Experience with local lymph node assay performance standards using standard radioactivity and nonradioactive cell count measurements

Abstract

The local lymph node assay (LLNA) is the preferred test for identification of skin-sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. To facilitate acceptance of nonradioactive variants, validation authorities have published harmonized minimum performance standards (PS) that the alternative endpoint assay must meet. In the present work, these standards were applied to a variant of the LLNA based on lymph node cell counts (LNCC) run in parallel as a control with the standard LLNA with radioactivity measurements, with threshold concentrations (EC3) being determined for the sensitizers. Of the 22 PS chemicals tested in this study, 21 yielded the same results from standard radioactivity and cell count measurements; only 2-mercaptobenzothiazole was positive by LLNA but negative by LNCC. Of the 16 PS positives, 15 were positive by LLNA and 14 by LNCC; methylmethacrylate was not identified as sensitizer by either of the measurements. Two of the six PS negatives tested negative in our study by both LLNA and LNCC. Of the four PS negatives which were positive in our study, chlorobenzene and methyl salicylate were tested at higher concentrations than the published PS, whereas the corresponding concentrations resulted in consistent negative results. Methylmethacrylate and nickel chloride tested positive within the concentration range used for the published PS. The results indicate cell counts and radioactive measurements are in good accordance within the same LLNA using the 22 PS test substances. Comparisons with the published PS results may, however, require balanced analysis rather than a simple checklist approach.

Further experience with the local lymph node assay using standard radioactive and nonradioactive cell count measurements

“…The calculations could be completed whenever a positive dose–response crossed the classification threshold and often it was also possible on some occasions by extrapolation according to published guidance (Ryan et al ., ). Table adds the results from the previous publication (Basketter et al ., ). Overall, for 101 studies where EC3 and EC1.5 values could be determined and/or extrapolated these could be compared.…”

“…Each substance was tested in a separate study carried out under good laboratory practice (GLP) conditions, according to OECD, EC and US EPA test guidelines (OECD, , ; EU, ; US EPA), and published protocols and modifications of the Murine Local Lymph Node Assay (LLNA) (Basketter et al ., ; Gamer et al ., ). For each study, the mice were randomized into individual groups of five to six animals each.…”

Immunophenotyping does not improve predictivity of the local lymph node assay in mice

“…() (surfactants), Basketter et al . () and Kolle et al . (, ) (LLNA PS) without in‐depth discussion of immunophenotyping (see Table ).…”

“…Detailed descriptions and modifications of the Murine LLNA are published by Gamer et al . and Basketter et al ., . A short description in the following: Three groups of five mice were treated with the three pre‐determined concentrations of the test substances in vehicle and one group was treated with vehicle alone (vehicle control).…”

“…The responses to test substances exposure were characterized by 3 H‐thymidine incorporation into the LN cells (dpm) and ear (piece) weight (EW). The skin sensitizing potential of a test substance is indicated by an increase in the stimulation index (SI) of 3 H‐thymidine incorporation by a factor of > 3 compared with the concurrent vehicle control group (Basketter et al ., ). The estimated concentration (EC) for the SI = 3 was calculated by linear regression (Ryan et al ., ).…”