Abstract  The National Toxicology Program rodent cancer bioassay program design evolved from that of the National Cancer Institute in the 1970s. Groups of 50 or more mice are assigned to control or treatment groups. Test substances are given at three dose levels by intubation, dietary or drinking water consumption, or dermal or inhalation exposure. Dosing starts at age 5-6 weeks and lasts for 2 years, when surviving animals receive a complete histopathologic examination. Statistical approaches accommodate survival differences and no longer require differentiation between fatal and incidental tumors. Photocarcinogenicity studies, employing SKH-1 hairless mice, evaluate onset of skin papillomas and incidences at 1 year. Top doses are chosen to expose animals to a minimally toxic challenge and lower doses to operate within the linear range of kinetics. This dosing allows comparison of results across studies. Bioassay and ancillary studies successfully identify tumor-causing agents in rodents, provide information on dose-response, and characterize other chemical-related toxicities. NTP and Ramazzini Foundation bioassay designs differ in several aspects, but bioassays at both institutions provide chemical-specific information for predicting human carcinogens, thus providing for protection of public health. Bioassays constitute an essential information reference set for new assay development and further investigations into mechanisms of action. The scientific community and the public owe a huge debt of gratitude to Dr. Cesare Maltoni of the European Foundation of Oncology and Environmental Sciences and to Dr. David P. Rall of the National Institute of Environmental Health Sciences for their foresight and wisdom in creating and nurturing these bioassay programs.

Abstract  Lymphomas were reported to be induced in rats in bioassays of aspartame, methyl-tertiary-butyl ether (MTBE), and other chemicals conducted by a nonprofit cancer research organization. European regulatory authorities concluded that lymphomas in the aspartame study were caused by Mycoplasma pulmonis and suggested that this also was the case for the MTBE bioassay. To assess the role of M. pulmonis in these bioassays, we reviewed the tumor data for the aspartame and MTBE bioassays and, additionally, the organization's bioassay of methanol. For all 3 studies, the most frequently reported hematopoietic neoplasm was lympho-immunoblastic lymphoma, the most frequently affected organ was the lung, and, in almost half of the rats with this diagnosis, the lung was the only affected organ. Lesions diagnosed as lymphoma in published illustrations had pleomorphic cellular morphology and appeared to contain neutrophils. Information from these reports and other sources indicated that lesions typical of M. pulmonis disease were prevalent among the aspartame and MTBE study rats and that the rats were not specific-pathogen?free. Because the lymphoma type, cellular morphology, and organ distribution reported in these studies are atypical of lymphoma in rats, because lymphocyte and plasma cell accumulation in the lung is characteristic of M. pulmonis disease, and because M. pulmonis disease can be exacerbated by experimental manipulations, including chemical treatment, we suggest that a plausible alternative explanation for the reported results of these bioassays is that the studies were confounded by M. pulmonis disease and that lesions of the disease were interpreted as lymphoma.

Abstract  Use of laboratory animals to identify carcinogenic potential of chemicals, mixtures, and other agents has a modern history of greater than 40 years from which much useful scientific and public health information can be derived. While laboratory animals differ from humans in some respects that may affect responses to hazardous exposures, use of such models is based on experimental evidence indicating that there are more genetic, genomic, physiological, biochemical, and metabolic similarities than differences among mammalian species. Issues of concordance of responses between rodent species and between rodents and humans as well as repeatability and site-specificity are important considerations in evaluating laboratory animal carcinogenicity results. Variables in experimental design such as animal strain, diet, route of exposure, and study duration as well as single-site versus multisite carcinogenic responses all influence interpretation and intelligent use of study data. Similarities and differences in site-specific laboratory animal and corresponding human cancers should also be considered in study evaluation. Recent attempts to explore genetically engineered mice and to humanize the mouse for more relevant identification of carcinogen hazard identification have yielded mixed results. In the end we are confronted by the realization that virtually all animal cancer models are useful but imperfect surrogates for humans. Assuming the percentage of chemicals currently in commerce that are estimated to be potent animal or human carcinogens is quite low, the task of identifying agents with significant carcinogenic potential is daunting and important. The biological conundrum of scientific debate regarding the relevance of carcinogenicity studies in laboratory animals is likely to continue. Nonetheless public health considerations must take precedence when deciding human safety issues.

Abstract  Aspartame (APM) or L-aspartyl-L-phenylalanine, and a 3 : 1 combination of APM with its decomposition product, 5-benzyl-3,6-dioxo-2-piperazine acetic acid (DKP), were incorporated at levels of up to 4 g/kg in the diet of male and female Wistar rats from 6 weeks to 104 weeks of age. There was a dose-dependent depression of body weight gain at 2 and 4 g/kg AMP and at 4 g/kg APM + DKP in males, and at all dose levels in females, correlated with decreased food consumption and attributed to liberation of amino acids from hydrolysis of APM. Increases in urinary specific gravity and pH, with increase of relative kidney weight, were attributed to the urinary excretion DKP and acidic metabolites of APM. A dose-related increase in urinary calcium probably reflected increased calcium absorption, as from high protein diets. A slight decrease in serum cholesterol and an increase in relative spleen weight appeared to be without adverse effect. It is concluded that the treatments were without toxic effect.

Abstract  An international, interdisciplinary working group of expert scientists met in June 2004 to develop IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans (IARC Monographs) on formaldehyde, 2-butoxyethanol, and 1-tert-butoxy-2-propanol. Each IARC Monograph includes a critical review of the pertinent scientific literature and an evaluation of an agent's potential to cause cancer in humans. After a thorough discussion of the epidemiologic, experimental, and other relevant data, the working group concluded that formaldehyde is carcinogenic to humans, based on sufficient evidence in humans and in experimental animals. In the epidemiologic studies, there was sufficient evidence that formaldehyde causes nasopharyngeal cancer, "strong but not sufficient" evidence of leukemia, and limited evidence of sinonasal cancer. The working group also concluded that 2-butoxyethanol and 1-tert-butoxy-2-propanol are not classifiable as to their carcinogenicity to humans, each having limited evidence in experimental animals and inadequate evidence in humans. These three evaluations and the supporting data will be published as Volume 88 of the IARC Monographs.

Abstract  These guidelines revise and replace the U.S. Environmental Protection Agency’s (EPA’s, or the Agency’s) Guidelines for Carcinogen Risk Assessment, published in 51 FR 33992, September 24, 1986 (U.S. EPA, 1986a) and the 1999 interim final guidelines (U.S. EPA, 1999a; see U.S. EPA 2001b). They provide EPA staff with guidance for developing and using risk assessments. They also provide basic information to the public about the Agency's risk assessment methods.

Abstract  Imaging mass spectrometry is becoming a key technology for the investigation of the molecular content of biological tissue sections in direct correlation with the underlying histology. Much of our work has been done with fresh-frozen tissue sections that has undergone minimal protein degradation between the time a tissue biopsy is sampled and the time it is snap-frozen so that no preserving or fixing agents need to be added to the frozen biopsy. However, in many sampling environments, immediate flash freezing may not be possible and so we have explored the use of ethanol-preserved, paraffin-embedded tissue specimens for proteomic analyses. Solvent-only preserved tissue specimens provide long-term preservation at room temperature, generation of high quality histological sections and little if any chemical alteration of the proteins. Using mouse organs, several key steps involved in the tissue dehydration process have been investigated to assess the potential of such preserved specimens for profiling and imaging mass spectrometry investigations.

Abstract  Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

Abstract  Peer review of histopathology findings in safety assessment studies involving rodents and other animals is a relatively recent procedure in toxicologic pathology. It serves to ensure the integrity of the pathology evaluation in safety studies, encourages consistency of diagnostic criteria and use of common terminology, and provides a method of continuing education for participants. The use of a standardized system of pathology nomenclature and diagnostic criteria, such as the Society of Toxicologic Pathologist's Guides for Toxicologic Pathology, is of great value in the procedure. Pathology reviews may involve government-sponsored bioassay programs, in-house industrial corporations, or individual peer reviews suggested or required by government regulatory agencies. Pathology Working Groups can be an integral part of the review process. The extent of the peer review is primarily dependent on the study results; however, other variables such as confidence of the data, study size and duration, complexity, and purpose are also important considerations. Essential components of any peer review, however, include selection of tissues/lesions for review, by a reviewing pathologist, discrepancy resolution, data modification, and documentation of all aspects of the review process. Specific procedures for pathology peer review are discussed. Disagreements among pathologists discovered in peer reviews can be resolved by several methods and examples will be presented. The entire pathology peer review process should be a learning experience for all involved and can help ensure the integrity of animal toxicology studies used for important regulatory decisions involving the use of chemicals in our society.

Abstract  The authors recently assessed the likelihood that lifetime cancer bioassays of aspartame, methanol, and methyl tertiary butyl ether conducted with conventional (not specific pathogen free) Sprague-Dawley rats were compromised by Mycoplasma pulmonis disease. From the tumor data and other information, the authors concluded that the rats used in these bioassays likely had M pulmonis disease and that lesions of the disease were plausibly interpreted as lymphoma. Subsequently, they analyzed the nonneoplastic lesion data from these bioassays for occurrence of inflammatory lesions and found that 2,267 of 2,960 rats (76.6%) were reported to have bronchitis, the signature lesion of M pulmonis disease, and that 633 rats (21.4%) were reported to have otitis, another common lesion of the disease. Also, documentation is now available containing serologic evidence of mycoplasma infection in the rats. In contrast, the reports of 6 National Toxicology Program bioassays based on specific pathogen-free Sprague-Dawley rats listed no instances of bronchitis or otitis. These findings provide substantial additional evidence that the bioassays in question were compromised by M pulmonis disease. Therefore, the reported induction of lymphoma in these studies should not be considered in cancer risk assessments. The authors also found that inflammatory lesions were prevalent in lymph nodes, thymus, pleura, and brain. Finally, they found that of all 328 cases of lymphoimmunoblastic lymphoma affecting the lung (the primary form of lymphoma reported), 218 (66.5%) occurred within the first 104 weeks of the studies, showing that occurrence of such lesions was not due to appearance in rats surviving beyond that interval.

Abstract  Thirteen-week and one-year toxicity studies of methyl tertiary-butyl ether (MTBE) administered in drinking water to Wistar rats were conducted. Male and female rats were exposed to MTBE in drinking water at 0.5, 3, 7.5 and 15 mg ml(-1) for 13 weeks and at 0.5, 3 and 7.5 (males) or 0.5, 3 and 15 mg ml(-1) (females) for 1 year. Body weights were reduced only in males following 13 weeks of exposure. Reduced water consumption and urine output were observed in males and females exposed to MTBE. Kidney cell replication and α(2u)-globulin levels in males were increased at 1 and 4 weeks of MTBE exposure and tubular cell regeneration was increased in male kidneys exposed to MTBE concentrations of 7.5 mg ml(-1) or greater for 13 weeks. Wet weights of male kidneys were increased following 13 weeks, 6 months and 1 year of exposure to MTBE concentrations of 7.5 mg ml(-1) or greater. Kidney wet weights were increased in females at MTBE concentrations of 15 mg ml(-1) for 13 weeks. Tertiary-butyl alcohol blood levels increased linearly with dose in males and females following 1 year of exposure. Chronic progressive nephropathy (CPN), of minimal to mild severity, increased in males, but not females, with 1 year of MTBE exposure. In summary, exposure of Wistar rats to MTBE in the drinking water resulted in minimal exposure-related effects including limited renal changes in male rats suggestive of α(2u)-globulin nephropathy following 13 weeks of exposure and an exacerbation of CPN in males at the end of 1 year of exposure.

Abstract  Long-term experimental carcinogenicity bioassay was carried out on MS 4A and MS 5A zeolites. The materials were tested on Sprague-Dawley rats by intraperitoneal, intrapleural, and subcutaneous injection of 25 mg in 1 ml of H2O. The results of this bioassay (the onset of one peritoneal mesothelioma in a male rat treated by intraperitoneal injection of zeolite MS 4A) calls for further and more sophisticated tests on zeolites used as detergents.

Abstract  PURPOSE: The European Association of Hematopathologists and the Society for Hematopathology have developed a new World Health Organization (WHO) classification of hematologic malignancies, including lymphoid, myeloid, histiocytic, and mast cell neoplasms. DESIGN: Ten committees of pathologists developed lists and definitions of disease entities. A clinical advisory committee (CAC) of international hematologists and oncologists was formed to ensure that the classification would be useful to clinicians. The CAC met in November 1997 to discuss clinical issues related to the classification. RESULTS: The WHO uses the Revised European-American Lymphoma (REAL) classification, published in 1994 by the International Lymphoma Study Group, to categorize lymphoid neoplasms. The REAL classification is based on the principle that a classification is a list of "real" disease entities, which are defined by a combination of morphology, immunophenotype, genetic features, and clinical features. The relative importance of each of these features varies among diseases, and there is no one gold standard. The WHO Neoplasms recognizes distinct entities defined by a combination of morphology and cytogenetic abnormalities. At the CAC meeting, which was organized around a series of clinical questions, participants reached a consensus on most of the questions posed. They concluded that clinical groupings of lymphoid neoplasms were neither necessary nor desirable. Patient treatment is determined by the specific type of lymphoma, with the addition of grade within the tumor type, if applicable, and clinical prognostic factors, such as the International Prognostic Index. CONCLUSION: The WHO classification has produced a new and exciting degree of cooperation and communication between oncologists and pathologists from around the world, which should facilitate progress in the understanding and treatment of hematologic malignancies.

Abstract  The European Food Safety Authority (EFSA) released a 2006 report questioning the relationship of aspartame exposure with increased incidence of lymphomas/leukemias in a European Ramazzini Foundation (ERF) rat study. The EFSA report suggested that the lymphoma/leukemia findings were most likely explained by infection in the rat colony. The ERF has also conducted the only available long-term oral study of methyl tertiary-butyl ether (MTBE). Thus, using the EFSA report as support, some have now raised questions about the human relevance of MTBE-associated hemolymphoreticular tumors reported by the ERF in female rats as well as whether their incidence was elevated above background levels. In this report, we discuss the hypothesized mode of action (MOA) of infection-induced lymphoma and its relevance to MTBE-associated lymphomas. We address the relationship of rat strain and study duration to lymphoma susceptibility and review evidence of low background rates of this tumor in control animals at the ERF, similar survival rates for female rats at the ERF and National Toxicology Program (NTP), and chemical- and gender-specificity of tumor induction for this type of tumor in studies at the ERF. We find that the background incidence of hemolymphoreticular tumors in female rats in the MTBE study is consistent with contemporaneous studies at the ERF and that there is an exposure-related effect, which is unlikely to be due to infections. We examine more recent tumor classification schemes for lymphomas, which support the combination of lymphoblastic leukemias and lymphomas reported by Belpoggi et al. ([1995] Toxicol Ind Health 11:119-149; [1998] Eur J Oncol 3:201-206).