ToxSci Advance Access originally published online on December 22, 2005
Toxicological Sciences 2006 90(2):440-450; doi:10.1093/toxsci/kfj081
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Phenolphthalein and Bisacodyl: Assessment of Genotoxic and Carcinogenic Responses in Heterozygous p53 (+/–) Mice and Syrian Hamster Embryo (SHE) Assay
* Stoll & Associates, LLC, Storrs Mansfield, Connecticut 06268; Department of Toxicology and Safety Assessment, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877; Pfizer, Inc., Ann Arbor, Michigan 48108; Chrysalis, Inc., Richmond, Virginia 23113; and ¶ Campbell University, Buies Creek, North Carolina 27506
1 To whom correspondence should be addressed at Stoll & Associates, LLC, 38 Homestead Drive, Storrs Mansfield, CT 06268-3102. Fax: (860) 429-1418. E-mail: rstoll{at}prodigy.net.
Received September 9, 2005; accepted December 20, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phenolphthalein (800 and 2400 mg/kg/day by gavage and 2400 mg/kg/day by diet) and bisacodyl (800–500, 4000–2000, and 8000 mg/kg/day by gavage) were administered to 15 male and 15 female and 20 male and 20 female p53+/– mice respectively for 26 weeks to investigate the potential carcinogenicity of each compound. Toxicokinetic analyses confirmed systemic exposure. p-Cresidine was administered by gavage (400 mg/kg/day) and served as the positive control agent in each study. Dietary phenolphthalein reduced survival in both sexes and early deaths were attributed to thymic lymphoma. No bisacodyl-related neoplasms were observed. Regardless of route of administration to p53+/– mice, phenolphthalein but not bisacodyl was unequivocally genotoxic, causing increased micronuclei in polychromatic erythrocytes. In the Syrian hamster embryo (SHE) cell transformation assay, phenolphthalein caused increases in morphologically transformed colonies, thereby corroborating NTP's earlier reports, showing phenolophthalein has potential carcinogenic activity. Bisacodyl was negative in the SHE assay. Results of these experiments confirm an earlier demonstration that dietary phenolphthalein causes thymic lymphoma in p53+/– mice and show that (1) phenolphthalein causes qualitatively identical results in this transgenic model regardless of route of oral administration, (2) phenolphthalein shows evidence of micronucleus induction in p53+/– mice for up to 26 weeks, (3) phenolphthalein induced transformations in the in vitro SHE assay, and (4) bisacodyl in p53+/– mice induces neither drug-related neoplasm, nor micronuclei in polychromatic erythrocytes, and did not induce transformations in the in vitro SHE assay.
Key Words: phenolphthalein; bisacodyl; carcinogenicity; p53 transgenic; laxatives; SHE assay.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phenolphthalein and bisacodyl have been used as laxatives in over-the-counter (OTC) products for decades, and in their use were considered safe and effective. These substances were "generally recognized as safe and effective" (GRAS), and given a Category I in the Tentative Final Monograph for over-the-counter laxatives. In 1995, the National Toxicology Program (NTP) presented for Peer Review draft reports on the results of two-year bioassays conducted on phenolphthalein in rats and mice. They were finalized the next year (NTP, 1996
). While positive tumor responses were observed in both species (Dunnick and Hailey, 1996), two tumor types described in the mouse as histiocytic sarcomas and thymic origin lymphomas, were of regulatory concern. The scientific and regulatory communities agreed that further work was necessary to determine the relevance of these bioassay findings, and additional work was undertaken to investigate phenolphthalein in numerous tests, including the Syrian hamster embryo (SHE) assay, and a six-month in vivo p53+/– transgenic mouse carcinogenicity test. Dunnick et al. (1997) reported that phenolphthalein was carcinogenic in the heterozygous p53+/– transgenic mouse as evidenced by the induction of thymic lymphomas. In addition, the incidence of micronuclei were increased at all dose levels tested (Tice et al., 1998). The Food and Drug Administration (FDA) published on 19 June 1998, a proposed amendment to the Tentative Final Monograph for the OTC laxative drug products (FDA, 1998), proposing a reclassification of bisacodyl, among other stimulant laxatives, from Category I to Category III. Drugs classified as Category III substances are considered to require further testing to support classification with respect to human safety (further testing is required). When, based on the results of the phenolphthalein reports, bisacodyl was also moved to Category III, we reviewed our extensive body of unpublished mutagenicity data. Those data, all of which were negative with respect to mutagenic potential, included the Ames test, repeated dose micronucleus, and CHO (HPRT) tests. We then investigated bisacodyl in the SHE assay and in the heterozygous p53+/– transgenic mouse, and performed the same two tests on phenolphthalein within our laboratories under GLPs (Good Laboratory Practices).
These experiments, reported in this communication were conducted to (1) corroborate the NTP reported effects of dietary phenolphthalein on the hematopoietic systems of p-53+/– mice, (2) extend those findings by assessing the influence of route of administration (gavage vs. dietary), (3) assess the carcinogenic potential of phenolphthalein in the SHE assay, and in a micronucleus test which we incorporated into the six-month carcinogenesis transgenic p53+/– mouse study, and (4) assess the carcinogenic potential of bisacodyl in an in vitro transformation test (SHE) and (5) assess bisacodyl in the p53+/–carcinogenesis assay which we supplemented with four replicates of the micronucleus test (micronucleus test performed on peripheral blood samples in Drug Days 39, 92, 137, and 183 of the study). The results of our studies support our working hypothesis that bisacodyl does not possess carcinogenic potential.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and husbandry.
Male and female heterozygous (C57BL/6TacBR-[KO]p53[TSG-p53] (Taconic Farms, Germantown, NY) for p53 (p53+/–) and C57BL/6NCrLBr (Charles River Laboratories, Raleigh, NC) mice were used throughout these experiments, with the exception of the 13 week range-finding bisacodyl mouse study, during which Crl:CD-1 (ICRBR)VAF+ mice (Charles River, Raleigh, NC) were used. Animals, approximately 6 weeks of age upon receipt, were uniquely identified by sc implantation of radio signal transponders (Biomedic Data Systems, Seaford, DE), housed individually in suspended stainless steel wire bottom cages, and acclimated to laboratory conditions for approximately two weeks prior to initiation of each study. Controlled animal room conditions included 72–75°F at 50 ± 20% relative humidity and a 12 h on/off fluorescent light cycle (light 6 A.M. to 6 P.M.). Mice had free access to rodent chow (PMI Feed no. 5002), except approximately 4 h prior to terminal necropsy, and potable municipal water delivered via an automatic watering system.
Drugs, chemicals, and dose selections.
Phenolphthalein (CAS No. 77-09-8), and p-cresidine (CAS No. 120-71-8) were purchased from Aldrich Chemical Company (Milwaukee, WI). Bisacodyl (CAS No. 603-50-9) was obtained from Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT). Dimethyl sulfoxide (DMSO) (CAS No. 67-68-5) and benzo(a)pyrene, both used in the Syrian Hamster Embryo (SHE) assay were purchased from Sigma-Aldrich Company (St. Louis, MO). Mitomycin C (CAS No. 50-07-7) was purchased from Sigma-Aldrich Comapny (St. Louis, MO).
When phenolphthalein or bisacodyl were administered by oral gavage, they were dispersed in carboxymethyl cellulose (CMC) suspension or solution (0.5% containing 0.2% Tween 80) and p-cresidine was suspended in corn oil (Mazola). CMC vehicle with Tween 80 was made fresh weekly and kept refrigerated; bisacodyl and phenolphthalein were stored at room temperature and ambient humidity. The p-cresidine was stored under nitrogen at room temperature. Dosing solutions/suspensions of phenolphthalein, bisacodyl, and p-cresidine were made fresh daily. When phenolphthalein was administered by diet it was blended into ground rodent chow. Dietary and dosing solution/suspension concentrations of phenolphthalein or p-cresidine were confirmed by HPLC analyses.
Bisacodyl blood concentrations were determined on the basis of the glucuronide conjugate in plasma by HPLC with ultra-violet (UV) detection and concentrations of phenolphthalein were determined by HPLC.
The dietary dose of phenolphthalein was based on the results of an earlier feeding study in p53+/– mice (Dunnick et al., 1997) where animals consumed diets containing up to 12,000 ppm of phenolphthalein. That concentration afforded a mean daily dose of approximately 2400 mg/kg body weight. The gavage dose of phenolphthalein was based on the results of a four-week oral gavage dose range finding test in which groups of 10 male and 10 female C57BL/6NCrlBr mice received daily doses of 0, 600, 2400, or 3600 mg/kg. The dose levels of bisacodyl investigated in the p53+/– six-month study were based on the results of a four-week dose range finding test in which groups of 10 male and 10 female C57BL/6NCrlBr mice received daily doses of 0, 40, 4000, and 8000 mg/kg. Additionally, a 13-week oral gavage range finding study was performed in which 10 male and 10 female CD-1 mice received daily bisacodyl doses of 0, 5/4000, 10/400/2000/8000 (8000 mg/kg/day from Drug Week 4 through termination of the study) or 40 mg/kg.
Twenty-Six Week Studies in Male and Female p53+/– Mice
Carcinogenesis study.
Groups of 15 male and 15 female p53+/– mice were administered phenolphthalein (0, 800, 2400 mg/kg/day) or p-cresidine (400 mg/kg/day) by oral gavage (volume dose of 10 ml/kg body weight) or phenolphthalein 2400 mg/kg/day by diet. In another study within the same laboratories, groups of 20 male and 20 female p53+/– mice were administered bisacodyl (0, 800, 4000, 8000 (4000 mg/kg b.i.d.) mg/kg/day) or p-cresidine (400 mg/kg/day) in oral gavage (volume doses of 20 ml/kg body weight). In the beginning of Drug Week 2, the 800 and 4000 mg/kg/day dose levels were lowered to 500 and 2000 mg/kg/day per recommendation of the FDA CAC (Carcinogenicity Assessment Committee), and the dose volume was lowered for both dose levels to 10 ml/kg of body weight (the design and dose level selections were approved by the FDA CAC committee on 8 October 1998, Drug Week 1 of the bisacodyl study). Dietary concentrations required to achieve the daily phenolphthalein dose, as well as the gavage dose levels of phenolphthalein, bisacodyl, and p-cresidine were based on mean body weights. Mice were observed for grossly observable signs and symptoms twice daily and incidences of palpable masses were recorded weekly. All animals which survived to scheduled termination were subjected to a detailed necropsy and organ weights (liver with gallbladder, heart, spleen, brain, testes, thymus, kidneys, and ovaries) were recorded. Animals which died spontaneously were necropsied, but organ weights were not recorded. More than 45 organs and tissues were collected from all animals necropsied and detailed histopathologic examinations were performed. No statistical analyses were performed on histopathologic data since the responses were considered to be biologically significant. Diagnoses and interpretations from both studies were peer reviewed by a board certified pathologist. Quantitative in-life and post mortem data were compared for statistical significance of differences by an analysis of variance (ANOVA) followed with a Dunnett's test.
Dunnett's test was done regardless of the outcome of the ANOVA, and significance was established at p 
0.05 or p 0.01.
Toxicokinetic analyses.
Blood samples (retro-orbital sinus) for toxicokinetic analysis were obtained from 4 to 5 animals per sex per group, inclusive of control animals, during Drug Weeks 8, 15, 19, and 26 of the phenolphthalein study, and in Drug Weeks 1 (Drug Day 1), 6, 14, and 26 of the bisacodyl study. Samples were routinely collected approximately 2 h post gavage dosing on the day of blood collection, and 2 h after the second dose of bisacodyl at the 8000 mg/kg/day, as well as at 0700 h for the dietary group. In Drug Week 19 of the phenolphthalein study, trough concentrations of phenolphthalein were approximated by collecting samples immediately prior to gavage dosing and at 1700 h for the dietary group.
Micronucleus assay.
Blood samples (retro-orbital sinus) were obtained from the first l0 surviving mice on Drug Days 39, 92, 137, and 183 in the phenolphthalein experiment, and from the 5 toxicokinetic animals on Drug Days 39, 92, 137, and 183 in the bisacodyl experiment. For the positive control with Mitocycin C, daily fresh test material was prepared, injected ip 30 min post reconstituting Mitocycin C in solution, and samples were collected from animals 24 h post the last treatment. Tail bleeds were performed on the positive control p-cresidine treated animals 24 h post treatment. Slides were then fixed in absolute methanol and air dried. Before evaluation, at least one slide of each animal was stained for approximately 1.5 min with acridine orange (125 ug/ml in phosphate buffer).
Slides were evaluated from all dose groups including the vehicle control and positive control groups. Slides were scored under fluorescence optics. Up to 2000 polychromatic erythrocytes (PCE) and 2000 normochromatic erythrocytes (NCE) were scored for the presence of micronuclei for each animal. Also, the ratio of PCE to NCE in up to 2000 erythrocytes was determined as an indicator of alteration of erythropoiesis. For data that were clearly within expected background levels, no statistical analysis was performed. For marginal data where interpretation was not obvious, statistical analyses were conducted according to the procedures of Margolin and Risko (1998) or Kastenbaum and Bowman (1970). The hematocrit of the first 10 surviving animals per sex were determined on Drug Days 39, 92, 137, and 183.
Syrian Hamster Embryo (SHE) in Vitro Cell Transformation Assay
The procedures used were based on the method of Kerchaert et al. (1996a) and aseptic techniques were used throughout the studies. Syrian hamster embryo cells were prepared in the Boehringer Ingelheim Pharmaceuticals, Inc. investigative toxicology laboratories and were maintained in a liquid nitrogen freezer until used. The growth medium used was Dulbecco's modified Eagle's medium-LeBoeuf (DMEM-L) supplemented with L-glutamine and fetal bovine serum (10% by volume).
Cells derived from 13-day embryos from timed pregnant Syrian hamsters were plated at a clonal density on an irradiated (5000R) feeder layer. Phenolphthalein or bisacodyl were added for either 24 h or 8 days. After the addition of treatment, the colonies were fixed in methanol, stained with Giemsa and scored for morphological transformation.
Relative plating efficiency was derived from the average plating efficiency of treated cultures as compared to the average plating efficiency of concurrent control cultures. The test article was considered positive if it caused a statistically significant increase (p 0.05; Fisher's Exact Test) in morphological transformation in at least two dose levels, compared to concurrent controls, or a significant increase in one dose with a statistically significant (p 0.05; Cochran-Armitage Trend Test) positive dose-response trend.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dose Range Finding Studies
The results of the preliminary phenolphthalein, four-week (gavage) dose range-finding test in C57BL/6 mice (parent strain) revealed that gavage doses of 600, 2400, or 3600 mg/kg/day were without effect on survival, food consumption, or hematocrit. The 3600 mg/kg/day dose caused significant reduction in body weight gain in males but not females. These results led to selection of 800 and 2400 mg/kg by gavage and 2400 mg/kg/day by dietary administration as dose levels for the 26-week carcinogenesis and micronucleus experiments in p53+/– mice.
The daily gavage dose of p-cresidine (400 mg/kg) was previously determined within the Boehringer Ingelheim labs in separate studies, whereby both 400 and 800 mg/kg/day were investigated, since previous investigators routinely administered p-cresidine only by dietary admixture. Due to potential worker safety issues, gavage was a safer way of reducing worker exposure to a carcinogen. The sole purpose of including p-cresidine in the p53+/– 26-week carcinogenesis studies, was to serve as a known positive control for induction of preneoplastic and neoplastic changes in the urinary bladder. A comparable bisacodyl four-week (gavage) dose range-finding test in C57BL/6 mice revealed that a dose level up to 8000 mg/kg/day (4000 mg/kg, b.i.d.) was well tolerated and produced no evidence of systemic toxicity. The dose of 8000 mg/kg/day was determined to be the maximum feasible dose of bisacodyl. Plasma concentrations of bisacodyl ranged from 215.5 to 369.8 µg/ml at the 8000 mg/kg/day dose level. Clinical signs included diarrhea, soft stools, and light colored stools, but no changes in food consumption or body weight gain were noted and no histopathological drug-related changes were observed.
The results of a 13-week bisacodyl (gavage) range-finding CD-1 mouse test were very similar to the previous 4-week experiment using the C57BL/6 mouse. A dose level of up to an escalated dose of 8000 mg/kg/day for 9 consecutive weeks showed no significant toxic signs, where drug plasma concentrations ranged from 310.4 to 536.9 µg/ml. Although we rarely conduct chronic studies at dose levels greater than 2000 mg/kg/day as a maximal feasible dose, this particular study deviated from this policy to maximize the chance of eliciting a tumorigenic response if in fact one did exist for bisacodyl. Thus, 8 g/kg/day (4000 mg/kg, b.i.d.), considered by most investigators as a huge dose level, was selected as the top dose to be tested in the p53+/– 26 week carcinogenesis experiment and micronucleus test.
Carcinogenesis and Micronucleus Studies: Phenolphthalein
Survival and clinical observations.
Survival and causes of phenolphthalein interim deaths are summarized in Table 1. Overall survival of mice administered phenolphthalein or p-cresidine by the gavage route was similar to that of control but dietary phenolphthalein reduced survival of both sexes by nearly 50%. Greater than 50% of the interim deaths (71 and 86% in males and females, respectively) in the dietary group were cancer-related and attributed to thymic lymphomas. Although survival in mice dosed with phenolphthalein by the gavage route was unaffected by treatment, most interim deaths in this group were also attributed to thymic lymphomas. None of the early deaths in either the vehicle- or positive groups were associated with hematopoietic tumors.
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Food consumption of phenolphthalein-dosed mice was unaffected and based on group mean body weight and food consumption data the mean doses of phenolphthalein administered via the diet were 2188 and 2333 mg/kg/day in males and females, respectively. The most consistent and drug-related clinical observation was light colored stools in mice dosed with phenolphthalein by gavage (but not by diet). Food consumption was reduced in both sexes dosed with p-cresidine and 14–15% decrements in body weight gains were observed (data not shown). p-Cresidine dosed animals also excreted dark colored urine. Assessment of hematocrit values on experimental days 39, 92, 137, and 183 revealed no changes attributable to phenolphthalein, however, p-cresidine dosed animals demonstrated a significant decrease of 19.7 and 14.3% on average as a function of time for males and females, respectively (data not shown).
Toxicokinetics and suspension analysis.
Toxicokinetic analyses revealed substantial systemic exposure to phenolphthalein regardless of route of administration (Table 2). Plasma concentrations were highly variable in all groups and there was no evidence of proportionality between the gavage doses. When administered by gavage the peak to trough swings were generally 80 to 90% while during dietary administration peak to trough differentials were smaller, being in the range of 20 to 30%. On Drug Day 1, the concentration of phenolphthalein at 2400 mg/kg/day gavage suspension was 125% of theoretical and the concentration of p-cresidine dosing solution was 123% of theoretical. These slightly high values were likely the result of the collection procedure, since a positive displacement pipette was not utilized. All subsequent test article analyses of gavage dosing suspensions/solutions were sampled with a displacement pipette and found to be within 84–118% of theoretical; concentrations of phenolphthalein in the diet throughout the study were within 98–110% of theoretical (data not shown).
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Micronucleus test.
The results of the micronucleus assays are summarized in Table 3. Neither gavage nor dietary administration of phenolphthalein affected the percent of erythrocytes existing as PCEs, but the percent of PCEs was consistently elevated in p-cresidine dosed mice. The p-cresidine associated elevations, although ranging from 2.0- to 3.1-fold, were not statistically significant (p > 0.05). Both phenolphthalein and p-cresidine caused statistically significant increases in the incidences of micronucleated PCEs. The effects of both phenolphthalein and p-cresidine were evident at all four time intervals and afforded unequivoval evidence of micronucleus induction.
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Assessment of hematocrit values on experimental days 39, 92, 137, and 183 were not affected by administration of phenolphthalein. However, p-cresidine was associated with significantly decreased hematocrit values, which may reflect an incidence of methemoblobinemia known to be associated with p-cresidine administration in rodents, in both sexes at all observation periods (data not shown).
Postmortem observations.
Neither absolute nor relative organ weights were affected by administration of either phenolphthalein or p-cresidine. Macroscopic examination at necropsy revealed an increased incidence of thymic masses in phenolphthalein-dosed mice and the presence of these masses was corroborated microscopically with the diagnosis of neoplasms.
The incidences of thymic lymphomas in phenolphthalein-dosed mice are shown in Table 4. Lymphoblastic lymphomas were composed of cells with large nuclei with single to multiple basophilic nucleoli and scant cytoplasm. The cells replaced and sometimes completely effaced the normal thymic cellular architecture and adjacent mediastinal structures. Abundant mitotic figures were present and macrophages containing phagoctosed cellular debris were usually present in the neoplasms. In Table 4, lymphoma, NOS was described as a discrete cell neoplasm but the degree of autolysis in affected tissues precluded a more definitive cellular identification. The cells of the neoplasm appeared to be monomorphic with non-lobulated nuclei. Thymic lymphomas were not observed in either control or p-cresidine-dosed mice, but all doses of phenolphthalein increased the incidence of the neoplasms in both sexes. While the incidence of total thymic lymphomas appeared to be dose-related in animals dosed by gavage, still higher incidences (80 and 93% in males and females, respectively) were observed in mice dosed by the dietary route. Microscopic evaluation of other organs and tissues revealed an increased incidence and severity of seminiferous tubule degeneration in the testes of males dosed with phenolphthalein by the dietary route. No additional evidence of systemic toxicity or phenolphthalein-associated carcinogeniesis was observed. Microscopic changes caused by the administration of p-cresidine are shown in Table 5. All p-cresidine associated lesions were located in the urinary tract, as expected. Five dosed males and one female each exhibited interstitial cell nephritis of the kidney and two animals of each sex had hydronephrosis. In the urinary bladder, hyperplasia (spindle and transitional cell) was present in 80 to 100% of mice and squamous- and transitional-cell neoplasms were also observed in dosed, but not in control mice, and indicated that p-cresidine served as a positive control in this test system. While the incidence of frank neoplasms was higher in males than in females (10/15, 67% in males vs. 2/15, 13% in females) the incidence of preneoplastic changes was high and essentially equal in both sexes (15/15, 100% in males and 14/15, 93% in females).
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SHE Cell Transformation Assay: Phenolphthalein
The SHE cells were treated with phenolphthalein over a dose range of 1.25 to 15.0 µg/ml for 8 days and 2.5 to 20 µg/ml for 24 h and results are given in Table 6. The plating efficiency of cultures treated with 15 µg/ml for 8 days was reduced to 55%. The average number of colonies per plate was outside the optimal range of 25–45 colonies/plate and this dose level was not evaluated for morphological transformation. Although there was minimal toxicity at 10.0 µg/ml, there was a statistically significant increase in morphologically transformed colonies (Fisher's exact test). Over the dose range evaluated, 1.25 to 10.0 µg/ml, there was a positive dose-related trend in transformed colonies (Cochran Armitage test).
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When cells were treated for 24 h with phenolphthalein over the dose range of 2.5 to 20 µg/ml, there was a significant increase in transformed colonies at dose levels greater than or equal to 10.0 µg/ml and a positive dose-related trend. Dose levels of 0, 1.25, 2.50 µg/ml were tested at a cell density of 80 cells/dish, whereas dose levels of 5.0 µg/ml or higher, 120 cells/dish were evaluated for transformation (cell densities based on range-finding plating efficiencies previously generated; data not shown). These results were consistent with those published by Kerchaert et al. (1996b)
, which indicated that phenolphthalein has potential carcinogenic activity.
Carcinogenesis and Micronucleus Studies: Bisacodyl
Survival and clinical observations.
Survival and causes of interim deaths are summarized in Table 7. Overall survival of mice administered bisacodyl was similar to that of controls. Of the total 16 animals that died prior to scheduled termination one Mid-dose bisacodyl animal died as a result of thymic lymphoma, and one animal in the bisacodyl (800–500 mg/kg), bisacodyl (4000–2000 mg/kg), and p-cresidine groups died as a result of sarcoma.
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Clinical observations related to bisacodyl included light colored stools and thinness in the 4000–2000 and/or 8000 mg/kg bisacodyl dose groups. Dark colored urine (may reflect methemogobinemia, however methemoglobin was not determined in this study) and decreased motor activity was found to be associated with p-cresidine administration. Bisacodyl decreased mean body weight (approximately 10%) in the female 8000 mg/kg dose level, and an approximate decrease of 17% in male and 9% in female animals administered p-cresidine (data not shown). Food consumption was decreased in male and female animals (approximately 22%) administered p-cresidine (data not shown).
Toxicokinetics and suspension analysis.
Toxicokinetic analyses revealed substantial systemic exposure at all bisacodyl dose levels (Table 8), and dose proportionality was observed throughout the study, except for Drug Day 1. No bisacodyl conjugate was detected in samples taken from control animals.
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Dosing solutions/suspensions of bisacodyl and p-cresidine were analyzed throughout various time points in the study and concentrations were observed to be 84 to 116% of theoretical (data not shown). No bisacodyl was observed in control vehicle solutions throughout the study with the exception of a sample taken from the first dosing session in Drug Week 25 (0.0074 mg/ml). Control animals were dosed first each day, and for the amount of drug noted, contamination was significantly lower than the Low dose level concentration, which indicated contamination occurred either during transfer of serum to cyro tubes or contamination occurring during sample preparation for analysis. Control samples were also taken from the same bulk reservoir (daily preparation) for both dosing sessions. Bisacodyl was not detected in samples taken from the second dosing session on that same day, and therefore the contamination was considered to be due to residual from the sampling vessels.
Micronucleus test.
The results of the micronucleus assays are summarized in Table 9. No evidence of drug induced PCE alterations in animals administered bisacodyl was observed. p-Cresidine was not assessed in this assay. Instead, mitomycin C was evaluated as a positive control in both C57B1/6 and p53+/– mice at Day 183, the last of four time points monitored of which an increased percent of micronucleated PCEs was observed. Under the conditions of this study, bisacodyl did not produce any evidence of clastogenic effects in mice following repeated oral dosing for up to 26 weeks.
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Postmortem observations.
Increased liver absolute organ weight, % relative to body weight, and % relative to brain weight ratios were observed for the male p-cresidine and female 8000 mg/kg bisacodyl dose groups compared to the respective control (10–35% and 21–25%, respectively. The increase in liver weight was attributed to hepatocellular hypertrophy noted in the histopathology examination.
No drug-related neoplasm findings were observed in bisacodyl treated animals in comparison to the drug-related thymic lymphomas observed with phenolphthalein. Discoloration of the urinary bladder was observed in several male and female p-cresidine animals, and correlated with mucosal proliferative findings microscopically, including spindle cell hyperplasia, transitional epithelial hyperplasia, or transitional cell carcinomas. Collectively, microscopic findings observed in the urinary bladder of p-cresidine treated animals gave a positive urinary bladder carcinogenic response which consisted of mixed mucosal infiltrates, squamous metaplasia, and transitional cell apoptosis (Table 10). Hyperplasia was more commonly observed than the two types of neoplasms, and often in combination with the other drug-related findings. Transitional cell papillomas were seen in two males and transitional cell carcinomas were observed in two females. In our laboratories within the p53+/– transgenic mouse, pre-neoplastic or neoplastic changes have not been observed in the urinary bladder spontaneously.
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SHE Cell Transformation Assay: Bisacodyl
The SHE cells were treated with bisacodyl over a dose range of 30 to 55 µg/ml for 8 days and 45 to 100 µg/ml for 24 h and results are given in Table 11. Bisacodyl precipitated upon addition to the treatment media at all dose levels ranging from 30 to 100 µg/ml. However, the precipitate dissipated after addition to the medium in the cultures at all dose levels except at the 100 µg/ml dose level. There was a dose-related decrease in plating efficiency at doses greater than or equal to 75 µg/ml when cells were treated with bisacodyl for 24 h. At the highest dose level, 100 µg/ml, the relative plating efficiency was reduced to 61% of current controls. Cells treated with bisacodyl for 8 days showed a reduction in plating efficiency at all dose levels.
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Whether the cells were treated for 24 h or 8 days with bisacodyl, there was no increase in transformation frequency at any dose level.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tice et al. (1998)
reported that chronic ingestion of phenolphthalein increased the frequency of micronucleated erythrocytes in female p53+/– mice. The interpretation of those results was complicated however, by the presence of treatment associated increases in PCE:NCE ratios, which might be indicative of anemia. Anemia per se has been shown under some conditions to increase the frequency of micronuclei in PCEs (Holden et al., 1999). The results of the phenolphthalein p53+/– 26-week carcinogenesis study showing no hematocrit change along with the consistently unaffected PCE:NCE ratios demonstrates unequivocal increases in the frequency of micronucleated PCE's at each observation period. These data indicate that phenolphthalein is associated with in vivo genotoxicity and the induction of DNA damage in p53+/– mice. We have tested bisacodyl at the maximum feasible dose (8000 mg/kg/day) in an assay similar to that used for assessing the carcinogenic potential of phenolphthalein. Under the conditions of our tests the dose of bisacodyl afforded unequivocal systemic challenge but revealed evidence of neither genotoxicity nor induction of carcinogenesis. Administration of p-cresidine decreased hematocrit in both sexes (16 to 25% and 12 to 15% in males and females respectively) and, although not statistically significant, increased the frequency of PCEs during the micronucleus test. These hematologic changes were accompanied by an increased frequency of micronuclei in PCEs. We are unable to separate p-cresidine's demonstrated hematologic effects from its intrinsic genotoxic activity in the micronucleus test.
The incidence of thymic lymphoma was higher in phenolphthalein animals dosed by the dietary route than by the gavage route and thymic lymphoma was detected earlier in dietary animals. Eleven animals (5 males and 6 females) fed dietary phenolphthalein, died with thymic lymphoma prior to scheduled study termination, while only four mice (two of each sex) administered 2400 mg/kg/day of phenolphthalein by gavage with lymphoma. In addition, the earliest deaths attributed to lymphoma occurred in mice dosed via diet (Drug Weeks 18 and 16 in males and females, respectively). The earliest lymphoma death in gavage-dosed mice occurred in a low-dose female during Drug Week 19. Lymphoma was not observed in either the negative or positive control groups. Since 23 and 33% of males and females, respectively at the low-dose phenolphthalein groups had a neoplasm, we were unable to project a non-carcinogenic dose for phenolphthalein in this transgenic model. Dunnick and Hailey (1996) reported that chronic administration of phenolphthalein to female B6C3F1 mice also caused ovarian tumors. Ovarian tumors were not observed in p53+/– females dosed with phenolphthalein by either diet (Dunnick et al., 1997) or daily gavage (current study).
In the bisacodyl 26-week p53+/– study none of the animals that survived to scheduled termination exhibited thymic lymphomas. One mid-dose animal (4000–2000 mg/kg/day), which died in Drug Week 11, had a thymic lymphoma. Worth noting, the same pathologist was the responsible pathologist on both the phenolphthalein and bisacodyl 26-week studies, and both studies were "peer" reviewed by a second board certified pathologist.
Thymic lymphoma does not often occur spontaneously in p53+/– mice used in 26-week carcinogenesis studies. Storer et al. (2001) noted that no thymic lymphoma was observed in 433 male and 434 female negative control p53+/– mice. However, Mahler et al. (1998) have observed spontaneous thymic lymphoma in the p53+/– male and female mouse at an incidence of 2/108 males and 2/109 females. Whereas all bisacodyl animals received drug by gavage, in the phenolphthalein study route-associated differences in response to phenolphthalein are most likely secondary to differences in absorption from the GI tract and the resulting extent of systemic exposure. Mice administered phenolphthalein by gavage consistently exhibited light colored stools, as did the mid- and high-dose levels of bisacodyl, and although no attempt was made to identify the white-pigment, it was most likely unabsorbed drug. Animals dosed with phenolphthalein via the diet did not exhibit colored stools, suggesting more complete systemic absorption of dose.
Toxicokinetic analyses revealed that both bisacodyl and phenolphthalein were absorbed from the GI tract, irrespective of the oral route of administration of phenolphthalein.
Daily administration of p-cresidine to p53+/– mice caused a high incidence of proliferative change in the urinary bladder, which was the intended organ for identification of a positive control agent in this test model. The carcinogenic response was more pronounced in the study conducted on phenolphthalein compared to bisacodyl. Although we see variability, the p-cresidine is consistently carcinogenic in our laboratories. The same batch/lot of p-cresidine was used in both studies. Higher dose levels investigated in our laboratories were judged toxic and lethal in p53+/– mice. Our results with p-cresidine were considered to be positive and similar to those obtained in 18/19 studies in p53+/– mice in which the chemical was employed as a positive control agent (Storer et al., 2001). In those experiments 400 mg/kg/day of p-cresidine, administered by either gavage or diet, caused proliferative changes of the urinary bladder, including hyperplasia and squamous- and transitional-cell neoplasms. Neither concurrent controls exhibited these urinary bladder changes.
CONCLUSIONS
The results of these 26-week duration experiments increase the body of knowledge about the utility of the p53+/– transgenic mouse strain for identifying genotoxic chemicals possessing carcinogenic potential. As well, the 26-week phenolphthalein results, along with the micronucleus test period results corroborates the work of Dunnick et al. (1997). The results of the SHE assay indicate that phenolphthalein is potentially carcinogenic whereas in a similarly designed study testing bisacodyl, no evidence of potential carcinogencity was observed. Collectively, the results of the three tests conducted on phenolphthalein (p53+/–, micronucleus and SHE tests) show phenolphthalein possesses carcinogenic potential, whereas the same battery of three tests revealed no evidence of carcinogenic potential attributable to bisacodyl.
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ACKNOWLEDGMENTS |
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We thank Mr. Mark Raymond, Michael Lipinski, Damian Bohler, and Ms. Amy Hudak, Debbie Studwell, Claudia Giannetto, Dee Lawrenia, Drs. John McCaffrey and Joseph Pav for all of their technical support in conducting these studies.
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REFERENCES |
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Dunnick, J. K., and Hailey, J. R. (1996). Phenolphthalein exposure causes multiple carcinogenic effects in experimental systems. Cancer Res. 56, 4922–4926.
Dunnick, J. K., Hardisty, J. F., Herbert, R. A., Seely, J. C., Furedi-Machacek, E. M., Foley, J. F., Lacks, G. D., Stasiewicz, S., and French, J. E. (1997). Toxicol. Pathol. 25, 533–540.[ISI][Medline]
Food and Drug Administration (1998). Tentative final monograph for over-the-counter (OTC) laxative drug products. Federal Register 63, 33592–33595.
Holden, H. E., Studwell, D. B., and Majeska, J. B. (1999). Anemia-induced micronucleus formation in mice. Environ. Mol. Mutat. 33, 32.
Kastenbaum, M. A., and Bowman, K. O. (1970). Tables for determining the statistical significance of mutation frequencies. Mutat. Res. 9, 527–549.[ISI][Medline]
Kerchaert, G. A., Isfort, R. J., Carr, G. J., Aardema, M. J., and LeBoeuf, R. A. (1996a). A Comprehensive protocol for conducting the Syrian hamster embryo cell transformation assay at pH 6.70. Mutat. Res. 356, 65–84.[ISI][Medline]
Kerchaert, G. A., Brauninger, R., LeBoeuf, R. A., and Isfort, R. J. (1996b). Use of the Syrian hamster embryo cell transformation assay for carcinogencity prediction of chemicals currently being tested by the National Toxicology Program in rodent bioassays. Environ. Health Perspect. 104, 1075–1083.
Mahler, J. F., Flagler, N. D., Malarkey, D. E., Mann, P. C., Haseman, J. K., and Eastin, W. (1998). Spontaneous and chemically induced proliferative lesions in Tg.AC transgenic and p53-heterozygous mice. Toxicol. Pathol. 26, 501–511.[ISI][Medline]
Margolin, B. H., and Risko, K. J. (1988). The statistical analysis of in vivo genotoxicity data; case studies of the rat hepatocyte UDS and mouse bone marrow micronucleus assays. In Evaluation of Short-Term Tests for Carcinogens. Report of the International Programme on Chemical Safety's Collaborative Study on In Vivo Assays (J. Ashby, F. deSerres, M. D. Shelby, B. H. Margolin, M. Ishidate, and G. C. Becking, Eds.), pp. 29–42. Press Syndicate of the University of Cambridge, England.
National Toxicology Program (1996). NTP toxicology and carcinogenesis studies of Phenolphthalein (CAS No. 77-09-8) in F344/N rats and B6C3F1 Mice (feed studies). Natl. Toxicol. Program Tech Rep. Ser. 465, 1–354.[Medline]
Storer, R. D., French, J. E., Haseman, J., Hajian, G., LeGrand, E. K., Long, G. G., Mixon, L. A., Ochoa, R., Sagartz, J. E., and Soper, K. A. (2001). P53+/– hemizygous knockout mouse: Overview of available data. Toxicol. Pathol. 29(Suppl.), 30–50.
Tice, R. R., Furedi-Machacek, M., Satterfield, D., Udumaundi, A., Vassquez, M., and Dunnick, J. K. (1998). Measurement of micronucleated erythrocytes and DNA damage during chronic ingestion of phenolphthalein in transgenic female mice heterozygous for the p53 gene. Environ. Mol. Mutat. 31, 113–124.[CrossRef][ISI][Medline]
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