Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals

We validated a two-tiered murine local lymph node assay (LLNA) with a panel of standard contact (photo)allergens and (photo)irritants with the aim of improving the discrimination between contact (photo) allergenic potential a nd true skin (photo)irritation potential. We determined ear weights to corr elate chemical-induced skin irritation with the ear-draining lymph node (LN ) activation potential. During tier I LLNAs, a wide range of concentrations were applied on three consecutive days to the dorsum of both ears. Mice we re exposed to UVA light immediately after topical application to determine the photoreactive potential of some test chemicals. Mice were killed 24 h a fter the last application to determine ear and LN weights and LN cell count s. It was possible to classify the tested chemicals into three groups accor ding to their threshold concentrations for LN activation and skin irritatio n: (1) chemicals with a low LN activation potential and no or very low skin irritation potential; (2) chemicals with a marked LN activation potential higher than a distinct skin irritation potential; and (3) chemicals with LN activation potential equal to or lower than their skin irritation potentia l. Group I consisted only of contact allergens, indicating that LN activati on in the absence of skin irritation points to a contact allergenic activit y. Since groups 2 and 3 comprised irritants and contact allergens, a tier I I LLNA protocol was used to finally differentiate between true irritants an d contact allergens. Briefly, mice were pretreated with mildly to moderatel y irritating concentrations of the chemical to the shaved back and after 12 days were challenged on the ears as described above in order to elicit a c ontact allergenic response in the ear skin and the ear-draining LN. With th is approach, tier II LLNAs have to be conducted only in cases for which ski n irritation potential is in the range of LN activation potential and no st ructure-activity relationship data indicating a contact allergenic hazard a re available.