Further evaluation of the local lymph node assay in the final phase of an international collaborative trial

E.I. du Pont de Nemours, Inc. Haskell Laboratory, Newark, DE 19711-0050, USA.
Toxicology (Impact Factor: 3.62). 05/1996; 108(1-2):141-52. DOI: 10.1016/0300-483X(95)03279-O
Source: PubMed


The local lymph node assay (LLNA) is a method used for the prospective identification in mice of chemicals that have the potential to cause skin sensitization. We report here the results of the second and final phase of an international trial in which the performance of the assay has been evaluated using seven test materials in five independent laboratories. The additional chemicals examined here included compounds which are considered less potent allergens than some of those tested in the first phase of the investigation, and includes hexylcinnamic aldehyde (HCA), a chemical recommended by the Organization for Economic Cooperation and Development (OECD) as a positive control for skin sensitization studies. In each laboratory all skin sensitizing chemicals examined (2,4-dinitrochlorobenzene {DNCB}, HCA, oxazolone, isoeugenal and eugenol) elicited positive responses of comparable magnitude as judged by the derived lowest concentration of test chemical required to elicit a 3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls. We observed that sodium lauryl sulphate, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. Para-aminobenzoic acid (pABA), a nonsensitizing chemical, was negative at all test concentrations in each laboratory. Some laboratories incorporated minor modifications into the standard assay procedure, including the evaluation of lymph nodes pooled from individual mice rather than treatment groups and the use of statistical analyses. The use of statistics did not markedly change the determination of the lowest concentration yielding a positive response. These data confirm that the local lymph node assay is robust and yields equivalent results when performed independently.

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    • "EC3 value is the estimated concentration that induces a 3-fold induction of cell proliferation in the LLNA (Gerberick et al., 2005; Loveless et al., 1996; van Och et al., 2000); b The elicitation dose is expressed as the actual dose applied (w/v %) and the % of the sensitization dose in brackets. with 0.4% DNCB, cell proliferation was still enhanced at day 23, as shown in mice that were challenged with the vehicle. "
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    ABSTRACT: The concept that thresholds exist for the induction of allergic contact dermatitis by chemicals with skin sensitizing properties has been used for a quantitative risk assessment approach. In this approach the potency of skin sensitizers as determined in the Local Lymph Node Assay is used to calculate the threshold for induction of sensitization. These are then used to estimate safe exposure levels for consumers. Whether these exposure levels will protect subjects that are already sensitized is unknown. The elicitation of allergic contact dermatitis supposedly occurs above a certain threshold as well and this threshold is most likely lower than that for the induction. It is unclear if induction thresholds can be extrapolated to elicitation thresholds. The aim of this study was to assess the potency of sensitizers with different sensitizing potencies in the elicitation phase in a mouse model for elicitation. Mice were sensitized by topical application on days 0 and 7 using equipotent concentrations of oxazolone, 2,4-dinitrochlorobenzene (DNCB) and eugenol to ensure that the sensitization strength would not influence the elicitation potency. Mice were challenged on day 21 by topical application on the ears in a dose-dependent manner and dose-response data were used to calculate the elicitation potency. Unexpectedly, sensitizers with different sensitizing potencies induced not the same dose-response curves in sensitized mice. The most potent sensitizer in the elicitation phase was oxazolone, followed by DNCB and eugenol. Similar to the induction phase, under equipotent sensitization conditions strong sensitizers such as oxazolone and DNCB elicit allergic reactions at lower concentrations than weak sensitizers such as eugenol. Our results indicate that elicitation thresholds cannot be readily deduced from sensitization thresholds.
    Full-text · Article · May 2012 · Toxicology
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    • "It is a threshold set as a precautionary measure to try and account for background fluctuations in lymphocyte proliferation [15]. For example, topical application of the well-studied surfactant sodium lauryl sulfate (SLS) has been shown to test positive in the LLNA with SI values above the threshold limit (3-fold increase) [9, 17, 31–34]. In contrast to the scenario presented for SLS, when numerous nonsensitizing skin irritants were evaluated using the LLNA, the majority tested negative [35]. "
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    ABSTRACT: Allergic contact dermatitis is the second most commonly reported occupational illness, accounting for 10% to 15% of all occupational diseases. This highlights the importance of developing rapid and sensitive methods for hazard identification of chemical sensitizers. The murine local lymph node assay (LLNA) was developed and validated for the identification of low molecular weight sensitizing chemicals. It provides several benefits over other tests for sensitization because it provides a quantitative endpoint, dose-responsive data, and allows for prediction of potency. However, there are also several concerns with this assay including: levels of false positive responses, variability due to vehicle, and predictivity. This report serves as a concise review which briefly summarizes the progress, advances and limitations of the assay over the last decade.
    Full-text · Article · Jun 2011 · Journal of Allergy
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    • "Oxa, on the other hand, did not exert cytotoxicity at extreme concentrations but nonetheless appeared to behave as an outlier in the VITOSENS potency classification. No data could be retrieved on the skinsensitizing potency in humans, whereas in the LLNA, Oxa was identified as an extreme sensitizer based on its EC3 stimulation (Loveless et al., 1996). In our setup, however, Oxa showed up merely as a moderate sensitizer. "
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    ABSTRACT: The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34+ progenitor-derived dendritic cells (CD34-DC), which allows to classify chemicals as skin (non-)sensitizing. However, skin sensitization is not an all-or- none phenomenon, and up to now, the assessment of relative potency can only be derived using the in vivo local lymph node assay (LLNA). In our study, we analyzed the feasibility to predict the sensitizing potency, i.e., the LLNA EC3 values, of 15 skin sensitizers using in vitro data from the CD34-DC-based assay. Hereto, we extended the in vitro-generated gene expression data set by an additional source of information, the concentration of the compound that causes 20% cell damage (IC20) in CD34-DC. We statistically confirmed that this IC20 is linearly independent from the gene expression changes but that it does correlate with LLNA EC3 values. In a further analysis, we applied a robust linear regression with both IC20 and expression changes of CREM and CCR2 as explanatory variables. For 13 out of 15 compounds, a high linear correlation was established between the in vitro model and the LLNA EC3 values over a range of four orders of magnitude, i.e., from weak to extreme sensitizers. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected] /* */
    Full-text · Article · Apr 2010 · Toxicological Sciences
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