Developments in making in vitro tests available for REACH

Transcription

1 Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 1 Developments in making in vitro tests available for REACH S. Bremer, S.Casati, S. Coecke, R. Corvi, L.Gribaldo, M.Halder, P.Prieto,V.Zuang In Vitro Toxicology/European Centre for the Validation of Alternative Methods (ECVAM) IHCP, EC JRC, Ispra, Italy Susanne.Bremer@jrc.it

2 Overview Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 2 Legislative background on the use of alternative methods and their validation Update on ECVAM s activities

3 REGULATION (EC) No 1907/2006 Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 3 Art. 1: ensure a high level of protection of human health and the environment..... should also promote the development of alternative methods for the assessment of hazards of substances. Art. 40: The Commission, Member States, industry and other stakeholders should continue to contribute to the promotion of alternative test methods including computer supported methodologies, in vitro methodologies,... The Community's strategy to promote alternative test methods is a priority and the Commission should ensure that within its future Research Framework Programmes and initiatives such as the Community Action Plan on the Protection and Welfare of Animals this remains a priority topic. Art. 47 In accordance with Directive 86/609/EEC, it is necessary to replace, reduce or refine testing on vertebrate animals. The use of animals should be avoided by recourse to alternative methods validated by the Commission or international bodies, or recognised by the Commission or the Agency as appropriate to meet the information requirements under this Regulation.. To this end, the Commission, following consultation with relevant stakeholders, should propose to amend the future Commission Regulation on test methods or this Regulation, where appropriate, to replace, reduce or refine animal testing. The Commission and the Agency should ensure that reduction of animal testing is a key consideration in the development and maintenance of guidance for stakeholders and in the Agency's own procedures.

4 TITLE XII INFORMATION Article 117 Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 4 3. Every three years the Agency, in accordance with the objective of promoting non-animal testing methods, shall submit to the Commission a report on the status of implementation and use of nonanimal test methods and testing strategies used to generate information on intrinsic properties and for risk assessment to meet the requirements of this Regulation. The first report shall be submitted by 1 June The amount and distribution of funding made available by the Commission for the development and evaluation of alternative test methods. The first report shall be published by 1 June 2012.

5 ANNEX XI GENERAL RULES FOR ADAPTATION OF THE STANDARD TESTING REGIME SET OUT IN ANNEXES VII TO X Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium In vitro methods Results obtained from suitable in vitro methods may indicate the presence of a certain dangerous property or may be important in relation to a mechanistic understanding, which may be important for the assessment. this context, "suitable" means sufficiently well developed according to internationally agreed test development criteria (e.g. the European Centre for the Validation of Alternative Methods (ECVAM)) criteria for the entry of a test into the prevalidation process). Depending on the potential risk, immediate confirmation requiring testing beyond the information foreseen.. If the results obtained from the use of such in vitro methods do not indicate a certain dangerous property, the relevant test shall nevertheless be carried out at the appropriate tonnage level to confirm the negative result.. Such confirmation may be waived, if the following conditions are met: 1) results are derived from an in vitro method whose scientific validity has been established by a validation study, according to internationally agreed validation principles; 2) results are adequate for the purpose of classification and labelling and/or risk assessment; and 3) adequate and reliable documentation of the applied method is provided.

6 OECD GD 34 Guidance document on the validation and international Acceptance of new or updated test methods for hazard assessment Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 6 Screening tests rapid, simple tests for the prioritizing or grouping substances in general categories of potential models of actions preliminary decision Definitive test method (sufficient data to characterise the hazard of the substance final decision Adjunct tests (add to the data set or support interpretation of data derived from other tests) Test batteries: number of tests generally performed at the same time and they are complementing each other

7 Validation Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 7 Test Method validation is the process based on scientifically sound principles by which the reliability and relevance of a particular test, approach, method, or process are established for a specific purpose (art.11; OECD GD 34).. Not reliable, relevant? Reliable but not relevant Reliable and relevant (1) The Amden CAAT/ERGATT workshop (ATLA 18, , 1990) (2) Frazier s report to the OECD (OECD Environment Monograph no. 36) (3) OECD GD 34 Guidance document on the validation and international Acceptance of new or updated test methods for hazard assessment

8 Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 8 acute systemic 35% skin sensitisation 5% repeated dose 10% PREDICT-IV chronic toxicity 17% eye irritation 1% toxicokinetics 2% skin irritation 1% phototoxicity 3% skin-eye corrosion 3% endocrine disruptions 2% reproductive toxicity 13% carcinogenicity 4% mutagenicity 4%

9 Topical Toxicity Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 9 Test method evaluations: Eye irritation: HCE, EpiOcular (broad applicability domain), EpiOcular (surfactants and surfactant-based products), Slug Mucosal Irritation assay, Irritection Peer reviews: Skin irritation: SkinEthic (similar assay), updated EpiDerm assay Skin corrosion: EST-1000 (Cellsystems) Eye irritation: LVET Validation: Eye irritation: 4 cytotoxicity, cell-function-based assays (RBC, FL, NRR, CM) Organotypic assays (BCOP, HET-CAM, IRE, ICE) for other ranges of irritancy Regulatory acceptance: Skin irritation: Reconstituted human epidermis assay (EPISKIN) BCOP HET-CAM human epidermis

10 Acute Toxicity Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 10 R&D activities: Strategy to replace acute oral toxicity testing - FP 6 IP A-Cute Tox Validation: Prediction of non-toxic substances in vitro by the Balb 3T3/NRU cytotoxicity test

11 Acute Toxicity: R&D Activities Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 11 Title: Optimisation and prevalidation of an in vitro test strategy for predicting human acute toxicity Integrated Project of the 6 th 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 Coordination: Leila Risteli, Oulu University/F

12 Acute Toxicity: R&D Activities Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium reference chemicals Generation of an in vivo database WP 1 and establishment of a depository of reference WP 2 compounds Generation of an in vitro database Analysis and integration of in vitro/in vivo data Alerts and correctors in toxicity screening WP 4 New cell systems, new endpoints WP 5 WP 5 Role of ADE WP 3 Iterative amendment of the testing strategy WP 6 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

13 Acute Toxicity: R&D Activities Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium reference chemicals Generation of an in vivo database WP 1 and establishment of a depository of reference WP 2 compounds 28 outliers identified Analysis and integration of in vitro/in vivo data Generation of an in vitro database Alerts and correctors in toxicity screening Set of cytotoxicity data on 96 chemicals WP 4 New cell systems, new endpoints WP 5 WP 5 Role of ADE WP 3 Iterative amendment of the testing strategy WP 6 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

14 Acute Toxicity: R&D Activities Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium reference chemicals Generation of an in vivo database WP 1 and establishment of a depository of reference WP 2 compounds 28 outliers identified Analysis and integration of in vitro/in vivo data Generation of an in vitro database Alerts and correctors in toxicity screening Set of cytotoxicity data on 96 chemicals WP 4 New cell systems, new endpoints WP 5 WP 5 Role of ADE in vitro data generated with 57 chemicals WP 3 Iterative amendment of the testing strategy WP 6 WP 7 WP 6 Role of metabolism Role of target organ toxicity 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

15 Acute Toxicity: R&D Activities Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium reference chemicals Generation of an in vivo database WP 1 and establishment of a depository of reference WP 2 compounds 28 outliers identified Analysis and integration of in vitro/in vivo data Generation of an in vitro database Alerts and correctors in toxicity screening Set of cytotoxicity data on 96 chemicals WP 4 New cell systems, new endpoints WP 5 WP 5 Role of ADE in vitro data generated with 57 chemicals WP 3 Iterative amendment of the testing strategy WP 6 WP 7 WP 6 Role of metabolism Role of target organ toxicity 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 2009 Blind testing additional chemicals

16 Validation Study: Non-Toxic substances Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 16 Chemicals (%) LD50 (mg/kg) 0.1 < 25 3 > > > 2000 New Chemical Database (TCS-ECB) January % of all new industrial chemicals are in this class Chemicals Selection Committee P. Prieto T. Cole A. Kinsner M. Liebsch Buying & Coding Chemicals- ECVAM A. Kinsner Validation of the 3T3 NRU assay for the prediction of nontoxic compounds Management Team ECVAM Pilar Prieto, Agnieszka Kinsner Independent Data Analysis - ECVAM Distribution of chemicals SIGMA 56 chemicals Lab 1 HSL, UK R. Gibson Lab 2 ECVAM/NMI JRC S. Coecke M. Whelan Lab 3 IIVS, US R. Curren Validated 3T3/NRU Manual protocol Validated 3T3/NRU Automated Protocol Abbreviated Protocol of Validated 3T3/NRU

17 Sensitisation Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 17 R&D activities: Novel Testing Strategies for In Vitro Assessment of Allergens - FP 6 IP Sens-it-iv Validation: Sensitisation: Direct Peptide-Binding Assay, Human Cell Line Activation Test (hclat), Myeloid U937 Skin Sensitisation Test (MUSST) Regulatory acceptance: Sensitisation: Cut Down Local Lymph Node Assay

18 R&D activities Sensitisation Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 18 Sens-it-iv Title: Novel Testing Strategies for In Vitro Assessment of Allergens Integrated Project of the 6th Framework Programme of the European Commission Consortium: 28 partners from 10 Member States Start October 2005-September 2010 Coordination: E. Roggen, Novozymes/ Denmark

19 Sens-it-iv: Overview on the project activities Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 19 Goal: to develop strategies to replace animal experimentation with in vitro assays for identifying skin and respiratory sensitisers WP1: Compound Selection Technology module (Months 18-60) WP7: Data Management WP 10: Management Science module (Months 0-42) WP3: DC T-cell interactions and biology WP4: Genomics WP2: EC- DC interactions and biology WP6: Metabonomics WP5: Proteomics WP9: Technology transfer & Dissemination (Months 0-60) WP8: In vitro assay development

20 Local Lymph Node Assay (LLNA) Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 20 Revised approach of the standard LLNA to reduce animal use in skin sensitisation testing Standard LLNA 3 concencentrations test chemical + vehicle control 4-5 mice/group Cut-down LLNA top concentration test chemical + vehicle control 4-5 mice/group mice 8-10 mice Does not provide information on potency For risk assessment purposes a Standard LLNA should be conducted

21 Genotoxicity Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 21 R&D FP6 STREP COMICS Testing Strategy Evaluation False positives in genotox testing: Chemical selection (Kirkland et al., Mut. Res., 2008 Reduction opportunities in Regulatory Genotoxicity Testing (e.g. integrating into other regulatory tests or optimizing current test) Validation Validation COMET Assay (coordinated by JaCVAM) Prevalidation Genotoxicity assays in 3D-skin model (coordinated by COLIPA) Regulatory acceptance Retrospective validation of the micronucleus test in vitro (OECD draft TG 487)

22 Carcinogenicity Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 22 R&D FP 6 IP carcinogenomics Validation Cell transformation assays (CTA) ECVAM prevalidation of CTAs: SHE low ph SHE standard ph Balb/c 3T3 Complete results of prevalidation (foreseen January 2009) Prevalidation of CTA BHAS42 coordinated by JaCVAM (Japan)

23 Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 23 CARCINOGENOMICS Development of a high throughput genomics-based test for assessing genotoxic and carcinogenic properties of chemical compounds in vitro EC contribution partners Starting date: Duration: 60 month Website: ECVAM contact: Raffaella Corvi@jrc.it

24 Reproductive and developmental Toxicity Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 24 R&D activities Reproductive toxicity: FP 6-IP ReProTect, FP7-IP ESNATS (developmental) Neurotoxicity: FP7-IP ESNATS Test evaluations: Reproductive toxicity tests: bovine female maturation/fertilisation test, CASA, Sertoli cell test, leydig cell test, endocrine disrupters Validation: Endocrine disruption: PANVERA -ER binding Test, HeLa ER antagonist, LUMICELL ER Agonist and Antagonist, ERa & AR CALUX (Human osteosarcoma osteoblastic U2-OS cells) MELN (ERa human breast cancer MCF7 cells), PALM (har human prostate adenocarcinoma PC-3 cells) Regulatory acceptance: Endocrine disruption: OECD test guideline on the HeLa agonist ER validation study Reproductive toxicity: draft OECD test guideline ext F1 generation study

25 Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 25 ReProTect Development of a novel approach in hazard and risk assessment of reproductive toxicity by a combination and application of in vitro, tissue and sensor technologies EC contribution partners Starting date: Duration: 60 month Website: ECVAM contact: Cristian.Pellizzer@jrc.it, Susanne.Bremer@jrc.it

26 Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 26 Embryonic stem cell-based novel alternative testing strategies EC contribution partners Starting date: Duration: 60 month Coordinator: Juergen Hescheler: University of Cologne/D ECVAM contact: Susanne.Bremer@jrc.it ECVAM/NIBSC WORKSHOP ; Ispra; hesc Technology for Toxicology and Drug Development: Current Status and Recommendations for Best Practice and Standardization

27 New Testguideline: ext.one generation study Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 27 Advantages in comparison to OECD 416 (2600 animals): Waiving of second generation depending of trigger/waiver (reduction: ~1200 animals ) Limit dose concept : (reduction.~ 700 animals, two dosage groups) Additional information on DNT and DIT if these modules are mandatory. Possibility to use F1 animals for prenatal toxicity study (reduction:~ 80 animals/chemical) Under discussion: Availability, relevance and sensitivity of trigger for DIT and DNT Practicability and use of the complex study design vs 2 generation study

28 Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 28 Ecotoxicity Acute aquatic toxicity Fish Embryo Test OECD ad hoc Expert Group collaborative study planned Threshold Approach Proposal as appendix to OECD TG203 Bioconcentration/accumulation Validation (Module 1-3) of in vitro trout S9 fraction assay started in January 2008 in vitro information on metabolic stability of substances will be used to refine bioaccumulation/bioconcentration models for derivation of BAF/BCF values International collaboration HESI (Sub)Committees on animal alternative needs ERA, bioaccumulation

29 Conclusion Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 29 The development, use and monitoring of non-animal testing methods is a main objective of REACH. REACH is distinguishing between validated and suitable in vitro methods Different types of tox. tests such as screening tools, adjunct tests, definite tests

30 Conclusion Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 30 Relevant parameter to know if an toxicological test is used in a testing strategy in order to judge uncertainty of data: Biological relevance of in vitro tests for various human health endpoints intra- and interlaboratory variability Performance of a test (sensitivity and specificity) Applicability domain Performance standards (similar tests) VALIDATION

31 Integrated testing strategies for REACH 1 st SETAC Europe Special Science Symposium 31