In vitro to in vivo extrapolation to derive a metabolism factor for estimating the aggregate exposure to salicylic acid after dermal exposure of its esters.

As part of the safety assessment of salicylate esters in cosmetics, we developed a metabolism factor based on in vitro to in vivo extrapolation (IVIVE) to provide a better estimation of the aggregate internal exposure to the common metabolite, salicylic acid. Optimal incubation conditions using human liver S9 were identified before measuring salicylic acid formation from 31 substances. Four control substances, not defined as salicylic esters but which could be mistaken as such due to their nomenclature, did not form salicylic acid. For the remaining substances, higher in vitro intrinsic clearance (CLint, in vitro) values generally correlated with lower LogP values. A "High-Throughput Pharmacokinetic" (HTPK) model was used to extrapolate CLint, in vitro values to human in vivo clearance and half-lives. The latter were used to calculate the percentage of substance metabolised to salicylic acid in 24 h in vivo following human exposure to the ester, i.e. the "metabolism factor". The IVIVE model correctly reproduced the observed elimination rate of 3 substances using in silico or in vitro input parameters. For other substances, in silico only-based predictions generally resulted in lower metabolism factors than when in vitro values for plasma binding and liver S9 CLint, in vitro were used. Therefore, in vitro data input provides the more conservative metabolism factors compared to those derived using on in silico input. In conclusion, these results indicate that not all substances contribute equally (or at all) to the systemic exposure to salicylic acid. Therefore, we propose a realistic metabolism correction factor by which the potential contribution of salicylate esters to the aggregate consumer exposure to salicylic acid from cosmetic use can be estimated.

Gene profiling in active dermatitis lesions strengthens the diagnosis of allergic contact dermatitis.

BACKGROUND: Distinguishing between allergic and nonallergic forms of Contact Dermatitis (CD) is challenging and requires investigations based on patch-testing. Early detection of allergy biomarkers in active CD lesions could refine and simplify the management of CD patients. OBJECTIVE: To characterize the molecular signatures of active CD lesions. METHODS: We studied the expression of 12 allergy biomarkers by qRT-PCR in active lesions of 38 CD patients. Allergic CD (ACD) was diagnosed based on patch test (PT) results and exposure assessment. Molecular signatures of active lesions, as well as positive PT reactions, were compared with those of reference chemical allergens and irritants. RESULTS: Nineteen of the 38 CD patients reacted positively upon patch-testing and exposure assessment confirmed ACD diagnosis for 17 of them. Gene profiling of active CD lesions revealed 2 distinct molecular patterns: patients harboring signatures similar to reference allergens (n = 23) or irritants (n = 15). Among the 23 patients with an "allergy signature," we found the 17 patients with confirmed ACD, while no culprit allergen was identified for the 6 other patients. Interestingly, the 15 patients without biomarker induction had negative PT, suggesting that they developed nonallergic CD reactions. CONCLUSION: Molecular signatures from active skin lesions may help to stratify CD patients and predict those suffering from ACD.

Expansion of the Cosmetics Europe skin sensitisation database with new substances and PPRA data.

The assessment of skin sensitisation is a key requirement in all regulated sectors, with the European Union's regulation of cosmetic ingredients being most challenging, since it requires quantitative skin sensitisation assessment based on new approach methodologies (NAMs). To address this challenge, an in-depth and harmonised understanding of NAMs is fundamental to inform the assessment. Therefore, we compiled a database of NAMs, and in vivo (human and local lymph node assay) reference data. Here, we expanded this database with 41 substances highly relevant for cosmetic industry. These structurally different substances were tested in six NAMs (Direct Peptide Reactivity Assay, KeratinoSens™, human Cell Line Activation Test, U-SENS™, SENS-IS, Peroxidase Peptide Reactivity Assay). Our analysis revealed that the substances could be tested without technical limitations, but were generally overpredicted when compared to reference results. Reasons for this reduced predictivity were explored through pairwise NAM comparisons and association of overprediction with hydrophobicity. We conclude that more detailed understanding of how NAMs apply to a wider range of substances is needed. This would support a flexible and informed choice of NAMs to be optimally applied in the context of a next generation risk assessment framework, ultimately contributing to the characterisation and reduction of uncertainty.

Identifying a reference list of respiratory sensitizers for the evaluation of novel approaches to study respiratory sensitization.

The induction of immunological responses that trigger bio-physiological symptoms in the respiratory tract following repeated exposure to a substance, is known as respiratory sensitization. The inducing compound is known as a respiratory sensitizer. While respiratory sensitization by high molecular weight (HMW) materials is recognized and extensively studied, much less information is available regarding low molecular weight (LMW) materials as respiratory sensitizers. Variability of symptoms presented in humans from such exposures, limited availability of (and access to) documented reports, and the absence of standardized and validated test models, hinders the identification of true respiratory sensitizers. This review aims to sort suspected LMW respiratory sensitizers based on available compelling, reasonable, inadequate, or questionable evidence in humans from occupational exposures and use this information to compose a reference list of reported chemical respiratory sensitizers for scientific research purposes. A list of 97 reported respiratory sensitizers was generated from six sources, and 52 LMW organic chemicals were identified, reviewed, and assigned to the four evidence categories. Less than 10 chemicals were confirmed with compelling evidence for induction of respiratory sensitization in humans from occupational exposures. Here, we propose the reference list for developing novel research on respiratory sensitization.

Development of a next generation risk assessment framework for the evaluation of skin sensitisation of cosmetic ingredients.

All cosmetic products placed onto the market must undergo a risk assessment for human health to ensure they are safe for consumers, including an assessment of skin sensitisation risk. Historically, in vivo animal test methods were used to identify and characterise skin sensitisation hazard, however non-animal and other new approach methodologies (NAMs) are now the preferred and mandated choice for use in risk assessment for cosmetic ingredients. The experience gained over the last three decades on how to conduct risk assessments based upon NAMs has allowed us to develop a non-animal, next generation risk assessment (NGRA) framework for the assessment of skin sensitisers. The framework presented here is based upon the principles published by the International Cooperation on Cosmetic Regulation (ICCR) and is human relevant, exposure led, hypothesis driven and designed to prevent harm. It is structured in three tiers and integrates all relevant information using a weight of evidence (WoE) approach that can be iterated when new information becomes available. The initial tier (TIER 0) involves a thorough review of the existing information including; identification of the use scenario/consumer exposure; characterisation of the chemical purity and structure; in silico predictions; existing data pertaining to skin sensitisation hazard (historical or non-animal); the identification of suitable read-across candidates with supporting hazard identification/characterisation information and application of exposure-based waiving. Considering all information identified in TIER 0, the next step is the generation of a hypothesis (TIER 1). All data are considered in an exposure-led WoE approach, taking into account an initial view on whether a chemical is likely to be a skin sensitiser or not, choice of defined approach (DA) and availability of read-across candidates. If existing information is insufficient for concluding the risk assessment, the generation of additional information may be required to proceed (TIER 2). Such targeted testing could involve refinement of the exposure estimation or generation of data from in vitro or in chemico NAMs. Once sufficient information is available, the final stage of the NGRA framework is the determination of a point of departure (POD), characterising uncertainty and comparing to the consumer exposure in a WoE. Thorough evaluation of the sources of uncertainty is essential to ensure transparency and build trust in new risk assessment approaches. Although significant progress has been made, industry must continue to share its experience in skin sensitisation NGRA via case studies to demonstrate that this new risk assessment approach is protective for consumers. Dialogue and collaboration between key stakeholders, i.e. risk assessors, clinicians and regulators are important to gain mutual understanding and grow confidence in new approaches.

NF-kappaB plays a major role in the maturation of human dendritic cells induced by NiSO(4) but not by DNCB.

Dendritic cell (DC) activation is a critical event for the induction of an immune response to haptens. Although signaling pathways such as mitogen-activated protein kinase (MAPK) family members have been reported to play a role in DC activation by haptens, little is known about the implication of the nuclear factor kappa B (NF-kappaB) pathway. In this work, we showed that NiSO(4) induced the expression of HLA-DR, CD83, CD86, and CD40 and the production of interleukin (IL)-8, IL-6, and IL-12p40 in human DCs, whereas DNCB induced mainly the expression of CD83 and CD86 and the production of IL-8. NiSO(4) but not DNCB was able to activate the degradation of IkappaB-alpha leading to the binding of the p65 subunit of NF-kappaB on specific DNA probes. Inhibition of the NF-kappaB pathway using BAY 11-7085 prevents both CD40 and HLA-DR expression and cytokine production induced by NiSO(4). However, BAY 11-7085 only partially inhibited CD86 and CD83 expression induced by NiSO(4). In addition, p38 MAPK and NF-kappaB were independently activated by NiSO(4) since SB203580 did not inhibit NF-kappaB activation by NiSO(4). Interestingly, we also showed that DNCB inhibited the degradation of IkappaB-alpha induced by tumor necrosis factor-alpha leading to alteration of CD40, HLA-DR, and CD83 expression but not of CD86 and CCR7. Extensive modifications of DC phenotype by NiSO(4) in comparison to DNCB are probably the consequence of NF-kappaB activation by NiSO(4) but not by DNCB.

Implication of the MAPK pathways in the maturation of human dendritic cells induced by nickel and TNF-alpha.

After application of haptens to the skin, immature dendritic cells (DC), also named Langerhans cells (LC), come into a maturation process, which include disappearance of specific molecules such as E-cadherin and Langerin and the expression of new molecules such as CD83, CD86 and CCR7. The involvement of p38 MAPK in DC maturation induced by haptens and TNF-alpha has already been shown, however, the role of the other MAPK, ERK and JNK, is less described. In this study, we demonstrated on human CD34(+)-derived DC that the three MAPK are participating to the expression of CD83, CD86 and CCR7 induced by nickel (NiSO(4)) but not to the down-regulation of E-cadherin and Langerin. In contrast, following TNF-alpha stimulation, only p38 MAPK is involved in CD83 and CCR7 expressions and ERK inhibits DC maturation while JNK inhibition had no effect. Our results also suggest that activation of p38 MAPK by TNF-alpha could partially suppress ERK activation and abrogates the inhibitory effect of ERK on DC maturation. In summary, the three MAPK pathways regulate DC maturation induced by haptens while only p38 MAPK seems to play a key role after TNF-alpha addition.

Nickel and DNCB induce CCR7 expression on human dendritic cells through different signalling pathways: role of TNF-alpha and MAPK.

After application of haptens on the skin, immature dendritic cells (DC), also named Langerhans cells (LC), migrate to the draining lymph node to sensitize naïve T-lymphocytes. Migration of DC involves many factors including the Cys-Cys chemokine receptor, CCR7. We investigated the effects of two well-known haptens, dinitrochlorobenzene (DNCB) and nickel (NiSO(4)), on the expression of CCR7 on human DC derived from CD34(+) progenitor cells. Both haptens were able to induce CCR7 expression and DC migration in response to Cys-Cys chemokine ligand, CCL19. Since interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha have been shown to participate in LC migration during contact hypersensitivity, we tested the effect of their neutralization on CCR7 expression. Neutralization of IL-1beta activity did not modify CCR7 expression in response to both haptens. CCR7 expression was strongly dependent on TNF-alpha in the case of DNCB, however, neutralization of TNF-alpha only partially reduced CCR7 expression upon NiSO(4) treatment. DNCB, NiSO(4) and TNF-alpha activated p38 mitogen-activated protein kinases (MAPK) and c-jun N-terminal kinase (JNK). Both p38 MAPK and JNK participated to TNF-alpha production induced by DNCB. Inhibition of both p38 MAPK and JNK affected significantly CCR7 expression upon nickel treatment whereas only inhibition of p38 MAPK but not of JNK downregulated CCR7 in the case of TNF-alpha stimulation. These results suggest that MAPK are necessary for haptens to induce CCR7 expression. NiSO(4), however, activates directly CCR7 expression through p38 MAPK and JNK activation whereas DNCB needs TNF-alpha whose secretion is also regulated by p38 MAPK and JNK.