Safety and Pharmacokinetics of Intravenous 10-1074 and VRC01LS in Young Children.

BACKGROUND: Broadly neutralizing monoclonal antibodies (bNAbs) suppress HIV-1 RNA and may deplete residual viral reservoirs. We evaluated the safety and pharmacokinetics (PK) of dual intravenous VRC01LS and 10-1074 in very early-treated children with HIV-1 on suppressive antiretroviral treatment (ART). SETTING: Botswana. METHODS: Children with HIV-1 (median age 3.1 years) on ART from <7 days old were enrolled. In phase A, 6 children received 10-1074 (30 mg/kg at day 0, 28, and 56) and 6 children received VRC01LS (30 mg/kg at day 0, 10 mg/kg at days 28 and 56) by intravenous infusion. In phase B, 6 children received the 2 bNAbs combined (with higher VRC01LS maintenance dose, 15 mg/kg) every 4 weeks for 32 weeks with PK evaluations over 8 weeks. Population PK models were developed to predict steady-state concentrations. RESULTS: BNAb infusions were well tolerated. There were no infusion reactions nor any bNAb-related grade 3 or 4 events. The median (range) first dose Cmax and trough (day 28) combined from both phases were 1405 (876-1999) µg/mL and 133 (84-319) µg/mL for 10-1074 and 776 (559-846) µg/mL and 230 (158-294) µg/mL for VRC01LS. No large differences in bNAb clearances were observed when given in combination. The estimated VRC01LS half-life was shorter than in adults. Predicted steady-state troughs [median (90% prediction interval)] were 261 (95-565) and 266 (191-366) µg/mL for 10-1074 and VRC01LS, respectively, when given in combination. CONCLUSIONS: 10-1074 and VRC01LS were safe and well-tolerated among children receiving ART. Troughs exceeded minimal targets with every 4-week administration of 10-1074 at 30 mg/kg and VRC01LS at 15 mg/kg.

Development of a quantitative morphological assessment of toxicant-treated zebrafish larvae using brightfield imaging and high-content analysis.

One of the rate-limiting procedures in a developmental zebrafish screen is the morphological assessment of each larva. Most researchers opt for a time-consuming, structured visual assessment by trained human observer(s). The present studies were designed to develop a more objective, accurate and rapid method for screening zebrafish for dysmorphology. Instead of the very detailed human assessment, we have developed the computational malformation index, which combines the use of high-content imaging with a very brief human visual assessment. Each larva was quickly assessed by a human observer (basic visual assessment), killed, fixed and assessed for dysmorphology with the Zebratox V4 BioApplication using the Cellomics® ArrayScan® V(TI) high-content image analysis platform. The basic visual assessment adds in-life parameters, and the high-content analysis assesses each individual larva for various features (total area, width, spine length, head-tail length, length-width ratio, perimeter-area ratio). In developing the computational malformation index, a training set of hundreds of embryos treated with hundreds of chemicals were visually assessed using the basic or detailed method. In the second phase, we assessed both the stability of these high-content measurements and its performance using a test set of zebrafish treated with a dose range of two reference chemicals (trans-retinoic acid or cadmium). We found the measures were stable for at least 1 week and comparison of these automated measures to detailed visual inspection of the larvae showed excellent congruence. Our computational malformation index provides an objective manner for rapid phenotypic brightfield assessment of individual larva in a developmental zebrafish assay. Copyright © 2016 John Wiley & Sons, Ltd.

Fluoxetine alters adult freshwater mussel behavior and larval metamorphosis.

We used acute and partial-lifecycle tests to examine the effects of the pharmaceutical fluoxetine on freshwater mussels (Unionida). In acute tests lasting 24-48 h, we determined median effective concentrations (EC50s) for fluoxetine with larval (glochidia viability) and juvenile (survival) life-stages of fatmucket (Lampsilis siliquoidea) and black sandshell (Ligumia recta). In a 28-d behavioral test we exposed brooding adult female wavy-rayed lampmussels (Lampsilis fasciola) to 0.37 and 29.3 µg/L fluoxetine to determine effects on adult behavior (foot protrusion, mantle lure display and glochidia parturition). We also assessed the effects of 24-h exposure of 1 and 100 µg/L fluoxetine on glochidia viability duration and metamorphosis success for the wavy-rayed lampmussel. Fluoxetine EC50s ranged from 62 µg/L for juveniles (96 h) to 293 µg/L for glochidia (24 h). In adults, statistically significant increases were observed in foot protrusion at 0.37 and 29.3 µg/L fluoxetine and lure display rates at 29.3 µg/L; glochidia parturition was not significantly affected at any test concentration. Twenty-four hour exposure of glochidia to fluoxetine did not affect viability duration, but likelihood of metamorphosis to the juvenile stage significantly increased with 1 and 100 µg/L treatments. Our results demonstrated effects of fluoxetine to unionid mussels at concentrations less than previously reported and approaching concentrations measured in surface waters.

Incorporating exposure information into the toxicological prioritization index decision support framework.

The Toxicological Prioritization Index (ToxPi) decision support framework was previously developed to facilitate incorporation of diverse data to prioritize chemicals based on potential hazard. This ToxPi index was demonstrated by considering results of bioprofiling related to potential for endocrine disruption. However, exposure information is required along with hazard information to prioritize chemicals for further testing. The goal of this analysis is to demonstrate the utility of the ToxPi framework for incorporating exposure information to rank chemicals and improve understanding of key exposure surrogates. The ToxPi tool was applied to common exposure surrogates (i.e., fate parameters, manufacturing volume, and occurrence measurements) and the relationship between resulting rankings and higher-tiered exposure estimates was investigated. As information more directly relevant to human exposure potential is incorporated, relative rank of chemicals changes. Binned ToxPi results are shown to be consistent with chemical priorities based on crude measures of population-level exposure for a limited set of chemicals. However, these bins are not predictive of higher tiered estimates of exposure such as those developed for pesticide registration. Although rankings based on exposure surrogates are used in a variety of contexts, analysis of the relevance of these tools is challenging. The ToxPi framework can be used to gain insight into the factors driving these rankings and aid identification of key exposure metrics. Additional exposure data is required to build confidence in exposure-based chemical prioritization.

Partial life-cycle and acute toxicity of perfluoroalkyl acids to freshwater mussels.

Freshwater mussels are among the most sensitive aquatic organisms to many contaminants and have complex life-cycles that include several distinct life stages with unique contaminant exposure pathways. Standard acute (24-96 h) and chronic (28 d) toxicity tests with free larva (glochidia) and juvenile mussels are effective at generating data on contaminant effects at two discrete life stages but do not incorporate effects on brooded glochidia. We developed a novel partial life-cycle assay that incorporates exposures to brooding adult female mussels and used this method in combination with acute toxicity tests to assess adverse effects of perfluoroctanesulfonic acid (PFOS) and perfluoroctanoic acid (PFOA) on freshwater mussels. Fatmucket (Lampsilis siliquoidea) were exposed to PFOS at two life stages: brooding glochidia (in marsupia) for 36 d and free glochidia in water for 24 h. In standard acute tests with glochidia (24-48 h exposures) and juveniles (48-96 h exposures) of fatmucket and black sandshell (Ligumia recta), glochidia were 8 to 25 times more sensitive than juveniles. Perfluoroctanesulfonic acid significantly reduced the duration of glochidia viability and reduced probability of metamorphosis at concentrations 3,000 times lower than the most sensitive acute endpoint (24-h EC50). The partial life-cycle test is adaptable to a variety of endpoints and research objectives and is useful for identifying adverse effects at contaminant concentrations below those required for an acute lethal response.

Aggregating data for computational toxicology applications: The U.S. Environmental Protection Agency (EPA) Aggregated Computational Toxicology Resource (ACToR) System.

Computational toxicology combines data from high-throughput test methods, chemical structure analyses and other biological domains (e.g., genes, proteins, cells, tissues) with the goals of predicting and understanding the underlying mechanistic causes of chemical toxicity and for predicting toxicity of new chemicals and products. A key feature of such approaches is their reliance on knowledge extracted from large collections of data and data sets in computable formats. The U.S. Environmental Protection Agency (EPA) has developed a large data resource called ACToR (Aggregated Computational Toxicology Resource) to support these data-intensive efforts. ACToR comprises four main repositories: core ACToR (chemical identifiers and structures, and summary data on hazard, exposure, use, and other domains), ToxRefDB (Toxicity Reference Database, a compilation of detailed in vivo toxicity data from guideline studies), ExpoCastDB (detailed human exposure data from observational studies of selected chemicals), and ToxCastDB (data from high-throughput screening programs, including links to underlying biological information related to genes and pathways). The EPA DSSTox (Distributed Structure-Searchable Toxicity) program provides expert-reviewed chemical structures and associated information for these and other high-interest public inventories. Overall, the ACToR system contains information on about 400,000 chemicals from 1100 different sources. The entire system is built using open source tools and is freely available to download. This review describes the organization of the data repository and provides selected examples of use cases.

Effects of lead on Na⁺, K⁺-ATPase and hemolymph ion concentrations in the freshwater mussel Elliptio complanata.

Freshwater mussels are an imperiled fauna exposed to a variety of environmental toxicants such as lead (Pb) and studies are urgently needed to assess their health and condition to guide conservation efforts. A 28-day laboratory toxicity test with Pb and adult Eastern elliptio mussels (Elliptio complanata) was conducted to determine uptake kinetics and to assess the toxicological effects of Pb exposure. Test mussels were collected from a relatively uncontaminated reference site and exposed to a water-only control and five concentrations of Pb (as lead nitrate) ranging from 1 to 245 µg/L in a static renewal test with a water hardness of 42 mg/L. Endpoints included tissue Pb concentrations, hemolymph Pb and ion (Na⁺, K⁺, Cl⁻, Ca²âº) concentrations, and Na⁺, K⁺-ATPase enzyme activity in gill tissue. Mussels accumulated Pb rapidly, with tissue concentrations increasing at an exposure-dependent rate for the first 2 weeks, but with no significant increase from 2 to 4 weeks. Mussel tissue Pb concentrations ranged from 0.34 to 898 µg/g dry weight, were strongly related to Pb in test water at every time interval (7, 14, 21, and 28 days), and did not significantly increase after day 14. Hemolymph Pb concentration was variable, dependent on exposure concentration, and showed no appreciable change with time beyond day 7, except for mussels in the greatest exposure concentration (245 µg/L), which showed a significant reduction in Pb by 28 days, suggesting a threshold for Pb binding or elimination in hemolymph at concentrations near 1000 µg/g. The Na⁺, K⁺-ATPase activity in the gill tissue of mussels was significantly reduced by Pb on day 28 and was highly correlated with tissue Pb concentration (R² = 0.92; P = 0.013). The Na⁺, K⁺-ATPase activity was correlated with reduced hemolymph Na⁺ concentration at the greatest Pb exposure when enzyme activity was at 30% of controls. Hemolymph Ca²âº concentration increased significantly in mussels from the greatest Pb exposure and may be due to remobilization from the shell in an attempt to buffer the hemolymph against Pb uptake and toxicity. We conclude that Na⁺, K⁺-ATPase activity in mussels was adversely affected by Pb exposure, however, because the effects on activity were variable at the lower test concentrations, additional research is warranted over this range of exposures.

The exposure data landscape for manufactured chemicals.

The U.S. Environmental Protection Agency is developing chemical screening and prioritization programs to evaluate environmental chemicals for potential risk to human health in a rapid and efficient manner. As part of these efforts, it is important to catalog available information on chemical toxicity and exposure from widely dispersed sources. The main objective of this analysis is to define important aspects of the exposure space and to catalog the available exposure information for chemicals being considered for analysis as part of the U.S. EPA ToxCast™ screening and prioritization program. Publicly available exposure data have been extracted into ACToR (Aggregated Computational Toxicology Resource), which combines information for hundreds of thousands of chemicals from >600 public sources. We use data from ACToR to assess the exposure data landscape for environmental chemicals. Of the roughly 100,000 chemicals that have at least limited toxicity information available, less than one-fifth also have exposure information - and for most of these the information is of limited utility (e.g., production volume). Readily accessible data on concentrations in exposure-related media are only available for a much smaller fraction. Among these, the largest number of chemicals is measured in water with over 1150 unique compounds, followed by 788 substances measured in soil, and 670 in air. These small numbers clearly reflect a focus of resources on those substances previously identified as possibly posing a hazard to human health. Exposure to a much broader number of chemicals will need to be measured in order to fully realize the envisioned goal of using exposure information to guide toxicity testing.

Acute and chronic toxicity of glyphosate compounds to glochidia and juveniles of Lampsilis siliquoidea (Unionidae).

Native freshwater mussels (family Unionidae) are among the most imperiled faunal groups in the world. Factors contributing to the decline of mussel populations likely include pesticides and other aquatic contaminants; however, there is a paucity of data regarding the toxicity of even the most globally distributed pesticides, including glyphosate, to mussels. Therefore, the toxicity of several forms of glyphosate, its formulations, and a surfactant (MON 0818) used in several glyphosate formulations was determined for early life stages of Lampsilis siliquoidea, a native freshwater mussel. Acute and chronic toxicity tests were performed with a newly established American Society of Testing and Materials (ASTM) standard guide for conducting toxicity tests with freshwater mussels. Roundup, its active ingredient, the technical-grade isopropylamine (IPA) salt of glyphosate, IPA alone, and MON 0818 (the surfactant in Roundup formulations) were each acutely toxic to L. siliquoidea glochidia. MON 0818 was most toxic of the compounds tested and the 48-h median effective concentration (0.5 mg/L) for L. siliquoidea glochidia is the lowest reported for any aquatic organism tested to date. Juvenile L. siliquoidea were also acutely sensitive to MON 0818, Roundup, glyphosate IPA salt, and IPA alone. Technical-grade glyphosate and Aqua Star were not acutely toxic to glochidia or juveniles. Ranking of relative chronic toxicity of the glyphosate-related compounds to juvenile mussels was similar to the ranking of relative acute toxicity to juveniles. Growth data from chronic tests was largely inconclusive. In summary, these results indicate that L. siliquoidea, a representative of the nearly 300 freshwater mussel taxa in North America, is among the most sensitive aquatic organisms tested to date with glyphosate-based chemicals and the surfactant MON 0818.

Acute and chronic toxicity of pesticide formulations (atrazine, chlorpyrifos, and permethrin) to glochidia and juveniles of Lampsilis siliquoidea.

Freshwater mussels are among the most imperiled faunal groups in North America; approximately 67% of the nearly 300 native freshwater mussel species (family Unionidae) are listed as endangered, threatened, or of special concern. Despite evidence that glochidia and juvenile life stages are highly sensitive to some chemical contaminants, the effects of pesticides on early life stages of unionid mussels are largely unknown. In the United States, pesticide registration is based on toxicity data of the active ingredient, not formulations as they are sold and applied. Some pesticide formulations, however, are more toxic than their active ingredient (technical-grade pesticide) alone because of the presence of surfactants, adjuvants, or other ingredients in the formulation. The objective of the present study was to compare the toxicity of active ingredients of several current-use pesticides (atrazine, chlorpyrifos, and permethrin) to the toxicity of pesticide formulations to glochidia and juvenile life stages of a freshwater mussel (Lampsilis siliquoidea). The atrazine formulation (Aatrex) was more toxic than technical-grade atrazine in chronic tests with juvenile L. siliquoidea. For other pesticides, acute and chronic toxicity of technical-grade pesticides were similar to the toxicity of pesticide formulations. Median effective concentrations for chlorpyrifos were 0.43 mg/L for glochidia at 48 h, 0.25 mg/L for juveniles at 96 h, and 0.06 mg/L for juveniles at 21 d. Atrazine and permethrin as well as their formulations did not cause significant acute toxicity in glochidia or juveniles at exposure concentrations approaching water-solubility limits. Additional research is needed on other pesticides with different modes of action, on the role of different routes of exposure, and with other species of unionid mussels to evaluate similarities of toxic response.