Replacement alternatives. The rule of 3R and the alternative methods. Alternative methods and REACH

Size: px

Start display at page:

Download "Replacement alternatives. The rule of 3R and the alternative methods. Alternative methods and REACH"

August Tate
5 years ago
Views:

ALTERNATIVE METHODS FOR ANIMAL EXPERIMENTATION IN TOXICOLOGY Francelyne MARANO Laboratory of Molecular and Cellular Responses to Xenobiotics University Paris Diderot Photo J.

Refine the technics for the improvement of the animal welfare The european legislation on animal welfare EU Directive 86/69/EEC An experiment shall not be performed on an animal, if another

The European Commission and the EU Member States must actively encourage and support the development, validation and acceptance of methods which could reduce, refine or replace the use of laboratory

1 ALTERNATIVE METHODS FOR ANIMAL EXPERIMENTATION IN TOXICOLOGY Francelyne MARANO Laboratory of Molecular and Cellular Responses to Xenobiotics University Paris Diderot Photo J.J. Poirault The rule of 3R and the alternative methods Russel and Burch in 1959 An alternative method permits: to Replace the animal experimentation to Reduce the use of animal in some assays to Refine the technics for the improvement of the animal welfare The european legislation on animal welfare EU Directive 86/69/EEC An experiment shall not be performed on an animal, if another scientifically satisfactory method of obtaining the result sought, is reasonably and practicably available. The European Commission and the EU Member States must actively encourage and support the development, validation and acceptance of methods which could reduce, refine or replace the use of laboratory animals. Alternative methods and REACH 3 From T.Hartung Washington 211 The in vitro methods : -isolated organs - tissue slices - cell cultures - abiotic assays -«OMICS» The in silico methods (computer simulations, mathematical models, QSAR). The development of alternative methods during the last 3 years Replacement alternatives In vitro methods are not primarily replacements of in vivo methods. Typically these methods have different roles in research, and they are complementary for each others. Depending on the objective of the study in vitro methods may be the most appropriate methods for certain areas of interest, because they can more accurately provide the information required (e.g. cellular and molecular events). From M.Viluksela, University of Kuopio 29

European Centre for the Validation of Alternative Methods Current use of in vitro methods Established in 1991 (Directive 86/69/EEC ) Located at the Joint Research Centre, Ispra, Italy Part of the

2 European Centre for the Validation of Alternative Methods Current use of in vitro methods Established in 1991 (Directive 86/69/EEC ) Located at the Joint Research Centre, Ispra, Italy Part of the Institute for Health and Consumer Protection (IHCP) Safety testing of chemicals, generally on cell lines: Local toxicity Genotoxicity Screening tests Mechanistical studies on differenciated cells and tissues, often human tissues 7 Safety testing of chemicals Replacement alternatives in use for testing: Genotoxicity: mutagenicity chromosomal effects Local tolerance: phototoxicity skin corrosivity skin irritation eye irritation ( 6th Amendment Directive 76/768/EEC) Toxicological screening: general cytotoxicity Percutaneous absorption The problem of the validation and the registration Validation of a new test or method: Evaluation: of the fiability, of the reproductibility, of the strenght Importance of the inter-laboratory studies at the european level. From M. Viluksela 29 More complex in vitro models for mechanistical studies: Well differenciated human tissues in primary culture and co-cultures. Cell lines to be differenciated in vitro Specific end-points. Repeated doses. One example: human airway epithelium The human airway epithelium: target of atmospheric pollutants. In vitro models available for studying atmospheric particles (ex: Diesel Particles, PM, nanoparticles).

Diesel particles Fraction Organique Soluble (SOF) spheres of carbone NP (.1-.8 µm) which are agglomerated (.5-1 µm) adsorbed hydrocarbures Condensed hydrocarbures 2 µm Soots ( 9% PM2.

cardiovascular diseases Respiratory symptoms Increased mortality Cardiopulmonary mortality Lung cancer ( Pope 2) Sulfates What biological effects of these particles in the lung?

Particles characteristics involved in their biological effects Size Penetration Deposition Epuration Retention (Churg, 2) surface reactivity Composition Metals Organic compounds Endotoxins IAL

treatments: -PM 2.5 sampled in Paris on back ground and kerbside stations.

3 Diesel particles Fraction Organique Soluble (SOF) spheres of carbone NP (.1-.8 µm) which are agglomerated (.5-1 µm) adsorbed hydrocarbures Condensed hydrocarbures 2 µm Soots ( 9% PM2.5 traffic) Organic compounds Metals Endotoxins Allergenes Epidemiological studies suggest that particulate matter is implicated in: Increased morbidity Hospital admissions for asthma and cardiovascular diseases Respiratory symptoms Increased mortality Cardiopulmonary mortality Lung cancer ( Pope 2) Sulfates What biological effects of these particles in the lung? Controlled human exposure to DEP Increase in number of inflammatory cells in BAL Increase of cytokines in nasal and bronchial lavages (Diaz-Sanchez et al. 1997, Salvi et al. 1999) PM Effects? Particles characteristics involved in their biological effects Size Penetration Deposition Epuration Retention (Churg, 2) surface reactivity Composition Metals Organic compounds Endotoxins IAL primary cultures of human airway epithelial cells Cell cultures: -Human bronchial cell line 16-HBE 14 o- - Human nasal and bronchial cells in primary culture PM, DEP Particules used for in vitro treatments: -PM 2.5 sampled in Paris on back ground and kerbside stations. -Diesel Particles: SMR 165( NIST) -Organic and aqueous extracts of PM and PDi Organic compounds Metals Carboneous core Phagocytosis Organic compounds desorption Transduction pathways Gene Transcription ROS Cytokines Dissociated cells from human nasal or bronchial mucosa Mucociliary differenciation in two weeks IAL confluence + RA Boland et al., AJP 2, Bonvallot et al., AJRCMB 22, Baulig et al. AJP 23

4 DEP PM phagocytosis Cytokine gene Carbon core Organic compounds 16 cellule HBE épithéliale cells respiratoire Boland et al., Am. J. Physiol., pg of GM-CSF/mL Cytokines IL1 IL8 GM-CSF pg of GM-CSF/mL GM-CSF 24h 2, DEP (µg/cm 2 ) Control ndep Ex sdep CB : different from the control The secretions of TNF-alpha and GM-CSF after exposure to DEP and PM are mostly at the apical surface pg/ml sécrétion du GM-CSF Témoin DPL Pdi 1 µg/cm2 apical basolatéral pg/ml sécrétion du TNF-alpha Témoin DPL Pdi 1 µg/cm2 apical basolatéral Amphiregulin, a ligand of EGFR PM 2.5 EGFR Airway epithelium Role of EGFR ligands in airway inflammation induced by PM EGFR PM 2.5 cytokines (GM-CSF) GM-CSF (pg/ml) C PM + AG1478 1µM amphiregulin Airway epithelial cell EGF receptor ligands : Amphiregulin, HB- EGF, TGFα, Betacellulin (Blanchet S, Ramgolam K, AJRCMB, 24) (Auger F, TAP, 26) EGFR ligands (amphiregulin, TGF, HB-EGF) Autocrine effect EGFR ligands: mediation of a PM 2.5 -induced proinflammatory response The " particle stress " ROS Signal Transduction Transcription factors Modulation of gene expression Genes of growth factors, Genes of inflammation Inflammatory response Advantages of in vitro tests Controlled testing conditions Lack of systemic effects Reduction of variability between experiments Testing is fast (and cheap) Small amount of test material is required Human cells and tissues can be used Transgenic cells carrying human genes can be used Reduction of testing in animals

effects cannot be tested THANK YOU FOR YOUR ATTENTION Réponses Moléculaires et Cellulaires aux Xénobiotiques CNRS EAC 759 Armelle BAEZA-SQUIBAN Augustin

5 Limitations of in vitro tests General toxicity profile of a chemical cannot be assessed In vivo dose-responses cannot be obtained (for human risk assessment) Systemic effects cannot be evaluated Interactions between tissues and organs cannot be tested Pharmacokinetics cannot be evaluated Chronic effects cannot be tested THANK YOU FOR YOUR ATTENTION Réponses Moléculaires et Cellulaires aux Xénobiotiques CNRS EAC 759 Armelle BAEZA-SQUIBAN Augustin BAULIG Véronique BONVALLOT Sonja BOLAND Francelyne MARANO INSERM U7 Michel Aubier IUF DÜSSELDORF Seema SINGH Roël SCHINS Financements: Primequal 2, ADEME, AFSSET, ANR

LSHB-CT

LSHB-CT Implementation of the results from the EU-FP6 project Sens-it-iv ---------- Integrated Data Analysis of In Vitro Testing Approaches Advancing BioTech Product Development and Safety ELRo_Sens-it-iv_PATC