Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency

The assessment of the potency of a skin sensitizing chemical is a key starting point for its subsequent risk assessment/management. The Local Lymph Node Assay can provide information on the relative skin sensitizing potency of contact allergens by interpolation from the dose response curve the concentration of a chemical required to elicit a threshold positive response (EC3 value). However, interpolation requires that the dose response curve have at least one stimulation index (SI) value above and one SI value below the threshold value of 3. For instances where all test concentrations result in SI values above 3, there was a need to develop a method that would permit estimation of EC3 values. This has been achieved by log-linear extrapolation using the two lowest test concentrations from the dose response curve. Before applying this approach, it is important that data quality is assessed. The dose response must include concentrations on the linear portion of the curve and, ideally, the SI induced by the lowest dose should approach 3. Judicious use of this approach for extrapolating EC3 values can provide information on a likely potency classification for use in risk assessment and may avoid the need for repeat animal testing.     

Potency values from the local lymph node assay: Application to classification,labelling and risk assessment

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: ‘extreme’, ‘strong’, ‘moderate’ and ‘weak’. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment.     

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.     

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.     

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.     

Evaluation of an ex vivo murine local lymph node assay: multiple endpoint comparison

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity.     

Local lymph node assay responses to paraphenylenediamine: intra- and inter-laboratory evaluations.

The murine local lymph node assay (LLNA) is a method for the prospective identification of skin sensitizing chemicals. Proliferative responses induced in lymph nodes draining the site of topical application of the test chemical are measured and those chemicals that induce a stimulation index of three or more compared with concurrent vehicle-treated controls are considered to have the potential to cause skin sensitization. Dose-response data from the LLNA may be used to derive an estimate of relative skin sensitizing potency, based upon derivation of the concentration of chemical required to cause a stimulation index of 3 (EC3 value) as calculated by linear interpolation. The purpose of the present investigations was to examine the stability of LLNA responses and the consistency of derived EC3 values induced by the contact allergen paraphenylenediamine (PPD). Analyses were conducted once a month over a 4-month period in each of two independent laboratories. In all assays, and in both laboratories, PPD elicited a positive response. Although some minor differences in responses between and within laboratories were observed, the derived EC3 values were generally very consistent. In Laboratory 1, EC3 values varied between 0.06 and 0.09% PPD, whereas in Laboratory 2 the range was 0.09-0.20%. These EC3 values are consistent with clinical experience of this material insofar as it is a common and relatively potent cause of allergic contact dermatitis in humans. Taken together, these data confirm the stability of LLNA responses both with time and between laboratories and provide additional support for the use of derived EC3 values in the assessment of relative skin sensitizing potency.     

Measurement of allergenic potency using the local lymph node assay

Chemicals that can act as contact allergens have been identified successfully using guinea-pig models. However, contact allergy is still common, probably because of, at least in part, failures of risk assessment. A new method, the local lymph node assay, replaces the guinea-pig as a tool for hazard identification and offers the real prospect of accurate prediction of allergen potency, the missing link in skin sensitization risk assessment.     

Comparison of mouse strains using the local lymph node assay

The local lymph node assay (LLNA), as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), only allows for the use of CBA mice. The objective of these studies was to begin to assess the response of chemical sensitizers in the LLNA across six strains of female mice (C57BL/6, SJL/J, BALB/c, B6C3F1, DBA/2 and CBA). The moderate sensitizer alpha-hexylcinnamaldehyde (HCA) was chosen as the test chemical, while toluene diisocyanate (TDI) and 2,4-dinitrofluorobenzene (DNFB) were evaluated at single concentrations as positive controls. Draining lymph node cell proliferation following acetone exposure varied across strains. SJL mice had a significantly higher degree of proliferation with 2111 d.p.m./2 nodes. The remaining five strains demonstrated responses which ranged from 345 to 887 dpm/2 nodes. DBA/2, B6C3F1, BALB/c and CBA mice had essentially equal levels of lymph node proliferation following exposure to the three chemicals. While C57BL/6 mice gave similar results as CBA mice following DNFB and HCA administration, the LLNA response to TDI was considerably lower. SJL mice provided low stimulation indexes (SI) values for all three chemicals evaluated. Regardless of the level of LLNA response, all six mouse strains identified the sensitization potential of HCA, TDI or DNFB. Based on these studies, DBA/2, B6C3F1 and BALB/c mice are good choices for continued evaluation as additional mouse strains for use in the LLNA.     

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.     

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.     

ICCVAM evaluation of the murine local lymph node assay. The ICCVAM review process.

New test methods are being developed to improve the prediction of human and environmental risks and to benefit animal welfare by reducing, refining, and replacing animal use. Regulatory adoption of new test methods is often a complex and protracted process, requiring test method validation, regulatory acceptance, and implementation. Assessments of new test methods have not always been uniform within or among regulatory agencies. Thus, there have been increased pressures for a harmonized approach to test method evaluation and acceptance. In 1997, in response to these pressures and to U.S. Public Law 103-43, the National Institute of Environmental Health Sciences (NIEHS) established the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) to coordinate interagency consideration of new and revised test methods. This article describes the validation and acceptance criteria and process used for the first test method evaluated by ICCVAM, the murine local lymph node assay (LLNA). Based on ICCVAM's conclusions and recommendations, the LLNA has been accepted by U.S. regulatory agencies as a stand-alone assay for allergic contact dermatitis. Two related articles in this series of three present the results of the independent peer review evaluation of the LLNA and summarize the performance characteristics of the database substantiating the validity of the LLNA.     

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.     

Development of a non-radioactive endpoint in a modified local lymph node assay.

A murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for contact sensitization testing. Although the LLNA appears to be a little less sensitive than the most stringent of guinea pig assays, it provides a rapid, objective, quantitative and cost-effective method for screening strong contact sensitizers and has advantages with respect to animal welfare. However, a potential disadvantage is the need for the use of radioactive material. We have reported previously that an ex vivo assay based on similar principles to the original in vivo LLNA, but using a non-radioactive endopoint, was valid for the prediction of strong sensitizers. This ex vivo assay was not sensitive enough to allow prediction of moderately potent ones. In this study, we propose a new parameter, Corrected IL-2 Index (CII), for the prediction of moderate sensitizers. To obtain CII the IL-2 release in the supernatant of the cell culture is corrected for lymph node weight ratio and ratio of CD4-positive subset. We found that CII predicted the allergenicity of moderate sensitizers, including the ones recommended by the OECD in guideline 406, such as mercaptobenzothiazole and hexyl cinnamic aldehyde. The allergenicity of metal salts, such as potassium dichromate, ammonium tetrachloroplatinate and cobalt chloride, was also predicted by the CII. We conclude that the use of CII as an index significantly increases the sensitivity of the ex vivo method so that moderate sensitizers may also be detected.     

Use of the local lymph node assay in assessment of immune function

The murine local lymph node assay (LLNA) was originally developed as a predictive test method for the identification of chemicals with sensitizing potential. In this study we demonstrated that an adapted LLNA can also be used as an immune function assay by studying the effects of orally administered immunomodulating compounds on the T-cell-dependent immune response induced by the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). C57Bl/6 mice were treated with the immunotoxic compounds cyclosporin A (CsA), bis(tri-n-butyltin)oxide (TBTO) or benzo[a]pyrene, (B[a]P). Subsequently, cell proliferation and interferon-gamma (IFN-gamma) and interleukin (IL)-4 release were determined in the auricular lymph nodes (LNs) after DNCB application on both ears. Immunosuppression induced by CsA, TBTO and B[a]P was clearly detectable in this application of the LLNA. Cytokine release measurements proved valuable to confirm the results of the cell proliferation assay and to obtain an indication of the effect on Th1/Th2 balance. We believe to have demonstrated the applicability of an adapted LLNA as an immune function assay in the mouse.     

The reduced local lymph node assay: the impact of group size

The local lymph node assay (LLNA) is a skin sensitization test that provides animal welfare benefits. To reduce animal usage further, a modified version (rLLNA) was proposed. Conducting the rLLNA as a screening test with a single high dose group and vehicle control differentiated accurately between skin sensitizers and non-sensitizers. This study examined whether a reduction in animal number/group is feasible. Historical data were utilized to examine the impact of conducting the rLLNA with two mice/group. To assess the effect on the stimulation index (SI) 41 datasets with individual animal data derived using five mice/group were analysed. SIs were calculated on all possible combinations of two control and two high dose group disintegrations per minute (dpm) values. For 25 of 33 sensitizer datasets, > 96% of possible dpm combinations resulted in a calculated SI > 3. The lowest percentages of positive SIs were observed with weak allergens when, in the standard LLNA, the mean SIs would have been nearer to the threshold value of 3. The results indicate that moderate, strong and extreme allergens are more likely than weak allergens to be identified as sensitizers when group sizes of two mice are used within the rLLNA. It is concluded that a rLLNA with two mice/group would display decreased sensitivity and is inappropriate for use in hazard identification.     

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.     

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.     

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.     

Immunophenotyping does not improve predictivity of the local lymph node assay in mice

The local lymph node assay (LLNA) is a regulatory accepted test for the identification of skin sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. However, there is evidence that LLNA is overestimating the sensitization potential of certain substance classes in particular those exerting skin irritation. Some reports describe the additional use of flow cytometry‐based immunophenotyping to better discriminate irritants from sensitizing irritants in LLNA. In the present study, the 22 performance standards plus 8 surfactants were assessed using the radioactive LLNA method. In addition, lymph node cells were immunophenotyped to evaluate the specificity of the lymph node response using cell surface markers such as B220 or CD19, CD3, CD4, CD8, I‐A^κ and CD69 with the aim to allow a better discrimination above all between irritants and sensitizers, but also non‐irritating sensitizers and non‐sensitizers. However, the markers assessed in this study do not sufficiently differentiate between irritants and irritant sensitizers and therefore did not improve the predictive capacity of the LLNA. Copyright © 2014 John Wiley & Sons, Ltd.