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  • EUROPEAN COMMISSION JOINT RESEARCH CENTRE Institute for Health and Consumer Protection European Union Reference Laboratory for Alternative Methods to Animal Testing (ECVAM)

    Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report Page 1 of 74

    Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report

    January 2012

  • Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report Page 2 of 74

    SUMMARY.............................................................................................................................................................. 4

    BACKGROUND...................................................................................................................................................... 5

    MANAGEMENT OF THE STUDY....................................................................................................................... 8 1. STUDY OBJECTIVES ...................................................................................................................................... 8 2. PROJECT PLAN ........................................................................................................................................... 8

    a. Structure of the study.............................................................................................................................. 9 b. Validation Management Group .......................................................................................................... 10 c. Laboratories......................................................................................................................................... 11 d. Quality Systems of the Participating Laboratories ............................................................................. 12

    3. STUDY EXPERIMENTAL DESIGN AND SAMPLE SIZE ................................................................................. 13 4. SELECTION OF TEST CHEMICALS ............................................................................................................ 14 5. CHEMICALS PURCHASE, CODING AND DISTRIBUTION ............................................................................. 20

    a. Solvent Compatibility assessment........................................................................................................ 20 b. Coding/Decoding ................................................................................................................................. 20

    6. DATA MANAGEMENT ................................................................................................................................ 21 7. STATISTICAL ANALYSIS OF EXPERIMENTAL DATA ................................................................................ 22

    a. Analysis performed on the experimental data .................................................................................... 22 b. Criteria for Assessing the Study Outcome .......................................................................................... 22

    TEST DEFINITION (MODULE 1) .................................................................................................................... 24 1. INTENDED PURPOSE OF THE TEST METHOD ............................................................................................. 24 2. EVIDENCE DEMONSTRATING THE NEED OF THE TEST METHOD.............................................................. 24 3. STATUS OF DEVELOPMENT OF THE TEST METHOD .................................................................................. 24 4. SCIENTIFIC BASIS BIOLOGICAL AND/OR MECHANISTIC RELEVANCE OF THE DPRA........................... 25 5. PROTOCOL OF THE TEST METHOD ........................................................................................................... 25 CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 1 ............................................................ 31

    TRANSFERABILITY (MODULE 3) .................................................................................................................. 33 1. GENERAL ASPECTS ................................................................................................................................... 33 2. TRAINING.................................................................................................................................................. 33 3. TRANSFER OF THE TEST METHOD TO THE NAVE LABORATORIES.......................................................... 35 4. QUALIFICATION RUNS FOR A SUCCESSFUL COMPLETION OF THE TRANSFER PHASE ............................. 37 CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 3 ............................................................ 39

    WITHIN-LABORATORY REPRODUCIBILITY (MODULE 2) .................................................................... 41 1. P&G.......................................................................................................................................................... 41

    a. Reproducibility (concordance in predictions).................................................................................... 41 b. Reproducibility of depletion values for cysteine and lysine................................................................ 42 c. Reproducibility of the controls values................................................................................................. 44

    2. RICERCA ................................................................................................................................................ 46 a. Reproducibility (concordance in predictions).................................................................................... 46 b. Reproducibility of depletion values for cysteine and lysine................................................................ 47 c. Reproducibility of controls values....................................................................................................... 48

    3. IVMU ....................................................................................................................................................... 50 a. Reproducibility of the controls values................................................................................................. 50 b. Reproducibility (concordance in predictions).................................................................................... 52 c. Reproducibility of depletion values for cysteine and lysine................................................................ 53

    CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 2 ............................................................ 54 BETWEEN LABORATORY REPRODUCIBILITY (MODULE 4)................................................................ 55

    1. REPRODUCIBILITY (CONCORDANCE IN PREDICTIONS) ........................................................................... 57 2. REPRODUCIBILITY OF DEPLETION VALUES FOR CYSTEINE AND LYSINE (FOR THE 24 CHEMICALS) ......... 60 CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 4 ............................................................ 64

    PREDICTIVE CAPACITY (MODULE 5) ......................................................................................................... 65 CONCLUSION OF THE VALIDATION MANAGEMENT TEAM ON MODULE 5 ............................................................ 67

  • Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report Page 3 of 74

    VMG OVERALL CONCLUSIONS AND RECOMMENDATIONS ............................................................... 69 OVERALL CONCLUSIONS ................................................................................................................................... 69 RECOMMENDATIONS ......................................................................................................................................... 71

    LIST OF APPENDICES....................................................................................................................................... 72

    LIST OF REFERENCES...................................................................................................................................... 73

  • Direct Peptide Reactivity Assay (DPRA) ECVAM Validation Study Report Page 4 of 74

    Summary The evaluation of the skin sensitisation potential represents an important component of the safety assessment of new and existing substances. Traditionally this is achieved with animal tests such as the Local Lymph Node Assay (LLNA) and the guinea pig tests (Buehler Test and Guinea Pig Maximisation Test). To date, there is a pressing need for having alternative non-animal methods available to reduce and eliminate the need for animals for this endpoint as advocated by some European regulations aiming at the protection of human health and the environment (i.e. Cosmetics Regulation and REACH). Several mechanistically-based non-animal test methods for the assessment of skin sensitisation are currently under development/evaluation. The validation of the Direct Peptide Reactivity Assay (DPRA) was part of a study coordinated by ECVAM in which two other methods (the human Cell Line Activation Test (h-CLAT) and the Myeloid U937 Skin Sensitisation Test (MUSST)) are still in the evaluation process. The study was designed to allow sound conclusions to be drawn on the transferability and reproducibility (within- and between-laboratories) of the DPRA in view of its future use as part of a non-animal testing strategy for skin sensitisation hazard assessment. Having reviewed the information generated in the study, the Validation Management Group (VMG) concluded that this appropriatel