Although previous studies suggest that H. hepaticus
infection is associated with hepatitis and increased liver neoplasms in male mice (Ward, et al., 1994a
; Fox, et al., 1996
; Hailey, et al., 1998
), to our knowledge, the present studies of TEA provide the only example of a chemical tested for carcinogenic activity in both infected mice and in mice free of infection. In H. hepaticus
infected mice exposed to TEA (NTP, 1999
), the absence of increased neoplasms at sites other than the liver made the interpretation of the carcinogenic activity of TEA difficult. As a result, the study was repeated in animals free of infection. The objectives of this report were to compare the results of the initial (NTP, 1999
) and repeat (NTP, 2004
) NTP chronic bioassays of TEA in B6C3F1 mice and to examine the impact of H. hepaticus
infection on study outcome and interpretation.
The primary treatment-related effect in males and females in both studies was an increase in both neoplastic and non-neoplastic lesions of the liver. Several approaches were used to determine the effects of H. hepaticus or differences in protocol between studies on the TEA bioassay in mice. A comprehensive examination of individual and combined liver neoplasm incidences in each study was undertaken. The incidences of liver neoplasms were then compared at each dose between studies. Finally, control liver tumor incidences in both studies were compared to those predicted by logistic regression modeling; these models took animal source and diet, the two primary differences in protocol between studies, into account. These analyses revealed that the observed differences in the incidences of liver lesions between studies were likely influenced by infection in males or differences in diet and animal source in females. Alternatively, these differences may have been the result of normal variability or the rederivation of the mouse colony and weak carcinogenic activity of TEA. There were also differences in neoplasm incidences at other sites between studies at various doses; however, these lesions were not increased over controls in either study.
In males, the neoplastic response in the liver was greater in mice infected with H. hepaticus
. In initial study males, there were lesions indicative of hepatitis (Ward, 1994a
; Fox, et al, 1996
; Hailey, et al., 1998
), which occurred at similar incidences across dose groups, including controls. At 2,000 mg/kg TEA, there were also increases in hepatocellular neoplasms. In contrast, neither increases in hepatocellular neoplasms nor hepatitis were observed in repeat study males. Although liver hemangiosarcomas were increased at 630 mg/kg, this increase was marginal, there was no increase in the next and highest dose group (2,000 mg/kg), or in any of the dosed groups of repeat study females, or in initial study mice of either sex. Comparison of neoplasm incidences between studies revealed that although there were fewer adenomas in the repeat study, the incidences of control liver neoplasms were not statistically different between studies, and that there were significantly fewer hepatocellular neoplasms in the repeat study at 2,000 mg/kg. Logistic regression modeling of control liver tumor incidences did not reveal any differences between observed and expected tumor incidences in either study; however, fewer control hepatocellular adenomas were expected in the repeat study compared to the initial study. Collectively, these observations suggest that the increase in hepatocellular neoplasms at 2,000 mg/kg in the initial study resulted from the combined stimulus of TEA and H. hepaticus
. The ability of H. hepaticus
to increase liver neoplasms in male mice has been demonstrated previously, both in untreated controls (Ward, et al., 1994a
; Fox, et al., 1996
; Hailey, et al., 1998
) and following treatment with genotoxic carcinogens (Diwan, et al., 1997
; Nagamine, et al., 2007), and likely involves increased cell proliferation (Fox, et al., 1996
; Ward, et al., 1994b
; Nyska, et al., 1997
In females, the dose-response of hepatocellular neoplasms was strengthened in the absence of H. hepaticus
infection. Hepatocellular neoplasms were increased at all dose levels in the repeat study, while these neoplasms were increased over controls only at the high dose in the initial study. Statistical comparison of tumor incidences between studies revealed that the control incidences of liver neoplasms were lower in the repeat study. Comparison of observed control liver lesions with those predicted by the logistic regression model revealed that in both studies, combined incidences of control liver neoplasms were similar to those expected. In addition, the expected incidences of these control neoplasms were higher in the initial study compared to the repeat study. Collectively, these observations suggest that H. hepaticus
did not influence the neoplastic response in the initial study and that differences in both diet and animal source may have contributed to the lower control liver neoplasm incidence in the repeat study, providing more sensitivity to the statistical tests. The absence of an effect of H. hepaticus
on the induction of hepatocellular neoplasms in TEA exposed females is consistent with the general absence of hepatitis in infected females. The NTP-2000 diet was designed in part to reduce tumor burden. The results of one study suggest that liver neoplasm incidences are lower in control mice from NTP feed and inhalation studies that were fed the NTP-2000 compared to control mice fed the NIH-07 diet (Rao and Crockett, 2003
); however, the studies used for the NIH-07 were more recent than the studies included in the historical control database used for the initial study of TEA. Furthermore, examination of the historical databases (all routes) for each study revealed that in H. hepaticus
free mice, control hepatocellular neoplasms incidences were lower in mice obtained from Taconic Labs and fed the NTP-2000 diet compared to mice obtained from Simonsen Labs and fed the NIH-07 diet; however, because control incidences of neoplasms vary with exposure route and TEA study was the only dermal study that fit these souce and diet conditions, a further analysis of these differences was not possible.
The ability of TEA to induce changes in survival or body weight was not modified by H. hepaticus or differences in protocol between studies, as survival was not affected in either study and treatment-related reductions in body weight gain were observed in males at 2,000 mg/kg in both studies. Although in both studies there were increases in non-neoplastic lesions of the skin at the site of application, which were more apparent in males, the extent of this damage was greater in the repeat study. The greater extent of skin damage in males, compared to females, in both studies may have been a result of the higher doses administered to males. The increased skin damage in the repeat study relative to the initial study in both males and females could not be explained by differences in protocol or failure to observe lesions in the initial study.
Because of the H. hepaticus infection, the initial study of TEA in B6C3F1 mice was considered inadequate. As a result, the NTP used the data from the repeat study to assess the carcinogenic activity of TEA and concluded that TEA was hepatocarcinogenic in female mice, based on increased hepatocellular neoplasms at all doses and may have been hepatocarcinogenic in male mice, based on an increase in hemangiosarcomas at the mid dose.
In conclusion, the data suggest that the increase in hepatocellular tumors in initial study male B6C3F1 mice resulted from the combined effects of H. hepaticus and TEA, while the increase in hepatocellular neoplasms at lower doses in repeat study female mice than in initial study females resulted from the reduction in control liver neoplasms and the effect of TEA without influence of H. hepaticus.