Advancing botanical safety: A strategy for selecting, sourcing, and characterizing botanicals for developing toxicological tools.

Increases in botanical use, encompassing herbal medicines and dietary supplements, have underlined a critical need for an advancement in safety assessment methodologies. However, botanicals present unique challenges for safety assessment due to their complex and variable composition arising from diverse growing conditions, processing methods, and plant varieties. Historically, botanicals have been largely evaluated based on their history of use information, based primarily on traditional use or dietary history. However, this presumption lacks comprehensive toxicological evaluation, demanding innovative and consistent assessment strategies. To address these challenges, the Botanical Safety Consortium (BSC) was formed as an international, cross-sector forum of experts to identify fit-for purpose assays that can be used to evaluate botanical safety. This global effort aims to assess botanical safety assessment methodologies, merging traditional knowledge with modern in vitro and in silico assays. The ultimate goal is to champion the development of toxicity tools for botanicals. This manuscript highlights: 1) BSC's strategy for botanical selection, sourcing, and preparation of extracts to be used in in vitro assays, and 2) the approach utilized to characterize botanical extracts, using green tea and Asian ginseng as examples, to build confidence for use in biological assays.

Evaluation of the herbicide glyphosate, (aminomethyl)phosphonic acid, and glyphosate-based formulations for genotoxic activity using in vitro assays.

Glyphosate, the most heavily used herbicide world-wide, is applied to plants in complex formulations that promote absorption. The National Toxicology Program reported in 1992 that glyphosate, administered to rats and mice at doses up to 50,000 ppm in feed for 13 weeks, showed little evidence of toxicity, and no induction of micronuclei was observed in the mice in this study. Subsequently, mechanistic studies of glyphosate and glyphosate-based formulations (GBFs) that have focused on DNA damage and oxidative stress suggest that glyphosate may have genotoxic potential. However, few of these studies directly compared glyphosate to GBFs, or effects among GBFs. To address these data gaps, we tested glyphosate, glyphosate isopropylamine (IPA), and (aminomethyl)phosphonic acid (AMPA, a microbial metabolite of glyphosate), 9 high-use agricultural GBFs, 4 residential-use GBFs, and additional herbicides (metolachlor, mesotrione, and diquat dibromide) present in some of the GBFs in bacterial mutagenicity tests, and in human TK6 cells using a micronucleus assay and a multiplexed DNA damage assay. Our results showed no genotoxicity or notable cytotoxicity for glyphosate or AMPA at concentrations up to 10 mM, while all GBFs and herbicides other than glyphosate were cytotoxic, and some showed genotoxic activity. An in vitro to in vivo extrapolation of results for glyphosate suggests that it is of low toxicological concern for humans. In conclusion, these results demonstrate a lack of genotoxicity for glyphosate, consistent with observations in the NTP in vivo study, and suggest that toxicity associated with GBFs may be related to other components of these formulations.

Comparative toxicokinetics of bisphenol S and bisphenol AF in male rats and mice following repeated exposure via feed.

We investigated the plasma toxicokinetic behavior of free (parent) and total (parent and conjugated forms) of bisphenol S (BPS) and bisphenol AF (BPAF) in plasma of adult male rats and mice following exposure via feed for 7 days to BPS (338, 1125, and 3375 ppm) or BPAF (338, 1125, and 3750 ppm). In rats, the exposure concentration-normalized maximum concentration [Cmax/D (ng/mL)/(ppm)] and area under the concentration time curve [AUC/D (h × ng/mL)/(ppm)] for free was higher for BPS (Cmax/D: 0.476-1.02; AUC/D: 3.58-8.26) than for BPAF (Cmax/D: 0.017-0.037; AUC/D:0.196-0.436). In mice, the difference in systemic exposure parameters between free BPS (Cmax/D: 0.376-0.459; AUC/D: 1.52-2.54) and free BPAF (Cmax/D: 0.111-0.165; AUC/D:0.846-1.09) was marginal. Elimination half-lives for free analytes (4.41-10.4 h) were comparable between species and analogues. When systemic exposure to free analyte was compared between species, in rats, BPS exposure was slightly higher but BPAF exposure was much lower than in mice. BPS and BPAF were highly conjugated; total BPS AUC values (rats ≥18-fold, mice ≥17-fold) and BPAF (rats ≥127-fold, mice ≥16-fold) were higher than corresponding free values. Data demonstrated that there are analogue and species differences in the kinetics of BPS and BPAF.

An investigation of systemic exposure to bisphenol AF during critical periods of development in the rat.

Due to structural similarity to bisphenol A and lack of safety data, the National Toxicology Program (NTP) is evaluating the potential toxicity of bisphenol AF (BPAF) in rodent models. The current investigation reports the internal exposure data for free (unconjugated BPAF) and total (free and conjugated forms) BPAF during critical stages of development following perinatal dietary exposure in Hsd:Sprague Dawley®SD® rats to 0 (vehicle control), 338, 1125, and 3750 ppm BPAF from gestation day (GD) 6 to postnatal day (PND) 28. Free and total BPAF concentrations in maternal plasma at GD 18, PND 4, and PND 28 increased with the exposure concentration; free BPAF concentrations were ≤ 1.61% those of total BPAF demonstrating extensive first pass metabolism of BPAF following dietary exposure in adults. Free and total BPAF were quantified in GD 18 fetuses and PND 4 pups with free concentrations 11.7-53.4% that of corresponding total concentrations. In addition, free concentrations were higher (130-571%) and total concentrations were lower (1.71-7.23%) than corresponding concentrations in dams, demonstrating either preferential transfer of free BPAF and/or inability of fetuses and pups to conjugate BPAF. Free and total concentrations in PND 28 pups were similar to maternal concentrations demonstrating direct exposure of pups via feed and that conjugating enzymes are developed in PND 28 pups. In conclusion, these data demonstrate considerable gestational and lactational transfer of parent aglycone from the mother to offspring. Since the ontogeny of conjugating enzymes in humans is similar to that of rodents, the data from rodent BPAF studies may be useful in predicting human risk from exposure to BPAF.

The Tox21 10K Compound Library: Collaborative Chemistry Advancing Toxicology.

Since 2009, the Tox21 project has screened ∼8500 chemicals in more than 70 high-throughput assays, generating upward of 100 million data points, with all data publicly available through partner websites at the United States Environmental Protection Agency (EPA), National Center for Advancing Translational Sciences (NCATS), and National Toxicology Program (NTP). Underpinning this public effort is the largest compound library ever constructed specifically for improving understanding of the chemical basis of toxicity across research and regulatory domains. Each Tox21 federal partner brought specialized resources and capabilities to the partnership, including three approximately equal-sized compound libraries. All Tox21 data generated to date have resulted from a confluence of ideas, technologies, and expertise used to design, screen, and analyze the Tox21 10K library. The different programmatic objectives of the partners led to three distinct, overlapping compound libraries that, when combined, not only covered a diversity of chemical structures, use-categories, and properties but also incorporated many types of compound replicates. The history of development of the Tox21 "10K" chemical library and data workflows implemented to ensure quality chemical annotations and allow for various reproducibility assessments are described. Cheminformatics profiling demonstrates how the three partner libraries complement one another to expand the reach of each individual library, as reflected in coverage of regulatory lists, predicted toxicity end points, and physicochemical properties. ToxPrint chemotypes (CTs) and enrichment approaches further demonstrate how the combined partner libraries amplify structure-activity patterns that would otherwise not be detected. Finally, CT enrichments are used to probe global patterns of activity in combined ToxCast and Tox21 activity data sets relative to test-set size and chemical versus biological end point diversity, illustrating the power of CT approaches to discern patterns in chemical-activity data sets. These results support a central premise of the Tox21 program: A collaborative merging of programmatically distinct compound libraries would yield greater rewards than could be achieved separately.

Comparison of phytochemical composition of Ginkgo biloba extracts using a combination of non-targeted and targeted analytical approaches.

Ginkgo biloba extract (GbE) is a dietary supplement derived from an ethanolic extract of Ginkgo biloba leaves. Unfinished bulk GbE is used to make finished products that are sold as dietary supplements. The variable, complex composition of GbE makes it difficult to obtain consistent toxicological assessments of potential risk. The National Toxicology Program (NTP) observed hepatotoxicity in its rodent studies of a commercially available, unfinished GbE product, but the application of these results to the broader GbE supplement market is unclear. Here, we use a combination of non-targeted and targeted chromatographic and spectrophotometric methods to obtain profiles of 24 commercially available finished GbE products and unfinished standardized and unstandardized extracts with and without hydrolysis, then used principal component analysis to group unfinished products according to their similarity to each other and to National Institute of Standards and Technology (NIST) standard reference materials (SRM), and the finished products. Unfinished products were grouped into those that were characteristic and uncharacteristic of standardized GbE. Our work demonstrates that different analytical approaches produced similar classifications of characteristic and uncharacteristic products in unhydrolyzed samples, but the distinctions largely disappeared once the samples were hydrolyzed. Using our approach, the NTP GbE was most similar to two unfinished GbE products classified as characteristic, finished products, and the NIST GbE SRM. We propose that a simple analysis for the presence, absence, or amounts of compounds unique to GbE in unhydrolyzed samples could be sufficient to determine a sample's authenticity.Graphical abstract.

Using Tox21 High-Throughput Screening Assays for the Evaluation of Botanical and Dietary Supplements.

Introduction: Recent nationwide surveys found that natural products, including botanical dietary supplements, are used by ∼18% of adults. In many cases, there is a paucity of toxicological data available for these substances to allow for confident evaluations of product safety. The National Toxicology Program (NTP) has received numerous nominations from the public and federal agencies to study the toxicological effects of botanical dietary supplements. The NTP sought to evaluate the utility of in vitro quantitative high-throughput screening (qHTS) assays for toxicological assessment of botanical and dietary supplements. Materials and Methods: In brief, concentration-response assessments of 90 test substances, including 13 distinct botanical species, and individual purported active constituents were evaluated using a subset of the Tox21 qHTS testing panel. The screen included 20 different endpoints that covered a broad range of biologically relevant signaling pathways to detect test article effects upon endocrine activity, nuclear receptor signaling, stress response signaling, genotoxicity, and cell death signaling. Results and Discussion: Botanical dietary supplement extracts induced measurable and diverse activity. Elevated biological activity profiles were observed following treatments with individual chemical constituents relative to their associated botanical extract. The overall distribution of activity was comparable to activities exhibited by compounds present in the Tox21 10K chemical library. Conclusion: Botanical supplements did not exhibit minimal or idiosyncratic activities that would preclude the use of qHTS platforms as a feasible method to screen this class of compounds. However, there are still many considerations and further development required when attempting to use in vitro qHTS methods to characterize the safety profile of botanical/dietary supplements.

Screening for Developmental Neurotoxicity at the National Toxicology Program: The Future Is Here.

The National Toxicology Program (NTP) receives requests to evaluate chemicals with potential to cause adverse health effects, including developmental neurotoxicity (DNT). Some recent requests have included classes of chemicals such as flame retardants, polycyclic aromatic compounds, perfluoroalkyl substances, and bisphenol A analogs with approximately 20-50 compounds per class, many of which include commercial mixtures. However, all the compounds within a class cannot be tested using traditional DNT animal testing guideline studies due to resource and time limitations. Hence, a rapid and biologically relevant screening approach is required to prioritize compounds for further in vivo testing. Because neurodevelopment is a complex process involving multiple distinct cellular processes, one assay will unlikely address the complexity. Hence, the NTP sought to characterize a battery of in vitro and alternative animal assays to quantify chemical effects on a variety of neurodevelopmental processes. A culmination of this effort resulted in a NTP-hosted collaborative project with approximately 40 participants spanning across domains of academia, industry, government, and regulatory agencies; collaborators presented data on cell-based assays and alternative animal models that was generated using a targeted set of compounds provided by the NTP. The NTP analyzed the assay results using benchmark concentration (BMC) modeling to be able to compare results across the divergent assays. The results were shared with the contributing researchers on a private web application during the workshop, and are now publicly available. This article highlights the overview and goals of the project, and describes the NTP's approach in creating the chemical library, development of NTPs data analysis strategy, and the structure of the web application. Finally, we discuss key issues with emphasis on the utility of this approach, and knowledge gaps that need to be addressed for its use in regulatory decision making.

How similar is similar enough? A sufficient similarity case study with Ginkgo biloba extract.

Botanical dietary supplements are complex mixtures that can be highly variable in composition and quality, making safety evaluation difficult. A key challenge is determining how diverse products in the marketplace relate to chemically and toxicologically characterized reference samples (i.e., how similar must a product be in order to be well-represented by the tested reference sample?). Ginkgo biloba extract (GBE) was used as a case study to develop and evaluate approaches for determining sufficient similarity. Multiple GBE extracts were evaluated for chemical and biological-response similarity. Chemical similarity was assessed using untargeted and targeted chemistry approaches. Biological similarity was evaluated using in vitro liver models and short-term rodent studies. Statistical and data visualization methods were then used to make decisions about the similarity of products to the reference sample. A majority of the 26 GBE samples tested (62%) were consistently determined to be sufficiently similar to the reference sample, while 27% were different from the reference GBE, and 12% were either similar or different depending on the method used. This case study demonstrated that approaches to evaluate sufficient similarity allow for critical evaluation of complex mixtures so that safety data from the tested reference can be applied to untested materials.

Characterization of Zinc Carbonate Basic as a Source of Zinc in a Rodent Study Investigating the Effects of Dietary Deficiency or Excess.

Zinc deficiency and excess can result in adverse health outcomes. There is conflicting evidence regarding whether excess or deficient zinc in the diet can contribute to carcinogenicity. The objective of this study was to characterize zinc carbonate basic for use as a source of dietary zinc in a rodent toxicity and carcinogenicity study investigating the effects of zinc deficiency and excess. Because of the complex chemistries of zinc carbonate basic compounds, inconsistent nomenclature, and literature and reference spectra gaps, it was necessary to employ multiple analytical techniques, including Karl Fischer titration, combustion analysis, inductively coupled plasma-optical emission spectrometry, X-ray diffraction, infrared spectroscopy, X-ray fluorescence spectrometry, and thermogravimetric analysis to characterize the test article. Based on the collective evidence and through the process of elimination, the test article was found to be composed mainly of zinc carbonate basic with zinc oxide as a minor component. The zinc content was determined to be 56.6% (w/w) with heavy metals such as arsenic, cadmium, mercury and lead below the limit of quantitation of less than or equal to 0.01%. The test material was stable at ambient temperature. Based on the work described in this manuscript, the test article was suitable for use as a source of zinc in studies of deficiency and excess in the diet.

The estrogenic content of rodent diets, bedding, cages, and water bottles and its effect on bisphenol A studies.

The lowest observed adverse effect level for bisphenol A (BPA) in mice and rats is currently poorly defined due to inconsistent study designs and results in published studies. The objectives of the current study were to (1) compare the estrogenic content of rodent diets, bedding, cages, and water bottles to evaluate their impact on the estrogenic activity of BPA and (2) review the literature on BPA to determine the most frequently reported diets, beddings, cages, and water bottles used in animal studies. Our literature review indicated that low-dose BPA animal studies have inconsistent results and that factors contributing to this inconsistency are the uses of high-phytoestrogen diets and the different routes of exposure. In 44% (76 of 172) of all reports, rodents were exposed to BPA via the subcutaneous route. Our literature review further indicated that the type of diet, bedding, caging, and water bottles used in BPA studies were not always reported. Only 37% (64 of 172) of the reports described the diet used. In light of these findings, we recommend the use of a diet containing low levels of phytoestrogen (less than 20 µg/g diet) and metabolizable energy (approximately 3.1 kcal/g diet) and estrogen-free bedding, cages, and water bottles for studies evaluating the estrogenic activity of endocrine-disrupting compounds such as BPA. The oral route of BPA exposure should be used when results are to be extrapolated to humans.

Mechanistic insights from the NTP studies of chromium.

Hexavalent chromium (Cr(VI)) is a contaminant of water and soil and is a human lung carcinogen. Trivalent chromium (Cr(III)), a proposed essential element, is ingested by humans in the diet and in dietary supplements such as chromium picolinate (CP). The National Toxicology Program (NTP) demonstrated that Cr(VI) is also carcinogenic in rodents when administered in drinking water as sodium dichromate dihydrate (SDD), inducing neoplasms of the oral cavity and small intestine in rats and mice, respectively. In contrast, there was no definitive evidence of toxicity or carcinogenicity following exposure to Cr(III) administered in feed as CP monohydrate (CPM). Cr(VI) readily enters cells via nonspecific anion channels, in contrast to Cr(III), which cannot easily pass through the cell membrane. Extracellular reduction of Cr(VI) to Cr(III), which occurs primarily in the stomach, is considered a mechanism of detoxification, while intracellular reduction is thought to be a mechanism of genotoxicity and carcinogenicity. Tissue distribution studies in additional groups of male rats and female mice demonstrated higher Cr concentrations in tissues following exposure to Cr(VI) compared to controls and Cr(III) exposure at a similar external dose, indicating that some of the Cr(VI) escaped gastric reduction and was distributed systemically. The multiple potential pathways of Cr-induced genotoxicity will be discussed.

Toxicology and carcinogenesis study of senna in C3B6.129F1-Trp53 tm1Brd N12 haploinsufficient mice.

Senna is a pod or leaf of Senna alexandrina P. Mill and is used as a stimulant laxative. In the large intestine, bacterial enzymes reduce sennosides to rhein-9-anthrone, the active form for the laxative effect. To determine the potential toxic effects of senna, a 5-week dose range finding study in the C57BL/6N mouse and a 40-week toxicology and carcinogenesis study in the C3B6.129F1-Trp53 (tm1Brd) N12 haploinsufficient (p53(+/-)) mouse were conducted. In the 5-week study, C57BL/6N mice were exposed to up to 10,000 ppm senna in feed. Increased incidences of epithelial hyperplasia of the cecum and colon were observed in males and females exposed to 5,000 or 10,000 ppm senna. These intestinal lesions were not considered to be of sufficient severity to cause mortality and, thus, in the p53(+/-) mouse 40-week study, the high dose of 10,000 ppm was selected. Significant increases in the incidences of epithelial hyperplasia of the colon and cecum were observed at 10,000 ppm in p53(+/-) males and females, and the incidence of hyperplasia of the colon was significantly increased at 3,000 ppm in females. In conclusion, the large intestine was the major target of senna-induced toxicity in both wild-type and the p53(+/-) mouse model. There was no neoplastic change when senna was administered to p53(+/-) mouse.

Immunotoxicological profile of chloramine in female B6C3F1 mice when administered in the drinking water for 28 days.

Monochloramine has been used to provide a disinfecting residual in water distribution systems where it is difficult to maintain an adequate free-chlorine residual or where disinfection by-product formation is of concern. The goal of this study was to characterize the immunotoxic effects of chloramine in female B(6)C(3)F(1) mice when administered via the drinking water. Mice were exposed to chloramine-containing deionized tap water at 2, 10, 20, 100, or 200 ppm for 28 days. No statistically significant differences in drinking water consumption, body weight, body weight gain, organ weights, or hematological parameters between the exposed and control animals were noted during the experimental period. There were no changes in the percentages and numbers of total B-lymphocytes, T-lymphocytes, CD4(+) and CD8(+) T-lymphocytes, natural killer (NK) cells, and macrophages in the spleen. Exposure to chloramine did not affect the IgM antibody-forming cell response to sheep red blood cells (SRBC) or anti-SRBC IgM antibody production. Minimal effects, judged to be biologically insignificant, were observed in the mixed-leukocyte response and NK activity. In conclusion, chloramine produced no toxicological and immunotoxic effects in female B(6)C(3)F(1) mice when administered for 28 days in the drinking water at concentrations ranging from 2-200 ppm.

Determination of L-ephedrine, pseudoephedrine, and caffeine in rat plasma by liquid chromatography-tandem mass spectrometry.

A rapid and simple liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of L-ephedrine, pseudoephedrine, and caffeine in male Fisher-344 rat plasma at nanogram-per-milliliter concentrations for use in support of toxicology studies. Only 25 µL of plasma is required, and extraction is performed using a simple, single-step protein precipitation. The method was validated over a range of 2.09 to 5460 ng/mL for L-ephedrine, 2.09 to 5050 ng/mL for pseudoephedrine and 2.03 to 5340 ng/mL for caffeine. A binary gradient elution at 0.3 mL/min was used with a Waters XBridge Phenyl (2.1 × 150 mm, 3.5 µm) column and a Waters XBridge Phenyl 2.1- × 10-mm guard column at ambient temperature. The mobile phase consisted of 10 mM ammonium acetate in water (pH 5.0) and methanol. Caffeine trimethyl-(13)C(3) was used as the internal standard. The method was evaluated for linearity, recovery, precision, accuracy, and stability, and it was successfully applied in toxicokinetic studies of ephedrine, administered alone, in combination with caffeine, and in the herbal source Ma Huang.

Effects of the PPARα Agonist and Widely Used Antihyperlipidemic Drug Gemfibrozil on Hepatic Toxicity and Lipid Metabolism.

Gemfibrozil is a widely prescribed hypolipidemic agent in humans and a peroxisome proliferator and liver carcinogen in rats. Three-month feed studies of gemfibrozil were conducted by the National Toxicology Program (NTP) in male Harlan Sprague-Dawley rats, B6C3F1 mice, and Syrian hamsters, primarily to examine mechanisms of hepatocarcinogenicity. There was morphologic evidence of peroxisome proliferation in rats and mice. Increased hepatocyte proliferation was observed in rats, primarily at the earliest time point. Increases in peroxisomal enzyme activities were greatest in rats, intermediate in mice, and least in hamsters. These studies demonstrate that rats are most responsive while hamsters are least responsive. These events are causally related to hepatotoxicity and hepatocarcinogenicity of gemfibrozil in rodents via peroxisome proliferator activated receptor-α (PPARα) activation; however, there is widespread evidence that activation of PPARα in humans results in expression of genes involved in lipid metabolism, but not in hepatocellular proliferation.

Exposure to hexavalent chromium resulted in significantly higher tissue chromium burden compared with trivalent chromium following similar oral doses to male F344/N rats and female B6C3F1 mice.

In National Toxicology Program 2-year studies, hexavalent chromium [Cr(VI)] administered in drinking water was clearly carcinogenic in male and female rats and mice, resulting in small intestine epithelial neoplasms in mice at a dose equivalent to or within an order of magnitude of human doses that could result from consumption of chromium-contaminated drinking water, assuming that dose scales by body weight(3/4) (body weight raised to the 3/4 power). In contrast, exposure to trivalent chromium [Cr(III)] at much higher concentrations may have been carcinogenic in male rats but was not carcinogenic in mice or female rats. As part of these studies, total chromium was measured in tissues and excreta of additional groups of male rats and female mice. These data were used to infer the uptake and distribution of Cr(VI) because Cr(VI) is reduced to Cr(III) in vivo, and no methods are available to speciate tissue chromium. Comparable external doses resulted in much higher tissue chromium concentrations following exposure to Cr(VI) compared with Cr(III), indicating that a portion of the Cr(VI) escaped gastric reduction and was distributed systemically. Linear or supralinear dose responses of total chromium in tissues were observed following exposure to Cr(VI), indicating that these exposures did not saturate gastric reduction capacity. When Cr(VI) exposure was normalized to ingested dose, chromium concentrations in the liver and glandular stomach were higher in mice, whereas kidney concentrations were higher in rats. In vitro studies demonstrated that Cr(VI), but not Cr(III), is a substrate of the sodium/sulfate cotransporter, providing a partial explanation for the greater absorption of Cr(VI).

Validation and application of a method for the determination of total chromium in rat tissues by inductively coupled plasma mass spectrometry.

The validation of a method for the determination of chromium (Cr) in F-344/N rat tissues by inductively coupled plasma-mass spectrometry is described. Samples were analyzed after a rapid, open-vessel microwave digestion procedure. Performance of the method was evaluated using kidney tissue across a concentration range of 0.50-5.00 microg Cr/g tissue. Data for method linearity, accuracy, precision, digest stability, and storage stability are presented along with limits of detection and quantitation data. Data from a method cross-validation for B6C3F1 mouse kidney tissue are also presented. After validation, the method was applied to analyze samples collected in support of two chronic toxicity and carcinogenesis studies conducted by the National Toxicology Program.