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Congenital abnormalities of the urinary tract are some of the most common human developmental abnormalities. Several genetically engineered mouse models have been developed to mimic these abnormalities and aim to better understand the molecular mechanisms of disease. This atlas has been developed as an aid to pathologists and other biomedical scientists for identification of abnormalities in the developing murine urinary tract by cataloguing normal structures at each stage of development. Hematoxylin and eosin- and immunohistochemical-stained sections are provided, with a focus on E10.5-E18.5, as well as a brief discussion of postnatal events in urinary tract development. A section on abnormalities in the development of the urinary tract is also provided, and molecular mechanisms are presented as supplementary material. Additionally, overviews of the 2 key processes of kidney development, branching morphogenesis and nephrogenesis, are provided to aid in the understanding of the complex organogenesis of the kidney. One of the key findings of this atlas is the histological identification of the ureteric bud at E10.5, as previous literature has provided conflicting reports on the initial point of budding. Furthermore, attention is paid to points where murine development is significantly distinct from human development, namely, in the cessation of nephrogenesis.

In histopathology, the presence of a tissue change that does not represent the tissue's normal appearance can often lead to an incorrect diagnosis and interpretation. These changes are collectively known as "artifacts" resulting from postmortem autolysis, improper fixation, problems with tissue handling or slide preparation procedures. Most tissue artifacts are obvious, yet some artifacts may be subtle, occur in relatively well-fixed tissue, and demand careful observation to avoid confusion with real biological lesions. The kidney often contains artifacts that may be observed throughout all regions of the renal parenchyma. Cortical tubule artifacts present the greatest challenge when discerning an artifact versus an induced lesion following exposure to a xenobiotic. However, confounding artifacts observed at the tip of the renal papilla may also be problematic for the pathologist. An uncommon artifact involving tinctorial alteration and rarefaction affecting the papillary tip of the rat kidney is described here and differentiated from treatment induced lesions of renal papillary necrosis.

Congenital uterine wall cysts arising from paramesonephric (Müllerian) and mesonephric (Wolffian) ducts are typically incidental findings in most species. We used immunohistochemistry to characterize and determine the origin of uterine cysts in Sprague-Dawley (SD) rats from multigeneration studies conducted by the National Toxicology Program. Subserosal uterine cysts were observed in 20 of the 2,400 SD rats evaluated in five studies, and 10 cysts were characterized for this study. Single cysts were unilocular, fluid-filled, and occurred throughout the uterus. Microscopically, all cysts had a well-developed smooth muscle wall, lined by flattened to cuboidal, sometimes ciliated, epithelium that stained intensely positive for cytokeratin 18 and paired box protein 8 (PAX8). Most cyst epithelia displayed weak to moderate positivity for progesterone receptor (PR) and/or estrogen receptor α (ER-α), as well as were negative for GATA binding protein 3 (GATA3). Cyst lumens contained basophilic flocculent material. The cysts appeared to be developmental anomalies arising from paramesonephric tissue based on positive PAX8 and ER-α and/or PR staining. Additionally, 70% of the cysts lacked GATA3 expression. Taken together, the subserosal uterine cysts observed in adult rats in these studies most likely arose from the paramesonephric duct.

Discovery and qualification of novel biomarkers with improved specificity and sensitivity for detection of xenobiotic-induced injuries is an area of active research across multiple sectors. However, the majority of efforts in this arena have used genetically limited rodent stocks that lack variability in xenobiotic responses inherent to genetically heterogeneous human populations. In this study, genetically diverse Diversity Outbred (DO) mice were used as a surrogate for human clinical populations to investigate performance of urinary kidney biomarkers against classical preclinical kidney injury biomarkers (blood urea nitrogen [BUN] and serum creatinine). In this study, cisplatin was used as a paradigm kidney toxicant, with female adult DO mice exposed to a single injection (5 mg/kg) of cisplatin or vehicle and necropsied 72 h post-exposure and 18 h following overnight urine collection. Interindividual variability in kidney toxicity was observed, with DO mice experiencing either no tubule cell necrosis or minimal-mild necrosis. A panel of urinary protein biomarkers and profiled miRNAs were assessed by receiver-operating characteristic curves as to their ability to distinguish non-responder versus responder animals, as defined by histopathological evidence of renal tubule cell necrosis. A surprising outcome of these studies was that BUN was elevated alongside of urinary miRNA and protein biomarkers in animals with grade 2 proximal tubular cell necrosis; but not elevated with grade 1 necrosis. These studies demonstrate a novel approach for using a rodent population to better assess sensitivity of candidate biomarkers, especially for proposed clinical applications. Impact statement Recent studies have indicated that several urinary proteins and miRNA species may be suitable as biomarkers for acute kidney injury. A major focus on biomarker qualification is demonstrating improved specificity and sensitivity over gold standard tests. In this study, a mouse population model, Diversity Outbred mice, was used to demonstrate that neither the urinary protein markers nor the miRNA species assayed in urine could reliably detect low severity kidney injury better than blood urea nitrogen. This study has implications for use of these biomarkers in the clinic, where interindividual heterogeneity is present within patient populations and for which the underlying tissue pathology may not be known.

Nonclinical juvenile animal tests perform a valuable role in determining adverse drug effects during periods of organogenesis and/or functional maturation. Developmental anatomic and functional maturation time points are important to consider between juveniles and adults when regarding different organ toxicities in response to drug administration. The kidney is an example of a major organ that has differences in these time points in comparing juveniles to adults and in contrasting humans to laboratory animal species. Toxicologic pathologists, involved in juvenile studies, need to be aware of these time points which are age-related exposure periods of sensitivity to drug toxicity. Age-related developmental anatomic and functional maturation are factors which can affect the way that a drug is absorbed, distributed, metabolized, and excreted (ADME). Changes to any component of ADME may alter drug toxicity resulting in kidney abnormalities, nephrotoxicity, or maturational disorders. Juvenile animal kidneys may either be less resistant or more resistant to known adult nephrotoxic drug effects. Furthermore, drug toxicity observed in juvenile animal kidneys may not always correspond to similar toxicities in humans. Juvenile animal nonclinical toxicology studies targeting the kidneys have to be carefully planned to attain the maximum knowledge from each study.

In order to harmonize diagnostic terminology, confirm diagnostic criteria, and describe aspects of tumor biology characteristic of different tumor types, a total of 165 cases of mesenchyme-related tumors and nephroblastomas of the rat kidney were reexamined from the National Toxicology Program (NTP) Archives. This survey demonstrated that renal mesenchymal tumor (RMT) was the most common spontaneous nonepithelial tumor in the rat kidney, also occurring more frequently in the NTP studies than nephroblastoma. Renal sarcoma was a distinct but very rare tumor entity, representing a malignant, monomorphous population of densely crowded, fibroblast-like cells, in which, unlike RMT, preexisting tubules did not persist. Nephroblastoma was characterized by early death of affected animals, suggesting an embryonal origin for this tumor type. Male and female rats were equally disposed to developing RMT, but most of the cases of nephroblastoma occurred in female rats and liposarcoma occurred mostly in male rats. This survey confirmed discrete histopathological and biological differences between the mesenchyme-related renal tumor types and between RMT and nephroblastoma. Statistical analysis also demonstrated a lack of any relationship of these renal tumor types to test article administration in the NTP data bank., (© 2016 by The Author(s) 2016.)

We report renal tubular adenomas and a carcinoma in 26-week Tg.rasH2 mouse carcinogenicity studies, which have not been reported to date either at our facility or in other published data. However, during the year 2014, renal tubular tumors were present in 4 studies conducted at our facility. Because of their morphological similarity to the amphophilic-vacuolar (AV) phenotypic variant of renal tubule tumors noted in Sprague-Dawley and Fischer 344 rats, which are thought to be familial, as well as the genetic homogeneity of Tg.rasH2 mice, we tracked the parents of these mice with tumors in each study. The origin of these tumors could not be traced back to any of the parents or even an animal barrier, and these tumors were not attributed to the vehicle or test article. Although the exact mechanism of tumorigenesis was not known, based on the available information, the development of renal tumors in these mice was considered random and spontaneous., (© The Author(s) 2016.)

The upper portion of the rat kidney pelvis has specialized anatomic structures referred to as fornices. Fornices have a role in urine concentration. Spontaneous lesions including mineralization, epithelial hyperplasia, and inflammatory cell infiltration may occur in the area of the fornices. However, little information regarding specific historical control data or the spontaneous development of these findings in male and female fornices is known. Understanding spontaneous age-related lesions in the area of the fornices versus other portions of the kidney pelvis may be relevant in the identification of test article-induced changes. A retrospective study was conducted of male and female Sprague-Dawley rat kidney fornices over several time points to determine the incidence and severity of mineralization, epithelial hyperplasia, and inflammatory cell infiltration. Based on this investigation, these lesions appeared to increase over time and, in general, occurred earlier and with a greater incidence in females. Regarding those chemicals that may result in lesions of the kidney pelvis, it may be important for pathologists to separately diagnose lesions of the fornices from other portions of the kidney pelvis to help differentiate between any spontaneous age-related and induced changes., (© The Author(s) 2016.)

Renal tubule lesions often prove troublesome for toxicologic pathologists because of the diverse nature and interrelated cell types within the kidney and the presence of spontaneous lesions with overlapping morphologies similar to those induced by renal toxicants. Although there are a number of guidance documents available citing straightforward diagnostic criteria of tubule lesions for the pathologist to refer to, most are presented without further advice on the when to or to the why and the why not of diagnosing one lesion over another. Documents presenting diagnostic perspectives and recommendations derived from an author's experience are limited since guidance documents are generally based on descriptive observations. In this Regulatory Forum opinion piece, the authors attempt to dispel confusing renal tubule lesion terminology in laboratory animal species by suggesting histological advice on the recognition and interpretation of these complex entities., (© 2015 by The Author(s).)

The spontaneous incidence of foci of oncocytic proliferation (oncocytic hyperplasia and oncocytoma) was assessed in a histopathological reevaluation of the kidneys of 2,391 male and female Fischer 344 (F344) groups of control rats from long-term carcinogenicity studies (involving 24 chemicals) that had been conducted by the National Toxicology Program. The overall incidence of oncocytic proliferation was 0.3%, with a male preponderance over females at 0.5% (6/1,236) versus 0.09% (1/1,155), respectively. In males, there appeared to be an association of oncocytic proliferation with advanced spontaneous chronic progressive nephropathy. Oncocytoma or oncocytic hyperplasia appear to be rare lesions in F344 rats, and observations from these carcinogenicity studies suggest that they are slow growing and tend to occur late in a rodent's life span., (© 2014 by The Author(s).)

Two critical components of the validation of any in vivo screening assay are to demonstrate sensitivity and specificity. Although the Endocrine Disruptor Screening Program's Tier 1 Male and Female Pubertal Protocols have been shown to be sensitive assays for the detection of weak endocrine disrupting chemicals (EDCs), there are concerns that the assays lack specificity for EDC effects when a chemical induces systemic toxicity. A lack of specificity, or the ability to correctly identify an inactive or "negative" chemical, would increase the probability of identifying false positives. Here, we orally exposed rats to hydroxyatrazine (OH-ATR), a biotransformation by-product of the chlorotriazine herbicides that produced nephrotoxicity following a 13-week dietary exposure. Based on a previous study in our laboratory, males were dosed with 11.4 to 183.4 mg/kg OH-ATR and females were dosed with 45.75 to 183.4 mg/kg OH-ATR. Following exposure in both sexes, there was a dose-response increase in mean kidney weights and the incidence and severity of kidney lesions. These lesions included the deposition of mineralized renal tubule concretions, hydronephrosis, renal tubule dilatation, and pyelonephritis. However, no differences in body weight, liver weight, or reproductive tissue weights, reproductive or thyroid histology, hormone concentrations or the age of pubertal onset were observed. Therefore, the results demonstrate that the endpoints included in the pubertal assay are useful for nonendocrine (systemic) effects that define an no observable effect level (NOEL) or lowest observable effect level (LOEL) and provide one example where an impact on kidney function does not alter any of the endocrine-specific endpoints of the assay., (© 2013 Wiley Periodicals, Inc.)

Spontaneous renal tubule tumors (RTTs), with a distinctive morphological phenotype, were present in three Sprague-Dawley rats, 1 male and 2 females, out a total of 120 animals of each sex from untreated and placebo control groups in a 2-year carcinogenicity study. One female had one carcinoma, adenoma and hyperplasia, and the other female had five adenomas and many hyperplastic lesions; the male case had one carcinoma. From these cases, a biological continuum of hyperplasia, adenoma and carcinoma could be recognized. The tumors were present in the renal cortex and appeared as solid lobulated growths with occasional central necrosis. The lobules were divided by a small amount of fibrovascular tissue. Occasionally the larger tumors contained a cystic area. Tumor cells appeared distinctive and exhibited variable amounts of eosinophilic/amphophilic and vacuolated cytoplasm. Nuclei were round to oval with a prominent nucleolus. Mitotic figures were uncommon, and no distant metastasis was noted. The tumors were seen as multiple and bilateral lesions in two animals and had no apparent relationship to chronic progressive nephropathy (CPN). Foci of tubule hyperplasia were also noted to contain the same type of cellular morphology. The morphological and biological features of these 3 cases resembled the amphophilic-vacuolar (AV) variant of RTT that has been posited to be of familial origin. This is a report of spontaneous familial renal tumors in Sprague-Dawley rats from Japan.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the urinary tract of rats and mice. The standardized nomenclature of urinary tract lesions presented in this document is 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 developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for urinary tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

From the archives of the National Toxicology Program, National Institutes of Health, kidney sections from twenty-four carcinogenicity studies (representing twenty-three chemicals) in male and female F344 rats were histopathologically re-evaluated to grade the severity of chronic progressive nephropathy (CPN) on an expanded scale of 0-8, and to record the presence of renal tubule tumors (RTT) and their precursor, atypical tubule hyperplasia (ATH). The data were statistically analyzed using SAS software for logistic regression analysis. This histopathological survey of 2,436 F344 rats showed clear evidence of a qualitative and statistically significant association between advanced stages of CPN severity and the development of low-grade RTT and ATH. Advanced CPN severity therefore represents a risk factor for the development of RTT and appears to be an underlying basis for spontaneous occurrence of RTT in the F344 rat. The difference in incidence and severity of CPN between the sexes also explains the 9:1 male-to-female sex difference in the spontaneous occurrence of ATH and RTT observed here. The regulatory significance of this finding is that chemicals exacerbating CPN as their only renal effect are likely to show a numerical increase in RTT with dose, which does not represent a direct tumorigenic effect of the chemical.

Dietary bisphenol A (BPA) was evaluated in a mouse two-generation study at 0, 0.018, 0.18, 1.8, 30, 300, or 3500 ppm (0, 0.003, 0.03, 0.3, 5, 50, or 600 mg BPA/kg/day, 28 per sex per group). A concurrent positive control group of dietary 17beta-estradiol (0.5 ppm; 28 per sex) confirmed the sensitivity of CD-1 mice to an endogenous estrogen. There were no BPA-related effects on adult mating, fertility or gestational indices, ovarian primordial follicle counts, estrous cyclicity, precoital interval, offspring sex ratios or postnatal survival, sperm parameters or reproductive organ weights or histopathology (including the testes and prostate). Adult systemic effects: at 300 ppm, only centrilobular hepatocyte hypertrophy; at 3500 ppm, reduced body weight, increased kidney and liver weights, centrilobular hepatocyte hypertrophy, and renal nephropathy in males. At 3500 ppm, BPA also reduced F1/F2 weanling body weight, reduced weanling spleen and testes weights (with seminiferous tubule hypoplasia), slightly delayed preputial separation (PPS), and apparently increased the incidence of treatment-related, undescended testes only in weanlings, which did not result in adverse effects on adult reproductive structures or functions; this last finding is considered a developmental delay in the normal process of testes descent. It is likely that these transient effects were secondary to (and caused by) systemic toxicity. Gestational length was increased by 0.3 days in F1/F2 generations; the toxicological significance, if any, of this marginal difference is unknown. At lower doses (0.018-30 ppm), there were no treatment-related effects and no evidence of nonmonotonic dose-response curves for any parameter. The systemic no observable effect level (NOEL) was 30 ppm BPA (approximately 5 mg/kg/day); the reproductive/developmental NOEL was 300 ppm (approximately 50 mg/kg/day). Therefore, BPA is not considered a selective reproductive or developmental toxicant in mice.

Nitrapyrin has been registered as a nitrogen stabilizer in the United States for many years based on a robust set of regulatory data. These data demonstrated that nitrapyrin was not genotoxic and that there were no tumors elicited in rats or mice that were relevant for human risk assessment. A repeat carcinogenicity study in B6C3F1 mice, conducted at two substantially higher-dose levels (0, 125 or 250 mg/kg/day) than the original study (0, 5, 25 or 75 mg/kg/day) identified liver, stomach, epididymal and Harderian gland tumors. In order to assess the relevance of these findings for human risk assessment, a Scientific Advisory Group (SAG) examined relevant microscopic changes in these tissues and also evaluated genotoxicity and mechanistic data. The SAG determined that the maximum tolerated dose had been exceeded in mice given 125 or 250 mg/kg/day, based on 26-33% decreased body weight gains (males-250 mg/kg/day), hepatocellular necrosis and compensatory hepatocellular proliferation (males and females-125 and 250 mg/kg/day). The SAG believed that the increased incidences of hepatocellular foci of alteration and hepatocellular neoplasms represented an epigenetic response to hepatocellular necrosis and increased mitogenesis. Increased incidences of proliferative lesions in the forestomach mucosa were likely secondary to the irritant effects of nitrapyrin. Neither the liver nor forestomach effects were interpreted to be a direct carcinogenic effect. Higher incidences of Harderian gland adenomas (females) and undifferentiated sarcomas in the epididymis represented normal biological variations in incidence and were unrelated to nitrapyrin. Therefore, it was the SAG's opinion that nitrapyrin exposure that does not produce target organ toxicity in exposed individuals would not be expected to increase the risk of cancer.

Although exposure to drugs or toxicants can affect children and adults very differently, many compounds lack specific safety information for children. Studies in juvenile animals can help researchers assess pediatric patients' potential response to certain chemicals. Juvenile studies are highly sensitive to animal age, sex and species and must be planned with care to prevent misinterpretation of experimental data. The author reviews considerations for the design of these studies, focusing on toxicological and pathological aspects.

The Toxicology Data Management System (TDMS) of the National Toxicology Program, National Institutes of Environmental Health Sciences, National Institutes of Health, was surveyed for occurrence and distribution of a distinctive renal tubule tumor type in rats. The hallmark features of this tumor included eosinophilic/amphophilic staining, large finely granular cells, and numerous vacuoles and/or minilumens. It is referred to here as the amphophilic-vacuolar (AV) variant of renal tubule tumor. Of 154 studies in which renal tubule tumors had been recorded in the standard single sections of kidney in the TDMS, there were collectively 1012 rats with renal adenomas, carcinomas, or adenocarcinomas, and of these, 100 displayed the distinctive AV morphology, representing 74 studies involving mostly the F344 rat, but also the Sprague-Dawley and Wistar strains. The AV tumors (mainly adenomas but also some carcinomas) occurred usually as solitary lesions in the affected animals. However, they were multiple and bilateral in a few cases. They were equally distributed between the sexes, did not metastasize (at least to the lung), and were not associated with chronic progressive nephropathy. The distribution of this renal tumor type was random across studies and dose groups, underscoring the likelihood that it was of spontaneous origin and not chemically induced. Accordingly, it is suggested that this distinctive renal tumor phenotype be recorded as a separate category from conventional RTT when assessing the carcinogenic potential of a test compound.

No information exists on reproductive/developmental effects in mice exposed to dietary 17beta-estradiol (E2) over multiple generations. Therefore, under OECD Test Guideline 416 with enhancements, CD-1 mice (F0 generation, 25 mice/sex/group) were exposed to dietary E2 at 0, 0.001, 0.005, 0.05, 0.15, or 0.5 ppm ( approximately 0, 0.2, 1, 10, 30, or 100 mug E2/kg body weight/day) for 8 weeks prebreed, 2 weeks mating, approximately 3 weeks gestation, and 3 weeks lactation. At weaning, selected F1 offspring (F1 parents; 25/sex/group) and extra retained F1 males (one per litter) were exposed to the same dietary concentrations and durations as the F0 generation; study termination occurred at F2 weaning; F1/F2 weanlings (up to three per sex per litter) were necropsied with organs weighed. At 0.5 ppm, effects were increased F1/F2 perinatal loss, prolonged F0/F1 gestational length, reduced numbers of F2 (but not F1) litters/group, reduced F1/F2 litter sizes, accelerated vaginal patency (VP) and delayed preputial separation (PPS), increased uterus + cervix + vagina weights (UCVW) in F0/F1 adults and F1/F2 weanlings, and decreased testes and epididymides weights (TEW) in F1/F2 weanlings. At 0.15 ppm, effects were increased UCVW in F0/F1 adults and F1/F2 weanlings, accelerated VP, delayed PPS, and reduced TEW in F1/F2 weanlings. At 0.05 ppm, UCVW were increased in F1/F2 weanlings, and PPS was delayed only in extra retained F1 males. There were no biologically significant or treatment-related effects on F0/F1 parental body weights, feed consumption, or clinical observations, or on F0/F1 estrous cyclicity, F0/F1 andrology, or F1/F2 anogenital distance at any dose. The no observable effect level was 0.005 ppm E2 ( approximately 1 mug/kg/day). Therefore, the mouse model is sensitive to E2 by oral administration, with effects on reproductive development at doses of 10- 100 mug/kg/day.

Renal histopathology in the most recent 2-year carcinogenicity bioassay of quercetin, in Fischer 344 rats, was re-evaluated in an attempt to determine a mode of action underlying a small increase in renal tubule tumors reported in the males (). The re-evaluation confirmed the reported increase in renal tumors in mid- and high-dose males, including a single carcinoma in a high-dose male, as well as an exacerbation of spontaneous, chronic progressive nephropathy (CPN) in male rats only. The re-evaluation also showed that there were no cellular alterations in the kidney indicative of chemical toxicity at 6 months, 15 months, or 2 years. The evidence linked the occurrence of the predominant basophilic adenomas and foci of atypical tubule hyperplasia (ATH) with the exacerbation of CPN to advanced grades of severity, supporting a mode of action involving quercetin interaction with CPN. This mode of action represents a secondary mechanism for renal tumor development, with no relevance for extrapolation to humans. In addition, the single carcinoma present in the high-dose males, along with 4 other lesions ranging from ATH to adenoma in male and female groups, were considered to have a unique phenotype associated previously with neoplasms of spontaneous and familial origin.

This study evaluated the potential for dietary para-nonylphenol (NP; CAS No. 84852-15-3) to affect parental fertility and growth and development of three offspring generations in CD (Sprague-Dawley [SD]) rats, including sperm counts across generations to determine the validity of equivocal reductions observed in the F2 generation by R. E. Chapin et al. (1999, Toxicol. Sci. 52, 80-91). Male rat kidney toxicity was also examined based on inconsistent observations in NP-exposed rats at 2000 ppm but not at 200 or 650 ppm in Purina 5002 (H. C. Cunny et al., 1997, Regul. Toxicol. Pharmacol. 26, 172-178) and at all of these NP concentrations in NIH-07 diet (R. E. Chapin et al., 1999, Toxicol. Sci. 52, 80-91). Concentrations were 0, 20, 200, 650, and 2000 ppm NP in Purina 5002 diet and 0 and 650 ppm NP in NIH-07 diet. 17beta-estradiol (E2) was used as a positive control at 2.5 ppm in Purina 5002 diet. There were no NP effects on any reproductive parameters in any generation, including sperm counts. Kidney toxicity (histopathology) occurred at 650 and 2000 ppm with no clear difference for the two diets. Ovarian weight was decreased at 2000 ppm NP in all generations, with no effect on reproduction. Dietary E2 at 2.5 ppm caused renal, reproductive, and developmental (lactational and peripubertal) toxicity in all generations. This study confirmed that dietary NP is not a selective reproductive toxicant with an no observable adverse effect level (NOAEL) of > 2000 ppm ( approximately > 150 mg/kg/day) and provided an NOAEL for male rat kidney toxicity of 200 ppm NP (approximately 15 mg/kg/day).

Mutations in both p53 and BRCA2 are commonly seen together in human tumors suggesting that the loss of both genes enhances tumor development. To elucidate this interaction in an animal model, mice lacking the carboxy terminal domain of Brca2 were crossed with p53 heterozygous mice. Females from this intercross were then irradiated with an acute dose of 5 Gy ionizing radiation at 5 weeks of age and compared to nonirradiated controls. We found decreased survival and timing of tumor onsets, and significantly higher overall tumor incidences and prevalence of particular tumors, including stomach tumors and squamous cell carcinomas, associated with the homozygous loss of Brca2, independent of p53 status. The addition of a p53 mutation had a further impact on overall survival, incidence of osteosarcomas and stomach tumors, and tumor latency. The spectrum of tumors observed for this Brca2 germline mouse model suggest that it faithfully recapitulates some human disease phenotypes associated with BRCA2 loss. In addition, these findings include extensive in vivo data demonstrating that germline Brca2 and p53 mutations cooperatively affect animal survivals, tumor susceptibilities, and tumor onsets.

Recently, guidelines were suggested for discriminating proliferative-appearing tubule profiles encountered in advanced spontaneous chronic progressive nephropathy (CPN) of rats, from hyperplastic precursors of renal tubule tumors (Hard and Seely, 2005). These recommendations were based on histological evaluation of a large number of cases of severe to end-stage CPN in male F344 rats from 8 separate 2-year carcinogenicity studies held in the Archives of the National Toxicology Program, NIEHS. This work has now been extended to characterize the various lesions further, mainly by serial sectioning to track their origin and fate within the adjacent renal tissue, but also by applying special staining procedures such as immunohistochemical assessment of proliferative activity, as well as fluorescence microscopy, to seek further differences from atypical tubule hyperplasia. The results obtained from these additional investigations support the contention that certain tubule profiles with a misleading proliferative appearance, sometimes found in advanced CPN, should be distinguished from preneoplastic tubule foci, and regarded as components of the nephropathy process.

There is little guidance in the literature on the spectrum of proliferative tubule lesions in the kidneys of aging rats affected with spontaneously occurring, chronic progressive nephropathy (CPN), or their interpretation. Through accessing 2-year carcinogenicity studies in male F344 rats held in the Archives of the National Toxicology Program, NIEHS, a large number of cases of advanced CPN have been surveyed histopathologically for proliferative tubule lesions, and an attempt made to provide guidelines for discrimination of lesions common to the CPN process, from those representing precursors of neoplasia. Several proliferative lesions were identified as common in advanced CPN with no apparent evidence supporting a role in renal tubule carcinogenesis. It is recommended that these lesions be viewed generically as CPN tubule profiles, and not recorded separately from the diagnosis of CPN. Criteria were developed to distinguish these CPN-associated lesions from atypical tubule hyperplasia, a precursor of adenoma, both of which were also represented in this survey of advanced CPN.

Butyl benzyl phthalate (BBP) was administered in the diet at 0, 750, 3750, and 11,250 ppm ad libitum to 30 rats per sex per dose for two offspring generations, one litter/breeding pair/generation, through weaning of F2 litters. Adult F0 systemic toxicity and adult F1 systemic and reproductive toxicity were present at 11,250 ppm (750 mg/kg per day). At 11,250 ppm, there were reduced F1 and F2 male anogenital distance (AGD) and body weights/litter during lactation, delayed acquisition of puberty in F1 males and females, retention of nipples and areolae in F1 and F2 males, and male reproductive system malformations. At 3750 ppm (250 mg/kg per day), only reduced F1 and F2 offspring male AGD was present. There were no effects on parents or offspring at 750 ppm (50 mg/kg per day). The F1 parental systemic and reproductive toxicity no observable adverse effect level (NOAEL) was 3750 ppm. The offspring toxicity NOAEL was 3750 ppm. The offspring toxicity no observable effect level (NOEL) was 750 ppm, based on the presence of reduced AGD in F1 and F2 males at birth at 3750 ppm, but no effects on reproductive development, structures, or functions.

Under Office of Prevention, Pesticides and Toxic Substances draft guidelines, CD weanling F0 rats (30 of each gender per group) inhaled tertiary amyl methyl ether vapor at 0, 250, 1500 or 3000 ppm 5 days a week and 6 h a day for 10 weeks, with vaginal cytology evaluated for weeks 8-10. The F0 animals then produced F1 offspring, with exposure 7 days a week from mating through to lactation. During the F1 prebreed exposure period, vaginal patency, preputial separation (PPS) and vaginal cytology were evaluated. The F1 animals were mated, with F2 anogenital distance measured on postnatal day zero. At F2 weaning 30 of each gender per group were selected for postwean retention, with no exposures, through vaginal patency and PPS. Body weights, feed consumption and clinical signs were recorded throughout the study. Adult F0 and F1 systemic toxicity was present at 1500 and 3000 ppm. Minor adult male reproductive toxicity was present at 3000 ppm. There were no adult effects on vaginal cyclicity, estrous cycle length, mating, fertility, pregnancy, gestational length or ovarian and uterine weights. There were no treatment-related gross or histopathologic findings in parental male or female systemic or reproductive organs. The F1 and F2 offspring toxicity was present at 1500 and 3000 ppm. The no-observable-adverse-effect level for adult systemic and offspring toxicity was 250 ppm and 1500 ppm for male reproductive toxicity (females at >3000 ppm)., (Copyright 2003 John Wiley & Sons, Ltd.)

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

Bisphenol A (BPA) was evaluated at concentrations of 0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm ( approximately 0.001, 0.02, 0.3, 5, 50, and 500 mg/kg/day of BPA) administered in the diet ad libitum to 30 CD((R)) Sprague-Dawley rats/sex/dose for 3 offspring generations, 1 litter/generation, through F3 adults. Adult systemic toxicity at 750 and 7500 ppm in all generations included: reduced body weights and body weight gains, reduced absolute and increased relative weanling and adult organ weights (liver, kidneys, adrenals, spleen, pituitary, and brain), and female slight/mild renal and hepatic pathology at 7500 ppm. Reproductive organ histopathology and function were unaffected. Ovarian weights as well as total pups and live pups/litter on postnatal day (PND) 0 were decreased at 7500 ppm, which exceeded the adult maximum tolerated dose (MTD). Mating, fertility, gestational indices; ovarian primordial follicle counts; estrous cyclicity; precoital interval; gestational length; offspring sex ratios; postnatal survival; nipple/areolae retention in preweanling males; epididymal sperm number, motility, morphology; daily sperm production (DSP), and efficiency of DSP were all unaffected. At 7500 ppm, vaginal patency (VP) and preputial separation (PPS) were delayed in F1, F2, and F3 offspring, associated with reduced body weights. Anogenital distance (AGD) on PND 0 was unaffected for F2 and F3 males and F3 females (F2 female AGD was increased at some doses, not at 7500 ppm, and was considered not biologically or toxicologically relevant). Adult systemic no observed adverse effect level (NOAEL) = 75 ppm (5 mg/kg/day); reproductive and postnatal NOAELs = 750 ppm (50 mg/kg/day). There were no treatment-related effects in the low-dose region (0.001-5 mg/kg/day) on any parameters and no evidence of nonmonotonic dose-response curves across generations for either sex. BPA should not be considered a selective reproductive toxicant, based on the results of this study.

Diet is one of the most important environmental factors influencing growth, survival, and appearance of age-associated diseases in rodents. NIH-07 open formula rodent diet was the selected diet for the National Toxicology Program studies from 1980 to 1994. After a number of experimental diets were evaluated, a new one designated as NTP-2000 was selected for rodents in NTP studies beginning in 1994. This report summarizes the results of dosed feed and inhalation studies for differences in growth, survival, and severity of kidney and heart lesions in Fischer 344 rats fed NTP-2000 or NIH-07 diets. In the dosed feed studies, male rats group housed and fed the NTP-2000 diet grew slightly slower, attained maximum body weight later, and lost less body weight by the end of the 2-year studies compared to the groups fed NIH-07. Female rats group housed and fed the NTP-2000 diet in dosed feed studies had significantly slower growth, with lower maximum body weight compared to the groups fed the NIH-07 diet. In the inhalation studies, male rats individually housed and fed the NTP-2000 diet had slightly higher maximum body weight and significantly higher final body weight, with lower loss of weight when compared to similarly housed groups fed the NIH-07 diet. In inhalation studies, female rats fed the NTP-2000 diet and individually housed had significantly slower growth. The NTP-2000 diet significantly increased the survival of male and female rats, with a dramatic increase in survival of males in inhalation studies. This diet also caused significant decreases in severity of nephropathy and cardiomyopathy, and the decrease was marked in males. These observations indicate that diets balanced for nutrients, such as the NTP-2000, could markedly improve the health and increase survival of the rats used in chronic studies.

The National Toxicology Program is conducting a chemical class study to investigate the structure-activity relationships for the toxicity of alpha,beta-unsaturated ketones. Ethyl vinyl ketone (EVK) was selected for study because it is a representative straight-chain aliphatic alpha,beta-unsaturated ketone with extensive use and widespread exposure. Short-term inhalation studies of EVK were conducted to provide toxicity data for comparison with the related alpha,beta-unsaturated ketones 2-cyclohexene-1-one (CHX) and methyl vinyl ketone (MVK). These data will be used in designing chronic toxicity and carcinogenicity studies of these ketones. Male and female F344 rats and B6C3F1 mice were exposed to 0, 2, 4, or 8 ppm EVK 6 h/day, 5 days/wk for 13 wk. The nasal cavity was the major target organ of EVK in both rats and mice. Pathologic findings in both the olfactory and respiratory epithelium were observed. Lesions consisted primarily of olfactory epithelial necrosis, atrophy and regeneration, and/or hyperplasia and squamous metaplasia of the respiratory epithelium. Squamous metaplasia of the respiratory epithelium was present in all rats and mice exposed to 4 and 8 ppm EVK, and these lesions were more severe in rats than in mice. Few systemic effects were observed in rats and mice exposed to EVK. A transient decrease in total leukocytes due to decrements in lymphocyte and monocyte populations was present in male rats after exposure to 8 ppm for 3 and 21 days; however, this effect was not present after exposure for 13 wk. There were no chemical-related effects on micronucleus formation in mice, or on sperm motility and vaginal cytology in either species. EVK, like other alpha,beta-unsaturated ketones, is a reactive, direct-acting gaseous irritant with toxicity limited primarily to the upper respiratory tract.

Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other sites. Recent studies have suggested a role for the APC I1307K allele as a low-penetrance breast cancer susceptibility gene that enhances the phenotypic effects of BRCA1 and BRCA2 mutations. To model the consequences of inheriting mutant alleles of the BRCA2 and APC tumor suppressor genes, we examined tumor outcome in C57BL/6 mice with mutations in the Brca2 and Apc genes. We hypothesized that if the Brca2 and Apc genes were interacting to influence mammary tumor susceptibility, then mammary tumor incidence and/or multiplicity would be altered in mice that had inherited mutations in both genes. Female and male offspring treated with a single IP injection of 50 mg/kg N-ethyl-N-nitrosourea (ENU) at 35 days of age developed mammary adenoacanthomas by 100 days of age. The female Apc-mutant and Brca2/Apc double-mutant progeny had mean mammary tumor multiplicities of 6.7+/-2.8 and 7.2+/-2.7, respectively, compared to wild-type and Brca2-mutant females, which had mean mammary tumor multiplicities of 0.1+/-0.4 and 0.3+/-0.5, respectively. Female ENU-treated Apc-mutant and Brca2/Apc double heterozygotes were also susceptible to premature ovarian failure. Thus, the inheritance of an Apc mutation predisposes ENU-treated female and male mice to mammary tumors and, in the case of female mice, to ovarian failure. These results indicate that mammary tumor development in Apc-mutant mice can progress independently of ovarian hormones. The Apc mutation-driven phenotypes were not modified by mutation of Brca2, perhaps because Brca2 acts in a hormonally dependent pathway of mammary carcinogenesis.

The National Toxicology Program is conducting a chemical class study to investigate the structure-activity relationships for the toxicity of alpha,beta-unsaturated ketones. Methylvinyl ketone (MVK) was selected for study because it is a representative straight-chain aliphatic alpha,beta-unsaturated ketone and because of its extensive use and widespread exposure. Short-term inhalation studies of MVK were conducted to provide toxicity data for comparison with other alpha,beta-unsaturated ketones and for use in designing chronic toxicity and carcinogenicity studies. In 2-week studies, rats and mice were exposed to 0, 0.25, 0.5, 1, 2, 4, or 8 ppm MVK 6 h/day, 5 days/week for 12 exposures. Morbidity and early deaths occurred in all male and female rats after 1 exposure and in 2 male mice after 10 exposures to 8 ppm. Rats exhibited nasal cavity toxicity and lung necrosis at 4 ppm. No toxicity was observed in animals exposed to less than 2 ppm. Based on these results a 13-week study was conducted at 0, 0.5, 1, and 2 ppm MVK. As observed in the 2-week study, the nasal cavity was the main target organ and rats were more sensitive than mice. Respiratory and olfactory epithelial necrosis were prominent by day 21 in the rat. At study termination these lesions were still evident but not as severe as noted earlier. Additionally, changes such as olfactory epithelial regeneration and metaplasia (respiratory) as well as respiratory epithelial hyperplasia and metaplasia (squamous) were clearly evident. Nasal lesions in mice were limited to a subtle squamous metaplasia of transitional and/or respiratory epithelium covering predominantly the tips of naso- and maxilloturbinates in Levels I and II. A transient, leukopenia was observed in rats exposed to 2 ppm, however, this effect was not present after 13 weeks of exposure. In mice, leukocyte counts were significantly decreased at all exposure concentrations after 13 weeks of exposure. Absolute testicular and epididymal weights and sperm counts were decreased at the high dose only. MVK can be characterized as a reactive, direct-acting gaseous irritant. MVK exposure causes the same nasal cavity lesions as the cyclic alpha,beta-unsaturated ketone, 2-cyclohexen-1-one, although at lower exposure concentrations.

Women who inherit mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are predisposed to the development of breast and ovarian cancer. We used mice with a Brca1 mutation on a BALB/cJ inbred background (BALB/cB1+/- mice) or a Brca2 genetic alteration on the 129/SvEv genetic background (129B2+/- mice) to investigate potential gene-environment interactions between defects in these genes and treatment with the highly estrogenic compound diethylstilbestrol (DES). Beginning at 3 weeks of age, BALB/cB1+/-, 129B2+/-, and wild-type female mice were fed a control diet or a diet containing 640 ppb DES for 26 weeks. DES treatment caused vaginal epithelial hyperplasia and hyperkeratosis, uterine inflammation, adenomyosis, and fibrosis, as well as oviductal smooth muscle hypertrophy. The severity of the DES response was mouse strain specific. The estrogen-responsive 129/SvEv strain exhibited an extreme response in the reproductive tract, whereas the effect in BALB/cJ and C3H/HeN(MMTV-) mice was less severe. The Brca1 and Brca2 genetic alterations influenced the phenotypic response of BALB/cJ and 129/SvEv inbred strains, respectively, to DES in the mammary gland and ovary. The mammary duct branching morphology was inhibited in DES-treated BALB/cB1+/- mice compared with similarly treated BALB/cB1+/+ littermates. In addition, the majority of BALB/cB1+/- mice had atrophied ovaries, whereas wild-type littermates were largely diagnosed with arrested follicular development. The mammary ductal architecture in untreated 129B2+/- mice revealed a subtle inhibited branching phenotype that was enhanced with DES treatment. However, no significant differences were observed in ovarian pathology between 129B2+/+ and 129B2+/- mice. These data suggest that estrogenic compounds may modulate mammary gland or ovarian morphology in BALB/cB1+/- and 129B2+/- mice. These observations are consistent with the hypothesis that compromised DNA repair processes in cells harboring Brca1 or Brca2 mutations lead to inhibited growth and differentiation compared with the proliferative response of wild-type cells to DES treatment.

Octylphenol (OP) is a commercial intermediate used primarily for the production of octylphenol polyethoxylate surfactants. To determine potential reproductive toxicity of OP, a two-generation reproduction study was conducted according to U.S. EPA OPPTS Guideline 870.3800 (draft 1996). Additional measurements were made on retained F2 offspring. OP was administered ad libitum to five groups of rats (30/sex) at dietary concentrations of 0, 0.2, 20, 200, or 2000 ppm. The 0.2 ppm concentration was included to evaluate potential low dose effects. Effects were observed only at 2000 ppm, including decreased body weights in adults and during the latter portion of lactation in offspring and minor body weight-related delays in acquisition of vaginal opening and preputial separation. No effects on reproductive parameters, testes, prostate, or ovary weights or morphology, on sperm counts, motility, morphology, production, or on estrous cyclicity were observed. No estrogen-like effects were evident. The NOAELs for systemic and postnatal toxicity were 200 ppm and at or above 2000 ppm for reproductive toxicity. This study supports the increasing evidence that screening assays for estrogenic activity or studies with limited numbers of animals and/or unrealistic dose regimens are inappropriate for use in the assessment of human health and environmental risk. It does not support previous preliminary data on low dose effects of OP., (Copyright 1999 Academic Press.)

Chemicals, by virtue of their varied interactions with biological molecules, are expected to differ in the way they may alter female reproduction. Reproductive toxicity may reflect effects either on the female germ cells or on various maternal processes such as ovulation, implantation, pregnancy, and parturition. In either case, the ultimate manifestation of chemical toxicity on female reproduction is a decrease in the number of normal young born. Very little information is available on the effects of chemicals that are nonhormonal in nature on the long-term ability of treated females to produce offspring. This report presents the results of long-term female total reproductive capacity (TRC) tests on 29 chemicals, including pharmaceuticals, pesticides, and alkylating and industrial agents. For each chemical, the minimum test involved an evaluation of the maximum tolerated dose administered as a single intraperitoneal injection. Females were single-pair mated with an untreated male for most of the female's reproductive life span (a minimum of 347 days posttreatment) and scored for the number of live births produced during this period. Confirmatory dominant lethal experiments or histological examinations for numbers of small follicles were carried out when mutagenic effects or cytotoxicity, respectively, were suspected as the basis for reduced fertility. Of the 29 chemicals studied, 17 had reproductive effects which may be grouped into one of three classes: (1) those that reduced the total number of young and litters per female, (2) those that reduced the total number of young but not of litters, and (3) those that had no significant effect on the total number of young produced but reduced the size of the first and/or second litters. The TRC provides a capacity for detecting a range of toxic insults upon female reproduction. Many of the chemicals were indeed shown to affect the reproductive performance of females through mutagenic and/or cytotoxic effects on follicles. In some cases, however, no causative mechanism could be identified for the observed reduction in reproductive performance. Nevertheless, with this report the number of chemicals tested by this TRC procedure has been quadrupled and the categories of chemicals tested have been substantially broadened.

Epidemiology studies have indicated that many human cancers are influenced by environmental factors. Genetically altered mouse model systems offer us the opportunity to study the interaction of chemicals with genetic predisposition to cancer. Using the heterozygous p53-deficient (+/-) mouse, an animal model carrying one wild type p53 gene and one p53 null allele, we studied the effects of phenolphthalein on tumor induction and p53 gene alterations. Earlier studies showed that phenolphthalein caused carcinogenic effects in Fisher 344 rats and B6C3F1 mice after a 2-yr dosing period (Dunnick and Hailey, Cancer Res. 56: 4922-4926, 1996). The p53 (+/-) mice received phenolphthalein in the feed at concentrations of 200, 375, 750, 3,000, or 12,000 ppm (approximately 43, 84, 174, 689, or 2,375 mg/kg body weight/day or 129, 252, 522, 2,867, or 7,128 mg/m2 body surface area/day) for up to 6 mo. A target organ cancer site that accumulated p53 protein in the B6C3F1 mouse (i.e., thymic lymphoma) was also a target site for cancer in the p53 (+/-) mouse. In the p53 (+/-) mouse, treatment-related atypical hyperplasia and malignant lymphoma of thymic origin were seen in the control and dosed groups at a combined incidence of 0, 5, 5, 25, 100, and 95%, respectively. Twenty-one of the thymic lymphomas were examined for p53 gene changes, and all showed loss of the p53 wild type allele. Chemical-induced ovarian tumors in the B6C3F1 mouse showed no evidence for p53 protein accumulation and did not occur in the p53 (+/-) mouse. The p53-deficient (+/-) mouse model responded to phenolphthalein treatment with a carcinogenic response in the thymus after only 4 mo of dosing. This carcinogenic response took 2 yr to develop in the conventional B6C3F1 mouse bioassay. The p53-deficient (+/-) mouse is an important model for identifying a carcinogenic response after short-term (< 6 mo) exposures. Our studies show that exposure to phenolphthalein combined with a genetic predisposition to cancer can potentiate the carcinogenic process and cause p53 gene alterations, a gene alteration found in many human cancers.

Tributyl phosphate (TBP) was tested for reproductive toxicity in rats. Thirty weanlings/sex (F0) were exposed to TBP in the diet ad libitum at 0, 200, 700, or 3000 ppm for 10 weeks and then randomly mated within groups for 3 weeks with continued exposure. F0 parents and 10 F1 weanlings/sex/dose were necropsied, and adult reproductive organs, urinary bladders (both sexes), kidneys (males), and livers (females) were evaluated histologically. Thirty F1 weanlings/sex/dose continued exposure for 11 weeks and were bred as described above. F1 parents and F2 weanlings, 10/sex/dose, were then necropsied as described above. Adult toxicity was observed in both sexes and generations at 700 and 3000 ppm; observations included reduced body weights, weight gain and feed consumption, urinary bladder epithelial hyperplasia (both sexes), renal pelvis epithelial hyperplasia only at 3000 ppm (male kidneys), and centrilobular hypertrophy (female livers). At 200 ppm, transient reductions in body weight were observed in F0 and F1 females, with urinary bladder epithelial hyperplasia in F0 males and females and in F1 males. There was no evidence of reproductive toxicity, of reproductive organ pathology, or of effects on gestation or lactation at any dose tested. Postnatal toxicity was evidenced by consistent reductions in F1 and F2 pup body weights at 3000 ppm and by occasional weight reductions in F2 litters at 700 ppm, and was associated with maternal toxicity observed at these doses and times. Under the conditions of this study, a NOAEL was not determined for adult toxicity; the NOAEL for reproductive toxicity was at least 3000 ppm and the NOAEL for postnatal toxicity was approximately 200 ppm.

Boric acid (BA), an ingredient of many pharmaceutical, cosmetic, and pesticide products, was previously shown to induce reproductive and developmental toxicity in laboratory rodents. In this study, BA (0, 62.5, 125, or 250 mg/kg/day, po) was administered on Gestational Days (GD) 6-19 to New Zealand White rabbits (18-23 pregnant/group). Maternal body weight, food consumption, and clinical condition were monitored at regular intervals throughout gestation. At termination (GD 30), the numbers of uterine implantations, resorptions, dead fetuses, and live fetuses were determined. Fetuses were weighed, and live fetuses examined for external, visceral, and skeletal defects. Maternal food intake decreased during treatment at 250 mg/kg/day and increased at >/=125 mg/kg/day after treatment. Maternal body weight (GD 9-30), weight gain during treatment, gravid uterine weight, and number of ovarian corpora lutea decreased at 250 mg/kg/day. In contrast, maternal corrected gestational weight gain increased at >/=125 mg/kg/day. Maternal liver weight was not affected. Relative (but not absolute) maternal kidney weight increased at 250 mg/kg/day, and microscopic evaluation revealed no treatment-related renal pathology. At 250 mg/kg/day, prenatal mortality was increased (90% resorptions/litter vs 6% for controls), the proportion of pregnant females with no live fetuses was increased (73% vs 0%), and live litter size was reduced (2.3 fetuses/litter vs 8.8). As a result, there were only 14 live fetuses (6 live litters) available for evaluation in the high-dose group, compared to 153-175 live fetuses (18-23 live litters) in the other groups. The percentage malformed fetuses/litter was increased at 250 mg/kg/day, primarily due to cardiovascular defects in 72% of high-dose fetuses vs 3% of controls. The most prevalent cardiovascular malformation (interventricular septal defect) was observed in 57% of high-dose fetuses compared to 0.6% among controls. At 250 mg/kg/day, average fetal body weight/litter was 92% of the average control weight (not statistically significant). In summary, no definitive maternal or developmental toxicity was observed at 62.5 or 125 mg/kg/day BA. Mild maternal effects and severe developmental toxicity were observed at 250 mg/kg/day.

Methylethyl ketoxime (CAS No. 96-29-7; MEKO; 2-butanone oxime), an antioxidant agent used in paints, resins, and adhesives, was tested for reproductive toxicity in a two-generation study with CD (Sprague-Dawley) rats. Thirty-eight-week-old rats/sex/group (F0) were administered MEKO in water, by gavage, at 0, 10, 100, or 200 mg/kg/day (at a dosing volume of 2 ml/kg), 5 days/week for 10 weeks with vaginal cytology evaluation (VCE) of F0 females during the last 3 weeks of the prebreed period. Animals were mated within groups for 3 weeks with dosing during mating, gestation, and lactation for 7 days/week. F0 parents and F1 weanlings, 10/sex/dose, were necropsied (after a 2-week postwean VCE in F0 females) with hematologic evaluation (including methemoglobin) and histology of adult livers, spleens, and reproductive organs. F1 weanlings, 30/sex/dose, were dosed for 11 weeks and mated as described above. Because of poor reproductive performance, not treatment related, F1 animals with no F2a litters were rebred to produce F2b litters. F1 parents and F2a weanlings, 10/sex/dose, were necropsied and evaluated as described above. Inguinal mammary glands were examined histologically from all nonselected F1 and F2 (a and b) female weanlings. Adult toxicity was observed in both generations and both sexes at all doses. Treatment-related parental deaths occurred at 200 mg/kg/day. At 100 and 200 mg/kg/day, parents exhibited dose-related reduced body weights and weight gains, reduced feed consumption, clinical signs of toxicity, and anemia with concomitant extramedullary hematopoiesis and hemosiderosis in livers and spleens (and increased spleen weights). At 10 mg/kg/day, only adult liver and spleen histologic effects were present. There was no evidence of reproductive organ or mammary glad pathology or of reproductive or postnatal toxicity at any dose tested. There was no adult "no observable adverse effect level" (NOAEL) established; the NOAEL for reproductive and postnatal toxicity was at least 200 mg/kg/day for rats in this study.

Increases in the use of methanol (MeOH) as a transportation fuel would result in greater potential for inhalation exposure. Because oral exposure to MeOH potentiates the hepatotoxicity of carbon tetrachloride (CCl4), we examined the ability of inhaled MeOH to potentiate CCl4 hepatotoxicity and the time course of injury and recovery. Adult male F-344 rats were exposed to 0 or to 10,000 ppm MeOH by inhalation for 6 h and gavaged with 0.075 ml CCl4/kg 24 h later. Hepatotoxicity was assessed 0.5, 1, 1.5, 2, 3, 7, 15, 30, and 61 d after CCl4 exposure. For CCl4 alone, hepatotoxicity was most severe at 0.5 and 1 d, when minimal centrilobular hepatocellular necrosis and predominately mild centrilobular hepatocellular vacuolar degeneration occurred. By d 3, the livers from the CCl4 rats were histologically normal. For MeOH+CCl4, peak severity of hepatic injury was at 1 and 1.5 d, when moderate centrilobular necrosis and moderate/marked centrilobular degeneration occurred. MeOH+CCl4 resulted in serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) that were increased, relative to CCl4 alone, 171- and 113-fold, respectively, on d 1, and 166- and 140-fold, respectively, on d 1.5. Significant serum elevations in MeOH+CCl4 rats, relative to CCl4 alone rats, were present until d 7 and d 15 for AST and ALT, respectively. By d 3 and d 7, degeneration and necrosis, respectively, due to MeOH+CCl4 were essentially resolved. On d 7, the MeOH+CCl4 hepatic injury consisted mainly of chronic inflammation and centrilobular fibrosis. By d 30, the livers of MeOH+CCl4 rats were histologically normal. These data demonstrate that inhaled MeOH potentiates the hepatotoxicity of orally ingested CCl4, increasing the severity of CCl4 hepatotoxicity as well as the time required for recovery.

Because groundwater contamination is an important environmental concern, we examined the hepatic and renal effects of repeated exposure to a mixture of 25 chemicals frequently found in groundwater near hazardous-waste disposal sites and the effect of such exposure on carbon tetrachloride (CCI4) toxicity. Adult male F-344 rats received ad libitum deionized water and feed (Ad Lib Water) or ad libitum 10% MIX (referring to 10% of a technically achievable stock mixture) and feed for 14 d. Because exposure to the 25-chemical mixture via the drinking water resulted in decreased water and feed consumption, restricted deionized water and feed controls (Restricted Water) were included. On d 14, rats were gavaged with 0, 0.0375, 0.05, 0.075 or 0.15 ml CCl4/kg, and hepatic and renal toxicity assessed 24 h later. Little or no hepatic and renal toxicity was observed in rats exposed to 10% MIX alone. No hepatic or renal lesions occurred that could be attributed to 10% MIX alone. Slight but statistically significant alterations, of uncertain biological significance, resulted from the water treatments: 10% MIX increased alanine aminotransferase, urea nitrogen (BUN), and BUN/creatinine ratio; Restricted Water increased 5'-nucleotidase and decreased alkaline phosphatase. Relative kidney weight was increased by both 10% MIX and Restricted Water. CCI4 resulted in significant dosage-dependent hepatotoxicity in all three water treatment groups but had little or no effect on renal indicators of toxicity. Relative to Ad Lib Water, significantly greater hepatotoxicity occurred in both 10% MIX and Restricted Water rats. The response to CCI4 in the Restricted Water rats was similar to that of 10% MIX rats, indicating that a substantial portion of the effect of 10% MIX on CCI4 hepatotoxicity is due to decreased water and feed intake.

The carcinogenic water disinfection byproduct, bromodichloromethane (BDCM), produces renal and hepatic toxicity in rodents in acute and subchronic studies. In the present investigation, female rats and mice (n = 6) were dosed daily for 5 consecutive days with BDCM (dissolved in an aqueous, 10% Emulphor solution) by gavage. Rats received 75, 150 and 300 mg BDCM/kg body weight/day and mice received 75 and 150 mg BDCM/kg body weight/day. Two rats in the 300 mg/kg/day treatment group died on day 5. On day 6, the animals were sacrificed and serum samples were taken for analysis of indicators of hepatic and renal toxicity. Livers and kidneys were excised and samples taken for histopathological evaluation. Portions of the livers were also utilized to produce microsomes for analysis of cytochrome P450 enzyme activities and total P450 content. Total hepatic cytochrome P450 was decreased in rats dosed with 150 and 300 mg BDCM/kg body weight/day, but was not significantly affected in BDCM-treated mice. Serum lactate (LDH) and sorbitol (SDH) dehydrogenase, aspartate aminotransferase (AST), creatinine and blood urea nitrogen were increased above those of controls in rats dosed with 300 mg BDCM/kg/day. These data suggested that hepatic and renal damage had occurred in this treatment group. This was confirmed by histopathological analyses which revealed that lesions occurred in both hepatic and renal tissues from rats dosed with 150 and 300 mg BDCM/kg/day. The hepatic lesions were centrilobular and primarily consisted of vacuolar degeneration. The hepatotoxicity indicators alanine aminotransferase (ALT) and SDH were increased in mice dosed with 150 mg BDCM/kg/day. However, no histopathological lesions were observed in these animals. This study shows that BDCM is both hepatotoxic and nephrotoxic to female rats after repeated dosing, but is only weakly hepatotoxic to female mice at the administered doses. Also, reduced activities of hepatic cytochrome P450 were observed in rats, but not mice. These species differences in toxicity and xenobiotic metabolizing enzyme inhibition caused by BDCM suggest that an understanding of the mechanism of toxicity of this compound will be critical when extrapolating rodent toxicity data to humans for this environmental pollutant.

4-Vinylcyclohexene (VCH), a dimer of 1,3-butadiene, was evaluated for reproductive toxicity in Swiss (CD-1) mice using the continuous breeding protocol (NTP, 1989). VCH in corn oil was administered by gavage at doses of 0, 100, 250, and 500 mg/kg/day to animals that were housed in same sex pairs for 1 week and then cohabited in breeding pairs for 14 weeks. During cohabitation, newborn litters were euthanized immediately after evaluation on postnatal Day (PND) 0. Litters born after Week 15 were reared until PND 21, when all F0 animals and low- and mid-dose F1 weanlings were humanely killed without a necropsy. At PND 74 +/- 10, control and high-dose F1 animals were cohabited within groups for 1 week and necropsied after delivery of the litters. In F0 breeding pairs, VCH did not affect measures of reproductive competence, including initial fertility, litters per pair, live litter size, or the proportion of pups born alive. Pup weight was decreased (4%) in the high-dose group relative to controls. High-dose F0 females exhibited slight general toxicity, manifested as an 8% difference in body weight compared to controls. VCH did not adversely affect preweaning growth or survival in the F1 generation. VCH had no effect on the reproductive competence of the F1 generation. High-dose F1 adult males and females had decreased body weight. At necropsy, increased relative liver weight (males 9% and females 8%) and sperm motility (although not thought to be biologically significant) were observed in the 500 mg/kg VCH group.(ABSTRACT TRUNCATED AT 250 WORDS)

Artificially inseminated New Zealand white (NZW) rabbits were administered ethylene glycol (EG) by gavage on Gestational Days (GD) 6 through 19 at doses of 0, 100, 500, 1000, or 2000 mg/kg/day, with 23-24 inseminated animals per group. Clinical signs were recorded and water consumption was measured daily; does were weighed on GD 0, 6-19, 25, and 30. At necropsy (GD 30), maternal liver, kidney, and gravid uterine weights were recorded. Histopathologic examination was performed on kidneys from 10 does/dose and for all unscheduled deaths. Ovarian corpora lutea were counted and uterine implantation sites (total sites, resorptions, dead and live fetuses) were recorded. All live fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and variations. EG resulted in profound maternal toxicity at 2000 mg/kg/day (42% mortality; three early deliveries and one spontaneous abortion) associated with renal pathology and unaccompanied by any other indicators of maternal toxicity. Renal lesions at 2000 mg/kg/day involved the cortical renal tubules and included intraluminal oxalate crystals, epithelial necrosis, and tubular dilatation and degeneration. No dose-related maternal toxicity occurred at 100-1000 mg/kg/day. There was no indication of developmental toxicity at any dose tested, including no effects on pre- or postimplantation loss, number of fetuses, fetal body weight, or sex ratio (% male fetuses) per litter, and no evidence of teratogenicity. The "no observable adverse effect level" (NOAEL) for maternal toxicity was therefore 1000 mg/kg/day and the NOAEL for developmental toxicity was at least 2000 mg/kg/day in this study. The sensitivity of NZW rabbits relative to that of Sprague-Dawley rats and Swiss mice for maternal and developmental toxicity from gavage administration of EG during organogenesis can be determined for maternal toxicity: rabbits > mice > rats, and for developmental toxicity, mice >> rats >> rabbits.

This paper explores the controversy among scientists on whether microscopic evaluation of tissue slides should be done in an open or blind fashion. Definitions are given and discussed that provide a better focus to the problem. An experiment was conducted in which hepatocellular degeneration and necrosis in rats were assessed both openly and blindly. The results indicate that 'simple bias' is present when the slides are read openly. Valid comparisons among treatment groups are possible in the presence of simple bias, provided appropriate control groups have been incorporated into the experimental design.

The potential reproductive toxicity of boric acid (BORA) in CD-1 mice (Swiss) was evaluated using the Reproductive Assessment by Continuous Breeding (RACB) Protocol. BORA was administered in the feed for 27 weeks to male and female Swiss (CD-1) mice at concentrations of 0, 1000, 4500, or 9000 ppm. Estimated doses, based on feed consumption and body weight, averaged 152, 636, and 1262 mg/kg body wt during Week 1 for males for 1000, 4500, and 9000 ppm, respectively. During 14 weeks of cohabitation, fertility of F0 mice was partially reduced at 4500 ppm and totally eliminated at 9000 ppm. No litters, dead or alive, were produced by 9000 ppm cohabited pairs. Among the litters born at 4500 ppm, live litter size and body weight were significantly reduced. A crossover mating trial of control and 4500 ppm groups confirmed the male as the affected sex, with fertility rates and the mating index significantly lower in the 4500 male x 0 ppm female group. At necropsy, after 27 weeks of BORA exposure, dose-related changes were present in F0 males for reduced body and reproductive organ weights, increased incidence of abnormal sperm, decreased sperm concentration and motility, and seminiferous tubule degeneration. In the 4500 ppm females, dietary BORA for 27 weeks caused significantly decreased weights of kidney/adrenals and livers; kidney/adrenal weight was also reduced in 4500 ppm males. The last litters of the control and 1000 ppm females, born in the 14-week breeding phase, were reared to 74 days of age and then mated in nonsibling pairs within treatment groups. These F1 mice had normal fertility, but the adjusted mean body weight of F2 pups was decreased. These data establish the reproductive toxicity of BORA in CD-1 mice and demonstrate that the male is the most sensitive sex.

Due to the ubiquitous presence of p-xylene in air and the existing uncertainty regarding its hepatotoxic potential, we examined the effect of acute and short-term exposure to inhaled p-xylene on the liver. Male F-344 rats were exposed to 0 or to 1600 ppm p-xylene, 6 h/d, for 1 or 3 d. Exposure to inhaled p-xylene caused no histopathological evidence of hepatic damage and had little or no effect on the serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, ornithine carbamyl transferase, alkaline phosphatase, and total bilirubin. Exposure to p-xylene for 1 or 3 d resulted in an increase in relative liver weight on d 1 post-exposure. The concentration of hepatic cytochrome P-450 was increased by both p-xylene exposure regimens on d 1 postexposure and had returned to control levels by d 3 following the single p-xylene exposure and by d 2 following the 3-d exposure. These observations provide consistent evidence that acute and short-term exposure to 1600 ppm p-xylene by inhalation did not produce overt hepatotoxicity but resulted in a significant increase in the concentration of hepatic cytochrome P-450, the principal enzyme system involved in the metabolic biotransformation of xenobiotics.