A comparison of the procedures and outcomes of two cancer bioassays of TCDD was made using previously published information. The assays were conducted in a similar manner but differences existed. provides an overview of similarities and differences in the experimental procedures used in the NTP and Dow studies. The method by which the chemical used in each of the studies was produced was not reported but the source of material was different as indicated in the Table. The purity of the chemicals was reported to be > 98% in each case and similar methods of analysis were used. Of potential importance is the fact that a different stocks of Sprague-Dawley rats from different suppliers was used in each study. Male and female animals were exposed to the chemical in the Dow study study whereas the NTP study only female rats were exposed to TCDD. However, in the latter study a sufficient number of male animals were housed with the females to ensure the continuation of estrus.
Administration of TCDD was different in each study with gavage occurring 5 days of each week in the NTP study whereas in the Dow study the chemical was mixed in lab chow to provide 7 days of exposure. The range of daily administered TCDD dose levels (estimated in Kociba et al., actual in NTP) used in each study was similar (1 to 100 ng/kg of body weight) with the 10 and 100 ng/kg dose levels being used in both studies. Given that the aim of the NTP study was to compare the tumor responses, the study was designed in an attempt to maximize the number of doses in the range where tumors were expected, and therefore, given that 1ng/kg was NOEL in the Dow study, this dose was omitted in the NTP study in favor of additional dose groups at higher doses. Its is of note that while the daily administered doses were similar, the weekly averaged daily exposures in the NTP study are lower since animals were treated with only 5 daily gavage doses (Monday to Friday) within each week rather than exposed 7 days per week via dosed feed (). The dosing media, corn oil acetone in the NTP and lab chow for the Dow study were routinely analyzed to confirm the exposure levels in each study.
Some differences in caging of animals, method of sacrifice, and body weight measurement protocol are noted in . Several categories of measurements were made in one study but not in the other and selected examples are shown in the . Measurements of endogenous components of blood and urine were not measured in the NTP study but results of these tests were reported by Kociba et al.
Urinary components did not change with treatment but changes in some hematology measurements occurred particularly at the highest dose level. Thyroid hormones, cytochrome P-450 enzymes [20
] and cell proliferation were evaluated in selected animals in the NTP study but were not made in the Dow study [9
A comparison of results in indicate that cumulative survival of animals were not altered in any treatment group in the NTP study whereas Kociba et al. reported a decrease in survival in the highest dose group during the latter half of the study. Both studies reported lower body weight gain at less than the highest dose (100 ng/kg). Measurement of TCDD in fat and liver at termination was conducted in both studies ( and ). The results shown indicate that tissue levels in the Dow study were approximately twice those observed in the NTP study, indicative of a higher internal exposure at equivalent daily averaged doses ().
Figure 1 Comparison of liver TCDD concentrations at 2 years in the NTP study (solid line) and the Dow study  (dotted line). Data are plotted as daily averaged doses relative to the wet weight liver concentration.
The incidences of neoplastic and non neoplastic lesions induced by TCDD in the NTP study, with comparison to the incidences noted in the Dow study are presented in Tables and . Treatment-related neoplasms were seen in the two studies in the liver, lungs, and oral cavity, arising in each organ from the same cell type. However, a striking difference between the two studies was the lack of cholangiocarcinoma and /or cholangiofibrosis in the Dow study. Proliferative lesions of the bile duct (e.g. bile duct hyperplasia, cholangiofibrosis and cholangiocarcinoma) were not specifically diagnosed in the Dow study study, although oval cell proliferation, which is suggested to be of bile duct origin, was noted. Two cases of bile duct adenoma were noted only in females treated with the high dose in the Dow study. According to Goodman and Sauer [19
], who peer-reviewed the liver slides from Dow 2-year study, regenerative hyperplasia of the liver was part of the process of “hepatotoxicity” in the Dow study, in contrast to the NTP study, where the term “nodular hyperplasia” was used, and was distinguished and reported separately from “toxic hepatopathy”, a term used to indicate a distinct spectrum of lesions that was seen in the livers of rats treated with TCDD and with other “dioxin-like” chemicals in the NTP study series [21
]. Nodular hyperplasia was generally composed of larger than normal hepatocytes (hepatocytic hypertrophy) sometimes mixed with areas of increased numbers of small hepatocytes (hepatocytic hyperplasia). Areas of nodular hyperplasia blended with the surrounding parenchyma, although often they had a distinct border. Large, focal to multifocal areas of nodular hyperplasia were sometimes seen that caused compression of surrounding tissue, and/or bulging of the capsular surface. Bile duct hyperplasia and portal areas were usually present within nodular hyperplasia. The nodular hyperplasia was considered to be the result of the presence of a proliferative stimulus. Evaluation of the lower doses of the present study as well as livers from other NTP TEF studies indicated that the nodular hyperplasia was sometimes seen in animals where the toxic hepatopathy was minimal or non-existent. According to Goodman and Sauer [19
], there was a “distinct correlation between the presence of overt hepatotoxicity and development of hepatocellular neoplasms”. These authors used “hepatotoxicity” as a comprehensive terminology to include similar range of non-neoplastic changes, except of the above-mentioned bile duct proliferative lesions, as described in the NTP studies under the term “toxic hepatopathy”.
Incidence of treatment-related neoplastic lesions in the NTP and Dow studies
Incidence of treatment-related non-neoplastic lesions in the NTP and Dow studies
Although the lung responded in both studies with proliferative squamous cell lesions, the NTP reported that all squamous cell tumors were benign cystic keratinizing epithelioma (CKE), while in the Dow study the squamous tumors were interpreted as malignant squamous cell carcinomas (SCC) (Tables and ). In addition the NTP reported increased acinar pancreatic tumors and uterine SCC, in contrast to no change noted in the incidence of these tumors in the Dow study. Both studies reported significant decreased incidences of thyroidal C-cell tumors, pituitary and mammary tumors. Kociba et al.
reported decreased benign tumors of the uterus[9
], in contrast to no change noted in the incidence of these tumors in the NTP study.
The NTP study reported additional changes in other organs (), but due to insufficient details included in the report of the Dow study [9
], more complete comparison was not possible for some of the findings.
The incidence of pulmonary bronchiolar metaplasia of alveolar epithelium increased in all 2-year NTP study treated groups. This change consisted of replacement of the normal alveolar epithelium by cuboidal to columnar, sometimes ciliated cells, and was often accompanied by abundant mucus production in the affected area [22
]. The lesion generally diffusely affected the epithelium located at the bronchiolar-alveolar junction and adjacent alveoli. The incidence of squamous metaplasia of alveolar epithelium increased in the 46, 100 and 100 ng/kg stop groups. The incidence of histiocytic infiltration increased in the 22, 46, 100 ng/kg treated groups. Alveolar hyperplasia was seen only in the control group. For the Dow study, Kociba et al.
reported treatment-related hyperplastic changes only in the 100 ng/kg-dosed groups.
Increased incidences of minimal to mild multifocal cardiomyopathy were seen in rats of the NTP 2-year study, administered 10 ng/kg or greater[23
]. The incidence of cardiomyopathy was lower in the 100-ng/kg stop-exposure groups compared to the 100-ng/kg core study group, but was greater than in the vehicle controls. Cardiomyopathy had the typical microscopic appearance of spontaneous cardiomyopathy as seen in aging F344/N rats [23
]. The heart was also considered a target organ in the Dow study, reporting an increase above the background incidence of myocardial degenerative changes.
Increased incidence of chronic arteriopathy was noted in all treated groups in the pancreas and mesenteric arteries of the NTP 2-year study. Kociba et al. indicated increased incidence of hemorrhage in the brain and spinal cord, as well as increased incidence of periarteritis in the mesenteric and thoracic blood vessels of animals treated with the 100 ng/kg.
The incidence and/or severity of thymic atrophy were significantly increased in the 46 and 100 ng/kg treated groups in the NTP 14, 31, and 53 weeks interim sacrifices, and in the two-year 22 ng/kg treated group and higher. The incidence in the 100ng/kg stop-exposure group was greater than those in the vehicle controls. Atrophy consisted varying degrees of loss of lymphoid cells from the cortex. The thymus was also a target organ in the 10 ng/kg dosed group of the Dow study.
Increased incidence of minimal thyroid follicular cell hypertrophy was noted in the NTP 2-year rats administered 22 ng/kg or greater [24
]. The incidence of this lesion was lower in the 100 ng/kg stop-exposure group compared to the 100 ng/kg core study group. Follicular cell hypertrophy was a localized to diffuse change, characterized by follicles that were decreased in size and contained decreased amounts of colloid in which aggregates of amphophilic, flocculant appearing material were often present.
In the uterus, the incidence of SCC in the 46 ng/kg treated group of the NTP study was greater than that in the vehicle controls, and there were two SCCs in the 100 ng/kg stop-exposure group. The tumor was characterized by irregular cords and clusters of atypical stratified squamous epithelial cells that invaded the underlying myometrium. The incidence of squamous metaplasia was lower in the 100 ng/kg core study group compared to vehicle controls and was less than that in the 100 ng/kg stop-exposure group. Squamous metaplasia was generally a minimal to mild, multifocal change, consisting of tubular structures within the endometrium that were lined by stratified squamous epithelium.
The incidence of gingival SCC increased in the NTP 2-year 46, 100 and 100 ng/kg stop groups[25
]. The incidence of gingival squamous hyperplasia was increased in all dosed groups, including the stop-exposure group. The SCC occurred within the oral mucosa of the palate and was located adjacent to the molars teeth in nasal section III. It was characterized by invading cords and clusters of stratified epithelium. The squamous hyperplasia was seen in the same location as the tumors, and consisted of varying degrees of thickened epithelium. Kociba et al.
also reported increased incidence of SCC of the hard palate/nasal turbinate region in the group treated with 100 ng/kg [9
]. They also reported increased incidence of SCC of the tongue noted in the 100 ng/kg treated group, which was not a target organ in the NTP study.
In the NTP study in the pancreas, the incidence of acinar cytoplasmic vacuolation were increased in the 100 ng/kg treated groups of the 31-, and 53-week interim sacrifices, and chronic active inflammation and acinar atrophy were present in 100 ng/kg at the 14 and 53 weeks [26
]. In the 2 year treated groups of the same study acinar adenoma and/or carcinomas were seen only in the 100 ng/kg and 100 ng/kg stop study treated groups. The incidence of acinar cytoplasmic vacuolation was increased in the 46 and 100 ng/kg core study groups, and the incidence of chronic inflammation, and acinar atrophy were increased in the 100 ng/kg core study group [26
]. Kociba et al.
did not report treatment-related change in the acinar cell tumors among the females in the Dow study, but indicated increased incidence of atrophy and fibrosis of the acinar tissue of animals treated with 100 ng/kg, which was or was not associated with increased incidence of periarteritis.
The incidence of adrenal cortical atrophy and hyperplasia increased in the NTP 2-year in groups treated with 10 ng/kg and higher. The incidence of cytoplasmic vacuolation was increased in the 22 ng/kg or greater groups. The incidence of cortical cystic degeneration increased in the 10 and 22 ng/kg groups when compared to the concurrent controls. Kociba et al. reported decreased incidence of medullary hyperplasia (which was not seen in the NTP study), and increased incidence of cortical necrosis and hemorrhage, changes which may be consistent with the cortical atrophy and cystic degeneration observed in the NTP study.
In contrast to the Dow study, the NTP investigation included a 100 ng/kg stop treatment group following 30 weeks of TCDD treatment. The majority of the treatment-related neoplasms were not seen in the stop treatment group, except for the oral SCC. These data suggest that there is need for a longer period than 6 months of exposure in order that the TCDD-related neoplasms will develop and/or acquire potential of autonomous growth, except for the gingival tumors, that apparently were initiated at early stage of exposure, and did not regress even after relatively long period of withdrawal of treatment.
Dose response comparison of the two studies indicated marked differences. The data from the Dow study has been shown to be essentially low dose linear, the Hill shape parameter for hepatocellular neoplasms essentially being close to 1 [10
]. In contrast, modeling of the tumor data from the NTP study in general gave higher estimated Hill shape parameters greater than 2.0 [8
] and as such were more low-dose non-linear in nature.