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I Kimber,
J Hilton,
R J Dearman,
G F Gerberick,
C A Ryan,
D A Basketter,
L Lea, R V House,
G S Ladics,
S E Loveless,
K L Hastings
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ABSTRACT: The murine local lymph node assay (LLNA) is a method for the predictive identification of chemicals that have a potential to cause skin sensitization. Activity is measured as a function of lymph node cell (LNC) proliferative responses stimulated by topical application of test chemicals. Those chemicals that induce a threefold or greater increase in LNC proliferation compared with concurrent vehicle controls are classified as skin sensitizers. In the present investigations we have evaluated further the reliability and accuracy of the LLNA. In the context of an international interlaboratory trial the sensitization potentials of six materials with a history of use in topical medicaments have been evaluated: benzoyl peroxide, hydroquinone, penicillin G, streptomycin sulfate, ethylenediamine dihydrochloride, and methyl salicylate. Each chemical was analyzed in the LLNA by all five laboratories. Either the standard LLNA protocol or minor modifications of it were used. Benzoyl peroxide and hydroquinone, both human contact allergens, elicited strong LLNA responses in each laboratory. Penicillin G, another material shown previously to cause allergic contact dermatitis in humans, was also positive in all laboratories. Streptomycin sulfate induced equivocal responses, in that this material provoked a positive LLNA response in only one of the five laboratories, and then only at the highest concentration tested. Ethylenediamine dihydrochloride dissolved in a 3:1 mixture of acetone with water, or in 4:1 acetone:olive oil (one laboratory), was uniformly negative. However, limited further testing with the free base of ethylene diamine yielded a positive LLNA response when applied in acetone:olive oil (AOO). Finally, methyl salicylate, a nonsensitizing skin irritant, was negative at all test concentrations in each laboratory. Collectively these data serve to confirm that the local lymph node assay is sufficiently robust to yield equivalent results when performed independently in separate laboratories and indicate also that the LLNA is of value in assessing the skin sensitization potential of topical medicaments.
Journal of Toxicology and Environmental Health Part A 05/1998; 53(7):563-79. · 1.83 Impact Factor
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ABSTRACT: 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.
Toxicology 05/1996; 108(1-2):141-52. · 3.68 Impact Factor
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ABSTRACT: 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.
Toxicology 12/1995; 103(1):63-73. · 3.68 Impact Factor
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ABSTRACT: Allergic reactions can be defined as the adverse, tissue-damaging, and sometimes fatal consequences of specific immune responses, usually to exogenous antigens. In the context of toxicology it is allergic reactions resulting from immune responses to chemicals and drugs which are of greatest relevance. The allergy may take a variety of forms including contact hypersensitivity (allergic contact dermatitis), respiratory hypersensitivity (with symptoms ranging from mild rhinitis to severe asthma), and various types of comparatively ill-defined reactions which in many respects resemble autoimmunity. Of these contact hypersensitivity is the most frequently encountered health problem resulting from the interaction of chemicals with the immune system. A wide variety of chemicals are able to induce contact sensitization. Some of these are, in addition, known to cause respiratory hypersensitivity, a less frequent, but no less important, form of chemical allergy.
Fundamental and Applied Toxicology 12/1992; 19(4):479-83.
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ABSTRACT: The purpose of this study was to evaluate the utility of the murine local lymph node assay (LLNA) for contact sensitization risk assessment. Cellular proliferative activity in draining lymph nodes was determined for individual animals on Day 5 following four daily epicutaneous applications of the test chemical to the ears. Seventeen chemicals were tested, covering a range of materials including preservatives, drug actives, and perfume raw materials. The assay was found to be useful for identifying strong, moderate, and some weak sensitizers as defined by other testing methods (guinea pig, human). For evaluating the antigen specificity of the LLNA proliferative response, an in vitro blastogenesis assay was used. Dendritic cells (DC) isolated from lymph nodes of mice treated 24 hr earlier with trinitrochlorobenzene (TNCB) were capable of in vitro stimulation of lymphocytes from TNCB-sensitized mice, but not lymphocytes from mice sensitized to the preservative mixture of 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone (MCI/MI). Conversely, DC from mice treated 24 hr earlier with MCI/MI were capable of stimulating lymphocytes from MCI/MI-sensitized mice, but were unable to stimulate lymphocytes from TNCB-sensitized mice, demonstrating the specificity of the response. The results of these studies support the use of the murine LLNA for both investigative and predictive contact sensitization testing. The LLNA offers the advantages of requiring less time for completion, incorporating an objective endpoint, requiring approximately half the number of animals, and being less costly than most currently employed guinea pig test methods. In addition, we believe the murine LLNA is a useful test to incorporate into a scheme for contact sensitization risk assessment.(ABSTRACT TRUNCATED AT 250 WORDS)
Fundamental and Applied Toxicology 11/1992; 19(3):438-45.
