Interlaboratory Study on Zebrafish in Toxicology: Systematic Evaluation of the Application of Zebrafish in Toxicology's (SEAZIT's) Evaluation of Developmental Toxicity.

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

Natural mineral fibers: conducting inhalation toxicology studies-part B: development of a nose-only exposure system for repeat-exposure in vivo study of Libby amphibole aerosol.

BACKGROUND: Exposure to asbestos is associated with malignant and nonmalignant respiratory disease. To strengthen the scientific basis for risk assessment on fibers, the National Institute of Environmental Health Sciences (NIEHS) has initiated a series of studies to address fundamental questions on the toxicology of naturally occurring asbestos and related mineral fibers after inhalation exposure. A prototype nose-only exposure system was previously developed and validated. The prototype system was expanded to a large-scale exposure system in this study for conducting subsequent in vivo rodent inhalation studies of Libby amphibole (LA) 2007, selected as a model fiber. RESULTS: The exposure system consisting of six exposure carousels was able to independently deliver stable LA 2007 aerosol to individual carousels at target concentrations of 0 (control group), 0.1, 0.3, 1, 3, or 10 mg/m3. A single aerosol generator was used to provide aerosol to all carousels to ensure that exposure atmospheres were chemically and physically similar, with aerosol concentration as the only major variable among the carousels. Transmission electron microscopy (TEM) coupled with energy dispersive spectrometry (EDS) and selected area electron diffraction (SAED) analysis of aerosol samples collected at the exposure ports indicated the fiber dimensions, chemical composition, and mineralogy were equivalent across exposure carousels and were comparable to the bulk LA 2007 material. CONCLUSION: The exposure system developed is ready for use in conducting nose-only inhalation toxicity studies of LA 2007 in rats. The exposure system is anticipated to have applicability for the inhalation toxicity evaluation of other natural mineral fibers of concern.

Natural mineral fibers: conducting inhalation toxicology studies - part A: Libby Amphibole aerosol generation and characterization method development.

BACKGROUND: Asbestos has been classified as a human carcinogen, and exposure may increase the risk of diseases associated with impaired respiratory function. As the range of health effects and airborne concentrations that result in health effects across asbestos-related natural mineral fiber types are not fully understood, the National Institute of Environmental Health Sciences has established a series of research studies to characterize hazards of natural mineral fibers after inhalation exposure. This paper presents the method development work of this research project. RESULTS: A prototype nose-only exposure system was fabricated to explore the feasibility of generating natural mineral fiber aerosol for in vivo inhalation toxicity studies. The prototype system consisted of a slide bar aerosol generator, a distribution/delivery system and an exposure carousel. Characterization tests conducted using Libby Amphibole 2007 (LA 2007) demonstrated the prototype system delivered stable and controllable aerosol concentration to the exposure carousel. Transmission electron microscopy (TEM) analysis of aerosol samples collected at the exposure port showed the average fiber length and width were comparable to the bulk LA 2007. TEM coupled with energy dispersive spectrometry (EDS) and selected area electron diffraction (SAED) analysis further confirmed fibers from the aerosol samples were consistent with the bulk LA 2007 chemically and physically. CONCLUSIONS: Characterization of the prototype system demonstrated feasibility of generating LA 2007 fiber aerosols appropriate for in vivo inhalation toxicity studies. The methods developed in this study are suitable to apply to a multiple-carousel exposure system for a rat inhalation toxicity testing using LA 2007.

Short-term toxicity studies of thallium (I) sulfate administered in drinking water to Sprague Dawley rats and B6C3F1/N mice.

Thallium is a heavy metal that is known to induce a broad spectrum of adverse health effects in humans including alopecia, neurotoxicity, and mortality following high dose acute poisoning events. Widespread human exposure to thallium may occur via consumption of contaminated drinking water; limited toxicity data are available to evaluate the corresponding public health risk. To address this data gap, the Division of Translational Toxicology conducted short-term toxicity studies of a monovalent thallium salt, thallium (I) sulfate. Thallium (I) sulfate was administered via dosed drinking water to time-mated Sprague Dawley (Hsd:Sprague Dawley® SD®) rats (F0 dams) and their offspring (F1) from gestation day (GD) 6 until up to postnatal day (PND) 28 at concentrations of 0, 3.13, 6.25, 12.5, 25, or 50 mg/L, and adult male and female B6C3F1/N mice for up to 2 weeks at concentrations of 0, 6.25, 12.5, 25, 50, or 100 mg/L. Rat dams in the 50 mg/L exposure group were removed during gestation, and dams and offspring in the 25 mg/L exposure group were removed on or before PND 0 due to overt toxicity. Exposure to thallium (I) sulfate at concentrations ≤ 12.5 mg/L did not impact F0 dam body weights, maintenance of pregnancy, littering parameters, or F1 survival (PND 4-28). However, in F1 pups, exposure to 12.5 mg/L thallium (I) sulfate resulted in decreased body weight gains relative to control rats and onset of whole-body alopecia. Measurement of thallium concentrations in dam plasma, amniotic fluid, fetuses (GD 18), and pup plasma (PND 4) indicated marked maternal transfer of thallium to offspring during gestation and lactation. Mice exposed to 100 mg/L thallium (I) sulfate were removed early due to overt toxicity, and mice exposed to ≥ 25 mg/L exhibited exposure concentration-related decreases in body weight. Lowest-observed-effect levels of 12.5 mg/L (rats) and 25 mg/L (mice) were determined based on the increased incidence of clinical signs of alopecia in F1 rat pups and significantly decreased body weights for both rats and mice.

Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT): Developing a Data Analysis Pipeline for the Assessment of Developmental Toxicity with an Interlaboratory Study.

The embryonic zebrafish is a useful vertebrate model for assessing the effects of substances on growth and development. However, cross-laboratory developmental toxicity outcomes can vary and reported developmental defects in zebrafish may not be directly comparable between laboratories. To address these limitations for gaining broader adoption of the zebrafish model for toxicological screening, we established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) program to investigate how experimental protocol differences can influence chemical-mediated effects on developmental toxicity (i.e., mortality and the incidence of altered phenotypes). As part of SEAZIT, three laboratories were provided a common and blinded dataset (42 substances) to evaluate substance-mediated effects on developmental toxicity in the embryonic zebrafish model. To facilitate cross-laboratory comparisons, all the raw experimental data were collected, stored in a relational database, and analyzed with a uniform data analysis pipeline. Due to variances in laboratory-specific terminology for altered phenotypes, we utilized ontology terms available from the Ontology Lookup Service (OLS) for Zebrafish Phenotype to enable additional cross-laboratory comparisons. In this manuscript, we utilized data from the first phase of screening (dose range finding, DRF) to highlight the methodology associated with the development of the database and data analysis pipeline, as well as zebrafish phenotype ontology mapping.

Systemic exposure and urinary excretion of vanadium following perinatal subchronic exposure to vanadyl sulfate and sodium metavanadate via drinking water.

Vanadium is a ubiquitous environmental contaminant although there are limited data to assess potential adverse human health impact following oral exposure. In support of studies investigating the subchronic toxicity of vanadyl sulfate (V4+) and sodium metavanadate (V5+) following perinatal exposure via drinking water in male and female rats, we have determined the internal exposure and urinary excretion of total vanadium at the end of study. Water consumption decreased with increasing exposure concentration following exposure to both compounds. Plasma and urine vanadium concentration normalized to total vanadium consumed per day increased with the exposure concentration of vanadyl sulfate and sodium metavanadate suggesting absorption increased as the exposure concentration increased. Additionally, females had higher concentrations than males (in plasma only for vanadyl sulfate exposure). Animals exposed to sodium metavanadate had up to 3-fold higher vanadium concentration in plasma and urine compared to vanadyl sulfate exposed animals, when normalized to total vanadium consumed per day, demonstrating differential absorption, distribution, metabolism, and excretion properties between V5+ and V4+ compounds. These data will aid in the interpretation of animal toxicity data of V4+ and V5+ compounds and determine the relevance of animal toxicity findings to human exposures.

Whole-body inhalation exposure to 2-ethyltoluene for two weeks produced nasal lesions in rats and mice.

OBJECTIVE: Ethyltoluenes are isolated during crude oil refinement for use in gasoline and commercial products and are ubiquitous in the environment. However, minimal toxicity data are available. Previously, we identified 2-ethyltoluene (2-ET) as the most potent isomer via nose-only inhalation exposure in rodents. Here, we expanded the hazard characterization of 2-ET in two rodent models using whole-body inhalation exposure and evaluated the role of prenatal exposure. METHODS: Time-mated Hsd:Sprague Dawley® SD® rats were exposed to 0, 150, 300, 600, 900, or 1200 ppm 2-ET via inhalation starting on gestation day 6 until parturition. Rat offspring (n = 8/exposure/sex) were exposed to the same concentrations as the respective dams for 2 weeks after weaning. Adult male and female B6C3F1/N mice (n = 5/exposure/sex) were exposed to the same concentrations for 2 weeks. RESULTS AND DISCUSSION: Exposure to ≥600 ppm 2-ET produced acute toxicity in rats and mice characterized by large decreases in survival, body weight, adverse clinical observations, and diffuse nasal olfactory epithelium degeneration (rats) or necrosis (mice). Due to the early removal of groups ≥600 ppm, most endpoint evaluations focused on lower exposure groups. In 150 and 300 ppm exposure groups, reproductive performance and littering were not significantly changed and body weights in exposed rats and mice were 9-18% lower than controls. Atrophy of the olfactory epithelium and nerves was observed in all animals exposed to 150 and 300 ppm. These lesions were more severe in mice than in rats. CONCLUSION: Nasal lesions were observed in all animals after whole-body exposure up to 600 ppm 2-ET for 2 weeks. Future studies should focus on 2-ET metabolism and distribution to better understand species differences and refine hazard characterization of this understudied environmental contaminant.

Internal dose of vanadium in rats following repeated exposure to vanadyl sulfate and sodium orthovanadate via drinking water.

Vanadium is a ubiquitous environmental contaminant that exists in multiple oxidation states. Humans are exposed to vanadyl (V4+) and vanadate (V5+) from dietary supplements, food, and drinking water and hence there is a concern for adverse human health. The current investigation is aimed at identifying vanadium oxidation states in vitro and in vivo and internal concentrations following exposure of rats to vanadyl sulfate (V4+) or sodium metavanadate (V5+) via drinking water for 14 d. Investigations in simulated gastric and intestinal fluids showed that V4+ was stable in gastric fluid while V5+ was stable in intestinal fluid. Analysis of rodent plasma showed that the only vanadium present was V4+, regardless of the exposed compound suggesting conversion of V5+ to V4+ in vivo and/or instability of V5+ species in biological matrices. Plasma, blood, and liver concentrations of total vanadium, after normalizing for vanadium dose consumed, were higher in male and female rats following exposure to V5+ than to V4+. Following exposure to either V4+ or V5+, the total vanadium concentration in plasma was 2- to 3-fold higher than in blood suggesting plasma as a better matrix than blood for measuring vanadium in future work. Liver to blood ratios were 4-7 demonstrating significant tissue retention following exposure to both compounds. In conclusion, these data point to potential differences in absorption and disposition properties of V4+ and V5+ salts and may explain the higher sensitivity in rats following drinking water exposure to V5+ than V4+ and highlights the importance of internal dose determination in toxicology studies.

Butylparaben multigenerational reproductive assessment by continuous breeding in Hsd:Sprague Dawley SD rats following dietary exposure.

Butylparaben (BP) is an antimicrobial agent utilized for decades as a preservative in numerous consumer products. The safety of parabens has recently come under scrutiny based on reports of estrogenic activity and suggested adverse effects upon the reproductive system. Due to the limited availability of studies that address the potential for BP exposure to induce reproductive toxicity, and clear evidence of human exposure, the National Toxicology Program conducted a multigenerational continuous breeding study to evaluate the impact of dietary BP-exposure at 0, 5000, 15,000, or 40,000 ppm on reproductive and developmental parameters in Hsd:Sprague Dawley SD rats. BP-exposure was not associated with adverse alterations of fertility, fecundity, pubertal attainment, or reproductive parameters in F0, F1, or F2 generations. Exposure-dependent increases in liver weights, and incidences of non-neoplastic liver lesions suggest the liver is a target organ of BP toxicity. No findings were observed that would support the purported mechanism of BP-induced endocrine disruption in perinatally-exposed rodents.

Finding the bad actor: Challenges in identifying toxic constituents in botanical dietary supplements.

Botanical-derived dietary supplements have widespread use in the general population. The complex and variable nature of botanical ingredients and reports of adverse responses have led to concern for negative human health impacts following consumption of these products. Toxicity testing of the vast number of available products, formulations, and combinations is not feasible due to the time and resource intensive nature of comprehensive testing. Methods are needed to assess the safety of a large number of products via more efficient frameworks. Identification of toxicologically-active constituents is one approach being used, with many advantages toward product regulation. Bioassay-guided fractionation (BGF) is the leading approach used to identify biologically-active constituents. Most BGF studies with botanicals focus on identifying pharmacologically-active constituents for drug discovery or botanical efficacy research. Here, we explore BGF in a toxicological context, drawing from both efficacy and poisonous plant research. Limitations of BGF, including loss of mixture activity and bias toward abundant constituents, and recent advancements in the field (e.g., biochemometrics) are discussed from a toxicological perspective. Identification of active constituents will allow better monitoring of market products for known toxicologically-active constituents, as well as surveying human exposure, two important steps to ensuring the safety of botanical dietary supplements.

Comparative inhalation toxicity of ethyltoluene isomers in rats and mice.

The C9 alkylbenzenes, composed mostly of ethyltoluenes and trimethylbenzenes, comprise 75-90% of the naphtha fraction of crude oil. Occupational and environmental exposure to C9 alkylbenzenes occur via inhalation. We conducted short-term inhalation studies on the ethyltoluene isomers (2-, 3- or 4-) to select one isomer for more comprehensive studies. Male Hsd:Sprague Dawley rats and female B6C3F1/N mice (n = 10) were exposed by nose-only inhalation to 2-, 3- or 4-ethyltoluene (0, 1000 or 2000 ppm) or cumene (a reference compound: 0, 500 or 1000 ppm) 3 h/day, 5 days/week, for 2 weeks. Clinical observations included abnormal gait and delayed righting reflex. Rats and mice exposed to 2000 ppm 2-ethyltoluene and mice exposed to 2000 ppm 4-ethyltoluene were euthanized early in moribund condition; no exposure-related deaths were observed with 3-ethyltoluene or cumene. Histopathology of selected tissues revealed that the nose and liver (rats and mice) and lung (mice only) to be toxicity targets. In the mouse lung, all compounds except 4-ethyltoluene produced bronchial and bronchiolar hyperplasia. In rats and mice, 2-ethyltoluene was the only compound to produce lesions in the nose and liver: in mice, squamous metaplasia and neutrophilic inflammation of the respiratory epithelium and atrophy and degeneration of the olfactory epithelium were observed in the nose and centrilobular hypertrophy and necrosis were observed in the liver. In rats, 2-ethyltoluene exposure produced atrophy of the olfactory epithelium in the nose and centrilobular necrosis in the liver. Based on mortality, body weight effects and histopathology, the 2-ethyltoluene isomer was the most potent isomer.

14-Day Toxicity Studies of Tetravalent and Pentavalent Vanadium Compounds in Harlan Sprague Dawley Rats and B6C3F1/N Mice via Drinking Water Exposure.

BACKGROUND: The National Toxicology Program (NTP) performed short-term toxicity studies of tetra- and pentavalent vanadium compounds, vanadyl sulfate and sodium metavanadate, respectively. Due to widespread human exposure and a lack of chronic toxicity data, there is concern for human health following oral exposure to soluble vanadium compounds. OBJECTIVES: To compare the potency and toxicological profile of vanadyl sulfate and sodium metavanadate using a short-term in vivo toxicity assay. METHODS: Adult male and female Harlan Sprague Dawley (HSD) rats and B6C3F1/N mice, 5 per group, were exposed to vanadyl sulfate or sodium metavanadate, via drinking water, at concentrations of 0, 125, 250, 500, 1000 or 2000 mg/L for 14 days. Water consumption, body weights and clinical observations were recorded throughout the study; organ weights were collected at study termination. RESULTS: Lower water consumption, up to -80% at 2000 mg/L, was observed at most exposure concentrations for animals exposed to either vanadyl sulfate or sodium metavanadate and was accompanied by decreased body weights at the highest concentrations for both compounds. Animals in the 1000 and 2000 mg/L sodium metavanadate groups were removed early due to overt toxicity. Thinness was observed in high-dose animals exposed to either compound, while lethargy and abnormal gait were only observed in vanadate-exposed animals. CONCLUSIONS: Based on clinical observations and overt toxicity, sodium metavanadate appears to be more toxic than vanadyl sulfate. Differential toxicity cannot be explained by differences in total vanadium intake, based on water consumption, and may be due to differences in disposition or mechanism of toxicity.

Exposure to butyl paraben during gestation and lactation in Hsd:Sprague dawley SD rats via dosed feed.

Butyl paraben (BPB) is an antimicrobial used in a variety of consumer products. Due to widespread human exposure and reported estrogenic activity, the National Toxicology Program quantified internal exposure during critical periods of development. Time-mated female Hsd:Sprague Dawley SD rats were administered 0, 1500, 5000 or 15,000 ppm BPB via NIH-07 feed, ad libitum, from gestation day (GD) 6 to postnatal day (PND) 28. Dam plasma, amniotic fluid and fetuses were collected on GD18 and pup and dam plasma were collected on PNDs 4, 10, 14, 21 and 28 and analyzed for free (unconjugated) and total (unconjugated and conjugated) BPB using liquid chromatography-tandem mass spectrometry. Free BPB was below the limit of quantitation in fetuses (LOQ 1.91 ng BPB/g fetus) and amniotic fluid (LOQ 0.17 ng BPB/mL amniotic fluid) at 1500 ppm. Analyte levels in amniotic fluid were less than 1% of maternal plasma, suggesting limited placental transfer. Total BPB in PND4 pup plasma was less than 5% of dam plasma in all exposure groups, suggesting low lactational transfer. However, at nearly all time points and exposure groups, there were higher levels of free BPB in pup versus dam plasma, suggesting limited conjugation in pups. Pup conjugation of BPB was age-dependent, not reaching the percent-conjugation in dams (>99%) until PNDs 21 to 28. These data illustrate low placental and lactational transfer of dietary BPB and that poor conjugation in pups during early lactation results in higher exposure to free BPB in pups compared to dams.