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

www.pcb.ub.cat/ceretox

http://goo.gl/VBsu7

http://goo.gl/BGLwt