NATIONAL TOXICOLOGY PROGRAM 

Technical Report Series 

No. 465 

TOXICOLOGY AND CARCINOGENESIS 

STUDIES OF PHENOLPHTHALEIN 

(CAS NO. 77-09-8) 

IN F344/N RATS AND B6C3Fl MICE 

(FEED STUDIES) 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 

Public Health Service 

National Institutes of Health 
FOREWORD 

The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of 

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relevant programs, staff, and resources from these Public Health Service agencies relating to basic and 

applied research and to biological assay development and validation. 

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and 

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primary prevention of disease. 

The studies described in this Technical Report were performed under the direction of the NIEHS and were 

conducted in compliance with NTP laboratory health and safety requirements and must meet or exceed all 

applicable federal, state, and local health and safety regulations. Animal care and use were in accordance 

with the Public Health Service Policy on Humane Care and Use of Animals. The prechronic and chronic 

studies were conducted in compliance with Food and Drug Administration (FDA) Good Laboratory 

Practice Regulations, and all aspects of the chronic studies were subjected to retrospective quality 

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Chemicals selected for NTP toxicology and carcinogenesis studies are chosen primarily on the bases of 

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World Wide Web site: http://ntp-server.niehs.nih.gov. 
NTP TECHNICAL REPORT 

ON THE 

TOXICOLOGY AND CARCINOGENESIS 

STUDIES OF PHENOLPHTHALEIN 

(CAS NO. 77-09-8) 

IN F344/N RATS AND B6C3Fl MICE 

(FEED STUDIES) 

NATIONAL TOXICOLOGY PROGRAM 

P.O. Box 12233 

Research Triangle Park, NC 27709 

November 1996 

NTP TR 465 

NIH Publication No. 97-3390 

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 

Public Health Service 

National Institutes of Health 
2 

~~ ~ ~~~~ 

Phenolphthalein, NTP TR 465 

CONTRIBUTORS 

National Toxicology Program 

Evaluated and interpreted results and reported findings 

J.K. Dunnick, Ph.D., Study Scientist 

G.A. Boorman, D.V.M., Ph.D. 

D.A. Bridge, B.S. 

J.R. Bucher, Ph.D. 

L.T. Burka, Ph.D. 

R.E. Chapin, Ph.D. 

J.R. Hailey, D.V.M. 

J.K. Haseman, Ph.D. 

G.N. Rao, D.V.M;, Ph.D. 

J.H. Roycroft, Ph.D. 

G.S. Travlos, D.V.M. 

D.B. Walters, Ph.D. 

K.L. Witt, M.S., OakRidgeAssociated Universities 

Cannon Laboratories, Inc. 

Conducted 14-day studies, evaluated pathology findings 

A.K. Reddy, Ph.D., Principal Investigator 

L. Billups, D.V.M. 

J.T. Ravert 

Microbiological Associates, Inc. 

Conducted 13-week studies, evaluated pathology findings 

L.T. Mulligan, Ph.D., Study Director 

R. Filler, Ph.D. 

M.A. Stedham, D.V.M., M.S. 

TSI Mason Laboratories, Inc. 

Conducted 2-year studies, evaluated pathology findings 

M.R. Osheroff, Ph.D., Principal Investigator 

C. Gamba-Vitalo, Ph.D. 

F.A. Voelker, M.S., D.V.M. 

Experimental Pathology Laboratories, Inc. 

Provided pathology quality assurance 

J.F. Hardisty, D.V.M., Principal Investigator 

S.Botts, M.S., D.V.M., Ph.D. 

E.T. Gaillard, MS., D.V.M. 

NTP Pathology Working Group 

Evaluated slides, prepared pathology report on rats 

(15 September 1995) 

J.C. Seely, D.V.M., Chairperson 

PATHCO, Inc. 

G.A. Boorman, D.V.M., Ph.D. 

National Toxicology Program 

S.Botts, MS., D.V.M., Ph.D. 

Experimental Pathology Laboratories, Inc. 

L. Bowman, B.S., Observer 

North Carolina State University 

J.M. Cullen, V.M.D., Ph.D. 

North Carolina State University 

D. Dixon, D.V.M., Ph.D. 

National Toxicology Program 

J.R. Hailey, D.V.M. 

National Toxicology Program 

J. Hellman, D.V.M., Ph.D., Observer 

Merck KGaA 

R.C. Sills, D.V.M., Ph.D. 

National Toxicology Program 

Evaluated slides, prepared pathology report on mice 

(26 September 1995) 

M.P. Jokinen, D.V.M, Chairperson 

Pathology Associates, Inc. 

P. Blackshear, D.V.M., Observer 

North Carolina State University 

A. Enomot, D.V.M., Ph.D. 

National Toxicology Program 

E.T. Gaillard, M.S., D.V.M. 

Experimental Pathology Laboratories, Inc. 

J.R. Hailey, D.V.M. 

National Toxicology Program 

R. Miller, D.V.M., Ph.D. 

North Carolina State University 

A. Radovsky, D.V.M., Ph.D. 

National Toxicology Program 

R.C.Sills, D.V.M., Ph.D. 

National Toxicology Program 

M. Torii, D.V.M., Ph.D. 

National Toxicology Program 
Phenolphthalein,NTP TR 465 3 

Dynamac Corporation 

Prepared quality assurance audits 

S. Brecher, Ph.D., Principal Investigator 

Analytical Sciences, Inc. 

Provided statistical analyses 

R.W. Morris, M.S., Principal Investigator 

N.G. Mintz, B.S. 

S. Rosenblum, M.S. 

Biotechnical Services, Inc. 

Prepared Technical Repon 

S.R. Gunnels, M.A., Principal Investigator 

G. Gordon, M.A. 

L.M. Harper, B.S. 

M.J. Nicholls, B.S. 

D.C. Serbus, P1I.D. 

S.M. Swift, B.S. 
4 

CONTENTS 

ABSTRACT ......................................................... 5 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY ........... 12 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE ............................. 13 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS ......... 14 

INTRODUCTION ..................................................... 17 

MATERIALS AND METHODS ............................................ 27 

RESULTS .......................................................... 39 

DISCUSSION AND CONCLUSIONS ......................................... 71 

REFERENCES ........................................................ 81 

APPENDIXA Summary of Lesions in Male Rats in the 2-Year Feed Study of Phenolphthalein .... 93 

APPENDIX B Summary of Lesions in Female Rats in the 2-Year Feed Study of Phenolphthalein ... 137 

APPENDIX C Summary of Lesions in Male Mice in the 2-Year Feed Study of Phenolphthalein .... 175 

. . 219 

........................................... 259 

.................. 269 

........................... 275 

...................... 281 

........... 291 

.................. 295 

... 309 

APPENDIXD Summary of Lesions in Female Mice in the 2-Year Feed Study of Phenolphthalein 

APPENDIXE Genetic Toxicology 

APPENDIXF Organ Weights and .Organ.Weight.to.Bod y.Weight Ratios 

APPENDIX G Hematology and Clinical Chemistry Results 

APPENDIX H Determinations of Total Phenolphthalein in Plasma 

APPENDIXI Reproductive Tissue Evaluations and Estrous Cycle Characterization 

APPENDIXJ Chemical Characterization and Dose Formulation Studies 

APPENDIXK Feed and Compound Consumption in the 2-Year Feed Studies of Phenolphthalein 

APPENDIX L Ingredients. Nutrient Composition. and Contaminant Levels 

in NIH-07 Rat and Mouse Ration .................................. 315 

APPENDIX M Sentinel Animal Program ....................................... 319 

APPENDIXN Continuous Breeding Study in Swiss (CD-la) Mice ....................... 323 

APPENDIX 0 Single-Dose Toxicokinetic Studies in M44/N Rats and B6C3Fl Mice ............ 339 
5 

ABSTRACT 

0 

OH 

OH 

PHENOLPHTHALEIN 

CAS NO.77-09-8 

Chemical Formula: C,,,H,,O, Molecular Weight: 318.33 

Synonyms: 3,3-Bis(4-hydroxyphenyl)-1(3H)-isobenzofranone; 3,3-bis@-hydroxyphenyl)phthalide; cu-@-hydroxyphenyl)-ar-(4-oxo2,5-cyclohexadien-l-ylidene)-o-toluicacid 

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" 

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 thelackofadequatetesting for 

carcinogenicity in experimental animals.Maleand 

female F344/Nrats and B6C3F,mice were exposed 

to phenolphthalein (98% to 99% pure) in feed for 

14 days, 13 weeks, or 2 years. Genetictoxicology 

studies were conducted in Salmonella typhimurium, 

cultured Chinese hamster ovary cells, and mouse 

peripheral blood. 

14-DAY STUDY IN RATS 

Groupsof five male and five female F344/N rats 

were given 0, 6,250, 12,500, 25,000, 50,000, or 

100,000ppmphenolphthalein in feed for 14 days. 

All rats survived to the end of the study. The final 

meanbodyweightsofallexposed groups of rats 

were similar to those of the controls. No chemicalrelated gross or microscopic lesions were observed. 

14-DAY STUDY IN MICE 

Groupsof five male and five female B6C3F1mice 

were given 0, 6,250, 12,500, 25,000, 50,000, or 
6 Phenolphthalein, NTP TR 465 

100,000ppm phenolphthalein in feed for 14 days. 

All mice survived to the end of the study. The final 

meanbodyweights of allexposed groups of mice 

were similar to those of the controls. No chemicalrelated gross or microscopic lesions were observed. 

13-WEEK STUDY IN RATS 

Groups of 10 male and 9 or 10 female F344/Nrats 

were or weights of 12,000, 25,000,and 50,000ppmmales given 0, 3,000, 6,000, 12,000, 25,000, 

50,000ppm phenolphthalein (equivalent to average 

daily doses of approximately 200, 400,800, 1,600, 

or 3,500mg phenolphthalein/kg body weight to 

males and 200, 400,800, 1,700,or 3,600mg/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 50,000ppm phenolphthalein in feed until day 21. All core study rats survived to the end of the study. The final mean body 

weight of the ofhypoplasiaof the bone marrow in malesand 50,000ppm females and the mean body 

weight gains of the 25,000and 50,000ppm 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 12,000ppm males 

was significantly greater than that in the controls, but 

no other significant differences in sperm morphology 

or vaginal cytology between exposedand control 

groups were observed. Absoluteandrelative liver 

weights of 25,000 and 50,000ppmmales were 

significantly greater than those of the. controls. No 

chemical-related gross or microscopic lesions were 

observed. 

13-WEEK STUDY IN MICE 

Groups of 10male and 10female B6C3F1mice were 

given 0, 3,000,6,000,12,000,25,000, or 

50,000ppm phenolphthalein (equivalent to average 

daily doses of approximately 500, 1,000, 2,OOO, 

4,100,or 9,000 mg phenolphthaleirdkg body weight 

to males and 600, 1,200, 2,400, 5,000, or 

10,500mg/kg to females) in feed for 13 weeks. All 

micesurviveduntiltheendof the study. The final 

mean body weights and mean body weight gains of 

allexposed groups were similar to those of the 

controls. There was no cathartic action or any other 

clinical finding attributed to exposure to phenolphthalein.Theabsolute right cauda weight of the 

12,000ppm males and the absolute right epididymis 

weresignificantlylessthan those of the conuols. 

Thepercentagesof abnormal sperm in 12,000, 

25,000,and 50,000ppmmales were significantly 

greater than that in the control group, and the sperm 

concentrations in 12,000and 50,000ppm males were 

significantly less than that of the control group. The 

absoluteand relative right testis weights of males 

exposed to 6,000ppm or greater and the absolute 

right testis weight of 3,000ppm males were significantly less than those of the controls. The incidences 

femalesexposed to 12,000ppm or greater were 

significantly greater than those in the controls. The 

incidences of hematopoiesis of the spleen in 25,000 

and 50,000ppm males were significantly greater than 

that in the controls. 

%YEAR STUDY IN RATS 

Groups of 50 male and 50 female F344/Nrats were 

given 0, 12,000, 25,000,or 50,000ppm phenolphthalein(equivalent to average daily doses of 

approximately 500, 1,W, or 2,000mg phenolphthalein/kg body weight to males and500, 1,000, or 

2,500mg/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 

exposedmaleswere 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 

thoseofthe controls from about week 16 until the 

endof the study. Clinical findings attributed to 

phenolphthalein exposure included thin appearance 

and ruffled fur in all exposed groups of males. 
Phenolphthalein, NTP TR 465 

Determinations of Total Phenolphthalein 

in Plasma 

The meanplasma concentrations of total phenolphthalein (free and conjugated) after 2yearsof 

exposure varied little with timeof day. Plasma 

concentrations of total were phenolphthalein

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 inthe controls and 

occurred with a significant positive trend. The 

incidencesbenign ofpheochromocytoma in

12,000ppmfemalesand of benign or malignant 

pheochromocytoma in (combined) 12,000 and 

25,000ppm females were significantly greater than 

those in the controls. The numbers of exposed males 

withbilateralbenignpheochromocytomasexceeded 

the number of controls with theseneoplasms. The 

incidences of malignant pheochromocytomasin 

exposed rats were similar to those inthe controls. 

The incidences offocalhyperplasia of the adrenal 

medulla in the 12,000and 50,000 ppmmaleswere 

significantly greater than in the controls. 

The incidencesrenal adenoma oftubule in 

50,000ppm 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. Althoughtheincreasedincidenceswere 

predominantly of renal tubule adenoma, four carcinomas were observed in exposed males (0 ppm, 0/50; 

12,000ppm, 1/50; 25,000 ppm, 1/50;50,000 ppm, 

2/50). The incidences ofrenaltubuleneoplasmsin 

exposed groups of femalesweresimilar 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 standardevaluation.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 malesandin 25,000 and 50,000 ppm 

females was significantly greater than in the controls. 

The incidences of diffuse hyperplasia ofthe parathyroidgland (0/41, 16/48, 14/49, 14/46), fibrous 

7 

glandular stomach in exposed groups of males were 

generallysignificantly greater than those in the 

controls. Theincidencesof hyperplasia of the 

thyroidglandC-cells (13/50, 3/50, 9/49,in 4/49) 

12,000and 50,000ppm males were significantly less 

than in the controls. Theselesions are commonly 

observed in male rats with more advanced nephropathyand are considered to be associated with a 

calcium/phosphorus imbalance created by compromised functional capacity of the kidney. 

&YEAR STUDY IN MICE 

Groups of 50 male and 50 female B6C3F, mice were 

given 0, 3,000, 6,000,or 12,000ppm phenolphthalein(equivalent to average daily doses of 

approximately 300, 600, or 1,200mg phenolphthaleidkg body weight to males and 400,800, or 

1,500mg/kg to females) in feed for 2 years. 

Survival, Body Weights, 

and Clinical Findings 

Survival of the 12,000ppm females was significantly 

lower than that of the controls; survival of all other 

exposed groups of micewas similar to that of the 

controls. Themeanbodyweightsof 12,000ppm 

maleswereslightly less than those of the controls 

beginningatweek 93 ofthe study, and the mean 

body weights of the 3,000,6,000, and 12,000ppm 

females were less than those of the controls during 

most of the secondyear 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 timeof day. Plasma 

concentrations of total phenolphthalein were approximatelythesamebetween exposure groups and between males and females. 

Pathology Findings 

Theincidences of histiocytic sarcoma in 6,000and 

12,000ppmmalesandfemaleswere significantly 

greater than those in the controls and occurred with 

a significant positive trend. In this study, histiocytic 

osteodystrophy of the bone (0150, 17/50, 14/50, sarcoma was consistently observed in the liver with 

12/50),and mineralization (0/50,11/50, 5/50, several 5/49) other sites (e.g., spleen, lung, bone marrow, 

and degeneration (0/50,11/50, 5/50, 4/49) of the and various lymph nodes) involved less frequently. 
8 Phenolphthalein, NTP TR 465 

The incidences ofalltypesofmalignantlymphomathan in the exposed groups. The incidences of clear 

andoflymphomaofthymic origin in allexposedcelland eosinophilic foci in all exposed groups of 

groups of femalesweresignificantly greater than malesandofmixedcell foci in 12,000 ppm males 

those in the controls and occurredwithsignificantweresignificantlylessthan those in the controls. 

positive trends, while theincidencesofalltypesofTheincidencesof eosinophilic foci inexposed 

malignant lymphoma in all exposed groups of males 

were similar to that inthe controls. Theincidences 

of lymphoma of thymic origin were increased in 

exposed groups of males, but weresignificantly 

increasedonlyinthe 6,000 ppm group. TheinciOf Of the thymus in 6yooo 

and 12,000 PPm males andinexposed all groups of 

femaleswere significantly greater than thoseinthe 

controls. 

The incidences of benign sex-cord stromal tumors of 

the ovary in allexposed groups of femaleswere 

significantly greater than inthe controls. Theincidencesof hyperplasia of theovaryin 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 

maleswere significantly greater than thatinthe 

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 exposedfemales. There were also 

increasedincidencesofpigmentation of minimal to 

mild severity in thebonemarrowof 6,000 and 

12,000 ppm males (0150, 2/50, 5/50, 16/49) and 

females (2/50, 3/50,11/50,11/50). 

Also, the incidences of hematopoietic cell proliferationin the red pulpofthespleen (10/50, 22/50, 

28/50, 21/49) in allexposed groups of males were 

significantly greater than that in the controls, and the 

severity of this lesion increasedwithincreasing 

exposure concentration. 

The incidences ofhepatocellularadenomainall 

exposed groups of malesandfemalesand of 

hepatocellular adenoma or carcinoma (combined) in 

6,000 and 12,000 ppm males and all exposed groups 

offemalesweresignificantlylessthanthoseinthe 

controls, and these lesions occurred withsignificant 

negative trends. Multiplehepatocellularadenomas 

were observed more frequently in the control groups 

groups of femalesweresignificantly less than in the 

controls. 

GENETICT~XIC~L~GY 

Phenolphthalein,in tested two laboratories, was not 

in any of four strains of Salmonella 

typhimurium with or without S9 metabolic activation 

enzymes,andnoinductionof sister chromatid 

exchanges was observed in cultured Chinese hamster 

ovarycellstreatedwith phenolphthalein with or 

without S9. However, significant increases in 

chromosomal aberrations ‘were observed after treatmentofcultured Chinese hamster ovary cells with 

phenolphthaleininthe presence of S9, and the 

frequenciesofmicronucleated erythrocytes were 

increased in peripheral blood samples from male and 

female mice administered phenolphthalein in feed for 

13 weeks. 

CONCLUSIONS 

Underthe conditions of these 2-year feed studies, 

there was clear evidence of carcinogenic activity* of 

phenolphthalein in male F344/N rats based on markedlyincreasedincidences of benign pheochromocytomas of the adrenal medulla and of renal 

tubule adenomas and adenomas or carcinomas (combined). There was someevidence of carcinogenic 

activity of phenolphthalein in female F344/N rats 

based on the increasedincidencesof benign pheochromocytomasofthe adrenal medullain the 

12,000 ppm group andof benign or malignant 

pheochromocytomas (combined) in the 12,000 and 

25,000 ppm groups. There was clearevidence of 

carcinogenicactivity of phenolphthalein in male 

B6C3F, mice based on increased incidences of 

histiocytic sarcomas and of malignant lymphomas of 

thymic origin. There was clear evidence of carcinogenicactivity of phenolphthalein in female B6C3F1 

micebasedonincreasedincidencesof 

sarcomas, malignant lymphomas of all 

phomasofthymic origin, andbenign 

stromal tumors of the ovary. 

histiocytic 

types, lymsex-cord 
Phenolphthalein, NTP TR 465 9 

Exposure of rats to phenolphthalein infeed for germinalepitheliumof the testis in males, and 

2 years resulted in increasedincidences of focal ovarian hyperplasia in females. 

hyperplasia of the adrenal medullainmalesandin 

increased incidences and/or severity of nephropathy 

of the kidney inmalesand females. Exposure of Exposure of mice to phenolphthalein in feed for 

mice to phenolphthalein in feed for 2 yearsresulted 2 yearsresultedindecreasedincidencesofhepatoin increased incidences of atypical hyperplasia of the cellular neoplasms and nonneoplastic lesions in males 

thymus in malesand females, degeneration of theandfemales. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 12. A summary of the Technical Reports Review Subcommittee 

comments and the public discussion on this Technical Report appears on page 14. 
10 Phenolphthalein,NTP TR 465 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studiesof Phenolphthalein 

Doses 

Body weights 

2-Year survival 

rates 

Nonneoplastic 

effects 

Neoplastic effects 

Female 

B6C3F, Mice 

0,3,OOO. 6,OOO.or 

12,OOOppm in feed 

(approximately 400, 

800, or 1,500mglkg 

per day) 

Exposed groups lower 

than the control group 

39/50, 31/50, 34/50. 

28/50 

Thvmus: atypical 

hyperplasia (0148, 

7/44, 6/49, 5/45) 

Ovary: hyperplasia 

(4150, 11/49,10150, 

17/50) 

All organs: histiocytic 

sarcoma (0150, 2/50, 

7/50. 7/50); malignant 

lymphoma (all types) 

(15150,28/50, 33/50, 

25/50);malignant 

lymphoma (thymic 

origin) (l/SO. 9/50. 

10150,7/50) 

Ovary: benign 

sex-cord stromal 

tumor (0150, 7/49. 

6/50,5/50) 

Male 

F344lN Rats 

0,12,000. 25.000, or 

50,000ppm in feed 

(approximately 500, 

1.000,or 2,OOOmglkg 

per day) 

Exposed groups lower 

than the control group 

21/50, 15/50. 15/50, 

13/50 

Adrenal medulla: focal 

hyperplasia (13/50, 

Female 

F344lN Rats 

0,12,000, 25,000. or 

50,OOO ppm in feed 

(approximately500. 

1,OOO, or 2,500mglkg 

per day) 

Exposed groups lower 

than the control group 

30150, 38/50, 32/50, 

38/50 

m:nephropathy 

Male 

B6C3FIMice 

0,3,OOO. 6,OOO.or 

12,OOOppm in feed 

(approximately 300. 

600, or 1,200mglkg 

per day) 

12,OOOppm group 

slightly lower than the 

control group 

40150, 33/50, 36/50, 

36/49 

Thvmus: atypical 

(34150, 45/50, 43/50,hyperplasia (0143, 

22/50, 18/50, 23/50)44\50); severity of 3/46,7/44,7/42) 

Kidney: severity of nephropathy (1.2. 1.4. Testis: germinal 

nephropathy (1.8, 2.9, 1.5,1.5) -

epithelial degeneration 

3.1, 3.1) (1150, 49/50. 50150, 

47/48) 

Adrenal medulla: Adrenal medulla: All organs: histiocytic 

benign benign sarcoma (1150, 3/50. 

pheochromocytoma pheochromocytoma 11/50. 12/49); 

(17/50, 34/50, 34/50.(3/50,11/50, 9/50, malignant lymphoma 

34/50)

m:renal tubule 

adenoma (standard 

evaluation - 0150. 

1/50, 2/50. 6/50; 

extended evaluation -

1\50, 7/50, 15/50. 

11/50;standard and 

extended evaluations 

combined - 1/50. 

10150, 15\50, 15/50); 

renal tubule adenoma 

or carcinoma (standard 

evaluation - O/SO, 

5/50, 3/50. 7/50; 

extended evaluation -

1/50, 7/50, 15/50. 

11/50; standard and 

extended evaluations 

combined - 1/50, 

10/50, 16/50, 16/50) 

2/49);benign or (thymic origin) (0150, 

malignant 4/50, 7/50.2/49) 

pheochromocytoma 

(3150,12/50. 10150, 

2/49) 
Phenolphthalein, NTP TR 465 11 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Phenolphthalein (continued) 

Decreased 

incidences 

Level of evidence 

of carcinogenic 

activity 

Genetic toxicology 

Male 

F344lN Rats 

None 

Clear evidence 

Salmonella ryphimurium gene mutations: 

Sister chromatid exchanges 

Cultured Chinese hamster ovary cells in vitro: 

Chromosomal aberrations 

Cultured Chinese hamster ovary cells in vim: 

Micronucleated erythrocytes 

Mouse peripheral blood in vivo: 

Female 

F344lN Rats 

None 

Some evidence 

Male 

B6C3Fl Mice 

Liver: hepatocellular 

adenoma (22/50. 

12/50, 8/50, 10/49); 

hepatocellular 

adenoma or carcinoma 

(27150. 20/50, 11/50. 

16/49); clear cell 

focus (24150, 6/50. 

1/50. 0/49); 

eosinophilic focus 

(22/50, 6/50. 1/50. 

1/49); mixed cell 

focus (6150, 2/50, 

1/50, 0/49) 

-

Clear evidence 

Female 

B6C3Fl Mice 

Liver: hepatocellular 

adenoma (17/50, 2/50, 

6/50. 1/50); 

hepatocellular 

adenoma or carcinoma 

(21150, 3/50, 6/50. 

2/50); eosinophilic 

focus (20/50, 4/50, 

2/50. 1/50) 

-

Clear evidence 

Negative in strains TA98, TAW, TA1535, and TA1537 with 

and without S9 

Negative with and without S9 

Positive with S9; negative without S9 

Positive 
12 Phenolphthalein, NTP TR 465 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY 

The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the evidence 

for conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of neoplasia than control 

animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of 

conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under the conditions of the study and indicate 

that exposure to the chemical has the potential for hazard to humans. Other organizations, such as the International Agency for Research on 

Cancer, assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studies such as 

those conducted by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from 

chemicals found to be carcinogenic in laboratory animals requires a wider analysis that extends beyond the purview of these studies. 

Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence 

observed in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain findings 

(equivocal evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated 

because of major flaws (inadequate study). These categories of interpretative conclusions were first adopted in June 1983 and then revised 

in March 1986 for use in the Technical Report series to incorporate more specifically the concept of actual weight of evidence of 

carcinogenic activity. For each separate experiment (male rats, female rats, male mice, female mice), one of the following five categories is 

selected to describe the findings. These categories refer to the strength of the experimental evidence and not to potency or mechanism. 

l Clear evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related 

(i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked increase 

of benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to malignancy. 

l Some evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a chemical-related 

increased incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than that 

required for clear evidence. 

l Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal increase 

of neoplasms that may be chemical related. 

l N o evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related 

increases in malignant or benign neoplasms. 

l Inadequate study of carcinogenic activity is demonstrated by studies that, because of major qualitative or quantitative 

limitations, cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity. 

When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the actual 

boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and current 

understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the borderline 

between two adjacent levels. Theseconsiderationsshouldinclude: 

l adequacy of the experimental design and conduct; l occurrence of common versus uncommon neoplasia; l progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions; l some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, it is 

impossible to identify the difference. Therefore, where progression is known to be a possibility, the most prudent course 

is to assume that benign neoplasms of those types have the potential to become malignant; 

l combining benign and malignant tumor incidence known or thought to represent stages of progression in the same organ or 

tissue; 

l latency in tumor induction; 

l multiplicity in site-specific neoplasia; 

l metastases; 

l supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments (same 

lesion in another sex or species); l presence or absence of dose relationships; l statistical significance of the observed tumor increase; 

l concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm; l survival-adjusted analyses and false positive or false negative concerns; 

l structure-activity correlations; and l in some cases, genetic toxicology. 
Phenolphthalein, NTP TR 465 13 

NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE 

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on phenolphthalein on 

December 5, 1995, are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, 

company, or governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing NTP studies: 

l to ascertain that all relevant literature data have been adequately cited and interpreted, l to determine if the design and conditions of the NTP studies were appropriate, l to ensure that the Technical Report presents the experimental results and conclusions fully and clearly, l to judge the significance of the experimental results by scientific criteria, and l to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses. 

Arnold L. Brown, M.D., Chairperson 

University of Wisconsin Medical School 

Madison, WI 

Gary P. Carlson, Ph.D. 

School of Health Sciences 

Purdue University 

West Lafayette, IN 

Thomas L. Goldsworthy, Ph.D., Principal Reviewer 

Department of Experimental Pathology and Toxicology 

Chemical Industry Institute of Toxicology 

Research Triangle Park, NC 

Robert LeBoeuf, Ph.D. 

Corporate Professional and Regulatory Services 

Human Safety Department 

The Procter & Gamble Company 

Cincinnati. OH 

Janardan K. Reddy, M.D.* 

Department of Pathology 

Northwestern University Medical School 

Chicago, IL 

* Did not attend 

Irma Russo, M.D. 

Fox Chase Cancer Center 

Philadelphia, PA 

Louise Ryan, Ph.D. 

Division of Biostatistics 

Dana-Farber Cancer Institute 

Boston, MA 

Robert E.Taylor, M.D., Ph.D., 

Department of Pharmacology 

Principal Reviewer 

Howard University College of Medicine 

Washington, DC 

Frederick L. Tyson, Ph.D. 

St. Mary’s Hospital and Research Center 

Cancer Research Institute 

Grand Junction. CO 

Jerrold M. Ward, D.V.M., Ph.D., 

National Cancer Institute 

Frederick, MD 

Principal Reviewer* 
14 Phenolphthalein; NTP TR 465 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS 

On December 5, 1995, the draft Technical Report on 

the toxicology and carcinogenesis studies of phenolphthalein received public review bytheNational 

Toxicology Program’s Board ofScientificCounselors’ Technical Reports Review Subcommittee. 

The review meeting was held at the National Institute 

of Environmental Health Sciences, Research Triangle 

Park, NC. 

Dr. J.K. Dunnick, NIEHS, introduced the toxicology 

and carcinogenesis studies of phenolphthaleinby 

describing the uses of the chemical and rationale for 

the study, describing the experimental design, reporting on survival and bodyweight effects, and commenting on chemical-related neoplasms and nonneoplastic lesions in maleandfemaleratsandmice. 

Dr. Dunnick noted that molecular biology studies in 

collaboration with NIEHS intramural scientists are in 

progress or planned, including studies of the chemicalin transgenic mouse models. Pharmacokinetic 

studies are in progress. Dr.J.R. Hailey, NIEHS, 

presented photomicrographs of lesions of the hematopoietic system, examples ofhistiocytic sarcoma, 

malignant lymphoma of thymic origin and associated 

hyperplasia, ovarian proliferative lesions, and testicular atrophy. The proposed conclusionswere clear 

evidence of carcinogenic activity of phenolphthalein 

in male F344/N rats, some evidence of carcinogenic 

activity ofphenolphthaleininfemale F344/N rats, 

and clear evidence of carcinogenic activity in male 

and female B6C3F, mice. 

Dr. Goldsworthy, a principal reviewer, agreed with 

the proposed conclusions. He stated that the Technical Report should provide assurance thatbiological 

responses observed, especially related those to

estrogenic effects, are attributable to phenolphthalein 

and not to lipophilic impurities. Small quantities of 

such impurities have been shown to account for the 

estrogenic activity in commercial preparations of the 

sulfonatedanalogof phenolphthalein, phenolsulfonphthalein or phenol red. Dr. Dunnick responded that 

thephenolphthaleinused in thesestudies was 99% 

pure. She noted that Dr. M.D. Shelby, NIEHS, is 

developing assays for estrogenic activity andanticipated that the estrogenic potential of phenolphthalein 

could be compared with those of phenol redandof 

lipophilic impurities. Dr. Goldsworthy said there 

should be a comprehensive treatment of the estrogenic responses; for example, discussion on decreased of incidences liver lesions mice. in 

Dr. Dunnick proposed isolating the 1 % impurity and 

examining its carcinogenic and estrogenic activity in 

short-term model systems such as transgenics and 

MCF-7 cells. 

Dr. Ward, the second principal reviewer, was unable 

to attend the meeting but had submitted his review, 

which Dr. L.G. Hart, NIEHS, read into the record. 

Dr. Ward agreed with the proposed conclusions. He 

askedifthe myelofibrosis reported was thetypical 

lesionfoundinaging B6C3F, mice and probably 

estrogenic in origin. Dr. Hailey said that it wasthe 

lesion associated with aging. 

Dr. Taylor, the third principal reviewer, agreed with 

the proposed conclusions. He noted that pharmacokinetic data on phenolphthalein are rare in the literature and recommended that the pharmacokinetic data, 

particularly the lack of a dose-response relationship, 

inAppendix 0 bediscussedinthebodyof the 

Technical Report. Dr. Dunnick said that these 

studies were limited, but that she would try to give 

moreemphasistothe findings. Dr. Goldsworthy 

hopedthatfollow-up studies would look at phenolphthaleinlevelsin organs such as the ovaries. 

Dr.G.W. Lucier, NIEHS, reported that follow-up 

pharmacokinetic studies will span a very wide dose 

rangeandwillenable a better definition of the 

leveling off of tissue or blood levels of phenolphthalein in relation to dose. 

Dr. Russo commented that there did not seem to be 

anyinfluenceofchemical treatment on pituitary 

gland or mammary gland lesions, with an even lower 

incidence of thyroid gland C-cell lesions than in 

control animals, all speaking against an estrogenic 

effect. Dr. Dunnick agreed, noting that another 

chemicalwith estrogenic activity, zearalenone, had 

not produced increases in mammary gland lesions in 

rats or mice. Dr. LeBoeuf asked whether the in vitro 

increases in chromosomal aberrations and in vivo 

increases in the frequency of micronucleated erythrocytes were consistent with other chemicals that may 
Phenolphthalein, NTP TR 465 15 

have estrogen-like properties and can form quinones 

with metabolism, such as diethylstilbestrol. 

Dr. Shelby repliedthat the database is too small to 

reachany general conclusion. However, these 

increases along with the 2-year study results are fully 

consistent with the endocrine disrupting estromimetic 

compounds. Dr. W.T. Allaben,NationalCenter for 

Toxicological Research and Food and Drug Administration (FDA), thanked the NTP, and particularly 

Dr. Dunnick, for sharing information duringthe 

studyandreview process and noted that the additional studies mentioned will be helpful to the FDA 

inits further review towardmaking an assessment 

regarding potential human risk. 

Dr. Goldsworthy moved that the Technical Report on 

phenolphthalein be acceptedwith the revisions 

discussedandwiththe conclusions as written. 

Dr. Taylor seconded the motion, which was accepted 

with six yes votes and one abstention (Dr. LeBoeuf). 
16 Phenolphthalein, NTP TR 465 
17 

INTRODUCTION 

0 

OH 

PHENOLPHTHALEIN 

CAS NO.77-09-8 

Chemical Formula: c2fi1404 Molecular Weight: 318.33

Synonyms: 3,3-Bis(4-hydroxyphenyl)-1(3H)-isobenzofnone; 3,3-bis@-hydroxyphenyl)phthalide; a-@-hydroxyphenyl)-a-(4-oxo2,5-cyclohexadien-l-ylidene)-o-toluicacid 

Tradenames: Agoral", Alophen", Colax", Correctol", Dialose", Doxidan", Espotabs", Evac-U-Gena, Evac-U-Lax", Ex-Lax", 

Feen-A-Mint", FemiLax", Kondremul", Laxcaps", Lax-Pills@, Medilax", Modane", Phenolax", PntleP 

CHEMICAL AND 

PHYSICAL PROPERTIES 

Phenolphthalein is a white or yellowishwhite, 

odorless, tastelesspowderconsisting of minute 

triclinic crystals (often twinned). It has a specific 

gravity of 1.277 at 32/4" C and a melting point range 

of 262" to 263" C (Weast, 1987) or 258" to 262" C 

(Merck Index, 1989), depending on therelative 

amounts of associated impurities. Althoughnot 

flammable, phenolphthalein emitsacridsmokeand 

fumes when heated to decomposition (Sa's, 1992). 

Phenolphthalein is readily soluble in alcohol (1 g 

dissolves in 12 to 15 mL) or ether (1 g dissolves in 

approximately 100 mL) and very slightly soluble in 

chloroform. It is almost insoluble in water; however, 

itssolubility is increased in physiologic buffered 

solutions intestinal simulating contents, i.e.,

7.8 mg/dL in Krebs-Ringer-bicarbonate solution, 

pH 7.4 (Sharaiha et al., 1983). The solubility of 

aqueousphenolphthaleinis also pH dependent and 

doesnotexceed 6 mg/dL without the addition of 

ethanol until the pH is above the physiologic range, 

i.e., greater than 9 (Fantus and Dyniewicz, 1937; 

Hubacher, 1945). Solutions containing phenolphthalein are colorless to pH 8.5 andpink to deep 

red above pH 9 (Figure 1). Phenolphthalein is used 

as a laboratory reagent and an acid-base indicator in 

titrations of mineral and organic acids and most 

alkalies (Merck Index, 1989). 
18 Phenolphthalein, NTP TR 465 

heat' znc12 a;o 

0 Phenol I1

0 Phthalic anhydride 

Phenolphthalein Phenolphthalein 

0 

Colorless Red Colorless 

Phenolphthalein 

FIGURE 1 

Phenolphthalein Synthesis and Reactions as an Indicator (Morrison and Boyd, 1966) 

PRODUCTION, USE, 

AND HUMANEXPOSURE 

Phenolphthalein is synthesizedcommerciallyby the 

condensation of phthalic anhydride with phenol in the 

presence of a dehydrating agent (sulfuric acid) 

(Figure 1). After the mixture is heated at 120" C for 

10 to 12 hours, the phenolphthalein residue isextracted with boiling water andthen dissolved indilute 

sodium hydroxide solution, filtered, and precipitated 

with acid (Remington3, 1990). Approximately

250 tons of phenolphthalein are producedinthe 

United States annuallyby a single manufacturer, 

Sigma-Aldrich Corporation (ChemicalEconomics 

Handbook, 1992). 

Early studies in humansassessingthesafetyof 

phenolphthalein when used as a pH-dependent coloring agent in adulterated wine led to recognition of its 

cathartic action (von Vhossy, 1908). Phenolphthalein is available worldwide in numerous proprietary 

over-the-counter laxative preparations including 

tablets, powders, suspensions, capsules, and chewables; the fact that phenolphthaleinis tasteless makes 

it desirable for marketing in the form of chocolate 

candy or chewing gum (AHFS, 1995). 

The usualdaily oral laxative doses for white or 

yellow phenolphthalein are 30 to 270 mg for adults 

and children 12 years of age and older, 30 to 60 mg 

for children 6 to 11 years of age, and 15 to 30 mg 

for children 2 to 5 years of age, although the use of 

stimulant laxatives is generally not recommended in 

children younger than 6 years of age (Fed. Regis?., 

1975; AHFS, 1995). 

Uses of Phenolphthalein as a laxative-cathartic may 

include: as bowel evacuants prior to bowel surgery 
Introduction 19 

or radiologic, proctoscopic, and colonoscopic procedures; to alleviate pain of elimination from an episiotomywound,anal fissures, perianal abscesses, or 

after surgery; to reduce intra-abdominal pressure 

during elimination in patients with hernias, anorectal 

stenosis, or abnormalities of the cerebral or coronary 

arterial vessels; to relieve constipation during pregnancy or the puerperium; to modifytheeffluentin 

ileostomy and colostomy patients; to compensate for 

lost abdominal and perineal muscle tone in geriatric 

patientswith poor eating habits;and for altered 

bowel motility in patients receiving anticholinergicor 

narcotictherapy (Pietrusko, 1977; Goodmanand 

Gilman’s, 1990). 

However, laxative-cathartics, which are habit forming (i.e., chronic usage resultsindependence), are 

commonly used to self-medicate symptoms of constipationand are frequentlyabusedinan effort to 

control weight (Pietrusko, 1977; Goodmanand 

Gilman’s, 1990). Ten percent of college students 

who participated in a 1981 survey admitted to purging behavior, i.e., laxative useandself-induced 

vomiting(Halmi el al., 1981). Purging behavioris 

alsoassociatedwiththeeating disorders anorexia 

nervosandbulimia nervosa; the prevalenceof 

laxative abuse in patientswithbulimianervosahas 

been reported as 38% to 63 % (VanRooyenand 

Ziady, 1972; Pyle et al., 1981; Johnsonet al., 1982; 

Bo-Linn et al., 1983; Mitchell et al., 1983; Fairburn 

and Cooper, 1984). A reviewbyCummings(1974) 

indicated that over 90% of chronic laxative abusers 

are women. 

It not ispossible to quantitate exposure to

phenolphthalein via medication because of the large 

number of over-the-counter laxative preparations 

(Fleischer et al., 1969; Van Rooyenand Ziady, 

1972; Cummings et al., 1974; LaRusso and McGill, 

1975; Bytzer et al., 1989). In the UnitedStates, 

Great Britain, and Australia, the rate of regular 

laxative use in populations who are apparently well 

is approximately 17% to 20% (Connell et al., 1965; 

Dent et al., 1986; Wu et al., 1987; Kune, 1993). In 

1975, 130 million dollars were spentintheUnited 

States on over-the-counter laxativepreparations 

(Binderand Donowitz, 1975). In 1980, overall 

United States sales increasedtoapproximatelyone 

billion dollars (Curry, 1990). 

According to the NationalOccupational Exposure 

Survey, 75,243 workers (26% of whom were female) 

werepotentiallyexposedto phenolphthalein in the 

years1981 to 1983. Of the potentially exposed 

workers, 20,122 (65 % female) were employed in the 

health services (NIOSH, 1990). 

REGULATORY STATUS 

Based ona review of over-the-counter laxativecathartics conducted by a Food and Drug Administrationpanel as part of a drug efficacy study 

implementation project, phenolphthalein was 

approved for human use and considered to be “generally recognized as safe” (Fed. Regist., 1975). 

PHARMACOKINETICS, ABSORPTION, 

DISTRIBUTION, IMETABOLISM, 

AND EXCRETION 

Experimental Animals 

Phenolphthalein is absorbed from the intestine and is 

excretedinthe bile, urine, feces, and milk(Visek 

et al., 1956; AHFS, 1995). Once phenolphthalein is 

absorbed, it is conjugated with glucuronic acid via 

uridine glucuronosyltransferase diphosphate 

(UDPGT; a phospholipid-dependent enzyme system) 

in the liver and intestine (Sund and Hillestad, 1982) 

and is distributed throughout the body in the blood 

and lymph (Visek et al., 1956). Studies reported in 

the literature provide evidence for enzyme multiplicity of UDPGT. and Steroidal nonsteroidal

UDPGT may have different membrane environments, 

and certain tissues suchas the kidney and uterus have 

beenshown to have a lower UDPGTactivity for 

xenobiotic estrogenic compounds such as phenolphthalein(Lucierand McDaniel, 1977). Minor 

amounts of sulfate conjugate metabolites have also 

beendetectedinmucosalsheetsisolated from the 

jejunum and colon of theguineapig(Sundand 

Lauterbach, 1986). 

Phenolphthalein conjugation enzyme activity (uridine 

diphosphatase glucuronosyltransferase) is absent or 

verylowinmicrosomes from fetal or neonatal rat 

and guinea pig liver compared to activity from adult 

livers (Jondorf et al., 1958; Wishart, 1978a). 

Glucuronideconjugationactivityin adult rats is 
20 Phenolphthalein, NTP TR 465 

stimulatedby phenobarbital andunaffectedby 

3-methylcholanthrene (Wishart, 1978b). 

Pharmacokinetic and tissue distribution studies in 

mice (strain not specified) and dogs(breednot 

specified) given 4.8 mg [14C]-phenolphthalein/kg 

(uniformly labeled) indicated thatphenolphthalein 

waswidelyandevenly distributed throughout the 

body, with levelsofradioactivityparallel to the 

concentration in the blood. In dogs, approximately 

50% of an oral dose was recovered in the feces and 

36% in the urine after 72 hours (Visek et al., 1956). 

In the mouse studies (Visek et al., 1956), no respiratory [14C]-carbon dioxide was recovered following administration of phenolphthalein labeled with14C 

on the nonaromatic carbon of the lactone ring, 

indicating that the bonds to the labeled carbon atoms 

probably were not broken. Within 48 hours approximately 96% of theadministeredradioactivitywas 

recovered in the urine and feces (56% inthe urine 

and 38 % in the feces following an oral dose; 30 % in 

the urine and 68% in thefecesfollowingan intravenous dose). At 30 minutes to 6 hours, the highest 

levels of radioactivity appearedinthe liver, gallbladder, and small intestine. On a total organ basis, 

the small intestine, which plays a major role in the 

excretion of phenolphthalein, contained more radioactivelabelthandidthe large intestine at all 

measurement intervals. However, when equatedon 

a unit weight basis, the radioactivity of the. large 

intestine and its contents increasedsubstantially 

6 hours after dosing. 

Whole-body autoradiography studiesin male 

B0M:NMRI micegivenan intragastric dose of 

1 mL/kg [14C]-phenolphthalein (10 pCi/lOO g) 

showedinitialhighlevels of radioactivityinthe 

stomach, gallbladder, and smallintestineat 10 and 

20 minutes. Radioactivity was observed in peripheral 

organs (including the kidney, liver, and skin) showing that the drug was absorbed from the gastrointestinal tract. The radiolabel moved along the intestinal 

tract, reaching the large intestine after 2 hours and 

showingmaximumactivityintherectumafter 

4 hours. No radiolabel was detectedinautoradiograms 2 days after dosing (Sund et al., 1986). 

Phenolphthalein undergoes extensive first pass 

metabolismintheintestinalepitheliumand liver, 

whichresultsinalmostcomplete conversion of 

phenolphthalein to its glucuronide (Parker et al., 

1980). Studiesby Colburn et al. (1979) demonstrated that 6 hours after intravenous administration 

of [3H]-phenolphthalein to female Wistar rats, all 

radioactivity in the systemic circulation was present 

asthe conjugate. In addition, a secondary peak in 

bloodradioactivity occurred 5 to 6 hours after 

intravenous administration and coincided with the 

absorption of [3H]-phenolphthalein from the intestine 

followingbacterialB-glucuronidase hydrolysis of 

[3H]-phenolphthalein glucuronide excreted in the bile. 

Hydrolysis of phenolphthalein glucuronide to the 

aglyconeis a rate-limiting step in enterohepatic 

recirculation (Berganet al., 1982); pretreatment with 

antibiotics to suppress intestinal microflora decreased 

the absorption of [3H]-phenolphthalein from 85% to 

22% in female Wistar rats following intraduodenal 

administrationof[3H]-phenolphthalein glucuronide 

(Parker et al., 1980). 

In a study withbeagle dogs, two males were given 

8-or 10-hour intravenous infusions of phenolphthaleinat a rate of 177 pglminute per kg body 

weight,andfoodwasintroduced at 2, 4,6, and 

8 hours during the study to stimulate gallbladder 

contraction. Steady-state serum levels were 7 pg/mL 

for phenolphthalein and 30 pg/mL for phenolphthalein glucuronide, and significant secondary peaks 

occurred for both compounds, an observation consistent with enterohepatic recirculation (Wilhelm et al., 

1992). 

Phenolphthalein has been used as a model compound 

for enterohepatic recirculation studies in rats 

(Colburn et al., 1979), and surgical cannulation of 

the bile duct has been used to evaluate the extent of 

enterohepatic recirculation in rats and dogs. In bileduct-cannulatedfemale Wistar rats, 95% ofan 

intraperitoneal dose of 25 mg [3H]-phenolphthalein/kg 

was recovered in the bile as the glucuronide within 

24 hours, while only 0.2% was recovered in the 

urine. Administration of the same dose to intact rats 

resultedinrecoveryof 86% in the feces, predominantly as the parent drug, and 10% in the urine 

astheglucuronide (Parker et al., 1980). In another 

study in female Wistaratswith biliary fistulae, 

phenolphthalein was completely eliminated in the bile 

(loo%), almost entirely in the form of phenolphthalein glucuronide (98%) (Millburn et al., 1967). The 
Introduction 21 

plasma disappearance and biliary excretion kinetics of 

phenolphthalein glucuronide in therathavebeen 

characterized by Mehendale (1 990). Male SpragueDawley rats of the CR-1 strain were intravenously 

administered 3, 30, or 60 mg phenolphthalein or 3, 

30, or 100 mg phenolphthalein glucuronide, and the 

femoral vein, artery, and common bile duct were 

cannulated. After administration of phenolphthalein, 

99.5% ofthe dose was eliminatedinthebileas 

phenolphthalein glucuronide with onlytracequantities (0.5%) as phenolphthalein. Followingadministration of phenolphthalein glucuronide, phenolphthalein was undetectableinthe bile. Biliary excretion 

was saturable at higher doses of both compounds. 

Female dogs (breed not specified)given 4.8 mg 

['4C]-phenolphthalein/kg excreted 5 1% and 36 % of 

the administered radioactivity in the feces and urine 

within 72 hours after an oral dose and 54 % and 37% 

in the feces and urine following an intravenous dose. 

Following surgical cannulation of thebile duct, 

recovery of orallyadministeredradiolabelfromthe 

same dogs over a 72-hour period was: feces, 31 %; 

urine, 38% ; and bile, 22%. Thecorresponding 

figures following an intravenous dose were: feces, 

11%; urine, 35%; and bile, 43% (Visek etal., 

1956). 

The investigations of Visek et al. (1956) also 

demonstrated that phenolphthalein radiolabeled 

crossed the placenta of mice, resulting in recovery of 

radiolabel from fetal tissues at 6, 24, and 96 hours 

after administration that paralleled that of maternal 

blood. In the dog, however, analysis of blood and 

tissue samples of puppies born 50 hours after the 

mother received a 4.8 mg/kg oral dose indicated less 

than 0.03% of the administered dose in the liver and 

gallbladder and no radiolabel in the blood, which was 

interpreted by the authors as exceedinglylimited 

passage of the drug across the placenta. 

Humans 

Phenolphthalein absorption in humanshasbeen 

estimated to be 15 % of an oral dose (Goodman and 

Gilman's, 1990). The absorbed is compound

excreted primarily the in urine as phenolichydroxyglucuronide or sulfate conjugates, and the 

urine becomes pink or red if it issufficientlyalkaline. Some conjugated compound is also excreted in 

the feces via the bile, and the resulting enterohepatic 

recirculation probably contributes to prolongation of 

the laxative effect, a hypothesis supported by the 

observationthatphenolphthalein is ineffective as a 

laxative in jaundiced patientsor experimental animals 

withligatedcommonbile ducts (Steigmann et aZ., 

1938; Goodmanand Gilman's, 1990). 

Smalldoses of phenolphthaleininhumans (30 to 

60 mg) are excreted entirely as conjugated metabolites in urine or feces, while larger doses (300 mg) 

resultin excretion of both the free and conjugated 

drug (Williams, 1959). 

Diarrhea in infants may be caused by phenolphthalein 

usagebythemother during breast feeding (Tyson 

et al., 1937). 

PHARMACOLOGY 

Followingoraladministrationof phenolphthalein, 

evacuation usually occurs within 4 to 8 hours, and a 

single dose may produce laxation for several days 

(AHFS, 1995). The major site of cathartic action for 

phenolphthaleininhumansis considered to be the 

large intestine with minor changes occurring in the 

smallintestine (Saunders et al., 1978; Sund, 1983). 

Phenolphthaleinhas choleretic properties and has 

been shown to accelerate bile secretions in male rats 

(Takeda and Aburada, 1981). 

Phenolphthalein, a diphenylmethane derivative, 

belongs to the class of stimulant or irritant laxativecathartics originally thought to stimulate peristalsis by 

irritation of the colonic mucosa (Binder, 1977). 

Current views of the mechanism of action of phenolphthaleinindicatethatithas a hydrophoric effect, 

which alters intestinal fluid and electrolyte movement 

and causes a net accumulation of luminal fluid and 

increasedfluidityoftheintestinal contents (Binder 

andDonowitz, 1975; Gaginella ndBass, 1978; 

Sund, 1983). This effect on the sodium pump, which 

has been demonstrated in an isolated rabbit ileal loop 

model invivo usingmeasurementof "Na flux, 

involves inhibition of Na+ ion transport from the gut 

lumen to the plasma.Accumulationof Na+ ions in 

thelumenresultsinsecondaryaccumulationof 

anions (chiefly C1- and HCO,-) to maintain electroneutralityat a pH ofapproximately 8 withluminal 

waterretention (Phillips et al., 1965). Inhibition of 

intestinal water absorption has been demonstrated in 
22 Phenolphthalein, NTP TR 465 

experimental animal modelsandinhumans. An 

in vivo model using isolated jejunal, ileal, and 

colonic segments oftheintestine of male SpragueDawley rats was usedtodemonstratethatphenolphthaleininhibitednetwater transport to thesame 

extent in all regions of the intestine (Saunders et al. , 

1978). A study of six ileostomy patients by the same 

Autore et al. (1984) attributed the laxative effect of 

a 16 mg/kg intragastric dose of phenolphthalein in 

maleWistar rats to stimulation of histamineand 

5-hydroxytryptamine production and increased

formation of prostaglandins, effects which were 

reducedby pretreatment with indomethacin. They 

concluded that the stimulation of biologically active 

investigators indicated that inhibition of water absorp-amines may be due to altered gut motility, an overtion in both the large and small intestine contributes 

to the laxative effect of phenolphthalein; 100 mg 

given four times a dayincreased the weight of 

ileostomy output by 30% and Na+ output by 39.%. 

In addition to inhibition of active transport of sodium 

across the rabbit ileum and frog skin (Phillips et al., 

1965), phenolphthalein inhibitedhe sodiumdependent uptake of 3-methyl-D-glucoseby the 

hamster small intestine in vitro (AdamiCand Bihler, 

1967) and reduced intestinal glucose absorption in the 

rat (Hart and McColl, 1967). Chignell (1968) 

investigated the effects of various purgative drugs on 

Na+, K+-adenosine triphosphatase activity in microsomes from the intestinal brush border of male 

Sprague-Dawley rats. Phenolphthalein was the most 

potent enzyme inhibitor of all the purgatives studied, 

blocking 70% of enzyme activity, which suggests that 

inhibition of sodium transport is a direct result of 

inhibition of Na+, K+-adenosine triphosphatase. 

Phenolphthalein has been shown to stimulate prostaglandin biosynthesis in the colon of female SpragueDawley rats, with release of E-series prostaglandins 

into the gut lumen (Cohen, 1982). The extent of 

prostaglandin release correlated with thenetwater 

flux (laxative action) inducedbythe drug, and 

pretreatment with inhibitors of prostaglandin synthesis (indomethacin or aspirin) reduced thelaxative 

effect in rats and mice(Beublerand Juan, 1978; 

Capasso et al., 1984). In in vitro studies in male rat 

intestinal homogenates, phenolphthalein (3 14 pM) 

significantly increased the conversion of arachidonic 

acid, a prostaglandin precursor, to prostaglandins and 

5-hydroxy-eicosatetraynoicacidinthe colon and to 

leukotriene B4 in the jejunum and colon. Similarly, 

in human colon homogenates, 100 pg phenolphthaleinlml increased conversion of arachidonic 

acid to prostaglandins and leukotriene B4 (Capasso 

et al., 1987). 

production of prostaglandin-like material, or a nonrelated direct effect of phenolphthalein. In a more 

recent study, there was an increase in kininogen 

content(measured as bradykinin equivalentdg wet 

tissue weight) in the colon of phenolphthalein-treated 

male Wistar rats, thus implicating kinins in the 

inductionof laxation (Autore et al., 1990). Augmentation of muscle contractions induced by prostaglandin &, demonstrated in the rat stomach and the 

longitudinal muscle of theguinea pig ileum and colon 

following in vitro administration of 10 pg phenolphthalein/mL, was suggested as a possible contributing factor in phenolphthalein laxation (Capasso 

et al., 1988). 

Although the laxative effect of phenolphthalein in 

monkeys is similar to that in humans, the dose 

required to induce laxation (25 mg/kg) is approximately 10 times that required for humans (Loewe and 

Hubacher, 1 94 1 ). 

TOXICITY 

Experimental Animals 

Phenolphthalein, given intravenously in dogs (breed 

not specified), was classified as relatively nontoxic in 

early studies (Abel and Rowntree, 1909). However, 

in rats it is regarded as moderately toxic by the 

intraperitoneal route, with an LD, of 500 mg/kg 

(Sax’s, 1992). 

In oral studies, female mice (strain not specified) fed 

5, 25, or 50 mg phenolphthaleinlkg per day for 

135 days showed no toxic manifestations or evidence 

of histopathologic changes in the liver, kidney, or 

gastrointestinal tract (Visek et al., 1956). 

Phenolphthalein,at doses of 25 and 50 pglmL, 

caused cytotoxic effects in cultured Chang liver cells 
Introduction 23 

characterized by decreased cell growth and increased 

anaerobic glycolysis, i.e., increasedglucoseconsumption and lactate production (Nishikawa, 198 1). 

Phenolphthalein has alsobeenshowntoinhibit 

growth of strains of anaerobic bacteria in vitro and 

cause leakage of potassium ions from both anaerobes 

and aerobes, findings consistent with the antibacterial 

properties of phenolic compounds (Bergan et al., 

1982; Sund, 1983). 

Humans 

Under normal conditions, phenolphthalein is regarded 

as nontoxic and safe for consumption, although 

therapeutic oral doses may occasionallyproduce 

abdominal discomfort, diarrhea, nausea, decreased 

blood pressure, faintness, and red urine and feces 

(AHFS, 1995). Serious side effects havebeen 

reported in cases of habitual phenolphthaleinconsumption under conditions of abuse (Cooke, 1977; 

Pietrusko, 1977); hypersensitivity reactions have been 

limited to susceptible or allergic individuals (Davies, 

1985). 

The primary reported organ for phenolphthalein 

toxicity is the intestine; indiscriminate use of phenolphthalein results in chronic constipation and laxative 

dependence, loss of normalbowel function, and 

bowel irritation. Habitual use for several years may 

cause a ‘‘cathartic colon, ’’ i.e., a poorly functioning, 

atonic dilation of the colon, especially oftheright 

side, resulting in extensive bowel retention. The 

clinical condition, which resembles chronic ulcerative 

colitis both radiologically andpathologically,involves thinning of the intestinal wall and loss of the 

normalmucosal pattern of thetrminalileum 

(Cummings, 1974; Cooke, 1977; Pietrusko, 1977; 

AHFS, 1995). 

Anecdotalcases of long-term use or overdose of 

phenolphthalein have been associated with abdominal 

pain, diarrhea, vomiting, electrolyte imbalance 

(hypokalemia, hypocalcemia, and/or metabolic 

acidosis or alkalosis), dehydration, malabsorption, 

protein-losing gastroenteropathy, steatorrhea, anorexia, weight loss, polydipsia, polyuria, cardiac 

arrhythmias, muscle weakness, prostration, and 

histopathologic lesions (Heizer et al., 1968; 

Velentzasand Ikkos, 197 1 ; Cummings,1974; 

LaRusso and McGill, 1975; Pohl andLowe,1978; 

AHFS, 1995). Kidney, muscle, and centralnervous 

system disturbances are thought to be due to electrolyteimbalance.Intestinalsodiumandwater loss 

stimulatescompensatoryrenin production and secondary aldosteronism, leading to sodium conservation 

andpotassium loss by thekidney (hypokalemia). 

Hypokalemia contributes to renal insufficiency, 

sometimes associated with rhabdomyolysis 

(Copeland, 1994). 

Abuse of phenolphthalein-containing laxatives has 

beenassociatedwith gastrointestinal bleeding and 

iron deficient anemia (Weiss and Wood, 1982), acute 

pancreatitis(Lambrianidesand Rosin, 1984), and 

multiple organ damage incasesofmassive overdosages, including fulminant hepatic failure and 

disseminated intravascular coagulation (Sidhu et al., 

1989). hypersensitivity Individual reactions to

phenolphthaleinhave also been reported to cause 

renal irritation, encephalitis, cardiac arrest, respiratory disturbances, and death (AHFS, 1995). 

Phenolphthaleinallergyis often manifestedby 

cutaneousinflammatoryreactions or fixed drug 

eruptions, i.e., solitary or multiple, well-defined, 

erythematous macules that may progress to vesicles 

and/or bullae. These lesions characteristically recur 

inthesamelocationwitheach subsequent dose of 

phenolphthaleinand generallyleave residual hyperpigmentationthat increases in intensitywitheach 

exposure; numerous melanin-containing dermal 

macrophages have been demonstrated in pigmented 

areas (Wyatt et al., 1972; Davies, 1985; Stroud and 

Rosio, 1987; Zanolli et al., 1993). In extreme cases, 

recurrences have involved progressively more severe 

lesions characterized as bullous erythema multiforme, 

with focal hemorrhage and necrosis and perivascular 

lymphocytic infiltration (Shelley et al., 1972), and in 

one case report, toxic epidermal necrolysis (Kar 

et al., 1986). 

A review of 204 casesof phenolphthalein ingestion in 

children 5 years of age and younger reported to the 

Pittsburgh Poison Center over a 30-month period 

indicated that ingestion of 1 g or less was associated 

with minimal risk of developing dehydration caused 

by excessive diarrhea and resulting fluid loss (Mrvos 

et al., 1991). However, despite the low acute 

toxicity profile documented by the Pittsburgh study, 

cases of fatal phenolphthalein poisoning of children 

havebeen reported; symptoms of pulmonary and 
24 Phenolphthalein, NTP TR 465 

cerebral edema, multiple organ toxicity, and encephalitis wereattributed to hypersensitivityreactions 

(Cleves, 1932; Kendall, 1954; Sarcinelli et al., 

1970). Repeated administration of phenolphthaleincontaining laxatives to children has ledtoserious 

illnessesandmultiplehospitalizations (Fleisher and 

Ament, 1977; Meadow, 1977; Devore et al., 1982; 

Rosenberg, 1987; Sugar et al., 1991; Ayass et al., 

1993). 

REPRODUCTIVE AND 

DEVELOPMENTALTOXICITY 

Experimental Animals 

In a three-generation study involving 678 births, no 

drug-related teratogenic or reproductive deficits were 

observed in mice (strain not specified) fed approximately 250 mg phenolphthaleirdkg per day in chocolate (Stockinger, 1965). 

Phenolphthalein has weak estrogenic activity. Ina 

study in immature Wistar rats, the minimum effective 

dose of phenolphthalein required to cause an estrogenic response (increase in glycogen content) in the 

uterus was 4 mg, compared to a 0.1 mg minimum 

effective dose of diethylstilbestrol (Bitman and Cecil, 

1970). Subcutaneous injection of phenolphthalein 

also stimulated growth of the immature rat uterus in 

a similar study (Nieto et al., 1990). 

Humans 

Phenolphthalein competed with estrogen for binding 

to cultured MCF-7 human breast cancer cells, with a 

relative binding affinity that of estradiol, and 

stimulated cell growth as measured byDNAand 

protein assays. Actingasan estrogen agonist, 

phenolphthalein induced elevatedlevelsof progesterone receptor in the MCF-7 cells. Growth 

stimulation by both phenolphthalein and estradiol was 

blockedby the anti-estrogen, 4-hydroxytamoxifen 

(Ravdin et al., 1987). No other information related 

to the reproductive or developmentaltoxicity of 

phenolphthalein in humans has been reported in the 

literature. 

CARCINOGENICITY 

Experimental Animals 

No definitive information regarding the carcinogenic 

potential of phenolphthalein in experimental animals 

wasfoundinthe literature. However, phenolphthalein phosphate was inactive as an inducer of tumors 

at the site of subcutaneous injections in mice when 

givenonceweekly for 13 weeks. Following an 

8-month, treatment-free period, no tumors were 

observedwhenthe experiment was terminatedat 

11 months with a 50% survival rate (10/20). The 

value of this study as an indicator of the carcinogenic 

potential of phenolphthalein was limited because the 

compoundwastested ata single unspecified dose 

level (Haddow and Horning, 1960). 

Humans 

In a study conductedin Melbourne, Australia with 

1,408 subjects, there was no statistically significant 

increased risk for colorectal cancer in phenolphthalein laxative users (Kune, 1993). The cases included 

inthisstudyincluded all histologically confirmed 

new patients in “The Melbourne Colorectal Cancer 

Study” from April 1980 to April 1981. Community 

controls were agehex frequency-matched with the 

clinicalcasesandwererandomlyselected from the 

same geographic area. In another study following 

11,888 residents of a retirement community in 

California for 4.5 years, the association between 

laxative use and risk of colorectal cancer was not 

significant(Wu et al., 1987). Information on other 

cancer sites was not reported. 

GENETICTOXICITY 

The mutagenicity of phenolphthalein has been investigatedand the data indicate that the chemical, althoughnotmutagenicin bacteria, is capable of 

inducing chromosomal damage in mammalian cells 

in vitro and in vivo. Phenolphthalein did not induce 

DNAdamagein repair-deficient strains of Bacillus 

subtilis (Kada et al., 1972; Fujita et al., 1976), nor 

was it mutagenic in Salmonella typhimurium (Bonin 

et al., 1981; Mortelmans et al., 1986). However, 
Introduction 25 

blood samples obtained from the mice at the end of 

the I3-week toxicity studywithphenolphthalein 

showed significant increasesinmicronucleated 

polychromatic erythrocytes andnormochromatic 

erythrocytes in malesandfemales(Dietz et al., 

1992). Efforts to confirm the genotoxicity of phenolphthalein in mice resulted in a series of experiments 

doses of phenolphthalein (120 mg/kg per day) were 

highly effective in inducing micronucleated erythrocytes when administered over a longer period of time 

(14 weeks) in Swiss (CD-l@) mice. Phenolphthalein 

alsoinduceddose-relatedincreasesin chromosomal 

aberrations in cultured Chinese hamster ovary cells 

treated in the presence of induced rat liver S9 (Witt 

that investigated the influence of various experimentalet al., 1995). These in vitro results provide additionparameters (route of administration, frequency of 

dosing, duration of exposure, carrier vehicle) on the 

phenolphthalein-inducedmicronucleus response in 

mice(Witt et al., 1995). It was concludedthat 

phenolphthalein at relatively high doses (greater than 

or equal to 2,000 mg/kg per day for at least 2 days) 

administered either in feed or by gavageinduced 

micronuclei in erythrocytes of B6C3F, mice and that 

thisindication of chromosomal damagecould be 

detectedin either bone marrow or blood. Lower 

al evidence of the genotoxicity of phenolphthalein. 

STUDYRATIONALE 

TheNationalCancer Institute nominated phenolphthalein for study because of its widespread use as 

a component of numerous over-the-counter laxative 

preparations, and thelackofadequatetesting for 

carcinogenicity in experimental animals. 
26 Phenolphthalein, NTP TR 465 
27 

MATERIALS AND METHODS 

PROCUREMENTAND 

CHARACTERIZATION 

OF PHENOLPHTHALEIN 

Phenolphthalein was obtained from Air Products and 

Chemicals, Inc. (Allentown, PA), in one lot 

(127-7809) and from Pharmco Laboratories, Inc. 

(Titusville, FL), twoin lots (P3186-D5and 

P9189-Jl). Lot 127-7809 was used during the

14-day studies, lot P3186-D5 was usedduringthe 

13-week studies, and lot P9189-J1 was usedduring 

the 2-year studies. Identity, purity, and stability 

analyses were conducted by the analytical chemistry 

laboratory, Midwest Research Institute (Kansas City, 

MO) (Appendix J). Reports onanalysesperformed 

in support of the phenolphthalein studies are on file 

at the National Institute of Environmental Health 

Sciences (NIEHS). 

All lots of the chemical, a yellow powder, were 

identified as phenolphthalein by infrared, ultraviolet/ 

visible, and nuclear magnetic resonance spectroscopy 

andbymelting point. The purity of all lots was 

determined by elemental analyses, Karl Fischer water 

analysis, functional group titration, thin-layer chromatography, and high-performance liquid chromatography. For lot 127-7809, elemental analyses for 

carbon, hydrogen, and oxygenwereinagreement 

with the theoretical values for phenolphthalein. Karl 

Fischer water analysisindicated 0.36% f 0.02% 

water.Functional group titration indicated a purity 

of 98.8% f 0.8%. Thin-layer chromatography by 

two systems indicated a major spot, a minor impurity, a trace impurity, and a slight race impurity. 

High-performance liquid chromatographywith one 

systemrevealed a major peakand five impurities 

with a combined area of 2.19% relative to the major 

peak area; high-performance liquid chromatography 

with a second system revealed a major peak and four 

impurities with a combined area of 2.28 % relative to 

themajorpeak area. The purity overall of 

lot 127-7809 was determinedtobe greater than or 

equal to 98 % . For lot P3 186-D5, elemental analyses 

for carbon and hydrogen were in agreement with the 

theoreticalvalues for phenolphthalein. Karl Fischer 

wateranalysisindicated 0.193% f 0.004% water. 

Functional group titration indicated a purity of 

99.5% f 0.4% . Thin-layer chromatography indicated a major spot, a minor impurity, and a trace 

impurity by one system and a major spot and a trace 

impurityby a second system. High-performance 

liquidchromatographyrevealed a major peakand 

threeimpuritieswith a combined area of 1.4% 

relative to the majorpeak area, and majorpeak 

comparisonsof lot P3 186-D5with lot 127-7809 

indicated a purity of 100.3% f 0.6% for 

lotP3186-D5relative to lot 127-7809. The overall 

purity of lot P3186-D5 was determined to be greater 

than or equal to 98 % . For lot P9189-J1, elemental 

analyses for carbon and hydrogen were in agreement 

with the theoretical values for phenolphthalein. Karl 

Fischerwateranalysisindicated 0.12% f 0.05% 

water.Functional group titration indicated a purity 

of 99.9% If: 0.5% . Thin-layer chromatography 

indicated a major spotand a trace impurity by one 

system and a major spot by a second system. Highperformance liquid chromatography revealed a major 

peakandthreeimpuritieswith a combined areaof 

1.2% relative to the major peak area, and major peak 

comparisons of lot P9189-J1 with lot 127-7809 

indicated a purity of 100.9% f 0.2% for 

lot P9189-J1 relative to lot 127-7809. The overall 

purity of lot P9189-J1 was determined to be greater 

than or equal to 99 % . 

Stability studies of lot 127-7809 were performed by 

theanalyticalchemistry laboratory using highperformanceliquid chromatography. These studies 

indicatedthatphenolphthaleinwas stable as a bulk 

chemical for 2 weeks when stored protected from 

light at temperatures up to 60" C. To ensure stability, the bulk chemical wastored protected from light 

at25 " C in sealed containers during the 13-week 

studiesand was stored protected from light at or 

below 27" C insealed containers during the 2-year 

studies. 
28 Phenolphthalein, NTP TR 465 

Stabilitywasmonitored during the 13-weekand 

2-year studies using high-performance liquid chromatography. No degradation of the bulk chemical 

was detected. 

PREPARATION AND ANALYSIS 

OF DOSEFORMULATIONS 

The dose formulations for all studies were prepared 

weeklymixingphenolphthaleinfeed by with 

(Table Jl). Stabilitystudies of the 6,000 ppm dose 

formulation of lot 127-7809 were performed by the 

analytical chemistry laboratory using high-performanceliquid chromatography. Thestabilityofthe 

dose formulation was confirmed for at least 2 weeks 

when stored at temperatures up to 25" C. Homogeneityandstability studies of the 500 ppm dose 

formulation of lot P3186-D5 were performed by the 

analytical chemistry laboratory using hghperformance liquid chromatography. Homogeneity 

was confirmed and the stability of the dose formulation was confirmed for at least 3 weeks when stored 

protected from light at room temperature. 

Periodic analyses of the dose formulations of phenolphthalein were conducted at the study laboratories 

using high-performance liquid chromatography. 

During the 13-week studies, the formulationswere 

analyzed every 6 weeks (Table 52). Duringthe 

2-year studies, the formulations wereanalyzed 

approximately every 8 weeks (Table J3). Allof the 

dose formulations analyzed during the13-week 

studies were within 10 % of the target concentrations. 

Of the dose formulations analyzed during the 2-year 

studies, 99% (122/123) were within 10% of the 

target concentrations; the one formulation out of 

specificationswasremixedand was within 10% of 

the target coricentration. During the 2-yearstudies, 

89% (40/45) of the animal room samples were within 

10%of the target concentrations. Results of periodic 

referee analyses performed during the13-week 

studies by the analytical chemistry laboratory agreed 

with the results obtained bythestudylaboratory 

(Table 54). 

14-DAY STUDIES 

Male and female F344/N rats and B6C3F, mice were 

obtained from Harlan Industries (Indianapolis, IN), 

andanimals were quarantined for 19 days. Before 

initiation of the studies, two male and two female rats 

and mice were randomly selected for parasite evaluation and gross observation for evidence of disease. 

Groupsof five male and five female rats and mice 

were fed diets containing 0, 6,250, 12,500, 25,000, 

50,000, or 100,000 ppm phenolphthalein. Feed and 

water were available ad libitum. Rats and mice were 

housed five per cage. Clinical findings were recordeddaily for rats and mice.Feed consumption 

wasrecorded on days 7 and 14 and whenever the 

feed had to be replenished due to an inadequate feed 

supply. Theanimalswereweighed initially, on 

day 7, and at the endof the studies. Details of the 

study design and animal maintenance are summarized 

in Table 1. 

At the end of the 14-day studies, a necropsy was 

performed on all rats and mice. Histopathologic 

examinationswere performed on one male control 

rat, two female control rats, two male control mice, 

onefemale control mouse, and two males and one 

female from the 100,000 ppm rats and mice. <Table 1 

lists the tissues and organs examined. 

WWEEK STUDIES 

The 13-week studies were conducted to evaluate the 

cumulativetoxic effects of repeated exposure to 

phenolphthaleinand to determine the appropriate 

exposures to be used in the 2-year studies. 

Male and female F344/N rats and B6C3F, mice were 

obtained from Taconic Farms (Germantown, NY). 

On receipt, the rats and mice were 4 weeks old. 

Animals were quarantined for 12 to 14 days (rats) or 

for 14 days (mice) and were 6 weeks old on the first 

dayofthestudies.Before initiation of the studies, 

five male and five female rats and mice were randomlyselected for parasite evaluation and gross 

observation for evidence of disease. At the end of 

the studies, serologic analyses were performed on 

five male and five female sentinel rats and on five 

male and five female control mice using the protocols 

of the NTP Sentinel Animal Program (Appendix M). 

Groupsof 10 male and 9 or 10 female rats and 

micewerefed diets containing 0, 3,000, 6,000, 

12,000, 25,000, or 50,000 ppm phenolphthalein. 

Additional groups of 10 male and 10 female rats 

designated for clinical pathology evaluations were 
Materials and Methods 29 

fed diets containing 0,3,000, 6,000, 12,000,25,000, 

or50,000 ppm phenolphthalein. Feedandwater 

were available ad libitum. Rats were housed five per 

cage, and micewerehousedindividually.Clinical 

findings were recordedweekly for rats and mice. 

Feed consumption was recorded The weekly. 

animalswereweighed initially, weekly, andatthe 

end of the studies. Details of the studydesignand 

animal maintenance are summarized in Table 1. 

On days 5 and 21, all clinicalpathology group rats 

wereanesthetizedwith carbon dioxide, and blood 

was collected from the retroorbital sinus for 

hematologyandclinicalchemistryanalyses. At the 

end of the 13-week study, all core study ratswere 

anesthetizedwith carbon dioxide, and bloodwas 

collected from the retroorbital sinus for hematology 

and clinical chemistry Blood analyses. for

hematology, determinations wasplacedintubes 

containing potassium EDTAastheanticoagulant. 

Hematology evaluations for hematocrit, hemoglobin, 

erythrocyte and reticulocyte counts, mean cellvolume, mean cell hemoglobin, and leukocytecounts 

and differentials were performed according to standard hematology methods using a Baker 7000 (Baker 

Instruments, Allentown, PA)hematology.analyzer. 

Platelet counts were performed using a Baker 

Model 810 (Baker Instruments, Allentown,PA) 

platelet analyzer. In addition, erythrocyte and 

platelet morphology were evaluated microscopically. 

For clinical chemistry analyses, samples were collectedinplastic centrifuge tubes. Serumsamples 

wereanalyzedusing a Centrifichem 400 (Baker 

Instruments, Allentown, PA). The hematology and 

clinical chemistry parameters measured are listed in 

Table 1. 

At the endofthe13-week studies, samples were 

collected from rats (core study) and mice exposed to 

0, 12,000,25,000,or 50,000 ppmphenolphthalein 

for sperm morphology and vaginal cytology evaluations. The parameters evaluated are listedin 

Table 1. Methods used werethosedescribedin 

NTP's sperm morphology andvaginalcytology 

evaluations protocol (NTP, 1984a). For 7 consecutive days prior to the end of the studies, the vaginal 

vaults of the females were moistened with saline, if 

necessary,andaspirated ,samples of vaginal fluids 

and cells were transferred to slides, air dried, fixed, 

andstained.Relative numbers of leukocytes, nucleatedepithelial cells, and large squamous cellswere 

determinedtoascertain estrus cycle stage (i.e., 

diestrus, proestrus, estrus, or metestrus). Male 

animalswereevaluated for sperm morphology, 

count, and motility. The right testis and right epididymis were isolated, weighed, and fixed and preserved 

for histopathology. The tail of the epididymis (cauda 

epididymis)wasthenremoved from the epididymal 

body (corpus epididymis) and weighed.Testyolk 

(rats) or modified Tyrode's buffer (mice) was applied 

to slides, and a small incision was made at the distal 

border of the cauda epididymis. The sperm effluxing 

from the incision were dispersed in the buffer on the 

slides, and thenumbersofmotileandnonmotile 

spermatozoa were counted in five fields per slide by 

two observers. Following completion of the sperm 

motility estimates, each right cauda epididymis was 

placedinbufferedsaline solution. Caudaewere 

finelyminced,andthetissuewasincubated in the 

saline solution. Sampleswerethenremoved for 

morphology evaluation, and the remainder were heat 

fixedat 65" C. Sperm density wasdetermined 

microscopically using a hemacytometer. Four sperm 

morphologyslideswere prepared for each animal 

evaluated. An aliquot of killed sperm suspension was 

stained in a test tube, spread on a microscope slide, 

coverslipped, and examined. Results of reproductive 

tissue evaluations and estrous cycle characterization 

are given in Appendix I. 

A necropsy was performed on all core study rats and 

on all mice. The brain, heart, right kidney, liver, 

lung, right testis, and thymus were weighed. Tissues 

for microscopic examinationwerefixed and preservedin 10% neutral buffered formalin, processed 

and trimmed, embedded in paraffin, sectioned to a 

thickness of 5 to 6 pm, and stained with hematoxylin 

and eosin. A complete histopathologic examination 

was performed on control and 50,000 ppm groups of 

rats and mice. Additionally, the liver (females), lung 

(males), and ovaries of rats were examined to a no 

effect level; the bone marrow and spleen (males) of 

micewereexamined to a no effect level. Table 1 

lists the tissues and organs routinely examined. 
30 Phenolphthalein, NTP TR 465 

%YEAR STUDIES 

Study Design 

Groups of 50 male and 50 female rats were fed diets 

containing0, 12,000,25,000, or50,000 ppm phenolphthalein. Groups of 50 maleand 50 femalemice 

werefed diets containing 0, 3,000,6,000, or 

12,000 ppm phenolphthalein. 

Source and Specification of Animals 

Male and female F344/N rats and B6C3FI mice were 

obtained from Taconic Farms (Germantown, NY) for 

use in the 2-year studies. Rats were quarantined for 

13 days (males) or 15 days (females) before the 

beginning of the study. Micewerequarantined for 

12 days (males) or13 days (females) before the 

beginning of the study. Five male andfivefemale 

rats and micewereselected for parasite evaluation 

and gross observation of disease. Male and female 

rats and female mice were approximately7 weeks old 

at the beginning of the studies; male micewere 

approximately 6 weeks oldatthebeginningofthe 

study. Serology samples were collected from up to 

five male and five female rats and mice for viral 

screening before the beginning of the study. Serology samples were collected from up to five male and 

five female sentinel rats at 5, 9, 12, 17, 18, and 

23 months and from up to three male and three 

female sentinel rats and up to five maleand five 

female 25,000 ppm rats at theendofthe study. 

Serology samples were collected from up to five male 

and five female sentinel mice at 5, 12, and 18 months 

and from up to five male and five female 6,000 ppm 

mice at the end of the study. The health of the 

animals was monitored during the studies according 

to the protocols of the NTP Sentinel Animal Program 

(Appendix M) . 

Animal Maintenance 

Rats were housed five per cage and micewere 

housed individually. Feed andwaterwereavailable 

ad libitum. Feed consumption was measured approximately every 4 weeks by cage. Cagesandracks 

were rotated every 2 weeks. Further details of 

animalmaintenance are given in Table 1. Information on feed composition and contaminants is 

provided in Appendix L. 

Clinical Examinations and Pathology 

Allanimalswereobservedtwice daily. Clinical 

findingswererecorded every 4 weeks, andbody 

weightswererecorded initially, weekly for the first 

13 weeks, and every 4 weeks thereafter. 

Onthe last day of the 2-year studies, blood was 

collected from the retroorbital sinus of three male 

and three female anesthetized rats and mice from 

each exposed group at five time points (6 and 9 a.m. 

and 1, 4, and 9 p.m.) for the determination of plasma 

concentrations of total phenolphthalein (free and 

conjugated). The phenolphthalein concentration was 

notmeasuredin the plasma of control animals 

because it was not expected to be a normal constituent of plasma and was not detected in plasma from 

untreated animals during development of the analytical procedure. Dosedfeedwasavailable ad libitum 

during the collection period. The plasma was stored 

at -20" C or lower until analysis. Plasmasamples 

wereanalyzedusing a procedure developed during 

the single-dose toxicokinetic studies (Appendix 0)for 

the analysis of concentrations of total phenolphthalein 

(free and conjugated). Plasma samples were treated 

with @glucuronidase and sulfatase enzyme preparation. Solid-phase extraction was used to isolate 

phenolphthalein, and the samples were analyzed using 

high-performanceliquid chromatography. The 

average plasma concentrations of total phenolphthaleinand standard deviations were calculated. The 

logarithms of these values were plotted as a function 

of time. Results of analyses of plasma concentrations 

for total phenolphthaleinat the end of the 2-year 

studies are given in Appendix H. 

A complete necropsyand microscopic examination 

were performed on all rats and mice. At necropsy, 

all organs and tissueswere examined for grossly 

visible lesions, and all major tissues were fixed and 

preserved in 10% neutral buffered formalin, 

processedand trimmed, embedded in paraffin, 

sectioned to a thickness of 5 to 6 pm, and stained 

with hematoxylin and eosin for microscopic examination. For all paired organs (i.e., adrenal gland, 

kidney, ovary), samples from each organ were 

examined. For the xtended evaluation of renal 

tubule proliferative lesions, kidneys ofmaleand 
Materials and Methods 31 

female rats were step sectionedat 1 mm intervals, 

and five or six additional sections were obtainedfrom 

eachanimal. Tissues examined microscopically are 

listed in Table 1. 

Microscopic evaluations were completed by the study 

laboratory pathologist, and the pathology data were 

entered into the Data Toxicology Management

System. The microscopic slides, paraffin blocks, and 

residual wet tissues were sent tothe NTP Archives 

for inventory, slide/block match, andwettissue 

audit. The slides, individual animaldata records, 

and pathology tables were evaluated by an independent quality assessment laboratory. Theindividual 

animal records and tables were compared for accuracy, the slide and tissue counts were verified, and the 

histotechnique was evaluated. For the 2-year studies, 

a quality assessment pathologist reviewed the adrenal 

gland and kidney of male and female rats; the seminalvesicleandspleen of male rats; the ovaryof 

female rats; the bone, bone marrow, liver, thymus, 

lymph nodes, and spleen of male andfemalemice; 

thetestis of male mice;andthe ovary, pituitary 

gland, thyroid gland, and uterus of female mice. 

The quality assessment report and the reviewed slides 

weresubmitted to the NTP Pathology Working 

Group (PWG) chairperson, who reviewed all tissues 

andaddressedanyinconsistenciesinthediagnoses 

madebythe laboratory and qualityassessment 

pathologists. Representativehistopathologyslides 

containingexamples of lesionsrelatedtochemical 

administration, examples ofdisagreementsindiagnoses between the laboratory and quality assessment 

pathologist, or lesions of generalinterestwere 

presented by the chairperson to the PWG for review. 

The PWG consisted of the quality assessment pathologistand other pathologists experiencedinrodent 

toxicologic pathology. This group examined the 

tissueswithoutanyknowledge of dose groups or 

previously rendered diagnoses. WhenthePWG 

consensus differed from the opinion of the laboratory 

pathologist, the diagnosis was changed. Thus, the 

final diagnoses represent a consensus of quality 

assessment pathologists, the PWG chairperson, and 

thePWG.Details of these reviewprocedureshave 

been described, in part, by MaronpotandBoorman 

(1982) and Boorman et al. (1 985). For subsequent 

analyses of the pathology data, the diagnosed lesions 

for each tissue type were evaluatedseparately or 

combined according to the guidelines of McConnell 

et al. (1986). 

STATISTICAL METHODS 

Survival Analyses 

Theprobabilityof survival was estimatedbythe 

product-limit procedure of Kaplan and Meier (1958) 

andispresentedinthe form of graphs. Animals 

found dead of other than naturalcauses or missing 

werecensored from the survival analyses; animals 

dying from naturalcauseswere not censored. 

Statisticalanalyses for possible dose-related effects 

on survival used Cox’s (1972) method for testing two 

groups for equality and Tarone’s (1975) life table test 

to identify dose-related trends. All reported P values 

for the survival analyses are two sided. 

Calculation of Incidence 

The incidences of neoplasms or nonneoplastic lesions 

as presented in Tables Al, A5, B1, B5, C1, C5, Dl, 

and D5 are given as the number of animals bearing 

suchlesionsat a specific anatomic site and the 

number of animals with that site examined microscopically. For calculation of statistical significance, 

theincidencesofmostneoplasms (Tables A3, B3, 

C3, and D3) and all nonneoplastic lesions are given 

asthenumbers of animals affectedateach site 

examinedmicroscopically. However, when macroscopic examination was required to detect neoplasms 

incertaintissues (e.g., harderian gland, intestine, 

mammary gland, andskin) before microscopic 

evaluation, or when neoplasms had multiple potential 

sites of occurrence (e.g., leukemia or lymphoma), the 

denominatorsconsistofthenumberofanimals on 

which a necropsy was performed. Tables A3, B3, 

C3, and D3 also give the survival-adjusted neoplasm 

rate for each group and each site-specific neoplasm, 

i.e., theKaplan-Meierestimateof the neoplasm 

incidence that would have been observed at the end 

of the study in the absence of mortality from all other 

competing risks (Kaplan and Meier, 1958). 

Analysis of Neoplasm Incidences 

Themajority of neoplasms inthese studies were 

considered to be incidental to the cause of death 

or not rapidly lethal. Thus, the primary statisticalmethodusedwas logistic regression analysis, 

whichassumedthatthediagnosedneoplasmswere 

discovered as the result of death from an unrelated 
32 Phenolphthalein, NTP TR 465 

cause and thus did notaffecttheriskofdeath. In 

this approach, neoplasm prevalence was modeled as 

a logistic function of chemical exposure and time. 

Both linear and quadratic terms intimewere incorporated initially, and the quadratic term waseliminated if the fit of themodelwasnotsignificantly 

enhanced. The neoplasm incidences of exposed and 

control groups were compared on the basis of the 

likelihood score test for the regression coefficient of 

dose. This method ofadjusting for intercurrent 

mortality is the prevalence analysis of Dime and 

Lagakos (1983), further described and illustrated by 

Dime and Haseman (1986). Whenneoplasms are 

incidental, this comparison ofthetime-specific 

neoplasm prevalences also provides a comparison of 

the time-specific neoplasm incidences (McKnight and 

Crowley, 1984). 

In addition to logistic regression, other methods of 

statistical analysis were used, and the results of these 

tests are summarizedintheappendixes.These 

methods include the life table test (Cox, 1972; 

Tarone, 1975), appropriate for rapidly lethalneoplasms, and the Fisher exact testandthe CochranArmitage trend test (Armitage, 197 1; Gart et al., 

1979), procedures based on the overall proportion of 

neoplasm-bearing animals. 

Tests of significance included pairwise comparisons 

of each exposed group with controls and a test for an 

overall dose-related trend. Continuity-corrected tests 

were used in the analysis of neoplasm incidence, and 

reported P values are one sided. The procedures 

described in the preceding paragraphs were also used 

to evaluate nonneoplastic selected lesions. For

further discussion of these statistical methods, refer 

to Haseman (1984). 

Analysis of Nonneoplastic Lesion Incidences 

Becauseallnonneoplastic lesions in this studywere 

considered to be incidental to the cause of death or 

not rapidly lethal, the primary statistical analysis used 

was a logistic regression analysis inwhichnonneoplasticlesion prevalence was modeledas a logistic 

function of chemical exposure and time. 

Analysis of Continuous Variables 

Two approaches were employed to assess the significance of pairwise comparisons between exposed and 

control groups in the analysis of continuous variables.Organandbodyweight data, which have 

approximatelynormal distributions, were analyzed 

using the parametric multiple comparison procedures 

of Dunnett (1 955) andWilliams (197 1, 1972). 

Clinical chemistry, hematology, spermatid, and 

epididymalspermatozoal data, which havetypically 

skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley 

(1 977) and Dunn (1964). Jonckheere’s test

(Jonckheere, 1954) wasused to assess the significance of thedose-related trends and to determine 

whether a trend-sensitive test (Williams’ or Shirley’s 

test) was more appropriate for pairwise comparisons 

than a test that does not assume a monotonic doserelatedtrend (Dunnett’s or Dunn’s test). Prior to 

analysis, extreme values identified by the outlier test 

of Dixon and Massey (1 95 1) were examined by NTP 

personnel, and implausible values were eliminated 

from the analysis.Average severity values were 

analyzed for significance using the Mann-Whitney U 

test (Hollanderand Wolfe, 1973). Because the 

vaginal cytology data are proportions (the proportion 

ofthe observation period that an animalwasin a 

given estrous stage), an arcsine transformation was 

used to bring the data into closer conformance with 

a normality assumption. Treatment effects were 

investigated by applying a multivariate analysis of 

variance (Morrison, 1976) to the transformed data to 

test for simultaneous equality of measurements across 

exposure concentrations. 

Historical Control Data 

Although the concurrent control group is always the 

first and most appropriate control group used for 

evaluation, historical control data can be helpful in 

the overall assessment ofneoplasmincidencein 

certain instances. Consequently, neoplasm incidences 

fromthe NTP historical control database (Haseman 

et al., 1984, 1985) are included inthe NTP reports 

for neoplasms appearing to show compound-related 

effects. 

QUALITYASSURANCEMETHODS 

The13-weekand 2-year studies were conductedin 

compliance with Food and Drug Administration Good 

LaboratoryPracticeRegulations (21 CFR, Part 58). 

In addition, as records from the 2-year studies were 
Materials and Methods 33 

submitted to the NTP Archives, these studieswere 

audited retrospectively by anindependentquality 

assurance contractor. Separate covering audits

completeness and accuracy of .the pathology data, 

pathology specimens, final pathology tables, and a 

draft of this NTP Technical Report were conducted. 

Audit procedures and findings are presented inthe 

reports and are on file at NIEHS. The audit findings 

werereviewedandassessedbyNTP staff, so all 

commentshadbeenresolved or wereotherwise 

addressed during the preparation of this Technical 

Report. 

GENETICTOXICOLOGY 

The genetic toxicity of phenolphthalein was assessed 

bytestingtheabilityofthechemicaltoinduce 

mutationsin various strains of Salmonella 

typhimurium, sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary 

cells, andincreasesinthefrequency of micronucleated erythrocytes in mouseperipheral blood. 

The protocols for these studiesandtheresults are 

given in Appendix E. 

The genetic toxicity studiesofphenolphthalein are 

part of a larger effort by theNTP to develop a 

database that would permit the evaluation of carcinogenicityinexperimentalanimalsfromthe structure 

and responses of the chemical in short-term in vitro 

and invivo genetic toxicity tests. Thesegenetic 

toxicitytestswere originally developed to study 

mechanisms of chemically induced DNA damage and 

topredictcarcinogenicityin animals, based on the 

electrophilictheoryofchemical carcinogenesis and 

the somatic mutation theory (Miller and Miller, 1977; 

Straus, 1981; Crawford, 1985). 

There is a strong correlation between a chemical’s 

potentialelectrophilicity (structural alert to DNA 

reactivity), mutagenicity in Salmonella, and carcinogenicityin rodents. The combination of electrophilicityand Salmonella mutagenicityishighly 

correlatedwiththeinduction of carcinogenicity in 

rats and mice and/or at multiple tissue sites (Ashby 

and Tennant, 1991). Other in vitro genetictoxicity 

tests do not correlate well with rodent carcinogenicity 

(Tennant et al., 1987; Zeiger et al., 1990), although 

these other tests can provide information on the types 

of DNA and chromosome effects that can be induced 

by the chemical being investigated. Data from NTP 

studies show that a positive response in Salmonella is 

currently the most predictive in vitro test for rodent 

carcinogenicity (89% of the Salmonella mutagens 

were rodent carcinogens), and that there is no complementarity among thein vitro genetic toxicity tests. 

That is, nobattery of testshatincludedthe 

Salmonella testimprovedthe predictivity of the 

Salmonella test alone. The predictivity for carcinogenicity of a positive response in bone marrow 

chromosome aberration or micronucleus tests is not 

yet defined. 
34 Phenolphthalein, NTP TR 465 

TABLE1 

Experimental Design and Materials and Methods in the Feed Studies of Phenolphthalein 

14-Day Studies 

Study Laboratory 

Cannon Laboratories, Inc. 

(Reading, PA) 

Strain and Species 

F344lN rats 

B6C3F, mice 

Animal Source 

Harlan Industries 

(Indianapolis, IN) 

Time Held Before Studies 

19 days 

Average Age When Studies Began 

Not available 

Date of First Dose 

4 June 1979 

Duration of Dosing 

14 days 

Date of Last Dose 

17 June 1979 

Necropsy Dates 

Rats: 19-20 June 1979 

Mice: 19 June 1979 

Average Age at Necropsy 

Not available 

13-Week Studies 

Microbiological Associates, Inc. 

(Bethesda, MD) 

F344lN rats 

B6C3F, mice 

Taconic Farms 

(Germantown, NY) 

Rats: 12 to 14 days 

Mice: 14 days 

6 weeks 

Rats: 28 April 1987 

Mice: 30 April 1987 

13 weeks 

Rats: 28-29 July 1987 (core study) or 

18 May 1987 (clinical pathology 

groups)

Mice: 30-31 July 1987 

Rats: 28-29 July 1987 

Mice: 30-31 July 1987 

19 weeks 

2-Year Studies 

TSI Mason Laboratories, Inc. 

(Worcester, MA) 

F344lN rats 

B6C3F, mice 

Taconic Farms 

(Germantown, NY) 

Rats: 13 days (males) or 

15 days (females) 

Mice: 12 days (males) or 

13 days (females) 

7 weeks (male and female rats and 

female mice) or 6 weeks (male mice) 

Rats: 13 March (males) or 

15 March (females) 1991 

Mice: 27 November (males) or 

28 November (females) 1990 

104 weeks (males) or 105 weeks 

(females) 

Rats: 10 March (males) or 

18-19 March (females) 1993 

Mice: 24-25 November (males) or 

3-4 December (females) 1992 

Rats: 10 March (males) or 

18-19 March (females) 1993 

Mice: 24-25 November (males) or 

3-4 December (females) 1992 

110 weeks (male mice), 111 weeks 

(male rats), or 112 weeks (female rats 

and mice) 

Sue of Study Groups 

5 males and 5 females 10 males and 9 or 10 females 50 males and 50 females 
Materials and Methods 35 

TABLE1 

Experimental Design and Materials and Methods in the Feed Studiesof Phenolphthalein(continued) 

14-Day Studies 

Method of Distribution 

Animals were distributed randomly into 

groups of approximately equal initial 

mean body weight 

Animals per Cage 

5 

Method of Animal Identification 

Ear tag 

Diet 

NIH-07 open formula meal diet 

(Zeigler Brothers, Inc., Gardners, PA), 

available ad liblrum, changed daily 

Water Distribution 

Tap water (Reading municipal supply) 

via water bottles changed weekly, 

available ad libitum 

Cages 

Polycarbonate cages with stainless steel 

tops, changed twice per week 

Bedding 

Absorb-Dri@ hardwood chips, changed 

twice per week 

Cage Filters 

Reemay@ spun-bonded polyester filter 

sheet (Andico, Birmingham, AL) 

Racks 

Racks changed every 2 weeks 

13-WeekStudies 

Same as 14-day studies 

Rats: 5 

Mice: 1 

Ear punch and toe clip 

Same as 14-day studies, changed 

weekly 

Tap water (Bethesda municipal supply) 

via automatic watering system 

(Edstrom Industries, Inc., Waterford, 

WI), available'ad libirum, changed 

every 2 weeks 

Polycarbonate suspended cages (Lab 

Products, Inc., Rochelle Park, NJ), 

changed twice per week 

Beta-chips@ heat-treated hardwood 

chips (Northeastern Products Corp., 

Warrensburg, NY), changed twice per 

week 

Spun-bonded polyester filter sheet 

(Snow Filtration Co., Cincinnati, OH), 

changed every 2 weeks 

Stainless steel racks (Lab Products, 

Inc., Rochelle Park, NJ), changed 

every 2 weeks 

2-Year Studies 

Same as 14-day studies 

Rats: 5 

Mice: 1 

Tail tattoo 

Same as 14day studies, changed twice 

weekly 

Tap water (Worcester municipal 

supply) via automatic watering system 

(Edstrom Industries, Inc., Waterford. 

WI), available ad libirum, changed 

every 2 weeks 

Polycarbonate, solid-bottom cages (Lab 

Products, Inc., Rochelle Park, NJ), 

changed 3 times per week (male rats), 

twice per week (female rats), or weekly 

(male and female mice) 

Sani-chips@ heat-treated hardwood 

chips (P.J. Murphy Forest Products, 

Montville, NJ), changed 3 times per 

week (male rats), twice per week 

(female rats), or weekly (male and 

female mice) 

Nonwoven fiber filters (Snow 

Filtration, Cincinnati, OH), changed 

every 2 weeks 

Same as 13-week studies 
36 Phenolphthalein, NTP TR 465 

TABLE1 

Experimental Design and Materials and Methods in the Feed Studies of Phenolphthalein(continued) 

14-Day Studies 

Animal Room Environment 

Temperature: 22" to 24" C 

Relative humidity: 45 % to 55 % 

Fluorescent light: 12 hours/day 

Room air: 10 to 15 changes/hour 

Doses 

0, 6,250. 12,500. 25,000. 50,000, or 

100.000 ppm in feed, available 

ad libitum 

Type and Frequency of Observation 

Observed once daily; animals were 

weighed initially, on day 7, at the endweighed 

of the studies; clinical findings were 

recorded daily. Feed consumption was 

recorded by cage on days 7 and 14 and 

whenever to hadfeedthe be 

replenished due to an inadequate feed 

supply. 

Method of Sacrifice 

Asphyxiation with carbon dioxide 

Necropsy 

Necropsy was performed on all 

animals. 

Clinical Pathology 

None 

13-Week Studies 

Temperature: 21 to 23" C 

Relative humidity: 50% to 67% 

Fluorescent light: 12 houdday 

Room air: minimum of 

10 changeslhour 

0, 3,000, 6,000, 12,000, 25,000. or 

50,000 ppm in feed, available 

ad libitum 

Observed twice daily; animals were 

initially, weekly, and at the 

end ofthe studies; clinical findings 

were recorded weekly. Feed 

consumption was recorded weekly by 

cage. 

Asphyxiation with carbon dioxide 

Necropsy was performed on all core 

study rats and on all mice. Organs 

weighed were brain, heart, right 

kidney, liver, lung, right testis, and 

thymus. 

Blood was collected from the 

retroorbital sinus of rats in the clinical 

pathology groups on days 5 and 21 and 

of core study rats at the end of the 

study for hematology and clinical 

chemistry. 

Hematology: hematocrit, hemoglobin, 

erythrocyte and reticulocyte counts, 

mean cell volume, mean cell 

hemoglobin, mean cell hemoglobin 

concentration, platelet count, and total 

leukocyte counts and differentials. 

Clinical chemistry: urea nitrogen, 

creatinine, total protein, albumin, 

alanine aminotransferase, alkaline 

phosphatase, creatine kinase, sorbitol 

dehydrogenase, and bile acids. 

2-Year Studies 

Temperature: 21" to 23" C 

Relative humidity: 37% to 58% 

F!uorescent light: 12 houdday 

Room air: 10changedhour 

Rats: 0, 12,000. 25.000, or 

50,000 ppm in feed, available 

ad libitum 

Mice: 0, 3.000, 6,OOO, or 12,000 ppm 

in feed, available ad libitum 

Observed twice daily; animals were 

weighed initially, weekly for 13 weeks, 

monthly thereafter, and at the end of 

the studies; clinical findings were 

recorded every 4 weeks. Feed 

consumption was recorded 

approximately monthly by cage. 

Asphyxiation with carbon dioxide 

Necropsy was performed on all 

animals. 

None 
Materials and Methods 37 

TABLE1 

Experimental Design and Materials and Methods in the Feed Studies of Phenolphthalein(continued) 

14-Day Studies 

Histopathology 

Complete histopathology was 

performed on one male rat and 

two female rats from the control 

groups, on two male mice and 

one female mouse from the control 

groups, and on two males and 

one female from the 100.000 ppm rats 

and mice. In addition to gross lesions 

and tissue masses, the tissues examined 

included: adrenal glands, bone 

marrow, brain, ears (external and 

middle), esophagus, eyes, gallbladder 

(mice), heart, large intestine (colon and 

rectum), small intestine (duodenum, 

jejunum, and ileum) kidneys, larynx, 

liver, lungs (and mainstem bronchi), 

lymph nodes (mandibular and 

mesenteric), mammary gland, nose, 

ovaries, pancreas, parathyroid glands, 

pituitary gland, prostate gland, rib (and 

costochondral junction), salivary gland, 

skin, spinal cord (and sciatic nerve), 

spleen, stomach, testes (with seminal 

vesicle), thigh muscle, thymus, thyroid 

glands, trachea, urinary bladder, and 

uterus. 

Sperm Morphology and Vaginal Cytology 

None 

Determinations of Total Phenolphthalein 

None 

13-Week Studies 

Complete histopathology was 

performed on 0 and 50,000 ppm rats 

andmice.Inaddition to gross lesions 

and tissue masses, the tissues examined 

included: adrenal gland, brain, clitoral 

gland (rats), esophagus, femur 

(including marrow), heart, gallbladder 

(mice), large intestine (cecum, colon, 

and rectum), small intestine 

(duodenum, jejunum, and ileum), 

kidney, liver, lungs (and mainstem 

bronchi), lymph nodes (mandibular and 

mesenteric), mammary gland, nose, 

ovaries, pancreas, parathyroid gland, 

pituitary gland, preputial gland (rats), 

prostate gland, salivary gland, skin, 

spleen, stomach (including forestomach 

and glandular stomach), testes (with 

epididymis and seminal vesicle), 

thymus, thyroid gland, trachea, urinary 

bladder, and uterus. In rats, the liver 

(females), lung (males), and ovaries 

were examined to a no effect level. In 

mice, the bone marrow and spleen 

(males) were examined to a no effect 

level. 

Evaluations 

At the end of the study, sperm samples 

were collected from male rats (core 

study) and male mice in the 0, 12,000. 

25,000. and 50,000 ppm groups for 

sperm morphology evaluations. The 

parameters evaluated included sperm 

density, morphology, and motility. 

The right cauda, right epididymis, and 

right testis were weighed. Vaginal 

fluid samples were collected for up to 

7 consecutive days prior to the end of 

the studies from female rats (core 

study) and female mice in the 0, 

12,000. 25.000, and 50.000 ppm 

groups. The parameters evaluated 

were relative frequency of estrous 

stages and estrous cycle length. 

inPlasma 

None 

2-Year Studies 

Complete histopathology was 

performed on all rats and mice. In 

addition to gross lesions and tissue 

masses, the tissues examined included: 

adrenal glands, brain (3 sections), 

clitoral gland, esophagus, femur 

(including marrow), heart, large 

intestine (cecum, colon, and rectum), 

small intestine (duodenum, jejunum, 

and ileum), kidneys, liver, lung (and 

mainstem bronchi), lymph nodes 

(mandibular and mesenteric), mammary 

gland, nose (3 sections), ovary, 

pancreas, parathyroid gland, pituitary 

gland, preputial gland, prostate gland, 

salivary glands, spleen, stomach 

(forestomach and glandular), testis 

(with epididymis and seminal vesicle), 

thymus, thyroid gland, trachea, urinary 

bladder, and uterus. 

None 

On the last day, blood was taken from 

three animals from each exposed group 

at 6 and 9 a.m. and 1, 4, and 9 p.m. 

for determination of plasma 

concentrations of total phenolphthalein. 
38 Phenolphthalein, NTP TR 465 
39 

RESULTS 

RATS 

14-DAY STUDY 

All rats survived to the end of the study(Table 2). 

The final mean body weights of all exposed groups 

of rats were similar to those of the controls. Feed 

consumption by 100,000ppm males and females was 

greater than that bythe controls; feed consumption 

byall other exposed groups of rats was similar to 

that by the controls. Dietary levels of 6,250, 12,500, 

25,000, 50,000, or 100,000ppmphenolphthalein 

TABLE2 

resulted in average daily doses of approximately 500, 

1,000, 4,500,10,500 2,000, or mg phenolphthaleidkg body weight to males and 500, 1,000, 

2,000, 4,000, or 11,000 mg/kg to females. The 

fecesof 100,000 ppm malesandfemaleswere 

abnormally lighter in color than those of the other 

groups. This discoloration was observed on day 3 

and continued throughout the study. There were no 

chemical-related gross or microscopic lesions observed in rats. 

Survival, Mean Body Weights, and Feed Consumption of Rats in the 14-Day Feed Study 

of Phenolphthalein 

Final Weight 

Mean Body Weightb (g) Feed Relative 

Dose Survivala Initial Final to Controls Cnsumption'

(PPd ("/.I Week 1 Week 2 

Male 

0 515 123 f 9 174 f 12 12 14 

6,250 515 128 f 13 13 168 f 13 97 11 

12,500 515 

25.000 515 

125 f 6 167 f 10 96 11 

125 f 8 13 167 f 11 96 11 

50,000 515 127 f 11 15 169 f 10 97 12 

100,000 515 127 f 13 18 167 f 12 96 13 

10 10 

Female 

0 515 109 f 4 136 f 4 

6,250 515 110 f 6 10 136 f 8 100 9 

12.500 515 110 f 6 10 133 f 6 98 10 

25,000 515 108 f 5 9 130 f 6 96 9 

50,000 515 111 f 7 10 135 f 7 99 10 

100.000 515 111 f 6 15 132 f 5 97 12 

a Number of animals surviving at 14 dayslnumber initially in group. Differences from the control group are not significant by Williams' 

or Dunnett's test. 

Weights are given as mean f standard deviation. ' Feed consumption is expressed as grams of feed consumed per animal per day. 

13 
40 Phenolphthalein, NTP TR 465 

ISWEEK STUDY 

All rats survived to the end of the study(Table 3). 

Thefinalmeanbodyweight of the 50,000 ppm 

females and the body weight gains of the 25,000 and 

50,000 ppmfemalesweresignificantlylowerthan 

thoseof the controls. The final mean body weights 

andbodyweightgainsofallotherexposed groups 

were similar to those of the controls. Feed consumptionbyexposed groups was similartothatbythe 

controls. Dietarylevels of 3,000, 6,000, 12,000, 

25,000, or 50,000 ppm phenolphthalein resulted in 

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. There was no cathartic action or any 

other clinical finding attributedto exposure to 

phenolphthalein. 

The few differences in the hematology and clinical 

chemistry parameters were sporadic and werenot 

considered to be chemical related (Table Gl). 

TABLE3 

The percentage of motile sperm in the 12,000 ppm 

maleswasignificantly greater than that in the 

controls, but no other significant differences in sperm 

morphology or vaginal cytology were observed 

between exposed and control groups (Table 11). 

The absolute and relative liver weights of 25,000 and 

50,000 ppmmalesand the relative liver weights of 

12,000 ppm malesandof 25,000 and 50,000 ppm . 

females were significantly greater than those of the 

controls (Table Fl). No chemical-related gross or 

microscopic lesions were observed. 

Dose Selection Rationale: Based on the absenceof 

histopathologic effects after 13 weeks of exposure to 

up to 50,000 ppm phenolphthalein, the highest 

phenolphthalein exposure concentration selected for 

the 2-year feed study in rats was 50,000 ppm. 

Survival, Mean Body Weights, and Feed Consumption of Rats in the 13-Week Feed Study 

of Phenolphthalein 

Final Weight 

Mean Body Weightb(g) Feed Relative 

Dose Survivala Initial Final Change to Consumption' Controls 

(PPd (%I Week 1 Week 13 

Male 

0 10110 132 f 4 351 f 5 219 6 16 18 

3.000 10110 128 f 3 346 f 6 218 f 6 99 16 18 

6,000 10110 124 f 2 349 f 6 225 f 5 99 15 17 

12,000 10110 129 f 3 345 f 7 216 f 6 98 16 18 

25.000 10110 136 It 4 344 f 8 208 f 6 98 16 19 

50.000 10110 129 f 3 334 f 5 205 It 5 95 15 20 

Female 

0 10/10 109 f 3 210 f 2 101 f 2 13 11 

3.000 10110 105 rt 2 202 f 5 97 f 4 97 12 11 

6,000 10110 105 f 2 208 f 2 103 f 2 99 13 12 

12,000 919 107 f 1 206 f 3 100 f 2 98 12 11 

25,000 10110 109 f 3 198 f 2 90 f 2* 95 12 12 

50.000 10110 106 f 3 198 f 3* 92 f 3* 95 12 12 

* Significantly different (P~0.05)from the control group by Dunnett's test 

a Number of animals surviving at 13 weeks/number initially in group 

Weights and weight changes are given as mean f standard error. 

Feed consumption is expressed as grams of feed consumed per animal per day. 
Results 41 

2-YEAR STUDY from about week 16 until theend of the study 

(Figure 3, Tables 5 and 6). Feed consumption by all Survival exposed groups of rats was similar to that by the Estimates of 2-year survival probabilities for male controls (Tables K1 and K2). Dietary levels of andfemale rats are shown inTable 4 andinthe 12,000,25,000,or 50,000 ppm phenolphthalein Kaplan-Meier survival curves (Figure 2). Survival of resulted in average daily doses of approximately 500, exposed males and females was similar to that of the 1,000, or 2,000 mg phenolphthaleidkg body weight controls. to males and 500, 1,000, or 2,500 mg/kg to females. 

The estimated equivalentdaily doses of phenol- Body Weights, Feed and Compound phthalein for the last year of the study ranged from 

Consumption, and Clinical Findings 430 to 1,900 mg/kg for male ratsand from 480 to 

The mean body weights of exposed males were less 2,100 mg/kg for female rats. Clinical findings 

than those of the controls through most of the second attributed to phenolphthalein exposure included thin 

year of the study, and themeanbodyweights of appearance and ruffled fur in all exposed groups of 

exposed females were less than those of the controls males. 

TABLE4 

Survival of ,Rats in the 2-Year Feed Study of Phenolphthalein 

12,000 ppm 25,000 ppm 50,000 ppm 

Male 

Animals initially in study 50 50 50 50 

Moribund 15 15 13 16 

Natural deaths 14 20 22 21 

Animals surviving to study termination 21 15 1gd 13 

Percent probability of survival at end of studya 42 30 30 26 

Mean survival (days)b 640 652 658 662 

Survival analysis' P=O.463 P=O.492 P=O.626 P=O.405 

Female 

Animals initially in study 50 50 50 50 

Moribund 9 8 I 8 

Natural deaths 11 4 11 4 

Animals surviving to study termination 30 38 32 38 

Percent probability of survival at end of study 60 76 64 76 

Mean survival (days) 680 713 701 686 

Survival analysis P=0.278N P=0.108N P=0.679N P=0.166N , 

a Kaplan-Meier determinations 

Mean of all deaths (uncensored, censored, and terminal sacrifice). ' The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparisons (Cox, 

1972) with the controls are in the exposed columns. A negative trend or lower mortality in an exposure group is indicated by N. 

Includes one animal that died during the last week of the study. 
42 Phenolphthalein, NTP TR 465 

1 .: 

0.s 

-I 0.1 < 

L 

0.7 

LL 

0 

t 0.6 

k

=’ 

m

3 0.5 

0 

a 

a 0.4 

0.2 

0.2 

15 30 45 b0 DO 1iS 120 

WEEKS ON STUDY 

;5 

FIGURE 2 

Kaplan-Meier Survival Curves for Rats Administered Phenolphthalein in Feed for 2 Years 
Results 43 

.-" 0 :5 30 A5 SO 75 90 105 0 

WEEKS ONSTUDY 

0 :I 30 4s 60 75 OD 105 

WEEKS ON STUDY 

FIGURE 3 

Growth Curves for Rats Administered Phenolphthalein in Feed for 2 Years 
44 Phenolphthalein, N" TR 465 

TABLE5 

Mean Body Weights and Survival of Male Rats in the 2-YearFeed Study of Phenolphthalein 

on 

Weeks 

Av. Wt. No. of 

0 ppm 

Av. Wt. Wt. (% of 

12,000 ppm 

No. of Av. Wt. Wt. (% of No. of 

25,000 ppm 

Av. Wt. Wt. (% of No. of 

50,000 ppm 

Study (8) Survivors (8) controls) Survivors (8) controls) Survivors (9) controls) Survivors 

1 135 50 135 100 50 136 100 50 133 98 50 

2 170 50 168 99 50 168 99 50 161 95 50 

3 208 50 203 98 50 204 98 50 197 94 50 

4 237 50 229 96 50 233 98 50 227 96 50 

5 258 50 250 97 50 254 98 50 249 97 50 

6 280 50 272 97 50 270 96 50 269 96 50 

7 294 50 286 97 50 286 97 50 283 96 50 

8 306 50 289 94 50 276 90 50 292 95 50 

9 320 50 301 94 50 294 92 50 306 95 50 

10 330 50 315 96 50 293 89 50 316 96 50 

11 341 50 325 95 50 317 93 50 326 95 50 

12 344 50 318 93 50 312 91 50 332 97 50 

13 344 50 327 95 50 326 95 50 333 97 50 

17 382 50 364 95 50 362 95 50 365 95 50 

21 400 50 381 95 50 386 97 50 385 96 50 

25 415 50 398 96 50 402 97 50 405 98 50 

29 425 50 406 95 50 410 97 50 409 96 50 

33 437 50 41 1 94 50 42 1 96 50 420 96 50 

37 449 49 428 95 50 432 96 50 434 97 50 

41 459 49 437 95 50 442 96 50 443 97 50 

45 460 49 440 96 50 442 96 50 443 96 50 

49 463 48 438 95 50 446 97 50 446 96 50 

53 464 48 443 96 50 445 96 50 447 96 50 

57 45 9 48 439 96 50 441 96 48 442 96 50 

61 466 47 439 94 50 444 95 47 439 94 50 

65 470 47 443 94 50 443 94 46 447 95 50 

69 466 47 440 95 48 445 96 46 442 95 50 

73 46 1 44 442 96 47 44 1 96 45 442 96 49 

77 465 41 436 94 46 439 94 45 437 94 47 

81 457 38 433 95 41 43 1 94 45 43 1 94 45 

85 455 36 423 93 40 422 93 44 428 94 42 

89 450 31 419 93 35 404 90 42 415 92 39 

93 457 29 423 93 28 397 87 35 390 86 34 

97 45 1 27 405 90 25 395 88 26 409 91 24 

101 439 26 375 85 20 364 83 21 372 85 19 

104 434 21 367 85 15 341 79 15 342 79 13 

Mean for weeks 

1-13 274 263 96 259 95 263 96 

14-52 432 411 95 416 96 417 97 

53-104 457 423 93 418 91 420 92 
Results 45 

TABLE6 

Mean Body Weights and Survival of Female Rats in the 2-Year Feed Study of Phenolphthalein 

Weeks 0 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

onAv.Wt. No. of Av.Wt.Wt. (% of No. of Av.Wt.Wt. (% of No. of Av.Wt.Wt. (% of No. of 

Study (9) Survivors (9) controls) Survivors (9) controls) Survivors (9) controls) Survivors 

1 113 50 114 100 50 113 100 50 113 100 50 

2 129 50 129 100 50 128 99 50 129 100 50 

3 139 50 138 100 50 138 100 50 141 102 50 

4 142 50 147 104 50 146 103 50 151 107 50 

5 157 50 157 100 50 155 99 50 159 102 50 

6 169 50 168 99 50 167 99 50 168 99 50 

7 172 50 173 100 50 171 100 50 170 98 50 

8 179 50 177 99 50 172 96 50 174 97 50 

9 184 50 182 99 50 179 97 50 180 98 50 

10 187 50 184 98 50 185 99 50 184 99 50 

11 194 50 191 98 50 189 98 50 185 95 50 

12 200 50 194 97 50 193 97 50 186 93 50 

13 20 1 50 195 97 50 196 98 50 191 95 50 

16 214 50 204 95 50 202 94 50 200 93 50 

21 222 50 211 95 50 209 94 50 206 93 50 

25 229 50 220 96 50 216 94 50 214 93 50 

29 234 50 222 95 50 219 94 50 216 93 50 

33 240 50 225 94 50 223 93 50 219 91 50 

37 252 50 234 93 50 230 91 50 228 90 50 

41 261 50 240 92 50 238 91 50 234 90 49 

45 267 50 244 91 50 242 91 50 237 89 49 

49 28 1 50 254 90 50 249 89 50 245 87 48 

53 287 50 258 90 50 254 88 50 249 87 48 

57 296 48 262 89 50 256 87 49 25 1 85 48 

61 304 47 265 87 50 260 86 49 259 85 48 

65 314 46 272 87 49 269 86 49 265 84 41 

69 322 46 282 88 49 280 87 49 270 84 47 

73 325 46 287 89 49 284 87 48 275 85 45 

77 332 45 290 87 49 288 87 48 28 1 85 43 

81 342 44 298 87 48 293 86 48 284 83 43 

85 346 44 303 88 47 296 86 47 292 85 42 

89 351 40 301 86 47 295 84 44 291 83 42 

93 345 40 299 87 46 294 85 42 293 85 41 

97 346 37 290 84 45 298 86 40 290 84 41 

101 348 34 301 87 41 300 86 37 296 85 40 

Mean for weeks 

1-13 167 165 99 164 98 164 98 

14-52 244 228 93 225 92 222 91 

53-101 328 285 87 282 86 277 84 
46 Phenolphthalein, NTP TR 465 

Determinations of Total Phenolphthalein Pathology and Statistical Analyses 

in Plasma Thissection describes the statistically significant or 

On the last day ofthe2-year study, blood was biologically noteworthy changes in the incidences of 

collected from the retroorbital sinus of three male neoplasms and/or nonneoplastic lesions of the adrenal 

and three female anesthetized rats from each group at medulla, kidney, seminal vesicle, spleen, mammary 

five time points for the determination of plasma gland, pituitary gland, and other organs. Summaries 

concentrations of totalphenolphthalein (free and of the incidencesofneoplasmsandnonneoplastic 

conjugated). The plasma concentrationsoftotal lesions, individual animal tumor diagnoses, statistical 

phenolphthalein and their standard deviations in rats analyses of primary neoplasms that occurred with an 

are given in Table H1. The semilogarithmic plots of incidence of at least 5 % in at least one animal group, 

plasma concentrations of total phenolphthalein (free and historical incidences for the neoplasms mentioned 

and conjugated) versus time for male and female rats in this section are presented in Appendix A for male 

administered 12,000, 25,000, and 50,000 ppm for rats and Appendix B for female rats. 

2 years are shown and Figures H1 through H5. In 

general, the meanplasmaconcentrationsoftotal Adrenalmedulla: The incidences of benign pheophenolphthalein varied little with time of day. There chromocytoma of the adrenal medulla in all exposed 

were no significant differences in plasma concentra- groups of malesweresignificantly greater than 

tions of total phenolphthalein between exposure thatinthe controls and occurred with a significant 

groups or between males and females. positive trend (Tables 7 and A3). The incidences of 

TABLE7 

Incidences of Neoplasms and NonneoplasticLesions of the Adrenal Medulla in Rats 

in the 2-Year Feed Studyof Phenolphthalein 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Male 

Number Examined Microscopically 50 50 50 50 

Focala Hyperplasia, 13 22* 18 23** 

Benign Bilateral Pheochromocytoma, 3 19** 19** 15**

Benign Pheochromocytomab (Includes Bilateral) 

Overallrate' 17/50 (34%) 34/50 (68%) 34/50 (68%) 34/50 (68X) 

Adjustedrated 62.1% 88.6% 93.7% 87.0% 

Terminalratee 11/21(52%) 11/15 (73%) 13/15 (87%) 9/13 (69%) 

First incidence (days) 608 485 372 528 

Logistic regression test' P=O.O06 P<O.ool P<O.OOl P=O.ool 

Malignant Pheochromocytoma 

(Includes Bilateral) 1 1 1 2 

Benign or Malignant Pheochromocytoma 

Overallrate 18/50 (36%) 35/50 (70%) 35/50 (70%) 35/50 (70%) 

Adjustedrate 62.9% 88.9% 93.9% 90.3% 

Terminalrate 11/21 (52%) 11/15 (73X) 13/15 (87%) 10113 (77%) 

First incidence (days) 330 485 372 528 

Logistic regression test P=O.O05 P<O.OOl P<O.ool P=O.ool 

(continued) 
Results 47 

TABLE7 

Incidences of Neoplasms and Nonneoplastic Lesions of the Adrenal Medulla in Rats 

in the 2-Year Feed Study of Phenolphthalein (continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Female 

Number Examined Microscopically 50 50 50 49 

Hyperplasia, Focal 10 18 15 11 

Benign Pheochromocytoma, Bilateral 1 2 0 0 

Benign Pheochromocytomag (Includes Bilateral) 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Logistic regression test 

Malignant yheochromocytoma 

Benign or Malignant Pheochromocytomah 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Logistic regression test 

(T)Terminal sacrifice 

3/50 (6%) 11/50 (22%) 9/50 (18%) 2/49 (4%) 

10.0% 25.8% 23.7 % 5.3% 

3/30 (10%) 7/38 (18%) 5/32 (16%) 2/38 (5%) 

735 (T) 549 608 735 (T) 

P=0.197N P=O.O31 P=O.O74 P=0.392N 

0 1 1 0 

3/50 (6%) 12/50 (24%) 10150 (20%) 2/49 (4%) 

10.0% 28.2% 25.3% 5.3% 

3/30 (10%) 8/38 (21 %) 5/32 (16%) 2/38 (5%) 

735 (T) 549 568 735 (T) 

P=0.189N P=O.O19 P=O.O41 P=0.392N 

* Significantly different (P10.05)from the control group by the logistic regression test 

** P10.01 

Number of animals with lesion 

Historical incidence for 2-year NTP feed studies with untreated controls (mean f standard deviation): 396/1,283 (30.9% f 12.1%); 

range, 10%-63% 

Number of animals with neoplasm per number of animals with adrenal medulla examined microscopically 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence in animals surviving until the end of the study 

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to 

the pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions in animals dying 

prior to terminal kill as nonfatal. A negative trend or lower incidence in an exposure group is indicated by N. 

Historical incidence: 5OA.274 (3.9% f 2.4%); range, 0%-8% 

Historical incidence: 64/1,274 (5.0% f 2.7%); range, 2%-12% 

benignpheochromocytomain 12,000 ppm females 

andof benign or malignantpheochromocytoma 

(combined) in 12,000 and 25,000 ppm females were 

significantly greater thanthoseinthe controls 

(Tables 7 and B3). The incidences of benign pheochromocytoma in all exposed groups of males and of 

benign pheochromocytoma and benign or malignant 

pheochromocytoma in (combined) 12,000 and

benign pheochromocytomas exceeded the number of 

controls with theseneoplasms (Tables 7 and Al). 

The incidences of malignant pheochromocytomas in 

exposed rats were similar to those in the controls 

(Tables 7, A1, and Bl). The incidences of focal 

hyperplasiaof the adrenal medulla in 12,000 and 

50,000 ppm males were significantly greater than that 

inthe controls (Tables 7 and A5); however, the 

25,000 ppm females exceeded the ranges in historicaldiagnosisoffocal hyperplasia in an adrenal gland 

controls in NTP 2-year feed studies (Tables 7, A4a, wasmadeonlyintheabsenceof a diagnosis of a 

and B4a). The numbers of males withbilateral neoplasm in that adrenal gland. Therefore, exposed 
48 Phenolphthalein, NTP TR 465 

groups were less at risk for diagnosis of hyperplasia. 

Focal hyperplasia, benign pheochromocytoma,and 

malignant pheochromocytoma are considered to be a 

morphologic continuum in theadrenalmedulla. 

Focal hyperplasia consisted of irregular, small foci of 

smalltonormal-sizedmedullarycellsarrangedin 

packets or solid clusters slightly larger than normal; 

compression of surrounding parenchyma was minimal 

or absent. Benignpheochromocytomaswerewelldelineated masses often with altered architecture and 

variable compression of the surrounding parenchyma. 

Neoplastic cells were arrangedinvariablysized 

aggregates, clusters, and trabecular cords of varying 

thickness. Larger neoplasms exhibited usually

greater cellular pleomorphism and atypia than smaller 

neoplasms. The few malignantpheochromocytomas 

were identified when there wasinvasion of,or 

beyond, the adrenal capsule. 

Kidney: The incidences of proliferative lesions of the 

renal tubule epithelium in male rats were greater in 

allexposed groups than inthe controls (Tables 8, 

A3, and AS). The incidences of renal tubule adenoma in 50,000 ppm males andof renal tubule adenoma 

or carcinoma (combined) in 12,000 and 50,000 ppm 

males were significantly greater than thoseinthe 

controls (Tables 8 and A3). Although the neoplasms 

were predominantly adenomas, a fewcarcinomas 

were observed in exposed males (Tables 8 and Al). 

The incidences of renal tubule adenoma and adenoma 

or carcinoma (combined) in 12,000 and 50,000 ppm 

malesexceededthe ranges in historical controls in 

NTP 2-year feed studies (Tables 7, A3, and A4b). 

Bothrenaltubuleadenomaandcarcinoma are relatively uncommon neoplasms in the maleF344/N rat. 

Hyperplasia, adenoma, and carcinoma are thought to 

represent a continuum in the progression of proliferative lesions of the renal tubule epithelium. Hyperplasias were generally focal lesions characterized by 

increased numbers of renaltubuleepithelial cells 

forming multiple layers that partially or totally filled 

the renal tubule lumen and usually caused dilation of 

the tubule. Generally, adenomas were discrete, 

expansile masses thatwere larger (greater than the 

diameter of five renal tubules) than the hyperplasias 

and had a more complex structure. Carcinomas were 

less discrete and larger than adenomas with hemorrhage, necrosis, and locally invasive growth patterns 

commonly observed. 

Onerenaltubuleadenomawas observed in a 

50,000 ppmfemaleatthe standard evaluation 

(Tables 8 and Bl). Because an effect was observed 

inmalesandrenaltubuleaden.omas are rare in the 

female F344/N rat, an extended histologic evaluation 

(stepsections) of the kidneys wasconducted in 

females (Table 8). In this extended evaluation, no 

additional neoplasms were observed in the 25,000 or 

50,000 ppm females, while neoplasms were identified 

inthe 12,000 ppm group and the controls. Therefore, the adenoma in the 50,000 ppm female was not 

considered to be chemical related. For completeness, 

an extended evaluation was also conducted for male 

rats (Tables 8 andA3). In the expanded evaluation 

of males,additionaladenomas were observed in 

exposed groups of males; only one additional adenomawas observed in the control males. This finding 

provides additional support that the increased incidences of renal tubule proliferative lesions in males were 

associated with the administration of phenolphthalein. 

The incidences of nephropathy in all exposed groups 

of femaleswere significantly greater than in the 

controls (Tables 8 andB5),and the severity of 

nephropathy in all exposed groups of malesand in 

25,000 and 50,000 ppm females wassignificantly 

greater than in the controls (Table 8). Nephropathy 

is a common spontaneously occurring lesion in aging 

F344/N rats, particularly males, and the typical 

morphologicalchangesassociatedwith nephropathy 

were present in this study. Changes consisted of the 

followingspectrum of lesions: varying degrees of 

tubular dilation and distortion with cyst formation; 

proteinaceous tubular casts; atrophy; regeneration and 

hypertrophy of the renal tubule epithelium; thickeningoftherenaltubuleand glomerular basement 

membranes;interstitial fibrosis; scattered foci of 

suppurative inflammation, primarily within degenerating renal tubules; and varying numbers and aggregates of mononuclear inflammatory cells within the 

interstitium. Marked renal tubule dilatation with cyst 

formation and transitional epithelial hyperplasia of 

therenalpelvisoccur more commonly in kidneys 

withthemost severe nephropathy, as was observed 

in exposed groups of males (Tables 8 and A5). The 

absence of a dose response for these two lesions is 

consistentwiththe similar severity of nephropathy 

observed in exposed groups. 
Results 49 

TABLE8 

Incidences of Neoplasms and Nonneoplastic Lesions of the Kidney in Rats in the 2-Year Feed Study 

of Phenolphthalein 

Male 

Number Examined Microscopically 

Single Sections (Standard Evaluation) 

cysta 

Nephropathy, Chronic 

Pelvis, Transitional Epithelium, Hyperplasia 

Renal Tubule, Hyperplasia 

Renal Tubule, Adenoma' 

Overall rated 

Adjusted ratee 

Terminal ratef 

First incidence (days) 

Logistic regression testg 

Renal Tubule, Carcinoma 

Adenoma or Carcinoma' 

Overall rate 

Adjusted rate 

Terminal rate 

0 PPm 

50 

1 (l.O)b 

47 (1.8)

4 (1.5) 

0 

0150 (0%) 

0.0% 

0121 (0%) - 577 

P=O.O26 

0 

0150 (0%) 

0.0% 

0121 (0%) 

12,000 ppm 

50 

19** (1.8)

49 (2.9)** 

31** (1.1)

6** (2.3) 

4/50 (8%) 

20.4% 

2/15 (13%) 

542 692

P=O.O60 

1 

5/50 (10%) 

24.0% 

2/15 (13%) 

First incidence (days) - 577 542 692

Logistic regression test 

Step Sections (Extended Evaluation) 

Renal Tubule, Hyperplasia 

Renal Tubule, Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Logistic regression test 

Renal Tubule, Carcinoma 

Renal Tubule, Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Logistic regression test 

Single Sections and Step Sections (Combined) 

Renal Tubule, Hyperplasia 

Renal Tubule, Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

P=O.O20 

3 

1/50 (2%) 

4.8% 

1/21 (5%) 

729 (T) 

P=O.O03 

0 

1/50 (2%) 

4.8% 

1/21 (5%) 

729 (T) 

P=O.O03 

3 

1/50 (2%) 

4.8% 

1/21 (5%) 

P=O.O31 

23** 

7/50 (14%) 

36.4% 

4/15 (27%) 

691 664 

P=O.O17 

0 

7/50 (14%) 

36.4% 

4/15 (27%) 

691 664 

P=O.O17 

25** 

10150 (20%) 

46.9% 

5/15 (33%) 

25,000 ppm 

50 

21** (1.7) 

50 (3.1)**

34** (1.2)

7** (2.3) 

2/50 (4%) 

10.6% 

1/15 (7%) 

P=O.233 

1 

3/50 (6%) 

16.1% 

1/15 (7%) 

P=O. 106 

29** 

15\50 (30%) 

64.9% 

8/15 (53%) 

542 

P<O.OOl 

1 

15/50 (30%) 

64.9% 

8/15 (53%) 

542 

P<O.OOl 

29** 

15/50 (30%) 

64.9% 

8/15 (53%) 

50,000 ppm 

50 

22** (1.9)

50 (3.1)**

29** (1.3)

2 (3.0) 

6/50 (12%) 

22.4% 

1/13 (8%) 

P=O.O18 

2 

7/50 (14%) 

24.7% 

1/13 (8%) 

P=O.O09 

27** 

11/50 (22%) 

51.1% 

5/13 (38%) 

P=O.O03 

0 

11/50 (22%) 

51.1% 

5/13 (38%) 

P=O.O03 

27** 

15\50 (30%) 

61.8% 

6/13 (46%) 

First incidence (days) 729 (T) 664577 542 

Logistic regression test P<O.OOl P=O.O03 P<O.OOl P<O.ool 

Renal Tubule, Carcinoma 0 1 2 2 

(continued) 
50 

~~ 

Phenolphthalein,NTP TR 465 

TABLE8 

Incidences of Neoplasms and NonneoplasticLesions of the Kidney in Rats in the 2-YearFeed Study 

of Phenolphthalein (continued) 

-

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Male (continued) 

Single Sections and Step Sections (Combined) (continued) 

Renal Tubule, Adenoma or Carcinoma 

Overall rate 1/50 (2%) 10150 (20%) 16/50 (32%) 16/50 (32%) 

Adjusted rate 4.8% 46.9% 67.1X 62.9% 

Terminal rate 1/21 (5%) 5/15 (33%) 8/15 (53%) 6/13 (46%) 

First incidence (days) 729 (T) 577664 542 

Logistic regression test P<O.OOl P=O.O03 P<O.OOl P<O.ool 

Female 

Number Examined Microscopically 50 50 50 50 

Single Sections (Standard Evaluation) 

Nephropathy, Chronic 34 (1.2) 45* (1.4) 43* (1.5)* 44* (1.5)* 

Renal Tubule, Adenomd 0 0 0 1 

Step Sections (Extended Evaluation) 

Renal Tubule, Hyperplasia 1 4 3 3 

Renal Tubule, Adenoma 1 2' 0 0 

Single Sections and Step Sections (Combined) 

Renal Tubule, Hyperplasia 1 4 3 3 

Renal Tubule, Adenoma 1 2 0 1 

(T)Terminal sacrifice 

* Significantly different (PSO.05) from the control group by the logistic regression test (incidence data) or the Mann-Whitney U test 

(severity of nephropathy)

** PCO.01 a Number of animals with lesion 

b Average severity of lesions in affectedanimals: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked C Historical incidence for 2-year NTP feedstudieswithuntreated controls (mean f standard deviation): 911,301 (0.7% f 1.5%); 

range, 0%-6% 

d Number of animals with neoplasm per number of animals with kidney examined microscopically e Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality f Observed incidence in animals surviving until the end of the study 

I2 In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to 

the pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions in animals dying 

prior to terminal kill as nonfatal. 

h Not applicable; no neoplasms in animal group i Historical incidence: 12/1,301 (0.9% f 1.5%); range, 0%-6% 

j Historical incidence: 0/1,298 (0.0%) 
Results 51 

Other organs: The incidences of diffuse hyperplasia 

of the parathyroid gland, fibrous osteodystrophy of 

the bone, and mineralization and degeneration of the 

glandular stomach in exposed groups of males were 

generally significantly greater than thoseinthe 

controls (Tables 9 and A5). Theselesions are 

commonly observed in male rats with more advanced 

nephropathyand are associatedwith a calcium/ 

phosphorus imbalance by createdcompromised 

TABLE9 

functional capacity of the kidney. The incidences of 

hyperplasia of the thyroidgland C-cells in 12,000 

and 50,000 ppm malesweresignificantlylessthan 

that in the controls and were also considered related 

to the calcium/phosphorus imbalance (Tables 9 and 

A5). Additionally, the incidence of mineralization of 

theaortain 12,000 ppm maleswassignificantly 

greater than that in the controls and was considered 

secondary to the renal disease. 

Incidences of Selected Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of Phenolphthalein 

Blood Vessela 

Aorta, Media, Mineralizationb 

Bone 

Femur, Fibrous Osteodystrophy 

Parathyroid Gland 

Hyperplasia, Diffuse 

Stomach, Glandular 

Degeneration 

Mineralization 

Thyroid Gland 

C-cell, Hyperplasia, Focal 

0 PPm 

50 

0 

50 

0 

41 

0 

50 

0 

0 

50 

13 

12,000 ppm 25,000 ppm 50,000 ppm 

49 50 50 

9** 3 4 

50 50 50 

17** 14** 12** 

48 49 46 

16** 14** 14** 

50 50 49 

11** 5* 4 

11** 5* 5* 

50 49 49 

3** 9 4* 

* Significantly different (P 50.05) from the control group by the logistic regression test 

** P10.01 

a Number of animals with tissue examined microscopically 

Number of animals with lesion 

Seminal vesicle: There were minimal increases in the 

incidencesof atrophy of theseminalvesicleinexposed groups of males (0 ppm, 33/50; 12,000 ppm, 

44/50;25,000 ppm, 45/50;50,000 ppm, 47/50; 

Table A5). The severity of the lesion was similar 

amongexposedand control groups (2.5, 2.5,2.8, 

2.6). The atrophy consisted of reduced lengthof 

acinar branching, decreased size of secretory epithelial cells, increased fibrosis within theperiacinarsmooth muscle, and a decreased amount of 

eosinophilic secretory material. Sex glands are

hormone dependent, and mild atrophy of the seminal 

Evaluationand interpretation of effects in primary 

sex organs and accessory sex glands ina 2-year study 

of male F344/N rats is complicated by the extremely 

high incidence of testicular interstitial cell neoplasms 

and associated degenerative changes. However, this 

effectwouldalso be expectedof an estrogenic 

exposure. The effect is consistent with a biological 

effect of phenolphthalein, but could also be explahed 

by other changes intheanimals.Chemical-related 

lesionswerenotobserved in other accessory sex 

glands (e.g., prostate gland) in this study. The 

significance of this marginal increase and its associavesicle associated with declining function of the testestionwiththeadministrationof phenolphthalein are 

is a common finding in agedmale F344/N rats. uncertain. 
52 Phenolphthalein, NTP TR 465 

Spleen: The incidences of lymphoid follicle atrophy 

of the spleen inexposed groups of maleswere 

greater than that inthe controls (2/50, 8/50, 9/50, 

12/49; Table A5). Macroscopically, affected spleens 

were observed to be smallerthan normal, and histologically, there was a reduction in the mantle layer 

of lymphocytes. This change wasobserved predominantly in animals that died early or were killed 

moribund. 

Mammary gland: The incidences of mammary gland 

fibroadenoma were decreased in exposed groups of 

females (0 ppm, 30150; 12,000 ppm, 25/50; 

25,000 ppm, 17/48; 50,000 ppm, 24/49; Table B3); 

however, the incidenceinthe control group (60%) 

slightlyexceededthe range in historical controls in 

NTP 2-year feed studies [524/1,301 (40% f 13%); 

range, 8%-58%]. There is a knownassociation 

betweentheincidenceofthisneoplasmandbody 

weight in female F344/N rats. The decreased incidencesof this neoplasm were considered to be due 

primarily to the lower body weightsofexposed 

groups and theunusuallyhighincidenceinthe 

control group. 

Pituitarygland: The incidence of adenoma of the 

pars distalis of the pituitaryglandin 50,000 ppm 

females was significantly less than that inthe controls 

(35150, 32/49, 29/50, 25/47; Table B3), and the 

incidenceofthis lesion occurred with a significant 

negative trend. Pituitaryglandadenomainfemale 

F344/N rats occurs spontaneously at a highand 

variable rate in historical controls in NTP 2-year feed 

studies[666/1,290(52% f 13%);range, 30%-74%]. 

In this study, the incidence in the control group 

(70%) was at the upper end of the range for the 

historical controls, whileincidence the inthe 

50,000 females (53%) was similar to themean 

historical control incidence. Further, the dose-related 

decreaseisinconsistentwiththeabsenceof a dose 

responseinplasma phenolphthalein concentrations 

and other chemical-related effects in this study. 

Therefore, the marginal decrease of pituitary gland 

adenoma was not considered related to the administration of phenolphthalein. 
Results 53 

MICE feed and compound consumption could not be made. 

WDAYSTUDY The feces of 100,000 ppm males and females were 

All mice survived to the end of the study (Table lo). abnormally lighter in colorthanthose of the other 

The final mean body weights of all exposed groups groups.Thisdiscoloration was observed on day 3 

of mice were similar to those of the controls. and continued throughout thestudy. No chemicalBecause of feed spillage, accurate measurements of related gross or microscopic lesions wereobserved. 

TABLE10 

Survival and Mean Body Weights of Mice in the 14-Day Feed Study of Phenolphthalein 

Mean Body Weightb (g) Final Weight 

Dose Survivala Initial Final Relative to Controls 

Male 

0 515 24.4 f 2.2 27.9 f 3.2 

6,250 515 25.2 f 1.8 28.2 f 2.0 101 

12,500 515 25.6 f 1.8 29.6 f 2.1 106 

25.000 515 25.4 f 1.9 28.9 f 1.8 104 

50,000 515 25.5 f 1.7 28.5 f 1.2 102 

100.000 515 25.9 f 2.3 26.5 f 2.6 95 

Female 

0 515 19.9 f 0.9 23.0 f 1.0 

6,250 5/5 20.1 f 1.0 22.8 * 1.1 99 

12,500 515 19.9 f 0.8 22.7 f 0.8 99 

25.000 515 20.3 f 1.5 21.8 f 0.6 95 

50.000 515 19.9 f 0.7 22.2 f 0.8 97 

100,000 515 19.9 f 0.8 22.4 f 0.8 97 

a Number of animals surviving at 14 dayslnumber initially in group. Differences from the control group are not significant by Williams’ 

or Dunnett’s test. 

Weights are given as mean f standard deviation. 
54 Phenolphthalein, NTP TR 465 

13-WEEK STUDY 50,000 ppmphenolphthaleinresulted in average 

Allmicesurviveduntiltheend of thestudy dailydosesofapproximately 500, 1,000,2,000, 

(Table 11). The final meanbodyweightsandbody 4,100, or 9,000 mg phenolphthaleidkg body weight 

weight gains of allexposed groups were similar to males to and 600, 1,200, 2,400, 5,000, or 

those of the controls. Feed consumption by exposed 10,500 mg/kg to females. There was no cathartic 

groups of micewas similar to that bythe controls. actionnorany other clinical finding attributed to 

Dietary levels of 3,000,6,000, 12,000, 25,000, or exposure to phenolphthalein. 

TABLE11 

Survival, Mean Body Weights, and Feed Consumption of Mice in the 13-Week Feed Study 

of Phenolphthalein 

Final Weight 

Mean Body Weightb (g) Relative Feed 

Dose Survivala Initial Final ChangeControlsto Consumption' 

(PPm) (%I Week 1 Week 13 

Male 

0 10/10 27.8 20.4 f 0.7 f 0.8 7.4 f 0.5 3.7 3.9 

3.00010110 19.8 25. f 0.5 f 0.6 936.1 f 0.5 4.0 4.3 

6,000 10110 20.3 f 0.5 26.8 f 0.6 6.6 f 4.40.3 3.8 97 

f 0.4 99 3.9 4.1 

f 0.4 99 4.4 4.3 

f 0.4 95 4.3 4.6 

12,000 10110 19.9 f 0.5 27.4 7.5 f 0.5 

f 0.5 27.6 7.9 f 0.7 

10/10 50,000 20.0 26.5 f 0.4 6.5 f 0.6 

Female 

0 10110 15.8 22.9 f 0.7 7.1 f 0.6 f 0.3 3.5 4.5 

3.0001011016.6 23.9 f 0.5 f 0.7 104 7.3 f 0.4 4.4 4.5 

6,000 10110 17.7 24.1f 0.4* f 0.5 6.4 f 0.3 LO5 4.6 4.0 

1011012.000 16.9 f 0.4 23.8 f 0.4 6.9 f 0.3 104 4.0 4.6 

25.000 10/1017.1 f 0.3 24.0 f 0.4 6.9 f 0.3 105 4.8 4.1 

50.000 10/1017.0 f 0.4 23.6 6.7 f 0.5 f 0.3 103 4.1 5.0 

* Significantly different (P50.05) from the control group by Dunnett's test 

a Numberofanimals surviving at 13 weekslnumber initially in group 

Weights and weight changes are given as mean f standard error. 

Feed consumption is expressed as gramsof feed consumed per animal per day. 

The absolute right cauda weight of the 12,000 ppm in the control group, and the sperm concentrations in 

males was significantly less than that of the controls 12,000 and 50,000ppm males were significantly less 

(Table 12). The absolute right epididymis weights of than that in the control group. The estrous cyclicity 

12,000, 25,000, and 50,000ppm males were signifi- appeared normal in females (Table 12). In some 

cantly less than that of the controls. The percentages groups of females, there was a slight lengthening of 

of abnormal sperm in 12,000, 25,000, and the cycle, but a specific phase could not be identified 

50,000 ppm males were significantly greater than that as being responsible. 

1011019.6 25.000
Results 55 

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 males were significantlylessthan ofcontrol those the group

(Table F2). Other organ weight differences inmice 

were attributed to changes inbodyweightsof exposed mice and were not considered to be related to 

exposure to phenolphthalein. Although a very subtle 

lesion in most instances, some seminiferous tubules 

lacked one or more generations of germ cellsin 

portions or the entirety of a seminiferous tubule. 

This could be seen by the presence of spermatogonia, 

early spermatocytes, and pachytenespermatocytes, 

but no round or elongated spermatids. Similarly, 

other seminiferous tubules contained Sertoli nuclei, 

spermatogonia, early spermatocytes, andelongating 

TABLE12 

spermatids, but no pachytene spermatocytes or round 

spermatids. Occasionally, seminiferous tubules 

containedonly Sertoli cells. Often an affected 

seminiferous tubule would be surrounded by tubules 

that looked normal. 

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 

(Table 12). This lesion was characterized by less 

cellularmarrowwith a decreased myeloid to erythroid ratio, vascular congestion, and individual cell 

necrosis. The identification of the necrotic cell type 

was not always possible, but in many instances, these 

cells appeared to be myeloid precursors. In addition, 

there was a slight increase in pigment interpreted as 

Incidences of Selected Nonneoplastic Lesions in Mice in the 13-Week Feed Study of Phenolphthalein 

0 ppm 3,000 ppm 6,000 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

BoneMarrowa 10 10 10 10 10 10 

Hypoplasiab 0 0 1 (l.O)C lo** (1.0) lo** (1.0) lo** (2.0) 

Necrosis 0 0 1 (1.0) lo** (1.0) lo** (1.O) lo** (1 .O) 

Spleen 10 -

d - 10 10 10 

Hematopoiesis 0 - - 0 9** (1.0) lo** (1.0) 

Female 

BoneMarrow 10 - 10 10 10 10 

Hypoplasia 0 - 0 8** (1.0) lo** (1 .O) lo** (1.O) 

Necrosis 0 - 0 8** (1.0) lo** (1.O) lo** (1 .O)

** Significantly different (P10.01) from the control group by the Fisher exact test 

a Number of animals withtissueexaminedmicroscopically 

Number of animals with lesion 

Averageseverity of lesions inaffectedanimals: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked 

Tissue not examined at this exposure concentration 

hemosiderin. The incidences of hematopoiesis of the Dose Selection Rationale: Based on the minimal 

spleenin 25,000 and 50,000 ppm malesweresignifi-severity of bone marrowand splenic lesions after 

cantly greater than that inthe controls (Table 12). 13 weeks of exposure to 12,000 ppm, the highest 

The bone marrowandspleenfindingswereminimalphenolphthalein exposure concentration selected for 

to mild in severity. the 2-year feed study in mice was 12,000 ppm. 
56 

&YEAR STUDY 

Survival 

Estimatesof 2-year survival probabilities for male 

and female mice are shown inTable13andinthe 

Kaplan-Meier survival curves (Figure 4). Survival of 

the 12,000 ppm females was significantly lower than 

thatof the controls; survival of all other exposed 

groups of mice was similar to that of the controls. 

Body Weights, Feed and Compound 

Consumption, and Clinical Findings 

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 (Figure 5, Tables 14 and 15). Feed 

consumption by allexposed groups of mice was 

similar to that by the controls (Tables K3 and K4). 

Dietary levels of 3,000, 6,000, or12,000 ppm 

phenolphthalein resulted in average daily dosesof 

approximately 300, 600, or 1,200 mg phenolphthaleidkg body weight to males and 400, 800, or 

1,500 mg/kg to females. The estimated equivalent 

daily doses of phenolphthalein for the last year of the 

Phenolphthalein, NTP TR 465 

study ranged from 290 to 1,200 mg/kg for male mice 

and from 310 to 1,300 mg/kg for female mice. In 

exposed mice, there were no clinical findings related 

to phenolphthalein exposure. 

Determinations of Total Phenolphthalein 

in Plasma 

Onthelastdayofthe 2-year study, blood was 

collected from the retroorbital sinus of three male 

and three female anesthetized mice from each group 

at five time points for the determination of plasma 

concentrations of total phenolphthalein (free and 

conjugated). Theplasma concentrations of total 

phenolphthalein and their standard deviations in mice 

are given in Table H2. The semilogarithmic plots of 

plasma concentration of total phenolphthalein (free 

and conjugated) versus time data for male and female 

mice administered3,000, 6,000, and 12,000 ppm for 

2 years are shown in Table H2 and Figures H6 

through H10. In general, the meanplasma concentrations of total phenolphthalein variedlittle with time 

of day. There were no significant differences in 

plasma concentrations of total phenolphthalein 

between exposure groups or betweenmalesand 

females. 
Results 

TABLE13 

Survival of Mice in the 2-Year Feed Study of Phenolphthalein 

3,000 ppm 6,000 ppm 12,000 ppm 

Male 

initially Animals in study 50 50 50 50

Missinga 0 0 0 1 

Moribund 6 13 11 3 

Natural deaths 4 4 3 10 

Animals surviving to study termination 40 33 36 36 

Percent probability of survival at end of studyb 80 66 72 I4 

Mean survival (days)c 701 695 695 681 

Survival analysisd P=O.826 P=O.186 P=O.461 P=O.600 

Female 

Animals initially in study 50 50 50 50 

Moribund 5 13 9 12 

Natural deaths 6 6 I 10 

Animals surviving termination to study39e 31 34 28

Percent probability of survival at end ofstudy 78 62 68 56 

Mean survival (days) 697 665 687 663 

Survival analysis P=O.O47 P=O.O97 P=O.331 P=O.O24 

a Censored from survival analyses 

Kaplan-Meier determinations 

Mean of all deaths (uncensored, censored, and terminal sacrifice). 

The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table painvise comparisons (Cox, 

1972) with the controls are in the exposed columns. 

e Includes one animal that died during the last week of the study. 
58 Phenolphthalein, NTP TR 465 

1.o 

0.9 

4

2 

1 

2

z 0.8 

& 

iz 

=I 0.7 

m 

0,

0 

63 

0.6 n 0.w 

0 3000 SPY 

C, 6000 PPY 

c.s 15 30 45 60 75 90 105 0 

H’EEKS ON STUDY 

1.0 

0.9 

c

.J 

L

t

5 0.8 

b 

t:

3 0.7 

z 

m 

0 

0.6 

0.1 

;5 30 45 60 75 90 105 10 

WEEKS ON STUDY 

FIGURE 4 

Kaplan-Meier Survival Curves for Mice Administered Phenolphthalein in Feed for 2 Years 
n 

Results 59 

................ :.......... i............ ;............... : ...................... I... ....... ..f .............. 

sa-. .......... ..i. ........... ................ ................. 

15_._............... .............. ................ 

do-. .............. ........ ........ ....... ........... ............ 

..... .., ............................ 

...... ...................... ................ 

............... ........... ......... ... 

. - I 

0 15 30 45 60 75 90 1os !O 

WEEKS ON STUDY 

60 

: mrn 5s ... ....................... . ...; ....... ......... 8 ,rn ,.: .......... 

8 

53 ................ ............. ................. ............... ..........

.......... ................. ................. .................. ................ 

AD ................ i................. ......................... .................. ................ i.................................. 

A5 

................ ....... .................................... i................ j .............. :.................. j................ 35 

10 ............... ;a.............. ;................. 1.. ............ ..i................ :............................... i................

.; .................. > ................................. : i .................. ....... 25 l CPPY 

0 3OoOPPY 

PO .......... i................. .i................ 1.................. i................. ...... A 6WOPPY .... 

0 lloWPPY 

15 

i5 30 4s 60 75 BO iD5 !O 

WEEKS ON STUDY 

mGURE 5 

Growth Curves for Mice Administered Phenolphthalein in Feed for 2 Years 
60 Phenolphthalein, NTP TR 465 

TABLE14 

Mean Body Weights and Survival of Male Mice in the 2-Year Feed Study of Phenolphthalein 

Weeks 0 ppm 3,000 ppm 6,000 ppm 12.000 ppm 

onAv.Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av.Wt.Wt. (% of No. of 

Study (9) Survivors (g) controls) Survivors (g) controls) Survivors (8) controls) Survivors 

1 24.1 50 23.4 97 50 23.2 96 50 23.2 96 50 

2 25.2 50 24.9 99 50 24.8 98 50 24.8 98 49 

3 25.8 50 25.5 99 50 25.5 99 50 25.5 99 49 

4 26.4 50 26.4 100 50 26.4 100 50 26.8 102 49 

5 27.7 50 27.4 99 50 27.1 98 50 26.9 97 48 

6 28.7 50 28.1 98 50 28.3 99 50 28.0 98 48 

7 28.9 50 28.5 99 50 28.6 99 50 28.8 100 48 

8 29.5 50 28.8 98 50 29.5 100 50 29.2 99 48 

9 29.5 50 29.2 99 50 29.4 100 50 28.8 98 48 

10 30.7 50 30.4 99 50 30.6 100 50 30.8 100 48 

11 31.5 50 31.5 100 50 31.1 99 50 30.7 98 48 

12 32.5 50 32.4 100 50 32.5 100 50 32.4 100 48 

13 32.6 50 32.8 101 50 32.6 100 50 32.5 100 48 

17 35.2 50 35.0 99 50 34.7 99 50 34.2 97 48 

21 40.5 50 39.1 97 50 39.7 98 50 38.5 95 48 

25 42.8 50 41.6 97 50 41.8 98 50 41.2 96 48 

29 44.8 50 43.9 98 50 44.0 98 50 43.2 96 48 

33 46.5 50 45.7 98 50 45.9 99 50 43.8 94 48 

37 46.8 49 46.0 98 50 45.7 98 50 43.9 94 48 

41 46.2 49 45.0 97 50 45.3 98 50 44.9 97 48 

45 47.1 49 47.O 100 50 46.4 99 50 45.6 97 48 

49 48.7 49 48.2 99 50 47.7 98 50 46.9 96 48 

53 48.7 49 48.3 99 49 48.3 99 50 47.0 97 48 

57 49.8 49 49.0 98 49 48.9 98 50 47.5 95 48 

61 49.6 49 48.9 99 49 47.9 97 50 47.3 95 48 

65 51.7 49 50.5 98 49 49.8 96 49 49.8 96 48 

69 52.0 49 50.9 98 49 49.8 96 49 49.8 96 48 

73 51.7 49 50.7 98 49 51.0 99 47 50.3 97 48 

77 51.6 48 50.7 98 49 50.3 98 46 49.8 97 47 

81 51.1 47 50.2 98 47 51.0 100 44 49.8 98 46 

85 51.1 46 50.2 98 45 51.5 101 44 50.1 98 45 

89 51.5 45 49.5 96 43 50.8 99 44 49.4 96 45 

93 50.0 44 49.1 98 41 50.2 100 44 47.1 94 44 

97 50.4 43 49.1 97 40 50.1 99 41 47.3 94 41 

101 49.9 42 49.0 98 39 50.2 101 38 46.3 93 40 

104 49.4 40 47.9 97 33 49.1 99 36 45.9 93 36 

Mean for weeks 

1-13 28.7 28.4 99 28.4 99 28.3 99 

14-52 44.3 43.5 98 43.5 98 42.5 96 

53-104 50.6 49.6 98 49.9 99 48.4 96 
Results 61 

TABLE15 

Mean Body Weights and Survival of Female Mice in the 2-YearFeed Study of Phenolphthalein 

Weeks 0 ppm 3,000 ppm 6.000 ppm 12,000 ppm 

on Av.Wt. No. of Av. Wt. Wt. (% of No. of Av.Wt.Wt. (% of No. of Av.Wt.Wt. (% of No. of 

Study (8) Survivors (8) controls) Survivors (8) controls) Survivors (8) controls) Survivors 

1 18.2 50 17.9 98 50 17.8 98 50 18.7 103 50 

2 19.4 50 19.5 98 50 19.6 99 50 19.6 99 50 

3 20.9 50 20.3 97 50 20.4 98 50 21.o 101 50 

4 21.3 50 21.1 99 50 21.o 99 50 21.1 99 50 

5 21.8 50 21.8 100 50 21.7 100 50 21.9 101 50 

6 23.4 50 23.0 98 50 23.2 99 50 23.4 100 50 

7 23.0 50 22.7 99 50 22.7 99 50 23.3 101 49 

8 24.O 50 23.5 98 50 23.8 99 50 24.0 100 49 

9 24.9 50 24.3 98 50 24.7 99 50 24.9 100 49 

10 25.4 50 24.5 97 50 25.2 99 50 25.1 99 49 

11 26.5 50 25.5 96 50 26.0 98 50 26.4 100 49 

12 27.6 49 26.9 98 50 26.7 97 50 26.9 98 49 

13 27.0 49 26.1 97 50 26.9 100 50 27.4 102 49 

17 30.6 49 29.1 95 50 29.3 96 50 29.6 97 49 

21 34.0 49 33.6 99 49 33.6 99 50 34.3 101 49 

25 36.2 48 35.2 97 49 35.1 97 50 35.8 99 49 

29 38.8 48 37.5 97 49 37.8 97 50 37.5 97 49 

33 42.2 48 40.3 96 49 40.3 96 50 39.9 95 49 

37 42.1 48 40.6 96 49 40.7 97 50 40.2 96 49 

41 44.5 48 42.8 96 48 42.1 95 50 41.7 94 49 

45 45.2 48 43.1 95 48 42.9 95 50 43.1 95 49 

49 46.6 48 44.6 96 48 43.7 94 49 43 .O 92 49 

53 47.8 48 46.1 96 48 45.5 95 48 45 .O 94 49 

57 49.0 48 46.9 96 48 47.1 96 48 46.5 95 49 

61 50.2 48 48.0 96 48 47.6 95 48 46.8 93 49 

65 53.6 48 50.7 95 47 51.1 95 48 49.7 93 48 

69 55.6 48 52.3 94 46 51.0 92 48 50.8 91 46 

73 55.6 48 52.9 95 45 51.7 93 47 50.9 92 46 

77 54.6 48 52.4 96 44 51.4 94 47 50.3 92 44 

81 55.6 47 52.7 95 43 51.5 93 46 50.3 91 44 

85 56.3 47 52.9 94 39 52.5 93 44 51.2 91 39 

89 56.5 44 52.6 93 37 52.7 93 41 52.9 94 35 

93 55.2 43 51.4 93 36 51.O 92 39 51.7 94 34 

97 55.6 43 52.5 94 33 51.8 93 37 51.8 93 32 

101 54.9 42 50.3 92 33 52.0 95 34 51.6 94 31 

Mean for weeks 

1-13 23.4 22.9 98 23.1 99 23.4 100 

14-52 40.0 38.5 96 38.4 96 38.3 96 

53-101 53.9 50.9 94 50.5 94 50.0 93 
62 Phenolphthalein, NTP TR 465 

Pathology and Statistical ,Analyses least one animal group, and historical incidences for 

This section describes the statisticallysignificant or the neoplasms mentioned in this section are presented 

biologically noteworthy changes in the incidences of inAppendix C for male miceandAppendix D for 

histiocytic sarcoma and malignantlymphomaand female mice. 

neoplasms and/or nonneoplastic lesions of the thymus, ovary, testis, bone marrow, kidney, spleen, All organs: The incidences of histiocytic sarcoma in 

liver, tooth, pituitary gland, andthyroid gland. 6,000 and 12,000 ppm males and females were sigSummaries of theincidences of neoplasmsand nificantly greater than those in the controls and 

nonneoplastic lesions, individual tumor animal occurred with a significant positive trend (Tables 16,

diagnoses, statistical analyses of primary neoplasms C3, and D3). The incidences of histiocytic sarcoma 

that occurred with an incidence of at least 5% inat inallexposed groups of males and in 6,000 and 

TABLE16 

Incidences of Histiocytic Sarcoma in Mice in the 2-Year Feed Study of Phenolphthalein 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

Malea 

Overall rateb 1/50 (2%) 3/50 (6%) 11/50 (22%) 12/49 (24%) 

Adjusted rate' 2.4% 7.9% 28.0% 27.5% 

Terminal rated 0140 (0%) 0133 (0%) 8/36 (22 X) 5/36 (14%) 

First incidence (days) 727 658 665 549 

Life table teste P<O.OOl P=O.255 P=O.O02 P=O.o02 

Logistic regression teste P<O.OOl P=O.302 P=O.O02 P=O.o01 

Female' 

Overall rate 0/50 (0%) 2/50 (4%) 7/50 (14%) 7/50 (14%) 

Adjusted rate 0.0% 6.5% 17.6% 18.0% 

Terminal rate 0139 (0%) 2/31 (6%) 3/34 (9%) 0128 (0%) 

First incidence (days) 2 737 (T) 567 588 

Life table test P=O.O02 P=O.189 P=O.O07 P=O.o05 

Logistic regression test P=O.O09 P=O.189 P=O.OlO P=O.O11 

~ 

(T)Terrninal sacrifice 

Historical incidence for 2-year NTP feed studies with untreated controls (mean f standard deviation): 6/1,474 (0.4% f 1.0%); 

range, 0%-2% 

Number of animals with neoplasm per number of animals necropsied 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence in animals surviving until the end of the study 

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to 

the painvise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to 

terniinal kill as being (directly or indirectly) the cause of death. The logistic regression test regards lesions in animals dying prior to 

terminal kill as nonfatal. 

Historical incidence: 19/1,473 (1.3% f 1.6%);range, 0%-4% 

Not applicable; no neoplasms in animal group 
Results 63 

12,000 ppm females exceeded the ranges in historical 

controls in NTP 2-year feed studies (Tables 16, C4a, 

and D4a). 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. In the liver, 

hepatic sinusoids were by expandedneoplastic

histiocytes, which frequently formed small nodules, 

andoccasionalthick bands, whichofteneffaced 

hepatic parenchyma (Plates 1 and 2). Nucleiof 

neoplastic cells varied from rounded to kidneyshaped, and cells contained abundantpaleeosinophilic cytoplasm. 

TABLE17 

The incidences of all typesof malignant lymphoma in 

allexposed groups of emales were significantly 

greater than thatinthe controls and exceeded the 

range in historical controls in NTP 2-year feed 

studies (Tables 17, D3, and D4b). The incidences in 

all exposed groups of males were similar to that in 

the controls (Tables 17 and C3). 

In this study, many proliferative lesions (atypical 

hyperplasia and lymphoma) that clearly arose within 

the thymus wereobservedinexposedmalesand 

females.Lymphomaswere considered to be of 

thymic origin when they were observed only in the 

Incidences of Malignant Lymphoma andof Selected Thymic Neoplasms and Nonneoplastic Lesions in Mice 

in the 2-YearFeed Study of Phenolphthalein 

Male 

Malignant Lymphoma, All Typesa 

overall rateb 

Adjustedrate' 

Terminalrated 

First incidence (days) 

Life table teste 

Logistic regression teste 

Thymusf 

Atypical Hyperplasiag 

Lymphoma,PrimarySiteThymus 

Overallrate 

Adjustedrate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Lymphoma, Primary Site Thymus 

Overallrate 

Adjustedrate 

Terminalrate 

First incidence (days) 

Life table test 

Logisticregressiontest 

(continued) 

0 PPm 

6/50 (12%) 

14.5% 

5/40 (1 3 %) 

662 

P=O.299 

P=O.324 

43 

0 

0150 (0%) 

0.0% 

0140 (0%)

-h

P=O.357 

P=O.412 

or Probably Thymus 

0/50 (0%) 

0.0% 

0140 (0%) 

-

P=O.317 

P=O.359 

3,000 ppm 6,000 ppm 12,000 ppm 

8/50 (16%) 12/50 (24%) 8/49 (16%) 

19.1% 27.2% 22.2% 

3/33 (9%) 6/36 (17%) 8/36 (22%) 

347 423 729 (T) 

P=O.300 P=O.O82 P=O.309 

P=O.396 P=O.101 P=O.353 

46 44 42 

3 I** I** 

2/50 (4%) 7/50 (14%) 1/49 (2 %) 

4.9% 15.4% 2.8% 

0133 (0%) 2/36 (6%) 1/36 (3X) 

583 423 729 (T) 

P=O.223 P=O.OlO P=O.479 

P=O.234 P=O.O06 P=O.479 

4/50 (8 %) 7/50 (14%) 2/49 (4%) 

9.8% 15.4% 5.6% 

1/33 (3%) 2/36 (6%) 2/36 (6%) 

583 423 729 (T) 

P=O.O55 P=O.OlO P=O.215 

P=O.O61 P=O.O06 P=O.215 
64 Phenolphthalein, NTP TR 465 

TABLE17 

Incidences of Malignant Lymphoma and of Selected Thymic Neoplasms and Nonneoplastic Lesions in Mice 

in the 2-Year Feed Study of Phenolphthalein (continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

Female 

Malignant Lymphoma, All Types' 

Overall rate 15/50 (30%) 28/50 (56%) 33/50 (66%) 25/50 (50%)

Adjusted rate 34.3% 60.2% 74.6% 65.8% 

Terminal rate 11/39 (28%) 13/3 1 (42 %) 23/34 (68%) 16/28 (57%) 

First incidence (days) 156 266 338 447 

Life table test P=O.OlO P=O.O03 P<O.OOl P=O.O04 

Logistic regression test P=O.114 P=O.O51 P<O.ool P=O.O35 

Thymus 48 44 49 45 

Atypical Hyperplasia 0 7** 6** 5** 

Lymphoma, Primary Site Thymus 

Overall rate 0/50 (0%) 5/50 (10%) 5/50 (10%) 4/50 (8%) 

Adjusted rate 0.0% 13.9% 13.8% 12.6% 

Terminal rate 0/39 (0%) 3/31 (10%) 4/34 (12%) 3/28 (1 1%) 

First incidence (days) - 266 604 450 

Life table test P=O.O86 P=O.O23 P=O.O25 P=O.O37 

.Logistic regression test P=O.157 P=O.O38 P=O.O30 P=O.O65 

Lymphoma, Primary Site Thymus or Probably Thymus 

Overall rate 1/50 (2%) 9/50 (18%) 10150 (20%) 7/50 (14%) 

Adjusted rate 2.6% 24.9% 25.8% 23.1% 

Terminal rate 1/39 (3 %) 6/31 (19%) 7/34 (21 %) 6/28 (21 %) 

First incidence (days) 737 (T) 266 55 1 450 

Life table test P=O.O44 P=O.O05 P=O.O04 P=O.O11 

Logistic regression test P=O.106 P=O.O11 P=O.O05 P=O.O25 

(T)Terminal sacrifice 

** Significantly different (PSO.Oi) from the control group by the logistic regression test 

Historical incidence for 2-year NTP feed studies with untreated controls (mean f standard deviation): 130/1,474 (8.8% f 6.0%); 

range, 2%-24% 

Number of animals with neoplasm per number of animals necropsied 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence in animals surviving until the end of the study 

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to 

the pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to 

terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards lesions in animals dying prior to 

terminal kill as nonfatal. 

Number of animals with thymus examined microscopically 

Number of animals with lesion 

Not applicable; no neoplasms in animal group 

Historical incidence: 339/1,473 (23.0% f 11.9%); range, 6%-44% 
Results 65 

thymus or within the thymus and metastatic only to 

other sites within the chest cavity. The incidences of 

lymphomaofthymic origin wereincreasedin 

exposed groups of males, but weresignificantly 

increasedonlyinthe 6,000 ppm group, and the 

incidences of lymphoma of thymic origin in all 

exposed groups of females were significantly greater 

(bythelifetabletest)thanthatinthe controls 

(Table 17). In the other exposed groups, where the 

lymphomawaslargestinthethymuswithminimal 

involvementof other sites, the thymus was consideredthe “probable” site of origin. Additionally, 

the incidences of atypical hyperplasia of the thymus 

in 6,000 and 12,000 ppm malesandinallexposed 

groups of femalesweresignificantly greater than 

those in the controls. 

After puberty, there is a gradual involution of the bilobedthymictissuewithadvancing age, and while 

the amount of thymic tissue is reduced at2 years, the 

morphological structure of the outer cortex and inner 

medullaisgenerallymaintained.Thethymiccortex 

is generally composed of smallerhyperchromatic 

lymphocytes, whileeosinophilic more staining 

lymphocytesandepithelialcells are major constituents of the medulla. In this study, animals with 

hyperplasia of the thymus often had a normal appearing lobe along with an abnormal lobe(Plates 3 and 

4). The abnormal lobe wasgenerallysmallerthan 

the normal lobe and lacked a distinct cortex and 

medulla. Rather, abnormal lobeswerecomposed of 

sheets of large lymphocytes combined with variable 

numbers of smaller, more normal appearing lymphocytes (Plate 5). The large lymphocytes hadstippled 

chromatin, scant to moderate amounts of cytoplasm, 

and one or two fairly distinct nucleoli.Themitotic 

,ndex was variable. In the earliest lesions diagnosed 

as malignant lymphomas, a fairlyhomogeneous 

population of lymphocytes (as described previously) 

extendedbeyondtheconfinesofnormalthymic 

tissue, and mitotic figures were common. Larger 

thymic lymphomas occupied a larger portion of the 

chest cavity. 

Ovary: There were increasedincidencesofbenign 

ovarianneoplasmsandassociated hyperplasia of a 

rather distinctive morphology in all exposed groups 

of females (Tables 18, Dl, D3, and D5). The 

incidences of benign sex-cord stromal tumors of the 

ovary in all exposed groups of females were significantly greater than that in the controls and exceeded 

the range for ovarian luteomas in historical controls 

inNTP 2-year feed studies (Tables 18, D3, and 

D4c). The incidences of hyperplasia of the ovary in 

3,000 and 12,000 ppm femalesweresignificantly 

greaterthanthatinthe controls. The neoplasms 

varied from occupying most of the ovary to markedly 

expanding it (three to four times) with compression 

of the surrounding parenchyma (Plates 6 and 7). 

Neoplasms were usually composed of sheets of round 

to oval cellswithabundant cytoplasm that varied 

from finely granular and eosinophilic to vesiculated 

(luteinized). In most neoplasms, the cells were rather 

homogeneous with a low mitotic index; however, in 

some of the larger neoplasms, there was a modest 

degree of cellular pleomorphism and a higher mitotic 

rate. Hyperplasiaswereobservedwithin the ovarianstromaandwerecomposed of cells that were 

morphologicallysimilar to those in the neoplasms 

(Plates 8 and 9). However, hyperplasias were 

smaller, and componentcellsusuallyblendedwith 

the surrounding parenchyma with little or no compression. The histomorphology of the granulosa cell 

tumorsidentifiedin a control female and in a 

3,000 ppm femalewastypicalof granulosa cell 

tumors and was distinctly different from the benign 

sex-cordstromal tumors observed exposed in

females. 
66 Phenolphthalein, NTP TR 465 

TABLE18 

Incidences of Neoplasms and Nonneoplastic Lesions of the Ovary in Female Mice 

in the 2-Year Feed Study of Phenolphthalein 

Number Examined Microscopically 50 

Hyperplasiaa 4 (1.5)b 

Benign Sex-Cord Stromal Tumor, Bilateral 0 

Benign Sex-Cord Stromal Tumor' (Includes Bilateral) 

Overall rated 

Adjusted ratee 

Terminal ratef 

First incidence (days) 

Logistic regression testg 

Benign Granulosa Cell Tumor 

(T)Terminal sacrifice 

0150 (0%) 

0.0% 

0139 (0%) - 668 

P=O.O66 

1 

3,000 ppm 6,000 ppm 12,000 ppm 

11* (2.0) 

49 

10 

50 

(2.6) 17** 

50 

(2.2) 

1 0 0 

P=O.O04 

737 (T) 

6/31 (19%) 

21.7% 

7/49 (14%) 

P=O.Oll 

737 (T) 

6/34 (18%) 

17.6%17.9% 

6/50 (12%) 

P=O.O12 

5/28 (18%) 

5/50 (10%) 

1 0 0 

* Significantly different (PSO.05)from the control group by the logistic regression test 

** PSO.01 

a Number of animals with lesion 

Average severity of lesions in affected animals: 1 = minimal; 2 = mild; 3 = moderate: 4 = marked 

Historical incidence of ovarian luteoma for 2-year NTP feed studies with untreated controls (mean f standard deviation): 5/1,436 

(0.4%f 1.0%);range, 0%-4% 

Number of animals with neoplasm per number of animals with ovary examined microscopically 

e Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence in animals surviving until the end of the study 

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to 

the pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions in animals dying 

prior to terminal kill as nonfatal. 

Not applicable; no neoplasms in animal group 

Testis: The incidences of germinal epithelial degeneration of the testis in all exposed groups of males 

were significantly greater than inthe controls 

(Tables 19 and C5). The lesion varied from involvement of a few to all seminiferous tubules. Within the 

tubules, there was variable loss of sperm and germinal epithelial cells (spermatogonia, spermatocytes, 

and spermatids), often with only Sertoli cells remaining (Plates 10 and 11). In the most severe cases, the 

cross-sectional diameter of the testis was decreased. 

Although more severe, this lesion is consistent with 

that observed in the 13-week study. 

Bone marrow: There were increasedincidencesof 

myelofibrosisofthebone marrow in 12,000 ppm 

males (Tables 19 and C5) and an increased severity 

of this lesion in exposed females (Table 19). In this 

study, the fibro-osseous lesion generally partially 

replaced the bone marrow cavity and was composed 

of minimal to mild proliferation of spindle cells in an 

eosinophilic, often fibrillar, matrix. In advanced 

lesions, the spindle cell proliferation was more 

extensive, and there was more osteosclerosis. 

Additionally, as observed in the 13-week study, there 

were increased incidences of pigmentation (probably 
Results 67 

TABLE19 

Incidences of Selected Nonneoplastic Lesions in Mice in the 2-Year Feed Study of Phenolphthalein 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

Male 

Bone Marrowa 50 50 50 49 

Myelofibrosisb 3 (1.O)C 8 (1.3) 8 (1.3) 19**(1.3) 

Pigmentation 0 2 (1.0) 5* (1.8) 16** (1.8) 

Kidney 50 50 50 49 

Renal Tubule, Accumulation, 

Hyaline Droplet 0 2(2.5) 5* (2.6) 5* (2.8) 

Spleen 50 50 50 49 

Hematopoietic Cell Proliferation 10(1.9) 22* (2.3) 28** (2.5) 21* (2.6) 

Testes 50 50 50 48 

Germinal Epithelium, Degeneration 1 (1.0) 49** (2.0) 50** (3.0) 47** (2.7) 

Female 

Bone Marrow 50 50 50 50 

Myelofibrosis 34 (1.4) 34 (2.7) 38 (2.0) 36 (2.3) 

Pigmentation 2 (1.5) 3(1.3) 11**(1.9) 11** (2.2) 

Kidney 50 50 50 50 

Renal Tubule, Accumulation, 

Hyaline Droplet 0 1(1.0) 3 (2.0) 8** (2.0) 

Spleen 50 50 50 50 

Hematopoietic Cell Proliferation 13 (2.8) 14 (2.5) 20 (2.7) 21 (2.5) 

* Significantly different (PlO.05) from the control group by the logistic regression test 

** PSO.01 

a Number of animals with tissue examined microscopically 

Number of animals with lesion 

Average severity of lesions in affected animals: 1 = minimal; 2 

hemosiderin) of minimal to mild severity in the bone 

marrow of 6,000 and 12,000 ppm males and females 

(Tables 19, C5, and D5). 

Kidney: The incidences of hyaline droplets within 

the renal tubules in 6,000 and 12,000 ppm males and 

in12,000ppm females were significantly greater 

than those in the controls (Tables 19, C5, and DS). 

Mildtomoderatenumbers of eosinophilic, intracytoplasmichyalinedroplets were observed within 

theproximal convoluted renal tubules. Generally, 

mice with renal hyaline droplets also had histiocytic 

sarcomainotherorgans. This renal change is not 

= mild; 3 = moderate; 4 = marked 

uncommon in mice with histiocytic sarcomas. It has 

been shown that histiocytic sarcomas may produce 

excessive amounts of lysozyme, which may accumulate in the proximal renal tubule (Barsoum et al., 

1984;Hard and Snowden, 1991 ; LuzandMurray, 

1991). 

Spleen: The incidences of hematopoietic cell proliferation in the red pulp of the spleen in all exposed 

groups of males were significantly greater than in the 

controls (Tables 19 and D5), and the severity of this 

lesion increased with increasing exposure concentration. Hematopoiesis is aprocessthat normally 
cusa Cell 

Focus 

us Cell 

68 

occurs to some extent in the spleen of B6C3F, mice; 

therefore, in this study, the diagnosis was generally 

made when the amount of hematopoietic cell proliferationexceededanestablished threshold. Thisminimal change is consistent with thatidentifiedinthe 

13-week study and is considered related to administration of phenolphthalein. 

Liver: The incidences ofhepatocellularadenomain 

allexposed groups of malesandfemalesandof 

hepatocellular adenoma or carcinoma (combined) in 

6,000 and 12,000 ppm males and all exposed groups 

offemalesweresignificantlylessthanthoseinthe 

controls, and the incidences of these lesions occurred 

with significant negative trends (Tables 20, C3, and 

D3). The incidences ofhepatocellularadenoma or 

carcinoma (combined) in all control and exposed 

groups were within the ranges in historical controls 

in NTP 2-year feed studies(TablesC4candD4d). 

TABLE20 

Phenolphthalein, NTP TR 465 

Multiplehepatocellularadenomaswereobserved 

morefrequentlyinthe control groups than in the 

exposed groups (Tables C1 and Dl). The incidences 

of clear celland eosinophilic foci in allexposed 

groups of malesof cell andmixed foci in 

12,000 ppm males were significantly less than those 

in the controls (Table C5). The incidences of eosinophilic foci in exposed groups of females were significantly less than that in the controls (Table DS). Foci 

of cellular alteration, adenomas, and carcinomas 

generally represent a spectrum in the progression of 

neoplasiainthe liver of B6C3F, mice. Although 

thereis a known associationoftheincidences of 

theseneoplasmswithbodyweightinthe B6C3F, 

mouse,theslightly lower mean bodyweightsof 

exposed groups could not account for the decreased 

incidencesobservedin this study. These decreases 

wereconsideredrelatedtothe administration of 

phenolphthalein. 

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice in the 2-Year Feed Study 

of Phenolphthalein 

Male 

Number Examined Microscopically 

Clear 

Eosinophilic 

Mixed 

Hepatocellular Adenoma, Multiple 

Hepqtocellular Adenoma (Includes Multiple) 

Overallrateb 

Adjustedrate' 

Terminalrated 

Firstincidence(days) 

Logisticregressionteste 

Hepatocellular Carcinoma (Includes Multiple) 

Overallrate 

Adjustedrate 

Terminalrate 

First incidence (days) 

Logistic regression test 

Hepatocellular Adenoma or Carcinomaf 

Overallrate 

Adjustedrate 

Terminalrate 

First incidence (days) 

Logistic regression test 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

6 

22 

24 

50 

2 

6** 

6** 

50 50 

1 

1** 

1** 

49 

O* 

1** 

0** 

12 1 0 1 

P=O.OION 

582 

21/40 (53%) 

53.5% 

22/50 (44%) 12/50 (24%) 

29.3% 

6/33 (18%) 

563 

P=0.032N 

8/50 (16%) 

19.6% 

5/36 (14%) 

495 

P=0.003N P=O.Ol IN 

522 

7/36 (19%) 

25.0% 

10/49 (20%) 

P=O.533 

727 

7/40 (1 8 %) 

19.5% 

8/50 (16%) 10/50 (20%) 

25.O % 

5/33 (15%) 

560 

P=O.385 

5/50 (10%) 

11.8% 

2/36 (6%) 

500 

P=0.273N P=O.474 

639 

5/36 (14%) 

22.0% 

9/49 (1 8 %) 

27/50 (54%) 

64.2% 

25/40 (63%) 

582 

P=O.O16N 

20/50 (40%) 

45.3% 

10133 (30%) 

560 

P=0.123N 

11/50 (22%) 

25.8% 

6/36 (17%) 

495 

P=O.OOlN P=0.026N 

522 

11/36 (31%) 

38.4% 

16/49 (33%) 

(continued) 
Results 69 

TABLE20 

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice in the 2-Year Feed Study 

of Phenolphthalein (continued) 

Female 

Number Examined Microscopically 

Clear Cell Focus 

Eosinophilic Focus 

Mixed Cell Focus 

Hepatocellular Adenoma, Multiple 

Hepatocellular Adenoma (Includes Multiple) 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Logistic regression test 

Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Logistic regression test 

Hepatocellular Adenoma or CarcinomJ’ 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Logistic regression test 

0 PPm 

50 

1 

20 

3 

5 

17/50 (34%) 

41.2% 

15/39 (38%) 

591 

P<O.OOlN 

6/50 (12%) 

14.9% 

5/39 (13%) 

708 

P=0.042N 

2 1/50 (42 %) 

49.8% 

18/39 (46%) 

597 

P<O.OOlN 

3,000 ppm 

50 

2 

4** 

L 

0 

2/50 (4%) 

6.5% 

2/31 (6%) 

737 (T) 

P<O.OOlN 

1/50 (2%) 

3.2% 

1/31 (3%) 

737 (T) 

P=0.099N 

3/50 (6%) 

9.7% 

3/31 (10%) 

737 (T) 

P<O.OOlN 

6,000 ppm 

50 

0 

2** 

1 

0 

6/50 (12%) 

17.1% 

5/34 (15%) 

693 

P=0.015N 

0150 (0%) 

0.0% 

0134 (0%)

2 

P=0.025N 

6/50 (12%) 

17.1% 

5/34 (15%) 

693 

P=0.002N 

12,000 ppm 

50 

0 

1** 

0 

0 

1/50 (2%) 

3.6% 

1/28 (4%) 

737 (T) 

P<0.001N 

1/50 (2%) 

3.6% 

1/28 (4%) 

737 (T) 

P=O. 11 1N 

2/50 (4%) 

7.1 % 

2/28 (7%) 

737 (T) 

P<0.001N 

(T)Terminal sacrifice 

* Significantly different (P10.05)from the control group by the logistic regression test 

** P50.01 

a Number of animals with lesion 

Number of animals with neoplasm per number of animals with liver examined microscopically 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence in animals surviving until the end of the study 

e In the control column are the P values associated with thetrend test. In the exposed group columns are the P values corresponding to 

the pairwise comparisons between the controls and that exposed group. The logistic regression test regards lesions in animals dying 

prior to terminal kill as nonfatal. A negative trend or lower incidence in an exposure group is indicated by N. 

Historical incidence for 2-year NTP feed studies with untreated controls (mean f standard deviation): 596/1,465 (40.7% f 14.5%); 

range, 10%-68% 

Not applicable; no neoplasms in animal group 

Historical incidence: 313/1,464 (21.4% f 13.0%); range, 3%-56% 

Tooth: There were decreasedincidencesofdegen- formation, and hyperplasia of various cellular eleeration of theteeth in all exposed groups of male ments associatedwith tooth formation. Because this 

mice (0 ppm, 22/49; 3,000 ppm, 12/50; 6,000 ppm, lesion occurs more frequently in males, it is possible 

4/50; 12,000 ppm, 1/49;Table C5) Degenerationof that male hormones play a role in its development or 

theteethis a common spontaneouslesioninmale that female hormones are protective. The decrease in 

B6C3F, mice andischaracterized by deformity and theincidence of this lesionwas considered to be 

distortion of dental profiles, excessive dentine related to phenolphthalein administration. 
70 Phenolphthalein, NTP TR 465 

Pituitary gland: The incidencesof hyperplasia of the 

pars distalis of the pituitaryglandin 6,000 and 

12,000 ppm females were significantly less than that 

in the controls (0 ppm, 11/49; 3,000 ppm, 4/50; 

6,000 ppm, 1/49; 12,000 ppm, 1/48; Table D5). 

Becauseofthe central role thepituitaryglandin 

hormonal homeostasis, it is probablethatthis 

decrease in the incidence of hyperplasia is related to 

the administration of phenolphthalein.However,its 

significance and mechanismof relationship to phenolphthalein administration are not understood. 

Thyroid gland: There were decreasedincidences of 

follicular cell hyperplasia of the thyroid gland in all 

exposed groups of females (27/50, 8/50, 3/50, 7/50; 

Table D5). However, all hyperplasiasobservedin 

the control mice wereofminimal severity. As with 

the pituitary gland, this change isofquestionable 

biological significance, but is also probably related to 

exposure to phenolphthalein. 

GENETICTOXICOLOGY 

Phenolphthalein, tested independently in two laboratories, was not mutagenic inSalmonella typhimurium 

strain TA98, TA100, TA1535, or TA1537, with or 

withoutinduced rat or hamster liver S9 metabolic 

activation enzymes (Mortelmans et al., 1986; 

Table El). In cytogenetic tests with cultured Chinese 

hamster ovary cells, no induction of sister chromatid 

exchanges was observed after treatment with 0.5 to 

50 pg/mL phenolphthaleinwith or without S9 

(Table E2). Cytotoxicity was noted at the 50 pg/mL 

dose level, and culture times werextended to 

maximize the proportion of cells available for analysis at this dose level. Results of the in vitro chromosomal aberrations test with phenolphthalein were 

positive for the two trials conducted with S9; no 

increase in chromosomal aberrations was observed in 

the absence of S9 (Witt et al., 1995; Table E3). At 

the 50 pg/mL dose level, approximately one quarter 

of all cells contained aberrations, and most of these 

were chromosome breaks located at the distal end of 

X,. The significance of this preferential breakage is 

asyet unknown, but it hasbeennotedwith other 

chemicals, and it occurs only in the presence of S9. 

A discussion of possible mechanisms for this 

phenomenon is presented in Galloway et aZ. (1987). 

Results from the in vivo mouse peripheral blood 

micronucleustestwere positive for both male and 

female mice (Dietz et al., 1992; Table E4). Significant increases in micronuclei were noted for both the 

normochromaticand polychromatic erythrocyte 

populations. The increases in micronucleated normochromatic erythrocytes, which indicate the effects of 

chronic exposure, occurred at all exposure concentrationsinmalesand females. The effects on polychromatic erythrocytes, which indicate chromosomal 

damageinducedwithinthe 48 hours preceding 

analysis, occurred only at 25,000 and 50,000 ppm, 

the two highest exposure concentrations. 
PLATE 1 

Histiocytic sarcoma of the liver of a male B6C3Fl mouse exposed to 

12,000 ppm phenolphthalein in feed for 2 years. Note the thick band 

of neoplastic histiocytes within the liver. H&E; 50 X 

PLATE 3 

Thymus of a male B6C3F, mouse exposed to 12,OOO ppm 

phenolphthalein in feed for 2 years. The section to the left is normal 

thymic tissue with a darkly stained peripheral cortex (large arrow) and 

an inner medullary area (small arrow). The section to the right is 

abnormal without a distinct cortex or medulla. H&E; lox 

PLATE 2 

A higher magnification of the histiocytic sarcoma shown in Plate 1. 

The upper half demonstrates the variability in nuclear shape and the 

abundant cytoplasm present in the neoplastic cells. In the lower half, 

neoplastic cells (large arrow) are present within expanded hepatic 

sinusoids. Also note few remaining hepatocytes (small arrow). 

H&E; 13OX 

PLATE 4 

Normal thymus of a control male B6C3Fl mouse in the 2-year feed 

study of phenolphthalein. The cortex is to the left and the medulla in 

the upper right. H&E; 13OX 
PLATE 5 

Higher magnification of Plate 3. Note the population of large 

lymphocytes with rounded to oval nuclei and scant cytoplasm. 

Compare to normal thymic tissue in Plate 4. H&E; 130~ 

PLATE 7 

Relatively normal ovary of a control female B6C3Fl mouse in the 

2-year feed study of phenolphthalein. H&E; 20 X 

PLATE 6 

Ovary ofa female B6C3F, mouse exposed to 12,OOO ppm 

phenolphthalein in feed for 2 years. Note the expansile mass composed 

of a homogeneous sheet of cells within and expanding the ovary. 

Compare with normal ovary in Plate 7 at much higher magnification. 

H&E; 6X 

PLATE 8 

Ovary ofa female B6C3Fl mouse exposed to 6,OOO ppm 

phenolphthalein in feed for 2 years. Note the area of hyperplasia 

(arrows) within the ovary. H&E; 20 X 
PLATE 9 

A higher magnification of Plate 8. Note the component cells with 

abundant finely granular (right) or vesiculated (left) cytoplasm. 

H&E; 130~ 

PLATE 10 

Marked degeneration of the seminiferous tubules in the testis of a male 

B6C3F, mouse exposed to 12,OOO ppm phenolphthalein in feed for 

2 years. variable loss Note the of germinal epithelium within tubules. 

Compare to a normal testis in Plate 11. H&E; 65 X 

PLATE 11 

Testis of a control male B6C3F, mouse in the 2-year feed study of 

phenolphthalein. Note normal appearing germinal epithelium. 

H&E; 65x 
71 

DISCUSSION AND CONCLUSIONS 

Phenolphthalein wasnominated by theNational 

Cancer Institute for toxicityandcarcinogenicity 

studies because it is an over-the-counter laxative for 

which no carcinogenesis bioassay has been reported 

inthe literature. Phenolphthalein is usuallytakenas 

a 60 mg oral dose, four times a day, for an approximatedaily dose of 4 to 5 mg/kg. Theprobable 

mechanism for the laxative effect is stimulation of the 

mucosal nerve plexus inthe colon, combined with 

water and electrolyte secretion or inhibition of water 

absorption in both large and smallintestines,and 

decreased glucose absorption (Pietrusko, 1977; 

Saunders et al., 1978; Goodmanand Gilman’s, 

1990). 

In the 13-weekfeed studies, rats andmicewere 

exposedto phenolphthalein at concentrationsup to 

50,000 ppm. All rats and micesurvivedtotheend 

ofthe studies. No chemical-related clinical signs of 

toxicity were observed in rats or mice, and there was 

no evidence of a laxative effect in these animals. In 

the 13-week studies, there were no chemical-related 

gross or microscopic lesions inrats.However, 

hypoplasia of the bone marrow occurred in male and 

female mice exposed to 12,000 ppm or greater. This 

lesion was characterizedbydecreasedamounts of 

hematopoietictissuewith a decreased myeloidto 

erythroid ratio. Splenic hematopoiesis was observed 

inmalemiceexposed to 25,000 or 50,000 ppm. 

There were also effects on the malereproductive 

system in mice. There was no evidence of reproductivetoxicityinfemaleB6C3F,mice or inmale or 

female F344/N rats. Lower epididymal weightsand 

sperm density and an increased incidence of abnormal sperm were observed in male mice at all exposure concentrations evaluated (12,000, 25,000, and 

50,000 ppm). In some seminiferoustubulesone or 

more generations of germ cells in portions of or in 

the entire tubule were missing. Oftensuchtubules 

were surrounded by seminiferous tubules that looked 

normal. This suggested an intermittent commitment 

of spermatogonia to differentiation, producing a 

“blank space” in the progression of cells through 

spermatogenesis. The lesion may be mimicked by 

dosingwith a toxicant for a limited periodoftime 

followed by quick recovery of the testis, as has been 

observedin rats administered 2-methoxyethanolin 

drinkingwater (NTP, 1993a). However, this is the 

only instance in the history of the NTP where continued exposure to a chemical has apparently caused 

stops and starts in spermatogenesis. 

In order to further analyze the reproductive toxic 

effectsinmice, a continuous breeding study of 

phenolphthaleinwasconductedinSwiss (CD-l@) 

miceaccording to a protocol previously described 

(Lamb, 1985; Heindel e? al., 1989). In this study, 

maleandfemalemicewere given 1,000, 7,000, or 

30,000 ppm phenolphthaleininfeed for a 98-day 

cohabitation period, and fertility during this period 

was assessed (Appendix N; NTP, 1991). Phenolphthaleincausedlower fertility in the 7,000 and 

30,000 ppm groups. Overall, the mean numbers of 

litters per pair in the 7,000 and 30,000 ppm groups 

were 24 % and 50% lower than in the controls. The 

numbers of live pups per litter in these groups were 

58% and 59% lower than in the controls. There was 

alsopostnataltoxicityinthe F, generation, as indicated by a 30% to 7 1% lower survival than that of 

the controls (all of the deaths occurred during the 

first 4 days of life). 

Phenolphthalein can cross the placental barrier in 

mice (strain not specified) (Visek e?al., 1956). The 

lower survival in the F, generation observed in this 

continuousbreedingstudyinSwiss (CD-l@) mice 

maybe attributed to effects to the developing fetus 

from in utero exposure, postnatal exposure via the 

milk, or from a combination of these exposures. No 

reproductive or teratogenic effects were observed in 

mice (strain notspecified) exposed to 250 mg 

phenolphthaleidkg body weight per day in feed 

(Stockinger, 1965), but this study was conducted at 

lower doses than those used in the continuous breeding study. In the continuous breeding study, the 

exposure concentrations of 7,000 and 30,000 ppm 

wereequivalent to daily doses of approximately 

1,056 and 4,530 mg/kg, respectively. 
72 

In a crossover mating trial with Swiss (CD-l@) mice 

(control males mated to 7,000 ppm females or control 

females mated to 7,000 ppm males), it was found that 

the lower fertility, as measured by a lower number of 

pups born per litter, resulted from effects inthe 

female mice. It should be noted that the 7,000 ppm 

male Swiss (CD-l@) mice exhibited approximately a 

20% lower sperm count. relative to the controls, but 

the lower sperm count did not affect fertility. The 

relationship between lower sperm counts and fertility 

in a number of species is an area of continuing 

investigation (Medical Research Council, 1995). 

In summary, phenolphthalein produced significant 

reproductive toxicity inbothSwiss (CD-I@) and 

B6C3F, mice as measuredbylowerepididymal 

weightand sperm density. Exposed female 

Swiss (CD-l@) mice mated to control male

Swiss (CD-l@) mice had fewer than half the number 

of live pups per litter as controls. 

The mechanism for the reproductive toxicity 

observed in mice (but notin rats) is notfully 

understood. Phenolphthalein contains a triphenylmethane structure, and this structure may actas a 

nonsteroidal estrogen agonist or antagonist through 

interaction with the estrogen receptor protein (Ravdin 

et al., 1987). Studies by other groups are underway 

to assess whether or not environmental estrogenic 

compounds play a significant role in reproductive 

disorders in humans (Jobling etal., 1995; Medical 

Research Council, 1995). 

In the 2-year feed studies of phenolphthalein, there 

were no chemical-related effects on survival in male 

or female rats or male mice; there was marginally 

lower survival in the 12,000 ppm femalemice.No 

chemical-related clinical signs were observed. 

Mean body weights of exposed groups of rats were 

5 % to 10% lower than those of the controls during 

most of the study, but toward the end of the study, 

the body weight differences became more pronounced. The body weight differences in mice were 

less than those in rats. 

Feed consumption by exposed groups was similar to 

that by the controls. Comparisons of doses based on 

body surface area are provided in Table 21. 

Phenolphthalein, NTP TR 465 

At the end of the 2-year study, organ toxicity and/or 

carcinogeniceffectswere observed in the adrenal 

gland of rats. These effects developed late in the 

study, and no toxicity was observed in these organs 

in the 13-week rat study. 

The incidences of benign pheochromocytoma of the 

adrenalmedullaweresignificantlyincreasedin 

all groups of exposed male rats and in 12,000 ppm 

female rats. The incidences of benign pheochromocytomainall groups of exposedmale rats and in 

12,000 and 25,000 ppm female rats exceeded the 

rangesinhistorical controls in NTP 2-year feed 

studies. The incidence of benign or malignant 

pheochromocytoma (combined) of the adrenal medulla in 12,000 and 25,000 ppm female rats was significantly greater than that of the controls and exceeded 

therangeinhistorical controls in NTP 2-year feed 

studies. In the NTP database of approximately 

450 chemicals, a chemical-induced neoplasm

response is observed in the adrenal gland of rats two 

to three times more frequently than in mice. These 

increases in the incidences of pheochromocytoma of 

the adrenal medulla were considered to be related to 

exposure to phenolphthalein because the effect was 

observedinmalesand females, and in the groups 

cited above the neoplasm rates were greater than 

those of the concurrent and historical control rates. 

However, increased incidences were not observed in 

50,000 ppm female rats. 

The incidence of chronic nephropathy, which is 

commoninaging rats (particularly males), was 

increased in exposed groups of female rats, and the 

severityofnephropathywas increased in exposed 

groups of male rats and slightly increased in exposed 

groups of female rats. In males, changes secondary 

to the exacerbatedrenal disease and indicative of 

compromisedrenal function (hyperplasia of the 

parathyroid gland, fibrous osteodystrophy of the 

bone, and mineralization and degeneration of the 

glandular stomach) were also increased, but not in a 

dose-related manner. Additionally, there were 

increasesintheincidencesof hyperplasia and neoplasms of the renal tubule epithelium. These lesions 

were confirmed by an extended evaluation of the 

kidneys. No mechanism by which these renal proliferative lesions developedwasreadily apparent, nor 

werethesestudiesdesigned to determine a mechanism. Onepossiblemodeof action is consistent 
Discussion and Conclusions 73 

TABLE21 

Summary of Feed and Compound Consumption by Rats and Mice in the 2-YearFeed Studies 

of Phenolphthalein 

Male Rats 

Mean for Weeks 1-13 

(g feedlday) 

(mglkg per day) 

(mglm’ per day)a 

Mean for Weeks 53-104 

(g feedlday) 

(mglkg per day) 

Female Rats 

Mean for Weeks 1-13 

(g feedlday) 

(mglkg Per day) 

(mglm’ per day) 

Mean for Weeks 53-105 

(g feedlday) 

(mglkg per day) 

MaleMice 

Mean for Weeks 1-13 

(g feedlday) 

(mglkg Per day) 

(mglm’ per day) 

Mean for Weeks 53-104 

(g feedlday) 

(mgk per day) 

Female Mice 

Mean for Weeks 1-13 

(g feedlday) 

(mgk per day) 

(mglm’ per day) 

Mean for Weeks 53-105 

(g feedlday) 

(mglkg per day) 

12,000 ppm 25,000 ppm 50,000 ppm 

16.3 16.2 17.0 16.6 

0 789 1,714 3,375 

0 4,102 8,912 17.550 

14.4 15.3 15.9 15.8 

0 434 953 1,880 

10.7 10.4 10.4 11.0 

0 787 1,655 3,423 

0 4,092 8,603 17,799 

11.4 11.3 11.4 11.8 

0 477 1.013 2,125 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

4.3 4.3 4.4 4.2 

0 453 940 1.802 

0 1.359 2,820 5.406 

4.8 4.8 4.9 4.8 

0 291 589 1,181 

5.1 4.9 5.3 5.1 

0 65 1 1.407 2,640 

0 1,953 4,221 7.920 

5.3 5.3 5.2 5.2 

0 311 617 1.255 

a Calculation for body surface area dose based on Freireich et nl., 1966; mglm’=K,,, x (dose in mglkg), where K,,,is 37 for humans, 

5.2 for rats, and 3.0 for mice. (K,, is a conversion based on average height-to-body weight ratio.) Compound consumption values for 

humans=5 mglkg and 185 mglm’. 
74 

with the theory of increased cell replication providing 

a “fertile ground” for increased mutation rates and 

neoplasm development. To a certain point, increased 

kidney damage is thought to increase the amount of 

renal tubule epithelial regenerationviacellreplication. In one study, r3H]-thymidine labelingdemonstratedincreasedlevels ofDNA synthesistobe 

directly proportional to increased severityofnephropathy in aging female F344/NCr rats (Konishi and 

Ward, 1989). 

The findings in themaleratkidney are similar to 

those observed with other chemicals including quercetin (NTP, 1992), coumarin (NTP, 1993b), and 

3,4-dihydrocoumarin (NTP, 1993~). With ese 

chemicals, there was no evidence for toxicity to the 

kidney in the 13-week studies, but toward the end of 

the 2-year studies, theseverity of nephropathy 

increasedinmale rats (and to a lesserextentin 

female rats), and there were a few kidney neoplasms 

inmale rats. The greater sensitivity of the malerat 

to this kidney toxicity is apparently due to a greater 

susceptibility of male rats to spontaneous nephropathy 

during aging and the exacerbation of this disease by 

chemical administration. Changesglomerular in 

permeability, resulting in proteinuria, progressive 

glomerular sclerosis, tubule damage, inflammation, 

and interstitial fibrosis, are associatedwiththe 

process of aging in rats. 

There was a decrease in the incidence of mammary 

gland fibroadenomas in groups of exposed female 

rats. Previous studies have shownthatdecreasesin 

theincidences of somenaturally occurring benign 

neoplasms, such as mammary gland fibroadenoma, 

are associated withlowerbodyweightsrelativeto 

those of the controls (Rao etal., 1987; Seilkop, 

1995). The lower incidences of female rat mammary 

gland neoplasms in this study were also thought to be 

related to the lower body weights. 

Attheend of the 2-year mouse study, carcinogenic 

effects were observed in the hematopoietic system of 

malesandfemalesandtheovary of females. There 

were increases in the incidences of histiocytic sarcomas and malignant lymphomas of thymic origin in all 

exposed groups of male andfemalemice.These 

effects were clearly related to exposure to phenolphthalein. The incidences of histiocytic sarcoma in 

all groups of exposed male and female mice exceeded 

Phenolphthalein, NTP TR 465 

the ranges in historical controls in NTP 2-year feed 

studies. Theincidencesofmalignantlymphoma 

(all types) in all exposed groups of female mice also 

exceededtherangesinhistorical controls in NTP 

2-year feed studies. 

Chemical-associatedincreasesin the incidences of 

histiocyticsarcoma are uncommon in NTP studies; 

however, increased incidences have been observed in 

thestudieswith 1,3-butadiene (NTP, 1984b, 1993d) 

(an increase in thymic lymphomas also occurred) and 

tetrafluoroethylene (NTP, 1996a). Spontaneous 

histiocyticsarcoma occurs two to three times more 

frequentlyinfemalemicethaninmale mice. Similarly, in the tetrafluoroethylene mouse study, incidences in groups of females were about twice those 

observedin groups of males. The reverse was true 

inthe current study. Histiocytic sarcomas occur 

most commonly in the liver, but are often widespread 

andinvolvenumerous tissues. In the current study, 

histiocytic sarcoma was identified in the liver of all 

but three mice diagnosedwith histiocytic sarcoma. 

Histiocytic sarcomas are generally considered to arise 

from a macrophage/histiocyte including the specializedKupffercellofthe liver, but definitive data 

relativetothesiteof origin are lacking. In studies 

wherehistiocytic sarcomas were transplanted, the 

localesionsgenerallyremained small, while the 

liverbecamequite large (Frith et al., 1980). There 

is also evidence that many of these neoplasms produce excessive amountsof lysozyme, which can 

result in the accumulation of hyaline droplets in the 

proximal renal tubules as apparently occurred in the 

current study. 

Exposuretophenolphthaleinwasassociatedwith a 

clear increase in the incidence of malignant lymphoma (alltypes)in groups of exposed female mice. 

However, significant increased incidences ofproliferativelymphocyticlesions that arose withinthe 

thymus were observed in both male andfemale mice. 

A number of classification schemes for malignant 

lymphomas (Dunn, 1954; Pattengale and Taylor, 

1983; Wogan, 1984), primarily based upon histomorphology, have been proposed. However, it is 

oftendifficult to categorize consistently the various 

lymphomas. Further, as more is learned, it appears 

that there is little biological relevance for some of the 

classificationschemes. Therefore, in mostrecent 

NTP studies, all lymphomashavebeencombined 
Discussion and Conclusions 75 

underthediagnosis of malignant lymphoma. However, under certain circumstances, subclassification of 

lymphomas may be necessary to better understand the 

effects associated with an administered agent. In the 

B6C3F,mouse,the vast majority of spontaneous 

lymphomasarisefrom within the spleen or lymph 

nodes and are of B-cell origin. Lymphomas of the 

T-cells are much less common, and lymphomas 

arisingfromthe thymus areuncommon. With 

phenolphthalein,1,3-butadiene, and 2’,3’-dideoxycytidine (ddC) (Sanderset al., 1995), the lymphomas 

originated primarily in the thymus.In this study, 

many proliferative lesions (atypical hyperplasias and 

lymphomas)clearlyarose within the thymus of 

exposed males and females. Lymphomas were considered of thymic origin when they were observed 

only in the thymusorin the thymus and metastatic 

only to other sites within the chest cavity. Additionally, there was an increase in the incidence of atypical hyperplasia of the thymus in exposed males and 

females. It was apparent that, in general, the proliferative lesions of the thymus represented a morphological continuum in which the latter stage is malignant lymphoma. Whilethere is a high probability 

that the lesions diagnosed as lymphomas are malignant neoplasms,there is less certainty as to the 

biological behavior of the smaller lesions (atypical 

hyperplasia).These smaller proliferative lesions are 

atleastpre-neoplastic lesions and at most an early 

lymphoma.Many of the mice in this study had 

advanced lymphoma involving multiple tissues and 

organs.Inthosemice,there was less confidence in 

determiningthesite of origin; however, because of 

the numerous thymic lesions observed in this study, 

it was expected that a number of those widespread 

lymphomaswere of thymic origin. Proliferative 

lesions (atypical hyperplasia and malignant lymphoma) of the thymusin exposed males and females 

were considered related to phenolphthalein exposure. 

Phenolphthalein,1,3-butadiene, and ddC are genotoxicin in vivo tests, suggesting that genotoxicity 

may be acontributingfactor to the observation of 

thymiclymphomas.However,furtherwork is in 

progress to see if phenolphthalein accumulates in the 

thymus and to identify possible mechanisms. Although mutations that activate ras proto-oncogenes 

arecommon in thymic lymphomas induced by a 

number of environmental agents including N-methylnitrosurea(Warren et al., 1990;Corominas ef al., 

1991) and radiation (Janowski et al., 1990), ras 

mutations have not been commonly observed in the 

ddC-induced thymic lymphomas(Wiseman et al., 

1994) and were observed in only 2 of 11 thymic 

lymphomas from mice exposed to1,3-butadiene 

(Goodrow et al., 1994). Ras mutations were more 

common in 1,3-butadiene-inducedlung and liver 

tumorsthaninthe 1,3-butadiene-induced thymic 

lymphomas examined (6/7 lung tumors had K-ras 

mutations; 3/7 liver tumors had K-ras mutations; and 

4/7 liver tumors had H-ras mutations) (Goodrow 

et al., 1994). 

In mice, exposureto phenolphthalein increased the 

incidence of ovarianhyperplasia in3,000 and 

12,000 ppm females and ovarian neoplasmsin all 

groups of female mice. Generally,ovarian neoplasms fall into three categories: 1)epithelial, 

2) germ cell, or 3) sex-cord stromal (e.g., granulosa 

cell tumors and luteomas). In the B6C3F,mouse, 

ovarian neoplasms areuncommon; epithelial cell 

tumors are slightly more common than granulosacell 

tumors in control females.Themorphology of the 

proliferative lesions of the ovary in the female mice 

in this study was somewhat distinctive from those 

typically observed in controls. It was apparent that 

cells composing proliferative lesions wereoften 

luteinized and were of sex-cord stromalorigin. 

However, it was uncertain if component cellswere 

granulosa, thecal, interstitial, or combination. a

Non-specific categorization of these ovarian neoplasms as sex-cord stromal reflects that uncertainty. 

Ovariantumors (granulosa1 and epithelial) have 

previously been reported in mice in NTP studies after 

administration of eight other chemicals: benzene 

(NTP,1986a),1,3-butadiene (NTP,1984b, 1993d), 

N-methylolacrylamide (NTP, 1989a), 5-nitroacenaphthene (NCI, 1978),nitrofurantoin (NTP, 

1989b),nitrofurazone (NTP, 1988), 4-vinylcyclohexene (NTP, 1986b),and 4-vinyl- l-cyclohexene 

diepoxide (NTP, 1989~). The mechanism forthe 

induction of ovarian tumors with some of these 

chemicals may involve atrophy of the ovaryand 

oocyte destruction followed by a subsequent decrease 

in estrogen production, which leads toacompensatory increase in pituitary gland gonadotropin 

release. The increased stimulation by gonadotropins 

is thoughtto stimulate cell proliferationinthe 

ovary and to promote the eventual development of 
76 Phenolphthalein, NTP TR 465 

tumors. In the current study, noatrophy was 

observed, and the tumors are notsimilar to the 

epithelial and granulosa cell tumors observed in the 

previous NTP studies. The observation that phenolphthalein interacts with estrogen receptors (Ravdin 

et al., 1987) may be one means by which this chemical stimulates cellular proliferation of the ovary. It 

has previously been demonstrated that estradiol can 

stimulate ovary cells to proliferate (including granulosa and theca cells) (Rao et al., 1978); phenolphthalein may mimic the action of this steroid hormone by 

interacting with estrogen receptors of ovary cells. 

Alternatively, phenolphthalein may act as an antiestrogen in the pituitary gland and lead to an increase 

in gonadotropin production. 

No chemical-related ovarian tumors havebeen 

reported in rats in any oftheNTP studies. In the 

phenolphthalein study in rats, there was notumor 

response at this site. Therefore, in our model systems, the mouse is more sensitive to a chemicalinduced ovarian tumor response than the rat. 

Cancer of the ovary is the fourth most common 

cancer in Americanwomen. There were an estimated 26,600 new casesdiagnosedin1995and 

14,500 deaths from this disease(SEER, 1995). In 

humans, approximately 80% ofthe ovarian tumors 

are of epithelial origin; other ovarian tumors may be 

of sex-cord stromal origin. Likemany cancers, 

ovarian cancer is a disease of aging, with almost half 

ofnew cases occurring in womenage 65 or older 

(SEER, 1995). In these mouse studies, ovarian 

effects were notobserved in the 13-weekstudybut 

developedtowardtheendofthe2-year study. The 

earliest occurrence of ovarian tumors in micewas 

noted on day 668. In this study, and inmicein 

general, the ovarian tumors were not fatal. 

Environmental factors thatmight contribute to 

ovarian cancer in humans have not been identified. 

It is known that the rate for ovarian cancer in American women is two to three times higher than that for 

women in other parts of the world (Parkin et al., 

1993). It is not knownifthis difference in the 

ovarian cancer rate is due to environmental or genetic 

factors. 

Like phenolphthalein, which has been shown to bind 

competitively to the estrogen receptor in MCF-7 cells 

(Ravdin et al., 1987), other chemicals tested in the 

NTP bioassay have also been shown to interfere with 

estrogen binding at the estrogen receptor and to have 

mitogeniceffects on breast cancer cells (Jobling 

et al., 1995). Theseinclude zearalenone (NTP, 

1982a), 2,4-dichlorophenol (NTP, 1989d), and butyl 

benzyl phthalate (NTP, 1982b, 1996b). However, of 

these chemicals, only phenolphthalein was shown to 

cause ovarian neoplasms in mice. Thus, demonstrationof a chemical-induced estrogenic response (as 

reported from in vitro and/or in vivo studies) does not 

necessarily correlate with ovarian neoplasm or 

mammary gland neoplasm response in the F344/N rat 

and B6C3F, mouse studies. Further, these NTP 

chemicalswith estrogenic activity show different 

neoplasm patterns in other tissues. This suggests that 

phenolphthalein-inducedneoplasmsmay be due to 

multiple factors, which might include specific metabolismand distribution of the chemical, estrogenic 

effects of the chemical, or genotoxic properties of the 

chemical. 

Estrogenic activity is found with compounds having 

different chemical structures, including estradiol, zearalenone, coumestrol, o,p’-DDT, kepone, 

bisphenol A, and impurities in phenol red 

(Katzenellenbogen, 1995). More information is 

needed on the molecular details of the three-dimensional structure of the estrogen receptor and its 

interaction with ligands to understand how these 

variedchemicals interact with the receptor. Studies 

with phenol red (phenolsulfonphthalein) have shown 

that lipophilic impurities in this dye have more 

estrogenic activity than the parent compound (Bindal 

andKatzenellenbogen, 1988; Bindal et al., 1988), 

suggesting that the lipophilicity of chemicals is an 

important property in reaching or binding to the 

estrogen receptor. 

The incidences of germinal epithelial degeneration 

of the testis in all exposed groups of male mice were 

significantly greater than that in the controls. The 

lesion varied from involvement of a few to all seminiferous tubules. Within the tubules, there was 

variable loss of sperm and germinal epithelial cells 
Discussion and Conclusions 77 

(spermatogonia, spermatocytes, and spermatids), 

often with only Sertoli cells remaining. In the most 

severe cases, the cross-sectional diameter of the testis 

was decreased. Although more severe, this lesion is 

consistent with that observed in the 13-week study. 

There was an increased incidence of myelofibrosis of 

the bone marrow of 12,000 ppm maleandfemale 

miceandanincreasedseverity of this lesionin 

exposedfemalemice.Thiswascharacterizedby 

focal to multifocalreplacement of bone marrow 

elementsby clusters of loosely arranged spindle cells. 

The finding that phenolphthaleinaffects the bone 

marrow cells of mice in both the 13-week and 2-year 

studies shows that, in both short-term and long-term 

exposures, thebonemarrowis a targettissue. 

Fibro-osseous lesions are observed intheflatand 

long bones of a number of strains of mice as spontaneousandinduced lesions, and theselesions are 

very common in female B6C3F, mice. Femalesex 

hormones, especially estradiol, appear toplay a 

major role in the spontaneous development of myelofibrosis. Estrogen has been reported to cause neutropenia, thrombocytopenia, decreased bonemarrow 

cellularity, andincreased .incidences of splenic 

hematopoiesis in mice (Fried et al., 1974; Adler and 

Trobaugh, 1978). Witt et al. (1995) have also shown 

that phenolphthalein can induce micronuclei in bone 

marrow erythrocytes of mice. Micronuclei are 

formed from acentric chromosomal fragments or 

whole chromosomes generated through a variety of 

mechanismssuch as mitotic loss of acentric fragments, mechanical consequencesofchromosomal 

breakage and exchange events resulting in abnormal 

anaphase separation, or mitoticloss of whole 

chromosomes due to centromere or spindle failures. 

Because phenolphthalein induced chromosomal

aberrations in cultured Chinese hamster ovary cells, 

induction of chromosomal breakage may be one of 

the mechanisms for the observed alterations in mouse 

bone marrow following exposure to phenolphthalein. 

The decreases in the incidences of liver neoplasms 

andnonneoplastic lesions and thedecreases in the 

incidences of proliferative lesions of the pituitary 

glandin female mice, the thyroidglandinfemale 

mice, and 'the tooth of male mice were considered to 

be chemical related. 

There was no indication that phenolphthalein caused 

an increase in the incidences of lesions of the colon 

or rectuminrats or mice. In one limited study 

conductedinAustraliatoidentify risk factors for 

colorectal cancer in humans, there was no indication 

that phenolphthaleintaken as a laxative causedany 

diseaseinthecolon or rectum (Kune, 1993). This 

study did not examine the potential of phenolphthaleintocausetoxic or carcinogenic effects at other 

sites. 

After oral administration, phenolphthalein is absorbed 

by the small intestine and is conjugated in the liver to 

phenolphthalein glucuronide, which passes into the 

colon where it isdeconjugatedandtheactivecompound, phenolphthalein, is released. In humans, the 

laxativeresponse may occurafter an oral dose of 

phenolphthalein or the phenolphthalein glucuronide 

(Anand et al., 1994). 

In the 2-year studies, the plasma concentrations of 

totalphenolphthaleinwere similar for all exposure 

concentrations. There was no consistent difference 

inplasmaconcentrations of total phenolphthalein 

betweenmalesandfemales. The findings that the 

plasma concentrations of total phenolphthalein were 

approximatelythesame (approximately 100 to 

200 pg/mL plasma in rats and mice) for the exposure 

concentrations used in the 2-year studies may explain 

why dose-response increases in the incidences of 

kidney neoplasms in male rats, histiocytic sarcoma in 

maleandfemalemice,malignant lymphoma in 

femalemice, or ovarian neoplasms in female mice 

didnot occur. However, in the single-dose toxicokineticstudies of phenolphthalein, the elimination 

half-lives in mice were approximately half those of 

rats (Appendix 0;NTP, 1994). 

The use of phenolphthalein as a laxative began early 

inthetwentiethcentury (von Vtimossy, 1908), and 

systematicstudiesplasma of concentrations of

phenolphthalein or its metabolites in humans have not 

been reported in the literature. 

Phenolphthaleinwasnegative in the Salmonella 

mutagenicityassaywithandwithoutmetabolic 

activation, but was positiveinan in vitro chromosomal aberrations test conductedwith S9 metabolic 
78 Phenolphthalein, NTP TR 465 

activation. In conjunction withthe13-weekfeed 

studyin mice, blood sampleswereanalyzed atthe 

endofstudy for the presence of micronucleated 

erythrocytes, and these were found to be significantly 

increasedinmaleandfemalemice at exposure 

concentrations of 6,000 ppm phenolphthalein or 

higher (Witt et ul., 1995). In feed studies in Swiss 

(CD- 1 @)mice, phenolphthalein was found to increase 

the frequency of micronucleated erythrocytes in mice 

receiving 1,000 ppm phenolphthaleininfeed for 

14 weeks (Witt et al., 1995). 

Metabolism appears to be needed for genotoxicity 

with phenolphthalein. This hypothesis is supported 

bythe requirement for S9 metabolicactivation 

enzymes for the positive response in the chromosomal aberrations test inculturedChinesehamster 

ovary cells. However, the predominanttypeof 

chromosomal damage observed in cultured Chinese 

hamster ovary cells treated with phenolphthalein was 

X chromosome breakage, a phenomenon that is not 

well understood, has beennotedwithonly a few 

chemicals, and is only observedincell cultures 

treated in the presence of S9. Therefore, the significance of the cultured Chinese hamster ovarycell 

results to interpretation of the invivo responsesis 

unclear. It is possible that the geneticdamage 

inducedby phenolphthalein is limited to chromosomal breakage (phenolphthalein, if converted to a 

quinoid, induces free oxygen radicals) and that point 

mutations and/or small deletions, such as are detected 

in the Salmonella assay, as well as other types of 

genetic damage, are not induced. For example, 

phenolphthalein did not induce sister chromatid 

exchanges in cultured Chinese hamster ovarycells 

with or without S9. It is unusual for a chemical to 

induce chromosomal aberrations but not sister chromatid exchanges in cultured Chinese hamster ovary 

cells, and this pattern of activity further supports 

the specificity of genetic damage induced by phenolphthalein. 

In summary, phenolphthalein caused toxicity to 

the reproductive system ofmaleandfemalemice. 

Phenolphthalein also caused neoplasms and nonneoplastic lesions in various tissues and organs in 

rats and mice (Tables 24 and 25). Themechanisms 

for these effects are not known; however, phenolphthalein has been shown to have estrogenic activity 

in vitro (Ravdin et al., 1987) and in vivo (Nieto 

et al., 1990) and genotoxicactivity in vivo (Witt 

et al., 1995). As is shown in Figure 1, phenolphthalein is capable of being converted to a quinoid 

structure. Quinoids are highly reactivechemicals 

capable of reacting with sulfhydryl groups and amino 

groups and forming oxygen free radicals (Brunmark 

and Cadenas, 1989; Degen and Metzler, 1989; 

Thompson et al., 1990). It is not known at what 

specific sites in the cell these reactions might occur. 

Specific microenvironment conditions (e.g., pH) 

might be conducive to the formation of the phenolphthalein quinoid. Multiple mechanisms maybe 

involved in causing the toxic/carcinogenic responses 

to phenolphthalein in the rodent. 

CONCLUSIONS 

Underthe conditions of these 2-year feed studies, 

there was clear evidence of carcinogenic activity* of 

phenolphthalein in male F344/N rats based on markedlyincreasedincidences 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 increasedincidencesof benign pheochromocytomasof the adrenal medulla in the 

12,000 ppm group andof benign or malignant 

pheochromocytomas (combined) in the 12,000 and 

25,000 ppm groups. There was clear evidence of 

carcinogenicactivity of phenolphthalein in male 

B6C3Fl mice based on increased incidences of 

histiocytic sarcomas and of malignant lymphomas of 

thymic origin. There was clear evidence of carcinogenicactivity of phenolphthalein in female B6C3Fl 

micebased on increasedincidences of histiocytic 

sarcomas, malignant lymphomas of all types, lymphomasofthymic origin, and benign sex-cord 

stromal tumors of the ovary. 

Exposure of rats to phenolphthalein in feed for 

2 years resultedinincreasedincidencesof focal 

hyperplasiaofthe 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 
Discussion and Conclusions 79 

inincreasedincidences of atypical hyperplasiaofthe Exposure of mice to phenolphthalein infeed for 

thymusinmalesand females, degeneration of the 2 yearsresultedindecreasedincidences of hepatogerminalepithelium of the testisinmales,andcellularneoplasmsandnonneoplastic lesions in males 

ovarian hyperplasia in females. and females. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 12. A summary of the Technical Reports Review Subcommittee 

comments and the public discussion on this Technical Report appears on page 14. 
80 Phenolphthalein, NTP TR 465 
81 

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92 Phenolphthalein, NTP TR 465 
93 

TABLEA1 

TABLEA2 

TABLEA3 

TABLEA4a 

TABLEA4b 

TABLEA5 

APPENDIX A 

SUMMARY OF LESIONS IN MALE RATS 

IN THE 2-YEAR FEED STUDY 

OF PHENOLPHTHALEIN 

Summary of the Incidence of Neoplasms in Male Rats 

in the 2-Year Feed Study of Phenolphthalein ........................... 94 

Individual Animal Tumor Pathology of Male Rats 

in the 2-Year Feed Study of Phenolphthalein ........................... 98 

Statistical Analysis of Primary Neoplasms in Male Rats 

in the 2-Year Feed Study of Phenolphthalein ........................... 122 

Historical Incidence of Adrenal Medulla Pheochromocytoma 

in Untreated Male F344/N Rats .................................... 129 

Historical Incidence of Renal Tubule Neoplasms in Untreated Male F344/N Rats .... 129 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats 

in the 2-Year Feed Study of Phenolphthalein ........................... 130 
94 Phenolphthalein, NTP TR 465 

TABLEA1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Phenolphthaleina -

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

DispositionSummary 

Animals initially in study 50 50 50 50 

Early deaths 

Moribund 15 I5 13 16 

Natural deaths 14 20 22 21 

Survivors 

Died last week of the study 1 

Terminal sacrifice 21 15 14 13 

Animals examined microscopically 50 50 50 50 

AlimentarySystem 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum : 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Oral mucosa 

Pharyngeal, squamous cell carcinoma 

Pharyngeal, squamous cell papilloma 

Pancreas 

Histiocytic sarcoma 

Acinus, adenoma 

Acinus, adenoma, multiple 

Salivaly glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Heart 

SchwaMoma malignant 

Endocrine System 

Adrenal cortex (50) 

Adenoma 1 (2%) 

Adrenal medulla (50) (50) (50) (50) 

Pheochromocytoma malignant 1(2%) 1 (2%) 2(4%) 

Pheochromocytoma benign 14 (28%) 15 (30%) 15 (30%) 19 (38%) 

Bilateral, pheochromocytoma malil nant 1(2%) 

Bilateral, pheochromocytoma benign 3(6%) 19 (38%) 19 (38%) 15 (30%) 

Islets, pancreatic (50) (50) (50) (48) 

Adenoma 3 (6%) 2(4%) 1(2%) 

Carcinoma 1 (2%) 2(4%) 1(2%) 2 (4%) 
Lesions in Male Rats 

TABLEA1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Phenolphthalein (continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Endocrine System (continued) 

Parathyroid gland 

Adenoma 

Pituitary gland 

Pars distalis, adenoma 

(50) 

13 (26%) 

Pars intermedia, adenoma 1 (2%) 

Bilateral, C-cell, adenoma 

Thyroid gland (50) 

1 (2%) 

Bilateral, follicular cell, adenoma 1 (2%) 

C-cell, adenoma 8 (16%) 2 (4%) 3 (6%) 4(8%) 

C-cell, carcinoma 1 (2%) 2 (4%) 

Follicular cell, adenoma 2 (4%) 3 (6%) 

Follicular cell, carcinoma 1 (2%) 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 1 (2%) 

Bilateral, carcinoma 

Prostate 

Adenoma 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell. adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Mediastinal, carcinoma, metastatic, kidney 

Renal, carcinoma, metastatic, kidney 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Fibrosarcoma 

Spleen (50) 

1 (2%) 

(49) 

Hemangioma 

Histiocytic sarcoma 

Histiocytic sarcoma 

Thymus (49) (47) 

Thymoma benign 1 (2%) 
96 Phenolphthalein, NTP TR 465 

TABLEA1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Phenolphthalein (continued) 

Integumentary System 

Mammary gland 

Adenoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Squamous cell carcinoma 

Pinna, schwannoma malignant 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibroma, multiple 

Subcutaneous tissue, histiocytic sarcoma 

Subcutaneous tissue, schwannoma malignant 

Musculoskeletal System 

Bone 

Cranium, osteosarcoma 

Skeletal muscle 

Osteosarcoma 

Nervous System 

Brain 

Cerebrum, oligodendroglioma malignant 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveoladbronchiolar carcinoma 

Carcinoma, metastatic, kidney 

Chordoma, metastatic, uncertain primary site 

Histiocytic sarcoma 

Osteosarcoma, metastatic, bone 

Osteosarcoma, metastatic, skeletal muscle 

Nose 

Squamous cell carcinoma, metastatic, oral 

mucosa 

Special Senses System 

Zymbal’s gland 

Adenoma 

Carcinoma 

0 PPm 

(43) 

1 (2%) 

5 (12%) 

1 (2%) 

(49) 

2 (4%) 

4(8%) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

(2) 

(50) 

1 (2%) 

(50) 

12,000 ppm 25,000 ppm 50,000 ppm 

(50) (50) (50) 

1 (2%) 

2 (4%) 3 (6%) 1 (2%) 

1 (2%) 

2 (4%) 

2 (4%) 1 (2%) 2 (4%) 

(50) 

(3) 

1 (2%) 
Lesions in Male Rats 97 

TABLEA1 

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Phenolphthalein (continued) 

0 PPm 12,000 ppm 

Urinary System 

Kidney 

Liposarcoma 

Renal tubule, adenoma 

Renal tubule, carcinoma 

Renal tubule, oncocytoma benign 

Transitional epithelium, carcinoma 

Urinary bladder 

Transitional epithelium, carcinoma 

Systemic Lesions 

Multiple organsb (50) (50) 

Histiocytic sarcoma 

Leukemia mononuclear 31 (62%) 27 (54%) 

Mesothelioma malignant 2 (4%) 2 (4%) 

NeoplasmSummary 

Total animals with primary neoplasms' 49 50 

Total primary neoplasms 159 155 

Total animals with benign neoplasms 47 50 

Total benign neoplasms 117 116 

Total animals with malignant neoplasms 35 36 

Total malignant neoplasms 42 39 

Total animals with metastatic neoplasms 1 2 

Total metastatic neoplasms 3 14 

Total animals with malignant neoplasms 

of uncertain primary site 1 

a Number of animals examined microscopically at the site andthe number of animals with neoplasm 

Number of animals with any tissue examined microscopically 

Primary neoplasms: all neoplasms except metastatic neoplasms 

25,000 ppm 

(50) 

1 (2%) 

24 (48%) 

50 

157 

49 

122 

31 

35 

2 

2 

50,000 ppm 

(50) 

29 (58%) 

2 (4%) 

50 

166 

50 

121 

33 

45 

4 

7 
98 Phenolphthalein, NTP TR 465 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Mesentery 

Mesothelioma malignant, metastatic, 

epididymis 

Oral mucosa 

Pharyngeal, squamous cell papilloma 

Pancreas 

Acinus, adenoma, multiple 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Bilateral, C-cell, adenoma 

C-cell, adenoma 

Follicular cell, carcinoma 

General Body System 

Tissue NOS 

+: Tissue examined microscopically 

A: Autolysis precludes examination 

2344445555555556666666667 

3308990223567790011144780 

8076086094311781845739154 

0000000000000000000000000 

1153433022314234223132344 

1701328274434069250698517 

......................... 

++++++++++++++++++++++A++ 

++++++++++++++++++++A+A++ 

++++++++++++A+++++++A+AA+ 

++++++++A+++A+++A++++++++ 

++++++++++++++++++++++AA+ 

++++++++++++A+++A+++A++A+

+++++++++++++++++%+++++++ 

++ + + + + + ++ 

+ 

X 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... X 

X xx 

X 

......................... 

++++++++M+M+MMM++++++++M+ 

......................... X X X X X 

......................... 

X 

X 

X X 

+ 

M:Missingtissue X: Lesion present 

I: Insufficient tissue Blank: Not examined 
Lesions in Male Rats 99 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Mesentery 

Mesothelioma malignant, metastatic, 

epididymis 

Oral mucosa 

Pharyngeal, squamous cell papilloma 

Pancreas 

Acinus, adenoma, multiple 

,Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Bilateral, C-cell, adenoma 

C-cell, adenoma 

Follicular cell, carcinoma 

General Body System 

Tissue NOS 

~ ~~~~~~ 

7177111711117111111771111 

0122222222222222222222222 

8541999999999999999999999 

0000000000000000000000000 Total 

4414000000001111122223344 Tissues/ 

5088134567890245913693726 Tumors 

......................... 50 

A++++++++++++++++++++++++ 48 

......................... 48 

A++A+++++++++++++++++++++ 44 

......................... 47 

+A+++++++++++++++++++++++ 47 

A++A+++++++++++++++++++++ 44 

......................... 50 

X 1 

+ + ++ + + ++++ + M 20 

X 1 

1 

1 

......................... 50 

X 1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

1 

x x	 x xxx X x x 14 

X X 3 

......................... 50 

X X X 3 

X 1 

+++++++++++M++M+++++++++M 41 

......................... 50 

xx X X x x x X 13 

X 1 

......................... 50 

X 1 

X X X x x 8 

1 

1 
100 Phenolphthalein, NTP TR 465 

TABLEA2 

Individual Animal Tumor Pathology 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Bilateral, adenoma 

Prostate 

Adenoma 

Seminal vesicle 

Testes 

Mesothelioma malignant, metastatic, 

epididymis 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

HematopoieticSystem 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

IntegumentarySystem 

Mammaly gland 

Adenoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Pinna, schwannoma malignant 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibroma, 

multiple 

Subcutaneous tissue. schwannoma 

malignant 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Bmin 

Cerebrum, oligodendroglioma malignant 

Peripheral nerve 

spinal cord 

of Male Rats in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

2344445555555556666666667 

3308990223567790011144780 

8076086094311781845739154 

0000000000000000000000000 

1153433022314234223132344 

1701328274434069250698517 

......................... 

......................... X 

......................... 

......................... 

......................... 

x x x x x x x xx x xx x x x xx x x 

X X 

++++++++++++++++++++A+A++ 

+++ + + + ++++ + + ......................... 

......................... 

......................... 

......................... 

++M++++M++M+++++M+++++M++ 

X X 

++++++++++++++++++++++M++ 

X 

X 

X 

X 

......................... + + 

......................... X 

+ + 

+ + 
Lesions in Male Rats 101 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Bilateral, adenoma 

Prostate 

Adenoma 

Seminal vesicle 

Testes 

Mesothelioma malignant, metastatic, 

epididymis 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

HematopoieticSystem 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

IntegumentarySystem 

Mammary gland 

Adenoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Pinna, schwannoma malignant 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibroma, 

multiple 

Subcutaneous tissue, schwannoma 

malignant 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Cerebmm, oligodendroglioma malignant 

Peripheral nerve 

Spinal cord 

7777777777777777777777777 

0122222222222222222222222 

8547999999999999999999999 

0000000000000000000000000 Total 

4414000000001111122223344 Tissues/ 

5088134567890245913693726 Tumors 

......................... 50 

......................... 50 

X X X 4 

X 1 

......................... 50 

X X 2 

......................... 50 

......................... 50 

X 1 

x x xxxxxxxxxxxxxxxxxxxxx 42 

x x 4 

......................... 48 

+++ + + 17 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

+++M++++++++++++++++++++M 43 

X 1 

X X X 5 

X 1 

......................... 49 

X 2 

xx X 4 

X 1 

X X 2 

1 

1 

......................... 50 

2 

......................... 50 

1 

2 

2 
102 Phenolphthalein, NTP TR 465 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

23444455555.55556666666667 

Number of Days on Study 3308990223567790011144780 

8076086094311781845739154 

0000000000000000000000000 

Carcass ID Number 1153433022314234223132344 

1701328274434069250698517 

~~ ~~_____ ~~~~ ~~ 

Respiratory System 

Lung ......................... Alveolar/bronchiolar adenoma 

Mesothelioma malignant, metastatic, 

epididymis 

Nose ......................... Trachea ......................... 

Special Senses System 

Ear + 

Zymbal’s gland + 

Carcinoma X 

Urinary System 

Kidney ......................... Liposarcoma 

Urinary bladder ,++++++++++++++++++++++A++ 

Transitional epithelium, carcinoma 

Systemic Lesions 

Multiple organs ......................... t Leukemia mononuclear x xxx x x x xx x x xx x x x 

Mesothelioma malignant X 
Lesions in Male Rats 103 

TABLEA2 

Individual Animal Tumor Pathology 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Mesothelioma malignant, metastatic, 

epididymis 

Nose 

Trachea 

Special Senses System 

Ear 

Zymbal’s gland 

Carcinoma 

Urinary System 

Kidney 

Liposarcoma 

Urinary bladder 

Transitional epithelium, carcinoma 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

of Male Rats in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

7777777777777777777777777 

0122222222222222222222222 

8547999999999999999999999 

0000000000000000000000000 Total 

4414000000001111122223344 Tissues/ 

5088134567890245913693726 Tumors 

......................... 50 

X 1 

X 1 

......................... 50 

......................... 50 

1 

1 

1 

......................... 50 

X 1 

......................... 49 

X 1 

......................... 50 

x xx xx xx xxxxxx X X 3 1

X 2 
104 Phenolphthalein, NTP TR 465 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Intestine small, duodenum 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Intestine small, jejunum 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Intestine small, ileum 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Liver 

Hepatocellular adenoma 

Mesentery 

Mesothelioma malignant, metastatic, 

epididymis 

Pancreas 

Mesothelioma malignant, metastatic, 

epididymis 

Salivary glands 

Stomach, forestomach 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Adenoma 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Bilateral, follicular cell, adenoma 

C-cell, adenoma 

4445555555555666666666666 

5183345661999111223333451 

8051838117124048462368600 

0000000000000000000000000 

7181899556656178151655589 

6763385466154415590223782 

......................... 

++++++++++++++++++++++++A 

......................... 

++++AA++++++++AA+A++++++A 

X 

......................... 

X 

++++++++++++A+A+++A+A+++A 

X 

+++++AA+++++A+A+++A+++++A 

X 

......................... X X 

+++ + + + 

X 

......................... 

X 

++++++++++++M++++++++++++ 

......................... 

X 

......................... 

++++++++++++++++++++++++M 

++++++++++++++++++++++++M 

......................... 

......................... X 

x x X xx xx X 

x xx xx X 

......................... 

X 

++M++++++++++++M+++++++++ 

X 

......................... xx ......................... X 

X 
Lesions in Male Rats 105 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Intestine small, duodenum 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Intestine small, jejunum 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Intestine small, ileum 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Liver 

Hepatocellular adenoma 

Mesentery 

Mesothelioma malignant, metastatic, 

epididymis 

Pancreas 

Mesothelioma malignant, metastatic, 

epididymis 

Salivary glands 

Stomach, forestomach 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Adenoma 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Bilateral, follicular cell, adenoma 

C-cell, adenoma 

6666677777777777777777777 

7899901122222222222222222 

9513410538999999999999999 

000000000.0000000000000001 Total 

5687996779566668888899990 Tissues/ 

1199648280803590124713790 Tumors 

......................... 50 

......................... 49 

++A++++++++++++++++++++++ 49 

++AA++A++++++++++++++++++ 41 

1 

......................... 50 

1 

++AA++A++A+++++++++++++++ 41 

1 

++A++++AAA+++++++++++++++ 40 

1 

......................... 50 

X 3 

+ 7 

1 

......................... 50 

1 

......................... 49 

......................... 50 

1 

......................... 50 

......................... 49 

......................... 49 

......................... 50 

......................... 50 

1 

X X X xx x x 15 

xx xxx X x xx x x x x 19

......................... 50 

X X 2 

X 2 

......................... 48 

1 

......................... 50 

X xx x xx X xx 11 

......................... 50 

1 

X 2 
106 Phenolphthalein, NTP TR 465 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

General Body System 

None 

Genital System 

Epididymis 

' Preputial gland 

Adenoma 

Carcinoma 

Bilateral, carcinoma 

Prostate 

Mesothelioma malignant, metastatic, 

epididymis 

Seminal vesicle 

Mesothelioma malignant, metastatic, 

epididymis 

Testes 

Mesothelioma malignant, metastatic, 

epididymis 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

HematopoieticSystem 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma 

Mesothelioma malignant, metastatic. 

epididymis 

Thymus 

Thymoma benign 

IntegumentarySystem 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Squamous cell carcinoma 

Subcutaneous tissue, fibroma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Mesothelioma malignant, metastatic, 

epididymis 

Nervous System 

Brain 

Peripheral nerve 

spinal cord 

4445555555555666666666666 

5183345667999111223333457 

8051838111124048462368600 

0000000000000000000000000 

1181899556656118151655589 

6763385466754415590223182 

......................... 

+++++++++++M+++++++++++++ 

X X X 

X 

......................... 

X ......................... 

X 

......................... 

X 

x xx x x xx xx xx x x x x x x x x 

X X X 

......................... 

+++ + ++ ++ + + + + +'+ 

++++++++++++M++++++++++++ 

......................... 

......................... 

X 

+++++++++++++++++++M+++++ 

++++++++++M+++++++++++M++ 

......................... 

X X 

......................... + + ++ 

X 

......................... + + 

+ + 
Lesions in Male Rats 107 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Bilateral, carcinoma 

Prostate 

Mesothelioma malignant, metastatic, 

epididymis 

Seminal vesicle 

Mesothelioma malignant, metastatic, 

epididymis 

Testes 

Mesothelioma malignant, metastatic, 

epididymis 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

HematopoieticSystem 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma 

Mesothelioma malignant, metastatic, 

epididymis 

Thymus 

Thymoma benign 

IntegumentarySystem 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Squamous cell carcinoma 

Subcutaneous tissue, fibroma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Mesothelioma malignant, metastatic, 

epididymis 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

6666677777777777777777777 

7899901122222222222222222 

9513410538999999999999999 

OOOOOOOOOOOOOOOOOOOOOOOOl Total 

5687996779566668888899990 Tissues/ 

1199648280803590124713790 Tumors 

......................... 50 

......................... 49 

X 4 

X 2 

X 1 

......................... 50 

1 

......................... 50 

1 

......................... 50 

1 

xxxx xxxxxxxx xxxxxxxxxxx 42 

X 4 

......................... 50 

++ + + + ++ + 22 

......................... 49 

......................... 50 

......................... 50 

X 1 

1 

......................... 49 

X 1 

+++++++++++++M+++++++++++ 47 

X X 2 

......................... 50 

X X 2 

X 1 

2 

......................... 50 

+ 5 

1 

......................... 50 

+ 3 

+ 3 
108 Phenolphthalein, NTP TR 465 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Chordoma, metastatic, uncertain 

primary site 

Nose 

Trachea 

Special Senses System 

EYe 

Urinary System 

Kidney 

Renal tubule, adenoma 

Renal tubule, carcinoma 

Urinary bladder 

Serosa, mesothelioma malignant, 

metastatic, epididymis 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

4445555555555666666666666 

5783345667999111223333457 

8051838117124048462368600 

0000000000000000000000000 

7787899556656778757655589 

6763385466754415590223782 

......................... 

......................... 

......................... 

......................... X 

......................... 

X 

......................... 

xxx xx xxx xxx xx xxx X 

x x 
Lesions in Male Rats 109 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Chordoma,metastatic,uncertain 

primarysite 

Nose 

Trachea 

Special Senses System 

EY e 

Urinary System 

Kidney 

Renaltubule,adenoma 

Renal tubule, carcinoma 

Urinary bladder 

Serosa,mesotheliomamalignant, 

metastatic,epididymis 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

~_____ ~~_____ ~~ 

6666677777777777777777777 

78999.01122222222222222222 

9513410538999999999999999 

0000000000000000000000001 Total 

5687996779566668888899990 Tissues/ 

1199648280803590124713790 Tumors 

......................... 50 

X 1 

X 1 

X 1 

......................... 50 

......................... 50 

+ 1 

......................... 50 

X X X 4 

X 1 

......................... 50 

1 

......................... 50 

xx x xx x xxxx 27 

2 
110 Phenolphthalein, NTP TR 465 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 25,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ilium 

Liver 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Oral mucosa 

Pharyngeal, squamous cell carcinoma 

Pancreas 

Histiocytic sarcoma 

Acinus, adenoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Bilateral, pheochromocytoma malignant 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, adenoma 

General Body System 

None 

_____~~~~ ~ ~~ ~ ~~~ ~~ ~ _______ 

334445566,6666666666666666 

7814889022233334566667778 

2364241123323465411460111 

1111111111111111111111111 

4334404211333345312223141 

1417329123957220842433809 

......................... 

+++++++++++++++++++++A+++ 

......................... 

++++++++++++++++AAMA+A+++ 

+++++++++++++++++A+++++++ 

++++++++++++++++++A++++++ 

+++++++++A+A+A++AAAA+++++ 

......................... 

+ + 

+ 

X ......................... 

......................... 

......................... 

......................... _______~ ~ 

......................... 

......................... 

......................... 

......................... 

X X xxx X 

X 

X xx x x x x x ......................... 

......................... 

......................... X X X 

......................... X 
Lesions in Male Rats 111 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 25,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Oral mucosa 

Pharyngeal, squamous cell carcinoma 

Pancreas 

Histiocytic sarcoma 

Acinus, adenoma 

Sajivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Bilateral, pheochromocytoma malignant 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, adenoma 

General Body System 

None 

6666777777777777777777777 

89990011222222222222222'22 

1278140307999999999999999 

1111111111'111111111111111 Total 

2124140300000001112222344 Tissues/ 

0195083098145675675678646 Tumors 

......................... 50 

......................... 49 

......................... 50 

+++A+++++++++++++++++++++ 44 

......................... 49 

+++A+++A+++++++++++++++++ 47 

A+A++++A+++++++++++++++++ 40 

......................... 50 

X 1 

+ 3 

X 1 

1 

1 

......................... 50 

X 1 

xx 2 

+++++++M+++++++++++++++++ 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

......................... 50 

xx X xx xx x x 15 

1 

X xx X xx xx xxx 19

......................... 50 

X 1 

+++++++M+++++++++++++++++ 49 

......................... 50 

X x xxx x x X xx x xx 16 

+++++++M+++++++++++++++++ 49 

X X 3 

X 1 

X X 2 
112 Phenolphthalein, NTP TR 465 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 25,000 ppm (continued) 

Number of Days on Study 

CarcassIDNumber 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

HematopoieticSystem 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangioma 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

IntegumentarySystem 

Mammary gland 

Adenoma 

Fibroadenoma 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, histiocytic 

sarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Osteosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Histiocytic sarcoma 

Osteosarcoma, metastatic, skeletal 

muscle 

Nose 

Squamous cell carcinoma, metastatic, 

oral mucosa 

Trachea 

3344455666666666666666666 

7814889022233334566667718 

2364241123323465411460111 

1111111111111111111111111 

4334404211333345312223141 

1417329123957220842433809 

......................... 

......................... X 

......................... 

......................... 

......................... xxxxxxxxxxxxxxxxxxxxxxx 

X 

......................... 

+ +++++++ ++ ++++ + ......................... 

++++++++++++++++++M++++++ 

......................... X 

+M+++++++++++++++++M+++++ 

+++++M+++++++++++++++++++ 

......................... 

X 

......................... 

+ + 

......................... 

+ + 

+ + 

......................... 

......................... 

X 

......................... 
Lesions in Male Rats 113 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangioma 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Adenoma 

Fibroadenoma 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, histiocytic 

sarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Osteosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Histiocytic sarcoma 

Osteosarcoma, metastatic, skeletal 

muscle 

Nose 

Squamous cell carcinoma, metastatic, 

oral mucosa 

Trachea 

6666777777777777777777777 

8999001122222222222222222 

1278140307999999999999999 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total 

2124140300000001112222344 Tissues/ 

0195083098145675675678646 Tumors 

......................... 50 

......................... 50 

X X X 4 

xx 2 

......................... 50 

......................... 50 

......................... 50 

xxxxxxxxxxxxxxxxx xxxxxx 46

xx 3 

......................... 50 

+++ ++++ + + + ++ 27 

+++++++M+++++++++++++++++ 49 

+++M+++++++++++++++M+++++ 47 

X 1 

......................... 50 

1 

X 1 

+++++++++++++++++++++++M+ 47 

X 1 

++++++M++++++++++++++++++ 48 

X 1 

X X 2 

......................... 50 

X 1 

X X 3 

X 1 

X 1 

......................... 50 

+ + 4 

X 1 

......................... 50 

+ 3 

+ 3 

......................... 50 

X 1 

X 1 

......................... 50 

1 

......................... 50 
114 Phenolphthalein,NTP TR 465 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Feed Study of Phenolphthalein: 25,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

Zymbal’s gland 

Adenoma 

Urinary System 

Kidney 

Renal tubule, adenoma 

Renal tubule, carcinoma 

Transitional epithelium, carcinoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

~ ~~ ~ 

3344455666666666666666666 

7814889022233334566667778 

2364241123323465411460111 

1111111111111111111111111 

4334404211333345312223141’ 

1417329123957220842433809 

+ 

......................... 

X ......................... 

......................... 

X xxxxxx X x x xx x x 
Lesions in Male Rats 115 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 25,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

Zymbal’s gland 

Adenoma 

Urinary System 

Kidney 

Renal tubule, adenoma 

Renal tubule, carcinoma 

Transitional epithelium, carcinoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

6666777777171777771771177 

8999001122222222222222222 

1278140307999999999999999 

1111111111111111111111111 Total 

2124140300000001112222344 Tissues/ 

0195083098145675675618646 Tumors 

1 

+ 1 

X 1 

......................... 50 

X X 2 

X 1 

1 

......................... 50 

......................... 50 

X 1 

x x x xx x x xx x 24 
116 Phenolphthalein, NTP TR 465 

TABLE A2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 50,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Mesothelioma malignant, metastatic, 

epididymis 

Acinus, adenoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Hean 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, adenoma 

General Body System 

None 

4555555555666666666666666 

8224467899122233455566667 

5481230514224636524944581 

1111111111111111111111111 

5869876968955796866678969 

9070594467326583382189596 

......................... 

+++++++++A++++++++++++M++ 

......................... 

+A+++++++A+++++++++A++MA+ 

+A+++++++A++++++++++++M++ 

+A++A++++A+++A++++++++MA+ 

+A+++++++A+++++++A++++MAA 

......................... X 

+ + 

+A++++++++++++++++++++MA+ 

X 

++++++++++++++++++M++++++ 

......................... 

......................... 

+ 

......................... 

......................... 

......................... 

......................... 

x x xx x xxx x x xx 

X X x x xx X 

+A++++++++++++++++++++M++ 

X 

++++++++++++++++++M+++M++ 

......................... X X X X 

+++++++++++t++++++M++++++ 

X X X 
Lesions in Male Rats 117 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 50,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple * 

Mesentery 

Pancreas 

Mesothelioma malignant, metastatic, 

epididymis 

Acinus, adenoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, adenoma 

General Body System 

None 

6666677777777777777777777 

7789901111122222222222222 

3353600117809999999999999 

1111111111111111111111112 Total 

5759687599875556777788890 Tissues1 

8312586419413570024712670 Tumors 

......................... 50 

......................... 48 

......................... 50 

A+++A+++AAA++++++++++++++ 40 

......................... 47 

++++A++++A+++++++++++++++ 42 

A+++A+++++A++++++++++++++ 41 

......................... 50 

1 

X 1 

+ 3 

......................... 41 

1 

X 1 

......................... 49 

......................... 49 

......................... 49 

1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X X 2 

X X xx X X X 19 

xx x x X X xx 15 

......................... 48 

1 

xx 2 

M+++++++++M++++++++++++++ 46 

......................... 50 

X x xx xx X 11 

......................... 49 

X 4 

X X 2 

X X X 3 
118 Phenolphthalein, NTP TR 465 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed study of Phenolphthalein: 50,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Prostate 

Seminal vesicle 

Testes 

Mesothelioma malignant, metastatic, 

epididymis 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

HematopoieticSystem 

Bone marrow 

Lymph node 

Mediastinal, carcinoma, metastatic, 

kidney 

Renal, carcinoma, metastatic, kidney 

Lymph node, mandibular 

. Lymph node, mesenteric 

Spleen 

Thymus 

IntegumentarySystem 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Pinna, schwannoma malignant 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, schwannoma 

malignant 

Musculoskeletal System 

Bone 

Cranium, osteosarcoma 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Carcinoma, metastatic, kidney 

Osteosarcoma, metastatic, bone 

Nose 

Trachea 

Special Senses System 

EYe Zymbal’s gland 

Carcinoma 

4555555555666666666666666 

822446789912.2233455566667 

5481230514224636524944581 

1111111111111111111111111 

5869876968955796866678969 

9010594467326583382189596 

......................... 

......................... X 

......................... 

......................... 

......................... 

xx 

xxxxxxxxxxxxxxxxxxxxx 

X x x 

+A+++++++++++++++++++++++ 

++ + + ++ + + ++++ + 

++++++++++++++++++M++++++ 

++++++++++++++++++++++MA+ 

++++++++++++++++++++++M++ 

++++M++++++++++M+++++++++ 

++M+++++++M++++++++++++++ 

......................... X 

X 

X 

......................... X 

+ 

......................... 

......................... 

X ......................... .......................... 

+ 

+ 

X 
Lesions 119 in Male Rats 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Prostate 

Seminal vesicle 

Testes 

Mesothelioma malignant, metastatic, 

epididymis 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Mediastinal, carcinoma, metastatic, 

kidney 

Renal, carcinoma, metastatic, kidney 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Pinna, schwannoma malignant 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, schwannoma 

malignant 

Musculoskeletal System 

Bone 

Cranium, osteosarcoma 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

Lung

Alveolar/bronchiolar adenoma 

Carcinoma, metastatic, kidney 

Osteosarcoma, metastatic, bone 

Nose 

Trachea 

Special Senses System 

Eye

Zymbal’s gland 

Carcinoma 

6666677777777777777777777 

7789901111122222222222222 

3353600177809999999999999 

1111111111111111111111112 Total 

5759687599875556777788890 Tissues/ 

8312586419413570024712670 Tumors 

......................... 50 

+++++++++M+++++++++++++++ 49 

X x x 4 

......................... 50 

......................... 50 

......................... 50 

2 

xxxxxxxxxxxxxxxxxxxxxxxx 45 

X 4 

......................... 49 

+ + + ++++ 20 

X 1 

X 1 

......................... 49 

......................... 48 

......................... 49 

++++++++++++++++M++++++++ 47 

++++M++++++++++++++++++++ 41 

X 1 

......................... 50 

1 

X 2 

xx 2 

1 

......................... 50 

1 

1 

......................... 50 

......................... 50 

X 1 

X 1 

1 

......................... 50 

......................... 50 

+ 2 

+ 2 

X 2 
120 Phenolphthalein,NTP TR 465 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 50,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Renaltubule,adenoma 

Renaltubule,carcinoma 

Renal tubule, oncocytoma benign 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

4555555555666666666666666 

8224467899122233455566667 

5481230514224636524944581 

1111111111111111111111111 

5869876968955796866678969 

9070594467326583382189596 

......................... X X X X 

X 

......................... 

......................... x x xx x x xx x xxx x xx 

xx 
Lesions in Male Rats 121 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of Phenolphthalein: 50,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Renal tubule, adenoma 

Renal tubule, carcinoma 

Renal tubule, oncocytoma benign 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

6666677777777777777777777 

7789901111122222222222222 

3353600177809999999999999 

1111111111111111111111112 Total 

5759687599875556777788890 Tissues/ 

8312586419413570024712670 Tumors 

......................... 50 

X X 6 

X 2 

X 1 

......................... 50 

......................... 50 

X X X x xxx x xxx xxx 29 

2 
122 Phenolphthalein, NTP TR 465 

TABLEA3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-YearFeed Study of Phenolphthalein 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

AdrenalMedulla:BenignPheochromocytoma 

Overall ratea 17/50 (34%) 34/50 (68 k) 34/50 (68 46) 34/50 (68%) 

Adjusted rateb 62.1X 88.6% 93.7% 87.0% 

Terminal rate' 11/21 (52%) 11/15 (73%) 13/15 (87%) 9/13 (69%) 

First incidence (days) 608 485 372 528 

Life table testd P=O.O03 P<O.ool P<O.ool P<O.ool 

Logistic regression testd P=O.O06 P<O.ool P<O.ool P=O.OOl 

Cochran-Armitage testd P=O.O03 

Fisher exact testd P<O.ool P<O.ool P<O.ool 

AdrenalMedulla:Benign or MalignantPheochromocvtoma 

Overall rate 18/50 (36%) 35/50 (70%) 35/50 (70%) 35/50 (70%) 

Adjusted rate 62.9% 88.9% 93.9% 90.3% 

Terminal rate 11/21 (52%) 11/15 (73%) 13/15 (87%) 10113 (77%) 

First incidence (days) 330 485 372 528 

Life table test P=O.O03 P<O.ool P<O.ool P<O.OOl 

Logistic regression test P=O.O05 P<O.ool P<O.ool P=O.ool 

Cochran-Armitage test P=O.O03 

Fisher exact test P<O.ool P<O.ool P<O.ool 

Kidney(Renal Tubule): Adenoma (SingleSections) 

Overall rate 0150 (0%) 4/50 (8%) 2/50 (4%) 6/50 (12%) 

Adjusted rate 0.0% 20.4% 10.6% 22.4% 

Terminal rate 0121 (0%) 2/15 (13%) 1/15 (7%) 1/13 (8%) 

First incidence (days) -e 577 692 542 

Life table test P=O.O15 P=O.o40 P=O.195 P=O.O17 

Logistic regression test P=O.O26 P=O.O60 P=O.233 P=O.O18 

Cochran-Armitage test P=O.O23 

Fisher exact test P=O.O59 P=O.247 P=O.O13 

Kidney(Renal Tubule): Adenoma (Step Sections) 

Overall rate 1/50 (2%) 7/50 (14%) 15/50 (30%) 11/50 (22%) 

Adjusted rate 4.8% 36.4% 64.9% 51.1% 

Terminal rate 1/21 (5%) 4/15 (27%) 8/15 (53%) 5/13 (38%) 

First incidence (days) 729 (T) 69 1 664 542 

Life table test P<O.ool P=O.O12 P<O.ool P<O.ool 

Logistic regression test P=O.O03 P=O.O17 P<O.ool P=O.O03 

Cochran-Armitage test P=O.O05 

Fisher exact test P=O.O30 P<O.ool P=O.oo2 

Kidney (Renal Tubule): Adenoma (Single and Step Sections) 

Overall rate 1/50 (2%) 10150 (20%) 15/50 (30%) 15/50 (30%) 

Adjusted rate 4.8% 46.9% 64.9% 61.8% 

Terminal rate 1/21 (5%) 5/15 (33%) 8/15 (53%) 6/13 (46%) 

First incidence (days) 729 (T) 577 664 542 

Life table test P<O.OOl P=O.ool P<O.ool P<O.ool 

Logistic regression test P<O.OOl P=O.O03 P<O.ool P<O.ool 

Cochran-Armitage test P<O.ool 

Fisher exact test P=O.o04 P<O.ool P<O.ool 
Lesions in Male Rats 123 

TABLEA3 

§tatistical Analysis of Primary Neoplasms in Male Rats in the2-YearFeed Study of Phenolphthalein(continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Kidney(Renal Tubule): Adenoma orCarcinoma(SingleSections) 

Overall rate 0150 (0%) 

Adjusted rate 0.0% 

Terminal rate 0121 (0%) 

First incidence (days) -

Life table test P=O.O11 

Logistic regression test P=O.O20 

Cochran-Armitage test P=O.O17 

Fisher exact test 

Kidney(Renal Tubule): Adenoma orCarcinoma(StepSections) 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Kidne$(RenalTubule): Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver:HepatocellularAdenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

MammaryGland:Fibroadenoma 

Overall rate 

Adjusted rate 

Terminal rate 

1/50 (2%) 

4.8% 

1/21 (5%) 

729 (T) 

P=O.O12 P<O.OOl 

P=O.O17 P=O.O03 

P=O.O05 

5/50 (10%) 

24.0% 

2/15 (13%) 

577 

P=O.O20 

P=O.O31 

P=O.O28 

7/50 (14%) 

36.4% 

4/15 (27%) 

69 1 

P=O.O30 

or Carcinoma(Singleand Step Sections) 

1/50 (2%) 10150 (20%) 

4.8% 46.9% 

1/21 (5%) 5/15 (33%) 

729 (T) 577 

P<O.OOl P=O.OOl 

P<O.OOl P=O.O03 

P<O.OOl 

P=O.O04 

1/50 (2%) 3/50 (6%) 

4.5% 9.4% 

0/21 (0%) 0/15 (0%) 

I27 577 

P=O.285 P=O.532 

P=O.571 P=O.307 

P=O.562 

P=O.309 

6/50 (12%) 2/50 (4%) 

22.8% 10.9% 

3/21 (14%) 1/15 (7%) 

3/50 (6%) 

16.1X 

1/15 (7%) 

692 

P=O.O84 

P=O.106 

P=O.121 

15/50 (30%) 

64.9% 

8/15 (53%) 

664 

P<O.OOl 

P<O.OOl 

P<O.ool 

16/50 (32%) 

67.1% 

8/15 (53%) 

664 

P<O.ool 

P<O.OOl 

P <0.001 

0/50 (0%) 

0.0% 

0115 (0%) -

P=0.564N 

P=0.554N 

P=0.500N 

2/50 (4%) 

13.3% 

2/15 (13%) 

7/50 (14%) 

24.7% 

1/13 (8%) 

542 

P=O.O11 

P=O.O09 

P=O.O06 

11/50 (22%) 

51.1% 

5/13 (38%) 

542 

P<O.OOl 

P=O.O03 

P=O.oo2 

16/50 (32%) 

62.9% 

6/13 (46%) 

542 

P<O.ool 

P<O.ool 

P<O.001 

2/50 (4%) 

8.8% 

0113 (0%) 

668 

P=O.409 

P=O.495 

P=O.500 

1/50 (2%) 

7.7% 

1/13 (8%) 

First incidence (days) 693 534 729 (T) 729 (T) 

Life table test P=0.092N P=0.231N P=0.235N P=0.131N

Logistic regression test P=0.041N P=0.138N P=0.120N P=0.054N 

Cochran-Armitage test P=0.043N 

Fisher exact test P=0.134N P=0.134N P=0.056N 
124 Phenolphthalein, NTP TR 465 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

MammaryGland:Fibroadenoma or Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pancreatic Islets: Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pancreatic Islets: Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pituitary Gland (Pars Distalis):Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

PreputialGland:Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

7/50 (14%) 

26.0% 

3/21 (14%) 

534 

P=0.075N 

P=0.030N 

P=0.030N 

3/50 (6%) 

13.6% 

2/21 (10%) 

727 

P=0.242N 

P=0.169N 

P=0.171N 

4/50 (8 %) 

18.2% 

3/21 (14%) 

727 

P=0.466N 

P=0.344N 

P=0.361N 

13/50 (26%) 

42.9% 

6/21 (29%) 

407 

P=O.362 

P=0.390N 

P=0.449N 

5/50 (10%) 

21.9% 

4/21 (19%) 

67 1 

P=O.543 

P=0.439N 

P=0.458N 

2/50 (4%) 

10.9% 

1/15 (7%) 

693 

P=0.162N 

P=0.084N 

P=0.080N 

2/50 (4%) 

11.9% 

1/15 (7%) 

715 

P=0.644N 

P=0.612N 

P=0.500N 

4/50 (8%) 

19.1% 

1/15 (7%) 

638 

P=O.483 

P=O.593 

P=0.643N 

11/50 (22%) 

50.8% 

6/15 (40%) 

610 

P=O.555 

P=0.391N 

P=0.408N 

4/49 (8 %) 

13.5% 

0115 (0%) 

485 

P=0.613N 

P=0.502N 

P=0.513N 

3/50 (6%) 

20.0% 

3/15 (20%) 

729 (T) 

P=0.295N 

P=0.142N 

P=0.159N 

0150 (0%) 

0.0% 

0/15 (0%) -

P=0.186N 

P=O. 175N 

P=0.121N 

1/50 (2%) 

3.8% 

0115 (0%) 

68 1 

P=0.271N 

P=0.199N 

P=0.181N 

16/50 (32%) 

67.3% 

8/15 (53%) 

623 

P=O.169 

P=O.388 

P=O.330 

4/50 (8 %) 

18.4% 

1/15 (7%) 

416 

P=O.625 

P=0.472N 

P=0.500N 

1/50 (2%) 

7.7% 

1/13 (8%) 

729 (T) 

P=0.088N 

P=0.030N 

P=0.030N 

1/48 (2%) 

2.5% 

0113 (0%) 

612 

P=0.425N 

P=0.311N 

P=0.324N 

3/48 (6%) 

12.2% 

0/13 (0%) 

612 

P=O.635 

P=0.525N 

P=0.523N 

11/50 (22%) 

51.3% 

5/13 (38%) 

542 

P=O.528 

P=0.367N 

P=0.408N 

4/49 (8%) 

22.9% 

2/13 (15%) 

585 

P=O.561 

P=0.539N 

P=0.513N 
Lesions in Male Rats 125 

TABLEA3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Phenolphthalein (continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

PreputialGland:Carcinoma 

Overall rate 0150 (0%) 3/49 (6%) 2/50 (4%) 0149 (0%) 

Adjusted rate 0.0% 15.8% 13.3% 0.0% 

Terminal rate 0121 (0%) 2/15 (13%) 2/15 (13%) 0113 (0%) 

First Incidence (days) - 618 729 (T) - Life table test P=0.498N P=O.O92 P=O.166 - Logistic regression test P=0.419N P=O.114 P=O.166 -

Cochran-Armitage test P=0.405N 

Fisher exact test P=O.117 P=O.247 -

PreputialGland:Adenoma or Carcinoma 

Overall rate 5/50 (10%) 7/49 (14%) 5/50 (10%) 4/49 (8 %) 

Adjusted rate 21.9% 27.2% 24.2% 22.9% 

Terminal rate 4/21 (19%) 2/15 (13%) 2/15 (13%) 2/13 (15%) 

First incidence (days) 671 485 416 585 

Life table test P=0.491N P=O.263 P=O.466 P=O.561 

Logistic regression test P=0.316N P=O.375 P=0.602N P=0.539N 

Cochran-Armitage test P=0.339N 

Fisher exact test P=O.365 P=0.630N P=0.513N 

Skin: Keratoacanthoma 

Overall rate 4/50 (8%) 2/50 (4%) 3/50 (6%) 1/50 (2%) 

Adjusted rate 17.5% 10.9% 15.4% 2.1 % 

Terminal rate 3/21 (14%) 1/15 (7%) 2/15 (13%) 0113 (0%) 

First incidence (days) 685 693 622 528 

Life table test P=0.245N P=0.468N P=0.614N P=0.276N 

Logistic regression test P=0.153N P=0.391N P=0.493N P=0.168N 

Cochran-Armitage test P=0.164N 

Fisher exact test P=0.339N P=0.500N P=0.181N 

Skin: Keratoacanthoma or Squamous Cell Carcinoma 

Overall rate 4/50 (8%) 3/50 (6%) 3/50 (6%) 1/50 (2%) 

Adjusted rate 17.5% 15.2% 15.4% 2.1 % 

Terminal rate 3/21 (14%) 1/15 (7%) 2/15 (13%) 0113 (0%) 

First incidence (days) 685 693 622 528 

Life table test P=0.212N P=0.640N P=0.614N P=0.276N 

Logistic regression test P=0.125N P=0.556N P=0.493N P=O.t68N 

Cochran-Armitage test P=0.136N 

Fisher exact test P=0.500N P=0.500N P=0.181N 

Skin: Keratoacanthoma, Basal Cell Adenoma, or Squamous CellCarcinoma 

Overall rate 5/50 (10%) 3/50 (6%) 3/50 (6%) 1/50 (2%) 

Adjusted rate 22.0% 15.2% 15.4% 2.1% 

Terminal rate 4/21 (19%) 1/15 (7%) 2/15 (13%) 0/13 (0%) 

First incidence (days) 693 685 622 528 

Life table test P=0.141N P=0.512N P=0.484N P=0.190N 

Logistic regression test P=0.073N P=0.416N P=0.353N P=0.095N 

Cochran-Armitage test P=0.080N 

Fisher exact test P=0.357N P=0.357N P=O.lMN 
- - 

126 Phenolphthalein, NTP TR 465 

TABLEA3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Phenolphthalein(continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Skin (Subcutaneous Tissue): Fibroma 

Overall rate 3/50 (6%) 2/50 (4%) 1/50 (2 %) 2/50 (4%) 

Adjusted rate 10.8% 4.8% 6.7% 8.0% 

Terminal rate 1/21 (5%) 0115 (0%) 1/15 (7%) 0113 (0%) 

First incidence (days) 534 561 729 (T) 673 

Life table test P=0.448N P=0.526N P=0.385N P=0.568N 

Logistic regression test P=0.407N P=0.518N P=0.292N P=0.488N 

Cochran-Armitage test P=0.404N 

Fisher exact test P=0.500N P=0.309N P=0.500N 

Skin (Pinna and Subcutaneous Tissue): Malignant Schwannoma 

Overall rate 2/50 (4%) 0150 (0%) 0/50 (0%) 3/50 (6%) 

Adjustedrate 7.6% 0.0% 0.0% 11.9% 

Terminal rate 1/21 (5%) 0115 (0%) 0/15 (0%) 1/13 (8%) 

First incidence (days) 608 570 

Life table test P=O.220 P=0.266N P=0.251N P=O.457 

Logistic regression test P=O.234 P=0.235N P=0.231N P=O.493 

Cochran-Armitage test P=O.241 

Fisher exact test P=0.247N P=0.247N P=O.500 

Testes:Adenoma 

Overall rate 46/50 (92%) 46/50 (92%) 49/50 (98%) 49/50 (98 %) 

Adjusted rate 100.0% 97.8% 100.0% 100.0% 

Terminal rate 21/21 (100%) 14/15 (93%) 15/15 (100%) 13/13 (100%) 

First incidence (days) 486 45 8 372 485 

Life table test P=O.112 P=O.233 P=O.180 P=O.103 

Logistic regression test P=O.223 P=0.365N P=O.225 P=O.680 

Cochran-Armitage test P=O.O76 

Fisher exact test P=0.643N P=O.181 P=O.181 

ThyroidGland (C-cell): Adenoma 

Overall rate 9/50 (18%) 2/50 (4%) 3/49 (6%) 4/49 (8%) 

Adjusted rate 35.3% 8.2% 13.7% 12.3% 

Terminal rate 6/21 (29%) 0115 (0%) 1/15 (7%) 0113 (0%) 

First incidence (days) 571 53 1 623 570 

Life table test P=0.216N P=0.061N P=O.lllN P=0.214N 

Logistic regression test P=0.137N P=0.026N P=0.055N P=O. 108N 

Cochran-Armitage test P=0.152N 

Fisher exact test P=0.026N P=0.065N P=0.125N 

ThyroidGland(C-cell):Adenoma or Carcinoma 

Overall rate 9/50 (1 8%) 2/50 (4%) 4/49 (8 %) 6/49 (12%) 

Adjusted rate 35.3% 8.2% 19.9% 22.2% 

Terminal rate 6/21 (29%) 0115 (0%) 2/15 (13%) 0113 (0%) 

First incidence (days) 57 1 53 1 623 570 

Life table test P=0.532N P=0.061N P=0.200N P=0.459N 

Logistic regression test P=0.386N P=0.026N P=0.106N P=0.271N 

Cochran-Armitage test P=0.417N 

Fisher exact test P=0.026N P=0.125N P=0.303N 
Lesions in Male Rats 127 

TABLEA3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Phenolphthalein (continued) 

ThyroidGland (Follicular Cell): Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

ThyroidGland(FollicularCell):Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Mononuclear Cell Leukemia 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Benign Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Malignant Neoplasms 

Overall rate 

Adjustedrate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0 PPm 

0150 (0%) 

0.0% 

0/21 (0%) -

P=O.O39 

P=O.O57 

P=O.O59 

or Carcinoma 

1/50 (2%) 

2.4% 

0121 (0%) 

529 

P=O.121 

P=O.168 

P=O.158 

31/50 (62%) 

76.0% 

12/21 (57%) 

330 

P=O.448 

P=0.457N 

P=0.416N 

47/50 (94%) 

100.0% 

21/21 (100%) 

407 

P=O.148 

P=O.354 

P=O.O89 

35/50 (70%) 

80.2% 

13/21 (62%) 

330 

P=O.467 

P=0.297N 

P=0.294N 

12,000 ppm 

1/50 (2%) 

3.6% 

0115 (0%) 

646 

P=O.493 

P=O.500 

P=O.500 

1/50 (2%) 

3.6% 

0115 (0%) 

646 

P=0.752N 

P=O.735 

P=0.753N 

27/50 (54%) 

67.6% 

5/15 (33%) 

458 

P=0.503N 

P=0.291N 

P=0.272N 

50150 (100%) 

100.0% 

15/15 (100%) 

458 

P=O.l3G 

P=O.456 

P=O.121 

36/50 (72 %) 

84.2% 

9/15 (60%) 

458 

P=O.297 

P=O.486 

P=O.500 

25,000 ppm 

2/49 (4%) 

13.3% 

2/15 (13%) 

729 (T) 

P=O.166 

P=O.166 

P=O.242 

2/49 (4%) 

13.3% 

2/15 (13%) 

729 (T) 

P=O.430 

P=O.500 

P=O.492 

24/50 (48 %) 

65.0% 

5/15 (33%) 

383 

P=0.284N 

P=O.l16N 

P=O.114N 

49/50 (98 %) 

100.0% 

15/15 (100%) 

372 

P=O.219 

P=O.480 

P=O.309 

31/50 (62%) 

81.6% 

9/15 (60%) 

383 

P=0.491N 

P=0.253N 

P=0.263N 

50,000 ppm 

3/49 (6%) 

16.1% 

1/13 (8%) 

685 

P=O.O76 

P=O.110 

P=O.117 

3/49 (6%) 

16.1% 

1/13 (8%) 

685 

P=O.241 

P=O.305 

P=O.301 

29/50 (58 %) 

87.4% 

10/13 (77%) 

524 

P=O.412 

P=0.421N 

P=0.419N 

50/50 (100%) 

100.0% 

13/13 (100%) 

485 

P=O.107 

P=O.602 

P=O.121 

33/50 (66%) 

89.6% 

10/13 (77%) 

524 

P=O.399 

P=0.420N 

P=0.415N 
128 Phenolphthalein, NTP TR 465 

TABLEA3 

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Phenolphthalein (continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

All Organs:BenignorMalignantNeoplasms 

Overall rate 49/50 (98 %) 50150 (100%) 50/50 (100%) 50/50 (100%)

Adjusted rate 100.0%100.0% 

Terminal rate 21/21 (100%) 15/15 (100%) 15/15 (100%) 13/13 (100%) 

100.0% 100.0% 

First incidence (days) 330 485 458 372 

Life table test P=O.201 P=O.206 P=O.268 P=O.167 

Logistic regression test -f - - -

Cochran-Armitage test P=O.309 

Fisher exact test P=O.500 P=O.500 P=O.500 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animalslnumber of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

kidney, liver, pancreatic islets, pituitary gland, preputial gland, testes, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 

painvise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being 

(directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group 

Value of statistic cannot be computed. 
Lesions in Male Rats 129 

TABLEA4a 

Historical Incidence of Adrenal Medulla Pheochromocytoma in Untreated Male F344/NRatsa 

Incidence in Controls 

Study Benign .Malignant Benign or Malignant 

Historical Incidence at EG&G Mason Research Institute/TSI Mason Laboratories, Inc. 

l-Amino-2,4dibromoanthraquinone 

Acetaminophen 

HC Yellow 4 

Methylphenidate Hydrochloride 

Pentaerythritol Tetranitrate 

Quercetin 

Turmeric Oleoresin 

Overall Historical Incidence 

Total 

Standard deviation 

Range 

16/44 

12/50 

1/44 

1 /so 

17144 

13/50 

19150 2150 19150 

17149 1/49 18/49 

19/49 0149 19149 

12150 1150 13/50 

14/47 0147 14/47 

39611,283 (30.9%) 

12.1% 

3911.283 

3.1% 

(3.0%) 42111.283 (32.8%) 

11.2% 

10%-63% 0%-12% 14%-63% 

a Data as if 12 May 1995;includes data for complex pheochromocytoma 

TABLEA4b 

Historical Incidence of Renal Tubule Neoplasms in Untreated Male F344/NRatsa 

Incidence in Controls 

Study Adenoma Carcinoma Adenoma or Carcinoma 

Historical Incidence at EG&G Mason Research Institute/TSI Mason Laboratories, Inc. 

l-Amino-2,4-dibromoanthraquinone 

Acetaminophen 

HC Yellow 4 

Methylphenidate Hydrochloride 

Pentaerythritol Tetranitrate 

Quercetin 

Turmeric Oleoresin 

Overall Historical Incidence 

Total 

Standard deviation 

Range 

a Data as of 12 May 1995 

2/50 0150 2150 

3/50 0150 3/50 

1150 0150 1 I50 

0149 0149 0149 

0149 0149 0149 

0150 0150 01.50 

0150 0150 0150 

911.301(0.7%)311,301(0.2%) 1211.301 (0.9%) 

1.5% 0.7% 1.5% 

0%-6% 0%-2% 0%-6% 
130 Phenolphthalein, NTP TR 465 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of Phenolphthaleina 

Disposition Summary 

Animals initially in study 

Early deaths 

Moribund 

Natural deaths 

Survivors 

Died last week of the study 

Terminal sacrifice 

Animals examined microscopically 

AlimentarySystem 

Intestine large, colon 

Parasite metazoan 

Muscularis, necrosis, focal 

Intestine large, rectum 

Necrosis 

Parasite metazoan 

Intestine large, cecum 

Edema 

Erosion, focal 

Inflammation, acute 

Inflammation, chronic 

Necrosis 

Artery, inflammation, chronic 

Epithelium, hyperplasia 

Intestine small, duodenum 

Artery, inflammation, chronic 

Muscularis, necrosis, focal 

Intestine small, jejunum 

Inflammation, chronic active 

0 PPm 

50 

15 

14 

21 

50 

(48) 

1(2%) 

(48) 

1(2%) 

(44) 

(47) 

(47) 

1(2%) 

12,000 ppm 

50 

15 

20 

15 

50 

(49) 

2 (4%) 

(49) 

1(2%) 

2(4%) 

(41) 

3(7%) 

2 (5%) 

(50) 

(41 

25,000 ppm 

50 

13 

22 

1 

14 

50 

(49) 

4(8%) 

(50) 

(44) 

1 (2%) 

1(2%) 

1(2%) 

1(2%) 

(49) 

1 (2%) 

(47) 

50,000 ppm 

50 

16 

21 

13 

50 

(48) 

3 (6%) 

1(2%) 

(50) 

(40) 

1(3%) 

1(3%) 

1 (3%) 

1 (3%) 

1(3%) 

(47) 

1 (2%) 

(42) 

(41) Intestine small, ileum (40) (44) (40) 

Inflammation, chronic 1 (3%) 

Liver (50) (50) (50) (50)

1 (2%) 

9 (18%) 21 (42%) 

Angiectasis, focal 7 (14%) 1(2%) 1 (2%)

Basophilic focus 17 (34%) 18 (36%) 

Clear cell focus 2(4%) 2 (4%) 1 (2%) 1(2%)

Degeneration, cystic, focal 

Eosinophilic focus 

Hematopoietic cell proliferation 

Hepatodiaphragmatic nodule 

Mixed cell focus 

Necrosis 

Bile duct, hyperplasia 

Centrilobular, necrosis 

Hepatocyte, vacuolization cytoplasmic 

Vein, thrombosis 

4(8%) 

8 (16%) 

2(4%) 

4(8%) 

4(8%) 

1 (2%) 

10 (20%) 

2(4%) 

3 (6%) 

2(4%) 

1 (2%) 

10 (20%) 

1(2%) 

8 (16%) 

1 (2%) 

1(2%) 

12 (24%) 

1(2%) 

6(12%) 

I (14%) 

2(4%) 

1 (2%) 

8 (16%) 

5 (10%) 

2(4%) 

1(2%) 

13 (26%) 

5 (10%) 

14 (28%) 

1 (2%) 

2(4%) 

3 (6%) 

4(8%) 

2 (4%) 

10 (20%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
Lesions in Male Rats 131 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Studyof Phenolphthalein (continued) 

Alimentary System (continued) 

Mesentery 

Metaplasia, osseous 

Polyarteritis 

Artery, inflammation, chronic 

Fat, necrosis 

Pancreas 

Infiltration cellular, focal, lymphocyte 

Necrosis, diffuse 

Pigmentation, focal 

Acinus, atrophy, diffuse 

Acinus, atrophy, focal 

Acinus, hyperplasia, focal 

Artery, inflammation, chronic 

Salivary glands 

Atrophy 

Duct, sublingual gland, hyperplasia, focal 

Parotid gland, atrophy 

Parotid gland, atrophy, focal 

Parotid gland, inflammation, chronic active 

Sublingual gland, atrophy 

Sublingual gland, inflammation, chronic 

Submandibular gland, atrophy 

Stomach, forestomach 

Edema 

Erosion, focal 

Inflammation, acute 

Inflammation, chronic 

Ulcer, focal 

Epithelium, hyperplasia 

Muscularis, mineralization 

Stomach, glandular 

Degeneration 

Edema 

Erosion, focal 

Inflammation, acute 

Mineralization 

Mineralization, focal 

Ulcer, focal 

Glands, cyst, focal 

Glands, inflammation, acute, focal 

Muscularis, mineralization 

Tooth 

Necrosis 

0 PPm 

(20) 

19 (95%) 

(50) 

1 (2%) 

1 (2%) 

22 (44%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

2 (4%) 

4 (8%) 

(50) 

8 (16%) 

1 (2%) 

12,000 ppm 

(7) 

4 (57%) 

(50) 

1 (2%) 

1 (2%) 

18 (36%) 

5 (10%) 

3 (6%) 

(49) 

2 (4%) 

(50) 

3 (6%) 

2 (4%) 

2 (4%) 

2 (4%) 

(50) 

11 (22%) 

6 (12%) 

11 (22%) 

1 (2%) 

1 (2%) 

~~ 

25,000 ppm 

(3) 

1 (33%) 

1 (33%) 

1 (33%) 

(50) 

1 (2%) 

1 (2%) 

25 (50%) 

4 (8%) 

(49) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

5 (10%) 

4 (8%) 

12 (24%) 

2 (4%) 

(50) 

5 (10%) 

1 (2%) 

4 (8%) 

5 (10%) 

1 (2%) 

2 (4%) 

1 (2%) 

2 (4%) 

50,000 ppm 

(3) 

1 (33%) 

2 (67%) 

(47) 

31 (66%) 

2 (4%) 

(49) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

4 (8%) 

2 (4%) 

2 (4%) 

3 (6%) 

9 (18%) 

(49) 

4 (8%) 

6 (12%) 

1 (2%) 

5 (10%) 

1 (2%) 

3 (6%) 

(1) 

1 (100%) 
132 Phenolphthalein, NTP TR 465 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-YearFeed Study of Phenolphthalein (continued) 

Cardiovascular System 

Blood vessel 

Aorta, media, mineralization 

Heart 

Cardiomyopathy 

Fibrosis 

Artery, mineralization 

Atrium, thrombosis 

Atrium, epicardium, hyperplasia, focal 

Atrium, epicardium, pigmentation 

Myocardium, inflammation, acute 

Myocardium, mineralization 

Valve, inflammation, acute 

Valve, inflammation, chronic active 

Endocrine System 

Adrenal cortex 

Hematopoietic cell proliferation 

Hyperplasia, focal 

Hypertrophy, focal 

Inflammation 

Necrosis 

Vacuolization cytoplasmic, focal 

Bilateral, hematopoietic cell proliferation 

Bilateral, necrosis 

Bilateral, vacuolization cytoplasmic, focal 

Adrenal medulla 

Hyperplasia, focal 

Infiltration cellular, lymphocyte 

Bilateral, hyperplasia, focal 

Islets, pancreatic 

Hyperplasia, focal 

Parathyroid gland 

Hyperplasia, diffuse 

Hyperplasia, focal 

Bilateral, hyperplasia 

Bilateral, hyperplasia, diffuse 

Pituitary gland 

Hyperplasia, focal 

Pars distalis, angiectasis, focal 

Pars distalis, cyst 

Pars distalis, hemorrhage, focal 

Pars distalis, hyperplasia, focal 

Pars distalis, hypertrophy, focal 

Pars nervosa, hyperplasia 

Rathke’s cleft, pigmentation 

Thyroid gland 

Necrosis 

Ultimobranchial cyst 

C-cell, hyperplasia, focal 

Follicle, cyst 

Follicular cell, hyperplasia 

0 PPm 

(50) 

(50) 

39 (78%) 

3 (6%) 

1 (2%) 

1 (2%) 

(50) 

4 (8%) 

2 (4%) 

6 (12%) 

(50) 

12 (24%) 

1 (2%) 

1 (2%) 

(50) 

2 (4%) 

(41) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

9 (18%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

13 (26%) 

3 (6%) 

12,000 ppm 

(49) 

9 (18%) 

(49) 

36 (73%) 

1 (2%) 

2 (4%) 

(50) 

1 (2%) 

1 (2%) 

11 (22%) 

4 (8%) 

2 (4%) 

(50) 

21 (42%) 

1 (2%) 

(50) 

(48) 

6 (13%) 

10 (21%) 

(50) 

4 (8%) 

6 (12%) 

1 (2%) 

(50) 

1 (2%) 

3 (6%) 

4 (8%) 

25,000 ppm 

(50) 

3(6%) 

(50) 

41 (82%) 

2 (4%) 

2 (4%) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

2 (4%) 

4 (8%) 

3(6%) 

2 (4%) 

(50) 

16 (32%) 

2 (4%) 

(50) 

(49) 

3(6%) 

11 (22%) 

(50) 

1 (2%) 

5 (10%) 

4 (8%) 

(49) 

1 (2%) 

9 (18%) 

7 (14%) 

50,000 ppm 

(50) 

4 (8%) 

(50) 

38 (76%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

6(12%) 

1 (2%) 

1 (2%) 

7 (14%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

19 (38%) 

4 (8%) 

(48) 

2 (4%) 

(46) 

8(17%) 

1 (2%) 

6(13%) 

(50) 

1 (2%) 

5 (10%) 

1 (2%) 

11 (22%) 

(49) 

4 (8%) 

6 (12%) 

1 (2%) 

Follicular cell, hyperplasia, focal 1 (2%) 
Lesions in Male Rats 133 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Studyof Phenolphthalein (continued) 

General Body System 

Tissue NOS 

Mediastinum, hemorrhage 

Genital System 

Epididymis 

Granuloma sperm, focal 

Inflammation, chronic 

Polyarteritis 

Preputial gland 

Atrophy 

Cyst 

Hyperplasia, focal 

Inflammation, acute 

Inflammation, chronic 

Inflammation, chronic active 

Bilateral, inflammation, chronic 

Bilateral, inflammation, chronic active 

Duct, hyperplasia 

Prostate 

Atrophy 

Inflammation, acute 

Inflammation, chronic 

Inflammation, chronic active 

Epithelium, hyperplasia, focal 

Seminal vesicle 

Atrophy 

Inflammation, acute 

Testes 

Mineralization, focal 

Bilateral, artery, inflammation, chronic 

Bilateral, interstitial cell, hyperplasia, 

focal 

Bilateral, germinal epithelium, atrophy 

Germinal epithelium, atrophy 

Interstitial cell, hyperplasia, focal 

Hematopoietic System 

Bone marrow 

Atrophy 

Fibrosis 

Hemorrhage 

Hyperplasia 

Myelofibrosis 

Necrosis 

3(6%) 

14 (28%) 

1(2%) 

21 (42%) 

6(12%) 

(50) 

14 (28%) 

6(12%) 

1(2%) 

3 (6%) 

1(2%) 

(50) 

33 (66%) 

1 (2%) 

(50) 

1(2%) 

2(4%) 

4 (8%) 

2(4%) 

2(4%) 

(48) 

1 (2%) 

15 (31%) 

1 (2%) 

1 (2%) 

12,000 ppm 

2(4%) 

(49) 

1 (2%) 

2(4%) 

1(2%) 

14 (29%) 

2(4%) 

21 (43%) 

8 (16%) 

(50) 

8 (16%) 

5 (10%) 

2(4%) 

7 (14%) 

2 (4%) 

(50) 

44 (88%) 

1 (2%) 

1 (2%) 

3 (6%) 

2(4%) 

(50) 

1 (2%) 

18 (36%) 

25,000 ppm 

2(4%) 

8 (16%) 

4 (8%) 

25 (50%) 

9(18%) 

1(2%) 

(50) 

9(18%) 

4 (8%) 

1 (2%) 

8 (16%) 

5 (10%) 

(50) 

45 (90%) 

1(2%) 

1(2%) 

3 (6%) 

(50) 

1 (2%) 

21 (42%) 

2(4%) 

50,000 ppm 

(49) 

1(2%) 

13 (27%) 

4 (8%) 

20(41%) 

4 (8%) 

(50) 

13 (26%) 

10 (20%) 

4 (8%) 

5 (10%) 

3(6%) 

(50)

47 (94%) 

1(2%) 

2(4%) 

2(4%) 

3 (6%) 

(49) 

1(2%) 

1 (2%) 

2(4%) 

26 (53%) 

1(2%) 
134 Phenolphthalein,NTP TR 465 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the2-YearFeed Study of Phenolphthalein (continued) 

Hematopoietic System (continued) 

Lymph node 

Infiltration cellular, histiocyte 

Iliac, hyperplasia, lymphoid 

Iliac, infiltration cellular, histiocyte 

Inguinal, hyperplasia, lymphoid 

Mediastinal, hematopoietic cell proliferation 

Mediastinal, hemorrhage 

Mediastinal, hyperplasia, lymphoid 

Mediastinal, infiltration cellular, 

histiocyte 

Mediastinal, pigmentation 

Pancreatic, fibrosis 

Pancreatic, hematopoietic cell proliferation 

Pancreatic, hemorrhage 

Renal, congestion 

Renal, hemorrhage 

Renal, hyperplasia, lymphoid 

Renal, infiltration cellular, histiocyte 

Renal, pigmentation 

Lymph node, mandibular 

Degeneration, cystic 

Hemorrhage 

Hyperplasia, lymphoid 

Infiltration cellular, histiocyte 

Inflammation, chronic 

Necrosis 

Pigmentation 

Lymph node, mesenteric 

Atrophy 

Degeneration, cystic 

Degeneration, cystic, focal 

Ectasia 

Hemorrhage 

Hyperplasia, lymphoid 

Infiltration cellular, histiocyte 

Inflammation, chronic 

Spleen 

Congestion 

Fibrosis 

Hematopoietic cell proliferation 

Inflammation, acute 

Inflammation, chronic active 

Pigmentation 

Capsule, degeneration, cystic 

Lymphoid follicle, atrophy 

Thymus 

Angiectasis, focal 

Cyst . 

Hemorrhage 

Necrosis, diffuse 

Epithelial cell, hyperplasia 

Thymocyte, atrophy 

1(6%) 

1 (6%) 

1(6%) 

2 (12%) 

(50) 

9(18%) 

1(2%) 

7(14%) 

2(4%) 

1(2%) 

(50) 

4(8%) 

1(2%) 

33 (66%) 

(50) 

1(2%) 

3 (6%) 

6(12%) 

3(6%) 

24 (48%) 

33 (66%) 

12,000 ppm 

1 (5%) 

3(14%) 

2 (9%) 

1 (5%) 

1 (5%) 

(49) 

6 (12%) 

6 (12%) 

1(2%) 

1(2%) 

2(4%) 

2 (4%) 

18 (36%) 

8 (16%) 

5 (10%) 

1(2%) 

1(2%) 

8 (16%) 

(49) 

1(2%) 

1(2%) 

25 (51%) 

33 (67%) 

25,000 ppm 

1(4%) 

2 (7%) 

3(11%) 

4(15%) 

2 (7%) 

1(4%) 

1(4%) 

5 (19%) 

3 (11%) 

1(4%) 

1 (4%) 

(49) 

2(4%) 

12 (24%) 

2 (4%) 

1(2%) 

(47) 

1 (2%) 

2 (4%) 

2 (4%) 

22 (47%) 

1(2%) 

(50) 

6 (12%) 

6 (12%) 

5 (10%) 

1 (2%) 

4 (8%) 

9(18%) 

(47) 

1 (2%) 

31(66%) 

37 (79%) 

50,000 ppm 

1 (5%) 

1 (5%) 

1 (5%) 

1 (5%) 

2(10%) 

1 (5%) 

1 (5%) 

1 (5%) 

2(10%) 

2 (10%) 

2 (10%) 

2(10%) 

(49) 

5 (10%) 

7(14%) 

4 (8%) 

1 (2%) 

(48) 

1(2%) 

1 (2%) 

1 (2%) 

1(2%) 

23 (48%) 

(49) 

2(4%) 

4 (8%) 

6 (12%) 

2 (4%) 

12 (24%) 

(47) 

22 (47%) 

34 (72%) 
Lesions in Male Rats 135 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the2-YearFeed Study of Phenolphthalein(continued) 

Integumentary System 

Mammary gland 

Galactocele 

Hyperplasia 

Inflammation, chronic 

Mineralization, focal 

Pigmentation 

Epithelium, hyperplasia 

Skin 

Fibrosis, focal 

Hyperkeratosis 

Hyperplasia, basal cell 

Ulcer 

Artery, thrombosis 

Epidermis, hyperplasia 

Lip, inflammation, chronic active 

Lip, ulcer 

Subcutaneous tissue, inflammation, acute 

Subcutaneous tissue, inflammation, chronic 

active 

Vein, inflammation, acute 

Musculoskeletal System 

Bone 

Femur, fibrous osteodystrophy 

Femur, hyperostosis 

Joint, inflammation, chronic 

Nervous System 

Brain 

Hydrocephalus 

Cerebellum, gliosis, focal 

Cerebellum, inflammation, acute, focal 

Cerebrum, inflammation, acute 

Cerebrum, necrosis 

Choroid plexus, cerebrum, infiltration 

cellular, lymphocyte 

Hippocampus, cerebmm, necrosis, focal 

Meninges, hemorrhage 

Respiratory System 

Lung 

Congestion 

Foreign body 

Hemorrhage 

Infiltration cellular, focal, histiocyte 

Inflammation, chronic active 

Alveolar epithelium, hyperplasia, focal 

Artery, mineralization 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

(43) 

8 (19%) 

(47) 

9 (19%) 

(48) 

13 (27%) 

(47) 

8 (17%) 

1 (2%) 

1(2%) 

1(2%) 

5 (11%) 

2 (4%) 

(50) 

1 (2%) 

1(2%) 

1(2%) 

1 (2%) 

1(2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) (50) 

17 (34%) 

(50) 

14 (28%) 

(50) 

12 (24%) 

2 (4%) 1 (2%) 1 (2%) 1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 
136 Phenolphthalein, NTP TR 465 

TABLEA5 

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of Phenolphthalein (continued) 

Respiratory System (continued) 

Lung (continued) 

Interstitium, inflammation, acute 

Interstitium. inflammation, chronic 

Interstitium, inflammation, chronic active 

Nose 

Inflammation, acute 

Inflammation, chronic 

Inflammation, chronic active 

Metaplasia, focal, squamous 

Metaplasia, squamous 

Nasolacrimal duct, inflammation, acute 

Nasolacrimal duct, inflammation, chronic 

active 

Trachea 

Inflammation, chronic aytive 

Glands, cyst, focal 

Special Senses System 

EYe 

Cornea, inflammation, chronic 

Cornea, inflammation, chronic active 

Retina, atrophy 

UrinarySystem 

Kidney 

cyst 

Hydronephrosis 

Inflammation, acute 

Mineralization, focal 

Nephropathy, chronic 

Bilateral, cyst 

Bilateral, inflammation, acute 

Bilateral, mineralization, focal 

Bilateral, nephropathy, chronic 

Bilateral, pelvis, transitional epithelium, 

hyperplasia 

Bilateral, renal tubule, hyperplasia, focal 

Bilateral, renal tubule, pigmentation 

Pelvis, inflammation, acute 

Pelvis, transitional epithelium, hyperplasia 

Renal tubule, hyperplasia, focal 

Renal tubule, hyperplasia, oncocytic 

Vein, inflammation, chronic 

Vein, thrombosis 

Urinary bladder 

Hemorrhage 

Inflammation, acute 

Inflammation, chronic active 

Transitional epithelium, hyperplasia 

0 PPm 

3(6%) 

(50) 

1(2%) 

2(4%) 

14 (28%) 

1(2%) 

1 (2%) 

(50) 

1 (2%) 

(50) 

1 (2%) 

1(2%) 

2(4%) 

45 (90%) 

1 (2%) 

10 (20%) 

3 (6%) 

(49) 

2(4%) 

1 (2%) 

12,000 ppm 

2(4%) 

12 (24%) 

1 (2%) 

1 (100%) 

(50)

3(6%) 

1 (2%) 

1(2%) 

16 (32%) 

2(4%) 

48 (96%) 

21 (42%) 

1(2%) 

5 (10%) 

10 (20%) 

5 (10%) 

(50) 

1 (2%) 

1(2%) 

25,000 ppm 

1 (2%) 

15 (30%) 

1 (2%) 

1 (2%) 

(50)

8(16%) 

1 (2%) 

1 (2%) 

13 (26%) 

1 (2%) 

1 (2%) 

49(98%) 

20 (40%) 

7(14%) 

14 (28%) 

7 (14%) 

1 (2%) 

1(2%) 

(50) 

1(2%) 

1 (2%) 

50,000 ppm 

6(12%) 

(50) 

1 (2%) 

1 (2%) 

16 (32%) 

2(4%) 

1 (2%) 

1(2%) 

(50) 

(2) 

1 (50%) 

1 (50%) 

1 (50%) 

(50)

10 (20%) 

12 (24%) 

1(2%) 

50 (100%) 

17 (34%) 

6(12%) 

1(2%) 

12 (24%) 

2(4%) 

1 (2%) 

(50) 
137 

APPENDIX B 

SUMMARY OF LESIONS IN FEMALE RATS 

IN THE 2-YEAR FEED STUDY 

OF PHENOLPHTHALEIN 

TABLEB1 Summary of the Incidence of NeoplasmsinFemale Rats 

in Feed 2-Year the Study of Phenolphthalein ........................... 138 

TABLEB2 Individual Animal Tumor Pathology of Female Rats 

in Feed 2-Year the Study of Phenolphthalein ........................... 142 

TABLE B3 Statistical Analysis of Primary Neoplasms inFemale Rats 

Study in Feed 2-Year the of Phenolphthalein ........................... 162 

TABLE B4a Historical Incidence of Adrenal Medulla Pheochromocytoma 

F344/N in Untreated Female Rats .................................. 168 

TABLEB4b Historical Incidence of Renal Tubule Neoplasms in Untreated Female F344/N Rats .. 168 

TABLEB5 Summary of the Incidence of Nonneoplastic Lesions in Female Rats 

in the 2-Year Feed Study of Phenolphthalein ........................... 169 
138 Phenolphthalein,NTP TR 465 

TABLEB1 

Summary of the Incidence of Neoplasms in FemaleRats in the 2-YearFeed Study of Phenolphthaleina 

DispositionSummary 

Animals initially in study 

Early deaths 

Moribund 

Natural deaths 

Survivors 

Terminal sacrifice 

Animals examined microscopically 

AlimentarySystem 

Intestine large, colon 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Carcinoma, metastatic, uterus 

Oral mucosa 

Squamous cell papilloma 

Pancreas 

Carcinoma, metastatic, uterus 

Acinus, adenoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell carcinoma 

Tooth 

Adamantinoma malignant 

Cardiovascular System 

Heart 

Schwannoma malignant 

EndocrineSystem 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

50 50 50 50 

9 8 7 8 

11 4 11 4 

30 38 32 38 

50 50 50 50 

1 (2%) 1 (2%) 

(12) 

1 (8%) 

(49) 

2 (4%) 

1(2%) 

(49) 

2 (4%) 

3 (6%) 
Lesions in Female Rats 139 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Rats in the2-YearFeed Study of Phenolphthalein(continued) 

Endocrine System (continued) 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Ovary 

Cystadenoma 

Granulosa cell tumor malignant 

Granulosa cell tumor benign 

Granulosa-theca tumor benign 

Bilateral, tubulostromal adenoma 

Uterus 

Carcinoma 

Histiocytic sarcoma 

Schwannoma malignant 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Endometrium, sarcoma stromal 

Vagina 

Schwannoma malignant 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Mediastinal, carcinoma, metastatic, uterus 

Lymph node, mandibular 

Lymph node, mesenteric 

Carcinoma, metastatic, uterus 

Spleen 

Fibrosarcoma 

Histiocytic sarcoma, metastatic, 

bone marrow 

Thymus 

0 PPm 

(50) 

35 (70%) 

1 (2%) 

(50) 

7 (14%) 

1 (2%) 

3 (6%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

12,000 ppm 

(49) 

32 (65%) 

(50) 

5 (10%) 

3 (6%) 

(50) 

1 (2%) 

2 (4%) 

1 (2%) 

1 (2%) 

7 (14%) 

1 (2%) 

(49) 

25,000 ppm 

(50) 

29 (58%) 

(50) 

7 (14%) 

1 (2%) 

1 (2%) 

5 (10%) 

1 (2%) 

(49) 

50,000 ppm 

(47) 

25 (53%) 

(48) 

7 (15%) 

2 (4%) 

4 (8%) 

1 (2%) 

(1) 

1 (100%) 

(49) 

1 (2%) 
140 Phenolphthalein, NTP TR 465 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-YearFeed Study of Phenolphthalein (continued) 

IntegumentarySystem 

Mammary gland 

Adenoma 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Lip, basal cell adenoma 

Pinna, squamous cell papilloma 

Subcutaneous tissue. fibroma 

MusculoskeletalSystem 

Skeletal muscle 

Sarcoma 

Nervous System 

Brain 

Cerebellum, granular cell tumor malignant 

Cerebrum, astrocytoma malignant 

Spinal cord 

RespiratorySystem 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, uterus 

Histiocytic sarcoma, metastatic, 

bone marrow 

Nose 

Turbinate, adenoma 

Special Senses System 

Zymbal’s gland 

Carcinoma 

Urinary System 

Kidney 

Pelvis, transitional epithelium, papilloma 

Renal tubule, adenoma 

Urinary bladder 

Carcinoma, metastatic, uterus 

Transitional epithelium, papilloma 

SystemicLesions 

Multiple organsb 

Histiocytic sarcoma 

Leukemia mononuclear 

0 PPm 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

(1) 

(50) 

(49) 

1 (2%) 

(50) 

1 (2%) 

21 (42%) 

12,000 ppm 

2(4%) 

(3) 

1 (33%) 

(50) 

(49) 

(50) 

1 (2%) 

21 (42%) 

25,000 ppm 

11 (23%) 

6 (13%) 

(49) 

50,000 ppm 

3 (6%) 

19 (39%) 

5 (10%) 

(50) 

1 (2%) 

1 (2%) 

2(4%) 

(2) 

1 (2%) 

(50) 

1 (2%) 

14 (28%) 

(50) 

17 (34%) 
Lesions in Female Rats 141 

TABLEB1 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Studyof Phenolphthalein(continued) 

~ ~ ~~~~~ 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Neoplasm Summary 

Total animals with primary neoplasms' 49 50 50 49 

Total primary neoplasms 138 133 98 108 

Total animals with benign neoplasms 43 49 42 41 

Total benign neoplasms 102 102 76 82 

Total animals with malignant neoplasms 30 25 21 23 

Total malignant neoplasms 36 31 22 26 

Total animals with metastatic neoplasms 2 

Total metastatic neoplasms 8 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 

Number of animals with any tissue examined microscopically ' Primary neoplasms: all neoplasms except metastatic neoplasms 
142 Phenolphthalein, NTP TR 465 

Number of Days on Study 

CarcassIDNumber 

AlimentarySystem 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery 

Carcinoma, metastatic, uterus 

Pancreas 

Carcinoma, metastatic, uterus 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell carcinoma 

Tooth 

Adamantinoma malignant 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

None 

+: Tissue examined microscopically 

A: Autolysis precludes examination 

3344555666666667777777777 

7703159000455880002233333 

0049594566809370293455555 

2222222222222222222222222 

1545446523515455242322223 

8790133384294419785013696 

......................... 

+++++++A+++++++++A+++++++ 

++++++++++++++++++A++++++ 

+++++++AA+A++++++AA++++++ 

+++++++AA+A+++++++A++++++ 

+++++++AA+A+A++++AA++++++ 

+++++++AA+A++++++++++++++ 

......................... 

+ + + + + + + 

X 

++++++++A++++++++++++++++ 

X ......................... 

......................... 

++++++++A++++++++++++++++ 

+ + 

X X 

+ 

X 

......................... 

......................... 

......................... 

......................... X 

++++++++A++++++++++++++++ 

X 

......................... 

......................... 

X xx x x x x xx x x x

X 

......................... X X X X 

M:Missingtissue X: Lesion present 

I: Insufficient tissue Blank: Not examined 
Lesions 143 in Female Rats 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery 

Carcinoma, metastatic, uterus 

Pancreas 

Carcinoma, metastatic, utem 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell carcinoma 

Tooth 

Adamantinoma malignant 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

None 

5555555556666666666666666 

3333333333333333333333333 

7777777777777777777777777 

2222222222222222222222222 

3344455661122233333445666 

8905658026702412357276145 

Total 

Tissues1 

Tumors 

......................... 50 

......................... 48 

......................... 49 

......................... 45 

......................... 46 

......................... 44 

......................... 47 

......................... 50 

+++++ 12 

1 

......................... 49 

1 

......................... 50 

++++++++++++++++++++M++++ 49 

++++++++++++++++++++M++++ 48 

2 

2 

1 

1 

......................... 50 

......................... 50 

......................... 50 

X X 2 

......................... 50 

X 2 

X 1 

......................... 49 

x x 2 

xx 3 

+++++++++++MM+M+M++++++++ 46 

......................... 50 

xxxxxxxxxxxxxx x xxxx xx 35 

1 

......................... 50 

X X X 7 

X 1 
144 Phenolphthalein, NTP TR 465 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

Adenoma 

Bilateral, adenoma 

Ovary 

Bilateral, tubulostromal adenoma 

Uterus 

Carcinoma 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Endometrium, sarcoma stromal 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Mediastinal, carcinoma, metastatic, 

uterus 

Lymph ride, mandibular 

Lymph node, mesenteric 

Carcinoma, metastatic, uterus 

Spleen 

Histiocytic sarcoma, metastatic, 

bone marrow 

Thymus 

Integumentary System 

Mammary gland 

Adenoma 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Pinna, squamous cell papilloma 

Subcutaneous tissue, fibroma 

Musculoskeletal System 

Bone 

Nervous System 

Brain 

Cerebellum, granular cell tumor 

malignant 

Peripheral nerve 

Spinal cord 

3344555666666667717777777 

7703159000455880002233333 

0049594566809370293455555 

222222222222222222i222222 

1545446523515455242322223 

8790133384294419785013696 

......................... X X 

X 

......................... 

X 

......................... X 

X 

X 

X 

......................... 

+ + ++ ++ 

X 

......................... 

++++++++A++++++++++++++++ 

X 

......................... 

......................... 

......................... X X 

X 

X X xxxxx X 

X 

......................... 

X 

......................... 

......................... 

X 

+ 

+ 
Lesions in Female Rats 145 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

Adenoma 

Bilateral, adenoma 

Ovary 

Bilateral, tubulostromal adenoma 

uterus 

Carcinoma 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Endometrium, sarcoma stromal 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Mediastinal, carcinoma, metastatic, 

utellls 

Lymph node, mandibular 

Lymph node, mesenteric 

Carcinoma, metastatic, uterus 

Spleen 

Histiocytic sarcoma, metastatic, 

bone marrow 

Thymus 

Integumentary System 

Mammary gland 

Adenoma 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Pinna, squamous cell papilloma 

Subcutaneous tissue, fibroma 

Musculoskeletal System 

Bone 

Nervous System 

Brain 

Cerebellum, granular cell tumor 

malignant 

Peripheral nerve 

Spinal cord 

7777777777777777777777777 

3333333333333333333333333 

5555555556666666666666666 

~~ ~~ _______~ ~~ -

2222222222222222222222222 Total 

3344455661122233333445666 Tissues1 

8905658026702412357276145 Tumors 

......................... 50 

x x x xx x X 9 

1 

......................... 50 

1 

......................... 50 

1 

X X 3 

1 

1 

......................... 50 

X 1 

+ 7 

1 

......................... 50 

......................... 49 

1 

......................... 50 

X 1 

......................... 50 

......................... 50 

2 

X X 3 

x x xxxxx xx x xxx 21 

X xx x x x xx 9 

......................... 50 

X 1 

1 

......................... 50 

......................... 50 
146 Phenolphthalein, NTP TR 465 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Carcinoma, metastatic, uterus 

Histiocytic sarcoma,metastatic, 

bonemarrow 

Nose 

Turbinate,adenoma 

Trachea 

Special Senses System 

EYe 

Urinary System 

Kidney 

Urinarybladder 

Carcinoma,metastatic,uterus 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

3344555666666667117171777 

7703159000455880002233333 

00495.94566809370293455555 

2222222222222222222222222 

1545446523515455242322223 

8790133384294419185013696 

......................... X 

X 

......................... 

......................... 

+ + 

......................... 

++++++++M++++++++++++++++ 

X 

......................... 

X X xxxx x x X 
Lesions in Female Rats 147 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Carcinoma,metastatic,uterus 

Histiocytic sarcoma, metastatic, 

bone marrow 

Nose 

Turbinate,adenoma 

Trachea 

Special Senses System 

EYe 

Urinary System 

Kidney 

Urinarybladder 

Carcinoma,metastatic,uterus 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

1111171111117717777111111 

3333333333333333333333333 

555555555666666.6666666666 

2222222222222222222222222 Total 

3344455661122233333445666 Tissues1 

8905658026102412351216145 Tumors 

......................... 50 

X X 3 

1 

X 1 

......................... 50 

X 1 

......................... 50 

2 

......................... 50 

......................... 49 

1 

......................... 50 

X 1 

xx x x x x x x xx x x 21 
148 Phenolphthalein, NTP TR 465 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

Number of Days on Study 

Carcass ID Number 

AlimentarySystem 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Oral mucosa 

Squamous cell papilloma 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell carcinoma 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

Peritoneum 

4556666667777777777777777 

3472777770113333333333333 

5918345995785555555555555 

2322322223232222222222222 

9187088680706777899999999 

2366817732469235813456789 

......................... 

+A+++++++++++++++++++++++ 

+A+++++++++M+++++++++++++ 

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

......................... X 

A 

+ + + + 

+ 

X 

+A+++++++++++++++++++++++ 

......................... 

......................... 

......................... + 

......................... 

......................... 

......................... 

......................... 

X X X X 

X X 

+A+++++++++++++++++++++++ 

+++++M+++++++++++++M+++++ 

++++++++++++++++++M++++++ 

xx x x xxx xx x xxxx ......................... X X 

X X 
Lesions in Female Rats 149 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Oral mucosa 

Squamous cell papilloma 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell carcinoma ~~ ~ ~ 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

Peritoneum 

7777177177777777777711717 

3333333333333333333333333 

5555555555556666666666666 

3333333333332222222222222 Total 

0000000111116617777888889 Tissues/ 

0134579012456801789024590 Tumors 

......................... 50 

......................... 49 

++++++++++++++++++++++M++ 41 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 50 

1 

X 1 

1 

+ + + 7 

+ + 3 

X 2 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

++ 3 

X 1 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

X 1 

X X X X X 9 

2 

......................... 49 

+++++M++++++++++f+M++++++ 46 

......................... 49 

xx xxxxx xxxxxx xxxx X 32

......................... 50 

xx X 5 

X 3 

+ 1 
150 Phenolphthalein, NTP TR 465 

TABLEB2 

Individual Animal Tumor Pathologyof Female Rats in the 2-YearFeed Study of Phenolphthalein: 12,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

Adenoma 

Ovary 

Cystadenoma 

Granulosa cell tumor benign 

Granulosa-theca tumor benign 

uterus 

Histiocytic sarcoma 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Vagina 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Subcutaneous tissue, fibroma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Sarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

EYe 

Zymbal’s gland 

Carcinoma 

4556666667777777777777777 

3472177770113333333333333 

5918345995785555555555555 

232232222323222222222.2222 

9187088680706777899999999 

2366817732469235813456789 

......................... X xxx X 

......................... X 

......................... 

X X X X X 

+ 

......................... +++ + + +++ + ......................... 

......................... 

......................... 

+++++++++++M+++++++++++++ 

......................... 

X X X xx x x xx ......................... 

......................... + + + 

X 

......................... + + 

+ + 

......................... X 

++++++++++++++++++M++++++ 

......................... ~~~ ~~~ 
Lesions in Female Rats 151 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

Adenoma 

Ovary 

Cystadenoma 

Granulosa cell tumor benign 

Granulosa-thecatumor benign 

Uterus 

Histiocytic sarcoma 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Vagina 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Subcutaneoustissue, fibroma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Sarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

~~~~~~~ ~~~ 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

EYe Zymbal’s gland 

Carcinoma 

7777777777777777777777777 

3333333333333333333333333 

55555.55555556666666666666 

3333333333332222222222222 Total 

0000000111116677777888889 Tissues/ 

0134579012456801789024590 Tumors 

......................... 50 

X X 7 

......................... 50 

1 

X X 2 

X 1 

......................... 50 

X 1 

X X 7 

X 1 

1 

......................... 50 

+ + 11 

......................... 50 

......................... 50 

......................... 50 

......................... 49 

......................... 50 

x xx xx x x x x xx xx x x x 25 

......................... 50 

X X 2 

......................... 50 

3 

1 

......................... 50 

2 

2 

......................... 50 

x x 3 

X 1 

......................... 49 

......................... 50 

+ 

+ 

X 
Phenolphthalein,NTP TR 465 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

~~ ~~~ ~ ~~ 

4556666661111111111111111 

3472111170113333333333333 

5918345995185555555555555 

2322322223232222222222222 

9181088680106177899999999 

2366811132469235813456189 

......................... 

+A+++++++++++++++++++++++ 

......................... 

xxxxx x xxxx xxx 
Lesions in Female Rats 153 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

(continued) 

7717777777777777777777777 

Number of Days on Study 3333333333333333333333333 

5555555555556666666666666 

3333333333332222222222222 Total 

Carcass ID Number 0000000111116677777888889 Tissues/ 

0134579012456801789024590 Tumors 

Urinary System 

Kidney ......................... 50 

Urinary bladder ......................... 49 

Systemic Lesions 

Multiple organs ......................... 50 

Histiocytic sarcoma X 1 

Leukemia mononuclear X X x x x X X 21 
154 Phenolphthalein, NTP TR 465 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 25,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Ccell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Ovary 

Granulosa cell tumor malignant 

Uterus 

Schwannoma malignant 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

3556666666666111771711111 

9060012246899112233333333 

1381862411901116905555555 

3333333333333333333333333 

4331254634654425241122222 

6291403004528169906812518 

......................... 

++++++++++A+++++++++++++,+ 

++++++++++A++A+++++++++++ 

++A++++++AA++++++++++++++ 

++++++++++A++++++++++++++ 

+A++++++++A++A+++++++++++ 

++A+++A++AA++++A+++++++++ 

......................... 

+ + ++ + 

++++++++++A++++++++++++++ 

......................... 

......................... 

......................... + 

......................... 

......................... 

+++++A+++++++++++++++++++ 

......................... X 

x x X X 

++++++++++A++++++++++++++ 

X 

++++++++M+++++++M++++++++ 

......................... 

xx X X X X x xxxxx ......................... X xx 

+M+++++++++++++++++++++++ 

X 

......................... 

......................... X 

X X 
Lesions in Female Rats 155 

TABLEB2 

Individual Animal Tumor Pathology 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Bilateral, adenoma 

Ovary 

Granulosa cell tumor malignant 

Uterus 

Schwannoma malignant 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

of Female Rats in the 2-Year Feed Study of Phenolphthalein: 25,000 ppm 

7777777777777777777777777 

3333333333333333333333333 

5555555555555556666666666 

3333333333333333333333333 Total 

3333333444455661225555566 Tissues/ 

1345678125958139031346724 Tumors 

......................... 50 

......................... 49 

......................... 48 

......................... 47 

......................... 49 

......................... 47 

......................... 45 

......................... 50 

X 1 

+ 6 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

+ 2 

......................... 50 

......................... 50 

X 1 

......................... 49 

X 1 

......................... 50 

1 

X X X x x 9 

......................... 49 

1 

M++++++++++++++++++++++++ 47 

......................... 50 

x x xx x x x x xx x x x x x x x 29 

......................... 50 

X xx x 7 

......................... 49 

X X 3 

X 1 

X 1 

......................... 50 

X 1 

......................... 50 

1 

X X X 5 

X 1 
156 Phenolphthalein, NTP TR 465 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 25,000 ppm 

(continued) \. 

Number -of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Trachea 

Special Senses System 

EYe 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

3556666666666111777771717 

9060012246899112233333333 

1381862471907176905555555 

3333333333333333333333333 

4331254634654425241122222 

6297403004528769906812518 

......................... + + + ++ +++ ......................... 

++++++++++A++++++++++++++ 

......................... 

++++++++++++++++++++++++M 

++++M++++++++++++++++++++ 

xxx 

xx ......................... 

......................... + + + 

......................... + + 

+ + 

......................... X 

......................... 

......................... 

++ 

........................... 

++++++++++A++++++++++++++ 

......................... xx x x xx xx x x 
Lesions in Female Rats 157 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 25,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bonemarrow 

Lymphnode 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammarygland 

Fibroadenoma 

Fibroadenoma,multiple 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Peripheralnerve 

Spinalcord 

Respiratory System 

Lung 

Alveolarlbronchiolar adenoma 

Nose 

Trachea 

Special Senses System 

EYe 

Urinary System 

Kidney 

Urinarybladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

7777777777777777777777777 

3333333333333333333333333 

5555555555555556666666666 

3333333333333333333333333 Total 

3333333444455661225555566 Tissues/ 

1345678125958139031346724 Tumors 

......................... 50 

+ 9 

......................... 50 

......................... 49 

......................... 50 

......................... 49 

+++++M+++++++++++++++++++ 48 

X X X xxx x x 11 

X X xx 6 

+++++M+++++++++++++++++++ 49 

......................... 50 

3 

......................... 50 

2 

2 

......................... 50 

1 

......................... 50 

......................... 50 

2 

......................... 50 

......................... 49 

......................... 50 

X xx x x X X 17 
Phenolphthalein, NTP TR 465 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 50,000 ppm 

2344455566777777777777777 

Number of Days on Study I338901829123333333333333 

1044162121245555555555555 

3334433433333333333333333 

Carcass ID Number 6981071199796667777788999 

7370886407486890357929124 

Alimentary System 

Esophagus ++++++++++M++++++++++++++ 

Intestine large, colon ++++++++++A++++++++++++++ 

Intestine large, rectum ++A++++++++++++++++++++++ 

Intestine large, cecum ++A++++++A+++++++++++++++ 

Intestine small, duodenum ++A+++++++A++++++++++++++ 

Intestine small, jejunum ++A++++++++++++M+++++++++ 

Intestine small, ileum ++++++++++A++++++++++++++ 

Liver ++A++++++++++++++++++++++ 

Hepatocellular adenoma X 

Mesentery + + + ++ + 

Pancreas ++M+++++MAM++++++++++++++ 

Acinus, adenoma X 

Salivary glands ......................... 

Stomach, forestomach ......................... Stomach, glandular ++A+++++++M++++++++++++++ 

Tongue 

Cardiovascular System 

Blood vessel ......................... Heart ++++++++++M++++++++++++++ 

Endocrine System 

Adrenal cortex ++A++++++++++++++++++++++ 

Adenoma 

Adrenal medulla ++A++++++++++++++++++++++ 

Pheochromocytoma benign xx 

Islets, pancreatic ++++++++MAM++++++++++++++ 

Adenoma 

Carcinoma 

Parathyroid gland ++++++++++M++++++++++M++M 

Pituitary gland ++M+++++++M+++++++++++++M 

Pars distalis, adenoma x x xxx x xxx 

Thyroid gland ++A+++++++M++++++++++++++ 

C-cell, adenoma X x x 

C-cell, carcinoma xx 

General Body System 

None 

Genital System 

Clitoral gland +++++M+++++++++++++++++++ 

Adenoma X X X 

Carcinoma 

Ovary ......................... Oviduct 

Uterus ......................... Endometrium, polyp stromal X 

Endometrium, polyp stromal, multiple 

Vagina + 

Schwannoma malignant X 
Lesions in Female Rats 159 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 50,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Acinus, adenoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Ovary 

Oviduct 

UkNS 

Endometrium, polyp stromal 

Endometrium, polyp stromal, multiple 

Vagina 

Schwannoma malignant 

_______~~ 

1111111111711111,111117111 

3333333333333333333333333 

5555555555555555666666666 

3334444444444444333333333 Total 

9990000000001111118888888 Tissues/ 

5690123456191235120134568 Tumors 

......................... 49 

......................... 49 

......................... 49 

......................... 48 

......................... 48 

......................... 48 

......................... 49 

......................... 49 

1 

+ I 

......................... 46 

1 

......................... 49 

......................... 49 

......................... 48 

+ + 2 

......................... 50 

......................... 49 

......................... 49 

X X 2 

......................... 49 

n

L 

......................... 47 

X 1 

X 1 

+M+++++M++++++++++++M++++ 44 

......................... 41 

xxx xxxx x xxxx xxx x 25

......................... 48 

xx X X I 

2 

+M+++++++++++++++++++++++ 48 

X x x X I 

X 1 

......................... 50 

+ 1 

......................... 50 

X X X 4 

X 1 

1 

1 
160 Phenolphthalein, NTP TR 465 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 50,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Lip, basal cell adenoma 

Subcutaneous tissue, fibroma 

-

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Cerebrum, astrocytoma malignant 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Trachea 

Special Senses System 

Zymbal’s gland 

Urinary System 

Kidney 

Peivis, transitional epithelium, 

papilloma 

Renal tubule, adenoma 

Ureter 

Urinary bladder 

Transitional epithelium, papilloma 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

2344455566771117771717777 

7338901829123333333333333 

1044162127245555555555555 

3334433433333333333333333 

6981011199796667177788999 

1310886407486890357929124 

......................... 

++ + ++ + + 

++A+++++++M++++++++++++++ 

++A++++++++++++++++++++++ 

++++++++++M++++++++++++++ 

++++++++++M++++++++++++++ 

++++++++++++++++++++++++M 

X X X 

X x x xx x x x X 

X X ......................... 

......................... 

+ + 

......................... 

X 

+ + 

+ + 

......................... 

......................... 

++++++++++M++++++++++++++ 

+ 

......................... 

+++++++++M+++++++++++++++ 

X 

......................... 

xx x xx x X X 
Lesions in Female Rats 161 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of Phenolphthalein: 50,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Fibrosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Lip, basal cell adenoma 

Subcutaneous tissue, fibroma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Cerebrum, astrocytoma malignant 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Trachea 

Special Senses System 

Zymbal’s gland 

Urinary System 

Kidney 

Pelvis, transitional epithelium, 

papilloma 

Renal tubule, adenoma 

Ureter 

Urinary bladder 

Transitional epithelium, papilloma 

~ ___~ ____~~~ 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

7771777717771777177777777 

3333333333333333333333333 

5555555555555555666666666 

3334444444444444333333333 Total 

9990000000001111118888888 Tissues/ 

5690123456791235120134568 Tumors 

......................... 50 

X 1 

++ 9 

......................... 48 

++++++++++M++++++++++++++ 48 

......................... 49 

X 1 

++M++++++++++++++++++++++ 48 

......................... 49 

3 

X xx x x x x xx x 19 

X X X 5 

......................... 50 

X 1 

X 1 

......................... 50 

2 

......................... 50 

X 2 

2 

2 

......................... 50 

x x 2 

......................... 50 

......................... 49 

1 

......................... 50 

X 1 

X 1 

+ 1 

......................... 49 

1 

~~~~~~ ~~ ~~~ ~~ ~ 

......................... 50 

X 1 

X X xx x x 14 
162 Phenolphthalein, NTP TR 465 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-YearFeed Study of Phenolphthalein 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

AdrenalMedulla:BenignPheochromocytoma 

Overall ratea 3/50 (6%) 11/50 (22%) 9/50 (18%) 2/49 (4%) 

Adjusted rateb 10.0% 25.8% 23.7% 5.3% 

Terminal rate' 3/30 (10%) 7/38 (18%) 5/32 (16%) 2/38 (5 %) 

First incidence (days) 735 (T) 549 608 735 (T) 

Life table testd P=0.138N P=O.O60 P=O.O88 P=0.392N 

Logistic regression testd P=0.197N P=O.O31 P=O.O74 P=0.392N 

Cochran-Armita e testd P=0.207N

Fisher exact test % P=O.O20 P=O.O61 P=0.510N 

AdrenalMedulla:Benign or Malignant Pheochromocytoma 

Overall rate 3/50 (6%) 12/50 (24%) 10150 (20%) 2/49 (4%) 

Adjusted rate 10.0% 28.2% 25.3% 5.3% 

Terminal rate 3/30 (10%) 8/38 (21 X) 5/32 (16%) 2/38 (5%) 

First incidence (days) 735 (T) 549 568 735 (T) 

Life table test P=0.130N P=O.O40 P=O.O58 P=0.392N 

Logistic regression test P=0.189N P=O.O19 P=O.o41 P=0.392N 

Cochran-Armitage test P=0.195N 

Fisher exact test P=O.O11 P=O.O36 P=0.510N 

ClitoralGland:Adenoma 

Overall rate 10150 (20%) 7/50 (14%) 4/49 (8 %) 7/48 (15%) 

Adjusted rate 32.2% 17.8% 12.5% 18.9% 

Terminal rate 9/30 (30%) 6/38 (16%) 4/32 (13%) 7/37 (19%) 

First incidence (days) 723 705 735 (T) 735 (T) 

Life table test P=0.162N P=0.142N P=0.053N P=0.150N 

Logistic regression test P=0.176N P=0.157N P=0.044N P=0.162N 

Cochran-Armitage test P=0.275N 

Fisher exact test P=0.298N P=0.080N P=0.330N 

ClitoralGland:AdenomaorCarcinoma 

Overall rate 10/50 (20%) 7/50 (14%) 5/49 (10%) 7/48 (15 %) 

Adjusted rate 32.2% 17.8% 15.6% 18.9% 

Terminal rate 9/30 (30%) 6/38 (16%) 5/32 (16%) 7/37 (19%) 

First incidence (days) 723 705 735 (T) 735 (T) 

Life table test P=0.172N P=0.142N P=0.097N P=0.150N 

Logistic regression test P=0.186N P=0.157N P=0.083N P=0.162N 

Cochran-Armitage test P=0.291N 

Fisher exact test P=0.298N P=O. 140N P=0.330N 

Liver:HepatocellularAdenoma or Carcinoma 

Overall rate 0150 (0%) 3/50 (6%) 1/50 (2%) 1/49 (2%) 

Adjusted rate 0.0% 7.2% 3.1% 2.6% 

Terminal rate 0/30 (0%) 1/38 (3 %) 1/32 (3%) 1/38 (3%) 

First incidence (days) -e 679 735 (T) 735 (T) 

Life table test P=0.596N P=O.162 P=O.513 P=O.547 

Logistic regression test P=O.602 P=O.126 P=O.513 P50.547 

Cochran-Armitage test P=O.592 

Fisher exact test P=O.121 P=O.500 PsO.495 
Lesions in Female Rats 163 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-YearFeed Study of Phenolphthalein(continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Lung: Alveolarhronchiolar Adenoma 

Overall rate 3/50 (6%) 3/50 (6%) 1/50 (2%) 2/50 (4%) 

Adjusted rate 10.0% 7.9% 3.1 % 5.3% 

Terminal rate 3/30 (10%) 3/38 (8%) 1/32 (3 X) 2/38 (5%) 

First incidence (days) 735 (T) 735 (T) 735 (T) 735 (T) 

Life table test P=0.276N P=0.550N P=0.281N P=0.392N 

Logistic regression test P=0.276N P=0.550N P=0.281N P=0.392N 

Cochran-Armitage test P=0.341N 

Fisher exact test P=0.661N P=0.309N P=0.500N 

Lung: Alveolarhronchiolar Adenoma orCarcinoma 

Overall rate 3/50 (6%) 4/50 (8%) 1/50 (2%) 2/50 (4%) 

Adjusted rate 10.0% 10.5% 3.1% 5.3% 

Terminal rate 3/30 (10%) 4/38 (11%) 1/32 (3%) 2/38 (5%) 

First incidence (days) 735 (T) 735 (T) 735 (T) 735 (T) 

Life table test P=0.225N P=O.629 P=0.281N P=0.392N 

Logistic regression test P=0.225N P=O.630 P=0.281N P=0.392N 

Cochran-Armitage test P=0.288N 

Fisher exact test P=O.500 P=0.309N P=0.500N 

MammaryGland:Fibroadenoma 

Overall rate 30/50 (60%) 25/50 (50%) 17/50 (34%) 24/50 (48 %) 

Adjusted rate 83.2% 60.6% 50.0% 58.4% 

Terminal rate 24/30 (80%) 22/38 (58 %) 15/32 (47%) 21/38 (55%) 

First incidence (days) 606 435 729 506 

Life table test P=0.026N P=0.019N P=0.002N P=0.014N 

Logistic regression test P=0.060N P=O.OSON P<O.OOlN P=0.075N 

Fisher exact test 

Cochran-Armitage test P=0.125N 

P=O.ZllN P=0.008N P=0.158N 

MammaryGland:FibroadenomaorAdenoma 

Overall rate 32/50 (64%) 25/50 (50%) 17/50 (34%) 24/50 (48 %) 

Adjusted rate 86.3% 60.6% 50.0% 58.4% 

Terminal rate 25/30 (83 %) 22/38 (58%) 15/32 (47 %) 21/38 (55%) 

First incidence (days) 594 435 729 506 

Life table test P=O.OllN P=0.007N P<O.OOlN P=0.005N 

Logistic regression test P=0.047N P=0.021N P<0.001N P=0.033N 

Cochran-Armitage test P=0.070N 

Fisher exact test P=0.113N P=0.002N P=0.079N 

MammaryGland:Carcinoma 

Overall rate 3/50 (6%) 0/50 (0%) 0/50 (0%) 3/50 (6%) 

Adjusted rate 8.8% 0.0% 0.0% 6.7% 

Terminal rate 2/30 (7 %) 0/38 (0%) 0/32 (0%) 1/38 (3%) 

First incidence (days) 594 - - 330 

Life table test P=O.461 P=0.093N P=0.112N P=0.612N 

Logistic regression test P=O.492 P=0.122N P=0.120N P=0.662N 

Cochran-Amitage test P=O.438 

Fisher exact test P=0.121N P=0.121N P=0.661N 
164 Phenolphthalein, NTP TR 465 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of Phenolphthalein (continued) 

0 PPm 

MammaryGland:Adenoma or Carcinoma 

Overall rate 4/50 (8%) 

Adjusted rate 12.0% 

Terminal rate 3130 (10%) 

First incidence (days) 594 

Life table test P=0.554N 

Logistic regression test P=0.542N 

Cochran-Armitage test P=0.584N 

Fisher exact test 

MammaryGland: Fibroadenoma, Adenoma, or Carcinoma 

Overall rate 32/50 (64%) 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pancreatic Islets: Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pancreatic Islets: Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

P=0.070N 

P=0.071N 

3/49 (6%) 

9.2% 

2/30 (7%) 

683 

P=0.222N 

P=0.254N 

P=0.261N 

5/49 (10%) 

15.7% 

4/30 (13%) 

12,000 ppm 

0150 (0%) 

0.0% 

0138 (0%) 

-

P=0.043N 

P=0.058N 

P=0.059N 

25150 (50%) 

25,000 ppm 

0/50 (0%) 

0.0% 

0132 (0%) 

-

P=O.O56N 

P=0.059N 

P=0.059N 

17150 (34%) 

P=O.408 

P=O.500 

0149 (0%) 

0.0% 

0138 (0%) -

P=0.091N 

P=O.lOlN 

P=0.121N 

0149 (0%) 

0.0% 

0138 (0%) 

P=0.343N 

P=0.247N 

0149 (0%) 

0.0% 

0132 (0%) -

P=O.lllN 

P=O.llON 

P=0.121N 

1/49 (2%) 

3.1 % 

1/32 (3%) 

50,000 ppm 

3/50 (6%) 

6.7% 

1138 (3%) 

330 

P=0.435N 

P=0.500N 

P=0.500N 

25150 (50%) 

Adjusted rate 60.6% 86.3% 50.0% 59.2% 

Terminal rate 25/30 (83 X) 22138 (58%) 15/32 (47%) 21/38 (55%) 

First incidence (days) 435 594 

Life table test P=0.021N P<0.001N P=0.007N

729 330 

P=0.009N 

Logistic regression test P=0.043N P=0.021N P<O.OOlN P=0.073N 

Cochran-Armitage test P=0.103N 

Fisher exact test P=0.002N P=0.113N P=0.113N

Oral Cavity (Oral Mucosa or Tongue): Squamous Cell Papilloma or Squamous Cell Carcinoma 

Overall rate 2/50 (4%) 3/50 (6%) 0150 (0%) 0150 (0%) 

Adjusted rate 5.1 % 7.6% 0.0% 0.0% 

Terminal rate 0130 (0%) 2/38 (5%) 0132 (0%) 0138 (0%) 

First incidence (days) 717 370 - -

Life table test P=0.061N P=O.577 P=0.230N P=0.222N

P=0.216N, 

P=0.247N 

1147 (2%) 

2.6% 

1138 (3%) 

735 (T) 

P=0.238N 

P=0.297N 

P=0.324N 

1 /47 (2%) 

2.6% 

1/38 (3%) 

First incidence (days) 735 683 - (T) 735 (T)

Life table test P=0.069N P=0.019N P=0.089N P=0.063N 

Logistic regression test P=0.084N P=0.022N P=0.086N P=0.091N 

Fisher exact test 

Cochran-Armitage test P=0.095N 

P=0.028N P=O. 102N P=0.112N 
Lesions in Female Rats 165 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study of Phenolphthalein (continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Pituitary Gland (Pars Distalii): Adenoma 

Overall rate 35/50 (70%) 32/49 (65 %) 29/50 (58 %) 25/47 (51%) 

Adjusted rate 89.5% 77.7 % 75.6% 63.9% 

Terminal rate 26/30 (87 %) 28/37 (76%) 23/32 (72%) 23/37 (62%) 

First incidence (days) 370 57 1 391 506 

Life table test P=0.002N P=0.040N P=0.078N P=0.002N 

Logistic regression test P=0.032N P=0.154N P=0.090N P=0.034N 

Cochran-Armitage test P=0.044N 

Fisher exact test P=0.388N P=0.149N P=0.067N 

PituitaryGland(Pars Distalii): Adenoma or Carcinoma 

Overall rate 36/50 (72%) 32/49 (65 %) 29/50 (58 %) 25/47 (53 %) 

Adjusted rate 89.8% 77.7% 75.6% 63.9% 

Terminal rate 26/30 (87 %) 28/37 (76%) 23/32 (72 %) 23/37 (62 46) 

First incidence (days) 370 57 1 391 506 

Life table test P=O.OOlN P=0.027N P=0.055N P<0.001N 

Logistic regression test P=0.021N P=0.102N P=0.057N P=0.020N 

Cochran-Armitage test P=0.030N 

Fisher exact test P=0.308N P=0.104N P=0.044N 

ThyroidGland(C-cell):Adenoma 

Overall rate 7/50 (14%) 5/50 (10%) 7/50 (14%) 7/48 (15%) 

Adjusted rate 21.2% 12.6% 20.5% 18.4% 

First incidence (days) 

Terminal rate 

659 

5/30 (17%) 

679 

4/38 (1 1 %) 

717 

5/32 (16%) 

735 (T) 

7/38 (18%) 

Life table test P=0.524N P=0.241N P=0.557N P=0.440N 

Logistic regression test P=O.493 P=0.293N P=0.549N P=O.S61N 

Cochran-Armitage test P=O.435 

Fisher exact test P=0.380N P=0.613N P=O.581 

ThyroidGland(C-cell):Carcinoma 

Overall rate 1/50 (2%) 3/50 (6%) 0150 (0%) 2/48 (4%) 

Adjusted rate 3.3% 7.4% 0.0% 5.3% 

Terminal rate 1\30 (3%) 2/38 (5 %) 0132 (0%) 2/38 (5 %) 

First incidence (days) 735 (T) 674 - 735 (T) 

Life table test P=0.606N P=O.390 P=0.487N P=O.583 

Logistic regression test P=O.568 P=O.346 P=0.487N P=O.583 

Cochran-Armitage test P=O.547 

Fisher exact test P=O.309 P=0.500N P=O.485 

ThyroidGland(C-cell):Adenoma or Carcinoma 

Overall rate 8/50 (16%) 8/50 (16%) 7/50 (14%) 9/48 (19%) 

Adjusted rate 24.3 % 19.6% 20.5 % 23.7% 

Terminal rate 6/30 (20%) 6/38 (16%) 5/32 (16%) 9/38 (24%) 

Life table test 

First incidence (days) 

P=0.520N 

659 

P=0.419N 

674 

P=0.442N 

717 

P=O..514N 

735 (T) 

Logistic regression test P=O.471 P=0.503N P=0.429N P=O.574 

Cochran-Armitage test P=O.410 

Fisher exact test P=0.607N P=0.500N P=O.463 
166 Phenolphthalein, NTP TR 465 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female R,ats in the 2-Year Feed Study of Phenolphthalein (continued) 

Uterus:StromalPolyp 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Uterus:StromalPolyp or Stromal Sarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: MononuclearCellLeukemia 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Annitage test 

Fisher exact test 

AU Organs: Benign Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

AU Organs: MalignantNeoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0 PPm 

4/50 (8 %) 

12.6% 

3/30 (10%) 

700 

P=0.431N 

P=0.533N 

P=0.539N 

5/50 (10%) 

14.7% 

3\30 (10%) 

606 

P=0.338N 

P=0.434N 

P=0.437N 

21/50 (42%) 

54.3% 

13/30 (43 W) 

515 

P=0.040N 

P=0.107N 

P=0.058N 

43/50 (86%) 

100.0% 

30/30 (100%) 

370 

P=0.014N 

P=0.126N 

P-O.111N 

30/50 (60%) 

69.O % 

17/30 (57 %) 

370 

P=0.071N 

P=0.096N 

P=0.103N 

12,000 ppm 

8/50 (16%) 

19.6% 

6/38 (16%) 

435 

P=O.299 

P=O.197 

P=O.178 

8/50 (16%) 

19.6% 

6/38 (16%) 

435 

P=O.422 

P=O.277 

P=O.277 

21/50 (42%) 

44.4% 

12/38 (32%) 

549 

P=0.285N 

P=0.571N 

P=0.580N 

49/50 (98%) 

100.0% 

38/38 (100%) 

435 

P=0.289N 

P=O.147 

P=O.O30 

25/50 (50%) 

52.9% 

16/38 (42%) 

549 

P=0.066N 

P=0.239N 

P=0.211N 

25,000 ppm 

6/50 (12%) 

17.8% 

5/32 (16%) 

697 

P=O.412 

P=O.424 

P=O.370 

6/50 (12%) 

17.8% 

5/32 (16%) 

697 

P=O.547 

P=O.547 

P=O.500 

17/50 (34%) 

39.8% 

7/32 (22%) 

601 

P=0.212N 

P=0.224N 

P=0.268N 

42/50 (84%) 

95.4% 

30/32 (94%) 

391 

P=0.281N 

P=0.301N 

P=0.500N 

21/50 (42%) 

47.0% 

9/32 (28%) 

568 

P=0.058N 

P=0.061N 

P=0.055N 

50,000 ppm 

5/50 (10%) 

12.7% 

4/38 (1 1 %) 

697 

P30.632 

P=O.558 

P-0.500 

5/50 (10%) 

12.7% 

4/38 (11%) 

697 

P=0.504N 

P=0.606N 

P=0.630N 

14/50 (28%) 

31.4% 

8/38 (21 %) 

434 

P-0.046N 

P=O.lOlN 

P=0.104N 

41/50 (82%) 

95.3% 

36/38 (95%) 

506 

P=0.016N 

P=0.335N 

P=0.393N 

23/50 (46%) 

47.5% 

13/38 (34%) 

27I 

P=0.055N 

P=0.127N 

P=0.115N 
Lesions in Female Rats 167 

TABLEB3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Studyof Phenolphthalein(continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

All Organs: Benign or MalignantNeoplasms 

Overall rate 49/50 (98%) 50/50 (100%) 50/50 (100%) 49/50 (98%) 

Adjusted rate 100.0% 100.0% 100.0% 98.0% 

Terminal rate 30/30 (1 00 %) 38/38 (100%) 32/32 (100%) 37/38 (97%) 

First incidence (days) 370 435 391 27 1 

Life table test P=0.086N P=0.401N P=0.073N P=0.131N

Logistic regression test P=0.627N P=O.834 P=O.716 P=O.758 

Cochran-Armitage test P=0.591N 

Fisher exact test P=O.500 P=O.500 P=0.753N 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

clitoral gland, liver, lung, pancreatic islets, pituitary gland, thyroid gland, and uterus; for other tissues, denominator is number of animals necropsied. 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 

pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being 

(directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group 
168 Phenolphthalein, NTP TR 465 

TABLEB4a 

Historical Incidence of Adrenal Medulla Pheochromocytoma in Untreated Female F344/NRatsa 

Incidence in Controls 

Study Benign Malignant Benign or Malignant 

Historical Incidence at EG&G Mason Research InstitutelTSI Mason Laboratories, Inc. 

l-Amino-2,4-dibromoanthraquinone 2/47 0147 2/47 

Acetaminophen 3/39 0139 3/39 

HC Yellow 4 1 149 1149 2/49 

Methylphenidate Hydrochloride 3/50 0150 3/50 

Pentaerythritol Tetranitrate 2149 0149 2/49 

Quercetin 3/49 1/49 3/49 

Turmeric Oleoresin 3/50 0150 3/50 

Overall Historical Incidence 

Total 5011,274(3.9%)611,274 (0.5%) 6411,274(5.0%) 

Standard deviation0.9% 2.4% 2.7% 

Range 0%-8% 0%-2% 2%-12% 

a Data as of 12 May 1995;includes data for complex and unspecified pheochromocytoma 

TABLE B4b 

Historical Incidence of Renal Tubule Neoplasms in Untreated Female F344/N Ratsa 

Study AdenomaAdenoma Carcinoma

Historical Incidence at EG&G Mason Research InstitutelTSI 

l-Amino-2,4-dibromoanthraquinone 

Acetaminophen 

HC Yellow 4 

Methylphenidate Hydrochloride 

Pentaerythritol Tetranitrate 

Quercetin 

Turmeric Oleoresin 

Overall Historical Incidence 

Total 

Standard deviation 

Range 

0150 

0150 

0149 

0150 

0150 

0149 

0150 

011,298(0.0%) 

Incidence in Controls 

or Carcinoma 

Mason Laboratories, Inc. 

0150 0150 

0150 0150 

0149 0149 

0150 0150 

0150 0150 

0149 0149 

0150 0150 

111,298(0.1 %) 111,298 (0.1 %)

0.4% 0.4% 

0%-2% 0%-2% 

a Data as of 12 May 1995 
Lesions in Female Rats 169 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof Phenolphthaleina 

DispositionSummary 

Animals initially in study 

Early deaths 

Moribund 

Natural deaths 

Survivors 

Terminal sacrifice 

Animals examined microscopically 

AlimentarySystem 

Intestine large, colon 

Parasite metazoan 

Intestine large, rectum 

Edema 

Inflammation, acute 

Parasite metazoan 

Intestine large, cecum 

Edema 

Necrosis 

Intestine small, duodenum 

Inflammation, chronic 

Intestine small, jejunum 

Ulcer, focal 

Intestine small, ileum 

Necrosis 

Ulcer, focal 

Liver 

Angiectasis, focal 

Basophilic focus 

Clear cell focus 

Cytoplasmic alteration, focal 

Eosinophilic focus 

Fibrosis, focal 

Hematopoietic cell proliferation 

Hepatodiaphragmatic nodule 

Hyperplasia, focal 

Infiltration cellular, focal, lymphocyte 

Inflammation, chronic 

Mixed cell focus 

Necrosis 

Bile duct, hyperplasia, focal 

Centrilobular, necrosis 

Hepatocyte, vacuolization cytoplasmic 

Kupffer cell, hyperplasia, diffuse 

Mesentely 

Accessoly spleen 

Fat, necrosis 

Oral mucosa 

Ulcer 

0 PPm 

50 

9 

11 

30 

50 

(48) 

1(2%) 

(49) 

1(2%) 

1(2%) 

4 (8%) 

(45) 

1(2%) 

1(2%) 

(46) 

(44) 

(47) 

(50) 

1(2%) 

32 (64%) 

2(4%) 

12 (24%) 

1 (2%) 

3 (6%) 

3 (6%) 

I (14%) 

25 (50%) 

2(4%) 

1(2%) 

1(2%) 

3 (6%) 

1 (2%) 

(12) 

1 (8%) 

10 (83%) 

12,000.ppm 

50 

8 

4 

38 

50 

(49) 

2 (4%) 

(47) 

(49) 

3 (6%) 

(49) 

1(2%) 

(49) 

(49) 

(50) 

4 (8%) 

34 (68%) 

3(6%) 

1 (2%) 

12 (24%) 

2(4%) 

I (14%) 

1(2%) 

3 (6%) 

23 (46%) 

1 (2%) 

(7) 

7(100%) 

(3) 

1 (33%) 

25,000 ppm 

50 

7 

11 

32 

50 

(49) 

2(4%) 

(48) 

(47) 

(49) 

(47) 

(45) 

1 (2%) 

(50) 

4 (8%) 

34 (68%) 

3(6%) 

12 (24%) 

1(2%) 

6(12%) 

4(8%) 

31 (62%) 

1 (2%) 

3 (6%) 

(6) 

6 (100%) 

50,000 ppm 

50 

8 

4 

38 

50 

(49) 

2(4%) 

(49) 

(48) 

(48) 

(48) 

1(2%) 

(49) 

1(2%) 

(49) 

34 (69%) 

1(2%) 

4 (8%) 

1(2%) 

4 (8%) 

1(2%) 

4 (8%) 

33 (67%) 

6(12%) 

(7) 

1(14%) 

5 (71%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
170 Phenolphthalein, NTP TR 465 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the2-YearFeed Study of Phenolphthalein 

(continued) 

Alimentary System (continued) 

Pancreas 

Acinus, atrophy, diffuse 

Acinus, atrophy, focal 

Acinus, basophilic focus 

Acinus, hyperplasia, focal 

Salivary glands 

Atrophy, diffuse 

Infiltration cellular, focal, lymphocyte 

Parotid gland, atrophy, diffuse 

Parotid gland, atrophy, focal 

Submandibular gland, atrophy, diffuse 

Stomach, forestomach 

Inflammation, chronic 

Ulcer, focal 

. Epithelium, hyperplasia 

Stomach, glandular 

Erosion, focal 

Inflammation, chronic 

Glands, cyst, focal 

Serosa, inflammation, acute 

Tongue 

Capillary, erosion, focal 

Tooth 

Developmental malformation 

Gingiva, inflammation, chronic 

- ~ ~ ~~~ 

Cardiovascular System 

Blood vessel 

Aorta, media, mineralization 

Heart 

Cardiomyopathy 

Artery, inflammation, chronic 

Atrium, thrombosis 

Myocardium, inflammation, acute, focal 

Endocrine System 

Adrenal cortex 

Hematopoietic cell proliferation 

Hemorrhage 

Hyperplasia, focal 

Hypertrophy, focal 

Inflammation. acute, focal 

Pigmentation 

Vacuolization cytoplasmic, focal 

Bilateral, hematopoietic cell proliferation 

Bilateral, hyperplasia, focal 

Bilateral, necrosis 

Bilateral, vacuolization cytoplasmic, focal 

(49) 

4 (8%) 

17 (35%) 

2 (4%) 

4 (8%) 

(48) 

6 (13%) 

8 (17%) 

(50) 

1 (2%) 

(50) 

36 (72%) 

1 (2%) 

2 (4%) 

1 (2%) 

2 (4%) 

2 (4%) 

1 (2%) 

6 (12%) 

1(2%) 

2 (4%) 

12,000 ppm 

(49) 

1(2%) 

28 (57%) 

1(2%) 

1 (2%) 

3 (6%) 

(50) 

2 (4%) 

9 (18%) 

(3) 

1 (33%) 

(50) 

(50) 

32 (64%) 

5 (10%) 

1 (2%) 

17 (34%) 

1(2%) 

1(2%) 

2 (4%) 

25,000 ppm 

(49) 

1(2%) 

30 (61%) 

2 (4%) 

(50) 

2 (4%) 

(50) 

1(2%) 

3 (6%) 

4 (8%) 

(50) 

1 (2%) 

1 (2%) 

21 (42%) 

1 (2%) 

(2) 

1 (50%) 

1 (50%) 

(50) 

(50) 

34 (68%) 

1 (2%) 

5 (10%) 

3 (6%) 

18 (37%) 

1 (2%) 

1 (2%) 

2 (4%) 

50,000 ppm 

(50) 

(49) 

27 (55%) 

(49) 

1 (2%) 

6 (12%) 

3 (6%) 

1 (2%) 

10 (20%) 

1 (2%) 

1 (2%) 
Lesions in Female Rats 171 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof Phenolphthalein 

(continued) 

Endocrine System (continued) 

Adrenal medulla 

Hyperplasia, focal 

Infiltration cellular, lymphocyte 

Bilateral, hyperplasia, focal 

Islets, pancreatic 

Metaplasia, focal 

Parathyroid gland 

Hyperplasia, focal 

Pituitary gland 

Pars distalis, angiectasis, focal 

Pars distalis, cyst 

Pars distalis, hyperplasia, focal 

Pars distalis, pigmentation 

Rathke’s cleft, cyst 

Thyroid gland 

Ultimobranchial cyst , C-cell, hyperplasia, diffuse 

C-cell, hyperplasia, focal 

Follicle, cyst 

Follicle, hyperplasia, focal 

General Body System 

None 

Genital System 

1 (2%) 

(49) 

1 (2%) 

(46) 

1 (2%) 

(50) 

1 (2%) 

14 (28%) 

5 (10%) 

1 (2%) 

1 (2%) 

20 (40%) 

1 (2%) 

cyst 

Hyperplasia, focal 

Inflammation, chronic 

Inflammation, chronic active 

Bilateral, atrophy 

Bilateral, hyperplasia, focal 

Duct, hyperplasia, focal 

Duct, inflammation, acute 

Ovary 

cyst 

Bilateral, atrophy 

Bilateral, cyst 

Bilateral, pigmentation 

Bursa, cyst 

Follicle, cyst 

Periovarian tissue, cyst 

Oviduct 

cyst 

1 (2%) 

4 (8%) 

3 (6%) 

1 (2%) 

1 (2%) 

(50)

14 (28%) 

1 (2%) 

4 (8%) 

(49) 

1 (2%) 

(46) 

1 (2%) 

(49) 

1 (2%) 

17 (35%) 

2 (4%) 

1 (2%) 

(50) 

1 (2%) 

18 (36%) 

1(2%) 

1 (2%) 

(49) 

1 (2%) 

4 (8%) 

4 (8%) 

3 (6%) 

2 (4%) 

1 (2%) 

4 (8%) 

2 (4%) 

1 (2%) 

(50) 

15 (30%) 

(49) 

(47)

1 (2%) 

(50) 

1 (2%) 

16 (32%) 

2 (4%) 

1 (2%) 

17 (34%) 

2 (4%) 

Clitoral gland (50) (50) 

1 (2%) 

4 (8%) 

8 (16%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

(49) 

11 (22%) 

(47) 

(47) 

5 (11%) 

11 (23%) 

4 (9%) 

(48) 

2 (4%) 

9 (19%) 

1 (2%) 

3 (6%) 

7 (15%) 

5 (10%) 

1 (2%) 

1(2%) 

6 (12%) 

4 (8%) 

(1) 

1 (100%) 
172 Phenolphthalein,NTP TR 465 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Ratsin the 2-Year Feed Study of Phenolphthalein 

(continued) 

Genital System (continued) 

UkNS 

Hydrometra 

Inflammation, chronic active 

Necrosis 

Thrombosis 

Endometrium, atrophy 

Endometrium, cyst 

Endometrium, inflammation, acute 

Hematopoietic System 

Bone marrow 

Atrophy 

Hyperplasia 

Myelofibrosis 

Lymph node 

Iliac, hemorrhage 

Iliac, hyperplasia, lymphoid 

Mediastinal, hemorrhage 

Mediastinal, pigmentation 

Renal, infiltration cellular, histiocyte 

Lymph node, mandibular 

Degeneration, cystic 

Hemorrhage 

Hyperplasia, lymphoid 

Infiltration cellular, histiocyte 

Necrosis 

Pigmentation 

Lymph node, mesenteric 

Degeneration, cystic 

Ectasia 

Hemorrhage 

Hyperplasia, lymphoid 

Infiltration cellular, histiocyte 

Pigmentation 

Spleen 

Congestion 

Fibrosis 

Hematopoietic cell proliferation 

Pigmentation 

Lymphoid follicle, atrophy 

Thymus 

cyst 

Hemorrhage 

Hyperplasia, lymphoid 

Epithelial cell, hyperplasia 

Thymocyte, atrophy 

0 PPm 

(50) 

4(8%) 

1(2%) 

2(4%) 

1(14%) 

1(14%) 

(50) 

6(12%) 

2(4%) 

14 (28%) 

4(8%) 

1 (2%) 

1(2%) 

(49) 

2(4%) 

1(2%) 

1(2%) 

2(4%) 

30 (61%) 

(50) 

1(2%) 

9(18%) 

8(16%) 

3(6%) 

(50) 

1 (2%) 

1(2%) 

20 (40%) 

21 (42%) 

12,000 ppm 

(50) 

4(8%) 

1(2%) 

1(2%) 

1 (2%) 

(50) 

11 (22%) 

4(8%) 

3(6%) 

1(2%) 

(50) 

1 (2%) 

2(4%) 

33 (66%) 

1(2%) 

(50) 

7(14%) 

7 (14%) 

(49) 

1 (2%) 

34 (69%) 

19 (39%) 

25,000 ppm 

(50) 

3 (6%) 

1 (2%) 

(50) 

3(6%) 

4(8%) 

2(4%) 

(9) 

1 (11%) 

1(11%) 

(50) 

2(4%) 

6(12%) 

1(2%) 

(49) 

4(8%) 

2(4%) 

31 (63%) 

6 (12%) 

10 (20%) 

(49) 

1(2%) 

1 (2%) 

31 (63%) 

20 (41%) 

50,000 ppm 

(50) 

6(12%) 

1(2%) 

4 (8%) 

(50) 

5 (10%) 

6(12%) 

2(4%) 

(9) 

1(11%) 

1(11%) 

(48) 

8 (17%) 

1(2%) 

5 (10%) 

3 (6%) 

1(2%) 

(48) 

1 (2%) 

2 (4%) 

1 (2%) 

26(54%) 

(49) 

1(2%) 

6 (12%) 

6 (12%) 

(48) 

1(2%) 

23 (48%) 

23 (48%) 
Lesions in Female Rats 173 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Studyof Phenolphthalein 

(continued) 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

Integumentary System 

Mammary gland (50)

Galactocele 22 (44%) 15 (30%) 14 (29%) 17 (35%) 

Inflammation, chronic 2(4%) 

Epithelium, hyperplasia 1(2%) 

(50) (48) (49) 

Skin (50)

Subcutaneous tissue, fibrosis, focal 

Subcutaneous tissue, inflammation, acute 

Subcutaneous tissue, inflammation, chronic, 

focal 

Musculoskeletal System 

Bone 

Inflammation, acute 

Femur, fibrous osteodystrophy 

Femur, hyperostosis 

Nervous System 

Brain 

Hydrocephalus 

Cerebellum, hemorrhage, focal 

Cerebrum, hemorrhage, focal 

Meninges, cerebrum, hemorrhage 

Pons, hemorrhage, focal 

Thalamus, cerebrum, necrosis 

Peripheral nerve 

Sciatic, degeneration 

Respiratory System 

Lung 

Foreign body 

Hemorrhage 

Infiltration cellular, focal, lymphocyte 

Infiltration cellular, focal, histiocyte 

Inflammation, chronic active 

Alveolar epithelium, hyperplasia, focal 

Interstitium, inflammation, chronic 

Interstitium, inflammation, chronic active 

Serosa, inflammation, chronic 

Nose 

Hemorrhage 

Inflammation, acute 

Inflammation, chronic 

Inflammation, chronic active 

Nasolacrimal duct, inflammation, acute 

Nasolacrimal duct, inflammation, chronic 

Nasolacrimal duct, inflammation, chronic 

active 

(50) 

(50) 

1 (2%) 

7 (14%) 

(50) 

2(4%) 

1 (2%) 

4 (8%) 

6 (12%) 

5 (10%) 

1 (2%) 

1(2%) 

(50) 

1(2%) 

2(4%) 

1(2%) 

1 (2%) 

5 (10%) 

5 (10%) 

1 (2%) 

(49) 

1(2%) 

2(4%) 

6 (12%) 

2 (4%) 

(50) 

3 (6%) 

6 (12%) 

1(2%) 

(50) 

1 (2%) 

1(2%) 

2(4%) 

3 (6%) 

2 (4%) 

1 (2%) 

(50) 

1(2%) 

6 (12%) 

4 (8%) 

1 (2%) 

2(4%) 

1 (2%) 
174 Phenolphthalein,NTP TR 465 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of Phenolphthalein 

(continued) 

Respiratory System (continued) 

Nose (continued) 

Olfactory epithelium, cytoplasmic alteration 

Turbinate, congestion 

Trachea 

Inflammation, acute 

Inflammation, chronic 

Special Senses System 

EYe 

Atrophy 

Cornea, inflammation, chronic 

Lens, cataract 

Retina, atrophy 

Zymbal’s gland 

cyst 

UrinarySystem 

Kidney 

cyst 

Ectopic liver 

Fibrosis, focal 

Inflammation, acute 

Mineralization, focal 

Nephropathy, chronic 

Bilateral, mineralization, focal 

Bilateral, nephropathy, chronic 

Bilateral, pelvis, dilatation 

Bilateral, pelvis, inflammation, acute 

Bilateral, pelvis, inflammation, chronic 

active 

Bilateral, pelvis, mineralization, focal 

Bilateral, pelvis, transitional epithelium, 

hyperplasia 

Bilateral, renal tubule, accumulation, 

hyaline droplet 

Bilateral, renal tubule, pigmentation 

Papilla, necrosis 

Pelvis, dilatation 

Pelvis, inflammation, acute 

Pelvis, transitional epithelium, hyperplasia 

Renal tubule, degeneration 

Urinary bladder 

Infiltration cellular, focal, lymphocyte 

Inflammation, chronic 

0 PPm 

(50) 

3 (6%) 

1(2%) 

(50) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1(2%) 

3 (6%) 

4 (8%) 

7 (14%) 

30 (60%) 

1 (2%) 

1 (2%) 

1 (2%) 

2(4%) 

11 (22%) 

1 (2%) 

1 (2%) 

(49) 

(2%) 1

12,000 ppm 

(49) 

6 (12%) 

(50) 

3 (6%) 

(50) 

2 (4%) 

1(2%) 

3 (6%) 

1 (2%) 

5 (10%) 

44 (88%) 

9 (18%) 

2(4%) 

1 (2%) 

(49) 

25,000 ppm 

(50) 

3 (6%) 

(50) 

2(4%) 

(50) 

1(2%) 

5 (10%) 

9 (18%) 

43 (86%) 

1(2%) 

I (14%) 

1 (2%) 

1 (2%) 

(49) 

1(2%) 

50,000 ppm 

(50) 

2 (4%) 

(49) 

1(2%) 

(50) 

1 (2%) 

1 (2%) 

3 (6%) 

2(4%) 

5 (10%) 

42 (84%) 

2(4%) 

8 (16%) 

(49) 
175 

TABLEC1 

TABLEC2 

TABLEC3 

TABLEC4a 

TABLEC4b 

TABLEC4c 

TABLEC5 

APPENDIX C 

SUMMARY OF LESIONS IN MALEMICE 

IN THE 2-YEAR FEED STUDY 

OF PHENOLPHTHALEIN 

Summary of the Incidence of Neoplasmsin Male Mice 

in the 2-Year Feed Study of Phenolphthalein ........................... 177 

Individual Animal Tumor Pathology of MaleMice 

in the 2-Year Feed Study of Phenolphthalein ........................... 182 

Statistical Analysis of Primary Neoplasms in Male Mice 

in the 2-Year Feed Study of Phenolphthalein ........................... 208 

Historical Incidence of Histiocytic Sarcoma in Untreated Male B6C3F, 212 Mice ....... Historical Incidence of Malignant Lymphoma in Untreated Male B6C3Fl Mice212 ..... Historical Incidence of Hepatocellular Neoplasms in Untreated Male B6C3Fl Mice213... Summary of the Incidence of Nonneoplastic Lesions in Male Mice 

in the 2-Year Feed Study of Phenolphthalein ........................... 214 
176 Phenolphthalein, NTF' TR 465 
Lesions in Male Mice 177 

TABLEC1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Studyof Phenolphthaleina 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

DispositionSummary 

Animals initially in study 50 

Early deaths 

Moribund 6 

Natural deaths 4 

Survivors 

Terminal sacrifice 40 

Missing 

Animals examined microscopically 50 

AlimentarySystem 

Esophagus 

Gallbladder 

Fibrosarcoma, metastatic, mesentery 

Intestine large, rectum 

Fibrosarcoma, metastatic, mesehtery 

Intestine large, cecum 

Carcinoma 

Intestine small, duodenum 

Carcinoma 

Fibrosarcoma, metastatic, mesentery 

Histiocytic sarcoma 

Intestine small, jejunum 

Fibrosarcoma, metastatic, mesentery 

Osteosarcoma, metastatic, uncertain primary 

site 

Intestine small, ileum 

Liver 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Hemangiosarcoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Hepatocholangiocarcinoma 

Histiocytic sarcoma 

It0 cell tumor malignant 

Osteosarcoma, metastatic, uncertain primary 

site 

Sarcoma, metastatic, uncertain primary site 

Mesentery 

Fibrosarcoma 

Hemangiosarcoma 

Hepatocholangiocarcinoma, metastatic, liver 

Sarcoma, metastatic, uncertain primary site 

Pancreas 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma,metastatic, liver 

Histiocytic sarcoma 

Sarcoma, metastatic, uncertain primary site 

50 50 50 

4 

13 

3 

11 

10 

3 

33 36 

1 

36 

50 50 49 
178 Phenolphthalein, NTP TR 465 

TABLEC1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of Phenolphthalein (continued) 

Alimentary System (continued) 

Salivary glands 

Hemangiosarcoma 

Stomach, forestomach 

Squamous cell carcinoma 

Squamous cell papilloma 

Histiocytic sarcoma 

Stomach, glandular (50) 

1 (2%) 

Squamous cell carcinoma, metastatic, 

stomach, forestomach 1 (2%) 

Tongue 

Squamous cell carcinoma 

Tooth 

Sarcoma, metastatic, uncertain primary site 

Cardiovascular System 

Heart (50) (50) (50) (49) 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 1 (2%) 

Fibrosarcoma, metastatic, mesentery 1 (2%) 

Hemangiosarcoma 1 (2%) 

Histiocytic sarcoma 3 (6%) 

Sarcoma, metastatic, uncertain primary site 1 (2%) 

Endocrine System 

Adenoma 

Adrenal cortex (49) (50) 

1 (2%) 

(49) 

Hepatocholangiocarcinoma, metastatic, liver 1 (2%) 

Capsule, adenoma 1 (2%) 2 (4%) 

Capsule, sarcoma, metastatic, uncertain 

primary site 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

Follicular cell, carcinoma 

General Body System 

Tissue NOS 
Lesions in Male Mice 179 

TABLEC1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-YearFeed Study of Phenolphthalein(continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

Genital System 

Epididymis 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, liver 

Histiocytic sarcoma 

Leiomyoma 

Preputial gland 

Fibrosarcoma, metastatic, skin 

Prostate 

Fibrosarcoma, metastatic, mesentery 

Seminal vesicle 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, liver 

Testes 

Hemangiosarcoma 

Interstitial cell, adenoma 

HematopoieticSystem 

Bone marrow 

Hemangiosarcoma 

Histiocytic sarcoma 

Mast cell tumor malignant 

Lymph node 

Iliac, fibrosarcoma, metastatic, mesentery 

Iliac, histiocytic sarcoma 

Inguinal, mast cell tumor malignant, 

metastatic, bone marrow 

Mediastinal, carcinoma, metastatic, kidney 

Mediastinal, sarcoma, metastatic, uncertain 

primary site 

Pancreatic, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Renal, osteosarcoma, metastatic, uncertain 

primary site 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, liver 

Histiocytic sarcoma 

Spleen 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Fibrosarcoma, metastatic, mesentery 

Histiocytic sarcoma 

(50) 

(49) 

(50) 

(50) 

(50) 

1 (2%) 

(49) 

5 (10%) 

1 (2%) 

(9) 

1 (11%) 

1 (11%) 

1 (11%) 

(49) 

4 (8%) 

(49) 

7 (14%) 

(49) 

1 (2%) 

9 (18%) 

(42) 

2 (5%) 
180 Phenolphthalein,NTP TR 465 

Integumentary System 

Skin (50) (50) (50) (49)

Subcutaneous tissue, fibrosarcoma 1 (2%) 1 (2%) 

Subcutaneous tissue, hemangiosarcoma 1 (2%) 3 (6%) 

Subcutaneous tissue, histiocytic sarcoma 1 (2%) 1 (2%) 

Subcutaneous tissue, sarcoma, metastatic, 

uncertain primary site 1 (2%) 

Musculoskeletal System 

Bone (50)

Carcinoma, metastatic, kidney 1 (2%) 

Skeletal muscle (2) (1) (1) 

(50) (50) (48) 

(1)

Alveolar/bronchiolar carcinoma, metastatic, 

lung 1 (50%) 

Carcinoma, metastatic, kidney 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcjnoma, metastatic, liver 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar carcinoma, multiple 

Carcinoma, metastatic, harderian gland 

Carcinoma, metastatic, kidney 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Hepatocellular carcinoma, metastatic, liver 

Histiocytic sarcoma 

Sarcoma, metastatic, uncertain primary site 

Nose 

Sarcoma, metastatic, uncertain primary site 

Special Senses System 

Ear 

Harderian gland 

Adenoma 

Carcinoma 

1 (50%) 

3 (6%) 

1 (2%) 

1 (2%) 

2 (4%) 

(3) 

3 (100%) 

1(100%) 

5 (10%) 

1 (2%) 

3 (6%) 

2 (4%) 

1 (2%) 

(50) 

1 (2%) 

(2) 

(4) 

3 (75%) 

1 (100%) 

(50) 

4(8%) 

4(8%) 

1 (2%) 

1 (2%) 

4 (8%) 

(4) 

2 (50%) 

2 (50%) 

(49) 

10 (20%) 

2 (4%) 

4 (8%) 

1 (2%) 

3 (6%) 

9 (18%) 

(3) 

1 (33%) 

1 (33%) 
Lesions in Male Mice 181 

TABLEC1 

Summary of the Incidence of Neoplasms in Male Mice in the 2-YearFeed Study of Phenolphthalein(continued) 

3,000 ppm 6,000 ppm 12,000 ppm 

Urinary System 

Kidney (50) (50) (50) (49)

Histiocytic sarcoma 2 (4%) 2 (4%) 3 (6%) 

Sarcoma, metastatic, uncertain primary site 1 (2%) 

Squamous cell carcinoma, metastatic, 

stomach, forestomach 1 (2%) 

Renal tubule, adenoma 2 (4%) 

Transitional epithelium, carcinoma 1 (2%) 

Urinary bladder (50) (50) (48) (49) 

Systemic Lesions 

Multiple organsb (50) (50) (50) (49) 

Histiocytic sarcoma 1 (2%) 3 (6%) 11 (22%) 12 (24%) 

Lymphoma malignant 6(12%) 8 (16%) 12 (24%) 8 (16%) 

Neoplasm Summary 

Total animals with primary neoplasms' 40 40 42 38 

Total primary neoplasms 63 71 65 62 

Total animals with benign neoplasms 29 24 18 19 

Total benign neoplasms 37 31 20 23 

Total animals with malignant neoplasms 19 29 35 29 

Total malignant neoplasms 26 40 45 39 

Total animals with metastatic neoplasms 4 5 5 4 

Total metastatic neoplasms 9 34 13 6 

Total animals with malignant neoplasms 

of uncertain primary site 1 1 

a Number of animals examined microscopically at the site andthe number of animals with neoplasm 

Number of animals with any tissue examined microscopically ' Primaryneoplasms: aII neoplasms except metastatic neoplasms 
182 Phenolphthalein, NTP TR 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Ito cell tumor malignant 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell carcinoma 

Tooth 

Cardiovascular System 

Heart 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, carcinoma 

General Body System 

None 

+: Tissue examined microscopically 

A: Autolysis precludes examination 

2555566611111111111111117 

3358916122222222222222222 

2302932341999999999999999 

0000000000000000000000000 

4322301412000111111222223 

1480111212239023689136190 

......................... 

......................... 

......................... 

......................... 

......................... 

+++A+++++++++++++++++++++ 

+++A+++++++++++++++++++++ 

+++++++++A+++++++++++++++ 

......................... X 

X 

X 

X X X X 

xx xx 

X 

+ + + + ++ ......................... 

......................... 

......................... 

......................... + 

X .................... ++++ 

......................... 

X 

++++++M++++++++++++++++++ 

X 

++++++M++++++++++++++++++ 

......................... X 

+++++++++++++++M++++M++++ 

++++++M++++++++++++++++++ 

X 

++++++++++++++++++++M++++ 

X 

M: Missingtissue X: Lesion present 

I:Insufficienttissue Blank: Not examined 
Lesions in Male Mice 183 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Dayson Study 

Carcass ID Number 

AlimentarySystem 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma ' 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

It0 cell tumor malignant 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell carcinoma 

Tooth 

Cardiovascular System 

Heart 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

EndocrineSystem 

Adrenal cortex 

Capsule, adenoma 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, carcinoma 

General Body System 

None 

7777777777777777777777777 

222222223333333333.3333333 

9999999900000000000000000 

0000000000000000000000000 Total 

3333344400000112233444445 Tissues/ 

2358903914568454516145680 Tumors 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 49 

......................... 49 

......................... 49 

......................... 50 

1 

X xx X x x 7 

1 

x x x X X X 10 

X X x x x x X 12 

1 

X 1 

+ + + 9 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

1 

1 

......................... 49 

......................... 50 

1 

......................... 49 

1 

......................... 49 

......................... 50 

1 

+++++++++++++++++++++M+M+ 46 

++++++++M++++++++++++++++ 48 

1 

......................... 49 

1 
Phenolphthalein, NTP TR 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Penis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

Interstitial cell, adenoma 

HematopoieticSystem 

Bone marrow 

Lymph node 

Mediastinal, carcinoma, metastatic, 

kidney 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

~~~~~~~ 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Carcinoma, metastatic, kidney 

Skeletal muscle 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Carcinoma, metastatic, kidney 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, kidney 

Hemangiosarcoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

~___ ~ ~ ~~~~ ~~ ~~ 

2555566677777771717717777 

3358916122222222222222222 

2302932347999999999999999 

0000000000000000000000000 

4322301412000111111222223 

7480777212239023689136190 

......................... + ......................... 

......................... 

......................... 

......................... 

......................... +++ + + + 

X 

++++++++++++++++++++M++++ 

......................... 

......................... 

+++++M+++++++++++M++I++++ 

X 

MM+MMMMMMMMMMMMMMMMMMMMMM 

......................... 

......................... X 

+ + 

X 

X 

......................... + 

+ 

......................... X X X 

x x X 

X 

X 

X 

......................... 

......................... 
Lesions in Male Mice 185 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Penis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

Interstitial cell, adenoma 

HematopoieticSystem 

Bone marrow 

Lymph node 

Mediastinal, carcinoma, metastatic, 

kidney 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

IntegumentarySystem 

MammaIy gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Carcinoma, metastatic, kidney 

Skeletal muscle 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Carcinoma, metastatic, kidney 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, kidney 

Hemangiosarcoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

7777777177777777777777777 

2222222233333333333333333 

9999999900000000000000000 

0000000000000000000000000 Total 

3333344400000112233444445 Tissues/ 

2358903914568454516145680 Tumors 

......................... 50 

1 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

+ + 8 

1 

......................... 49 

......................... 50 

......................... 50 

+++++M+++M++++++I+++++++M 43 

1 

MMMMMMMMMMMMMMMMMMMMMMMMM 1 

......................... 50 

X 1 

X 1 

......................... 50 

1 

2 

1 

1 

......................... 50 

1 

1 

......................... 50 

X xx x x 8 

3 

1 

1 

X 2 

......................... 50 

......................... 50 
186 Phenolphthalein, NTF' TR 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-YearFeed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Transitional epithelium, carcinoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

2555566611111111111111111 

3358916122222222222222222 

2302932341999999999999999 

0000000000000000000000000 

4322301412000111111222223 

1480111212239023689136190 

+ + 

X X 

......................... X 

......................... 

......................... X 

X X 
Lesions in Male Mice 187 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Transitionalepithelium,carcinoma 

Urinarybladder 

Systemic Lesions 

Multipleorgans 

Histiocyticsarcoma 

Lymphomamalignant 

7711777117777711771711717 

2222222233333333333333333 

9999999900000000000000000 

0000000000000000000000000 Total 

3333344400000112233444445 Tissues/ 

2358903914568454516145680 Tumors 

+ 3 

X 3 

......................... 50 

1 

......................... 50 

......................... 50 

1 

xx X X 6 
188 Phenolphthalein,NTP TR 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm 

Number of Days on Study 

Carcass ID Number 

AlimentarySystem 

Esophagus 

Gallbladder 

Fibrosarcoma, metastatic, mesentery 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Carcinoma 

Intestine small, duodenum 

Fibrosarcoma, metastatic, mesentery 

Histiocytic sarcoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Hemangiosarcoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Hepatocholangiocarcinoma 

Histiocytic sarcoma 

Sarcoma, metastatic, uncertain 

primary site 

Mesentery 

Fibrosarcoma 

Hemangiosarcoma 

Hepatocholangiocarcinoma, metastatic, 

liver 

Sarcoma, metastatic, uncertain 

primary site 

Pancreas 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, 

liver 

Sarcoma, metastatic, uncertain 

primary site 

Salivary glands 

Stomach,- forestomach 

Stomach, glandular 

Histiocytic sarcoma 

Tongue 

Tooth 

Sarcoma, metastatic, uncertain 

primaly site 

3555556666611111171171111 

4466890335700012222222222 

7903372418637841499999999 

0000000000000000000000000 

6658196685196695655555667 

6835345414239311212568010 

......................... 

+++M+++++++++++A+M+++++++ 

X 

......................... 

......................... 

+++++++++A+++++A+++++++++ 

+++A+++++A+++++++++++++++ 

X 

X 

+++++++++A+++++++++++++++ 

......................... 

......................... X 

X 

X 

X X X X X 

X 

xxx X X x x X 

X 

X X X 

X 

+++ + + + 

X 

X 

X ......................... X 

X 

X ......................... 

......................... 

......................... X 

......................... 

X 
Lesions in Male Mice 189 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Fibrosarcoma, metastatic, mesentery 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Carcinoma 

Intestine small, duodenum 

Fibrosarcoma, metastatic, mesentery 

Histiocytic sarcoma 

Intestine small, jejunum 

Intestine small, ileum * Liver 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Hemangiosarcoma, multiple 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Hepatocholangiocarcinoma 

Histiocytic sarcoma 

Sarcoma, metastatic, uncertain 

primary site 

Mesentery 

Fibrosarcoma 

Hemangiosarcoma 

Hepatocholangiocarcinoma, metastatic, 

liver 

Sarcoma, metastatic, uncertain 

primary site 

Pancreas 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, 

liver 

Sarcoma, metastatic, uncertain 

primary site 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Histiocytic sarcoma 

Tongue 

Tooth 

Sarcoma, metastatic, uncertain 

primary site 

11111171171111111711~7171 

222222222222222223.3333333 

9999999999999999900000000 

~~_____ ~_______ ~~_______ ~______ ~_____ ~_____ 

0000000000000000100000000 Total 

1111888888899999056111899 Tissues/ 

1469023418905689091518621 Tumors 

......................... 50 

+++++++++++++++++M+++++++ 46 

1 

......................... 50 

......................... 50 

......................... 48 

X 1 

......................... 48 

1 

1 

......................... 49 

......................... 50 

......................... 50 

1 

1 

1 

X X X X 9 

1 

X x x 11 

X 1 

1 

3 

1 

+ + + 9 

1 

X X 2 

1 

1 

......................... 50 

1 

1 

1 

......................... 50 

......................... 50 

......................... 50 

1 

+ 1 

......................... 50 

1 
190 Phenolphthalein, NTP TR 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Cardiovascular System 

Heart 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Sarcoma, metastatic, uncertain 

primary site 

Endocrine System 

Adrenal cortex 

Adenoma 

Hepatocholangiocarcinoma, metastatic, 

liver 

Capsule, adenoma 

Capsule, sarcoma, metastatic, 

uncertain primary site 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

Tissue NOS 

Genital System 

Epididymis 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, 

liver 

Leiomyoma 

Preputial gland 

Prostate 

Fibrosarcoma, metastatic, mesentery 

Seminal vesicle 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, 

liver 

Testes 

HematopoieticSystem 

Bone marrow 

Hemangiosarcoma 

Histiocytic sarcoma 

Lymph node 

Iliac, fibrosarcoma, metastatic, 

mesentery 

Mediastinal, sarcoma, metastatic, 

uncertain primary site 

3555556666617717177771777 

446689033.5700012222222222 

7903372478637841499999999 

0000000000000000000000000 

6658796685796695655555667 

6835345414239317212568010 

......................... X 

X 

X 

......................... 

X 

X 

X ......................... 

......................... 

+++M++++++++++++++++++++M 

......................... X ......................... X 

+ 

......................... X 

X 

X ......................... 

......................... X 

......................... X 

X ......................... 

......................... X 

X 

+ + + + + 

X 

X 
Lesions in Male Mice 191 

Number of Days on Study 

Carcass ID Number 

Cardiovascular System 

Heart 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Sarcoma, metastatic, uncertain 

primary site 

Endocrine System 

Adrenal cortex 

Adenoma 

Hepatocholangiocarcinoma, metastatic, 

liver 

Capsule, adenoma 

Capsule, sarcoma, metastatic, 

uncertain primary site 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, adenoma 

~ ~ ~~ 

General Body System 

Tissue NOS 

Genital System 

Epididymis 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, 

liver 

Leiomyoma 

Preputial gland 

Prostate 

Fibrosarcoma, metastatic, mesentery 

Seminal vesicle 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, 

liver 

Testes 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Histiocytic sarcoma 

Lymph node 

Iliac, fibrosarcoma, metastatic, 

mesentery 

Mediastinal, sarcoma, metastatic, 

uncertain primary site 

7771117777111171111771111 

2222222222222222233333333 

9999999999999999900000000 

0000000000000000100000000 Total 

7771888888899999056117899 Tissues/ 

1469023418905689091518621 Tumors 

......................... 50 

1 

1 

1 

......................... 50 

X 1 

1 

X 2 

1 

......................... 50 

......................... 50 

......................... 48 

......................... 50 

1 

......................... 50 

1 

1 

......................... 50 

1 

1 

1 

......................... 50 

......................... 50 

1 

......................... 50 

1 

1 

......................... 50 

......................... 50 

1 

1 

5 

1 

1 
192 Phenolphthalein, NTP T R 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

HematopoieticSystem (continued) 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Spleen 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Fibrosarcoma, metastatic, mesentery 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, histiocytic 

sarcoma 

Subcutaneous tissue, sarcoma, 

metastatic, uncertain primary site 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hepatocholangiocarcinoma, metastatic, 

liver 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolarlbronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Fibrosarcoma, metastatic, mesentery 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Sarcoma, metastatic, uncertain 

primary site 

Nose 

Sarcoma, metastatic, uncertain 

primary site 

Trachea 

______~~ 

Special Senses System 

Ear 

Harderian giand 

Adenoma 

3555556666611171111117111 

4466890335100012222222222 

7903312418631841499999999 

0000000000000000000000000 

6658796685196695655555667 

6835345414239317212568010 

......................... X X 

......................... X 

X 

X X 

......................... X 

‘X X X 

X X 

+M++++++M++++++++++++++M+ 

X 

MMM++MM+MMMMMMMMM+MMMMMMM 

......................... 

X 

X 

......................... + 

X 

.......................... 

......................... X X X xx 

X X 

X 

X 

X X 

X 

......................... 

X 

......................... 

+ + 

+ + t 

X X 
Lesions in Male Mice 193 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System (continued) 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Fibrosarcoma, metastatic, mesentery 

Hepatocholangiocarcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Spleen 

Fibrosarcoma, metastatic. mesentery 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Fibrosarcoma, metastatic, mesentery 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, histiocytic 

sarcoma 

Subcutaneous tissue, sarcoma, 

metastatic, uncertain primary site 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hepatocholangiocarcinoma, metastatic, 

liver 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolarlbronchiolar carcinoma 

Fibrosarcoma, metastatic, mesentery 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Sarcoma, metastatic, uncertain 

primary site 

Nose 

Sarcoma, metastatic, uncertain 

primary site 

Trachea 

Special Senses System 

Ear 

Harderian gland 

Adenoma 

7777777777777777777777777 

2222222222222222233333333 

9999999999999999900000000 

0000000000000000100000000 Total 

7777888888899999056777899 Tissues/ 

1469023478905689091578627 Tumors 

......................... 50 

2 

......................... 50 

1 

1 

2 

......................... 50 

1 

x x 5 

1

L 

++++++++++++++++++M++++++ 46 

1 

+MMMMMMMM+MMMMMMMMMMMMMMM 6 

......................... 50 

1 

1 

......................... 50 

1 

1 

......................... 50 

.......................... 50 

X X x x X 10 

x x X 5 

1 

X X 3 

L 

1 

......................... 50 

1 

......................... 50 

2 

+ 4 

X 3 
194 Phenolphthalein, hTP TR 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Histiocytic sarcoma 

Sarcoma,metastatic,uncertain 

primarysite 

Urinarybladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

~ ~~ ~~ __ 

3555556666677777777777777 

4466890335700012222222222 

7903372478637841499999999 

0000000000000000000000000 

6658796685796695655555667 

683534541423931’7212568070 

......................... X X 

X ......................... 

......................... X X X 

X xx x X X 
Lesions in Male Mice 195 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the &Year Feed Study of Phenolphthalein: 3,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Histiocytic sarcoma 

Sarcoma,metastatic,uncertain 

primarysite 

Urinarybladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

7777777777777777771771777 

2222222222222222233333333 

9999999999999999900000000 

0000000000000000100000000 Total 

7777888888899999056717899 Tissues/ 

1469023478905689091578627 Tumors 

......................... 50 

2 

1 

......................... 50 

......................... 50 

3 

X X 8 
196 Phenolphthalein, NTP TR 465 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm 

Number of Days on Study 

CarcassIDNumber 

AlimentarySystem 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Fibrosarcoma, metastatic, mesentery 

Intestine large, cecum 

Intestine small, duodenum 

Carcinoma 

Intestine small, jejunum 

Fibrosarcoma, metastatic, mesentery 

Osteosarcoma, metastatic, uncertain 

primary site 

Intestine small, ileum 

Liver 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Histiocytic sarcoma 

Osteosarcoma, metastatic, uncertain 

primary site 

Mesentery 

Fibrosarcoma 

Pancreas 

Fibrosarcoma, metastatic, mesentery 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

4455556666611111111111171 

2901355567901122222222222 

3505561953603699999999999 

1111111111111111111111111 

02145102303212000001l1l1l 

9508096438718212351123456 

~~____ 

......................... 

+M+++++++++AA++++++++++M+ 

+++++++++++A+++++++++++++ 

......................... X 

++++++++A++AA++++++++++++ 

+++++++++++AA++++++++++++ 

X 

+++++++++++AA++++++++++++ 

A

.I 

X 

+++++++++++AA++++++++++++ 

......................... X 

X X X X 

X 

xx X X X 

X xx X X 

X 

+ + 

X 

......................... X 

......................... 

......................... 

......................... 

......................... 

......................... 

+++++++++++++++++M+++++++ 

f++++++++++++++++M+++++++ 

......................... 

++++++++++M++++++++++++++ 

+++++++++M+++++++++++++++ 

......................... X X 
Lesions in Male Mice 197 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Fibrosarcoma, metastatic, mesentery 

Intestine large, cecum 

Intestine small, duodenum 

Carcinoma 

Intestine small, jejunum 

Fibrosarcoma, metastatic, mesentery 

Osteosarcoma, metastatic, uncertain 

primary site 

Intestine small, ileum 

Liver 

Fibrosarcoma, metastatic, mesentery 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Histiocytic sarcoma 

Osteosarcoma, metastatic, uncertain 

primary site 

Mesentery 

Fibrosarcoma 

Pancreas 

Fibrosarcoma, metastatic, mesentery 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

1771117771171111777111111 

2222222222222222233333333 

9999999999999999900000000 

1111111111111111111111111 Total 

1222223333344444402333444 Tissues/ 

7036180246823456949159011 Tumors 

......................... 50 

......................... 46 

......................... 49 

......................... 50 

1 

......................... 41 

......................... 48 

1 

......................... 48 

1 

1 

......................... 48 

......................... 50 

1 

X X 2 

4 

1 

X X X 8 

X xx X X 10 

1 

+ + 4 

1 

......................... 50 

1 

......................... 50 

......................... 50 

X X 2 

......................... 50 

......................... 50 

......................... 50 

......................... 49 

......................... 49 

......................... 50 

++++++++M++++++++++++++++ 48 

......................... 49 

......................... 50 

2 
Phenolphthalein, NTP TR 465 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Fibrosarcoma, metastatic, mesentery 

Preputial gland 

Fibrosarcoma, metastatic, skin 

Prostate 

Seminal vesicle 

Fibrosarcoma, metastatic, mesentery 

Testes 

Hemangiosarcoma 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Histiocytic sarcoma 

Lymph node 

Iliac, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Renal, osteosarcoma, metastatic, 

uncertain primary site 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, histiocytic 

sarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Fibrosarcoma, metastatic, mesentery ~~ ~~~~ 

Nervous System 

Brain 

44555566666111711117111~1 

2901355561901122222222222 

350556195360,3699999999999 

1111111111111111111111111 

0214510230321200000111111 

9508096438118212351123456 

......................... X 

......................... X ......................... 

......................... X 

......................... 

......................... 

xx X 

++++ + + + 

X X 

X 

X 

......................... 

+++++++++M+A+++++++++++++ 

X X X X 

......................... X 

X X X X 

++++++++++++++MMM+++++M++ 

MM+MM+MMMMMMM+MMMMM+MMMMM 

......................... X 

X X X 

......................... + 

X _ _ ~ - ~ ~~__ ~~

......................... 
Lesions in Male Mice 199 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Fibrosarcoma, metastatic, mesentery 

Preputial gland 

Fibrosarcoma, metastatic, skin 

Prostate 

Seminal vesicle 

Fibrosarcoma, metastatic, mesentery 

Testes 

Hemangiosarcoma 

Hematopoietic System 

Bone marrow 

Hemangiosarcoma 

Histiocytic sarcoma 

Lymph node 

Iliac, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Renal, osteosarcoma, metastatic, 

uncertain primary site 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

~~ ~ ~~~~~~~ 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangiosarcoma 

Subcutaneous tissue, histiocytic 

sarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Fibrosarcoma, metastatic, mesentery 

Nervous System 

Brain 

7777777777777777777777171 

2222222222222222233333333 

999999999999999'9900000000 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total 

1222223333344444402333444 Tissues/ 

7036780246823456949159017 Tumors 

......................... 50 

1 

+++++++++++++++++M+++++++ 49 

1 

......................... 50 

......................... 50 

1 

......................... 50 

X 1 

......................... 50 

X 1 

X X 5 

+ + + +++ + 14 

2 

X 2 

1 

......................... 49 

......................... 48 

4 

......................... 50 

1 

X X 6 

+++++++++++++++++M+++M+++ 44 

MMMMMMMMMMMMMMMMMMMMMMMMM 4 

......................... 50 

1 

3 

X 1 

......................... 50 

1 

1 

......................... 50 
200 Phenolphthalein, NTP TR 465 

TABLE C2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveoladbronchiolar carcinoma 

Carcinoma, metastatic, harderian 

gland 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Histiocytic sarcoma 

Renal tubule, adenoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

4455556666611111111111111 

2901355561901122222222222 

3505561953603699999999999 

1111111111111111111111111 

0214510230321200000111111 

9508096438118212351123456 

......................... X X X 

X 

X 

X 

......................... 

......................... 

......................... X 

X 

+++++++++++AA++++++++++++ 

......................... X xx X x x 

x x x xx X x x 
Lesions in Male Mice 201 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

RespiratorySystem 

Lung 

Alveoladbronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Carcinoma,metastatic,harderian 

gland 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Histiocytic sarcoma 

Renal tubule, adenoma 

Urinary bladder 

Systemic Lesions 

Multiple organs r’ 

Histiocytic sarcoma 

Lymphoma malignant 

1111111111111111111111111 

2222222222222222233333333 

9999999999999999900000000 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

1222223333344444402333444 

1036180246823456949159011 

......................... X 

X xx 

X 

X X X 

......................... 

......................... 

+ ++ + 

xx 

X X 

......................... X 

X 

......................... 

......................... X xx X X 

xx X X 

Total 

Tissues/ 

Tumors 

50 

4 

4 

1 

1 

4 

50 

50 

4 

2 

2 

50 

2 

2 

48 

50 

11 

12 
202 Phenolphthalein, NTP TR 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Hemangiosarcoma 

Stomach, forestomach 

Squamous cell carcinoma 

Stomach, glandular 

Squamous cell carcinoma, metastatic, 

stomach, forestomach 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Histiocytic sarcoma 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Histiocytic sarcoma 

Preputial gland 

Prostate 

- Seminal vesicle 

Testes 

055566666777777777777777 

024834459012222222222222 

329595629340399999999999 

21'1111111111111111111111 

059989995967755555556666 

075239834043712356890126 

........................ 

A+++++A+AA+A+++++M+M++++ 

A+++++++++++++++++++++++ 

A+++++++++++++++++++++++ 

A+++++A+A++A++++++++++++ 

A+++++A++++A++++++++++++ 

A+++++A++++A++++++++++++ 

A+++++A++++A++++++++++++ 

........................ x x xx X 

X X X 

X x xxxx x X X 

+ + ......................... X x x X 

........................ X 

........................ 

A+++++++++++++++++++++++ 

........................ 

+ ........................ X X X 

........................ 

........................ 

........................ 

+++++++++M++++++++++++++ 

........................ 

........................ 

........................ X 

........................ 

........................ 

........................ 

+++++++++M++++++++++++++ i 
Lesions in Male Mice 203 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Hemangiosarcoma 

Stomach, forestomach 

Squamous cell carcinoma 

Stomach, glandular 

Squamous cell carcinoma, metastatic, 

stomach, forestomach 

Tooth 

Cardiovascular System 

Blood vessel 

Heart 

Histiocytic sarcoma 

EndocrineSystem 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Histiocytic sarcoma 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

7777777777777777777777777 

2222222222222333333333333 

9999999999999000000000000 

1111111111111111111111111 Total 

7778888888899666677777899 Tissues/ 

0460124567846358912589917 Tumors 

......................... 49 

+++++++++++++++++++M+++++ 41 

......................... 48 

......................... 48 

......................... 45 

......................... 46 

......................... 46 

......................... 46 

......................... 49 

X xx X 9 

xxxx X X 9 

X 1 

X X X 12 

+ 3 

......................... 49 

4 

......................... 49 

1 

......................... 49 

X 1 

......................... 48 

X 1 

......................... 49 

1 

......................... 49 

3 

++++++M++++++++++++++++++ 48 

++++++M++++++++++++++++++ 48 

......................... 49 

+M+++++M+++++M+++++++++++ 45 

......................... 48 

......................... 49 

......................... 49 

1 

......................... 49 

......................... 49 

......................... 49 

......................... 48 
204 Phenolphthalein, NTF' TR 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Mast cell tumor malignant 

Lymph node 

Iliac, histiocytic sarcoma 

Inguinal, mast cell tumor malignant, 

metastatic, bone marrow 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

~ ~~ ~ ~ ~~~ ~ ~ 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, 

multiple 

Alveolar/bronchiolar carcinoma 

Alveolarlbronchiolar carcinoma, 

multiple 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Carcinoma 

055566666111111111111111 

024834459012222222222222 

329595629340399999999999 

211111111111111111111111 

059989995961755555556666 

015239834043112356890126 

........................ X X X 

+ + + 

........................ X X X X 

........................ X X x x X X 

........................ 

X x xx x X X 

++++++M++++++++++M+++I++ 

x x 

MMM++MMMMM+MMMMMM+M+M+MM 

........................ 

++++++++++++ +++++++++++ 

+ 

........................ 

........................ X X xx 

X X 

X X 

xx 

X x xxxx X X 

........................ 

........................ 

+ 

X 
Lesions in Male Mice 205 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Mast cell tumor malignant 

Lymph node 

Iliac, histiocytic sarcoma 

Inguinal, mast cell tumor malignant, 

metastatic, bone marrow 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Skin 

~ ~~~~~ 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, 

multiple 

Alveolarlbronchiolar carcinoma 

Alveolarlbronchiolar carcinoma, 

multiple 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Carcinoma 

11177771,17771777777717717 

2222222222222333333333333 

9999999999999000000000000 

1111111111111111111111111 Total 

I778888888899666671777899 Tissues/ 

0460124567846358912589917 Tumors 

......................... 49 

X X 

+ +++ 

X 

X 

5 

X 1 

+ + 9 

1 

X 1 

1 

......................... 

......................... X 

......................... X 

X X 

++++++++++M+++++M++++++MI 

2 

42 

9 

1 

49 

7 

49 

4 

49 

MMMMMMMMM+MMMM+MMMMMMMMMM 

......................... 8 

49 

~ ~~~~ ~ ~~ ~~ ~~ 

......................... 1 48 

......................... 49 

X xxx X 

......................... X 10 

49 

X X 4 

2 

X 1 

X 

X 

......................... 

......................... 49 

49 

9 

3 

+ + 

X 
206 Phenolphthalein, NTP TR 465 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Histiocytic sarcoma 

Squamous cell carcinoma, metastatic, 

stomach, forestomach 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

055566666111111111111111 

02483445901,2222222222222 

329595629340399999999999 

211111111111111111111111 

059989995961155555556666 

015239834043112356890126 

........................ x x 

........................ 

........................ X x xxxx x X X 

X xx 
Lesions in Male Mice 207 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

UrinarySystem 

Kidney 

Histiocytic sarcoma 

Squamous cell carcinoma, metastatic, 

stomach,forestomach 

Urinarybladder 

SystemicLesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

7777777777777777777777777 

2222222222222333333333333 

9999999999999000000000000 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total 

7778888888899666677777899 Tissues/ 

0460124567846358912589917 Tumors 

......................... 49 

X 3 

X 1 

......................... 49 

......................... 49 

X X X 12 

X X X x x 8 
208 Phenolphthalein, NTP TR 465 

TABLEC3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Phenolphthalein 

AdrenalCortex:Adenoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armitage testd 

Fisher exact testd 

HarderianGland:Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

HarderianGland:Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0 PPm 

1/49 (2%) 

2.5% 

1/40 (3%) 

729 (T) 

P=0.168N 

P=0.158N 

P=0.156N 

3/50 (6%) 

7.5% 

3/40 (8%) 

729 (T) 

P=0.215N 

P=0.208N 

P=0.199N 

3/50 (6%) 

7.5% 

3/40 (8%) 

729 (T) 

P=0.451N 

P=0.444N 

P=0.431N 

22/50 (44%) 

53.5% 

21/40 (53%) 

582 

P=0.017N 

P=O.OlON 

P=O.OlON 

8/50 (16%) 

19.5% 

7/40 (18%) 

727 

P=O.508 

P=O.533 

P=O.533 

3,000 ppm 

3/50 (6%) 

8.2% 

2/33 (6%) 

634 

P=O.255 

P=O.308 

P=O.316 

3/50 (6%) 

8.7% 

2/33 (6%) 

714 

P=O.574 

P=O.613 

P=0.661N 

3/50 (6%) 

8.7% 

2/33 (6%) 

714 

P=O.574 

P=O.613 

P=0.661N 

12/50 (24%) 

29.3% 

6/33 (18%) 

563 

P=0.103N 

P=0.032N 

P=0.028N 

10/50 (20%) 

25.0% 

5/33 (15%) 

560 

P=O.275 

P=O.385 

P=O.398 

6,000 ppm 

0/49 (0%) 

0.0% 

0135 (0%) 

-e 

P=0.527N 

P=0.527N 

P=0.500N 

2/50 (4%) 

5.6% 

2/36 (6%) 

729 (T) 

P=0.548N 

P=0.548N 

P=0.500N 

4/50 (8%) 

11.1% 

4/36 (1 1 %) 

729 (T) 

P=O.442 

P=O.442 

P=0.500 

8/50 (16%) 

19.6% 

5/36 (14%) 

495 

P=0.006N 

P=0.003N 

P=0.002N 

5/50 (10%) 

11.8% 

2/36 (6%) 

500 

P=0.343N 

P=0.273N 

P=0.277N 

12,000 ppm 

0148 (0%) 

0.0% 

0135 (0%) 

-

P=0.527N 

P=0.527N 

P=0.505N 

1/49 (2%) 

2.8% 

1/36 (3%) 

729 (T) 

P=0.343N 

P=0.343N 

P=0.316N 

2/49 (4%) 

5.6% 

2/36 (6%) 

729 (T) 

P=0.548N 

P=0.548N 

P=0.510N 

10/49 (20%) 

25.0% 

7/36 (19%) 

522 

P=0.022N 

P=O.OllN 

P=O.OlON 

9/49 (18 %) 

22.0% 

5/36 (14%) 

639 

P=O.420 

P=O.474 

P=O.482 
Lesions in Male Mice 209 

TABLEC3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Phenolphthalein (continued) 

Liver:HepatocellularAdenoma or Carcinoma 

Overall rate 27/50 (54%) 20/50 (40%) 11/50 (22%) 16/49 (33%) 

Adjusted rate 64.2% 45.3% 25.8% 38.4% 

Terminal rate 25/40 (63 %) 10133 (30%) 6/36 (17%) 11/36 (31%) 

First incidence (days) 582 560 495 522 

Life table test P=0.033N P=0.341N P=0.005N P=0.060N 

Logistic regression test P=0.016N P=0.123N P=O.OOlN P=0.026N 

Cochran-Armitage test P=0.016N 

Fisher exact test P=0.115N P<O.OOlN P=0.026N 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 8/50 (16%) 10/50 (20%) 4/50 (8 %) 12/49 (24%) 

Adjusted rate 20.0% 27.0% 10.5% 30.6% 

Terminal rate 8/40 (20%) 7/33 (21 X) 3/36 (8SI) 9/36 (25 %) 

First incidence (days) 729 (T) 602 659 699 

Life table test P=O.211 P=O.255 P=0.229N P=O.162 

Logistic regression test P=O.225 P=O.352 P=0.203N P=O.185 

Cochran-Armitage test P=O.226 

Fisher exact test P=O.398 P-0.178N P=O.212 

Lung: Alveolar/bronchiolar Carcinoma 

Overall rate 3/50 (6%) 5/50 (10%) 4/50 (8 %) 5/49 (10%) 

Adjusted rate 6.5% 14.2% 11.1% 13.5% 

First incidence (days) 

Terminal rate 

550 

0140 (0%) 

637 

4/33 (12%) 

729 (T) 

4/36 (1 1 %) 

720 

4/36 (1 1 %) 

Life table test P=O.324 P=O.291 P=O.450 P=O.314 

Logistic regression test P=O.335 P=O.361 P=O.505 P=O.349 

Cochran-Armitage test P=O.337 

Fisher exact test P=O.357 P=O.500 P=O.346 

Lung: Alveolarlbronchiolar Adenoma orCarcinoma 

Overall rate 11/50 (22%) 15/50 (30%) 8/50 (16%) 16/49 (33%) 

Adjusted rate 25.2 % 39.7% 21.3% 40.9% 

Terminal rate 8/40 (20%) 11/33 (33%) 7/36 (19%) 13/36 (36%) 

Life table test 

First incidence (days) 

P=O.200 

550 

P=O.134 

602 

P=0.394N 

659 

P=O.126 

699 

Logistic regression test P=O.215 P=O.236 P=0.318N P=O.160 

Fisher exact test 

Cochran-Armitage test P=O.217 

P=O.247 P=0.306N P=O.168 

Overall rate 

Skin (Subcutaneous Tissue): Hemangiosarcoma 

1/50 (2%) 0150 (0%) 3/50 (6%) 0149 (0%) 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

P=0.515N 

729 (T) 

1/40 (3%) 

2.5% 

0133 (0%) 

0.0% 

P=0.538N 

-

P=O.275 

515 

1/36 (3 %) 

7.4% 

0/36 (0%) 

0.0% 

P=0.521N 

-

Logistic regression test P=0.496N P=0.538N P=O.307 P=0.521N 

Fisher exact test 

Cochran-Armitage test P=0.506N 

P=0.500N P=O.309 P=0.505N 
210 Phenolphthalein, NTP TR 465 

TABLEC3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Phenolphthalein(continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

Spleen:Hemangiosarcoma 

Overall rate 0150 (0%) 5/50 (10%) 1/50 (2%) 1 149 (2 %) 

Adjusted rate 0.0% 14.0% 2.7% 2.8% 

Terminal rate 0140 (0%) 3/33 (9%) 0136 (0%) 1/36 (3%) 

First incidence (days) 703 716 729 (T) 

Life table test P=O.O22 P=O.475 P=O.479 

Logistic regression test P=O.O26 P=O.494 P=O.479 

Cochran-Armitage test 

Fisher exact test P=O.O28 P=OSo0 P=O.495 

All Organs: Hemangiosarcoma 

Overall rate 3/50 (6%) 6/50 (12%) 7/50 (14%) 2/49 (4%) 

Adjusted rate 7.3% 16.9% 18.0% 5.6% 

Terminal rate 2/40 (5 %) 4/33 (12%) 5/36 (14%) 2/36 (6%) 

First incidence (days) 724 703 515 729 (T) 

Life table test P=0.380N P=O.166 P=O. 125 P=0.552N 

Logistic regression test P=0.351N P=O.197 P=O.154 P=0.534N 

Cochran-Armitage test P=0.350N 

Fisher exact test P=O.243 P=O.159 P=0.510N 

All Organs: Histiocytic Sarcoma 

Overall rate 1/50 (2%) 3/50 (6%) 11/50 (22%) 12/49 (24%) 

Adjusted rate 2.4% 7.9% 28.0% 27.5% 

Terminal rate 0/40 (0%) 0133 (0%) 8/36 (22%) 5/36 (14%) 

First incidence (days) 727 65 8 665 549 

Life table test P<O.OOl P=O.255 P=O.O02 P=O.o02 

Logistic regression test P<O.OOl P=O.302 P=O.o02 P=O.ool 

Fisher exact test 

Cochran-Armitage test P<O.OOl 

P=O.309 P=O.o02 P<O.ool 

All Organs: MalignantLymphoma 

Overall rate 6/50 (12%) 8/50 (16%) 12/50 (24%) 8/49 (16%) 

Adjusted rate 14.5% 19.1% 27.2% 22.2% 

Terminal rate 5/40 (13%) 3/33 (9%) 6/36 (17%) 8/36 (22 %) 

First incidence (days) 662 347 423 729 (T) 

Life table test P=O.299 P=O.300 P=O.O82 P=O.309 

Logistic regression test P=O.324 P=O.396 P=O.101 P=O.353 

Cochran-Armitage test P=O.310 

Fisher exact test P=O.387 P=O.O96 P=O.371 

Overall rate 

All Organs: Benign Neoplasms 

29/50 (58%) 24/50 (48%) 18/50 (36%) 19/49 (39%) 

Terminal rate 

Adjusted rate 

27/40 (68%) 

68.9% 

14/33 (42%) 

55.0% 

13/36 (36%) 

43.3% 

14/36 (39%) 

45.9% 

Life table test 

First incidence (days) 

P=0.058N 

582 

P=0.521N 

563 

P=0.066N 

495 

P=0.095N 

522 

Logistic regression test P=0.030N P=0.234N P=0.027N P=0.043N 

Fisher exact test 

Cochran-Armitage test P=0.030N 

P=0.212N P=0.022N P=0.043N 
Lesions in Male Mice 

TABLEC3 

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-YearFeed Study of Phenolphthalein(continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

All Organs: MalignantNeoplasms 

Overall rate 19/50 (38%) 29/50 (58%) 35/50 (70%) 29/49 (59%) 

Adjusted rate 41.2% 61.3% 71.4% 63.0% 

Terminal rate 13/40 (33 %) 15/33 (45 %) 22/36 (61 %) 19/36 (53%) 

First incidence (days) 423 550 347 549 

Life table test P=O.O30 P=O.O02 P=O.O21 P=O.O57 

Logistic regression test P=O.O35 P=O.OOl P=O.O39 P=O.O26 

Cochran-Armitage test P=O.O31 

Fisher exact test P=O.O36 P=O.ool P=O.O28 

All Organs: Benign or MalignantNeoplasms 

Overall rate 40150 (80%) 40150 (80%) 42/50 (84%) 38/49 (78%) 

Adjusted rate 84.0% 81.6% 83.3% 80.8% 

Terminal rate 32/40 (80%) 24/33 (73%) 28/36 (78%) 27/36 (75%) 

First incidence (days) 423 550 347 522 

Life table test P=O.194 P=O.516 P=O.211 P=O.459 

Logistic regression test P=0.461N P=O.590 P=O.392 P=0.502N 

Cochran-Armitage test P=0.443N 

Fisher exact test P=0.598N P=O.398 P=0.479N 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animalslnumber of animals examined. Denominator is number of animals examined microscopically for adrenal gland, liver, 

lung, and spleen: for other tissues, denominator is number of animals necropsied. 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 

pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being 

(directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group 
212 Phenolphthalein,NTP TR 465 

TABLE C4a 

Historical Incidence of Histiocytic Sarcoma in Untreated Male B6C3Fl Micea 

Study Incidence in Controls 

Historical Incidence at EG&G Mason Research Institute/TSI Mason Laboratories, Inc. 

l-Amino-2,4-dibromoanthraquinone 0150 

Acetaminophen 1I50 

HC Yellow 4 0150 

Methylphenidate Hydrochloride 0150 

Pentaerythritol Tetranitrate 0149 

Turmeric Oleoresin 0150 

Overall Historical Incidence 

Total 6/1,474 (0.4%) 

Standard deviation 1.O% 

Range 0%-2% 

a Dataas of 12 May 1995 

TABLE C4b 

Historical Incidence of Malignant Lymphoma in Untreated Male B6C3Fl Micea 

Study 

Historical Incidence at EG&G Mason Research Institute/TSI Mason 

l-Amino-2,4-dibromoanthraquinone 

Acetaminophen 

HC Yellow 4 

Methylphenidate Hydrochloride 

Pentaerythritol Tetranitrate 

Turmeric Oleoresin 

Overall Historical Incidence 

Total 

Standard deviation 

Range 

Incidence in Controls 

Laboratories, Inc. 

10150 

7/50 

2/50 

3/50 

4/49 

1I50 

130/1.474 (8.8%) 

6.0% 

2%-24% 

a Data as of 12 May 1995; includes data for histocytic, lymphocytic, mixed, unspecified, or undifferentiated cell type lymphomas 
Lesions in Male Mice 213 

TABLEC4c 

Historical Incidence of Hepatocellular Neoplasms in Untreated Male B6C3F, Micea 

Incidence in Controls 

Study Adenoma Carcinoma Adenoma or Carcinoma 

Historical Incidence at EG&G Mason Research Institute/TSI Mason Laboratories, Inc. 

l-Amino-2,4-dibromoanthraquinone 10150 9/50 18/50 

Acetaminophen 11/50 7/50 16/50 

HC Yellow 4 8/49 5/49 13/49 

Methylphenidate Hydrochloride 18/50 10150 24/50 

Pentaerythritol Tetranitrate 9/48 3/48 11/48 

Turmeric Oleoresin 25/50 12/50 30/50 

Overall Historical Incidence 

Total 413/1,465 252/1,465 596/1,465 (28.2%) (17.2%) (40.7%) 

Standard deviation 14.5% t 14.2% 7.1% 

Range 4%-60% 3 %-29% 10%-68% 

a Data as of 12 May 1995 
214 Phenolphthalein, NTP TR 465 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of Phenolphthaleina 

Disposition Summary 

Animals initially in study 

Early deaths 

Moribund 

Natural deaths 

Survivors 

Terminal sacrifice 

Missing 

Animals examined microscopically 

AlimentarySystem 

Gallbladder 

Metaplasia 

Intestine large, rectum 

Edema 

Intestine large, cecum 

Edema 

Serosa, inflammation, chronic 

Intestine small, jejunum 

Hemorrhage 

Necrosis 

Clear cell focus 

Congestion 

Eosinophilic focus 

Fibrosis 

Hematopoietic cell proliferation 

Inflammation, chronic 

Mineralization 

Mixed cell focus 

Necrosis 

Vacuolization cytoplasmic 

Bile duct, cyst 

Bile duct, inflammation, suppurative 

Oval cell, hyperplasia 

Mesentery 

Inflammation, chronic active 

Fat, necrosis 

50 

6 

4 

40 

50 

(50) 

1(2%) 

(50) 

(50) 

(49) 

1 (2%) 

1 (2%) 

24 (48%) 

22 (44%) 

1 (2%) 

4 (8%) 

6 (12%) 

4 (8%) 

7 (14%) 

1(2%) 

1 (2%) 

4 (8%) 

(9) 

7 (78%) 

3,000 ppm 

50 

13 

4 

33 

50 

(46) 

(50) 

(48) 

1(2%) 

1 (2%) 

(49) 

6,000 ppm 

50 

11 

3 

36 

50 

(46) 

(50) 

1 (2%) 

(47) 

(48) 

12,000 ppm 

50 

3 

10 

36 

1 

49 

(411 

(48) 

(45) 

(46) 

Liver (50) (50) (50) (49) 

Angiectasis 1 (2%) 1 (2%)

6 (12%) 

2 (4%) 

6 (12%) 

6 (12%) 

3(6%) 

2 (4%) 

4 (8%) 

4 (8%) 

2 (4%) 

(9) 

1(11%) 

3 (33%) 

1 (2%) 

1 (2%) 

6 (12%) 

7 (14%) 

1 (2%) 

1 (2%) 

4 (8%) 

2 (4%) 

(4) 

2 (50%) 

1(2%) 

2 (4%) 

5 (10%) 

1 (2%) 

2 (4%) 

4 (8%) 

1 (2%) 

1 (2%) 

(3) 

2 (67%) 

Pancreas (50) (50) (50) (49) 

Inflammation, chronic 1 (2%) 

Acinus, atrophy 1(2%) 1 (2%) 1(2%) 4 (8%) 

Duct, cyst 1 (2%) 

Inflammation, chronic 

Salivary glands (50) (50) (50) (49) 

1 (2%) 

Necrosis 1 (2%) 

Duct, cyst 1 (2%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
Lesions in Male Mice 215 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Studyof Phenolphthalein 

(continued) 

Alimentary System (continued) 

Stomach, forestomach 

Hyperkeratosis 

Hyperplasia, squamous 

Inflammation, chronic 

Mineralization 

Ulcer 

Serosa, inflammation, chronic 

Stomach, glandular 

cyst 

Erosion 

Mineralization 

Glands, atrophy, focal 

Glands, cyst 

Glands, hyperplasia 

Serosa, inflammation, chronic 

Tooth 

Degeneration 

Inflammation, chronic 

Inflammation, chronic active 

Cardiovascular System 

Heart 

Cardiomyopathy 

Inflammation, chronic 

Inflammation, chronic active 

Mineralization 

Myocardium, degeneration 

Valve, inflammation 

Endocrine System 

Adrenal cortex 

Hemorrhage 

Hyperplasia 

Hypertrophy, focal 

Capsule, hyperplasia 

Capsule, inflammation, chronic 

Adrenal medulla 

Hyperplasia 

Islets, pancreatic 

Hyperplasia 

Parathyroid gland 

cyst 

Pituitary gland 

Pars distalis, cyst 

Pars distalis, hyperplasia 

Thyroid gland 

cyst 

Follicular cell, hyperplasia 

0 PPm 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(49) 

22 (45%) 

(49) 

1 (2%) 

16 (33%) 

36 (73%) 

(49) 

1 (2%) 

(50) 

45 (90%) 

(46) 

5 (10%) 

3,000 ppm 

I (14%) 

2 (4%) 

3(6%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

12 (24%) 

1 (2%) 

1 (2%) 

1 (2%) 

14 (28%) 

37 (74%) 

1 (2%) 

(50) 

(50) 

45 (90%) 

(48) 

6,000 ppm 

1 (2%) 

1 (2%) 

(49) 

10 (20%) 

34 (69%) 

12,000 ppm 

(49) 

4 (8%) 

2 (4%) 

(49) 

1 (2%) 

2 (4%) 

(48) 

1 (2%) 

11 (23%) 

34 (71%) 
216 Phenolphthalein, N" TR 465 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of Phenolphthalein 

(continued) 

General Body System 

None 

Genital System 

Epididymis 

Granuloma sperm 

Inflammation, chronic 

Inflammation, granulomatous 

Spermatocele 

Penis 

Hemorrhage 

Preputial gland 

cyst 

Granuloma 

Hyperplasia, lymphoid 

Inflammation, acute 

Inflammation, chronic 

Inflammation, chronic active 

Prostate 

cyst 

Inflammation, chronic 

Seminal vesicle 

Atrophy 

Hypertrophy 

Testes 

Germinal epithelium, degeneration 

Interstitial cell, hyperplasia 

Hematopoietic System 

Bone marrow 

Angiectasis 

Myelofibrosis 

Pigmentation 

Thrombosis 

Myeloid cell, hyperplasia 

Lymph node 

Iliac, angiectasis 

Iliac, hyperplasia 

Iliac, hyperplasia, lymphoid 

Iliac, pigmentation 

Inguinal, hyperplasia, lymphoid 

Inguinal, pigmentation 

Mediastinal, hyperplasia, lymphoid 

Mediastinal, infiltration cellular, plasma 

cell 

Pancreatic, angiectasis 

Renal, angiectasis 

Renal, hyperplasia, lymphoid 

0 PPm . 3,000 ppm 

1 (100%) 

1 (2%) 

(49) 

(1) 

39 (80%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

49 (98%) 

(50) 

3(6%) 

3 (38%) 

1 (13%) 

1(13%) 

1 (13%) 

1 (13%) 

6,000 ppm 

(49) 

41(84%) 

1 (2%) 

1 (2%) 

2 (14%) 

2 (14%) 

1 (7%) 

1(7%) 

1(7%) 

1(7%) 

12,000 ppm 

(49) 

1 (2%) 

(49) 

47 (96%) 

3(6%) 

7 (14%) 

(49) 

(49) 

1 (2%) 

(48) 

47 (98%) 

(49) 

19 (39%) 

16 (33%) 

(9) 

1 (11%) 

1(11%) 

1(11%) 

1(11%) 
Lesions 217 in Male Mice 

Hematopoietic System (continued) 

Lymph node, mandibular 

Angiectasis 

Hyperplasia, lymphoid 

Infiltration cellular, histiocyte 

Lymph node, mesenteric 

Angiectasis 

Hyperplasia, lymphoid 

Infiltration cellular, polymorphonuclear 

Infiltration cellular, histiocyte 

Inflammation, chronic 

Spleen 

Angiectasis 

Hematopoietic cell proliferation 

Pigmentation 

Lymphoid follicle, atrophy 

Lymphoid follicle, hyperplasia 

Lymphoid follicle, necrosis 

Thymus 

Atrophy 

cyst 

Hyperplasia, atypical 

Hyperplasia, oncocytic 

Necrosis 

Integumentary System 

Skin 

Inflammation, acute 

Inflammation, chronic 

Subcutaneous tissue, edema 

Subcutaneous tissue, infiltration cellular, 

polymorphonuclear 

Subcutaneous tissue, inflammation, acute 

Subcutaneous tissue, inflammation, chronic 

Subcutaneous tissue, necrosis 

Musculoskeletal System 

None 

Nervous System 

Brain 

Hemorrhage 

Necrosis 

Thalamus, mineralization 

(49) 

2 (4%) 

(50) 

11 (22%) 

3(6%) 

2(4%) 

10 (20%) 

3 (6%) 

1(2%) 

2 (4%) 

1(2%) 

(43) 

13 (30%) 

1 (2%) 

1 (2%) 

(50) 

(50) 

1(2%) 

27 (54%) 

1(2%) 

(50) 

10 (20%) 

2 (4%) 

1(2%) 

(50) 

1(2%) 

22 (44%) 

5(10%) 

2 (4%) 

(46) 

11 (24%) 

1 (2%) 

3 (7%) 

1(2%) 

(50) 

1 (2%) 

24 (48%) 

(49) 

2 (4%) 

(48) 

13 (27%) 

4(8%) 

(44) 

16 (36%) 

I (16%) 

1(2%) 

(50) 

25 (50%) 

(49) 

1 (2%) 

1 (2%) 

1(2%) 

(49) 

12 (24%) 

3 (6%) 

(49) 

21(43%) 

4 (8%) 

(42) 

12 (29%) 

7(17%) 

1(2%) 

1 (2%) 

1(2%) 

(49) 

1 (2%) 

35 (71%) 
218 Phenolphthalein,NTP TR 465 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 

(continued) 

0 PPm 3,000 ppm 

Respiratory System 

Lung 

Congestion 

Hemorrhage 

Infiltration cellular, histiocyte 

Inflammation, granulomatous 

Alveolar epithelium, hyperplasia 

Nose 

Inflammation, acute 

Polyp, inflammatory 

Special Senses System 

Harderian gland 

Hypertrophy 

Urinary System 

Kidney 

cyst 

Hydronephrosis 

Infarct 

Infarct, chronic 

Inflammation, acute 

Metaplasia, osseous 1 (2%) 

Mineralization 7 (14%) 1 (2%) 

Nephropathy, chronic 47 (94%) 46 (92%) 

Pigmentation 

Papilla, necrosis 1 (2%) 

Pelvis, angiectasis 1 (2%) 

Renal tubule, accumulation, hyaline droplet 2(4%) 

Renal tubule, dilatation 1 (2%) 

Renal tubule, hyperplasia 1 (2%) 

Renal tubule, necrosis 

Transitional epithelium, hyperplasia 

Urinary bladder 

Hemorrhage 

Inflammation, acute 

Serosa, inflammation, chronic 

2-Year Feed Study of Phenolphthalein 

6,000 ppm 12,000 ppm 

(3) 

2 (67%) 

(49) 

4 (8%) 

1 (2%) 

1 (2%) 

1 (2%) 

2 (4%) 

42 (84%) 41(84%) 

1 (2%) 

5 (10%) 5 (10%) 

1 (2%) 2 (4%) 

1 (2%) 

1 (2%) 

(48) (49) 

1 (2%) 
219 

APPENDIX D 

SUMMARY OF LESIONS IN FEMALE MICE 

TABLEDl 

Study 

TABLED2 

TABLED3 

TABLED4a 

TABLED4b 

TABLED4c 

Mice 

TABLED4d 

252 

TABLED5 

IN.THE 2-YEAR FEED STUDY 

OF PHENOLPHTHALEIN 

Summary of the Incidence of Neoplasms in Female Mice 

in Feed 2-Year the of Phenolphthalein ........................... 220 

Individual Animal Tumor Pathology of Female Mice 

in Feed 2-Year the Study of Phenolphthalein ........................... 224 

Statistical Analysis of Primary Neoplasms in Female Mice 

in Feed 2-Year the Study of Phenolphthalein ........................... 246 

Historical Incidence of Histiocytic Sarcoma in Untreated Female B6C3Fl Mice251 ..... Historical Incidence of Malignant Lymphoma in Untreated Female. B6C3FlMice251 .... Historical Incidence s f Luteoma in the Ovary 

B6C3Fl of Untreated Female .................................. Historical Incidence of Hepatocellular Neoplasms 

in Untreated Female Mice B6C3Fl .................................. Summary of the Incidence of Nonneoplastic Lesions in Female Mice 

in Feed 2-Year the Study of Phenolphthalein ........................... 253 
220 Phenolphthalein, NTP TR 465 

TABLEDl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of Phenolphthaleina 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

Disposition Summary 

Animals initially in study 

Early deaths 

Moribund 

Natural deaths 

Survivors 

Died last week of the study 

Terminal sacrifice 

Animals examined microscopically 

Alimentary System 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Fibrosarcoma 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Fibrous histiocytoma 

Hemangiosarcoma, metastatic, spleen 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Fibrous histiocytoma 

Hemangiosarcoma 

Pancreas 

Fibrous histiocytoma 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Squamous cell carcinoma 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Heart 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Fibrous histiocytoma 

50 50 

5 13 

6 6 

1 

38 31 

50 50 

1 (2%) 

6 (12%) 1 (2%) 

12 (24%) 2 (4%) 

5 (10%) 

1 (2%) 

(16) (2) 

1 (6%) 

(50) 

1 (2%) 

1 (2%) 

(50) 

(50) (50) 

1 (2%) 

1 (2%) 

50 

9 

7 

34 

50 

6 (12%) 

6 (12%) 

(7) 

(49) 

50 

12 

10 

28 

50 

1 (2%) 

1 (2%) 

7(14%) 

(1) 

3 (6%) 

(50) 

(50) 

1 (2%) 
Lesions in Female Mice 221 

TABLEDl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Studyof Phenolphthalein(continued) 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

Tissue NOS 

Genital System 

Clitoral gland 

Ovary 

Cystadenoma 

Fibrous histiocytoma 

Granulosa cell tumor benign 

Hemangioma 

Histiocytic sarcoma 

Sex-cord stromal tumor, benign 

Teratoma benign 

Bilateral, sex-cord stromal tumor, benign 

UteNS 

Carcinoma 

Histiocytic sarcoma 

Polyp stromal 

Hematopoietic System 

Bone marrow 

Hemangioma 

Histiocytic sarcoma 

Lymph node 

Iliac, fibrous histiocytoma 

Iliac, histiocytic sarcoma 

Mediastinal, alveolar/bronchiolar carcinoma, 

metastatic, lung 

Mediastinal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Fibrous histiocytoma 

Histiocytic sarcoma 

0 PPm 3,000 ppm 

(49) 

3 (6%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 1 (2%) 

2 (4%) 

1 (2%) 

6 (12%) 

1(2%) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 2 (4%) 

2 (14%) 

6,000 ppm 

6 (12%) 

3 (6%) 

(49) 

3 (6%) 

(49) 

1(2%) 

3 (6%) 

12,000 ppm 

1 (2%) 

5 (10%) 

1(2%) 

3 (19%) 

1 (6%) 

1 (6%) 

2 (13%) 

(49) 

4 (8%) 

(50) 

4 (8%) 
222 Phenolphthalein,NTP TR 465 

TABLEDl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Studyof Phenolphthalein(continued) 

Hematopoietic System (continued) 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Fibrous histiocytoma 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Adenoacanthoma 

Skin 

Basal cell adenoma 

Trichoepithelioma 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 

Hemangiosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, uncertain primary site 

Fibrous histiocytoma 

Hepatocellular carcinoma, metastatic, liver 

Histiocytic sarcoma 

Osteosarcoma, metastatic, bone 

Nose 

Carcinoma, metastatic, harderian gland 

Hemangiosarcoma 

Osteosarcoma, metastatic, bone 

Trachea 

0 PPm 

(50) 

(48) 

1 (2%) 

(50) 

(1) 

1 (100%) 

4 (8%) 

1 (2%) 

2 (4%) 

(50) 

3,000 ppm 

(50) 

1 (2%) 

1 (2%) 

(44) 

(50) 

1 (2%) 

(1) 

1 (100%) 

4 (8%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

(50) 

6,000 ppm 

(50) 

2 (4%) 

3 (6%) 

(49) 

1 (2%) 

(50) 

(1) 

1 (100%) 

12,000 ppm 

(50) 

1 (2%) 

6 (12%) 

(45) 

1 (2%) 

2 (4%) 

(50) 

1 (2%) 

2 (4%) 

4 (8%) 

1 (2%) 

(50) 

1 (2%) 
Lesions in Female Mice 223 

TABLEDl 

Summary of the Incidence of Neoplasms in Female Mice in the 2-YearFeed Study of Phenolphthalein(continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

Special Senses System 

Harderian gland 

Adenoma 

Carcinoma 

UrinarySystem 

Kidney (50) (50) 

Alveolar/bronchiolar carcinoma, metastatic, 

lung 1 (2%) 

Histiocytic sarcoma 

Urinary bladder (50) (50) 

SystemicLesions 

Multiple organsb (50) (50) 

Histiocytic sarcoma 2(4%) 

Lymphoma malignant 15 (30%) 28 (56%) 

Neoplasm Summary 

Total animals with primary neoplasmsC 40 39 

Total primary neoplasms 70 63 

Total animals with benign neoplasms 26 20 

Total benign neoplasms 33 23 

Total animals with malignant neoplasms 25 34 

Total malignant neoplasms 37 40 

Total animals with metastatic neoplasms 3 4 

Total metastatic neoplasms 7 4 

Total animals with malignant neoplasms 

of uncertain primary site 1 

a Number of animals examined microscopically at the site andthe number of animals with neoplasm 

Number of animals with any tissue examined microscopically 

Primary neoplasms: a11 neoplasms exceptmetastaticneoplasms 

(50) 

7 (14%) 

33 (66%) 

47 

75 

23 

25 

41 

50 

1 (2%) 

(50) 

(50) 

7 (14%) 

25 (50%) 

43 

58 

17 

19 

37 

39 

3 

4 
______ 

TABLED2 

Individual Animal Tumor Pathology of 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, recNm 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Fibrous histiocytoma 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Fibrous histiocytoma 

Pancreas 

Fibrous histiocytoma 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

~~________ _____ ~~ 

Cardiovascular System 

Heart 

Alveolarlbronchiolar carcinoma, 

metastatic, lung 

Fibrous histiocytoma 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

+: Tissue examined microscopically 

A: Autolysis precludes examination 

Phenolphthalein, NTP TR 465 

Female Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm 

0155666677777777777777777 

7549112802333333333333333 

8667358683017777771777777 

2222222222222222222222222 

4554555252312222222333333 

3725863216780134789123689 

......................... 

A++A+++A++++M++++++++++++ 

......................... 

......................... 

......................... 

A+++++A++++++++++++++++++ 

A+++++'+++++++++++++++++++ 

A++++A+++++++++++++++++++ 

......................... 

X x x 

X xxx x 

X X X 

+ ++ + + 

......................... 

......................... 

......................... X 

......................... ~~~ ~~ _____ ~~ ~ ~~ _______ 

......................... 

X 

......................... 

......................... 

......................... 

+M+++++++++++++++M+++++++ 

+++++++++++++++++++M+++++ 

......................... X 

M:Missingtissue X: Lesion present 

I: Insufficient tissue Blank: Not examined 
Lesions in Female Mice 225 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Fibrous histiocytoma 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Fibrous histiocytoma 

Pancreas 

Fibrous histiocytoma 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

~~~ ~~ 

Cardiovascular System 

Heart 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Fibrous histiocytoma 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

7777777777777171711717777 

3333333333333333333333333 

7777777777788888888888888 

2222222222222222222222222 Total 

4444455666611123334445566 Tissues/ 

0128945123567950454670904 Tumors 

......................... 50 

+++M+++++++++++++++++++++ 45 

......................... 50 

......................... 50 

......................... 50 

......................... 48 

......................... 49 

......................... 48 

......................... 50 

X 1 

*X X X 6 

xx X x xx X 12 

X X 5 

+ + ++ + ++ + + + + 16 

X 1 

......................... 50 

X 1 

++++++++++++++++++M++++++ 49 

......................... 50 

1 

......................... 50 

~~~~ ~ ~ ~~ ~ 

......................... 50 

1 

X 1 

......................... 50 

......................... 50 

++++M++++++++++++++++++++ 49 

+++++++++++++++M+++++M+++ 46 

......................... 49 

X X X 3 

X 1 

......................... 50 

1 
226 Phenolphthalein, NTP TR 465 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Clitoral gland 

Ovary 

Fibrous histiocytoma 

Granulosa cell tumor benign 

Hemangioma 

Teratoma benign 

Uterus 

Carcinoma 

Polyp stromal 

Hematopoietic Systemb 

Bone marrow 

Lymph node 

Iliac, fibrous histiocytoma 

Mediastinal, alveolar/bronchiolar 

carcinoma, metastatic, lung 

Lymph node, mandibular 

Lymph node, mesenteric 

Fibrous histiocytoma 

Spleen 

Thymus 

Fibrous histiocytoma 

Integumentary System 

Mammary gland 

Skin 

Musculoskeletal System 

Bone 

Skeletal muscle 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Fibrous histiocytoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

0155666677777777777777777 

7549112802333333333333333 

8667358683077777777777777 

2222222222222222222222222 

4554555252312222222333333 

3725863216780134789123689 

M++++++++++++++M+++++++++ 

......................... 

X 

X 

X 

......................... 

......................... ++ +++ + + + 

X X 

......................... 

++++++++M++++++++++++++++ 

......................... 

++++M+++M++++++++++++++++ 

......................... 

......................... 

......................... + 

X 

......................... + 

+ 

......................... X X 

X X 

X 

......................... 

......................... 
Lesions in Female Mice 227 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm (conthed) 

Number of Days on Study 

Carcass ID Number 

GenitalSystem 

Clitoral gland 

Ovary 

Fibrous histiocytoma 

Granulosa cell tumor benign 

Hemangioma 

Teratoma benign 

Uterus 

Carcinoma 

Polyp stromal 

Hematopoietic System 

~~~ 

Bone marrow 

Lymph node 

Iliac, fibrous histiocytoma 

Mediastinal, alveolar/bronchiolar 

carcinoma, metastatic, lung 

Lymph node, mandibular 

Lymph node, mesenteric 

Fibrous histiocytoma 

Spleen 

Thymus 

Fibrous histiocytoma 

Integumentary System 

Mammary gland 

Skin 

MusculoskeletalSystem 

Bone 

Skeletal muscle 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Fibrous histiocytoma 

Hepatocellular carcinoma, metastatic, 

liver 

Nose 

Trachea 

7777771777777777777771777 

3333333333333333333333333 

1171777717788888888888888 

2222222222222222222222222 Total 

4444455666611123334445566 Tissues1 

0128945123567950454670904 Tumors 

......................... 48 

......................... 50 

X 1 

1 

X 2 

1 

......................... 50 

X 1 

X 1 

......................... 50 

++ + + + + 14 

X 1 

2 

++++++++++++++++++M++++++ 49 

......................... 49 

X 1 

......................... 50 

......................... 48 ..

A 1 

......................... 50 

......................... 50 

......................... 50 

1 

1 

......................... 50 

1 

1 

......................... 50 

X 3 

X X 4 

X 1 

X 2 

......................... 50 

......................... 50 
228 Phenolphthalein,NTP TR 465 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

Harderian gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant 

0155666617777777177711777 

7549112802333333333333333 

8661358683077777777717717 

2222222222222222222222222 

4554555252312222222333333 

3125863216180134189123689 

+ 

++ + 

X 

X X 

......................... 

X 

......................... 

......................... X xxx x x X X 
Lesions in Female Mice 229 

TABLED2 

Individual Animal Tumor Pathology 

Number of Days on Study 

Carcass ID Number 

Special Senses System 

Ear 

Harderian gland 

Adenoma 

Carcinoma 

Urinary System 

Kidney 

Alveolarlbronchiolar carcinoma, 

metastatic, lung 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Lymphoma malignant 

of Female Mice in the 2-Year Feed Study of Phenolphthalein: 0 ppm (continued) 

7777777711777117117717717 

1777177717788888888888888 

3333333333333333333333333 

2222222222222222222222222 

4444455666611123334445566 

0128945123561950454670904 

Total 

Tissues/ 

Tumors 

++ 

X 

......................... 50 

1 

......................... 50 

......................... 50 

X X X x xx X 15 
230 

TABLED2 

Individual Animal Tumor Pathology of 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Fibrosarcoma 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangiosarcoma, metastatic, spleen 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell carcinoma 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

Tissue NOS 

Genital System 

Clitoral gland 

Ovary 

Cystadenoma 

Granulosa cell tumor benign 

Histiocytic sarcoma 

Sex-cord stromal tumor, benign 

Bilateral, sex-cord stromal tumor, 

benign 

Uterus 

Histiocytic sarcoma 

Polyp stromal 

Phenolphthalein, NTP TR 465 

Female Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm 

1244555555566666677777777 

2635015778801456600333333 

6650010081185393838777777 

3232222222222233232222222 

0608887768998100909667777 

9612802199026702477780348 

......................... 

+A++++++++++A++A+++++++++ 

X 

++++++++++A+A++++++++++++ 

++++++++++A++++++++++++++ 

++++++++++A+A++++++++++++ 

+A++++++++A+A++++++++++++ 

++++++++++A+A++++++++++++ 

+A++++++++A+A++++++++++++ 

......................... 

X 

+ ......................... 

......................... 

......................... 

++++++++++A++++++++++++++ 

......................... 

......................... 

......................... 

......................... 

++++++++++++++M+++++++M++ 

......................... 

......................... X X 

+ 

M++++++++++++++++++++++++ 

+++++++++++++++I+++++++++ 

X 

X xx 

......................... X 

X 

X 
Lesions in Female Mice 231 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

AlimentarySystem 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Fibrosarcoma 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangiosarcoma, metastatic, spleen 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell carcinoma 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Heart 

EndocrineSystem 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

Tissue NOS 

GenitalSystem 

Clitoral gland 

Ovary 

Cystadenoma 

Granulosa cell tumor benign 

Histiocytic sarcoma 

Sex-cord stromal tumor, benign 

Bilateral, sex-cord stromal tumor, 

benign 

Uterus 

Histiocytic sarcoma 

Polyp stromal 

7177771777111177177171777 

3333333333333333333333333 

7771777777777717188888888 

2222222222333333322223333 Total 

1888899999000111177890011 Tissues/ 

9145713569458134556383602 Tumors 

......................... 50 

+++++++++++++M+++++++++++ 46 

1 

......................... 48 

......................... 49 

......................... 48 

X 1 

......................... 47 

......................... 48 

......................... 47 

.......................... 50 

1 

X 1 

X X 2 

1 

+ 2 

......................... 50 

......................... 50 

......................... 50 

X 1 

X 1 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

........................... 48 

.......................... 50 

X X 2 

......................... 50 

2 

1 

+++++M+++++++++++++++++++ 48 

......................... 49 

xx 3 

X 1 

1 

X x x 6 

X 1 

......................... 50 

1 

X 2 
232 Phenolphthalein, NTP TR 465 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

HematopoieticSystem 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

IntegumentarySystem 

Mammary gland 

Skin 

Basal cell adenoma 

MusculoskeletalSystem 

Bone 

Osteosarcoma 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

RespiratorySystem 

Lung 

Aheolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, uncertain 

primary site 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Nose 

Osteosarcoma, metastatic, bone 

Trachea 

Special Senses System 

Ear 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

1244555555566666677777777 

2635015778801456600333333 

6650010081185393838717777 

3232222222222233232222222 

06088877689987009096677’77 

9612802199026702477780348 

......................... X 

++ + +++++ +++ +++ ......................... 

......................... X 

......................... 

X 

+++++++++++M++MM+++++++++ 

......................... 

......................... 

......................... X 

++++++++++++++++M++++++++ 

......................... 

X 

X 

X 

......................... X 

......................... 

+ -t 

+ 

......................... 

......................... 

.......................... x x 

xxx xxxxxxxxx xxx x xxx
Lesions in Female Mice 233 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 3,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Basal cell adenoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Carcinoma, metastatic, uncertain 

primary site 

Hepatocellular carcinoma, metastatic, 

liver 

Histiocytic sarcoma 

Nose 

Osteosarcoma, metastatic, bone 

Trachea ~~ ~ 

Special Senses System 

Ear 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

7711771177171111111171711 

3333333333333333333333333 

1177771771117177188888888 

2222222222333333322223333 Total 

7888899999000111177890011 Tissues/ 

9145113569458134556383602 Tumors 

......................... 50 

1 

+ ++ 11 

......................... 50 

......................... 50 

1 

......................... 50 

X 1 

1 

+++++++++++++M++I+++++++M 44 

......................... 50 

......................... 50 

X 1 

......................... 50 

1 

+ 1 

X 1 

......................... 49 

......................... 50 

X 1 

X X X 4 

1 

X 1 

1 

......................... 50 

1 

......................... 50 

2 

+ 2 

X 1 

......................... 50 

......................... 50 

......................... 50 

2 

xx xx x xx xx 28 
234 Phenolphthalein, NTP TR 465 

TABLED2 

Individual Animal Tumor'Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Histiocytic sarcoma 

Mesentery 

Hemangiosarcoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

General Body System 

None 

Genital System 

Clitoral gland 

Ovary 

Sex-cord stromal tumor, benign 

Uterus 

Polyp stromal 

~~ ~~ 

3345556666666666777777777 

2375660012455889333333333 

9891172452514663777777777 

3333333333333333333333333 

5366243124645234112222233 

8724162650357981890247801 

......................... 

A++A++++++++A+A++++++++++ 

X 

A++++++++++A+++++++++++++ 

A++++++++++A+++++++++++++ 

A++++++++++A+++++++++++++ 

A++A+++++++A+++++++++++++ 

A++A+++++++A++A++++++++++ 

A++A+++++++A++A++++++++++ 

......................... X X 

xx X 

+ + + 

X 

......................... 

......................... 

......................... 

A++++++++++++++++++++++++ 

......................... 

......................... 

......................... 

......................... X 

++M++++++++++++++++++++++ 

......................... 

X 

......................... 

+++++++M+++++++M+++++++++ 

......................... X 

......................... X 
Lesions in Female Mice 235 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Carcinoma 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Histiocytic sarcoma 

Mesentery 

Hemangiosarcoma 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tooth 

Cardiovascular System 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

General Body System 

None 

Genital System 

Clitoral gland 

Ovary 

Sex-cord stromal tumor, benign 

Uterus 

Polyp stromal 

7177717771777777771111117 

3333333333333333333333333 

7777777777777111178888888 

3333333333333333333333333 Total 

3333444444555556661223555 Tissues/ 

3569234789145690151364023 Tumors 

......................... 50 

......................... 46 

1 

......................... 48 

......................... 48 

......................... 48 

......................... 41 

......................... 46 

X 1 

......................... 46 

......................... 50 

X X X X 6 

X X X 6 

+ + + + 7 

1 

......................... 50 

X 1 

......................... 50 

......................... 50 

......................... 49 

+ 1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

I 

+M++++M++++++++++++++++++ 47 

......................... 49 

1 

......................... 50 

......................... 48 

......................... 50 

X X X X X 6 

......................... 50 

X X 3 
236 Phenolphthalein, NTP TR 465 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Hemangioma 

Histiocytic sarcoma 

Lymph node 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Fibrous histiocytoma 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Adenoacanthoma 

Skin 

Trichoepithelioma 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, 

multiple 

Alveolar/bronchiolar carcinoma 

Histiocytic Sarcoma 

Nose 

Hemangiosarcoma 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

3345556666666666777777777 

2375660012455889333333333 

9891172452514663777777777 

3333333333333333333333333 

5366243124645234112222233 

8724162650357981890247801 

......................... X 

X 

+ + ++ ++ + + 

++++++++++++++++M++++++++ 

xx 

+++M+++++++++++++++++++++ 

X 

xx ......................... X 

X X 

......................... X 

......................... X 

......................... X 

......................... 

......................... + 

+ 

......................... xx 

X 

xx X ......................... 

......................... 
Lesions in Female Mice 237 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Hemangioma 

Histiocytic sarcoma 

Lymph node 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Fibrous histiocytoma 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Histiocytic sarcoma 

_____~~~ ~ __________~ ~~ 

Integumentary System 

Mammary gland 

Adenoacanthoma 

Skin 

Trichoepithelioma 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, 

multiple 

Alveolar/bronchiolar carcinoma 

Histiocytic sarcoma 

Nose 

Hemangiosarcoma 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

_____~~ ~~ ~ 

7717717117771117111117171 

3333333333333333333333333 

1177171777717777778888888 

3333333333333333333333333 Total 

3333444444555556661223555 Tissues/ 

3569234789145690157364023 Tumors 

......................... 50 

1 

X 2 

++ + + + 13 

......................... 49 

X 3 

......................... 49 

1 

X 3 

.......................... 50 

X 2 

X 3 

++++M+'+++++++++++++++++++ 49 

1 

______~~~~ ~~ ~ ~~~ ~ 

......................... 50 

1 

......................... 50 

1 

X 1 

......................... 50 

+ 1 

X 1 

......................... 50 

1 

1 

......................... 50 

X X 4 

X 1 

1 

X 4 

......................... 50 

X 1 

......................... 50 

+ 1 

X 1 
238 Phenolphthalein, NTP-TR 465 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Urinalybladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

3345556666666666777777777 

2315660012455889333333333 

9891172452514663777777777 

3333333333333333333333333 

5366243124645234112222233 

8724162650357981890247801 

......................... 

A++++++++++++++++++++++++ 

......................... X xx X 

x x xx x x xx x xx x xx x x x x 
Lesions in Female Mice 239 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 6,000 ppm (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Urinarybladder 

Systemic Lesions 

Multipleorgans 

Histiocytic sarcoma 

Lymphomamalignant 

7111111777771717777117171 

333333-3333333333333333333 

7177777777717777778888888 

3333333333333333333333333 Total 

3333444444555556661223555 Tissues1 

3569234189145690151364023 Tumors 

......................... 50 

......................... 49 

......................... 50 

X X X I 

xxxx x X xx xxxxx x x 33 
240 Phenolphthalein, NTP TR 465 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Heart 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Clitoral gland 

Ovary 

Cystadenoma 

Hemangioma 

Sex-cord stromal tumor, benign 

Uterus 

Polyp stromal 

0444555555555666666777777 

4445016778899004568123333 

0790548388948120270520777 

4333443333333343343434333 

1989119988998708618060667 

4809104783209817936687670 

......................... 

+++++++A+AA+AA++A++++++++ 

X 

++++++++++++AA+++++++++++ 

+++++++++++++A+++++++++++ 

+++++++++A++AA++A++++A+++ 

++++++++++++AA+++++++++++ 

A+++++++++++AA+++++++A+++ 

+++++++++A++AA+++++++++++ 

......................... 

x xxxx x X 

......................... X 

+++++++++++++++M+++++++++ 

......................... 

......................... 

......................... 

X 

......................... 

......................... 

......................... 

+++++M+++++++M+++++++++++ 

+++++++++++++++M+++++++++ 

......................... 

.......................... 

......................... X 

X ......................... 
Lesions in Female Mice 241 

TABLE D2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Histiocytic sarcoma 

Intestine large. colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Histiocytic sarcoma 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Heart 

Alveolar/bronchiolar carcinoma, 

metastatic,lung 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Clitoral gland 

Ovary 

Cystadenoma 

Hemangioma 

Sex-cord stromal tumor, benign 

Uterus 

Polyp stromal 

7777777777771777777777777 

3333333333333333333333333 

7777777777777777777788888 

3333333333333344444433444 Total 

7777777888899900001179000 Tissues/ 

1235679124535623492541058 Tumors 

......................... 50 

......................... 44 

1 

......................... 48 

......................... 49 

......................... 45 

......................... 48 

......................... 46 

......................... 41 

......................... 50 

A 1 

X 1 

7 

+ 1 

......................... 50 

1 

......................... 49 

......................... 50 

X X X 3 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

++++++++++++++++M++++++++ 47 

++++++++M++++++++++++++++ 48 

......................... 50 

......................... 50 

......................... 50 

1 

X 1 

X x x x 5 

......................... 50 

X 1 
242 Phenolphthalein, NTP TR 465 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Iliac, histiocytic sarcoma 

Mediastinal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Alveolar/bronchiolar carcinoma, 

metastatic. lung 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Histiocytic sarcoma 

Osteosarcoma, metastatic, bone 

Nose 

Carcinoma, metastatic, harderian 

gland 

Trachea 

Special Senses System 

Ear 

Harderian gland 

Adenoma 

Carcinoma 

0444555555555666666777777 

4445016778899004568123333 

0790548388948120270520177 

4333443333333343343434333 

1989119988998708618060667 

4809104783209817936687670 

......................... x x x X X 

+ ++ ++++ + + + + + 

X X X 

X 

X 

X X 

+++++++++++++++M+++++++++ 

X xx X 

......................... X X X X 

......................... X 

x xxx X X 

+M+++++++++M+++++++++++++ 

X 

X X 

......................... 

......................... X 

......................... X 

......................... 

......................... X 

X 

xxxx 

X 

......................... 

......................... 

+ + 

+ 

X 
Lesions in Female Mice 243 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Iliac, histiocytic sarcoma 

Mediastinal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Alveolar/bronchiolar carcinoma, 

metastatic, lung 

Histiocytic sarcoma 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Alveolar/bronchiolar carcinoma 

Histiocytic sarcoma 

Osteosarcoma, metastatic, bone 

Nose 

Carcinoma, metastatic, harderian 

gland 

Trachea 

Special Senses System 

Ear 

Harderian gland 

Adenoma 

Carcinoma 

7777777777777777777777777 

3333333333333333333333333 

7777777777777777777788888 

3333333333333344444433444 Total 

7777777888899900001179000 Tissues/ 

1235679124535623492541058 Tumors 

......................... 50 

5 

+ +++ 16 

3 

1 

1 

2 

......................... 49 l 4 

......................... 50 

4 

++++++++++Sf+++++++++++++ 50 

1 

6 

+++++++M+M+++++++M+++++++ 45 

1 

L 

......................... 50 

......................... 50 

1 

......................... 50 

1 

~~ ~~ ~ ~~ ~ ~~ ~ 

......................... 50 

......................... 50 

X xx xx 6 

X 2 

4 

1 

......................... 50 

X 1 

......................... 50 

+ 

X 
244 Phenolphthalein, NTP TR 465 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Histiocytic sarcoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

0444555555555666666777777 

4445016778899004568123333 

0790548388948120270520777 

4333443333333343343434333 

1989119988998708618060667 

4809104783209817936687670 

......................... X 

......................... 

......................... x xxxx x X 

x xxxx x x x x xxx 
Lesions in Female Mice 245 

TABLED2 

Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of Phenolphthalein: 12,000 ppm 

(continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Histiocyticsarcoma 

Urinarybladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

7777777777777777777788888 

3333333333333333333333333 

7777777777777777777777777 

3333333333333344444433444 

7777777888899900001179000 

1235679124535623492541058 

Total 

Tissues/ 

Tumors 

......................... 50 

1 

......................... 50 

......................... 50 

7 

xxxx x x X x x x xxx 25 
246 Phenolphthalein,NTP TR 465 

TABLED3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Phenolphthalein 

HarderianGland:AdenomaorCarcinoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armitage testd 

Fisher exact testd 

Liver:HepatocellularAdenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver:HepatocellularCarcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver:HepatocellularAdenomaorCarcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Amitage test 

Fisher exact test 

Lung: Alveolarhronchiolar Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0 PPm 

4/50 (8%) 

10.0% 

3/39 (8%) 

730 

P=0.422N 

P=0.414N 

P=0.313N 

17/50 (34%) 

41.2% 

15/39 (38%) 

597 

P<O.OOlN 

P<O.M)lN 

P<O.OOlN 

6/50 (12%) 

14.9% 

5/39 (13%) 

708 

P=0.046N 

P=0.042N 

P=0.025N 

21/50 (42%) 

49.8% 

18/39 (46%) 

597 

P<O.OOlN 

P<O.OOlN 

P<O.OOlN 

3/50 (6%) 

7.2% 

2/39 (5 %) 

615 

P=O.O32 

P=O.O41 

P=O.O80 

3,000 ppm 

1/50 (2%) 

3.2% 

1/31 (3%) 

737 (T) 

P=0.258N 

P=0.259N 

P=0.181N 

2/50 (4%) 

6.5% 

2/31 (6%) 

737 (T) 

P=O.OOlN 

P<O.M)lN 

P<O.OOlN 

1/50 (2%) 

3.2% 

1/31 (3%) 

737 (T) 

P=0.106N 

P=0.099N 

P=0.056N 

3/50 (6%) 

9.7% 

3/31 (10%) 

737 (T) 

P<O.OOlN 

P<O.OOlN 

P<O.OOlN 

1/50 (2 %) 

3.2% 

1/31 (3%) 

737 (T) 

P=0.385N 

P=0.328N 

P=0.309N 

6,000 ppm 

1/50 (2%) 

2.9% 

1/34 (3 %) 

737 (T) 

P=0.227N 

P=0.230N 

P=0.181N 

6/50 (12%) 

17.1X 

5/34 (15%) 

693 

P=0.022N 

P=0.015N 

P=0.008N 

0150 (0%) 

0.0% 

0134 (0%) -e

P=0.028N 

P=0.025N 

P=0.013N 

6/50 (12 %) 

17.1% 

5/34 (15%) 

693 

P=0.003N 

P=0.002N 

P<0.001N 

5/50 (10%) 

14.7% 

5/34 (15%) 

737 (T) 

P=O.289 

P=O.315 

P=O.357 

12,000 ppm 

2/50 (4%) 

7.1 % 

2/28 (7%) 

737 (T) 

P=0.497N 

P=0.485N 

P=0.339N 

1/50 (2%) 

3.6% 

1/28 (4%) 

737 (T) 

P<0.001N 

P<0.001N 

P<O.OOlN 

1/50 (2%) 

3.6% 

1/28 (4%) 

737 (T) 

P=0.128N 

P=O.lllN 

P=0.056N 

2/50 (4%) 

7.1% 

2/28 (7%) 

737 (T) 

P<O.OOlN 

P<O.OOlN 

P<O.OOlN 

6/50 (12%) 

20.7% 

5/28 (18%) 

730 

P=O.115 

P=O.154 

P=O.243 
Lesions in Female Mice 247 

TABLED3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Phenolphthalein(continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

Lung: Alveolarlbronchiolar Carcinoma 

Overall rate 4/50 (8 %) 4/50 (8 X) 1/50 (2%) 2/50 (4%) 

Adjusted rate 9.4% 12.3% 2.9% 5.6% 

Terminal rate 2/39 (5 %) 3/31 (10%) 0134 (0%) 1128 (4%) 

First incidence (days) 597 663 693 449 

Life table test P=0.276N P=O.514 P=0.231N P=0.459N 

Logistic regression test P=0.190N P=O.607 P=0.180N P=0.321N 

Cochran-Armitage test P=0.186N 

Fisher exact test P=0.643N P=0.181N P=0.339N 

Lung: Alveolar/bronchiolar Adenoma orCarcinoma 

Overall rate 6/50 (12%) 5/50 (10%) 6/50 (12%) 8/50 (16%) 

Adjusted rate 13.8% 15.4% 17.1% 25.7% 

Terminal rate 3/39 (8%) 4/31 (13%) 5/34 (15%) 6/28 (21 58) 

First incidence (days) 597 663 693 449 

Life table test P=O.133 P=O.597 P=O.522 P=O.198 

Logistic regression test P=O.213 P=0.542N P=O.603 P=O.354 

Cochran-Armitage test P=O.269 

Fisher exact test P=0.500N P=0.620N P=O.387 

Ovary:Cystadenoma 

Overallrate 0150 (0%) 3/49 (6%) Ol50 (0%) 1/50 (2%) 

Adjusted rate 0.0% 9.7% 0.0% 2.1% 

First incidence (days) 

Terminal rate 0/39 (0%) - 737 (T) 

3/31 (10%) 0134 (0%) - 449 

0/28 (0%) 

Life table test P=O.575 P=O.O84 - P=O.500 

Logistic regression test P=0.626N P=O.O84 - P=O.518 

Cochran-Armitage test P=0.631N 

Fisher exact test P=O.117 - P=O.500 

Ovary:BenignSex-CordStromalTumor 

Overall rate 0150 (0%) 7/49 (14%) 6/50 (12%) 5/50 (10%) 

Adjustedrate 0.0% 21.7% 17.6% 17.9% 

Terminal rate 0139 (0%) 6/31 (19%) 6/34 (18%) 5/28 (18%) 

First incidence (days) - 668 737 (T) 737 (T) 

Life table test P=O.O56 P=O.O04 P=O.Oll P=O.O12 

Logistic regression test P=O.O66 P=O.O04 P=O.Oll P=O.O12 

Cochran-Armitage test P=O.142 

Fisher exact test P=O.O06 P=O.O13 P=O.O28 

PituitaryGland (Pars Distalis): Adenoma 

Overallrate 3/49 (6%) 2/50 (4%) 1/49 (2%) 0148 (0%) 

Adjustedrate 7.9% 6.5% 2.9% 0.0% 

Terminal rate 3/38 (8%) 2/31 (6%) 1/34 (3 %) 0/27 (0%) 

First incidence (days) 737 (T) 737 (T) 737 (T) -

Life table test P=0.094N P=0.593N P=0.345N P=0.187N 

Logistic regression test P=0.094N P=0.594N P=0.345N P=0.187N 

Cochran-Armitage test P=0.064N 

Fisher exact test P=0.490N P=0.309N P=O.lZSN 
248 Phenolphthalein,NTP TR 465 

TABLED3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Phenolphthalein (continued) 

3,000 ppm 6,000 ppm 12,000 ppm 

Stomach (Forestomach): Squamous Cell Papilloma 

Overall rate 1/50 (2%) 1/50 (2%) 0150 (0%) 3/50 (6%) 

Adjusted rate 3.2% 2.6% 0.0% 10.7% 

Terminal rate 1/39 (3 %) 1/31 (3%) 0134 (0%) 3/28 (1 1%) 

First incidence (days) 737 (T) 737 (T) - 737 (T) 

Life table test P=O.710 P=O.105 P=0.527N P=O.195 

Logistic regression test P=O.710 P=O.105 P=0.527N P=O.195 

Cochran-Armitage test P=O.160 

Fisher exact test P=0.753N P=0.500N P=O.309 

Stomach (Forestomach): Squamous Cell Papilloma or Squamous Cell Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Uterus: Stromal Polyp 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Hemangiosarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Hemangioma or Hemangiosarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cocllran-Armitage test 

Fisher exact test 

1/50 (2%) 

2.6% 

1/39 (3%) 

737 (T) 

P=O.164 

P=O.164 

P=O.242 

1/50 (2%) 

2.6% 

1/39 (3 %) 

737 (T) 

P=O.521 

P=O.541 

P=0.577N 

0150 (0%) 

0.0% 

0/39 (0%) -

P=O.263 

P=O.312 

P=O.337 

2/50 (4%) 

4.8% 

1/39 (3 %) 

686 

P=O.368 

P=O.435 

P=O.477 

2/50 (4%) 

6.5% 

2/31 (6%) 

737 (T) 

Pe0.420 

P=O.420 

P-0.500 

2/50 (4%) 

6.2% 

1/31 (3%) 

703 

P=O.419 

P=O.440 

P=O.500 

1/50 (2%) 

3.2% 

1/31 (3%) 

737 (T) 

P=O.454 

P=O.454 

P=O.500 

1/50 (2%) 

3.2% 

1/31 (3%) 

737 (T) 

P=0.583N 

P=0.548N 

P=0.500N 

0150 (0%) 

0.0% 

0134 (0%) -

P=0.527N 

P=0.527N 

P=0.500N 

3/50 (6%) 

8.8% 

3/34 (9%) 

737 (T) 

P=O.257 

P=O.257 

P=O.309 

4/50 (8%) 

10.8% 

3/34 (9%) 

567 

P=O.O51 

P=O.O62 

P=O.O59 

4/50 (8 %) 

10.8% 

3/34 (9%) 

567 

P=O.285 

P=O.332 

P=O.339 

3/50 (6%) 

10.7% 

3/28 (1 1 %) 

737 (T) 

P=O. 195 

P=O.195 

P=O.309 

1/50 (2%) 

3.6% 

1/28 (4%) 

737 (T) 

P=O.686 

P=O.686 

P=0.753N 

1/50 (2%) 

3.2% 

0128 (0%) 

715 

P=O.444 

P=O.473 

P=O.500 

2/50 (4%) 

6.7% 

1/28 (4%) 

715 

P=O.575 

P=O.640 

P=0.69tN 
Lesions in Female Mice 249 

TABLED3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Phenolphthalein(continued) 

All Organs:Histiocytic Sarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs:MalignantLymphoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs:BenignNeoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs:MalignantNeoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0 PPm 

0/50 (0%) 

0.0% 

0/39 (0%) -

P=O.O02 

P=O.O09 

P=O.O04 

15/50 (30%) 

34.3% 

11/39 (28 %) 

136 

P=O.OlO 

P=O.114 

P=O.O60 

26/50 (52%) 

58.8% 

21/39 (54%) 

78 

P=0.372N 

P=0.208N 

P=0.067N 

25/50 (50%) 

55.1% 

19/39 (49%) 

156 

p=o.002 

P=O.O40 

P=O.OlO 

3,000 ppm 

2/50 (4%) 

6.5% 

2/31 (6%) 

737 (T) 

P=O.189 

P=O.189 

P=O.247 

28/50 (56%) 

60.2% 

13/31 (42%) 

266 

P=O.003 

P=O.O51 

P=O.O07 

20/50 (40%) 

58.7% 

17/31 (55%) 

663 

P=0.509N 

P=0.431N 

P=0.158N 

35/50 (70%) 

71.4% 

17/31 (55%) 

266 

P=O.Oll 

P=O.166 

P=O.O33 

6,000 ppm 

7/50 (14%) 

17.6% 

3/34 (9%) 

567 

P=O.O07 

P=O.OlO 

P-0.006 

33/50 (66%) 

74.6% 

23/34 (68 %) 

338 

PCO.001 

P<O.001 

P<O.ool 

23/50 (46%) 

61.7% 

20/34 (59%) 

479 

P~O.566 

P=0.559N 

P-0.345N 

41\50 (82%) 

85.4% 

27/34 (79%) 

338 

P<O.ool 

P<O.ool 

P<O.OOl 

12,000 ppm 

7/50 (14%) 

18.0% 

0128 (0%) 

588 

P=O.005 

P=O.O11 

P=O.006 

25/50 (50%) 

65.8% 

16/28 (57%) 

447 

P=O.004 

P=O.O35 

P=O.O33 

17/50 (34%) 

56.1% 

15/28 (54%) 

449 

P=0.389N 

P=0.199N 

P=0.053N 

37/50 (74%) 

78.5% 

18/28 (64%) 

447 

P=O.ool 

P=O.O58 

P=O.O11 
250 Phenolphthalein, NTP TR 465 

TABLED3 

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study of Phenolphthalein (continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

All Organs: Benign or Malignant Neoplasms 

Overall rate 40150 (80%) 40/50 (80%) 47/50 (94%) 43/50 (86%) 

Adjustedrate 83.3% 81.6% 95.9% 91.4% 

Terminal rate 31/39 (79%) 22/31 (71%) 32/34 (94%) 24/28 (86%) 

First incidence (days) 78 266 338 447 

Life table test P=O.O14 P=O.135P=O.O14 P=O.O24 

Logistic regression test P=O.230 P=0.566N P=O.O37 P=O.282 

Cochran-Armitage test P=O.158 

Fisher exact test P=0.598N P=O.298P=O.O36 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver, lung, ovary, 

pituitary gland, stomach, and uterus; for other tissues, denominator is number of animals necropsied. 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to 

pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being 

(directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. 

e Not applicable; no neoplasms in animal group 
Lesions in Female Mice 251 

TABLED4a 

Historical Incidence of Histiocytic Sarcoma in Untreated Female B6C3F, Micea 

Study 

Historical Incidence at EG&G Mason Research Institute/TSI Mason 

l-Amino-2,4-dibromoanthraquinone 

Acetaminophen 

HC Yellow 4 

Methylphenidate Hydrochloride 

Pentaerythritol Tetranitrate 

Turmeric Oleoresin 

Overall Historical Incidence 

Total 

Standard deviation 

Range 

a Data as of 12 May 1995 

TABLED4b 

Incidence in Controls 

Laboratories, Inc. 

0150 

1150 

0150 

2/49 

0150 

- 0150 

1911.473 (1.3%) 

1.6% 

0%4% 

Historical Incidence of Malignant Lymphoma in Untreated Female B6C3Fl Micea 

Study 

Historical Incidence at EG&G Mason Research Institute/TSI Mason 

l-Amino-2,4dibromoanthraquinone 

Acetaminophen 

HC Yellow 4 

Methylphenidate Hydrochloride 

Pentaerythritol Tetranitrate 

Turmeric Oleoresin 

Overall Historical Incidence 

Total 

Standard deviation 

Range 

Incidence in Controls 

Laboratories, Inc. 

11/50 

10150 

9150 

12/49 

20150 

9/50 

33911,473 (23.0%) 

11.9% 

6%4% 

a Data as of 12 May 1995;includes data for histocytic, lymphocytic, mixed, unspecified, or undifferentiated cell type lymphomas 
252 Phenolphthalein, NTP TR 465 

TABLED4c 

Historical Incidence of Luteoma in the Ovary of Untreated Female B6C3Fl Micea 

Study Incidence in Controls 

Historical Incidence at EG&G Mason Research Institute/TSI Mason Laboratories, Inc. 

l-Amino-2,4-dibromoanthraquinone 0/49 

Acetaminophen 0147 

HC Yellow 4 0150 

Methylphenidate Hydrochloride 0/46 

Pentaerythritol Tetranitrate 0150 

Turmeric Oleoresin 0150 

Overall Historical Incidence 

Total 5/1,436 (0.4%) 

Standard deviation 1.O% 

Range 0%-4% 

~~ 

a Data as of 12 May 1995 

TABLED4d 

Historical Incidence of Hepatocellular Neoplasms in Untreated Female B6C3Fl Micea 

Incidence in Controls 

Study Adenoma Carcinoma Adenoma Carcinoma or 

Historical Incidence at EG&G Mason Research Institute/TSI Mason Laboratories, Inc. 

l-Amino-2,4-dibromoanthraquinone 6/50 0150 6/50 

Acetaminophen 3/49 0149 3/49 

HC Yellow 4 5/50 1 I50 6/50 

Methylphenidate Hydrochloride 6/49 5/49 9/49 

Pentaerythritol Tetranitrate 5/49 1/49 6/49 

Turmeric Oleoresin 7/50 7/50 13/50 

Overall Historical Incidence 

Total 23111,464 (15.8%) 10811.464 (7.4%) 313/1,464 (21.4%) 

Standard deviation 10.6% 6.1% 13.0% 

Range 2%-50% 0%-20% 3%-56% 

a Data as of 12 May 1995 
Lesions in Female Mice 253 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of Phenolphthaleina 

0 PPm 3,000 ppm 

Disposition Summary 

Animals initially in study 50 50 

Early deaths 

Moribund 5 13 

Natural deaths 6 6 

Survivors 

Died last week of the study 1 

Terminal sacrifice 38 31 

Animals examined microscopically 50 50 

AIimentarySystem 

Intestine large, cecum 

Hemorrhage 

Epithelium, hyperplasia 

Hemorrhage 

Intestine small, jejunum (49) 

Angiectasis 

Liver (50) 

2 (4%) 

Clear cell focus 1 (2%) 2 (4%) 

Eosinophilic focus 20 (40%) 4 (8%) 

Fibrosis 1 (2%) 

Hematopoietic cell proliferation 

Inflammation, chronic 1 (2%) 

Mixed cell focus 3 (6%) 2 (4%) 

Necrosis 2 (4%) 2 (4%) 

Vacuolization cytoplasmic 3(6%) 

Centrilobular, necrosis 

Kupffer cell, hyperplasia 

Mesentery 

Inflammation, chronic 

Fat, congestion 

Fat, necrosis 

Pancreas 

Cytoplasmic alteration 

Inflammation, acute 

Inflammation, chronic 1 (2%) 

Necrosis 

Acinus, atrophy 

Stomach, forestomach 

Edema 

Hemorrhage 

Hyperkeratosis 1 (2%) 

Hyperplasia, squamous 3 (6%) 6 (12%) 

Inflammation, chronic 1 (2%) 

Ulcer 2 (4%) 

Muscularis, hyperplasia 1 (2%) 

~~ ~~ ~~~ ~~ 

a Number of animals examined mlcroscopically at the site andthe number of animals with lesion 

6,000 ppm 

50 

9 

7 

34 

50 

(48) 

1 (2%) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

2 (4%) 

4 (8%) 

1 (2%) 

12,000 ppm 

50 

12 

10 

28 

50 

1 (2%) 

(46) 

(50) 

4 (8%) 

1 (2%) 

1 (2%) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

2 (4%) 

3 (6%) 
Phenolphthalein,NTP TR 465 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the2-YearFeed Study of Phenolphthalein 

(continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 

Alimentary System (continued) 

Stomach, glandular 

Edema 

(50) 

Erosion 

Glands, hyperplasia 

Tooth 

Inflammation, chronic 

Cardiovascular System 

Heart 

Angiectasis 

Cardiomyopathy 2 (4%) 

Hemorrhage 

Inflammation, chronic 1 (2%) 

Inflammation, chronic active 

Mineralization 

Atrium, thrombosis 

Valve, inflammation, acute 

Valve, inflammation, chronic active 

Endocrine System 

Angiectasis 

Adrenal cortex (50) 

1 (2%) 

Degeneration, cystic 1 (2%) 

Hematopoietic cell proliferation 1 (2%) 1 (2%) 

Hypertrophy, focal 1 (2%) 

Capsule, hyperplasia 49 (98%) 

Hyperplasia 

Adrenal medulla 

2 (4%) 

(50) 

Hyperplasia 

Islets, pancreatic 

32 (65%) 

(49) 

Parathyroid gland (46) 

cyst 1 (2%) 

Hyperplasia 1 (2%) 

Pars distalis, angiectasis 

Pituitary gland (49) (49) 

Pars distalis, cyst 1 (2%) 

Pars distalis, hyperplasia 11 (22%) 1 (2%) 1 (2%) 

Rathke’s cleft, cyst 1 (2%) 

Thyroid gland (50) (50) 

Cyst 1 (2%) 

Inflammation, chronic 1 (2%) 

Follicular cell, hyperplasia 27 (54%) 8 (16%) 3 (6%) 7 (14%) 

General Body System 

None 
Lesions in Female Mice 255 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Studyof Phenolphthalein 

(continued) 

Genital System 

Clitoral gland 

cyst 

Inflammation, chronic active 

Pigmentation 

Ovary 

Angiectasis 

cyst 

Hemorrhage 

Hyperplasia 

Hyperplasia, papillary 

Inflammation, chronic 

Necrosis 

Thrombosis 

Periovarian tissue, necrosis 

UkNS 

Angiectasis 

Hemorrhage 

Hyperplasia, cystic 

Inflammation, chronic active 

Necrosis 

Hematopoietic System 

Bone marrow 

Hyperplasia, reticulum cell 

Myelofibrosis 

Pigmentation 

Lymph node 

Iliac, hyperplasia, lymphoid 

Mediastinal, hyperplasia, lymphoid 

Mediastinal, infiltration cellular, 

histiocyte 

Mediastinal, pigmentation 

Renal, infiltration cellular, histiocyte 

Renal, pigmentation 

Lymph node, mandibular 

Atrophy 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Lymph node, mesenteric 

Angiectasis 

Atrophy 

Hematopoietic cell proliferation 

Hemorrhage 

Hyperplasia, lymphoid 

Spleen 

Fibrosis 

Hematopoietic cell proliferation 

Pigmentation 

Lymphoid follicle, atrophy 

Lymphoid follicle, hyperplasia 

0 PPm 

(48) 

2(4%) 

2(4%) 

(50) 

1(2%) 

15 (30%) 

3 (6%) 

3 (6%) 

1(2%) 

1(2%) 

(50) 

3(6%) 

49 (98%) 

1 (2%) 

34 (68%) 

2(4%) 

(14) 

2 (14%) 

1 (7%) 

1 (7%) 

1 (7%) 

1(7%) 

1 (7%) 

(49) 

1 (2%) 

(49) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1(2%) 

13 (26%) 

4 (8%) 

6 (12%) 

3,000 ppm 

(48) 

2(4%) 

3 (6%) 

(49) 

8 (16%) 

1 (2%) 

10 (20%) 

1(2%) 

1 (2%) 

46 (92%) 

(50) 

14 (28%) 

1(2%) 

1(2%) 

5 (10%) 

6,000 ppm 

1(2%) 

(50) 

1(2%) 

16 (32%) 

3 (6%) 

10 (20%) 

1 (2%) 

43 (86%) 

(50) 

1(2%) 

38 (76%) 

11 (22%) 

(13) 

(49) 

1 (2%) 

(49) 

3 (6%) 

1(2%) 

(50) 

20(40%) 

2(4%) 

3 (6%) 

12,000 ppm 

(50) 

1 (2%) 

1 (2%) 

(50) 

12 (24%) 

2 (4%) 

17 (34%) 

45 (90%) 

1 (2%) 

(49) 

1 (2%) 

21 (42%) 

3 (6%) 

1 (2%) 
256 Phenolphthalein, NTP TR 465 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-YearFeed Study of Phenolphthalein 

(continued) 

Hematopoietic System (continued) 

Thymus 

Atrophy 

Fibrosis 

Hyperplasia,atypical 

Infiltration cellular, poymorphonuclear 

IntegumentarySystem 

Skin 

Inflammation,chronic 

Ulcer 

Subcutaneous tissue, edema 

Subcutaneoustissue,inflammation,chronic 

Musculoskeletal System 

None 

Nervous System 

Brain 

Demyelination 

Hemorrhage 

Thalamus,mineralization 

Peripheral nerve 

Demyelination 

Spinal cord 

Demyelination 

Respiratory System 

Lung 

Hemorrhage 

Infiltration cellular, histiocyte 

Thrombosis 

Alveolar epithelium, hyperplasia 

Special Senses System 

None 

0 PPm 

(48)

3 (6%) 

1 (2%) 

2 (4%) 

(50) 

7 (14%) 

2(4%) 

1 (2%) 

1(2%) 

3,000 ppm 

(44) 

1 (2%) 

7(16%) 

(50) 

2(4%) 

(50) 

4(8%) 

3 (6%) 

6,000 ppm 

(49) 

6 (12%) 

6(12%) 

3 (6%) 

(50) 

6(12%) 

1 (2%) 

12,000 ppm 

(45) 

9 (20%) 

5 (11%) 

1 (2%) 

3 (6%) 

1 (2%) 

(50) 

4 (8%) 

1 (2%) 
Lesions in Female Mice 257 

TABLED5 

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Studyof Phenolphthalein 

(continued) 

3,000 ppm 6,000 ppm 12,000 ppm 

Urinary System 

Kidney 

cyst 

Hydronephrosis 

Infarct 

Inflammation, chronic 

Mineralization 1 (2%) 

Necrosis 1 (2%) 1 (2%) 

Nephropathy, chronic 15 (30%) 16 (32%) 19 (38%) 14 (28%) 

Pigmentation 1 (2%) 

Pelvis, hemorrhage 1 (2%) 

Renal tubule, accumulation, hyaline droplet 1 (2%) 3 (6%) 8 (16%) 

Renal tubule, dilatation 2 (4%) 

Hyperplasia, lymphoid 

Urinary bladder (50) 

1 (2%) 

(49) (50) 

Infiltration cellular, lymphocyte 1 (2%) 
258 Phenolphthalein,NTP TR 465 
259 

APPENDIX E 

GENETIC TOXICOLOGY 

SALMONELLAMUTAGENICITY TEST PROTOCOL .. . . . . . . . . . . . . . . . .. . . . . . . . . . . ... . . . 260 

CHINESEHAMSTER OVARYCELL CYTOGENETICS PROTOCOLS .. . . . . . . . . . . ..... . . . . . . . . 260 

Mourn PERIPHERAL BLOODMICRONUCLEUS TESTPROTOCOL . . . . . . . . . . . .. . . . . . . . . . . . . 261 

RESULTS ........................................................... 262 

TABLEEl Mutagenicity of Phenolphthalein in Salmonella typhimurium . . . . . . . . . . . . . . ... . 263 

TABLEE2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

by Phenolphthalein . . . .... . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . .. . 266 

TABLEE3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells 

by Phenolphthalein . . . . . . . . . . . .. . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . 267 

TABLE4 Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes 

Following Treatment with Phenolphthalein in Feed for 13 Weeks . . . . . . . . . . . . . . . 268 
260 Phenolphthalein, NTP TR 465 

GENETIC TOXICOLOGY 

SALMONELLAMUTAGENICITYESTPROTOCOL 

Testing was performed as reported by Mortelmans et al. (1986). Phenolphthalein was sent to the 

laboratories as a coded aliquot from Radian Corporation (Austin, TX). It was incubated with the 

Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 either in buffer or S9 mix 

(metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or 

Syrian hamster liver) for 20 minutes at 37" C. Top agar supplemented with Z-histidineand d-biotin was 

added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar 

plates. Histidine-independent mutant colonies arising on these plates were counted following incubation 

for 2 days at 37 O C. 

Each trial consisted of triplicate plates of concurrent positive and negative controls and of at least five 

doses of phenolphthalein. The high dose was limited by toxicity. All trials were repeated. 

In this assay, a positive response is defined as a reproducible, dose-related increase in histidineindependent (revertant) colonies in any one straidactivation combination. An equivocal response is 

defined as an increase in revertants that is not dose related, not reproducible, or is not of sufficient 

magnitude to support a determination of mutagenicity. A negative response is obtained when no increase 

in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold 

increase required for a chemical to be judged positive or weakly positive. 

CHINESEHAMSTEROVARYCELLCYTOGENETICSPROTOCOLS 

Testing was performed as reported by Galloway et al. (1987) and by Witt et al. (1995). Phenolphthalein 

was sent to the laboratory as a coded aliquot by Radian Corporation. It was tested in cultured Chinese 

hamster ovary (CHO) cells for induction of sister chromatid exchanges (SCEs) and chromosomal 

aberrations (Abs) both in the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat 

liver S9 and cofactor mix. Cultures were handled under gold lights to prevent photolysis of 

bromodeoxyuridine-substituted DNA. Each test consisted of concurrent solvent and positive controls and 

of at least three doses of phenolphthalein; the high dose was limited by toxicity. A single flask per dose 

was used, and tests yielding equivocal or positive results were repeated. 

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 26 hours 

with phenolphthalein in McCoy's 5A medium supplemented with fetal bovine serum, I-glutamine, and 

antibiotics. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the 

medium containing phenolphthalein was removed and replaced with fresh medium plus BrdU and 

Colcemid, and incubation was continued for 2 hours. Cells were then.harvested by mitotic shake-off, 

fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with 

phenolphthalein, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced 

with medium containing serum and BrdU and no phenolphthalein, and incubation proceeded for an 

additional 26 hours with Colcemid present for the final 2 hours. Harvesting and staining were the same as 

for cells treated without S9. All slides were scored blind, and those from a single test were read by the 

same person. Fifty. second-division metaphase cells were scored for frequency of SCEdcell from each 

dose level. Because significant chemical-induced cell cycle delay was seen at the 50 pg/mL dose, 

incubation time was lengthened to 31 hours to ensure a sufficient number of scorable (second-division 

metaphase) cells. 
Genetic Toxicology 261 

Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose 

points (Galloway et al., 1987). An SCE frequency 20% above the concurrent solvent control value was 

chosen as a statistically conservative positive response. The probability of this level of difference 

occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at 

two dose points is less than 0.001. An increase of 20% or greater at any single dose was considered weak 

evidence of activity; increases at two or more doses resulted in a determination that the trial was positive. 

A highly significant trend (P 50.005) in the absence of any responses reaching 20% above background led 

to a call of equivocal. 

Chromosomal Aberrations Test: In the Abs test without S9, cells were incubated in McCoy’s 5A medium 

with phenolphthalein for 14.2 hours; Colcemid was added and incubation continued for 2 hours. The cells 

were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells 

were treated with phenolphthalein and S9 for 2 hours, after which the treatment medium was removed and 

the cells incubated for 12 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were 

harvested in the same manner as for the treatment without S9. The harvest time for the Abs test was 

based on the cell cycle information obtained in the SCE test; because cell cycle delay was anticipated in 

the absence of S9, the incubation period for these,cultures was extended. 

Cells were selected for scoring on the basis of good morphology and completeness of karyotype 

(21 f 2 chromosomes). All slides were scored blind, and those from a single test were read by the same 

person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations 

included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other 

(pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). 

Chromosomal aberration data are presented as percentage of cells with aberrations. To arrive at a 

statistical call for a trial, analyses were conducted on both the dose response curve and individual dose 

points. For a single trial, a statistically significant (PS 0.05) difference for one dose point and a 

significant trend (P 50.015) were considered weak evidence for a positive response; significant differences 

for two or more doses indicated the trial was positive. A positive trend in the absence of a statistically 

significant increase at any one dose point led to an equivocal call (Galloway et al., 1987). Ultimately, the 

trial calls were based on a consideration of the statistical analyses as well as the biological information 

available to the reviewers. 

MOUSEPERIPHERAL BLOODMICRONUCLEUS TESTPROTOCOL 

A detailed discussion of this assay is presented in Dietz et al. (1992). At the end of the 13-week study, 

blood was obtained from male and female mice, and smears were immediately prepared and fixed in 

absolute methanol. The methanol-fixed slides were stained with a chromatin-specific fluorescent dye 

mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983) and coded. Slides were scanned at a 

magnification of 630 X or 1000 X using a semi-automated image analysis system to determine the 

frequency of micronuclei in 2,000 polychromatic erythrocytes (PCEs) and 10,000 normochromatic 

erythrocytes (NCEs) in each of 10 animals per exposure group. The criteria of Schmid (1976) were used 

to define micronuclei, with the additional requirement that micronuclei exhibit the characteristic 

fluorescent emissions of DNA (blue with 360 nm and orange with 540 nm illumination); the minimum size 

limit was approximately one-twentieth the diameter of the nucleus. 

The frequency of micronucleated cells among PCEs was analyzed by the Cochran-Armitage trend test, and 

individual exposure groups were compared to the concurrent control groups by Kastenbaum-Bowman’s 

binomial test. Log transformation of the NCE data and testing for normality by the Shapiro-Wilk test and 

for heterogeneity of variance by Cochran’s test were performed before statistical analyses. The frequency 
262 Phenolphthalein, NTP TR 465 

of micronucleated cells among NCEs was analyzed by analysis of variance using the SAS GLM procedure. 

The NCE data for each exposure group were compared with the concurrent control group using Student’s 

t-test. 

RESULTS 

Phenolphthalein, tested independently in two laboratories, was not mutagenic in Salmonella typhimurium 

strain TA98, TA100, TA1535, or TA1537, with or without induced rat or hamster liver S9 metabolic 

activation enzymes (Table El; Mortelmans et al., 1986). In cytogenetic tests with cultured CHO cells, no 

induction of SCEs was observed after treatment with 0.5 to 50 pglmL phenolphthalein with or without S9 

(Table E2). Cytotoxicity was noted at the 50 pg/mL dose level, and culture times were extended to 

maximize the proportion of cells available for analysis at this dose level. Results of the in vitro Abs test 

with phenolphthalein were positive for the two trials conducted with S9; no increase in Abs was observed 

in the absence of S9 (Table E3; Witt et al., 1995). At the 50 pg/mL dose level, approximately one 

quarter of all cells contained aberrations, and most of these were chromosome breaks located at the distal 

end of X,. The significance of this preferential breakage is as yet unknown, but it has been noted with 

other chemicals, and it occurs only in the presence of S9. A discussion of possible mechanisms for this 

phenomenon is presented in Galloway et al. (1987). Results from the in vivo mouse peripheral blood 

micronucleds test were positive for both male and female mice (Table E4; Dietz et al., 1992). Significant 

increases in micronuclei were noted for both NCE and PCE populations. The increases in micronucleated 

NCEs, which indicate the effects of chronic exposure, occurred at all exposure concentrations in males 

and females. The effects on PCEs, which indicate chromosomal damage induced within the 48 hours 

preceding analysis, occurred only at 25,000 and 50,000 ppm, the two highest exposure concentrations. 
Genetic Toxicology 263 

TABLEEl 

Mutagenicity of Phenolphthalein in Salmonella @phimunuma 

Revertants/plateb 

Strain Dose -s9 +lo% hamster S9 +lo% rat S9 

Olglplate) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 

Study performed at EG&G Mason Research Institute 

TAlOO 0 153 f 7.9 176 f 6.2 144 f 5.7 197 f 6.9 125 f 5.6 169 f 6.8 

3.3 141 f 7.2 170 f 4.5 140 f 6.6 196 f 7.5 124 f 3.0 173 f 11.8 

10 131 f 2.1 180 f 4.7 145 f 8.3 211 f 8.5 150 f 7.6 166 f 14.7 

33 127 f 3.0 163 f 3.4 140 f 10.4 198 f 7.2 140 f 9.9 177 f 1.5 

100 157 f 7.4 184 f 9.0 162 f 1.5 204 f 8.8 148 f 6.6 191 f 7.2 

220 132 f 2.7' 226 f 3.5' 168 f 6.9' 

333 Toxic 94 f 6.4' 92 f 5.9' 

Trial summary Negative Negative Negative Negative Negative Negative 

Positive controld 984 f 41.6 1.072 f 48.0 1,109 f 32.7 691 f 21.4 908 f 46.0 617 f 8.8 

TA1535 0 31 f 3.8 28 f 7.0 13 f 1.2 11 f 1.2 15 f 1.9 14 f 0.6 

3.3 29 f 3.5 26 f 1.9 11 f 0.0 12 f 0.0 10 f 1.8 12 f 2.3 

10 21 f 4.7 19 f 3.3 9 f 2.0 12 f 1.9 11 f 3.3 11 f 0.7 

33 31 f 5.2 21 f 3.7 9 f 1.0 13 f 4.2 9 f 3.8 17 f 1.8 

100 28 f 4.0 25 f 2.6 11 f 0.9 14 f 0.6 12 f 2.3 13 f 1.2 

220 Toxic 14 f 0.6' 15 f 4.7' 

333 Toxic Toxic 6 f 1.0' 

Trial summary Negative Negative Negative Negative Negative Negative 

Positive control 972 f 21.3 840 f 53.4 148 f 15.2 68 f 1.5 117 f 6.6 59 f 0.9 

"A1537 0 5 f 2.3 7 f 1.2 12 f 1.8 9 f 2.0 7 f 1.5 9 f 2.1 

3.3 7 f 0.6 6 f 2.3 12 f 0.3 9 f 2.2 7 f 1.0 8 f 0.3 

10 7 f 2.3 7 f 0.7 8 f 1.2 7 f 1.2 9 f 0.3 10 f 1.0 

33 6 f 0.6 5 f 2.0 8 f 1.8 11 f 1.2 8 f 1.2 7 f 0.6 

100 6 f 1.2 11 f 1.5 9 f 1.5 9 f 0.3 8 f 0.9 11 f 1.8 

220 4 f 1.0' 10 f 1.8' 5 f 0.3' 

333 Toxic 5 f 1.7' Toxic 

Trial summary Negative Negative Negative Negative Negative Negative 

Positive control 393 f 85.6 188 f 17.2 102 f 2.3 54 f 2.5 73 f 1.5 42 f 2.5 

TA98 0 17 f 1.8 19 f 2.7 23 f 2.7 23 f 4.0 19 f 1.3 22 f 2.3 

3.3 21 f 2.1 18 f 2.3 29 f 3.2 23 f 1.0 26 f 0.9 28 f 3.1 

10 20 f 2.1 12 f 2.0 26 f 1.5 32 f 2.1 23 f 2.6 25 f 2.6 

33 21 f 2.7 14 f 3.8 27 f 2.0 25 f 2.0 25 f 4.5 25 f 0.6 

100 16 f 1.5 11 f 0.9 29 f 2.4 25 f 1.2 25 f 4.4 20 f 0.3 

220 10 f 2.9' 33 f 5.0' 25 f 2.6' 

333 Toxic 30 f 2.4' 29 f 2.6' 

Trial summary Negative Negative Negative Negative Negative Negative 

Positive control 1,335 f 20.7 1,335 f 27.2 1,231 f 9.5 467 f 30.1 1,001 f 10.1 402 f 49.7 
264 Phenolphthalein, NTP TR 465 

TABLEEl 

Mutagenicity of Phenolphthalein in Salmonella typhimunum (continued) 

Revertantslplate 

StrainDose -s9 +lo% hamster S9 

(pglplate) Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 

Study performed at Case Western Reserve University 

TAlOO 0 86 f 1.7 99 f 10.2 87 f 0.9 90 f 6.5104 f 8.0 113 f 12.8 

3.3 94 f 6.0. 82 f 1.2 94 f 2.4 96 f 5.6 

10 94 f 8.0 79 f 7.7 88 f 4.3 95 f 8.0 95 f 3.8 102 f 10.1 

33 90 f 6.085 f 5.9 82 f 7.8 102 f 6.0 113 f 9.0 89 f 6.7 

100 109 f 23.4 72 f 1.2 70 f 7.3 100 f 9.2 120 f 5.8 108 f 10.7 

333 Toxic Toxic Toxic Toxic 122 f 6.9 117 f 9.3 

1,000 Toxic 13 f 8.5 

Trial summary Negative Ngative Ngative Negative Ngative Ngative

Positive control 1,381 It 22.1 911 f 36.0 1,641 f 38.6 945 f 126.4 1,383 f 108.6 2,007 f 217.0 

+lo% rat S9 

Trial 1 Trial 2 

TAlOO 0 115 f 9.9 128 f 3.5 

(continued) 3.3 

10 92 f 5.9 107 f2.7 

33 90 f 1.8 128 f 2.9 

100 101 f 3.5 118 f 6.2 

333 90 f 3.9 126 f 4.0 

1.000 Toxic 103 f 15.4 

Trial summary Negative Ngative

Positive control 1,491 f 345.7 1,586 f 45.0 

-s9 +lo% hamster S9 

Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 

TA1535 0 7 f 0.9 14 f 1.5 5 f 1.8 10 f 1.0 13 f 0.3 

3.3 12 f 1.2 6 f 1.7 

10 10 f 0.9 9 f 1.5 7 * 1.5 13 f 1.5 12 f 3.5 

33 9 f2.3 8 f 1.5 5 f 0.7 9 f 1.2 10 f 1.7 

100 9 f 1.0 10 f 0.7 4 f 1.5 13 f 2.3 9 f 2.4

333 Toxic 9 f 1.8 Toxic 11 f 3.7 11 f 2.0 

1,ooo Toxic Toxic 12 * 3.3 

Trial summary Negative Negative Negative Negative Negative

Positive control 1,029 f 16.2 680 f28.7 1,081 f 144.9 308 f 26.5 133 f 6.8 
Genetic Toxicology 265 

TABLEEl 

Mutagenicity of Phenolphthalein in Salmonella typhimuriurn (continued) 

Revertantslplate 

Strain Dose +lo% rat S9 

Oldplate) Trial 1 Trial 2 

Study performed at Case Western Reserve University (continued) 

TA1535 0 10 f 4.0 11 f 1.2 

(continued) 3.3 

10 6 f 2.6 11 f 1.9 

33 7 f 1.2 7 f 1.0 

100 9 f 0.0 11 f 2.1 

333 8 f 1.7 10 f 2.1 

1,ml Toxic 16 f 2.3 

Trial summary Negative Ngative

Positive control 416 f 46.5 287 f 19.9 

-s9 +lo% hamster S9 +lo% rat S9 

Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 

TA1537 0 3 f 1.2 9 f 2.6 13 f 1.0 11 f 1.5 10 f 1.0 15 f 1.0 

10 4 f 0.3 9 f 1.8 10 f 3.2 9 f 1.5 9 f 2.2 11 f 0.6 

33 7 f 1.8 9 f 0.9 9 f 2.0 12 f 0.9 9 f 2.0 11 f 1.2 

100 9 f 1.5 10 f 1.2 11 f0.3 9 f 0.3 7 f 1.7 13 f 1.8 

333 4 f 2.4 7 f 0.9 10 f 1.2 9 f 2.1 8 f 1.3 13 f 1.8 

1,000 Toxic Toxic Toxic Toxic 10 f 3.5 Toxic 

Trial summary Negative Negative Negative Negative Negative Negative 

Positive control 109 f 9.6 113 f 39.5 68 f 9.6 194 f 5.9 105 f 8.0 158 f 41.7 

TA98 0 16 f 1.8 25 f 2.2 25 f 3.4 30 f 4.3 24 f 1.2 31 f 5.8 

10 13 f 3.8 21 f 2.9 22 f 4.8 30 f 4.2 22 f 6.0 30 f 1.5 

33 11 f 1.5 21 f 3.5 20 f 3.5 27 f 2.0 18 f 2.7 31 f 2.3 

I00 17 f 0.9 20 f 4.1 29 f 2.3 31 f 0.9 19 f 3.7 22 f 3.5 

333 16 f 3.8 17 f 0.9 25 f 2.6 23 f 4.0 25 f 3.5 19 f 2.8 

1,000 Toxic Toxic Toxic Toxic Toxic Toxic 

Positive control 

Trial sunmary 

146 f 17.8 

Negative 

92 f 4.0 

Negative 

554 f 93.2 

Negative 

966 f 33.4 

Negative Negative 

334 f 132.8 

Negative 

681 f 66.8 

a The detailed protocol and thesedata are presented in Mortelmans et al. (1986). 

Revertants are presented as mean f standard error from three plates. 

Slight toxicity 

The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535). 9-aminoacridine (TA1537). and 

4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-arninoanthracene. 
266 Phenolphthalein, NTP TR 465 

TABLEE2 

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Phenolphthaleina 

No. of SCEs/ Relative 

Dose Total Chromo- No. of Chromo- SCEsl Hrs Change of SCEs/ 

Cell SCEsCompoundCells somesomes(pg/mL) in Chromosomeb BrdU 

-s9 

Summary: Negative 

Dimethylsulfoxide 50 1,0430.37 

Mitomycin-C 0.001 50 1,045 677 0.64 13.5 26.0 72.38 

0.005 10 212 277 1.30 27.7 26.0 247.66 

Phenolphthalein 0.5 50 1.047 416 0.39 8.3 26.0 5.72 

392 7.8 26.0 

50 1.050 1.7 427 0.40 8.5 26.0 8.20 

5 50 1,044 379 0.36 7.6 26.0 -3.41 

17 50 1,047 417 0.39 8.3 26.0 5.97 

50 0' 31.Od 

P=0.419e 

+s9 

Summary: Negative 

Dimethylsulfoxide 50 407 0.3926.0

Cyclophosphamide 0.125 50 1,043 556 0.53 11.1 26.0 36.48 

0.5 10 208 170 0.81 17.0 26.0 109.25 

Phenolphthalein 1.7 50 1.047 389 0.37 7.8 26.0 -4.88 

5 50 1,050 383 0.36 7.7 26.0 -6.61 

17 50 1,042 361 0.34 7.2 26.0 -11.30 

50 50 1.042 464 0.44 9.3 31.0d 14.00 

P=O.O85 

a SNdy performed at SITEK Research Laboratories. A detailed description of the protocol is presented in Galloway et of. (1987). 

SCE=sister chromatid exchange; BrdU= bromodeoxyuridine. 

SCEslchromosome in treated cells versus SCEslchromosome in solvent control cells 

Dose was cytostatic. 

Because phenolphthalein induced a delay in the cell division cycle, harvest time was extended to maximize the proportion of 

second-division metaphase cells available for analysis. 

e Significance of relative SCEslchromosome tested by the linear regression trend test versus log of the dose 

1.042 8.1 
Genetic Toxicology 267 

TABLEE3 

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Phenolphthaleina 

-s9 +s9 

Dose Total No. of Abs/ Cells with Dose Total No. of Absl Cells with 

(pg/mL) Cells Abs Cell Abs (%) (pglmL) Cells Abs Cell Abs (%) 

Harvest time: 16.2 hoursb Trial 1 - Harvest time: 14.0 hours 

Summary: Negative Summary: Weakly positive 

Dimethylsulfoxide Dimethylsulfoxide 

200 2 0.01 1.o 200 3 0.02 1.o 

Mitomycin-C Cyclophosphamide 

0.1 200 67 0.34 25.0 5 200 47 0.24 17.0 

0.4 50 37 0.74 46.0 15 50 32 0.64 34.0 

Phenolphthalein Phenolphthalein 

11 200 

23 200 

0 0.00 0 

1.53 

11 200 1 0.01 0.5 

2 0.01 1.o 23 200 0 0 0

50 59

P=O.198' P<O.001 

Trial 2 - Harvest time: 14.0 hours 

Summary: Positive 

Dimethylsulfoxide 

200 2 0.01 1.o 

Cyclophosphamide 

5 200 27 0.14 13.0 

15 50 36 0.72 26.0 

Phenolphthalein 

50 200 0.02 200 0.30 25.0*

30 200 3 0.02 1.5 

40 0.05

50 77

200 9 4.5* 

200 0.39 29.0* 

Ps;O.001 

* Positive (PlO.05) 

a Study performed at SITEK Research Laboratories. The detailed protocol and these data are presented in Witt et al. (1995). 

Abs=aberrations. 

Because of chemical-induced cell cycle delay, incubation time prior to addition of Colcemid was lengthened to provide sufficient 

second-division metaphase cells at harvest. 

Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose 
268 Phenolphthalein, NTP TR 465 

TABLEE4 

Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes Following Treatment 

with Phenolphthalein in Feed for 13 Weeksa 

Dose Micronucleated Cells11.000 Cellsb Number of 

(PP) PCEs NCEs Mice 

Male 0 2.01 f 0.26 1.75 f 0.16 10 

6.000 2.08 f 0.49 2.51 f 0.23** 10 

12.000 2.29 f 0.52 2.63 f 0.24** 10 

25.000 5.36 f 0.67** 4.03 f 0.48** 10 

50.000 4.99 f 0.56** 4.72 f 0.25** 10 

P10.001C P10.001 

Female 0 1.49 f 0.24 1.32 f 0.08 10 

6,000 2.20 f 0.44 2.07 f 0.19** 10 

12,000 2.93 f 0.37 2.94 f 0.23** 10 

25.000 5.36 f 0.70** 4.41 f 0.23** 10 

50.000 4.94 f 0.64** 4.31 f 0.35** 10 

PSO.001 P10.001 

** PSO.01 by Kastenbaum-Bowman’s binomial test (PCEs) or Student’s t-test (NCEs) 

a Study performed at USDA Western Regional Center. The detailed protocol and these data are presented in Dietz et al. (1992); 

PCEs=polychromatic erythrocytes; NCEs=normochromatic erythrocytes; 2,000 PCEs and 10,OOO NCEs scored per animal. 

Data are presented as mean f standard error. 

Significance by Cochran-Armitage linear regression of proportions (PCEs) or linear contrasts from analysis of variance (NCEs) 

\ 
269 

ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS 

ORGAN WEIGHTS AND 

APPENDIX F 

TABLEF2 OrganWeightsandOrgan-Weight-to-Body-WeightRatiosforMice 

TABLEF1OrganWeightsandOrgan-Weight-to-Body-WeightRatiosforRats 

inthe 13-Week FeedStudy of Phenolphthalein . . . . . . . . ... . . .. . . . . . .. . . . . 

inthe13-WeekFeedStudy of Phenolphthalein . . . . . . . . . . . . . . . . . . . . . . . .. . 

270 

272 
270 Phenolphthalein, NTF' TR 465 

TABLEF1 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 13-Week Feed Study of Phenolphthaleina 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

Male 

n 10 10 10 10 10 10 

Necropsybodywt 360 f 6 356 f 7 360 f 6 356 f 6 359 f 10 352 f 6 

Brain 

Absolute 2.033 f 0.035 1.995 f 0.0251.995 f 0.017 2.005 f 0.026 2.005 f 0.018 2.015 f 0.033 

Relative 5.65 f 0.11 5.61 f 0.095.56 f 0.08 5.64 f 0.08 5.62 f 0.13 5.74 f 0.09 

Heart 

Absolute 1.115 f 0.016 1.115 f 0.0291.127 f 0.016 1.147 f 0.033 1.141 f 0.036 1.099 f 0,021 

Relative 3.10 f 0.05 3.13 f 0.063.14 f 0.06 3.22 f 0.09 3.19 f 0.07 3.13 f 0.04 

R. Kidney 

Absolute 1.344 f 0.032 1.309 f 0.0341.351 f 0.023 1.439 f 0.036 1.416 f 0.039 1.464 f 0,044 

Relative 3.73 f 0.05 3.67 f 0.063.76 f 0.06 4.04 f 0.07** 3.95 f 0.06 4.16 f 0.08** 

Liver 

Absolute 13.664 f 0.535 13.245 f 0.337 14.108 f 0.491 15.266 f 0.480 15.560 f 0.507* 16.577 f 0.485** 

Relative 37.82 f 0.83 37.18 f 0.49 39.17 f 1.00 42.85 f 1.10** 43.39 f 0.74** 47.16 f 1.06** 

Lung 

Absolute 1.916 f 0.085 1.940 f 0.1141.933 f 0.138 1.847 f 0.087 1.745 f 0.063 1.741 f 0.109 

Relative 5.34 f 0.28 5.46 f 0.335.40 f 0.43 5.19 f 0.24 4.88 f 0.17 4.97 f 0.33 

R.Testis 

Absolute 1.481 f 0.029 1.511 f 0.0311.488 f 0.025 1.502 f 0.030 1.525 f 0.042 1.517 f 0.029 

Relative 4.11 f 0.05 4.25 f 0.05 4.14 f 0.06 4.22 f 0.06 4.26 f 0.10 4.32 f 0.08 

Thymus 

Absolute 0.330 f 0.014 0.414 f 0.019** 0.346 f 0.010 0.349 f 0.018 0.324 f 0.016 0.335 f 0.017 

Relative 0.92 f 0.04 1.16 f 0.04** 0.96 f 0.03 0.98 f 0.04 0.90 f 0.03 0.95 f 0.05 
Organ Weight Analyses 271 

TABLEF1 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 13-Week Feed Studyof Phenolphthalein 

(continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

Female 

n 10 10 10 10 10 10 

Necropsy body wt 210 f 3 207 f 4 212 f 3 205 f 3 202 f 3 198 f 3* 

Brain 

Absolute 1.875 f 0.017 1.885 f 0.015 1.881 f 0.019 1.861 f 0.013 1.843 f 0.016 1.816 f 0.043 

Relative 8.96 f 0.08 9.12 f 0.19 8.88 f 0.08 9.10 f 0.10 9.13 f 0.10 9.16 f 0.15 

Heart 

Absolute 0.720 f 0.015 0.728 f 0.021 0.728 f 0.021 0.722 f 0.017 0.722 f 0.014 0.710 f 0.016 

Relative 3.44 f 0.05 3.51 f 0.08 3.44 f 0.09 3.53 f 0.08 3.57 f 0.07 3.58 f 0.05 

R.Kidney 

Absolute 0.806 f 0.016 0.798 f 0.014 0.817 f 0.027 0.809 f 0.013 0.807 f 0.022 0.800 f 0.022 

Relative 3.85 f 0.07 3.86 f 0.08 3.85 f 0.10 3.95 f 0.05 3.99 f 0.08 4.04 f 0.10 

Liver 

Absolute 6.911 f 0.122 7.055 f 0.214 7.110 f 0.196 7.315 f 0.336 7.423 f 0.214 7.244 f 0.179 

Relative 32.98 f 0.33 34.07 f 0.93 33.52 f 0.66 35.66 f 1.35 36.68 f 0.73* 36.54 f 0.62* 

Lung 

Absolute 1.385 f 0.050 1.224 f 0.030 1.445 f 0.103 1.273 f 0.039 1.199 f 0.045 1.247 f 0.070 

Relative 6.64 f 0.30 5.92 f 0.17 6.80 f 0.43 6.22 f 0.20 5.93 f 0.22 6.29 f 0.32 

Thymus 

Absolute 0.346 f 0.013 0.322 f 0.014 0.339 f 0.015 0.299 f 0.016 0.334 f 0.016 0.292 f 0.013* 

Relative 1.66 f 0.07 1.56 f 0.08 1.60 f 0.06 1.46 f 0.08 1.65 f 0.06 1.47 f 0.06 

* Significantly different (P SO.05) from the control group by Dunnett’s test 

** PSO.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight 

(mean f standard error). 
272 Phenolphthalein,NTP TR 465 

TABLEF2 

Organ Weights and Organ-Weight-to-Body-Weight Ratiosfor Mice in the 13-Week Feed Study of Phenolphthaleina 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

Male 

n 10 10 10 10 10 10 

Necropsy body wt 28.8 f 0.8 27.4 f 0.5 28.4 f 0.6 28.5 f 0.6 29.3 f 0.8 28.0 f 0.6 

Brain 

Relative 

Absolute 

16.52 f 0.32 

0.474 f 0.006 

17.52 * 0.42 

0.479 f 0.013 

17.03 f 0.39 

0.481 f 0.006 

16.85 f 0.32 

0.479 f 0.005 

16.91 f 0.74 

0.494 f 0.022 

16.74 f 0.31 

0.468 f 0.005 

Heart 

Absolute 0.155 f 0.005 0.149 f0.007 0.145 f 0.002 0.151 f 0.004 0.149 f 0.004 0.144 f 0.004 

Relative 5.39 f 0.09 5.46 f 0.26 5.13 f 0.10 5.30 f 0.12 5.11 f 0.11 5.14 f 0.11 

R. Kidney 

Absolute 0.307 f 0.01 1 0.292 f 0.009 0.337 f 0.026 0.301 f 0.009 0.331 f 0.014 0.297 f 0.008 

Relative 10.61 f 0.17 10.65 f 0.18 11.91 f 0.99 10.55 f 0.23 11.33 f 0.49 10.60 f 0.21 

Liver 

Absolute 1.435 f 0.046 1.511 f 0.043 1.503 f 0.053 1.4660.069 1.580 f 0.054 1.528 f 0.046 

Relative 49.91 f 1.60 55.26 f 1.26* 52.88 f 1.09 51.24 f 1.59 53.97 f 1.21 54.54 f 1.31 

Lung 

Absolute 0.236 f 0.020 0.219 f 0.012 0.237 f 0.014 0.204 f 0.013 0.207 f 0.009 0.215 f 0.011 

Relative 8.16 f 0.63 8.06 f 0.50 8.39 f 0.56 7.18 f 0.47 7.05 f 0.17 7.69 f 0.42 

R.Testis 

Absolute 0.121 f 0.003 0.112 f 0.002** 0.066 f 0.002** 0.063 f 0.002** 0.077 f 0.001** 0.079 f 0.002** 

Relative 4.21 f 0.05 4.11 f 0.13 2.34 f 0.06** 2.22 f 0.10** 2.63 f 0.07** 2.82 f 0.06** 

Thymus 

Absolute 0.046 f 0.003 0.041 f 0.003 0.042 f 0.003 0.039 f 0.003 0.041 f 0.002 0.040 f 0.002 

Relative 1.63 f 0.16 1.51 f 0.11 1.49 f 0.10 1.36 f 0.11 1.42 f 0.09 1.41 f 0.05 
Organ Weight Analyses 273 

TABLEF2 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Feed Studyof Phenolphthalein 

(continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

Female 

n 10 10 10 10 10 10 

Necropsy body wt 23.8 f 0.7 25.0 f 0.8 25.3 f 0.4 25.0 f 0.5 25.0 f 0.5 24.8 f 0.5 

Brain 

Absolute 0.474 f 0.006 0.486 f 0.006 0.481 f 0.005 0.493 f 0.008 0.499 f 0.008* 0.478 f 0.004 

Relative 20.06 f 0.49 19.59 f 0.44 19.08 f 0.32 19.74 f 0.38 20.02 f 0.44 19.31 f 0.34 

Heart 

Absolute 0.127 f 0.004 0.136 f 0.003 0.132 f 0.005 0.132 f 0.003 0.134 f 0.004 0.129 f 0.004 

Relative 5.34 f 0.14 5.48 f 0.11 5.22 f 0.14 5.29 f 0.14 5.37 f 0.15 5.21 f 0.18 

R.Kidney t 

Absolute 0.205 f 0.005 0.203 f 0.009 0.221 f 0.006 0.207 f 0.004 0.222 f 0.004 0.218 f 0.005 

Relative 8.68 f 0.28 8.14 f 0.21 8.74 f 0.15 8.27 f 0.14 8.91 f 0.17 8.77 f 0.16 

Liver 

Absolute 1.259 f 0.034 1.287 f 0.044 1.421 f 0.035* 1.322 f 0.049 1.392 f 0.040 1.396 f 0.046 

Relative 53.12 f 1.11 51.72 f 1.46 56.30 f 1.24 52.81 f 1.48 55.76 f 1.18 56.27 f 1.48 

Lung 

Absolute 0.206 f 0.012 0.209 f 0.014 0.231 f 0.020 0.220 f 0.008 0.214 f 0.010 0.214 f 0.013 

Relative 0.51 8.71 8.42 f 0.58 9.10 f 0.72 8.79 f 0.31 8.59 f 0.41 8.60 f 0.45

Thymus 

Absolute 0.050 f 0.004 0.056 f 0.005 0.051 f 0.003 0.048 f 0.003 0.053 f 0.003 0.053 f 0.003 

Relative 2.07 f 0.12 2.24 f 0.20 2.03 f 0.12 1.91 f 0.12 2.13 f 0.13 2.15 f 0.15 

* Significantly different (PSO.05) from the control group by Dunnett’s test 

** P-zO.01 

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weightlg body weight 

(mean f standard error). 
Phenolphthalein, NTP TR 465 
275 

RESULTS 

AND CLINICAL CHEMISTRY 

HEMATOLOGY 

APPENDIX G 

TABLEG1 HematologyandClinicalChemistryData for Rats in the 13-WeekFeedStudy 

of Phenolphthalein . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 276 
276 Phenolphthalein,NTP TR 465 

TABLEG1 

Hematology and Clinical Chemistry Data for Rats in the 13-Week Feed Study of Phenolphthaleina 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

Male 

n 10 10 10 10 10 10

Hematology 

Hematocrit (%) 

Day 5 38.3 f 1.2 40.1 f 0.9 39.2 f 0.6 39.4 f 0.7 38.8 f 0.9 40.6 f 0.8 

Day 21 43.1 f 0.8 40.5 f 1.1 43.1 f 0.9 44.1 f 0.7 43.5 f 0.5 43.1 f 1.1 

Week 13 44.1 f 0.5 43.2 f 0.6 44.4 f 0.4 43.9 f 0.4 43.3 f 0.7 43.5 f 0.6 

Hemoglobin (gldL) 

Day 5 14.5 f 0.2 14.5 f 0.2 14.4 & 0.2 14.4 f 0.2 14.5 f 0.2 14.6 f 0.1 

Day 21 15.7 f 0.1 15.6 f 0.1 15.7 f 0.1 15.8 f 0.1 15.5 f 0.1 15.6 f 0.1 

Week 13 15.9 f 0.2 15.5 f 0.2 16.0 f 0.4 16.2 f 0.4 15.5 f 0.2 15.6 f 0.2 

Erythrocytes (106/pL) 

Day 5 6.50 f 0.22 6.89 f 0.17 6.66 f 0.12 6.56 f 0.11 6.76 f 0.14 6.83 f 0.12 

Day 21 7.97 f 0.17 7.49 f 0.23 7.89 f 0.16 8.01 f 0.14 ,8.00f 0.11 7.78 f 0.20 

Week 13 9.17 f 0.11 8.90 f 0.19 9.13 f 0.10 9.04 f 0.07 8.84 f 0.15 8.93 f 0.13 

Reticulocytes (106/pL) 

Day 5 0.25 f 0.02b 0.29 f 0.03b 0.24 f 0.02b 0.23 f 0.03 0.22 f 0.02 0.23 f 0.03b 

Day 21 0.15 f 0.02' 0.14 f O.Olb 0.15 f O.Old 0.17 f O.Olb 0.14 f O.Olb 0.16 f 0.01' 

Week 13 0.13 f O.Ole 0.14 f O.Olf 0.13 f O.Old 0.12 f O.Olf 0.11 f 0.01f 0.15 f O.Olf 

Mean cell volume (tL) 

Day 5 59.1 f 1.0 58.1 f 0.4 59.0 f 0.6 60.1 f 0.6 57.4 f 0.9 59.4 f 0.9 

Day 21 54.2 f 0.3 54.0 f 0.3 54.8 f 0.3 54.8 f 0.3 54.4 f 0.4 55.3 f 0.4 

Week 13 48.2 f 0.3 48.8 f 0.5 48.4 f 0.3 48.6 f 0.3 49.0 f 0.2 48.7 f 0.4 

Mean cell hemoglobin (pg) 

Day 5 22.5 f 0.7 21.1 f 0.4 21.6 f 0.4 22.0 f 0.3 21.5 f 0.3 21.5 f 0.3 

Day 21 19.7 f 0.4 20.9 f 0.6 20.0 f 0.4 19.7 f 0.2 19.4 f 0.2 20.2 f 0.4 

Week 13 19.1 f 1.8 17.4 f 0.2 17.5 f 0.4 18.0 f 0.4 17.6 f 0.2 17.5 f 0.1 

Mean cell hemoglobin concentration (g/dL) 

Day 5 38.2 f 1.2 36.2 f 0.5 36.8 f 0.6 36.6 f 0.5 37.5 f 0.8 36.2 f 0.6 

Day 21 36.4 f 0.5 38.7 f 1.0 36.5 f 0.8 35.7 f 0.4 35.7 f 0.3 36.4 f 0.7 

Week 13 36.1 f 0.3 35.8 f 0.3 36.1 f 0.8 37.0 f 1.0 35.8 f 0.3 35.9 f 0.2 

Platelets (10~1~~) 

Day 5 998.2 f 18.5 967.8 f 18.4 915.8 f 23.7 898.3 f 46.3* 907.1 f 21.7* 916.4 f 25.2 

Day 21 760.7 f 13.0 814.0 f 13.3 778.7 f 8.2 791.8 f 18.1 817.8 f 13.3* 848.3 f 6.9** 

Week 13 608.1 f 11.7 665.3 f 14.8 645.2 f 9.7 709.3 f 22.4** 707.8 f 62.9* 684.9 f 11.9** 

Leukocytes (103/pL) 

Day 5 6.58 f0.28 5.55 f 0.26 6.08 f 0.35 6.15 0.45 5.72 f0.39 6.73 f 0.38 

Day 21 7.41 f 0.43 7.22 f 0.26 7.14 f 0.20 6.55 f 0.28 7.03 f 0.42 7.78 f 0.31 

Week 13 6.03 f 0.42 6.43 f 0.38 6.42 f 0.28 6.98 f 0.63 6.31 f 0.38 6.39 f 0.30 

Segmented neutrophils (103/pL) 

Day 5 0.74 f 0.08 0.81 f 0.11 0.82 f 0.11 0.69 f 0.07 0.60 f 0.04 0.85 f 0.08 

Day 21 0.95 f 0.09 1.00 f 0.12 1.00 f 0.08 0.81 f 0.13 0.68 f 0.08 0.81 f 0.09 

Week 13 1.21 f 0.07 1.14 f 0.17 1.08 f 0.13 1.34 f 0.16 1.03 f 0.11 1.19 f 0.13 

Lymphocytes (103/pL) 

Day 5 5.71 f 0.22 4.58 f 0.23* 5.07 f 0.26 5.31 f 0.46 4.95 f 0.37 5.73 f 0.40 

Day 21 6.33 f 0.39 6.16 f 0.23 6.06 f 0.23 5.67 f 0.27 6.26 f 0.35 6.87 f 0.26 

Week 13 4.63 f 0.37 5.12 f 0.25 5.19 f 0.34 5.44 f 0.53 5.13 f 0.33 5.04 f 0.26 
Hematology and Clinical Chemistry 277 

TABLEG1 

Hematology and Clinical Chemistry Data for Rats in the 13-Week Feed Study of Phenolphthalein (continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

Male (continued) 

n 10 10 10 10 10 10 

Hematology (continued) 

Monocytes (103/pL) 

Day 5 0.11 f 0.04 0.15 f 0.03 0.19 f 0.03 0.11 f 0.03 0.16 f 0.05 0.15 f 0.04 

Day 21 0.09 f 0.02 0.05 f 0.02 0.05 f 0.02 0.08 f 0.01 0.08 f 0.04 0.09 f 0.03 

Week 13 0.11 f 0.02 0.14 f 0.05 0.13 f 0.02 0.15 f 0.03 0.11 f 0.02 0.11 f 0.04 

Eosinophils (103/pL) 

Day 5 0.04 f 0.02 0.04 f 0.02 0.03 f 0.02 0.03 f 0.02 0.03 f 0.02 0.01 f 0.01 

Day 21 0.03 f 0.02 0.01 f 0.01 0.02 f0.01 0.01 f 0.01 0.01 f 0.01 0.02 f 0.01 

Week 13 0.08 f 0.02 0.06 f 0.02 0.05 f 0.02 0.07 f 0.02 0.06 f 0.02 0.09 f 0.01 

n 10 10 10 10 10 10 

Clinical Chemistry 

Urea nitrogen (mg/dL) 

Day 5 19.0 f 0.6 18.8 f 0.5 18.3 f 0.4 17.5 f 0.7 18.3 f 0.4 18.0 f 0.6 

Week 13 

Day 21 

20.3 f 0.5 

20.8 f 0.4 

20.1 f 0.6 

20.9 f 0.3 

19.6 f 0.8 

21.5 f0.4 

21.4 f 0.6 

21.9 f 0.5 

19.7 f 0.9 

21.9 f 1.1 

21.0 f 0.7 

22.8 f 0.6 

Creatinine (mgldL) 

Day 5 0.60 f 0.03 0.62 f 0.03 0.60 f 0.02 0.57 f 0.03 0.55 f 0.02 0.55 f 0.02 

Week 13 

Day 21 

0.81 * 0.07 

0.64 f 0.03 

0.72 f 0.05 

0.56 f 0.05 

0.66 f 0.04 

0.58 f 0.03 

0.60 f 0.02** 

0.61 f 0.02 

0.65 f 0.03 

0.59 f 0.03b 

0.64 f 0.03* 

0.58 f 0.02 

Total protein (g/dL) 

Day 5 6.4 f 0.1 6.4 f 0.1 6.4 f 0.1 6.3 f 0.1 6.2 f 0.1 6.2 f 0.1 

Week 13 

Day 21 

7.5 f 0.3 

7.1 f 0.1 

7.6 f 0.2 

7.1 f 0.1 

7.6 f 0.1 

7.2 f 0.2 

7.9 f 0.1 

7.4 f 0.1 

8.2 f 0.2 

7.3 f 0.1 

7.9 f 0.3 

7.5 f 0.1 

Day 5 

Albumin (gldL) 

4.2 f 0.2 4.3 f 0.0 4.3 f 0.0 4.3 f 0.1 4.2 f 0.1 4.2 f 0.1 

Week 13 

Day 21 

4.7 f 0.1 

4.5 f 0.0 

4.6 f 0.1 

4.5 f 0.1 

4.7 f 0.1 

4.6 f 0.1 

4.8 f 0.1 

4.7 f 0.1 

4.9 f 0.1 

4.6 f 0.1 

4.9 f0.1 

4.7 f 0.1 

Alanine aminotransferase (IU/L) 

Day 5 32 f 1 30 f 1 31 f 1 34 f 1 33 f 1 39 f 1' 

Week 13 

Day 21 

45 f 2 

37 f 2 

37 f 2 

41 f 3 

36 * 2 

37 f 2 

43 f 3 

37 f 1 

33 f 2** 

39 f 2 

43 f 6 

42 f 2 

Day 5 541 f 9 

Alkaline phosphatase (IUIL) 

565 f 14 550 f 6 556 f 9 535 f 12 557 f 13 

Day 21 376 f 10 379 f 16 379 f 5 386 f 7b 355 f 11 366 f 9 

Week 13 185 f 7 173 f 5 184 f 10 181 f 4 184 f 9 188 f 6 

Day 5 

Creatine kinase (IU/L) 

178 f 25 228 f 51 216 2 53 319 f 83 197 f 59 177 f 35 

Week 13 

Day 21 

212 f 40 

152 f 18 

134 f 17 

172 f 30 

148 f 19 

139 f 14 

223 f 43 

152 f 15b 

172 f 20 

158 f 21 

168 f 30 

132 f 9 

Sorbitol dehydrogenase (IUIL) 

Day 5 8f1 9fl 92.1 11 f 1 7f1 7f1 

Week 13 

Day 21 

8f.1 

14 f 2 

8fl 

9f1 

9f1 

10 f 1 

10 f 1 

11 f 1 

8fl 

10 f 1 

11 f2 

9 f 1* 

Week 13 

Day 21 

Day 5 

Bile acids (pmollL) 

20.8 * 8.3 

8.9 * 1.3 

25.2 f 4.1 17.5 f 3.7 

5.3 f 0.6* 

18.6 f 8.7 

5.3 f 0.4* 

8.3 f 0.7** 

14.8 f 6.9 19.9 f 7.4b 

9.1 f 3.2b 

9.2 f 2.0** 

23.0 f 8.4 

10.0 f 1.1 

5.0 f 0.8*b 6.2 f 0.3b 

8.9 f 0.9** 

19.5 f 9.6 
278 Phenolphthalein, NTP TR 465 

TABLEG1 

Hematology and Clinical Chemistry Data for Rats in the 13-Week Feed Study of Phenolphthalein (continued) 

0 PPm 3,000 ppm 6,000 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

Female 

n 10 10 10 9 10 10 

Hematology 

Hematocrit (%) 

Day 5 39.8 f 1.0 39.6 f 0.7 39.6 f 0.6 40.2 f 1.2 39.8 f 0.9 39.6 f 1.1 

Day 21 41.3 f 0.5 43.5 f 0.6 42.5 f 0.7 42.2 f 1.2 43.0 f 0.6 42.8 f 0.7 

Week 13 44.1 f 0.3 44.1 f 0.3 43.7 f 0.6 43.0 f 0.8 43.6 f 0.5 44.3 f 0.4 

Hemoglobin (gldL) 

Day 5 15.1 f 0.2 15.1 f 0.2 15.2 f 0.1 15.1 f 0.2 15.2 f 0.2 15.0 f 0.2 

Day 21 15.9 f 0.2 15.9 f 0.1 16.1 f 0.2 15.8 f 0.2 16.0 f 0.1 15.6 f 0.1 

Week 13 16.3 f 0.2 15.9 f 0.2 16.1 f 0.2 16.2 f 0.2 16.1 f 0.3 16.6 f 0.4 

Elythrocytes (106/pL) 

Day 21 

Day 5 

7.64 f 0.09 

7.05 f 0.23 

8.02 f 0.11 

7.01 f 0.13 

7.81 f 0.16 

7.00 f 0.11 

7.75 f 0.23 

7.08 f 0.23 

7.90 f 0.12 

7.12 f 0.19 

7.89 f 0.11 

6.98 f 0.18 

Week 13 8.61 f 0.08 8.60 f 0.07 8.54 f 0.12 8.36 f 0.18 8.50 f 0.12 8.61 f 0.08 

Reticulocytes (106/pL) 

Day 21 

Day 5 0.19 f 0.02 

0.12 f 0.01' 0.14 f 0.01' 

0.19 f 0.01 

0.13 f 0.01' 

0.18 f 0.02b 

0.13 f O.Old 

0.16 f 0.01' 

0.14 f 0.01' 

0.16 f O.Olb 

0.13 f 0.01' 

0.14 f 0.02' 

Week 13 0.13 f O.Olf 0.12 f 0.01f 0.12 f 0.01e 0.12 f 0.01g 0.12h 0.12 f Omf 

Mean cell volume (eL) 

Week 13 

Day 21 

Day 5 56.7 f 0.6 

51.1 f 0.2 

54.2 f 0.3 

51.2 f 0.3 

54.2 f 0.3 

56.5 f 0.2 

51.0 f 0.3 

54.4 f 0.4 

56.9 f 0.7 

51.4 f 0.2 

54.4 f 0.3 

56.8 f 0.4 

51.2 f 0.2 

54.2 f 0.3 

56.0 f 0.3 

51.3 f 0.2 

54.4 f 0.3 

56.6 f 0.7 

Mean cell hemoglobin (pg) 

Week 13 

Day 21 

Day 5 

18.9 f 0.2 

20.8 f 0.3 

21.6 f 0.6 

18.5 f 0.1 

19.9 f 0.3 

21.5 f 0.3 

18.8 f 0.1 

20.7 f 0.3 

21.8 f 0.3 

19.4 f 0.5 

20.6 f 0.7 

21.4 f 0.7 

18.9 f 0.3 

20.3 f 0.3 

21.4 f 0.4 

19.2 f 0.4 

19.8 f 0.3 

21.6 f 0.4 

Week 13 

Day 21 

Day 5 

36.9 f 0.4 

38.5 f 0.5 

38.1 f 0.8 

Mean cell hemoglobin concentration (gldL) 

36.0 f 0.3 

36.6 f 0.5* 

38.1 f 0.5 

36.7 f 0.2 

38.0 f 0.4 

38.5 f 0.4 

37.7 f 0.9 

37.8 f 1.2 

37.7 f 1.1 

37.0 f 0.6 

37.3 f 0.5 

38.2 f 0.6 

37.4 f 0.9 

36.5 f OS* 

38.0 f 0.8 

Week 13 

Day 21 

Day 5 

Platelets (1031p~) 

638.7 f 14.6 

721.3 f 10.2 

856.0 f 18.1 

652.1 f 10.0 

748.2 f 15.2 

863.0 f 12.2 

673.2 f 9.5 

711.3 f 26.3 

870.5 f 13.4 

672.2 f 8.6 

741.7 f 12.9 

867.0 f 11.7 

656.6 f 14.4 

746.5 f 8.5 

841.0 f 16.2 

673.1 f 14.2 

748.4 f 13.6 

823.1 f 17.6 

Week 13 

Day 21 

Day 5 

Leukocytes (103/pL) 

5.40 f 0.33 

6.11 f 0.20 

6.14 f 0.21 

5.52 f 0.22 

6.21 f 0.18 

6.17 f 0.30 

5.46 f 0.31 

6.70 f 0.28 

6.54 f 0.22 

6.18 f0.27' 

6.53 f 0.44' 

5.75 f 0.46' 

5.44 f 0.26 

7.49 f 0.26** 

6.07 f 0.18 

5.68 f0.42 

6.92 f 0.40 

5.91 f 0.29 

Week 13 

Day 21 

Day 5 

1.04 f 0.15 

0.57 f 0.03 

0.67 f 0.05 

Segmented neutrophils (103/pL) 

0.87 f 0.11 

0.72 f 0.06 

0.68 f 0.07 

1.11 f 0.16 

0.78 f 0.13 

0.65 f 0.06 

0.78 f 0.08' 

0.58 f 0.08' 

0.61 f 0.06' 

1.07 f 0.14 

0.93 f 0.07** 

0.69 f 0.07 

0.88 f 0.10 

0.70 f 0.08 

0.72 f 0.09 

Week 13 

Day 21 

Day 5 

Lymphocytes (1 03/pL) 

4.19 f 0.29 

5.43 f 0.20 

5.34 

*0.19 

4.53 f 0.20 

5.44 f 0.20 

5.24 f 0.28 

4.21 f 0.26 

5.80 f 0.22 

5.70 f 0.16 

5.30 f 0.27' 

5.85 f 0.41' 

4.99 f 0.40' 

4.21 f 0.22 

6.47 f 0.23* 

5.25 f 0.20 

4.66 f 0.37 

6.14 f 0.34 

5.05 f 0.28 
Hematology and Clinical Chemistry 279 

TABLEG1 

Hematology and Clinical Chemistry Data for Rats in the 13-Week Feed Study of Phenolphthalein (continued) 

3,000 ppm 6,000 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

Female (continued) 

n 10 10 10 9 10 10 

Hematology (continued) 

Monocytes (103/pL) 

Day 5 0.12 f 0.03 0.18 f 0.04 0.16 f 0.04 0.14 f 0.04' 0.11 f 0.03 0.07 f 0.02 

Day 21 0.09 f 0.03 0.08 f 0.02 0.10 f 0.02 0.09 f 0.02' 0.09 f 0.03 0.09 f 0.03 

Week 13 0.13 f 0.04 0.09 f 0.03 0.11 f 0.03 0.10 f 0.03' 0.12 f 0.04 0.11 f 0.03 

Eosinophils (103/pL) 

Day 5 0.04 f 0.02 0.06 f 0.02 0.06 f 0.02 0.03 f 0.02' 0.03 f 0.02 0.07 f 0.02 

Day 21 0.03 f 0.02 0.01 f 0.01 0.04 f 0.02 0.05 f 0.02' 0.01 f 0.01 0.01 f 0.01 

Week 13 0.04 f 0.02 0.04 f 0.02 0.06 f 0!02 0.03 f 0.02' 0.03 f 0.02 0.04 f 0.02 

n 

Day 5 10 10 9 9 10 10 

Day 21 10 10 10 9 10 10 

Week 13 10 10 10 9 10 10 

Clinical Chemistry 

Urea nitrogen (mg/dL) 

Day 5 21.1 f 0.7 18.6 f 0.5 19.9 f 0.5 18.5 f 0.6* 20.0 f 1.2 18.2 f 0.5** 

Day 21 21.5 f 0.7 20.4 f 0.6 20.4 f 0.5 20.8 f 0.5 22.0 f 0.5 21.3 f 0.5 

Week 13 19.0 f 0.8 20.5 f 0.9 19.7 f 0.9 20.1 f 1.1 20.2 f 0.7 19.5 f 0.8 

Creatinine (mgldL) 

Day 5 0.55 f 0.01 0.56 f 0.02 0.62 f 0.02 0.55 f 0.03 0.57 f 0.02 0.54 f 0.02 

Day 21 0.58 f 0.03 0.61 f 0.04 0.64 f 0.03 0.60 f 0.02 0.61 f 0.03 0.60f 0.03 

Week 13 0.71 f 0.02 0.70 f 0.02 0.74 f 0.02 0.68 f 0.03 0.70 f 0.02 0.67 f 0.02 

Total protein (gldL) 

Day 5 6.3 f 0.1 6.1 f 0.1 6.2 f 0.1 6.2 f 0.1 6.2 f 0.1 6.1 f 0.1 

Day 21 7.0 f 0.1 7.0 f 0.1 7.2 f 0.2 7.0 f 0.1 7.1 f 0.1 6.9 f O.lb 

Week 13 8.0 f 0.1 8.0 f 0.2 7.8 f 0.2 7.6 f 0.2 8.1 f 0.1 8.1 f 0.2 

Day 5 

Albumin (g/dL) 

4.6 f 0.1 4.4 f 0.1* 4.4 f 0.1 4.4 f 0.1 4.4 f 0.0 4.3 f o.o* 

Day 21 4.6 f 0.1 4.5 f 0.0 4.6 f 0.1 4.6 f 0.1 4.6 f 0.1 4.5 f 0.1 

Week 13 5.0 f 0.1 5.0 f 0.1 4.8 f 0.2 4.9 f 0.1 5.1 f 0.1 5.0 f 0.1 

Alanine aminotransferase (IU/L) 

Day 5 30 f 1 26 f 1 29 f 1 26 f 2 31 f 1 29 f 1 

Day 21 33 f 1 33 f lb 32 f 2 30 f 1 33 f 1 35 f 1 

Week 13 37 f 2 36 f 1 37 f 2 28 f 2 29 f 1 28 f 1* 

Alkaline phosphatase (IU/L) 

Day 5 441 f 11 420 f 9 428 f 11 420 f 12 432 f 14 405 f 9 

Day 21 294 f 9 294 f 14 300 f 12 294 f 11 295 f 6 293 f 6 

Week 13 159 f 7 160 f 5 159 f 7 148 f 8 162 f 6 148 f 6 

Creatine kinase (IUIL) 

Day 5 153 f 16 142 f 11 216 f 74 154 f 38 168 f 24 114 f 11 

Day 21 219 f 64 153 f 16 163 f 22 152 f 39 127 f 11 117 f 11 

Week 13 169 f 20 136 f 23 113 f 14 215 f 84 171 f 20 198 f 64 
280 Phenolphthalein, NTP TR 465 

TABLEG1 

Hematology and Clinical Chemistry Data for Rats in the 13-Week Feed Study of Phenolphthalein (continued) 

Female (continued) 

n 

Week 13 

Day 21 

Day 5 

10 

10 

10 

10 

10 

10 

10 

10 

9 

9 

9 

9 

10 

10 

10 

10 

10 

10 

Clinical chemistry (continued) 

6f0 

Sorbitol dehydrogenase (IU/L) 

Week 13 

Day 21 

Day 5 

Week 13 

Day 21 

Day 5 

7f1 8fl

9f2 

6f1 

21.3 f 4.9 

11.8f 2.4b 

23.5 f 5.2 

Bile acids (pmol/L) 

12 f 2 

11.9f 1.3 

7.5 f 1.0 

20.0 f 3.7 

8*0 

12 f 2 

17.3f 3.8 

9.7f 1.2 

20.9f 6.9 

7*1 

7f1 

11 f2 

9.3 f 1.3* 

8.6 f 1.5 

15.9 f 5.6 

11 f 1 

13.9 f 2.1 

9.4f 0.7 

21.6 f 9.1 

7f.1 

7f1 

7f0 

12 f 1 

10.4 f 2.2 

8.8f 1.0 

23.6 f 7.9 

a Mean f standard error. Statisticaltests were performed on unroundeddata. 

** PSO.01 

* Significantly different (P50.05) fromthecontrolgroupby Dum’s test 

n=7 

n=8 

n=9 

g n=4 

n=3 

e n=5 

n= 1: no standard error calculated 
281 

APPENDIX H 

DETERMINATIONS 

OF TOTAL PHENOLPHTHALEIN 

IN PLASMA 

TABLE H1 Plasma Concentrations and Estimated Area Under the Curve Values 

of Total Phenolphthalein in Rats in the 2-Year Feed Study 

of Phenolphthalein ............................................ 282 

TABLEH2 Plasma Concentrations and Estimated Area Under the Curve Values 

of Total Phenolphthalein in Mice in the 2-Year Feed Study 

of Phenolphthalein ............................................ 283 

FIGURE H1 Plasma Concentrations of Total Phenolphthalein in Male Rats After Exposure 

to 12,000, 25,000, or 50,000 ppm in Feed for 2 Years ..................... 284 

FIGURE H2 Plasma Concentrations of Total Phenolphthalein in Female Rats After Exposure 

to 12,000, 25,000, or 50,000 ppm in Feed for 2 Years ..................... 284 

FIGURE H3 Plasma Concentrations of Total Phenolphthalein in Male and Female Rats 

After Exposure to 12,000 ppm in Feed for 2 Years ....................... 285 

FIGURE H4 Plasma Concentrations of Total Phenolphthalein in Male and Female Rats 

After Exposure to 25,000 ppm in Feed for 2 Years ....................... 285 

FIGURE H5 Plasma Concentrations of Total Phenolphthalein in Male and Female Rats 

After Exposure to 50,000 ppm in Feed for 2 Years ....................... 286 

FIGURE H6 Plasma Concentrations of Total Phenolphthalein in Male Mice After Exposure 

to 3,000, 6,000, or 12,000 ppm in Feed for 2 Years ....................... 287 

FIGURE H7 Plasma Concentrations of Total phenolphthalein in Female Mice After Exposure 

to 3,000, 6,000, or 12,000 ppm in Feed for 2 Years ....................... 287 

FIGURE H8 Plasma Concentrations of Total Phenolphthalein in Male and Female Mice 

After Exposure to 3,000 ppm in Feed for 2 Years ........................ 288 

FIGURE H9 Plasma Concentrations of Total Phenolphthalein in Male and Female Mice 

After Exposure to 6,000 ppm in Feed for 2 Years ........................ 288 

FIGURE H10 Plasma Concentrations of Total Phenolphthalein in Male and Female Mice 

After Exposure to 12,000 ppm in Feed for 2 Years ....................... 289 
282 Phenolphthalein, NTP TR 465 

TABLEH1 

Plasma Concentrations and Estimated Area Under the Curve Values of Total Phenolphthalein 

in Rats in the 2-Year Feed Study of Phenolphthaleina 

12,000 ppm 25,000 ppm 50,000 ppm 

n 3 3 3 

Male 

Plasma Concentrationsb 

6 a.m. 107.80 f 32.81' 127.00 f 72.58 177.23 f 185.33 

9 a.m. 102.43 f 8.79 63.50 f 50.79 73.20 f 24.91 

1 p.m. 59.63 f 2.54 34.45 f 15.77' 157.47 f 181.13 

4 p.m. 57.40 f 9.93 101.23 * 86.08 224.40 f 232.78' 

9 p.m. 69.17 f 10.18 59.53 f 16.70 80.85 f 13.08' 

Area Under the Curve 

6 a.m.-9 p.m. 1,131 f 49 1,087 f 217 2,173 f 587 

Female 

Plasma Concentrations 

6 a.m. 94.13 f 25.01 90.10 f 12.05 104.23 f 32.87 

9 a.m. 92.77 f 23.48 110.10 41.79 108.33 f 3.51 

1 p.m. 68.17 f 9.92 101.37 f 13.45 104.07 f 34.28 

4 p.m. 97.50 f 32.15 72.83 f 23.25 78.37 f 17.45 

9 p.m. 69.23 f 12.96 110.77 f 40.25 83.30 f 10.38 

Area Under the Curve 

6 a.m.-9 p.m. 1,268 f 94 1,444 f 125 1,422 f 81 

a Data are presented as mean f standard deviation. Plasma concentrations are given in pglmL. Area under the curve values are given 

as pg-hourlml. Total phenolphthalein equals free and conjugated phenolphthalein. 

Samples were collected on the last day of the study. 

n=2 
Determinations of Total Phenolphthalein in Plasma 283 

TABLEH2 

Plasma Concentrations and Estimated Area Under the Curve Values of Total Phenolphthalein 

in Mice in the 2-Year Feed Study of Phenolphthaleina 

n 

Male 

Plasma Concentrations” 

6 a.m. 

9 a.m. 

1 p.m. 

4 p.m. 

9 p.m. 

Area Under the Curve 

6 a.m.-9 p.m. 

Female 

Plasma Concentrations 

6 a.m. 

9 a.m. 

1 p.m. 

4 p.m. 

9 p.m. 

Area Under the Curve 

6 a.m-9 p.m. 

3,000 ppm 6,000 ppm 

3 3 

128.67 f 13.50 149.00 f 33.15 

100.47 f 18.49 112.00 f 20.95 

177.00 f 90.54 106.43 f 13.98 

99.67 f 20.98 121.17 f 35.75 

102.03 f 6.94 121.33 f 15.88 

1,818 f 166 1,776 f 106 

112.00 f 57.00 180.63 f 17.86 

130.57 f 10.85 169.77 f 15.77 

127.40 f 32.08 135.93 f 14.59 

101.93 f 8.80 110.97 f 14.62 

134.53 f 32.10 112.27 f 48.05 

1,815 f 103 2.066 f 104 

12,000 ppm 

3 

174.0 f 9.0 

159.0 f 57.5 

105.3 f 17.3 

109.8 f 38.1 

175.7 f 11.4 

2,065 f 135 

233.13 f 34.49 

205.40 f 26.61 

149.93 f 3.90 

175.60 f 31.13 

203.27 f 114.67 

2,804 f 222 

a Data are presented as mean f standard deviation. Plasma concentrations are given in pglmL. Area under the curve values are given 

as pg-hourlml. Total phenolphthalein equals free and conjugated phenolphthalein. ” Samples were collected on the last day ofthe study. 
284 Phenolphthalein, NTP TR 465 

1000 J 

12000p?m 0 

2500Oppm El 

5OOOOppm H 

1 I I I I 

Time of Day FIGUREH1 

Plasma Concentrations of Total Phenolphthalein in Male Rats After Exposure 

to 12,000, 25,000, or 50,000 ppmin Feed for 2 Years 

030 -

-

- 12030pDm 0 -

- 25OOOppm 0 

- 50000ppm 0 

-

-

100, -

-

-

-

-

-

-

10 I 1 1 I I 

1 2 A M8 A M 4AM :2 PM 4 PM 8 PM 12Afvl

Time of Day FIGUREH2 

Plasma Concentrations of Total Phenolphthalein in Female Rats After Exposure 

to 12,000, 25,000, or 50,000 ppm in Feed for 2 Years 
Determinations of Total Phenolphthalein in Plasma 

1 

T T 

1 I 1 I I 

12AM 4AM 8 AM 12 PM 4 PM 8 PM 12Abl

Time of Day FIGURE H3 

Plasma Concentrations of Total Phenolphthalein in Male and Female Rats After Exposure 

to 12,000 ppm in Feed for 2 Years 

1000 1 J

-

- Female,25000ppm 0 -

- Male, 25000 ppm El

1 

- I 1 1 I 1 

12 AM 4 AM 8 AM 12 PM 4 PM 8 PM 12 AM 

FIGURE H4 Time of Day 

Plasma Concentrationsof Total Phenolphthalein in Male and Female Rats After Exposure 

to 25,000 ppm in Feed for 2 Years 
286 Phenolphthalein, NTP TR 465 

1000 -

-

-

- i - T

- c 

-

1oo-I:-

-

-

-

-

-

- Female, 5OGOOppm 0 

Male, 50000ppm I 

10 1 I I I I 

1.2~M 4 AM 8AM !2 Pfd 4 P!d 8 PM 12Ah! 

Time of Day FIGURE H5 

Plasma Concentrations of Total Phenolphthalein in Male and Female Rats After Exposure 

to 50,000 ppm in Feed for 2 Years 
Determinations 287 of Total Phenolphthalein in Plasma 

1000 4 

3000ppm cl 

6000ppm 0 

72000ppm n

! 1 1 I 

12'P.M 4,414 8 AM 12 PM 4 PM 8 PM 12AM 

Time of Day FIGURE H6 

Plasma Concentrationsof Total Phenolphthalein in Male Mice After Exposure 

to 3,000, 6,000, or 12,000 ppm in Feed for 2 Years 

4 nnn 

T T 

1 1 

I 72000ppm 0

Time of Day FIGURE H7 

Plasma Concentrationsof Total Phenolphthalein in Female Mice After Exposure 

to 3,000, 6,000, or 12,000 ppm in Feed for 2 Years 
288 Phenolphthalein, NTP TR 465 

!OOC -

J 

-

-

-

- -

2 

E - -

9 - c 

.-0

+ 

Q, -

r 5

S 

tu 

1007 

t------;

0 -

- c 

0 - 0 - E - v)

m - h Female, 3000ppm 0 

~afe,3000ppm q

10 I I I 1 I 

12AM 48AM 12PM 4PM 8PM 12AM

FIGURE H8 Time of Day 

Plasma Concentrations of Total Phenolphthalein in Male and Female Mice After Exposure 

to 3,000 ppm in Feed for 2 Years 

000 

Time of Day FIGURE H9 

Plasma Concentrations of Total Phenolphthalein in Male and Female Mice After Exposure 

to 6,000 ppm in Feed for 2 Years 
Determinations of Total Phenolphthalein in Plasma 289 

100i 

Female, i2000ppm 0 

Male, 72000ppm 

1 I I 1 I 

Time of Day FIGUREH10 

Plasma Concentrations of Total Phenolphthalein in Male and Female Mice After Exposure 

to 12,000 ppm in Feed for 2 Years 
290 Phenolphthalein, NTF' TR 465 
291 

APPENDIX I 

REPRODUCTIVE TISSUE EVALUATIONS 

AND ESTROUS CYCLE CHARACTERIZATION 

TABLE I1 Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization 

for Rats inthe13-WeekFeedStudyofPhenolphthalein . . .. . . . ... . . . . . . . .. . 292 

TABLE I2 Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization 

for-Miceinthe13-WeekFeedStudyofPhenolphthalein . . . . . . . . . . . . . . . .. . . . 293 
292 Phenolphthalein, NTP TR 465 

TABLEI1 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization for Rats 

in the 13-Week Feed Study of Phenolphthaleina 

Male 

n 

Weights (9) 

Necropsy body wt 

R. cauda 

R. epididymis 

R. testis 

Epididymal spermatozoal parameters 

Motility (%) 

Abnormal (W) 

Concentration 

(106/g cauda epididymal tissue) 

Female 

n 

Necropsy body wt (g) 

Estrous cycle length (days) 

Estrous stages (% of cycle) 

Diestrus 

Proestrus 

Estrus 

Metestrus 

0 PPm 12,000 ppm 25,000 ppm 50,000 ppm 

10 10 10 10 

360 f 6 356 f 6 359 f 10 352 f 6 

0.194 f 0.006 0.198 f 0.0060.203 f 0.005 0.207 f 0.005 

0.448 f 0.007.454 f 0.009.458 f 0.009.452 f 0.009 

1.481 f 0.0291.502 f 0.0301.525 f 0.042 1.517 f 0.029 

61.60 f 2.06 72.35 f 2.20* 67.19 f 2.49 67.84 f 4.23 

0.680 f 0.134 0.760 f 0.107 0.840 f 0.126 0.800 f 0.107 

368 f 14 352 f 32 381 f 18 386 f 22 

10 10 10 10 

210 f 3 205 f 3 202 f 3 198 f 3* 

4.90 f 0.104.60 f 0.164.80 f 0.20 4.70 f 0.15 

40.0 38.6 34.3 40.0 

18.6 20.0 14.3 20.0 

17.1 21.4 28.6 18.6 

24.3 20.0 22.9 21.4 

* Significantly different (P10.05)from the controls by Dunnett’s test (body weight) or by Dum’s test (motility) 

a Weights, epididymal spermatozoal parameters, and estrous cycle length are presented as mean f standard error. Differences from the 

control group for organ weights and estrous cycle length are not significant by Dum’s or Dunnett’s test. By multivariate analysis of 

variance, exposed females did not differ significantly from the control females in relative length of time spent in the estrous stages. 
Sperm Morphology and Vaginal Cytology 293 

TABLEI2 

Summary of Reproductive Tissue Evaluations and Estrous Cycle Characterization for Mice 

in the 13-Week Feed Study of Phenolphthaleina 

12,000 ppm 25,000 ppm 50,000 ppm 

Male 

n 10 10 10 10 

Weights (g) 

Necropsy body wt 28.8 * 0.8 28.5 f 0.6 29.3 f 0.8 28.0 f 0.6

R. cauda 0.022f 0.001 0.018 f 0.001* 0.019 f 0.001 0.019 f 0.001 

R. epididymis 0.049f 0.002 0.038 f 0.001** 0.040 f 0.001** 0.040 * 0.001** 

R. testis 0.121 f0.003 0.063 f 0.002** 0.077f 0.001** 0.079 f 0.002** 

Epididymal spermatozoal parameters 

Motility (%) 78.88f 1.95 19.22 f 1.9079.83 f 1.74 75.37f 1.71 

Abnormal (%) 1.30f0.10 5.74 f 0.63** 4.80 f 0.45** 5.74 f 0.26** 

Concentration 

(106/g cauda epididymal tissue) 590 f 54 349 f 33*417 f 50 288 f 32** 

Female 

n IO 10 10 10 

Necropsy body wt (9) 23.8 f 0.7 25.0f 0.5 25.0f 0.5 24.8 f 0.5 

Estrous cycle length (days) 4.00f 0.00 4.56* 0.29b 4.20f 0.20‘ 4.25 f 0.16d 

Estrous stages (% of cycle) 

Diestms 37.1 31.4 38.6 27.1 

Proestrus 25.7 21.4 15.7 15.7 

Estrus 28.6 34.3 34.3 41.1 

Metestrus 8.6 12.9 11.4 15.7 

* Significantly different (P10.05)from the controls by Williams’ or Dunnett’s test (weights) or by Dunn’s test (epididymal spermatozoal 

parameters)

** PSO.01 

a Weights, epididymal spermatozoal parameters, and estrous cycle length are presented as mean f standard error. Differences from the 

control group for estrous cycle length are not significant by Dunn’s or Dunnett’s test. By multivariate analysis of variance, exposed 

females did not differ significantly from the control females in relative length of time spent in the estrous stages. 

Estrous cycle was longer than 7 days or was unclear in 1 of 10 animals. 

Estrous cycle was longer than 7 days or was unclear in 5 of 10 animals. 

Estrous cycle was longer than 7 days or was unclear in 2 of 10 animals. 
294 Phenolphthalein, NTP TR 465 
295 

APPENDIX J 

CHEMICAL CHARACTERIZATION AND 

DOSE FORMULATION STUDIES 

PROCUREMENT AND CHARACTERIZATION OF PHENOLPHTHALEIN . . . . . . . . . . . . ... . . . . . . .. . 296 

PREPARATION AND ANALYSISOF DOSE FORMULATIONS . . . . . . .. . . . . . .. . . . . ... . . . . . ... 297

FIGURE J1 Infrared Absorption Spectrum of Phenolphthalein . . . . . . .. . . . . . .. . . . . ... . . 299 

FIGURE 52 Nuclear Magnetic Resonance Spectrum of Phenolphthalein .. . . . . . . . . . . . . . . . . 300 

TABLEJ1 Preparationand Storage of Dose Formulations in the Feed Studies 

of Phenolphthalein .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . ... . . . . .. . . . . 301 

TABLE52 Results of Analyses of Dose Formulations Administered to Rats and Mice 

in the 13-Week Feed Studies of Phenolphthalein . . . . . . . . . . .. .. . . . . . .... . . 302 

TABLE53 Results of Analyses of Dose Formulations Administered to Rats and Mice 

in the 2-Year Feed Studies of Phenolphthalein . . . . . . . . . . .. . . . . . . . .. . . . . . 303 

TABLE54 Results of Referee Analyses of Dose Formulations Administered to Rats and Mice 

in the 13-Week Feed Studies of Phenolphthalein . . . . . . .. .. . .. . . . . ... ... . . 308 
296 Phenolphthalein, NTP TR 465 

CHEMICAL CHARACTERIZATION AND 

DOSE FORMULATION STUDIES 

PROCUREMENT AND CHARACTERIZATION OF PHENOLPHTHALEIN 

Phenolphthalein was obtained from Air Products and Chemicals, Inc. (Allentown, PA), in one lot 

(127-7809) and from Pharmco Laboratories, Inc. (Titusville, FL), in two lots (P3186-D5 and P9189-Jl). 

Lot 127-7809 was used during the 14-day studies, lot P3186-D5 was used during the 13-week studies, and 

lot P9189-J1 was used during the 2-year studies. Identity, purity, and stability analyses were conducted 

by the analytical chemistry laboratory, Midwest ResearchInstitute(Kansas City, MO). Reports on 

analyses performed in support of the phenolphthalein studies are on file at the National Institute of 

Environmental Health Sciences. 

All lots of the chemical, a yellow powder, were identified as phenolphthalein by infrared, ultraviolet/ 

visible, and nuclear magnetic resonance spectroscopy and by melting point. All spectra were consistent 

with the literature spectra (Sadtler Standard Spectra) of phenolphthalein. The infrared and nuclear 

magnetic resonance spectra are presented in Figures J1 and 52. The melting points of all lots of the 

chemical were consistent with literature values (Merck Index, 1989). 

The purity of each lot was determined by elemental analyses, Karl Fischer water analysis, functional 

group titration, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). 

For functional group titration of lot 127-7809, samples were titrated with 0.1 N tetrabutylammonium 

hydroxide in 2-propanol. For functional group titration of lots P3 186-D5 and P9 189-J 1, samples were 

dissolved in dimethylformamide and reacted with excess iodine. The unreacted iodine was titrated with 

standardized 0.1 N sodium thiosulfate to a colorimetric endpoint using a starch indicator. TLC was 

performed on Silica Gel 60 F-254 plates for lots 127-7809 and P3186-D5 and on Whatman Silica 

Gel 60A K6F plates for lot P9189-31. Three solvent systems were used: 1) carbon tetrach1oride:diethyl 

ether:95% ethanol (53:42:5), 2) ch1oroform:ethyl acetate (80:20), and 3) acetone:methylene chloride 

(80:20). System 1 wasused for all lots, system 2 was used for lot 127-7809, and system 3 was used for 

lots P3 186-D5 and P9 189-J 1. For all lots, plates were examined under ultraviolet light (254 and/or 

366 nm) and with a spray of 1 N aqueous sodium hydroxide. a-Cresolphthalein (10 pg/pL in methanol) 

was used as a reference standard. For lot P3186-D5, plates were also examined under visible light. 

HPLC for all lots was performed with a Waters pl3ondapak C,, column using ultraviolet detection 

(280 nm) and a flow rate 1.O mL/minute. Mobile phases of water:methanol (45:55 and 55:45) were used 

for lot 127-7809. Mobile phases of water with 1% glacial acetic acid:methanol with 1% glacial acetic 

acid with ratios of 50:50 and 54:46 were used for lots P3 186-D5 and P9189-51, respectively. 

For lot 127-7809, elemental analyses for carbon, hydrogen, and oxygen were in agreement with the 

theoretical values for phenolphthalein. Karl Fischer water analysis indicated 0.36% f 0.02% water. 

Functional group titration indicated a purity of 98.8% +_ 0.8 %. TLC by systems 1 and 2 indicated a 

major spot, a minor impurity, a trace impurity, and a slight trace impurity. HPLC with a mobile phase 

ratio of 45:55 revealed a major peak and five impurities with a combined area of 2.19%relative to the 

major peak area. HPLC with a mobile phase ratio of 55:45 revealed a major peak and four impurities 

with a combined area of 2.28 % of the major peak area. The overall purity of lot 127-7809 was 

determined to be greater than or equal to 98 % . 

For lot P3 186-D5, elemental analyses for carbon and hydrogen were in agreement with the theoretical 

values for phenolphthalein. Karl Fischer water analysisindicated 0.193% rt 0.004% water. Functional 

group titration indicated a purity of .99.5 % f 0.4 % . TLC indicated a major spot, a minor impurity, and a 

trace impurity by system 1 and a major spot and a trace impurity by system 3. HPLC revealed a major 
Chemical Characterization and Dose Formulation 297 

peak and three impurities with a combined area of 1.4 % relative to the major peak area. Major peak 

comparisons of lot P3186-D5 with lot 127-7809 using the methods previously described but with a mobile 

phase of water:methanol (47: 53), 3.O mg/mL butyrophenone as the internal standard, and a flow rate of 

1.2 mWminute indicated a purity of 100.3 % & 0.6% for lot P3186-D5 relative to lot 127-7809. The 

overall purity of lot P3 186-D5 was determined to be greater than or equal to 98% . 

For lot P9189-J1, elemental analyses for carbon and hydrogen were in agreement with the theoretical 

values for phenolphthalein. Karl Fischerwateranalysisindicated 0.12% & 0.05% water. Functional 

group titration indicated a purity of 99.9% 0.5% . TLCindicated a major spot and a trace impurity by 

system 1 and only a major spot by system 3. HPLC revealed a major peak and three impurities with a 

combined area of 1.2% relative to the major peak area. Major peak comparisons of lot P9189-J1 with 

lot 127-7809 using the methods previously described but with a mobile phase of water:methanoi (5050) 

and 3.0 mg/mL butyrophenone as the internal standard indicated a purity of 100.9 f 0.2% for lot 

P9189-J1 relative to lot 127-7809. The overall purity of lot P9189-J1 was determined to be greater than 

or equal to 99 % . 

Stability studies of lot 127-7809 were performed by the analyti2al chemistry laboratory. Samples were 

dissolved in methanol (100 mL) containing n-butyrophenone (1.9 mg/mL) as an internal standard. HPLC 

was performed as described for the purity analyses but with a mobile phase ratio of 34:63. These studies 

indicated that phenolphthalein was stable as a bulk chemical for 2 weeks when stored protected from light 

at temperatures up to 60" C. To ensure stability, the bulk chemical was stored protected from light at 

25" C in sealed containers during the 13-week studies and was stored protected from light at or below 

27" C in sealed containers during the 2-year studies. Stability was monitored using HPLC approximately 

monthly during the 13-weekstudiesandapproximatelyevery 16 weeks during the 2-year studies. No 

degradation of the bulk chemical was detected. 

PREPARATION AND ANALYSIS OF DOSEFORMULATIONS 

The dose formulations for all studies were prepared weekly by mixing phenolphthalein with feed 

(Table Jl). For the 14-day studies, dose formulations were mixed by hand for 1 minute and then blended 

in a Vortex feed mixer for 20 minutes using an intensifier bar for the initial 5 minutes. Formulations 

were stored in plastic bags within metal containers at room temperature for up to 1 week. For the 

13-week and 2-year studies, a phenolphthaleidfeed premix was prepared by hand and was then blended 

with feed in a Patterson-Kelly twin-shell blender for 15 minutes using an intensifier bar for the initial 

5 minutes. For the 13-week studies, formulations were stored in double plastic bags at temperatures at or 

below -20" C for up to 3 weeks. For the 2-year studies, formulations were stored in polyethylene bags 

at room temperature protected from light for up to 3 weeks. 

Stability studies of the 6,000 ppm dose formulation of lot 127-7809 were performed by the analytical 

chemistry laboratory. Samples were extracted with 100 mL of acetonitri1e:water:hydrochloric acid 

(97:2: 1). The samples were sonicated for 1 minute and then shaken for 20 minutes on a Burrell wristaction shaker. After centrifugation, 5-mL aliquots were diluted to 50 mL with acetonitrile. The solutions 

were filtered and then analyzed using HPLC with the same system as described for the purity analyses but 

with a mobile phase of acetonitri1e:water (45:55). The stability of the dose formulation was confirmed for 

at least 2 weeks when stored at temperatures up to 25" C. 

Homogeneity studies of the 500 ppm dose formulation of lot P3186-D5 were performed by the analytical 

chemistry laboratory. Samples were extracted with 100 mL of acetonitri1e:water:hydrochloric acid 

(97:2: 1). The samples were shaken for 15 minutes on a wrist-action shaker. After centrifugation, 5-mL 

aliquots were mixed with 5 mL of internal standard solution (acetophenone, 0.09 mg/mL in water: 
298 Phenolphthalein, NTP TR 465 

acetonitrile [45:55]) and diluted to 50 mL with water:acetonitrile (44:55). The solutions were filtered and 

analyzed using HPLC with a Hamilton PRP-1 10 p column using ultraviolet detection (280 nm) and a 

mobile phase of water:acetonitrile (45:55). Theflowratewas 1.O mL/minute. Stability studies of the 

500 ppm dose formulation were also performed using the same HPLC methods used for the homogeneity 

analyses. Homogeneity was confirmed and the stability of the dose formulation was confirmed for at least 

3 weeks when stored protected from light at room temperature. 

Periodic analyses of the dose formulations of phenolphthalein were conducted at the study laboratories 

using HPLC with thesamemethodsused for the homogeneity analyses. During the 13-week studies, the 

formulations were analyzed every 6 weeks (Table 52). During the 2-year studies, the formulations were 

analyzed approximately every 8 weeks (Table 53). All of the dose formulations analyzed during the 

13-week studies were within 10% of the target concentrations. Of the dose formulations analyzed during 

the 2-year studies, 99% (122/123) were within 10% of the target concentrations. During the 2-year 

studies, one 25,000 ppm sample mixed on 6 November 1992 was determined to be 28% greater than the 

target concentration. This formulation was remixed on 10 November 1992; the remix was within 10% of 

the target concentration. During the 2-year studies, 89% (40145) of the animal room samples were within 

10%of the target concentrations. One sample collected on 24 February 1992 was 52% less than the 

target concentration; it was determined that the food container was mislabeled and that the animals 

received the correct dose formulation. The other four animal room samples were within 17% of the target 

concentrations. Results of periodic referee analyses performed during the 13-week studies by the 

analytical chemistry laboratory agreed with the results obtained by the study laboratory (Table 54). 
Chemical Characterization and Dose Formulation 299 

c i

0

0 

-(3 

-

0 

I 

0

0

0

N 

I 

0

0 

0 

?=- /I\/ 

0 

0 

P c N

N 

0

0 

N 

rY 

0

0 

L3 

m 

3

3 

s 0 0 0 0 N 0 N c c 

FIGURE J1 

Infrared Absorption Spectrum of Phenolphthalein 
w 

0

0 

n 

&\ON f’hnnnlpl~ll~nlc!i~~ 

t.01 NO.:1’9 109-JI 

I3alch No.: 03 

Task Designation: RE-2453 

Inslrumenl: Varian VXR-300 FT-NMn 

Solvenl: Deuterated dimethyl sulfoxide 

Internal t7eforence: Deuterated dimethyl sulfoxido blld 

Assignments ( ppm) -J lnlogralion 

(a) 6.73 4.09 

(b) 7.05 3.95 

(c) 7.63 

3.95 

0.37 

Impurities: 

-0.05 traco 

- 1.25 lrace 

a Partiallyexchanged. 

LU 

0.0 7.5 l Due to Sotvenl 

# I-ID0 J-

’ w 
Chemical Characterization and Dose Formulation 301 

TABLEJ1 

Preparation and Storage of Dose Formulations in the Feed Studies of Phenolphthalein 

14-Day Studies 

Preparation 

A premix of feed and phenolphthalein 

was prepared by hand and then blended 

in a Vortex feed mixer with the 

intensifier bar on for 5 minutes and off 

for 15 minutes. Dose formulations 

were prepared weekly. 

Chemical Lot Number 

127-7809 

Maximum Storage Time 

I week 

Storage Conditions 

Stored in plastic bags within metal 

containers at room temperature 

Study Laboratory 

Cannon Laboratories, Inc. 

(Reading, PA) 

Referee Laboratory 

None 

13-Week Studies 

A premix of feed and phenolphthalein 

was prepared, and then layered into the 

remaining feed and blended in a 

Patterson-Kelly twin-shell blender with 

the intensifier bar on for 5 minutes and 

off for 10 minutes. Dose formulations 

were prepared weekly. 

P3 186-D5 

3 weeks 

Stored in double plastic bags at or 

below -20" C 

Microbiological Associates, Inc. 

(Bethesda, MD) 

Midwest Research Institute 

(Kansas City, MO) 

2-Year Studies 

Same as 13-week studies 

P9189-J1 

3 weeks 

Stored in polyethylene bags protected 

from light at room temperature 

TSI Mason Laboratories 

(Worcester, MA) 

None 
302 Phenolphthalein, NTP TR 465 

TABLE52 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 13-Week Feed Studies 

of Phenolphthalein 

Date Prepared ConcentrationDate Analyzed Concentrationa 

20 April 1987b 20-21April 1987 

22 April 1987 24April 1987 

3 June 1987 3-4June 1987 

15 July 1987 16-17July 1987 

a Resultsof duplicate analyses 

Homogeneity analyses, not used for dosing 

Sample selection from top left of twin-shell blender 

Sample selection from top right of twin-shell blender 

e Sample selection from bottom oftwin-shell blender 

Target 

(PPm) 

3,OOOc 

3,OOOd 

3.000" 

50,000c 

50,000d 

50.000e 

3,000 

6,000 

12.000 

25.000 

5O.OOO 

3,000 

6,000 

12.000 

25.000 

50,000 

3,000 

6,000 

12,000 

25,OOO 

50,000 

Determined 

(PPm) 

2.910 

2,890 

2,940 

48.800 

50.200 

49.200 

3.040 

6,000 

11,800 

24,500 

50.400 

3.000 

5,860 

11,800 

26.300 

49,100 

2,990 

5,910 

1 1,200 

23,400 

46.800 

% Difference 

from Target 

-3 

-4 

-2 

-2 

0 

-2 

+1 

0 

-2 

-2 

+1 

0 

-2 

-2 

+5 

-2 

0 

-2 

-7 

-6 

-6 
Chemical Characterization and Dose Formulation 303 

TABLE53 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies 

of Phenolphthalein 

Date Prepared ConcentrationDate Analyzed Concentrationa 

Rats 

1 November 1990b 2 November 1990 

1 March 1991 4 March 1991 

1-2April 1991f 

4 March 1991 4 March 1991 

1-2April 1991f 

26 April 1991 29-30 April 1991 

21 June 1991 24-25June 1991 

16 August 1991 20 August 1991 

4 September 1991f 

Determined Target

(PPd 

3,oooc 

3,OOOd 

3,000e 

50,000c 

50,000d 

50,000e 

12,000 

12.000 

12,000 

12.m 

25,000 

25,000 

50,000 

50.000 

25,000 

25,000 

50,000 

50,000 

12,000 

12,000 

25,000 

25,000 

50,000 

50,000 

12,000 

12,000 

25,000 

25,000 

50,000 

50.000 

12.000 

12,000 

25,000 

25,000 

50,000 

50,000 

12,000 

12,000 

25.000 

25,000 

50.000 

50,000 

(PPm) 

2,980 

2,890 

2,870 

50,600 

50.400 

50,500 

12.300 

1 1.700 

1 1,200 

11.600 

25,OOO 

24,700 

49,800 

48,700 

25,300 

25.000 

50,200 

49,900 

12,000 

12,400 

25,600 

25,100 

50,000 

49,900 

12,100 

12,200 

25,OOO 

25,OOO 

50,400 

50,OOO 

11,900 

11,800 

25,500 

25,900 

50,500 

51,500 

11,500 

12.000 

25,400 

25,000 

49,500 

49,600 

% Difference 

from Target 

-1 

-4 

-4 

+1 

+1 

+1 

+3 

-3 

-7 

-3 

0 

-1 

0 

-3 

+1 

0 

0 

0 

0 

+3 

+2 

0 

0 

0 

+1 

+2 

0 

0 

+1 

0 

-1 

-2 

+2 

+4 

+1 

+3 

-4 

0 

+2 

0 

-1 

-1 
304 Phenolphthalein, NTP TR 465 

TABLE53 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies 

of Phenolphthalein (continued) 

Target Determined % Difference 

Date Prepared ConcentrationDe Analyzed Concentration from Target 

(PPlll) (PPm) 

Rats (continued) 

11 October 1991 15 October 1991 12,000 11,500 -4 

25.000 24,500 -2 

50,000 50.000 0 

5 December 1991 6 December 1991 12.000 1 1,700 -3 

12.000 11.700 -3 

25.000 25,100 0 

25.000 24,800 -1 

50,000 49,000 -2 

50.000 48,300 -3 

31 January 1992 3 Februaly 1992 12,000 12,300 +3 

25.000 25,400 +2 

50.000 49.700 -1 

24 February 1992' 12,000 12.100 +1 

25.000 11,900 -52g 

50.000 50,000 0 

27 March 1992 30March 1992 12,000 12.200 +2 

12.000 12,300 +3 

25.000 25,500 +2 

25.000 25.700 +3 

50.000 49,700 -1 

50.000 51,200 +2 

22May 1992 26 May 1992 12.000 12,100 +1 

12,000 12.200 +2 

25,000 24,600 -2 

25,000 24,200 -3 

50,000 47,000 -6 

50.000 47,500 -5 

17 July 1992 20July 1992 12.000 11,700 -3 

12,000 11,600 -3 

25,000 23,900 -4 

25,000 24,200 -3 

50,OOO 49,600 -1 

50,000 53,300 +7 

10-12August 1992f 12.000 12,Ooo 0 

12,000 11,800 -2 

25.000 24.900 0 

25,000 24,500 -2 

50,000 49,500 -1 

50,000 49,Ooo -2 
Chemical Characterization and Dose Formulation 305 

TABLE53 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies 

of Phenolphthalein (continued) 

Date Prepared 

Rats (continued) 

September 18 September1992 1992 21 

6 November 1992 

10 November 1992h 

4 January1993 

19 February 1993 

Mice 

1 November 1990b 

-

Determined Target % Difference

Date ConcentrationConcentration Analyzed

9 November 1992 

10 November 1992 

5 January1993 

25 January 1993' 

22February1993 

2 November 1990 

(PPW 

12,000 

12,000 

25.000 

25.000 

50,000 

50.000 

12.000 

12.000 

25.000 

25,000 

50,OOO 

50,000 

25,000 

12,000 

12,000 

25,000 

25.000 

50,000 

50.000 

12.000 

12,000 

25.000 

25,000 

50.000 

50,000 

12,000 

12.000 

25,000 

25.000 

50,000 

50.000 

3,000' 

3,OOOd 

3,OOOe 

50.000' 

50,000d 

50,000e 

(PPd 

12.000 

12,000 

24,900 

25,000 

50,100 

50.300 

12,100 

12.000 

25,200 

31.900 

49,800 

50,300 

26,200 

10,900 

10.800 

25.800 

25,100 

49,200 

49,400 

11,400 

11,300 

25,100 

24,700 

49,000 

50,OOO 

12,000 

12,400 

25,800 

25,400 

48,300 

48,500 

2,980 

2,890 

2,870 

50,600 

50,400 

50,500 

from Target 

0 

0 

0 

0 

0 

+1 

+1 

0 

+1 

+28 

0 

+1 

+5 

-9 

-10 

+3 

0 

-2 

-1 

-5 

-6 

0 

-1 

-2 

0 

0 

+3 

+3 

+2 

-3 

-3 

-1 

-4 

-4 

+1 

+1 

+1 
306 Phenolphthalein, NTP TR 465 

TABLE53 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies 

of Phenolphthalein (continued) 

Date Prepared 

Mice (continued) 

November 16 November1990 1990 19 

18 January 1991 

8 March1991 

26 April 1991 

21June1991 

16 August 1991 

11October1991 

5 December 1991 

ConcentrationConcentration Analyzed Date

11 December1990f 

21January1991 

11March1991 

29 April 1991 

16May 199if 

24June 1991 

20 August 1991 

15 October 1991 

30 October 1991f 

6 December 1991 

Determined Target

(PPm) 

3.000 

3,000 

6,000 

6,000 

12,000 

12,000 

3,000 

3.000 

6.000 

6.000 

12,000 

12,000 

3.000 

6.000 

12.000 

3.000 

6,000 

12.000 

3,000 

6,000 

12,000 

3,000 

6.000 

12.000 

3,000 

6,000 

12,000 

3.000 

6.000 

12,000 

3,000 

6,000 

12,000 

3,000 

6,000 

12,000 

3.000 

6,000 

12,000 

(PPm) 

2,970 

3,060 

6.020 

6,020 

12,040 

12,150 

2,500 

2,750 

5,370 

5,470 

10,080 

11,350 

3,060 

6,030 

12,040 

3,000 

6,100 

11.990 

3.090 

5,970 

11,870 

2,640 

5,520 

11,670 

3,050 

5.930 

11,960 

3.000 

6,050 

12,160 

2,940 

5,980 

11.950 

2,920 

5,800 

11,900 

3,010 

5,990 

11,900 

% Difference 

from Target 

-1 

+2 

0 

0 

0 

+I 

-17: 

-8: 

-11' 

-gi 

-16f 

-5' 

+2 

+I 

0 

0 

+2 

0 

+3 

-1 

-1 

-12i 

-8 

-3 

+2 

-1 

0 

0 

+.I 

+I 

-2 

0 

0 

-3 

-3 

-1 

0 

0 

-1 
Chemical Characterization and Dose Formulation 307 

TABLE53 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-YearFeed Studies 

of Phenolphthalein (continued) 

Determined Target % Difference 

oncentrationConcentration DatePreparedDateAnalyzed from Target 

(PPm) (PP@ 

Mice (continued) 

31 January 1992 3 February 1992 3,000 3,090 +3 

6.000 6,100 +2 

12.000 12.060 +1 

27 March 1992 30 March 1992 3,000 3.210 +7 

6,000 5,900 -2 

12,000 11,880 -1 

15-16 April 1992f 3,000 3,150 +5 

6,000 6,240 +4 

12,000 12,170 +I 

22 May 1992 22 May 1992 3,000 3,060 +2 

6,000 6,030 +1 

12.000 11.960 0 

17 July 1992 20 July 1992 3,000 2.920 -3 

6,000 6,050 +1 

12.000 11,890 -1 

18 September 1992 21 September 1992 3.000 3,080 +3 

6,000 6,050 +1 

12,000 12.020 0 

10 October 1992f 3.000 2.910 -3 

6,000 5,800 -3 

12.000 12.030 0 

6 November 1992 9 November 1992 3,000 3.040 +I 

6,000 6.130 +2 

12,000 12,080 +1 

a Resultsof duplicate analyses 

Homogeneity analyses, not used for dosing 

Sample selection from top left of twin-shell blender 

Sample selection from top right of twin-shell blender 

e Sample selection frombottom of twin-shellblender 

Animal room samples 

For the 25.000 ppm dose formulation prepared 31 January 1992, it was determined that a container of the 12.000 ppm dose formulation 

was mislabeled when the animal room samples were collected on 24 February 1992 and that the animals were not misdosed. 

h Resultsofremix

' Low values were caused bythe presence of feces, urine, and/or bedding in the animal room samples; it was determined that the animals 

were not misdosed. 
308 Phenolphthalein, NTP TR 465 

TABLE54 

Results of Referee Analyses of Dose Formulations Administered to Rats and Mice 

in the 13-Week Feed Studies of Phenolphthalein 

DeterminedConcentration (Dprn) 

Target Concentration Study Referee 

Date Prepared (PPm) Laboratorya Laboratoryb 

22 April 1987 6,000 6,m 6,100 f 60 

15 July 1987 25,000 23,400 25,500 f 300 

a Results of duplicate analyses 

Results of triplicate analyses (mean f standard error) 
309 

APPENDIX K 

FEED AND COMPOUND CONSUMPTION 

IN THE 2-YEAR FEED STUDIES 

OF PHENOLPHTHALEIN 

TABLEK1 FeedandCompoundConsumption by MaleRatsinthe2-YearFeedStudy 

of Phenolphthalein ............................................ 310 

TABLEK2 FeedandCompoundConsumption by FemaleRatsinthe2-YearFeedStudy 

of Phenolphthalein ............................................ 311 

TABLEK3 FeedandCompoundConsumption by MaleMiceinthe2-YearFeedStudy 

of Phenolphthalein ............................................ 312 

TABLEK4 FeedandCompoundConsumption by FemaleMice in the2-YearFeedStudy 

of Phenolphthalein ............................................ 313 
310 Phenolphthalein, NTP TR 465 

TABLEK1 

Feed and Compound Consumption by Male Rats in the 2-Year Feed Study of Phenolphthalein 

0 ppm 12.000 ppm 25,000 ppm 50.000 ppm 

BodyFeed BodyFeed Dose/ BodyFeed Dosel Feed Body Dosel 

(g/day)a Weight (glday) Weight Dayb (glday) Weight Day (glday) Weight Day

Week (g) (g) (mglkd (g) (mglkg) (8) (mgW 

2 12.7 12.7 168 161 3.818

4 17.6 19.6 229

5 17.5 18.2 250

9 17.4 320 16.3 301 65 1 16.5 294 1.403 18.0 306 2,949 

17.9 

17.3 

14.5 170 907 168 1,899 12.3 

17.3 237 924 233 2,105 18.4 227 4,051 

16.8 258 840 254 1.790 18.0 249 3,604 

327 16.3 333 2,453

364 

398 520 17.8 402 1,106 18.3 405 2,256 

1317.0 622 326 

1717.0

2517.3

2916.7

318.4 3

37 16.2 449 17.4 

15.5 344 1,372 

16.8 382 562 362 1,198 17.6 365 2,416 

16.0 415 

17.4 406 410 1,059 16.9 409 2,068

17.9 411

18.1 428

18.4 437

17.7 440

49 16.2 463 19.0 446 1,062 18.6 446 2,092 

53 15.3 464 16.7 443 45 1 17.6 445 988 17.9 447 1.999 

15.7 425 492 

15.8 437 537 42 1 1,062 18.0 420 2,136 

487 432 1,048 17.9 434 2,058 

4118.3 503 442 1,039 18.6 443 2,100 

45 15.9 460 17.5 

16.8 459 

476 442 1.002 18.0 443 2,029 

44 1 984 17.3 442 1,961 

444 1,018 17.7 439 2,023 

443 1,116 18.2 447 2,034 

445 93 6 16.9 442 1,906 

441 894 16.1 442 1,825 

439 907 16.1 437 1,843 

43 1 920 16.0 43 1 1,854 

422 923 15.6 428 1.821 

404 834 14.3 415 1,719 

397 884 13.7 390 1.758 

395 961 14.5 409 1.780 

364 979 13.5 372 1.822 

34 1 998 13.5 342 1.972 

57 

61 

65 

16.4 

16.1 

15.8 

459 

466 

470 

17.2 

16.9 

17.5 443 

439 

439 

19.8 

18.1 

17.4 

69 14.1 466 16.0 16.6 440

73 13.8 461 15.9 15.8 442

77 14.7 465 15.3 15.9 436

81 

8514.4 

13.9 

14.1 

457 

455 

14.1 433 

15.6 423

89 12.8 450 13.8 419 

469 

463 

475 

437 

433 

422 

15.9 3 92

409 

13.5 3 94

14.0 423

15.2 405

14.3 375

13.6 367 

Mean for weeks 

1-13 16.3 266 16.2 255 789 17.0 255 1,714 16.6 255 3,375 

14-52 16.2 436 17.5 412 511 17.9 420 1,072 18.0 42 1 2,144 

53-104 14.4 457 15.3 423 434 15.9 418 953 15.8 420 1,880 

a Gramsof feed consumed per animalperday 

Milligrams of phenolphthalein consumed per kilogram body weightper day 

9314.4 407 

97 

10413.7

14.1 457 

13.5 451 14.6 432 

101 14.0 439 14.0 448 

12.8 434 448 
Feed and Compound Consumption 311 

TABLEK2 

Feed and Compound Consumption by Female Rats in the 2-Year Feed Study of Phenolphthalein 

0 ppm 12,000 ppm 25,000 ppm 50,000 ppm 

Body Feed Body Feed Dose/ Body Feed Dose/ Feed Body Dose/ 

Weight (glday) Weight Dayb (glday) WeightDay (glday) WeightDay 

Week (g) (g) (mglkg) (g) (mgW (9) (mglkg) 

2 10.4 129 9.3 129 865 9.4 128 1.829 9.4 129 3,661 

4 9.7 142 11.1 147 907 11.1 146 1,896 11.6 151 3,844 

5 11.9 157 11.0 157 843 10.9 155 1,764 11.0 159 3,441 

9 11.0 184 10.6 182 702 10.5 179 1,461 11.3 180 3,144 

13 10.5 20 1 10.1 195 618 10.4 196 1.324 11.5 191 3.024 

16 10.7 214 11.0 204 647 10.6 202 1,313 11.1 200 2,775 

21 10.9 222 10.7 21 1 609 10.6 209 1,275 11.2 206 2,712 

25 10.7 229 11.3 220 615 11.1 216 1,289 11.4 214 2,677 

29 10.8 234 10.8 222 583 10.6 219 1,215 10.8 216 2,499 

33 10.1 240 11.1 225 593 11.0 223 1,235 11.2 219 2,566 

37 11.7 252 11.4 234 584 11.2 230 1,217 11.6 228 2,548 

41 11.8 26 1 12.2 240 613 11.8 238 1.241 12.3 234 2,624 

45 11.4 267 11.3 244 556 11.6 242 1,196 11.8 231 2.480 

49 11.4 28 1 12.1 254 572 11.8 249 1,189 12.2 245 2,497 

53 11.5 287 11.6 258 543 11.6 254 1,147 11.7 249 2,355 

57 11.8 296 11.6 262 530 11.5 256 1,122 12.0 25I 2,396 

61 12.0 304 11.5 265 520 11.6 260 1,119 12.0 259 2,323 

65 11.9 314 11.9 272 522 12.5 269 1,160 12.3 265 2,320 

69 11.4 322 11.4 282 485 11.7 280 1,047 11.5 '270 2,128 

73 11.9 325 11.7 287 490 11.9 284 1.050 12.1 275 2,198 

77 12.2 332 11.9 290 49 1 12.1 288 1,048 12.3 28 1 2.193 

81 12.8 342 12.5 298 503 12.2 293 1.039 11.9 284 2,090 

85 11.2 346 11.0 303 438 11.4 296 96 1 12.0 292 2.059 

89 11.3 35 1 10.9 301 436 10.7 295 902 11.2 291 1,918 

93 10.7 345 10.4 299 417 10.8 294 914 11.5 293 1.967 

97 9.7 346 9.9 290 410 10.6 298 893 11.3 290 1,940 

101 10.4 348 11.1 30 1 44 1 10.5 300 877 11.5 296 1,941 

105 10.5 337 11.4 302 455 10.9 303 904 11.3 294 1.923 

Mean for weeks 

1-13 10.7 162 10.4 1 62 787 10.4 161 1,655 11.0 162 3,423 

14-52 11.1 244 11.3 228 597 11.2 225 1.241 11.5 222 2,598 

53-105 11.4 328 11.3 286 477 11.4 284 1.013 11.8 278 2,125 

a Grams of feed consumed per animal per day 

Milligrams of phenolphthalein consumed per kilogram body weight per day 
312 Phenolphthalein, NTP TR 465 

TABLEK3 

Feed Compound Consumption by Male Mice in the 2-Year Feed Study of Phenolphthalein 

0 ppm 3,000ppm 6,000 pDm 12.000 ppm 

Body Feed Body Feed Dose/ Body Feed Dose/ Feed Body Dose/ 

Weight @/day) Weight Dayb (glday) WeightDay (glday) Weight Day 

Week (g) (9) (mg/kg) (g) (mglkg) (g) (mglkg) 

81 

I7 

73 

69 

65 

61 

57 

53 

49 

45 

41 

37 

33 

29 

25 

21 

17 

13 

9 

5 

2 

4.l 

4.7 

5.1 

4.8 

4.9 

5.2 

4.7 

4.6 

4.7 

4.4 

4.7 

4.6 

4.2 

4.2 

4.0 

4.3 

4.2 

4.1 

4.6 

4.2 

4.2 25.2 

27.7 

29.5 

32.6 

35.2 

40.5 

42.8 

44.8 

46.5 

46.8 

46.2 

47.1 

48.7 

48.7 

49.8 

49.6 

51.7 

52.0 

51.7 

51.6 

51.1 

4.4 

4.1 

4.3 

4.3 

4.3 

4.4 

4.1 

4.1 

4.4 

4.5 

4.7 

4.6 

4.8 

4.7 

4.7 

4.9 

5.0 

4.9 

5.1 

4.8 

4.6 50.2 

50.7 

50.7 

50.9 

50.5 

48.9 

49.0 

48.3 

48.2 

47.0 

45.0 

46.0 

45.7 

43.9 

41.6 

39.1 

35.0 

32.8 

29.2 

27.4 

24.9 530 

447 

441 

393 

367 

336 

295 

320 

287 

293 

313 

291 

301 

289 

288 

302 

298 

289 

303 

282 

277 

4.1 

4.2 

5.0 

4.4 

4.4 

4.3 

4.3 

4.6 

4.6 

4.6 

4.5 

4.8 

4.8 

4.9 

4.9 

5.3 

5.2 

5.O 

5.2 

4.7 

4.8 51.O 

50.3 

51.0 

49.8 

49.8 

41.9 

48.9 

48.3 

47.1 

46.4 

45.3 

45.7 

45.9 

44.0 

41.8 

39.7 

34.7 

32.6 

29.4 

27.1 

24.8 1,002 

933 

1.025 

801 

763 

644 

611 

630 

598 

598 

591 

616 

610 

608 

600 

659 

621 

601 

607 

565 

567 

4.1 

4.2 

4.3 

4.2 

3.8 

4.2 

4.2 

4.5 

4.6 

4.3 

4.6 

4.8 

4.1 

4.7 

4.7 

5.1 

4.9 

4.8 

4.9 

4.8 

4.7 

24.8 

26.9 

28.8 

32.5 

34.2 

38.5 

41.2 

43.2 

43.8 

43.9 

44.9 

45.6 

46.9 

47.0 

47.5 

47.3 

49.8 

49.8 

50.3 

49.8 

49.8 

1,999 

1,855 

1,809 

1,547 

1,328 

1,305 

1,228 

1,256 

1,248 

1,188 

1,231 

1,259 

1,206 

1,196 

1,194 

1,302 

1,191 

1,149 

1,179 

1,157 

1,130 

89 

85 

4.3 

4.6 51.1 

51.5 

4.8 

4.7 49.5 

50.2 281 

282 

4.9 

4.9 50.8 

51.5 575 

573 

4.8 

4.8 

50.1 

49.4 

1,153 

1,162 

104 

101 

97 

93 

4.6 

4.7 

4.9 

5.3 50.0 

50.4 

49.9 

49.4 

5.2 

4.9 

4.6 

4.4 47.9 

49.0 

49.1 

49.1 320 

298 

284 

279 

5.2 

4.8 

4.6 

4.3 49.1 

50.2 

50.1 

50.2 622 

575 

549 

525 

5.O 

4.9 

4.5 

4.1 

47.1 

47.3 

46.3 

45.9 

1,261 

1,238 

1,157 

1,062 

Mean for weeks 

1-13 4.3 28.8 4.3 28.6 453 4.4 28.5 940 4.2 28.3 1,802 

14-52 4.4 44.3 4.5 43.5 311 4.5 43.5 629 4.4 42.5 1,250 

53-104 4.8 50.6 4.8 49.6 291 4.9 49.9 589 4.8 48.4 1,181 

a Grams of feed consumed per animal per day 

Milligrams of phenolphthalein consumed per kilogram body weight per day 
Feed and Compound Consumption 313 

TABLEK4 

Feed and Compound Consumption by Female Mice in the 2-Year Feed Study of Phenolphthalein 

0 om 3,000 RRm 6.000 PRm 12,000 ppm 

Body Feed Body Feed Dose/ Body Feed Dose/ Feed Body Dose/ 

Weight (glday) Weight Dayb (glday) WeightDay (glday) Weight Day 

Week (8) (g) (mgW (8) (mglkg) (8) (mglkg) 

2 6.2 19.9 5.3 19.5 812 6.0 19.6 1,831 5.4 19.6 3,299 

5 4.7 21.8 4.8 21.8 658 4.7 21.7 1,301 4.4 21.9 2.409 

9 5.2 24.9 5.2 24.3 645 5.6 24.7 1,351 5.7 24.9 2,733 

13 4.2 27.O 4.3 26.1 489 5.1 26.9 1.144 4.8 27.4 2,118 

17 4.3 30.6 4.5 29.1 460 4.8 29.3 982 4.5 29.6 1,844 

21 4.7 34.0 4.9 33.6 436 4.7 33.6 843 4.8 34.3 1,666 

25 4.3 36.2 4.6 35.2 3 92 4.4 35.1 757 4.2 35.8 1,409 

29 4.7 38.8 4.7 37.5 373 4.7 37.8 750 4.6 37.5 1,475 

33 4.7 42.2 4.8 40.3 359 4.7 40.3 699 4.8 39.9 1,433 

37 4.7 42.1 4.7 40.6 346 4.8 40.7 701 4.7 40.2 1,407 

41 5.0 44.5 4.9 42.8 344 4.9 42.1 696 4.9 41.7 1,397 

45 5.1 45.2 5.3 43.1 366 5.1 42.9 719 5.2 43.1 1,444 

49 4.9 46.6 5.2 44.6 349 5.1 43.7 702 5.0 43.0 1,406 

53 5 .O 47.8 4.7 46.1 308 4.9 45.5 643 4.9 45.0 1,311 

57 5.1 49.0 5.2 46.9 334 5.1 47.1 654 5.1 46.5 1,326 

61 5.7 50.2 5.7 48.0 354 5.5 47.6 697 5.4 46.8 1,384 

65 5.5 53.6 5.6 50.7 332 5.6 51.1 656 5.3 49.7 1,270 

69 5.4 55.6 5.5 52.3 317 5.2 51 .O 615 5.4 50.8 1,271 

73 5.7 55.6 5.7 52.9 325 5.7 51.7 659 5.3 50.9 1,245 

77 5 .O 54.6 5.1 52.4 294 4.9 51.4 567 5.2 50.3 1,244 

81 5.1 55.6 4.9 52.7 282 5 .O 51.5 582 5.1 50.3 1.210 

85 5.2 56.3 5.2 52.9 295 5 .O 52.5 566 5 .O 51.2 1,169 

89 5.3 56.5 5.4 52.6 309 5.3 52.7 608 5.6 52.9 1,260 

93 5.8 55.2 5.6 51.4 330 5.6 51.0 659 5.5 51.7 1,287 

97 5.5 55.6 5.4 52.5 307 5.3 51.8 615 5.4 51.8 1,246 

101 5.1 54.9 4.8 50.3 288 4.8 52.0 550 5.1 51.6 1,190 

105 4.9 54.0 4.7 49.4 283 4.6 49.3 564 4.9 50.5 1,155 

Mean for weeks 

1-13 5.1 23.4 4.9 22.9 65 1 5.3 23.2 1.407 5.1 23.5 2,640 

14-52 4.7 40.0 4.8 38.5 381 4.8 38.4 761 4.7 38.3 1,498 

53-105 5.3 53.9 5.3 50.8 311 5.2 50.4 617 5.2 50.0 1,255 

a Grams of feed consumed per animal per day 

Milligrams of phenolphthalein consumed per kilogranl body weight per day 
314 Phenolphthalein, NTP TR 465 
315 

APPENDIX L 

INGREDIENTS, NUTRIENT COMPOSITION, 

AND CONTAMINANT LEVELS 

IN NIH-07 RAT AND MOUSE RATION 

TABLE L1 Ingredients of NIH-07 Rat and Mouse Ration ........................... 316 

TABLE L2 Vitamins and Minerals in NIH-07 Rat and Mouse Ration ................... 316 

TABLE L3 Nutrient Composition of NIH-07 Rat and Mouse Ration .................... 317 

TABLE L4 Contaminant Levels in NIH-07 Rat and Mouse Ration ..................... 318 
316 Phenolphthalein, NTF' TR 465 

TABLEL1 

Ingredients of NIH-07 Rat and Mouse Rationa 

Ingredientsb 

Ground #2 yellow shelled corn 

Ground hard winter wheat 

Soybean meal (49%protein) 

Fish meal (60%protein) 

Wheat middlings 

Dried skim milk 

Alfalfameal (dehydrated, 17%protein) 

Corn gluten meal (60%protein) 

Soy oil 

Dried brewer's yeast 

Dry molasses 

Dicalcium phosphate 

Ground limestone 

Salt 

Premixes (vitamin and mineral) 

a NCI, 1976; NIH, 1978 

~~ ~ 

Percent by Weight 

24.50 

23.00 

12.oo 

10.00 

10.00 

5 .OO 

4.OO 

3.OO 

2.50 

2.00 

1 SO 

1.25 

0.50 

0.50 

0.25 

Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed. 

TABLEL2 

Vitamins and Minerals in NIH-07 Rat and Mouse Rationa 

Vitamins 

A 

D3 

K3 

d-a-Tocopheryl acetate 

Choline 

Folic acid 

Niacin 

&Pantothenic acid 

Riboflavin 

Thiamine 

Bl2 

Pyridoxine 

Biotin 

Minerals 

Iron 

Manganese 

Zinc 

Copper 

Iodine 

Cobalt 

a Per ton (2,OOOIb) of finished product 

Amount 

5,500,000 IU 

4,600,000 IU 

2.8 g 

20,OOO IU 

560.0 g 

2.2 g 

30.0 g 

18.0 g 

3.4 g 

10.0 g 

4.000 c1g 

1.7 g 

140.0mg 

120.0g 

60.0 g 

16.0 g 

4.0 g 

1.4 g 

0.4 g 

Source 

Stabilized vitamin A palmitate or acetate 

D-activated animal sterol 

Menadione 

Choline chloride 

d-Calcium pantothenate 

Thiamine mononitrate 

Pyridoxine hydrochloride 

d-Biotin 

Iron sulfate 

Manganous oxide 

Zinc oxide 

Copper sulfate 

Calcium iodate 

Cobalt carbonate 
Feed Analyses 317 

TABLEL3 

Nutrient Composition of NIH-07 Rat and Mouse Ration 

Nutrient 

Protein (% by weight) 

Crude fat (% by weight) 

Crude fiber (% by weight) 

Ash (% by weight) 

Amino Acids (% of total diet) 

Arginine 

Cystine 

Glycine 

Histidine 

Isoleucine 

Leucine 

Lysine 

Methionine 

Phenylalanine 

Threonine 

Tryptophan 

Tyrosine 

Valine 

Essential Fatty Acids (% of total diet) 

Linoleic 

Linolenic 

Vitamins 

Vitamin A (IU/kg) 

Vitamin D (IU/kg) 

a-Tocopherol (ppm) 

Thiamine (ppm) 

Riboflavin (ppm) 

Niacin (ppm) 

Pantothenic acid (ppm) 

Pyridoxine (ppm) 

Folic acid (ppm) 

Biotin (ppm) 

Vitamin B12 (ppb) 

Choline (ppm) 

Minerals 

Calcium (%) 

Phosphorus (%) 

Potassium (9s) 

Chloride (%) 

Sodium (%) 

Magnesium (%) 

Sulfur (%) 

Iron (PPm)

Manganese (ppm) 

Zinc (ppm) 

Copper (ppm) 

Iodine (ppm) 

Chromium (ppm) 

Cobalt (ppm) 

Mean f Standard 

Deviation 

23.61 f 0.52 

5.28 f 0.24 

3.34 f 0.29 

6.54 f 0.21 

1.280 f 0.083 

0.308 f 0.071 

1.158 f 0.048 

0.584 f 0.027 

0.917 f 0.033 

1.975 f 0.051 

1.274 f 0.049 

0.437 f 0.109 

0.999 f 0.120 

0.904 f 0.058 

0.218 f 0.153 

0.685 f 0.094 

1.086 * 0.055 

2.407 f 0.227 

0.259 * 0.065 

6,976 f 1,367 

4,450 f 1,382 

36.12 f 9.15 

17.76 f 2.76 

7.83 f 0.923 

98.64 f 25.5 

30.55 f 3.52 

9.11 f 2.53 

2.46 f 0.63 

0.268 f 0.047 

40.5 f 19.1 

2,991 f 382 

1.18 f 0.09 

0.93 f 0.060 

0.886 f 0.063 

0.529 f 0.087 

0.316 f 0.033 

0.166 f 0.010 

0.272 f 0.059 

350.5 f 87.3 

92.48 f 5.14 

59.33 f 10.2 

11.81 f 2.50 

3.54 f 1.19 

1.66 f 0.46 

0.76 f 0.23 

Range 

22.6 -25.2 

4.80 -5.80 

2.60 -3.80 

6.23 -7.03

1.110 -1.390

0.181 -0.400 

1.060 -1.220

0.531 -0.630 

0.867 -0.965 

1.850 -2.040

1.200 - 1.370 

0.306 -0.699 

0.665 - 1.110 

0.824 -0.985 

0.107 -0.671 

0.564 -0.794 

0.962 -1.170

1.830 -2.570

0.100 -0.320

5,940 - 12,540 

3,000 - 6,300 

22.5 -48.9

12.0 -25.0 

6.10 -9.00 

65.0 - 150.0 

23.0 -34.6 

5.60 - 14.0 

1.80 - 3.70 

0.190 -0.354 

10.6 -65.0 

2,300 -3,430 

1.02 - 1.36 

0.770 - 1.03 

0.772 -0.971 

0.380 -0.635 

0.258 -0.371

0.148 -0.181

0.208 -0.420 

255.0 -523.0 

81.7 -99.4 

46.1 - 81.6 

8.09 - 15.4 

1.52 -5.83 

0.85 -2.09 

0.49 - 1.15 

Number of Samples 

25 

25 

25 

25 

11 

11 

11 

11 

11 

11 

11 

11 

11 

11 

11 

11 

11 

10 

10 

25 

4 

10 

25 

11 

10 

11 

11 

11 

11 

11 

10 

25 

25 

9 

9 

11 

11 

10 

11 

11 

11 

11 

10 

11 

7 
318 Phenolphthalein, NTP TR 465 

TABLEL4 

Contaminant Levels in NIH-07 Rat and Mouse Rationa 

Mean f Standard 

Deviation” Range Number of Samples 

Contaminants 

Arsenic (ppm) 

Cadmium (ppm) 

Lead (ppm) 

Mercury (ppm) 

Selenium (ppm) 

Aflatoxins (ppb) 

Nitrate nitrogen (ppm)‘ 

Nitrite nitro en (pprn)‘ 

BHA (ppm) % 

BHT (ppm)d

Aerobic plate count (CFUlg) 

Coliform (MPNlg) 

Escherichia coli (MPNlg) 

Salmonella (MPN/g) 

Total nitrosoamines (ppb)e 

N-Nitrosodimethylamine (ppb)e 

- N-Nitrosopyrrolidine (ppb)e 

Pesticides (ppm) 

a-BHC 

P-BHC 

7-BHC 

&BHC 

Heptachlor 

Aldrin 

Heptachlor epoxide 

DDE 

DDD 

DDT 

HCB 

Mirex 

Methoxychlor 

Dieldrin 

Endrin 

Telodrin 

Chlordane 

Toxaphene 

Estimated PCBs 

Ronnel 

Ethion 

Trithion 

Diazinon 

Methyl parathion 

Ethyl parathion 

Malathion 

Endosulfan I 

Endosulfan I1 

Endosulfan sulfate 

” 

0.50 f 0.20 0.10 -

0.14 f 0.08 0.04 - 0.31 f 0.22 0.10 -

0.02 f 0.00 0.02 -

0.35 f 0.08 0.10 -

<5.0 

7.20 f 3.29 1.80 -

0.17 f 0.12 0.02 - 1.40 f 0.91 1.00 -

1.36 f 1.22 1.OO - 135,520 f 167,289 10.000 -

75 f 232 3 -

<3 

Negative 

7.03 f 1.75 4.80 -

5.06 f 1.12 3.40 -

1.99 f 1.20 1.00 -

<0.01 

<0.02 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.05 

<0.01 

<0.01 

CO.01 

<0.05 

<0.10 

<0.20 

<0.01 

co.02 

<0.05 

<0.10 

<0.02 

<0.02 

0.20 f 0.18 0.05 -

<0.01 

<0.01 

<0.03 

~~ 

0.80 25 

0.20 25 

1.10 25 

0.03 25 

0.40 25 

25 

16.0 25 

0.50 25 

4.00 25 

7.00 25 

630,000 25 

1.100 25 

25 

25 

11.10 25 

7.90 25 

5.80 25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

25 

0.54 25 

25 

25 

25 

~~ ~ ~~ 

a CFU=colony forming units, MPN =most probable number, BHC is hexachlorocyclohexane or benzene hexachloride 

For values less than the limit of detection, the detection limit is given as the mean. 

Sources of contamination: alfalfa, grains, and fish meal 

Sources of contamination: soy oil and fish meal 

e All values were corrected for percent recovery. 
3 19 

APPENDIX M 

SENTINEL ANIMAL PROGRAM 

METHODS ........................................................... 320 

RESULTS ........................................................... 322 
320 Phenolphthalein, NTP TR 465 

SENTINEL ANIMAL PROGRAM 

METHODS 

Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in 

optimally clean facilities to eliminate potential pathogens that may affect study results. The Sentinel 

Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic 

evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via 

serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals 

are subject to identical environmental conditions. The sentinel animals come from the same production 

source and weanling groups as the animals used for the studies of chemical compounds. 

Serum samples were collected from randomly selected rats and mice during the 13-week and 2-year 

studies of phenolphthalein. Blood from each animal was collected and allowed to clot, and the serum was 

separated. The samples were processed appropriately and sent to Microbiological Associates, Inc. 

(Bethesda, MD), for determination of antibody titers. The laboratory serology methods and viral agents 

for which testing was performed are tabulated below; the times at which blood was collected during the 

studies are also listed. 

Method and Test Time of Analysis 

RATS 

13-Week Study 

ELISA 

PVM (pneumonia virus of mice) Study termination 

RCV/SDA (rat coronavirus/ 

sialodacryoadenitis virus) Study termination 

Sendai Study termination 

Hemagglutination Inhibition 

H-1 (Toolan’s H-1 virus) Study termination 

KRV (Kilham rat virus) Study termination 

2-Year Study 

ELISA 

Mycoplasma arthritidis Study termination 

Mycoplasma pulmonis Study termination 

PVM 5, 9, 12, 17, 18, and 23 months, study termination 

RCV/SDA 5, 9, 12, 17, 18, and 23 months, study termination 

Sendai 5, 9, 12, 17, 18, and 23 months, study termination 

Immunofluorescence Assay 

RCV/SDA Study termination 

Hemagglutination Inhibition 

H- 1 5, 9, 12,17,18, and 23 months, study termination 

KRV 5, 9, 12, 17, 18, and 23 months, study termination 
Sentinel Animal Program 321 

MICE 

13-Week Study 

ELISA 

Ectromelia virus 

GDVII (mouse encephalomyelitis virus) 

LCM (lymphocytic choriomeningitis virus) 

MVM (minute virus of mice) 

Mouse adenoma virus 

MHV (mouse hepatitis virus) 

PVM 

Reovirus 3 

Sendai 

Immunofluorescence Assay 

EDIM (epizootic diarrhea of infant mice) 

Hemagglutination Inhibition 

K (papovavirus) 

Polyoma virus 

2-Year Study 

ELISA 

Ectromelia virus 

EDIM 

GDVII 

LCM 

Mouse adenoma virus-FL 

MHV 

M.arthritidis 

M.pulrnonis 

PVM 

Reovirus 3 

Sendai 

Immunofluorescence Assay 

EDIM 

GDVII 

MHV 

Reovirus 3 

Hemagglutination Inhibition 

K 

MVM 

Polyoma virus 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

Study termination 

5, 12, and 18 months, study termination 

12 and 18 months, study termination 

5, 12, and 18 months, study termination 

5, 12, and 18 months, study termination 

5, 12, and 18 months, study termination 

5, 12, and 18 months, study termination 

Study termination 

Study termination 

5, 12, and 18 months, study termination 

5, 12, and 18 months, study termination 

5, 12, and 18 months, study termination 

5 months, study termination 

Study termination 

12 months 

12 months 

5, 12, and 18 months, study termination 

5, 12, and 18 months, study termination 

5, 12, and 18 months, study termination 
322 Phenolphthalein, NTP TR 465 

RESULTS 

One rat had a positive titer to M.arthritidis at the end of the 2-year study. Further evaluation of the 

serum positive for M. arthritidis by immunoblot and Western blot procedures indicated that the positive 

titer may have been due to a cross reaction with antibodies of nonpathogenic Mycoplasma or other agents. 

There were no clinical findings or histopathologic changes indicative of M. arthritidis infection in the rat 

with the positive titer. Accordingly, the M. arthritidis-positive titer was considered to be a false positive. 
323 

APPENDIX N 

CONTINUOUS BREEDING STUDY 

IN SWISS (CD-l@) MICE 

INTRODUCTION ....................................................... 324

MATERIALSAND METHODS ................................................ 324 

RESULTS ........................................................... 326 

TABLE N1 Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F, and F, Swiss (CD-1@) Mice in the Continuous Breeding Study 

of Phenolphthalein ............................................ 328 

TABLE N2 Litter and Body Weight Data for F, Swiss(CD-1@)Mouse Pups 

in the Continuous Breeding Study of Phenolphthalein ...................... 329 

TABLE N3 Survival and Body Weights of F, Swiss(CD-1@)Mouse Pups (Final Litter) 

in the Continuous Breeding Study of Phenolphthalein ...................... 330 

TABLE N4 Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F, and F, Swiss (CD-19 Mice in the Crossover Mating Trial 

of Phenolphthalein ............................................ 331 

TABLE N5 Organ Weights and Organ-Weight-to-Body-Weight Ratios 

for F,, Swiss (CD-1@) Mice Administered Phenolphthalein in Feed .............. 332 

TABLEN6 Sperm Parameters and Estrous Cycle Characterization 

for F, Swiss (CD-l@) Mice Administered Phenolphthalein in Feed .............. 333 

TABLEN7 Incidences of Selected Nonneoplastic Lesions in Male F, Swiss (CD-l@) Mice 

Administered Phenolphthalein in Feed ............................... 334 

TABLEN8 Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F, and F2Swiss (CD-l@) Mice in the Offspring Assessment Phase 

of the Continuous Breeding Study of Phenolphthalein ..................... 335 

TABLEN9 Organ Weights and Organ-Weight-to-Body-Weight Ratios for F, Swiss (CD-13 Mice 

in the Offspring Assessment Phase of the Continuous Breeding Study 

of Phenolphthalein ............................................ 336 

TABLEN10 Sperm Parameters and Estrous Cycle Characterization for F, Swiss (CD-1@) Mice 

in the Offspring Assessment Phase of the Continuous Breeding Study 

of Phenolphthalein ............................................ 337 

TABLEN11 Incidences of Selected Nonneoplastic Lesions in Male F, Swiss (CD-l@) Mice 

in the Offspring Assessment Phase of the Continuous Breeding Study 

of Phenolphthalein ............................................ 338 
324 Phenolphthalein, NTP TR 465 

CONTINUOUS BREEDING STUDY 

IN SWISS (CD-la) MICE 

INTRODUCTION 

The potential reproductive toxicity of phenolphthalein was evaluated in Swiss (CD-l@) mice because 

evidence in the literature suggests that phenolphthalein has a weak estrogenic activity (Bitman and Cecil, 

1970; Ravdin et al., 1987) and because data gathered at the end of the 13-week study in B6C3F, mice 

indicate chemical-related effects in male mice (Appendix I). The effects of exposure to phenolphthalein on 

reproduction were assessed with a continuous breeding study in Swiss (CD-I@) mice administered 

phenolphthalein in feed (NTP, 1991). 

Reproductive assessment by the methods presented in Lamb (1985) and Heindel et al. (1989) consists of 

four phases: dose range finding, continuous breeding, identification of the affected sex (crossover mating 

trial), and offspring assessment. A 2-week dose-finding phase is conducted to determine exposure 

concentrations of the continuous breeding phase. During the continuous breeding phase, the effects of the 

maximum tolerated exposure concentration estimated in the dose-finding phase and two lower exposure 

concentrations on fertility and reproduction of first-generation (F,,) animals are determined. If fertility is 

significantly affected during the continuous breeding phase, crossover mating trials are performed to 

determine if males, females, or both sexes are affected. Offspring assessment includes evaluation of 

reproductive performance of second-generation (F,) animals from the final litters of the continuous 

breeding phase. The F, animals are raised to sexual maturity while receiving the same exposure 

concentrations as their parents, are mated, and are allowed to deliver the third-generation (F,) offspring. 

In the phenolphthalein study, the exposure concentrations for the continuous breeding study were based on 

results of studies reported in the literature. A crossover mating trial was performed, and pups from all 

exposure groups were maintained for offspring assessment. 

MATERIALSAND METHODS 

Phenolphthalein was obtained from Pharmco Laboratories, Inc. (Titusville, FL) in one lot (P3186-D5), 

which was also used for the 13-week studies conducted at Microbiological Associates, Inc. (Bethesda, 

MD). Results of purity and stability analyses of lot P3 186-D5 are given in Appendix J. Dose 

formulations were prepared every 3 weeks, and analyses by Research Triangle Institute indicated that the 

dose formulations were homogeneous and within 10% of the target concentration. 

Male and female VAF Cr1:Swiss CD-1@ (1CR)BR outbred albino mice were obtained from Charles River 

Breeding Laboratories, Inc. (Kingston, NY). Upon receipt, serum samples werecollected from two males 

and two females, and the serum samples were analyzed for antibody titers to rodent titers. All serum 

samples were negative. Mice were quarantined for 2 weeks and were 11 weeks old at the start of the 

continuous breeding study. Mice werehousedtwopercagebysex during quarantine. NIH-07 open 

formula meal diet containing the appropriate concentrations of phenolphthalein and distilled water were 

available ad libitum for all phases of the study. 

During the continuous breeding phase, the mice were housed two per cage by sex for 1 week while being 

exposed to 0, 1,000, 7,000, or 30,000 ppm phenolphthalein; mice were then housed in breeding pairs for 

98 days while exposure continued. After the mating period, mice were housed separately for 

approximately 21 days while exposure continued to allow delivery of the final litter of pups. During the 

continuous breeding phase, clinical observations, feed consumption, pregnancy index, litters per pair, 

length of gestation, dam body weights, live pups per litter, proportion of pups born alive, sex of live 
Continuous Breeding Study 325 

pups, and pup body weights were recorded (Tables N1 and N2). For the last litter, pup survival and body 

weights were recorded on lactation days 0, 4, 7, 14, and 21 (Table N3). 

After the last litter of pups born during the holding period was weaned, crossover mating trials with the 

F, adult mice were performed. The following groups were mated: 0 ppmmales X 0 ppm females 

(18 pairs), 0 ppm males X 7,000 ppm females (19 pairs), and 7,000 ppm males X 0 ppm females 

(18 pairs). . The breeding pairs were housed together for 7 days or until a vaginal plug was observed, after 

which mice were housed separately while exposure continued. Clinical observations, feed consumption, 

mating index, pregnancy index, fertility index, dam body weights, length of gestation, live pups per litter, 

proportion of pups born aIive, sex of live pups, and pup body weights were recorded (Table N4). Before 

necropsy, estrous cycle data were collected (Table N6). At necropsy, sperm data were collected 

(Table N6), and the following organs were weighed: right cauda epididymis, right epididymis, kidneys 

(with adrenal glands), liver, right ovary, prostate gland, seminal vesicles, and right testis (Table N5). 

Selected organs were fixed in 10% neutral buffered formalin or Bouin’s fixative and imbedded in glycol 

methacrylate or paraffin. Sections were stained with hematoxylin and eosin (testis only) or PAS and 

hematoxylin. Histopathologic examination of selected organs was performed on 10 representative 0 and 

7,000 ppm males and females as well as on animals that died during the crossover mating trial (Table N7). 

To assess the offspring of treated mice, the final litter of pups born to each F, mouse in the 0, 1,000, and 

7,000 ppm groups was raised to sexual maturity. After weaning, siblings were housed two per cage by 

sex and were administered the same exposure concentrations as their parents. At sexual maturity 

(74 & 10 days of age), nonsibling male and female mice from within the same exposure group (20 pairs 

per group) were housed as breeding pairs for 7 days. Female mice were examined for a copulatory plug, 

and mice were then housed separately through the delivery of pups. Clinical observations, feed 

consumption, mating index, pregnancy index, fertility index, dam body weights, length of gestation, live 

pups per litter, proportion of pups born alive, sex of live pups, and pup body weights were recorded 

(Table N8). Before necropsy, estrous cycle datawerecollected (Table N10). At necropsy, sperm data 

were collected (Table NlO), andthefollowing organs were weighed:rightcauda epididymis, right . 

epididymis, kidneys (with adrenal glands), liver, right ovary, prostate gland, seminal vesicles, and right 

testis (Table N9). Selected organs were fixed in 10% neutral buffered formalin or Bouin’s fixative and 

imbedded in glycol methacrylate or paraffin. Sections were stained with hematoxylin and eosin (testis 

only) or PAS and hematoxylin. Histopathologic examination of selected organs was performed on 

10 representative 0 and 7,000 ppm males and females (Table N1 I). 

For data expressed as proportions (fertility, mating, and pregnancy indices), the Cochran-Armitage test 

(Armitage, 1971) was used to test for dose-related trends, and pairwise comparisons were performed with 

a chi-square test (Conover, 1971). A chi-square test for homogeneity wasused to identify overall 

differences in fertility across exposure groups and for pairwise comparisons in the crossover mating trial. 

The number of litters and the number of live pups per litter were determined per fertile pair and then 

exposure group means were determined. The proportion of live pups was defined as the number of pups 

born alive divided by the total number of pups produced by each pair. The sex ratio was expressed as the 

number of male pups born alive divided by the total number of live pups born to each fertile pair. 

Exposure group means for data with skewed distributions were analyzed by the nonparametric multiple 

comparisons methods of Shirley (1977) or Dunn (1964). Jonckheere’s test (Jonckheere, 1954) wasused to 

assess the significance of dose-response trends and to determine whether a trend-sensitive test (Shirley’s) 

was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related 

trend (Dunn’s). Inthe crossover mating trial, the Kruskal-Wallis test (Kruskal and Wallis, 1952) was used 
326 Phenolphthalein, NTP TR 465 

to test for overall differences among exposure groups, and multiple comparisons were made with Dum’s 

test or Wilcoxon’s test (Conover, 1971). 

Analyses of covariance (Neter and Wasserman, 1974) with average litter size as the covariate, were 

performed to remove the potential effect of number of pups per litter on average pup weight. Leastsquares estimates of exposure group means adjusted for litter size were tested for overall equality by an 

F-test and for pairwise equality by Dunnett’s test (Dunnett, 1955) or a t-test; these tests were performed 

on males, females, and males and females (combined) to analyze potential sex differences. 

For vaginal cytology data, an arcsine transformation was used to bring the data into closer conformance 

with normality assumptions. Treatment effects were investigated by applying a multivariate analysis of 

variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements 

across exposure concentrations (Tables N6 and N10). 

Incidences of lesions were analyzed by the Fisher exact test. 

RESULTS 

During the continuous breeding phase, exposure to phenolphthalein caused significant reproductive toxicity 

in F, mice. The pregnancy indices for the 7,000 and 30,000 ppm exposure groups were significantly 

lower than those of the controls for most litters, and only one pair in the 30,000 ppm exposure group 

delivered a fifth litter (Table Nl). The average numbers of litters per pair of the 7,000 and 30,000 ppm 

exposure groups were significantly lower than that of the controls. The cumulative days to litter for the 

7,000 and 30,000 ppm exposure groups were significantly greater than that for the controls from litter 2 

through litter 4. Although the body weights of exposed dams were generally similar to those of the 

controls at delivery (data not shown), the body weight of dams in the 30,000 ppm exposure group was 

significantly lower than that of the controls on lactation day 0. Body weights of dams in the 7,000 and 

30,000 ppm exposure groups during lactation of litter 5 were significantly lower than those of the controls 

from lactation days 4 to 14, and the body weight of dams in the 7,000 ppm exposure group was 

significantly lower than that of the controls on lactation day 21. The numbers of live male, live female, 

and total live pups per litter in the 7,000 and 30,000 ppm exposure groups were significantly lower than 

those in the controls (Table Nl). The adjusted total weight of live pups in the 30,000 ppm exposure 

group was significantly lower than that in the controls. 

The numbers of live pups per litter in the 7,000 and 30,000 ppm exposure groups were significantly lower 

than those in the controls for most litters and for the combined litters (Table N2). The adjusted total 

weight of live pups in litters 2, 3, and 4 and in the combined litters 1 through 5 in the 30,000 ppm 

exposure group were significantly lower than those of the controls. 

The survival of male pups in litter 5 in the 30,000 ppm exposure group was significantly lower than that 

in the controls on lactation days 4 through 21 (Table N3). The total survival of male and female pups in 

the 30,000 ppm exposure group was significantly lower than that of the controls on lactation days 7 and 

14. Body weights of male pups in the 7,000 ppm exposure group were significantly lower than those of 

the controls on lactation days 14 and 21; the single surviving pup in the 30,000 ppm exposure group also 

weighed less than the control pups. 

For dams in the 7,000 ppm exposure group of the crossover mating trial, the average body weight at 

delivery and the number of days to litter were significantly lower than those of the controls (Table N4). 

The numbers of live male, live female, and total live pups per litter by dams in the 7,000 ppm exposure 

group were significantly lower than those of the controls. There were no significant differences in 
Continuous Breeding Study 327 

fertility, reproductive performance, or length of gestation between 0 ppm dams mated with 7,000 ppm 

males and those mated with 0 ppm males. Functionally, males were affected substantially less than 

females by exposure to phenolphthalein. 

All mice in the control and 7,000 ppm F, groups were necropsied. In females, there were no treatmentrelated differences in body or organ weights (Table N5). There was no significant difference in the length 

or pattern of the estrous cycle between females in the control and 7,000 ppm groups (Table N6). The 

absolute and relative right epididymis and right testis weights of 7,000 ppm F, males were significantly 

lower than those of the controls (Table N5). The absolute weight of the kidneys (with adrenal glands) of 

the 7,000 ppm F, males was significantly greater than that of the controls. The sperm concentration in 

7,000 ppm F, males was significantly lower than that in the controls, and the percent of abnormal sperm 

in this group was significantly greater than that in the controls (Table N6). There was evidence of 

seminiferous tubule degeneration (9/lo), interstitial hyperplasia (4/10), and epididymal degeneration (7110) 

in males in the 7,000 ppm exposure group (Table N7). 

Because of excessive toxicity at 30,000 ppm, the reproductive effects on the F, mice in the offspring 

assessment phase were evaluated only at 0, 1,000, and 7,000 ppm. At the time of mating, there were no 

treatment-related differences in body weights (data not shown). The pregnancy index of the 7,000 ppm 

exposure group was significantly lower than that of the controls (Table N8). The length of gestation for 

the 7,000 ppm exposure group was significantly greater than that of the controls. In 7,000 ppm group, 

fewer than half of the pairs that mated delivered any live young. The numbers of live male pups and total 

live pups per litter and the sex ratio in the 7,000 ppm exposure group were significantly lower than those 

of the controls. The body weights of male, female, and total pups in the 7,000 ppm exposure group were 

significantly lower than those of the controls. 

The necropsy body weight and absolute liver weight of 7,000 ppm F, females were significantly lower 

than those of the controls (Table N9). There were no significant differences in estrous cycle parameters 

between control and exposed females (Table N10). The absolute and relative right epididymis and right 

testis weights of the 7,000 ppm F, males were significantly lower than those of the controls (Table N9), 

but the sperm counts were similar to that of the controls despite the lower epididymis and testis weights 

(Table N10). However, the percent abnormal sperm in the 7,000 ppm F, males was significantly greater 

than that in the controls. Incidences of lesions in the testis and epididymis were similar between control 

and 7,000 ppm males (Table N1 1). 

In summary, phenolphthalein at concentrations up to 30,000 ppm produced significant reproductive 

toxicity in the absence of changes in body .or somatic organ weights. While the second generation did not 

appear markedly more affected than the first, the pattern of effect was qualitatively different. F, males 

had functioning spermatogenesis at the start of the study. Toxicant-induced decreases in this process 

would translate into reduced sperm counts and testicular and epididymal weights, and these effects were 

observed in this study. In developing F, males, the toxic effect appeared as a larger decrease in testis 

weights (50% in F, males vs. 35% in F, males) with no significant decrease in sperm count and with no 

significant active lesions present in the seminiferous tubule epithelium. This pattern of response suggests 

an effect on the development of the testis, which would be consistent with an estrogenic effect, an antiandrogenic effect, or a thyroid gland effect. An anti-androgenic effect would not explain the effects on 

female reproduction that were observed, so the more likely explanation may be an estrogenic effect and/or 

interference with thyroid hormone economy. Additional studies are necessary to explore these effects. 
328 Phenolphthalein, NTP TR 465 

TABLEN1 

Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F, and F, Swiss (CD-la) Mice in the Continuous Breeding Study of Phenolphthaleina 

F, Adult Data 

Pregnancy indexb 

Litter 1 

Litter 2 

Litter 3 

Litter 4 

Litter 5 

Average littedpair 

Cumulative days to litter 

Litter 1 

Litter 2 

Litter 3 

Litter 4 

Litter 5 

Dam weight during lactation of litter 5 (g) 

n 

Lactation day 0 

Lactation day 4 

Lactation day 7 

Lactation day 14 

Lactation day 2 1 

F, Pup Data (Litters 1 Through 5) 

0 PPm 

38/38 (100%) 

38/38 (100%) 

38/38 (100%) 

38/38 (100%) 

34/38 (89%) 

4.9 f 0.1 

22.3 f 0.7 

42.4 f 0.7 

63.3 f 1.0 

84.3 f 1.2 

103.2 f 0.7 

37 

48.2 f 0.8 

50.4 f 0.9 

53.0 f 0.7 

54.6 f 0.9 

44.8 f 0.9d 

1,000 ppm 

18/18 (100%) 

18/18 (100%) 

18/18 (100%) 

18/18 (100%) 

18/18 (100%) 

5.0 f 0.0 

21.4 & 0.3 

41.2 f 0.3 

61.7 f 0.5 

82.8 f 0.9 

103.9 f 1.0 

18 

47.7 f 1.0 

49.0 f 1.0 

51.7 f 1.3 

53.2 f 1.6 

43.4 f l.Se 

7,000 ppm 

19/19 (100%) 

17/19 (89%)* 

16/19 (84%)* 

13/19 (68%)* 

5/19 (26%)* 

3.7 f 0.3* 

29.7 f 5.8 

47.9 f 3.1* 

74.8 f 3.5* 

94.7 f 3.6* 

101.8 f 0.8 

14 

45.3 f 1.0 

43.9 f 1.2* 

44.0 f 1.7* 

44.4 f 1.9* 

40.2 f 1.5*f 

30,000 ppm 

17/19 (89%)* 

14/19 (74%)* 

10/19 (53%)* 

4/19 (21 %)* 

1/19 (5%)* 

2.7 f 0.3* 

24.9 f 2.0 

51.1 f 3.3* 

76.2 f 5.0* 

93.5 f 3.9* 

113.0' 

4 

43.8 f 0.9* 

40.8 f 0.4* 

41.6 f 1.5* 

43.7 f 1.3* 

41.9 f 0.7 

Number of litters 38 17 . 

Live male pupsllitter 6.5 f 0.2 6.2 f 0.4 2.8 f 0.5*: 2.5 f Q.4* 

Live female pupdlitter 6.5 f 0.2 5.9 f 0.4 2.9 f 0.4*! 2.9 f 0.4* 

Total live pupsllitter 13.0 f 0.3 12.1 f 0.7 5.7 0:9*' 5.4 f 0.6* 

Live pupsllitter (%) 99 f 0 97 f 2 83 f 7' 93 f 4 

Sex ratiog (%) 50 f 1 51 f 1 49 f 3 48 f 5 

Male pup weight (9) 1.62 f 0.01 1.61 f 0.03 1.75 f 0.05 1.62 f O.Me 

Total live pup weight (g) 

Female pup weight (9) 

1.59 f 0.01 

1.56 f 0.01 

1.57 f 0.03 

1.53 f 0.03 

1.70 f 0.04 

1.65 f 0.03 

1.60 

*0.04 

1.56 f 0.04 

Adjusted total pup weigh8 (9) 1.66 f 0.02 1.62 f 0.03 1.61 f 0.03 1.49 f 0.04* 

* Significantly different (P 50.05) from the control group by a chi-square test (pregnancy indices), Shirley's test (average litterslpair, 

cumulative days to litter, dam weights, and live pups/litter), or Dunnett's test (adjusted pup weights) a Data for average litterslpair, cumulative days to litter, body weights, live pupsllitter, and sex ratio are given as mean f standard error. 

Differences from the control group for sex ratio and nonadjusted pup weights are not significant by DUM'Stest. 

b Pregnant femaleslcohabiting pairs C n= 1; no standard error calculated 

d n=36 e n= 16 

f n= 13 

g Live male pupdlive pups h Least-squares estimate of the mean of the average pup weight adjusted for average litter size I n= 19 

18 
Continuous Breeding Study 329 

TABLEN2 

Litter and Body Weight Data for F, Swiss (CD-13 Mouse Pups in the Continuous Breeding Study 

of Phenolphthaleina 

1,000 ppm 7,000 ppm 30,000 ppm 

Litter 1 

Number of pairs delivering 38 17 15 15 

Live pups/litterb 12.2 f 0.5 10.7 f l.Oe 6.4 f l.l*f 8.1 f 1.2*g 

Total live pup weightc (8) 1.60 f 0.02 1.64 f 0.06 1.72 f 0.06 1.63 f 0.05 

Adjusted total live pup weightd (9) 1.66 f 0.02 1.66 f 0.03 1.62 f 0.03 1.56 f 0.03 

Litter 2 

Number of pairs delivering 38 18 16 14 

Live pups/litter 13.4 f 0.5 12.2 f 1.0 6.9 f l.l*g 5.0 f 0.8* 

Total live pup weight (g) 1.59 f 0.02 1.56 f 0.04 1.71 f 0.05 1.63 f 0.06 

Adjusted total live pup weight (8) 1.66 f 0.03 1.62 f 0.04 1.61 f 0.04 1.46 f 0.05* 

Litter 3 

Number of pairs delivering 38 18 15 8 . 

Live pupdlitter 13.6 f 0.5 12.6 f 1.0 5.1 f l.O*h 3.4 f 1.1*’ 

Total live pup weight (g) 1.59 f 0.02 1.58 f 0.04 1.75 f 0.05* 1.48 f 0.08 

Adjusted total live pup weight (g) 1.67 f 0.02 1.63 f 0.03 1.59 f 0.04 1.30 f 0.05* 

Litter 4 

Number of pairs delivering 38 18 12 . 4 

Live pupdlitter 13.2 f 0.5 12.9 f 1.0 5.7 f 1.04 3.8 f 1.9* 

Total live pup weight (9) 1.61 f 0.02 1.57 f 0.04 1.68 f0.05 1.48 f 0.14 

Adjusted total live pup weight (g) 1.66 f 0.02 1.61 f 0.03 1.53 f 0.05 1.27 f 0.08* 

Litter 5 

Number of pairs delivering 34 18 5 1 

Live pupsllitter 12.6 f 0.6 12.4 f 0.8 10.2 f 0.6* 2.0k 

Total live pup weight (8) 1.66 f 0.03 1.62 f 0.03 1.59 f 0.07 1 .66k 

Adjusted total live pup weight (g) 1.68 f 0.02 1.62 f 0.03 1.53 f 0.06 1.36 f 0.14 

Litters 1 Through 5 

Number of pairs delivering 38 18 17 17 

Live pups/litter 13.0 f 0.3 12.1 f 0.7 5.7 f 0.9*f 5.4 f 0.6* 

Total live pup weight (g) 1.59 f 0.01 1.57 f 0.03 1.70 f 0.04 1.60 f 0.04 

Adjusted total live pup weight (g) 1.66 f 0.02 1.62 f 0.03 1.61 f 0.03 1.49 f 0.04* 

* Significantly different (P20.05) from the control group by DUM’S or Shirley’s test (live pupsllitter and total live pup weights) or 

Dunnett’s test (adjusted pup weights) a Data are given as mean f standard error. b Mean of average number of live pups per litter for each fertile pair C Mean of average live pup weight for each fertile pair d Least-squares estimate of mean pup weight adjusted for average litter size e n=18 

f n= 19 

g n= 17 

h n= 16 

i n= 10 

j n=13 

k n= 1; no standard error calculated 
330 Phenolphthalein, NTP TR 465 

TABLEN3 

Survival and Body Weights of F, Swiss (CD-la) Mouse Pups (Final Litter) 

in the Continuous Breeding Study of Phenolphthaleina 

0 PPm 1,000 ppm 7,000 ppm 30,000 ppm 

Day 0 

Number of litters 37b 18 12c 3d 

Male pup weight (9) 1.67 f 0.03 1.65 f 0.03 1.65 f 0.05 1.50 f 0.20 

Female pup weight (g) 1.63 f 0.03 1.59 f 0.03 1.63 f 0.04 1.49 f 0.09 

Day 4 

Male survival (%) 99 f 1 98 f 1 88 f 13 29 f 29* 

Female survival (%) 97 f 1 91 f6 75 f 13 67 f 33 

Total survival (%) 98 f 1 95 f 2 75 f 13 56 f 29 

Male pup weight (8) 3.34 f 0.08 3.37 f 0.09 3.28 f 0.25= 1.89' 

Female pup weight (g) 3.29 f 0.09 3.28 f 0.1Og 3.02 f 0.24h 2.39 f 0.65 

Day 7 

Male survival (%) 99 f 1 98 f 1 75 f 16 29 f 29* 

Female survival (%) 97 f 1 91 f6 67 f 14 67 f 33 

Total survival (X) 98 f 1 95 f 2 67f14 , 56 f 29* 

Male pup weight (g) 5.05 f 0.13b 5.09 f 0.14 5.53 f 0.23: 3.06' 

Female pup weight (g) 4.96 f 0.16 4.93 f 0.14g 4.93 f 0.36' 3.92 f 1.03k 

Day 14 

Male survival (%) 99 f 1 98 f 1 75 f 16 29 f 29* 

Female survival (%) 97 f 1 90 f 6 67 f 14 67 f 33 

Total survival (%) 98 f 1 95 f 2 67 f 14 , 56 f 29* 

Male pup weight (g) 7.68 f 0.22 7.88 f 0.39 9.18 f 0.56*' 6.35' 

Female pup weight (g) 7.72 f 0.30 7.52 f 0.3Og 8.68 f 0.47' 8.13 f 1.97k 

Day 21 

Male survival (%) 97 f 1 92 f 6 75 f 16 29 f 29* 

Female survival (X) 96 f 2 84 f 8 67 f 14 67 f 33 

Total survival (%) 97 f 1 89 f 6 67 f 14 , 56 f 29 

Male pup weight (g) 11.98 f 0.41' 12.84 f 0.748 14.16 f 0.64*' 8.02f 

Female pup weight (9) 11.49 f 0.41 11.76 f 0.54m 12.54 f 0.6d 10.43 f 3.4gk 

* Significantly different (P10.05)from the control group by Shirley's test a Data are given as mean f standard error. 

Because no live male pups were born in one litter, n=36 for male pup survival and body weights. 

Because no live male pups were born in four litters, n=8 for male pup survival and body weights. 

Because no live male pups were born in one litter, n=2 for male pup survival and body weights. 

e n=7 

n=l; no standard error calculated 

n=17 

h n=9

n=6 

j n=8 

n=2 ' n=35 

n=16 

I 
Continuous Breeding Study 331 

TABLEN4 

Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F, and F, Swiss (CD-l@) Mice in the Crossover Mating Trial of Phenolphthaleina 

Male Exposure Group 0 PPm 7,000 ppm 0 PPm 

Female Exposure Group 0 PPm 0 PPm 7,000 ppm 

F, Adult Data 

Mating indexb 14/18 (78%) 18/19 (95%) 13/18 (72%) 

Pregnancy index' 11/18 (61%) 14/19 (74%) 10/18 (56%) 

Fertility indexd 11/14 (79%) 14/18 (78%) 10113 (77%) 

Average dam weight at delivery (g) 46.8f 1.0 47.5 f 1.0 41.7f 1.0* 

Days to litter 19.0f 0.0 19.6f 0.2 19.9f 0.2* 

F, Pup Data 

Number of litters 11 14 IO 

Live male pupsllitter 6.0f 0.7 6.3f 0.9 2.3 f 0.5* 

Live female pupdlitter 6.0f 0.8 4.7f 0.6 3.2f 0.7* 

Total live pupsllitter 12.0 f 1.0 11.0f 1.3 5.5 f 1.2* 

Total live pupsllitter (%) 100 f 0 94 f 6 85 f 11 

Sex ratioe (%) 51 f5 52 f 6 38 f 5g , 

Male pup weight (g) 1.67f 0.04 1.69f 0.05h 1.81 f 0.10' 

Female pup weight (g) 1.60 f 0.03 1.63 f 0.06 1.74f 0.07g 

Total live pup weight (g) 1.64f 0.03 1.67f 0.05 1.77 f 0.08g 

Adjusted total live pup weight'(g) 1.70 f 0.04 1.71 f 0.04 1.63 f 0.05g 

* Significantly different (P10.05) from the control group by Dunn's test a Data for body weights, days to litter, live pupdlitter, and sex ratio are given as mean f standard error. Differences from the control 

group for mating, pregnancy, and fertility indices were not significant by a chi-square test; differences for sex ratio and pup weights 

were not significant by Dunn's test; and differences for adjusted pup weights were not significant by a t-test. 

Females with sperm pluglcohabiting pairs 

Pregnant females/cohabiting pairs 

Pregnant femaleslfemales with sperm plug 

e Live male pups/live pups 

Least-squares estimate of the mean pup weight adjusted for average litter size 

g n=9 

n=13 

n=8 

h

' 
332 Phenolphthalein, NTP TR 465 

TABLEN5 

Organ Weights and Organ-Weight-to-Body-Weight Ratios 

for F, Swiss (CD-l@)Mice Administered Phenolphthalein in Feeda 

Male 

n 

Necropsy body wt 

R. Cauda Epididymis 

Absolute 

Relative 

R.Epididymis 

Absolute 

Relative 

Kidneys and Adrenal Glands 

Absolute 

Relative 

Liver 

Absolute 

Relative 

Prostate Gland 

Absolute 

Relative 

Seminal Vesicles 

Absolute 

Relative 

R. Testis 

Absolute 

Relative 

Female 

n 

Necropsy body wt 

Kidneys and Adrenal Glands 

Absolute 

Relative 

Liver 

Absolute 

Relative 

R. Ovary 

Absolute 

Relative 

* 

0 PPm 7,000 ppm 

39 20 

42.4 f 0.71 42.0 f 0.95 

18.0 f 0.37 17.7 f 0.79 

0.43 f 0.01 0.42 f 0.02 

56.6 f 0.98 37.5 f 3.5* 

1.3 f 0.03 0.91 f 0.09* 

834.0 f 12.6 870.7 f 17.2* 

19.8 f 0.36 20.9 f 0.64 

2.2 f0.04 2.3 f 0.06 

51.8 f 0.76 54.1 f 0.69 

25.0 f 1.1 25.5 f 1.4 

0.59 f 0.03 0.61 f 0.04 

692.1 f 18.0 686.3 f 20.4 

16.4 f 0.37 16.4 f 0.43 

142.2 f 3.1 93.3 f 4.6* 

3.4 f 0.08 2.2 f 0.12* 

37 18 

40.5 f 0.70 39.0 f 0.66 

645.7 f 13.1 634.6 f 20.3 

16.0 f0.28 16.3 f 0.43 

2.3 f 0.05 2.3 f 0.06 

57.3 f 0.87 57.9 f 1.1 

11.3 f 0.44 11.2 f 0.65 

0.28 f 0.01 0.29 f 0.02 

Significantly different (PSO.05) from the control group by Wilcoxon’s test a Liver weights and body weights are given in grams; other organ weights are given in milligrams; organ-weight-to-body-weight ratios are 

given as mg organ weight/g body weight (mean f standard error). 
Continuous Breeding Study 333 

TABLEN6 

Sperm Parameters and Estrous Cycle Characterization 

for F, Swiss (CD-13 Mice Administered Phenolphthalein in Feeda 

0 PPm 7,000 ppm 

Male 

n 39 20 

Epididymal spermatozoal parameters 

Motility (%) 91.0 f 0.6 86.7 f 2.8b 

Abnormal (%) 2.6 f 0.3 4.1 f OS*‘ 

Concentration 

(106/g cauda epididymal tissue) 1,167 f 37 816 f 88* 

Female 

n 37 18 

Estrous cycle length (days) 4.92 f 0.12d 4.68 f 0.17e 

Estrous stages (% of cycle) 

Diestrus 47.1 45.8 

Proestrus 14.2 14.4 

Estrus 25.7 26.4 

Metestrus 13.1 13.4 

* Significantly different (P10.05) from the control group by Wilcoxon’s test 

a Epididymal spermatozoal parameters and estrous cycle lengths are given as mean f standard error. Differences from the control group 

for epididymal spermatozoal motility are not significant by Wilcoxon’s test. By multivariate analysis of variance, exposed females do 

not differ significantly from control females in cycle length or in the relative length of time spent in estrous stages. 

n=19 

n=18 

Estrous cycle length was longer than 7 days or was unclear in 7 of 37 animals. 

e Estrous cycle length was longer than 7 days or was unclear in 4 of 18 animals. 
334 Phenolphthalein, NTP TR 465 

TABLEN7 

Incidences of Selected Nonneoplastic Lesions in Male F, Swiss (CD-l@)Mice 

Administered Phenolphthalein in Feed 

7,000 ppm 

Testisa 

Interstitial Cell Hyperplasia 

Seminiferous Tubule, Degenerationb 

11 

2 

0 

(2.OY 

4* 

9** 

10 

(1.3) 

(1.9) 

Epididymis 

Presence of FluidlDegenerated Cell 

Epithelial Cell, Degeneration 

1 

5 

11 

(4.0) 

(1.0) 

7** (1.3) 

7 (1.1) 

10 

* Significantly different (P10.05) from the control group by the Fisher exact test 

** P10.01 

a Number of animals with organ examined microscopically 

Number of animals with lesion 

Average severity grade of lesions in affected animals: 1 = minimal; 2 = mild; 3 = moderate; 4 = severe 
Continuous Breeding Study 335 

TABLEN8 

Fertility, Reproductive Performance, Length of Gestation, and Body Weight Data 

for F, and F, Swiss (CD-l@) Mice in the Offspring Assessment Phase 

of the Continuous Breeding Study of Phenolphthaleina 

F, Adult Data 

Mating indexb 19/20 (95%) 20/20 (100%) 19/20 (95%) 

Pregnancy index‘ 17/20 (85%) 17/20 (85 %) 9/20 (45 %)* 

Fertility indexd 17/19 (89%) 17/20 (85 %) 9/19 (47%) 

Dam weight at delivery (g) 35.9 f 0.8 37.1 f 0.9 34.8 f 0.8 

Days to litter 19.1 f 0.2e 19.0 f 0.1 19.7 f 0.2* 

F2Pup Data 

Number of litters 17 17 9 

Live male pups/litter 4.7 f 0.6 5.5 f 0.5 1.6 f 0.4* 

Live female pupsllitter 4.5 f 0.5 6.3 f 0.6 3.3 f 0.4 

Total live pupsllitter 9.2 f 1.0 11.8 f 0.5 4.9 f 0.8* 

Total live pups/litter (%) 94 f 6 100 f 0 100 f 0 

Sex ratiof (%) 47 f 5e 47 f 4 25 f 7* , 

Male pup weight (9) 1.67 f 0.03h 1.60 f 0.03 1.88 f 0.05*’ 

Female pup weight (g) 1.61 f 0.04e 1.51 k 0.03 1.76 f 0.04* 

Total live pup weight (g) 1.66 f 0.04e 1.55 f 0.03 1.80 f 0.03* 

Adjusted total live pup weightg (9) 1.66 f 0.03e 1.61 f 0.03 1.67 f 0.05 

* Significantly different (P50.05)from the control group by a chi-square test (pregnancy index) or Dunn’s or Shirley’s test (days to litter, 

live pupsllitter, sex ratio, and pup weights) 

a Data for body weights, days to litter, live pupsllitter, and sex ratio are given as mean f standard error. Differences from the control 

group for mating and fertility indices are not significant by a chi-square test, differences for dam weights were not significant by Dunn’s 

test, and differences for adjusted pup weights are not significant by Dunnett’s test. 

Females with sperm pluglcohabiting pairs 

Pregnant females/cohabiting pairs 

Pregnant females/females with sperm plug 

e n=16 

Live male pupsllive pups 

g Least-squares estimate of mean pup weight adjusted for average litter size 

n=15 

n=6 
336 Phenolphthalein, NTP TR 465 

TABLEN9 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for F, Swiss (CD-la) Mice 

in the Offspring Assessment Phase of the Continuous Breeding Study of Phenolphthaleina 

0 PPm 1,000 ppm 7,000 ppm 

n 20 20 20 

Male 

Necropsy body wt 34.9 f 0.73 34.7 f 0.49 33.4 f 0.44 

R. Cauda Epididymis 

Absolute 14.7 f 0.55 15.2 f 0.50 13.2 f 0.53 

Relative 0.42 f 0.02 0.44 f 0.02 0.40 f 0.02 

R. Epididymis 

Absolute 45.1 f 1.5 47.6 f 0.96 37.4 f 1.4* 

Relative 1.3 f 0.05 1.4 f 0.03 1.1 f 0.03* 

Kidneys and Adrenal Glands 

Absolute 723.3 f 20.5 723.1 f 18.8 709.6 f 13.3 

Relative 20.8 f 0.50 20.9 f 0.50 21.3 f 0.41 

Liver 

Absolute 1.8 f 0.05 1.9 f 0.05 1.8 f 0.04 

Relative 51.6 f 1.0 54.0 f 1.0 53.8 f 1.0 

Prostate Gland 

Absolute 19.9 f 1.3b 18.1 f 1.5 16.0 f 1.2b 

Relative 0.57 f 0.03b 0.52 f 0.04 0.48 f 0.03b 

Seminal Vesicles 

Absolute 400.4 * 12.2 387.9 f 13.5 398.2 * 14.3 

Relative 11.5 f 0.32 11.2 f 0.38 11.9 f 0.41 

R. Testis 

Absolute 134.7 f 6.2 139.5 f 4.7 74.5 f 5.5* 

Relative 3.9 f 0.18 4.0 f 0.14 2.2 f 0.15* 

Female 

Necropsy body wt 30.9 f 0.61 30.7 f 0.67 29.0 f 0.55* 

Kidneys and Adrenal Glands 

Absolute 514.7 f 23.4 478.3 f 10.5 489.2 f 9.3 

Relative 16.7 f 0.67 15.6 f 0.24 17.0 f 0.38 

Liver 

Absolute 1.8 f 0.08 1.7 f 0.04 1.6 f 0.04*, 

Relative 57.5 f 2.1 56.2 f 0.99 54.8 f 1.4 

R. Ovary 

Absolute 9.1 f 0.60b 7.9 f 0.53 8.1 f 0.50 

Relative 0.30 f 0.02b 0.26 f 0.02 0.28 f 0.02 

* Significantly different (PIO.05) from the control group by Shirley’s test a Liver weights and body weights are given in grams; other organ weights are given in milligrams; organ-weight-to-body-weight ratios are 

given as mg organ weighug body weight (mean f standard error). 

n=19 
Continuous Breeding Study 337 

TABLEN10 

Sperm Parameters and Estrous Cycle Characterization for F, Swiss (CD-l@)Mice 

in the Offspring Assessment Phase of the Continuous Breeding Study of Phenolphthaleina 

1,000 ppm 7,000 ppm 

Male 

n 20 20 20 

Epididymal spermatozoal parameters 

Motility (%) 79.4 f 5.5 85.3 f 3.6 83.3 f 4.7 

Abnormal (%) 2.2 f 0.3 2.4 f 0.2 4.9 f0.8* 

Concentration 

(10'lg cauda epididymal tissue) 970 f 85 1.087 f 44 772 f 86 

Female 

n 20 20 20 

Estrous cycle length (days) 4.90 f 0.13 4.84 f 0.14b 5.03 f 0.12' 

Estrous stages (% of cycle) 

Diestrus 25.4 22.1 24.2 

Proestrus 18.3 18.3 16.7 

Estrus 39.2 42.9 45.8 

Metestrus 16.7 16.2 12.9 

Uncertain diagnoses 0.4 0.4 0.4 

* Significantly different (PSO.05) from the control group by Shirley's test 

a Epididymal spermatozoal parameters and estrous cycle lengths are given as mean f standard error. Differences from the control group 

for epididymal spermatozoal motility and concentration are not significant by Dunn's test. By multivariate analysis of variance, exposed 

females do not differ significantly from control females in cycle length or in the relative length of time spent in estrous stages. 

Estrous cycle length was longer than 7 days or was unclear in 1 of 20 animals. 

Estrous cycle length was longer than 7 days or was unclear in 3 of 20 animals. 
338 Phenolphthalein, NTP TR 465 

TABLEN11 

Incidences of Selected Nonneoplastic Lesions in Male F, Swiss (CD-l@) Mice 

in the Offspring Assessment Phase of the Continuous Breeding Study of Phenolphthalein 

0 PPm 7,000 ppm 

Testisa 10 10 

Seminiferous Tubule, Degenerationb 5 (1.21C 7 (1.3) 

Interstitial Cell Hyperplasia 0 1 (2.0) 

Epididymis 10 IO 

Epithelial Cell, Degeneration 4 (1.3) 6 (1.2) 

Presence of Fluid/Degenerated Cell 1 (1.0) 1 (2.0) 

a Number of animals with organ examined microscopically 

Number of animals with lesion 

Average severity grade of lesions in affected animals: 1 = minimal; 2 = mild; 3 = moderate: 4 = severe 
339 

APPENDIX 0 

SINGLE-DOSE TOXICOKINETIC STUDIES 

IN F344/N RATS AND B6C3Fl MICE 

INTRODUCTION ....................................................... 340 

MATERIALS AND METHODS . . . . . . . ... . . . . .. . . . . . . . . . . . . ... . . . . . . . . . . . ... . .. 340

RESULTS ........................................................... 341 

TABLE01 Plasma Concentrations of Total Phenolphthalein in F344/N Rats and B6C3Fl Mice 

After a Single GavageDose of Phenolphthalein . . . . . . .. . .. . . . . . . . . . .. . . .. 343 

FIGURE 01 Plasma Concentrations of Total Phenolphthalein in Male F344/N Rats 

After a Single Gavage Dose of 100 or 250 mg/kg . . . . . . . .. . . .. . . . . . . . . .. . . 344 

FIGURE 02 Plasma Concentrations of Total Phenolphthalein in Female F344/N Rats 

After a Single Gavage Dose of 100 or 250 mg/kg . . ... . . . . . . . . . . . . . . . . . . . . 344 

FIGURE03 Plasma Concentrations of Total Phenolphthalein in Male and Female F344/N Rats 

After a Single Gavage Dose of 100 mg/kg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 

FIGURE 04 Plasma Concentrations of Total Phenolphthalein in Male and Female F344/N Rats 

After a Single GavageDose of 250 mg/kg . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 345 

FIGURE 05 Plasma Concentrations of Total Phenolphthalein in Male B6C3Fl Mice 

After a Single Gavage Dose of 50 or 125 mg/kg . . .. . . . . . . .. . . .. . . . . . . . . . 346 

FIGURE 06 Plasma Concentrations of Total Phenolphthalein in Female B6C3Fl Mice 

After a Single Gavage Dose of 50 or 125 mg/kg . . . . . . . . . . . . . . . . . . . . . . ... 346 

FIGURE 07 Plasma Concentrations of Total Phenolphthalein in Male and Female B6C3Fl Mice 

After a Single Gavage Dose of 50 mg/kg . . . . . .... . . . ... . . . . . .. . ... . ... 347 

FIGURE 08 Plasma Concentrations of Total Phenolphthalein in Male and Female B6C3Fl Mice 

After a Single Gavage Dose of 125 mg/kg . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 347 

TABLE02 Pharmacokinetic Parameters in F344/N Rats After a Single Gavage Dose 

of Phenolphthalein . . . . . . . . . . . . . . . .. . . . . .. ... . . . . .. . . . .. . .. . . .. 348 

TABLE03 Pharmacokinetic Parameters in B6C3FlMiceAfter a Single Gavage Dose 

of Phenolphthalein ... . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . 348 
340 Phenolphthalein, NTP TR 465 

SINGLE-DOSE TOXICOKINETIC STUDIES 

IN F344/N RATS AND B6C3Fl MICE 

INTRODUCTION 

Phenolphthalein is an isobenzofuranone derivative that is used as a cathartic and laxative. The single-dose 

toxicokinetic studies conducted in F344/N rats and B6C3F, mice were performed to determine the 

pharmacokinetic profile and elimination kinetics of phenolphthalein in rats and mice following a single 

dose by gavage (NTP, 1994). 

MATERIALSAND METHODS 

Phenolphthalein was obtained from Pharmco Laboratories, Inc. (Titusville, FL) in one lot (P9189-J1), 

which was also used for the 2-year studies conducted at TSI Mason Laboratories (Worcester, MA). 

Results of purity and stability analyses of lot P9189-J1 are given in Appendix J. Dose formulations were 

prepared as needed by suspending appropriate amounts of phenolphthalein in 0.5 % aqueous 

methylcellulose, and analyses by the study laboratory using high-performance liquid chromatography 

(HPLC) indicated that the dose formulations were homogeneous and within 10.7% of the target 

concentrations. Stability studies of a 5 mg/mL dose formulation conducted by the study laboratory 

indicated that suspensions are stable for up to 14 days when stored at room temperature and protected 

from light in sealed vials. 

F344/N rats and B6C3F, mice wereobtainedfromTaconic Farms (Germantown, NY). Upon receipt, the 

rats and mice were observed for parasites and indications of disease. The rats were quarantined for 

140 (males) or 141 (females) days and were 17 1 (males) or 172 (females) days old at the start of the 

studies. The mice were quarantined for 145 (males) or 146 (females) days and were 177 (males) or 

178 (females) days old at the start of the studies. Filtered municipal water and NIH-07 open formula meal 

diet were available ad libitum. During the studies, the rats administered 100 mg phenolphthaleinlkg body 

weight were housed three per cage, and the rats administered 250 mg/kg were housed two per cage. The 

mice were housed separately. 

Doses and sampling time points for the single-dose toxicokinetic studies were selected based on analysis of 

data from preliminary toxicokinetic studies and in preparation for the 2-year toxicokinetic studies. 

Groups of 30 male and 30 female rats were administered a single dose of 100 mg phenolphthaleidkg body 

weight by gavage, and groups of 14 male and 16 female rats were administered 250 mg/kg. The dosing 

volume was 5 mL/kg. Groups of 30 male and 30 female mice were administered 50 mg/kg, and groups of 

14 male and 16 female micewereadministered125 mg/kg. The dosing volume was 10 mL/kg. The 

animals were anesthetized with a mixture of carbon dioxide and oxygen, and blood samples were collected 

from the retroorbital sinus. Blood samples were collected from three male and three female 100 mg/kg 

rats and from three male and three female 50 mg/kg mice per time point at 5, 15, and 30 minutes and at 

2, 4, 6, 10, 24, 34, and 48 hours after the administration of phenolphthalein. Blood samples were 

collected from two 250 mg/kg male rats and two 125 mg/kg male mice per time point at 30 minutes and at 

3, 5, 8, 24, 34, and 48 hours after the administration of phenolphthalein; blood samples were collected 

from two 250 mg/kg female rats and two 125 mg/kg female mice per time point at 15 and 30 minutes and 

at 3, 5, 8, 24, 34, and 48 hours after the administration of phenolphthalein. Blood samples were collected 

only once from each animal. The samples were processed by the study laboratory, and the plasma was 

stored at -20" C or lower until analysis. 
Single-Dose Toxicokinetic Studies 341 

All animals were observed twice per day, 7 days per week. Clinical observations were not recorded for 

these studies. Individual body weights were recorded on the first day of the studies for the calculation of 

dosing volumes. 

A plasma analysis procedure was developed and evaluated at the study laboratory for the analysis of 

plasma concentrations of total phenolphthalein (free and conjugated) at concentrations ranging from 0.5 to 

60 pg/mL. Concentrations less than the experimental level of quantitation (ELOQ=0.5 ,ug/mL) should be 

considered approximations. Plasma samples were treated with 0-glucuronidase and sulfatase enzyme 

preparations. Solid-phase extraction with octadecyl packing material was used to isolate total 

phenolphthalein and clean up the extract. The samples were then analyzed using HPLC with a Whatman 

pellicular ODS guard column, a Waters pBondapak 10 pm C,, column, ultraviolet detection (229 nm), and 

a mobile phase of methano1:water:O. 1 N hydrochloric acid (530:470: 10) with a 1.O mL/minute flow rate. 

The average plasma concentrations of total phenolphthalein and standard deviations were calculated. The 

logarithms of these values were plotted as a function of time. The areas under the plasma concentration 

versus time curves (AUC,) and standard errors were calculated using the trapezoid rule of the form 

AUC,= {(C,+C,-,)/2} X {t, -t,,-,}, where AUC, is the cumulative area under the curve to time t and 

Cl,-l and C, are successive concentrations at tn-* and t,,, respectively. The areas under the curves to 

infinity (AUC,") were calculated from AUC," = AUC,+ CJA, where C, is the last measured time point and 

X is the terminal rate constant determined from the slope of the terminal phase of the log plasma 

concentration-time profiles. Linear regression of the last three or four data points gave the slope (X). The 

half-lives (tlh) were calculated as 0.693/A. The total body clearance (Cl) was calculated as dose/AUC,". 

The clearance gives an estimate of the volumetric elimination of the drug from the body. The maximum 

observed concentration (C,,,) and corresponding time (t,,,) were determined from the plasma 

concentration-time data as the maximum observed concentration and corresponding time, respectively. 

RESULTS 

The plasma concentrations of total phenolphthalein and their standard deviations in rats are given in 

Table 01. The semilogarithmic plots of plasma concentration-time data for male and female rats 

administered 100 and 250 mg/kg are shown in Figures 01 through 04 and show linear kinetics in this 

dose range. The AUC,", t,h, C1, C,,, and t,, values for rats are reported in Table 02. The t, values 

were generally similar among groups. For rats administered 100 mg/kg, the t, values were 13.1 hours 

for males and 12.3 hours for females. For rats administered 250 mg/kg, the t, values were 13.8 hours 

for males and 28.3 hours for females. For rats administered 100 mg/kg, the C1 values were 

0.157 mL/hour/kg for males and 0.1 18 mL/hour/kg for females. For rats administered 250 mg/kg, the 

C1 values were 0.286 mL/hour/kg for males and 0.133 mL/hour/kg for females. The C,,, values were 

similar for the male and female rats administered 100 mg/kg. For rats administered 250 mg/kg, 

C,,, values were 49.4 mg/kg for males and 63.4 mg/kg for females. The values were hax generally 

similar for 100 and 250 mg/kg males and females and ranged from 2 to 5 hours. 

The plasma concentrations of total phenolphthalein and their standard deviations in mice are given in 

Table 01. The semilogarithmic plots of plasma concentration-time data for male and female mice 

administered 50 and 125 mg/kg are shown in Figures 05 through 08 and show linear kinetics in this dose 

range. However, the scatter on the 125 mg/kg females did not allow for the accurate estimation of the 

terminal slope, and the slope in this case was determined between the 8- and 48-hour time points. The 

AUC,", tth, C1, C,,, and h, values for mice are reported in Table 03. The t, values were similar 

among groups. For mice administered 50 mg/kg, the t,h values were 6.28 hours for males and 6.68 hours 

for females. For mice administered 125 mg/kg, the t, values were 6.52 hours for males and 6.96 hours 

for females. For mice administered 50 mg/kg, the C1 values were 0.104 mL/hour/kg for males and 
342 Phenolphthalein, NTP TR 465 

0.074 mL/hour/kg for females. For mice administered 125 mg/kg, the C1 values were 0.168 mL/hour/kg 

for males and 0.119 mL/hour/kg for females. For mice administered 50 mg/kg, C,,, values were 

29.5 mg/kg for males and 44.5 mg/kg for females. For mice administered 125 mg/kg, C,,, values were 

50.7 mg/kg for males and 79.0 mg/kg for females. The Laxvalues demonstrated an erratic trend. The 

50 mg/kg males showed a maximum at 4 hours, and the 125 mg/kg males showed a maximum at 

30 minutes. The 50 mg/kg females showed a maximum at 30 minutes, and the 125 mg/kg females 

showed a maximum at 5 hours. This could be due to one or a combination of interanimal, assay, and 

dosing variabilities. 

Statistical analysis of the data has not been performed; however, it appears that 1) the elimination halflives were longer for rats than for mice; 2) the AUC,"s were not dose proportional for either males or 

females or for rats or mice; and 3) the AUC,"s were larger for female rats and mice than for males. 

Thus, these data indicate that females are exposed to higher plasma concentrations of total phenolphthalein 

and that some process, possibly absorption, is saturated at the high dose. 
- - 

Single-Dose Toxicokinetic Studies 343 

TABLE01 

Plasma Concentrations of Total Phenolphthalein in F344/NRats and B6C3F, Mice 

After a Single Gavage Dose of Phenolphthaleina 

100 mg/kg 250 mgkg 50 mgkg 125 mgkg 

Rats Rats Mice Mice 

n 3 L 3 2 

Male 

Time After Dosing (minutes) 

5 1.77 f 0.85 -b 14.30 f 1.97 - 15 6.67 f 1.36 - 22.10 f 10.14 - 30 12.21 f 3.46 14.68 f 7.53 25.10 f 5.08 50.65 f 0.64 

120 27.90 f 0.92 - 28.27 f 3.92 - 180 - 39.00 f 7.78 - 45.55 f 4.17 

240 33.27 f 5.35 - 29.47 f 7.73 

300 - 49.40 f 0.42 - 40.90 f 1.56 

360 29.17 f 1.52 - 24.20 f 3.00 -

480 - 29.45 f 11.53 - 37.85 f 2.62 

600 20.27 f 5.12 24.37 f 3.96 

1,440 8.37 f 2.89 11.75 f 1.34 2.70 f 0.84 5.21 f 0.16 

2.040 6.12 f 2.00 7.94 f 2.18 1.35 f 0.18 1.96 f 0.63 

2,880 2.57 f 0.51 4.17 f 2.64 0.33 f 0.07' 0.61d 

Female 

Time After Dosing (minutes) 

5 2.50 f 0.10 - 16.47 f 0.50 -

15 9.39 f 1.53 10.83 f 4.77 37.20 f 6.49 67.25 f 6.01 

30 17.47 f 5.31 26.20 f 3.39 44.47 f 8.82 71.85 f 6.29 

120 31.00 f 2.46 - 44.20 f 3.63

180 - 57.30 f 11.17 - 74.50 f 1.56 

240 28.10 f 7.14 - 32.00 f 6.68 -

300 - 63.35 f 25.67 - 79.00 f 4.38 

360 27.73 f 4.65 - 40.23 f 9.09 -

480 - 45 .80d 47.40 f 0.14 

600 28.83 f 1.12 - 34.60 f 10.90

1.440 16.83 f 3.16 16.55 f 1.20 2.32 * 1.01 3.00 f 2.03 

2.040 6.44 f 2.00 11.80 f 3.54 0.97 f 0.15 5.95 f 0.33 

2.880 3.93 It 1.27 15.93 f 9.16 0.87 f 0.51e 0.57 f 0.08 

a Data are given in pg/mL asmean f standard deviation. Total phenolphthalein equals free and conjugated phenolphthalein. 

No samples were collected. 

' AI1 values were below the experimental limit of quantitation (0.5 pg/mL). 

n= 1; no standard deviation calculated 

e n=2 
344 Phenolphthalein, NTP TR 465 

100 f

-

- 7 00 mgkg 0 

250rngLkg 

ELOQ -

4 A 

1 

0.1 i

I 1 1 I I I 

0 500 1000 1500 2ODO 2500 3c 00 

Minutes Post Dosing 

FIGURE 01 

Plasma Concentrations of Total Phenolphthalein in Male F344/NRats 

After a Single Gavage Dose of 100 or 250 mg/kg 

Ivu 

10 

E L 00

1

........................................................................................... 

-

0 500 1000 1500 2000 2500 3000 

Minutes Post Dosing 

FIGURE 02 

Plasma Concentrations of Total Phenolphthalein in Female F344/NRats 

After a Single Gavage Dose of 100 or 250 mg/kg 
Single-Dose Toxicokinetic Studies 345 

100 

Male, Q l00mgkg 

Female, 100mgkg 0

2 EL00 - \

2 

E 

-

r 10 

.-0

c 

2 

c 

c 

Q)

0 

8 

$ 1 

E ........................................................................................... v )

Q

B 

0.1 n e 0 1000 1500 2000 2500 3: 30 

Minutes Post Dosing 

FIGURE 03 

Plasma Concentrationsof Total Phenolphthalein in Male and FemaleF344/NRats 

After a Single Gavage Dose of 100 mglkg 

I'00 

Plasma Concentrationsof Total Phenolphthalein in Male and Female F344/NRats 

After a Single Gavage Dose of 250 mg/kg 
346 Phenolphthalein, NTP TR 465 

I 

125 mgkg . 

EL00 -

10 

1 . -1.......................................................................................... 

I 0.1 I I I I I I 

0, 500 15001000 2000 2500 3( 

Minutes Post Dosing 

FIGURE 05 

Plasma Concentrations of Total Phenolphthalein in Male B6C3Fl Mice 

After a Single Gavage Dose of 50 or 125 mg/kg 

100 

10 

1 

........................................................................................h 

0.1 I I 1 I I I 

0 500 1000 1500 2000 2500 3000 

Minutes Post Dosing 

FIGURE 06 

Plasma Concentrations of Total Phenolphthalein in Female B6C3Fl Mice 

After a Single Gavage Dose of 50 or 125 mg/kg 
Single-Dose Toxicokinetic Studies 347 

Male, 50 mgkg 0 

Female, 50 mgn(Sl 

EL00 -

--

-

-

-

-

---

-

-

-

-

I I 1 I 1 I 

0 500 I OOG 1500 2000 2500 3000 

Minutes Post Dosing 

FIGURE07 

Plasma Concentrationsof Total Phenolphthalein in Male and Female B6C3Fl Mice 

After a Single Gavage Dose of 50 mg/kg 

1 

0.1 I I 1 1 1 

0 500 1000 1500 2000 2500 3C 00 

Minutes Post Dosing 

FIGURE OS 

Plasma Concentrationsof Total Phenolphthalein in Male and Female B6C3F, Mice 

After a Single Gavage Dose of 125 mg/kg 
348 Phenolphthalein, NTP TR 465 

TABLE02 

Pharmacokinetic Parameters in F344/N Rats After a Single Gavage Dose of Phenolphthaleina 

t,C Cld t, 

f 

(hours) (mL/hour/kg) (hours) 

Male 

100 638 f 38 13.1 0.157 33.3 4.00 

250 873 f 88 13.8 0.286 49.4 5.OO 

Female 

100 847 f 32 12.3 0.118 31.O 2.00 

250 1,879 f 275 28.3 0.133 63.4 5.00 

a The data were calculated from the plasma concentration-time curves, where each point represents the mean of three male or three female 

100 mg/kg rats or of two male or two female 250 mglkg rats. 

AUC; = area under the curve to infinity 

tH = elimination half-life 

CI = DoselAUCT 

e c,,, = maximummean concentration 

kax= time of maximum mean concentration (estimated from last four timepoints) 

TABLE03 

Pharmacokinetic Parameters in B6C3Fl Mice After a Single Gavage Dose of Phenolphthaleina 

Male 

50 482 f 23 6.28 0.104 29.5 4.00 

125 742 f 18 6.52 0.168 50.7 0.50 

Female 

50 676 f 55 6.68 0.074 44.5 0.50 

125 1,051 * 18 6.96 0.119 79.0 5.oo 

a The data were calculated from the plasma concentration-time curves, where each point represents the mean of three male or three female 

50 mg/kg mice or of two male or two female 125 mglkg mice. 

AUC; = area under the curve to infinity 

t,h = elimination half-life 

CI = Dose/AUC; 

f c,,,, = maximummean concentration 

t,,,,, = time of maximum mean concentration (estimated from last four timepoints) 

U.S. PRIWIIG OETIIX: 1997-421-514/60452 
DEPARTMENT OF 

HEALTH & HUMAN SERVICES 

~~ 

Public Health Service FIRST-CLASS MAIL

National Toxicology Program POSTAGE & FEES PAID 

Central Data Management DHHS/NIH

P.O. Box 12233, MD El-02 PERMIT NO. G-763 

Research Triangle Park, NC 27709 

Official Business 

Penalty for Private Use - $300 

NIH Publication No. 97-3390 

November 1996 

i