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

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

SCCS scientific opinion on Butylated hydroxytoluene (BHT) - SCCS/1636/21.

OPINION TO BE CITED AS: SCCS (Scientific Committee on Consumer Safety), scientific opinion on Butylated hydroxytoluene (BHT), preliminary version of September 27, 2021, final version of December 2, 2021, SCCS/1636/21.

SCCS Scientific Opinion on Acid Yellow 3 (submission II) - SCCS/1631/21.

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion on Acid Yellow 3 - C054 (CAS Number 8004-92-0, EC No 305-897-5), submission II, preliminary version of 7 May 2021, final version of 23 July 2021, SCCS/1631/21.

Occupational exposure to hexavalent chromium. Part II. Hazard assessment of carcinogenic effects.

Hexavalent chromium (Cr(VI)) compounds have been studied extensively and several agencies have described their toxicological profile. In the past, personnel of the Dutch Ministry of Defence may have been exposed to Cr(VI) during maintenance activities on NATO equipment. To investigate if this exposure may have caused irreversible adverse health effects, the Dutch National Institute for Public Health and the Environment (RIVM) summarized all available knowledge from previous evaluations. This information was complemented with a scoping review to retrieve new scientific literature. All scientific evidence was evaluated in workshops with external experts to come to an overview of irreversible adverse health effects that could be caused by occupational exposure to Cr(VI) compounds. This review provides the hazard assessment for occupational exposure to Cr(VI) and carcinogenic effects by integrating and weighting evidence provided by international agencies complemented with newly published studies. It was concluded that occupational exposure to Cr(VI) can cause lung cancer, nose and nasal sinus cancer in humans. Cr(VI) is suspected to cause stomach cancer and laryngeal cancer in humans. It is currently insufficiently clear if Cr(VI) can cause cancer of the small intestine, oral cavity, pancreas, prostate or bladder in humans.

Occupational exposure to hexavalent chromium. Part I. Hazard assessment of non-cancer health effects.

Hexavalent chromium (Cr(VI)) compounds have been studied extensively and several agencies have described their toxicological profile. In the past, personnel of the Dutch Ministry of Defence may have been exposed to Cr(VI) during maintenance activities. To investigate if this exposure may have caused irreversible adverse health effects, the Dutch National Institute for Public Health and the Environment (RIVM) summarized all available knowledge from previous evaluations. This information was complemented with a scoping review to retrieve new scientific literature. All scientific evidence was evaluated in workshops with external experts to come to an overview of irreversible adverse health effects that could be caused by occupational exposure to Cr(VI) compounds. This review focuses on non-cancer health effects. It was concluded that occupational exposure to Cr(VI) can cause perforation of the nasal septum by chromium ulcers, chronic lung diseases, including asthma, rhinitis, pulmonary fibrosis and COPD, skin ulcers and allergic contact dermatitis in humans. It is currently insufficiently clear if Cr(VI) can cause irreversible diseases due to disturbances of the immune system (other than allergic contact eczema, allergic asthma and rhinitis and chronic lung diseases) or adverse effects on fertility or prenatal development in humans.

Applying non-animal strategies for assessing skin sensitisation report from an EPAA/cefic-LRI/IFRA Europe cross sector workshop, ECHA helsinki, February 7th and 8th 2019.

Four years on since the last cross sector workshop, experience of the practical application and interpretation of several non-animal assays that contribute to the predictive identification of skin sensitisers has begun to accumulate. Non-animal methods used for hazard assessments increasingly are contributing to the potency sub-categorisation for regulatory purposes. However, workshop participants generally supported the view that there remained a pressing need to build confidence in how information from multiple methods can be combined for classification, sub-categorisation and potency assessment. Furthermore, the practical experience gained over the last few years, highlighted the overall high potential value of using the newly validated methods and testing strategies, but also that limitations for certain substance/product classes may become evident with further use as had been the case with other new regulatory methods. As the available information increases, review of the data and collated experience could further determine strengths and limitations leading to more confidence in their use. Finally, the need for a substantial and universally accepted dataset of non-sensitisers and substances of different sensitising potencies, based on combined human and in vivo animal data for validation of methods and test strategies was re-emphasised.

State of the art in non-animal approaches for skin sensitization testing: from individual test methods towards testing strategies.

The hazard assessment of skin sensitizers relies mainly on animal testing, but much progress is made in the development, validation and regulatory acceptance and implementation of non-animal predictive approaches. In this review, we provide an update on the available computational tools and animal-free test methods for the prediction of skin sensitization hazard. These individual test methods address mostly one mechanistic step of the process of skin sensitization induction. The adverse outcome pathway (AOP) for skin sensitization describes the key events (KEs) that lead to skin sensitization. In our review, we have clustered the available test methods according to the KE they inform: the molecular initiating event (MIE/KE1)-protein binding, KE2-keratinocyte activation, KE3-dendritic cell activation and KE4-T cell activation and proliferation. In recent years, most progress has been made in the development and validation of in vitro assays that address KE2 and KE3. No standardized in vitro assays for T cell activation are available; thus, KE4 cannot be measured in vitro. Three non-animal test methods, addressing either the MIE, KE2 or KE3, are accepted as OECD test guidelines, and this has accelerated the development of integrated or defined approaches for testing and assessment (e.g. testing strategies). The majority of these approaches are mechanism-based, since they combine results from multiple test methods and/or computational tools that address different KEs of the AOP to estimate skin sensitization potential and sometimes potency. Other approaches are based on statistical tools. Until now, eleven different testing strategies have been published, the majority using the same individual information sources. Our review shows that some of the defined approaches to testing and assessment are able to accurately predict skin sensitization hazard, sometimes even more accurate than the currently used animal test. A few defined approaches are developed to provide an estimate of the potency sub-category of a skin sensitizer as well, but these approaches need further independent evaluation with a new dataset of chemicals. To conclude, this update shows that the field of non-animal approaches for skin sensitization has evolved greatly in recent years and that it is possible to predict skin sensitization hazard without animal testing.

Developing a framework for assessing chemical respiratory sensitization: A workshop report.

Respiratory tract sensitization can have significant acute and chronic health implications. While induction of respiratory sensitization is widely recognized for some chemicals, validated standard methods or frameworks for identifying and characterizing the hazard are not available. A workshop on assessment of respiratory sensitization was held to discuss the current state of science for identification and characterization of respiratory sensitizer hazard, identify information facilitating development of validated standard methods and frameworks, and consider the regulatory and practical risk management needs. Participants agreed on a predominant Th2 immunological mechanism and several steps in respiratory sensitization. Some overlapping cellular events in respiratory and skin sensitization are well understood, but full mechanism(s) remain unavailable. Progress on non-animal approaches to skin sensitization testing, ranging from in vitro systems, -omics, in silico profiling, and structural profiling were acknowledged. Addressing both induction and elicitation phases remains challenging. Participants identified lack of a unifying dose metric as increasing the difficulty of interpreting dosimetry across exposures. A number of research needs were identified, including an agreed list of respiratory sensitizers and other asthmagens, distinguishing between adverse effects from immune-mediated versus non-immunological mechanisms. A number of themes emerged from the discussion regarding future testing strategies, particularly the need for a tiered framework respiratory sensitizer assessment. These workshop present a basis for moving towards a weight-of-evidence assessment.

A critical appraisal of the process of regulatory implementation of novel in vivo and in vitro methods for chemical hazard and risk assessment.

Regulatory toxicology urgently needs applicable alternative test systems that reduce animal use, testing time, and cost. European regulation on cosmetic ingredients has already banned animal experimentation for hazard identification, and public awareness drives toward additional restrictions in other regulatory frameworks as well. In addition, scientific progress stimulates a more mechanistic approach of hazard identification. Nevertheless, the implementation of alternative methods is lagging far behind their development. In search for general bottlenecks for the implementation of alternative methods, this manuscript reviews the state of the art as to the development and implementation of 10 diverse test systems in various areas of toxicological hazard assessment. They vary widely in complexity and regulatory acceptance status. The assays are reviewed as to parameters assessed, biological system involved, standardization, interpretation of results, extrapolation to human hazard, position in testing strategies, and current regulatory acceptance status. Given the diversity of alternative methods in many aspects, no common bottlenecks could be identified that hamper implementation of individual alternative assays in general. However, specific issues for the regulatory acceptance and application were identified for each assay. Acceptance of one-in-one replacement of complex in vivo tests by relatively simple in vitro assays is not feasible. Rather, innovative approaches using test batteries are required together with metabolic information and in vitro to in vivo dose extrapolation to convincingly provide the same level of information of current in vivo tests. A mechanistically based alternative approach using the Adverse Outcome Pathway concept could stimulate further (regulatory) acceptance of non-animal tests.

Anchoring molecular mechanisms to the adverse outcome pathway for skin sensitization: Analysis of existing data.

Allergic contact dermatitis (ACD) is a hypersensitivity immune response induced by small protein-reactive chemicals. Currently, the murine local lymph node assay (LLNA) provides hazard identification and quantitative estimation of sensitizing potency. Given the complexity of ACD, a single alternative method cannot replace the LLNA, but it is necessary to combine methods through an integrated testing strategy (ITS). In the development of an ITS, information regarding mechanisms and molecular processes involved in skin sensitization is crucial. The recently published adverse outcome pathway (AOP) for skin sensitization captures mechanistic knowledge into key events that lead to ACD. To understand the molecular processes in ACD, a systematic review of murine in vivo studies was performed and an ACD molecular map was constructed. In addition, comparing the molecular map to the limited human in vivo toxicogenomic data available suggests that certain processes are similarly triggered in mice and humans, but additional human data will be needed to confirm these findings and identify differences. To gain insight in the molecular mechanisms represented by various human in vitro systems, the map was compared to in vitro toxicogenomic data. This analysis allows for comparison of emerging in vitro methods on a molecular basis, in addition to mathematical predictive value. Finally, a survey of the current in silico, in chemico, and in vitro methods was used to indicate which AOP key event is modeled by each method. By anchoring emerging classification methods to the AOP and the ACD molecular map, complementing methods can be identified, which provides a cornerstone for the development of a testing strategy that accurately reflects the key events in skin sensitization.

Assessment of the risk of respiratory sensitization from fragrance allergens released by air fresheners.

Consumers using air fresheners are exposed to the emitted ingredients, including fragrances, via the respiratory tract. Several fragrances are known skin sensitizers, but it is unknown whether inhalation exposure to these chemicals can induce respiratory sensitization. Effects on the immune system were assessed by testing a selection of five fragrance allergens in the respiratory local lymph node assay (LLNA). The probability and extent of exposure were assessed by measuring concentrations of the 24 known fragrance allergens in 109 air fresheners. It was shown that the most frequently used fragrances in air fresheners were D-limonene and linalool. In the respiratory LLNA, these fragrances were negative. Of the other tested chemicals, only isoeugenol induced a statistically significant increase in cell proliferation. Consumer exposure was assessed in more detail for D-limonene, linalool, and isoeugenol by using exposure modeling tools. It was shown that the most frequently used fragrances in air fresheners, D-limonene, and linalool gave rise to a higher consumer exposure compared with isoeugenol. To evaluate whether the consumer exposure to these fragrances is low or high, these levels were compared with measured air concentrations of diisocyanates, known human respiratory sensitizers. This comparison showed that consumer exposure from air fresheners to D-limonene, linalool, and isoeugenol is considerably lower than occupational exposure to diisocyanates. By combing this knowledge on sensitizing potency with the much lower exposure compared to diisocyanates it seems highly unlikely that isoeugenol can induce respiratory sensitization in consumers using air fresheners.

Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals.

The currently available animal-free methods for the detection of skin sensitizing potential of chemicals seem promising. However, no single method is able to comprehensively represent the complexity of the processes involved in skin sensitization. To ensure a mechanistic basis and cover the complexity, multiple methods should be integrated into a testing strategy, in accordance with the adverse outcome pathway that describes all key events in skin sensitization. Although current majority voting testing strategies have proven effective, the performance of individual methods is not taken into account. To that end, we designed a tiered strategy based on complementary characteristics of the included methods, and compared it to a majority voting approach. This tiered testing strategy was able to correctly identify all 41 chemicals tested. In terms of total number of experiments required, the tiered testing strategy requires less experiments compared to the majority voting approach. On the other hand, this tiered strategy is more complex due the number of different alternative methods required, and predicted costs are similar for both strategies. Both the tiered and majority voting strategies provide a mechanistic basis for skin sensitization testing, but the strategy most suitable for regulatory decision-making remains to be determined.

Induction of skin sensitization is augmented in Nrf2-deficient mice.

Several in vitro DNA microarray studies have shown the importance of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in skin sensitization. Nevertheless, the exact in vivo role of the Nrf2-Keap1 pathway during the induction of skin sensitization remains unknown. To study the function of Nrf2, a local lymph node assay was performed in wild-type and Nrf2-deficient mice using 2,4-dinitrochlorobenzene. The Nrf2-deficient mice show a more pronounced response, indicating that Nrf2 is involved in dampening the induction of skin sensitization.

Evaluation of the performance of the reduced local lymph node assay for skin sensitization testing.

The local lymph node assay (LLNA) is the preferred method for classification of sensitizers within REACH. To reduce the number of mice for the identification of sensitizers the reduced LLNA was proposed, which uses only the high dose group of the LLNA. To evaluate the performance of this method for classification, LLNA data from REACH registrations were used and classification based on all dose groups was compared to classification based on the high dose group. We confirmed previous examinations of the reduced LLNA showing that this method is less sensitive compared to the LLNA. The reduced LLNA misclassified 3.3% of the sensitizers identified in the LLNA and misclassification occurred in all potency classes and that there was no clear association with irritant properties. It is therefore not possible to predict beforehand which substances might be misclassified. Another limitation of the reduced LLNA is that skin sensitizing potency cannot be assessed. For these reasons, it is not recommended to use the reduced LLNA as a stand-alone assay for skin sensitization testing within REACH. In the future, the reduced LLNA might be of added value in a weight of evidence approach to confirm negative results obtained with non-animal approaches.

Unraveling toxicological mechanisms and predicting toxicity classes with gene dysregulation networks.

The use of genes for distinguishing classes of toxicity has become well established. In this paper we combine the reconstruction of a gene dysregulation network (GDN) with a classifier to assign unseen compounds to their appropriate class. Gene pairs in the GDN are dysregulated in the sense that they are linked by a common expression pattern in one class and differ in this pattern in another class. The classifier gives a quantitative measure on this difference by its prediction accuracy. As an in-depth example, gene pairs were selected that were dysregulated between skin cells treated with either sensitizers or irritants. Pairs with known and novel markers were found such as HMOX1 and ZFAND2A, ATF3 and PPP1R15A, OXSR1 and HSPA1B, ZFP36 and MAFF. The resulting GDN proved biologically valid as it was well-connected and enriched in known interactions, processes and common regulatory motifs for pairs. Classification accuracy was improved when compared with conventional classifiers. As the dysregulated patterns for heat shock responding genes proved to be distinct from those of other stress genes, we were able to formulate the hypothesis that heat shock genes play a specific role in sensitization, apart from other stress genes. In conclusion, our combined approach creates added value for classification-based toxicogenomics by obtaining novel, well-distinguishing and biologically interesting measures, suitable for the formulation of hypotheses on functional relationships between genes and their relevance for toxicity class differences.

The way forward for assessing the human health safety of cosmetics in the EU - Workshop proceedings.

Although the need for non-animal alternatives has been well recognised for the human health hazard assessment of chemicals in general, it has become especially pressing for cosmetic ingredients due to the full implementation of testing and marketing bans on animal testing under the European Cosmetics Regulation. This means that for the safety assessment of cosmetics, the necessary safety data for both the ingredients and the finished product can be drawn from validated (or scientifically-valid), so-called "Replacement methods". In view of the challenges for safety assessment without recourse to animal test data, the Methodology Working Group of the Scientific Committee on Consumer Safety organised a workshop in February 2019 to discuss the key issues in regard to the use of animal-free alternative methods for the safety evaluation of cosmetic ingredients. This perspective article summarises the outcomes of this workshop and reflects on the state-of-the-art and possible way forward for the safety assessment of cosmetic ingredients for which no experimental animal data exist. The use and optimisation of "New Approach Methodology" that could be useful tools in the context of the "Next Generation Risk Assessment" and the strategic framework for safety assessment of cosmetics were discussed in depth.

Erratum to Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.

In this manuscript, which appeared in ALTEX (2019), 36(4), 682- 699, doi:10.14573/altex.1909271 , the affiliation of Hennicke Kamp should be Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany. Further, the reference to an article by Bal-Price et al. (2015) should have the following doi:10.1007/s00204-015-1464-2 .

An in vitro coculture system for the detection of sensitization following aerosol exposure.

The aim of the study was to develop an in vitro model that mimics the alveolar-capillary barrier and that allows assessment of the respiratory sensitizing potential of respiratory sensitizers. The 3D in vitro model cultured at the air liquid interface consists of alveolar type II epithelial cells (A549), endothelial cells (EA.hy926), macrophage-like cells (PMA-differentiated THP-1) and dendritic-like cells (non-differentiated THP-1). This alveolar model was exposed apically to nebulized chemical respiratory sensitizers (Phthalic Anhydride (PA) and TriMellitic Anhydride (TMA)) or irritants (Methyl Salicylate (MeSa) and Acrolein (Acr)) at concentrations inducing at maximum 25% of cytotoxicity. The exposure to respiratory sensitizers induced dendritic cells activation and a specific cytokine release pattern, while the irritants did not. In addition, the cell surface marker OX40L was determined for dendritic like cells activation to identify high molecular weight allergens. With this in vitro model we can postulate a set of promising markers based on the studied compounds that allow the discrimination of chemical respiratory sensitizers from irritants.

Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.

Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia). The majority of toxicity tests still falls into the category of non-guideline methods. Data from these tests may nevertheless be used to support regulatory decisions or to guide strategies to assess compounds (e.g., drugs, agrochemicals) during research and development if they fulfill basic requirements concerning their relevance, reproducibility and predictivity. Only a method description of sufficient clarity and detail allows interpretation and use of the data. To guide regulators faced with increasing amounts of data from non-guideline studies, the OECD formulated Guidance Document 211 (GD211) on method documentation for the purpose of safety assessment. As GD211 is targeted mainly at regulators, it leaves scientists less familiar with regulation uncertain as to what level of detail is required and how individual questions should be answered. Moreover, little attention was given to the description of the test system (i.e., cell culture) and the steps leading to it being established in the guidance. To address these issues, an annotated toxicity test method template (ToxTemp) was developed (i) to fulfill all requirements of GD211, (ii) to guide the user concerning the types of answers and detail of information required, (iii) to include acceptance criteria for test elements, and (iv) to define the cells sufficiently and transparently. The fully annotated ToxTemp is provided here, together with reference to a database containing exemplary descriptions of more than 20 cell-based tests.

Lactobacillus casei Shirota does not decrease the food allergic response to peanut extract in Brown Norway rats.

Probiotics are claimed to beneficially affect the immune system and their involvement in allergy prevention is being investigated extensively. However, the efficacy of probiotics in allergy prevention remains controversial. We investigated whether the probiotic Lactobacillus casei Shirota (LcS) could modulate the food allergic response against peanut extract (PE) in Brown Norway (BN) rats. For this purpose BN rats were sensitized to PE (0, 1 and 10 mg/(rat d)) by daily oral gavage and the LcS-groups were additionally orally dosed with 1 x 10(9) colony forming units LcS/(rat d). LcS administration had minor effects in animals that were not sensitized. LcS increased Th1-(PE-specific IgG1), whereas the Th1/Th2 ratio based on PE-specific IgG1/PE-specific IgG2a shifted towards Th2 dominance in rats sensitized to PE in the presence of LcS as compared to rats that were sensitized to PE only. LcS stimulated PE-specific IgG2a; but for PE-specific IgE the effect was less clear; whereas there was no overall effect, two rats did not show detectable specific IgE antibodies, whereas the remainder showed significantly increased levels. LcS also resulted in increased numbers of basophilic granulocytes in blood. Furthermore, LcS increased levels of both Th1-(IFN-gamma) and Th2-(IL-4) related cytokines in PE stimulated spleen and mesenteric lymph node (MLN) cells, but predominantly IL-4 levels in the supernatants of both spleens and MLNs. Our study does not support the hypothesis that LcS down-regulates food allergic responses in a BN rat model for food allergy to peanut.