Assessment of sensitization potential of monoterpenes using the rat popliteal lymph node assay

The popliteal lymph node assay (PLNA) has been proposed as a screening test for detecting chemicals with potential of inducing allergic and auto-immune-like reactions in humans. In the present study, we used the rat PLNA to evaluate the immuno-sensitizing potential of 10 monoterpenes found in the essential oils of a variety of aromatic, edible and medicinal plants. The primary or direct PLNA was performed with the monoterpenes, and chlorpromazine (CPZ) and barbital were used as positive and negative controls, respectively. Female, 7-8 week-old Wistar rats were injected subcutaneously (50 microL) with the test substance (0.5, 2.5 or 5mg) into the right hind footpad while the contralateral footpad was injected with the vehicle (DMSO) alone. Weight (WI) and cellularity (CI) indices for draining PLNs were determined 7 days after treatment. PLNA was positive (WI >or= 2 and CI >or= 5) for CPZ, citral, alpha-terpinene, beta-myrcene and (-)-alpha-pinene, and negative for barbital, DMSO, (-)-menthol, 1,8-cineole, (+/-) citronellal, (+)-limonene, (+/-) camphor and terpineol. A secondary PLNA, a T-cell priming test, was carried out with the four substances that had been positive in the primary assay. Six weeks after being locally primed with 5 mg/paw, rats were sc injected into the same footpad with a dose (0.5 mg/paw) of the substance that had been previously found to be insufficient to cause a positive response. WI and CI were then calculated 4 and 7 days after the second injection. CPZ was also positive in the secondary assay thereby confirming that it is a sensitizing agent. Citral, alpha-terpinene, beta-myrcene and (-)-alpha-pinene, however, were negative in the secondary assay. In summary, citral, alpha-terpinene, beta-myrcene and (-)-alpha-pinene induced a clear immuno-stimulatory response due to their irritant properties but no monoterpene proved to be a sensitizing agent in the PLNA.