Carcinogenicity Testing Research Articles

Design, synthesis, molecular prediction and biological evaluation of pyrazole-azomethine conjugates as antimicrobial agents

A series of Schiff bases linked with heterocyclic moiety (6a–c, 7a–c, 11a–c, 12a–c, and 13a–c) and ferrocenyl Schiff bases (15a–c) were designed, synthesized, and confirmed based on the spectral data. Also, evaluations of their in vitro antibacterial and antifungal activities were performed and some Schiff bases (7a, 13a, b, and 15b) show moderate activities. Moreover, the physicochemical, pharmacokinetic, lipophilicity properties, druglikeness, ADME prediction, and toxicity properties of the newly Schiff bases (6a–c, 7a–c, 11a–c, 12a–c, and 13a–c) were predicted in silico. The results exhibited that some Schiff bases agree with druglikeness rules, inhibition of some CYP isoenzymes, and predicted as negative values on the carcinogenicity test. Also, all the Schiff bases exhibited more than 70% on the human intestinal absorption test, moderate permeation on the in vitro Caco-2 cell permeability test, and low permeability on the in vitro MDCK cell permeability test.

Benchmark dose risk analysis with mixed-factor quantal data in environmental risk assessment.

Benchmark analysis is a general risk estimation strategy for identifying the benchmark dose (BMD) past which the risk of exhibiting an adverse environmental response exceeds a fixed, target value of benchmark response (BMR). Estimation of BMD and of its lower confidence limit (BMDL) is well understood for the case of an adverse response to a single stimulus. In many environmental settings, however, one or more additional, secondary, qualitative factor(s) may collude to affect the adverse outcome, such that the risk changes with differential levels of the secondary factor. This paper extends the single-dose BMD paradigm to a mixed-factor setting with a secondary qualitative factor possessing two levels. With focus on quantal-response data and using a generalized linear model with a complementary-log link function, we derive expressions for BMD and BMDL. We study the operating characteristics of six different multiplicity-adjusted approaches to calculate the BMDL, using Monte Carlo evaluations. We illustrate the calculations via an example data set from environmental carcinogenicity testing.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS

BackgroundConflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies).ResultsTwo of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay.ConclusionsThe usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

Comet Assay Evaluation of Lanthanum Nitrate DNA Damage in C57-ras Transgenic Mice.

Due to the wide application of rare-earth elements (REEs) in the last decades, lanthanum has increasingly entered the environment and has gradually accumulated in the human body through the food chain. Lanthanum is worth paying attention in terms of food safety. Although the genotoxicity of lanthanum has been studied in vitro, data on its DNA damage in vivo rodent are limited, moreover, which have also presented some controversy. This study aimed to conduct an in vivo rodent alkaline comet assay, and as a companion test to the lanthanum nitrate carcinogenicity test. We conducted an oral gavage experiment for 180days (26weeks) to test for the persistence of DNA damage of long-term low-dose accumulation of lanthanum nitrate (12.5, 25, and 50mg/kg body weight), in F1 hybrid C57-ras transgenic mice (CB6F1) by using alkaline comet assay in the blood and liver. The comet assay revealed that all the tested concentrations of lanthanum nitrate did not induce DNA damage in any of the tissues investigated, whereas DNA damage was induced in the positive control group. These results could indicate that lanthanum nitrate can accumulate in tissues and organs of the mice after exposure, and does not possess DNA damage in C57-ras transgenic mice after repeated treatments at oral doses up to 50mg/kg·BW for 26weeks; also, it did not cause pathological changes in the liver of the mice.

Characteristics of Medical Products Comprising Human Cells, Genes, or Tissues Developed in Japan and the European Union Compared via Public Assessment Reports.

Medical products comprising human cells, genes, and tissues have been developed for clinical applications worldwide, and their developmental environment has been established. These products can be imported and exported, but marketing authorization regulations are complicated among regions. This investigation was conducted to identify the characteristics of medical products comprising human cells, genes, and tissues. We used website data, books from survey companies, and reports from public agencies to conduct two investigations. We used website data to conduct a general information survey of 143 cell-therapy and gene-therapy products sold in 24 countries and public assessment reports to individually survey non-clinical and clinical developments of 18 cell-therapy and gene-therapy products developed in Japan and the European Union (EU). The first survey revealed that the numbers of products used in orthopedic surgery and dermatology have increased since 2000, and the numbers of hematological products have increased since 2011. The second investigation revealed that fewer orphaned products were developed in Japan than in the EU. The most appropriate dose was 1.2 × 108 cells per injection per adult. Clinical trials to determine the most appropriate dose were conducted in the EU but not in Japan. No non-clinical immunogenicity tests for autogenous products were conducted in Japan or the EU. Pharmacokinetics tests were not individually performed for sheet-form products. Both in vivo and in vitro pharmacological tests were more likely to be conducted in the EU, while only one or the other was conducted in Japan. Furthermore, in Japan, carcinogenicity tests were performed based on non-clinical technical guidance, while in the EU, these tests were determined according to each product's features. Fewer clinical trials were performed, and fewer subjects per product were used in Japan than in the EU. Many aspects of the clinical and non-clinical development of medical products comprising human cells, genes, and tissues differ between Japan and the EU. Analyzing these differences will enable the safe and rapid distribution of these products to clinical sites.

A rat subchronic study transcriptional point of departure estimates a carcinogenicity study apical point of departure

Considerations of human relevance and animal use are driving research to identify new approaches to inform risk assessment of chemicals and replace guideline-based rodent carcinogenicity tests. Here, the hypothesis was tested across four agrochemicals that 1) a rat 90-day transcriptome-based BEPOD is protective of a rat carcinogenicity study and 2) a subchronic liver or kidney BEPOD would approximate a cancer bioassay apical POD derived from other organs and a rat subchronic BEPOD would approximate a mouse cancer bioassay apical POD. Using RNA sequencing and BMDExpress software, liver and/or kidney BEPOD values were generated in male rats exposed for 90 days to either Triclopyr Acid, Pronamide, Sulfoxaflor, or Fenpicoxamid. BEPOD values were compared to benchmark dose-derived apical POD values generated from rat 90-day and rodent carcinogenicity studies. Across all four agrochemicals, findings showed that a rat 90-day study BEPOD approximated the most sensitive apical POD (within 10-fold) generated from the 90-day rat study and long-term rodent carcinogenicity studies. This study supports the conclusion that a subchronic transcriptome-based BEPOD could be utilized to estimate an apical POD within a risk-based approach of chronic toxicity and carcinogenicity agrochemical assessment, abrogating the need for time- and resource-intensive rodent carcinogenicity studies and minimizing animal testing.

Quantification of cancer driver mutations in human breast and lung DNA using targeted, error-corrected CarcSeq.

There is a need for scientifically‐sound, practical approaches to improve carcinogenicity testing. Advances in DNA sequencing technology and knowledge of events underlying cancer development have created an opportunity for progress in this area. The long‐term goal of this work is to develop variation in cancer driver mutation (CDM) levels as a metric of clonal expansion of cells carrying CDMs because these important early events could inform carcinogenicity testing. The first step toward this goal was to develop and validate an error‐corrected next‐generation sequencing method to analyze panels of hotspot cancer driver mutations (hCDMs). The “CarcSeq” method that was developed uses unique molecular identifier sequences to construct single‐strand consensus sequences for error correction. CarcSeq was used for mutational analysis of 13 amplicons encompassing >20 hotspot CDMs in normal breast, normal lung, ductal carcinomas, and lung adenocarcinomas. The approach was validated by detecting expected differences related to tissue type (normal vs. tumor and breast vs. lung) and mutation spectra. CarcSeq mutant fractions (MFs) correlated strongly with previously obtained ACB‐PCR mutant fraction (MF) measurements from the same samples. A reconstruction experiment, in conjunction with other analyses, showed CarcSeq accurately quantifies MFs ≥10−4. CarcSeq MF measurements were correlated with tissue donor age and breast cancer risk. CarcSeq MF measurements were correlated with deviation from median MFs analyzed to assess clonal expansion. Thus, CarcSeq is a promising approach to advance cancer risk assessment and carcinogenicity testing practices. Paradigms that should be investigated to advance this strategy for carcinogenicity testing are proposed.

Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action

Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound’s subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001–770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the “misleading” in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.

Abstract 6119: RNAi rat models for drug discovery

Abstract The cost of drug development has skyrocketed to an estimated $2.6B for every FDA approved drug primarily due to failures from lack of efficacy or safety. This suggests that our current preclinical validation process has been insufficient in predicting therapeutic potential and toxicity in humans. While genetically engineered mice have become the gold standard for dissecting cancer mechanisms and evaluating novel drug targets in vivo, the rat has historically been the major model species in many biomedical fields, notably toxicology and carcinogenicity testing; and for many scientists, the rat still remains the preferred rodent due to their larger size for surgical manipulation, repeat blood sampling, and their cognitive and physiological characteristics that more closely resemble humans than their mouse counterparts. Moreover, many hormone-dependent tumors cannot be engineered in mouse models and are better modeled in rats. Here, we take advantage of our two-step engineering approach and exploit the efficiency of CRISPR-based targeting to develop RNAi rat models that enable inducible and reversible gene silencing to simulate therapeutic regimes. We demonstrate that our approach allows us to rapidly generate RNAi rat models and mimic the function of the targeted small molecule inhibitors, such as BET inhibitors targeting Brd4. These results demonstrate that our high-throughput system currently used to generate RNAi mice is also applicable to the rat system and, by extension, other mammalian models. Inducible RNAi rat models will undoubtedly be powerful tools that can be used to model human cancers, to mimic the action of putative drugs, and to assess the potential of therapeutic targeting strategies in vivo prior to the costly drug development, ultimately guiding the development of safer and more effective drugs. Citation Format: Chia-Lin Wang, Yu-ting Yang, Ana Vasileva, Allison Maurice, Lukas Dow, Johannes Zuber, Scott Lowe, Prem K. Premsrirut. RNAi rat models for drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6119.

EVALUACIÓN DE LA CITOGENOTOXICIDAD DE UNA CERVEZA ELABORADA INDUSTRIALMENTE Y UNA CERVEZA DE ELABORACIÓN ARTESANAL

<p class="Default">Beer is a complex beverage, due to the extended variety of dissolved constituents which interact with each other and may change rapidly from the moment of exposure to the environment. Much has been said about the impact on human health on the consumption of beer; excessive intake can lead to health problems, on the contrary with the moderate intake can significantly reduce DNA damage. The importance of short-term tests for carcinogens and mutagens (micronucleus test) is feasible to be applied in risk analysis and quality control of commercial alcohol beverages. The objective of this study was to evaluate the genotoxic effects in vivo of two different types of beers using <em>Vicia faba</em>micronucleus test. Faba beans were used (<em>Vicia faba var. minor</em>) that were exposed for 24, 48, 72 and 120 hours to the chemical composition of a mass-produced beer and craft beer. The cytogenotoxic effect from the beers was evaluated in root meristematic cells. Cycloheximide (5 <em>u</em>g/ml), 3.6% ethanol (v/v) and 7.5% (v/v) were used as positive controls. Data were applied by analysis of variance with individual comparisons (Dunnett’s test) with a significance criterion of <em>p</em>< 0.05. The results allowed toestablishing that treatments for the mitotic index and the frequency of micronuclei in meristematic cells had significant differences compared to the negative control. In conclusion, the chemical components of these alcoholic beverages and acetaldehyde derived from metabolized ethanol by the root neristematic cells of <em>V.faba</em>did not induce high frequencies of micronuclei, in other words, they were slightly genotoxic and mildly toxic by decreasing or increasing of cell division on root meristem cells of <em>V. faba</em>.</p>

Tg.rasH2 Mouse Model for Assessing Carcinogenic Potential of Pharmaceuticals: Industry Survey of Current Practices.

The Tg.rasH2 mouse was developed as an alternative model to the traditional 2-year mouse bioassay for pharmaceutical carcinogenicity testing. This model has found extensive use in support of pharmaceutical drug development over the last few decades. It has the potential to improve quality and timeliness, reduce animal usage, and in some instances allow expedient decision-making regarding the human carcinogenicity potential of a drug candidate. Despite the increased use of the Tg.rasH2 model, there has been no systematic survey of current practices in the design, interpretation of results from the bioassay, and global health authority perspectives. Therefore, the aim of this work was to poll the pharmaceutical industry on study design practices used in the dose range finding and definitive 6-month studies and on results relative to the ongoing negotiations to revise The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S1 Guidance. Twenty-two member companies of International Consortium for Innovation and Quality in Pharmaceutical Development DruSafe Leadership Group participated in the survey, sharing experiences from studies conducted with 55 test compounds between 2010 and 2018. The survey results provide very useful insights into study design and interpretation. Importantly, the results identified several key opportunities for reducing animal use and increasing the value of testing for potential human carcinogenicity using this model. Recommended changes to study designs that would reduce animal usage include eliminating the requirement to include positive control groups in every study, use of nontransgenic wild-type littermates in the dose range finding study, and use of microsampling to reduce or eliminate satellite groups for toxicokinetics.

Biological Evaluation and Molecular Docking with In Silico Physicochemical, Pharmacokinetic and Toxicity Prediction of Pyrazolo[1,5-a]pyrimidines.

Pyrazolo[1,5-a]pyrimidines 5a–c, 9a–c and 13a–i were synthesized for evaluation of their in vitro antimicrobial properties against some microorganisms and their immunomodulatory activity. The biological activities of pyrazolo[1,5-a]pyrimidines showed that the pyrazolo[1,5-a]pyrimidines (5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h) displayed promising antimicrobial and immunomodulatory activities. Studying the in silico predicted physicochemical, pharmacokinetic, ADMET and drug-likeness properties for the pyrazolo[1,5-a]pyrimidines 5a–c, 9a–c and 13a–i confirmed that most of the compounds (i) were within the range set by Lipinski’s rule of five, (ii) show higher gastrointestinal absorption and inhibition of some CYP isoforms, and (iii) have a carcinogenicity test that was predicted as negative and hERG test that presented medium risk. Moreover, the molecular docking study demonstrated that the compounds 5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h are potent inhibitors of 14-alpha demethylase, transpeptidase and alkaline phosphatase enzymes. This study could be valuable in the discovery of a new series of drugs.

Community-based burden, warning signs, and risk factors of cancer using public-private partnership model in Kerala, India.

Background:According to the GBD report published in 2016, the burden of cancer in Kerala is 135.3/100,000 population in contrast to the national average of 100/100,000 population. Cancer is a complex disease that requires broad engagement of various departments and organizations to implement a community based health promotion strategy.Objective:To estimate the prevalence of diagnosed cancers, warning signs and selected risk factors of cancer in Niranam Panchayath of Pathanamthitta district, Kerala.Methodology:A total of 13,736 population was covered by door to door survey using a structured questionnaire. The questionnaire collected information on the sociodemographic variables of the residents, source of water supply, warning signs of cancer and details of diagnosed cancer cases.Results:The mean age of the population was 39.7 ± 21 years. The prevalence of diagnosed cases of cancer in our study was 652/100,000 population. Most common type of cancer identified was Breast cancer (37.3%). The prevalence of any warning sign among the study population was 400/100,000 population. Breast lump was the common warning sign identified. Increasing age, female gender and occupational status were the factors found to be significantly associated with cancer.Recommendations:Community based health education to increase awareness, screening for cancers and breast self-examination in the community could help in early diagnosis and prevention at primary level. Scientific study to assess the risk factors of cancers using case control design could be done in this population along with soil and water sample testing for carcinogens.

Gene expression and cytosine DNA methylation alterations in induced pluripotent stem-cell-derived human hepatocytes treated with low doses of chemical carcinogens.

The increasing number of man-made chemicals in the environment that may pose a carcinogenic risk emphasizes the need to develop reliable time- and cost-effective approaches for carcinogen detection. To address this issue, we have investigated the utility of human hepatocytes for the in vitro identification of genotoxic and non-genotoxic carcinogens. Induced pluripotent stem-cell (iPSC)-derived human hepatocytes were treated with the genotoxic carcinogens aflatoxin B1 (AFB1) and benzo[a]pyrene (B[a]P), the non-genotoxic liver carcinogen methapyrilene, and the non-carcinogens aflatoxin B2 (AFB2) and benzo[e]pyrene (B[e]P) at non-cytotoxic concentrations for 7days, and transcriptomic and DNA methylation profiles were examined. 1569, 1693, and 2061 differentially expressed genes (DEGs) were detected in cells treated with AFB1, B[a]P, and methapyrilene, respectively, whereas no DEGs were found in cells treated with AFB2 or B[e]P. In contrast to the profound cellular transcriptomic responses, exposure of iPSC-derived hepatocytes to the test chemicals resulted in minor random alterations in global DNA methylome, most of which were not associated with changes in gene expression. Overall, our results demonstrate that the major non-genotoxic effect of exposure to carcinogens, regardless of their mode of action, is a profound global transcriptomic response rather than global DNA methylome alterations, indicating the significance of transcriptomic alterations as an informative endpoint in short-term in vitro carcinogen testing.

Development of a Novel AOP for Cyp2F2-Mediated Lung Cancer in Mice.

Traditional methods for carcinogenicity testing rely heavily on the rodent bioassay as the standard for identification of tumorigenic risk. As such, identification of species-specific outcomes and/or metabolism are a frequent argument for regulatory exemption. One example is the association of tumor formation in the mouse lung after exposure to Cyp2F2 ligands. The adverse outcome pathway (AOP) framework offers a theoretical platform to address issues of species specificity that is consistent, transparent, and capable of integrating data from new approach methodologies as well as traditional data streams. A central premise of the AOP concept is that pathway progression from the molecular initiating event (MIE) implies a definable "response-response" (R-R) relationship between each key event (KE) that drives the pathway towards a specific adverse outcome (AO). This article describes an AOP for lung cancer in the mouse from an MIE of Cyp2F2-specific reactive metabolite formation, advancing through KE that include protein and/or nucleic acid adducts, diminished Club Cell 10 kDa (CC10) protein expression, hyperplasia of CC10 deficient Club cells, and culminating in the AO of mixed-cell tumor formation in the distal airways. This tumor formation is independent of route of exposure and our AOP construct is based on overlapping mechanistic events for naphthalene, styrene, ethyl benzene, isoniazid, and fluensulfone in the mouse. This AOP is intended to accelerate the explication of an apparent mouse-specific outcome and serve as a starting point for a quantitative analysis of mouse-human differences in susceptibility to the tumorigenic effects of Cyp2F2 ligands.

Health experts wary of EPA rush to revise carcinogen testing

Health experts wary of EPA rush to revise carcinogen testing

The Carcinogenome Project: In Vitro Gene Expression Profiling of Chemical Perturbations to Predict Long-Term Carcinogenicity.

Background:Most chemicals in commerce have not been evaluated for their carcinogenic potential. The de facto gold-standard approach to carcinogen testing adopts the 2-y rodent bioassay, a time-consuming and costly procedure. High-throughput in vitro assays are a promising alternative for addressing the limitations in carcinogen screening.Objectives:We developed a screening process for predicting chemical carcinogenicity and genotoxicity and characterizing modes of actions (MoAs) using in vitro gene expression assays.Methods:We generated a large toxicogenomics resource comprising expression profiles corresponding to 330 chemicals profiled in HepG2 (human hepatocellular carcinoma cell line) at multiple doses and replicates. Predictive models of carcinogenicity and genotoxicity were built using a random forest classifier. Differential pathway enrichment analysis was performed to identify pathways associated with carcinogen exposure. Signatures of carcinogenicity and genotoxicity were compared with external sources, including Drugmatrix and the Connectivity Map.Results:Among profiles with sufficient bioactivity, our classifiers achieved 72.2% Area Under the ROC Curve (AUC) for predicting carcinogenicity and 82.3% AUC for predicting genotoxicity. Chemical bioactivity, as measured by the strength and reproducibility of the transcriptional response, was not significantly associated with long-term carcinogenicity in doses up to . However, sufficient bioactivity was necessary for a chemical to be used for prediction of carcinogenicity. Pathway enrichment analysis revealed pathways consistent with known pathways that drive cancer, including DNA damage and repair. The data is available at https://clue.io/CRCGN_ABC, and a portal for query and visualization of the results is accessible at https://carcinogenome.org.Discussion:We demonstrated an in vitro screening approach using gene expression profiling to predict carcinogenicity and infer MoAs of chemical perturbations. https://doi.org/10.1289/EHP3986

Mechanisms of DNA-reactive and epigenetic chemical carcinogens: applications to carcinogenicity testing and risk assessment.

Chemicals with carcinogenic activity in either animals or humans produce increases in neoplasia through diverse mechanisms. One mechanism is reaction with nuclear DNA. Other mechanisms consist of epigenetic effects involving either modifications of regulatory macromolecules or perturbation of cellular regulatory processes. The basis for distinguishing between carcinogens that have either DNA reactivity or an epigenetic activity as their primary mechanism of action is detailed in this review. In addition, important applications of information on these mechanisms of action to carcinogenicity testing and human risk assessment are discussed.

Toxicology and carcinogenesis study of dietary zinc (CASRN 5263-02-5) in Sprague Dawley (Hsd:Sprague Dawley SD) rats (feed study).

Zinc is a naturally occurring element and is ubiquitous in the environment. Zinc itself is stable in dry air, but exposure to moist air results in the formation of zinc oxide or basic carbonate. Due to the reactivity of zinc metal, it is not found as a free element in nature but as a variety of different compounds including zinc chloride, zinc oxide, and zinc sulfate. Zinc and zinc compounds are used across a wide range of industries that include rubber production, animal feed supplementation, as a fertilizer additive, in cosmetics and drugs, as a paint pigment, in dental cements, as a wood preservative, in batteries, in galvanizing and metal work, in textile production, in television screens and watches, and in smoke bombs. Of the zinc compounds, zinc oxide is the most widely used. Zinc was nominated by the Agency for Toxic Substances and Disease Registry (ATSDR) for carcinogenicity and genotoxicity testing based on the increasing size of the population exposed to zinc through dietary supplements and the lack of studies examining the carcinogenicity of zinc. There was an additional nomination to investigate the tumorigenicity of zinc deficiency by private individuals as a result of data showing that deficiency of some vitamins and minerals in humans can cause DNA damage. Zinc carbonate basic was selected as the source of dietary zinc due to its use as the source of supplemental zinc in rodent diets. Male and female Hsd:Sprague Dawley SD rats were exposed to dietary zinc in feed for 2 years. Genetic toxicology studies were conducted in rat peripheral blood erythrocytes, peripheral blood leukocytes, and colon epithelial cells. (Abstract Abridged).

A decision tree-based integrated testing strategy for tailor-made carcinogenicity evaluation of test substances using genotoxicity test results and chemical spaces.

Genotoxicity evaluation has been widely used to estimate the carcinogenicity of test substances during safety evaluation. However, the latest strategies using genotoxicity tests give more weight to sensitivity; therefore, their accuracy has been very low. For precise carcinogenicity evaluation, we attempted to establish an integrated testing strategy for the tailor-made carcinogenicity evaluation of test materials, considering the relationships among genotoxicity test results (Ames, in vitro mammalian genotoxicity and in vivo micronucleus), carcinogenicity test results and chemical properties (molecular weight, logKow and 179 organic functional groups). By analyzing the toxicological information and chemical properties of 230 chemicals, including 184 carcinogens in the Carcinogenicity Genotoxicity eXperience database, a decision tree for carcinogenicity evaluation was optimised statistically. A decision forest model was generated using a machine-learning method-random forest-which comprises thousands of decision trees. As a result, balanced accuracies in cross-validation of the optimised decision tree and decision forest model, considering chemical space (71.5% and 75.5%, respectively), were higher than balanced accuracy of an example regulatory decision tree (54.1%). Moreover, the statistical optimisation of tree-based models revealed significant organic functional groups that would cause false prediction in standard genotoxicity tests and non-genotoxic carcinogenicity (e.g., organic amide and thioamide, saturated heterocyclic fragment and aryl halide). In vitro genotoxicity tests were the most important parameters in all models, even when in silico parameters were integrated. Although external validation is required, the findings of the integrated testing strategies established herein will contribute to precise carcinogenicity evaluation and to determine new mechanistic hypotheses of carcinogenicity.