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

Abstract

The local lymph node assay (LLNA) is used to assess the contact hypersensitivity potential of compounds. In the standard assay, mice are treated topically with test compound to the dorsum of both ears on Days 1-3. The induction of a hypersensitivity response is assessed on Day 6 by injecting [ 3 H]-thymidine into a tail vein and measuring thymidine incorporation into DNA of lymph node cells draining the ears. The ex vivo LLNA is conducted similarly except lymphocyte proliferation is assessed after in vitro incubation of lymph node cells with [ 3 H]-thymidine, which significantly reduces the amount of radioactive waste. The current study tested the use of this approach for hazard assessment of contact hypersensitivity and to estimate allergenic potency. Female BALB/c mice were treated on Days 1-3 with two nonsensitizers (4 -methoxyacetophenone, diethyl phthalate), three weak sensitizers (hydroxycitronellal, eugenol, citral), one weak-tomoderate sensitizer (hexylcinnamic aldehyde), two moderate sensitizers (isoeugenol, phenyl benzoate), and one strong sensitizer (dinitrochlorobenzene). On Day 6, isolated lymph node cells were incubated overnight with [ 3 H]-thymidine and thymidine incorporation was measured by liquid scintillation spectrophotometry. The ex vivo LLNA accurately distinguished the contact sensitizers from the nonsensitizing chemicals, and correctly ranked the relative potency of the compounds tested. The EC3 values, i.e., the effective concentration of test substance needed to induce a stimulation index of 3, were as follows: 4 -methoxyacetophenone (>50%), diethyl phthalate (>50%), hydroxycitronellal (20.4%), eugenol (13.6%), citral (8.9%), isoeugenol (3.8%), hexylcinnamic aldehyde (2.7%), phenyl benzoate (2%), and dinitrochlorobenzene (0.02%). In addition, low inter-animal and inter-experiment variability was seen with 25% hexyl-cinnamic aldehyde (assay positive control). The results of the ex vivo LLNA in the current study were consistent with published reports using the standard LLNA and provided further

Utilization of the Ex Vivo LLNA

“…These results are compatible with the previous reports. 39 -41 The European Food Safety Authority The Panel on Additives and Products or Substances used in Animal Feed considered isoeugenol as an irritant to the respiratory system, skin, and eyes and as a skin and respiratory sensitizer, based on the information available in the literature. 41…”

“…These results are compatible with the previous reports. [39][40][41] The European Food Safety Authority The Panel on Additives and Products or Substances used in Animal Feed considered isoeugenol as an irritant to the respiratory system, skin, and eyes and as a skin and respiratory sensitizer, based on the information available in the literature. 41 Increases in cytokine levels are related to T helper cell proliferation, which plays an important role in the induction and elicitation of contact sensitivity.…”

Local lymph node assay: 5-bromo-d-deoxyuridine-ELISA method for comparative study in assessing chemical potencies and skin sensitization in BALB/c and CBA/J strains

“…LLNA is based on measurement of proliferated responses in corporation of 3 H- methyl thymidine. However, special facilities and disposal procedures are required because it is a radioisotope (RI)-based method [5] . To avoid use of the radioisotope in LLNA, some modified LLNA protocols were developed [6] , [7] , [8] .…”