The murine local lymph node assay: results of an inter-laboratory trial

The local lymph node assay is a novel predictive test for the identification of contact allergens. The collaborative study reported here was performed to evaluate the reliability of the method when performed in independent laboratories. Eight chemicals were examined in each of 4 participating laboratories and results compared with predictions of skin-sensitizing activity made from concurrent Magnusson and Kligman guinea-pig maximization tests performed in a single laboratory. The local lymph node assay has as its theoretical basis the fact that contact allergens induce T-lymphocyte proliferative responses. In practice, predictions of contact-sensitizing potential are made following measurement of proliferation in lymph nodes draining the site of exposure to chemical, and derivation of a stimulation index using control values as the comparator. Although in the present study there was some variation between laboratories with respect to the absolute stimulation indices recorded, it was found that with all chemicals each laboratory made the same predictions of sensitizing activity. Six chemicals (2,4-dinitrochlorobenzene, formalin, eugenol, isoeugenol, p-phenylenediamine and potassium dichromate) yielded positive responses, and two (methyl salicylate and benzocaine) were negative, in each laboratory. Furthermore, with 7 of the 8 chemicals tested there was no significant difference between laboratories in terms of the characteristics of the dose-response relationships recorded. With the exception of one chemical (benzocaine), predictions made with the local lymph node assay were in accord with those derived from guinea-pig maximization tests. These inter-laboratory comparisons demonstrate that the local lymph node assay is a robust and reliable method for the identification of at least moderate and strong contact allergens.     

The local lymph node assay: results of a final inter-laboratory validation under field conditions.

The local lymph node assay (LLNA) assesses the sensitizing activity of chemicals by measurement of primary lymphocyte proliferation in lymph nodes draining the site of application. In this final inter-laboratory study the consistency of LLNA results between laboratories and with guinea pig maximization test (GPMT) data was examined under 'field' conditions. Nine chemicals were evaluated independently by each laboratory according to guidelines for test concentration and vehicle selection developed during previous validation studies to ensure assay optimization. Equivalent predictions of sensitization potential were obtained by all laboratories for eight chemicals. Five of seven chemicals identified as sensitizers in the GPMT were correctly identified in the LLNA--four by all laboratories and 1 (4-chloroaniline) by one laboratory only--although in this latter case, two other laboratories obtained clear dose responses, suggestive of sensitization. The LLNA identified correctly those chemicals predicted to be extreme or strong sensitizers in the GPMT. The remaining two chemicals were non-sensitizers in the guinea pig and failed to elicit positive proliferative responses in the LLNA. These data demonstrate that sensitivity and reliability of the LLNA is retained when chemicals are evaluated independently, and that it provides a reliable pre-screen for the identification of chemicals with significant sensitization potential.     

Local lymph node assay with non-radioisotope alternative endpoints

The local lymph node assay has recently been accepted by regulatory agencies as a stand-alone alternate method for predicting allergic contact dermatitis. To compare the sensitivity of non-radioisotope methods with that of the standard assay, we determined if these modified methods would affect evaluation of sensitization potency. For this reason, we used 2,4-dinitrochlorobenzene (DNCB) and benzocaine for different sensitizing criteria. Female CBA mice were treated for 3 days with a test compound or vehicle applied to each side of both ears. Bilateral auricular lymph node proliferative activity was assessed by the following endpoints with incorporation of 3H-methyl thymidine (3H-TdR), bromodeoxyuridine (BrdU) in vivo, and BrdU ex vivo, IL-2 production, and proliferating cell nuclear antigen (PCNA) expression. Ear thickness was also tested. The strong sensitizer DNCB was detectable by any of the non-radioisotope endpoints as well as by radioisotope-dependent standard assay. On the other hand, when evaluating the weak sensitizer benzocaine, significant changes were evident in BrdU incorporation ex vivo and in vivo, and IL-2 production. We believe that these non-radioisotope methods can assess allergic contact dermatitis caused by chemicals even in the laboratory, where it can be difficult to handle radioisotopes.     

An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round

The new OECD guideline 429 (skin sensitization: local lymph node assay) is based upon a protocol, which utilises the incorporation of radioactivity into DNA as a measure for cell proliferation in vivo. The guideline also enables the use of alternative endpoints in order to assess draining lymph node (LN) cell proliferation. Here we describe the first round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in seven laboratories. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products, Swissmedic. Statistical analyses of all data were performed by an independent centre at the University of Bern, Department of Statistics. Ear-draining, LN weight and cell count were used to assess proliferation instead of radioactive labeling of lymph node cells. In addition, the acute inflammatory skin reaction was measured by ear swelling and weight of circular biopsies of the ears to identify skin irritating properties of the test items. Hexylcinnamaldehyde (HCA) and three blinded test items were applied to female, 8--10 weeks old NMRI and BALB/c mice. Results were sent via the independent study coordinator to the statistician. The results of this first round showed that the alternative endpoints of the LLNA are sensitive and robust parameters. The use of ear weights added an important parameter assessing the skin irritation potential, which supports the differentiation of pure irritative from contact allergenic potential. There were absolute no discrepancies between the categorisation of the three test substances A--C determined by each single participating laboratories. The results highlighted also that many parameters do have an impact on the strength of the responses. Therefore, such parameters have to be taken into consideration for the categorisation of compounds due to their relative sensitizing potencies.     

Local lymph node assay - validation, conduct and use in practice.

The validation of alternative methods is a relatively new activity in toxicology. The local lymph node assay (LLNA), a novel method for the identification of chemicals that have the potential to cause skin sensitization, was the first test to pass through the formal regulatory validation process established in the USA under the auspices of ICCVAM, the Interagency Coordinating Committee on the Validation of Alternative Methods. ICCVAM approved the LLNA as an alternative to guinea pig tests for the identification of skin sensitisation hazards. In this report, we explore the nine recommendations made by ICCVAM and discuss their interpretation in relation to the new OECD Guideline 429, which describes the LLNA. In particular, the value and limitations of the use of statistical evaluation of data and of the inclusion of routine positive controls is examined. It is concluded that the OECD Guideline as currently written embodies the necessary flexibility to permit conduct of the LLNA in a manner necessary to meet the varying needs of regulatory agencies and toxicologists around the world.     

Anamnestic responses to contact allergens: application in the murine local lymph node assay

The murine local lymph node assay, an alternative predictive test for the identification of contact sensitizing chemicals, is based upon the fact that skin allergens induce proliferation in lymph nodes draining the site of application. In the present study we have examined whether pre-exposure to the test chemical at a distant site enhances subsequent draining lymph node cell proliferation and, thereby, the sensitivity of the assay. Experiments were performed using both in vitro and in situ measurement of induced lymph node cell proliferation. It was found that, with the exception of potent skin sensitizers such as picryl chloride and oxazolone, which impair subsequent proliferative activity as a consequence of induced immunoregulatory processes, pre-treatment with the test allergen resulted in enhanced proliferation. Evidence is presented that the local lymph node assay response to a variety of skin allergens (including eugenol, isoeugenol, dihydrocoumarin, 4-vinylpyridine, cinnamic aldehyde and 2,4,5-trichlorophenol) was augmented when mice received a single exposure to the same chemical 5 days earlier. It is concluded that the use of a modified protocol, incorporating pre-exposure to the test material, can enhance local lymph node assay responses to all but the most potent skin allergens, and may be of particular value when increased sensitivity is required.     

Differentiation of responses to allergenic and irritant compounds in mouse popliteal lymph node assay

The mouse popliteal lymph node assay (PLNA) has been proposed as an immunotoxicological test to predict allergenic chemicals. However, PLN response is also observed in association with non-specific activation induced by some irritants. We, therefore, examined the kinetics of the PLN cellularity indices of primary and secondary responses. Flow cytometric analysis was used to measure the proportions of T and B cells in PLN. Male ICR mice were subcutaneously injected with TNBS (an allergenic compound) or SDS (an irritant compound) in the left hind footpad, and with vehicle in the right one. On day 28 after first injection, mice were injected with 1/10 or 1/100 dose of the initial injection. On day 7 after first injection of TNBS, primary response reached maximal PLN cellularity index (16.5). On day 2 after second injection, secondary response reached maximal PLN cellularity index (13.1). A marked increase in proportion of B cells was observed in the PLN. On the other hand, after first injection of SDS, primary response reached maximal PLN cellularity index (2.8) on day 10, but neither secondary response nor increase in the B cell proportion were observed. These results suggest that the detection of secondary response and the flow cytometric analysis are useful in differentiating responses to allergenic and irritant compounds in the PLNA.     

Positive responses to imipramine in the popliteal lymph node assay are due to primary irritation

The popliteal lymph node (PLN) assay has long been proposed as a tool to detect immunotoxicants with the potential to induce systemic autoimmunity. A major problem hampering the further validation of this assay is the need to rule out irritants that cause false-positive PLN responses. The anti-depressant, imipramine, has not been reported to induce systemic autoimmune reactions in treated patients, but has been repeatedly found positive in the PLN assay, suggesting that this is a false-positive response. To test this hypothesis, the effects of imipramine were compared to those of 50% ethanol in C57B1/6 mice. Footpad edema was evidenced in the few days after injection of both ethanol and imipramine. T-cell depletion using monoclonal antibodies against either CD4+ or CD8+ T-lymphocytes prior to the PLN assay did not influence the responses to either ethanol or imipramine. Cytokine (TNFalpha, IL-1alpha, IL-1beta, IL-2R, IL-6, IL-12 and IFN-gamma) fingerprinting of the PLNs after injection of ethanol and imipramine evidenced the same pattern of responses. These results indicate a closely similar pattern of responses following the footpad injection of either imipramine or ethanol. The conclusion can be drawn that imipramine induces positive responses in the PLN assay via primary (nonspecific) irritation.     

Popliteal lymph node assay: facts and perspectives

The popliteal lymph node assay (PLNA) derives from the hypothesis that some supposedly immune-mediated adverse effects induced by certain pharmaceuticals involve a mechanism resembling a graft-versus-host reaction. The injection of many but not all of these compounds into the footpad of mice or rats produces an increase in the weight and/or cellularity of the popliteal lymph node in the treated limb (direct PLNA). Some of the compounds known to cause these adverse effects in humans, however, failed to induce a positive PLNA response, leading to refinements of the technique to include pretreatment with enzyme inducers, depletion of CD4(+) T cells or additional endpoints such as histological examination, lymphocyte subset analysis and cytokine fingerprinting. Alternative approaches have been used to improve further the predictability of the assay. In the secondary PLNA, the test compound is injected twice in order to illicit a greater secondary response, thus suggesting a memory-specific T cell response. In the adoptive PLNA, popliteal lymph node cells from treated mice are injected into the footpad of naive mice; a marked response to a subsequent footpad challenge demonstrates the involvement of T cells. Finally, the reporter antigens TNP-Ficoll and TNP-ovalbumin are used to differentiate compounds that induce responses involving neo-antigen help or co-stimulatory signals (modified PLNA). The PLNA is increasingly considered as a tool for detection of the potential to induce both sensitization and autoimmune reactions. A major current limitation is validation. A small inter-laboratory validation study of the direct PLNA found consistent results. No such study has been performed using an alternative protocol. Other issues include selection of the optimal protocol for an improved prediction of sensitization vs autoimmunity, and the elimination of false-positive responses due to primary irritation. Finally, a better understanding of underlying mechanisms is essential to determine the most relevant endpoints. The confusion resulting from use of the PLNA to predict autoimmune-like reactions as well as sensitization should be clarified. Interestingly, most drugs that were positive in the direct PLNA are also known to cause drug hypersensitivity syndrome in treated patients. This observation is expected to open new avenues of research.     

A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses.

Effective risk assessment and management of allergic contact dermatitis require three key factors: adequate hazard identification, measurement of the relative potency of identified hazards and an understanding of the nature, extent and duration of exposure. Suitable methods for hazard identification, such as the murine local lymph node assay (LLNA) and the guinea-pig maximization test, are well established and conditions of human exposure normally can be well anticipated. Thus, the need is for a robust and quantitative method for the estimation of relative skin sensitizing potency. One possible approach is via the analysis of LLNA dose-response data. In the LLNA, contact allergens are defined currently as those chemicals that cause a threefold or greater increase in lymph node cell proliferative activity compared with concurrent vehicle-treated controls. It is possible to estimate the concentration of a sensitizer required to generate a threefold stimulation of proliferation in draining lymph nodes; such a concentration is known as the EC3 value. Using a variety of statistical approaches to derive EC3 values from LLNA dose-response data for 10 chemicals, it has been demonstrated that simple linear interpolation between the values either side of the threefold stimulation index provides a robust assessment of the EC3 value without the need for recourse to more sophisticated statistical techniques. Provided that the appropriate concentrations of test chemical have been selected, EC3 values obtained in this way are reproducible both within and between laboratories and form the basis for examination of the utility of this approach for the estimation of relative skin sensitizing potency.     

Draining lymph node cell activation in guinea pigs: comparisons with the murine local lymph node assay

The local lymph node assay in the mouse is a novel predictive test for the identification of contact sensitizing chemicals. The purpose of the studies described was to determine whether a similar local lymph node assay could be performed successfully in guinea pigs; currently the species of choice for assessment of sensitizing potential for regulatory purposes. Ten sensitizing chemicals (oxazolone, picryl chloride, 2,4-dinitrofluorobenzene, benzocaine, cinnamic aldehyde, 2,4,-dinitrothiocyanobenzene, p-nitrosodimethylaniline, formaldehyde, p-phenylenediamine and cyanuric chloride) and equal concentrations of sodium lauryl sulphate were examined in a guinea pig local lymph node assay. Animals received three consecutive daily applications of various concentrations of the test chemical on the dorsum of both ears. Control animals were untreated. Five days following the initiation of exposure, draining auricular lymph nodes were excised and weighed. Suspensions of lymph node cells (LNC) were prepared and cultured for 24 or 48 h and proliferation measured by incorporation of [3H]thymidine. Exposure to at least one concentration of all sensitizing chemicals, other than benzocaine, induced proliferation by draining LNC. Responses were higher at 24 h rather than 48 h. Evidence is presented that guinea pig LNC proliferation may be enhanced or maintained by addition to culture of an exogenous source of the T cell growth factor interleukin 2 (IL-2). Draining lymph node weight was increased following exposure to some sensitizing chemicals but, compared with LNC proliferation, provided a less sensitive correlate of lymph node activation. Exposure to sodium lauryl sulphate failed to induce changes in either lymph node weight of LNC proliferation. Data are compared with three-day murine local lymph node assays performed concurrently. The available information indicates that the local lymph node assay may be performed in guinea pigs.     

Skin sensitisation, vehicle effects and the local lymph node assay.

Accurate risk assessment in allergic contact dermatitis is dependent on the successful prospective identification of chemicals which possess the ability to behave as skin sensitisers, followed by appropriate measurement of the relative ability to cause sensitisation; their potency. Tools for hazard identification have been available for many years; more recently, a novel approach to the quantitative assessment of potency--the derivation of EC3 values in the local lymph node assay (LLNA)--has been described. It must be recognised, however, that these evaluations of chemical sensitisers also may be affected by the vehicle matrix in which skin exposure occurs. In this article, our knowledge of this area is reviewed and potential mechanisms through which vehicle effects may occur are detailed. Using the LLNA as an example, it is demonstrated that the vehicle may have little impact on the accuracy of basic hazard identification; the data also therefore support the view that testing ingredients in specific product formulations is not warranted for hazard identification purposes. However, the effect on potency estimations is of greater significance. Although not all chemical allergens are affected similarly, for certain substances a greater than 10-fold vehicle-dependent change in potency is observed. Such data are vital for accurate risk assessment. Unfortunately, it does not at present appear possible to predict notionally the effect of the vehicle matrix on skin sensitising potency without recourse to direct testing, for example by estimation of LLNA EC3 data, which provides a valuable tool for this purpose.     

Local popliteal lymph node reactions to hexachlorobenzene and pentachlorobenzene: Comparison with systemic effects

The effects of the presumed autoimmunogenic chemical hexachlorobenzene (HCB), and the closely related non-autoimmunogenic pentachlorobenzene (PCB) in the local popliteal lymph node assay (PLNA) were investigated. To that end 1–5 mg of HCB, equimolar amounts of PCB or the vehicle only, were injected into the hind footpads of rats or mice, and the reaction in the draining lymph node was evaluated on days 7 and 21 after injection. PLN were isolated, weighed, and cell suspensions were prepared to determine PLN cell numbers, and antibody production of PLN cells with an ELISPOT assay or a line immunoassay. The extent of the lymphoproliferative effect was examined by detection of proliferating cells with the BrdU method, and by measurement of paracortex and follicle areas, by combined immunohistochemistry and morphometry of PLN cryosections. We demonstrate here that HCB elevated PLN weights and cell numbers of the rat PLN, by day 7 after injection, but no elevation of antibody production in the PLN. Moreover, HCB caused an enlargement of both the PLN paracortical and follicular areas, and an elevation of proliferating paracortical T cells. None of the HCB-induced effects were found on day 21. HCB caused the same effects in the mouse PLNA, but they tended to sustain at least until day 21. Hardly any of the HCB-induced changes were found when PCB was injected. Previously, we have shown that oral exposure of Wistar rats to HCB elevated the number of splenic T cells and B cells, but also the serum levels of (auto-)antibodies and the production of these antibodies in the spleen, which is thus only partly in accordance with the results of the local reaction to HCB described in this study. This seeming contradiction is discussed.     

Application of the popliteal lymph node assay in immunotoxicity testing: complementation of the direct popliteal lymph node assay with flow cytometric analyses

The popliteal lymph node assay (PLNA) has been proposed to measure the immunosensitizing potential of chemicals. The direct PLNA detects an immunomodulating effect but does not give insight into the mode of action of the chemical under test. Modifications of this test have been proposed, but they are difficult to perform in routine toxicity testing and require many animals. In the present investigation the direct PLNA was extended with the flow cytometric determinations of: (a) lymphoblasts; (b) the phenotyping of lymphoid subpopulations; (c) the determination of expression of proliferation/activation markers CD25, CD69 and CD62L/CD44 and (d) the analysis of intracellular cytokines interferon gamma, interleukin 2 and interleukin 4. Streptozotocin, hydrazine, HgCl2 and trinitrobenzene sulfonic acid were used as model chemicals. The different mode of action of these substances was well documented by the techniques applied. As the proposed flow cytometric methods can easily be performed and do not require additional test animals this complementation of the direct PLNA seems a promising approach in immunotoxicity testing.     

Local lymph node assay (LLNA): Comparison of different protocols by testing skin-sensitizing epoxy resin system components

Thirteen epoxy resin system components were tested in the LLNA with regard to their sensitizing potency. Lymph node stimulation was quantified not only by measuring the incorporation of [³H]-thymidine into the ear lymph nodes but also the counts of cells recovered from these organs. Equivalent figures were obtained with both endpoints used for the evaluation of lymph node cell proliferation if the reference stimulation indices were adjusted. When dissolved in acetone, all test substances showed skin-sensitizing potential, mainly on the boundary between “strong” and “moderate” according to common potency evaluation schemes. Replacing acetone with acetone/olive oil (4:1) as a vehicle for four selected test items, resulted in considerably lower estimated concentrations for sensitization induction. The challenges in comparing the results obtained by different LLNA variations are discussed.     

Local lymph node assay (LLNA): comparison of different protocols by testing skin-sensitizing epoxy resin system components

Thirteen epoxy resin system components were tested in the LLNA with regard to their sensitizing potency. Lymph node stimulation was quantified not only by measuring the incorporation of [³H]-thymidine into the ear lymph nodes but also the counts of cells recovered from these organs. Equivalent figures were obtained with both endpoints used for the evaluation of lymph node cell proliferation if the reference stimulation indices were adjusted. When dissolved in acetone, all test substances showed skin-sensitizing potential, mainly on the boundary between “strong” and “moderate” according to common potency evaluation schemes. Replacing acetone with acetone/olive oil (4:1) as a vehicle for four selected test items, resulted in considerably lower estimated concentrations for sensitization induction. The challenges in comparing the results obtained by different LLNA variations are discussed.     

Differential gene expression responses distinguish contact and respiratory sensitizers and nonsensitizing irritants in the local lymph node assay

Genomic approaches have the potential to enhance the specificity and predictive accuracy of existing toxicology endpoints, including those for chemical sensitization. The present study was conducted to determine whether gene expression responses can distinguish contact sensitizers (1-chloro-2,4-dinitrobenzene [DNCB] and hexyl cinnamic aldehyde [HCA]), respiratory sensitizers (ortho-phthalaldehyde and trimellitic anhydride [TMA]), and nonsensitizing irritants (methyl salicylate [MS] and nonanoic acid [NA]) in the local lymph node assay (LLNA). Female Balb/c mice received doses of each chemical as per the standard LLNA dosing regimen on days 1, 2, and 3. Auricular lymph nodes were analyzed for tritiated thymidine ((3)HTdR) incorporation on day 6 and for gene expression responses on days 6 and 10. All chemicals induced dose-dependent increases in stimulation index, which correlated strongly with the number of differentially expressed genes. A majority of genes modulated by the irritants were similarly altered by the sensitizers, consistent with the irritating effects of the sensitizers. However, a select number of responses involved with immune-specific functions, such as dendritic cell activation, were unique to the sensitizers and may offer the ability to distinguish sensitizers from irritants. Genes for the mast cell proteases 1 and 8, Lgals7, Tim2, Aicda, Il4, and Akr1c18 were more strongly regulated by respiratory sensitizers compared with contact sensitizers and may represent potential biomarkers for discriminating between contact and respiratory sensitizers. Collectively, these data suggest that gene expression responses may serve as useful biomarkers to distinguish between respiratory and contact sensitizers and nonsensitizing irritants in the LLNA.     

The murine local lymph node assay: a commentary on collaborative studies and new directions.

The murine local lymph node assay is a predictive test for the identification of contact allergens. This paper provides a historical background to the development of the assay and describes the performance of a recently completed interlaboratory trial designed to evaluate further the utility of the method as an alternative or adjunct to guinea-pig predictive tests. On the basis of these and supplementary investigations, a number of recommendations can be made regarding the use and interpretation of the local lymph node assay. Finally, a number of issues arising from recent studies are discussed, including comparisons of the local lymph node assay with guinea-pig methods.