This chapter, the introduction to the first edition of this book entitled Pathology of the Fischer Rat, Reference and Atlas, describes the role of the toxicologic pathologist in the evaluation of safety assessment studies. The approach to evaluation of short-term and carcinogenicity studies is discussed along with the approprate use of diagnostic terminology. Comparisons between the role of the diagnostic pathologist and the toxicologic pathologist are discussed.

Inhibitors of Bruton's tyrosine kinase (BTK) are under development as potential therapies for various autoimmune diseases. In repeat-dose toxicity studies, small-molecule BTK inhibitors (BTKi) have been reported to cause a constellation of histologic effects at the pancreatic endocrine-exocrine interface in male rats; however, similar findings were not reported in other species. Since the BTKi-induced pancreatic effect is morphologically similar to well-documented spontaneous changes (predominantly characterized by insular/peri-insular hemorrhage, pigment deposition, chronic inflammation, and fibrosis) that are known to vary by rat strain, we investigated potential strain-dependent differences in the pancreatic effects of a small-molecule BTKi, LY3337641. Following 13 weeks of LY3337641 treatment, Crl:CD(SD) rats were most sensitive, Crl:WI(Han) rats were of intermediate sensitivity, and Hsd:SD rats were least sensitive. These strain differences appear to be related to differences in rate of weight gain across strains and sexes; however, a definitive mechanism was not determined. This study demonstrated that BTKi-induced pancreatic effects were highly dependent on rat strain and correlated with differences in the incidence and severity of the spontaneous background change. When considered with the lack of pancreas effects in nonrat species, these changes in rats are unlikely predictive of similar changes in humans administered a BTK inhibitor.

Standard components of nonclinical toxicity testing for novel pharmaceuticals include clinical and anatomic pathology, as well as separate evaluation of effects on reproduction and development to inform clinical development and labeling. General study designs in regulatory guidances do not specifically mandate use of pathology or reproductive end points across all study types; thus, inclusion and use of these end points are variable. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) formed a Working Group to assess the current guidelines and practices on the use of reproductive, anatomic pathology, and clinical pathology end points in general, reproductive, and developmental toxicology studies. The Working Group constructed a survey sent to pathologists and reproductive toxicologists, and responses from participating organizations were collected through the STP for evaluation by the Working Group. The regulatory context, relevant survey results, and collective experience of the Working Group are discussed and provide the basis of each assessment by study type. Overall, the current practice of including specific end points on a case-by-case basis is considered appropriate. Points to consider are summarized for inclusion of reproductive end points in general toxicity studies and for the informed use of pathology end points in reproductive and developmental toxicity studies.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) project 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 and diagnostic criteria for nonproliferative and proliferative lesions in laboratory animals.The purpose of this publication is to provide a standardized nomenclature and diagnostic criteria for classifying lesions in the digestive system including the salivary glands and the exocrine pancreas of laboratory rats and mice.Most lesions are illustrated by color photomicrographs.The standardized nomenclature, the diagnostic criteria, and the photomicrographs are also available electronically on the Internet (http://www.goreni.org/).Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world.Content includes spontaneous and age related lesions as well as lesions induced by exposure to test items.Relevant infectious and parasitic lesions are included as well.A widely accepted and utilized international harmonization of nomenclature and diagnostic criteria for the digestive system will decrease misunderstandings 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.(

Standard components of nonclinical toxicity testing for novel pharmaceuticals include clinical and anatomic pathology, as well as separate evaluation of effects on reproduction and development to inform clinical development and labeling. General study designs in regulatory guidances do not specifically mandate use of pathology or reproductive end points across all study types; thus, inclusion and use of these end points are variable. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) formed a Working Group to assess the current guidelines and practices on the use of reproductive, anatomic pathology, and clinical pathology end points in general, reproductive, and developmental toxicology studies. The Working Group constructed a survey sent to pathologists and reproductive toxicologists, and responses from participating organizations were collected through the STP for evaluation by the Working Group. The regulatory context, relevant survey results, and collective experience of the Working Group are discussed and provide the basis of each assessment by study type. Overall, the current practice of including specific end points on a case-by-case basis is considered appropriate. Points to consider are summarized for inclusion of reproductive end points in general toxicity studies and for the informed use of pathology end points in reproductive and developmental toxicity studies.

In a two-year carcinogenicity study with administration of high doses of the partial nicotinic agonist varenicline (recently approved for smoking cessation), mediastinal hibernomas occurred in three male rats. To investigate potential mechanisms for partial and full nicotinic agonists to contribute to development of hibernomas, the effects of nicotine on rat brown adipose tissue (BAT) were studied. Male and female rats were administered nicotine at doses of 0, 0.3, and 1 mg/kg subcutaneously for fourteen days. Intrathoracic (mediastinal periaortic and mediastinal perithymic) BAT and interscapular BAT were examined microscopically, and determinations of uncoupling protein-1 (UCP-1) expression and norepinephrine (NE) content were made. Additionally, NE turnover was measured in mediastinal periaortic and perithymic BAT. Nicotine (1 mg/kg) administration resulted in decreased vacuolation only in mediastinal periaortic and mediastinal perithymic BAT of males and elevated UCP-1 in mediastinal periaortic BAT of males and females. Increased NE content occurred only in mediastinal periaortic BAT of males given 0.3 and 1 mg/kg doses, whereas NE turnover was decreased in both males and females given 1 mg/kg. Together, these data demonstrate that nicotine primarily affects mediastinal BAT in male rats, consistent with the gender and location of the hibernomas observed in the two-year carcinogenicity study.

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.

This is the second part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies, covering the respiratory, male and female genital, and the endocrine systems. The article is based on the experience of the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (Bahnemann et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and/or a macro-photograph showing the plane of section as well as a low magnification of the H&E stained slide demonstrating the optimum "end-product". The objectives of this work, as addressed in detail in the first part (Ruehl-Fehlert et al. 2003), are to standardize tissue sampling and trimming, to improve the comparability of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming. dardize tissue sampling and trimming, to improve the comparability of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming.

This chapter provided details related to performance of necropsy examination, tissue collection and fixation. Instructions for standard trimming and blocking of the common protocol-required tissues for microscopic evaluation are also provided. The necropsy examination, including tissue collection and preservation, represents one of the most critical phases for the histopathological evaluation of animals from toxicity and carcinogenicity studies. At the time of necropsy, fluids such as urine can frequently be obtained by using a tuberculin syringe and needle to aspirate the contents of the urinary bladder. There are various standard methods and sites for blood collection from rats during the in-life phase. They all require appropriate anesthesia and have certain limitations, depending on volume requirements and type of analyses to be performed on the sample. In exsanguination, the gauze is used primarily to prevent excessive blood staining and discoloration of surrounding tissues that might otherwise adversely affect the subsequent gross examination and tissue collection. Further, discussion addresses the most common fixatives and some of the basic rules governing good fixation. Most routine fixation is done by immersion. That is to say, the tissues are placed whole or in part into the fixative solution and fixed from the outside in. Perfusion is a much more effective method for rapid fixation. Perfusion may be done for selected organs of interest, or the whole body can be perfused. Normally, whole-body perfusion is done for neurotoxicity studies.

1,2-Dihydro-2,2,4-trimethylquinoline (TMQ) was evaluated in a 2-year study in which groups of 60 male or female F344 rats received 0, 36 or 60 mg kg(-1) (0, 0.022, or 0.037 mg cm(-2)) and groups of 60 male or female B6C3F1 mice received 0, 3.6 or 10 mg kg(-1) (0, 0.00136, 0.00435 mg cm(-2)) in acetone by topical administration. Survival of all treated groups was comparable to survival of controls. Mean body weights of female rats were lower than those of controls throughout the study but mean body weights of male rats and male and female mice were comparable to the mean body weights of controls. No neoplasms of the skin were observed in any group of rats or mice. Acanthosis at the site of application was increased in male and female rats that received 60 or 100 mg kg(-1) and hyperkeratosis was increased in female rats that received 60 mg kg(-1). The incidences of renal tubule adenoma and renal tubule adenoma or carcinoma were increased significantly in the 60 and 100 mg kg(-1) groups of male rats. There were no neoplastic or non-neoplastic lesions in mice associated with exposure to 1,2-dihydro-2,2,4-trimethylquinoline. In a 1-year initiation-promotion study, groups of 30 female SENCAR mice received an initiating dose of 50 mg kg(-1) 1,2-dihydro-2,2,4-trimethylquinoline followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), or an initiating dose of 7,12-dimethylbenzanthracene (DMBA) followed by promotion with 5, 10 or 25 mg kg(-1) 1,2-dihydro-2,2,4-trimethylquinoline. Other groups served as initiator control, promoter control, vehicle control and positive control (DMBA initiation, TPA promotion). In this system, 1,2-dihydro-2,2,4-trimethylquinoline-initiated skin was not promoted by TPA, and DMBA-initiated skin was not promoted by 1,2-dihydro-2,2,4-trimethylquinoline.

Diethanolamine is a high-production chemical used in cosmetics, in cutting fluids, as a dispersing agent for agricultural chemicals, and as an absorbent for acidic gases. Toxicology studies of diethanolamine were conducted in F344/N rats and B6C3F1 mice of both sexes for 2 weeks (5/sex/species/dose) and 13 weeks (10/sex/species/dose) to characterize and compare the effects of oral and dermal exposure. In addition to histopathology, evaluations included clinical pathology, urinalyses, and sperm morphology or vaginal cytology. In vitro genetic toxicity studies included assessments of mutagenicity in Salmonella typhimurium and mouse lymphoma L5178Y cells, analysis of chromosomal aberrations and sister chromatid exchange in Chinese hamster ovary cells, and determination of micronuclei formed in mice during the 13-week dermal exposure study. Groups of rats and mice received drinking water containing diethanolamine at concentrations of up to 10000 ppm during studies of 2 or 13 weeks duration. In the 2-week studies, rats and mice of both sexes received in the were 0, 630, 1250, 5000, and 10000 ppm diethanolamine in the drinking water. In the 13-week studies, rats received 0, 320, 630, 1250, 2500, and 5000 ppm (males) or 0, 160, 320, 630, 1250, and 2500 ppm (females) in drinking water; male and female mice received 0, 630, 1250, 2500, 5000, and 10000 ppm. All female rats in the 2 highest dose groups and 2 males in the 10000 ppm group in the 2-week study died before the end of the study. In the 13-week study, deaths of mice occurred in the 3 highest dose groups; 2 male rats in the top dose group also died. Surviving animals in the higher concentration groups in both studies exhibited depressed weight gains. Rats receiving diethanolamine developed a poorly regenerative, microcytic anemia in both studies. In the 2-week study, dosed male and female rats had increased kidney weights, renal tubular cell necrosis, and decreased renal function; rats in the 13-week study also showed increased incidences or severity of nephropathy, tubular necrosis, and mineralization. Degeneration of the seminiferous tubules of the testis was noted in dosed males in both the 2and 13-week studies, and sperm motility and count were decreased in the 13-week study. Demyelination in the brain (medulla oblongata) and spinal cord was observed in male and female rats in the 13-week study. In mice, dose-dependent increases in liver weight were observed in males and females in the 2-week study; cytologic alteration and 8 DIETHANOLAMINE, NTP TOXICITY REPORT NUMBER 20 necrosis of individual hepatocytes were observed in the highest dose group. In the 13-week drinking water study in mice, nephropathy and tubular necrosis were observed in males, and degeneration of cardiac myocytes, and hepatocellular necrosis were seen in males and females. Cytologic alteration in the submandibular salivary gland was noted in male and female mice. Hepatocyte cytologic alteration also was noted in all dosed groups of mice. In the 2-week dermal studies, groups of rats and mice were administered daily doses of diethanolamine in 95% ethanol, ranging from 160 to 2500 mg/kg for mice, and from 125 to 2000 mg/kg for rats, 5 days per week. In 13-week studies, dermal doses ranged from 32 to 500 mg/kg for rats, and from 80 to 1250 mg/kg for mice. In the 2-week study, early deaths of male rats and male and female mice occurred in the highest dose groups and in female rats in the 2 highest dose groups (1000 and 2000 mg/kg). Body weight gains were reduced in rats and mice in the higher dose groups. Early deaths in the 13-week study were observed in the highest dose groups of rats (500 mg/kg) and mice (1250 mg/kg). Body weight gains were reduced in rats and mice given the higher doses. Rats in the dermal studies exhibited dose-dependent hematologic and renal function changes similar to those observed in rats in the drinking water study. In addition, in the 2-week study, rats exhibited ulcerative skin lesions at the site of application, accompanied by inflammatory cell infiltration, hyperkeratosis, and acanthosis (hyperplasia) of the epidermis. Hyperkeratosis, without ulceration, was observed in some animals. Ulceration at the site of application was observed in male and female mice. Acanthosis, without ulceration or inflammatory cell infiltration, was observed in mice in all lower dose groups. In the 13-week study, skin lesions at the site of application included ulceration and inflammation, hyperkeratosis, and acanthosis. Liver weights were increased in male and female rats, but there were no associated histopathological changes. Other treatment-related effects observed in rats included demyelination in the brain and spinal cord, and nephropathy, renal tubular necrosis, and/or tubular mineralization; mice exhibited cytological alterations in the liver and/or hepatocellular necrosis, renal tubular epithelial necrosis, and cardiac myocyte degeneration. In in vitro genetic toxicity studies, diethanolamine was not mutagenic in Salmonella typhimurium or mouse L5178Y TK+/cells. Diethanolamine did not induce sister-chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells, nor did it induce micronuclei in peripheral blood erythrocytes in mice exposed by topical application for 13 weeks. All in vitro studies were conducted with and without S9 activation. Target organs of diethanolamine toxicity identified in these studies included bone marrow, kidney, brain, spinal cord, testis, and skin in rats, and liver, kidney, heart, salivary gland, and skin in mice. A no-observed-adverse-effect-level (NOAEL) was not achieved for hematological changes or nephropathy in rats (< 160 ppm), or for cytologic alteration of the liver in mice (< 630 ppm) in the drinking water studies. In the dermal studies, a NOAEL was not achieved for hematological changes, nephropathy, or hyperkeratosis of the skin in rats (< 32 mg/kg), or for cytologic alteration of the liver or acanthosis of the skin in mice (< 80 mg/kg). 9 DIETHANOLAMINE, NTP TOXICITY REPORT NUMBER 20

Diffuse hyperplasia, which may occur in prechronic toxicity studies, has been grossly described as goiter (Latin gutter, throat). This is characterized by diffuse, uniform, bilateral enlargement of the thyroid gland; the weight of a hyperplastic gland may be ten times that of the normal thyroid gland. The diffusely hyperplastic thyroid gland is generally a darker red-brown color than the glands of control rats. This color difference has been attributed to increased vascularity and decreased follicular colloid in the hyperplastic gland. In short-term studies the capsular surface of the gland may be smooth but may become lobulated in studies of longer duration. The lobulated appearance has been attributed to the capsular fibrosis that may occur in more severe cases of hyperplasia.

C-cell hyperplasia and adenoma are usually not visible grossly. C-cell carcinoma may appear as a unilateral or bilateral nodule or irregularly shaped enlargement of the thyroid. When incised, C-cell carcinoma is a moderately firm, uniform, white to tan mass that contrasts sharply with the red-brown color of the normal thyroid parenchyma.

Journal Article Relationship of Carcinogenicity and Cellular Proliferation Induced by Mutagenic Noncarcinogens vs Carcinogens: III. Organophosphate Pesticides vs Tris(2,3-dibromopropyl)phosphate Get access MICHAEL L. CUNNINGHAM, MICHAEL L. CUNNINGHAM 2 *Chemistry Branch, National Institute of Environmental Health SciencesResearch Triangle Park, North Carolina 27709 2 To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar MICHAEL R. ELWELL, MICHAEL R. ELWELL †Laboratory of Experimental Pathology, National Institute of Environmental Health SciencesResearch Triangle Park, North Carolina 27709 Search for other works by this author on: Oxford Academic PubMed Google Scholar H. B. MATTHEWS H. B. MATTHEWS *Chemistry Branch, National Institute of Environmental Health SciencesResearch Triangle Park, North Carolina 27709 Search for other works by this author on: Oxford Academic PubMed Google Scholar Toxicological Sciences, Volume 23, Issue 3, October 1994, Pages 363–369, https://doi.org/10.1093/toxsci/23.3.363 Published: 01 October 1994 Article history Received: 13 December 1993 Accepted: 04 April 1994 Published: 01 October 1994

The application of alpha-cyclodextrin (alpha-CD) as an alternative vehicle for water insoluble and volatile chemicals was investigated in toxicity studies of p-chloro-alpha, alpha, alpha-trifluorotoluene (CTFT). Groups of F344 rats and B6C3F1 mice of each sex were administered CTFT (97% pure) by gavage in either corn oil or alpha-CD aqueous formulations daily for 14 consecutive days. The dose levels used were 10 (mice only), 50, 400, and 1000 mg/kg for corn oil vehicle and 10, 50, and 400 mg/kg (maximum achievable dose at gavage volume of 5 ml/kg) for alpha-CD vehicle. With both vehicles CTFT and alpha 2u-globulin were found to accumulate in the male rat kidney after 14 days of exposure and a dose-related toxic nephropathy was observed at dose of 50 mg/kg or higher. The hepatocellular hypertrophy and cytoplasmic vacuolation of the adrenal cortex which appeared in dosed male and female rats were also found to be independent of vehicle. Clinical pathology findings suggested a mild anemia and cholestasis in rats. With both vehicles no tissue bioaccumulation of CTFT was found in male or female mice. Vehicle-independent hepatocellular hypertrophy and cholestasis were also observed in mice at doses of 400 and 1000 mg/kg. In conclusion, the alpha-CD vehicle does not affect the toxic responses of CTFT in both sexes of both species. The results of the studies suggest that alpha-CD may be an appropriate alternative vehicle for toxicity studies.

Thirteen-week toxicity studies of the flame retardant 2,2-bis(bromomethyl)-1,3-propanediol (BMP; dibromoneopentyl glycol; FR-1138; CAS No. 329690-0) were conducted in male and female F344N rats and B6C3F1 mice. The chemical was administered by oral gavage in corn oil 5 days per week for 13 weeks to rats at doses of 0, 50, 100, 200, 400, and 800 mg/kg and to mice at doses of 0, 25, 50, 100, 200, and 400 mg/kg, or in the feed for 13 weeks at concentrations of 0, 1250, 2500, 5000, 10,000, and 20,000 ppm for rats and at 0, 625, 1250, 2500, 5000, and 10,000 ppm for mice. There was a dose-related decrease in body weight gain in rats and mice after chemical administration. Mortality attributed to toxicity of BMP was seen in the gavage study in 210 high-dose (800 mg/kg) male rats and 310 high-dose (400 mg/kg) male mice; no dose-related mortality occurred in the feed study. Minimal degeneration in the renal papilla was seen in male rats at 800 mg/kg in the gavage study and at doses of 5000 ppm or more in the feed study. This was also present in one female rat at the 20,000 ppm dose. In male mice renal papillary necrosis occurred at 400 mg/kg after dosing by the gavage route and at 2500, 5000, and 10,000 ppm in the dosed-feed study. In female mice papillary necrosis occurred only at the 10,000 ppm dose in the feed study. Tubular cell regeneration of the renal cortex was also present in mice at the same dose levels at which the papillary necrosis was observed. Transitional cell hyperplasia of the urinary bladder was seen in male rats at 400 and 800 mg/kg and in both sexes of mice at 200 and 400 mg/kg. Hyperplasia of the urinary bladder was also seen when BMP was administered in the feed at doses of 20,000 ppm to male rats; at doses of 2500, 5000, and 10,000 ppm to male mice; and at doses of 5000 and 10,000 ppm to female mice. The kidney and urinary bladder are target organs when BMP is administered by gavage or the dosed-feed route; mice were more sensitive than rats for the development of kidney and bladder lesions. Male rats and mice were more sensitive than females for the development of renal papillary degeneration or necrosis.