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Expert Panel Reviews New Local Lymph Node Assay for Product Testing

By Debbie McCarley
June 2009

Wind, Stokes and Luster
Stokes, center, makes a point about the tests, while Wind, left, looks on and Luster, right, makes notes. (Photo courtesy of Ernie Branson and NIH)

Front row, left to right - Lovell, Luster, Wind, Olson, Jirova, and Stokes. Second row, left to right: Richmond, Api, Ullrich, Flournoy and Theran. Top row - Yoshida, Regal, Maibach, and Alepee.
Panel participants gathered in the NIH Natcher Auditorium. Front row, left to right - Lovell, Luster, Wind, Olson, Jirova, and Stokes. Second row, left to right: Richmond, Api, Ullrich, Flournoy and Theran. Top row - Yoshida, Regal, Maibach, and Alepee. Woolheiser was not pictured. (Photo courtesy of Ernie Branson and NIH)

An international scientific peer review panel of 15 expert scientists met on April 28-29 on the NIH Bethesda campus to review new test methods proposed to determine if chemicals and substances may cause allergic contact dermatitis. The panel was convened by the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) (http://iccvam.niehs.nih.gov/about/about_NICEATM.htm) and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). (http://iccvam.niehs.nih.gov/about/about_ICCVAM.htm)

The panel reviewed three new versions of the murine local lymph node assay (LLNA) and proposals to use the LLNA to test pesticide formulations and other products. Federal agencies accepted the LLNA in 1999 for safety testing, based on ICCVAM recommendations that the LLNA was a valid substitute for the traditional guinea pig test methods used to identify potential sensitizers. The LLNA has many advantages, including using fewer animals, eliminating the potential discomfort that can occur with positive results in the guinea pig methods and providing dose-response information. The LLNA can be completed in one week, as compared to four weeks for the guinea pig test.

Unlike the original LLNA, the three new versions use nonradioactive markers to measure lymphocyte proliferation. One of them measures increases in ATP content, while the other two measure the incorporation of bromodeoxyuridine (BrdU) by either ELISA or flow cytometry. Two of the methods were developed in Japan, while the BrdU-ELISA method was developed through an NIEHS Small Business Innovation Research grant with MB Research Laboratories in Pennsylvania.

The panel evaluated the validation status of each of the proposed alternative test methods and applications according to established federal and international criteria. (http://iccvam.niehs.nih.gov/docs/about_docs/validate.pdf) The panel also commented on draft ICCVAM recommendations regarding the usefulness and limitations of each proposed test method and application.

The Panel agreed that two of the three nonradioactive LLNA procedures reviewed could be used to identify substances as potential skin sensitizers or nonsensitizers, with certain limitations. The Panel supported the third method, contingent upon demonstration of adequate interlaboratory reproducibility in at least two other labs and a laboratory data audit to verify the summary data submitted for review. The Panel also concluded that the LLNA should be able to test any material, including pesticides and substances such as fragrances and dyes, unless the substance is thought to have properties that would interfere with the conduct or accuracy of the assay.

Acceptance by federal agencies of a nonradioactive version of the LLNA that can be applied to a broader range of chemicals and products should permit more widespread use of the LLNA, further reduce the number of animals needed and reduce animal discomfort associated with the guinea pig tests. The avoidance of radioactive materials will also provide a benefit to the environment. ICCVAM, which has members from 15 federal agencies, coordinates the technical evaluations of new, revised and alternative methods for regulatory safety testing of chemicals and products. ICCVAM will consider the Panel's report, (http://iccvam.niehs.nih.gov/) along with comments from the public and its scientific advisory committee in preparing final test method recommendations that will be forwarded to federal agencies later this year.

The goal of NICEATM and ICCVAM is to achieve the regulatory acceptance of alternative test methods that will benefit animal welfare by reducing, refining and replacing animal use - the 3Rs of alternative testing - and benefit public health by ensuring continued or improved protection of human and animal health and the environment.

(Debbie McCarley is the Special Assistant to Rear Admiral William Stokes, D.V.M., D.A.C.L.A.M., director of the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods.)

Key Figures in the Interagency Expert Panel Review

An ICCVAM Immunotoxicity Working Group (IWG) developed draft LLNA recommendations and draft Background Review Documents for review by the Panel with support from NICEATM. Abby Jacobs, Ph.D., of the Food and Drug Administration and Joanna Matheson, Ph.D., of the Consumer Product Safety Commission co-chaired the IWG.

The IWG includes scientists from the Consumer Product Safety Commission, the U.S. Environmental Protection Agency, the Food and Drug Administration, the National Institute for Occupational Safety and Health and NIEHS. NIEHS scientists Dori Germolec, Ph.D., William Stokes, D.V.M., and Raymond Tice, Ph.D., participated on the IWG. Stokes is director of NICEATM and executive director of ICCVAM. Marilyn Wind, Ph.D., of the Consumer Product Safety Commission, is the chair of ICCVAM.

The panel was chaired by Michael Luster, Ph.D., who recently retired from the National Institute for Occupational Safety and Health after serving for many years as a branch chief. The panel included scientists with expertise in immunotoxicology, clinical immunology, biostatistics, regulatory toxicology, occupational health and animal welfare science.

Former NIEHS postdocs David Lovell, Ph.D., a biostatistician in the Medical School at the University of Surrey in the U.K., and Michael Olson, Ph.D., director of Occupational Toxicology for GlaxoSmithKline, served on the panel, as did Takahiko Yoshida, M.D., Ph.D., a Professor at the Asahikawa Medical College in Japan and a former guest researcher at NIEHS.

Additional panelists included Nathalie Alépéé, Ph.D., of L'Oreal; Anne Marie Api, Ph.D., of the Research Institute for Fragrance Materials; Nancy Flournoy, Ph.D., of the University of Missouri - Columbia; Dagmar Jírová, M.D., Ph.D., of the Czech Republic National Institute of Public Health: Howard Maibach, M.D., of the University of California, San Francisco; Jean Regal, Ph.D., of the University of Minnesota - Duluth Campus Medical School; Jon Richmond, M.B. Ch.B., of the U.K. Home Office; Peter Theran, V.M.D., of the Masachusetts Society for the Prevention of Cruelty to Animals; Stephen Ullrich, Ph.D., of the University of Texas M.D. Anderson Cancer Center; and Michael Woolhiser, Ph.D., Dow Chemical Company.



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