An integrated in vitro carcinogenicity test that distinguishes between genotoxic carcinogens, non-genotoxic carcinogens, and non-carcinogens.

Chemical safety testing plays a crucial role in product and pharmacological development, as well as chemoprevention; however, in vitro genotoxicity safety tests do not always accurately predict the chemicals that will be in vivo carcinogens. If chemicals test positive in vitro for genotoxicity but negative in vivo, this can contribute to unnecessary testing in animals used to confirm erroneous in vitro positive results. Current in vitro tests typically evaluate only genotoxicity endpoints, which limits their potential to detect non-genotoxic carcinogens. The frequency of misleading in vitro positive results can be high, leading to a requirement for more informative in vitro tests. It is now recognized that multiple-endpoint genotoxicity testing may aid more accurate detection of carcinogens and non-carcinogens. The objective of this review was to evaluate the utility of our novel, multiple-endpoint in vitro test, which uses multiple cancer-relevant endpoints to predict carcinogenic potential. The tool assessed micronucleus frequency, p53 expression, p21 expression, mitochondrial respiration, cell cycle abnormalities and, uniquely, cell morphology changes in human lymphoblastoid cell lines, TK6 and MCL-5. The endpoints were used to observe cellular responses to 18 chemicals within the following categories: genotoxic carcinogens, non-genotoxic carcinogens, toxic non-carcinogens, and misleading in vitro positive and negative agents. The number of endpoints significantly altered for each chemical was considered, alongside the holistic Integrated Signature of Carcinogenicity score, derived from the sum of fold changes for all endpoints. Following the calculation of an overall score from these measures, carcinogens exhibited greater potency than non-carcinogens. Genotoxic carcinogens were generally more potent than non-genotoxic carcinogens. This novel approach therefore demonstrated potential for correctly predicting whether chemicals with unknown mechanism may be considered carcinogens. Overall, while further validation is recommended, the test demonstrates potential for the identification of carcinogenic compounds. Adoption of the approach could enable reduced animal use in carcinogenicity testing.

Carcinogenicity testing in drug development: Getting it right.

For a pharmaceutical drug, carcinogenicity testing occurs in rodents to identify its tumorigenic potential to allow assessment of the risk from its use in humans. Testing takes the form of 2-year studies in mice and rats and/or more recently, a 6-month study in transgenic mice. This paper examines the process of regulatory interaction regarding carcinogenicity testing, notably through the United States (US) Food and Drug Administration (FDA) Special Protocol Assessment (SPA) process to seek Executive Carcinogenicity Assessment Committee (ECAC) approval. The content of 37 submissions to CAC were examined. The paper also examines the outcome from such agency engagement, notably around study dose level selection as well as looking at the design of proposed carcinogenicity study protocols used in submissions (including numbers of animals, control group aspects and toxicokinetic [TK] evaluation). Overall, it was shown that the current process of regulatory interaction allows for studies acceptable to support eventual drug approval and marketing. However, it was established that areas exist to improve the content of submission documents and study design aspects.

Can a 12-gene expression signature predict the cell transforming potential of tumor promoting agents in Bhas 42 cells?

To date, long-term rodent carcinogenesis assays are the only assays recognized by regulators to assess non-genotoxic carcinogens, but their reliability has been questioned. In vitro cell transformation assays (CTAs) could represent an interesting alternative to animal models as it has the advantage of detecting both genotoxic and non-genotoxic transforming chemicals. Among them, Bhas 42 CTA uses a cell line that has been transfected with the oncogenic sequence v-Ha-ras. This sequence confers an "initiated" status to these cells and makes them particularly sensitive to non-genotoxic agents. In a previous work, transcriptomic analysis revealed that the treatment of Bhas 42 cells with transforming silica (nano)particles and 12-O-tetradecanoylphorbol-13-acetate (TPA) commonly modified the expression of 12 genes involved in cell proliferation and adhesion. In the present study, we assess whether this signature would be the same for four other soluble transforming agents, i.e. mezerein, methylarsonic acid, cholic acid and quercetin. The treatment of Bhas 42 cells for 48 h with mezerein modified the expression of the 12 genes of the signature according to the same profile as that of the TPA. However, methylarsonic acid and cholic acid gave an incomplete signature with changes in the expression of only 7 and 5 genes, respectively. Finally, quercetin treatment induced no change in the expression of all genes but exhibited higher cytotoxicty. These results suggest that among the transforming agents tested, some may share similar mechanisms of action leading to cell transformation while others may activate different additional pathways involved in such cellular process. More transforming and non-transforming agents and gene markers should be tested in order to try to identify a relevant gene signature to predict the transforming potential of non-genotoxic agents.

A Human Cell-based Assay to Assess the Induction of Vasculature Formation for Non-genotoxic Carcinogenicity Testing Purposes: A Pilot Study.

The induction of vasculature formation is proposed to be a significant mechanism behind the non-genotoxic carcinogenicity of a chemical. The vasculature formation model used in this study is based on the coculture of human primary HUVECs and hASCs. This model was used to develop an assay to assess the induction of vasculature formation. Three assay protocols, based on different conditions, were developed and compared in order to identify the optimal conditions required. Some serum supplements and growth factors were observed to be essential for initiating vasculature formation. Of the studied putative positive reference chemicals, aspartame, sodium nitrite, bisphenol A and nicotine treatment led to a clear induction of vasculature formation, but arsenic and cadmium treatment only led to a slight increase. This human cell-based assay has the potential to be used as one test within a next generation testing battery, to assess the non-genotoxic carcinogenicity of a chemical through the mechanism of vasculature formation induction.

The Cell Transformation Assay: A Historical Assessment of Current Knowledge of Applications in an Integrated Approach to Testing and Assessment for Non-Genotoxic Carcinogens.

The history of the development of the cell transformation assays (CTAs) is described, providing an overview of in vitro cell transformation from its origin to the new transcriptomic-based CTAs. Application of this knowledge is utilized to address how the different types of CTAs, variously addressing initiation and promotion, can be included on a mechanistic basis within the integrated approach to testing and assessment (IATA) for non-genotoxic carcinogens. Building upon assay assessments targeting the key events in the IATA, we identify how the different CTA models can appropriately fit, following preceding steps in the IATA. The preceding steps are the prescreening transcriptomic approaches, and assessment within the earlier key events of inflammation, immune disruption, mitotic signaling and cell injury. The CTA models address the later key events of (sustained) proliferation and change in morphology leading to tumor formation. The complementary key biomarkers with respect to the precursor key events and respective CTAs are mapped, providing a structured mechanistic approach to represent the complexity of the (non-genotoxic) carcinogenesis process, and specifically their capacity to identify non-genotoxic carcinogenic chemicals in a human relevant IATA.

Evaluation of functional candidate biomarkers of non-genotoxic hepatocarcinogenicity in human liver spheroid co-cultures.

Validated in vitro assays for testing non-genotoxic carcinogenic potential of chemicals are currently not available. Consequently, the two-year rodent bioassay remains the gold standard method for the identification of these chemicals. Transcriptomic and proteomic analyses have provided a comprehensive understanding of the non-genotoxic carcinogenic processes, however, functional changes induced by effects at transcriptional and translational levels have not been addressed. The present study was set up to test a number of proposed in vitro biomarkers of non-genotoxic hepatocarcinogenicity at the functional level using a translational 3-dimensional model. Spheroid cultures of human hepatocytes and stellate cells were exposed to 5 genotoxic carcinogenic, 5 non-genotoxic carcinogenic, and 5 non-carcinogenic chemical compounds and assessed for oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, apoptosis, and inflammation. The spheroid model could capture many of these events triggered by the genotoxic carcinogenic chemicals, particularly aflatoxin B1 and hydroquinone. Nonetheless, no clear distinction could be made between genotoxic and non-genotoxic hepatocarcinogenicity. Therefore, spheroid cultures of human liver cells may be appropriate in vitro tools for mechanistic investigation of chemical-induced hepatocarcinogenicity, however, these mechanisms and their read-outs do not seem to be eligible biomarkers for detecting non-genotoxic carcinogenic chemicals.

Short-term carcinogenicity study of N-methyl-N-nitrosourea in FVB-Trp53 heterozygous mice.

Carcinogenicity tests predict the tumorigenic potential of various substances in the human body by studying tumor induction in experimental animals. There is a need for studies that explore the use of FVB/N-Trp53em2Hwl/Korl (FVB-Trp53+/-) mice, created by TALEN-mediated gene targeting in Korea, in carcinogenicity tests. This study was performed to determine whether FVB-Trp53+/- mice are a suitable model for short-term carcinogenicity studies. To compare the carcinogenicity at different concentrations, 25, 50, and 75 mg/kg of N-methyl-N-nitrosourea (MNU), a known carcinogen, were administered intraperitoneally to FVB-Trp53+/- and wild-type male mice. After 26 weeks, the survival rate was significantly reduced in FVB-Trp53+/- mice compared to the wild-type mice in the 50 and 75 mg/kg groups. The incidence of thymic malignant lymphoma (TML) in the 50 and 75 mg/kg groups was 54.2 and 59.1% in FVB-Trp53+/- male mice, respectively. TML metastasized to the lungs, spleen, lymph nodes, liver, kidney, and heart in FVB-Trp53+/- male mice. Furthermore, the incidence of primary lung tumors, such as adenomas and adenocarcinomas, was 65.4, 62.5, and 45.4% in the FVB-Trp53+/- mice of the 25, 50, and 75 mg/kg groups, respectively. The main tumor types in FVB-Trp53+/- mice were TML and primary lung tumors, regardless of the dose of MNU administered. These results suggest that systemic tumors may result from malfunctions in the p53 gene and pathway, which is an important factor in the pathogenesis of human cancers. Therefore, FVB-Trp53 heterozygous mice are suitable for short-term carcinogenicity tests using positive carcinogens, and that the best result using MNU, a positive carcinogen, might have a single dose of 50 mg/kg.

Analyses of Transcriptomics Cell Signalling for Pre-Screening Applications in the Integrated Approach for Testing and Assessment of Non-Genotoxic Carcinogens.

With recent rapid advancement of methodological tools, mechanistic understanding of biological processes leading to carcinogenesis is expanding. New approach methodologies such as transcriptomics can inform on non-genotoxic mechanisms of chemical carcinogens and can be developed for regulatory applications. The Organisation for the Economic Cooperation and Development (OECD) expert group developing an Integrated Approach to the Testing and Assessment (IATA) of Non-Genotoxic Carcinogens (NGTxC) is reviewing the possible assays to be integrated therein. In this context, we review the application of transcriptomics approaches suitable for pre-screening gene expression changes associated with phenotypic alterations that underlie the carcinogenic processes for subsequent prioritisation of downstream test methods appropriate to specific key events of non-genotoxic carcinogenesis. Using case studies, we evaluate the potential of gene expression analyses especially in relation to breast cancer, to identify the most relevant approaches that could be utilised as (pre-) screening tools, for example Gene Set Enrichment Analysis (GSEA). We also consider how to address the challenges to integrate gene panels and transcriptomic assays into the IATA, highlighting the pivotal omics markers identified for assay measurement in the IATA key events of inflammation, immune response, mitogenic signalling and cell injury.

10% Body weight (gain) change as criterion for the maximum tolerated dose: A critical analysis.

The concept of the Maximum Tolerated Dose (MTD) was introduced in the seventies for carcinogenicity testing and was defined as the highest dose inducing clear toxicity, but not mortality by causes other than cancer. As estimation of the MTD in a carcinogenicity study, the highest dose that causes a 10% decrease in body weight compared to control animals over the course of a 90-day study, was formulated as a suitable criterion. This criterion was not seen as indicator of excessive toxicity but as a means to avoid false negative outcomes in a carcinogenicity study, as tumor formation may be reduced when body weight is significantly decreased. The body weight-based MTD criterion, however, turned up in carcinogenicity test guidelines and guidance (e.g., from OECD) as the highest dose that causes a 10% decrease in body weight gain relative to controls. Moreover, the 10% decrease in body weight gain criterion for MTD also ended up in test guidelines and guidances for toxicity endpoints other than carcinogenicity, so outside the context it was intended for. A 10% decrease in body weight gain relative to controls is however not a biologically relevant effect as it corresponds to less than 3% body weight reduction relative to controls in a 90-day study, which is within the normal variation in body weight. It therefore should certainly not be considered as a condition of excessive toxicity. Using the 10% lower weight gain criterion and incorrectly associating it with excessive toxicity has major implications for top dose selection in regulatory safety studies, resulting in tests performed at doses too low to elicit toxicity. This negatively impacts the reliability of studies and their regulatory usability; moreover, it results in a waste of experimental animals, which is ethically highly undesirable. Hence, our plea is to remove this MTD criterion for top dose selection in test guidelines and guidances for toxicity endpoints other than carcinogenicity and to reinstall the original 10% decrease in body weight criterion in test guidelines and guidances for carcinogenicity.

Gene Expression over Time during Cell Transformation Due to Non-Genotoxic Carcinogen Treatment of Bhas 42 Cells.

The Bhas 42 cell transformation assay (Bhas 42 CTA) is the first Organization for Economic Cooperation and Development (OECD)-certificated method used as a specific tool for the detection of the cell-transformation potential of tumor-promoting compounds, including non-genotoxic carcinogens (NGTxCs), as separate from genotoxic carcinogens. This assay offers the great advantage of enabling the phenotypic detection of oncotransformation. A key benefit of using the Bhas 42 CTA in the study of the cell-transformation mechanisms of tumor-promoting compounds, including non-genotoxic carcinogens, is that the cell-transformation potential of the chemical can be detected directly without treatment with a tumor-initiating compound since Bhas 42 cell line was established by transfecting the v-Ha-ras gene into a mouse fibroblast cloned cell line. Here, we analyzed the gene expression over time, using DNA microarrays, in Bhas 42 cells treated with the tumor-promoting compound 12-O-tetradecanoylphorbol-13-acetate (TPA), and NGTxC, with a total of three repeat experiments. This is the first paper to report on gene expression over time during the process of cell transformation with only a tumor-promoting compound. Pathways that were activated or inactivated during the process of cell transformation in the Bhas 42 cells treated with TPA were related not only directly to RAS but also to various pathways in the hallmarks of cancer.

In Silico Methods for Carcinogenicity Assessment.

Screening compounds for potential carcinogenicity is of major importance for prevention of environmentally induced cancers. A large sequence of predictive models, ranging from short-term biological assays (e.g., mutagenicity tests) to theoretical models, has been attempted in this field. Theoretical approaches such as (Q)SAR are highly desirable for identifying carcinogens, since they actively promote the replacement, reduction, and refinement of animal tests. This chapter reports and describes some of the most noted (Q)SAR models based on human expert knowledge and statistical approaches, aiming at predicting the carcinogenicity of chemicals. Additionally, the performance of the selected models has been evaluated, and the results are interpreted in details by applying these predictive models to some pharmaceutical molecules.

Carcinogenicity Evaluation of Baclofen in TgrasH2 Mice.

Baclofen is a γ-aminobutyric acid-B receptor agonist used for control of spastic muscle activity and as a treatment for alcohol abuse. The review of the nonclinical database suggested a data gap for potential carcinogenicity following long-term use. Regulatory requirements for pharmaceutical safety testing of cancer-causing potential have historically included 2-year rodent studies in rats and mice. The availability of transgenic models with greater specificity and sensitivity to carcinogens provides safety testing alternatives that align with the 3Rs. The carcinogenicity of baclofen was evaluated in CB6F1-TgrasH2 transgenic mice following daily oral administration at 45, 90, and 180 mg/kg/d for 26 weeks, preceded by a 2-week drug-conditioning period. There were no treatment-related palpable masses or neoplastic findings, and survival rates were not affected by the baclofen treatment. In conclusion, baclofen was considered as noncarcinogenic in CB6F1-TgrasH2 mice, which is consistent with results previously obtained in a 2-year rat study.

Employing an adverse outcome pathway framework for weight-of-evidence assessment with application to the ICH S1B guidance addendum.

Across industry, there is a paradigm shift occurring for carcinogenicity testing, with the focus moving from long term animal studies to alternative approaches. Based on the explorative work done in recent years, the International Council for Harmonization (ICH) recently published a draft addendum to the S1B guidance, which allows for a weight-of-evidence (WoE) assessment to be conducted based on data gathered throughout the pharmaceutical development process and literature to mitigate some testing in rodents if the body of evidence clearly shows undertaking an animal lifetime study would not add value to the risk assessment. While several alternative approaches already exist, and other new approach methodologies (NAMs) are being explored, all of which can contribute to this WoE, it is important that all the evidence can be combined in a meaningful and consistent way to reach a conclusion. Adverse outcome pathways have been advocated as a framework for organising evidence in an integrated approach to testing and assessment, which gives context to data and can aid reaching a conclusion as to the adverse outcome (AO). This approach can be combined with a reasoning methodology to give a prediction for an AO and applied to the factors which need to be considered for the ICH S1B WoE to predict for carcinogenicity. Using this approach to the WoE assessment, consistent, scientifically robust, and transparent calls can be made as to whether conducting an animal carcinogenicity study would add value to a human risk assessment and mitigate the need to run animal studies unnecessarily.

Opportunities and challenges related to saturation of toxicokinetic processes: Implications for risk assessment.

Top dose selection for repeated dose animal studies has generally focused on identification of apical endpoints, use of the limit dose, or determination of a maximum tolerated dose (MTD). The intent is to optimize the ability of toxicity tests performed in a small number of animals to detect effects for hazard identification. An alternative approach, the kinetically derived maximum dose (KMD), has been proposed as a mechanism to integrate toxicokinetic (TK) data into the dose selection process. The approach refers to the dose above which the systemic exposures depart from being proportional to external doses. This non-linear external-internal dose relationship arises from saturation or limitation of TK process(es), such as absorption or metabolism. The importance of TK information is widely acknowledged when assessing human health risks arising from exposures to environmental chemicals, as TK determines the amount of chemical at potential sites of toxicological responses. However, there have been differing opinions and interpretations within the scientific and regulatory communities related to the validity and application of the KMD concept. A multi-stakeholder working group, led by the Health and Environmental Sciences Institute (HESI), was formed to provide an opportunity for impacted stakeholders to address commonly raised scientific and technical issues related to this topic and, more specifically, a weight of evidence approach is recommended to inform design and dose selection for repeated dose animal studies. Commonly raised challenges related to the use of TK data for dose selection are discussed, recommendations are provided, and illustrative case examples are provided to address these challenges or refute misconceptions.

GOLPH3/CKAP4 promotes metastasis and tumorigenicity by enhancing the secretion of exosomal WNT3A in non-small-cell lung cancer.

Cancer metastasis is the main cause of mortality associated with non-small-cell lung cancer (NSCLC), accounting for up to 70% of deaths among patients. The mechanisms underlying distal metastasis remain largely unknown. Golgi phosphoprotein 3 (GOLPH3) correlates negatively with overall survival in multiple tumors. In this study, we evaluated the function of GOLPH3 in NSCLC distal metastasis. GOLPH3 was expressed at high levels in samples from patients with NSCLC and was positively associated with clinicopathologic characteristics including clinical stage (P < 0.001), T (P = 0.001), N (P = 0.007), and M (P = 0.001) classification. Functionally, Transwell and wound-healing assays suggested that GOLPH3 overexpression enhances NSCLC cell migration and invasion abilities. Tumor-sphere formation and flow cytometry assays demonstrated that GOLPH3 overexpression enhances a stem cell-like phenotype of NSCLC cells. Metastasis models established by tail vein and intracardiac injection confirmed the pro-metastatic function of GOLPH3 in vivo. A subcutaneous tumor formation model confirmed that GOLPH3 overexpression increased the tumorigenicity of NSCLC cells. Mechanistically, gene set enrichment analysis revealed a positive association of GOLPH3 mRNA expression with WNT-activated gene signatures. Luciferase-reporter and nuclear extract assays showed that GOLPH3 overexpression enhances metastasis and tumorigenicity through activation of the WNT/ß-catenin pathway. Immunoprecipitation-mass spectrometry and gene ontology analysis demonstrated that GOLPH3 interacts with cytoskeleton-associated protein 4 (CKAP4) in exosome-mediated distal metastasis. We found that GOLPH3 decreased the amount of plasma membrane-localized CKAP4 and increased the amount of exosome-localized CKAP4 to promote the formation of CKAP4-containing exosomes. Furthermore, we demonstrated that CKAP4 binds exosomal WNT3A to enhance its secretion. Therefore, the GOLPH3/CKAP4 axis plays a crucial role in promoting exosomal-WNT3A secretion to enhance and maintain the stem-like phenotype and metastasis in NSCLC, thus indicating the therapeutic potential of GOLPH3 in patients with NSCLC metastasis.

Dynamically monitoring cellular γ-H2AX reveals the potential of carcinogenicity evaluation for genotoxic compounds.

Amongst all toxicological endpoints, carcinogenicity might pose the greatest concern. Genetic damage has been considered an important underlying mechanism for the carcinogenicity of chemical substances. The demand for in vitro genotoxic tests as alternative approaches is growing rapidly with the implementation of new regulations for compounds. However, currently available in vitro genotoxicity tests are often limited by relatively high false positive rates. Moreover, few studies have explored carcinogenicity potential by in vitro genotoxicity testing due to the shortage of suitable toxicological biomarkers to link gene damage with cancer risk. γ-H2AX is a recently acknowledged attractive endpoint (biomarker) for evaluating DNA damage and can simultaneously reflect the DNA damage response and repair of cells. We previously reported an ultrasensitive and reliable method, namely stable-isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), for detecting cellular γ-H2AX and evaluating genotoxic chemicals. More importantly, our method can dynamically monitor the specific processes of genotoxic compounds affecting DNA damage and repair reflected by the amount of γ-H2AX. To clarify the possibility of using this method to assess the potential carcinogenicity of genotoxic chemicals, we applied it to a set of 69 model compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), with already-characterized genotoxic potential. Compared to conventional in vitro genotoxicity assays, including the Ames test, the γ-H2AX assay by MS has high accuracy (94-96%) due to high sensitivity and specificity (88% and 100%, respectively). The dynamic profiles of model compounds after exposure in HepG2 cells were explored, and a mathematical approach was employed to simulate and quantitatively model the DNA repair kinetics of genotoxic carcinogens (GCs) based on γ-H2AX time-effect curves up to 8 h. Two crucial parameters, i.e., k (rate of γ-H2AX decay) and t50 (time required for γ-H2AX from maximum decrease to half) estimated by the least squares method, were achieved. An open web server to help researchers calculate these two key parameters and profile simulated curves of the tested compound is available online ( http://ccb1.bmi.ac.cn:81/shiny-server/sample-apps/prediction1/ ). We detected a positive association between carcinogenic levels and k and t50 values of γ-H2AX in tested GCs, validating the potential of using this MS-based γ-H2AX in vitro assay to help preliminarily evaluate carcinogenicity and assess genotoxicity. This approach may be used alone or integrated into an existing battery of in vitro genetic toxicity tests.

Applicability of Scrape Loading-Dye Transfer Assay for Non-Genotoxic Carcinogen Testing.

Dysregulation of gap junction intercellular communication (GJIC) is recognized as one of the key hallmarks for identifying non-genotoxic carcinogens (NGTxC). Currently, there is a demand for in vitro assays addressing the gap junction hallmark, which would have the potential to eventually become an integral part of an integrated approach to the testing and assessment (IATA) of NGTxC. The scrape loading-dye transfer (SL-DT) technique is a simple assay for the functional evaluation of GJIC in various in vitro cultured mammalian cells and represents an interesting candidate assay. Out of the various techniques for evaluating GJIC, the SL-DT assay has been used frequently to assess the effects of various chemicals on GJIC in toxicological and tumor promotion research. In this review, we systematically searched the existing literature to gather papers assessing GJIC using the SL-DT assay in a rat liver epithelial cell line, WB-F344, after treating with chemicals, especially environmental and food toxicants, drugs, reproductive-, cardio- and neuro-toxicants and chemical tumor promoters. We discuss findings derived from the SL-DT assay with the known knowledge about the tumor-promoting activity and carcinogenicity of the assessed chemicals to evaluate the predictive capacity of the SL-DT assay in terms of its sensitivity, specificity and accuracy for identifying carcinogens. These data represent important information with respect to the applicability of the SL-DT assay for the testing of NGTxC within the IATA framework.

Categorizing the characteristics of human carcinogens: a need for specificity.

The International Agency for Research on Cancer (IARC) has recently proposed employing "ten key characteristics of human carcinogens" (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

Integration of data across toxicity endpoints for improved safety assessment of chemicals: the example of carcinogenicity assessment.

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.