Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation

Asthma resulting from sensitisation of the respiratory tract to chemicals is an important occupational health issue, presenting many toxicological challenges. Most importantly there are no recognised predictive methods for respiratory allergens. Nevertheless, it has been found that all known chemical respiratory allergens elicit positive responses in assays for skin sensitising chemicals. Thus, chemicals failing to induce a positive response in skin sensitisation assays such as the local lymph node assay (LLNA) lack not only skin sensitising activity, but also the potential to cause respiratory sensitisation. However, it is unclear whether it will be possible to regard chemicals that are negative in in vitro skin sensitisation tests also as lacking respiratory sensitising activity. To address this, the behaviour of chemical respiratory allergens in the LLNA and in recently validated non-animal tests for skin sensitisation have been examined. Most chemical respiratory allergens are positive in one or more newly validated non-animal test methods, although the situation varies between individual assays. The use of an integrated testing strategy could provide a basis for recognition of most respiratory sensitising chemicals. However, a more complete picture of the performance characteristics of such tests is required before specific recommendations can be made.     

Applying the skin sensitisation adverse outcome pathway (AOP) to quantitative risk assessment

As documented in the recent OECD report ‘the adverse outcome pathway for skin sensitisation initiated by covalent binding to proteins’ (OECD, 2012), the chemical and biological events driving the induction of human skin sensitisation have been investigated for many years and are now well understood. Several non-animal test methods have been developed to predict sensitiser potential by measuring the impact of chemical sensitisers on these key events (Adler et al., 2011; Maxwell et al., 2011); however our ability to use these non-animal datasets for risk assessment decision-making (i.e. to establish a safe level of human exposure for a sensitising chemical) remains limited and a more mechanistic approach to data integration is required to address this challenge.Informed by our previous efforts to model the induction of skin sensitisation (Maxwell and MacKay, 2008) we are now developing two mathematical models (‘total haptenated protein’ model and ‘CD8⁺ T cell response’ model) that will be linked to provide predictions of the human CD8⁺ T cell response for a defined skin exposure to a sensitising chemical. Mathematical model development is underpinned by focussed clinical or human-relevant research activities designed to inform/challenge model predictions whilst also increasing our fundamental understanding of human skin sensitisation. With this approach, we aim to quantify the relationship between the dose of sensitiser applied to the skin and the extent of the hapten-specific T cell response that would result. Furthermore, by benchmarking our mathematical model predictions against clinical datasets (e.g. human diagnostic patch test data), instead of animal test data, we propose that this approach could represent a new paradigm for mechanistic toxicology.     

Guinea pig maximisation test for skin sensitisation: the use of fewer test animals

Current European Community (Annex V) guidelines recommend the use of 20 test animals in the guinea pig maximisation test for skin sensitisation. The suitability, for classification and labelling purposes, of reducing the number of test animals has been examined by analysing the results of 40 studies submitted to the Health and Safety Executive, and by the use of a mathematical model. Our results suggest that in most cases an experiment with ten test animals can be used to determine satisfactorily whether a substance should be labelled with the risk phrase may cause sensitisation by skin contact. However, serious consideration should be given to the need for additional investigation if two or three of the ten test animals show a sensitisation response. The highest nonirritant concentration of a substance should be used at challenge. Clearer guidance in Annex V on evaluating challenge responses would be beneficial.     

Determinants of skin sensitisation potential

Skin sensitisation is an important toxicological endpoint. The possibility that chemicals used in the workplace and in consumer products might cause skin sensitisation is a major concern for individuals, for employers and for marketing. In European REACH (Registration, Evaluation, and Authorisation of Chemicals) legislation, the sensitising potential should therefore be assessed for chemicals below the 10 ton threshold. Development of methods for prediction of skin sensitisation potential without animal testing has been an active research area for some time, but has received further impetus with the advent of REACH and the EU Cosmetics Directive (EU 2003). This paper addresses the issue of non‐animal based prediction of sensitisation by a mechanistic approach. It is known that the sequence of molecular, biomolecular and cellular events between exposure to a skin sensitiser and development of the sensitised state involves several stages, in particular penetration through the stratum corneum, covalent binding to carrier protein, migration of Langerhans cells, presentation of the antigen to naïve T‐cells. In this paper each of these stages is considered with respect to the extent to which it is dependent on the chemical properties of the sensitiser. The evidence suggests that, although penetration of the stratum corneum, stimulation of migration and maturation of Langerhans cells, and antigen recognition are important events in the induction of sensitisation, except in certain specific circumstances they can be taken for granted. They are not important factors in determining whether a compound will be a sensitiser or not, nor are they important factors in determining how potent one sensitiser will be relative to another. The ability to bind covalently to carrier protein is the major structure‐dependent determinant of skin sensitisation potential. A chemistry‐based prediction strategy is proposed involving reaction mechanistic domain assignment, reactivity and hydrophobicity determination, and application of quantitative mechanistic modelling (QMM) or read‐across. Copyright © 2007 John Wiley & Sons, Ltd.     

Application of a weight of evidence approach to assessing discordant sensitisation datasets: Implications for REACH

The local lymph node assay (LLNA) is the assay of choice in European regulatory toxicology. As with other toxicology/sensitisation assays, it has a potential for false results, the anionic surfactant sodium lauryl sulphate (SLS) representing a classic example. In the work reported here, examples of false positives in the LLNA are compared to published “benchmarks” such as SLS. Clear false positives (e.g. oleic acid) are also contrasted with examples where data interpretation is more challenging. As the LLNA will be applicable to >30,000 chemicals under REACH, and in the light of animal welfare considerations to do no more than the absolute minimum of animal testing, results from a single LLNA often represent the only available data on sensitisation. This reinforces the need to ensure data from this assay are interpreted intelligently, using scientific analysis of results and considering the weight of evidence, before decisions are made on which substances should be classified as representing a skin sensitisation hazard. In chemical classes where the LLNA has been shown to be an inappropriate assay other standardised methods (e.g. the Buehler or Magnusson and Kligman guinea pig tests [OECD 406]) should be employed as the first choice assays.     

Risk assessment of local dermal effects and skin sensitisation under the EU Chemicals Regulation REACH: a proposal for a qualitative, exposure scenario specific, approach

Within the framework of REACH, an assessment regarding local dermal effects and skin sensitisation should be performed for substances. Quantitative hazard information for these effects is often not available. Furthermore, it is difficult to relate the way in which animals are exposed in dermal toxicity studies directly to dermal exposure in practice. In the absence of quantitative information, a qualitative assessment for dermal effects is the most reasonable option. The qualitative approach as proposed in the REACH guidance recommends only general risk management measures (RMM) for three categories with a low, moderate and high identified hazard, without specifying which RMM are needed for a specific exposure scenario. We propose to differentiate frequency of exposure based on differences in activities and to compare measured and estimated local skin exposure levels with rules of thumb for evaluation of control of risks per hazard category. For workers, specific RMM regimes are assigned to each combination of hazard category and process category (PROC). For consumers, a strategy in which RMM are arranged from product-integrated measures to the use of personal protective equipment (PPE) is presented. Our approach may be transferred into automated assessment tools like Chesar and CEFIC GES.     

In vitro permeation of platinum and rhodium through Caucasian skin

During platinum group metals (PGMs) refining the possibility exists for dermal exposure to PGM salts. The dermal route has been questioned as an alternative route of exposure that could contribute to employee sensitisation, even though literature has been focused on respiratory exposure. This study aimed to investigate the in vitro permeation of platinum and rhodium through intact Caucasian skin. A donor solution of 0.3 mg/ml of metal, K₂PtCl₄ and RhCl₃ respectively, was applied to the vertical Franz diffusion cells with full thickness abdominal skin. The receptor solution was removed at various intervals during the 24 h experiment, and analysed with high resolution ICP-MS. Skin was digested and analysed by ICP-OES. Results indicated cumulative permeation with prolonged exposure, with a significantly higher mass of platinum permeating after 24 h when compared to rhodium. The mass of platinum retained inside the skin and the flux of platinum across the skin was significantly higher than that of rhodium. Permeated and skin retained platinum and rhodium may therefore contribute to sensitisation and indicates a health risk associated with dermal exposure in the workplace.     

In vitro permeation of platinum and rhodium through Caucasian skin

During platinum group metals (PGMs) refining the possibility exists for dermal exposure to PGM salts. The dermal route has been questioned as an alternative route of exposure that could contribute to employee sensitisation, even though literature has been focused on respiratory exposure. This study aimed to investigate the in vitro permeation of platinum and rhodium through intact Caucasian skin. A donor solution of 0.3 mg/ml of metal, K₂PtCl₄ and RhCl₃ respectively, was applied to the vertical Franz diffusion cells with full thickness abdominal skin. The receptor solution was removed at various intervals during the 24 h experiment, and analysed with high resolution ICP-MS. Skin was digested and analysed by ICP-OES. Results indicated cumulative permeation with prolonged exposure, with a significantly higher mass of platinum permeating after 24 h when compared to rhodium. The mass of platinum retained inside the skin and the flux of platinum across the skin was significantly higher than that of rhodium. Permeated and skin retained platinum and rhodium may therefore contribute to sensitisation and indicates a health risk associated with dermal exposure in the workplace.     

Assessment of the skin sensitising potency of the lower alkyl methacrylate esters

There is continued interest in, and imperatives for, the classification of contact allergens according to their relative skin sensitising potency. However, achieving that end can prove problematic, not least when there is an apparent lack of concordance between experimental assessments of potency and the prevalence allergic contact dermatitis as judged by clinical experience. For the purpose of exploring this issue, and illustrating the important considerations that are required to reach sound judgements about potency categorisation, the lower alkyl methacrylate esters (LAM) have been employed here as a case study.     

Differential effects of dopaminergic agents on locomotor sensitisation and on the reinstatement of cocaine-seeking and food-seeking behaviour

Rationale The dopaminergic pathways are involved in natural and drug reward related processes.Objectives To compare the respective involvement of the dopaminergic receptors D₁, D₂ and D₃ in natural-seeking versus drug-seeking behaviour and evaluate any concomitant expression of locomotor sensitisation.Methods In separate experiments, male Wistar rats were trained to self-administer cocaine (0.25 mg/infusion) or to press a lever to obtain food pellets. Following a prolonged period of extinction, reinstatement of lever responding was measured following non-contingent food delivery, cocaine (15 mg/kg), D₁-like (SKF 82958, 0.25 mg/kg), D₂-like (quinelorane, 0.25 mg/kg) and D₃-like (7-OHDPAT, 0.25 mg/kg) agonists. To demonstrate parallel expression of behavioural sensitisation, locomotor activity was recorded during the reinstatement sessions.Results Cocaine and quinelorane administrations reinstated cocaine-seeking behaviour and induced the expression of locomotor sensitisation, whereas SKF 82958 and 7-OHDPAT had either no effect or non-specific effects. In the food-seeking experiment, we found that quinelorane and 7-OHDPAT did not reinstate lever pressing. Cocaine increased responding on both active and inactive levers, whereas SKF 82958 had a more specific effect with higher responding on the previously food-associated lever.Conclusions Our results indicate that expression of locomotor sensitisation and reinstatement of cocaine-seeking but not food-seeking behaviours are in part supported by common dopaminergic substrates, among which the D₂ receptors play a crucial role.     

Assessing the potency of respiratory allergens: uncertainties and challenges

In contrast to skin sensitisation, there are no accepted tests for the identification of chemicals or proteins with the potential to cause sensitisation of the respiratory tract. Although progress has been made, the assessment of respiratory sensitisation potential remains associated with significant challenges and uncertainties. Nevertheless, there is interest in determining whether it is possible to assess the relative potency of respiratory sensitisers. The second Adaptation to Technical Progress (ATP) to the EU Classification, Labelling and Packaging (CLP) Regulation recently introduced changes to criteria for classification and labelling of chemicals and preparations, bringing it in line with the 3rd revision to the UN Globally Harmonised System of Classification and Labelling of Chemicals (GHS). Among other things, the second ATP introduces sub-categories for respiratory and skin sensitisers, discriminating between strong sensitisers and other sensitisers. Here we examine whether such categorisation of protein and/or chemical respiratory allergens is realistic and/or feasible. For this purpose comparisons have been drawn with skin sensitisation, where potency categorisation has now been widely accepted and successfully integrated into the regulatory process. The conclusion drawn is that, on the basis of the currently available information, potency categorisation for respiratory sensitisers is premature and could potentially be misleading.     

Chemical reactivity indices and mechanism-based read-across for non-animal based assessment of skin sensitisation potential

The skin sensitisation potential of chemicals is currently assessed using in vivo methods where the murine local lymph node assay (LLNA) is typically the method of first choice. Current regulatory initiatives are driving the impetus for the use of in vitro/in silico alternative approaches to provide the relevant information needed for the effective assessment of skin sensitisation, for both hazard characterisation and risk assessment purposes. A chemical must undergo a number of steps for it to induce skin sensitisation but the main determining step is formation of a stable covalent association with carrier protein. The ability of a chemical to react covalently with carrier protein nucleophiles relates to both its electrophilic reactivity and its hydrophobicity. This paper focuses on quantitative indices of electrophilic reactivity with nucleophiles, in a chemical mechanism-of-action context, and compares and contrasts the experimental approaches available to generate reactivity data that are suitable for mathematical modelling and making predictions of skin sensitisation potential, using new chemistry data correlated against existing in vivo bioassay data. As such, the paper goes on to describe an illustrative example of how quantitative kinetic measures of reactivity can be usefully and simply applied to perform mechanism-based read-across that enables hazard characterisation of skin sensitisation potential. An illustration of the types of quantitative mechanistic models that could be built using databases of kinetic measures of reactivity, hydrophobicity and existing in vivo bioassay data is also given.     

Dendritic cells and the assessment in vitro of skin sensitizing potential

It is now well established that dendritic cells (DC) play pivotal roles in the initiation and orchestration of adaptive immune responses, including cutaneous immune responses to chemical allergens that drive the acquisition of skin sensitization. It is not unexpected, therefore, that a large number, and wide variety, of proposed approaches for the identification of skin sensitizing chemicals in vitro are based upon the use of cultured DC or DC-like cells. The use of DC in this context is legitimate. However, with our rapidly increasing understanding of the diversity of cutaneous DC with respect to both phenotype and function, it is timely now to review briefly the potential limitations and interpretive difficulties that are associated with the use of DC-based assays. Among the important considerations are the fact that chemical-induced changes in the characteristics and function of cultured DC will not necessarily reflect accurately the events that that support the development of skin sensitization in vivo. In addition, most DC-based assays are predicated on a view that cutaneous DC have as their primary function the initiation of adaptive immune responses. However, it is now appreciated that cutaneous DC, and in particular epidermal Langerhans cells (LC), may also play important immunoregulatory roles that serve to limit and contain skin immune responses. Notwithstanding these considerations there is reason to believe that at least some in vitro DC-based assays are of value, and indeed some are currently the subject of a formal validation process. However, it is appropriate that such assays are configured and interpreted carefully, and with an appreciation of the complexity of DC biology.     

An assessment of the skin sensitisation hazard of a group of polyfunctional silicones using a weight of evidence approach

Discordant results were observed when testing five prototype polyfunctional silicone materials for skin sensitization potential in the murine local lymph node assay (LLNA) and in the guinea pig maximization test (GPMT). While all five silicone materials were consistently negative in the GPMT, the testing in the LLNA revealed weak to moderate skin sensitisation potential for four of the five test materials. Neither study quality nor other known chemical factors could explain these findings. Further analysis did not provide sufficient evidence for a link between the LLNA responses and the irritancy of the test substances. Only in the case of one of the test materials, the occurrence of an excessive level of irritation could be linked to the positive LLNA result. Considering all existing information including physico–chemical and structure activity and animal data as well as existing human experience from silicone exposures at the workplace or their use in cosmetic products, the weight of evidence suggests that none of the examined silicone materials represents a significant skin sensitization hazard to humans. The suitability of the LLNA appears questionable for this class of materials. In case of any additional data needs for other or new silicone materials, the skin sensitization testing strategy will require careful evaluation and will need to be set up on a case by case basis.     

Skin tumorigenic potential of benzanthrone: Prevention by ascorbic acid

Benzanthrone (BA) exposed occupational workers have been found to exhibit toxicological manifestations in the skin, thus it is quite likely that long term exposure may lead to skin tumorigenicity. Thus, attempts were made to elucidate the tumor initiating and promoting potentials of pure (PBA) and commercial benzanthrone (CBA). Additionally, the preventive role of ascorbic acid (AsA) was also assessed. PBA showed tumor initiating activity while CBA demonstrated tumor initiating as well as promoting activities in two-stage mouse skin tumor protocol. Further, prior treatment of AsA to PBA and CBA followed by twice weekly application of 12-o-tetradecanoyl phorbal myristate acetate (TPA) resulted into delayed onset of tumor formation and similarly single application of 7,12-dimethylbenz [α] anthracene (DMBA) followed by twice weekly application of AsA and CBA showed an increase in the latency period. Thus, AsA showed a protective effect against CBA promoted skin tumor. Furthermore, the topical application of CBA significantly increased the levels of xenobiotic enzymes. The animals topically treated with AsA along with topical application of CBA, restored all the impairment observed in enzyme activities. Thus, this study suggested that AsA can be useful in preventing PBA and CBA induced skin tumorigenicity.     

儿童专科医院头孢菌素类药物皮肤试验管理实践效果分析

目的探讨儿童专科医院病儿开展头孢菌素类药物皮肤试验的规范化管理和成效。方法回顾性分析 2019年 3月至 2020年 2月和 2020年 6月至 2021年 5月青岛市妇女儿童医院取消头孢菌素类药物常规皮试(仅保留说明书要求的头孢噻肟、头孢美唑皮试)前后,儿童病人头孢菌素类药物使用数据、药品不良反应情况以及抗菌药物使用强度、病人次均药品费用等指标变化情况,并对儿童病人皮试成本进行分析。结果头孢菌素皮试人次明显下降;保留常规皮试的头孢美唑、头孢噻肟消耗量降幅为 6.58%、4.73%,取消常规皮试的药物中头孢曲松消耗量增幅最高为 8.30%;各类头孢菌素药品不良反应(ADR)报告发生率均有提高( P<0.05);取消头孢菌素皮试前后,头孢噻肟、头孢美唑与其他头孢菌素类药物间 ADR发生率相比,均差异无统计学意义;住院儿童皮试抗菌药物使用强度由 1.27 DDDs下降至 0.55 DDDs;门急诊、住院病儿次均皮试药品费用平均下降 1.00元、 10.41元;头孢菌素皮试总成本下降 80.87%。结论取消儿童病人常规头孢菌素皮肤试验后,有利于降低病人用药负担,减少抗菌药物使用的同时有效保证医疗质量与安全。     

Analysis for the potential of polystyrene and TiO2 nanoparticles to induce skin irritation, phototoxicity, and sensitization

The human skin equivalent model (HSEM) is well known as an attractive alternative model for evaluation of dermal toxicity. However, only limited data are available on the usefulness of a HSEM for nanotoxicity testing. This study was designed to investigate cutaneous toxicity of polystyrene and TiO₂ nanoparticles using cultured keratinocytes, a HSEM, and an animal model. In addition, we also evaluated the skin sensitization potential of nanoparticles using a local lymph node assay with incorporation of BrdU. Findings from the present study indicate that polystyrene and TiO₂ nanoparticles do not induce phototoxicity, acute cutaneous irritation, or skin sensitization. Results from evaluation of the HSEMs correspond well with those from animal models. Our findings suggest that the HSEM might be a useful alternative model for evaluation of dermal nanotoxicity.     

In chemico,in vitro and in vivo comparison of the skin sensitizing potential of eight unsaturated and one saturated lipid compounds

The applicability of the Direct Peptide Reactivity Assay (DPRA), the KeratinoSens™ assay and the human cell line activation test (OECD Test Guidelines 442C, 442D, 442E) in predicting the skin sensitising potential of nine lipid (bio)chemicals was investigated. The results from the three assays were integrated using a published prediction model (PM), by which skin sensitisation is predicted if at least two of the three assays yield positive results. Of the eight test substances that were classified as non-sensitisers using available Guinea Pig Maximisation Test (GPMT) data, only five were correctly predicted as ‘negative’ in the PM. (However, only two were correctly predicted as ‘negative’ in the murine Local Lymph Node Assay.) The one lipid (bio)chemical that tested positive in the GPMT was also positive applying the PM. Based upon the outcome of the present study, lipid (bio)chemicals with a log K_ow up to 7–8 appear amenable to the three assays. However, solubility problems, that were not evident initially, affected the performance of the DPRA. Further investigations are merited to address the conclusiveness of negative test results with concurrent lack of cytotoxicity in the in vitro assays, to evaluate if poorly soluble substances come into contact with the cells.     

A comparative study of the sensitivity of the 3-induction and 9-induction Buehler test procedures for assessing skin sensitisation potential.

Assessment of skin sensitization potential is a mandatory requirement for the registration or notification of most types of chemicals and products. Until recently, two methods using the guinea pig as test model were the most widely accepted; the guinea pig maximisation test and the Buehler test. In the case of agrochemical formulations, which constitute the final end use product in contact with operators, industry and also some regulatory authorities consider the Buehler method more appropriate as the methodology is more relevant to likely exposure in the field. However, certain European regulatory authorities have become concerned about the sensitivity of the Buehler test for this purpose and have requested that a modified method is used in which additional applications of test materials are used during the induction phase of the protocol (a total of 9 rather than the normal 3). This study was designed to assess whether this modification was justified. Six reference substances (formaldehyde, alpha-hexylcinnamaldehyde, fragrance mix, thimerosal, mercaptobenzothiazole and phthalic anhydride); all mild to moderate skin sensitizing chemicals, were assessed in a study, which compared the use of 3 and 9 induction applications. The results of this study demonstrated that, although most of these sensitisers were detected by both protocols, the modified method (9 induction applications) was no more sensitive than the standard method (3 induction applications). As the modified protocol is also potentially more stressful to the animals, it is concluded that the use of additional induction applications in the Buehler test cannot be justified from either a scientific or an animal welfare perspective.     

疱疹宁乳膏皮肤毒性实验研究

目的:考察疱疹宁乳膏对动物皮肤的毒性作用。方法:采用健康大白鼠进行皮肤急性毒性试验;用健康家兔进行皮肤刺激性试验;用健康豚鼠进行皮肤过敏试验。结果:疱疹宁乳膏对大鼠完整皮肤及破损皮肤无皮肤急性吸收毒性,对家兔完整皮肤和破损皮肤无刺激性,对豚鼠完整皮肤无致敏作用。结论:疱疹宁乳膏用于治疗单纯疱疹和带状疱疹是较安全的。