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Kinetic Direct Peptide Reactivity Assay (Kdpra): Validation Study Report

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Kinetic Direct Peptide Reactivity Assay (Kdpra): Validation Study Report Kinetic DPRA Validation Study Report 6 March 2020 Kinetic Direct Peptide Reactivity Assay (kDPRA): Validation Study Report Lead Laboratories and Study Management: BASF SE Britta Wareing Phone: +49 621 60-58107, Email: [email protected] Dr. Susanne Kolle Phone: +49 621 60-56731, Email: [email protected], Experimental Toxicology and Ecology Postal Address: BASF SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen am Rhein, Germany Responsibilities: Protocol authorship Coordination between laboratories Organisation of test chemical procurement Givaudan Schweiz AG Dr. Andreas Natsch Phone: +41 44 824 21 05, Email: [email protected]; Tina Haupt Phone: +41 44 824 25 15, Email: [email protected] Postal Address: Department of in vitro molecular screening, Givaudan Schweiz AG, Kemptpark 50, CH-8310 Kemptthal, Switzerland Responsibilities: Test chemical selection Optimisation of rate constant calculation in evaluation sheet and cut-off refinement Biostatistics Naïve Test Laboratories: Institute for In Vitro Sciences (IIVS), 30 West Watkins Mill Road Suite 100, Gaithersburg, MD 20878, USA: Contact persons: Erin H Hill ([email protected]), Hans Raabe ([email protected]), Rishil Kathawala ([email protected]) Procter & Gamble, Mason Business Center, DS3-812, 8700 Mason-Montgomery Road, Mason, OH, 45040, USA: Contact persons: Cindy Ryan ([email protected]) and Petra Kern ([email protected]) Charles River Laboratories Den Bosch BV, Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands: Contact persons: Walter Westerink ([email protected]), Sjoerd Verkaart ([email protected]) 1 | P a g e Kinetic DPRA Validation Study Report 6 March 2020 National Institute of Public Health, Srobarova 48, 10042 Praha, Czech Republic: Contact persons: Marian Rucki ([email protected]), Dvořáková Markéta; ([email protected]), Kejlová Kristina ([email protected]) L’Oréal Research & Innovation, Bâtiment 25-101S, 1 Avenue Eugène Schueller, 93600 Aulnay sous Bois, France: Contact persons: Nathalie Alepée ([email protected]) Fleur Tourneix ([email protected]) Test chemical procurement and blinding BioTeSys GmbH, Schelztorstrasse 54-56, 73728 Esslingen, Germany Contact persons: Dr. Dirk Dressler ([email protected]) 2 | P a g e Kinetic DPRA Validation Study Report 6 March 2020 1. Contents 1. Contents ............................................................................................................ 3 2. Abbreviations ...................................................................................................... 6 3. Introduction ........................................................................................................ 9 3.1. Background ................................................................................................... 9 3.2. Principle of the test ....................................................................................... 10 3.3. Log kmax Calculation Example DNCB ................................................................ 11 4. Goals set for the validation study ........................................................................ 13 5. Work packages ................................................................................................. 13 5.1. WP 3.1.: Preparation phase / protocol refinement ............................................ 15 5.2. WP3.2. Transfer phase of the refined protocol in experienced labs ...................... 16 5.2.1. Procedure ............................................................................................... 16 5.2.2. Results for the positive chemicals (Set A) ................................................... 17 5.2.3. Results for the positive control .................................................................. 18 5.2.4. Results for the chemicals of Set B ............................................................. 19 5.2.5. Conclusions testing in experienced labs ...................................................... 22 5.3. WP 3.3. Transfer phase to naïve labs (phase I) ................................................ 22 5.3.1. Goals and set-up ..................................................................................... 22 5.3.2. Setting up the test in different labs............................................................ 23 5.3.3. Reproducibility of log kmax ......................................................................... 24 5.3.4. Reproducibility of predicted GHS categories ................................................ 28 5.3.5. Results for positive control ....................................................................... 29 5.3.6. Discussion on transfer phase to naïve labs.................................................. 29 5.4. WP 3.4 Blind-coded Testing – Phase II ............................................................ 31 5.4.1. Goals and set-up ..................................................................................... 31 5.4.2. Rationale for test chemical selection for Phase II ......................................... 32 5.4.3. Overview of the different data evaluations performed: ................................. 34 5.4.4. Intra-laboratory reproducibility of log kmax .................................................. 36 5.4.5. Inter-laboratory reproducibility of log kmax .................................................. 39 5.4.6. Comparison of intra-laboratory and inter-laboratory reproducibility of log kmax 41 5.4.7. Prediction of GHS Categories .................................................................... 43 5.4.8. Intra-laboratory reproducibility of predicted GHS categories ......................... 43 3 | P a g e Kinetic DPRA Validation Study Report 6 March 2020 5.4.9. Inter-laboratory reproducibility of predicted GHS categories ......................... 44 5.4.10. Reproducibility of positive control ........................................................... 48 5.4.11. Interference from autofluorescence and potential fluorescence quenching .... 50 5.4.12. Intra-laboratory reproducibility of 24 h / 5 mM depletion value ................... 51 5.4.13. Inter-laboratory reproducibility of 24 h / 5 mM depletion value ................... 52 5.4.14. Comparison of kDPRA (24 h / 5 mM depletion value) to the classical DPRA .. 53 5.4.15. Congruency of solvent-decision .............................................................. 55 5.4.16. Intra-laboratory congruency of solvent-decision ....................................... 55 5.4.17. Inter-laboratory congruency of solvent-decision ....................................... 55 5.4.18. Conclusions on intra-and inter-laboratory reproducibility ........................... 58 5.5. WP 3.5. Evaluation of the predictive capacity for the dataset of Phase I and Phase II 58 5.6. WP 3.6. Building the database and testing the prediction cut-off ........................ 61 5.6.1. Deriving the optimal cut-off to discriminate GHS Cat 1A ............................... 62 6. General discussion ............................................................................................. 70 6.1. Test definition .............................................................................................. 70 6.2. Transferability .............................................................................................. 70 6.3. Intra-laboratory Reproducibility ...................................................................... 71 6.4. Inter-laboratory Reproducibility ...................................................................... 71 6.5. Predictive capacity to identify GHS Cat1A ........................................................ 71 6.6. Applicability domain ...................................................................................... 72 6.6.1. Technical limitations ................................................................................ 72 6.6.2. Predictive limitations................................................................................ 73 6.7. Relationship to DPRA 442C and advantages over the classical DPRA ................... 74 6.8. Potential use in tiered testing strategy ............................................................ 75 6.9. Potential use of numeric log kmax in defined approaches .................................... 75 6.10. Introduction as a test method into OECD 442C .............................................. 76 6.11. Potential improvements .............................................................................. 76 7. References ....................................................................................................... 76 Appendix 1: Detailed result tables of the blind-coded inter-laboratory testing (Phase II) . 77 Appendix 2: Result interpretation in case of fluorescence quenching and potential control experiments .......................................................................................................... 87 Non-linear peptide depletion observed for other chemicals when establishing the database ......................................................................................................................... 90 4 | P a g e Kinetic DPRA Validation Study Report 6 March 2020 Appendix 3. Extended dataset to evaluate predictivity and to determine optimal GHS 1A vs. GHS 1B/NC cut-off.................................................................................................. 93 Appendix 4: Statistical analysis for the contribution of continuous parameters from vali- dated
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