December 2001 Local Lymph Node Assay: Background Documents
November 26, 2001
FIFRA SCIENTIFIC ADVISORY PANEL (SAP)
December 11, 2001
FIFRA SAP WEB SITE: http://www.epa.gov/scipoly/sap/
OPP Docket Telephone: (703) 305-5805
The murine Local Lymph Node Assay (LLNA) is a test method for assessing the allergic contact dermatitis (skin sensitization) potential of chemicals and compounds. In 1998, upon review by the EPA FIFRA Science Advisory Panel (SAP), the LLNA was incorporated as a screen in OPPTS Test Guideline 870.2600 Skin Sensitization. Materials testing positive in the LLNA would be considered to be skin sensitizers, while those that were negative would need additional sensitization testing in the guinea pig. Significant work has been conducted to determine the correlation between LLNA test outcomes and test outcomes in the guinea pig test and in humans. An evaluation of the relevance and reproducibility of the LLNA was conducted by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The method was found to be valid as a stand-alone test for skin sensitization (i.e. for appropriate testing situations, a negative as well as a positive response could be accepted without the need for additional testing in the guinea pig). The Organization of Economic Cooperation and Development (OECD) has prepared and accepted a LLNA test guideline which is anticipated to appear in final form in approximately the next nine months. A workshop was held in Washington, DC by ICCVAM and the International Life Sciences Institute, Health and Environmental Sciences Institute (ICCVAM/ILSI-HESI) in January 2001 to inform scientists about the conduct and evaluation of the test. The workshop provided a forum to address scientific questions, issues or differences with regard to the LLNA. Scientific differences of concern to the EPA were resolved and have been addressed in the revised OPPTS Test Guideline for skin sensitization incorporating the LLNA as a stand-alone method. The OPPTS revised test guideline is fully harmonized with that accepted by OECD.
General Description of the LLNA
Test substance is applied to the dorsum of the mouse ear, and lymphocyte proliferation, as measured by in vivo radioisotope incorporation, is assessed in the draining auricular lymph node proximal to the site of application. The assay is based on the principle that skin sensitizers induce lymphocyte cellular proliferation as part of the induction phase and that, generally, the degree of proliferation is proportional to the dose applied. In contrast, the guinea pig tests measure the development of cutaneous dermatitis during the elicitation phase of a hypersensitization response.
ICCVAM Peer Review
ICCVAM is a standing committee of the federal government, established through the National Institute of Environmental Health Sciences and includes representation from 15 research and regulatory agencies, including EPA. The Committee facilitates the development, validation, and regulatory acceptance of new test methods that aid the ability to perform risk assessments and, where possible, reduce, refine and replace the use of animals in toxicological testing. ICCVAM has developed general criteria and a process for validation of test methods which includes considerations for their regulatory applicability (ICCVAM, 1997). In addition, ICCVAM has developed guidance for the submission of new test methods to ICCVAM for review (ICCVAM, 1999a).
Scientists representing three chemical companies (Procter and Gamble, Unilever, and Zeneca) submitted the LLNA to ICCVAM in 1997. An ICCVAM Working Group, composed of federal employees from various member agencies, interacted with the test sponsors to prepare the final submission package for peer review. An independent peer review panel assembled through ICCVAM deliberated a full day in a public meeting on September 17, 1998. The peer review panel was charged to evaluate the usefulness and limitations of the LLNA for assessing allergic contact dermatitis and with reaching conclusions and recommendations on each section of the LLNA test method submission package concerning the following:
(1) has the LLNA been evaluated sufficiently and is LLNA performance for assessing allergic contact dermatitis potential satisfactory to support its adoption as a stand-alone alternative to two methods currently accepted by regulatory authorities (Guinea Pig Maximization Test and the Buehler Assay); and
(2) does the LLNA offer advantages with respect to animal welfare considerations compared to the existing test methods?
Panel consensus was that LLNA could serve as a valid stand-alone method for the assessment of dermal sensitization potential if certain modifications were incorporated into the sponsors' original protocol. In addition, the panel concluded that the assay performed at least as well as currently accepted guinea pig methods (Guinea Pig Maximization Test and the Buehler Assay) for the hazard identification of strong to moderate chemical sensitizing agents and appeared to provide an equivalent prediction of the risk for human allergic contact dermatitis. Various advantages for animal welfare were also identified (e.g. less animal discomfort, shorter test duration, potentially fewer animals). Detailed conclusions, strengths, and possible limitations of the assay as determined by the panel are presented in the peer panel review report (ICCVAM, 1999b). Note, many of these findings are described below.
Subsequently, the ICCVAM Working Group reviewed and endorsed the peer review panel's findings and provided a modified protocol for the assay as an appendix to the peer panel review report. Following endorsement by the main body of ICCVAM, the peer panel report was sent to Federal agencies for their consideration of regulatory acceptance.
EPA Evaluation of the Regulatory Acceptance of LLNA
The EPA is in general agreement with the ICCVAM peer panel report and recommendations: a) the LLNA is considered to be a valid satisfactory alternative to existing tests in the guinea pig in applicable circumstances; and (b) it is the preferred test method for skin sensitization testing, when there are no reservations concerning its use (OSCP, 1999). This is because the LLNA is equivalent to the guinea pig sensitization tests in predicting human allergic contact dermatitis, provides not only qualitative but also quantitative data and an assessment of dose-response, gives consideration to animal welfare concerns, and is suitable for testing colored substances not easily tested in the guinea pig since there is no associated erythematous response.
There are also some limitations to the applicability of the LLNA. When it is not applicable or may provide unreliable or problematic results, the Agency's position is that the Guinea Pig Maximization Test or Buehler Tests are recommended for testing. Such cases include the following:
(a) Metallic compounds (e.g. metals, metal salts and organometallic materials) may produce unreliable results in the LLNA; (b) Runny liquids and wholly aqueous vehicles do not sufficiently adhere to the ear and therefore such test materials should be avoided in the LLNA; (c) Strong dermal irritants may yield false positive results in the LLNA;
The LLNA may have limitations in other circumstances (e.g. some test materials may not be readily absorbed by the skin, such as high molecular weight proteins and some weak skin sensitizers may not be accurately predicted, yielding false negative results). However, the guinea pig assays may have limitations in these areas as well.
The Agency agrees that the modifications to the original LLNA protocol proposed by the ICCVAM peer panel are reasonable. These include: (a) limiting the use of the assay to female CBA mice until a systematic comparison can be made with male mice and with other strains; (b) individually identifying animals; (c) collecting body weight data at the start and end of a study; (d) including a concurrent positive control in each study; (e) adding an isotope with a shorter half-life, 125I-iododeoxyuridine, as a possible alternative to 3H-methyl thymidine with which to perform the assay; (f) collecting individual animal lymphocyte proliferation data; (g) using the results of statistical analyses of the proliferation data, the stimulation index and dose response information as part of the process of identifying a positive response; and (h) adding illustrative materials as needed (although such illustrations can only be referenced in the EPA's harmonized guidelines).
The protocol for the LLNA provided in the revised EPA guideline is essentially the same as that modified by the ICCVAM to reflect the conclusions and recommendations of the independent peer review panel. Other changes have been incorporated to harmonize the test method with that prepared and accepted by OECD. Some changes have been made by EPA to the modified ICCVAM protocol for clarification or consistency with Agency guideline presentation and format.
How the LLNA Fits into OPP and OPPT Evaluations of Skin Sensitization
1. Skin sensitization testing with the LLNA or any other type of assay is not required if the test material is a known skin sensitizer in animals or humans. The test is not usually required for the active ingredients of microbial and biochemical pesticides but may be required in cases of repeated formulated product contact with human skin.
2. If it is suspected that the test material is a strong dermal irritant, skin sensitization testing with the LLNA or any other test method may not be required (see OPPTS 870.1000, Section (d)(2)(iii)).
3. When skin sensitization testing is needed, the LLNA is considered to be a valid stand-alone alternative to the standard guinea pig tests and is the preferred skin sensitization test method in applicable circumstances. However, EPA agrees with the ICCVAM Working Group and the ICCVAM peer review panel that there are certain situations that may necessitate the use of the traditional guinea pig tests.
4. The LLNA is not recommended for all types of test materials as was previously discussed, including but not limited to, metallic compounds, strong dermal irritants, and materials that do not sufficiently adhere to the ear (e.g. aqueous materials). Although the LLNA is not recommended for materials that are not readily absorbed through the skin, such as high molecular weight proteins, it is noted that the guinea pig assays may also have limitations in testing these substances as well.
5. A substance is regarded as a skin sensitizer in the LLNA if at least one concentration of the test material results in a 3-fold or greater increase in 3H-methyl thymidine or 125IU incorporation in the lymph node cells of test group lymph nodes relative to that recorded for solvent/vehicle control lymph nodes, as indicated by the stimulation index (SI). However, the magnitude of the SI should not be the sole factor used in determining the biological significance of a skin sensitization response. A quantitative assessment must be performed by statistical analysis of individual animal data in order to provide a more complete evaluation of the test substance. Factors to be considered in evaluating the biological significance of a response or outcome of the test include the results of the SI determinations, statistical analyses of the data, the strength of the dose-response relationship, chemical toxicity, solubility of the test material, and the consistency of the vehicle and positive control responses.
6. Any test substance determined in a well conducted LLNA to be negative by the analysis criteria noted above is generally not considered to be a skin sensitizer. However, given the complexity of such decisions, the final judgment belongs with the Agency.
7. Any test substance found to be positive in the LLNA will be labeled as a dermal sensitizer.
8. If LLNA test results are deemed to be equivocal, the Agency will err on the side of caution, in the absence of adequate information to the contrary.
ICCVAM. 1997. Validation and regulatory acceptance of toxicological test methods. A report of the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods. NIH publication No. 97-3981. Research Triangle park, NC: National Institute of Environmental Health Sciences. http://iccvam.niehs.nih.gov/docs/docs.htm#general
ICCVAM. 1999a. Evaluation of the Validation Status of Toxicological Methods: General Guidelines for Submissions to ICCVAM. NIH publication No. 99-4496. Research Triangle Park, NC: National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods. http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm
ICCVAM. 1999b. The murine local lymph node assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds. NIH publication No. 99-4494. Research Triangle park, NC: National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods. (http://iccvam.niehs.nih.gov/docs/docs.htm#llna)
OSCP Letter from Steven Galson, Office of Science Coordination and Policy, OPPTS to Kenneth Olden, National Institute of Environmental Health Sciences on the regulatory acceptability of the LLNA, October 12, 1999.LOCAL LYMPH NODE ASSAY: BACKGROUND DOCUMENT