1. TOXICIDAD IN VITRO
Joaquín de Lapuente
Coordinador CERETOX
jlapuente@pcb.ub.cat
JORNADA TOX®
1 de Febrero de 2013
Parc Científic de Barcelona

2. Uso animales EU
•Sexto Informe sobre las estadísticas relativas al número de animales
utilizados para experimentación y otros fines científicos en los Estados
miembros de la Unión Europea
120 millones de animales

3. Uso de animales en toxicología
DECISIÓ N DE EJECUCIÓ N DE LA COMISIÓ N por la que
se establece un formato común para la presentación de la información
prevista en la Directiva 2010/63/UE del Parlamento Europeo y del
Consejo, relativa a la protección de los animales utilizados para fines
científicos [C(2012) 8064] (EEE) (2012/707/UE)

4. Necesidad racional

5. 3 Rs
1960 Russel y Burch
“The Principle of Humane Experimental Technique
•Alternativas de Reemplazo
aluden a métodos que eviten o sustituyan el uso
de animales (reemplazos absolutos, reemplazos
relativos).
•Alternativas de Reducción
aluden a cualquier estrategia que tenga como resultado el uso de un
menor número de animales.
•Alternativas de Refinamiento
aluden a la modificación de la crí de animales o de los
a
procedimientos para minimizar el dolor y la angustia, asícomo para
mejorar el bienestar de los animales.
GreenFacts based on the Canadian Council on Animal Care, Three Rs introduction

6. Ni contigo, ni sin tí
REALIDAD
IN VIVO A
C
E
R
C
IN VITRO A
M
I
E
IN SILICO
N Perdida de
información
T
O

7. Tox in vitro
Investigación (R+D)
Toxicologí in vitro
a Screening
Regulatorio

8. Información
Fuente Web
NICEATM and ICCVAM http://iccvam.niehs.nih.gov/methods/methods.htm
EURL ECVAM http://ecvam.jrc.it/
Altweb 2 http://altweb.jhsph.edu/
AnimAlt-ZEBET http://www.dimdi.de/static/en/db/dbinfo/zt00.htm
OECD http://www.oecd.org/chemicalsafety/testing/animalwelfare.htm
NIH http://nccam.nih.gov/
AWI http://labanimals.awionline.org/Default.htm
PrimaleLit http://primatelit.library.wisc.edu/
Norina http://oslovet.norecopa.no/fag.aspx?fag=57&mnu=databases_1
EURCA http://www.eurca.org/
REMA http://www.remanet.net/
Altbib http://toxnet.nlm.nih.gov/altbib.html
INVITTOX http://www.invittox.com/
ILAR http://dels.nas.edu/ilar/
AWIC http://awic.nal.usda.gov/
FishBase http://www.fishbase.org/
InterNICHE http://www.interniche.org/
GTEMA http://aetox.es/secciones/gtema/
AltTOX http://alttox.org/ttrc/validation-ra/validated-ra-methods.html
ECOPA http://www.ecopa.eu/
SOT http://www.toxicology.org/isot/ss/ivss/index.htm

9. REACH (others) APPROVAL METHODS
EU
Acronym Method 3 Rs Method EU Regulation method OECD
Transcutaneous Electrical Resistance
TER Test Replacement In vitro Directive 2000/33/EC B 40 TG 430
In Vitro Skin Corrosion: Human Skin
EpiSkin™ Model Test Replacement In vitro Directive 2000/33/EC B 40 bis TG 431
In Vitro Skin Corrosion: Human Skin
SKIN EpiDerm™ Model Test Replacement In vitro Directive 2000/33/EC B 40 bis TG 431
CORROSION
In Vitro Skin Corrosion: Human Skin
SkinEthic™ RHE Model Test Replacement In vitro Directive 2000/33/EC B 40 bis TG 431
In Vitro Skin Corrosion: Human Skin
EST-1000™ Model Test Replacement In vitro Directive 2000/33/EC B 40 bis TG 431
In Vitro Membrane Barrier Test Partial
CORROSITEX Method for Skin Corrosion Replacement In vitro Stage 3 TG 435
Reconstructed Human Epidermis
EpiSkin™ Model Test Replacement In vitro Directive 761/2009/EC B 46 TG 439
Original Reconstructed Human Epidermis Partial Not included and not Not included and not
SKIN IRRITATION
EpiDerm™ Model Test Replacement In vitro foreseen B 46 foreseen
Modified Reconstructed Human Epidermis
EpiDerm™ SIT Model Test Replacement In vitro Directive 761/2009/EC B 46 TG 439
Reconstructed Human Epidermis
SkinEthic™ RHE Model Test Replacement In vitro Directive 761/2009/EC B 46 TG 439

10. REACH (others) APPROVAL METHODS
EU
Acronym Method 3 Rs Method EU Regulation method OECD
Partial
BCOP Bovine Cornea Opacity Test Replacement In vitro L 324 2010 B 47 TG 437
Partial
ICE Isolated chicken eye test Replacement In vitro L 324 2010 B 48 TG 438
Partial Not included and
IRE Isolated rabbit eye tes Replacement In vitro Positive results None not foreseen yet
EYE Hen's egg test - chorio-allantoic Partial Not included and
IRRITATION HET-CAM membrane Replacement In vitro Positive results None not foreseen yet
Cytosensor Microphysiometer Partial None
CM Test Replacement In vitro Not included yet Draft
Partial None
FL Fluorescein Leakage Test Replacement In vitro Not included yet Draft
Refinement/Re
LVET Low Volume Eye Test duction In vitro Not included None None
Refinement/Re Directive
LLNA rLLNA Local Lymph Node Assay duction in vivo 2004/73/EC B 42 TG 429
SKIN Refinement/Re
SENSITISATIO LLNA DA Local Lymph Node Assay: DA duction in vivo In progress B 50 TG 422a
N
LLNA BrdU- Local Lymph Node Assay: BrdU- Refinement/Re
Elisa Elisa duction in vivo In progress B 51 TG 422a

11. REACH APPROVAL METHODS
EU
Acronym Method 3 Rs Method EU Regulation method OECD
Regulation
MUTAGENICITY
Micronucleus In vitro Micronucleus Test Reduction In vitro 1907/2006/EC TG 487
Acute oral Toxicity - Fixed Dose Reduction and
FDP Procedure refinement in vivo Directive 2004/73/EC B1 bis TG 420
Acute oral toxicity - Acute Toxic
ATC Class Method Reduction in vivo Directive 2004/73/EC B1 tris TG 423
Up and Down Up and Down Method Reduction in vivo Stage 3 TG 425
ACUTE SYSTEM
Acute Inhalation Reduction and
TOXICITY
Toxicity Acute Inhalation Toxicity Method refinement in vivo Stage 3 TG 436
Reduction and
ATC Inhalation Acute Toxic Class Method refinement in vivo Stage 3 TG 404
Starting doses for Reduction and Guidance
acute oral toxicity using in vitro test refinement Not appropriate Document 129
REPEAT DOSE
TOXICITY Not available
Embryonic Stem Cell Test for Partial until comprehensive Not included and
EST Embryotoxicity Replacement In vitro valid testing strategy not foreseen yet
REPRODUCTIV until comprehensive Not included and
E TOXICITY MM Micromass Embryotoxicity Assay Refinement In vitro valid testing strategy not foreseen yet
Whole rat Embryo Embryotoxicity until comprehensive Not included and
WET Test Refinement In vitro valid testing strategy not foreseen yet
3T3NRU In vitro 3T3 NRU Phototoxicity
Phototoxicity Test Replacement In vitro Directive 2000/33/EC B 41 TG 432
Regulation
Skin absorption In vitro Skin Absorption Replacement In vitro 440/2008/EC B 45 TG 428
OTHER Stably Transfected Human
Estrogen Receptor-alpha
Transcriptional Activation Assay for
Detection of Estrogenic Agonist- Reduction and
STTA for EDs Activity of Chemicals refinement Stage 3 TG 436
ACUTE
TOXICITY TO Guidance
FISH FTA Fish Threshold Approach Reduction Strategy REACH Guidance Document 126

12. Status
Validation & Regulatory Acceptance
Status of Alternative Test Methods & http://alttox.org/ttrc/validation
Testing Strategies -ra/validated-ra-
methods.html
Test
Endpoint Method Name Type1 Endorsement of Scientific Validity Regulatory Acceptance
International(click h
ere for approved
Subsequent OECD TGs, here for National/ Regional (for
Endorsement(s Draft TGs, methods not yet accepted
Lead Authority ) and here for GDs) internationally)
Biologics & ELISA for erysipelas vaccines batch
Vaccines potency testing In vitro ESAC 14(2002) European Pharmacopeia
ELISA for human tetanus vaccines batch
potency testing In vitro ESAC 9(2000) European Pharmacopeia
Draft OECD TG
Carcinogenicity Cell transformation assays In vitro3 EURL ECVAM(2012) (2012)
Revised US EPA Pesticide Data
Chronic toxicity Ending 1-year dog studies of pesticides In vivo ESAC 26(2006) Requirements
Genotoxicity Bacterial reverse mutation (Ames) test In vitro OECD TG 471 (1997)
In vitro cell gene mutation test In vitro OECD TG 476 (1997)
In vitrochromosomal aberration test In vitro OECD TG 473 (1997)
ICCVAM
comments to
In vitromammalian cell micronucleus test In vitro ESAC 24(2006) OECD OECD TG 487 (2010)
In vitro sister chromatid exchange test In vitro OECD TG 479 (1986)
In vitrounscheduled DNA synthesis test In vitro OECD TG 482 (1986)
Saccharomyces cerevisiae gene
mutation assay In vitro OECD TG 480 (1986)
Saacharomyces cerevisiae mitotic
recombination assay In vitro OECD TG 481 (1986)
Hematotoxicity:
acute Colony forming unit granulocyte
neutropenia macrophage (CFU-GM) assay In vitro ESAC 18(2006) Submitted to EMEA
ICCVAM(2008)1 EMEA; European
Pyrogenicity Human whole blood IL-1 In vitro ESAC 19(2006) 3 Pharmacopeia;US agencies
ICCVAM(2008)1 EMEA; European
Human whole blood IL-6 In vitro ESAC 20(2006) 3 Pharmacopeia;US agencies
European Pharmacopeia
Limulus amebocyte lysate (LAL) test In vitro (5.0); US Pharmacopeia (85)

13. OECD
OECD- Last 6 month news Deadline
Draft Test Guidelines Stage Public Comments
Cytosensor Microphysiometer Test Method Early .In process February 2013
Draft revised TG 437 (BCOP) and TG 438 (ICE) In process February 2013
Draft revised TG 431 ( Reconstructed Human Epidermis (RhE) Test Method) In process February 2013
Draft TG 473 on an in vitro Mammalian Chromosome Aberration Test In process January 2013
Draft TG 487 on an in vitro Mammalian Cell Micronucleus Test PDF In process January 2013
Syrian Hamster Embryo (SHE) Cell Transformation Assay In process November 2012
TG 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method In process November 2012
TG 430: In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method
(TER) In process November 2012
TG 437: Bovine Corneal Opacity and Permeability In process October 2012
TG 438: Isolated Chicken Eye Test Method In process October 2013
TG 474 on an Mammalian Erythrocyte Micronucleus Test PDF In process September 2012
TG 475 on an Mammalian Bone Marrow Chromosomal Aberration Test PDF In process September 2012
Source: http://www.oecd.org/env/ehs/testing/section4healtheffects.htm

14. CERETOX
Parc Científic de Barcelona - Edifici Cluster
c/Baldiri Reixac, 10-12
08028 Barcelona
T. 93 403 71 83
www.pcb.ub.cat/ceretox
http://goo.gl/BGLwt
http://goo.gl/VBsu7