Extrapolation of in vitro effects to in vivo embryotoxicity Basic requirements for prediction model development Han van de Sandt, PhD
TNO: Netherlands Organisation for Applied Scientific Research Established in 1932 (by Act of Parliament) Independent RTO Revenue Generating - Not for Profit 5500 employees Serving 5 Core Areas 2
Method validation Assessment of reliability and relevance of a test procedure for a specific purpose Regulatory decision or In-house screening The intended use of the data generated by the method determines the required characteristics 3
REACH information requirements Annexes VII to X Higher tonnage more data Annex XI Rules for adaptation of the standard testing regimes Testing does not appear scientifically necessary Use of existing data Weight of evidence Qualitative or Quantitative Structure Activity Relationships In vitro methods ( suitable ; sufficiently well developed ) Grouping of substances and read-across approach Testing is technically not possible Substance-tailored exposure-driven testing 4
Validated methods or suitable methods? International validation of in vitro assays aims at: Making predictions for all chemicals General regulatory acceptance of method Hartung et al., ATLA 32, 467-472 (2004) 5
Validated methods or suitable methods? Suitable methods mentioned in Annex XI of REACH E.g. ECVAM criteria for entry of a test into the pre-validation process Mechanistic basis of the test method and endpoint(s) evaluated Purpose of the test method and proposed practical application Relevance of the test method (preliminary evidence on predictive capacity) Preliminary self-assessment of the protocol optimization level Information on the prediction model applied Discussions ongoing on requirements 6
Prediction Models Definition (Worth and Balls, 2004): Unambiguous algorithm for converting (in vitro) data into predictions of a pharmaco-toxicological endpoint in animals or humans Test system (scientific basis) Prediction model (predictive capacity) (partial) replacement assay 7
Prediction Models Sensitivity to variation Dependence on training set Availability and quality of in vivo data for establishing predictive value predicting what? (Dis)similarity of in vivo and in vitro read-outs use of surrogate markers defining mechanisms / processes Categories or exact quantification (e.g. NOAEL / LOAEL) Selection of the reference test / data 8
Improving prediction models Biology (mechanistic understanding) Data (statistical analysis) PM 9
In vivo prenatal/developmental study (OECD TG 414) Read-outs Interauterine death Altered growth Structural abnormalities Growth and development Sexual maturation Gamete production and release Post-natal development Fertilisation Classification & Labelling R61 - may cause harm to the unborn R63 - possible risk of harm to the unborn Parturition Foetal development Implantation Zygote transport Embryogenesis 10
Validation of in vitro assays for embryotoxicity Embryonic Stem cell Test (EST) Model: mouse ES differentiating to beating cardiomyocytes Exposure time: 10 days Endpoints: cytotoxicity and cell differentiation Whole Embryo Culture (WEC) Model: cultured rat embryos with 1-5 somites Exposure time: 2 days Endpoints: malformation and morphological score Micromass (MM) Model: limb bud cell cultures from rat embryo Exposure time: 7 days Endpoints: cytotoxicity and cell differentiation 11
Classification on the basis of in vitro assays ECVAM validation study Class 1: non-embryotoxic chemicals not developmentally toxic at maternally toxic exposure Class 2: weakly embryotoxic chemicals of intermediate activity (excluding receptor mechanisms) Class 3: strongly embryotoxic chemicals which are developmentally toxic in all species tested, including multiple developmental effects and with a high A/D ratio Variable data on in vivo effects; Classification of test chemicals on basis of expert judgment 12
Validation of in vitro assays for embryotoxicity General conclusions Good concordance in vitro in vivo classification (71-80%) 100% predictivity for strong embryotoxicants Lack of discrimination between none and weak embryotoxicity Limitations Number of chemicals in training and test set Availability of in vivo data Link to regulatory practice Kinetic aspects and metabolic (de)activation 13
Use of in vitro data in risk assessment What is the purpose? In house prioritizing / screening Classification & Labeling Regulatory risk assessment Full replacement: stand-alone test / test batteries Tiered approaches Integrated Testing Strategies Substance grouping and read-across 14
Full replacement: in vitro test batteries Reference test ( all chemicals) Sexual maturation Growth and development Gamete production and release Post-natal development Fertilisation Parturition Zygote transport Foetal development Implantation Embryogenesis 15
Full replacement: in vitro test batteries In vitro approach chem group A chem group B Sexual maturation Growth and development Gamete production and release Post-natal development Fertilisation Parturition Zygote transport Foetal development Implantation Embryogenesis 16
Tiered approaches (I) - Further testing of all toxic' substances - High sensitivity required for in vitro test In vitro test Non-toxic Toxic In vivo test Non-toxic Toxic 17
Tiered approaches (II) - Further testing of all non-toxic' substances - High specificity required for in vitro test In vitro test Toxic Non-toxic In vivo test Toxic Non-toxic 18
Integrated Testing Strategies (Q)SARs Exposure Scenarios Read Across Endpoint information? TESTING In vitro Existing information 19
Grouping of substances and read-across Similarities may be based on: a common functional group common precursor and/or breakdown products constant pattern in changing of potency of properties 20
In vitro read across: filling in vivo data gaps Classification No classification In vivo data point In vitro data point Functional or chemical descriptor 21
In vitro in vivo extrapolation: kinetic aspects Predicting systemic toxicity on the basis of in vitro tests Information on kinetics & metabolism of the compound is essential Local exposure CELL in vitro toxicity test ORGANISM in vitro toxicity test CELL ORGAN Systemic exposure 22
Dose (LOAEL) Estimation of corresponding dose PK-Model Systemic exposure levels at target tissue Estimation of relevant systemic exposure Pathological effect in target tissue In vitro toxicological effect (EC 50 ) 23
Classification of embryotoxicity on the basis of EST 2-ME 2-MAA 2-EE 2-EAA RA 5-FU MTX Toxic effect levels In vitro IC50 3T3 (mg/l) 1811 658 4055 1092 >10 0.204 0.034 In vitro IC50 ES- D3 (mg/l) 1788 633 2055 1214 0.0093 0.0104 0.073 In vitro ID50 (mg/l) 3462 139 1785 196 0.00031 0.032 0.038 Classification (strong, weak or nonembryotoxic) In vitro Non Weak Non Weak Strong Strong Strong In vivo Weak Weak Weak Weak Strong Strong Strong 5-fluorouracil (5-FU), 2-methoxyethanol (2-ME), 2-methoxyacetic acid (2-MAA), 2-ethoxyethanol (2-EE), 2-ethoxyacetic acid (2-EAA), Retinoic acid (RA), Methotrexate (MTX) 24
Toxic effect levels: comparison in vitro and in vivo data In vitro ID50 (mg/l) In vivo LOAEL 2-ME 3462 50 ppm 2-MAA 139-2-EE 1785 100 ppm 2-EAA 196 - RA 0.00031 6 mg/kg 5-FU MTX 0.032 0.038 10 mg/kg 0.1 mg/kg Direct translation in vitro to in vivo effect levels not possible Extrapolation steps necessary 25
In vitro/in silico prediction of effect level of 2-ME In vitro effect levels determined with EST (ID50) ID50 2-MAA = 1539 µm 1 Extrapolation rules ID50>ECplasma Extrapolation rule 1: ID50 = ECplasma Internal exposure (EC plasma) EC plasma 2-MAA = 1539 µm 2-ME 2-MAA Inhaled Exhaled air air Lung 2 In silico modeling PBPK Venous Blood Fat Richly perfused Poorly perf Liver Kegc Arterial Blood 2-MAA Kmaac Venous Blood Fat Richly perfused Poorly perfused Liver Arterial Blood Kex Predicted effect level, scaled from in vitro In vivo effect level 2-ME = 326 ppm 26
Comparison predicted and measured effect levels Route of exposure In vitro/in silico (predicted in vivo) In vivo (observed in vivo) Ratio pred/obs 2-ME Inhalation 326 ppm 50 ppm 6.5 2-EE Inhalation 354 ppm 100 ppm 3.5 RA Oral 5.45 mg/kg 6 mg/kg 0.9 5-FU Subcutaneous 0.05 mg/kg 10 mg/kg 0.005 MTX Intravenous 0.13 mg/kg 0.1 mg/kg 1.3 Verwei et al., Toxicology Letters (2006) 165 (1), 79-87. 27
In vitro in vivo extrapolation: further development and evaluation Extension of database Development of generic kinetic model for extrapolation from internal to external exposure levels Implementation of metabolic fraction in in vitro embryotoxicity tests 28
Conclusions Formal validation of new in vitro methods is preferred for maximal impact in regulatory risk assessment, but is costly and time consuming Proof of method suitability (limited application) will enhance introduction of innovations and reduce costs and animal use Use of in vitro data for prediction of embryotoxicity is feasible, provided that kinetics are taken into account. Proof-of-principle of in vitro / in silico approach has been shown, but needs to be further developed and evaluated 29
Acknowledgements TNO (NL) BfR/Zebet (D) RIVM (NL) Miriam Verwei, Cyrille Krul Dinant Kroese, Andreas Freidig Ine Waalkens-Berendsen, André Wolterbeek Mariska Tegelenbosch-Schouten Horst Spielmann, Andrea Seiler Aldert Piersma Dutch Ministry of Social Affairs and Employment EU 6 th Framework Programme ReProTect 30