Refine and Strengthen SAR-Based Read-Across by Considering Bioactivation and Modes of Action.

Structure-activity relationship (SAR)-based read-across is an important and effective method to establish the safety of a data-poor target chemical (structure of interest (SOI)) using hazard data from structurally similar source chemicals (analogues). Many methods use quantitative similarity scores to evaluate the structural similarity for searching and selecting analogues as well as for evaluating analogue suitability. However, studies suggest that read-across based purely on structural similarity cannot accurately predict the toxicity of an SOI. As mechanistic data become available, we gain a greater understanding of the mode of action (MOA), the relationship between structures and metabolism/bioactivation pathways, and the existence of "activity cliffs" in chemical chain length, which can improve the analogue rating process. For this purpose, the current work identifies a series of classes of chemicals where a small change at a key position can result in a significant change in metabolism and bioactivation pathways and may eventually result in significant changes in chemical toxicity that have a big impact on the suitability of analogues for read-across. Additionally, a series of SAR-based read-across case studies are presented, which cover a variety of chemical classes that commonly link to different toxic endpoints. The case study results indicate that SAR-based read-across can be refined and strengthened by considering MOAs or proposed reactive metabolite formation pathways, which can improve the overall accuracy, consistency, transparency, and confidence in evaluating analogue suitability.

Workshop report: Challenges faced in developing inhalation thresholds of Toxicological Concern (TTC) - State of the science and next steps.

A challenging step in human risk assessment of chemicals is the derivation of safe thresholds. The Threshold of Toxicological Concern (TTC) concept is one option which can be used for the safety evaluation of substances with a limited toxicity dataset, but for which exposure is sufficiently low. The application of the TTC is generally accepted for orally or dermally exposed cosmetic ingredients; however, these values cannot directly be applied to the inhalation route because of differences in exposure route versus oral and dermal. Various approaches of an inhalation TTC concept have been developed over recent years to address this. A virtual workshop organized by Cosmetics Europe, held in November 2020, shared the current state of the science regarding the applicability of existing inhalation TTC approaches to cosmetic ingredients. Key discussion points included the need for an inhalation TTC for local respiratory tract effects in addition to a systemic inhalation TTC, dose metrics, database building and quality of studies, definition of the chemical space and applicability domain, and classification of chemicals with different potencies. The progress made to date in deriving inhalation TTCs was highlighted, as well as the next steps envisaged to develop them further for regulatory acceptance and use.

Threshold of Toxicological Concern: Extending the chemical space by inclusion of a highly curated dataset for organosilicon compounds.

The Threshold of Toxicological Concern (TTC) for non-genotoxic substances, a risk assessment tool to establish safe exposure levels for chemicals with insufficient toxicological data, is based on the 5th percentile of cumulated distributions of Point of Departures in a high amount of repeat-dose, developmental and reproductive toxicity studies, grouped by Cramer Classes. The lack of organosilicon compounds in this dataset has resulted in regulatory concerns over the applicability of the TTC concept for this chemistry. We collected publicly available, scientifically robust oral repeat-dose and DART studies for 71 organosilicon substances for inclusion in the existing TTC dataset, using criteria for evaluation of studies and derivation of points of departure analogous to the Munro and COSMOS TTC publications. The resulting 5th percentile of this dataset was 13-fold higher than the 5th percentile for Cramer Class III compounds reported by Munro (which is the default for silicon-containing substances). Both the existing TTC for Cramer Class III compounds from Munro (1.5 µg/kg bw/day) and the COSMOS TTC (2.3 µg/kg bw/day), recommended by the SCCS for cosmetics-related substances, provide a conservative and sufficiently protective approach for this class of chemistry.

The Threshold of Toxicological Concern (TTC) is a pragmatic tool for the safety assessment: Case studies of cosmetic ingredients with low consumer exposure.

The Threshold of Toxicological Concern (TTC) is an internationally accepted pragmatic and conservative tool for the safety assessment of substances, which is used in a wide range of regulatory contexts. The TTC approach produces human exposure threshold values (TTC values) originally derived by Munro from oral toxicity data on cancer and non-cancer toxicity endpoints. This database has been recently substantially enlarged by the COSMOS database, an enhanced oral non-cancer TTC dataset on a larger chemical domain, thereby resulting in a new, transparent and public TTC database also including 552 cosmetics-related chemicals. The 5th percentile point of departure value for each Cramer Class was determined, from which human exposure TTC values have been derived. The combined COSMOS/Munro dataset provided TTC values of 46, 6.2 and 2.3 µg/kg bw/day for Cramer Classes I, II or III, respectively. In order to demonstrate the diverse scope and successful application of the TTC concept to cosmetic ingredients including hair dyes, fragrances and plant-derived ingredients, Cosmetics Europe has prepared several case studies. Overall, the TTC concept is not only useful to replace animal testing but can also successfully be applied to the safety evaluation of cosmetic ingredients in the marketed formulas with low human exposure.

Building institutional resilience in the context of climate change in Aurora, Philippines.

The role of local government units (LGUs) in disaster resilience is crucial for a hazard-prone country such as the Philippines. Although the country has its own institutional framework on disaster risk reduction, a number of issues limit LGUs' potential to perform its role. This study focused on building institutional resilience of LGUs towards building climate risk resilience in Aurora, Philippines by engaging key actors in the formulation of Local Climate Change Action Plans (LCCAP). The study adopted the shared learning process from the Climate Resilience Framework (CRF) to strengthen partnership and implement capacity building activities, aimed at developing the Climate and Disaster Risk Assessment (CDRA) and LCCAP beyond compliance. An institutional capacity assessment was administered through a survey involving 87 members of the Technical Working Group (TWG) from eight municipalities and provincial government. Institutional capacity was measured using 70 indicators representing access rights and entitlements, information flows, decision-making processes, application of new knowledge, capacity to anticipate risk, capacity to respond, as well as capacity to recover and change. Data were analyzed using descriptive statistics. Both Spearman Correlation and Cramer's V determined the interrelationship between socio-demographic variables and institutional characteristics. Results revealed that the LGUs performed better in risk response and management. A strong correlation between expertise and position vis-à-vis all resilient institution metrics was also observed, while gender is moderately correlated with all parameters except access rights and entitlements. Three key areas, not adequately articulated in current literature, need to be improved to enhance institutional resilience towards climate and disaster risks, namely: staffing and human resource; access to financial support from other sources; and development of knowledge management systems.

An interim internal Threshold of Toxicologic Concern (iTTC) for chemicals in consumer products, with support from an automated assessment of ToxCast™ dose response data.

Additional non-animal methods are urgently needed to meet regulatory and animal welfare goals. TTC is a broadly used risk assessment tool. TTC based on external dose has limited utility for multi-route exposure and some types of structure activity relationship assessments. An internal TTC (iTTC), where thresholds are based on blood concentration, would extend the applicability of TTC. While work is on-going to develop robust iTTC thresholds, we propose an interim conservative iTTC. Specifically, an interim iTTC of 1 µM, supported by the published experience of the pharmaceutical industry, a literature review of non-drug chemical/receptor interactions, and analysis of ToxCast™ data. ToxCast™ data were used to explore activity versus the 1 µM interim iTTC and recommendations for the analysis and interpretation of HTS data. Test concentration-based points of departure were classified to identify quality of fit to the Hill Model. We identified, for exclusion from the approach, estrogen receptor and androgen receptor targets as potent chemical/receptor interactions potentially associated with low dose exposure to non-pharmaceutical active ingredients in addition to the original TTC exclusions. With these exclusions, we conclude that a 1 µM plasma concentration is unlikely to be associated with significant biological effects from chemicals not intentionally designed for biological activity.

Bolstering the existing database supporting the non-cancer Threshold of Toxicological Concern values with toxicity data on fragrance-related materials.

The use of threshold of toxicological concern (TTC) supports the safety assessment of exposure to low levels of chemicals when toxicity data are limited. The Research Institute for Fragrance Materials (RIFM) delivers safety assessments for fragrance materials that result in safe products for consumer use. A major goal for the RIFM safety assessment program is to invest in alternative methods to animal testing for use in assessment of fragrance materials. This includes use of TTC, which provides a pragmatic approach for safety evaluation of fragrance materials in the absence of chemical-specific toxicity data and reduces the need to generate new animal data. To bolster the TTC approach for support of fragrance materials and specifically to strengthen the Cramer class II threshold, the RIFM database was reviewed with a goal of identifying fragrance materials with data that can be added to the existing TTC databases. The RIFM database identified a total of 476 chemicals that were added to the existing TTC databases. The chemicals were then individually assigned a Cramer class and 238, 76 and 162 chemicals in Cramer class I, II and III respectively were identified. The RIFM-TTC dataset was then combined with the COSMOS-Federated TTC dataset for a total of 421, 111 and 795 chemicals in Cramer class I, II and III respectively. The combined dataset further expands the chemical space thereby providing more robust 5th percentile thresholds. Moreover, the combined dataset bolsters the threshold for Cramer class II to include a total of 111 chemicals which is an improvement over the original (Munro) TTC dataset which only included 28 chemicals in Cramer Class II and the COSMOS Federated dataset which had 40 chemicals. This allows for a more reliable and robust 5th percentile NOAEL value for Cramer class II chemicals of 1.27 mg/kg bw/day. The 5th percentile NOAELs for Cramer class I, II and III from the combined dataset are 4.91, 1.27 and 0.29 mg/kg bw/day, which supports the threshold values derived from the original Munro dataset. This work confirms the adequacy of the existing TTC values and provides further support for the use of TTC as a tool to conduct safety assessments for fragrance materials. It further opens the future possibility of updating the existing values with more robust TTC values for fragrance and cosmetic materials.

Temporal and spatial trends in freshwater fish tissue mercury concentrations associated with mercury emissions reductions.

Mercury (Hg) concentrations were monitored from 1999 to 2011 in largemouth bass (LMB) and yellow perch (YP) in 23 lakes in Massachusetts USA during a period of significant local and regional Hg emissions reductions. Average LMB tissue Hg concentration decreases of 44% were seen in 13 of 16 lakes in a regional Hg "hotspot" area. YP in all lakes sampled in this area decreased 43% after the major emissions reductions. Comparative decreases throughout the remainder of the state were 13% and 19% for LMB and YP respectively. Annual tissue mercury concentration rate decreases were 0.029 (LMB) and 0.016 mg Hg/kg/yr (YP) in the hotspot. In lakes around the rest of the state, LMB showed no trend and YP Hg decreased 0.0068 mg Hg/kg/yr. Mercury emissions from major point sources in the hotspot area decreased 98%, and 93% in the rest of the state from the early 1990s to 2008. The significant declines in fish Hg concentrations in many lakes occurred over the second half of a two decade decrease in Hg emissions primarily from municipal solid waste combustors and, secondarily, from other combustion point sources. In addition to the substantial Hg emissions reductions achieved in Massachusetts, further regional, national and global emissions reductions are needed for fish Hg levels to decrease below fish consumption advisory levels.

Comparison between endocrine activity assessed using ToxCast/Tox21 database and human plasma concentration of sunscreen active ingredients/UV filters.

Sunscreen products are composed of ultraviolet (UV) filters and formulated to reduce exposure to sunlight thereby lessening skin damage. Concerns have been raised regarding the toxicity and potential endocrine disrupting (ED) effects of UV filters. The ToxCast/Tox21 program, that is, CompTox, is a high-throughput in vitro screening database of chemicals that identify adverse outcome pathways, key events, and ED potential of chemicals. Using the ToxCast/Tox21 database, octisalate, homosalate, octocrylene, oxybenzone, octinoxate, and avobenzone, 6 commonly used organic UV filters, were found to have been evaluated. These UV filters showed low potency in these bioassays with most activity detected above the range of the cytotoxic burst. The pathways that were most affected were the cell cycle and the nuclear receptor pathways. Most activity was observed in liver and kidney-based bioassays. These organic filters and their metabolites showed relatively weak ED activity when tested in bioassays measuring estrogen receptor (ER), androgen receptor (AR), thyroid receptor, and steroidogenesis activity. Except for oxybenzone, all activity in the endocrine assays occurred at concentrations greater than the cytotoxic burst. Moreover, except for oxybenzone, plasma concentrations (Cmax) measured in humans were at least 100× lower than bioactive (AC50/ACC) concentrations that produced a response in ToxCast/Tox21 assays. These data are consistent with in vivo animal/human studies showing weak or negligible endocrine activity. In sum, when considered as part of a weight-of-evidence assessment and compared with measured plasma concentrations, the results show these organic UV filters have low intrinsic biological activity and risk of toxicity including endocrine disruption in humans.

Identifying chemicals based on receptor binding/bioactivation/mechanistic explanation associated with potential to elicit hepatotoxicity and to support structure activity relationship-based read-across.

The liver is the most common target organ in toxicology studies. The development of chemical structural alerts for identifying hepatotoxicity will play an important role in in silico model prediction and help strengthen the identification of analogs used in structure activity relationship (SAR)- based read-across. The aim of the current study is development of an SAR-based expert-system decision tree for screening of hepatotoxicants across a wide range of chemistry space and proposed modes of action for clustering of chemicals using defined core chemical categories based on receptor-binding or bioactivation. The decision tree is based on âˆ¼ 1180 different chemicals that were reviewed for hepatotoxicity information. Knowledge of chemical receptor binding, metabolism and mechanistic information were used to group these chemicals into 16 different categories and 102 subcategories: four categories describe binders to 9 different receptors, 11 categories are associated with possible reactive metabolites (RMs) and there is one miscellaneous category. Each chemical subcategory has been associated with possible modes of action (MOAs) or similar key structural features. This decision tree can help to screen potential liver toxicants associated with core structural alerts of receptor binding and/or RMs and be used as a component of weight of evidence decisions based on SAR read-across, and to fill data gaps.

Chemical composition determination and evaluation of the antibacterial activity of essential oils from Ruellia asperula (Mart. Ex Ness) Lindau and Ruellia paniculata L. against oral streptococci.

This study investigated the chemical composition and evaluated the antibacterial and antibiofilm activities of essential oils (EOs) extracted from Ruellia asperula (EORA) and Ruellia paniculata (EORP) against oral streptococci. The EO constituents were analyzed by gas chromatography/mass spectrometry. The antimicrobial potential of EOs was evaluated using the minimum inhibitory concentration, minimum bactericidal concentration, and time-kill determination. Furthermore, the quantification of total biomass and the number of viable cells in the biofilms were evaluated. The major constituents of EORA were cariophylla-4(12)-8-(13)-dien-5ß-ol (14.1%), (ß)-caryophyllene (22.7%), and caryophyllene oxide (29.4%). For EORP, the major constituents were (ß)-caryophyllene (11.0%), spathulenol (13.1%), and δ-amorphene (14.9%). The tested EOs exhibited antibacterial activity against planktonic growth and biofilm formation. Thus, the EOs from R. asperula and R. paniculata prove to be promising alternatives for bacterial growth control and biofilm formation prevention of oral streptococci.

Casbane diterpene as a promising natural antimicrobial agent against biofilm-associated infections.

Croton nepetaefolius is a native plant from northeastern Brazil that belongs to the Euphorbiaceae family. The biological action of this plant has been extensively explored, being the secondary metabolites responsible for its properties alkaloids, diterpenes, and triterpenes. This study aimed to evaluate the ability of casbane diterpene (CD), isolated from the ethanolic extract of C. nepetaefolius, to inhibit microbial growth and biofilm formation of several clinical relevant species (bacteria and yeasts). It was found that CD possessed biocidal and biostatic activity against the majority of the species screened, with minimal active concentrations ranging between 125 and 500 µg/mL. In addition, it was observed that biofilm formation was inhibited even when the planktonic growth was not significantly affected. In conclusion, CD showed potential to be a natural tool for the treatment of diseases caused by different infectious microorganisms.

Vaccine hesitancy among general practitioners in Southern France and their reluctant trust in the health authorities.

Purpose: Vaccine hesitancy is common in France, including among general practitioners (GPs).  We aimed to understand vaccine hesitant GPs' views towards vaccines. Method: We conducted in-depth interviews that were thematically analysed. Result: We found that, facilitated by health scandals and vaccine controversies-that according to participants were not effectively handled by health authorities-the implicit contract existing between health authorities and GPs has been ruptured. This contract implies that health authorities support GPs in making vaccine recommendations by addressing GPs' own concerns, providing them with adequate and up-to-date information and advice, and involving them in vaccine decision-making. In turn, GPs encourage vaccination to reach vaccine coverage targets. Conclusion: The rupture of this implicit contract has led to a breach in trust in the health authorities and the vaccines that they recommend.

Hepatocyte growth factor inhibits epithelial to myofibroblast transition in lung cells via Smad7.

Idiopathic pulmonary fibrosis is a lethal parenchymal lung disease characterized by denudation of the lung epithelium, fibroblast proliferation, and collagen deposition. Cellular changes underlying disease progression involve injury to alveolar epithelial cells, epithelial to mesenchymal transition, proliferation of alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts and of fibroblasts resulting in enhanced deposition of extracellular matrix proteins. Hepatocyte growth factor (HGF) inhibits progression of bleomycin-induced pulmonary fibrosis in mice. The mechanism underlying the inhibitory effect of HGF was investigated in an in vitro model. We show that HGF markedly antagonizes basal and transforming growth factor (TGF)-beta-induced expression of myofibroblast markers such as alpha-SMA, collagen type 1, and fibronectin in rat alveolar epithelial cells. HGF also inhibited TGF-beta-induced alpha-SMA expression in primary murine alveolar epithelial cells. Since TGF-beta is known to regulate alpha-SMA expression, the effect of HGF on components of TGF-beta signaling was investigated. HGF induced expression of Smad7, an inhibitor of TGF-beta signaling, in a mitogen-activated protein kinase-dependent manner. HGF also induced the nuclear export of Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1) to the cytoplasm. HGF-dependent decrease in alpha-SMA was abolished with specific siRNAs targeted to Smad7. Thus, induction of Smad7 by HGF serves to limit acquisition of the myofibroblast phenotype in alveolar epithelial cells.

Cytotoxic diterpenoids from Croton argyrophylloides.

Two new diterpenes, 1 and 2, together with the known ent-15-oxo-kaur-16-en-18-oic acid (3), were isolated from the bark of Croton argyrophylloides. The structural characterization of 1 and 2 was determined on the basis of spectroscopic data interpretation. The cytotoxicity of each compound was evaluated against HL-60 (leukemia), MDAMB-435 (melanoma), SF-295 (glioblastoma), and HCT-8 (colon carcinoma) human tumor cell lines and against human peripheral blood mononuclear cells. The hemolytic potential in mouse erythrocytes was also tested for 1-3.

Pure-Tone Frequency Discrimination in Preschoolers, Young School-Age Children, and Adults.

Purpose: Published data indicate nearly adultlike frequency discrimination in infants but large child-adult differences for school-age children. This study evaluated the role that differences in measurement procedures and stimuli may have played in the apparent nonmonotonicity. Frequency discrimination was assessed in preschoolers, young school-age children, and adults using stimuli and procedures that have previously been used to test infants. Method: Listeners were preschoolers (3-4 years), young school-age children (5-6 years), and adults (19-38 years). Performance was assessed using a single-interval, observer-based method and a continuous train of stimuli, similar to that previously used to evaluate infants. Testing was completed using 500- and 5000-Hz standard tones, fixed within a set of trials. Thresholds for frequency discrimination were obtained using an adaptive, two-down one-up procedure. Adults and most school-age children responded by raising their hands. An observer-based, conditioned-play response was used to test preschoolers and those school-age children for whom the hand-raise procedure was not effective for conditioning. Results: Results suggest an effect of age and frequency on thresholds but no interaction between these 2 factors. A lower proportion of preschoolers completed training compared with young school-age children. For those children who completed training, however, thresholds did not improve significantly with age; both groups of children performed more poorly than adults. Performance was better for the 500-Hz standard frequency compared with the 5000-Hz standard frequency. Conclusions: Thresholds for school-age children were broadly similar to those previously observed using a forced-choice procedure. Although there was a trend for improved performance with increasing age, no significant age effect was observed between preschoolers and school-age children. The practice of excluding participants based on failure to meet conditioning criteria in an observer-based task could contribute to the relatively good performance observed for preschoolers in this study and the adultlike performance previously observed in infants.

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

Tocoyena sellowiana extract decreases bone loss in an experimental model of periodontitis in rats: Putative role for cyclooxygenase-2 and IL-1? inhibition.

Tocoyena sellowiana (Cham. & Schltdl.) K.Schum is one of the most important families of Brazilian medicinal plants. This study aimed to evaluate the effect of Tocoyena sellowiana (Cham. & Schltdl.) K.Schum ethanolic extract in a pre-clinical trial of periodontitis and to investigate possible mechanisms underlying such effects. Periodontitis was induced in Wistar rats by placing a nylon thread ligature around second upper left molars for 11 days. Rats received (per os) Tocoyena sellowiana (0.1, 1 or 10?mg?kg) or vehicle 1?h before ligature and daily until day 11. Macroscopic, histopathological, and COX-2 immunohistochemical analyses were performed to evaluate the periodontium. The gingival tissue was used to quantify the myeloperoxidase (MPO) activity and interleukin (IL)-1? levels by ELISA. Blood samples were collected to evaluate bone-specific alkaline phosphatase (BALP), the dosage of creatinine, aspartate and alanine transaminases. The liver, kidneys, spleen, and body mass variations were also evaluated. Tocoyena sellowiana decreased bone loss, reduced MPO, IL-1? levels as well as COX-2 immunostaining, and increased BALP activity. Moreover, Tocoyena sellowiana did not alter organs nor body weight. Tocoyena sellowiana reduced bone loss in rats and its efficacy was at least partially dependent upon both IL-1? and cyclooxygenase-2 inhibition.

Studies on the cytotoxicity of miscellaneous compounds from Eupatorium betonicaeforme (D.C.) Baker (Asteraceae).

A detailed study on the cytotoxic effects of five known constituents isolated from the flowers and roots of Eupatorium betonicaeforme is reported, including 2,2-dimethyl-6-vinylchroman-4-one (1), 2-senecioyl-4-vinylphenol (2), 6-acetyl-2,2-dimethylchroman-4-one (3), (4E)-8beta-angeloyloxy-9beta,10beta-dihydroxy-1-oxogermacra-4,11(13)-dien-12,6alpha-olide (4), and 3beta-hydroxyicosan-1,5beta-olide (5). The sesquiterpene lactone 4 exhibited the highest cytotoxicity, with IC50 values ranging from 3.9 to 9.9 microM, showing some degree of cell selectivity. The antiproliferative activity of 4 was examined towards HL-60 cells, and found to diminish cell viability in a dose-dependent manner. Moreover, at all concentrations tested, there was a decrease in the number of cells capable of incorporating 5-bromo-2'-deoxyuridine (BrdU), indicating disruption of DNA synthesis. The morphological changes induced by 4 were compatible with apoptotic cell death. This work, thus, corroborates the anticancer potential of Eupatorium secondary metabolites.