Inhibition of Acetyl-CoA Carboxylase Causes Malformations in Rats and Rabbits: Comparison of Mammalian Findings and Alternative Assays.

Acetyl-CoA carboxylase (ACC) is an enzyme within the de novo lipogenesis (DNL) pathway and plays a role in regulating lipid metabolism. Pharmacologic ACC inhibition has been an area of interest for multiple potential indications including oncology, acne vulgaris, metabolic diseases such as type 2 diabetes mellitus, and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. A critical role for ACC in de novo synthesis of long-chain fatty acids during fetal development has been demonstrated in studies in mice lacking Acc1, where the absence of Acc1 results in early embryonic lethality. Following positive predictions of developmental toxicity in the alternative in vitro assays (positive in murine embryonic stem cell [mESC] assay and rat whole embryo culture, but negative in zebrafish), developmental toxicity (growth retardation and dysmorphogenesis associated with disrupted midline fusion) was observed with the oral administration of the dual ACC1 and 2 inhibitors, PF-05175157, in Sprague Dawley rats and New Zealand White rabbits. The results of these studies are presented here to make comparisons across the assays, as well as mechanistic insights from the mESC assay demonstrating high ACC expression in the mESC and that ACC-induced developmental toxicity can be rescued with palmitic acid providing supportive evidence for DNL pathway inhibition as the underlying mechanism. Ultimately, while the battery of alternative approaches and weight-of-evidence case were useful for hazard identification, the embryo-fetal development studies were necessary to inform the risk assessment on the adverse fetal response, as malformations and/or embryo-fetal lethality were limited to doses that caused near-complete inhibition of DNL.

Characterizing sources of variability in zebrafish embryo screening protocols.

There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.

High-Mileage Light-Duty Fleet Vehicle Emissions: Their Potentially Overlooked Importance.

State and local agencies in the United States use activity-based computer models to estimate mobile source emissions for inventories. These models generally assume that vehicle activity levels are uniform across all of the vehicle emission level classifications using the same age-adjusted travel fractions. Recent fuel-specific emission measurements from the SeaTac Airport, Los Angeles, and multi-year measurements in the Chicago area suggest that some high-mileage fleets are responsible for a disproportionate share of the fleet's emissions. Hybrid taxis at the airport show large increases in carbon monoxide, hydrocarbon, and oxide of nitrogen emissions in their fourth year when compared to similar vehicles from the general population. Ammonia emissions from the airport shuttle vans indicate that catalyst reduction capability begins to wane after 5-6 years, 3 times faster than is observed in the general population, indicating accelerated aging. In Chicago, the observed, on-road taxi fleet also had significantly higher emissions and an emissions share that was more than double their fleet representation. When compounded by their expected higher than average mileage accumulation, we estimate that these small fleets (<1% of total) may be overlooked as a significant emission source (>2-5% of fleet emissions).

Reactive Nitrogen Species Emission Trends in Three Light-/Medium-Duty United States Fleets.

Repeated, fuel-specific, emission measurements in Denver (2005/2013), Los Angeles (LA) (2008/2013), and Tulsa (2005/2013) provide long-term trends in on-road reactive nitrogen emissions from three light-/medium-duty U.S. fleets. Reductions in oxides of nitrogen (NOx) emissions ranged from 21% in Denver (from 5.6 ± 1.3 to 4.4 ± 0.2 g of NOx/kg of fuel) to 43% in Tulsa (from 4.4 ± 0.3 to 2.5 ± 0.1 g of NOx/kg of fuel) since 2005, while decreases in fleet ammonia (NH3) emissions ranged from no change in Denver (from 0.45 ± 0.09 to 0.44 ± 0.02 g of NH3/kg of fuel) since 2005 to a 28% decrease in LA (from 0.80 ± 0.02 to 0.58 ± 0.02 g of NH3/kg of fuel) since 2008. The majority of the reduction in gasoline vehicle NOx emissions occurred prior to the full implementation of the Tier II emission standards in 2009. High in-use NOx emissions from small-engine diesel passenger vehicles produced a significant contribution to the fleet means despite their small numbers. NH3 emissions decreased at a slower rate than NOx emissions as a result of modest NH3 emission reduction among the newest vehicles and increased emissions from a growing number of older vehicles with active catalytic converters. In addition, the reactive nitrogen emissions from many new model year vehicles are now dominated by NH3.

Stem cell-derived systems in toxicology assessment.

Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and research applications. However, scientific and technical hurdles must be overcome before SCD in vitro methods undergo appropriate validation and become accepted in the regulatory arena.

On-road heavy-duty vehicle emissions monitoring system.

The introduction of particulate and oxides of nitrogen (NOx) after-treatment controls on heavy-duty vehicles has spurred the need for fleet emissions data to monitor their reliability and effectiveness. The University of Denver has developed a new method for rapidly measuring heavy-duty vehicles for gaseous and particulate fuel specific emissions. The method was recently used to collect 3088 measurements at a Port of Los Angeles location and a weigh station on I-5 in northern California. The weigh station NOx emissions for 2014 models are 73% lower than 2010 models (3.8 vs 13.9 gNOx/kg of fuel) and look to continue to decrease with newer models. The Port site has a heavy-duty fleet that has been entirely equipped with diesel particulate filters since 2010. Total particulate mass and black carbon measurements showed that only 3% of the Port vehicles measured exceed expected emission limits with mean gPM/kg of fuel emissions of 0.031 ± 0.007 and mean gBC/kg of fuel emissions of 0.020 ± 0.003. Mean particulate emissions were higher for the older weigh station fleet but 2011 and newer trucks gPM/kg of fuel emissions were nevertheless more than a factor of 30 lower than the means for pre-DPF (2007 and older) model years.

The recession of 2008 and its impact on light-duty vehicle emissions in three western United States cities.

The global economic recession of 2008-2010 severely depressed light-duty vehicle sales in the United States. On-road fleets observed with a remote vehicle exhaust sensor in 2013 at three historical sampling locations in Denver, Los Angeles, and Tulsa showed large reductions in the fleet fractions of 2009 model year vehicles of 40%, 38%, and 35%, respectively, when compared to prerecession 2007 levels with the light-duty truck category suffering the largest percentage declines. The fleet fraction for these ∼ 5 year old vehicles is normally reserved for vehicles more than twice their age. This resulted in a significant increase in the on-road freeway fleet age, which had been relatively stable. The fleet average age increased by two years in Denver and Los Angeles but only by one year in Tulsa, likely due to its faster economic recovery. Using fleet fractions from previous data sets, we estimated age-adjusted mean emissions increases for the 2013 fleet to be 17-29% higher for carbon monoxide, 9-14% higher for hydrocarbons, 27-30% higher for nitric oxide, and 7-16% higher for ammonia emissions than if historical fleet turnover rates had prevailed.

Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay.

A consortium of biopharmaceutical companies previously developed an optimized Zebrafish developmental toxicity assay (ZEDTA) where chorionated embryos were exposed to non-proprietary test compounds from 5 to 6 h post fertilization and assessed for morphological integrity at 5 days post fertilization. With the original 20 test compounds, this achieved an overall predictive value for teratogenicity of 88% of mammalian in vivo outcome [Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Interlaboratory assessment of a harmonized Zebrafish developmental toxicology assay-Progress report on phase I. Reprod. Toxicol. 33, 155-164]. In the second phase of this project, 38 proprietary pharmaceutical compounds from four consortium members were evaluated in two laboratories using the optimized method using either pond-derived or cultivated-strain wild-type Zebrafish embryos at concentrations up to 100µM. Embryo uptake of all compounds was assessed using liquid chromatography-tandem mass spectrometry. Twenty eight of 38 compounds had a confirmed embryo uptake of >5%, and with these compounds the ZEDTA achieved an overall predictive value of 82% and 65% at the two respective laboratories. When low-uptake compounds (≤ 5%) were retested with logarithmic concentrations up to 1000µM, the overall predictivity across all 38 compounds was 79% and 62% respectively, with the first laboratory achieving 74% sensitivity (teratogen detection) and 82% specificity (non-teratogen detection) and the second laboratory achieving 63% sensitivity (teratogen detection) and 62% specificity (non-teratogen detection). Subsequent data analyses showed that technical differences rather than strain differences were the primary contributor to interlaboratory differences in predictivity. Based on these results, the ZEDTA harmonized methodology is currently being used for compound assessment at lead optimization stage of development by 4/5 of the consortium companies.

Improving the odds of success in drug discovery: choosing the best compounds for in vivo toxicology studies.

A set of molecules that advanced into exploratory animal toxicology studies (two species) was examined to determine what properties contributed to success in these safety studies. Compounds were rigorously evaluated across numerous safety end points and classified as "pass" if a suitable in vivo therapeutic index (TI) was achieved for advancement into regulatory toxicology studies. The most predictive end point contributing to compound survival was a predicted human efficacious concentration (Ceff) of ≤250 nM (total drug) and ≤40 nM (free drug). This trend held across a wide range of CNS modes of action, encompassing targets such as enzymes, G-protein-coupled receptors, ion channels, and transporters.

Heavy-duty truck emissions in the South Coast Air Basin of California.

California and Federal emissions regulations for 2007 and newer heavy-duty diesel engines require an order of magnitude reduction in particulate matter and oxides of nitrogen spurring the introduction of new aftertreatment systems. Since 2008, four emission measurement campaigns have been conducted at a Port of Los Angeles location and an inland weigh station in the South Coast Air Basin of California. Fuel specific oxides of nitrogen emissions at the Port have decreased 12% since 2010 while infrared opacity (a measure of particulate matter) remained low, showing no diesel particulate filter deterioration. The weigh station truck's fuel specific oxides of nitrogen emission reductions since 2010 (18.5%) almost double the previous three year's reductions and are the result of new trucks using selective catalytic reduction systems. Trucks at the weigh station equipped with these systems have a skewed oxides of nitrogen emissions distribution (half of the emissions were from 6% of the measurements) and had significantly lower emissions than similarly equipped Port trucks. Infrared thermographs of truck exhaust pipes revealed that the mean temperature observed at the weigh station (225 ± 4.5 °C) was 70 °C higher than for Port trucks, suggesting that the catalytic aftertreatment systems on trucks at our Port site were often below minimum operating temperatures.

Toxicity assessments of nonsteroidal anti-inflammatory drugs in isolated mitochondria, rat hepatocytes, and zebrafish show good concordance across chemical classes.

To reduce costly late-stage compound attrition, there has been an increased focus on assessing compounds in in vitro assays that predict attributes of human safety liabilities, before preclinical in vivo studies are done. Relevant questions when choosing a panel of assays for predicting toxicity are (a) whether there is general concordance in the data among the assays, and (b) whether, in a retrospective analysis, the rank order of toxicity of compounds in the assays correlates with the known safety profile of the drugs in humans. The aim of our study was to answer these questions using nonsteroidal anti-inflammatory drugs (NSAIDs) as a test set since NSAIDs are generally associated with gastrointestinal injury, hepatotoxicity, and/or cardiovascular risk, with mitochondrial impairment and endoplasmic reticulum stress being possible contributing factors. Eleven NSAIDs, flufenamic acid, tolfenamic acid, mefenamic acid, diclofenac, meloxicam, sudoxicam, piroxicam, diflunisal, acetylsalicylic acid, nimesulide, and sulindac (and its two metabolites, sulindac sulfide and sulindac sulfone), were tested for their effects on (a) the respiration of rat liver mitochondria, (b) a panel of mechanistic endpoints in rat hepatocytes, and (c) the viability and organ morphology of zebrafish. We show good concordance for distinguishing among/between NSAID chemical classes in the observations among the three approaches. Furthermore, the assays were complementary and able to correctly identify "toxic" and "non-toxic" drugs in accordance with their human safety profile, with emphasis on hepatic and gastrointestinal safety. We recommend implementing our multi-assay approach in the drug discovery process to reduce compound attrition.

Multispecies remote sensing measurements of vehicle emissions on Sherman Way in Van Nuys, California.

UNLABELLED: As part of the 2010 Van Nuys tunnel study, researchers from the University of Denver measured on-road fuel-specific light-duty vehicle emissions from nearly 13,000 vehicles on Sherman Way (0.4 miles west of the tunnel) in Van Nuys, California, with its multispecies Fuel Efficiency Automobile Test (FEAT) remote sensor a week ahead of the tunnel measurements. The remote sensing mean gram per kilogram carbon monoxide (CO), hydrocarbon (HC), and oxide of nitrogen (NO(x)) measurements are 8.9% lower 41% higher, and 24% higher than the tunnel measurements, respectively. The remote sensing CGO/NO(x) and HC/NO(x) mass ratios are 28% lower and 20% higher than the comparable tunnel ratios. Comparisons with the historical tunnel measurements show large reductions in CO, HC, and NO(x) over the past 23 yr, but little change in the HC/NO(x) mass ratio since 1995. The fleet CO and HC emissions are increasingly dominated by a few gross emitters, with more than a third of the total emissions being contributed by less than 1% of the fleet. An example of this is a 1995 vehicle measured three times with an average HC emission of 419 g/kg fuel (two-stroke snowmobiles average 475 g/kg fuel), responsible for 4% of the total HC emissions. The 2008 economic downturn dramatically reduced the number of new vehicles entering the fleet, leading to an age increase (> 1 model year) of the Sherman Way fleet that has increased the fleet's ammonia (NH3) emissions. The mean NH3 levels appear little changed from previous measurements collected in the Van Nuys tunnel in 1993. Comparisons between weekday and weekend data show few fleet differences, although the fraction of light-duty diesel vehicles decreased from the weekday (1.7%) to Saturday (1.2%) and Sunday (0.6%). IMPLICATIONS: On-road remote sensing emission measurements of light-duty vehicles on Sherman Way in Van Nuys, California, show large historical emission reductions for CO and HC emissions despite an older fleet arising from the 2008 economic downturn. Fleet CO and HC emissions are increasingly dominated by a few gross emitters, with a single 1995 vehicle measured being responsible for 4% of the entire fleet's HC emissions. Finding and repairing and/or scrapping as little as 2% of the fleet would reduce on-road tailpipe emissions by as much as 50%. Ammonia emissions have locally increased with the increasing fleet age.

Comparison of the MOVES2010a, MOBILE6.2, and EMFAC2007 mobile source emission models with on-road traffic tunnel and remote sensing measurements.

UNLABELLED: The Desert Research Institute conducted an on-road mobile source emission study at a traffic tunnel in Van Nuys, California, in August 2010 to measure fleet-averaged, fuel-based emission factors. The study also included remote sensing device (RSD) measurements by the University of Denver of 13,000 vehicles near the tunnel. The tunnel and RSD fleet-averaged emission factors were compared in blind fashion with the corresponding modeled factors calculated by ENVIRON International Corporation using U.S. Environmental Protection Agency's (EPA's) MOVES2010a (Motor Vehicle Emissions Simulator) and MOBILE6.2 mobile source emission models, and California Air Resources Board's (CARB's) EMFAC2007 (EMission FACtors) emission model. With some exceptions, the fleet-averaged tunnel, RSD, and modeled carbon monoxide (CO) and oxide of nitrogen (NO(x)) emission factors were in reasonable agreement (+/- 25%). The nonmethane hydrocarbon (NMHC) emission factors (specifically the running evaporative emissions) predicted by MOVES were insensitive to ambient temperature as compared with the tunnel measurements and the MOBILE- and EMFAC-predicted emission factors, resulting in underestimation of the measured NMHC/NO(x) ratios at higher ambient temperatures. Although predicted NMHC/NO(x) ratios are in good agreement with the measured ratios during cooler sampling periods, the measured NMHC/NO(x) ratios are 3.1, 1.7, and 1.4 times higher than those predicted by the MOVES, MOBILE, and EMFAC models, respectively, during high-temperature periods. Although the MOVES NO(x) emission factors were generally higher than the measured factors, most differences were not significant considering the variations in the modeled factors using alternative vehicle operating cycles to represent the driving conditions in the tunnel. The three models predicted large differences in NO(x) and particle emissions and in the relative contributions of diesel and gasoline vehicles to total NO(x) and particulate carbon (TC) emissions in the tunnel. IMPLICATIONS: Although advances have been made to mobile source emission models over the past two decades, the evidence that mobile source emissions of carbon monoxide and hydrocarbons in urban areas were underestimated by as much as a factor of 2-3 in past inventories underscores the need for on-going verification of emission inventories. Results suggest that there is an overall increase in motor vehicle NMHC emissions on hot days that is not fully accounted for by the emission models. Hot temperatures and concomitant higher ratios of NMHC emissions relative to NO(x) both contribute to more rapid and efficient formation of ozone. Also, the ability of EPA's MOVES model to simulate varying vehicle operating modes places increased importance on the choice of operatingmodes to evaluate project-level emissions.

Embryo-fetal transfer of bevacizumab (Avastin) in the rat over the course of gestation and the impact of neonatal Fc receptor (FcRn) binding.

BACKGROUND: There is concern about embryo-fetal exposure to antibody-based biopharmaceuticals based on the increase of such therapies being prescribed to women of childbearing potential. Therefore, there is a desire to better characterize embryo-fetal exposure of these molecules. The pregnant rat is a standard model for evaluating the potential consequences of exposure but placental transfer of antibody-based biopharmaceuticals is not well understood in this model. METHODS: The relative embryo-fetal distribution of an antibody-based biopharmaceutical was evaluated in the rat. Bevacizumab (Avastin) was chosen as a tool antibody since it does not have significant target binding in the rat that might influence embryo-fetal biodistribution. Avastin was labeled with a fluorescent dye, characterized, and injected into pregnant rats at different gestation ages. Labeled Avastin in fetal tissues was visualized ex vivo using an IVIS 200 (Caliper, A PerkinElmer Company, Alameda, CA). RESULTS: Avastin localized to the fetus as early as 24-hr post intravenous injection of the dam, and was taken up by the fetus in a dose-dependent manner. Avastin was detectable in the developing embryo as early as gestation day 13 and continued to be transferred until the end of gestation. Fetal transfer of Avastins mutated in the portion of the antibody that binds the neonatal Fc receptor (FcRn) was tested in late gestation and was found to correlate with affinities of the mutant Avastin antibody to FcRn. CONCLUSIONS: The novel application of this imaging technology was used to characterize the onset and duration of Avastin maternal-fetal transfer in rats and the importance of FcRn binding.

Reproductive toxicity assessment of sunitinib, a multitargeted receptor tyrosine kinase inhibitor, in male and female rats.

BACKGROUND: Sunitinib (SUTENT, Pfizer Inc., New York, NY) is a multitargeted inhibitor of selected receptor tyrosine kinases, which produces an antiproliferative and antiangiogenic effect by blocking pathways fundamental to tumor growth and survival. We investigated the effects of sunitinib on male and female fertility and early embryonic development in the rat. METHODS: In the female fertility and early embryonic development phase, untreated males were paired with treated females dosed at 0 (control), 0.5, 1.5, and 5 mg/kg/day from 14 days premating, through mating, to gestation day 7. In the male fertility phase, the same males were then treated 58 days at doses of 0 (control), 1, 3, and 10 mg/kg/day, mated with untreated females, with continued daily dosing for a total of 74 days. RESULTS: There was no systemic toxicity- or treatment-related effects on fertility in female rats. Females exposed at 5 mg/kg/day had an increase in the number of early resorptions with associated decrease in viable embryos. In the males, body weight and food consumption were decreased at 10 mg/kg/day compared to the controls. Male reproductive capacity, as assessed by copulation, fertility, and conception indices, was not impacted at any dose level. Sperm morphology, concentration, and motility were also unaffected by treatment. CONCLUSIONS: There were no effects on male reproduction. An increase in corpora lutea and an increase in early resorptions with associated reduction in viable embryos was noted in the females dosed 5 mg/kg/day. Sunitinib at doses up to 1.5 and 10 mg/kg/day had no effects on female and male reproduction, respectively.

Emission changes resulting from the San Pedro Bay, California Ports Truck Retirement Program.

Recent U.S. Environmental Protection Agency emissions regulations have resulted in lower emissions of particulate matter and oxides of nitrogen from heavy-duty diesel trucks. To accelerate fleet turnover the State of California in 2008 along with the Ports of Los Angeles and Long Beach (San Pedro Bay Ports) in 2006 passed regulations establishing timelines forcing the retirement of older diesel trucks. On-road emissions measurements of heavy-duty diesel trucks were collected over a three-year period, beginning in 2008, at a Port of Los Angeles location and an inland weigh station on the Riverside freeway (CA SR91). At the Port location the mean fleet age decreased from 12.7 years in April of 2008 to 2.5 years in May of 2010 with significant reductions in carbon monoxide (30%), oxides of nitrogen (48%) and infrared opacity (a measure of particulate matter, 54%). We also observed a 20-fold increase in ammonia emissions as a result of new, stoichiometrically combusted, liquefied natural gas powered trucks. These results compare with changes at our inland site where the average ages were 7.9 years in April of 2008 and 8.3 years in April of 2010, with only small reductions in oxides of nitrogen (10%) being statistically significant. Both locations have experienced significant increases in nitrogen dioxide emissions from new trucks equipped with diesel particle filters; raising the mean nitrogen dioxide to oxides of nitrogen ratios from less than 10% to more than 30% at the Riverside freeway location.

Developmental toxicity testing for safety assessment: new approaches and technologies.

The ILSI Health and Environmental Sciences Institute's Developmental and Reproductive Toxicology Technical Committee held a 2-day workshop entitled "Developmental Toxicology-New Directions" in April 2009. The fourth session of this workshop focused on new approaches and technologies for the assessment of developmental toxicology. This session provided an overview of the application of genomics technologies for developmental safety assessment, the use of mouse embryonic stem cells to capture data on developmental toxicity pathways, dynamical cell imaging of zebrafish embryos, the use of computation models of development pathways and systems, and finally, high-throughput in vitro approaches being utilized by the EPA ToxCast program. Issues discussed include the challenges of anchoring in vitro predictions to relevant in vivo endpoints and the need to validate pathway-based predictions with targeted studies in whole animals. Currently, there are 10,000 to 30,000 chemicals in world-wide commerce in need of hazard data for assessing potential health risks. The traditional animal study designs for assessing developmental toxicity cannot accommodate the evaluation of this large number of chemicals, requiring that alternative technologies be utilized. Though a daunting task, technologies are being developed and utilized to make that goal reachable.

Not a walk in the park: the ECVAM whole embryo culture model challenged with pharmaceuticals and attempted improvements with random forest design.

BACKGROUND: The European Committee for the Validation of Alternative Methods (ECVAM) supported the development of a linear discriminant embryotoxicity prediction model founded on rat whole embryo culture (Piersma et al. (2004). Altern Lab Anim 32:275­307). Our goals were to (1) assess the accuracy of this model with pharmaceuticals, and (2) to use the data to develop a more accurate prediction model. METHODS: Sixty-one chemicals of known in vivo activity were tested. They were part of the ECVAM validation set (N513), commercially available pharmaceuticals (N531), and Pfizer chemicals that did not reach the market, but for which developmental toxicity data were available (N517). They were tested according to the ECVAM procedures. Fifty-seven of these chemicals were used for Random Forest modeling to develop an alternate model with the goal of using surrogate endpoints for simplified assessments and to improve the predictivity of the model. RESULTS: Using part of the ECVAM chemical test set, the ECVAM prediction model was 77% accurate. This approximated what was reported in the validation study (80%; Piersma et al. (2004). Altern Lab Anim 32:275­307). However, when confronted with novel chemicals, the accuracy of the linear discriminant model dropped to 56%. In an attempt to improve this performance, we used a Random Forest model that provided rankings and confidence estimates. Although the model used simpler endpoints, its performance was no better than the ECVAM linear discriminant model. CONCLUSIONS: This study confirms previous concerns about the applicability of the ECVAM prediction model to a more diverse chemical set, and underscores the challenges associated with developing embryotoxicity prediction models.

On-road emission measurements of reactive nitrogen compounds from three California cities.

The three California cities of San Jose, Fresno, and West Los Angeles (wLA) were visited during March 2008 to collect on-road emission measurements of reactive nitrogen compounds from light-duty vehicles. At the San Jose and wLA sites, comparison with historical measurements showed that emissions of carbon monoxide (CO), hydrocarbons (HC), and nitric oxide (NO) continue to decrease in the on-road fleet, yet the ratio of nitrogen dioxide (NO(2)) to NO in new diesel vehicles appears to be undergoing large increases. A small fleet of 2007 diesel ambulances measured in Fresno was found to have more than 60% of their emitted oxides of nitrogen as NO(2). Ammonia (NH(3)) emissions are shown to have a strong dependence on model year and vehicle specific power. NH(3) means are 0.49 +/- 0.02, 0.49 +/- 0.01, and 0.79 +/- 0.02 g/kg of fuel for San Jose, Fresno, and wLA, respectively, with the larger emissions at the wLA site likely due to driving mode. NH(3) at these locations was found to account for 25%, 22%, and 27% of the molar fixed nitrogen emissions, respectively. Using these mean values to construct a national fuel-based NH(3) inventory results in a range of 210000 to 330000 short tons of NH(3) annually from light-duty vehicles.

Portable emission measurements of Yellowstone Park snowcoaches and snowmobiles.

As part of the National Park Service's Temporary Winter Use Plans Environmental Assessment, the University of Denver has been collecting in-use tailpipe emissions data from snowcoaches and snowmobiles in Yellowstone National Park. During the winter of 2006, using a portable emissions monitoring system, tailpipe data were collected from 10 snowcoaches and 2 four-stroke snowmobiles. These vehicles were operated over a standard route within the park, and the snowcoaches all carried identical passenger loads. These snowcoaches were newer in age with more advanced fuel management technology than those studied earlier, and average emissions were lower as a result (120, 1.7, and 11 g/mi for carbon monoxide [CO], hydrocarbons [HC], and oxides of nitrogen [NOx]). Large emissions variability was still observed despite using a standardized route and equal passenger loading. A comparison between five nearly identically equipped snowcoaches that had CO emissions ranging between 12 and 310 g/mi suggests that snow and road conditions are the most important factors behind the large emissions variability observed between modern snowcoaches: The first comprehensive emission measurements, using a portable emissions measurement system, on two snowmobiles showed that computer-controlled fuel management systems have increased fuel economy (>25 mpg) and are a major reason that emissions from these winter vehicles have dropped so dramatically. Using all of the tailpipe emissions data collected to date shows that the two primary winter vehicles in Yellowstone National Park are now very similar in their per-passenger emissions.