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This study evaluated the effect of different protocols for Ca(OH)2 removal on the bond strength (BS) of epoxy resin-based sealer in long oval root canals. Forty-eight mandibular incisors with long oval root canals were selected by CBCT. Biomechanical preparation was performed, the samples were filled with Ca(OH)2 paste and distributed in four groups (n = 12): G-without Ca(OH)2 paste (C); G2-syringe/needle (SN); G3-SN + XP-Endo® Finisher (XPF); G4-SN + passive ultrasonic irrigation (PUI). After 7 days, Ca(OH)2 was removed and teeth were filled using AHPlus and guttapercha. In four samples from each group, the sealer was manipulated with 0.1% rhodamine to assess sealer penetration by fluorescent laser confocal microscopy (FLCM). Samples were sectioned in slices for BS, scanning electron microscopy, and FLCM analysis. The BS data were analyzed by ANOVA and Tukey tests (α = 0.05). The lowest BS values were found for the SN group (12.89 ± 4.36) compared to C (35.55 ± 10.05), while PUI group presented intermediate values (28.57 ± 9.35) and the XPF group (31.34 ± 9.8) showed values that were sometimes similar to C group and sometimes similar to PUI group (p > .05). The analysis of the adhesive interface showed evident gaps, with the presence of residues between the dentin and the filling material for the SN group, and for C, XPF and PUI groups juxtaposed adhesive interface. FLCM images showed sealer penetration in the dentinal tubules along the entire perimeter for C, XPF, and PUI groups. Ca(OH)2 removal with XPF and PUI from long oval root canals resulted in higher bond strength values compared to SN, besides better sealer penetration on dentinal tubules and juxtaposed adhesive interface.

The in vitro MultiFlow® DNA Damage Assay multiplexes γH2AX, p53, phospho-histone H3, and polyploidization biomarkers into a single flow cytometric analysis. The current report describes a tiered sequential data analysis strategy based on data generated from exposure of human TK6 cells to a previously described 85 chemical training set and a new pharmaceutical-centric test set (n = 40). In each case, exposure was continuous over a range of closely spaced concentrations, and cell aliquots were removed for analysis following 4 and 24 hr of treatment. The first data analysis step focused on chemicals' genotoxic potential, and for this purpose, we evaluated the performance of a machine learning (ML) ensemble, a rubric that considered fold increases in biomarkers against global evaluation factors (GEFs), and a hybrid strategy that considered ML and GEFs. This first tier further used ML output and/or GEFs to classify genotoxic activity as clastogenic and/or aneugenic. Test set results demonstrated the generalizability of the first tier, with particularly good performance from the ML ensemble: 35/40 (88%) concordance with a priori genotoxicity expectations and 21/24 (88%) agreement with expected mode of action (MoA). A second tier applied unsupervised hierarchical clustering to the biomarker response data, and these analyses were found to group certain chemicals, especially aneugens, according to their molecular targets. Finally, a third tier utilized benchmark dose analyses and MultiFlow biomarker responses to rank genotoxic potency. The relevance of these rankings is supported by the strong agreement found between benchmark dose values derived from MultiFlow biomarkers compared to those generated from parallel in vitro micronucleus analyses. Collectively, the results suggest that a tiered MultiFlow data analysis pipeline is capable of rapidly and effectively identifying genotoxic hazards while providing additional information that is useful for modern risk assessments-MoA, molecular targets, and potency. Environ. Mol. Mutagen. 60:513-533, 2019. © 2019 Wiley Periodicals, Inc.

The International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing established an Emerging Technologies and New Strategies Workgroup to review the current State of the Art in genetic toxicology testing. The aim of the workgroup was to identify promising technologies that will improve genotoxicity testing and assessment of in vivo hazard and risk, and that have the potential to help meet the objectives of the IVGT. As part of this initiative, HESI convened a workshop in Washington, DC in May 2008 to discuss mature, maturing, and emerging technologies in genetic toxicology. This article collates the abstracts of the New and Emerging Technologies Workshop together with some additional technologies subsequently considered by the workgroup. Each abstract (available in the online version of the article) includes a section addressed specifically to the strengths, weaknesses, opportunities, and threats associated with the respective technology. Importantly, an overview of the technologies and an indication of how their use might be aligned with the objectives of IVGT are presented. In particular, consideration was given with regard to follow-up testing of positive results in the standard IVGT tests (i.e., Salmonella Ames test, chromosome aberration assay, and mouse lymphoma assay) to add weight of evidence and/or provide mechanism of action for improved genetic toxicity risk assessments in humans.

The genotoxicity of zidovudine (AZT) based treatments was investigated in human H9 lymphoblastoid cells in an in vitro study and in red blood cells (RBCs) from perinatally exposed HIV-1-infected mothers and their infants in an observational cohort study. Exposure of H9 cells for 24 hr to AZT produced dose-dependent increases in Comet assay tail moment (TM) when electrophoresed at pH 13.0, but not at pH 12.1 or pH 8.0, suggesting that DNA damage was via alkali-labile lesions and not double-stranded DNA strand breaks. The TM dose response at pH 13.0 correlated directly with AZT-DNA incorporation determined by AZT-radioimmunoassay. Levels of DNA damage in utero, measured by Comet assay TM, were similar in cord blood mononuclear cells of nucleoside analog-exposed newborns (n = 43) and unexposed controls (n = 40). In contrast, the glycophorin A (GPA) somatic cell mutation assay (which screens for large-scale DNA damage in RBCs) showed clear evidence that GPA N/N variants, arising from chromosome loss and duplication, somatic recombination, and gene conversion, were significantly elevated in mother–child pairs receiving prepartum AZT plus lamivudine (3TC). Cord blood from newborns exposed to AZT-3TC had GPA N/N variant frequencies of 4.7 ± 0.7 (mean ± SE) × 10−6 RBCs (n = 26 infants) compared with 2.2 ± 0.3 × 10−6 RBCs for unexposed controls (n = 30 infants; P < 0.001). Elevations in GPA N/N variants generally persisted through 1 year of age in nucleoside analog-exposed children. Overall, the mutagenic effects found in mother–child pairs receiving AZT-based treatments justify their surveillance for long-term genotoxic consequences. Environ. Mol. Mutagen., 2007. © 2007 Wiley-Liss, Inc.