National Toxicology Program Studies Research Articles

Mixed reaction likely to fluoride cancer study

It is difficult to project the public response to the National Toxicology Program study about fluoride and cancer in rodents. But I think one has to be prepared for a rather stronger reaction than what one might expect based on the technical arguments [pro and con fluoridation]. This was the message Paul Slovic, president of Decision Research, conveyed during a symposium attended by more than 1000 dental researchers and dentists last week. (Decision Research is a nonprofit research institute that studies decision making under uncertainty.) Because of recent publicity about the new NTP study (C&EN, Jan. 29, page 17), the International Association for Dental Research (IADR) hastily organized the symposium to provide background on the study at its annual meeting in Cincinnati. On Jan. 22, NTP, which is run by the National Institute of Environmental Health Sciences, released preliminary data from a two-year rodent carcinogenicity study that showed that one of 50 male rats in ...

Effects of Mononuclear Cell Leukemia on Altered Hepatocellular Foci in Fischer 344 Rats

Quantitative stereologic analyses were conducted on hematoxylin and eosin-stained liver sections to investigate the potential effects of the presence of mononuclear cell leukemia in Fischer 344 rats on the occurrence of altered hepatocellular foci (AHF). The study consisted of 132 male and 144 female rats taken from control groups at the termination of seven, 2-year National Toxicology Program (NTP) carcinogenicity studies. A minimum of 10 male and 10 female rats were killed at 6, 9, 12, 15, and 18 months into the seven NTP studies to assess progressive development of AHF. At the end of the studies, 43 males and 35 females had histologic evidence of mononuclear cell leukemia in the liver. There were no differences in the morphologic features of AHF between leukemic and non-leukemic rats; however, there was a decreased incidence of clear AHF in both sexes and in basophilic, vacuolated, and mixed-cell AHF in males. Stereologic analysis revealed that there was also a 40 to 73% reduction in the density of basophilic, clear, vacuolated, and mixed-cell AHF in male rats with leukemia and a 31 and 70% reduction in basophilic and clear AHF, respectively, in females with leukemia. The number of eosinophilic AHF was statistically unchanged in both sexes. The eosinophilic AHF, however, were 2.3 times larger and occupied a 4.3-fold greater volume fraction of the liver in leukemic male rats. Changes in the incidence and density of AHF were directly associated with the severity of the leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Commentary on “Are there location/cage/systematic nontreatment effects in long-term rodent studies? A question revisited”

In the previous article, Young (1989) discusses “location/cage/systematic nontreatment effects” in long-term rodent studies. His article requires a response on several points, particularly those related to the evaluation of “local room effects” in the National Toxicology Program (NTP) eugenol study (NTP, 1983). In a previous paper, Young (1987) compared liver tumor rates between two shelf levels of male mice receiving a low dose of eugeno1 and found a statistically significant difference. On the basis of this single finding, Young (1987) made the general conclusion that “local room effects might change tumor rates from about 30 to 80%” and recommended that in long-term studies cage location be assigned at random, despite the logistical and other difficulties associated with a completely randomized housing scheme. It is dangerous to draw general inferences from a single isolated finding. Thus, in view of the hundreds of carcinogenicity studies carried out by the NTP and the multiplicity of tumor sites examined in each experiment, it is important to consider whether the “local room effect” found by Young in the eugenol study could be merely reflecting random variability. To address this issue, Haseman ( 1988a) examined all NTP studies showing evidence of hepatic carcinogenicity in mice and found that the frequency of “significant” shelf level effects, such as that reported by Young (1987) in the eugenol study, was virtually identical to chance expectation. Haseman (1988b) then expanded his analysis to include all carcinogenic effects observed in F344 rats in NTP studies and found similar results. Investigations of other large databases (e.g., Haseman et al., 1986) have also found little evidence that cage location influences tumor rates. Thus, although one cannot identify with absolute certainty the factors responsible for differences in tumor rates between shelf levels, these analyses suggest that the majority of such differences merely reflect random variability. For eugenol, an elevated tumor rate was observed only in animals housed in the fifth row of the front of the rack. It is difficult to envision a biologically meaningful local room effect that could produce such a response. In his latest paper Young (1989) redefines “cage” or “shelf’ effects to be a “catchall” for basically any systematic nontreatment effect. However, while no one would deny that systematic nontreatment effects may exist in some long-term studies, it is important to emphasize that Young’s evaluation of the eugeno1 data was limited to an assessment of a single factor: cage location. It is inappropriate to regard his comparison of tumor rates in

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.

Experimental design constraints on carcinogenic potency estimates.

The multistage model is used by U.S. regulatory agencies to calculate estimates of the carcinogenic potency (beta) of chemicals; the data for these estimates are generally obtained from chronic rodent bioassays. Three quantities characterize each group tested in the chronic bioassay: the dose level, the sample size, and the number responding to the dose. The dose levels tested are fixed by conventional protocols; the typical National Toxicology Program (NTP) experimental design calls for use of the maximum tolerated dose (MTD), one-half and one-fourth MTD, plus a control group. Only rarely are doses even one order of magnitude less than the MTD utilized in chronic bioassays. This experimental design constraint on dose selection limits the possible values of beta that can arise from multistage model analyses of chronic bioassay data. Sample size is also constrained by the experimental design of the chronic bioassay; the typical sample size in NTP studies is 50 animals. Occasionally, fewer animals are used, but only rarely are more. Thus, the multistage model which theoretically has three variable quantities with which to estimate carcinogenic potency, has in practice only one: the incidence of treatment-related response. Even this can vary within only a narrow range determined by sample size, the control incidence, and the level of statistical significance desired. The net result of these design constraints is that carcinogenic potency estimates derived from multistage-model analyses of chronic bioassay data may vary within only a narrow range surrounding the inverse maximum dose tested. We have illustrated this by calculating the largest possible finite potency estimates that could have arisen from the experimental designs used to test 82 mouse carcinogens in chronic bioassays. On average these maximum potency estimates were within one order of magnitude of the inverse maximum dose tested. We thus conclude that the chronic rodent bioassay, in and of itself, is altogether inadequate as a data source for estimating the risk to humans from exposure to carcinogenic chemicals.

Effects of nitrofurantoin on the primary and secondary reproductive organs of female B6C3F1 mice.

The National Toxicology Program (NTP) has reported female mice fed high doses of Nitrofurantoin (NFT) were found to have ovarian atrophy as diagnosed histologically and increased benign ovarian tumors after 24 months of exposure (30). This result contrasts with 4 other recent carcinogenicity assays in rodents with NFT, all with no evidence of an ovarian effect. An extensive database documents benign tubular adenomas develop secondary to ovarian atrophy in many mouse strains, including B6C3F1. The present study was initiated to confirm this mechanism could be responsible for the ovarian tumors in the NTP study and to investigate the time course of ovarian changes seen in female B6C3F1 mice. Mice were provided diet containing NFT at doses of 350 and 500 mg/kg body weight/day and examined after 4, 8, 13, 17, 43 and 64 weeks. A dose-related decrease in feed consumption, feed efficiency and body weight gain was seen and persisted throughout the study. Sexual maturity was delayed in a dose-related fashion, compatible with previously reported effects of reduced food consumption in rodents (12, 16). All groups of mice eventually did have normal estrous cycles, but cycle lengths were increased in a dose-related fashion. Both doses of NFT resulted in histological evidence of senile ovarian atrophy by week 43. Based on the reported association between sterility and ovarian tumors, we conclude the benign tubular adenomas seen at 2 yr in the NTP carcinogenicity study with NFT were secondary to the ovarian atrophy induced in this strain of mouse and not an indication NFT, itself, is a carcinogen.

Use of rat liver altered focus models for testing chemicals that have completed two-year carcinogenicity studies.

Partial hepatectomy (PH) and neonatal rat short-term liver focus models were used to examine the effects of selected chemicals that had been previously tested in the National Toxicology Program (NTP) 2-yr carcinogenicity studies. C.I. Solvent Yellow 14, monuron, chlorendic acid, and 4-hydroxyacetanilide were tested for initiating and promoting activity in the PH model. Chlorendic acid, 4,4'-oxydianiline, 1-amino-2,4-dibromoanthraquinone (ADBAQ), and 4-hydroxyacetanilide were similarly tested in a neonatal rat liver focus model. With the exception of 4-hydroxyacetanilide which was not carcinogenic in the NTP studies, all chemicals tested showed clear evidence of hepatocarcinogenicity. While none of the chemicals showed initiating activity in either the PH or neonatal models, promoting activity, as indicated by increased number, size, or volume fraction of histochemically detected hepatic foci of cellular alteration, was evident for all chemicals with previously demonstrated hepatocarcinogenicity. Liver tumor incidence was documented at 14 months in the PH model and at 300 days in the neonatal model. On the basis of the results obtained from these few chemicals, it is suggested that the use of short-term rat liver focus models may represent a reliable means for identifying chemicals with hepatocarcinogenic potential.

Uncertainty in quantitative carcinogenic risk assessment procedures for 2,3,7,8-tetrachlorodibenzo-p-dioxin

Various high-to-low dose extrapolation models (Probit, Logit, Extreme-Value, One-Hit, Multistage) are applied to all 6 available data sets (Kociba et al. study, 1978: male and female Sprague-Dawley rats; National Toxicology Program study, 1982: male and female Osborne-Mendel rats, male and female B6C3F1 mice), in order to assess 2,3,7,8 TCDD-related carcinogenic risk at low doses. Our results show that risk assessment at low doses, based on experimental data, is limited by a huge amount of uncertainty (10 6 at least), according to both models and species under consideration.

Pathological and biochemical effects of dimethyl hydrogen phosphite in Fischer 344 rats

In a chronic study by the National Toxicology Program (NTP), dimethyl hydrogen phosphite (DMHP) caused neoplastic and nonneoplastic changes in the lungs and forestomach of F344 N rats following gavage administration for 2 years. The current investigation was designed to study the effect of a short-term exposure on a series of biochemical systems in target and nontarget tissues which may be involved in the metabolism and/or the manifestation of DMHP toxicity. Rats were treated daily with a dose similar to that used in the NTP study (200 mg/kg) for 4, 5, or 6 weeks. Two groups of animals were also treated for 4 weeks and then treatment was discontinued and the rats were allowed to recover for 1 or 2 weeks. An equal number of animals was treated similarly with the vehicle and used as control. The microsomal and soluble fractions were separated from liver, lungs, kidneys, forestomach, and glandular stomach from the 6-week treatment group. Another group of rats treated for 6 weeks was prepared for pathology examination of the lungs, forestomach, and glandular stomach. There was a significant increase in the weight of the forestomach of rats treated for 4, 5, or 6 weeks relative to control animals, while no significant difference was observed in the weight of liver, lungs, kidneys, and glandular stomach. The forestomach weight of rats treated for 4 weeks returned to the control value after 1 week of recovery. Microscopic examination of the forestomach of rats treated for 6 weeks revealed a thickened stratified squamous epithelium characterized by hyperplasia, hyperkeratosis, and subepithelial inflammation and edema. There were no microscopic changes in the lungs or glandular stomach of animals treated for 6 weeks. The activity of angiotensin converting enzyme in the serum of rats treated for 4, 5, or 6 weeks was significantly increased over that of control animals. The activity of this enzyme returned to near levels seen in the control animals after 1 week of recovery following 4 weeks of treatment. No treatment-related effect was observed in the activities of the microsomal p-nitroanisole demethylase, soluble glutathione S-transferase, and soluble superoxide dismutase in the five tissues studied. There was a significant increase in the level of nonprotein soluble sulfhydryls in the forestomach but in no other tissue of rats treated for 6 weeks. Also the activity of soluble carboxylesterase was significantly reduced in the lungs and forestomach, but not in any other tissue of the 6-week-treated rats. Results of this study indicate that early pathological and biochemical changes are detected in target tissues of rats exposed to DMHP.

Pathological and Biochemical Effects of Dimethyl Hydrogen Phosphite in Fischer 344 Rats

Pathological and Biochemical Effects of Dimethyl Hydrogen Phosphite in Fischer 344 Rats. NOMEIR, A. A., AND URAIH, L. C. (1988). Fundam. Appl. Toxicol 10, 114–124. In a chronic study by the National Toxicology Program (NTP), dimethyl hydrogen phosphite (DMHP) caused neoplastic and nonneoplastic changes in the lungs and forestomach of F344/N rats following gavage administration for 2 years. The current investigation was designed to study the effect of a short-term exposure on a series of biochemical systems in target and nontarget tissues which may be involved in the metabolism and/or the manifestation of DMHP toxicity. Rats were treated daily with a dose similar to that used in the NTP study (200 mgkg) for 4, 5, or 6 weeks. Two groups of animals were also treated /for 4 weeks and then treatment was discontinued and the rats were allowed to recover for 1 or 2 weeks. An equal number of animals was treated similarly with the vehicle and used as control. The microsomal and soluble fractions were separated from liver, lungs, kidneys, forestomach, and glandular stomach from the 6-week treatment group. Another group of rats treated for 6 weeks was prepared for pathology examination of the lungs, forestomach, and glandular stomach. There was a significant increase in the weight of the forestomach of rats treated for 4, 5, or 6 weeks relative to control animals, while no significant difference was observed in the weight of liver, lungs, kidneys, and glandular stomach. The forestomach weight of rats treated for 4 weeks returned to the control value after 1 week of recovery. Microscopic examination of the forestomach of rats treated for 6 weeks revealed a thickened stratified squamous epithelium characterized by hyperplasia, hyperkeratosis, and subepithelial inflammation and edema. There were no microscopic changes in the lungs or glandular stomach of animals treated for 6 weeks. The activity of angiotensin converting enzyme in the serum of rats treated for 4, 5, or 6 weeks was significantly increased over that of control animals. The activity of this enzyme returned to near levels seen in the control animals after 1 week of recovery following 4 weeks of treatment. No treatment-related effect was observed in the activities of the microsomal p-nitroanisole demethylase, soluble glutathione S-transferase, and soluble superox-ide dismutase in the five tissues studied. There was a significant increase in the level of nonpro-tein soluble sulfhydryls in the forestomach but in no other tissue of rats treated for 6 weeks. Also the activity of soluble carboxylesterase was significantly reduced in the lungs and forestomach, but not in any other tissue of the 6-week-treated rats. Results of this study indicate that early pathological and biochemical changes are detected in target tissues of rats exposed to.

Ovarian toxicity and carcinogenicity in eight recent National Toxicology Program studies.

Ovarian toxicity and/or carcinogenicity has been documented for at least eight chemicals recently tested in National Toxicity Program prechronic and chronic rodent studies. The chemicals that yielded treatment-related ovarian lesions were 1,3-butadiene, 4-vinylcyclohexene, vinylcyclohexene deipoxide, nitrofurantoin, nitrofurazone, benzene, delta-9-tetrahydrocannabinol, and tricresylphosphate. Typical nonneoplastic ovarian changes included hypoplasia, atrophy, follicular necrosis, and tubular hyperplasia. The most commonly observed treatment-related neoplasms were granulosa cell tumors and benign mixed tumors. A relationship between antecedent ovarian hypoplasia, atrophy, and hyperplasia and subsequent ovarian neoplasia is supported by some of these National Toxicology Program studies. Pathologic changes in other tissues such as the adrenal glands and uterus were associated with the treatment-related ovarian changes.

Ovarian Toxicity and Carcinogenicity in Eight Recent National Toxicology Program Studies

Ovarian toxicity and/or carcinogenicity has been documented for at least eight chemicals recently tested in National Toxicity Program prechronic and chronic rodent studies. The chemicals that yielded treatment-related ovarian lesions were 1,3-butadiene, 4-vinylcyclohexene, vinylcyclohexene deipoxide, nitrofurantoin, nitrofurazone, benzene, delta-9-tetrahydrocannabinol, and tricresylphosphate. Typical nonneoplastic ovarian changes included hypoplasia, atrophy, follicular necrosis, and tubular hyperplasia. The most commonly observed treatment-related neoplasms were granulosa cell tumors and benign mixed tumors. A relationship between antecedent ovarian hypoplasia, atrophy, and hyperplasia and subsequent ovarian neoplasia is supported by some of these National Toxicology Program studies. Pathologic changes in other tissues such as the adrenal glands and uterus were associated with the treatment-related ovarian changes.

Issues in carcinogenicity testing: Dose selection

Dose selection in testing chemicals for possible carcinogenicity in rodents continues to be an area of scientific debate. In this paper the definition of “maximum tolerated dose” (MTD) is considered, and the advantages and disadvantages of using MTDs are given. There is no universally accepted definition of an MTD, and as a result, objections to utilizing high doses in carcinogenicity testing may reflect differing definitions of an MTD rather than basic disagreements in dose selection philosophy. Data from 52 National Toxicology Program (NTP) carcinogenicity studies indicate that while dose selection has caused difficulties in certain studies using the gavage route of chemical administration, there is little evidence that this has been a problem in NTP studies using the dietary (feed) route of exposure. These data also indicate that more than two-thirds of the carcinogenic effects detected in feeding studies would have been missed has the high dose been reduced from the estimated MTD to 1 2 MTD. The inherent insensitivity of laboratory animal studies for detecting weak-to-moderate carcinogenic responses also argues against reducing the highest dose level. The addition of a third, lower-dosed group provides for a margin of safety against the possibility of over-estimating the MTD. Primary emphasis should be given to improving procedures for estimating the MTD, particularly for gavage studies. Efforts should also be increased to obtain pharmacokinetic and metabolism data for the test chemical that might be factored into the dose selection and study evaluation processes.