National Toxicology Program Rodent Research Articles

Systemic exposure to Ginkgo biloba extract in male F344/NCrl rats: Relevance to humans

Ginkgo biloba extract (GBE) is a popular botanical dietary supplement used worldwide and the safety of use is a public health concern. While GBE is a complex mixture, the terpene trilactones and flavonol glycosides are believed to elicit the pharmacological and/or toxicological effects of GBE. In a National Toxicology Program (NTP) 2-year rodent bioassay with GBE, hepatotoxicity was observed in rodents (≥100 mg/kg in rats, ≥ 200 mg/kg in mice). Subsequently, questions arose about whether or not the GBE used in NTP studies was representative of other GBE products and how rodent doses are related to human doses. To address these, we generated systemic exposure data for terpene trilactones in male rats following oral administration of 30, 100, and 300 mg/kg GBE test article from the 2-year bioassay. Dose-normalized Cmax and AUC∞ for terpene trilactones from the current study were within 5-fold of published rodent studies using a standardized GBE preparation. Comparison of our rat systemic exposure data at 100 mg/kg GBE to published human data following ingestion of 240 mg GBE-containing product showed that the rat/human exposure multiple was 3–22, for terpene trilactones. These data demonstrate the relevance of NTP rodent toxicity data to humans.

Evaluating consistency in the interpretation of NTP rodent cancer bioassays: An examination of mouse lung tumor effects in the 4-MEI study

The potential carcinogenicity of 4-methylimidazole (4-MEI) was evaluated in a National Toxicology Program (NTP) rodent cancer bioassay in Fischer 344 rats and B6C3F1 mice (NTP, 2007; Chan et al., 2008). The NTP concluded that there was “clear evidence of carcinogenic activity” in male and female mice, based on an increased incidence of lung tumors. The “category of evidence” that the NTP assigns to a rodent cancer bioassay outcome can have significant regulatory implications. This is especially important for 4-MEI, which forms in caramel colorings and other foods during cooking, with potential widespread human exposure in a broad spectrum of food and beverage products. A detailed analysis of all NTP mouse-lung-tumor-only carcinogens reveals that the proper call for lung tumors in the 4-MEI study should have been “some evidence” rather than “clear evidence” of carcinogenic activity for both male and female mice in order to be consistent with the NTP’s interpretation of other mouse lung carcinogens showing a similar strength of response. Suggestions are given as to measures the NTP should consider in the preparation of some or all future Technical Reports in order to enhance consistency of interpretation of experimental results.

Commentary: Update on Animal Models for NTP Studies

Based on recommendations of participants at the National Toxicology Program (NTP) workshop "Animal Models for the NTP Cancer Bioassay: Strains and Stocks-Should We Switch?" some modifications to the NTP rodent cancer bioassay were made. The B6C3F1 remains the mouse model used in the cancer bioassay. The use of multiple strains of mice will be explored through a new branch within the NTP, the Host Susceptibility Branch. Several rat models were evaluated; the Harlan Sprague Dawley rat is currently being used in the NTP studies.

NTP Workshop: Animal Models for the NTP Rodent Cancer Bioassay: Stocks and Strains—Should We Switch?

The National Toxicology Program (NTP) hosted a workshop, "Animal Models for the NTP Rodent Cancer Bioassay: Strains and Stocks--Should We Switch?" on June 16-17, 2005, at the National Institute of Environmental Health Sciences (NIEHS) in Research Triangle Park, North Carolina. The workshop's objectives were to determine (1) whether the currently used models, the F344/N rat and B6C3F1/N mouse, continue to be appropriate to identify substances that may pose a carcinogenic hazard for humans and (2) whether the NTP should consider conducting cancer bioassays using multiple strains of rats and/or mice to better capture the range of genetic variability. Workshop participants advised the NTP to discontinue using the current F344/N strain due to the recent issues with fertility, seizure activity, and chylothorax and provided several options on how the program should approach identifying and selecting a new rat model. Participants believed that the B6C3F1/N mouse is still appropriate for use by the NTP, but suggested the NTP take steps to better understand and address increases in background rates of liver tumors in this strain. Finally, the participants supported the NTP exploring the use of the multiple strain approach, although they raised many questions concerning data interpretation and feasibility. This article also outlines the NTP's next steps in pursuing the workshop recommendations.

Ochratoxin a: Its Cancer Risk and Potential for Exposure

Ochratoxin A (OA) is a naturally occurring mycotoxin known to contaminate a variety of foods and beverages. The cancer risk posed by OA was reviewed as relevant to human exposure, regulatory activities, and risk management efforts occurring worldwide, particularly in Europe. OA moves through the food chain and has been found in the tissues and organs of animals, including human blood and breast milk. Results from the National Toxicology Program's rodent bioassays show significantly increased incidence of mammary gland tumors in female rats and kidney tumors in male and female rats given OA orally. Liver tumors in female mice fed OA in the diet have also been observed. In humans, OA exposure has been most often associated with the kidney disease Balkan endemic nephropathy (BEN), symptoms of which include tumors of the kidney and urinary tract. No epidemiological studies have yet adequately evaluated the cancer risk of OA in human populations. Studies have shown OA to be genotoxic as well as immunotoxic, although its mode of action is not fully understood. Organizations and agencies in many countries are currently promulgating standards for OA in foods and beverages. Increased efforts in farm management and food safety are being made to mitigate the risks to public health posed by OA. The U.S. Food and Drug Administration (FDA) is currently evaluating data on OA levels in domestic and imported commodities but has not established official regulations or guidelines for OA in the U.S. food supply. Funding for this work was provided by the US Department of Agriculture Cooperative State Research, Education and Extension Service. Any findings, conclusions or recommendations are those of the authors and do not necessarily reflect the views of the U.S. Department of Agriculture.

Chemical-Induced Atrial Thrombosis in NTP Rodent Studies

Cardiac thrombosis, one of the causes of sudden death throughout the world, plays a principal role in several cardiovascular diseases, such as myocardial infarction and stroke in humans. Data from studies of induction of chemical thrombosis in rodents help to identify substances in our environment that may contribute to cardiac thrombosis. Results for more than 500 chemicals tested in rodents in 2-year bioassays have been published as Technical Reports of the National Toxicology Program (NTP) http://ntp-server.niehs.nih.gov/index. We evaluated atrial thrombosis induced by these chemical exposures and compared it to similarly induced lesions reported in the literature. Spontaneous rates of cardiac thrombosis were determined for control Fischer 344 rats and B6C3F1 mice: 0% in rats and mice in 90-day studies and, in 2-year studies, 0.7% in both genders of mice, 4% in male rats, and 1% in female rats. Incidences of atrial thrombosis were increased in high-dosed groups involving 13 compounds (incidence rate: 20-100%): 2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3'-dimethoxybenzidine dihydrochloride, hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4'-thiobis(6-t-butyl-m-cresol). The main localization of spontaneously occurring and chemically induced thromboses occurred in the left atrium. The literature survey suggested that chemical-induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis, hypercoagulability, and impaired atrial mechanical activity, such as atrial fibrillation, which could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis. Supplementary data referenced in this paper are not printed in this issue of Toxicologic Pathology. They are available as downloadable files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 33(5), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

How many high production chemicals are rodent carcinogens? Why should we care? What do we need to do about it?

High production volume (HPV) chemicals are produced in or imported to the US in amounts greater than 1 million pounds per chemical per year. The EPA has identified thousands of HPVs. Due to their abundance, such chemicals bring a substantial risk for human exposure, and as a result some level of adverse consequences to health are to be expected. In order to examine the potential for cancer risk associated with HPVs, this paper examines HPVs that have been tested in the National Toxicology Program's rodent cancer bioassay. The chemicals tested in the bioassay represent a small sample of the universe of environmental chemicals to which people are exposed. Unexpectedly, 60% of the 128 HPVs evaluated in the bioassay proved to be rodent carcinogens. This value compares to a predicted prevalence of only 16.5% carcinogens in a previous study. The previous study concluded that HPVs were less likely to be toxic by a variety of other test criteria as well. However, the approach involved identifying putative carcinogens using quantitative chemical structure-activity relationships (QSAR) in contrast to the direct tabulation of bioassay test results performed here. Detailed examination of bioassay results reveals that test outcomes depend heavily on route of administration as well as on dose level, sex, strain, and species used. Since most of these factors have little or no apparent relationship to chemical structure, results of this study suggest that QSAR, as well as virtually all other alternative methods, may not generally provide accurate predictions of carcinogenic potential. As we wait for efficient and effective methods for predicting carcinogens to be developed, people continue to be exposed to environmental carcinogens. Progress on regulating environmental carcinogens as well as on developing more effective test methods has been minimal since "war on cancer" began approximately 30 years ago. The present study questions whether the current strategy for dealing with environmental carcinogens will ever be successful. Close examination of rodent cancer test results seems to suggest that almost all chemicals may have carcinogenic potential in some genotypes under some exposure circumstances. If this hypothesis is correct, it would explain the general lack of progress in developing methods to differentiate carcinogens from noncarcinogens. A completely new strategy for dealing with cancer caused by exposures to environmental chemicals seems to be needed.

Carcinogenicity Testing and the Evaluation of Regulatory Requirements for Pharmaceuticals

The results of rat and mouse carcinogenicity studies for 282 human pharmaceuticals in the FDA database were analyzed and compared as part of an International Conference on Harmonization (ICH) evaluation of rodent carcinogenicity studies and their utility for carcinogenicity testing. A majority of the carcinogenicity studies in the FDA database were carried out in Sprague–Dawley-derived rats and Swiss–Webster-derived CD-1 mice in contrast to Fisher 344 rats and B6C3F1 mice employed in National Toxicology Program (NTP) studies. Despite the differences in rodent strains, the relative proportion of compounds with positive findings (44.3%) and the degree of overall concordance between rats and mice (74.1%) in the FDA database were similar to the NTP rodent carcinogenicity database. Carcinogenicity studies in two rodent species are necessary primarily to identify trans-species tumorigens, which are considered to pose a relatively greater potential risk to humans than single species positive compounds. Two-year carcinogenicity studies in both rats and mice may not be the only means of identifying trans-species tumorigens. Sufficient experience is now available for some alternativein vivocarcinogenicity models to support their application as complementary studies in combination with a single 2-year carcinogenicity study to identify trans-species tumorigens. Our analysis of the rodent carcinogenicity studies supports such an approach for assessing carcinogenic potential without compromising the public health.

The rodent bone marrow micronucleus assay: contrast between its sensitivity to human carcinogens and its insensitivity to NTP rodent carcinogens

The rodent bone marrow micronucleus (MN) assay occupies a critical position in the accompanying schemes to detect potential human carcinogens and germ cell mutagens ( Shelby, 1996; Ashby et al., 1996: for reviews of the MN assay see Heddle et al., 1983; Schlegel and MacGregor, 1984; CSGMT, 1990; Mavournin et al., 1990; Tinwell, 1990; Gatehouse, 1994; Asanami et al., 1995). The intention of this article is to note two perceptional problems currently associated with the MN assay. The first concerns how it should be used — as a screening assay, or as a means to evaluate the genetic toxicity in vivo of genotoxins defined in vitro. The second relates to its sensitivity to the rodent carcinogens defined by the US National Toxicology Program (NTP).

Cell proliferation: concluding remarks

In beginning this overview, I want to acknowledge the fact that, even though I disagree with most of his conclusions, Bruce Ames has played an important role in further stimulating interest in cell proliferation in carcinogenesis. This meeting has provided a balanced discussion of the current state of knowledge in this Eleld. It has also highlighted the complexities of cell proliferation, the need for further mechanistic studies, and the desirability of developing new methods that can be used to precisely quantitate the various parameters related to cell proliferation in the intact organism. At this meeting and in a recent paper (1), Ames has put forth several postulates that I believe represent an oversimplification of the multistage carcinogenic process. These postulates include the following: a) the aging process per se is a major factor in human cancer causation; b) oxidative damage due to endogenous factors plays a major role in human cancer causation; c) mitogenesis (induced cell division) plays a major role in carcinogenesis by increasing mutagenesis; d) the standard National Toxicology Program (NTP) rodent bioassay frequently yields unreliable results with respect to predicting human carcinogens; and e) synthetic pesticides and certain other synthetic chemicals pose lesser carcinogenic risks than nature's pesticides and other naturally occurring chemicals in various food stuffs. Elsewhere, I have reviewed evidence that calls into question these conclusions (2,3). Some of this evidence was discussed at this meeting and will be briefly reviewed here. Some of Ames' postulates may apply in specific cases. I do not believe, however, that there is sufElcient evidence at the present time to use these postulates as a general basis for discarding current methods of carcinogen detection and risk extrapolation or for altering current guidelines for regulating potential carcinogens. With respect to Ames' criticism of the standard NTP rodent carcinogen bioassay, we have heard evidence

Results and conclusions of the national toxicology program's rodent carcinogenicity studies with sodium fluoride

The US National Toxicology Program (NTP) has conducted toxicity and carcinogenicity studies with sodium fluoride administered in the drinking water to F344/N rats and B6C3F1 mice. The drinking water concentrations used in the 2-year studies were 0, 25, 100, or 175 ppm sodium fluoride (equivalent to 0, 11, 45 or 79 ppm fluoride). Survival and weight gains of rats and mice were not affected by fluoride treatment. Animals receiving sodium fluoride developed effects typical of dental fluorosis, and female rats given 175 ppm had increased osteosclerosis. There were no increases in neoplasms in female rats or in male or female mice that were attributed to sodium fluoride administration. There was equivocal evidence of carcinogenic activity of sodium fluoride in male rats based on the occurrence of a small number of osteosarcomas in treated animals.

Metabolite-based internal doses used in a risk assessment of benzene.

Risk assessments of benzene have been based upon both human and animal studies. In this paper, metabolite information is used to construct an internal dose (a surrogate of the biologically effective dose) for a given administered dose. The relationship between the administered dose and this internal dose is nonlinear and is well described by a Michaelis-Menten function. The administered doses from the National Toxicology Program's rodent carcinogenicity study of benzene are transformed into internal doses, and these internal doses are used in conjunction with a multistage model to compare previous estimated virtually safe doses (VSD) associated with small added health risks. The ratio of VSD for the administered dose risk assessment to the VSD from the internal dose risk assessment was approximately 1.0 for the F344/N rats and ranged from 2.5 to 5.0 for B6C3F1 mice in the National Toxicology Program study. For an occupational exposure of 1 ppm, a risk estimate of 0.7 excess cancers/1000 exposed with an upper bound of 3.5/1000 was obtained for a total metabolite internal dose risk assessment. Risk estimates based upon internal doses constructed from levels of the toxic metabolites of benzene are also presented. The implication of a dose-rate study of benzene metabolism for risk assessment is discussed, and finally, suggestions for better characterization of the dose-response function for benzene are provided.