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). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

Development and application of consensus in silico models for advancing high-throughput toxicological predictions.

Computational toxicology models have been successfully implemented to prioritize and screen chemicals. There are numerous in silico (quantitative) structure-activity relationship ([Q]SAR) models for the prediction of a range of human-relevant toxicological endpoints, but for a given endpoint and chemical, not all predictions are identical due to differences in their training sets, algorithms, and methodology. This poses an issue for high-throughput screening of a large chemical inventory as it necessitates several models to cover diverse chemistries but will then generate data conflicts. To address this challenge, we developed a consensus modeling strategy to combine predictions obtained from different existing in silico (Q)SAR models into a single predictive value while also expanding chemical space coverage. This study developed consensus models for nine toxicological endpoints relating to estrogen receptor (ER) and androgen receptor (AR) interactions (i.e., binding, agonism, and antagonism) and genotoxicity (i.e., bacterial mutation, in vitro chromosomal aberration, and in vivo micronucleus). Consensus models were created by combining different (Q)SAR models using various weighting schemes. As a multi-objective optimization problem, there is no single best consensus model, and therefore, Pareto fronts were determined for each endpoint to identify the consensus models that optimize the multiple-criterion decisions simultaneously. Accordingly, this work presents sets of solutions for each endpoint that contain the optimal combination, regardless of the trade-off, with the results demonstrating that the consensus models improved both the predictive power and chemical space coverage. These solutions were further analyzed to find trends between the best consensus models and their components. Here, we demonstrate the development of a flexible and adaptable approach for in silico consensus modeling and its application across nine toxicological endpoints related to ER activity, AR activity, and genotoxicity. These consensus models are developed to be integrated into a larger multi-tier NAM-based framework to prioritize chemicals for further investigation and support the transition to a non-animal approach to risk assessment in Canada.

Isolated cryptococcal osteomyelitis in the setting of immune reconstitution inflammatory syndrome.

Cryptococcal infections, though rare, must be considered in all immunocompromised patients. Patients with HIV/AIDS on antiretrovirals may have a treatment course complicated by immune reconstitution inflammatory syndrome. Here we present a case of a 38-year-old woman with HIV/AIDS with knee pain who only began to experience severe pain after induction of antiretroviral therapy. She was found to have cryptococcal osteomyelitis without dissemination to the central nervous system, an unusual presentation for immunocompromised patients. She was treated with oral fluconazole with a resolution of symptoms. This case report suggests conservative management of isolated cryptococcal infection with fluconazole, regardless of immune status.

Conservative treatment of isolated cryptococcus infection in a patient with a weakened immune system Cryptococcus neoformans is a fungus found in the soil which care rarely infect humans, especially those who have a weakened immune system, like those with HIV infection. The treatment of HIV in people with 'secondary infections', like cryptococcal infections, may cause patients to get worse before they get better as the immune system starts to function and attack the secondary infection. In this case report, we look at a patient who had untreated HIV who only began to develop symptoms of a secondary cryptococcal infection once treatment for HIV was started. However, because the cryptococcal infection was only in her bone and not throughout her body and nervous system, we were able to treat her with a conservative, oral regimen. In patients with severe cryptococcal infection or with infection of their nervous system, they often need to be treated with medications that can cause a lot of unwanted side effects. The key takeaway from this article is that conservative treatment of Cryptococcus may be effective, even in people with weakened immune systems, as long as the cryptococcal infection is isolated.

Smooth vs Textured Expanders: Patient Factors and Anatomic Plane Are Greater Factors in Determining First-Stage Breast Reconstruction Outcomes.

BACKGROUND: Textured implants and expanders are associated with an increased risk of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL). As a result, plastic surgeons are utilizing smooth expanders, but many perceive these produce undesirable outcomes including infection, seroma, and lateral displacement. OBJECTIVES: The aim of this study was to compare clinical outcomes of smooth and textured expanders. METHODS: Breast reconstruction patients from January 2018 to May 2021 were retrospectively reviewed. Included patients underwent placement of tissue expanders at the time of mastectomy. Primary outcomes included postoperative seroma, infection, malposition, days to final reconstruction, explantation, and the need for capsulorrhaphy. RESULTS: In total, 233 patients were reviewed, of whom 167 met both inclusion and exclusion criteria. There was no statistically significant difference in poor outcomes comparing smooth and textured expanders. Days to final reconstruction was lower with smooth expanders per breast (P = .0424). The subpectoral group was associated with an increased likelihood of undergoing capsulorrhaphy (P = .004). Prepectoral placement was associated with more seromas (P = .0176) and infections (P = .0245). Demographic factors included older age as a protective factor for undergoing capsulorrhaphy (odds ratio [OR] = 0.962, P = .038), obesity increased the risk of infection (OR = 5.683, P = .0279) and malposition (OR = 6.208, P = .0222), and radiation was associated with malposition (OR = 3.408, P = .0246). CONCLUSIONS: There was no significant difference in poor outcomes between smooth and textured expanders. Patient demographics and anatomical plane placement had greater effects on infection, seroma, and the need for capsulorrhaphy compared with tissue expander texturing.

MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement.

Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

Interpretation of in vitro concentration-response data for risk assessment and regulatory decision-making: Report from the 2022 IWGT quantitative analysis expert working group meeting.

Quantitative risk assessments of chemicals are routinely performed using in vivo data from rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.

Application of a new approach methodology (NAM)-based strategy for genotoxicity assessment of data-poor compounds.

The conventional battery for genotoxicity testing is not well suited to assessing the large number of chemicals needing evaluation. Traditional in vitro tests lack throughput, provide little mechanistic information, and have poor specificity in predicting in vivo genotoxicity. New Approach Methodologies (NAMs) aim to accelerate the pace of hazard assessment and reduce reliance on in vivo tests that are time-consuming and resource-intensive. As such, high-throughput transcriptomic and flow cytometry-based assays have been developed for modernized in vitro genotoxicity assessment. This includes: the TGx-DDI transcriptomic biomarker (i.e., 64-gene expression signature to identify DNA damage-inducing (DDI) substances), the MicroFlow® assay (i.e., a flow cytometry-based micronucleus (MN) test), and the MultiFlow® assay (i.e., a multiplexed flow cytometry-based reporter assay that yields mode of action (MoA) information). The objective of this study was to investigate the utility of the TGx-DDI transcriptomic biomarker, multiplexed with the MicroFlow® and MultiFlow® assays, as an integrated NAM-based testing strategy for screening data-poor compounds prioritized by Health Canada's New Substances Assessment and Control Bureau. Human lymphoblastoid TK6 cells were exposed to 3 control and 10 data-poor substances, using a 6-point concentration range. Gene expression profiling was conducted using the targeted TempO-Seq™ assay, and the TGx-DDI classifier was applied to the dataset. Classifications were compared with those based on the MicroFlow® and MultiFlow® assays. Benchmark Concentration (BMC) modeling was used for potency ranking. The results of the integrated hazard calls indicate that five of the data-poor compounds were genotoxic in vitro, causing DNA damage via a clastogenic MoA, and one via a pan-genotoxic MoA. Two compounds were likely irrelevant positives in the MN test; two are considered possibly genotoxic causing DNA damage via an ambiguous MoA. BMC modeling revealed nearly identical potency rankings for each assay. This ranking was maintained when all endpoint BMCs were converted into a single score using the Toxicological Prioritization (ToxPi) approach. Overall, this study contributes to the establishment of a modernized approach for effective genotoxicity assessment and chemical prioritization for further regulatory scrutiny. We conclude that the integration of TGx-DDI, MicroFlow®, and MultiFlow® endpoints is an effective NAM-based strategy for genotoxicity assessment of data-poor compounds.

Quantitative in vitro to in vivo extrapolation of genotoxicity data provides protective estimates of in vivo dose.

Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, and aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points-of-departures (PODs) for setting exposure limits. The more recent higher-throughput in vitro genotoxicity assays, many of which also provide mechanistic information, offer a powerful approach for determining defined PODs for potency ranking and risk assessment. In order to obtain relevant human dose context from the in vitro assays, in vitro to in vivo extrapolation (IVIVE) models are required to determine what dose would elicit a concentration in the body demonstrated to be genotoxic using in vitro assays. Previous work has demonstrated that application of IVIVE models to in vitro bioactivity data can provide PODs that are protective of human health, but there has been no evaluation of how these models perform with in vitro genotoxicity data. Thus, the Genetic Toxicology Technical Committee, under the Health and Environmental Sciences Institute, conducted a case study on 31 reference chemicals to evaluate the performance of IVIVE application to genotoxicity data. The results demonstrate that for most chemicals considered here (20/31), the PODs derived from in vitro data and IVIVE are health protective relative to in vivo PODs from animal studies. PODs were also protective by assay target: mutations (8/13 chemicals), micronuclei (9/12), and aneugenicity markers (4/4). It is envisioned that this novel testing strategy could enhance prioritization, rapid screening, and risk assessment of genotoxic chemicals.

Establishing a quantitative framework for regulatory interpretation of genetic toxicity dose-response data: Margin of exposure case study of 48 compounds with both in vivo mutagenicity and carcinogenicity dose-response data.

Quantitative relationships between carcinogenic potency and mutagenic potency have been previously examined using a benchmark dose (BMD)-based approach. We extended those analyses by using human exposure data for 48 compounds to calculate carcinogenicity-derived and genotoxicity-derived margin of exposure values (MOEs) that can be used to prioritize substances for risk management. MOEs for 16 of the 48 compounds were below 10,000, and consequently highlighted for regulatory concern. Of these, 15 were highlighted using genotoxicity-derived (micronucleus [MN] dose-response data) MOEs. A total of 13 compounds were highlighted using carcinogenicity-derived MOEs; 12 compounds were overlapping. MOEs were also calculated using transgenic rodent (TGR) mutagenicity data. For 10 of the 12 compounds examined using TGR data, the results similarly revealed that mutagenicity-derived MOEs yield regulatory decisions that correspond with those based on carcinogenicity-derived MOEs. The effect of benchmark response (BMR) on MOE determination was also examined. Reinterpretation of the analyses using a BMR of 50% indicated that four out of 15 compounds prioritized using MN-derived MOEs based on a default BMR of 5% would have been missed. The results indicate that regulatory decisions based on in vivo genotoxicity dose-response data would be consistent with those based on carcinogenicity dose-response data; in some cases, genotoxicity-based decisions would be more conservative. Going forward, and in the absence of carcinogenicity data, in vivo genotoxicity assays (MN and TGR) can be used to effectively prioritize substances for regulatory action. Routine use of the MOE approach necessitates the availability of reliable human exposure estimates, and consensus regarding appropriate BMRs for genotoxicity endpoints.

ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells.

Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.

Narcissism, Empathy, and Rape Myth Acceptance Among Heterosexual College Males.

The perpetration of rape and sexual assault on college campuses is a pervasive problem that has been linked to narcissism and rape myth acceptance. Studies evaluating empathy priming-based prevention programs have yielded mixed results, and empathy priming has not been examined specifically among high-risk populations. The present study sought to address this gap in the literature by exploring how empathy priming interacts with narcissistic traits to predict heterosexual college males' (n = 74) rape myth acceptance. Participants read a vignette depicting a date rape and were either primed to be empathetic or objective. Results showed that baseline empathy and narcissism were negatively and positively associated with rape myth acceptance, respectively. After priming, participants low on narcissistic traits had lower rape myth acceptance when they were in the empathy (vs. the objective) condition, whereas individuals high in narcissistic traits had higher rape myth acceptance when they were in the empathy priming condition. Findings suggest that males who were at higher risk of perpetration more strongly endorsed problematic beliefs about rape after being asked to empathize with a fictional rape victim. Future prevention and intervention studies should incorporate measures of personality traits and continue to explore the possibility that empathy priming may produce the opposite of the intended effect among high-risk males.

The neuronal-specific isoform of BIN1 regulates ß-secretase cleavage of APP and Aß generation in a RIN3-dependent manner.

Genome-wide association studies have identified BIN1 (Bridging integrator 1) and RIN3 (Ras and Rab interactor 3) as genetic risk factors for late-onset Alzheimer's disease (LOAD). The neuronal isoform of BIN1 (BIN1V1), but not the non-neuronal isoform (BIN1V9), has been shown to regulate tau-pathology and Aß generation via RAB5-mediated endocytosis in neurons. BIN1 directly interacts with RIN3 to initiate RAB5-mediated endocytosis, which is essential for ß-secretase (BACE1)-mediated ß-secretase cleavage of ß-amyloid precursor protein (APP) to generate Amyloid-ß (Aß), the key component of senile plaques in AD. Understanding the regulatory roles of BIN1 (neuronal BIN1V1) and RIN3 in ß-secretase mediated cleavage of APP and Aß generation is key to developing novel therapeutics to delay or prevent AD progression. Neuronal and non-neuronal isoforms of BIN1 (BIN1V1 and BIN1V9, respectively) were introduced with RIN3 into an in vitro cell-based system to test RIN3-dependent effects of neuronal BIN1V1 and non-neuronal BIN1V9 on ß-secretase-mediated cleavage of APP and Aß generation. Confocal microscopy was performed to examine RIN3-dependent subcellular localization of BIN1V1 and BIN1V9. Western blot analysis was performed to assess the effects of RIN3 and BIN1V1/BIN1V9 on ß-secretase mediated processing of APP. We enriched cells expressing BIN1V1 without or with RIN3 via FACS to measure Aß generation using Aß ELISA assay, and to evaluate APP internalization by chasing biotinylated or antibody-labeled cell surface APP. Neuronal BIN1V1 containing the CLAP domain and non-neuronal BIN1V9 lacking the CLAP domain are the major isoforms present in the brain. Employing confocal microscopy, we showed that RIN3 differentially regulates the recruitment of both BIN1V1 and BIN1V9 into RAB5-endosomes. We further showed that BIN1V1, but not BIN1V9, downregulates ß-secretase (BACE1)-mediated processing of APP in a RIN3-dependent manner. Overexpression of BIN1V1 also attenuated Aß generation in a RIN3-dependent manner. Using cell-based internalization assays, we show BIN1V1, but not BIN1V9, delays the endocytosis of APP, but not of BACE1, into early endosomes, thereby spatially and temporally separating these two proteins into different cellular compartments, resulting in reduced cleavage of APP by BACE1 and reduced Aß generation-all in a RIN3-dependent manner. Finally, we show that RIN3 sequesters BIN1V1 in RAB5-positive early endosomes, likely via the CLAP-domain, resulting in attenuated ß-secretase processing of APP and Aß generation by delaying endocytosis of APP. Our findings provide new mechanistic data on how two AD-associated molecules, RIN3 and BIN1 (neuronal BIN1V1), interact to govern Aß production, implicating these two proteins as potential therapeutic targets for the prevention and treatment of AD.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD)-Inducible Poly-ADP-Ribose Polymerase (TIPARP/PARP7) Catalytic Mutant Mice (TiparpH532A) Exhibit Increased Sensitivity to TCDD-Induced Hepatotoxicity and Lethality.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-adenosine diphosphate (ADP)-ribose polymerase (TIPARP/PARP7), an aryl hydrocarbon receptor (AHR) target gene and mono-ADP-ribosyltransferase, acts as part of a negative feedback loop to repress AHR signaling. This process is prevented by a single H532A mutation in TIPARP that destroys its catalytic activity. We hypothesized that the loss of TIPARP catalytic activity would increase sensitivity to TCDD-induced toxicity in vivo. To test this, we created a catalytically deficient mouse line (TiparpH532A) by introducing a single H532A mutation in TIPARP. Treatment of mouse embryonic fibroblasts or hepatocytes isolated from TiparpH532A mice confirmed the increased TCDD-induced expression of the AHR target genes Cyp1a1, Cyp1b1, and Tiparp. TiparpH532A mice given a single injection of 10 µg/kg TCDD, a nonlethal dose in Tiparp+/+ mice, did not survive beyond day 10. All Tiparp+/+ mice survived the 30-day treatment. TCDD-treated TiparpH532A mice displayed increased expression of AHR target genes, increased steatohepatitis and hepatotoxicity. Hepatic RNA-sequencing revealed 7-fold more differentially expressed genes in TiparpH532A mice than in Tiparp+/+ mice (4542 vs 647 genes) 6 days after TCDD treatment. Differentially expressed genes included genes involved in xenobiotic metabolism, lipid homeostasis and inflammation. Taken together, these data further support TIPARP as a critical negative regulator of AHR activity and show that loss of its catalytic activity is sufficient to increase sensitivity to TCDD-induced steatohepatitis and lethality. Since TIPARP inhibition has recently emerged as a potential anticancer therapy, the impact on AHR signaling, TCDD and polycyclic aromatic hydrocarbon toxicity will need to be carefully considered under conditions of therapeutic TIPARP inhibition.

A not-so-simple twist of fate.

Multiciliated cells are considered terminally differentiated, yet tissues bearing them are remodeled during development and after injury. In this issue of Developmental Cell, Tasca et al. (2021) show that multiciliated epithelial cells are lost via two different Notch-dependent processes, apoptosis and transdifferentiation, during developmental remodeling of the Xenopus epidermis.

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure.

At cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus and minus ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

Microneedle manipulation of the mammalian spindle reveals specialized, short-lived reinforcement near chromosomes.

The spindle generates force to segregate chromosomes at cell division. In mammalian cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle anchors kinetochore-fibers in space and time to move chromosomes. Yet, how it does so remains poorly understood as we lack tools to directly challenge this anchorage. Here, we adapt microneedle manipulation to exert local forces on the spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals the preservation of local architecture in the spindle-center over seconds. Sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting chromosome stretching. Further, pulled kinetochore-fibers pivot around poles but not chromosomes, retaining their orientation within 3 µm of chromosomes. This local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. Together, these observations indicate short-lived, specialized reinforcement in the spindle center. This could help protect chromosome attachments from transient forces while allowing spindle remodeling, and chromosome movements, over longer timescales.

In vitro mutagenicity of selected environmental carcinogens and their metabolites in MutaMouse FE1 lung epithelial cells.

Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide -S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide -S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP -S9, 4-ABP ±S9 and N-OH-4-ABP -S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.

Quantitative Interpretation of Genetic Toxicity Dose-Response Data for Risk Assessment and Regulatory Decision-Making: Current Status and Emerging Priorities.

The screen-and-bin approach for interpretation of genotoxicity data is predicated on three false assumptions: that genotoxicants are rare, that genotoxicity dose-response functions do not contain a low-dose region mechanistically characterized by zero-order kinetics, and that genotoxicity is not a bona fide toxicological endpoint. Consequently, there is a need to develop and implement quantitative methods to interpret genotoxicity dose-response data for risk assessment and regulatory decision-making. Standardized methods to analyze dose-response data, and determine point-of-departure (PoD) metrics, have been established; the most robust PoD is the benchmark dose (BMD). However, there are no standards for regulatory interpretation of mutagenicity BMDs. Although 5-10% is often used as a critical effect size (CES) for BMD determination, values for genotoxicity endpoints have not been established. The use of BMDs to determine health-based guidance values (HBGVs) requires assessment factors (AFs) to account for interspecies differences and variability in human sensitivity. Default AFs used for other endpoints may not be appropriate for interpretation of in vivo mutagenicity BMDs. Analyses of published dose-response data showing the effects of compensatory pathway deficiency indicate that AFs for sensitivity differences should be in the range of 2-20. Additional analyses indicate that the AF to compensate for short treatment durations should be in the range of 5-15. Future work should use available data to empirically determine endpoint-specific CES values; similarly, to determine AF values for BMD adjustment. Future work should also evaluate the ability to use in vitro dose-response data for risk assessment, and the utility of probabilistic methods for determination of mutagenicity HBGVs. Environ. Mol. Mutagen. 61:66-83, 2020. © 2019 Her Majesty the Queen in Right of Canada.