A Proposal for New Strategies in Dose Selection for 26-Week Tg.Rash2 Carcinogenicity Studies.

Our experience indicates that extrapolation of doses from the maximum tolerated doses (MTD) derived from 4-week dose range finding (DRF) studies conducted in CByB6F1 may overpredict tolerability and undermine utility of the high-dose groups in 26-week carcinogenicity studies conducted in Tg.rasH2. In the 26-week carcinogenicity studies conducted in Tg.rasH2 mice, we analyzed the initial body weights, food consumption (FC), terminal body weights, body weight gain (BWG), mortality, and tumor incidence for vehicle and test article-treated dose groups for 26 studies conducted from 2014 to 2018. Although not statistically significant compared to the control dose group, the % BWG decreased in male mice of mid- and high-dose groups by >10%, whereas in females there were no differences. The mortality increased in a statistically significant manner for medium and high doses of males. In female mice, the mortality increased in the high-dose group but not in a statistically significant manner. When the cause of death (COD) was analyzed in all dose groups of both sexes, the COD due to tumors was highest in the control groups, whereas it was lowest in high-dose groups of both sexes. At the same time, the COD due to undetermined causes, which is possible indication of test article-induced toxicity, was highest in high-dose groups of both sexes. These findings together indicate that MTD derived from earlier DRF studies was exceeded when applied to 26-week carcinogenicity studies and did not serve any purpose in the outcome of these studies.

In Vivo Mutagenicity Testing of Arylboronic Acids and Esters.

Arylboronic acids and esters (referred to collectively as arylboronic compounds) are commonly used intermediates in the synthesis of pharmaceuticals but pose a challenge for chemical syntheses because they are often positive for bacterial mutagenicity in vitro. As such, arylboronic compounds are then typically controlled to levels that are acceptable for mutagenic impurities, that is, the threshold of toxicological concern (TTC). This study used ICH M7 guidance to design and conduct a testing strategy to investigate the in vivo relevance of the in vitro positive findings of arylboronic compounds. Eight arylboronic compounds representing a variety of chemical scaffolds were tested in Sprague Dawley and/or Wistar rats in the in vivo Pig-a (peripheral blood reticulocytes and mature red blood cells) and/or comet assays (duodenum and/or liver). Five of the eight compounds were also tested in the micronucleus (peripheral blood) assay. The arylboronic compounds tested orally demonstrated high systemic exposure; thus the blood and bone marrow were adequately exposed to test article. One compound was administered intravenously due to formulation stability issues. This investigation showed that arylboronic compounds that were mutagenic in vitro were not found to be mutagenic in the corresponding in vivo assays. Therefore, arylboronic compounds similar to the scaffolds tested in this article may be considered non-mutagenic and managed in accordance with the ICH Q3A/Q3B guidelines. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

A Comparison of Spontaneous Tumors in Tg.rasH2 Mice in 26-week Carcinogenicity Studies Conducted at a Single Test Facility during 2004 to 2012 and 2013 to 2018.

This article presents the historical control data of spontaneous tumors in Tg.rasH2 published in 2013 (2004-2012) and compares and contrasts it to more recent data collected from 2013 to 2018, reporting differences in the average percentage incidences or incidence ranges as well as the incidence of new tumors. In 2013, we published a comprehensive review of spontaneous tumors in Tg.rasH2 mice used in 26-week carcinogenicity studies, which included data from control dose groups from 26 studies and a total of 710 mice per sex. The total database, now including the more recent data, has nearly doubled the number of animals, completing to date a total of 52 studies in males and 51 studies in females for a total of 1,615 male mice and 1,560 female mice, respectively. In this article, we compare the data collected from 2004 to 2012 against the data collected from 2013 to 2018 and the overall tumor incidence change.

The Antitussive Benzonatate Is Not Tumorigenic in Rodent Carcinogenicity Studies.

Benzonatate is a peripheral oral antitussive that dampens the activity of cough stretch receptors. Rodent carcinogenicity studies were performed in Tg.rasH2 mice and Wistar Han rats. Mice were orally gavaged benzonatate at 10, 30, 75, and 100 mg/kg/day for males and 5, 15, and 50 mg/kg/day for females. Rats were gavaged at 10, 30, and 90 mg/kg/day for males and 5, 15, and 50 mg/kg/day for females. Higher doses in males were due to differences in maximum tolerated doses in dose-ranging studies. In both species, benzonatate was not detected in plasma because of rapid ester hydrolysis producing 4-(butylamino) benzoic acid (BBA) and methylated polyethylene glycol polymer. This metabolism was similar in human plasma; therefore, plasma BBA was used to show systemic exposure. Both species had no evidence of a benzonatate-related increase in any neoplasm. A slight increase in nasal cavity exudative inflammation was present in benzonatate-dosed male mice. Retinal atrophy was observed in male rats at ≥30 mg/kg/day, but the incidence was within historical control data range and not related to benzonatate. In conclusion, benzonatate and its 2 major metabolites were not carcinogenic in rodent carcinogenicity studies at BBA exposures of ≥32 and 70 times a 200 mg human benzonatate dose, respectively.

Genotoxicity assessment of the novel antitussive agent Benzonatate and its major metabolite.

Benzonatate (TESSALON®) is a peripherally acting oral antitussive. It undergoes rapid ester hydrolysis producing 4-(butylamino) benzoic acid (BBA) and methylated polyethylene glycol (MPG) metabolites, which are eliminated in urine and feces. The nonclinical and clinical efficacy of Benzonatate has been demonstrated over the last 60 years, but its safety was not fully assessed. In this study, we tested the genotoxicity of Benzonatate and its major metabolite BBA in an in vitro bacterial reverse mutation and in vivo micronucleus assays. A chromosomal aberration assay was also performed on Benzonatate and BBA. In the reverse mutation assay, Benzonatate and BBA doses 1.5-5000 µg/plate ±â€¯S9 metabolic activation were used and the numbers of revertants/plate were compared to various controls. Chromosomal aberration assays with human peripheral blood lymphocytes used Benzonatate and BBA concentrations 25-2000 and 62.5-1930 µg/mL, respectively. A CByB6F1 mouse bone marrow micronucleus assay was performed as part of a 28-day oral toxicology study at up to 250 mg/kg/day. The frequencies of micronuclei in polychromatic erythrocytes in treated groups were compared with the control group. Neither Benzonatate nor BBA induced significant mutagenicity in any of the bacterial strains, with or without metabolic activation. They also did not produce any biologically relevant structural or numerical aberrations in human chromosomes. Benzonatate and its BBA and MPG metabolites rapidly produced from esterase activity did not produce any significant increase in the incidence of micronucleated polychromatic erythrocytes. In conclusion, Benzonatate and its major metabolite BBA were not mutagenic and did not cause numerical or structural chromosome alterations. While the MPG metabolite was not tested, studies on structural analogues indicated it was also unlikely to be genotoxic. This was supported by oral rodent carcinogenicity assays showing no increase in malignancies.

Tg.rasH2 Mice and not CByB6F1 Mice Should Be Used for 28-Day Dose Range Finding Studies Prior to 26-Week Tg.rasH2 Carcinogenicity Studies.

Our recent retrospective analysis of data, collected from 29 Tg.rasH2 mouse carcinogenicity studies, determined how successful the strategy of choosing the high dose for the 26-week studies was based on the estimated maximum tolerated dose (EMTD) derived from earlier 28-day dose range finding (DRF) studies conducted in CByB6F1 mice. Our analysis demonstrated that the high doses applied at EMTD in the 26-week Tg.rasH2 studies failed to detect carcinogenic effects. To investigate why the dose selection process failed in the 26-week carcinogenicity studies, the initial body weights, terminal body weights, body weight gains, food consumption, and mortality from the first 4 weeks of 26-week studies with Tg.rasH2 mice were compared with 28-day DRF studies conducted with CByB6F1 mice. Both the 26-week and the earlier respective 28-day studies were conducted with the exact same vehicle, test article, and similar dose levels. The analysis of our results further emphasizes that the EMTD and subsequent lower doses, determined on the basis of the 28-day studies in CByB6F1 mice, may not be an accurate strategy for selecting appropriate dose levels for the 26-week carcinogenicity studies in Tg.rasH2 mice. Based on the analysis presented in this article, we propose that the Tg.rasH2 mice and not the CByB6F1 mice should be used in future DRF studies. The Tg.rasH2 mice demonstrate more toxicity than the CByB6F1 mice, possibly because of their smaller size compared to CByB6F1 mice. Also, the Tg.rasH2 males appear to be more sensitive than the female Tg.rasH2 mice.

Progression of Serosal Vascular Proliferative Lesions to Hemangiosarcomas in the Uterus of the 26-Week Tg.rasH2 Mice Carcinogenicity Studies.

Tg.rasH2 mice are predisposed to hemangiosarcomas. Following the spleen, the uterus is the second most commonly affected organ in the female mice. Female mice are also predisposed to spontaneous vascular proliferative lesions on the serosal surface of the uterus, in which there is proliferation of normal vessels that are lined by well-differentiated endothelial cells. The hemangiosarcomas and vascular proliferative lesions can occur independently. In our facility, we have recorded a total of 47 uterine hemangiosarcomas in 3,985 female Tg.rasH2 mice assigned to various groups in 38 studies. Of these 47 cases, we have seen 22 (46.8%) cases where there was a clear progression of the serosal uterine vascular proliferative lesion into a hemangiosarcoma. In the remaining 25 (53.2%) cases, the uterine hemangiosarcomas involved myometrium and endometrium, but there was no serosal vascular proliferation. Based on the retrospective analysis of our data, we demonstrate that the vascular proliferative lesions noted on the serosal surfaces can progress to hemangiosarcomas and therefore these vascular proliferative lesions should be considered as preneoplastic lesions.

Nonclinical Safety Profile of Etelcalcetide, a Novel Peptide Calcimimetic for the Treatment of Secondary Hyperparathyroidism.

Etelcalcetide is a novel d-amino acid peptide that functions as an allosteric activator of the calcium-sensing receptor and is being developed as an intravenous calcimimetic for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease on hemodialysis. To support clinical development and marketing authorization, a comprehensive nonclinical safety package was generated. Primary adverse effects included hypocalcemia, tremoring, and convulsions. Other adverse effects were considered sequelae of stress associated with hypocalcemia. Cardiovascular safety evaluations in the dog revealed an anticipated prolongation of the corrected QT interval that was related to reductions in serum calcium. Etelcalcetide did not affect the human ether-a-go-go gene ion channel current. Etelcalcetide was mutagenic in some strains of Salmonella, however, based on the negative results in 2 in vitro and 2 in vivo mammalian genotoxicity assays, including a 28-day Muta mouse study, etelcalcetide is considered nongenotoxic. Further support for a lack of genotoxicity was provided due to the fact that etelcalcetide was not carcinogenic in a 6-month transgenic rasH2 mouse model or a 2-year study in rats. There were no effects on fertility, embryo-fetal development, and prenatal and postnatal development. All of the adverse effects observed in both rat and dog were considered directly or secondarily related to the pharmacologic activity of etelcalcetide and the expected sequelae associated with dose-related reductions in serum calcium due to suppression of parathyroid hormone secretion. These nonclinical data indicate no safety signal of concern for human risk beyond that associated with hypocalcemia and associated QT prolongation.

Renal Tumors in 26-Week Tg.Rash2 Mice Carcinogenicity Studies.

We report renal tubular adenomas and a carcinoma in 26-week Tg.rasH2 mouse carcinogenicity studies, which have not been reported to date either at our facility or in other published data. However, during the year 2014, renal tubular tumors were present in 4 studies conducted at our facility. Because of their morphological similarity to the amphophilic-vacuolar (AV) phenotypic variant of renal tubule tumors noted in Sprague-Dawley and Fischer 344 rats, which are thought to be familial, as well as the genetic homogeneity of Tg.rasH2 mice, we tracked the parents of these mice with tumors in each study. The origin of these tumors could not be traced back to any of the parents or even an animal barrier, and these tumors were not attributed to the vehicle or test article. Although the exact mechanism of tumorigenesis was not known, based on the available information, the development of renal tumors in these mice was considered random and spontaneous.

Comparison of integrated genotoxicity endpoints in rats after acute and subchronic oral doses of 4-nitroquinoline-1-oxide.

During interlaboratory validation trials for the Pig-a gene mutation assay we assessed the genotoxicity of 4-nitroquinoline-1-oxide (4NQO) across endpoints in multiple tissues: induction of Pig-a mutant red blood cells (RBCs) and reticulocytes (RETs); micronucleated RETs (MN RETs); and DNA damage in blood and liver via the alkaline Comet assay (%tail intensity [TI]). In a previous subchronic toxicity study with 28 daily doses, biologically meaningful increases were observed only for Pig-a mutant RBCs/RETs while marginal increases in the frequency of MN RET were observed, and other clastogenic endpoints were negative. Follow up acute studies were performed using the same cumulative doses (0, 35, 70, 105, and 140 mg/kg) administered in a bolus, or split over three equal daily doses, with samples collected up to 1 month after the last dose. Both of the acute dosing regimens produced similar results, in that endpoints were either positive or negative, regardless of 1 or 3 daily doses, but the three consecutive daily dose regimen yielded more potent responses in TI (in liver and blood) and Pig-a mutant frequencies. In these acute studies the same cumulative doses of 4NQO induced positive responses in clastogenic endpoints that were negative or inconclusive using a subchronic study design. Additionally, a positive control group using combination doses of cyclophosphamide and ethyl methanesulfonate was employed to assess assay validity and potentially identify a future positive control treatment for integrated genetic toxicity studies.

Cyclophosphamide and etoposide canine studies demonstrate the cross-species potential of the flow cytometric peripheral blood micronucleated reticulocyte endpoint.

Erythrocyte-based micronucleus tests have traditionally been performed with bone marrow specimens, since, in most preclinical animal models, the spleen can efficiently remove aberrant erythrocytes from the circulation. Even so, evidence is mounting that by examining tens of thousands of young (CD71-positive) circulating reticulocytes for the presence of micronuclei via flow cytometry, a sensitive assay of cytogenetic damage is realized. The work described herein was designed to test this hypothesis further, using an important preclinical toxicology model, the beagle dog. In these experiments, purebred male beagles were treated for five consecutive days with cyclophosphamide (0, 6.25, 12.5 or 25mg/m(2)/day) or for two consecutive days with etoposide (0, 1.56, 6.25 or 12.5mg/m(2)/day). Before treatment, and on each day of administration, blood specimens were collected and processed for flow cytometric scoring of micronucleated reticulocyte (MN-RET) frequency. Twenty-four hours after the final administration, blood MN-RET frequencies were determined via flow cytometry, and frequencies of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) were determined using acridine orange and May-Grunwald Giemsa staining. In the case of cyclophosphamide, elevated blood MN-RET frequencies were observed 2 days after treatment began, and the maximal frequency was achieved 1 day later. Similarly, etoposide-induced blood MN-RET were not evident 1 day after administration began, but a robust effect was apparent 2 days after treatments were initiated. Twenty-four hours after the final administrations, dose-related micronucleus responses were evident for both agents and in both blood and bone marrow compartments. Good overall agreement between MN-RET and MN-PCE frequencies was evidenced by high Spearman's correlation coefficients-0.89 for blood flow cytometry versus bone marrow acridine orange staining and 0.83 for blood flow cytometry versus bone marrow May-Grunwald Giemsa staining. Taken together, these results provide further support for the cross-species utility of flow cytometry-based blood MN-RET measurements.

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.

5-androstenediol stimulates multilineage hematopoiesis in rhesus monkeys with radiation-induced myelosuppression.

Total body ionizing irradiation (TBI) between 2-8 Gy causes the hematopoietic component of the acute radiation syndrome (ARS) in humans. Here we report on an exploratory study with 5-androstenediol (AED) in rhesus monkeys exposed to 4 Gy (60)Co gamma TBI. In this study, the effects of two formulations administered 3-4 h after irradiation were evaluated. After radiation, severe neutropenia (<500 neutrophils/microL), thrombocytopenia (<50,000 platelets/microL), and anemia (hemoglobin <8.0 g/dL) occurred in 6, 6, and 5 of the 6 control animals, respectively. In these 6 control animals, the median time to first day of each defined cytopenia was 8.5, 13, and 20 days and the median time to last occurrence was 22.5, 19.5 and 29.5 days, respectively. All treated groups had a decrease in the duration of severe neutropenia relative to vehicle control. All but one dosing regimen decreased the duration of thrombocytopenia and anemia. Five consecutive days of a 15 mg/kg intramuscular (IM) micro-particle preparation and a once weekly 15 mg/kg subcutaneous (SC) nanoparticle suspension generally provided the greatest radiation protection. AED, as a single agent, promotes multilineage hematopoietic recovery of the bone marrow. These data suggest that it may play an important therapeutic role in the management of acute radiation syndrome.