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PubMed1992

Comparison of the local lymph node assay with the guinea-pig maximization test for the detection of a range of contact allergens.

The guinea-pig maximization test (GMPT) has been in use as a method for the prediction of skin sensitization potential for over 20 years, and is widely accepted by regulatory authorities because of its reliable detection of a wide variety of potential human contact allergens. Nevertheless, the method has some limitations and drawbacks, including the use of an adjuvant, the injection of the test substance at induction thus bypassing the normal skin barrier and metabolic function, a subjective endpoint, interference by irritant and/or coloured chemicals, and a relatively long and complex protocol. To address these points, an alternative technique, the local lymph node assay (LLNA), has been proposed and has become the focus of much attention. Recent data from interlaboratory trials have shown a good level of agreement between test facilities and with existing guinea-pig data. The present work investigated the correlation between LLNA results and those derived from the GPMT for 40 chemicals covering a range of chemical types and levels of skin sensitization potential. The LLNA assay was capable of detecting chemicals that exhibit a strong sensitization potential in the GPMT. For chemicals classified as moderate sensitizers in the GPMT, the LLNA was usually positive or provided an indication of sensitizing activity (that was not sufficient to satisfy the current criteria for regarding the result as positive). Weaker sensitizers in the GPMT were usually not detected by the LLNA. With the single exception of copper chloride, non-sensitizers were not positive in the LLNA. The results support the view that the LLNA can provide a rapid and objective screening test for strong sensitizers.

Allergens↗
PubMed1999

Structure-activity relationships in the murine local lymph node assay for skin sensitization: alpha,beta-diketones.

The biological activity of skin-sensitizing chemicals is related to their ability to react, either directly or after metabolic activation, with appropriate skin proteins. For direct acting electrophilic compounds, this ability can be modelled, using the RAI (relative alkylation index) approach, by a combination of electrophilicity and hydrophobicity parameters. Several structure-activity relationships based on this approach have been reported, but most of them cover guinea pig sensitization test data on what chemists would classify as relatively soft electrophilic chemicals. In the present work, an electrophilicity parameter based on Taft substituent constants is derived for hard electrophiles having a reactive carbonyl group, and is used to calculate RAI values for the analysis of sensitization test data obtained in the murine local lymph node assay (LLNA) for a series of alpha, beta-diketones. The sensitization potential of these reactive hard electrophilic carbonyl compounds in the LLNA shows a good correlation with the RAI. Overall, the findings reaffirm our view that physical organic chemistry is the key to understanding why some chemicals sensitize more strongly than others, while some do not sensitize at all, and provide further evidence of the value of the LLNA for SAR studies.

Animals↗
PubMed1998

A modified murine local lymph node assay for the differentiation of contact photoallergy from phototoxicity by analysis of cytokine expression in skin-draining lymph node cells.

Since predictive differentiation of photoallergenic from phototoxic reactions, induced by low molecular weight compounds, represents a current problem, we tried to improve the differentiation between the two reactions by using a modified protocol of the local lymph node assay (LLNA). Briefly, groups of female BALB/c mice received compound solution or vehicle alone on the dorsum of both ears on 3 consecutive days. Immediately after compound application indicated groups of mice were exposed to a UVA light-dose of 10 J/cm2. Auricular lymph nodes draining the ear tissue were excised 24 h following the last exposure. Evaluation consisted of assessing lymph node weights and cell counts to monitor organ hyperplasia and in vivo-proliferative events following substance application. Furthermore, we analysed cytokine gene transcription in freshly prepared lymph node cells (LNC) and the cytokine release in vitro by restimulated CD4+ T-cells and antigen presenting cells (APC), both purified from the skin-draining lymph nodes. Both contact (photo) allergenic (oxazolone and tetrachlorosalicylanilide) and phototoxic substances (8-methoxypsoralen and acridine) caused a dose dependent increase in lymph node weights and cell counts pointing to an inflammatory process in the lymph nodes. Analysis of cytokine gene transcription ex vivo and cytokine release in vitro revealed that during the induction phase of contact (photo) allergy CD4+ T-cells produced IL-2 and IFN-gamma as well as IL-4 and IL-10, whereas IL-6 was derived from APC. In contrast, phototoxic reactions caused only an upregulation of IL-2 and IFN-gamma. Furthermore, we demonstrate that the release of IL-4 and IL-10 by CD4+ T-cells was clearly increased, whereas IL-6 and IFN-gamma expression was reduced or not changed following a challenge with contact (photo) allergens revealing an allergy-indicative shift in cytokine expression. In conclusion, our results show that contact photoallergenic reactions could be differentiated from phototoxic events by analysis of LNC cytokine expression patterns.

Animals↗
PubMed1994

The performance of the local lymph node assay with chemicals identified as contact allergens in the human maximization test.

For many years, tests in the guinea pig have been the favoured option for the identification of the skin sensitization potential of chemicals. However, the mouse has been used widely in immunology research and can represent a viable alternative. A variety of murine assays have been described, including several methods based on ear swelling as an endpoint. Another option is to assess induced lymph node cell proliferation and it is this which forms the basis of the murine local lymph node assay (LLNA). The LLNA has undergone several successful interlaboratory validations and compares well with standard guinea pig assays. In the present study, the performance of the LLNA was examined with chemicals tested previously in the human maximization test (HMT). 30 chemicals, 23 of which proved positive in the HMT and seven of which were negative, have been tested. All but four of the materials found positive in the HMT also tested positive in the LLNA. Of these four, sulfanilamide and paraben esters would not classify as skin sensitizers in the guinea pig maximization test and nickel has been found to yield variable results in a number of predictive animal tests. Of the seven substances which proved negative in the HMT, six were also negative in the LLNA, the exception being sodium dodecyl sulfate. These data demonstrate that the LLNA is able to identify accurately chemicals which have the potential to cause significant allergic contact dermatitis in humans.

Allergens↗
PubMed1993

Influence of irritants on lymph node cell proliferation and the detection of contact sensitivity to metal salts in the murine local lymph node assay.

Dimethyl sulfoxide (DMSO) and sodium lauryl sulfate (SLS) are known to cause irritation of the skin, and to enhance the penetration of chemicals into the epidermis. In the present study, the lymph node cell (LNC) proliferative response following exposure to irritants, such as SLS and DMSO, was examined in the murine local lymph node assay (LLNA). Exposure to DMSO or SLS aqueous solution induced a small increase in lymph node cell proliferation compared with aqueous solution alone. Exposure to SLS in DMSO caused a significant increase in LNC proliferation. Further, the effect of addition of the irritants in a vehicle on the detection of contact sensitivity to metal allergens was examined. Application of potassium dichromate and nickel sulfate in DMSO or SLS aqueous solution caused increases in LNC proliferation. Exposure to metal allergen with SLS in DMSO also induced a significant LNC proliferative response, but did not induce a significant increase in stimulation index (increase in 3H-thymidine incorporation relative to vehicle-treated control group). This was because of increased 3H-thymidine incorporation following exposure to SLS-DMSO in the control group. These results suggest that irritants enhance the LNC proliferative responses to metal allergens. The use of SLS in aqueous solution is effective for the detection of sensitivity to water-soluble allergens, such as metal allergens, in the LLNA, as well as the use of DMSO as an application vehicle.

Animals↗
PubMed2007

Electrophilic chemistry related to skin sensitization. Reaction mechanistic applicability domain classification for a published data set of 106 chemicals tested in the mouse local lymph node assay.

This article presents an overview of electrophilic reaction mechanisms relevant to skin sensitization, with reference to a published skin sensitization test data set for 106 chemicals. Where appropriate to aid the interpretation, additional data on a small number of further compounds are also discussed. It is shown that there is a close correspondence in the way differences and similarities in skin sensitization potency of chemicals relate to differences and similarities in their physical organic chemistry and electrophilic reaction mechanistic chemistry. The 106 chemicals are classified into their reaction mechanistic applicability domains, and reactivity-sensitization trends are analyzed for each domain: the Michael acceptor and pro-Michael acceptor electrophile domain; the SNAr electrophile domain; the SN2 electrophile domain; the Schiff base electrophile domain; the acyl transfer electrophile domain; and the non-electrophilic non-pro-electrophilic domain. The last of these domains should be populated mainly by non-sensitizers. Classification of 87 of the 106 compounds, using these domains, was straightforward. In most of the domains and subdomains where there are sufficient compounds, clear trends can be seen, in conformity with the Relative Alkylation Index (RAI) model, between sensitization potential and reactivity/hydrophobicity. Of the remaining 19 compounds, 7 are alpha-X-methyl-gamma-lactones that on the basis of published organic chemistry studies and guinea pig sensitization data can be classed as pro-Michael acceptors by elimination of HX but that are mostly negative in the LLNA, indicating a difference in bioactivation capabilities between mice and guinea pigs. The other 12 compounds, whose chemistry was not immediately obvious, were found after further analysis and literature research to fit into appropriate mechanistic domains that rationalize their skin sensitizing properties.

Animals↗
PubMed2002

Cytokine production induced by low-molecular-weight chemicals as a function of the stimulation index in a modified local lymph node assay: an approach to discriminate contact sensitizers from respiratory sensitizers.

In general, contact sensitizers have been shown to selectively induce Th1 immune responses, such as interferon-gamma (IFN-gamma) production, whereas Th2 responses, such as interleukin (IL)-4 production, were seen after exposure to respiratory allergens. However, these features may be dependent on the dose of the particular allergen. Therefore, the aim of the present study was to investigate the distinction between contact sensitizers and respiratory allergens, by establishing dose-dependent cytokine profiles. The contact allergens 2,4- dinitrochlorobenzene (DNCB), hexyl cinnamic aldehyde (HCA), and oxazolone (OXA, 4-ethoxymethylene 2-phenyloxazol-5-one) as well as the respiratory allergens fluorescein 5-isothiocyanate, phthalic anhydride, toluene diisocyanate, and trimellitic anhydride were tested. For a range of concentrations, both proliferative responses and cytokine production were established. Estimated concentrations were derived at several stimulation indices (SIs). From the estimated concentrations, IFN-gamma, IL-4, and IL-10 production as a function of stimulation indices were plotted. All four respiratory allergens showed significantly higher IL-4 and IL-10 production patterns compared to the contact allergens. Positive identification of DNCB, HCA, and OXA as contact allergens on the basis of IFN-gamma production was observed only at very high stimulation indices (SI >or= 35) for DNCB and OXA and at low SIs (SI <or= 7) for HCA. We propose that, by direct linkage of proliferation and cytokine production in a dose-response manner, distinguishing contact allergens from respiratory allergens may be improved compared to present approaches.

Administration, Inhalation↗
PubMed1996

Effect of topical cis-urocanic acid on local lymph node activation during contact sensitization in mouse, rat and guinea-pig.

Cis-urocanic acid (cUCA) has been suggested as a mediator of impairment of contact hypersensitivity induction by ultraviolet B (UVB) irradiation. We ascertained whether topical cUCA influences local lymph node activation during induction of contact hypersensitivity. Topical cUCA or vehicle was applied during the local lymph node assay to oxazolone. Local lymph node weight and cell number were assessed in all animals. Additionally, cell proliferation rate was studied in Hartley guinea-pigs and CBA/Ca mice, whereas activation of antigen-presenting cells was quantified in NMRI mice and Wistar rats. Topical cUCA suppressed all parameters of local lymph node activation due to oxazolone application in guinea-pigs. No effect, with the exception of a suppression of antigen-presenting cell activity, was seen in mice. No effect was seen in rats. The study shows that topical cUCA may suppress local lymph node activation during contact sensitization and suggests that differences between the effect of cUCA in different animal species may exist.

Animals↗
PubMed2002

Sodium metasilicate hypersensitivity in BALB/c mice.

BACKGROUND: Sodium metasilicate (SMS) is a key ingredient for a number of industrial and consumer products. Although little is known about potential for this chemical to cause allergic reactions, a similar silicate compound, sodium silicate, was reported to elicit IgE-mediated contact urticaria. OBJECTIVE: The aim of this study was to evaluate the potential for sodium metasilicate to elicit an allergic response in female BALB/c mice after dermal exposure. METHODS: The primary irritancy assay (IA), local lymph node assay (LLNA), and a mouse ear swelling test (MEST) were used to evaluate the hypersensitivity response elicited by SMS exposure. An evaluation of lymph node subpopulations, cytokine mRNA expression, and serum IgE levels was also conducted. RESULTS: SMS caused significant dermal irritation at concentrations >or=6% and an allergic response after mice were sensitized with 4% SMS then challenged with 6% SMS in the MEST. Lymph node cell proliferation was not observed in the LLNA after treatment with SMS (2% to 6% SMS). Increases in lymph node cellularity, the percentage of B cells, and the expression of certain cytokine mRNAs were observed in mice treated with SMS. Changes in the concentration of serum IgE after SMS treatment, however, were not observed. CONCLUSIONS: SMS appears to elicit a chemical hypersensitivity response in mice, as indicated by the MEST, but not by the LLNA. Increases in auricular lymph node cellularity, the percentage of B cells, and certain cytokine mRNAs support classifying SMS as a weak chemical allergen.

Allergens↗
PubMed2005

Quantitative comparison of the results obtained by the multiple-dose guinea pig maximization test and the non-radioactive murine local lymph-node assay for various biocides.

We compared the results of the multiple-dose guinea pig maximization test (GPMT) and the non-radioactive murine local lymph-node assay (LLNA) for various biocides. Thirteen out of 17 positive biocides in the GPMT gave positive results in the LLNA. In the GPMT, the minimum first induction doses ranged over four orders (0.00005-0.5%), while elicitation-threshold doses, which were evaluated using an optimally sensitized group of animals in the multiple-dose studies, ranged over five orders (0.00006-2.8%). In the LLNA, minimum induction doses ranged over more than three orders (0.01-30%). With respect to 13 biocides that were positive in both the GPMT and the LLNA, results were quantitatively compared. When compared after conversion to corresponding area doses (microg/cm), the minimum doses required to elicit skin reaction in guinea pigs were always lower than that for induction in mice with all biocides. Correlation between minimum induction doses from the GPMT and the LLNA seemed poor (r=0.57), while that between minimum induction doses in the LLNA and elicitation-threshold doses in the GPMT was relatively good (r=0.73). The results suggest the possibility to estimate human elicitation-threshold doses, which are definitely lacking in the process of risk assessment for skin-sensitizers, from the data of the LLNA.

Administration, Topical↗
PubMed1994

Assessment of contact sensitivity of four thiourea rubber accelerators: comparison of two mouse lymph node assays with the guinea pig maximization test.

Contact sensitivity of four thiourea rubber accelerators, diphenylthiourea (DPTU), dilaurylthiourea (DLTU), dibutylthiourea (DBTU) and diethylthiourea (DETU), was evaluated by a new sensitive mouse lymph node assay (SLNA) and the murine local lymph node assay (LLNA). The results of the SLNA and LLNA were compared with the data of the guinea pig maximization test (GPMT). In the LLNA and SLNA, the sensitizing activity was measured as a function of draining lymph node activation following application of the test chemicals. Of these four thioureas, three (DETU, DBTU and DPTU) were not classified as skin sensitizers in the LLNA. The SLNA successfully detected the sensitivity of all thioureas tested. This result indicated that the SLNA was, in these cases, more sensitive than the LLNA for identification of contact allergens. The order of sensitization potential observed from the SLNA was DPTU (greatest), DLTU, DBTU and then DETU (least). The predictions of sensitizing potential and the order of the sensitizing capacity of four thioureas by the SLNA and the GPMT are very similar.

Administration, Topical↗
PubMed2000

Sensitizing potential of chlorothalonil in the guinea pig and the mouse.

The fungicide chlorothalonil is used extensively under several tradenames for the protection of various horticultural and fruit crops and bananas against fungal infections. It is also used as fungicide in wood preservation and as a preservative in paints. Clinical experience has shown chlorothalonil to be a contact allergen and several cases of allergic contact dermatitis attributed to chlorothalonil have been described. 2 previous guinea pig maximization test studies have shown the sensitizing potential of chlorothalonil to be high. The sensitizing property of chlorothalonil was studied by us with the predictive test methods the local lymph node assay and the cumulative contact enhancement test. In the local lymph node assay, chlorothalonil induced a dose-dependent increase in proliferation with a maximal stimulation index of 19.2 and 27.2. In the cumulative contact enhancement test, a statistically significant dose-dependent high sensitization rate was seen with a maximal sensitization rate of 100%. In conclusion, it is evident that chlorothalonil is an extremely potent contact allergen, inducing sensitization using only topical exposure on intact skin.

Animals↗
PubMed2003

Allergenicity evaluation of p-chloro-m-cresol and p-chloro-m-xylenol by non-radioactive murine local lymph-node assay and multiple-dose guinea pig maximization test.

p-Chloro-m-cresol (PCMC) and p-chloro-m-xylenol (PCMX) are known to cause allergic contact dermatitis. For risk assessment of skin sensitizers, information on dose-response profiles in the induction and elicitation phases and cross-reactivity with analogous chemicals are important. In the non-radioactive local lymph-node assay (LLNA) using 5-bromo-2'-deoxyuridine instead of 3H-methyl thymidine, significant effect on lymph node cell proliferation was detected at 10% PCMC and 25% PCMX, while in the multiple-dose guinea pig maximization test (GPMT) at least one animal tested in the group was sensitized at a 5 ppm induction dose of either chemical. When mean skin reaction score in an animal group maximally sensitized with each allergen with the GPMT was plotted against log challenge concentration, linear regression lines with high correlations were obtained in both cases. The calculated elicitation threshold was lower for PCMC than PCMX. The area under the linear regression line between the threshold point and 1% of the elicitation concentration, another index of relative elicitation potency, was also greater for PCMC. Bidirectional cross-reactivity between PCMX and PCMC was detected in the GPMT. PCMC was thus identified in both LLNA and GPMT as a stronger sensitizer than PCMX in both the induction and elicitation phases. These results suggest that the non-radioactive LLNA is a simple and useful method for evaluating allergenicity in the induction phase, while the GPMT using a maximally sensitized animal group is more suitable for assessing the dose-response profile and cross-reactivity in the elicitation phase.

Allergens↗