High information content assays for genetic toxicology testing: A report of the International Workshops on Genotoxicity Testing (IWGT).

We live in an era of 'big data', where the volume, velocity, and variety of the data being generated is increasingly influencing the way toxicological sciences are practiced. With this in mind, a workgroup was formed for the 2017 International Workshops on Genotoxicity Testing (IWGT) to consider the use of high information content data in genetic toxicology assessments. Presentations were given on adductomics, global transcriptional profiling, error-reduced single-molecule sequencing, and cellular phenotype-based assays, which were identified as methodologies that are relevant to present-day genetic toxicology assessments. Presenters and workgroup members discussed the state of the science for these methodologies, their potential use in genetic toxicology, current limitations, and the future work necessary to advance their utility and application. The session culminated with audience-assisted SWOT (strength, weakness, opportunities, and threats) analyses. The summary report described herein is structured similarly. A major conclusion of the workgroup is that while conventional regulatory genetic toxicology testing has served the public well over the last several decades, it does not provide the throughput that has become necessary in modern times, and it does not generate the mechanistic information that risk assessments ideally take into consideration. The high information content assay platforms that were discussed in this session, as well as others under development, have the potential to address aspect(s) of these issues and to meet new expectations in the field of genetic toxicology.

Improving dose selection and identification of aneugens in the in vitro chromosome aberration test by integration of flow cytometry-based methods.

Previously, this laboratory reported on the development of a flow cytometry-based method that automates the assessment of the mitotic index (MI) and numerical chromosome changes in chemically treated cultures of human lymphocytes [Muehlbauer PA and Schuler MJ, 2003, 2005]. With this method, testing design can easily include numerous well-spaced doses to better define the shape of MI dose response curves. In addition, the hypodiploid, hyperdiploid, and polyploid mitotic populations are available simultaneously to determine the biological relevance of polyploidy effects during the conduct of the assay. The current work describes the integration of this flow cytometry-based method into the routine conduct of good laboratory practice structural chromosome aberration assays in vitro, and discusses improvements in evaluating cytotoxicity and polyploidy endpoints. Additional methods for simultaneous assessment of cell death (sub-G1 DNA) are shown in combination with the MI to provide a more complete evaluation of cytotoxic conditions. A total of 30 pharmaceutical compounds were assayed in compliance with Organization for Economic Cooperation and Development and International Conference on Harmonization guidelines. The inclusion of numerous well-spaced doses improved high dose selection and resulted in fewer high dose artifacts. Only 1 compound in 30 produced a positive response in structural aberrations. In comparison, polyploidy induction was observed in 22 of 30 (73%) compounds, with no apparent increases in numerical chromosomal aberrations. These studies show that flow cytometry-based methods can be used to better characterize cytotoxicity dose-response relationships and improve the detection of aneugens.

Histone markers identify the mode of action for compounds positive in the TK6 micronucleus assay.

The in vitro micronucleus assay with TK6 cells is frequently used as part of the genotoxicity testing battery for pharmaceuticals. Consequently, follow-up testing strategies are needed for positive compounds to determine their mode of action, which would then allow for deployment of appropriate in vivo follow-up strategies. We have chosen 3 micronucleus positive compounds, the clastogen etoposide, the aneugen noscapine and the cytotoxicant tunicamycin to evaluate different approaches to determine their aneugenic or clastogenic properties. Each of the three compounds were evaluated following 4 and 24h of continuous treatment by flow cytometry for micronucleus induction, the aneugenicity markers phosphorylated-histone 3 (p-H3) and polyploidy, the clastogenicity marker γH2AX and the apoptosis marker cleaved caspase 3. They were further evaluated by Western blot for mono-ubiquitinated and γH2AX. Results show that the clastogen etoposide produced a dose related increase in γH2AX and mono-ubiquitinated H2AX and a dose related decrease in p-H3 positive mitotic cells. Conversely, the aneugen produced increases in p-H3 and polyploidy with no significant increases seen in mono-ubiquitinated H2AX or γH2AX. Lastly, the cytotoxicant tunicamycin induced neither an increase in p-H3 nor γH2AX. All three compounds produced dose-related increases in cleaved caspase 3. The results from this study provide evidence that adding clastogenicity and aneugenicity markers to the in vitro micronucleus assay in TK6 cells could help to identify the mode of action of positive compounds. The combination of endpoints suggested here needs to be further evaluated by a broader set of test compounds.

IWGT report on quantitative approaches to genotoxicity risk assessment I. Methods and metrics for defining exposure-response relationships and points of departure (PoDs).

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.

IWGT report on quantitative approaches to genotoxicity risk assessment II. Use of point-of-departure (PoD) metrics in defining acceptable exposure limits and assessing human risk.

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.

Interpreting in vitro micronucleus positive results: simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents.

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.

Evaluation of the Pig-a, micronucleus, and comet assay endpoints in a 28-day study with ethyl methanesulfonate.

Ethyl methanesulfonate (EMS) was evaluated as part of the validation effort for the rat Pig-a mutation assay and compared with other well-established in vivo genotoxicity endpoints. Male Sprague-Dawley (SD) rats were given a daily dose of 0, 6.25, 12.5, 25, 50, or 100 mg/kg/day EMS for 28 days, and evaluated for a variety of genotoxicity endpoints in peripheral blood, liver, and colon. Blood was sampled pre-dose (Day 1) and at various time points up to Day 105. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies. The first statistically significant increases in mutant frequencies were seen in RETs on Day 15 and in RBCs on Day 29 with the maximum RET(CD59-) on Day 29 and of RBC(CD59-) on Day 55. The lowest dose producing a statistically significant increase of RET(CD59-) was 12.5 mg/kg on Day 55 and 25 mg/kg for RBC(CD59-) on Day 55. EMS also induced significant increases in % micronucleated RETs (MN-RETs) in peripheral blood on Days 3, 15, and 28. No statistically significant increases in micronuclei were seen in liver or colon. Results from the in vivo Comet assay on Day 29 showed generally weak increases in DNA damage in all tissues evaluated with little evidence for accumulation of damage seen over time. The results with EMS indicate that the assessment of RBC(CD59-) and/or RET(CD59-) in the Pig-a assay could be a useful and sensitive endpoint for a repeat dose protocol and complements other genotoxicity endpoints.

Phenolphthalein induces centrosome amplification and tubulin depolymerization in vitro.

Aneuploidy is a major cause of human reproductive failure and plays a large role in cancer. Phenolphthalein (PHT) induces tumors in rodents but its primary mechanism does not seem to be DNA damage. In heterozygous TSG-p53(®) mice, PHT induces lymphomas and also micronuclei (MN), many containing kinetochores (K), implying chromosome loss (aneuploidy). The induction of aneuploidy would be compatible with the loss of the normal p53 gene seen in the lymphomas. In this study, we confirm PHT's aneugenicity and determine the aneugenic mechanism of PHT by combining traditional genetic toxicology assays with image and flow cytometry methods. The data revealed that PHT induces tubulin polymerization abnormalities and deregulates the centrosome duplication cycle causing centrosome amplification. We also show that one of the consequences of these events is apoptosis.

Concordance analysis of an in vitro micronucleus screening assay and the regulatory chromosome aberration assay using pharmaceutical drug candidates.

The in vitro micronucleus assay is under consideration by regulatory agencies as a suitable alternative to the in vitro chromosome aberration (CA) assay. At Pfizer, we utilized a non-Good Laboratory practices cytokinesis-block in vitro micronucleus (CBMN) assay in CHO cells as a screen to predict the regulatory outcome of the human lymphocyte CA assay, and we have retrospectively analyzed a highly select set of 112 internal drug candidates to measure concordance. Overall, our exploratory CBMN correctly classified 97 of 112 (86.6%) compounds in the CA assay. Specificity was high with 87 of 92 (94.6%) CA negative compounds correctly classified by CBMN. Sensitivity was low at 50% with 10 of 20 CA positive compounds correctly classified by CBMN; this may be attributed to the low number of CA positives in the select set. In an attempt to improve sensitivity, we increased the number of CA positives by combining our internal set with an industrial data set previously published (Miller B et al. 1997: Mutat Res 392:45-59). When combined, concordance was 86.7% (143/165), specificity was 91.2% (114/125), and sensitivity increased to 72.5% (29/40). Because cytotoxicity is considered a confounding factor of in vitro test systems, we also examined, within the Pfizer data set, the influence of cytotoxicity in the CBMN assay, and the results indicated that seemingly low (<50%) or excessively high (>70%) levels of cytotoxicity did not significantly alter predicted CA outcome. These collective analyses contribute to growing evidence that the CBMN assay is a suitable regulatory option in the standard battery of genetic toxicology tests.