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.

Spontaneous Cholangiofibrosis in a Wistar Rat.

In a 2-year carcinogenicity study, we identified a spontaneous cholangiofibrosis in a control male Wistar rat. This lesion has long been considered as a compound-related change, with no spontaneous cases reported in the Wistar rat. In addition to routine hematoxylin and eosin stains evaluation, we applied Masson's trichrome staining, Alcian blue-periodic acid-Schiff staining, and OV-6 immunohistochemistry staining. The special staining demonstrated the fibrous component in the interstitium and intestinal metaplasia of the epithelium (presence of goblet cells), while the positive anti-OV-6 reaction indicated the bile duct origin of the epithelium. These results help to confirm the diagnosis of cholangiofibrosis in this case. We report this rare case to alert pathologists that spontaneous cholangiofibrosis does occur in Wistar rats.

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.

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.

Is It Adverse, Nonadverse, Adaptive, or Artifact?

One of the principal challenges facing a toxicologic pathologist is to determine and differentiate a true adverse effect from a nonadverse or an adaptive response. Recent publications from the Society of Toxicologic Pathology (STP) and the European STP provide guidance for determining and communicating adversity in nonclinical toxicology studies. In order to provide a forum to inform and engage in a discussion on this important topic, a continuing education (CE) course was held during the 2016 STP Annual meeting in San Diego, CA. The lectures at this course provided guidance on determining and communicating adversity using case studies involving both clinical pathology and anatomic pathology. In addition, one talk also focused on data quality, study design, and interpretation of artifacts that could hinder the determination of adversity. The CE course ended with a talk on understanding adversity in preclinical studies and engaging the regulatory agencies in the decision-making process. This manuscript is designed to provide brief summaries of all the talks in this well-received CE course.

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.

The role of the pathologist in GLP studies.

This continuing education course presented at the Society of Toxicologic Pathology's 31st Annual Symposium explored and defined the many roles that toxicologic pathologists serve Good Laboratory Practice (GLP)-conducted toxicology and carcinogenicity studies.

Pathology working groups.

Pathology Working Groups (PWG) are a specialized form of review consisting of a panel of expert pathologists who provide an independent, unbiased assessment of specific questions concerning study results. PWGs may concentrate on pivotal studies with controversial end points, address questions that are of concern to a regulatory agency, or compare the results of multiple studies that may have been conducted and evaluated by different laboratories and/or pathologists. The PWG does not review the entire study but always includes a preliminary review by a peer review pathologist. The PWG chairperson needs to thoroughly understand the issue in question, reviews all relevant data and study results, and is responsible for the organization and conduct of the PWG. The members of the PWG examine coded slides without knowledge of treatment group or previous diagnosis and arrive at a consensus diagnosis by majority vote. Once the results are decoded, the PWG evaluates the results and provides discussion and conclusions that are reflected in the final PWG report. Specific examples of PWG issues are provided.

International harmonization of toxicologic pathology nomenclature: an overview and review of basic principles.

The International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice is a global project that is publishing criteria for both proliferative and nonproliferative changes in laboratory animals. This paper presents a set of general suggestions for terminology across systems. These suggestions include the use of diagnostic versus descriptive terms, modifiers, combination terms, and grading systems; and the use of thresholds, synonyms, and terminology for some processes that are common to several organ systems. The purpose of this paper is to help the reader understand some of the basic principles underlying the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice process.

Hematopoietic proliferative lesions in the spleen of rasH2 transgenic mice treated with MNU.

In this study, rasH2-Tg mice treated with N-methyl-N-nitrosurea (MNU) developed exuberant hematoproliferative changes in the spleen that included dysplasia and features of neoplasia. Hematoproliferative change was characterized as exuberant proliferation of hematopoietic cells within the spleen that distorted but did not displace normal splenic morphologic features. The hematopoietic cells were of mixed lineage, but one type, often erythroid, predominated. Cellular atypia was present in all mice with hematoproliferative change, and dysplasia was present in five of eight examined. Hematoproliferative neoplasia was characterized by similar cytologic features but also resulted in displacement/disruption of normal splenic architecture and increased numbers of unidentified blast cells. One case was differentiated toward myeloid proliferation, suggesting granulocytic leukemia. Affected mice had other neoplasms, such as lymphoma and anemia. These proliferative and dysplastic lesions of the spleen in rasH2-Tg mice treated with MNU require additional characterization to definitively differentiate them from the reactive hematopoiesis that can occur in response to inflammatory, neoplastic, or hematopoietic insults in mice.

Histopathology of hemangiosarcomas in mice and hamsters and liposarcomas/fibrosarcomas in rats associated with PPAR agonists.

Peroxisome proliferator-activated receptors (PPAR) are involved in the pathogenesis of insulin resistance, diabetes, and related complications. Consequently, the identification of PPAR subtypes and the potential for their activation provides promising therapeutic targets for the management of type 2 diabetes mellitus. Available data from rodent carcinogenicity studies, however, demonstrate that PPAR agonists can be tumorigenic in one or more species of rodents at multiple sites. In 2005, the Health and Environmental Sciences Institute (HESI) PPAR Agonist Project Committee was established by a group of pharmaceutical companies to advance research on and to understand the modes of action and human relevance of this emerging rodent tumor data for PPAR agonists. Since the most commonly observed tumor types reported in rodents are hemangiosarcomas, fibrosarcomas and liposarcomas, the PPAR Agonist Project Committee approved a Pathology Working Group (PWG) to develop consensus of morphologic criteria for tumor diagnoses and consistency of diagnoses across multiple studies for hemangiosarcomas in mice and hamsters and liposarcomas/fibrosarcomas in rats. Therefore, the focus of the PWG review was to establish consistent tumor diagnostic criteria, to assess evidence of potentially preneoplastic changes and to identify distinguishing morphologic differences which may exist between spontaneous changes present in control animals with similar changes from treated animals. Specific diagnostic criteria and nomenclature are recommended for the classification of proliferative vascular lesions which may be present in mice or hamsters and for proliferative mesenchymal changes in rats in studies that are conducted with PPAR agonists.

Acute adverse effects of the indenopyridine CDB-4022 on the ultrastructure of sertoli cells, spermatocytes, and spermatids in rat testes: comparison to the known sertoli cell toxicant Di-n-pentylphthalate (DPP).

Acute effects of CDB-4022 on testicular ultrastructure were determined. Rats were treated orally with vehicle or a maximally effective single dose of CDB-4022 or Di-n-pentylphthalate (DPP). Preserved testes were processed for transmission electron microscopy. Sertoli and germ cells of vehicle-treated rats demonstrated normal morphological characteristics. Disruption of Sertoli cell ultrastructure was apparent in CDB-4022-treated rats by 3 hours. A decrease in the presence of nucleoli, an increase in the amount and diameter of swollen smooth endoplasmic reticulum, and decreases in cytoplasmic ground substance were observed. The severity of these degenerative effects increased at 6 and 12 hours: Vacuoles were apparent; increased cellular debris, swollen mitochondria, and phagocytic structures were observed; and membranes became more disorganized. Similar ultrastructural changes were observed in the Sertoli cells of DPP-treated rats. By 3 hours, spermatocytes and spermatids were adversely affected by CDB-4022 treatment with swelling of the nuclear envelope. The Step 8 spermatids were especially noteworthy; chromatin was more diffuse and rarefied, the nuclear envelopes were incomplete or broken, and the position of the spermatid nucleus within the cell and relative to Sertoli cell cytoplasm was unusual. Fusion of spermatids to form giant cells was observed by 12 hours. CDB-4022 acts acutely on Sertoli cells to induce marked cellular rarefaction and degeneration, but not necrosis. A rapid and direct effect of CDB-4022 on spermatocytes and spermatids was observed. The antispermatogenic activity of CDB-4022 appears to be a consequence of direct effects on Sertoli and germ cells.

Subchronic feeding study of DAS-59122-7 maize grain in Sprague-Dawley rats.

59122 is a transgenic maize line containing event DAS-59122-7 that expresses the corn rootworm (CRW) specific pesticidal Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis (Bt) Berliner strain PS149B1 and the phosphinothricin-N-acetyltransferase (PAT) protein from Streptomyces viridochromogenes for tolerance to the herbicidal ingredient glufosinate-ammonium. For the current study, 59122 maize grain, non-transgenic near-isogenic maize grain (091), and a commercially available non-transgenic reference maize grain (33R77) were grown under conditions simulating commercial farming practices. Adult Sprague-Dawley rats (12/sex/group) were fed diets formulated with 35% maize grain from either 59122, 091, or 33R77, or one of two separate lots of commercially available rodent chow prepared with commercially available corn (35%) in accordance with the standards of Purina Mills Labdiet 5002 for approximately 90 days. All diets possessed similar nutritional and contaminant profiles. The transgenic proteins were detected only in diets prepared with 59122 maize grain and were stable over the course of the study. Compared to control groups, no adverse diet-related differences were observed in rats fed diets formulated with 59122 maize grain with respect to body weight/gain, food consumption/efficiency, clinical signs of toxicity, mortality, ophthalmology, neurobehavioral (FOB and motor activity) assessments, clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis), and pathology (organ weights and gross and microscopic pathology). Results from this study indicate that 59122 maize grain is nutritionally equivalent to and as safe as conventional maize grain.

Evaluation of the carcinogenic potential of clofibrate in the p53+/- mouse.

This study was conducted as part of International Life Sciences Institute (ILSI) program to evaluate the carcinogenic potential of clofibrate, a nongenotoxic, peroxisome proliferator-activated receptor (PPAR) alpha agonist, following oral administration to p53+/- heterozygous mice for a minimum of 26 weeks. p-Cresidine, a urinary bladder carcinogen, was given orally at 400 mg/kg/day as a positive control. Initial clofibrate doses were 50, 250, and 400 mg/kg/day for males and 50, 200, and 500 mg/kg/day for females. Due to unexpected mortality during the first week of dosing, clofibrate doses were lowered to 25, 75, and 100 mg/kg/day for males and 25, 75, and 125 mg/kg/day for females. Clinical signs and mortality were greater in p53+/- than wild-type (WT) mice. With the exception of liver weights, no marked differences in any other parameters either between the sexes or between WT and p53+/- mice were noted. Moderate increases in liver weights noted in WT males given 100 mg/kg/day clofibrate were not associated with any microscopic changes. No neoplastic response was observed in p53+/- mice after 6 months of exposure to clofibrate at doses up to 100 mg/kg/day for males and 125 mg/kg/day for females. Transitional-cell hyperplasia and carcinoma of the urinary bladder were noted in both sexes given p-cresidine, demonstrating that the p53+/- mouse responded to a known mouse carcinogen as expected. Clofibrate produced non-neoplastic findings in the adrenals, pancreas, and prostate, whereas p-cresidine affected the kidney, liver, pancreas, and spleen.

Confounders in interpreting pathology for safety and risk assessment.

The contribution of pathology to toxicity assessment is invaluable but often not clearly understood. Pathology endpoints are the central response around which human health risk assessment is frequently determined; therefore, it is important that the general toxicology community understand current concepts and nomenclature of toxicologic pathology. Toxicologic pathology encompasses the study of changes in tissue morphology that help define the risk of exposure to xenobiotics. Toxicologic pathology is a discipline that has changed and adapted over time including methods of analysis and nomenclature of lesions. As risk assessments are updated for chemicals in commerce, frequently the older literature must be reviewed and reevaluated. When interpreting pathology data from animal studies, it is important to consider the biological significance of a lesion as well as its relationship to the ultimate adverse health effect. Assessing the potential for a chemical to cause harm to humans must include the examination of the entire pathology database in context of the study design, the mode of action of the chemical of concern, and using the most current interpretation of a lesion to determine the significance for human health effects of a particular tissue response.

Exposure-disease continuum for 2-chloro-2'-deoxyadenosine (2-CdA), a prototype teratogen: induction of lumbar hernia in the rat and species comparison for the teratogenic responses.

BACKGROUND: The purine analog 2-chloro-2'-deoxyadenosine (2-CdA) caused ocular and limb defects in the mouse and rabbit. The current study examined the teratogenic potential of this drug in the rat and compared the adverse developmental outcomes with the other species. METHODS: Timed-pregnant Sprague-Dawley rats were given a single intraperitoneal injection of various doses of 2-CdA ranging from 5-60 mg/kg, at gestational day (GD) 9.5 and GD 14. 2-CdA concentrations in maternal serum and embryos were measured by HPLC and termed fetuses were prepared for teratological examination. RESULTS: Full-litter resorption was seen in dams receiving 50 mg/kg of 2-CdA at GD 9.5, whereas post-implantation loss was significantly increased and fetal weights significantly reduced at 40 mg/kg. Gross examination of the surviving fetuses revealed microphthalmia, a shortened body trunk and lumbar hernia, manifested by a soft mass protrusion at the lumbar region on one or both sides of the spine. Incidence of these defects increased in a dose-dependent fashion. Histological examination indicated that the hernia was associated with hypoplasia of the body wall, poorly developed skeletal muscle bundles surrounding the vertebral column in the lumbar region, and an absence of the lateral muscle groups that allowed protrusion of the abdominal viscera. The lumbar hernia was generally accompanied by spina bifida, deformed ribs and a wide spectrum of soft tissue-abnormalities that included kidney, genitourinary and heart defects. At GD 14, exposure to 2-CdA at 60 mg/kg produced oligodactyly in one of six litters. CONCLUSIONS: 2-CdA produced similar ocular defects in the rat and mouse, although the incidence was much lower in the former species. In contrast, the drug-induced lumbar hernia was only seen in the rat. These apparent disparities were not readily explained by species differences in pharmacokinetic parameters. the similarities between the teratological features of 2-CdA-induced lumbar hernia in the rat and the clinical description of lumbocostovertebral syndrome, however, may provide a key to unlock the etiology of this rare birth defect in humans.

Suggested Standard Operating Procedures (SOPs) for the preparation of electron microscopy samples for toxicology/pathology studies in a GLP environment.

We provide a set of Standard Operating Procedures (SOPs) for preparing samples for electron microscopic evaluation that allow storage of samples in the primary fixative for at least 17 years without noticeable degradation, do not compromise the ability to prepare the same samples for standard light microscopic evaluation, and provide tips for orientation of samples that may be necessary for evaluation. Guidelines for proper sample size, buffer composition, and fluid concentrations during processing are given. The impact of these procedures on specimen quality, ability to produce truly comparable samples for drug development studies, and ways to minimize time spent by technicians preparing these samples during necropsies is evaluated. Although many laboratories routinely employ most of these techniques, this compilation will facilitate the simultaneous light and electron microscopic preparation by the pathologist of comparable specimens that can be stored long-term at 4 degrees C in McDowell's and Trump's 4F:1G fixative (4F:1G).