Mouse local lymph node assay

The only in vitro studies fully validated and included in the EU test methods (a.2) are for skin corrosivity and phototoxicity, but efforts are underway to speed up validation for other endpoints. An original aim was that there would be no animal studies for the basic level of registration, but as there is currently no suitable in vitro test for skin sensitisation, the mouse local lymph node assay is included in Annex V of the Regulation (Table 9). The in vitro tests for skin and eye irritation are not fully validated, so as a compromise they are accepted for registration at up to 10 tonnes per annum (i.e., they are in Annex V), but if the results are negative they have to be confirmed by a standard animal test at 10 tonnes per annum (i.e., the Annex VI data, as shown in Table 10). 

FIGURE 15.7. The mouse local lymph node assay. 

FIGURE 15.8. Mouse local lymph node assay (LLNA) (ICVAM protocol). Modification using flow cytometry instead of radiolabeling is preferable. 

Sensitization can be evaluated in the well-known delayed contact hypersensitivity assay or the mouse local lymph node assay. 

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. 

Dose selection for the mouse IgE test is an important issue. In practice the policy currently is to examine test chemicals first in the local lymph node assay, a mouse predictive test for the identification of contact allergens (Kimber and Basketter, 1992 Kimber et al., 1994). Wherever possible application concentrations are selected for use in the mouse IgE test that elicit a positive... 

While the use of IgGl as an immunogenicity endpoint to attempt to predict the relative allergenicity of enzymes/proteins is open to criticism as mechanistically irrelevant, the true value of the method will lie in its performance relative to other preclinical test methods and human sensitization patterns. Many newly emerging methods in immunoloxicology rely on surrogate endpoints or endpoints that measure only a component of the response process (e.g. skin equivalent cultures for skin and eye irritation, local lymph node assay for contact sensitization, mouse IgE test for chemical respiratory sensitization). Their value lies in their... 

In the local lymph node assay, a threefold increase in the proliferation of auricular lymph node cells was observed in mice treated topically with the compound bixin (1-25% w/v) at concentrations of 5 to 25%. In the mouse ear swelling test, a significant increase in percent ear swelling was observed in animals treated with 5 to 10% bixin. Bixin was classified as a nonirritant at concentrations of 1 to 25% (w/v) but was considered a contact sensitizer. In these same tests, the compound norbixin (1-20% w/v) showed no irritation or contact sensitization (Auttachoat et al. 2005). 

Several in vivo methods exist and are used as tools to determine the potential for contact, and potentially respiratory, hypersensitivity (e.g., the local lymph node (LENA) (Kimber et al., 2002) and popliteal lymph node (PLNA) (Pieters, 2001) assays) and the mouse drug allergy model, which is a modification of the LENA, demonstrates promise as a screening tool in that a number of drugs which cause idiosyncratic hypersensitivity reactions in humans were positive in this assay (Whritenour et al., 2014 Zhu et al., 2014). However, there are no fully predictive animal models of drug allergy in humans. 

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