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Current Validation Status of the Use of In Vitro Test Methods for Identifying Eye Injury Hazard Potential
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Overview Test Method Evaluation Report

Test Method Evaluation Report

ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products

NIH Publication No. 10-7553
Download complete report:
Volume 1 (Main Body and Appendices A-D - 654 pages, 47 MB) [PDF]
Volume 2 (Appendices E-J - 670 pages, 12 MB) [PDF]

Large files may take some time to download: times will vary based on the speed of the online connection. The complete report files may not be accessible to all readers; files of individual sections of the report linked below may provide improved accessibility. If you would like assistance in accessing this document, please send an email to the ICCVAM Webmaster [Send Email] or call NICEATM at 919-316-4729.

Correction to the test method evaluation report (October 2011) [PDF]

The correction affects the BCOP false negative substances for the EPA classification system, specifically the contents of Table 2-1 of the main body of the report (page 5 of Volume 1) and Table 6-10 of the Background Review Document (Appendix C; page C-58 of Volume 1) and the associated text. If you have a hard copy of this document (NIH Publication No. 10-7553) you may wish to print out the PDF document above and keep it with the hard copy. In addition, please note that the electronic versions of the main body and BRD linked below do not include these updates.

 Cover of the In Vitro Methods TMER

View Test Method Evaluation Report by Section

Front Matter [PDF]
Includes Table of Contents, Lists of Tables and Figures, List of Abbreviations and Acronyms, Acknowledgements, Preface, and Executive Summary

Main Body [PDF]

  • Section 1: Introduction
  • Section 2: The Bovine Corneal Opacity and Permeability Test Method
  • Section 3: The Cytosensor Microphysiometer Test Method
  • Section 4: The Hen’s Egg Test–Chorioallantoic Membrane Test Method
  • Section 5: The Isolated Chicken Eye Test Method
  • Section 6: The Isolated Rabbit Eye Test Method
  • Section 7: ICCVAM Consideration of Public, SACATM, and ICATM Comments
  • Section 8: References

Appendix A: ICCVAM Evaluation Timeline [PDF]

Appendix B: ICCVAM-recommended Test Method Protocols [PDF]

  • Appendix B1: ICCVAM-Recommended Protocol for Future Studies Using the Bovine Corneal Opacity And Permeability (BCOP) Test Method
  • Appendix B2: ICCVAM-Recommended Protocol for Future Studies Using the Cytosensor Microphysiometer (CM) Test Method
  • Appendix B3: ICCVAM-Recommended Protocol for Future Studies Using the Hen's Egg Test–Chorioallantoic Membrane (HET-CAM) Test Method
  • Appendix B4: ICCVAM-Recommended Protocol for Future Studies Using the Isolated Chicken Eye (ICE) Test Method
  • Appendix B5: ICCVAM-Recommended Protocol for Future Studies Using the Isolated Rabbit Eye (IRE) Test Method

Appendix C: Background Review Document – Current Status of In Vitro Test Methods for Identifying Mild/Moderate Ocular Irritants: Bovine Corneal Opacity and Permeability Test Method

  • Main Body [PDF]
    • Front matter: Includes Table of Contents, Acknowledgements, Preface, and Executive Summary
    • Section 1: Introduction
    • Section 2: Bovine Corneal Opacity and Permeability Test Method Protocol Components
    • Section 3: Substances Used for Validation of the Bovine Corneal Opacity and Permeability Test Method
    • Section 4: In Vivo Reference Data Used for an Assessment of Test Method Accuracy
    • Section 5: Bovine Corneal Opacity and Permeability Test Method Data and Results
    • Section 6: Bovine Corneal Opacity and Permeability Test Method Accuracy
    • Section 7: Bovine Corneal Opacity and Permeability Test Method Reliability
    • Section 8: Bovine Corneal Opacity and Permeability Test Method Data Quality
    • Section 9: Reports in the Peer-Reviewed Literature
    • Section 10: Animal Welfare Considerations (Reduction, Refinement, and Replacement)
    • Section 11: Practical Considerations
    • Section 12: References
    • Section 13: Glossary
  • Annex I: Characterization of Substances Tested in the BCOP Test Method
    1. Chemical and Product Classes of Substances Tested in the BCOP Assay [PDF]
    2. Components of Formulations Tested in Gettings et al. (1996) [PDF]
    3. Components of Formulations Tested in Swanson et al. (1995) [PDF]
    4. Components of Formulations Tested in Swanson and Harbell (2000) [PDF]
    5. Components of AMCP Formulations Tested [PDF]
  • Annex II: In Vitro Data for Substances Tested in the BCOP Test Method
    1. BCOP Data Sorted by Reference [PDF]
    2. BCOP Data Sorted by Substance Name [PDF]
  • Annex II: Comparison of In Vivo and In Vitro Ocular Irritancy Classifications
    1. BCOP Data Sorted by Reference [PDF]
    2. BCOP Data Sorted by Substance Name [PDF]

Appendix D: Background Review Document of Existing Methods for Eye Irritation Testing: Silicone Microphysiometer and Cytosensor Microphysiometer
Provided to ICCVAM by the European Centre for the Validation of Alternative Methods

  • Main Body [PDF]
    • Front Matter: Includes Table of Contents, List of Tables, List of Figures, List of Annexes, List of Abbreviations, and Acknowledgements
    • Section 1: Data Collections
    • Section 2: Test Definition (Module 1)
    • Section 3: Within-Laboratory Reproducibility (Module 2)
    • Section 4: Transferability (Module 3)
    • Section 5: Between-Laboratory Reproducibility (Module 4)
    • Section 6: Predictive Capacity (Module 5)
    • Section 7: Applicability Domain (Module 6)
    • Section 8: Supporting Materials
  • Annexes [PDF]
    Please Note: File is 39 MB in size and may take some time to download: time will vary based on the speed of the online connection.
    • Annex A: Protocols
    • Annex B: Physicochemical Properties of Chemicals
    • Annex C: Physicochemical Properties of Formulations
    • Annex D: Chemicals and Formulations
    • Annex E: Study and Test Method Relationship
    • Annex F: Study Reports
    • Annex G: References Containing Data
    • Annex H: Raw Cytosensor Data
    • Annex I: CTFA Phase II Study Animal Data
    • Annex J: COLIPA Animal Data
    • Annex K: Company #1 Animal Data
    • Annex L: EC/HO Animal Data

Appendix E: Background Review Document – Current Status of In Vitro Test Methods for Identifying Mild/Moderate Ocular Irritants: The Hen's Egg Test–Chorioallantoic Membrane (HET-CAM) Test Method

  • Main Body [PDF]
    • Front matter: Includes Table of Contents, Acknowledgements, Preface, and Executive Summary
    • Section 1: Introduction
    • Section 2: Hen’s Egg Test–Chorioallantoic Membrane Test Method Protocol Components
    • Section 3: Substances Used for Validation of the HET-CAM Test Method
    • Section 4: In Vivo Reference Data Used for an Assessment of HET-CAM Test Method Accuracy
    • Section 5: Hen’s Egg Test–Chorioallantoic Membrane Test Method Data and Results
    • Section 6: Hen’s Egg Test–Chorioallantoic Membrane Test Method Accuracy
    • Section 7: HET-CAM Test Method Reliability
    • Section 8: Test Method Data Quality
    • Section 9: Other Scientific Reports and Reviews
    • Section 10: How the HET-CAM Test Method Will Refine, Reduce, or Replace Animal Use
    • Section 11: Practical Considerations
    • Section 12: References
    • Section 13: Glossary
  • Annex I: Chemical and Product Class Information for the Substances Tested in the HET-CAM Test Method [PDF]
    Originally published as Appendix B of ICCVAM Background Review Document on the HET-CAM Test Method, NIH Publication No. 06-4515
  • Annex II: In Vitro Data for the IS(A) Analysis Method
    1. In Vitro Data for the IS(A) Analysis Method: by Reference [PDF]
    2. In Vitro Data for the IS(A) Analysis Method: by Substance [PDF]
  • Annex III: Comparison of In Vivo and In Vitro Ocular Irritancy Classifications
    1. Comparison of In Vivo and In Vitro Ocular Irritancy Classifications: Sorted by Reference [PDF]
    2. Comparison of In Vivo and In Vitro Ocular Irritancy Classifications: Sorted by Substance Name [PDF]

Appendix F: Background Review Document – Current Status of In Vitro Test Methods for Identifying Mild/Moderate Ocular Irritants: The Isolated Chicken Eye (ICE) Test Method

  • Main Body [PDF]
    • Front matter: Includes Table of Contents, Acknowledgements, Preface, and Executive Summary
    • Section 1: Introduction
    • Section 2: Isolated Chicken Eye Test Method Protocol Components
    • Section 3: Substances Used for Validation of the ICE Test Method
    • Section 4: In Vivo Reference Data Used to Assess Isolated Chicken Eye Test Method Accuracy
    • Section 5: Isolated Chicken Eye Test Method Data and Results
    • Section 6: Isolated Chicken Eye Test Method Accuracy
    • Section 7: Isolated Chicken Eye Test Method Reliability
    • Section 8: Isolated Chicken Eye Test Method Data Quality
    • Section 9: Other Scientific Reports and Reviews
    • Section 10: Animal Welfare Considerations (Reduction, Refinement, and Replacement)
    • Section 11: Practical Considerations
    • Section 12: References
    • Section 13: Glossary
  • Annex I: Chemical and Product Class Information for the Substances Tested in the ICE Test Method [PDF]
    Originally published as Appendix B of ICCVAM Background Review Document on the ICE Test Method, NIH Publication No. 06-4513
  • Annex II: In Vitro Data for Substances Tested in the ICE Test Method
    1. In Vitro Data for Substances Tested in the ICE Test Method: Sorted by Reference [PDF]
    2. In Vitro Data for Substances Tested in the ICE Test Method: Sorted by Substance Name [PDF]
  • Annex III: Comparison of In Vivo and In Vitro Ocular Irritancy Classifications
    1. Comparison of In Vivo and In Vitro Ocular Irritancy Classifications: Sorted by Reference [PDF]
    2. Comparison of In Vivo and In Vitro Ocular Irritancy Classifications: Sorted by Substance Name [PDF]

Appendix G: Independent Scientific Peer Review Panel Assessment

  • Appendix G1: Summary Minutes from the Peer Review Panel Meeting on May 19-21, 2009 [PDF]
  • Appendix G2: Independent Scientific Peer Review Panel Report: Evaluation of the Validation Status of Alternative Ocular Safety Testing Methods and Approaches [PDF]

Appendix H: Federal Register Notices and Public Comments

  • Appendix H1: Federal Register Notices
    • 70 FR 13512 (March 21, 2005): Request for Data on Non-Animal Methods and Approaches for Determining Skin and Eye Irritation Potential of Antimicrobial Cleaning Product Formulations; Request for Nominations for an Independent Expert Panel [PDF]
    • 72 FR 26396 (May 9, 2007): Request for Data on the Use of Topical Anesthetics and Systemic Analgesics for In Vivo Eye Irritation Testing [PDF]
    • 72 FR 31582 (June 7, 2007): Request for Ocular Irritancy Test Data From Human, Rabbit, and In Vitro Studies Using Standardized Testing Methods [PDF]
    • 73 FR 18535 (April 4, 2008): Non-Animal Methods and Approach for Evaluating Eye Irritation Potential for Antimicrobial Cleaning Products (AMCPs): Request for Nominations for an Independent Expert Panel and Submission of Relevant Data [PDF]
    • 74 FR 14556 (March 31, 2009): Announcement of an Independent Scientific Peer Review Panel on Alternative Ocular Safety Testing Methods; Availability of Draft Background Review Documents (BRD); Request for Comments [PDF]
    • 74 FR 19562 (April 29, 2009): Meeting of the Scientific Advisory Committee on Alternative Toxicological Methods (SACATM) [PDF]
    • 74 FR 33444 (July 13, 2009): Independent Scientific Peer Review Panel Report: Evaluation of the Validation Status of Alternative Ocular Safety Testing Methods and Approaches: Notice of Availability and Request for Public Comments [PDF]

  • Appendix H2: Public Comments Received in Response to Federal Register Notices

  • Appendix H3: Scientific Advisory Committee on Alternative Toxicological Methods (SACATM) Comments: SACATM Meeting on June 25-26, 2009
    View relevant excerpts from SACATM Minutes [PDF]
    Full meeting minutes available on U.S. National Toxicology Program website: refer to materials for 2009 meeting Exiting the ICCVAM website

  • Appendix H4: ECVAM Comments on ICCVAM Recommendations for the BCOP Test Method [PDF]

Appendix I: Relevant U.S. Federal and International Ocular Toxicity Regulations, Labeling, and Test Guidelines

  • Appendix I1: Table of Relevant U.S. Federal and International Ocular Testing Regulations for Hazard Classification and Labeling [PDF]
  • Appendix I2: U.S. Environmental Protection Agency Health Effects Test Guidelines: OPPTS 870.2400 - Acute Eye Irritation [PDF]
  • Appendix I3: U.S. Environmental Protection Agency Office of Pesticide Programs Label Review Manual
    View on U.S. Environmental Protection Agency website Exiting the ICCVAM website
  • Appendix I4: Organisation for Economic Co-operation and Development Test Guideline 405: Acute Eye Irritation/Corrosion [PDF]

Appendix J: NICEATM Analysis: Reduced Eye Hazard Labeling Resulting from Using Globally Harmonized System (GHS) Instead of Current U.S. Regulatory Classification Criteria [PDF]

  • Main Body
    • Front Matter: Includes Table of Contents, List of Tables, and Summary
    • Section 1: Background
    • Section 2: Overview of NICEATM and ICCVAM Analyses
    • Section 3: Analysis of the ECETOC Eye Irritation Database
    • Section 4: Analysis of the 1999 OECD Detailed Review Document on Classification Systems for Eye Irritation/Corrosion in OECD Member Countries
    • Section 5: Summary of Analyses
    • Section 6: Possible Options for GHS Hazard Categories for Classification and Labeling of Reversible Eye Irritation
    • Section 7: References
    • Section 8: Isolated Chicken Eye Test Method Data Quality
    • Section 9: Other Scientific Reports and Reviews
    • Section 10: Animal Welfare Considerations (Reduction, Refinement, and Replacement)
    • Section 11: Practical Considerations
    • Section 12: References
    • Section 13: Glossary
  • Annex I: OECD Series on Testing and Assessment - Number 14: Detailed Review Document on Classification Systems for Eye Irritation/Corrosion in OECD Member Countries (ENV/JM/MONO(99)4)
  • Annex II: Grades for Ocular Lesions
  • Annex III: Consideration of the Minimum Number of Animals with Positive Eye Injury Responses Required for Classification of a Chemical as an Eye Irritation Hazard
  • Annex IV: Nature, Duration, and Severity of Ocular Lesions for 31 ECETOC Substances that are EPA Irritants (Category I, II, or III) or FHSA Irritants but Not Classified as Ocular Hazards by GHS Classification Criteria
  • Annex V: OECD Database: Comparison of Percent Irritant Classification Using EPA, FHSA, and GHS Classification Systems
  • Annex VI: Nature, Duration, and Severity of Ocular Lesions for 33 OECD DRD Substances that are EPA Irritants (Category I, II, or III) or FHSA Irritants but Not Classified as Ocular Hazards by GHS Classification Criteria
  • Annex VII: Representative Rabbit Eye Test Data Used in the NICEATM Evaluation of the GHS Classification System

Appendix K: ECVAM Scientific Advisory Committee Statement oon the Scientific Validity of Cytotoxicity/Cell-Function Based In Vitro Assays for Eye Irritation Testing [PDF]
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