Published Papers

England et al. (2024). The key characteristics of cardiotoxicity for the pervasive pollutant phenanthrene. Journal of Hazardous Materials. Volume 469, 5 May 2024, 133853. PMID: 38503207. DOI: 10.1016/j.jhazmat.2024.133853.

Abstract. The key characteristic (KCs) framework has been used previously to assess the carcinogenicity and cardiotoxicity of various chemical and pharmacological agents. Here, the 12 KCs of cardiotoxicity are used to evaluate the previously reported cardiotoxicity of phenanthrene (Phe), a tricyclic polycyclic aromatic hydrocarbon (PAH), and major component of fossil fuel-derived air pollution. Phe is a semi-volatile pollutant existing in both the gas phase and particle phase through adsorption onto or into particulate matter (PM). Phe can translocate across the airways and gastrointestinal tract into the systemic circulation, enabling body-wide effects. Our evaluation based on a comprehensive literature review, indicates Phe exhibits 11 of the 12 KCs for cardiotoxicity. These include adverse effects on cardiac electromechanical performance, the vasculature and endothelium, immunomodulation and oxidative stress, and neuronal and endocrine control. Environmental agents that have similarly damaging effects on the cardiovascular system are heavily regulated and monitored, yet globally there is no air quality regulation specific for PAHs like Phe. Environmental monitoring of Phe is not the international standard with benzo[a]pyrene being frequently used as a proxy despite the two PAH species exhibiting significant differences in sources, concentration variations and toxic effects. The evidence summarised in this evaluation highlights the need to move away from proxied PAH measurements and develop a monitoring network capable of measuring Phe concentration. It also stresses the need to raise awareness amongst the medical community of the potential cardiovascular impact of PAH exposure. This will allow the production of mitigation strategies and possibly the development of new policies for the protection of the societal groups most vulnerable to cardiovascular disease.

Rusyn and Wright (2024). Ten Years of Using Key Characteristics of Human Carcinogens to Organize and Evaluate Mechanistic Evidence in IARC Monographs on the Identification of Carcinogenic Hazards to Humans: Patterns and Associations. Toxicol Sci 2024 Feb 28;198(1):141-154.   PMID: 38141214. DOI: 10.1093/toxsci/kfad134

Abstract. Systematic review and evaluation of mechanistic evidence using the Key Characteristics approach was proposed by the International Agency for Research on Cancer (IARC) in 2012 and used by the IARC Monographs Working Groups since 2015. Key Characteristics are ten features of agents known to cause cancer in humans. From 2015 to 2022 a total of 19 Monographs (73 agents combined) used Key Characteristics for cancer hazard classification. We hypothesized that a retrospective analysis of applications of the Key Characteristics approach to cancer hazard classification using heterogenous mechanistic data on diverse agents would be informative for systematic reviews in decision-making. We extracted information on the conclusions, data types, and the role mechanistic data played in the cancer hazard classification from each Monograph. Statistical analyses identified patterns in the use of Key Characteristics, as well as trends and correlations among Key Characteristics, data types, and ultimate decisions. Despite gaps in data for many agents and Key Characteristics, several significant results emerged. Mechanistic data from in vivo animal, in vitro animal and in vitro human studies was most impactful for concluding that an agent could cause cancer via a Key Characteristic. To exclude the involvement of a Key Characteristic, data from large-scale systematic in vitro testing programs such as ToxCast were most informative. Overall, increased availability of systemized data streams, such as human in vitro data, would provide the basis for confident conclusions about both positive and negative associations and constitute relative contributions of various sources of weight in expert judgments.

Ricker et al., (2024). Application of the Key Characteristics of Carcinogens to Bisphenol A. Int J Toxicol 2024 Jan 10:10915818231225161. doi: 10.1177/10915818231225161. PMID: 38204208. DOI: 10.1177/10915818231225161

Abstract. The ten key characteristics (KCs) of carcinogens are based on characteristics of known human carcinogens and encompass many types of endpoints. We propose that an objective review of the large amount of cancer mechanistic evidence for the chemical bisphenol A (BPA) can be achieved through use of these KCs. A search on metabolic and mechanistic data relevant to the carcinogenicity of BPA was conducted and web-based software tools were used to screen and organize the results. We applied the KCs to systematically identify, organize, and summarize mechanistic information for BPA, and to bring relevant carcinogenic mechanisms into focus. For some KCs with very large data sets, we utilized reviews focused on specific endpoints. Over 3000 studies for BPA from various data streams (exposed humans, animals, in vitro and cell-free systems) were identified. Mechanistic data relevant to each of the ten KCs were identified, with receptor-mediated effects, epigenetic alterations, oxidative stress, and cell proliferation being especially data rich. Reactive and bioactive metabolites are also associated with a number of KCs. This review demonstrates how the KCs can be applied to evaluate mechanistic data, especially for data-rich chemicals. While individual entities may have different approaches for the incorporation of mechanistic data in cancer hazard identification, the KCs provide a practical framework for conducting an objective examination of the available mechanistic data without a priori assumptions on mode of action. This analysis of the mechanistic data available for BPA suggests multiple and inter-connected mechanisms through which this chemical can act.

Kay et al., (2024). Application of the Key Characteristics Framework to Identify Potential Breast Carcinogens Using Publicly Available in Vivo, in Vitro, and in Silico Data. Environ Health Perspect 2024 Jan;132(1):17002. doi: 10.1289/EHP13233. Epub 2024 Jan 10. PMCID: PMC10777819. DOI: 10.1289/EHP13233

Abstract. Background: Chemicals that induce mammary tumors in rodents or activate estrogen or progesterone signaling are likely to increase breast cancer (BC) risk. Identifying chemicals with these activities can prompt steps to protect human health. Objectives: We compiled data on rodent tumors, endocrine activity, and genotoxicity to assess the key characteristics (KCs) of rodent mammary carcinogens (MCs), and to identify other chemicals that exhibit these effects and may therefore increase BC risk. Methods: Using authoritative databases, including International Agency for Research on Cancer (IARC) Monographs and the US Environmental Protection’s (EPA) ToxCast, we selected chemicals that induce mammary tumors in rodents, stimulate estradiol or progesterone synthesis, or activate the estrogen receptor (ER) in vitro. We classified these chemicals by their genotoxicity and strength of endocrine activity and calculated the overrepresentation (enrichment) of these KCs among MCs. Finally, we evaluated whether these KCs predict whether a chemical is likely to induce mammary tumors. Results: We identified 279 MCs and an additional 642 chemicals that stimulate estrogen or progesterone signaling. MCs were significantly enriched for steroidogenicity, ER agonism, and genotoxicity, supporting the use of these KCs to predict whether a chemical is likely to induce rodent mammary tumors and, by inference, increase BC risk. More MCs were steroidogens than ER agonists, and many increased both estradiol and progesterone. Enrichment among MCs was greater for strong endocrine activity vs. weak or inactive, with a significant trend. Discussion: We identified hundreds of compounds that have biological activities that could increase BC risk and demonstrated that these activities are enriched among MCs. We argue that many of these should not be considered low hazard without investigating their ability to affect the breast, and chemicals with the strongest evidence can be targeted for exposure reduction. We describe ways to strengthen hazard identification, including improved assessments for mammary effects, developing assays for more KCs, and more comprehensive chemical testing.

Keller et al. (2023). In silico approaches in carcinogenicity hazard assessment: case study of pregabalin, a nongenotoxic mouse carcinogen. Frontier Toxicol 2023 Nov 13:5:1234498. PMCID: PMC10679394. DOI: 10.3389/ftox.2023.1234498

Abstract. In silico toxicology protocols are meant to support computationally-based assessments using principles that ensure that results can be generated, recorded, communicated, archived, and then evaluated in a uniform, consistent, and reproducible manner. We investigated the availability of in silico models to predict the carcinogenic potential of pregabalin using the ten key characteristics of carcinogens as a framework for organizing mechanistic studies. Pregabalin is a single-species carcinogen producing only one type of tumor, hemangiosarcomas in mice via a nongenotoxic mechanism. The overall goal of this exercise is to test the ability of in silico models to predict nongenotoxic carcinogenicity with pregabalin as a case study. The established mode of action (MOA) of pregabalin is triggered by tissue hypoxia, leading to oxidative stress (KC5), chronic inflammation (KC6), and increased cell proliferation (KC10) of endothelial cells. Of these KCs, in silico models are available only for selected endpoints in KC5, limiting the usefulness of computational tools in prediction of pregabalin carcinogenicity. KC1 (electrophilicity), KC2 (genotoxicity), and KC8 (receptor-mediated effects), for which predictive in silico models exist, do not play a role in this mode of action. Confidence in the overall assessments is considered to be medium to high for KCs 1, 2, 5, 6, 7 (immune system effects), 8, and 10 (cell proliferation), largely due to the high-quality experimental data. In order to move away from dependence on animal data, development of reliable in silico models for prediction of oxidative stress, chronic inflammation, immunosuppression, and cell proliferation will be critical for the ability to predict nongenotoxic compound carcinogenicity.

Zahm et al., (2023). Carcinogenicity of perfluorooctanoic acid and perfluorooctanesulfonic acid. Lancet Oncol 2024 Jan;25(1):16-17. PMID: 38043561. DOI: 10.1016/S1470-2045(23)00622-8.

Abstract. None available.

Berridge et al., (2023). Enabling novel paradigms: A biological questions-based approach to human chemical hazard and drug safety assessment. Toxicol Sci 2023 Dec 22:kfad124. PMID: 38134427. DOI: 10.1093/toxsci/kfad124

Abstract. Throughput needs, costs of time and resources, and concerns about the use of animals in hazard and safety assessment studies are fueling a growing interest in adopting new approach methodologies for use in product development and risk assessment. However, current efforts to define “next-generation risk assessment” vary considerably across commercial and regulatory sectors, and an a priori definition of the biological scope of data needed to assess hazard is generally lacking. We propose that the absence of clearly defined questions that can be answered during hazard assessment is the primary barrier to the generation of a paradigm flexible enough to be used across varying product development and approval decision contexts. Herein, we propose a biological questions-based approach (BQBA) for hazard and safety assessment to facilitate fit-for-purpose method selection and more efficient evidence-based decision making. The key pillars of this novel approach are bioavailability, bioactivity, adversity, and susceptibility. This BQBA is compared with current hazard approaches and is applied in scenarios of varying pathobiological understanding and/or regulatory testing requirements. To further define the paradigm and key questions that allow better prediction and characterization of human health hazard, a multidisciplinary collaboration among stakeholder groups should be initiated.

Borgert CJ (2023). Issue analysis: key characteristics approach for identifying endocrine disruptors. Arch Toxicol 97(10):2819-2822.

PMCID: PMC10474976. DOI: 10.1007/s00204-023-03568-3

Abstract: For more than a decade, weight of evidence (WoE) evaluations have been the standard method for determining whether a chemical meets the definition of an endocrine disrupting chemical (EDC). WoE methods consider all data pertinent to satisfying the EDC definition and evaluating those data with respect to relevance, reliability, strength, and coherence with established endocrine physiology and pharmacology. A new approach for identifying EDC hazards has been proposed that organizes and evaluates data according to ten so-called “Key Characteristics (KCs) of EDCs”. The approach claims to address the lack of a widely accepted, systematic approach for identifying EDC hazards, but completely ignores the WoE literature for EDCs. In contrast to WoE methods, the KC approach fails to apply the consensus definition of EDC and is not amenable to empirical testing or validation, is fungible and ensures inconsistent and unreliable results, ignores principles of hormone action and characteristics of dose-response in endocrine pharmacology and toxicology, lacks a means of distinguishing endocrine-mediated from non-endocrine mediated mechanisms, lacks a means to reach a negative conclusion about a chemical’s EDC properties or to distinguish EDCs from non-EDCs, and provides no means for developing a valid consensus among experts nor provides a means of resolving conflicting interpretations of data. Instead of shortcuts like the KC approach, which are prone to bias, error, and arbitrary conclusions, identifying EDCs should rely on WoE evaluations that supply the critical components and scientific rigor lacking in the proposed KCs for EDCs.

 Meek, MEB and Wikoff, D (2023). The need for good practice in the application of mechanistic constructs in hazard and risk assessment. Toxicol Sci 194(1):13-22.

PMID: 37074944. DOI: 10.1093/toxsci/kfad039

Abstract: A series of recent and proposed workshops address the interface between key characteristics and mechanistic pathway descriptions (adverse outcome pathways and mode of action) to identify commonalities and potential for complementary application. Informed by different communities, these constructs have collective potential to increase confidence to support the application of mechanistic data in hazard assessment. This forum article summarizes concepts, introduces evolving understanding, and invites future collaboration to contribute to better common understanding and development of good practice in the use of mechanistic data in hazard assessment.

 Muncke J et al., (2023). A vision for safer food contact materials: Public health concerns as drivers for improved testing. Environ Int 180:108161.

PMID: 37758599. DOI: 10.1016/j.envint.2023.108161

Abstract: Food contact materials (FCMs) and food contact articles are ubiquitous in today’s globalized food system. Chemicals migrate from FCMs into foodstuffs, so called food contact chemicals (FCCs), but current regulatory requirements do not sufficiently protect public health from hazardous FCCs because only individual substances used to make FCMs are tested and mostly only for genotoxicity while endocrine disruption and other hazard properties are disregarded. Indeed, FCMs are a known source of a wide range of hazardous chemicals, and they likely contribute to highly prevalent non-communicable diseases. FCMs can also include non-intentionally added substances (NIAS), which often are unknown and therefore not subject to risk assessment. To address these important shortcomings, we outline how the safety of FCMs may be improved by (1) testing the overall migrate, including (unknown) NIAS, of finished food contact articles, and (2) expanding toxicological testing beyond genotoxicity to multiple endpoints associated with non-communicable diseases relevant to human health. To identify mechanistic endpoints for testing, we group chronic health outcomes associated with chemical exposure into Six Clusters of Disease (SCOD) and we propose that finished food contact articles should be tested for their impacts on these SCOD. Research should focus on developing robust, relevant, and sensitive in-vitro assays based on mechanistic information linked to the SCOD, e.g., through Adverse Outcome Pathways (AOPs) or Key Characteristics of Toxicants. Implementing this vision will improve prevention of chronic diseases that are associated with hazardous chemical exposures, including from FCMs.

Rana, I et al., (2023). Mapping the key characteristics of carcinogens for glyphosate and its formulations: A systematic review. Chemosphere 339:139572.

PMID: 37474029. DOI: 10.1016/j.chemosphere.2023.139572

Abstract: Glyphosate was classified as a probable human carcinogen (Group 2A) by the International Agency for Research on Cancer (IARC) partially due to strong mechanistic evidence in 2015. Since then, numerous studies of glyphosate and its formulations (GBF) have emerged. These studies can be evaluated for cancer hazard identification with the newly described ten key characteristics (KC) of carcinogens approach. Our objective was to assess all in vivo, ex vivo, and in vitro mechanistic studies of human and experimental animals (mammals) that compared exposure to glyphosate/GBF with low/no exposure counterparts for evidence of the ten KCs. A protocol with our methods adhering to PRISMA guidelines was registered a priori (INPLASY202180045). Two blinded reviewers screened all in vivo, ex vivo, and in vitro studies of glyphosate/GBF exposure in humans/mammals reporting any KC-related outcome available in PubMed before August 2021. Studies that met inclusion criteria underwent data extraction conducted in duplicate for each KC outcome reported along with key aspects of internal/external validity, results, and reference information. These data were used to construct a matrix that was subsequently analyzed in the program R to conduct strength of evidence and quality assessments. Of the 2537 articles screened, 175 articles met inclusion criteria, from which we extracted >50,000 data points related to KC outcomes. Data analysis revealed strong evidence for KC2, KC4, KC5, KC6, KC8, limited evidence for KC1 and KC3, and inadequate evidence for KC7, KC9, and KC10. Notably, our in-depth quality analyses of genotoxicity (KC2) and endocrine disruption (KC8) revealed strong and consistent positive findings. For KC2, we found: 1) studies conducted in humans and human cells provided stronger positive evidence than counterpart animal models; 2) GBF elicited a stronger effect in both human and animal systems when compared to glyphosate alone; and 3) the highest quality studies in humans and human cells consistently revealed strong evidence of genotoxicity. Our analysis of KC8 indicated that glyphosate’s ability to modulate hormone levels and estrogen receptor activity is sensitive to both exposure concentration and formulation. The modulations observed provide clear evidence that glyphosate interacts with receptors, alters receptor activation, and modulates the levels and effects of endogenous ligands (including hormones). Our findings strengthen the mechanistic evidence that glyphosate is a probable human carcinogen and provide biological plausibility for previously reported cancer associations in humans, such as non-Hodgkin lymphoma. We identified potential molecular interactions and subsequent key events that were used to generate a probable pathway to lymphomagenesis.

 Rusyn, I and Wright, A (2023). Ten Years of Using Key Characteristics of Human Carcinogens to Organize and Evaluate Mechanistic Evidence in IARC Monographs on the Identification of Carcinogenic Hazards to Humans: Patterns and Associations. bioRxiv 2023.07.11.548354.

PMCID: PMC10369858. DOI: 10.1101/2023.07.11.548354

Abstract: Systematic review and evaluation of the mechanistic evidence only recently been instituted in cancer hazard identification step of decision-making. One example of organizing and evaluating mechanistic evidence is the Key Characteristics approach of the International Agency for Research on Cancer (IARC) Monographs on the Identification of Carcinogenic Hazards to Humans. The Key Characteristics of Human Carcinogens were proposed almost 10 years ago and have been used in every IARC Monograph since 2015. We investigated the patterns and associations in the use of Key Characteristics by the independent expert Working Groups. We examined 19 Monographs (2015-2022) that evaluated 73 agents. We extracted information on the conclusions by each Working Group on the strength of evidence for agent-Key Characteristic combinations, data types that were available for decisions, and the role mechanistic data played in the final cancer hazard classification. We conducted both descriptive and association analyses within and across data types. We found that IARC Working Groups were cautious when evaluating mechanistic evidence: for only ∼13% of the agents was strong evidence assigned for any Key Characteristic. Genotoxicity and cell proliferation were most data-rich, while little evidence was available for DNA repair and immortalization Key Characteristics. Analysis of the associations among Key Characteristics revealed that only chemical’s metabolic activation was significantly co-occurring with genotoxicity and cell proliferation/death. Evidence from exposed humans was limited, while mechanistic evidence from rodent studies in vivo was often available. Only genotoxicity and cell proliferation/death were strongly associated with decisions on whether mechanistic data was impactful on the final cancer hazard classification. The practice of using the Key Characteristics approach is now well-established at IARC Monographs and other government agencies and the analyses presented herein will inform the future use of mechanistic evidence in regulatory decision-making.

Zhang, L. et al., (2023). A systematic evidence map of chronic inflammation and immunosuppression related to per- and polyfluoroalkyl substance (PFAS) exposure. Environ Res 220:115188.

PMID: 36592815. PMCID: PMC10044447 (available on 2024-03-01). DOI: 10.1016/j.envres.2022.115188

Abstract: Background: The ability to induce chronic inflammation and immunosuppression are two key characteristics of carcinogens and important forms of immunotoxicity. The National Toxicology Program (NTP) evaluated the immunotoxicity of two per- and polyfluoroalkyl substances (PFASs), PFOA (perfluorooctanoic acid) and PFOS (perfluorooctane sulfonate), in 2016. However, the potential pro-inflammatory and immunosuppressive effects of other PFASs remain largely uncharacterized.

Methods: We developed an expanded set of search terms pertaining to the chronic inflammatory and immunosuppressive effects of PFASs based on those of the International Agency for Research on Cancer (IARC) and NTP. To confirm searching effectiveness and scope, we compared our search term results with those of IARC and NTP for both PFASs and two other known carcinogens, chromium (VI) and benzene. Systematic evidence maps (SEMs) were also produced using Tableau to visualize the distribution of study numbers and types reporting immunotoxic effects and specific biomarkers elicited by PFAS exposures. Results: In total, 1155 PFAS studies were retrieved, of which 321 qualified for inclusion in our dataset. Using our search terms, we identified a greater number of relevant studies than those obtained using IARC and NTP’s search terms. From the SEM findings, increased cytokine production strengthened an association between PFAS exposure and chronic inflammation, and decreased B-cell activation and altered levels of T-cell subtypes and immunoglobulins confirmed PFAS-induced immunosuppression. Conclusion: Our SEM findings confirm that several PFASs commonly found in both in the environment, including those that are lesser-known, may induce immunosuppression and chronic inflammation, two key characteristics of carcinogens. This approach, including development of search terms, study screening process, data coding, and evidence mapping visualizations, can be applied to other key characteristics of chemical carcinogens.

Germolec et al. (2022). “Consensus on the Key Characteristics of Immunotoxic Agents as a Basis for Hazard Identification”. Environ Health Perspect. 130(10): 105001. PMCID: PMC9536493. DOI: 10.1289/EHP10800

Abstract: Background: Key characteristics (KCs), properties of agents or exposures that confer potential hazard, have been developed for carcinogens and other toxicant classes. KCs have been used in the systematic assessment of hazards and to identify assay and data gaps that limit screening and risk assessment. Many of the mechanisms through which pharmaceuticals and occupational or environmental agents modulate immune function are well recognized. Thus KCs could be identified for immunoactive substances and applied to improve hazard assessment of immunodulatory agents. Objectives: The goal was to generate a consensus-based synthesis of scientific evidence describing the KCs of agents known to cause immunotoxicity and potential applications, such as assays to measure the KCs. Methods: A committee of 18 experts with diverse specialties identified 10 KCs of immunotoxic agents, namely, 1) covalently binds to proteins to form novel antigens, 2) affects antigen processing and presentation, 3) alters immune cell signaling, 4) alters immune cell proliferation, 5) modifies cellular differentiation, 6) alters immune cell-cell communication, 7) alters effector function of specific cell types, 8) alters immune cell trafficking, 9) alters cell death processes, and 10) breaks down immune tolerance. The group considered how these KCs could influence immune processes and contribute to hypersensitivity, inappropriate enhancement, immunosuppression, or autoimmunity. Discussion: KCs can be used to improve efforts to identify agents that cause immunotoxicity via one or more mechanisms, to develop better testing and biomarker approaches to evaluate immunotoxicity, and to enable a more comprehensive and mechanistic understanding of adverse effects of exposures on the immune system. https://doi.org/10.1289/EHP10800.

DeWitt and Walker (2022). “Invited Perspective: Key Characteristics as a Starting Point for Improved Hazard Identification of Immunotoxic Agents”. Environ Health Perspect. 130(10: 101301. DOI: 10.1289/EHP11726.

Abstract: None.
Companion of: Germolec et al. (2022) PMCID: PMC9536493. DOI: 10.1289/EHP10800

Scholten et al. (2022). “Automated Network Assembly of Mechanistic Literature for Informed Evidence Identification to Support Cancer Risk Assessment”. Environ Health Perspect. 130(3): 37002. PMCID: PMC8893280. DOI: 10.1289/EHP9112

Abstract. Background: Mechanistic data is increasingly used in hazard identification of chemicals. However, the volume of data is large, challenging the efficient identification and clustering of relevant data. Objectives: We investigated whether evidence identification for hazard assessment can become more efficient and informed through an automated approach that combines machine reading of publications with network visualization tools. Methods: We chose 13 chemicals that were evaluated by the International Agency for Research on Cancer (IARC) Monographs program incorporating the key characteristics of carcinogens (KCCs) approach. Using established literature search terms for KCCs, we retrieved and analyzed literature using Integrated Network and Dynamical Reasoning Assembler (INDRA). INDRA combines large-scale literature processing with pathway databases and extracts relationships between biomolecules, bioprocesses, and chemicals into statements (e.g., “benzene activates DNA damage”). These statements were subsequently assembled into networks and compared with the KCC evaluation by the IARC, to evaluate the informativeness of our approach. Results: We found, in general, larger networks for those chemicals which the IARC has evaluated the evidence to be strong for KCC induction. Larger networks were not directly linked to publication count, given that we retrieved small networks for several chemicals with little support for KCC activation according to the IARC, despite the significant volume of literature for these specific chemicals. In addition, interpreting networks for genotoxicity and DNA repair showed concordance with the IARC KCC evaluation. Discussion: Our method is an automated approach to condense mechanistic literature into searchable and interpretable networks based on an a priori ontology. The approach is no replacement of expert evaluation but, instead, provides an informed structure for experts to quickly identify which statements are made in which papers and how these could connect. We focused on the KCCs because these are supported by well-described search terms. The method needs to be tested in other frameworks as well to demonstrate its generalizability. https://doi.org/10.1289/EHP9112.

Reisfeld et al. (2022). “kc-hits: A tool to aid in the evaluation and classification of chemical carcinogens”. Bioinformatics. btac189. Accepted manuscript (March 28). PMID: 35348625. DOI: 10.1093/bioinformatics/btac189

Abstract. Motivation: The evaluation of chemicals for their carcinogenic hazard requires the analysis of a wide range of data and the characterization of these results relative to the key characteristics of carcinogens. The workflow used historically requires many manual steps that are labor-intensive and can introduce errors, bias, and inconsistencies. Results: The automation of parts of the evaluation workflow using the kc-hits software has led to significant improvements in process efficiency, as well as more consistent and comprehensive results. Availability and implementation: https://gitlab.com/i1650/kc-hits.git. Supplementary information: Supplementary information is available from the project repository at https://gitlab.com/i1650/kc-hits.git.

Singh and Hsieh (2021). Exploring Potential Carcinogenic Activity of Per- and Polyfluorinated Alkyl Substances Utilizing High-Throughput Toxicity Screening Data. Int J Toxicol. 2021 Jul-Aug;40(4):355-366. PMID: 33944624. DOI: 10.1177/10915818211010490

Abstract. Per- and polyfluorinated alkyl substances (PFAS) are ubiquitous, persistent, and toxic chemicals that pose public health risks. Recent carcinogenicity concerns have arisen based on epidemiological studies, animal tumor findings, and mechanistic data. Thousands of PFAS exist; however, current understanding of their toxicity is informed by studies of a select few, namely, perfluorooctanoic acid and perfluorooctanesulfonic acid. Hence, the computational, high-throughput screening tool, the US EPA CompTox Chemical Dashboard’s ToxCast, was utilized to explore the carcinogenicity potential of PFAS. Twenty-three major PFAS that had sufficient in vitro ToxCast data and covered a range of structural subclasses were analyzed with the visual analytics software ToxPi, yielding a qualitative and quantitative assessment of PFAS activity in realms closely linked with carcinogenicity. A comprehensive literature search was also conducted to check the consistency of analyses with other mechanistic data streams. The PFAS were found to induce a vast range of biological perturbations, in line with several of the International Agency for Research on Cancer-defined key carcinogen characteristics. Patterns observed varied by length of fluorine-bonded chains and/or functional group within and between each key characteristic, suggesting some structure-based variability in activity. In general, the major conclusions drawn from the analysis, that is, the most notable activities being modulation of receptor-mediated effects and induction of oxidative stress, were supported by literature findings. The study helps enhance understanding of the mechanistic pathways that underlie the potential carcinogenicity of various PFAS and hence could assist in hazard identification and risk assessment for this emerging and relevant class of environmental toxicants.

Mesnage et al. (2021). “Genotoxicity evaluation of 2,4-D, dicamba and glyphosate alone or in combination with cell reporter assays for DNA damage, oxidative stress and unfolded protein response”. Food and Chemical Toxicology. 157: 112601. November. PMID: 34626751 DOI: 10.1016/j.fct.2021.112601.

Abstract. The current generation of carcinogenicity tests is often insufficient to predict cancer outcomes from pesticide exposures. In order to facilitate health risk assessment, The International Agency for Research on Cancer identified 10 key characteristics which are commonly exhibited by human carcinogens. The ToxTracker panel of six validated GFP-based mouse embryonic stem reporter cell lines is designed to measure a number of these carcinogenic properties namely DNA damage, oxidative stress and the unfolded protein response. Here we present an evaluation of the carcinogenic potential of the herbicides glyphosate, 2,4-D and dicamba either alone or in combination, using the ToxTracker assay system. The pesticide 2,4-D was found to be a strong inducer of oxidative stress and an unfolded protein response. Dicamba induced a mild oxidative stress response, whilst glyphosate did not elicit a positive outcome in any of the assays. The results from a mixture of the three herbicides was primarily an oxidative stress response, which was most likely due to 2,4-D with dicamba or glyphosate only playing a minor role. These findings provide initial information regarding the risk assessment of carcinogenic effects arising from exposure to a mixture of these herbicides.

Tice et al. (2021). “In silico approaches in carcinogenicity hazard assessment: Current status and future needs”. Computational Toxicology. 20:100191. Online September 23. https://doi.org/10.1016/j.comtox.2021.100191

Abstract. Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. In silico models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved in silico carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making. As part of an international effort to develop in silico toxicological protocols, a consortium of toxicologists, computational scientists, and regulatory scientists across several industries and governmental agencies evaluated the extent to which in silico models exist for each of the recently defined 10 key characteristics (KCs) of carcinogens. This position paper summarizes the current status of in silico tools for the assessment of each KC and identifies the data gaps that need to be addressed before a comprehensive in silico carcinogenicity protocol can be developed for regulatory use.

Baan and Straif (2021). “The Monographs Programme of the International Agency for Research on Cancer. A brief history of its Preamble”. ALTEX. Epub on Jun 16. PMID: 34164695 doi: 10.14573/altex.2004081

Abstract: Since the early 1970s, the Monographs published by the International Agency for Research on Cancer (IARC) apply rigorous procedures for the scientific review and evaluation of carcinogenic hazards. The Preamble to the IARC Monographs describes the objective and scope of the Programme, the scientific principles and procedures used in developing a Monograph, the types of evidence considered, and the scientific criteria that guide the evaluations. This article presents an overview of the historical development of the Preamble from the time it began to take shape in the late 1970s up to and including the most recent update in 2019. Over the years, the IARC Monographs Programme has taken account of scientific and procedural advances in identifying, reviewing, evaluating and integrating evidence to define causes of human cancer. Since the previous edition of the Preamble in 2006, the new developments include a stronger emphasis on mechanistic evidence based on key characteristics of carcinogens; greater consideration of exposure assessment methods in epidemiological studies; and integration of the streams of evidence on cancer in humans, cancer in experimental animals and mechanisms in reaching the overall evaluations. Thus, the Preamble now allows an evaluation process in the absence of data from animal studies, and the evidence on key characteristics of cancer may be contributed by new approach methodologies, thus potentially reducing or avoiding the use of experimental animals.

Ali et al. (2021). “Application of Text Mining in Risk Assessment of Chemical Mixtures: A Case Study of Polycyclic Aromatic Hydrocarbons (PAHs)”. Environmental Health Perspectives. 129(6): 067008. Online Jun 24. PMID: 34165340 doi: 10.1289/EHP6702

Abstract: Background: Cancer risk assessment of complex exposures, such as exposure to mixtures of polycyclic aromatic hydrocarbons (PAHs), is challenging due to the diverse biological activities of these compounds. With the help of text mining (TM), we have developed TM tools—the latest iteration of the Cancer Risk Assessment using Biomedical literature tool (CRAB3) and a Cancer Hallmarks Analytics Tool (CHAT)—that could be useful for automatic literature analyses in cancer risk assessment and research. Although CRAB3 analyses are based on carcinogenic modes of action (MOAs) and cover almost all the key characteristics of carcinogens, CHAT evaluates literature according to the hallmarks of cancer referring to the alterations in cellular behavior that characterize the cancer cell. Objectives: The objective was to evaluate the usefulness of these tools to support cancer risk assessment by performing a case study of 22 European Union and U.S. Environmental Protection Agency priority PAHs and diesel exhaust and a case study of PAH interactions with silica. Methods: We analyzed PubMed literature, comprising 57,498 references concerning priority PAHs and complex PAH mixtures, using CRAB3 and CHAT. Results: CRAB3 analyses correctly identified similarities and differences in genotoxic and nongenotoxic MOAs of the 22 priority PAHs and grouped them according to their known carcinogenic potential. CHAT had the same capacity and complemented the CRAB output when comparing, for example, benzo[a]pyrene and dibenzo[a,l]pyrene. Both CRAB3 and CHAT analyses highlighted potentially interacting mechanisms within and across complex PAH mixtures and mechanisms of possible importance for interactions with silica. Conclusion:

These data suggest that our TM approach can be useful in the hazard identification of PAHs and mixtures including PAHs. The tools can assist in grouping chemicals and identifying similarities and differences in carcinogenic MOAs and their interactions. https://doi.org/10.1289/EHP6702 

*Lind et al. (2021). “Commentary: Key Characteristics of Cardiovascular Toxicants”. Environmental Health Perspectives. 129(9): 095001. Epub Sept 24. PMID: 34558968 doi:10.1289/EHP9321

Abstract: Background: The concept of chemical agents having properties that confer potential hazard called key characteristics (KCs) was first developed to identify carcinogenic hazards. Identification of KCs of cardiovascular (CV) toxicants could facilitate the systematic assessment of CV hazards and understanding of assay and data gaps associated with current approaches. Objectives: We sought to develop a consensus-based synthesis of scientific evidence on the KCs of chemical and nonchemical agents known to cause CV toxicity along with methods to measure them. Methods: An expert working group was convened to discuss mechanisms associated with CV toxicity. Results: The group identified 12 KCs of CV toxicants, defined as exogenous agents that adversely interfere with function of the CV system. The KCs were organized into those primarily affecting cardiac tissue (numbers 1-4 below), the vascular system (5-7), or both (8-12), as follows: 1) impairs regulation of cardiac excitability, 2) impairs cardiac contractility and relaxation, 3) induces cardiomyocyte injury and death, 4) induces proliferation of valve stroma, 5) impacts endothelial and vascular function, 6) alters hemostasis, 7) causes dyslipidemia, 8) impairs mitochondrial function, 9) modifies autonomic nervous system activity, 10) induces oxidative stress, 11) causes inflammation, and 12) alters hormone signaling. Discussion: These 12 KCs can be used to help identify pharmaceuticals and environmental pollutants as CV toxicants, as well as to better understand the mechanistic underpinnings of their toxicity. For example, evidence exists that fine particulate matter [PM ≤2.5μm≤2.5μm in aerodynamic diameter (PM2.5PM2.5)] air pollution, arsenic, anthracycline drugs, and other exogenous chemicals possess one or more of the described KCs. In conclusion, the KCs could be used to identify potential CV toxicants and to define a set of test methods to evaluate CV toxicity in a more comprehensive and standardized manner than current approaches. 

Guyton and Schubauer-Berigan (2021). “Invited Perspective: Prioritizing Chemical Testing and Evaluation Using Validated in Vitro Assays Relevant to Key Characteristics”. Environmental Health Perspectives. 129(7): 71303. Epub Jul 21. PMID: 34287027. PMCID: PMC8312475. doi: 10.1289/EHP9507. 

            Abstract: None available.

Rusyn et al. (2021). “Key Characteristics of Human Hepatotoxicants as a Basis for Identification and Characterization of the Causes of Liver Toxicity”. Hepatology. 2021 Jun 9. Online ahead of print. PMID: 34105804 DOI: 10.1002/hep.31999

Abstract: Hazard identification regarding adverse effects on the liver is a critical step in safety evaluations of drugs and other chemicals. Current testing paradigms for hepatotoxicity rely heavily on pre-clinical studies in animals and human data (epidemiology and clinical trials). Mechanistic understanding of the molecular and cellular pathways that may cause or exacerbate hepatotoxicity is well advanced, and holds promise for identification of hepatotoxicants. One of the challenges in translating mechanistic evidence into robust decisions about potential hepatotoxicity is the lack of a systematic approach to integrate these data to help identify liver toxicity hazards. Recently, marked improvements were achieved in the practice of hazard identification of carcinogens, female and male reproductive toxicants, and endocrine disrupting chemicals using the key characteristics approach. Here, we describe the methods by which key characteristics of human hepatotoxicants were identified and provide examples for how they could be used to systematically identify, organize and utilize mechanistic data when identifying hepatotoxicants.

Barupal et al., (2021). “Prioritizing cancer hazard assessments for IARC Monographs using an integrated approach of database fusion and text mining”. Environ Int. 156:106624. Online ahead of print May 10. PMID: 33984576 doi: 10.1016/j.envint.2021.106624.

Abstract: Background: Systematic evaluation of literature data on the cancer hazards of human exposures is an essential process underlying cancer prevention strategies. The scope and volume of evidence for suspected carcinogens can range from very few to thousands of publications, requiring a complex, systematically planned, and critical procedure to nominate, prioritize and evaluate carcinogenic agents. To aid in this process, database fusion, cheminformatics and text mining techniques can be combined into an integrated approach to inform agent prioritization, selection, and grouping.

Results: We have applied these techniques to agents recommended for the IARC Monographs evaluations during 2020-2024. An integration of PubMed filters to cover cancer epidemiology, key characteristics of carcinogens, chemical lists from 34 databases relevant for cancer research, chemical structure grouping and a literature data-based clustering was applied in an innovative approach to 119 agents recommended by an advisory group for future IARC Monographs evaluations. The approach also facilitated a rational grouping of these agents and aids in understanding the volume and complexity of relevant information, as well as important gaps in coverage of the available studies on cancer etiology and carcinogenesis.

Conclusion: A new data-science approach has been applied to diverse agents recommended for cancer hazard assessments, and its applications for the IARC Monographs are demonstrated. The prioritization approach has been made available at www.cancer.idsl.me site for ranking cancer agents.

Madia et al., (2021). “Integration of data across toxicity endpoints for improved safety assessment of chemicals: the example of carcinogenicity assessment”. Arch Tox. 95(6):1971-1993 PMID: 33830278. doi: 10.1007/s00204-021-03035-x. Online ahead of print.

Abstract: In view of the need to enhance the assessment of consumer products called for in the EU Chemicals Strategy for Sustainability, we developed a methodology for evaluating hazard by combining information across different systemic toxicity endpoints and integrating the information with new approach methodologies. This integrates mechanistic information with a view to avoiding redundant in vivo studies, minimising reliance on apical endpoint tests and ultimately devising efficient testing strategies. Here, we present the application of our methodology to carcinogenicity assessment, mapping the available information from toxicity test methods across endpoints to the key characteristics of carcinogens. Test methods are deconstructed to allow the information they provide to be organised in a systematic way, enabling the description of the toxicity mechanisms leading to the adverse outcome. This integrated approach provides a flexible and resource-efficient means of fully exploiting test methods for which test guidelines are available to fulfil regulatory requirements for systemic toxicity assessment as well as identifying where new methods can be integrated.

*Rider CV et al. (2021). “Using the Key Characteristics of Carcinogens to Develop Research on Chemical Mixtures and Cancer”. Environmental Health Perspectives. 129(3):35003. Epub 2021 Mar 30. PMID: 33784186. doi: 10.1289/EHP8525.

Background: People are exposed to numerous chemicals throughout their lifetimes. Many of these chemicals display one or more of the key characteristics of carcinogens or interact with processes described in the hallmarks of cancer. Therefore, evaluating the effects of chemical mixtures on cancer development is an important pursuit. Challenges involved in designing research studies to evaluate the joint action of chemicals on cancer risk include the time taken to perform the experiments because of the long latency and choosing an appropriate experimental design. Objectives: The objectives of this work are to present the case for developing a research program on mixtures of environmental chemicals and cancer risk and describe recommended approaches. Methods: A working group comprising the coauthors focused attention on the design of mixtures studies to inform cancer risk assessment as part of a larger effort to refine the key characteristics of carcinogens and explore their application. Working group members reviewed the key characteristics of carcinogens, hallmarks of cancer, and mixtures research for other disease end points. The group discussed options for developing tractable projects to evaluate the joint effects of environmental chemicals on cancer development. Results and Discussion: Three approaches for developing a research program to evaluate the effects of mixtures on cancer development were proposed: a chemical screening approach, a transgenic model-based approach, and a disease-centered approach. Advantages and disadvantages of each are discussed.

McKeon et al. (2021). “Environmental exposomics and lung cancer risk assessment in the Philadelphia metropolitan area using ZIP code–level hazard indices”. Environmental Science and Pollution Research. PMID: 33611735. doi: 10.1007/s11356-021-12884-z.  Online ahead of print.

Abstract: To illustrate methods for assessing environmental exposures associated with lung cancer risk, we investigated anthropogenic based air pollutant data in a major metropolitan area using United States-Environmental Protection Agency (US-EPA) Toxic Release Inventory (TRI) (1987-2017), and PM_2.5 (1998-2016) and NO₂ (1996-2012) concentrations from NASA satellite data. We studied chemicals reported according to the following five exposome features: (1) International Agency for Research on Cancer (IARC) cancer grouping; (2) priority EPA polycyclic aromatic hydrocarbons (PAHs); (3) component of diesel exhaust; (4) status as a volatile organic compound (VOC); and (5) evidence of lung carcinogenesis. Published articles from PubChem were tallied for occurrences of 10 key characteristics of cancer-causing agents on those chemicals. Zone Improvement Plan (ZIP) codes with higher exposures were identified in two ways: (1) combined mean exposure from all features, and (2) hazard index derived through a multi-step multi-criteria decision analysis (MMCDA) process. VOCs, IARC Group 1 carcinogens consisted 82.3% and 11.5% of the reported TRI emissions, respectively. ZIP codes along major highways tended to have greater exposure. The MMCDA approach yielded hazard indices based on imputed toxicity, occurrence, and persistence for risk assessment. Despite many studies describing environmental exposures and lung cancer risk, this study develops a method to integrate these exposures into population-based exposure estimates that could be incorporated into future lung cancer screening trials and benefit public health surveillance of lung cancer incidence. Our methodology may be applied to probe other hazardous exposures for other cancers.

Gualtieri AF (2021). “Bridging the gap between toxicity and carcinogenicity of mineral fibres by connecting the fibre crystal-chemical and physical parameters to the key characteristics of cancer”. Current Research in Toxicology. 2:42-52. PMCID: PMC8320635. https://doi.org/10.1016/j.crtox.2021.01.005

Airborne fibres and particularly asbestos represent hazards of great concern for human health because exposure to these peculiar particulates may cause malignancies such as lung cancer and mesothelioma. Currently, many researchers worldwide are focussed on fully understanding the patho-biological mechanisms leading to carcinogenesis prompted by pathogenic fibres. Along this line, the present work introduces a novel approach to correlate how and to what extent the physical/crystal-chemical and morphological parameters (including length, chemistry, biodurability, and surface properties) of mineral fibres cause major adverse effects with an emphasis on asbestos. The model described below conceptually attempts to bridge the gap between toxicity and carcinogenicity of mineral fibres and has several implications: 1) it provides a tool to measure the toxicity and pathogenic potential of asbestos minerals, allowing a quantitative rank of the different types (e.g. chrysotile vs. crocidolite); 2) it can predict the toxicity and pathogenicity of “unregulated” or unclassified fibres; 3) it reveals the parameters of a mineral fibre that are active in stimulating key characteristics of cancer, thus offering a strategy for developing specific cancer prevention strategies and therapies.

Chappell et al. (2021). “Lack of potential carcinogenicity for steviol glycosides – Systematic evaluation and integration of mechanistic data into the totality of evidence.” Food Chem Toxicol.  2021 Feb 12;112045. PMID: 33587976. DOI: 10.1016/j.fct.2021.112045

Steviol glycosides are present in the leaves of the Stevia rebaudiana plant, have a sweet taste, and have been used as a sweetener for centuries. To build on previous authoritative safety assessments of steviol glycosides, a systematic assessment of mechanistic data related to key characteristics of carcinogens (KCCs) was conducted. Over 900 KCC-relevant endpoints from peer-reviewed literature and high-throughput screening data (ToxCast/Tox21) were identified across individual steviol glycosides and derivatives, metabolites, and whole leaf extracts. Most data (both in vivo and in vitro, including human cells), showed inactivity. Studies were weighted according to quality and relevance. Although data were available for eight of the ten KCC, genotoxicity, oxidative stress, inflammation, and cell proliferation/cell death represent the KCCs with the most data. The data for these KCC primarily show beneficial activity (anti-inflammatory, antioxidant, and anti-proliferative). Following integration across all data, and accounting for study quality and relevance, the totality of the evidence demonstrated an overall lack of genotoxic and carcinogenic activity for steviol glycosides. This is in agreement with previous regulatory decisions, and is consistent with the lack of tumor response in two-year rodent cancer bioassays. The findings support prior conclusions that steviol glycosides are unlikely to be carcinogenic in humans.

Chappell et al. (2021). Assessment of mechanistic data for hexavalent chromium-induced rodent intestinal cancer using the key characteristics of carcinogens. Toxicol Sci. Jan 6;kfaa187.  PMID: 33404626. DOI: 10.1093/toxsci/kfaa187

Oral exposure to hexavalent chromium (Cr(VI)) induces intestinal tumors in mice. Mutagenic and non-mutagenic modes of action (MOAs) have been accepted by different regulatory bodies globally, the latter involving cytotoxicity-induced regenerative cell proliferation. However, concerns persist that all possible MOAs have not been fully considered. To address the potential for alternative MOAs, mechanistic data not represented in the existing two MOAs were evaluated. Relevant data were identified and organized by key characteristics of carcinogens (KCCs); literature related to epigenetics, immunosuppression, receptor-mediated effects, and immortalization were reviewed to identify potential key events associated with an alternative MOA. Over 200 references were screened for these four KCCs and further prioritized based on relevance to the research objective (i.e., in vivo, oral exposure, gastrointestinal tissue). Minimal data were available specific to the intestine for these KCCs, and there was no evidence of any underlying mechanisms or key events that are not already represented in the two proposed MOAs. For example, while epigenetic dysregulation of DNA repair genes has been demonstrated, epigenetic effects were not measured in intestinal tissue, and it has been shown that Cr(VI) does not cause DNA damage in intestinal tissue. High-throughput screening (HTS) data related to the KCCs were also evaluated, with activity generally limited to the two recognized MOAs. Collectively, no plausible alternative MOAs (or key events) were identified in addition to those previously proposed for Cr(VI) SI tumors.

Vandenberg and Bugos (2021). “Assessing the Public Health Implications of the Food Preservative Propylparaben: Has This Chemical Been Safely Used for Decades?” Curr Environ Health Rep. doi: 10.1007/s40572-020-00300-6. PMID: 33415721. DOI: 10.1007/s40572-020-00300-6

Purpose: Parabens are chemicals containing alkyl-esters of p-hydroxybenzoic acid, which give them antimicrobial, antifungal, and preservative properties. Propylparaben (PP) is one paraben that has been widely used in personal care products, cosmetics, pharmaceuticals, and food. In this review, we address the ongoing controversy over the safety of parabens, and PP specifically. These chemicals have received significant public attention after studies published almost 20 years ago suggested plausible associations between PP exposures and breast cancer.

Recent findings: Here, we use key characteristics, a systematic approach to evaluate the endocrine disrupting properties of PP based on features of “known” endocrine disruptors, and consider whether its classification as a “weak” estrogen should alleviate public health concerns over human exposures. We also review the available evidence from rodent and human studies to illustrate how the large data gaps that exist in hazard assessments raise concerns about current evaluations by regulatory agencies that PP use is safe. Finally, we address the circular logic that is used to suggest that because PP has been used for several decades, it must be safe. We conclude that inadequate evidence has been provided for the safe use of PP in food, cosmetics, and consumer products.

Vandenberg et al. (2020). “Agrochemicals with estrogenic endocrine disrupting properties: Lessons Learned?” Mol Cell Endocrinol. Dec 1;518:110860. doi: 10.1016/j.mce.2020.110860. PMID: 32407980. DOI: 10.1016/j.mce.2020.110860

Many agrochemicals have endocrine disrupting properties. A subset of these chemicals is characterized as “estrogenic”. In this review, we describe several distinct ways that chemicals used in crop production can affect estrogen signaling. Using three agrochemicals as examples (DDT, endosulfan, and atrazine), we illustrate how screening tests such as the US EPA’s EDSP Tier 1 assays can be used as a first-pass approach to evaluate agrochemicals for endocrine activity. We then apply the “Key Characteristics” approach to illustrate how chemicals like DDT can be evaluated, together with the World Health Organization’s definition of an endocrine disruptor, to identify data gaps. We conclude by describing important issues that must be addressed in the evaluation and regulation of hormonally active agrochemicals including mixture effects, efforts to reduce vertebrate animal use, chemical prioritization, and improvements in hazard, exposure, and risk assessments.

Guyton and Smith (2020). “Identifying carcinogens from 10 key characteristics: A new approach based on mechanisms”. In: World Cancer Report: Cancer Research for Cancer Prevention, Chapter 3.11, pp.177-183, Wild CP, Weiderpass E, Stewart BW, editors, Published by the International Agency for Research on Cancer, Lyon, France. Available from: http://publications.iarc.fr/586.

*Guo et al. (2020). “Benzene-associated immunosuppression and chronic inflammation in humans: a systematic review.” Occup Environ Med 2020;oemed-2020-106517.

Objective: Recent evidence has accumulated that the immune system is intimately intertwined with cancer development. Two key characteristics of carcinogens in which the immune system plays a central role are chronic inflammation and immunosuppression. In this systematic review, we investigated the association of chronic inflammatory and immunosuppressive outcomes with benzene, a widely used industrial chemical. Benzene has been confirmed to cause acute myeloid leukaemia and suspected to cause non-Hodgkin lymphoma, two cancers of the blood-forming system that affect immune cells. Methods: We systematically searched PubMed and Embase for all relevant studies using a combination of Medical Subject Headings (MeSH) and selected key words. The detailed review protocol, including search strategy, was registered with PROSPERO, the international prospective register of systematic reviews (#CRD42019138611). Results: Based on all human studies selected in the final review, we report new evidence of a benzene-induced immunosuppressive effect on the adaptive immune system and activation of the innate immune system to cause inflammation. In particular, benzene significantly lowers the number of white blood cells, particularly lymphocytes such as CD4⁺ T-cells, B-cells and natural killer cells, and increases proinflammatory biomarkers at low levels of exposure.

Conclusion: To the best of our knowledge, this is the first comprehensive review of benzene’s immunotoxicity in humans. Based on results obtained from this review, we propose two potential immunotoxic mechanisms of how benzene induces leukaemia/lymphoma: (1) cancer invasion caused by proinflammatory cytokine production, and (2) cancer promotion via impaired immunosurveillance. Further studies will be required to confirm the connection between benzene exposure and its effects on the immune system.

Sone et al. (2020). “Hazard evaluation of air pollution by using the key characteristics approach.” IOP Conf. Ser.: Earth Environ. Sci. 496 012004.

The key characteristic (KC) as a new approach based on mechanistic evidence has been shown by the IARC Monographs Program working group. Human carcinogens like exhibit one or more key characteristics that are related to how they cause cancer, and different carcinogenic agents exhibit different spectra of these key characteristics. Air pollution is one of the major concerns of human health for the people who lives in Asia because air pollution contains many different human carcinogens including heavy metals, volatile compounds. electrophile compounds and polyaromatic hydrocarbon compounds. In this study, weinvestigated air pollution mixtures derived from diesel and gasoline engine exhausts by a mechanism-based approach. We first conducted a KC analysis of organic compounds of diesel exhaust in IARC monograph 105 using data of biological test results from the PubChem database. As a result, it was found that some PAH among those compounds is highly responsive to aryl hydrocarbon receptor, nuclear factor erythroid 2 like 2, NR1I2/3 nuclear receptor subfamily 1 group I member 2/3. Further, we analyzed KCs of water-extract mixtures from diesel exhaust particle (DEP-WM) using gene expression values in the mouse lung responded induced by exposures to DEP-WM. The KC of DEP-WM showed lipid related-nuclear receptor signaling and apoptotic pathway, suggesting that DEP-WM affects lipid metabolism in lung tissues. Thus, the KC method will be useful for high-precision assessment of a mixture of air pollution.

Calaf et al. (2020). “Endocrine disruptors from the environment affecting breast cancer (Review).” Oncology Letters 20: 19-32.

Evaluation of carcinogenic substances from the environment is a challenge for scientists. Recently, a novel approach based on 10 key characteristics of human carcinogens classified by the International Agency for Research on Cancer (IARC) has emerged. Carcinogenesis depends on different mechanisms and factors, including genetic, infectious (bacteria, viruses) and environmental (chemicals) factors. Endocrine disruptors are exogenous chemicals that can interfere and impair the function of the endocrine system due to their interaction with estrogen receptors or their estrogen signaling pathways inducing adverse effects in the normal mammary development, originating cancer. They are heterogeneous chemicals and include numerous synthetic substances used worldwide in agriculture, industry and consumer products. The most common are plasticizers, such as bisphenol A (BPA), pesticides, such as dichlorodiphenyltrichloroethane, and polychlorinated biphenyls (PCBs). Xenoestrogens appear to serve an important role in the increased incidence of breast cancer in the United States and numerous other countries. Several studies have demonstrated the role of organochlorine xenoestrogens in breast cancer. Therefore, the overall cumulative exposure of women to estrogens results in an increased risk for this type of cancer. Factors like lifestyle and diet also serve a role in the increased incidence of this disease. The aim of the present study was to analyze these chemical compounds based on the key characteristics given by the IARC, with a special focus on breast cancer, to establish whether these compounds are carcinogens, and to create a model for future analysis of other endocrine disruptors. doi: 10.1007/s10911-013-9275-7

Chappell G. A. et al. (2020). Lack of potential carcinogenicity for acesulfame potassium–Systematic evaluation and integration of mechanistic data into the totality of the evidence. Food and Chemical Toxicology 141: 111375

The safety of low- and no-calorie sweeteners remains a topic of general interest. Substantial evidence exists demonstrating a lack of carcinogenicity of the no-calorie sweetener acesulfame potassium (Ace K). The objective of this evaluation was to conduct a systematic assessment of available mechanistic data using a framework that quantitatively integrates proposed key characteristics of carcinogens (KCCs) into the totality of the evidence. Over 800 KCC-relevant endpoints from a variety of in vitro and in vivo assays were assessed for quality, relevance, and activity, and integrated to determine the overall strength of the evidence for plausibility that Ace K acts through the KCC. Overall, there was a lack of activity across the KCCs (overall integrated score <0 and no “strong” categorization for evidence of activity) in which data were identified. Together with the absence of treatment-related tumor effects in rodent bioassays, these results support the conclusion that Ace K is unlikely to induce a carcinogenic response. This assessment employed a weight of the evidence analysis that includes the consideration of factors such as reliability, strength of the model system, activity, and dose in a complex and heterogeneous dataset, and the ultimate integration of multiple data streams in the cancer hazard evaluation.

*La Merrill, M. A., et al. (2020). “Consensus on the key characteristics of endocrine-disrupting chemicals as a basis for hazard identification.” Nat Rev Endocrinol 16(1): 45-57.

Endocrine-disrupting chemicals (EDCs) are exogenous chemicals that interfere with hormone action, thereby increasing the risk of adverse health outcomes, including cancer, reproductive impairment, cognitive deficits and obesity. A complex literature of mechanistic studies provides evidence on the hazards of EDC exposure, yet there is no widely accepted systematic method to integrate these data to help identify EDC hazards. Inspired by work to improve hazard identification of carcinogens using key characteristics (KCs), we have developed ten KCs of EDCs based on our knowledge of hormone actions and EDC effects. In this Expert Consensus Statement, we describe the logic by which these KCs are identified and the assays that could be used to assess several of these KCs. We reflect on how these ten KCs can be used to identify, organize and utilize mechanistic data when evaluating chemicals as EDCs, and we use diethylstilbestrol, bisphenol A and perchlorate as examples to illustrate this approach.

https://doi.org/10.1038/s41574-019-0273-8

*Smith, M. T. et al. (2020). “The Key Characteristics of Carcinogens: Relationship to the Hallmarks of Cancer, Relevant Biomarkers, and Assays to Measure Them.” Cancer Epidemiol Biomarkers Prev. https://doi.org/10.1158/1055-9965.EPI-19-1346

Background: The key characteristics (KCs) of human carcinogens provide a uniform approach to evaluating mechanistic evidence in cancer hazard identification. Refinements to the approach were requested by organizations and individuals applying the KCs. Methods: We assembled an expert committee with knowledge of carcinogenesis and experience in applying the KCs in cancer hazard identification. We leveraged this expertise and an examination of the literature to more clearly describe each KC; identify current and emerging assays and in vivo biomarkers that can be used to measure them; and, make recommendations for future assay development. Results: We found that the KCs are clearly distinct from the Hallmarks of Cancer, that interrelationships among the KCs can be leveraged to strengthen the KC approach (and an understanding of environmental carcinogenesis), and that the KC approach is applicable to the systematic evaluation of a broad range of potential cancer hazards in vivo and in vitro. We identified gaps in coverage of the KCs by current assays. Conclusion: Future efforts should expand the breadth, specificity and sensitivity of validated assays and biomarkers that can measure the 10 KCs. Impact: Refinement of the KC approach will enhance and accelerate carcinogen identification, a first step in cancer prevention.

Temkin, A. M. et al. (2020). “Application of the key characteristics of carcinogens to Per and Polyfluoroalkyl Substances.” Int Journal Env Res and Public Health 17, 1668.

Per- and polyfluoroalkyl substances (PFAS) constitute a large class of environmentally persistent chemicals used in industrial and consumer products. Human exposure to PFAS is extensive, and PFAS contamination has been reported in drinking water and food supplies as well as in the serum of nearly all people. The most well-studied member of the PFAS class, perfluorooctanoic acid (PFOA), induces tumors in animal bioassays and has been associated with elevated risk of cancer in human populations. GenX, one of the PFOA replacement chemicals, induces tumors in animal bioassays as well. Using the Key Characteristics of Carcinogens framework for cancer hazard identification, we considered the existing epidemiological, toxicological and mechanistic data for 26 different PFAS. We found strong evidence that multiple PFAS induce oxidative stress, are immunosuppressive, and modulate receptor-mediated effects. We also found suggestive evidence indicating that some PFAS can induce epigenetic alterations and influence cell proliferation. Experimental data indicate that PFAS are not genotoxic and generally do not undergo metabolic activation. Data are currently insufficient to assess whether any PFAS promote chronic inflammation, cellular immortalization or alter DNA repair. While more research is needed to address data gaps, evidence exists that several PFAS exhibit one or more of the key characteristics of carcinogens. https://doi.org/10.3390/ijerph17051668

Wikoff, D. S. et al. (2020). Lack of potential carcinogenicity for aspartame–Systematic evaluation and integration of mechanistic data into the totality of the evidence. Food and Chemical Toxicology 135: 110866

Despite repeated confirmation of aspartame safety in a variety of foods and beverages, there continues to be interest in researching the potential carcinogenic risk associated with its consumption. The objective of this evaluation was to conduct a systematic assessment of available mechanistic data using a framework for quantitatively integrating the key characteristics of carcinogens (KCCs). For aspartame, 1332 endpoints were appraised for quality and relevance and quantitatively integrated using an algorithm to determine the potential for individual KCC activity based on all available evidence, and subsequently assessed in the context of human and animal evidence streams. An overall lack of activity (integrated scores <0 and no “strong” categorizations) was observed for all KCCs except oxidative stressor (#5), for which activity was determined to be unlikely to be related to a carcinogenic response. Overall, the KCC-based analysis, together with the lack of consistent evidence of carcinogenicity in experimental animals, continue to support lack of carcinogenicity from aspartame consumption. This comprehensive evaluation of available mechanistic data demonstrates the need for a systematic approach to identify and appraise all avaialble data as part of weight-of-evidence determinations related use of KCC in evaluations of potential human carcinogenicity.

*Arzuaga, X., et al. (2019). “Proposed Key Characteristics of Male Reproductive Toxicants as an Approach for Organizing and Evaluating Mechanistic Evidence in Human Health Hazard Assessments.” Environ Health Perspect 127(6): 65001.

BACKGROUND: Assessing chemicals for their potential to cause male reproductive toxicity involves the evaluation of evidence obtained from experimental, epidemiological, and mechanistic studies. Although mechanistic evidence plays an important role in hazard identification and evidence integration, the process of identifying, screening and analyzing mechanistic studies and outcomes is a challenging exercise due to the diversity of research models and methods and the variety of known and proposed pathways for chemical-induced toxicity. Ten key characteristics of carcinogens provide a valuable tool for organizing and assessing chemical-specific data by potential mechanisms for cancer-causing agents. However, such an approach has not yet been developed for noncancer adverse outcomes. OBJECTIVES: The objective in this study was to identify a set of key characteristics that are frequently exhibited by exogenous agents that cause male reproductive toxicity and that could be applied for identifying, organizing, and summarizing mechanistic evidence related to this outcome. DISCUSSION: The identification of eight key characteristics of male reproductive toxicants was based on a survey of known male reproductive toxicants and established mechanisms and pathways of toxicity. The eight key characteristics can provide a basis for the systematic, transparent, and objective organization of mechanistic evidence relevant to chemical-induced effects on the male reproductive system. https://doi.org/10.1289/EHP5045.

Atwood, S. T., et al. (2019). “New Perspectives for Cancer Hazard Evaluation by the Report on Carcinogens: A Case Study Using Read-Across Methods in the Evaluation of Haloacetic Acids Found as Water Disinfection By-Products.” Environ Health Perspect 127(12): 125003.

BACKGROUND: Due to the large number of chemicals not yet tested for carcinogenicity but to which people are exposed, the limited number of human and animal cancer studies conducted each year, and the frequent need for a timely response, mechanistic data are playing an increasingly important role in carcinogen hazard identification. OBJECTIVES: To provide a targeted approach to identify relevant mechanistic data in our cancer evaluation of haloacetic acids (HAAs), we used several approaches including systematic review, the 10 key characteristics of carcinogens (KCs), and read-across methods. Our objective in this commentary is to discuss the strengths, limitations, and challenges of these approaches in a cancer hazard assessment. METHODS: A cancer hazard assessment for 13 HAAs found as water disinfection by-products was conducted. Literature searches for mechanistic studies focused on the KCs and individual HAAs. Studies were screened for relevance and categorized by KCs and other relevant data, including chemical properties, toxicokinetics, and biological effects other than KCs. Mechanistic data were organized using the KCs, and strength of evidence was evaluated; this information informed potential modes of action (MOAs) and read-across-like approaches. Three read-across options were considered: evaluating HAAs as a class, as subclass(es), or as individual HAAs (analog approach). DISCUSSION: Because of data limitations and uncertainties, listing as a class or subclass(es) was ruled out, and an analog approach was used. Two brominated HAAs were identified as target (untested) chemicals based on their metabolism and similarity to source (tested) chemicals. In addition, four HAAs with animal cancer data had sufficient evidence for potential listing in the Report on Carcinogens (RoC). This is the first time that the KCs and other relevant data, in combination with read-across principles, were used to support a recommendation to list chemicals in the RoC that did not have animal cancer data. https://doi.org/10.1289/EHP5672.

Chappell, G. A., et al. (2019). “Lack of potential carcinogenicity for sucralose – Systematic evaluation and integration of mechanistic data into the totality of the evidence.” Food Chem Toxicol: 110898.

Sucralose is widely used as a sugar substitute. Many studies and authoritative reviews have concluded that sucralose is non-carcinogenic, based primarily on animal cancer bioassays and genotoxicity data. To add to the body of knowledge on the potential carcinogenicity of sucralose, a systematic assessment of mechanistic data was conducted. This entailed using a framework developed for the quantitative integration of data related to the proposed key characteristics of carcinogens (KCCs). Data from peer-reviewed literature and the ToxCast/Tox21 database were evaluated using an algorithm that weights data for quality and relevance. The resulting integration demonstrated an overall lack of activity for sucralose across the KCCs, with no “strong” activity observed for any KCC. Almost all data collected demonstrated inactivity, including those conducted in human models. The overall lack of activity in mechanistic data is consistent with findings from animal cancer bioassays. The few instances of activity across the KCC were generally accompanied by limitations in study design in the context of either quality and/or dose and model relevance, highlighted upon integration of the totality of the evidence. The findings from this comprehensive and integrative evaluation of mechanistic data support prior conclusions that sucralose is unlikely to be carcinogenic in humans. https://doi.org/10.1016/j.fct.2019.110898

Iyer, S. et al. (2019). “An Integrated Approach Using Publicly Available Resources for Identifying and Characterizing Chemicals of Potential Toxicity Concern: Proof-of-

Concept With Chemicals That Affect Cancer Pathways.” Toxicol Sci, 169(1), 2019, 14–24.

We developed an integrated, modular approach to predicting chemical toxicity relying on in vitro assay data, linkage of molecular targets to disease categories, and software for ranking chemical activity and examining structural features (chemotypes). We evaluate our approach in a proof-of-concept exercise to identify and prioritize chemicals of potential carcinogenicity concern. We identified 137 cancer pathway-related assays from a subset of U.S. EPA’s ToxCast platforms. We mapped these assays to key characteristics of carcinogens and found they collectively assess 5 of 10 characteristics. We ranked all 1061 chemicals screened in Phases I and II of ToxCast by their activity in the selected cancer pathway-related assays using Toxicological Prioritization Index software. More chemicals used as biologically active agents (eg, pharmaceuticals) ranked in the upper 50% versus lower 50%. Twenty-three chemotypes are enriched in the top 5% (n 1⁄4 54) of chemicals; these features may be important for their activity in cancer pathway-related assays. The biological coverage of the ToxCast assays related to cancer pathways is limited and short-term assays may not capture the biology of some key characteristics. Metabolism is also minimal in the assays. The ability of our approach to identify chemicals with cancer hazard is limited with the current input data, but we expect that our approach can be applied with future iterations of ToxCast and other data for improved chemical prioritization and characterization. The novel approach and proof-of- concept exercise described here for ranking chemicals for potential carcinogenicity concern is modular, adaptable, and amenable to evolving data streams. https://doi.org/10.1093/toxsci/kfz017

*Luderer, U., et al. (2019). “Proposed Key Characteristics of Female Reproductive Toxicants as an Approach for Organizing and Evaluating Mechanistic Data in Hazard Assessment.” Environ Health Perspect 127(7): 75001.

BACKGROUND: Identification of female reproductive toxicants is currently based largely on integrated epidemiological and in vivo toxicology data and, to a lesser degree, on mechanistic data. A uniform approach to systematically search, organize, integrate, and evaluate mechanistic evidence of female reproductive toxicity from various data types is lacking. OBJECTIVE: We sought to apply a key characteristics approach similar to that pioneered for carcinogen hazard identification to female reproductive toxicant hazard identification. METHODS: A working group of international experts was convened to discuss mechanisms associated with chemical-induced female reproductive toxicity and identified 10 key characteristics of chemicals that cause female reproductive toxicity: 1) alters hormone receptor signaling; alters reproductive hormone production, secretion, or metabolism; 2) chemical or metabolite is genotoxic; 3) induces epigenetic alterations; 4) causes mitochondrial dysfunction; 5) induces oxidative stress; 6) alters immune function; 7) alters cell signal transduction; 8) alters direct cell-cell interactions; 9) alters survival, proliferation, cell death, or metabolic pathways; and 10) alters microtubules and associated structures. As proof of principle, cyclophosphamide and diethylstilbestrol (DES), for which both human and animal studies have demonstrated female reproductive toxicity, display at least 5 and 3 key characteristics, respectively. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), for which the epidemiological evidence is mixed, exhibits 5 key characteristics. DISCUSSION: Future efforts should focus on evaluating the proposed key characteristics against additional known and suspected female reproductive toxicants. Chemicals that exhibit one or more of the key characteristics could be prioritized for additional evaluation and testing. A key characteristics approach has the potential to integrate with pathway-based toxicity testing to improve prediction of female reproductive toxicity in chemicals and potentially prevent some toxicants from entering common use. https://doi.org/10.1289/EHP4971.

NIEHS Superfund Research Program Research Brief 297, Sept 4, 2019

Samet, J. M. (2019). The IARC Monographs: Updated Procedures for Modern and Transparent Evidence Synthesis in Cancer Hazard Identification. J National Cancer Institute 112 (1): 30-37.

The Monographs produced by the International Agency for Research on Cancer (IARC) apply rigorous procedures for the scientific review and evaluation of carcinogenic hazards by independent experts. The Preamble to the IARC Monographs, which outlines these procedures, was updated in 2019, following recommendations of a 2018 expert advisory group. This article presents the key features of the updated Preamble, a major milestone that will enable IARC to take advantage of recent scientific and procedural advances made during the 12 years since the last Preamble amendments. The updated Preamble formalizes important developments already being pioneered in the Monographs program. These developments were taken forward in a clarified and strengthened process for identifying, reviewing, evaluating, and integrating evidence to identify causes of human cancer. The advancements adopted include the strengthening of systematic review methodologies; greater emphasis on mechanistic evidence, based on key characteristics of carcinogens; greater consideration of quality and informativeness in the critical evaluation of epidemiological studies, including their exposure assessment methods; improved harmonization of evaluation criteria for the different evidence streams; and a single-step process of integrating evidence on cancer in humans, cancer in experimental animals, and mechanisms for reaching overall evaluations. In all, the updated Preamble underpins a stronger and more transparent method for the identification of carcinogenic hazards, the essential first step in cancer prevention. https://doi.org/10.1093/jnci/djz169

*Smith, M.T. (2019). “Key characteristics of carcinogens.” Chapter 10 in Tumor site concordance and mechanisms of carcinogenesis. Robert A. Baan, Bernard W. Stewart and Kurt Straif, Eds. IARC Scientific Publication No. 165. IARC, Lyons, France.

*Fielden, M. R., et al. (2018). “Modernizing Human Cancer Risk Assessment of Therapeutics.” Trends Pharmacol Sci 39(3): 232-247.

Cancer risk assessment of therapeutics is plagued by poor translatability of rodent models of carcinogenesis. In order to overcome this fundamental limitation, new approaches are needed that enable us to evaluate cancer risk directly in humans and human-based cellular models. Our enhanced understanding of the mechanisms of carcinogenesis and the influence of human genome sequence variation on cancer risk motivates us to re-evaluate how we assess the carcinogenic risk of therapeutics. This review will highlight new opportunities for applying this knowledge to the development of a battery of human-based in vitro models and biomarkers for assessing cancer risk of novel therapeutics. https://doi.org/10.1016/j.tips.2017.11.005

Guyton, K. Z., et al. (2018). “Key Characteristics Approach to Carcinogenic Hazard Identification.” Chem Res Toxicol 31(12):1290-1292

Evaluating carcinogenic mechanisms is a challenging part of hazard identification, as mechanistic data are both voluminous and diverse. An evaluation approach based on 10 key characteristics of human carcinogens provides a holistic and unbiased way to tackle this challenge. https://doi.org/10.1021/acs.chemrestox.8b00321

*Guyton, K. Z., et al. (2018). “Application of the key characteristics of carcinogens in cancer hazard identification.” Carcinogenesis 39(4): 614-622.

Smith et al. (Env. Health Perspect. 124: 713, 2016) identified 10 key characteristics (KCs), one or more of which are commonly exhibited by established human carcinogens. The KCs reflect the properties of a cancer-causing agent, such as ‘is genotoxic,’ ‘is immunosuppressive’ or ‘modulates receptor-mediated effects,’ and are distinct from the hallmarks of cancer, which are the properties of tumors. To assess feasibility and limitations of applying the KCs to diverse agents, methods and results of mechanistic data evaluations were compiled from eight recent IARC Monograph meetings. A systematic search, screening and evaluation procedure identified a broad literature encompassing multiple KCs for most (12/16) IARC Group 1 or 2A carcinogens identified in these meetings. Five carcinogens are genotoxic and induce oxidative stress, of which pentachlorophenol, hydrazine and malathion also showed additional KCs. Four others, including welding fumes, are immunosuppressive. The overall evaluation was upgraded to Group 2A based on mechanistic data for only two agents, tetrabromobisphenol A and tetrachloroazobenzene. Both carcinogens modulate receptor-mediated effects in combination with other KCs. Fewer studies were identified for Group 2B or 3 agents, with the vast majority (17/18) showing only one or no KCs. Thus, an objective approach to identify and evaluate mechanistic studies pertinent to cancer revealed strong evidence for multiple KCs for most Group 1 or 2A carcinogens but also identified opportunities for improvement. Further development and mapping of toxicological and biomarker endpoints and pathways relevant to the KCs can advance the systematic search and evaluation of mechanistic data in carcinogen hazard identification. https://doi.org/10.1093/carcin/bgy031

Smith, M. T. et al. (2016). Key Characteristics of Carcinogens as a Basis for Organizing Data on Mechanisms of Carcinogenesis. Environmental Health Perspectives 124 (6): 713-721.

Background: A recent review by the International Agency for Research on Cancer (IARC) updated the assessments of the > 100 agents classified as Group 1, carcinogenic to humans (IARC Monographs Volume 100, parts A–F). This exercise was complicated by the absence of a broadly accepted, systematic method for evaluating mechanistic data to support conclusions regarding human hazard from exposure to carcinogens. Objectives and Methods: IARC therefore convened two workshops in which an international Working Group of experts identified 10 key characteristics, one or more of which are commonly exhibited by established human carcinogens. Discussion:

These characteristics provide the basis for an objective approach to identifying and organizing results from pertinent mechanistic studies. The 10 characteristics are the abilities of an agent to 1) act as an electrophile either directly or after metabolic activation; 2) be genotoxic; 3) alter DNA repair or cause genomic instability; 4) induce epigenetic alterations; 5) induce oxidative stress; 6) induce chronic inflammation; 7) be immunosuppressive; 8) modulate receptor-mediated effects; 9) cause immortalization; and 10) alter cell proliferation, cell death, or nutrient supply. Conclusion: We describe the use of the 10 key characteristics to conduct a systematic literature search focused on relevant end points and construct a graphical representation of the identified mechanistic information. Next, we use benzene and polychlorinated biphenyls as examples to illustrate how this approach may work in practice. The approach described is similar in many respects to those currently being implemented by the U.S. EPA’s Integrated Risk Information System Program and the U.S. National Toxicology Program.