Novel approach for classifying chemicals according to skin sensitizing potency by non‐radioisotopic modification of the local lymph node assay

Abstract

The murine local lymph node assay (LLNA) is currently recognized as a stand-alone sensitization test for determining the sensitizing potential of chemicals, and it has the advantage of yielding a quantitative endpoint that can be used to predict the sensitization potency of chemicals. The EC3 has been proposed as a parameter for classifying chemicals according to the sensitization potency. We previously developed a non-radioisotopic endpoint for the LLNA based on 5-bromo-2'-deoxyuridine (BrdU) incorporation (non-RI LLNA), and we are proposing a new procedure to predict the sensitization potency of chemicals based on comparisons with known human contact allergens. Nine chemicals (i.e. diphencyclopropenone, p-phenylenediamine, glutaraldehyde, cinnamicaldehyde, citral, eugenol, isopropyl myristate, propyleneglycol and hexane) categorized as human contact allergen classes 1-5 were tested by the non-RI LLNA with the following reference allergens: 2,4-dinitrochlorobenzene (DNCB) as a class 1 human contact allergen, isoeugenol as a class 2 human contact allergen and alpha-hexylcinnamic aldehyde (HCA) as a class 3 human contact allergen. Consequently, nine test chemicals were almost assigned to their correct allergen class. The results suggested that the new procedure for non-RI LLNA can provide correct sensitization potency data. Sensitization potency data are useful for evaluating the sensitization risk to humans of exposure to new chemical products. Accordingly, this approach would be an effective modification of LLNA with regard to its experimental design. Moreover, this procedure can be applied also to the standard LLNA with radioisotopes and to other modifications of the LLNA.

Use of an Ex Vivo Local Lymph Node Assay to Assess Contact Hypersensitivity Potential

“…In summary, the results of the current study and others (van Och et al, 2000;Woolhiser et al, 2000;De Jong et al, 2002;Ehling, et al, 2005;Takeyoshi et al, 2004Takeyoshi et al, , 2005van den Berg et al, 2005;Piccotti et al, 2006) collectively demonstrate the robustness of the murine LLNA, in that the hypersensitivity potential of compounds could be evaluated in different mouse stains and by measuring lymph node cell proliferation by multiple methods. Although additional positive and negative chemicals need to be tested in the ex vivo LLNA, the current study also provided some evidence indicating that this alternative approach could be used to estimate allergenic potency.…”

“…Several reports have been published describing the development and use of alternative methods to evaluate lymphocyte proliferation in the LLNA, including ex vivo [ 3 H]-thymidine incorporation and assessment of non-radioactive approaches such as in vivo bromodeoxyuridine (BrdU) incorporation (van Och et al, 2000;De Jong et al, 2002;Takeyoshi et al, 2004Takeyoshi et al, , 2005van den berg et al, 2005). We initially investigated the sensitivity of ex vivo [ 3 H]-thymidine incorporation by testing the contact sensitizers hexylcinnamic aldehyde and oxazolone, and the nonsensitizer methyl salicylate (Piccotti et al, 2006).…”

The LLNA: A Brief Review of Recent Advances and Limitations

“…As with all the validated tests discussed, both positive and negative (solvent) controls must be run in parallel to the test substance. The concept for the LLNA: BrdU method (OECD 429B) is similar to that of the standard LLNA and is based on the incorporation and quantification of BrdU into proliferating cells in the auricular lymph nodes following topical chemical exposure [67]. BrdU incorporation is measured by peroxidase-labeled BrdU-specific antibody.…”

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).…”