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PubMed1999

The impact of vehicle on assessment of relative skin sensitization potency of 1,4-dihydroquinone in the local lymph node assay.

BACKGROUND: The murine local lymph node assay (LLNA) has been validated as an alternative method for the identification of skin sensitization hazards. Contact allergens are identified as a function of proliferative responses induced in draining lymph nodes. The quantitative nature of the LLNA data also provides the opportunity of assessing relative potency by reference to dose response analyses. OBJECTIVE: In the current investigations, the influence of vehicle on the skin sensitization potency of a known skin sensitizer (1,4-dihydroquinone) was assessed. METHODS: 1, 4-dihydroquinone was tested in the LLNA using 7 different vehicle systems in each of 2 independent laboratories. RESULTS: Results from the 2 laboratories were almost identical. LLNA dose response data were interpolated to derive the estimated concentration (EC) of 1, 4-dihydroquinone necessary to cause a three-fold stimulation of proliferation compared with controls, the EC3 value. The vehicles used and mean EC3 values obtained were: methyl ethyl ketone 0.07%, acetone 0.08%, acetone/olive oil (80/20 v/v) 0.15%, dimethyl formamide 0.22%, dimethyl sulfoxide 0.4%, and propylene glycol and acetone/saline (50/50 v/v) vehicles gave negative results. However, when tested at higher concentrations, positive results were obtained in these vehicles. CONCLUSION: These data reveal that the vehicle in which a chemical is encountered in the skin can have a significant impact on a quantitative measure of skin sensitization potency. The implication is that accurate assessment of risk to humans will require an understanding of the matrix in which skin exposure is likely to occur.

Animals↗
PubMed2005

An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round.

The original local lymph node assay (LLNA) is based on the use of radioactive labelling to measure cell proliferation. Other endpoints for the assessment of proliferation are also authorized by the OECD Guideline 429 provided there is appropriate scientific support, including full citations and description of the methodology (OECD, 2002. OECD Guideline for the Testing of Chemicals; Skin Sensitization: Local Lymph Node Assay, Guideline 429. Paris, adopted 24th April 2002.). Here, we describe the outcome of the second round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in nine laboratories in Europe. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products (Swissmedic) in Bern. Ear-draining lymph node (LN) weight and cell counts were used to assess LN cell proliferation instead of [3H]TdR incorporation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears to identify skin irritation properties of the test items. The statistical analysis was performed in the department of statistics at the university of Bern. Similar to the EC(3) values defined for the radioactive method, threshold values were calculated for the endpoints measured in this modification of the LLNA. It was concluded that all parameters measured have to be taken into consideration for the categorisation of compounds due to their sensitising potencies. Therefore, an assessment scheme has been developed which turned out to be of great importance to consistently assess sensitisation versus irritancy based on the data of the different parameters. In contrast to the radioactive method, irritants have been picked up by all the laboratories applying this assessment scheme.

Animals↗
PubMed2005

The local lymph node assay being too sensitive?

The local lymph node assay (LLNA) and modifications thereof were recently recognized by the OECD as stand-alone methods for the detection of skin-sensitizing potential. However, although the validity of the LLNA was acknowledged by the ICCVAM, attention was drawn to one major problem, i.e., the possibility of false positive results caused by non-specific cell activation as a result of inflammatory processes in the skin (irritation). This is based on the fact that inflammatory processes in the skin may lead to non-specific activation of dendritic cells, cell migration and non-specific proliferation of lymph node cells. Measuring cell proliferation by radioactive or non-radioactive methods, without taking the irritating properties of test items into account, leads thus to false positive reactions. In this paper, we have compared both endpoints: (1) cell proliferation alone and (2) cell proliferation in combination with inflammatory (irritating) processes. It turned out that a considerable number of tests were "false positive" to the definition mentioned above. By excluding such false positive results the LLNA seems not to be more sensitive than relevant guinea pig assays. These various methods and results are described here.

Allergens↗
PubMedDate not supplied

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.

Acrolein↗
PubMedDate not supplied

Temporal stability of local lymph node assay responses to hexyl cinnamic aldehyde.

The local lymph node assay is an alternative method for the prospective identification of chemicals that have the potential to cause skin sensitization. Activity in the assay is measured as a function of proliferative responses by draining lymph node cells induced by topical exposure of mice to the test chemical. Positive responses are defined as those where a test chemical, at one or more application concentrations, is able to induce a stimulation index of 3 or greater compared with concurrent vehicle-treated control values. Although the method has been evaluated extensively, the stability over time of responses induced in the local lymph node assay has not previously been addressed formally. It was the purpose of the investigations described here to consider this issue and, to this end, responses provoked in the local lymph node assay by hexyl cinnamic aldehyde (HCA)--a skin sensitizing chemical of moderate potency--were assessed in five separate experiments conducted in a single laboratory over a 10-month period. In each case, HCA elicited a positive response. Although some significant inter-experimental variation was recorded, this was attributable entirely to the stimulation by HCA of slightly more vigorous responses in one of the five experiments. When the results of this experiment were excluded from the data set, significant variations were lost. Finally, for each experiment an EC3 value was derived, this being the estimated concentration of test chemical required to induce a stimulation index of 3. Similar EC3 values were derived in each experiment. These data demonstrate the relative stability over time of activity in the local lymph node assay.

Acrolein↗
PubMed1998

Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation.

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.

Administration, Topical↗
PubMed2002

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

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

Allergens↗
PubMed2002

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

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

Allergens↗
PubMed1995

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

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

Allergens↗
PubMed1995

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

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

Analysis of Variance↗
PubMed1991

The murine local lymph node assay: results of an inter-laboratory trial.

The local lymph node assay is a novel predictive test for the identification of contact allergens. The collaborative study reported here was performed to evaluate the reliability of the method when performed in independent laboratories. Eight chemicals were examined in each of 4 participating laboratories and results compared with predictions of skin-sensitizing activity made from concurrent Magnusson and Kligman guinea-pig maximization tests performed in a single laboratory. The local lymph node assay has as its theoretical basis the fact that contact allergens induce T-lymphocyte proliferative responses. In practice, predictions of contact-sensitizing potential are made following measurement of proliferation in lymph nodes draining the site of exposure to chemical, and derivation of a stimulation index using control values as the comparator. Although in the present study there was some variation between laboratories with respect to the absolute stimulation indices recorded, it was found that with all chemicals each laboratory made the same predictions of sensitizing activity. Six chemicals (2,4-dinitrochlorobenzene, formalin, eugenol, isoeugenol, p-phenylenediamine and potassium dichromate) yielded positive responses, and two (methyl salicylate and benzocaine) were negative, in each laboratory. Furthermore, with 7 of the 8 chemicals tested there was no significant difference between laboratories in terms of the characteristics of the dose-response relationships recorded. With the exception of one chemical (benzocaine), predictions made with the local lymph node assay were in accord with those derived from guinea-pig maximization tests. These inter-laboratory comparisons demonstrate that the local lymph node assay is a robust and reliable method for the identification of at least moderate and strong contact allergens.

Allergens↗
PubMedDate not supplied

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

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

Animals↗
PubMed1994

The local lymph node assay: developments and applications.

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

Allergens↗
PubMed1990

Identification of contact allergens using the murine local lymph node assay: comparisons with the Buehler occluded patch test in guinea pigs.

A murine local lymph node assay has been developed for the identification of contact sensitizing chemicals. In the present study, the performance of the local lymph node assay has been evaluated with twenty-four coded chemicals of previously unknown skin sensitizing potential and the results compared with predictions made from concurrent occluded patch tests (Buehler tests) in guinea pigs. The data presented demonstrate that the local lymph node assay successfully identified those chemicals that were classified as moderate or strong skin sensitizers in the Buehler test. In the present series of experiments, chemicals predicted to be mild sensitizers in the Buehler test were classified as 'not strong sensitizers' in the local lymph node assay. In the majority of instances, the Buehler test and local lymph node assay were in agreement with regard to the identification of non-sensitizing chemicals. However, two chemicals that were classified as non-sensitizers in the guinea pig test exhibited positive responses in the local lymph node assay and were predicted to be sensitizers. Some coloured chemicals resulted in obscured Buehler readings and, here, assessment was based upon histological examination of the challenge site. These compounds were examined also in the local lymph node assay and similar predictions of sensitizing potential were made. Taken together, the data reveal close, but not absolute, concordance between the local lymph node assay and the Buehler test. The relative merits of these predictive test methods are discussed.

Allergens↗
PubMed1992

The murine local lymph node assay: a commentary on collaborative studies and new directions.

The murine local lymph node assay is a predictive test for the identification of contact allergens. This paper provides a historical background to the development of the assay and describes the performance of a recently completed interlaboratory trial designed to evaluate further the utility of the method as an alternative or adjunct to guinea-pig predictive tests. On the basis of these and supplementary investigations, a number of recommendations can be made regarding the use and interpretation of the local lymph node assay. Finally, a number of issues arising from recent studies are discussed, including comparisons of the local lymph node assay with guinea-pig methods.

Allergens↗
PubMed2005

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.

Administration, Oral↗
PubMed2004

Differences in responsiveness of mouse strain against p-benzoquinone as assessed by non-radioisotopic murine local lymph node assay.

The non-radioisotopic modification of murine local lymph node assay (LLNA) by using 5-bromo-2'-deoxyuridine (BrdU) was conducted to investigate the strain-related difference of the responsiveness of mice to p-benzoquinone (PBQ) with BALB/cAnN, CBA/JN and CD-1 mouse strains. Strain and dose related differences were analyzed by two-way analysis of variance (two-way ANOVA). CBA/JN was considered to be the highest responsive strain to PBQ, and interaction was detected between CD-1 and each of the other inbred strains. These results support the recommendation in the OECD test guideline 429 and the skin sensitization test guideline of US-EPA with regard to the selection of mouse strain for LLNA.

Analysis of Variance↗
PubMed2002

Local lymph node assay with non-radioisotope alternative endpoints.

The local lymph node assay has recently been accepted by regulatory agencies as a stand-alone alternate method for predicting allergic contact dermatitis. To compare the sensitivity of non-radioisotope methods with that of the standard assay, we determined if these modified methods would affect evaluation of sensitization potency. For this reason, we used 2,4-dinitrochlorobenzene (DNCB) and benzocaine for different sensitizing criteria. Female CBA mice were treated for 3 days with a test compound or vehicle applied to each side of both ears. Bilateral auricular lymph node proliferative activity was assessed by the following endpoints with incorporation of 3H-methyl thymidine (3H-TdR), bromodeoxyuridine (BrdU) in vivo, and BrdU ex vivo, IL-2 production, and proliferating cell nuclear antigen (PCNA) expression. Ear thickness was also tested. The strong sensitizer DNCB was detectable by any of the non-radioisotope endpoints as well as by radioisotope-dependent standard assay. On the other hand, when evaluating the weak sensitizer benzocaine, significant changes were evident in BrdU incorporation ex vivo and in vivo, and IL-2 production. We believe that these non-radioisotope methods can assess allergic contact dermatitis caused by chemicals even in the laboratory, where it can be difficult to handle radioisotopes.

Allergens↗