ICCVAM-Recommended Test Method Protocols
This page contains links to current ICCVAM-recommended protocols for specific test methods. It also includes links to the ICCVAM Test Method Evaluation Reports where the protocols were originally published and to pages on this website that contain additional information about the evaluation of the methods.
Acute Systemic Toxicity
Revised Up-and-Down Procedure for Acute Oral Toxicity Testing
- Protocol for the Revised Up-and-Down Procedure
Published as Appendix B to ICCVAM Peer Review Evaluation Report, NIH Document No. 02-4501, 2001
- More information about the ICCVAM evaluation of the oral up-and-down procedure
In Vitro Cytotoxicity Test Methods for Estimating Starting Doses for Acute Oral Systemic Toxicity Tests
- Protocol for the BALB/c 3T3 NRU Cytotoxicity Test Method
- Protocol for the NHK NRU Cytotoxicity Test Method
Published as Appendix C to ICCVAM Test Method Evaluation Report, NIH Document No. 07-4519, 2006
- More information about the NICEATM validation study of in vitro cytotoxicity test methods
Protocols for the BG1Luc Estrogen Receptor Transactivation Assays
- BG1Luc ER TA — Agonist Protocol
- BG1Luc ER TA — Antagonist Protocol
- More information about the ICCVAM evaluation of the BG1Luc ER TA assay
Note: Beginning in 2016, the BG1Luc ER TA assay will be referred to as the VM7Luc ER TA assay. This reflects new information regarding the identity of the cell line used in the assay. Refer to the webpage summarizing the assay validation for more details.
Eye Corrosion and Irritation
In Vitro Test Methods for Assessing Potential to Cause Chemically Induced Eye Injuries
The most recent ICCVAM evaluation of these test methods was published in the ICCVAM Test Method Evaluation Report, NIH Document No. 10-7553, 2010.
- Protocol for the Bovine Corneal Opacity and Permeability Test Method
- Protocol for the Cytosensor Microphysiometer Test Method
- Protocol for the Hen's Egg Test — Chorioallantoic Membrane Test Method
- Protocol for the Isolated Chicken Eye Test Method
- Protocol for the Isolated Rabbit Eye Test Method
- More information about the ICCVAM evaluation of in vitro ocular testing methods
Note: Some of the protocols listed are recommended only for non-regulatory, validation, or optimization studies. Such limitations are noted on the title pages of the relevant protocols.
In Vitro Test Methods Proposed for Assessing Pyrogenicity of Pharmaceuticals and Other Products
Protocols were published as Appendix C to ICCVAM Test Method Evaluation Report, NIH Document No. 08-6392.
- Protocol for the Human Whole Blood/Interleukin-1β Test Method
- Protocol for the Human Whole Blood/Interleukin-1β Test Method: Application of Cryopreserved Human Whole Blood
- Protocol for the Human Whole Blood/Interleukin-6 Test Method
- Protocol for the Human Peripheral Blood Mononuclear Cell/Interleukin-6 Test Method
- Protocol for the Mono Mac 6/Interleukin-6 Test Method
- More information about the ICCVAM evaluation of in vitro pyrogen testing methods
Note: Availability of the Mono Mac 6 cell line for commercial testing purposes may be limited. Go to the in vitro pyrogen test method evaluation page for details.
In Vitro Test Methods for Identifying Potential Skin Sensitizers
- KeratinoSens assay (OECD Test Guideline 442D)
- Direct peptide reactivity assay (DPRA: OECD Test Guideline 442C)
These two assays were evaluated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). In a 2014 statement, ICCVAM concurred with EURL ECVAM that the methods can be used as part of an integrated testing strategy to identify potential skin sensitizers, but emphasized that neither method should be used as a standalone method for the identification of potential skin sensitizers or to determine skin sensitization potency categories. Test guidelines describing the methods were accepted in 2015 by the Organisation for Economic Co-operation and Development (OECD).
Updated Protocol for the Murine Local Lymph Node Assay (LLNA) for Assessing Allergic Contact Dermatitis Hazard Potential
This protocol is also recommended for use with the reduced LLNA. Learn more about the ICCVAM evaluation of the reduced LLNA.
- Updated Protocol for the Murine Local Lymph Node Assay (LLNA)
Published as Appendix A to ICCVAM-recommended Performance Standards for the LLNA, NIH Document No. 09-7357, 2009
- More information about development of performance standards for the LLNA
Nonradiolabeled Murine Local Lymph Node Assay (LLNA) Methods for Assessing Allergic Contact Dermatitis Hazard Potential
- Protocol for the LLNA: DA
Published as Appendix B to ICCVAM Test Method Evaluation Report, NIH Document No. 10-7551, 2010
- Protocol for the LLNA: BrdU-ELISA
Published as Appendix B to ICCVAM Test Method Evaluation Report, NIH Document No. 10-7552, 2010
- More information about the ICCVAM evaluation of nonradiolabeled LLNA methods
Note: In guidance issued in February 2020, FDA stated that it no longer recommends that sponsors conduct the murine local lymph node assay to assess the sensitization potential of topical drug products due to the limitations of the assay. As an alternative to accepted guinea pig tests, FDA will consider a battery of in silico, in chemico, and in vitro studies that have been shown to adequately predict human skin sensitization with an accuracy similar to existing in vivo methods.