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PubMed2002

Determination of the sensitising activity of the rubber contact sensitisers TMTD, ZDMC, MBT and DEA in a modified local lymph node assay and the effect of sodium dodecyl sulfate pretreatment on local lymph node responses.

A modified local lymph node assay (LLNA) was used to determine the sensitising activity of four chemicals used for the production of natural rubber latex products. Tetramethylthiuramdisulfide (TMTD), 2-mercaptobenzothiazole (MBT) and zincdimethyldithiocarbamate (ZDMC), three moderate human sensitisers, and diethylamine (DEA) a known human sensitiser, were epicutaneously administered on the ear and the proliferating activity in the draining (auricular) lymph node (LN) was determined by ex vivo (3)H-thymidine incorporation. Consistent results were obtained for TMTD and ZDMC with stimulation indices (SI) above 3, identifying these compounds as sensitiser, while for DEA and MBT inconsistent results were obtained. For all parameters determined such as LN weight, LN cell number, cell proliferation per 2 x 10(6) cells, and cell proliferation per LN statistical significant increases were observed. The SI, expressed as cellular proliferation per LN or per animal (left and right LN combined), was the most sensitive parameter with an optimum at day 5 after start of treatment.Furthermore, we investigated whether the use of sodium dodecyl sulfate (SDS) was able to enhance weak responses in the LLNA. SDS treatment with dosages of 10% and higher resulted in a SI above 3, while a dosage of 1% SDS showed no activity. Pretreatment with 1% SDS 1 h before application of the rubber chemicals enhanced the responses to these chemicals consistently, identifying also DEA and MBT as sensitisers. Our results indicate that SDS had synergistic activity on the LN responses of the administered rubber chemicals in the LLNA. For the moderately responding sensitisers TMTD and ZDMC both IFN-gamma and IL-4 production was observed. For the weakly responding sensitisers DEA and MBT both IFN-gamma and IL-4 cytokine production was only observed after pretreatment of the animals with 10% SDS. For 10% and 20% SDS, inducing approximately a SI of 20 in the LLNA, no induction of cytokines was observed.

Allergens↗

PubMed2002

The local lymph node assay: past, present and future.

The local lymph node assay (LLNA) was developed originally as a method for the identification of chemicals that have the potential to cause skin sensitization and allergic contact dermatitis. The assay is based on an understanding that the acquisition of contact sensitization is associated with, and dependent upon, the stimulation by chemical allergens of lymphocyte proliferative responses in skin-draining lymph nodes. Those chemicals that provoke a defined level of lymph node cell (LNC) proliferation (a 3-fold or greater increase compared with concurrent vehicle controls) are classified as skin sensitizers. Following its original inception and development, the LLNA was the subject of both national and international interlaboratory collaborative trials, and of very detailed comparisons with other test methods and with human skin sensitization data. The assay has now been validated fully as a stand-alone test for the purposes of hazard identification. In recent years, there has been a growing interest also in the use of the LLNA to assess the potency of contact allergens and in risk assessment. There is reason to believe that the extent of skin sensitization achieved is associated with the vigour of LNC proliferation induced in draining nodes. Given this relationship, the relative potency of skin sensitizing chemicals is measured in the LLNA by derivation of an EC3 value, this being the concentration of chemical required to provoke a 3-fold increase in the proliferation of LNC compared with controls. Experience to date indicates that relative potency as determined using this approach correlates closely with what is known of the activity of skin sensitizing chemicals in humans. In this article, we review the development, evaluation and validation of the LLNA for the purposes of hazard identification, and the more recent application of the method for evaluation of potency in the context of risk assessment. In addition, we consider what new applications and modifications are currently being investigated.

Allergens↗

PubMed1995

A modification of the local lymph node assay for contact allergenicity screening: measurement of interleukin-2 as an alternative to radioisotope-dependent proliferation assay.

The local lymph node assay is an effective prediction method for contact allergenicity, but employs radioisotopes. We investigated whether measurement of interleukin-2 (IL-2) production by lymph node cells could be used instead to predict contact allergenicity of chemicals. Test chemicals were applied for three consecutive days to both ears of Balb/c mice and the auricular lymph nodes were obtained on either the fourth or fifth day after the first application. Both IL-2 concentration in supernatant of the suspension and proliferative activity of lymph node cells were determined after 24-, 48-, 72-h cell culture in RPMI-1640 medium by ELISA and by measuring [3H]methylthymidine incorporation, respectively. These two methods detected allergenicity similarly except in the case of TNCB and oxazolone, which showed excessive proliferation-inducing capacity as compared to IL-2 release-increasing effect. Flow cytometry showed that these two chemicals also increased the percentage of Iad-positive cells in the lymph nodes, suggesting that these chemicals might induce not only cellular immunity but also humoral immunity. We conclude that interleukin-2 assay is a convenient and dependable method for screening strong contact allergens without using radioisotopes.

Allergens↗

PubMed1995

An international evaluation of the murine local lymph node assay and comparison of modified procedures.

The murine local lymph node assay is a predictive test for the identification of skin-sensitizing chemicals. The method has been the subject both of national inter-laboratory studies and of extensive comparisons with guinea pig tests. In the investigations reported here, the local lymph node assay has been evaluated further in the context of an international study comprising five independent laboratories. In addition, the influence of minor modifications to the standard assay procedure on the performance of the test has been examined. The modified procedures investigated were exposure of mice for 4 rather than 3 consecutive days, excision of lymph nodes 4 rather than 5 days after the initiation of exposure and the use of an alternative isotope. All five laboratories, irrespective of whether the standard or a modified protocol was used, were able to identify accurately, and with comparable sensitivity, potassium dichromate and 2,4-dinitrochlorobenzene as skin sensitizers. Using standard criteria, none of the laboratories recorded positive responses with methyl salicylate, a non-sensitizer. In the standard protocol, lymph nodes are pooled for each experimental group and the vigor of responses measured as a stimulation index relative to vehicle controls. A stimulation index of 3 or greater is considered to indicate skin-sensitizing potential. One further modification adopted by three of the laboratories was to analyze nodes from individual animals and, thereby, permit statistical evaluation. This allowed a direct comparison of statistical significance with the conventional stimulation index as criteria for a positive response. The data indicate that, while statistical evaluation may provide, in some instances, for small increases in sensitivity, this may be at the expense of some loss of selectivity. There are, however, insufficient data presently to draw firm conclusions regarding the relative value of statistical analysis. These studies demonstrate that the local lymph node assay is sufficiently robust to accommodate minor procedural and technical modifications without material changes in test performance.

Analysis of Variance↗

PubMed1991

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.

Allergens↗

PubMedDate not supplied

Evaluation of lymphocyte proliferation by immunohistochemistry in the local lymph node assay.

A modified version of the local lymph node assay (LLNA) is presented, using bromodeoxyuridine to label proliferating lymphocytes. Cell counting is done on mid-sagittal sections of individual nodes under light microscopy. Two irritants (sodium lauryl sulfate and salicylic acid), four allergens of various sensitizing potential (potassium dichromate, 4-chloroaniline, neomycin sulfate and nickel sulfate) and one chemical of unknown sensitizing potential (ethyl 3-aminobenzoate) were tested either in the short protocol using three daily topical applications and/or in the long protocol with a pre-exposure step under an occluded patch. A weak T cell proliferation was noted with both irritants in the short protocol, but not in the long protocol. Potassium dichromate induced a strong proliferative response in the short protocol. A lesser sensitizing potential was detected for 4-chloroaniline, ethyl 3-aminobenzoate and neomycin sulfate but only in the long protocol. Nickel sulfate was negative in both protocols. The long protocol was the most valuable for weak or moderate sensitizers. Histological examination of nodes ruled out intercurrent processes. The present procedure offers several advantages. The use of a non-isotopic marker enables this test to be run in a routine safety assessment department and allows the preparation of permanent slides. An increased specificity is obtained by restricting cell counting to the paracortex. Moreover, the collection of individual data permits statistical analysis of the results. This method is sensitive and reproducible and may be viewed as a useful adjunct to the LLNA.

Animals↗

PubMed1994

The local lymph node assay: developments and applications.

The murine local lymph node assay is a predictive test method for the identification of contact allergens in which sensitizing activity is measured as a function of induced proliferative responses in lymph nodes draining the site of application. In this article the development and validation of the assay are described and comparisons with guinea pig predictive test methods discussed. In addition we examine the advantages and limitations of the method and consider new opportunities and applications of the assay in the context of the toxicological evaluation of sensitizing potential.

Allergens↗

PubMed1990

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.

Allergens↗

PubMed1992

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.

Allergens↗

PubMed2002

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.

Allergens↗

PubMed1994

Experience with the murine local lymph node assay: inability to discriminate between allergens and irritants.

The murine local lymph node assay is a new predictive test for identifying contact sensitizers. It measures the proliferative response in the lymph nodes during the sensitization phase. In the present study, moderate-to-extreme allergens (from human and guinea pig experience) gave clearly positive results in this assay. However, irritants tested, i.e. sodium dodecyl sulphate (SDS), chloroform/methanol, oxalic acid, triton X-100 and methylsalicylate, also gave positive results, not distinguishable from the results with low-grade/moderate allergens. Two allergens were also tested in the presence of 10% SDS. The effect on proliferation was additive in the first case and synergistic in the second. The local lymph node assay in its present design and with the criteria used for a positive response requires further validation studies and perhaps further development before it can be accepted as an alternative to guinea pig tests for allergenicity. Substances with exclusively irritating properties could falsely be classified as allergens by the method or, alternatively, the allergenicity of chemicals with both allergenic and irritating properties could be overestimated.

Allergens↗

PubMed1996

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

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.

Animals↗

PubMedDate not supplied

Local lymph node assay: use in hazard and risk assessment.

The murine local lymph node assay (LLNA) was developed as an alternative method for the identification of chemicals that have the ability to cause skin sensitization and allergic contact dermatitis. The assay now has been evaluated extensively in the context of both national and international inter-laboratory collaborative trials and has been the subject of detailed comparisons with guinea pig test methods and human skin sensitization data. On the basis of these evaluations the LLNA has been endorsed recently by the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) as a stand-alone method for skin sensitization testing. The assay offers a number of important benefits compared with conventional guinea pig test methods, among these being provision of an objective and quantitative endpoint. Moreover, the LLNA provides advantages in the context of animal welfare; compared with guinea pig tests, fewer animals are required and these animals are subject to less trauma. It is important now that the validation status of the LLNA is recognized and the method applied widely so that these advantages may be realized. Hazard identification represents only the first step in the risk assessment process. A full toxicological evaluation of skin sensitization activity requires an understanding of relative potency. Guinea pig methods do not lend themselves readily to assessment of potency, and interest recently has focused on the utility of the LLNA for this purpose. Contained within this review article are brief descriptions of the history of the LLNA and the immunobiological basis for the method, together with detailed accounts of the conduct and interpretation of the assay. Procedural modifications to, and alternative endpoints for, the LLNA are considered also. Finally, the current regulatory status of the LLNA is summarized and the application of the method for the purposes of defining relative potency and developing risk assessments is reviewed.

Allergens↗

PubMed2001

The local lymph node assay and potential application to the identification of drug allergens.

The local lymph node assay (LLNA) is a method for the identification of skin sensitization hazard. The method is based upon measurement of proliferative responses induced in draining lymph nodes following topical exposure of mice to the test chemical. More recently the LLNA has also been used for the evaluation of relative skin sensitization potency in the context of risk assessment. Idiosyncratic drug reactions resulting from the stimulation of allergic or autoimmunogenic responses are poorly understood but represent an important clinical problem. In this article, the potential utility of the LLNA, either in a conventional modified configuration, to provide information of value in assessment the potential for systemic allergenicity is considered.

Allergens↗

PubMed2003

Assessment of statistic analysis in non-radioisotopic local lymph node assay (non-RI-LLNA) with alpha-hexylcinnamic aldehyde as an example.

The murine local lymph node assay (LLNA) is used for the identification of chemicals that have the potential to cause skin sensitization. However, it requires specific facility and handling procedures to accommodate a radioisotopic (RI) endpoint. We have developed non-radioisotopic (non-RI) endpoint of LLNA based on BrdU incorporation to avoid a use of RI. Although this alternative method appears viable in principle, it is somewhat less sensitive than the standard assay. In this study, we report investigations to determine the use of statistical analysis to improve the sensitivity of a non-RI LLNA procedure with alpha-hexylcinnamic aldehyde (HCA) in two separate experiments. Consequently, the alternative non-RI method required HCA concentrations of greater than 25% to elicit a positive response based on the criterion for classification as a skin sensitizer in the standard LLNA. Nevertheless, dose responses to HCA in the alternative method were consistent in both experiments and we examined whether the use of an endpoint based upon the statistical significance of induced changes in LNC turnover, rather than an SI of 3 or greater, might provide for additional sensitivity. The results reported here demonstrate that with HCA at least significant responses were, in each of two experiments, recorded following exposure of mice to 25% of HCA. These data suggest that this approach may be more satisfactory-at least when BrdU incorporation is measured. However, this modification of the LLNA is rather less sensitive than the standard method if employing statistical endpoint. Taken together the data reported here suggest that a modified LLNA in which BrdU is used in place of radioisotope incorporation shows some promise, but that in its present form, even with the use of a statistical endpoint, lacks some of the sensitivity of the standard method. The challenge is to develop strategies for further refinement of this approach.

Acrolein↗

PubMedDate not supplied

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.

Allergens↗

PubMed1999

Local lymph node assay: differentiating allergic and irritant responses using flow cytometry.

The murine local lymph node assay (LLNA) is a method for assessing the contact sensitization potential of chemicals. Based on events that occur during the induction phase of a contact sensitization response, the LLNA measures the in vivo proliferation of cells in the draining lymph nodes (DLNs) of mice following topical exposure to chemicals. In terms of predictive identification of important skin sensitizers, the LLNA has been shown to be at least as sensitive as, and much more reliable than, current guinea pig tests. However, proliferation has also been observed following treatment with some irritants. In an attempt to distinguish allergic from irritant-induced proliferation, flow cytometric techniques have been used to examine the phenotype of lymphocyte subsets in the DLNs as well as markers of T-lymphocyte activation and memory. Mice were treated on the ears for 3 consecutive days with allergens or irritants. The DLNs were harvested 72 h after the final treatment. Single-cell suspensions were prepared, counted, and stained for analysis of the percentages of T cells and B cells and T-cell expression of two adhesion molecules that have been associated with differentiating naïve and activated/memory T cells, CD62L (L-selectin) and CD44 (H-cam). Increases in lymph node cellularity were observed in both allergen- and irritant-treated mice relative to naïve and vehicle-treated animals. Mice treated with allergens showed a preferential increase in the percentage of B220(+) B cells compared with irritant-treated mice. Treatment with allergens, but not irritants, resulted in a selective increase in the percentages of CD4(+) and CD8(+) cells expressing the T-cell activation/memory phenotype CD62L(lo)CD44(hi). Taken together, flow cytometric analysis of cell phenotype and expression of T-cell activation/memory markers may provide important information for differentiating allergen- and irritant-induced proliferative responses in the DLNs of chemically treated mice.

Allergens↗

PubMed1999

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.

Animal Testing Alternatives↗