Local Lymph Node Assay LLNA

PURPOSE AND RATIONALE LLNA consists of a topical induction followed by measurement of the mitotic activity of the draining lymph node. It involves application of multiple topical in vivo doses of the material of interest to mouse ear, followed by in vitro evaluation of mitotic activity of cells from the draining lymph nodes. 

Experimental animals of either sex, 8-12 weeks of age are selected. Each individual assay should use a single sex only. At least 3 dose levels are evaluated in separate groups of diluents include 4 1 acetone/olive, methyl ethyl ketone, dimethylformamide, propylene glycol, dimethyl sulphoxide, and 2.5% hydroxypropyl cellulose in methanol. Three consecutive concentrations from the series 100 %, 50 %, 25 %, 10 %, 5 %, 2.5 %, 1.0%, 0.5%, 0.25%, 0.1%, 0.05% and 0.001% are tested. 

Standardized validating predictive assays in mice have been developed in the last 10 years. LLNA as one of such assays offers advantages in the low number of animals used, lower cost, and less time required for conducting the assay in comparison to various guinea pig assays. 

CRITICAL ASSESSMENT OF THE METHOD LLNA is unique of all predictive assays in only evaluating the response of the efferent phase of the response. 

National Toxicology Program (1999) The murine local lymph node assay A test method for assessing the allergic contact dermatitis potential of chemicals/compounds. National Institutes of Health Publication 99 1494. Research Triangle Park, NC 

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The most important advance in ACD testing has been the development of the murine local lymph node assay (LLNA). Although the general concept originated in the 1960s,... 

Finally, multiparameter flow cytometry has the potential to improve existing methodologies for the evaluation of immunotoxicological endpoints such as the natural killer cell (NK) assay and the local lymph node assay (LLNA) to evaluate innate immunity and chemical-induced allergic disease. Accepted methodologies typically utilize radio-... 

NIEHS, Interagency Coordinating Committee for Validation of Alternative Methods (IC-CVAM) Peer Review Panel Evaluation of the Murine Local Lymph Node Assay (LLNA) Report, 1999. 

There are currently three OECD test guideline methods for test of skin sensitization in animals. These include the Guinea-Pig Maximization Test (GPMT), the Buehler test, and the murine Local Lymph Node Assay (LLNA). 

This test uses an in vitro human dendritic cell culture to obtain information of the potential for various chemicals to induce allergic contact dermatitis. This test is used as an alternative to the Local Lymph node assay (LLNA) to minimize or replace the use of live animal testing for predicting skin sensitization (Kimber et al. 2002, see below). The test allows for evaluation of skin sensitization by examining the presence of cell surface markers on Periperal Blood Mononuclear Cell (PBMC)-derived dendritic cells (DC) that are known to be involved in the development of allergic contact dermatitis. 

Recently, a more economical, less subjective test for contact hypersensitivity has been developed using mice. This test, the local lymph node assay (LLNA), assesses the proliferative response of lymphocytes in the draining lymph node following appli-... 

In addition to the above testing protocols, the local lymph node assay (LLNA) has been validated and accepted to assess the skin sensitization potential of chemicals in animals. This does not replace the guinea pig maximization test but is considered to be an equivalent. This in vivo method helps to reduce the number of animals used for contact sensitization activity. This test is based on the principle that sensitizer can induce a primary proliferation of lymphocytes in the lymph node draining the site of chemical application. This provides a quantitative measurement in which the proliferation is proportional to the dose applied. 

Piperazine was evaluated in the local lymph node assay (LLNA) and the guinea pig maximization test. In both studies, piperazine was a mild sensitizer. In the LLNA, piperazine did not induce markers indicative of respiratory sensitization. In laboratory studies, piperazine has exhibited cross-sensitization reactions with diethylenetriamine. 

Rovida C, Ryan C, Cinelli S, Basketter D, Dearman R, Kimber I (2012) The local lymph node assay (LLNA) (Chapter 20). Curr Protoc Toxicol (Suppl. 51) 20.7.1-20.7.14... 

Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama M, Itagaki H (2010) A comparative evaluation of in vitro skin sensitisation tests the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). ATLA 38 275-284... 

Up to now, pulmonary sensitization cannot be examined in animal tests. For the examination of dermal sensitization, different test methods with different sensitiveness are available. For the examination of industrial chemicals, the so-caUed patch test applied to guinea pigs is usually sufficiently sensitive. In this, the test substance is administered directly to the skin, as in the human exposure situation. The maximization test, also called the Magnusson-Kligmann test (see Fig. 2.7) and developed originally for cosmetics and pharmaceuticals, is nowadays more and more used for chemicals also. A recently developed test method is the local lymph node assay (LLNA), which can be used to examine the potency of an allergen. 

In recent years, the mouse local lymph node assay (LLNA) has been approved as an alternative to the guinea pig test. It is based on the proliferation of lymphocytes (a type of immune cell) in lymph nodes draining the site of contact with the chemical. In the LLNA, the substance is applied to the mouse s ear on days 1-3 of the test but withheld on days 4 and 5 to give the immune system a chance to respond. On day 6, the mouse is injected in its tail vein with a small amount of a radioactive DNA base such as H-thymidine (tritiated thymidine) to label newly formed immune cells. The mouse is sacrificed, its auricular lymph nodes (located near the ears) are cut out, and their radioactivity is measured. Increased radioactivity in the lymph nodes of treated animals compared with controls indicates that the test chemical sensitizes the immune system and can cause contact dermatitis. The LLNA is more objective than the traditional guinea pig test because the amount of immune cell proliferation can be determined as a function of dose. 

The third commonly used sensitization test method is called the mouse local lymph node assay (LLNA). The LLNA is an acceptable test method, but device manufacturers have been using the guinea pig tests for decades and are satisfied with the results obtained using those methods. The LLNA, like any test, has its pros and cons. On the positive side, it is shorter in duration, uses animals of a lower phylum, and requires far less test material. However, it also generates radioactive waste and is not accurate in distinguishing a sensitization response from an irritation response, which increases the possibility of a false-positive outcome. 

Immunotoxicity/skin Local lymph node assay (LLNA) In vivo... 

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