Differences of draining lymph node cell proliferation among mice, rats and guinea pigs following exposure to metal allergens.

Contact sensitivities of three well known metal allergens (nickel sulfate, potassium dichromate and cobalt chloride) were examined using the local lymph node assay in CBA/N mice, F344 rats and Hartley guinea pigs. The effect of various species sera on lymph node cell (LNC) proliferation was also investigated. Exposure to potassium dichromate and cobalt chloride induced significant LNC proliferative responses in the three species. The LNC responses to potassium dichromate in the rats were higher than those in the mice and guinea pigs. Mice exhibited the highest response to cobalt chloride among the three species, whereas, exposure to nickel sulfate failed to induce a marked LNC proliferation. Increased draining lymph node weights and LNC numbers were also observed following exposure to the metal salts. However, these parameters were less sensitive compared with the LNC proliferative response. There was a large difference in the lymph node weight between individual guinea pigs. The [methyl-3H]thymidine incorporation into LNC of each species cultured in the presence of the homologous serum in vitro was lower than in the presence or absence of fetal calf serum. However, there was no significant difference in stimulation indices among the different culture conditions. The local lymph node assay may be performed in rats as well as in mice for the detection of metal allergens.     

A sensitive mouse lymph node assay with two application phases for detection of contact allergens

A predictive test using mice for the identification of contact sensitizing chemicals was developed. Contact sensitizing activity is measured as a function of draining lymph node activation following application of test chemical. Experimental conditions for assessment of induced lymph node cell (LNC) responses have been optimized. BALB/c mice were initially treated with intradermal injections of test chemical in Freund's complete adjuvant emulsion. Five days after intradermal injection, mice were exposed topically to chemical in vehicle on the ears daily for 3 consecutive days. Next day following the final exposure, changes in lymph node weight, total cell number in the draining lymph nodes and LNC proliferation for 24 h culture were assessed. The performance of the method was evaluated with ten sensitizing chemicals and a non-sensitizing irritant, sodium lauryl sulfate (SLS). The LNC proliferation induced by combination of intradermal injection and topical application of sensitizing chemicals was more clearly increased than that following only topical application. With the single exception of sulfanilic acid, the method developed was able to detect the sensitizing capacity of chemicals that failed to induce sensitization in the local lymph node assay. Under the conditions used, SLS did not induce measurable lymph node responses. These results suggest that the mouse lymph node assay can provide a sensitive screening test for weak to moderate sensitizers. In addition, the assay offers the advantages of objective and quantitative endpoints, and is suitable for the evaluation of colored or irritant chemicals.     

A murine local lymph node assay for the identification of contact allergens

The development of an alternative predictive test for the identification of contact sensitizing chemicals is described. The method is based upon the fact that, following epicutaneous application, sensitizing chemicals initiate a primary immunological response in the draining lymph node(s) which is characterized by lymphocyte proliferation. Experimental conditions for the measurement in vitro of the induced lymph node cell proliferative response have been optimized. On the basis of the data presented a local lymph node assay was developed in which CBA/Ca strain mice were exposed daily, for 3 consecutive days, to various concentrations of the test chemical, or to vehicle alone, on the dorsum of the ear. Lymph node activation was measured subsequently as a function of increased node weight, the frequency of large pyroninophilic cells and lymphocyte proliferation in the presence or absence of an exogenous source of interleukin 2 (IL-2). The results of a validation study are reported in which 22 well-characterized sensitizing chemicals of varying potency were examined. With the exception of three chemicals where water was used as the application vehicle, positive responses, defined as a substantial increase in lymphocyte proliferative activity, were recorded with all these test materials. Under the conditions employed non-sensitizing chemicals, including non-sensitizing irritant chemicals, failed to influence the immunological status of the draining lymph node. Taken together, the data suggest that the local lymph node assay provides the basis for a rapid and cost-effective alternative to the currently available guinea pig predictive test methods. The local lymph node assay may be of particular value for the evaluation of coloured or irritant chemicals.     

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.     

Identification of contact allergens using the murine local lymph node assay: comparisons with the Buehler occluded patch test in guinea pigs

A murine local lymph node assay has been developed for the identification of contact sensitizing chemicals. In the present study, the performance of the local lymph node assay has been evaluated with twenty-four coded chemicals of previously unknown skin sensitizing potential and the results compared with predictions made from concurrent occluded patch tests (Buehler tests) in guinea pigs. The data presented demonstrate that the local lymph node assay successfully identified those chemicals that were classified as moderate or strong skin sensitizers in the Buehler test. In the present series of experiments, chemicals predicted to be mild sensitizers in the Buehler test were classified as 'not strong sensitizers' in the local lymph node assay. In the majority of instances, the Buehler test and local lymph node assay were in agreement with regard to the identification of non-sensitizing chemicals. However, two chemicals that were classified as non-sensitizers in the guinea pig test exhibited positive responses in the local lymph node assay and were predicted to be sensitizers. Some coloured chemicals resulted in obscured Buehler readings and, here, assessment was based upon histological examination of the challenge site. These compounds were examined also in the local lymph node assay and similar predictions of sensitizing potential were made. Taken together, the data reveal close, but not absolute, concordance between the local lymph node assay and the Buehler test. The relative merits of these predictive test methods are discussed.     

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.     

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.     

ICCVAM evaluation of the murine local lymph node assay. Conclusions and recommendations of an independent scientific peer review panel.

The validation status of the murine local lymph node assay (LLNA), a method for assessing the allergic contact dermatitis potential of chemicals, was evaluated by an independent peer review panel (Panel) convened by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The LLNA measures lymphocyte proliferation using incorporation of radioactive thymidine or iododeoxyuridine into cells of the draining lymph nodes of mice topically exposed to a test article. The Panel concluded that the assay performed as well as currently accepted guinea pig methods [guinea pig maximization test (GPMT)/Buehler assay (BA)] for the hazard identification of strong to moderate chemical sensitizing agents, but that it might not correctly identify all weak sensitizers or metals (potential false negative response) or all strong irritants (potential false positive response). The Panel concluded also that the LLNA involves less pain and distress than conventional guinea pig methods. The Panel unanimously recommended the LLNA as a stand-alone alternative for contact sensitization hazard assessment, provided that certain protocol modifications were made. These included collection of individual, rather than pooled, animal response data; the inclusion of a concurrent positive control; and consideration of dose-response information and statistical analyses. A standardized LLNA protocol is provided.     

Threshold for classification as a skin sensitizer in the local lymph node assay: a statistical evaluation

For more than 15 years, the murine local lymph node assay (LLNA) has undergone development, evaluation and validation as an alternative approach to the predictive identification of skin sensitizing chemicals. The criteria by which sensitizing chemicals are distinguished from those without significant skin sensitising hazard were developed empirically and were based on experience rather than a mathematical formula or statistical method. The current practice is to classify, as skin sensitizers, those chemicals which at one or more test concentrations stimulate a threefold or greater increase in the proliferative activity in draining lymph node cells. Despite the apparent confirmation of the utility of this approach from the extensive data available, there has not previously been any attempt to substantiate the accuracy of this criterion. In this present investigations, data from 134 chemicals tested in the LLNA and in the guinea pig and/or for which there exists clear evidence relating to human skin sensitization potential, have been subjected to a rigorous statistical evaluation using Receiver Operating Characteristic (ROC) curves. Whether the analysis is based on a comparison with guinea pig or human data, the results indicate that the empirically derived threefold threshold is an acceptable practical value for hazard identification.     

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.     

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.     

Detection of contact sensitivity of metal salts using the murine local lymph node assay.

The local lymph node assay (LLNA) is a predictive test for the detection of contact allergens. Nickel and chromium sensitization are common cases in man. However, in a previous study topical application of nickel sulfate and potassium dichromate in aqueous solution failed to induce activation in the draining lymph node. This study describes the application of LLNA to evaluate the contact sensitivity of metal salts. The metal salts were applied in dimethylsulfoxide or aqueous ethanol solution. In some experiments, the skin of the ears was gently abraded using a needle prior to application of metal salts. The ability of seven metal salts to induce lymph node cell (LNC) proliferation was compared. Nickel, cobalt, chromium and copper salts increased LNC proliferation, whereas zinc, manganese and iron salts failed to induce LNC proliferation in this assay.     

Cytokine endpoints for the local lymph node assay: consideration of interferon-gamma and interleukin 12.

The murine local lymph node assay (LLNA) is a method for the prospective identification of contact allergens. Skin sensitization potential is assessed as a function of induced proliferative responses in lymph nodes draining the site of topical exposure measured in situ by incorporation of radiolabelled thymidine ([3H]thymidine). The results of previous investigations have demonstrated that the analysis of [3H]thymidine incorporation represents a robust and reliable endpoint for the LLNA for the assessment of skin sensitizing activity for strong and moderate allergens and, in addition, many weaker sensitizers. The aim of the current experiments was to explore the utility of the production of the cytokines interferon-gamma (IFN-gamma) and interleukin 12 (IL-12) by draining lymph node cells (LNC) as alternative readouts for the LLNA. Animals were exposed to a range of skin sensitizers at two application concentrations. The first of these was chosen on the basis of results from previous investigations to stimulate a strong proliferative response (tenfold or greater increase in proliferation compared with concurrent vehicle controls). The second concentration of test material in each case was the amount of chemical estimated to be necessary mathematically for elicitation of a stimulation index of 3 (EC3 value); the induction of a threefold or greater increase in proliferation is the current criterion for a positive response in the LLNA. In addition, analyses were conducted with para-aminobenzoic acid (PABA), a non-sensitizing chemical shown previously not to induce LLNA responses. Secretion of IFN-gamma and the p40 subunit of IL-12 by draining LNC was measured by cytokine-specific enzyme-linked immunosorbent assay. In parallel experiments, LNC activity was assessed as a function of [3H]thymidine incorporation in situ. All the chemical allergens tested provoked robust proliferative responses, with the stimulation indices recorded at both test concentrations reflecting only small changes in activity compared with previously recorded data. Exposure to vehicle (4:1 acetone:olive oil, AOO) alone resulted in detectable, although variable, expression of both IFN-gamma and IL-12. Treatment with chemical allergen in each case caused a marked increase in IFN-gamma secretion, with particularly vigorous production of cytokine being stimulated following exposure to oxazolone or hexyl cinnamic aldehyde. In contrast, application of chemical allergens was not generally associated with elevated IL-12 p40 secretion. Exposure of mice to PABA did not result in increased IFN-gamma or IL-12 production compared with vehicle-treated controls. In general, however, cytokine secretion did not correlate closely with the induction of LNC proliferation. These data indicate that expression by allergen-activated LNC of IFN-gamma or IL-12 does not provide a reliable or sufficiently sensitive endpoint for the LLNA compared with [3H]thymidine incorporation in situ.     

Evaluation of lymphocyte subpopulations in draining lymph node cells following allergen and irritant

The murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for the assessment of the contact sensitization potential. However, there is a need to develop a non-radioisotopic endpoint for the LLNA, because of the radioisotopic method's requiring the use of special facilities. In this study, we investigated to evaluate the lymphocyte subpopulations in the lymph node cells following allergen and irritant treatment. Female Balb/c mice were treated by the topical application on the dorsum of both ears with sensitizers, 2,4-dinitrochlorobenzene (DNCB), toluene diisocyanate (TDI), and α-hexylcinnamaldehyde (HCA), and an irritant, sodium lauryl sulfate (SLS), once daily for three consecutive days. The lymph node (LN) cells were harvested 72h after the final treatment. Phenotypic analysis of lymphocytes subsets was performed with a flow cytometry. The allergens DNCB, TDI, and HCA and an irritant, SLS increased cell number compared to the vehicle. Mice were treated with DNCB, HCA, and TDI showed a preferential increase in the percentage of B220+CD40+ cells compared with vehicle and irritant-treated mice. There was an increase in B220+CD86+ cells of mice treated with DNCB, TDI, and HCA, but no significant increases were observed in mice treated with SLS. Mice were treated with DNCB and TDI showed an increase in the percentage of B220+CD23+ cells compared with vehicle and irritant-treated mice. These results suggest that analysis of B cell activation marker, CD40 on B cells may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice.     

Assessment of cumulative allergen-activated lymph node cell proliferation using flow cytometry.

The murine local lymph node assay (LLNA) is a method for the prospective identification of chemical contact allergens. The current validated protocol assesses lymphocyte proliferation induced in the draining lymph node as a function of in situ incorporation of radiolabeled thymidine. We have explored the potential utility of an alternative nonradioisotopic marker of cell division, the cytoplasmic dye carboxyfluoresein succinimidyl ester (CFSE). Using this method, the cell phenotype and the number of divisions each cell has undergone can be tracked using flow cytometry. BALB/c strain mice were exposed topically to various concentrations of the contact allergens 2,4-dinitrochlorobenzene (DNCB), oxazolone (ox) or hexyl cinnamic aldehyde (HCA), or to the nonsensitizing skin irritant methyl salicylate (MS). Five days later, lymph node cells (LNC) were labeled with CFSE, cultured for 96 h, then incubated with fluorescent labeled anti-CD4 (T helper) and -CD8 (T cytotoxic) cell antibodies, and proliferating CD4+ and CD8+ cells analyzed by flow cytometry. In LNC populations derived from vehicle-treated animals, less than 1% of either cell population had undergone one cell division or more. Topical exposure to MS (2.5 to 20%) did not increase the frequencies of proliferating cells. Exposure to all three allergens, however, resulted in a marked increase in the percentages of both CD4+ and CD8+ cells undergoing division, with up to 5% and 3% of these cells, respectively, proliferating in response to DNCB and oxazolone, and with lower levels of proliferation stimulated by HCA. These preliminary data suggest that this method may be applied to provide the basis of a nonradioisotopic end point for the LLNA, particularly for the identification of potent contact allergens.     

ICCVAM evaluation of the murine local lymph node assay. Data analyses completed by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods.

To evaluate the reliability of the murine local lymph node assay (LLNA), a test for allergic contact dermatitis activity, the inter- and intralaboratory consistency statistics (h and k, respectively) were calculated for validation studies testing multiple chemicals. The analysis indicated the absence of excessive variability in the dose calculated to induce a threefold or greater increase in the stimulation index (SI). To assess the appropriateness of using an SI of 3 as the decision criteria for identifying a sensitizing compound, LLNA results based on SI values of 2.0, 2.5, 3.0, 3.5, and 4.0 were compared with guinea pig or human results. The results supported the use of an SI of 3 as the decision criteria. Assay performance was determined by comparing LLNA results to results obtained for guinea pigs or humans. The accuracy of the LLNA was 89% when compared with results from the guinea pig maximization test (GPMT)/Buehler assay (BA). The performance of the LLNA and the GPMT/BA was similar when each was compared to human maximization test results plus substances included as human patch test allergens. The LLNA offered advantages over the GPMT in respect to both the time required to conduct the test and the assay cost.     

Interlaboratory Evaluation of the Local Lymph Node Assay with 25 Chemicals and Comparison with Guinea Pig Test Data

Abstract

Summary: The murine local lymph node assay (LLNA) has been developed as an alternative method for the identification of skin sensitizing chemicals. Measurement is made of the proliferation of lymphocytes within lymph nodes draining the site of exposure to the test chemical. This report describes a collaborative study in which 25 test chemicals were evaluated in each of four participating laboratories and the results compared with existing data from guinea pig predictive tests. Nineteen chemicals were predicted to be sensitizers in the guinea pig. Of these, 14 were correctly identified in the LLNA (9 by all laboratories and 5 by two or three laboratories). Five chemicals predicted to be contact allergens by guinea pig tests failed to elicit positive LLNA responses. With adoption of a 5 day rather than a 4 day exposure period to the test chemical and administration of maximum soluble test concentrations, positive reactions could be obtained with each of the chemicals initially negative in the LLNA. The LLNA and guinea pig tests were in agreement for all three chemicals predicted to be nonsensitizers in the guinea pig. Positive LLNA responses were obtained with four chemicals (including a re-evaluation of one initially negative in the LLNA) for which guinea pig results were equivocal in three cases and negative in another. These results suggest that the LLNA may provide a rapid and reliable elective prescreen for the identification of contact allergens.     

A COMBINED MURINE LOCAL LYMPH NODE AND IRRITANCY ASSAY TO PREDICT SENSITIZATION AND IRRITANCY POTENTIAL OF CHEMICALS

In an effort to establish a single, rapid screening procedure for the sensitization and irritancy potential of new chemicals, the parameters of a murine Local Lymph Node Assay and a mouse ear swelling irritancy assay were combined. To validate this assay, a range of chemical irritants and sensitizers were evaluated for their ability to elicit responses in B6C3F1 female mice. Chemicals were administered for four consecutive days to the dorsal and ventral surfaces of each ear. An increasein ear thickness served to predict irritancy, while [3H]thymidine uptake by cervical draining lymph nodes suggested sensitization. All chemicals known to be potent chemical sensitizers (oxazolone, 2,4-dinitrofluorobenzene, toluene diisocyanate) produced a marked lymph node cell proliferation in this assay. Animals exposed to irritating agents (sodium lauryl sulfate, croton oil, tetradecane, nonanoic acid, and benzalkonium chloride) experienced a significant increase in ear swelling. In addition, these irritating agents elicited lowlevel lymphocyteproliferation.In cases wherechemicals areconsidered tobeboth potent sensitizers and irritants (2,4-dinitrofluorobenzene, toluene diisocyanate, and benzalkonium chloride), robust increases in [3H]thymidine incorporation and ear swelling were demonstrated. Results were dose-responsive for all chemicals tested. The combined LLNA/ear swelling assay appears to be a reliable predictor of sensitization and irritancy potential, since it identified the activity of all eight chemicals tested. The advantages of this approach include a further reduction in the number of animals and time required to screen chemicals for both irritancy and/or sensitization potential. Although this assay does not have the capacity to discriminate between nonspecific lymph node proliferation and weak sensitizing ability of strong irritants, the information gained by the irritation component of the assay provides additional information when evaluating the significance of low-level lymphocyte proliferation in the LLNA. With further validation this assay could be useful as a common screening tool in the research and development of new chemical products.     

Evaluation of cell proliferation in ear and lymph node using BrdU immunohistochemistry for mouse ear swelling test

The mouse ear swelling test (MEST) was developed as an alternative to guinea pig models for measuring the contact sensitization potential. However, the MEST relies on the quantitative measurement of ear swelling by micrometer as the means of determining the endpoint. The purpose of this study was to investigate the possibility of using cell proliferation in the ear and lymph node by bromodeoxyuridine (BrdU) immunohistochemistry as a reliable marker for MEST. Female Balb/c mice were treated by the topical application of various sensitizers, 2,4-dinitrochlorobenzene (DNCB), toluene diisocyanate (TDI) and α-hexylcinnamaldehyde (HCA) and an irritant, sodium lauryl sulfate (SLS) following the protocol of MEST. The proliferation of cells in the ear and auricular lymph node was analyzed by BrdU incorporations into cells. There were significant increases in the cell proliferations of the ear and auricular lymph node in mice treated with DNCB and TDI compared to the vehicle control. All allergens and the irritant were correctly identified by the MEST using BrdU immunohistochemistry of lymph node responses. The standard MEST assay showed positive results in the case of the strong sensitizers, DNCB and TDI. However, HCA and SLS were not correctly identified in the ear swelling assay. These results suggest that the measurement of cell proliferation in the auricular lymph node using BrdU immunohistochemistry could provide a reliable marker for MEST.     

ICCVAM Evaluation of the Murine Local Lymph Node Assay: II. Conclusions and Recommendations of an Independent Scientific Peer Review Panel

The validation status of the murine local lymph node assay (LLNA), a method for assessing the allergic contact dermatitis potential of chemicals, was evaluated by an independent peer review panel (Panel) convened by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The LLNA measures lymphocyte proliferation using incorporation of radioactive thymidine or iododeoxyuridine into cells of the draining lymph nodes of mice topically exposed to a test article. The Panel concluded that the assay performed as well as currently accepted guinea pig methods [guinea pig maximization test (GPMT)/Buehler assay (BA)] for the hazard identification of strong to moderate chemical sensitizing agents, but that it might not correctly identify all weak sensitizers or metals (potential false negative response) or all strong irritants (potential false positive response). The Panel concluded also that the LLNA involves less pain and distress than conventional guinea pig methods. The Panel unanimously recommended the LLNA as a stand-alone alternative for contact sensitization hazard assessment, provided that certain protocol modifications were made. These included collection of individual, rather than pooled, animal response data; the inclusion of a concurrent positive control; and consideration of dose–response information and statistical analyses. A standardized LLNA protocol is provided.