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PubMed2002

Local lymph node assay - validation, conduct and use in practice.

The validation of alternative methods is a relatively new activity in toxicology. The local lymph node assay (LLNA), a novel method for the identification of chemicals that have the potential to cause skin sensitization, was the first test to pass through the formal regulatory validation process established in the USA under the auspices of ICCVAM, the Interagency Coordinating Committee on the Validation of Alternative Methods. ICCVAM approved the LLNA as an alternative to guinea pig tests for the identification of skin sensitisation hazards. In this report, we explore the nine recommendations made by ICCVAM and discuss their interpretation in relation to the new OECD Guideline 429, which describes the LLNA. In particular, the value and limitations of the use of statistical evaluation of data and of the inclusion of routine positive controls is examined. It is concluded that the OECD Guideline as currently written embodies the necessary flexibility to permit conduct of the LLNA in a manner necessary to meet the varying needs of regulatory agencies and toxicologists around the world.

Animals↗
PubMed1992

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 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)

Animals↗
PubMed2005

Investigation of the dermal sensitization potential of various essential oils in the local lymph node assay.

Essential oils are commonly used fragrance ingredients. The oils themselves are complex mixtures, which may contain naturally occurring contact sensitizers. The local lymph node assay was used to evaluate the dermal sensitization potential of basil, citronella, clove leaf, geranium, litsea cubeba, lemongrass, and palmarosa oils. Three of the major components--citral, eugenol, and geraniol--were included to investigate any difference in sensitization potential arising from their exposure in a mixture. Each fragrance material was tested at five concentration ranging from 2.5% to 50% w/v in 1:3 ethanol:diethyl phthalate. The stimulation index (SI) values were calculated for each dose level, an SI > or = 3 was considered a positive response. The estimated concentration (EC3) required to elicit a positive was calculated and taken as a measure of relative potency. The EC3 values and potency classification for basil, clove leaf, litsea cubeba, lemongrass and palmarosa oils were calculated to be <2.5% (> or = moderate), 7.1% (weak), 8.4% (weak), 6.5% (weak) and 9.6% (weak), respectively. Citronella and geranium oils were negative. The individual components citral, eugenol and geraniol resulted in EC3 values of 6.3%, 5.4% and 11.4%, respectively. In general, the potency of each essential oil did not differ significantly from that observed for its main individual component.

Acyclic Monoterpenes↗
PubMed1991

The identification of photoallergic potential in the local lymph node assay.

Guinea pig test methods are the most commonly used and reliable of predictive models for contact photoallergenicity of chemicals. The murine local lymph node assay (LLNA) has been developed recently as an alternative method for the identification of skin-sensitizing chemicals. Sensitization potential is measured from an assessment of the proliferation of lymphocytes in lymph nodes draining the site of exposure to the test chemical. This work investigates the activity of 6 widely reported photoactive chemicals in a modified LLNA (a photo-LLNA). The photoallergens tetrachlorosalicylanilide and fentichlor elicited positive ultraviolet radiation (UV)-dependent proliferative responses that were greater than their positive UV-independent responses, suggesting that they are both contact and photoallergic in the mouse. The lack of a proliferative response to 6-methylcoumarin and the absence of a reproducible response to musk ambrette suggest that the assay is insufficiently sensitive to identify weak photoallergic potential. Positive UV-dependent responses to acridine and anthracene, both photoirritants, cast doubt on the specificity of the photo-LLNA. Positive LLNA responses to these chemicals may be due to skin protein modification, based on evidence from the in vitro photo-chemical protein-binding assay. These results demonstrate that the photo-LLNA is able to detect at least moderate photoallergic potential.

Acridines↗
PubMed1997

A local lymph node assay to analyse immunosuppressive effects of topically applied drugs.

Topical glucocorticosteroids represent the mainstay of antiinflammatory therapy in the treatment of inflammatory skin diseases. Their clinical use, however, is limited by local and systemic side-effects. Thus, in dermatopharmacology there is a large demand for alternative non-steroidal antiinflammatories. Other than transplantation models, most of the frequently used in vivo test systems for assessment of drug-induced immunosuppression measure changes in inflammatory skin responses by means of skin erythema and edema after challenge of sensitized animals. The aim of this study was to develop an alternative mouse model to detect and analyse immunosuppressive effects of topically applied drugs. On the basis of a modified local lymph node assay, we analysed effects of topical hydrocortisone, dexamethasone, mometasone furoate and FK506 (tacrolimus) during the induction phase of contact hypersensitivity. On 4 consecutive days, NMRI mice were treated on the dorsal surfaces of both ears with increasing concentrations of test compound. During the last 3 days, the mice received in addition the contact sensitizer, oxazolone (1%). On day 5, draining auricular lymph nodes were removed in order to assess lymph node cell counts and perform flow cytometric analysis of lymph node cell subpopulations (CD4+/CD25+, Ia+/CD69+, Ia+/B220+). All test compounds proved to exert significant immunosuppressive effects after topical application, but showed differences in their immunomodulatory potential. In conclusion, the local lymph node assay serves as an appropriate model to characterize immunosuppressive effects of topically applied drugs by measuring immunologically relevant end-points.

Administration, Topical↗
PubMed2001

Measurement of allergenic potency using the local lymph node assay.

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

Allergens↗
PubMedDate not supplied

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.

Allergens↗
PubMed2006

Sensitising potential of four textile dyes and some of their metabolites in a modified local lymph node assay.

We studied the sensitising and allergenic potentials of the textile dyes disperse yellow 3, disperse orange 30, disperse red 82, disperse yellow 211 and two metabolites of disperse yellow 3, 4-aminoacetanilide and 2-amino-p-cresol, using modified protocols of the murine "local lymph node assay" (LLNA). Test substances were applied either to the dorsum of the mice ears (sensitisation protocol) or they were first applied to the skin of their backs and 2 weeks later to their ears (sensitisation-challenge protocol). In addition to the endpoints weight and cell number of the draining ear lymph nodes we analysed lymphocyte subpopulations by flow cytometry. In the sensitisation protocol, disperse yellow 3 and its metabolite 4-aminoacetanilide did not induce significant effects, whereas in the sensitisation-challenge protocol cell number and lymph node weight increased significantly indicating a sensitising potential in NMRI mice. Hence, two-phase treatment (skin of the back, ear) increased the sensitivity of this assay. The second metabolite of disperse yellow 3, 2-amino-p-cresol, showed distinct effects in both treatment protocols; this applied mainly to the parameters cell number and lymph node weight. The dye disperse red 82 caused ambiguous increases in lymph node weight and cell number in the sensitisation protocol which were not reproduced in the sensitisation-challenge protocol, ruling out a relevant sensitising potential for this dye in NMRI mice. Disperse yellow 211 and disperse orange 30 did not induce relevant changes under our experimental conditions. Phenotyping of lymphocytes did not influence the assessment of these dyes.

Allergens↗
PubMed2006

Skin sensitization potency of methyl methacrylate in the local lymph node assay: comparisons with guinea-pig data and human experience.

There is compelling evidence that contact allergens differ substantially (by 4 or 5 orders of magnitude) with respect to their inherent skin-sensitizing potency. Relative potency can now be measured effectively using the mouse local lymph node assay (LLNA) and such data form the basis of risk assessment and risk management strategies. Such determinations also facilitate distinctions being drawn between the prevalence of skin sensitization to a particular contact allergen and inherent potency. The distinction is important because chemicals that are implicated as common causes of contact allergy are not necessarily potent sensitizers. One example is provided by nickel that is undoubtedly a common cause of allergic contact dermatitis, but is a comparatively weak sensitizer in predictive tests. In an attempt to explore other examples of contact allergens where there may exist a discrepancy between prevalence and potency, we describe here analyses conducted with methyl methacrylate (MMA). Results of LLNA studies have been interpreted in the context of historical clinical data on occupational allergic contact dermatitis associated with exposure to MMA.

Allergens↗
PubMed1992

Sulphanilic acid: divergent results in the guinea pig maximization test and the local lymph node assay.

The guinea pig maximization test (GPMT) has proven to be a valuable tool for the identification of the skin sensitization potential of chemicals. The method identifies a hazard which can lead in the EC to compulsory labelling of that chemical. In the present study, data on sulphanilic acid derived from the GPMT has been compared with results from a second guinea pig assay (the cumulative contact enhancement test) and the murine local lymph node assay, both of which require only topical application of chemical. Except for the GPMT, no test identified any sensitizing activity associated with exposure to sulphanilic acid. These latter results are consistent with the experience gained from substantial human exposure in an occupational setting and from which no cases of allergic contact dermatitis to sulphanilic acid have arisen over a 20-year period. In consequence, it is questioned which test protocol in practice has given the more accurate identification of sensitization hazard relevant to man.

Administration, Topical↗
PubMed2005

Potency and risk assessment of a skin-sensitizing disperse dye using the local lymph node assay.

Disperse Blue 106 is an acknowledged skin-sensitization hazard. However, information about the relative sensitization potency of this chemical is lacking, and to provide this information was the purpose of the investigations described here. The approach taken was to measure dose-response relationships for C.I. Disperse Blue in the local lymph node assay, a method for the assessment of skin-sensitization potential in which activity is measured as a function of lymphocyte proliferative responses induced in draining lymph nodes. From these data, it was possible to derive EC3 values (such being the estimated concentration of chemical required to elicit a 3-fold increase in proliferation) that have been shown previously to reflect the relative sensitizing potency of contact allergens. These analyses revealed that Disperse Blue 106 had a relatively low EC3 value (0.01%), comparable to that measured concurrently for 2,4-dinitrochlorobenzene, a potent contact allergen. Collectively, these data reveal that Disperse Blue 106 represents a significant skin-sensitization hazard, and, in combination with information on dye migration and percutaneous penetration from various types of fabric and use conditions provide a basis for the development of effective and accurate risk assessments.

Allergens↗
PubMed1993

Evaluation of contact sensitivity of rubber chemicals using the murine local lymph node assay.

The sensitizing abilities of 4 rubber additives, tetramethylthiuram disulfide (TMTD). 2-mercaptobenzothiazole (MBT), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) and zinc diethyldithiocarbamate (ZDEC), were evaluated using the murine local lymph node assay. Exposure to IPPD induced a significant increase of lymph node cell proliferation in the draining lymph nodes even at a low concentration. Exposure to TMTD and MBT induced moderate proliferative responses, while ZDEC induced a weak proliferation even at the higher concentrations. The sensitizing potency of each chemical was described in terms of the concentration that increased lymph node cell proliferation by a factor of 2 over that in the vehicle-treated control group. The concentrations of IPPD, TMTD, MBT and ZDEC were 0.14%, 0.35%, 4.2% and > 10%, respectively.

Animals↗
PubMed2001

Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals.

We validated a two-tiered murine local lymph node assay (LLNA) with a panel of standard contact (photo)allergens and (photo)irritants with the aim of improving the discrimination between contact (photo) allergenic potential and true skin (photo)irritation potential. We determined ear weights to correlate chemical-induced skin irritation with the ear-draining lymph node (LN) activation potential. During tier I LLNAs, a wide range of concentrations were applied on three consecutive days to the dorsum of both ears. Mice were exposed to UVA light immediately after topical application to determine the photoreactive potential of some test chemicals. Mice were killed 24 h after the last application to determine ear and LN weights and LN cell counts. It was possible to classify the tested chemicals into three groups according to their threshold concentrations for LN activation and skin irritation: (1) chemicals with a low LN activation potential and no or very low skin irritation potential; (2) chemicals with a marked LN activation potential higher than a distinct skin irritation potential; and (3) chemicals with LN activation potential equal to or lower than their skin irritation potential. Group 1 consisted only of contact allergens, indicating that LN activation in the absence of skin irritation points to a contact allergenic activity. Since groups 2 and 3 comprised irritants and contact allergens, a tier II LLNA protocol was used to finally differentiate between true irritants and contact allergens. Briefly, mice were pretreated with mildly to moderately irritating concentrations of the chemical to the shaved back and after 12 days were challenged on the ears as described above in order to elicit a contact allergenic response in the ear skin and the ear-draining LN. With this approach, tier II LLNAs have to be conducted only in cases for which skin irritation potential is in the range of LN activation potential and no structure-activity relationship data indicating a contact allergenic hazard are available.

Administration, Topical↗
PubMed2001

Contact allergenic potency: correlation of human and local lymph node assay data.

BACKGROUND: Effective toxicologic evaluation of skin sensitization requires that potential contact allergens are identified and that the likely risks of sensitization among exposed populations are assessed. By definition, chemicals that are classified as contact sensitizers have the capacity to cause allergic contact dermatitis (ACD) in humans. However, this hazard is not an all-or-nothing phenomenon; clear dose-response relationships can be discerned and thresholds identified for both the induction of sensitization and the elicitation of ACD. Commonly, these parameters are grouped under the heading of potency, the determination of which is vital for risk assessment. Preclinical testing for sensitization potential is critically important for hazard assessment before human exposure. The murine local lymph node assay (LLNA) is the most recently accepted test method for sensitization hazard assessment. OBJECTIVE: The aim was to compare potency estimations derived from LLNA data with clinical determinations of relative potency based on human data. METHODS: No-effect levels (NOELs) for a range of 21 chemicals were determined from nondiagnostic human repeat patch test studies as reported in the literature. These levels were compared with LLNA EC(3) values, the estimated concentration required to produce a 3-fold increase (positive response) in draining lymph node cell (LNC) proliferative activity. RESULTS: Using available human repeat patch test data, together with expert judgment, the compounds were classified as strong, moderate, weak, extremely weak, or nonsensitizing. Additionally, the potency of each chemical was classified independently based on its LLNA EC(3) value. The results show clearly that LLNA EC(3) values are very comparable with the NOELs calculated from the literature. Moreover, the potency rankings based upon LLNA EC(3) data support their human classification. CONCLUSION: The present investigations show that the LLNA can be used to provide quantitative estimates of relative skin sensitizing potency EC(3) values that correlate closely with NOELs established from human repeat patch testing and from our clinical experience.

Allergens↗
PubMed2000

A quantitative method for assessing the sensitizing potency of low molecular weight chemicals using a local lymph node assay: employment of a regression method that includes determination of the uncertainty margins.

Risk assessment of sensitizing chemicals requires, besides hazard identification, the assessment of potency. To examine the sensitizing capacity of low molecular weight chemicals, a murine local lymph node assay (LLNA) was used. The sensitizing capacity of known allergens was quantified by dose-response modeling. At a stimulatory index (SI) of 3, the corresponding estimated concentration was calculated (EC(3)), together with a confidence interval to take account of the quality of the particular data set. We tested ten allergens (ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), 4-ethoxymethylene 2-phenyl oxazol-5-one (oxazolone), phthalic anhydride (PA), toluene diisocyanate (TDI), trimellitic anhydride (TMA), tetramethylthiuramdisulfide (TMTD) and zincdimethyldithiocarbamate (ZDMC)). Oxazolone showed the strongest sensitizing potency followed in this order by DNCB, TDI, TMA, PA, TMTD, ZDMC, MBT, benzocaine and DEA. The approach performed in this study is a way to accurately assess the potency of sensitizing chemicals and thus a possibility for classification.

Allergens↗
PubMed2003

Contact allergy: the local lymph node assay for the prediction of hazard and risk.

The prospective identification of potential contact allergens and their subsequent safety assessment are pivotal in successful management of this risk to human health. Although much can be learned from the chemical and physical properties of a substance, the definitive information in respect of sensitizing hazard/risk derives from an assessment of the integrated response of the immune system. In recent years, the focus for such assessments has begun to switch from the guinea pig to the mouse, notably to the local lymph node assay (LLNA). In this paper, the current value of the LLNA for hazard identification is reviewed and its regulatory status defined. Once a potential contact allergen has been identified, however, the vital clue to accurate safety evaluation is the assessment of the potency of the allergen. How this can be achieved using the LLNA and employed in safety evaluation is discussed in detail, together with practical suggestions for the deployment of such processes in regulatory toxicology.

Allergens↗
PubMedDate not supplied

Application of the random forest method in studies of local lymph node assay based skin sensitization data.

The random forest and classification tree modeling methods are used to build predictive models of the skin sensitization activity of a chemical. A new two-stage backward elimination algorithm for descriptor selection in the random forest method is introduced. The predictive performance of the random forest model was maximized by tuning voting thresholds to reflect the unbalanced size of classification groups in available data. Our results show that random forest with a proposed backward elimination procedure outperforms a single classification tree and the standard random forest method in predicting Local Lymph Node Assay based skin sensitization activity. The proximity measure obtained from the random forest is a natural similarity measure that can be used for clustering of chemicals. Based on this measure, the clustering analysis partitioned the chemicals into several groups sharing similar molecular patterns. The improved random forest method demonstrates the potential for future QSAR studies based on a large number of descriptors or when the number of available data points is limited.

Algorithms↗
PubMed2001

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

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

Animal Welfare↗