2009 NICEATM-ICCVAM Activity: Test Method Validation and Regulatory Acceptance in the US

To facilitate the replacement of existing toxicity tests with new ones, national and international authorities have developed processes and criteria for evaluating new test methods. The major steps for partially or fully replacing an existing regulatory test with a new method are:

• Test method development
• Protocol optimization
• Multi-laboratory validation studies
• Peer review / independent assessment
• Regulatory acceptance consideration (national and international levels)

National validation centers prefer to liaise with test method developers during all phases of development and validation. Once adequate validation studies have been completed for a new or revised method, it can be submitted to one of the validation centers for an assessment of its scientific validity, including its demonstrated usefulness and limitations for the specific proposed use. In addition to submission from an external organization, the centers are often involved in a variety of ways in the validation process from test development to peer review/assessment, although the details of their processes may vary.

This article provides a summary of 2009 activities at the US-based National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods-Interagency Coordinating Committee on the Validation of Alternative Methods (NICEATM-ICCVAM). It is the second part of our updates on validation and regulatory acceptance activities that took place in 2009 at three national validation centers. The first part covered the Japanese Center for the Validation of Alternative Methods (JaCVAM); the third part will cover the European Centre for the Validation of Alternative Methods (ECVAM).

NICEATM-ICCVAM 2009

Table 1 summarizes NICEATM and ICCVAM activities related to the validation of new or revised toxicity test methods that were ongoing or completed in 2009. Table 2 summarizes regulatory acceptance activities in 2009 related to test methods that have been endorsed by the ICCVAM.

Dr. William Stokes has been the Director of NICEATM since its establishment in 1998. NICEATM, located in Research Triangle Park, NC, US, is the organization that provides support for the ICCVAM. NICEATM's activities include evaluating new test method submissions and nominations for completeness and adherence to ICCVAM guidelines, and determining the suitability of a test method for ICCVAM evaluation of its validation status. NICEATM has recently started to conduct validation studies. The ICCVAM is comprised of representatives from 15 US Federal agencies who participate in the decision-making regarding "which assays are ready for the validation process," and make recommendations on test methods to US Federal agencies following a formal peer review process.

NICEATM continued in 2009 to provide stakeholder access to their activities and to documents related to methods under NICEATM-ICCVAM review on the NICEATM-ICCVAM website. The expanded compilation of validation and regulatory acceptance information into tables facilitates understanding and access.

In 2009, NICEATM-ICCVAM conducted two public meetings of independent scientific peer review panels to assess the validation status of alternative test methods. The first meeting, convened in April 2009 (Updated Evaluation of New Versions and Applications of the Murine Local Lymph Node Assay on alternative test methods for skin sensitization testing. The second meeting, convened in May 2009 (Alternative Ocular Safety Testing Methods focused on alternative tests methods for eye irritation testing. ICCVAM has considered the conclusions and recommendations from these international independent experts and the public comments provided during this evaluation in developing final test method recommendations that will be forwarded to Federal agencies in 2010.

The murine local lymph node assay (LLNA) is a test method to detect potential skin sensitizing substances. ICCVAM forwarded recommendations for: (1) the reduced LLNA (rLLNA), (2) an updated LLNA test method protocol, and (3) LLNA performance standards to Federal agencies in September 2009. Using the rLLNA will reduce animal use by 40% compared to the traditional multidose LLNA. ICCVAM is also recommending performance standards that can be used to efficiently evaluate the validity of improved and modified versions of the LLNA that are mechanistically and functionally similar to the traditional LLNA. The updated LLNA test method protocol reduces animal use by 20% compared to the original LLNA.

NICEATM and ICCVAM continued to work on various international cooperative efforts such as participating in international validation studies and contributing to the international acceptance of validated methods. NICEATM-ICCVAM is participating in international validation efforts for in vitro assays that evaluate potential endocrine disruptor activity. A NICEATM-coordinated international validation study of the LUMI-CELL^® ER assay (an in vitro estrogen receptor transcriptional activation assay) is currently nearing completion. The study includes three laboratories sponsored by NICEATM, ECVAM, and JaCVAM. This is the first joint validation study sponsored by all three of these validation organizations. An international independent scientific peer review of the LUMI-CELL^® ER assay is planned for June 2010.

In April 2009, an ICCVAM initiative fostered an international agreement between the United States, Canada, Japan, and the European Union that is expected to further reduce animal use in product toxicity testing worldwide by working toward harmonization of critical components of the validation process. This agreement, the International Cooperation on Alternative Test Methods (ICATM), promotes enhanced international cooperation and coordination on the scientific validation of non- and reduced-animal toxicity testing methods. These efforts are expected to improve the efficiency of validation and regulatory acceptance of new test methods.

After the BCOP and ICE test methods for identifying severe/corrosive eye irritants were accepted last year by US Federal agencies, NICEATM and ICCVAM drafted test guidelines in collaboration with validation organizations in Europe and Japan for submission to the Organisation for Economic Cooperation and Development (OECD). The new test guidelines, which have been designated Test Guideline (TG) 437 (BCOP) and TG 438 (ICE), were formally adopted by the OECD Council in September. This represents the most rapid adoption of any new TG by the OECD Test Guidelines Programme. The prompt acceptance was due in large part to the comprehensive NICEATM-ICCVAM evaluation of the validation status of the test methods and the involvement of international validation partners in the development of the TGs.

The OECD convened an expert consultation meeting on October 20-22 to evaluate modifications of the LLNA. This meeting was hosted in Bethesda, Maryland by the Consumer Products Safety Commission (CPSC), NICEATM, and ICCVAM. The expert group reviewed proposed revisions to the OECD test guideline for the LLNA, TG 429. The proposed revisions provide multiple mechanisms for reducing the number of animals in each test by up to 40%. They also provide a standardized approach and improved guidance on establishing the highest test dose, and performance standards that can be used to expedite the validation of modified versions of the LLNA. All of the proposed revisions are based on the recommendations recently forwarded to US Federal agencies by ICCVAM.

The expert group also reviewed drafts of two new proposed test guidelines for nonradioisotopic versions of the LLNA. These test methods, the LLNA: DA (which measures ATP content) and LLNA: BrdU-ELISA (which measures BrdU incorporation), do not require radioisotopes to measure lymphocyte proliferation, which is a requirement in the traditional LLNA. The availability of OECD TGs for these test methods will enable them to be used where use of radioisotopes is not permitted. The draft TGs are also based on an ICCVAM evaluation of the LLNA: DA and LLNA: BrdU-ELISA test methods. ICCVAM recommendations on the usefulness and limitations of these test methods are currently being finalized and will be forwarded to US Federal agencies in early 2010.

Other NICEATM-ICCVAM activities included the development of a plan for implementing the NICEATM-ICCVAM Five-year Plan, which was presented at the June 2009 meeting of ICCVAM's Scientific Advisory Committee on Alternative Toxicological Methods (SACATM). The Five-year Plan, developed in 2008, describes priority areas determined in collaboration with relevant US agencies for research, development, and validation of new test methods that will support regulatory agency needs. The highest priority areas are test methods for biologics/vaccines, ocular toxicity, dermal toxicity, and acute toxicity.

NICEATM and ICCVAM continue to support the translation of key research advances into new test methods. To this end, ICCVAM established a Research and Development Working Group in 2009 to help ICCVAM and NICEATM promote research incorporating new technologies expected to support future test method development. The Research and Development Working Group includes scientists from ICCVAM member agencies who know about the scope and range of research and development activities within their agencies that are relevant to alternative test methods. ICCVAM and NICEATM are also monitoring the progress of a collaboration between the NTP, the Environmental Protection Agency (EPA), and the National Institutes of Health (NIH) Center for Chemical Genomics to develop high-throughput technologies for toxicological testing. NICEATM-ICCVAM has nominated nearly 1000 commercially-available reference substances for use in this effort.

NICEATM staff and ICCVAM members presented four posters at the 2009 Annual Meeting of the Society of Toxicology in Baltimore, MD. These presentations described ICCVAM-recommended performance standards for the LLNA, ICCVAM test method recommendations for the rLLNA and an updated LLNA test method protocol, establishment of laboratory historical databases for the international validation study of the LUMI-CELL^® test method, and the NICEATM-ICCVAM International Scientific Workshop on Acute Chemical Safety Testing held in February 2008.

NICEATM and ICCVAM scientists presented highlights of their progress at the 2009 7^th World Congress on Alternatives and Animal Use in the Life Sciences. Four plenary sessions addressed the recently signed agreement on ICATM; a session on recent progress and future directions of the national validation centers in the US (NICEATM-ICCVAM), Europe (ECVAM), and Japan (JaCVAM); a session on evolving concepts, opportunities, and challenges in validation; and a session providing an update on the recent acceptance of in vitro pyrogen tests in the US and Europe.

NICEATM and ICCVAM scientists also presented eight posters highlighting recent progress. These presentations, summarized test method evaluation activities relevant to skin and eye corrosivity and irritation, skin sensitization, pyrogenicity, and endocrine disruptors. These posters also highlighted international cooperative activities, including the development of internationally harmonized test method performance standards, international validation studies, and facilitating international regulatory acceptance of in vitro alternatives.

Several key activities coming up in 2010 include: 1) coordinating an inter-laboratory validation study on the MCF-7 Cell Proliferation Assay, and 2) holding a NICEATM-ICCVAM Workshop on "Alternative Methods for Vaccine Potency and Safety Testing" in September 2010.

Table 1. Toxicity test methods in some stage of assessment by NICEATM-ICCVAM during 2009.

Table 2. Regulatory acceptance activities during 2009 for test methods endorsed by ICCVAM.

Acknowledgements

We would like to thank the NICEATM staff for providing feedback on the accuracy and completeness of the information provided in this article.