Seis-ing up the Super-Morrison formation sauropods.

The Upper Jurassic Morrison Formation sauropods Diplodocus (formerly "Seismosaurus") hallorum and Supersaurus vivianae are quantifiably the largest dinosaurian taxa from the formation, as well as being among the largest dinosaurs in the world. Their extreme body size (in particular body length, c. 50+ m) has fascinated the paleontological community since their discoveries and has sparked an ongoing discussion on the trends and limits of Morrison Formation sauropod body size. Although not an undeviating proxy, often the largest and skeletally most mature specimens are among the rarest (as exemplified in Triceratops). While their body size has no phylogenetic bearing, the extreme size and potential eco and biological significance of these two sauropod taxa are frequently discussed. Whether these rare and titanically proportioned sauropod specimens are large-bodied, senescent or both is an often-repeating rhetoric. To definitively make maturational inferences about these taxa, we osteohistologically sampled the holotype of D. hallorum (NMMNH P-25079) and the second known specimen of S. vivianae (WDC DMJ-021). Our age-determinant and maturational assessments indicate that both specimens were skeletally mature at their respective age of death. Retrocalculation methods for D. hallorum NMMNH P-25079 produce a maximum age-at-death estimation of 60 years, whereas S. vivianae WDC DMJ-021 lived well past skeletal maturity-so much so that reliable retrocalculated ages cannot be accurately determined at this time. Additionally, the rarity of such large sauropods within the Morrison Formation might be more parsimoniously explained as relating to their maturity as opposed to representing aberrant taxa on the Morrison landscape.

Hepatitis A Virus Infection in Cynomolgus Monkeys Confounds the Safety Evaluation of a Drug Candidate.

In a 3-month toxicity study in cynomolgus monkeys at a European contract laboratory, animals were infected with HAV, initially resulting in hepatic injury being incorrectly attributed to the test compound. Elevated serum ALT/AST/GLDH (5- to 10-fold) were noted in individual animals from all groups including controls, with no apparent dose, exposure, or time-related relationship. Liver histopathology revealed minimal to slight inflammatory cell accumulation in periportal zones of most animals, and minimal to slight hepatocyte degeneration/necrosis in 10/42 animals from all groups. As these findings were more pronounced in 6 drug-treated animals, including 2/6 in the low dose group, the draft report concluded: "treatment-related hepatotoxicity at all dose levels precluded determination of a NOAEL." However, the unusual pattern of hepatotoxicity suggested a factor other than drug exposure might have caused the hepatic effects. Therefore, snap-frozen liver samples were tested for hepatitis viruses using a PCR method. Tests for hepatitis B, C, and E virus were negative; however, 20/42 samples were positive for hepatitis A virus (HAV). Infection was strongly associated with increased serum ALT/GLDH, and/or hepatocyte degeneration/necrosis. Re-evaluation of the study in light of these data concluded that the hepatic injury was not drug-related. A subsequent 6-month toxicology study in HAV-vaccinated cynomolgus monkeys confirmed the absence of hepatotoxicity. Identification of HAV infection supported progression of the drug candidate into later clinical trials. Although rarely investigated, subclinical HAV infection has occasionally been reported in laboratory primates, including those used for toxicology studies and it may be more prevalent than the literature indicates.

"Dragons" on the landscape: Modeling the abundance of large carnivorous dinosaurs of the Upper Jurassic Morrison Formation (USA) and the Upper Cretaceous Dinosaur Park Formation (Canada).

Counts of the number of skeletal specimens of "adult" megaherbivores and large theropods from the Morrison and Dinosaur Park formations-if not biased by taphonomic artifacts-suggest that the big meat-eaters were more abundant, relative to the number of big plant-eaters, than one would expect on the basis of the relative abundance of large carnivores and herbivores in modern mammalian faunas. Models of megaherbivore population density (number of individuals per square kilometer) that attempt to take into account ecosystem productivity, the size structure of megaherbivore populations, and individual megaherbivore energy requirements, when combined with values of the large theropod/megaherbivore abundance ratio, suggest that large theropods may have been more abundant on the landscape than estimates extrapolated from the population density versus body mass relationship of mammalian carnivores. Models of the meat production of megaherbivore populations and the meat requirements of "adult" large theropods suggest that herbivore productivity would have been insufficient to support the associated number of individuals of "adult" large theropods, unless the herbivore production/biomass ratio was substantially higher, and/or the large theropod meat requirement markedly lower, than expectations based on modern mammals. Alternatively, or in addition to one or both of these other factors, large theropods likely included dinosaurs other than megaherbivores as significant components of their diet.

Accumulation of molybdenum in major organs following repeated oral administration of bis-choline tetrathiomolybdate in the Sprague Dawley rat.

Molybdenum is an essential dietary trace element required for several critical enzyme systems. High intake is associated with toxicity in ruminants and animal studies. The proposed therapeutic use of molybdenum-based drugs poses a potential risk for accumulation through chronic administration of therapeutic doses of this element. The current experiment was designed to study the effect of daily dosing of a molybdenum compound, bis-choline tetrathiomolybdate (TTM), in Sprague Dawley rats using laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-ToF-MS) and two dosing levels of TTM for up to 3 months. To investigate if molybdenum accumulation was associated with tissue toxicity, histopathology, haematology and clinical biochemistry markers of toxicity were incorporated into the study design. There were no behavioural signs of toxicity to the rats, and no clinical or anatomic pathology was associated with treatment. The current data did show a progressive accumulation of molybdenum within the adrenal gland, kidneys, liver, spleen, brain and testes. Although this was not associated with tissue toxicity within the 3-month study design, greater exposure over a longer period of time has the potential for producing adverse pathophysiological cellular function. Tissue toxicity, as a result of local excessive accumulation of molybdenum over time, has clear implications for the therapeutic use of molybdenum in humans and demands sensitive monitoring of tissue molybdenum levels to avoid toxicity. The current study highlights the shortcomings of conventional biomonitoring approaches to detect molybdenum accumulation with the goal of avoiding molybdenum-associated toxicity.

A toxicological evaluation of geranylgeraniol.

Geranylgeraniol (GGOH) is an isoprenoid compound found in annatto seeds and an intermediate of the mevalonate pathway found within organisms serving various functions. Toxicological studies on its safety profile are not readily available. To assess the safety of GGOH, a molecularly distilled, food grade annatto oil, consisting of approximately 80% trans-GGOH, was subjected to a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test in order to investigate its genotoxic potential and a 90-day repeated-dose oral toxicity study in rats in order to investigate its potential subchronic toxicity and identify any target organs. No evidence of mutagenicity or genotoxic activity was observed under the applied test systems. In the 90-day study, male and female Hsd. Han Wistar rats were administered daily doses of 0, 725, 1450, and 2900 mg/kg bw/day by gavage. Treatment-related adverse effects were observed in the forestomach at all dose levels and in the liver at the intermediate- and high-dose levels. Based on these results, the lowest observed adverse effect level (LOAEL) for local effects and the no observed adverse effect level (NOAEL) for systemic effects were determined as 725 mg/kg bw/day.

Nootkatone Is an Effective Repellent against Aedes aegypti and Aedes albopictus.

We tested a nootkatone product for insecticide activity against the most prominent vectors of Zika virus (ZIKV), Aedes aegypti, and Aedes albopictus. We tested the permethrin-resistant (PERM-R) Vergel strain of A. aegypti and the permethrin-susceptible (PERM-S) New Orleans strain of A. aegypti to determine if insecticide resistance affected their susceptibility to nootkatone. Bottle bioassays showed that the PERM-S strain (New Orleans) was more susceptible to nootkatone than the confirmed A. aegypti permethrin-resistant (PERM-R) strain, Vergel. The A. albopictus strain ATM-NJ95 was a known PERM-S strain and Coatzacoalcos permethrin susceptibility was unknown but proved to be similar to the ATM-NJ95 PERM-S phenotype. The A. albopictus strains (ATM-NJ95 and Coatzacoalcos) were as susceptible to nootkatone as the New Orleans strain. Bottle bioassays conducted with ZIKV-infected mosquitoes showed that the New Orleans (PERM-S) strain was as susceptible to nootkatone as the mock-infected controls, but the PERM-R strain was less susceptible to nootkatone than the mock-infected controls. Repellency/irritancy and biting inhibition bioassays (RIBB) of A. aegypti determined whether the nootkatone-treated arms of three human subjects prevented uninfected A. aegypti mosquitoes from being attracted to the test subjects and blood-feeding on them. The RIBB analyses data calculated the spatial activity index (SAI) and biting inhibition factor (BI) of A. aegypti at different nootkatone concentrations and then compared the SAI and BI of existing repellency products. We concluded that nootkatone repelled mosquitoes at a rate comparable to 7% DEET or 5% picaridin and has the potential to be an efficacious repellent against adult A. aegypti mosquitoes.

A review of species differences in the control of, and response to, chemical-induced thyroid hormone perturbations leading to thyroid cancer.

This review summarises the current state of knowledge regarding the physiology and control of production of thyroid hormones, the effects of chemicals in perturbing their synthesis and release that result in thyroid cancer. It does not consider the potential neurodevelopmental consequences of low thyroid hormones. There are a number of known molecular initiating events (MIEs) that affect thyroid hormone synthesis in mammals and many chemicals are able to activate multiple MIEs simultaneously. AOP analysis of chemical-induced thyroid cancer in rodents has defined the key events that predispose to the development of rodent cancer and many of these will operate in humans under appropriate conditions, if they were exposed to high enough concentrations of the affecting chemicals. There are conditions however that, at the very least, would indicate significant quantitative differences in the sensitivity of humans to these effects, with rodents being considerably more sensitive to thyroid effects by virtue of differences in the biology, transport and control of thyroid hormones in these species as opposed to humans where turnover is appreciably lower and where serum transport of T4/T3 is different to that operating in rodents. There is heated debate around claimed qualitative differences between the rodent and human thyroid physiology, and significant reservations, both scientific and regulatory, still exist in terms of the potential neurodevelopmental consequences of low thyroid hormone levels at critical windows of time. In contrast, the situation for the chemical induction of thyroid cancer, through effects on thyroid hormone production and release, is less ambiguous with both theoretical, and actual data, showing clear dose-related thresholds for the key events predisposing to chemically induced thyroid cancer in rodents. In addition, qualitative differences in transport, and quantitative differences in half life, catabolism and turnover of thyroid hormones, exist that would not operate under normal situations in humans.

The Cumulative Risk Assessment of Hepatotoxic Chemicals: A Hepatic Histopathology Perspective.

The increased concern on the consequence of exposure to multiple chemical combinations has led national regulatory authorities to develop different concepts to conduct risk assessments on chemical mixtures. Pesticide residues were identified as "problem formulation" in the respective European regulations and in this context, the European Food and Safety Authority has suggested to group pesticidal active ingredients (AIs) into cumulative assessment groups (CAGs) based on the toxicological properties of each AI. One proposed CAG, on the liver, currently consists of 15 subgroups, each representing a specific hepatotoxic effect observed in toxicity studies. Dietary cumulative risk assessments would then have to be conducted assuming dose additivity of all members of each CAG subgroup. The purpose of this publication is to group AIs based upon the knowledge of the pathogenesis of liver effects to discriminate between primary end points (direct consequence of chemical interaction with a biological target) and secondary end points (which are a consequence of, or that arise out of, a previous pathological change). Focusing on the relevant primary end points strengthens and simplifies the selection of compounds for cumulative risk assessment regarding the liver and better rationalizes the basis for chemical grouping. Relevant dose additivity is to be expected at the level of the primary/leading pathological end points and not at the level of the secondary end points. We recognize, however, that special consideration is needed for substances provoking neoplasia, and this category is included in the group of primary end points for which chemicals inducing them are grouped for risk assessment. Using the pathological basis for defining the respective CAGs, 6 liver subgroups and 2 gallbladder/bile duct groups are proposed. This approach simplifies the cumulative assessment calculation without obviously affecting consumer safety.

Evaluation of a novel West Nile virus transmission control strategy that targets Culex tarsalis with endectocide-containing blood meals.

Control of arbovirus transmission remains focused on vector control through application of insecticides directly to the environment. However, these insecticide applications are often reactive interventions that can be poorly-targeted, inadequate for localized control during outbreaks, and opposed due to environmental and toxicity concerns. In this study, we developed endectocide-treated feed as a systemic endectocide for birds to target blood feeding Culex tarsalis, the primary West Nile virus (WNV) bridge vector in the western United States, and conducted preliminary tests on the effects of deploying this feed in the field. In lab tests, ivermectin (IVM) was the most effective endectocide tested against Cx. tarsalis and WNV-infection did not influence mosquito mortality from IVM. Chickens and wild Eurasian collared doves exhibited no signs of toxicity when fed solely on bird feed treated with concentrations up to 200 mg IVM/kg of diet, and significantly more Cx. tarsalis that blood fed on these birds died (greater than 80% mortality) compared to controls (less than 25% mortality). Mosquito mortality following blood feeding correlated with IVM serum concentrations at the time of blood feeding, which dropped rapidly after the withdrawal of treated feed. Preliminary field testing over one WNV season in Fort Collins, Colorado demonstrated that nearly all birds captured around treated bird feeders had detectable levels of IVM in their blood. However, entomological data showed that WNV transmission was non-significantly reduced around treated bird feeders. With further development, deployment of ivermectin-treated bird feed might be an effective, localized WNV transmission control tool.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild-type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats.

The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats). The treatment of WT rats for 7 d with 500 ppm NaPB in the diet and 100 mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats. Male C57BL/6 J (WT) and CAR KO/PXR KO mice were given 1000 ppm NaPB in the diet for 7 d. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes. These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.

The association between chemical-induced porphyria and hepatic cancer.

The haem biosynthetic pathway is of fundamental importance for cellular metabolism both for the erythroid and nonerythroid tissues. There are several genetic variants of the pathway in the human population that cause dysfunction of one or other of the enzymes resulting in porphyrias of varying severity. Serious chronic hepatic and systemic diseases may result. Some of these can be precipitated by exposure to drugs including hormones, barbiturates and antibiotics, as well as alcohol and particular chlorinated aromatic chemicals. In experimental animals some of the steps of this pathway can also be severely disrupted by a variety of environmental chemicals, potential drugs and pesticides, especially in the liver, leading to the accumulation of uroporphyrins derived from the intermediate uroporphyrinogens or protoporphyrin IX, the immediate precursor of haem. With some of these chemicals this also leads to cholestasis and liver cell injury and eventually hepatic tumours. The review evaluates the available evidence linking hepatic porphyria with carcinogenesis in naturally occurring human genetic conditions and in chemically-induced porphyrias in laboratory animals. The existing data showing gender, strain, and species differences in sensitivity to the chemical-induced porphyrias, liver injury and liver tumours are discussed and the role that transgenically altered mouse models have played in defining the varying mechanisms. Finally, the review proposes a novel, unifying hypothesis linking the hepatotoxicity induced by the accumulation of various porphyrins, with the increased risk of developing hepatic cancer as a long term consequence.

Neutron scanning reveals unexpected complexity in the enamel thickness of an herbivorous Jurassic reptile.

Eilenodontines are one of the oldest radiation of herbivorous lepidosaurs (snakes, lizards and tuatara) characterized by batteries of wide teeth with thick enamel that bear mammal-like wear facets. Unlike most reptiles, eilenodontines have limited tooth replacement, making dental longevity particularly important to them. We use both X-ray and neutron computed tomography to examine a fossil tooth from the eilenodontine Eilenodon (Late Jurassic, USA). Of the two approaches, neutron tomography was more successful and facilitated measurements of enamel thickness and distribution. We find the enamel thickness to be regionally variable, thin near the cusp tip (0.10 mm) but thicker around the base (0.15-0.30 mm) and notably greater than that of other rhynchocephalians such as the extant Sphenodon (0.08-0.14 mm). The thick enamel in Eilenodon would permit greater loading, extend tooth lifespan and facilitate the establishment of wear facets that have sharp edges for orally processing plant material such as horsetails (Equisetum). The shape of the enamel dentine junction indicates that tooth development in Eilenodon and Sphenodon involved similar folding of the epithelium but different ameloblast activity.

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats.

A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies.

Evaluation of Uracil, Sodium Ascorbate, and Rosiglitazone as Promoters of Urinary Bladder Transitional Cell Carcinomas in Male Sprague-Dawley Rats.

The purpose of this study was to establish a 2-stage model of urinary bladder carcinogenesis in male Sprague-Dawley rats to identify tumor promoters. In phase 1 of the study, rats ( n = 170) were administered 100 mg/kg of the tumor initiator, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), twice weekly by oral gavage (po) for a period of 6 weeks. Phase 2 consisted of dividing rats into 4 groups ( n = 40 per group) and administering one of the following for 26 weeks to identify putative tumor promoters: (1) vehicle po, (2) 25 mg/kg/day rosiglitazone po, (3) 5% dietary sodium l-ascorbate, and (4) 3% dietary uracil. Rats were necropsied after 7.5 months, and urinary bladders were evaluated by histopathology. BBN/vehicle treatments induced the development of urothelial hyperplasia (83%) and papillomas (15%) but no transitional cell carcinomas (TCCs). Rosiglitazone increased the incidence and severity of papillomas (93%) and resulted in TCC in 10% of treated rats. Uracil was the most effective tumor promoter in our study and increased the incidence of papillomas (90%) and TCC (74%). Sodium ascorbate decreased the incidence of urothelial hyperplasia (63%) and did not increase the incidence of urothelial papillomas or TCC. These data confirm the capacity of our 2-stage model to identify urinary bladder tumor promoters.

Descendants of the Jurassic turiasaurs from Iberia found refuge in the Early Cretaceous of western USA.

A new, largely complete eusauropod dinosaur with cranial and postcranial elements from two skeletons, Mierasaurus bobyoungi gen. nov., sp. nov. from the lower Yellow Cat Member (Early Cretaceous) of Utah (USA), is the first recognized member of Turiasauria from North America. Moreover, according to our phylogenetic results, Moabosaurus utahensis from the lower Yellow Cat Member of Utah (USA) is also a member of this clade. This group of non-neosauropod eusauropods, which now includes five genera (Losillasaurus, Turiasaurus, Mierasaurus, Moabosaurus and Zby), was previously known only from the Jurassic of Europe. These recent discoveries in Utah suggest that turiasaurs as a lineage survived the Jurassic-Cretaceous extinction boundary and expanded their known range, at least, into western North America. The revised spatiotemporal distribution of turiasaurs is consistent with the presence of a land connection between North America and Europe sometime during the late Tithonian to Valanginian (c.147-133 Ma). Mierasaurus and Moabosaurus are the only non-neosauropod eusauropods known from North America, despite being younger than the classic neosauropods of the Morrison Formation (c.150 Ma).

The first specimen of Camarasaurus (Dinosauria: Sauropoda) from Montana: The northernmost occurrence of the genus.

A partial skeleton from the Little Snowy Mountains of central Montana is the first referable specimen of the Morrison Formation macronarian sauropod Camarasaurus. This specimen also represents the northernmost occurrence of a sauropod in the Morrison. Histological study indicates that, although the specimen is relatively small statured, it is skeletally mature; this further emphasizes that size is not a undeviating proxy to maturity in dinosaurs, and that morphologies associated with an individual's age and stature may be more nebulous in sauropods.

Trichloroethylene-induced formic aciduria in the male C57 Bl/6 mouse.

1, 1, 2-Trichloroethylene (TCE) is of environmental concern, due to evaporation while handling, chemical processing and leakage from chemical waste sites, leading to its contamination of ground water and air. For several decades there has been issues about possible long term health effects of TCE but recently the International Agency for Research on Cancer (IARC) and the US Environmental Protection Agency classified TCE as a human carcinogen. Links having been established between occupational exposures and kidney cancer and possible links to non-Hodgkin lymphoma and liver cancer, but there is more still more to learn. In male rats, TCE produces a small increase in the incidence of renal tubule tumours but not in female rats or mice of either sex. However, chronic renal injury was seen in these bioassays in both sexes of rats and mice. The mechanism of kidney injury from TCE is thought to be due to reductive metabolism forming a cysteine conjugate that is converted to a reactive metabolite via the enzyme cysteine conjugate ß-lyase. However, TCE also produces a marked and sustained formic aciduria in male rats and it has been suggested that long term exposure to formic acid could lead to renal tubule injury and regeneration. In this study we have determined if TCE produces formic aciduria in male mice following a single and repeat dosing. Male C57 Bl/6OlaHsd mice were dosed with 1000mg/kg by ip injection and urine collected overnight 24, 48, 72 and 96h after dosing. Formic acid was present in urine 24h after dosing, peaked around 48h at 8mg formic acid excreted/mouse, and remained constant over the next 24h and was not back to normal 96h after dosing. This was associated with a marked acidification of the urine. Plasma creatinine and renal pathology was normal. Plasma kinetics of formic acid showed it was readily cleared with an initial half-life of 2.42h followed by a slower rate with a half-life of 239h. Male mice were then dosed twice/week at 1000mg/kg TCE for 56days, as anticipated there was a marked and sustained formic aciduria over the duration of the study. This was associated with acidification of the urine, mild diuresis and a marked fall in urinary ammonia. Six biomarkers of renal injury KIM-1, NGAL, NAG, Cystatin-c, Albumin and IL-18 were measured in urine over time and they all showed a small increase at the later time points indicative of early markers of renal injury. However, there was no histological evidence of renal damage or renal tubule cell proliferation after 8 weeks' exposure to TCE. The concentration of formic acid in plasma at the end of the study was 1.05±0.61mM compared to control, 0.39±0.17mM. In the liver, formic acid was present at a concentration of 1mM in both control and treated mice while in the kidney it was higher at 2mM in both treated and controls. We also report that trichloroacetic acid (TCA) a metabolite of TCE also causes formic aciduria, at doses likely to arise in vivo after 1000mg/kg TCE namely 16 and 32mg/kg. Urinary formic acid peaked 24h after dosing at 4mg formic acid excreted/mouse. Thus, as in male and female rats (Yaqoob et al., 2013) male mice show a marked formic aciduria following TCE which after 8 weeks' exposure did not produce renal injury, but the small rise in renal biomarkers suggest renal damage may occur following longer exposure. Thus, TCE-induced formic aciduria may be a contributor factor in the chronic renal injury seen in male and female rats and mice.

Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop.

The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.

Regulatory Forum Opinion Piece*: The Value of Publishing Negative Scientific Study Data.

Historically it has been easier to publish positive scientific results than negative data not supporting the research hypothesis. This appears to be increasing, with fewer negative studies appearing in the literature across many disciplines. Failure to recognize the value of negative results has important implications for the toxicology community. Implications include perpetuating scientific fields based upon selective or occasionally erroneous, positive results. One example is decreased vaccination rates and increased measles infections that can lead to childhood mortality following one erroneous positive study linking vaccination to adverse effects despite multiple negative studies. Publication of negative data that challenges existing paradigms enhances progress by stopping further investment in scientifically barren topics, decreases the use of animals, and focuses research in more fruitful areas. The National Toxicology Program (NTP) publishes both positive and negative rodent data. Retrospective analysis of the NTP database has provided insights on the carcinogenic process and in the gradual acceptance of using fewer animals in safety studies. This article proposes that careful publication of both positive and negative data can enhance product safety assessment, add robustness to safety determinations in the regulatory decision-making process, and should be actively encouraged by those determining journal editorial policy.

Quantification of Drug-Induced Inhibition of Canalicular Cholyl-l-Lysyl-Fluorescein Excretion From Hepatocytes by High Content Cell Imaging.

We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between <1 and 794 µM). Thirteen drugs that inhibited CLF efflux also inhibited hepatocellular uptake of the probe substrate [(3)H]-taurocholate. Although no clear correlation between the potencies of inhibition of the 2 processes was evident, these data highlight the need to consider possible uptake transporter inhibition when interpreting hepatocyte CLF inhibition data. It has been reported that CLF is transported by MRP2. The CLF efflux inhibition data correlated closely with published data on inhibition by the drugs of the bile salt export pump (Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk.