The suitability of hexyl cinnamic aldehyde as a calibrant for the murine local lymph node assay

The murine local lymph node assay (LLNA) for the prospective identification of contact allergens assesses skin sensitization potential as a function of proliferative activity induced in lymph nodes draining the site of topical exposure to test chemical. This method has been endorsed recently as a stand alone test for the identification of contact allergens. We have now examined the suitability of hexyl cinnamic aldehyde (HCA), a recommended positive control for skin sensitization testing, as a calibrant for comparing the consistency of LLNA responses with time, and between laboratories, and thus for the routine assessment of assay reliability. Standard LLNAs were performed with CBA strain mice in 3 independent laboratories over a period of 8 years. Dose-response curves were used to derive mathematically the EC3 value (the estimated concentration of chemical necessary to cause a stimulation index (SI) of 3 compared with proliferation induced by concurrent vehicle controls). In each laboratory, 6 separate experiments were conducted using a single concentration of HCA (25%). Very similar stimulation indices were achieved, with mean values of 9.0, 6.5 and 6.6 recorded. A total of 10 dose-response experiments were performed independently in the 3 laboratories and these revealed that there was very little inter-laboratory, or temporal, variation in EC3 values. These data confirm that HCA responses in the LLNA are very stable and demonstrate that HCA provides a suitable calibrant for determining assay sensitivity and performance.