Examination of a vehicle for use with water soluble materials in the murine local lymph node assay

Abstract

The murine local lymph node assay (LLNA) is a validated method for identifying skin sensitization hazard. Vehicle choice can influence the sensitization potential of haptens in both the LLNA and in humans, therefore selection of an appropriate vehicle is important. Suggested vehicles for the LLNA include organic solvents and organic–aqueous mixtures. However, due to its high surface tension and poor wetting qualities, water is not recommended and therefore testing aqueous soluble materials can be problematic. The aims of this investigation were to identify a water-based vehicle that possesses better skin wetting properties than water alone, and to assess its performance relative to other solvents in the LLNA using aqueous soluble haptens. The selected wetting agent was the surfactant Pluronic® L92 (L92). Concentrations of L92 of up to 50% did not induce positive responses in the LLNA. 1% aqueous L92 was chosen for further examination. Dose–response analyses were performed with dinitrobenzene sulfonic acid (DNBS) and formaldehyde formulated either in water, 1% L92, dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). Potassium dichromate (PDC) and nickel sulfate were tested in 1% L92, DMSO or DMF. The highest concentration of potassium dichromate was retested in each vehicle and in water to assess the effect of the wetting agent. Estimates of the relative sensitizing potency in each vehicle were determined by calculation of EC3 values (the estimated concentration required to induce a threshold positive response). While DNBS and formaldehyde produced positive responses in all four vehicles, their relative potency varied among the vehicles. The rank ordering of potencies for both materials was, from highest to lowest, DMF⩾DMSO>1% L92>water. Compared with water, use of 1% L92 resulted in >2-fold increase in potency for DNBS and >3-fold increase for formaldehyde. PDC was positive in DMF, DMSO and 1% L92. The potency ranking was DMF⩾DMSO>1% L92. Re-evaluation of 0.5% PDC confirmed that formulations of both DMSO and DMF induced strong proliferative responses, whereas somewhat less proliferation was recorded with the 1% L92 vehicle. PDC in water was without activity. The performance of 1% L92 as a vehicle for nickel sulfate was assessed relative to DMSO and DMF. In DMSO, nickel sulfate produced a stimulation index (SI) >3 at only the highest level. Testing in DMF induced low levels of proliferation, but failed to produce a SI of 3 at any concentration tested. When formulated in 1% L92, nickel sulfate caused a SI of 3 when tested at 2.5%. Based on the results of these experiments, for identification of sensitization hazard of aqueous soluble materials using the LLNA, DMF and DMSO are the preferred vehicles. However, if a test material is not soluble in DMF or DMSO, or if higher test concentrations can be achieved in an aqueous vehicle, then 1% L92 may provide a better alternative to water alone in terms of improved assay performance.