GERMAN FEDERAL INSTITUTE NAMs- prospects, challenges and limits FOR RISK ASSESSMENT Tewes Tralau
Regulatory Status Quo : Where do we stand? Consumer products / Contaminants: Heterogeneous ( REACH, product-specific regulations, ALARA ), cave: in vivo ban (cosmetics), nanotox, EDCs, mixtures ... Pesticides & Biocides: Harmonised guidelines (OECD, EU), mostly in vivo , mechanistic data only as “add-on”. REACH & CLP: Harmonised guidelines, mostly in vivo , mechanistic studies currently treated as “add- on”. Rely on data-sharing & "read across" (SAR) Food & Feed: Most testing is done in vivo , non-animal methods comprise PBTK modelling At the same time: and TTC. - Pressure to move away from in vivo testing. - Arbitrary political deadlines for doing so range from 2025 (NL) to 2035 (US-EPA). - Successful use of alternatives for hazard ranking/prioritisation and read across. T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 2
Regulatory requirements Primary objective is public health protection! This means safety testing must allow • clear conclusions regarding the potential adversity in an intact organism , • clear conclusions regarding dose-response relationships , • provide legal certainty , • refer to set and accepted testing guidelines, • and, ideally, should have earned some trust ( e.g. , by standing the test of time). T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 3
Regulatory requirements Primary objective is public health protection! b) mostly harmonised legal framework, Current system - PROs: This means safety testing must allow a) Established methods, • clear conclusions regarding the potential adversity in an intact organism , e) predictability of legal procedures. c) long experience, • clear conclusions regarding dose-response relationships , d) trusted results, • provide legal certainty , • refer to set and accepted testing guidelines, • and, ideally, should have earned some trust ( e.g. , by standing the test of time). T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 4
Regulatory requirements Current system - CONs: Primary objective is public health protection! > 80,000 chemicals, increasing a) Capacity issues: addressed/addressable ( e.g. , residues, a) Not all questions or endpoints mixtures, low dose effects, neurotoxicity, This means safety testing must allow by ~ 2,000/a • clear conclusions regarding the potential adversity in an intact organism , b) Worries about species specific effects • clear conclusions regarding dose-response relationships , ED- or epigenetics…). (metabolism/microbiome). • provide legal certainty , c) Societal pressure/ethics. • refer to set and accepted testing guidelines, • and, ideally, should have earned some trust ( e.g. , by standing the test of time). T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 5
Case for change – “classical” testing vs. NAMs Adapted from Tralau & Luch, Arch. Toxicol. 2015, 89: 819-21 T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 6
NAMs – basic considerations & concepts NAM approaches allow to: • Characterise molecular targets and the respective pathways affected. • Classify and rank substances according to their potential for cellular interference. • Identify substances with no effect . • Identify biomarkers and signatures. • Build testing strategies by using a versatile modular setup. Inherent issues: • Limited compatibility to the grown histopathological picture of adversity. • They do not easily translate into established quantitative measures of risk assessment. • Prone to over predictability … T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 7
NAMs – considerations & concepts “Advanced Outcome Pathways” Identification of endpoint-specific events & signalling cascades suitable to define adverse outcome Figure by M. Whelan “Mechanistic Integrated Testing Strategies” Tralau et al. , EHP 2012, 120: 1498-94 “ Relevant Pathways of Toxicological Concern ” Entity of pathways with the potential for adversity, the network of which leads to the “ toxome ” T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 8
Current state of the art Cellular and biochemical assays for ≥ 600 endpoints This includes: Molecular & biochemical assays • Cell lines • Primary cells • Designed cells (transformed) • Adapted from Tralau & Luch, TiPS 2012, 33: 353-64 T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 9
Where are we on the way to a Tox-test dummy? Current use of NAMs for: - Biomarker identification. - Substantiating read across. - Screening & hazard identification, prioritisation. - Exclusion of risk and hence further testing ( e.g. mutagenicity, genotoxicity or skin corrosion). - Substance classification ( e.g., skin corrosion and irritation). - As part of tiered testing strategies ( e.g., tier 1 for “endocrine disruption”). - Use of organotypic (cell and tissue) cultures to assess aspects of organ toxicity ( e.g., skin). - Use of PBTK models for internal exposure assessment ( in vivo & in vitro ). T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 10
Where are we on the way to a Tox-test dummy? Tralau et al. , EHP 2012, 120: 1498-94 From a scientific perspective current state of the art NAMs ( in silico , in vitro , “omics“…) Are fit to deliver data suitable for assessing many aspects of potential organ toxicity and, to a limited extent , also parts of chronic toxicity . However, more complex endpoints often still need further development in terms of system stability and complexity (tissue interaction, metabolism, immunocompetence) as well as with regard to in vivo to in vitro extrapolation. T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 11
Principal open issues – regulatory side (Regulatory) acceptance Validation does not necessarily warrant acceptance … The latter also depends on experience and • trust into the method. This should be addressed by blinded cross-validation trials a longside established assays. Also, there often still is a substantial skill gap on both sides (regulatory as well as on the “science” side). Adversity & Plasticity Cellular and biochemical Need to convert molecular measures into quantitative (or probabilistic) measures suitable for • assays for ≥ 600 endpoints risk assessment. Need for (pre)adverse “signatures” and databases thereof . Need to (partly) rethink adversity (biomarker based vs. histopathology-based). • How to address plasticity ? Can one agree on biomarkers that define the ‘point-of-no-return’ ? • Need for improved in vitro / in vivo correlations . • Test System Physiology This includes: Test systems used should be adequate and sufficiently complex (long term testing, metabolism). • - Molecular & biochemical assays - Cell lines Test System Validation - Primary cells How do we validate and do we do so against the right standards? How “golden” are they? - Designed cells (transformed) • Informed decisions on method performance and testing reliability require sound comparable performance parameters. T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 12
Principal open issues – regulatory side (Regulatory) acceptance Validation does not necessarily warrant acceptance … The latter also depends on experience and • trust into the method. This should be addressed by blinded cross-validation trials a longside established assays. Also, there often still is a substantial skill gap on both sides (regulatory as well as on the “science” side). Adversity & Plasticity Cellular and biochemical Need to convert molecular measures into quantitative (or probabilistic) measures suitable for • assays for ≥ 600 endpoints risk assessment. Need for (pre)adverse “signatures” and databases thereof . Need to (partly) rethink adversity (biomarker based vs. histopathology-based). • How to address plasticity ? Can one agree on biomarkers that define the ‘point-of-no-return’ ? • Need for improved in vitro / in vivo correlations . • Test System Physiology This includes: Test systems used should be adequate and sufficiently complex (long term testing, metabolism). • - Molecular & biochemical assays - Cell lines Test System Validation - Primary cells How do we validate and do we do so against the right standards? How “golden” are they? - Designed cells (transformed) • Informed decisions on method performance and testing reliability require sound comparable performance parameters. T. Tralau, July 2 nd 2020, 76 th meeting of the EFSA advisory forum page 13
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