In the risk assessment of agrochemicals, there has been a historical paucity of using data to refine the default adjustment factors, even though large datasets are available to support this. The current state of the science for addressing uncertainty regarding animal to human extrapolation (AFA) is to develop a "data-derived" adjustment factor (DDEF) to quantify such differences, if data are available. Toxicokinetic (TK) and toxicodynamic (TD) differences between species can be utilized for the DDEF, with human datasets being ideal yet rare. We identified a case for a currently registered herbicide, mesotrione, in which human TK and TD are available. This case study outlines an approach for the development of DDEFs using comparative human and animal data and based on an adverse outcome pathway (AOP) for inhibition of 4-hydroxyphenol pyruvate dioxygenase (HHPD). The calculated DDEF for rat to human extrapolation (AFA) for kinetics (AFAK = 2.5) was multiplied by the AFA for dynamics (AFAD = 0.3) resulting in a composite DDEF of similar to 1 (AFA = 0.75). This reflects the AOP and available scientific evidence that humans are less sensitive than rats to the effects of HPPD inhibitors. Further analyses were conducted utilizing in vitro datasets from hepatocytes and liver cytosols and extrapolated to whole animal using in vitro to in vivo extrapolation (IVIVE) to support toxicodynamic extrapolation. The in vitro datasets resulted in the same AFAD as derived for in vivo data (AFAD = 0.3). These analyses demonstrate that a majority of the species differences are related to toxicodynamics. Future work with additional in vitro/in vivo datasets for other HPPD inhibitors and cell types will further support this result. This work demonstrates utilization of all available toxicokinetic and toxicodynamic data to replace default uncertainty factors for agrochemical human health risk assessment.

INTRODUCTION:Acetyl-coenzyme A carboxylase (ACCase) inhibition is an attractive herbicide target. However, issues with fetal developmental toxicity identified at the late stages of the development process can halt progression of previously promising candidates. OBJECTIVES:To select and verify predictive lipid biomarkers of ACCase inhibition activity in vivo using liver samples obtained from early stage 7 day repeat dose studies in non-pregnant female Han Wistar rats that could be translated to developmental toxicity endpoints discovered during late-stage studies to provide an early screening tool. METHODS:Liver samples from eight rat repeat dose studies, exposed to six ACCase inhibitors from three different chemistries and one alternative mode of action (MoA) that also perturbs lipid biochemistry, were analysed using liquid chromatography - high resolution accurate mass - mass spectrometry. Multivariate and univariate data analysis methods were used for biomarker discovery and validation. RESULTS:A biomarker signature consisting of sixteen lipids biomarkers were selected. Verification of the signature as indicative of ACCase inhibition was established by demonstrating consistent perturbations in the biomarkers using two different ACCase inhibitor chemistries and the lack thereof with an alternate MoA. The fold change profile pattern was predictive of which test substance doses would or would not cause developmental toxicity. CONCLUSION:A strategy for selecting and verifying a robust signature of lipid biomarkers for predicting a toxicological end point has been described and demonstrated. Differences in lipidomic profiles correlated with developmental toxicity suggesting that indicators of a molecular initiation event resulting in pup developmental toxicity can be predicted from short term, toxicity studies conducted in non-pregnant adult female Han Wistar rats.

Species differences in hepatic metabolism of thyroxine (T4) by uridine diphosphate glucuronosyl transferase (UGT) and susceptibility to thyroid hormone imbalance could underlie differences in thyroid carcinogenesis caused by hepatic enzyme inducers in rats and humans. To investigate this hypothesis we examined profiles of hepatic UGT induction by the prototypical CAR activator phenobarbital (PB) in rat and human liver 3D microtissues. The rationale for this approach was that 3D microtissues would generate data more relevant to humans. Rat and human liver 3D microtissues were exposed to PB over a range of concentrations (500 u M - 2000 u M) and times (24-96 hr). Microarray and proteomics analyses were performed on parallel samples to generate integrated differentially expressed gene (DEG) datasets. Bioinformatics analysis of DEG data, including CAR response element (CRE) sequence analysis of UGT promoters, was used to assess species differences in UGT induction relative to CAR-mediated transactivation potential. A higher proportion of human UGT promoters were found to contain consensus CREs compared to the rat homologs. UGTs 1a6, 2b17 and 2b37 were upregulated by PB in rat liver 3D microtissues, but unaltered in human liver 3D microtissues. By contrast, human UGTs 1A8, 1A10 and 2B10 showed higher levels of induction (RNA and /or protein) compared to the rat homologs. There was general concordance between the presence of CREs and the induction of UGT RNA. As UGT1A and 2B isoforms metabolise T4, these results suggest that differences in UGT induction could contribute to differential susceptibility to CAR-mediated thyroid carcinogenesis in rats and humans.

Characterisation of the mode of action (MOA) of constitutive androstane receptor (CAR)-mediated rodent liver tumours involves measurement 5 key events including activation of the CAR receptor, altered gene expression, hepatocellular proliferation, clonal expansion and increased hepatocellular adenomas/carcinomas. To test whether or not liver 3D microtissues (LiMTs) recapitulate CAR- mediated procarcinogenic key events in response to the prototypical CAR activator phenobarbital (PB) we performed hepatocyte proliferation (LI%) analysis in rat and human LiMTs using a microTMA technology in conjunction with integrated transcriptomics (microarray) and proteomics analysis. The rationale for this approach was that LiMTs containing parenchymal and non-parenchymal cells (NPCs) are more physiologically representative of liver and thus would generate data more relevant to the in vivo situation. Rat and human LiMTs were treated with PB over a range of concentrations (500 uM - 2000 uM) and times (24 h - 96 h) in a dose-response/time-course analysis. There was a dose-dependent induction of LI% in rat LiMTs, however there was little or no effect of PB on LI% in human LiMTs. ATP levels in the rat and human LiMTs were similar to control in all of the PB treatments. There was also a dose- and time-dependent PB-mediated RNA induction of CAR regulated genes CYP2B6/Cyp2b2, CYP3A7/Cyp3a9 and UGT1A6/Ugt1a6 in human and rat LiMTs, respectively. These CAR regulated genes were also upregulated at the protein level. Ingenuity pathways analysis (IPA) indicated that there was a significant (Z score > 2.0;-log p value >) activation of CAR by PB in both human and rat LiMTs. These results indicate that human and rat LiMTs showed the expected responses at the level of PB-induced hepatocyte proliferation and enzyme induction with rat LiMTs showing significant dose-dependent effects while human LiMTs showed no proliferation response but did show dose-dependent enzyme induction at the RNA and protein levels. In conclusion LiMTs serve as a model to provide mechanistic data for 3 of the 5 key events considered necessary to establish a CAR-mediated MOA for liver tumourigenesis and thus can potentially reduce the use of animals when compiling mechanistic data packages.

Carotenoids are the pigments responsible for conferring the characteristic deep red colour to chilli pepper. The post-harvest retention of this colour is a key trait that governs the price of the produce. Determining colour retention and the associated underlying biochemical mechanisms are important issues that require investigation. In this present study, the ability of image analysis to determine colour change in ground chilli fruit was evaluated. This method enabled differentiation of extreme retention phenotypes whilst also reducing the duration of storage required to make accurate determinations. The analysis of volatiles indicated different levels of lipid and carotenoid derived volatiles in lines with different retention properties. Metabolite profiling of intermediary metabolism supported these findings, with increased levels of unsaturated fatty acids present in lines with low retention properties. Collectively, these data have led us to propose that in chilli fruit lipid peroxidation is one of the progenitors of carotenoid degradation.

Experimental data demonstrate a mode of action (MOA) for liver tumors in male rats and mice treated with sedaxane that starts with activation of CAR, followed by altered expression of CAR-responsive genes, increased cell proliferation, and eventually clonal expansion of preneoplastic cells, leading to the development of altered foci and tumors. This MOA is nonrelevant to human risk assessments. Methods and results in the MOA work for sedaxane illustrate promising directions that future MOA studies may be able to employ, in the spirit of "Tox21" and reduction of in vivo animal use: (1) currently available in vitro CAR and PXR reporter assays demonstrated that sedaxane is a direct CAR activator in mice and rats, and a weak PXR activator in rats; (2) mouse liver microarray results compared with a published CAR biomarker signature (based on 83 genes) showed a clear, statistical match, and a lack of correlation to similar biomarker signatures for AhR, PPARα, and STAT5B; (3) Ki67 immunohistochemistry and zonal image analysis showed significant increases in this marker of cell proliferation in mouse liver, without the need to dose a DNA labeling agent; and (4) toxicokinetic analysis of Cmax levels of sedaxane in blood showed a marked species difference between mice and rats that helps to explain differences in sensitivity to sedaxane. Incorporating these tools into the study plan for a new agrochemical or drug during development offers a promising alternative to the traditional need to conduct later, specialized MOA studies after the results of chronic bioassays are known.

Children show unique features concerning chemical hazards and risks, due to different exposure scenarios, age-related metabolic capacity and biological susceptibility linked to post-natal development. Chemical Regulatory frameworks state the need of children risk characterization. Current testing guidelines covering post-natal development are not routinely required by regulatory applications other than pesticides and biocides. Juvenile toxicity studies are foreseen for paediatric drugs: the toxicological repeated-dose tests don't allow accurate evaluations of effects upon direct exposure of immature organism. The paper discusses a testing approach aimed to address regulatory requirements of chemical hazard identification/characterization in a children-specific perspective. Juvenile toxicity test could be performed primarily on chemicals that may have relevant modes of action and/or age-related toxicokinetic differences and/or lead to important children exposure. This could be pursued by updating existing guidelines/test protocols with triggers for endpoints relevant to juvenile toxicity.

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.