The skin sensitization potential of resorcinol: experience with the local lymph node assay

Resorcinol is a simple aromatic chemical (1,3-benzenediol) that has found widespread use, particularly as a coupler in hair dyes. Clinical experience clearly shows that resorcinol is a (albeit uncommon) skin sensitizer. By contrast, predictive methods, both animal and human, have previously failed to identify resorcinol as such. Here, we describe the outcome of a recent local lymph node assay performed in accordance with Organisation for Economic Co-operation and Development guideline 429, which correctly identified resorcinol as a skin sensitizer. Clear evidence of a dose response was apparent, and an EC3 value of approximately 6% was calculated. This suggests that the skin-sensitizing potency of resorcinol is approximately 2 orders of magnitude lower than that of p-phenylenediamine but similar to that of hexyl cinnamic aldehyde. These data show the importance of adherence to test guidelines and aligns the clinical experience with resorcinol with that obtained in predictive animal methods.     

Dinitrohalobenzenes: evaluation of relative skin sensitization potential using the local lymph node assay

The dinitrohalobenzenes are known to cause skin sensitization and have been used in many seminal investigations of the relationships between physicochemical characteristic and sensitizing potential. The electrophilic theory of skin sensitization implies that contact allergic potential should correlate positively with the ability of chemicals to react with proteins to form immunogenic hapten-protein conjugates. It is intriguing, therefore, that previous studies in guinea pigs and mice have suggested that such correlations do not apply to dinitrohalobenzenes. To address this, we have examined, using the murine local lymph node assay (LLNA), the sensitizing activity of 2,4-dinitroiodobenzene (DNFB), 2,4-dinitrochlorobenzene (DNCB), 2,4-dinitrobromo-bezene (DNBB) and 2,4-dinitroiodobenzene (DNIB). In contrast to previous investigations, it was found that the ability of these chemicals to provoke responses in the LLNA correlated closely with their reported protein reactivity. On the basis of these data, it is proposed that dinitrohalobenzenes conform to the electrophilic theory of skin sensitization and that they should be regarded as direct acting haptens.     

Hapten-protein binding: from theory to practical application in the in vitro prediction of skin sensitization

In view of the forthcoming European Union ban on in vivo testing of cosmetic and toiletry ingredients, following the publication of the 7th amendment to the Cosmetics Directive, the search for practical, alternative, non-animal approaches is gathering pace. For the end-point of skin sensitization, the ultimate goal, i.e. the development and validation of alternative in vitro/in silico assays by 2013, may be achieved through a better understanding of the skin sensitization process on the cellular and molecular levels. One of the key molecular events in skin sensitization is protein haptenation, i.e. the chemical modification of self-skin protein(s) thus forming macromolecular immunogens. This concept is widely accepted and in theory can be used to explain the sensitizing capacity of many known skin sensitizers. Thus, the principle of protein or peptide haptenation could be used in in vitro assays to predict the sensitization potential of a new chemical entity. In this review, we consider some of the theoretical aspects of protein haptenation, how mechanisms of protein haptenation can be investigated experimentally and how we can use such knowledge in the development of novel, alternative approaches for predicting skin sensitization potential in the future.     

Structure-activity relationships for skin sensitization: recent improvements to Derek for Windows

Derek for Windows (DfW) is a knowledge-based expert system that predicts the toxicity of a chemical from its structure. Its predictions are based in part on alerts that describe structural features or toxicophores associated with toxicity. Recently, improvements have been made to skin sensitization alerts within the DfW knowledge base in collaboration with Unilever. These include modifications to the alerts describing the skin sensitization potential of aldehydes, 1,2-diketones, and isothiazolinones and consist of enhancements to the toxicophore definition, the mechanistic classification, and the extent of supporting evidence provided. The outcomes from this collaboration demonstrate the importance of updating and refining computer models for the prediction of skin sensitization as new information from experimental and theoretical studies becomes available.     

Testing for skin sensitization according to the notification procedure for new chemicals: the Magnusson and Kligman test

The notification procedure for new chemicals in the European Union (called the Chemicals Act in Germany) requires a skin sensitization test when the amount of a new chemical produced exceeds 100 kg/year. The preferred test is that of Magnusson and Kligman; more than 90% of the tests submitted are performed with it. Though the Magnusson and Kligman test is described in the literature, and in the test guidelines of the European Union and of the OECD, discrepancies do occur in the performance of the test between test laboratories. In this paper, recommendations are given for standardized performance of the Magnusson and Kligman test.     

Anti‐hapten antibodies in response to skin sensitization

Whereas T lymphocyte (T cell) activation is the key event in the acquisition of skin sensitization and subsequent elicitation of allergic contact dermatitis, the humoral component of immune responses to organic contact allergens has received little consideration. There is evidence that, in experimental animals, topical exposure to potent contact allergens is associated with B cell activation and proliferation, and hapten‐specific antibody production. However, there is very limited evidence available for anti‐hapten antibody responses being induced following topical exposure of humans to contact allergens. Nevertheless, it is important to appreciate that there are almost no negative studies in which evidence for antibody production as the result of skin sensitization has been sought and not found. That is, there is absence of evidence rather than evidence of absence. Furthermore, exposure to chemical respiratory allergens, in which the skin has been implicated as a potential route of sensitization, results in anti‐hapten antibody responses. It is proposed that skin sensitization to contact allergens will normally be accompanied by antibody production. The phenomenon is worthy of investigation, as anti‐hapten antibodies could potentially influence and/or regulate the induction of skin sensitization. Moreover, such antibodies may provide an informative correlate of the extent to which sensitization has been acquired.     

Quantitative structure-activity relationships for skin sensitization potential of urushiol analogues

The relative alkylation index (RAI), a theoretically derived parameter intended to quantify the relative extent of carrier haptenation resulting from a given dose of a given sensitizer, has previously been successfully applied to the analysis of relative sensitization potential and dose-response data for a variety of contact allergens which are directly electrophilic. Here the RAI concept is applied to analysis of data on compounds related to urushiol (i.e., 3-substituted catechols), the naturally occurring mixture of allergens responsible for contact allergy to poison ivy and poison oak. These allergens are believed to act as pro-electrophiles, being oxidized to electrophilic orthoquinones in vivo. It is found that the various types of urushiol derivatives fit the same sort of RAI-sensitization relationships as expected theoretically and as found previously with direct acting electrophiles. There is evidence that in many cases, the test conditions were such that overload effects, whereby the degree of sensitization induced decreases with increasing carrier haptenation, applied. It is also concluded that the question as to the relative sensitization potencies of the naturally occurring urushiols remains open. The commonly held view that with these materials, sensitization potential increases with increasing unsaturation in the 3-hydrocarbyl chain of the 3-hydrocarbyl catechols, is based on evidence that is capable of alternative interpretation.     

Nothing is perfect, not even the local lymph node assay: a commentary and the implications for REACH

For many regulatory authorities, the local lymph node assay (LLNA) is the preferred assay for the predictive identification of skin‐sensitizing chemicals. It is the initial requirement for sensitization testing within the new REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) regulations in the European Union. The primary reasons for the preferment of the LLNA are the animal welfare benefits it provides compared with traditional guinea‐pig methods (refinement and reduction of animal usage) and the general performance characteristics of the assay with regard to overall reliability, accuracy, and interpretation. Moreover, a substantial published literature on the LLNA is available making it appropriate for use as a benchmark against which new approaches, including in vitro alternatives, can be evaluated and validated. There is, therefore, a view that the LLNA represents the ‘gold standard’ for skin sensitization testing. However, although this is probably correct, it is important to recognize and acknowledge that in common with all other predictive tests (whether they be validated or not), the LLNA has limitations, in addition to strengths, some of which were mentioned above. Arguably, it is the limitations (e.g., the occurrence of false positive and false negative results) of test methods that are most important to understand. With respect to the LLNA, these limitations are similar to those associated with guinea‐pig skin sensitization methods. Among these are the occurrence of false positive and false negative results, susceptibility of results to changes in vehicle, and the possibility that interspecies differences may confound interpretation. In this commentary, these issues are reviewed and their impact on the utility of the LLNA for identification, classification, and potency assessment of skin sensitizers are considered. In addition, their relevance for the future development and validation of novel in vitro and in silico alternatives is explored.     

The impact of exposure variables on the induction of skin sensitization

Whereas many investigations of the variables associated with the elicitation of allergic contact dermatitis have been undertaken, to the point where we can begin to predict the likelihood of elicitation occurring in a given situation, the same is not true for the induction of skin sensitization. Studies have demonstrated that increasing dose has an impact; in an experimental setting, a number of variables received attention some decades ago. However, in the work reported here, the relative importance of the frequency and the duration of exposure is highlighted. In an investigation using a human repeated insult patch test, it was demonstrated that reduction of the exposure duration from 48 hr to 5 min decreased the rate of sensitization to 1% p-phenylenediamine (PPD) from 54% to 3%. However, in an extended clinical study, it was observed that infrequent but longer duration and higher concentration exposure to PPD was significantly less likely to induce sensitization compared to more frequent, short duration, and lower concentration exposure. Detailed statistical analysis of the results indicated that the most important factor driving the induction of skin sensitization was the number of exposures.     

Possible origin of the skin sensitization potential of isoeugenol and related compounds

Although many simple chemicals can give rise to the phenomenon of allergic contact dermatitis, it is rare that the mechanism of reaction between the chemical hapten and skin protein is known. A further complication is that metabolic processes may produce substantial changes to a chemical penetrating skin. Thus the skin contactant may be regarded as a prohapten which will give rise to the true hapten in vivo. In this study, the possible reaction mechanisms for a number of related simple aromatic chemicals have been investigated. The approach taken was to evaluate potential reaction mechanisms by assessing the degree to which chemicals could cross-react in sensitization tests. By careful choice of chemicals, it was then possible to confirm (or reject) options. Using this approach, a number of reaction schemes were investigated for eugenol, isoeugenol, dihydroeugenol, anethole and several related chemicals. The patterns of sensitization obtained and the cross-reactions observed indicated clearly that electrophile/nucleophile interactions were unlikely to provide a complete explanation of the sensitization processes. Eugenol and isoeugenol are not mutually cross-reactive, yet both cross-reacted with dihydroeugenol. Examination of the possible reaction mechanisms allows the speculation that eugenol reacts in part via a phenolic radical mechanism, whilst isoeugenol reacts largely via formation of an orthoquinone. Both reaction mechanisms are proposed for dihydroeugenol.     

Skin sensitization: strategies for the assessment and management of risk

Allergic contact dermatitis (ACD) is to a considerable extent a preventable disease. Limitation can be achieved by correct identification of skin sensitizers, characterization of their potency, understanding human skin exposure and application of good risk assessment/management strategies. Various methods exist which are accurate for the predictive identification of chemicals that possess skin-sensitizing properties. These are enshrined in regulations that aim to provide a harmonized approach to hazard identification. One of the methods, the local lymph node assay, also delivers information on the relative potency of sensitizers. Efforts are continuing in the European Union and at the Organization for Economic Cooperation and Development to use elements of this information for regulatory categorization of skin sensitizers. However, greater use can be made of this potency information in the application of quantitative risk assessments. Such assessments depend also on the availability of accurate data on human skin exposure, one aspect where legislation has little role to play. Management of risks by restriction of skin exposure is, in contrast, a key point where legislation can play an important role, helping to establish a level playing field for industry and setting good standards based on the legislator's ability to access all data. Ultimately, the combination of accurate hazard identification, potency measurement, risk assessment and management, underpinned by enabling legislation, will lead to reduction of ACD. For individuals who do still develop contact allergy, avoidance of ACD should continue to be a goal, based on raising awareness of skin protection, allergen labelling and other skincare strategies.     

Skin sensitization: Uncertainties,challenges, and opportunities for improved risk assessment

At the ESCD congress held in Manchester in 2016, a session was organized to encourage more dialogue between clinicians with expertise in skin sensitization and toxicologists seeking to provide effective risk assessment to prevent human health issues. That session focused on the remaining uncertainties regarding the induction and regulation of skin sensitization in humans, and the opportunities and challenges associated with the refinement and improvement of risk assessment methodologies. This short article, prompted by those discussions, debates what the authors regard as being among the most important and most intriguing uncertainties about skin sensitization and allergic contact dermatitis in humans, and the most significant opportunities for improving risk assessment. The aim has been to provide a basis for mapping out the areas that might benefit from a closer alignment between the relevant clinical community and toxicologists charged with the responsibility of ensuring that skin sensitization risks are understood and managed.     

Comparative analysis of skin sensitization potency of acrylates (methyl acrylate, ethyl acrylate, butyl acrylate, and ethylhexyl acrylate) using the local lymph node assay

There are currently available no systematic experimental data on the skin sensitizing properties of acrylates that are of relevance in occupational settings. Limited information from previous guinea-pig tests or from the local lymph node assay (LLNA) is available; however, these data are incomplete and somewhat contradictory. For those reasons, we have examined in the LLNA 4 acrylates: butyl acrylate (BA), ethyl acrylate (EA), methyl acrylate (MA), and ethylhexyl acrylate (EHA). The LLNA data indicated that all 4 compounds have some potential to cause skin sensitization. In addition, the relative potencies of these acrylates were measured by derivation from LLNA dose-response analyses of EC3 values (the effective concentration of chemical required to induce a threefold increase in proliferation of draining lymph node cells compared with control values). On the basis of 1 scheme for the categorization of skin sensitization potency, BA, EA, and MA were each classified as weak sensitizers. Using the same scheme, EHA was considered a moderate sensitizer. However, it must be emphasized that the EC3 value for this chemical of 9.7% is on the borderline between moderate (<10%) and weak (>10%) categories. Thus, the judicious view is that all 4 chemicals possess relatively weak skin sensitizing potential.     

Influence of application vehicle on skin sensitization to methylchloroisothiazolinone/methylisothiazolinone: an analysis using the local lymph node assay

The murine local lymph node assay (LLNA) is a method for the identification of skin sensitizing chemicals in which activity is measured as a function of proliferative responses induced in draining lymph nodes following topical exposure of mice to the test material. More recently, the LLNA has also been used for the determination of relative skin sensitizing potency based upon the mathematical derivation of an EC3 value, this being the estimated concentration of test chemical necessary to provoke a 3-fold increase in lymph-node cell-proliferative activity compared with concurrent vehicle-treated controls. Here we describe the use of the LLNA to determine the influence of vehicle on the skin-sensitizing potency of methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), the active ingredient of preservatives such as Kathon CG. To this end, LLNA responses to MCI/ MI were measured using the vehicles 4:1 acetone:olive oil (AOO), methyl ethyl ketone, dimethylsulfoxide, dimethylformamide, propylene glycol (PG) and acetone. It was found that the vehicle in which MCI/MI was applied had a substantial impact on activity, with derived EC3 values varying from 0.0049% with AOO to 0.048% with PG. With the other vehicles, EC3 values ranged from 0.0068 to 0.0076%. The skin sensitizing potency of MCI/MI as judged from LLNA responses is consistent with what is known of the requirements for sensitization in humans. It is proposed that the LLNA not only provides a method for determination of relative skin sensitizing potency, but is also appropriate for assessing the influence of vehicle matrix on sensitizing activity.     

Does occupational exposure to glass-fibres increase the general skin reactivity to irritants?

A total of 33 workers at a glass-wool factory were tested (patch tests with glass-fibres and chemical irritants, epicutaneous tests with Trafuril and intracutaneous tests with histamine) before starting to work at the factory and then re-tested after at least 4 weeks of exposure to glass-fibres. No statistically significant differences in the intensity of the skin reactions before and after exposure were found. Thus no general increased irritancy of the normal skin was induced by the continuous exposure to glass-fibres.     

Proactive surveillance of contact allergies: an important component of the risk management strategy for skin sensitizers

Risk assessment serves to ensure that dermal exposure to skin sensitizers does not result in the acquisition of allergic skin disease. Traditionally, the approach adopted was one of comparative analysis, involving benchmarking against other allergens of known potency. More recently, efforts have been made to embrace a quantitative risk assessment (QRA) approach. However, the accuracy of any risk assessment is reflected in the extent to which it meets the fundamental objective stated above. Thus, clinical experience is of key importance. There exists the possibility for the originators of risk assessments relating to chemicals that possess skin-sensitizing potential to work directly with the clinical community to proactively obtain this experience through specific surveillance programmes. This forms the focus of this review article. The current status of the QRA approach is considered initially. A recently published example of one such surveillance programme that was undertaken as a collaborative initiative between industry and the clinical community is then reviewed. Finally, a possible strategy for the future is presented, in which it is suggested that surveillance strategies might be deployed in certain situations as an adjunct to the initial risk assessment. It is hoped that such a framework might further improve the efficacy of future approaches to skin sensitization risk assessment.     

Experimental study of the potential for contact sensitization and cross-reaction of imidazole antifungals

We examined the potential for contact sensitization of miconazole nitrate and croconazole hydrochloride and the cross-reaction between them in guinea pigs by the maximization test of Magnusson and Kligman. Contact sensitivity was induced by croconazole hydrochloride in 5 out of 7 animals which, after being injected with 5% croconazole hydrochloride, underwent a closed patch with 25% croconazole hydrochloride. Contact sensitivity was not induced by miconazole nitrate. The 5 animals sensitized to croconazole hydrochloride were tested with 8 other imidazole antifungals and positive reactions were observed to oxiconazole nitrate in 2 of the 5 animals. This response may be a cross-reaction.