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A recent article by Gaus (2014) demonstrates a serious misunderstanding of the NTP’s statistical analysis and interpretation of rodent carcinogenicity data as reported in Technical Report 578 ( Ginkgo biloba ) ( NTP, 2013 ), as well as a failure to acknowledge the abundant literature on false positive rates in rodent carcinogenicity studies. The NTP reported Ginkgo biloba extract to be carcinogenic in mice and rats. Gaus claims that, in this study, 4800 statistical comparisons were possible, and that 209 of them were statistically significant ( p < 0.05) compared with 240 (4800 × 0.05) expected by chance alone; thus, the carcinogenicity of Ginkgo biloba extract cannot be definitively established. However, his assumptions and calculations are flawed since he incorrectly assumes that the NTP uses no correction for multiple comparisons, and that significance tests for discrete data operate at exactly the nominal level. He also misrepresents the NTP’s decision making process, overstates the number of statistical comparisons made, and ignores the fact that the mouse liver tumor effects were so striking (e.g., p < 0.0000000000001) that it is virtually impossible that they could be false positive outcomes. Gaus’ conclusion that such obvious responses merely “generate a hypothesis” rather than demonstrate a real carcinogenic effect has no scientific credibility. Moreover, his claims regarding the high frequency of false positive outcomes in carcinogenicity studies are misleading because of his methodological misconceptions and errors. [ABSTRACT FROM AUTHOR]

A rat carcinogenicity bioassay (CaBio) of quinacrine was reanalyzed to investigate its mode of tumor induction. Quinacrine’s effects in the rat uterus when administered as a slurry in methylcellulose were contrasted with the human clinical experience which uses a solid form of the drug, to determine the relevance of the tumors produced in the rat to safe clinical use of quinacrine for permanent contraception (QS). A review was performed of the study report, dose feasibility studies, and clinical evaluations of women who had undergone the QS procedure. The top three doses of quinacrine in the CaBio exceeded the maximum tolerated dose, and produced chronic damage, including inflammation, resulting in reproductive tract tumors. Chronic inflammation was significantly correlated with the tumors; there was no evidence of treatment-related tumors in animals without chronic inflammation or other reproductive system toxicity. Because such permanent uterine damage and chronic toxicity have not been observed in humans under therapeutic conditions, we conclude that this mode of action for tumor production will not occur at clinically relevant doses in women who choose quinacrine for permanent contraception. [ABSTRACT FROM AUTHOR]

Abstract: U.S. Federal Hazardous Substances Act (FHSA) regulations specify eye safety testing procedures and hazard classification criteria for substances regulated by the U.S. Consumer Product Safety Commission (CPSC). Current regulations require up to three sequential 6-animal tests. Testing consistent with the Organisation for Economic Co-operation and Development (OECD) test guideline for eye irritation/corrosion, which specifies 3 animals, can also be submitted to US agencies. However, current FHSA regulations do not provide criteria to classify results from 3-animal tests. An analysis was conducted to determine criteria using results from 3-animal tests that would provide equivalent labeling to FHSA regulations. The frequency that FHSA requirements identify substances as ocular irritants was compared with the frequency that a criterion of either ⩾1/3 or ⩾2/3 positive animals would identify these substances. A database of rabbit eye tests was also used to estimate over- and underprediction rates for each criterion. In each instance, a criterion of ⩾1/3 positive animals more closely matched the expected outcome based on FHSA requirements, while a criterion of ⩾2/3 positive animals identified far fewer irritants. Using a classification criterion of ⩾1/3 positive animals provided equivalent or greater eye hazard labeling as current FHSA requirements, while using 50–83% fewer animals. [Copyright &y& Elsevier]

Abstract: The original Organisation for Economic Co-operation and Development Test Guideline 429 (OECD TG 429) for the murine local lymph node assay (LLNA) required five mice/group if mice were processed individually. We used data from 83 LLNA tests (275 treated groups) to determine the impact on the LLNA outcome of reducing the group size from five to four. From DPM measurements, we formed all possible four- and five-mice combinations for the treated and control groups. Stimulation index (SI) values from each four-mice combination were compared with those from five-mice combinations, and agreement (both SI<3 or both SI⩾3) determined. Average agreement between group sizes was 97.5% for the 275 treated groups. Compared test-by-test, 90% (75/83) of the tests had 100% agreement; agreement was 83% for the remaining eight tests. Disagreement was due primarily to variability in animal responses and closeness of the SI to three (positive response threshold) rather than to group size reduction. We conclude that using four rather than five mice per group would reduce animal use by 20% without adversely impacting LLNA performance. This analysis supported the recent update to OECD TG 429 allowing a minimum of four mice/group when each mouse is processed individually. [ABSTRACT FROM AUTHOR]

Regression models are routinely used in many applied sciences for describing the relationship between a response variable and an independent variable. Statistical inferences on the regression parameters are often performed using the maximum likelihood estimators (MLE). In the case of nonlinear models the standard errors of MLE are often obtained by linearizing the nonlinear function around the true parameter and by appealing to large sample theory. In this article we demonstrate, through computer simulations, that the resulting asymptotic Wald confidence intervals cannot be trusted to achieve the desired confidence levels. Sometimes they could underestimate the true nominal level and are thus liberal. Hence one needs to be cautious in using the usual linearized standard errors of MLE and the associated confidence intervals. [ABSTRACT FROM AUTHOR]

The literature evidencing the role of iron in promoting a range of neoplasms in humans and animals prompted us to search for a possible association between chemically induced hemosiderosis and hemangiosarcomas in the liver of mice in selected studies conducted by the National Toxicology Program (NTP). Its historical control database was examined for studies in which treatment-related liver hemangiosarcoma was noted; 130 consecutive NTP studies in B6C3F1 mice from Technical Report (TR)-340 to TR-493 were evaluated. Three compounds (2-butoxyethanol, p -nitroaniline, and para-chloroaniline) were associated with a relatively high incidence of Kupffer cell pigmentation consisting of hemosiderin in both sexes; only the male mice developed a relatively low incidence of treatment-related hemangiosarcoma. With a fourth compound ( o -nitroanisole), a relatively low incidence (16/50, high-dose males) of chemical-related hemosiderosis was noted, with no associated increase of hemangiosarcoma. Two chemicals (pentachlorophenol and tetrafluoroethylene) increased the incidence of liver hemangiosarcoma in male and female mice, with no increase in Kupffer cell pigmentation. The overall association between liver hemangiosarcoma and Kupffer cell pigmentation was highly significant ( p < 0.001). The cause for hemosiderosis in all cases was the erythrocytic hemolytic effect of the compounds. The reason for the sex-increased susceptibility for development of hemangiosarcoma is unknown but may be due to a hormone-related, reduced antioxidative defensive capacity through modulation of the activities of antioxidative enzymes. [ABSTRACT FROM AUTHOR]

Diet is an important environmental factor affecting body weight, survival, and age-related diseases of rodents. The NIH-07 open formula diet was the diet used in the National Toxicology Program's (NTPs) rodent carcinogenicity studies from 1980 to 1994. In 1994 the NTP began using a new diet designated the NTP-2000 diet. This paper compares body weight, survival, tumor incidence, and nephropathy severity in untreated control groups of Fischer 344 (F344) rats fed the NTP-2000 or NIH-07 diets, using data from 22 separate 2-year feed and inhalation studies. The feed studies were conducted in 3 different facilities, and all the inhalation studies were conducted in a single facility. During feed studies, rats were group housed in polycarbonate cages and fed diets in powder (mash) form, while in inhalation studies, rats were housed individually in wire mesh cages, and fed diets in pelleted form. Survival was significantly ( p < 0.05) higher in groups fed NTP-2000 diet compared to the corresponding groups fed NIH-07 diet, irrespective of sex or housing conditions. Use of the NTP-2000 diet was also associated with a decreased incidence of pituitary gland tumors in both sexes and decreased incidences of adrenal pheochromocytoma and preputial gland tumors in males. The incidence and severity of nephropathy was also decreased in animals receiving the NTP-2000 diet, especially males. The decreased nephropathy severity and the decreased incidence of pituitary gland tumors are likely the major factors contributing to the improved survival of rats receiving the NTP-2000 diet relative to those given the NIH-07 diet. These data also support earlier findings that decreased incidences of adrenal pheochromocytoma are associated with reduced nephropathy severity in male F344 rats. Throughout the two-year study female rats receiving the NTP-2000 diet were significantly ( p < 0.05) lighter than those receiving the NIH-07 diet. However, it is uncertain if this difference can be attributed to the NTP-2000 diet, since implementation of this diet by the NTP approximately coincided with changes in the F344 rat production colony that resulted in somewhat lighter animals being provided to the NTP. Controls from inhalation studies and feed studies differed significantly ( p < 0.01) in the incidence of a variety of tumors, irrespective of diet. This suggests that differences in animal care and housing protocols may impact tumor incidence in F344 rats, most notably pituitary gland and testis tumors. [ABSTRACT FROM AUTHOR]

These investigations of riddelliine analyzed potential carcinogenesis and the utility of the female-rat/male-mouse design in bioassays and dose–response. Groups of 50 Fischer rats and B6C3F1 mice were gavage-administered riddelliine 5 days per week for 105 weeks. The dose levels for male rats were 0 or 1.0 mg/kg body weight; female rats 0, 0.01, 0.033, 0.1, 0.33, or 1.0 mg/kg; male mice 0, 0.1, 0.3, or 1.0, 3.0 mg/kg; and female mice 0 or 3.0 mg/kg. The dose groups were purposely designed to evaluate the dose–response relationship only in female rats and male mice. In rats, liver hemangiosarcoma, hepatocellular adenoma, and mononuclear cell leukemia were significantly increased in the 1.0 mg/kg male and female dose groups. Non-neoplastic lesions occurred in the liver and kidney of male and female rats. In mice, hemangiosarcomas increased significantly in the liver of males in the 3.0 mg/kg dose group. Alveolar/bronchiolar neoplasms in the 3.0 mg/kg dose group of female mice were significantly increased. Hepatocellular neoplasms were significantly decreased in the 1.0 mg/kg dose group of male and 3.0 mg/kg dose groups of male and female mice. Non-neoplastic lesions occurred in the liver and kidney of male and female, and lung and arteries of female mice. These studies demonstrate toxicity and carcinogenicity of riddelliine in rats and mice, and a dose–response relationship in female rats and male mice under the experimental conditions employed. [Copyright &y& Elsevier]

A study was conducted to determine the effects of dietary factors in natural ingredient and purified diets on uterine weights of immature CD-1 mice used in uterotrophic bioassays. Factors evaluated included body weight gain, dietary phytoestrogen content, total metabolizable energy, and percent crude fiber. Fifteen to 147 mice per group, housed 5 per cage, were randomly assigned to each of the 20 test diets. The test diets were fed for 7 days to 15-day old immature female CD-1 mice and their body weight gain and uterine weights were determined. Analysis of covariance procedures were used to evaluate differences in uterine weights, after adjusting for body weight and time-related trends. Fisher’s least significant difference test was used to compare adjusted uterine weights and weight gains among the test diets. Additionally, multiple linear regression procedures were used to identify those characteristics of the rodent diet that were most predictive of the adjusted uterine weights. Total metabolizable energy was significantly (P<0.01) correlated with and was predictive of uterine weights. The following dietary variables were not significantly predictive of uterine weights: total daidzein and genistein content, percent protein, fat, N-FE (carbohydrates) or percent crude fiber. We concluded that: (1) total metabolizable energy (ME) in natural ingredient or purified diets has a significant (P<0.01) effect on the uterine weights of immature mice used in 7-day uterotrophic bioassays; (2) a standardized, estrogen-free diet with a constant level of ME should be used for conducting uterotrophic assays when comparing results between different laboratories or when determining the estrogenic or anti-estrogenic activity of endocrine disruptor compounds; (3) the mouse uterotrophic assay remains a sensitive bioassay for assessing chemicals for estrogenic activity or for the detection of total estrogenic activity in rodent diets that may be contaminated with estrogenic compounds, and (4) chemical assays should be used to detect or measure low levels of the phytoestrogens in rodent diets. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Systemic hypoxemia, occurring in space-occupying lung pathologies such as inflammation and neoplasms, reduces the gas exchange area and stimulates catecholamine secretion from the adrenal medulla where chronic endocrine hyperactivity may lead to hyperplasia and neoplasia. We investigated the possible correlation between nonneoplastic chronic pulmonary lesions and adrenal pheochromocytoma in 9 recent, NTP, 2-year particulate inhalation studies in male F344 rats. Re-evaluation for chronic active inflammation, interstitial fibrosis, alveolar epithelial hyperplasia, squamous metaplasia, proteinosis, and histiocytosis revealed significant associations of pheochromocytoma only with the severity of inflammation and fibrosis. Nickel oxide, cobalt sulfate, indium phosphide, talc, and nickel subsulfide studies showed chemical-related incidences of adrenal pheochromocytoma and significant (p < 0.01) associations with inflammation and fibrosis. Gallium arsenide, vanadium pentoxide, molybdenum trioxide, and nickel sulfate hexahydrate studies revealed an increased incidence and/or severity of nonneoplastic lung lesions, but no increased incidence of pheochromocytoma. Although gallium arsenide and molybdenum trioxide showed no dose-related increase in pheochromocytoma, a significant (p < 0.01) correlation of the latter with the severity of fibrosis and inflammation occurred. In the vanadium pentoxide and nickel sulfate hexahydrate studies, no relationship between nonneoplastic lung lesions and pheochromocytoma was manifested. Our investigation assessed the strength of these various associations and supports the possible roles of 2 chronic pulmonary lesions—fibrosis and inflammation—and hypoxemia in the induction of pheochromocytoma in the F344 male rat. [ABSTRACT FROM AUTHOR]

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

Reports on the dietary restriction experience in the U.S. national toxicology program. Influence of body weights on tumor incidence; Rodent bioassay; Implications for rodent carcinogenicity studies.

Investigates the four widely used in vitro assays for genetic toxicity for their ability to predict the carcinogenicity of chemicals evaluated in long-term rodent studies by the National Toxicology Program (NTP). Methods used in the study; Results of the study; Discussion of the study; Conclusions.

Presents a response by Joseph K. Haseman to the letters to the editor from K.K. Rozman and W.J. Waddell, about his criticism of their log linear threshold-based extrapolation model.

The NTP has a long history of using Fischer rats and has compiled a large database of incidences of lesions seen in control animals. Such a database is lacking for Harlan Sprague–Dawley (SD) rats. The intention of this paper is to report spontaneous lesions observed in female vehicle control Harlan SD rats, and to compare the incidence in 2 strains of rats (Fischer and Harlan SD) used in NTP studies. Female Harlan SD rats served as the test animals for a special series of 2-year studies. Male rats were not used in these studies. Complete histopathology was performed on all animals, and the pathology results underwent comprehensive NTP pathology peer review. The most commonly observed neoplasms in these female control Harlan SD rats were mammary gland fibroadenoma (71%), tumors of the pars distalis of the pituitary (41%) and thyroid gland C-cell tumors (30%). Female Fischer rats had incidences of 44% for mammary gland fibroadenomas, 34% for tumors of the pars distalis, and 16% for thyroid gland C-cell tumors. Fischer rats had a 15% incidence of clitoral gland tumors, while the Harlan SD rats had an incidence of [ABSTRACT FROM AUTHOR]

Dipropylene glycol (DPG) is a component of many commercial products such as antifreeze, air fresheners, cosmetic products, solvents, and plastics. Male and female F344/N rats and B6C3F1 mice were exposed to DPG in the drinking water for 2 weeks, 3 months, or 2 years. In the 2-week and 3-month studies, rats and mice were exposed to 0, 5000, 10,000, 20,000, 40,000, or 80,000ppm DPG. There was no mortality in the 2-week studies. In the 3-month rat study, all animals survived to the end of the study. Liver weights of rats exposed to 10,000ppm or greater and kidney weights of rats exposed to 40,000 and 80,000ppm were greater than those of the controls. The incidences of liver and kidney lesions were significantly increased in males exposed to 20,000ppm or greater and females exposed to 80,000ppm. Focal olfactory epithelial degeneration was present in all rats exposed to 80,000ppm. In males, the incidences of testicular atrophy, epididymal hypospermia, and preputial gland atrophy were significantly increased in the 80,000ppm group. In the 3-month mouse study, three males and one female exposed to 80,000ppm died. Liver weights were increased, as was the incidence of centrilobular hypertrophy in males exposed to 40,000ppm and males and females exposed to 80,000ppm. In the 2-year studies, exposure groups were 0, 2500 (rats only), 10,000, 20,000 (mice only) or 40,000ppm DPG. Survival of male rats exposed to 40,000ppm and mean body weights of males and females exposed to 40,000ppm were significantly less than controls. In male rats, exposure to DPG resulted in increased incidences and severities of nephropathy and secondary lesions in the parathyroid and forestomach. Increased incidences of focal histiocytic and focal granulomatous inflammation of the liver were also observed. In male and female rats, there were increased incidences of bile duct hyperplasia and changes in the olfactory epithelium of the nose. In mice, survival of males and females was similar to controls. Mean body weights and water consumption of males exposed to 40,000ppm were less than that of the controls. Treatment-related nonneoplastic lesions did not occur in mice. Treatment-related neoplastic lesions did not occur in rats or mice. [Copyright &y& Elsevier]

Presents a study which focused on the grading scheme for the assessment of proliferative lesions of the mouse prostate in the Transgenic Adenocarcinoma of Mouse Prostate model. Information on the grading system; Experimental design; Discussion.

Epidemiological studies suggest an association between ambient particulate matter and cardiopulmonary diseases in humans. The mechanisms underlying these health effects are poorly understood. Tobetter understand the potential relationship between particulate-matter-induced inflammation and vascular disease, a 2-phase retrospective study was conducted. Phase one included the review of heart, lung, and kidney tissues from high-dose and control male B6C3F1 mice exposed by inhalation to 9 particulate compounds for a 2-year period. The results showed that high-dose males developed significantly increased incidences of coronary and renal arteritis over controls in 2 of the 9 studies (indium phosphide and cobalt sulfate heptahydrate), while marginal increases in arteritis incidence was detected in 2 additional studies (vanadium pentoxide and gallium arsenide). In contrast, arteritis of the muscular arteries of the lung was not observed. Morphological features of arteritis in these studies included an influx of mixed inflammatory cells including neutrophils, lymphocytes, and macrophages. Partial and complete effacement of the normal vascular wall architecture, often with extension of the inflammatory process into the periarterial connective tissue, was observed. Phase 2 evaluated the heart, lung, kidney, and mesentery of male and female B6C3F1 mice from the 90-day studies of the 4 compounds demonstrating arteritis after a 2-year period. The results showed arteritis did not develop in the 90-day studies, suggesting that long-term chronic exposure to lower-dose metallic particulate matter may be necessary to induce or exacerbate arteritis. [ABSTRACT FROM AUTHOR]

Because sampling of the paired lobes (ventral, dorsal, lateral, and anterior) of the mouse prostate has often been inconsistent, comparisons among different investigations have lacked validity.The absence of site identification for prostatic lesions has made reported incidences relatively nonspecific. We present here the lobe-specific incidences and degree of severity of spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands in 612 control B6C3F[sub 1] mice from 12 recent National Toxicology Program 2-year carcinogenicity and toxicity studies conducted in 1 of 4 different laboratories. Lymphocytic infiltration, inflammation, epithelial hyperplasia, mucinous cyst, and mucinous metaplasia were observed in the dorsolateral lobes. Lymphocytic infiltration, inflammation, epithelial hyperplasia, and edema were present in the ventral lobes. Lymphocytic infiltration, acinar dilatation, inflammation, epithelial hyperplasia, and atrophy occurred in the coagulating glands. No neoplastic lesions were observed in the prostate or coagulating gland. Lymphocytic infiltration, acinar dilatation, inflammation, atrophy, adenoma, adenocarcinoma, and a granular cell tumor were observed in the seminal vesicles. Lymphocytic infiltration was also present in the ampullary glands. The results of our survey indicate thatthe amounts of glandular tissues were not present consistently in slides from the different laboratories.Landmarks for uniform tissue trimming are needed. We therefore suggest an optimal trimming and embedding method for mouse prostate and seminal vesicles to ensure adequate, consistent sampling. [ABSTRACT FROM AUTHOR]

Peroxisome proliferators are non-mutagenic carcinogens in the liver of rodents, acting both as initiators and promoters. The National Toxicology Program (NTP) conducted a study of several peroxisome proliferators (PPs), including Wyeth (WY)-14643 as a prototypical PP and 2,4-dichlorophenoxyacetic acid (2,4-D) as a weak PP, in Sprague-Dawley rats, B6C3F1 mice, and Syrian hamsters. In the kidney, an unusual change was observed in the outer stripe of the outer medulla, especially in rats treated with 2,4-D or WY-14643. This change was characterized by foci of tubules that were partially or completely lined by basophilic epithelial cells with decreased cytoplasm and high nuclear density. Changes typical of chronic nephropathy such as interstitial fibrosis or basement membrane thickening were not associated with these foci. Results of immunohistochemical staining for catalase and cytochrome P-450 4A in the kidney indicated increased staining intensity in renal tubular epithelial cells primarily in the region where the affected tubules were observed; however, the altered cells were negative for both immunohistochemical markers. Ultrastructurally, affected cells had long brush borders typical of the P3 tubule segment. The most distinguishing ultrastructural change was a decreased amount of electronlucent cytoplasm that contained few differentiated organelles and, in particular, a prominent reduced volume and number of mitochondria; changes in peroxisomes were not apparent. In addition to the lesion in rats, mice treated with the highest dose of 2,4-D, but not WY-14643, manifested similar renal tubular changes as seen by light microscopy. Neither chemical induced renal tubular lesions in hamsters. Hepatocellular changes characteristic of PPs were present in all 3 species treated with WY-14643, but not 2,4-D. These results indicate that the rat is the species most sensitive to the nephrotoxic effects of PPs and there is a site specificity to this toxicity related to areas of PP-related enzyme induction. Although 2,4-D is considered a weak PP for the liver, it was the most effective at inducing renal lesions, indicating that the toxic potency of various PPs will depend on the target organ. [ABSTRACT FROM AUTHOR]

The carcinogenic potential of a non-genotoxic pyrethroid imiprothrin was examined in rats and mice. There was no carcinogenicity in rats up to a maximum dose of 5000 ppm of the diet. There was a higher (p = 0.03) incidence of lung adenocarcinomas at 7000 ppm in males, and females showed an increasing (p < 0.01) trend in the incidence of lung adenomas and combined lung adenoma/adenocarcinomas. Additional step sections of lung demonstrated no significant increases in any tumor at p < 0.05, although an increasing trend with dose was observed among females. We argue that, the 7000 ppm dose exceeded the Maximum Tolerated Dose (MTD) for both sexes, based on systemic toxicity as evidenced by body weight gain reduction (both sexes) and high mortality (females). If the 7000 ppm dose is therefore removed from consideration, there are not significant (p < 0.05) increases in tumor formation. Moreover, a consideration of multiple comparisons reveals that the lung tumor increases observed are totally consistent with what would be expected by chance alone. Based on high susceptibility of this mouse strain for the appearance of lung tumors and the lack of a statistically significant increase in tumors by appropriate analysis, the mouse study does not indicate a carcinogenic effect of imiprothrin, and thus no classification for carcinogenicity is warranted. Image 1 • There was no carcinogenic activity in rats up to 5000 ppm of the diet. • The Maximum Tolerated Dose was exceeded at 7000 ppm for both male and female mice. • Increases in lung adenocarcinoma was observed in male mice at the highest dose. • Increased trends of lung tumor was due to a toxic high dose that exceeded the MTD. • Multiple comparisons reveal that the mouse lung tumors were consistent with chance. [ABSTRACT FROM AUTHOR]

BACKGROUND: The optimum test diet and rodent species/strain for evaluating endocrine-disrupting compounds (EDCs) are critical. OBJECTIVES: We conducted studies to evaluate rodent species sensitivity and the effects of diets varying in phytoestrogen content on the time of vaginal opening (VO) in CD-1 mice, Fischer 344 (F344) rats, and CD Sprague-Dawley (S-D) rats. METHODS: Mice were weaned on postnatal day (PND) 15 and rats on PND19 and randomly assigned to control or test diets. Body weights, food consumption, and time of VO were recorded. RESULTS: The time of VO was significantly advanced in F344 rats fed diets containing daidzein and genistein, whereas these same diets did not advance VO in S-D rats. When animals were fed the AIN-76A diet spiked with genistein, time of VO was significantly advanced at all doses in CD-1 mice, at the two highest doses in F344 rats, and at the highest dose in S-D rats. The time of VO in F344 rats was more highly correlated with the phytoestrogen content than with the total metabolizable energy (ME) of 12 diets. CONCLUSIONS: The S-D rat is less sensitive to dietary phytoestrogens compared with the F344 rat or the CD-1 mouse, suggesting that the S-D rat is not the ideal model for evaluating estrogenic activity of EDCs. The profound effects of dietary phytoestrogens on the time of VO, an estrogensensitive marker, indicate that a standardized open-formula phytoestrogen-free diet containing a low ME level should be used to optimize the sensitivity of estrogenic bioassays. [ABSTRACT FROM AUTHOR]

Abstract: In a 2-year NTP bioassay, Bromoethane (BE) was found to induce endometrial neoplasms in the uterus of B6C3F1 mice [; ]. In women, hormonal influences, such as “unopposed” estrogenic stimulus, have been implicated as important etiologic factors in uterine cancer. BE, however, does not affect the serum concentrations of sex hormones in female B6C3F1 mice [] and the mechanism of BE-induced uterine carcinogenesis still remains unclear. In the present study, we examined the estrogenic effects of BE on the uterus of ovariectomized B6C3F1 mice and on Ishikawa cells. Groups of 6 mice were given daily s.c. injections of 0, 100, 500 or 1000 mg BE/kg for 3 consecutive days. Mice treated with 17β-estradiol served as positive controls. Mice were necropsied 24 h after the final injection, and uteri were weighed and examined histologically and immunohistochemically along with the vagina. Changes observed in the estrogen-treated mice included increased uterine weights, edema and inflammation of the endometrium, increased epithelial layers of the uterine and vaginal lumens and keratinization of the vaginal epithelium. In the BE-treated mice, no such changes occurred; however, immunohistochemical staining of the uterus revealed a significant increase in immunoexpression of the estrogen receptor alpha (ERα) in the two higher dose groups. Analysis of mRNA also showed slightly increased uterine ERα expression in these groups. Upregulated expression of ERα was confirmed in BE-treated Ishikawa cells, in which Western blotting analyses identified an intense signal at approximately 66 kDa, which is consistent with ERα. These data suggest that upregulated expression of ERα may be important in the induction of endometrial neoplasms in BE-treated mice. [Copyright &y& Elsevier]

Use of the dioxin toxic equivalency factor (TEF) approach in human risk assessments assumes that the combined effects of dioxin-like compounds in a mixture can be predicted based on a potency-adjusted dose-additive combination of constituents of the mixture. In this study, we evaluated the TEF approach in experimental 2-year rodent cancer bioassays with female Harlan Sprague-Dawley rats receiving 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of the three compounds. Statistically based dose-response modeling indicated that the shape of the dose-response curves for hepatic, lung, and oral mucosal neoplasms was the same in studies of the three individual chemicals and the mixture. In addition, the dose response for the mixture could be predicted from a combination of the potency-adjusted doses of the individual compounds. Finally, we showed that use of the current World Health Organization dioxin TEF values adequately predicted the increased incidence of liver tumors (hepatocellular adenoma and cholangiocarcinoma) induced by exposure to the mixture. These data support the use of the TEF approach for dioxin cancer risk assessments. [ABSTRACT FROM AUTHOR]

Propylene glycol mono-t-butyl ether (PGMBE) is used as a solvent in a variety of commercial applications. Male and female F344/N rats and B6C3F1 mice were exposed to PGMBE by whole-body inhalation for 2 or 14 weeks (0, 75, 150, 300, 600, or 1200 ppm) or 2 years (0, 75, 300, or 1200 ppm); male NBR rats were exposed for 2 weeks. The kidney and the liver were targets of PGMBE toxicity in rats. Renal lesions suggestive of α2u-globulin nephropathy were observed in male F344/N, in the 2 and 14-week studies, no kidney lesions were seen in NBR rats. In the 2-year study, male rats displayed exposure-related nonneoplastic lesions in the kidney, and may have shown marginal increases in tubular neoplasms. In the liver, the incidences of hepatocellular adenomas occurred with a positive trend in male rats, and may have been related to PGMBE exposure. In mice of both sexes, the major target of PGMBE toxicity was the liver. In the 2-week study, liver weights and in the 14-week study, liver weights and the incidences of centrilobular hypertrophy were increased. In the 2-year study, the incidences of exposure-related hepatocellular adenoma, adenoma or carcinoma combined, and hepatoblastoma occurred with a positive trend, and were significantly increased in 1200 ppm groups. In summary, exposure to PGMBE resulted in nonneoplastic lesions of the kidney characteristic of α2u-globulin nephropathy, and may have increased renal tubular neoplasms in male rats. Exposure to PGMBE also produced increases in hepatic tumors in male and female mice. [Copyright &y& Elsevier]

We evaluated the effect of chronic exposure to dioxin and dioxin-like compounds on the pancreas in female Harlan Sprague-Dawley rats. This investigation represents part of an ongoing National Toxicology Program initiative to determine the relative potency of chronic toxicity and carcinogenicity of polychlorinated dioxins, furans, and biphenyls. Animals were treated by gavage for up to 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a toxic-equivalency-factor (TEF) mixture of these agents; control animals received corn oil-acetone vehicle alone. A complete necropsy was performed on all animals, and a full complement of tissues was collected and examined microscopically. Administration of each of the four compounds was associated with increased incidences of several nonneoplastic changes in the exocrine pancreas, including cytoplasmic vacuolation, chronic active inflammation, atrophy, and arteritis. Low incidences, but higher than those in the historical database, of pancreatic acinar adenoma and carcinoma were seen in the TCDD, PeCDF, and TEF-mixture groups. These results indicate that the pancreatic acini are target tissues for dioxin and certain dioxin-like compounds. [ABSTRACT FROM AUTHOR]

2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) affects the thyroid morphologically and/or functionally in adult animals. Recently, the National Toxicology Program conducted a 2-year gavage study of TCDD in female Harlan Sprague-Dawley rats. The only treatment-related alterations found in thyroid follicles were decreased luminal size and increased height of the follicular epithelial cells, without prominent protrusion into the lumen. The present study elucidated the nature of these follicular lesions. Thyroid glands of 10 rats each from the control, high (100 ng/kg/day)-dose, and stop-study (100 ng/kg/day, 30 weeks; vehicle to study termination) groups in the 2-year study were evaluated microscopically. Twenty randomly selected follicles were measured morphometrically in each animal. TCDD treatment significantly decreased the mean ratio of luminal/epithelial areas and increased the mean sectional epithelial height of the high-dose group compared to controls. Thyroid sections were immunostained with antibody against minichromosome maintenance (MCM) proteins, a novel cell-cycle biomarker. The MCM labeling index of the high-dose group was significantly higher than that of the control; however, the TUNEL labeling index was also higher in the high-dose group than the control. All data from the stop group were comparable to those from controls. These results indicate that the follicular cell response was hypertrophic and reversible. This information should contribute to diagnosis of nonneoplastic thyroid follicular lesions in rats. [ABSTRACT FROM AUTHOR]

The present study characterized the immunohistochemical localization of β-catenin protein in hepatocellular neoplasms and hepatoblastomas in B6C3F1 mice exposed to diethanolamine (DEA) for 2 years and evaluated genetic alterations in the Catnb and H-ras genes which are known to play important roles in the pathogenesis of liver malignancies. Genomic DNA was isolated from paraffin sections of each liver tumor. Catnb exon 2 (corresponds to exon 3 in human) genetic alterations were identified in 18/18 (100%) hepatoblastomas from DEA exposed mice. Deletion mutations (15/18, 83%) were identified more frequently than point mutations (6/18, 33%) in hepatoblastomas. Eleven of 34 (32%) hepatocellular adenomas and carcinomas from DEA treated mice had mutations in exon 2 of the β-catenin gene, while only 1 of 10 spontaneous neoplasms had a deletion mutation of codon 5–6. Common to all liver neoplasms (hepatocellular adenomas, carcinomas and hepatoblastomas) was membrane staining for the β-catenin protein, while cytoplasmic and nuclear staining was observed only in hepatoblastomas. The lack of H-ras mutations in hepatocellular neoplasms and hepatoblastomas suggests that the ras signal transduction pathway is not involved in the development of liver tumors following DEA exposure which is different from that of spontaneous liver tumors that often contain H-ras mutations. [Copyright &y& Elsevier]

In this article, we examine existing data on the use of transgenic mouse models for identification of human carcinogens. We focus on the three most extensively studied of these mice, Trp53+/-, Tg/AC, and RasH2, and compare their performance with the traditional 2-year rodent bioassay. Data on 99 chemicals were evaluated. Using the International Agency for Research on Cancer/Report on Carcinogens determinations for the carcinogenicity of these chemicals to humans as the standard for comparison, we evaluated a variety of potential testing strategies ranging from individual transgenic models to combinations of these three models with each other and with traditional rodent assays. The individual transgenic models made the "correct" determinations (positive for carcinogens; negative for noncarcinogens) for 74-81% of the chemicals, with an increase to as much as 83% using combined strategies (e.g., Trp53+/- for genotoxic chemicals and RasH2 for all chemicals). For comparison, identical analysis of chemicals in this data set that were tested in the 2-year, two-species rodent bioassay yielded correct determinations for 69% of the chemicals. However, although the transgenic models had a high percentage of correct determinations, they did miss a number of known or probable human carcinogens, whereas the bioassay missed none of these chemicals. Therefore, we also evaluated mixed strategies using transgenic models and the rat bioassay. These strategies yielded approximately 85% correct determinations, missed no carcinogens, and cut the number of positive determinations for human noncarcinogens in half. Overall, the transgenic models performed well, but important issues of validation and standardization need further attention to permit their regulatory acceptance and use in human risk assessment. [ABSTRACT FROM AUTHOR]

The TRAMP model and human prostatic cancer (PCA) cell lines DU145 and PC3 are useful for chemopreventive studies. We compared the efficacy of 3 anti-oxidants [a water-soluble natural anti-oxidant, NAO (200 mg/kg), found in spinach leaves; epigallocatechin-3 gallate, EGCG (200 mg/kg), a major green tea polyphenol; and N-acetylcysteine, NAC (125 mg/kg)] plus vehicle in slowing spontaneous tumorigenic progression in TRAMP and wild-type male mice. Sacrifices occurred on weeks 5, 9, and 13. Prostatic histopathology and oxidative-stress blood markers were evaluated. Hyperplasias were ranked by a combination of severity grade and distribution (focal, multifocal, and diffuse). The effectivity of each tested compound in reducing the severity/focalness of hyperplasia varied from lobe to lobe. NAO exerted a significant effect on the dorsal and lateral lobes; NAC, on the anterior and ventral lobes, and EGCG, on the ventral lobe. When the most severe hyperplasia in all 4 lobes of TRAMPs was evaluated, only NAO reduced hyperplasia at weeks 9 and 13. Plasma peroxide levels in TRAMPs were reduced following oral administration of NAO or NAC for 13 weeks; EGCG only slightly reduced these levels. In NAO-treated DU145 and PC3 PCA cells, inhibition of cellular proliferation occurred in a dose-dependent manner, increasing numbers of G 1 cells and reducing ROS levels. The anti-oxidative and antiproliferative properties of NAO may explain its efficacy in slowing the spontaneous prostatic carcinogenic process in the TRAMP and its effects in the cell lines. [ABSTRACT FROM AUTHOR]

Diazoaminobenzene (DAAB), a manufacturing intermediate metabolized primarily to the known carcinogens benzene and aniline, has been identified as an impurity in a number of dyes and coloring agents that are components of cosmetics, food products, and pharmaceuticals. Several structural analogs of DAAB are carcinogenic as well. DAAB was selected for metabolism and toxicity studies by the National Toxicology Program (NTP) based on the potential for human exposure, positive Salmonella data, and lack of adequate toxicological data. In the toxicology studies in mice, DAAB exhibited properties similar to benzene and aniline. Because both these metabolites induce micronuclei (MN) in rodent bone marrow erythrocytes, DAAB was tested for induction of micronuclei in male B6C3F1 mice. DAAB was administered twice by corn oil gavage at 24 h intervals, at doses of 25, 50, and 100 mg/kg per day. In addition, comparative micronucleus tests were conducted with benzene, aniline, and a mixture of benzene plus aniline; doses were based on the respective molar equivalents of each metabolite to DAAB. It was hypothesized that any observed increase in micronuclei seen in DAAB-treated mice would be due primarily to the effects of the benzene metabolite, as benzene is a more potent inducer of chromosomal damage than aniline. Results of this study showed that DAAB and benzene were effective inducers of micronuclei, with stronger responses noted for DAAB at higher doses. Positive results were also obtained with the mixture of benzene and aniline, although the magnitude of the response was lower than for DAAB. Aniline gave a weak positive response at doses exceeding its molar equivalent to 100 mg/kg DAAB. Overall, the data indicated that DAAB is a potent inducer of micronuclei in mice, and its activity appears to be closely related to the activity of benzene, one of its primary metabolites. The results are consistent with a prediction of carcinogenicity for DAAB. [Copyright &y& Elsevier]

Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3′ exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol[sup +] Exo[sup +]) enzyme, an exonuclease-deficient mutator variant (Pol[sup +] Exo[sup -]), mutator variants with substitutions at Tyr[sup 567] in the polymerase active site (Pol[sup M] Exo[sup +]), and the double mutator Pol[sup M] Exo[sup -]. Comparing the mutational spectra of the Pol[sup +] Exo[sup -] and Pol[sup +] Exo[sup +] enzymes revealed the patterns and efficiencies of proofreading, while Tyr[sup 567] was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rams in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others. [ABSTRACT FROM AUTHOR]

Over the last 8 years, a 5-fold increase in the incidence of mice with spontaneous hepatoblastomas and a moderate increase in the incidence of chemically induced hepatoblastomas in B6C3F1 mice occurred in 2-year NTP studies compared to the previous 7 years. There was a positive association between an increased incidence of mice with hepatoblastoma and an increased incidence of mice with hepatocellular tumors in the treated mice. The rate of pulmonary metastases for hepatoblastoma was similar to that of pulmonary metastasis for hepatocellular carcinomas. Although a variety of chemicals caused an increased incidence of mice with hepatoblastoma, there was no apparent association between a specific chemical structure or a biological class of compounds and their capacity to induce hepatoblastomas. Hepatoblastomas frequently arose within hepatocellular carcinomas or adenomas and were induced by the same compounds that induced hepatocellular neoplasms. Therefore, it seems reasonable to combine the incidence of mice with hepatoblastomas and the incidence of mice with hepatocellular carcinomas in hazard identification studies. [ABSTRACT FROM AUTHOR]

Presents information on a study which investigated the effects of antiretrovirals on lymphocyte and sperm chromosomes and semen quality. Methodology; Results and discussion.

Because the paired lobes (ventral, dorsal, lateral, and anterior) of the rat prostate have not been consistently sampled in many carcinogenicity and toxicity studies, comparison among different investigations has been compromised. The lack of specific site identification for prostatic lesions further lessens the value of incidences reported. We present here the lobe-specific incidences and degree of severity of background prostatic, seminal vesicular, and ampullary glandular lesions in 1768 control Fischer-344 rats from 35 recent National Toxicology Program 2-year carcinogenicity and toxicity studies conducted in 4 laboratories. The dorsal and lateral lobes were combined and considered the dorsolateral lobe where inflammation, epithelial degeneration, mucinous cysts, and edema were observed. Inflammation in the dorsolateral lobes was significantly associated with pituitary gland adenoma whose prolactin was suggested to play an important role in pathogenesis of prostatic inflammation. Epithelial degeneration, epithelial hyperplasia, inflammation, edema, and adenoma were conspicuous in the ventral lobes. Inflammation and edema occurred in the anterior lobes (coagulating glands). Inflammation, dilatation, epithelial hyperplasia, edema, and adenoma were observed in the seminal vesicles. Inflammation was also present in the ampullary glands. We suggest an optimal embedment and trimming method in rat prostate and seminal vesicle to ensure adequate, consistent sampling. [ABSTRACT FROM AUTHOR]

Four widely used in vitro assays for genetic toxicity were evaluated for their ability to predict the carcinogenicity of selected chemicals in rodents. These assays were mutagenesis in Salmonella and mouse lymphoma cells and chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Seventy-three chemicals recently tested in 2-year carcinogenicity studies conducted by the National Cancer Institute and the National Toxicology Program were used in this evaluation. Test results from the four in vitro assays did not show significant differences in individual cobcordance with the rodent carcinogenicity results; the concordance of each assay was approximately 60 percent. Within the limits of this study there was no evidence of complementarity among the four assays, and no battery of tests constructed from these assays improved substantially on the overall performance of the Salmonella assay. The in vitro assays which represented a range of three cell types and four end points did show substantial agreement among themselves, indicating that chemicals positive in one in vitro assay tended to be positive in the other in vitro assays. [ABSTRACT FROM AUTHOR]