title: NTP Hepatotoxicity Studies of the Liver Carcinogen Methapyrilene Hydrochloride (CAS No. 135-23-9) Administered in Feed to Male F344/N Rats.
abstract: Methapyrilene hydrochloride is a histamine H(1)-receptor antagonist that was an active ingredient in many over-the-counter cold and allergy medications. In the mid- to late 1970s, studies in rats suggested that methapyrilene hydrochloride was a hepatocarcinogen, and the drug was removed from these preparations. In most cases, methapyrilene hydrochloride was replaced by pyrilamine maleate, a structurally similar analogue. As part of a program to investigate mechanisms of toxicity whereby structurally similar chemicals produce different toxicities, these chemicals were studied for induction of cell proliferation and protein alterations by two-dimensional gel electrophoresis in the liver of F344/N rats. A complete toxicologic evaluation was not needed for this research-oriented study. Rather, the goal of the present study was to provide retrospective data from subchronic toxicity studies with the known rat carcinogen methapyriline hydrochloride that could then be used to predict the potential carcinogenicity of unknown chemical agents and that could also be compared with similar data on the structural analogue pyrilamine maleate. Pyrilamine maleate differs from methapyrilene hydrochloride in the substitution of the thienyl ring with a paramethoxyphenyl ring. Pyrilamine maleate has been shown to produce an equivocal increase in the incidences of liver neoplasms in rats in 2-year feed studies, but only at 2,000 ppm, indicating that its potency, if any, to produce neoplasms is much less than that of methapyriline hydrochloride. The hepatocarcinogenic peroxisome proliferator Wy-14,643 was included in this study as a positive control that is known to induce cell proliferation, as well as protein alterations, in the liver. In the 14-week study of methapyrilene hydrochloride, groups of 40 male F344/N rats were given 0, 50, 100, 250, or 1,000 ppm methapyrilene hydrochloride, 1,000 ppm pyrilamine maleate (negative control), or 50 ppm Wy-14,643 ( positive control) in feed. Rats in all groups were administered bromodeoxyuridine (BrdU) by osmotic minipump for the assessment of hepatocyte proliferation. Ten rats from each group were evaluated on days 15, 29, and 43 and at 14 weeks. At these times, samples of liver tissue were analyzed for evidence of cell proliferation via BrdU labeling and proliferating cell nuclear antigen (PCNA) labeling. There were no exposure-related deaths. Low mean body weights were generally observed in the 1,000 ppm methapyriline hydrochloride group and in the positive control group. Final mean body weights and mean body weight gains of rats exposed to 1,000 ppm methapyrilene hydrochloride were significantly less than those of the untreated control group at all time points. The final mean body weights of rats in the positive control group were significantly less than those of the untreated control group for rats evaluated on days 29 and 43 and at week 14; the mean body weight gains of rats in the positive control group were significantly less than those of the untreated control group on day 29 and at week 14. Feed consumption by rats exposed to 1,000 ppm methapyrilene hydrochloride was significantly less than that by the untreated control group throughout the study. The predominant clinical observation related to methapyrilene hydrochloride exposure was thinness in rats exposed to 1,000 ppm; this finding was first observed on day 29. On days 29 and 43 and at 14 weeks, the absolute liver weights of rats exposed to 1,000 ppm methapyrilene hydrochloride were significantly less than those of the untreated control group. At all time points, the relative liver weights of rats exposed to 1,000 ppm methapyrilene hydrochloride and the absolute and relative liver weights of positive control rats were significantly greater than those of the untreated control group. No significant differences in liver weights were observed between the negative and untreated control groups at any time point. Hepatic lesions were observed predominantly in the 250 and 1,000 ppm methapyrilene hydrochloride groups and in the positive control group. The incidences of bile duct hyperplasia, hepatocyte necrosis, hepatocyte mitosis, and hepatocyte hypertrophy in rats in the 1,000 ppm group were significantly greater than those in the untreated control group at all time points. The severities of hepatocyte hypertrophy and hepatocyte mitosis in 1,000 ppm rats were generally mild to moderate; the lesions occurring in 250 ppm animals were less severe. At each time point, the incidence of bile duct hyperplasia in 250 ppm rats was significantly greater than that in the untreated control group. The incidences of hepatocyte mitosis on days 15 and 29 and the incidences of hepatocyte necrosis on days 29 and 43 in rats in the 250 ppm group were significantly greater than those in the untreated control group. Incidences of pigmentation in the 250 and 1,000 ppm methapyrilene hydrochloride groups were significantly greater than those in the untreated control group on days 29 and 43 and at 14 weeks. In the positive control group, the incidences of granulomatous inflammation were significantly greater than those in the untreated control group on days 15, 29, and 43. The incidences of hepatocyte hypertrophy and hepatocyte mitosis in the positive control group were significantly greater than those in the untreated control group on days 15, 29, and 43. The incidence of hepatocyte hypertrophy was also significantly increased in the positive control group at 14 weeks. The severity of hepatocyte hypertrophy in the 1,000 ppm methapyrilene hydrochloride group was generally greater than that in the positive control group at each time point. In general, methapyriline hydrochloride produced a dramatic and sustained increase in hepatic cell proliferation over 14 weeks, whereas pyrilamine maleate at the same concentration produced few if any effects. Wy-14,643 also induced a large increase in cell proliferation which declined over time, as has been observed in previous studies. The mean BrdU labeling indexes of the 250 and 1,000 ppm methapyrilene hydrochloride groups were generally significantly greater than those of the untreated controls at all time points. In the negative control group, the BrdU labeling index was significantly less than that of the untreated control group on day 29. The BrdU labeling index in the positive control group was significantly greater than that of the untreated control group at all time points. On day 43 and at week 14, the mean PCNA labeling indexes of the 1,000 ppm methapyrilene hydrochloride group were significantly greater than those of the untreated control group. The mean PCNA labeling indexes of the negative control group were significantly less than those of the untreated control group on days 29 and 43. On day 29, the mean PCNA labeling index of the positive control group was significantly greater than that of the untreated control group. The mitotic indexes of the 1,000 ppm methapyrilene hydrochloride group were significantly greater than those of the untreated control group at all time points. The mitotic indexes of the 250 ppm group were significantly greater than those of the untreated control group on day 43 and at week 14. At least 32 proteins underwent significant abundance changes at the highest exposure concentration of methapyrilene hydrochloride, and 39 protein changes were observed in the positive control group. Many, but not all, of the protein changes in the methapyrilene hydrochloride-exposed animals also occurred in the positive control group. Treatment with pyrilamine maleate produced no significant quantitative protein changes, as judged by the same criteria used for methapyrilene hydrochloride and Wy-14,643. Methapyrilene hydrochloride produced covalent modification of mitochondrial proteins as measured by the charge modification index. PCNA abundance in liver samples from the 250 and 1,000 ppm methapyrilene hydrochloride exposure groups on day 43 was significantly greater than that of the untreated control group. Results of tests for induction of mutagenicity by methapyrilene hydrochloride were negative in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and in L5178Y mouse lymphoma cells, with and without S9 metabolic activation. However, positive responses were obtained in cytogenetic tests with cultured Chinese hamster ovary cells, in which methapyrilene hydrochloride induced sister chromatid exchanges and chromosomal aberrations. The increases in sister chromosome exchanges were obtained with and without S9, but chromosomal aberrations were increased only in the presence of S9. In summary, the significance of the increased hepatic cell proliferation and the protein alterations observed in this study is not definite, but may be of predictive value for assessing the toxicity and carcinogenicity of chemicals in preclinical assays. A chemical which does not produce an increase in cell proliferation or a large number of protein changes may be considered safer than a similar chemical that produces many such changes.