James R. Hailey

Spontaneous Neoplasm Incidences in Fischer 344 Rats and B6C3F1 Mice in Two-Year Carcinogenicity Studies: A National Toxicology Program Update

Spontaneous neoplasm rates were determined for control Fischer 344 (F344) rats and B6C3F, mice from 2-yr rodent carcinogenicity studies carried out by the National Toxicology Program (NTP). The most frequently occurring neoplasms in untreated male F344 rats were testicular adenoma (89.1%), mononuclear cell leukemia (50.5%), adrenal gland pheochromocytoma (31.9%), and pituitary gland neoplasms (30.4%). For untreated female F344 rats, the most frequently occurring neoplasms were pituitary gland neoplasms (54.2%), mammary gland fibroadenoma (41.2%), and mononuclear cell leukemia (28.1%). The most frequently occurring neoplasms in untreated male B6C3F, mice were liver adenoma/carcinoma (42.2%), lung adenoma/carcinoma (20.5%), and malignant lymphoma (8.3%). For untreated female B6C3F, mice, the most frequently occurring neoplasms were liver adenoma/carcinoma (23.6%), malignant lymphoma (20.9%), and pituitary gland adenoma/carcinoma (1.4.8%). The tumor rates observed in feeding study (untreated) and inhalation study (chamber) control rats were generally similar. The major exceptions were pituitary gland tumors and testicular adenoma in male F344 rats. The overall incidence of testicular adenoma was much lower in chamber controls (69.4%) than in feeding study controls (89.1%), whereas pituitary gland neoplasms showed the opposite trend (60.7% vs 30.4%). The most likely explanation for this difference is related to the individual housing of chamber controls and the group housing of feeding study controls. Differences in diagnostic criteria may influence reported tumor rates. To ensure consistency and comparability of tumor diagnosis from study to study, the NTP uses rigorous histopathology quality assurance and peer review procedures. Biological factors such as body weight may also affect tumor incidence. For example, increased body weights are associated with increased incidences of certain site-specific neoplasms, especially pituitary gland and mammary gland neoplasms in rats and liver tumors in mice. The presence of Helicobacter hepaticus has been associated with an increased incidence of liver neoplasms in male B6C3F, mice. Other factors that may produce differences in control tumor rates from study to study include diet, environmental factors, genetic drift, study duration, and survival differences. The NTP database provides historical control data that may be useful in the evaluation of possible chemically related changes in tumor incidence. However, it is essential that the study being evaluated be comparable to those in the NTP database with respect to those factors that are known to influence tumor occurrence.

Dose-Additive Carcinogenicity of a Defined Mixture of “Dioxin-like Compounds”

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.

An Analysis of Pharmaceutical Experience with Decades of Rat Carcinogenicity Testing: Support for a Proposal to Modify Current Regulatory Guidelines

Data collected from 182 marketed and nonmarketed pharmaceuticals demonstrate that there is little value gained in conducting a rat two-year carcinogenicity study for compounds that lack: (1) histopathologic risk factors for rat neoplasia in chronic toxicology studies, (2) evidence of hormonal perturbation, and (3) positive genetic toxicology results. Using a single positive result among these three criteria as a test for outcome in the two-year study, fifty-two of sixty-six rat tumorigens were correctly identified, yielding 79% test sensitivity. When all three criteria were negative, sixty-two of seventy-six pharmaceuticals (82%) were correctly predicted to be rat noncarcinogens. The fourteen rat false negatives had two-year study findings of questionable human relevance. Applying these criteria to eighty-six additional chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome. These analyses support a proposal to refine regulatory criteria for conducting a two-year rat study to be based on assessment of histopathologic findings from a rat six-month study, evidence of hormonal perturbation, genetic toxicology results, and the findings of a six-month transgenic mouse carcinogenicity study. This proposed decision paradigm has the potential to eliminate over 40% of rat two-year testing on new pharmaceuticals without compromise to patient safety.

Impact of Helicobacter hepaticus Infection in B6C3F1 Mice from Twelve National Toxicology Program Two-Year Carcinogenesis Studies

Male and female B6C3F1 mice from 12 National Toxicology Program (NTP) 2-yr carcinogenesis studies were found to be infected with Helicobacter hepaticus. Many of the male mice from 9 of these studies had an associated hepatitis (affected studies). Helicobacter hepaticus has been reported to be associated with an increased incidence of hepatitis and hepatocellular neoplasms in the A/JCr male mouse. We attempted to determine if the data from the Helicobacter-affected NTP B6C3F1 mouse studies were compromised and unsuitable for cancer hazard identification. The incidences of neoplasms of the liver (both hepatocellular and hemangiosarcoma) but not of other organs in control male B6C3F1 mice were increased in affected studies as compared with control males from unaffected studies. The increased incidence of hepatocellular neoplasms was observed in those males exhibiting H. hepaficus-associated hepatitis. Other observations further differentiated control male mice from affected and unaffected studies. H-ras codon 61 CAA to AAA mutations were less common in liver neoplasms from males from affected studies as compared with historical and study controls. In addition, increases in cell proliferation rates and apoptosis were observed in the livers of male mice with H. hepaticus-associaled hepatitis. These data support the hypothesis that the increased incidence of liver neoplasms is associated with H. hepaticus and that hepatitis may be important in the pathogenesis. Therefore, interpretation of carcinogenic effects in the liver of B6C3F1 mice may be confounded if there is H. hepaticus-associaled hepatitis.

Chronic Toxicity/Oncogenicity Evaluation of 60 Hz (Power Frequency) Magnetic Fields in F344/N Rats

A 2-yr whole-body exposure study was conducted to evaluate the chronic toxicity and possible oncogenicity of 60 Hz (power frequency) magnetic fields in rats. Groups of 100 male and 100 female F344/N rats were exposed continuously to pure, linearly polarized, transient-free 60 Hz magnetic fields at flux densities of 0 Gauss (G) (sham control), 20 milligauss (mG), 2 G, and 10 G; an additional group of 100 male and 100 female F344/N rats received intermittent (1 hr on/1 hr off) exposure to 10 G fields. Mortality patterns, body weight gains throughout the study, and the total incidence and number of malignant and benign tumors in all groups exposed to magnetic fields were similar to those found in sex-matched sham controls. Statistically significant increases in the combined incidence of C-cell adenomas and carcinomas of the thyroid were seen in male rats chronically exposed to 20 mG and 2 G magnetic fields. These increases were not seen in male rats exposed continuously or intermittently to 10 G fields or in female rats at any magnetic field exposure level. No increases in the incidence of neoplasms, which have been identified in epidemiology studies as possible targets of magnetic field action (leukemia, breast cancer, and brain cancer), were found in any group exposed to magnetic fields. There was a decrease in leukemia in male rats exposed to 10 G intermittent fields. The occurrence of C-cell tumors at the 2 lower field intensities in male rats is interpreted as equivocal evidence of carcinogenicity; data from female rats provides no evidence of carcinogenicity in that sex. These data, when considered as a whole, are interpreted as indicating that chronic exposure to pure linearly polarized 60 Hz magnetic fields has little or no effect on cancer development in the F344/N rat.

The utility of multiple-section sampling in the histopathological evaluation of the kidney for carcinogenicity studies

In a recent review of 379 carcinogenicity studies in rodents conducted under the auspices of the National Cancer Institute and, later, the National Toxicology Program (NTP), the kidneys were the third most frequent site for chemical-related neoplasia. While some potent carcinogens induced high incidences of renal neoplasms with shortened latency in Fischer-344 (F-344) rats or B6C3F1 mice, other usually nonmutagenic compounds produced marginally increased incidences of renal neoplasms that were difficult to interpret. As an aid to the interpretation of 16 recent studies, additional kidney sections from rats or mice were prepared and examined microscopically. The remaining pieces of formalin-fixed kidney were embedded and sectioned at intervals of 1 mm (rats) or 0.5 mm (mice) to produce an additional 6-8 (rats) or 4-6 (mice) H&E-stained sections per kidney per animal for microscopic examination. The average number of additional sections per animal was similar between dosed and control groups to avoid sampling bias. The supplemental evaluation of these additional kidney sections was clearly useful in determining potential renal carcinogenicity in male F-344 rats in these NTP studies. Of the 13 studies in male rats in which step-sections of kidney were evaluated, the supplemental data demonstrated conclusively an association between chemical administration and renal tubule hyperplasia, adenoma, or both in 9 studies. For 3 chemicals, the evidence of an association with renal proliferative lesions in male rats remained uncertain. In contrast, the supplemental evaluation of step-sections was less useful for female rats, male mice, and female mice, largely because such evaluations generally revealed few if any additional neoplasms. For these sex-species groups, there were only two instances, both involving male mice, in which the additional data confirmed an association with kidney neoplasia.

The Effect of Chronic Progressive Nephropathy on the Incidence of Renal Tubule Cell Neoplasms in Control Male F344 Rats

Chronic progressive nephropathy (CPN) is the most frequently diagnosed lesion in the rat kidney. It has many component s including degeneration and regeneration of renal tubule (RT) epithelium, glomerular lesions and interstitial inflammation and fi brosis. 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 defi nitively 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 signifi cant 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.

Chronic Toxicity/Oncogenicity Evaluation of 60 Hz (Power Frequency) Magnetic Fields in B6C3F1 Mice

A 2-yr whole-body exposure study was conducted to evaluate the chronic toxicity and possible oncogenicity of 60 Hz (power frequency) magnetic fields in mice. Groups of 100 male and 100 female B6C3F1 mice were exposed to pure, linearly polarized, transient-free 60 Hz magnetic fields at flux densities of 0 Gauss (G) (sham control), 20 milligauss (mG), 2 G, and 10 G; an additional group of 100 male and 100 female B6C3F1 mice received intermittent (1 hr on/1 hr off) exposure to 10 G fields. A small but statistically significant increase in mortality was observed in male mice exposed continuously to 10 G fields; mortality patterns in all other groups of mice exposed to magnetic fields were comparable to those found in sex-matched sham controls. Body weight gains and the total incidence and number of malignant and benign tumors were similar in all groups. Magnetic field exposure did not increase the incidence of neoplasia in any organ, including those sites (leukemia, breast cancer, and brain cancer) that have been identified in epidemiology studies as possible targets of magnetic field action. A statistically significant decrease in the incidence of malignant lymphoma was observed in female mice exposed continuously to 10 G fields, and statistically significant decreases in the incidence of lung tumors were seen in both sexes exposed continuously to 2 G fields. These data do not support the hypothesis that chronic exposure to pure, linearly polarized 60 Hz magnetic fields is a significant risk factor for neoplastic development in mice.

Peer review in toxicologic pathology.

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 Pathologists 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.

A Retrospective Analysis of Background Lesions and Tissue Accountability for Male Accessory Sex Organs in Fischer-344 Rats

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 specifi c site identifi cation 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. Infl ammation in the dorsolateral lobes was signifi cantly associated with pituitary gland adenoma whose prolactin was suggested to play an important role in pathogenesi s of prostatic inflammation. Epithelial degeneration, epithelial hyperplasia, infl ammation, edema, and adenoma were conspicuous in the ventral lobes. Infl ammation and edema occurred in the anterior lobes (coagulating glands). Inflammation, dilatation, epithelial hyperplasia, edema, and adenoma were observed in the seminal vesicles. Infl ammation 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.