GARDskin dose-response assay and its application in conducting Quantitative Risk Assessment (QRA) for fragrance materials using a Next Generation Risk Assessment (NGRA) framework.

Development of New Approach Methodologies (NAMs) capable of providing a No Expected Sensitization Induction Level (NESIL) value remains a high priority for the fragrance industry for conducting a Quantitative Risk Assesment (QRA) to evaluate dermal sensitization. The in vitro GARDskin assay was recently adopted by the OECD (TG 442E) for the hazard identification of skin sensitizers. Continuous potency predictions are derived using a modified protocol that incorporates dose-response measurements. Linear regression models have been developed to predict human NESIL values. The aim of the study was to evaluate the precision and reproducibility of the continuous potency predictions from the GARDskin Dose-Response (DR) assay and its application in conducting QRA for fragrance materials using a Next Generation Risk Assessment (NGRA) framework. Results indicated that the GARDskin Dose-Response model predicted human NESIL values with a good degree of concordance with published NESIL values, which were also reproducible in 3 separate experiments. Using Isocyclocitral as an example, a QRA was conducted to determine its safe use levels in different consumer product types using a NGRA framework. This study represents a major step towards the establishment of the assay to derive NESIL values for conducting QRA evaluations for fragrance materials using a NGRA framework.

A novel three-dimensional Nrf2 reporter epidermis model for skin sensitization assessment.

Skin sensitization assessment has progressed from the use of animal models towards the application of New Approach Methodologies (NAMs). Several skin sensitization NAMs are accepted for regulatory use, but a majority relies on submerged in vitro cell cultures that limit their applicability domain, posing challenges for testing hydrophobic chemicals and mixtures. A newly developed three-dimensional (3D) Nrf2 reporter epidermis model for skin sensitization assessment is reported. This NAM may help to overcome these limitations. The NAM combines the in vivo-like biology and exposure conditions of 3D epidermis models with the reliability, convenience, and cost-effectiveness of secreted reporter gene technology. The Keap1-Nrf2-ARE pathway was chosen as the reporter gene read-out, as it is induced by most skin sensitizers and already adopted in OECD Test guideline 442D. Immortalized human primary keratinocytes (Ker-CT) were stably transfected with the pIGB-Nrf2-SEAP vector to construct a Nrf2 reporter cell line. Ker-CT Nrf2 reporter cells showed negligible basal expression of the Secreted Embryonic Alkaline Phosphatase (SEAP) reporter, which was induced 13.5-fold by exposure to the skin sensitizer cinnamic aldehyde (CA). Co-exposure to CA and the Nrf2 inhibitor glucocorticoid clobetasol propionate significantly suppressed the CA-induced SEAP expression, confirming dependance of the SEAP expression on Nrf2 activation. Using air-liquid interface and animal constituent free culture conditions, the Ker-CT Nrf2 reporter cells differentiated to stratified 3D epidermis models with an in vivo-like skin architecture and functional skin barrier. Evaluation of a Ker-CT Nrf2 reporter cell-based 2D assay by testing 10 conventional reference chemicals showed a predictive accuracy for skin sensitization potential of 80% and 70% compared to LLNA and human data in two independent laboratories and a high intra- and interlaboratory reproducibility. Moreover, the 3D epidermis models predicted 3 sensitizing and 2 non-sensitizing reference chemicals correctly in a first proof-of-concept study. Further investigations foresee the testing of additional chemicals, including hydrophobic compounds and mixtures to confirm the potential of the 3D epidermis models to broaden the applicability domain for NAM-based skin sensitization assessment.

The skin sensitization adverse outcome pathway: exploring the role of mechanistic understanding for higher tier risk assessment.

For over a decade, the skin sensitization Adverse Outcome Pathway (AOP) has served as a useful framework for development of novel in chemico and in vitro assays for use in skin sensitization hazard and risk assessment. Since its establishment, the AOP framework further fueled the existing efforts in new assay development and stimulated a plethora of activities with particular focus on validation, reproducibility and interpretation of individual assays and combination of assay outputs for use in hazard/risk assessment. In parallel, research efforts have also accelerated in pace, providing new molecular and dynamic insight into key events leading to sensitization. In light of novel hypotheses emerging from over a decade of focused research effort, mechanistic evidence relating to the key events in the skin sensitization AOP may complement the tools currently used in risk assessment. We reviewed the recent advances unraveling the complexity of molecular events in sensitization and signpost the most promising avenues for further exploration and development of useful assays.

Use of guinea pig data to obtain starting points for skin sensitisation risk assessment - A commentary.

The increasing drive to understand the likelihood of skin sensitisation from plant protection products (PPPs) in workers and the general public has resulted in recent initiatives to establish a quantitative risk assessment (QRA) methodology applicable to these products and their exposure scenarios. The effective evaluation of skin sensitising substances requires not only the identification of that toxicological hazard, but also determination of relative sensitising potency. Typically, this has been achieved by interpretation of local lymph node assay (LLNA) dose response data, delivering what is known as the EC3 value. This permitted regulatory division of skin sensitisers into defined potency sub-categories, but more importantly enabled derivation of a no expected sensitisation induction level (NESIL) as the point of departure for QRA. However, for many existing substances there is no LLNA data, only older guinea pig results exist. To avoid additional (in vivo) testing, an approach has been outlined to employ guinea pig data and existing regulatory guidelines on the determination of potency sub-categorisation to provide a guinea pig based NESIL. The approach adopts a conservative extrapolation from LLNA NESIL benchmarks to deliver points of departure as the basis for the type of QRA process already in successful use by other industries.

Development of an in silico evaluation system that quantitatively predicts skin sensitization using OECD Guideline No. 497 ITSv2 defined approach for skin sensitization classification.

The Integrated Testing Strategy version 2 (ITSv2) Defined Approach, which is a reliable skin sensitization hazard and multi-step risk assessment method, does not support quantitative risk assessment such as local lymph node assay EC3 values. In this study, we developed a high-performance in silico evaluation system that quantitatively predicts the EC3 values of chemical substances by combining the ITSv2 Defined Approach for hazard identification (ITSv2 HI) with machine learning models. This system uses in chemico/in vitro test data, molecular descriptors, and distance information based on read-across concepts as explanatory variables. The system achieves an R2 value of 0.617 on external-validation data. Substances misclassified in ITSv2 HI are considered to have properties that do not match the correspondence between tests expressing the adverse outcome pathway assumed in the ITSv2 Defined Approach and skin sensitization. Therefore, ITSv2 HI is assumed to be correct within the applicability domains of this system. When using only substances within the applicability domains to reconstruct CatBoost models, the R2 value reached 0.824 on the external-validation data, representing an improvement in system performance. The results demonstrate the utility of explanatory variables that reflect the read-across concept and the advantages of integrating multiple prediction methods.

Quality of patch testing patient's own material in patients with suspected occupational skin diseases throughout Germany: Interim results of the German Social Accident Insurance (DGUV) research project FB 317b.

BACKGROUND: Due to limited availability of commercial test preparations, patch testing patient's own material (POM) is of great importance to diagnose occupational allergic contact dermatitis. OBJECTIVES: To assess the quality of performance and documentation of patch testing with POM in patients with suspected occupational skin diseases (OSD) in Germany. METHODS: Retrospective-prospective analysis of protocols of patch tests with POM was conducted between 2013 and 2021 in patients with suspected OSD and submitted to statutory accident insurance institutions. Assessments were done by predefined criteria. RESULTS: Three thousand and four patch tests with POM from 460 patients were included. A full description of the POM was provided in 73.3% of all tests. The test concentration, test vehicle and pH value were documented in 74.3%, 70.5% and 42.2% of tests, for which the respective parameters were considered relevant. One hundred and sixty-one positive reactions to POM were documented. In 72%, sufficient patch testing with commercial test substances was conducted to investigate the positive reaction. In 30.4%, consecutive patch testing of all ingredients of the POM was done. CONCLUSIONS: The results not only show considerable shortcomings mainly in documentation but also to some extent performance of patch tests with POM in patients with suspected OSD in Germany.

Challenges integrating skin sensitization data for assessment of difficult to test substances.

Difficult to test substances, including poorly soluble, mildly irritating, or UVCBs (unknown or variable composition complex reaction products or biological materials), producing weak or borderline in vivo results, face additional challenges in in vitro assays that often necessitate data integration in a weight of evidence (WOE) approach to inform skin sensitization potential. Here we present several case studies on difficult to test substances and highlight the utility of the toxicological priority index (ToxPi) as a data visualization tool to compare skin sensitization biological activity. The case study test substances represent two poorly soluble substances, tetrakis (2-ethylbutyl) orthosilicate and decyl palmitate, and two UVCB substances, alkylated anisole and hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2 undecanone. Data from key events within the skin sensitization adverse outcome pathway were gathered from publicly available sources or specifically generated. Incorporating the data for these case study test substances as well as data on chemicals of a known sensitization class (sensitizer, irritating non-sensitizer, and non-sensitizer) into ToxPi produced biological activity profiles which were grouped using unsupervised hierarchical clustering. Three of the case study test substances concluded to lack skin sensitization potential by traditional WOE produced biological activity profiles most consistent with non-sensi­tizing substances, whereas the prediction was less definitive for a substance considered positive by traditional WOE. Visualizing the data using bioactivity profiles can provide further support for WOE conclusions in certain circumstances but is unlikely to replace WOE as a stand-alone prediction due to limitations of the method including the impact of missing data points.

Non-animal test methods to detect chemicals that cause skin allergies are accepted alternatives to animal testing for this purpose. However, some chemicals are difficult to test using these methods, e.g., substances that cause skin irritation, are not water soluble or are mixtures of different compo­nents. We compiled existing and new data on how four such chemicals activate key elements of the biological pathway leading to allergic skin reactions and compared the resulting patterns with respective patterns of many chemicals confirmed to cause skin allergy, skin irritation or neither. The patterns were visualized and analyzed with a computer software tool. The tool confirmed that three substances were non-sensitizers but did not confirm that the fourth substance was a skin sensitizer as predicted by the standard assessment. This approach, which incorporates all available data types into the assessment of difficult to test chemicals, may further reduce unnecessary animal testing.

Assessment of Contact Allergens in "Hypoallergenic" Athletic Shoes by Mass Spectrometry.

Background: Identification of athletic shoes for patients with contact allergy is difficult. Company reports of allergen content are often incorrect. Objectives: To determine whether chemical analysis of 4 athletic shoes, previously reported to be free of the most common contact allergens, contain quantifiable allergen levels. Methods: Samples from the uppers and insoles of 4 shoes believed to be free of common allergens were assessed by mass spectrometry. A total of 4 rubber accelerators and 2 adhesives were directly quantified and additional 7 rubber accelerators were assessed using semiquantitative measures. Results: Aside from carbamates (assayed as 59 ppm zinc in insoles) in SeaVee's Sixty-Six sneakers, para-tertiarybutylphenol formaldehyde resin (PTBFR) (assayed as 7.6 ppm paratertiary butylphenol or 4-tertiary butylphenol [4TBP] in uppers) in Allbirds Tree Runners and rosin (assayed as 628 ppm sodium abietate in uppers) and carbamates (24 ppm zinc in uppers) in Saucony Jazz sneakers, these shoes had low levels of all allergens assayed in this study. Tom's Carlo sneakers contained rosin (127 ppm sodium abietate in insoles), PTBFR (6.5 ppm 4TBP in uppers), and carbamates (112 ppm sodium abietate in insoles) but had low levels of all other assayed allergens. Conclusions: Although identifying allergen-free shoes is challenging, the results of this analysis will help patch testing physicians recommend athletic shoes to patients with specific allergies.

Quantitative risk assessment of skin sensitising pesticides: Clinical and toxicological considerations.

Like many other consumer and occupational products, pesticide formulations may contain active ingredients or co-formulants which have the potential to cause skin sensitisation. Currently, there is little evidence they do, but that could just reflect lack of clinical investigation. Consequently, it is necessary to carry out a safety evaluation process, quantifying risks so that they can be properly managed. A workshop on this topic in 2022 discussed how best to undertake quantitative risk assessment (QRA) for pesticide products, including learning from the experience of industries, notably cosmetics, that already undertake such a process routinely. It also addressed ways to remedy the matter of clinical investigation, even if only to demonstrate the absence of a problem. Workshop participants concluded that QRA for skin sensitisers in pesticide formulations was possible, but required careful justification of any safety factors applied, as well as improvements to the estimation of skin exposure. The need for regulations to stay abreast of the science was also noted. Ultimately, the success of any risk assessment/management for skin sensitisers must be judged by the clinical picture. Accordingly, the workshop participants encouraged the development of more active skin health monitoring amongst groups most exposed to the products.

Skin sensitisation prediction using read-across, an illustrative next generation risk assessment (NGRA) case study for vanillin.

Skin sensitisation is a key adverse human health effect to be addressed in the safety assessment of cosmetic ingredients. Regulatory demands and scientific progress have led to the development of a Next Generation Risk Assessment (NGRA) framework, relying on the use of New Approach Methodologies (NAM) Defined Approaches (DA) and read-across instead of generating animal data. This case study illustrates the application of read-across for the prediction of the skin sensitisation potential of vanillin at the hypothetical use concentration of 0.5% in a shower gel and face cream. A three-step process was applied to select the most suitable analogues based on their protein reactivity, structural characteristics, physicochemical properties, skin metabolism profile and availability of skin sensitisation data. The applied read-across approach predicted a weak skin sensitiser potential for vanillin corresponding with a Local Lymph Node Assay EC3 value of 10%. Based on this EC3 value a point of departure of 2500 µg/cm2 was derived, resulting in an acceptable exposure level (AEL) of 25 µg/cm2. Because the consumer exposure levels (CEL) for the face cream (13.5 µg/cm2) and shower gel (0.05 µg/cm2) scenarios were lower than the AEL, the NGRA concluded both uses as safe.

Quantitative Risk Assessment of Dermal Sensitization Potential Following Use of Shampoo Products Containing the Formaldehyde Releasing Preservative DMDM Hydantoin.

Historically, formaldehyde was used as a preservative in personal care products to extend product shelf-life; however, given its skin sensitization potential it has been phased out of use and replaced with formaldehyde-releasing preservatives, such as Dimethyloldimethyl hydantoin (DMDMH). A relationship has been established between positive patch test results following exposure to DMDMH and previous sensitization to formaldehyde. Upon direct contact with the skin, formaldehyde can react with skin proteins and cause an acute inflammatory reaction, which may progress to skin sensitization following repeated exposure. This quantitative risk assessment (QRA) aimed to assess the risk of skin sensitization induction following use of shampoo products containing the maximum allowable concentrations of DMDMH in formulation (1% w/v), translating to a free formaldehyde concentration of 0.02%. To determine a margin of safety (MOS) for exposure to DMDMH from use of shampoo products, consumer exposure levels (CEL) were estimated based on typical use scenarios and then benchmarked against an acceptable exposure level (AEL). The AEL was derived using a weight of evidence approach where a range of no expected sensitization induction levels (NESILs) was utilized. The MOS values for a shampoo product containing 1% DMDMH (.02% formaldehyde) was above 1 for the typical use scenario indicating a low likelihood of skin sensitization induction among healthy individuals. Thus, it can be concluded that shampoo products containing DMDMH at or below current allowable concentrations are not expected to increase the risk of skin sensitization induction.

Integrated skin sensitization assessment based on OECD methods (III): Adding human data to the assessment.

Skin sensitizer potency assessment based on new approach methodologies is key to deriving a point of departure (PoD) for risk assessment. Regression models to predict a PoD based on OECD-validated in vitro tests and trained on local lymph node assay (LLNA) data were previously presented, and results from human tests were recently compiled. To integrate both data sources, the Reference Chemical Potency List (RCPL), which provides potency values (PV) for 33 chemicals integrating LLNA and human data in a structured weight-of-evidence approach, was developed. When calculating regression models vs PV or LLNA data, different weights for the input parameters were noted. As the RCPL is based on too few chemicals to train robust statistical models, the list of human data was extended to a larger set of PV (n = 139) with associated in vitro data. This database was used to retrain the regression models and to compare regression models trained vs (i) LLNA, (ii) PV or (iii) human DSA04 values. Using the PV as a target, predictive models of similar predictivity to the LLNA-based models were obtained, which mainly differ in a lesser weight of cytotoxicity and a higher weight of cell activation and reactivity parameters. Analysis of the human DSA04 dataset indicates a similar pattern but also shows that the human dataset is too small and biased to be a key dataset for potency prediction. Hence, an enlarged set of PV values appears to be a complementary tool to train predictive models next to an LLNA-only database.

Retrospective quantitative risk assessment of skin sensitization caused by an antimicrobial agent used in a consumer product: 2,3,5,6-Tetrachloro-4-(methylsulfonyl) pyridine.

BACKGROUND: Serious cases of allergic contact dermatitis (ACD) caused by exposure to 3,5,6-tetrachloro-4-(methylsulfonyl)pyridine (TCMSP; CAS no. 13108-52-6) used as an antimicrobial agent for desk mats have been reported in Japan. OBJECTIVE: A quantitative risk assessment (QRA) of TCMSP contained in desk mats was performed retrospectively. MATERIALS AND METHODS: A local lymph node assay (LLNA): BrdU-ELISA was conducted to determine a reliable EC1.6 value for TCMSP. The acceptable exposure level (AEL) for TCMSP was derived from the EC1.6 value by applying sensitization assessment factors (SAFs). The exposure level was estimated based on the assumption referring to the use conditions of thiabendazole in the same purpose. Then, the estimated exposure level was compared with the AEL to evaluate the skin sensitization risk. RESULTS: The AEL was calculated as 0.00458 µg/cm2 based on the EC1.6 value (0.011%, 2.75 µg/cm2 ) by applying a composite SAF of 600. The estimated TCMSP exposure level from the desk mat was 500 times greater than the AEL, indicating a serious skin sensitization risk. CONCLUSIONS: Assessments of skin sensitization potencies of chemicals to be used in consumer products are strongly recommended, and QRAs should be performed at the pre-marketing stage to avoid the skin sensitization risk in consumers.

Plant extracts, polymers and new approach methods: Practical experience with skin sensitization assessment.

Over the last decade, research into methodologies to identify skin sensitization hazards has led to the adoption of several non-animal methods as OECD test guidelines. However, predictive accuracy beyond the chemical domains of the individual validation studies remains largely untested. In the present study, skin sensitization test results from in vitro and in chemico methods for 12 plant extracts and 15 polymeric materials are reported and compared to available in vivo skin sensitization data. Eight plant extracts were tested in the DPRA and h-CLAT, with the 2 out of 3 approach resulting in a balanced accuracy of 50%. The balanced accuracy for the 11 plant extracts assessed in the SENS-IS was 88%. Excluding 5 polymers inconclusive in vitro, the remainder, assessed using the 2 out of 3 approach, resulted in 63% balanced accuracy. The SENS-IS method, excluding one polymeric material due to technical inapplicability, showed 68% balanced accuracy. Although based on limited numbers, the results presented here indicate that some substance subgroups may not be in the applicability domains of the method used and careful analysis is required before positive or negative results can be accepted.

Assessment of Comorbidities Associated With Allergic Contact Dermatitis in the United States: A Retrospective Claims-Based Study.

BACKGROUND: Allergic contact dermatitis (ACD) is a common dermatologic disease. Patch testing remains the criterion standard for diagnosis. In clinical practice, avoidance may be limited by patient occupation or noncompliance, the pervasive nature of the culprit agent, or barriers to expert care because of socioeconomic, cultural, or geographic factors. Thus, ACD is frequently chronic and/or recurrent; however, the comorbidities associated with ACD are not well characterized. OBJECTIVE: The aim of the study is to identify associations between ACD and psychiatric, sleep health, cardiovascular, and infectious conditions. METHODS: In this study, we used a large US claims database to identify comorbidities associated with ACD diagnosed after patch testing, including psychiatric, sleep health, cardiovascular, and infectious conditions. We also stratified these associations by chronicity of disease. RESULTS: We identified associations between ACD and psychiatric, sleep-related, cardiovascular, and infectious comorbidities. We also found that more chronic ACD was associated with more infectious comorbidities. All of these associations remained significant on further subanalysis when patients with AD and venous stasis were excluded. CONCLUSIONS: Allergic contact dermatitis is associated with multiple comorbidities. Further study is required to corroborate these findings, determine causality, and to explore the impact of possible interventions in the workup and management of this common and often debilitating disease.

Quantitative risk assessment of allergens leaching from menstrual hygiene products.

Allergic contact dermatitis (ACD) often associated with the topical use of perfumed products, remains one of the most common chronic skin disorders in Western countries. Since labelling of scented menstrual hygiene products (MHPs) is not mandatory, women might be unknowingly exposed to allergens. Given that vaginal mucosae lack the vital barrier function of the skin, skin allergens can easily penetrate and become systemically available and hence women may experience adverse effects in the anogenital region. The aim of this study was therefore to investigate whether women using scented MHPs are at risk of sensitization and hence developing ACD. Hereto, a Quantitative Risk Assessment (QRA) is performed on four well-known skin sensitizing chemicals (α-isomethyl ionone, benzyl salicylate, hexyl cinnamaldehyde and heliotropine) that were previously found leaching from five different scented MHPs including tampons and sanitary pads. The amounts of heliotropine, leached by one of the investigated tampons, exceeded acceptable exposure levels determined with the QRA and could induce sensitization. In addition, although no sensitization is expected for the other three compounds, an allergenic reaction might be provoked in women who are already sensitized. Labelling of allergens on scented MHPs would therefore help consumers to prevent adverse effects linked to ACD.