Hollaender award 2023: Adventures in applied genetic toxicology.

This work describes career "adventures" into applied research in environmental mutagenesis. Surprising and interesting results, as well as publications in Nature and Science, may counter an assumption that applied research is not as exciting or impactful as basic research. The narrative is described in terms of "mentors," whose advice had a lasting impact and resonated in many ways.

Optimizing the detection of N-nitrosamine mutagenicity in the Ames test.

Accurately determining the mutagenicity of small-molecule N-nitrosamine drug impurities and nitrosamine drug substance-related impurities (NDSRIs) is critical to identifying mutagenic and cancer hazards. In the current study we have evaluated several approaches for enhancing assay sensitivity for evaluating the mutagenicity of N-nitrosamines in the bacterial reverse mutagenicity (Ames) test. Preincubation assays were conducted using five activation conditions: no exogenous metabolic activation and metabolic activation mixes employing both 10% and 30% liver S9 from hamsters and rats pretreated with inducers of enzymatic activity.

Developing a pragmatic consensus procedure supporting the ICH S1B(R1) weight of evidence carcinogenicity assessment.

The ICH S1B carcinogenicity global testing guideline has been recently revised with a novel addendum that describes a comprehensive integrated Weight of Evidence (WoE) approach to determine the need for a 2-year rat carcinogenicity study. In the present work, experts from different organizations have joined efforts to standardize as much as possible a procedural framework for the integration of evidence associated with the different ICH S1B(R1) WoE criteria. The framework uses a pragmatic consensus procedure for carcinogenicity hazard assessment to facilitate transparent, consistent, and documented decision-making and it discusses best-practices both for the organization of studies and presentation of data in a format suitable for regulatory review.

Medical device report analyses from MAUDE: Device and patient outcomes, adverse events, and sex-based differential effects.

Post-market medical device-associated failures and patient problems are reported in Medical Device Reports (MDRs) to the US Food and Drug Administration. Reports are accessible through Manufacturer and User Facility Device Experience (MAUDE), a database including both required and voluntary submissions. We present an overview of >10 million MDRs received from 2011 to 2021.

Test method for evaluating the photocytotoxic potential of fluorescence imaging products.

Various fluorescence imaging agents are currently under clinical studies. Despite significant benefits, phototoxicity is a barrier to the clinical translation of fluorophores. Current regulatory guidelines on medication-based phototoxicity focus on skin effects during sun exposure.

Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays.

Propranolol is a widely used β-blocker that can generate a nitrosated derivative, N-nitroso propranolol (NNP). NNP has been reported to be negative in the bacterial reverse mutation test (the Ames test) but genotoxic in other in vitro assays. In the current study, we systematically examined the in vitro mutagenicity and genotoxicity of NNP using several modifications of the Ames test known to affect the mutagenicity of nitrosamines, as well as a battery of genotoxicity tests using human cells.

micronucleus assay: Method for assessment of nanomaterials using cytochalasin B.

The micronucleus (MNvit) assay is used to evaluate the aneugenic and clastogenic potential of a test material based upon its ability to induce micronuclei in the cells. This protocol is provided for testing of nanomaterials (NM) with standard cell lines in the absence of metabolic activation. The use of cytochalasin B (CytoB) and the analysis of binucleated cells in the cytokinesis-block version of the micronucleus assay ensures that cells analyzed have undergone cell division, which is required for expression of DNA damage and micronucleus formation.

Thymidine Kinase Mammalian Cell Mutagenicity Assays for Assessment of Nanomaterials.

The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM characterization, sample preparation and dose selection, relevant to all genotoxicity assays, are found in an accompanying paper. The present paper describes methods for evaluation of mutagenicity in the mammalian (mouse) () gene occurring in L5178Y mouse lymphoma (ML) cells and in the designated gene in human lymphoblastoid TK6 cells.

Mammalian Alkaline Comet Assay: Method Adapted for Genotoxicity Assessment of Nanomaterials.

The Comet assay measures the generation of DNA strand breaks under conditions in which the DNA will unwind and migrate to the anode in an electrophoresis assay, producing comet-like figures. Measurements are on single cells, which allows the sampling of a diversity of cells and tissues for DNA damaging effects. The Comet assay is the most common method for genotoxicity assessment of nanomaterials (NM).

Common Considerations for Genotoxicity Assessment of Nanomaterials.

Genotoxicity testing is performed to determine potential hazard of a chemical or agent for direct or indirect DNA interaction. Testing may be a surrogate for assessment of heritable genetic risk or carcinogenic risk. Testing of nanomaterials (NM) for hazard identification is generally understood to require a departure from normal testing procedures found in international standards and guidelines.

Quantifying the Photochemical Damage Potential of Contrast-Enhanced Fluorescence Imaging Products: Singlet Oxygen Production.

The benefits of contrast-enhancing imaging probes have become apparent over the past decade. However, there is a gap in the literature when it comes to the assessment of the phototoxic potential of imaging probes and systems emitting visible and/or near-infrared radiation. The primary mechanism of fluorescent agent phototoxicity is thought to involve the production of reactive molecular species (RMS), yet little has been published on the best practices for safety evaluation of RMS production levels for clinical products.

Approaches In Carcinogenicity Hazard Assessment: Current Status and Future Needs.

Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making.

Appropriate in vivo follow-up assays to an in vitro bacterial reverse mutation (Ames) test positive investigational drug candidate (active pharmaceutical ingredient), drug-related metabolite, or drug-related impurity.

Kirkland et al. [Mutation Research/Genetic Toxicology and Environmental Mutagenesis 847 (2019) 403035, https://doi.org/10.

The Photoinitiator Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate with Exposure to 405 nm Light Is Cytotoxic to Mammalian Cells but Not Mutagenic in Bacterial Reverse Mutation Assays.

Lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP) is a free radical photo-initiator used to initiate free radical chain polymerization upon light exposure, and is combined with gelatin methacryloyl (GelMA) to produce a photopolymer used in bioprinting. The free radicals produced under bioprinting conditions are potentially cytotoxic and mutagenic. Since these photo-generated free radicals are highly-reactive but short-lived, toxicity assessments should be conducted with light exposure.

Revisiting the bacterial mutagenicity assays: Report by a workgroup of the International Workshops on Genotoxicity Testing (IWGT).

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent.

Demonstrating laboratory proficiency in bacterial mutagenicity assays for regulatory submission.

The bacterial reverse mutation test is a mainstay for evaluation of mutagenicity predicting the carcinogenic potential of a test substance and is recommended by regulatory agencies across the globe. The popularity of the test is due, in part, to the relatively low cost, rapid results and small amount of test material required compared to most other toxicological tests as well as the near universal acceptance of the toxicological significance of a clear positive or negative result. Most laboratories follow the Organization for Economic Cooperation and Development Test Guideline 471 (TG471) or national guidelines based on TG471.

Are all bacterial strains required by OECD mutagenicity test guideline TG471 needed?

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to seek consensus on difficult or conflicting approaches to genotoxicity testing based upon experience, available data, and analysis techniques. At the 2017 IWGT meeting in Tokyo, one working group addressed the sensitivity and selectivity of the bacterial strains specified in the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471 to recommend possible modification of the test guideline. Three questions were posed: (1) Although TA100 is derived from TA1535, does TA1535 detect any mutagens that are not detected by TA100? (2) Among the options of Salmonella TA1537, TA97 or TA97a, are these strains truly equivalent? (3) Because there is a choice to use one of either E.

Rationale and Roadmap for Developing Panels of Hotspot Cancer Driver Gene Mutations as Biomarkers of Cancer Risk.

Cancer driver mutations (CDMs) are necessary and causal for carcinogenesis and have advantages as reporters of carcinogenic risk. However, little progress has been made toward developing measurements of CDMs as biomarkers for use in cancer risk assessment. Impediments for using a CDM-based metric to inform cancer risk include the complexity and stochastic nature of carcinogenesis, technical difficulty in quantifying low-frequency CDMs, and lack of established relationships between cancer driver mutant fractions and tumor incidence.

Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods.

Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays).

Next generation testing strategy for assessment of genomic damage: A conceptual framework and considerations.

For several decades, regulatory testing schemes for genetic damage have been standardized where the tests being utilized examined mutations and structural and numerical chromosomal damage. This has served the genetic toxicity community well when most of the substances being tested were amenable to such assays. The outcome from this testing is usually a dichotomous (yes/no) evaluation of test results, and in many instances, the information is only used to determine whether a substance has carcinogenic potential or not.

Performance characteristics of the AmpliSeq Cancer Hotspot panel v2 in combination with the Ion Torrent Next Generation Sequencing Personal Genome Machine.

Next-Generation Sequencing is a rapidly advancing technology that has research and clinical applications. For many cancers, it is important to know the precise mutation(s) present, as specific mutations could indicate or contra-indicate certain treatments as well as be indicative of prognosis. Using the Ion Torrent Personal Genome Machine and the AmpliSeq Cancer Hotspot panel v2, we sequenced two pancreatic cancer cell lines, BxPC-3 and HPAF-II, alone or in mixtures, to determine the error rate, sensitivity, and reproducibility of this system.

Silver nanoparticles: correlating nanoparticle size and cellular uptake with genotoxicity.

The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage.

Assessment of titanium dioxide nanoparticle effects in bacteria: association, uptake, mutagenicity, co-mutagenicity and DNA repair inhibition.

Due to their unique properties, the use of nanoparticles (NPs) is expanding; these same properties may affect their potential risk to humans. However, standard methods for genotoxicity assessment may not be adequate for NPs; altered tests reported here have been developed to address perceived inadequacies. The bacterial reverse mutation assay is an essential part of the battery of tests to determine genotoxicity.

Genotoxicity of nanomaterials: refining strategies and tests for hazard identification.

A workshop addressing strategies for the genotoxicity assessment of nanomaterials (NMs) was held on October 23, 2010 in Fort Worth Texas, USA. The workshop was organized by the Environmental Mutagen Society and the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute. The workshop was attended by more than 80 participants from academia, regulatory agencies, and industry from North America, Europe and Japan.

Harnessing genomics to identify environmental determinants of heritable disease.

Next-generation sequencing technologies can now be used to directly measure heritable de novo DNA sequence mutations in humans. However, these techniques have not been used to examine environmental factors that induce such mutations and their associated diseases. To address this issue, a working group on environmentally induced germline mutation analysis (ENIGMA) met in October 2011 to propose the necessary foundational studies, which include sequencing of parent-offspring trios from highly exposed human populations, and controlled dose-response experiments in animals.