Slide_template_IHCP.ppt - Hacettepe

Slide_template_IHCP.ppt - Hacettepe

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Key area Systemic toxicity Agnieszka Kinsner and Pilar Prieto 1 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 2 Key area - Systemic toxicity Rationale: Knowledge of the effects of acute and chronic exposure to unknown compounds is necessary for hazard and risk assessment The aim of this key area: to identify and validate in vitro tests relevant for target organ (e.g. lung, liver, kidney) and target systemspecific toxicities (e.g. the haemopoietic system and the immune system) to be incorporated into testing strategies for the estimation of human systemic toxicity The final goal: to provide cheaper, more ethical and more scientifically-based testing strategies. Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Key area - Systemic toxicity ECVAMs activities in the area of systemic toxicity cover: Acute systemic toxicity Haematotoxicology Immunotoxicology Chronic systemic toxicity Pulmonary toxicity Biological barriers (intestinal and blood-brain barrier) 4 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 5 Acute oral toxicity - OECD test guidelines Deletion of TG 401 in 2000

Adoption of TG420, TG423 and TG425 by OECD represents a significant reduction in the number of animals used per test: from ~45 to ~8 animals per substance Recommendations: to take into account the in vitro methods for the determination of the starting dose OECD TG420: Acute Oral Toxicity - Fixed Dose Procedure OECD TG423: Acute Oral toxicity - Acute Toxic Class Method OECD TG425: Acute Oral Toxicity - Up-and-Down Procedure 6 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Strategy to Replace Acute Toxicity Testing Registry of Cytotoxicity MEIC ICCVAM/ECVAM validation study In vitro cytotoxicity test: Relatively good correlation (~70%) Certain number of misclassifications 3 log LD50 [mmol/kg] 2 1 Further needs: Improve the in vitro - in vivo correlation by evaluating existing outliers in order to introduce further parameters (ADE, metabolism, organ specificity). 0 -1 -2

-3 -4 -6 -4 -2 0 log IC 50 [mmol/l] 2 4 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Title: Optimisation and prevalidation of an in vitro test strategy for predicting human acute toxicity Integrated Project of the Sixth Framework Programme of the European Commission 35 Partners from 13 European states: Universities, SME, Research Institutes, Industries, Foundations, JRC Start: January 2005; End: December 2009 7 8 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP 1 Generation of an in vivo database and establishment of a depository of reference compounds WP 2 Generation of an in vitro database

Analysis and integration of in vitro/in vivo data WP 4 New cell systems, new endpoints WP 3 Alerts and correctors in toxicity screening WP 5 WP 5 Role of ADE WP 6 WP 6 Role of metabolism Iterative amendment of the testing strategy Role of target organ toxicity WP 7 WP 7.1 neurotoxicity WP 7.2 nephrotoxicity WP 8 Technical optimisation of the amended test strategy WP 9 Prevalidation of the testing strategy

WP 7.3 hepatotoxicity 9 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 97 reference chemicals kinetics (in vitro, in silico) animal in vivo data human data in vitro data Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 10 WP1: Selection of reference chemicals and collection of in vivo data 97 reference chemicals were selected within a wide range of acute toxicity and generic uses Generation of the in vivo database (animal and human) 12 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP1: Evaluation of in vivo animal data variability Distribution of SD for log-transformed LD50 (rat oral studies: 62 chemicals) 20 log-transformed SD <0.5 for majority of chemicals, i.e., excluding 5 chemicals exhibiting extreme variability Interpolation: overall median log-transformed SD ~0.2

(0.17 for rat, 0.21 for mouse) 15 number of chemicals 10 Range comparable to intervals (log-scale) defining EU/GHS toxicity classifications/categories Inter-species comparison: rat vs mouse mean LD50 (n=40): highly correlated (near overlap with line of identity) 5 0 0.0 0.2 0.4 0.6 0.8 1.0 SD of log-transformedLD50 (rat, oral) 1.2 1.4 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP1: Evaluation of in vivo human data calc. of LC50 values The database contains human acute toxicity data from a single poisoning, consisting of: sub-lethal blood concentrations

lethal blood concentrations post-mortem blood concentrations 13 14 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP1: Estimation of LC50 human Example: Acetaminophen approximate LC0 and LC100 and LC50 Investigation: 01-acetaminophen-lethal Lethal blood concentratio~ RawData Plot 3,80 3,60 3,40 . 65 66 8 39 15 55 4262 53 3,20 log (Lethal blood concentration mikro M) 9

33 51 2763 47 26 19 49 7 23 18 21 121 48 68 52 74 50 10 28 11 69 73 46 72 3,00 17 2,80 31 59 46 75 58 60 38

56 54 2,60 25 57 14 2 2,40 29 40 71 64 61 67 34 3 24 32 45 36 22 70 20 44 16 2,20 30

5 35 37 2,00 13 1,80 41 0 10 20 30 40 43 50 60 70 80 time (h) LC100 = 3.40 LC0 = 3.35 LC50 = (3.35+ 3.40)/2= 3.37 in microM Converted to M LC50=-2.63 90

100 15 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP 1 Generation of an in vivo database and establishment of a depository of reference compounds WP 2 Generation of an in vitro database Analysis and integration of in vitro/in vivo data WP 4 New cell systems, new endpoints WP 3 Alerts and correctors in toxicity screening WP 5 WP 5 Role of ADE WP 6 WP 6 Role of metabolism Iterative amendment of the

testing strategy Role of target organ toxicity WP 7 WP 7.1 neurotoxicity WP 7.2 nephrotoxicity WP 8 Technical optimisation of the amended test strategy WP 9 Prevalidation of the testing strategy WP 7.3 hepatotoxicity Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP2: Generation of in vitro basal cytotoxicity data Assessment of basal cytotoxicity in: BALB/3T3 (NRU) NHK (NRU) HL-60 (ATP) HepG2 (NRU, total protein) Fa32 (NRU, total protein) Generation of an in vitro database for 97 selected reference chemicals 16 17 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP 1

Generation of an in vivo database and establishment of a depository of reference compounds WP 2 Generation of an in vitro database Analysis and integration of in vitro/in vivo data WP 4 New cell systems, new endpoints WP 3 Alerts and correctors in toxicity screening WP 5 WP 5 Role of ADE WP 6 WP 6 Role of metabolism Iterative amendment of the testing strategy Role of target organ toxicity WP 7 WP 7.1 neurotoxicity WP 7.2 nephrotoxicity

WP 8 Technical optimisation of the amended test strategy WP 9 Prevalidation of the testing strategy WP 7.3 hepatotoxicity Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP3: Evaluation of in vitro cytotoxicity data 6 basal cytotoxicity tests: similar information i.e. similar ranking The validated 3T3/NRU seems to be the best candidate 18 19 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Plot observed rat vs predicted LD50 From in vitro 3T3/NRU, PLS regression analysis Richards12var28june07clean-MSj+phys+in vivo-del.M6 (PLS) YPred[Last comp.](log(mean LD50mol/kg)rat)/YVar(log(mean LD50mol/kg)rat) y=1*x-0,01449 R2=0,457 -1 Log LD50 (mol/kg rat, exp. YVar(log(meanb.w.) LD50mol/kg)rat)

formaldehy hexachloro xylene acetaminop carbamazep chlormethi pyrene chloramphe methanol ethanol glycerol diethylene ethylene g acetonitri isopropyl sodium sodiumchl bicbenzene dimethylfo potassium dichlorome chloroform lithium su isoniazid acetylsali glufosinat tetracycli procainami tert-butyl phenol 1,2,3,4-te sodium lau meprobamat5,5-diphen sodium val malathion amiodarone pentachlor diazepam 17a-ethyny

phenanthre chloralsodium hy flu maprotilin glutethimi thioridazi propranolo rifampicin codeine 2,4-dichlo 5-fluorour haloperido caffeine theophylli amitriptyl cyclospori orphenadri atropine s chloroquin disopyrami phenobarbi chlorproma cadmium (I acrylaldeh diquat dib quinidine lindane paraquatpentachlor d arsenic tr nicotine dichlorvos hexachloro ()-verapa methadone mercury (I D-amphetam potassium (-)-epinep cis-diammi ochratoxin w arfarin thallium s

strychnine digoxin parathion sodium sel physostigm -2 -3 -4 -5 cyclohexim -4 -3 -2 -1 YPred[1](log(mean LD50mol/kg)rat) Log LD50 (mol/kg b.w.), predicted with 3T3/NRU RMSEE = 0,784482 SIMCA-P+ 11 - 2007-06-29 08:53:49 20 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Plot observed LC50 humans vs predicted in vitro variables Djurnsdata.M2 (PLS) YPred[Last comp.](human logLC50corr+-,2 (log M))/YVar(human logLC50corr+-,2 (log M)) -1 -2

YVar(human logLC50corr+-,2 (log M)) Log human LC50 -3 -4 -5 -6 -7 y=1*x-0,02498 R2=0,7104 sodium chloride ethanol sodium bicarbon isopropyl alcoh methanol ethylene glycol paraldehyde acetonitrile chlor glufosinate-amm sodium valproatpotassiumdimethylformami acetylsalicylic lithium sulfate 2,4-dichlorophe acetaminophen pentachlorophen phenol chloral hydrate hy procainamide sodium fluoride caffeine isoniazid theophylline chloramphenicol

meprobamate chlormethiazole 5,5-diphenylhyd diquat dibromid iron II sulfate 5-fluorouracil carbamazepine rifampicine warfarin glutethimide sodium pentobar potassium cyani phenobarbital disopyramide orphenadrine hy diazepam sodium selenate quinidine sulfa cis-diamminepla mercury (II) ch amiodarone hydr paraquat dichlo diphenhydramine thioridazine hydip propranolol hyd nicotine chloroquine thallium sulpha strychnine codeine ()-verapamil h arsenic trioxid amitriptyline h

maprotiline atropine sulfat malathion methadone hydro lindane chlorpromazine cyclosporine A cadmium (II) ch colchicine digoxin -8 17a-ethynylestr -6 -5 -4 -3 -2 -1 Log predicted YPred[1](human logLC50corr+-,2LC50 (log M)) RMSEE = 0,900934 SIMCA-P+ 11 - 2007-06-30 10:49:21 Chemicals with poor human data 21 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

WP 1 Generation of an in vivo database and establishment of a depository of reference compounds WP 2 Generation of an in vitro database Analysis and integration of in vitro/in vivo data WP 4 New cell systems, new endpoints WP 3 Alerts and correctors in toxicity screening WP 5 WP 5 Role of ADE WP 6 WP 6 Role of metabolism Iterative amendment of the testing strategy Role of target organ toxicity WP 7

WP 7.1 neurotoxicity WP 7.2 nephrotoxicity WP 8 Technical optimisation of the amended test strategy WP 9 Prevalidation of the testing strategy WP 7.3 hepatotoxicity 22 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP4: Cytokine secretion plasma PBMC Ficoll Granulocytes & macrophages PBMC Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP4: Haematopoiesis 23 24 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP 1

Generation of an in vivo database and establishment of a depository of reference compounds WP 2 Generation of an in vitro database Analysis and integration of in vitro/in vivo data WP 4 New cell systems, new endpoints WP 3 Alerts and correctors in toxicity screening WP 5 WP 5 Role of ADE WP 6 WP 6 Role of metabolism Iterative amendment of the testing strategy Role of target organ toxicity WP 7 WP 7.1 neurotoxicity WP 7.2 nephrotoxicity

WP 8 Technical optimisation of the amended test strategy WP 9 Prevalidation of the testing strategy WP 7.3 hepatotoxicity 25 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP5: Role of ADE (in vitro/in silico) Measurement of the transport across the intestinal barrier and the blood-brain barrier using in vitro concentration of compounds in the in vitro systems. Generic biokinetic model for the interpretation of in vitro toxic concentrations in relation to the in vivo acute toxic dose under development special barriers Measurement (n=3) and modelling of free absorption stability, lipophilicity (n=42) Concentration at target Free concentration and metabolism protein binding

Measurement of protein binding, microsomal elimination: excretion models and neuronal networks (n=21) 26 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP5: Oral absorption H = High ; HIA > 80 % computerCaco-2 Caco-2 M = Moderate ; HIA < 20-70 % P = Poor ; HIA < 20 % Papp 10-6cm/s < 1= Poor (P) Papp 10-6cm/s< 1 - 10 = Moderate (M) Papp 10-6cm/s > 10 = High (H) 72% overall accuracy 27 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP5: Blood-brain barrier Log BB > -0.7 Poor (P) -0.7 < Log BB < -0.3 Moderate (M) Log BB > -0.3 High (H) Luminal compartment (Blood) Coated microporous membrane Abluminal compartment (Brain)

73% overall accuracy 28 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP6: Role of metabolism 100 Hepatocyte Hepatoma 0 Cytotoxicity (%) A B Bioactivated IC50A < IC50A < IC50B Concentration 100 A B IC50B 0 IC50hepat/IC50 HepG2 Compound Cas no Atropine sulfate 5908-99-6 Mercury II

7487-94-7 Pentachlorophen 87-86-5 Rifampicine 13292-46-1 Tetracycline HCl 64-75-5 Orphenadrine HCl 341-95-5 Diazepam 439-14-5 Malathion 121-75-5 Amiodarone HCl 1951-25-3 SLS 151-2-3 Digoxin 20830-75-5 ()-Verapamil HCl 152-11-4 P15 >1E-03 0,04 0,97 0,85 >1E-03 1,34 1,25 1,46 1,35 1,63 908,72 8,85 P23 0,06 0,18 0,04 0,56

0,31 1,55 1,50 1,46 1,02 0,42 ?? 2,75 P31 0,53 0,75 0,84 1,18 0,06 0,25 1,24 >=1E-03 1,10 1,69 >=1000 0,31 Cytotoxicity (%) IC50A Bayer 0,01 0,18 1,28 0,67 1,13 0,56 0,85 >1E-03 1,54 1,45 1,70 NotIC50

Bioactivated A IC50A = IC50B Concentration Mean 0,20 0,29 0,78 0,82 0,50 0,93 1,21 1,46 1,25 1,30 >=1000 3,40 Comparison hepatocyes vs HepG2 More toxic to hepatocytesthan to HepG2 More toxic to hepatocytesthan to HepG2 Slightly more toxic to hepatocytes than to HepG2 Slightly more toxic to hepatocytes than to HepG2 Slightly more toxic to hepatocytes than to HepG2 Similar toxicity to hepatocytes than to HepG2 Similar toxicity to hepatocytes than to HepG2 Similar toxicity to hepatocytes than to HepG2 Similar toxicity to hepatocytes than to HepG2 Similar toxicity to hepatocytes than to HepG2 Less toxic to hepatocytes than to HepG2 Less toxic to hepatocytes than to HepG2 Reported bioactivation YES NO YES NO NO

NO NO YES NO NO NO NO 29 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP7.1: Neurotoxicity Basal cytotoxicity General cell physiology (energy status, glycolytic activity, Ca2+ homeostasis, cell and mitochondrial membrane potential, oxidative stress (ROS) Neurochemistry Voltage operated ion channels Receptor function Neurotransmitter synthesis/degradation Neurotransmitter uptake Neurotransmitter release Global electrical activity Human neuroblastoma SH-SY5Y cell line Serum-free aggregating rat brain cell cultures Primary cultures of rat cerebellar granule cells 30 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP7.2: Nephrotoxicity

TER: sensitive indicator of nephrotoxicity TER: greater sensitivity for nephrotoxic chemicals Compounds requiring metabolism (diethylene glycol) did not show toxicity at concentrations used ions Cells: LLC-PK1 BARRIER FUNCTION Grown on permeable supports Current across epithelium Rate of flux of ions Electrical resistance REMS MACHINE TER ----------- Permeability Loss of barrier function 31 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP7.3 Hepatotoxicity Hepatoma Non-hepatic cell

IC50A IC50B IC50C 10 0 A B C Effect (%) Hepatocyte 0 Concentration IC50(A) < IC50 (B) IC50(C): hepatotoxic (bioactivable) alert IC50(A) IC50 (B) < IC50(C): hepatotoxic alert IC50(A) IC50 (B) IC50(C): no hepatotoxic no alert 32 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 WP 1 Generation of an in vivo database and establishment of a depository of reference compounds WP 2 Generation of an in vitro

database Analysis and integration of in vitro/in vivo data WP 4 New cell systems, new endpoints WP 3 Alerts and correctors in toxicity screening WP 5 WP 5 Role of ADE WP 6 WP 6 Role of metabolism Iterative amendment of the testing strategy Role of target organ toxicity WP 7 WP 7.1 neurotoxicity WP 7.2 nephrotoxicity Last year of the project WP 8 Technical optimisation of the amended test strategy WP 9

Prevalidation of the testing strategy WP 7.3 hepatotoxicity Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Haematotoxicology The Colony Forming Unit-Granulocyte/Macrophage (CFUGM) Assay for predicting acute neutropenia in humans has been scientifically validated A validation study on CFU-GM assay with rat progenitor cells is ongoing, aiming to increase the accuracy of determining the maximum permissible exposure limit for xenobiotics. Pessina A. et al. Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics. Toxicol Sci. 2003 75(2):355-67. 33 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 34 Immunotoxicology A multi-laboratory study has been carried out to evaluate the most promising endpoints for immune-suppression and immunotoxicity As a follow-up, a validation study is currently ongoing, with the aim of evaluating reproducibility and predictivity of a set of in vitro assays to detect immunotoxicity, to be used as animals replacement Carfi' M et al. In vitro tests to evaluate immunotoxicity: a preliminary study.Toxicology. 2007 229(1-2):11-22. 35 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Barrier models: blood-brain barrier Study on evaluation of the performance of five selected in vitro blood-brain barrier models for predicting absorption/uptake into the brain

In vitro models Model 1: Primary culture bovine brain capillary endothelial cells co-cultured with rat astrocytes Model 2: Porcine primary endothelial cells cultured in hydrocortisone-conditioned medium Model 3: 4-days BBB culture of bovine brain capillary endothelial cells in conditioned medium Model 4: Cell line HCMEC/D3 Model 5: MDCKmdr-1 cells Transport experiments (Papp values) in 5 labs using 16 coded reference chemicals; 3 independent experiments 36 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Barrier models intestinal barrier Prevalidation of in vitro models for the prediction of gastrointestinal absorption 10 Reference Chemicals High Absorption Intermediate absorption Low absorption Isopropanol (reference) Cimetidine (reference) Atenolol (reference)

Ethylen glycol (chemical) Paraquat (pesticide) Cupric sulphate (chemical) Sodium Valproate (drug) Paracetamol (drug) Colchicine (drug) Acetylsalicylic acid (drug) - Two testing laboratories - Two in vitro models: Caco-2, TC7 clone - Permeability assays (Paap): at non toxic concentration during 60 min, samples taken at 15, 30, 45 and 60 min Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 37 Pulmonary toxicity Marie Curie Research Training Network that aims to elucidate basic molecular, cellular and tissuerelated mechanisms involved in the generation of various acute and chronic pulmonary diseases. diseases Two in vitro models are under investigation at ECVAM: human airway epithelial cell line Calu3 and the human alveolar type cell line NCI H441

38 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Key area - Systemic toxicity Haematotoxicology [email protected] Immunotoxicology Chronic systemic toxicity [email protected] Pulmonary toxicity Biological barriers Acute systemic toxicity [email protected] [email protected] Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 39 46 Woirkshop on Alternative Methods - Ankara, 12-13 November 2007 Toxicity prevalence of new industrial Chemicals in the EU Chemicals (%) LD50 (mg/Kg) 0 < 25 3

> 25 200 21 > 200 2000 76 > 2000 In these 2 classes 97% of all new industrial chemicals Evaluation of the 3T3 NRU assay for the prediction of nontoxic compounds

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