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Objectives To investigate the formation, detection, mutagenicity, and control strategies of nitrosamine drug substance-related impurities (NDSRIs) in pharmaceutical formulations, emphasizing regulatory compliance, risk mitigation, and the establishment of acceptable intake (AI) limits for enhanced drug safety. Methods This study reviews the NDSRI formation and mutagenicity assessment methods, including in silico, in vitro , and in vivo assays. It also explores mitigation strategies and approaches for determining AI limits. Results The findings indicate that NDSRIs are primarily formed through the nitrosation of APIs containing amine groups, with key risk factors including reactive functional groups and interactions between drugs and excipients. Mutagenicity evaluation revealed that while in silico and in vitro assays provide initial insights, in vivo assays offer more comprehensive and biologically relevant data by capturing complex metabolic processes and systemic interactions. Effective mitigation strategies, such as optimizing the manufacturing conditions and using nitrosation inhibitors, are crucial in reducing NDSRI formation. Approaches like the carcinogenic potency categorization (CPCA) and read-across methods are proposed for determining AI limits, facilitating safer exposure thresholds and supporting regulatory compliance. Conclusion A multifaceted approach is vital for managing NDSRIs in pharmaceuticals. Comprehensive mutagenicity testing, especially in vivo assays, provides biologically relevant insights into NDSRI-associated risks. Implementing control strategies and, determining AI limits are key to minimizing exposure. Strengthening regulatory frameworks and industry practices improves drug safety, quality, and public health protection.

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.

The increasing size and frequency of wildland fires are leading to greater potential for cardiopulmonary disease and cancer in exposed populations; however, little is known about how the types of fuel and combustion phases affect these adverse outcomes. We evaluated the mutagenicity and lung toxicity of particulate matter (PM) from flaming vs. smoldering phases of five biomass fuels, and compared results by equal mass or emission factors (EFs) derived from amount of fuel consumed. A quartz-tube furnace coupled to a multistage cryotrap was employed to collect smoke condensate from flaming and smoldering combustion of red oak, peat, pine needles, pine, and eucalyptus. Samples were analyzed chemically and assessed for acute lung toxicity in mice and mutagenicity in . The average combustion efficiency was 73 and 98% for the smoldering and flaming phases, respectively. On an equal mass basis, PM from eucalyptus and peat burned under flaming conditions induced significant lung toxicity potencies (neutrophil/mass of PM) compared to smoldering PM, whereas high levels of mutagenicity potencies were observed for flaming pine and peat PM compared to smoldering PM. When effects were adjusted for EF, the smoldering eucalyptus PM had the highest lung toxicity EF (neutrophil/mass of fuel burned), whereas smoldering pine and pine needles had the highest mutagenicity EF. These latter values were approximately 5, 10, and 30 times greater than those reported for open burning of agricultural plastic, woodburning cookstoves, and some municipal waste combustors, respectively. PM from different fuels and combustion phases have appreciable differences in lung toxic and mutagenic potency, and on a mass basis, flaming samples are more active, whereas smoldering samples have greater effect when EFs are taken into account. Knowledge of the differential toxicity of biomass emissions will contribute to more accurate hazard assessment of biomass smoke exposures. https://doi.org/10.1289/EHP2200.

Benzophenone (BPs) and 4-Methylbenzylidene Camphor are used as ultraviolet (UV) filters to protect the skin and hair in personal care products. The discharging of the three chemicals may endanger the receiving water ecosystem. In the present study, the mutagenicity of BP-6, BP-8, and 4-Methylbenzylidene Camphor was tested using the Salmonella typhimurium reverse mutation test (Ames test) in the system with and without rat liver microsomal preparations (S9). Four S . typhimurium strains, TA97, TA98, TA100, and TA102 were employed in the Ames tests. The mutagenicity was detected from all three chemicals. The addition of S9 increased the mutation ratios of three chemicals to four strains, except BP-6 to TA100 strain and 4-MBC to TA97 and TA98 strain. In the mixed experiment, all positive effects were detected in the absence of S9. However, the results all became negative in the presence of S9. For the mixture of BP-6 and 4-MBC, positive results were detected on four tester strains except for the TA100 strain. For the mixture of BP-6, BP-8, and 4-MBC, positive results were detected on four strains. The mixture test results showed antagonism in mutagenicity for the mixture of BP-6 and 4-MBC to TA98 and TA100 strains and the mixture of BP-6, BP-8, and 4-MBC to TA100 and TA102 strains.

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

Concerns have recently increased that the integrity of some scientific research is questionable due to the inability to reproduce the claimed results of some experiments and thereby confirm that the original researcher's conclusions were justified. This phenomenon has been described as 'reproducibility crisis' and affects various fields from medicine to basic applied sciences. In this context, the REPLICA project aims to replicate previously conducted in vitro studies on the toxicity of cigarette smoke and e-cigarette aerosol, sometimes adding experiments or conditions where necessary, in order to verify the robustness and replicability of the data. In this work the REPLICA Team replicated biological and toxicological assessment published by Rudd and colleagues in 2020. As in the original paper, we performed Neutral Red Uptake (NRU) assay for the evaluation of cytotoxicity, Ames test for the evaluation of mutagenesis and In Vitro Micronuclei (IVMN) assay for the evaluation of genotoxicity on cells treated with cigarette smoke or e-cigarette aerosol. The results showed high cytotoxicity, mutagenicity and genotoxicity induced by cigarette smoke, but slight or no cytotoxic, mutagenic and genotoxic effects induced by the e-cigarette aerosol. Although the two studies presented some methodological differences, the findings supported those previously presented by Rudd and colleagues.

Goji (Lycium barbarum L.) leaves are emphasized as a functional tea or as dietary supplements. The phenolic compound profile, antioxidant, enzyme inhibitory, antimicrobial, and antimutagenic activities of leaf extracts from two selected cultivars in comparison with wild-growing plants have been evaluated. HPLC-DAD/ESI-ToF-MS analysis revealed the presence of phenolic acids and flavonoids with chlorogenic acid and rutin being the dominant compounds in the cultivated plants, whereas rutin and kaempeferol-3-O-rutinoside for wild growing ones. In particular, cv. Erma contained the highest amount of chlorogenic acid and showed a strong tyrosinase-inhibitory effect. Staphylococcus aureus, Listeria monocytogenes, and Penicillium funiculosum were the most sensitive strains when exposed to extracts from cultivated plants. Antimutagenic activity was evaluated by Ames' test. The tested extracts provided high protection against mutagenicity induced by 2-anthramine (2-AA) to Salmonella typhimurium strains TA 98 and TA 100 (max. inhibition (%) 88% and 74.2%, respectively). Overall, Goji leaves are a rich source of bioactive compounds with functional properties that need further risk/benefit evaluation when used in foods or health-promoting formulations.

OBJECTIVES: According to the 2007 National Research Council report Toxicology for the Twenty-First Century, modern methods (e.g., \"omics,\" in vitro assays, high-throughput testing, computational methods) will lead to the emergence of a new approach to toxicology. The Salmonell. mammalian microsome mutagenicity assay has been central to the field of genetic toxicology since the 1970s. Here we document the paradigm shifts engendered by the assay, the validation and applications of the assay, and how the assay is a model for future in vitro toxicology assays. DATA SOURCES: We searched PubMed, Scopus, and Web of Knowledge using key words relevant to the Salmonella assay and additional genotoxicity assays. DATA EXTRACTION: We merged the citations, removing duplicates, and categorized the papers by year and topic. DATA SYNTHESIS: The Salmonella assay led to two paradigm shifts: that some carcinogens were mutagens and that some environmental samples (e.g., air, water, soil, food, combustion emissions) were mutagenic. Although there are > 10,000 publications on the Salmonella assay a covering tens of thousands of agents, data on even more agents probably exist in unpublished form, largely as proprietary studies by industry. The Salmonell. assay is a model for the development of 21st century in vitro toxicology assays in terms of the establishment of standard procedures, ability to test various agents, transferability across laboratories, validation and testing, and structure—activity analysis. CONCLUSIONS: Similar to a stethoscope as a first-line, inexpensive tool in medicine, the Salmonell. assay can serve a similar, indispensable role in the foreseeable future of 21st century toxicology.