D. A. Basketter

Use of the local lymph node assay for the estimation of relative contact allergenic potency.

The effective toxicological evaluation of skin sensitization demands that potential contact allergens are identified and that the likely risks of sensitization among exposed populations assessed. By definition, chemicals which possess the toxicological property of skin sensitization potentially are capable of causing allergic contact dermatitis (ACD) in humans. However, this hazard is not an all‐or‐none phenomenon; clear dose‐response relationships can be discerned and thresholds identified for both the induction of sensitization and the elicitation of contact dermatitis. Commonly, these parameters are grouped under the heading of potency, determination of which is vital for risk assessment. In the present investigation, the local lymph node assay (LLNA) has been employed to determine the relative potency of a range of 20 chemicals. The parameter used is the estimated concentration required to produce a 3‐fold increase in draining lymph‐node cell proliferative activity, the EC3 value. These measurements have been compared with an assessment of the human sensitizing potency of the 20 selected chemicals, each being assigned to 1 of 5 classes based on their human sensitizing potency. The EC3 value, derived from LLNA work carried out in acetone/olive oil vehicle, correlated well with the human classification, with the strongest sensitizers having low EC3 values (<0.1%), weaker sensitizers having EC3 values generally in the 1–10% range, and non‐sensitizing chemicals having EC3 values in excess of 100%. In conclusion, the derivation of the EC3 for a chemical provides an objective and quantitative estimate of potency that is of considerable utility for skin sensitization risk assessment.

Classification of contact allergens according to potency: proposals

It is clear that contact allergens vary substantially with regard to the relative potency with which they are able to induce skin sensitisation. Considerations of potency will in the future become a significant factor in the classification of skin sensitising chemicals. It is therefore appropriate to establish what is known of potency and thresholds in the induction of skin sensitisation and the elicitation of allergic contact dermatitis, and to identify approaches that might be available for assessment of relative potency for the purposes of categorising chemical allergens. This paper was prepared by an ECETOC (European Centre for Ecotoxicology and Toxicology) Task Force that had the objective of recommending approaches for the measurement of potency and definition of thresholds for both the induction and elicitation of contact sensitisation. The deliberations recorded here build upon recommendations made previously by an ECETOC Task Force that considered the conduct of standard skin sensitisation test methods for the purposes of hazard identification and risk assessment (ECETOC, Monograph No. 29, Brussels, 2000). The emphasis in this present paper is also on standard and accepted methods for the assessment of skin sensitisation, and for which OECD guidelines are available: the local lymph node assay (LLNA), the guinea pig maximisation test and the occluded patch test of Buehler. For various reasons, discussed in detail herein, attention focused primarily upon consideration of categorisation of chemical allergens and the identification of thresholds with respect to the induction of skin sensitisation, rather than the elicitation of allergic contact dermatitis. It is concluded that although the LLNA is the method of choice for the determination of skin sensitisation potency for the purposes of categorisation, if data are already available from appropriate guinea pig tests then their judicious interpretation may provide information of value in determinations of potency and categorisation. Included here are detailed and specific recommendations for how best the results of the three test methods considered can be used for the categorisation of chemical allergens as a function of skin sensitisation potency.

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.

Contact allergy, irritancy and ‘danger’

Conventional models of the immune response are based on distinguishing self and non‐self. However, we consider that the more recently proposed ‘danger’ model may be an illuminating alternative for studying allergic contact dermatitis. In this model, an antigenic signal on its own would tend to produce tolerance. In contrast, in the presence of a ‘danger’ signal, which, in the case of allergic contact dermatitis, we suggest is usually cutaneous irritancy, the immune system would become activated, leading first to the induction of sensitization and then subsequently to the elicitation of a contact hypersensitivity response. In most cases, both the antigenic signal and irritant signal will come from the hapten, although, e.g., in an occupational setting, traumiterative dermatitis would be the source of the ‘danger’ signal. Typically, the irritant signal tends to be more concentration‐dependent and thus is the overriding factor in the determination of the effective sensitizing and eliciting concentrations of the hapten. A further prediction of this hypothesis is that successful experiments demonstrating low‐dose tolerance with contact allergens may be explained by the loss of the irritant effect at lower dilutions, whilst an antigenic stimulus remains present.

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.

Threshold for classification as a skin sensitizer in the local lymph node assay: a statistical evaluation.

For more than 15 years, the murine local lymph node assay (LLNA) has undergone development, evaluation and validation as an alternative approach to the predictive identification of skin sensitizing chemicals. The criteria by which sensitizing chemicals are distinguished from those without significant skin sensitising hazard were developed empirically and were based on experience rather than a mathematical formula or statistical method. The current practice is to classify, as skin sensitizers, those chemicals which at one or more test concentrations stimulate a threefold or greater increase in the proliferative activity in draining lymph node cells. Despite the apparent confirmation of the utility of this approach from the extensive data available, there has not previously been any attempt to substantiate the accuracy of this criterion. In this present investigations, data from 134 chemicals tested in the LLNA and in the guinea pig and/or for which there exists clear evidence relating to human skin sensitization potential, have been subjected to a rigorous statistical evaluation using Receiver Operating Characteristic (ROC) curves. Whether the analysis is based on a comparison with guinea pig or human data, the results indicate that the empirically derived threefold threshold is an acceptable practical value for hazard identification.

Ranking of hair dye substances according to predicted sensitization potency: quantitative structure–activity relationships

Allergic contact dermatitis following the use of hair dyes is well known. Many chemicals are used in hair dyes and it is unlikely that all cases of hair dye allergy can be diagnosed by means of patch testing with p‐phenylenediamine (PPD). The objectives of this study are to identify all hair dye substances registered in Europe and to provide their tonnage data. The sensitization potential of each substance was then estimated by using a quantitative structure–activity relationship (QSAR) model and the substances were ranked according to their predicted potency. A cluster analysis was performed in order to help select a number of chemically diverse hair dye substances that could be used in subsequent clinical work. Various information sources, including the Inventory of Cosmetics Ingredients, new regulations on cosmetics, data on total use and ChemId (the Chemical Search Input website provided by the National Library of Medicine), were used in order to identify the names and structures of the hair dyes. A QSAR model, developed with the help of experimental local lymph node assay data and topological sub‐structural molecular descriptors (TOPS‐MODE), was used in order to predict the likely sensitization potential. Predictions for sensitization potential were made for the 229 substances that could be identified by means of a chemical structure, the majority of these hair dyes (75%) being predicted to be strong/moderate sensitizers. Only 22% were predicted to be weak sensitizers and 3% were predicted to be extremely weak or non‐sensitizing. Eight of the most widely used hair dye substances were predicted to be strong/moderate sensitizers, including PPD – which is the most commonly used hair dye allergy marker in patch testing. A cluster analysis by using TOPS‐MODE descriptors as inputs helped us group the hair dye substances according to their chemical similarity. This would facilitate the selection of potential substances for clinical patch testing. A patch‐test series with potent, frequently used, substances representing various chemical clusters is suggested. This may prove useful in diagnosing PPD‐negative patients with symptoms of hair dye allergy and would provide some clinical validation of the QSAR predictions.

Evaluation of a human patch test for the identification and classification of skin irritation potential

Current regulations require that the skin irritation/corrosion potential of new chemicals is assessed in the rabbit Draize test, although there are opportunities to use alternative methods to identify the most aggressive materials. Previously, we have proposed that it is possible la employ a strategy that avoids the use of animals and at the same time delivers a more relevant assessment of skin irritation‐corrosion potential. The approach is to identify corrosive materials in vitro and then proceed to human volunteer testing for skin irritation. In this study, the human 4‐h patch test, its interpretation, and results with 29 test materials are presented. Using 14 materials not classified as “Irritating to skin” by EU criteria. 13 classified as “Irritating to skin” by suppliers, and 2 as “Corrosive – causes burns”, it is demonstrated that, by evaluating these on human skin in vitro and in vivo, a significant proportion are either over‐classified or under‐classified. In conclusion, we are convinced that by application of the approach described in detail here, it is possible to avoid the use of animals, whilst at the name time obtaining an assessment of skin irritation/corrosion potential that is more relevant to man and which, if required, may he used directly for classification and labelling of substances and preparations within the European Union.

Influence of sodium lauryl sulphate on 2,4-dinitrochlorobenzene-induced lymph node activation

The influence of the anionic surfactant sodium lauryl sulphate (SLS) on the ability of the contact allergen 2,4-dinitrochlorobenzene (DNCB) to provoke draining lymph node cell proliferative responses, a correlate of skin sensitizing potential, has been examined in mice. Topical application of 10% SLS with 0.1% DNCB caused a more vigorous proliferative response than did exposure to 0.1% DNCB alone. Lower concentrations (0.1% or 1%) of SLS were ineffective and 10% SLS failed to influence proliferative responses to higher concentrations (0.5% or 1%) of DNCB. Using an in vitro model for measurement of percutaneous absorption 10% SLS was shown not to increase the skin penetration of 0.1% DNCB. We therefore examined the influence of SLS on the accumulation of dendritic cells (DC) in lymph nodes draining the site of exposure, an important early event during the induction phase of skin sensitization. The frequency of DC in draining nodes was measured following topical application of SLS, DNCB or a combination of both. Epicutaneous exposure to 0.1% DNCB caused only a modest increase in the number of lymph node DC. However, 10% SLS or a mixture of 10% SLS with 0.1% DNCB each resulted in a significant elevation of DC numbers. It is proposed that SLS augments the skin sensitizing potential of sub-irritant concentrations of DNCB via an increase in the number of immunostimulatory DC which reach the draining nodes.

Individual, ethnic and seasonal variability in irritant susceptibility of skin: the implications for a predictive human patch test.

Since irritants are the major cause of contact dermatitis, it is important to identify those chemicals that possess significant ability to cause skin irritation. This process must then be followed by risk assessment and risk management. Historically, animal tests have played a major rôle in this process, but human volunteer studies are of increasing importance in the field. Where the appropriate safely and ethical controls are in place, human testing can give data that identities skin irritation hazard. To be of widest value, these human studies must not he flawed due to inter‐individual, inter‐ethnic or seasonal variation. We conducted a large dose‐response study and studied the impact of summer and winter weather on a predictive human assay. Sodium dodecyl sulphate (SDS) was tested at 0.1%‐20% in 3 national groups of approximately 100 volunteers, using 25 mm Hill Top chambers loaded with 0.2 ml solution and applied to the tipper outer arm for 4 h. Reactions were scored at 24. 48 and/or 72 h after patch application. The German and Chinese studies were completed in a few weeks under. similar winter conditions, whereas the UK work was spread fairly evenly over about a 15‐month period Some relatively minor differences were observed in the dose‐response curves obtained, probably due to weather conditions. The effect of me weather on the intensity, but not the put tern, of irritant reactivity was also evident in the smaller specific study that assessed reactions to SDS in summer and in winter. Whereas 45% of the panel reacted to 20% SDS in summer, 9l% reacted in the winter. However, in both studies, substantial inter‐individual variations in response to SDS dominated the pattern of response. When designing a human patch test to discriminate skin irritant substances from those that are of minimal effect, it is this inter‐individual variability, rather than any small inter‐ethnic or seasonal variation, which must be taken into account. This can be achieved by the routine inclusion of a suitable positive irritant control, which then calibrates each human volunteer panel.