Assessing human carcinogenicity risk of agrochemicals without the rodent cancer bioassay.

The rodent cancer bioassays are conducted for agrochemical safety assessment yet they often do not inform regulatory decision-making. As part of a collaborative effort, the Rethinking Carcinogenicity Assessment for Agrochemicals Project (ReCAAP) developed a reporting framework to guide a weight of evidence (WOE)-based carcinogenicity assessment that demonstrates how to fulfill the regulatory requirements for chronic risk estimation without the need to conduct lifetime rodent bioassays. The framework is the result of a multi-stakeholder collaboration that worked through an iterative process of writing case studies (in the form of waivers), technical peer reviews of waivers, and an incorporation of key learnings back into the framework to be tested in subsequent case study development. The example waivers used to develop the framework were written retrospectively for registered agrochemical active substances for which the necessary data and information could be obtained through risk assessment documents or data evaluation records from the US EPA. This exercise was critical to the development of a framework, but it lacked authenticity in that the stakeholders reviewing the waiver already knew the outcome of the rodent cancer bioassay(s). Syngenta expanded the evaluation of the ReCAAP reporting framework by writing waivers for three prospective case studies for new active substances where the data packages had not yet been submitted for registration. The prospective waivers followed the established framework considering ADME, potential exposure, subchronic toxicity, genotoxicity, immunosuppression, hormone perturbation, mode of action (MOA), and all relevant information available for read-across using a WOE assessment. The point of departure was estimated from the available data, excluding the cancer bioassay results, with a proposed use for the chronic dietary risk assessment. The read-across assessments compared data from reliable registered chemical analogues to strengthen the prediction of chronic toxicity and/or tumorigenic potential. The prospective case studies represent a range of scenarios, from a new molecule in a well-established chemical class with a known MOA to a molecule with a new pesticidal MOA (pMOA) and limited read-across to related molecules. This effort represents an important step in establishing criteria for a WOE-based carcinogenicity assessment without the rodent cancer bioassay(s) while ensuring a health protective chronic dietary risk assessment.

Developing prototypes of a modernized approach to assess crop protection chemical safety.

In 2019, the US EPA Administrator issued a directive directing the agency away from reliance on vertebrate tests by 2035, whilst maintaining high-quality human health and environmental risk assessments. There is no accepted approach to achieve this. The decade-long duration of the crop protection (CP) chemical R&D process therefore requires both the invention and application of a modernized approach to those CP chemical projects entering corporate research portfolios by the mid-2020s. We conducted problem formulation discussions with regulatory agency scientists which created the problem statement: "Develop, demonstrate, and implement a modern scientifically sound and robust strategy that applies appropriate and flexible exposure and effects characterization without chemical specific vertebrate tests to reliably address risk, uncertainties, and deficiencies in data and its interpretation with equivalent confidence as do the currently accepted test guidelines and meet the regulatory needs of the agencies". The solution must provide the knowledge needed to confidently conclude human health and environmental protective risk assessments. Exploring this led to a conceptual model involving the creation and parallel submission of a new approach without reliance on chemical-specific vertebrate tests. Assessment in parallel to a traditional package will determine whether it supports some, or all, of the necessary risk management actions. Analysis of any deficiencies will provide valuable feedback to focus development of tools or approaches for subsequent iterations. When found to provide sufficient information, it will form the technical foun­dation of stakeholder engagement to explore acceptance of a new approach to CP chemical risk assessment.

The US EPA, and other regulatory agencies, aim to reduce the use of vertebrate animal tests for assessing risks of crop protection chemicals. There is currently no accepted way to do this. We outline a proposal to perform both the assessment using traditional vertebrate testing and a set of new non-animal methods. These data sets must each be combined with a calculated estimate of user exposure to the pesticide based on its intended use. Comparing the outcome of these two assess­ments will show whether the set of non-animal methods needs to be improved further. When the new approach appears to reliably predict the risks, the different stakeholders must be brought together to assess whether the non-animal methods package is acceptable and can replace the tests on vertebrate animals while maintaining the same level of protection of human health and the environment.

Towards replacement of animal tests with in vitro assays: a gene expression biomarker predicts in vitro and in vivo estrogen receptor activity.

High-throughput transcriptomics (HTTr) has the potential to support efforts to reduce or replace some animal tests. In past studies, we described a computational approach utilizing a gene expression biomarker consisting of 46 genes to predict estrogen receptor (ER) activity after chemical exposure in ER-positive human breast cancer cells including the MCF-7 cell line. We hypothesized that the biomarker model could identify ER activities of chemicals examined by Endocrine Disruptor Screening Program (EDSP) Tier 1 screening assays in which transcript profiles of the same chemicals were examined in MCF-7 cells. For the 62 chemicals examined including 5 chemicals examined in this study using RNA-Seq, the ER biomarker model accuracy was 1) 97% for in vitro reference chemicals, 2) 76-85% for guideline uterotrophic assays, and 3) 87-88% for guideline and nonguideline uterotrophic assays. For the same chemicals, these accuracies were similar or slightly better than those of the ToxCast ER model based on 18 in vitro assays. The performance of the ER biomarker model indicates that HTTr interpreted using the ER biomarker correctly identifies active and inactive ER reference chemicals. As part of the HTTr screening program the approach could rapidly identify chemicals with potential ER bioactivities for additional screening and testing.

Rethinking chronic toxicity and carcinogenicity assessment for agrochemicals project (ReCAAP): A reporting framework to support a weight of evidence safety assessment without long-term rodent bioassays.

Rodent cancer bioassays have been long-required studies for regulatory assessment of human cancer hazard and risk. These studies use hundreds of animals, are resource intensive, and certain aspects of these studies have limited human relevance. The past 10 years have seen an exponential growth of new technologies with the potential to effectively evaluate human cancer hazard and risk while reducing, refining, or replacing animal use. To streamline and facilitate uptake of new technologies, a workgroup comprised of scientists from government, academia, non-governmental organizations, and industry stakeholders developed a framework for waiver rationales of rodent cancer bioassays for consideration in agrochemical safety assessment. The workgroup used an iterative approach, incorporating regulatory agency feedback, and identifying critical information to be considered in a risk assessment-based weight of evidence determination of the need for rodent cancer bioassays. The reporting framework described herein was developed to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode-of-action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

Genomic comparisons between hepatocarcinogenic and non-hepatocarcinogenic organophosphate insecticides in the mouse liver.

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice. To provide insights into the adverse outcome pathway (AOP) that underlies these tumors, effects of methidathion in the male mouse liver were examined after 7 and 28 day exposures and compared to those of two other OPs that either do not increase (fenthion) or possibly suppress liver cancer (parathion) in mice. None of the chemicals caused increases in liver weight/body weight or histopathological changes in the liver. Parathion decreased liver cell proliferation after 7 and 28 days while the other chemicals had no effects. There was no evidence for hepatotoxicity in any of the treatment groups. Full-genome microarray analysis of the livers from the 7 and 28 day treatments demonstrated that methidathion and fenthion regulated a large number of overlapping genes, while parathion regulated a unique set of genes. Examination of cytochrome P450 enzyme activities and use of predictive gene expression biomarkers found no consistent evidence for activation of AhR, CAR, PXR, or PPARα. Parathion suppressed the male-specific gene expression pattern through STAT5b, similar to genetic and dietary conditions that decrease liver tumor incidence in mice. Overall, these findings indicate that methidathion causes liver cancer by a mechanism that does not involve common mechanisms of liver cancer induction.

A Gene Expression Biomarker Identifies Chemical Modulators of Estrogen Receptor α in an MCF-7 Microarray Compendium.

Identification of chemicals that affect hormone-regulated systems will help to predict endocrine disruption. In our previous study, a 46 gene biomarker was found to be an accurate predictor of estrogen receptor (ER) α modulation in chemically treated MCF-7 cells. Here, potential ERα modulators were identified using the biomarker by screening a microarray compendium consisting of ∼1600 gene expression comparisons representing exposure to ∼1200 chemicals. A total of ∼170 chemicals were identified as potential ERα modulators. In the Connectivity Map 2.0 collection, 75 and 39 chemicals were predicted to activate or suppress ERα, and they included 12 and six known ERα agonists and antagonists/selective ERα modulators, respectively. Nineteen and eight of the total number were also identified as active in an ERα transactivation assay carried out in an MCF-7-derived cell line used to screen the Tox21 10K chemical library in agonist or antagonist modes, respectively. Chemicals predicted to modulate ERα in MCF-7 cells were examined further using global and targeted gene expression in wild-type and ERα-null cells, transactivation assays, and cell-free ERα coregulator interaction assays. Environmental chemicals classified as weak and very weak agonists were confirmed to activate ERα including apigenin, kaempferol, and oxybenzone. Novel activators included digoxin, nabumetone, ivermectin, and six progestins. Novel suppressors included emetine, mifepristone, niclosamide, and proscillaridin. Our strategy will be useful to identify environmentally relevant ERα modulators in future high-throughput transcriptomic screens.

Activation of Nrf2 in the liver is associated with stress resistance mediated by suppression of the growth hormone-regulated STAT5b transcription factor.

The transcription factor Nrf2 (encoded by Nfe2l2) induces expression of numerous detoxifying and antioxidant genes in response to oxidative stress. The cytoplasmic protein Keap1 interacts with and represses Nrf2 function. Computational approaches were developed to identify factors that modulate Nrf2 in a mouse liver gene expression compendium. Forty-eight Nrf2 biomarker genes were identified using profiles from the livers of mice in which Nrf2 was activated genetically in Keap1-null mice or chemically by a potent activator of Nrf2 signaling. The rank-based Running Fisher statistical test was used to determine the correlation between the Nrf2 biomarker genes and a test set of 81 profiles with known Nrf2 activation status demonstrating a balanced accuracy of 96%. For a large number of factors examined in the compendium, we found consistent relationships between activation of Nrf2 and feminization of the liver transcriptome through suppression of the male-specific growth hormone (GH)-regulated transcription factor STAT5b. The livers of female mice exhibited higher Nrf2 activation than male mice in untreated or chemical-treated conditions. In male mice, Nrf2 was activated by treatment with ethinyl estradiol, whereas in female mice, Nrf2 was suppressed by treatment with testosterone. Nrf2 was activated in 5 models of disrupted GH signaling containing mutations in Pit1, Prop1, Ghrh, Ghrhr, and Ghr. Out of 59 chemical treatments that activated Nrf2, 36 exhibited STAT5b suppression in the male liver. The Nrf2-STAT5b coupling was absent in in vitro comparisons of chemical treatments. Treatment of male and female mice with 11 chemicals that induce oxidative stress led to activation of Nrf2 to greater extents in females than males. The enhanced basal and inducible levels of Nrf2 activation in females relative to males provides a molecular explanation for the greater resistance often seen in females vs. males to age-dependent diseases and chemical-induced toxicity.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Synthesis, antioxidant and antichagasic properties of a selected series of hydroxy-3-arylcoumarins.

Oxidative stress is involved in several parasitic diseases such as Chagas. Agents able to selectively modulate biochemical processes involved in the disease represent promising multifunctional agents for the delay or abolishment of the progression of this pathology. In the current work, differently substituted hydroxy-3-arylcoumarins are described, exerting both antioxidant and trypanocidal activity. Among the compounds synthesized, compound 8 showed the most interesting profile, presenting a moderate scavenging ability for peroxyl radicals (ORAC-FL=2.23) and a high degree of selectivity towards epimastigotes stage of the parasite T. cruzi (IC50=1.31µM), higher than Nifurtimox (drug currently used for treatment of Chagas disease). Interestingly, the current study revealed that small structural changes in the hydroxy-3-arylcoumarin core allow modulating both activities, suggesting that this scaffold has desirable properties for the development of promising classes of antichagasic compounds.

Moving Toward Integrating Gene Expression Profiling Into High-Throughput Testing: A Gene Expression Biomarker Accurately Predicts Estrogen Receptor α Modulation in a Microarray Compendium.

Microarray profiling of chemical-induced effects is being increasingly used in medium- and high-throughput formats. Computational methods are described here to identify molecular targets from whole-genome microarray data using as an example the estrogen receptor α (ERα), often modulated by potential endocrine disrupting chemicals. ERα biomarker genes were identified by their consistent expression after exposure to 7 structurally diverse ERα agonists and 3 ERα antagonists in ERα-positive MCF-7 cells. Most of the biomarker genes were shown to be directly regulated by ERα as determined by ESR1 gene knockdown using siRNA as well as through chromatin immunoprecipitation coupled with DNA sequencing analysis of ERα-DNA interactions. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression datasets from experiments using MCF-7 cells, including those evaluating the transcriptional effects of hormones and chemicals. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% and 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) ER reference chemicals including "very weak" agonists. Importantly, the biomarker predictions accurately replicated predictions based on 18 in vitro high-throughput screening assays that queried different steps in ERα signaling. For 114 chemicals, the balanced accuracies were 95% and 98% for activation or suppression, respectively. These results demonstrate that the ERα gene expression biomarker can accurately identify ERα modulators in large collections of microarray data derived from MCF-7 cells.