In Vitro Skin Models for Skin Sensitisation: Challenges and Future Directions

Predictive testing in contact allergy: facts and future.

The murine local lymph node assay (LLNA) is a predictive test for the identification of chemicals that have the potential to cause skin sensitization. Since its original development, the assay has been the subject of national and international evaluation studies and extensive comparisons with guinea pig tests and human data. On the basis of these investigations, the LLNA has recently been endorsed by ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) as a stand-alone method for skin sensitization hazard identification. At the same time, ICCVAM confirmed that, although the LLNA is not an in vitro method, it does represent a refinement in the way animals are used and can provide a means for reducing the number of animals used in sensitization hazard assessment. The investigations described here were designed to explore further the ability of the LLNA to identify accurately those chemicals that cause allergic contact dermatitis in humans. To that end we have measured, amongst 3 independent laboratories, LLNA responses induced by a total of 18 test chemicals, 11 of which are known to cause skin sensitization and 7 of which are believed not to be associated with any significant evidence of allergic contact dermatitis in humans. The LLNA correctly classified 16 of the 18 materials. The 11 chemicals tested which are associated with allergic contact dermatitis in humans were found to be positive in the LLNA. Of the 7 materials believed to be non-sensitizers, 5 were negative in the LLNA and 2 produced positive results. Collectively, these data provide additional evidence that the LLNA is able to discriminate skin sensitizers from those chemicals which do not possess a significant skin sensitization potential and thus provides a method for hazard identification that offers important animal welfare benefits.

許多類型的農藥，諸如有機磷類及胺基甲酸鹽類，具有較高的風險過度暴露足以危害到人類健康，可能導致過敏性氣喘，或者是過敏性接觸性皮膚炎。近幾年，過敏性接觸性皮膚炎已成為人類生活中一重要課題。為了檢測潛在的化學過敏物，研究人員發展出一種小鼠局部淋巴結分析 (local lymph node assay, LLNA)的方法，提供了定量且客觀的數據，適合做為劑量反應關係評估的標準。本研究中，利用LLNA檢測納乃得、谷速松以及四種混合農藥潛在皮膚過敏反應，並以2,4-dinitrochlorobenzene (DNCB)為陽性對照組，選用10% dibutyl phthalate (DBP)添加做為提升淋巴結細胞增生反應的增敏劑 (sensitizing agent)。實驗結果顯示，納乃得、谷速松及其他三種混合農藥 (包括陶斯寧、益保扶以及益達胺+第滅寧)具有較低或者陰性皮膚過敏反應。DNCB組及貝芬菲克利組之刺激指數 (stimulated index, SI值)皆大於3。然而，在添加10% DBP增敏劑後，僅造成部分組別輕微提升細胞增生活性，甚至有些組別加入DBP抑制了原有的細胞增生反應；針對貝芬菲克利混合農藥之兩單一農藥原體，在進行LLNA結果顯示SI值皆小於3。綜合本實驗結果，顯示納乃得及谷速松可能不具皮膚過敏反應；此外，DBP無法對任何過敏物質皆具良好的增敏效應，應為一種部分促進物；對於貝芬非克利混合農藥，我們發現到兩種非過敏原的農藥原體在相互調配混合後，可能影響原有的皮膚過敏特性。為了避免直接暴露於農藥過敏原之噴灑環境當中，建議農友及相關使用者應做好適當的防護措施，且建議農藥申請者在販售及註冊混合農藥時，對於皮膚過敏此一選項，應提供詳盡的實驗數據讓使用者做為參考，而非以現有之單一農藥原體本身的檢測結果做為主要依據。

The murine local lymph node assay (LLNA) was developed as an alternative method for the identification of chemicals that have the ability to cause skin sensitization and allergic contact dermatitis. The assay now has been evaluated extensively in the context of both national and international inter-laboratory collaborative trials and has been the subject of detailed comparisons with guinea pig test methods and human skin sensitization data. On the basis of these evaluations the LLNA has been endorsed recently by the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) as a stand-alone method for skin sensitization testing. The assay offers a number of important benefits compared with conventional guinea pig test methods, among these being provision of an objective and quantitative endpoint. Moreover, the LLNA provides advantages in the context of animal welfare; compared with guinea pig tests, fewer animals are required and these animals are subject to less trauma. It is important now that the validation status of the LLNA is recognized and the method applied widely so that these advantages may be realized. Hazard identification represents only the first step in the risk assessment process. A full toxicological evaluation of skin sensitization activity requires an understanding of relative potency. Guinea pig methods do not lend themselves readily to assessment of potency, and interest recently has focused on the utility of the LLNA for this purpose. Contained within this review article are brief descriptions of the history of the LLNA and the immunobiological basis for the method, together with detailed accounts of the conduct and interpretation of the assay. Procedural modifications to, and alternative endpoints for, the LLNA are considered also. Finally, the current regulatory status of the LLNA is summarized and the application of the method for the purposes of defining relative potency and developing risk assessments is reviewed.

Background: Chemicals vary considerably in their intrinsic ability to cause allergic contact dermatitis. Presently, there are no experimental methods available for the quantitative assessment of the relative sensitizing potency of chemical allergens. Objective: The objective of the investigations described here was to evaluate the use of the local lymph node assay for determining the relative skin sensitizing potential of chemicals. This has been addressed by comparing the sensitizing potency of formaldehyde with glutaraldehyde. Methods: Dose responses induced by formaldehyde and glutaraldehyde in the local lymph node assay, using either acetone or dimethylformamide (DMF) as the application vehicle, have been measured. Relative skin sensitizing potency was estimated as a function of the amount of chemical required to induce a threefold increase in lymph node cell proliferative activity, a mathematically derived EC3 value (estimated concentration required to induce a stimulation index of 3). Results: In both vehicles, glutaraldehyde induced substantially more vigorous responses in the local lymph node assay (EC3 values of 0.006mol/L in acetone and 0.002mol/L in DMF) than did formaldehyde (EC3 values of 0.18mol/L in acetone and 0.11mol/L in DMF). Conclusions: These results demonstrate that glutaraldehyde has a considerably greater potential to induce skin sensitization than does formaldehyde; the data are consistent with what is known of the ability of these chemicals to cause allergic contact dermatitis in humans. Using formaldehyde and glutaraldehyde as examples, the results here illustrate the utility of EC3 values derived from local lymph node assay responses for the estimation of the relative potency of skin sensitizing chemicals.

Estimation of relative skin sensitizing potency using the local lymph node assay: A comparison of formaldehyde with glutaraldehyde

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.

Propylene glycol (PG) has widespread use in pharmaceuticals, cosmetics, fragrances and personal care products. PG is not classified as hazardous under the Globally Harmonised System of Classification and Labelling of Chemicals (GHS) but poses an intriguing scientific and regulatory conundrum with respect to allergic contact dermatitis (ACD), the uncertainty being whether and to what extent PG has the potential to induce skin sensitisation. In this article we review the results of predictive tests for skin sensitisation with PG, and clinical evidence for ACD. Patch testing in humans points to PG having the potential to be a weak allergen under certain conditions, and an uncommon cause of ACD in subjects without underlying/pre-disposing skin conditions. In clear contrast PG is negative in predictive toxicology tests for skin sensitisation, including guinea pig and mouse models (e.g. local lymph node assay), validated in vitro test methods that measure various key events in the pathway leading to skin sensitisation, and predictive methods in humans (Human Repeat Insult Patch and Human Maximisation Tests). We here explore the possible scientific basis for this intriguing inconsistency, recognising there are arguably no known contact allergens that are universally negative in, in vitro, animal and human predictive tests methods.

Quantitative relationship between the local lymph node assay and human skin sensitization assays

Allergic Contact Dermatitis (ACD) is a common work-related skin disease that often develops as a result of repetitive skin exposures to a sensitizing chemical agent. A variety of experimental tests have been suggested to assess the skin sensitization potential. We applied a method of Quantitative Structure-Activity Relationship (QSAR) to relate measured and calculated physical-chemical properties of chemical compounds to their sensitization potential. Using statistical methods, each of these properties, called molecular descriptors, was tested for its propensity to predict the sensitization potential. A few of the most informative descriptors were subsequently selected to build a model of skin sensitization. In this work sensitization data for the murine Local Lymph Node Assay (LLNA) were used. In principle, LLNA provides a standardized continuous scale suitable for quantitative assessment of skin sensitization. However, at present many LLNA results are still reported on a dichotomous scale, which is consistent with the scale of guinea pig tests, which were widely used in past years. Therefore, in this study only a dichotomous version of the LLNA data was used. To the statistical end, we relied on the logistic regression approach. This approach provides a statistical tool for investigating and predicting skin sensitization that is expressed only in categorical terms of activity and nonactivity. Based on the data of compounds used in this study, our results suggest a QSAR model of ACD that is based on the following descriptors: nDB (number of double bonds), C-003 (number of CHR3 molecular subfragments), GATS6M (autocorrelation coefficient) and HATS6m (GETAWAY descriptor), although the relevance of the identified descriptors to the continuous ACD QSAR has yet to be shown. The proposed QSAR model gives a percentage of positively predicted responses of 83% on the training set of compounds, and in cross validation it correctly identifies 79% of responses.

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 EC3 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 EC3 value. The results show clearly that LLNA EC3 values are very comparable with the NOELs calculated from the literature. Moreover, the potency rankings based upon LLNA EC3 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 EC3 values that correlate closely with NOELs established from human repeat patch testing and from our clinical experience.

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.

The purpose of this article is to review, and make recommendations for, the use of relevant skin sensitization test methods, for the purposes of determination of relative potency and the threshold dose necessary for the induction of skin sensitization, and for risk assessment. In addressing the first area, the utility of three guinea pig tests (the guinea pig maximization test, the occluded patch test, and the open epicutaneous test) of the local lymph node assay (LLNA) and of human volunteer testing for the assessment of relative potency and identification of thresholds for sensitization were considered. The following conclusions were drawn. (1) Although attempts have been made to modify the guinea pig maximization test for the purposes of deriving dose-response relationships, this method is usually unsuitable for determination of relative sensitizing potency. (2) Guinea pig methods that do not require the use of adjuvant and which employ a relevant route of exposure (the occluded patch test and the open epicutaneous test) are more appropriate for the assessment of relative skin-sensitizing potency. (3) The LLNA is suitable for the determination of relative skin sensitizing potency, and the adaptation of this method for derivation of comparative criteria such as EC3 values (the estimated concentration of test chemical required to induce a stimulation index of 3 in the LLNA) provides an effective and quantitative basis for such measurements. (4) For all the methods identified above, potency is assessed relative to other chemical allergens of known skin sensitizing potential. The estimation of likely threshold concentrations is dependent upon the availability of suitable benchmark chemicals of known potency for human sensitization. (5) Human testing (and specifically, the Human Repeat Insult Patch Test) can provide information of value in confirming the absence of skin sensitizing activity of formulations and products under specific conditions of use and exposure. Based on the above, the following recommendations are made. (1) If results are already available from suitable guinea pig tests, then judicious interpretation of the data may provide information of value in assessing relative skin sensitizing potency. This option should be explored before other analyses are conducted. (2) The LLNA is the recommended method for new assessments of relative potency, and/or for the investigation of the influence of vehicle or formulation on skin sensitizing potency. (3) Whenever available, human skin sensitization data should be incorporated into an assessment of relative potency. With respect to risk assessment, the conclusion drawn is that all the available data on skin-sensitizing activity in animals and man should be integrated into the risk-assessment process. Appropriate interpretation of existing data from suitable guinea pig studies can provide valuable information with respect to potency, as the first step in the development of a risk assessment. However, for de novo investigations, the LLNA is the method favored for providing quantitative estimations of skin-sensitizing potency that are best suited to the risk assessment process. Finally, human testing is of value in the risk assessment process, but is performed only for the purposes of confirming product safety.

The Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TG) adopted the murine local lymph node assay (LLNA) and guinea pig maximization test (GPMT) as stand-alone skin sensitization test methods. However, unsaturated carbon-carbon double-bond and/or lipid acids afforded false-positive results more frequently in the LLNA compared to those in the GPMT and/or in human subjects. In the current study, oleic, linoleic, linolenic, undecylenic, fumaric, maleic, and succinic acid and squalene were tested in a modified LLNA with an elicitation phase (LLNA:DAE), and in a direct peptide reactivity assay (DPRA) to evaluate their skin-sensitizing potential. Oleic, linoleic, linolenic, undecylenic and maleic acid were positive in the LLNA:DAE, of which three, linoleic, linolenic, and maleic acid were positive in the DPRA. Furthermore, the results of the cross-sensitizing tests using four LLNA:DAE-positive chemicals were negative, indicating a chemical-specific elicitation response. In a previous report, the estimated concentration needed to produce a stimulation index of 3 (EC3) of linolenic acid, squalene, and maleic acid in the LLNA was < 10%. Therefore, these chemicals were classified as moderate skin sensitizers in the LLNA. However, the skin-sensitizing potential of all LLNA:DAE-positive chemicals was estimated as weak. These results suggested that oleic, linoleic, linolenic, undecylenic, and maleic acid had skin-sensitizing potential, and that the LLNA overestimated the skin-sensitizing potential compared to that estimated by the LLNA:DAE.