Post on 11-Jun-2015
TOXICIDAD IN VITRO
Joaquín de Lapuente
Coordinador CERETOX
jlapuente@pcb.ub.cat
JORNADA TOX®
1 de Febrero de 2013
Parc Científic de Barcelona
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
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)
Necesidad racional
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 cría de animales o de los
procedimientos para minimizar el dolor y la angustia, así como para
mejorar el bienestar de los animales.
GreenFacts based on the Canadian Council on Animal Care, Three Rs introduction
Ni contigo, ni sin tí
REALIDAD
A
C
E
R
C
A
M
I
E
N
T
O
IN SILICO
IN VITRO
IN VIVO
Perdida de
información
Tox in vitro
Toxicología in vitro
Investigación (R+D)
Screening
Regulatorio
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
REACH (others) APPROVAL METHODS
Acronym Method 3 Rs Method EU Regulation
EU
method OECD
SKIN
CORROSION
TER Transcutaneous Electrical Resistance
Test Replacement In vitro Directive 2000/33/EC B 40 TG 430
EpiSkin™ In Vitro Skin Corrosion: Human Skin
Model Test Replacement In vitro Directive 2000/33/EC B 40 bis TG 431
EpiDerm™ In Vitro Skin Corrosion: Human Skin
Model Test Replacement In vitro Directive 2000/33/EC B 40 bis TG 431
SkinEthic™ RHE In Vitro Skin Corrosion: Human Skin
Model Test Replacement In vitro Directive 2000/33/EC B 40 bis TG 431
EST-1000™ In Vitro Skin Corrosion: Human Skin
Model Test Replacement In vitro Directive 2000/33/EC B 40 bis TG 431
CORROSITEX In Vitro Membrane Barrier Test
Method for Skin Corrosion
Partial
Replacement In vitro Stage 3 TG 435
SKIN IRRITATION
EpiSkin™ Reconstructed Human Epidermis
Model Test Replacement In vitro Directive 761/2009/EC B 46 TG 439
Original
EpiDerm™ Reconstructed Human Epidermis
Model Test
Partial
Replacement In vitro
Not included and not
foreseen B 46 Not included and not
foreseen
Modified
EpiDerm™ SIT Reconstructed Human Epidermis
Model Test Replacement In vitro Directive 761/2009/EC B 46 TG 439
SkinEthic™ RHE Reconstructed Human Epidermis
Model Test Replacement In vitro Directive 761/2009/EC B 46 TG 439
REACH (others) APPROVAL METHODS
Acronym Method 3 Rs Method EU Regulation
EU
method OECD
EYE
IRRITATION
BCOP Bovine Cornea Opacity Test
Partial
Replacement In vitro L 324 2010 B 47 TG 437
ICE Isolated chicken eye test
Partial
Replacement In vitro L 324 2010 B 48 TG 438
IRE Isolated rabbit eye tes
Partial
Replacement In vitro Positive results None
Not included and
not foreseen yet
HET-CAM Hen's egg test - chorio-allantoic
membrane
Partial
Replacement In vitro Positive results None
Not included and
not foreseen yet
CM Cytosensor Microphysiometer
Test
Partial
Replacement In vitro Not included
None
yet Draft
FL Fluorescein Leakage Test
Partial
Replacement In vitro Not included
None
yet Draft
LVET Low Volume Eye Test
Refinement/Re
duction In vitro Not included None None
SKIN
SENSITISATIO
N
LLNA rLLNA Local Lymph Node Assay
Refinement/Re
duction in vivo
Directive
2004/73/EC B 42 TG 429
LLNA DA Local Lymph Node Assay: DA
Refinement/Re
duction in vivo In progress B 50 TG 422a
LLNA BrdU-
Elisa Local Lymph Node Assay: BrdU-
Elisa
Refinement/Re
duction in vivo In progress B 51 TG 422a
REACH APPROVAL METHODS
Acronym Method 3 Rs Method EU Regulation
EU
method OECD
MUTAGENICITY Micronucleus In vitro Micronucleus Test Reduction In vitro
Regulation
1907/2006/EC TG 487
ACUTE SYSTEM
TOXICITY
FDP Acute oral Toxicity - Fixed Dose
Procedure
Reduction and
refinement in vivo Directive 2004/73/EC B1 bis TG 420
ATC Acute oral toxicity - Acute Toxic
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 Inhalation
Toxicity Acute Inhalation Toxicity Method
Reduction and
refinement in vivo Stage 3 TG 436
ATC Inhalation Acute Toxic Class Method
Reduction and
refinement in vivo Stage 3 TG 404
Starting doses for
acute oral toxicity using in vitro test
Reduction and
refinement Not appropriate
Guidance
Document 129
REPEAT DOSE
TOXICITY Not available
REPRODUCTIV
E TOXICITY
EST Embryonic Stem Cell Test for
Embryotoxicity
Partial
Replacement In vitro
until comprehensive
valid testing strategy
Not included and
not foreseen yet
MM Micromass Embryotoxicity Assay Refinement In vitro
until comprehensive
valid testing strategy
Not included and
not foreseen yet
WET Whole rat Embryo Embryotoxicity
Test Refinement In vitro
until comprehensive
valid testing strategy
Not included and
not foreseen yet
OTHER
3T3NRU
Phototoxicity In vitro 3T3 NRU Phototoxicity
Test Replacement In vitro Directive 2000/33/EC B 41 TG 432
Skin absorption In vitro Skin Absorption Replacement In vitro
Regulation
440/2008/EC B 45 TG 428
STTA for EDs
Stably Transfected Human
Estrogen Receptor-alpha
Transcriptional Activation Assay for
Detection of Estrogenic Agonist-
Activity of Chemicals
Reduction and
refinement Stage 3 TG 436
ACUTE
TOXICITY TO
FISH FTA Fish Threshold Approach Reduction Strategy REACH Guidance
Guidance
Document 126
Status Validation & Regulatory Acceptance
Status of Alternative Test Methods &
Testing Strategies
http://alttox.org/ttrc/validation
-ra/validated-ra-
methods.html
Endpoint Method Name
Test
Type1 Endorsement of Scientific Validity Regulatory Acceptance
Lead Authority
Subsequent
Endorsement(s
)
International(click h
ere for approved
OECD TGs, here for
Draft TGs,
and here for GDs)
National/ Regional (for
methods not yet accepted
internationally)
Biologics &
Vaccines
ELISA for erysipelas vaccines batch
potency testing In vitro ESAC 14(2002) European Pharmacopeia
ELISA for human tetanus vaccines batch
potency testing In vitro ESAC 9(2000) European Pharmacopeia
Carcinogenicity Cell transformation assays In vitro3 EURL ECVAM(2012)
Draft OECD TG
(2012)
Chronic toxicity Ending 1-year dog studies of pesticides In vivo ESAC 26(2006)
Revised US EPA Pesticide Data
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)
In vitromammalian cell micronucleus test In vitro ESAC 24(2006)
ICCVAM
comments to
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
neutropenia
Colony forming unit granulocyte
macrophage (CFU-GM) assay In vitro ESAC 18(2006) Submitted to EMEA
Pyrogenicity Human whole blood IL-1 In vitro ESAC 19(2006)
ICCVAM(2008)1
3
EMEA; European
Pharmacopeia;US agencies
Human whole blood IL-6 In vitro ESAC 20(2006)
ICCVAM(2008)1
3
EMEA; European
Pharmacopeia;US agencies
Limulus amebocyte lysate (LAL) test In vitro
European Pharmacopeia
(5.0); US Pharmacopeia (85)
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
CERETOX Parc Científic de Barcelona - Edifici Cluster
c/Baldiri Reixac, 10-12
08028 Barcelona T. 93 403 71 83
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