* National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709-2233; and
Triangle Pharmaceuticals, Inc., Durham, North Carolina 27707
Received December 29, 1999; accepted April 14, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: phenolphthalein; toxicokinetics; phenolphthalein-glucuronide; AUC; iv; gavage; feed; multiple routes; rat; mouse.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PTH has been a widely used active ingredient in prescription and over-the-counter laxative products (PDR, 1982; PDR, 1991
), predominantly in chewing gum and candy formulations. Human exposure therefore has occurred primarily by the oral route. Several studies have shown that, in humans, PTH is partially absorbed from the small intestine and conjugated in the liver to phenolphthalein glucuronide (PTH-G). PTH-G is excreted in bile and passes into the colon, where it is de-glucuronidated by the bacterial flora (Anand et al., 1994
; Morotomi et al., 1985
). In humans, approximately 7090% of an ingested dose is eliminated in the feces in the free or conjugated form, with renal excretion accounting for the remaining 1030% of the dose in the free or conjugated form (Kok and Faber, 1981
).
Past studies in rodents have indicated that no free PTH (PTH-F) circulates in the systemic blood. Colburn et al. (1979) reported that, because of the efficiency with which the liver conjugates this compound, PTH-F was found only in blood samples taken from the portal vein. Systemic samples collected from the carotid artery contained only the glucuronide (PTH-G) at 6 h after an intravenous dose (25 mg/kg) in female Wistar rats. Although the initial dose was administered intravenously as PTH-F, only the conjugated form was found in the systemic circulation during enterohepatic recycling. The conjugated form is thought to be biologically inactive and of no toxicological significance until it is deconjugated, which occurs during enterohepatic recycling.
Clark and Cooke (1978) reported that 85% of an iv dose of PTH, after 60 min, was excreted in the bile as the conjugate. No PTH-F was detected in the bile and no more than 3% of the dose was found in the urine. Griffin et al. (1998) have reported the glucuronide conjugate, as well as a diglucuronide conjugate, in F344 rats and B6C3F1 mice. PTH-derived radioactivity was recovered primarily in the feces in rats, whereas in mice 3040% of the administered radioactivity was found in the urine. They report that PTH was excreted in the feces in mice primarily as PTH-F (~90%), with some PTH-sulfate (~6%) and hydroxy-PTH (~4%). In rats, PTH was excreted in feces as PTH-F (~98%), with the remainder excreted as PTH-sulfate. No PTH-derived radioactivity was found to accumulate in any tissue. Sipes et al. (1997) report that both PTH-F and PTH-G enhanced free radical production and may be a significant source of oxidative stress in physiological systems. PTH-G was found to have a lower activity than PTH-F; however, this effect was offset by the larger amount of PTH-G present in the system.
In this paper, we compare plasma levels and kinetic parameters of PTH in B6C3F1 mice and F344 rats exposed to PTH for 2 years (Dunnick and Hailey, 1996); in p53 (+/) deficient mice exposed to PTH for 6 months (Dunnick et al., 1997
); and in B6C3F1 mice, C57BL mice, and F344 rats exposed to PTH and sampled over several days to obtain information for toxicokinetic evaluations. These studies provide a basis for comparing exposures to PTH across species.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
Toxicokinetic studies.
Male and female F344 rats and B6C3F1 mice were purchased from Taconic Farms, Inc. (Germantown, NY). C57BL mice were purchased from Charles River Laboratories (Raleigh, NC). The animals were quarantined upon arrival for ca. 7 days. Throughout the study, animals were individually housed in solid-bottom, polycarbonate cages with stainless steel wire lids. Certified NIH-07 feed (Zeigler Brothers, Gardeners, PA) was available ad libitum (pelleted, iv studies or ground chow, feed studies). Drinking water consisting of tap water (Durham, NC, or Worchester, MA) was made available ad libitum. Animal rooms were kept at 72 ± 3°C, with relative humidity of 4070% and a 12-h light cycle. Food consumption was recorded daily.
Two-year and transgenic studies.
The B6C3F1 mice, F344 rats, and p53 (+/) mice used in these studies were as previously described (Dunnick and Hailey, 1996; Dunnick et al., 1997
).
Chemicals
PTH (lot P9189-J1) was obtained from Pharmco Laboratories, Inc. (Titusville, FL), for the 2-year studies. For the single-dose iv and oral studies and the 2- and 26-week dosed-feed studies, Aldrich (Milwaukee, WI) supplied PTH (lot 13427 LF). The chemical was characterized by reverse-phase HPLC/UV and found to be 99% pure in lot P9189-J1 (NTP, 1996), or 97% pure in lot 13427 LF (Sparacino and Handy, 1996
). Both lots met all USP requirements. The structures of PTH and PTH-G are given in Figure 1
. Formulated diets were prepared by mixing appropriate amounts of PTH with NIH-07 feed. All diets fed to animals in these studies were analyzed and found to be within 10% of the targeted concentrations of 200, 375, 750, 3000, 12,000, and 50,000 ppm. Intravenous formulations were prepared by mixing appropriate amounts of PTH with a vehicle consisting of Emulphor®: ethanol: water (1:1:8). All intravenous formulations were analyzed and found to be within 10% of the targeted concentration that would produce a dose of 25 mg/kg. Oral doses were prepared by mixing appropriate amounts of PTH with water to achieve concentrations resulting in doses of 50, 100, 125, and 250 mg/kg. All dose preparations were analyzed and found to be within 10% of the targeted concentrations.
|
Single-oral-dose studies in F344 rats and B6C3F1 mice.
Male and female rats and mice received a single oral dose of PTH. Blood samples were obtained from three animals per time point at 5, 15, and 30 min, and at 2, 4, 6, 10, 24, 34, and 48 h after dosing. Total phenolphthalein (PTH-T) concentration was measured in plasma after incubation with glucuronidase/sulfatase.
Two-week dosed-feed studies in F344 rats and B6C3F1 mice.
Male and female rats and mice received PTH in the diet ad libitum for 2 weeks. PTH concentrations in feed were 375, 750, 3000, 12,000, and 50,000 ppm for rats and 200, 375, 750, 3000, and 12,000 ppm for mice. No controls were included in the study. At day 14, beginning at 10:00 A.M., blood was obtained from 3 animals sacrificed every 2 h until 8:00 A.M. on day 15. PTH-F and PTH-G concentrations were measured in plasma.
Two-week dosed-feed studies in C57BL mice.
Female C57BL mice received PTH in the diet ad libitum for 2 weeks. PTH concentrations in feed were 200, 750, and 12,000 ppm. No controls were included in the study. At day 14, beginning at 6:00 A.M., blood was obtained from three animals sacrificed every 2 h until midnight. PTH-F and PTH-G concentrations were measured in plasma.
Two-year dosed-feed studies in F344 rats and B6C3F1 mice.
Male and female rats and mice received PTH in the diet ad libitum for 2 years. PTH concentrations in feed were 0, 12,000, 25,000, and 50,000 ppm for rats and 0, 3000, 6000, and 12,000 ppm for mice. On the last day of the study, 3 animals from each dosed group, but not controls, were sacrificed at each specified time (6:00 A.M., 9:00 A.M., 1:00 P.M., 4:00 P.M., and 9:00 P.M.), and blood was obtained. PTH-T concentration was measured in plasma after incubation with glucuronidase/sulfatase.
Twenty-six-week dosed-feed studies in transgenic mice.
Groups of 20 female p53 (+/) mice received PTH in the diet ad libitum for 26 weeks. PTH concentrations in feed were 0, 200, 375, 750, 3000, and 12,000 ppm. At terminal sacrifice, blood was obtained from each animal and centrifuged at 3400 rpm. to obtain plasma. PTH-F and PTH-G concentrations were measured in plasma.
Sample Analysis
Sample collection.
For each study, blood samples were collected via cardiac puncture in heparinized tubes and centrifuged to obtain plasma. The separated plasma was drawn off and placed in a silanized vial, sealed with a Teflon-faced septum, and stored at 70°C until analysis.
Sample preparation.
Each plasma sample was thawed, and an aliquot of each sample was placed into a silanized 1-dram vial. Distilled water (20 µl) and internal standard solution (20 µl; 1.20 mg bromcresol purple/ml acetone) were added to each vial. The samples were vortexed briefly and allowed to equilibrate at room temperature for 15 min. Acetonitrile (0.3 ml) was added to each vial. The samples were vortexed for 1 min, sonicated for 10 min, and centrifuged for 15 min. The supernatant was placed in a silanized 1-dram vial and stored at 20°C prior to analysis.
Sample analysis.
On the day of analysis, a 200-µl aliquot of each extract was transferred to a silanized 1-dram vial, diluted with distilled water (250 µl), and mixed with a silanized Pasteur pipette. The sample was transferred to a silanized 400-µl limited-volume insert and centrifuged for 10 min. If precipitate was noted in the insert, the sample was transferred to a clean silanized 400-µl limited-volume insert. Samples were analyzed by reverse-phase HPLC-UV (230 nm) consisting of a Waters 845 HPLC (Millford, MA) with a Zorbax C18 (25 cm x 4.6 mm ID) column and Zorbax RX-18 guard column (Mac-Mod Analytical, Inc., Chadds Ford, PA). The system used an acetonitrile/0.02 M aqueous sodium phosphate, pH 4.0 gradient (22/78 for 6 min, to 32/68 in 0.5 min, hold 24.5 min, then to 22/78 in 1 min, hold 23 min), resulting in retention times of 2627.9 min for PTH-F, 6.1 6.9 min for PTH-G, and 18.622.7 min for the internal standard, bromocresol purple. The limit of detection for the method was found to be 0.001 µg/ml plasma for PTH-F and 0.045 µg/ml for PTH-G.
Data Analysis
Calculation of mean PTH-F, PTH-G, and PTH-T concentrations in plasma at each time point.
For 2-year dosed-feed studies and single-oral-dose studies, PTH-T concentration was determined in plasma after incubation with glucuronidase/sulfatase (Sigma, St. Louis, MO). For female p53 (+/) mice in the 26-week dosed-feed study, all 2-week dosed-feed studies, and single-dose iv studies, concentrations of PTH-F and PTH-G were measured in plasma. Mean plasma concentrations of PTH-F, PTH-G, and PTH-T were calculated from replicate plasma samples at each time point. When a peak in a sample chromatogram corresponded to a calculated concentration that was less than the limit of detection for the analytical method, a concentration of zero was assigned and used in the calculation of the mean. For studies in which PTH-F and PTH-G were measured separately, PTH-T was calculated by summing the concentrations of PTH-F and PTH-G.
Analysis of single-iv-dose and oral-dose data.
WinNonlin (Version 1.5, SCI Software, Morrisville, NC) was used for noncompartmental pharmacokinetic analysis of mean PTH-F (where applicable) and PTH-T concentration versus time data from the single-iv-dose (WinNonlin Model 201) and oral-dose (WinNonlin Model 200) studies in F344 rats and B6C3F1 mice. Data were uniformly weighted, and the program was allowed to determine the range for estimation of the terminal elimination phase in all but the following data sets: PTH-F in male and female B6C3F1 mice after the iv dose; PTH-T in male and female B6C3F1 mice and male and female rats after the iv dose; PTH-T in male and female B6C3F1 mice after the low oral dose; PTH-T in female rats and male and female mice after the high oral dose. The parameters determined for PTH-F were area under the plasma concentration-versus-time curve from 0 h to infinity (AUC), elimination half-life (t
), and clearance (Cl). The parameters determined for PTH-T were AUC
and t
.
Analysis of dosed-feed data.
Plasma samples were collected over different time intervals for the different feed studies (see Study Design). An effort was made to normalize the data from all studies for a 24-h collection interval. In cases where a 24-h sample was not obtained, the concentrations of PTH-F, PTH-G, and PTH-T at T24 were assumed to be the same as those found at T0. Mean concentrations over the 24-h period were then calculated for PTH-F (when applicable) and PTH-T. Area under the plasma concentration-versus-time curve for the 24-h period (AUC24h) was determined for PTH-F (when applicable) and PTH-T.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
As shown in Figure 2D, PTH-T was rapidly absorbed and eliminated from mouse plasma after both oral doses (50 and 125 mg PTH/kg). As with rats, female mice received a higher internal dose of PTH-T than did the males (Table 2
). Elimination of PTH-T was similar for male and female mice, with an elimination half-life of ca. 67 h. As in rats, PTH-T kinetics after oral administration to mice were not dose-proportional; bioavailability decreased and clearance increased as dose increased.
Dosed-Feed Studies
The toxicokinetic parameters calculated for PTH-F in male and female F344 rats, male and female B6C3F1 mice, female p53 (+/) mice, and female C57BL mice in 2-week dosed-feed studies are summarized in Table 3. The mean PTH-F concentration in F344 rat plasma (Fig. 3A
) and internal dose (AUC24h) (Table 3
) were similar for males and females in the same dose group, and were similar for all dose groups except 375 ppm. As in rats, the mean PTH-F concentration in B6C3F1 mouse plasma (Fig. 3B
) and AUC24h (Table 3
) were similar for males and females in the same dose group. In contrast to rats, the mean PTH-F concentration in plasma and AUC24h increased with dose through the 3000-ppm dose group, but leveled off between the 3000- and 12,000-ppm dose groups in B6C3F1 mice. PTH-F was not detected in plasma from male and female B6C3F1 mice in the 200-ppm dose group, but was present in plasma from female C57BL mice at concentrations similar to those observed for female B6C3F1 mice in the 750-ppm dose group (Fig. 3B
, Table 3
). AUC24h for PTH-F in female C57BL mice in the 200-ppm dose group was similar to that for female B6C3F1 mice in the 750- to 3000-ppm dose groups (Table 3
).
|
|
|
|
|
For PTH-T the relative extent of absorption (Q) was estimated as follows: the dose-normalized AUC24h for the mean plasma concentration time course following feeding was divided by the dose-normalized AUC from the iv study. In feeding studies, Q for PTH-T was generally lower in mice than in rats, with a range of 1066% compared to 591%, respectively. In both species, Q values decreased with increasing dose. In rats, PTH-T at the highest dose was 6% and 9% absorbed in females and males, respectively. By comparison, in mice it was 1.3% and 1% absorbed in females and males, respectively.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our studies provide information for comparing pharmacokinetic parameters for F344 rats, B6C3F1 mice, p53 (+/) mice, and C57BL mice. Mean plasma concentrations of PTH-F were similar for mice and rats at all dose concentrations following feeding. Identical oral PTH doses among the various duration feeding studies in rodents resulted in similar mean steady state PTH-T concentrations () for each species or strain. For feeding studies in F344 rats, B6C3F1 mice, p53 (+/) mice, and C57BL mice, plasma PTH-T concentrations were within <2.5 times each other for a given oral dose of PTH, indicating that length of dosing had no effect on the steady state concentrations of PTH-T.
The NTP is developing the p53 (+/) mouse for use in identifying the carcinogenic properties of chemicals, and these studies also provide an indication that the p53 (+/) mouse is exposed to approximately the same amount of PTH as the B6C3F1 mouse, the mouse that has traditionally been used in the NTP bioassay. Thus, there is correspondence between PTH exposures in the p53 (+/) and B6C3F1 mouse, which suggests that exposures to other similarly metabolized chemicals will also result in similar internal doses to both strains.
Following oral gavage, AUC values calculated for plasma PTH-T concentrations in B6C3F1 mice at the higher doses were 60% of those which would be expected if the system were dose proportional. In F344 rats, a similar relationship between AUC
and dose was observed, with AUC
values at the higher gavage doses approximately 50% of those expected from a dose-linear relationship for males and 55% for females. Half-life (t
) is not significantly different at any dose for either species, which suggests the lack of dose proportionality for PTH-T plasma concentrations was due to nonlinear absorption, rather than saturation of metabolic pathways at the higher doses.
Estimation of F following feeding studies is difficult due to the pattern of feed consumption in rodents. Caution must be exercised in interpretation of F values following feeding since F may be altered by saturable absorption, induction of metabolizing enzymes, or altered GI tract physiology. Welling (1986) has demonstrated that AUC over a dosing interval, (AUC0
), following repeated oral dosing, is identical to AUC0
following a single oral dose. Moreover, the identity of AUC0
with AUC0
following a single oral dose is independent of the dosing interval. Yuan (1993) has shown that plasma concentration data following a feeding study may be treated as a continuous series of small bolus oral doses. F is normally calculated as the dose-normalized AUC0
of the free drug following a single oral dose divided by the dose-normalized AUC0
of the free drug following a single iv dose. For PTH-F following feeding, F may be calculated by substituting dose-normalized AUC0
for AUC0
. Direct calculation of F for PTH-T is not feasible, due to the inclusion of metabolite plasma concentrations in the reported values for PTH-T. However AUC of PTH-T is a valid measure of the internal exposure of the animal to PTH arising from the applied feed dose. We have defined a parameter, Q, calculated from the quotient of the dose-normalized AUC24h following feeding and the dose-normalized AUC
following an iv dose, which will allow the comparison of relative absorption of PTH-T between doses.
For PTH-T, Q following feeding was as high as 90% but decreased with increasing dose to 1% in mice and 9% in rats. The decrease in Q at the highest doses likely results from decreasing absorption of PTH from feed at doses over 500 ppm. PTH-F concentrations were very low in both species following an iv dose, representing less than 16% of the PTH-T circulating in rats and less than 5% in mice, which reflected the extensive glucuronidation of this chemical. F for PTH-F following feeding was very low (<1% for mice or <5% for rats), and similar across all doses, resulting from efficient glucuronidation of the small amounts of PTH presented to the liver at any one time. Since glucuronidation of the parent PTH does not become saturated, the amount of PTH-F does not rise with dose, even at the high doses used in the feed studies. It is therefore likely that the low F values seen at the high doses result from less efficient absorption, which may explain the consistency in mean plasma concentration noted for the high doses in the 2 week feeding study and the bioassay.
In our studies, enterohepatic circulation of PTH is evident in the B6C3F1 mouse plasma concentration time profile for PTH-T (Fig. 2C) as a broad plateau beginning at about 2 h after iv administration and continuing through 12 h after dosing. Male and female rat iv data for PTH-F and PTH-T show evidence of extensive enterohepatic recycling with a period of ~4 h (Figs. 2A and 2C
). Following oral gavage, the plasma concentration time course demonstrates enterohepatic circulation times up to 10 h in B6C3F1 mice and F344 rats, and is probably responsible for the long half-lives seen in the oral studies (ca. 6.5 h mice, 16 h rats).
A study conducted in four human volunteers given a dose of 2 mg/kg (74 mg/m2) (FDA, 1997; FDA, 1998
) measured PTH-T in plasma over a period of 10 days after dosing. In two of the four subjects, PTH-T was still present in plasma 10 days after dose administration. Based on data provided by the FDA, PTH-T kinetic parameters were estimated using WinNonlin. The non-compartmental analysis of the plasma concentration vs. time data resulted in an elimination half-life of 21.9 ± 3.3 h (mean ± SD), a mean residence time of 31.6 ± 3.5 h, an AUC24h of 226 ± 62 h x µmol/l, an apparent Cl of 0.030 ± 0.001 l/h/kg, and a mean plasma PTH-T Cmax concentration of 8.069 ± 1.02 µmol/l.
In the rodent feeding studies, for doses of 24.92990 mg/kg (11515,220 mg/m2) was 590 times higher than those found in the human study. Normalized to dose based on body surface area,
in the mouse 2 week feeding study was 0.95.9 times the value in the human study, and tended to decrease with increasing dose. Similar trends were seen in normalized
values for both rats and mice in all studies. For the rodent feeding studies in which AUC24h was calculated, AUC24h was found to be 478 times the average value for the human study.
In the two-year studies, for doses of 2912125 mg/kg (87311050 mg/m2), the in rats was 2858 times the value in the human study; AUC24h was 2446 times the value in the human study. For mice
was 4778 times the value in the human study; AUC24h was 4066 times the value in the human study.
Table 6 gives a summary of the dose levels where carcinogenic and/or in vivo genotoxicity activity was seen in mice along with selected pharmacokinetic parameters at these dose levels. Genotoxic results were seen at all applied PTH doses, while thymic lymphomas were seen at the three highest applied PTH doses. These three doses correspond to doses at which absorption becomes saturated, which may explain the similarity of response seen in the 3000- and 12,000-ppm doses. At the lowest dose found to cause an in vivo genotoxic effect in mice (200 ppm), internal dose measured by
is approximately 9 times the human
after a bolus, oral dose of 2 mg/kg. At the lowest dose where a rodent carcinogenic effect is seen (3000 ppm in mice),
is approximately 30 times the value in the human study. The dose administered in the human study, 2 mg/kg, is lower than the recommended human dose of 5 mg/kg. Assuming linear kinetics between the administered dose and the recommended human dose, the internal dose at the lowest dose where a rodent carcinogenic effect was seen would be less than 20 times the expected human value.
|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clark, A. G., and Cooke, R. (1978). The effect of route of administration on the biliary excretion of phenolphthalein and its glucuronide. J. Pharm. Pharmacol. 30, 382383.[ISI]
Colburn, W. A., Hirom, P. C., Parker, R. J., and Milburn, P. (1979). A pharmacokinetic model for enterohepatic recirculation in the rat: Phenolphthalein, a model drug. Drug Metab. Dispos. 7, 100102.[Abstract]
Dunnick, J. K., and Hailey, J. R. (1996). Phenolphthalein exposure causes multiple carcinogenic effects in experimental model systems. Cancer Res. 56, 49224926.[Abstract]
Dunnick, J. K., Hardisty, J. F., Herbert, R. A., Seely, J., Furedi-Machacek, E., Foley, J. F., Lacks, G. J., Stasiewicz, S., and French, J. E. (1997). Phenolphthalein induces thymic lymphomas accompanied by loss of the p53 wild type allele in heterozygous p53-deficient (+/) mice. Toxicol. Pathol. 25, 533540.[ISI][Medline]
FDA (U.S. Food and Drug Administration) (1997). U. S. Food and Drug Administration Laxative Drug Products for Over-the-Counter Human Use; Proposed Amendment to the Tentative Final Rule [Docket No. 78N036L]. U.S. Food and Drug Administration, 4622346227.
FDA (U.S. Food and Drug Administration) (1998). U. S. Food and Drug Administration Laxative Drug Products for Over-the-Counter Human Use; Proposed Amendment to the tentative final monograph [Docket No. 78N-036]. 3359233595.
Griffin, R. J., Godfrey, V. B., and Burka, L. T. (1998). Metabolism and disposition of phenolphthalein in male and female F344 rats and B6C3F1 mice. Toxicol. Sci. 42, 7381.[Abstract]
Kok, R. M., and Faber, D. B. (1981). Qualitative and quantitative analysis of some synthetic, chemically acting laxatives in urine by gas chromatography-mass spectrometry. J. Chromatogr. 222, 389398.[Medline]
Morotomi, M., Nanno, M., Watanabe, T., Sakurai, T., and Mutai, M. (1985). Mutagenic activation of biliary metabolites of 1-nitropyrene by intestinal microflora. Mutat. Res. 149, 171178.[ISI][Medline]
NTP (National Toxicology Program) (1996). Toxicology and carcinogenesis studies of phenolphthalein (CAS No. 77098) in F344/N rats and B6C3F1 mice (feed studies). National Toxicology Program; Report Number 465.
PDR. (Physicians` Desk Reference) (1982). Physicians' Desk Reference for Nonprescription Drugs. Medical Economics Company Inc., Oradell, NJ.
PDR. (Physicians` Desk Reference) (1991). Physicians' Desk Reference. Medical Economics Company Inc., Oradell, NJ.
Sipes, H. J., Jr., Corbett, J. T., and Mason, R. P. (1997). In vitro free radical metabolism of phenolphthalein by peroxidases. Drug Metab. Dispos. 25, 468480.
Sparacino, C. M., and Handy, R. W. (1996). Bulk chemical comprehensive analysis report: phenolphthalein. RTI Project No. 60U-6430071. Research Triangle Institute, Research Triangle Park, NC.
Tice, R. R., Furedi-Machacek, M., Satterfeld, D., Udumudi, A., Vasquez, M., and Dunnick, J. K. (1998). Genotoxicity of chronically ingested phenolphthalein in female heterozygous TSG-p53 mice. Environ. Mol. Mutagen. 31, 113124.[ISI][Medline]
Welling, P. G. (1986). Pharmacokinetics. American Chemical Society, Washington, DC.
Yuan, J. H. (1993). Modeling blood/plasma concentrations in dosed feed and dosed drinking water toxicology studies. Toxicol. Appl. Pharmacol. 119, 131141.[ISI][Medline]