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.

Application of the adverse outcome pathway framework to genotoxic modes of action.

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.

A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals.

Suitability of Long-Term Frozen Rat Blood Samples for the Interrogation of Pig-a Gene Mutation by Flow Cytometry.

The rodent blood Pig-a assay has been undergoing international validation for use as an in vivo hematopoietic cell gene mutation assay, and given the promising results an Organization for Economic Co-operation and Development (OECD) Test Guideline is currently under development. Enthusiasm for the assay stems in part from its alignment with 3Rs principles permitting combination with other genotoxicity endpoint(s) and integration into repeat-dose toxicology studies. One logistical requirement and experimental design limitation has been that blood samples required antibody labeling and flow cytometric analysis within one week of collection. In the current report, we describe the performance of freeze-thaw reagents that enable storage and subsequent labeling and analysis of rat blood samples for at least seven months. Data generated from three laboratories are presented that demonstrate rat erythrocyte recoveries in the range of 80-90%. Despite some loss of erythrocytes, Pearson coefficients and Bland-Altman analyses based on fresh blood vs. frozen/thawed matched pairs indicate that mutant cell and reticulocyte frequencies are not significantly affected, as the measurements are highly correlated and exhibit low bias. Collectively, these data support the effectiveness and suitability of a freeze-thaw procedure that endows the assay with several new advantageous characteristics that include: flexibility in scheduling personnel/instrumentation; reliability when shipping samples from in-life facilities to analytical sites; 3Rs-friendly, as blood from positive control animals can be stored frozen to serve as analytical controls; and ability to defer a decision to generate Pig-a data until more toxicological information becomes available on a test substance. Environ. Mol. Mutagen. 60:47-55, 2019. © 2018 Wiley Periodicals, Inc.

Birth Weight and Risk of Childhood Acute Lymphoblastic Leukemia in Arizona, Illinois, and Kentucky.

OBJECTIVE: To confirm the previously reported increased risk of leukemia among macrosomic children (those with birth weight >4 kg). METHODS: Birth certificates of Arizona, Illinois, and Kentucky children diagnosed as having acute lymphoblastic leukemia (ALL) before age 5 years were matched with birth certificates from leukemia-free children of the same sex, race, and ethnicity who were born in the same county on or about the same day. Odds ratios (ORs) for ALL among children of low (<2.5 kg) or high (>4 kg) birth weight were calculated by conditional logistic regression. RESULTS: Children with high birth weight had an elevated risk of ALL in the first 5 years of life (OR 1.28, 95% confidence interval [CI] 1.01-1.61). The excess risk was confined to non-Hispanic whites (OR 1.77, 95% CI 1.27-2.48), both boys (OR 1.57, 95% CI 1.01-2.45) and girls (OR 2.10, 95% CI 1.26-3.52). CONCLUSIONS: This study confirms the association between high birth weight and ALL previously reported by other studies in children of European ancestry. The literature on maternal risk factors for both macrosomia and ALL is reviewed, with maternal overnutrition emerging as a plausible risk factor for both outcomes.

ErbB2 promotes endothelial phenotype of human left ventricular epicardial highly proliferative cells (eHiPC).

The adult human heart contains a subpopulation of highly proliferative cells. The role of ErbB receptors in these cells has not been studied. From human left ventricular (LV) epicardial biopsies, we isolated highly proliferative cells (eHiPC) to characterize the cell surface expression and function of ErbB receptors in the regulation of cell proliferation and phenotype. We found that human LV eHiPC express all four ErbB receptor subtypes. However, the expression of ErbB receptors varied widely among eHiPC isolated from different subjects. eHiPC with higher cell surface expression of ErbB2 reproduced the phenotype of endothelial cells and were characterized by endothelial cell-like functional properties. We also found that EGF/ErbB1 induces VEGFR2 expression, while ligands for both ErbB1 and ErbB3/4 induce expression of Tie2. The number of CD31posCD45neg endothelial cells is higher in LV biopsies from subjects with high ErbB2 (ErbB2high) eHiPC compared to low ErbB2 (ErbB2low) eHiPC. These findings have important implications for potential strategies to increase the efficacy of cell-based revascularization of the injured heart, through promotion of an endothelial phenotype in cardiac highly proliferative cells.

Integration of Pig-a, micronucleus, chromosome aberration, and Comet assay endpoints in a 28-day rodent toxicity study with 4-nitroquinoline-1-oxide.

As part of the Stage III Pig-a multilaboratory validation trial, we examined the induction of CD59-negative reticulocytes and total red blood cells (RET(CD59-) and RBC(CD59-) , respectively) in male Sprague Dawley(®) rats treated with 4-nitroquinoline-1-oxide (4NQO), for 28 consecutive days by oral gavage, at doses of 1.25, 2.50, 3.75, 5.00, and 7.50 mg kg(-1) day(-1) (the high dose group was sacrificed on Day 15 due to excessive morbidity/mortality). Animals also were evaluated for: micronucleated reticulocytes (mnRET) by flow cytometry; DNA damage in peripheral blood, liver, and stomach using the Comet assay; and chromosome aberrations (CAb) in peripheral blood lymphocytes (PBL). All endpoints were analyzed at two or more timepoints where possible. Mortality, body and organ weights, food consumption, and clinical pathology also were evaluated, and demonstrated that the maximum tolerated dose was achieved at 5.00 mg kg(-1) day(-1) . The largest increases observed for the genetic toxicology endpoints (fold-increase compared to control, where significant; all at 5.00 mg kg(-1) day(-1) on Day 29) were: RET(CD59-) (21X), RBC(CD59-) (9.0X), and mnRET (2.0X). In contrast, no significant increases were observed for the CAb or Comet response, in any tissue analyzed, at any timepoint. Because 4NQO is a well known mutagen, clastogen, and carcinogen, the lack of response for these latter endpoints was unexpected. These results emphasize the extreme care that must betaken in dose and endpoint selection when incorporating genotoxicity endpoints into routine toxicity studies as has been recommended or is under consideration by various regulatory and industrial bodies.

Another duel in the sun: weighing the balances between sun protection, tanning beds, and malignant melanoma.

OBJECTIVE: The purpose of this report is to put the dueling factors of risk and prevention for melanoma in perspective for the thoughtful pediatric specialist to facilitate preteen preventive health counseling. STUDY DESIGN: We examined the rate of malignant melanoma among Kentucky residents and compared this rate with indicators of tanning bed prevalence in a large metropolitan area and sunscreen sales from a major distributor. We obtained malignant melanoma annual incidence data from the Kentucky Cancer Registry, which recorded Kentucky population incidence rates over the years 1995 to 2004. The rates reflected 2 malignant melanoma classifications: pre-invasive cancer only, or both invasive and noninvasive cancers combined. RESULTS: The age-adjusted incidence rate per hundred thousand for combined invasive and pre-invasive malignant melanoma swelled from 21.9 in 1995 to 31.3 in 2004. The respective invasive-only malignant melanoma incidence rates increased less dramatically, from 17.3 to 20.7, during this same 10-year time period. Since 1983, the number of separate tanning bed businesses increased from 1 in 1983 to 119 by the mid-1990s, and then declined to about 74 separate businesses by 2003. Sunscreen sales data is uneven between states and is currently inconclusive. CONCLUSIONS: Although current data cannot draw a precise link between melanoma and the use of tanning beds, the associated risk is implicit, as the ultraviolet A (UVA) and ultraviolet B (UVB) radiation in tanning bed usage is a well-established melanoma risk factor. In advising patients, the pediatric specialist should consider that melanoma rates are poised as a balance of some known risk factors and a few potential preventive factors.

Automated human blood micronucleated reticulocyte measurements for rapid assessment of chromosomal damage.

This study evaluated the utility of human blood micronucleated reticulocyte (MNCD71+) frequency measurement as a cytogenetic damage biomarker. The analytical methodology was flow cytometry in conjunction with a previously described three color fluorescence labeling technique that includes anti-CD71 to focus analyses on the most immature fraction of reticulocytes [S.D. Dertinger, K. Camphausen, J.T. MacGregor, M.E. Bishop, D.K. Torous, S. Avlasevich, et al., Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood, Environ. Mol. Mutagen. 44 (2004) 427-435]. Blood specimens from 50 self-reported healthy adult volunteers were studied. In addition to MNCD71+ measurements, blood plasma folate and B12 levels were assessed, since these variables tend to influence other indices of cytogenetic damage. Time-course data are also provided for 10 cancer patients undergoing treatment. For these subjects, frequency of MNCD71+ was measured immediately before therapy, and daily during the first week of chemotherapy and/or fractionated radiotherapy. For the group of healthy volunteers, the variables of age, and folate and B12 levels demonstrated no significant effect on MNCD71+ frequency. In addition, no difference was observed between pre-treatment MNCD71+ values for cancer patients compared with healthy volunteers. Regarding chemotherapy and/or partial body radiotherapy, elevated frequencies were observed upon initiation of treatment for 9 of the 10 patients studied. Maximal effects were observed 3-5 days following initiation of therapy. The largest increases in frequency of MNCD71+ (up to 25.9-fold) were observed in those patients exposed to anti-neoplastic drugs, presumably due to the systemic red marrow exposure provided by these agents. Taken together, these data support the hypothesis that the MNCD71+ endpoint represents a valuable biomarker of cytogenetic damage that does not require cell culture or microscopy-based scoring.