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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.

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.

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.

In humans, hibernoma is a very rare, benign neoplasm of brown adipose tissue (BAT) that typically occurs at subcutaneous locations and is successfully treated by surgical excision. No single cause has been accepted to explain these very rare human tumors. In contrast, spontaneous hibernoma in rats is rare, often malignant, usually occurs in the thoracic or abdominal cavity, and metastases are common. In recent years, there has been an increased incidence of spontaneous hibernomas in rat carcinogenicity studies, but overall the occurrence remains relatively low and highly variable across studies. There have only been four reported examples of pharmaceutical-induced hibernoma in rat carcinogenicity studies. These include phentolamine, an alpha-adrenergic antagonist; varenicline, a nicotine partial agonist; tofacitinib, a Janus kinase (JAK) inhibitor; and hydromorphone, an opiod analgesic. Potential non-genotoxic mechanisms that may contribute to the pathogenesis of BAT activation/proliferation and/or subsequent hibernoma development in rats include: (1) physiological stimuli, (2) sympathetic stimulation, (3) peroxisome proliferator-activated receptor (PPAR) agonism, and/or (4) interference or inhibition of JAK/Signal Transducer and Activator of Transcription (JAK/STAT) signaling. The evaluation of an apparent increase of hibernoma in rats from 2-year carcinogenicity studies of novel pharmaceutical therapeutics and its relevance to human safety risk assessment is complex. One should consider: the genotoxicity of the test article, dose/exposure and safety margins, and pathophysiologic and morphologic differences and similarities of hibernoma between rats and humans. Hibernomas observed to date in carcinogenicity studies of pharmaceutical agents do not appear to be relevant for human risk at therapeutic dosages.

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.

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.

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.

Accurate interpretation of microscopic changes in tissues is critical in hazard identification and risk assessment. To address a possible confounder, the effects of postmortem interval on hepatocyte vacuolation and liver weight were studied in fasted and nonfasted Sprague-Dawley rats. Male and female rats (5/sex/interval) were euthanized with CO2, weighed, and necropsied either immediately or after remaining in the closed CO2 chamber for 5, 10, or 25 minutes after respirations ceased. The liver was removed, weighed, and fixed for light microscopy, immunohistochemistry, and electron microscopy. The liver weight and liver to body weight ratio increased significantly in both male and female rats. Postmortem hepatocellular vacuolation was more prominent in males than in females. Both fasted and nonfasted males were similarly affected, however, fasted females were affected more than nonfasted females at the 25-minute interval. Ultrastructurally, intracytoplasmic vacuoles in hepatocytes and/or endothelial cells contained electron-lucent material that was morphologically similar to plasma in sinusoidal spaces. Results of our study suggest that hepatocyte vacuoles were formed in a postmortem time-dependent manner as a result of plasma influx into the cytoplasm. This change was associated with hepatic sinusoidal congestion and increases in liver weight. Males were more sensitive than females to postmortem hepatocyte vacuolation.

The objective of this study was to characterize the renal toxicity and carcinogenicity of p-nitrobenzoi c acid in F344 rats. Dose levels in 13-week and 2-year studies ranged from 630—10,000 ppm and 1,250—5,000 ppm, respectively. At 13 weeks, renal lesions included minimal to mild hyaline droplet accumulation in male rats and karyomegaly in male and female rats. At 2 years, renal lesions included proximal tubule epithelial cell hyperplasia in male rats and oncocytic hyperplasia in high-dose male and female rats, and a decreased severity of nephropathy in males and females. The hyaline droplets in renal tubular epithelial cells of male rats at 13 weeks were morphologically similar to those described in α2u-globulin nephropathy. Using immunohistochemica l methods, α2u-globulin accumulation was associated with the hyaline droplets. In addition, at 13 weeks, cell proliferation as detected by PCNA immunohistochemistry was signifi cantly increased in males exposed to 5,000 and 10,000 ppm when compared to controls. Cytotoxicity associated with α2u-globulin nephropathy such as single-cell necrosis of the P2 segment epithelium or accumulation of granular casts in the outer medulla did not occur in the 13-week study. In addition, chronic treatment related nephrotoxic lesions attributed to accumulation of α 2u-globulin such as linear foci of mineralization within the renal papilla, hyperplasia of the renal pelvis urothelium and kidney tumors were not observed. Although there was histologic evidence of α2u-globulin accumulation in male rats at 13 weeks, the minimal severity of nephropathy suggests that the degree of cytotoxicity was below the threshold, which would contribute to the development of renal tumors at 2 years.

Several brominated chemicals have been shown to be multisite-multispecies carcinogens in laboratory animals, and in this paper we report that the flame retardant, 2,2-bis(bromomethyl)-1,3-propanediol (BMP) is also a multisite carcinogen in both sexes of Fischer 344 rats and B6C3F, mice. BMP was administered continuously in the diet for up to 2 yr to rats at doses of 0, 2,500, 5,000, or 10,000 ppm and to mice at doses of 0, 312, 625, or 1,250 ppm. Interim groups of rats were examined at 15 mo. An additional recovery group of male rats received the chemical for 3 mo at 20,000 ppm in the feed, and then the control diet for the remainder of the study. Chemical exposure caused neoplasms of the skin, subcutaneous tissue, mammary gland, Zymbal's gland, oral cavity, esophagus, forestomach, small intestine, large intestine, mesothelium, kidney, urinary bladder, lung, thyroid gland, seminal vesicle, hematopoietic system, and pancreas in the male rat; mammary gland, oral cavity, esophagus, and thyroid gland in the female rat; lung, kidney, and Harderian gland in male mice; and subcutaneous tissue, lung, and Harderian gland in the female mouse. The recovery group of male rats presented with the same spectrum of treatment-related neoplasms as in the core study. In this recovery group, BMP (at 20,000 ppm) caused irreversible effects at numerous sites after 90 days of exposure that was not detectable by histologic examination, but without further exposure resulted in carcinogenic responses at 2 yr. BMP is mutagenic in the salmonella test, but it was not determined if the BMP-induced effects that eventually lead to development of neoplasms at multiple sites are the same in both species and in all organ systems affected.

Previous studies from our laboratory found that when boric acid (BA) was administered in the diet to rats, boron levels in bone were approximately fourfold greater than serum levels. The current studies were undertaken to determine if these elevations produced adverse effects on several bone-related measures, including serum electrolyte levels, bone structure, and bone strength. Data from two studies are presented: in the first study, young adult male rats consumed a powdered diet containing 0, 3000, 4500, 6000, or 9000 ppm BA for 9 weeks. Endpoints were serum calcium, phosphorous, potassium, and chloride, as well as blood and bone boron concentrations ([B]) measured weekly during the 9-week exposure period, and at 8, 16, 24, and 32 weeks after the end of exposure. In the second study, the male and female young adult rats diet contained 0, 200, 1000, 3000, or 9000 ppm BA for 12 weeks; endpoints measured weekly were serum levels of calcium, phosphorous, and magnesium, bone [B], and bone structure (humerus) and strength (tibia, femur, and lumbar vertebrae). In treated rats, calcium was reduced in the first study but not the second. Serum phosphorous was reduced in both studies; potassium was unchanged, chloride was increased by 1%, and magnesium was reduced in all BA-exposed groups in the second study, to a maximal 19% reduction. Bone [B] was consistently increased in all treated groups, to concentrations approximately fourfold those of serum. After cessation of exposure, serum and urinary boron concentrations dropped to within control values within a week. However, even 32 weeks after the end of exposure, bone [B] remained threefold greater than controls. Male tibia and femur resistance to bending was unchanged. However, vertebral strength in compression was significantly increased by 5-10% in all dose groups (200 to 9000 ppm). The pattern was substantially similar in females. Only the humerus was examined by light microscopy and was found to be unchanged at any level of BA consumption. These data show that, despite a reduction in some serum electrolyte levels, BA consumption increased vertebral resistance to crush force, without detectably altering the microscopic structure of the humerus or the resistance of femur and tibia to a bending load. This increase in compression resistance occurred at exposure levels substantially below those that were previously reported to be reproductively toxic.

Three drugs that affect the neuroendocrine system (amphetamine, methylphenidate, and codeine) caused decreases in body weights and in the incidence of spontaneously occurring mammary gland neoplasms in the female F344/N rat in 2-year carcinogenicity studies. Using a mathematical model that relates body weight changes to the incidence of mammary gland neoplasms, we find that the decrease in mammary gland tumours seen in female rats cannot be fully explained by body weight decreases relative to control animals. Further, the observed decreases in body weight in treated female rats were not a function of differences in feed consumption between treated and control groups. These pharmaceuticals are thought to affect the biologic system through interaction with membrane receptors. This interaction and/or subsequent cell signaling events may play a role in the observed decrease in spontaneously occurring mammary gland neoplasms in the female rat treated with amphetamine, methylphenidate, or codeine.

Chloroprene (2-chloro-1,3-butadiene) is a high production chemical used almost exclusively in the production of polychloroprene (neoprene) elastomer. Because of its structural similarity to isoprene (2-methyl-1,3-butadiene) and to 1,3-butadiene, a potent trans-species carcinogen, inhalation studies were performed on chloroprene to characterize its toxicological potential and to provide a basis for selecting exposure concentrations for chronic toxicity and carcinogenicity studies. Thirteen-week inhalation toxicology studies were conducted in male and female F344 rats and B6C3F(1) mice at exposure concentrations of 0, 5, 12, 32 or 80 ppm (6 h/day; 5 days/week). A 200 ppm exposure group was also included for rats only, because a previous study showed that this concentration of chloroprene is lethal to mice. In mice, exposure to 80 ppm chloroprene caused a marginal decrease in body weight gain in males and epithelial hyperplasia of the forestomach in males and females. This lesion has been observed in mice exposed to isoprene or 1,3-butadiene. In rats, exposure to 80 ppm chloroprene or higher concentrations caused degeneration and metaplasia of the olfactory epithelium and exposure to 200 ppm caused anemia, hepatocellular necrosis and reduced sperm motility. These lesions have not been observed in rats exposed to isoprene or 1,3-butadiene. The profile of toxic effects of chloroprene is considerably different from that of isoprene or 1,3-butadiene; this may be due to differences in exposure concentrations that were used in toxicology studies of these compounds and /or to the influence of the chlorine substitution on the toxicokinetics of these compounds, on their biotransformation, or on the reactivity of metabolic intermediates with tissue macromolecules.

Incidences of breast cancer change in populations as people migrate from one area of the world to another, suggesting that environmental factors contribute to this disease. There is a continuing effort to identify these environmental factors and the role that exposures to specific chemicals play in this disease. Results from experimental studies show that chemicals identified to cause mammary gland cancer in rodents are frequently mutagenic chemicals, suggesting that genetic damage is an important mechanism for the induction of this cancer. Prevalent classes of chemicals that were identified to cause mammary gland cancer in rodents in studies by the National Toxicology Program include halogenated hydrocarbons, aromatic amino/nitro compounds and epoxide-forming chemicals. Results from these experimental studies will help to elucidate mechanisms and possible causes of breast cancer in humans.

Because of the potential increased use of phosphine (PH3) as a fumigant and the lack of adequate toxicity data, short-term inhalation studies were conducted to characterize the toxicity of PH3 for Fischer 344 (F344) rats and B6C3F1 mice. Male rats and mice were exposed to 0, 1, 5, or 10 ppm PH3 for up to 4 days, and males and females to 0, 1.25, 2.5, or 5 ppm for 2 wk. In the 4-day study, all rats died by the end of the third exposure to 10 ppm, and all mice were euthanized in moribund condition after the fourth exposure to 10 ppm. Clinical pathology data were obtained only for mice, due to early mortality of rats. There were no significant treatment-related effects in hematological indices in mice exposed to 1 or 5 ppm; at 10 ppm there was a moderate anemia, and leukocyte counts were significantly decreased. There were significant biologically relevant increases in serum activity of alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) and in the concentration of urine nitrogen (UN) at ...

Nitrotoluenes are high-production-volume chemicals used in the synthesis of agricultural chemicals and in various dyes. Because of differences in the metabolism of the three isomers and their capabilities to bind to DNA, comparative toxicity studies of o-, m-, and p-nitrotoluene were conducted in F344 rats and B6C3F1 mice. o-, m-, or p-Nitrotoluene was administered in the feed to male and female rats and mice at doses ranging from 625 to 10,000 ppm for 13 weeks. These doses delivered approximately 40 to 700 mg/kg body wt/day for rats and 100 to 1700 mg/kg/day for mice. There were no treatment-related effects on survival in any of the studies. Decreased body weights relative to controls occurred in dosed rats and mice in all studies at the higher dose levels and were most pronounced in rats receiving o-nitrotoluene. Mesotheliomas of the tunica vaginalis were observed in 3 of 10 male rats receiving o-nitrotoluene at 5000 ppm, and mesothelial cell hyperplasia was observed in 2 of 10 male rats receiving o-nitrotoluene at 10,000 ppm. Kidney toxicity was observed in male rats receiving o-, m-, or p-nitrotoluene and included hyaline droplet nephropathy and an associated increase in the renal concentration of alpha 2U-globulin. Evidence of liver toxicity in the male rats receiving o-nitrotoluene included hepatocyte vacuolization, oval cell hyperplasia, and increased serum bile acids, sorbitol dehydrogenase, and alanine aminotransferase. Although there was no histopathologic evidence of hepatic toxicity in male or female rats given the m- or p-isomers or in female rats given the o-isomer, treatment-related hepatic effects were detected in these groups, as measured by an increase in the relative liver weights and by elevations in serum bile acids and liver-specific enzymes. The spleens of treated male and female rats had a mild increase in hematopoiesis, hemosiderin deposition, and/or congestion. These splenic changes were slightly more prominent in rats administered the o- and p-isomers. Administration of o-, m-, or p-nitrotoluene impaired testicular function in the rat, as shown by testicular degeneration and reduction in the density, motility, and number of sperm cells. Administration of each isomer to rats caused increases in the length of the estrus cycle. The only histopathologic evidence for treatment-related toxicity in mice in the 13-week studies occurred in animals receiving the o-nitrotoluene isomer where the chemical caused degeneration and metaplasia of the olfactory epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship of Carcinogenicity and Cellular Proliferation Induced by Mutagenic Noncarcinogens vs Carcinogens. III. Organophosphate Pesticides vs Tris(2,3-dibromopropyl)phosphate. Cunningham, M. L., Elwell, M. R., and Matthews, H. B. (1994). Fundam. Appl. Toxicol. 23, 363-369. Our laboratory has been examining the mechanisms whereby chemicals produce mutagenicity in short-term in vitro assays yet fail to produce carcinogenesis in 2-year rodent bioassays. Previous studies indicated that some mutagenic hepatocarcinogens increased cell proliferation in the target organ, the liver, while other structurally related mutagens that were noncarcinogenic failed to do so. We demonstrate in this report that another mutagenic carcinogen, tris(2,3-dibromopropyl phosphate), increased cell proliferation that was localized in the outer medulla of the kidney. This was also the target site for carcinogenesis in a 2-year bioassay and is another example of the association between chemically induced cell proliferation and carcinogenesis. This study also reports the absence of increased cell proliferation in the liver or kidney after exposure in the diet to the mutagenic organophosphate insecticides dimethoate, dioxathion, and dichlorvos following dietary exposure for 2 weeks at the same dose levels and routes of exposure that did not increase the tumor incidence in either organ in 2-year carcinogenesis assays. The present studies support the tenet that chemically induced cell proliferation may be a necessary prerequisite for chemical carcinogenesis, since in rat liver and kidney there was neither cell proliferation after 2 weeks nor tumor development after 2 years dietary exposure to the mutagenic organophosphate insecticides dimethoate, dioxathion, and dichlorvos.

Our laboratory has been examining the mechanisms whereby chemicals are mutagenic in short-term in-vitro assays yet are not carcinogenic in 2-year rodent bioassays. Previous studies indicated that mutagenic carcinogens increased the amount of cell turnover in the target organ, but that mutagenic noncarcinogens failed to do so. The present study compares the incidence of cell proliferation in specific regions of the kidney, which is the site of carcinogenicity, with cell proliferation induced in a nontarget tissue, the liver, by the mutagenic renal tubular carcinogen tris(2,3-dibromopropyl)phosphate (TRIS). Renal tubular adenocarcinoma induced by TRIS was the only tumor type identified in male F344 rats, and it was localized in the outer medulla. Male F344 rats were fed a diet containing 0, 50, or 100 ppm TRIS for 14 days. These doses were identical to the doses given in the National Toxicology Program cancer bioassay. Replicating cells were labeled with bromodeoxyuridine administered by an osmotic minipump and identified in tissue sections from liver and kidney using immunohistochemical techniques. Examination of liver sections showed no chemically related increases in cell proliferation above control for either dose group. However, in the kidney, TRIS induced significant cell proliferation that was localized in the renal outer medulla region, the target area for carcinogenesis. The labeling index (number of labeled cells/total number of cells counted) in the kidneys of TRIS-exposed rats was increased approximately 4-fold in the outer medulla and was not increased in the cortex or inner medulla. The results of this study suggest an association between the chemically-induced renal cell proliferation and the renal carcinogenicity of TRIS. Images FIGURE 2. FIGURE 2.

A study of the potential effects of microencapsulation on the toxicity of citral was conducted in 14-day continuous feeding studies with both sexes of F344 rats and B6C3F 1 mice. Toxicity by the feeding route was compared with that from bolus doses of the neat chemical in corn oil administered by gavage. Both sexes of rats and mice were given diet containing 0, 0.63, 1.25, 2.5, 5 and 10% citral microcapsules. These feed formulations were equivalent to daily doses of 0, 142, 285, 570, 1140 and 2280 mg citral/kg body weight for rats and 0, 534, 1068, 2137, 4275 and 8550 mg citral/kg body weight for mice. The daily gavage doses were 0, 570, 1140 and 2280 mg citral/kg body weight for both sexes of rats, and 0, 534, 1068 and 2137 mg citral/kg body weight for both sexes of mice. Citral microcapsules administered in the diet did not cause mortality in mice or rats. Toxicity was confined to decreases in body weight at the 10% concentration in mice, at the 5 and 10% concentrations in rats, and decreases in absolute weights of the liver, kidney and spleen at the 10% concentration in rats. The only histopathological change observed was minimal to mild hyperplasia and/or squamous metaplasia of the respiratory epithelium in the anterior portion of the nasal passages of rats fed 5 or 10% citral microcapsules. By contrast, citral gavage caused mortality in five out of five male and female mice at 2137 mg/kg body weight, and in two out of five male mice at 1068 mg/kg body weight. There were dose-related increases in absolute liver weights of male and female mice. Cytoplasmic vacuolization of hepatocytes occurred in all female mice gavaged with 1068 and 2137 mg citral/kg body weight, and in male mice from the 2137 mg/kg dose group. Necrosis, ulceration and/or acute inflammation of the forestomach occurred in the high-dose mice of both sexes. Inflammation and/or hyperplasia of the forestomach occurred in about half of the male and female mice dosed with 1068 mg citral/kg. Citral gavage at doses that were equivalent to up to 10% in the diet (2280 mg/kg body weight) did not cause toxicity in rats, except for minimal hyperplasia of the squamous epithelium of the forestomach in high-dose males. Microencapsulated citral given in the diet has distinct advantages as an alternative route of administration for long-term studies: the microencapsulation process prevents the normally rapid degradation of the chemical; continual ingestion from the diet mimics the human route of exposure; and higher doses of the chemical can be attained in the animal model to maximize the detection of potential toxic or carcinogenic responses.

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.

Phenolphthalein is a cathartic agent that is widely used in over-the-counter laxatives. Thirteen-week toxicity studies of phenolphthalein were performed using F 344 N rats and B6C3F1 mice. Rats and mice were fed ad libitum with a NIH 07 diet containing 0; 3000; 6000; 12,000; 25,000; or 50,000 ppm phenolphthalein. On a milligram per kilogram body weight basis, rats and mice fed 50,000 ppm phenolphthalein ingested more drug than would be expected during human laxative abuse. Phenolphthalein produced little evidence of toxicity in rats. There was slingtly lower weight gain among the 25,000 and 50,000 ppm groups. Treated rats showed elevated relative kidney weights (males only) and elevated absolute and relative liver weights at 12,000–50,000 ppm phenolphthalein. Rat serum bile acids were depressed early (Days 5 and 6) by phenolphthalein treatment. Several treatment-related toxic effects, however, were identified in mice who received more phenolphthalein per unit body weight than rats. Although there were no effects on body weight gain, elevated liver weights were noted in female mice receiving 6000–50,000 ppm phenolphthalein. The primary treatment-related findings that occurred during the mouse studies involved the reproductive and hematopoietic systems. Reproductive changes including depressed testis and right epididymal weights and sperm density, an elevated production of abnormal sperm, and morphologic alterations in seminiferous tubules occurred at all levels of exposure (3000–50,000 ppm). Hematopoietic changes included bone marrow hypoplasia (12,000–50,000 ppm), increased splenic hematopoiesis (males only; 25,000 and 50,000 ppm), and an elevated incidence of micronucleated erythrocytes (6000–50,000 ppm).

Long-term rodent bioassays with chemicals administered at maximum tolerated doses identify noncarcinogens as well as carcinogens. Thirty-one chemicals recently evaluated for carcinogenic potential by the National Toxicology Program provide unique data on the relationships between mutagenicity, toxicity, and carcinogenicity. Twenty-two substances were classified as carcinogens, and nine showed no evidence of carcinogenicity. Although cellular proliferation does play an intrinsic role in neoplastic processes, the responses associated with chronic toxicity in these studies were not always sufficient to induce neoplasia. Regardless of their mutagenic potential, 19 carcinogens induced toxic effects at sites that did not show neoplastic changes; similar toxic lesions were also seen among the mutagenic and nonmutagenic noncarcinogens. Although many nonmutagens induced neoplasia at sites that showed toxic effects, some of the same chemicals also exhibited toxicity at other sites that showed no neoplastic effect. These results suggest that for some chemicals, properties other than mutagenicity or toxicity may be responsible for their carcinogenic potential.

Groups of 10 F344/N rats and B6C3F1 mice of each sex were exposed to cobalt sulfate heptahydrate aerosols of 0, 0.3, 1.0, 3.0, 10, or 30 mg/m3, 6 hr per day, 5 days per week, for 13 weeks. All rats and female mice and all but 2/10 male mice exposed at the top concentration survived to the end of the studies. Polycythemia was observed in exposed rats but not in mice. Sperm motility was decreased in mice exposed at 3 mg/m3 (the lowest concentration evaluated) and at higher concentrations, and increased numbers of abnormal sperm and decreased testis and epididymal weights occurred in mice exposed to 30 mg/m3. Cobalt content in the urine of rats increased with increasing atmospheric cobalt exposure. Primary histopathologic effects were limited to the respiratory tract. Lesions in rats and mice included degeneration of the olfactory epithelium, squamous metaplasia of the respiratory epithelium, and inflammation in the nose; inflammation, necrosis, squamous metaplasia, ulcers (rats), and inflammatory polyps (rats) of the larynx; metaplasia of the trachea (mice); and fibrosis, histiocytic infiltrates, bronchiolar epithelial regeneration, and epithelial hyperplasia in the alveoli of the lung. The most sensitive tissue was the larynx, with squamous metaplasia observed in rats and mice at the lowest exposure concentration of 0.3 mg/m3. Thus, a no-observed-adverse-effect level was not reached in these studies.

Studies were conducted to compare the toxicity of ethylene dichloride (EDC) in F344/N rats, Sprague-Dawley rats, and Osborne-Mendel rats. Ten rats/sex/group were exposed to EDC in drinking-water at 0, 500, 1000, 2000, 4000 and 8000 ppm for 13 wk. The highest concentration was limited by the maximum solubility of EDC in water (about 9000 ppm). In addition, F344/N rats (10/sex/group) were administered EDC in corn oil by gavage to compare toxicity resulting from bolus administration with that of continuous exposure in drinking-water. Gavage doses of EDC were within the range of total daily doses (in mg/kg body weight/day) resulting from exposure in drinking-water. EDC administered by gavage resulted in greater toxicity to F344/N rats than did administration of similar doses in drinking-water. All males receiving 240 and 480 mg/kg body weight and 9/10 females receiving 300 mg/kg body weight by gavage died before the end of the study. Necrosis of the cerebellum was observed in the brains of 3 males receiving 240 mg/kg body weight and 3 females receiving 300 mg/kg body weight. Hyperplasia and inflammation of the forestomach mucosa were observed in 8 male and 3 female rats that died or were killed in moribund condition. EDC caused minimal toxicity to F344/N, Sprague-Dawley and Osborne-Mendel rats at the drinking-water concentrations used in these studies; only female F344/N rats had EDC-related renal lesions. Based on mortality and EDC-related lesions, the no-effect levels for EDC administered by gavage to F344/N rats were 120 mg/kg body weight for males and 150 mg/kg body weight for females.

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 HE 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.

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

itance play important roles in the pathogenesis of this disease. The National Toxicology Program reported that trihalomethanes, which are by-products of water chlorination, induce colorectal cancers in rats (3). Epidemiological studies also suggest an association between consumption of chlorination by-products in drinking water and an increased risk for colorectal cancer in humans (4). Although disinfection of drinking water by chlorination has been a major disease prevention treatment, these findings raise an important public health concern because of the large number of people who consume chlorinated water. In recent years there has been extensive study of the molecular events involved in the development of human colorectal cancer. Consequently, the molecular genetics of colorectal carcinoma are among the best understood of any common human cancer. To foster an exchange of information and ideas on potential interactions of environmental factors and molecular genetic events that may occur in the development of colorectal cancer, a workshop, "Trihalomethanes and Other Environmental Factors That Contribute to Colorectal Cancer," was held at the National Institute of Environmental Health Sciences in Research Triangle Park, North Carolina, 14 September 1993. The objectives of the workshop were 1) to review the current status of research on

Inhalation Toxicology of Decamethylcyc lopentasiloxane (D5) Following a 3-Month Nose-Only Exposure in Fischer 344 Rats. Burns-Naas, L. A., Mast, R. W., Meeks, R. G., Mann, P. C, and Thevenaz, P. (1998). Toxicol. Sci. 43, 230-240. D5 is a low-molecular-weight cyclic siloxane used for industrial and consumer product applications. The objective of the present study was to evaluate the subchronic toxicity of D 5 following a 3-month nose-only inhalation exposure. In addition, animals from both sexes of the control and high dose groups were allowed a 4-week recovery period to observe reversibility, persistence, or delayed occurrence of any potential adverse effects. Male and female Fischer 344 rats were exposed for 6 h/day, 5 days/week for 3 months to target concentrations of 0 (30/sex/group), 26 (20/sex/ group), 46 (20/sex/group ), 86 (20/sex/group), and 224 (30/sex/ group) ppm D5. Recovery groups (0 and 224 ppm) comprised 10 rats/sex/group. Body weights and food consumption were moni- tored at least twice weekly over the course of exposures. Approx- imately 16 h preceding euthanasia, animals were transferred into metabolism caging for urine collection and were fasted. Rats were anesthetized with pentobarbital and euthanized by exsanguina- tion. Blood was collected for hematological and clinical biochem- ical analyses. Selected organ weights were measured and a com- plete set of tissues was taken for histopathological examination. There were several minor changes observed in clinical biochemis- try parameters; the most notable was an increase in gamma glu- tamyl transferase (y-GT) in both sexes at the high dose. In fe- males, this effect was dose-related (46-224 ppm) and did not recover upon cessation of exposure. Additionally, there was an decrease in serum lactate dehydrogenase (LDH) observed in fe- males at 86 and 224 ppm which was not resolved during recovery. There was an increase in absolute and/or relative liver weight in rats of both sexes. Taken together, these data suggest that the female rat is more sensitive to the actions of D 5 on the liver. Exposure-related increases in absolute and relative lung weights were observed in both sexes at terminal necropsy. This observation was not noted in males in the recovery phase, but was still present in females. Finally, histopathological evidence indicated the pri- mary target organ following D5 inhalation exposure is the lung, with an increase in focal macrophage accumulation and intersti- tial inflammation in the lungs of male and female rats exposed to 224 ppm D5. This observation did not appear to resolve at the end of a 1-month period of nonexposure. The incidence of these changes was also slightly increased in rats of both sexes exposed to 86 ppm D5. These data suggest that nose-only D5 vapor inhalation provokes minimal changes in the lung which are similar in inci- dence and severity to spontaneously occurring changes in control animals after nose-only exposures. There were no histopathologi- cal findings noted in the livers which support this organ as a target in this study, despite the observed changes in organ weight and in some serum chemistry parameters, o is>98 Sociay of Toxicology.

Publisher Summary 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.

Copper gallium diselenide (CGS), copper indium diselenide (CIS), and cadmium telluride (CdTe) are novel compounds used in the photovoltaic and semiconductor industries. This study was conducted to characterize the relative toxicities of these compounds and to evaluate the pulmonary absorption and distribution after intratracheal instillation. Female Sprague–Dawley rats were administered a single equimolar dose (70 m m ) of CGS (21 mg/kg), CIS (24 mg/kg), CdTe (17 mg/kg), or saline by intratracheal instillation. Bronchoalveolar lavage fluid (BALF) protein, fibronectin, inflammatory cells, lung hydroxyproline, and tissue distribution were measured 1, 3, 7, 14, and 28 days after instillation. Relative lung weights were significantly increased in CIS- and CdTe-treated rats at most time points. Inflammatory lesions in the lungs consisting of an influx of macrophages, lymphocytes, and PMNs were most severe in CdTe-treated rats, intermediate in CIS-treated rats, and minimal in rats receiving CGS. Hyperplasia of alveolar type 2 cells was present in CIS- and CdTe-treated rats and was greatest in CdTe-treated rats. Pulmonary interstitial fibrosis was observed in CdTe-treated rats at all time points. All three compounds caused marked increases in total BALF cell numbers, with the greatest increase observed in CIS-treated rats. BALF protein, fibronectin, and lung hydroxyproline were significantly increased in all treated animals and were highest in CdTe-treated animals. There was no apparent pulmonary absorption or tissue distribution of CGS. Indium levels increased in extrapulmonary tissues of CIS-treated rats, although Cu and Se levels remained unchanged. CdTe was absorbed from the lung to a greater extent than CGS and CIS. Cd and Te levels decreased in the lung and increased in extrapulmonary tissues. Of these compounds CdTe presents the greatest potential health risk because it causes severe pulmonary inflammation and fibrosis and because it is readily absorbed from the lung may potentially cause extrapulmonary toxicity.

Administration of 500 mg/kg acetaminophen (APAP) to female B6C3F1 mice resulted in well-documented pathophysiological changes in the liver manifested as increased serum concentration of liver enzymes (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and serum sorbitol dehydrogenase), centrilobular congestion, and hepatocellular degeneration and necrosis. The role of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha) and interleukin 1 alpha (IL-1 alpha), on the hepatotoxicity of APAP was examined at 4, 8, 12, and 24 hr following APAP administration. Neutralization of TNF-alpha or IL-1 alpha with specific antibodies partially prevented the hepatotoxic effects of APAP at the 4- and 8-hr time points. In addition, prior administration of anti-TNF-alpha antibodies shortened the recovery time following APAP treatment. While IL-1 receptor antagonist (IL-1ra) had only a modest protective effect against APAP-induced liver damage, as determined by serum enzyme release, IL-1ra had no effect on the degree of hepatic congestion or necrosis at any of the time points examined. On the other hand, administration of antibodies against IL-1ra exacerbated APAP-induced liver toxicity. These results suggest that TNF-alpha and IL-1 alpha play an important role in the degree of damage and recovery that the liver undergoes following APAP intoxication.

A significant treatment-related decrease in the incidence of mononuclear cell leukemia (MCL) was identified in Fischer-344/N rats for 20 chemicals tested in the National Toxicology Program's 2-yr carcinogenicity bioassay. Fourteen of the 20 chemicals caused decreases of MCL in both male and female rats; 6 of the 20 caused a significant decrease only in males and a marginal or no decrease in female rats. Seventeen of the chemicals associated with a decrease in MCL had a free aromatic amine or nitro functional groups that could be metabolized to free amines. With 1 exception, all 14 chemicals causing a decrease in MCL in both sexes produced spleen toxicity in the 13-wk studies. Reduced body weight and decreased survival, related either to toxicity or to an increase in other types of lethal neoplasms, did not contribute to the decreases in MCL observed in chemical exposure groups. Thirteen of the 20 chemicals were positive in Salmonella tests, and 15 were associated with increases in neoplasms at other sites in rats and/or mice, suggesting that different metabolites could be responsible for these varied biological effects.

The relative sensitivities of eight commonly used clinical chemistry end points and histopathology to detect potential toxic effects in liver and kidney were evaluated for a series of 61 13-week rat toxicity studies conducted for the National Toxicology Program. The data consisted of 1-,2- to 3-, and 13 week clinical chemistry measurements and 13-week histopathological assessments of liver and kidney. Except for serum alkaline phosphatase, treatment-related alterations of individual clinical chemistry variables occurred in 20-48% of the studies, depending on the analyte, sampling time, and sex. Liver and kidney lesions were reported for 31% and 41% of the studies respectively. There was an association between treatment-related increases in alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities and histopathological changes in the liver. SDH activity had greater positive and negative predictive values than similar changes in ALT; by week 1 in females and weeks 2-3 in both sexes. SDH predicted morphological hepatic change at study termination with 75% or better accuracy. If increases in activities of both enzymes occurred simultaneously, however, terminal histopathological changes could be predicted, in both sexes, with 75% accuracy by week 1, increasing to 100% by weeks 2-3. There also was an association between treatment-related increases in urea nitrogen (UN) and creatinine (Cre) concentrations and morphological kidney change. Cre concentration had greater positive predictive values than similar changes in UN; by weeks 2-3 in males and week 13 in both sexes. Cre predicted morphological renal change at study termination with 56% or better accuracy. UN concentration was associated and predictive of morphological kidney change only in females at week 13. Depending on time point and sex, serum alkaline phosphatase activity increased in 5-22% of the studies. Increases in total bile acid concentration occurred in 33-48% of the studies. Because both tests are used as markers of cholestasis, this marked discrepancy was unexpected. Treatment-related decreases in alkaline phosphatase activity occurred, however, in 39-56% of the studies; serum alkaline phosphatase may be more useful as an indicator of decreased food intake (decreased activity) than of cholestasis (increased activity). In summary, treatment-related alterations of clinical chemistry and histopathology occurred frequently in this series of toxicity studies in rats. Changes in the chemistry end points also occurred frequently at interim time points, indicating that clinical chemistry evaluations can be useful for detecting potential treatment effects throughout a study. This observation is important, since histopathological evaluations are limited to animal termination and not useful for detecting transient responses or the onset of treatment-related effects.

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.

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.

In a previous 13-week study of o-nitrotoluene, a chemical-related increase in liver weight, hepatocellular vacuolization, and oval cell hyperplasia in male F344 rats was reported. In this study, the occurrence and change in number and size of hepatic foci in male F344 rats fed a diet containing 5000 ppm o-nitrotoluene or a control diet for 13 weeks, 26 weeks, and 13 weeks followed by a 13-week recovery period (26-week stop-exposure) were evaluated. The livers were stained immunohistochemically for placental glutathione S-transferase (PGST), a marker of hepatic preneoplasia, and quantified stereologically using computer-assisted image analysis. Exposure to o-nitrotoluene induced PGST-positive (PGST-positive (PGST+) liver foci in all treatment groups. The 26-week continuous-exposure group produced more PGST+ liver foci (961.4 foci/cm3 versus 445.4 foci/cm3) and greater mean focus volume (4.34 microns3 versus 1.34 microns3) than the 13-week continuous-exposure group. In the 26-week stop-exposure group, there were fewer PGST+ liver foci (181.4 foci/cm3) than observed with continuous exposure at 13 weeks or 26 weeks; however, the mean focal volume in the stop-exposure group at 26 weeks (5.33 microns3) was greater than that at 13 weeks (1.34 microns3) or 26 weeks of continuous exposure (4.34 microns3). These findings demonstrate that (1) PGST+ foci are observed after only 13 weeks of exposure to o-nitrotoluene; (2) the number and size of foci increase with continued exposure for 26 weeks; and (3) although the number of PGST+ foci decreases with time after chemical exposure is discontinued, many PGST+ foci do not regress but increase in size during the recovery of 13 weeks. The persistence and increase in size of these foci, even in the absence of chemical exposure, suggest the potential for a hepatocarcinogenic effect in long-term studies for o-nitrotoluene.

The incidence of all spontaneously occurring histologic lesions was determined for control Fischer-344 (F-344) rats from 90-day (13-wk) prechronic National Toxicology Program (NTP) toxicity studies. A total of 319 rats, represented by control groups of 10 males and 10 females each from dosed feed ( n = 8), inhalation ( n = 4), and gavage ( n = 4) studies were included in the review. All protocol required tissues routinely collected for evaluation were reexamined for potential nonneoplastic and neoplastic lesions. Histopathologic findings in tissues included a spectrum of degenerative and inflammatory lesions. The most common lesions in male rats were nephropathy [145/160 (90.6%)] and cardiomyopathy [125/158 (79.1%)]. These changes were also present in the female rats, however, at much lower incidence rates [nephropathy = 30/157 (19.1%); cardiomyopathy = 36/158 (22.8%)]. Other less frequently occurring lesions included inflammation of the preputial [36/152 (23.7%)] and clitoral [34/155 (21.9%)] glands and inflammation of the liver consisting of either foci of mononuclear inflammatory cells [19/159 (11.9%) in males and 33/159 (20.8%) in females] or focal granulomatous inflammation [1/159 (0.6%) in males and 14/159 (8.8%) in females]. Pancreatic acinar cell atrophy occurred in both males [11/160 (6.9%)] and females [8/159 (5.0%)]. A variety of other less common nonneoplastic lesions were identified in both sexes of rats. Also recorded in this review are histologic changes generally considered to be components of the normal morphology of a particular tissue or organ for the F-344 rat (i.e., extramedullary hematopoiesis and hemosiderin deposition in the spleen, renal mineralization, uterine dilation, etc.). These findings were included and discussed due to potential treatment effects that may result in an increase or decrease in these changes compared to controls. Neoplasms were not observed in rats from the prechronic studies evaluated.

A total of 121 spontaneous amelanotic melanomas of the skin were identified in 70 of 11,171 male and 51 of 10,927 female Fischer-344/N rats in 63 2-yr carcinogenicity studies conducted by the National Cancer Institute's Carcinogenicity Testing Program/National Toxicology Program. Amelanotic melanomas had characteristic anatomical locations and histologic features distinguishable from Schwann cell tumors. Of the 121 tumors, 84, 19, 10, and 8 cases occurred in the pinna, eyelid, scrotum, and perianal region (anus and tail), respectively. Amelanotic melanomas originated from the dermis and consisted of spindle cells arranged in an interlacing fascicular pattern often with a perivascular orientation; epithelioid cells were rarely seen. Only the tumors arising in the pinna metastasized to the lung and/or mandibular lymph nodes. The metastatic rate was 19% (16/84) of the tumors and was clearly increased with an increase in tumor size. Most metastasizing tumors had focal areas consisting of anaplastic spindle cells with an increased number of mitosis. The tumor cells stained positive for S-100 protein but negative for melanin. Ultrastructurally, the tumors were diagnosed as amelanotic melanomas based on the identification of numerous, intracytoplasmic pre-melanosomes without melanin formation in the tumor cells which were not enveloped by pericytoplasmic basal laminae. One localized amelanotic melanoma of the pinna was successfully transplanted to the subcutaneous tissue in the flank of 3 Fischer-344/N rats.

Chloroethane and bromoethane have been shown to cause a marked uterine tumor response in B6C3F1 mice exposed for 2 years. These chemicals are nearly unique in this regard among the nearly 400 chemicals studied by the National Toxicology Program, and the reasons for this carcinogenic activity are unclear. The possible relationship of changes in blood concentrations of sex hormones to this response was evaluated by examining the estrous cycle of mice prior to and during a 21-day exposure to concentrations of the haloethanes which resulted in the tumorigenic response in the 2-year studies. Serum concentrations of estradiol and progesterone were determined at the termination of the exposures and compared to exposure group and stage of the estrous cycle. No consistent patterns of change were found in estrous cyclicity or in blood concentrations of sex hormones. Thus, the findings suggest that early changes in circulating sex hormones are not important contributing factors in the uterine neoplasia caused by these chemicals.

Comparative Carcinogenicity of Polybrominated Biphenyls with or without Perinatal Exposure in Rats and Mice. Chhabra, R. S., Bucher, J. R., Haseman, J. K., Elwell, M. R., Kurtz, P. J., and Carlton, B. D. (1993). Fundam. Appl. Toxicol. 21, 451-460. Chronic toxicity and carcinogenicity studies of a polybrominated biphenyl mixture (PBB) were conducted in F344/N rats and B6C3F1 mice of each sex. The major objective of the study was to determine if exposure to PBB during the perinatal period, in addition to conventional exposure of animals for 2 years, enhances the sensitivity of the bioassay to identify the carcinogenic potential of this chemical. The studies were designed to determine the toxic and carcinogenic effects of dietary PBB in rats and mice receiving (i) perinatal exposure up to 8 weeks of age followed by control diet for 2 years, (ii) exposure for 2 years beginning at the age of 8 weeks, and (iii) combined perinatal/adult exposure to PBB (perinatal exposure to 8 weeks of age followed by adult exposure for 2 years). During the perinatal period, rats were exposed to PBB at close levels ranging from 1 to 10 ppm and adult exposure concentrations ranged from 3 to 30 ppm in the diet. In the mice, the dose levels ranged from 3 to 30 ppm in both perinatal and adult exposure portions of the chronic studies. A total of eight dose groups (including controls) were used with 60 animals in each group. Liver was the major target organ of PBB toxicity. Perinatal exposure alone (through dietary administration of 10 ppm PBB to the dams) had no effect on the incidences of neoplasms in female F344/N rats, but in male rats, perinatal exposure was associated with a marginally increased incidence of hepatocellular adenomas that may have been related to chemical administration. In male and female B6C3F1 mice, perinatal exposure to 30 ppm PBB resulted in significantly increased incidences of hepatocellular neoplasms. In adult-only dietary exposure studies, PBB was carcinogenic in male and female F344/N rats and male and female B6C3F1 mice based on increased incidences of hepatocellular neoplasms. Combined perinatal and adult dietary exposure to PBB confirmed the findings of the adult-only exposures for the increased incidences of hepatocellular neoplasms in rats and mice. In male rats, there were no enhancing effects of combined perinatal and adult exposure. However, perinatal exposure enhanced the susceptibility of female rats receiving adult exposure of 10 or 30 ppm to the induction of liver neoplasms. For male and female rats, a combined analysis of the incidences of leukemia in the adult-only, perinatal-only, and combined perinatal and adult exposure groups revealed an apparent association between increasing incidences of mononuclear cell leukemia and exposure to PBB. In male and female mice, it was not possible to adequately assess the enhancing effects of combined perinatal and adult exposure on hepatocellular tumors, because adult-only exposure to 10 or 30 ppm PBB resulted in high incidences (84-98%) of liver neoplasms. However, with increased perinatal exposure, there were increases in the numbers of mice with hepatocellular carcinomas and in the numbers of mice with multiple hepatocellular adenomas, suggesting that combined perinatal and adult exposure increases PBB-related hepatocellular carcinogenicity relative to adult-only exposure.

The effects of acute poisoning by cupric sulfate in a number of species are well known; however, the effects of chronic low-level ingestion of cupric sulfate are less well characterized. Because exposure of humans to cupric sulfate may occur through drinking water, food, soil, or ambient air, subchronic toxicity studies were conducted in male and female F344/N rats and B6C3F1 mice by the drinking water (2-week exposure) and dosed feed (2- and 13-week exposure) routes. Animals were evaluated for histopathology, clinical pathology, reproductive toxicity, and tissue metal accumulation, and target organs were examined by a variety of special stains and by electron microscopy to characterize the observed lesions. In drinking water, cupric sulfate concentrations of 300 to 100 ppm produced no ill effects, whereas concentrations of 3000 to 30,000 ppm were lethal to rats and mice within 2 weeks. In feed, cupric sulfate concentrations of 4000 to 16,000 ppm caused significant reductions in body weight gain in both species in the 2- and 13-week studies. Hyperplasia and hyperkeratosis of the limiting ridge of the forestomach were present in both species in the 2- and 13-week studies. Rats in the dosed feed studies had a dose-related increase in inflammation in the liver and changes in clinical chemistry parameters which were indicative of hepatocellular damage and cholestasis. Histologic changes in the kidneys of rats consisted of a dose-related increase in the number and size of eosinophilic protein droplets in the epithelial cytoplasm and the lumina of the proximal convoluted tubules. Droplets were larger and more numerous in males than in females. Urinalysis results were suggestive of renal tubular epithelial damage. Iron staining of spleens from treated animals indicated a marked depletion of iron stores in both male and female rats, but not in mice, while hematologic and clinical chemistry alterations in rats in the 13-week study, along with histologic changes in bone in the 2-week dosed feed study, were indicative of a microcytic anemia. Cupric sulfate produced no adverse effects on any of the reproductive parameters measured in rats or mice of either sex. These results indicate that cupric sulfate at high exposure levels is a hepatic and renal toxicant, as well as an inducer of anemia in rodents, with rats more sensitive than mice following subchronic exposure.