B cell increases and ex vivo IL-2 production as secondary endpoints for the detection of sensitizers in non-radioisotopic local lymph node assay using flow cytometry

Non-radioisotopic local lymph node assay (LLNA) using 5-bromo-2′-deoxyuridine (BrdU) with flow cytometry (FCM) is gaining attention since it is free from the regulatory issues in traditional LLNA (tLLNA) accompanying in vivo uses of radioisotope, ³H-thymidine. However, there is also concern over compromised performance of non-radioisotopic LLNA, raising needs for additional endpoints to improve the accuracy. With the full 22 reference substances enlisted in OECD Test Guideline No. 429, we evaluated the performance of LLNA:BrdU-FCM along with the concomitant measurements of B/T cell ratio and ex vivo cytokine production from isolated lymph node cells (LNCs) to examine the utility of these markers as secondary endpoints. Mice (Balb/c, female) were topically treated with substances on both ears for 3 days and then, BrdU was intraperitoneally injected on day 5. After a day, lymph nodes were isolated and undergone FCM to determine BrdU incorporation and B/T cell sub-typing with B220⁺ and CD3e⁺. Ex vivo cytokine production by LNCs was measured such as IL-2, IL-4, IL-6, IL-12, IFN-γ, MCP-1, GM-CSF and TNFα. Mice treated with sensitizers showed preferential increases in B cell population and the selective production of IL-2, which matched well with the increases in BrdU incorporation. When compared with guinea pig or human data, BrdU incorporation, B cell increase and IL-2 production ex vivo could successfully identify sensitizers with the accuracy comparable to tLLNA, suggesting that these markers may be useful for improving the accuracy of LLNA:BrdU-FCM or as stand-alone non-radioisotopic endpoints.     

Development of non-radio isotopic endpoint of murine local lymph node assay based on 5-bromo-2'-deoxyuridine (BrdU) incorporation

Allergic contact dermatitis is a serious health problem. Over the last decade, the murine local lymph node assay (LLNA) has been developed to detect chemical allergens, and international validation studies have been conducted. We have tried to establish an alternative non-radioisotopic endpoint for the LLNA by using 5-bromo-2'-deoxyuridine (BrdU) incorporation in place of radioisotopes, such as [3H]thymidine, employed in the standard method. BrdU was given as a single administration at 5 mg/animal 2 days following three consecutive daily applications of a test chemical. BrdU incorporation into draining lymph node cells was measured using an enzyme immunosorbent assay technique. In this study, p-benzoquinone(PBQ), trimellitic anhydride (TMA), citral(CT) and dextran (DEX) were used as pilot chemicals. PBQ, TMA and CT, which are classified as moderate to strong sensitizers in the guinea pig maximization test and were positive in the original LLNA, were also found to elicit positive responses in the alternative LLNA using BrdU incorporation. In contrast, DEX tested negative in the modified assay consistent with previous guinea pig and LLNA data. Consequently, the modified LLNA endpoint using BrdU incorporation may represent a useful alternative to the standard assay in situations, where there is a need to avoid the use of radioisotopes.     

Inter‐laboratory validation of the modified murine local lymph node assay based on 5‐bromo‐2′‐deoxyuridine incorporation

The murine local lymph node assay (LLNA) is a well‐established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. ( 2001 ) has developed a modified LLNA based on the 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation (LLNA:BrdU‐ELISA). The LLNA:BrdU‐ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU‐ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical‐treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut‐off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU‐ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively. Copyright © 2010 John Wiley & Sons, Ltd.     

Advantage of using CBA/N strain mice in a non-radioisotopic modification of the local lymph node assay

The murine local lymph node assay (LLNA) is currently recognized as a stand-alone test method for determining the skin sensitizing potential of chemicals. It has been incorporated into the official test guidelines published by some authorities, including the OECD. To avoid the use of radioisotopes, efforts have been made recently to develop non-radioisotopic modifications of the LLNA. A non-radioisotopic modification of the LLNA was developed previously using 5-bromo-2'-deoxyuridine (BrdU) incorporation (non-RI LLNA). However, the non-RI LLNA was found to be somewhat less sensitive than the standard assay. This study reports the advantage of using mice of the CBA/N strain in the non-RI LLNA to improve the sensitivity of this method. The non-RI LLNA was performed using CBA/JN and CBA/N mice exposed to one of four confirmed skin sensitizers, 2,4-dinitrochlorobenzene (DNCB), eugenol (EG), isoeugenol (IEG) or alpha-hexylcinnamic aldehyde (HCA), and to one non-sensitizer, propylene glycol (PG). The EC3 values for DNCB, IEG, EG, HCA and PG were calculated to be 0.1%, 9.6%, 40.6%, 45.5% and >50% in CBA/JN mice and 0.08%, 1.9%, 10.7%, 20.3% and >50% in CBA/N mice, respectively. The EC3 values for DNCB, IEG, EG, HCA and PG in the standard LLNA using CBA/Ca mice and radioisotopes were reported elsewhere as being 0.08%, 1.3%, 13.0%, 8.0% and >50%, respectively. The EC3 values derived from the CBA/N mice in the non-RI LLNA were nearly equivalent to the EC3 values obtained using the standard radioisotopic LLNA with CBA/Ca mice. These data suggest that the use of CBA/N mice may provide a realistic opportunity to develop a version of the LLNA that does not have a requirement for the use of radioisotopes, but which nevertheless has sensitivity approaching, or comparable to, the standard method.     

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.     

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.     

Assessment of the skin sensitization potency of eugenol and its dimers using a non-radioisotopic modification of the local lymph node assay

Allergic contact dermatitis is a serious health problem. There is a need to identify and characterize skin sensitization hazards, particularly with respect to relative potency, so that accurate risk assessments can be developed. For these purposes the murine local lymph node assay (LLNA) was developed. Here, we have investigated further a modi fi cation of this assay, non-radioisotopic LLNA, which in place of tritiated thymidine to measure lymph node cell proliferation employs incorporation of 5-bromo-2'-deoxyuridine. Using this method we have examined the skin sensitizing activity of eugenol, a known human contact allergen, and its dimers 2,2'-dihydroxyl-3,3'-dimethoxy-5,5'-diallyl-biphenyl (DHEA) and 4,5'-diallyl-2'-hydroxy-2,3'-dimethoxy phenyl ether (DHEB). Activity in the guinea pig maximization test (GPMT) also measured. On the basis of GPMT assays, eugenol was classified as a mild skin sensitizer, DHEA as a weak skin sensitizer and DHEB as an extreme skin sensitizer. In the non-radioisotopic LLNA all chemicals were found to give positive responses insofar as each was able to provoke a stimulation index (SI) of >or=3 at one or more test concentrations. The relative skin sensitizing potency of these chemicals was evaluated in the non-radioisotopic LLNA by derivation of an ec(3) value (the concentration of chemical required to provoke an SI of 3). The ec(3) values calculated were 25.1% for eugenol, >30% for DHEA and 2.3% for DHEB. Collectively these data suggest that assessments of relative potency deriving from non-radioisotopic LLNA responses correlate well with evaluations based on GPMT results. These investigations provide support for the proposal that the non-radioisotopic LLNA may serve as an effective alternative to the GPMT where there is a need to avoid the use of radioisotopes.     

Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers

The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [³H]methyl thymidine. A more recent non‐isotopic variation of the assay utilizes bromodeoxyuridine (BrdU) incorporation in vivo. To further improve the utility of this assay, we developed an ex vivo BrdU labeling procedure eliminating the need for in vivo injections. The results of this assay correctly identified a strong sensitizer (i.e., trimellitic anhydride) as well as weak/moderate sensitizers (i.e., eugenol, cinnamaldehyde and hexylcinnaminic aldehyde). As anticipated, neither non‐sensitizers isopropanol and lactic acid nor the false negative chemical nickel II sulfate hexahydrate induced a positive threshold response in the assay. The results of this assay are in close agreement with those of the in vivo LLNA:BrdU‐enzyme‐linked immunosorbent assay labeling procedure. We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate, ammonium tetrachloroplatinate and cis‐diamminedichloroplatinum(II) and the assay correctly identified them as sensitizers based on the calculation of EC₂ values. We conclude that this ex vivo BrdU labeling method offers predictive capacity comparable to previously established LLNA protocols while eliminating animal injections and the use of radioisotope. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

A nonradioisotopic endpoint for measurement of lymph node cell proliferation in a murine allergic contact dermatitis model,using bromodeoxyuridine immunohistochemistry

INTRODUCTION: The murine local lymph node assay (LLNA) was developed as an alternative to guinea pig models for the assessment of the xenobiotic contact sensitization potential. However, it would be advantageous to have an alternative endpoint to the usual radioisotopic-dependent measures. In the present study, we investigated the development of a nonradioisotopic endpoint for LLNA using immunohistochemistry. METHODS: Female Balb/c mice were treated by the topical application of strong sensitizers, 2,4-dinitrochlorobenzene (DNCB) and toluene diisocyanate (TDI), and a strong irritant, sodium lauryl sulfate (SLS), on the dorsum of both ears once daily for three consecutive days. The proliferation of cells in the auricular lymph node and ears was analyzed by means of the labeling index (LI) of bromodeoxyuridine (BrdU) incorporation into cells. RESULTS: Skin reactions, consisting of increased ear thickness and the presence of inflammatory cell infiltrates, were observed in mice treated with DNCB and TDI. The cell number and the weight of the lymph nodes in the mice treated with the allergens, DNCB and TDI, were increased compared to vehicle control. We observed an increase in the areas of the B220(+) cells in the lymph nodes of mice treated with allergens, as determined by immunohistochemistry. There was an increase in the percentage of B220(+) cells in mice treated with DNCB and TDI compared to the vehicle control, but not in those treated with SLS. Because we observed an increase in the percentage of B cells in the allergen-treated group, we measured the stimulation index (SI) in the cortex and medulla (C+M) of the lymph node. The SI values of the C+M in the lymph nodes of the mice treated with DNCB and TDI were increased more than threefold compared with that of the control. However, the SI of the C+M in the lymph nodes of the mice exposed to 25% SLS was not significantly increased compared to the vehicle control, although the lymph node weight of the SLS group was significantly increased. DISCUSSION: In Balb/c mice, BrdU immunohistochemistry showed its potential use for the identification and differentiation of chemicals with the capacity to induce irritation and sensitization. The results suggest that the measurement of the SI in the cortex and medulla of the lymph node using BrdU immunohistochemistry could provide a useful method to screen irritants and allergens.     

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.     

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.     

Novel approach for classifying chemicals according to skin sensitizing potency by non-radioisotopic modification of the local lymph node assay

The murine local lymph node assay (LLNA) is currently recognized as a stand-alone sensitization test for determining the sensitizing potential of chemicals, and it has the advantage of yielding a quantitative endpoint that can be used to predict the sensitization potency of chemicals. The EC3 has been proposed as a parameter for classifying chemicals according to the sensitization potency. We previously developed a non-radioisotopic endpoint for the LLNA based on 5-bromo-2'-deoxyuridine (BrdU) incorporation (non-RI LLNA), and we are proposing a new procedure to predict the sensitization potency of chemicals based on comparisons with known human contact allergens. Nine chemicals (i.e. diphencyclopropenone, p-phenylenediamine, glutaraldehyde, cinnamicaldehyde, citral, eugenol, isopropyl myristate, propyleneglycol and hexane) categorized as human contact allergen classes 1-5 were tested by the non-RI LLNA with the following reference allergens: 2,4-dinitrochlorobenzene (DNCB) as a class 1 human contact allergen, isoeugenol as a class 2 human contact allergen and alpha-hexylcinnamic aldehyde (HCA) as a class 3 human contact allergen. Consequently, nine test chemicals were almost assigned to their correct allergen class. The results suggested that the new procedure for non-RI LLNA can provide correct sensitization potency data. Sensitization potency data are useful for evaluating the sensitization risk to humans of exposure to new chemical products. Accordingly, this approach would be an effective modification of LLNA with regard to its experimental design. Moreover, this procedure can be applied also to the standard LLNA with radioisotopes and to other modifications of the LLNA.     

Modulation of intracellular cytokines 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 populations of intracellular cytokine producing cells and to analyze the expression of mRNA levels in the lymph node (LN) cells following allergen and irritant. Female Balb/c mice were treated by the topical application on the dorsum of both ears with strong sensitizers, 2,4-dinitrochlorobenzene (DNCB) and toluene diisocyanate (TDI) and a strong irritant, sodium lauryl sulfate (SLS), once daily for 3 consecutive days. The lymph node cells were harvested 72h after the final treatment. The analysis of intracellular cytokine cell in LN cells was performed with a flow cytometry. Mice were treated with DNCB and TDI showed a preferential increase in the percentage of CD4+IL-2+ cells compared with vehicle and irritant-treated mice. There was an increase in CD4+IFN-g+ cells of mice treated with DNCB and TDI, 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 CD4+IL-4+ cells compared with vehicle and irritant-treated mice. There was an increase in the mRNA level for interleukin 4 (IL-4) in mice treated with DNCB and TDI, but no significant increases were observed in mice treated with SLS. These results suggest that the population of interferon-gamma (IFN-g+) and IL-4+ cells on CD4+ cells and the mRNA expression for IL-4 in lymphocytes could be selectively modulated in allergen-treated mice.     

Comparison of BALB/c and CBA/J mice for the local lymph node assay using bromodeoxyuridine with flow cytometry (LLNA: BrdU-FCM)

The local lymph node assay using 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA that is used to identify skin sensitizers by counting BrdU-incorporated lymph node cells (LNCs) with flow cytometry. Unlike other LLNA methods (OECD TG 429, 442A and 442B) in which the CBA/J mouse strain is used, LLNA: BrdU-FCM was originally designed to be compatible with BALB/c, a mouse strain that is more widely used in many countries. To justify the substitution of CBA/J for BALB/c, the equivalence of the test results between two strains shall be established prior to the official implementation of LLNA: BrdU-FCM. This study aims to compare the test results of LLNA: BrdU-FCM produced in BALB/c mice with those in CBA/J mice for 18 reference substances, including 13 sensitizers and 5 non-sensitizers, listed in OECD Test Guideline 429. Based on the LLNA: BrdU-FCM test procedure, we selected an appropriate solvent and then performed preliminary tests to determine the non-irritating dose ranges for the main study, which revealed the difference in the irritation responses to 8 of the 18 chemicals between the two strains. In the main study, we measured the changes in the number of total LNCs, which indicated differences in the responses to test chemicals between the two strains. However, the stimulation index obtained with the counts of BrdU-incorporated LNCs with 7-AAD using flow cytometry yielded comparable results and 100% concordance between the BALB/c and CBA/J mouse strains was achieved, suggesting that the performance of LLNA: BrdU-FCM using BALB/c mice was equivalent to that with CBA/J mice.     

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.     

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.     

An in vitro human skin test for assessing sensitization potential

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold‐standard method for identification and characterization of skin‐sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in‐vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro‐haptens, respiratory sensitizers, non‐sensitizing chemicals (including skin‐irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non‐sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in‐vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.     

Increased cell proliferation in spleen and lymph nodes peripheral to contact allergen application site

The local lymph node assay (LLNA) is widely used to identify chemicals that are contact sensitizers. The assay involves dosing mice with the chemical on both ears and pooling the superficial parotid lymph nodes for assessment of lymphocyte proliferation as a marker of sensitization. The present study explored potential reduction in animal usage by dosing one ear with the allergen and the other with vehicle-only. The respective draining lymph nodes were processed separately for tritiated thymidine (³H-TdR) incorporation. Cell proliferation in proper axillary and renal nodes, as well as in the spleen was also assessed. Cross-contamination of the chemicals from the dosed ears to other parts of the body via preening was prevented by dosing restrained animals and washing off the residual chemical with saline after 4 h. Dosing the left ear with 0.02% oxazolone (OX) on unrestrained animals resulted in marked cell proliferation in its draining lymph node (stimulation index, SI = 12.8) and in the lymph node draining the contra-lateral vehicle-dosed ear (SI = 6), as well as the proper axillary lymph nodes (SI = 3.3). Increased ³H-TdR incorporation was not observed in the renal lymph nodes (SI = 1.1). Similar stimulation of cells was observed in the lymph node draining the ear contra-lateral to the 30% hexylcinnamaldehyde (HCA)-dosed ear. Increased proliferative activity was observed in contra-lateral draining lymph nodes of restrained mice demonstrating that these results cannot be attributed to cross-contamination of adjacent skin. A significant increase in proliferation of splenocytes was also observed. It is concluded that dermal application of a contact allergen, as exemplified by OX and HCA, may induce cell proliferation in the neighboring lymph nodes and spleen indicative of hapten and/or haptenated proteins diffusing through the skin to peripheral nodes and the blood to produce systemic sensitization. It is also possible that lymphatic capillaries may communicate between the left and right side of the mouse head. Thus the contra-lateral draining superficial parotid node cannot be used as a control for application of contact allergen to a single ear in a modified LLNA.     

Examination of the Local Lymph Node Assay for Use in Contact Sensitization Risk Assessment

The purpose of this study was to evaluate the utility of themurine local lymph node assay (LLNA) for contact sensitizationrisk assessment. Cellular proliferative activity in draininglymph nodes was determined for individual animals on Day 5 followingfour daily epicutaneous applications of the test chemical tothe ears. Seventeen chemicals were tested, covering a rangeof materials including preservatives, drug actives, and perfumeraw materials. The assay was found to be useful for identifyingstrong, moderate, and some weak sensitizers as defined by othertesting methods (guinea pig, human). For evaluating the antigenspec ificity of the LLNA proliferative response, an in vitroblastogenesis assay was used. Dendritic cells (DC) isolatedfrom lymph nodes of mice treated 24 hr earlier with trinitrochlorobenzene(TNCB) were capable of in vitro stimulation of lymphocytes fromTNCB-sensitized mice, but not lymphocytes from mice sensitizedto the preservative mixture of 5-chloro-2-methylisothiazolinoneplus 2-methylisothiazolinone (MCI/MI). Conversely, DC from micetreated 24 hr earlier with MCI/MI were capable of stimulatinglymphocytes from MCI/MI-sensitized mice, but were unable tostimulate lymphocytes from TNCB-sensitized mice, demonstratingthe specificity of the response. The results of these studiessupport the use of the marine LLNA for both investigative andpredictive contact sensitization testing. The LLNA offers theadvantages of requiring less time for completion, incorporatingan objective endpoint, requiring approximately half the numberof animals, and being less costly than most currently employedguinea pig test methods. In addition, we believe the murineLLNA is a useful test to incorporate into a scheme for contactsensitization risk assessment. The major advantage of this approachis that the LLNA will provide information which will allow oneto proceed directly to confirmatory human predictive testingwithout performing guinea pig testing.