A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 2 of 2).

To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.

A Diagnostic Approach for Rodent Progressive Cardiomyopathy and Like Lesions in Toxicology Studies up to 28 Days in the Sprague Dawley Rat (Part 1 of 2).

Spontaneous rodent progressive cardiomyopathy (PCM) in the Sprague Dawley rat may confound identification and/or interpretation of potential test article (TA)-related cardiotoxicity. Pathologists apply diagnostic term(s) and thresholds for diagnosing and assigning severity grades for PCM and/or PCM-like (PCM/like) lesions consistently within a study, which is necessary to identify and interpret TA-related findings. Due to differences in training and/or experiences, diagnostic terms and thresholds may vary between pathologists. Harmonized terminology and thresholds across studies will generate better historical control data, will likely enhance interpretation of study data, and may further enhance our understanding of the spontaneous change. An assessment of the diagnostic approaches of a group of 37 pathologists identified an approach that is relatively easily applied; and if adopted, it could enhance diagnostic consistency across studies. This approach uses the single "slash" term "necrosis/inflammatory cell infiltrate (NICI)" as the diagnosis for the spectrum of lesions seen in younger rats, uses no threshold for diagnosis (e.g., diagnose all lesions clearly identifiable as PCM/like), and uses aggregate lesion size of approximately ≥45% of the field of view (FOV) using a 10×/22 eyepiece and the 40× objective or approximately ≥100% of the FOV using the 60× objective as the criterion separating minimal from mild severities.

Proceedings of the 2015 National Toxicology Program Satellite Symposium.

The 2015 Annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri" was held in Minneapolis, Minnesota, at the American College of Veterinary Pathologists/American Society for Veterinary Clinical Pathology/Society of Toxicologic Pathology combined meeting. The goal of this symposium is to present and discuss diagnostic pathology challenges or nomenclature issues. Because of the combined meeting, both laboratory and domestic animal cases were presented. This article presents summaries of the speakers' talks, including challenging diagnostic cases or nomenclature issues that were presented, along with select images that were used for audience voting and discussion. Some lesions and topics covered during the symposium included hepatocellular lesions, a proposed harmonized diagnostic approach to rat cardiomyopathy, crop milk in a bird, avian feeding accoutrement, heat exchanger in a tuna, metastasis of a tobacco carcinogen-induced pulmonary carcinoma, neurocytoma in a rat, pituicytoma in a rat, rodent mammary gland whole mounts, dog and rat alveolar macrophage ultrastructure, dog and rat pulmonary phospholipidosis, alveolar macrophage aggregation in a dog, degenerating yeast in a cat liver aspirate, myeloid leukemia in lymph node aspirates from a dog, Trypanosoma cruzi in a dog, solanum toxicity in a cow, bovine astrovirus, malignant microglial tumor, and nomenclature challenges from the Special Senses International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Group.

Recommendations from the INHAND Apoptosis/Necrosis Working Group.

Historically, there has been confusion relating to the diagnostic nomenclature for individual cell death. Toxicologic pathologists have generally used the terms "single cell necrosis" and "apoptosis" interchangeably. Increased research on the mechanisms of cell death in recent years has led to the understanding that apoptosis and necrosis involve different cellular pathways and that these differences can have important implications when considering overall mechanisms of toxicity, and, for these reasons, the separate terms of apoptosis and necrosis should be used whenever differentiation is possible. However, it is also recognized that differentiation of the precise pathway of cell death may not be important, necessary, or possible in routine toxicity studies and so a more general term to indicate cell death is warranted in these situations. Morphological distinction between these two forms of cell death can sometimes be straightforward but can also be challenging. This article provides a brief discussion of the cellular mechanisms and morphological features of apoptosis and necrosis as well as guidance on when the pathologist should use these terms. It provides recommended nomenclature along with diagnostic criteria (in hematoxylin and eosin [H&E]-stained sections) for the most common forms of cell death (apoptosis and necrosis). This document is intended to serve as current guidance for the nomenclature of cell death for the International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Groups and the toxicologic pathology community at large. The specific recommendations are:Use necrosis and apoptosis as separate diagnostic terms.Use modifiers to denote the distribution of necrosis (e.g., necrosis, single cell; necrosis, focal; necrosis, diffuse; etc.).Use the combined term apoptosis/single cell necrosis whenThere is no requirement or need to split the processes, orWhen the nature of cell death cannot be determined with certainty, orWhen both processes are present together. The diagnosis should be based primarily on the morphological features in H&E-stained sections. When needed, additional, special techniques to identify and characterize apoptosis can also be used.

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.

Biliary proliferative lesions in the Sprague-Dawley rat: adverse/non-adverse.

Whether biliary proliferative lesions in nonclinical species are predictive of potential hepatotoxicity in humans depends, at least in part, on the nature and severity of such changes in the nonclinical species. We reviewed published literature (clinical and nonclinical) and experimental data from rat toxicology studies conducted by GlaxoSmithKline and the National Institute of Environmental Health Sciences' National Toxicology Program in an effort to better characterize the relative risk of hepatobiliary effects in humans. Available evidence supports the interpretation that minimal "typical" appearing bile duct hyperplasia limited to the portal triads may be considered non-adverse in the rat and is of little to no concern to humans. The toxicological relevance of mild to moderate "typical" hyperplasia is less certain, and may be considered adverse in the rat and potentially pose a risk for humans, particularly if accompanied by evidence of hepatobiliary injury or functional compromise. In addition, any proliferative lesion that includes atypical or dysplastic epithelial changes, oval cell proliferation, and/or significant extension beyond the portal tracts is considered more ominous and may be considered adverse in the rat.

The role of the peer review pathologist in good laboratory practices studies: a sponsor perspective.

While the study pathologist (SP) signs the pathology report and is ultimately accountable for the data, the sponsor peer review pathologist (SPRP) generally signs a peer review (PR) statement indicating agreement with the overall pathology data and the associated interpretations. Additionally, the SPRP is often the initial contact to field internal and regulatory pathology data queries. Therefore, the SPRP should conduct as comprehensive a pathology PR as necessary to have complete confidence in the data and interpretations. Ideally, the SPRP should be involved at study design, as the study evolves, through completion of the overall study report. The SPRP should prepare as much as possible before the actual PR start date to include a review of all available data that may impact the PR. This review should focus on identifying findings not already identified in the draft pathology report that may need further interrogation. While all discrepancies between the SPRP and SP may be discussed for mutual learning, the emphasis should be on resolving issues that impact study interpretation. The final pathology report should reflect the consensus between the SP and SPRP and appropriately communicate the study findings. This article provides further background and example scenarios to illustrate these PR perspectives.

Effects of Solutol (Kolliphor) and cremophor in polyethylene glycol 400 vehicle formulations in Sprague-Dawley rats and beagle dogs.

When conventional vehicles (eg, methylcellulose and water) impart inadequate physical, chemical, and/or biological properties for proper toxicological assessment of test article formulations, nonconventional vehicles may be considered. Often toxicity data for nonconventional vehicle formulations are limited. Studies were conducted to collect toxicity data from a rodent and a non-rodent species given 2 nonconventional vehicles, Solutol HS15/polyethylene glycol (PEG) 400 and Cremophor RH40/PEG 400, with differing formulations and dose volumes (10 mL/kg for rats; 2 or 5 mL/kg for dogs). In rats, both vehicles caused increase in kidney weights (males only) and decrease in thymic weights (males only) without concurrent microscopic findings; altered urine electrolytes, minimally decreased serum electrolytes (males only), and increased serum total cholesterol (females only) were also present. The Cremophor formulation was also associated with increased serum urea (males only) and urine phosphorus: creatinine. For rats given the Solutol formulation, both genders had decreased urine glucose parameters and males had increased urine volume. In dogs, loose/watery feces and emesis were present given either vehicle, and mucus-cell hyperplasia of the ileum was present given the Solutol formulation. Increased red blood cell mass and decreased urine volume in dogs given 30% Solutol/70% PEG 400 (5 mL/kg/d) were likely due to subclinical dehydration and hemoconcentration. For the Cremophor formulations, dose volume-dependent increased incidence of minimal subepithelial gastric hemorrhage was noted in dogs, and dogs given 5 mL/kg/d showed increased serum urea nitrogen. Overall, regardless of the formulation or dose volume, neither vehicle produced overt toxicity in either species, but the Solutol formulation produced fewer effects in rats. Generally, lower dose volumes minimized the severity and/or incidence of findings.

An analysis of pharmaceutical experience with decades of rat carcinogenicity testing: support for a proposal to modify current regulatory guidelines.

Data collected from 182 marketed and nonmarketed pharmaceuticals demonstrate that there is little value gained in conducting a rat two-year carcinogenicity study for compounds that lack: (1) histopathologic risk factors for rat neoplasia in chronic toxicology studies, (2) evidence of hormonal perturbation, and (3) positive genetic toxicology results. Using a single positive result among these three criteria as a test for outcome in the two-year study, fifty-two of sixty-six rat tumorigens were correctly identified, yielding 79% test sensitivity. When all three criteria were negative, sixty-two of seventy-six pharmaceuticals (82%) were correctly predicted to be rat noncarcinogens. The fourteen rat false negatives had two-year study findings of questionable human relevance. Applying these criteria to eighty-six additional chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome. These analyses support a proposal to refine regulatory criteria for conducting a two-year rat study to be based on assessment of histopathologic findings from a rat six-month study, evidence of hormonal perturbation, genetic toxicology results, and the findings of a six-month transgenic mouse carcinogenicity study. This proposed decision paradigm has the potential to eliminate over 40% of rat two-year testing on new pharmaceuticals without compromise to patient safety.

Proceedings of the 2009 National Toxicology Program Satellite Symposium.

The National Toxicology Program (NTP) Satellite Symposium is a one-day meeting that is held in conjunction with the annual Society of Toxicologic Pathology (STP) meeting. The topic of the 2009 Symposium was "Tumor Pathology and INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Nomenclature." The goal of this article is to provide summaries of each speaker's presentation, including the diagnostic or nomenclature issues that were presented, along with a few select images that were used for voting. The results of the voting process and interesting points of discussion that were raised during the presentation are also provided. A supplemental file with voting choices and voting results for each case presented at the symposium is available at http://tpx.sagepub.com/supplemental.

alpha 2u-globulin nephropathy and renal tumors in national toxicology program studies.

Chemically induced renal neoplasms in male rats, developed coincident with alpha(2u)-globulin nephropathy, are not considered predictive of risk to humans by the International Agency for Research on Cancer (IARC) and the U.S. Environmental Protection Agency. Criteria have been defined to establish the role of alpha(2u)-globulin nephropathy in renal carcinogenesis, based on a proposed mode of action involving sustained tubular cell proliferation resulting from alpha(2u)-induced nephropathy, with consequent development of neoplastic lesions. Recent NTP studies demonstrated inconsistencies with this proposed mechanism, including in some cases, far weaker kidney tumor responses than expected based on the extent of alpha(2u)-globulin nephropathy. NTP studies with decalin, propylene glycol mono-t-butyl ether and Stoddard solvent IIC included extended evaluations of alpha(2u)-related nephropathy, and were thus used in assessing the linkage between key events in 90-day studies with renal tumors in 2-year studies. This review revealed no or at best weak associations of tumor responses with renal alpha(2u)-globulin concentrations, indices of cell turnover, or microscopic evidence of alpha(2u)-associated nephropathy in prechronic studies. While tumor responses corresponded somewhat with a measure of cumulative alpha(2u)-associated nephropathy (linear mineralization of the papilla) at the end of the 2-year studies, the severity of chronic nephropathy was generally in best agreement with the pattern of tumor response. These results suggest that while alpha(2u)-globulin nephropathy may contribute to the renal tumor response, the critical component(s) of the nephropathy most closely associated with the development of tumors could not be clearly identified in this review.

Topical application of representative multifunctional acrylates produced proliferative and inflammatory lesions in F344/N rats and B6C3F1 mice, and squamous cell neoplasms in Tg.AC mice.

Widespread human exposure to multifunctional acrylates is of concern, due to their inherent reactivity and irritating properties. Trimethylolpropane triacrylate (TMPTA) and pentaerythritol triacrylate (PETA) are industrially important representatives of multifunctional acrylates. The current studies characterized the toxicity of 3-month topical administration of technical grade TMPTA and PETA in F344/N rats and B6C3F1 mice, and evaluated the carcinogenic potential of TMPTA and PETA in hemizygous Tg.AC (v-Ha-ras) transgenic mice. Administration of 0.75, 1.5, 3, 6, and 12 mg/kg TMPTA and PETA for 3 months resulted in hyperplastic, degenerative, and necrotic lesions, accompanied by chronic inflammation of the skin, with severities generally increasing with dose. Lesions were slightly more severe in rats, when compared with mice, and illustrate the irritant potential of TMPTA and PETA. A similar dosage regimen was used for the 6-month study with Tg.AC mice. Topical application of TMPTA and PETA to Tg.AC mice showed dose-dependent increases in squamous cell papillomas at the site of application, with decreases in the latency of their appearance in mice receiving 3 mg/kg or greater. Papillomas, the reporter phenotype in Tg.AC mice, were accompanied by a few squamous cell carcinomas, along with hyperplastic and inflammatory lesions. Although chronic inflammation might have contributed to the development of the skin lesions, the dose-related nature of the induction of the skin papillomas in Tg.AC mice by TMPTA and PETA may reflect a potential for carcinogenicity.

Classification of proliferative hepatocellular lesions in harlan sprague-dawley rats chronically exposed to dioxin-like compounds.

Over the years, the most appropriate classification scheme for nodular proliferative lesions of the hepatocyte has been heavily debated. In the most recent guidelines there appears to be a consensus for classifying these lesions as hepatocellular adenoma, hepatocellular carcinoma, or regenerative hyperplasia. Also, large foci of cellular alteration may appear somewhat nodular. Some nodular hepatocellular lesions from a group of 7 studies of dioxin and dioxin-like compounds conducted by the National Toxicology Program did not readily fit into these categories. Some of these lesions had morphologic features consistent with hyperplasia. However, there was not sufficient morphological or biological evidence to conclude that the entire response was regenerative. In other instances, these lesions had some features resembling adenoma, but contained a prominent component of biliary epithelium and/or oval cells. This component does not appear to be well described in the literature, and while its presence suggested a nodule to be nonneoplastic, this is inconclusive. This paper describes the morphology of these lesions, as well as the diagnostic approach taken in this series of studies.

Dose-additive carcinogenicity of a defined mixture of "dioxin-like compounds".

Use of the dioxin toxic equivalency factor (TEF) approach in human risk assessments assumes that the combined effects of dioxin-like compounds in a mixture can be predicted based on a potency-adjusted dose-additive combination of constituents of the mixture. In this study, we evaluated the TEF approach in experimental 2-year rodent cancer bioassays with female Harlan Sprague-Dawley rats receiving 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3 ,4,4 ,5-pentachlorobiphenyl (PCB-126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of the three compounds. Statistically based dose-response modeling indicated that the shape of the dose-response curves for hepatic, lung, and oral mucosal neoplasms was the same in studies of the three individual chemicals and the mixture. In addition, the dose response for the mixture could be predicted from a combination of the potency-adjusted doses of the individual compounds. Finally, we showed that use of the current World Health Organization dioxin TEF values adequately predicted the increased incidence of liver tumors (hepatocellular adenoma and cholangiocarcinoma) induced by exposure to the mixture. These data support the use of the TEF approach for dioxin cancer risk assessments.

Inhalation toxicology and carcinogenesis studies of propylene glycol mono-t-butyl ether in rats and mice.

Propylene glycol mono-t-butyl ether (PGMBE) is used as a solvent in a variety of commercial applications. Male and female F344/N rats and B6C3F(1) mice were exposed to PGMBE by whole-body inhalation for 2 or 14 weeks (0, 75, 150, 300, 600, or 1200 ppm) or 2 years (0, 75, 300, or 1200 ppm); male NBR rats were exposed for 2 weeks. The kidney and the liver were targets of PGMBE toxicity in rats. Renal lesions suggestive of alpha(2u)-globulin nephropathy were observed in male F344/N, in the 2 and 14-week studies, no kidney lesions were seen in NBR rats. In the 2-year study, male rats displayed exposure-related nonneoplastic lesions in the kidney, and may have shown marginal increases in tubular neoplasms. In the liver, the incidences of hepatocellular adenomas occurred with a positive trend in male rats, and may have been related to PGMBE exposure. In mice of both sexes, the major target of PGMBE toxicity was the liver. In the 2-week study, liver weights and in the 14-week study, liver weights and the incidences of centrilobular hypertrophy were increased. In the 2-year study, the incidences of exposure-related hepatocellular adenoma, adenoma or carcinoma combined, and hepatoblastoma occurred with a positive trend, and were significantly increased in 1200 ppm groups. In summary, exposure to PGMBE resulted in nonneoplastic lesions of the kidney characteristic of alpha(2u)-globulin nephropathy, and may have increased renal tubular neoplasms in male rats. Exposure to PGMBE also produced increases in hepatic tumors in male and female mice.

Hepatoblastomas in mice in the US National Toxicology Program (NTP) studies.

Over the last 8 years, a 5-fold increase in the incidence of mice with spontaneous hepatoblastomas and a moderate increase in the incidence of chemically induced hepatoblastomas in B6C3F1 mice occurred in 2-year NTP studies compared to the previous 7 years. There was a positive association between an increased incidence of mice with hepatoblastoma and an increased incidence of mice with hepatocellular tumors in the treated mice. The rate of pulmonary metastases for hepatoblastoma was similar to that of pulmonary metastasis for hepatocellular carcinomas. Although a variety of chemicals caused an increased incidence of mice with hepatoblastoma, there was no apparent association between a specific chemical structure or a biological class of compounds and their capacity to induce hepatoblastomas. Hepatoblastomas frequently arose within hepatocellular carcinomas or adenomas and were induced by the same compounds that induced hepatocellular neoplasms. Therefore, it seems reasonable to combine the incidence of mice with hepatoblastomas and the incidence of mice with hepatocellular carcinomas in hazard identification studies.

Association of adrenal pheochromocytoma and lung pathology in inhalation studies with particulate compounds in the male F344 rat--the National Toxicology Program experience.

Systemic hypoxemia, occurring in space-occupying lung pathologies such as inflammation and neoplasms. reduces the gas exchange area and stimulates catecholamine secretion from the adrenal medulla where chronic endocrine hyperactivity may lead to hyperplasia and neoplasia. We investigated the possible correlation between nonneoplastic chronic pulmonary lesions and adrenal pheochromocytoma in 9 recent, NTP, 2-year particulate inhalation studies in male F344 rats. Re-evaluation for chronic active inflammation, interstitial fibrosis, alveolar epithelial hyperplasia, squamous metaplasia, proteinosis, and histiocytosis revealed significant associations of pheochromocytoma only with the severity of inflammation and fibrosis. Nickel oxide, cobalt sulfate, indium phosphide, talc, and nickel subsulfide studies showed chemical-related incidences of adrenal pheochromocytoma and significant (p < 0.01) associations with inflammation and fibrosis. Gallium arsenide, vanadium pentoxide, molybdenum trioxide, and nickel sulfate hexahydrate studies revealed an increased incidence and/or severity of nonneoplastic lung lesions, but no increased incidence of pheochromocytoma. Although gallium arsenide and molybdenum trioxide showed no dose-related increase in pheochromocvtoma, a significant (p < 0.01) correlation of the latter with the severity of fibrosis and inflammation occurred. In the vanadium pentoxide and nickel sulfate hexahydrate studies, no relationship between nonneoplastic lung lesions and pheochromocytoma was manifested. Our investigation assessed the strength of these various associations and supports the possible roles of 2 chronic pulmonary lesions-fibrosis and inflammation-and hypoxemia in the induction of pheochromocytoma in the F344 male rat.

The effect of chronic progressive nephropathy on the incidence of renal tubule cell neoplasms in control male F344 rats.

Chronic progressive nephropathy (CPN) is the most frequently diagnosed lesion in the rat kidney. It has many components including degeneration and regeneration of renal tubule (RT) epithelium, glomerular lesions and interstitial inflammation and fibrosis. The incidence and severity of CPN is strain, age, and sex dependent and may be altered by a number of factors including exposure to xenobiotics. In National Toxicology Program (NTP) 2-year bioassays, xenobiotic-associated increased severity (exacerbation) of CPN often occurs in association with a marginal increased incidence of renal tubule cell neoplasms (RTCN). The relationship between CPN and RTCN development has not been definitively determined. The present study evaluated the association between severity of CPN and the occurrence of RTCN in control male F344 rats. A slight but statistically significant increase in CPN severity was present in those animals with RTCN compared to aged-matched controls without RTCN. Although these data suggest there is a positive correlation between CPN and RTCN, cause and effect were not determined. This marginal association suggests that the number of RTCNs that may develop secondary to chemically exacerbated nephropathy would be few.