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Introduction Objectives Methods Results Conclusions Patch test results may be influenced by age‐related factors. However, there is still discordant evidence between age and patch test results.We aim to evaluate the patch test results reflecting skin sensitisation, their relevance and association with clinical features by age group.Prospective multicentric study of all patients patch tested with the Spanish baseline series in participating centres. Age groups were pre‐defined as children (0‐ to 11‐years), adolescents (12‐ to 18‐years), young adults (19‐ to 30‐years), middle‐aged adults (31‐ to 65‐years) and older adults (≥66‐years). Occurrence of sensitisation, relevance and clinical features were compared by age group. Factors associated with skin sensitisation were investigated with multivariate logistic regression.A total of 13 368 patients were patch‐tested. Differences in positive patch test results and relevance by age were detected with the highest proportion in middle‐aged adults. Age‐related trend differences were found for nickel, potassium dichromate, caines, colophony, Myroxylon pereirae resin, 2‐hydroxyethyl methacrylate and limonene hydroperoxide. The multivariate logistic analysis (adjusted for sex, atopic dermatitis, body location and occupational dermatitis) showed an association between the age group of 31–65 (OR: 1.41, 95% CI: 1.26–1.58) and above 66‐years (OR: 1.15, 95% CI: 1.01–1.32) with a higher proportion of positive results, compared with young adults.Positive patch test results vary according to age, with the highest occurrence in middle‐aged adults. Most haptens did not present age‐related differences, reinforcing the use of baseline series regardless of age. [ABSTRACT FROM AUTHOR]

Skin sensitisation is a critical adverse effect assessed to ensure the safety of compounds and materials exposed to the skin. Alongside the development of new approach methodologies (NAMs), defined approaches (DAs) have been established to promote skin sensitisation potency assessment by adopting and integrating standardised in vitro, in chemico, and in silico methods with specified data analysis procedures to achieve reliable and reproducible predictions. The incorporation of additional NAMs could help increase accessibility and flexibility. Using superior algorithms may help improve the accuracy of hazard and potency assessment and build confidence in the results. Here, we introduce two new DA models, with the aim to build DAs on freely available software and the newly developed kDPRA for covalent binding of a chemical to skin peptides and proteins. The new DA models are built on an existing Bayesian network (BN) modelling approach and expand on it. The new DA models include kDPRA data as one of the in vitro parameters and utilise in silico inputs from open-source QSAR models. Both approaches perform at least on par with the existing BN DA and show 63% and 68% accuracy when predicting four LLNA potency classes, respectively. We demonstrate the value of the Bayesian network's confidence indications for predictions, as they provide a measure for differentiating between highly accurate and reliable predictions (accuracies up to 87%) in contrast to low-reliability predictions associated with inaccurate predictions. [ABSTRACT FROM AUTHOR]

Background: Skin sensitisation is one of the important regulatory endpoints used to determine whether a given test chemical can elicit an allergic response in susceptible individuals. The in-vitro approaches for predicting skin sensitisation potential for chemicals have been well-developed and widely accepted. Continuous efforts are being made to identify novel solutions to overcome the limitations of the existing approaches. Purpose: The herbal preparations used in dermal therapeutics are frequently found to elicit skin sensitisation, requiring caution. The herbal preparations tend to contain more than one phytochemical. The expected active ingredient may not possess skin sensitisation. However, the other phytochemical traces could pose synergistic and potentiating effects towards enhanced/suppressed skin sensitisation. In this regard, isolation, purification, yield, and in-vitro / in-vivo evaluation of skin sensitisation for all the traces would be time, cost, and animal-consuming. Hence, the regulatory agencies appreciate using computational tools to substantiate in-vitro methods. The regulators have also endorsed using in-silico approaches like quantitative structure-activity relationships (QSAR) and read-across. These approaches are limited to structure-based predictions and in combination with system-level approaches, which comprehensively facilitate a better understanding of complex biological systems. Study design: In the present study, 603 phytochemicals were screened in-silico using OECD QSAR Toolbox. The limitations of the QSAR Toolbox in predicting skin sensitisers were identified. Method: The systems biology-based approach to complement the QSAR-based prediction was developed, and 41 biomarkers were identified as a determinant of skin sensitisation. The molecules attributed to these biomarkers were predicted as skin sensitisers. Results: As a result, 31 phytochemicals were predicted as skin sensitisers. Acrolein, eugenol, formaldehyde, and glycerol were selected for experimental validation of the in-silico predictions using direct peptide reactivity assay (DPRA), KeratinoSens™, and h-CLAT assays representing each key event in skin sensitisation. Further, identified biomarkers were validated in THP-1 and KeratinoSens cell lines using qRT-PCR. Among all genes, IL-8 and CCL4 were upregulated by 6.4 and 5.6 folds, respectively, in THP-1, while AKR1C2 and AKR1C3 were upregulated by 7.8 and 11.1 folds, respectively, in KeratinoSens. IL-8 level was analysed by ELISA and found to be in line with qRT-PCR results. Conclusion: The study proposes a possible strategy based on the “iSiCiV” approach to enhance the prediction of the dermal sensitisation potential of a test compound. The approach offers a robust platform and highlights an opportunity to shift the paradigm of skin sensitisation assay with a better understanding of toxicology mechanisms.

Background Atopic eczema is a common childhood skin problem linked with asthma, food allergy and allergic rhinitis that impairs quality of life. Objectives To determine whether advising parents to apply daily emollients in the first year can prevent eczema and/or other atopic diseases in high-risk children. Design A United Kingdom, multicentre, pragmatic, two-arm, parallel-group randomised controlled prevention trial with follow-up to 5 years. Setting Twelve secondary and four primary care centres. Participants Healthy infants (at least 37 weeks’ gestation) at high risk of developing eczema, screened and consented during the third trimester or post delivery. Interventions Infants were randomised (1 : 1) within 21 days of birth to apply emollient (Doublebase Gel®; Dermal Laboratories Ltd, Hitchin, UK or Diprobase Cream®) daily to the whole body (excluding scalp) for the first year, plus standard skin-care advice (emollient group) or standard skin-care advice only (control group). Families were not blinded to allocation. Main outcome measures Primary outcome was eczema diagnosis in the last year at age 2 years, as defined by the UK Working Party refinement of the Hanifin and Rajka diagnostic criteria, assessed by research nurses blinded to allocation. Secondary outcomes up to age 2 years included other eczema definitions, time to onset and severity of eczema, allergic rhinitis, wheezing, allergic sensitisation, food allergy, safety (skin infections and slippages) and cost-effectiveness. Results One thousand three hundred and ninety-four newborns were randomised between November 2014 and November 2016; 693 emollient and 701 control. Adherence in the emollient group was 88% (466/532), 82% (427/519) and 74% (375/506) at 3, 6 and 12 months. At 2 years, eczema was present in 139/598 (23%) in the emollient group and 150/612 (25%) in controls (adjusted relative risk 0.95, 95% confidence interval 0.78 to 1.16; p = 0.61 and adjusted risk difference −1.2%, 95% confidence interval −5.9% to 3.6%). Other eczema definitions supported the primary analysis. Food allergy (milk, egg, peanut) was present in 41/547 (7.5%) in the emollient group versus 29/568 (5.1%) in controls (adjusted relative risk 1.47, 95% confidence interval 0.93 to 2.33). Mean number of skin infections per child in the first year was 0.23 (standard deviation 0.68) in the emollient group versus 0.15 (standard deviation 0.46) in controls; adjusted incidence rate ratio 1.55, 95% confidence interval 1.15 to 2.09. The adjusted incremental cost per percentage decrease in risk of eczema at 2 years was £5337 (£7281 unadjusted). No difference between the groups in eczema or other atopic diseases was observed during follow-up to age 5 years via parental questionnaires. Limitations Two emollient types were used which could have had different effects. The median time for starting emollients was 11 days after birth. Some contamination occurred in the control group (< 20%). Participating families were unblinded and reported on some outcomes. Conclusions We found no evidence that daily emollient during the first year of life prevents eczema in high-risk children. Emollient use was associated with a higher risk of skin infections and a possible increase in food allergy. Emollient use is unlikely to be considered cost-effective in this context. Future research To pool similar studies in an individual patient data meta-analysis. Trial registration This trial is registered as ISRCTN21528841. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 12/67/12) and is published in full in Health Technology Assessment; Vol. 28, No. 29. See the NIHR Funding and Awards website for further award information. Plain language summary Eczema is a troublesome itchy skin condition affecting 1 in 5 children and 1 in 10 UK adults. There is no cure and affected children are more likely to develop food allergies. We wanted to see if we could prevent eczema by protecting the skin of babies at higher risk of developing eczema (with an immediate relative with eczema, asthma or hay fever) with moisturisers used to treat dry skin. Previous research suggested that protecting the skin barrier might also prevent food allergy. One thousand three hundred and ninety-four families took part in a study; half of them were asked to apply moisturiser every day to their newborn baby for the first year and half to look after their baby’s skin in the normal way. At the age of 2 years, we did not see any difference in how common eczema was between the two groups: 23% had eczema in the moisturiser group and 25% in the normal care group. It did not matter how we defined eczema – whether examined by a researcher or parent report. We did not find any differences in related conditions like asthma or hay fever either. We found that children using moisturisers had seen their doctor slightly more often for mild skin infections. There was a hint that food allergy might have been increased in the moisturiser group, but there was not enough data to be sure. We followed up the children to age 5 years, but we still did not find any benefits from using moisturisers in early life. Since this study, other similar research has been done using newer types of moisturisers, but their results are the same. This study shows that using daily moisturisers on healthy babies with a high risk of eczema does not prevent eczema. It is one less thing for busy families to worry about. Scientific summary Background Eczema, also known as atopic eczema (AE) or atopic dermatitis, is a common inflammatory skin disease that typically starts in early life. Eczema affects around 20% of children and 10% of adults in developed countries. Although most cases are mild, moderate to severe disease can have a major impact on the quality of life of an affected child and their family. The main symptom of this systemic inflammatory disease is itching which leads to scratching, bleeding, secondary infection as well as sleep loss and the social stigma of a visible skin disease. Genetic factors such as mutations in the gene encoding filaggrin (FLG) – a protein that is important for maintaining skin barrier function – have been shown to be important in increasing eczema risk, severity and persistence. Epidemiological studies showing that eczema is more common in smaller families, migrant populations and in higher socioeconomic status also suggest that the environment is critically important in determining disease expression. Eczema is closely related to other ‘atopic’ conditions including asthma, hay fever and food allergy. Some evidence suggests that the impaired skin barrier caused by eczema in early life is an important route for the development of food allergy as a result of sensitisation through the skin. Treatment of eczema depends on severity: mild eczema can be treated with emollients (moisturisers) and weak topical corticosteroids. Moderate eczema usually needs potent topical corticosteroids and other anti-inflammatory treatments such as topical tacrolimus, whereas severe eczema is treated by ultraviolet light or systemic treatments including ciclosporin, methotrexate, dupilumab and an emerging pipeline of new biologics. While good progress has been made with treatment, prevention of eczema remains challenging. Strategies include avoidance of allergens (foods and house dust mite) during pregnancy and early life, exclusive breastfeeding, or different timing of introducing solids, but these have not shown convincing preventative effects. Probiotics and prebiotics have shown some benefit, but the exact strain or combination and timing of intervention remain unclear. One previously unexplored eczema prevention strategy is enhancement of the skin barrier in early life. The hypothesis is that enhancing the skin barrier could interrupt an early cascade of inflammatory events that can lead to chronic auto-immune eczema and potentially prevent skin sensitisation to common allergens that can lead to food allergy. Two small pilot studies suggested that protecting the skin of babies who had a first-degree relative with eczema, asthma or hay fever with emollients could prevent the development of eczema in the first year of life. We wanted to see whether daily emollient application for the first year of life in such high-risk babies can prevent eczema and other allergic diseases in a sustained and convincing way by means of a definitive pragmatic randomised controlled trial. Objectives The primary objective was to determine whether advising parents to apply emollient daily for the first year of life could prevent eczema at 2 years of age when compared with standard skin-care advice alone in children at high risk of developing eczema. Secondary objectives included evaluating whether prophylactic use of emollients would delay the onset of eczema or reduce eczema severity when compared to standard skin-care advice, whether any preventive effect at age 2 years was sustained up to age 5 years and to determine the safety and cost-effectiveness of such a strategy. We also sought to determine whether emollients could prevent the development of other associated allergic conditions including food allergy, asthma and hay fever up to the age of 5 years. Methods We conducted a multicentre, pragmatic, two-arm parallel group randomised controlled prevention trial which recruited participants from 12 hospitals and 4 general practices in the UK. Participants were infants of at least 37 weeks’ gestation at high risk of developing eczema defined by having at least one first-degree relative with a parental report of doctor-diagnosed eczema, allergic rhinitis or asthma. Mothers had to be 16 years or older, and the consenting adult had to understand English. We excluded babies with a severe widespread skin condition that would make eczema assessment difficult; a serious health issue that would make it difficult for the family to take part in the trial; or a condition that would make the use of emollient inadvisable. Informed consent was obtained from mothers during pregnancy, or mother, father or guardian post delivery. The intervention group was advised to use one of two study emollients (Doublebase Gel® or Diprobase Cream®) at least once daily to the whole body (excluding the scalp) until the child reached 1 year, plus standard skin-care advice (designated the ‘emollient group’). The control group was advised to use standard skin-care advice only. Standard skin-care guidance, in booklet and video format at the time of randomisation, provided advice to use mild cleansers and shampoos specifically formulated for infants, and to avoid soap, bubble bath and baby wipes, and was given to both groups. Intervention adherence was measured at 3, 6 and 12 months and defined as satisfactory if emollients were applied at least three to four times per week to most of the child’s body. A similar definition was used to define contamination in the control group. The primary outcome was presence of eczema in the last year when the baby was aged 2 years, defined as meeting the United Kingdom Working Party (UKWP) Diagnostic Criteria for AE for children under 4 years. Secondary outcomes encompassed other ways of defining eczema including any parental report of a clinical diagnosis of eczema up to 2 years, parent completion of UKWP criteria at 1 and 2 years, and presence of visible eczema at 2 years recorded by a nurse masked to treatment allocation. Other eczema secondary outcomes included time to onset of eczema (based on first parent report of clinician diagnosis and first topical corticosteroid or immunosuppressant prescription); severity of eczema measured by trained nurses using the Eczema Area and Severity Index at 2 years and parent-reported Patient-Oriented Eczema Measure at 1 and 2 years. Other secondary outcomes included parent-reported wheezing and allergic rhinitis between 1 and 2 years; allergic sensitisation (masked skin prick tests) to milk, egg, peanut, cat dander, grass pollen or dust mite at 2 years; parent-reported food allergy; parental report of clinical diagnosis of food allergy at 1 and 2 years; and allergy to milk, egg or peanut at 2 years confirmed either by oral food challenge or for cases in which no oral food challenge was done, an expert panel of experienced paediatric allergists masked to treatment allocation. The panel decisions were made using an algorithm, validated using data from a previous trial, which incorporates all available data including skin prick test results, previous reaction history, frequency of food ingestion and allergy tests done outside the trial. The main economic outcome measure was incremental cost per percentage decrease in risk of eczema at 2 years in a cost-effectiveness analysis (CEA). Cost–utility analyses using quality-adjusted life-years (QALYs) derived from the proxy Child Health Utility instrument-9 domains (CHU-9D) for the child was also conducted as a secondary analysis. Safety outcomes were parent-reported skin infections (parents were asked what the doctor called the infection) and emollient-related infant slippages during the intervention period (year 1). Tertiary outcomes at 3, 4 and 5 years were to evaluate if emollients in early life could also prevent later-onset allergic diseases including asthma, hay fever and food allergy, and to see whether any early possible benefits of eczema prevention were sustained. Outcomes included parental-reported presence of eczema; severity of eczema; wheezing; allergic rhinitis; food allergy symptoms and clinical diagnoses of asthma, allergic rhinitis and food allergy. Randomisation (1 : 1) within 21 days of birth was performed using computer-generated pseudo-random code with permuted blocks of randomly varying size and concealed from the trial investigators via a web-based randomisation system. Randomisation was stratified by recruiting centre and number of first-degree relatives with eczema, asthma or hay fever (1, 2 or > 2). Research nurses conducting outcome assessments were masked to participant treatment allocation. Families were not masked to the allocated interventions and were reminded not to reveal allocation to research nurses. The trial was powered to detect a relative reduction of 30% in the primary outcome at the 5% significance level (two-sided) with 90% power assuming that 30% of children in the control group would have eczema and 20% attrition, resulting in a sample size of 1282. The target sample size was reached faster than anticipated at which point the Trial Steering Committee permitted that all pregnant mothers who had already consented to the trial could be randomised on the birth of the baby giving a maximum possible sample size of 1400. We analysed participants as randomised regardless of adherence with allocation using available data (i.e. without imputation for missing data) with sensitivity analyses for missing data. The adjusted relative risk (RR) and risk difference for the primary outcome were estimated using generalised estimating equations with the binomial family and log/identity link respectively, with an exchangeable correlation matrix to account for randomisation stratification by centre and number of immediate family members with atopic disease (one, two, or more than two) included as a covariate. A number of planned subgroup analyses for the primary outcome were conducted including according to FLG genotype, to test the hypothesis that FLG null genotype affects response to intensive emollient use from birth. Results One thousand three hundred and ninety-four newborns were randomised between 19 November 2014 and 18 November 2016; 693 to the emollient group and 701 to the control group. Primary outcome data at 2 years were collected for 1210 infants (87%). Unblinding of research nurses prior to skin examination occurred for 12 infants in the intervention group and 6 in the control group. Adherence in the emollient group was 88% (466/532) at 3 months, 82% (427/519) at 6 months and 74% (375/506) at 12 months. In the control group, contamination due to self-directed use of emollients was reported for 18% (82/457), 17% (62/372) and 15% (49/324) at 3, 6 and 12 months, respectively, for infants who did not have a parental report of a doctor diagnosis of eczema. At age 2 years, eczema was present in 139 (23%) of 598 infants in the emollient group and in 150 (25%) of 612 infants in the control group [adjusted RR 0.95, 95% confidence interval (CI) 0.78 to 1.16; p = 0.61; adjusted risk difference −1.2%, 95% CI −5.9% to 3.6%]. All sensitivity analyses conducted, including using multiple imputation for missing primary outcome data, were consistent with the primary analysis. There was no evidence of an interaction effect with allocated group for the primary eczema outcome in any of the subgroup analyses (including FLG genotype). Other eczema definitions supported the results of the primary analysis. Eczema severity and time to onset of eczema were also similar in the two groups. Mean number of skin infections per child in year 1 was 0.23 [standard deviation (SD) 0.68] in the emollient group versus 0.15 (SD 0.46) in the control group; adjusted incidence rate ratio 1.55 (95% CI 1.15 to 2.09). Infant slippage incidents were reported for 15/584 (2.6%) in the emollient group and 11/584 (1.9%) in the control group. Food allergies to milk, egg or peanut at 2 years were confirmed in 41/547 (7.5%) infants in the emollient group and 29/568 (5.1%) in the control group (adjusted RR 1.47, 95% CI 0.93 to 2.33). The largest difference was in the proportion of infants with confirmed food allergy to egg, with an adjusted RR of 1.56 (95% CI 0.92 to 2.65). Results of other measures of food allergy and food sensitisation were similar. The proportion of infants with allergic rhinitis, wheezing and allergic sensitisation to cat dander, grass pollen and dust mite was similar between groups at 2 years. The differences in quality of life (using CHU-9D for the child and EuroQol-5 Dimensions, five-level version for the main carer) between the two groups were very small. Although the emollient intervention period was the first year of life, parents in the emollient group continued to report more frequent moisturiser application through to 5 years than in the control group. By 5 years, 188/608 (31%) parents in the emollient group had reported a clinical diagnosis of eczema in their child since 12 months, compared with 178/631 (28%) in the control group (adjusted RR 1.10, 95% CI 0.93 to 1.30). A diagnosis of food allergy by 5 years was reported for 92/609 (15%) allocated to emollients and 87/632 (14%) allocated to control (adjusted RR 1.11, 95% CI 0.84 to 1.45). Similarly, the percentage of parents reporting that their child had a clinical diagnosis of asthma or allergic rhinitis by 5 years were similar in the two groups. In the complete-case CEA mean cost was £398.23 (SD 1408.39) per child in the emollient group (n = 598) and £312.16 (SD 1105.04) in the control group (n = 610). When intervention use was combined with other health resource use, the adjusted incremental cost was £87.45 (95% CI −54.31 to 229.27). The adjusted difference in the proportion of children without eczema at 2 years was 0.0164 (95% CI −0.0329 to 0.0656) higher in the emollient group compared to the control group. The adjusted incremental cost per percentage decrease in risk of eczema was £5337 at 2 years. Adjusted QALYs for children were very slightly improved (i.e. higher) in the emollient group at 2 years. Conclusions We found no evidence of a useful preventive effect of emollients for eczema at our primary outcome time of 2 years. The failure to show a reduction in eczema was consistent regardless of how eczema was assessed. Some evidence of an increase in skin infections during the intervention period and a possible increase in food allergy at age 2 years was observed. No benefit was observed for time to onset of eczema or eczema severity, and no benefits were observed for eczema, asthma, hay fever or food allergy in longer-term follow-up to 5 years. Emollient use is unlikely to be considered cost effective in this context. Inclusion of individual patient data from all similar eczema prevention studies in further meta-analysis may provide a clearer assessment of whether emollients can prevent eczema and related diseases and provide more certainty about potential harms. Implications for health care The study does not support the use of emollients to prevent eczema and has found a small signal of possible harms, so this intervention cannot be recommended for health care or public health use. As the study relates to prevention of eczema, emollients should continue to be used as part of standard treatment for eczema. Trial registration This trial is registered as ISRCTN21528841. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 12/67/12) and is published in full in Health Technology Assessment; Vol. 28, No. 29. See the NIHR Funding and Awards website for further award information.

An introduction is presented in which the editor discusses articles within the issue on topics including integration of data from in silico, in chemico and in vitro methods for skin sensitization Adverse Outcome Pathway, new approach methodologies for research on skin color changes in response to tobacco and air pollution, and association of drug exposure with T-cell mediated cutaneous hypersensitivity reactions.

Skin sensitisation is a critical adverse effect assessed to ensure the safety of compounds and materials exposed to the skin. Alongside the development of new approach methodologies (NAMs), defined approaches (DAs) have been established to promote skin sensitisation potency assessment by adopting and integrating standardised in vitro, in chemico, and in silico methods with specified data analysis procedures to achieve reliable and reproducible predictions. The incorporation of additional NAMs could help increase accessibility and flexibility. Using superior algorithms may help improve the accuracy of hazard and potency assessment and build confidence in the results. Here, we introduce two new DA models, with the aim to build DAs on freely available software and the newly developed kDPRA for covalent binding of a chemical to skin peptides and proteins. The new DA models are built on an existing Bayesian network (BN) modelling approach and expand on it. The new DA models include kDPRA data as one of the in vitro parameters and utilise in silico inputs from open-source QSAR models. Both approaches perform at least on par with the existing BN DA and show 63% and 68% accuracy when predicting four LLNA potency classes, respectively. We demonstrate the value of the Bayesian network’s confidence indications for predictions, as they provide a measure for differentiating between highly accurate and reliable predictions (accuracies up to 87%) in contrast to low-reliability predictions associated with inaccurate predictions.

Established in vitro assays for regulatory testing of skin sensitisation partly suffer from only moderate sensitivity, specificity, and predictivity when testing specific groups of chemicals. This may be due to limited biomarker response in vitro in cell types that interact as crucial players of in vivo skin sensitisation pathogenesis. Here, we propose a molecular approach to overcome this limitation. In our model, we apply genome editing and blocking of immunoregulatory molecules to increase the range of biomarker modulation by sensitising chemicals. To this end, aryl hydrocarbon receptor (AhR) knockout was done by CRISPR/Cas9 technology in THP-1 cells and combined with Programmed Cell Death-Ligand (PD-L)1 blockade. AhR-knockout THP-1 in coculture with HaCaT keratinocytes showed increased CD54 expression compared to wild type cells after stimulation with 10 µmol/L dinitrochlorobenzene (DNCB) that was further enhanced by anti-PD-L1. After stimulation of AhR-knockout THP-1 with 200 µmol/L mercaptobenzothiazol or 10 µmol/L DNCB, cocultivated Jurkat T cells significantly increased expression of T cell receptor-associated CD3. No such increase was detected after prior treatment of THP-1 with 150 µmol/L of irritant sodium lauryl sulphate. Additionally, higher levels of inflammatory cytokines MIP-3α, MIP-1β, TNF-α, and IL-8 were found in supernatants of enhanced loose-fit co-culture based sensitisation assay (eLCSA) after substance treatment. Hence, eLCSA allowed to discriminate between sensitisers and non-sensitisers. Thus, inhibition of immunoinhibitory pathway signalling by combining AhR knockout and PD-L1 antibody blockage into an assay involving main acting cell types in skin sensitisation may increase sensitivity and specificity of such assays and allow potency derivation. [ABSTRACT FROM AUTHOR]

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Purpose: Of the several selenized yeasts authorised for use as feed additives in the EU, Saccharomyces cerevisiae CNCM I-3060 inactivated' (Sel-Plex®), was the first to be approved for use, in 2006. The additive has a concentration of selenium between 2000 and 2400 mg/kg and a selenomethionine content greater than 63%. Previous toxicological and safety studies have shown Sel-Plex® to be safe for use for target animal species, consumers, users and the environment. A new formulation of Sel-Plex® was recently developed however, with a minimum selenium content of 3000 mg/kg. The increase in selenium in this product, Sel-Plex® 3000, presented the need to assess the risk for workers and users and to establish if there would be any eye and/or skin irritancy and skin sensitisation effects associated with the product. The purpose of this paper is to present the methodology and results of the user safety skin and eye studies performed on Sel-Plex® 3000. Materials & Methods: In vitro skin and eye models were used to assess skin and eye irritancy, while skin sensitisation was examined using an in vivo method. The acute eye irritation was evaluated using a Reconstructed human Cornea-like Epithelium (RhCE) model, which followed the OECD guideline 492. The skin irritation was assessed based on its ability to induce cell death in a commercial reconstructed human epidermis (RhE) model (EPISKIN™) according to the OECD Guideline No. 439. The skin sensitising potential was evaluated in the Guinea pig in line with OECD Guideline 406, and measured the extent and degree of skin reaction to a challenge exposure following previous topical exposure of a substance on the skin. Results: The skin and eye irritation test results showed that Sel-Plex® 3000 was a non-irritant in both cases. The skin sensitisation study showed that the additive did not generate a sensitisation response in the guinea pig and should not be considered a skin sensitiser. Conclusion: These results indicate that Sel-Plex® 3000 is safe to use for workers in an industrial setting when handling the product and the studies may be further used to support regulatory compliance in respective markets. [ABSTRACT FROM AUTHOR]

Oil body (OB), a subcellular organelle that stores oil in plant seeds, is considered a new transdermal drug delivery system. With the increasing understanding of the OB and its main protein (oleosin), numerous studies have been conducted on OB as "carrier" for the expression of exogenous proteins. In our previous study, oil body fused with aFGF (OLAF) was obtained using a plant oil body expression system that had been preliminarily proven to be effective in accelerating the healing of skin wounds. However, no dermal toxicological information on OLAF is available. To ensure the dermal safety of OLAF, a series of tests (the acute dermal toxicity test, 21-day repeat dermal toxicity test, dermal irritation test and skin sensitisation test) were conducted after optimising the extraction protocol of OLAF. To improve the extraction rate of OLAF, response surface methodology (RSM) was first employed to optimise the extraction conditions. Then, Wistar rats were exposed to OLAF (400 mg·kg−1 body weight) in two different ways (6 hours/time for 24 hours and 1 time/day for 21 days) to evaluate the acute dermal toxicity and 21-day repeated dermal toxicity of OLAF. In the acute dermal toxicity test, clinical observations were conducted to evaluate the toxicity, behaviour, and health of the animals for 14 consecutive days. Similarly, the clinical signs, body weight, haematological and biochemical parameters, histopathological changes and other indicators were also detected during the 21 days administration. For the dermal irritation test, single and multiple doses of OLAF (125 mg·kg−1 body weight) were administered to albino rabbits for 14 days (1 time/day). The irritation reaction on the skin of each albino rabbit was recorded and scored. Meanwhile, skin sensitisation to OLAF was conducted using guinea pigs for a period of 28 days. Suitable extraction conditions for OLAF (PBS concentration 0.01, pH of PBS 8.6, solid–liquid ratio 1:385 g·mL−1) were obtained using RSM. Under these conditions, the extraction rate and particle size of OLAF were 7.29% and 1290 nm, respectively. In the tests of acute dermal toxicity and 21-day repeated dermal toxicity, no mortality or significant differences were observed in terms of clinical signs, body weight, haematological parameters, biochemical parameters and anatomopathological analysis. With respect to the dermal irritation test and skin sensitisation test, no differences in erythema, oedema or other abnormalities were observed between treatment and control groups on gross and histopathological examinations. The results of this study suggest that OLAF does not cause obvious toxicity, skin sensitisation or irritation in animals. [ABSTRACT FROM AUTHOR]

Recently, alternatives to animal testing have been used to evaluate skin sensitisers in cosmetic products. However, testing is still complicated and expensive. To develop a simpler, cost-effective and more accurate evaluation method for the skin sensitising chemicals, we employed cell-based and RT-PCR-based assay. Representative sensitiser specific gene expression in THP-1 cells was analysed by microarray. Gene ontology (GO) analysis revealed that 26 genes induced by the sensitisers were associated with immune function. First, seven of the 26 genes were chosen arbitrarily as candidate markers for our sensitisation assay. Then, THP-1 cells were exposed to 13 reference chemicals with known sensitising potential, and real-time RT-PCR assays targeting the candidate marker genes were performed. Among them, six markers were able to properly evaluate the sensitisation potential by classifying the gene induction rates with appropriate criteria. Especially, the results of the assay using TREM1 and TNFRSF12A gene markers showed 100% sensitivity and specificity. An existing test method, h-CLAT, requires a flow cytometer and is complicated to operate. In contrast, our method is relatively simpler and more cost-effective. Therefore, our method is a promising one to evaluate sensitising chemicals. [ABSTRACT FROM AUTHOR]

Skin sensitisation, one of the most frequent forms of human immune toxicity, is authenticated to be a significant endpoint in the field of drug discovery and cosmetics. Due to the drawbacks of traditional animal testing methods, in silico methods have advanced to study skin sensitisation. In this study, mechanism-based binary and ternary classification models were constructed with a comprehensive data set. 1007 compounds were collected to develop five series of local and global models based on mechanisms. In each series, compounds were classified into five groups according to EC3 values, and applied as training sets, test sets and external validation sets. For each of the five series, 81 binary classification models and 81 ternary classification models were acquired via 9 molecular fingerprints and 9 machine learning methods using a novel KNIME workflow. Meanwhile, the applicability domains for the best 10 models were figured out to certify the rationality of prediction effect. In addition, 8 toxic substructures probably causing skin sensitisation were identified to speculate whether a compound is a skin sensitiser. The mechanism-based prediction models and the toxic substructures can be applied to predict the skin sensitising potential and potency of compounds. • The number of compounds collected in this study is the largest among all published papers about skin sensitisation so far. • The models can be applied to predict the skin sensitising potential and potency, and contribute to mechanism determination. • 8 toxic substructures probably causing skin sensitisation were identified. [ABSTRACT FROM AUTHOR]

Skin sensitisation associated with allergic contact dermatitis is an important occupational and environmental disease. The identification of skin sensitisation hazards was traditionally performed using animal tests; originally guinea pig assays and subsequently the murine local lymph node assay (LLNA). More recently there has, for a variety of reasons, been an increased interest in, and requirement for, non-animal assays. There are now available both validated in vitro assays and a variety of approaches based on consideration of quantitative structure-activity relationships (QSAR). With the increased availability and use of non-animal alternatives for skin sensitisation testing there is a continuing need to monitor the performance of these approaches using series of chemicals that do not normally form part of validation exercises. Here we report studies conducted with 11 methacrylate esters and methacrylic acid in which results obtained with 3 validated in vitro tests for which there are OECD guidelines (the Direct Peptide Reactivity Assay, DPRA; ARE-Nrf2 luciferase test methods, and – with some chemicals – a dendritic cell activation test, the myeloid U937 Skin Sensitisation test [U-SENS] assay) have been compared with QSAR approaches (DEREK and TIMES-SS), and with LLNA and guinea pig maximisation test (GPMT) data. The conclusions drawn from these data are that – with this series of chemicals at least – there is a strong correlation between the results of animal tests and the in vitro assays considered, but not with either DEREK or TIMES-SS. • Skin sensitising activity of methacrylate esters. • Comparison of animal data with data from alternative methods. • Performance of in vitro and in silico approaches. [ABSTRACT FROM AUTHOR]

The amino acid derivative reactivity assay (ADRA) is a novel in chemico alternative to animal testing for assessment of skin sensitization potential. The conventional ADRA protocol stipulates that test chemical solutions should be prepared to a specific molar concentration, allowing only for use of test chemicals with known molecular weights. Since many potential test substances are prepared by weight concentration or contain multiple unknown chemicals, this study was conducted to verify if it is possible to accurately assess the sensitization potential of test chemical solutions prepared at a specific weight concentration. (1) Test chemical solutions for 82 chemicals were prepared at four different weight concentrations. Results were evaluated for agreement with in vivo results. (2) A liquid mixture comprising ten different non-sensitizers was prepared at 1 mg/mL. Ten different sensitizers of varying sensitization potencies were added individually to this mixture. The resulting pseudobinary mixtures were tested to confirm that the sensitizers could be detected. (1) The accuracies for test chemical solutions prepared at 0.5 and 0.2 mg/mL were 87.8% and 86.6%, respectively, which were roughly equivalent to the accuracy of 86.6% achieved with a solution prepared at the conventional molar concentration of 1 mM. In contrast, the accuracies for solutions prepared at 0.1 and 0.05 mg/mL were 82.9% and 74.4%, respectively, both of which were lower than that obtained with the conventional method. (2) Sensitizers added to the liquid mixture at 0.5 mg/mL were all correctly detected. Preparing test chemical solutions at a weight concentration of 0.5 mg/mL decreased false negatives and increased false positives while improving prediction accuracy, which suggests that the sensitization potential of mixtures can also be assessed with this method. [ABSTRACT FROM AUTHOR]

Abstract Electronic cigarettes (e-cigarettes) are an increasingly popular alternative to combustible tobacco cigarettes among smokers worldwide. A growing body of research indicates that flavours play a critical role in attracting and retaining smokers into the e-cigarette category, directly contributing to declining smoking rates and tobacco harm reduction. The responsible selection and inclusion levels of flavourings in e-liquids must be guided by toxicological principles. Some flavour ingredients, whether natural extracts or synthetic, are known allergens. In this study, we used the Genomic Allergen Rapid Detection (GARD) testing strategy to predict and compare the respiratory and skin sensitising potential of three experimental and two commercial e-liquids. These novel, myeloid cell-based assays use changes in the transcriptional profiles of genomic biomarkers that are collectively relevant for respiratory and skin sensitisation. Our initial results indicate that the GARD assays were able to differentiate and broadly classify e-liquids based on their sensitisation potential, which are defined mixtures. Further studies need to be conducted to assess whether and how these assays could be used for the screening and toxicological assessment of e-liquids to support product development and commercialisation. Highlights • Flavoured e-liquids are a vital part of the vaping experience. • Some flavour ingredients, whether natural extracts or synthetic, are known allergens. • Novel assays were used to predict the skin and respiratory sensitisation potential of e-liquids. • Experimental and commercial e-liquids were tested in respiratory and dermal GARD assays. • Our results indicate that these methods can differentiate and broadly classify e-liquids. [ABSTRACT FROM AUTHOR]

Background: Allergic sensitisation to foods may occur in infancy without prior oral exposure to the offending food, leading to the assumption that food allergy sensitisation may occur through the skin. Concerns have been raised regarding the safety of use of personal care products containing hydrolysed wheat proteins, since these products have been shown to induce allergy through the skin, and even cause an abrogation of an already established oral tolerance. Objective: To establish an animal model for food allergy skin sensitisation and compare the sensitising capacity of an unmodified and an acid-hydrolysed gluten product via slightly damaged skin in naïve versus tolerant rats. Methods: Gluten products were applied on the slightly damaged skin of naïve or tolerant Brown Norway (BN) rats without adjuvant 3 times per week for 3 or 5 consecutive weeks. The effect of the skin applications was evaluated by means of different ELISAs and immunoblotting. Results: A robust animal model was developed for food allergy skin sensitisation. In naïve rats, both gluten products were able to induce a statistically significant level of specific antibodies and sensitise through the skin, but in the wheat-tolerant rats, only the acid-hydrolysed gluten was able to sensitise through the skin, albeit at a level much lower than in the naïve rats. Results showed that new epitopes had been developed as a result of acid hydrolysis but original epitopes were maintained. This may explain why only the acid-hydrolysed gluten could induce specific antibody responses in the tolerant animals. Conclusions: This study showed that it is possible to sensitise BN rats through slightly damaged skin, and that the sensitising capacity is heavily influenced by the tolerance status of their immune system and the degree of modification of the wheat products. [ABSTRACT FROM AUTHOR]

Several New Approach Methodologies (NAMs) for hazard assessment of skin sensitisers have been formally validated. However, data regarding their applicability on certain product classes are limited. The purpose of this project was to provide initial evidence on the applicability domain of GARD™skin and GARD™potency for the product class of agrochemical formulations. For this proof of concept, 30 liquid and 12 solid agrochemical formulations were tested in GARDskin for hazard predictions. Formulations predicted as sensitisers were further evaluated in the GARDpotency assay to determine GHS skin sensitisation category. The selected formulations were of product types, efficacy groups and sensitisation hazard classes representative of the industry's products. The performance of GARDskin was estimated by comparing results to existing in vivo animal data. The overall accuracy, sensitivity, and specificity were 76.2% (32/42), 85.0% (17/20), and 68.2% (15/22), respectively, with the predictivity for liquid formulations being slightly higher compared to the solid formulations. GARDpotency correctly subcategorized 14 out of the 17 correctly predicted sensitisers. Lack of concordance was justifiable by compositional or borderline response analysis. In conclusion, GARDskin and GARDpotency showed satisfactory performance in this initial proof-of-concept study, which supports consideration of agrochemical formulations being within the applicability domain of the test methods. • Tested 42 agrochemical formulations to expand applicability domain of GARD. • GARDskin showed good accuracy (76.2%), sensitivity (85.0%) and specificity (68.2%). • GARDpotency correctly subcategorized 14/17, correctly predicted sensitisers. • GARD satisfactory for Key Event 3 characterisation of agrochemical formulations. [ABSTRACT FROM AUTHOR]

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. • Effective safety evaluation of sensitisers requires quantitative risk assessment. • Older guinea pig tests do not lend themselves to sensitiser potency measurement. • LLNA results give potency sub-categories and risk assessment points of departure. • Regulatory guidelines detail potency sub-categorisation based on guinea pig results. • The sub-categories can be extrapolated to give guinea pig based points of departure. [ABSTRACT FROM AUTHOR]

Abstract A decision tree-based defined approach (DA) has been designed using exclusion criteria based on applicability domain knowledge of in chemico/in vitro information sources covering key events 1–3 in the skin sensitisation adverse outcome pathway and an in silico tool predicting the adverse outcome (Derek Nexus). The hypothesis is that using exclusion criteria to de-prioritise less applicable assays and/or in silico outcomes produces a rational, transparent, and reliable DA for the prediction of skin sensitisation potential. Five exclusion criteria have been established: Derek Nexus reasoning level, Derek Nexus negative prediction, metabolism, lipophilicity, and lysine-reactivity. These are used to prioritise the most suitable information sources for a given chemical and results from which are used in a '2 out of 3' approach to provide a prediction of hazard. A potency category (and corresponding GHS classification) is then assigned using a k-Nearest Neighbours model containing human and LLNA data. The DA correctly identified the hazard (sensitiser/non-sensitiser) for 85% and 86% of a dataset with reference LLNA and human data. The correct potency category was identified for 59% and 68% of chemicals, and the GHS classification accurately predicted for 73% and 76% with reference LLNA and human data, respectively. Graphical abstract Image 1 Highlights • A decision tree-based defined approach predicting human sensitisation hazard and potency was developed. • Combines in silico predictions with in chemico/in vitro data after considering the applicability domain. • Correctly identifies sensitisers/non-sensitisers for 85% (LLNA) and 86% (human) when compared to reference data. • The potency category was predicted correctly for 59% of chemicals with LLNA data and 68% with human data. • The GHS classification was accurately predicted for 73% and 76% with reference LLNA and human data, respectively. [ABSTRACT FROM AUTHOR]

In all current in vitro skin sensitisation assays, DMSO is used to dissolve water-insoluble chemicals. However, our previous study suggested the superiority of the modified IL-8 Luc assay (mIL-8 Luc), in which X-VIVOTM 15 is used to dissolve chemicals, over the original assay using DMSO (oIL-8 Luc). In this study, to confirm the superiority of the mIL-8 Luc, we first increased the number of chemicals examined and demonstrated the superiority of the mIL-8 Luc, in which the mIL-8 Luc provided 87.6% of sensitivity, 74.2% of specificity, and 84.6% of accuracy. Next, to clarify the cause of false negative judgment by the mIL-8 Luc, we examined the effects of physical properties of chemicals on judgment. The results demonstrated that high molecular weight, high LogKo/w, or poor water solubility, did not cause false negative judgment. When it was accepted as an OECD test guideline, the criteria of the mIL-8 Luc to determine sensitisers were modified to further decrease false negative judgment by poor solubility. By applying the new criteria, the test guideline IL-8 Luc assay (tgIL-8 Luc) improved sensitivity but decreased specificity and increased the number of chemicals that cannot be judged. To overcome this problem, we examined a simple combination of the tgIL-8 Luc with direct peptide reactive assay (DPRA), which could improve specificity and decrease the number of the chemicals that cannot be judged. These data suggest that the tgIL-8 Luc is a promising in vitro skin sensitisation assay in combination with other in vitro or in chemico methods. [ABSTRACT FROM AUTHOR]

Abstract Tests for identification of chemical skin sensitisation hazard have been available for decades, evolving from guinea pig assays, through the first validated method, the local lymph node assay, to several validated in vitro methods. These methods successfully identify the hazard for chemicals, with an accuracy in the region of 85%. However, in some regulations, consideration may be given to their application to proteins. Here, the scientific relevance of the use of skin sensitisation tests for the assessment of the allergenic potential of proteins is reviewed and considered in the context of both: (a) what is known of the allergenic properties of proteins compared with chemicals, and (b) current understanding of the extent to which proteins actually give rise to contact allergy. There is no doubt that many foreign proteins can behave as respiratory sensitisers and food allergens, and that certain proteins can also cause cutaneous allergy via skin contact, typically mediated via immunoglobulin E (IgE) antibody. However, the absence of any specificity in predictions from existing skin sensitisation test methods, together with the lack of a suitable body of either positive or negative controls, dictates that use of these tests with proteins is without any scientific justification or predictive merit. Highlights • Proteins, including enzymes, have the potential to cause allergy, but specific predictive methods are lacking. • Do tests designed for skin sensitising chemicals have merit for the assessment of protein allergenic potential? • It is concluded skin sensitisation tests fail as they cannot distinguish between protein immunogenicity and allergenicity. • Ultimately, novel approaches are needed, for which a key first step would be establishment of suitable performance standards. [ABSTRACT FROM AUTHOR]

Abstract In the context of a larger testing programme that aimed at assessing the skin sensitisation potential of functional polysiloxanes and silanes, this investigation complements the available in vitro and in vivo data with data in the SENS-IS assay, a human in vitro 3D skin-based model. The SENS-IS assay allowed testing of all functional polysiloxanes and silanes without any solubility issues or limitations related to the multiconstituent nature of the commercial grade test substances. It appeared to encompass skin metabolism, a factor which we considered important for the skin sensitisation hazard assessment particularly of aminofunctionalised siloxanes and silanes. These three technical aspects posed significant challenges in the first part of the in vitro programme with the OECD-validated in vitro assays. The SENS-IS assay delivered promising results for this group of substances. On its own, it was the best performing model, as it did not pose any technical issues with the assay and it matched all in vivo outcomes. Considering its performance and avoidance of any limitations due to lack of solubility or chemical composition aspects, we concluded that the SENS-IS assay to be a suitable starting point for an integrated testing strategy for skin sensitisation for the group of functional polysiloxanes and silanes. [ABSTRACT FROM AUTHOR]

Predictive testing to characterise substances for their skin sensitisation potential has historically been based on animal models such as the Local Lymph Node Assay (LLNA) and the Guinea Pig Maximisation Test (GPMT). In recent years, EU regulations, have provided a strong incentive to develop non-animal alternatives, such as expert systems software. Here we selected three different types of expert systems: VEGA (statistical), Derek Nexus (knowledge-based) and TIMES-SS (hybrid), and evaluated their performance using two large sets of animal data: one set of 1249 substances from eChemportal and a second set of 515 substances from NICEATM. A model was considered successful at predicting skin sensitisation potential if it had at least the same balanced accuracy as the LLNA and the GPMT had in predicting the other outcomes, which ranged from 79% to 86%. We found that the highest balanced accuracy of any of the expert systems evaluated was 65% when making global predictions. For substances within the domain of TIMES-SS, however, balanced accuracies for the two datasets were found to be 79% and 82%. In those cases where a chemical was within the TIMES-SS domain, the TIMES-SS skin sensitisation hazard prediction had the same confidence as the result from LLNA or GPMT. [ABSTRACT FROM AUTHOR]

A previously published fragmentation method for making reliable negative in silico predictions has been applied to the problem of predicting skin sensitisation in humans, making use of a dataset of over 2750 chemicals with publicly available skin sensitisation data from 18 in vivo assays. An assay hierarchy was designed to enable the classification of chemicals within this dataset as either sensitisers or non-sensitisers where data from more than one in vivo test was available. The negative prediction approach was validated internally, using a 5-fold cross-validation, and externally, against a proprietary dataset of approximately 1000 chemicals with in vivo reference data shared by members of the pharmaceutical, nutritional, and personal care industries. The negative predictivity for this proprietary dataset was high in all cases (>75%), and the model was also able to identify structural features that resulted in a lower accuracy or a higher uncertainty in the negative prediction, termed misclassified and unclassified features respectively. These features could serve as an aid for further expert assessment of the negative in silico prediction. [ABSTRACT FROM AUTHOR]

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. • Quantitative risk assessment is pre-eminent for skin sensitiser safety evaluation. • Plant protection products may contain sensitising actives and/or co-formulants. • An expert workshop reviewed clinical and toxicological aspects of the issue. • The workshop recommended PPP safety assessment evolve to use QRA. • The workshop noted the paucity of skin allergy to PPPs required an evidence base. [ABSTRACT FROM AUTHOR]

Skin sensitisation is the regulatory endpoint that has been at the centre of concerted efforts to replace animal testing in recent years, as demonstrated by the Organisation for Economic Co-operation and Development (OECD) adoption of five non-animal methods addressing mechanisms under the first three key events of the skin sensitisation adverse outcome pathway. Nevertheless, the currently adopted methods, when used in isolation, are not sufficient to fulfil regulatory requirements on the skin sensitisation potential and potency of chemicals comparable to that provided by the regulatory animal tests. For this reason, a number of defined approaches integrating data from these methods with other relevant information have been proposed and documented by the OECD. With the aim to further enhance regulatory consideration and adoption of defined approaches, the European Union Reference Laboratory for Alternatives to Animal testing in collaboration with the International Cooperation on Alternative Test Methods hosted, on 4–5 October 2016, a workshop on the international regulatory applicability and acceptance of alternative non-animal approaches, i.e., defined approaches, to skin sensitisation assessment of chemicals used in a variety of sectors. The workshop convened representatives from more than 20 regulatory authorities from the European Union, United States, Canada, Japan, South Korea, Brazil and China. There was a general consensus among the workshop participants that to maximise global regulatory acceptance of data generated with defined approaches, international harmonisation and standardisation are needed. Potential assessment criteria were defined for a systematic evaluation of existing defined approaches that would facilitate their translation into international standards, e.g., into a performance-based Test Guideline. Informed by the discussions at the workshop, the ICATM members propose practical ways to further promote the regulatory use and facilitate adoption of defined approaches for skin sensitisation assessments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Azodicarbonamide (ADCA) is widely used by industry in the manufacture of a variety of products. ADCA has been classified as a respiratory allergen, and the purpose of this article was to consider whether this classification is appropriate based upon the available data. Here both clinical experience and relevant experimental data have been reviewed. Although there have been reports of an association between workplace exposure to ADCA and symptoms of respiratory allergy and occupational asthma, the evidence is less than persuasive, with in many instances a lack of properly controlled and executed diagnostic procedures. In addition, ADCA fails to elicit positive responses in mouse and guinea pig predictive tests for skin sensitisation; a lack of activity that is regarded as being inconsistent with respect to respiratory sensitising potential. Collectively, the data reviewed here do not provide an adequate basis for the classification of ADCA as a respiratory allergen. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • NGRA case study using read-across predicting the skin sensitisation potential of 0.5% vanillin in shower gel and face cream. • Five analogues based on protein reactivity, structure, physicochemical properties, skin metabolism and sensitisation data. • Read-across predicted a weak skin sensitiser potential for vanillin corresponding to a PoD of 2500 μg/cm2. • NGRA concluded both consumer exposure scenarios as safe. [ABSTRACT FROM AUTHOR]

Historically, allergic contact dermatitis (ACD) to chemicals encouraged hazard identification improvements, more sophisticated risk assessment and implementation of regulatory strategies, including banning of specific sensitising substances. The validation process applied to hazard identification methods demonstrates their accuracy; their use to characterise sensitiser potency facilitates quantitative and transparent risk assessment. Diagnostic patch testing at dermatology clinics worldwide delivers feedback showing where risk assessment/management has been insufficient or did not target the exposure of concern, thereby facilitating improvements. When urgent action to protect human health was required, regulations limited/banned, specific skin sensitisers. This can be seen in practice with the fragrance industry, a known source of ACD, thus requiring risk management, usually restrictions to limit allergy induction, and very rarely specific bans on ingredients. Experience and development of more sophisticated tools, e.g. to assess aggregate exposure from multitude of consumer product types, has led to repeated adaptation of risk assessment and promulgation of updated fragrance use limits. Although targeted control may not always lead to rapid change in the overall clinical picture, it is preferable to a blanket undifferentiated regulatory control of all sensitisers, resulting in unwarranted restrictions for many uses of no health concern, with consequent substantial socio-economic impacts. • Skin sensitisers are regulated largely on the basis of intrinsic hazard. • Evidence that this has been beneficial for human health is absent. • In contrast risk-based regulation of specific skin sensitisers has a positive impact. • Any future legislation therefore should be specific and evidence-based, not generic. [ABSTRACT FROM AUTHOR]

Animal testing has been prohibited for the safety assessment of cosmetic ingredients or finished products. Thus, alternative non-animal methods, followed by confirmatory clinical studies on human volunteers, should be used as the sole legally acceptable approach within the EU. The safety assessment of cosmetic products requires the involvement of multiple scientific disciplines, including analytical chemistry and biomedicine, as well as in chemico , in vitro and in silico toxicology. Recent data suggest that fragrance components may exert multiple adverse biological effects, e.g. cytotoxicity, skin sensitisation, (photo)genotoxicity, mutagenicity, reprotoxicity and endocrine disruption. Therefore, a pilot study was conducted with selected samples of fragrance-based products, such as deodorant, eau de toilette and eau de parfum, with the aim of integrating results from a number of alternative non-animal methods suitable for the detection of the following toxicological endpoints: cytotoxicity (with 3T3 Balb/c fibroblasts); skin sensitisation potential ( in chemico method, DPRA); skin sensitisation potential (LuSens in vitro method, based on human keratinocytes); genotoxicity potential ( in vitro Comet assay with 3T3 Balb/c cells); and endocrine disruption ( in vitro YES/YAS assay). The presence of twenty-four specific known allergens in the products was determined by using GC-MS/MS. The strategies for estimation of the NOAEL of a mixture of allergens, which were proposed by the Scientific Committee on Consumer Products in their 'Opinion on Tea tree oil' document and by the Norwegian Food Safety Authority in their 'Risk Profile of Tea tree oil' report, were used as models for the NOAEL estimation of the mixtures of allergens that were identified in the individual samples tested in this study.

Since March 2013, it is no longer possible to market in the European Union (EU) cosmetics containing new ingredients tested on animals. Although several in silico alternatives are available and achievements have been made in the development and regulatory adoption of skin sensitisation non-animal tests, there is not yet a generally accepted approach for skin sensitisation assessment that would fully substitute the need for animal testing. The aim of this work was to build a defined approach ( i.e. a predictive model based on readouts from various information sources that uses a fixed procedure for generating a prediction) for skin sensitisation hazard prediction (sensitiser/non-sensitiser) using Local Lymph Node Assay (LLNA) results as reference classifications. To derive the model, we built a dataset with high quality data from in chemico (DPRA) and in vitro (KeratinoSens™ and h-CLAT) methods, and it was complemented with predictions from several software packages. The modelling exercise showed that skin sensitisation hazard was better predicted by classification trees based on in silico predictions. The defined approach consists of a consensus of two classification trees that are based on descriptors that account for protein reactivity and structural features. The model showed an accuracy of 0.93, sensitivity of 0.98, and specificity of 0.85 for 269 chemicals. In addition, the defined approach provides a measure of confidence associated to the prediction. [ABSTRACT FROM AUTHOR]

There is a pressing need for non-animal methods to predict skin sensitisation potential and a number of in chemico and in vitro assays have been designed with this in mind. However, some compounds can fall outside the applicability domain of these in chemico/in vitro assays and may not be predicted accurately. Rule-based in silico models such as Derek Nexus are expert-derived from animal and/or human data and the mechanism-based alert domain can take a number of factors into account (e.g. abiotic/biotic activation). Therefore, Derek Nexus may be able to predict for compounds outside the applicability domain of in chemico/in vitro assays. To this end, an integrated testing strategy (ITS) decision tree using Derek Nexus and a maximum of two assays (from DPRA, KeratinoSens, LuSens, h-CLAT and U-SENS) was developed. Generally, the decision tree improved upon other ITS evaluated in this study with positive and negative predictivity calculated as 86% and 81%, respectively. Our results demonstrate that an ITS using an in silico model such as Derek Nexus with a maximum of two in chemico/in vitro assays can predict the sensitising potential of a number of chemicals, including those outside the applicability domain of existing non-animal assays. [ABSTRACT FROM AUTHOR]

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

In silico models are often built solely on publicly available data which may mean that they are less predictive for proprietary chemical space. Data sharing initiatives can improve the performance of such models, but organisations are often unable to share their data due to the need to protect their business interests and maintain the confidentiality of the chemicals in their research and development programmes. In silic o models like Derek Nexus, which use expert knowledge to develop structural alerts based on chemical toxicity, can use proprietary data to identify new areas of chemical space and/or refine existing alerts whilst still preserving the privacy of the confidential data. Five hundred and thirty seven proprietary chemicals with skin sensitisation data were shared which led to the implementation of 7 new alerts and 5 modified alerts, with a concomitant 19% increase in sensitivity and 3% increase in specificity of the model. [Display omitted] • Proprietary data can be used to create structural alerts whilst preserving privacy. • A dataset of 537 proprietary chemicals was shared to improve Derek Nexus. • 7 new skin sensitisation alerts were developed, and 5 existing alerts were modified. • The sensitivity and specificity of the model increased by 19% and 3%, respectively. • After data sharing, the overall balanced accuracy of the model increased by 12%. [ABSTRACT FROM AUTHOR]

The U-SENS™ is a test method based on the human myeloid U937 cell line to assess the skin sensitisation potential of substances. To demonstrate its robustness, a multicentre validation study with four laboratories testing 24 coded substances has been conducted according to internationally agreed principles. The primary objective of the study was to enlarge the U-SENS™'s reproducibility database. Secondary objectives were to provide additional evidence on its transferability and its predictive capability. Reproducibility within laboratories was approximately 92%, while the reproducibility between laboratories was 87.5%. Predictivity for the 24 validation substances was high, with sensitivity, specificity and accuracy being on average at least 93.8%. Similar performances are obtained for 38 substances when combining the study results with those of an earlier multicentre study, as well as with an automated version of the U-SENS™. With reliability and relevance similar to comparable non-animal skin sensitisation test methods, which have achieved regulatory acceptance, it is concluded that the U-SENS™ is a well reproducible and predictive test method. This profiles the U-SENS™ as a valuable addition to the suite of non-animal testing methods for skin sensitisation with the potential to significantly contribute to the development of integrated testing strategies. [ABSTRACT FROM AUTHOR]

It is well established that some fragrance substances have the potential to cause skin sensitisation associated with the development of allergic contact dermatitis (ACD). Fragrances are invariably relatively volatile leading to the consideration that inhalation of fragrances might be a relevant route for either the induction of allergic sensitisation or the elicitation of allergic reactions. Moreover, there has been increasing recognition that allergic sensitisation of the respiratory tract can be induced by topical exposure to certain chemical allergens. Here the central question addressed is whether inhalation exposure to fragrance allergens has the potential to cause skin and/or respiratory sensitisation via the respiratory tract, or elicit allergic symptoms in those already sensitised. In addressing those questions, the underlying immunobiology of skin and respiratory sensitisation to chemicals has been reviewed briefly, and the relevant experimental and clinical evidence considered. The essential mechanistic differences between skin and respiratory allergy appear consistent with other sources of information, including the phenomenon of ACD that can arise from topical exposure to airborne allergens, but in the absence of accompanying respiratory effects. The conclusion is that, in contrast to topical exposure (including topical exposure to airborne material), inhalation of fragrance sensitisers does not represent a health risk with respect to allergy. [ABSTRACT FROM AUTHOR]

In the two years since the last workshop report, the environment surrounding the prediction of skin sensitisation hazards has experienced major change. Validated non-animal tests are now OECD Test Guidelines. Accordingly, the recent cross sector workshop focused on how to use in vitro data for regulatory decision-making. After a review of general approaches and six case studies, there was broad consensus that a simple, transparent stepwise process involving non-animal methods was an opportunity waiting to be seized. There was also strong feeling the approach should not be so rigidly defined that assay variations/additional tests are locked out. Neither should it preclude more complex integrated approaches being used for other purposes, e.g. potency estimation. All agreed the ultimate goal is a high level of protection of human health. Thus, experience in the population will be the final arbiter of whether toxicological predictions are fit for purpose. Central to this is the reflection that none of the existing animal assays is perfect; the non-animal methods should not be expected to be so either, but by integrated use of methods and all other relevant information, including clinical feedback, we have the opportunity to continue to improve toxicology whilst avoiding animal use. [ABSTRACT FROM AUTHOR]

An essential step in ensuring the toxicological safety of chemicals used in consumer products is the evaluation of their skin sensitising potential. The sensitising potency, coupled with information on exposure levels, can be used in a Quantitative Risk Assessment (QRA) to determine an acceptable level of a given chemical in a given product. Where consumer skin exposure is low, a risk assessment can be conducted using the Dermal Sensitisation Threshold (DST) approach, avoiding the need to determine potency experimentally. Since skin sensitisation involves chemical reaction with skin proteins, the first step in the DST approach is to assess, on the basis of the chemical structure, whether the chemical is expected to be reactive or not. Our accompanying publication describes the probabilistic derivation of a DST of 64 μg/cm 2 for chemicals assessed as reactive. This would protect against 95% of chemicals assessed as reactive, but the remaining 5% would include chemicals with very high potency. Here we discuss the chemical properties and structural features of high potency sensitisers, and derive an approach whereby they can be identified and consequently excluded from application of the DST. [ABSTRACT FROM AUTHOR]

The evaluation of chemicals for their skin sensitising potential is an essential step in ensuring the safety of ingredients in consumer products. Similar to the Threshold of Toxicological Concern, the Dermal Sensitisation Threshold (DST) has been demonstrated to provide effective risk assessments for skin sensitisation in cases where human exposure is low. The DST was originally developed based on a Local Lymph Node Assay (LLNA) dataset and applied to chemicals that were not considered to be directly reactive to skin proteins, and unlikely to initiate the first mechanistic steps leading to the induction of sensitisation. Here we have extended the DST concept to protein reactive chemicals. A probabilistic assessment of the original DST dataset was conducted and a threshold of 64 μg/cm 2 was derived. In our accompanying publication, a set of structural chemistry based rules was developed to proactively identify highly reactive and potentially highly potent materials which should be excluded from the DST approach. The DST and rule set were benchmarked against a test set of chemicals with LLNA/human data. It is concluded that by combining the reactive DST with knowledge of chemistry a threshold can be established below which there is no appreciable risk of sensitisation for protein-reactive chemicals. [ABSTRACT FROM AUTHOR]

In June 2021 the Organisation for Economic Co-operation and Development published Guideline No. 497 on Defined Approaches for Skin Sensitisation (DASS GL). There are two DAs published, known as the 2o3 and the ITS. The 2o3 uses two concordant results from either the DPRA, KeratinoSens™, or the h-CLAT assays to predict hazard (sensitiser/non-sensitiser). The ITS applies a score to results from the DPRA, the h-CLAT and an in silico model to predict United Nations Globally Harmonized System (GHS) sub-categories (1A/1B/Not Classified). The ITS can use Derek Nexus as the in silico model (known as ITSv1) or use OECD QSAR Toolbox (known as ITSv2). As limitations of the individual in chemico/in vitro assays and in silico predictions are carried through to the DAs, inconclusive predictions are possible for chemicals with results in the borderline range, and chemicals with out of domain results. However, these inconclusive predictions can be resolved by applying a weight of evidence approach. Herein, four case studies are presented, each 'inconclusive' for skin sensitisation potential according to both DAs. A weight of evidence approach was applied to each using a robust scientific approach to provide a conclusive prediction, where possible, based on several additional, non-animal lines of evidence. [Display omitted] • The OECD has published two defined approaches (DA) in Guideline No. 497. • These DAs may generate an inconclusive skin sensitisation prediction. • Inconclusive results may be resolved by applying a weight of evidence approach. • Case studies on four "inconclusive" substances are presented. [ABSTRACT FROM AUTHOR]