GruBadresse zum Welcome address to the 13th MEGAT-Congress ... · Nicht zuletzt nirnrnt die Prasentation und Diskussion neuer Entwicklungen zur Irnplementierung der 3R in der biornedizi

LINZ 2006 ~ __

GruBadresse zum 13. MEGAT Kongress uber ,,Alternativen zum Tierexperiment"

Sehr geehrte Kongressteilnehmer(innen),

im Namen von zet, der osterreichischen nationalen Plattform fur Alternativen zum Tierexperiment, darf ich Sie herzlich zum diesjahrigen .Linzer Kongress" begrufien, Die Vorverlegung des Treffens von September auf Juni hat

mit der Elf-Ratsprasidentschaft Osterreichs zu tun. Anlasslich der letzten (ersten) Ratsprasidentschaft hat Oster

reich ein Symposium zur .Jmplementierung der 3R in der Europaischen Union in Wissenschaft und Industrie" veranstal tet. Diese Aktivitat wird nun anlasslich der diesjahrigen Rats prasidentschaft mit dern ,,13. MEGAT/zet-Kongress iiber Alter nativen zurn Tierexperiment" fortgesetzt. Das Organisationskornitee hat sich deshalb bemiiht, das Pro

gramm so zu gestalten, dass ein moglichst groBer Kreis von An wendern alternativer Methoden in ganz Europa angesprochen wird und hat deshalb Englisch als Kongresssprache gewahlt. Die Tagungsteilnehmer werden uber die Bemuhungen der EU

zur Implementierung alternativer Methoden in Grundlagen forschung, lndustrie einschlieBlich der Kosrnetik- Industrie, und auf Behordenebene, informiert. Dabei werden, wie bisher, ethische und rechtliche Aspekte von Tierversuchen ebenso wie die mit der Etablierung standardisierter in vitro Verfahren (Zell kulturtechniken) verbundenen Problerne behandelt. Wichtig erschien es uns auch, die neuesten Entwicklungen

beziiglich der Richtlinie fur die neue Chemikalienpolitik der EU (REACH) im Zusarnrnenhang rnit dem Einsatz von Alter nativen zu beleuchten. Nicht zuletzt nirnrnt die Prasentation und Diskussion neuer

Entwicklungen zur Irnplementierung der 3R in der biornedizi nischen Grundlagenforschung und der angewandten Forschung breiten Raum ein. Wie bisher wird bewusst auf Parallelsitzungen am Kongress

verzichtet, dafiir aber den Posterprasentationen mehr Zeit ge widmet, sodass die prasentierten Ergebnisse intensiv diskutiert werden konnen. Die besten und innovativsten Poster werden rnit einern Preis ausgezeichnet, urn rnehr junge Wissenschafter zur Weiterentwicklung der 3R Prinzipien zu motivieren.

Wir hoffen, Ihnen ein attraktives Prograrnrn in einer der inno vativsten osterreichischen Stadte bieten zu konnen und freuen uns auf eine zahlreiche Teilnahme!

74

Welcome address to the 13th MEGAT-Congress on u Alternatives to Animal Experimentation"

Dear Participant,

on behalf of zet (Centre for Alternative and Complementary Methods to Animal Testing), the Austrian national platform on Alternatives, I cordially welcome you to this year's "Linz Congress". The unusual date, June instead of September, was chosen to lie within the Austrian EU-Presidency. On occasion of the previous, first Austrian EU-presidency, a

symposium on "Implementation of the 3R Targets in the EU, in Science and Industry" was held by the Austrian authorities. This activity will be continued during the current EU-presidency of our country in form of the "13th Congress on Alternatives to Animal Experimentation". The organising committee has invested all its efforts in order to

address the largest possible number of scientists from all over Europe that are potentially interested in this field and has thus, for the first time, selected English as the official congress language. The meeting participants will be updated on the European

Community's and the member states' efforts to implement the 3R principles into academic, i.e. basic biomedical, and applied, i.e. industrial (including the cosmetics industry), research and on the status of implementation by European Regulatory Authorities. In this context, the programme will focus on legal and ethical aspects as well as on problems arising from efforts to standardise and harmonise in vitro approaches, specifically cell culture techniques. We further thought it of importance to review the newest

developments in the guidelines on European chemicals and cos metics with regard to the use of alternatives. Last but not least the programme will pay special attention to

new technologies and methods promising to expand our reper toire of 3R related experimental approaches. As in the past, we will consciously avoid parallel sessions at

the congress, but instead give a higher value than in the past to poster presentations. The best and most innovative posters will receive awards, which should help to convince and motivate young scientists to contribute effectively to the development of alternative methods in the future.

We strongly hope to be able to provide you with an attractive programme in one of the most innovative cities of Austria. We are looking forward to welcoming a large number of participants and scientists interested in promoting 3R related research.

ALTEX 23, 2/06

Congress Linz 2006 Preliminary program

12.45-13.00

Friday, 02.06.2006

Addresses of welcome

Session I

13.00-13.20

13.20-13.40

13.40-14.00

14.00-14.15

14.15-14.30

14.30-14.45

14.45-15.00

15.00-15.40

Session II

15.40-16.00

16.00-16.15

16.15-16.30

ALTEX 23, 2/06

REACH, cosmetics and toxicology Chair: pfaller Walter, A-Innsbruck N.N.

Sauer Ursula, D-Neubiberg Finalizing REACH -An evaluation of the European Parliament's and the Council's position from the point of view of the German Animal Welfare Federation

Hartung Thomas, I-Ispra Finalizing REACH -An evaluation of the European Parliament's and the Council's position from the point of view of European Commission/ECVAM

Lucaroni Beatrice, B- Brussels N.N.

Hendriksen Coenraad, NL-Bilthoven Towards eliminating the use of animals for regulatory required vaccine quality control

Mueller Stefan 0., D-Darmstadt Alternatives in pharmaceutical toxicology: global and focussed approaches

Westmoreland Carl, UK-Shambrook N.N. (Amendment to the Cosmetics Directive)

Ruhdel Irmela, D-Neubiberg N.N. (Amendment to the Cosmetics Directive)

Coffee-break and posters

Ecotoxicology Chair: Cervinka Miroslav, CS-Hradec Kralove

Scheiwiller Susanne, CH·Zurich

Hoet Peter, B-Leuven Nanoparticles - a new challenge to toxicology

Kahru Anne, EST-Tallinn Ecotoxicological tests and recombinant luminescent microbial models in toxicity studies: contribution to 3Rs

Tonkopii Valerii, RUS-St. Petersburg Daphnia magna as alternative bioobject in ecotoxicology

75

LINZ 2006 ~ __

Session Ill

16.30-16.50

16.50-17.05

17.05-17.20

17.20-17.35

17.35-19.30

20.00

Education Chair: Cervinka Miroslav, CS-Hradec Kralove

Scheiwiller Susanne, CH-Zurich

Dewhurst David, UK-Edinburgh Computer-based alternatives - past, present and future

Boumans Iris, NL-Utrecht Humane endpoints in laboratory animal experimentation: an interactive CD ROM

Martinsen Siri, S-Glava Veterinary training without the use of laboratory animals - an example from Norway

Jukes Nick, UK-Leicester Internationalising alternatives in higher education

Postersession I

Reception (at the congress venue)

Session IV

Saturday, 03.06.2006

09.00-09.15

09.15-09.30

09.30-09.45

09.45-10.00

10.00-10.15

10.15-10.30

10.30-10.45

10.45-11.15

Session V

11.15-11.30

76

Chronic toxicity Chair: Hartung Thomas, 1-lspra

N.N.

Runge Dieter, D-Schwerin Use of a standardized and validated long-term human hepatocyte culture system for repetitive analyses of drugs: Repeated administrations of acetaminophen reduces albumin and urea secretion

Thedinga Elke, D-Rostock Using of an in vitro system for prediction of hepatotoxic effects in primary human hepatoctyes

Cervinka Miroslav, CS-Hradec Kralove Microscope-assisted cytometry as an in vitro alternative for sub-chronic toxicity assessment

Slaughter Mark, UK-Sandwich 10-day medium throughput in vitro screen to assess chronic cytotoxicity of nucleoside analogues

Giese Christoph, D-Berlin Human organoids for immunogenicity and immunotoxicity testing

Huang Song, CH-Geneva A novel in vitro cell model of muco-ciliated human airway epithelium for long term toxicity testing

Poth Albrecht, D-Rossdorf The Bhas42 cell transformation assay as an predictor of carcinogenicity


Acute and target organ toxicity Chair: Spielmann Horst, D·Berlin

N.N.

Telang Nitin, USA-New York Cell culture model for colon carcinogenesis, cemoprevention and organ-selective-toxicity

ALTEX 23, 2/06

11.30-11.45

11.45-12.00

12.00-12.15

12.15-12.30

12.30-14.00

Session VI

14.00-14.20

14.20-14.40

14.40-15.00

15.00-15.20

15.20-15.40

15.40-16.00

16.00-18.00

18.15

20.00

Szameit Sandra, A-Seibersdorf Application of DNA microarrays for immunotoxicological testing

Scholz Gabriele, CH-Lausanne Hazard identification in chemical food safety: use of modeling, primary hepatocyte cultures and microarrays to address the level of concern for potential process contaminants of unknown toxicity

Hoffmann Jens J., D-St. Katharinen Increased reproducibility in toxicity testing using Epidermal Skin Test 1000

Haltner-Ukomadu Eleonore, D-Saarbriicken Assessment of applicability and tolerability of drugs and excipients on lung cell models

Lunch break

Ethical and legal aspects Chair: Roman Kolar, D·Neubiberg

Mayr Petra, A-Linz

Van der Valk Jan, NL-Utrecht Criteria for expert assessment by animal experiments committees

Kuil Jaime, NL-The Hague Regulatory animal testing

Ferrari Arianna, D-Ttibingen Schwierigkeiten und Dringlichkeit der Evaluierung der gentechnischen Veranderung von Tieren nach den 3R-Prinzipien

Lindi Toni, D-Munich Animal experiments in biomedical research. An evaluation of the clinical relevance of approved animal experimental projects: No evident implementation in human medicine within more than lO years

Luy Joerg, D-Berlin Ein Lebensrecht for Tiere ~ ethisch zu rechtfertigen?

Balluch Martin, A-Vienna Tiere haben ein Lebensrecht

Postersession 11

MEGAT - general assembly

Social evening & awards ceremony

Session VII

Sunday, 04.06.2006

09.00-09.10

09 .10-09 .30

ALTEX 23, 2/06

Good Cell Culture Practice Chair: Gstraunthaler Gerhard, A-Innsbruck

Fischer Rene, CH-Zurich

Gstraunthaler Gerhard, A-Innsbruck Safety in the cell culture laboratory

Merten Otto-Wilhelm, F-Evry Contaminations caused by viruses and prions

77

09.30-09.50

09.50-10.10

Session VIII

10.10-10.25

10.25-10.40

10.40-10.55

10.55-11.10

11. 10- 11.40

Session tx

11.40-12.00

12.00-12.20

12.20-12.40

12.40-13.00

13.00-13.20

13.20-13.35

13.35-13.50

13.50

78

Weiss Richard, A-Salzburg Viral expression vectors: biosafety considerations

Fischer Rene, CH-Zurich A new high potent freezing medium closes the last gap in the "serum free cell culture technique"

Free communications Chair: Gstraunthaler Gerhard, A-Innsbruck

Fischer Rene, CH-Zurich

Vuia Alexander, D-Berlin Results of the validation study on percutaneous absorption via reconstructed human epidermis

Visan Anke, D-Berlin Estimation of the embryotoxic potency of valproic acid derivatives in vitro by using the Embryonic Stem Cell Test (EST)

Eder Claudia, A-Vienna A modified HET-CAM approach for biocompatibility testing of medical devices

Falkner Erwin, A-Vienna HET-CAM model for melanom tumorgenicity studies


New methods Chair: Haltner-Ukomadu Eleonore, D-Saarbrucken

N.N.

Schrattenholz Andre, D-Mainz Stem cell- based in vitro models as a basis to test efficacies of preclinical phases of anti-neurodegenerative treatments

Curren Roger D., USA-Gaithersburg A novel, non-animal genetic toxicology assay - the reconstructed skin micronucleus assay using EpiDerm™

Humphery-Smith Ian, UK-Newcastle Knowledge of the Human genome (total DNA sequence) as an opportunity to build better drugs and concomitantly reduce the need for animal experimentation

Pfaller Walter, A- Innsbruck EpiFlow perfusion culture improves in vitro assessment of acute and chronic toxicity

Vedani Angelo, CH-Basel The challenge of predicting drug toxicity in silica

Weitzer Georg, A-Wien An in vitro model for early embryonic development

Hayess Katrin, D-Berlin Expansion of the Embryonic Stem Cell Test: Differentiation into neural cells

Closing words

ALTEX 23, 2/06

ALTEX 23, 2/06 79

Linz 2006

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

Some years ago, prior to human use ofnew cosmetic products, in vivo animaltests were conducted in order to screenfor safety and efficacy. The tenets of eth-ical animal testing call for practice of thethree R\’s of refinement, reduction andreplacement of animal tests. Therefore, agrowing need exists for rapid and reliablein vitro methods for safety and efficacyscreening of new dermato-cosmetologi-cal products. Three-dimensional organ-otypic skin models exhibit in vivo-likemorphological, metabolical, and growthcharacteristics which are uniform andhighly reproducible. These skin-like tis-sues consists of organized basal, spinous,granular and cornified epidermal layersanalogous to those found in vivo, andthey can provide alternative models to re-place animal and human experiments. In

the present study, these organotypic cul-tures have allowed to determine the ef-fects of the test product in the basementmembrane of skin, also called dermo-epidermal junction, which is responsibleof the contact between epidermis anddermis, and plays an important role in themaintenance of tissue architecture andnormal skin functions. In the epidermisof the skin, basal keratinocytes adhere tothe basement membrane through α6β4integrin which binds to Laminin-5, an in-teraction necessary for hemidesmosomeformation and basement membrane sta-bility. Hemidesmosomes are anchoringjunctions of the cell to a non-cellularsubstrate that play a critical role in sta-bilising the association of the dermiswith the epidermis. The importance ofthe hemidesmosome junctions for the

epidermis attachment is clearly demon-strated by the fact that in a variety of blis-tering diseases, there is a disruption ofthe hemidesmosomal complex. Severalstudies also confirm that there are changes occurring at this dermal-epidermaljunction during the course of age. Serile-sine is a Laminin-like peptide that wasfound to be active in promoting fibroblastand keratinocyte proliferation and there-fore this peptide may play an importantrole in skin. In this study, Serilesine hasbeen tested in order to determine its ef-fects on the basement membrane proteinsand on hemidesmosome formation. Tothis purpose, the effect of Serilesine onLaminin-5 and α6-integrin expressionand hemidesmosome formation was de-termined in the skin model.

Poster

Use of an in vitro skin model to study the dermo-epidermal junctionNúria Almiñana and Eva Martinez Cell Culture Laboratory, R&D Department, Lipotec S.A., Gava, SpainE-mail: [email protected]

Keywords: skin models, dermo-epidermal junction, laminin, integrin

2_06-S.079-103-AltexLinz.qxd 07.05.2006 10:44 Uhr Seite 79

There is much current debate surround-ing the use of non-human primates (NH-Ps) in medical research and drug devel-opment, largely from an ethicalperspective. Despite a significant pro-portion of the European public objectingto NHP experiments regardless of any

potential human benefits, the generalconsensus seems to be supportive pro-viding that it results in tangible medicalprogress and if there is no alternative.This review, stimulated by calls for evi-dence from UK-based inquiries intoNHP research, takes a critical view in or-

der to provide some important balanceagainst papers supporting it and callingfor it to be expanded. We show that thereis a paucity of evidence to demonstratethe positive contribution and translationof NHP research to human medicine,that there is a great deal of often over-

Poster

Non-human primates in medical research and drugdevelopment: A critical review Jarrod BaileyEuropeans for Medical Progress, London, United Kingdom E-mail: [email protected]

LINZ 2006

ALTEX 23, 2/0680

Biopredic International Rennes, FranceHuman primary hepatocytes and hep-atoma cells are widely used for in vitropharmaco-toxicological studies. Althoughprimary hepatocytes represent the mostpertinent system, they have limitationsdue to scarce and unpredictable availabil-ity, early phenotypic changes and lowgrowth activity and life-span. Hepatomacell lines have indefinite proliferativegrowth but they usually lack a variety offunctions, especially the major CYPs in-volved in xenobiotic metabolism, the con-stitutive androstane receptor (CAR) andseveral drug transporters. We have anal-ysed the drug metabolic capacity of theHepaRG cell line derived from a humanhepatocellular carcinoma (1). When Hep-aRG cells are plated at low density, theyactively divided and after having reachedconfluence, form typical hepatocyte-likeclusters surrounded by biliary epithelial-like cells. Transcripts encoding a number

of genes related to drug metabolism wereestimated by RT-qPCR. All were detectedin confluent HepaRG cells and for most ofthem, at levels comparable to those mea-sured in primary human hepatocyte cul-tures; they included various CYPs (1A2,2B6, 2C9, 2D6, 2E1 and 3A4), phase IIenzymes (UGT1A1, GSTA1, A4 andM1), nuclear receptors (AhR, ) and sinu-soidal (OCT1, OATP-C, NTCP andBSEP) and canalicularaPXR, CAR andPPAR (MDR1, MRP2 and MRP3) drugtransporters. The levels of several tran-scripts were strongly increased whenHepaRG cells were maintained for 2weeks at confluence in the presence of 2%DMSO. By contrast, in confluent HepG2cells tra nscripts of CYP1A2, CYP2B6,CYP2E1, CYP3A4, CAR, OCT1, OATP-C, NTCP and BSEP were barely ex-pressed in any (2,3). Basal CYP activitiesand response to prototypical inducers aswell as activities of drug transporters and

regulation by known inducers confirmedthe functional resemblance of HepaRGcells to primary human hepatocytes.These results show for the first time thatthe major CYPs, CAR and hepatic drugtransporters can remain expressed in a hu-man hepatoma cell line, leading to theconclusion that HepaRG cells represent aunique tool for the study of human livermetabolism and toxicity of chemicals.

1) Gripon, P. et al. 2002. Infection of ahuman hepatoma cell line by hepatitis Bvirus. Proc. Natl. Acad. Sci. U S A 99,15655-15660. 2) Aninat, C. et al. 2006.Expression of cytochromes P-450, conju-gating enzymes and nuclear receptors inhuman hepatoma HepaRG cells. Drug.Metab. Dispos. 34, 75-83. 3) Le Vee, M. etal. Functional expression of sinusoidaland canalicular hepatic drug transportersin the differentiated human hepatomaHepaRG cell line; Europ. J. Pharmaceut.Sci. in press.

Keywords: hepatic cell line, metabolism and toxicity studies

Poster

The human hepatoma HepaRG cells : A new in vitro model for xenobiotic metabolism and toxicity studies in human liverCaroline Aninat, Denise Glaise, Marc Levee, Christophe Chesne, Fabrice Morel. Olivier Fardel,Christiane Guguen-Guillouzo and André GuillouzoINSERM U620 and INSERM U522, University of Rennes, FranceE-mail: [email protected]


LINZ 2006

ALTEX 23, 2/06 81

looked data showing NHP research to beirrelevant, unnecessary, even hazardousto human health and to have little or nopredictive value and application to hu-

man medicine. We briefly discuss thereasons why this may be so, reflect uponthe consequences for future medicalprogress, and on the basis of our findings

suggest a more scientifically robust andpromising way forward.

Keywords: Primates, NHP, monkeys

Keywords: Animal experiment, stress, fear, handling, blood collection, gavage

Keywords: animal experiment, laboratory animal housing, stress, environmental enrichment

Eighty published studies were reviewedto document the potential stress associat-ed with three routine laboratory proce-dures commonly performed on animals:handling, blood collection, and gavage.Handling was defined as any non-inva-sive manipulation that is part of routinehusbandry, such as picking up an animal,and/or cleaning or moving an animal’scage. Significant changes in stress indi-cators (e.g., concentrations of corticos-terone, glucose, growth hormone or pro-

lactin, heart rate, blood pressure, and/orbehavior) were associated with all threeprocedures in the reviewed studies (re-porting primarily on rats, mice, monkeys,dogs, rabbits, hamsters, bats, or birds).Studies showed that animals respondedwith rapid, pronounced, and statisticallysignificant elevations in stress-related re-sponses to each of the procedures exam-ined. Changes from baseline or controlmeasures typically ranged from 20 to100 percent or more and lasted from 30

to 60 min or more. These findings indi-cate that laboratory routines are associat-ed with stress, and that animals do notreadily habituate to them. The data sug-gest that significant fear, stress, and pos-sibly distress are predictable conse-quences of routine laboratoryprocedures, and that these phenomenahave substantial scientific and humaneimplications for the use of animals inlaboratory research.

Poster

Laboratory routines cause animal stress [NB: recently published in Contemporary Topics in Laboratory Animal Science Nov. 2004;43(6):42-51]

Jonathan Balcombe, Neal Barnard and Chad Sandusky. Physicians Committee for Responsible Medicine, Washington DC, US. [to be presented at Linz by Andrew Knight from Animal Consultants International if no co-authors can attend] E-mail: [email protected]

Laboratory housing conditions have sig-nificant physiological and psychologicaleffects on rodents, raising both scientificand humane concerns. Published studiesof rats, mice and other rodents were re-viewed to document behavioural andpsychological problems attributable topredominant laboratory housing condi-tions. Studies indicate that rats and mice

value opportunities to take cover, buildnests, explore, gain social contact, andexercise some control over their socialmilieu, and that the inability to satisfythese needs is physically and psycholog-ically detrimental, leading to impairedbrain development and behaviouralanomalies (e.g., stereotypies). To the ex-tent that space is a means to gain access

to such resources, spatial confinementlikely exacerbates these deficits. Addingenvironmental “enrichments” to smallcages reduces but does not eliminatethese problems, and we argue that sub-stantial changes in housing and hus-bandry conditions would be needed tofurther reduce them.

Poster

Laboratory environments and rodents’ behavioural needs: A review [NB: to be published in Laboratory Animals 2006. In press.]

Jonathan BalcombePhysicians Committee for Responsible Medicine, Washington DC, US. [to be presented at Linz by Andrew Knight from AnimalConsultants International if Dr Balcombe is unable to attend] E-mail: [email protected]


LINZ 2006

ALTEX 23, 2/0682

Staat und Kirche sind zu trennen, und da-her dürfen sich auch die Gesetzgebungund das Rechtssystem nicht an religiösenDogmen orientieren. Sie müssen auf einerempirisch-rationalen Ethik basieren, diekonsensfähig ist. Dazu bedarf es zunächstzweierlei. Erstens muss das Bewusstseinnaturwissenschaftlich gefasst und be-schrieben werden. Und zweitens muss derAnthropozentrismus und das daraufbasierende Mensch-Tier Bild der Aufk-lärung kritisiert und auf seine faktischeBasis hin untersucht werden. Dabei zeigtsich, dass Bewusstsein an sich durch die

Fähigkeit zu Autonomie bestimmt ist.Und Bewusstsein hat eine evolutionäreKontinuität, d.h. es gibt keinen grundsät-zlichen Unterschied zwischen men-schlichem und tierlichem Bewusstsein.Eine implizite Voraussetzung dafür, au-tonom handeln zu können, ist, am Lebenzu bleiben. Das Leben ist von höchsteminstrumentellem Wert für ein autonomesLebewesen, um seine Autonomie umset-zen zu können, und liegt daher in seinemgrößten Interesse. Umgekehrt ist daherder Tod von größtem Schaden für diesesLebewesen. Weil ich als autonomes Lebe-

wesen autonom handeln will, fordere ichu.a. von der Gesellschaft ein Recht aufLeben ein. Wenn ich rational konsistentbin, dann muss ich dasselbe Recht auchfür alle anderen autonomen Lebewesen,die autonom handeln wollen, fordern.Mein Recht auf Leben – ein Menschen-recht – wiegt also genauso viel, wie dasRecht auf Leben für alle anderen Wesenmit Bewusstsein. Welche Wesen außer mirnoch ein Bewusstsein haben, bleibt alsrein empirische Frage zu klären, aber zu-mindest alle Wirbeltiere und Kopffüßerdürften darunter fallen.

Lecture

[Animals have a right to live]Tiere haben ein LebensrechtMartin BalluchVerein gegen Tierfabriken, Wien, ÖsterreichE-mail: [email protected]

Keywords: Tierrechte, Bewusstsein, Mensch-Tier Beziehung

The Embryonic Stem Cell Test (EST)determines the embryotoxic potential ofchemicals. The aim of this study is to adda static co-culture system with metaboliccompetence to this test to be able todetect pro-teratogens. The EST is basedon the capacity of embryonic stem cellsto differentiate in vitro into contractingcardiomyocytes which can be detected bymicroscopic analysis. It is the only invitro reproductive toxicity test withoutusing test animals and has been scientifi-cally validated by the European Centrefor the Validation of Alternative Methods(ECVAM). In order to be accepted byinternational authorities, the EST must beable to detect pro-teratogens which needmetabolic activation to exert a toxicaction.

Different metabolic systems weretested: primary hepatocytes from rat andmouse, and the hepatoma cell linesH4IIE (rat), Hepa 1-6 (mouse) andHepG2 (human). They were selectedconsidering their tolerance of the testenvironment, and a high metabolic com-petence. Investigations included the timeintervals in which metabolic activationhas to take place, and different co-culturetechniques at different test phases.Cyclophosphamide, a known pro-terato-gen, was used as a first model substance.

We found that metabolic activationmust take place for the whole test period.Changing the test duration or the point oftreatment reduces the test sensitivity astested in the EST with 5-fluorouracil and 6-aminonicotinamide. In hepatocyte

medium, embryonic stem cells do not dif-ferentiate into beating cardiomyocytes,and primary hepatocytes do not tolerateembryonic stem cell medium. Hepa 1-6cells show abnormal morphology on co-culture membranes. Hence, H4IIE- orHepG2 cells are the most promising can-didates. With these cells, the maintenanceof a co-culture in the EST is possible.First results suggest a metabolic activa-tion of cyclophosphamide.

It could be shown that in the EST, a co-cultivation with hepatoma cells is possi-ble. The test protocol has yet to beoptimised to reliably meet all validity cri-teria. In further studies, valpromide andretinol will be tested in the metaboliccompetent EST (mEST) to test its properfunction.

Poster

A metabolic activation system for the embryonicstem cell test Markus Binder, Jutta Volland, Frauke Meyer, Ulrich Hübel and Paul-Georg GermannALTANA Pharma AG, Institute for Preclinical Drug Safety; Hamburg, GermanyE-mail: [email protected]

Keywords: embryonic stem cell test, metabolic activation, hepatocytes, co-culture


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ALTEX 23, 2/06 83

Humane endpoints are an example of arefinement alternative (one of the 3Rs:Replacement, Reduction and Refine-ment). The aim of a humane endpoint isto terminate, minimise or reduce an ani-mal’s pain and/or distress. The CD ROM“Humane endpoints in laboratory animalexperimentation” is an interactive pro-gram for educational and training pur-poses and is intended to be part of thetrainings program of animal welfare offi-cers, researchers, animal technicians andanimal caretakers. The CD ROM focuseson more aspects than only the title doesexpect. The first part of the CD ROMcontains chapters on normal behavior ofa mouse or rat, pain and distress and

recognition of general clinical signs aswell as clinical signs typical for a num-ber of specific biomedical research areas(e.g. cancer research). Another part of theCD ROM offers information aboutpathology, which can be a useful controltool for the correct assessment of suffer-ing and the quality of the experiment it-self. This part is followed by the maintopic of the CD ROM: Humane end-points, which describes the notion “hu-mane endpoints” and gives an overviewof parameters for applying humane end-points. The responsibilities and valida-tion of humane endpoints are discussed,and relevant national and internationallaws, guidelines and reports are also in-

cluded. The last part gives the opportuni-ty to test the acquired knowledge by anumber of interactive tests about differ-ent subjects. Furthermore the CD ROMincludes a glossary and more than onehundred additional images and videoclips are available. The Dutch version ofthe CD ROM has been distributed in theNetherlands since 2004 and is nowadaysbeing used by the research communityand also in a few courses (e.g. Laborato-ry Animal Science Course). The Englishversion is finished in the beginning of2006 and will be available at the 13thCongress on Alternatives to Animal Test-ing in Linz.

Lecture

Humane endpoints in laboratory animal experimentation: an interactive CD ROM Iris J. M. M. Boumans and Coenraad F. M. HendriksenNetherlands Centre Alternatives to animal use (NCA), Utrecht University, Utrecht, The Netherlands E-mail: [email protected]

Keywords: Humane endpoints, refinement, education, rodents

Annual figures on the use of laboratoryanimals in biomedical research are thenet result of all factors that determine an-imal studies and that increase or decreasetotal numbers. Based on financial sup-port of the Sophia Vereeniging; a liberalanimal welfare organisation in theNetherlands, we evaluated trends in ani-mal use over the years, particularly forthe Netherlands, but also for the UK,Switzerland and the EU. Parameters in-cluded total animal numbers, speciesused, purposes, degree of pain and suf-fering, etc. We also looked at factors thatinfluence the use of animals and currentdevelopments in these factors. We identi-

fied four main factors: society in generaland NGO organisations in particular, in-dustry, regulatory bodies and govern-mental/political organisations. Generally,influences are complex and quite oftenalso conflicting. For example, society asa whole favours the EU programmeREACH but is quite indifferent with re-gard to the number of chemicals to betested. However, individual NGO’s lobbyto increase the number (such as Environ-mental organisations), while animal wel-fare organisations want just the opposite.As the number of animals is related tothe number of chemicals tested, animalusage will be influenced by those activi-

ties. Specific attention is given to theThree R’s. The route of a Three Rmethod from the stage of development tovalidation and final implementation isbeing analysed and potential obstaclesare identified. The report includes manyrecommendations for a more efficientThree R’s strategy. We believe that a bet-ter understanding of all those factors thatinfluence laboratory animals use, as wellas knowledge of the processes that un-derlie Three R’s output will be beneficialfor a more specific and effective policytowards smaller numbers of animals andhigher welfare standards for those ani-mals still to be used.

Poster

The animal use barometer Iris Boumans 1, 2 and Coenraad Hendriksen 2

1Sophia Vereeniging, Amsterdam and 2Netherlands Centre Alternatives to Animal Use, Utrecht University, The NetherlandsE-mail: [email protected]

Keywords: three Rs, development animal use


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Zellsysteme gewinnen als Alternativezum Tierversuch immer mehr an Bedeu-tung. Solche 3D-Modelle wurden bereitsfür die Cornea und die Haut etabliert undeignen sich aufgrund ihrer organspezi-fischen Eigenschaften zur Testung ver-schiedenster Substanzen hinsichtlichihrer Biokompatibilität. Auch dieAtemwege stellen eine wichtige Ein-trittspforte für verschiedenste Krankheit-serreger und Umwelteinflüsse dar. Ziel-gerichtet wollen wir ein tubuläresTestsystem entwickeln, in dem die Zellenunter physiologischen Bedingungen ineinem Bioreaktor kultiviert werden. ZumAufbau eines 3D-Modells der Tracheawerden zunächst porcine Zellen mit un-

terschiedlichen enzymatischen Method-en isoliert und eine Kollagen I-Träger-struktur beschichtet. Die Besiedelung erfolgt anschließend bei 37° C und 5%CO2. Zur Versorgung der Zellen wirdAirway Epithelial Cell Growth Mediummit verschiedenen Supplementen einge-setzt. Die Zellcharakterisierung wirdüber histologische und immunhistologi-sche Methoden und die Vitalität überLive Dead Essays festgestellt. ZurIsolierung der Trachea-Zellen eignet sichunter anderem die Auswachsmethode,eine weitere Möglichkeit stellt die enzy-matische Isolierung der Zellen dar. Dabeiwurden die verschiedenen enzymatis-chen Methoden unter Verwendung von

Trypsin, Collagenase und Protease ver-glichen. Bereits 24 h nach der Isolationlässt sich die charakteristische Kino-zilienbewegung erkennen. Unser Ziel istder Aufbau eines komplexen 3D-Modellseiner bioartifiziellen Trachea in tubulärerForm, durch die im Bioreaktor unter zell-physiologischen Bedingungen die At-mung simuliert werden kann. DiesesModell könnte dann als Ersatz zuTierversuchen oder auch als biologischesImplantat, welches die Gewebefunktionerhält, eingesetzt werden. Erste Arbeitenzur Langzeitkultivierung von porcinenTrachea-Zellen ex-vivo liegen bereits vor,eine Umsetzung des Modells mit huma-nen Zellen wird angestrebt.

Poster

[Construction of a three-dimensional tubular trachea-model]Aufbau eines dreidimensionalen tubulären Trachea-ModellsMartina Brenner, Heike Mertsching und Herwig BrunnerFraunhofer Institut für Grenzflächen- und Bioverfahrenstechnik, Stuttgart, GermanyE-mail: [email protected]

Keywords: Trachea, Dreidimensionales Testsystem, primäre Zellen

Sub-chronic and chronic toxicity ofxenobiotics is standartly assessed usinglaboratory animals and, so far, no alter-native methods have been generally ac-cepted to replace this testing strategy.This area of research is therefore werechallenging, in particular in view of therecent developments in the field of alter-natives. Of all proposed and evaluated al-ternative models, the development of asuitable in vitro system for long term (5and more days) toxicity assessment ofxenobiotics by monitoring various cellu-lar functions represents an attractive

challenge. In order to develop and suc-cessfully validate and implement such anin vitro system, several questions, whichmay be divided roughly into two groups,are to be solved. Firstly, it is the biologi-cal nature of any in vitro system whichhas to be considered, in particular itsgrowth properties, need for passagingand maintenance of integrity. Secondly,technical aspects of thus proposed modelrequire the use of a very sophisticatedmonitoring system capable of non-inva-sive observation of cellular behavior andvarious cellular functions. In ideal case,

long term monitoring of selected cellularfunctions and subsequent data miningshould combine various optical methods(light microscopy – phase contrast, dif-ferential interference contrast) followingbasic parameters such as cell morpholo-gy, cell proliferation, motility or celldeath. Furthermore, this system shoulduse fluorescence to determine other as-pects of cellular functions (oxygen con-sumption, mitochondrial activity, gene expression and so forth) and their poten-tial perturbation after exposure to studiedxenobiotics. In our presentation, we will

Lecture

Microscope-assisted cytometry as an in vitroalternative for sub-chronic toxicity assessment M. Cervinka, Z. Cervinkova and E. Rudolf Department of Medical biology and genetics, Charles University Faculty of Medicine, Hradec Kralove, Czech RepublicE-mail: [email protected]


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The 7th amendment to the CosmeticsDirective precludes the use of in vivoassays for genotoxicity assessments ofcosmetic ingredients. This (among otheranimal testing prohibitions) poses aproblem for the complete safety assess-ment of such materials. For most cos-metics, skin has the highest exposure;therefore we are developing an in vitrohuman skin micronucleus assay usingthe 3-D EpiDerm™ skin model (MatTekCorp, Ashland, MA). Theoretically sucha model could approximate the complex-ities typical of in vivo exposures, e.g.absorption, tissue specificity, meta-bolism, etc., and at the same time reflecthuman-specific responses in theseparameters. Our assay is based on apply-ing two 10 ul doses of test material to the

present and discuss advantages anddrawbacks of a combination of severalapproaches – classical phase contrasttime-lapse recording and advanced cy-tometry observation with Live CellImaging System (CellR, Olympus) and

laser scanning confocal microscope withspectral analysis (C1si, Nikon). We willalso focus on other associated technicalproblems including the use of optimalcultivation chambers, maintenance ofstandard experimental conditions and ro-

bustness of this approach towards sub-chronic and chronic in vitro toxicity ex-perimentation.

This work was supported by Ministryof Education Research Project MSM0021620820.

surface of the EpiDerm™ tissue 24hours apart, and harvesting 24 hoursafter the last dose. Using this procedurewe show dose related increases in bothcytotoxicity and micronuclei inductionfor several model genotoxins includingmitomycin C (maximum micronucleusresponse [MMR] ~8% at 0.6 ug totaldose), vinblastine sulfate (MMR ~4% at0.01 ug total dose), methylmethane sul-fonate (MMR ~0.6% at 20 ug totaldose), and N-methyl-N’-nitro-N-nitro-soguanidine (MMR ~0.7% at 40 ug totaldose). The average background fre-quency of micronuclei is low at <0.2%(N=25). Three chemicals known to benon-skin carcinogens in rodents (4-nitrophenol, 1,2-epoxydedocane andtrichloroethylene) do not induce micro-

nuclei in our assay even when tested atcytotoxic dosesAs the first step in investigating whetherour model will respond to genotoxinsrequiring metabolic activation, we haveshown that EpiDerm™ cultures fromdifferent donors express numerous genesassociated with xenobiotic metabolismthat are also found in normal humanskin. We have also shown the inducibil-ity of several of these genes by 3-methylcholanthrene. We believe thisnovel assay system holds excellentpromise for future use as a human “invivo-like” genotoxicity model for theassessment of both cosmetic and non-cosmetic materials.

Lecture

A novel, non-animal genetic toxicology assay – the 3-D reconstructed skin micronucleus assayusing EpiDerm™Rodger D. Curren1, Marilyn J. Aardema2, Patrick J. Hayden3, Greg Mun1 and Ting Hu2

1Institute for In Vitro Sciences, Inc., Gaithersburg, MD, USA; 2The Procter & Gamble Co., Cincinnati, OH, USA; 3MatTek Corporation, Ashland, MA, USAE-mail: [email protected]

Keywords: micronucleus, skin, in vitro, genetox, non-animal testing

Keywords: sub-chronic toxicity, in vitro alternative, cytometry, time-lapse, microscopy


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Induced liver preparations known as S9fractions are required for in vitro genotox-icity studies to activate indirect genotox-ins. The use of S9 preparations, withmammalian cell cultures, raise toxicityproblems. Moreover bias may arise due tothe kinetic profiles (intra-cellular penetra-tion) of short half-lives metabolites, pro-duced by the extra-cellular S9 enzymes.In order to circumvent these limits, sever-al genetically engineered immortal cellslines (ex: V79) have been developed witha stably expression of several functionallymammalian cytochromes P-450. We pro-pose another approach, based on the useof a well equipped hepatic cell line. Wehave analysed the drug metabolic capaci-ty of the HepaRG cell line derived from a

human hepatocellular carcinoma, to es-tablish its future potential in genotoxicitystudies. When HepaRG cells are plated atlow density, they actively divide and afterhaving reached confluence, form typicalhepatocyte-like clusters surrounded bybiliary epithelial-like cells. Transcripts en-coding a number of genes related to drugmetabolism were estimated by RT-qPCR.All were detected in confluent HepaRGcells and for most of them, at levels com-parable to those measured in primary hu-man hepatocyte cultures; they includedvarious CYPs (1A2, 2B6, 2C9, 2D6, 2E1and 3A4), phase II enzymes (UGT1A1,GSTA1, A4 and M1), nuclear receptors(AhR, ) and sinusoidal (OCT1, OATP-C,NTCP and BSEP) and αPXR, CAR and

PPAR canalicular (MDR1, MRP2 andMRP3) drug transporters. Basal CYP ac-tivities and response to prototypical in-ducers as well as activities of drug trans-porters and regulation by known inducersconfirmed the functional resemblance ofHepaRG cells to primary human hepato-cytes. These results show that the majorCYPs, CAR and hepatic drug transporterscan remain expressed in this human hep-atoma cell line. HepaRG cells represent apromising tool for genotoxicity studies ofenvironmental chemicals, more especiallywhen the mutagenic potential of a chemi-cal is related to its metabolites, or whenthe cellular enzymatic pathways, involvedin the production of the genotoxins, areunder evaluation.

Poster

The human hepatoma HepaRG cells: A promisingtool for genotoxic studies of environmental chemicalsYann Courbebaisse and Christophe Chesné. Biopredic Rennes, FranceE-mail: [email protected]

Keywords: HepaRG, metabolism, genotoxicity, in vitro

When explaining to faculty that alterna-tives to animal use in education arepreferable not only from an ethical pointof view but also from an educationalpoint of view, we need systematic assess-ments of the alternative models. This isconsistent with evidence-based practice.Teachers are mostly interested inwhether resources meet their learningobjectives, but also in whether resourcesare interactive, realistic and user-friend-ly. Evaluations such as exams or assign-ments conducted to assess studentachievement of learning objectives needto take into consideration the teaching

methodology used, the contents, and thegrouping of students. These didacticcomponents are also included in an inde-pendent review process previously devel-oped by the author at the European Re-source Centre for Alternatives to animaluse (EURCA). The review process is de-signed to enable teachers to systematical-ly assess the educational value of alterna-tive models. Other features examinedinclude the co mparison between the al-ternative model and the animal laborato-ry, ease of use of the alternative model,the contribution of the resource to aware-ness of the 3Rs, and the level of service

provided by the supplier. Several reviewsof, mostly electronic, alternative modelsare used as examples. Generally, the re-viewers are positive about the effective-ness of alternative models, but there isroom for improvement both at didacticlevel, as well as the user level (naviga-tion, presentation and level of interac-tion). Through teachers’ assessments ofeducational models, alternative resourcesare becoming more accepted in main-stream education, which is likely to pos-itively influence the attitudes towards the3Rs of scientists, professionals and a newgeneration of science teachers.

Poster

Didactic assessments of educational models used as alternatives to harmful animal use Jasmijn de BooCambridge E-learning Institute (CEI) Orwell, UK E-mail: [email protected]

Keywords: Alternative model, assessment, education, review


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The use of computer-based alternatives tousing animals in the teaching of disci-plines such as pharmacology and physiol-ogy dates back to the early 1980’s. Thenthere were two main approaches to devel-opment. Some programs were designed tosimulate an animal preparation using amathematical algorithm which predicted,based on known data, how a tissue wouldrespond to drugs or drug combinations,and factors such as electrical stimuli. Thistype of program encouraged learning byexploration and was best suited to a learn-ing environment in which a tutor was pre-sent to guide students. Another approachwas to create a tutorial program around aset of data derived from a set of real ex-periments which had been designed by aknowledgeable tutor well acquainted withhow a particular animal preparation could

be used to teach major principles and fac-tual knowledge. This second approach re-sulted in programs which could be usedindependent of tutor supoport though of-ten learning would be enhanced by thepresence of a tutor freed from trouble-shooting technical problems with equip-ment. Each approach fosters differentlearning outcomes and both have beendemonstrated to be extremely successfulin differerent learning or teaching situa-tions. Several studies have been carriedout to evaluate the effectiveness of bothtypes of program and evidence from thesewill be presented together with a descrip-tion of successful strategies designed toenable integration of these resources intomainstream teaching.

Many of these computer programs arenow quite old and, although the content

and underlying pedagogical approach isstill sound, the technology underpinningthe delivery of the programs is rapidlybecoming obsolete. A project to preservethe educational value of these programsin the face of rapidly changing deliverytechnologies is underway in Edinburgh.This aims to more effectively manage thecomponent learning and information as-sets (text, images, animations, self-as-sessments, video and audio), provideteachers with easy-to-use authoring tem-plates to enable them to build their ownresources from these assets, and build fu-ture-proofed delivery engines compatiblewith delivery over the Internet or onCDROM. Examples of specific e-alterna-tives will be used to illustrate this pro-cess.

Lecture

Computer-based alternatives – past, present and futureDavid DewhurstLearning Technology Section, College of Medicine & Veterinary Medicine, University of Edinburgh, UKE-mail: [email protected]

Keywords: computer-assisted learning (CAL), alternatives in teaching, computer simulations, animal experiments in teaching

Introduction: The HET-CAM (Hen EggTest - Chorionallantoic Membrane) testsystem offers a partially immunodeficient,borderline in vitro/in vivo test system al-lowing the simulation of transplantationexperiments prior to actual animal testing.Material and Methods: A collagen scaffoldwas seeded with sheep primary meniscuscells at different concentrations. Prelimi-nary incubation in vitro was varied be-

tween three and fourteen days. Transplan-tation success and cell migration in ovowere evaluated using the HET-CAM as-say. Results: Increasing cell seeding densi-ty from 2x105 to up to 5x106 cells perscaffold did not result in satisfying tissueformation at the transplantation site. In-stead, the high cell density led to changedmaterial properties of the scaffold, leadingto poorer tissue integration and CAM ves-

sel bleeding. Raising the preliminary incu-bation time to two weeks led to stable tis-sue formation and reduced cell migrationin ovo. Conclusion: Sufficient cell deliveryto the transplantation site is dependant onthe preliminary incubation period ratherthan the initial cell load. Protocols intend-ed for in vivo evaluation can be tested andoptimised prior to their in vivo applicationusing the HET-CAM assay.

Poster

Simulation of in vivo cell transplantation experiments using the HET-CAM bioassay Claudia Eder 1, Erwin Falkner 1,2, Michael Mickel 3, Udo M. Losert 2 and Harald Schöffl4

1BioMed – Society for the Promotion of Biomedical and Medical Technological Research in Upper Austria, Linz; 2Core Unit for Biomedical Research, Medical University Vienna; 3Karl Landsteiner Institute for Cardiovascular Research, Vienna;4zet – Centre for Alternative and Complementary Methods to Animal Testing, Linz, AustriaE-mail: [email protected]

Keywords: HET-CAM, cell transplantation, animal experiment


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Introduction: The implantation of newbiomedical devices into living animalswithout any previous toxicity or biocom-patibility evaluation is possible based oncurrent legislation. The HET-CAM (HenEgg Test - Chorionallantoic Membrane)test system offers a partially immunode-ficient, borderline in vitro/in vivo testsystem allowing the simulation of trans-plantation experiments to obtain biocom-patibility data prior to animal testing.Material and Methods: A collagen type

I/III scaffold designed for tissue regener-ation was tested for angiogenetic proper-ties and biocompatibility patterns. Bio-compatibility was then altered byincubation in Acridine Orange/EthidiumBromide and the test was repeated. Re-sults: The native material demonstratedgood biocompatibility patterns and a sig-nificant angiogenetic stimulus caused bythe collagen scaffold material was ob-served. Altering biocompatibility pat-terns by incubation with Acridine Or-

ange/Ethidium Bromide led to severevessel thrombosis and a foreign body tis-sue response. Conclusion: CAM-Testingof biomaterials and tissue engineeredproducts allows a selection of the mostsuitable biomaterial and an exclusion ofimproper materials from animal experi-ments, leading to refined testing proce-dures and a reduction of animal numbersrequired for biocompatibility testing.

Lecture

A modified HET-CAM approach for biocompatibilitytesting of medical devices Claudia Eder 1, Erwin Falkner 1,2, Michael Mickel3, Harald Schöffl1, Stefan Nehrer 4 and Udo M. Losert 2

1zet – Centre for Alternative and Complementary Methods to Animal Testing, Linz; 2Core Unit for Biomedical Research, Medical University Vienna; 3Karl Landsteiner Institute for Cardiovascular Research, Vienna; 4Department of OrthopaedicSurgery, Medical University Vienna, Austria E-mail: [email protected]

Keywords: biocompatibility testing, HET-CAM, tissue engineering

Introduction: The HET-CAM (Hen EggTest - Chorionallantoic Membrane) testhas originally been validated for toxicityand irritation studies and is under in-creasing attention for biomaterial evalua-tion purposes. The high vascularity of theCAM and the rapid development of con-nective tissue and vessel system offer anattractive environment for tissue reactionstudies, which are often evaluated bymacroscopical examination only.

Materials & methods: Various bio-degradable scaffolds were applied ontothe CAM on incubation day 7 and main-tained in ovo for 3 days prior to digital

documentation, macroscopical biocom-patibility evaluation and subsequent his-tological analysis. A collagen sponge,two different Collagen Type I/III scaf-folds (Chondro-Gide®, Bio-Gide®) and aCollagen Type II membrane (Chondro-cell®) were tested. Macroscopic scoringcriteria were embryo viability, vessel re-actions, bleeding and infection. Histolog-ical criteria were angiogenesis inductionand alteration of CAM structure.

Results: Collagen sponge: Macroscop-ic analysis demonstrated extreme rapiddegradation, spontaneous bleedings inthe surrounding of the implant and a ves-

sel retraction from the implantation site.Histological analysis, in contrast,demonstrated an increase in blood vesselcontent compared to control CAMs. Aforeign body tissue reaction was ob-served. Chondro-Gide®: Macroscopicevaluation showed excellent integrationand biocompatibility patterns whichwere confirmed by histology. A tissue re-action was not observed and the scaffoldshowed excellent angiogenetic proper-ties. Bio-Gide®: Macroscopic observa-tion showed excellent integration andsignificant induction of angiogenesis,which was confirmed by histology. An

Poster

Macroscopic evaluation of HET-CAM biomaterial testing: How reliable is macroscopicalscoring without histology ? Claudia Eder 1, Erwin Falkner 1,2, Harald Schöffl3, Stefan Nehrer 4 and Udo M. Losert 2

1BioMed – Society for the Promotion of Biomedical and Medical Technological Research in Upper Austria, Linz; 2Core Unit for Biomedical Research, Medical University Vienna; 3zet – Centre for Alternative and Complementary Methods to AnimalTesting, Linz; 4Dept. of Orthopaedic Surgery, Medical University Vienna, AustriaE-mail: [email protected]


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inflammatory infiltrate was observed.Chondrocell®: Macroscopic evaluationshowed good integration of the biomate-rial, which was confirmed by histology.Spontaneous bleedings at the implanta-tion site as well as vessel retraction andaltered vessel courses were observedmacroscopically. Histological evaluation

The cultivation of cells in vitro is an es-sential tool for biomedical research andproduction purposes. For many tasks thesupplementation of mammalian cell cul-ture media with serum (-components) ofanimal origin remains still standard, pro-viding for e.g. nutrition, shear protection,growth factors and cytokines. Because of

undefined/varying composition, risk ofcontamination with prions/viruses/bacte-ria, the cost factor and also animal wel-fare considerations concerning the pro-duction of sera, the change to serum freealternatives is promoted by regulatoryauthorities, the industry and the researchcommunity in general. A growing num-

ber of alternatives exists for cell lines andprimary cultures: chemically defined me-dia, additives of non-serum origin, non-animal derived proteins and also opti-mized adaptation/sampling/processingprotocols and production systems/biore-actors for serum-free usage of all scales.A regularly updated product guide (cur-

demonstrated good biocompatibility pat-terns, the absence of an inflammatory in-filtrate and good angiogenetic properties.

Conclusions: Macroscopical scoringonly partially correlated to histologicalevaluation: Tissue integration could beassessed with both methods. The impactof spontaneous bleedings and vessel path

alteration was often overestimated and aforeign body tissue reaction could not bedetected after macroscopical evaluation.HET-CAM biomaterial testing shouldtherefore be combined with histologicalevaluation to receive the full force of ex-pression of the _CAM model.

Poster

Invitrotrain: practical training courses on alternative(non-animal) test methods Maria Engelke, Burkhard Kleuser and Monika Schäfer-Korting Institute for Pharmacy, Freie Universität Berlin, GermanyE-mail: [email protected]

Poster

Discussing serum-free cell culture – the product data base 2006 Erwin Falkner1,2, Helmut Appl3, Claudia Eder2, Udo M. Losert1, Harald Schöffl2 and Walter Pfaller2

1Core Unit for Biomedical Research, Medical University, Vienna; 2Biomed – Society for the Promotion of Biomedical Researchin Upper Austria; 3zet – CentreAlternative and Complementary Methods to Animal Testing, AustriaE-mail: [email protected]

The Invitrotrain project covers the devel-opment, validation and – most important-ly – the demonstration of in vitro meth-ods for testing of chemicals andprediction of toxicity. The Free Universi-ty of Berlin offers practical training ofscientifically validated in vitro methodsthat have gained regulatory acceptancefor specific purposes. The practical train-ing courses focus on • skin models, skincorrosion and phototoxicity • acute eyeirritation, • penetration models, • repro-

ductive toxicology • ecotoxicology. Allin vitro methods are hands-on laboratoryexercise. The participants will performthe tests and evaluate the results accord-ing to Standard Operation Procedures orInvittox Protocols. The aims of the train-ing courses are to provide the attendeeswith sufficient experience, so that theymay apply the techniques to their ownneeds and to promote the use of in vitroalternative methods. The theoreticalbackground of each test method will be

introduced during seminars. Moreover,general aspects concerning the 3R’s con-cept and validation studies will be ad-dressed. The courses will take placebiannually at the Institute for Pharmacy.The objectives of the Invitrotrain projectare in line with the future EU policies oncosmetics and chemicals which call forthe use of animal alternatives and newtesting strategies. The project is spon-sored by the European Commission.

Keywords: skin models, eye irritation, reproduction toxicology

Keywords: HET-CAM, biomaterial testing


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rently 2006) can be downloaded as PDFor HTML for free at: www.zet.or.at.Serum free media do not require supple-mentation with serum, nevertheless theymay contain various undefined proteinsand/or protein hydrolysates. Protein free

media supporting growth and expressionwithout the presence of proteins, protein-free media have eliminated many of theconcerns regarding animal proteins inmedia supplementation. Some animalorigin materials may remain in the pro-

tein-free product, such as amino acids.Chemically Defined Media All compo-nents have a known chemical structure,resulting in consistent product perfor-mance and the elimination of lot-to-lotperformance variability.

Poster

Standardization o f the HET-CAM Assay forBiomedical Research: Analysis Software Erwin Falkner1,2, Claudia Eder 2, Udo M. Losert 1 and Harald Schoeffl 3

1Core Unit for Biomedical Research, Medical University, Vienna; 2Biomed – Society for the Promotion for Biomedical Researchin Upper Austria; 3zet – Centre for Alternative and Complementary Methods E-mail: [email protected]

Keywords: fetal calf serum, cell culture, serum free

Keywords: HET-CAM, Software, Standardization

Incubated chicken eggs of early phasesform an immunodeficient transition be-tween in vitro and in vivo situations. TheChorionallantoic Membrane (CAM) - anextraembryonal membrane deriving fromthe fusion of chorion and allantois - pro-vides an easily available, cheap and forcertain applications validated testing en-vironment. The test-system, originallyconceived as alternative method for toxi-

city and irritation studies, has been sug-gested for tissue engineering tasks, bio-compatibility testing and also as celltransplantation model. Evaluation of re-sults often is performed just macroscopi-cally by different members of workinggroup. If digital documentation is per-formed, commercially available all-pur-pose software packages are used. There-fore interpretation/originality can be

questioned, because images can be al-tered/modified easily. The authors usestandardized biomedical software origi-nally intended for other applications.Nevertheless, these programmes can beused/adapted for HET-CAM Bioassaysthereby improving quality of data outputand standardizing of HET-CAM Tests.

Lecture

HET-CAM model for melanom tumorgenicity studies Erwin Falkner1,2, Claudia Eder 2, Michael Mickel3, Harald Schöffl 4, Udo M. Losert1 and M. Seltenhammer 5

1Core unit for Biomedical Research, Medical University Vienna; 2Biomed – Society for the Promotion of Biomedical Research in Upper Austria; 3Karl Landsteiner Institute for Cardiovascular Research; 4zet – Centre for Alternative and ComplementaryMethods to Animal Methods; 5Klinik für Chirurgie und Augenheilkunde, Veterinärmedizinische Universität Wien, AustriaE-mail: [email protected]

Keywords: HET-CAM, melanom, tumor model, 3R

The authors present a updated version ofthe Hen Egg Test-Chorioallantois Mem-brane (HET-CAM) angiogenesis test sys-tem which was originally designed andfor certain applications validated for irri-tation/toxicology studies. New experi-

mental setups and first data/results showsuitability for innovative melanom tumorcell research: tumor formation, trans-membrane malignant migration and pos-sibilities of intervention/treatment in ovoand the potential consequences for con-

servative in vivo melanom models usingrodent animal models/scientific animalwelfare according to the 3Rs are dis-cussed in detail.


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The Hen’s Egg Test ChorioallantoisMembrane (HET-CAM) angiogenesissystem, originally developed for irrita-tion studies, was used for evaluation ofeffects of exponation of early breedingstage chicken egg CAMs (up to incub-tion day 10) to carbon monoxide (CO):

quantitative/qualitative effects of lowdoses (50 - 500 ppm) upon developmentof vessels were evaluated. CO applica-tion of doses at this concentration are re-ported to effect cell cultures in vitro,modulating/triggering expression ofheme oxygenase enzymes and therby ef-

fecting angiogenesis in vivo. The suit-ability of the in ovo HET-CAM systemfor e.g. angiogenesis/hypoxia but alsoCO-induced embryo toxicity testing isdiscussed.

Poster

Carbon monoxide in angiogenesis research and the HET-CAM bioassay Erwin Falkner1,2, G. Holzer 3, Claudia Eder 2, O. Wagner3, Harald Schoeffl 4 and Udo M. Losert 1

1Core Unit for Biomedical Research, Medical University Vienna; 2Biomed – Society for the Promotion of Biomedical Researchin Upper Austria; 3Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Vienna; 4zet – Centrefor Alternative and Complementary Methods to Animal Testing, AustriaE-mail: [email protected]

Keywords: carbon monoxide, HET-CAM, angiogenesis, 3R

Als in den 90er Jahren das Verfahren dergentechnischen Modifikation eine breiteAnwendung in der experimentellen biome-dizinischen Forschung fand, sahen zahlrei-che begeisterte Wissenschaftler in gentech-nisch veränderten Versuchstieren denSchlüssel für die künftige tierexperimentel-le Forschung sowie für die Eröffnung neuervielversprechender Forschungsgebiete (z.B. Xenotransplantation). Trotzdem fehlenspezifische Richtlinien zur Haltung undNutzung gentechnisch veränderter Ver-suchstiere und ihren speziellen Bedürfnis-sen und nur wenige Berichte über die Bela-stung ihres Wohlbefindens sind bis jetztveröffentlicht worden. Insbesondere man-gelt es an spezifischen Analysen auf der Ba-sis der 3R-Prinzipien bezüglich der Impli-kationen der Verwendung von gentechnischveränderten Tieren. Bei der Bewertung der

gentechnischen Veränderung von Tieren inder Forschung haben sich zwei gegenläufigeStrömungen entwickelt: Auf der einen Seitegibt es große Vorbehalte wegen der massi-ven Leidzufügung und dem großen „Tier-verbrauch“. Auf der anderen Seite wird indiesen Techniken ein enormes Potenzial füreine drastische Verminderung der gesamtenAnzahl der Tierversuche gesehen (einigesprechen von der gentechnischen Verände-rung als einer Alternativmethode). Unklar-heiten bestehen noch in Bezug auf die ange-messene Methode für eine Evaluierung derForschungsbereiche. Die Vielfältigkeit die-ser Bereiche und die Heterogenität derZwecke der Verwendung gentechnisch ver-änderter Versuchstiere erschweren eine all-gemeine Bewertung im Sinne der 3R-Prin-zipien. Zusätzliche Probleme verursacht dergroße Mangel an spezifischen Daten über

den Tierverbrauch und die Belastungen inden unterschiedlichen Phasen der Herstel-lung und Nutzung. In den meisten Tierver-suchsstatistiken ist nämlich keine spezifi-sche Kategorie für gentechnisch veränderteTiere enthalten. In diesem Artikel werdenzwei zentrale Fragen auch anhand einerAnalyse empirischer Daten diskutiert: 1) obdie gentechnische Veränderung von Tiereneinen Beitrag zur Verminderung der gesam-ten Zahl von Tierversuchen in der For-schung, bzw. zur Verbesserung der Effizienzder tierexperimentellen Forschung, leistenkann; 2) was für Konsequenzen für die ge-samte Bewertung des Forschungsfeldes dieKollision zwischen den Prinzipien Reduc-tion und Refinement hat, welche sich bei derAnalyse vieler Anwendungen gentechnischveränderter Tiere (z. B. bei Krankheitsmo-dellen) ergibt.

Lecture

[Difficulties and urgency of evaluating the genetic modifications ofanimals according to the three Rs principle]Schwierigkeiten und Dringlichkeit der Evaluierung der gentechnischen Veränderung von Tieren nach den 3R-PrinzipienArianna FerrariLehrstuhl für Ethik in den Biowissenschaften, GK Bioethik, Universität Tübingen, GermanyE-mail: [email protected]

Keywords: 3R-Prinzipien, Ethik, gentechnische Veränderung, gentechnisch veränderte Tiere


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Sera are generally obtained from drawnand collected whole blood from adult,calf or fetal animals. Following the natu-ral clotting process, which may take sev-eral hours at 4 degree C, the blood con-sists of serum and blood clot containing:95% of red blood cells, 5% platelets, lessthan 1% and numeorus amounts of fibrinstrands. In comparison to a PRP (Plateletrich plasma) blood clot containin 4% ofred blood cells, 95% pletlets and 1% ofwhite cells. The specific cell-growth pro-moting components are the platelet de-rived growth factor (PDGF) and thetransforming growth factor β (TGF β).Both of them are contained in the α gran-ules of the platelets. Fibronectin and vit-ronectin are also the components of thePRP. They are the cell adhesionmolecules found in plasma and fibrin it-self. The experiments presented hereinwere aimed to isolate, characterise and totest in vitro on different cell cultures the

growth promoting material from theswine blood clot. The swine blood wascollected and allowed to form the clot.Afterward the whole content was cen-trifuged at 2500 RPM for 20 minutes andthe supernatant (serum) was aspiratedoff. The sediment (»clot«) was quicklywashed with the sterile buffered salinepH=6.9 for 20 minutes, and centifugedfor 25 minutes at 2500 RPM for 25 min-utes. The supernatant (Fraction I) wascollected and frozen. To the remaining»clot« the PBS pH=7.2 was added andleft for 1 hour at +4oC. After the cen-trifugation of the suspension at 2500RPM for 25 minuts, the supernatant(Fraction II) was collected and frozen. Tothe sediment (»clot«) the PBS pH = 7.4was than added for 18 hours (FractionIII) . All the fractions were sterilised by0.2 membrane filtration. The content wasanalysed by PAG-SDS electrophoresis.The cell growth promotion/inhibition ac-

tivity in the comparison to the SR-2.055P(Serum replacement based on porcineocular fluid) and FCS (Fetal calf serum)was tested on the Chicken embryonal fi-broblasts, WISH, HAC-3/T2 (Humanamniotic cell lines), PLA-2 (Adult pigkidney cell line), Bovine intestinal ep-ithelial cell line, WiREF (Wistar rat em-brional fibroblastoid cell line) and CaCo-2(Colon cancer carcinoma cell line). Theresults of the experiments shows: (1) Theattachment and growth of primary cul-tures (Chicken embryonal fibroblasts) isnot affected after the use of procineblood clot fractions (2) Fractions (I-IV)shows the growth promotion, but differ-ent according to the group of cells usedin the test. (3) The strongest growth en-hancement was found when transformedcells (WiREF, CaCo-2) were tested. (4)The optimal content was 8-10% in Ea-gle’s medium. In this range up to 95%value of the SR-2.55P could be obtained.

Poster

Cell-growth promoting fractions originated fromporcine blood clotBratko Filipic1, Srec’ko Sladoljev 2, Ferenc Somogyvari 3,Lidija Gradisnik4, Natasa Pipenbaher 4,Avrelija Cencic4, Sandor Toth4, Eva Ruzic-Sabljic1 and Srecko Koren1

1Institute of Microbiology and Immunology, University of Ljubljana, Slowenia; 2Institute of Immunology, Zagreb, Croatia;3Institute of Clinical Microbiology, Medical Faculty, University of Szeged, Hungary; 4Faculty of Agriculture, University ofMaribor, SI; 5Bekes County Hopsital, Blood transfusion unit, Oroshaza, Hungary E-mail: [email protected]

Poster

Questioning the ethical justification of primate experiments by launching a campaign for a legal banCorina Gericke, Marion Selig and Christiane Baumgartl-SimonsPeople for Animal Rights Germany, D-Aachen E-mail: [email protected]

Keywords: swine, blood clot, growth factors, cell growth

Primates are sentient beings with highcognitive abilities. It is increasingly recog-nized by the public, scientists and politi-cians that their use in experiments causesextreme suffering. By laying down the an-imal welfare into the German constitutionin 2002 Germany took an important steptowards protecting animals for ethical rea-

sons, which is now a governmental obliga-tion. But until today it is not clear whatthis means concerning animal experimen-tation and which experiments are nolonger ethically justifyable under this leg-islation. In October 2004 People for Ani-mal Rights Germany have started the cam-paign “It’s my life” to stop primate

experiments in Germany. The campaignincludes informing the public and workingon juridical and political level. We alsowant to raise the question whether primateexperiments are ethically justifyable underArticle 20a of the German constitution.Our aim is to show that they are not – andtherefore convince politicians to make use


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Preclinical evaluation of biological drugcandidates, e.g. antibodies, vaccines andgrowth factors, demands novel predictivecell based assays. Species specificity ofthose tests is crucial for valid efficacyand side effect data. Three-dimensionaltissue culture of the respective species,e.g. human, seems to be mandatory. Incontrast to chemical drugs, any injectionof biologics induce a light to severe im-

mune response. In case of vaccines thisparticular response is the most wanted.For therapeutical antibodies for example,immunogenicity is a no go criteria. Wewill present concepts of organoid culture,to address vaccine efficacy and immuno-genicity of biologics. Complex co-cul-tures of human dendritic cells and lym-phocytes were performed in miniaturisedperfusion cell culture devices. Cytokine

patterns, histological analyses and cellimaging are used to evaluate functionali-ty and responsiveness of these tissue cul-tures. First attempts were made to multi-plex, miniaturise and automatise thesystems for later high content. Processperformance was evaluated for robust-ness, consistency and standardisation.

Lecture

Human organoids for immunogenicity andimmunotoxicity testingChristoph GieseProBioGen AG, AG Tissue Engineering, Berlin, GermanyE-mail: [email protected]

Keywords: cell based assay, immunogenicity, lymphatic organoid, perfusion culture

In the recently completed project spon-sored by FFVFF Zurich, we adapted sev-eral cell lines frequently used by biotech-nologists and molecular biologists tochemically fully defined culture media.We followed the growth behaviour be-fore and during the adaptation phase. After a stringent quality control and au-thentication, the cell lines are now avail-able for the scientific community by theEuropean Collection of Cell Cultures(ECACC). Cells cultured without FBS(fetal bovine serum) are in general more

sensitive to chemical agents and mechan-ical stress. The proteins in FBS, consist-ing mainly of albumins, create a bal-anced viscosity in the cells’ environmentand bind added reagents, as well as toxicmetabolites. These facts are especiallyimportant in the process of cryo-conser-vation as it is often done using a mixtureof culture medium, FBS and DMSO asanti-freeze agent. The main part of ourpresentation deals with the evaluation ofa freezing protocol designed for cellscultured in absence of FBS. Therefore

we developed a new chemically fully de-fined cryo-medium with Pluronic F68™as active ingredient. Using FILOCETHas freezing medium with Pluronic F68™medium we demonstrate the recovery ofliving cells after thawing of a cryo-con-served cell sample being factor 1.5 high-er as compared to the standard FBS/DMSO mixture. We also show the influ-ence of different cooling rates on the re-covery rate of frozen cells.

Lecture

A new high potent freezing medium closes the lastgap in the “serum free” cell culture techniqueYanela González Hernandez1, Susanne Scheiwiller2, Franz Gruber 2 and René W. Fischer 1,1Swiss Federal Institute of Technology Zurich, Institute of Organic Chemistry ETH Zurich, Switzerland; 2FFVFF Fonds fürVersuchstierfreie Forschung, Zurich, Switzerland E-mail: [email protected]

Keywords: cryoprotectant, chemically fully defined cell freezing medium, chemically fully defined medium

Keywords: primates, ban, German Animal Welfare Law

of their governmental obligation byamending the German Animal WelfareLaw to outlaw primate experiments. Insome EU countries (Austria, Sweden,

Netherlands) experiments at least on greatapes have been made illegal. In Germanyno experiments on great apes have beenconducted since 1991. Our campaign

wants to see all primate experiments in-cluded in a future ban and is also seen inthe light of the currently ongoing amend-ment of the Directive 86/609.


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Cell culture models play a pivotal role inlaboratory research of various fields. Ap-part of existance of many various celllines, there is a substantial lack of good in-testinal cell culture models. In spite of thefact, that human colon tumorigenic celllines, like Caco-2, are widely used as amodel of a small intestine, such cell linesdo not represent the normal small intestine

of the man. It is accepted, that pig is muchbetter animal model than rodents in hu-man research , therefore we have estab-lished a functional cell model of an adultpig small intestine, consistent of two pigintestinal epithelial cell lines (PSI andCLAB) and macrophages from the pig pe-ripheral blood (PoMac). All newly estab-lished cell lines were were spontaneously

transformed to the continous cell line andcharacterised by morphological, immuno-chemical and functional characteristics.Results obtained proved, that the estab-lished functional cell model of the pigsmall intestine can be successfully us edin various studies involving the small in-testinal tract, as well as in studies of localor systemic immunity.

Poster

Establishment of a functional cell culture model of the pig small intestine Lidija Gradisnik 1, Bratko Filipic2, Christiane de Vaureix 3, Francois Lefevre3, Claude LaBonnardiere3 and Avrelija Cencic 1,4

1University of Maribor, Faculty of Agriculture, Dept. of Microbiology, Biochemistry and Biotechnology, Maribor, Slovenia;2University of Ljubljana, Medical Faculty, Institute of Microbiology and Immunology, Ljubljana, Slovenia; 3INRA, Unite de Virologie et Immunologie Moleculaires, Jouy-en-Josas, France; 4University of Maribor, Medical Faculty, Dept. of Biochemistry, Maribor, SloveniaE-mail: [email protected]

Keywords: cell line, functional cell model, small intestine, pig

Germany, Austria and Switzerland rep-resent the taillights of Europe with re-gard to citizens’ information rights. Inmany countries worldwide, citizens caninform themselves on how the statespends their tax money. This is not so inthe three alpine republics.

This form of access to informationhas enjoyed its longest tradition in Swe-den. Already in 1766, the citizens at-tained the right to study official docu-ments based on the freedom of thepress. This right was anchored in theSwedish constitution. What is called„Offentlighetsprincipen“ in Sweden isnow internationally known as „Freedomof Information“ or „The Principle ofPublic Access“.

In the USA this right was extended byBill Clinton in 1996, by provisions for

information requests via Internet. It has been investigated where in the

world this principle has been intro-duced, where it is planned and where itis absent (Harvard-Banisar-Study,2002). In Germany, a first attempt wasstarted on 1.1.2006 on the federal level,which must still be tested from the ani-mal protection side. From Austria wehave not yet heard of any kind of politi-cal approach towards this subject.

In Switzerland, it is often not evenrecognized as a problem by the politi-cians. The secretiveness of the officialsin Switzerland is so pronounced thatmembers of the cantonal committees foranimal experiments may not even en-gage experts for certain subjects as thiswould breach official secrecy require-ments.

Who is protected by this official se-crecy?

Is it the privacy of the scientist whocould otherwise perhaps not defendhim- or herself against public hostility?

Is it to protect the intellectual proper-ty of the scientists?

Does the agency itself need to be pro-tected?

Regulatory agencies in many wayssimply choose to believe in science. Atleast this is less work than evaluatingthe truth and probability content of aca-demic claims. The committees for ani-mal experiments are not in a position todo this because of the official secrecyrequirements. It is easier to leave thepublic in doubt about what experimentsare planned and what arguments speakfor or against them. Fear of (ill-)in-

Poster

On the status of citizens’ rights to information inGerman-speaking countriesFranz P. Gruber 1 and Susanne Scheiwiller2

1Doerenkamp-Zbinden Foundation, Zurich; 2Fonds für versuchstierfreie Forschung (FFVFF), Zurich, SwitzerlandE-mail: [email protected]


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formed laypersons may also play a role,even though sufficient well-educatedspecialists are now available who canread and interpret applications and eval-

uate them with regard to animal protec-tion aspects.

Animal protection requires that at leastthose animal experiments that are fi-

nanced with public funds should be ac-cepted by the public. Starting at a mediumlevel of severity they should be discussedin a suitable manner prior to approval.

Lecture

Safety in the cell culture laboratory Gerhard Gstraunthaler Dept. of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria E-mail: [email protected]

Keywords: risk assessment, biosafety level, biohazard

Safety is a prerequisite in every work-place. Identifying and evaluating risks,and taking appropriate action to avoid orminimize them, are the basis on whichsafety is built in any laboratory.

Risk assessment in cell culture labora-tories demands special awareness of po-tential hazards associated with the biolog-ical materials that are handled, and of thespecific physical and chemical hazards re-lated to cell and tissue culture work.

The risks associated with cultured cellsare dependent upon the species and themode of origin (primary cultures or celllines), and the procedures and techniquesapplied. Thus, the biosafety level of thecultured cells have to be defined. Cellcultures presenting the highest risks, forexample, are those of human and primateorigin. As a consequence, the cultured

cells should be handled at a containmentlevel in compliance with national bio-safety regulations. Other potential bio-logical hazards that should be consideredinvolve biological components of culturemedia (serum, tissue extracts, growthsupplements) and/or culture products,e.g. in eukaryotic expression systems,virus propagation or vaccine production.In addition, techniques and protocols togenerate genetically modified cells, forexample in mutagenesis, in vitro trans-formation, cell transfection, or cell hy-bridization can be hazardous.

The design and management of a cellculture laboratory are important consid-erations with regard to safety. Contain-ment is the most obvious means of re-ducing risk from cell cultures thatdemands the use of microbiological safe-

ty cabinets or laminar flow hoods. Formost cell lines, the appropriate biosafetylevel is Level 2 requiring a Class II mi-crobiological safety cabinet.

The cell culture laboratory is not a par-ticularly dangerous place to work withregard to chemical hazard, although gen-eral safety standards should always bemaintained to protect workers and theenvironment.

Physical hazards in cell and tissue cul-ture are directly related to the mainte-nance and storage of cultured cells.These include “heat” (e.g. autoclave anddry-heat oven), pressure and/or vacuumin sterile filtration, and extreme “cold” inhandling liquid nitrogen at freezing, stor-age, and thawing of cells.

Finally, appropriate safety measures inwaste disposal should always be applied.

Lecture

Assessment of applicability and tolerability ofdrugs and excipients on lung cell models withoutanimal testing Eleonore Haltner1, Udo Bock1, Manfred, Keller2, Ellen Bitterle2, Markus Tservistas 2, Karin Steinfuehrer 2 and Aslihan Akkar2

1Across Barriers GmbH, Saarbrücken, Germany; 2PARI GmbH, Munich, Germany E-mail: [email protected]

Introduction: Processes of drug develop-ment are growing more complex andtime consuming. Therefore, smart con-cepts should be integrated into the for-mulation development prior to clinical

studies to streamline the development interms of costs and risks. In-vitro cell cul-ture studies instead of animal studiesprove to be a fast and cost-effective toolto assess the applicability of a formula-

tion in the early development stage. Thispaper summarizes results with airwayand lung cells as tool for an excipient andformulation screening. Methods: Calu-3cells were cultivated in Minimum Essen-

Keywords: freedom of information, regulatory agencies, committees for animal experiments, tax money


The embryonic stem cell test (EST) is avalidated in vitro assay that has been established to classify compounds withrespect to their embryotoxic potential.The current experimental procedure involves differentiation of murine embry-onic stem cells (D3) into contracting car-diomyocytes. However, potentially em-bryotoxic drugs may effect primarilyother tissues than the myocardium. Con-sequently, this consideration promptedus to expand the EST to other major tar-get tissues. Here, we present a protocolfor differentiation of murine embryonicstem cells into neurones designed with

special regard to the testing of chemicals.This modified protocol is based on amonolayer differentiation procedure andoffers the advantage of a reproducibledevelopment of neural cells in a compar-atively short time. The differentiation ofD3 cells into neural cells was charac-terised by analysis of neurone-specificmarker gene expression using flow cy-tometry. In addition, the developing neu-rones were examined by immunofluores-cence staining using neurone-specificantibodies. As a result, we were able todefine neurone-specific molecular end-points for the detection of chemical

effects on embryonic development. Pre-liminary chemical testing revealed differ-ent sensitivities between neural cells andcardiomyocytes for a limited number oftest chemicals. The expansion of the EST to more than one target tissue willconsiderably improve the accuracy ofthis predictive screen by preventing falsenegative results. Moreover, the differenti-ation of murine embryonic stem cells into neurones might prove to be a usefultool in developmental neurotoxicity test-ing as well as in pharmacological testing.

Keywords: in vitro, embryotoxicity, embryonic stem cell test, differentiation, neurons, developmental neurotoxicity

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tial Medium (MEM) with Earl’s Saltsand L- supplemented with 10% FCS. Pri-mary cell were isolated as described inSteimer et al. 2005. For toxicity and per-meability studies cells were seeded onTranswell clear® filter inserts. The cellcultures were standardized by verifyingthe TransEpithelial Electrical Resistance(TEER) and transport markers for highand low permeable transport. The trans-port characteristics of drug substancesand the toxic effect of formulations andexcipients have been investigated. Re-

sults: Different formulations with thesame drug (e.g. Cyclosporin A) showedtoxic effect on cell monolayers depen-dent on the used excipients as well asconcentrations of the drug and/or excipi-ent. Additionally the transport mecha-nism of drug substances and the in-fluence of formulation could be investi-gated. Conclusions: Based on the cell results data, we conclude that cell culturemodels, are a useful tool to study perme-ation as well as irritation or toxic effectsof drugs, formulations and/or common

excipients selected to be used for inhala-tion. The tests offer the possibility to in-vestigate potential effects of the drugsand formulations in fast and cost effec-tive way before further development ef-forts and money are invested in more ex-pensive development steps or preclinicaland clinical evaluation of drug formula-tions with a markedly decrease of animalstudies. References: Steimer, A., Haltner, E. et.Al., J. Pharm. Res., submitted.

Lecture

Expansion of the embryonic stem cell test:Differentiation into neural cellsKatrin Hayess, Anke Visan, Roland Buesen, Katharina Schlechter, Birgitta Slawik, Horst Spielmann and Andrea SeilerFederal Institute for Risk Assessment, Berlin, GermanyE-mail: [email protected]

Keywords: drug development, inhalation, toxicity, permeability, transport mechanism, aerosols


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Traditionally, regulatory required vac-cine quality control relies heavily on theuse of laboratory animals. Both batchtesting for safety and testing for potencyis based on animal models and quite of-ten these models include procedures thatinduce severe pain and distress. Thus, itis estimated that for instance potencytesting of tetanus and diphtheria toxoidvaccines requires more than one millionanimals per year world-wide and as achallenge procedure with tetanus toxin ispart of the protocol, half of the animals is expected to die from tetanus. Thesefigures have urged the development of3R methods. In this presentation an

overview will be given of some major3Rs achievements and breakthroughsthat ultimately will allow batch qualitycontrol without the use of laboratory an-imals. These include the replacement ofchallenge procedures by serologicalmethods, the reduction of numbers of an-imals by single dose instead of multi-dose testing, and the developments in thearea of in vitro models and physicochem-ical procedures. Central in progress to-wards elimination of animal use is theacceptance of the consistency approach.The redline in the consistency approachis that a new batch of vaccine is nolonger seen as a unique product but as

only one of a series of batches producedfrom the same seed lot. As a conse-quence, a batch of vaccine producedshares many of the characteristics of theprevious batches that were producedfrom the same seed lot. This allows for anew strategy of vaccine quality control,giving emphasis to aspects such as inprocess testing, the implementation ofGMP principles and to quality assurance.These approaches particularly rely onnon-animal test models. An outline willbe given of a protocol for the consistencyapproach and potentials and possible pit-falls will be discussed.

Lecture

Towards eliminating the use of animals forregulatory required vaccine quality control Coenraad Hendriksen1 and Marlies Leenaars2

1Netherlands Vaccine Institute (NVI, NL); 2Netherlands Centre Alternatives to Animal Use (NCA, NL), Utrecht University ,Bilthoven, The NetherlandsE-mail: [email protected]

Keywords: vaccine, quality control, toxoid, consistency, in vitro

There is a demand for novel analgesics toprovide relief for different types of pain.During development of new analgesicstraditional pa in tests are performed mea-suring behavioural reactions of awakeanimals which consequently suffer fromthese painful stimuli. A solution couldcome from measuring responses topainful stimuli in anesthetized animalsnon-invasively by functional magneticresonance imaging (fMRI). This methodprovides highly resolved objective func-tional information on the processing ofnociceptive stimuli throughout the wholebrain as has already been demonstratedin human pain studies. Consequently,

this method could also improve objectivemeasurements of modulatory effects ofanalgesics. Moreover, optimized data-analysis strategies can help to reduce thenumber of experiments needed to obtaina representative activatin maps e.g. fordifferent analgesics. We established sucha fMRI testing system in anesthetizedrats using a mild noxious heat stimula-tion applied to rat hindpaws. Because thetesting is applied to animals under anaes-thesia with mild stimulation (tempera-ture: 34 - 45°C, suitable for humans too)we are minimizing the stress for the ani-mal. Moreover, we were able to 1) obtainreliable objective information of differ-

ent pain competent structures along thepain pathway 2) by applying modern im-age processing and data analysis tech-niques we were able to obtain represen-tative group average results at a minimalnumber of experiments. By doing so wecould define the degree of pain suppres-sion of different conventional analgesics.Such a system can further be applied forinvestigating (chronic) pain processes.This would open a new avenue for re-search on pain chronification and it maycontribute to the evaluation of novelmore specific analgesics intended to in-hibit or even reverse chronic pain.

Poster

Measuring nociception by fMRI in anesthetisedanimalsAndreas HessFAU Erlangen NürnbergE-mail: [email protected]

Keywords: fMRI, rat, human, computer, image processing


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Mitochondrial dysfunction is a majortoxicity of nucleoside reverse transcrip-tase inhibitors (NRTIs), used for treat-ment of human immunodeficiency virus(HIV) infection. NRTIs induce mito-chondrial dysfunction mainly through in-hibition of mitochondrial (mt) DNA syn-thesis which could lead to a wide rangeof severe adverse effects. Lactic acidosisand hepatic steatosis are increasinglyrecognized in NRTI treated patients.Since at present no clinically relevant invitro model exists to investigate mito-chondrial toxicity, in vivo studies aremostly used to confirm preliminary invitro data. There is a need for developingnon-animal models to detect mitochon-drial toxicity induced by NRTIs. The aim

of this study was to establish an in ovomodel for the investigation of mitochon-drial toxicity. Incubated hens’ eggs weretreated with the NRTIs zalcitabine (ddC),didanosine (ddI), stavudine (d4T), andzidovudine (AZT) between days 5 and 9by air cell administration. Ethidium bro-mide (EtBr), an agent known to depletemtDNA in cell cultures, was used as pos-itive control. Mitochondrial toxicity wasinvestigated at day 11 of incubation,when the embryo’s nervous system is notyet completely established. Mitochondri-al function was analysed by measuringblood lactate levels and ATP content inerythrocytes. mtDNA content in relationto nDNA content was determined in em-bryos’ livers using a novel dual colour re-

al-time PCR based assay. Treatment withAZT, ddI, ddC, and EtBr resulted in sig-nificant increase in blood lactate levels.A significant decrease of ATP contentwas only observed for AZT and EtBr. Inthe liver a significant depletion of mtD-NA was found for ddI, ddC, and EtBr butnot for AZT, a weak inhibitor of mtDNAsynthesis. d4T did not induce significantchanges on the parameters tested. The re-sults are in good agreement with those ofother published data from in vitro and in vivo studies. The in ovo model is asimple, sensitive, and rapidly respondingalternative model to animal testing, applicable for studying mitochondrialtoxicity of NRTIs and other agents.

Poster

Investigating mitochondrial toxicity of nucleoside reverse transcriptase inhibitors andethidium bromide in ovoDoris Höschele, Inmaculada Garcia Moreno and Martina WiertzFederal Institute for Drugs and Medical Devices, Bonn, GermanyE-mail: [email protected]

Keywords: in ovo, chick embryo, mitochondrial toxicity, nucleoside reverse transcriptase inhibitor, ethidium bromide, alternativeto animal testing

The reliability of skin corrosivity testingusing epidermal models was shown al-ready with different reconstituted sys-tems. The successful classification in thisfield is required for the more subtle task ofcorrect identification of substances withskin irritative properties. To facilitate cor-rect classification in those studies theskinmodels were reconstituted of neonatalforeskin keratinocytes as it is widely ac-cepted that using those cells enhance re-producibility. Here we present data on the

reproducible and reliable classification ofall twelve reference compounds of OECDTG 431: “In Vitro Skin Corrosion: HumanSkin Model Test” using Epidermal SkinTest 1000 (EST-1000). The results are ob-tained in a blinded multicenter trial fulfil-ing all the criteria to be peer reviewed according to the ECVAM validationstrategies. To support our data the propermorphology of EST-1000 is demonstratedby an extensive histological and immuno-histochemical characterization. Further-

more the intact barrier function is con-firmed by showing the resistance againstseveral solutions of detergents over a longperiod and for different batches. Addition-ally, we present promising data using thisadult Epidermal Skin Test in testing of irritative as well as phototoxic properties.With these data we demonstrate the adap-tion of this skin model to the actual pur-pose. In respect to animal testing thisadaptability is one major advantage of invitro skin models.

Lecture

Increased reproducibility in toxicity testing usingEpidermal Skin Test 1000Jens J. Hoffmann, Eckhard Heisler, Jan Losse, Diemo Thomas and Horst W. FuchsCellSystems Biotechnologie Vertrieb GmbH, R & D, St. Katharinen, Germany E-mail: [email protected]

Keywords: skin, epidermal model, skin corrosion, skin irritation, reconstructed skin


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Most of the in vitro cell models for longterm testing of chemicals suffer of at leasttwo shortcomings: 1)The failure of repro-ducing the in vivo physiological character-istics of the corresponding tissues, such isthe case for the immortalised cell lines. 2)A limited shelf-life, for example, thefreshly established primary cell cultures.Our company has developed and commer-cialises a novel in vitro cell model of thehuman airway epithelium which is free ofthese limitations. The epithelium is notonly morphologically and functionallydifferentiated; it can also remain at ahomeostatic state for more than sixmonths. Indeed, under the electronic mi-

croscope, the typical ultrastructures of thehuman airway epithelium, such as thetight junctions, the cilia, the basal cells,the mucuous cells, could be observed. Theepithelium is resistant when measured bytrans-epithelial /cm2. The ionΩelectricalresistance (TEER): the mean value isabout 450 channels like the sodium chan-nel and chloride channel are fully func-tional and respond normally to their spe-cific inhibitors and activators whenmeasured in modified Ussing chambers.Moreover, the epithelial cells react to pro-inflammatory mediators such as TNF-α ina physiological manner: the amount of se-creted interleukin-8 (a marker of inflam-

matory reaction of the airway epithelium)increased significantly after TNF-stimula-tion, and then decreased gradually to thebasal level after 5 days of recovery. Re-markably, the epithelium has a strong ca-pacity of regeneration after mechanical orchemical injuries. Furthermore, our in vit-ro cell model, when generated with cellsisolated from patients suffering of geneticdiseases (cystic fibrosis, primary ciliarydyskinesia), reproduces the pathologicalphenotypes of the disease. The fidelityand the longevity of our in vitro humanairway epithelium cell model make it anew valuable tool for the long term toxic-ity testing of molecules.

Lecture

A novel in vitro cell model of muco-ciliated humanairway epithelium for long term toxicity testing Song Huang, Ludovic Wiszniewski, Karim Khatib and Jean-Paul Derouette Epithelys, Geneva, SwitzerlandE-mail: [email protected]

Keywords: primary cell culture, airway epithelium, long term toxicity testing

Although laudable and to some extentcost-effective for the pharmaceutical in-dustry, the latter will not set about large-scale reduction of animal testing unlessmajor financial incentives and viable al-ternatives are forthcoming. A detailedknowledge of the Human genome andpost-genomic systems integration offerconcrete potential to achieve both. For thefirst time in the history of the pharmaceu-tical industry, the scientific communityhas access to an accurate ‘Parts-List’ ofall the drug targets in the Human body.However, because this translates intosome 20 billion distinct drug-binding

sites, each needing to be assessed ‘in sili-co’, the task is highly computationally in-tensive. Encouragingly, the last decadehas also witnessed significant advances incomputing power. As in other scientificdisciplines, the biomedical sciences anddrug development must become increas-ingly driven by computation as part of ef-forts to more accuratel y prediction Ad-verse Drug Effects prior to enteringanimal testing and clinical trials. Both hu-mans and animals will benefit directlyfrom such approaches. Concrete exam-ples will be drawn from the recent failureof Vioxx TM, the >27,000 heart attacks

and sudden cardiac deaths registered bythe FDA in association with Vioxx TMduring its stay on the market and the $10 billion (USD) negative impact on theMarket Capitalisation of the Merck Cor-poration as proof of concept. A new era ofenhanced target selectivity (better drugs)can run hand-in-hand with short term re-duction of animal testing due to what istermed: ‘Early Cull’, namely killing po-tentially deleterious compounds well inadvance of animal and clinical testing.Successful delivery of such can also re-sult is large-scale economies of both costand time during drug development.

Lecture

Knowledge of the human genome (total DNA sequence)as an opportunity to build better drugs and concomi-tantly reduce the need for animal experimentation Ian Humphery-SmithBiosystems Informatics Institute, Newcastle-upon-Tyne, UKE-mail: [email protected]

Keywords: enhanced target selectivity, proteomics, biocomputing, in silico modelling, drug screening, adverse side effects


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Awareness of animal suffering and possi-ble replacements of animal experimentshave been receiving great attention bycommunities around the world. The threeRs of Russell and Burch introduced dur-ing the 50s put profound influence onscientific communities. With the direc-tive “The Council Directive for the Pro-tection of Vertebrae Animals Used forExperimental and other Scientific Pur-poses -” in 1986, the EU countries are re-quired to adopt new regulations. Besidesgoverning the Member States, the direc-tive 86/609/EEC also influenced indi-rectly other European countries includ-ing Turkey. Interactions between Turkishresearchers with European counterpartsplayed a crucial role in this sense.Turkey, an EU candidate country, madesignificant progresses recently as far as

laws and regulations on animal experi-mentation. Turkish Parliaments voted thenew Animal Rights Act on the 24th ofJune 2004. In this sense, directives con-cerning animal experiments, ethics com-mittees, and lab animal husbandry havebeen prepared and under review. The di-rective of the Ministry of Agriculture hasalready proclaimed a new directive onlaboratory animal medicine so that theuse of animal has been regulated by legalacts. The Scientific and Technical Re-search Council of Turkey, the main grantprovider for scientific studies in Turkey,no longer grants fund for animal experi-mentations without approval from an eth-ic committee. Alternatives to animal ex-perimentations concept is fairly a newconcept in Turkey. Ethic committees nowhave been questioning if possibly the an-

imal experiments can be replaced or re-duced to some degree with cell culture orwith lower vertebrae. For example, inGulhane Medical Academy (GATA), thenematod Caenorhabditis elegans hasbeen used in aging and genetic studies.There are no formal lectures or educationon alternatives yet. However, awarnessamong researchers and lab animal scien-tists are the driving forces at this point.There has been an awaking among Turk-ish colleagues for alternative methods.However, investment on education of in-vestigators and development of alterna-tive methods are crucially important. Inaddition, transferring experience of Eu-ropean colleagues is equally important.The bond between international and Eu-ropean organisations such as ECVAMand ICLAS should be strengthened.

Poster

Progress in alternative concepts in Turkey Tayfun Ide 1 and Siyami Karahan2

1Department of Lab. Animal Health, Center for Research and Development, Gulhane Military Medical Academy, Etlik, Ankara,Turkey; 2Department of Basic Sciences, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale, Turkey E-mail: [email protected]

Keywords: alternatives, animal experiments, Turkey, directive, law regulations

Active ingredients of herbal origin be-came a regular part of cosmetic prod-ucts, especially in formulations intendedfor consumers with sensitive or dry skin,with the aim to improve skin conditionand appearance. Herbal ingredients arereported to promote physiological func-tions of the skin and may offer a bal-anced complex of health effects as moisturising, free radical scavenging,calming and anti-inflammatory, improv-ing skin elasticity, anti-aging, healingsunburn or chemical induced irritation.

The aim of the study was to assess thesignificance of addition of selectedherbal ingredients, used in formulationsof cleaning products in order to min-imise possible adverse irritative effectsof tensides, e.g. Sodium Dodecyl Sulfate(SDS). The protective effects of selectedactive ingredients were tested in vitro inthe cell culture of 3T3 fibroblasts and inthe human reconstructed skin model(EpiDerm ™) and subseqently evaluatedin vivo by testing in a group of volun-teers by means of closed epicutaneous

patch test according to COLIPA Guide-lines. Protective effects against SDS cytotoxicity were demonstrated in thecell culture in case of a number of natural substances, e.g. Green Tea, AloeVera, Pronalen Sunlife, Pronalen Cereal,Sea Silk, Pronalen Sensitive Skin andChamomile. The effects of the mostpromising substances were confirmed inthe 3D human skin model. Results fromthe in vitro systems were compared toresults obtained in an experimentalstudy comprising epicutaneous test in a

Poster

Herbal cosmetic ingredients – protective effectsassessed in vitro and proved in vivoDagmar Jírová 1, Kristina Kejlová 1, Hana Bendová1, Hana Kolárová 2 and Marek Brabec1

1National Institute of Public Health, Prague, Czech Republic; 2Faculty of Medicine, Palacky University, Olomouc, CzechRepublic E-mail: [email protected]


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group of volunteers. Although all the se-lected herbal substances, in accordancewith results obtained in vitro, exhibitedprotective effect against SDS-inducedskin irritation in human volunteers, the

highest degree of protection in vivo wasproved for Pronalen Sensitive Skin andChamomile. The in vitro test systemswere found to be a useful tool forscreening of biological effects and be-

came a valuable part of regular safetyand efficacy testing of cometics, em-ployed before confirmatory testing inhuman volunteers.

InterNICHE has been working interna-tionally to promote and implement alter-natives in higher education for 18 years,facilitating the replacement of harmfulanimal use and building a broad network

with contacts in over 50 countries. Fromthe InterNICHE experience, successfulinternational work requires qualities andpractices from organisations that include:a bold and positive vision, and an aware-

ness of the links between issues; a com-mitment to pro-actively catalyse sustain-able change and create win-win solu-tions; the design of organisationalstructures conducive to participatory

Poster

Facilitating the implementation of best practice education through access to and training in alternatives Nick Jukes 1 and Monika Percic2

1InterNICHE, Leicester, England; 2InterNICHE Alternatives Loan System, Ljutomer, Slovenia E-mail: [email protected]

Lecture

Internationalising alternatives in higher education Nick Jukes InterNICHE, Leicester, England E-mail: [email protected]

Keywords: alternatives, animals, best practice, education, India, InterNICHE, library, students, teachers, training

Access to alternatives plays a crucial rolein familiarising teachers with the diversi-ty and quality of tools that are availableto support best practice life science edu-cation. Equally important is training thatallows for a more detailed exploration ofspecific alternative tools and approaches.To meet the need for access to and train-ing in alternatives, InterNICHE main-tains an Alternatives Loan System, andorganises training seminars across theworld to provide expert training. The Al-ternatives Loan System is an evolving li-brary of alternatives available for freeloan worldwide. It was established dur-ing 2001-2002 and includes over 100CD-ROMs, videos, simulators and train-ing mannekins, chosen for their peda-gogical value and potential to replacecommon dissections and animal experi-

ments. Borrowers include teachers, stu-dents, animal ethics committees, govern-ment ministries, organisations and cam-paigners in over 40 countries. Thefacility has serviced over 200 loans,comprising over 4000 usages of individ-ual alternatives. As a tool for facilitatingimplementation, the value of the LoanSystem is indicated by significant teach-er use and the high number and wide ge-ographical range of loans, subsequentpurchase and implementation of prod-ucts, direct replacement of harmful ani-mal use, and providing an internationalresource for campaigners. Small-scale‘micro-Loan Systems’ have been estab-lished in Brazil, Russia, Ukraine, Indiaand Japan. Since its inception in 1988,InterNICHE has organised demonstra-tions and training at annual conferences

and dedicated training seminars. The al-ternatives used are selected for their rele-vance to specific national and cultural re-alities in order to maximise theopportunities for replacement. Using theAlternatives Loan System and the skillsof local teacher trainers, over 400 univer-sity teachers were trained in alternativesand animal welfare during August-September 2004 at seminars in over 10cities across India. This project was or-ganised by InterNICHE in conjunctionwith the World Society for the Protectionof Animals (WSPA) and many commit-ted local organisations, and was the firstof its kind worldwide that provided train-ing at a national level. The network isplanning further alternatives training for2006, including across Latin America,the Middle East and North Africa.

Keywords: herbal ingredients, cell culture, 3D skin model, cytotoxicity, epicutaneous test, irritation


Due to industrial development and therapid growth of human population, therole of chemicals in the everyday life has

considerably increased. Ecotoxicologyaddresses the environmental hazards ofxenobiotics already accumulated in the

environment and also of newly producedchemicals. The political awareness onknowledge on human safety and environ-

The objective of this work is to define apractical sequence scheme for a fast andreasonable development of semisolidformulations without animal testing. Thedevelopment of new topical formulationis depending on drug and skin specificproperties. The way from active com-pound to a proper semisolid formulationis a challenge. For this purpose four ac-tive compounds were cho-sen from thecorticosteroid group: Betamethasonevalerate, Clobetasol propionate, Hydro-corti-sone and Mometasone furoate. TwoOECD-markers for in vitro percutaneousabsorption: Caf-feine and Testosteronewere taken additionally as controls.Three different semisolid formulationswith an active agent concentration of

0.1 % were used as delivery system (am-phiphilic cream DAC, hydrophobic an-hydrous wool-fat ointment DAB and hy-drophilic carbomer hydrogel DAB).Physicochemical profiling and the in vit-ro release and permeability profile acrosshuman skin of the model drugs testos-terone and caffeine will be here present-ed. The transport studies showed thatpermeation from formulations withtestosterone and caffeine is dependent onformulation type. For both analyzed sub-stances we could see the highest re-leaserate from the hydrogel. For the lipophiliccompound, testosterone, the maximumabsorp-tion rate was observed from thehydrophilic hydrogel. In contrast, the re-sults of the in vitro permeation studies

for the hydrophilic caffeine from threedifferent formulations show the highestpermeation rate from the hydrophobicointment, which shows the lowest releaserate. For both test substances the lowesttransport through the analyzed humanmembranes was seen from the am-phiphilic cream. The observed releaseprofiles of testosterone and caffeine showthat the release rate of the drug from theformulation to the skin surface is not al-ways the rate-limiting step in the overalldrug diffusion process. We hope to makesome contributions to the reduction ofthe animal tests in the development offormulations for topical application byusing meaningful in vitro alternativesand physicochemical methods.

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democracy, alliance building and the or-ganic growth of the network; the practiceof solidarity and support for local initia-tives rather than empire building; and theprovision of resources and training foraction and capacity building. The presen-tation will draw on examples of Inter-NICHE projects such as the productionand multi-language translations of print-

ed, video, and website resources; the Alternatives Loan System for trial of soft-ware, mannekins and simulators any-where in the world; the international Hu-mane Education Award for localdevelopment and implementation of al-ternatives, including freeware; supportfor student conscientious objectors; andconferences, outreach visits, and training

in alternatives for teachers. An overviewof the international momentum towardsfull replacement of harmful animal use ineducation will be presented. The chal-lenges met within such work will also beexplored, and suggestions of how toovercome them will be given.

Poster

Physicochemical profile of testosterone and caffeineand permeability across human skin in vitroMonika Kaca1, Udo Bock1, Eleonore Haltner-Ukomadu1 and Rolf Daniels 2

1Across Barriers GmbH, Saarbruecken, Germany; 2Dept. of Pharmaceutical Technology, Eberhard Karls University of Tübingen, GermanyE-mail: [email protected]

Lecture

Ecotoxicological tests and recombinant luminescentmicrobial models in toxicity studies: contribution to 3Rs Anne Kahru and Angela Ivask National Institute of Chemical Physics and Biophysics, Tallinn, Estonia E-mail: [email protected]

Keywords: alternatives, animals, education, freeware, grant, internet, InterNICHE, library, students, training, translation

Keywords: semisolid formulations, alternative methods, physicochemical metohods, skin absorption in vitro


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mental effects of chemicals is reflectedby the new EC chemical policy REACH.Both, the development of new chemicalsand environmental protection requiretoxicity testing that up to now implies theusage of a very large number of laborato-ry animals. The majority of the so farproposed in vitro tests – although greatlyreducing the number of experimental an-imals – involve the use of cell and tissuecultures derived from vertebrate species.The role of the whole non-animal organ-isms (e.g. bacteria, crustaceans, proto-zoa, plants, yeasts) in human health riskassessment, especially for regulatorypurposes, is still relatively small. Al-though many assays using non-animalsas test organisms, e.g. Vibrio fischeri bi-oluminescence inhibition assay, Daphniamortality assay, algal growth inhibitiontest etc., have been validated and even

accepted on regulatory level (OECD,ISO), these tests have mainly been usedin ecotoxicity testing. The application ofecotoxicological tests (e.g., luminescentbacteria, protozoa, crustaceans) at leastat the early screening stage of all areas oftoxicological research should be serious-ly considered, as this could save a lot oftime, money, manpower and lives of ex-perimental animals. The single non-spe-cific toxicity tests, although unreplacablein evaluation of integral toxic effects, arenot able to identify mechanisms of toxic-ity. However, competent interprepationof a test battery data may lead to a betterunderstanding of toxic action. Differentnon-vertebrate tests have shown to besensitive and of reasonable predictivepower for a number of organic and inor-ganic chemicals and even nanoparticles –new emerging toxicants. Last but not

least, specific microbial assays may beset up by genetically modifying bacteria(prokaryotic models) or yeasts (eukary-otic models). It is possible to incorporateinto these organisms special reporter ele-ments which synthesis will be triggeredas a response to specified target bioavail-able chemical(s) and thus reporting ontheir transfer into the cell and resultingbiological effects in vivo. Moreover, the microbial models may be chosen according to their medical significance,amenability to genetic manipulations,physiological simplicity and ease of han-dling. Recombinant luminescent bacteriathat increase their light production in response to bioavailable heavy metalsand phenols have been constructed andused as powerful tools in analysis of thebioavailability of these compounds incomplex matrices.

Acute fish toxicity is an important end-point for ecotoxicological hazard identi-fication and assessment of chemicals. Ithas been shown that fish embryo testinghas the potential to serve as a replace-ment for in vivo testing on fish. In orderto build up methodologies that can beused as screening tools during substancedevelopment or to prioritize in vivo tox-icity testing, a screening protocol usingembryos of zebra fish was developed andvalidated in our laboratory. The screen-ing test was conducted in 24-well platesusing one fertilized zebra fish embryoper well. The embryos were maintainedin our ecotoxicology facilities. Morethan 40 test substances with knownacute fish toxicity covering a wide range

of chemistry, including agrochemicalswere used to validate this test system.The substances were either dissolved in water, or acetone used for highlylipophilic compounds as a solvent, re-spectively. Four different dose groupscovering a concentration range of threeto four orders of magnitudes were usedwith 20 embryos each. Additionally, an-other 20 embryos at a time served as sol-vent (water, where necessary acetone)and positive (3,4-dichloraniline) con-trols. All test compounds have been test-ed three times at minimum. LC50 valueswere estimated from defined lethal end-points as described in the literature andcompared to acute fish LC50 values. Theresults derived from testing on fish em-

bryos using this screening protocol cor-related well with the acute toxicity infish and showed good reproducibility.Therefore, this test system can at least beused as a tool for screening and priori-tizing purposes. Additionally, we pursuethe development, validation and regula-tory acceptance of alternative methods(3R concept of Russell and Burch,1959), which are highly needed regard-ing the new EU Chemicals RegulationREACH and the Cosmetic Directive aswell as for other regulatory purposes.Thus, further efforts are undertaken tocontribute to a full validation of the fishembryo test that may become a replace-ment for acute tests using adult fish.

Poster

Inhouse validation of a screening test for the determination of acute fish toxicity using embryos of zebra fishHennicke G. Kamp, Volker Hisgen, Edgar Leibold, Kristin Radke, Sabine Zok and Bennard van Ravenzwaay BASF Aktiengesellschaft, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany E-mail: [email protected]

Keywords: Vibrio fischeri, protozoa, crustaceans, recombinant bacterial sensors, bioavailability, REACH, nanoparticles, 3Rs

Keywords: fish embryo, acute fish toxicity, screening, alternative methods, validation


Animal use resulting in harm or deathhas historically played an integral role inveterinary education, in disciplines suchas surgery, physiology, biochemistry,anatomy, pharmacology, and parasitolo-gy. However, the last decade has seen arapid increase in the availability of non-harmful alternatives, such as computersimulations, high quality videos, ‘ethi-cally-sourced cadavers’ such as thosefrom animals euthanased for medicalreasons, preserved specimens, modelsand surgical simulators, non-invasive

self-experimentation and supervisedclinical experiences. However, experi-ence has shown that many veterinaryfaculty remain opposed to such teachingmethods, usually citing teaching efficacyas their main concern. Consequentlystudies were reviewed comparing learn-ing outcomes generated by non-harmfulteaching methods with those achievedby harmful animal use. Of ten studiesfrom 1989 to 2000, nine assessed surgi-cal training – historically the disciplineinvolving greatest harmful animal use.

30% (3/10) demonstrated superior learn-ing outcomes using more humane alter-natives. 60% (6/10) demonstrated equiv-alent learning outcomes, and only onestudy demonstrated inferior learningoutcomes. Eleven additional studies inwhich comparison with harmful animaluse did not occur illustrated other benefits of humane teaching methods,namely; time and cost savings, increasedrepeatability and flexibility of use, customization of the laboratory experi-ence, more active learning, facilitation

Poster

Humane teaching methods demonstrate efficacy in veterinary education Andrew KnightAnimal Consultants International, London, UKE-mail: [email protected]

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Due to the increased need for informationon the safety of chemicals (driven mainlyby new the chemicals policy of the Euro-pean Commission (REACH)) a lot of at-tention is paid to development and valida-tion of in vitro methods replacing orreducing the use of test animals. Recon-structed human skin models offer a possi-bility to satisfy regulatory requirementsfor testing of local toxicity and at the samethey contribute to the 3R approach. For invitro skin irritation and corrosion testingseveral test assays have been successfullydeveloped in the past applying diverse re-constructed human epidermal (RHE)models. However, since several RHEmodels disappeared from the market aftervalidation (and with them the validated as-says), a common test protocols, applicableto all well developed and standardised

RHE models had to be developed. Forskin corrosion and acute phototoxicity, theusefulness of a common protocol has beenalready proven (Liebsch et al., 1997;Liebsch et al., 1999). Recently an attempthas been made to develop a \"commonskin irritation protocol\" for EpiDerm andEPISKIN RHE models (Cotovio et al.,2005; Kandárová et al., 2005). This proto-col is currently evaluated in an ECVAMSkin Irritation Validation Study. To showthat the \"common protocol concept\"works ZEBET has in 2004 performed twoadditional studies employing the SkinEth-ic RHE model. First, in a co-operationwith BASF AG (Germany) and Safe-Pharm (UK) we applied the common skincorrosion protocol of OECD TG 431.Than in a co-operation with SkinEthicLaboratories (France) and SCHERING

AG Germany) we applied the \"commonskin irritation\" protocol to SkinEthicRHE. After minimal adaptations specificfor SkinEthic RHE model almost identicalresults compared to EpiDerm andEPISKIN were obtained in both assays. Inthe above mentioned studies the reliabili-ty of the common protocol and similaritybetween three reconstructed human epi-dermal models have been evaluated andconfirmed. Minor, probably model-relateddifferences in prediction have been ob-served. However, taken into account largevariability of in vivo responses, the abovementioned RHE models and protocols canprovide reliable prediction of irritationand corrosion potential of chemicals, thatare concordant between laboratories andover time.

Keywords: skin irritation, skin corrosion, in vitro, common protocol, validation, RHE models

Poster

Development and evaluation of a robust, common protocols for reconstructed human skinmodels: In vitro skin irritation/corrosion testing Helena Kandárová, Manfred Liebsch and Horst Spielmann Fachgruppe 37 – Alternativmethoden zum Tierversuch (ZEBET), Bundesinstitut für Risikobewertung (BfR), Berlin, Germany E-mail: [email protected]

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of autonomous and life-long learning,improved attitudes towards computersand alternatives to animal use, and in-creased employer perception of comput-

er literacy. The results indicate that vet-erinary educators can best serve theirstudents and animals, while minimizingfinancial and time burdens upon their

faculties, by introducing well-designedteaching methods not reliant upon harm-ful animal use.

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

Keywords: REACH, alternative, animal experiment, animal test, bioassay, cancer prevention, carcinogenicity, chemicalclassification, chemical safety, computer simulation, in vitro, risk assessment

The 2001 European Commission propos-al for the Registration, Evaluation and Au-thorisation of Chemicals (REACH) aimsto improve public and environmentalhealth by assessing the toxicity of, and re-stricting exposure to, potentially toxicchemicals. The greatest benefits are ex-pected to accrue from decreased cancerincidences. Hence the accurate identifica-tion of chemical carcinogens must be atop priority for the REACH system. Dueto a paucity of human clinical data, theidentification of potential human carcino-gens has conventionally relied on animaltests. However, our survey of the US En-vironmental Protection Agency’s (EPA’s)

toxic chemicals database revealed that, fora majority of the chemicals of greatestpublic health concern (93/160, i.e.58.1%), the EPA found animal carcino-genicity data to be inadequate to supportclassifications of probable human carcino-gen or non-carcinogen. A wide variety ofspecies were used, with rodents predomi-nating; a wide variety of routes of admin-istration were used; and a particularlywide variety of organ systems were affect-ed. These factors raise serious biologicalobstacles that render the meaningful ex-trapolation to humans profoundly diffi-cult. Furthermore, that InternationalAgency for Research on Cancer assess-

ments for the same chemicals are signifi-cantly different, indicates that the true hu-man predictivity of animal carcinogenici-ty data is even poorer than is indicated bythe EPA figures alone. Consequently, wepropose the replacement of animal car-cinogenicity bioassays with a tiered com-bination of non-animal assays, which canbe expected to yield a weight-of-evidencecharacterisation of carcinogenic risk withsuperior human predictivity. Additionaladvantages include substantial savings offinancial, human and animal resources,and potentially greater insights into mech-anisms of carcinogenicity.

Poster

Animal carcinogenicity studies: Implications for the REACH System [Full paper to be published 2006 in Alternatives to Laboratory Animals 34, suppl., in press.]

Andrew Knight1, Jarrod Bailey2 and Jonathan Balcombe 3

1Animal Consultants International, London, UK; 2Faculty of Medical Sciences, University of Newcastle upon Tyne, UK; 3Physicians Committee for Responsible Medicine, Washington, DC, USAE-mail: [email protected]

The regulation of human exposures topotentially carcinogenic chemicals con-stitutes society’s most important use ofanimal carcinogenicity data. Environ-mental contaminants of greatest U.S.

concern are listed in the EnvironmentalProtection Agency’s (EPA’s) IntegratedRisk Information System (IRIS) chemi-cals database. However, of the 160 IRISchemicals lacking even limited human

exposure data but possessing animal dataas of January 1, 2004, we found that inmost cases (58.1%; 93/160) the EPAconsidered animal carcinogenicity datainadequate to support a classification of

Poster

Animal carcinogenicity studies: 1) Poor human predictivity [Full paper published 2006 in Alternatives to Laboratory Animals 34, 19-27.]


1Animal Consultants International, London, UK; 2Faculty of Medical Sciences, University of Newcastle upon Tyne, UK;3Physicians Committee for Responsible Medicine, Washington, DC, USAE-mail: [email protected]


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probable human carcinogen or non-car-cinogen. For the 128 chemicals with hu-man or animal data also assessed by theWorld Health Organization’s Internation-al Agency for Research on Cancer(IARC), human carcinogenicity classifi-cations were compatible with EPA classi-fications only for those 17 having at leastlimited human data (p=0.5896). Forthose 111 primarily reliant on animal da-

ta, the EPA was much likelier than theIARC to assign carcinogenicity classifi-cations indicative of greater human risk(p<0.0001). The IARC is a leading in-ternational authority on carcinogenicityassessments, and its significantly differ-ent human carcinogenicity classificationsof identical chemicals indicate that: (i) inthe absence of significant human data theEPA is over-reliant on animal carcino-

genicity data, (ii) as a result, the EPAtends to over-predict carcinogenic risk,and (iii) the true predictivity for humancarcinogenicity of animal data is evenpoorer than indicated by EPA figuresalone. EPA policy erroneously assumingthat tumours in animals are indicative ofhuman carcinogenicity is implicated as aprimary cause.

Keywords: animal experiment, animal test, bioassay, cancer prevention, carcinogenicity, chemical classification, chemical safety,risk assessment

Due to limited human exposure data,risk classification and the consequentregulation of exposures to potential car-cinogens has conventionally reliedmainly upon animal tests. However, sev-eral investigations have revealed animalcarcinogenicity data to be lacking in hu-man predictivity. To investigate the rea-sons, we surveyed the 160 chemicalspossessing animal but not human expo-sure data within the U.S. EnvironmentalProtection Agency chemicals databasethat had received human carcinogenicityassessments by January 1, 2004. Wefound a wide variety of species used,with rodents predominating; a wide vari-

ety of routes of administration used, anda particularly wide variety of organ sys-tems affected. The likely causes of thepoor human predictivity of rodent car-cinogenicity bioassays include (i) theprofound discordance of bioassay resultsbetween rodent species, strains and gen-ders, and further, between rodents andhuman beings; (ii) the variable yet sub-stantial stresses caused by handling andrestraint, and the stressful routes of ad-ministration common to carcinogenicitybioassays, and their effects on hormonalregulation, immune status and carcino-genesis predisposition; (iii) differencesin rates of absorption and transport

mechanisms between test routes of ad-ministration and other important humanroutes of exposure; (iv) the considerablevariability of organ systems in responseto carcinogenic insults, between andwithin species; and (v) the predisposi-tion of chronic high dose bioassays to-wards false positive results, due to theoverwhelming of physiological de-fences, and the unnatural elevation ofcell division rates during ad libitumfeeding studies. Such factors render at-tempts to accurately extrapolate humancarcinogenic hazards from animal dataprofoundly difficult.

Poster

Animal carcinogenicity studies: 2) Obstacles to extrapolation of data to humans[Full paper published 2006 in Alternatives to Laboratory Animals 34, 29-38.]



Keywords: animal experiment, animal test, bioassay, cancer prevention, carcinogenicity, chemical classification, chemical safety,extrapolation, risk assessment


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Conventional animal carcinogenicitytests take around three years to design,conduct and interpret. Consequently, on-ly a tiny fraction of the thousands of in-dustrial chemicals in use have so far beentested for carcinogenicity. Despite thecost of hundreds of millions of dollars,millions of skilled personnel hours, andmillions of animal lives, several investi-gations have revealed animal carcino-genicity data to lacking in human speci-ficity (ability to identify humannon-carcinogens), which severely limitsits human predictivity. Causes includethe scientific inadequacies of many car-cinogenicity bioassays, and numerous

serious biological obstacles, which ren-der attempts to accurately extrapolate hu-man carcinogenic hazards from animaldata profoundly difficult. Proposed mod-ifications to conventional bioassays haveincluded the elimination of mice as a sec-ond species, the use of genetically-al-tered or neonatal mice, decreased studydurations, initiation-promotion models,greater incorporation of toxicokineticand toxicodynamic assessments, struc-ture-activity relationship (computerised)systems, in vitro assays, cDNA microar-rays for detecting genetic expressionchanges, limited human clinical trials,and epidemiological research. Potential

advantages of non-animal assays whencompared to bioassays include superiorhuman specificity results, substantiallyreduced timeframes, and greatly reduceddemands on financial, personnel and ani-mal resources. Inexplicably, however,regulatory agencies have been frustrat-ingly slow to adopt alternative protocols.In order to decrease cancer losses to so-ciety, a substantial redirection of re-sources away from excessively slow andresource-intensive rodent bioassays, intothe further development and implemen-tation of non-animal assays, is bothstrongly justified and urgently required.

Poster

Animal carcinogenicity studies: 3) Alternatives to the bioassay [Full paper very recently published 2006 in Alternatives to Laboratory Animals 34 (1), 39-48.]



Keywords: alternative, animal experiment, animal test, bioassay, cancer prevention, carcinogenicity, chemical classification,chemical safety, computer simulation, in vitro, risk assessment

Biomedical research on captive chim-panzees incurs substantial animal wel-fare, ecological, ethical and financialcosts. Advocates of such research claimthese costs are outweighed by substantialadvancements in biomedical knowledge.To assess the accuracy of such claims we examined the disciplines investigatedin 749 studies of captive chimpanzees or chimpanzee tissues conducted from1995-2004. 48.5% (363/749) were biological experiments conducted in

nine disciplines, with cognition/neu-roanatomy/neurology (36.6%, 133/363)and behaviour/communication (20.7%,75/363) arising most frequently. 41.5%(311/749) were investigations of 30 viralgroups, with hepatitis C virus and humanimmunodeficiency virus, which bothcomprised 31.2% (97/311) of all virologyexperiments, arising most frequently.Therapeutic investigations comprised3.5% (26/749) of all chimpanzee experi-ments, of which 61.5% (16/26) explored

the pharmacological properties of variouscompounds. Other investigations in-cluded the testing of surgical techniquesor prostheses, anaesthesiology and toxi-cology experiments. Investigations ofeight parasitic species comprised 3.1%(23/749) of all chimpanzee experiments,of which the most frequent were the malaria protozoa Plasmodiumfalciparum and P. ovale (26.1%, 6/23),the roundworm Onchocerca volvulus(21.7%, 5/23), and the flatworm

Poster

Chimpanzee research: 1) Questionable contributions to biomedicalknowledgeAndrew Knight1, Jarrod Bailey2 and Jonathan Balcombe 3



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Schistosoma mansoni (17.4%, 4/23).Other diseases and miscellaneous experi-ments comprised 3.5% (26/749) of allchimpanzee experiments when combined,of which the most frequent were investi-gations of laboratory/husbandry tech-niques (42.3%, 11/26) and endotoxaemia(30.1%, 8/26). Radiation studies werealso conducted, and four other diseases

were also investigated, namely benignprostatic hyperplasia, Creutzfeldt-Jakobdisease, gastrointestinal bacteriology(Bacillus thuringiensis), and tuberculosis(Mycobacterium tuberculosis). To assessthe value of these 749 chimpanzee stud-ies, we randomly selected 100 and deter-mined the frequency with which theywere cited by other published papers.

Citations were not available for four stud-ies, however, 49.0% (47/96) of theremainder were not cited by any otherpapers. Research of lesser value is noteven published. These results indicate thatthe majority of chimpanzee research gen-erates data of very little use, and con-tributes very little to the advancement ofbiomedical knowledge.

Keywords: animal experiment, animal research, chimpanzee, bonobo, Pan troglodytes, Pan paniscus

Advocates of increased expenditure onchimpanzee research claim their geneticsimilarities to humans enables thisresearch to make critical contributions tocombating human diseases. To assess thevalidity of such claims, we randomlyexamined 100 studies of captive chim-panzees from a population of 749 studiesconducted between 1995 and 2004.Citations were not available for 4 chim-panzee studies. 49.0% of the remainder(47/96) were not cited by any otherpapers, demonstrating minimal contribu-tion towards the advancement of bio-medical knowledge generally. Onechimpanzee study was cited only by apaper for which no abstract was avail-able, and was discarded. Of the 95

remaining chimpanzee studies, 36.8%(35/95) were cited only by 116 papersthat clearly did not describe well devel-oped diagnostic or therapeutic methodsfor human diseases. Instead, abstractsfocused primarily on a surprising array ofnon-human species, including a largevariety of primates; or on human subjectsin relation to a variety of biological disci-plines other than pathology; or on exam-inations of the aetiological or otheraspects of human diseases. Only 14.7%(14/95) of applicable chimpanzee studieswere cited by a total of 27 papers thatappeared to describe well developed pro-phylactic, diagnostic or therapeutic meth-ods for combating human diseases.However, detailed examination of these

27 human medical papers revealed that invitro studies, human clinical and epi-demiological studies, molecular assaysand methods, and genomic studies, con-tributed most to their development.63.0% (17/27) of these medical paperswere found to be wide-ranging reviewsof 26-300 (median 104) references, towhich the cited chimpanzee study madeonly a small contribution. Duplication ofhuman outcomes, inconsistency withother human or primate data, and othercauses resulted in the absence of anychimpanzee study able to demonstrate anessential contribution, or, in most cases, asignificant contribution of any kind,towards the development of the humanmedical method described.

Poster

Chimpanzee research: 2) Lack of efficacy in combating human diseaseAndrew Knight1, Jarrod Bailey2 and Jonathan Balcombe 3




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The advanced sensory, cognitive, socialand communicative abilities of chim-panzees also confer upon them a pro-found ability to suffer when capturedfrom the wild or born into unnatural cap-tive environments, and when subse-quently subjected to confinement, socialdisruption, and involuntary participationin potentially harmful biomedicalresearch. Advocates justify such researchbased on the crucial contributions theyclaim it has made towards the advance-ment of biomedical knowledge, and, inparticular, towards combating major

human diseases. However, our systematicreview of 749 biomedical studies of cap-tive chimpanzees conducted during arecent decade revealed that almost halfwere not cited by any subsequent paperswithin the comprehensive bibliographicdatabases examined, suggesting minimalcontribution towards the advancement ofbiomedical knowledge generally. Giventhat research of lesser significance is notpublished at all, these results are of con-siderable concern. Furthermore, closerexamination of a subset of 100 randomly-selected chimpanzee studies failed to

identify any that made an essential contri-bution, or, in a disturbing majority ofcases, a significant contribution of anykind, towards papers describing well-developed prophylactic, diagnostic ortherapeutic methods for combatinghuman diseases, including major diseasessuch as AIDS, hepatitis and cancer.Consequently, we therefore call for, andbelieve it eminently reasonable to call for,the banning of biomedical research oncaptive chimpanzees in those remainingcountries, notably the US, that continueto conduct it.

Poster

Chimpanzee research: 3) The necessity of a banAndrew Knight1, Jarrod Bailey2 and Jonathan Balcombe 3



The acute zebra fish embryo test is an ac-cepted bioassay to assess the toxicity ofwaste water which may be used for the re-placement of testing with adult fish. It isalso suggested for chemical hazard as-sessment although only a few groups ofsubstances have been studied yet. Specifi-cally acting substances like neurotoxic in-secticides pose a potentially hazard fornon-target fish. To establish whether theproposed zebra fish embryo test protocol

and the inhibition of cholinesterases(acetylcholinesterase EC 3.1.1.7, propi-onylcholinesterase EC 3.1.1.8) and car-boxylesterase (EC 3.1.1.1)enzymes canbe used in a similar fashion for risk as-sessment, two types of experiments wereconducted. Visual ffects of exposure to theorganophosphate paraoxon-methyl after24 and 48 h in the zebra fish embryo testsystem were analysed with the use of aninverse microscope(rate of mortality, de-

velopmental disturbances, heart rate andothers). The inhibition to cholinesterasesand carboxylesteras e was also measured.Enzyme inhibition as a biomarker wasabout 70 times more sensitive than the ef-fects in the zebra fish embryo test with anIC50 below 1.2 µmol compared to an EC50

of 91 µmol. Significant overt effects couldonly be seen at concentrations at which al-ready 80% of the activities of the differentesterases were inhibited.

Poster

Sensitivity of the zebrafish embryo (danio rerio) topesticides: Visible effects versus cholin- andcarboxylesterase enzyme biomarkers of insecticideexposure Eberhard Küster and Rolf Altenburger UFZ Centre for Environmental Research Leipzig-Halle in the Helmholtz-Association, Department of BioanalyticalEcotoxicology, Leipzig, Germany E-mail: [email protected]

Keywords: cholinesterase, carboxylesterase, zebra fish embryo test, insecticide, bi omarker, paraoxon-methyl


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Rats are widely used as animal modelsfor human diseases. However, due totheir relatively small body size, bloodsampling is difficult, invasive and thereby might seriously interfere withendocrine functions and animal welfareaspects. Therefore a non-invasive tech-nique to monitor stress hormones inthese animals is highly desired. Ourstudy aimed to gain information on cor-ticosterone metabolism and to validate a 5α-pregnane-3β,11β,21-triol-20-oneenzyme immunoassay (EIA) to monitorcorticosterone metabolites (CM) in faecal samples of laboratory rats. Sixrats of each sex were administered 2.4 MBq of 3H-corticosterone intra-

venously and three of each sex receivedthe labeled steroid per os. Subsequently,all voided excreta were collected for fivedays. Peak concentrations in urine appeared after 1.7 ± 0.6 h and after 6.0 ± 3.5 h in males and females, re-spectively. In faeces, excretion maximawere observed after 14.7 ± 2.4 h in bothsexes. In principal the route and delayfor both administration methods was thesame, except a delay of peak concentra-tion in urine (4.5 ± 2.1 h) in per os administered males. About 75% of therecovered CM was found in the faeces.Using high performance liquid chro-matography (HPLC) substantial infor-mation about the biochemical character-

istics of faecal 3H-CM was revealed anddifferences between the sexes werefound. In both sexes, corticosterone washeavily metabolized. While malesshowed only minor variations in theirCM patterns, those of females differedlargely between individuals. To validatethe EIA, we conducted an ACTH chal-lenge test, a dexamethasone suppressiontest and investigated effects of the diur-nal variation (DV) of glucocorticoids,using six male and six female rats each.Our results demonstrated that pharmaco-logical stimulation, suppression and theDV of adrenocortical activity were re-flected accurately by means of CM mea-surement in faeces. Therefore, our find-

Poster

Non-invasive measurement of adrenocortical activity in male and female ratsMichael Lepschy1, Chadi Touma2, Robert Hruby 3 and Rupert Palme 1

1Institute of Biochemistry, Department of Natural Sciences, University of Veterinary Medicine, Vienna, Austria; 2Department of Behavioral Neuroendocrinology, Max Planck Institute of Psychiatry, Munich, Germany; 3Institute of Toxicology,Austrian Research Centers, Seibersdorf, Austria E-mail: [email protected]

Trans-Epithelial Electrical Resistance(TEER) is a useful parameter to quantifybarrier function in endothelial and ep-ithelial cells and has been demonstratedto be a sensitive endpoint for determiningthe toxicity of compounds to epithelialcells in vitro. The aim of this study wasto establish TEER measurement to mon-itor barrier function of epithelial cells un-der continuous perfusion conditions. Ep-ithelial cell lines, exhibiting different

degrees of monolayer tightness, werecultivated on microporous growth sup-ports in a perfusion apparatus (Epi-Flow®) with an implemented TEER mea-suring unit. Confluent cell monolayerswere intoxicated with various toxic con-centrations of CdCl2 (0-15 µM). The tox-ic influence on cell layer integrity wasmonitored via TEER measurement andadditionally controlled by measuring lac-tate dehydrogenase (LDH) activity in the

out-flowing medium. During formationof the epithelial monolayer, TEER in-creased to a steady state value. CdCl2 ex-posure resulted in a gradual collapse ofTEER, which correlated with an in-creased LDH release. TEER is a sensi-tive, simple, non-invasive online measur-ing tool of cell monolayer integrity.TEER monitoring increases the usabilityof perfusion technology for the determi-nation of time resolved toxicity.

Poster

Trans-Epithelial Electrical Resistance monitoring in perfusion culture is a reliable indicator of monolayer integrity and barrier functionThomas Lechleitner, Paul Jennings and Walter PfallerDepartment of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria E-mail: [email protected]

Keywords: TEER, EpiFlow, perfusion culture, epithelial cells


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Keywords: non-invasive, stress, glucocorticoids, rats

Retrospective evaluation of the ethicalreview process for animal experimenta-tion has been demanded for years. Onemain aspect in evaluating this system isin investigating whether the prospectivecost-benefit-analysis that served as thebasis for granting licenses for procedurescan be regarded realistic when comparedto the actual outcome of the scientific re-search. Previous investigations have indi-cated that the distress for the animals hascommonly been underestimated in appli-cations for granting a license, and thatthe majority of investigated research pro-jects failed to deliver the envisioned spe-cific scientific results. In the presentstudy, we looked at the clinical signifi-cance of research projects in those caseswhere applicants suggested a concretebenefit for the cure of human diseases.

ings confirm the suitability of this EIAto non-invasively monitor adrenocorticalactivity in rats of both sexes, which can

open new perspectives for biomedicaland pharmacological investigations aswell as animal welfare related issues.

This study was funded by the AustrianFederal Ministry for Education, Scienceand Culture (GZ 80.102/2-BrGT/2004).

Only those projects were taken into ac-count where previous studies had shownthat the results of the animal research ap-plied had confirmed the hypotheses ofthe researchers. A retrospective citationanalysis for 12 years was performed andthe results were unambiguous: after anal-ysis of more than 1000 scientific articles:It has become evident that none of the 17analysed applications at three Germanuniversities led to any new therapies orhad any clinical impact. According to Di-rective 86/609 (Art. 12/2), painful animalexperimentation need to be “of sufficientimportance for meeting the essentialneeds of man or animal” to be licensed.The results of our study verify that inspecific cases the practice of licensing ofanimal research in Germany fails com-pletely. With regard to the “benefit”-fac-

tor, authorities have to apply much morestringent criteria when certifying the im-portance of the proposed research. Forthis “cost”-factor, as indicated in previ-ous studies, the basis for prospective as-sessment of pain, suffering, and distresshas to be objectified. The authoritieshardly ever raise any serious doubtsabout the significance of the proposed re-search. Our study confirms the need tochange perspectives, approaches, andquestions so they can be addressed bynon-animal methods. This notion shouldbe enforced by the authorities. From ananimal welfare perspective, the mainparadigm of the licensing practice has tobe turned upside down: when there is anyquestion about the relevance of an atleast painful animal research the decisionin doubt should be for the animal.

Lecture

Animal experiments in biomedical research. An evaluation of the clinical relevance of approvedanimal experimental projects: No evidentimplementation in human medicine within more than 10 years Toni Lindl 1, Manfred Völkel 2 and Roman Kolar3

1Inst. Applied Cell Culture Ltd., Munich, Germany; 2Ethical Commission of Northern Bavaria, Würzburg, Germany; 3Animal Welfare Academy, Neubiberg, Germany E-mail: [email protected]

Keywords: animal experiments, ethical review, ethical evaluation, ethical committees, cost-benefit-analysis, citation analysis,clinical relevance


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In Germany, according to the GermanAnimal Welfare Act, scientists must pro-vide prior to undertaking an animal ex-periment an ethical and scientific justifi-cation in their applications to thelicensing authority. In such justificationsreference is made to lacking knowledgewith regard to development of humandiseases or the need of better and evennew therapies for humans. Basis of thepresent study is the literature research,based on applications of biomedicalstudy groups of three universities inBavaria (Germany) between 1991 and1993 (Lindl et al. ALTEX, 18, 171-178,2001). Here we show the aim of each re-search (n = 51), the animal model, thereasons had been given to the choice ofthe animal model and whether the re-search has led or not led to a publicationfound in DIMDI or PUBMED. These ap-plications have been classified according

to their publications as successful in theanimal model (Lindl et al. 2001). We in-vestigated here in greater detail the fre-quency of citations, the course of cita-tions, and the question in which type ofresearch the primary citations have beentaken up: In subsequent animal basedstudies, in In-vitro-studies, in review ar-ticles or in clinical studies. The criterionwe applied was whether the scientistssucceeded to reach the goal that they pos-tulated in their applications: To con-tribute to new therapies or to gain resultsof direct clinical impact. The outcomewas unambiguous: even though 97 clini-cally orientated publications in which theabove mentioned publications were citedcould be tracked (8 % of all citations),only in 4 publications a direct correlationbetween the results from animal experi-ments and observations in humans couldbe noted (0,3 %). But even in these 4 cas-

es the hypotheses that had been verifiedsuccessfully in the animal experimentfailed in any respect. The implications ofour findings may lead to demands con-cerning improvement of the licensingpractice in Germany. AcknowledgementFirst results of this study were presentedas Poster at the 12th Congress on Alter-natives to Animal Experiments at the uni-versity of Linz, Austria and at the FifthWorld Congress on Alternatives & Ani-mal Use, Berlin, August 2005. ThePoster was honoured with the Poster-award \"LINZ 2004\" We gratefullythank the Austrian \"Zentrum für Ersatz-und Ergänzungsmethoden zu Tierver-suchen\" (zet) and the \"VIER PFOTEN-Stiftung für Tierschutz\" for the award.We also thank Dr. Ingrid Weichenmeierand Dr. Birgit Lewandowski for theirhelpful investigations into literature.

Keywords: animal experiments, ethical review, ethical evaluation, ethical Postercommittees, citation analysis, clinical relevance

Poster

Study of the clinical relevance of 51 applications onanimal experiments in biomedical research. Toni Lindl 1, Manfred Völkel 2 and Roman Kolar3

1Inst. Applied Cell Culture Ltd., Munich, Germany; 2Ethical Commission of Northern Bavaria, Würzburg, Germany; 3Animal Welfare Academy, Neubiberg, Germany E-mail: [email protected]

Poster[Vascularized liver test system as an alternative to animal tests]Vaskularisiertes Lebertestsystem als Alternative zuTierversuchenKirstin Linke, Johanna Schanz und Heike MertschingFraunhofer Institut für Grenzflächen- und Bioverfahrenstechnik (IGB), Stuttgart, GermanyE-mail: [email protected]

Die Entwicklung von Alternativen zuTierversuchen ist essentiell, um neueWirkstoffe früh in der Entwicklungspha-se an humanen Zellen untersuchen zukönnen und somit eine bessere Übertrag-barkeit von Ergebnissen zu gewährlei-sten. Die Leber spielt eine zentrale Rolleim Stoffwechsel. Deshalb sind Leber-zellen (Hepatozyten) bzw. das gesamte

Organ von besonderem Interesse alsAnalysesystem für Substanzen undWirkstoffe sowie ihre Metaboliten. Exi-stierende in vitro Testsysteme sind sehrartifiziell, die Zellen werden nicht unterphysiologischen Bedingungen kultiviertund zeigen deshalb nur einen kleinen An-teil der biologischen Reaktionen im Kör-per. Wir entwickeln ein Testsystem auf

der Basis einer vaskularisierten porcinenMatrix, die in einem Bioreaktormoduldie physiologische Versorgung von He-patozyten in Co-Kultur mit Endothelzel-len ermöglicht. Im ersten Schritt wird einStück porcines Jejunum mit erhaltenemGefäßsystem ( mit arteriellem Zuflußund venösem Abfluß) chemisch azellula-risiert (1). Das Gefäßsystem dieser Ma-


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trix wird mit porcinen Endothelzellenaus der Leber rebesiedelt. Die Besied-lung erfolgt unter pulsatilem Fluß für 7Tage bei 37°C und 5% CO2. Nach einerWoche Kultur wird die Co-Kultur mitHepatozyten gestartet.Die Zellen werdenimmunhistologisch (z.B. CD31, CKLP34, Anti Human Hepatocyte) untersuchtund ihre Vitalität (Live Dead Assay )überprüft. Es werden Harnstoff- und Al-buminsynthese, LDH-Aktivität und Ga-laktoseabbau der Hepatozyten analysiert.Endothelzellen konnten erfolgreich indas Gefäßsystem integriert werden.

Nach zweiwöchiger Kultur sind sie vitalund exprimieren endothelzellspezifischeMarker. Die Kultur von Hepatozyten aufder azellulären, vaskularisierten Matrixzeigt bei einem Kulturzeitraum von 2Wochen gute Ergebnisse für das Wachs-tum der Zellen und den Erhalt leberspe-zifischer Funktionen. Die in das Ma-trixlumen aufgebrachten Leberzellensind vital, exprimieren leberspezifischeMarker und beginnen in die Matrix ein-zuwandern. Die Hepatozyten bilden aufder Matrix zwei bis drei Zelllagen in typischer Lebermorphologie. Unser Ziel

ist die Entwicklung eines vaskularisier-ten Lebermoduls für verschiedene Appli-kationen. Durch die Entwicklung einesLebertestsystems auf Basis der vaskula-risierten Matrix wäre es erstmals mög-lich Wirkstoffe arteriell zu applizierenund venös zu analysieren. Ein derartigesModell kann als Testsystem zur Identifi-zierung von potentiellen Wirkstoffenoder toxischen Metaboliten herangezo-gen werden und so zur Reduzierung oderzum Ersatz von Tierversuchen beitragen.Biomaterials 2005; 26, 6610-6617

Poster

Gene expression profiling of an in vitro tissue model for cartilage destruction Carsten Lübke1, Kristin Andreas1, Mehdi Yahyawi1, Veit Krenn2,3, Lars Morawietz 2, Jochen Ringe1, Gerd-Rüdiger Burmester1, Thomas Häupl1, Christian Kaps4 and Michael Sittinger1

1Tissue Engineering Laboratory, Department of Rheumatology, Charité – Universitätsmedizin Berlin; 2Institute of Pathology,Charité – Universitätsmedizin Berlin; 3Institute of Pathology, Trier, 4TransTissue Technologies GmbH, Berlin, Germany E-mail: [email protected]

Keywords: Vaskularisiertes Testsystem, Leber, Co-Kultur

Keywords: cartilage, rheumatoid arthritis, in vitro model, microarray, inflammation, drug screening

An rheumatoid arthritis (RA) in vitropannus tissue model was developed tostudy molecular processes of cartilagedestruction by synovial fibroblasts. Hu-man articular chondrocytes were cul-tured in alginate beads for 2 weeks toform neo-cartilage. Immortalized RA(RASF) and normal (NDSF) human syn-ovial fibroblasts were cultured for 48 hand supernatants were collected. Thisconditioned synovial fibroblast mediumwas used to stimulate neo-cartilage algi-nate beads for 48h. Normal cell culturemedium served as controls. Gene expres-sion profiling of stimulated alginatebeads was performed with Affymetrixoligonucleotide microarrays. In addition,RASF and NDSF were treated with thefrequently used anti-rheumatic drugsmethotrexate, prednisolone or diclofenac

and used for subsequent microarray geneexpression profiling. The expression pro-files of selected genes were verified bysemi- quantitative real-time PCR and byELISA. Neo-cartilage alginate beadsstimulated with RASF conditioned medi-um showed differential expression of 150genes compared to beads cultured in thepresence of NDSF conditioned medium.Most of these genes are associated withcartilage formation and destruction (e.g.cartilage oligomeric matrix protein,cathepsin S), inflammation and immuneresponse (e.g. interleukins: IL1alpha andIL8) as well as cell adhesion (e.g. beta 8-integrin, vascular cell adhesion molecule1 (VCAM1)) known from RA. In RASF,the cytostatic drug methotrexate resultsin the reversion of the RA-related ex-pression profile of genes associated with

growth and apoptosis (e.g. IGFB3),whereas the glucocorticoid prednisolonereverted the RA-related profile of genesthat are known from inflammation (e.g.IL1beta and IL8). The non-steroidal anti-inflammatory drug diclofenac showed noeffects on the RA-related gene expres-sion. Mediators produced by RASF pro-mote destruction of articular cartilage asknown from rheumatoid arthritis pannustissue. Moreover, RA-related mediatorsand gene expression profiles of RASFcan be reverted to normal by commonanti-rheumatic drugs. Therefore, RASFand neo-cartilage alginate beads mayrepresent a useful in vitro RA pannusmodel for screening of putative anti-rheumatic compounds.


Recently, a new organotypic, three-di-mensional corneal equivalent based onSV40-immortalized human cell lines wasreconstructed by embedding keratocytes(HCK) in a collagen matrix, layering ep-ithelial cells (HCE) on top and endothe-lial cells (EC) below (Zorn- Kruppa etal., 2005). The aim of this work was toevaluate this cornea model as a tool for

eye irritation testing, permeation studiesand wound healing processes by charac-terising EC and HKC. The main attributeof EC is to build a functional monolayer,while the phenotype of HCK as well astheir ability to transform into a fibroticphenotype (myofibroblasts) is known tobe crucial in the wound healing process(Matsuda et al., 1973). Five culture

mediums were tested in order to evaluatethe effect of serum and calcium concen-tration on cellular response. For EC char-acterization cells were tested by fluores-cence activated cell sorting (FACS) andimmunocytochemistry (ICC). Antibodiesfor the nuclear markers Ki-67 andp27KIP1 were used to evaluate cellularproliferation and contact inhibition re-

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Innerhalb der Tierschutzethik ist wohlkeine Frage zu finden, die so kontroversdiskutiert wird wie die des Lebensrechts.Selbst unter „tierfreundlichen“ Philoso-phen gibt es den größten Dissens, unterwelchen Voraussetzungen eine Tiertö-tung ethisch gerechtfertigt werden kann.Die aktuelle Diskussion um die Tötungs-frage resultiert aus der Speziesismus-Kritik der frühen 1970er Jahre. Peter Sin-ger (*1946) gehörte damals zu denersten, die den üblichen Umgang mitMensch und Tier als speziesistisch, d.h.als ohne plausibles Differenzierungskri-terium die Vertreter der eigenen Spezies(Homo sapiens) bevorzugend, „entlarvt“hat. In Weiterführung eines Gedankensvon Jeremy Bentham (1748-1832), dererstmals die Leidensfähigkeit als mora-lisch entscheidende Eigenschaft be-schrieb, bemüht sich die Ethik seitherdarum, neben dem Menschen auch alleleidensfähigen Tiere zu integrieren (sog.

Pathozentrische Ethik). Die Speziesis-mus-Kritiker argumentieren im Sinne ei-ner Beweislastumkehr; was de m Men-schen Recht ist, soll – bis zum Nachweisfehlender Voraussetzungen – den Tierenbillig sein. Die aus der Speziesismus-Kritik entstandenen Argumente zur Tö-tung bauen auf dem (fragwürdigen) Po-stulat auf, eine Tötung sei deswegenunmoralisch, weil sie gegen das Interesseder Person verstoße, weiterzuleben. InAnlehnung an Leonard Nelson (1882-1927) wird so aus Tierrechtler- und Ve-ganerkreisen für ein Lebensrecht allerTiere argumentiert. Peter Singer meintjedoch, dass das Argument, solche Tierenicht zu töten, die weiterleben wollen,nur auf diejenigen Tiere anwendbar sei,die diesen Gedanken tatsächlich zu den-ken im Stande sind, also ein reales „In-teresse am Weiterleben“ haben; er nenntals Beispiele für eine solche „Präferenz“u.a. Affen, Hunde und Katzen. Bestärkt

wird seine Einschätzung durch Tom Re-gan (*1938), der – als Kritiker der Sin-ger’schen Methodik – zu einer ganz ähn-lichen Teilung des Tierreichs in derTötungsfrage gelangt. Die Anerkennungeines Lebensrechts b ei kognitiv höherentwickelten Arten wurde dann z.B. vonUrsula Wolf (*1951) und Jean-ClaudeWolf (*1953) jeweils im Rahmen pater-nalistischer Interpretation des erwähntenGrundgedankens wieder auf alle bewus-st-empfindungsfähigen Tiere ausgedehnt.Von früheren Philosophen wurde im An-schluss an die antike Philosophie ge-schlussfolgert, dass Tiertötungen angst-und schmerzlos durchgeführt werdenmüssen, jedoch kein Lebensrecht derTiere hergeleitet werden könne. – Diebeiden Argumentationsgruppen werdenvorgestellt und ihre Stärken undSchwächen beleuchtet.

Lecture

[The question of right to life in animal ethics]Ein Lebensrecht für Tiere – ethisch zu rechtfertigen? Joerg LuyInstitut für Tierschutz und Tierverhalten, FU Berlin, Deutschland [email protected]

Poster

Characterisation of two im mortalized human corneal cell lines used for building a new organotypic corneal equivalentAnna K. Manzer, Simone Lombardi-Borgia, Monika Schäfer-Korting and Maria EngelkeInstitut of Pharmacy, Free University of Berlin, Berlin, GermanyE-mail: [email protected]

Keywords: animal killing, animal welfare, bioethics, professional ethics


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spectively. Primary human corneal en-dothelial cells were used as positive con-trol. In all the mediums tested EC failedto build a constant monolayer. The ex-pression of Ki-67 did not show a signifi-cant decrease after confluence, while lowexpression of p27KIP1 confirmed the ab-sence of contact inhibition. In conclu-sion, growth conditions of the EC cells

still have to be optimized. The HCK-phenotype was assessed in culture, withand without stimulation through TGF-β,as well as in cryosections of the stromalmatrix. The expression of α- smoothmuscle actin (α-SMA), a specific markerfor the myofibroblast phenotype, was de-tected by ICC. Primary human fibrob-lasts were used as positive control. As re-

sult, HCK did only show significant _-SMA expression after TGF-β stimula-tion. In conclusion, the immortalizedHCK proved to be the keratocyte pheno-type which can be stimulated to differen-tiate into myfibroblast form. Hence, theHCK are suitable for the assessment ofeye injury and wound healing.

Lecture

Veterinary training without the use of laboratoryanimals – an example from Norway Siri MartinsenInterNICHE Norway, Oslo, Norway E-mail: [email protected]

Keywords: cornea model, immortalized keratocytes, immortalized endothelial cells, immunocytochemistry

Keywords: alternatives, animals, animal experimentation, clinical, dissection, ethically-sourced, InterNICHE, veterinary education

This presentation describes the steps tak-en by a Norwegian veterinary student tosuccessfully complete her veterinary ed-ucation using alternatives to laboratoryanimals. This included the use of com-puter simulations, student self-experi-ments in physiology, dissections onwaste material from the pathology de-

partment and naturally dead animals, andsurgical training through beneficial pro-cedures in veterinary clinics. The presen-tation offers an example of how a veteri-nary student can complete her educationwith commitment to the principle of\"First, do no harm\", and addresses howthis principle relates to the use of animal

experiments. Describing practical solu-tions as well as discussing the reasons foran approach without the harmful use ofanimals, the author argues that this ap-proach can and should be implementedas the standard method of education forveterinary students.

Poster

The InterNICHE policy on the use of animals andalternatives in education Siri Martinsen1 and Nick Jukes 2

1InterNICHE Norway, Oslo, Norway; 2InterNICHE, Leicester, England E-mail: [email protected]

The InterNICHE ‘Policy on the Use ofAnimals and Alternatives in Education’is a comprehensive document in 10 sec-tions that addresses all aspects of workwith animals and alternatives in life sci-ence education. The Policy presentsguidelines to ensure effective and fullyethical acquisition of knowledge andskills. It includes a definition of alterna-tives in education and of harm, and pre-sents individual policies on dissection,

the sourcing of animal cadavers and tis-sue, work with live animals for clinicalskills and surgery training, and ethicalfield studies. It also addresses the use ofanimals for the production of alterna-tives themselves. While the ideal ‘re-placement alternative’ is defined as‘non-animal’ within the 3Rs philosophyof Russell and Burch (1959), the Policyhighlights a shortcoming of the 3Rs ap-proach for education. Not only is there a

requirement for some students to workwith animals, animal tissue and clinicalprocedures in their education, there iswide spread evidence of the ability tofully meet all teaching objectives inways that are neutral or beneficial to in-dividual animals and that do not involveanimal experimentation or killing. Aswell as non-animal learning tools likemultimedia computer simulation, digitalvideo, training models and mannekins,


Experimental animal studies are per-formed to gain further knowledge aboutbiological processes. They have to bejustified and properly designed to cometo sound conclusions, to gain as much re-liable information as possible and tokeep the number of animals as small as

possible. Sound knowledge of study de-sign aspects (e.g. parallel groups, factori-al designs, block designs, …), statisticalanalyses strategies and sample size cal-culation should be a matter of course forstudy protocols, not only in clinical trialsinvolving humans, but also in animal tri-

als. The importance of these points andfrequent problems/misunderstandingsare presented and discussed with a spe-cial focus on real problems of submittedstudies to animal ethics committees inAustria.

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replacement alternatives also include theuse of ethically-sourced animal cadaversfor dissection and skills training, and ap-prenticeship into clinical practice with

animal patients. A definition of ‘ethical-ly-sourced’, and of ethical educationalopportunities within clinical work, areincluded in the Policy which demon-

strates the possibilities for full replace-ment of harmful animal use in educa-tion.

Poster

Statistical aspects of experimental animal studies Martina MittlböckCore Unit for Medical Statistics and Informatics; Medical University of Vienna, Austria E-mail: [email protected]

Keywords: 3Rs, alternatives, animals, education

Keywords: study design, sample size calculation

The design, operation and performancesof a biosensor based on immobilized liv-ing chromatophores isolated from Bettasplendens Siamese fighting fish are pre-sented in this paper. Development of thecell immobilization technique as an im-portant biosensor enabling technology,and integration of the immobilized cellsin the biosensor system is described. Thebiosensor was used to test several classesof biologically active agents (environ-mental and bacterial toxins) categorized

in the following classes: 1) neurotrans-mitters; 2) adenyl cyclase activators;3)cytoskeleton effectors; 4) cell membraneeffectors; and 5) protein synthesis in-hibitors. The obtained responses wereanalyzed and quantified by measuringthe change in cell area covered by toxin,by using image analysis. Particular re-sponse features such as mode of re-sponse, its magnitude; kinetics, sensitivi-ty and dose dependence were monitoredand may be used as a basis for mathe-

matical modeling of the responses.Streptococcus pyogenes streptolysin Sand streptolysin O, Clostridium tetanitetanolysin, Staphylococcus aureus al-pha-toxin, and Vibrio parahemolyticushemolysin, all bacterial toxins which acton cell membrane, elicited a strong re-sponse from chromatophores. The resultsindicated that the biosensor has a greatpotential for the use in food and watertesting and in pharmacy.

Poster

Biosensor for environmental and bacterial toxinsbased on immobilized fish chromatophores Ljiljana Mojovic1 and Goran Jovanovic 2

1Faculty of Technology and Metallurgy, University of Belgrade, SCG; 2Oregon State University, Corvallis, OR, U.S.A. E-mail: [email protected]

Keywords: fish chromatophores, biosensor, environmental toxins, bacterial toxins, biologically active agents


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Testing for the safety of a potential drughas been and maintains to be a challeng-ing task for the toxicologists in the phar-maceutical industry. The emerging “newtechnologies” such as “omics” have in-creased the possibilities but also the ex-pectations to assess relevant and species-specific toxicities of drug candidates.Nevertheless, the rapid pace of techno-logic development has not acceleratedsafety testing to the expected extent yet.In this review, I will give two examplesfor the application of target-specific cellmodels to detect and assess species-spe-cific toxicities. Here, global and focussed

analyses of gene-, protein and cellularchanges have been successfully em-ployed for the characterization of adrenaland hepatic toxicities. In the first exam-ple, we have used adrenal cell modelsbased on primary as well as a permanenthuman adrenal cell line. Both cell sys-tems enabled a good prediction ofadrenal effects in rodents, non-rodents aswell as humans. The second examplemade use of primary hepatocytes. In thisproject, a drug development candidateshowed unexpected toxicities as well asspecies-specific cytochrome P450 induc-tion (CYP) in vivo. We therefore ana-

lyzed CYP induction and potential toxic-ity signatures in rat and human hepato-cytes as well as in samples from in vivotoxicity studies. By this approach, the rathepatocyte model predicted correctly theeffects observed in rodents. By compar-ing effects observed in vitro and in vivo,a solid extrapolation for effects in hu-mans was possible. These examplesshow that an intelligent testing strategyusing alternative methods can permit ameaningful safety assessment for hu-mans if a “tailor-made” range of tech-nologies along with “classical” toxicolo-gy is employed.

Lecture

Alternatives in pharmaceutical toxicology: global and focussed approachesStefan O. MuellerInstitute of Toxicology, Merck KGaA, Darmstadt, GermanyE-mail: [email protected]

Keywords: expression profiling, in vitro models, primary cells, target-specific tests, mechanistic toxicology, explanatorytoxicology

Bei gesetzlich vorgeschriebenen toxiko-logischen Untersuchungen in Verbin-dung mit dem Inverkehrbringen vonPharmawirkstoffen, der Herstellung vonMedizingütern und der Abschätzung vonChemikaliengefahren wird weltweit einegroße Anzahl von verschiedenen Testsdurchgeführt, um Aussagen über eineeventuelle schädliche Wirkung auf denmenschlichen Organismus machen zukönnen. Zu diesem Zweck kommen injüngster Zeit vermehrt photometrisch basierende Zytotoxizitätstets zum Ein-satz. In dieser Arbeit wurde eine vorhan-dene Methodik der elektrochemischen

Bioaktivitätssensorik als in vitro Scree-ningmethode zur Bestimmung der Zyto-toxizität eingesetzt. Zur Anwendung kamdabei ein amperometrisches Messsystem(Dreielektrodensystem). Bei einem be-stimmten Potential wurden so die reduzierenden Eigenschaften stoffwech-selaktiver Organismen unter Einsatz von Redox-Mediatoren elektrochemischerfasst. In Abhängigkeit der Menge vondurch Organismen/Zellen reduziertenMediatormolekülen entsteht ein Strom-signal. Das Stromsignal gibt je nach Ausgestaltung der Versuchsbedingungenentweder Aufschluss über die Anzahl

von lebenden Organismen/Zellen oderbei vorgegebener Menge an Organismen/Zellen Aussagen über deren Stoffwech-selzustand bzw. Stoffwechselaktivität.Bei entsprechender Zugabe einer Test-substanz lässt sich hierbei in Abhängig-keit von der jeweiligen Zelltoxizität dieser Substanz auf das eingesetzte Zell-system eine Verminderung des Stromsig-nals im Vergleich mit einem entsprechen-den Blindwert ablesen. Bei einerhinreichend genau abgestuften Konzen-trationsreihe einer Testsubstanz kann soschnell eine Zytotoxizitätskurve aufge-nommen werden. Die Besonderheit der

Poster

[Cytotoxical investigations with an electrochemical in vitromeasuring method]Zytotoxikologische Untersuchungen mit einemelektrochemischen in vitro Messverfahren M. Pescheck, C. Glaß und D. SellDECHEMA e.V., Karl-Winnacker-Institut, 60486 Frankfurt/M, Germany E-mail: [email protected]


In contrast to conventional static cell cul-tures perfusion cultures provide steadystate conditions for nutrients andmetabolites, organotypic conditions,over prolonged periods of time. In addi-tion toxins can be administered at con-stant rates and concentrations either con-tinuously or repeatedly. Perfusion culturefurther allows time series analyses oftoxic effects as the outflowing culturemedium can be collated at high time res-olution. The cytotoxicity exerted by sev-eral toxic compounds (CdCl2, diquat andcyclosporine A) on epithelial monolayer

cultures was investigated in static andperfusion culture using the EpiFlowTMsystem and compared to each other. Theendpoints assessed over the whole intox-ication interval were the release of lactatedehydrogenase (LDH), adenylate kinase(AK) and the potential of cells to reduceresazurin. The results showed a greatersensitivity of epithelial cells towardscontinuous toxin administration in perfu-sion cultures as compared to single or re-peated dose administration in conven-tional static cultures. These differencescan be ascribed to the different delivery

rates of toxins to the cellular site of ac-tion. The cellular accumulation of Cd-Cl2, for example, was higher in cells keptunder perfusion conditions than in cellskept under standard static culture condi-tions. Time series analyses of LDH re-lease into the culture medium were per-formed for both culture methods andevaluated by a newly developed deter-ministic population balance model of celldeath, which delivers insight to the dy-namics of cell deathduring toxin expo-sure.

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dabei zum Einsatz kommenden elektro-chemischen Messzelle ist, dass währendder Messung alle Substanzen und Zellenin einem Polysaccharidgel immobilisiertwerden. Dies Gel begünstigt zum einen,dass die Zellen, Mediator und Testsub-stanz gleichmäßig im Volumen der Mes-szelle verteilt bleiben, zum anderen wirddurch das aufgequollene Gel das Ein-

dringen von Luft in den Testansatz er-schwert, was eine essenzielle Bedingungfür eine stabile Messungen mit diesemMesssystem darstellt. Die Immobilisie-rung der Substanzen und Zellen durchdas Gel macht es zudem möglich, dasspulvrige Mediatoren und Testsubstanzenmit lipophilem Charakter ohne einen ent-sprechenden Lösungsvermittler direkt

für die Tests eingesetzt werden können.Mit dieser Messzelle wurden erste Testsan Modellsubstanzen durchgeführt, wel-che den vergleich mit etablierten Zytoto-xizitätstests stand hielten. Das Projektwird aus Mitteln des Bundesministeriumfür Wirtschaft und Technologie über dieAiF gefördert.

Carcinogenesis has been shown to be amultistep process, which involves se-quential genetic alterations in a singletarget cell, which cause subtle alterationsin growth control and culminate in cellsthat are able to form malignant tumours.Genetic changes can result from sponta-

neous or carcinogen-induced alterationsin DNA. Non-genotoxic mechanisms,that are at least initially independent ofdirect DNA damage, can play a causalrole in carcinogenesis. In vitro cell trans-formation tests using BALB/c3T3 orC3H10T1/2 cells can simulate the pro-

cess of animal two-stage carcinogenesis.A limitation of these methods however isthe required time (4-8 weeks) for the ex-pression of focus formation as an indica-tion of transformation. In order to im-prove the experimental conditions Sasakiet. al developed a cell line, Bhas42 cells,

Lecture

EpiFlow perfusion culture improves in vitroassessment of acute and chronic toxicityWalter Pfaller, Thomas Abberger and Sinikka PrajcerDept. Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, AustriaE-mail: [email protected]

Lecture

The Bhas42 Cell Transformation Assay as a predictor of carcinogenicty Albrecht Poth1, Susanne Kunz1, Andreas Heppeheimer 1 and Dieter Pollet 2

1RCC Cytotest Cell Research GmbH, Rossdorf, Germany; 2Hochschule Darmstadt, Darmstadt, GermanyE-mail: [email protected]

Keywords: perfusion culture, epithelial cells, cadmium chloride

Keywords: Zytotoxizitätstets, Zellen, Amperometrie, Immobilisierung, Bioaktivitätssensorik


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which was established for BALB/c 3T3cells transfected with v-Ha-ras onco-gene. Transformed foci can be efficientlyinduced in a single culture of the cells by treatment with initiating and promotingagents within a period of not more than 2weeks. The Bhas-system was establishedby using the standard reference carcino-gen, 3-methylcholantrene. A repro-ducible dose-dependent increase in trans-formed foci (type III) was obtained withconcentrations ranging from 0.05 to 3.0

µg/ml 3-methylcholanthrene. Also someof the type III foci were isolated by atrypsination technique. Growth charac-teristics of the type III cells were com-pared to that of the normal BALB/c 3T3cells. FACS-analysis indicate a greaterproportion of S-phase cells from thetransformed phenotype compared to thatof the parent cells. This indicates a dis-tinctly higher proliferation rate of thetransformed cells compared to normalcells. The Bhas-system could be easily

established in our laboratory with the ref-erence carcinogene 3-methylcholan-threne and the obtained results were re-producible and stable. Based on thedifferent characteristics of transformedand non-transformed cells we are nowcommitted in seeking molecular markersof morphological transformation whichwill lead to a more objective scoring ofthe different types of foci (type I, II andIII) and a greater acceptance of the trans-formation systems.

Poster

An in vitro model to test cosmetics and drugs on human hair folliclesEdoardo Raposio, Chiara Guida, Ilaria Baldelli and PierLuigi SantiDICM I - Tissue Engineering Lab, University of Genova, ItalyE-mail: [email protected]

Keywords: cell transformation, in vitro carcinogenicity, Bhas42 cells, alternative method

Normal human occipital scalp samplesare obtained, from healthy male patients,during routine excision of benign scalplesions (e.g. , naevi and cysts). Isolationof anagen hair follicles is achieved by us-ing a surgical blade, microscissors andwatchmakers forceps under a stereo dis-secting microscope. As in micrograftpreparation for hair transplantation proce-dures, each follicle is isolated intact as awhole, always maintaining a significantamount of tissue (epidermis, dermis, sub-cutaneous fat) around the entire length ofthe follicle. Alternative techniques for therecovery of follicles (as by digesting theskin with collagenase) have failed to yieldfollicles capable of supporting hairgrowth, being the maintenance of thestructural integrity of the follicle (and re-lated structures) an essential requirementfor growth in vitro. Any follicle visiblydamaged during dissection is discarded.The obtained follicles are divided are ran-domly assigned to a control Group or a nexperimental Group. Follicles from both

Groups are stored in 500 µl of Williams E medium with supplements as follows:1% fetal calf serum, 10µg/ml transferring,10 µg/ml insulin, 10 ng/ml sodium selen-ite, 10 ng/ml hydrocortisone, 100 U/mlpenicillin, 100 µg/ml streotomycin, 2.5 µg/ml fungizone, and cultured for tendays. Supplemented medium is preparedfresh prior to experiment, and changedevery 72 hours. In the culture medium ofthe experimental Group is added the sub-stance to be tested. Follicles are main-tained free floating in individual wells of24 well multiwell plates in an atmosphereof 37°C, 5% CO2/95% air, and 100% hu-midity. This allows detailed measure-ments to be made on the length of indi-vidual hair follicles. The length of eachfollicle is measured at magnification x20 immediately following the 5-hourtest period and at the end of the 10-dayculture period, using a microscope with acalibrated eye-piece graticule. Total folli-cle length is computed as the distancefrom the base of the bulb to the end of the

shaft. Follicles which loose normal follic-ular architecture due to degeneration latein the culture period are computed as notsurvived. Histology is accomplished, atthe end of the ten day culture period, byfixing the follicles in phosphate buffersaline (pH 7.4) containing 10% para-formaldehyde, embedding in paraffinwax, sectioning at 10µm thickness, andstaining with the Heidenhein’s “Azantrichromic” modified protocol: this pro-cedure results in a red staining of the nu-clei and of the internal root sheath, pinkstaining of the follicular papilla, dark bluestaining of the matrix, violet staining ofcollagenous areas, orange staining ofmuscular tissues, and light yellow stain-ing of the cortex of the hair, permitting adetailed analysis of the histology of cul-tured follicles. In our experience, the de-scribed method allows to collect some re-liable data regarding the acute toxicity ofpeculiar cosmetics and drugs without theuse of animal models.

Keywords: cosmetics, drugs, hair follicles


Poster

Baby-cream blues: An in vitro investigation into the estrogenic potential of infant skin-care productsAndrew Rastall, Kati Graf, Marcus Frey and Lothar ErdingerInstitute for Hygiene and Medical Microbiology, University of Heidelberg, GermanyE-mail: [email protected]

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Numerous studies have reported the ad-verse effects of airborne pollutants onhuman health. However, only a smallnumber have attempted to examine theimmunomodulative potential of environ-mental pollutants and have tended to fo-cus on the effects of single compounds.There appears to be a paucity of dataconcerning both the immunomodulativeeffects of complex environmental sam-ples and the identity of causal agents.When investigations have been under-taken, these have often utilised animalmodels or sacrificial animal organ/celldonors. Our aim is therefore to developan alternative in vitro methodology basedon donated human cells to detect andidentify airborne pollutants with im-munomodulative potential in humans.

Here, we outline our approach and reportprogress towards the development of ourmethodology. For initial validation, anumber of common airborne pollutantswere selected and assessed for their abil-ity to modulate the proliferative responseof human T and B cells to known mito-gens. Isolated peripheral blood mononu-clear cells were stimulated with eitherphytohemagglutinin or pansorbin and ex-posed to test compounds. Results wereplotted to give the percentage increase ordecrease in mitogenesis as a function oftest compound concentration. Similarmethods were then used to examine theimmunomodulative potential of a num-ber of samples of airborne particulatematter collected during and following anatmospheric temperature inversion in

early 2006. Our results show that somecommon airborne pollutants includingcertain polycyclic aromatic hydrocar-bons significantly inhibit the normal re-sponse of both human T and B cells tomitogenic stimulation. Extracts of air-borne particulate material displayedhighly significant inhibitive effects atconcentrations equivalent to less than theamount of material present in one cubicmetre of air. Samples collected duringthe temperature inversion displayed sig-nificantly higher immunomodulative po-tential than those collected during nor-mal atmospheric conditions. Future workwill involve the application of bioassay-directed chemical analysis and investiga-tions into mechanisms of action of thecausal agents.

Recent research has shown a number ofchemicals commonly added to so-called‘personal care products’ (PCPs) such assunscreens, shampoos and deodorantsdisplay significant estrogenic potential.Infant skin-care products (ISCPs or ‘ba-by-creams’) can be defined as PCPs ap-plied to infant skin mainly for cosmeticreasons. In common with other PCPs,many ISCP formulations contain com-pounds that have been individuallyshown to display estrogenic potential in

vitro. The often routine and extensive ap-plication of these potentially estrogeniccompounds to immature human skin maytherefore represent an important route ofexposure. Our aim was to develop a suit-able analytical methodology and applythis to commercially available ISCPs togain an insight into the estrogenic poten-tial of this group of products. Thirteendifferent ISCPs were purchased off-the-shelf and extracted by sealing inside 50cm sections of pre-cleaned low-density

polyethylene tubing and dialysing in n-hexane. Interferants were removed fromthe extracts using size exclusion chro-matography and the extracts were as-sessed for estrogenic potential using ayeast-based estrogen screen (YES). Tenof the tested samples displayed signifi-cant concentration-response activitywhereas no activity was observed in anyprocess controls. Most sample curveswere biphasic with higher concentrationsof sample eliciting cytotoxic effects on

Poster

Something in the air? Progress towards thedevelopment of an analytical methodology for the detection and identification of immunomodulativeairborne pollutants Andrew Rastall, Holger Bartz, Lothar Erdinger and Klaus Heeg Institute for Hygiene and Medical Microbiology, University of Heidelberg, Germany E-mail: [email protected]

Keywords: immunomodulative, airborne, pollutants


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the yeast. 17b-estradiol equivalents (E2-EQ) were calculated for each of the ac-tive samples. These ranged from 5.5 to170 ng/ml suggesting a high estrogenicpotential associated with some ISCP for-mulations. Several of the most activesamples were subsequently subjected toreverse phase chromatographic fraction-

ation (HPLC) and re-tested using theYES. Charts of estrogenic activity as afunction of retention time showed activi-ty to be concentrated in the early elutingfractions. Initial analysis suggests thatthe majority of observed estrogenic ac-tivity is associated with para-substitutedhydroxybenzoate esters (parabens) added

to many PCPs as preservatives. Futurework will involve assessing the estro-genic potency of the ISCP formulationsusing an estrogen responsive human cellline (MCF-7) and analytical confirmationof the active compounds.

Human hepatocytes are the in vitro sys-tem of choice to study drug-induced pro-cesses in man. Here, we present HEP-AC2: A standardised and validated

culture system in which human hepato-cytes are maintained in HHMM (HumanHepatocyte Maintenance Medium) withHGF (hepatocyte growth factor) and EGF

(epidermal growth factor). Cellular via-bility and hepatocellular functions weremonitored daily. Albumin and urea pro-duction remained on a relatively constant

Poster

Comparison of three methods in the sensitivityassessment of the in vitro micronucl eus testGregory Rodrigo, Margit Oppong and Thomas Becker BSL Bioservice GmbH, Planegg (Munich), Germany E-mail: [email protected]

Lecture

Use of a standardised and validated long-term humanhepatocyte culture system for repetitive analyses of drugs: Repeated administrations of acetaminophenreduces albumin and urea secretion Dieter Runge1, Christine Berg 1, Jan Hengstler 2 and Anett Ullrich1

1Primacyt Cell Culture Technology GmbH, FRG, Schwerin; 2Center for Toxicology, University Leipzig, FRG, Leipzig, GermanyE-mail: [email protected]

Keywords: estrogenic, baby-creams, YES

Many chemicals interact with genetic ma-terial, leading to chromosome aberra-tions. Such substances are classified asaneugenic or clastogenic. Different testsallow the characterisation and the classi-fication of these chemicals. Among them,the chromosome aberration test and the micronucleus test permit detection of chromosome aberrations. Due tometaphase analysis, the first test presentssome disadvantages. The second test iseasier, the preparations can be scoredmore quickly and be assessed more ob-jectively. Despite the fact that this type of

assay is well-accepted and validated as anin vivo test (OECD Guideline 474), the invitro method is not reflected in officiallyaccepted test guidelines at OECD, just adraft is published to date (OECD draftproposal for a new guideline 487) andmany disparities in the conductance ofthis test have become obvious. To con-tribute to standardisation, three differentmethodological approaches of the mi-cronucleus test in vitro with V79 cells andcytokinesis block were compared at BSLBioservice GmbH : GIEMSA staining,Acridine Orange staining (fluorescence)

and flow cytometry. V79 cells were treat-ed with three known clastogens (Ethylmethanesulfonate, Mytomycin C and Cy-clophosphamide) and one aneugen (Col-cemid®). All three assays yielded positiveresponse for all test substances. However,sensibility and specificity differences be-tween the three methods were noted.Thus, all three methods are appropriatefor micronucleus detection, but variationsbetween each other exist, in terms of re-sult interpretation.

Keywords: in vitro micronucleus genotoxicity V79 Flow Cytometry


Based upon the framework depicted inthe White Paper “Strategy for a futureEU chemicals policy” published inFebruary 2001, in October 2003 the Eu-ropean Commission put forward the draftfor the new REACH regulation to theCouncil and the European Parliament fortheir decision. The aims of the so-calledREACH system (Registration, Evalua-tion and Authorisation of Chemicals) de-picted therein are better to protect hu-mans and the environment fromunwanted effects of chemical substanceswhile at the same time taking into ac-count economic considerations. In ac-knowledgement of the importance ofthese aims, the German Animal WelfareFederation submitted detailed commentson how they can be met without per-forming new animal tests. From the pointof view of animal welfare, animal testingfor the safety evaluation of chemical sub-

stances can be avoided, if the draftREACH regulation, amongst other is-sues, is amended to better ensure that allexisting information related to hazardouseffe cts of chemicals is made availableand shared, that all available means tocollect information without animal test-ing are made full use of and that onlysuch information is collected that is re-quired to ensure the safe use of a specif-ic substance. After long and extensivedebates, on 17 November 2005 the Euro-pean Parliament voted on its amend-ments to the draft REACH regulation,and the Council adopted its opinion on13 December 2005. Unfortunately, thechanges decided upon by the Council donot reveal a clear tendency to improveanimal welfare issues, and also theamendments adopted by the Parliamentdo not go far enough to address all con-cerns of animal welfare. For instance,

while both sides did adopt provisions tostrengthen the rules on data sharing, nei-ther set of rules go far enough to makedata sharing mandatory without excep-tion. And while the provisions to ensurethat only such data will be collected thatare necessary for the safe handling of agiven substance were improved, both theCouncil and the European Parliamentfailed to adapt the testing strategies to thestate-of-the-art concerning availablemeans to determine chemical hazardwithout animal testing. In the presenta-tion an overview will be given over theanimal welfare relevant amendmentsadopted by Council and Parliament aswell as the common position currently inpreparation, and these relevant docu-ments will be commented on from thepoint of view of animal welfare.

Keywords: REACH, data sharing, flexible step-by-step non-animal testing strategy, regulatory toxicity testing, 3R, risk assessment

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level for up to 2-3 weeks. Based on this, astandard protocol was established that al-lows repeated exposure of hepatocytes totest substances for studying drugmetabolism. We used acetaminophen(AAP) to assay the feasibility of this sys-tem. Hepatocytes were exposed to AAP(100-2815 mg/l) for 24 h. Subsequently,the culture medium was replaced bymedium without AAP and the same expo-sure scenario was repeated in intervals of

4 days. High doses of AAP (2815 mg/l)diminished urea production by 15-30%and albumin secretion by 70-80%. Thiseffect was reversible. After removal ofAAP, secretion of urea and albumin re-turned to control levels. AAP hepatotoxi-city is caused by its biotransformation tothe reactive metabolite N-acetyl-p-benzo-quinone imine that is mediated byCYP2E1 and CYP1A2. Obviously, theAAP activating enzymes were active for

at least 21 days and the activity was main-tained during at least four repeated cyclesof exposure to AAP. In conclusion, thesedata demonstrate the suitability of ourlong-term culture system to serve as atool for repetitive screening of drug-me-diated changes on hepatocellular func-tions. Thereby, this culture technique mayhelp to overcome the sparse availabilityof human hepatocytes for testing drug-mediated responses in man.

Lecture

Finalizing REACH – an evaluation of the European Parliament’s and the Council’s positionfrom the point of view of the German Animal Welfare Federation Ursula G. SauerGerman Animal Welfare Federation – Deutscher Tierschutzbund, Animal Welfare Academy – Akademie für Tierschutz,Neubiberg, Germany E-mail: [email protected]

Keywords: human hepatocytes, acetaminophen, toxicity, hepatocellular metabolism


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A total of 577 scientific articles on re-search projects with transgenic animalsperformed in Germany published in theyears 2001 -2003 were collected with theaim of obtaining an overview over thegoals and the contents of such funda-mental research. According to the topicscovered by the publications, main areasof biomedical research with transgenicanimals can be found in the fields of neu-robiology, immunology, cardiology, em-bryology and oncology. However theiruse can be discerned in all other areas offundamental biomedical research aswell. The vast majority of transgenic an-imals used were mice, followed by ratsand pigs. Additionally, singular researchprojects with fish, rabbits and chickenwere recorded. A high percentage of therats were used in cardiovascular re-search, whereas transgenic pigs wereproduced and bred as organ donors in

xenotransplantation research. As a rule,transgenic animals are being used in invivo experiments to examine gene func-tions, their regulation or the contributionof genetic alterations to the developmentof diseases. Many transgenic animals al-ready are affected in their wellbeing dueto the genetic modification alone regard-less of the procedures performed withthem. An ethical evaluation showed thatit is to be questioned whether the experi-mental use of transgenic animals led toresults that were of such outstanding sci-entific relevance that they legitimated thesuffering of the animals. In order to pointto possible approaches to avoiding theuse of transgenic animals in the areas ofresearch identified, subsequent investiga-tions aimed at collecting information onnon-animal test methods that might beapplied in pursuing the aforesaid ques-tions. In particular, these were non-ani-

mal test methods that make use of genet-ic techniques. Amongst these are in vitrocell culture methods with geneticallymodified cells, such as the so calledTransfected Cell Array, as well as in vitro test methods, in which specificallytargeted genes can be turned on or off selectively for example with help of theso-called RNA interference technique.Since such technologies can also be ap-plied to cell cultures with human cells,investigations with these methods enabledirect information on the function of hu-man genes. From the point of view of an-imal welfare the broad spectrum of al-ready available non animal test methodswith which to study the function of genesand genetically caused pathophysiologi-cal reactions shows that waiving of ani-mal tests with transgenic animals is pos-sible without impeding biomedicalresearch as such.

Poster

The use of transgenic animals in biomedical researchin Germany – status report 2001-2003, ethicalevaluation and examination of indispensability Ursula G. Sauer Animal Welfare Academy, Neubiberg, GermanyE-mail: [email protected]

Lecture

Regulatory animal testingMarie-Jeanne Schiffelers 1, Bas J. Blaauboer 2, Martje Fentener van Vlissingen3, Coenraad F.M.Hendriksen 4, Janne Kuil 5, René Remie6, Joop W.G.M. Thuring7 and Manon A. Vaal8

1Utrecht School of Governance, Utrecht University; 2Interfaculty Institute for Risk Assessment Sciences (IRAS); UtrechtUniversity; 3Erasmus Animal Experimentation Centre (EDC) Erasmus University Rotterdam; 4Netherlands Centre forAlternatives to Animal Use (NCA), Utrecht University/ Dutch Vaccine Institute (NVI), Bilthoven; 5Dutch Society for theProtection of Animals , The Hague; 6Solvay Pharmaceuticals, Weesp; 7Notox (Contract Research Laboratory), Den Bosch,8Science Shop for Biology, Utrecht University, all in The NetherlandsE-mail: [email protected]

Keywords: transgenic animals, genetic engineering, literature survey, cost-benefit-analysis, ethical evaluation, 3Rs, animalexperimentation, gene technological non-animal test methods

Thirty percent of the animal tests con-ducted annually in Europe are performedto meet regulatory requirements pertain-ing to the authorization and release of asubstance or product onto the Europeanmarket. Regulatory animal testing is of-

ten repetitive in nature and more likely tocause severe suffering than other types ofanimal testing due to the proceduresused. Because of these characteristics,regulatory animal testing is very interest-ing in terms of the 3R policy (Replace,

Reduce and Refine). A survey (2005)was conducted into the actors and factorsthat influence the use of animals in test-ing to comply with regulatory require-ments. Wherever possible, the researchfocussed on animal testing required by


Nowadays many people prefer drugs ofnatural sources instead using synthetics.But often the knowledge of their pharma-cological activities and safety is limitedor based on historical experiences. So wedecided to check different supposedpharmacological activities of several es-sential oils and their single componentswith a modern scientific method. A useful model was supposed to be theHET-CAM-Assay (Hen’s Egg Test –Chorioallantoic Membrane) utilizing theCAM’s capillary system of breeded heneggs. We used different modifications ofthe HET-CAM-Assay to screen for theantiangiogenic, the antiinflammatory ef-fect and also the irritant side effect of es-sential oils and their single components.Irritant side effect identification was

done by determining the irritation thresh-old concentrations which is the first con-centration causing bleeding of CAM´svessels. Antiinflammatory activity waschecked by ranking the phenomenons“star like vascularisation” and “granulo-ma formation”. In addition a cyclooxyge-nase (COX) assay was done, an enzymeinvolved in inflammation process. An-tiangiogenic activity was identified bydetecting vessel free areas on the CAM.Ranking essential oils and their singlecomponents by their irritation thresholdwas very successful. All essential oils orcomponents despite of sesquiterpenescaused haemorrhage on the CAM at leastin case of undiluted application. Identify-ing an antiinflammatory effect of essen-tial oils or their components using the

HET-CAM-Assay failed. Is was not pos-sible to create reproducible data not evenfor established antiinflammatory drugslike hydrocortison. Remarkalbe is that itwas possible to show that essential oilsdo have inhibition potential on COX, andso on inflammation processes, especiallyoils containing phenylpropanes. Someessential oils and single componentswere also able to create vessel free areason the CAM means to inhibit physiolog-ical angiogenesis. In conclusion theHET-CAM-Assay is a very useful detec-tion model to determine the irritating andthe antiangiogenic effect of essential oils.We also postulate that the HET-CAM-Assay is not able to give any antiinflam-matory response because of the matureimmune system of the chicken embryo.

Keywords: HET-CAM; essential oils; irritation; angiogenesis

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protocol for the authorization and releaseof pharmaceuticals. Regulatory animaltesting is a persistent element in the as-sessment procedures for registering asubstance or product for release onto themarket. Even though the number of alter-native test methods keeps increasing,these new methods are not automaticallyincluded in assessment procedures. In or-der to increase the use of alternativemethods to comply with regulatory re-

quirements, a number of obstacles mustfirst be overcome. Technical factors: Inthis field the greatest gain is expectedfrom so-called strategic test approaches,data sharing and retrospectivelyanalysing existing data. Political / admin-istrative and social factors: Harmoniza-tion of legislation and regulations is aprecondition for reducing regulatory ani-mal testing. This survey identifies anddescribes the various opportunities and

threats for the implementation of the 3Rsin regulatory animal testing. Further rec-ommendations are: * use risk communi-cation in order to influence the level of risk acceptance, * make the costs ofconducting animal tests transparent, * widely publicize available alternatives,* improve communication betweenstakeholders, * strengthen the policy network.

Poster

Usage of modified HET-CAM-assays to determineseveral phamacological activities of essential oils Jürgen Schneele and Jürgen ReichlingUniversity of Heidelberg, Institute of Pharmacy and Molecular Biotechnology, Dep. Biology, Heidelberg, GermanyE-mail: [email protected]

Keywords: regulatory requirements, animals, testing, technical factors, political/administrative factors, social factors,harmonization


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The discovery in 2002 of acrylamide for-mation in heated foods triggered a stillongoing worldwide research effort to ad-dress human health significance. Al-though formation pathways, food levelsand human exposure are currently large-ly established, no exposure- or food lim-its have been established to date. We hadpreviously initiated a study that wasbased on the assumption that dependenton precursors present in food (free aminoacids and reducing sugars), processesthat trigger the formation of acrylamide(Maillard reaction) may similarly triggerthe formation of other compounds thatmight raise concern. Modeling of forma-tion, food levels, exposure and toxicity

had led to the identification of severalcompounds that were prioritized by theirpotential level of concern based on mar-gin of exposure (MOE). Using this ap-proach, acrylamide turned out as thecompound with the highest level of con-cern, whereas all other compounds sig-nificantly ranked behind. In order to con-firm the results of the modeling study, 2compounds that revealed a relatively lowMOE and for which toxicological infor-mation was lacking, were selected fortesting in an in vitro study using primaryhepatocytes: 2- and 3-butenamide werestudied in comparison to acrylamide. Weused biochemical endpoints (cytotoxicityand glutathion depletion) as quantitative

cytotoxicity parameters and Affymetrixmicroarrays to address qualitative as-pects of the response to the compounds.Results will be presented that demon-strate that, in spite of very similar chem-ical structure compared to acrylamide, 2-and 3-butenamide are of much less cyto-toxic potency. In addition, the two com-pounds induce changes in the gene ex-pression profile that significantly differfrom the response induced by acry-lamide. We conclude that based on mod-eling and in vitro studies, potential pro-cessing contaminants can be rankedaccording to safety concern. Such an ap-proach may allow to set priorities for fur-ther research.

Lecture

Hazard identification in chemical food safety: use of modeling, primary hepatocyte cultures andmicroarrays to address the level of concern forpotential process contaminants of unknown toxicity Gabriele Scholz 1, Gabriela Guignard 1, Clothilde Verguet 1, Sofiane Lariani 2, Marie-Camille Zwahlen2 and Benoît Schilter 1

1Quality & Safety; 2Bioanalytical Science, Nestlé Research Center (CH), Vers-chez-les-Blanc, 1000 Lausanne 26 E-mail: [email protected]

Keywords: process contaminants, modeling, acrylamide, primary hepatocytes, microarrays

Next to toxicity tests, efficacy is the ma-jor issue before compounds can be trans-ferred to clinical phases. Related animalmodels for some of the most importantareas of human disease, e.g. cancer andneurodegeneration, require very labori-ous, sophisticated but often extremelydisruptive, irksome and even cruel proce-dures. Functional, molecular and be-havioural read-outs are an absolute ne-cessity for pre-clinical therapeutic trials.Among examples there are xenograft

models for various human cancers; mod-els for stroke which induce severe cere-bral ischemia by permanent occlusion ofcerebral arteries in test animals (MCAO);various transgenic rodent models forneurodegenerative diseases, or extremelytough experimental models for autoim-mune encephalomyelitis induced by vac-cination of test animals with autoanti-gens as preclinical models of multiplesclerosis (MOG-EAE). Despite a highdegree of sophistication, there is a gener-

al sense of caution towards these models,because often their read-outs are insuffi-cient or misleading. Taken together, inthe area of efficacy testing, there is an ur-gent need for the development of novelin vitro methods, which can balance dis-advantages in terms of organ specificbarriers and metabolism with advantagesregarding the higher relevance of human-ised systems, more precise functionaland molecular read-outs and potential ofhigher throughput. In the given situation

Lecture

Stem cell-based in vitro models as a basis to test efficacies of preclinical phases of anti-neurodegenerative treatmentsAndré Schrattenholz ProteoSys AG, Mainz, GermanyE-mail: [email protected]


Institute of Pharmacy, Free University ofBerlin, G-Berlin The corneal epithelialmultilayer represents the principal barri-er in the ocular pathway and is involvedin corneal inflammation and wound heal-ing processes. An immortalised humancorneal cell (HCE) line (Araki-Sasaki,1995) has been used to reconstruct thecorneal epithelium. This model hasproved to be a useful tool for both drugtoxicity and permeation studies. It wasthe aim of this study to optimise the mor-phology and barrier properties of the ep-ithelial layer with respect to different cul-ture conditions. HCE were cultivated in

cell culture inserts. The effect of medi-ums containing different serum and cal-cium concentrations were followed for16 days. Growing rate, number of celllayers and epithelial differentiation wereevaluated by cryosectioning and light mi-croscopy. Barrier properties were deter-mined by transepithelial electrical resis-tance (TEER) together with immuno-histochemistry of the tight junction relat-ed protein claudin-1. Epitheliums culti-vated in high serum concentrationshowed hyper-proliferation (about 13cell layers at day 8 and 23 cell layers atday 16). In contrast, serum-free culture

conditions using mediums supplementedwith calcium resulted in a morphologywhich is closer to in vivo conditions (5layers at day 8 and 7 layers at day 16).According to our TEER measurementsand claudin-1 expression, high serumconcentration reduced the barrier proper-ties compared to serum-free conditions,whereas calcium seemed to increasethem. In conclusion, serum free cultureconditions paralleled by high calciumconcentrations are the most promisingconditions for adequate number of celllayers and effective barrier function ofreconstructed epithelia.

Keywords: human corneal epithelial model, immortalised epithelial cells, transepithelial electrical resis tance,immunohistochemistry

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of regulatory frameworks in the EC andelsewhere, again the purely diagnosticuse of human embryonic stem cells mod-els appears to develop into a highly at-tractive alternative. They can be differen-tiated to organotypic cell cultures andserve as flexible substrate for a variety offunctional molecular endpoints. More-over they are ideal for validation purpos-

es using modern silencing technologiesin a framework of genetically homoge-neous differentiations to various “tis-sue”-like cell culture substrates like neu-ral cells, cardiomyocytes, various typesof muscle cells and adipocytes, etc. Herewe show results from initial phases of aproject which attempts to explore theoutstanding potential of corresponding

human embryonic stem cell (hESC)-based in vitro models. Related resultsfrom corresponding murine ESC-screen-ing systems, in combination with quanti-tative differential proteomic display techniques, have already provided bio-markers for potential replacement ofsome related animal models.

Sandwich Nucleoside analogues (NUC)are a major class of antiviral drugs thattarget viral DNA polymerases. They arewell established in the treatment of HIV

infection and are also under evaluation asnewer treatment strategies against thehepatitis B and C viruses. Long-termtreatment with NUC is however, associ-

ated with adverse side effects often at-tributed to inhibition of mitochondrialDNA (mtDNA) synthesis that results ingradual impairment of mitochondrial

Poster

Characterisation of morphology and barrierproperties of a new human corneal epithelial modelJudith Wanda Seeber, Simone Lombardi-Borgia, Monika Schäfer-Korting and Maria EngelkeInstitute of Pharmacy, Free University of Berlin, GermanyE-mail: [email protected]

Lecture

10-day medium throughput in vitro screen to assesschronic cytotoxicity of nucleoside analogues Mark R. Slaughter 1, Claire Hougham1, Jonathan M. Hitchcock1, Aisling N. Potter1, Philip Brain2 and Peter J. O’Brien1

1Safety Sciences Europe and 2Clinical Research and Development, Pfizer Ltd, United KingdomE-mail: [email protected]

Keywords: embryonic stem cells, MCAO, MOG-EAE, neuroprotection, neurodegeneration


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function. As clinical toxicity normallydevelops only after long-term NUC ther-apy, in vitro investigations tend to in-volve chronic modelling for periods ofup to 30 days. We required a suitable andefficient in vitro toxicity screen for NUCthat could be applied at the drug discov-ery stage as part of a strategy to reducethe rate of late-stage attrition of new drugcandidates. Accordingly we designed achronic cytotoxicity assessment model(CCAM) with HepG2 human hepatomacells grown in 96-well microplates with-out the nee d to subculture, and which al-lows co-measurement of cell count/via-bility and mtDNA. The format includes

generation of nine-point dose-responsecurves using triplicate wells per drugconcentration with treatment replenish-ment three times per week. After 4, 7 and10-days’ treatment, cells are assessed forgrowth and viability with nuclear dyes(Guava ViaCount)and counted on a Gua-va PCA-96 flow cytometer after whichmtDNA (day 4) is measured by PCR.Degree of toxicity is determined by therate of decrease in therapeutic index(IC50:Cmax or IC50:antiviral IC50 ratios)with time and/or whether a threshold TIis exceeded. Dependence of toxicity onmtDNA depletion is determined by rela-tive displacement of dose-response

curves for cell count and mtDNA levels.Five anti-AIDS NUC were used to vali-date the model, for which there was closeconcordance between degree of in vitrotoxicity and severity of clinical toxicity.After 4 days’ treatment, the relative dis-placement of mtDNA do se-responsecurves from those for cell count accu-rately predicted the differential depen-dence of toxicity of NUC on mtDNA de-pletion. CCAM offers considerablesavings over animal studies, only re-quires a total of 10-15 mg compound,and is conducive to the 3Rs principle. Wenow use CCAM for routine screening.

Lecture

An in vitro model for early embryonic development Martina Stary, Waltraud Pasteiner, Georg Weitzer and Max F. Perutz Laboratories, Medical University of Vienna, Austria E-mail: [email protected]

Lecture

Application of DNA microarrays for immunotoxicological testingSandra Szameit, Markus Mansfeld, Waltraud Novak, Helga Tuschl and Christa Nöhammer Molecular Diagnostics Unit, ARC Seibersdorf research GmbH, Seibersdorf, AustriaE-mail: [email protected]

Keywords: chronic cytotoxicity screen, nucleoside analogues, HepG2 cells, cell growth, mitochondria, mtDNA

Embryonic stem cells give rise to embry-oid bodies which undergo developmentalprocesses at the molecular, cellular andmorphological level reminiscent of eu-therian embryonic development [1]. De-fined culture conditions and epigeneticstatus of embryonic stem cells allow tomimic pregastrulation development andearly gastrulation in vitro. We coulddemonstrate that extra-embryonic endo-derm forms properly and influences ear-ly gastrulation giving rise to all threegerm layers [2]. Specific modulation of

primitive mesoderm by extra-embryonicendoderm derived growth factors allowsreproducible development of large num-bers of cardiomyocytes [3-5], which sofar continues to proliferate for 35 daysand to contract rhythmically for 150days. This model allows to study earlydevelopmental processes in vitro in basicscience avoiding some experiments onliving embryos, and provide a source ofcardiomyocytes which may be used forapplied long term toxicological studies.Further development of this model be-

yond gastrulation into organogenesis,however, challenges the questionwhether this ends justify the means?

[1] Weitzer, G. (2006). Handb. Exp.Pharmacol., 174, 21-51 ; [2] Bader, A. etal. (2001). Differentiation, 68, 31-43 ; [3]Bader, A. et al. (2000). Circ. Res. 86,787-794 ; [4] Lauss, M. et al. (2005).Biochem. Biophys. Res. Com., 1577-1586; [5] Stary, M. et al. (2005). Exp.Cell Res. 310, 331-343.

Keywords: embryonic stem cells, embryoid bodies, embryogenesis, gastrulation, cardiomyogenesis, growth factors

The goal of our present research is thedevelopment of a DNA microarray as anin vitro toxicological test system to re-

veal the sensitising potential of chemi-cals. Dendritic cells from human donorsare used to analyse gene expression after

treatment with different potential aller-gens. Furthermore, the microarray shallbe used to discriminate between contact


Agrochemicals must undergo numeroustoxicological tests before registration.Using animals in toxicological screeningis a controversial issue. The Draize eyeirritation test is one of the most criti-cized methods because of the injuries in-flicted on the test animals. Several in vit-ro methods have been used to investigatethe toxicity of potential eye irritants witha view to replacing in vivo eye irritationtesting. In the HET-CAM test chemicalsare placed in direct contact withchorioallantoic membrane of the hen’segg. The occurrence of vascular injuryor coagulation in response to a com-pound is the basis for employing thistechnique as an indication of the likeli-

hood that a chemical would damage mu-cous membranes (especially the eye) invivo. The CAM is a complete tissue in-cluding arteries, capillaries and veins,and is technically easy to study. It re-sponds to injury with a complete inflam-matory reaction, similar to the tissue ofthe rabbit eye. In our studies compara-tive screening was performed with a setof agrochemicals to establish paralelldata on in vitro (HET-CAM) and in vivo(Draize) results. The test materials were:Totril (ioxynil), Omite 57 E (propargite),Actellic 50 EC (pirimiphos methyl),Stomp 330 EC (pendimethalin), Mospi-lan 3 EC (acetamiprid), Alirox 80 EC(EPTC), Thionex 35 EC (endosulfan),

Pyrinex 48 EC (chlorpyrifos). Agro-chemicals to be tested are added to themembrane and left in contact for 5 min-utes and the membrane is examined forvascular damage at set time periods. Irri-tancy is scored according to the severityand speed at which damage occurs pro-viding an indication of the likely irritanteffect of the compound. Our studyshowed good correlation between resultsobtained by the HET-CAM test andthose of the Draize rabbit eye test mostcases. The present form of the HET-CAM test can be proposed as a pre-screen method of eye irritation tests,therefore the number of test animals canbe reduced.

Keywords: agrochemicals, pesticides, in vitro, ocular irritation, chorioallantoic membrane

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allergens and respiratory allergens. Wedeveloped a DNA microarray containing65 immune genes, housekeeping genes,negative controls and external spike con-trols for normalization. Different la-belling techniques were tested in order tooptimize the system, especially takinginto account the limited amount of im-mune cells and RNA available for the mi-croarray assay. For protocol optimizationthe human monocytic cell line THP-1was used, which can be cultivated easilyand leads to higher RNA amounts com-pared to immune cells. The cells werestimulated with LPS and gene expression

in LPS-treated cells was compared togene expression in untreated cells. Ap-plying a direct labelling protocol, in-creased expression of several immunerelevant genes could be shown with ei-ther 10 µg or only 2 µg of total RNA.However, more upregulated genes couldbe detected with an indirect labellingmethod. Again, specific signals could al-ready be obtained after using only 2 µgof total RNA. Based on these results, wenow use the indirect labelling protocolfor the microarray analysis. In order tofind candidate genes for the identificationof immunotoxic chemicals, nickel sulfate

and other model allergens are applied todendritic cells and gene expression iscompared to gene expression in untreat-ed cells. For the analysis of yet unchar-acterized chemicals, a general treatmentprocedure has to be established and atime point for expression analysis has tobe defined. Therefore, expression pat-terns at different time points after appli-cation of known allergens are compared.Our preliminary results indicate that themicroarray technology could provide anin vitro alternative to animal test methodscurrently available to predict the sensitiz-ing effects of chemicals.

Poster

Examination of ocular irritancy by the HET-CAM test Judit Tavaszi and Péter Budai University of Veszprém, Georgikon Faculty of Agriculture, Institute of Plant Protection, Department of Hygiene, Keszthely,HungaryE-mail: [email protected]

Keywords: DNA microarrays, dendritic cells, immunotoxicological testing


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Lecture

Cell culture model for colon carcinogenesis,chemoprevention and organ-selective toxicityNitin Telang and Meena KatdareStrang Cancer Prevention Center and Weill Medical College of Cornell University, New York, USAE-mail: [email protected]

Germ line mutation in the tumor sup-pressor adenomatous polyposis coli(APC) gene represents a primary geneticdefect in the clinical familial adenoma-tous polyposis (FAP) syndrome, predis-posing for colon cancer. Targeted chaintermination mutation on codon 1638 ofthe mouse APC gene promotes small in-testinal carcinogenesis predominantly inthe small intestine. Administration of achemical carcinogen or a tumor promot-ing high fat/ low micronutrient diet toAPC mutant mice, however, inducescolon carcinogenesis. A reliable cell cul-ture model established from appropriatetarget organ that expresses relevant ge-netic defect and quantifiable risk for car-cinogenesis should provide an innovativecomplementary approach to long termanimal studies on colon carcinogenicity,chemoprevention and organ selective

toxicity. The objective of this study wasto develop a reliable preclinical cell cul-ture model for human FAP syndrome andvalidate this model as a rapid mecha-nism-based approach for screening effi-cacious preventive/therapeutic com-pounds.

The newly developed epithelial cellline 1638N COL from morphologicallynormal colon of haplo-insufficient APC1638N +/- mutant mouse exhibited aber-rant cell cycle progression, down-regu-lated apoptosis, enhanced risk for car-cinogenesis in vitro and tumorigenesis invivo. The aberrantly proliferative tumori-genic APC mutant cells also exhibitedabout a 2-4 fold higher constitutive ex-pression of β-catenin, cyclin D1 andCOX-2 proteins, relative to that in thewild type colon epithelial cells express-ing the APC +/+ genotype. Treatment of

APC mutant cells with low dose, non-toxic combinations of mechanisticallydistinct chemopreventive/ therapeuticagents resulted in about a 3 fold higherefficacy for cytostatic growth arrest, andabout a 20-35% higher efficacy for inhi-bition of the risk for carcinogenesis, rel-ative to that exhibited by these agentsused independently.

These data establish a novel cell cul-ture model for clinical FAP syndromeand validate a rapid, mechanism-basedscreening approach to prioritize effica-cious combinations of preventive/ thera-peutic agents for long term in vivo ani-mal studies and future clinical trials oncolon cancer prevention. [Support: TheIrving Weinstein Foundation and NCIMAO # CN-75029-63].

Keywords: epithelial cell culture, gene mutation, cancer prevention

With the intensifying demand on alter-natives to animal experiments in indus-try and to address fundamental scientificquestions, the need for qualified techni-cal and scientific personnel is steadilygrowing. It will become increasingly im-portant to educate technicians special-ized to develop and perform alternativesmodels and have a critical view on ex-perimental designs of animal studies foroptimal use of animal data. In order toaddress this need a course on alterna-tives to animal use in life sciences re-

search will start in September 2006. Stu-dents engaged in bachelor-level educa-tion in life sciences can participate inthis course. The aim of the course is toacquaint students with both theoreticaland practical aspects. The focus will benot only on development of single re-placements for individual animal experi-ment, but to educate students in innova-tive testing strategies resulting inoptimal benefit from animal and non-an-imal data. The up to date 6-month courseconsists of several thematically and in-

terdisciplinary modules, including thethree R’s principle, Legislation, in vitroand in silico alternatives in Efficacy &Safety testing, Toxicological risk assess-ment in industry, Validation issues, Datamanagement, bioinformatics and com-puter modelling, Ethics and Public opin-ion. One important feature of the coursewill be the project \'Mission Alterna-tive\'. Throughout the course partici-pants will work together with researchinstitutes and industry to solve an exist-ing problem or answer a scientific ques-

Poster

Innovative testing strategies as alternatives toanimal testing, an international course Marc Teunis1and Cyrille Krul 2

1Utrecht University of Professional Education, Utrecht, The Netherlands; 2TNO Quality of Life, Zeist, The Netherlands E-mail: [email protected]


Ethics is a philosophical state of mind,required to make a correct judgment.Each country has a specific moral disci-pline related to their cultural and reli-gious background. In the Sri Lankan con-text, being a majority Buddhist nation,animal welfare as well as animal rightsissues take on added prominence. Ac-cording to the paper titled Animal Wel-fare Legislation in Sri Lanka (proposalfor law reforms on animal welfare legis-lation), of the 16 enactments that dealwith animal and animal welfare, onlyfour statutes have been passed during thepost independence era (1948). A recentupsurge in interest in these issues has led

to the development of a Draft Act await-ing parliamentary approval in Sri Lanka,which seeks to replace the antiquatedPrevention of Cruelty to Animal Ordi-nance No. 13 of 1907 and also to imple-ment several lapses in other legislationsrelating to animal welfare such as thestatute to regulate the use of animals forscientific research. The Sri Lankan pub-lic is mostly unaware of animal experi-ments. However, it is the duty of the sci-entific community to carry out animalresearch where needed for the well beingof the society; the society being com-prised of both humans and animals. Thusthere is a requirement for proper ethical

control of animal experiments. If this as-pect is not given adequate consideration,as soon as the public does become moreaware of animal experimentation a hugecontroversy is bound to arise. As can beclearly seen in the above discussion, inaddition to ethical controls to animal ex-perimentation, alternative techniques areimportant to enhance our research ef-forts. We look forward eagerly to learn asmuch as possible about this relativelynew and developing area of alternativesfor animal experiments, as we in SriLanka feel it is especially relevant andappropriate to the local cultural situation.

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tion, regarding alternatives to animaluse. Currently, we are attracting lectur-ers from abroad to enhance the interna-tional character of the course. To facili-tate participation for international

Prediction of toxicity and compound re-sponses in hepatocytes continues to be amajor concern for the pharmaceutical in-dustry. Therefore, in vitro studies of livercells are important investigations for tox-icity and drug compatibility and can helpto reduce animal trials. Furthermore an

important question is: How compatibleare results of animal testing with humanresponse? The advantage of in vitro sys-tems is the usability of human cells. Butimmortalised human cell lines do not be-have like primary cells. Non-immor-talised primary cells may best represent

normal physiology. In the present studyprimary human hepatocytes were usedfor an in vitro test in our Bionas® 2500analysing system. This analysing systemis able to monitor oxygen consumption,acidification and adhesion on six siliconchips in parallel. Modifications of these

students this course will be developed inclose collaboration with the internation-al office of our university and foreigncontacts. The course has been set up inclose collaboration with the Dutch Orga-

nization for Applied Scientific Research,TNO. This resulted in an excellent pro-gram on both conceptual and practicalaspects.

Poster

Ethical and legal aspects in animal experiments in Sri LankaMayuri Thammitiyagodage1, Sharmini Jayasekera 1 and Nalinika Obeyesekera 2

1Medical Research Institute, Colombo 8, Sri Lanka; 2Pet Vet Clinic, Malalasekera Mawatha, Colombo 7, Sri Lanka. E-mail: [email protected]

Lecture

Using of an in vitro system for prediction ofhepatotoxic effects in primary human hepatoctyes Elke Thedinga1, Anett Ullrich2, Sabine Drechsler 1, Ricarda Niendorf 1, Axel Kob1, Dieter Runge 2,Andreas Keuer 1, Ingo Freund 3, Mirko Lehmann3 and Ralf Ehret 1

1Bionas GmbH, Rostock; 2Primacyt GmbH, Schwerin, 3Micronas GmbH, Freiburg, GermanyE-mail: [email protected]

Keywords: education, alternatives

Keywords: Sri Lanka, animal welfare


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parameters in response to test com-pounds reflect effects on the cellmetabolism. All parameters are detectedcontinuously and online during the longterm measurement (up to several days).As an additional advantage regenerationand recovery effects can also be moni-tored respectively. In this study we havecultured primary human hepatocytes oncollagen pre-coated chips in chemicallydefined Human Hepatocyte Maintenance

Medium and, for comparison, in conven-tional two-dimensional cultures. The op-timised medium allows cultivation offunctionally differentiated hepatocytesfor several weeks. In this study we com-pared the sensor chip based in vitro re-sults with standard assays for hepato-cytes like albumin release and urearelease. We have investigated effects ofacetaminophen (AAP) on primary hepa-tocytes. The hepatocytes were exposed to

AAP (50-2815 mg/l) for 24 h. Primarily,cell respiration was obviously inhibitedby AAP concentrations from 500 mg/l.Whereas cell adhesion was marginallyreduced. In conventional cultures AAPadministration had no effect on cellularviability. High doses of AAP (2815 mg/l)diminished urea pr oduction by 15-30%and albumin secretion by 70-80%.

Lecture

Daphnia magna as alternative bioobject inecotoxicology Valerii TonkopiiInstitute of Limnology, St. Petersburg, Russia E-mail: [email protected]

Keywords: human primary hepatocytes, in vitro, online monitoring, cell metabolism

We have been developing non-tradition-al methods of the identification of pollu-tants, using various hydrobionts as bio-logical objects and the study of themechanism of toxic action of xenobi-otics. The experiments were carried outwith using of Daphnia magna. Daphniamagna is a Crustacean in the order ofCladocera. This aquatic animal exten-sively used as a test organism in aquatictoxicology due to their small size, shortlife cycle and amenability to lab culture.Daphnia magna is the most sensitivetest-object in relation of different pollu-tants among all known biological objectsincluding experimental animals. Experi-ments were performed with a 2-days oldculture of Daphnia magna. The toxicityof xenobiotics was determined by thevalue of LC50, a concentration of the

compounds causing death to 50% of hy-drobionts during incubation with toxi-cants for 24 hours. In the first stage ofthe work, toxicity of organophosphates(Dipterex, DFP, DDVP, Paraoxon,Malathion, Malaoxon), heavy metalsions (Hg, Pb, Cu, Co, Cd, Cr, As, Al),organochlorines (Aldrin, Dieldrin, En-drin, Aroclor, DDT, Lindane, PCBsetc.), cyanides (sodium cyanide) andpyrethroids (Cypermethrin, Fenvalerate,Deltamethrin, Permethrin, Allethrin,Resmethrin, Phenothrin, Kadethrin,Cyphenothrin) was determined. The ef-fects of a number of antagonists on thetoxicity of xenobiotics were studied. Atthe first time we discovered that in ex-periments to Daphnia magna some mus-carinic cholinoreceptor blockers (at-ropine, amyzil etc.) reduced a toxic the

effect of organophosphates. In the caseof heavy metals the chelating agents(EDTA , Dithioethylcarbamate, Unithi-olum, Sodium thiosulphuricum, L-As-partic acid) were effective, for certainorganochlorine poisonings - anticonvul-sive drugs (diazepam, phenobarbital),for cyanide poisoning sodium nitrite andanticyane. In the case of pyrethroid’spoisonings the antagonist of glutamatereceptor (ketamine) and agonists of GA-BA-receptor (phenazepam, ethanol) re-duced the toxicity of xenobiotics. As faras these antidotes have a specific treat-ment action only against definite classesof pollutants, we have elaborated thesensitive express-methods of bioidentifi-cation of pollutants.

Keywords: daphia magna, pesticides, bioidentification


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In the experiments on the rats we havedeveloped methodological approach tothe evaluation of the selectivity of mus-carinic cholinergic receptors (M-ChR)antagonists action in the whole organismconditions. According the results ob-tained during investigation the protectiveeffect of M cholinolytics during acutepoisonings of organophosphates (DDVP,DFP etc.) depends on M1 subtype ChRoccupation. The efficiency of antago-nists in inhibition of tremor reactioncaused by M-ChR agonist arecoline ad-ministration associates with interactionof M2 subtype of ChR. It was estab-lished by the method of linear regres-sion, that there was a high degree of correlation (r=0.99) for different M

cholinolytics between the ratios of ED50of M antagonists in the tests with areco-line and organophosphates and the ratiosof dissociation constants of antagonistscomplexes with M-ChR from the ho-mogenates of rat\’s cerebral cortex andheart containing M1 and M2 ChR sub-types respectively. Thus, the ratio ofED50 arecoline/ ED50 DDVP is serve as ameasure of the selectivity of drugs ac-tion. Pharmacological analysis of Daph-nia magna cholinergic system with theuse of anticholinesterase compounds,and M-ChR agonists and antagonistswas carried out. On the experiments toDaphnia magna the effects of some nonselective, mainly M1 and M2 ChR an-tagonists on the toxicity of DDVP and

arecoline were studied. There was astrong correlation between the ED50 ofantagonists in the tests with arecolineand DDVP in the experiments on ratsand the EC50 of antagonists in experi-ments on Daphnia magna. For the firsttime in the experiments on Daphniamagna it was shown that a ratio of theaverage effective concentrations (EC50)of M antagonists in the tests with areco-line and organophosphates also may beused as a measure of the selectivity ofM-ChR antagonists action. The principalsimilarity in action of muscarinic antag-onists to Daphnia magna and rats allowto recommend the Daphnia for screeningof selective muscarinic receptor antago-nists.

Poster

The usage of Daphnia magna for screening ofselective muscarinic cholinergic receptors antagonistsValerii Tonkopii Institute of Limnology, Russian Academy of Sciences, St.Petersburg, Russia. E-mail: [email protected]

Keywords: Daphnia magna, screening,cholinilytics

Directive 86/609/EEC on the protectionof animals used for experimental andother scientific purposes is currently be-ing revised. The Technical Expert Work-ing Sub-Group on ethical review pro-poses that part of the ethical reviewprocess should take place at the locallevel. Suggestions are also made withrespect to the competencies of the par-ticipants in the ethical review process.In the Netherlands, ethical review ismandatory since 1997. The Dutch Acton Animal Experimentation (1996) re-quires that animal experiments commit-tees (AECs) should review animal ex-periments and balance the scientific and

societal interests of the experimentsagainst the suffering of the experimentalanimals. According to the Dutch regula-tions the AECs have to be composed ofat least seven members. The AECsshould equally represent competencieson experimental animals, alternatives tolaboratory animals, ethics and finally onanimal welfare and protection. Criteriathat have to be met in order to be re-garded as expert in one or more of theseareas have not been described. The com-petence of the AEC members can there-fore not be guaranteed. Recently, theDutch Act has been evaluated. The eval-uation committee recommends that cri-

teria have to be developed for the com-petence of members of AECs In thisstudy, representatives of the four com-petencies were consulted in order todraft criteria which include both ade-quate knowledge and competence butthat also allow a sufficient number ofpeople to qualify for satisfying the com-position of the AECs. Furthermore, it isclaimed that in order to keep the knowl-edge of each expert up-to-date, compul-sory continuing education of AECmembers is required. Education shouldcover general items like statistics, alter-natives, ethics, legislation, discussiontechniques, and the importance of con-

Lecture

Criteria for expert assessment by animal experiments committeesJan van der Valk and Sandra Swart Netherlands Centre Alternatives to animal use, Nl, Utrecht E-mail: [email protected]


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sensus. In addition, compulsory coursesare proposed to regularly update eachcompetence on developments in their

respective areas. The results of thisstudy may serve as a starting point forfurther discussions on criteria for AECs

members both in the Netherlands and inother countries.

Poster

Relevance of the in vitro and in vivo studies ingenotoxicological research on fibresCsaba Varga and Katalin SzendiDepartment of Environmental Health, Institute of Public Health Medicine, University of Pécs, Pécs, HungaryE-mail: [email protected]

Keywords: fibres, asbestos, mesothelioma, genotoxicity

Asbestos fibres can be considered asarchetypes of toxic mineral fibres. Long-term exposure to the fibres can initiateasbestosis, bronchial carcinoma andpleural or peritoneal mesothelioma. Ox-idative damage caused by the fibres maystudied in in vitro genotoxicity tests in-volving analyses of bacterial mutagenic-ity or cytogenetic alterations in culturedcells. But the crucial endpoint (mesothe-lioma) is very difficult to study in an in

vitro system. Mesothelial cell culturesshow very short proliferative lifespanand early onset of senescence. Humanperitoneal mesothelial cells were foundonly few (appr. 6) population doublingsprior to senescence. Cells of the agingcultures generate more reactive oxygenespecies and display increased quantity ofoxidatively modified DNA. Thereforethey are not suitable for testing similarinduced effects by fibres and long-term

exposures. We developed an in vivomesothelioma-induction test for researchon fibrous materials. The test involvesone single treatment and simple surgicalmethods by using minimal number ofrats. Our model provides the opportunityof new classes of fibrous materials liketubes produced by nanotechnology. Weconcluded that only a battery of in vitroand in vivo studies can provide relevantdata for the human risk assessment.

Lecture

The challenge of predicting drug toxicity in silicoAngelo Vedani, Max Dobler and Markus A. Lill Biographics Laboratory 3R, CH-Basel E-mail: [email protected]

Poor pharmacokinetics and toxicity arenot only frequent causes of late-stagefailures in drug development but also asource for unnecessary animal tests. Indrug discovery and for the assessment ofthe toxic potential of chemicals, in silicotechniques are nowadays considered asvaluable alternatives to in vivo ap-proaches. Computer-based concepts al-low to study both existing and hypothet-ical compounds; the methods are fast,reproducible, and typically based on hu-man bioregulators, making the questionof transferability obsolete. In the recentpast, our laboratory contributed towardsthe development of in silico concepts (→ multi- dimensional QSAR) and vali-

dated a series of “virtual test kits” basedon the estrogen, androgen, thyroid, andaryl hydrocarbon receptor (endocrinedisruption, receptor-mediated toxicity)as well as on the enzyme cytochromeP450 (metabolic transformations, drug-drug interactions). The test kits arebased on the three-dimensional structureof their target protein (i.e. ERαβ, AR,TRαβ, CYP450 3A4) or a surrogatethereof (AhR) and were trained using arepresentative selection of 362 sub-stances. Subsequent evaluation of 107compounds different therefrom showedthat binding affinities are predicted closeto experimental uncertainty. These re-sults suggest that our approach is suited

for the in silico identification of adverseeffects triggered by drugs and chemicalsand encouraged us to compile an Inter-net Database for the virtual screening ofdrugs and chemicals for toxic effects.

References: Vedani, A., Dobler, M., Lill,M. A. (2005). Combining protein model-ing and 6D-QSAR – Simulating thebinding of structurally diverse ligands tothe estrogen receptor. J. Med. Chem. 48,3700–3703. Lill, M. A., Winiger, F.,Vedani, A. and Ernst, B. (2005). Impactof induced fit on the ligand binding tothe androgen receptor: A multidimen-sional QSAR study to predict endocrine-disrupting effects of environmental

Keywords: animal ethics committees, ethical review process, criteria, expertises


The Biographics Laboratory 3R has developed and validated a series of virtu-al test kits to identify the toxic potentialof both existing and hypothetical chemi-cals. Presently, our pilot simulation (cf. http://www.biograf.ch/GIFS/ToxDataBase.gif) focusses on endrocrinedisruption (estrogen, androgen, thyroidand aryl hydrocarbon receptor) as well ason metabolic events mediated by cytochrome P450 (cf. http://www. biograf.ch/projects.html). The models

were trained using a representative selec-tion of 362 substances. Subsequent eval-uation of 107 compounds different there-from showed that binding affinities arepredicted close to experimental uncer-tainty. These results suggest that our ap-proach is suited for the in silico identifi-cation of adverse effects triggered bydrugs and chemicals and encouraged usto compile an Internet Database for thevirtual screening of drugs and chemicalsfor toxic effects.

References: Vedani, A., Dobler, M., Lill,M. A. (in press). The challenge of pre-dicting drug toxicity in silico. Pharma-cology & Toxicology. Vedani, A., Dobler,M., Lill, M. A. (2005). Virtual test kitsfor predicting harmful effects triggeredby drugs and chemicals mediated by spe-cific proteins. ALTEX 22, 123–134.Vedani, A., Dobler, M., Lill, M. A.(2005). In silico prediction of harmful ef-fects triggered by drugs and chemicals.Toxicol. Appl. Pharmacol. 207, 398–407.

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chemicals. J. Med. Chem. 48,5666–5674. Vedani, A., Dobler, M., Lill,M. A. (2005). Virtual test kits for pre-dicting harmful effects triggered by

drugs and chemicals mediated by specif-ic proteins. ALTEX 22, 123–134. Lill,M. A., Dobler, M., Vedani, A (2006).Prediction of small-molecule binding to

Cytochrome P450 3A4: Flexible dock-ing combined with multidimensionalQSAR. Chem. Med. Chem. 1, 73–81.

Poster

Virtual test kits for predicting chemical toxicity in silico Angelo Vedani, Max Dobler and Markus A. Lill Biographics Laboratory 3R, CH-Basel E-mail: [email protected]

Keywords: ToxDataBase, in silico, reduction of animal models

Due to the misunderstanding of the termalternatives and the confusion of manypeople who think that it refers only toreplacement, it is of great interest, thedevelopment of a special course on al-ternatives and the 3Rs principle, in or-der to clarify concepts. A trainingcourse in the 3Rs principle has been de-veloped in the last years in the Facultyof Pharmacy of the University ofBarcelona. This is a 30 hours post-grad-uated course based on theoretic andpractical classes. The people attendantit, are principally, post-graduates in

Pharmacy and Biology, but it is open toother graduates. The objectives of thecourse are to introduce students on the3Rs principle and the use of alternativesto animals according to the EU policieson cosmetics and chemicals, the so-called REACH (Registration, Evalua-tion and Authorisation of Chemicals).After an introduction on the history ofthe 3Rs we focused the course in themost relevant alternative methods oftoxicology in vitro. Especial attention ispaid on the performance of some scien-tifically validated in vitro methods

which are accepted by regulatory toxi-cology and the statistical evaluation ofthe test results. Among these test, thechorioallantoic membrane based test,the red blood cell haemolysis and thecytotoxicity in cell lines are evaluated.The search of alternatives and the infor-mation, on the more relevant webs, con-stitutes an important part of this course,providing the media to develop a searchstrategy using the more appropriate key-words and concepts. The students areintroduced in the most relevant institu-tions involved in the development and

Poster

Training course on the 3Rs principle Maria Pilar Vinardell and Montserrat Mitjans Dep. Fisiologia, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain E-mail: [email protected]

Keywords: virtual test kits, in silico prediction of harmful effects triggered by drugs and chemicals, reducing animal testing


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validation of alternatives. Special atten-tion is paid on alternatives in educationand their advantages in front of the tra-

ditional practices with animals. We pro-vide to our students experience to applythe different methodologies to their own

research and especially, to promote theuse of alternative methods.

Poster

In vitro alternative to evaluate the potentialantioxidant capacity of polyphenols from differentsources Maria Pilar Vinardell 1, Vanessa Ugartondo1, Montserrat Mitjans 1, Sonia Touriño2

and Josep Lluis Torres 2

1Dep. Fisiologia, Facultat de Farmacia, Universitat de Barcelona, Barcelona; 2IIQAB-CSIC, Barcelona, Spain E-mail: [email protected]

Keywords: 3Rs, alternatives, course, education

The evaluation of the antioxidant capaci-ty of new compounds is usually tested inanimals. The in vivo study is performedin rats pre-treated by gavage with theproduct and the determination of theserum levels of the hepatic enzymemarkers aspartate aminotransferase andalanine aminotransferase and the indica-tors of oxidative stress in the liver, suchas the glutathine disulfide content andlipid peroxidation are measured. In otherdeterminations rat liver microsomes areused. A possible alternative to the animaluse, is the chemical determination, nev-ertheless, biological determinations havebeen demonstrated to be more useful. Wehave studied the potential antioxidant ac-tivity of polyphenolic fractions obtainedfrom pine, hamammelis, and grape. The

mixtures contain mainly flavanols (cate-chins) of similar degree of polymerisa-tion and different percentage of pyrogal-lol groups in the following order:hammamelis > grape > pine. The in vitrooxidative haemolysis of human red bloodcells (RBCs) was used as a model tostudy the free radical-induced damage ofbiological membranes and the protectiveeffect of the flavanols. The haemolysis ofRBCs was induced by a water-solublefree radical initiator 2,2\'-azobis(2-methylpropionamidine) dihydrochloride(AAPH). Whole blood obtained fromhealthy volunteers was analysed in thisexperiment. Erythrocytes were separatedfrom plasma by centrifugation, washedand suspended in PBS solution. The ad-dition of AAPH (a peroxyl radical initia-

tor) to the RBC suspension causes theoxidation of lipids and proteins in cellmembrane and thereby induces haemoly-sis. It is known that AAPH-inducedhaemolysis in RBC is a function of incu-bation time and is proportional to theconcentration of free radicals. The in-hibitory effect on RBC haemolysis is al-so proportional to the concentration ofantioxidants in the incubation mixture.The incubation mixture was shaken gen-tly in a water bath at 37°C for 2.5 h. Ourstudy demonstrated that the biologicaltest in vitro is an excellent model for theevaluation of antioxidant capacity of pu-tatively biologically active compoundsobtained from different natural sources,without the use of laboratory animals.

Keywords: antioxidants, red blood cells, haemolysis


The REACH initiative of the EuropeanCommunity will increase the efforts fortoxicological testing, hazard analysis andrisk assessment. Since the skin is an im-

portant exposure route for chemicals, re-constructed human epidermis (RHE)models are of increasing interest for reg-ulatory testing. Nine laboratories plus the

ZEBET ran a validation study to qualifyRHE for in vitro percutaneous absorptionstudies.Nine substances of varyingphysicochemical characteristics includ-

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The Embryonic Stem Cell Test (EST)takes advantage of the potential ofmurine embryonic stem (ES) cells to dif-ferentiate in culture to test embryotoxici-ty in vitro. The test is based on the mostimportant mechanisms in embryotoxicity– cytotoxicity and inhibition of differen-tiation - as well as on differences in sen-sitivity between differentiated and em-bryonic tissues. For the classification ofcompounds according to their embry-otoxic potential we analyse the beatingcardiomyocytes in embryoid body (EB)outgrowths and compare the results tocytotoxic effects on undifferentiated ES

cells and differentiated 3T3 fibroblasts.Through a number of prevalidation andvalidation studies the EST was demon-strated to be a reliable alternative methodfor differentiating of test compounds be-tween three classes of embryotoxicity:strongly, weakly and non embryotoxic.Recent improvements of the EST proto-col using flow cytometry analysisshowed that differential expression ofcardiac specific proteins is a usefulmarker for detecting the embryotoxic po-tential of test compounds. The aim of thepresent study is to investigate whetherthe EST is sensitive enough to discrimi-

nate between structurally related com-pounds with gradually different embry-otoxic potential. VPA is a potentantiepileptic drug with known terato-genic impact in humans and rodents.VPA derivatives, which were synthesisedand highly purified at the University ofVeterinary Medicine Hannover, exhibitdifferent embryotoxic potentials, from invivo highly to non teratogenic. Our re-sults show that we are able to reflectstructure-activity relationships of theVPA derivatives in vitro by using the val-idated and the new established molecularendpoints of the EST.

Lecture

Results of the validation study on percutaneousabsorption via reconstructed human epidermisAlexander Vuia1, Udo Bock2, Walter Diembeck3, Hans-Jürgen Düsing3, Armin O. Gamer4,Eleonore Haltner-Ukomadu2, Christine Hoffmann5, Monika Kaca2, Hennicke G. Kamp4, Silke Kersen6, Manfred Kietzmann7, Hans-Christian Korting8, Hans-Udo Krächter 9, Claus-Michael Lehr10, Manfred Liebsch11, Annette Mehling9, Christel Müller-Goymann5, Frank Netzlaff 10, Frank Niedorf 7, Maria Rübbelke8, Ulrich F. Schäfer10, Elisabeth Schmidt11, Horst Spielmann11, Michaela Weimer6 and Monika Schäfer-Korting1

1Freie Universität Berlin, Pharmazie, Berlin; 2Across Barriers GmbH, Saarbrücken; 3Beiersdorf AG, Hamburg; 4BASF AG,Experimental Toxicology and Ecology, Ludwigshafen; 5Technische Universität Carolo-Wilhelmina-Braunschweig, PharmazeutischeTechnologie, Braunschweig; 6Fraunhofer, Institut für Grenzflächen- und Bioverfahrenstechnik, Stuttgart; 7Stiftung TierärztlicheHochschule Hannover, Pharmakologie, Toxikologie und Pharmazie, Hannover; 8Ludwig-Maximilians-Universität München, Klinikund Poliklinik für Dermatologie und Allergologie, München; 9Cognis Deutschland GmbH, Düsseldorf; 10Universität des Saarlandes,Biopharmazie und Pharmazeutische Technologie, Saarbrücken; 11ZEBET, BfR, Berlin, Germany E-mail: [email protected]

Keywords: Embryotoxicity, Embryonic Stem Cell Test, in vitro, Valproic acid

Lecture

Estimation of the embryotoxic potency of valproic acid derivatives in vitro by using the embryonic stem cell testAnke Visan 1, Roland Büsen1, Birgitta Slawik1, Katharina Schlechter 1, Daniel Eikel 2, Heinz Nau 2,Horst Spielmann1 and Andrea Seiler 1

1Federal Institute for Risk Assessment (BfR), National Center for Documentation and Evaluation of Alternative Methods toAnimal Experiments (ZEBET), Berlin, Germany; 2University of Veterinary Medicine Hannover, Centre of Food Science, Sectionfor Food Toxicology, Hannover, GermanyE-mail: [email protected]


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ing the OECD standard substances caf-feine, testosterone and benzoic acid wereapplied to human epidermis sheets(HES), pig skin and RHE (EpiDerm™,EPISKIN™ and SkinEthic®) mountedinto Franz cells. Permeation experimentswere run in parallel in the partner labora-tories based on a standardised protocol.Finite dose experiments were finally con-

ducted. Although some variability wasobserved in the data, the results showedan acceptable inter- and intra-laboratoryreproducibility. The permeation of testsubstances agreed well with the expecta-tion, high molecular weight substancesas well as hydrophilic agents showed lowpenetration. However, RHE was morepermeable than pig skin and HES. Due to

enormous scatter of benzoic acid perme-ation of HES and RHE, which were notbased on analytical or stability problems,benzoic acid was judged to be unsuitableas a standard compound. Based on the re-sults, it appears possible to develop aprediction model for correlating RHE tonative human and pig skin.

The ability to permit cross-talk betweendermal fibroblasts and epidermal ker-atinocytes is a main advantage of three

dimensional full skin models versusmonolayer cultures. Both cell speciescan influence each other, triggered re-

sponses correlate closely to in vivo mon-itored responses. For the evaluation ofthe anti-inflammatory activities of gluco-

Poster

Acute toxicity of selected chemicals in adult zebrafish (Danio rerio) and its early life stages – thecomparative studyDagmar Zeljenková, Jevgenij A. Kovriznych and Elena SzabováSlovak Medical University in Bratislava, Bratislava, SlovakiaE-mail: [email protected]

Poster

Monitoring cell – cell interaction and the anti-inflammatory activity of glucocorticoids with a new full thickness skin modelNadja Zoeller1,2, Karsten Mewes2, Martina Raus2, Kerstin Draeger2, Juergen Bereiter-Hahn3,Roland Kaufmann1 and August Bernd1

1ZDV, University Hospital Frankfurt/Main, Frankfurt/Main, Germany; 2Phenion GmbH & Co. KG, Frankfurt/Main, Germany;3Kinematic Cell Research Group, J. W. Goethe University, Frankfurt/Main, Germany E-mail: [email protected]

Keywords: skin absorption; reconstructed human epidermis; alternative methods; human skin models

One of the methods for determination ofacute toxicity of chemical substances istheir assessment on fish. A possible wayto accelerate the testing of chemicals onfish can be the use its early life stages(embryos in eggs). We compared acutetoxicity data of eight different chemicalsin adult zebrafish Danio rerio to fisheggs. Tests on zebrafish eggs were per-formed according to the OECD Guidelinefor Testing of Chemicals 203 and 210. 96-h value of LC50s of adult fish compareto fish embryos were following: ace-

tochlor 0.37 mg/L and 0.61 mg/L; acry-lamide 59.0 mg/L and 160.0 mg/L; ben-zene 560 mg/L and 320 mg/L; colchicine18.5 mg/L and 39 mg/L; diethylene gly-col 45000 mg/L and 30000 mg/L;diethylnitrosamine 560 mg/L and 1200mg/L; methanol 22300 mg/L and 290mg/L; Triton X-100 13 mg/L and 17 mg/L.Results of tested substances were compa-rable for 7 of 8 chemicals for both lifefish stages (adult organisms and embryosin eggs). Our toxicity data on Danio

rerio eggs showed comparable resultsand confirmed their alternative use inacute toxicity tests.This method showed following advan-tages:- 100 to 250 fold lower quantity of com-pounds can be used - the capacity of the laboratory for thebiological testing can increased cca 2000times- the method enables to use acute toxicityalong with teratogenicity

Keywords: zebrafish, acute toxicity, early life stages, chemicals


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corticoids and direct monitoring of pro-inflammatory cytokines we used a newfull skin model. Primary fibroblasts andkeratinocytes were isolated from fore-skin of boys less than four years of age.They were seeded on a collagen matrix(Phenion, Frankfurt, Germany) and culti-vated according to common protocol. Astratified epidermis containing stratumbasale, -spinosum, -granulosum and -corneum developed on a fibroblast richdermis. Release of pro-inflammatory cy-tokines was induced by irradiating theskin models using 50mJ/cm2 to

300mJ/cm2 UVB. The release of IL-6and IL-8 was measured using commer-cial ELISA (R&D, Wiesbaden, Ger-many). The IL-6 and IL-8 concentrationsin the cell supernatants increased depen-dent on the used UVB intensity. To mon-itor which cells are responsible for therelease of the pro-inflammatory cy-tokines, skin model sections were stainedwith anti-IL-6 and anti-IL-8 (abcam,Cambridge, UK). Both cytokines werepredominantly located in the region ofvital keratinocytes. Furthermore wecould show that Betnesol®, topically ap-

plied, generated comparable results tosystemic treatment with 20 ((mikro))MBetamethason-17-valerate. In both casesthe UVB induced release of IL-6 and IL-8 and their basal levels were suppressed.These results suggest that full skin mod-els offer the opportunity to investigateglucocorticoid mediated anti-inflamma-tory effects in vitro including topical ap-plication. Apart from analysing super-natants, they enable IHC studies ontissue that resembles human skin.

Keywords: full thickness skin model, cytokines, UVB


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GruBadresse zum Welcome address to the 13th MEGAT-Congress ... · Nicht zuletzt nirnrnt die Prasentation und Diskussion neuer Entwicklungen zur Irnplementierung der 3R in der biornedizi