We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow™ DNA Damage Kit— p53, γH2AX, Phospho-histone H3. For these experiments seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and non-genotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hrs. At 4 and 24 hrs cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all inter-laboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or non-genotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals’ predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay.

This report summarizes the discussion, conclusions, and points of consensus of the IWGT Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (QWG) based on a meeting in Foz do Iguaçu, Brazil October 31-November 2, 2013. Topics addressed included (1) the need for quantitative dose-response analysis, (2) methods to analyze exposure-response relationships & derive point of departure (PoD) metrics, (3) points of departure (PoD) and mechanistic threshold considerations, (4) approaches to define exposure-related risks, (5) empirical relationships between genetic damage (mutation) and cancer, and (6) extrapolations across test systems and species. This report discusses the first three of these topics and a companion report discusses the latter three. The working group critically examined methods for determining point of departure metrics (PoDs) that could be used to estimate low-dose risk of genetic damage and from which extrapolation to acceptable exposure levels could be made using appropriate mode of action information and uncertainty factors. These included benchmark doses (BMDs) derived from fitting families of exponential models, the No Observed Genotoxic Effect Level (NOGEL), and "threshold" or breakpoint dose (BPD) levels derived from bilinear models when mechanistic data supported this approach. The QWG recognizes that scientific evidence suggests that thresholds below which genotoxic effects do not occur likely exist for both DNA-reactive and DNA-nonreactive substances, but notes that small increments of the spontaneous level cannot be unequivocally excluded either by experimental measurement or by mathematical modeling. Therefore, rather than debating the theoretical possibility of such low-dose effects, emphasis should be placed on determination of PoDs from which acceptable exposure levels can be determined by extrapolation using available mechanistic information and appropriate uncertainty factors. This approach places the focus on minimization of the genotoxic risk, which protects against the risk of the development of diseases resulting from the genetic damage. Based on analysis of the strengths and weaknesses of each method, the QWG concluded that the order of preference of PoD metrics is the statistical lower bound on the BMD > the NOGEL > a statistical lower bound on the BPD. A companion report discusses the use of these metrics in genotoxicity risk assessment, including scaling and uncertainty factors to be considered when extrapolating below the PoD and/or across test systems and to the human.

This is the second of two reports from the International Workshops on Genotoxicity Testing (IWGT) Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (the QWG). The first report summarized the discussions and recommendations of the QWG related to the need for quantitative dose-response analysis of genetic toxicology data, the existence and appropriate evaluation of threshold responses, and methods to analyze exposure-response relationships and derive points of departure (PoDs) from which acceptable exposure levels could be determined. This report summarizes the QWG discussions and recommendations regarding appropriate approaches to evaluate exposure-related risks of genotoxic damage, including extrapolation below identified PoDs and across test systems and species. Recommendations include the selection of appropriate genetic endpoints and target tissues, uncertainty factors and extrapolation methods to be considered, the importance and use of information on mode of action, toxicokinetics, metabolism, and exposure biomarkers when using quantitative exposure-response data to determine acceptable exposure levels in human populations or to assess the risk associated with known or anticipated exposures. The empirical relationship between genetic damage (mutation and chromosomal aberration) and cancer in animal models was also examined. It was concluded that there is a general correlation between cancer induction and mutagenic and/or clastogenic damage for agents thought to act via a genotoxic mechanism, but that the correlation is limited due to an inadequate number of cases in which mutation and cancer can be compared at a sufficient number of doses in the same target tissues of the same species and strain exposed under directly comparable routes and experimental protocols.

The specificity of in vitro mammalian cell genotoxicity assays is low, as they yield a high incidence of positive results that are not observed in animal genotoxicity and carcinogenicity tests, that is, "misleading" or "irrelevant" positives. We set out to develop a rapid and effective follow-up testing strategy that would predict whether apparent in vitro micronucleus-inducing effects are due to a clastogenic, aneugenic, or secondary irrelevant mode(s) of action. Priority was given to biomarkers that could be multiplexed onto flow cytometric acquisition of micronucleus frequencies, or that could be accomplished in parallel using a homogeneous-type assay. A training set of 30 chemicals comprised of clastogens, aneugens, and misleading positive chemicals was studied. These experiments were conducted with human TK6 cells over a range of closely spaced concentrations in a continuous exposure design. In addition to micronucleus frequency, the following endpoints were investigated, most often at time of harvest: cleaved Parp-positive chromatin, cleaved caspase 3-positive chromatin, ethidium monoazide bromide-positive chromatin, polyploid nuclei, phospho-histone H3-positive (metaphase) cells, tetramethylrhodamine ethyl ester-negative cells, cellular ATP levels, cell cycle perturbation, and shift in γ-H2AX fluorescence relative to solvent control. Logistic regression was used to identify endpoints that effectively predict chemicals' a priori classification. Cross validation using a leave-one-out approach indicated that a promising base model includes γ-H2AX shift and change in phospho-histone H3-positive events (25/30 correct calls). Improvements were realized when one or two additional endpoints were included (26-30/30 correct calls). These models were further evaluated with a test set of 10 chemicals, and also by evaluating 3 chemicals at a collaborating laboratory. The resulting data support the hypothesis that a matrix of high throughput-compatible biomarkers can effectively delineate two important modes of genotoxic action as well as identify cytotoxicity that can lead to irrelevant positive results.

It was by way of total synthesis that the issues concerning the stereostructure of leiodermatolide (1) have recently been solved; with the target now being unambiguously defined, the mission of synthesis changes as to secure a meaningful supply of this exceedingly scarce natural product derived from a deep-sea sponge. To this end, a scalable route of 19 steps (longest linear sequence) has been developed, which features a catalytic asymmetric propargylation of a highly enolizable β-keto-lactone, a ring closing alkyne metathesis and a modified Stille coupling as the key transformations. Deliberate digression from this robust blueprint brought a first set of analogues into reach, which allowed the lead qualities of 1 to be assessed. The acquired biodata show that 1 is a potent cytotoxin in human tumor cell proliferation assays, distinguished by GI50 values in the ≤3 nM range even for cell lines expressing the Pgp efflux transporter. Studies with human U2OS cells revealed that 1 causes mitotic arrest, micronucleus induction, centrosome amplification and tubulin disruption, even though no evidence for direct tubulin binding has been found in cell-free assays; moreover, the compound does not seem to act through kinase inhibition. Indirect evidence points at centrosome declustering as a possible mechanism of action, which provides a potentially rewarding outlook in that centrosome declustering agents hold promise of being inherently selective for malignant over healthy human tissue.