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.

Use of silicone wristbands to measure firefighters' exposures to polycyclic aromatic hydrocarbons (PAHs) during live fire training.

Firefighters experience exposures to carcinogenic and mutagenic substances, including polycyclic aromatic hydrocarbons (PAHs). Silicone wristbands (SWBs) have been used as passive samplers to assess firefighters' exposures over the course of a shift but their utility in measuring short term exposures, source of exposure, and correlations with other measurements of exposure have not yet been investigated. In this study, SWBs were used to measure the concentrations of 16 priority PAHs inside and outside of firefighters' personal protective equipment (PPE) while firefighting. SWBs were placed on the wrist and jacket of 20 firefighters conducting live fire training. Correlations were made with matching data from a sister project that measured urinary concentrations of PAH metabolites and PAH concentrations from personal air samples from the same participants. Naphthalene, acenaphthylene and phenanthrene had the highest geometric mean concentrations in both jacket and wrist SWB, with 1040, 320, 180 ng/g SWB for jacket and 55.0, 4.9, and 6.0 ng/g SWB for wrist, respectively. Ratios of concentrations between the jacket and wrist SWBs were calculated as worker protection factors (WPFs) and averaged 40.1 for total PAHs and ranged from 2.8 to 214 for individual PAHs, similar to previous studies. Several significant correlations were observed between PAHs in jacket SWBs and air samples (e.g., total and low molecular weight PAHs, r = 0.55 and 0.59, p < 0.05, respectively). A few correlations were found between PAHs from SWBs worn on the wrist and jacket, and urinary concentrations of PAH metabolites and PAH concentrations in air samples. The ability of the SWBs to accurately capture exposures to various PAHs was likely influenced by short sampling time, high temperatures, and high turbulence. Future work should further examine the limitations of SWBs for PAH exposures in firefighting, and other extreme environments.

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.

Effectiveness of dermal cleaning interventions for reducing firefighters' exposures to PAHs and genotoxins.

Firefighters are exposed to carcinogenic and mutagenic combustion emissions, including polycyclic aromatic hydrocarbons (PAHs). Fire service and firefighter cancer advocacy groups recommend skin cleaning using wipes or washing with detergent and water after exposure to smoke, although these strategies have not been proven to reduce exposures to harmful combustion products such as PAHs. This study assessed dermal decontamination methods to reduce PAH exposures by firefighters participating in live fire training scenarios. Study participants (n = 88) were randomly assigned to an intervention group (i.e., two types of commercial skin wipes, detergent and water, or a control group who did not use any skin decontamination). PAHs were measured in personal air (during the fire) and dermal wipe samples (before and after fire suppression and after dermal decontamination). PAH metabolites and mutagenicity were measured in urine samples before and after fire suppression. Airborne PAH concentrations during the fire ranged between 200 and 3,970 µg/m3 (mean = 759 µg/m3, SD = 685 µg/m3). Firefighters had higher total PAHs and high-molecular-weight PAHs on their skin after the fire compared to before (1.3- and 2.2-fold, respectively, p < 0.01). Urinary PAH metabolites increased significantly following exposure to the training fires by 1.7 to 2.2-fold (depending on the metabolite, p < 0.001). Urinary mutagenicity did not differ significantly between pre- and post-fire for any of the decontamination methods. Detergent and water was the only intervention that removed a significant amount of total PAHs from the skin (0.72 ng/cm2 preintervention vs. 0.38 ng/cm2 postintervention, p < 0.01). However, fold changes in urinary PAH metabolites (i.e., pre- vs. post-exposure levels) did not differ among any of the dermal decontamination methods or the control group. These data suggest that despite on-site attempts to remove PAHs from firefighters' skin, the examined interventions did not reduce the internal dose of PAHs. Future work should investigate preventing initial exposure using other interventions, such as improved personal protective equipment.

Comprehensive interpretation of in vitro micronucleus test results for 292 chemicals: from hazard identification to risk assessment application.

Risk assessments are increasingly reliant on information from in vitro assays. The in vitro micronucleus test (MNvit) is a genotoxicity test that detects chromosomal abnormalities, including chromosome breakage (clastogenicity) and/or whole chromosome loss (aneugenicity). In this study, MNvit datasets for 292 chemicals, generated by the US EPA's ToxCast program, were evaluated using a decision tree-based pipeline for hazard identification. Chemicals were tested with 19 concentrations (n = 1) up to 200 µM, in the presence and absence of Aroclor 1254-induced rat liver S9. To identify clastogenic chemicals, %MN values at each concentration were compared to a distribution of batch-specific solvent controls; this was followed by cytotoxicity assessment and benchmark concentration (BMC) analyses. The approach classified 157 substances as positives, 25 as negatives, and 110 as inconclusive. Using the approach described in Bryce et al. (Environ Mol Mutagen 52:280-286, 2011), we identified 15 (5%) aneugens. IVIVE (in vitro to in vivo extrapolation) was employed to convert BMCs into administered equivalent doses (AEDs). Where possible, AEDs were compared to points of departure (PODs) for traditional genotoxicity endpoints; AEDs were generally lower than PODs based on in vivo endpoints. To facilitate interpretation of in vitro MN assay concentration-response data for risk assessment, exposure estimates were utilized to calculate bioactivity exposure ratio (BER) values. BERs for 50 clastogens and two aneugens had AEDs that approached exposure estimates (i.e., BER < 100); these chemicals might be considered priorities for additional testing. This work provides a framework for the use of high-throughput in vitro genotoxicity testing for priority setting and chemical risk assessment.

The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals.

The Organisation for Economic Co-Operation and Development Test Guideline 488 (TG 488) uses transgenic rodent models to generate in vivo mutagenesis data for regulatory submission. The recommended design in TG 488, 28 consecutive daily exposures with tissue sampling three days later (28 + 3d), is optimized for rapidly proliferating tissues such as bone marrow (BM). A sampling time of 28 days (28 + 28d) is considered more appropriate for slowly proliferating tissues (e.g., liver) and male germ cells. We evaluated the impact of the sampling time on mutant frequencies (MF) in the BM of MutaMouse males exposed for 28 days to benzo[a]pyrene (BaP), procarbazine (PRC), isopropyl methanesulfonate (iPMS), or triethylenemelamine (TEM) in dose-response studies. BM samples were collected + 3d, + 28d, + 42d or + 70d post exposure and MF quantified using the lacZ assay. All chemicals significantly increased MF with maximum fold increases at 28 + 3d of 162.9, 6.6, 4.7 and 2.8 for BaP, PRC, iPMS and TEM, respectively. MF were relatively stable over the time period investigated, although they were significantly increased only at 28 + 3d and 28 + 28d for TEM. Benchmark dose (BMD) modelling generated overlapping BMD confidence intervals among the four sampling times for each chemical. These results demonstrate that the sampling time does not affect the detection of mutations for strong mutagens. However, for mutagens that produce small increases in MF, sampling times greater than 28 days may produce false-negative results. Thus, the 28 + 28d protocol represents a unifying protocol for simultaneously assessing mutations in rapidly and slowly proliferating somatic tissues and male germ cells.

The influence of demographic and lifestyle factors on urinary levels of PAH metabolites-empirical analyses of Cycle 2 (2009-2011) CHMS data.

Polycyclic aromatic hydrocarbons (PAHs) are a group of compounds formed during the incomplete combustion of organic matter. Several are mutagenic carcinogens; the magnitude of exposure can be assessed by examining urinary levels of PAH metabolites. Data from biomonitoring studies that record urinary PAH metabolite levels, as well as demographic and lifestyle information, can be used to investigate relationships between PAH exposure and variables, such as smoking status, workplace smoking restrictions, age, sex, household income, home age, and occupation. This study analysed creatinine-adjusted urinary PAH metabolite concentrations and questionnaire data from ~1200 individuals aged 16 years and older surveyed in Cycle 2 of the Canadian Health Measures Survey (CHMS). Statistical analyses revealed that smoking status, age, and sex are associated with urinary concentrations of a pyrene metabolite (1-OHP), phenanthrene metabolites (ΣOH-Phen), fluorene metabolites (ΣOH-Flu) and naphthalene metabolites (ΣOH-Nap). More specifically, smoking status, age and sex can collectively account for 30, 24, 52, and 34% of the observed variations in 1-OHP, ΣOH-Phen, ΣOH-Flu and ΣOH-Nap metabolites, respectively (p < 0.001). Analyses of non-smokers revealed weak but significant effects of age, sex, home age, and occupation on urinary levels of selected PAH metabolites (i.e., <7% of observed variation, p < 0.05). The unexplained variation in PAH metabolite levels is most likely related to diet, which was not examined. Although the results revealed significant relationships between urinary PAH metabolite levels and several lifestyle and/or demographic variables, robust examinations of selected effects (e.g., sex, home age, occupation) will require datasets that are balanced with respect to the other highlighted variables. The results can be used to identify remedial measures to reduce exposure and concomitant risk, and/or design follow-up studies to test hypotheses regarding the causes of exposure differences empirically related to sex, age, home age, and occupation.

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.

Mutation as a Toxicological Endpoint for Regulatory Decision-Making.

Mutations induced in somatic cells and germ cells are responsible for a variety of human diseases, and mutation per se has been considered an adverse health concern since the early part of the 20th Century. Although in vitro and in vivo somatic cell mutation data are most commonly used by regulatory agencies for hazard identification, that is, determining whether or not a substance is a potential mutagen and carcinogen, quantitative mutagenicity dose-response data are being used increasingly for risk assessments. Efforts are currently underway to both improve the measurement of mutations and to refine the computational methods used for evaluating mutation data. We recommend continuing the development of these approaches with the objective of establishing consensus regarding the value of including the quantitative analysis of mutation per se as a required endpoint for comprehensive assessments of toxicological risk. Environ. Mol. Mutagen. 61:34-41, 2020. © 2019 Wiley Periodicals, Inc.

The IARC Monographs: Updated Procedures for Modern and Transparent Evidence Synthesis in Cancer Hazard Identification.

The Monographs produced by the International Agency for Research on Cancer (IARC) apply rigorous procedures for the scientific review and evaluation of carcinogenic hazards by independent experts. The Preamble to the IARC Monographs, which outlines these procedures, was updated in 2019, following recommendations of a 2018 expert advisory group. This article presents the key features of the updated Preamble, a major milestone that will enable IARC to take advantage of recent scientific and procedural advances made during the 12 years since the last Preamble amendments. The updated Preamble formalizes important developments already being pioneered in the Monographs program. These developments were taken forward in a clarified and strengthened process for identifying, reviewing, evaluating, and integrating evidence to identify causes of human cancer. The advancements adopted include the strengthening of systematic review methodologies; greater emphasis on mechanistic evidence, based on key characteristics of carcinogens; greater consideration of quality and informativeness in the critical evaluation of epidemiological studies, including their exposure assessment methods; improved harmonization of evaluation criteria for the different evidence streams; and a single-step process of integrating evidence on cancer in humans, cancer in experimental animals, and mechanisms for reaching overall evaluations. In all, the updated Preamble underpins a stronger and more transparent method for the identification of carcinogenic hazards, the essential first step in cancer prevention.

Polycyclic aromatic hydrocarbon (PAH) and metal contamination of air and surfaces exposed to combustion emissions during emergency fire suppression: Implications for firefighters' exposures.

This study examined occupational exposures of Ottawa firefighters to combustion by-products and selected metals. We measured exposures to polycyclic aromatic hydrocarbons (PAHs), antimony, cadmium, and lead using (1) personal air samplers worn by firefighters during emergency fire suppression; (2) wipe samples from skin, personal clothing, and personal protective equipment (PPE) collected before and after emergency firefighting (n = 29); and (3) air samples collected in three fire stations vehicle bays, truck cabs, and one administration office. We assessed OFS PPE decontamination procedures using wipe samples collected before and after laundering (n = 12). Air concentrations exceeded occupational exposure limits at two fire events for lead and nine for PAHs. After fire suppression, PAH concentrations were significantly higher on skin and PPE (p < 0.001), skin, clothing, and PPE for antimony (p < 0.001, 0.01, and 0.05, respectively), and skin and PPE for lead (p < 0.001). Air concentrations of PAHs and antimony were significantly higher in vehicle bays compared to the office (p < 0.05), but significantly lower compared to fire truck cabs (p < 0.05). Washing PPE was effective in removing, on average, 61% of PAHs, 55% of antimony, 97% of lead, and 90% of cadmium. These results indicate that firefighters are significantly exposed, via multiple routes, to combustion by-products during on-shift fire suppression.

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.

Utility of a next generation framework for assessment of genomic damage: A case study using the industrial chemical benzene.

We recently published a next generation framework for assessing the risk of genomic damage via exposure to chemical substances. The framework entails a systematic approach with the aim to quantify risk levels for substances that induce genomic damage contributing to human adverse health outcomes. Here, we evaluated the utility of the framework for assessing the risk for industrial chemicals, using the case of benzene. Benzene is a well-studied substance that is generally considered a genotoxic carcinogen and is known to cause leukemia. The case study limits its focus on occupational and general population health as it relates to benzene exposure. Using the framework as guidance, available data on benzene considered relevant for assessment of genetic damage were collected. Based on these data, we were able to conduct quantitative analyses for relevant data sets to estimate acceptable exposure levels and to characterize the risk of genetic damage. Key observations include the need for robust exposure assessments, the importance of information on toxicokinetic properties, and the benefits of cheminformatics. The framework points to the need for further improvement on understanding of the mechanism(s) of action involved, which would also provide support for the use of targeted tests rather than a prescribed set of assays. Overall, this case study demonstrates the utility of the next generation framework to quantitatively model human risk on the basis of genetic damage, thereby enabling a new, innovative risk assessment concept. Environ. Mol. Mutagen. 61:94-113, 2020. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

The IWGT in vitro Mammalian Cell Gene Mutation (MCGM) assays working group-Introductory remarks & consensus statements.

Assays for gene mutations in cultured mammalian cells, i.e., Mammalian Cell Gene Mutation (MCGM) assays, are widely used in genetic toxicology laboratories worldwide; over the past four decades they have been commonly employed in safety assessment studies, and studies designed to address hypothesis-driven research questions. Despite many advances in the fields of cellular and molecular biology over the past four decades, the MCGM assays commonly used for regulatory evaluations continue to be those developed in the 1970s, including assays that enumerate induced mutations at the hprt or tk loci of several commonly used cell lines. Consequently, the Steering Committee of the 7th International Workshops on Genotoxicity Testing (IWGT) convened a working group (WG) to critically assess the state-of-the-science in regards to the current and emerging tools for the detection of mutagens using cultured mammalian cells. The WG was divided into four sub-groups that evaluated the state-of- the-science with respect to the: (1) in vitro Pig-a gene mutation assay, (2) in vitro assays based on cells from transgenic rodents, (3) technologies and innovations to improve MCGM assays using TK6 cells, and (4) novel and emerging technologies and approaches for detection and enumeration of gene mutations in mammalian cells. Each of these sub-groups critically reviewed the scientific literature, along with other unpublished data, to develop consensus statements on the status of the test systems in their respective focus areas. These reviews, with their associated consensus statements, are presented in the accompanying works by Bemis and Heflich., White et al., Honma et al., and Evans et al. The MCGM assay WG, in consultation with the entire IWGT, formulated consensus statements regarding the overall utility of MCGM assays for identification and assessment of mutagenic hazard.

In vitro mammalian cell mutation assays based on transgenic reporters: A report of the International Workshop on Genotoxicity Testing (IWGT).

Chemical safety evaluations require assessment of genetic toxicity. Transgenic rodent (TGR) assays permit enumeration of mutations in chromosomally-integrated targets contained in shuttle vectors. In order to improve in vitro mutagenicity assessment, and to substantially reduce animal use, in vitro assays using transgenic reporters have been developed. These assays are based on cells derived from TGRs, or cells transfected with transgenic shuttle vectors containing a mutation target. As part of the 7th International Workshop on Genotoxicity Testing, an In Vitro Mammalian Cell Gene Mutation Assay working group reviewed all published information pertaining to in vitro transgene mutagenicity assays; the utility, advantages and disadvantages of the assays were evaluated and discussed. The review revealed that over 20 TGR-based in vitro assays have been used to assess the mutagenic activity of over 150 agents. Overall, the Working Group considered in vitro transgene mutagenicity assays pragmatic tools for the safety evaluation of new and existing substances. A formal SWOT (strengths, weaknesses, opportunities, threats) analysis revealed advantages including the use of established scoring protocols, avoidance of laborious clone isolation and enumeration, ability to use metabolically competent primary cells, ability to detect different types of genetic damage, large dynamic range, and complementarity to in vivo TGR endpoints. Disadvantages include lack of validation and little consistency in protocols, the use of specialised reagents, the time and effort required for mutant enumeration, the use of some cell lines that lack metabolic capacity, and the need for multiple assays to cover all mutational mechanisms. Several assays have been partially validated, indicating promising reliability, reproducibility and applicability domain. Once in vitro transgene mutagenicity assays have been more thoroughly validated, they are well placed to augment or replace existing in vitro mammalian cell mutagenicity assays, particularly in cases where the in vivo TGR mutation assay is intended for follow-up.

The mutagenic activity of select azo compounds in MutaMouse target tissues in vivo and primary hepatocytes in vitro.

The mutagenicity of Direct Black 38, Sudan I, and Para Red were evaluated in the in vivo MutaMouse assay and the in vitro MutaMouse primary hepatocyte (PH) assay. Direct Black 38 is an International Agency for Research on Cancer (IARC) Group 1 carcinogen and a prototypical benzidine-based azo compound that requires azo-reduction to yield a DNA-reactive metabolite. Sudan I and Para Red are structurally related azo compounds that have been detected as illegal contaminants in foods. Sudan I is an in vivo mutagen, and both it and Para Red are known to be mutagenic in vitro. Sudan I is oxidized by hepatic and/or bladder enzymes to yield a mutagenic metabolite, but little is known about Para Red. In the present study, Direct Black 38 elicited a significant mutagenic response in the bone marrow, glandular stomach, small intestine and colon in vivo, and in PHs in vitro. Sudan I elicited a weak positive response in the bone marrow and a marginally significant treatment effect in the bladder (p = 0.059); it did not elicit a significant response in PHs in vitro. Para Red elicited a positive response in the colon, as well as in PHs in vitro, albeit at a cytotoxic concentration. The findings are well aligned with the known mechanisms of action of Direct Black 38 and Sudan I; they suggest that intestinal azo-reduction plays an important role in the activation of Para Red. The MutaMouse pH results illustrate the ability of this assay to detect chemicals requiring azo-reduction; however, they also demonstrate a gap in applicability domain, as MutaMouse PHs elicit a negative response following exposure to Sudan I. Elucidation of the mechanisms underlying this gap will require further study.

Integrated in silico and in vitro genotoxicity assessment of thirteen data-poor substances.

The Canadian Domestic Substances List (DSL) contains chemicals that have not been tested for genotoxicity as their use pre-dates regulatory requirements. In the present study, (quantitative) structure-activity relationships ((Q)SAR) model predictions and in vitro tests were conducted for genotoxicity assessment of 13 data-poor chemicals from the DSL (i.e. CAS numbers 19286-75-0, 13676-91-0, 2478-20-8, 6408-20-8, 74499-36-8, 26694-69-9, 29036-02-0, 120-24-1, 84696-48-9, 4051-63-2, 5718-26-3, 632-51-9, and 600-14-6). First, chemicals were screened by (Q)SAR models in Leadscope® and OASIS TIMES; two chemicals were excluded from (Q)SAR as they are complex mixtures. Six were flagged by (Q)SAR as potentially mutagenic and were subsequently confirmed as mutagens using the Ames assay. Of nine chemicals with clastogenic (Q)SAR flags, eight induced micronuclei in TK6 cells. Benchmark dose analysis was used to evaluate the potency of the chemicals. Four chemicals were bacterial mutagens with similar potencies. Three chemicals were more potent in micronuclei induction than the prototype alkylating agent methyl methanesulfonate and three were equipotent to the mutagenic carcinogen benzo[a]pyrene in the presence of rat liver S9. Overall, 11 of the 13 DSL chemicals demonstrated at least one type of genotoxicity in vitro. This study demonstrates the application of genotoxic potency analysis for prioritizing further investigations.

Glucagon-Like Peptide-1-Mediated Cardioprotection Does Not Reduce Right Ventricular Stunning and Cumulative Ischemic Dysfunction After Coronary Balloon Occlusion.

Stunning and cumulative ischemic dysfunction occur in the left ventricle with coronary balloon occlusion. Glucagon-like peptide (GLP)-1 protects the left ventricle against this dysfunction. This study used a conductance catheter method to evaluate whether the right ventricle (RV) developed similar dysfunction during right coronary artery balloon occlusion and whether GLP-1 was protective. In this study, the RV underwent significant stunning and cumulative ischemic dysfunction with right coronary artery balloon occlusion. However, GLP-1 did not protect the RV against this dysfunction when infused after balloon occlusion.

Correction to: Re: Gi et al. 2018, In vivo positive mutagenicity of 1,4-dioxane and quantitative analysis of its mutagenicity and carcinogenicity in rats, Archives of Toxicology 92:3207-3221.

The publisher would like to apologize for the failed cross-linking to the following Letter to the Editor by Paul A.