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Since 2020, the REACh regulation requires toxicological data on nanoforms of materials, including the assessment of their skin-sensitizing properties. Small molecules' skin sensitization potential can be assessed by new approach methodologies (NAMs) addressing three key events (KE: protein interaction, activation of dendritic cells, and activation of keratinocytes) combined in a defined approach (DA) described in the OECD guideline 497. In the present study, the applicability of three NAMs (DPRA, LuSens, and h-CLAT) to nine materials (eight inorganic nanomaterials (NM) consisting of CeO 2 , BaSO 4 , TiO 2 or SiO 2 , and quartz) was evaluated. The NAMs were technically applicable to NM using a specific sample preparation (NANOGENOTOX dispersion protocol) and method modifications to reduce interaction of NM with the photometric and flowcytometric read-outs. The results of the three assays were combined according to the defined approach described in the OECD guideline No. 497; two of the inorganic NM were identified as skin sensitizers. However, data from animal studies (for ZnO, also human data) indicate no skin sensitization potential. The remaining seven test substances were assessed as "inconclusive" because all inorganic NM were outside the domain of the DPRA, and the achievable test concentrations were not sufficiently high according to the current test guidelines of all three NAMs. The use of these NAMs for (inorganic) NM and the relevance of the results in general are challenged in three ways: (i) NAMs need modification to be applicable to insoluble, inorganic matter; (ii) current test guidelines lack adequate concentration metrics and top concentrations achievable for NM; and (iii) NM may not cause skin sensitization by the same molecular and cellular key events as small organic molecules do; in fact, T-cell-mediated hypersensitivity may not be the most relevant reaction of the immune system to NM. We conclude that the NAMs adopted by OECD test guidelines are currently not a good fit for testing inorganic NM.

The ongoing transition from chemical hazard and risk assessment based on animal studies to assessment relying mostly on non-animal data, requires a multitude of novel experimental methods, and this means that guidance on the validation and standardisation of test methods intended for international applicability and acceptance, needs to be updated. These so-called new approach methodologies (NAMs) must be applicable to the chemical regulatory domain and provide reliable data which are relevant to hazard and risk assessment. Confidence in and use of NAMs will depend on their reliability and relevance, and both are thoroughly assessed by validation. Validation is, however, a time- and resource-demanding process. As updates on validation guidance are conducted, the valuable components must be kept: Reliable data are and will remain fundamental. In 2016, the scientific community was made aware of the general crisis in scientific reproducibility-validated methods must not fall into this. In this commentary, we emphasize the central importance of ring trials in the validation of experimental methods. Ring trials are sometimes considered to be a major hold-up with little value added to the validation. Here, we clarify that ring trials are indispensable to demonstrate the robustness and reproducibility of a new method. Further, that methods do fail in method transfer and ring trials due to different stumbling blocks, but these provide learnings to ensure the robustness of new methods. At the same time, we identify what it would take to perform ring trials more efficiently, and how ring trials fit into the much-needed update to the guidance on the validation of NAMs., (© 2024. The Author(s).)

The acyclic linear monoterpenes Linalool (Lin) and Linalyl acetate (LinAc) occur in nature as major constituents of various essential oils such as lavender oils. A potential endocrine activity of these compounds was discussed in literature including premature thelarche and prepubertal gynecomastia due to lavender product use. This study aims to follow-up on these critical findings reported by testing Lin and LinAc in several studies in line with current guidance and regulatory framework. No relevant anti-/ER and AR-mediated activity was observed in recombinant yeast cell-based screening tests and guideline reporter gene in vitro assays in mammalian cells. Findings in the screening test suggested an anti-androgenic activity, which could not be confirmed in the respective mammalian cell guideline assay. Mechanistic guideline in vivo studies (Uterotrophic and Hershberger assays) with Lin did not show significant dose related changes in estrogen or androgen sensitive organ weights and a guideline reproductive toxicity screening study did not reveal evident effects on sex steroid hormone sensitive organ weights, associated histopathological findings and altered sperm parameters. Estrous cycling and mating/fertility indices were not affected and no evident Lin-related steroid hormone dependent effects were found in the offspring. Overall, the initial concerns from literature were not confirmed. Findings in the yeast screening test were aberrant from follow-up guideline in vitro and in vivo studies, which underlines the need to apply careful interpretation of single in vitro test results to support a respective line of evidence and to establish a biologically plausible link to an adverse outcome., (© 2023. The Author(s).)

Multiple in vitro eye irritation methods have been developed and adopted as OECD health effects test guidelines. However, for predicting the ocular irritation/damage potential of agrochemical formulations there is an applicability domain knowledge gap for most of the methods. To overcome this gap, a retrospective evaluation of 192 agrochemical formulations with in vivo (OECD TG 405) and in vitro (OECD TG 437, 438, and/or 492) data was conducted to determine if the in vitro methods could accurately assign United Nations Globally Harmonized System for Classification and Labelling of Chemicals (GHS) eye irritation hazard classifications. In addition, for each formulation the eye irritation classification was derived from the classification of the contained hazardous ingredients and their respective concentration in the product using the GHS concentration threshold (CT) approach. The results herein suggest that the three in vitro methods and the GHS CT approach were highly predictive of formulations that would not require GHS classification for eye irritation. Given most agrochemical formulations fall into this category, methods that accurately identify non-classified agrochemical formulations could significantly reduce the use of animals for this endpoint., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

In regulatory toxicology, in vivo studies are still prevailing, and human-derived in vitro models are mostly used in testing for local toxicity to the skin and the eyes. A single in vitro model may be limited to address one or few molecular or cellular events leading to adverse outcomes. Hence, in many instances their regulatory use involves the combination of several in vitro models to assess the hazard potential of test substance. A so-called defined approach combines different testing methods and a 'data interpretation procedure' to obtain a comprehensive overall assessment which is used for the regulatory hazard classification of the test substance.Validation is a prerequisite of regulatory acceptance of new testing methods: This chapter provides an overview of the method development from an experimental method to a test guideline via application of GIVIMP (good in vitro method practice), standardization, validation to the regulatory adoption as an OECD test guidelines. Quandaries associated with the validation towards reference data from in vivo animal studies with limited accuracy and limited human relevance are discussed, as well as uncertainty and limitations arising from restricted applicability and technical and biological variance of the in vitro methods.This chapter provides an overview of human-derived in vitro models currently adopted as OECD test guidelines: From the first skin corrosion tests utilizing reconstructed human epidermis models (RhE), to models to test for skin irritation, phototoxicity, eye irritation, and skin sensitization. The latter is using a battery of different methods and defined approaches which are still under discussion for their regulatory adoption. They will be a vanguard of future applications of human-derived models in regulatory toxicology. RhEs for testing of genotoxicity and of dermal penetration and absorption, have been developed, underwent validation studies and may soon be adopted for regulatory use; these are included in this chapter.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Susanne N. Kolle reports a relationship with BASF SE that includes: employment. Melanie Flach reports a relationship with BASF SE that includes: employment. Marcus Kleber reports a relationship with BASF Personal Care and Nutrition GmbH that includes: employment. David A. Basketter reports a relationship with BASF SE that includes: consulting or advisory. David A. Basketter reports serving in an editorial capacity for the journal the manuscript is submitted to. Britta Wareing reports a relationship with BASF SE that includes: employment. Annette Mehling reports a relationship with BASF Personal Care and Nutrition GmbH that includes: employment. Lars Hareng reports a relationship with BASF SE that includes: employment. Nico Watzek reports a relationship with BASF SE that includes: employment. Steffen Bade reports a relationship with BASF SE that includes: employment. Dorothee Funk-Weyer reports a relationship with BASF SE that includes: employment. Robert Landsiedel reports a relationship with BASF SE that includes: employment., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

For hazard classifications of chemicals, continuous data from animal- or nonanimal testing methods are often dichotomized into binary positive/negative outcomes by defining classification thresholds (CT). Experimental data are, however, subject to biological and technical variability. Each test method's precision is limited resulting in uncertainty of the positive/negative outcome if the experimental result is close to the CT. Borderline ranges (BR) around the CT were suggested, which represent ranges in which the study result is ambiguous, that is, positive or negative results are equally likely. The BR reflects a method's precision uncertainty. This article explores and compares different approaches to quantify the BR. Besides using the pooled standard deviation, we determine the BR by means of the median absolute deviation (MAD), with a sequential combination of both methods, and by using nonparametric bootstrapping. Furthermore, we quantify the BR for different hazardous effects, including nonanimal tests for skin corrosion, eye irritation, skin irritation, and skin sensitization as well as for an animal test on skin sensitization (the local lymph node assay, LLNA). Additionally, for one method (direct peptide reactivity assay) the BR was determined experimentally and compared to calculated BRs. Our results demonstrate that (i) the precision of the methods is determining the size of their BRs, (ii) there is no "perfect" method to derive a BR, alas, (iii) a consensus on BR is needed to account for the limited precision of testing methods., (© 2021 The Authors. Risk Analysis published by Wiley Periodicals LLC on behalf of Society for Risk Analysis.)

The availability of reference data is a key requirement for the development of new approach methods (NAM), i.e., in vitro, in chemico and in silico methods and integrated approaches, like defined approaches (DA), which combine these data sources. Reference data are of even greater importance for regulatory acceptance. In contrast to most other adverse effects, human skin sensitization data on many chemicals are available, next to data from animal studies, such as the local lymph node assay (LLNA). Skin sensitization NAM data can therefore be compared to different reference datasets. Recent publications and validation at the OECD focused on human and LLNA reference data. The “2 out of 3” DA (2o3 DA) is the first DA for skin sensitization solely based on experimental data from validated tests and was recently adopted as an OECD test guideline. Here we review the predictivity of the 2o3 DA on multiple human and LLNA reference datasets. Concomitantly, we compare the predictivity of the LLNA for human data within the same datasets. Comparing predictivity of methods not only bilaterally (NAM or DA vs. animal method) but including human data in a triangle “NAM data – animal data – human data” offers a comprehensive assessment of the NAM’s and DA’s predictivity. In all these assessments, the 2o3 DA was superior to the LLNA in predicting human skin sensitization hazard. This highlights the importance of a holistic view of reference data instead of limiting validation of NAMs and DAs to data from a single animal test only.

While single non-animal methods have been adopted in OECD test guidelines, combinations of methods (so called defined approaches, DA) are not. Hardly any animal study can be replaced by a single non-animal method, rather DA are needed. The OECD published the Adverse Outcome Pathway (AOP) on skin sensitization in 2012 and is currently discussing the implementation of DA into a guideline. Obviously, it takes thorough considerations and evaluations to validate such DA. Currently we see four preconditions for a proper and expedient implementation of DA in a guideline: (i) The reference data should be selected to allow meaningful evaluations and must not replicate the limitations of the murine local lymph node assay (LLNA) (ii) Methods and prediction models should be validated before they are used in an OECD-adopted DA, (iii) An OECD-adopted DA should follow the respective AOP and (iv) acknowledge regulatory needs and successful toxicological practice. These points still need to be considered in the current discussion at the OECD. A guideline for skin sensitization DA is setting the scene for regulatory acceptance of all new approaches (for any toxicological endpoint) in the future. In this commentary, we are expounding these preconditions to allow a scientifically valid and sustainable application of modern (no-animal) approaches in regulatory toxicology., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SNK and RL are employees of BASF SE and AN is employee of Givaudan SA, both companies use the described DA to develop and register substances., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Several in vitro OECD test guidelines address key events 1-3 of the adverse outcome pathway for skin sensitization, but none are validated for sensitizer potency assessment. The reaction of sensitizing molecules with skin proteins is the molecular initiating event and appears to be rate-limiting, as chemical reactivity strongly correlates with sensitizer potency. The kinetic direct peptide reactivity assay (kDPRA), a modification of the DPRA (OECD TG 442C), allows derivation of rate constants of the depletion of the cysteine-containing model peptide upon reaction with the test item. Its reproducibility was demonstrated in an inter-laboratory study. Here, we present a database of rate constants, expressed as log kmax, for 180 chemicals to define the prediction threshold to identify strong sensitizers (classified as GHS 1A). A threshold of log kmax -2 offers a balanced accuracy of 85% for predicting GHS 1A sensitizers according to the local lymph node assay. The kDPRA is proposed as a stand-alone assay for identification of GHS 1A sensitizers among chemicals identified as sensitizers by other tests or defined approaches. It may also be used for the prediction of sensitizer potency on a continuous scale, ideally in combination with continuous parameters from other in vitro assays. We show how the rate constant could be combined with read-outs of other in vitro assays in a defined approach. A decision model based on log kmax alone has, however, a high predictivity and can be used as stand-alone model for identification of GHS 1A sensitizers among chemicals predicted as sensitizers.

While the skin sensitization hazard of substances can be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide are evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times. The reaction is stopped by addition of monobromobimane, which forms a fluorescent complex with the free cysteine of the model peptide. The relative remaining non-depleted amount of peptide is determined. Kinetic rate constants are derived from the depletion vs concentration and time matrix and used to distinguish between UN GHS sub-category 1A sensitizers and test substances in sub-category 1B/not classified test substances. In this study, we present a ring trial of the kDPRA with 24 blind-coded test substances in seven laboratories. The intra- and inter-laboratory reproducibility were 96% and 88%, respectively (both for differentiating GHS Cat 1A sensitizers from GHS Cat 1B/not classified). Following an independent peer review, the kDPRA was considered to be acceptable for the identification of GHS Cat 1A skin sensitizers. Besides GHS Cat 1A identification, the kDPRA can be used as part of a defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment. For this aim, next to reproducibility of classification, the quantitative reproducibility and variability of the rate constants were quantified in this study.

There has been significant progress in recent years in the development and application of alternative methods for assessing the skin sensitization potential of chemicals. The pathways involved in skin sensitization have been described in an OECD adverse outcome pathway (AOP). To date, a single non-animal test method is not sufficient to address this AOP so numerous approaches involving the use of 2 or more assays are being evaluated for their performance. The 2 out of 3 approach is a simple approach that has demonstrated very good sensitivity, specificity and overall accuracy numbers for predicting the skin sensitization potential of chemicals. Chemicals with at least two positive results in tests addressing Key events 1-3 are predicted sensitizers, while chemicals with none or only one positive outcome are predicted non-sensitizers. In this report we have thoroughly reviewed the discordant results of 29 chemicals with 1 out of 3 positive results to understand better what led to the results observed and how this information might impact our hazard assessments of these chemicals. We initially categorized each chemical using a weight of evidence approach as positive, negative or indeterminate based on review of available human and animal data as well as what skin sensitization alerts were triggered using two versions of OECD Toolbox and DEREK Nexus. We determined that 4 of the 29 chemicals should be classified as indeterminate and not included in analysis of method performance based on insufficient, borderline and/or conflicting data to confidently categorized the chemicals as allergens or non-allergens. Of the 29 chemicals included in this analysis, 17 were classified as negative and would be correctly identified using a 2 out of 3 approach while 8 chemicals were classified as positive in vivo and would be false-negative with this approach. For some of these chemicals, the outcomes observed can be explained by in vitro borderline results (13 chemicals) or in some instances there is mechanistic understanding of why a chemical is positive or negative in a particular assay (9 chemicals). Thus, when comparing the performance of different defined approaches, one should attempt to only include chemicals which demonstrate clear evidence to be categorize as allergens or non-allergens. Finally, when interpreting the results obtained for an individual unknown chemical it is critical that the in vitro skin sensitization data is reviewed critically and there is a good understanding of the variance and applicability domain limitations for each assay being used., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The selection of reference and proficiency chemicals is an important basis for method validation and proficiency evaluations. Reference chemicals are a set of test substances used by a method developer to evaluate the reliability and relevance of a new method, in comparison to reference data (usually to a validated reference method). Proficiency chemicals, as defined in OECD Guidance Document on Good In Vitro Method Practices, are defined post validation as a subset of the reference chemicals or other chemicals with sufficient supporting data that are used by naïve laboratories to demonstrate technical competence with a validated test method. Proficiency chemicals should cover different physical states, several chemical classes within the applicability domain of the method and yield the full range of responses (in the validated reference method and in vivo), they shall be commercially available (at non-prohibitive costs) and have high quality reference data. If reference and subsequent proficiency chemicals are chosen without sufficient evidence for their inclusion, both test method evaluation and demonstration of technical proficiency can be hampered. In this report we present cases in which the selection of reference chemicals led to problems in the reproduction of the reference results and demonstration of technical proficiency: The variability of results was not always taken into account in selection of several reference substances of the LLNA (OECD TG 429). Based on the available reference data one proficiency chemical for the Corrositex skin corrosion test (OECD TG 435) should be replaced. Likewise, the expected in vitro result for one of the proficiency chemicals for the BCOP (OECD TG 437) was difficult to reproduce in several labs. Furthermore, it was not possible to obtain one of the proficiency chemicals for the Steroidogenesis Assay (OECD TG 456) at non-prohibitive costs at a reasonable purity. Based on these, we recommend changes of current proficiency chemicals lists with established OECD Test Guidelines and provide recommendations for developing future sets of reference chemicals., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While the skin sensitization hazard of substances can already be identified using non-animal methods, the classification of potency sub-categories GHS-1A and 1B is still challenging. Potency can be measured by the dose at which an effect is observed; since the protein-adduct formation is determining the dose of the allergen in the skin, peptide reactivity was used to assess the potency. The Direct Peptide Reactivity Assay (DPRA; one concentration and reaction-time) did not sufficiently discriminate between sub-categories 1A and 1B (56% accuracy compared to LLNA data, n=124). An extended protocol termed 'quantitative DPRA' (three concentrations and one reaction-time), discriminated sub-categories GHS 1A and 1B with an accuracy of 81% or 57% compared to LLNA (n=36) or human (n=14) data, respectively. The analysis of the Cys-adducts was already sufficient; additional analysis of Lys-adducts did not improve the predictivity. An additional modification, the 'kinetic DPRA' (several concentrations and reaction-times) was used to approximate the rate constant of Cys-peptide-adduct formation. 35 of 38 substances were correctly assigned to the potency sub-categories (LLNA data), and the predictivity for 14 human data was equally high. These results warrant the kinetic DPRA for further validation in order to fully replace in vivo testing for assessing skin sensitization including potency sub-classification., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Several in vitro methods have gained regulatory acceptance for the prediction of skin irritation and corrosion. However, the test guidelines for the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. Hence, we would like to share the results from our routine skin corrosion and irritation testing of agrochemical formulations in which both in vitro (according to OECD TG 431 and OECD TG 439) and in vivo (according to OECD TG 404) tests were conducted as regulatory requirements. The in vitro skin irritation test did not correlate well with the CLP classification by in vivo results (44% sensitivity, 60% specificity, and 54% accuracy, based on 65 data pairs). This indicates a lack of applicability of the current protocol of the in vitro skin irritation test for agrochemical formulations. The data set did not contain formulations which were skin corrosive in vivo and hence its applicability could not be assessed. The correlation of in vitro skin corrosion testing to formulations which were not corrosive in vivo was, however, high (95% specificity based on 81 data pairs)., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

In vitro methods have gained regulatory acceptance for the prediction of serious eye damage (UN GHS Cat 1). However, the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. This manuscript presents a study of up to 27 agrochemical formulations tested in three in vitro assays (three versions of the bovine corneal opacity and permeability test (BCOP, OECD TG 437) assay, the isolated chicken eye test (ICE, OECD TG 438) and the EpiOcular™ ET-50 assay). The results were compared with already-available in vivo data. In the BCOP only one of the four, one of five in the ICE and six of eleven tested formulations in the EpiOcular™ ET-50 Neat Protocol resulted in the correct UN GHS Cat 1 prediction. Overpredictions occurred in all assays. These data indicate a lack of applicability of the three in vitro methods to reliably predict UN GHS Cat 1 of agrochemical formulations. In order to ensure animal-free identification of seriously eye damaging agrochemical formulations testing protocols and/or prediction models need to be modified or classification rules should be tailored to in vitro testing rather than using in vivo Draize data as a standard., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

In the EU, chemicals with a production or import volume in quantities of one metric ton per year or more have to be tested for skin sensitizing properties under the REACH regulation. The murine Local Lymph Node Assay (LLNA) and its modifications are widely used to fulfil the data requirement, as it is currently considered the first-choice method for in vivo testing to cover this endpoint. This manuscript describes a case study highlighting the importance of understanding the chemistry of the test material during testing for 'skin sensitization' of MCDA (mixture of 2,4- and 2,6-diamino-methylcyclohexane) with particular focus on the vehicle used. While the BrdU-ELISA modification of the LLNA using acetone/olive oil (AOO) as vehicle revealed expectable positive results. However, the concentration control analysis unexpectedly revealed an instability of MCDA in the vehicle AOO. Further studies on the reactivity showed MCDA to rapidly react with AOO under formation of various imine structures, which might have caused the positive LLNA result. The repetition of the LLNA using propylene glycol (PG) as vehicle did not confirm the positive results of the LLNA using AOO. Finally, a classification of MCDA as skin sensitizer according to the Globally Harmonized System (GHS) was not justified., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Test methods to assess the skin sensitization potential of a substance usually use threshold criteria to dichotomize continuous experimental read-outs into yes/no conclusions. The threshold criteria are prescribed in the respective OECD test guidelines and the conclusion is used for regulatory hazard assessment, i.e., classification and labelling of the substance. We can identify a borderline range (BR) around the classification threshold within which test results are inconclusive due to a test method's biological and technical variability. We quantified BRs in the prediction models of the non-animal test methods DPRA, LuSens and h-CLAT, and of the animal test LLNA, respectively. Depending on the size of the BR, we found that between 6% and 28% of the substances in the sets tested with these methods were considered borderline. When the results of individual non-animal test methods were combined into integrated testing strategies (ITS), borderline test results of individual tests also affected the overall assessment of the skin sensitization potential of the testing strategy. This was analyzed for the 2-out-of-3 ITS: Four out of 40 substances (10%) were considered borderline. Based on our findings we propose expanding the standard binary classification of substances into "positive"/"negative" or "hazardous"/"non-hazardous" by adding a "borderline" or "inconclusive" alert for cases where test results fall within the borderline range.

The molecular initiating event (MIE) of skin sensitization is the binding of a hapten to dermal proteins. This can be assessed using the in chemico direct peptide reactivity assay (DPRA) or in silico tools such as the QSAR Toolbox and TIMES SS. In this study, the suitability of these methods was analyzed by comparing their results to in vivo sensitization data of LLNA and human studies. Compared to human data, 84% of non-sensitizers and sensitizers yielded consistent results in the DPRA. In silico tools resulted in 'no alert' for 83%-100% of the non-sensitizers, but alerted only 55%-61% of the sensitizers. The inclusion of biotic and abiotic transformation simulations yielded more alerts for sensitizers, but simultaneously dropped the number of non-alerted non-sensitizers. In contrast to the DPRA, in silico tools were more consistent with results of the LLNA than human data. Interestingly, the new "DPRA profilers" (QSAR Toolbox) provided unsatisfactory results. Additionally, the results were combined in the '2 out of 3' prediction model with in vitro data derived from LuSens and h-CLAT. Using DPRA results, the model identified 90% of human sensitizers and non-sensitizers; using in silico results (including abiotic and biotic activations) instead of DPRA results led to a comparable high predictivity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In general, no single non-animal method can cover the complexity of any given animal test. Therefore, fixed sets of in vitro (and in chemico) methods have been combined into testing strategies for skin and eye irritation and skin sensitisation testing, with pre-defined prediction models for substance classification. Many of these methods have been adopted as OECD test guidelines. Various testing strategies have been successfully validated in extensive in-house and inter-laboratory studies, but they have not yet received formal acceptance for substance classification. Therefore, under the European REACH Regulation, data from testing strategies can, in general, only be used in so-called weight-of-evidence approaches. While animal testing data generated under the specific REACH information requirements are per se sufficient, the sufficiency of weight-of-evidence approaches can be questioned under the REACH system, and further animal testing can be required. This constitutes an imbalance between the regulatory acceptance of data from approved non-animal methods and animal tests that is not justified on scientific grounds. To ensure that testing strategies for local tolerance testing truly serve to replace animal testing for the REACH registration 2018 deadline (when the majority of existing chemicals have to be registered), clarity on their regulatory acceptance as complete replacements is urgently required., (2016 FRAME.)

Because of ethical and regulatory reasons, several nonanimal test methods to assess the skin sensitization potential of chemicals have been developed and validated. In contrast to in vivo methods, they lack or provide limited metabolic capacity. For this reason, identification of pro-haptens but also pre-haptens, which require molecular transformations to gain peptide reactivity, is a challenge for these methods. In this study, 27 pre- and pro-haptens were tested using nonanimal test methods. Of these, 18 provided true positive results in the direct peptide reactivity assay (DPRA; sensitivity of 67%), although lacking structural alerts for direct peptide reactivity. The reaction mechanisms leading to peptide depletion in the DPRA were therefore elucidated using mass spectrometry. Hapten-peptide adducts were identified for 13 of the 18 chemicals indicating that these pre-haptens were activated and that peptide binding occurred. Positive results for five of the 18 chemicals can be explained by dipeptide formations or the oxidation of the sulfhydryl group of the peptide. Nine of the 27 chemicals were tested negative in the DPRA. Of these, four yielded true positive results in the keratinocyte and dendritic cell based assays. Likewise, 16 of the 18 chemicals tested positive in the DPRA were also positive in either one or both of the cell-based assays. A combination of DPRA, KeratinoSens, and h-CLAT used in a 2 out of 3 weight of evidence (WoE) approach identified 22 of the 27 pre- and pro-haptens correctly (sensitivity of 81%), exhibiting a similar sensitivity as for directly acting haptens. This analysis shows that the combination of in chemico and in vitro test methods is suitable to identify pre-haptens and the majority of pro-haptens.

Background: Assessment of eye irritation hazard has long been a core requirement in any chemical legislation. Nevertheless, publications focussing on the eye damaging potential of nanomaterials are scarce. Traditionally, eye irritation testing was performed using rabbits. The OECD Test Guideline 437 Bovine Corneal Opacity and Permeability (BCOP) test method allows determining severely irritating substances without animals, and the recently adopted OECD Test Guideline 492 Reconstructed human cornea-like epithelium test method allows identifying chemicals that neither induce eye irritation nor serious eye damage. For substances applicable to these tests, huge progress has been made in replacing animal testing., Methods: The in vitro eye irritation potential of 20 nanosized and 3 non-nanosized materials was investigated in a 2-tier EpiOcular™ Eye Irritation Test (EpiOcular™-EIT) and BCOP testing strategy including histopathology of the bovine corneas. Furthermore, applicability of the testing strategy for nanomaterials was assessed. Test materials encompassed OECD representative nanomaterials (metals (Ag), metal oxides (ZnO, TiO2, CeO2), amorphous SiO2 and MWCNTs), three organic pigments, quartz, and talc., Results: None of the dry-powder nanomaterials elicited eye irritation in either the EpiOcular™-EIT or the BCOP assay. Likewise, an amorphous SiO2 nanomaterial that was supplied as suspension was tested negative in both assays. By contrast, in the EpiOcular™-EIT, the silver nanomaterial that was supplied as dispersion was tested positive, whereas its surfactant-containing dispersant was borderline to negative. In the BCOP assay, the silver nanomaterial elicited highly variable results and dark-brown patches remained on the corneal surface, whereas the results for its dispersant alone were borderline to positive, which was assessed as inconclusive due to high inter-assay variability., Conclusion: The present study points to the low eye irritation potential of a spectrum of nanomaterials, which is consistent with available in vivo data for the same test materials or for nanosized or bulk materials of the same composition.

In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (μM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Bovine Corneal Opacity and Permeability (BCOP) test is commonly used for the identification of severe ocular irritants (GHS Category 1), but it is not recommended for the identification of ocular irritants (GHS Category 2). The incorporation of human reconstructed tissue model-based tests into a tiered test strategy to identify ocular non-irritants and replace the Draize rabbit eye irritation test has been suggested (OECD TG 405). The value of the EpiOcular™ Eye Irritation Test (EIT) for the prediction of ocular non-irritants (GHS No Category) has been demonstrated, and an OECD Test Guideline (TG) was drafted in 2014. The purpose of this study was to evaluate whether the BCOP test, in conjunction with corneal histopathology (as suggested for the evaluation of the depth of the injury( and/or the EpiOcular-EIT, could be used to predict the eye irritation potential of agrochemical formulations according to the UN GHS, US EPA and Brazil ANVISA classification schemes. We have assessed opacity, permeability and histopathology in the BCOP assay, and relative tissue viability in the EpiOcular-EIT, for 97 agrochemical formulations with available in vivo eye irritation data. By using the OECD TG 437 protocol for liquids, the BCOP test did not result in sufficient correct predictions of severe ocular irritants for any of the three classification schemes. The lack of sensitivity could be improved somewhat by the inclusion of corneal histopathology, but the relative viability in the EpiOcular-EIT clearly outperformed the BCOP test for all three classification schemes. The predictive capacity of the EpiOcular-EIT for ocular non-irritants (UN GHS No Category) for the 97 agrochemical formulations tested (91% sensitivity, 72% specificity and 82% accuracy for UN GHS classification) was comparable to that obtained in the formal validation exercise underlying the OECD draft TG. We therefore conclude that the EpiOcular-EIT is currently the best in vitro method for the prediction of the eye irritation potential of liquid agrochemical formulations., (2015 FRAME.)

The local lymph node assay (LLNA) is a regulatory accepted test for the identification of skin sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. However, there is evidence that LLNA is overestimating the sensitization potential of certain substance classes in particular those exerting skin irritation. Some reports describe the additional use of flow cytometry-based immunophenotyping to better discriminate irritants from sensitizing irritants in LLNA. In the present study, the 22 performance standards plus 8 surfactants were assessed using the radioactive LLNA method. In addition, lymph node cells were immunophenotyped to evaluate the specificity of the lymph node response using cell surface markers such as B220 or CD19, CD3, CD4, CD8, I-A(κ) and CD69 with the aim to allow a better discrimination above all between irritants and sensitizers, but also non-irritating sensitizers and non-sensitizers. However, the markers assessed in this study do not sufficiently differentiate between irritants and irritant sensitizers and therefore did not improve the predictive capacity of the LLNA., (Copyright © 2014 John Wiley & Sons, Ltd.)

Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Whilst western diet and sedentary lifestyles heavily contribute to the global obesity epidemic, it is likely that chemical exposure may also contribute. A substantial body of literature implicates a variety of suspected environmental chemicals in metabolic disruption and obesogenic mechanisms. Chemically induced obesogenic metabolic disruption is not yet considered in regulatory testing paradigms or regulations, but this is an internationally recognised human health regulatory development need. An early step in the development of relevant regulatory test methods is to derive appropriate minimum chemical selection lists for the target endpoint and its key mechanisms, such that the test method can be suitably optimised and validated. Independently collated and reviewed reference and proficiency chemicals relevant for the regulatory chemical universe that they are intended to serve, assist regulatory test method development and validation, particularly in relation to the OECD Test Guidelines Programme. To address obesogenic mechanisms and modes of action for chemical hazard assessment, key initiating mechanisms include molecular-level Peroxisome Proliferator-Activated Receptor (PPAR) α and γ agonism and the tissue/organ-level key event of perturbation of the adipogenesis process that may lead to excess white adipose tissue. Here we present a critical literature review, analysis and evaluation of chemicals suitable for the development, optimisation and validation of human PPARα and PPARγ agonism and human white adipose tissue adipogenesis test methods. The chemical lists have been derived with consideration of essential criteria needed for understanding the strengths and limitations of the test methods. With a weight of evidence approach, this has been combined with practical and applied aspects required for the integration and combination of relevant candidate test methods into test batteries, as part of an Integrated Approach to Testing and Assessment for metabolic disruption. The proposed proficiency and reference chemical list includes a long list of negatives and positives (20 chemicals for PPARα, 21 for PPARγ, and 11 for adipogenesis) from which a (pre-)validation proficiency chemicals list has been derived. [ABSTRACT FROM AUTHOR]

Plant protection measures are necessary to prevent pests and diseases from attacking and destroying crop plants and to meet consumer demands for agricultural produce. In the last decades the use of chemical pesticides has largely increased. Farmers are looking for alternatives. Biopesticides should be considered a sustainable solution. They may be less toxic than chemical pesticides, be very specific to the target pest, decompose quickly, and be less likely to cause resistance. On the other hand, lower efficacy and higher costs are two disadvantages of many biopesticides. Biopesticides include macroorganisms, natural compounds and microorganisms. Microbial pesticides are the most widely used and studied class of biopesticides. The greatest difference between microbial and chemical pesticides is the ability of the former to potentially multiply in the environment and on the crop plant after application. The data requirements for the European Union and the United States Environmental Protection Agency are highlighted, as these regulatory processes are the most followed in regions where local regulations for biopesticide products are not available or vague. New Approach Methods already proposed or harmonized for chemical pesticides are presented and discussed with respect to their use in evaluating microbial pesticide formulations. Evaluating the microbials themselves is not as simple as using the same validated New Approach Methods as for synthetic pesticides. Therefore, the authors suggest considering New Approach Method strategies specifically for microbials and global harmonization with acceptability with the advancements of such approaches. Further discussion is needed and greatly appreciated by the experts. [ABSTRACT FROM AUTHOR]

We genetically manipulated HaCaT cells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL-8) and ubiquitin-C (UBC) promoters, respectively. Subsequently, we generated a three-dimensional (3D) skin-like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non-woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real-time bioluminescence monitoring, allowing the assessment of time- and dose-dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Chemically induced skin sensitization, or allergic contact dermatitis, is a common occupational and public health issue. Regulatory authorities require an assessment of potential to cause skin sensitization for many chemical products. Defined approaches for skin sensitization (DASS) identify potential chemical skin sensitizers by integrating data from multiple non-animal tests based on human cells, molecular targets, and computational model predictions using standardized data interpretation procedures. While several DASS are internationally accepted by regulatory agencies, the data interpretation procedures vary in logical complexity, and manual application can be time-consuming or prone to error. Results: We developed the DASS App, an open-source web application, to facilitate user application of three regulatory testing strategies for skin sensitization assessment: the Two-out-of-Three (2o3), the Integrated Testing Strategy (ITS), and the Key Event 3/1 Sequential Testing Strategy (KE 3/1 STS) without the need for software downloads or computational expertise. The application supports upload and analysis of user-provided data, includes steps to identify inconsistencies and formatting issues, and provides predictions in a downloadable format. Conclusion: This open-access web-based implementation of internationally harmonized regulatory guidelines for an important public health endpoint is designed to support broad user uptake and consistent, reproducible application. The DASS App is freely accessible via https://ntp.niehs.nih.gov/go/952311 and all scripts are available on GitHub (https://github.com/NIEHS/DASS). [ABSTRACT FROM AUTHOR]

Biological evaluation of resin-based dental composites has traditionally been based on in vitro endpoint tests with different methods to determine loss of cell viability and cell morphology changes after exposure to the material or monomer constituents. The data reveals a potential for biological effects, but clinical relevance of such data is limited. Positive allergy tests and allergic clinical reactions to dental monomers are observed in dental personnel and patients. The aim of this review is to address newer research on molecular events caused by exposure to resin-based composites to have a better understanding of the potential for clinical adverse effects. A more accurate understanding of the biological aspects of dental composite materials has been found after studying parameters like glutathione depletion, oxidative stress, genotoxicity, and immunomodulatory key effects in various cell culture models. Using omics-based approaches allow for a broader and non-specified search of changes caused by methacrylate exposure. Defense mechanisms and adaption are observed in cells exposed to monomer concentrations relevant to clinical exposure. The above-mentioned methods are the foundations for modified testing strategies. The clinical relevance of most available in vitro endpoint tests is of limited relevance for the patient. Research focusing on molecular mechanisms has given new insight into methacrylate toxicity in exposed cells. Using this knowledge from mechanistic studies to develop standardized in vitro biocompatibility tests will likely improve their clinical relevance. [ABSTRACT FROM AUTHOR]

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Background: Fragrances are important contact allergens; however, investigation of their skin sensitization potency has been challenging in new approach methods (NAMs). Many fragrance chemicals are susceptible to autoxidation or can be metabolized by enzymes constitutively expressed in skin keratinocytes. Strong sensitizers can be formed in both of these processes. Further, keratinocytes can modulate the dendritic cell (DC) activation and maturation potential, a key event in the acquisition of contact allergy. Objectives: To evaluate the 2D coculture model consisting of keratinocytes and DCs using different weak to moderate sensitizing fragrance chemicals. Further, to investigate fragrances and related oxidation products in the in vitro model and compare to in vivo data. Methods: Chemicals were tested in the coculture activation test (COCAT), consisting of HaCaT keratinocytes and THP‐1 cells. THP‐1 cell surface expression of costimulatory and adhesion molecules (CD86 and CD54) collected after 24 h incubation with the chemicals was analysed using flow cytometry. Results: Twenty‐four molecules were tested positive, three were negative (n = 27). Four pairs were evaluated, with aldehydes showing a 6‐ to 13‐fold stronger responses compared to their corresponding alcohols. Conclusions: Results provide insight into the activation of DC in their natural environment of keratinocytes. α,β‐Unsaturated alcohols were classified as weaker sensitizers compared to their corresponding aldehydes. In sum, testing of fragrances retrieved results in good agreement with in vivo data. [ABSTRACT FROM AUTHOR]

Particularly since the wide-ranging health effects of asbestos exposure became known, great emphasis has been placed on detailed toxicity testing of known but also newly developed fiber materials. Exposure to respirable pollutants like fibers can lead to tissue injury causing lung diseases such as pulmonary fibrosis or cancer. In order to detect the toxic potential of such aerosols at an early stage, the development of suitable test systems is essential. In this study, we illustrate the development of an advanced in vitro cell model closely resembling the physiological structure of the alveoli, and we highlight its advantages over simpler models to predict pro-fibrotic changes. For this reason, we analyzed the cytotoxic effects of fiber-like multi-walled carbon nanotubes after 24 and 48 h exposure, and we investigated inflammatory, genotoxic and pro-fibrotic changes occurring in the developed triple culture consisting of lung epithelial cells, macrophages and fibroblasts compared to a co-culture of epithelial cells and fibroblasts or a mono culture of epithelial cells. In summary, the triple culture system is more precisely able to detect a pro-fibrotic phenotype including epithelial-mesenchymal transition as well as secondary genotoxicity, even if exhibiting lower cytotoxicity in contrast to the less advanced systems. These effects might be traced back to the complex interplay between the different cell types, all of which play an important role in the inflammatory response, which precedes wound healing, or even fibrosis or cancer development. [ABSTRACT FROM AUTHOR]

Testing of all manufactured products and their ingredients for eye irritation is a regulatory requirement. In the last two decades, the development of alternatives to the in vivo Draize eye irritation test method has substantially advanced due to the improvements in primary cell isolation, cell culture techniques, and media, which have led to improved in vitro corneal tissue models and test methods. Most in vitro models for ocular toxicology attempt to reproduce the corneal epithelial tissue which consists of 4-5 layers of non-keratinized corneal epithelial cells that form tight junctions, thereby limiting the penetration of chemicals, xenobiotics, and pharmaceuticals. Also, significant efforts have been directed toward the development of more complex three-dimensional (3D) equivalents to study wound healing, drug permeation, and bioavailability. This review focuses on in vitro reconstructed 3D corneal tissue models and their utilization in ocular toxicology as well as their application to pharmacology and ophthalmic research. Current human 3D corneal epithelial cell culture models have replaced in vivo animal eye irritation tests for many applications, and substantial validation efforts are in progress to verify and approve alternative eye irritation tests for widespread use. The validation of drug absorption models and further development of models and test methods for many ophthalmic and ocular disease applications is required.

Preclinical research struggles with its predictive power for drug effects in patients. The clinical success of preclinically approved drug candidates ranges between 3% and 33%. Regardless of the approach, novel disease models and test methods need to prove their relevance and reliability for predicting drug effects in patients, which is usually achieved by method validation. Nevertheless, validating all models appears unrealistic due to the variety of diseases. Thus, novel concepts are needed to increase the quality of preclinical research.Herein, we introduce qualification as a minimal standard to establish the relevance of preclinical models and test methods. Qualification starts with prioritizing and translating scientific requirements into technical parameters by quality function deployment. Qualified models use authenticated cells, which resemble the corresponding cells in humans in morphology and drug target expression. Moreover, disease models differ from normal models in the expression of relevant biomarkers. As a result, qualified test methods can discriminate effects of treatment standards and the effects of weakly effective or ineffective substances. Observer-blind readout, adequate data documentation, dropout inclusion, and a priori power studies are as crucial as realistic dosage regimens for qualified approaches. Here, we showcase the implementation of qualification. Adjusting the level of model complexity and qualification to three defined phases of preclinical research assures the optimal level of certainty at each step.In conclusion, qualification strengthens the researchers' impact by defining basic requirements that novel approaches must fulfill while still allowing for scientific creativity. Qualification helps to improve the predictive power of preclinical research. Applied to human cell-based models, qualification reduces animal testing, since only effective drug candidates are subjected to final animal testing and subsequently to clinical trials.

The setting of concentrations for testing substances in ecotoxicological studies is often based on fractions of the concentrations that cause 50% mortality (LC50 or LD50) rather than environmentally relevant levels. This practice can result in exposures to animals at test concentrations that are magnitudes of order greater than those experienced in the environment. Often, such unrealistically high concentrations may cause non-specific biochemical or morphologic changes that primarily reflect the near-lethal health condition of the animal subjects, as opposed to effects characteristic of the particular test compound. Meanwhile, it is recognized that for many chemicals, the toxicologic mode of action (MOA) responsible for lethality may differ entirely from the MOAs that cause various sublethal effects. One argument for employing excessively high exposure concentrations in sublethal studies is to ensure the generation of positive toxicological effects, which can then be used to establish safety thresholds; however, it is possible that the pressure to produce exposure-related effects may also contribute to false positive outcomes. The purpose of this paper is to explore issues involving some current usages of acute LC50 data in ecotoxicology testing, and to propose an alternative strategy for performing this type of research moving forward. Toward those ends, a brief literature survey was conducted to gain an appreciation of methods that are currently being used to set test concentrations for sublethal definitive studies. [ABSTRACT FROM AUTHOR]

This review addresses the need for a framework to increase the consistency, objectivity and transparency in the regulatory assessment of respiratory sensitisers and associated uncertainties. Principal issues are considered and illustrated through a case study (with methyl methacrylate). In the absence of test methods validated for regulatory use, formal documentation of the weight-of-evidence for hazard classification both at the level of integration of individual studies within lines of evidence and across a broad range of data streams was agreed to be critical for such a framework. An integrated approach is proposed to include not only occupational studies and clinical evidence for the regulatory assessment of respiratory sensitisers, but also information on structure and physical and chemical factors, predictive approaches such as structure activity analysis and in vitro and in vivo mechanistic and toxicokinetic findings. A weight-of-evidence protocol, incorporating integration of these sources of data based on predefined considerations, would contribute to transparency and consistency in the outcome of the assessment. In those cases where a decision may need to be taken on the basis of occupational findings alone, conclusions should be based on transparent weighting of relevant data on the observed prevalence of occupational asthma in various studies taking into account all relevant information including the range and nature of workplace exposures to the substance of interest, co-exposure to other chemicals and study quality. [ABSTRACT FROM AUTHOR]

E-cigarette consumption is under scrutiny by regulatory authorities due to concerns about product toxicity, lack of manufacturing standards, and increasing reports of e-cigarette- or vaping-associated acute lung injury. In vitro studies have demonstrated cytotoxicity, mitochondrial dysfunction, and oxidative stress induced by unflavored e-cigarette aerosols and flavoring additives. However, e-cigarette effects on the complex lung parenchyma remain unclear. Herein, the impact of e-cigarette condensates with or without menthol flavoring on functional, structural, and cellular responses was investigated using mouse precision cut lung slices (PCLS). PCLS were exposed to e-cigarette condensates prepared from aerosolized vehicle, nicotine, nicotine + menthol, and menthol e-fluids at doses from 50 to 500 mM. Doses were normalized to the glycerin content of vehicle. Video-microscopy of PCLS revealed impaired contractile responsiveness of airways to methacholine and dampened ciliary beating following exposure to menthol-containing condensates at concentrations greater than 300 mM. Following 500 mM menthol-containing condensate exposure, epithelial exfoliation in intrabronchial airways was identified in histological sections of PCLS. Measurement of lactate dehydrogenase release, mitochondrial water-soluble-tetrazolium salt-1 conversion, and glutathione content supported earlier findings of nicotine or nicotine + menthol e-cigarette-induced dose-dependent cytotoxicity and oxidative stress responses. Evaluation of PCLS metabolic activity revealed dose-related impairment of mitochondrial oxidative phosphorylation and glycolysis after exposure to menthol-containing condensates. Taken together, these data demonstrate prominent mentholinduced pulmonary toxicity and impairment of essential physiological functions in the lung, which warrants concerns about e-cigarette consumer safety and emphasizes the need for further investigations of molecular mechanisms of toxicity and menthol effects in an experimental model of disease. [ABSTRACT FROM AUTHOR]

Plant protection products (PPPs) consist of pesticide active substances and co-formulants. Generally, active substance effects are assumed to dominate in PPP toxicity. Nevertheless, co-formulants may well affect the toxicity of PPPs via toxicodynamic and toxicokinetic interaction. To account for potential mixture effects and improve PPP data requirements for application in risk assessment, a tiered test strategy is proposed. The strategy is based on a comparison of PPP and active substance toxicity, which enables the prioritisation of PPPs for further testing, adaptation of the toxicological threshold value or removal of toxic co-formulants from the PPP. Moreover, it focuses on the integrative assessment of existing information and newly generated data using alternative test methods. The proposed strategy will improve PPP toxicological assessment by accounting for mixture toxicity, providing a set of regulatory options for risk assessment and the necessary data for hazard assessment. The predictivity of alternative methods for PPPs will improve by evaluation of their reliability and uncertainty. © 2020 The Authors. Pest Management Science published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2020 The Authors. Pest Management Science published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.)

Background: Chemical-induced allergies at workplace represent a significant occupational health issue. These substances must be properly identified as sensitizers. In previous studies, an original model using mouse bone marrow-derived dendritic cells (BMDC) was developed for this purpose., Objectives: The aim of this study was to evaluate the predictive capacity of the BMDC model with a large panel of sensitizers (including pre- and pro-haptens) and non-sensitizers., Methods: The readout from the BMDC model is based on expression levels of six phenotypic markers measured by flow cytometry., Results: The results indicate that 29 of the 37 non-sensitizers, and 81 of the 86 sensitizers were correctly classified compared to the Local Lymph Node Assay (LLNA). Statistical analysis revealed the BMDC model to have a sensitivity of 94%, a specificity of 78%, and an accuracy of 89%. The EC2 (Effective Concentration) values calculated with this model allow sensitizers to be categorized into four classes: extreme, strong, moderate and weak., Conclusions: These excellent predictive performances show that the BMDC model discriminates between sensitizers and non-sensitizers with outstanding precision equal to or better than existing validated alternative models. Moreover, this model allows to predict sensitization potency of chemicals. The BMDC test could therefore be proposed as an additional tool to assess the sensitizing potential and potency of chemicals., (© 2023 The Authors. Contact Dermatitis published by John Wiley & Sons Ltd.)

Skin irritancy to topically applied chemicals is a significant problem that affects millions of people worldwide. New or modified chemical entities must be tested for potential skin irritancy by industry as part of the safety and toxicity profiling process. Many of these tests have now moved to a non‐animal‐based format to reduce experiments on animals. However, these tests for irritancy potential often rely on monolayer cultures of keratinocytes that are not representative of the skin architecture or tissue‐engineered human skin equivalents (HSE) using complex multi‐gene expression panels that are often cumbersome and not amenable for high throughput. Here, we show that human skin equivalents increase abundance of several phosphorylated kinases (c‐Src, c‐Jun, p53, GSK3α/β) in response to irritant chemical stimulation by phosphokinase array analysis. Specific phosphorylation of c‐SrcY419 was confirmed by immunoblotting and was plasma membrane‐associated in basal/spinous cells by phospho‐specific immunohistochemistry. Moreover, c‐SrcY419 phosphorylation in response to the irritants lactic acid and capsaicin was inhibited by the c‐Src inhibitors KB‐SRC and betaine trimethylglycine. These data provide the first evidence for c‐Src specific activation in response to chemical irritants and point to the development of new modes of rapid testing by immunodetection for first‐pass screening of potential irritants. This study demonstrates that specific kinases, in particular c‐Src, are activated in HSE in response to chemical irritants. The rapid increase in specific phosphorylated signal transduction molecules upon exposure to irritants may prove ideal targets to rapidly identify and differentiate potential chemical irritants from non‐irritants. [ABSTRACT FROM AUTHOR]

The skin is the organ that is most susceptible to the impact of the exposome. Located at the interface with the external environment, it protects internal organs through the barrier function of the epidermis. It must adapt to the consequences of the harmful effects of solar radiation, the various chemical constituents of atmospheric pollution, and wounds associated with mechanical damage: oxidation, cytotoxicity, inflammation, and so forth. In this biological context, a capacity to adapt to the various stresses caused by the exposome is essential; otherwise, more or less serious conditions may develop accelerated aging, pigmentation disorders, atopy, psoriasis, and skin cancers. Nrf2‐controlled pathways play a key role at this level. Nrf2 is a transcription factor that controls genes involved in oxidative stress protection and detoxification of chemicals. Its involvement in UV protection, reduction of inflammation in processes associated with healing, epidermal differentiation for barrier function, and hair regrowth, has been demonstrated. The modulation of Nrf2 in the skin may therefore constitute a skin protection or care strategy for certain dermatological stresses and disorders initiated or aggravated by the exposome. Nrf2 inducers can act through different modes of action. Keap1‐dependent mechanisms include modification of the cysteine residues of Keap1 by (pro)electrophiles or prooxidants, and disruption of the Keap1–Nrf2 complex. Indirect mechanisms are suggested for numerous phytochemicals, acting on upstream pathways, or via hormesis. While developing novel and safe Nrf2 modulators for skin care may be challenging, new avenues can arise from natural compounds‐based molecular modeling and emerging concepts such as epigenetic regulation. [ABSTRACT FROM AUTHOR]