[show abstract][hide abstract] ABSTRACT: In a previous EPAA-Cefic LRI workshop in 2011, issues surrounding the use and interpretation of results from the local lymph node assay were addressed. At the beginning of 2013 a second joint workshop focused greater attention on the opportunities to make use of non-animal test data, not least since a number of in vitro assays have progressed to an advanced position in terms of their formal validation. It is already recognised that information produced from non-animal assays can be used in regulatory decision-making, notably in terms of classifying a substance as a skin sensitiser. The evolution into a full replacement for hazard identification, where the decision is not to classify, requires the generation of confidence in the in vitro alternative, e.g. via formal validation, the existence of peer reviewed publications and the knowledge that the assay(s) are founded on key elements of the Adverse Outcome Pathway for skin sensitisation. It is foreseen that the validated in vitro assays and relevant QSAR models can be organised into formal testing strategies to be applied for regulatory purposes by the industry. To facilitate progress, the European Partnership for Alternative Approaches to animal testing (EPAA) provided the platform for cross-industry and regulatory dialogue, enabling an essential and open debate on the acceptability of an in vitro based integrated strategy. Based on these considerations, a follow up activity was agreed upon to explore an example of an Integrated Testing Strategy for skin sensitisation hazard identification purposes in the context of REACH submissions.
Regulatory Toxicology and Pharmacology 10/2013; · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Abstract There is considerable interest in the immunobiological processes through which the development of allergic sensitization to chemicals is initiated and orchestrated. One of the most intriguing issues is the basis for the elicitation by chemical sensitizers of different forms of allergic reaction; that is, allergic contact dermatitis or sensitization of the respiratory tract associated with occupational asthma. Studies in rodents have revealed that differential forms of allergic sensitization to chemicals are, in large part at least, a function of the selective development of discrete functional sub-populations of CD4(+) and CD8(+) T-lymphocytes. Evidence for a similar association of chemical allergy in humans with discrete T-lymphocyte populations is, however, limited. It is of some interest, therefore, that two recent articles from different teams of investigators have shed new light on the role of polarized T-lymphocyte responses in the development of allergic contact dermatitis and occupational asthma in humans. The implications for understanding of chemical allergy in humans are explored in this Commentary.
Journal of Immunotoxicology 09/2013; · 1.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ability to be sensitized to experimental contact allergens declines significantly with increasing age, from as early as age 40 years. In contrast, the rate of contact allergy to chemical allergens (haptens) in cosmetic products significantly increases with age. This has been explained previously on the basis of greater cumulative exposure in the older age groups. However, outbreaks of contact allergy to preservatives in cosmetic products recorded soon after their introduction to the market have also shown a significantly higher rate among older adult age groups. This association with increasing age cannot be readily explained by exposure history or pattern, and is not compatible with a sensitizing/stimulatory reaction that degrades with age as the sole immune response. From this, the existence of a second, tolerizing/regulatory arm to the immune response to cutaneous haptens that possibly becomes less effective with age at a higher rate than the sensitizing/stimulatory arm can be inferred. This reinforces the view that current clinical and experimental observations of allergic contact dermatitis are best explained by an immune system with the functional ability to produce both sensitizing/stimulatory and tolerizing/regulatory responses.
[show abstract][hide abstract] ABSTRACT: One explanation for the large increase in the prevalence of atopic disease during the last 50 years in developed countries is the 'hygiene hypothesis'. This proposes that a reduced exposure to pathogenic microorganisms at a key period(s) during development result in the maintenance or acquisition of an atopic phenotype. Alternatively, or additionally, we have postulated that increased exposures to chemicals generally, and to irritant/haptenic chemicals in particular, during critical windows of pregnancy/ early life development have also contributed to changes in the prevalence of atopic disease. Having previously reviewed the potential roles of oral and cutaneous exposures to chemicals on the subsequent diagnosis of atopic disease, we here consider possible evidence of a role for exposure to airborne chemicals as a contributory factor in acquired susceptibility to atopic allergy. After controlling for known confounders five specific maternal occupations during pregnancy have been implicated as being associated with subsequent atopic disease in offspring. Each of these occupations is characterised by high and persistent exposure to airborne chemicals. High level exposure to volatile organic compounds in the domestic environment either during maternal pregnancy or in early life, is also associated with development of childhood atopic disease. Similarly, sustained exposure to airborne chlorinated chemicals from swimming pools during childhood has been associated with the development of atopic allergy. A possible immunological basis for these associations is that exposure to certain airborne chemicals, even at low levels, can result in the delivery of 'danger' signals that, in turn, bias the immune response toward the selective induction or maintenance of preferential Th2-type immune responses consistent with the acquisition of allergic sensitisation. This article is protected by copyright. All rights reserved.
British Journal of Dermatology 08/2013; · 3.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background. Dimethylfumarate (DMF) was the cause of a major outbreak of allergic contact dermatitis as a consequence of its use as an antifungal agent in leather products, particularly in furniture, with what became known as 'toxic sofa dermatitis'. Objectives. To determine whether the frequency and severity of reactions to DMF arose as a function of its intrinsic potency and/or the nature and extent of exposure. Methods. The intrinsic potency of DMF was measured with the standard local lymph node assay (LLNA), with determination of an EC3 value, which is the threshold in the LLNA and serves as an indicator of relative skin-sensitizing potency in humans. Results. The EC3 value for DMF was 0.35% when tested in dimethylformamide as a vehicle, indicating that DMF is a strong, but not an extreme, skin sensitizer in this mouse model. Conclusions. DMF appears to have a sensitizing potency in the mouse that is very similar to that of formaldehyde, which is also a strong human skin sensitizer. However, the frequency and intensity of allergic contact dermatitis reactions to DMF suggest that it was the prolonged, repeated and occlusive exposure to this chemical over large skin areas, combined with the strong sensitizing potency, that generated the 'perfect storm' conditions that caused the DMF epidemic.
[show abstract][hide abstract] ABSTRACT: The skin immune system's propensity to produce allergic contact dermatitis (ACD) to harmless chemicals, whilst otherwise being an efficient defence system, represents a dermatological paradox. We postulate that a major role in signaling in ACD is played by Toll-like receptor (TLR) 2 and TLR4 that arises from their activation by extracellular DAMPs (danger associated molecular patterns). Ligand activation of TLR4/2 results in the expression of interleukins (IL) IL-1β, IL-6, IL-12, IL-18 and IL-23, tumour necrosis factor-α (TNF-α) and interferon-α (IFN-α). These cytokines promote acquisition of sensitisation, and facilitate elicitation of contact allergy, via multiple mechanisms, including the recruitment of CD4+ T helper (Th) cells, Th1 and Th17 cells. As Th1 cells secrete large amounts of Danger Associated Molecular Pattern molecules (DAMPs), a DAMP immune circuit (positive feedback loop) is created. This is an important driver of skin sensitisation and skin inflammation. Pathogenic extracellular bacteria, but not commensal bacteria, produce PAMPs (Pathogen-Associated Molecular Pattern molecules), that stimulate the expression of Th1 and Th17-promoting cytokines via TLR 2 and 4. This also induces an immune circuit. The ability of the skin immune system to activate host defence mechanisms and to distinguish between pathogenic bacteria and commensals provides an explanation why skin sensitisation and ACD develops; processes that rely on the same biological pathways. These pathways may also shed light on pathogenesis of chronic pustular inflammatory dermatoses (e.g. acne vulgaris). The existence of safety signals from commensal bacteria that prevent initiation of these pathways may represent opportunities for novel therapeutic approaches to the treatment of inflammatory skin diseases.
British Journal of Dermatology 02/2013; · 3.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: To encourage the development and validation of alternative toxicity test methods, the effort required for validation of test methods proposed for regulatory purposes should be minimised. Performance standards (PS) facilitate efficient validation by requiring limited testing. Based on the validated method, PS define accuracy and reliability values that must be met by the new similar test method. The OECD adopted internationally harmonized PS for evaluating new endpoint versions of the local lymph node assay (LLNA). However, in the process of evaluating a lymph node cell count alternative (LNCC), simultaneous conduct of the regulatory LLNA showed that this standard test may not always perform in perfect accord with its own PS. The LNCC results were similar to the concurrent LLNA. Discrepancies between PS, LLNA and LNCC were largely associated with "borderline" substances and the variability of both endpoints. Two key lessons were learned: firstly, the understandable focus on substances close to the hazard classification borderline are more likely to emphasise issues of biological variability, which should be taken into account during the evaluation of results; secondly, variability in the results for the standard assay should be considered when selecting reference chemicals for PS.
Regulatory Toxicology and Pharmacology 12/2012; · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: The identification of chemicals possessing the intrinsic ability to cause sensitization via skin contact or inhalation, commonly referred to as skin and respiratory sensitizers, is a key endpoint in regulatory toxicology. Predictive assays for this purpose exist only for skin sensitizers, but, for both types of sensitizer, human evidence can be used to determine whether a substance should be classified. Furthermore, the use of human evidence for subcategorization according to sensitization potency is also accommodated within the regulations. Normally, this is based on the prevalence of sensitization in relation to the degree of exposure in the context of the size of the population exposed. However, the regulations also indicate that the severity of (allergic) reactions may be taken into account. In this article, we consider whether this is appropriate and whether there is evidence that reaction severity can inform decisions on classification and/or potency categorization. The conclusion drawn is that the severity of an allergic reaction does not correlate with, or serve as an indicator of, the sensitizing potency of a chemical. In reality, it reflects the overall extent of sensitization that an individual has acquired, in concert with the concentration of the causative allergen to which they have been exposed.
[show abstract][hide abstract] ABSTRACT: Enzymes used in cleaning products have an excellent safety profile, with little ability to cause adverse responses in humans. For acute toxicity, genotoxicity, sub-acute and repeated dose toxicity, enzymes are unremarkable. Reproductive toxicity and carcinogenicity are also not endpoints of concern. Exceptions are the ability of some proteases to produce irritating effects at high concentrations and more importantly, the intrinsic potential of these bacterial/fungal proteins to act as respiratory sensitizers. It is a reasonable assumption that the majority of enzyme proteins possess this hazard. However, methods for characterising the respiratory sensitisation hazard of enzymes are lacking and the information required for risk assessment and risk management, although sufficient, remains limited. Previously, most data was generated in animal models and in in vitro immunoassays that assess immunological cross-reactivity. Nevertheless, by the establishment of strict limits on airborne exposure (based on a defined minimal effect limit of 60ng active enzyme protein/m(3)) and air and health monitoring, occupational safety can be assured. Similarly, by ensuring that airborne exposure is kept similarly low, coupled with knowledge of the fate of these enzymes on skin and fabrics, it has proven possible to establish a long history of safe consumer use of enzyme containing products.
Regulatory Toxicology and Pharmacology 06/2012; 64(1):117-23. · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: As toxicology in the 21st century progresses towards a future which aims at avoiding the use of in vivo testing, the endpoint of skin sensitisation can now be found in the front line. Accordingly, it was appropriate for several industry sectors to meet and review what has been learned from the currently most widely used in vivo method, the local lymph node assay (LLNA), and to consider the status of progress as we attempt to move beyond that test. No toxicology test is perfect, an experience brought into focus by issues of false positives and, to a lesser extent, false negatives in the LLNA. Use of weight of evidence arguments for classification and labelling, as well as for risk assessment was emphasised and it was also noted that a sufficient body of evidence now exists for conduct of methods other than the LLNA for carefully defined chemical classes. In terms of in vitro alternatives, progress towards methods which will deliver mainly hazard identification is being made, with some entering the final stages of validation, whereby (Q)SAR tools still need improvement to be used on a large scale in practise. As various other challenges also remain, e.g. testing lipophilic substances, as well as the development of non-animal methods which deliver reliable information on potency for risk assessment, these will remain a topic for continuing research and development.
Regulatory Toxicology and Pharmacology 06/2012; 64(1):9-16. · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Cutaneous and Ocular Toxicology 06/2012; · 1.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: In a previous study, the predictive capacity of a modified local lymph node assay (LLNA) based on cell counts, the LNCC, was demonstrated to be closely similar to that of the original assay. In addition, a range of substances, including some technical/commercial materials and a range of agrochemical formulations (n = 180) have also been assessed in both methods in parallel. The results in the LNCC and LLNA were generally consistent, with 86% yielding an identical classification outcome. Discordant results were associated with borderline data and were evenly distributed between the two methods. Potency information derived from each method also demonstrated good consistency (n = 101), with 93% of predictions being close. Skin irritation was observed only infrequently and was most commonly associated with positive results; it was not associated with the discordant results. Where different vehicles were used with the same test material, the effect on sensitizing activity was modest, consistent with historical data. Analysis of positive control data indicated that the LNCC and LLNA displayed similar levels of biological variation. When taken in combination with the previously published results on LLNA Performance Standard chemicals, it is concluded that the LNCC provides a viable non-radioactive alternative to the LLNA for the assessment of substances, including potency predictions, as well as for the evaluation of preparations.
Journal of Applied Toxicology 05/2012; 32(8):597-607. · 2.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: There exists considerable historic experience of the relationship between exposure and both the induction of sensitization and the elicitation of respiratory symptoms from industrial enzymes of bacterial and fungal origin used in a wide variety of detergent products. The detergent industry in particular has substantial experience of how the control of exposure leads to limitation of sensitization with low risk of symptoms. However, the experience also shows that there are substantial gaps in knowledge, even when the potential occupational allergy problem is firmly under control, and also that the relationship between exposure and sensitization can be hard to establish. The latter aspect includes a poor appreciation of how peak exposures and low levels of exposure over time contribute to sensitization. Furthermore, while a minority of workers develop specific IgE, essentially none appear to have symptoms, a situation which appears to contradict the allergy dogma that, once sensitized, an individual will react to much lower levels of exposure. For enzymes, the expression of symptoms occurs at similar or higher levels than those that cause induction. In spite of some knowledge gaps, medical surveillance programs and constant air monitoring provide the tools for successful management of enzymes in the occupational setting. Ultimately, the knowledge gained from the occupational setting facilitates the completion of safety assessments for consumer exposure to detergent enzymes. Such assessments have been proven to be correct by the decades of safe use both occupationally and in consumer products.
Journal of Immunotoxicology 03/2012; 9(3):314-9. · 1.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Detergent enzymes have a very good safety profile, with almost no capacity to generate adverse acute or chronic responses in humans. The exceptions are the limited ability of some proteases to produce irritating effects at high concentrations, and the intrinsic potential of these bacterial and fungal proteins to act as respiratory sensitizers, demonstrated in humans during the early phase of the industrial use of enzymes during the 1960s and 1970s. How enzymes generate these responses are beginning to become a little clearer, with a developing appreciation of the cell surface mechanism(s) by which the enzymatic activity promotes the T-helper (T(H))-2 cell responses, leading to the generation of IgE. It is a reasonable assumption that the majority of enzyme proteins possess this intrinsic hazard. However, toxicological methods for characterizing further the respiratory sensitization hazard of individual enzymes remains a problematic area, with the consequence that the information feeding into risk assessment/management, although sufficient, is limited. Most of this information was in the past generated in animal models and in vitro immunoassays that assess immunological cross-reactivity. Ultimately, by understanding more fully the mechanisms which drive the IgE response to enzymes, it will be possible to develop better methods for hazard characterization and consequently for risk assessment and management.
Journal of Immunotoxicology 03/2012; 9(3):320-6. · 1.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Contact allergies are complex diseases, and one of the important challenges for public health and immunology. The German 'Federal Institute for Risk Assessment' hosted an 'International Workshop on Contact Dermatitis'. The scope of the workshop was to discuss new discoveries and developments in the field of contact dermatitis. This included the epidemiology and molecular biology of contact allergy, as well as the development of new in vitro methods. Furthermore, it considered regulatory aspects aiming to reduce exposure to contact sensitisers. An estimated 15-20% of the general population suffers from contact allergy. Workplace exposure, age, sex, use of consumer products and genetic predispositions were identified as the most important risk factors. Research highlights included: advances in understanding of immune responses to contact sensitisers, the importance of autoxidation or enzyme-mediated oxidation for the activation of chemicals, the mechanisms through which hapten-protein conjugates are formed and the development of novel in vitro strategies for the identification of skin-sensitising chemicals. Dendritic cell cultures and structure-activity relationships are being developed to identify potential contact allergens. However, the local lymph node assay (LLNA) presently remains the validated method of choice for hazard identification and characterisation. At the workshop the use of the LLNA for regulatory purposes and for quantitative risk assessment was also discussed.
Cellular and Molecular Life Sciences CMLS 03/2012; 69(5):763-81. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: Skin-sensitizing chemicals that cause allergic contact dermatitis do so by reacting with self-proteins such that the modified structure becomes antigenic. The reaction chemistry involved is well characterized, but there are exceptions, such as the occasional allergen sodium metabisulfite.
To identify the potential in cutaneo reaction chemistry of sodium metabisulfite.
The established protein reaction chemistry associated with aqueous sulfite chemistry was explored in the context of the protein modification stage in allergic contact dermatitis.
A probable mechanism for the in cutaneo modification of proteins by sodium metabisulfite involves the sulfite di-anion, acting as a nucleophile towards electrophilic centres in proteins, which is a rare mechanism, as most known skin-sensitizing chemicals behave as electrophiles.
Sodium metabisulfite is an unusual but not infrequent contact allergen whose chemistry suggests a previously unrecognized protein modification mechanism involving nucleophilic attack by sulfite di-anions on target electrophilic centres in skin proteins. The chemical properties required for sensitization by nucleophilic attack on skin proteins are quite restrictive, so the domain of nucleophilic sensitizers is expected to be small. Thiourea derivatives are among the sensitizers likely to act by this mechanism.
[show abstract][hide abstract] ABSTRACT: The murine local lymph node assay (LLNA) is a widely accepted method for assessing the skin sensitization potential of chemicals. Compared with other in vivo methods in guinea pig, the LLNA offers important advantages with respect to animal welfare, including a requirement for reduced animal numbers as well as reduced pain and trauma. In addition to hazard identification, the LLNA is used for determining the relative skin sensitizing potency of contact allergens as a pivotal contribution to the risk assessment process. The LLNA is the only in vivo method that has been subjected to a formal validation process. The original LLNA protocol is based on measurement of the proliferative activity of draining lymph node cells (LNC), as determined by incorporation of radiolabeled thymidine. Several variants to the original LLNA have been developed to eliminate the use of radioactive materials. One such alternative is considered here: the LLNA:BrdU-ELISA method, which uses 5-bromo-2-deoxyuridine (BrdU) in place of radiolabeled thymidine to measure LNC proliferation in draining nodes.
Current protocols in toxicology 02/2012; Chapter 20:Unit 20.7.