It has been shown that exposure of mice to contact allergens induces B cell activation in the draining lymph nodes (DLN), as seen by an increase in the percentage of B220+ or IgG/IgM+ cells. We have now examined whether the measurement of the percentage of B220+ cells could be used as an alternative or supplementary endpoint for the local lymph node assay (LLNA) to differentiate between allergenic responses and those few irritants that induce low-level proliferation in the DLN. Mice were treated on the ears, daily for 3 consecutive days, with various allergens (1-chloro-2,4-dinitrobenzene, alpha-hexylcinnamaldehyde, trinitrochlorobenzene, isoeugenol, and eugenol) or irritants (benzalkonium chloride, methyl salicylate, salicylic acid, and sodium lauryl sulfate). The DLN were excised 72 h following the final topical treatment, and the cells were prepared for B220 analysis using flow cytometry. The percentage of B220+ cells in lymph nodes derived from test and vehicle-treated animals was determined for 5 allergens and 4 irritants tested in multiple experiments (n = 3 to 17). As expected, the percentage of B220+ B cells was increased with each of the allergens tested, whereas irritant treatment did not cause similar increases. Moreover, the method was reproducible. For example, the strong allergen, 1-chloro-2,4-dinitrobenzene and the weak allergen, alpha-hexylcinnamaldehyde were identified as allergens in 17 of 17 and in 12 of 13 experiments, respectively. The percentage of B220 values for each chemical treatment (41 observations for allergens; 28 observations for irritants) versus the percentage of B220 values for the concurrent vehicle controls were plotted, and a classification tree model was developed that defined a B220 test:vehicle ratio cutoff of 1.25 for discriminating between allergens (>1.25) and irritants (<1.25). Using this B220 test:vehicle ratio of 1.25 in 93% of the 69 independent observations made, the allergens and irritants tested were identified correctly. Finally, to evaluate the performance of this model in a second independent laboratory, 3 allergens and 2 irritants were tested. Each of the allergens and irritants were classified correctly using the B220 test:vehicle ratio cutoff of 1.25. These data demonstrate that analysis of B220 expression in DLN may be useful in differentiating between allergen and irritant responses induced in chemically treated mice.
The murine local lymph node assay (LLNA) is a predictive test for the identification of chemicals that have the potential to cause skin sensitization. Since its original development, the assay has been the subject of national and international evaluation studies and extensive comparisons with guinea pig tests and human data. On the basis of these investigations, the LLNA has recently been endorsed by ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) as a stand-alone method for skin sensitization hazard identification. At the same time, ICCVAM confirmed that, although the LLNA is not an in vitro method, it does represent a refinement in the way animals are used and can provide a means for reducing the number of animals used in sensitization hazard assessment. The investigations described here were designed to explore further the ability of the LLNA to identify accurately those chemicals that cause allergic contact dermatitis in humans. To that end we have measured, amongst 3 independent laboratories, LLNA responses induced by a total of 18 test chemicals, 11 of which are known to cause skin sensitization and 7 of which are believed not to be associated with any significant evidence of allergic contact dermatitis in humans. The LLNA correctly classified 16 of the 18 materials. The 11 chemicals tested which are associated with allergic contact dermatitis in humans were found to be positive in the LLNA. Of the 7 materials believed to be non-sensitizers, 5 were negative in the LLNA and 2 produced positive results. Collectively, these data provide additional evidence that the LLNA is able to discriminate skin sensitizers from those chemicals which do not possess a significant skin sensitization potential and thus provides a method for hazard identification that offers important animal welfare benefits.
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