Jerry F. Hardisty

Defining a Noncarcinogenic Dose of Recombinant Human Parathyroid Hormone 1–84 in a 2-Year Study in Fischer 344 Rats

The carcinogenic potential of human parathyroid hormone 1–84 (PTH) was assessed by daily subcutaneous injection (0, 10, 50, 150 μg/kg/day) for 2 years in Fischer 344 rats. Histopathological analyses were conducted on the standard set of soft tissues, tissues with macroscopic abnormalities, selected bones, and bones with abnormalities identified radiographically. All PTH doses caused widespread osteosclerosis and significant, dose-dependent increases in femoral and vertebral bone mineral content and density. In the mid- and high-dose groups, proliferative changes in bone increased with dose. Osteosarcoma was the most common change, followed by focal osteoblast hyperplasia, osteoblastoma, osteoma and skeletal fibrosarcoma. The incidence of bone neoplasms was comparable in control and low-dose groups providing a noncarcinogenic dose for PTH of 10 μg/kg/day at a systemic exposure to PTH that is 4.6-fold higher than for a 100 μg dose in humans. The ability of PTH to interact with and balance the effects of both the PTH-1 receptor and the putative C-terminal PTH receptor, may lead to the lower carcinogenic potential observed with PTH than reported previously for teriparatide.

Phenolphthalein Induces Thymic Lymphomas Accompanied by Loss of the p53 Wild Type Allele in Heterozygous p53-Deficient (±) Mice

Epidemiology studies have indicated that many human cancers are influenced by environmental factors. Genetically altered mouse model systems offer us the opportunity to study the interaction of chemicals with genetic predisposition to cancer. Using the heterozygous p53-deficient (±) mouse, an animal model carrying one wild type p53 gene and one p53 null allele, we studied the effects of phenolphthalein on tumor induction and p53 gene alterations. Earlier studies showed that phenolphthalein caused carcinogenic effects in Fisher 344 rats and B6C3F, mice after a 2-yr dosing period (Dunnick and Hailey, Cancer Res. 56: 4922-4926, 1996). The p53 (±) mice received phenolphthalein in the feed at concentrations of 200, 375, 750, 3,000, or 12,000 ppm (approximately 43, 84, 174, 689, or 2,375 mg/kg body weight/day or 129, 252, 522, 2,867, or 7,128 mg/m2 body surface area/day) for up to 6 mo. A target organ cancer site that accumulated p53 protein in the B6C3F, mouse (i.e., thymic lymphoma) was also a target site for cancer in the p53 (±) mouse. In the p53 (±) mouse, treatment-related atypical hyperplasia and malignant lymphoma of thymic origin were seen in the control and dosed groups at a combined incidence of 0, 5, 5, 25, 100, and 95%, respectively. Twenty-one of the thymic lymphomas were examined for p53 gene changes, and all showed loss of the p53 wild type allele. Chemical-induced ovarian tumors in the B6C3F, mouse showed no evidence for p53 protein accumulation and did not occur in the p53 (±) mouse. The p53-deficient (±) mouse model responded to phenolphthalein treatment with a carcinogenic response in the thymus after only 4 mo of dosing. This carcinogenic response took 2 yr to develop in the conventional B6C3F, mouse bioassay. The p53-deficient (±) mouse is an important model for identifying a carcinogenic response after short-term (<6 mo) exposures. Our studies show that exposure to phenolphthalein combined with a genetic predisposition to cancer can potentiate the carcinogenic process and cause p53 gene alterations, a gene alteration found in many human cancers.

Quality Review Procedures Necessary for Rodent Pathology Databases and Toxicogenomic Studies: The National Toxicology Program Experience

Accuracy of the pathology data is crucial since rodent studies often provide critical data used for setting human chemical exposure standards. Diagnoses represent a judgment on the expected biological behavior of a lesion and peer review can improve diagnostic accuracy and consistency. With the conduct of 500 2-year rodent studies, the National Toxicology Program (NTP) has refined its process for comprehensive review of the pathology data and diagnoses. We have found that careful judgment can improve and simplify the review, whereas simply applying a set review procedure may not assure study quality. The use of reviewing pathologists and pathology peer review groups is a very effective procedure to increase study quality with minimal time and cost. New genomic technology to assess differential gene expression is being used to predict morphological phenotypes such as necrosis, hyperplasia, and neoplasia. The challenge for pathologists is to provide uniform pathology phenotypes that can be correlated with the gene expression changes. The lessons learned in assuring data quality in standard rodent studies also applies to the emerging field of toxicogenomics.

Histopathology of nasal olfactory mucosa from selected inhalation toxicity studies conducted with volatile chemicals.

In recent years, histopathologic changes have been reported in the olfactory mucosa of rodents exposed, by inhalation, to a variety of volatile chemicals. In order to better characterize these lesions, a panel of experienced pathologists reviewed microscopic lesions of the olfactory epithelium of rats reported in 10 inhalation studies conducted with 8 different chemicals. The objectives were to determine if the olfactory epithelial lesions are morphologically similar or different for the chemicals of interest, to develop and recommend appropriate diagnostic criteria and nomenclature to characterize the morphology of these olfactory lesions, and to provide specific criteria for judging the degree of severity of the olfactory changes in these studies. The results indicated that the distribution and nature of the lesions were similar in all the examined studies in which olfactory changes were observed. Recommended standardized nomenclature and diagnostic criteria and a uniform method for scoring lesion severity based on the extent of distribution and severity of tissue damage are presented.

Recommendations for Pathology Peer Review

Pathology peer review verifies and improves the accuracy and quality of pathology diagnoses and interpretations. Pathology peer review is recommended when important risk assessment or business decisions are based on nonclinical studies. For pathology peer review conducted before study completion, the peer-review pathologist reviews sufficient slides and pathology data to assist the study pathologist in refining pathology diagnoses and interpretations. Materials to be reviewed are selected by the peer-review pathologist. Consultations with additional experts or a formal (documented) pathology working group may be used to resolve discrepancies. The study pathologist is solely responsible for the content of the final pathology data and report, makes changes resulting from peer-review discussions, initiates the audit trail for microscopic observations after all changes resulting from peer-review have been made, and signs the final pathologist’s report. The peer-review pathologist creates a signed peer-review memo describing the peer-review process and confirming that the study pathologist’s report accurately and appropriately reflects the pathology data. The study pathologist also may sign a statement of consensus. It is not necessary to archive working notes created during the peer-review process.

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.

Comparative hepatic toxicity: Prechronic/chronic liver toxicity in rodents

The morphologic assessment of the gross and microscopic appearance of the liver can provide a broad base of knowledge concerning the potential toxicity of a drug or chemical. This information may either lead to an understanding of the underlying mechanism of toxicity or guide further study to discern the mode of action of the hepatotoxicity. In standard regulatory bioassays, toxicity studies are conducted during phase 1 and phase 2 of the development process to define the acute, subchronic and chronic toxicity of the test compound. In the liver, there are a limited number of morphologic changes that can be identified using conventional light microscopy. These morphologic alterations are often characterized as “adaptive,” consisting of an exaggerated normal physiologic response; “pharmacologic,” consisting of an expected alteration in response to the desired action of the test article; or “adverse,” consisting of morphologic alterations that are generally undesired, progressive and deleterious to the normal function of the cell(s) involved. Morphologic evidence of adverse effects may involve hepatocytes, the biliary system, hepatic vasculature, Kupffer cells, or stellate cells (Ito cells). In drug discovery and development programs, it is necessary to utilize a multidisciplinary approach, using different endpoints, to investigate the same or similar biological responses in the liver. This results in large amounts of data that must be organized in a retrievable fashion. In order for such a multidisciplinary approach to succeed, each discipline must organize and generate their data in a manner that is easily used by others in the process. The toxicologic pathologist must develop and use standardized nomenclature and diagnostic criteria when examining the liver so that data from various investigators can be compared in a useful manner.

Comparative Prevalence, Multiplicity, and Progression of Spontaneous and Vinyl Carbamate-Induced Liver Lesions in Five Strains of Male Mice

The overall and age-specific prevalences and multiplicities of spontaneous and chemically induced hepatocellular neoplasia were compared among male B6D2F1, B6C3F1, C3H (C3H/HeNCrl MTV-), B6CF1, and C57BL/6 (C57BL/6NCrl) mice following a single intraperitoneal injection of 0.03 μM vinyl carbamate (VC)/g body weight or vehicle alone at 15 days of age. Additional groups of B6C3F1, C3H, and C57BL/6 males received 0.15 μM VC/g body weight at 15 days of age. For male B6D2F1, B6C3F1, C3H, B6CF1, and C57BL/6 mice, the estimated overall prevalences (and multiplicities) of hepatocellular adenomas or carcinomas in vehicle controls were 14.1% (0.19), 12.3% (0.15), 8.2% (0.10), 7.2% (0.09), and 2.4% (0.02), respectively. The analogous estimates in the low-dose group were 59.2% (1.19), 72.9% (4.07), 48.6% (1.99), 22.8% (0.29), and 43.9% (0.82). Analogous estimates for B6C3F1, C3H, and C57BL/6 mice in the high-dose group were 45.3% (4.29), 59.7% (6.63), and 46.8% (1.74), respectively. Age-specific multiplicity estimates suggested a progression from altered hepatocellular foci (AHF) to hepatocellular neoplasms. Further evidence of progression was provided by the temporal occurrence of hepatocellular adenomas before carcinomas, and the apparent origination of carcinomas within adenomas. Pulmonary metastases were observed in many of the mice with hepatocellular carcinomas. These findings confirm previous observations of strain differences in liver neoplasm response, suggest a progressive development from AHF to adenomas, and ultimately to carcinomas, and show sensitivity to VC-induced hepatocarcinogenesi s in all 5 strains.

Propylene glycol monomethyl ether (PGME): inhalation toxicity and carcinogenicity in Fischer 344 rats and B6C3F1 mice.

A series of inhalation studies with propylene glycol monomethyl ether (PGME) vapor were undertaken to characterize its subchronic toxicity in mice and chronic toxicity/oncogenicity in rats and mice. Groups of male and female Fischer 344 rats and B6C3F1 mice were exposed to 0,300, 1,000, or 3,000 ppm vapor from 1 week to 2 years. Primary treatment-related effects included: initial sedation of animals exposed to 3,000 ppm and its subsequent resolution correlating with induction of hepatic mixed function oxidase activity and S-phase DNA synthesis; elevated mortality in high-exposure male rats and mice (chronic study); elevated deposition of alpha2U-globulin (α 2U-G) and associated nephropathy and S-phase DNA synthesis in male rat kidneys; accelerated atrophy of the adrenal gland X-zone infemale mice (subchronic study only); and increased occurrence and/or severity of eosinophilic foci of altered hepatocytes in male rats. No toxicologically relevant statistically significant increases in neoplasia occurred in either species. A numerical increase in the incidence of kidney adenomas occurred in intermediate-exposure male rats; however, the association with α 2U-G nephropathy, a male rat specific effect, indicated a lack of relevance for human risk assessment.

A commentary on the process of peer review and pathology data locking.

JENNIFER S. MCKAY, ERIO BARALE-THOMAS, BRAD BOLON, CATHERINE GEORGE, JERRY HARDISTY, SUNAO MANABE, FRÉDÉRIC SCHORSCH, MUNEHIRO TERANISHI, AND KLAUS WEBER AstraZeneca, Macclesfield, SK10 4TG, United Kingdom Johnson and Johnson PRD, 2340 Beerse, Belgium GEMpath Inc., Longmont, CO 80503-2339, USA Ipsen, 91966 Les Ulis Cedex, France Experimental Pathology Laboratories, Research Triangle Park NC 22709, USA Daiichi Sankyo Co., Ltd., Japan Bayer CropScience, 69009 Lyon, France Harlan Laboratories, 4452 Itingen, Switzerland