Sunu 6

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007
Key area Systemic toxicity

Agnieszka Kinsner and Pilar Prieto
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.

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)
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
Strategy to Replace Acute Toxicity Testing

Registry of Cytotoxicity
3 2
MEIC
ICCVAM/ECVAM validation study

In vitro cytotoxicity test: Relatively good correlation (~70%) Certain number of misclassifications Further needs: Improve the in vitro - in vivo correlation by evaluating existing outliers in order to introduce further parameters (ADE, metabolism, organ specificity).
log LD50 [mmol/kg]
1 0 -1 -2 -3 -4 -6 -4 -2 0 log IC 50 [mmol/l] 2 4
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
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
Alerts and correctors in toxicity screening
WP 5 WP 6 WP 3
Iterative amendment of the testing strategy
WP 5 Role of ADE WP 6 Role of metabolism 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 7.3 hepatotoxicity
WP 9
Prevalidation of the testing strategy
97 reference chemicals
kinetics (in vitro, in silico)
animal in vivo data
human data
in vitro data
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
11 chemicals GHS cat.5 2000 < LD50 5000 mg/kg
7 chemicals GHS Not Classified LD50 > 5000 mg/kg
10 chemicals GHS cat.1 LD50 5 mg/kg 11 chemicals GHS cat.2 5 < LD50 50 mg/kg
Pesticides: 12 other: 5
Industrial chemicals: 30
Drugs: 50
Generation of the in vivo database (animal and human)
12
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
15
number of chemicals
Interpolation: overall median log-transformed SD ~0.2 (0.17 for rat, 0.21 for mouse) 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)
10
0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
SD of log-transformedLD50 (rat, oral)
13
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
14
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
.
73 46 72 17 5 0
65 8 33 51 15 42 62 53 27 63 47 26 9 39 55 25 57 58 60 56 54 14 2 16 29 40 71 64 61 67 3 45 24 32 70 30 37 35 38 34 22 44 36 20 59 46 75 66
3,20
) log (Lethal blood concentration mikro M
3,00 2,80
19 49 7 23 18 21 121 48 68 52 74 50 10 28 11 69 31
2,60 2,40 2,20
2,00 1,80 41 10 20 30 40
13
43 50 60 70 80 90 100
time (h)
LC100 = 3.40 LC0 = 3.35
LC50 = (3.35+ 3.40)/2= 3.37 in microM Converted to M LC50=-2.63
15
WP 1
WP 2

WP 5 WP 6 WP 3
WP 7
WP 8
WP 9
16
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
17
WP 1
WP 2

WP 5 WP 6 WP 3
WP 7
WP 8
WP 9
18
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
19
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 methanol ethanol glycerol diethylene ethylene g acetonitri isopropyl sodium sodiumchl bicbenzene dimethylfo potassium dichlorome
(mol/kg b.w.) rat, exp. Log LD50 YVar(log(mean LD50mol/kg)rat)
formaldehy hexachloro acetaminop carbamazep chlormethi pyrene chloramphe
xylene
-2
-3
-4
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 chloral hy flu sodium 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 paraquat d arsenic tr pentachlor nicotine dichlorvos hexachloro ()-verapa methadone mercury (I D-amphetam potassium (-)-epinep cis-diammi ochratoxin w arfarin thallium s strychnine digoxin parathion sodium sel physostigm cyclohexim -4 -3 -2 -1
-5
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
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)) y=1*x-0,02498 R2=0,7104
sodium chloride ethanol sodium bicarbon isopropyl alcoh methanol
Chemicals with poor human data

03-atropine sulfate monohydrate 08-diazepam 13-pentachlorophenol 14-phenobarbital 21-cadmium chloride 28-amiodarone hydrochloride 30-rifampicine 41-glufosinate ammonium 47-17-ethynylestradiol 51-dimethylformamide 56-phenol 67-w arfarin 89-chlorpromazine hydrochloride 90-paraldehyde 33-nicotine 34-lindane 91-sodium selenate 92-acetonitrile 93-sodium bicarbonate 84-diphenhydramine 85-chlormethiazole 87-procainamide hydrochloride 57-sodium chloride
-1
YVar(human logLC50corr+-,2 (log M))
-2 -3 -4 -5 -6 -7 -8
ethylene glycol paraldehyde acetonitrile chlor glufosinate-amm sodium valproatpotassium dimethylformami 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 rifampicine warfarin glutethimide carbamazepine 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 17a-ethynylestr -6 -5 -4 -3 -2 -1
Log human LC50
Log predicted LC50 YPred[1](human logLC50corr+-,2 (log M))

RMSEE = 0,900934
SIMCA-P+ 11 - 2007-06-30 10:49:21
21
WP 1
WP 2

WP 5 WP 6 WP 3
WP 7
WP 8
WP 9
22
WP4: Cytokine secretion
plasma PBMC Ficoll Granulocytes & macrophages
PBMC
23
WP4: Haematopoiesis
24
WP 1
WP 2

WP 5 WP 6 WP 3
WP 7
WP 8
WP 9
25
WP5: Role of ADE (in vitro/in silico)

Measurement of the transport across the intestinal
barrier and the blood-brain barrier using in vitro

models and neuronal networks (n=21)
elimination: excretion
Measurement of protein binding, microsomal

special barriers absorption
protein binding
stability, lipophilicity (n=42) Measurement (n=3) and modelling of free concentration of compounds in the in vitro systems. Generic biokinetic model for the interpretation of in
Concentration at target Free concentration
vitro toxic concentrations in relation to the in vivo

acute toxic dose under development
and metabolism
26
WP5: Oral absorption

H = High ; HIA > 80 %
computer Caco-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
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
WP6: Role of metabolism

Cytotoxicity (%)
100
Hepatocyte
Hepatoma
Bioactivated IC50A < IC50A < IC50B
0 Concentration 100
Cytotoxicity (%)
IC50A
IC50B
A B
Not Bioactivated IC50 A IC50A = IC50B
0 Concentration
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
Bayer 0,01 0,18 1,28 0,67 1,13 0,56 0,85 >1E-03 1,54 1,45 1,70
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
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
WP7.2: Nephrotoxicity
ions
Cells: LLC-PK1
TER: sensitive indicator of nephrotoxicity TER: greater sensitivity for nephrotoxic chemicals Compounds requiring metabolism (diethylene glycol) did not show toxicity at concentrations used
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
WP7.3 Hepatotoxicity
Hepatocyte
Hepatoma
Non-hepatic cell
10 0
IC50A
IC50B
IC50C
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
WP 1
WP 2

WP 5 WP 6 WP 3
WP 7
WP 8
Last year of the project
WP 9
33
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.
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
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
Barrier models intestinal barrier

Prevalidation of in vitro models for the prediction of gastrointestinal absorption
10 Reference Chemicals
High Absorption Isopropanol (reference) Ethylen glycol (chemical) Intermediate absorption Cimetidine (reference) Paraquat (pesticide) Low absorption Atenolol (reference) Cupric sulphate (chemical)
- 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
Sodium Valproate (drug)
Paracetamol (drug)
Colchicine (drug)
Acetylsalicylic acid (drug)
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. 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

Haematotoxicology Immunotoxicology Chronic systemic toxicity Pulmonary toxicity Biological barriers Acute systemic toxicity maria.prieto-pilar@jrc.it agnieszka.kinsner@jrc.it maria.prieto-pilar@jrc.it laura.gribaldo@jrc.it
39
46
Toxicity prevalence of new industrial Chemicals in the EU
Chemicals (%) 0 3 21 76
LD50 (mg/Kg) < 25 > 25 200 > 200 2000 > 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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Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Key area Systemic toxicity

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Key area - Systemic toxicity

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Key area - Systemic toxicity

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Acute oral toxicity - OECD test guidelines

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Strategy to Replace Acute Toxicity Testing

ICCVAM/ECVAM validation study

log LD50 [mmol/kg]

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Generation of an in vivo database and establishment of a depository of reference compounds

Generation of an in vitro database

Analysis and integration of in vitro/in vivo data WP 4

Alerts and correctors in toxicity screening

WP 5 Role of ADE WP 6 Role of metabolism Role of target organ toxicity

WP 7.1 neurotoxicity WP 7.2 nephrotoxicity

Technical optimisation of the amended test strategy

Prevalidation of the testing strategy

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

kinetics (in vitro, in silico)

animal in vivo data

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

WP1: Selection of reference chemicals and collection of in vivo data

11 chemicals GHS cat.5 2000 < LD50 5000 mg/kg

7 chemicals GHS Not Classified LD50 > 5000 mg/kg

36 chemicals GHS cat.4 300 < LD50 2000 mg/kg

22 chemicals GHS cat.3 50 < LD50 300 mg/kg

Generation of the in vivo database (animal and human)

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

WP1: Evaluation of in vivo animal data variability

0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

SD of log-transformedLD50 (rat, oral)

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

WP1: Evaluation of in vivo human data calc. of LC50 values

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

WP1: Estimation of LC50 human

3,80 3,60 3,40

2,60 2,40 2,20

LC100 = 3.40 LC0 = 3.35

LC50 = (3.35+ 3.40)/2= 3.37 in microM Converted to M LC50=-2.63

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Generation of an in vivo database and establishment of a depository of reference compounds

Generation of an in vitro database

Analysis and integration of in vitro/in vivo data WP 4

Alerts and correctors in toxicity screening

WP 5 Role of ADE WP 6 Role of metabolism Role of target organ toxicity

WP 7.1 neurotoxicity WP 7.2 nephrotoxicity

Technical optimisation of the amended test strategy

Prevalidation of the testing strategy

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Generation of an in vitro database for 97 selected reference chemicals

Woirkshop on Alternative Methods - Ankara, 12-13 November 2007

Generation of an in vivo database and establishment of a depository of reference compounds

Generation of an in vitro database

Analysis and integration of in vitro/in vivo data WP 4

Alerts and correctors in toxicity screening

WP 5 Role of ADE WP 6 Role of metabolism Role of target organ toxicity

WP 7.1 neurotoxicity WP 7.2 nephrotoxicity