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The European Society of Toxicologic Pathology organized an expert workshop in May 2018 to address adversity considerations related to thyroid follicular cell hypertrophy and/or hyperplasia (FCHH), which is a common finding in nonclinical toxicity studies that can have important implications for risk assessment of pharmaceuticals, food additives, and environmental chemicals. The broad goal of the workshop was to facilitate better alignment in toxicologic pathology and regulatory sciences on how to determine adversity of FCHH. Key objectives were to describe common mechanisms leading to thyroid FCHH and potential functional consequences; provide working criteria to assess adversity of FCHH in context of associated findings; and describe additional methods and experimental data that may influence adversity determinations. The workshop panel was comprised of representatives from the European Union, Japan, and the United States. Participants shared case examples illustrating issues related to adversity assessments of thyroid changes. Provided here are summary discussions, key case presentations, and panel recommendations. This information should increase consistency in the interpretation of adverse changes in the thyroid based on pathology findings in nonclinical toxicity studies, help integrate new types of biomarker data into the review process, and facilitate a more systematic approach to communicating adversity determinations in toxicology reports.

Lysosomes have a central role in cellular catabolism, trafficking, and processing of foreign particles. Accumulation of endogenous and exogenous materials in lysosomes represents a common finding in nonclinical toxicity studies. Histologically, these accumulations often lack distinctive features indicative of lysosomal or cellular dysfunction, making it difficult to consistently interpret and assign adverse dose levels. To help address this issue, the European Society of Toxicologic Pathology organized a workshop where representative types of lysosomal accumulation induced by pharmaceuticals and environmental chemicals were presented and discussed. The expert working group agreed that the diversity of lysosomal accumulations requires a case-by-case weight-of-evidence approach and outlined several factors to consider in the adversity assessment, including location and type of cell affected, lysosomal contents, severity of the accumulation, and related pathological effects as evidence of cellular or organ dysfunction. Lysosomal accumulations associated with cytotoxicity, inflammation, or fibrosis were generally considered to be adverse, while those found in isolation (without morphologic or functional consequences) were not. Workshop examples highlighted the importance of thoroughly characterizing the biological context of lysosomal effects, including mechanistic data and functional in vitro readouts if available. The information provided here should facilitate greater consistency and transparency in the interpretation of lysosomal effects.

Chromated copper arsenate (CCA) is used as a wood preservative worldwide. Exposure to it may adversely affect human health. Some events have increased human exposure to CCA, including the Great East Japan Earthquake, which generated a large amount of lumber debris from CCA-treated woods. We elucidated the toxicity due to daily exposure to CCA over a 4-week period at doses of 0, 8, 40, and 80 mg/kg/day in Wistar Hannover rats. Chromium (Cr) and arsenic (As), but not copper, were detected in the plasma samples of rats treated with various doses of CCA. Males and females showed sedation, and males had poor body weight gain. The clinical pathologies observed in both sexes included hypochromic and microcytic anemia, hepatic and renal dysfunction, and changes in lipid and glucose levels. Histopathologically, males and females showed forestomach hyperkeratosis, mucosal epithelial hyperplasia in the small intestine, rectal goblet cell hypertrophy, and lipofuscin deposition in the proximal renal tubule. Females showed diffuse hepatocellular hypertrophy with increased 8-hydroxydeoxyguanosine levels. These results indicated that oral administration of CCA mainly affected hematopoietic, gastrointestinal, hepatic, and renal systems owing to the toxic effects of As and/or Cr. Major toxic effects were observed in both sexes receiving 40 and 80 mg/kg/day.

A panel of pathologists (Panel) was formed to evaluate the pathogenesis and human relevance of tumors that developed in the fundic region of rat stomachs in carcinogenicity and mechanistic studies with alachlor and butachlor. The Panel evaluated stomach sections stained with hematoxylin and eosin, neuron-specific enolase, and chromogranin A to determine the presence and relative proportion of enterochromaffin-like (ECL) cells in the tumors and concluded all tumors were derived from ECL cells. Biochemical and pathological data demonstrated the tumor formation involved a nongenotoxic threshold mode of action (MOA) initially characterized by profound atrophy of the glandular fundic mucosa that affected gastric glands, but not surface epithelium. This resulted in a substantial loss of parietal cells and a compensatory mucosal cell proliferation. The loss of parietal cells caused a marked increase in gastric pH (hypochlorhydria), leading to sustained and profound hypergastrinemia. The mucosal atrophy, together with the increased gastrin, stimulated cell growth in one or more ECL cell populations, resulting in neoplasia. ECL cell autocrine and paracrine effects led to dedifferentiation of ECL cell tumors. The Panel concluded the tumors develop via a threshold-dependent nongenotoxic MOA, under conditions not relevant to humans.

The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.

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 when There is no requirement or need to split the processes, or When the nature of cell death cannot be determined with certainty, or When 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.

Preclinical toxicity studies have demonstrated that exposure of laboratory animals to liver enzyme inducers during preclinical safety assessment results in a signature of toxicological changes characterized by an increase in liver weight, hepatocellular hypertrophy, cell proliferation, and, frequently in long-term (life-time) studies, hepatocarcinogenesis. Recent advances over the last decade have revealed that for many xenobiotics, these changes may be induced through a common mechanism of action involving activation of the nuclear hormone receptors CAR, PXR, or PPARα. The generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investigation, have now demonstrated that sensitivity to many of these effects is rodent-specific. These data are consistent with the available epidemiological and empirical human evidence and lend support to the scientific opinion that these changes have little relevance to man. The ESTP therefore convened an international panel of experts to debate the evidence in order to more clearly define for toxicologic pathologists what is considered adverse in the context of hepatocellular hypertrophy. The results of this workshop concluded that hepatomegaly as a consequence of hepatocellular hypertrophy without histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a non-adverse reaction. This conclusion should normally be reached by an integrative weight of evidence approach.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and differential diagnosis for classifying microscopic lesions observed in the hepatobiliary system of laboratory rats and mice, with color microphotographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available for society members electronically on the internet (http://goreni.org). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions of the hepatobiliary system in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Hepatic enzyme induction is generally an adaptive response associated with increases in liver weight, induction of gene expression, and morphological changes in hepatocytes. The additive growth and functional demands that initiated the response to hepatic enzyme induction cover a wide range of stimuli including pregnancy and lactation, hormonal fluctuations, dietary constituents, infections associated with acute-phase proteins, as well as responses to exposure to xenobiotics. Common xenobiotic enzyme inducers trigger pathways involving the constitutive androstane receptor (CAR), the peroxisome proliferator-activated receptor (PPAR), the aryl hydrocarbon receptor (AhR), and the pregnane-X-receptor (PXR). Liver enlargement in response to hepatic enzyme induction is typically associated with hepatocellular hypertrophy and often, transient hepatocyte hyperplasia. The hypertrophy may show a lobular distribution, with the pattern of lobular zonation and severity reflecting species, strain, and sex differences in addition to effects from specific xenobiotics. Toxicity and hepatocarcinogenicity may occur when liver responses exceed adaptive changes or induced enzymes generate toxic metabolites. These undesirable consequences are influenced by the type and dose of xenobiotic and show considerable species differences in susceptibility and severity that need to be understood for assessing the potential effects on human health from similar exposures to specific xenobiotics.

Myopericytoma is a perivascular tumor that has been recently described in humans, but not in laboratory rodents. The authors encountered an intra-abdominal tumor resembling human malignant myopericytoma in a Fischer rat. Grossly, the tumor was found as two brown-colored masses located in the mesentery of rectum. Microscopically, the tumor was composed of oval to spindle-shaped cells, which were arranged in sheets around numerous thin-walled branching vessels and partly showed a concentric perivascular growth pattern. Mitoses were frequently seen, and the tumor cells showed a local invasion. Immunohistochemically, the tumor cells were strongly positive for alpha-smooth muscle actin and weakly positive for vimentin and desmin. Ultrastructurally, the tumor cells had dendritic processes, actin-like thin filaments with dense bodies, basement membranes, hemidesmosomes, and micropinocytotic vesicles. These findings suggest that the most appropriate term for diagnosis of the present case could be a malignant myopericytoma.

The INHAND Proposal (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) has been operational since 2005. A Global Editorial Steering Committee (GESC) manages the overall objectives of the project and the development of harmonized terminology for each organ system is the responsibility of the Organ Working Groups (OWG), drawing upon experts from North America, Europe and Japan.Great progress has been made with 9 systems published to date – Respiratory, Hepatobiliary, Urinary, Central/Peripheral Nervous Systems, Male Reproductive and Mammary, Zymbals, Clitoral and Preputial Glands in Toxicologic Pathology and the Integument and Soft Tissue and Female Reproductive System in the Journal of Toxicologic Pathology as supplements and on a web site – www.goreni.org. INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in rodent toxicity and carcinogenicity studies. The guides provide representative photo-micrographs of morphologic changes, information regarding pathogenesis, and key references. During 2012, INHAND GESC representatives attended meetings with representatives of the FDA Center for Drug Evaluation and Research (CDER), Clinical Data Interchange Standards Consortium (CDISC), and the National Cancer Institute (NCI) Enterprise Vocabulary Services (EVS) to begin incorporation of INHAND terminology as preferred terminology for SEND (Standard for Exchange of Nonclinical Data) submissions to the FDA. The interest in utilizing the INHAND nomenclature, based on input from industry and government toxicologists as well as information technology specialists, suggests that there will be wide acceptance of this nomenclature. The purpose of this publication is to provide an update on the progress of INHAND.

A Sarcoma arising in the abdominal cavity in an aged Fischer-344 rat was studied by immunohistochemistry and electron microscopy. The white-yellow soft mass was located on the lumbosacral vertebrae, compressing adjacent parenchymal organs. The tumor was made up of spindle shaped cells situated in a background of myxoid substance and a small amount of reticulin and collagen fibers. The tumor cells grew in a loose storiform pattern and often adhered to each other by their cell processes to form ovoid or slitlike spaces. Immunohistochemically, the tumor cells were strongly positive for vimentin but negative for keratin, macrophage ED1 antigen, α-smooth muscle actin, Factor VIII-related antigen, and S100 protein. Electron microscopy demonstrated the endothelial differentiation of the tumor cells, such as occasional luminal spaces, a small number of micropinocytotic vesicles, and interdigitating junctions with desmosomes between cell processes of adjacent cells. Furthermore, its endothelial origin was suggested by the presence of electron-dense rods resembling Weibel-Palade bodies. Instead of a definitive basement lamina surrounding the tumor cells, there were extracellular thin "anchoring filaments" that were attached to the cell surface at areas of increased electron density. These findings indicate that the tumor is of lymphatic vessel type rather than blood vessel type.

An epithelioid cell type of spontaneous amelanotic melanoma in the pinna of an aged albino Fischer-344 (F-344) rat is described. When the rat was necropsied at 109 wk of age, the pinnal tumor was recognized as a white spherical mass of 4 mm in diameter. Histologically, the tumor was observed in the dermis and composed of large round or polyhedral epithelioid eosinophilic cells that were arranged in various-sized cell nests. Immunohistochemistry demonstrated that the tumor cells were positive for S-100 protein and vimentin but negative for keratin and a rat macrophage antigen. Ultrastructurally, a few single membrane-bound premelanosomes that were accompanied by many primary lysosome-like bodies containing very fine granules were present in the cytoplasm of the tumor cells. This study has disclosed that, in addition to spindle cell variants of amelanotic melanomas, an epithelioid cell type of amelanotic melanoma may occur spontaneously in the skin of F-344 rats.

Dimethylarsinic acid (DMAV), the major excreted metabolite of inorganic arsenic, is carcinogenic to the rat urinary bladder. Oxidative stress has been proposed as one possible mechanism of DMAV-induced carcinogenesis. The authors determined whether the antioxidant N-acetylcysteine (NAC) modifies DMAV-induced urinary bladder injury in rats. The treatment solutions—DMAV at 10 mg/kg, NAC at 90 or 1.6 mg/kg (high or low dose, respectively), and their combination—were intravesically instilled into female F344 rats over two hours under pentobarbital anesthesia. The treatment was conducted twice with an interval of three days. All animals were euthanized one day after the second treatment. NAC (low dose) alone did not induce histopathological changes or increase 5-bromo-2′-deoxyuridine (BrdU) labeling index in urothelial cells. Both DMAV and NAC (high dose) induced a weak neutrophil infiltration and an increase in the BrdU labeling index; these pathological changes were enhanced by the combined treatment of DMAV and NAC (high or low dose). Increased oxidative stress and urothelial cell hyperplasia with evidence of activated p44/42 MAPK (ERK1/2) and cyclin D1 were found in the DMAV and NAC (high dose) cotreated group. These results suggest that cotreatment with NAC enhanced DMAV-induced urinary bladder injury and that the effects may be mediated by excess oxidative stress and ERK signaling.

Carcinogenic potential of ethylenethiourea (ETU) in combination with sodium nitrite was investigated in ICR mice of both sexes. Groups of 30 males and 30 females each were given 10 weekly oral administrations of ETU and sodium nitrite with the following combinations of dosing (ETU vs sodium nitrite, mg/kg/wk): 0 vs 0, 100 vs 0, 0 vs 70, 25 vs 17.5, 50 vs 35, and 100 vs 70. Thereafter, the animals were allowed to live without treatment up to 18 mo after the first administration. Concurrent administration of ETU and sodium nitrite caused earlier development of tumors and/or dose-dependent increases in the incidences of tumors in the lymphatic tissue, lung, forestomach, Harderian gland, and uterus, whereas treatment with either ETU or sodium nitrite failed to show carcinogenic activity. In addition, carcinomas in the forestomach and uterine horn were limited to mice receiving concurrent administrations of ETU and sodium nitrite. These results indicate that ETU is most probably converted in vivo into N-nitroso ETU and that the N-nitroso ETU has a greater carcinogenic potential in mice than ETU alone.

Time-related changes in potential factors involved in hepatocarcinogenesis by DDT were investigated in a 4-week and a 2-year feeding studies of p, p'-DDT with F344 rats. In the 4-week study with males at doses of 50, 160, and 500 ppm, cell proliferation and gap junctional intercellular communication (GJIC) were examined after 1, 2, 3, 7, 14, and 28 days. Cell proliferation was enhanced within 3 days at any dose level, but returned to normal after 7 days, whereas GJIC was inhibited throughout the study. In the 2-year study with both sexes at doses of 5, 50, and 500 ppm, cell proliferation, GJIC, enzyme induction, and oxidative stress were investigated after 26, 52, 78, and 104 weeks. Males and females showed an inhibition of GJIC and increases in P450 isozymes (CYP2B1 and CYP3A2) in a dose-dependent manner at all time points, but no significant change in cell proliferation. Lipid peroxide for males at 50 and 500 ppm and 8-hydroxydeoxyguanosine for both sexes at 500 ppm were elevated throughout the study. Histologically, eosinophilic foci and hepatocellular adenomas increased in males at 50 ppm and both sexes at 500 ppm. Hepatocellular carcinomas also developed in males at 500 ppm. These results indicate that DDT may induce eosinophilic foci as a result of oxidative DNA damage and leads them to neoplasms in combination with its mitogenic activity and inhibitory effect on GJIC. Oxidative stress could be a key factor in hepatocarcinogenesis by DDT.

Quantitative evaluation of altered hepatocellular foci (AHF), followed by stereological analysis was performed on standard hematoxylin and eosin-stained liver sections from control Fischer 344 (F344) rats of both sexes from seven 2-yr carcinogenicity studies conducted by the National Toxicology Program (NTP). Liver samples were collected at 6, 9, 12, 15, 18, and/or 24 months on study. Although AHF had a broad spectrum of morphological features, they could be classified into the following 5 types using previously published criteria: basophilic, eosinophilic, clear, vacuolated, and mixed cell foci. Approximately 50% of the animals had foci at 6 months, and the incidence reached nearly 100% at 15 months in both sexes. The number, size and volume fraction of AHF increased with age in both sexes; these changes were most evident for basophilic and clear cell foci. The number of basophilic foci was significantly greater in females than in males while clear cell foci were more numerous in males. This sex difference was observed at each time point. Mean number of all types of AHF in males and females at 24 months was 547 and 460 per cubic centimeter of liver, respectively. Despite the high incidence of AHF in control rats, the incidence of hepatocellular neoplasms is low. The implication is that most foci do not progress to neoplasia in control F344 rats used in 2-yr studies.

Retrospective characterization of morphological and stereological features of altered hepatocellular foci (AHF) in hematoxylin & eosin (H&E)-stained sections was performed on 6 conventional 2−yr carcinogenicity studies conducted in Fischer 344 (F344) rats by the National Toxicology Program (NTP). In 3 of these studies where there was clear evidence of hepatocarcinogenicity [1−amino−2,4−dibromoanthraquinone (ADBAQ), C.I. Acid Red 114, methyl carbamate], there was greater morphological variability in AHF than in the studies of chemicals that were not hepatocarcinogenic [4−hydroxyacetanilide, epinephrine, dimethoxane]. In addition to having the expected types of AHF, rats treated with ADBAQ, C.I. Acid Red 114, and methyl carbamate had atypical basophilic AHF. In addition, atypical eosinophilic AHF were present in rats treated with ADBAQ. Both types of atypical AHF showed a morphological spectrum and sequential changes suggesting they could develop into hepatocellular neoplasms. For the 3 liver tumor positive studies, there were dose-and time-dependent increases in stereological parameters for the atypical as well as commonly occurring clear, vacuolated, and mixed cell AHF. Consistent stereological changes were not found for commonly occurring basophilic and eosinophilic AHF. Aside from some decreases in stereological measurements in some rats treated with 4−hydroxyacetanilide and epinephrine, there were no significant quantitative changes in AHF in the three liver tumor negative studies. These results show that hepatocarcinogens may induce unique types of AHF in conventional 2−yr carcinogenicity/toxicity studies in rats and may cause quantitative increases in commonly occurring clear, vacuolated, and mixed cell AHF. Such qualitative and quantitative changes are potentially useful predictors of hepatic neoplasia.

Altered hepatocellular foci (AHF) can be reliably identified in hematoxylin and eosin (H&E)-stained sections of liver from interim and final sacrifice intervals in 2-yr carcinogenicity studies in rats. While most AHF can be categorized on the basis of a defined set of descriptive terms, viz., basophilic, eosinophilic, clear, vacuolated, and mixed foci, exposure to hepatocarcinogenic agents may induce unique types of AHF which should be distinguished from those that occur more commonly. It is proposed that unique treatment-associated AHF be classified as atypical AHF and that they be completely described in the pathology narrative accompanying the study. Since profound changes in the number and size of AHF have been documented in Fischer 344 rats with mononuclear cell leukemia, it is recommended that liver focus data from leukemic animals be censored in assessing potential effects of treatment on AHF. At the present time, there are insufficient data to allow routine use of AHF in regulatory decision-making in the absence of a liver tumor response. However, such data may form part of weight-of-evidence considerations used by regulatory bodies when accompanied by a concomitant liver tumor response.