Phenolphthalein is used as a laboratory reagent and acid-base indicator and in over-the-counter laxative preparations. The National Cancer Institute nominated phenolphthalein for study because of its widespread use as a component in numerous laxative preparations and the lack of adequate testing for carcinogenicity in experimental animals. Male and female F344/N rats and B6C3F1 mice were exposed to phenolphthalein (98% to 99% pure) in feed for 14 days, 13 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and mouse peripheral blood. 14-DAY STUDY IN RATS: Groups of five male and five female F344/N rats were given 0, 6,250, 12,500, 25,000, 50,000, or 100,000 ppm phenolphthalein in feed for 14 days. All rats survived to the end of the study. The final mean body weights of all exposed groups of rats were similar to those of the controls. No chemical-related gross or microscopic lesions were observed. 14-DAY STUDY IN MICE: Groups of five male and five female B6C3F1 mice were given 0, 6,250, 12,500, 25,000, 50,000, or 100,000 ppm phenolphthalein in feed for 14 days. All mice survived to the end of the study. The final mean body weights of all exposed groups of mice were similar to those of the controls. No chemical-related gross or microscopic lesions were observed. 13-WEEK STUDY IN RATS: Groups of 10 male and 9 or 10 female F344/N rats were given 0, 3,000, 6,000, 12,000, 25,000, or 50,000 ppm phenolphthalein (equivalent to average daily doses of approximately 200, 400, 800, 1,600, or 3,500 mg phenolphthalein/kg body weight to males and 200, 400, 800, 1,700, or 3,600 mg/kg to females) in feed for 13 weeks. Additional groups of 10 male and 10 female rats designated for clinical pathology evaluations were also given 0, 3,000, 6,000, 12,000, 25,000, or 50,000 ppm phenolphthalein in feed until day 21. All core study rats survived to the end of the study. The final mean body weight of the 50,000 ppm females and the body weight gains of the 25,000 and 50,000 ppm females were significantly lower than those of the controls. The final mean body weights and mean body weight gains of all other exposed groups were similar to those of the controls. There was no cathartic action or any other clinical finding attributed to exposure to phenolphthalein. The few differences in the hematology and clinical chemistry parameters were sporadic and were not considered to be chemical related. The percentage of motile sperm in the 12,000 ppm males was significantly greater than that in the controls, but no other significant differences in sperm morphology or vaginal cytology between exposed and control groups were observed. Absolute and relative liver weights of 25,000 and 50,000 ppm males were significantly greater than those of the controls. No chemical-related gross or microscopic lesions were observed. 13-WEEK STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were given 0, 3,000, 6,000, 12,000, 25,000, or 50,000 ppm phenolphthalein (equivalent to average daily doses of approximately 500, 1,000, 2,000, 4,100, or 9,000 mg phenolphthalein/kg body weight to males and 600, 1,200, 2,400, 5,000, or 10,500 mg/kg to females) in feed for 13 weeks. All mice survived until the end of the study. The final mean body weights and mean body weight gains of all exposed groups were similar to those of the controls. There was no cathartic action or any other clinical finding attributed to exposure to phenolphthalein. The absolute right cauda weight of the 12,000 ppm males and the absolute right epididymis weights of 12,000, 25,000, and 50,000 ppm males were significantly less than those of the controls. The percentages of abnormal sperm in 12,000, 25,000, and 50,000 ppm males were significantly greater than that in the control group, and the sperm concentrations in 12,000 and 50,000 ppm males were significantly less than that of the control group. The absolute and relative right testis weights of males exposed to 6,000 ppm or greater and the absolute right testis weight of 3,000 ppm mof 3,000 ppm males were significantly less than those of the controls. The incidences of hypoplasia of the bone marrow in males and females exposed to 12,000 ppm or greater were significantly greater than those in the controls. The incidences of hematopoiesis of the spleen in 25,000 and 50,000 ppm males were significantly greater than that in the controls. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were given 0, 12,000, 25,000, or 50,000 ppm phenolphthalein (equivalent to average daily doses of approximately 500, 1,000, or 2,000 mg phenolphthalein/kg body weight to males and 500, 1,000, or 2,500 mg/kg to females) in feed for 2 years. Survival, Body Weights, and Clinical Findings: Survival of exposed males and females was similar to that of the controls. The mean body weights of exposed males were less than those of the controls through most of the second year of the study, and the mean body weights of exposed females were less than those of the controls from about week 16 until the end of the study. Clinical findings attributed to phenolphthalein exposure included thin appearance and ruffled fur in all exposed groups of males. Determinations of Total Phenolphthalein in Plasma: The mean plasma concentrations of total phenolphthalein (free and conjugated) after 2 years of exposure varied little with time of day. Plasma concentrations of total phenolphthalein were approximately the same between exposure groups and between males and females. Pathology Findings: The incidences of benign pheochromocytoma of the adrenal medulla in all exposed groups of males were significantly greater than those in the controls and occurred with a significant positive trend. The incidences of benign pheochromocytoma in 12,000 ppm females and of benign or malignant pheochromocytoma (combined) in 12,000 and 25,000 ppm females were significantly greater than those in the controls. The numbers of exposed males with bilateral benign pheochromocytomas exceeded the number of controls with these neoplasms. The incidences of malignant pheochromocytomas in exposed rats were similar to those in the controls. The incidences of focal hyperplasia of the adrenal medulla in the 12,000 and 50,000 ppm males were significantly greater than in the controls. The incidences of renal tubule adenoma in 50,000 ppm male rats and of renal tubule adenoma or carcinoma (combined) in 12,000 and 50,000 ppm male rats were significantly greater than those in the controls. Although the increased incidences were predominantly of renal tubule adenoma, four carcinomas were observed in exposed males (0 ppm, 0/50; 12,000 ppm, 1/50; 25,000 ppm, 1/50; 50,000 ppm, 2/50). The incidences of renal tubule neoplasms in exposed groups of females were similar to those in the controls. The findings from an extended evaluation (step section) of the kidneys of female rats were similar to those from the standard evaluation. The incidences of nephropathy in all exposed groups of females were significantly greater than in the controls, and the severity of nephropathy in all exposed groups of males and in 25,000 and 50,000 ppm females was significantly greater than in the controls. The incidences of diffuse hyperplasia of the parathyroid gland (0/41, 16/48, 14/49, 14/46), fibrous osteodystrophy of the bone (0/50, 17/50, 14/50, 12/50), and mineralization (0/50, 11/50, 5/50, 5/49) and degeneration (0/50, 11/50, 5/50, 4/49) of the glandular stomach in exposed groups of males were generally significantly greater than those in the controls. The incidences of hyperplasia of the thyroid gland C-cells (13/50, 3/50, 9/49, 4/49) in 12,000 and 50,000 ppm males were significantly less than in the controls. These lesions are commonly observed in male rats with more advanced nephropathy and are considered to be associated with a calcium/phosphorus imbalance created by compromised functional capacity of the kidney. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were given 0, 3,000, 6,000, or 12,000 ppm phenolphthalein (equivalent to average daily doses of approximately 300, 600, or 1,200 mg phenolphthalein/kg body weight to males and 400, 800, or 1,500 mg/kg to females) in feed for 2 years. Survival, Body Weights, and Clinical Findings: Survival of the 12,000 ppm females was significantly lower than that of the controls; survival of all other exposed groups of mice was similar to that of the controls. The mean body weights of 12,000 ppm males were slightly less than those of the controls beginning at week 93 of the study, and the mean body weights of the 3,000, 6,000, and 12,000 ppm females were less than those of the controls during most of the second year of the study. In exposed mice, there were no clinical findings related to phenolphthalein exposure. Determinations of Total Phenolphthalein in Plasma: The mean plasma concentrations of total phenolphthalein (free and conjugated) after 2 years of exposure varied little with time of day. Plasma concentrations of total phenolphthalein were approximately the same between exposure groups and between males and females. Pathology Findings: The incidences of histiocytic sarcoma in 6,000 and 12,000 ppm males and females were significantly greater than those in the controls and occurred with a significant positive trend. In this study, histiocytic sarcoma was consistently observed in the liver with several other sites (e.g., spleen, lung, bone marrow, and various lymph nodes) involved less frequently. The incidences of all types of malignant lymphoma and of lymphoma of thymic origin in all exposed groups of females were significantly greater than those in the controls and occurred with significant positive trends, while the incidences of all types of malignant lymphoma in all exposed groups of males were similar to that in the controls. The incidences of lymphoma of thymic origin were increased in exposed groups of males, but were significantly increased only in the 6,000 ppm group. The incidences of atypical hyperplasia of the thymus in 6,000 and 12,000 ppm males and in all exposed groups of females were significantly greater than those in the controls. The incidences of benign sex-cord stromal tumors of the ovary in all exposed groups of females were significantly greater than in the controls. The incidences of hyperplasia of the ovary in 3,000 and 12,000 ppm females were significantly greater than in the controls. The incidences of germinal epithelial degeneration of the testis in all exposed groups of males were significantly greater than that in the controls. There were increased incidences of myelofibrosis of the bone marrow in 12,000 ppm males (0 ppm, 3/50; 3,000 ppm, 8/50; 6,000 ppm, 8/50; 12,000 ppm, 19/49) and an increased severity but not incidence of this lesion in exposed females. There were also increased incidences of pigmentation of minimal to mild severity in the bone marrow of 6,000 and 12,000 ppm males (0/50, 2/50, 5/50, 16/49) and females (2/50, 3/50, 11/50, 11/50). Also, the incidences of hematopoietic cell proliferation in the red pulp of the spleen (10/50, 22/50, 28/50, 21/49) in all exposed groups of males were significantly greater than that in the controls, and the severity of this lesion increased with increasing exposure concentration. The incidences of hepatocellular adenoma in all exposed groups of males and females and of hepatocellular adenoma or carcinoma (combined) in 6,000 and 12,000 ppm males and all exposed groups of females were significantly less than those in the controls, and these lesions occurred with significant negative trends. Multiple hepatocellular adenomas were observed more frequently in the control groups than in the exposed groups. The incidences of clear cell and eosinophilic foci in all exposed groups of males and of mixed cell foci in 12,000 ppm males were significantly less than those in the controls. The incidences of eosinophilic foci in exposed groups of females were significantly less than that in the controls. GENETIC TOXICOLOGY: Phenolphthalein, tested in two laboratories, was not mutagenic in any of four strains of Salmonella typhimurium with or without S9 metabolic activation enzymes, and no induction of sister chromatid exchanges was observed in cultured Chinese hamster ovary cells treated with phenolphthalein with or without S9. However, significant increases in chromosomal aberrations were observed after treatment of cultured Chinese hamster ovary cells with phenolphthalein in the presence of S9, and the frequencies of micronucleated erythrocytes were increased in peripheral blood samples from male and female mice administered phenolphthalein in feed for 13 weeks. CONCLUSIONS: Under the conditions of these 2-year feed studies, there was clear evidence of carcinogenic activity of phenolphthalein in male F344/N rats based on markedly increased incidences of benign pheochromocytomas of the adrenal medulla and of renal tubule adenomas and adenomas or carcinomas (combined). There was some evidence of carcinogenic activity of phenolphthalein in female F344/N rats based on the increased incidences of benign pheochromocytomas of the adrenal medulla in the 12,000 ppm group and of benign or malignant pheochromocytomas (combined) in the 12,000 and 25,000 ppm groups. There was clear evidence of carcinogenic activity of phenolphthalein in male B6C3F1 mice based on increased incidences of histiocytic sarcomas and of malignant lymphomas of thymic origin. There was clear evidence of carcinogenic activity of phenolphthalein in female B6C3F1 mice based on increased incidences of histiocytic sarcomas, malignant lymphomas of all types, lymphomas of thymic origin, and benign sex-cord stromal tumors of the ovary. Exposure of rats to phenolphthalein in feed for 2 years resulted in increased incidences of focal hyperplasia of the adrenal medulla in males and in increased incidences and/or severity of nephropathy of the kidney in males and females. Exposure of mice to phenolphthalein in feed for 2 years resulted in increased incidences of atypical hyperplasia of the thymus in males and females, degeneration of the germinal epithelium of the testis in males, and ovarian hyperplasia in females. Exposure of mice to phenolphthalein in feed for 2 years resulted in decreased incidences of hepatocellular neoplasms and nonneoplastic lesions in males and females. Synonyms: 3,3-Bis(4-hydroxyphenyl)-1(3H)-isobenzofuranone; 3,3-bis(p-hydroxyphenyl)phthalide; a-p -hydroxyphenyl)-a- (4-oxo-2,5-cyclohexadien-1-ylidene)- o-toluic acid Trade names: Agoral®, Alophen®, Colax®, Correctol®, Dialose®, Doxidan®, Espotabs®, Evac-U-Gen®, Evac-U-Lax®, Ex-Lax®, Feen-A-Mint®, FemiLax®, Kondremul®, LaxCaps®, Lax-Pills®, Medilax®, Modane®, Phenolax®, Prulet®

A patient with chronic constipation developed liver injury and leukopenia following the ingestion of Doxidan, a combination drug consisting of danthron and dioctyl calcium sulfosuccinate. Evidence is presented that dioctyl calcium sulfosuccinate may have potentiated the toxicity. The liver injury was associated with deposition of IgE in the Kupffer cells. The mechanism of toxicity remains unclear.