Botanicals have long been used to promote health and treat diseases, but the safety of many currently marketed botanicals has not been adequately evaluated. Given the chemical complexity of botanicals, which often contain numerous unknown constituents, and their widespread use, comprehensive toxicity assessments are needed. The Botanical Safety Consortium was established to address this challenge. This international group of experts in toxicology, chemistry, bioinformatics, and pharmacognosy is developing a toolkit of assays to generate reliable toxicological profiles for botanicals. Genotoxicity assessment is especially critical, because, unlike other toxicities, genotoxicity is not adequately identified by adverse event and history-of-use reports, and genotoxicity is directly linked to health consequences such as cancer and birth defects. The Consortium's Genotoxicity Technical Working Group is exploring a genotoxicity testing strategy based on the use of in silico modeling and the bacterial reverse mutation and in vitro micronucleus assays and including several options for additional tests to further characterize genotoxicity and mode of action when indicated. The effectiveness of this testing strategy is being evaluated using 13 well-characterized botanicals with existing toxicological data as case studies. A brief overview of each of these 13 botanicals is provided. The final strategy for developing comprehensive genotoxicity profiles of botanicals will incorporate published genotoxicity data, chemical composition information, in silico and in vitro test data, and human exposure data, reducing the need for animal testing.

Cytochrome P450 (CYP) enzymes are membrane-bound hemoproteins crucial for drug and xenobiotic metabolism. While more than 50 CYPs have been identified in humans, the isoforms from CYP1, 2, and 3 families contribute to the metabolism of about 80% of clinically approved drugs. To evaluate the effects of environmental chemicals on the activities of these important CYP enzyme families, we screened the Tox21 10K compound library to identify chemicals that inhibit CYP1A2, 2C9, 2C19, 2D6, and 3A4 enzymes. The data obtained from these five screenings were analyzed to reveal the structural classes responsible for inhibiting multiple and/or selective CYPs. Some known structural compound classes exhibiting pan-CYP inhibition, such as azole fungicides, along with established clinical inhibitors of CYPs, including erythromycin and verapamil inhibiting CYP3A4 and paroxetine and terbinafine inhibiting CYP2D6, were all confirmed in the current study. In addition, some selective CYP inhibitors, previously unknown but with potent activity (IC50 values < 1 µM), were identified. Examples included yohimbine, an indole alkaloid, and loteprednol, a corticosteroid, which showed inhibitory activity in CYP2D6 and 3A4 assays, respectively. These findings suggest that assessment of a candidate compound’s impact on CYP function may allow pre-emptive mitigation of potential adverse reactions and toxicity during drug development or toxicological characterization of environmental chemicals.

There has been an alarming trend toward earlier puberty in girls, suggesting the influence of an environmental factor(s). As the reactivation of the reproductive axis during puberty is thought to be mediated by the hypothalamic neuropeptides kisspeptin and gonadotropin-releasing hormone (GnRH), we asked whether an environmental compound might activate the kisspeptin (KISS1R) or GnRH receptor (GnRHR). We used GnRHR or KISS1R-expressing HEK293 cells to screen the Tox21 10K compound library, a compendium of pharmaceuticals and environmental compounds, for GnRHR and KISS1R activation. Agonists were identified using Ca2+ flux and phosphorylated extracellularly regulated kinase (p-ERK) detection assays. Follow-up studies included measurement of genes known to be upregulated upon receptor activation using relevant murine or human cell lines and molecular docking simulation. Musk ambrette was identified as a KISS1R agonist, and treatment with musk ambrette led to increased expression of Gnrh1 in murine and human hypothalamic cells and expansion of GnRH neuronal area in developing zebrafish larvae. Molecular docking demonstrated that musk ambrette interacts with the His309, Gln122, and Gln123 residues of the KISS1R. A group of cholinergic agonists with structures similar to methacholine was identified as GnRHR agonists. When applied to murine gonadotrope cells, these agonists upregulated Fos, Jun, and/or Egr1. Molecular docking revealed a potential interaction between GnRHR and 5 agonists, with Asn305 constituting the most conservative GnRHR binding site. In summary, using a Tox21 10K compound library screen combined with cellular, molecular, and structural biology techniques, we have identified novel environmental agents that may activate the human KISS1R or GnRHR.

The Ames test is required by regulatory agencies worldwide for assessing the mutagenic and carcinogenic potential of chemical compounds. This test uses several strains of bacteria to evaluate mutation induction: positive results in the assay are predictive of rodent carcinogenicity. As an initial step to understanding how well the assay may detect mutagens present as constituents of complex mixtures such as botanical extracts, a cross-sector working group examined the within-laboratory reproducibility of the Ames test using the extensive, publicly available National Toxicology Program (NTP) Ames test database comprising more than 3000 distinct test articles, most of which are individual chemicals. This study focused primarily on NTP tests conducted using the standard Organization for Economic Co-operation and Development Test Guideline 471 preincubation test protocol with 10% rat liver S9 for metabolic activation, although 30% rat S9 and 10 and 30% hamster liver S9 were also evaluated. The reproducibility of initial negative responses in all strains with and without 10% S9, was quite high, ranging from 95% to 99% with few exceptions. The within-laboratory reproducibility of initial positive responses for strains TA98 and TA100 with and without 10% rat liver S9 was >= 90%. Similar results were seen with hamster S9. As expected, the reproducibility of initial equivocal responses was lower, <50%. These results will provide context for determining the optimal design of recommended test protocols for use in screening both individual chemicals and complex mixtures, including botanicals.

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.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures.

The use of error-corrected Next Generation Sequencing (ecNG) to determine mutagenicity has been a subject of growing interest and potentially a disruptive technology that could supplement, and in time, replace current testing paradigms in preclinical safety assessment. Considering this, a Next Generation Sequencing Workshop was held at the Royal Society of Medicine in London in May 2022, supported by the United Kingdom Environmental Mutagen Society (UKEMS) and TwinStrand Biosciences (WA, USA), to discuss progress and future applications of this technology. In this meeting report, the invited speakers provide an overview of the Workshop topics covered and identify future directions for research. In the area of somatic mutagenesis, several speakers reviewed recent progress made with correlating ecNGS to classic in vivo transgenic rodent mutation assays as well as exploring the use of this technology directly in humans and animals, and in complex organoid models. Additionally, ecNGS has been used for detecting off-target effects of gene editing tools and emerging data suggest ecNGS potential to measure clonal expansion of cells carrying mutations in cancer driver genes as an early marker of carcinogenic potential and for direct human biomonitoring. As such, the workshop demonstrated the importance of raising awareness and support for advancing the science of ecNGS for mutagenesis, gene editing, and carcinogenesis research. Furthermore, the potential of this new technology to contribute to advances in drug and product development and improve safety assessment was extensively explored.

ABSTRACT Error-corrected Duplex Sequencing (DuplexSeq) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DuplexSeq to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N -ethyl- N -nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DuplexSeq mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A robust increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (R 2 =0.95). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DuplexSeq for characterizing chemical mutagenicity in both research and regulatory evaluation.

Error-corrected next-generation sequencing (ecNGS) is an emerging technology with the potential to revolutionize the field of genetic toxicology. Here, we present recommendations from an expert working group convened to discuss potential applications, advantages and challenges associated with implementing ecNGS in nonclinical safety studies.

The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate "sufficient" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki-67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required.

Historical negative control data (HCD) have played an increasingly important role in interpreting the results of genotoxicity tests. In particular, Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines recommend comparing responses produced by exposure to test substances with the distribution of HCD as one of three criteria for evaluating and interpreting study results (referred to herein as "Criterion C"). Because of the potential for inconsistency in how HCD are acquired, maintained, described, and used to interpret genotoxicity testing results, a workgroup of the International Workshops for Genotoxicity Testing was convened to provide recommendations on this crucial topic. The workgroup used example data sets from four in vivo tests, the Pig-a gene mutation assay, the erythrocyte-based micronucleus test, the transgenic rodent gene mutation assay, and the in vivo alkaline comet assay to illustrate how the quality of HCD can be evaluated. In addition, recommendations are offered on appropriate methods for evaluating HCD distributions. Recommendations of the workgroup are:When concurrent negative control data fulfill study acceptability criteria, they represent the most important comparator for judging whether a particular test substance induced a genotoxic effect.HCD can provide useful context for interpreting study results, but this requires supporting evidence that (i) HCD were generated appropriately, and (ii) their quality has been assessed and deemed sufficiently high for this purpose.HCD should be visualized before any study comparisons take place; graph(s) that show the degree to which HCD are stable over time are particularly useful.Qualitative and semi-quantitative assessments of HCD should also be supplemented with quantitative evaluations. Key factors in the assessment of HCD include: (i) the stability of HCD over time, and (ii) the degree to which inter-study variation explains the total variability observed.When animal-to-animal variation is the predominant source of variability, the relationship between responses in the study and an HCD-derived interval or upper bounds value (i.e., OECD Criterion C) can be used with a strong degree of confidence in contextualizing a particular study's results.When inter-study variation is the major source of variability, comparisons between study data and the HCD bounds are less useful, and consequentially, less emphasis should be placed on using HCD to contextualize a particular study's results.The workgroup findings add additional support for the use of HCD for data interpretation; but relative to most current OECD test guidelines, we recommend a more flexible application that takes into consideration HCD quality. The workgroup considered only commonly used in vivo tests, but it anticipates that the same principles will apply to other genotoxicity tests, including many in vitro tests.

1. alpha-Pinene caused a concentration-responsive increase in bladder hyperplasia and decrease in sperm counts in rodents following inhalation exposure. Additionally, it formed a prospective reactive metabolite, alpha-pinene oxide. 2. To provide human relevant context for data generated in animal models and explore potential mechanism, we undertook studies to investigate the metabolism of alpha-pinene to alpha-pinene oxide and mutagenicity of alpha-pinene and alpha-pinene oxide. 3. alpha-Pinene oxide was formed in rat and human microsomes and hepatocytes with some species differences. Based on area under the concentration versus time curves, the formation of alpha-pinene oxide was up to 4-fold higher in rats than in humans. 4. While rat microsomes cleared alpha-pinene oxide faster than human microsomes, the clearance of alpha-pinene oxide in hepatocytes was similar between species. 5. alpha-Pinene was not mutagenic with or without induced rat liver S9 in Salmonella typhimurium or Escherichia coli when tested up to 10000 mu g/plate while alpha-pinene oxide was mutagenic at >= 25 mu g/plate. 6. alpha-Pinene was metabolised to alpha-pinene oxide under the conditions of the bacterial mutation assay although the concentration was approximately 3-fold lower than the lowest alpha-pinene oxide concentration that was positive in the assay, potentially explaining the lack of mutagenicity observed with alpha-pinene.

Black cohosh (BC; Actaea racemosa L.), a top‐selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose‐related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross‐sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC‐naïve women. Twenty‐three women were enrolled in the BC‐exposed group and 28 in the BC‐naïve group. Use of any brand of BC‐only supplement for at least 3 months was required for inclusion in the BC‐exposed group. Supplements were analyzed for chemical composition to allow cross‐product comparisons. All participants were healthy, with no known exposures (e.g., x‐rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings.