transgenic mice used inthe EU and overseas. In 1997, anECVAM Workshop on The Use ofTransgenic Animals in the EU was held,and a Workshop Report together with a

series of recommendations was publishedin 1998 (Mepham et al., 1998). Furtherrecommendations on welfare of GM miceand on reduction of animal numbers havebeen published elsewhere (Robinson etal., 2003). Among these it was clearlystated, “that adequate and clear statisticson the numbers, species, types, produc-tion, breeding and uses of transgenic ani-mals should be collected and published atleast bi-annually by each EU Member

State, and by the European Commissionto promote transparency and accountabil-ity” (Mepham et al., 1998). It is the pri-mary aim of the present contribution, toprovide the latest statistics on the num-bers of transgenic animals and to createawareness of this steadily increasingproblem in the scientific community andin the public.

ReferencesKnight, J. and Abbot, A. (2002). Full

house. Nature 417, 785-786. Mepham, T. B. et al. (1998). The use of

transgenic animals in the EuropeanUnion. The report and recommenda-tions of ECVAM Workshop 28. ATLA26, 21-43.

Robinson, V. et al. (2003). Refinementand reduction in production of geneti-cally modified mice. Lab. Anim. 37(Suppl. 1), 1-51.


Use of an alternative test battery to predict ocular irritancy of agrochemicalsJudit Tavaszi, P. Budai and Á. PálovicsUniversity of Pannonia, Georgikon Faculty of Agriculture (Keszthely) (HU)e-mail: [email protected]

Keywords: transgenic animals, animal numbers, animal statistics, animal protection law

Keywords: alternative, HET-CAM, MTT, agrochemicals, eye irritancy

The Draize rabbit eye test, or some modi-fication of this test, is essentially the onlymethod for determining ocular irritationthat is acceptable to the various regulatoryagencies. Several in vitro methods havebeen used to investigate the toxicity of po-tential eye irritant with a view to replacingin vitro eye irritation testing. This studyreports the results of an alternative ap-proach for predicting irritation potential ofagrochemicals. The approach was a two-stage test battery in vitro. The first stagewas a cytotoxicity test, the MTT assay.The second stage was the HET-CAM test.The chick chorioallantoic membrane

(CAM), being a connective tissue sheetwith a visible blood supply, has been pro-posed as a substrate to identify the eye ir-ritation potential of chemicals. During theHET-CAM test the chemicals are placeddirectly onto the chorioallantoic mem-brane. The changes of the vascular injury(haemorrhage, lysis or coagulation) areindications of the potential of the chemi-cal to damage mucous membranes in vivo.MTT assay is a simple method to deter-mine the viability / number of cells in cul-ture, through the formation of a colouredproduct to which the cell membrane is im-permeable. Determination of the ability of

cells to reduce MTT to the formazanproduct after exposure to test compoundsenables the relative toxicity of test chemi-cals to be assessed. In our studies compar-ative screening was performed with 6agrochemicals to establish parallel data onalternative test battery (HET-CAM, MTT)and in vivo (Draize) results. In most cases,this study showed a good correlation be-tween in vitro and in vivo data. By theseresults the present form of alternative testbattery (HET-CAM and MTT) can be pro-posed as a pre-screen method for predict-ing ocular irritancy.

2000 2001 2002 2003 2004 2005 %

Germany: transgenic mice 149,859 200,776 218,072 244,588 302,143 348,399 233 %

Switzerland: transgenic animals 59,000 61,000 68,500 63,500 81,000 94,000 159 %(>99 % mice)

UK: transgenic animals 581,740 630,759 709,979 764,095 914,023 957,451 165 %(>95 % mice)

Nanoparticles are already in our environ-ment; we use them in our cosmetics prod-ucts and medical researchers use them innovel drug delivery systems. Because oftheir small size they have the potential tocross biological barriers and produceexponentially greater effects than theirnon-nano counterparts. There is thereforeincreasing concern about the toxicity ofnanomaterials and increasing pressure on

governments and regulators to ensure thatappropriate testing methods are in place.The European Scientific Committee onEmerging and Newly Identified HealthRisks have recently assessed the appropri-ateness of the current chemical testingguidelines for nanomaterials and, in theUK, DEFRA have proposed a voluntaryreporting scheme for the results of nan-otoxicity tests. Animal protection organi-

sations have an important role to play inhighlighting the unreliability of existinganimal models for the safety testing ofnon-nanomaterials (and by inference theinadequacy of these for nanomaterials)and the ethical objection to increased ani-mal use. The relatively new field of nan-otoxicity is ideally placed to change thecurrent testing paradigm away from ani-mal models. This already appears to be


In vitro techniques and nanotoxicity – the wayforward Katy TaylorBUAV (British Union for the Abolition of Vivisection) (London) (GB)e-mail: [email protected]

In general, azole fungicides have a lowacute toxicity, but we have only littleknowledge about their potential healthrisks at low chronic exposures. Previously,we have shown that prochloraz has multi-ple potential mechanisms of action in cell-based assays, and prochloraz possessedantiestrogenic (Andersen et al., 2002) andantiandrogenic effects both in vitro and invivo (Vinggaard et al., 2002). Two otherazole fungicides, tebuconazole and epoxi-conazole, have now been investigated forantiandrogenic effects in vitro and in vivoas well. The fungicides were screened intwo well-established cell assays, includingtesting for agonistic and antagonisticeffects on AR in transfected CHO cells,using an AR reporter gene assay. The com-pounds were also analyzed for effects onsteroidogenesis in H295R cells, a humanadrenocorticocarcinoma cell line, used todetect effects on steroid production. Invitro tebuconazole and epoxiconazole

proved to be antagonists of the AR, and inthe H295R cell assay, they were able toinhibit testosterone and estradiol levels,and increase progesterone levels. In an invivo study, designed to test for develop-mental effects on rat offspring after prena-tal exposure, the effects on hormone levelsin male fetuses and morphological signs offeminization of the male offspring wereinvestigated. Tebuconazole caused anincrease in testicular 17alfa-hydroxypro-gesterone and progesterone levels, and adecrease in testosterone levels in malefetuses. Epoxiconazole had no effect onany of the mesured hormonelevels.Furthermore, tebuconazole increased theAGD in female pups and resulted in anincreased number of nipples in male pups,a tendency that was also seen for epoxi-conazole, though it was not statisticallysignificant (Taxvig et al., subm.). In con-clusion the results obtained in vitro are ingood agreement with the effects observed

in vivo. Tebuconazole showed antiandro-genic effects both in vitro and in vivo.Antiandrogenic effects were also seen forepoxiconazole in vitro, however the domi-nating effect observed in vivo was a highfrequency of stillbirths at the highest dose.

ReferencesAndersen, H. R., Vinggaard, A. M.,

Rasmussen, T. H. et al. (2002). Effects ofcurrently used pesticides in assays forestrogenicity, androgenicity, and aro-matase activity in vitro. Toxicol. Appl.Pharmacol. 179(1), 1-12.

Taxvig, C., Hass, U., Axelstad, M. et al.(2007). Endocrine disrupting activities invivo of the fungicides tebuconazole andepoxiconazole. Submitted to Toxicologi-cal Sciences.

Vinggaard, A. M., Nellemann, C.Dalgaard, M. et al. (2002). Antiandro-genic effects in vitro and in vivo of thefungicide prochloraz. Toxicol. Sci. 69(2),344-353.

Poster: endocrine disruptors

In vitro and in vivo screening of azole fungicides for antiandrogenic effects Camilla Taxvig0, Anne Marie Vinggaard1, Ulla Hass1, Marta Axelstad1 and Christine Nellemann1

0 National Food Institute, Technical University of Denmark, (Søborg) (DK); 1 National Food Institute, Technical University ofDenmark, Department of Toxicology and Risk Assessment (Søborg) (DK)e-mail: [email protected]

Keywords: tebuconazole, epoxiconazole, reproductive toxicity, azole fungicides, endocrine disrupters

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happening, with a greater proportion of invitro studies being published. For exam-ple, techniques have been developed toinvestigate the translocation of nanoparti-cles through the blood-brain barrier, therespiratory epithelia and the skin, com-pletely in vitro. Such methods have theadvantage in that they enable not only the

indication of likely cytotoxic effects butthe mechanism by which they might occur(e.g. oxidative stress). In addition, the useof diseased human tissues can identify anylikely enhanced toxicity due to increasedsensitivity and deterioration in cellularand tissue functions, without the addi-tional problems of extrapolation from ani-

mal models. An intelligent or tiered test-ing strategy has been adopted underREACH and can form the basis for a sim-ilar strategic approach to testing nanoma-terials. An example for how these methodsmay fit into a testing strategy for nanotox-icity is offered. In addition, priorities forresearch in the future are discussed.

The European Coalition to End AnimalExperiments (ECEAE) is an alliance ofanimal protection organisations from 17European countries. The revision of thedirective relating to animals used forexperimental and other scientific pur-poses (Directive 86/609/EEC) is anopportunity for increased co-operation

amongst European stakeholders towardsthe achievement of an eventual end toanimal testing in the EU. A primary aimof the ECEAE in this revision is to seekan end to the licensing of experimentsthat use non-human primates. This is pro-posed on both scientific and ethicalgrounds, for which there is considerable

support from members of the public.Secondary aims include seeking a ban onthe licensing of experiments relating towarfare, xenotransplantation, education,tobacco, alcohol and household products.The scope of the Directive should also beextended to cover foetuses, cephalopodsand all possible harmful uses of animals

Keywords: nanotechnology, nanotoxicology, in vitro, non-animal methods, testing strategy

A large proportion of laboratory animal useis in fundamental or basic research (35% inthe EU). This is not carried out in directrelation to the testing of medical productsbut to understand more about the body andhow, and why, it may malfunction. Underthe proposed revision of European legisla-tion for laboratory animals (EEC86/609),each research project may have to undergoa “cost: benefit” assessment prior to autho-risation. In some countries (such as theUK) this already occurs but is done in arather rudimentary manner. Although

under-appreciated, more confidence isplaced on our ability to assess the “cost” toanimal. In contrast, the assessment of the“benefit” of animal research has been littlestudied, with the result that unsubstantiatedclaims to possible improvements to humanhealth may be used in licence applications.This paper critically evaluates how it ispossible to assess the value of fundamentalresearch that uses animals retrospectively,particularly in relation to actual humanhealth advances. A range of techniques aredescribed including assessment of publica-

tion rate, citation analysis and systematicreview. Additional problems that arise withthe assessment of genuinely fundamentalprojects are also discussed. In particular,the possibility that applied benefits may notbe realised for a number of years (if at all)or may be difficult to trace, given the non-linear nature of research and citation. Thisraises the genuine question of whether pro-cedures, in which the cost to the animal canbe predicted quantitatively but the benefitto humans cannot, should logically, be per-missible.


How do we assess the value of fundamentalresearch using animals?Katy TaylorBUAV (British Union for the Abolition of Vivisection) (London) (GB)e-mail: [email protected]

Poster: revision of Directive 86/609/EEC

ECEAE recommendations for the revision ofDirective 86/609/EEC relating to animals in scienceKaty Taylor, Sandra Hannen and Gill LangleyBUAV (British Union for the Abolition of Vivisection) (London) (GB)e-mail: [email protected]

Keywords: ethical review, basic research, citation analysis, systematic review, publication

Keywords: alternatives, EU legislation, animal experiments

Keywords: human breast epithelial cell culture, HER-2 oncogene, growth arrest, apoptosis, cancer prevention

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in laboratories (e.g. tissue supply, educa-tion, breeding). If animal testing is notended by the revision, a number ofimprovements are supported by theECEAE. These include the formalisationof a transparent system of ethical review,including lay and animal protection rep-

resentation. Prospective and retrospectiveassessment by independent experts of theseverity of harm caused to individual ani-mals during their lifetime should also bemandatory. This will allow for a moreaccurate assessment of the harm: benefitof each research project, which currently

underpins its authorisation. Targets forthe replacement of animals used in scien-tific procedures should also be set.Finally, a responsibility on individuals aswell as member states to develop non-animal methods should also be incorpo-rated into the legislation.


Novel cell culture model for prevention of estrogen receptor negative, chemotherapy-resistant breast cancer: an alternative approach for animal experimentationNitin Telang and Meena KatdareStrang Cancer Prevention Center and Weill-Cornell Medical College (New York) (USA)e-mail: [email protected]

In progressive pathogenesis of clinical breastcancer, comedo ductal carcinoma in situ(DCIS) represents a high risk lesion forchemo-endocrine therapy-resistant breastcancer. This preinvasive lesion is character-ized by the presence of HER-2+, EGFR+,p53+, and ER- / PR- cells. Human tissue-derived cell culture models expressing clini-cally relevant genetic defects and exhibitingquantifiable carcinogenic risk should pro-vide a facile paradigm to reduce, refineand/or replace the use of existing preclinicalanimal models involving xenotransplants ofhuman breast cancer cells in athymic mice.

The EGFR+, p53+, and ER-/PR- humanbreast epithelial cells with stable expres-

sion of HER-2 oncogene (184-B5/HERcell line) represented the model for comedoDCIS. Status of cell proliferation, cellcycle progression and cellular apoptosis,represented the modifiable mechanistic endpoint biomarkers.

Aberrantly proliferative preneoplastic184-B5/HER cells exhibited defectivehomeostatic growth control as evidenced byenhanced cell proliferation and down-regu-lated cellular apoptosis. Acute treatment of184-B5/HER cells with synthetic chemo-preventive agent, select dietary phytochem-icals, or their active components/structuralanalogues, either inhibited cell cycle pro-gression, or induced cellular apoptosis, thus

re-establishing homeostatic growth control,predominantly via cytostatic growth arrest.Long-term treatment with these chemopre-ventive test compounds inhibited anchor-age-dependent and anchorage-independentcolony formation, thereby decreasing therisk for carcinogenesis.

These data validate an alternative andcomplementary approach to animal experi-ments for rapid prioritization of dietaryphytochemicals for primary prevention oradjuvant therapy of ER- /HER-2+ breastcancer [Support: NCI CN-5029-63, DOD-BCRP DAMD 17-94-J-4208 and IrvingWeinstein Foundation].

Caco-2 bi-dimensional system allows theevaluation of chemicals ability to crossthe intestinal barrier, as well as to study

their transport mechanism. Several stud-ies, comparing Caco-2 permeability val-ues of a large number of compounds

(mainly drugs) with different physico-chemical characteristics with the corre-sponding in vivo human oral fraction


Towards the validation of Caco-2 cell line as modelof intestinal absorption Valentina Tirelli, Annalaura Stammati and Isabella De Angelis Istituto Superiore di Sanità, Environment and Primary Prevention Dept. (Rome) (IT)e-mail: [email protected]

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The global market of engineered nanopar-ticles is consequent and in perpetualprogress, this exponential proliferation ofnanomaterials may generate a new classof risk on health and environment not onlyfor workers chronically exposed tonanoparticles, but also for population ingeneral indirectly exposed. In order tobetter determine the potential harmfuleffects of nanoparticles on health, a newfield of toxicology defined as nanotoxicol-ogy has emerged worldwide to investigatethe risks of exposition to nanomaterials.The questioning and the awareness ofauthorities, industries and scientific com-munity encourage investigations of thepotential hazards of nanoparticles to avoida similar disaster as with the use of

asbestos which happened in the past. Ifthe literature dealing with nanoparticleshas been exploding during the last decade,there is no sufficient analysis of the toxic-ity of such particles till now due to inade-quate understanding of a very complexreality and the absence of relevant stan-dards. Therefore, additional toxicologicalstudies are necessary to understand inter-actions between nanoparticles and livingcells. The purpose of the Nanotoxico pro-ject managed by an excellence centre con-sists in the toxicological study of threetypes of nanoparticles having a real eco-nomic benefit in Belgium (carbon nan-otubes, exfoliated nanoclays, silicon andtitanium carbides). The multidisciplinaryresearch team including chemists, physi-

cists, biologists, physicians and pharma-cists has five years to develop representa-tive in vitro models using cell culturesmimicking tissues exposed to nanomateri-als. These in vitro tissues models includeskin, lung and intestine, which constitutethe main potential routes of contact withnanoparticles. With this project, a 3Dreconstituted human epidermis (fully dif-ferentiated) and new relevant cell modelsof reconstituted respiratory and gastroin-testinal epithelia (with differentiatedintestinal epithelia absorption cells andFollicle Associated Epithelium) has beendeveloped to characterise the origin of thetoxicity related to nanoparticles using his-tology, cytotoxicity and genotoxicitytests.

absorbed (Fa) values, have shown a goodcorrelation between in vitro and in vivodata. Based on these results, the Caco-2cell model, already extensively used indifferent laboratories to predict the oralabsorption and permeability of differentcompounds in humans, is a good candi-date for its validation for regulatory purposes. Recently, two different inter-laboratory studies have been founded byECVAM: the first one was aimed at eval-uating the best Caco-2 line and/or clone interms of reproducibility and sensitivity.The latter (still in progress) is running intwo different laboratories, with the pur-pose to evaluate the predictive capacity ofthis in vitro model (e.g., sensitivity, speci-

ficity, concordance), that is, its ability topredict unknown oral Fa. An appropriatenumber of compounds, for which goodhuman and/or animal oral absorption dataare available, were chosen as representa-tives of different in vivo response and/oruse category (industrial chemicals anddrugs), and have been tested either on theparental Caco-2 cell line (ATCC-derived)or on the TC7 clone (derived from latepassage of parental Caco-2), in strictlydefined experimental conditions. Ab-sorption of the chemicals through Caco-2monolayer, cultured on insert, is deter-mined in a two-steps procedure: a) thehighest non toxic concentration on cellu-lar barrier is calculated for each com-

pound using 14C-mannitol permeability,in presence of the chemical, as marker ofintegrity; b) the absorption kinetic (influxand efflux direction) is evaluated at theconcentration selected in the previous step and expressed as permeability coeffi-cient (Papp = dQ/dt*1/(A*C0). Resultsobtained in the present study with selectedreference compounds for high, mediumand low absorption (propanolol, cimeti-dine and atenolol respectively) are inagreement with the ranking reported forhuman oral Fa values, and look promisingfor the validation of Caco-2 system. Thisstudy is supported by ISS- ECVAM studycontract CCR.IHCP.C431224.X0


Nanotoxicology: In vitro model development fornanomaterial toxicity assessment on human healthOlivier Toussaint, Sébastien Vankoningsloo, Jean-Pascal Piret, Christelle SaoutResearch Unit on Cellular Biology, Department of Biology, University of Namur (Namur) (BE)e-mail: [email protected]

Keywords: nanoparticles, 3D cell cultures, skin, intestine, lung, histology, cytotoxicity, genotoxicity

Keywords: Caco-2, intestinal absorption, validation

The follicular penetration of two differentliposomal formulations (rigid and flexi-ble) was compared. The critical parame-ters occlusion, body area and humiditywere examined. The Franz Diffusion Cell(FD-C) is an in vitro method often usedfor skin absorption tests. In this study, anew in vitro method was developed tomimic the dermal application of formula-tions with this test system. After donorapplication, human full thickness skinfixed in the FD-C was massaged for 3minutes (female subjects, plasticsurgery). The liposome membranes werelabelled with Lissamine Rhodamine Band the core contained carboxyfluores-

ceine, allowing the analysis of the pene-tration pathway using Confocal LaserScanning Microscopy. The recovery ratewas determined for each skin layer(donor, heat separated epidermis, dermisand receptor). If the humidity was 75%(Organisation for Economic Co-opera-tion and Development Test guideline(OECD TG) 428, skin absorption), it wasnot possible to detect differences betweenocclusion and no occlusion. If the skin isobserved as an entity, no body area dif-ferences were detectable, while signifi-cant differences were found between theskin layers. If the humidity was varied, asignificant influence for the liposomal

formulation was detectable. The influ-ence of the liposomal formulationsdepends on humidity (summer vs. wintersituations in living quarters) as well as thedifferent body areas in the deeper skinlayers (as the follicle act as reservoirs forliposomal dermal applications). The fol-licular pathway was influenced indirectlyby the humidity and the properties of theexamined liposomes.

Remark: Trauer Sindy: Center of Experi-mental and Applied Cutaneous Physiology,Department of Dermatology, Univer-sitätsmedizin Charité and ZEBET at theFederal Institute of Risk Assessment (BfR)


Follicular penetration – in vitro testing of rigid and flexible liposomesSindy Trauer0, Heike Richter1, Rolf Bütemeyer1, Jürgen Lademann1, Michael Linscheid 2, Judith Kuntsche3, Alfred Fahr3 and Manfred Liebsch0

0 ZEBET at the Federal Institute of Risk Assessment (BfR) (Berlin) (DE); 1 Center of Experimental and Applied CutaneousPhysiology, Department of Dermatology, Universitätsmedizin Charité (Berlin) (DE); 2 Institute of Chemistry of the HumboldtUniversity (Berlin) (DE); 3 Institute of Pharmaceutical Technology of the Friedrich-Schiller-University (Jena) (DE)e-mail: [email protected]

Keywords: follicular penetration, in vitro skin absorption test, rigid and flexible liposomes, in vitro massage technique

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Drug efflux transporters like the mul-tidrug resistance protein MDR1 can sig-nificantly affect oral absorption andbioavailability, tissue distribution excre-tion, pharmacodynamics of drugs andtake part in the drug-drug interactions.Bidirectional transport experiments usingcell culture models that mimics the char-acteristics of physiological barriers such

as intestines have shown good perfor-mance for passively transported drugs,but for drug molecules with carrier medi-ated transport there is still a need for anaccurate and repeatable method for theprediction of in vivo absorption and drug-drug interactions. In this study our aim isto establish and pre-validate an in vitrobi-directional cell culture model using

MDCKII, MDCKII-Pgp and Caco-2 cellsfor the prediction of in vivo absorption,drug-drug interactions and pharmacoki-netics of efflux system substrates. In bi-directional transport experimentsdesigned to evaluate the suitability of theCaco-2 model used in this study low,middle and high molecular markers: pro-pranolol, atenolol and mannitol, respec-


Development and pre-validation of an in vitromethod for the prediction of drug-drug interactionsand intestinal absorptions of P-glycoproteinsubstrates using cell monolayersAkif Emre Tuereli, Udo Bock, Bernd Baumstuemmler, Annette Amann and Eleonore Haltner-UkomaduAcross Barriers GmbH (Saarbruecken) (DE)e-mail: [email protected]

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Within the light of revised EU directive(86/609/EEC) it is of utmost importanceto develop new in vitro methods as a pos-sible replacement for laboratory animals.Calu-3 cells have been described to havenumerous features to be used as suitablein vitro model for upper epithelial airway(Foster et al., 2000) but there are no vali-dated methods available. We report herethe influence of DMSO, pH on Calu-3cells and selection of a suitable buffer for transport experiment. TEER (Trans-epithelial resistance) and epithelial trans-port were the parameters we focused on.DMSO, a popular solvent for lipophiliccompounds, was found to have consider-able influence over TEER and transportof compounds across the monolayer in aconcentration and time dependent man-ner. When applied apically DMSO hadmodest effect on TEER, decrease inTEER was more prominent when DMSO

was present only in the basolateral side.When DMSO was present in both com-partments the TEER values were compa-rable to negative controls. DMSOincreases transport of rhodamine (RHO)from basolateral to apical direction buthad no influence over fluorescein (FLU).It is known that pH influences tight junc-tions, which in turn govern the TEER. Weobserved TEER values over a range of pH(6.6-8.0) in two buffers Hanks’ BalancedSalt Solution (HBSS) and Krebs-RingerBicarbonate Buffer (KRB). TEER valueswere more stable in HBSS over the entirerange. Whereas KRB showed moreanomalous behavior as compared toHBSS, and TEER values showed incon-sistent increase and decrease over differ-ent pH values moreover at low pH values(6.8-7.0), TEER values were relativelyhigher. TEER values were stable at pH7.4 in both buffers. Based on these

results, HBSS was chosen as a buffer forfuture experiments. Our findings clearlydemonstrate that even apparently simplesteps could have profound influence overcell behavior and can govern the fate offuture experiments. Buffer selection,effect of pH and DMSO, are the land-marks, which will help better validationand establishment of Calu-3 as in vitromodels for possible replacement of ani-mal trials in studying pathogenesis of thelung and assessing potential toxins aswell as future drugs. Financial support byEU project PULMONET is gratefullyacknowledged.

ReferencesFoster, K. A., Avery, M. L., Yazdanian M.

and Audus, K. L. (2000). Characteriza-tion of the Calu-3 cell line as a tool toscreen pulmonary drug delivery. Int. J.Pharm. 208 (1-2), 1-11.


Establishment and pre-validation of a pulmonary invitro model for transport and toxicity studiesShariq Mahmood Usmani, Andreas Kraft, Bernd Baumstuemmler, Udo Bock, Tawfik Jalal and Eleonore Haltner-UkomaduAcross Barriers GmbH (Saarbruecken) (DE)e-mail: [email protected]

tively were used and apparent permeabil-ity coefficient (Papp) clearly decreasedwith the increasing molecular weight ofthe markers. The low permeability coeffi-cients of mannitol observed throughoutall experiments demonstrated the tight-ness and tight junction integrity of theCaco-2 cell monolayers used during thisstudy. Functional expression of the P-gpin this cell line was shown with the bidi-rectional transport experiments done withRhodamine 123 which resulted in the

asymmetric transport of the substancewith an efflux ratio of 16.5. In the exper-iments with Digoxin (1 µM), the effluxratio was found to be 21.7 which isdecreased to 1.24 and 1.07 in the pres-ence of Cyclosporine A (12 µM) andVerapamil (200 µM), respectively.Substrates of P-gp, Fexofenadine,Saquinavir, Paclitaxel, Loperamide,Quinidine, Talinolol and Celiprolol alongwith the specific inhibitor LY-335979 willbe used as reference substances in the

further experiments in compliance withrecently relieved draft FDA Guideline onin vitro testing of drug-drug interactions.Nowadays many animal experiments areconducted in order to be able to deter-mine the leading drugs from a large num-ber of drug candidates thus this study willallow reduction in the number of animalexperiments in testing the drug-druginteractions. Financial support from theEU project MEMTRANS is gratefullyacknowledged.

Keywords: efflux proteins, permeability, drug-drug interaction, cell monolayers

Keywords: lung, Calu-3, epithelial barrier, permeability, DMSO toxicity, TEER

Education in the field of biology, medicineand veterinary science was always accom-panied by experiments on the animalswhich were far from humane and ethicalgrasps. Both students and lecturers criti-cized such educational methods.

During lessons there are protestsamong students who don’t want to takepart in severe experiments on animalsbecause of moral, ethical and religiousreasons. However, students don’t have a

choice and are obliged to attend suchlessons, because otherwise they won’t beadmitted to the examinations. Such situa-tion is contrary to the principles of free-dom of student’s conscience and breakshis human rights.

This problem has been unsolvable formany years. But today, owing to activity ofthe numerous nongovernmental organiza-tions, there are libraries of alternatives toexperiences on animals. Alternative meth-

ods imply thematic computer programs,modeling, plaster casts which substituteuse of animals.

Now National Center on Bioethics suc-cessfully realizes the project on applica-tion of progressive educational methods,humane alternative programs, which arementioned above, in education system ofuniversities of Armenia.


New bioethical forms of educational process in universities of ArmeniaArmen VardapetyanNational Center on Bioethics (Yerevan) (AM)e-mail: [email protected]

Keywords: education, Armenia

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Today, on a large number of chemicals welack information on their adverse effectson the developing nervous system.Existing in vivo test methods are timeconsuming, laborious, expensive andrequire high numbers of laboratory ani-mals. Thus, standardized, predictive invitro screens for the evaluation of devel-opmental neurotoxicity need to be avail-able. The long-term goal is to increaseefficiency in terms of reduced animal useand higher throughput compared towhole-animal testing using the existingregulatory guidelines. In this study we

established a method for differentiation ofmouse embryonic stem cells into neuralcells, designed with special regard to thetesting of chemicals. It is based on a mod-ified protocol for adherent monolayer cul-tures in a defined medium and offers theadvantage of a reproducible developmentof neural cells in a comparatively shorttime. The differentiation of D3 cells intoneural cells was determined by analysis ofneuron-specific marker protein expressionusing flow cytometry. In addition, thedeveloping neurons were characterized byimmunofluorescence staining using neu-

ron- and glia-specific antibodies. In thisway, we also identified neurotransmittersubtypes. As a result, we were able todefine neural-specific molecular end-points for the detection of chemicaleffects on neural development. To investi-gate the assay performance, we assessedconcentration-dependent effects on neu-rons and glial cells using a limited numberof model substances. This in vitro methodmight prove to be a useful component of amore complex modular in vitro test sys-tem assessing the impact of neurotoxi-cants on the developing nervous system.


Towards an in vitro test system for developmental neurotoxicity: a new test methodusing mouse embryonic stem cellsAnke Visan, Katrin Hayess, Birgitta Slawik, Horst Spielmann and Andrea SeilerFederal Institute for Risk Assessment (BfR) (Berlin) (DE)e-mail: [email protected]

Keywords: in vitro, developmental neurotoxicity, embryonic stem cells, neurons, differentiation, test method

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Under the new EU chemicals legislationREACH 30,000 existing chemicals shouldbe evaluated within a period of 15 years.About 3,000 high production volumechemicals have to be assessed during thefirst 3 years. For most of these chemicalslarge data gaps must be closed by conduct-ing various studies, repeated dose studiesin particular. According to estimates of theEuropean Commission, around 70% of allexperimental animals are used to testchemicals for toxicity on reproduction.This is also where about 70% of the costsare generated – particularly for testing sub-stances on impairment of fertility and

reproductive ability, because REACH pre-scribes a two-generation test in rats(OECD Test Guideline 416) as the stan-dard procedure for chemicals with produc-tion volumes of more than 1,000 tonnes.Around 3,000 animals are needed to testone substance using OECD TG 416.ZEBET proposes a procedure with a con-siderable lower number of animals: Thestandard test could be the one-generationtest. There is growing evidence that theexamination of only one generation hardlyleads to any loss of information for deci-sion making. However, before a one-gen-eration test could be used for regulatory

purposes, the corresponding OECD TG415 would have to be updated. Preliminarywork for the use of a test of this kind in agraduated programme of pesticide testingwas done by the International Life ScienceInstitute/Health and EnvironmentalSciences, USA and by ZEBET. If the stan-dard testing requirement could be limitedto a one-generation study, around 1,400experimental animals for each substancetested would be saved. With an estimated2,000 substances that have to be testedover the next three years under REACH,this would mean a 2.8 million reduction inthe number of laboratory animals used.

Lecture: EU-chemicals policy (REACH)

The need for improvement of the integrated testingstrategy for reproductive toxicity under REACHRichard Vogel and Horst SpielmannFederal Institute for Risk Assessment (BfR), ZEBET (Berlin) (DE)e-mail: [email protected]

Keywords: REACH, reproductive toxicology, testing strategy, generation study

Protection against contact allergy beginswith the collection of reliable data aboutthe sensitizing potential of chemicals.Today, the LLNA (local lymph nodeassay) in mice is widely used to identifysensitizing substances. For several rea-sons, an in vitro assay could be preferableto animal experiments. We propose an invitro test for the detection of a sensitizingpotential of a chemical composed of asingle layer of human non-differentiatingkeratinocytes and of allogenic floatingmonocytes which are cocultured inserum-free medium in the presence of a

cytokine cocktail. Within days, the cocul-ture develops to an allergen-sensitive sys-tem consisting of activated keratinocytesand of mobile dendritic cell-related cells.The sensitizing potential can be deter-mined by analyzing the expression of thedendritic cell maturation marker CD86.For the model contact allergens tested sofar (trinitrobenzenesulfonic acid (TNBS),phenylendiamine (PPD), 4-aminoacet-anilide), the strength of the reaction wasin concordance with results from theLLNA. Sensitivity of the assay allowedtesting at concentrations without general

cytotoxicity. Thus, a differentiationbetween allergens and irritants was possi-ble. Regarding cytokine secretion, theassay distinguished between the allergenTNBS and the Toll-like receptor ligandLPS (lipopolysaccharide). The coculturecan be set up from cryopreserved cells.The assay is easy to perform and repro-ducible. Donor-variance is negligible.This in vitro assay based on a loose-fitcoculture is a reasonable approach toscreen for the sensitizing potential ofxenobiotics and might partially replacethe LLNA and other animal tests.


LCSA, loose-fit coculture-based sensitization assayReinhard WannerTeSens (Berlin) (DE)e-mail: [email protected]

Keywords: in vitro assay, sensitization, contact allergen, cell culture

A wide range of ophthalmic drugs arebeing used in veterinary ophthalmology.Their dosage regime is often directlytransferred from humans to companionanimals without formal testing. To pro-vide a model to be used for in vitro stud-ies on drug effects in dogs, this study wasconducted to establish a protocol for theprimary culture of canine corneal cells(i.e. endothelium, fibroblasts, epithelium)and the construction of a three-dimen-sional cornea equivalent. Pharmacologi-cal effects of dexamethasone were usedto test the functionality of this equivalent.Corneal cells were isolated using a com-bined enzymatic and mechanical tech-nique. In culture, the different cell typeswere verified with phase contrastmicroscopy, immunofluorescense andwestern blotting. The cornea equivalent

was constructed step by step in mem-brane inserts of a six-well plate. Stromalfibroblast in a collagen matrix wereseeded onto a confluent endothelial celllayer and cultured for 6-8 days. Thenepithelial cells were added and grown toconfluence in a submerged culture.Finally, the equivalent was lifted to theair-liquid-interface for two more weeks toallow a differentiation of the epithelialcells. To study the effects of dexametha-sone, the glucocorticoid receptor wasinvestigated in the cells and the equiva-lents using RT-PCR and immunohisto-chemistry. Furthermore, both systemswere stimulated with lipopolysaccharide(LPS) and treated with dexamethasone;the effects of the treatment were mea-sured as a change in prostaglandin E2(PGE2) concentration in the culture

medium. A protocol for the isolation andculture of canine corneal cells as well astheir reassembly in a vital cornea equiva-lent was successfully established. Thecornea equivalents could be cultured for atotal of five weeks. The glucocorticoidreceptor was detected in both the culturedcells and the cornea equivalents. Dexa-methasone led to a dose-dependentreduction of PGE2 in both systems. Theprimary culture of the canine cornealcells and the cornea equivalent are inter-esting systems to test drug effects oncorneal cells. Interestingly the corneaequivalents were more sensitive to thedexamethasone treatment than the singlecell cultures. Studies using the equivalentmay reveal further insights on pathophys-iological and therapeutic mechanisms inocular disease.


Construction and functional testing of a three-dimensional cornea equivalent using primary canine corneal cellsAnke Werner, Michael Braun and Werner KietzmannDepartment of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, Foundation (Hannover) (DE)e-mail: [email protected]

Keywords: cornea equivalent, dexamethasone, glucocorticoid receptor, LPS, canine

LINZ 2007

ALTEX 24, 3/07240

The European Union supports researchand development activities coveringalmost all scientific disciplines.Framework programmes are the mainfinancial tool for RTD support. The cur-rent Seventh Framework Programme(FP7) is organised in four programmescorresponding to four basic componentsof European research: Coorperation,Ideas, People and Capacities. Within

the Cooperation Programme life sciencerelated research is supported by theHealth Theme, the Environment Themeand the Food, Agriculture and Fisheriesand Biotechnology Theme. TheEuropean Union is Europe's major spon-sor of research in alternative testingstrategies. Joint European research inthis area is expected to mobilise and integrate research excellence necessary

for the promotion of new strategies foralternative testing. The research shouldprogress towards alternative methods in order to reduce, refine or replace substantially the number of tests and laboratory animals involved in pharma-ceutical discovery and development.Obtained results should establish a basisfor international validation.


EC funding of alternative testing strategiesChristian WimmerEuropean Commission (Brussels) (BE)e-mail: [email protected]

Keywords: European Commission, FP7, collaborative research

LINZ 2007

ALTEX 24, 3/07

241

Metal oxides are generally considerednon-harmful primarily because of theirlow solubility. It is known however, thatif reduced to nanoscale (<100 nm), chem-icals start to feature characteristics thatdiffer from those of the bulk material.The greater specific surface area renders

nanoparticles (NPs) biologically moreactive. Currently the research concerningenvironmental hazards of engineered NPsis limited: There are no standardizedmethods and the question remainswhether the techniques used for the toxi-city testing of chemicals are applicable

for NPs. Insolubility and aggregation ofparticles in aqueous media make the test-ing technically difficult and give rise to the dilemma whether to simulate envi-ronmentally relevant conditions or usesolvents/dispergants to stabilize the sus-pensions.


Ecotoxicological hazard of ZnO, TiO2 and CuO (nano)powders: effects on bacteria, crustaceansand protozoaMargit Heinlaan1,2, Irina Blinova1, Angela Ivask1, Monika Mortimer1, Henri-CharlesDubourguier2 and Anne Kahru1

1 National Institute of Chemical Physics and Biophysics, Tallinn 12618, Estonia; 2 Estonian University of Life Sciences, Instituteof Agricultural and Environmental Sciences, Tartu 51014, Estoniae-mail: [email protected]

The screening for estrogenicity andandrogenicity of existing and new chemi-cals has been assigned priority in thedetermination of endocrine activity ofcompounds. Insight into the mechanismof action of steroid hormones has pro-vided tools to develop alternatives to clas-sical in vivo assays. Several in vitroassays are available and prevalidation fortwo estrogen receptor (ER-) and twoandrogen receptor (AR-) gene reporterassays is going on as part of the EU 6thFP project REPROTECT, coordinated byECVAM. VITO has been appointed as theleading lab for the MELN assay andresults of the 1st & 2nd module of theECVAM validation scheme are pre-sented. For this assay, which makes useof estrogen-sensitive human breast cancercells (MCF-7) stably transfected with anestrogen-responsive Luc-gene (Balagueret al., 1999), test procedures were opti-mised and intralaboratory variability wasevaluated. As cytotoxicity should be eval-

uated adjacent to estrogen receptor trans-activation, the Cyto-TOX-OneTM homo-genous membrane integrity assay, asensitive fluorescent LDH-leakage testwas included for cost-efficient dual end-point measurements using the same cells(Berckmans et al., 2007). Standard oper-ating procedures considering cell cultureconditions and evaluation of estrogenicactivity are available. For chemicals withunknown properties, first a pre-screenprocedure should be performed. The lat-ter, based on a broad range finding from10-13 M up to 10-5 M, does include a testfor mode of action (agonist or antago-nist), a test for evaluation of cytotoxicityand a test for unspecific toxicity. Next,the non-toxic working range for estro-genicity is defined, and the test procedurefor agonistic mode using -estradiol as apositive control or for antagonistic modeusing OH-Tamoxifenβ17 as a positivecontrol can be applied. The test results fora panel of 16 chemicals evaluated accord-

ing to these procedures of the MELNassay will be presented. EC50 calculationswill be made and used (1) to evaluateintralaboratory performance and (2) toclassify the chemicals according to theirpotency and compare these with availableliterature data. The other steps in prevali-dation, the transferability of the proce-dures and evaluation of interlaboratoryvariability have started, and data will beavailable by the end of this year.

ReferencesBalaguer, P., François, F., Comunale, F. et

al. (1999). Reporter cell lines to studythe estrogenic effects of xenoestrogens.Sci. Total Environ. 233(1-3), 47-56.

Berckmans, P., Leppens, H., Vangen-echten, C. and Witters, H. (2007).Screening of endocrine disruptingchemicals with MELN cells, an ER-transactivation assay combined withcytotoxicity assessment. Toxicol InVitro, (Epub ahead of print).

Poster: endocrine disruptors

Prevalidation of the MELN assay, a reporter cell lineto screen for estrogenic activityHilda Witters, Katrien Smits, Clea Vangenechten and Pascale BerckmansFlemish Institute for Technological Research (VITO), Centre of Expertise in Environmental Toxicology (Mol) (BE)e-mail: [email protected]

Keywords: estrogens, MELN cell line, prevalidation

LINZ 2007

ALTEX 24, 3/07242

Keywords: ecotoxicology, nanoparticles,CuO, TiO2, ZnO, bacteria, protozoa

The aim of this study was to evaluatethe influence of size and hydrolysis ofmetal oxides on ecotoxicity with bacterial(Vibrio fischeri) luminescence andgrowth inhibition, crustacean (Daphniamagna, Thamnocephalus platyurus)mortality and protozoan (Tetrahymenathermophila) growth inhibition. Testedchemicals were nanosized ZnO (50-70nm), TiO2 (25-70 nm), CuO (30 nm).Bulk forms of the metal oxides and ionicforms of the metals (ZnSO4*7H2O andCuSO4) were assessed in parallel.Bioavailable ions from hydrolysis ofoxides as potential contributors to thetoxic effects were quantified by lumines-cent recombinant metal-specific E.colisensor bacteria. The metal oxide suspen-sions were studied for particle size distri-bution by using Nanosight LM10nanoparticle characterisation system.

Our study is the first comparison oftoxicity of nanosized and bulk ZnO andCuO for V. fischeri, D. magna, T. platyu-rus and T. thermophila – representativesof different trophic levels. Our datashowed that in case of all organisms nanoand bulk ZnO exhibited practically thesame toxicity, whereas nano CuO was upto100-fold more toxic than its bulk form.Nearly all the toxicity of ZnO and a greatpart of toxicity of CuO was due to dis-solved toxic concentrations of metal ions.TiO2 had no effect (up to 20.000 mg/l) forall the test organisms. Visual appearanceof test solutions and Nanosight LM10analysis revealed that NPs had aggre-gated to the size of their bulk forms,which may also occur in natural condi-tions.

» 4th International SkinEthicWorkshop,September 6-7, 2007,Nice, France.http://www.skinethic.com

» 18th Meeting of the InternationalSociety for Livestock Husbandry(Internationale Gesellschaft fürNutztierhaltung, IGN),September 20-21, 2007,Giessen/Germany.

» 14th Congress on Alternatives toAnimal Testing – Linz 2007,September 28-30, 2007,University of Linz, Austria.http://www.zet.or.at/kongress/Linz2007/index.html

» EUROTOX 2007,October 7-10,Amsterdam, The Netherlands.http://www.eurotox2007.org

» First International Forum Towardsan Evidence-Based Toxicology (BT),October 15-18, 2007,Spazio Villa Erba, near Cernobbio,Lake of Como, Italy.http://www.ebtox.org, [email protected]

» AALAS National Meeting 2007,October 14-18, 2007,Charlotte, NC.http://www.aalas.org/index.aspx

» 2. REACH-Symposium,November 22, 2007,Berlin, Germany.http://www.gd-online.de

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