Integration of the rat liver micronucleus assay into a 28-day treatment protocol: testing the genotoxicity of four small-molecule nitrosamines with different carcinogenic potencies and tumor target specificities.

Several potent carcinogenic nitrosamines, including N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA), induce micronuclei in the micronucleated hepatocyte (MNHEP) assay but not in the micronucleated reticulocyte (MNRET) assay. However, the MNHEP assay is not as frequently used as the MNRET assay for evaluating in vivo genotoxicity. The present study evaluated MN formation in the liver of Big Blue transgenic rats exposed to four small-molecule nitrosamines, NDMA, N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisoporpylamine (NEIPA), and N-nitrosomethylphenylamine (NMPA), using a repeat-dose protocol typically used for in vivo mutagenicity studies.

Mutagenicity and genotoxicity evaluation of 15 nitrosamine drug substance-related impurities in human TK6 cells.

Nitrosamine drug substance-related impurities (NDSRIs) are a sub-category of N-nitrosamine drug impurities that share structural similarity to the corresponding active pharmaceutical ingredient. The mutagenicity of NDSRIs is poorly understood. We previously tested a series of NDSRIs using the Enhanced Ames Test (EAT).

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.

Repeat treatment of organotypic airway cultures with ethyl methanesulfonate causes accumulation of somatic cell mutations without expansion of bronchial-carcinoma-specific cancer driver mutations.

The human in vitro organotypic air-liquid-interface (ALI) airway tissue model is structurally and functionally similar to the human large airway epithelium and, as a result, is being used increasingly for studying the toxicity of inhaled substances. Our previous research demonstrated that DNA damage and mutagenesis can be detected in human airway tissue models under conditions used to assess general and respiratory toxicity endpoints. Expanding upon our previous proof-of-principle study, human airway epithelial tissue models were treated with 6.

Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF).

Erythrocyte PIG-A mutant frequencies in cancer patients receiving cisplatin.

Background: Cisplatin is a primary chemotherapy choice for various solid tumors. DNA damage caused by cisplatin results in apoptosis of tumor cells. Cisplatin-induced DNA damage, however, may also result in mutations in normal cells and the initiation of secondary malignancies.

Error-corrected next generation sequencing - Promises and challenges for genotoxicity and cancer risk assessment.

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.

Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models.

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates.

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.

Assessing the quality and making appropriate use of historical negative control data: A report of the International Workshop on Genotoxicity Testing (IWGT).

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.

Key Challenges and Recommendations for Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol.

Genotoxicity evaluation of nitrosamine impurities using human TK6 cells transduced with cytochrome P450s.

Many nitrosamines are recognized as mutagens and potent rodent carcinogens. Over the past few years, nitrosamine impurities have been detected in various drugs leading to drug recalls. Although nitrosamines are included in a 'cohort of concern' because of their potential human health risks, most of this concern is based on rodent cancer and bacterial mutagenicity data, and there are little data on their genotoxicity in human-based systems.

90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure.

Effect of life stage and target tissue on dose-response assessment of ethyl methane sulfonate-induced genotoxicity.

In order to investigate the possibility that treatment age affects the genotoxic response to ethyl methane sulfonate (EMS) exposure, we dosed gpt-delta neonatal mice on postnatal days 1-28 with 5-100 mg/kg/day of EMS and measured micronucleus (MN) induction in peripheral blood and gpt gene mutation in liver, lung, bone marrow, small intestine, spleen, and kidney. The data were compared to measurements from similarly exposed adult gpt-delta mice. Our results indicate that the peripheral blood MN frequencies in mice treated as neonates are not substantially different from those measured in mice treated as adults.

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.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020).

Recommendations for conducting the rodent erythrocyte Pig-a assay: A report from the HESI GTTC Pig-a Workgroup.

The rodent Pig-a assay is a flow cytometric, phenotype-based method used to measure in vivo somatic cell mutation. An Organization for Economic Co-operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93).

Molecular analysis of GPI-anchor biosynthesis pathway genes in rat strains used for the Pig-a gene mutation assay.

Recent studies support the assumption that mutation of the X-linked Pig-a gene is most likely responsible for the mutant phenotype of the cells deficient in glycosylphosphatidylinositol (GPI)-anchored proteins quantified in the rodent Pig-a gene mutation assay. In humans, however, mutations in both alleles of one of the 30 other genes involved in GPI-anchor synthesis, e.g.

Invited review: human air-liquid-interface organotypic airway tissue models derived from primary tracheobronchial epithelial cells-overview and perspectives.

The lung is an organ that is directly exposed to the external environment. Given the large surface area and extensive ventilation of the lung, it is prone to exposure to airborne substances, such as pathogens, allergens, chemicals, and particulate matter. Highly elaborate and effective mechanisms have evolved to protect and maintain homeostasis in the lung.

Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7 International Workshop on Genotoxicity Testing (IWGT).

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a "Use of 3D Tissues in Genotoxicity Testing" working group (WG) met at the 7 IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy.