A quantitative method for assessing the sensitizing potency of low molecular weight chemicals using a local lymph node assay: employment of a regression method that includes determination of the uncertainty margins.

Risk assessment of sensitizing chemicals requires, besides hazard identification, the assessment of potency. To examine the sensitizing capacity of low molecular weight chemicals, a murine local lymph node assay (LLNA) was used. The sensitizing capacity of known allergens was quantified by dose-response modeling. At a stimulatory index (SI) of 3, the corresponding estimated concentration was calculated (EC(3)), together with a confidence interval to take account of the quality of the particular data set. We tested ten allergens (ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), 4-ethoxymethylene 2-phenyl oxazol-5-one (oxazolone), phthalic anhydride (PA), toluene diisocyanate (TDI), trimellitic anhydride (TMA), tetramethylthiuramdisulfide (TMTD) and zincdimethyldithiocarbamate (ZDMC)). Oxazolone showed the strongest sensitizing potency followed in this order by DNCB, TDI, TMA, PA, TMTD, ZDMC, MBT, benzocaine and DEA. The approach performed in this study is a way to accurately assess the potency of sensitizing chemicals and thus a possibility for classification.

Comparison of dose-responses of contact allergens using the guinea pig maximization test and the local lymph node assay.

The guinea pig maximization test (GPMT) has been used as a method for the prediction of skin sensitizing potential for over 30 years. Besides hazard identification, risk assessment of sensitizing chemicals requires the assessment of potency. For the determination of potency based on lowest effective dose levels, dose-response studies are required. In the standard GPMT a single concentration is used for intracutaneous and topical induction and the assay provides a qualitative assessment of allergenicity. This paper presents data derived from quantitative evaluation of the sensitizing potency of chemicals in the GPMT, based on multiple concentrations. We performed the GPMT in accordance with the original procedure of Magnusson and Kligman; and included in this procedure a range of intradermal and topical concentrations for induction. Three allergens with different sensitizing potencies, diethylamine (DEA), tetramethyl thiuram disulfide (TMTD) and zinc dimethyl dithiocarbamate (ZDMC) were tested. The data obtained with this test procedure were compared to data we previously obtained using the local lymph node assay (LLNA). Both the GPMT and the LLNA showed dose response relationships for the three chemicals tested. For the chemicals tested, both tests differed in the relative potencies based on benchmark concentrations. While both tests ranked DEA as the least potent allergen, the GPMT ranked ZDMC more potent than TMTD, the reverse being found in the LLNA. The nature of the data provided in the LLNA makes it likely that benchmarks as defined with this test are more reliable than that defined in the GPMT. However, further validation with human data is necessary.

Comparison of three different in vitro mutation assays used for the investigation of cytochrome P450-mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons.

Three different in vitro mutation assays were used to investigate the involvement of cytochrome P450 enzymes in the activation of the nitro-polycyclic aromatic hydrocarbons (nitroPAHs) 1-nitropyrene and 2-nitrofluorene and their reduced metabolites amino-polycyclic aromatic hydrocarbons (aminoPAHs) 1-aminopyrene and 2-aminofluorene. Mutagenicity was investigated at the HPRT locus in Chinese hamster V79 cells with (V79-NH) or without (V79-MZ) endogenous acetyltransferase activity, stably expressing human cytochrome P450 cDNAs; in NIH/3T3 control or stably expressing human CYP1A2 cells, in combination with a shuttle vector containing a reporter gene; and in Salmonella typhimurium TA98, by inhibition of cytochrome P450 enzymes in rat liver S9 mix. Both the HPRT assay and the Ames test did not show any involvement of CYP3A in the activation of 1-nitropyrene to a mutagenic metabolite. In addition, a clear involvement of CYP1A2 in the activation of the nitroPAH 1-nitropyrene was demonstrated in both mutation assays using eukaryotic cells. However, no activation of 1-nitropyrene was seen in the eukaryotic cell lines when expressing only CYP1A2 (V79-MZ1A2) or acetyltransferase (V79-NH, 3T3-LNCX). The reduced metabolite of 1-nitropyrene, 1-aminopyrene, was also shown to be activated to a mutagenic metabolite by CYP1A2, using 3T3-1A2 cells in combination with a shuttle vector, and the Amestest in combination with the specific CYP1A2 inhibitor furafylline. No clear involvement of cytochrome P450 could be demonstrated for activation of 2-nitrofluorene to a mutagenic metabolite, whereas a role for CYP1A2 in the bioactivation of 2-aminofluorene is suggested. In the present study, we have demonstrated the complementary value of the three in vitro mutation assays in the examination of promutagen activation pathways.