Development of LLNA:DAE: a new local lymph node assay that includes the elicitation phase, discriminates borderline-positive chemicals, and is useful for cross-sensitization testing

Abstract

-We developed a new local lymph node assay (LLNA) that includes the elicitation phase termed LLNA:DAE for discrimination of borderline-positive chemicals as classified by the LLNA modified by Daicel based on ATP content (LLNA:DA) and for cross-sensitization testing. Although the LLNA:DA method could help identify skin sensitizers, some skin irritants classified as non-sensitizers by the LLNA were classified as borderline positive. In addition, the evaluation for the cross-sensitization potential between chemicals was impossible. In the LLNA:DAE procedure, test group of mice received four applications of chemicals on the dorsum of the right ear for induction and one application on the dorsum of the left ear for elicitation. Control group of mice received one chemical application on the dorsum of the left ear. We evaluated the sensitizing potential by comparing the weights of the lymph nodes from the left ears between the two groups. The results of using the LLNA:DAE method to examine 24 chemicals, which contained borderline-positive chemicals, were consistent with those from the LLNA method, except for nickel chloride (NiCl 2 ). Two chemical pairs, 2,4-dinitrochlorobenzene (DNCB) with 2,4-dinitrofluorobenzene (DNFB) and hydroquinone (HQ) with p-benzoquinone (p-BQ), showed clear cross-sensitization with each other, while another chemical pair, DNFB with hexylcinnamic aldehyde (HCA) did not. Taken together, our results suggest that the LLNA:DAE method is useful for discriminating borderline-positive chemicals and for determining chemical cross-sensitization.

Further development of LLNA:DAE method as stand-alone skin-sensitization testing method and applied for evaluation of relative skin-sensitizing potency between chemicals

“…This result was obtained in a mouse treated with 0.06% DNCB. This weight (2.7 mg) was similar to the lymph node weight of naïve mice (Yamashita et al, 2014). Therefore, we concluded that this dose of (0.06% DNCB) was not suitable as the test dose for the primary LLNA:DAE study, due to the possibility of a false negative result from the variability in lymph node weights between mice.…”

“…The test dose selected for the validation study was the dose that gave the highest left ear lymph node weight or the dose that gave a left ear lymph node weight of at least 4 mg. For glycerol and sulfanilamide, the left ear lymph node weights obtained from all dose groups were in the same range as the lymph node weights of naïve mice; therefore, the highest concentrations were selected. The lymph node weights for naïve mice were reported in our previous study (Yamashita et al, 2014). When 1.56% glutaraldehyde, 6.25% propyl gallate, 25.0% resorcinol, 25% NiSO 4 , 12.5% cinnamic aldehyde, 25.0% abietic acid, and 50.0% hydroxycitronellal were tested, a clear elicitation response was observed.…”

“…The left ear lymph node weight data used in this analysis were obtained from the results of this study and were cited from our previous study (Yamashita et al, 2014).…”

“…To resolve this issue, we developed a local lymph node assay with an elicitation phase (LLNA:DAE) method and published the results (Yamashita et al, 2014). The LLNA:DAE method evaluated the skin sensitizing potential of chemicals by elicitation responses.…”

“…The SI value was affected by the skin irritation potential of the chemicals (Karen et al, 2010;Montelius et al, 1998). However, the LLNA:DAE method evaluates skin sensitization potential in the elicitation phase and the influence of skin irritation potential of the chemicals is eliminated in the evaluation process of skin sensitization potential (Yamashita et al, 2014). Thus, the results of LLNA:DAE may have the potential to provide new information regarding the sensitizing potency of chemicals.…”