Local lymph node assay with non-radioisotope alternative endpoints.

Abstract

-The local lymph node assay has recently been accepted by regulatory agencies as a standalone alternate method for predicting allergic contact dermatitis. To compare the sensitivity of non-radioisotope methods with that of the standard assay, we determined if these modified methods would affect evaluation of sensitization potency. For this reason, we used 2,4-dinitrochlorobenzene (DNCB) and benzocaine for different sensitizing criteria.Female CBA mice were treated for 3 days with a test compound or vehicle applied to each side of both ears. Bilateral auricular lymph node proliferative activity was assessed by the following endpoints with incorporation of 3 H-methyl thymidine (3H-TdR), bromodeoxyuridine (BrdU) in vivo, and BrdU ex vivo, IL-2 production, and proliferating cell nuclear antigen (PCNA) expression. Ear thickness was also tested.The strong sensitizer DNCB was detectable by any of the non-radioisotope endpoints as well as by radioisotope-dependent standard assay. On the other hand, when evaluating the weak sensitizer benzocaine, significant changes were evident in BrdU incorporation ex vivo and in vivo, and IL-2 production. We believe that these non-radioisotope methods can assess allergic contact dermatitis caused by chemicals even in the laboratory, where it can be difficult to handle radioisotopes.

Evaluation of auricular lymph node cell lymphocyte proliferation and cytokine production as non-radioactive endpoints during murine contact allergy

“…In our study, since DI values were > 1 for all concentrations, DNCB is an allergic compound. These results are compatible with those of Suda et al (2002).…”

“…Flow cytometric analysis and immunohistochemical assays have been developed to count BrdU incorporation into lymph node cells (Boussiquet-Leroux et al, 1995;Lee et al, 2002, Suda et al, 2002. Takeyoshi et al (2001) investigated BrdU incorporation into lymph node cells by using a cell proliferation ELISA kit but their results for a number of allergens suggested an insufficient sensitivity because the increase in lymph node cell counts was not taken into account when calculating SI values (Takeyoshi et al, 2006).…”

Assessment of the sensitizing potential of textile disperse dyes and some of their metabolites by the loose-fit coculture-based sensitization assay (LCSA)

“…Several modiWcations of the LLNA have been suggested over the past years to improve its reliability and practicability. Non-radioactive endpoints, such as lymph node weight, cell proliferation and ear swelling, have been described by several authors (Ehling et al 2005a, b;Suda et al 2002;for review: Anderson et al 2011). Two versions of the LLNA with non-radioactive endpoints, the LLNA: DA and the BrdU-ELISA-version, are currently validated and available as draft OECD guidelines (Omori et al 2008;Kojima et al 2011).…”

Evaluation of non-radioactive endpoints ofex vivolocal lymph node assay-BrdU to investigate select contact sensitizers

“…The use of this method is difficult in any laboratory because such a radioisotope (RI)-based method requires special facilities and handling procedures. Several authors have conducted investigations into the development of alternative non-RI methods for performing LLNA (Takeyoshi et al, 2001(Takeyoshi et al, , 2005(Takeyoshi et al, , 2006Lee et al, 2002;Suda et al, 2002;Ehling et al, 2005a, b;Idehara et al, 2008).…”