3.2　DNCB Protocol

The protocol was designed as an observational study of topical DNCB application by the study subjects.DNCB was supplied in an acetone solution in concentrations of 10%，2%，0.2%and 0.02%（Healing Alternatives Foundation，San Francisco，CA）.Once subjects were enrolled in the study，baseline blood samples were drawn for lymphocyte subset testing，and serum was banked at-70℃.Each subject then underwent patch testing with a 2%DNCB solution applied to a 5 mm skin site.The patch test was used to rule out prior sensitization to DNCB.If the patch test was negative，each subject then proceeded to the sensitization step by applying the 10%DNCB solution to a 2.5 cm area of skin，usually on the forearm.Reactivity was achieved when contact dermatitis occurred at the site within 72 h of DNCB application.If reactivity did not occur with the original 10%solution，subjects repeated this dose at weekly intervals until sensitization occurred.

Once reactivity was demonstrated，subjects began weekly application of 2%DNCB to a 2.5 cm site located on the forearms or upper arms.With repeated application，a‘reflare’（reddening or itching at the original application site）was seen.This response confirmed systemic sensitization to the DNCB.Subjects then decreased the weekly dose to the lowest amount that elicited a localized contact dermatitis.(https://www.daowen.com)

Subjects kept a log of all symptoms during DNCB use.Clinical evaluation was performed every two months by the protocol coordinator.