Glyphosate is an herbicide found worldwide in glyphosate-based formulations (GBFs). Although glyphosate appears to have a low toxicity profile for humans and mammals, conflicting reports exist regarding the risk for cancer in humans. US-EPA and European regulatory agencies have described glyphosate as unlikely to pose a carcinogenic hazard to humans. However, the International Agency for Research on Cancer (IARC) classified glyphosate as "probably carcinogenic to humans (Group 2A)," citing "mechanistic data provide strong evidence for genotoxicity and oxidative stress." Given these discrepancies, the Division of Translational Toxicology at NIEHS designed an experimental strategy to expand mechanistic evidence and address critical gaps within existing literature (e.g. mechanistic evaluations of glyphosate alongside GBFs, inclusion of context-defining positive controls). Cell morphology, viability, H2O2, and γH2AX formation were assayed in human keratinocytes (HaCaT), previously cited by IARC, and human hepatocytes (HepaRG) to derive benchmark concentrations and fold-change response metrics. Our findings revealed glyphosate alone was weakly and inconsistently bioactive for oxidative stress and DNA damage when compared with positive controls. In contrast, most of the 13 GBFs evaluated were more clearly bioactive with no apparent correlation to varied glyphosate concentrations. Hierarchical clustering of biological responses revealed some bioactive GBFs to cluster near well-characterized positive controls for oxidative stress, whereas 4 GBFs clustered more similarly to negative controls and glyphosate. Collectively, this study provides a robust dataset with context-defining results that advance our understanding of the hazard potential of GBFs while revealing that glyphosate is likely not a primary driver of oxidative stress from GBF exposures.

2,2'-Dimorpholinodiethyl ether (DMDEE) is a tertiary amine catalyst used in the production of polyurethane foams. Human exposure may occur occupationally through inhalation of DMDEE vapors. B6C3F1/N mice (5-6 weeks old) were exposed to 0-1000 mg DMDEE/kg in water by oral gavage daily for 4 weeks. Time-mated Harlan Sprague Dawley (HSD) rats (12-14 weeks old) were exposed to 0-500 mg DMDEE/kg in water from gestation day (GD) 6 through post-natal day (PND) 27 by oral gavage; their pups were directly dosed from PND12 through PND27. Blood was collected from mice (4-weeks), rat dams (GD18, PND4, PND28) and rat pups (PND 4, PND 28) for hematology, clinical chemistry, and micronucleus evaluation; mouse tissues were analyzed for histopathology. DMDEE (500-6000 μg/plate) was evaluated for mutagenicity using Salmonella typhimurium (TA98, TA100) or Escherichia coli WP2 uvrA pKM101. No DMDEE-related effects were observed on pregnancy or litter parameters in HSD rats. The mouse and rat micronucleus tests and the bacterial reverse mutation tests were negative. At 1000 mg DMDEE/kg, behavioral observations of circling, repetitive head lifting, and abnormal gait were accompanied by cytoplasmic vacuolization in epithelial cells of the brain choroid plexus, and in the renal cortical tubules in male and female mice. Cytoplasmic vacuolization was also observed in the epithelial cells in the coagulating gland, prostate, and epididymis of male mice. These data indicate deficits in the cellular structure and function of neurological and renal tissues in male and female mice, as well as male reproductive tissues at higher doses and suggest caution in the industrial use of DMDEE.

Polycyclic aromatic hydrocarbons (PAHs) are well-known for their mutagenic and carcinogenic effects. Benzo[b]fluoranthene (BbF) is one of 16 PAHs prioritized by the US Environmental Protection Agency for toxicological evaluation due to pervasive human exposure. As part of a multi-stakeholder consortium, the genotoxic effects of BbF were evaluated in MutaMouse males exposed to five doses of BbF or a vehicle control via repeated oral gavage for 28, 60, 90, 120, or 180 days, with dose ranges adjusted by duration of exposure. Mutagenesis was evaluated in lung tissue (n = 4) at 28, 90, and 180 days using Duplex Sequencing (DS), and chromosomal damage (n = 8) was evaluated using the micronucleus assay in peripheral blood at all time points. Dose- and time-dependent increases in total mutation frequency (MF) and C:G > A:T mutations were observed in lung tissue after 28, 90, and 180 days of exposure. By 28 days, BbF exposure produced lung cancer-associated mutational signatures linked to tobacco smoking. Mutations accumulated over time in lung, whereas chromosomal damage in peripheral blood erythrocytes reached a steady state by 28 days. Benchmark dose (BMD) confidence intervals (CIs) narrowed with extended exposure only for MF. Collectively, the data demonstrate that BbF is a potent mutagen capable of inducing cancer-relevant mutations in lung, supporting its potential role in human lung carcinogenesis. By distinguishing early mutagenic responses from cumulative mutation effects over time, these findings highlight the value of integrating mutagenicity assessment into extended-duration studies to better inform the potential health effects of chronic genotoxic exposures.

Duplex Sequencing (DS) is an ultra-accurate, error-corrected next generation sequencing (ecNGS) technology for mutation analysis. A working group (WG) within Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee is investigating the suitability of ecNGS for regulatory mutagenicity testing, using DS as a model. Initial steps to promote acceptance require demonstrating technical reproducibility across DS-experienced and inexperienced laboratories and establishing the method's sensitivity relative to conventional tests. Thus, the WG conducted a 'reconstruction experiment' to evaluate the transferability, reproducibility, and sensitivity of DS. TwinStrand Biosciences first applied DS to establish mutation frequency (MF) in DNA samples extracted from the livers of an untreated Sprague Dawley rat, or rats treated with either 100 mg/kg/day benzo[a]pyrene (B[a]P) for ten days or 40 mg/kg/day N-ethyl-N-nitrosourea (ENU) for three days. Using the measured MF in these original samples, mixtures were then constructed using the B[a]P- and ENU-treated samples to create "MF standards" with target MFs 1.2-, 1.5-, and 2-fold greater than the untreated control. Aliquots of these standards were distributed to seven laboratories in North America and Europe. DS libraries were prepared by each laboratory and TwinStrand. All eight laboratories met library preparation and assay performance metrics to yield high quality sequencing data with MF in the expected 'MF standard' range. The measured MF and mutation spectra were nearly identical across the laboratories and a 2-fold increase in MF could readily be identified in all labs relative to the untreated controls. The results confirm the high reproducibility and sensitivity of DS for mutagenicity assessment.

Error-corrected sequencing (ECS) is a transformative method for in vivo mutagenicity assessment, enabling direct, highly sensitive measurement of mutation frequency and spectrum. ECS addresses key limitations of the transgenic rodent (TGR) assay, including lack of integration into standard toxicity studies, restricted model availability, and limited alignment with the 3R principles. To support regulatory acceptance, an expert workgroup of the International Workshops on Genotoxicity Testing (IWGT) reviewed ECS technologies and developed consensus recommendations for its inclusion into Organisation for Economic Co-operation and Development (OECD) test guidelines. The working group agreed that ECS: produces results that are concordant with validated TGR assays; can be incorporated into standard ≥28-day repeat-dose toxicity studies; and, data interpretation should be based on overall mutation frequency compared with concurrent vehicle controls. The working group emphasized harmonized data reporting aligned with OECD principles and endorsed study designs that enable quantitative risk assessment. Overall, the working group agreed that ECS offers a significant advancement over current mutagenicity assays by enabling the use of diverse models beyond conventional TGR systems described in OECD test guideline 488. The working group fully supports the application of ECS to generate in vivo mutagenicity data for regulatory submissions and recommends its inclusion in future OECD test guidelines.

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.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that originate from the incomplete combustion of organic materials. We investigated the clastogenicity and mutagenicity of benzo[b]fluoranthene (BbF), one of 16 priority PAHs, in MutaMouse males after a 28 day oral exposure. BbF causes robust dose-dependent increases in micronucleus frequency in peripheral blood, indicative of chromosome damage. Duplex sequencing (DS), an error-corrected sequencing technology, reveals that BbF induces dose-dependent increases in mutation frequencies in bone marrow (BM) and liver. Mutagenicity is increased in intergenic relative to genic regions, suggesting a role for transcription-coupled repair of BbF-induced DNA damage. At higher doses, the maximum mutagenic response to BbF is higher in liver, which has a lower mitotic index but higher metabolic capacity than BM; however, mutagenic potency is comparable between the two tissues. BbF induces primarily C:G > A:T mutations, followed by C:G > T:A and C:G > G:C, indicating that BbF metabolites mainly target guanines and cytosines. The mutation spectrum of BbF correlates with cancer mutational signatures associated with tobacco exposure, supporting its contribution to the carcinogenicity of combustion-derived PAHs in humans. Overall, BbF's mutagenic effects are similar to benzo[a]pyrene, a well-studied mutagenic PAH. Our work showcases the utility of DS for effective mutagenicity assessment of environmental pollutants.

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.

Supplementary Figures 1-6 from Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

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.

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.

Supplementary Table 2 from Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

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.